Possible Role of Osteoblasts in Regulating the Initiation ... · PDF fileEndochondral Repair Process during Fracture ... Chapter 1 General Introduction ... in Regulating the Initiation
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Possible Role of Osteoblasts in Regulating the Initiation of Endochondral Repair Process during Fracture Healing
by
Yasha Amani Andabili
A thesis submitted in conformity with the requirements for the degree of Master of Science
Department of Laboratory Medicine and Pathobiology University of Toronto
Chapter 2 Possible Role of Osteoblasts in Regulating the Initiation of Endochondral Repair Process during Fracture Healing ............................................................................. 60
2.3.1 Pretreatment of DTK transgenic mice with GCV leads to ablation of osteoblasts ............................................................................................................ 65
2.3.2 Osteoblast depletion delays initiation of endochondral bone repair .............. 66
2.3.3 Continuous ablation of osteoblasts hinders the progression of endochondral ossification past the soft callus stage ......................................... 67
Figure 1: Genetic Control of Skeletal Cell Type Differentiation.
Figure 2: Bone Formation Occurs Through Two Different Modes of Ossification Processes.
Figure 3: Model of Fracture Healing Stages and Cellular Participants Involved During the Process of Repair.
Figure 4: Continuous GCV Treatment Leads to Non-union of the Fractured Bone.
Figure 5: In Vivo Experimental Design for Studying the Role of Osteoblasts During Fracture Repair Process.
Figure 6: GCV Pretreatment is Effective in Ablating Pre-existing Osteoblasts.
Figure 7: GCV Treatment of the DTK Transgenic Mice Leads to a Decrease in Bone-Lining Osteoblast Population.
Figure 8: Continuous GCV Treatment Leads to a Delay in Initiation and Progression of Endochondral Ossification Process.
Figure 9: Matrix Metalloproteinase 13 Gene Expression is Reduced in Absence of Osteoblasts.
Figure 10: Osteocytes are Unaffected by GCV Treatment.
Figure 11: Upon GCV Withdrawal Bones are Able to Replenish the Osteoblast Population as Early as 7 Days Post Fracture.
Figure 12: Osteoblast Ablation Leads to a Decrease in TRAP Positive Osteoclasts During the Remodeling Stage of Fracture Healing, and the Decrease in Osteoclast Population Hinders the Healing Process.
Figure 13: In Absence of Osteoblasts There is a Lack of Collagen Type X Matrix Resorption.
Figure 14: Continuous Depletion of Osteoblasts Results in Unresorbed Cortical Bone and Cartilage Matrix at the Fracture Site.
Figure 15: Fluorescent and Immunohistochemistry staining for GFP-tagged SKP-injected into fracture site of continuous GCV treated DTK mice.
vii
List of Abbreviations
ALP – Alkaline phosphatase
bHLH – A basic helix-loop-helix domain present in two Twist genes (Twist-1 and -2)
BMP – Bone morphogenetic protein
BSP – Bone sialoprotein
BV – Bone volume
CAT – Chloramphenicol acetyltransferase
Cbfa1 – Core binding factor 1
Col-I – Collagen type I
Col-II – Collagen type II
Col-X – Collagen type X
CV – Cartilage volume
CXCR4 – A CXC chemokine receptor (type 4) in the G-protein coupled receptor family specific
for stromal cell-derived factor-1.
DAPI – A polpular flourecent stain (4’, 6-diamidino-2-phenylindole) used for nuclear
counterstain in immunohistochemistry.
DTK – Refers to the transgenic mouse model expressing the HSV-tk gene under the control of a
2.3 kilobase fragment of the rat α1 type I collagen promoter (Col2.3∆tk)
ECM – Extra cellular matrix
EDTA – Ethylenediaminetetraacetic acid is a chelating agent used for bone decalcification.
FGF – Fibroblast growth factor
GCV – Ganciclovir
GFP – Green fluorescent protein
HE – Haematoxylin eosin
HIF – Hypoxia inducible factor
HSV-TK – Herpes simplex virus thymidine kinase
HTC – Hypertrophic chondrocyte
IGF – Insulin-like growth factor
Ihh – Indian hedgehog
IL – Interleukin
M-CSF – Macrophage colony stimulating factor
viii
MMP – Matrix metalloproteinase
MSC – Mesenchymal stromal cells
NF-κB – Nuclear factor-κB
NOD-SCID – Refers to the non-obese diabetic severe combined immunodeficiency mouse
model
OC – Osteocalcin
OG2 – Osteocalcin gene 2
OCIL – Osteoclast inhibitory lectin
OPG – Osteoprotegerin
OPN – Osteopontin
Osx – Osterix
PDGF – Platelet-derived growth factor
PGE2 – Prostaglandin E2
PTH – Parathyroid hormone
PTHrP – Parathyroid hormone related peptide
RANK – Receptor activator of nuclear factor-κB
RANKL – Receptor activator of nuclear factor-κB ligand
RFP – Red fluorescent protein
Runx2 – Runt-related transcription factor 2
SDF-1 – Stromal cell-derived factor-1
SKP – Skin derived precursors
SO – Safranin O
TGF – Transforming growth factor
TNF – Tumor necrosis factor
TRAF – TNF receptor-associated factor
TRAP – Tartrate-resistant acid phosphatase
TV – Tissue volume
VEGF – Vascular endothelial growth factor
1
Chapter 1 General Introduction
1.1 Bone Formation
Bone is composed of two distinct tissues, cartilage and bone, each of which includes
specific cell types that fulfill unique functions that are critical for the growth, maintenance and
integrity of the skeleton (Ducy, 1998; Ducy, 2000). Chondrocytes, found in cartilage, are
essential for longitudinal growth of bone from the two metaphyseal ends, and synthesize the
cartilaginous templates during development and postnatal skeletogenesis, onto which osteoblasts
can deposit the bone matrix (Ducy, 2000). Osteoblasts and osteoclasts, found in bone, closely
collaborate in basic multicellular units (BMU) to coordinate the anabolic build-up (synthesis)
and catabolic break down (resorption) of the bone tissue, which occurs regularly throughout the
skeleton at micro scales during adult life (Hadjidakis, 2006; Hill, 1998; Neumann, 2007; Proff,
2009).
Certain genes are critical for regulating the progression of skeletal formation through the
stage of chondrogenesis, angiogenesis, and osteogenesis to the process of remodelling. The
initial phase of mesenchymal condensation involves the expression of transcription factors core
binding factor 1/runt-related transcription factor 2 (Cbfa1/Runx2) and Sox9 genes, which play a
role in determining the population of cells that will occupy and contribute to the condensation
(Hall, 1992; Hall, 1995; Zhou, 2006). Expression of certain growth factors, such as transforming
growth factor beta (TGFβ) superfamily, regulate the proliferation of mesenchymal progenitors
and their differentiation into chondrocytes (Mundlos, 1997a; Mundlos, 1997b). The cartilage
anlage is developed by the secretion of various structural proteins such as syndecan-3, versican
and tenascin by cells within the condensate (Koyama, 1995; Koyama, 1996).
The master regulator, Runx2/Cbfa1, also plays important role in chondrocyte maturation,
angiogenesis and the remodelling of extracellular matrix, as well as being required for
intramembranous ossification (Ferguson, 1999; Yoshida, 2005). Furthermore, it is essential for
fetal osteogenesis (Komori, 1997; Otto, 1997), as deletion of the cbfa1 gene in homozygous null
mutants (cbfa1-/-) results in an almost complete lack of endochondral and intramembranous
2
ossification, while development of the cartilage scaffold proceeds normally (Komori, 1997;
Mundlos, 1997a; Mundlos, 1997b; Otto, 1997).
This transcription factor regulates the maturation of chondrocytes by controlling the
expression of osteocalcin and osteopontin (OPN), which despite being known as osteoblast-
specific markers, do get expressed in hypertrophic chondrocytes (Gerstenfeld, 1996; Lian, 1993;
Nomura, 1989). By controlling the expression of OPN, which is responsible for the attachment
of cells to the extracellular matrix (Miyauchi, 1991; Reinholt, 1990; Somerman, 1987),
Runx2/Cbfa1 indirectly affects terminal differentiation and apoptosis of chondrocytes. It is also
responsible for directly regulating the expression of matrix metalloproteinase 13 (MMP13), a
collagenase that is involved in remodelling of extracellular matrix that is laid down by
hypertrophic chondrocytes (Vu, 1998).
Another population of cells, the osteoblasts, synthesize a matrix rich in type I collagen
which eventually becomes mineralized, and osteoclasts function to resorb this mineralized
matrix, allowing the remodelling of the rigid bone tissue (Ducy, 2000). Aside from the
transcription factor Runx2/Cbfa1 (Maruyama, 2007), another important signalling molecule
during skeletogenesis is the Wnt-family of molecules, which functions during both embryology
and adult bone repair by regulating the differentiation of mesenchymal precursors into
osteoblasts and the subsequent osteoblastic bone formation (Chen, 2009; Topol, 2009).
Given the biologic importance of these three cell types, and the nonredundancy of their
functions, a defect in differentiation and function of any of these cells leads to severe
repercussions during development that manifests as various diseases of bone and cartilage
(Ducy, 2000). Therefore, knowledge of the genetic network controlling their differentiation and
function (Figure 1) becomes important for developing new and effective therapeutic strategies.
1.1.1 Endochondral Ossification
Endochondral ossification, an anabolic response driven by osteoblasts, includes the
formation of a cartilaginous template that is eventually replaced with bone matrix (Schindeler,
2009). It is initiated by the condensation of mesenchymal cells and their differentiation into
chondrocytes, which secrete a type-II collagen and aggrecan-rich cartilage matrix. Cells
surrounding the condensate form the perichondrium, while cells within the cartilage template
3
further differentiate into hypertrophic chondrocytes, which requires a cessation of proliferation
and exit from the cell cycle (Hartmann, 2009). These cells produce type-X collagen, as well as
the angiogenic vascular endothelial growth factor (VEGF) and various osteoblast-specific
proteins including alkaline phosphatase (ALP) and OPN, which results in recruitment of new
blood vessels to the avascular cartilage and the mineralization of the cartilage matrix (Iyama,
1991; Kronenberg, 2003). This matrix is eventually resorbed, and the degradation of this
cartilage matrix during endochondral ossification is dependent on the activity of MMPs,
particularly MMP9 (gelatinase B) and MMP13, that regulate remodeling and neovascularization
of the cartilage anlage (Johansson, 1997; Vu, 1998). The invasion of blood vessels into the
cartilage matrix leads to recruitment of osteoblast and osteoclast precursors.
Development of adequate vasculature is critical during the endochondral ossification, as
it determines the rate of bone formation by coupling the processes of chondrogenesis and
osteogenesis (Gerber, 2000). It has been determined that the coordination of metaphyseal and
epiphyseal vascularization, ossification and cartilage formation during endochondral process are
dependent on functions of VEGF isoforms (Maes, 2002). These factors are secreted by the
developing hypertrophic chondrocytes, and lead to recruitment of osteoblasts, osteoclasts and
haematopoietic cells.
Osteoblasts residing in the perichondrium will create a collar of compact bone around the
middle region of the cartilage template, referred to as diaphysis, the site of primary ossification
center. At the two ends of the developing bone, known as epiphyses, secondary ossification
centers are formed, and the cartilaginous plate separating the diaphysis and epiphysis will
become the growth plate, which regulates the longitudinal growth of the long bones (Collin-
1985), while fibroblasts are unresponsive to these hormones. Furthermore, fibroblastic cells
produce a variety of collagen types (I, III, IV, V and VI), whereas fully differentiated osteoblasts
almost exclusively synthesize type I collagen (Fessler, 1981; Liau, 1985; Olsen, 1989;
Pihlajaniemi, 1989). Finally, a retroviral insertion in the first intron of the COL1A1 gene of
MOV13 mouse, does not affect collagen transcription in osteoblasts while resulting in a
complete block of collagen transcription in fibroblasts (Kratochwil, 1989). This ability to
override the inhibitory effect of the retroviral insertion suggest the presence of a cis-active DNA
sequence within the COL1A1 gene in osteoblast and odontoblasts that differ from those used in
fibroblastic cells.
The modular organization of the Col1a1 promoter allows selection of specific domains
for use in different type I collagen-producing tissues (Bedalov, 1995; Bogdanovic, 1994;
28
Rossert, 1995). For expression of the transgene in the osteoblast layer lining newly formed
calvarial bone, a homeodomain binding TAAT sequence localized between the -1670 and -1683
base pairs have been shown to be important (Dodig, 1996). The 3.6-kilobase (kb) rat Col1a1
promoter have previously been used in conjunction with the chloramphenicol acetyl transferase
(CAT) gene, and shown high level of transgene expression in bone, tendon, and developing tooth
germ with lower levels in skin (Pavlin, 1992). Truncation of the 3.6-kb promoter to 2.3-kb
fragment caused no change in the level of CAT activity in calvaria while a 2- to 4-fold decrease
in the transgene expression was observed in tendon and a greater loss in the skin (Bogdanovic,
1994; Krebsbach, 1993). In other studies, similar cell-specific pattern of expression was detected
in transgenic mice harboring a 2.3-kb human COL1A1 promoter driving the expression of
bovine growth hormone (Liska, 1994), as well as in mice carrying a murine 2.3-kb Col1a1
promoter fragment fused to either the β-galactosidase or luciferase gene (Rossert, 1995).
One method for studying the lineage relationships and cell functions of a population of
differentiating cells is the use of tissue-specific transgenic expression of herpes simplex virus
thymidine kinase (HSV-tk) to conditionally deplete a specific cell population (Heyman, 1989).
Targeted cell ablation using this strategy requires high level synthesis of enzyme in a subset of
rapidly proliferating cells. The expression of the transgene can be directed to restricted cell types
via the use of tissue-specific promoters and enhancers that allow the physiological manipulation
of the cell and organs of interest (Hammer, 1985; Palmiter, 1982). The HSV-tk, although not
deleterious in mammalian cells by itself, becomes toxic in the presence of nucleoside analogs
like ganciclovir (GCV), leading to inhibition of DNA synthesis and cell ablation (Heyman,
1989). This is a useful approach in evaluating the contribution of a cell type to a particular
developmental program, while allowing one to control the precise timing of the toxic insult, the
degree of ablation, and the potential for recovery upon termination of drug-induced toxicity
(Heyman, 1989). Upon administration and delivery of specific nucleoside analogs to target cells,
the HSV-tk is capable of monophosphorylating the benign substrate. The nucleoside
monophosphate is further phosphorylated by cellular kinases to nucleoside triphosphate,
producing a toxic product that becomes incorporated into DNA during differentiation and leads
to inhibition of DNA synthesis and cell ablation (Elion, 1977; Furman, 1980; Fyfe, 1978). This
ablation technique has been utilized previously using an immunoglobulin promoter/enhancer to
target the expression of HSV-tk to the lymphoid system (Heyman, 1989). In transgenic animals
29
the activity of the enzyme was restricted to the spleen, lymph nodes, bone marrow and thymus,
where the expression of HSV-tk resulted in massive depletion of B and T lymphocytes after drug
treatment (Heyman, 1989).
Following the initial use of this method for the haematopoietic and immune cell
pathways, strategies have been developed to study the relationship of different cell types in bone
tissue. In regards with osteoblasts, the use of a highly tissue specific promoter, such as
osteocalcin, leads to the expression of the tk gene late in the osteoblast lineage when cell division
has ceased and the rate of cell proliferation is reduced (Corral, 1998). Previous use of the mouse
osteocalcin gene 2 (OG2) promoter, a marker of mature osteoblasts, to drive expression of the
HSV-tk gene, showed a nearly complete absence of cells with morphologic features of
osteoblasts (Corral, 1998). Due to late tk expression in the OG2 mice, osteoclast activity was still
persistent and bone resorption remained unaffected despite a reduction in the osteoblast
population. This would suggest that the surviving osteoblast progenitors that are unaffected by
the GCV treatment in OG2-tk mice could express signals that would regulate osteoclast function
(Corral, 1998).
In contrast to the osteocalcin promoter, the modular design of the Col1a1 promoter
allows the creation of promoter-reporter constructs that can be used in various type I collagen-
producing tissues (Dodig, 1996). In an in vitro study by Dacic and colleagues (Dacic, 2001)
different Col1a1 promoter fragments were used to drive the chloramphenicol acetyltransferase
(CAT) gene. In this model, the activity of a 3.6 Col1a1 fragment was detected in osteoblast
progenitors concurrent with type I collagen mRNA and ALP expression. In contrast, the 2.3
Col1a1 fragment became active coincident with expression of the early bone-specific marker
bone sialoprotein (BSP) but distinctly prior to osteocalcin mRNA, which is produced at a later
stage in the osteoblast lineage (Dacic, 2001). In another study, Visnjic and colleagues generated
transgenic mice that express the HSV-tk gene under 2.3-kb fragments of the collagen type I
promoter, which was found to be active in concurrent with other genetic markers of early
differentiating osteoblast (Visnjic, 2001). Treatment of these mice with GCV resulted in an
extensive destruction of the bone lining cells, decreased osteoclast numbers and a decrease in
bone marrow elements. The reduction in the bone marrow cellularity was more prominent in the
metaphyseal regions and areas adjacent to the endosteal surface of the diaphyseal bone, which
normally house cells of osteoblastic lineage. Termination of the GCV treatment resulted in a
30
pronounced response of new cortical and trabecular bone formation as well as the replenishment
of the bone marrow cells. Their findings suggest that early differentiating osteoblasts are
necessary for the maintenance of haematopoiesis and osteoclastogenesis (Visnjic, 2001).
Therefore, in contrast to the other osteoblast specific promoters mentioned, the 2.3-kb fragment
of type I collagen promoter provides a useful osteoblast-specific model to define and analyze the
interrelation of osteoblasts with the other bone cell types during the different modes of bone
formation and fracture healing.
31
Figure 1: Genetic Control of Skeletal Cell Type Differentiation.
(A) Differentiation of mesenchymal progenitor cells down an osteogenic or chondrogenic lineage and expression of the transcription factors and cytokines regulating each pathway is shown. Transcription factor Runx2/Cbfa1 is the earliest marker of osteoblast differentiation and is also involved in promoting hypertrophic differentiation of chondrocytes. Sox9 transcription factor, along with Sox5 and Sox6 regulate the expression of various genes involved in chondrogenesis and induction of cartilage matrix proteins such as type II collagen. (Refer to ‘Abbreviation List’, for acronyms).
