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Rib resection and healing in the mouse: a new model of bone repair

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Rib resection and healing in the mouse: a new model of bone repair

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RIB RESECTION AND HEALING IN THE MOUSE:
A NEW MODEL OF BONE REPAIR.

By

Nikita Tripuraneni

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLGOY AND IMMUNOLOGY)

August 2013
Nikita Tripuraneni

ii
Acknowledgements
I would like to take this opportunity to thank people who have helped me in each and
every stage of acquiring my master’s degree.
Firstly, I would like to thank my wonderful P.I, Dr. Francesca Mariani, for her valuable
guidance, support and mentorship.
Secondly, I would like to thank my family for always being there for me and encouraging
me all through my graduate studies.
Lastly, I would like to thank my friends for keeping me sane during the toughest times
and bringing a smile on my face even when everything went wrong.

iii
Table of Contents
Acknowledgements.............................................................................................................ii
List of Figures......................................................................................................................v
Abstract...............................................................................................................................vi
Chapter 1.0 Introduction..................................................................................................... 1
1.1 Bone as a Tissue............................................................................................................ 2
1.1.1 Osteoblasts..................................................................................................... 2
1.1.2 Osteoclasts......................................................................................................2
1.1.3 Remodeling.....................................................................................................4
1.2 Bone as an organ............................................................................................................4
1.2.1 Endochondral Ossification..............................................................................4
1.2.2 Intramembranous Ossification........................................................................6
1.3 Periosteum .....................................................................................................................7
1.3.1 Structure of the Periosteum.............................................................................7
1.3.2 Role in Bone Repair........................................................................................7
1.4 Remarkable Capabilities of Ribs ..................................................................................8
Chapter 2.0 Materials and Methods.................................................................................. 10
2.1 Surgery Method...........................................................................................................10
2.2 X-Gal Staining.............................................................................................................11
2.3 Skeletal Preparation.....................................................................................................11
2.4 Histological Analysis...................................................................................................12
2.5 Analysis of Healing..................................................................................................... 13
Chapter 3.0 Results........................................................................................................... 14
3.1 Removal of Both Periosteum and Bone.......................................................................14
3.1.1 Whole mount Skeletal Analysis ...................................................................14
3.1.2 Histological Analyses...............................................................................................15
3.2 Segmental Defects leaving the Periosteum behind......................................................16
3.2.1 Whole Mount Skeletal Analysis ..................................................................16

iv
3.2.2 Histological Analyses ..................................................................................17
3.3 Growth of the Healed portion: Quantification.............................................................20
3.4 X-Gal: Osteoclast Activity..........................................................................................21
Chapter 4.0 Discussion..................................................................................................... 23
4.1 Summary..................................................................................................................... 23
4.2 A comparison with fracture healing ........................................................................... 24
4.3 Is the new bon endochondral or directly formed........................................................ 25
4.3.1 Cut Ends .......................................................................................................25
4.3.2 Within the resection zone .............................................................................26
4.4 Model for how repair is occurring in the rib removal experiments............................ 27
4.5 Role of periosteum in repair....................................................................................... 28
4.6 Role of osteoclasts...................................................................................................... 29
Chapter 5.0 Figure Legend................................................................................................31
References......................................................................................................................... 60

v

List of Figures
Figure 1 Skeletal Preparation of control surgery (8 day healing)..................................... 31
Figure 2 Skeletal Preparation of control surgery (91 days healing) .................................31
Figure 3 H& E staining of control surgery (91 day healing).............................................32
Figure 4 H& E Staining of gap region of control surgery (91 day healing)......................33
Figure 5 Analysis of the rib resected from control surgeries............................................ 33
Figure 6 Skeletal Preparation of surgery (0 day healing)................................................. 34
Figure 7 Skeletal Preparation of surgery (8 day healing)................................................. 34
Figure 8 Skeletal Preparation of surgery (10 day healing)............................................... 34
Figure 9 Skeletal Preparation of surgery (16 day healing)............................................... 35
Figure 10 Skeletal Preparation of surgery (19 day healing)............................................. 35
Figure 11 Skeletal Preparation of surgery (38 and 57 day healing)................................. 35
Figure 12 Skeletal Preparation of surgery (78 day healing)………................................. 36
Figure 13 H&E Staining of surgery (8 day healing)......................................................... 37
Figure 14 H& E staining of surgery (10 day healing)...................................................... 38
Figure 15 H& E staining of surgery (16 day healing).......................................................38
Figure 16 H& E staining of surgery (57 day healing).......................................................39
Figure 17 Trichrome staining of surgery (8 day healing)..................................................40
Figure 18 Trichrome staining of surgery (10 day healing)............................................... 40
Figure 19 Trichrome staining of surgery (16 day healing)............................................... 40
Figure 20 Analysis of the rib resected from surgery ....................................................... 41
Figure 21 X-Gal staining on surgeries…………………………...................................... 41
Figure 22 Schematic representation of the hypothetical pattern of repair.........................42
Figure 23. Drawing depicting the regions of a typical fracture of a rib in the rabbit.........43
Figure 24 Osteoclast activity in calvarial defects..............................................................43
Summary of all samples.....................................................................................................44
Summary of all surgeries...................................................................................................56

vi

Abstract
Bone is one of the very few tissues in our body that has the capacity for repair without
leaving a scar. However, there are instances when bone repair fails. In injuries or surgical
situations where a large portion of bone is missing, healing can be completely
compromised. Anecdotal reports in humans have suggested that the rib may be one of the
only long bones in the body that has the capacity to repair large defects. The aim of this
project is to study the healing of a segmental defect in murine ribs to determine if healing
occurs with the help of innate factors. Surgeries were performed on adult CD-1 mice
where a piece of the vertebral rib was removed leaving the periosteum intact. Control
surgeries were also performed where both the rib and the surrounding periosteum were
resected. Animals were sacrificed at various time points and the rib cages were dissected
to analyze the healed region. We found that when the periosteum was left behind, the
resected region would be completely filled in within 1-2 months. The healed bone had
restored the original morphology with only some minor irregularities. In the control
surgeries there was no sign of regeneration except modestly at the cut ends. Based on our
studies, we found that the mouse model can be effectively used to study large defects.
Furthermore, we believe that the periosteum plays a key role in the healing process and
may serve as a repository for stem cells that can mediate the repair. Eventually our hope
is that this research will give a better insight in developing ways to stimulate large defect
repair in other locations of the body.
1

Chapter 1.0 Introduction
The healing of the bone is a remarkable process. Under optimal conditions, injured bone
can be reconstituted without a scar and be almost identical to its original shape. However,
not all fractures heal completely. Achieving a complete union between broken ends can
sometimes be impossible. Furthermore, resolving large segmental defects caused by,
tumor resection and trauma is still a major treatment challenge. Current methods to treat
segmental defects involve the use of prosthetics, bone grafts and distraction osteogenisis
(where an external device is used to move two segments of bone apart in such a way that
new bone fills in the gap). However, these methods are unsatisfactory because they yield
poor results (bone of inadequate strength) or involve severe morbidity risks. One way to
develop innovative ways to treat segmental defects is to study situations in which large-
scale repair occurs naturally. Amphibians famously can regenerate skeletal elements
while mammals are considered limited in this ability. There is one exception to this in
that there have been anecdotal and rare reports of regeneration in the human rib (Philip et
al. 2005, Taggard et al. 2001, Munro et al. 1981). Hence, the focus of my project is to
determine if a mouse model can be used to determine how segmental defects in ribs can
be repaired and if this can then be used to identify the necessary innate factors involved
for the complete union of the bones.
To outline the parameters involved in repair, I will first introduce the major cell types
involved in normal bone homeostasis. I will then discuss how bone develops as some of
these events can repeat themselves during repair (Colnot et al. 2012). The role of the
periosteum, a connective tissue surrounding the bone will be introduced, and finally I will
discuss segmental injury in the rib as model system for repair.
2

1.1 Bone as a tissue
Bone is constantly being remodeled and for this to happen two major cell types are
involved: Osteoblasts that deposit bone and osteoclasts that resorb bone.

