Revascularization and bone remodeling of frozen allografts stimulated by intramedullary sustained delivery of FGF‐2 and VEGF

Frozen bone allografts are susceptible to nonunion and fracture due to limited revascularization and incomplete bone remodeling. We aim to revascularize bone allografts by combining angiogenesis from implanted arteriovenous (AV) bundles with delivery of fibroblast growth factor (FGF‐2) and/or vascular endothelial growth factor (VEGF) via biodegradable microspheres. Rat femoral diaphyseal allografts were frozen at ?80°C, and heterotopically transplanted over a major histocompatibility mismatch. A saphenous AV bundle was inserted into the intramedullary canal. Growth factor was encapsulated into microspheres and inserted into the graft, providing localized and sustained drug release. Forty rats were included in four groups: (I) phosphate‐buffered saline, (II) FGF‐2, (III) VEGF, and (IV) FGF‐2 + VEGF. At 4 weeks, angiogenesis was measured by the hydrogen washout method and microangiography. Bone remodeling was evaluated by quantitative histomorphometry and histology. Bone blood flow was significantly higher in groups III and IV compared to control (p < 0.05). Similarly, bone remodeling was higher in VEGF groups. FGF‐2 had little effect on allograft revascularization. No synergistic effect was observed with use of both cytokines. Delivered in microspheres, VEGF proved to be a potent angiogenic cytokine, increasing cortical bone blood flow and new bone formation in frozen allografts revascularized with an implanted AV bundle. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1431–1436, 2011     

Recipient‐derived angiogenesis with short term immunosuppression increases bone remodeling in bone vascularized composite allotransplantation: A pilot study in a swine tibial defect model

Current vascularized composite allotransplantation (VCA) transplantation protocols rely upon life‐long immune modulation to maintain tissue perfusion. Alternatively, bone‐only VCA viability may be maintained in small animal models using surgical angiogenesis from implanted autogenous vessels to develop a neoangiogenic bone circulation that will not be rejected. This study tests the method's efficacy in a large animal model as a bridge to clinical practice, quantifying the remodeling and mechanical properties of porcine tibial VCAs. A segmental tibial defect was reconstructed in Yucatan miniature swine by transplantation of a matched tibia segment from an immunologically mismatched donor. Microsurgical repair of nutrient vessels was performed in all pigs, with simultaneous intramedullary placement of an autogenous arteriovenous (AV) bundle in Group 2. Group 1 served as a no‐angiogenesis control. All received 2 weeks of immunosuppression. After 16 weeks, micro‐CT and histomorphometric analyses were used to evaluate healing and remodeling. Axial compression and nanoindentation studies evaluated bone mechanical properties. Micro‐CT analysis demonstrated significantly more new bone formation and bone remodeling at the distal allotransplant/recipient junction and on the endosteal surfaces of Group 2 tibias (p = 0.03). Elastic modulus and hardness were not adversely affected by angiogenesis. The combination of 2 weeks of immunosuppression and autogenous AV‐bundle implantation within a microsurgically transplanted tibial allotransplant permitted long‐term allotransplant survival over the study period of 16 weeks in this large animal model. Angiogenesis increased bone formation and remodeling without adverse mechanical effects. The method may allow future composite‐tissue allotransplantation of bone without the risks associated with long‐term immunosuppression. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1242–1249, 2017.

     

Surgical angiogenesis: a new approach to maintain osseous viability in xenotransplantation

