Endochondral Fracture Healing Research Articles

Rosiglitazone induces adipogenesis of both marrow and periosteum derived mesenchymal stem cells during endochondral fracture healing

BackgroundType 2 diabetes mellitus (T2DM) afflicts about six percent of the global population, and these patients suffer from a two-fold increased fracture risk. Thiazolidinediones (TZDs), including rosiglitazone, are commonly used medications in T2DM because they have a low incidence of monotherapy failure. It is known that rosiglitazone is associated with secondary osteoporosis, further increasing the fracture risk in an already susceptible population. However, it is not yet understood how rosiglitazone impacts endochondral bone healing after fracture. The aim of this study is to elucidate how rosiglitazone treatment impacts endochondral fracture healing, and how rosiglitazone influences the differentiation of skeletal stem and progenitor cells from the bone marrow and the periosteum. MethodsAn in-vivo mouse femur fracture model was employed to evaluate differences in fracture healing between mice treated with and without rosiglitazone chow. Fracture healing was assessed with histology and micro computed tomography (μCT). In-vitro assays utilized isolated mouse bone marrow stromal cells and periosteal cells to investigate how rosiglitazone impacts the osteogenic capability and adipogenicity of these cells. ResultsThe in-vivo mouse femur fracture model showed that fracture callus in mice treated with rosiglitazone had significantly more adipose content than those of control mice that did not receive rosiglitazone. In addition, μCT analysis showed that rosiglitazone treated mice had significantly greater bone volume, but overall greater porosity when compared to control mice. In-vitro experimentation showed significantly less osteogenesis and more adipogenesis in bone marrow derived progenitor cells that were cultured in osteogenic media. In addition, rosiglitazone treatment alone caused significant increases in adipogenesis in both bone marrow and periosteum derived cells. ConclusionRosiglitazone impairs endochondral fracture healing in mice by increasing adipogenesis and decreasing osteogenesis of both bone marrow and periosteum derived skeletal progenitor cells.

What's New in Musculoskeletal Basic Science.

What's New in Musculoskeletal Basic Science.

Locally delivered adjuvant biofilm-penetrating antibiotics rescue impaired endochondral fracture healing caused by MRSA infection.

Infection is a devastating complication following an open fracture. We investigated whether local rifampin-loaded hydrogel can combat infection and improve healing in a murine model of methicillin-resistant Staphylococcus aureus (MRSA) osteomyelitis. A transverse fracture was made at the tibia midshaft of C57BL/6J mice aged 10-12 weeks and stabilized with an intramedullary pin. A total of 1 × 106 colony-forming units (CFU) of MRSA was inoculated. A collagen-based hydrogel containing low-dose (60 μg) and high-dose (300 μg) rifampin was applied before closure. Postoperative treatment response was assessed through bacterial CFU counts from tissue and hardware, tibial radiographs and microcomputed tomography (μCT), immunohistochemistry, and histological analyses. All untreatedMRSA-infected fractures progressed to nonunion by 28 days with profuse MRSA colonization. Infected fractures demonstrated decreased soft callus formation on safranin O stain compared to controls. Areas of dense interleukin-1β stain were associated with poor callus formation. High-dose rifampin hydrogels reduced the average MRSA load in tissue (p < 0.0001) and implants (p = 0.041). Low-dose rifampin hydrogels reduced tissue bacterial load by 50% (p = 0.021). Among sterile models, 88% achieved union compared to 0% of those infected. Mean radiographic union scale in tibia scores improved from 6 to 8.7 with high-dose rifampin hydrogel (p = 0.024) and to 10 with combination local/systemic rifampin therapy (p < 0.0001). μCT demonstrated reactive bone formation in MRSA infection. Histology demonstrated restored fracture healing with bacterial elimination. Rifampin-loaded hydrogels suppressed osteomyelitis, prevented implant colonization, and improved healing. Systemic rifampin was more effective at eliminating infection and improving fracture healing. Further investigation into rifampin-loaded hydrogels is required to correlate these findings with clinical efficacy.

A quantitative serum biomarker of circulating collagen X effectively correlates with endochondral fracture healing.

