Role of mechanical loading in healing of massive bone autografts

We assessed healing of a 3.5 cm autograft transport segment, denuded of periosteum, and docked to the healthy distal femur with an intramedullary nail. We hypothesized that healing relates to proximity to the healthy distal femur and to mechanical loading patterns. Total bone area, area of new bone apposition, and quality of new bone formed in the 2 weeks after surgery, and area and degree of perfusion 16 weeks after surgery were measured as a function of proximity and loading patterns (as defined by the major and minor centroidal axes, CA). At 16 weeks, no significant differences in early bone apposition or perfusion were observed as a function of distance from the healthy distal femur. Qualitatively, bone was well perfused, both vascularly and pericellularly, and highly remodeled. When cross‐sections were pooled from distal to proximal through the docking zone and normalized for total bone area, significant differences in the amount of early proliferative woven bone were related to loading patterns. In contrast, no differences in normalized perfusion area were attributable to loading patterns. Furthermore, early bone apposition and perfusion decreased with increasing radial distance from the bone surface toward the intramedullary nail. Finally, no differences were observed in areas of resorption within the docking zone compared to baseline levels measured in the control (in bone removed to create the defect zone at the time of surgery). Interestingly, infilling of resorption spaces within docking zone specimens related significantly to predominant loading patterns, where areas within the major CA exhibited significantly more infilling. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1657–1664, 2010     

振动力学刺激对骨质疏松性骨折愈合的影响

目的本研究旨在探讨高频率低能量的振动力学刺激对骨质疏松性骨折愈合的影响。方法59只大鼠建立大鼠卵巢切除骨质疏松模型,建模成功后构建股骨骨折模型,并随机分为振动力学刺激组和对照组。骨折后第5天开始对振动组使用高频率低能量振动平台(35Hz,峰振幅0.3重力加速度)行每天20min的振动力学刺激,对照组则行假治疗。每周摄X线片观察骨折愈合的情况,并采用显微计算机断层扫描和生物力学测试评估骨痂生成的情况及其力学属性。结果 X线随访显示,振动组的骨折愈合速度和愈合率均优于对照组。前者新生矿化骨痂的体积在治疗后第2周和第4周时均显著高于后者。第8周时,振动组成熟骨痂的体积以及整体的力学强度均显著优于对照组。结论高频率低能量振动力学刺激可促进骨质疏松性骨折的愈合。     

Optimization of intramedullary nailing by numerical simulation of fracture healing

Due to the annular gap between intramedullary (IM) nails and the endosteal surface, high interfragmentary movement can occur under loading. This could prolong the healing time, particularly for thin IM nails that are often used for unreamed IM nailing. The aims of our study were to determine the influence of the nail diameter on the healing time of human tibial shaft fractures and to investigate whether the healing time could be shortened by increasing the stiffness of the implant material. Therefore, a corroborated numerical model for simulating the fracture healing process in humans was used to simulate the healing process of human tibial fractures treated with IM nails. The calculated healing time (up to 71 weeks) was longest for transverse fractures treated with thin IM nails made of titanium. That the healing time was disproportionately long depended on the nail diameter, and could be greatly reduced by using a thicker nail or using steel instead of titanium. To avoid a prolonged healing time, the nail should be thick, and the annular gap should be as narrow as possible. Alternatively, using steel instead of titanium may also help to avoid a prolonged healing time.     

Low dose erythropoietin stimulates bone healing in mice

Beyond its classical role in regulation of erythropoiesis, erythropoietin (EPO) has been shown to exert protective and regenerative actions in a variety of non‐hematopoietic tissues. However, little is known about potential actions in bone regeneration. To analyze fracture healing in mice, a femoral 0.25 mm osteotomy gap was stabilized with a pin‐clip technique. Animals were treated with 500 U EPO/kg bw per day or with vehicle only. After 2 and 5 weeks, fracture healing was analyzed biomechanically, radiologically and histologically. Expression of PCNA and NFκB was examined by Western blot analysis. Vascularization was analyzed by immunohistochemical staining of PECAM‐1. Circulating endothelial progenitor cells were measured by flow‐cytometry. Herein, we demonstrate that EPO‐treatment significantly accelerates bone healing in mice. This is indicated by a significantly greater biomechanical stiffness and a higher radiological density of the periosteal callus at 2 and 5 weeks after fracture and stabilization. Histological analysis demonstrated significantly more bone and less cartilage and fibrous tissue in the periosteal callus. Endosteal vascularization was significantly increased in EPO‐treated animals when compared to controls. The number of circulating endothelial progenitor cells was significantly greater in EPO‐treated animals. The herein shown acceleration of healing by EPO may represent a promising novel treatment strategy for fractures with delayed healing and non‐union formation. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:165–172, 2011     

