University of Southern California Dissertations and Theses

Craniofacial skull joint and temporomandibular joint (TMJ) in homeostasis and disease

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Craniofacial skull joint and temporomandibular joint (TMJ) in homeostasis and disease

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Content Craniofacial skull joint and temporomandibular joint (TMJ)
in homeostasis and disease
By
Supawadee Jariyasakulroj
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CRANIOFACIAL BIOLOGY)
May 2024
Copyright 2024 Supawadee Jariyasakulroj

ii
Dedication
This dissertation is dedicated to my beloved parents, Mr. Somsak and Mrs. Laddawan
Jariyasakulroj, as well as to my sisters and brother, who have continually supported me
throughout my PhD journey.

iii
Acknowledgements
I would like to express my gratitude to my PhD mentor Dr. Jian-Fu (Jeff) Chen, for his
guidance, support, and expert mentorship throughout my PhD study. His commitment to
excellence and constant encouragement have played a crucial role in shaping my academic and
professional development. Additionally, I would like to express my gratitude to all PhD committee
members, Dr. Yang Chai, Dr. Amy Merrill-Brugger, Dr. Pinghui Feng, and the late Dr. Baruch
Frenkel. Their expertise, perspectives, and helpful guidance have greatly enhanced my research
and academic experience. I extend my appreciation to Dr. Michael Paine for being supportive and
addressing my questions during the early stages of PhD training. I would like to acknowledge
Mahidol University, Thailand along with the CBY program for providing me with a scholarship and
continued financial support.
I am also grateful to my friends and colleagues in Dr. Chen's lab, Dr. Wei Zhang and Dr.
Li Ma, for their collaboration, guidance and helpful suggestions; Qing Chang, Ziying Lin, and
Paofen Ko for their continuous support, sharing knowledge, helping me throughout PhD projects,
and creating a positive and joyful work environment; Jingyi Chen and Yang Shu for their kindness
and assistance; previous lab members, Dr. Binbin Li, Dr. Can Wang, and Jotham Sadan for their
help and full support during my PhD studies.
In addition, I am thankful to members of Dr. Chai’s lab for teaching me various lab
techniques, sharing their knowledge, and providing assistance throughout PhD projects,
especially Dr. Jifan Feng, Dr. Takahiko Yamada, Dr. Tingwei Guo, and Dr. Eva Janeckova. I also
would like to thank Dr. Jiang Qian and Prerna Sehgal for their kindness and guidance in molecular
biology techniques. I am thankful to Dr. Rucha Arun Bapat, Dr. Nicha Ungvijanpunya, and
Gayathri Visakan for their advice during the oral examination, dissertation preparation, and PhD
in the CBY program.

iv
I would like to express my appreciation to our collaborators, Dr. Zhipeng Lu, Dr. Jianhui
Bai, and Dr. Minjie Zhang for their contributions and bioinformatic analysis in my calvarial suture
MSC project. Furthermore, I am thankful to Dr. Zhen Zhao and Dr. Feixiang Chen for their
generous support in providing biomaterials for the TMJ project. I am grateful for the suggestions
and guidance provided by Dr. Zhaoyang Liu and her lab members, Fangzhou (Jo) Bian and
Colleen Feng, for their help and support in the TMJ project.
Additionally, I would like to express my gratitude to Thach-Vu Ho for his advice on microCT analysis, for always being supportive, and for effectively resolving all the lab technical issues
I encountered. I also would like to thank Janice Bea, Linda Hattemer, Zhounan Liu, Jian-Bao Xie,
Gina Nieto, Deanne Aiau, Kimi Nakaki, and all the staff and faculty members of the Center for
Craniofacial Molecular Biology at USC for creating a supportive environment and promptly
addressing all issues.
Lastly, I would like to express my deepest gratitude to my family for their constant support
throughout my PhD journey. Their encouragement, understanding, and belief in me helped me
overcome the challenges, and I would not have come this far without their support.

v
Table of Contents
Dedication......................................................................................................................................ii
Acknowledgements.......................................................................................................................iii
List of Figures...............................................................................................................................vii
Abstract..........................................................................................................................................x
Chapter 1: Introduction...................................................................................................................1
1.1 Cranial suture and craniosynostosis…………………………………………………….………….2
1.2 Temporomandibular joint (TMJ) and degeneration…………………..……………………………4
Chapter 2: Ribosome biogenesis controls cranial suture MSC fate via the complement
pathway in mouse and human iPSC models…………………………….…………………..………..7
2.1 Background………………………………………………….………………………………………..7
2.1.1 Cranial suture mesenchymal stem cells (MSCs)………………………………………7
2.1.2 The role of suture MSCs in suture development, homeostasis, and regeneration...8
2.1.3 Ribosome biogenesis……………….…………………………………………………….9
2.1.4 Dynamic translation in stem cells……………………………………………………….10
2.1.5 Hypothesis……………….………………………………………………………………..11
2.2 Materials and Methods……………………………………………………………………...………12
2.2.1 Mouse models……………….…………….……………………………………………..12
2.2.2 Micro-CT analysis ……………….…………….….……………………………………..12
2.2.3 Histology ……………………..…………….……………………………………………..13
2.2.4 Immunostaining………………………...….……………………………………………..13
2.2.5 RNAscope staining..…………………...….……………………………………………..14
2.2.6 TUNEL assay..……….………………...….………………………………………….….14
2.2.7 Tartrate-resistant acid phosphatase (TRAP) staining…………………………………14
2.2.8 Western blot analysis…………………………………………………………………….15
2.2.9 RNA isolation and quantitative RT-PCR……………………………………………….15
2.2.10 Ribosome profiling……………………………………………………………………...16
2.2.11 Bulk RNA-seq library preparation……………………………………………….........16
2.2.12 Suture explant ex-vivo. ………………………………………………………….........16
2.3 Results……………………….………………,,……………………………………………….…….17
2.3.1 Snord118 mutant mice exhibited craniosynostosis-like defects…………………….17
2.3.2 Ribosome biogenesis disruption in Gli1+ MSCs leads to p53 activation,
cell death, proliferation reduction, and loss of mesenchymal stromal cells………..…….18
2.3.3 Snord118 depletion results in premature osteogenic differentiation of MSCs
and osteoclast loss……………………………………………….…………………………….20
2.3.4 Human iPSC-derived mesenchymal stem cells exhibit impaired self-renewal and
differentiation…………………………….…………………………………………...…………23
2.3.5 Ribosome profiling revealed a translational downregulation of ribosomal protein
genes in mutant MSCs…………………………………………………………………………25
2.3.6 Snord118 deletion in MSCs leads to transcriptional dysregulation of genes
encoding complement pathway components………….…………………..……...………...26
2.3.7 Disruption of complement pathway exacerbates suture defects in Snord118
mutant mice……………………………………………..………………………………………28
2.3.8 Activation of complement pathway could rescue suture defects in Snord118

vi
mutant calvarial suture explants……………………………………………………………….30
2.4 Discussion and Future Direction…………….…………………………………………………….32
Chapter 3: Mechanisms and treatments of temporomandibular joint degeneration with pain…..35
3.1 Background…………………………………..……………..……………………………………….35
3.1.1 Structures of the TMJ……………………….…………………………………….……..36
3.1.2 Uniqueness of the TMJ……………………….…………………………………….……39
3.1.3 Etiology and risk factors of TMD…………………………………..……….……………41
3.1.4 Signs and symptoms of TMD……………………….……………………………...……42
3.1.5 Diagnostic criteria of arthrogenous TMD ………………………………………………43
3.1.6 Pathogenesis of TMJOA ………………………………………………………………..45
3.1.7 Neural processing of painful TMJ ………………………………………………………48
3.1.8 Treatment of TMJ degeneration…………………………………………..…………….52
3.1.9 Animal models in TMJOA…………………………………………..……………………55
3.2 Materials and Methods………………………………………………………………………..……60
3.2.1 Mouse model…………………………………………..…………………………………60
3.2.2 CFA intra-articular injection…………………………………………..…………………61
3.2.3 Nociceptive behavior assessment……………………………………………………...61
3.2.4 Micro-CT analysis………………...……………………………………………………...62
3.2.5 Histology………...………………...……………………………………………………...62
3.2.6 Safranin O and fast green staining………………………………………………….....63
3.2.7 Immunostaining………………………………………………………………………......63
3.2.8 RNAscope staining…………………………………………………………...……….....64
3.3 Results…………………………………………………………...……………………….......…......64
3.3.1 Established painful TMJ degeneration mouse model…………………………….…..64
3.3.2 Inflammatory response surrounding TMJ after CFA injection……………………….68
3.3.3 Neuroimmune interaction in TMJ degeneration and pain……………………………69
3.3.4 Distinct anatomical features of synovial lining cells in TMJ……………..……………73
3.3.5 Increased nerve innervation in fibrous tissue surrounding TMJ……………...………75
3.4 Discussion and Future Direction………………….………………………………………………..78
Bibliography…………………………………………,,…………………………………………………..81

vii
List of Figures
Figure 1.1. The craniofacial skeleton is formed by cranial and facial bones………………………...1
Figure 1.2. Muscle of mastication and facial expression…………………………………….……….2
Figure 1.3. Comparative anatomy of human and mouse skull……………………………………….3
Figure 1.4. Anatomy of the TMJ………………………………………………………………………...5
Figure 2.1. The distribution pattern of suture MSCs, including Gli1+ MSCs, Axin2+ MSCs,
Prrx1+ MSCs, and Ctsk+ MSCs……………………………………………………………..……………8
Figure 2.2. Gli1+ suture MSCs contribute to several components of the cranial suture…………..9
Figure 2.3. The structure of Snord118, a small nucleolar RNA (snoRNA) encodes the box
C/D snoRNA U8………….……………………………………………………………..……………….12
Figure 2.4. Disruption of ribosome biogenesis in MSCs results in reduced cranial suture
volume and craniosynostosis-like defects.………….………………………………………...……...19
Figure 2.5. Snord118 depletion in Gli1+ MSCS leads to p53 upregulation, cell death,
proliferation reduction, and loss of mesenchymal stromal cells. ………….……………………….21
Figure 2.6. Disruption of ribosome biogenesis leads to premature osteogenic differentiation
and loss of osteoclasts. ………….………………………………………………………………….….22
Figure 2.7. Mutant iPSC-MSCs exhibit impaired self-renewal, differentiation, and translational
dysregulation of ribosomal protein genes……………………………...……………………………..24
Figure 2.8. Snord118 depletion results in transcriptional and translational dysregulation of
genes encoding complement pathway………………………………………………………………..27
Figure 2.9. Complement pathway disruption leads to more severe suture defects in Snord118
mutant mice….………….……………………………………………………...………………….…….29
Figure 2.10. Activation of complement pathway activation could rescue suture defects in
calvarial suture explants of Snord118 mice………………………………………………….……….31

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Figure 3.1. TMD symptoms encompass both pain and dysfunction affecting the TMJ and
masticatory muscles…………………………………...…………………………….………….…...…35
Figure 3.2. TMJ and associated structures…..………………………………………………….……36
Figure 3.3. Major factors and the associated genes that increase the risk for an individual to
develop TMD symptoms……………………………………………………………..…………………42
Figure 3.4. Diagnostic criteria for arthritis or arthralgia………………………………………………44
Figure 3.5. Diagnostic criteria for TMJOA or DJD……………………………………………………45
Figure 3.6. Overview of the trigeminal nerve (CN V) and trigeminal ganglion…………………...50
Figure 3.7. Pain transmission from the trigeminal ganglion (first-order neuron) to the spinal
trigeminal nucleus (second-order neuron), thalamus (third-order neuron), and sensory cortex..51
Figure 3.8. Minimally invasive procedures and the compounds delivered with an intra-articular
technique…………………………………..……………………………..……………………………...53
Figure 3.9. (A)Arthrocentesis procedure.(B)The location of the two needles for arthrocentesis...55
Figure 3.10. Mouse TMJ intra-articular injection technique…………………………………………61
Figure 3.11. Increased subchondral bone loss in the CFA intra-articular injection group………65
Figure 3.12. Cartilage remodeling in CFA-treated mice…………………………………………….66
Figure 3.13. Inflammation in synovial tissue after 4 weeks of CFA injection……………………..66
Figure 3.14. Nociceptive behavior assessment……………………………………………………...67
Figure 3.15. The infiltration of inflammatory cells surrounding TMJ after CFA injection……….68-69
Figure 3.16. Increased sensory nerve innervation in the CFA group. …………………………….70
Figure 3.17. Increased neurovascular structures surrounding TMJ area after CFA injection……71
Figure 3.18. Neuroimmune response after CFA injection with increased CGRP nociceptive
neurons and Iba1+ macrophages in TG as well as microglia activation in SpVc………..……….72
Figure 3.19. Synovial lining cells in TMJ in normal condition………………………………………..74
Figure 3.20. Synovial fibroblasts in TMJ.……………………………………………..…………..74-75

ix
Figure 3.21. Increased nerve innervation and fibrous tissue in CFA-induced inflammatory
TMJOA……………………………………..……………………………..……………………………...76
Figure 3.22. Increased IL-33 expression in CFA-induced inflammatory TMJOA….………...……77

x
Abstract
Disruption of ribosome biogenesis selectively affects craniofacial biology and disease,
including craniosynostosis, but the underlying mechanisms remain poorly understood. Suture
mesenchymal stem cells (MSCs) serve as the engine that drives calvarial suture development,
homeostasis, and regeneration. Its loss leads to craniosynostosis, a craniofacial disorder
characterized by premature suture closure. Ribosome biogenesis, historically thought to be a
static housekeeping process, is now known to have tissue-specific roles. However, the functional
specificity of ribosome biogenesis in suture MSCs remains largely unexplored. Here, we
genetically perturbed ribosome biogenesis in MSCs using Snord118, a small nucleolar RNA
(snoRNA) required for ribosomal RNA (rRNA) maturation. Snord118 mutant mice exhibited
craniosynostosis-like defects with p53 activation, increased cell death, reduced proliferation, and
premature osteogenic differentiation of MSCs. We established a method to induce human induced
pluripotent stem cells (iPSCs) into suture MSCs and used CRISPR genome editing to generate
SNORD118 mutations in iPSCs. Ribosome profiling of human iPSC-MSCs revealed that
SNORD118 deficiency in MSCs causes global translation dysregulations and downregulation of
complement pathway. Further loss-of-function of complement pathway using complement C3a
receptor 1 (C3ar1) KO mice exacerbated cellular defects leading to suture fusion in Snord118
mutant mice, while complement pathway activation rescued MSC cell fate and suture growth
defects. Thus, ribosome biogenesis is crucial for the regulation of MSC fate via the complement
pathway.
Temporomandibular joint (TMJ) osteoarthritis (OA) is a common and debilitating disease
characterized by joint degeneration with synovitis, cartilage remodeling, and subchondral bone
destruction along with painful conditions. Disease-driving pain mechanisms are poorly
understood, and current treatments cannot provide effective and long-term therapeutic effects.
Here we established an inflammatory TMJOA mouse model via intra-articular injection of CFA

xi
(Complete Freund’s Adjuvant). TMJOA mice exhibited cartilage remodeling, bone loss, synovial
inflammation, and orofacial pain behaviors which recapitulate clinical characteristics in TMJOA
patients. Immunofluorescence and RNAscope staining revealed neuroimmune interaction in
painful TMJ degeneration and distinct anatomical features of synovial lining cells in TMJ.
Additionally, sensory innervation was robustly induced surrounding TMJ coupled with the
expansion of fibroblasts and macrophages, contributing to OA pain generation and progression.
An increased nerve innervation in fibrous tissue surrounding TMJ points toward a potential
direction to further investigate the functional importance of fibroblasts in TMJ pain and
degeneration. Together, we established mouse models and provided cellular mechanisms of
TMJOA and pain, which help identify treatment strategies to improve TMJ pain management and
restore TMJ functions.

1
Chapter 1 Introduction
Craniofacial structures are composed of several components and specialized tissues such
as the cranium, brain, sensory organs, cartilage, muscles, ligaments, and teeth. Each structure
develops into its unique form and shape to facilitate its purpose and coordinate with other
structures to provide vital living functions, including digestive and respiratory. Muscle and jaw
movements are crucial for speech, facial expression, and mastication. The craniofacial skeleton
is also an important structure that helps protect the brain and sensory organs which serve their
function in vision, hearing, smell, and taste (Du et al., 2021). Disruption in their development or
any disturbance to their function could result in structural malformation and dysfunction with the
consequence of impaired quality of life.
The craniofacial skeleton is the bony structure of the head that can be subdivided into
cranial bones and facial bones. Cranial bones are formed by frontal, occipital, ethmoid, sphenoid,
parietal, and temporal bones. Their important function is to protect the brain from impact. The
facial bones provide the framework to form the nasal cavity, enclose the eyeballs, and support
the teeth. They consist of lacrimal, nasal, palatine, inferior turbinate, vomer, zygomatic, maxillary,
and mandibular bones (Figure 1.1). Both cranial and facial bones are the sites of attachment for
the muscles of facial expression and masticatory muscles which function in facial expression and
jaw movement (Figure 1.2) (Du et al., 2021; Tse et al., 2014).
Figure 1.1. The craniofacial skeleton is formed by cranial and facial bones (Tse et al., 2014).