Currently, there are no standardized methods for quantitatively measuring fracture repair. Physicians rely on subjective physical examinations and qualitative evaluation of radiographs to detect mineralized tissue. Since most fractures heal indirectly through a cartilage intermediate, these tools are limited in their diagnostic utility of early repair. Prior to converting to the bone, cartilage undergoes hypertrophic maturation, characterized by the deposition of a provisional collagen X matrix. The objective of this study was to characterize the kinetics of a novel collagen X biomarker relative to other biological measurements of fracture healing using a murine model of endochondral fracture repair in which a closed, mid-shaft tibia fracture was created using the classic drop-weight technique. Serum was collected 5 to 42 days post-fracture in male and female mice and compared to uninjured controls (n = 8-12). Collagen X in the serum was quantified using a recently validated ELISA-based bioassay ("Cxm")1 and compared to genetic and histological markers of fracture healing and inflammation. We found the Cxm biomarker reliably increased from baseline to a statistically unique peak 14 days post-fracture that then resolved to pre-fracture levels by 3 weeks following injury. The shape and timing of the Cxm curve followed the genetic and histological expression of collagen X but did not show a strong correlation with local inflammatory states. Assessment of fracture healing progress is crucial to making correct and timely clinical decisions for patients. This Cxm bioassay represents a minimally invasive, inexpensive technique that could provide reliable information on the biology of the fracture to significantly improve clinical care.

CTRP3 Regulates Endochondral Ossification and Bone Remodeling During Fracture Healing.

C1q/TNF-related protein 3 (CTRP3) is a cytokine known to regulate a variety of metabolic processes. Though previously undescribed in the context of bone regeneration, high throughput gene expression experiments in mice identified CTRP3 as one of the most highly upregulated genes in fracture callus tissue. Hypothesizing a positive regulatory role for CTRP3 in bone regeneration, we phenotyped skeletal development and fracture healing in CTRP3 knockout (KO) and CTRP3 overexpressing transgenic (TG) mice relative to wild-type (WT) control animals. CTRP3 KO mice experienced delayed endochondral fracture healing, resulting in abnormal mineral distribution, the presence of periosteal marrow compartments, and a nonunion-like state. Decreased osteoclast number was also observed in CTRP3 KO mice, whereas CTRP3 TG mice underwent accelerated callus remodeling. Gene expression profiling revealed a broad impact on osteoblast/osteoclast lineage commitment and metabolism, including arrested progression toward mature skeletal lineages in the KO group. A single systemic injection of CTRP3 protein at the time of fracture was insufficient to phenocopy the chronic TG healing response in WT mice. By associating CTRP3 levels with fracture healing progression, these data identify a novel protein family with potential therapeutic and diagnostic value. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:00-19966, 2020.

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions.

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur.

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and µCT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

Exogenous hedgehog antagonist delays but does not prevent fracture healing in young mice

Fracture healing recapitulates many aspects of developmental osteogenesis. The hedgehog (Hh) signaling pathway, essential to skeletal development, is upregulated during fracture healing, although its importance is unclear. Our goal was to assess the functional importance of Hh signaling in endochondral fracture healing. We created closed, transverse diaphyseal femur fractures in mice, stabilized with an intramedullary pin, and administered a systemic Hh inhibitor or vehicle. Because Hh pathway activation is mediated by the receptor Smoothened (Smo), we used the Smo antagonist GDC-0449 (GDC, 50mg/kg, twice daily) to target the pathway. First, in vehicle-treated 10-wk. female C57BL/6 mice we confirmed that Hh signaling was increased in fracture callus compared to intact bone, with >5-fold upregulation of target genes Ptch1 and Gli1. Additionally, using 10-wk. male and female Gli1 reporter mice, we saw a strong activation of the reporter in the osseous regions of the fracture callus 7–10days after fracture. GDC treatment significantly blunted these responses, indicating effective inhibition of fracture-induced Hh signaling in bone. Moreover, microCT analysis revealed that GDC treatment significantly reduced cancellous and cortical bone volume at non-fracture sites (tibial metaphysis and diaphysis), suggesting that the drug inhibited normal bone formation. GDC treatment had a modest effect on fracture healing, with evidence of delayed callus mineralization radiographically (significantly lower Goldberg score at day 14) and by microCT (reduced callus vBMD at 14days), and a delay in the recovery of torsional rotation to normal (elevated rotation-at-peak torque at 21days). On the other hand, GDC treatment did not inhibit qPCR or morphological measures of chondrogenesis or angiogenesis, and did not impair the recovery of failure torque (at day 14 or 21), a measure of biomechanical competence. In summary, GDC treatment inhibited Hh signaling, which delayed but did not prevent fracture healing in young mice. We conclude that Hh signaling is strongly induced after fracture and may play a role in early callus mineralization, although it does not appear to be required for eventual healing.