(B) Molecular regulation of osteoclast differentiation from haematopoietic stem cells and expression of enzymes, secreted molecules, and transcription factors acting at different stages of differentiation/activation are depicted. The activation of osteoclast involves the formation of ruffled membrane and secretion of catabolic enzymes such as cathepsin K, TRAP and H+ into resorption pits for degradation of bone matrix. (Refer to ‘Abbreviation List’ for acronyms).
32
Figure 2: Bone Formation Occurs Through Two Different Modes of Ossification Processes.
(A) The endochondral bone formation begins with the condensation of mesenchymal progenitor cells at sites of future skeletal development, a process that is recapitulated during the early stages of fracture repair. The cells at the central region of the aggregate then differentiate along a chondrogenic lineage and produce a cartilaginous template. In the region adjacent to the hypertrophic chondrocyte (HTC), the first osteoblast precursors differentiate and secrete a non-mineralized extracellular matrix that becomes organized into compact mineralized bone. During this process osteoblasts that become entrapped in the bone differentiate into osteocytes. Osteoclasts are required for remodelling and resorption of the bone matrix.
(B) Intramembranous ossification involves the direct differentiation of osteoblasts from the condensed mesenchyme at the site of bone formation such as the flat bones of the skull. In this process bone is made without the need for an intermediate cartilaginous template.
(Fig 2A adapted from Hartmann, 2006 and Fig 2B adapted from Hartmann, 2009)
33
Figure 3: Model of Fracture Healing Stages and Cellular Participants Involved During the Process of Repair.
(A) Representative images of a fracture healing model. The process of fracture healing is partitioned into four overlapping stages. Upon damage to the bone there is an immediate inflammation response followed by the formation of a soft callus, which undergoes remodeling prior to the hard callus formation stage. The final stage of repair involves the remodeling of the hard callus back into the original cortical bone configuration.
(B) Diagram showing the corresponding cellular contributors to the healing process at various stages.
(Adapted from Schindeler, 2008. 19; 460).
34
References
Adams CS, Shapiro IM. 2002. The fate of the terminally differentiated chondrocyte: evidence for microenvironmental regulation of chondrocyte apoptosis. Crit Rev Oral Biol Med 13(6): 465-473.
Adams GB, Scadden DT. 2006. The hematopoietic stem cell in its place. Nat Immunol 7: 333-337.
Akiyama H, Chaboissier MC, Martin JF, Schedl A, de Crombrugghe B. 2002. The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev 16(21): 2713-2828.
Al-Aql ZS, Alagl AS, Graves DT, Gerstenfeld LC, Einhorn TA. 2008. Molecular mechanisms controlling bone formation during fracture healing and distraction. Osteogenesis. J Dent Res 87: 107-18.
Alagiakrishnan K, Juby A, Hanely D, Tymchak W, Sclater A. 2003. Role of vascular factors in osteoporosis. J Gerontol A Biol Sci Med Sci 58: 362-366.
Alcobendas M, Baud CA, Castanet J. 1991. Structural changes of the preosteocytic area in Vipera aspis (L.) (Ophidia, Viperidae) bone tissue in various physiological conditions. Calc Tissue Int 49(1): 53-57.
Allgower M, Speigel PG. 1979. Internal fixation of fractures: evolution of concepts. Clin Orthop 138: 26-29.
Althoff J, Quint P, Krefting ER, Hohling HJ. 1982. Morphological studies on the epiphyseal growth plate combined with biochemical and X-ray microprobe analyses. Histochemistry 74: 541-552.
Andersen TL, del Carent Ovejero M, Kirkegaard T, Lehhard T, Foged NT, Delaisse JM. 2004. A scrutiny of matrix metalloproteinases in osteoclasts: Evidence for heterogeneity and for the presence of MMPs synthesized by other cells. Bone 35:110-1119.
Arai F, Hirao A, Ohmura M, Sato H, Matsuoka S, Takubo K, Ito K, Koh GY, Suda T. 2004. Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 118: 149-161.
Aubin JE, Liu F. 1996. The osteoblast lineage. Principles of Bone Biology 51-67.
Aubin JE. 1998. Advances in the osteoblast lineage. Biochem Cell Biol 76(6): 899-910.
Augustin G, Antabak A, Davila S. 2007. The periosteum. Part 1: Anatomy, histology and molecular biology. Injury 38(10): 1115-1130.
Bafico A, Liu G, Yaniv A, Gazit A, Aaronson SA. 2001. Novel mechanism of Wnt signaling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow. Nature Cell Biol 3(7): 683-686.
Barnes GL, Kostenuik PJ, Gerstenfeld LC, Einhorn TA. 1999. Growth factor regulation of fracture repair. J Bone Miner Res 14: 1805-15.
Bedalov A, Salvatori R, Dodig M, Kronenberg MS, Kapural B, Bogdanovic Z, Kream BE, Woody CO, Clark SH, Mack K. 1995. Regulation of COL1A1 expression in type I
35
collagen producing tissues: Identification of a 49 base pair region which is required for transgene expression in bone of transgenic mice. J Bone Miner Res 10: 1443-1451.
Beertsen W, Brekelmans M, Everts V. 1978. The site of collagen resorption in the periodontal ligament of the rodent molar. Anat Rec 192: 305-318.
Behonick DJ, Xing Z, Lieu S, Buckley JM, Lotz JC, Marcucio RS, Werb Z, Miclau T, Colnot C. 2007. Role of matrix metalloproteinase 13 in both endochondral and intramembranous ossification during skeletal regeneration. PLoS One 2: e1150.
Belanger LF. 1977. The skeletal tissues. In: Weiss L, Greep RO, editors. Histology, 4th ed. New York: McGraw-Hill Book Company.
Bi W, Deng JM, Zhang Z, Behringer RR, de Crombrugghe B. 1999. Sox9 is required for cartilage formation. Nat Genet 22: 85-89.
Bialek P, Kern B, Yang X, Schrock M, Sosic D, Hong N, Wu H, Yu K, Ornitz DM, Olson EN, Justice MJ, Karsenty G. 2004. A twist code determines the onset of osteoblast differentiation. Dev Cell 6: 423-435.
Bianco P, Fisher LW, Young MF, Termine JD, Robey PG. 1991. Expression of bone sialoprotein (BSP) in developing human tissues. Calcif Tissue Int 49: 421-426.
Billinghurst RC, Dahlberg L, Ionescu M, Reiner A, Bourne R, Rorabeck C, Mitchell P, Hambor J, Diekmann O, Tschesche H, Chen J, van Wart H, Poole AR. 1997. Enhanced cleavage of type II collagen by collagenases in osteoarthritic articular cartilage. J Clin Invest 99: 1534-1545.
Birkedal-Hansen H, Moore WG, Bodden MK, Windsor LJ, Birkedal-Hansen B, Decarlo A, Engler JA. 1993. Matrix metalloproteinases: a review. Crit Rev Oral Biol Med 4: 197-250.
Blair HC, Teitelbaum SL, Ghiselli R, Gluck S. 1989. Osteoclastic bone-resorption by a polarized vacuolar proton pump. Science 245(4920): 855-857.
Blavier L, Delaisse JM. 1995. Matrix metalloproteinases are obligatory for the migration of preosteoclasts to the developing marrow cavity of primate long bones. J Cell Sci 108: 3649-3659.
Boast S, Su MW, Ramirez F, Sanchez M, Avvedimento EV. 1990. Functional analysis of cis-acting DNA sequences controlling transcription of the human type I collagen genes. J Biol Chem 265: 13351-13356.
Bogdanovic Z, Bedalov A, Krebsbach PH, Pavlin D, Woody CO, Clark SH, Thomas HF, Rowe DW, Kream BE, Lichtler AC. 1994. Upstream regulatory elements necessary for expression of the rat COL1A1 promoter in transgenic mice. J Bone Miner Res 9: 285-292.
Bolander ME. 1992. Regulation of fracture repair by growth factors. Proc Soc Exp Biol Med 200(2): 165-170.
Bonewald LF, Dallas SL. 1994. Role of active and latent transforming growth factor beta gene in bone formation. J Cell Biochem 55: 350-357.
36
Bostrom MPG, Lane JM, Tomin E, Browne M, Berberian W, Turek T, Smith J, Wozney J, Schildhauer T. 1996. The use of bone morphogenetic protein-2 in the rabbit ulnar nonunion model. Clin Orthop 327: 272-282.
Bradley EW, Drissi MH. 2011. Wnt5b regulates mesenchymal cell aggregation and chondrocyte differentiation through the planar cell polarity pathway. J Cell Physiol 226: 1683-1693.
Brighton CT, Lorich DG, Kupcha R, Reilly TM, Jones AR, Woodbury RA, 1992. The pericyte as a possible osteoblast progenitor cell. Clin Orthop Relat Res 275: 287-299.
Brinckerhoff, CE and Matrisian LM. 2002. Matrix metalloproteinases: a tail of a frog that became a prince. Nat Rev Mol Cell Biol 3: 207-214.
Brunet LJ, McMahon JA, McMahon AP, Harland RM. 1998. Noggin, cartilage morphogenesis, and joint formation in the mammalian skeleton. Science 280: 1455-1457.
Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, Capparelli C, Scully S, Tan HL, Xu W, Lacey DL, Boyle WJ, Simonet WS. 1998. Osteoprotegerin-deficeint mice develop early onset osteoporosis and arterial calcification. Genes Dev 12: 1260-1268.
Burger EH, Klein-Nulend J, Smit TH. 2003. Strain-derived canalicular fluid flow regulates osteoclast activity in a remodelling osteon: a proposal. J Biomech 36: 1453-1459.
Burkhardt R, Kettner G, Bohm W, Schmidmeier M, Schlag R, Frisch B, Mallmann B, Eisenmenger W, Gilg T. 1987. Changes in trabecular bone, hematopoiesis and bone marrow vessels in aplastic anemia, primary osteoporosis, and old age: a comparative histomorphometric study. Bone 8: 157-164.
Cadigan KM, Nusse R. 1997. Wnt signaling: A common theme in animla development. Genes Dev 11: 3286-3305.
Canalis E. 1980. Effect of insulin-like growth factor I on DNA and protein synthesis in cultured rat calvaria. J Clin Invest 66: 709-719.
Canalis E, McCarthy T, Centrella M. 1988. Growth factors and the regulation of bone remodeling. J Clin Invest 81: 277-281.
Cao Y, Mori S, Mashiba T, Westmore MS, Ma L, Sato M, Akiyama T, Shi L, Komatsubara S, Miyamoto K, Norimatsu H. 2002. Raloxifene, estrogen, and alendronate affect the processes of fracture repair differently in ovariectomized rats. J Bone Miner Res 17: 2237-2246.
Capdevila J, Johnson RL. 1998. Endogenous and ectopic expression of noggin suggests a conserved mechanism for regulation of BMP function during limb and somite patterning. Dev Biol 197(2): 205-217.
Carmeliet P, Collen D. 2000. Molecular basis of angiogenesis. Role of VEGF and VE-cadherin. Ann N Y Acad Sci 902: 249-262.
Carmeliet P. 2003. Angiogenesis in health and disease. Nature Med 9(6): 653-660.
Carter DR, Beaupre GS, Giori NJ, Helms JA. 1998. Mechanobiology of skeletal regeneration. Clinical Orthop Relat Res Suppl. 355: S41-S55.
37
Castanon I, Baylies MK. 2002. A Twist in fate: evolutionary comparison of Twist structure and function. Gene 287: 11-22.
Celeste AJ, Iannazzi JA, Taylor RC, Hewick RM, Rosen V, Wang EA, Wozney JM. 1990. Identification of transforming growth factor beta family members present in bone-inductive protein purified from bovine bone. Proc Natl Acad Sci USA 87: 9843-9847.
Centrella M, Horowitz MC, Wozney JM, McCarthy TL. 1994. Transforming growth factor-beta gene family members and bone. Endocr Rev 15: 27-39.
Chambers TJ, Fuller K. 1985a. Bone cells predispose bone surfaces to resorption by exposure of mineral to osteoclastic contact. J Cell Sci 76: 155-165.
Chambers TJ, Darby JA, Fuller K. 1985b. Mammalian collagenase predisposes bone surfaces to osteoclastic resorption. Cell Tissue Res 241: 671-675.
Cheifetz S. 1999. BMP receptors in limb and tooth formation. Crit Rev Oral Biol Med 10(2): 182-198.
Chen J, Shapiro HS, Sodek J. 1992. Development expression of bone sialoprotein mRNA in rat mineralized connective tissues. J Bone Miner Res 7: 987-997.
Chen Y, Alman BA. 2009. Wnt pathway, an essential role in bone regeneration. J Cell Biochem 106(3): 353-362.
Childs SC. 2005. The future of bone healing. Clin Podiatr Med Surg 22: 631-641.
Chin JR, Werb Z. 1997. Matrix metalloproteinases regulate morphogenesis, migration and remodeling of epithelium, tongue skeletal muscle and cartilage in the mandibular arch. Development 124: 1519-1530.
Cho TJ, Gerstenfeld LC, Einhorn TA. 2002. Differential temporal expression of members of the transforming growth factor beta superfamily during murine fracture healing. J Bone Miner Res 17: 513-520.
Choe I, Aycock RS, Raghow R, Myers JC, Seyer JM, Kang AH. 1987. A hepatic fibrogenic factor stimulates the synthesis of types I, III and V procollagens in cultured cells. J Biol Chem 262: 5408-5413.
Cole AA, Wlaters LM. 1987. Tartrate-resistant acid phosphatise in bone and cartilage following decalcification and cold-embedding in plastic. J Histochem Cytochem 35: 203-206.
Collin-Osdoby P. 1994. Role of vascular endothelial cells in bone biology. J Cell Biochem 55: 304-309.
Colnot C, Thompson Z, Miclau T, Werb Z, Helms JA. 2003. Altered fracture repair in the absence of MMP9. Development 130: 4123-4133.
Colnot C, de la Fuente L, Huang S, Hu D, Lu C, St-Jacques B, Helms JA. 2005. Indian hedgehog synchronizes skeletal angiogenesis and perichondrial maturation with cartilage development. Development 132: 1057-1067.
Cook SD, Baffes GC, Wolfe MW, Sampath TK, Rueger DC. 1994. Recombinant human bone morphogenetic protein-7 induces healing in a canine long-bone segmental defect model. Clin Orthop 301: 302-312.
38
Cook SD. 1997. Acceleration of bone healing with OP-1 in a canine noncritical size defect model. Second International OP-1 Conference, Boston, MA. Abstract
Corral DA, Amling M, Priemel M, Loyer E, Fuchs S, Ducy P, Baron R, and Karsenty G. 1998. Dissociation between bone resorption and bone formation in osteopenci transgenic mice. Proc Natl Acad Sci. USA 95: 13835-13840.
Cottler-Fox MH, Lapidot T, Petit I, Kollet O, DiPersio JF, Link D, Devine S. 2003. Stem cell mobilization. Hematology (Am Soc Hematol Educ Program) 419-437.
D’Souza RN, Aberg T, Gaikwas J, Cavender A, Owen M, Karsenty G, Thesleff I. 1999. Cbfa1 is required for epithelial-mesenchymal interactions regulating tooth development in mice. Development 126: 2911-2920.
Dacic S, Kalajzic I, Visnjic D, Lichtler AC, Rowe DW. 2001. Col1a1-driven transgenic markers of osteoblast lineage progression. J Bone Miner Res 16(7): 1228-1236.
Davidson G, Mao BY, Barrantes ID, Niehrs C. 2002. Kremen proteins interact with Dickkopf1 to regulate anteroposterior CNS patterning. Development 129(24): 5587-5596.
Davis RJ. 2000. Signal transduction by the JNK group of MAP kinases. Cell 103(2): 239-252.
de Saint-Georges L, Miller SC, Bowman BM, Jee WS. 1989. Ultrastructural features of osteoclasts in situ. Scanning Microsc 3: 1201-1206.
DeLacure MD. 1994. Physiology of bone healing and bone grafts. Otolaryngol Clin N Am 27(5): 859-874.
Delaisse JM, Boyde A, Maconnachie E, Ali NN, Sear CHJ, Eeckhout Y, Vaes G, Jones SJ. 1987. The effects of inhibitors of cysteine-proteinases and collagenase on the resorptive activity of isolated osteoclasts. Bone 8: 305-313.
Delaisse JM, Eeckhout Y, Neff L, Francois-Gillet C, Henriet P, Su Y, Vaes G, Baron R. 1993. (Pro)collagenase (matrix metalloproteinase-1) is present in rodent osteoclasts and in the underlying bone-resorbing compartment. J Cell Sci 106: 1071-1082.
Dellavalle A, Sampaolesi M, Tonlorenzi R, Tagliafico E, Sacchetti B, Perani L, Innocenzi A, Galvez BG, Messina G, Morosetti R, Li S, Belicchi M, Peretti G, Chamberlain JS, Wright WE, Torrente Y, Ferrari S, Bianco P, Cossu G, 2007. Pericytes of human askeletal muscle are myogenic precursors distinct from satellite cells. Nat Cell Biol 9: 255-267.
den Boer FC, Bramer JAM, Blokhius TJ, Van Soest EJ, Jenner JM, Patka P, Bakker FC, Burger EH, Haarman HJ. 1997. The effect of recombinant human osteogenic protein-1 on the healing of fresh closed diaphyseal fracture. Second International OP-1 Conference, Boston MA. Abstract.
Desbois C, Seldin MF, Karsenty G. 1994. Localization of the osteocalcin gene-cluster on mouse chromosome-3. Mamm Genome 5(5): 321-322.
Diegelmann RF, Peterkofsky B. 1971. Collagen biosynthesis during connective tissue development in the chick embryo. Dev Biol 28: 443-453.
39
Dodds RA, Connor JR, James IE, Rykaczewski EL, Appelbaum E, Dul E, Gowen M. 1995. Human osteoclasts, not osteoblasts, deposit osteopontin onto resorptin surfaces: An in vitro and ex vivo study of remodelling. J Bone Miner Res 10: 1666-1680.
Dodig M, Kronenberg MS, Bedalov A, Kream BE, Gronowicz G, Clark SH, Mack K, Liu YH, Maxon R, Pan ZZ, Upholt WB, Rowe DW, Lichtler AC. 1996. Identification of a TAAT-containing motif required for high level expression of the COL1A1 promoter in differentiated osteoblasts of transgenic mice. J Biol Chem 271: 16422-16429.
