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     

Increased fracture callus mineralization and strength in cathepsin K knockout mice

Cathepsin K (CatK) is a cysteine protease, expressed predominantly in osteoclasts (OC) which degrades demineralized bone matrix. Novel selective inhibitors of CatK are currently being developed for the treatment of postmenopausal osteoporosis. Pharmacological inhibition of CatK reduces OC resorption activity while preserving bone formation in preclinical models. Disruption of the CatK gene in mice also results in high bone mass due to impaired bone resorption and elevated formation. Here, we assessed mid-shaft femoral fracture healing in 8–10 week old CatK knock-out (KO) versus wild type (WT) mice. Fracture healing and callus formation were determined in vivo weekly via X-ray, and ex vivo at days 14, 18, 28 and 42 post-fracture by radiographic scoring, micro-computed tomography (μCT), histomorphometry and terminal mechanical four point bend strength testing. Radiological evaluation indicated accelerated bone healing and remodeling for CatK KO animals based on increased total radiographic scores that included callus opacity and bridging at days 28 and 42 post-fracture. Micro-CT based total callus volume was similar in CatK KO and WT mice at day 14. Callus size in CatK KO mice was 25% smaller than that in WT mice at day 18, statistically significant by day 28 and exhibited significantly higher mineralized tissue volume and volumetric BMD as compared to WT by day 18 onward. Osteoclast surface and osteoid surface trended higher in CatK KO calluses at all time-points and osteoblast number was also significantly increased at day 28. Increased CatK KO callus mineral density was reflected in significant increases in peak load and stiffness over WT at day 42 post-fracture. Regression analysis indicated a positive correlation (r = 0.8671; p < 0.001) between callus BMC and peak load indicating normal mineral properties in CatK KO calluses. Taken together, gene deletion of cathepsin K in mice accelerated callus size resolution, significantly increased callus mineralized mass, and improved mechanical strength as compared to wild type mice.     

Mechanical strength of fracture callus in osteopenic bone at different phases of healing

OBJECTIVES: To quantify and compare peak bending force and stiffness of fractured femurs during healing of ovariectomized (OVX) and sham-operated (SHAM) rats. DESIGN: Temporal biomechanical animal study. SETTING: Rat femurs were fractured and surgically fixed by a qualified surgeon. The inherent instability of the fixation system employed produced delayed union of the fracture. All biomechanical assessments were performed with servohydraulic test machines (Instron Inc., Canton, MA, U.S.A.; and MTS Corp., Eden Prairie, MN, U.S.A.). INTERVENTION: OVX was performed sixteen weeks before femur fracture, and the effect of OVX on healing fractures was determined. MAIN OUTCOMES: Peak bending force and stiffness of the healing femurs at four, six, and eight weeks after fracture. RESULTS: Peak bending loads of the healing fractured femurs in the OVX and SHAM animals were not significantly different. Peak bending loads for the OVX animals at four and six weeks were significantly lower than the peak load at eight weeks (p < 0.05), whereas no difference was found in the peak load with respect to time for the SHAM animals. Both SHAM and OVX animals had greater bending stiffness of the healing fractured femur after eight weeks of healing than at four weeks (p < 0.05). CONCLUSIONS: OVX is known to reduce cancellous bone mass and strength, but the effect of OVX on healing of fractures in cortical bone is controversial. This study, using a delayed-union model, found no significant differences between OVX and SHAM animals in the breaking strength of healing fractures.     

Zoledronic acid suppresses callus remodeling but enhances callus strength in an osteoporotic rat model of fracture healing