Evaluation of high-resolution In Vivo MRI for longitudinal analysis of endochondral fracture healing in mice.

Mice are extensively used for experimental bone-healing studies. However, there are few established nondestructive in vivo techniques for longitudinal fracture-healing analysis in mice, including in vivo micro-computed tomography (μCT) and radiography. Importantly, none of the established methods can discriminate between non-mineralized fibrous tissue and cartilage in the soft fracture callus. Therefore, the objective was to establish high-resolution in vivo magnetic resonance imaging (MRI) for the longitudinal assessment of soft callus formation during bone healing in mice. C57BL/6J mice received a femur osteotomy stabilized using an external fixator and were randomly assigned to five groups. Group 1 mice were scanned three times longitudinally during fracture healing using an optimized MRI scanning protocol to establish an algorithm to characterize the different fracture-callus tissues. Mice of groups 2–4 were scanned once on day 10, 14 or 21, respectively, euthanized after scanning and their femurs subjected to ex vivo μCT and histomorphometric analysis to compare the data assessed by MRI with μCT and histology. Control group 5 mice were not scanned. After 28 days, mice of groups 1 and 5 were euthanized and the fracture-healing outcome was evaluated by bending-test, μCT and histology to determine whether the repeated anesthesia, handling and the MRI measurements themselves influenced fracture healing. The callus-tissue values determined by MRI were mostly comparable to those obtained by μCT and histomorphometric analysis. However, at time points characterized by small relative bone or cartilage areas, MRI measurements were weakly comparable to histomorphometric data, possibly due to the inferior spatial resolution. Importantly, at the early and intermediate phases of healing, cartilage and fibrous-tissue values obtained by MRI were highly accurate. Furthermore, repeated anesthesia, handling and MRI scans did not impact bone healing. Therefore, we demonstrated the feasibility of high-resolution in vivo MRI for longitudinal assessment of soft callus formation during murine endochondral fracture healing.

Effect of osteoporosis medications on fracture healing.

Antiosteoporotic medications are often used to concurrently treat a patient's fragility fractures and underlying osteoporosis. This review evaluates the existing literature from animal and clinical models to determine these drugs' effects on fracture healing. The data suggest that these medications may enhance bone healing, yet more thorough prospective studies are warranted. Pharmacologic agents that influence bone remodeling are an essential component of osteoporosis management. Because many patients are first diagnosed with osteoporosis when presenting with a fragility fracture, it is critical to understand how osteoporotic medications influence fracture healing. Vitamin D and its analogs are essential for the mineralization of the callus and may also play a role in callus formation and remodeling that enhances biomechanical strength. In animal models, antiresorptive medications, including bisphosphonates, denosumab, calcitonin, estrogen, and raloxifene, do not impede endochondral fracture healing but may delay repair due to impaired remodeling. Although bisphosphonates and denosumab delay callus remodeling, they increase callus volume and result in unaltered biomechanical properties. Calcitonin increases cartilage formation and callus maturation, resulting in improved biomechanical properties. Parathyroid hormone, an anabolic agent, has demonstrated promise in animal models, resulting in accelerated healing with increased callus volume and density, more rapid remodeling to mature bone, and improved biomechanical properties. Clinical data with parathyroid hormone have demonstrated enhanced healing in distal radius and pelvic fractures as well as postoperatively following spine surgery. Strontium ranelate, which may have both antiresorptive and anabolic properties, affects fracture healing differently in normal and osteoporotic bone. While there is no effect in normal bone, in osteoporotic bone, strontium ranelate increases callus bone formation, maturity, and mineralization; forms greater and denser trabeculae; and improves biomechanical properties. Further clinical studies with these medications are needed to fully understand their effects on fracture healing in order to simultaneously treat fragility fractures and underlying osteoporosis.