2
Figure 1.2. Muscle of mastication and facial expression (Du et al., 2021).
Skeletal joints are the sites of articulation between two bones. Joints can be classified into
three types by tissue components: fibrous, cartilaginous, and synovial tissues. They also can be
categorized into three types by their function, or the degree of movement permitted: synarthrosis
(little or no movement), amphiarthrosis (slight movement), and diarthrosis (free movement). Major
skeletal joints that present in craniofacial structures are fibrous joints (skull-type sutures) and
synovial joints (temporomandibular joints) (Lawry and Bewyer, 2008).
This study focuses on two important skeletal joints of craniofacial structures. One focal
point is cranial sutures where disruption of suture homeostasis can lead to craniosynostosis. The
second focal point is the temporomandibular joint (TMJ) and associated structures with pain and
degeneration.
1.1 Cranial suture and craniosynostosis
Cranial suture is a fibrous joint where calvarial bones join together and allow little or no
movement (synarthrosis) (Anderson et al., 2024; Lawry and Bewyer, 2008). There are four major
cranial sutures in the skull vault. The metopic or frontal suture separates the two paired frontal

3
bones. The sagittal suture is located between the parietal bones. The coronal suture separates
the frontal and the left/right parietal bones. The most posterior suture is the lambdoid suture that
lies between the left/right parietal and occipital bones in humans, or interparietal bone in rodents
(Figure 1.3) (Ang et al., 2022) (Li et al., 2021b).
Figure 1.3. Comparative anatomy of human and mouse skull. The major calvarial bones and
cranial sutures are labeled. Fb, frontal bone; CS, coronal suture; FS, frontal suture; Ipb,
interparietal bone; LS, lambdoid suture; MS, metopic suture; Nb, nasal bone; Ob, occipital bone;
Pb, parietal bone; SS, sagittal suture; SqS, squamosal suture (Ang et al., 2022).
The growth and development of cranial sutures play a crucial role in brain development.
At birth, the sutures remain open to enable the skull to expand in concert with the rapidly growing
brain. Each cranial suture undergoes fusion at a different time. In humans, the metopic suture is
the first suture that usually fuses within three to nine months after birth, whereas other sutures
remain patent until 20-40 years old. However, the posterior part of the frontal suture in mice starts
to fuse within one month postnatally, while other sutures remain patent throughout their life (Li et
al., 2021b; Menon et al., 2021).
Despite the discrepancy in the time point of suture fusion, cranial mouse sutures have
several similar fundamental features to humans. The four principal components of suture include:
the osteogenic fronts of the approximating bone plates, the suture mesenchyme within the suture,
the overlying periosteum, and the underlying dura mater. Under normal conditions, bone
resorption and new bone formation occur at osteogenic fronts, while mesenchymal stem cells

4
(MSCs) within the suture stay undifferentiated, which allows the suture to remain patent and the
skull to enlarge evenly in alignment with brain growth. It has been well-studied that loss of suture
stem cells disrupts suture homeostasis and consequently leads to cranial suture fusion or
craniosynostosis (Li et al., 2021b; Zhao et al., 2015).
Craniosynostosis is a congenital defect that occurs as a consequence of premature cranial
suture fusion. This craniofacial abnormality can result in developmental delays, neurological
dysfunction, facial deformities, and psychosocial issues. Several genetic and environmental
factors have been implicated as potential initiating factors for the development of craniosynostosis
(Kajdic et al., 2018; Zhao et al., 2015).
The current standard treatment of craniosynostosis involves several surgical
interventions, such as a strip craniectomy, cranial vault expansion, and a reshaping procedure.
The major goal of these complex surgeries is to correct deformity and create suture patency,
which is essential to reduce intracranial pressure and provide more space for the brain to grow.
However, premature suture fusion can happen again during the follow-up period leading to
surgical revision. Moreover, postoperative surgical complications can occur, such as bleeding,
hematoma, hypovolemic shock, cerebrospinal fluid fistula, and complications from the general
anesthesia (Li et al., 2021b; Stanton et al., 2022) Therefore, understanding the mechanisms of
craniosynostosis and investigating associated risk factors could benefit those afflicted by the
malformation and disfunction with an improved therapeutic approach.
1.2 Temporomandibular joint (TMJ) and degeneration
The TMJ is a synovial joint that can be categorized as a diarthrosis joint performing
complicated jaw movement. Most of synovial joints share the same important structural
components, including cartilage covering the bone surfaces, a fibrous articular capsule uniting all
the joint components together, supporting ligaments, and synovial membrane lining the inner
surface of the capsule. However, there is no synovial membrane covering the articular cartilage.

5
TMJ structure comprises of the mandibular condyle that fits into the glenoid fossa of the
temporal bone, surrounded by capsular ligaments (Figure 1.4). The capsular ligament is a fibrous
membrane that covers all TMJ structures. It attaches to the articular eminence, disc, and the neck
of the mandibular condyle. The complex movement of TMJ is facilitated by masticatory muscles,
which are the group of muscles associated with jaw movements, such as chewing, biting, and
mouth opening. There are four muscles, including temporalis, masseter, lateral pterygoid, and
medial pterygoid muscles (Chung et al., 2023a; Langendoen et al., 1997).
Figure 1.4. Anatomy of the TMJ. (A) TMJ is formed by the mandibular condyle and the temporal
bone of the cranium. (B) The articular capsule or capsular ligament covers all the TMJ
components. Masticatory muscles associated with jaw movement; temporalis, masseter, lateral
pterygoid (not shown), and medial pterygoid (not shown) muscles
(https://www.encinosleepandtmj.com/tmj-disorder/).
The articular surface of TMJ is covered by cartilage, a connective tissue with special
viscoelastic properties. The main function of cartilage is to provide a smooth and lubricated
surface for low-friction articulation. Additionally, it possesses the ability to resist compressive
forces and provides support for underlying subchondral bone. However, overloading on the TMJ
surface or any excessive force beyond the adaptability of TMJ structures can result in cartilage
damage, subchondral bone remodeling, and chronic inflammation in the synovial tissues. The
A B

6
inflammatory response that occurs as part of TMJ degeneration or osteoarthritis (OA) can cause
pain and dysfunction leading to impaired quality of life (Chung et al., 2023a; Wang et al., 2015).
Current management of TMJ degeneration is mainly symptomatic treatment to suppress
inflammatory processes, relieve pain, restore TMJ function, and prevent further deformity. The
initial treatment of painful TMJOA includes medication and behavioral management. Pain relief is
a major part of treatment since painful symptom is the primary chief complaint for TMJ disorders.
The most common medications prescribed for painful TMJOA are NSAIDs (nonsteroidal antiinflammatory drugs) which can reduce both pain and inflammation. However, NSAIDs can cause
several side effects, such as gastrointestinal distress (indigestion, ulcers, and bleeding in the
stomach), raised liver enzymes, drowsiness, and other complications from drug interaction. Some
patients are also at higher risk of developing serious complications from taking NSAIDs, including
patients who are over 65 years old, pregnant, breastfeeding, have asthma, high blood pressure,
or have any problems related to heart, liver, or kidney disease. Although the alternative pain
medication is paracetamol which is safe for most people to ingest, it is only used for mild pain and
has no anti-inflammatory effect. In addition, the effectiveness of intra-articular injection and
arthrocentesis, which are usually provided for moderate or severe TMJOA, is still inconclusive.
These injections do not provide long-term pain relief or structural regeneration. Additionally, joint
replacement for severe TMJ damage is an invasive treatment and can cause serious
complications from surgery (Al-Ani, 2021; Sperry et al., 2019). Thus, there remains a demand in
research to identify novel targets and enhance effective approaches, aiming to address the unmet
clinical needs associated with TMJ degeneration.

7
Chapter 2
Ribosome biogenesis controls cranial suture MSC fate via
the complement pathway in mouse and human iPSC models
2.1 Background
2.1.1 Cranial suture mesenchymal stem cells (MSCs)
The complex interplay between bone formation and resorption is essential for maintaining
suture patency or homeostasis. Disruption of the osteogenic balance which increases bone
deposit at osteogenic fronts can subsequently lead to premature suture fusion or
craniosynostosis.
Previous studies have shown that suture mesenchymal stem cells (MSCs) play a crucial
role in maintaining suture patency. Several MSCs have been identified residing in the cranial
sutures, such as Gli1-positive (Gli1+
) cells, Axin2-expressing (Axin2+
) cells, Cathepsin K
expressing (Ctsk+
) cells, and postnatal Prrx1-expressing (Prrx1+
) cells (Doro et al., 2017;
Maruyama et al., 2016; Zhang et al., 2020) (Figure 2.1). These suture stem cells exhibit selfrenewal and multipotent differentiation potential and interact with neighboring cells to regulate
osteogenic balance in the suture homeostasis (Menon et al., 2021; Zhao et al., 2015). The
different distribution of these stem cells has been found in cranial suture mesenchyme. Axin2+
cells are expressed in the midline of the suture mesenchyme from postnatal day 10, while Gli1+
cells cover a larger area and broadly distribute within the suture mesenchyme, periosteum, and
dura. Prrx1+ cells are also detected in calvarial sutures from two weeks of age until late adulthood
in mouse models. However, both Prrx1+ and Axin2+ cells are not observed in the periosteum and
dura mater. Additionally, all of these stem cells could not be detected in the posterior frontal suture
(Doro et al., 2017; Maruyama et al., 2016). Studies on gene expression profiles in suture stem
cells have found the overlap expression of Gli1 and Axin2 in stem cell populations. The distinct

8
stem cell populations and differential localizations of Axin2+
, Gli1+
, and Prrx1+ cells might
contribute to regulatory mechanisms to maintain suture homeostasis and promote suture
regeneration in different calvarial areas (Li et al., 2021b; Maruyama et al., 2016).
Figure 2.1. The distribution pattern of suture MSCs, including Gli1+ MSCs, Axin2+ MSCs, Prrx1+
MSCs, and Ctsk+ MSCs (Li et al., 2021b).
2.1.2 The role of suture MSCs in suture development, homeostasis, and regeneration
Stem cells are essential for normal suture development and maintaining suture patency
(Doro et al., 2017). The deletion of Gli1+
cells could result in severe craniofacial phenotypes, suture
fusion, reduced body size, and severe osteoporosis. The numbers of Gli1+
cells are reduced in all
the sutures in Twist1+/-
craniosynostosis mice compared to wild-type control, suggesting that loss
of stem cells could lead to craniosynostosis (Zhao et al., 2015). Similarly, premature cranial suture
fusion has been found in mice with deletion of Axin2+
cells. A decrease in Axin2-expressing cells
is also observed in the posterior frontal suture. These findings indicated that suture stem cells
developmentally regulated the posterior frontal suture (Maruyama et al., 2016). Additionally,
ablation of Prrx1+ cells consequently leads to incomplete bone formation and calvarial bone
healing impairment (Doro et al., 2017). Moreover, the crucial role of suture MSCs in suture
homeostasis and regeneration has been highlighted in the study of MSC suture implantation.
Previous studies have shown that Gli1+ MSCs implantation can regenerate suture defects and
mitigate neurocognitive defects in craniosynostosis mice, indicating a crucial role of suture MSCs

9
in suture homeostasis and regeneration (Yu et al., 2021). Therefore, suture stem cells are
required for development, homeostasis, and regeneration.
Gli1 is a transcriptional factor of the Hedgehog signaling pathway. Gli1+ cells have been
found as the main stem cell population in the suture mesenchyme in adult craniofacial bone. They
can give rise to periosteum and dura (Figure 2.2.) (Ang et al., 2022; Zhao et al., 2015).
Figure 2.2. Gli1+ suture MSCs contribute to several components of the cranial suture. (A) Gli1+
MSCs in suture mesenchyme can give rise to osteogenic fronts, periosteum, and dura to support
suture homeostasis. (B) The expression of Gli1 in calvarial bones from whole-mount LacZ staining
(blue) at different postnatal time points of Gli1–LacZ mice (Zhao et al., 2015).
Gli1+ cells are expressed in the entire periosteum, dura mater, and suture mesenchyme
at postnatal day 0 (P0) in mouse calvarial bones, then they are gradually restricted to the midline
of sutures from postnatal day 21 (P21) onwards, and still detected in the suture mesenchyme,
periosteum, dura and parts of the calvarial bone in later stage (Figure 2.2). Ablation of Gli1+ cells
can disrupt skull growth and consequently lead to craniosynostosis. Therefore, Gli1+ MSCs are
indispensable for the suture homeostasis (Zhao et al., 2015).
2.1.3 Ribosome biogenesis
In addition to the importance of MSCs in suture homeostasis, the role of ribosome
biogenesis has been highlighted in craniofacial development (Ross and Zarbalis, 2014).
Ribosome is an essential organelle that functions in protein synthesis. It consists of ribosomal
RNA (rRNA) and ribosomal proteins (RPs). Ribosome biogenesis is the process of making

10
ribosome, which is tightly regulated and begins in the nucleolus of eukaryotic cells. There are
three major processes: rRNA synthesis, rRNA processing, and ribosome maturation. This process
begins with transcription of both 47S pre-rRNA, containing 18S, 5,8S, and 28S rRNA, by RNA
Polymerase I (RNA Pol I), and 5S rRNA by RNA Polymerase III (RNA Pol III). Then, the 47S prerRNA is modified, processed and cleaved into 18S, 5,8S, and 28S rRNAs. The 18S rRNA and
small subunit RPs will form the 40S subunit, which functions in decoding mRNA sequence. The
5S, 5.8S, and 28S along with large subunit RPs will form the 60S subunit, which links amino acid
through peptide bonds. These ribosomal subunits are transported from the nucleolus to the
cytoplasm and form mature 80S ribosome to function in protein translation (Saba et al., 2021;
Yelick and Trainor, 2015). Therefore, ribosome biogenesis is a fundamental cellular process
related to mRNA translation and protein synthesis which is essential for all cells.
Although ribosome biogenesis is universally required for all cells and tissues, perturbation
in ribosome biogenesis or ribosomopathies caused by mutations in ribosomal proteins and
ribosome biogenesis factors results in tissue-specific disorders, such as craniofacial deformities
in Treacher Collins syndrome (TCS). Moreover, disruption in ribosome biogenesis also results in
a decrease in protein production and subsequently reduces the advancement of osteogenic fronts
in the frontal suture development (Holmes et al., 2020; Neben et al., 2014; Neben et al., 2017).
These findings indicate the crucial role of ribosome biogenesis in craniofacial development.
2.1.4 Dynamic translation in stem cells
Furthermore, protein synthesis and translation in stem cells during proliferation and
differentiation are dynamic and sensitive to ribosome biogenesis. Recent findings have showed
global translation is maintained at a low level in undifferentiated stem cells, while translation
efficiency is increased during stem cell differentiation, suggesting that a tightly regulated
translation rate is required to coordinate with the differentiation and commitment status of stem
cells (Gabut et al., 2020; Saba et al., 2021).

11
Moreover, low protein synthesis and high ribosome biogenesis in stem cells could play an
important role in maintaining stem cell self-renewal and differentiation. Stem cells maintain their
undifferentiated state via global translation repression resulting in a low abundance of proteins.
When stem cells receive signals to differentiate, an old proteome must be removed and replaced
with the new correct proteins to support the differentiation process. Thus, a low level of proteins
might support stem cells to erase the old proteins rapidly, while upregulated ribosome biogenesis
could provide a large pool of ribosomes for stem cells to synthesize new proteins and enable
efficient stem cell differentiation. Therefore, an overabundance of proteins or inappropriate
numbers of ribosomes could negatively affect the onset of differentiation (Saba et al., 2021).
In conclusion, tight regulation of protein synthesis machinery, including ribosome and its
associated factors, is essential for stem cell self-renewal and differentiation.
2.1.5 Hypothesis
Based on previous studies, Gli1+
MSCs within the suture play an important role in
maintaining suture homeostasis, and disruption of ribosome biogenesis is associated with cranial
suture defects. However, the underlying mechanisms by which dysregulation of ribosome
biogenesis in MSCs contributes to craniosynostosis remain largely unknown. Therefore, we
hypothesized that ribosome biogenesis is essential for MSC function to maintain suture
homeostasis.
To further elucidate the functions of ribosome biogenesis in MSCs during cranial suture
homeostasis, Snord118 was used as a genetic tool to perturb ribosome biogenesis in MSCs.
Snord118 is a small nucleolar RNA (snoRNA) that encodes the box C/D snoRNA U8 (Figure 2.3).
It acts as a ribosome biogenesis factor that guides chemical modifications of ribosomal RNA
(rRNAs) and is required for rRNA maturation in the ribosome biogenesis (Jenkinson et al., 2016;
Peculis and Steitz, 1993).