Chung Y‐G, Bishop AT, Giessler GA, Suzuki O, Platt JL, Pelzer M, Friedrich PF, Kremer T. Surgical angiogenesis: a new approach to maintain osseous viability in xenotransplantation. Xenotransplantation 2010; 17: 38–47. © 2010 John Wiley & Sons A/S. Abstract: Background: Large segmental osseous defects are challenging clinical problems. Current reconstructive methods, using non‐viable allografts, vascularized autografts or prostheses have significant rates of serious complications and failure. These include infection, stress fracture and non‐union (frozen structural allogenic bone); loosening and implant failure (prosthetic replacement); limited availability, poor match of size and shape and donor site morbidity (vascularized autograft bone). In the future, microvascular transplantation of living allogenic or xenogenic bone could solve some of these issues, combining the advantages of living bone autografts (capability of primary osseous healing, remodeling, and fracture resistance) with the ability to match size and shape, provide immediate stability and avoid donor site morbidity. Xenotransplants would be particularly attractive, as they could be readily available, if long‐term bone survival could be achieved without unacceptable morbidity. Here, we present a preliminary study to evaluate a new and unique method to maintain xenogenic bone circulation without need for long‐term immune modulation that depends upon generation of a neo‐angiogenic circulation within the transplanted bone from recipient‐derived vessels. Thus, only short‐term immunosuppression would be required to achieve bone survival. Methods: One hundred and forty‐one hamster femora were microsurgically transplanted to rats, restoring nutrient vessel circulation with standard microvascular anastomoses. At the same time, a host‐derived arteriovenous bundle (AVB) was placed within the medullary canal. Two independent variables were evaluated: use of tacrolimus/cyclophosmamid immunosuppression (IS) and patency of the implanted AVB. Rats were therefore randomized to four groups; group 1—no IS + patent AVB; group 2—no IS + ligated AVB; group 3—IS + patent AVB; group 4—IS + ligated AVB. Rats were sacrificed after 1 or 2 weeks. We evaluated bone blood flow (microsphere entrapment), neoangiogenesis (microangiography and quantification of capillary density), bone necrosis rate (osteocyte counts) and nutrient pedicle rejection (microsurgical anastomotic patency). Statistical Analysis was performed with two‐way ANOVA with Bonferroni adjustment. Differences were considered significant when P < 0.05. Results: Capillary density was significantly increased with a patent intramedullary AVB (groups 1/3) compared to groups with ligated AVBs (groups 3/4). Capillary sprouting was predominantly restricted to the endosteal layer. Most nutrient pedicles (78.7%) stayed patent in groups with IS (groups 3 and 4). Consequently, bone blood flow was significanty higher in groups 3 and 4 compared to groups 1 and 2. Similary, a patent AV bundle improved flow in group 1 when compared to group 2. The bone necrosis rate was not influenced by the presence of patent AVBs but was significantly reduced in groups 3 and 4. Conclusions: Surgical angiogenesis occurs when patent arteriovenous bundles are placed in the medullary canal of xenogenic bone and leads to increased bone blood flow. Bone viability was not significantly influenced by neoangiogenesis. Although capillary sprouting was restricted to the endosteal layer in this short term study, more complete cortical revascularization might be observed in a long‐term study. Such a study should further evaluate whether these new vessels supply sufficient blood flow to maintain long‐term bone viability and allow remodeling.     

Effect of intramedullary polymethylmethacrylate and autogenous cancellous bone on healing of frozen segmental allografts

This study was designed to compare bone mineral density, periosteal callus production. new bone formation, bone porosity, histologic appearance, and union of mid-diaphyseal segmental allografts of the femur stabilized with an interlocking nail technique in a canine model 4 months after the procedure. An in vivo study was performed to compare the effects of augmenting interlocking nail fixation with an interlocking nail alone, intramedullary polymethylmethacrylate, intramedullary polymelhylmethacrylate and autogenous cancellous bone applied to the periosteal surface of the host-allograft junction, autogenous cancellous bone applied to the endosteal surface of the allograft, autogenous cancellous bone applied to the periosteal surface of the host-allograft junction, and autogenous cancellous bone applied to the periosteal surface of the hostallograft junction and to the endosteal surface of the allograft. There were no differences among treatments for bone mineral density at any time interval. Callus area 4 weeks after the procedure was greater along the lateral and cranial surfaces for treatments with periosteal cancellous bone (p < 0.05). New bone within the allograft segment did not differ among treatments and was reduced compared with the host-allograft junctions (p < 0.05). The amount and quality of bone tissue at the host-allograft junctions were greatest with treatments of intramedullary polymethylmethacrylate and autogenous cancellous bonc applied to the periostcal surface of the host-allograft junction and of autogenous cancellous bone applicd to the periosteal surface of the host-allograft junction and to the endosteal surface of the allograft (p < 0.05). The rate of bone union was lower, and there was a greater gap (non-bone tissue) remaining between host and allograft bone with treatment involving just intramedullary polymethylmethacrylate than with other treatments (p < 0.05). The results suggest that augmenting interlocking nail fixation with intramedullary polyrnethylmethacrylate by itself offers no advantage but that a combination of intramedullary polymethylmethacrylate and cancellous bone at the periosteal surface or of cancellous bone within the medullary canal and at the periosteal surface improves the quality of healing at 6 months.     