Doherty MJ, Ashton BA, Walsh S, Beresford JN, Grant ME, Canfield AE, 1998. Vascular pericytes express osteogenic potential in vitro and in vivo. J Bone Miner Res 13: 828-838.
Doty SB, Nunez EA. 1985. Activation of osteoclasts and the repopulation of bone surfaces following hibernation in the bat, Myotis lucifugus. Anat Rec 213: 481-495.
Ducy P, Desbois C, Boyce B, Pinero G, Story B, Dunstan C, Smith E, Bonadio J, Goldstein S, Gundberg C, Bradley A, Karsenty G. 1996. Increased bone formation in osteocalcin-deficient mice. Nature 382(6590): 448-452.
Ducy P, Karsenty G. 1998. Genetic control of cell differentiation in the skeleton. Curr Opin Cell Biol 10: 614-619.
Ducy P. 2000. Cbfa1: A molecular switch in osteoblast biology. Dev Dyn 219:461-471.
Duprez D, Bell EJ, Richardson MK, Archer CW, Wolpert L, Brickell PM, Francis-West PH. 1996. Overexpression of BMP-2 and BMP-4 alters the size and shape of developing skeletal elements in the chick limb. Mech Dev 57(2): 145-157.
Einhorn TA, Majeska RJ, Rush EB, Levine PM, Horowtiz MC. 1995. The expression of cytokine activity by fracture callus. J Bone Miner Res 10: 1272-1281.
Einhorn TA, Majeska RJ, Oloumi G. 1997. Enhancement of experimental fracture healing with a local percutaneous injection of rhBMP-2. American Academy of Orthopaedic Surgeons Annual Meeting. San Francisco CA 64: 216. Abstract
Einhorn TA. 1998. The cell and molecular biology of fracture healing. Clin Orthop Relat Res 355: S7-21.
Einhorn T, Lee C. 2001. Bone regeneration: new findings and potential clinical applications. J Am Acad Orthop Surg 9: 157-165.
Ekanayake S, Hall BK. 1987. The development of acellularity of the vertebral bone of the Japanese Medaka, Oryzias latipes (Teleostei, Cyprinidontidae). J Morph 193: 253-261.
Ekanayake S, Hall BK. 1988. Ultrastructure of the osteogenesis of acellular vertebral bone in the Japanese Medaka, Oryzias latipes (Teleostei, Cyprinidontidae). Am J Anat 182: 241-249.
Elion GB, Furman PA, Fyfe JA, de Miranda P, Beauchamp L, and Schaeffer HJ. 1977. Selectivity of action of an anti-herpetic agent, 9-(2-Hydroxyethoxymethyl) Guanine. Proc Natl Acad Sci USA. 74: 5716-5720.
Ellingsworth LR, Brennan JE, Fok K, Rosen DM, Bentz H, Piez KA, Seyedin SM. 1986. Antibodies to the N-terminal portion of cartilage-inducing factor A and transforming
40
growth factor beta: Immunohistochemical localization and association with differentiating cells. J Biol Chem 261: 12362.12367.
Engsig MT, Chen QJ, Vu TH, Pedersen AC, Therkidsen B, Lund LR, Henriksen K, Lenhard T, Foged NT, Werb Z, Delaisse JM. 2000. Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones. J Cell Biol 151: 879-890.
Enomoto H, Enomoto-Iwamoto M, Iwamoto M, Nomura S, Himeno M, Kitamura Y, Kishimoto T, Komori T. 2000. Cbfa1 is a positive regulatory factor in chondrocyte maturation. J Biol Chem 275: 8695-8702.
Enomoto H, Shiojiri S, Hoshi K, Furuichi T, Fukuyama R, Yoshida CA, Kanatani N, Nakamura R, Mizuno A, Zanma A, Yano K, Yasuda H, Higashio K, Takada K, Komori T. 2003. Induction of osteoclast differentiation by Runx2 through receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin regulation and partial rescue of osteoclastogenesis in Runx2-/- mice by RANKL transgene. J Biol Chem 278: 23971-23977.
Erlebacher A, Filvaroff EH, Ye JQ, Derynck R. 1998. Osteoblastic responses to TGF-beta during bone remodelling. Mol Biol Cell 9: 1903-1918.
Everts V, Delaisse JM, Korper W, Jansen DC, Tiggelaar-Gutter W, Saftig P, Beertsen W. 2002. The bone lining cell: its role in cleaning Howship’s lacunae and initiating bone formation. J Bone Miner Res 17: 77-90.
Ferguson C, Alpern E, Miclau T, Helms JA. 1999. Does adult fracture repair recapitulate embryonic skeletal formation? Mech Dev 87: 57-66.
Ferrara N, Gerber HP. 2001. The role of vascular endothelial growth factor in angiogenesis. Acta Haematol 106: 148-156.
Fessler LI, Robinson WJ, Fessler JH. 1981. Biosynthesis of procollagen [(proα1 V)2 (proα2 V)] by chick tendon fibroblasts and procollagen (proα1 V)3 by hamster lung cell cultures. J Biol Chem 256: 9646-9651.
Franz-Odendaal TA, Hall BK, Witten PE. 2006. Buried alive: How osteoblasts become osteocytes. Dev Dyn 235(1): 176-190.
Franzoso G, Carlson L, Xing L, Poljak L, Shores EW, Brown KD, Leonardi A, Tran T, Boyce BF, Siebenlist U. 1997. Requirement for NF- κB in osteoclast and B-cell development. Genes Dev 11: 3482-3496.
Frost HM. 1960. In vivo osteocyte death. J Bone Joint Surg 42A: 138-143.
Frost HM. 1989. The biology of fracture healing. And overview for clinicians. Part I. Clin Orthop 248: 283-293.
Furman PA, McGujirt PV, Keller PM, Fyfe JA and Elion GB. 1980. Inhibition by acyclovir of cell-growth and DNA-synthesis of cells biochemically transformed with herpesvirus genetic information. Virology 102: 420-430.
Fyfe JA, Keller PM, Furman PA, Miller RL and Elion GB. 1978. Thymidine kinase from herpes-simplex virus phosphorylates the new anti-viral compound, 9-(2-Hydroxyethoxymethyl) Guanine. J Biol Chem 253: 8721-8727.
41
Gack S, Vallon R, Schmidt J, Grigoriadis A, Tuckermann J, Schenkel J, Weiher H, Wagner EF, Angel P. 1995. Expression of interstitial collagenase during skeletal development of the mouse is restricted to osteoblast-like cells and hypertrophic chondrocytes. Cell Growth Differ 6: 759-767.
Garant PR. 1976. Collagen resorption by fibroblasts. A theory of fibroblastic maintenance of the periodontal ligament. J Periodontol 47: 380-390.
Gerber HP, Vu TH, Ryan AM, Kowalski J, Werb Z, Ferrara N. 1999. VEGF couples hypertrophic cartilage remodelling, ossification, and angiogenesis during endochondral bone formation. Nat Med 5: 623-628.
Gerber HP, Ferrara N. 2000. Angiogenesis and bone growth. Trends Cardiovasc Med 10: 223-228.
Gerstenfeld LC, Shapiro FD. 1996. Expression of bone-specific genes by hypertrophic chondrocytes: implication of the complex functions of the hypertrophic chondrocyte during endochondral bone development. J Cell Biochem 62: 1-9.
Gerstenfeld LC, Cullinane DM, Barnes GL, Graves DT, Einhorn TA. 2003. Fracture healing as a post-natal developmental process: molecular, spatial, and temporal aspects of its regulation. J Cell Biochem 88(5): 873-884.
Gerstenfeld LC, Sacks DJ, Pelis M, Mason ZD, Graves DT, Barrero M, Ominsky MS, Kostenuik PJ, Morgan EF, Einhorn TA. 2009. Comparison of effects of the bisphosphonate alendronate versus the RANKL inhibitor denosumab on murine fracture healing. J Bone Miner Res 24: 196-208.
Gillet C, Eeckhout Y, Vaes G. 1977. Purification of procollagenase and collagenase by affinity chromatography on Sepharose-collagen. FEBS Lett 74:126-128.
Giannoudis PV, Jones E, Einhorn TA. 2011. Fracture healing and bone repair. Injury 42(6):549-550.
Glass DA 2nd, Karsenty G. 2006a. Canonical Wnt signaling in osteoblasts is required for osteoclast differentiation. Ann N Y Acad Sci 1068: 117-130.
Glass DA 2nd, Karsenty G. 2006b. Molecular bases of the regulation of bone remodeling by the canonical Wnt signaling pathway. Curr Top Dev Biol 73: 43-84.
Goto T, Yamaza T, Tanaka T. 2003. Cathepsins in the osteoclast. J Electron Micros 52(6): 551-558.
Green E, Lubahn JE, Evans J. 2005. Risk factors, treatment, and outcomes associated with non-union of the midshaft humerus fracture. J Surg Orhop Adv 14(2): 64-72.
Guo J, Jin J, Cooper LF. 2008. Dissection of sets of genes that control the character of wnt5a-deficient mouse calvarial cells. Bone 43: 961-971.
Habas R, Dawid IB, He X. 2003. Coactivation of Rac and Rho by Wnt/Frizzled signaling is required for vertebrate gastrulation. Genes Dev 17(2): 295-309.
Habuchi H, Conrad HE, Glaser JH. 1985. Coordinate regulation of collagen and alkaline phosphatase levels in chick embryo chondrocytes. J Biol Chem 260: 13029-13034.
42
Hadjidakis DJ, Androulakis II. 2006. Bone remodeling. Ann NY Acad Sci 1092: 385-396.
Hall BK. 1988. The embryonic development of bone. Am Sci 76: 174-181.
Hall BK, Miyake T. 1992. The membranous skeleton: the role of cell condensations in vertebrate skeletogenesis. Anat Embryol (Berl) 186: 107-124.
Hall BK, Miyake T. 1995. Divide, accumulate, differentiate: cell condensation in skeletal development revisited. Int J Dev Biol 39: 881-893.
Hall BK, Witten PE. 2005. The origin and plasticity of skeletal tissues in vertebrate evolution and development. In: Anderson JS, Sues H-D, editors. Major transitions in vertebrae evolution. Festschrift for Dr. Robert L. Carroll. Bloomington, IN: Indiana University Press. 1-53.
Hall AP, Westwood FR, Wadsworth PF. 2006. Review of the effects of anti-angiogenic compounds on the epiphyseal growth plate. Toxicol Pathol 34: 131-147.
Haller AC, Zimny ML. 1978. Effects of hibernation on interradicular alveolar bone. J Dent Res 56: 1552-1557.
Hammer RE, Brinster RL, Rosenfeld MG, Evans RM and Mayo KE. 1985. Expression of human growth hormone-releasing factor in transgenic mice results in increased somatic growth. Nature 315: 413-416.
Han Y, Lefebvre V. 2008. L-Sox5 and Sox6 drive expression of the aggrecan gene in cartilage by securing binding of Sox9 to a far-upstream enhancer. Mol Cell Biol 28(16): 4999-5013.
Harris WH, Heaney RP. 1969. Skeletal renewal and metabolic bone diseases. N Engl J Med 280: 193-202, 253-259,303-311.
Harris SE, Bonewald LF, Harris MA, Sabatini M, Dallas S, Feng JQ, Chosh-Choudhury N, Wozney J, Mundy GR. 1994. Effects of transforming growth factor beta on bone nodule formation and expression of bone morphogenetic protein 2, osteocalcin, osteopontin, alkaline phosphatase, and type I collagen mRNA in long-term cultures of fetal rat calvarial osteoblasts. J Bone Miner Res 9: 855-863.
Hartmann C. 2006. A Wnt canon orchestrating osteoblastogenesis. Trends Cell Biol 16(3): 151-158.
Hartmann C. 2009. Transcriptional networks controlling skeletal development. Curr Opin Genet Dev 19(5): 437-443.
Hauschka PV, Wians FH. 1989. Osteocalcin-hydroxyapatite interaction in the extracellular organic matrix of bone. Anat Rec 224(2): 180-188.
Hayden JM, Mohan S, Baylink DJ. 1995. The insulin-like growth factor system and the coupling of formation to resorption. Bone 17: 93S-98S.
Heissig B, Tejeda R, Friedrich M, Werb Z, Rafii S, Hattori K. 2002a. MMP9 mediated activation and release of pro-angiogenic factors following irradiation. Blood 100(11): 124 Abstract.
Heissig B, Hattori K, Dias S, Friedrich M, Ferris B, Hackett NR, Crystal RG, Besmer P, Lyden D, Moor MAS, Werb Z, Rafii S. 2002. Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand. Cell 109: 625-637.
43
Hermey DC, Popoff SN, Marks SC Jr. 1996a. Reduce bone resorption in toothless (osteopetrotic) rats: An abnormally of osteoblasts related to their inability to activate osteoclast activity in vitro. Connet Tissue Res 35: 273-278.
Hermey DC, Popoff SN, Marks SC Jr. 1996b. Differential gene expression in bone from toothless (osteopetrotic) rats. J Bone Miner Res 11: S652-S652.
Heyman RA, Borreli E, Lesley J, Anderson D, Richman DD, Baird SM, Hyman R, Evans RM. 1989. Thymidine kinase obliteration: creation of transgenic mice controlled immune deficiency. Proc Natl Acad Sci USA 86(8): 2698-2702.
Hill PA. 1998. Bone remodeling. Br J Orthod 25: 101-107.
Hill TP, Spater D, Taketo MM, Birchmeier W, Hartmann C. 2005. Canonical Wnt/beta-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev Cell 8: 727-738.
Hofbauer LC, Khosla S, Dunstan CR, Lacey DL, Boyle WJ, Riggs BL. 2000. The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption. J Bone Miner Res 15: 2-12.
Holliday LS, Welgus HG, Gliszar CJ, Veith GM, Jeffrey JJ, Gluck SL. 1997. Initiation of osteoclast bone resorption by interstitial collagenase. J Biol Chem 272: 22053-22058.
Holtrop ME. 1990. Light and electron microscopical structure of bone forming cells. In: Hall BK, editor. Bone, Vol. 1: the osteoblast and osteocyte. Caldwell, NJ: The Telford Press. 1-40.
Hui M, Li SQ, Holmyard D, Cheng P. 1997. Stable transfection of nonosteogenic cell lines with tissue nonspecific alkaline phosphatase enhances mineral deposition both in the presence and absence of beta-glycerophosphate: Possible role for alkaline phosphatase in pathological mineralization. Calcif Tissue Int 60: 467-472.
Ingram RT, Bonde SK, Riggs BL, Fitzpatrick LA. 1994. Effects of transforming growth factor beta and 1,25 dihydroxyvitamin D3 on the function, cytochemistry, and morphology of normal osteoblast-like cells. Differentiation 55: 153-163.
Inada M, Yasui T, Nomura S, Miyake S, Deguchi K, Himeno M, Sato M, Yamagiwa H, Kimua T, Yasui N, Ochi T, Endo N, Kitamura Y, Kishimoto T, Komori T. 1999. Maturational disturbance of chondrocytes in Cbfa1-deficeint mice. Dev Dyn 214: 279-290.
Inada M, Wang Y, Byrne MH, Rahman MU, Miyaura C, Lopez-Otin C, Krane SM, 2004. Critical roles for collagenase-3 (Mmp13) in development of growth plate cartilage and in endochondral ossification. Proc Natl Acad Sci USA 101: 17192-17197.
Ishida N, Hayashi K, Hoshijima M, Ogawa T, Koga S, Miyatake Y, Kumegawa M, Kimura T, Takeya T. 2002. Large scale gene expression analysis of osteoclastogenesis in vitro and elucidation of NFAT2 as a key regulator. J Biol Chem 277: 41147-41156.
Itagaki T, Honma T, Takahashi I, Echigo S, and Sasano Y. 2008. Auantitative analysis and localization of mRNA transcripts of type I collagen, osteocalcin, MMP2, MMP8, and MMP13 during bone healing in a rat calvarial experimental defect model. Anat Rec 291: 1038-1046.
44
Iwaki A, Jingushi S, Oda Y, Izumi T, Shida JI, Tsuneyoshi M, Sugioka Y. 1997. Localization and quantification of proliferating cells during rat fracture repair: detection of proliferating cell nuclear antigen by immunohistochemistry. J Bone Miner Res 12: 96-102.
Iyama K, Ninomiya Y, Olsen BR, Linsenmayer TF, Trelstad RL, Hayashi M. 1991. Spatiotemporal pattern of type X collagen gene expression and collagen deposition in embryonic chick vertebrae undergoing endochondral ossification. Anat Rec 229(4): 462-472.
Jilka RL, Weinstein RS, Bellido T, Parfitt AM, Manolagas SC. 1998. Osteoblast programmed cell death (apoptosis): modulation by growth factors and cytokines. J Bone Miner Res 13(5): 793-802.
Jilka RL, Weinstein RS, Parfitt AM, Manolagas SC. 2007. Quantifying osteoblast and osteocyte apoptosis: Challenges and rewards. J Bone Miner Res 22(10): 1492-1501.
Jimenez MJG, Balbin M, Lopez JM, Alvarez J, Komori T, Lopez-Orin C. 1999. Collagenase-3 is a target of Cbfa1, a transcription factor of the runt gene family involved in bone formation. Mol Cell Biol 19: 4431-4442.
Johansson N, Saarialho-Kere U, Airola K, Herva R, Nissinen L, Westermarck J, Vuorio E, Heino J, Kahari VM. 1997. Collagenase-3 (MMP13) is expressed by hypertrophic chondrocytes, periosteal cells, and osteoblasts during human fetal bone development. Dev Dyn 208: 387-397.
Jonason JH, Xiao G, Zhang M, Xing L, Chen D. 2009. Post-translational regulation of Runx2 in bone and cartilage. J Dent Res 88(8): 693-703.
Jones SJ, Boyde A, Ali NN. 1986. The interface of cells and their matrices in mineralized tissues: A review. Scan Electron Microsc 4: 1555-1569.
Jones CM, Lyons KM, Lapan PM, Wright CV, Hogan BL. 1992. DVR-4 (bone morphogenetic protein-4) as a posterior-ventralizing factor in Xenopus mesoderm induction. Development 115, 639-647.
Joyce ME, Jingushi S, Bolander ME. 1990a. Transforming growth factor-beta in the regulation of fracture repair. Orthop Clin North Am 21: 199-209.
Joyce ME, Terek RM, Jingushi S, Bolander ME. 1990b. Role of transforming growth factor-beta in fracture repair. Ann N Y Acad Sci 593: 107-123.
