Host-derived neoangiogenesis with short-term immunosuppression allows incorporation and remodeling of vascularized diaphyseal allogeneic rabbit femur transplants

The purpose of this study was to demonstrate that living bone allotransplants can incorporate, remodel, and maintain mechanical properties without long-term immunosuppression in a fashion comparable to living autotransplants. For this, viability is maintained by repair of nutrient vessels and neovascularization from implanted host-derived vasculature. Microsurgically revascularized femoral diaphysis allotransplants were transferred from young male New-Zealand-White (NZW) into 4 groups of male Dutch-Belted (DB) rabbits. Short-term immunosuppression by tacrolimus (IS, groups 4 and 5) and host-derived neovascularization (NV) from implanted fascial flaps was used to maintain viability (groups 3 and 5) as independent variables. Group 2 received neither IS nor NV. Vascularized pedicled autotransplants were orthotopically transplanted in group 1. After 16 weeks, transplants were evaluated using radiologic, histologic, biomechanical, and histomorphometric parameters. Vascularized bone allotransplants treated with both short-term IS and host-derived NV (group 5) healed in a fashion similar to pedicled autotransplants (group 1). Their radiographic scores were higher than other groups. Groups with patent fascial flaps (3 and 5) showed significantly greater neoangiogenesis than ligated controls (2 and 4). Tacrolimus administration did not affect neoangiogenesis. Elastic modulus and ultimate stress were significantly greater in autogenous bone than in allotransplanted femora. Biomechanical properties were not significantly different among allotransplants. Bone turnover was decreased with IS, but increased with NV by the implanted fascial flaps. Living allogeneic femoral allotransplants treated with short-term IS and host-derived neoangiogenesis can lead to stable transplant incorporation in this rabbit model. The combination of both factors optimizes bone healing. Transplant mineralization is improved with neoangiogenesis but diminished with IS. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 763–770, 2009     

Effects of Surgical Angiogenesis on Segmental Bone Reconstruction With Cryopreserved Massive‐Structural Allografts in a Porcine Tibia Model

Cryopreserved bone allografts (CBA) used to reconstruct segmental bone defects provide immediate structural stability, but are vulnerable to infection, non‐union and late stress fracture as the majority of the allograft remains largely avascular. We sought to improve the bone vascularity and bone formation of CBAs by surgical angiogenesis with an implanted arteriovenous (AV) bundle, using a porcine tibial defect model. Cryopreserved tibial bone allografts were transplanted in swine leukocyte antigen (SLA) mismatched Yucatan minipigs to reconstruct a 3.5 cm segmental tibial defect. A cranial tibial AV‐bundle was placed within its intramedullary canal to induce angiogenesis. The AV bundle was patent in eight pigs and ligated in a control group of eight pigs. At 20 weeks neo‐angiogenesis was evaluated by micro‐angiography. Bone formation was measured by quantitative histomorphometry and micro‐computed tomography. Seven of eight AV‐bundles in the revascularized group were patent. One had thrombosed due to allograft displacement. Total vascular volume was higher in the revascularized allografts compared to the ligated group (p = 0.015). Revascularized allografts had increased levels of bone formation on the allograft endosteal surface compared to the ligated control group (p = 0.05). Surgical angiogenesis of porcine tibial CBAs by intramedullary implantation of an AV‐bundle creates an enhanced autogenous neoangiogenic circulation and accelerates active bone formation on allograft endosteal surfaces. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1698–1708, 2019     

A modified vascularized whole knee joint allotransplantation model in the rat

Previous papers have shown surgical neoangiogenesis to allow long‐term bone allotransplant survival without immunosuppression. Whole joint composite tissue allotransplants (CTA) might be treated similarly. A novel rat knee CTA model is described for further study of the roles of neoangiogensis in joint allotransplant survival and adjustment of immunosuppression. Microvascular knee CTA was performed in nine rats across a major histocompatibility barrier with both pedicle repair and implantation of host‐derived arteriovenous (“a/v”) bundles. In the control group (N = 3), the pedicle was ligated. Immunosuppression was given daily. Joint mobility, weight‐bearing, pedicle patency, bone blood flow, and sprouting from a/v bundles were assessed at 3 weeks. All but the nonrevascularized control knees had full passive motion and full weight bearing. One nutrient pedicle thrombosed prematurely. Blood flow was measurable in transplants with patent nutrient pedicles. Implanted a/v bundles produced new vascular networks on angiography. This new rat microsurgical model permits further study of joint allotransplantation. Patency of both pedicles and implanted a/v bundles was maintained, laying a foundation for future studies. © 2010 Wiley‐Liss, Inc. Microsurgery, 2010.     