1.1.1 Osteoblasts
Osteoblasts are mesenchymal in origin and differentiate from a pre-osteoblastic stromal
cell (Aubin, 2001). They are cuboidal cells with a single nucleus (Mark et al. 2002) and
can be found on the surface of the bone in characteristic rows. Upon differentiation into
osteoblasts, the cells release an osteoid matrix that predominantly consists of collagen I
along with other proteins such as proteoglycans, glycoproteins and γ-carboxylated
proteins (Mackie, 2003). Osteoblasts have different fates. Once they deposit the matrix
they can either become inactive or line the quiescent mineralized bone-matrix surface
(Goltzman, 2002) or they become encased within the osteoid matrix giving rise to
osteocytes (Mackie, 2003). Osteocytes are abundant in the bone and they communicate
with each other and the surrounding medium via their cellular extensions (Knothe et al.
2004, Manolagas 2000)

1.1.2 Osteoclasts
Unlike the osteoblasts, osteoclasts are multinucleated and have a monocyte/macrophage
origin and their role is to erode or resorb the osteoid matrix deposited by osteoblasts
(Teitelbaum, 2000). The pre-osteoblastic stromal cells also receive signals from bone
resorptive inducers like parathyroid hormone (Rouleau, et al.1988), cytokines,
prostaglandins etc. (Goltzman, 2002) and play a role in bone resorption by releasing
3

RANKL and m-CSF which are necessary factors to help activate the osteoclasts (Suda et
al. 2001).
Osteoclasts are hematopoietic in origin. Monocytes or macrophage lineage cells
proliferate, differentiate and fuse to form osteoclasts (Goltzman, 2002). Sphingosine-1-
phosphate, which is a lipid mediator rich in blood (Rosen 2005, Cyster 2005), plays a
vital role in homing the osteoclast precursor cells to the bone. It induces chemotaxis and
regulates the migration of these precursor cells where they fuse and become terminally
differentiated into osteoclasts (Ishii et al. 2005). In addition, even inflammatory
cytokines like tumor necrosis factor (TNF), IL-1, and IL-6 play a role in direct osteoclast
formation (Axmann et al. 2009). Studies have showed that by blocking the IL6-R,
osteoclast differentiation and formation was significantly reduced suggesting Il-6 might
have a direct role in osteoclast formation (Axmann et al. 2009).
RANKL, which is released by osteoblasts, binds to the cognate receptor RANK on
osteoclasts or the monocytes and stimulate their production and differentiation (Theill et
al. 2002). Once they are terminally differentiated osteoclasts bind to the surface of the
bone via alpha v beta 3 integrins where they secrete acid and proteases, which degrade
the bone matrix proteins (Goltzman, 2002). They release several enzymes like carbonic
anhydrase, cathepsin K, and cysteine proteases which efficiently degrade the type I
collagen previously deposited by the osteoblasts (Stroup et al., 2001).
Osteoblasts can also inhibit osteoclast differentiation by releasing osteoprotegrin, which
binds to the RANK inhibiting RANKL- RANK interaction (Yasuda et al. 1999)

4

1.1.3 Remodeling
Bone is a dynamic tissue and it is continuously built, broken down and built again via the
process of bone remodeling (Zaidi 2007, Cohen 2006). This process occurs throughout
life and allows for replacement of primary infantile bone to secondary bone, which is
more mechanically competent (Rucci, 2008). In order for proper remodeling to occur,
there should be a balance between the bone deposition activity of the osteoblasts and
bone resorption activity of the osteoclasts. If this balance is disrupted, the bone mass is
compromised leading to skeletal disorders like osteoporosis and osteopetrosis (Rucci,
2008). Osteoblasts and osteoclasts also play a role when external forces injure bone.
During a bone injury the body’s innate immune response creates an inflammation. During
this phase inflammatory mediators such as prostaglandins E1 and E2 are speculated to be
responsible for early resorption activity by osteoclasts and differentiation of
osteoprogenitor cells to osteoblasts (Millis 1999).

1.2 Bone as an organ
Bone as an organ is formed during embryonic and post-natal development via 2 main
processes namely endochondral ossification and intramembranous ossification.
Endochondral ossification forms via a cartilage intermediate while in intramembranous
ossification bone forms directly.

1.2.1 Endochondral ossification
Endochondral ossification begins with the condensation of the mesoderm. Cell adhesion
molecules and numerous growth factors and proteins are involved in this critical process.
5

During condensation there is dense packing of cells rather than cell proliferation (Long et
al. 2013). N-CAM’s and N-Cadherin are the main cell adhesion molecules known to
mediate the condensation process. BMP’s and TGF beta superfamily in general are the
important players that allow for mesenchyme condensation (Long & Ornitz, 2013).
BMP’s bind to type I and II Ser/Thr kinases phosphorylating them. The phosphorylated
kinases then bind and phosphorylate SMAD receptors 1, 5 and 8. Phosphorylated SMAD
then enters the nucleus for gene regulation (Massague et al. 2005). BMP signaling is
vital for condensations to form and this has been revealed by micromass cultures of limb
bud mesenchyme containing Noggin, an antagonist of BMP (Pizette & Niswander, 2000).
Genetic deletions and mutations have also revealed that other factors like Noggin, Sox-9
and FGF to contribute to mesenchyme condensation (Long & Ornitz, 2013).
Condensation is followed by chondrocyte differentiation. Cells of the condensation begin
to differentiate into chondrocytes, which are the cartilage-forming cells. These cells
produce large amounts of type II, IX and XI collagen and they suppress type I collagen,
which is the main component of the bone. However, the cells of the periphery of the
condensation mass express collagen I and form the perichondrium which is the boundary
for the developing skeletal element. It separates it from the surrounding mesenchyme.
(Caplan et al. 1987). For chondrocyte differentiation to occur many extracellular signals
are involved.
As the chondrocytes are formed from the mesenchyme they undergo rapid proliferation.
This step allows for the linear growth of the skeleton. At a specific time point, for
example in a mouse tibia at ~E14.5, the chondrocytes at the center undergo maturation,
become hypertrophic, and die and during this time they express type X collagen (Poole,
1991). As chondrocytes undergo hypertrophy, osteoblasts start to differentiate from the
6

perichondrium and continue towards the marrow cavity (Long & Ornitz, 2013). While the
hypertrophic chondrocytes undergo apoptosis they express the protease MMP13. This
protease degrades the matrix of the cartilage, which allows for vascular invasion (Inada et
al. 2004). The arteries bring in osteoprogenitor cells, which deposit new bone matrix
eventually creating a primary ossification center (Maes et al. 2010). Vascular invasion
also allows for the formation of the chondroclasts, a resorptive cell type similar to
osteoclasts, which release MMP9, which further degrades the cartilage matrix (Vu et al.
1998). Their formation is due to the release of RANKL by the hypertrophic chondrocytes
(Xiong 2011). The less mature chondrocytes continue to proliferate at the articular ends
(epiphysis) and then undergo hypertrophy and die at the middle region of the skeletal
element (or diaphysis). The existing cartilage matrix is replaced with trabecular bone at
the diaphysis. The growth plate is thus displaced and longitudinal growth of each skeletal
element occurs (Long 2012). Thus during endochondral ossification a cartilage template
is first formed and is completely replaced by bone by the dual action of cartilage
resorptive cells and bone forming cells.

1.2.2 Intramembranous Ossification
Unlike endochondral ossification, during intramembranous ossification, the mesenchyme
directly differentiates into osteoblasts (Takarada et al., 2013). Capillaries invade the
mesenchyme where they bring in osteoprogenitor cells that differentiate into osteoblasts
and start depositing bone matrix. Initially trabecular bone is formed which is weak,
disorganized and contain high number of osteocytes. It is then replaced by lamellar bone,
which is stronger and has more organized osteocytes (Kanczler et al., 2008). Many cell-
signaling pathways are involved to allow for ossification to occur. For example, in cranial
7

bones, which undergo intramembranous ossification, loss of function studies have shown
that hedgehog signaling plays a vital role in calvarial ossification. (Pan et al., 2013).

1.3 Periosteum
1.3.1 Structure of the Periosteum
The periosteum is a thin layer of connective tissue surrounding almost every bone in the
body with the exceptions of the intra-articular surface and sesamoid bones (Dwek 2009).
It is separated into two distinct layers: the inner cambium layer (Dwek 2009) and the
outer fibrous layer. The inner cambium layer, which is closest to the bone, consists
mainly of the osteogenic and chondrogenic progenitor cells, which have been proposed to
play a key role in bone development and repair (Colnot et al. 2009). Unlike the cellular
rich cambium layer, the outer layer of the periosteum is highly fibrous and rich in
collagen (Squier et al.1990). Along with collagen, the outer layer also has elastin, which
provides it with a mechanical role (Colnot et al. 2009).