Size and habit of mineral particles in bone and mineralized callus during bone healing in sheep

Bone healing is known to occur through the successive formation and resorption of various tissues with different structural and mechanical properties. To get a better insight into this sequence of events, we used environmental scanning electron microscopy (ESEM) together with scanning small‐angle X‐ray scattering (sSAXS) to reveal the size and orientation of bone mineral particles within the regenerating callus tissues at different healing stages (2, 3, 6, and 9 weeks). Sections of 200 µm were cut from embedded blocks of midshaft tibial samples in a sheep osteotomy model with an external fixator. Regions of interest on the medial side of the proximal fragment were chosen to be the periosteal callus, middle callus, intercortical callus, and cortex. Mean thickness (T parameter), degree of alignment (ρ parameter), and predominant orientation (ψ parameter) of mineral particles were deduced from resulting sSAXS patterns with a spatial resolution of 200 µm. 2D maps of T and ρ overlapping with ESEM images revealed that the callus formation occurred in two waves of bone formation, whereby a highly disordered mineralized tissue was deposited first, followed by a bony tissue with more lamellar appearance in the ESEM and where the mineral particles were more aligned, as revealed by sSAXS. As a consequence, degree of alignment and mineral particle size within the callus increased with healing time, whereas at any given moment there were structural gradients, for example, from periosteal toward the middle callus. © 2010 American Society for Bone and Mineral Research.     

Mechanical stimulation alters tissue differentiation and molecular expression during bone healing

Further understanding of how mechanical cues modulate skeletal tissue differentiation can identify potential means of enhancing repair following injury or disease. Prior studies examined the effects of mechanical loading on osteogenesis, chondrogenesis, and fibrogenesis in an effort to enhance bony union. However, exploring how mechanical stimuli can divert the bone healing process towards formation of other mesenchymal tissues, as an endpoint, may elucidate new avenues for repair and regeneration of tissues such as cartilage and fibrous tissue. This study investigated the use of mechanical stimulation to promote cartilage rather than bone formation within an osteotomy. Our overall goal was to define skeletal tissue distribution and molecular expression patterns induced by the stimulation. Retired breeder Sprague‐Dawley rats (n = 85) underwent production of a mid‐diaphyseal, transverse femoral osteotomy followed by external fixation. Beginning on postoperative day 10 and continuing for 1, 2, or 4 weeks, a cyclic bending motion (+35°/?25° at 1 Hz) was applied in the sagittal plane for 15 min/day for 5 consecutive days/week. Control animals experienced continuous rigid fixation. Histological and molecular analyses indicated that stimulation substantially altered normal bone healing. Stimulated specimens exhibited an increase in cartilage volume over time, while control specimens demonstrated bony bridging. Stimulation induced upregulation of cartilage‐related genes (COL2A1 and COL10A1) and downregulation of bone morphogenetic proteins (BMPs) ‐4, ‐6 and ‐7. However, BMP‐3 was upregulated with stimulation. These findings illustrate that mechanical cues can selectively modulate osteogenesis and chondrogenesis in vivo, and suggest a potential basis for treatment regimens for injured or diseased cartilaginous tissues. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res     

Neuromuscular electrical stimulation enhances fracture healing: results of an animal model.

Neuromuscular electrical stimulation (NMES) could simulate physiological muscle functions known to be associated with the normal bone healing process. The object of the present study was to evaluate the effect of NMES on fracture healing, using an animal model. Thirty rabbits received unilateral, transverse, mid-tibial, 3-mm gapped osteotomies that were stabilized with double-bar external fixators. The femoral vein was ligated to induce venous stasis. From the fourth post-operative day, the study group was treated with 1 h daily of NMES for four weeks, while the control group was treated without NMES. For NMES, two surface electrodes were used: one above the patellar tendon and another around the lateral thigh. Callus area and mineral content at the osteotomy gap were measured, biweekly, using computerized tomographic examinations. Biomechanical properties of healing were evaluated with a torsion test, eight weeks after the index operation. Osteotomies treated with NMES exhibited 31% (p=0.01) higher mineral content and 27% (p=0.009) larger callus area than control osteotomies at eight weeks. The maximum torque, torsional stiffness, angular displacement at maximum torque, and energy required to failure of specimens in the study group were 62% (p=0.006), 29% (p=0.03), 34.6% (p=0.008), and 124% (p<0.0001) higher, respectively, than those in the control group at eight weeks. The results of the present study demonstrated that the use of NMES can enhance callus development and mineralization, with the consequent improvement in biomechanical properties of the healing bone.     