An experimental study on a new artificial vertebral body

This study was undertaken to develop a new artificial vertebral body (AVB) for metastatic tumors, and to confirm its feasibility for practical use. (1) In mechanical tests using bovine cancellous bone and the vertebrae of fresh human cadavers, six ways of fixing the AVBs to cancellous bone were compared. A rectangular plate with a blunt-toothed surface fixed into a bone groove with cement proved to be the best fixation method. (2) In experiments using seven dogs, small AVBs were applied to the 4th lumbar vertebra and the plate portions were fixed into the grooves made in the 3rd and 5th vertebrae. Undecalcified specimens of these vertebrae were examined 4, 8, 12, and 16 weeks after the operations. New bone formation was found around the grooves and endplates. The height of AVBs could be adjusted. By endurance tests AVBs were well tolerated and remained unchanged. The results of these experiments have indicated that the new AVB appeared to be feasible for clinical application.     

Fibroblast growth factor‐2 and vascular endothelial growth factor mediated augmentation of angiogenesis and bone formation in vascularized bone allotransplants

We previously demonstrated recipient‐derived neoangiogenesis to maintain viability of living bone allogeneic transplants without long‐term immunosuppression. The effect of cytokine delivery to enhance this process is studied. Vascularized femur transplantation was performed from Dark Agouti to Piebald Virol Glaxo rats. Poly(d,l ‐lactide‐co‐glycolide) microspheres loaded with buffer (N = 11), basic fibroblast growth factor (FGF2) (N = 10), vascular endothelial growth factor (VEGF) (N = 11), or both (N = 11) were inserted intramedullarly alongside a recipient‐derived arteriovenous bundle. FK‐506 was administered for 2 weeks. At 18 weeks, bone blood flow, microangiography, histologic, histomorphometric, and alkaline phosphatase measurements were performed. Bone blood flow was greater in the combined group than control and VEGF groups (P = 0.04). Capillary density was greater in the FGF2 group than in the VEGF and combined groups (P < 0.05). Bone viability, growth, and alkaline phosphatase activity did not vary significantly between groups. Neoangiogenesis in vascularized bone allotransplants is enhanced by angiogenic cytokine delivery, with results using FGF2 that are comparable to isotransplant from previous studies. Further studies are needed to achieve bone formation similar to isotransplants. © 2014 Wiley Periodicals, Inc. Microsurgery 34:301–307, 2014.     

Short-term immunosuppression and surgical neoangiogenesis with host vessels maintains long-term viability of vascularized bone allografts.

Currently available methods to reconstruct large skeletal defects have limitations. These include nonunion and stress fractures in structural allografts, and inability to match the size, shape, and/or strength of most recipient sites using vascularized fibular autografts. Prosthetic diaphyseal replacements may loosen or produce periprosthetic fractures. Transplantation of living allogenic bone would enable matching donor bone to the recipient site, combined with the desirable healing and remodeling properties of living bone. We propose a novel method by which the transplantation of such tissue might be done without the risks of life-long immunosuppression, using surgical neoangiogenesis to develop a new host-derived osseous blood supply. We performed vascularized femoral allografts from 86 female Dark Agouti donor rats to male Piebald Virol Glaxo recipients across a major histocompatibility (MHC) barrier. In addition to microvascular reconstruction of the nutrient vessel, we surgically implanted a host arteriovenous (AV) bundle into the medullary canal to promote host vessel neoangiogenesis. Independent variables included patency of the implanted AV bundle, and use of 2 weeks' FK-506 immunosuppression. After 18 weeks, bone blood flow was measured, and neoangiogenic capillary density quantified. Bone blood flow and capillary density were significantly greater in transiently immunosuppressed recipients with a patent AV pedicle. We conclude that neoangiogenesis from implanted host-derived AV-bundles, combined with short-term immunosuppression maintains blood flow in vascularized bone allografts, and offers potential for clinical application.     