1.3.2 Role in Bone Repair
The periosteum is highly vascular and it is vital that this layer is preserved to allow for
normal bone repair (Colnot et al. 2009). During fracture repair, depending on if it’s a
stable or a non-stable fracture a variable sized cartilaginous callus is formed. Unstabilized
fractures have a larger callus and a higher periosteal reaction compared to stabilized
fractures and lineage analyses based on bone grafting has revealed that the cells required
for this callus formation mainly come from the periosteum rather than the bone marrow
and the endosteum (inner lining of the bone facing the marrow) (Colnot et al. 2009). Just
8

within the past decade more research has been conducted to study the regenerative
capabilities of periosteum as it is speculated that it plays a larger role in healing than the
bone marrow cells.

1.4 Remarkable capabilities of ribs
For numerous reconstruction surgeries, there is a long history of using rib material for
autogenous grafts. For example, since 1968 rib grafts were used to treat mandibular
condyle defects (Ware et al.1968). After a mandibular tumor resection there are often
instances of osseous destruction or loss of major portion of the mandibular bone. To
repair this loss, rib grafts were used to fill in the gap. Postoperative analyses have showed
improved facial contours and appearance (Eckardt et al. 2010). Rib grafts were also used
for maxillary reconstructions in patients with facial trauma (Anantanarayanan et al. 2013).
Even in rhinoplasty surgeries like the repair of saddle nose deformity, ribs have been
used with excellent success. (Isac et al. 1990, Kim et al. 2013). However, very little is
known in literature about the donor site (Philip et al. 2005, Taggard et al. 2001, Munro et
al. 1981). Laurie et al. 1984, show donor-site morbidity results in 44 rib donor sites. Early
morbidity results showed no incidence of chest infections or excessive hemorrhage. Late
morbidity results showed that majority of the patients had mild chest-wall contour effect
and no patients had a palpable contour deficit of the removed rib. However, although
segmental defects in the rib may repair well, little is known about the cellular and
molecular mechanisms that might be involved. Therefore, we decided the study the
healing mechanism of ribs in the mouse to better understand the anecdotal reports of rib
repair by surgeons doing the above procedures. Should ribs repair well in a mouse model,
our ultimate main goal is to use what we learn from this large scale repair to develop
9

ways to stimulate large defect repair in other locations of the body. In our study ~0.3cm –
0.5cm of the rib was surgically excised under two different conditions: one leaving the
periosteum intact and the other, where the both the bone and periosteum were removed.
Thus, we could evaluate the potential role of the periosteum in the healing process.

10

Chapter 2.0 Materials and Methods
2.1 Surgery Method
36 adult male CD-1 mice (Charles River Laboratories, MA) were used in this study. All
procedures performed were in accordance with an animal protocol approved by the
Institutional Animal Care and Use Committee (IACUC). A standard surgical technique
was devised to resect the vertebral rib of the mouse. The mice were anesthetized with
isoflurane inhalation vapor along with oxygen. Buprenorphine (0.1mg/kg) was injected
subcutaneously for post-surgery pain. By palpation, the area above the 10
th
and 11
th
rib
was detected and shaved. A 3cm transverse incision was made on the skin and underlying
muscle and fat layers were cut using a medium sized microsurgery scissors. With the help
of a retractor all the 3 layers were held in place to optimize the surgical area while
minimizing the incision size. Using a Sharpoint
TM
5.0mm scalpel a vertical incision was
made through the connective tissue , periosteum, along the length of the 11
th
rib. Using
two Dumont 55 forceps the periosteum was separated from the bone and approximately
0.3cm of the naked rib was excised at both ends using iridectomy scissors. This is a very
delicate step as removing the bone swiftly can lead to a tear in the pleural membrane
causing a pneumothorax which is hard to manage in a mouse. After the rib resection, the
intercostal muscles were sutured over the top of remaining periosteal sleeve, using 9.0
nylon sutures. The sutures were placed right above the cut sites of the rib and were used
as indicator sutures for the surgery location and length of piece removed. The overlying
fat and muscle were also sutured using 9.0 nylon sutures. The skin was sutured with 7.0
prolene sutures to close the incision and then secured with suture glue. The mouse was
then taken off isoflurane and placed under a heat lamp until regaining consciousness
(typically 8 to 10 minutes). The mouse was then maintained in the cage with free access
11

to food and medicated water that contained ( 1.7ug /ml) Oral Meloxicam. The removed
rib piece was fixed immediately in 4% PFA for 3-4 days. At various points, the mice
were euthanized for analysis.
In addition control surgeries were also performed on an additional eleven CD-1 female
mice that included the same procedure described above except that the rib piece was
resected with the periosteum covering it.

2.2 X-Gal Staining
After euthanasia, the entire rib cage was dissected from each mouse and fixed in 4% PFA
for 30 minutes in ice. The rib cages were then transferred to a 2% X-Gal Solution
containing 5mM K3Fe(CN)6, 5mM K4Fe(CN)6, 0.24 mg/ml X-gal and 1mM MgCl2 for
2 to 3 days at 37 degrees. Samples can be left longer in the X-Gal solution for an
enhanced deep blue staining. Once satisfied staining was achieved the rib cages were
fixed again in 4% PFA for 30 min on ice and then transferred to 95% ethanol for skeletal
preparation.

2.3 Skeletal Preparation
Rib cages in 95% ethanol had skeletal preparation performed on them. Skeletal staining
often includes both Alcian blue and Alizarin red steps. Alcian blue is retained by
cartilage and Alizarin red detects mineralized tissue. However, in our method the Alcian
blue step was skipped to avoid confusion with the blue staining produced during the X-
Gal stain.
After several days of dehydration in 95% Ethanol the rib cages were transferred to
Alizarin red solution that contained Alizarin red S (Sigma. 0.06mg/ml) and water for 2
12

days. Once satisfactory staining had been achieved. the rib cages were placed in 70%
Ethanol overnight and then transferred to 50% Glycerol: 50% Water for photography. No
KOH step was included in order to preserve the sample for future histological analysis.

2.4 Histological Analyses
After whole mount photography, the vertebral ribs from 10
th
to 12
th
were dissected from
the rib cages (at least one above and one below the resected rib) and placed in 20%EDTA
for 2 weeks for demineralization. The fixed excised rib pieces, which were previously
resected, were also subjected to demineralization. After 20% EDTA treatment the rib
cage pieces were dehydrated in ascending alcohol concentrations, cleared with xylene
and embedded in paraffin. Sections of 10um were cut along the length of the rib on a
microtome (Shandon Finesse® ME+ Microtome, Thermo Scientific). The sections were
deparaffinized in xylene and dehydrated in ascending alcohol concentrations and then
stained by the following two techniques.

Hematoxylin and Eosin (H&E)
H&E staining is a commonly used staining method in histology and can be used to detect
multiple cell types. Hematoxylin is a basic dye and its stains acidic structures in the cell.
Hence the nucleus is stained blue. On the other hand Eosin is an acidic dye that stains
basic structures in the cell. Therefore, the cytoplasm is stained red. (Histology guide,
University of Leeds). A standard protocol was used to stain the slides.

13

Trichrome Stain
Trichrome stains are used to differentiate between collagen and smooth muscles. The
staining was done using the Gomori One Step, Aniline Blue Kit (Newcomer Supply CAT
#9176). In this method the cytoplasm, muscle fibers are stained red, collagen and mucus
are stained blue and finally the nuclei blue-black to black.
Once the rib sections were stained with H&E and trichrome they were photographed
using the Wide Field Fluorescent Microscope Zeiss AxioImager.A1.

2.5 Analysis of healing
Image J software was used to measure the exact length of the resected rib and the gap
region. With these values the percent healed was calculated by the following equation:

Using the healing time and % healed from all the surgeries (excluding the controls) a
graph was then deduced to find out the average time required for complete union.

X
100

Size
of
the
resected
rib
(mm)

Gap
Size
(mm)

100

%
Healed
=

14

Chapter 3.0 Results
3.1 Removal of both the periosteum and the bone
In order to determine the degree to which the rib bone could repair in the absence of the
periosteum, 11 CD-1 females were used where a segment (average length = 3.24mm) of
the 11
th
vertebral rib was removed along with its surrounding periosteum (see Materials
and Methods). The mice were sacrificed at various time points and subjected to Alizarin
Red staining and histological sectioning analysis.