Osteoblast and osteocyte‐specific loss of Connexin43 results in delayed bone formation and healing during murine fracture healing

Connexin43 (Cx43) plays an important role in osteoblastic differentiation in vitro, and bone formation in vivo. Mice with osteoblast/osteocyte‐specific loss of Cx43 display decreased gap junctional intercellular communication (GJIC), bone density, and cortical thickness. To determine the role of Cx43 in fracture healing, a closed femur fracture was induced in Osteocalcin‐Cre+; Cx43^flox/flox (Cx43cKO) and Cre‐; Cx43^flox/flox (WT) mice. We tested the hypothesis that loss of Cx43 results in decreased bone formation and impaired healing following fracture. Here, we show that osteoblast and osteocyte‐specific deletion of Cx43 results in decreased bone formation, bone remodeling, and mechanical properties during fracture healing. Cx43cKO mice display decreased bone volume, total volume, and fewer TRAP+ osteoclasts. Furthermore, loss of Cx43 in mature osteoblasts and osteocytes results in a significant decrease in torsional rigidity between 21 and 35 days post‐fracture, compared to WT mice. These studies identify a novel role for the gap junction protein Cx43 during fracture healing, suggesting that loss of Cx43 can result in both decreased bone formation and bone resorption. Therefore, enhancing Cx43 expression or GJIC may provide a novel means to enhance bone formation during fracture healing. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:147–154, 2012     

Efficacy of a sclerostin antibody compared to a low dose of PTH on metaphyseal bone healing

We compared the effect of a sclerostin antibody to that of a clinically relevant dose of parathyroid hormone (PTH) in a rat model for metaphyseal bone healing. Screws of steel or poly methyl methacrylate (PMMA) were inserted bilaterally into the proximal tibia of young male rats. During 4 weeks the animals then received injections of either phosphate buffered saline (control), sclerostin antibody (25 mg/kg, twice weekly) or PTH (5 µg/kg, daily). The healing response around the screws was then assessed by mechanical testing and X‐ray microtomography (µCT). To distinguish between effects on healing and general effects on the skeleton, other untraumatized bone sites and serum biomarkers were also assessed. After 4 weeks of treatment, PTH yielded a 48% increase in screw pull‐out force compared to control (p = 0.03), while the antibody had no significant effect. In contrast, the antibody increased femoral cortical and vertebral strength where PTH had no significant effect. µCT showed only slight changes that were statistically significant for the antibody mainly at cortical sites. The results suggest that a relatively low dose of PTH stimulates metaphyseal repair (screw fixation) specifically, whereas the sclerostin antibody has wide‐spread effects, mainly on cortical bone, with less influence on metaphyseal healing. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:471–476, 2014.     

Effects of macrophage activation on bone healing

In this study the effect of macrophage activation on bone healing was investigated in rats. In three groups of rats, an osteotomy of the femoral bone was performed and then nailed. Macrophages were activated by semisoluble aminated glucan. In one group of animals this was applied locally, in another group it was applied systemically (intraperitoneally), and the third group served as control. Eight rats in each group were killed after 4, 8, and 12 weeks, and the mechanical characteristics of the healing osteotomies were evaluated. We found that local activation of macrophages induced an immature hypertrophic callus with reduced biomechanical characteristics, as evaluated by bending moment, rigidity, and energy absorption. There were no significant differences between the rats subjected to systemic macrophage activation and the control rats. We conclude that local macrophage activation during the initial phase of bone repair impairs healing. Received for publication on Feb. 8, 1999; accepted on Oct. 8, 1999     

Oxysterols enhance osteoblast differentiation in vitro and bone healing in vivo.