可调式中空钛合金人工椎体的生物力学评价

目的 测试并评价可调式中空肽合金人工椎体的生物力学性能。方法 27具小牛胸腰椎标本随机分为对照组(IS)、髂骨植骨组(IBG)及人工椎体组(AVB)3组，测试屈曲、伸展、侧屈及扭转状态下的载荷—应变、载荷—位移关系变化以及最大载荷时的应力强度及刚度。IBG组、AVB组分别附加前路钢板及后路椎弓根螺钉进行第2次、第3次测试，各组间进行比较，并进行统计学处理。结果 髂骨植骨组不同状态下应变及位移均大于原始标本，人工椎体组小于原始标本，且差异具显著性(P＜0．05)；附加内固定后应变及位移均较不附加内固定者减小(P＜0．05)，且人工椎体组附加内固定后分别小于髂骨植骨组(P＜0．05)。人工椎体附加或不附加内固定时的应力强度及轴向刚度均大于或接近原始标本，而单纯髂骨植骨组伸展及侧屈状态下，以及附加前路或后路固定后的侧屈状态下的应力强度低于原始标本；轴向压缩刚度亦小于原始标本，附加前路钢板后有所提高，但只有附加后路椎弓根螺钉后才与原始标本相当。人工椎体组的扭转强度大于髂骨组，扭转刚度与原始标本接近。而髂骨植骨组即使附加内固定后的扭转刚度亦远小于原始标本。结论 单纯人工椎体替代基本可满足生物力学性能要求，附加前路或后路固定后，可满足包括扭转性能在内的生物力学要求；单纯髂骨植骨后不满提供足够的生物力学稳定性、必须辅加内固定，同时应注意术后保护。     

Preoperative low-dose irradiation promotes long-term allograft acceptance and induces regulatory T cells in a porcine model of pulmonary transplantation

BACKGROUND: A simplified conditioning protocol including single-dose preoperative thymic and low-dose whole body irradiation with or without subsequent donor bone marrow transplantation (BMTx) can be applied in porcine lung transplantation. We hypothesized that this protocol would prolong allograft survival. METHODS: Left-sided single lung transplantation from major histocompatibility complex (MHC)-mismatched donors was performed in 27 minipigs. Recipients received whole body (1.5 Gy) and thymic irradiation (7 Gy) before transplantation (IRR group, n=6), intravenous immunosuppression with methylprednisolone, cyclosporine, and azathioprine for 27 postoperative days (IS group, n=5) or both (IRR+IS group, n=10). BMTx group recipients were treated with irradiation, immunosuppression and a donor bone marrow infusion on postoperative day 1. Peripheral blood leukocyte phenotype and donor cell chimerism were monitored by flow cytometry. Purified CD25+ T cells from long-term survivors or rejecting animals were used for in vitro MLR suppression assays. RESULTS: Median graft survival was: IRR 12 days, IS 55 days, IRR+IS 239 days, and BMTx 80 days (P<0.0001). Early peripheral blood macrochimerism was substantial in both the IRR+IS and the BMTX group but was lost in all groups after day 80. The frequency and suppressive function of CD4+CD25+ T cells were enhanced in IRR+IS group long-term survivors. CONCLUSION: Although donor bone marrow infusion was not beneficial in our model, a substantial proportion of the animals treated with irradiation and a 28-day course of immunosuppression accepted their lung allografts long term. The mechanism involved in maintaining allograft tolerance may be based on peripheral T-cell regulation.     

