Microcomputed tomography imaging in a rat model of delayed union/non‐union fracture

We aimed to develop a clinically relevant delayed union/non‐union fracture model to evaluate a cell therapy intervention repair strategy. Histology, three‐dimensional (3D) microcomputed tomography (micro‐CT) imaging and mechanical testing were utilized to develop an analytical protocol for qualitative and quantitative assessment of fracture repair. An open femoral diaphyseal osteotomy, combined with periosteal diathermy and endosteal excision, was held in compression by a four pin unilateral external fixator. Three delayed union/non‐union fracture groups established at 6 weeks—(a) a control group, (b) a cell therapy group, and (c) a group receiving phosphate‐buffered saline (PBS) injection alone—were examined subsequently at 8 and 14 weeks. The histological response was combined fibrous and cartilaginous non‐unions in groups A and B with fibrous non‐unions in group C. Mineralized callus volume/total volume percentage showed no statistically significant differences between groups. Endosteal calcified tissue volume/endosteal tissue volume, at the center of the fracture site, displayed statistically significant differences between 8 and 14 weeks for cell and PBS intervention groups but not for the control group. The percentage load to failure was significantly lower in the control and cell treatment groups than in the PBS alone group. High‐resolution micro‐CT imaging provides a powerful tool to augment characterization of repair in delayed union/non‐union fractures together with outcomes such as histology and mechanical strength measurement. Accurate, nondestructive, 3D identification of mineralization progression in repairing fractures is enabled in the presence or absence of intervention strategies. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:729–736, 2008     

Are polymorphisms of molecules involved in bone healing correlated to aseptic femoral and tibial shaft non‐unions?

Fracture healing is a well‐organized process between several molecules and mediators. As known from other diseases, genetic polymorphisms may exhibit different expression patterns in these mediators. Concerning fracture healing, this may lead to an extended healing process or non‐union. We investigated the incidence of polymorphisms in patients with aseptic non‐unions after femoral and tibial shaft fractures as compared to patients with uneventful healing. Exclusion criteria were smoking, diabetes, bilateral fractures, systemic corticoid therapy, and septic non‐unions. Analysis of allele frequencies and genotype distribution of various mediators were carried out following PCR. Clinical parameters such as injury severity and in‐hospital were analyzed. Fifty patients following non‐union (group NU) were enrolled, the control group consisted of 44 patients (group H). A significant association of a PDGF haplotype and non‐unions following fracture could be observed. There was a significantly increased in‐hospital time and amount of surgical procedures in group NU. Polymorphisms within the PDGF gene seem to be a genetic risk factor for the development of non‐unions of the lower extremity following fracture. The early identification of high risk patients could result in an adapted therapeutical strategy and might contribute to a significant decrease of posttraumatic non‐unions. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1724–1731, 2011     

Distinct frequency dependent effects of whole‐body vibration on non‐fractured bone and fracture healing in mice

Low‐magnitude high‐frequency vibration (LMHFV) provokes anabolic effects in non‐fractured bone; however, in fracture healing, inconsistent results were reported and optimum vibration conditions remain unidentified. Here, we investigated frequency dependent effects of LMHFV on fracture healing. Twelve‐week‐old, female C57BL/6 mice received a femur osteotomy stabilized using an external fixator. The mice received whole‐body vibrations (20 min/day) with 0.3g peak‐to‐peak acceleration and a frequency of either 35 or 45 Hz. After 10 and 21 days, the osteotomized femurs and intact bones (contra‐lateral femurs, lumbar spine) were evaluated using bending‐testing, µ‐computed tomography, and histomorphometry. In non‐fractured trabecular bone, vibration with 35 Hz significantly increased the relative amount of bone (+28%) and the trabecular number (+29%), whereas cortical bone was not influenced. LMHFV with 45 Hz failed to provoke anabolic effects in trabecular or cortical bone. Fracture healing was not significantly influenced by whole‐body vibration with 35 Hz, whereas 45 Hz significantly reduced bone formation (?64%) and flexural rigidity (?34%) of the callus. Although the exact mechanisms remain open, our results suggest that small vibration setting changes could considerably influence LMHFV effects on bone formation in remodeling and repair, and even disrupt fracture healing, implicating caution when treating patients with impaired fracture healing. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1006–1013, 2014.

