Friedrich, Patricia F.

IntroductionVascularized bone allotransplantation may provide new options for reconstruction of segmental defects if problems of long‐term immune modulation can be solved. The current literature lacks an orthotopic large animal model, limited to bone and without the confounding effects of other tissue types, permitting a multifaceted evaluation before new methods are used clinically. The purpose of this study was to develop a large animal model for vascularized bone allotransplantation.Materials and methodsEight porcine hind limbs were dissected. Length, diameter, and location of all hindlimb vessels were measured and a single nutrient vessel supplying the tibial diaphysis identified enabling its use as a vascularized bone allotransplant. Four Yucatan minipigs were divided into two pairs with a major swine leukocyte antigen mismatch. A 3.5 cm tibial segment including its nutrient pedicle was raised simultaneously from each pig and transplanted into the matched defect of the other animal. Microarterial anastomosis of the pedicle and 3‐drug immunosuppression maintained VCA viability. Bone healing and limb function were followed for 16 weeks.ResultsA consistent tibia diaphyseal nutrient artery arose from the caudal tibial artery to enter bone a mean 2.8 mm distal to the tibial tubercle with a pedicle length of 6.6 ± 3.3 mm and diameter of 1.6 ± 0.2 mm. Using this pedicle, we reconstructed a 3.5 cm tibial defect with a vascularized bone allotransplant in four animals. Immediate weightbearing as well as progressive bone healing was demonstrated.ConclusionWe have developed a vascularized tibial bone allotranplantation large‐animal model suitable for future bone‐only allotranplantation research in mini‐pigs.

BackgroundThe purpose of this study was to identify which triple immunosuppressive protocols, currently used for vascularized composite allotransplantation in the clinic, will have the best effect on motor function recovery following nerve allograft reconstruction.MethodsEighty‐eight Lewis rats underwent a 1‐cm sciatic nerve allograft transplantation and skin graft from 44 Brown‐Norway rats. Group I received 0.9% isotonic saline (control); Group II, 2 mg/kg FK506; Group III, 1 mg/kg FK506 with 15 mg/kg mycophenolate mofetil (MMF); and Group IV, 2 mg/kg FK506 with 30 mg/kg MMF and prednisone. Each group consisted of 11 rats. After 12 weeks, motor function recovery was evaluated with isometric tetanic force, muscle mass, ankle contracture angle, electrophysiology, and nerve histomorphometry. Adequacy of immunosuppression was monitored with the transplanted skin graft. All data are expressed as a percentage of the contralateral side.ResultsIsometric tetanic force showed significantly better functional recovery in all groups treated with immunosuppression compared to control. Within the immunosuppression groups no significant difference was found: 42.1 ± 6.4% (Group I), 56.1 ± 12.4% (Group II), 58.4 ± 10.7% (Group III), and 61.3 ± 11.2% (Group IV). Group IV was superior to all other groups regarding ankle contracture (P < .05) and electrophysiology (P < .001). Skin graft rejection occurred in 41 and 0% (Groups III and IV, respectively).ConclusionsFK506 significantly enhanced motor recovery after allograft reconstruction. This effect was comparable between combination treatment (low‐dose FK506 and MMF) and triple therapy (high‐dose FK506 and MMF plus prednisolone). However, triple therapy was more effective in suppressing skin rejection.

OBJECTIVE Commercially available processed nerve allografts have been shown to be inferior to autografts in previous animal studies. The authors hypothesized that combining different processing and storage techniques will result in improved nerve ultrastructure preservation, lower immunogenicity, and minimized cellular debris. Different processing protocols were evaluated using chemical detergents, enzymes, and irradiation, with the addition the of enzyme elastase, were used. Additionally, the difference between cold and frozen storage was investigated. The goal of this study was to create an optimized nerve allograft. METHODS Fifty rat nerves were decellularized with modifications of previous protocols and the addition of elastase. Subsequently, the nerve segments were stored at either 4°C or −80°C. Both processed and fresh control nerves were analyzed with confocal microscopy using immunohistochemical staining on the basal lamina (laminin γ-1), Schwann cells (S100 protein), and immunogenicity using major histocompatibility complex–I (MHCI) staining. Morphology of the ultrastructure and amount of cellular debris were analyzed on cross-sections of the nerves stained with toluidine blue and H & E, and by using electron microscopy. RESULTS Nerve ultrastructure was preserved with all decellularization protocols. Storage at −80°C severely altered nerve ultrastructure after any decellularization method. Elastase was found to significantly reduce the immunogenicity and amount of Schwann cells, while maintaining good structural properties. CONCLUSIONS Reduced immunogenicity, diminished cellular debris, and the elimination of Schwann cells was observed when elastase was added to the nerve processing while maintaining ultrastructure. Storage at −80°C after the decellularization process heavily damaged the nerve ultrastructure as compared with cold storage. Further in vivo studies are needed to prove the nerve regenerative capacity of these optimized allografts.

