Vascularized bone transplant chimerism mediated by vascular endothelial growth factor.

BACKGROUND: Vascular endothelial growth factor (VEGF) induces angiogenesis and osteogenesis in bone allotransplants. We aim to determine whether bone remodeling in VEGF-treated bone allotransplants results from repopulation with circulation-derived autogenous cells or survival of allogenic transplant-derived cells. METHODS: Vascularized femoral bone transplants were transplanted from female Dark Agouti rats (DA;RT1(a) ) to male Piebald Viral Glaxo (PVG;RT1(c) ). Arteriovenous bundle implantation and short-term immunosuppression were used to maintain cellular viability. VEGF was encapsulated in biodegradable microspheres and delivered intramedullary in the experimental group (n = 22). In the control group (n = 22), no VEGF was delivered. Rats were sacrificed at 4 or 18 weeks. Laser capture microdissection of bone remodeling areas was performed at the inner and outer cortex. Sex-mismatched genes were quantified with reverse transcription-polymerase chain reaction to determine the amount of male cells to total cells, defined as the relative expression ratio (rER). RESULTS: At 4 weeks, rER was significantly higher at the inner cortex in VEGF-treated transplants as compared to untreated transplants (0.622 ± 0.225 vs. 0.362 ± 0.081, P = 0.043). At 4 weeks, the outer cortex in the control group had a significantly higher rER (P = 0.038), whereas in the VEGF group, the inner cortex had a higher rER (P = 0.015). Over time, in the outer cortex the rER significantly increased to 0.634 ± 0.106 at 18 weeks in VEGF-treated rats (P = 0.049). At 18 weeks, the rER was >0.5 at all cortical areas in both groups. CONCLUSIONS: These in vivo findings suggest a chemotactic effect of intramedullary applied VEGF on recipient-derived bone and could imply that more rapid angiogenesis of vascularized allotransplants can be established with microencapsulated VEGF.

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 µm(3)/µm(2)/year versus 44 ± 43 µm(3)/µm(2)/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 µm(3)/µm(2)/year versus 13 ± 16 µm(3)/µm(2)/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 µm(3)/µm(2)/year versus 19 ± 21 µm(3)/µm(2)/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.

Cell lineage in vascularized bone transplantation.

BACKGROUND: The biology behind vascularized bone allotransplantation remains largely unknown. We aim to study cell traffic between donor and recipient following bone auto-, and allografting. METHODS: Vascularized femoral transplantation was performed with arteriovenous bundle implantation and short-term immunosuppression. Twenty male Piebald Virol Glaxo (PVG; RT1(c) ) rats received isotransplants from female PVG (RT1(c) ) rats and 22 male PVG rats received allografts from female Dark Agouti rats (DA, RT1(a) ), representing a major histocompatibility mismatch. Both groups were randomly analyzed at 4 or 18 weeks. Bone remodeling areas (inner and outer cortical samples) were labeled and laser capture microdissected. Analysis of sex-mismatch genes by real-time reverse transcription-polymerase chain reaction provided the relative Expression Ratio (rER) of donor (female) to recipient (male) cells. RESULTS: The rER was 0.456 ± 0.266 at 4 weeks and 0.749 ± 0.387 at 18 weeks (p = 0.09) in allotransplants. In isotransplants, the rER was 0.412 ± 0.239 and 0.467 ± 0.252 at 4 and 18 weeks, respectively (p = 0.21). At 4 weeks, the rER at the outer cortical area of isotransplants was significantly lower in isotransplants as compared with allotransplants (0.247 ± 0.181 vs. 0.549 ± 0.184, p = 0.007). Cells in the inner and outer cortical bone remodeling areas in isotransplants were mainly donor derived (rER < 0.5) at 18 weeks, whereas allotransplants contained mainly recipient-derived cells (rER > 0.5) at 18 weeks. CONCLUSIONS: Applying novel methodology, we describe detailed cell traffic in vascularized bone transplants, elaborating our comprehension on bone transplantation.

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.

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 mm(3) versus 0.07 mm(3)) 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.

Description and validation of isometric tetanic muscle force test in rabbits.