Effect of rhBMP‐2 and VEGF in a vascularized bone allotransplant experimental model based on surgical neoangiogenesis

We have demonstrated survival of living allogeneic bone without long‐term immunosuppression using short‐term immunosuppression and simultaneous creation of an autogenous neoagiogenic circulation. In this study, bone morphogenic protein‐2 (rhBMP‐2), and/or vascular endothelial growth factor (VEGF), were used to augment this process. Femoral diaphyseal bone was transplanted heterotopically from 46 Dark Agouti to 46 Lewis rats. Microvascular repair of the allotransplant nutrient pedicle was combined with intra‐medullary implantation of an autogenous saphenous arteriovenous (AV) bundle and biodegradable microspheres containing buffer (control), rhBMP‐2 or rhBMP‐2 + VEGF. FK‐506 given daily for 14 days maintained nutrient pedicle flow during angiogenesis. After an 18 weeks survival period, we measured angiogenesis (capillary density) from the AV bundle and cortical bone blood flow. Both measures were greater in the combined (rhBMP‐2 + VEGF) group than rhBMP‐2 and control groups (p < 0.05). Osteoblast counts were also higher in the rhBMP‐2 + VEGF group (p < 0.05). A trend towards greater bone formation was seen in both rhBMP2 + VGF and rhBMP2 groups as compared to controls (p = 0.059). Local administration of VEGF and rhBMP‐2 augments angiogenesis, osteoblastic activity and bone blood flow from implanted blood vessels of donor origin in vascularized bone allografts. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 561–566, 2013     

Host-derived angiogenesis maintains bone blood flow after withdrawal of immunosuppression

A novel method of living bone allotransplantation combining microvascular repair of the nutrient circulation, implantation of host-derived arteriovenous (AV) bundles, and short-term immunosuppression is described. We hypothesized that neoangiogenesis from the implanted vessels would maintain graft viability and circulation after withdrawal of FK506 (Tacrolimus) immunosuppression. Vascularized femoral transplantation was performed between DA and PVG rats. In addition to microsurgical pedicle anastomoses, a saphenous AV bundle from the recipient animal was implanted in the medullary space. Ninety-seven rats were randomly allocated to groups differing in immunosuppression and AV bundle patency. Implanted vessels significantly improved capillary density and bone blood flow in nonimmunosuppressed and immmunosuppressed groups, respectively. A lower incidence of spontaneous AV bundle thrombosis was found with Tacrolimus treatment. More viable osteocytes were seen at 4 weeks when the AV bundle was patent. Further investigations may confirm host-derived neoangiogenesis as an alternative to tolerance induction or immunosuppression in bone allotransplantation.     

Repopulation of vascularized bone allotransplants with recipient‐derived cells: Detection by laser capture microdissection and real‐time PCR

Mechanisms underlying successful composite tissue transplantation must include an analysis of transplant chimerism, which is little studied, particularly in calcified tissue. We have developed a new method enabling determination of lineage of selected cells in our model of vascularized bone allotransplantation. Vascularized femoral allotransplantation was performed from female Dark Agouti (DA) donor rats to male Piebald Virol Glaxo (PVG) recipients, representing a major histocompatibility mismatch. Four groups differed in use of immunosuppression (±2 weeks Tacrolimus) and surgical revascularization, by implantation of either a patent or a ligated saphenous arteriovenous (AV) bundle. Results were assessed at 18 weeks. Bone blood flow was measured by the hydrogen washout technique and transverse specimens were prepared for histology. Real‐time PCR was performed on DNA from laser capture microdissected cortical bone regions to determine the extent of chimerism. To do so, we analyzed the relative expression ratio of the sex‐determining region Y (Sry) gene, specific only for recipient male rat DNA, to the cyclophilin housekeeper gene. Substantial transplant chimerism was seen in cortical bone of all groups (range 77–97%). Rats without immunosuppression and with a patent AV bundle revealed significantly higher chimerism than those with immunosuppression and a ligated AV bundle, which maintained transplant cell viability. We describe a new method to study the extent of chimerism in rat vascularized bone allotransplants, including a sex‐mismatched transplantation model, laser capture microdissection of selected bone regions, and calculation of the relative expression ratio. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1514–1520, 2009     

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     

Neo-Angiogenesis,Transplant Viability,and Molecular Analyses of Vascularized Bone Allotransplantation Surgery in a Large Animal Model