Kahn AJ, Patridge NC. 1987. New concepts in bone remodelling: An expanding role for the osteoblast. Am J Otolaryngol 8: 258-264.
Kaltschmidt JA, Lawrence N, Morel V, Balayo T, Fernandez BG, Pelissier A, Jacinto A, Martinez Arias A. 2002. Planar polarity and actin dynamics in the epidermis of Drosophila. Nat Cell Biol 4: 937-944.
Karaplis AC, Luz A, Glowaski J, Bornson RT, Tybulewicz VL, Kronenberg HM, Mulligan RC. 1994. Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene. Genes Dev 8: 277-289.
Karsdal MA, Fjording MS, Foged NT, Delaisse JM, Lochter A. 2001. Transforming growth factor-beta-induced osteoblast elongation regulates osteoclastic bone resorption through a
45
p38 mitogen0activated protein kinase- and matrix metalloproteinase-dependent pathway. J Biol Chem 276: 39350-8.
Karsdal MA, Larsen L, Engsig MT, Lou H, Ferreras M, Lochter A, Delaisse JM, Foged NT. 2002. MMP dependent activation of latent TGF-beta controls the conversion of osteoblasts into osteocytes by blocking osteoblast apoptosis. J Biol Chem 277(46): 44061-44068.
Karsenty G, de Crombrugghe B. 1990. Two different negative and one positive regulatory factors interact with a short promoter segment of the α1(I) collagen gene. J Biol Chem 265: 9934-9942.
Karsenty G and Wangner EF. 2002. Reaching a genetic and molecular understanding of skeletal development. Dev Cell 2: 389-406.
Katoh M. 2002. Wnt and FGF gene clusters (Review). Int J Oncol 21(6): 1269-1273.
Katoh M. 2005. Wnt/PCP signaling pathway and human cancer (Review). Oncol Rep 14(6): 1583-1588.
Kauffman JM, Taelman P, Vermeulen A, Vandeweghe M. 1992. Bone mineral status in growth hormone-deficient males with isolated and multiple pituitary deficiencies of childhood onset. J Clin Endocrinol Metab 74: 118-123.
Ketenjian AY, Arsenis C. 1975. Morphological and biochemical studies during differentiation and calcification of fracture callus cartilage. Clin Orthop Relat Res 107: 266-273.
Khillan JS, Schmidt A, Overbeek PA, de Crombrugghe B, Westphal H. 1986. Developmental and tissue-specific expression directed by the α2 type I collagen promoter in transgenic mice. Proc Natl Acad Sci USA 83: 725-729.
Kim IS, Otto F, Abel B, Mundlos S, 1999. Regulation of chondrocyte differentiation by Cbfa1. Mech Dev 80: 159-170.
Kim HJ, Kim JH, Bae SC, Choi JY, Kim HJ, Ryoo HM. 2003. The protein kinase C pathway plays a central role in the fibroblast growth factor-stimulated expression and transactivation activity of Runx2. J Biol Chem 278: 319-326.
Kirker-Head CA, Gerhart TN, Schelling SH, Hennig GE, Wang E, Holtrop ME. 1995. Long-term healing of bone using recombinant human bone morphogenetic protein 2. Clin Orthop 318: 222-230.
Knauper V, Lopez-Otin C, Smith B, Knight G, Murphy G. 1996. Biochemical characterization of human collagenase-3. J Biol Chem 271: 1544-1550.
Knauper V, Cowell S, Smith B, Lopez-Otin C, O’Shea M, Morris H, Zardi L, Murphy G. 1997. The role of the C-terminal domain of human collagenase-3 (MMP-13) in the activation of procollagense-3, substrate specificity, and tissue inhibitor of metalloproteinase interaction. J Biol Chem 272: 7608-7616.
Knothe Tate ML, Adamson JR, Tami AE, Bauer TW. 2004. The osteocyte. Int J Biochem Cell Biol 36: 1-8.
Kollet O, Shivitel S, Chen YQ, Suriawinata J, Thung SN, Dabeva MD, Kahn J, Spiegel A, Dar A, Samira S, Goichberg P, Kalinkovich A, Arenzana-Seisdedos F, Nagler A, Hardan I,
46
Revel M, Shafritz DA, Lapidot T. 2003. HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver. J Clin Invest 112(2): 160-169.
Kollet O, Dar A, Shivtiel S, Kalinkovich A, Lapid K, Sztainberg Y, Tesio M, Samstein RM, Goichberg P, Spiegel A, Elson A, Lapidot T. 2006. Osteoclasts degrade endosteal component and promote mobilization of hematopoietic progenitor cells. Nat Med 12(6): 657-664.
Komatsubara S, Mori S, Mashiba T, Nonaka K, Seki A, Akiyama T, Miyamoto K, Cao Y, Manabe T, Norimatsu H. 2005. Human parathyroid hormone (1-34) accelerates the fracture healing process of woven to lamellar bone replacement and new cortical shell formation in rat femora. Bone 36: 678-687.
Komori T, Yagi H, Nomura S, Yamaguchi H, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao YH, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T. 1997. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts Cell 89: 755-764.
Kosaki N, Takaishi H, Kamekura S, Kimura T, Okada Y, Minqi L, Amizuka N, Chung UI, Nakamura K, Kawagucchi H, Toyama Y, D’Armiento J. 2007 Impaired bone fracture healing in matrix metalloproteinase-13 deficient mice. Biochem Biophys Res Commun 354: 846-851.
Koyama E, Leatherman JL, Shimazu A, Nah HD. 1995. Syndecan-3, tenascin-C, and the development of cartilaginous skeletal elements and joints in chick limbs. Dev Dyn 203: 152-162.
Koyama E, Shimazu A, Leatherman JL, Golden EB, Nah HD, Pacifici M. 1996. Expression of syndecan-3 and tenascin-C: possible involvement in periosteum development. J Orthop Res 14: 403-412.
Kramer I, Halleux C, Keller H, Pequrri M, Gooi JH, Weber PB, Feng JQ, Bonewald LF, Kneissel M. 2010. Osteocyte Wnt/beta-catenin signaling is required for normal bone homeostasis. Mol Cell Biol 30(12): 3071-3085.
Kratochwill, K, von der Mark K, Kollar EJ, Jaenisch R, Mooslehner K, Schwarz M, Haase K, Gmachi I, Harbers K. 1989. Retrovirus-induced insertional mutation in Mov13 mice affects collagen I expression in a tissue-specific manner. Cell 57: 807-816.
Kream BE, Rowe DW, Gworek G, Raisz LG. 1980. Parathyroid hormone alters collagen synthesis and procollagen mRNA levels in fetal rat calvaria. Proc Natl Acad Sci USA 77: 5654-5658.
Kream BE, Simth MD, Canalis E, Raisz LG. 1985. Characterization of the effect of insulin on collagen synthesis in fetal rat bone. Endocrinology 116: 1922-1928.
Krebsbach PH, Harrison JR, Lichtler AC, Woody CO, Rowe DW, Kream BE. 1993. Transgenic expression of COL1A1-cholarmphenicol acetyltransferase fusion genes in bone: Differential utilization of promoter elements in vivo and in cultured cells. Mol Cell Biol 13: 5168-5174.
Kronenberg HM. 2003. Developmental regulation of the growth plate. Nature 15: 423(6937): 332-336.
47
Kuhl M. 2004. The Wnt/calcium pathway: biochemical mediators, tools and future requirements. Front Biosci 9: 967-974.
Kwiecinski GG. 1985. Bone remodelling and its regulation in Myotis lucifugus. Prog Zool 30: 45-47.
Kwiecinski GG, Krook L, Wimsatt WA. 1987. Annual skeletal changes in the little brown bat, Myotis lucifungus, with particular references to pregnancy and lactation. Am J Anat 178: 410-420.
Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian YX, Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J, Boyle WJ. 1998. Osteoprotegerin (OPG) ligand is a cytokine that regulates osteoclasts differentiation and activation. Cell 93(2): 165-176.
Lanske B, Karaplis AC, Lee K, Luz A, Vortkamp A, Pirro A, Karperien M, Defize LHK, Ho C, Mulligan RC, Defize LH, Ho C, Mulligan RC, Abou-Samra AB, Juppner H, Segre GV, Kronenberg HM. 1996. PTh/PThrP receptor in early development and Indian hedgehog-regulated bone growth. Science 273: 663-666.
Lapidot T, Dar A, Kollet O. 2005. How do stem cells find their way home? Blood 106: 1901-1910.
Lazzarini L, De Lalla F, Mader JT. 2002. Long bone osteomyelitis. Curr Infect Dis Rep 4: 439-445.
Le AX, Miclau T, Hu D, Helms JA. 2001. Molecular aspects of healing in stabilized and non-stabilized fractures. J Orthop Res 19: 78-84.
Lee MH, Javed A, Kim JH, Shin JI, Gutierrez S, Choi JY, Rosen JL, van Wijnen AJ, Stein GS, Lian JB, Ryoo HM. 1999. Transient upregulation of Cbfa1 in response to bone morphogenetic protein-2 and transforming growth factor beta1 in C2C12 myogenic cells coincides with suppression of the myogenic phenotype but is not sufficient for osteoblast differentiation. J Cell Biochem 73: 114-125.
Lee MH, Kim YJ, Kim HJ, Park HD, Kang AR, Kyung HM, Sung JH, Wozney JM, Kim HJ, Ryoo HM. 2003. BMP-2-induced Runx2 expression is mediated by Dlx5, and TGF-beta 1 opposes the BMP-2-induced osteoblast differentiation by suppression of Dlx5 expression. J Biol Chem 278(36): 34387-34394.
Lefebvre V, Huang W, Harley VR, Goodfellow PN, de Crombrugghe B. 1997. Sox9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha1 (II) collagen gene. Mol Cell Biol 17(4): 2336-2346.
Lefebvre V, de Crombrugghe B. 1998a. Toward understanding Sox9 function in chondrocyte differentiation. Matrix Biol 16(9): 529-540.
Lefebvre V, Li P, de Crombrugghe B. 1998b. A new long form of Sox5 (L-Sox5), Sox6 and Sox9 are coexpressed in chondrogenesis and cooperatively activate the type II collagen gene. EMBO J 17(19): 5719-5733.
Levesque JP, Hendy J, Takamatsu Y, Simmons PJ, Bendall LJ. 2003. Disruption of the CXCR4/CXCL12 chemotactic interaction during hematopoietic stem cell mobilization induced by G-CSF or cyclophosphamide. J Clin Invest 111: 187-196.
Li J, Mori S, Kaji Y, Mashiba T, Kawanishi J, Norimatsu H. 1999. Effect of bisphosphonate (incadronate) on fracture healing of long bones in rats. J Bone Miner Res 14: 969-979.
Li L, Cserjesi P, Olsen BR. 1995. Dermo-1: a novel twist-related bHLH protein expressed in the developing dermis. Dev Biol 172: 280-292.
Li M, Amizuka N, Oda K, Tokunaga K, Ito T, Takeuchi K, Takagi R, Maeda T. 2004. Histochemical evidence of the initial chondrogenesis and osteogenesis in the periosteum of a rib fractured model: implications of osteocyte involvement in periosteal chondrogenesis. Microsc Res Tech 64: 330-342.
Lian JB, McKee MD, Todd AM, Gerstenfeld LC. 1993. Induction of bone-related proteins, osteocalcin and osteopontin, and their matrix ultrastructural localization with development of chondrocyte hypertrophy in vitro. J Cell Biochem 52: 206-219.
Liau G, Yamada Y, de Crombrugghe B. 1985. Coordinate regulation of the levels of type III and type I collagen mRNA in most but not all mouse fibroblasts. J Biol Chem 260: 531-536.
Lichteler A, Stover ML, Angily J, Kream B, Rowe DW. 1989. Isolation and characterization of the rat α(1) collagen promoter. J Biol Chem 264: 3072-3077.
Liska DJ, Reed MJ, Sage EH, Bornstein P 1994 Cell-specific expression of alpha 1(I) collagen-hGH minigenes in transgenic mice. J Cell Biol 125: 695–704.
Liu Z, Luyten FP, Lammens J, Degueker J. 1999. Molecular signalling in bone fracture healing and distraction osteogenesis.Histol Histopathol 14(2): 587-595.
Lopez E, Macintyre I, Martelly E, Lallier F, Vidal B. 1980. Paradoxical effect of 1,25-dihydroxycholecalciferol on osteoblastic and osteoclastic activity in the skeleton of the eel Anguilla Anguilla L. Calc Tissue Int 32: 83-87.
Macias D, Garian Y, Sampath TK, Piedra ME, Ros MA, Hurle JM. 1997. Role of BMP-2 and OP-1 (BMP-7) in programmed cell death and skeletogenesis during chick limb development. Development 126(6): 1109-1117.
Madeddu P. 2005. Therapeutic angiogenesis and vasculogenesis for tissue regeneration. Exp Physiol 90: 315-326.
Madison M, Martin RB. 1993. Fracture healing, in Operative Orthopaedics (Chapman, M. W., ed.), Lippincott, Philadelphia, 221-228.
Maes C, Carmeliet P, Moermans K, Stockmans I, Smets N, Collen D, Bouillon R, Carmeliet G. 2002. Impaired angiogenesis and endochondral bone formation in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188. Mech Dev 111: 61-73.
Manolagas SC. 2000. Birth and death of bone cells: basic regulatory mechanisms and implications for pathogenesis and treatment of osteoporosis. Endocrinol Rev 21: 115-137.
Mao B, Wu W, Davidson G, Marhold J, Li M, Mechler BM, Delius H, Hoppe D, Stannek P, Walter C, Glinka A, Niehrs C. 2002. Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signaling. Nature 417(6889): 664-667.
49
Mark MP, Butler WT, Prince CW, Finkelman RD, Ruch JV. 1988. Developmental expression of 44-kDa bone phosphoprotein (osteopontin) and bone gamma-carboxyglutamic acid (Gla)-containing protein (osteocalcin) in calcifying tissues of rat. Differentiation 37: 123-136.
Marotti G. 1996. The structure of bone tissues and the cellular control of their deposition. Italian J Anat Embryol 101: 25-79.
Marsell R, Einhorn TA. 2009. The role of endogenous bone morphogenetic proteins in normal skeletal repair. Injury 40(3): S4-7.
Marsell R, Einhorn TA. 2011. The biology of fracture healing. Injury 42: 551-555.
Marsh DR, Li G. 1999. The biology of fracture healing: optimising outcome. Br Med Bull 55(4): 856-869.
Martin TJ, Ng KW. 1994. Mechanisms by which cells of the osteoblast lineage control osteoclast formation and activity. J Cell Biochem 56: 357-366.
Martin JF, Sims NA. 2005. Osteoclast-derived activity in the coupling of bone formation to resorption. Trends Mol Med 11: 76-81.
Maruya Y, Sasano Y, Takahashi I, Kagayama M, Mayanagi H. 2003. Expression of extracellular matrix molecules, MMPs and TIMPs in alveolar bone, cementum and periodontal ligaments during rat tooth eruption. J Electon Microsc 52: 593-604.
Maruyama Z, Yoshida CA, Furuichi T, Amizuka N, Ito M, Fukuyama R, Miyazaki T, Kitaura H, Nakamura K, Fujita T, Kanatani N, Moriishi T, Yamana K, Liu W, Kawaguchi H, Nakamura K, Komori. 2007. Runx2 determines bone maturity and turnover rate in postnatal bone development and is involved in bone loss in estrogen deficiency. Dev Dyn 236: 1876-1890.
Marzia M, Sims NA, Voit S, Migliaccio S, Taranta A, Bernardini S, Faraggiana T, Yoneda T, Mundy GR, Boyce BF, Baron R, Teti A. 2000. Decreased c-Src expression enhances osteoblast differentiation and bone formation. J Cell Biol 151: 311.320.
Massaque J. 1990. The transforming growth factor-beta family. Ann Rev Cell Biol 6: 597-609.
Mattot V, Raes MB, Henriet P, Eeckhout Y, Stehelin D, Vandenbunder B, Desbiens X. 1995. Expression of interstitial collagenase is restricted to skeletal tissue during mouse embryogenesis. J Cell Sci 108 (pt2): 529-535.
McCarthy TL, Centrella M, Canalis E. 1988. Further biochemical and molecular characterization of primary rat parietal bone cell cultures. J Bone Miner Res 3: 401-406.
McCulloch CAG, Heersche JNM. 1988. Lifetime of the osteoblast in mouse periodontium. Anat Rec 222(2): 128-135.
McDonald MM, Dulai S, Godfrey C, Amanat N, Sztynda T, Little DG. 2008. Bolus or weekly zoledronic acid administration does not delay endochondral fracture repair but weekly dosing enhances delays in hard callus remodeling. Bone 43: 653-662.
McKee MD, Glimcher MJ, Nanci A. 1992. High-resolution immunolocalization of osteopontin and osteocalcin in bone and cartilage during endochondral ossification in the chicken tibia. Anat Rec 234: 479-492.
McKibbin B. 1978. The biology of fracture healing in long bones. J Bone Joint Surg Br 60B(2): 150-162.
50
Merino R, Ganan Y, Macias D, Economides AN, Sampath KT, Hurle JM. 1998. Morphogenesis of digits in the avian limb is controlled by FGFs, TGFβs, and Noggin through BMP signaling. Dev Biol 200: 35-45.
Mersell R, Einhorn TA. 2011. The biology of fracture healing. Injury 42: 551-555.
Meunier FJ. 1989. The acellularisation process in Osteichthyan bone. Prog Zool 35: 443-445.
Miyauchi A, Alvarez J, Greefield EM, Teti A, Grano M, Colucci S, Zambonin-Zallone A, Ross FP, Teitelbaum SL, Cheresh D, Hruska KA. 1991. Recongnition of osteopontin and related peptides by an alpha v beta 3 integrin stimulates immediate cell signals in osteoclasts. J Biol Chem 266: 20369-20374.
Moen RC, Rowe DW, Palmiter RD. 1979. Regulation of procollagen synthesis during the development of chick embryo calvaria. J Biol Chem 254: 3526-3530.
Mohan S, Baylink DJ. 1991. The role of IGF-II in the coupling of bone formation to resorption. In modern concepts of insulin-like growth factors (ed. Spencer, EM) 19-174.
Mori K, Kitazawa R, Kondo T, Maeda S, Yamaguchi A, Kitazawa S, 2006. Modulation of mouse RANKL gene expression by Runx2 and PKA pathway. J Cell Biochem 98: 1629-1644.