Isometric tetanic muscle force has been described in a rat model to evaluate motor recovery in a segmental sciatic nerve defect reconstructions. However, to test longer nerve defects, an alternative and larger animal model is necessary. The purpose of this study is to describe and validate a technique for isometric force measurement of the tibialis anterior (TA) muscle in New Zealand rabbits. Muscle preload and electrical stimulation parameters were optimized to obtain the highest tetanic contraction bilaterally in 10 animals. Electrophysiology, muscle weight, peroneal nerve length, and histomorphometry were also analyzed. Only the peroneal nerve length and the ratio of highest muscle force/muscle weight demonstrated the equivalence between the sides. A small variability of TA muscle force and TA muscle weight was observed between the sides suggesting dominance. Optimization of electrical stimulation and preload as well as the use of correct anesthesia were fundamental to acquire the highest muscle force.

Vascularized bone grafting in a canine carpal avascular necrosis model.

BACKGROUND: Limited experimental research has been performed on the treatment of avascular necrosis (AVN) by vascularized bone grafting. QUESTIONS/PURPOSES: A new model simulating carpal AVN was created to investigate surgical revascularization of necrotic bone. METHODS: In seven mongrel dogs, AVN was induced by removal of the radial carpal bones bilaterally, deep-freezing, coating in cyanoacrylate, and reimplantation. A reverse-flow vascularized bone graft from the distal radius was implanted in the avascular radial carpal bone. The contralateral side served as an untreated ischemic control. Bone blood flow, bone volume, radiography, histomorphometry, histology, and MRI were analyzed at 4 weeks. RESULTS: Blood flow was substantially higher in grafted bones when compared with controls (14.68 ± 15.43 versus 0.27 ± 0.28 mL/minute/100 g). Blood flow correlated with increased osteoid formation and higher levels of bone turnover. T1 and T2 signals on MRI did not correlate with quantitative bone blood flow measurements. Necrotic bones with no blood flow had normal T1 and T2 signals, whereas revascularized bones had signal changes when compared with adjacent carpal bones. No major collapse occurred in any radiocarpal bone. CONCLUSION: In a canine experimental model, investigation of carpal AVN shows the ability of vascularized bone grafting to revascularize and remodel avascular bone. CLINICAL RELEVANCE: Surgical revascularization of necrotic bone induced by vascularized bone grafting results in increased bone perfusion and bone remodeling as compared with untreated necrotic bone. MRI T1 and T2 signals can be normal in necrotic avascular bone.

Clinical Challenges in Resection and Reconstruction of a Verrucous Carcinoma of the Right Calcaneus With a Medial Plantar Artery Perforator Flap: A Case Report.

A verrucous carcinoma is a rare, low-grade variant of a well-differentiated squamous cell carcinoma (SCC). It frequently occurs in Caucasian males aged 50 to 60. The tumour is locally destructive, grows into muscle, nerves and bones, but rarely metastasizes. Here we report a patient with verrucous carcinoma on the right calcaneus with the uncommon symptom of a haemorrhagic plaque at the centre and also an exophytic component. A 52-year-old man presented with a 10-year-old, progressive, painful, pruritic, exophytic growing, hyperkeratotic and haemorrhagic plaque of 5.2 x 3.5cm on the right calcaneus. The lesion emerged after extensive burns after blast trauma as a child. Excisional biopsy with 2mm margin in combination with clinical presentation favoured a verrucous carcinoma. The pathology report showed that the tumour was not excised radically; therefore, re-excision with 5mm margin was required and was performed two months later. The wound was closed with a medial plantar artery perforator (MPAP) flap. The donor site was closed with a full-thickness skin graft (FTSG) from the medial side of the right upper leg and was covered by a tie-over bandage. Verrucous carcinoma is a rare tumour and can be adequately surgically treated by excision with clear margins. In this case, the verrucous carcinoma of the right calcaneus was excised and reconstructed with an MPAP flap with decent results, despite a challenging postoperative course.

Complications in the Treatment of Delayed Union and Underlying Chronic Osteomyelitis After Right Crural Fracture Treated With Anterolateral Thigh Flap and Double-Barrelled Vascularized Fibula Graft.

BACKGROUND: Segmental bone defects pose a major, unsolved clinical challenge and may be the result of high-energy trauma, infection, and tumour resection or revision surgery. Several options exist to reconstruct, including Ilizarov bone transport, Masquelet technique, cylindrical mesh technique, allografts, and vascularized bone autografts. We present a patient with a delayed union of the tibia with concomitant chronic osteomyelitis treated with anterolateral thigh (ALT) flap and double-barrelled vascularized fibula graft. CASE PRESENTATION: A 60-year-old male with a chronic pretibial wound with underlying osteomyelitis of the right leg presented himself at the emergency department and was admitted to the surgical ward. He had complex chronic osteomyelitis of a tibial non-union after an earlier right crural fracture (a previous work-related accident). He was treated with an ALT flap and double-barrelled vascularized fibula graft, which was complicated with an additional fracture and breakage of osteosynthesis material. CONCLUSION: Segmental bone defects pose a major, unsolved clinical challenge in orthopaedic, trauma-surgical, and plastic surgical practice. Concomitant infections and fractures can be part of the postoperative course. Patients with complex segmental bone defects need to be treated by a multidisciplinary team including at least an (orthopaedic) trauma surgeon, a plastic surgeon, and an infectiologist.