Vascularized composite allotransplantation of bone is a possible alternative treatment for large osseous defects but requires life-long immunosuppression. Surgical induction of autogenous neo-angiogenic circulation maintains transplant viability without this requirement, providing encouraging results in small animal models [1–3]. A preliminary feasibility study in a swine tibia model demonstrated similar findings [4, 5]. This study in swine tibial allotransplantation tests its applicability in a pre-clinical large animal model. Previously, we have demonstrated bone vascularized composite allotransplantation (VCA) survival was not the result of induction of tolerance nor an incompetent immune system [1]. Fourteen tibia vascularized bone allotransplants were microsurgically transplanted orthotopically to reconstruct size-matched tibial defects in Yucatan miniature swine. Two weeks of immunosuppression was used to maintain allotransplant pedicle patency during angiogenesis from a simultaneously implanted autogenous arteriovenous bundle. The implanted arteriovenous bundle was patent in group 1 and ligated in group 2 (a neo-angiogenesis control). At twenty weeks, we quantified the neo-angiogenesis and correlated it with transplant viability, bone remodeling, and gene expression. All patent arteriovenous bundles maintained patency throughout the survival period. Micro-angiographic, osteocyte cell count and bone remodeling parameters were significantly higher than controls due to the formation of a neo-angiogenic autogenous circulation. Analysis of gene expression found maintained osteoblastic and osteoclastic activity as well as a significant increase in expression of endothelial growth factor-like 6 (EGFL-6) in the patent arteriovenous bundle group. Vascularized composite allotransplants of swine tibia maintained viability and actively remodeled over 20 weeks when short-term immunosuppression is combined with simultaneous autogenous neo-angiogenesis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:288-296, 2020     

钻孔复合浓缩骨髓对兔结构性骨移植愈合的影响

目的 观察异体骨钻孔并复合自体浓缩骨髓移植对兔骨缺损的修复和重建能力,为临床结构性骨移植提供新方法.方法 将36只日本大耳白兔随机分成三组,每组12只,制成左侧股骨中段3 cm缺损,分别采用不同方式处理过的同种异体骨进行移植,A组:植入未打孔异体骨+骨泥;B组:植入打孔异体骨+骨泥;C组:植入打孔异体骨+骨泥浓缩骨髓复合物.术后3个月分别行x线片、组织学观察、骨密度测定和生物力学测试. 结果 ①放射学检查:A组仅在供.宿结合部可见有骨痂包绕,异体骨周围无明显骨痂;B组及C组整个移植骨段周围均可见明显骨痂,C组骨痂延伸范围更广.②组织学观察:三组异体骨供.宿结合部均完全吸收,被大量骨痂取代且塑形良好;中间横断面A组异体骨周围可见少量内骨痂,B组可见少量内骨痂及外骨痂且被大量纤维结缔组织所分隔;C组异体骨可见大量内外骨痂生长,髓腔内充满骨母细胞,外骨痂骨小梁已相互融合形成编织网状骨,但与异体骨间仍被纤维结缔组织间断分隔.③平均骨密度值测定:C组高于A、B组及对侧正常股骨,差异有统计学意义(P＜0.05).④力学性能检测:A、B、C三组间最大载荷比较,差异无统计学意义(P＞0.05),A、B、C组均低于对侧正常股骨(P＜0.05). 结论 钻孔并复合浓缩骨髓能促进大段移植骨的早期活化,提高新骨产量.     

Canine bone blood flow estimated with microspheres

Blood flow rate in the periosteal and endosteal cortices of the diaphysis of long bones was determined in eight anesthetized adult dogs using the radioactively labeled microsphere method. The flow rate in endosteal and periosteal cortices is not significantly different unless the endosteal cortex contains cancellous bone from the medullary cavity. The highest flow rate is in the ulna, with 6.50 ml/min/100 g for the endosteal cortex and 5.15 ml/min/100 g for the periosteal cortex. The lowest flow rate is in the femur, with 2.89 ml/min/100 g for the endosteal cortex and 2.29 ml/min/100 g for the periosteal cortex. The results of this study indicate that variation of blood flow does exist between bones of dogs. However, the flow rates of individual bone on the left and right sides in the same dog are not significantly different. This indicates that the data are reproducible and reliable, and differences are not an inaccuracy of the radioactively labeled microsphere method. The results also demonstrate that there are no significant differences in bone blood flow as measured with small numbers (fewer than 400 microspheres per sample) and large numbers (more than 400 microspheres per sample) of microspheres. The adequate number of microspheres in each bone sample is 150-250. Therefore, a dose of 0.5 x 10(6) spheres/kg body weight can be sufficient.     