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     

Surgical Revascularization in Structural Orthotopic Bone Allograft Increases Bone Remodeling

Background

Osseous defects reconstructed with cryopreserved structural allografts are poorly revascularized and therefore are prone to nonunion, infection, deterioration of mechanical properties, and fracture. Whether this can be mitigated by specific interventions such as intramedullary surgical revascularization has been incompletely evaluated.

Questions/purposes

We aimed to study surgical revascularization as a means to improve bone remodeling in cryopreserved allograft. Second, we questioned whether spatial histomorphometric differences occur in cortical bone areas after intramedullary surgical revascularization. Third, biomechanical properties of the graft-recipient construct in surgically revascularized allograft were compared with those of conventional allografts.

Methods

Allografts were harvested from 10 Brown Norway rats, cryopreserved, and transplanted orthotopically in a 10-mm defect in two groups of 10 Lewis rats each (major histocompatibility mismatch). In the control group, no surgical revascularization was performed, whereas in the experimental group, a saphenous arteriovenous bundle was transposed in the bone marrow cavity. Bone remodeling was measured with histomorphometry, histology, and microcomputed tomography at 16 weeks. Spatial differences were analyzed with histomorphometry. To determine biomechanical properties, load at failure and structural stiffness in bending were evaluated by the three-point bend testing. In both groups, normal values of the contralateral femur also were analyzed.

Results

Surgically revascularized allografts had increased bone remodeling (bone formation rate to bone surface ratio: 130 ± 47 µm³/µm²/year versus 44 ± 43 µm³/µm²/year, p = 0.006) and higher cortical osteocyte counts (18.6% ± 12.7% versus 3.1% ± 2.8%, p = 0.002) than nonrevascularized grafts. In nonrevascularized grafts, the bone formation rate to bone surface ratio was 35% of the contralateral normal values, whereas in surgically revascularized grafts, the bone formation rate to bone surface ratio in the grafts exceeded the contralateral values (110%). Microcomputed tomography did not show differences in bone volume between groups, however in both groups, bone volume was less in grafts compared with the contralateral femurs. Inner cortical bone formation rate to bone surface ratio was greater in surgically revascularized grafts (65 ± 30 µm³/µm²/year versus 13 ± 16 µm³/µm²/year in the control group, p = 0.012). Outer cortical bone formation rate to bone surface ratio also increased in surgically revascularized grafts (49 ± 31 µm³/µm²/year versus 19 ± 21 µm³/µm²/year, p = 0.032). No differences were found in load at failure and structural stiffness between both groups. In the control group, load at failure and structural stiffness were lower in grafts than in the contralateral femurs (p = 0.004 and p = 0.02, respectively). In the experimental group, surgically revascularized grafts also had lower load at failure and structural stiffness than the contralateral femurs (p = 0.008 and p = 0.02, respectively).

Conclusions

Surgical revascularization of large segmental allografts improved bone remodeling and viability without an adverse effect on total bone volume or bending strength and stiffness in this short-term analysis.

Clinical Relevance

Cryopreserved allografts remain largely necrotic and are associated with a high rate of complications. Surgical revascularization increases graft healing which could contribute to graft survival with time.     

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     

Autogenous Arteriovenous Bundle Implantation Maintains Viability Without Increased Immune Response in Large Porcine Bone Allotransplants

