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     

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.

     

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     

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.     

Augmentation of surgical angiogenesis in vascularized bone allotransplants with host‐derived a/v bundle implantation,fibroblast growth factor‐2, and vascular endothelial growth factor administration

We have previously shown experimental transplantation of living allogeneic bone to be feasible without long‐term immunosuppression by development of a recipient‐derived neoangiogenic circulation within bone. In this study, we examine the role of angiogenic cytokine delivery with biodegradable microspheres to enhance this process. Microsurgical femoral allotransplantation was performed from Dark Agouti to Piebald Virol Glaxo rats. Poly(D,L‐lactide‐co‐glycolide) microspheres loaded with buffer, basic fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), or both, were inserted intramedullarly along with a recipient‐derived arteriovenous (a/v) bundle. FK‐506 was administered daily for 14 days, then discontinued. At 28 days, bone blood flow was measured using hydrogen washout. Microangiography, histologic, and histomorphometric analyses were performed. Capillary density was greater in the FGF+VEGF group (35.1%) than control (13.9%) (p < 0.05), and a linear trend was found from control, FGF, VEGF, to FGF+VEGF (p < 0.005). Bone formation rates were greater with VEGF (p < 0.01) and FGF+VEGF (p < 0.05). VEGF or FGF alone increased blood flow more than when combined. Histology rejection grading was low in all grafts. Local administration of vascular and fibroblast growth factors augments angiogenesis, bone formation, and bone blood flow from implanted blood vessels of donor origin in vascularized bone allografts after removal of immunosuppression. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1015–1021, 2010     

Clinical experience in allogeneic vascularized bone and joint allografting

The allotransplantation of vascularized femoral diaphyses and total knee joints is a novel approach in orthopedic surgery. Allogeneic femoral diaphyses were transplanted into three patients suffering from chondrosarcoma or posttraumatic defects. Total knee joints allografts were transplanted in five patients with large bone defects of the knee and loss of the extensor apparatus caused either by major trauma alone or infection after a major trauma. Bone segments and total joints were harvested from multi-organ donors, perfused with UW-solution and transplanted within cold ischemia times of 18-25 h. Patients were immunosuppressed postoperatively primarily with cyclosporine (Cyclosporin A) and azathioprine. Two allografts (1 femur, 1 knee) were lost due to infections. Seven of the eight patients are able to walk with full weight-bearing posttransplant. Two of the patients with transplanted joint allografts subsequently received total knee arthroplasty implantations. Vascularized bone and joint allotransplantation may serve as a last line of defense treatment before considering lower limb amputation.     

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     

VEGF-promoted surgical angiogenesis in necrotic bone

The ability of vascular endothelial growth factor (VEGF) to accelerate neoangiogenesis from implanted arterovenous (AV) bundles in necrotic bone was evaluated. A saphenous AV bundle was placed in a necrotic segment of rabbit ilium. In group II, VEGF (100 ng/h x 3 days) was administered by continuous infusion. Bone blood flow was measured with radioactive-labeled microspheres, and capillary density was determined by microangiography combined with Sp?lteholtz bone clearing at 1, 2, and 4 weeks. Neovascularization was observed along the implanted vascular bundle in both groups. One week after surgery, bone blood flow and vessel area were significantly higher in VEGF-treated animals (P < 0.05). No significant difference was observed at later times. Direct VEGF administration increased surgical angiogenesis and improved blood flow and neovascularization in necrotic bone 1 week after AV bundle implantation. Thereafter, a robust angiogenic response from the AV bundle was seen in both groups.     

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.     

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.     

Rabbit articular cartilage defects treated by allogenic chondrocyte transplantation

Articular cartilage defects have a poor capacity for repair. Most of the current treatment options result in the formation of fibro-cartilage, which is functionally inferior to normal hyaline articular cartilage. We studied the effectiveness of allogenic chondrocyte transplantation for focal articular cartilage defects in rabbits. Chondrocytes were cultured in vitro from cartilage harvested from the knee joints of a New Zealand White rabbit. A 3 mm defect was created in the articular cartilage of both knees in other rabbits. The cultured allogenic chondrocytes were transplanted into the defect in the right knees and closed with a periosteal flap, while the defects in the left knees served as controls and were closed with a periosteal flap alone, without chondrocytes. Healing of the defects was assessed at 12 weeks by histological studies. Allogenic chondrocyte transplantation significantly increased the amount of newly formed repair tissue (P=0.04) compared with that found in the control knees. The histological quality score of the repair tissue was significantly better (P=0.05), with more hyaline characteristics in the knees treated with allogenic chondrocytes than in the control knees. Articular cartilage defects treated with allogenic chondrocyte transplantation result in better repair tissue formation with hyaline characteristics than those in control knees.     

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.     