     

Disruption of thrombospondin‐2 accelerates ischemic fracture healing

Thrombospondin‐2 (TSP2) is a matricellular protein that is highly up‐regulated during fracture healing. TSP2 negatively regulates vascularity, vascular reperfusion following ischemia, and cutaneous wound healing. As well, TSP2‐null mice show increased endocortical bone formation due to an enhanced number of mesenchymal progenitor cells and show increased cortical thickness. Mice deficient in TSP2 (TSP2‐null) show an alteration in fracture healing, that is unrelated to their cortical bone phenotype, which is characterized by enhanced vascularization with a shift towards an intramembranous healing phenotype; thus, we hypothesized that there would be enhanced ischemic fracture healing in the absence of TSP2. We investigated whether an absence of TSP2 would enhance ischemic fracture healing utilizing Laser doppler, µCT and histological analysis. Ischemic tibial fractures were created in wildtype (WT) and TSP2‐null mice and harvested 10, 20, or 40 days post‐fracture. TSP2‐null mice show enhanced vascular perfusion following ischemic fracture. At day 10 post‐fracture, TSP2‐null mice have 115% greater bone volume than WT mice. This is associated with a 122% increase in vessel density, 20% increase in cell proliferation, and 15% decrease in apoptosis compared to WT. At day 20, TSP2‐null mice have 34% more bone volume, 51% greater bone volume fraction, and 37% more bone tissue mineral density than WT. By 40 days after fracture the TSP2‐null mice have a 24% increase in bone volume fraction, but other parameters show no significant differences. These findings indicate TSP2 is a negative regulator of ischemic fracture healing and that in the absence of TSP2 bone regeneration is enhanced. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 935–943, 2013     

Colchicine inhibits fracture union and reduces bone strength—in vivo study

Introduction: Recent studies have demonstrated that Colchicine (CO) prevents heterotopic ossification (HO) after total hip replacement in patients suffering from familial Mediterranean fever (FMF). Other investigators have proved that CO is an in vitro inhibitor of proliferation of osteoblasts and osteosarcoma cells, and is a non-selective mitosis inhibitor and selective inhibitor of mineralization.Methods: A double blind prospective study comprised four groups of adult rats. The left posterior tibia in each rat was fractured except in one of the control groups. The study groups were treated with CO 1 mg/kg/day 1 week before, or on the fracture day. The control groups did not receive CO treatment. Six weeks after fracture induction the groups were compared radiographically mechanically and histologically.Results: Prolonged CO treatment had a significant negative influence on fracture healing according to radiological, clinical, mechanical (p < 0.02), and pathological parameters (p < 0.0001).Conclusions: We were able to demonstrate that prolonged CO treatment reduced bone healing.     

Selective and non‐selective cyclooxygenase inhibitors delay stress fracture healing in the rat ulna

Anti‐inflammatory drugs are widely used to manage pain associated with stress fractures (SFxs), but little is known about their effects on healing of those injuries. We hypothesized that selective and non‐selective anti‐inflammatory treatments would retard the healing of SFx in the rat ulna. SFxs were created by cyclic loading of the ulna in Wistar rats. Ulnae were harvested 2, 4 or 6 weeks following loading. Rats were treated with non‐selective NSAID, ibuprofen (30 mg/kg/day); selective COX‐2 inhibition, [5,5‐dimethyl‐3‐3 (3 fluorophenyl)‐4‐(4 methylsulfonal) phenyl‐2 (5H)‐furanone] (DFU) (2.0 mg/kg/day); or the novel c5a anatagonist PMX53 (10 mg/kg/day, 4 and 6 weeks only); with appropriate vehicle as control. Quantitative histomorphometric measurements of SFx healing were undertaken. Treatment with the selective COX‐2 inhibitor, DFU, reduced the area of resorption along the fracture line at 2 weeks, without affecting bone formation at later stages. Treatment with the non‐selective, NSAID, ibuprofen decreased both bone resorption and bone formation so that there was significantly reduced length and area of remodeling and lamellar bone formation within the remodeling unit at 6 weeks after fracture. The C5a receptor antagonist PMX53 had no effect on SFx healing at 4 or 6 weeks after loading, suggesting that PMX53 would not delay SFx healing. Both selective COX‐2 inhibitors and non‐selective NSAIDs have the potential to compromise SFx healing, and should be used with caution when SFx is diagnosed or suspected. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 235–242, 2013     