Mountziaris PM, Mikos AG. 2008. Modulation of the inflammatory response for enhanced bone tissue regeneration. Tissue Eng B Rev 14(2): 179-186.
Mundlos S, Olsen BR. 1997a. Heritable diseases of the skeleton. Part I: Molecular insights into skeletal development-transcription factors and signalling pathways. FASEB J 11: 125-132.
Mundlos S, Olsen BR. 1997b. Heritable diseases of the skeleton. Part II: Molecular insights into skeletal development-matrix components and their homeostasis. FASEB J 11: 227-233.
Nakamura H, Sato G, Hirata A, Yamamoto T. 2004. Immunolocalization of matrix metalloproteinase-13 on bone surface under osteoclasts in rat tibia. Bone 34: 48-56.
Nakamura A, Ly C, Cipetic M, Sims NA, Vieusseux J, Kartsogiannis V, Bouralexis S, Saleh H, Zhou H, Price JT, martin TJ, Ng KW, Gillespie MT, Quinn JM. 2007. Osteoclast inhibitory lectin (OCIL) inhibits osteoblast differentiation and function in vitro. Bone 40: 305-315.
Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, de Crombrugghe B. 2002. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108: 17-29.
Nefussi JR, Sautier JM, Nicolas V, Forest N. 1991. How osteoblasts become osteocytes: a decreasing matrix forming process. J Biol Buccale 19: 75-82.
Nemeth GG, Bolander ME, Martin GR. 1988. Growth factors and their role in wound and fracture healing. Prog Clin Biol Res 266:1-17.
51
Neumann E, Schett G. 2007. Bone metabolism: molecular mechanisms. Z Rheumatol 66: 286-289.
Niehrs C. 2006. Function and biological roles of the Dickkopf family of Wnt modulators. Oncogene 25(57): 7469-7481.
Noble BS, Stevens H, Loveridge N, Reeve J. 1997. Identification of apoptotic changes in osteocytes in normal and pathological human bone. Bone 20: 273-282.
Nomura S, Wills AJ, Edwards DR, Heath JK, Hogan BL. 1989. Expression of genes for non-collagenous proteins during embryonic bone formation. Connect Tissue Res 21: 31-39.
Olsen DR, Peltonen J, Jaakkola S, Chu ML, Uitto J. 1989. Collagen gene expression by cultured human skin fibroblasts. Abundant steady-state levels of type VI procollagen messenger RNAs. J Clin Invest 83: 791-795.
Oreffo RO, Bonewald L, Kukita A, Garrett IR, Seyedin SM, Rosen D, Mundy GR. 1990. Inhibitory effects of the bone-derived growth factors osteoinductive factor and transforming growth factor-beta on isolated osteoclasts. Endocrinology 126: 3069-3075.
Ortega N, Behonick D, Stickens D. 2003. How proteases regulate bone morphogenesis. Ann N Y Acad Sci 995, 109-116.
Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR, Stamp GW, Beddinton RS, Mundlos S, Olsen BR, Selby PB, Owen MJ. 1997. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 89: 765-771.
Oursler MJ. 1994. Osteoblast synthesis and secretion and activation of latent transforming growth factor β. J Bone Miner Res 9: 443-452.
Palmiter RD, Brinster RL, Hammer RE, Trumbauer ME, Rosenfeld MG, Birnberg NC and Evans RM. 1982. Dramatic growth of mice that develop from eggs micro-injected with metallothioneine-growth hormone fusion genes. Nature 300: 611-615.
Palumbo C, Palazzini S, Zappe D, Marotti G. 1990a. Osteocyte differentiation in the tibia of newborn rabbits: an ultrastructural study of the formation of cytoplasmic processes. Acta Anat 137: 350-358.
Palumbo C, Palazinni S, Marotti G. 1990b. Morphological study of intercellular junctions during osteocyte differentiation. Bone 11, 401-406.
Pape HC, Giannoudis PV, Grimme K, van Griensven M, Krettek C. 2002. Effects of intramedullary femoral fracture fixation: what is the impact of experimental studies in regards to the clinical knowledge? Shock 18(4): 291-300.
Parfitt AM. 1990. Bone-forming cells in clinical conditions. In: Hall BK, editor. Bone, Vol. 1: the osteoblast and osteocyte. Boca Raton, FL: Telford Press and CRC Press. 351-429.
Pavlin D, Lichtler AC, Bedalov A, Kream BE, Harrison JR, Thomas HF, Gronowicz GA, Clark SH, Woody CO, Rowe DW 1992. Differential utilization of regulatory domains within the alpha 1(I) collagen promoter in osseous and fibroblastic cells. J Cell Biol116:227–236.
52
Pelissier P, Bareille R, Masquelet A, Pelissier SM, Amedee J. 2003. Induced membranes secrete growth factors including vascular and osteoinductive factors and could stimulate bone regeneration. J Bone Miner Res 18(2): S212-S213.
Peng HR, Wright V, Usas A, Gearhart B, Shen HC, Cummins J, Huard J, 2002. Synergistic enhancement of bone formation and healing b y stem cell-expressed VEGF and bone morphogenetic protiein-4. J Clin Invest 110(6): 751-759.
Perren SM, Huggler A, Russenberger S. 1969. Cortical bone healing. Acta Orthop Scand Suppl 125.
Perren SM. 1979. Physical and biological aspects of fracture healing with special reference to internal fixation. Clin Orthop 138: 175-196.
Perren SM. 2002. Evolution of the internal fixation of long bone fracture. The scientific basis of biological internal fixation: choosing a new balance between stability and biology. J Bone Joint Surg Br 84(8): 1093-1110.
Petit I, Szyper-Kravitz M, Nagler A, Lahav M, Peled A, Habler L, Ponomaryov T, Taichman RS, Arenzana-Seisdedos F, Fujii N, Sandbank J, Zipori D, Lapidot T. 2002. G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4. Nat Immunol 3: 687-694.
Pfeilschifter J, Wolf O, Naumann A, Minne HW, Mundy GR, Ziegler R. 1990. Chemotactic response of osteoblast-like cells to transforming growth factor β. J Bone Miner Res 5: 825-830.
Pihlajaniemi T, McKeon J, Gay S, Gay R, DeWet WJ, Myers JC, Prockop DJ. 1989. Increased expression of the gene for the pro-alpha-1(IV) chain of basement-membrane procollagen in cultured skin fibroblasts from two variants of osteogenesis imperfect. Biochem J 257: 439-445.
Ponomaryov T, Peled A, Petit I, Taichman RS, Habler L, Sandbank J, Arenzana-Seisdedos F, Maqerus A, Caruz A, Fujii N, Nagler A, Lahav M, Szyper-Kravitz M, Zipori D, Lapidot T. 2000. Induction of the chemokine stromal-derived factor-1 following DNA damage improves human stem cell function. J Clin Invest 106, 1331-1339.
Poplich LS, Slafeld SL, Rueger DC, Tucker M, Cook SD. 1997. Critical and noncritical size defects healing with osteogenic protein-1. Trans Orthop Res Soc 22: 600. Abstract
Porte D, Tuckermann J, Becker M, Baumann B, Teurich S, Higgins T, Owen MJ, Schorpp-Kistner M, Angel P. 1999. Both AP-1 and Cbfa-1-like factors are required for the induction of interstitial collagenase by parathyroid hormone. Oncogene 18: 667-678.
Probst A and Spiegel HU. 1997. Cellular mechanisms of bone repair. J Invest Surg. 10: 77-86.
Proff P, Romer P. 2009. The molecular mechanism behind bone remodeling: A review. Clin Oral Invest 13: 355-362.
Puzas JE, Ishibe M. 1992. Osteoblast/osteoclast coupling. In: Rifkin BR, Gay CV (eds.) Biology and Physiology of the Osteoclast. CRC Press, Boca Ratom, FL, USA, pp. 337-356.
53
Rahn BA. 2002. Bone healing: histologic and physiologic concepts. In: Fackelman GE, editor. Bone in clinical orthopaedics. Stuttgart, NY: Thieme. 287-326.
Ramirez F, Di Liberto M. 1990. Complex and diversified regulatory programs control the expression of vertebrate collagen genes. FASEB J 4: 1616-1623.
Reddi AH. 1994. Cartilage morphogenesis role of bone and cartilage morphogenetic proteins, homeobox genes and extracellular matrix. Matrix Biol 14: 599.
Reinholt FP, Hultenby K, Oldberg A, Heinegard D. 1990. Osteopontin – a possible anchor of osteoclasts to bone. Proc Natl Acad Sci USA 87: 4473-4475.
Rifkin BR, Heijl L. 1979. The occurrence of mononuclear cells at sites of osteoclastic bone resorption in experimental periodontitis. J Periodontol 50: 636-640.
Rippe RA, Lorenzen SI, Brenner DA, Breindl M. 1989. Regulatory elements in the 5’-flanking region and the first ntron contribute to transcriptional control of the mouse alpha 1 type I collagen gene. Mol Cell Biol 9: 2224-2227.
Robert Josse. 2009. Bone Biology and the Role of RANK/RANKL/OPG Pathway. Geriatrics and Aging Conference Report from the 2009 CGS Annual Scientific Meeting: Satellite Symposium 30-31.
Robey PG, Young MF, Flanders KC, Roche NS, Kondaiah P, Reddi AH, Termine JD, Sporn MB, Roberts AB. 1987. Osteoblasts synthesize and respond to transforming growth factor-type beta (TGF-beta) in vitro. J Cell Biol 105: 457-463.
Rodan GA. 1997. Mechanical loading, estrogen deficiency, and the coupling of bone formation to bone resorption. J Bone Miner Res 6: 527-530.
Rossant J, Howard L. 2002. Signaling pathways in vascular development. Annu Rev Cell Dev Biol 18: 541-573.
Rossert J, Eberspaecher H, de Cromrugghe B. 1995. Separate cis-acting DNA elements of the mouse pro-alpha 1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice. J Cell Biol 129: 1421-1432.
Rossi P, de Crombrugghe B. 1988. A nuclear factor 1 binding site mediates the transcription activation of a type I collagen promoter by transforming growth factor-beta. Cell 52: 405-414.
Rothe L, Collin-Osdoby P, Chen Y, Sunyer T, Chaudhary L, Tsay A, Goldring S, Avioli L, Osdoby P. 1998. Human osteoclasts and osteoclast-like cells synthesize and release high basal and inflammatory stimulated levels of the potent chemokine interleukin-8. Endocrinology 139(10): 4353-4363.
Rowe DW, Kream BE. 1982. Regulation of collagen synthesis in fetal rat calvaria by 1,25-dihydroxyvitamin D3. J Biol Chem 257: 8009-8015.
Rubin C, Judex S, Hadjiargyrou M. 2002. Skeletal adaptation to mechanical stimuli in the absence of formation or resorption of bone. J Musculoskelet Neuronal Interact 2: 264-267.
Sandberg MM, Aro HT, Vuorio EI. 1993. Gene expression during fracture repair. Clin Orthop 289: 292-312.
54
Sasano Y, Zhu JX, Tsubota M, Takahashi I, Onodera K, Mizoguchi I, Kagayama M. 2002. Gene expression of MMP8 and MMP13 during embryonic development of bone and cartilage in the rat mandible and hind limb. J Histochem Cytochem 50: 325-332.
Sato T, Foged NT, Delaisse JM. 1998. The migration of purified osteoclasts through collagen is inhibited by matrix metalloproteinase inhibitors. J Bone Miner Res 13: 59-66.
Schaller S, Henriksen K, Sveigaard C, Heegaard AM, Helix N, Stahlhut M, Ovejero MC, Johansen JV, Solberg H, Andersen TL, Hougaard D, Berryman M, Shiodt CB, Sorensen BH, Lichtenberg J, Christophersen P, Foged NT, Delaisse JM, Engsig MT, Karsdal MA. 2004. The chloride channel inhibitor NS3736 prevents bone resorption in ovariectomized rats without changing bone formation. J Bone Miner Res 19: 1144-1153.
Schatzker J, Waddell J, Stoll JE. 1989. The effects of motion on the healing of cancellous bone. Clin Orthop 245: 282-287.
Schell H, Lienau J, Epari DR, Seebeck P, Exner C, Muchow S, Bragulla H, Haas NP, Duda GN. 2006. Osteoclastic activity begins early and increases over the course of bone healing. Bone 38: 547-554.
Schenk R, Willenegger H. 1967. Morphological findings in primary fracture healing: callus formation. Simp Biol Hungar 7: 75-80.
Schenk R. 1987. Cytodynamics and histodynamics of primary bone repair, in Fracture Healing (Lang JM, ed.) Churchill Livingstone, New York, 23-32.
Schindeler A, McDonald MM, Bokko P, Little DG. 2008. Bone remodelling during fracture repair: The cellular picture. Semin Cell Dev Biol 19:459-466.
Schindeler A, Liu R, Little DG. 2009. The contribution of different cell lineages to bone repair: exploring a role for muscle stem cells. Differentiation 77: 12-18.
Schmidt A, Rossi P, de Crombrugghe B. 1986. Transcriptional control of the mouse α2(I) collagen gene: functional deletion analysis of the promoter and evidence for cell-specific expression. Mol Cell Biol 6: 347-354.
Schor AM, Allen TD, Canfield AE, Sloan P, Schor SL. 1990. Pericytes derived from the retinal microvasculature undergo calcification in vitro. J Cell Sci 97: 449-461.
Schweizer L, Varmus H. 2003. Wnt/Wingless signaling through beta-catenin requires the function of both LRP/Arrow and frizzled classes of receptors. BMC Cell Biol 4(4).
Selvamurugan N, Chou W-Y, Pearman AR, Pulumamti MR, Partridge NC. 1998. Parathyroid hormone regulates the rat collagenase-3 promoter in osteoblastic cells through the cooperative interaction of the activator protein-1 site and the runt domain binding sequence. J Biol Chem 273: 19647-10657.
Shapiro F. 2008. Bone development and its relation to fracture repair. The role of mesenchymal osteoblasts and surface osteoblasts. Eur Cell Mater 15: 53-76.
Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, Nguyen HQ, Wooden S, Bennett L, Boone T, Shimamoto G, DeRose M, Elliott R, Colombero A, Tan HL, Trail G, Sullivan J, Davy E, Bucay N, Renshaw-Gegg L, Hughes TM, Hill D, Pattison W, Campbell P, Sander S, Van G, Tarpley J, Derby P, Lee R, Boyle WJ. 1997.
55
Osteoprotegerin: A novel secreted protein involved in the regulation of bone density. Cell 89: 309-319.
Simon AM, Manigrasso MB, O’Connor JP. 2002. Cyclo-oxygenase 2 function is essential for bone fracture healing. J Bone Miner Res 17: 963-976.
Slack JL, Liska DJ, Bornstein P. 1991. An upstream regulatory region mediates high-level, tissue-specific expression of the human α1(I) collagen gene in transgenic mice. Mol Cell Biol 11: 2066-2074.
Somerman MJ, Prince CW, Sauk JJ, Foster RA, Butler WT. 1987. Mechanism of fibroblast attachment to bone extracellular matrix: role of a 44 kilodalton bone phosphoprotein. J Bone Mineral Res 2: 259-265.
Sporn MB, Roberts AB. 1989. Transforming growth factor-beta. Multiple actions and potential clinical applications. JAMA 262: 938-941.
St-Jacques B, Hammerschmidt M, McMahon AP. 1999. Indian hedgehog signalling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev 13: 2072-2086.
Steinberg B, Singh IJ, Mitchell OG. 1981. The effects of cold-stress, hibernation and prolonged inactivity on bone dynamics in the Golden Hamster, Mesodricetus auratus. J Morph 167: 43-51.
Sternlight, MD and Werb Z. 1999. Matrix metalloproteinases. In Guidebook to the Extracellular Matrix, Anchor and Adhesion Proteins, 519-538. Sambrook and Tooze (Oxford University Press). London.
Stickens D, Behonick DJ, Ortega N, Heyer B, Hartenstein B, Yu Y, Fosang AJ, Schorpp-Kistner M, Angel P, Werb Z, 2004. Altered endochondral bone development in matrix metalloproteinase 13-deficient mice, Development 131: 5883-5895.
Strong DD, Beachler AL, Wergedal JE, Linkhart TA. 1991. Insulin-like growth factor II and transforming growth factor beta regulate collagen expression in human osteoblast-like cells in vitro. J Bone Miner Res 6: 15-23.
Suda T, Nakamura I, Jimi E, Takahashi N. 1997. Regulation of osteoclast function. J Bone Miner Res 12: 869-879.
Takamatsu Y, Simmons PJ, Moore RJ, Morris HA, To LB, Levesque JP. 1998. Osteoclast-mediated bone resorption is stimulated during short-term administration of granulocyte colony-stimulating factor but is not responsible for hematopoietic progenitor cell mobilization. Blood 92: 3465-3473.
Takamoto M, Tsuji K, Yamashita T, Sasaki H, Yano T, Taketani Y, Komori T, Nifuji A, Noda M. 2003. Hedgehog signaling enhances core-binding factor a1 and receptor activator of nuclear factor-kappa B ligand (RANKL) gene expression in chondrocytes. J Endocrinol 177: 413-421.
Takayanagi H, Kim S, Koga T, Nishina H, Isshiki M, Yoshida H, Saiura A, Isobe M, Yokochi T, Inoue J, Wagner EF, Mak TW, Kodama T, Taniguchi T. 2002. Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev Cell 3: 889-901.
56
Takeda S, Bonnamy JP, Owen MJ, Ducy P, Karsenty G, 2001. Continuous expression of Cbfa1 in non-hypertrophic chondrocytes uncovers its ability to induce hypertrophic chondrocyte differentiation and partially rescues Cbfa1-deficient mice. Genes Dev 15: 467-481.
Tally-Ronsholdt DJ, Lajiness E, Nagodawithana K. 1995. Transforming growth factor-beta inhibition of mineralization by neonatal rat osteoblasts in monolayer and collagen gel culture. In Vitro Cell Dev Biol 31: 274-282.
Tammela T, Enholm B, Alitalo K, Paavonen K. 2005. The biology of vascular endothelial growth factors. Cardiovasc Res 65: 550-563.
Tazawa T, Hoshi K, Kawamoto S, Tanaka M, Ejiri S, Ozawa H. 2004. Osteocytic osteolysis observed in rats to which parathyroid hormone was continuously administered. J Bone Miner Metab 22(6): 524-529.
Teitelbaum SL, Ross FP. 2003. Genetic regulation of osteoclast development and function. Nature Review 4; 638-649.