BackgroundTransplantation of living allogeneic bone segments may permit reconstruction of large defects, particularly if viability is maintained without immunosuppression. Development of a new autogenous osseous blood supply accomplishes this goal in rodent experimental models. This study evaluates potential systemic and local inflammatory responses to this angiogenesis in a large-animal model.MethodsVascularized allogeneic tibia segments were transplanted orthotopically into matched tibial defects in Yucatan minipigs. Microvascular anastomoses of bone nutrient artery and vein were supplemented by intramedullary placement of an autogenous arteriovenous (AV) bundle in group 1. Group 2 served as a no-angiogenesis control. A 3-drug immunosuppression regimen was withdrawn after 2 weeks. During the 20-week survival period, periodic leukocyte counts and inflammatory cytokine levels were measured. Thereafter, osteocyte survival was quantified and transplant rejection graded by histologic examination and quantitative real-time polymerase chain reaction of immunologic markers.ResultsBoth groups developed an initial systemic response, which resolved after 4 to 6 weeks. No differences were seen in blood cytokine levels. Interleukin 2 expression was diminished in group 1 tibiae. As expected, nutrient pedicles had thrombosed without sustained immunosuppression, occluded by intimal hyperplasia. In group 1, angiogenesis from the autogenous AV bundle resulted in significantly less osteonecrosis (P = .04) and fibrosis (P = .02) than group 2 allotransplants.ConclusionsSystemic immune responses to large-bone allotransplants were not increased by generation of an autogenous osseous blood supply within porcine tibial bone allotransplants. Implanted AV bundles diminished inflammation and fibrosis and improved bone viability when compared to no-angiogenesis controls.     

Surgical Revascularization Induces Angiogenesis in Orthotopic Bone Allograft

Background

Remodeling of structural bone allografts relies on adequate revascularization, which can theoretically be induced by surgical revascularization. We developed a new orthotopic animal model to determine the technical feasibility of axial arteriovenous bundle implantation and resultant angiogenesis.

Questions/purposes

We asked whether arteriovenous bundles implanted in segmental allografts would increase cortical blood flow and angiogenesis compared to nonrevascularized frozen bone allografts and contralateral femoral controls.

Methods

We performed segmental femoral allotransplantation orthotopically from 10 Brown Norway rats to 20 Lewis rats. Ten rats each received either bone allograft reconstruction alone (Group I) or allograft combined with an intramedullary saphenous arteriovenous flap (Group II). At 16 weeks, we measured cortical blood flow with the hydrogen washout method. We then quantified angiogenesis using capillary density and micro-CT vessel volume measurements.

Results

All arteriovenous bundles were patent. Group II had higher mean blood flow (0.12 mL/minute/100 g versus 0.05 mL/minute/100 g), mean capillary density (23.6% versus 2.8%), and micro-CT vessel volume (0.37 mm³ versus 0.07 mm³) than Group I. Revascularized allografts had higher capillary density than untreated contralateral femora, while vessel volume did not differ and blood flow was lower.

Conclusions

Axial surgical revascularization in orthotopic allotransplants can achieve strong angiogenesis and increases cortical bone blood flow.

Clinical Relevance

Poor allograft revascularization results in frequent complications of nonunion, infection, and late stress fracture. The presented technique of surgical revascularization could therefore offer a beneficial adjunct to clinical segmental bone allografting.

     

骨膜瓣复合异体骨移植修复大段骨缺损

目的探讨带血管薄层皮质骨-骨膜瓣嵌入开窗的异体骨修复大段骨缺损的效果。方法将兔胫骨去抗原后制备异体骨标本,制作大段骨缺损动物模型。以带血管薄层皮质骨-骨膜瓣复合开窗的异体骨进行修复。将60只健康家兔随机分为3组:实验组、对照组及空白对照组。实验组以带血管薄层皮质骨-骨膜瓣嵌入开窗的异体骨进行修复;对照组以骨膜瓣直接包裹异体骨进行修复;空白对照组以异体骨直接进行修复。术后观察骨缺损大体标本及X线影像,对移植物及其周围软组织行组织学和免疫组织化学观察。结果实验组骨缺损骨痂形成并改造塑形,新生血管长入骨缺损处,其血管化、新骨形成和骨单位成熟均较对照组早。结论以带血管薄层皮质骨-骨膜瓣嵌入开窗的异体骨修复大段骨缺损的手术方法可缩短骨缺损修复的时间,优于以骨膜瓣直接包裹异体骨的方法。     