Strongly enhanced levels of sclerostin during human fracture healing

Sclerostin (SOST), an antagonist of Wnt signaling, is an important negative regulator of bone formation. However, no data on the role of SOST in the human fracture healing have been published so far. This study addressed this issue. Seventy‐five patients with long bone fractures were included into the study and divided in two groups. The first group contained 69 patients with normal fracture healing. Six patients with impaired fracture healing formed the second group. Thirty‐four volunteers donated blood samples as control. Serum samples were collected over a period of 1 year following a standardized time schedule. In addition, SOST levels were measured in fracture hematoma and serum of 16 patients with bone fractures. Fracture hematoma contained significantly higher SOST concentrations compared to patient's serum. SOST levels in fracture hematoma and in patient's serum were both significantly higher than in the serum of controls. Highly elevated SOST serum concentrations were found in patients with physiological fracture healing. SOST levels were decreased in patients with impaired fracture healing. However, this difference was not statistically significant. This is the first study to provide evidence of strongly enhanced SOST levels in patients with bone fracture. The results indicate local and systemic involvement of SOST in humans during fracture healing. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1549–1555, 2012     

The impact of colony‐stimulating factor‐1 on fracture healing: An experimental study

The role of colony stimulating factor‐1 (CSF‐1) in the regulation of osteoclasts and bone remodeling suggests that CSF‐1 may also be involved in regulation of bone healing. The ability of CSF‐1 to promote healing of bone defects was tested in a rabbit model. Twenty‐four New Zeeland rabbits were included in the study. Animals were assigned to two groups: the control group (n = 12) was treated by plate fixation. The animals in the second group (n = 12) were also stabilized by conventional plating and received additionally CSF‐1 for 2 weeks systemically. Histologic, histomorphometric, and radiologic examinations were performed to evaluate the healing process at 4, 8, and 12 weeks following surgery. Animals that were treated by CSF‐1 produced a significantly higher amount of mineralized bone over the first 8 weeks after fracture compared to the control animals. Furthermore, a higher number of osteoclasts was found in CSF‐1‐treated animals within the first 8 weeks, compared to the controls. The present data emphasize for the first time the importance of CSF‐1 in the bone healing. The use of CSF‐1 in addition to conventional fixation might be a novel approach for the treatment of bone defects. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:36–41, 2009     

Systemic treatment with the sphingosine‐1‐phosphate analog FTY720 does not improve fracture healing in mice

Sphingosine‐1‐phosphate (S1P) has recently been recognized as a crucial coupling molecule of osteoclast and osteoblast activity provoking osteoanabolic effects. Targeting S1P receptors could, therefore, be a potential strategy to support bone formation in osteopenic diseases or in fracture repair. Here we investigated whether systemic treatment with the S1P analog FTY720 (Fingolimod) could improve fracture healing. Twelve‐week‐old, female C57BL/6 mice received an osteotomy of the femur, which was stabilized using an external fixator. The mice received a daily subcutaneous injection of either FTY720 (6 mg/kg) or vehicle from the third postoperative day. Fracture healing was evaluated after 10 and 21 days using biomechanical testing, µ‐computed tomography, and histomorphometry. Because FTY720 is supposed to influence osteoclast recruitment, osteoclasts were identified in the fracture callus by staining for tartrate resistant acid phosphatase (TRAP). There were no significant differences in callus mechanical properties, tissue composition and osteoclast number between the groups, suggesting that systemically applied FTY720 did not influence bone regeneration in this model of regular fracture healing. Even if further studies should test the potency of FTY720 under unfavorable healing conditions, we conclude that the effect of systemically applied FTY720 on fracture healing might be inferior compared to other anabolic treatments. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1845–1850, 2013     

BMPR1A antagonist differentially affects cartilage and bone formation during fracture healing

A soluble form of BMP receptor type 1A (mBMPR1A‐mFC) acts as an antagonist to endogenous BMPR1A and has been shown to increase bone mass in mice. The goal of this study was to examine the effects of mBMPR1A‐mFC on secondary fracture healing. Treatment consisted of 10 mg/kg intraperitoneal injections of mBMPR1A‐mFC twice weekly in male C57BL/6 mice. Treatment beginning at 1, 14, and 21 days post‐fracture assessed receptor function during endochondral bone formation, at the onset of secondary bone formation, and during coupled remodeling, respectively. Control animals received saline injections. mBMPR1A‐mFC treatment initiated on day 1 delayed cartilage maturation in the callus and resulted in large regions of fibrous tissue. Treatment initiated on day 1 also increased the amount of mineralized tissue and up‐regulated many bone‐associated genes (p = 0.002) but retarded periosteal bony bridging and impaired strength and toughness at day 35 (p < 0.035). Delaying the onset of treatment to day 14 or 21 partially mitigated these effects and produced evidence of accelerated coupled remodeling. These results indicate that inhibition of the BMPR1A‐mediated signaling has negative effects on secondary fracture healing that are differentially manifested at different stages of healing and within different cell populations. These effects are most pronounced during the endochondral period and appear to be mediated by selective inhibition of BMPRIA signaling within the periosteum. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2096–2105, 2016.     