Thompson Z, Miclau T, Hu D, Helms JA. 2002. A model for intramembranous ossification during fracture healing. J Orthop Res 20: 1091-1098.
Topol L, Chen W, Song H, Day TF, Yang Y. 2009. Sox9 inhibits Wnt signaling by promoting beta-catenin phosphorylation in the nucleus. J Biol Chem 284(5): 3323-3333.
Tou L, Quibria N, Alexander JM. 2001. Regulation of human Cbfa1 gene transcription in osteoblasts by selective estrogen receptor modulators (SERMs). Mol Cell Endocrinol 183: 71-79.
Tran Van P, Vignery A, Baron R. 1982. An electron microscopic study of the bone-remodeling sequence in the rat. Cell Tissue Res 225: 283-292.
Tsuji K, Ito Y, Noda M. 1998. Expression of the PEBP2 alpha A/AML3/CBFA1 gene is regulated by BMP4/7 heterodimer and its overexpression suppresses type I collagen and osteocalcin gene expression in osteoblastic and nonosteoblastic mesenchymal cells. Bone 22(2): 87-92.
Turek TJ, Bostrom MPG, Camacho N, Blake CA, Palmer R, Seeherman HJ, Wozney JM. 1997. Acceleration of bone healing in a rabbit ulnar osteotomy model with recombinant human bone morphogenic protein 2. Clin Orthop 318: 222-230.
Ueta C, Iwamoto M, Kanatani N, Yoshida C, Liu Y, Enomoto-Iwamoto M, Ohmori T, Enomoto H, Nakata K, Takada K, Kurisu K, Komori T. 2001. Skeletal malformations caused by overexpression of Cbfa1 or its dominant negative form in chondrocytes. J Cell Biol 153: 87-100.
Urist MR. 1965. Bone formation by autoinduction. Science 150: 893-899.
Ushiku C, Adams DJ, Jiang X, Wang L, Rowe DW. 2010. Long bone fracture repair in mice harboring GFP reporters for cells within the osteoblastic lineage. J Orthop Res 28(10): 1338-1347.
Usui M, Xing L, Drissi H, Zuscik M, O’Keefe R, Chen D, Boyce BF. 2008. Murine and chicken chondrocytes regulate osteoclastogenesis by producing RANKL in response to BMP2. J Bone Miner Res 23: 314-325.
57
Uusitalo H, Hiltunen A, Soderstrom M, Aro HT, Vuorio E, 2000. Expression of cathepsins B, H, K, L, and S and matrix metalloroteinases 9 and 13 during chondrocyte hypertrophy and endochondral ossification in mouse fracture callus. Calcif Tissue Int 67: 382-390.
Vainio S, Karavanova I, Jowett A, Thesleff I. 1993. Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissues during early tooth development. Cell 75: 45-85.
Visnjic D, Kalajzic I, Gronowicz G, Aguila HL, Clark SH, Lichtler AC, Rowe DW. 2001. Conditional ablation of the osteoblast lineage in Col2.3Δtk transgenic mice. J Bone Miner Res 16(12): 2222-2231.
Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ. 1996. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 273: 613-622.
Vortkamp A, Pathi S, Peretii GM, Caruso EM, Zaleske DJ, Tabin CJ. 1998. Recapitulation of signals regulating embryonic bone formation during postnatal growth and in fracture repair. Mech Dev 71, 65-76.
Vu TH, Shipley JM, Bergers G, Berger JE, Helms JA, Hanahan D, Shapiro SD, Senior RM, Werb Z. 1998. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93(3): 411-422.
Vu TH, Werb Z. 2000. Matrix metalloproteinases: effectors of development and normal physiology. Genes Dev 14: 2123-2133.
Vuorio E, de Crombrugghe B. 1990. The family of collagen genes. Annu Rev Biochem 59: 837-872.
Wang Y, Wan C, Gilbert SR, Clemens TL, 2007a. Oxygen sensing and osteogenesis. Ann N Y Acad Sci 1117: 1-11.
Wang Y, Wan C, Deng L, Liu X, Cao X, Gilbert SR, Bouxsein ML, Faugere MC, Guldberg RE, Gerstenfeld LC, Haase VH, Johnson RS, Schipani E, Clemens TL, 2007b. The hypoxia-inducible factor alpha pathway couples angiogenesis to oseogenesis during skeletal development. J Clin Invest 117: 1616-1626.
Wagner EF and Karsenty G, 2001. Genetic control of skeletal development. Curr Opin Genet Dev 11: 527-532.
Walsh MJ, LeLeiko NS, Sterling JM. 1987. Regulation of types I, III, and IV procollagen mRNA synthesis in glucocorticoid-mediated intestinal development. J Biol Chem 262: 10814-10818.
Watanabe T, Suzuya H, Onishi T, Kanai S, Kaneko M, Watanabe H, Nakagawa R, Kawano Y, Takaue Y, Kuroda Y, Talmadge JE. 2003. Effects of granulocyte colony-stimulating factor on bone metabolism during peripheral blood stem cell mobilization. Int J Hematol 77: 75-81.
Wergedal JE, Matsuyama T, Strong DD. 1992. Differentiation of normal human bone cells by transforming growth factor beta and 1,25(OH)2 Vitamin D3. Metabolism 41: 42-48.
Westendorf JJ, Kahler RA, Schroeder TM. 2004. Wnt signaling in osteoblasts and bone diseases. Gene 341: 19-39.
58
White AAI, Punjabi MM, Southwick WO. 1977. The four biomechanical stages of fracture healing. J Bone Joint Surg 59A, 188.
Witten PE. 1997. Enzyme histochemical characteristics of osteoblasts and mononucleated osteoclasts in a teleost fish with acellular bone (Oreochromis niloticus, Cichlidae). Cell Tissue Res 287: 591-599.
Witten PE, Villwock W, Peters N, Hall BK. 2000. Bone resorption and bone remodelling in juvenile carp (Cyprinus carpio). J Appl Ichthy 16: 254-261.
Wolf C, Thisse C, Stoetzel C, Thisse B, Gelinger P, Perrin-Schmitt F. 1991. The M-twist gene of Mus is expressed in subsets of mesodermal cells and is closely related to the Xenopus X-twi and the Drosophila twist genes. Dev Biol 143: 363-373.
Wong GL. 1982. Characterizations of the subpopulations of OC and OB bone cells obtained by sedimentation at unit gravity. Calcif Tissue Int 34: 67-75.
Wright E, Hargrave MR, Christiansen J, Cooper L, Kun J, Evans T, Gangadharan U, Greenfield A, Koopman P. 1995. The Sry-related gene Sox9 is expressed during chondrogenesis in mouse embryos. Nat Genet 9: 15-20.
Wu CW, Tchetina EV, Mwale F, Hasty K, Pidoux I, Reinder A, Chen J, can Wart HE, Poole AR. 2002. Proteolysis involving matrix metalloproteinase 13 (Collagenase-3) is required for chondrocyte differentiation that is associated with matrix mineralization. J Bone Miner Res 17(4): 639-651.
Yajima T, Sakakura Y, Tsuruga E, Hirai T, Ikeda Y, Fujii S, Shide N. 1999. Collagen-phagocytosing ability of periodontal osteoblasts at the bone surface. Arch Histol Cytol 62:17-25.
Yancopoulos GD, Davis S, Gale NW, Rudge JS, Wiegand SJ, Holash J. 2000. Vascular-specific growth factors and blood vessel formation. Nature 407: 551-566.
Yang Y, Topol L, Lee H, Wu J. 2003. Wnt5a and Wnt5b exhibit distinct activities in coordinating chondrocyte proliferation and differentiation. Development 130: 1003-1015.
Yasko AW, Lane JM, Fellinger EJ, Rosen V, Wozney JM, Wang EA. 1992. The healing of segmental bone defects induced by recombinant human bone morphogenetic protein (rhBMP-2): A radiographic, histological, and biomechanical study in rats. J Bone Joint Surg 74(7): 659-670.
Yasuda H, Shima N, Nakagawa N, Mochizuki SI, Yano K, Fujise N, Sato Y, Goto M, Yamaguchi K, Kuriyama M, Kanno T, Murakami A, Tsuda E, Morinaga T, Higashio K. 1998. Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): a mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology 139: 1329-1337.
Yee JA, Yan L, Dominguez JC, Allan EH, Martin TJ. 1993. Plasminogen-dependent activation of latent transforming growth factor beta by growing cultures of osteoblast-like cells. J Cell Physiol 157: 528-534.
Yin T, Li L. 2006. The stem cell niches in bone. J Clin Invest 116(5): 1195-1201.
59
Yoshida H, Hayashi S, Kunisada T, Ogawa M, Nishikawa S, Okamura H, Sudo T, Shultz LD, Nishikawa S. 1990. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 345: 442-444.
Yoshida CA, Komori T. 2005. Role of Runx proteins in chondrogenesis.Crit Rev Eukaryot Gene Expr 15(3): 243-254.
Zhang X, Bao K, Dai K. 2000. Stress-relaxation plates and the remodelling of callus and cortex under the palte in rabbits. Chin Med J (Eng) 113(9): 805-809.
Zhou YX, Xu X, Chen L, Li C, Brodie SG, Deng CX. 2000. A Pro250Arg substitution in mouse Fgfr1 causes increased expression of Cbfa1 and premature fusion of calvarial sutures. Hum Mol Genet 9: 2001-2008.
Zhou G, Zhen Q, Engin F, MUnivez E, Chen Y, Sebald E, Krakow D, Lee B. 2006. Dominance of Sox9 function over Runx2 during skeletogenesis. Proc Natl Acad Sci USA 103(50): 19004-194009.
Zou H, Niswander L. 1996. Requirement for BMP signaling in interdigital apoptosis and scale formation. Science 272: 738-741.
60
Chapter 2 Possible Role of Osteoblasts in Regulating the Initiation of
Endochondral Repair Process during Fracture Healing
2 Summary
Fracture healing is a regenerative process requiring precise coordination of a variety of
skeletal, haematopoietic, vascular and immune cells which are necessary for successful repair
process and union of damaged bone. Previously, osteoblasts have been shown to be important in
osteoclastogenesis during the remodelling phase of fracture repair. However, their role during the
initiation phase of endochondral fracture repair has been unclear. Therefore, we hypothesized
that ablation of osteoblasts would lead to a delay in the initiation of chondrogenesis and
progression of the endochondral bone formation. The objective for this project was to analyze
the various stages of endochondral repair process, in order to determine the relative progression
of the fracture repair in the absence of osteoblasts. A transgenic mouse model that expresses a
truncated form of herpes simplex virus thymidine kinase (HSV-tk) gene under the control of a
2.3-kilobase fragment of rat α1 type I collagen promoter (Col2.3∆tk; DTK), was used to drive
the expression of the gene in early differentiating osteoblasts. A nucleoside analog, ganciclovir
(GCV), was used to ablate the cells in osteogenic lineage and fracture calluses were examined at
3, 7 and 21 days post fracture. Our results show that osteoblast depletion delays the initiation of
endochondral bone repair and the process of chondrogenesis. Continuous ablation of osteoblasts
for 21 days post fracture hindered the progression of endochondral ossification at the soft callus
stage with abundant deposition of cartilage matrix at the fracture site of DTK transgenic mice.
Upon cessation of the drug treatment, the bone was able to heal uneventfully similar to the
wildtype controls.
2.1 Introduction
Fracture healing is a complex physiological process that recapitulates certain aspects of
normal skeletal development and growth. It is a regenerative event that involves the coordination
of a variety of cells, ranging from haematopoietic and immune cells within the bone marrow in
conjunction with vascular and skeletal cell precursors that are recruited to the fracture site, to
ensure proper repair of the damaged bone. Within the microstructure hierarchy of fracture repair,
61
the predominant cells involved include the chondrocytes, osteocytes, osteoblasts, and osteoclasts.
Thus understanding the interplay between these different cell types during the healing process
would be useful for improving the development of cell-based therapies.
The repair process is comprised of four overlapping phases, initiated by (1) an
inflammatory response that results in the recruitment of mesenchymal progenitor cells that
differentiate into chondrocytes, which produce cartilage and osteoblasts, the bone forming cells
(Schindeler, 2008). Thereafter, (2) the fracture is bridged by soft callus and (3) later hard callus
until mechanical instability is restored to the fracture site (Schindeler, 2008). The final stage of
fracture repair involves the (4) remodeling of the hard callus by osteoclasts, the bone resorbing
cells, and osteoblasts into the original cortical and/or trabecular bone configuration (Gerstenfeld,
2003).
During development, bone tissue is formed by two distinct ossification processes.
Intramembranous bone formation is mediated through the inner periosteal osteogenic layer with
bone made initially without the intervention of a cartilage phase (Shapiro, 2008). Most fractures
possess some level of mechanical instability and heal by the process of endochondral
ossification, which occurs in unstable regions external to the periosteum immediately adjacent to
the fracture site (Dimitriou, 2005). The eventual bridging of the hard callus across the fracture
gap provides the initial stabilization and regaining of biomechanical function.
Previous works have shown that close coordination and interplay between the different
cell types involved in fracture repair is important for successful repair process. For example,
expression of RANKL by osteoblasts coordinates bone remodeling by stimulating bone
resorption by local osteoclasts through binding of RANKL to its cellular receptor RANK on
osteoclasts (Lacey, 1998; Li, 2000; Nakagawa, 1998). This close coordination between the two
cell types is further regulated through the expression of the decoy receptor osteoprotegerin by
osteoblasts, which binds RANKL and prevents its interaction with RANK receptors present on
osteoclasts (Roodman, 2006; Udagwa, 2000).
This coordination between the different cell types that are involved in fracture healing is
further evident by the role of various matrix metalloproteinases (MMPs) that degrade most
components of the extracellular matrix. Besides participating in degradation of ECM to allow
cell migration, MMPs are involved in modulation of biologically active molecules by direct
62
cleavage and regulation of the activity of other proteases (Ortega, 2003). Two critical MMPs that
are highly expressed during endochondral ossification include MMP9 and MMP13.
Preosteoclasts and other chondroclastic cells express MMP9 during the start of
neovascularization of the cartilage anlage (Holmbeck, 1999; Zhou, 2000), and MMP13 is
expressed in the terminal hypertrophic chondrocytes and in the newly recruited osteoblasts (Vu,
1998). Together, these two MMPs account for most of the protease-dependent steps of
endochondral ossification (Engsig, 2000) and along with the angiogenic factor, VEGF, are key
regulators of the remodeling of the skeletal tissue (Ortega, 2003).
Long bone fracture has been used for many years as the model for evaluating the
induction of osteogenic and chondrogenic lineages for the initial skeletal stabilization as well as
the subsequent coordination of the osteoblast and osteoclast lineages to remodel the immature
callus back to a cortical bone (Ushiku, 2010). Although the role of osteoblasts during the
remodeling phase of fracture repair has been previously studies, their role during chondrogenesis
has not been well investigated. Few studies investigating the interplay between these two cell
populations have shown that soluble factors expressed by chondrocytes selectively promote
osteogenesis of mesenchymal stromal cells (Gerstenfeld, 2003a), and in co-culturing of
osteoblasts and chondrocytes the differentiation level of osteoblasts influenced the proliferation
and differentiation levels of chondrocytes (Nakoka, 2006). Therefore, we hypothesized that
ablation of osteoblasts would lead to a delay in the initiation of chondrogenesis and progression
of the endochondral bone formation. The objective for this project was to analyze the various
stages of endochondral repair process, in order to determine the relative progression of the
fracture repair in the absence of osteoblasts.
One method for studying the lineage relationships and cell functions of a population of
differentiating cells is the use of tissue-specific transgenic expression of herpes simplex virus
thymidine kinase (HSV-tk) to conditionally deplete a specific cell population. As such, in order
to study the role of osteoblasts during bone fracture repair, we used a transgenic mouse model
expressing the HSV-tk gene under the control of a 2.3-kilobase fragment of the rat α1 type I
collagen promoter (Col2.3∆tk; DTK), which is active in early differentiating osteoblasts.
Expression of the tk gene renders the cells susceptible to a variety of nucleoside analogs such as
GCV, which leads to ablation of cells in osteoblastic lineage. In our study, fractures were
generated in the tibia of three-month-old male DTK mice that were pretreated with GCV for two
63
weeks. After surgery, one set of DTK transgenic animals were given GCV treatment for an
additional 3, 7 and 21 days, while another set received saline treatments (Figure 4). Control
animals negative for the DTK transgene were treated with GCV for above time point as controls.
Furthermore, in order to evaluate the potential side effects of the GCV drug during the healing
process, additional untreated animals (both transgenic and wildtype) were analyzed for the same
timepoints (data not shown). Our results show that osteoblast depletion delays the initiation of
endochondral bone repair and the process of chondrogenesis, with continuous ablation of
osteoblasts for 21 days post fracture hindering the progression of endochondral ossification past
the soft callus stage fracture site of DTK transgenic mice. Upon cessation of the drug treatment,
the bone was able to heal uneventfully similar to the wildtype controls.
2.2 Methods and Materials
2.2.1 Generation of fractures:
All animal procedures were approved by the animal care committee of Hospital for Sick
Children. Tibial fracture of control and DTK transgenic mice was performed as previously
described (Chen, 2007). Briefly, after anaesthetizing the animals with isoflurane, a small incision
was made close to the left leg to expose the head of the tibia. A suture needle was used to
puncture the head of the tibia to allow the insertion of a non-corrosive metal pin in the marrow
cavity in order to stabilize the leg prior to fracture generation. The fracture was generated using a
surgical scissor after the skeletal muscles, juxtaposed to the tibia, were pulled away from the
bone to prevent unnecessary damages to this tissue. The skin was then closed using silk sutures
and metal staples and the animals were allowed to recover. After recovery, an analgesic
(ibumorphine) was administered for the first three days post surgery. The fractured left tibia and
the intact right tibia were then harvested at 3, 7 and 21 days time-points post surgery (Figure 5).
2.2.2 Mechanism of action of GCV:
The DTK transgenic mice express a truncated form of HSV-tk gene whose expression is under
the control of the rat α1 type I collagen gene promoter that restricts TK expression in early
differentiating osteoblasts. Upon treatment of animals with GCV, a nucleoside analogue of
guanosine, and a homologue of acyclovir, the thymidine kinase present in target cells
monophosphorylates the GCV drug. The drug is further phosphorylated by endogenous kinases
64
and competitively inhibits the incorporation of dGTP into DNA, resulting in inhibition of DNA
replication and cell ablation.