Zoledronate reduces unwanted bone resorption in intercalary bone allografts

Bone allografts are often hampered by graft incorporation and poor host bone formation. Bisphosphonates, synthetic pyrophosphate analogs, have shown promise in inhibiting bone resorption in human and animal trials. Some in vitro studies have suggested that high dose bisphosphonate may also inhibit bone formation, leading to our hypothesis that an ideal dose of bisphosphonate in allografts could protect allografts from resorption. We transplanted intercalary allografts in to the segmental defect of the rat femurs after soaking each allograft in zoledronate solution (30 μM) and then analysed bone density of the allografts six to 12 weeks after transplantation. At six and 12 weeks, the bone mineral density was higher in the experimental group compared with the control group. Qualitative radiographic and histological analysis also revealed more allograft resorption in the control group than in the zoledronate-treated group. Our data indicate that pharmacological modification of intercalary allografts with zoledronate solution can decrease osteoclast-mediated allograft resorption.     

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     

Perforations of cortical bone allografts improve their incorporation

The incorporation of perforated cortical bone allografts was compared with non-perforated allografts. A 5-cm circumferential defect in the middiaphysis at the tibia was created in adult sheep. A frozen tibial allograft was implanted and fixed with a locked nail for 6 months. There was no postoperative immobilization. Group I consisted of eight sheep with non-perforated allografts, whereas Group II was comprised of 10 sheep with perforated allografts. Union was evaluated radiographically, whereas the central part of the allograft had a densitometric evaluation. Creeping substitution was assessed on microradiographs from cross-sections of the central 3 cm of graft by measurement of porosity and percentage of new and old bone area within the confines of the graft. The width of periosteal and endosteal callus also was determined. There was no statistical difference between both groups for the union score and bone density. However, the cortical bone graft porosity and the amount of new bone within the cortical bone differed significantly between the perforated allografts and the non-perforated ones. Periosteal callus was similar in both groups, whereas endosteal callus was significantly more wide and extended in the perforated allografts. Perforation of a cortical bone substantially improved the amount of newly formed bone by the host when compared with a non-perforated bone. The creation of channels seemed to increase the interface between living soft tissues of the host and the allografted bone with a resulting enhanced incorporation.     

Intercalary tibial allografts following tumor resection: the role of fibular centralization

Options to reconstruct intercalary tibial defects include allografts, vascularized bone transfers, autogenous cortical grafts, endoprostheses, and Ilizarov bone transport. Five patients underwent intercalary bulk allograft reconstruction following en bloc resection of tibial sarcomas. Two patients underwent immediate fibular centralization and iliac crest bone grafting in addition to the allograft. Two patients who underwent fibular centralization during primary reconstruction united uneventfully. The remaining three patients developed nonunion, of which one was successfully salvaged by fibular centralization. A combined allograft transplant and fibular centralization with iliac crest bone grafting is an effective procedure to reconstruct the tibial diaphysis, as well as a salvage procedure for allograft nonunion.     

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.     

Induction of angiogenesis and osteogenesis in surgically revascularized frozen bone allografts by sustained delivery of FGF‐2 and VEGF

Large conventional bone allografts are susceptible to fracture and nonunion due to incomplete revascularization and insufficient bone remodeling. We aim to improve bone blood flow and bone remodeling using surgical angiogenesis combined with delivery of fibroblast growth factor (FGF‐2) and vascular endothelial growth factor (VEGF). Frozen femoral allografts were heterotopically transplanted in a rat model. The saphenous arteriovenous bundle was implanted within the graft medullary canal. Simultaneously, biodegradable microspheres containing phosphate buffered saline (control), FGF‐2, VEGF, or FGF‐2 + VEGF were placed within the graft. Rats were sacrificed at 4 and 18 weeks. Angiogenesis was determined by quantifying bone capillary density and measuring cortical bone blood flow. Bone remodeling was assessed by histology, histomorphometry, and alkaline phosphatase activity. VEGF significantly increased angiogenesis and bone remodeling at 4 and 18 weeks. FGF‐2 did not elicit a strong angiogenic or osteogenic response. No synergistic effect of FGF‐2 + VEGF was observed. VEGF delivered in microspheres had superior long‐term effect on angiogenesis and osteogenesis in surgically revascularized frozen bone structural allografts as compared to FGF‐2 or FGF‐2 + VEGF. Continuous and localized delivery of VEGF by microencapsulation has promising clinical potential by inducing a durable angiogenic and osteogenic response in frozen allografts. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1556–1562, 2012     