Mini-abstractIn this study, we demonstrated that the use of zoledronic acid does not impair fracture healing, but results in superior callus size and resistance at the fracture site, which could be the consequence of a lower rate of bone turnover due to its anti-catabolic effect.ObjectiveTo investigate the effect of inhibition of bone remodeling by the bisphosphonate, zoledronic acid, on callus properties in an osteoporotic rat model of fracture healing.MethodsOvariectomized (OVX) rats were randomly divided into four treatment groups (n = 24 per group): saline control (CNT); and three systemic zoledronic acid-injected groups (0.1 mg/kg), administered 1 day (ZOLD1), 1 week (ZOLW1), and 2 weeks (ZOLW2) after fracture. Rats were killed at either 6 or 12 weeks postoperatively. Postmortem analyses included radiography, microcomputed tomography, histology, histomorphometry, biomechanical tests, and nanoindentation tests.ResultsTreatment with zoledronic acid led to a significant increase in trabecular bone volume within the callus, as well as in callus resistance, compared to those in the saline control rats; delayed administration (ZOLW2) reduced intrinsic material properties, including ultimate stress and elastic modulus, and microarchitecture parameters, including bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), and connectivity density (Conn.D), compared with ZOLD1 at 12 weeks after surgery. OVX had a negative effect on the progression of endochondral ossification at 6 weeks. Zoledronic acid administration at an early stage following fracture may bind to early callus, and thus not affect subsequent callus formation and endochondral ossification, while delayed administration (ZOLW2) mildly suppresses bony callus remodeling.ConclusionThe superior results obtained with zoledronic acid (ZOLD1, ZOLW1, and ZOLW2) compared to CNT in terms of callus size and resistance could be the consequence of a lower rate of bone turnover at the fracture site due to the anti-catabolic effect of zoledronic acid. Mild suppression of callus remodeling by delayed administration did not impair the initial phase of the fracture healing process.     

Metabolic changes in the cells of the callus during fracture healing in the rat

Summary Unfixed, undecalcified sections from closed fractures of rat metatarsals have been studied by quantitative cytochemistry from the first week after fracture to late in the fracture-healing process. Alkaline phosphatase activity appeared to be related to calcification. Glucose 6-phosphate dehydrogenase activity was associated with proliferation and with calcification. Studies made with the critical electrolyte Alcian blue technique suggested that whether the ossification process is of the endochondral or membranous type might be predetermined in the relatively undifferentiated callus.     

Studies on the mechanism of callus cartilage differentiation and calcification during fracture healing

The morphologic and biochemical events during fracture callus cartilage differentiation and calcification are presented. 1. Histologic studies have demonstrated that unimmobilized fractures heal through endochondral ossification. 2. Biochemical studies have demonstrated an increase in the activities of alkaline phosphatase, enzymes involved with aerobic glucose metabolism, and lysosomal enzymes and a decrease in activities of enzymes involved with glycogen synthesis and anaerobic glycolysis. Hexosamines and hydroxyproline show a net decrease with cartilage differentiation. 3. Electron microscopic studies have demonstrated the intracellular origin and aggregation of collagen molecules, the cellular origin of matrix vesicles, and the early sites of calcification in the fracture callus.     

Evolution of substance P immunofluorescent nerves in callus tissue during fracture healing

Tibial fractures were experimentally produced in rats. The fractures were allowed to heal spontaneously. Samples of callus tissue were studied by a specific immunohistochemical method for substance P, the peptide which is found in sensory nerves and probably plays a role in nociceptive nerve transmission. The control periosteum contained a rich network of substance P immunofluorescent nerves. The early callus tissue also contained such sensory nerves, the peak frequency of which was at the end of the second week. It is proposed that the substance P-containing nerves play a protective nociceptive role during fracture healing.     

椎体压缩性骨折愈合过程中骨痂骨密度变化及其临床意义

目的观察椎体压缩性骨折骨痂骨密度变化与病程长度的关系,探讨双能X线骨密度仪术前定位陈旧性椎体骨折不愈合的可行性。方法本院2013年1月至2016年1月采用椎体后凸成形术(percutaneous kyphoplasty,PKP)治疗腰1椎体压缩性骨折(均经磁共振成像或SPECT-CT明确诊断)患者66例,年龄60~80岁,均为绝经后女性。按病程长度分为A组(对照组)、B组(新鲜骨折组)、C组(亚急性期骨折组)、D组(陈旧性骨折不愈合组)。运用方差分析观察骨密度比率(腰1椎体骨密度/腰2椎体骨密度×100%)与病程长度的关系。结果各组间年龄、身高、体重差异无统计学意义(P0.05),各组间骨密度比率差异有统计学意义(F=34.839,P0.05),在各组多重比较中,A组与B组间差异无统计学意义(P0.05),其余两组间差异有统计学意义(P0.05)。结论椎体压缩性骨折骨痂骨密度随病程延长而增加,陈旧性骨折不愈合期骨痂骨密度较邻近椎体明显增高,在条件有限的情况下,双能X线骨密度仪不失为术前定位"责任椎"的一种好方法。     