2.2.3 Real-time PCR:
Total RNA was isolated from the fracture callus at the 7 days time point and from intact
unfractured tibias at 3 and 7 days post-surgery. cDNAs were made from the extracted RNA
samples. Real-time PCR was performed to determine the level of osteoblastic (Type I collagen
and ALP), chondrogenic (Sox5 and Aggrecan) and MMP13 gene expression as compared with
GAPDH (a housekeeping gene control). At least 3 animals in each treatment group were
examined. PCR primers (Type I Collagen, ALP, Sox5, Aggrecan and Matrix metalloproteinase
DTK continuous GCV treated = 4n;3n. A p-value below 0.05 was considered statistically
significant.
2.3 Results
2.3.1 Pretreatment of DTK transgenic mice with GCV leads to ablation of osteoblasts
To study the role of osteoblasts during bone fracture repair DTK transgenic animals and
control mice were pretreated with GCV for two weeks prior to fracture generation. In order to
examine if the GCV pretreatment of the DTK transgenic animal was effective in ablation of the
osteoblast cell lineage, tibial fractures were collected at 3 days post fracture. Histological
analysis of safranin O stained sections of these bones showed the presence of bone lining cells at
the metaphyseal area near the site of longitudinal growth adjacent to the growth plate in the
control animals (Figure 6A’). These cells were absent in the DTK transgenic animals in both the
non-continuous and continuous GCV treated groups (Figure 6B’, C’). The effect of the GCV
treatment on osteoblasts was found to be consistent in all the time points tested (3, 7, and 21 days
post-fracture). A marked decrease in mono-nucleated bone-lining cell population was observed
around the trabecular bones with the GCV drug treatment, and the cessation of the treatment was
followed by a repopulation of these cell types around the metaphyseal trabecular niche (Figure
66
7). This finding is consistent with the original study done by Vinsnjic et al that showed a strong
expression of the thymidine kinase protein localized in osteoblasts lining the endosteal,
periosteal, and trabecular surfaces (Visnjic, 2004). Furthermore, similar to their study, transgenic
mice treated with GCV had a marked loss of bone marrow cellularity as compared to the non-
transgenic controls that showed no evidence of GCV-induced toxicity (Visnjic, 2004) (data not
shown).
2.3.2 Osteoblast depletion delays initiation of endochondral bone repair
During development, the onset of endochondral ossification begins with the condensation
of mesenchymal progenitor cells at sites of future skeletal development (Hall, 1995), a process
that is recapitulated during the early stages of fracture repair (Hiltunen, 1993) Shortly after the
condensation stage, cells in the central region of the aggregation begin to differentiate along a
cartilaginous lineage (Iwasaki, 1997) in response to growth factors and cytokines released by
platelets, inflammatory cells, and neighboring cells and tissues (Bolander, 1992; Bruder, 1994).
In order to establish a baseline for comparison of repair process at a stage when
osteoblasts have not yet been recruited to the fracture callus, fractured tibias were harvested at 7
days post surgery from GCV treated controls as well as continuous and non-continuous GCV
treated DTK mice. As previously reported (Visnjic, 2004) it was hypothesized that at 7 days
following the fracture, most of the callus would be composed of cartilage. As expected,
histological analysis of the safranin-o stained sections showed an extensive collagen matrix
production in the fractures of the wildtype controls (Figure 8D). However, there was a lack of
collagen deposition at the fracture site of continuous GCV treated transgenic mice (Figure 8F).
The growth plate collagen staining was used as an internal control and collagen matrix
production was identified by the red staining of proteoglycans at the site of fracture.
In addition, fracture calluses from a separate group of GCV-treated control and
continuous GCV-treated DTK mice were harvested at 7 day time point for RNA analysis. To
control for any contamination that might occur during the harvesting of the calluses, mainly from
the muscle tissue surrounding the fracture site that would need to be collected along with the
fracture callus, intact tibias from the same animals were also collected as a reference. The muscle
67
tissue was easily removed from these bones and the entire tibia containing the growth plate was
flash frozen in liquid nitrogen for further processing.
Since the expression of the tk transgene is under type I collagen promoter, and GCV
treatment should lead to ablation of dividing cells expressing type I collagen, the expression level
of this gene along with ALP (another early osteoblast marker) was examined in the fracture
calluses and intact tibia of both the control and DTK transgenic animals. Real-time PCR analysis
shows a decrease in the expression of these genes in the fracture callus of the continuous GCV
treated mice as compared to the wildtype controls (Figure 9).
Type I collagen is also expressed in osteocytes (Kamiya, 2001), but since these cells are
not dividing the GCV treatment should not lead to death of osteocytes in the cortical bone
(Figure 10). Furthermore, it has been previously reported that the distance between osteocytes
can be calculated (Sugawara, 2005). Thus it was assumed that there would be similar number of
osteocytes in each of the intact tibias collected, thereby setting a baseline threshold, where any
decrease in type I collagen in continuous GCV-treated transgenic mice could be hypothesized to
be due to the ablation of osteoblasts. Similar decrease in type I collagen expression was observed
in the intact tibias of the continuous GCV-treated mice as compared to the wildtype controls
(data not shown).
Other genes that were examined in the 7 day old calluses included genes involved in
chondrogenesis and cartilage matrix production (Sox5 and aggrecan, respectively), as well as
collagenase MMP13, which is expressed by hypertrophic chondrocytes and osteoblasts during
endochondral ossification. A significant decrease was seen in all above genes in the continuous
GCV-treated groups as compared to wildtype controls (Figure 9). These results, along with the
histology data suggests that the continuous ablation of osteoblasts with GCV leads to a lack of
cartilage deposition in these animals at the chondrogenic stage of fracture healing.
2.3.3 Continuous ablation of osteoblasts hinders the progression of endochondral ossification past the soft callus stage
To determine the role of osteoblasts during endochondral repair process DTK transgenic
animals were pretreated with GCV for duration of two weeks prior to fracture generation. One
set of transgenic animals was injected with GCV continuously for 21 days, whereas another set
68
of DTK animals received saline for the same duration (Figure 5). Wildtype control mice
receiving GCV for 21days were used as controls. Additionally, to examine the natural fracture
repair process in absence of GCV treatment, calluses were harvested from untreated wildtype
controls and DTK transgenic mice at 21 day time point (data not shown).
To verify the effect of GCV treatment on osteocytes, various locations in the cortical
bone were examined in the different treatment groups at all time points. Osteocytes were
identified by their elongated morphology and their parallel orientation to the long axis of cortical
bone. In the representative pictures (Figure 10) osteocytes are seen present in round to oval
shaped lacunae spaces in the bone (arrow). Adjacent lacuanes are linked by multiple small canals
known as canaliculi that house the osteocyte cell processes (Palumbo, 1990). This lacunar-
canalicular intraosseous system plays a key role in providing the bone cells with nutrients from
blood vessels and allows intercellular communication of bone cells for biophysical control
mechanisms essential to tissue development and maintenance (Shapiro, 2008). Any injury to the
bone that leads to a break of this communication system would result in the death of osteocytes
that lie in close proximity to the damaged site. This is apparent by the empty lacuanae spaces
found in the cortical bone adjacent to fracture site of the different treatment and control groups,
where the osteocytes closest to the damaged area have died off (Figure 11E).
Safranin-O staining was performed on paraffin-embedded tibial sections of 21 day old
fractures from the different treatment group for detection of osteoid and cartilage deposition at
the site of fracture (Figure 8G-I). Representative fracture were selected from each treatment
group and the red staining of cartilage at the growth plate was used a reference control. Other
than the continuous GCV-treated animals (Figure 8I), the callus of the various control and
treatment groups was primarily composed of new bone at 21 days post fracture. Multiple
condensation sites were still present in the fracture callus of continuous GCV-treated animals,
suggesting that osteoblast ablation delays the endochondral repair process.
In addition, HE staining of selected 21 day old fracture callus section of the various
treatment groups showed osteoblasts lining the periosteum or along the trabecular bone within
the interim callus of the untreated control animals (data not shown) as well as the GCV-treated
wildtype and non-continuous GCV-treated DTK mice (Figure 11D-F). Osteoblasts were
identified by their mononucleated morphology and their single array lining of the surface of
69
newly synthesized bone at the site of fracture. Active osteoblasts were identified by their
polarized orientation at sites of bone mineralization.
Endochondral bone formation was observed at the site of fracture repair on mineralized
cartilage remains as would be seen in normal metaphyseal bone formation. The initially
synthesized woven bone at the site of repair can be seen surrounded by darker staining more
organized lamellar bone that is then immediately covered with a layer of surface osteoblasts.
Higher power view of the fracture callus (Figure 11D’-F’) shows the presence of active
osteoblasts at sites of osteoid deposition (arrows). In addition, areas of osteoblast-to-osteocyte
transformation can be observed in the fracture callus as some osteoblasts have become embedded
within the bone matrix produced by neighboring osteoblasts (Figure 11E’).
To determine the level of remodeling at 21 days post fracture, sections from the various
treatment groups were stained with TRAP. Almost no TRAP staining was observed in the
continuous GCV-treated group whereas GCV-treated control animals and non-continuous GCV-
treated transgenic animals exhibited TRAP positive multinucleated cells scattered among the
newly formed trabeculae at the fracture site (Figure 12). Osteoclasts, which are a member of the
monocyte/macrophage family that involved in bone resorption during normal bone turnover as
well as in the remodeling phase of fracture repair, can be seen along the newly deposited bone.
They are multinucleated cells that lie in close proximity to the bone surface at site of bone
resorption and stain positive for tartrate-resistant acid phosphatase (TRAP).
Staining for collagen type X showed that at 7 day time point, the fracture callus of the
continuous GCV-treated DTK mice lacked the hypertrophic chondrocyte specific type X
collagen matrix production, while in control animals abundant matrix deposition was observed
(Figure 13A-C). At 21 day time point there was still an abundant amount of collagen type X
matrix present in the fracture callus of the continuous GCV-treated DTK mice (Figure 13D-F).
These data taken together with the safranin O staining showing no new bone deposition at the
fracture site, suggest that the endochondral repair process of these callus is delayed at 7 day time
point and is still in the early chondrogenic stages at the 21 time point stage.
Finally, to quantify the level of bone and cartilage deposition at the fracture site,
histomorphometric measurements were performed on safranin O stained section of 7 day and 21
day old tibial fractures of the different treatment groups (Figure 14). Tissue areas selected for
70
analysis were determined by the two farthest edges of the callus where the periosteal new bone
formation meets the adjacent cortical bone. Various bone and cartilage parameters were
examined and the data shows lower cartilage matrix synthesis in 7 day old fractures of
continuous GCV treated transgenic mice in comparison to wildtype controls (Figure 14A).A
significant decrease in new osteoid formation and a significantly higher cartilage deposition was
observed in continuous GCV-treated group as compared to the GCV-treated control animals and
non-continuous GCV-treated DTK mice at the 21 day time point (Figure 14B). Overall bone
volume (mineralized bone plus the newly synthesized osteoid) did not change across the three
treatment groups, however a significantly higher mineralized bone volume per tissue volume was
observed in the continuous GCV-treated DTK mice (Figure 14B).
2.4 Discussion
The data gathered from the 7 day continuous group taken together with how the process
of chondrogenesis progresses at the site of fracture, as observed in the 21 day fractures, suggests
that osteoblasts may play a role in the initiation and advancement of endochondral ossification.
The initiation of endochondral repair seems to be delayed, and as expected, in the absence of
osteoblasts the process does not appear to be able to advance to the next phase of hard callus
formation. One explanation for the delay and eventual cessation of this process could be because
the calcified cartilage matrix at the site of the fracture is not being resorbed to allow for the
advancement of vasculature into the fracture callus. As already mentioned, bone development
requires a complex remodeling of the extracellular matrix, which is in large part mediated by the
function of MMPs. Osteoblastic cells have been shown to express several MMPs implicated in
bone morphogenesis (Ortega, 2003), unmineralized matrix degradation (Uchida, 2001),
osteoblast and chondrocyte migration (Blavier, 1995) and cell invasion (Javed, 2005).
In addition, angiogenesis requires locally restricted extracellular proteolysis, which is
achieved by a tight balance of MMPs and MMP inhibitors. MMP2 and MMP9 are of particular
interest for angiogenesis (Bergers, 2000; Itoh, 1998). MMP9 is specially required for the
invasion of osteoclasts and endothelial cells into the mineralized hypertrophic cartilage that is
deposited within the fracture callus, whereas other MMPs, principally MMP13, are expressed in
early osteoblastic cells and required for the passage of cells through the unmineralized type I
collagen and play a role in the resorption of mineralized matrix (Itagaki, 2008). Therefore, a
71
finely tuned coordination between these various MMPs that are expressed by the different cell
types involved during fracture healing is required for successful bone development and
remodelling (Engsig, 2000). In addition, other osteoblastic factors such as TNF-α, have been
shown to regulate the expression of specific angiogenic factors and MMPs during fracture
healing (Lehmann, 2005).
In this study, the ablation of osteoblasts with GCV is accompanied by delayed
mesenchymal condensation and hypertrophic chondrocyte type X collagen matrix production at
the site of injury at 7 days post-fracture. In the absence of osteoblasts there is lower production
of the necessary MMP13 (Figure 9), which is required for the degradation of the collagen
matrix. The process of endochondral ossification was found to be delayed in the absence of
osteoblasts, where the production of soft callus was observed to occur at a time when the
wildtype fractures have already advanced to the stage of new osteoid production along with bone
remodelling. Furthermore, in the continuous GCV treatment model, there is an accumulation of
the collagen matrix at the fracture site at the 21 day time point. This can be due to the continued
ablation of osteoblasts and a lower availability of MMPs required for collagen degradation.
Furthermore, decreased osteoclastic activity was seen with continuous ablation of
osteoblasts. As osteoclasts also produce the necessary MMP9 involved in matrix degradation,
this would lead to further depletion of the MMP pools that are available for extracellular matrix
resorption. In an uneventful healing process, the necrotic dead cortical bone adjacent to the
fracture site is normally resorbed by osteoclasts. However, with continuous administration of the
GCV drug, the resulting lower osteoclastic activity due to osteoblast ablation leads not only to
unresorbed cartilage matrix, but also to decreased removal of the damaged cortical bone. This is
evident by the significantly higher mineralized bone and cartilage volume in the continuous
GCV treated mice as compared to wildtype and non-continuous GCV treated groups (Figure
14B). The activity of osteoclasts during bone repair has been previously explored using various
mouse models. Strain-dependent mouse knockout models with defective osteoclast function
show an osteopetrotic phenotype where a defect in either the regulation of osteoclast activation
(Lacey, 1998), their attachment to the bone at resorption sites (Kollet, 2006), or the secretion of
degradative enzymes (Frattini, 2000), leads to reduced bone resorption.
72
Moreover, since the longitudinal bone growth occurs at the two metaphyseal ends of the
bone, majority of the active osteoblast can be assumed to reside at the site of active
skeletogenesis. In contrast, the central diaphyseal region of the bone contains mostly quiescent
bone-lining cells that are not associated with mineralization (Ushiku, 2010). Upon damage to the
bone, the first response is seen in the population of osteoblasts present near the vicinity of the
fracture site, which become active and migrate toward the site of injury (Ushiku, 2010). In our
study, the pretreatment of the DTK transgenic mice with GCV leads to ablation of osteoblasts
throughout the tibial bone. Upon cessation of the drug at the time of fracture generation, the bone
is seen to be able to repopulate the osteoblast pool within the bone and progress toward
formation of soft callus, resorption of the deposited cartilage matrix, deposition of new osteoid as
well as exhibiting similar osteoclastic activity compared to the wildtype model. From these
observations, along with the notion that a correct homeostasis needs to be achieved for
successful recovery from pathologic conditions, it can be hypothesized that the repopulation of
the osteoblasts would occur at the site of injury in order to accelerate the healing process. Also,
from the shear number of osteoblasts seen at the site of fracture compared to the population of
pre-existing osteoblast present at the metaphyseal zone, along with the accelerated rate of the
healing process versus the rate of normal longitudinal growth, the involvement and influence of
pre-existing osteoblast toward fracture repair needs to be further studied.
Overall, from the data gather in our study, it can be argued that perhaps due to the lack of
resorption of the calcified matrix at the site of fracture, there is a lack of blood vessel invasion
and as a result the endochondral process does not advance to the hard callus stage. However,
osteoid deposition at the site of fracture site would not be possible in the absence of osteoblasts
in the continuous GCV-treated transgenic animals. It is nonetheless interesting that at the early
phase of endochondral repair process, the presence of osteoblasts (whether pre-existing or newly
differentiated osteoblasts) is required for regulating the event of chondrogenesis. This would
suggest that osteoblasts are not only required in the later stages of fracture repair as the medium
for osteoid deposition and osteoclast activation, which is necessary for remodelling, but also for
the initiation of the endochondral ossification process.
73
2.5 Figures
Figure 4: Continuous GCV Treatment Leads to Non-union of the Fractured Bone.
Radiographic analysis from GCV treated wildtype control mice (A,D,F) as well as non-continuous (B,G) and continuous (C,E,H) GCV-treated DTK mice at various time points post fracture. Arrows indicate the fracture site. At least 3 animals were generated for each treatment group. Only one animal was examined for 7 days non-continuous GCV-treated transgenic group.
74
Figure 5: In Vivo Experimental Design for Studying the Role of Osteoblasts During Fracture Repair Process.
DTK transgenic and wildtype animals were pretreated with GCV for duration of two weeks prior to fracture generation. After surgery, one set of transgenic animals was injected with GCV continuously for 3, 7 and 21 days, whereas another set of DTK animals received saline for the same duration. Wildtype littermate mice receiving GCV were used as controls.
75
Figure 6: GCV Pretreatment is Effective in Ablating Pre-existing Osteoblasts.
(A-C) Safranin O staining of metaphyseal area near the growth plate showing a marked reduction in the bone marrow cellularity with GCV pretreatment. GP: growth plate.
(A’-C’) Higher magnification images showing the ablation of bone lining cells in the DTK transgenic animals with GCV pretreatment at 3 days time point. Arrows (A’) indicate the osteoblasts lining bone near the growth plate.
76
Figure 7: GCV Treatment of the DTK Transgenic Mice Leads to a Decrease in Bone-Lining Osteoblast Population.
Quantification of mononucleated bone-lining cells for the three treatment groups at 3days (A), 7 days (B), and 21 days (C) post-fracture. The cessation of the GCV treatment leads to repopulation of the osteoblast in the fractured tibia.
Error bars represent standard deviation.