Cryopreservation of osteochondral allografts: dimethyl sulfoxide promotes angiogenesis and immune tolerance in mice

BACKGROUND: Although transplantation of cryopreserved bone allografts has become a routine procedure in orthopaedic surgery, biological and immunological impairment remains an unsolved problem that causes clinical failures. Experimental and clinical evidence has indicated that bone grafts that are revascularized early remain viable and contribute to union at the recipient site. Unprotected cryopreservation, used in most bone banks to reduce graft antigenicity, is associated with complete loss of graft viability, potentially contributing to graft failure. The differences in the survival of various cell types during cryopreservation with use of dimethyl sulfoxide, particularly the increased sensitivity of leukocytes to fast freezing, has resulted in a new approach to modulate immunogenicity. On the basis of this concept, it was proposed that a reduction in the immune response and enhanced revascularization of osteochondral allografts could be achieved by rapid cryopreservation with dimethyl sulfoxide. To test this hypothesis, angiogenesis and immune tolerance were quantified in a murine model with use of intravital microscopy. METHODS: Fresh osteochondral tissue and osteochondral tissue that had been cryopreserved with and without dimethyl sulfoxide was transplanted into dorsal skinfold chambers as isografts and as allografts in presensitized and nonsensitized recipient mice. To quantify angiogenesis, the onset of hemorrhages in the vicinity of the grafts and the revascularization of the grafts were determined by means of intravital fluorescence microscopy. To determine the recipient's intravascular immune response to the grafts, the leukocyte-endothelium interaction was assessed on the twelfth day after transplantation. RESULTS: Nine of nine fresh isografts were revascularized at a mean (and standard deviation) of 57 +/- 33 hours, eight of nine isografts that had been cryopreserved with dimethyl sulfoxide were revascularized at 98 +/- 50 hours, and zero of nine isografts that had been cryopreserved without dimethyl sulfoxide were revascularized. Seven of seven fresh allografts were revascularized at 53 +/- 6 hours, and ten of ten allografts that had been cryopreserved with dimethyl sulfoxide were revascularized at 82 +/- 29 hours. However, signs of revascularization faded in four of the seven fresh allografts whereas reperfusion was maintained in the majority (seven) of the ten grafts frozen in the presence of dimethyl sulfoxide. Similar to the findings associated with unprotected frozen isografts, zero of ten unprotected frozen allografts were revascularized. None of the allografts that had been transplanted into presensitized recipients were revascularized, regardless of whether they had been implanted fresh (nine grafts) or had been implanted after protected (eight grafts) or unprotected (nine grafts) freezing. Quantification of the leukocyte-endothelium interaction revealed a reduction in the intravascular immune response to frozen allografts (both protected and unprotected) compared with fresh allografts. CONCLUSION: Osteochondral allografts that had been pretreated by cryopreservation with dimethyl sulfoxide demonstrated improved angiogenesis induction and enhanced immune tolerance compared with unprotected frozen grafts. A selective reduction in donor passenger leukocytes is the proposed mechanism underlying this phenomenon. Clinical Relevance: In the absence of presensitization, cryopreservation with dimethyl sulfoxide appears to reduce the immune response to allografts and to enhance their revascularization; in the presence of presensitization, alternatives to allograft transplantation should be considered since the allografts will be exposed to a deleterious immune response.     

Effect of unreamed, limited reamed, and standard reamed intramedullary nailing on cortical bone porosity and new bone formation

OBJECTIVE: To compare the effects of unreamed nail insertion and reamed nail insertion with limited and standard canal reaming on cortical bone porosity and new bone formation. DESIGN: A canine segmental tibial fracture was created in fifteen adult dogs. The tibiae were stabilized with a statically locked 6.5-millimeter intramedullary nail without prior canal reaming (n = 5), after limited reaming to 7.0 millimeters (n = 5), or after standard canal reaming to 9.0 millimeters (n = 5). Porosity, new bone formation, and the mineral apposition rate of cortical bone were directly compared between the three nailing techniques. RESULTS: A significant increase in cortical bone porosity and new bone formation was seen in all three groups of experimental animals compared with the control tibiae. The overall lowest porosity levels were measured in the limited reamed group, with similar porosity levels measured in the unreamed and standard reamed groups. Porosity was lower in the limited reamed group in the entire cortex of the segmental and distal cross sections, as well as the endosteal, anterior, and posterior cortices along the length of the tibia. Overall, there was no difference in the amount of new bone formation or the mineral apposition rate between the three groups of animals at eleven weeks after surgery. DISCUSSION: The results of this study suggest that limited intramedullary reaming is a biologically sound alternative for the treatment of tibial diaphyseal fractures in which the circulation is already compromised.     

