Wouter F. Willems

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.

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.

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.

Knee joint transplantation combined with surgical angiogenesis in rabbits – a new experimental model

Purpose: We have previously described a means to maintain bone allotransplant viability, without long‐term immune modulation, replacing allogenic bone vasculature with autogenous vessels. A rabbit model for whole knee joint transplantation was developed and tested using the same methodology, initially as an autotransplant. Materials/Methods: Knee joints of eight New Zealand White rabbits were elevated on a popliteal vessel pedicle to evaluate limb viability in a nonsurvival study. Ten additional joints were elevated and replaced orthotopically in a fashion identical to allotransplantation, obviating only microsurgical repairs and immunosuppression. A superficial inferior epigastric facial (SIEF) flap and a saphenous arteriovenous (AV) bundle were introduced into the femur and tibia respectively, generating a neoangiogenic bone circulation. In allogenic transplantation, this step maintains viability after cessation of immunosuppression. Sixteen weeks later, X‐rays, microangiography, histology, histomorphometry, and biomechanical analysis were performed. Results: Limb viability was preserved in the initial eight animals. Both soft tissue and bone healing occurred in 10 orthotopic transplants. Surgical angiogenesis from the SIEF flap and AV bundle was always present. Bone and joint viability was maintained, with demonstrable new bone formation. Bone strength was less than the opposite side. Arthrosis and joint contractures were frequent. Conclusion: We have developed a rabbit knee joint model and evaluation methods suitable for subsequent studies of whole joint allotransplantation.

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.

Cell lineage in vascularized bone transplantation.

The biology behind vascularized bone allotransplantation remains largely unknown. We aim to study cell traffic between donor and recipient following bone auto‐, and allografting.

Vascularized bone transplant chimerism mediated by vascular endothelial growth factor

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.