3.1.1 Whole mount skeletal analysis
Animals were euthanized after a ~1 week healing period (N=2) and photographed.
Figure 1 shows a whole-mount preparation viewed from the external lateral surface of the
rib cage. A yellow bar (to scale) has been placed to indicate the length of the portion
removed as measured directly from the resected piece. In both the samples, abnormal
mineralization was observed within the resection gap surgery (Fig 1). In addition,
minimal growth was apparent from the cut ends by comparing the length of the resected
piece to the length of the gap.

However, later the abnormal ossification that was noticed during the early repair period
had cleared and only a few spots of mineralization were visible in the gap region. The
sites of mineralization could either be ‘new’ bone activity as the piece removed was in
one intact piece or could be the residual bone pieces left behind when making the initial
cut on the ends of the bones. After ~2 weeks healing period (N=3) the gap was still
evident and the ends continued to show modest growth. Similar results were noticed in
15

the surgeries with 27 days to 44 days of healing (N=4) and the gap size remained
relatively similar in length to the resected piece (average of 16% healing). In a sample
with 68 days of healing one of the ends acquired a tapered morphology while the other
and the gap showed small areas of spotty mineralization (N=1). Similar results were also
seen in a sample with 3 months of healing time where the ends continued to show drastic
decrease of the gap size (27 % healing) and only of the ends acquired a tapered
morphology (N=1). (Fig 2)

3.1.2 Histological Analysis
Histological analysis was performed on the sample with the longest healing time point
(~3 months). Ribs 10-12 were embedded and sectioned. This allowed visualization of
both the resected rib and the control un-resected rib in the same preparation. The sections
were stained with H & E.

H and E Analysis
After 3 months healing, the tapered ends were distinctly visible and a thick periosteum
was present surrounding the cut ends (Fig 3). In addition, the new bone had a
disorganized marrow cavity. The gap region consisted mainly of fat cells and granulation
tissue was detected mainly around the sutures. (Fig 4)

16

Analysis of the segment removed
The rib piece was resected in one piece and was fixed immediately. Hand E stain was
performed on longitudinal sections of the piece and it confirmed that the periosteum was
left behind covering the bone. (Fig 5)

3.2 Segmental defects leaving the periosteum behind
35 CD-1 female mice were used where a portion of the vertebral rib was removed
(average length = 3.08mm) carefully leaving most of the periosteum intact. The mice
were sacrificed at various time points and subjected to whole mount Alizarin Red
staining and histological analysis.

3.2.1 Whole mount skeletal analysis
To determine how well the surgery was carried out, some animals (N=6) were euthanized
immediately after surgery. For all 6 animals, skeletal prep revealed the gap region where
the bone was resected (example shown, Fig 6A). In addition, the piece resected is in one
whole piece and has most of the periosteum removed (Fig 6B). These results show that
none or very little bone was left behind during the surgery. Thus, any significant new
bone to form is most likely newly generated.
After about one week of healing time, samples (N=2) showed evidence of healing in that
the gap that was present was smaller in size compared to the length of rib removed. This
could indicate healing from either of the cut ends. The cut ends took up a slightly swollen
morphology (Fig 7).
17

In animals with 10 to 20 days week healing period (N=6), drastic growth was seen from
the ends. However, in one sample a region of mineralization were noticed within the gap
area (Fig 8). There were 2 pieces of varying sizes. Some samples showed an apparent
piece in the middle, which seemed like it fused to the growing ends (Figure 9) and the
rest showed robust growth at the ends. (Figure 10)
In animals with 22 to 45 days of healing time (N=6) complete union was first noticed
around 5 weeks after the surgery (Fig 11) when the gap was closed and the healed region
had a larger diameter appeared enlarged and irregular, possible due to callous formation.
After longer time points i.e. at 52 days to 155 days (N=21) most of the samples showed
further remodeling as the healed bone was morphologically indistinguishable in
comparison to the unresected normal bone (Fig 12).

3.2.2 Histological Analysis
Histological analyses were performed where the vertebral ribs from 10
th
to 12
th
were
dissected and embedded in paraffin. The sections were stained with H & E and Gomori’s
Trichrome.

H and E Analysis
After one week healing time the gap area was filled with abundant granulation tissue. At
the sides of cut ends, clusters of cells that resembled chondrocytes were noticed with a
perichondrium surrounding it (Fig 13). At the bone ends, osteoid deposition was seen at
the tips and a very unorganized marrow activity. The cut sites also showed a thickened
periosteum in comparison to the unresected normal rib (Fig 13C)
18

After 10 days healing time, isogenous groups of cartilage were detected in the gap region
as well as at the sides of the ends. At the gap region hypertrophic chondrocytes were
detected whose matrix coloration was lighter from the mineralized regions (Fig 14). As
the days progressed, the cartilage seemed like it was being replaced by the newly formed
immature trabecular bone (Figure 15). Potentially
undifferentiated
populations
were
seen spreading towards the periphery
and
the
more
differentiated
towards the center.
Right adjacent to the hypertrophic chondrocytes trabecular bone was detected which
inferred that as the chondrocytes were reaching an end stage, bone or osteoid matrix was
being formed. The trabeculae had osteoblasts lining the surface and cells, likely
osteocytes, embedded in them. The marrow tissue was unevenly distributed around the
bony trabeculae.
Around 5 weeks when complete union took place, the new bone had a more organized
marrow cavity and periosteum. However, the size of the marrow cavity was around 2X
smaller in the newly formed bone. The periosteum adhering to the lateral side of the bone
showed a varied difference in thickness as well. Some regions of the healed bone showed
a thicker periosteum (approximately 2x compared to the normal) and in some regions no
significant difference was seen. (Fig 16)

Trichrome Stain
Trichrome stain revealed a high number of collagen fibrils surrounding the cut sites at
the periosteal layers during early repair period (Fig 17). As healing progressed the newly
formed ossification spots were stained red but acquired a boundary lined by collagen.
Collagen fibrils were also detected at the cartilaginous callus sites as expected and at the
19

periosteal layer (Fig 18). The newly formed immature trabecular bone and its
surrounding was stained blue thus inferring collagen activity. (Fig 19)

Measurements
The thickness of the periosteum was measured using the scale in the Wide Field
Fluorescent Microscope Zeiss AxioImager.A1.
The thickness of the periosteum is uneven throughout the bone. Hence an approximate
range values were calculated.
At day 8 healing the healing bone showed a periosteal thickness of 34.2µm to 35.57µm.
The periosteum at this point showed an obvious inner cambium layer but the outer
fibrous layer was very thin to undetectable. The unresected normal bone showed an
average thickness of 16.01 µm 20.11µm.
As the days progressed the thickness of the periosteum increased as well. The outer
fibrous layer was evident at this stage. The thickness of the periosteum in the healing
bone was from 72.03µm to 68.3µm whereas the normal rib showed a thickness of range
14.3µm to 35.43µm.
However at a 16 day time point the thickness of the periosteum slightly reduced to a
range of 44µm to 65µm at the healing bone whereas the normal bone continued to
showed a range of 18.03µm to 37.52µm.
Around 5 weeks where the segmental defect was completely filled in with new bone the
periosteum’s thickness reduced to a range of 26.23µm to 31.31 µm. However it was still
20

thicker compared to the overlying unresected normal rib which had a range of 12.12µm
to 15.79µm.
In summary as the bone is healing the periosteum becomes thick because the cellular
content and as well as the outer fibrous content increases. Once the remodeling stage is
achieved the periosteum’s thickness is reduced comparatively but still thicker than the
normal rib.
Analysis of the piece removed
The resected rib was removed in one intact piece and had barely any periosteum covering
it. H and E staining of the cross section of piece confirmed that only little periosteum was
retained on the bone. (Fig 20). However, as only one sample was analyzed there might
be variation in the amount of periosteum left behind.

3.3 Growth of the healed portion: quantification
As mentioned earlier, the percent healed was calculated for each sample using the
following formula:

A graph was then plotted using this value against the healing period (the time at which
the mouse was sacrificed). 33 of the 35 surgeries are represented in this graph.
X
100

Size
of
the
resected
rib
(mm)

Gap
Size
(mm)

100
%
Healed
=

21

-‐40

-‐20

0

20

40

60

80

100

120

0
50
100
150
200

Percent
Healed

Healing
Period
(days)

percent

healed

Log.

(percent

healed)

Two surgeries were not included as the measurements for these 2 samples were not done.
From the graph it can be inferred that active repair of the defect mainly takes place within
first three weeks after the surgery. Some samples even reach 90% healing around this
time. Shortly after, complete healing was detected around 5 weeks. After one month
most of the samples reached the 90% -100% healing point with few exceptions. These
exceptions could be due to difference in the amount of periosteum left behind or
difference in the length of the rib removed.