12
Figure 2.3. The structure of Snord118, a small nucleolar RNA (snoRNA) encodes the box C/D
snoRNA U8 (Jenkinson et al., 2016; Peculis and Steitz, 1993).
2.2 Materials and Methods
Snord118, a snoRNA involved in ribosome biogenesis, was used as a genetic and
molecular tool to disrupt ribosome biogenesis in MSCs by knocking out Snord118 in human iPSCderived MSCs and Gli1-CreERT2;Snord118f/f mouse model.
2.2.1 Mouse models
Gli1-CreERT2 (JAX#007913), tdTomato (JAX#007905), C3aR1-/- (JAX#033904), and
C57BL/6J (JAX#000664) mouse lines were obtained from Jackson Laboratory. For induction of
Cre lines, tamoxifen (Sigma T5648) was suspended in corn oil (Sigma C8267) at 20mg/mL and
injected intraperitoneally at a dose of 1 mg per 10 g body weight for 2 consecutive days. Mice
were injected with tamoxifen at P20 and their sutures collected at the indicated time points after
induction. C57BL/6J mice were used as controls. All mice were housed in specific pathogen-free
conditions under a 12 h light/dark cycle, with controlled temperature and humidity. Mice were
euthanized via carbon dioxide overdose followed by cervical dislocation. All studies were
performed with the approval of the Institutional Animal Care and Use Committee of the University
of Southern California.
2.2.2 Micro-CT analysis
Calvarial bones were dissected and fixed in 4% paraformaldehyde at 4C overnight.
Samples were radiographed using Skyscan. Images were collected at a resolution of 20 µm using

13
a 70 kVp and 114 µA X-ray source. AVIZO 9.4.0 (Thermo Fisher Scientific) was used to perform
3D reconstruction and measure coronal and sagittal suture volume. At least 3 mice from
independent mouse litters were used to calculate suture volume for each group or genotype. Each
dot in the graph quantification represents each sample (n). Student’s t-test and ANOVA were
used for statistical analysis. A significant level was set at p-value of 0.05.
2.2.3 Histology
Calvarial bone samples were dissected and fixed in 4% paraformaldehyde at 4C
overnight. The samples were decalcified in 10% EDTA for one week and passed through serial
concentrations of ethanol and paraffin embedding. Haematoxylin and eosin staining was
performed on deparaffinized sections of 12 µm.
For cryosections, the samples were decalcified in 10% EDTA for one week and
dehydrated gradually in 15% sucrose for 2 hours, followed by 30% sucrose for 2 hours, and 60%
sucrose/OCT (Tissue-Tek, Sakura) (1:1) at 4C overnight. Then, samples were embedded in OCT
compound, frozen in dry ice, and sectioned at 12 µm thickness using cryostat (Leica CM1850).
2.2.4 Immunostaining
Coronal and sagittal cryosections were used for immunofluorescence staining following
standard protocols. Antigen unmasking solution (Vector, H-3300) was used for antigen retrieval.
The primary antibodies were as follows: Runx2 (Cell Signaling Technology; #12556, 1:100), Sp7
(Abcam; ab209484, 1:100), Cathepsin K (CTSK; 11239-1-AP; 1:200), Gli1 (Novus, NBP1–78259,
1:50), C1R (Proteintech; 17346-1-AP; 1:100), C1S (Proteintech;14554-1-AP; 1:100), MASP1
(Proteintech; 21837-1-AP; 1:100), C3/C3b/C3c (Proteintech; 21337-1-AP; 1:100), and p53 (Leica;
p53 Protein (CM5); 1:200). Pdgfr-a (Cell Signaling Technology; #3174S, 1:200), CD31 (R&D
Systems; AF3628; 1:200), βIII-Tubulin (Cell Signaling Technology; #5666S, 1:200). Alexa Fluor
488/568/647 (Invitrogen; 1:200) were used as secondary antibodies. DAPI (Invitrogen; 62248)

14
was used for nuclear staining. The percentage of positive immunofluorescence signals was
determined using ImageJ software. Quantification was performed at 20X magnification, 3 to 5
sagittal or coronal suture sections were analyzed per mouse. Each dot presented in the graph
quantification represents the mean value of each sample or mouse in that group. At least three
mice from independent litters were analyzed for each group or genotype. The quantified area is
the total area of suture mesenchyme in the cranial suture. Student’s t-test and ANOVA were used
for statistical analysis. A significant level was set at p-value of 0.05.
2.2.5 RNAscope staining
Sample preparation and RNAscope staining were performed according to standard ACD
protocol: The RNAscope® Multiplex Fluorescent Reagent Kit v2 (Cat. No. 323100). The probes
used for this study were as follows: Mm-Gli1 (311001-C1) and Mm-Twist1 (1266861-C1). A
negative control probe (320871) was used when staining and taking images to remove
background signals. Quantification of RNAscope staining by ImageJ was performed at 20X
magnification and interpreted according to ACD scoring guidelines. At least three mice from
independent litters were analyzed for each group or genotype. The quantified area is the total
area of suture mesenchyme in the cranial suture. Student’s t-tests were used for statistical
analysis with a significant level p-value of 0.05.
2.2.6 TUNEL assay
Cryosection slides were dried at room temperature for 30 minutes. Apoptotic cells were
detected using a TUNEL assay (ApopTag Fluorescein In Situ Apoptosis Detection Kit, SigmaAldrich, S7110) following the manufacturer’s protocols. Quantification of TUNEL-positive cells
was analyzed using the same method as IHC staining.
2.2.7 Tartrate-resistant acid phosphatase (TRAP) staining

15
TRAP staining was performed on cryosections using a commercial acid phosphatase
leucocyte (TRAP) kit following the standard protocols (Sigma-Aldrich, 387A). Quantification was
analyzed in the same method as IHC staining. In addition, TRAP-positive multinucleated cells
were defined by TRAP+ signals with more than three nuclei in one cell as determined by light
microscopy at 20X magnification.
2.2.8 Western blot analysis
Protein lysates from Human iPSCs-derived MSCs were prepared with lysis buffer
(Pierce RIPA Buffer #89900) and a protease and phosphatase inhibitor cocktail (Pierce Protease
and Phosphatase Inhibitor Mini Tablets #A32959). Then, lysates were separated by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to PVDF
membranes. The membranes were incubated with the following primary antibodies: C1R
(Proteintech; 17346-1-AP; 1:1000), C1S (Proteintech;14554-1-AP; 1:1000), MASP1 (Proteintech;
21837-1-AP; 1:1000), C3/C3b/C3c (Proteintech; 21337-1-AP; 1:1000), β-Actin as a loading
control (Cell Signaling Technology; β-actin antibody #4967; 1:2000), and followed by horseradish
peroxidase (HRP)-labeled secondary antibodies (Bio-Rad Goat Anti-Rabbit IgG (H+L)- HRP
conjugate, # 172-1019). The densitometry of individual blot signals was normalized to respective
controls of their primary antibodies followed by Student’s t-test statistical analyses.
2.2.9 RNA isolation and quantitative RT-PCR
Trizol (Invitrogen;15596026) was used to extract total RNA from human iPSCs-derived
MSCs. One microgram of RNA was reversely transcribed. Quantitative real-time PCR (qPCR)
was performed to determine gene expression levels using specific primers: C3
(Hs00163811_m1), C1R (Hs00357637_m1), MASP1 (Hs00373559_m1), and C1S
(Hs01043803_g1). Reaction conditions for amplification followed the manufacturer’s protocols.
The relative values in each sample were calculated and normalized to a housekeeping gene
(GADPH).

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2.2.10 Ribosome profiling
Ribosome profiling was performed as previously described (Ingolia et al., 2009) with minor
modifications in the condition of RNase digestion. Two replicates for each sample were collected
and for each replicate, three 10-cm dishes were collected. When cells were ready, cell media was
aspirated, and cells were washed with ice-cold PBS. After removing PBS thoroughly, the dish was
added 400 uL ice-cold lysis buffer, which was then placed on wet ice for 5 min. Cells were then
scraped off to a 1.5 mL tube and added NP40 (10%) for complete lysis for 10 min on ice. Lysates
were homogenized by pipetting and triturated ten times through a 25-gauge needle if the cell
lysates were not clear. The lysate was cleared by centrifugation at 2,000 g 4°C for 10 min and
then 20,000 g 4°C for 10 min. The supernatant was transferred to a fresh microfuge tube. Lysate
RNA was measured with Qubit HS RNA kit and certain amounts of lysates were used for digestion
at concentration (600 ng Rnase A + 75 U Rnase T1/ug RNA) at 25°C for 30 min(Liu et al., 2019).
The reaction was stopped by chilling on ice and adding 50 U SUPERase In RNase inhibitor (2.5
uL/sample) and 0.5 M EDTA (100x). Following nuclease digestion, monosomes were purified
using ultracentrifugation in a SW55i rotor at 55,000 rpm at 4°C for 5 h. PNK treatment was
performed after pellet RNA recovery. Then 15% polyacrylamide TBE-Urea gel was used to isolate
RPFs (26-34 nt) and recovered RPFs were quantified for library preparation. The reagents and
steps of library preparation were previously described in detail (Zhang et al., 2021). Ribosomal
rRNA depletion was performed after circularization step using pre-synthesized biotinylated
probes. The libraries prepared were pooled and sequenced.
2.2.11 Bulk RNA-seq library preparation
Bulk RNA library preparation was performed following the protocol given in NEBNext®
Ultra™ II RNA Library Prep Kit for Illumina® (E7775S). 1 ug total RNA was used for starting
material for library preparation. rRNA Depletion Kit v2 (Human/Mouse/Rat) (E7405L) was used
for the depletion of ribosomal rRNA. The libraries prepared from total RNA for different conditions

17
were pooled and sequenced. For data analysis, the adapter sequences of the sequencing data
were removed with trimmomatic (v0.36). After data preprocessing, the reads were aligned to
GRCh38 with HISAT2 (v2.2.1). FeatureCounts (v2.0.1) was used to generate counts of reads
mapping to exons of these transcripts for total RNA. The RNAseq data have been deposited to
GEO under accession code GSE223614.
2.2.12 Suture explant ex-vivo
Ex-vivo suture explant experiments were modified from previous protocols (Menon et al.,
2021). WT and Gli1-CreERT2;Snord118f/f mice were injected with tamoxifen at postnatal day 3 (P3).
Their calvarial bones were collected at P4 and placed on a wire mesh in a 24-well plate, and
cultured for ten days in DMEM, GlutaMax, 10% fetal bovine serum (FBS), and 1% penicillin-strep.
For the rescue group, calvarial bones from Gli1-CreERT2;Snord118f/f mice were cultured in the
medium with 10 uM C3a Receptor agonist (Cayman Chemical, 21683). After ten days in culture,
calvarial bones were washed with PBS and fixed with 4%PFA at 40
C overnight. The samples were
decalcified with 10% EDTA for 3 days and processed for paraffin sections and cryosections as
mentioned above. The suture area for quantification was defined by the total area of suture
mesenchyme in suture sections and quantified by ImageJ.
2.3 Results
Results of the presented work have been published in Stem Cell Reports, 2023
(https://doi.org/10.1016/j.stemcr.2023.10.015).
2.3.1 Snord118 mutant mice exhibited craniosynostosis-like defects.
To investigate the functions of ribosome biogenesis in MSCs during cranial suture
homeostasis, we generate Snord118 MSC-specific heterozygous and homozygous conditional
knockout (cKO) mice by crossing Snord118f/f mice with Gli1-CreERT2 mice and performing
tamoxifen injection at postnatal day 20 (P20) to specifically disrupted Snord118 in Gli1+
MSCs.

18
Suture morphology and suture volume of Gli1-CreERT2;Snord118f/f and Gli1-CreERT2;Snord118f/+
mice were examined and compared to wild-type mice. The volume of coronal and sagittal sutures
was significantly reduced in Gli1-CreERT2;Snord118f/f mice compared to wild-type mice at one
month after tamoxifen induction. Histological analysis also showed narrower suture gaps in Gli1-
CreERT2;Snord118f/f mice compared to control (Figures 2.4A-2.4D). In addition, coronal suture
malformation in Snord118 mutant mice resembles suture defects in Twist1+/- craniosynostosis
mice. Therefore, we further examined Twist1 expression and found that Twist1+ cells are
significantly reduced in Snord118 mutant coronal sutures (Figures 2.4E-2.4F). To examine the
relative long-term consequence of suture growth defects, we analyzed homozygous cKO mice at
3 months post-tamoxifen induction and found craniosynostosis-like partial suture fusion defects,
which reduced both coronal and sagittal suture volume compared to controls (Figures 2.4G-2.4J).
These results suggest that Snord118-dependent ribosome biogenesis is essential for MSCs to
prevent craniosynostosis defects.
2.3.2 Ribosome biogenesis disruption in Gli1+ MSCs leads to p53 activation, cell death,
proliferation reduction, and loss of mesenchymal stromal cells.
Since sagittal homozygous cKO mice showed consistent suture defects and more severe
phenotypes than heterozygous mice, we focused on sagittal homozygous cKO mice for the
downstream analysis. Previous studies have reported that loss of Gli1+ MSCs could result in
premature suture fusion (Zhao et al., 2015). To investigate the causes of suture defects, we
examined Gli1+ MSCs in homozygous cKO mice. Gli1-CreERT2;Ai-14 and Gli1-CreERT2; Snord118f/f;
Ai-14 mice were used to label Gli1+ cells. One month after tamoxifen induction at P20, Gli1-

19
tdTomato+ cells were significantly reduced in Gli1-CreERT2;Snord118f/f;Ai-14 mice compared to
control mice (Figures 2.5A and 2.5C). However, Gli1- tdTomato+ cells could contain both Gli1+
MSCs and differentiated mesenchymal cells. To ensure Gli1+ MSC changes, we performed Gli1
RNAscope staining and found that the percentage of Gli1+ MSCs was significantly reduced in
mutant sutures compared with controls. Previous studies have also found that p53 is activated
Figure 2.4. Disruption of ribosome biogenesis in MSCs results in reduced cranial suture volume
and craniosynostosis-like defects. (A, B) Micro-CT analysis and H&E staining of coronal and
sagittal sutures from WT, Gli1-CreERT2;Snord118f/+
, and Gli1-CreERT2;Snord118f/f mice after one
month of tamoxifen injection (1mpt), Scale bar 1 mm (A), 100 um (B). (C-D) Quantification of
cranial suture volume. (E) RNAscope staining of Twist1 (green) in the coronal suture (1mpt).
Scale bar 100 um. (F) Quantification of Twist1 expression in coronal suture mesenchyme. (G-H)
Micro-CT analysis of coronal and sagittal sutures from WT control and Gli1-CreERT2;Snord118f/f
mice after 3months of tamoxifen injection (3mpt). Blue arrowheads indicate the area of partial
suture fusion. (I-J) Quantification of the coronal and sagittal suture volume. Scale bar 1 mm, and
50 µm. Values represent mean ± SEM. *p-value <0.05, ** p-value <0.01, **** p-value <0.0001,
Student’s t-test and one-way ANOVA with Tukey post hoc tests.

20
upon impairment of ribosomal biogenesis (Golomb et al., 2014; Ross and Zarbalis, 2014).
Therefore, we hypothesized that depletion of Snord118 in Gli1+ MSCs may lead to p53 activation
and cell death. Results showed that p53 activation and TUNEL-positive Gli1+ MSCs were
increased in Snord118 mutant mice (Figures 2.5B, 2.5D, 2.5E, and 2.5G). Next, we examined cell
proliferation and found that Ki67-positive Gli1+MSCs were decreased in mutant sutures compared
with controls (Figures 2.5F and 2.5H), suggesting reduced cell proliferation of Gli1+ MSCs after
Snord118 depletion.
In addition, cranial suture patency is maintained by mesenchymal cells, which are derived
from Gli1+ MSCs. Thus, we further examined suture mesenchymal cells, which are labeled by
platelet-derived growth factor receptor alpha (Pdgfr-α). Consistent with Gli1+ MSC loss, Snord118
cKO mice displayed a decrease in Pdgfr-α+ mesenchymal cells (Figures 2.5 I and 2.5M). These
findings suggested that perturbation in ribosome biogenesis via knockout Snord118 in Gli1+
MSCs
could affect MSCs and their niche cells in the suture. To determine the specificity of MSC and
mesenchymal cell loss, we used CD31 and βIII-Tubulin (TUBB3) to label blood vessel endothelial
cells and neurons, respectively. There were no significant changes in the numbers of CD31+
endothelial cells and TUBB3+ neurons in Snord118 mutant mice (Figures 2.5J - 2.5L).
2.3.3 Snord118 depletion results in premature osteogenic differentiation of MSCs and
osteoclast loss.
Calvarial suture homeostasis is maintained by the interaction of MSCs with the niche cells
and the balance between the osteogenesis and osteoclastogenesis activity. Previous studies
have found that disruption of bone remodeling process and cell-cell interaction in the suture
resulted in craniosynostosis (Guo et al., 2018; Zhao et al., 2015). To elucidate the cellular
mechanisms responsible for the suture defects in Snord118 mutant mice, we examined bone
turnover at the osteogenic front involved in suture homeostasis. Since Gli1+
MSCs can differentiate
into the osteoblast lineage, Runx2 and Sp7 were used to monitor the differentiation of individual
MSCs into osteoblasts. Results from lineage tracing of tdTomato-positive Gli1+ MSCs

21
Figure 2.5. Snord118 depletion in Gli1+ MSCS leads to p53 upregulation, cell death, proliferation
reduction, and loss of mesenchymal stromal cells. (A) Immunofluorescence staining of Gli1-
tdTomato+ cells. (B) p53 staining in Gli1-CreERT2;Ai-14 (Control) and Gli1-CreERT2;Snord118f/f;Ai14 mice. (C,D) Quantification of Gli1-tdTomato+ cells and p53+
;Gli1-tdTomato+ cells in the suture
mesenchyme. (E,F) TUNEL and Ki67 staining with Gli1-tdTomato+ cells. (G,H) Quantification of
TUNEL- or Ki67-positive Gli1-tdTomato+ cells. (I,J) Immunostaining of Pdgfra, CD31, and TUBB3.
(K-M) Quantification of Pdgfra+
, CD31+
, and TUBB3+ cells. Scale bar 100 µm. Values represent
mean ± SEM, *p-value <0.05, **p-value <0.01, ***p-value <0.001, ****p-value <0.0001, nsnonsignificant.
showed that Runx2-and Sp7- tdTomato-Gli1-double-positive cells were significantly increased in
the mutant cranial sutures compared with controls (Figures 2.6A-2.6D). In contrast, tartrateresistant acid phosphatase (TRAP) and Cathepsin K, markers for osteoclasts, were significantly
reduced at the osteogenic fronts of the cranial suture in Gli1-CreERT2;Snord118f/f mice compared

22
Figure 2.6. Disruption of ribosome biogenesis leads to premature osteogenic differentiation
and loss of osteoclasts. (A-D) Immunofluorescence staining and quantification of Runx2 and
Sp7 in sagittal suture of Gli1-CreERT2;Ai-14 (Control) and Gli1-CreERT2;Snord118f/f;Ai-14 mice.
(E,F) Immunofluorescence staining of Cathepsin K (left panels) and TRAP staining (right
panels) in the suture mesenshyme of WT (Control) and Gli1-CreERT2;Snord118f/f mice. (G,H)
Quantification of Cathepsin K+ and TRAP+ cells in the suture mesenshyme, Scale bar 100 µm.
Values represent mean ± SEM. *p-value <0.05, **p-value <0.01, ***p-value <0.001. Student’s
t-test.