Oxysterols, naturally occurring cholesterol oxidation products, can induce osteoblast differentiation. Here, we investigated short-term 22(S)-hydroxycholesterol + 20(S)-hydroxycholesterol (SS) exposure on osteoblastic differentiation of marrow stromal cells. We further explored oxysterol ability to promote bone healing in vivo. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity, osteocalcin (OCN) mRNA expression, mineralization, and Runx2 DNA binding activity. To explore the effects of osteogenic oxysterols in vivo, we utilized the critical-sized rat calvarial defect model. Poly(lactic-co-glycolic acid) (PLGA) scaffolds alone or coated with 140 ng (low dose) or 1400 ng (high dose) oxysterol cocktail were implanted into the defects. Rats were sacrificed at 6 weeks and examined by three-dimensional (3D) microcomputed tomography (microCT). Bone volume (BV), total volume (TV), and BV/TV ratio were measured. Culture exposure to SS for 10 min significantly increased ALP activity after 4 days, while 2 h exposure significantly increased mineralization after 14 days. Four-hour SS treatment increased OCN mRNA measured after 8 days and nuclear protein binding to an OSE2 site measured after 4 days. The calvarial defects showed slight bone healing in the control group. However, scaffolds adsorbed with low or high-dose oxysterol cocktail significantly enhanced bone formation. Histologic examination confirmed bone formation in the defect sites grafted with oxysterol-adsorbed scaffolds, compared to mostly fibrous tissue in control sites. Our results suggest that brief exposure to osteogenic oxysterols triggered events leading to osteoblastic cell differentiation and function in vitro and bone formation in vivo. These results identify oxysterols as potential agents in local and systemic enhancement of bone formation.     

The basic science of peri-implant bone healing

Given the popularity of cementless orthopedic implants, it is imperative for orthopedic surgeons to have a basic understanding of the process of peri-implant bone healing. Contact and distance osteogenesis have been used to explain peri-implant bone healing. In contact osteogenesis, de novo bone forms on the implant surface, while in distance osteogenesis, the bone grows from the old bone surface toward the implant surface in an appositional manner. Contact osteogenesis may lead to bone bonding if the surface of the implant displays the appropriate surface topography. The early stage of peri-implant bone healing is very important and involves the body’s initial response to a foreign material: protein adsorption, platelet activation, coagulation, and inflammation. This results in the formation of a stable fibrin clot that is a depot for growth factors and allows for osteoconduction. Osteoconduction is the migration and differentiation of osteogenic cells, such as pericytes, into osteoblasts. Osteoconduction allows for contact osteogenesis to occur at the implant surface. The late stage of healing involves the remodeling of this woven bone. In many respects, this process is similar to the bone healing occurring at a fracture site.     

Impact of mechanical stress and tension-stress on angiogenesis in wound healing

Angiogenesis plays a fundamental role in the development of the embryonic vascular tree as well as in several normal and pathologic conditions during postnatal life. Blood supply, established by neovascularization. is imperative for histogenesis during wound healing as well as the limb lengthening applied extensively in the treatment of skeletal trauma sequalae. But little attention has been paid to this area. This review aims to summarize angiogenesis regulation, the process of angiogenesis in wound healing and angiogenesis under mechanical stress, particularly in association with the tension-stress principle.     

Strontium(II) and mechanical loading additively augment bone formation in calcium phosphate scaffolds

Calcium phosphate cements (CPCs) are widely used for bone‐defect treatment. Current developments comprise the fabrication of porous scaffolds by three‐dimensional plotting and doting using biologically active substances, such as strontium. Strontium is known to increase osteoblast activity and simultaneously to decrease osteoclast resorption. This study investigated the short‐ and long‐term in vivo performances of strontium(II)‐doted CPC (SrCPC) scaffolds compared to non‐doted CPC scaffolds after implantation in unloaded or load‐bearing trabecular bone defects in sheep. After 6 weeks, both CPC and SrCPC scaffolds exhibited good biocompatibility and osseointegration. Fluorochrome labeling revealed that both scaffolds were penetrated by newly formed bone already after 4 weeks. Neither strontium doting nor mechanical loading significantly influenced early bone formation. In contrast, after 6 months, bone formation was significantly enhanced in SrCPC compared to CPC scaffolds. Energy dispersive X‐ray analysis demonstrated the release of strontium from the SrCPC into the bone. Strontium addition did not significantly influence material resorption or osteoclast formation. Mechanical loading significantly stimulated bone formation in both CPC and SrCPC scaffolds after 6 months without impairing scaffold integrity. The most bone was found in SrCPC scaffolds under load‐bearing conditions. Concluding, these results demonstrate that strontium doting and mechanical loading additively stimulated bone formation in CPC scaffolds and that the scaffolds exhibited mechanical stability under moderate load, implying good clinical suitability. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:106–117, 2018.     

