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     

Moderate soft tissue trauma delays new bone formation only in the early phase of fracture healing.

The aim of this study was to investigate the effect of a moderate soft tissue trauma to the course of fracture healing in a standardized animal model. Thirty-eight Wistar rats were randomly divided into a fracture group (F, n = 19) and a group with a fracture and a soft tissue trauma (F + STT, n = 19). The fracture and the soft tissue trauma were created using an impact device with a standardized energy. All fractures were stabilized by two Kirschner wires. Three rats were measured for blood flow and sacrificed at days 1, 3, 7, and 14, and seven rats at day 28, from both groups. A three-point bending test was performed on the healed tibia after 28 days. During the first 24 h there was a reduction in blood flow, which was more pronounced in the F + STT group than in the F group. From histological sections, the shape of the callus formation, as well as the tissue distribution of newly formed bone, fibrous cartilage and fibrous connective tissue were determined. Distinctly more periosteal new bone formed and a larger callus formed at days 3 and 7 in group F compared to group F + STT. However, by days 14 and 28, the ossification and overall callus size no longer showed differences between the two groups. A fast recovery of blood flow and callus formation took place in the F + STT group, which led to similar histological and biomechanical results in fracture healing observed after 28 days between the two groups.     

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     

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     

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.     

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.     

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.     

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。     

Parecoxib impairs early metaphyseal bone healing in rats

Introduction Cox2 inhibitors decrease prostaglandin production and therefore influence bone healing especially in unstable long bone models. It is unclear to what extent implant fixation in stable metaphyseal bone is impaired. Method Male rats numbering 30 and female rats numbering 40 received a stainless steel screw in the metaphyseal bone of the proximal tibia. Half of the rats were treated with 6.4 mg/kg BW parecoxib by continuous release from a subcutaneous mini pump during 7 or 14 days. After treatment, the pull out force, stiffness, and pull out energy of the screw were measured. Results No effect of parecoxib on the pull out force was found for male rats. In female rats the pull out force was decreased by 16% (P = 0.03) after 7 days treatment with parecoxib. This effect had disappeared after 14 days. Conclusion Adverse effects of parecoxib on the early phase healing of metaphyseal bone in female rats are small and were not detectable after 14 days. No effect was seen in male rats, possibly due to a faster metabolic elimination of the drug.     

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.     

Wound healing in the bone chamber 1. Neoosteogenesis during transition from the repair to the regenerative phase in the rabbit tibial cortex

The optical bone chamber technique that includes intravital microscopy is described and is then applied to measuring primary wound healing neoostogenesis in rabbit tibia cortical bone during the period when fibrovascular tissue is being replaced by regenerating osteovascular tissue. The small population of rabbits sampled allowed only tentative conclusions. The quantitative measurements, a direct from of histomorphometry, are applied to determine the consistency of observations, with the hypothesis that healing into the bone chamber slit-gap follows the pattern of primary bone regeneration established as "characteristic" by other studies. The results supported the hypothesis and showed that bony ingrowth started during the third postoperative week (W3) with a maximum linear growth rate of 85.5 micron/day for a bone front. Evidence for remodeling by W6 was also obtained. While the bone chamber environment for tissue ingrowth is artificial, it can generate quantitative data that may provide a statistically valid basis for modeling pathophysiologic processes associated with bone wound healing.     

Evidence for a cellular andmolecular decline in bone healing with age

Aging is a process of cumulative effects. Aging effects sometimes appear random in sequence and consequence. We propose that considerable complexity underlies changes associated with aging. Apparent simplicity masks the interrelated events at a molecular, cellular, systemic, and organism level. Clinical observation suggests the majority of age-related events are initially constructive, optimal, and conducive to maximized survivability. Then, in subtle, poorly understood mechanisms, the fate of the organism progresses toward degeneration. Clinical experiences give rise to the impression that there might be differences in fracture healing in different age groups. Minimal knowledge exists in the literature objectively evaluating this impression. In this review, we focus on cellular and molecular evidence that suggests that changes occur in the aging skeletons capacity to heal. The medical community is faced with an aging population, steadily increasing fracture numbers in the elderly population, increasing treatment costs, and the most important, quality-of-life issues. An understanding of age-related changes in bone healing is necessary as a basis for the development of future research strategies. Understanding bone healing in an aging individual devoid of pathological complications is a challenging first step toward rationalizing therapies to restore the aged skeleton to health.     