Bmp2 conditional knockout in osteoblasts and endothelial cells does not impair bone formation after injury or mechanical loading in adult mice

Post-natal osteogenesis after mechanical trauma or stimulus occurs through either endochondral healing, intramembranous healing or lamellar bone formation. Bone morphogenetic protein 2 (BMP2) is up-regulated in each of these osteogenic processes and is expressed by a variety of cells including osteoblasts and vascular cells. It is known that genetic knockout of Bmp2 in all cells or in osteo-chondroprogenitor cells completely abrogates endochondral healing after full fracture. However, the importance of BMP2 from differentiated osteoblasts and endothelial cells is not known. Moreover, the importance of BMP2 in non-endochondral bone formation such as intramembranous healing or lamellar bone formation is not known. Using inducible and tissue-specific Cre-lox mediated targeting of Bmp2 in adult (10–24week old) mice, we assessed the role of BMP2 expression globally, by osteoblasts, and by vascular endothelial cells in endochondral healing, intramembranous healing and lamellar bone formation. These three osteogenic processes were modeled using full femur fracture, ulnar stress fracture, and ulnar non-damaging cyclic loading, respectively. Our results confirmed the requirement of BMP2 for endochondral fracture healing, as mice in which Bmp2 was knocked out in all cells prior to fracture failed to form a callus. Targeted deletion of Bmp2 in osteoblasts (osterix-expressing) or vascular endothelial cells (vascular endothelial cadherin-expressing) did not impact fracture healing in any way. Regarding non-endochondral bone formation, we found that BMP2 is largely dispensable for intramembranous bone formation after stress fracture and also not required for lamellar bone formation induced by mechanical loading. Taken together our results indicate that osteoblasts and endothelial cells are not a critical source of BMP2 in endochondral fracture healing, and that non-endochondral bone formation in the adult mouse is not as critically dependent on BMP2.

Endochondral fracture healing with external fixation in the Sost knockout mouse results in earlier fibrocartilage callus removal and increased bone volume fraction and strength

Sclerostin deficiency, via genetic knockout or anti-Sclerostin antibody treatment, has been shown to cause increased bone volume, density and strength of calluses following endochondral bone healing. However, there is limited data on the effect of Sclerostin deficiency on the formative early stage of fibrocartilage (non-bony tissue) formation and removal. In this study we extensively investigate the early fibrocartilage callus. Closed tibial fractures were performed on Sost−/− mice and age-matched wild type (C57Bl/6J) controls and assessed at multiple early time points (7, 10 and 14days), as well as at 28days post-fracture after bony union. External fixation was utilized, avoiding internal pinning and minimizing differences in stability stiffness, a variable that has confounded previous research in this area.Normal endochondral ossification progressed in wild type and Sost−/− mice with equivalent volumes of fibrocartilage formed at early day 7 and day 10 time points, and bony union in both genotypes by day 28. There were no significant differences in rate of bony union; however there were significant increases in fibrocartilage removal from the Sost−/− fracture calluses at day 14 suggesting earlier progression of endochondral healing. Earlier bone formation was seen in Sost−/− calluses over wild type with greater bone volume at day 10 (221%, p<0.01). The resultant Sost−/− united bony calluses at day 28 had increased bone volume fraction compared to wild type calluses (24%, p<0.05), and the strength of the fractured Sost−/− tibiae was greater than that that of wild type fractured tibiae.In summary, bony union was not altered by Sclerostin deficiency in externally-fixed closed tibial fractures, but fibrocartilage removal was enhanced and the resultant united bony calluses had increased bone fraction and increased strength.