23
with controls (Figures 2.6E-2.6H). Osteoclasts originate from HSCs or erythromyeloid progenitors
(EMPs) but not from Gli1+ MSCs (Yahara et al., 2022). The osteoclast reduction in mutant sutures
implies a non-cell autonomous role of ribosome biogenesis in suture MSCs. Thus, these findings
suggest that Snord118 depletion in Gli1+ MSCs results in premature osteogenic differentiation of
MSCs and decreased osteoclast-driven bone resorption, which could contribute to premature
suture fusion.
2.3.4 Human iPSC-derived mesenchymal stem cells exhibit impaired self-renewal and
differentiation.
To further investigate the mechanism underlying suture growth defects Snord118 cKO
mice, we examined the cell autonomous roles of Snord118 in MSCs and established a platform
to differentiate human iPSCs into suture human iPSC-derived mesenchymal stem cells (iMSCs).
Snord118 cKO mice exhibited consistent and dose-dependent growth defects in sagittal sutures,
which are derived from cranial neural crest cells (cNCCs) in early development. Thus, we focused
on cNCC-derived suture MSCs. Human iPSCs were used to generate embryonic bodies (EBs),
which were then cultured in N2B27 medium in the presence of insulin, epidermal growth factor
(EGF), and basic fibroblast growth factor (bFGF) on uncoated dishes without Matrigel (Bajpai et
al., 2010). Under these culture conditions, neural progenitor cells detached from uncoated culture
dishes and were washed away, resulting in highly purified NCCs attached to cultured dishes.
Then we used an a-MEM medium in the presence of fetal bovine serum (FBS) to generate GLI1+
MSCs (Figures 2.7A-B). Together, human iPSC-derived MSCs allowed us to have sufficient
materials for ribosome profiling.
Using CRISPR-Cas9 approaches, we generated three independent iPSC lines with
heterozygous SNORD118 mutations, since homozygous mutations led to cell death without the
production of stable iPSC lines. DNA sequencing identified specific nucleotide deletions. RT-PCR
confirmed SNORD118 RNA downregulation in mutant iPSCs. Single-cell clonal analysis showed

24
Figure 2.7. Mutant iPSC-MSCs exhibit impaired self-renewal, differentiation, and translational
dysregulation of ribosomal protein genes. (A) Diagram of procedures of induced MSC generation
from human iPSC via cranial neural crest cells. (B) Immunofluorescence staining of iPSC-derived
NCCs and iMSCs. (C) Bright-field images of sphere formation. Scale bar 100 100 µm. (D,E)
Quantification of the number of spheres from iMSC starting with 1 x 104 and sphere diameter. N=
60 spheres analyzed for three independent cell lines. (F) Images of EdU incorporation (red) in
the spheres. Scale bar, 100 µm. (G) Quantification of the percentage of EdU+ cells in spheres.
N=18 spheres analyzed for each group. (H) RUNX2 staining under iMSC osteogenic
differentiation at day 3. Scale bar, 50 µm. (I, J) Quantification of the percentage of RUNX2+ cells
or EdU+ cells in a 2D culture of iMSCs. (K) Alizarin Red S staining for iMSCs at osteogenic
differentiation day 21. Scale bar, 1cm. (L, M) Images of adipogenic differentiation (Oil red O
staining, top) and chondrocyte differentiation (Alcian blue staining, bottom) for iMSCs at
differentiation day 14. Scale bar, 100 µm. (N) Correlation between mutant iPSC-MSCs and
controls in mRNA expression and translation efficiency. (O) Analysis of RNA-seq data from total
mRNAs and ribosome protected RNAs reveals translational downregulation of genes encoding
ribosomal proteins (RPs) in mutant iPSC-MSCs. n=3 different independent iPSC lines containing
control and SNORD118+/- for the generation of iMSCs. Values represent mean ± SEM. *p-value
<0.05, ** p-value <0.01, *** p-value <0.001, Student’s t-test.

25
that iMSCs can effectively self-renew to form spheres. After 14 days, the spheres were
dissociated and seeded as the single-cell suspension for the culture of the next passage.
SNORD118+/-
mutant iMSCs showed a significant decrease in the sphere numbers out of 1 x 104
single cells as well as the diameter of the spheres at the primary, secondary, and tertiary
passages (Figures 2.7C-2.7E). Additionally, EdU labeling showed a reduced percentage of EdU+
cells in the mutant spheres compared to controls (Figures 2.7F-2.7G), which is consistent with
decreased self-renewal in mutant iMSCs. The 2D culture also identified a decrease in EdU+ cells
in mutant iMSCs compared to controls (Figure 2.7J). In addition to cell proliferation, we examined
iMSC at osteogenic differentiation day 3 and found that mutant iMSCs have an increased
percentage of RUNX2+ cells (Figures 2.7H-2.7I), suggesting premature osteogenic differentiation.
To examine the multipotency of iMSCs, we performed tri-lineage differentiation. Alizarin Red S
(ARS) staining showed that SNORD118+/- mutant cells have reduced calcium deposit in
osteogenic differentiation (Figure 2.7K). Oil Red staining and Alcian blue staining were used to
assay adipogenesis and chondrogenesis, respectively, and failed to reveal substantial differences
between control and mutant cells (Figures 2.7L-2.7M). Together, these results suggest that
SNORD118+/- mutant iMSCs exhibit impaired self-renewal and osteogenic differentiation in vitro.
2.3.5 Ribosome profiling revealed a translational downregulation of ribosomal protein
genes in mutant MSCs.
To further study the molecular mechanisms underlying the cell fate changes of mutant
MSCs, we examined translational regulation using ribosome profiling (Ingolia et al., 2019). This
approach compares the levels of ribosome-protected fragments (RPFs) to the total expression
levels of mRNA in individual genes to measure translational efficiency (TE). Ribosome profiling
requires a substantial number of MSCs that are technically challenging to obtain from mutant
mice. Therefore, we used human iPSC-derived iMSCs to perform ribosome profiling and
RiboToolkit to decode mRNA translation (Liu et al., 2020). Then, we compared the levels of RPFs

26
over total mRNA between control and mutant iPSC-MSCs (Figure 2.7N). The bioinformatic
analyses reveal that the top translational downregulated genes encode ribosomal proteins (RPs),
although the mRNA levels are relatively normal (Figure 2.7O). These results suggest that RPs
are co-regulated at the translational level to maintain RP stoichiometry. This is consistent with
findings in human hematopoiesis that ribosome levels co-translationally regulate RPs (Khajuria
et al., 2018).
2.3.6 Snord118 deletion in MSCS leads to transcriptional dysregulation of genes encoding
complement pathway components.
Next, we analyzed transcriptional dysregulation in mutant iMSCs and found that
SNORD118 mutations lead to the downregulation of complement pathway genes, including C1S,
C1R, and C1RL in iPSC-derived MSCs (Figure 2.8A). Previous findings have shown that
mutations in complement pathway components in humans selectively affect craniofacial structure.
Specifically, mutations in MASP1/3 and COLEC10/11 cause 3MC syndrome (Mingarelli,
Malpeuch, Michels, and Carnevale syndrome) with craniosynostosis, cleft lip/palate, or facial
dysmorphism (Munye et al., 2017; Rooryck et al., 2011). However, the molecular and cellular
mechanisms underlying craniosynostosis in 3MC syndrome remain unknown. Therefore, we
further investigated the complement pathway. Western blots (WB) confirmed that mutant iPSCMSC cells have reduced protein levels in MASP1 (Figures 2.8B-2.8C), the mutation of which leads
to 3MC syndrome. Simultaneously, there were no significant mRNA expression level changes in
MASP1 (Figure 2.8D), suggesting a potential translational dysregulation of MASP1 in mutant
iPSC-MSCs. In addition, WB and RT-PCR confirmed protein and mRNA downregulation of
complement pathway components C3, C1R, and C1S (Figures 2.8E-2.8G). To examine
complement pathway dysregulations in vivo, we examined Snord118 MSC cKO mice.
Immunofluorescence staining revealed that a decrease in the expression of complement pathway
components, including MASP1, C1S, and C3 in the suture mesenchyme of Snord118 mutant mice

27
(Figures 2.8H-2.8M). Together, these results indicate that perturbation of Snord118 ribosome
biogenesis in MSCs results in transcriptional dysregulation of genes in the complement pathway.
Figure 2.8. Snord118 depletion results in transcriptional and translational dysregulation of
genes encoding complement pathway. (A) Log2-fold changes in up- and down-regulated
genes from bulk RNA-seq reads in the control and SNORD118+/- iMSCs. The downregulated
C1S, C1R, and C1RL in mutant iMSCs are highlighted. (B,C,E,F) Western blot of protein
levels and quantification of MASP1, C3, C1R, and C1S in control and SNORD118 mutant
iPSC-MSCs. b-actin serves as the negative control. (D,G) RT-PCR and quantification of
mRNA levels of MASP1, C3, C1R, and C1S in control and SNORD118 mutant iPSC-MSCs.
(H-J) Immunostaining of complement pathway proteins MASP1, C1S, and C3 in the sagittal
suture of control and Gli1-CreERT2;Snord118f/f mice. (K-M) Quantification of relative
fluorescence intensity of complement proteins. Scale bars: 100 μm. Values represent mean
± SEM. *p-value < 0.05, ** p-value < 0.01, *** p-value <0.001; ns represents non-significant.
Student’s t-tests.

28
2.3.7 Disruption of complement pathway exacerbates suture defects in Snord118 mutant
mice.
Previous studies have found that complement pathway plays a regulative role in bone
metabolism. MSCs, osteoblasts, and osteoclasts can generate C3 and express C3a receptor
(C3aR). C3a/C3aR axis is a central component of complement pathway, which is involved in the
bi-directional communication between osteoclasts and osteoblasts to maintain bone homeostasis
(Mödinger et al., 2018; Schoengraf et al., 2013). However, the functions of Snord118-mediated
complement pathway in Gli1+ MSCs and the importance of complement pathway in suture
homeostasis are not fully understood. To further examine the functional importance of
complement pathway in mediating Snord118 functions during suture homeostasis, we generated
C3aR1 genetic deletion in Snord118 MSC cKO mice by crossing Gli1-CreERT2;Snord118f/+ mice
with C3aR1-/- mice. Suture morphology and suture volume of Gli1-CreERT2;Snord118f/+;C3aR1-/-
were examined and compared to wild-type and Gli1-CreERT2;Snord118f/+ mice. Loss-of-function
perturbation using C3aR1 KO model showed that heterozygous KO mice exhibited a decrease in
coronal and sagittal suture volumes, which were further exacerbated by the deletion of C3aR1
Supporting data from micro-CT analysis also revealed significantly reduced suture volume
(Figures 2.9A-2.9D). These findings suggest that there is a genetic interaction between C3aR1
and Snord118 in suture growth. To investigate the cellular basis of C3aR1 deletion-induced
exacerbation of suture defects in Snord118 cKO mice, we examined Gli1+ MSCs. We did not
detect notable defects in Gli1+ MSCs in C3aR1-/- KO mice (data not shown), which is consistent
with their normal suture growth. In contrast, complement pathway disruption by C3aR1-/- deletion
induced a drastic reduction of Gli1+ MSCs in Gli1-CreERT2;Snord118f/+ heterozygous KO mice
(Figures 2.9E and 2.9F). To investigate the causes of Gli1+ MSC loss, we examined p53 activation
and found that there is a substantial increase in p53+ cells in the cranial sutures of Gli1-
CreERT2;Snord118f/+;C3aR1-/- mice compared to heterozygous KO mice and WT controls (Figures
2.9G and 2.9H). In addition, cranial sutures exhibited an increase of TUNEL-positive cells in

29
double mutant mice compared to heterozygous KO mice and WT controls (Figures 2.9I and 2.9J),
suggesting that increased cell death underlies the MSC loss. Thus, these results indicate that
complement pathway disruption by C3aR1-/- KO exacerbates MSC cellular defects and leads to
more severe suture defects in Snord118 cKO mice.

30
Figure 2.9. Complement pathway disruption leads to more severe suture defects in Snord118
mutant mice. (A) H&E staining of coronal and sagittal sutures from control, C3aR1-/-
, Gli1-
CreERT2;Snord118f/+, and Gli1-CreERT2;Snord118f/+,C3aR1-/- mice. (B-D) Micro-CT analysis and
quantification of suture volume. (E, G, I) Immunostaining of Gli1, p53, and TUNEL in the suture
mesenchyme of control, Gli1-CreERT2;Snord118f/+, and Gli1-CreERT2;Snord118f/+,C3aR1-/-
mice. (F,
H, J) Quantification of the percentage of Gli1+
, p53+
, and TUNEL+ cells per section. Scale bars
100 μm. Values represent mean ± SEM. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001,
****p-value <0.0001; ns represents non-significant. One-way ANOVA with Tukey post hoc tests.
2.3.8 Activation of complement pathway could rescue suture defects in Snord118 mutant
calvarial suture explants.
We next investigated whether activation of complement pathway could restore cellular
defects and thereby rescue the craniosynostosis phenotype. To address this question, C3aR
agonist was used to promote the function of complement pathway. To exclude C3aR agonistinduced complications in other non-suture organs, suture explant ex vivo culture model was
performed. This model has been well-established to study suture growth in postnatal but not adult
stages (Menon et al., 2021). Coronal and sagittal sutures explanted from postnatal day 4 (P4) WT
and Gli1-CreERT2;Snord118f/f mice were treated with C3aR agonist for 10 days. Results from
histological analysis showed that WT sutures remained patent, while mutant cranial sutures
showed bony fusions (Figure 2.10A). Importantly, agonist-treated sutures from Snord118 cKO
mice remained patent, suggesting complement pathway activation can rescue suture growth
defects. Statistical analysis of suture area showed that C3aR agonist-mediated complement
pathway promotion can partially rescue the suture growth defects in Snord118 cKO mice (Figures
2.10B-2.10C). To investigate the cellular basis of C3aR agonist’s beneficial effects, we used
tdTomato Ai14 mice to genetically label Gli1+ MSCs and found that the C3aR agonist significantly
rescued MSC loss in Snord118 cKO mice (Figures 2.10D-2.10E). When treated with the C3aR
agonist, the percentage of p53+ cells in suture mesenchyme was significantly reduced in the
mutant suture explants compared to Gli1-CreERT2;Snord118f/f mutant explants treated with DMSO
controls (Figures 2.10F-2.10G). Both WT and C3aR agonist-treated mutant suture explants
showed minimal cell death labeled by TUNEL staining, whereas untreated Snord118 mutant

31
sutures showed significant cell apoptosis (Figures 2.10H-2.10I). The activation of the complement
pathway partially rescued cellular and suture growth defects caused by Snord118 deletion.
Together, these results suggest that the complement pathway is a key mediator of Snord118
function in MSC behaviors and suture homeostasis.
Figure 2.10. Activation of complement pathway activation could rescue suture defects in calvarial
suture explants of Snord118 mice. (A) H&E staining of coronal and sagittal sections of suture
explants from control, Gli1-CreERT2;Snord118f/f , and Gli1-CreERT2;Snord118f/f mice treated with
C3aR agonist (rescue group). Blue arrowheads indicate the area of bony fusion. (B, C)
Quantification of coronal and sagittal suture area of calvarial suture explants. (D-I)
Immunostaining and Quantification of Gli1-tdTomato and p53, or TUNEL staining in suture
explants from control, Gli1-CreERT2;Snord118f/f;Ai-14, and Gli1-CreERT2;Snord118f/f;Ai-14 mice
treated with C3aR agonist. Scale bars 100 μm. Values represent mean ± SEM, One-way ANOVA
with Tukey post hoc tests. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, ****p-value
<0.0001; ns represents non-significant.

32
2.4 Discussion and Future Direction
Ribosome biogenesis is a fundamental cellular process and universally required in all
eukaryotic cells. Although extensive research has shown that perturbed ribosome
biogenesis results in tissue-specific defects, including craniofacial deformities, how ribosome
biogenesis disruption preferentially affects craniofacial tissues leading to craniofacial deformities
remains poorly understood. Findings from this study showed that global ribosome biogenesis
selectively regulates the complement pathway to control suture MSC cell fate, disruption of which
impairs suture growth and results in craniosynostosis.
Protein synthesis and mRNA translation in stem/progenitor cells during tissue
development is dynamic and sensitive to changes in ribosome biogenesis. Tight regulation of
protein synthesis machinery, including ribosomes and their associated factors, is essential for
stem cell self-renewal and differentiation (Saba et al., 2021). Compared to other tissue stem cells,
ribosome biogenesis in suture MSCs is poorly understood. It is possible that a global disruption
of ribosome biogenesis selectively affects suture MSCs since they serve as the engine of calvarial
growth, suture homeostasis, and regeneration. Loss of suture MSCs is one of the major causes
of craniosynostosis (Menon et al., 2021; Zhao et al., 2015). Similarly, Snord118 mutant mice
showed a significant decrease of Gli1+ MSCs in the suture mesenchyme and developed
craniosynostosis-like defects in the later stage. The MSC loss could be due to an increased p53
activation leading to apoptosis. These findings are consistent with previous studies showing that
perturbed ribosome homeostasis causes p53 activation resulting in cell cycle arrest and apoptosis
(Golomb et al., 2014; Jones et al., 2008).
It is well known that MSCs are multipotent stem cells that can differentiate into various cell
types. Our studies found that tdTomato-labeled Gli1+ MSCs in mutant mice prematurely
differentiate into osteoblast lineage cells. It is possible that ribosome biogenesis disruption might
abnormally upregulate activators of osteogenic differentiation. Another possibility is that ribosome

33
biogenesis disruption inhibits suppressors of osteogenic differentiation, which is illustrated by
Twist1 downregulation in mutant cranial sutures. Twist1 represses pro-osteogenic differentiation
factor Runx2 to maintain mesenchymal cells in an undifferentiated state (Bialek et al., 2004).
Twist1 downregulation could relieve the repression of Runx2 and lead to premature osteogenic
differentiation in Snord118 mutant MSCs. Meanwhile, osteoclasts are reduced in Snord118
mutant mice, which might result in more osteoblast cells contributing to premature suture fusion
and craniosynostosis.
Interestingly, our data revealed that the complement pathway is not only involved in
inflammation and immune response but also plays a crucial role in suture homeostasis. Moreover,
complement pathway has emerging roles in stem cells, tissue homeostasis, and injury repair
(Rutkowski et al., 2010). Mutation of complement pathway components in humans selectively
affects the craniofacial structure and leads to 3MC syndrome (Mingarelli, Malpeuch, Michels, and
Carnevale syndromes), which is characterized by facial dysmorphism, cleft lip/palate, and
craniosynostosis (Munye et al., 2017; Rooryck et al., 2011). Snord118 depletion results in reduced
expression of complement pathway components in cranial suture mesenchyme coupled with
craniosynostosis defects. Importantly, functional studies suggest that the complement pathway is
a key mediator of Snord118-controlled ribosome biogenesis functions in regulating MSC fate.
Thus, complement pathway disruption might contribute to craniosynostosis in 3MC syndrome.
Future studies should investigate what cellular functions the complement pathway might have in
suture development. Additionally, complement factor C3a has been shown to play critical roles in
neural crest cell migration (Carmona-Fontaine et al., 2011), and disruption of C3a might occur in
3MC syndrome. All things considered, it is important to further investigate how a global disruption
of ribosome biogenesis selectively affects the complement pathway.
In addition, the human iPSC-derived MSC model in our studies can provide a valuable
platform to study cranial suture development, disease pathogenesis, and potential treatments for
craniofacial disorders. These iMSCs are capable of clonal self-renewal and multiple differentiation

34
towards osteogenic, adipogenic, and chondrogenic cell fate. Human iPSC cells coupled with
CRISPR-Cas9 genome editing, cross-validated that SNORD118 mutations cause decreased
MSC proliferation and premature osteogenic differentiation, which is consistent with mouse
genetic studies. Importantly, iMSCs can provide unlimited cell materials for cell-numbers
demanding research such as ribosome profiling and bulk RNA-seq analysis. Future direction will
evaluate and compare human iPSC-derived MSCs with Gli1-tdTomato+ cells from Gli1-CreERT2;Ai14 mice.
In conclusion, this study provides mechanistic insights into the specificity of ribosome
biogenesis in craniofacial tissue development and disorders. Findings from human iPSC and
mouse genetic studies cross-validated that suture MSCs are particularly sensitive to the disruption
of global ribosome biogenesis which results in a change in their cell fate. Ribosome biogenesismediated regulation and the crucial roles of the complement pathway in MSC to maintain suture
homeostasis, which has implications for the pathogenesis of craniofacial abnormalities in 3MC
syndrome.