Resurfacing of the humeral head: An analysis of the bone stock and osseous integration under the implant

Cementless‐surface‐replacement‐arthroplasty (CSRA) of the shoulder aims for functional joint restoration with minimal bone loss. Good clinical results have been reported, but due to the radiopaque metal shell no data is available on the structure, osseous integration, and bone stock under the implant. 14 hemi‐CSRAs (4 manufacturers) with two geometries (crown [n = 7]/ stem [n = 7] fixation) were retrieved from patients undergoing revision due to glenoidal erosion. Histological sections cutting through the implant centre and bone were analysed. Quantitative histomorphometry evaluated the bone‐implant‐contact and compared the bone‐area to native humeral retrievals (n = 7). The bone‐implant‐interface was further assessed by scanning‐electron‐microscopy (SEM) and energy‐dispersive‐x‐ray (EDX). Qualitative histology revealed a reduced and inhomogeneous bone stock. Obvious signs of stress shielding were observed with bone predominantly visible at the stem and implant rim. Quantitative histomorphometry confirmed the significantly reduced bone‐area (9.2 ± 3.9% [crown 9.9 ± 4.3%, stem 8.6 ± 3.6%]) compared to native humeri (21.2 ± 9.1%; p < 0.05). Bone‐implant‐contact was 20.5 ± 5.8% (crown 21.8 ± 6.2%, stem 19.2 ± 5.6%) which was confirmed by SEM and EDX. Altogether, CRSA shows satisfactory bone ingrowth at the interface suggesting sufficient primary stability to allow osseous integration. However, clear signs of stress shielding with an inhomogeneous and reduced bone stock were observed. The impact on the long‐term‐results is unclear requiring further investigation. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1382–1390, 2015.     

Effects of Up to 5 Years of Denosumab Treatment on Bone Histology and Histomorphometry: The FREEDOM Study Extension

Denosumab reduced bone resorption, increased bone mineral density (BMD), and decreased new vertebral, hip, and nonvertebral fracture risk in postmenopausal women with osteoporosis in the FREEDOM trial. Consistent with its mechanism of action, transiliac crest bone biopsies from subjects treated with denosumab for 1 to 3 years demonstrated reduced bone turnover that was reversible upon treatment cessation. Long‐term denosumab treatment for up to 6 years in the FREEDOM extension provides sustained bone turnover reduction and continued low fracture incidence. Here, we evaluate 5 years of denosumab treatment on bone remodeling at the tissue level. Transiliac crest bone biopsies were obtained from 41 subjects (13 cross‐over and 28 long‐term from the FREEDOM placebo and denosumab groups, respectively) at year 2 of the FREEDOM extension, representing up to 5 years of denosumab treatment. Demographics for this subset were comparable to the overall extension cohort. The mean (SD) duration from the last denosumab dose to the first dose of tetracycline was 5.7 (0.5) months. Qualitative bone histology assessed in all biopsy samples was unremarkable, showing normally mineralized lamellar bone. Structural indices, including trabecular bone volume, number, and surface, were similar between cross‐over and long‐term groups. Bone resorption was decreased as reflected by eroded surface in cross‐over and long‐term subjects. A total of 11 of 13 (85%) cross‐over subjects and 20 of 28 (71%) long‐term subjects had specimens with double or single tetracycline label in trabecular and/or cortical compartments; specimens from 5 cross‐over subjects and 10 long‐term subjects were evaluable for dynamic trabecular bone parameters. Dynamic remodeling indices were low for both groups and consistent with reduced bone turnover with denosumab. In conclusion, denosumab treatment through 5 years resulted in normal bone quality with reduced bone turnover. These observations are consistent with its mechanism of action and associated with continued BMD increases and low fracture incidence. © 2014 American Society for Bone and Mineral Research.     