3.4 X-Gal: Osteoclasts Activity
Tartrate resistant acid phosphatase (TRAP) is a classical staining method for detecting
osteoclasts. However, in this study we used 5-bromo-4-chloro-3-indolyl-beta-D-
Graph
1

22

galactopyranoside (X-gal) to detect osteoclasts as it can be performed on whole mount
samples and is a relatively faster and an easier detection method.
Osteoclasts are known to have endogenous β-galactosidase (β-gal) activity which can be
detected by the chromogenic substrate X-Gal. β-gal is known to cleave X-Gal and this
yields a blue color which is a classic detection tool used in plasmid and bacterial colony
screening assays (Silhavy et al. 1985).
This method has been confirmed to detect osteoclast as TRAP enzyme localizes with β-
gal activity. (Odgren
et
al.
2006)
X-Gal staining during early repair period showed high numbers of cells with ß-
galactosidase activity. Cells with blue staining were highly abundant mostly at the cut
sites. However, as the healing period increased, the number of cells that stained blue
reduced in number and seemed more spread out. Finally the cell numbers eventually
returned to normal in samples with complete healing (Fig 21).
Since osteoclasts are multi-nuclear H and E stain were also used to detect their location
as well. During early repair period abundant osteoclasts were seen right underneath the
periosteum at the cut sites (Fig 13 A). As the trabecular bone was being formed they were
seen in fewer numbers towards the endosteal surface of the bone (Fig 15). Finally as
complete union took place, their presence was much harder to detect.

23

Chapter 4.0 Discussion
4.1 Summary of the results
Remarkable healing of a 3-5mm segmental defect was achieved in the surgeries where
the periosteum was left behind. The majority of the repair occurred within a 1-2 month
period where signs of repair were detected at the cut ends and as well as in the gap region.
During the first week of repair the region at the cut ends undertook a swollen morphology
and increased both in diameter and in length. The periosteum was thickened at the ends
and the presence of trabecular bone at the tips suggested that new bone was being formed.
Signs of repair were simultaneously seen in the gap region where patches of Alizarin Red
staining suggested that new sites of mineralization were present. Cells with
chondroblast/chondrocyte morphology were seen underneath the periosteum at the ends
and seemingly also streaming into the gap region. In addition, the juxtaposition of this
cartilage tissue in the gap region with trabecular bone suggested that terminally
differentiated chondrocytes were being replaced by immature trabecular bone through an
endochondral process. However this would need to be tested more directly. Around 2
weeks of healing, most of the gap was filled with trabecular bone with little cartilage
present. This further suggested that the cartilage template was almost completely replaced
by trabecular bone. Finally in a month’s time the immature trabecular bone underwent
remodeling to form cortical bone. Samples that had completely healed (gap filled in)
(mostly after 4-5 weeks of healing) were similar in morphology in comparison to the
unresected normal rib with a few differences. The healed rib showed a thicker periosteum
(1.96x) and a much smaller marrow cavity compared to normal. However, there was
variation in healing where some samples underwent only 63% healing even after 113
days of repair (percent healed vs. healing period graph). This irregularity is speculated to
24

happen because some samples could have had a lot more periosteum retained on the
resected piece than the ones that have undergone complete fusion. On the contrary, the
control surgeries showed very little or no healing at all. Most samples showed tapered
growth at the ends and even after 3 months of healing no significant growth were seen.

4.2 A comparison with fracture healing
Fracture healing is a complex process which involves numerous cell signaling pathways,
growth factors, extracellular matrix elements etc. “A most elaborate classification of
fracture healing processes would be hematoma formation, inflammation,
neovascularization and granulation tissue formation, fibrous tissue formation,
fibrocartilage, hyaline cartilage (soft callus), cartilage mineralization, woven bone (hard
callus), and finally remodeling” (Kolar et al., 2010). However these events don’t
necessarily happen in order all the time.
Callus formation in fractures is significantly different than the callus seen in our
segmental defects. In fractures, for a callus to form micro movement is highly essential.
Studies have shown that the more dynamic the fracture site the larger the callus formation
is. (Yamaji et al. 2002). At the fracture site a periosteal callus is formed on the periosteal
surface extending on both sides of the broken bone on top of which is an external callus.
In addition there is also an interfragmentary callus formation with a medullary callus
bridging the gap (Figure 23) (Brighton et al. 1991). However, in our study an apparent
periosteal callus was detected at the cut sites as cells resembling
chondroblast/chondrocyte morphology were detected right underneath the periosteum and
the width of the periosteum at this site was significantly larger (Figure 13A). Furthermore
this callus did not extend all the way to the other cut end to bridge the gap.
25

In fractures, healing occurs either via endochondral ossification or intramembranous
ossification depending on the stability. During an unstable fracture the periosteal cells
undergo rapid activity where they form a cartilaginous callus at the break region which is
eventually converted to bone (Colnot et al., 2012) On the other hand; during a stable
fracture the periosteum is comparatively less stimulated. Hence very minimal or no callus
is formed and the defect is repaired by direct ossification (Colnot et al., 2012). Lineage
analysis and protein studies have concluded that these differences in healing are mainly
due to the characteristics of the periosteum. (Colnot et al., 2012)
However, we hypothesize that in our healing process both forms of ossifications are
occurring simultaneously.

4.3 Is new bone endochondral or directly formed?
4.3.1 Cut ends
Within a week’s time of the surgery, the cut ends took up a swollen morphology. As the
healing time increased the cut ends seem to increase both in length and diameter as well
suggesting that new bone was being deposited at the cut sites facing the gap region.
Histological stains showed immature trabecular bone directly at the cut sites (Figure 13)
in the absence of any cartilage. As stated in the introduction, in intramembranous
ossification bone is formed directly by the differentiation of mesenchymal cells. We
believe that it is by this form of ossification that the bone is being deposited at the ends.
Similar situation was also seen in our control surgeries where very minimal growth was
evident from the cut sites. However the only difference being that some of the samples
undertook a tapered morphology. Although these samples showed no cartilage, an
26

examination of healing time points prior to 1 week would be necessary to support our
claim of direct or intramembranous ossification.
4.3.2 Within the resection zone
As the ends appeared to be lengthening with the formation of new bone, the resection
zone simultaneously showed repair activity. Within 10 days of healing, sites of
mineralization were detected (Figure 14 ). These sites were mostly likely new
mineralization since osteoid could be detected via Trichrome stain (Figure 18 As the days
progressed these sites seemed to increase in size (ie. Longer healing periods correlated
with larger/longer pieces) and they seemed to eventually fuse with the growing cut ends.
In addition to the mineralization, cartilage was detected as well. Cells of
chondroblast/chondrocyte morphology could be found within the resection zone.
Chondrocytes with a hypertrophic profile were also found surrounded what appeared to
be a mineralized matrix. These observations support the idea that a cartilage template was
initially laid at the gap and that the cartilage underwent mineralization The location of
trabecular bone close to this suggested that terminally differentiated chondrocytes were
likely undergoing apoptosis, osteoprogenitors were migrating in and osteoid deposition
was taking place (Figure 14 & 15). Thus, although it is possible that direct ossification is
occurring within the resection zone, the presence of tissues representing all the steps of
endochondral ossification suggests that this process is a major contributor to healing
within the gap.
In summary, though during development, bone either develops by intramembranous
ossification or endochondral ossification, during the repair of segmental defects in the rib
both these processes may interplay simultaneously to fill in the gap i.e. intramembranous
at the cut sites and endochondral at the resection zone.
27

4.4 Model for how repair is occurring in the rib removal experiments
In our surgeries, the periosteum is critical for repair since if it is not left behind, repair is
compromised. The inner cambium layer of the periosteum has been proposed to contain
undifferentiated mesenchymal cells that allow for development and repair. During early
repair period the periosteum not only thickens but becomes hypercellularized. A
reasonable hypothesis is that osteochondral cells needed to facilitate repair are replicating
and then leaving the periosteum. One possibility is that chondrogenic precursors are first
pumped into the gap region from the periosteum where they differentiate into
chondrogenic cells and deposit a cartilage matrix. Vascularization would then bring in
osteoprogenitors either derived from the circulation or the periosteum as cell proliferation
and cell expression studies have shown that during a fracture the periosteum acts as a
source of osteoprogenitors (Maes et al. 2006). New bone at the cut ends could be
generated from osteoprogenitors derived from the periosteum or from bone marrow stem
cells/endosteum.
The periosteum is also highly vascular in nature and it has the ability to bring in
osteo(chondro)clasts that erode the cartilage matrix as angiogenesis is essential in
recruiting osteo(chondro)clast precursors (Yang et al. 2012) . In addition, research has
also shown that the periosteum has VEGF activity, which is essential for angiogenesis
(Yao et al., 2004). Thus, aside form being a potential source of cells for repair, the
periosteum may have an additional function in providing the vasculature to bring
osteoclasts into the repair site. This may facilitate both the endochondral process and the
final remodeling of trabecular bone into cortical bone.
Figure 22 shows a schematic of how this hypothetical pattern of repair is occurring.
28