35
Chapter 3
Mechanisms and treatments of temporomandibular joint
degeneration with pain
3.1 Background
Temporomandibular disorders (TMD) are defined as a group of musculoskeletal and
neuromuscular disorders affecting the temporomandibular joint (TMJ), masticatory muscles, and
associated structures (Figure 3.1). About 10% of the U.S. population experiences symptoms of
TMD (Wadhwa and Kapila, 2008), including pain and dysfunction in the TMJ and/or the muscles
that control their movement. In addition, the prevalence of TMD is higher in women than in men
(Maixner et al., 2011). Approximately 80% of TMD patients present with signs and symptoms of
joint diseases, suggesting that an understanding of the underlying pathology of TMJ would be
beneficial to a large proportion of TMD patients (Wadhwa and Kapila, 2008).

Figure 3.1. TMD symptoms encompass both pain and dysfunction affecting the TMJ and
masticatory muscles (https://www.nidcr.nih.gov/health-info/tmd#overview).

36
3.1.1 Structure of the TMJ
As mentioned in Chapter 1, the temporomandibular joints are the most used synovial joint
in the body during daily function. They are formed by the mandibular condyle that fits into the
glenoid fossa of the temporal bone, while an articular disc is between the condylar head and
temporal bone. Capsular ligaments surround the whole TMJ structure. The main components are
the articular fossa of the temporal bone, mandibular condyle, articular disc, capsular ligament or
joint capsule, and muscle (Figure 3.2) (Tuncer, 2020).
Figure 3.2. TMJ and associated structures. (Pandarakalam and Khalaf, 2014; Tuncer, 2020)
Articular fossa of the temporal bone
The articular fossa or glenoid fossa of the temporal bone is the concavity that forms the
roof of the TMJ. Anterior to this fossa is the articular eminence and the posterior part is the
tympanic plate. The articular fossa is lined with thin articular tissues, while the articular eminence
is covered by thick, firm fibrous tissues. These characteristics can facilitate loading between the
disc and articular eminence in the TMJ (Tuncer, 2020).
Articular disc and retrodiscal tissues
The articular disc in humans is a biconcave fibrocartilage which consists of dense collagen
fibers. An intermediate articular disc, which is attached partially to the bones and the capsule,

37
separates the joint cavity into superior and inferior joint cavities. The disc is avascular and lacks
nerve supply. The anterior portion of the disc attaches to the joint capsule and lateral pterygoid
muscle. The intermediate zone of the disc attaches to the medial/lateral collateral ligaments,
which attach to the medial and lateral poles of the condylar head. The posterior attachment of the
disc is the bilaminar zone and is referred to as retrodiscal tissue. The bilaminar zone is divided
into the superior retrodiscal lamina composed of elastic tissue and the inferior retrodiscal lamina,
which is collagenous. In contrast to the disc, the retrodiscal tissue is highly vascularized and
innervated. Inflammation or compression to retrodiscal tissues can result in painful TMJ (Figure
3.2) (Langendoen et al., 1997).
The articular disc is a fibrocartilage consisting of 70% fibroblast-like cells and 30%
chondrocyte-like cells. Although the disc contains glycosaminoglycans and various types of
collagens, type I collagen predominates in the extracellular matrix (ECM). These components
facilitate its function as a cushion or stress absorber to endure long-term stress and transmit
contact force during the TMJ movement. Therefore, the main function of disc is to stabilize the
joint, provide smooth joint movement, and prevent bone-to-bone contact. This function is essential
in minimizing frictional forces that have the potential to cause damage to both the condylar head
and articular fossa (Lawry and Bewyer, 2008; Pandarakalam and Khalaf, 2014; Tuncer, 2020).
Capsular ligament and synovial tissues
TMJ structures are surrounded by the fibrous capsule, a highly vascular and innervated
fibroelastic tissue. The joint capsule thickens medially and laterally which is considered as the
medial and lateral collateral ligament of the TMJ. The collateral ligaments function in limiting
excessive medial and lateral movement of the disc and TMJ.
In addition to the collateral ligament, there are the temporomandibular, stylomandibular,
and sphenomandibular ligaments that facilitate TMJ movement. The temporomandibular ligament
functions in stabilizing the lateral side of the capsule, limiting joint rotation, and preventing

38
posterior displacement of the condyle. Stylomandibular and sphenomandibular ligaments are the
accessory ligaments supporting the mandibular movement (Tuncer, 2020).
The fibrous capsule covering the TMJ can be divided into two parts: an outer fibrous layer
and an inner synovial membrane. The synovial membrane is composed of two layers: a surface
layer of synovial lining cells and a connective sub-lining layer. It covers the entire inner surface of
the joint capsule, except the surface of the articular disc and condylar cartilage. Two types of
synovial lining cells have been identified, including macrophage-like type A and fibroblast-like B
cells in the synovial membrane of TMJ (Nozawa-Inoue et al., 2003). The macrophage-like type A
cells function in absorbing and degrading extracellular components, antigens, and cell debris in
the synovial fluid, while fibroblast-like type B cells play a crucial role in a secretory function. They
can produce and secrete collagens, fibronectin, and glycosaminoglycans including hyaluronic
acid into the synovial membrane and synovial fluid (Smith, 2011).
The synovial fluid within the joint acts as a lubricant and shock absorber which facilitates
movement within the joint by reducing friction and cushioning the articular cartilage. It also serves
as a medium for the transportation of nutrients and waste products from articular surfaces
(Nozawa-Inoue et al., 2003; Smith, 2011).
Mandibular condyle
The articular surface is the upper and anterior surface of the mandibular condyle. It is
covered by thin but dense fibrocartilage. The fibro-articular cartilage of the mandibular condyle
can be divided into two distinct zones, the outer fibrous zone and the inner fibrocartilage zone.
Fibroblasts are predominated in the fibrous zone. The ECM in this zone consists of type I collagen,
type II collagen, and chondroitin sulfate-based proteoglycan. The fibrocartilage zone is divided
into proliferative and hypertrophic zones. Fibroblasts and chondrocytes make up the
fibrocartilage zone, and the ECM is mostly composed of type II collagen but also includes type I,
type X, and aggrecan (Wadhwa and Kapila, 2008).

39
Muscle
TMJ movement is achieved by contraction and relaxation of muscles of mastication. The
masticatory muscles are the paired masseter, the temporalis, the medial pterygoid, and the lateral
pterygoid muscles. These muscles work in conjunction with other muscles of the face and
ligaments to facilitate jaw function, such as chewing, speaking, and jaw opening-closing. Any
disturbance or excessive forces beyond the adaptive capacity of these structures may result in
TMJ disorders (Han and Lieblich, 2022; Tuncer, 2020).
Innervation of the TMJ
The TMJ and surrounding structures are innervated by the mandibular nerve (V3), the
third division of the trigeminal nerve (Cranial nerve V). The mandibular nerve contains both
sensory and motor components. It receives sensory information from the facial skin in the lower
third of the face, mandibular teeth, gingiva, the anterior two-thirds of the tongue, and TMJ. The
auriculotemporal branch of V3 is composed of both sensory and secretory-motor parasympathetic
fibers. The sensory part innervates the anterior tympanic membrane, the anterior external meatus,
the anterior part of the auricle, and the lateral part of the temple. TMJ is innervated by the
auriculotemporal nerve, which supplies the lateral joint capsule, posterior attachment or
retrodiscal tissue, and the anterior and medial regions of the TMJ. The medial joint capsule is also
innervated by the deep temporal and the masseteric nerves. In addition to the sensory functions
of V3, the motor axons innervate several muscles, including the masticatory muscles, the anterior
belly of the digastric muscle, the mylohyoid, the tensor tympani, and the tensor veli palatini
muscles (Han and Lieblich, 2022; Tuncer, 2020).
3.1.2 Uniqueness of the TMJ
The temporomandibular joint (TMJ) is unique when compared with other systemic joints
in functional, morphological, and developmental aspects. Unlike most synovial joints in the body
which are hyaline cartilage, the articular surfaces of TMJ are lined with dense fibrocartilage. The

40
special characteristic of fibrocartilage is that it contains both type I and type II collagen, whereas
hyaline cartilage only contains type II collagen. These compositions make fibrocartilage a superior
material for withstanding sheer forces and enduring the large amount of occlusal load on the TMJ.
The fibers in fibrocartilage are tightly packed, enhancing its ability to withstand the forces
associated with jaw movement. As a result, it is less likely to break down over time and possesses
a greater ability to repair itself (Sperry et al., 2019; Wadhwa and Kapila, 2008).
In terms of development and cartilage formation, the TMJ is secondary cartilage,
compared to other joints which are primary cartilage. The development of secondary cartilage is
related to specific bones formed by intra-membranous ossification after the bones are already
formed. The cartilage in other joints is formed by endochondral ossification, in which the formation
of cartilage precedes bone formation, referred to as primary cartilage.
In the development of primary cartilage, the cartilage cells within the central layer of an
epiphyseal plate undergo mitosis. As a result, the two daughter cells contain the total amount of
genetic formation from their original cells. During the phase of epiphyseal growth, the new cells
enlarge to the size of the original and secrete extracellular matrix causing the cells to drift away
from each other. These cells can become new progenitor cells or be replaced by bone. The new
cartilage growth that occurs within existing tissue is called interstitial growth. Therefore, the growth
of primary cartilage occurs in the middle part of an epiphyseal plate of the long bone.
Secondary cartilage growth in the mandibular condyle is different from primary growth in
the long bone. In the developmental stages, cartilage growth begins with undifferentiated cells
which are mesenchymal tissue covering the prenatal or postnatal condyle. The mesenchymal
cells start to split and become smaller cells, but eventually attain full size. Then, these cells
migrate into the interior condyle and differentiate into immature cartilage cells. Thus, the growth
of secondary cartilage occurs from the exterior (appositional growth) through differentiation of
mesenchymal tissue rather than mitosis of cartilage progenitor cells.

41
In conclusion, TMJ demonstrates specific organization, anatomic, and developmental
differences from other joints. This implies that making broad assumptions based on knowledge
about other joints may not be sufficient to justify a correlation to TMJ diseases. Further studies
into the pathology and mechanisms of TMJ disorders are required, as the management of TMJ
disorders may differ from that of other systemic joints in the body.
3.1.3 Etiology and risk factors of TMD
TMD is a multifactorial disorder. There is no single etiologic factor or single gene mutation
that can explain the development of TMD. Loss of TMJ structural integrity, altered function,
biomechanical stresses, or any disturbance in the system that prevents the coordinated
functioning of structures can compromise adaptability and increase the likelihood of developing
TMD. In addition, psychosocial factors have the potential to significantly diminish the adaptive
capacity of the masticatory system, resulting in the development of TMD. Previous studies have
also shown that TMD involves a complex interplay of biological, physiological, psychological, and
environmental factors leading to a wide range of possible symptoms and impairing patients’
quality of life (Figure 3.3). For example, direct trauma that exceeds the typical functional loading
on the mandible, TMJ, or masticatory structures. Microtrauma from postural imbalances or
parafunctional habits that create muscle and joint strain leading to musculoskeletal pain in TMD.
Genetic susceptibility and gene–environment interaction can contribute to alterations in gene
expression, serving as contributing factors in the onset of painful TMD (Smith et al., 2011).
Additionally, psychological factors, such as stress, depression, and anxiety, can adversely affect
the pain intensity in TMD patients (Fillingim et al., 2013; Maixner et al., 2011).
Due to the complex nature of TMD, an effective treatment should encompass multiple
modalities including pharmaceuticals, physical therapy, and behavioral modification. Therefore,
understanding the etiology and mechanisms that influence the development of TMD will be
beneficial for establishing proper diagnosis and successful management.

42
Figure 3.3. Major factors and the associated genes that increase the risk for an individual to
develop TMD symptoms (Maixner et al., 2011).
3.1.4 Signs and symptoms of TMD
TMD symptoms involve pain and dysfunction of TMJ, masticatory muscles, and
associated structures. The most frequent presenting symptom that causes patients to seek
treatment is pain, usually localized in the muscles of mastication and the preauricular area.
Chewing or other jaw movement activities usually aggravate the pain. In addition to pain, patients
often report limited mandibular movements and TMJ sounds, such as clicking, popping, and
crepitus (Ohrbach et al., 2013).
As mentioned above, approximately 80% of TMD patients present with signs and
symptoms of the joint disorder, suggesting that an understanding of the underlying pathology of
TMJ would be beneficial to a large proportion of TMD patients. Therefore, painful TMJ disorders
or arthrogenous TMD will be the focus of this study.

43
3.1.5 Diagnostic criteria of arthrogenous TMD
Arthrogenous TMD is a joint-related disorder that results from inflammation, dysfunction,
and degeneration of the hard or soft tissues within the TMJ. One of the most common and painful
arthrogenous disorders of the TMJ that deteriorates quality of life is degenerative arthritis or TMJ
osteoarthritis (TMJOA) (International Classification of Orofacial Pain, 1st edition (ICOP), 2020;
Gremillion and Klasser, 2017).
TMJOA is defined as a progressive degenerative disease in the TMJ and surrounding
tissues. Although degenerative TMJ disease can occur at any age, it occurs with greater
frequency as age increases. This affects multiple structures, including cartilage, subchondral
bone, synovial membrane, and other hard and soft tissues causing articular cartilage abrasion,
subchondral bone loss, TMJ remodeling, and inflammation in joint capsules and synovial
membrane. Patients with TMJOA usually have a history of joint pain and joint sound during jaw
movement (Ohrbach et al., 2013; Sperry et al., 2019).
Joint pain (arthritis, synovitis, or capsulitis)
According to the Research Diagnostic Criteria for Temporomandibular Disorders
(RDC/TMD) and American Academy of Orofacial Pain (AAOP) guidelines, the diagnosis of
arthritis, synovitis, or capsulitis is defined as joint pain with clinical characteristics of inflammation
or infection, including edema, erythema, and/or increased temperature (Figure 3.4). TMJ arthritis
can be triggered by trauma and repetitive or excessive forces loading to TMJ. Associated
symptoms can include occlusal changes from unilateral intra-articular swelling. However, there
should be no history of systemic inflammatory disease in the localized TMJ arthritis (Ohrbach and
Dworkin, 2016; Schiffman et al., 2014)

44
Figure 3.4. Diagnostic criteria for arthritis or arthralgia (Schiffman et al., 2014)
Degenerative joint disease (DJD)
Osteoarthritis (or osteoarthrosis: TMJOA without pain) is defined as a degenerative
condition of the joint characterized by deterioration and abrasion of articular tissue and
concomitant remodeling of the underlying subchondral bone due to overload of the remodeling
mechanism. Loss of articular TMJ cartilage is caused by an imbalance between predominantly
chondrocyte-controlled reparative and degradative processes. The rate of disease progression is
associated with proteoglycan depletion, collagen fiber network disintegration, and fatty
degeneration weakening the functional capacity of the cartilage (Ibi, 2019).
Localized osteoarthritis or primary TMJOA is a degenerative disorder that involves only
TMJ and does not involve other systemic joints. Clinical characteristics of TMJOA are
deterioration of articular tissue with concomitant bony changes in the condyle and articular
eminence (Schiffman et al., 2014). The current gold standard for diagnosis of TMJOA is using
computed tomography (CT) or Cone-beam computed tomography (CBCT) for TMJ imaging
combined with history taking, as shown in Figure 3.5

45
Figure 3.5. Diagnostic criteria for TMJOA or DJD (Schiffman et al., 2014).
However, the early stages of TMJ degeneration are frequently under-detected from
radiographic findings since bone erosion can be detected when more than 60% demineralization
occurs in the condyle. In addition, pain and inflammatory response in the early stage are usually
the results of synovitis, while osseous change typically lags behind articular tissue changes.
Therefore, the early changes in the synovial tissue, fibrous tissue, and cartilage degradation could
not be seen from CBCT. They can be only detectable with biopsy and arthroscopy (Al-Ani, 2021).
3.1.6 Pathogenesis of TMJOA
TMJOA is an important subtype of TMD which can occur secondary to functional overload,
direct major trauma, microtrauma, developmental abnormality, and generalized joint systemic
diseases. The etiology of TMJOA is complex and multifactorial. The majority of TMJ degeneration
is the consequence of the imbalance in anabolic and catabolic processes involving chondrocyte
initiation, proliferation, differentiation, and matrix synthesis and degradation. The early phase of
TMJOA may be sub-clinical; therefore, the onset of symptoms may not present until later stages
of degeneration. Increasing evidence showed that there are various causes or contributing factors
leading to degenerative TMJ diseases. The etiopathogenesis of TMJOA involves inflammatory
and immune response, altered mechanical loading, hormonal changes, and degeneration of the
extracellular matrix (Kalladka et al., 2014; Sperry et al., 2019; Wang et al., 2015).
Inflammatory responses