Bone and cartilage allotransplantation. A review of 14 years of research and clinical studies

The authors review their experience of over 14 years in the field of osteochondral allotransplantation. Experimental studies demonstrated the immunogenicity of bone and cartilage, immunosuppression in skeletal transplantation models, and a subset of myeloid cells within bone marrow with strong immunogenic properties. Clinical results of knee joint resurfacing with fresh small-fragment osteochondral allografts have been best in posttraumatic joints. Experience with allograft reconstructions of skeletal defects after tumor surgery has been gratifying as a limb salvage procedure. Microvascular fibular autografts have been an important adjunct in massive reconstructive osteochondral transplantation.     

二膦酸盐对兔膝关节不稳早期软骨 下骨生化改变的影响

 目的观察兔膝关节不稳早期软骨下骨基质金属蛋白酶-9(matrix metallo proteinase-9,MMP-9)和组织蛋白酶K(cathepsinK,CK)的表达变化及二膦酸盐的抑制作用,探讨其机制及二膦酸盐的作用途径。方法健康雄性新西兰大白兔50只,随机数字表法分为对照组(n=10)、模型组(n=20)和二膦酸盐组(n=20)。采用Hulth兔膝关节不稳模型的制作方法右侧膝关节造模,分别于造模后2周和10周行空气栓塞处死,各时相点模型组和二膦酸盐组各取10个右膝关节的股骨内侧髁,对照组取5个右膝关节的股骨内侧髁,用SP免疫组织化学染色方法检测各组在2周和10周时软骨下骨MMP-9和CK的表达变化,并进行比较分析。结果对照组、二膦酸盐组和模型组在兔膝关节不稳造模后2周时均有MMP-9和CK阳性表达细胞,MMP-9阳性表达率分别为4%、10%和92%,CK阳性表达率分别为8%、12%和90%;10周时亦均有MMP-9和CK阳性表达细胞,MMP-9阳性表达率分别为4%、8%和72%,CK阳性表达率分别为8%、10%和68%。二膦酸盐能明显减少MMP-9和CK阳性表达;与模型组比较差异有统计学意义。 结论二膦酸盐可阻抑兔膝关节不稳早期软骨下骨MMP-9和CK的表达,抗骨吸收以减少软骨下骨的病理改变,从而保护关节软骨。     

Bilateral hand transplantation: bone healing under immunosuppression with tacrolimus, mycophenolate mofetil, and prednisolone

PURPOSE: Little is known about bone healing after composite tissue transplantation that requires pharmacologic immunosuppression. Bone integration and callus development were assessed in bilateral hand transplantation. METHODS: In this study the course of callus development and callus maturation were assessed by color Doppler sonography and radiography in a double hand transplant and compared with forearm replantation. RESULTS: After hand transplantation, ingrowth of small vessels at the bone junction was observed at week 3, calcified callus became visible at month 4, and bone union was completed at month 11. A similar time course of bone integration was observed after replantation. Plating offered sufficient stability. A recipient periostal flap is thought to have improved blood supply and favored development and induction of callus. CONCLUSIONS: Bone healing after hand transplantation under immunosuppression with tacrolimus, mycophenolate mofetil, and prednisolone is identical to that after forearm replantation.     

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     

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.     

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     

Cell-based treatment of osteochondral defects in the rabbit knee with natural and synthetic matrices: cellular seeding determines the outcome

Introduction: Matrix-associated transplantation of cartilage constructs is an appealing method in cartilage repair. Three different matrices seeded with allogenic chondrocytes were compared in an osteochondral defect model in the rabbit. An investigation was conducted to identify the best matrix for cell-based treatment of osteochondral defects in the rabbit knee joint. Materials and methods: Osteochondral defects (diameter 3 mm) were created in the trochlea and the femoral condyles of 33 New Zealand White rabbits, which were then treated with bioartificial cartilage constructs. The cartilage constructs were created in vitro using three different resorbable carrier materials (two fleece matrices: one of PLLA, and one composite of polydioxanon/polyglactin, as well as one consisting of lyophilized dura) cultured with isolated allogenic chondrocytes. The defects were evaluated macroscopically, by histological and immunhistological techniques, and by scanning electron microscopy after 6 weeks, 6 months, and 12 months. The chondrocyte-seeded constructs were compared to defects treated with carrier material alone as well as to untreated control defects. Results: There was a significant improvement in defect repair quality in the transport materials, which were cultured with chondrocytes prior to implantation (P<0.0005). No significant differences were observed between the three carrier matrices, and no significant differences were seen between the unseeded matrices and the untreated control defects. Conclusion: There is no difference in the outcome between the three tested matrices in the treatment of osteochondral defects in the rabbit knee. The results of this in vitro experiment are promising and with refinement may lead to useful clinical therapies.