Odanacatib increases mineralized callus during fracture healing in a rabbit ulnar osteotomy model

The effects of the cathepsin K inhibitor odanacatib (ODN) on fracture healing were monitored for ～6 and 15 weeks post‐fracture in two separate studies using the unilateral transverse mid‐ulnar osteotomy model in skeletally mature female rabbits. Rabbits were pre‐treated for 3–4 weeks with vehicle (Veh), ODN (2 mg/kg, po, daily), or alendronate (ALN) (0.3 mg/kg, sc, twice‐weekly) prior to osteotomy. In Study 1, the animals were maintained on the same respective treatment for ～6 weeks. In Study 2, the animals were also continued on the same therapy or switched from Veh to ODN or ODN to Veh for 15 weeks. No treatment‐related impairment of fracture union was seen by qualitative histological assessments in the first study. Cartilage retention was detected in the calluses of ALN‐treated rabbits at week‐6, while calluses in the ODN and Veh groups contained bony tissue with significantly less residual cartilage. ODN treatment also markedly increased the number of cathepsin K‐(+) osteoclasts in the callus, indicating enhanced callus remodeling. From the second study, ex vivo DXA and pQCT confirmed that ODN treatment pre‐ and post‐osteotomy increased callus bone mineral content and bone mineral density (BMD) versus Veh (p < 0.001) and discontinuation of ODN post‐surgery returned callus BMD to Veh. Peak load of ODN‐ or ALN‐treated calluses were comparable to Veh. ODN increased callus yield load (20%, p = 0.056) and stiffness (26%, p < 0.05) versus Veh. These studies demonstrated that ODN increased mineralized callus during the early phase of fracture repair without impairing callus formation or biomechanical integrity at the fracture site. © 2015 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 34:72–80, 2016.     

脑外伤促进骨折局部骨痂快速形成机制的研究进展

合并脑外伤的骨折患者其骨痂形成速度乃至骨折愈合速度通常快于单纯骨折患者,本文综述体液因素、神经营养因子及神经肽等在脑外伤后骨折愈合加速过程中的作用。单唾液酸四己糖神经节苷脂作为脑外伤的常用治疗药物,有可能在脑外伤后骨折愈合加速这一过程中起作用。     

Determination of variations in gene expression during fracture healing

The genetic make-up and physiological state of a cell or tissue in an organism interact to determine the level at which specific genes are expressed. Identifying genes differentially expressed between 2 genetic or physiological states often gives insight into the molecular mechanisms controlled by the process in question. Various methods have been devised to identify differentially expressed genes and to quantify the expression of differentially regulated genes at the RNA or protein level. These methods are most accurate when the experimental samples are derived from highly controlled and reproducible sources, such as cultured cells. However, no simple in vitro models have been developed to study all biological processes and some are still best studied in the context of the whole organism. Using bone fracture healing as a model, we quantified the expression of 2 housekeeping and 2 regulatory genes during this complex biological process to determine the statistical parameters required to study differential gene expression in tissue samples derived from entire organisms. Our analysis shows that 5 samples in each group are needed to identify genes differentially expressed by a factor of 3 between 2 physiological or genetic states.     

Determination of variations in gene expression during fracture healing

The genetic make-up and physiological state of a cell or tissue in an organism interact to determine the level at which specific genes are expressed. Identifying genes differentially expressed between 2 genetic or physiological states often gives insight into the molecular mechanisms controlled by the process in question. Various methods have been devised to identify differentially expressed genes and to quantify the expression of differentially regulated genes at the RNA or protein level. These methods are most accurate when the experimental samples are derived from highly controlled and reproducible sources, such as cultured cells. However, no simple in vitro models have been developed to study all biological processes and some are still best studied in the context of the whole organism. Using bone fracture healing as a model, we quantified the expression of 2 housekeeping and 2 regulatory genes during this complex biological process to determine the statistical parameters required to study differential gene expression in tissue samples derived from entire organisms. Our analysis shows that 5 samples in each group are needed to identify genes differentially expressed by a factor of 3 between 2 physiological or genetic states.     