4.5 Role of periosteum in repair
The periosteum’s cambium layer is rich in cells which are both chondrogenic and
osteogenic in nature. While we have not looked at markers for the cambium layer to see
if this is the portion that has undergone expansion, we believe that the precursor cells
come mainly from the periosteum which helps in repairing the bone defect both in the
gap and at the cut ends. These cells presumably proliferate in response to injury and then
enter the repair zone to do their course of action. Markers for proliferation could be used
to confirm this idea.
On the contrary, if not providing the defect with the building blocks, the periosteum
could possibly be providing an ideal niche for repair cells. In addition, the periosteum
could be providing the defect area with a necessary vascular environment that can favor
the activity of the precursor cells.
The periosteum also has interesting mechanical properties. Studies have shown that the
periosteum shrinks more in the axial than in the circumferential direction when stripped
from the bone, which indicates that the tissue is already under stress. (McBride et al.,
2011). It also exhibits strain stiffening during trauma conditions. These mechanical
properties are believed to influence the bone biology during its development and repair.
(McBride et al., 2011)
In addition, the periosteum also exhibits barrier properties where it swells under isotonic
conditions and physiological pH. (McBride et al., 2011). The barrier properties are
exhibited by the periosteum because the cells from them express zona occludens 1 and N-
29

cadherin which are both vital tight junction proteins and allow for the formation of tight
junctions between cells. (Evans et al., 2013)

4.6 Role of osteoclasts
Lastly, the X-Gal method was used to detect osteoclasts. As stated previously, strong
staining was seen both at a distance from the injury and at the cut ends during the early
repair period. As healing progressed the staining was reduced. These observations
suggest that in response to injury, monocytes are stimulated to differentiate into
osteoclasts. After the resection zone has been filled in and remodeling of trabecular bone
completed, the number of osteoclasts appears to return to normal levels. Further studies
using TRAP staining and evaluating nuclear number would be needed to confirm this
idea. Previous studies have shown that osteoclastogenisis is directly influenced by pro-
inflammatory cytokines (Axmann et al. 2009) and during any injury our bodies’ innate
immune response is inflammation. This tells us how osteoclasts get there but their precise
role is yet undefined. Interestingly, similar results were also seen in calvarial defects
where in a 10 day healing, high osteoclast activity was detected. However, after a 20 day
healing the intensity of X-Gal stain reduced (Figure 24). However, relatively the X-Gal
activity was much more modest in the calvarial defects.
One possibility is that the osteoclasts are clearing the debris at the cut sites and preparing
the stage for the osteoblasts to deposit their osteoid matrix. On the other hand, since
osteoblasts are needed for osteoclast development and maturation (Boyle et al., 2003)
another possibility is that the abundance of osteoclasts at the cut sites is simply due to
their induction by the numerous osteoblasts involved in the healing process.
30

On the other hand mature osteoclasts stimulate chemotaxis of osteoblasts that would
suggest that the intense osteoclast activity during early repair period is to allow for
osteoblast recruitment. (Sanchez-Fernandez et al.,2008).
However, all the above is speculation and further research is needed to investigate the
role of osteoclasts during all stages of repair.
In summary, from this project it can be safely stated, that leaving the periosteum intact
when rib bone is excised, can yield better healing results compared to rib excised along
with its surrounding periosteum. Extending this idea to bone repair in other locations,
like the appendicular skeleton, it may be that in order to achieve complete repair of a
segmental defect, the periosteum may be required.