Comparison of meniscal allograft transplantation techniques using a preclinical canine model

Meniscal allograft transplantation (MAT) can be a safe, effective treatment for meniscal deficiency resulting in knee dysfunction, leading to osteoarthritis (OA) without proper treatment with 5‐year functional success rates (75%‐90%). While different grafts and techniques have generally proven safe and effective, complications include shrinkage, extrusion, progression of joint pathology, and failure. The objective of this study was to assess the functional outcomes after MAT using three different clinically‐relevant methods in a preclinical canine model. The study was designed to test the hypothesis that fresh meniscal‐osteochondral allograft transplantation would be associated with significantly better function and joint health compared with fresh‐viable or fresh‐frozen meniscus‐only allograft transplantations. Three months after meniscal release to induce meniscus‐deficient medial compartment disease, research hounds (n = 12) underwent MAT using meniscus allografts harvested from matched dogs. Three MAT conditions (n = 4 each) were compared: frozen meniscus–fresh‐frozen meniscal allograft with menisco‐capsular suture repair; fresh meniscus–fresh viable meniscal allograft (Missouri Osteochondral Preservation System (MOPS)‐preservation for 30 days) with menisco‐tibial ligament repair; fresh menisco‐tibial–fresh, viable meniscal‐tibial‐osteochondral allografts (MOPS‐preservation for 30 days) with menisco‐tibial ligament preservation and autogenous bone marrow aspirate concentrate on OCA bone. Assessment was performed up to 6 months after MAT. Pain, comfortable range of motion, imaging, and arthroscopic scores as well histological and cell viability findings were superior (P < .05) for the fresh menisco‐tibial group compared with the two other groups. Novel meniscal preservation and implantation techniques with fresh, MOPS‐preserved, viable meniscal‐osteochondral allografts with menisco‐tibial ligament preservation appears to be safe and effective for restoring knee function and joint health in this preclinical model. This has the potential to significantly improve outcomes after MAT.     

PTH‐enhanced structural allograft healing is associated with decreased angiopoietin‐2–mediated arteriogenesis,mast cell accumulation,and fibrosis

Recombinant parathyroid hormone (rPTH) therapy has been evaluated for skeletal repair in animal studies and clinical trials based on its known anabolic effects, but its effects on angiogenesis and fibrosis remain poorly understood. We examined the effects of rPTH therapy on blood vessel formation and osseous integration in a murine femoral allograft model, which caused a significant increase in small vessel numbers, and decreased large vessel formation (p < 0.05). Histology showed that rPTH also reduced fibrosis around the allografts to similar levels observed in live autografts, and decreased mast cells at the graft‐host junction. Similar effects on vasculogenesis and fibrosis were observed in femoral allografts from Col1caPTHR transgenic mice. Gene expression profiling revealed rPTH‐induced angiopoietin‐1 (8‐fold), while decreasing angiopoietin‐2 (70‐fold) at day 7 of allograft healing. Finally, we show anti‐angiopoietin‐2 peptibody (L1‐10) treatment mimics rPTH effects on angiogenesis and fibrosis. Collectively, these findings show that intermittent rPTH treatment enhances structural allograft healing by two processes: (1) anabolic effects on new bone formation via small vessel angiogenesis, and (2) inhibition of angiopoietin‐2–mediated arteriogenesis. The latter effect may function as a vascular sieve to limit mast cell access to the site of tissue repair, which decreases fibrosis around and between the fractured ends of bone. Thus, rPTH therapy may be generalizable to all forms of tissue repair that suffer from limited biointegration and excessive fibrosis. © 2013 American Society for Bone and Mineral Research.