Enhancement of Long Bone Fracture Healing by Local Soft Tissue Compression1

Endochondral fracture healing, the process in which callus bridges a fracture, can be enhanced using a brace with a deforming element. This deforming element acts to locally increase pressure at the fracture site. In this paper, we describe a bracing device, which has the capability of controlling blood flow in targeted regions of an extremity. Controlling the blood flow around a fracture site induces a mechanism that enhances fracture healing. We hypothesize that, since local oxygen tension is lowered by means of controlling the blood flow at the fracture site, fracture healing is accelerated and bony union is more likely. Using the results of several previous studies, we will show that increased mechanical pressure in the soft tissues over the fracture site enhances fracture healing.

Mice Lacking Pten in Osteoblasts Have Improved Intramembranous and Late Endochondral Fracture Healing

The failure of an osseous fracture to heal (development of a non-union) is a common and debilitating clinical problem. Mice lacking the tumor suppressor Pten in osteoblasts have dramatic and progressive increases in bone volume and density throughout life. Since fracture healing is a recapitulation of bone development, we investigated the process of fracture healing in mice lacking Pten in osteoblasts (Ocn-cretg/+;Ptenflox/flox). Mid-diaphyseal femoral fractures induced in wild-type and Ocn-cretg/+;Ptenflox/flox mice were studied via micro-computed tomography (µCT) scans, biomechanical testing, histological and histomorphometric analysis, and protein expression analysis. Ocn-cretg/+;Ptenflox/flox mice had significantly stiffer and stronger intact bones relative to controls in all cohorts. They also had significantly stiffer healing bones at day 28 post-fracture (PF) and significantly stronger healing bones at days 14, 21, and 28 PF. At day 7 PF, the proximal and distal ends of the Pten mutant calluses were more ossified. By day 28 PF, Pten mutants had larger and more mineralized calluses. Pten mutants had improved intramembranous bone formation during healing originating from the periosteum. They also had improved endochondral bone formation later in the healing process, after mature osteoblasts are present in the callus. Our results indicate that the inhibition of Pten can improve fracture healing and that the local or short-term use of commercially available Pten-inhibiting agents may have clinical application for enhancing fracture healing.

Osteoblast-targeted disruption of glucocorticoid signalling does not delay intramembranous bone healing

Objective Glucocorticoids at pharmacological doses have been shown to interfere with fracture repair. The role of endogenous glucocorticoids in fracture healing is not well understood. We examined whether endogenous glucocorticoids affect bone healing in an in vivo model of cortical defect repair. Methods Experiments were performed using a well characterised mouse model in which intracellular glucocorticoid signalling was disrupted in osteoblasts through transgenic overexpression of 11β-hydroxysteroid-dehydrogenase type 2 (11β-HSD2) under the control of a collagen type I promoter (Col2.3-11β-HSD2). Unicortical bone defects (∅0.8 mm) were created in the tibiae of 7-week-old male transgenic mice and their wild-type littermates. Repair was assessed via histomorphometry, immunohistochemistry and microcomputed tomography (micro-CT) analysis at 1–3 weeks after defect creation. Results At week 1, micro-CT images of the defect demonstrated formation of mineralized intramembranous bone which increased in volume and density by week 2. At week 3, healing of the defect was nearly complete in all animals. Analysis by histomorphometry and micro-CT revealed that repair of the bony defect was similar in Col2.3-11β-HSD2 transgenic animals and their wild-type littermates at all time-points. Conclusion Disrupting endogenous glucocorticoid signalling in mature osteoblasts did not affect intramembranous fracture healing in a tibia defect repair model. It remains to be shown whether glucocorticoid signalling has a role in endochondral fracture healing.