31
Figure 1. Skeletal Preparation of control surgery (8 day healing). Mini-
mal growth was seen at the two ends. Intense alizarin red staining was seen
at the gap region (arrow head) which did not assume any particular shape
and is spread out around the gap region.
The bar (to scale) is the size of the rib resected
Figure 2 Skeletal Preparation of control surgery (91 days healing).
The cut ends showed growth and acquired a tapered morphology (black
arrows). Tiny areas of mineralization were spotted at the gap region
(black arrow head) .
The bar (to scale) is the size of the rib removed.
Chapter 5.0 FIGURE LEGEND
32
Figure 3 H& E staining of control surgery (91 day healing). A and B
are showing the cut ends with a thickend periosteum.
Skeletal prep placed right above the pics shows the area (square) which is stained.
(H&E 10x )
A
B
33
Figure 4: H& E Staining of gap region of control surgery (91 day
healing). Granulation tissue was seen surrounding the suture regions
(black arrows) and only fat (black arrow head) and muscle cells (red
arrow head) were seen predominantly at the gap region. Skeletal prep
placed right above the pics shows the area (square) which is stained.
(H&E 2.5x)
Figure 5 Analysis of the rib resected from control surgeries.
A: Resected rib piece from a control surgery with periosteum covering
it.
B: Longitudnal section of the resected piece showing the bone covered
with periosteum . (H&E 10x)
A
B
34
Figure 6 Skeletal Preparation of surgery (0 day healing):
A: Skeletal prep reveals the gap region and the cut ends are shown by ar-
row head. The unresected normal bone shown by arrows.The bar (to scale)
is the size of the rib resected. Arrow head point to the dissected ends and
arrows point to the unresected normal ribs.
B: Rib resected with most of the periosteum removed.
A
B
Figure 7 Skeletal Preparation of
surgery (8 day healing). Growth
was seen at the two ends. Initial
size of the defect was 2.38mm
and after a week the defect size
reduced to 1.127mm. One of the
cut ends took up a slightly swol-
len morphology (arrow head).
Bar (to scale) is the size of rib re-
moved
Figure 8 Skeletal Preparation of
surgery (10 day healing). Notice-
able mineralization within in the gap
region occurred. Healing also seem
to occur from the cut ends as the to-
tal gap size was reduced by 76.31%.
Mineralization in the gap was irregu-
larly shaped (arrow head) compared
to the unresected bone.
Bar (to scale) is the size of rib re-
moved
35
Figure 11 Skeletal Preparation of surgery (38 and 57 day healing)
A: 38 days after the injury the segmental defect was completely repaired.
Though new bone (black arrow) has completely filled in the gap the healed
portion is not similar to the unresected portion (red arrow). Repair region is
irregularly shaped and slightly bent in morphology. B: 57 days of healing
where the segmental defect is completely repaired however the new bone
is swollen and bent (black arrow) in comparison to the unresected normal
bone ( red arrow).
Bar (to scale) represents to size of the bone removed.
A B
Figure 9. Skeletal Preparation of sur-
gery (16 day healing). Growth was seen
at the two ends. This sample showed an
apparent piece in the middle which likely
fused to the cut end. One of the ends took
up a bulbous morphology.
Bar (to scale) is the size of rib removed
Figure 10.Skeletal Preparation
of surgery (19 day healing ).
Growth was seen at the two ends.
One of the ends acquired a ta-
pered morphology at the growing
tip.
Bar (to scale) is the size of the rib
removed.
36
Figure 12 Skeletal Preparation of surgery (78 day healing). The seg-
mental defect was completely repaired. The new bone (black arrow)is
morphologically similar in comparison to the unresected bone (red arrow).
The bar (to scale) represents the size of the rib removed.
37
Figure 13 H&E Staining of surgery (8 day healing)
A. Longitudinal section of the cut ends show cells resembling chondroblast/
chondrocyte morphology (arrow heads) at their periosteal surface which are
streamlined by a thick perichondrium ( red arrow). The tip shows osteoid
deposition with an unorganized marrow activity (black arrows). Area in the
box shows X-Gal positive cells stained blue (H&E 10X)
B: Granulation tissue is detected at the gap region (a small area is shown in
the square) . Arrows point to one of the dissected ends.
C: Longitudnal section of the unresected normal bone showing a compari-
tively thinner periosteum.
Skeletal prep placed right above the pics shows the area (square) which is
stained.
(H&E 10X) .
D. Higher magnification emphasising the thickness of the periosteum in the
healing bone. (20x)
A
C
D
B
38
Figure 14 H& E staining of sur-
gery (10 day healing). Longitu-
dinal section of the repair region
show isogenous groups containing
hypertrophic chondrocytes (arrow
head) undergoing mineralization
(M) . Adjacent to the differentiated
chondrocytes are the mineralization
spots (OS). Thick periosteum is
seen surrounding the repair region
(arrow).
Skeletal prep placed right above the
pics shows the area (square) which
is stained.
H&E 10x
Figure 15 H& E staining of sur-
gery (16 day healing). Longitudinal
sections of the repair region show-
ing Trabecular bone (TB). TB is
being formed on both sides of the
cartilage. The cartilage has a thick
perichondrium (black arrow) and
consists of Hypertrophic chondro-
cytes at the center (blue circle) and
the less differentiated chondrocytes
spreading towards to periphery
(black circle). The trabecular bone
has bone lining cells (red arrow
head) and osteocytes embedded in
them (blue arrow head).
Area in the box shows X-Gal posi-
tive cells stained blue
Skeletal prep placed right above the
pics shows the area (square) which
is stained.
H&E 10X
39
Figure 16. H& E staining of surgery (57 day healing): Longitudnal sec-
tions of the newly healed bone (A) vs the unresected bone (B) shows the
difference in the thickness of the periosteum. A has a thicker periosteum
(2x) compared to B. In addition the marrow cavity is smaller in the newly
formed bone.
Skeletal prep placed right above the pics shows the area (square) which is
stained.
(H&E 10X)
C and D are higher magnifications (20X).
A B
C D
40
Figure 18 Trichrome staining of
surgery (10 day healing). Collagen
fibrils are seen at the the periosteum
(arrow heads) and seemed to be spred-
ing out from the cut site. The mineral-
ization spot (OS) is stained red but has
a boundary lined by collagen.
Skeletal prep placed right above the
pics shows the area (square) which is
stained
Trichrome 10X
Figure 17. Trichrome staining
of surgery (8 day healing). Col-
lagen fibrils are detected at the cut
sites (Arrow heads).
Skeletal prep placed right above
the pics shows the area (square)
which is stained
Trichrome 10X
Figure 19 Trichrome staining
of surgery (16 day healing). The
trabecular bone (TB) is stained with
blue inferring collagen activity and
the marrow cells are stained red
(green arrow head). The cartilage
area (C) also shows collagen activity
along with its surrounding perichon-
drium (arrow head).
Skeletal prep placed right above the
pics shows the area (square) which is
stained
Trichrome 10X
41
Figure 21 X-Gal staining on surgeries.
X-Gal activity is seen in both unresected
(red arrow head) and in the bone under-
going repair (black arrow head). How-
ever a intense activity is seen at th repair
regions during early repar period. As the
bone is under going repair the intensity
of X-Gal staining is reduced and the
staining is more spread out. (A to D ar-
ranged in ascending healing period).
A B C
D
Figure 20 Analysis of the rib resected from surgery.
A: Rib piece resected from the surgery showing slight periosteum being
retained.
B: Cross section of the rib piece showing residual periosteum .
H&E 10x
A
B
42
Chondrogenic precursors
Osteogenic precursors
A
B
C
D
E
F
G
Figure 22: Schematic representation of the hypothetical pattern of re-
pair.
A. Normal rib bone.
B. Rib bone with a segmental defect leaving the periosteum intact.
C. Once the defect is made the periosteum thickens and pumps in chondro-
genic precursors into the gap site.
D. The cartilage teplate is laid down and simultaneously repair also occurs
from the cut ends. Osteogenic precursors are the pumped in which start to
replace the cartilage template with the help of chondro(osteo)clasts (not
shown in figure).
E. Sites of mineralization are seen in the middle which eventually replaces
the cartilage template (F) and fuses with the cut ends to form the newly
healed bone (G). The new bone is slightly swollen in morphology in com-
parison to the unresected normal.
periosteum
Cartilage template
43
Day 10 Day 21
Figure 24: Osteoclast activity in calvarial defects.
5mm cranial injury was performed on rats were the bone was removed along with the
periosteum. The cranium was then subjected to X-Gal followed by skeletal prep.
Day 10 shows X-Gal activity at the cut sites but as the healing progressed to 21 days in-
tensity of X-Gal staining was relatively reduced. However, compared to the rib resection
model the osteoclast activity is very modest.
Hole drilled on the cra-
nium where the bone
was removed along
with the eperiosteum
Figure 23: Drawing depicting the regions of a typical fracture of a rib
in the rabbit. Adapted from Carl et.1991
44
Summary of all samples
Sample 159
Surgery: 8/13/2012
Sacrifice: 11/8/2012
Healing: 91 Days
Sample 139
Surgery: 7/2/2012
Sacrifice: 9/7/2012
Healing: 68 Days
Sample 184
Surgery: 9/29/2012
Sacrifice: 11/11/2012
Healing: 44 Days
Sample 188
Surgery: 10/18/2012
Sacrifice: 11/29/2012
Healing: 43 Days
Control Surgeries
45
Sample 194
Surgery: 11/5/2012
Sacrifice: 12/13/2012
Healing: 39 Days
Sample 192
Surgery: 10/26/2012
Sacrifice: 11/21/2012
Healing: 27 Days
Sample 195
Surgery: 11/6/2012
Sacrifice: 11/20/2012
Healing: 15 Days
Sample 200
Surgery: 11/21/2012
Sacrifice: 12/5/2012
Healing: 15 Days
46
Sample 204
Surgery: 11/30/2012
Sacrifice: 12/14/2012
Healing: 15 Days
Sample 198
Surgery: 11/12/2012
Sacrifice: 11/19/2012
Healing: 8 Days
Sample 202
Surgery: 11/28/2012
Sacrifice: 12/5/2012
Healing: 8 Days
47
Periosteum Retained Surgeries
Sample 132
Surgery: 6/7/2012
Sacrifice: 11/8/2012
Healing: 155 Days
Sample 128
Surgery: 5/25/2012
Sacrifice: 10/8/2012
Healing: 137 Days
Sample 141
Surgery: 7/9/2012
Sacrifice: 11/8/2012
Healing: 123 Days
Sample 144
Surgery: 7/14/2012
Sacrifice: 11/8/2012
Healing: 118 Days
48
Sample: 146
Surgery: 7/17/2012
Sacrifice: 11/11/2012
Healing: 118 Days
Sample 134
Surgery: 6/14/2012
Sacrifice: 10/8/2012
Healing: 117 Days
Sample 148
Surgery: 7/19/2012
Sacrifice: 11/8/2012
Healing: 113 Days
Sample 156
Surgery: 8/8/2012
Sacrifice: 11/8/2012
Healing: 96 Days
49
Sample 140
Surgery: 7/9/2012
Sacrifice: 10/8/2012
Healing: 92 Days
Sample 143
Surgery: 7/12/2012
Sacrifice: 10/8/2012
Healing: 89 Days
Sample 135
Surgery: 6/20/2012
Sacrifice: 9/7/2012
Healing: 80 Days
Sample 171
Surgery: 8/22/2012
Sacrifice: 11/8/2012
Healing: 79 Days
50
Sample 151
Surgery: 7/23/2012
Sacrifice: 10/8/2012
Healing: 78 Days
Sample 173
Surgery: 8/29/2012
Sacrifice: 10/8/2012
Healing: 75 Days
Sample 157
Surgery: 8/10/2012
Sacrifice: 10/8/2012
Healing: 60 Days
51
Sample 185
Surgery: 10/3/2012
Sacrifice: 11/28/2012
Healing: 57 Days
Sample 186
Surgery: 10/3/2012
Sacrifice: 11/28/2012
Healing: 57 Days
Sample 187
Surgery: 10/4/2012
Sacrifice: 11/29/2012
Healing: 57 Days
Sample 164
Surgery: 8/15/2012
Sacrifice: 10/8/2012
Healing: 55 Days
52
Sample 181
Surgery: 9/20/2012
Sacrifice: 11/11/2012
Healing: 53 Days
Sample 180
Surgery: 9/18/2012
Sacrifice: 11/8/2012
Healing: 52D Days
Sample 182
Surgery: 9/25/2012
Sacrifice: 11/8/2012
Healing: 45 Days
Sample 172
Surgery: 8/28/2012
Sacrifice: 10/8/2012
Healing: 42 Days
53
Sample 126
Surgery: 5/21/2012
Sacrifice: 6/27/2012
Healing : 38 Days
Sample 138
Surgery: 6/30/2012
Sacrifice: 7/27/2012
Healing: 28 Days
Sample 190
Surgery: 10/25/2012
Sacrifice: 11/21/2012
Healing: 28 Days
Sample 197
Surgery: 11/9/2012
Sacrifice: 11/30/2012
Healing: 22 Days
54
Sample 175
Surgery: 9/5/2012
Sacrifice: 9/24/2012
Healing: 20 Days
Sample 179
Surgery: 9/14/2012
Sacrifice: 10/2/2012
Healing: 19 Days
Sample 142
Surgery: 7/10/2012
Sacrifice: 7/25/2012
Healing: 16 Days
Sample 196
Surgery: 11/7/2012
Sacrifice: 11/21/2012
Healing: 15 Days
55
Sample 203
Surgery: 11/29/2012
Sacrifice: 12/13/2012
Healing: 15 Days
Sample 183
Surgery: 9/29/2012
Sacrifice: 10/8/2012
Healing: 10 Days
Sample 199
Surgery: 11/20/2012
Sacrifice: 11/27/2012
Healing: 8 Days
Sample 201
Surgery: 11/27/2012
Sacrifice12/4/2012
Healing: 8 Days