The challenges of promoting osteogenesis in segmental bone defects and osteoporosis

Conventional clinical management of complex bone healing scenarios continues to result in 5–10% of fractures forming non‐unions. Additionally, the aging population and prevalence of osteoporosis‐related fractures necessitate the further exploration of novel ways to augment osteogenesis in this special population. This review focuses on the current clinical modalities available, and the ongoing clinical and pre‐clinical research to promote osteogenesis in segmental bone defects, delayed unions, and osteoporosis. In summary, animal models of fracture repair are often small animals as historically significant large animal models, like the dog, continue to gain favor as companion animals. Small rodents have well‐documented limitations in comparing to fracture repair in humans, and few similarities exist. Study design, number of studies, and availability of funding continue to limit large animal studies. Osteoinduction with rhBMP‐2 results in robust bone formation, although long‐term quality is scrutinized due to poor bone mineral quality. PTH 1‐34 is the only FDA approved osteo‐anabolic treatment to prevent osteoporotic fractures. Limited to 2 years of clinical use, PTH 1‐34 has further been plagued by dose‐related ambiguities and inconsistent results when applied to pathologic fractures in systematic human clinical studies. There is limited animal data of PTH 1‐34 applied locally to bone defects. Gene therapy continues to gain popularity among researchers to augment bone healing. Non‐integrating viral vectors and targeted apoptosis of genetically modified therapeutic cells is an ongoing area of research. Finally, progenitor cell therapies and the content variation of patient‐side treatments (e.g., PRP and BMAC) are being studied. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1559–1572, 2018.     

Expression and localization of ossification-related factors in fracture healing: Comparative study of experimental union and non-union models

The immunohistochemical expression and localization of some ossification-related factors, including a bone growth factor (bone morphogenetic protein, BMP), non-collagenous proteins (osteocalcin, BGP; bone sialoprotein, BSP), and type II collagen, were compared in union and non-union models of fracture healing in rats. In the union model, fracture healing was seen by day 25, whereas the non-union model did not show healing even on day 32. In both models, BMP, BGP, BSP, and type II collagen were expressed mainly in chondrocytes, with proliferating cell nuclear antigen (PCNA)-positive reaction in the soft callus. In the non-union model, the expression of BMP, BGP, BSP, type II collagen, and PCNA disappeared earlier than in the union model. In fracture healing, PCNA-positive cells expressed the ossification-related factors, these being essential for ossification.     

Induction of fracture repair by mesenchymal cells derived from human embryonic stem cells or bone marrow

Development of novel therapeutic approaches to repair fracture non‐unions remains a critical clinical necessity. We evaluated the capacity of human embryonic stem cell (hESC)‐derived mesenchymal stem/stromal cells (MSCs) to induce healing in a fracture non‐union model in rats. In addition, we placed these findings in the context of parallel studies using human bone marrow MSCs (hBM‐MSCs) or a no cell control group (n = 10–12 per group). Preliminary studies demonstrated that both for hESC‐derived MSCs and hBM‐MSCs, optimal induction of fracture healing required in vitro osteogenic differentiation of these cells. Based on biomechanical testing of fractured femurs, maximum torque, and stiffness were significantly greater in the hBM‐MSC as compared to the control group that received no cells; values for these parameters in the hESC‐derived MSC group were intermediate between the hBM‐MSC and control groups, and not significantly different from the control group. However, some evidence of fracture healing was evident by X‐ray in the hESC‐derived MSC group. Our results thus indicate that while hESC‐derived MSCs may have potential to induce fracture healing in non‐unions, hBM‐MSCs function more efficiently in this process. Additional studies are needed to further modify hESCs to achieve optimal fracture healing by these cells. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1804–1811, 2011     

BMP‐6 and BMPR‐1a are up‐regulated in the growth plate of the fractured tibia

Bone overgrowth is a known phenomenon occurring after fracture of growing long bones with possible long‐term physical consequences for affected children. Here, the physeal expression of bone morphogenetic proteins (BMPs) was investigated in a fracture‐animal model to test the hypothesis that a diaphyseal fracture stimulates the physeal expression of these known key regulators of bone formation, thus stimulating bone overgrowth. Sprague–Dawley rats (male, 4 weeks old), were subjected to a unilateral mid‐diaphyseal tibial fracture. Kinetic expression of physeal BMP‐2, ‐4, ‐6, ‐7, and BMP receptor‐1a (BMPR‐1a) was analyzed in a monthly period by quantitative real time‐polymerase chain reaction and immunohistochemistry. On Days 1, 3, 10, and 14 post‐fracture, no changes in physeal BMPs gene‐expression were detected. Twenty‐nine days post‐fracture, when the fracture was consolidated, physeal expression of BMP‐6 and BMPR‐1a was significantly upregulated in the growth plate of the fractured and contra‐lateral intact bone compared to control (p < 0.005). This study demonstrates a late role of BMP‐6 and BMPR‐1a in fracture‐induced physeal growth alterations and furthermore, may have discovered the existence of a regulatory “cross‐talk” mechanism between the lower limbs whose function could be to limit leg‐length‐discrepancies following the breakage of growing bones. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 357–363, 2013     