Effect of human amniotic fluid on bone healing

BACKGROUND: Bone healing continues to pose challenges for researchers and clinicians working in the field of plastic surgery. Complete bone regeneration cannot be obtained in critical size osseous defects without the application of osteogenic or osteoinductive bone material. In this study, we hypothesized that because extracellular matrix components are known to play a major role in the first steps of healing during bone or injury healing and because hyaluronic acid as chondroitin sulfate is recognized as an osteogenic compound without osteoinductive activity, human amniotic fluid, which contains high concentrations of hyaluronic acid, gyaluronic acid -stimulating activator, and other factors, might accelerate bone healing when applied subperiosteally to rabbit calvarial defects. MATERIALS AND METHODS: We created 20 calvarial defects in 10 12-week-old New Zealand white rabbits who were divided into 2 groups. Group 1 defects were instilled with human amniotic fluid, whereas the group with contralateral defects, i.e., group 2, were given with same amount of normal saline solution. We then measured the density of the bone that formed over the defects using computed tomography at the third, fourth, fifth, and sixth weeks postoperatively. After this period, the defects were harvested for histopathologic evaluation. RESULTS: The defects from group 1, which were treated with human amniotic fluid, showed significantly higher ossification than the group 2 defects, which were instilled with saline solution. Histological examination at 6 weeks postoperatively revealed that the defects treated with human amniotic fluid (group 1) had superior ossification compared with the control group defects (group 2). CONCLUSION: Because of its positive effects on bone healing and also because of its ability to be stored in deep freeze if made cell-free, human amniotic fluid would appear to be a useful adjunct in the treatment of bone healing.     

The systemic angiogenic response during bone healing

Introduction  Angiogenesis is known to be a critical and closely regulated step during bone formation and fracture healing driven by a complex interaction of various cytokines. Delays in bone healing or even nonunion might therefore be associated with altered concentrations of specific angiogenic factors. These alterations might in turn be reflected by changes in serum concentrations. Method  To determine physiological time courses of angiogenic cytokines during fracture healing as well as possible changes associated with failed consolidation, we prospectively collected serum samples from patients who had sustained surgical treatment for a long bone fracture. Fifteen patients without fracture healing 4 months after surgery (nonunion group) were matched to a collective of 15 patients with successful healing (union group). Serum concentrations of angiogenin (ANG), angiopoietin 2 (Ang-2), basic fibroblast growth factor (bFGF), platelet derived growth factor AB (PDGF-AB), pleiotrophin (PTN) and vascular endothelial growth factor (VEGF) were measured using enzyme linked immunosorbent assays over a period of 24 weeks. Results  Compared to reference values of healthy uninjured controls serum concentrations of VEGF, bFGF and PDGF were increased in both groups. Peak concentrations of these cytokines were reached during early fracture healing. Serum concentrations of bFGF and PDGF-AB were significantly higher in the union group at 2 and 4 weeks after the injury when compared to the nonunion group. Serum concentrations of ANG and Ang-2 declined steadily from the first measurement in normal healing fractures, while no significant changes over time could be detected for serum concentrations of these factures in nonunion patients. PTN serum levels increased asymptotically over the entire investigation in timely fracture healing while no such increase could be detected during delayed healing. Conclusion  We conclude that fracture healing in human subjects is accompanied by distinct changes in systemic levels of specific angiogenic factors. Significant alterations of these physiologic changes in patients developing a fracture nonunion over time could be detected as early as 2 (bFGF) and 4 weeks (PDGF-AB) after initial trauma surgery. Authors Stefan Weiss and Gerald Zimmermann contributed equally to this work.     

螺旋CT扫描重建对经椎弓根伤椎植骨骨愈合的评估价值

目的探讨64排螺旋CT扫描后多平面重建（multi-planner reformation,MPR）和3D成像对经椎弓根伤椎植骨骨愈合的评估价值。方法2005年5月至2010年5月行椎弓根螺钉内固定基础上经椎弓根伤椎成形椎体内植骨62例68椎,术后1—3年应用64排螺旋CT扫描做冠状面、矢状面等多平面和3D重建成像,以骨愈合、部分骨愈合、骨不愈合3种标准评估伤椎植骨后的骨愈合情况,与X线片的评估结果相比较。结果全部获得随访,随访时间1～3年,平均2年,末次随访CT扫描重建的62例68椎中57椎骨愈合,11椎部分骨愈合,无一椎发生骨不愈合,3D成像示伤椎形态恢复满意,皮质骨均骨性愈合,骨愈合率为89．7％。同期的x线片评估结果示伤椎均骨愈合,骨愈合率为100％。结论64排螺旋CT扫描和多平面及3D成像技术是评估脊柱骨折伤椎骨性愈合的可靠指标之一。