Mechanical stimulation enhanced estrogen receptor expression and callus formation in diaphyseal long bone fracture healing in ovariectomy-induced osteoporotic rats

Summary

Estrogen receptor (ER) in ovariectomy-induced osteoporotic fracture was reported to exhibit delayed expression. Mechanical stimulation enhanced ER-α expression in osteoporotic fracture callus at the tissue level. ER was also found to be required for the effectiveness of vibrational mechanical stimulation treatment in osteoporotic fracture healing.

Introduction

Estrogen receptor(ER) is involved in mechanical signal transduction in bone metabolism. Its expression was reported to be delayed in osteoporotic fracture healing. The purpose of this study was to investigate the roles played by ER during osteoporotic fracture healing enhanced with mechanical stimulation.

Methods

Ovariectomy-induced osteoporotic SD rats that received closed femoral fractures were divided into five groups, (i) SHAM, (ii) SHAM-VT, (iii) OVX, (iv) OVX-VT, and (v) OVX-VT-ICI, where VT stands for whole-body vibration treatment and ICI for ER antagonization by ICI 182,780. Callus formation and gene expression were assessed at 2, 4, and 8 weeks postfracture. In vitro osteoblastic differentiation, mineralization, and ER-α expression were assessed.

Results

The delayed ER expression was found to be enhanced by vibration treatment. Callus formation enhancement was shown by callus morphometry and micro-CT analysis. Enhancement effects by vibration were partially abolished when ER was modulated by ICI 182,780, in terms of callus formation capacity at 2–4 weeks and ER gene and protein expression at all time points. In vitro, ER expression in osteoblasts was not enhanced by VT treatment, but osteoblastic differentiation and mineralization were enhanced under estrogen-deprived condition. When osteoblastic cells were modulated by ICI 182,780, enhancement effects of VT were eliminated.

Conclusions

Vibration was able to enhance ER expression in ovariectomy-induced osteoporotic fracture healing. ER was essential in mechanical signal transduction and enhancement in callus formation effects during osteoporotic fracture healing enhanced by vibration. The enhancement of ER-α expression by mechanical stimulation was not likely to be related to the increased expression in osteoblastic cells but rather to the systemic enhancement in recruitment of ER-expressing progenitor cells through increased blood flow and neo-angiogenesis. This finding might explain the observed difference in mechanical sensitivity of osteoporotic fracture to mechanical stimulation.

     

冲击波对骨折愈合过程中骨痂生成及血管内皮生长因子和碱性成纤维细胞生长因子蛋白表达的影响

目的研究冲击波对骨折愈合过程中骨痂生成及血管内皮生长因子(VEGF)和碱性成纤维细胞生长因子(bFGF)蛋白表达的影响。方法选用新西兰大白兔60只,随机分为6组:3 d、1周、2周、3周、4周和6周组,每组10只。制备双侧桡骨横断骨折模型,左侧为对照侧,右侧为冲击波治疗侧(14 kV,2000次)。分别于术后1、2、4和6周摄X线片,并评价骨痂生成量;于术后3 d、1周、2周、3周、4周和6周取材行病理检查,观察骨折愈合情况,并做免疫组化染色检测VEGF和bFGF蛋白的表达水平。对检测结果进行统计学分析。结果X线片示术后1、2、4、6周冲击波治疗侧骨痂生成量多于对照侧,差异均有统计学意义(P＜0．01)。病理检查显示术后4周治疗侧皮质骨生成量较对照侧多且致密,差异有统计学意义(P＜0．01),术后6周治疗侧皮质骨生成量较对照侧多,差异有统计学意义(P＜0．01)。免疫组化检测显示术后1、2和3周治疗侧VEGF阳性指数均较对照侧高,差异均有统计学意义(P＜0．05);术后3 d、1周、2周和3周bFGF阳性指数治疗侧均高于对照侧,差异均有统计学意义(P＜0．05)。结论冲击波能促进骨折愈合过程中VEGF、bFGF蛋白的表达,并能促进骨痂生成,加速骨折愈合。