Bone morphogenetic proteins in clinical applications

The role of bone morphogenetic proteins (BMPs) in bone healing has been shown in numerous animal models. To date, at least 20 BMPs have been identified, some of which have been shown in vitro to stimulate the process of stem cell differentiation into osteoblasts in human and animal models. Having realized the osteoinductive properties of BMPs and having identified their genetic sequences, recombinant gene technology has been used to produce BMPs for clinical application - most commonly, as alternatives or adjuncts in the treatment of cases in which fracture healing is compromised. BMP-2 and BMP-7 are approved for clinical use in open fractures of long bones, non-unions and spinal fusion. However, despite significant evidence of their potential benefit to bone repair and regeneration in animal and preclinical studies, there is, to date, a dearth of convincing clinical trials. The purpose of this paper is to give a brief overview of BMPs and to critically review the clinical data currently available on the use of BMP-2 and BMP-7 in fracture healing.     

Can OP‐1 stimulate union in a rat model of pathological fracture post treatment for soft tissue sarcoma?

The goal of soft tissue sarcoma management in the extremities is limb preservation, often combining surgery and external beam radiation. In patients who have undergone this therapy in the thigh, pathologic fracture is a serious, late complication. Non‐union rates of 80–90% persist. No reliable biologic solution exists. A rat model combining one 18 Gy dose of radiation and diaphyseal periosteal excision reliably generates atrophic non‐union of femoral fractures. We hypothesized that augmentation with OP‐1 would increase union rate. Female Sprague‐Dawley retired breeder rats were randomized to Control, Disease (external beam radiotherapy and periosteal stripping), Control + OP‐1 (80 µg) and Disease + OP‐1 groups. Animals underwent prophylactic fixation and controlled left femur fracture. Twenty‐eight, 35, and 42 days post‐fracture were end‐points. Femora were analyzed using MicroCT, Back Scattered Electron Microscopy, and Histomorphometry. We observed a 2% union rate in the Disease groups (±OP‐1 treatment). The union rate in Control groups was 97%. MicroCT demonstrated a lack of callus volume in Disease groups. Heterotopic ossification was observed in some OP‐1 treated animals. The ineffectiveness of OP‐1 in stimulating fracture union in this model suggests the endogenous repair mechanism has been compromised beyond the capabilities of osteoinductive biologics. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1252–1263, 2014.     

Recombinant human BMP-7 effectively prevents non-union in both young and old rats.

The purpose of this study was to evaluate the influence of age on the effectiveness of rhBMP-7 treatment in a fracture with severe periosteal damage that is known to result in non-union formation. Closed stabilized femur fractures were produced in 3-month-old and 18-month-old rats. The fracture site was exposed and 2 mm of the periosteum cauterized circumferentially to impair normal fracture healing. The cauterized fracture site was immediately treated with either 100 microg rhBMP-7 (BMP-7 group), or with 25 microL of vehicle alone (control group). Fracture healing was evaluated with radiographs taken at 3 and 6 weeks. Animals were sacrificed at 3 and 6 weeks and specimens subjected to biomechanical and histological evaluation. In both age groups, none of the control animals healed throughout the 6 weeks experimental duration. All of the rhBMP-7-treated 3-month-old animals were radiographically healed at 3 weeks. In comparison, only 56% (9/16) of the rhBMP-7-treated 18-month-old animals were radiographically healed at 3 weeks. At 6 weeks, however, all of the 18-month-old rhBMP-7-treated animals had healed. Histology revealed slower healing in the 18-month-old animals. Treatment with rhBMP-7 significantly increased all of the biomechanical properties in both age groups. In the 3-month-old animals the mechanical strength approached that of the intact femur at 3 weeks, while in the 18-month-old animals this did not occur until 6 weeks. In conclusion, rhBMP-7 can effectively stimulate fracture repair in both young (3-month-old) and old (18-month-old) rats. However, the effect of rhBMP-7 on the rate of fracture healing is greater in young rats compared to old rats.