77
Figure 8: Continuous GCV Treatment Leads to a Delay in Initiation and Progression of Endochondral Ossification Process.
Safranin-O staining of fracture calluses at 3 days (A-C), 7 days (D-F) and 21 days (G-I) time points. Growth plate staining of selected sections as an internal reference control. CB: cortical bone. At 7 days post-fracture, the wildtype callus shows abundant proteoglycans deposition at the fracture site (red staining), while in the continuous GCV treated group this collagen matrix deposition is absent. At 21 days post-fracture, which traditionally marks the hard callus phase of healing, the continuous GCV treated group exhibits multiple condensation sites (red staining), while in the wildtype and non-continuous GCV treatment groups the cartilage matrix is gradually resorbed.
78
Figure 9: Matrix Metalloproteinase 13 Gene Expression is Reduced in Absence of Osteoblasts.
Real-time PCR analysis of the fracture callus at 7 days post surgery shows a decrease in osteoblastic and chondrogenic gene expression. Type I collagen (Col1), Alkaline phosphatase (ALP), MMP13, Sox5 and Aggrecan (Agcn) gene expression in fracture callus of continuous GCV-treated DTK mice is shown compared to wildtype control mice. The data is shown as fold change normalized to GAPDH housekeeping gene expression.
* is for p<0.05, error bars represent standard deviation (3 animals in each treatment group were examined).
79
Figure 10: Osteocytes are Unaffected by GCV Treatment.
Random sites of cortical bone were examined at 3 days (A-C), 7 days (D-F) or 21 days (G-I). 400X magnification of cortical bone. Osteocytes are shown with arrows.
80
Figure 11: Upon GCV Withdrawal Bones are Able to Replenish the Osteoblast Population as Early as 7 Days Post Fracture.
(A-C) 200X magnification showing osteoblasts lining along the newly synthesized bone (arrows) in wildtype GCV treated mice and their absence in the non-continuous and continuous treated animals.
(D-F) HE staining of the fracture callus and metaphyseal area (F) from the various treatment groups. 100X maganification, white box (E) shows areas of necrotic bone where osteocytes have fallen off.
(D’-F’) 400X magnification images of selected areas. Arrows point to osteoblasts, gray asterix (E’) shows areas of osteoblast-to-osteocyte transition.
Figure 12: Osteoblast Ablation Leads to a Decrease in TRAP Positive Osteoclasts During the Remodeling Stage of Fracture Healing, and the Decrease in Osteoclast Population Hinders the Healing Process.
(A-C) TRAP staining was done on 21 days post fracture sections to look at the parameters of remodeling and to identify TRAP positive cells (arrows). By 21 days post-fracture the decrease in TRAP activity leads to a reduction in osteoclast-mediate degradation of the hypertrophic chondrocyte deposited type X collagen matrix.
(A’-C’) 400X magnification images of selected areas.
82
Figure 13: In Absence of Osteoblasts There is a Lack of Collagen Type X Matrix Resorption.
Staining for type X collagen deposition at the site of fracture at 7days (A-C) and 21 days (D-F) post surgery. Type X collagen in only produced by hypertrophic chondrocytes. Hypertophic matrix remodelling is delayed in continuous GCV treated DTK mice. Arrows show sites of staining.
(A’-F’) 100X magnification images of selected areas.
83
Figure 14: Continuous Depletion of Osteoblasts Results in Unresorbed Cortical Bone and Cartilage Matrix at the Fracture Site.
Histomorphic analysis shows a significant decrease in bone regeneration and an increase in collagen deposition in DTK mice after continuous GCV treatment as compared to control groups. (A) 7 days and (B) 21 days old fractures. Measurements of bone volume (BV), mineralized volume (Md.V), osteoid volume (OV) and cartilage volume (CV) are expressed as a percentage of total callus tissue volume (TV).
* is for p<0.05, error bars represent standard deviation.
84
References
Bergers G, Brekken R, McMahon G, Vu TH, Itoh T, Tamaki K, Tanzawa K, Thorpe P, Itohara S, Werb Z and Hanahan D. 2000. Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat. Cell Biol. 2, 737-744.
Blavier L, Delaisse JM. 1995. Matrix metalloproteinases are obligatory for the migration of preosteoclasts to the developing marrow cavity of primate long bones. J Cell Sci 108: 3649-3659.
Bolander ME. 1992. Regulation of fracture repair by growth factors. Protocol Soc. Exp. Biol. Med. 200, 165-170.
Bruder SP, Fink DJ, Caplan AI. 1994. Mesenchymal stem cells in bone development, bone repair, and skeletal regeneration therapy. J Cell. Biochem. 56, 283-294.
Chen Y, Whetstone HC, Lin AC, Nadesan P, Wei Q, Poon R, and Alman BA. 2007. Beta-catenin signaling plays a disparate role in different phases of fracture repair: Implications for therapy to improve bone healing. PLoS Medicine 4 (7), 1216-1229.
Dimitriou R, Tsiridis E, Giannoudis PV. 2005. Current concepts of molecular aspects of bone healing. Injury-Int. J. of Care of Injured. 36, 1392-1404.
Engsig MT, Chen QJ, Vu TH, Pedersen AC, Therkidsen B, Lund LR, Henriksen K, Lenhard T, Foged NT, Werb Z, Delaisse JM. 2000. Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones. J Cell Biol 151: 879-890.
Frattini A, Orchard PJ, Sobacchi C, Giliani S, Abinun M, Mattsson JP, Keeling DJ, Andersson AK, Wallbrandt P, Zecca L, Notarangelo LD, Vezzoni P, Villa A. 2000. Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis. Nature Genet 25: 343-346.
Gerstenfeld LC, Barnes GL, Shea CM, Einhorn TA. 2003a. Osteogenic differentiation is selectively promoted by morphogenetic signals from chondrocytes and synergized by a nutrient rich growth environment. Connect Tissue Res 44(Suppl 1): 85-91.
Gerstenfeld LC, Cullinane DM, Barnes GL, Graves DT, Einhorn TA. 2003b. Fracture healing as a post-natal developmental process: molecular, spatial, and temporal aspects of its regulation. J Cell Biochem 88(5): 873-884.
Hall BK, Miyake T. 1995. Divide, accumulate, differentiate: cell condensation in skeletal development revisited. Int. J. Dev. Biol. 39, 881-893.
Hiltunen A, Vuorio E, and Aro HT. 1993. A standardized experimental fracture in the mouse tibia. J. Orthop. Res. 11, 305-312.
Holmbeck K, Bianco P, Caterina J, Yamada S, Kromer M, Kuznetsov SA, Mankani M, Robey PG, Poole AR, Pidoux I, Ward JM and Birkedal-Hansen H. 1999. MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover. Cell. 99, 81-92.
Itagaki T, Honma T, Takahashi I, Echigo S, and Sasano Y. 2008. Quantitative analysis and localization of mRNA transcripts of type I collagen, osteocalcin, MMP2, MMP8, and
85
MMP13 during bone healing in a rat calvarial experimental defect model. The Anatomical Record 291: 1038-1046.
Itoh T, Tanioka M, Yoshida H, Yoshioka T, Nishimoto H, Itohara S. 1998. Reduced angiogenesis and tumor progression in gelatinase A-deficient mice. Cancer Res. 58, 1048-1051.
Iwasaki M, Le AX, Helms JA. 1997. Expression of Indian hedgehog, bone morphogenetic protein 6 and gli during skeletal morphogenesis. Mech. Dev. 69, 197-202.
Javed A, Barnes GL, Pratap J, Antkowiak T, Gerstenfeld LC, van Wijnen AJ, Stein JL, Lian JB, Stein GS. 2005. Impaired intranuclear trafficking of Runx2 (AML3/CBFA1) transcription factors in breast cancer cells inhibits osteolysis in vivo. Proceedings of the National Academy of Sciences of the USA, 102, 1454-1459.
Kamiya N, Takagi M. 2001. Differential expression of dentin matrix protein 1 type I collagen and osteocalcin bone in rat developing mandibular bone. Histochemical J. 33 (9), 545-552.
Kollet O, Dar A, Shivtiel S, Kalinkovich A, Lapid K, Sztainberg Y, Tesio M, Samstein RM, Goichberg P, Spiegel A, Elson A, Lapidot T. 2006. Osteoclasts degrade endosteal component and promote mobilization of hematopoietic progenitor cells. Nature Medicine 12(6): 657-664.
Lavoie JF, Biernaskie JA, Chen Y, Bagli D, Alman B, Kaplan DR, Miller FD. 2009. Skin-derived precursors differentiate into skeletogenic cell types and contribute to bone repair. Stem Cells Dev 18(6): 893-906.
Lehmann W, Edgar CM, Wang K, Cho TJ, Barnes GL, Kakar S, Graves DT, Rueger JM, Gerstenfeld LC and Einhorn TA. 2005. Tumor necrosis factor alpha (TNF-alpha) coordinately regulates the expression of specific matrix metalloproteinases (MMPS) and angiogenic factors during fracture healing. Bone. 36 (2), 300- 10.
Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian YX, Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J, Boyle WJ. 1998. Osteoprotegerin (OPG) ligand is a cytokine that regulates osteoclasts differentiation and activation. Cell 93(2): 165-176.
Li, J, Sarosi, I, Yan, XQ, Morony, S, Capparelli, C, Tan, HL, McCabe, S, Elliott, R, Scully, S, Van, G, Kaufman, S, Juan, SC, Sun, Y, Tarpley, J, Martin, L, Christensen, K, McCabe J, Kostenuik, P, Hsu, H, Fletcher, F, Dunstan, DR, Lacey, DL, and Boyle, WJ. 2000. RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. Proc. Natl Acad. Sci. USA 97, 1566-1571.
Nakagawa, N, Kinosaki, M, Yamaguchi, K, Shima, N, Yasuda, H, Yano, K, Morinaga, T, Higashio, K. 1998. RANK is the essential signaling receptor for osteoclast differentiation factor in osteoclastogenesis. Biochem. Biophys. Res. Commun. 253, 395-400.
Ortega N, Behonick D, Stickens D. 2003. How proteases regulate bone morphogenesis. Ann. N.Y. Acad. Sci. 995, 109-116.
86
Palumbo C, Palazinni S, Marotti G. 1990. Morphological study of intercellular junctions during osteocyte differentiation. Bone 11, 401-406.
Roodman, G. D. 2006. Regulation of osteoclast differentiation. Ann N Y Acad Sci 1068, 100-109.
Schindeler A, McDonald MM, Bokko P, Little DG. 2008. Bone remodelling during fracture repair: The cellular picture. Seminars in Cell and Developmental Biology 19:459-466.
Shapiro F. 2008. Bone development and its relation to fracture repair. The role of mesenchymal osteoblasts and surface osteoblasts. Eur Cell Mater 15: 53-76.
Sugawara Y, Kamioka H, Honjo T, Tezuka K, Takano-Yamamoto T. 2005. Three-dimensional reconstruction of chick calvarial osteocytes and their cell processes using confocal microscopy. Bone. 36 (5), 877-883.
Uchida M, Shima M, Chikazu D, Fujieda A, Obara K, Suzuki H, Nagai Y, Yamato H and Kawaguchi H. 2001. Transcriptional induction of matrix metalloproteinase-13 (collagenase-3) by 1alpha,25-dihydroxyvitamin D3 in mouse osteoblastic MC3T3-E1 cells. J. Bone Miner. Res. 16, 221-230.
Udagwa, N, Takahashi, N, Yasuda, H, Misuno, A, Itoh, K, Ueno, Y, Shinki, T, Gillespie, MT, Martin, TJ, Higashio, K and Suda, T. 2000. Osteoprotegrin produced by osteoblasts is an important regulator in osteoclast development and function. Endocrinology. 141 (9), 3478-3484.
Ushiku C, Adams DJ, Jiang X, Wang L, Rowe DW. 2010. Long bone fracture repair in mice harboring GFP reporters for cells within the osteoblastic lineage. J Orthop Res 28(10): 1338-1347.
Visnjic D, Kalajzic Z, Rowe DW, Katavic V, Lorenzo J, Aguila HL. 2004. Hematopoiesis is severely altered in mice with an induced osteoblast deficiency. Blood 103(9): 3258-3264.
Vu TH, Shipley JM, Bergers G, Berger JE, Helms JA, Hanahan D, Shapiro SD, Senior RM, Werb Z. 1998. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93(3): 411-422.
Zhou YX, Xu X, Chen L, Li C, Brodie SG, Deng CX. 2000. A Pro250Arg substitution in mouse Fgfr1 causes increased expression of Cbfa1 and premature fusion of calvarial sutures. Hum Mol Genet 9: 2001-2008.
87
Chapter 3 Summary and Conclusions
Endochonral ossification is a regenerative process that is seen in most of the generated
bone traumas. Previous studies on the mechanisms of activation and differentiation have
elucidated the interplay between osteoclasts and chondrocytes during the matrix degradation
stage of bone formation, as well as the interaction between osteoblasts and osteoclasts during the
hard callus remodelling phase of bone healing. However, the relations between osteoblast and
chondrocytes have not been previously studied in vivo. Our study show that in the absence of
osteoblasts, there is a delay in the initiation of endochondral ossification process at the soft callus
formation, and an obstruction in the progression toward the hard callus formation. This can be
argued to be in part due to a lack of matrix metalloproteinase enzymes that help degrade the
cartilage matrix during the soft callus remodelling stage, which allow the invasion of vasculature
and the recruitment of osteoblastic and osteoclastic precursors. Due to the close interaction
between osteoblasts and osteoclasts, this phenotype is exacerbated with the continuous ablation
of osteoblasts, which also leads to decreased osteoclastic activity and reduced production of
necessary osteoclastic enzymes. Furthermore, our data also suggest that osteoblasts also play a
role, either directly or indirectly, during chondrogenesis.
88
Chapter 4 Future Directions
Several questions that arise from this study include:
1. What are the contributions of pre-existing osteoblasts versus the newly
differentiated osteoblasts? In this study the HSV-tk was expressed in osteoblastic
lineages under the control of the type I collagen gene promoter. Therefore, the
administration of the GCV drug resulted in ablation of both pre-existing osteoblasts and
the newly differentiated osteoblasts at the stage when they begin to express type I
collagen. Upon damage to the bone, the number of newly differentiated osteoblasts
greatly exceeds those of pre-existing osteoblasts present at the growth plate regions of the
bone. Our study shows that in the absence of pre-existing osteoblasts, the contribution of
newly differentiated osteoblast at the site of fracture is sufficient for normal endochondral
ossification process. However, in order to examine the contribution of pre-existing
osteoblasts versus the newly differentiated osteoblasts, a mouse model in which the latter
population of cells is ablated without affecting the pre-existing osteoblasts needs to be
established.
2. Can administration of exogenous sources of MMP13 or MMP9 aid in
revascularization of the fracture callus at the site of injury? The importance of these
two MMPs produced by osteoblasts and osteoclast has been already established in the
literature, and in our model the delayed ossification process with continuous osteoblast
ablation can be attributed to a decreased pool of available MMPs required for matrix
degradation. Therefore, the contribution of vasculature to the endochondral ossification
process can be studied by administration of exogenous sources of MMPs that would aid
in cartilage degradation and allow the invasion of endothelial cells into developed soft
callus.
3. What are the interactions and interplay between osteoblasts and chondrocytes that
contribute to soft callus formation? From our study the requirement of osteoblasts for
the initiation of endochondral ossification process at the stage of soft callus formation
suggest that osteoblasts play a role, either directly or indirectly, in the process of
89
chondrogenesis. The exact mechanism for this interaction is still not known and needs to
be further studied.
4. Can transplantation of stem cells capable of osteogenic/chondrogenic differentiation
rescue the phenotype seen with continuous ablation of osteoblasts? A recent study by
Lavoie and colleagues demonstrated that rodent and human foreskin-derived SKPs (Skin
Derived Precursors) are able to differentiate into ALP-positive, collagen type-I positive,
mineralizing osteoblasts, as well as into collagen type-II positive chondrocytes in culture
(Lavoie, 2009). In addition, this study showed that upon transplantation into a NOD/SCID
tibial bone fracture model, GFP-tagged rat SKPs behaved, morphogenically and
phenotypically, similar to the endogenous mesenchymal cells during bone healing and were
able to participate in bone healing process (Lavoie, 2009). To build on this study, we were
interested in whether SKPs are able to contribute functionally to fracture healing in animals
that lack the necessary osteogenic lineage cells. Sections from preliminary work on GFP-
tagged SKPs injection into fracture of GCV-treated DTK transgenic mice were used to
stain for GFP protein. Cells positive for GFP were observed at the site of fracture
(Figure15). To show SKPs are able to differentiate into osteoblasts, even in the harsh
microenvironment that is created by the ablation of osteoblasts, sections were stained for
osterix. The experiment was repeated twice, but since the antibody was found to be non-
specific, no conclusions could be made (data not shown). Staining for SKP markers are
needed to identify these cells as SKP cells. This preliminary data could suggest that the
microenvironment niche within the callus is beneficial but not necessary for the survival of
the SKPs, as was previously thought. Further optimization of the experimental design is
needed to maximize the survival and differentiation of SKPs after injection. Scaffolds may
be used to increase the chance of survival of injected cells while decreasing the spillage of
cells into surrounding tissues (muscle, skin, etc..). GFP+ SKPs and RFP+ MSCs can be
used separately or in combination in order to show and compare the degree of contribution
of each cell type to fracture healing.
90
Appendix
91
Figure 15: Fluorescent and Immunohistochemistry staining for GFP-tagged SKP-injected into fracture site of continuous GCV treated DTK mice.
Arrows point to GFP positive SKP cells within the fracture callus.
(A-B) Fluorescent staining for GFP positive SKP cells. (Panels – TopBottom) DAPI statining showing nuclei; GFP stain; Red Channel showing background autofluorescence; and merged DAPI images with Red and GFP flourescence images.
(C-E”) Immunohistochemistry staining of GFP positive cells. (C) Positive GFP staining of wound tissue. (D) Safranin O staining of the fracture site.(E) 50X magnification of the selected region in fracture area (D) showing GFP positive cells in the center (box). (E’-N) 200X magnification of negative control of the selected region.
(E’-P) 200X magnification and (E”) 400X magnification of selected area at the fracture site. GFP positive cells are shown with arrows.
92
Acknowledgements
For their helpful discussion and valuable guidance, both technically and scientifically, I
want to thank members of Kaplan/Miller lab’s SKP group, especially Jean-Francois Lavoie, Jeff