Dermal fibroblast‐mediated BMP2 therapy to accelerate bone healing in an equine osteotomy model

This study evaluated healing of equine metacarpal/metatarsal osteotomies in response to percutaneous injection of autologous dermal fibroblasts (DFbs) genetically engineered to secrete bone morphogenetic protein‐2 (BMP2) or demonstrate green fluorescent protein (GFP) gene expression administered 14 days after surgery. Radiographic assessment of bone formation indicated greater and earlier healing of bone defects treated with DFb with BMP2 gene augmentation. Quantitative computed tomography and biomechanical testing revealed greater mineralized callus and torsional strength of DFb‐BMP2‐treated bone defects. On the histologic evaluation, the bone defects with DFb‐BMP2 implantation had greater formation of mature cartilage and bone nodules within the osteotomy gap and greater mineralization activity on osteotomy edges. Autologous DFbs were successfully isolated in high numbers by a skin biopsy, rapidly expanded without fastidious culture techniques, permissive to adenoviral vectors, and efficient at in vitro BMP2 protein production and BMP2‐induced osteogenic differentiation. This study demonstrated an efficacy and feasibility of DFb‐mediated BMP2 therapy to accelerate the healing of osteotomies. Skin cell‐mediated BMP2 therapy may be considered as a potential treatment for various types of fractures and bone defects. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:403–411, 2010     

Early mechanical stimulation only permits timely bone healing in sheep

Bone fracture healing is sensitive to the fixation stability. However, it is unclear which phases of healing are mechano‐sensitive and if mechanical stimulation is required throughout repair. In this study, a novel bone defect model, which isolates an experimental fracture from functional loading, was applied in sheep to investigate if stimulation limited to the early proliferative phase is sufficient for bone healing. An active fixator controlled motion in the fracture. Animals of the control group were unstimulated. In the physiological‐like group, 1 mm axial compressive movements were applied between day 5 and 21, thereafter the movements were decreased in weekly increments and stopped after 6 weeks. In the early stimulatory group, the movements were stopped after 3 weeks. The experimental fractures were evaluated with mechanical and micro‐computed tomography methods after 9 weeks healing. The callus strength of the stimulated fractures (physiological‐like and early stimulatory) was greater than the unstimulated control group. The control group was characterized by minimal external callus formation and a lack of bone bridging at 9 weeks. In contrast, the stimulated groups exhibited advanced healing with solid bone formation across the defect. This was confirmed quantitatively by a lower bone volume in the control group compared to the stimulated groups.The novel experimental model permits the application of a well‐defined load history to an experimental bone fracture. The poor healing observed in the control group is consistent with under‐stimulation. This study has shown early mechanical stimulation only is sufficient for a timely healing outcome. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1790–1796, 2018.

     

Principles of management of long bone fractures and fracture healing

This article describes the management of long bone fractures to the lower limb. It covers initial management in the emergency department through to definitive treatment by the orthopaedic surgeon. Bone healing is covered and how different treatment techniques can influence bone healing.     

Augmentation of bone healing in delayed and atrophic nonunion of fractures of long bones by partially decalcified bone allograft (decal bone)

Background:

Autograft from iliac crest is considered as gold standard for augmentation of bone healing in delayed and nonunion of fractures. Bone demineralized with 0.6N hydrochloric acid has shown to retain its osteoinductive capacity. We report the outcome of partially decalcified bone allograft (decal bone) in the treatment of delayed union and atrophic nonunions of bones.