46
TMJ degeneration is a slow progress driven by inflammation. Several inflammatory
mediators are increased in the synovial fluid of TMJOA patients, such as Interleukin (IL) –12, IL1β, IL-6, and tumor necrosis factor (TNF)–α. Previous studies in chronic inflammation of rodent
TMJs model found a decreased biomechanical property of the disc and increased expression of
IL-1β and TNF-α, implying that these inflammatory cytokines could be the cause of degenerative
changes in TMJ and chronic inflammation could impair the adaptive capability of the TMJ structure
(Sperry et al., 2019; Wang et al., 2015). Inflammatory cell infiltration was also reported in TMJOA.
Findings from inflamed synovial tissues and fluids of patients with OA showed an increase of
monocyte chemoattractant protein (MCP)–1, a chemokine that regulates migration and recruits
monocytes and macrophages to inflamed synovial tissues. Although several inflammatory
mediators have been identified to date, there is still no potential prognostic indicator or diagnostic
marker for TMJOA.
Excessive mechanical stress
The articular surface of TMJ is covered by fibrocartilage and sensitive to mechanical stress
resulting in extensive remodeling. Appropriate loading on the TMJ is required for chondrocyte
proliferation, ECM synthesis, and the development of mandibular condyle (Zhao et al., 2022).
Although normal mechanical loading on TMJ and masticatory function are essential for
maintaining normal growth and morphology of TMJ, aberrant mechanical loading or excessive
mechanical stress that surpasses the adaptive capacity of TMJ is one of the important factors
leading to cartilage degradation in the TMJ both in mouse models and TMJOA patients (Tanaka
et al. 2008).
Microtrauma from sustained and repetitious adverse loading of the masticatory system
through parafunctional habits such as tooth grinding and bruxism, which contribute to excessive
joint loading, are considered as perpetuating factors for TMJ degeneration. Several studies in
animal models have investigated the molecular mechanisms and signal pathways related to

47
mechanical stress–induced TMJOA. They demonstrated that excessive mechanical stress could
induce activation of the plasminogen activator (PA) system and result in proteolysis of ECM
leading to cartilage destruction (Wang et al., 2015). Furthermore, mechanical loading animal
models have revealed an increased expression of SEMA4D in early-stage TMJOA and
consequently decreased osteoblast activity in the subchondral bone. Mechanical stress could
also increase HIF-1 in mature chondrocytes, which repress OPG expression, and activate
osteoclastogenesis resulting in TMJ degeneration (Chung et al., 2023b; Wang et al., 2015; Zhao
et al., 2022). Thus, excessive joint loading could be an initiation factor and play a crucial role in
the progression of TMJOA.
Hormonal pathogenesis
Epidemiologic studies on the prevalence of TMD show a higher prevalence of TMJOA in
women which occurs mainly after puberty during the reproductive years, implying a possible role
of female hormones in an increased susceptibility of individuals to developing TMJOA (Kalladka
et al., 2014; Maixner et al., 2011; Ohrbach et al., 2013). Studies have found that estrogen and
progesterone receptors are localized in the TMJ (Wadhwa and Kapila, 2008). Proinflammatory
metabolites from the conversion of estrone/17β-estradiol were found in OA synovial cells of the
knee joint; this finding implies that proinflammatory metabolites in synoviocytes may be an
important mechanism underlying the pro-inflammatory effects of estradiol in the inflamed TMJ
(Wang et al., 2015). Previous studies also found that estrogen could inhibit chondrocyte
proliferation and aggravate the degradation of cartilage and destruction of subchondral bone
(Chen et al., 2014). In addition, estrogen and relaxin may contribute to TMJ degeneration by
increasing the expression of tissue-degrading enzymes, matrix metalloproteinase (MMP) family
from TMJ fibrocartilage (Wadhwa and Kapila, 2008). These findings suggest that estrogen plays
a role in the sexual dimorphism of TMJOA. However, the role of estrogen in TMJOA is still
inconclusive since previous studies found that the expression of nitric oxide can be inhibited by

48
estrogen, which has a protective effect on TMJ chondrocytes (Hu et al., 2013). Moreover, there
is no direct evidence that links female hormones to TMJ degeneration (Wang et al., 2015).
Degenerative changes in the extracellular matrix.
Collagen fibers and large proteoglycans are the main components of cartilage's
extracellular matrix (ECM). The ECM functions as a protective framework against elastic and
shear forces for cartilage and regulates chondrocyte behavior. The expression of matrix
metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs
(ADAMTS) in the cell could result in ECM degradation in TMJOA (Li et al., 2014; Yu et al., 2019).
Furthermore, collagen type II (COL2A1) is not only a structural component of the cartilage matrix
but also an extracellular signaling molecule that plays an important role in chondrocyte
proliferation and differentiation. hypertrophy. The decrease in COL2A1 could enhance
chondrocyte hypertrophy resulting in initiating and promoting TMJOA progression (Lian et al.,
2019). In addition, the previous findings have identified several signaling molecules involved in
the ECM degeneration in TMJOA, including β-catenin, Notch, and NF- κB signaling pathway (Li
et al., 2021a; Lian et al., 2019).
3.1.7 Neural Processing of painful TMJ
Degenerative TMJ disease does not only result in anatomical changes in the TMJ, but it
also leads to joint pain from the alteration in the peripheral and central pain mechanisms. Pain is
defined as “an unpleasant sensory and emotional experience associated with actual or potential
tissue damage, or described in terms of such damage” (Treede et al., 2019). Nociceptors are
sensory receptors that have high-threshold nerve endings detecting signals from damaged tissue
and sending impulses from fast-conducting Aδ fibers and slow-conducting C-fibers to the central
nervous system (CNS) to interpret as pain perception. As a result, several brain regions are
activated and subsequently modify behavioral responses to avoid further harmful stimuli (Ferrillo
et al., 2022).

49
Peripheral innervation and trigeminal ganglia
Patients with degenerative changes in TMJ often have TMJ capsulitis/ synovitis presenting
with significant pain and dysfunction. The development and progression of TMJOA involve
inflammation which is considered a part of the nociceptive input that activates trigeminal nerves
resulting in peripheral and central sensitization (Yi et al., 2021).
Nociceptors in the TMJ can be stimulated by various noxious stimuli including mechanical
and chemical stimuli, such as inflammatory mediators (bradykinins and prostaglandin E2) and
neuropeptides (calcitonin gene-related peptide (CGRP) and substance P) (Xu et al., 2019). The
noxious stimuli generated by the degenerative changes in TMJ can activate free nerve endings
surrounding TMJ and send pain signals through Aδ fibers and C-fibers of the trigeminal nerve to
their cell bodies in the trigeminal ganglion (O'Neill and Felson, 2018; Sperry et al., 2019).
Numerous nerve endings are densely innervated surrounding the joint capsule, articular disc, and
periosteal bone, except TMJ condylar cartilage. However, chemical mediators released from
macrophages and mast cells when cartilage is damaged can also activate nociceptors
surrounding TMJ and generate painful sensations (Sperry et al., 2019; Yi et al., 2021).
In addition, repeated activation of nerve endings by inflammatory mediators and
neurotrophic factors can cause peripheral sensitization. Peripheral sensitization is a state in which
the pain threshold is reduced and/or the magnitude of response at the peripheral ends of sensory
nerve fibers is increased. Thus, even non-painful stimuli, such as normal jaw movement and light
touch, can induce pain (Sperry et al., 2019).
When nociceptors from surrounding TMJ are activated, the painful sensory inputs are
carried along the auriculotemporal nerve, a branch of the mandibular nerve (V3; third branch of
the trigeminal nerve), and conveyed on first-order neurons through the trigeminal ganglion. The
central branch of the first-order neurons synapses with secondary neurons in the spinal trigeminal
nucleus in the brainstem (Figure 3.6) (O'Neill and Felson, 2018; Wilson-Pauwels, 2014).

50
Figure 3.6. Overview of the trigeminal nerve (CN V) and trigeminal ganglion (first-order neuron).
General sensory afferents are labeled in blue-green. Motor efferent is labeled in yellow (WilsonPauwels, 2014).
Trigeminal Nucleus and central sensitization
Nociceptive activation from the TMJ area is further modulated at the subnucleus caudalis
of the trigeminal nucleus. Neuropeptides and other inflammatory agents are released in the spinal
trigeminal nucleus in response to nociceptive signals and consequences in the excitation of
neurons and glial cells. Supporting evidence showed that inflammation in the TMJ can result in
microglia activation at the spinal trigeminal nucleus leading to the release of proinflammatory
cytokines. As a result, these inflammatory mediators could further regulate synaptic pain
transmission related to the development of acute and chronic pain. The acute pain condition can
resolve if the neuron activation is temporary and pain signaling returns to baseline after the
removal of stimuli. However, chronic pain can develop due to prolonged nociception, an increase
in neuronal activity, and loss of inhibitory neurons in the spinal trigeminal nucleus, which
subsequently leads to central sensitization. At this stage, patients could even feel pain from

51
nonpainful stimuli or allodynia with increased pain sensitivity/response from painful stimuli or
hyperalgesia (Figure 3.7) (O'Neill and Felson, 2018; Yi et al., 2021).
Thalamus, somatosensory cortex, and pain perception
Pain signals from the second-order neuron at the trigeminal nucleus can be transmitted to
higher brain regions to interpret pain perception. The third-order neuron is in the thalamus, which
receives nociceptive signals from the periphery and mostly connects to the ventral medioposterior
(VPM) region, and projects to the sensory cortex with the basal ganglia and the limbic system to
interpret nociceptive input (Figure 3.7) (Sperry et al., 2019).
Figure 3.7. Pain transmission from the trigeminal ganglion (first-order neuron) to the spinal
trigeminal nucleus (second-order neuron), thalamus (third-order neuron), and sensory cortex
(Wilson-Pauwels, 2014).
Increasing evidence showed that pain sensation is associated with psychological factors.
Some psychological traits, such as anxiety and depression, are frequently found to be comorbidity
chronic TMJ disorders. Currently, the biopsychosocial model is a widely accepted theory as an
etiology of painful TMD. Neuroimaging studies in TMD patients with pain also revealed the
alterations in cortical regions, such as increased activation of the anterior insula and anterior

52
cingulate cortex (ACC), affective brain regions, that integrate interoceptive input with emotional
salience and influence on pain perception resulting in increased attention to the pain sensations
(Yin et al., 2020).
3.1.8 Treatment of TMJ degeneration
Management of TMJOA requires multidisciplinary care due to the complexity of the TMJ
degeneration and painful symptoms, which involve both biological and psychological factors.
Although various combinations of contributing factors lead to TMJOA in individuals, the treatment
strategy for TMJOA aims to identify potential major causes and eliminate them. The therapeutic
goals of TMJOA should be directed at relieving pain, suppressing an active inflammatory process,
reducing joint loading, and restoring normal jaw function. The current clinical management for
TMJ degeneration is contingent on the severity. It can be categorized from the least to the most
invasive procedures, including conservation, minimally invasive, and invasive procedures (Ferrillo
et al., 2022; Kalladka et al., 2014; Tanaka et al., 2008).
Noninvasive treatment
Conservative or reversible treatment is a noninvasive method that is endorsed for the
initial care of nearly all TMD. Management of TMJOA also starts with conservative treatment
which aims to control inflammation and TMJOA progression, reduce pain, and eventually improve
joint function. The treatment covers a wide range of management, including occlusal stabilization
splints, physical treatment, and pharmacotherapy(Al-Ani, 2021; Maixner et al., 2011).
Occlusal stabilization splints and physical therapy, such as self-management instructions,
soft diet, and behavioral modification, play a crucial role in protecting TMJ from excessive
mechanical loading (Al-Ani, 2021; Ferrillo et al., 2022).
Pharmacologic approaches for pain relief form the major parts of TMJOA management
since joint pain is the main factor that causes patients to seek treatment. Several medications
have been used to control pain and inflammation in TMJ degeneration, including corticosteroids,

53
nonsteroidal anti-inflammatory drugs (NSAIDs), muscle relaxants, antidepressants, and
anticonvulsants. Non-steroidal anti-inflammatory drugs (NSAIDs) are usually prescribed for mild
to moderate joint pain, and they have a dual effect of reducing pain and reducing inflammation.
However, some patients have to avoid using NSAIDs and corticosteroids due to the side effects.
(Ferrillo et al., 2022; Kalladka et al., 2014). Therefore, studies to identify potential new drugs with
more specific targets and fewer side effects are required for the management of TMJ
degeneration.
Minimally invasive treatment
TMJOA patients with moderate or severe pain who do not respond to oral medication and
conservative treatment, minimally invasive therapies may be used. There are various types of
minimally invasive treatment, including intra-articular injection, arthrocentesis, and arthroscopy.
The intra-articular injections are widely used in patients with moderate or severe painful TMJOA
whose symptoms did not improve by oral medication and conservation treatment. NSAIDs,
corticosteroids, platelet-rich plasma (PRP) from blood, and hyaluronic acid (HA) have been used
as injectable drugs into the TMJ to relieve pain and improve joint function (Figure 3.8) (Juan et
al., 2019).
Figure 3.8. Minimally invasive procedures and the compounds delivered with an intra-articular
technique (Juan et al., 2019).

54
Although intra-articular injection of a steroid can reduce inflammation and improve
localized clinical symptoms, the limitation of this treatment is the side effects of injected
biomolecules, such as dizziness, dry mouth, and possible drug dependency. Furthermore,
repeated injections are required in some cases due to the rapid clearance of the injected
biomolecules and drugs. This can result in infection, fibrosis, and consequently damage to TMJ
structures (Juan et al., 2019).
In addition to steroid injection, sodium hyaluronate, an alternative agent with fewer side
effects, has been used to treat patients whose symptoms did not respond to conservative
treatment. Hyaluronate acid is a viscosupplement that can increase joint lubrication and augment
synovial fluid viscosity. However, the literature showed that the effectiveness of hyaluronate acid
injections is still inconclusive. Sodium hyaluronate could be helpful for joint clicking or disc
displacement with reduction, but not help joint-locking, arthritis, and cartilage or bone regeneration
in TMJOA (Al-Ani, 2021; Manfredini et al., 2010).
Although there are limitations of current injectable molecules, intra-articular injection is still
considered a more promising strategy to deliver drugs or biomolecules to the target site with fewer
side effects (Juan et al., 2019). Therefore, further studies in biomaterial-based carriers that can
deliver better therapeutic molecules into the specific anatomical area and sustained release are
required.
TMJ arthrocentesis is a less invasive surgical procedure that can be offered to patients
with TMJOA who are unresponsive to other conservation treatments. The procedure involves joint
lavage which is flushing the superior joint space with a sterile saline solution or a lactated Ringers
solution to remove tissue breakdown products and inflammatory mediators (Figure 3.9). This
method also generates pressure during irrigation which benefits in release of joint adhesions.
However, repeated joint lavage might be required since the source of inflammation is inside the
intra-articular tissues (Ferrillo et al., 2022; Lee et al., 2021).

55
Figure 3.9. (A) Arthrocentesis procedure. (B) The location of the two needles for arthrocentesis
(Lee et al., 2021).
TMJ arthroscopy is more invasive than an arthrocentesis. An endoscope will allow the
surgeon to visualize inside the TMJ and can perform joint lavage and some surgical procedures
to remove the pathology in intra-articular tissue. Since arthroscopy involves a surgical procedure,
as a result, injuries related to adjacent structures can occur, such as otological and neurovascular
complications.
Invasive treatment
Invasive surgical procedures involve open joint surgery and joint replacement. This
method is only performed for patients suffering from severe TMJ problems with limited joint
movement. However, post-operative complications from surgery can occur, such as bleeding,
infection, numbness from anesthesia, damage to the facial nerve, fibrosis, or scar tissue from
surgery. Therefore, early diagnosis and proper management to prevent disease progression are
essential to avoid complicated and invasive treatment.
3.1.9 Animal models in TMJOA
Since it is challenging to collect clinical samples from patients with painful TMJOA, animal
models have become a critical approach to study the pathogenesis of TMJOA and investigate
new potential therapeutic interventions. Several types of animal mouse models have been
developed to replicate various etiologies of TMJ degeneration and understand the pathogenesis
A B

56
(Zhao et al., 2022). The review in the following sections will focus on studies exploring the
mechanisms of TMJ pain and degeneration in rodent models. The existing animal models related
to painful TMJOA can be categorized into inflammatory-induced, surgical induction, mechanical
loading, naturally occurring, and genetically modified models.
Inflammatory-induced TMJOA model
Inflammation plays a crucial role in the pathogenesis of TMJ degeneration and painful
symptoms. The intra-articular injection of chemical reagents into the TMJ to induce inflammation
is a well-characterized model for the TMJOA study. Chemical drug injections that are commonly
used to induce inflammatory pain in TMJOA include monosodium iodoacetate (MIA), complete
Freund’s adjuvant (CFA), and formalin. Intra-articular injection models are mainly used to study
pain and inflammatory response since it is a reliable method producing measurable nociceptive
behaviors that start quickly and last for a relatively long period.
Previous studies found that the degenerative changes in patients with TMJOA are not
always correlated with pain. However, there is stronger correlation of pain with tissue changes
such as synovitis and joint effusion. In addition, various mechanisms contribute to the onset,
progression, and maintenance of TMJ pain and degeneration. Therefore, it is challenging to
develop an ideal animal model that can mimic the etiologies and pathogenesis of TMJOA for both
TMJ degenerative changes and pain development (Chung et al., 2023b).
Nevertheless, the CFA-induced arthritis model has been extensively used to study
physiological, biochemical, and histopathological in TMJOA (Wang et al., 2017a; Zhao et al.,
2022). This model is also well characterized and frequently used for screening novel compounds
or drugs targeted to treat inflammatory pain, including osteoarthritis pain. CFA is an oil-based
solution that consists of inactivated and dried mycobacteria which consequently activate immune
response. Acute inflammatory reactions from rapid activation of immune cells result in pain,
edema as well as synovial hyperplasia. Following that, degenerative changes in the TMJ, such