Visualization of Different Tissues Involved in Endochondral Ossification With Alcian Blue Hematoxylin and Orange G/Eosin Counterstain

The histologic evaluation of endochondral ossification is critical to the study of fracture healing, developmental biology, and comparative histology. A modification of Sayers' alcian blue hematoxylin staining technique is described in which a different counterstain is applied. Paraffin sections are stained in alcian blue hematoxylin for 30 min, and then placed in orange G-eosin counterstain for 1 min, 30 s. This method can differentiate cartilage, mature bone, and immature bone found in various stages of endochondral ossification and fracture callus. Increasing the hematoxylin content to 0.5% produced a more striking contrast between the bone and cartilaginous aspects of the healing fracture callus. Inclusion of orange G (instead of acid fuchsin) in the counterstain provides better demarcation of mature and immature bone. Alcian blue hematoxylin/orange G-eosin consistently stains cartilage blue, mature bone orange, and immature bone mauve and is particularly suited to the study of endochondral ossification, fracture healing, and bone remodeling. (The J Histotechnol 31:19, 2008)Submitted May 29, 2007; accepted with revisions October 1, 2007

The Science of Fracture Healing

Fracture healing is a unique repair process in which the events of endochondral and intramembranous bone formation follow a specific time sequence. There are many ways in which these events can be disrupted, leading to unsuccessful fracture healing. This article summarizes the series of cellular and molecular events that take place in the process of endochondral fracture healing, based on observations obtained from an animal model of a femoral fracture.

Microvascular invasion during endochondral ossification in experimental fractures in rats

In this study morphologic techniques have been used to detail the angiogenic response that accompanies endochondral fracture healing in a clinically relevant, reproducible rat model. In this displaced fracture, the gap fills with cartilage that later is replaced by bone, via endochondral ossification. A transient periosteal circulation, followed by a permanent medullary circulation accompany this progression. From 2 to 6 weeks, vessels grow out from the periosteal tissue and give rise to vascular buds, which abut directly onto the avascular zone corresponding to the fracture defect. From 3 weeks onwards, a second wave of vessels grows out from the marrow to the cartilage-filled fracture defect, terminating as vascular buds and loops lined by endothelial and perivascular cells. The loops and buds stain strongly for laminin but transmission electron microscopy does not demonstrate an identifiable basement membrane, pointing to a region of active extracellular matrix turnover. These vessels are intimately associated with osteoblasts and newly formed woven bone forming finger-like composite structures that protrude into the mineralized cartilage matrix with which they form a clearly demarcated interface. Invading vessels and woven bone successively replace the cartilage matrix to mediate repair. Both the vascular structures and progression of endochondral ossification observed, closely resemble those described in the normal epiphyseal growth plate, indicating that the fundamental processes are similar. However, there is a difference in the spatial orientation of cells such that the healing front in the fracture model is relatively disorganized, compared to the orderly linear array of cells at the epiphyseal growth plate.

Spatial and temporal gene expression in chondrogenesis during fracture healing and the effects of basic fibroblast growth factor

Chondrogenesis is an essential component of endochondral fracture healing, though the molecular and cellular events by which it is regulated have not been fully elucidated. In this study, we used a rat model of closed fracture healing to determine the spatial and temporal expression of genes for cartilage-specific collagens. Furthermore, to determine the effects of basic fibroblast growth factor (bFGF) on chondrogenesis in fracture healing, we injected 100 μg recombinant human bFGF into the fracture site immediately after fracture. In normal calluses, pro-α1(II) collagen mRNA (COL2A1) was detected in proliferative chondrocytes beginning on day 4 after the fracture, and pro-α1(X) collagen mRNA (COL10A1) in hypertrophic chondrocytes beginning on day 7. In FGF-injected calluses, the cartilage enlarged in size significantly. On day 14, both COL2A1- and COL10A1-expressing cells were more widely distributed, and the amounts of COL2A1 and COL10A1 mRNAs were both approximately 2-fold increased when compared with uninjected fractures. Temporal patterns of expression for these genes were, however, identical to those found in normal calluses. The number of proliferating cell nuclear antigen-positive cells was increased in the non-cartilaginous area in the bFGF-injected calluses by day 4. The present molecular analyses demonstrate that a single injection of bFGF enhances the proliferation of chondroprogenitor cells in fracture callus, and thus contributes to the formation of a larger cartilage. However, maturation of chondrocytes and replacement of the cartilage by osseous tissue are not enhanced by exogenous bFGF, and this results in the prolonged cartilaginous callus phase. We conclude that, in the healing of closed fractures of long bones, exogenous bFGF has a capacity to enlarge the cartilaginous calluses, but not to induce more rapid healing.