Materials and Methods:

Twenty patients with clinicoradiological diagnosis of delayed union or atrophic nonunion of long bone fractures were included in this retrospective study. Patients at extreme of ages (<18 years and >60 years), pathological fractures, metabolic bone diseases, infected nonunion, hypertrophic nonunion and those having systemic illness like diabetes mellitus and on drugs that impair fracture healing were excluded from the study. Decal bone was prepared in the bone bank and maintained in department of orthopedics. Allografting was done in 20 patients of delayed union (9/20) and atrophic nonunion (11/20) of long bone fractures with mean age of 34 years (range 18–55 years). The bones involved were humerus (8/20), tibia (7/20) and femur (5/20). Fourteen patients underwent treatment in the form of internal fixation and allografting and six patients were operated with osteoperiosteal allografting.

Results:

Nineteen patients achieved union in mean time of 14.9 weeks range (range 8–20 weeks). Eight patients had serous discharge from the operative site that subsided in 11 days (range 4–21 days). One patient had pus discharge that required repeat debridement and antibiotics for 6 weeks. The fracture healed in 16 weeks.

Conclusion:

The partially decalcified bone allograft is an effective modality for augmentation of bone healing without complication associated with autograft like donor site morbidity, increased blood loss and increase in the surgical time.     

The effect of rhPTH on the healing of tendon to bone in a rat model

Successful rotator cuff tendon repair depends on secure tendon‐to‐bone healing. Recombinant human parathyroid hormone (rhPTH) has been shown in multiple studies to accelerate bone healing. Recent studies have also shown that rhPTH is chondrogenic by increasing chondrocyte recruitment and differentiation. We hypothesized that rhPTH would improve tendon‐to‐bone healing in a rat rotator cuff repair model. One hundred and fourteen Sprague Dawley rats underwent division and repair of the supraspinatus tendon. Fifty seven rats received daily subcutaneous injections of 10 µg/kg of rhPTH. Rats were sacrificed at 3, 7, 14, 28, and 56 days for histologic and immunohistochemical analysis. In addition, rats in each group were sacrificed at 14, 28, and 56 days for biomechanical testing and micro CT analysis. At 2 weeks the controls had a significantly higher load to failure than the rhPTH group. At 28 and 56 days there were no differences in load to failure. rhPTH specimens had significantly higher stiffness at 56 days. MicroCT analysis showed that the rhPTH group had significantly greater total mineral content at all time points, as well as significantly higher bone volume (BV) at 14 and 28 days. Histologically, the rhPTH specimens had more fibrocartilage, osteoblasts, and blood vessels at all timepoints, with significantly better collagen fiber orientation at 28 and 56 days. Although treatment with rhPTH resulted in an increase in bone and mineralized fibrocartilage formation, as well as better collagen fiber organization, this did not translate into improved biomechanical properties. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:769–774, 2012     

Local administration of alendronate reduced peri‐tunnel bone loss and promoted graft‐bone tunnel healing with minimal systemic effect on bone in contralateral knee

Continued systemic administration of alendronate was reported to reduce peri‐tunnel bone resorption and promoted graft‐bone tunnel healing at the early stage post‐anterior cruciate ligament (ACL) reconstruction. However, systemic increase in bone mineral density (BMD) in the contralateral intact knee was observed. We tested if single local administration of alendronate into the bone tunnel during ACL reconstruction could achieve similar benefits yet without the systemic effect on bone. Seventy‐two rats with unilateral ACL reconstruction were divided into three groups: saline, low‐dose (6 μg/kg) and mid‐dose (60 μg/kg) alendronate. For local administration, alendronate was applied to the bone tunnels for 2 min before graft insertion and repair. At weeks 2 and 6, the reconstructed complex was harvested for high‐resolution computed tomography (vivaCT) imaging followed by biomechanical test or histology. Our results showed that local administration of low‐dose alendronate showed comparable benefits on the reduction of peri‐tunnel bone loss, enhancement of bone tunnel mineralization, tunnel graft integrity, graft osteointegration and mechanical strength of the reconstructed complex at early stage post‐reconstruction, yet with minimal systemic effect on mineralized tissue at the contralateral intact knee. A single local administration of alendronate at the low‐dose therefore might be used to promote early tunnel graft healing post‐reconstruction. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1897–1906, 2013