57
as articular cartilage degradation and bone erosion, occur at a later stage after a few weeks of
CFA injection. Findings from the CFA-induced arthritis model are also consistent with the natural
course of TMJOA. In patients with painful TMJOA, pain is prominent in the initial phase of OA and
has been suggested to be associated with over-joint loading and the activation of inflammatory
processes in the synovium, joint, and peri-articular structures. Radiographic features of TMJ
destruction can be detected at the intermediate and late stages of TMJOA when more than 60%
demineralization occurs (Al-Ani, 2021). By carefully reviewing the strengths and limitations of
each animal model of TMJOA, the CFA-induced TMJ arthritis model was chosen to study TMJ
degeneration with pain in our study.
Mechanical loading
The TMJ is unique in its versatile ability to accommodate with wide dynamic range of
movement. However, excessive mechanical stress loading on TMJ, which is beyond its
physiological adaptation, can result in pain and structure remodeling. Clinical studies showed that
prolonged mouth opening during dental procedures, excessive force from trauma or injury, and
microtrauma from parafunctional habits are contributing factors associated with degenerative
changes in TMJ. Therefore, forced mouth opening and abnormal TMJ loading models are useful
to study the mechanism of pathological changes since they can mimic excessive loading and
trauma pathogenesis of TMJOA in humans. In the experimental rodent models, mechanical
hyperplasia is observed on day1 after sustained passive mouth opening in a mouse model and
can last for 4 weeks when the procedure is performed for 1.5-3 hours per day for 5 consecutive
days. TMJ structural degeneration can be detected two weeks after sustained mouth opening.
However, the onset and severity of pain phenotype vary depending on the amount of force and
time of prolonged jaw opening. The decreased mechanical hyperplasia, such as head withdrawal
to mechanical stimuli, could be confounded by an increase in the muscle fatigue of temporal and
masseter muscles. Furthermore, mechanical loading in this model might not be completely

58
equivalent to loading that occurs in human TMJ since TMJ structures, such as articular disc,
temporal fossa, and occlusion, in rodent animals are different from humans which influence joint
movement and mechanical force loading on TMJ (Chung et al., 2023a; Zhao et al., 2022).
Surgical approaches
Surgery is a widely used approach to destabilize TMJ structures by removing the entire
(discectomy) or some portion (partial discectomy) of the articular disc, surgical perforation of the
disc, surgical anterior disc displacement, and injury in the condylar surface. The articular disc in
the TMJ plays a crucial role in TMJ function as it serves as a stress absorber or distributor and
facilitates TMJ movement. The surgical interventions aiming to damage the disc can affect the
ability of the condylar surface to withstand loading onto the TMJ leading to cartilage destruction
and bone remodeling in the TMJOA model(Lei et al., 2022; Zhao et al., 2022).
Different surgical approaches can cause different effects on joint loading leading to
different changes in disease progression rate and severity. Surgical models are mainly used to
investigate degenerative mechanisms and are useful for regenerative strategies in TMJOA, such
as stem cell transplantation and tissue engineering. Although TMJ structural changes from
surgical models can mimic the advanced or late phase of TMJ degeneration in humans, severe
injuries are not a common initiating factor in general TMJOA (Chung et al., 2023b).
In addition, pain phenotypes were not well-characterized in the surgically induced TMJOA
model. The limitation of the surgical approach is that the procedure itself can cause pain and
inflammation inside the TMJ. It is challenging to distinguish the effects of acute or post-operative
alterations from the disease progression occurring from surgical disc manipulation on the TMJ.
Moreover, postoperative pain is difficult to differentiate from the pain caused by TMJ
degeneration. Thus, it could be challenging to use surgical models for investigating the TMJ paindegeneration relationship.
Genetic models of TMJOA

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Studies of genetically modified mice are useful for studying the precise molecular
pathways involved in TMJOA development and disease progression. Several specific genetically
modified models have been developed to target different pathologies in TMJOA such as genes
encoding regulators of chondrocyte homeostasis, inflammatory mediators, and extracellular
matrix components (Chung et al., 2023b). These mouse models allow us to understand their
critical contributions to the development and degeneration of the TMJ
Although genetic models are reliable tools that can eliminate other interferences and no
intervention is needed to induce TMJOA, the limitation is that they only act on specific genes
(Chung et al., 2023b; Sperry et al., 2019; Zhao et al., 2022). As mentioned above, the
pathogenesis of TMJOA is complex and influenced by multiple genes and environmental
interactions. Furthermore, most genetic models are focused on the time course of degenerative
changes within the TMJ, and pain-related phenotypes in these genetic models are still unknown.
Naturally occurring models
TMJOA is a slow progressive inflammatory joint disease with the prevalence and severity
increasing with age. Some animal models can develop OA-like lesions which is similar to the
natural progression of TMJOA in humans. TMJ articular cartilage and bone changes can be found
in aging animals, such as mice, guinea pigs, and horses. Thus, these models are ideally used for
studying cartilage degradation and bone remodeling in TMJOA (Chung et al., 2023b; Lim et al.,
2009). The advantage of these models is that no external interventions are required. They can
mimic primary TMJOA in humans without any induction. However, the naturally occurring models
are not widely used since they require an extremely long period of study and high-cost (Zhao et
al., 2022).
Although there are no ideal animal models that can mimic the etiology and natural course
of painful TMJ degeneration in humans, they still can provide platforms to investigate mechanisms
underlying TMJ degeneration and pain. Different animal models could generate different TMJOA

60
lesions, but each model has its limitations. Thus, selecting appropriate animal models is key to
finding evidence that answers research questions.
Based on previous studies and literature review, several studies using animal models have
investigated the pathogenesis of TMJ degeneration and pain. For example, the CFA-induced
inflammatory mouse model is a widely accepted animal model to study pain mechanisms related
to TMJ disorders, since it is a reliable method producing measurable pain behaviors and
degenerative changes in condylar and surrounding TMJ structures that occur in TMJOA patients
(Chung et al., 2023b; Xu et al., 2019). However, there is still a lack of innovative targeted therapies
that can provide long-term treatment effects, restore TMJ structures, and minimize complications.
Therefore, research findings to fully understand the mechanisms underlying painful TMJ
degeneration and improve the limitations of the current management of painful TMJOA are
required.
To further investigate and develop novel therapeutic agents to improve painful TMJ
degeneration, the CFA-induced inflammatory mouse model was used to study the pathogenesis
and characterization of painful TMJ degeneration, as well as evaluate potential therapeutic
interventions in our study.
3.2 Materials and Methods
3.2.1 Mouse model
C57BL/6J (JAX#000664) mouse lines were obtained from Jackson Laboratory. Eight
week-old female mice were used in this study. All mice were housed in specific pathogen-free
conditions under a 12 h light/dark cycle, controlled temperature, and humidity. Mice were
euthanized via carbon dioxide overdose followed by cervical dislocation. All studies were
performed with the approval of the Institutional Animal Care and Use Committee of the University
of Southern California.

61
3.2.2 CFA intra-articular injection
Two month-old female mice were randomly assigned into control and experimental
groups. Mice were anesthetized with isoflurane. TMJ inflammation was induced by bilateral TMJ
intra-articular injection with 10 uL CFA (5mg/mL; Chondrex, Inc) according to the previous studies
(Chen et al., 2013), and the control group received bilateral injections with 10 uL sterile PBS.
Then, mice stayed on the heat pad until they fully recovered.
To validate the accuracy of the injection, we initially verified the injection location by
injecting 10 uL of fast green and dissecting the masseter muscle to check the accuracy of the fast
green injection, following the protocol in the previous studies (Figure 3.10) (Chen et al., 2013;
Morel et al., 2019).
Figure 3.10. Mouse TMJ intra-articular injection technique. (A-B) The injection site (yellow circle)
is located anterior to the ear canal and below the zygomatic arch. (C-D) Fast green was injected
surrounding the TMJ (Morel et al., 2019).
3.2.3 Nociceptive behavior assessment
The mice were acclimated to the testing environment for at least 1 hour before behavioral
testing. The baseline measurement of bite force and head withdrawal threshold were recorded
before starting to induce inflammation. Mice were prepared for at least 3 days during the training
period.
A B
C D

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Bite force measurement: A bite force transducer (YFM Bite force sensor) was used to
measure the biting force, following the protocol in the previous studies (Chen et al., 2013; Guo et
al., 2019; Wang et al., 2017b). The voluntary biting was recorded for 2 minutes per session and
the top five of bite force amplitude were averaged for further analysis.
Measurement of head withdrawal threshold: Von Frey filament was used to determine the
threshold causing a head withdrawal response. The head withdrawal threshold is defined as the
minimum force from the Von Frey filament that can elicit a withdrawal reflex. The measurement
was recorded at least five tests for each mouse at an interval of 10 seconds. The average of these
values was identified as the head withdrawal threshold.
3.2.4 Micro-CT analysis
Micro-CT live imaging was performed on a Scanco Medical μCT 50 scanner (Scanco
Medical, Switzerland) at the University of Southern California Molecular Imaging Center (90 kVp,
78 µA, 10 µm pixel size). AVIZO 9.4.0 (Thermo Fisher Scientific) were used to perform 3D
reconstruction of TMJ. The microarchitecture parameters of the subchondral bone, including bone
volume over total volume (BV/TV), trabecular spacing (TbSp), trabecular thickness (TbTh), and
trabecular number (TbN) were analyzed using VGStudio Max3.3 (Volume Graphics, Inc., USA)
based on previous literature (Jiao et al., 2010; Jiao et al., 2014; Lei et al., 2022). For each sample,
three spherical regions of interest (ROI) with a radius of 0.1 mm were selected at the midpoints
of the anterior, middle, and posterior condyle for all measurements. Then, the values of each bone
parameter listed above were averaged. At least 5 mice from independent mouse litters were used
to calculate each parameter. Each dot in the graph quantification represents each sample (n).
Student’s t-test was used for statistical analysis. A significant level was set at p-value of 0.05.
3.2.5 Histology
Mice were euthanized and performed perfusion with 20 mL of PBS, followed by 20 mL of
4% paraformaldehyde (4% PFA). TMJ and surrounding structures were dissected and fixed in 4%

63
PFA at 4C overnight. The samples were decalcified in 14% EDTA for at least 20 days and passed
through serial concentrations of ethanol and paraffin for the paraffin section. Haematoxylin and
eosin staining was performed on deparaffinized sections of 8 µm.
Brain and trigeminal ganglion were isolated from the cranium and fixed with 4% PFA at
4C overnight. Then, samples were dehydrated with 30% sucrose at 4C overnight followed by 30%
sucrose/OCT (1:1) at 4C overnight, and embedded tissues with OCT on dry ice.
For cryosections of TMJ, the samples were decalcified in 14% EDTA for at least 10 days
and dehydrated gradually in 15% sucrose for 2 hours, followed by 30% sucrose for 2 hours, and
60% sucrose/OCT (Tissue-Tek, Sakura) (1:1) at 4C overnight. Then, samples were embedded in
OCT compound, frozen in dry ice, and sectioned at 14 µm thickness using a cryostat (Leica
CM1850).
3.2.6 Safranin O and fast green staining
Paraffin sections were used to perform Safranin O and fast green staining following the
standard protocols (IHCWORLD, #IW-3011).
3.2.7 Immunostaining
Brain, trigeminal ganglion, and TMJ sections were used for immunofluorescence staining
following standard protocols. Antigen unmasking solution (Vector, H-3300) was used for antigen
retrieval. The primary antibodies were as follows: Cathepsin K (CTSK; 11239-1-AP; 1:200), Pdgfra (Cell Signaling Technology; #3174S, 1:200), CD31 (R&D Systems; AF3628; 1:200), βIII-Tubulin
(Cell Signaling Technology; #5666S, 1:200). Alexa Fluor 488/568/647 (Invitrogen; 1:200) were
used as secondary antibodies. DAPI (Invitrogen; 62248) was used for nuclear staining. The
percentage of positive immunofluorescence signals and area fraction were determined using
ImageJ software. Quantification was performed at 20X magnification, 3 to 5 sections were
analyzed per mouse. Each dot presented in the graph quantification represents the mean value

64
of each sample or mouse in that group. At least three mice from independent mouse litters were
analyzed for each group or genotype. Student’s t-tests were used for statistical analysis. A
significant level was set at p-value of 0.05.
3.2.8 RNAscope staining
Sample preparation and RNAscope staining were performed according to standard ACD
protocol: The RNAscope® Multiplex Fluorescent Reagent Kit v2 (Cat. No. 323100). The probes
used for this study were as follows: Mm-Thy1 (430661-C1), Mm-Prg4 (437661-C2), and Mm-Il1b
(316891). A negative control probe (320871) was used when staining and imaging to remove
background signals. Quantification of RNAscope staining by ImageJ was performed at 20X
magnification and interpreted according to ACD scoring guidelines. At least three mice from
independent litters were analyzed for each group or genotype. The quantified area is the total
area of suture mesenchyme in the cranial suture. Student’s t-tests were used for statistical
analysis with a significant level p-value of 0.05.
3.3 Results
3.3.1 Established painful TMJ degeneration mouse model.
The CFA-induced inflammatory TMJOA mouse model was used to investigate the
mechanism underlying TMJ degeneration with pain. To examine and validate whether CFA could
induce degenerative processes in our mouse model, micro-CT imaging was used to analyze bone
architecture. Findings from micro-CT analysis revealed substantial subchondral bone loss after
3-4 weeks of CFA injection compared to PBS control (Figures 3.11A-3.11E), including decreased
bone volume and trabecular thickness, while trabecular space and trabecular number increased.
Immunofluorescence staining also showed an increase in Cathepsin K+ cells, a marker for
osteoclast, in the subchondral bone after CFA injection compared to control (Figures 3.11F3.11G). ln addition to subchondral bone change, articular cartilage degradation determined by

65
Osteoarthritis Research Society International (OARSI) scoring systems was found at 2 weeks and
increased severity at 4 weeks after CFA injection (Figures 3.12A-3.12C).
Figure 3.11. Increased subchondral bone loss in the CFA intra-articular injection group. (A)
Sagittal view of mandibular condyle from Micro-CT images. Scale bar 500 μm. (B-E)
Quantification analysis of microarchitecture parameters of the subchondral bone. Bone
volume/total volume; BV/TV. (F) Immunofluorescence staining of Cathepsin K in TMJ sections.
(G) Quantification of Cathepsin K+ cells in subchondral bone of TMJ condylar head. Scale bar
100 μm. Values represent mean ± SEM, Student’s t-test, *p-value < 0.05, **p-value < 0.01, ****pvalue <0.0001.
Cathepsin K

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Figure 3.12. Cartilage remodeling in CFA-treated mice. (A) Safranin O and fast green staining
showed reduced glycosaminoglycan (red) in the CFA group compared to the control. (B)
Hematoxylin and eosin (H&E) staining revealed loss of fibrocartilage and unclear boundary of
cartilage layers after 4 weeks of CFA injection (C) Quantification of OARSI score. Scale bar 100
μm. Values represent mean ± SEM, Student’s t-test, *p-value < 0.05.

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Figure 3.13. Inflammation in synovial tissue after 4 weeks of CFA injection. (A) Overview of TMJ
structure and inflammation surrounding TMJ. (B) Synovial hyperplasia was indicated by black
arrowhead (upper panels) in CFA treated group and inflammatory cell infiltration (black
arrowdead) in synovial tissue (lower panels). (C) Quantification of synovitis scoring in the synovial
membrane. (D) Quantification of inflammatory cell infiltration in synovial tissue. Scale bar 500 μm.
(A) and 100 μm. (B). Values represent mean ± SEM, Student’s t-test. *p-value < 0.05.
Furthermore, inflammation in the soft tissue surrounding TMJ was observed, including
synovial hyperplasia and inflammatory cell infiltration in synovial tissue (Figures 3.13A-3.13D).
These results suggest that CFA-induced inflammation in TMJ could result in bone degeneration,
cartilage degradation, and synovitis in TMJ which are consistent with pathological findings in
TMJOA patients.
The CFA-induced inflammatory TMJOA model has been widely used to study TMJ pain
mechanisms (Chung et al., 2023a; Xu et al., 2019). To investigate whether the CFA intra-articular
injection can induce painful symptoms, we measured nociceptive behaviors, including bite force
and head withdrawal threshold. CFA injection significantly reduced bite force and lowered the
head withdrawal threshold (Figures 3.14A-3.14B). These findings demonstrate that the CFAinduced inflammatory TMJOA model can generate painful symptoms and TMJ degeneration
which resemble clinical characteristics in TMJOA patients.
Figure 3.14. Nociceptive behavior assessment. (A) Bite force measurement. (B) Measurement of
head withdrawal threshold by Von Frey filament test. Values represent mean ± SEM, Student’s ttest. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, ****p-value <0.0001.

68
3.3.2 Inflammatory response surrounding TMJ after CFA injection.
TMJOA is a multifactorial disease and one of the etiologies leading to TMJ pain and
degeneration is an inflammatory process (Sperry et al., 2019; Tanaka et al., 2008). To further
expand on this observation, we examined the inflammatory response on our mouse model. After
CFA injection at three weeks, we found there was significant cartilage damage, subchondral bone
loss, and synovitis at four weeks, compared to two weeks after CFA injection which showed mild
phenotype with no obvious bone change. Immunostaining results showed that inflammatory cells
such as neutrophils and macrophages infiltrated surrounding the TMJ in CFA-treated mice (Figure
3.15A). Ly6b is expressed on neutrophils, inflammatory monocytes, and some activated
macrophages, while Iba1 is well accepted as a pan-macrophage marker (Rehg et al., 2012).
Additionally, the expression of IL-1β, a pro-inflammatory cytokine, was significantly increased in
synovial tissue and colocalized with Ly6b+ and Iba1+ cells in CFA-treated mice (Figures 3.15A3.15C). Our findings are consistent with previous studies showing that IL-1β was related to TMJ
pain and inflammation in TMD patients (Ibi, 2019; Ulmner et al., 2022). Therefore, these findings
suggest that CFA can induce an inflammatory response in TMJ related to the pathology of
degenerative joint disease in patients.