56
Summary of all surgeries
ID Size of rib
removed
(mm)
Gap
Size
(mm)
Healing
Time
(Days)
Percent
healed
Analysis Resecte
d Piece
Healed
Paraffin/
Plastic
embedde
d
S
e
c
t
i
o
n
e
d
Par
affi
n
em
bed
ded
S
e
c
t
i
o
n
e
d
132 3.1 0 155 100 Skeletal
Prep
Plastic (Y) N N N
128 3 0.7 137 76.6666 X-Gal
and
Skeletal
Prep
Plastic (Y) N N N
141 2.5 0.18
97
123 92.412 Skeletal
Prep
Plastic (Y) N N N
144 3.1 0 118 100 Skeletal
Prep
Plastic (Y) N N N
146 3.5 0.77
56
118 77.84 X-Gal
and
Skeletal
Prep
Plastic (Y) N N N
134 3 0 117 100 X-Gal
and
Skeletal
Prep
Plastic (Y) N N N
148 3 1.10
2
113 63.2666 Skeletal
Prep
Plastic (Y) N N N
156 3 0.82
9
96 72.3666 X-Gal
and
Skeletal
Prep
Paraffin
(Y)
N N N
140 3 0.41
7
92 86.1 X-Gal
and
Skeletal
Prep
Plastic (Y) N N N
143 3 0 89 100 X-Gal
and
Skeletal
Plastic (Y) N N N

57
135 3.9 0.84
1
80 78.4358 X-Gal Plastic (Y) N N N
171 5 0 79 100 Skeletal
Prep
Paraffin
(Y)
N N N
151 2.5 0 78 100 X-Gal
and
Skeletal
Prep
lost piece
while
embeddin
g
N
A
N N
173 3.1 0.67
8
75 78.1290 X-Gal
and
Skeletal
Prep
Paraffin
(Y)
N N N

157

2.687

0.09
3

60

96.538
8

X-Gal
and
Skeletal
Prep

Paraffin
(Y)

N

N

N
185 2.84 0 57 100 X-Gal
and
Skeletal
Prep
Paraffin
(Y)
N Y Y
186 3 3.54 57 -18 X-Gal
and
Skeletal
Prep
N N N N
187 3 0 57 100 X-Gal
and
Skeletal
Prep
N N Y N
164 4.5 1.66
8
55 62.9333 X-Gal
and
Skeletal
Prep
Paraffin
(Y)
N
A
N N
180 3 0 52 100 Skeletal
Prep
Paraffin
(Y)
N N N
182 2 0.35
3
45 82.35 Skeletal
Prep
Paraffin
(Y)
N Y N
126 2.9 0 38 100 X-Gal
and
Skeletal
Prep
Plastic (Y) N Y N
138 3 1.34 28 55.3333 X-Gal
and
Skeletal
Prep
Plastic (Y) N N N
190 4 0.16 28 96 X-Gal Paraffin N N N
197 2 0.69 22 65.3 X-Gal N N N N

58

CONTROL SURGERIES
175 3.21 1.68
5
20 47.5077 X-Gal Paraffin N N N
179 3.589 0.36
99
19 89.6935 X-Gal
and
Skeletal
Prep
Paraffin
(Y)
Y N N
142 2.5 0.29
5
16 88.2 X-Gal
and
Skeletal
Prep
Plastic (Y) N Y Y
196 2.2 1.24
6
15 43.3636 X-Gal
and
Skeletal
Prep
N N Y N

203

2

0.45

15

77.5

X-Gal
and
Skeletal
Prep

N

N

N

N
183 2.739 0.64
9
10 76.3052 X-Gal
and
Skeletal
Prep
Paraffin
(Y)
N Y Y
199 2.2 2.03
1
8 7.68181 X-Gal
and
Skeletal
Prep
N N Y Y
201 3 1.49
1
8 50.3 X-Gal
and
Skeletal
Prep
Paraffin
(Y)
N N N
ID Size of
rib
remove
d(mm)
Gap
Size
(mm)
Healing
Time
(Days)
Percent
healed
Analysis Resec
ted
Rib

Heale
d
Porti
on

Par
affi
n/
Pla
stic
em
bed
S
ec
ti
o-
n
e
d
Paraf
fin
embe
dded
S
ec
ti
o-
n
e
d

59

159 2.97 2.149 91 27.6430 X-Gal
and
Skeletal
Par
affi
n
(Y)
N Y Y

139

NA

2.103

68

NA

X-Gal
and
Skeletal
Prep

Pla
stic
(Y)

N

N

N
184 3.358 3.025 44 9.91661 X-Gal
and
Skeletal
Prep
Par
affi
n
(Y)
N N N
188 3.721 3.529 43 5.15990 X-Gal
and
Skeletal
Prep
Par
affi
n
(Y)
N N N
194 4.267 3.466 39 18.7719 X-Gal
and
Skeletal
Prep
Par
affi
n
(Y)
Y N N
192 4.049 2.762 27 31.7856 X-Gal
and
Skeletal
Prep
Par
affi
n
(Y)
N N N
195 3.287 2.252 15 31.4876 X-Gal
and
Skeletal
Prep
Par
affi
n
(Y)
N N N
200 3.31 2.676 15 19.1540 X-Gal N N N N
204 2.215 1.57 15 29.1196 Skeletal N N N N
198 2.605 1.531 8 41.2284 Skeletal N N N N
202 2.662 1.521 8 42.862 X-Gal Par
affi
n
N N N

60

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Abstract (if available)

Abstract Bone is one of the very few tissues in our body that has the capacity for repair without leaving a scar. However, there are instances when bone repair fails. In injuries or surgical situations where a large portion of bone is missing, healing can be completely compromised. Anecdotal reports in humans have suggested that the rib may be one of the only long bones in the body that has the capacity to repair large defects. The aim of this project is to study the healing of a segmental defect in murine ribs to determine if healing occurs with the help of innate factors. Surgeries were performed on adult CD-1 mice where a piece of the vertebral rib was removed leaving the periosteum intact. Control surgeries were also performed where both the rib and the surrounding periosteum were resected. Animals were sacrificed at various time points and the rib cages were dissected to analyze the healed region. We found that when the periosteum was left behind, the resected region would be completely filled in within 1-2 months. The healed bone had restored the original morphology with only some minor irregularities. In the control surgeries there was no sign of regeneration except modestly at the cut ends. Based on our studies, we found that the mouse model can be effectively used to study large defects. Furthermore, we believe that the periosteum plays a key role in the healing process and may serve as a repository for stem cells that can mediate the repair. Eventually our hope is that this research will give a better insight in developing ways to stimulate large defect repair in other locations of the body.

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Asset Metadata

Creator Tripuraneni, Nikita (author)

Core Title Rib resection and healing in the mouse: a new model of bone repair

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 07/29/2013

Defense Date 07/29/2013

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

Tag endochondral ossification,OAI-PMH Harvest,rib

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Mariani, Francesca (committee chair), DePaolo, R. William (committee member), Machida, Keigo (committee member)

Creator Email nikitatc@gmail.com,tripuran@usc.edu

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