Surgical angiogenesis with short-term immunosuppression maintains bone viability in rabbit allogenic knee joint transplantation.

BACKGROUND: Vascularized composite allotransplantation has the potential for reconstruction of joint defects but requires lifelong immunosuppression, with substantial risks. This study evaluates an alternative, using surgical angiogenesis from implanted autogenous vessels to maintain viability without long-term immunotherapy. METHODS: Vascularized knee joints were transplanted from Dutch Belted donors to New Zealand White rabbit recipients. Once positioned and revascularized microsurgically, a recipient-derived superficial inferior epigastric fascial flap and a saphenous arteriovenous bundle were placed within the transplanted femur and tibia, respectively, to develop a neoangiogenic, autogenous circulation. There were 10 transplants in group 1. Group 2 (n = 9) consisted of no-angiogenesis controls with ligated flaps and arteriovenous bundles. Group 3 rabbits (n = 10) were autotransplants with patent implants. Tacrolimus was used for 3 weeks to maintain nutrient flow during angiogenesis. At 16 weeks, the authors assessed bone healing, joint function, bone and cartilage mechanical properties, and histology. RESULTS: Group 1 allotransplants had more robust angiogenesis, better healing, improved mechanical properties, and better osteocyte viability than ligated controls (group 2). All three groups developed knee joint contractures and arthritic changes. Cartilage thickness and quality were poorer in allograft groups than in autotransplant controls. CONCLUSIONS: Surgical angiogenesis from implanted autogenous tissue improves bone viability, healing, and material properties in rabbit allogenic knee transplants. However, joint contractures and degenerative changes occurred in all transplants, regardless of antigenicity or blood supply. Experimental studies in a larger animal model with improved methods to maintain joint mobility are needed before the merit of living joint allotransplantation can be judged.

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.

Return of motor function after segmental nerve loss in a rat model: comparison of autogenous nerve graft, collagen conduit, and processed allograft (AxoGen).

BACKGROUND: An effective alternative to nerve autograft is needed to minimize morbidity and solve limited-availability issues. We hypothesized that the use of processed allografts and collagen conduits would allow recovery of motor function that is equivalent to that seen after the use of autografts. METHODS: Sixty-five Lewis rats were divided into three experimental groups. In each group, a unilateral 10-mm sciatic nerve defect was repaired with nerve autograft, allograft treated by AxoGen Laboratories, or a 2.0-mm-inner-diameter collagen conduit. The animals were studied at twelve and sixteen weeks postoperatively. Evaluation included bilateral measurement of the tibialis anterior muscle force and muscle weight, electrophysiology, assessment of ankle contracture, and peroneal nerve histomorphometry. Muscle force was measured with use of our previously described and validated method. Results were expressed as a percentage of the values on the contralateral side. Two-way analysis of variance (ANOVA) corrected by the Ryan-Einot-Gabriel-Welsch multiple range test was used for statistical investigation (α = 0.05). RESULTS: At twelve weeks, the mean muscle force (and standard deviation), as compared with that on the contralateral (control) side, was 45.2% ± 15.0% in the autograft group, 43.4% ± 18.0% in the allograft group, and 7.0% ± 9.2% in the collagen group. After sixteen weeks, the recovered muscle force was 65.5% ± 14.1% in the autograft group, 36.3% ± 15.7% in the allograft group, and 12.1% ± 16.0% in the collagen group. Autograft was statistically superior to allograft and the collagen conduit at sixteen weeks with regard to all parameters except histomorphometric characteristics (p < 0.05). The collagen-group results were inferior. All autograft-group outcomes improved from twelve to sixteen weeks, with the increase in muscle force being significant. CONCLUSIONS: The use of autograft resulted in better motor recovery than did the use of allograft or a collagen conduit for a short nerve gap in rats. A longer evaluation time of sixteen weeks after segmental nerve injuries in rats would be beneficial as more substantial muscle recovery was seen at that time.

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.

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.