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Figure 3.15. The infiltration of inflammatory cells surrounding TMJ after CFA injection. (A) Overview
of inflammation in TMJ. (B) RNAscope of IL-1β (red) and immunofluorescence staining of Ly6b
(white) and Iba1 (green) in different regions surrounding TMJ. (C) Quantification of the area
fraction of IL-1β+
, Ly6b+
, and Iba1+ cells in the TMJ area. Scale bar 100 μm. Values represent
mean ± SEM, Student’s t-test. *p-value < 0.05, **p-value < 0.01,***p-value < 0.001
3.3.3 Neuroimmune interaction in TMJ degeneration and pain.
Inflammation from TMJ synovitis can cause painful symptoms in TMD patients since
immune response and inflammatory cytokines are the main factors that act as noxious stimuli
leading to peripheral sensitization (O'Neill and Felson, 2018; Xu et al., 2019; Yi et al., 2021). To
investigate neuroimmune interaction in the CFA-induced inflammatory pain model, TMJ,
trigeminal ganglion (TG), and spinal trigeminal nucleus caudalis (SpVc) were collected to perform
immunostaining. Our studies revealed an increased nerve innervation surrounding the condylar
head of TMJ in CFA-treated mice compared to the control. Additionally, nociceptive fibers labeled
by CGRP colocalized with TUBB3 were increased in the CFA group (Figures 3.16C-3.16D).

70
Figure 3.16. Increased sensory nerve innervation in the CFA group. (A) Overview of nerve
innervation surrounding TMJ. (B) Immunofluorescence staining of CGRP (green) and TUBB3
(red) in different regions (C-E) Quantification of CGRP+
; TUBB3+ area fraction surrounding TMJ.
Scale bar 100 μm. Values represent mean ± SEM, Student’s t-test. *p-value < 0.05, **p-value <
0.01, ***p-value < 0.001
In addition, neurovascular supply plays a crucial role in maintaining tissue homeostasis
and inflammatory response. Although angiogenesis is a tightly controlled process in normal
healthy tissues, some pathological conditions such as inflammation are associated with aberrant
angiogenesis in granulation tissue. Results from immunofluorescence staining of TUBB3 for nerve
fibers and CD31 for blood vessels showed that nerves run along blood vessels in synovial tissue
and increase their innervation with newly formed blood vessels in inflammatory tissue (Figures
3.17A-3.17C).

71
Figure 3.17. Increased neurovascular structures surrounding TMJ area after CFA injection. (A)
Overview of neurovascular structures in TMJ. (B) Immunofluorescence staining of TUBB3 (red)
and CD31 (green) in different regions surrounding TMJ. (C) Quantification of the area fraction of
TUBB3+ and CD31+ area fraction in the TMJ area. Scale bar 100 μm. Values represent mean ±
SEM, Student’s t-test. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001.

72
Next, we investigated the immune response in the peripheral and central nervous systems
related to the pain process. Immunostaining results in trigeminal ganglion showed that there was
an increased expression of CGRP+ neurons and Iba1+ cells in V3 of the trigeminal ganglion,
implying an increase of nociceptive neurons and macrophage in the peripheral nervous system
after CFA injection (Figures 3.18A-3.18C).
Figure 3.18. Neuroimmune response after CFA injection with increased CGRP nociceptive
neurons and Iba1+ macrophages in TG as well as microglia activation in SpVc. (A)
Immunofluorescence staining of CGRP (green) and Iba1 (red) in V3 of the trigeminal ganglion (B)
Quantification of CGRP+ neurons and Iba1+ area fraction in V3 of the trigeminal ganglion. Scale
bar 100 μm. (D) Immunofluorescence staining of microglia activation with Iba1(red) and CD68
(green) in spinal trigeminal nucleus caudalis. (E) Quantification of Iba1+ cells in SpVc area. (F)
Quantification of the number of primary processes per microglia. (G) Quantification of the total
length of processes per microglia. Scale bar 100 μm. (right panels), 20 μm. (left panels). Values
represent mean ± SEM, Student’s t-test. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001.
Furthermore, we found that the number of microglia cells, which are labeled Iba1, were
significantly increased in SpVc in the CFA-treated group compared to the control. In addition to
D E F
G

73
the increase in the number of microglia, the number of primary processes increased, whereas the
length of the process of microglia was decreased in CFA group. Increased expression of CD68+
microglia, a marker for microglia activation, was also found in SpVc of CFA-treated mice (Figures
3.18D-3.18G). Together, these findings suggested that there were neuroimmune interactions and
microglia activation in the TMJ degenerative pain mouse model.
3.3.4 Distinct anatomical features of synovial lining cells in TMJ.
The synovial membrane is an important structure covering the inner surface of the joint
capsule, except articular cartilage. It functions in the production, secretion, and resorption of
synovial fluids. Patients often experience TMJ pain which is associated with inflammation in the
synovial membrane or synovitis (Nozawa-Inoue et al., 2003; Smith, 2011). Previous findings in
knee joints have revealed specialized subsets of immune-regulatory macrophages and fibroblasts
(Croft et al., 2019; Knab et al., 2022). The synovial membrane consists of a lining layer and
sublining interstitial synovial tissue. The superficial lining surface is enriched in macrophages and
fibroblasts, while the sublining connective tissues contain nerves, blood vessels, lymphocytes,
mast cells, adipocytes, and heterogeneous populations of interstitial macrophages and
fibroblasts. Lining macrophages and fibroblasts act as a synovial barrier to maintain joint integrity
and homeostasis. However, inflammation can disrupt the lining integrity resulting in inflammatory
immune cell infiltration in the sublining layer and the joint cavity (Knab et al., 2022). To examine
the cellular phenotype of the synovial membrane in the TMJ, RNAscope and immunostaining
were performed to identify specific subtypes of major cells in the synovial tissues. During steadystate, TMJ synovial lining macrophages express CX3CR1, while lining fibroblasts strongly
express Prg4 (Figures 3.19A-3.19B). In addition, recent studies have identified Thy1+ cells
(express CD90) as sublining fibroblasts. However, this sublining fibroblasts could undergo a major
expansion in the inflammatory joint disease (Wei et al., 2020). Similarly, our studies found an

74
increased expression of Thy1+ cells in the sublining layer, while superficial lining fibroblasts that
expressed Prg4 were disrupted in CFA-induced inflammatory TMJOA (Figures 3.20A-3.20C).
Figure 3.19. Synovial lining cells in TMJ in normal condition. (A) The expression of CX3CR1+
macrophage (green) in the superficial lining of the synovial membrane. Arrows indicate lining
cells. (B) CX3CR1+ lining macrophages (green) are next to Prg4+ lining fibroblasts (red), while
Thy1 (white) is expressed in the sublining synovial tissue. Scale bar 100 μm.

75
Figure 3.20. Synovial fibroblasts in TMJ. (A) Overview of the expression of Thy1+ and Prg4+
synovial fibroblasts in the surrounding TMJ. (B) RNAscope staining of Thy1 (green) and Prg4
(red). (C) Quantification of Thy1+ and Prg4+ area fraction in different regions surrounding TMJ.
Scale bar 100 μm. Values represent mean ± SEM, Student’s t-test. *p-value < 0.05, **p-value <
0.01, ***p-value < 0.001.
3.3.5 Increased nerve innervation in fibrous tissue surrounding TMJ.
TMJ structures are covered by fibrous joint capsules with synovial tissue lined inside the
joint capsule. These mesenchymal tissues are mainly composed of fibroblasts. As mentioned
above, we found an increased nerve innervation in the connective tissue surrounding the condylar
head of TMJ after three weeks of CFA injection (Figures 3.16 and 3.17). These results lead us to
further examine which cell types are closely related to nerve innervation in TMJ pain. We found
an expression of platelet-derived growth factor receptor-α (Pdgfr-α) around nerve innervation
(Figures 3.21A-3.21C). Pdgfr-α marker is expressed broadly in fibroblasts, which can become
excessive proliferation during inflammation. As a result, synovial fibrosis, one of the
characteristics of synovitis, can occur and contribute to joint pain and stiffness in the TMJOA
(Croft et al., 2019; Kendall and Feghali-Bostwick, 2014). In addition, previous studies have

76
revealed that fibroblasts produce and secrete several cytokines, such as IL-33, which play a
crucial role in mediating joint pain and inflammation (Kendall and Feghali-Bostwick, 2014; Palmer
et al., 2009). Therefore, we performed immunofluorescence staining to identify the expression
pattern of IL-33 in TMJ structures and found that the expression of IL-33 significantly increased
surrounding TMJ in CFA-treated mice (Figures 3.22A-3.22C). Together, these findings suggest
that Pdgfr-α+ fibroblasts and IL-33+ cells could play an important role in TMJ degeneration and
pain.
Figure 3.21. Increased nerve innervation and fibrous tissue in CFA-induced inflammatory
TMJOA. (A-B) The expression of Pdgfr-α+ fibroblasts (green) and βIII-Tubulin (red). (C)
Quantification of the area of Pdgfr-α+ fibroblasts surrounding βIII-Tubulin. Scale bar 100 μm.
Values represent mean ± SEM, Student’s t-test. *p-value < 0.05, **p-value < 0.01.

77
Figure 3.22. Increased IL-33 expression in CFA-induced inflammatory TMJOA. (A-B) The
expression of IL-33+ cells (green). (C) Quantification of IL-33+ cells in different regions surrounding
TMJ. Scale bar 100 μm. Values represent mean ± SEM. Student’s t-test. **p-value < 0.01, ***pvalue < 0.001.

78
3.4 Discussion and Future Direction
Temporomandibular joint osteoarthritis is a common degenerative joint disease with high
prevalence and a significant impact on patient’s quality of life, primarily due to TMJ pain and
dysfunction. However, pain management is a challenging task, because of the difficulty in
precisely targeting underlying mechanisms. Since inflammation is an important factor in the
development of TMJ pain and the progression of TMJOA, we further investigated the mechanisms
contributing to painful TMJ degeneration using a CFA-induced TMJOA mouse model. Intraarticular injection of CFA can generate painful symptoms, osseous changes, and inflammation in
the TMJ which represent cardinal symptoms and pathology in TMJOA patients.
The pain behaviors in our TMJOA mouse model showed a significantly lower head
withdrawal threshold and reduced bite force, particularly at the early stage. The purpose of the
Von Frey filament test is to measure the lowest threshold force that could lead to head withdrawal
behavior indicating pain response and hyperalgesia state of the orofacial region (O'Neill and
Felson, 2018). Reduced head withdrawal threshold could be interpreted as joint tenderness and
reduced pressure pain threshold presenting in patients with TMJ pain (Hansdottir and Bakke,
2004; Ohrbach et al., 2013). Additionally, we found a substantially decreased bite force after CFA
injection, which can be related to TMJ pain, since TMJ is one of the key structures involved in all
jaw movement, such as mouth opening-closing, biting, and chewing. The dominant clinical feature
of TMD is chewing or mastication-related pain. Consistent with clinical studies, patients with joint
pain had lower bite force compared to healthy individuals. In addition, reduced bite force in TMJ
pain patients was significantly correlated with their lower pressure pain threshold. Together, CFA
induced TMJOA mouse model, and measurement of bite force and head withdrawal threshold,
can provide a reliable method for pain behavior assessment.
Although TMJ pain and degeneration encompass a broad spectrum of conditions, the
disease initially starts with TMJ pain from capsulitis or synovitis and eventually ends with

79
degenerative joint disease resulting in osteoarthritic changes (Al-Ani, 2021; Chung et al., 2023b;
O'Neill and Felson, 2018). Our findings suggested that CFA-induced TMJOA mice also exhibited
similar disease progression to humans when experiencing severe pain at the early stage;
whereas, at the later stage osseous changes appeared. As the disease progresses into an
intermediate and late phase of TMJ degeneration, pain subsides, and bone remodeling occurs.
However, TMJ pain can be triggered again by abnormal joint loading and inflammation (Al-Ani,
2021). Therefore, future studies identifying potential therapeutic targets to inhibit or reduce pain
severity in the early phase while preventing relapse in the late phase of TMJ degeneration, would
be beneficial for TMJOA management.
The injection of CFA intra-articularly induces inflammation, resulting in synovitis. In our
mouse model, the histological findings mirror the pathology observed in humans, characterized
by the hyperplasia or thickening of the synovial membrane, increased stromal vascularization,
and infiltration of inflammatory cells (Ohrbach et al., 2013; Xu et al., 2019). These pathological
conditions contribute to the main characteristics of TMJOA, such as joint pain in patients.
Once the synovial tissue undergoes hyperplasia and inflammation, inflammatory
mediators can invade and compromise joint integrity, ultimately resulting in cartilage degradation
and subchondral bone loss in the later stages (O'Neill and Felson, 2018; Rim and Ju, 2020; Xu
et al., 2019). This could be a plausible explanation supporting our research, where we found
synovitis within two weeks and obvious osseous changes in four weeks following CFA injection.
Our investigation revealed a significant increase of IL-1β+
and IL-33+ cells in inflammatory
synovial tissues. This corresponds with prior clinical studies, where IL-1β and IL-33 were identified
in synovial fluids of osteoarthritis patients (Ibi, 2019; Ulmner et al., 2022; Yi et al., 2021). IL-1β
was also found to be linked with TMJ pain upon palpation and synovitis score. Additionally, studies
demonstrated a strong correlation between synovial tissue cytokines and inflammatory factors
(Ulmner et al., 2022). Previous research also showed that inhibition of IL-33 could alleviate both
joint pain and neuropathic pain (He et al., 2020; Kimura et al., 2022; Palmer et al., 2009). Thus,

80
a future study focusing on IL-33 could be a promising therapeutic approach for managing TMJ
pain.
In addition to pain and inflammation, our study has detected an expansion of fibroblasts,
characterized by Thy+ and Pdgfr-α+ cells, in the surrounding area of the TMJ. It has been
demonstrated that a specific subset of fibroblasts in the sublining undergoes significant expansion
in osteoarthritis, and this expansion is associated with disease activity (Croft et al., 2019; Knab et
al., 2022; Wei et al., 2020). Intriguingly, we found the cluster of IL-33+ and Pdgfr-α+ cells align with
the nerve fibers, indicating a potential connection between pain sensation and fibrous
inflammation. Therefore, our future studies will explore the molecular mechanisms by which
Pdgfr-α+ fibroblasts drive pain, ultimately leading to TMJ degeneration.
Furthermore, increasing evidence indicates the alterations in the TMJ microenvironment
linked to the progression of pain and TMJ osteoarthritis (Ulmner et al., 2022; Yi et al., 2021). The
temporal-spatial changes in TMJ pain and degeneration cannot be adequately explained by the
current rough cell type classification, since there is a complex cellular network driving the
progression of TMJ disease. Therefore, we will perform a single-cell RNA sequencing (scRNAseq) analysis of mouse TMJ and surrounding structures using the CFA-induced inflammatory
TMJOA model. Understanding these complex tissues at the single-cell level can provide valuable
information and new insights into the pain initiation and progression of TMJOA, potentially
advancing the development of innovative and effective therapeutic targets.

81
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Abstract (if available)

Abstract Disruption of ribosome biogenesis selectively affects craniofacial biology and disease, including craniosynostosis, but the underlying mechanisms remain poorly understood. Suture mesenchymal stem cells (MSCs) serve as the engine that drives calvarial suture development, homeostasis, and regeneration. Its loss leads to craniosynostosis, a craniofacial disorder characterized by premature suture closure. Ribosome biogenesis, historically thought to be a static housekeeping process, is now known to have tissue-specific roles. However, the functional specificity of ribosome biogenesis in suture MSCs remains largely unexplored. Here, we genetically perturbed ribosome biogenesis in MSCs using Snord118, a small nucleolar RNA (snoRNA) required for ribosomal RNA (rRNA) maturation. Snord118 mutant mice exhibited craniosynostosis-like defects with p53 activation, increased cell death, reduced proliferation, and premature osteogenic differentiation of MSCs. We established a method to induce human induced pluripotent stem cells (iPSCs) into suture MSCs and used CRISPR genome editing to generate SNORD118 mutations in iPSCs. Ribosome profiling of human iPSC-MSCs revealed that SNORD118 deficiency in MSCs causes global translation dysregulations and downregulation of complement pathway. Further loss-of-function of complement pathway using complement C3a receptor 1 (C3ar1) KO mice exacerbated cellular defects leading to suture fusion in Snord118 mutant mice, while complement pathway activation rescued MSC cell fate and suture growth defects. Thus, ribosome biogenesis is crucial for the regulation of MSC fate via the complement pathway.

Temporomandibular joint (TMJ) osteoarthritis (OA) is a common and debilitating disease characterized by joint degeneration with synovitis, cartilage remodeling, and subchondral bone destruction along with painful conditions. Disease-driving pain mechanisms are poorly understood, and current treatments cannot provide effective and long-term therapeutic effects. Here we established an inflammatory TMJOA mouse model via intra-articular injection of CFA (Complete Freund’s Adjuvant). TMJOA mice exhibited cartilage remodeling, bone loss, synovial inflammation, and orofacial pain behaviors which recapitulate clinical characteristics in TMJOA patients. Immunofluorescence and RNAscope staining revealed neuroimmune interaction in painful TMJ degeneration and distinct anatomical features of synovial lining cells in TMJ. Additionally, sensory innervation was robustly induced surrounding TMJ coupled with the expansion of fibroblasts and macrophages, contributing to OA pain generation and progression. An increased nerve innervation in fibrous tissue surrounding TMJ points toward a potential direction to further investigate the functional importance of fibroblasts in TMJ pain and degeneration. Together, we established mouse models and provided cellular mechanisms of TMJOA and pain, which help identify treatment strategies to improve TMJ pain management and restore TMJ functions.

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Creator Jariyasakulroj, Supawadee (author)

Core Title Craniofacial skull joint and temporomandibular joint (TMJ) in homeostasis and disease

School School of Dentistry

Degree Doctor of Philosophy

Degree Program Craniofacial Biology

Degree Conferral Date 2024-05

Publication Date 03/19/2024

Defense Date 03/11/2024

Publisher Los Angeles, California (original), University of Southern California (original), University of Southern California. Libraries (digital)

Tag craniosynostosis,OAI-PMH Harvest,osteoarthritis,ribosome biogenesis,suture homeostasis,temporomandibular joint

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Language English

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Advisor Chen, Jian-Fu (committee chair), Brugger, Amy Merrill (committee member), Chai, Yang (committee member), Feng, Pinghui (committee member)

Creator Email jariyasa@usc.edu,jariyasa079@gmail.com

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