Measurement of growth and resorption of bone in the seventh caudal vertebra of the rat

A method for measuring bone formation and resorption rates in the seventh caudal vertebra of young rats during the growth period from 25–90 g is reported. This bone site has unique advantages over those previously studied, being symmetrical in transverse section and uncomplicated by cortical drift. Utilising three different coloured ultraviolet fluorescent bone labelling substances administered at either 3, 5 or 7 day intervals, the nature of growth changes was determined from undecalcified central transverse sections of the seventh caudal vertebrae. Linear bone formation occurred at a mean rate of 10.0 μ/day and linear bone resorption at 6.1 μ/day. During the growth period studied, bone formation occurred exclusively on the periosteal surface and bone resorption on the endosteal surface. Bone existing in this site in 25 g animals was completely removed by endosteal resorption within 22–26 days. Evidence exists that in older rats endosteal bone formation occurs and renders the site unsuitable for long term studies.     

Cortical Bone Resorption in Osteoporosis

A study was made of 110 women: 35 healthy premenopausal, 40 healthy postmenopausal, and 35 women diagnosed as having postmenopausal osteoporosis. The postmenopausal women had similar ages and years since menopause (YSM). In all of the women, total bone mass was evaluated by dual-energy X-ray absorptiometry and metacarpal morphometry was evaluated by radiogrammetry on the second metacarpal of the nondominant hand, performed by computed radiography. An external metacarpal diameter of ≥7.4 mm was required as proof of having developed an adequate peak bone mass. The endosteal diameter, which is indicative of bone resorption in both groups of postmenopausal women, obtained in the postmenopausal groups was subtracted from the endosteal diameter obtained in the premenopausal group and the resulting figure was divided by the years since menopause to calculate the rate of cortical bone resorption/year for each group. The endosteal diameters values differed in the three groups studied (P < 0.0001): 3.2 ± 0.7 mm in the healthy premenopausal women; 3.9 ± 0.6 mm in the healthy postmenopausal women; and 4.7 ± 0.5 mm in the osteoporotic postmenopausal women. The rate of cortical bone resorption was 0.068 ± 0.002 mm/YSM (years since menopause) in the osteoporotic postmenopausal women and 0.033 ± 0.003 mm/YSM in the healthy postmenopausal women (P < 0.0001). These figures reflect the importance of bone resorption, as opposed to deficient bone formation, as a cause of osteoporosis. Received: 27 January 1995 / Accepted: 21 August 1996     

The Impact of Acute Kidney Injury With Temporary Dialysis on the Risk of Fracture

Acute kidney injury (AKI) has a negative impact on long‐term renal function and prognosis. However, the association between acute renal dysfunction and long‐term effects on bone disorders has not yet been characterized. Using a population‐based cohort study, we aimed to evaluate associations between AKI and long‐term effects on bone fractures. We identified relevant data of all hospitalized patients aged >18 years with histories of dialysis‐requiring AKI, with subsequent recovery and discharge, from the claim records of the Taiwan National Health Insurance database between 2000 and 2008. We determined long‐term de novo bone fracture and all‐cause mortality after patients' index‐hospitalization discharge using propensity score–adjusted Cox proportional hazard model. Varying‐time models were used to adjust for long‐term effects of end‐stage renal disease (ESRD) on main outcomes. Among 448 AKI patients who had dialysis and survived 90 days after index‐hospitalization discharge without reentering dialysis, 273 were male (60.9%) with a mean age of 61.4 ± 16.6 years. Controls included 1792 hospitalized patients without AKI, dialysis, or bone fracture history. In the AKI recovery group, bone fracture incidence was 320 per 10,000 person‐years and hazard ratio (HR) of long‐term bone fracture was 1.25 (p = 0.049) compared with the control group, independent of subsequent ESRD status (HR = 1.55; p = 0.01). Both AKI recovery status (HR = 2.31; p < 0.001) and time varying factor of bone fracture (HR = 1.43; p < 0.001) were independent predictors of mortality compared with controls. In conclusion, AKI requiring temporary dialysis independently increases long‐term risk of bone fracture, regardless of subsequent progression to ESRD. Long‐term bone fractures may negatively impact patient mortality. © 2014 American Society for Bone and Mineral Research.