Processed nerve allografts have become an alternative to repair segmental nerve defects, with results comparable with autografts regarding sensory recovery; however, they have failed to reproduce comparable motor recovery. The purpose of this study was to determine how revascularizaton of processed nerve allograft would affect motor recovery. Eighty‐eight rats were divided in four groups of 22 animals each. A unilateral 10‐mm sciatic nerve defect was repaired with allograft (group I), allograft wrapped with silicone conduit (group II), allograft augmented with vascular endothelial growth factor (group III), or autograft (group IV). Eight animals from each group were sacrificed at 3 days, and the remaining animals at 16 weeks. Revascularization was evaluated by measuring the graft capillary density at 3 days and 16 weeks. Measurements of ankle contracture, compound muscle action potential, tibialis anterior muscle weight and force, and nerve histomorphometry were performed at 16 weeks. All results were normalized to the contralateral side. The results of capillary density at 3 days were 0.99% ± 1.3% for group I, 0.33% ± 0.6% for group II, 0.05% ± 0.1% for group III, and 75.6% ± 45.7% for group IV. At 16 weeks, the results were 69.9% ± 22.4% for group I, 37.0% ± 16.6% for group II, 84.6% ± 46.6% for group III, and 108.3% ± 46.8% for group IV. The results of muscle force were 47.5% ± 14.4% for group I, 21.7% ± 13.5% for group II, 47.1% ± 7.9% for group III, and 54.4% ± 10.6% for group IV. The use of vascular endothelial growth factor in the fashion used in this study improved neither the nerve allograft short‐term revascularization nor the functional motor recovery after 16 weeks. Blocking allograft vascularization from surrounding tissues was detrimental for motor recovery. The processed nerve allografts used in this study showed similar functional motor recovery compared with that of the autograft. © 2014 Wiley Periodicals, Inc. Microsurgery 36:134–143, 2016.

BackgroundVascular 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.MethodsVascularized femoral bone transplants were transplanted from female Dark Agouti rats (DA;RT1a) to male Piebald Viral Glaxo (PVG;RT1c). 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).ResultsAt 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.ConclusionsThese 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. © 2014 Wiley Periodicals, Inc. Microsurgery 35:45–51, 2015.

The purpose of this study was to evaluate the effect of wrapping bioabsorbable nerve conduit around primary suture repair on motor nerve regeneration in a rat model. Forty rats were randomly divided into two experimental groups according to the type of repair of the rat sciatic nerve: group I had primary suture repair; group II had primary suture repair and bioabsorbable collagen nerve conduit (NeuraGen® 1.5 mm, Integra LifeSciences Corp., Plainsboro, NJ) wrapped around the repair. At 12 weeks, no significant differences in the percentage of recovery between the two groups were observed with respect to compound muscle action potentials, isometric muscle force, and muscle weight (P = 0.816, P = 0.698, P = 0.861, respectively). Histomorphometric analysis as compared to the non‐operative sites was also not significantly different between the two groups in terms of number of myelinated axons, myelinated fiber area, and nerve fiber density (P = 0.368, P = 0.968, P = 0.071, respectively). Perineural scar tissue formation was greater in primary suture repair group (0.36 ± 0.15) than in primary repair plus conduit wrapping group (0.17 ± 0.08). This difference was statistically significant (P < 0.001). Wrapping bioabsorbable nerve conduit around primary nerve repair can decrease perineural scar tissue formation. Although the scar‐decreasing effect of bioabsorbable nerve wrap does not translate into better motor nerve recovery in this study, it might have an effect on the functional outcome in humans where scar formation is much more evident than in rats. © 2014 Wiley Periodicals, Inc. Microsurgery 34:562–567, 2014.

Object Choline acetyltransferase (ChAT) is an enzyme synthesized within the body of a motor neuron whose role is to form the neurotransmitter acetylcholine. Quantification of ChAT levels in motor or mixed nerves has been proposed to provide information regarding the viability of a proximal nerve stump for motor neurotization following brachial plexus injury. To do so requires information regarding normal ChAT levels and those in injured nerves, as well as the correlation of ChAT level determined at surgery with eventual motor recovery. The purpose of this study was to determine ChAT activity in the normal and injured sciatic/peroneal nerve in a rat model, evaluate the correlation between ChAT and motor recovery, find the relationship between ChAT activity and isometric muscle force, and elucidate the parallel between ChAT activity and acetylcholinesterase (AChE) activity. Methods Sixty animals were divided into 3 groups. The sciatic nerves in Group 1 were transected without repair. Nerves in Group 2 were transected and repaired. Nerves in Group 3 sustained a crush injury followed by transection and reconstruction. All animals were allowed 12 weeks of recovery followed by evaluation of ChAT levels in the peroneal nerve, correlated with measures of maximal isometric tibialis anterior muscle force and muscle weight (the operated side normalized to the control side). Karnovsky AChE staining of peroneal nerve segments was also compared with radiochemical assay of ChAT activity in the same nerve. Results A significant difference in the tibialis anterior isometric tetanic force and the tibialis anterior muscle weight index (TAMI) was noted between Group 1 and Groups 2 and 3 (p < 0.0001); no significant difference was found comparing Group 2 with Group 3. The correlation between the force measurement and the TAMI was 0.382. Both AChE measurement and ChAT activity demonstrated significantly fewer fibers in the operated nerve compared with the contralateral nerve. Intergroup variability could also be illustrated using these tests. The correlation coefficient between the isometric tetanic force measurement and the ChAT analysis in Groups 1 and 2 was 0.468. The correlation for the AChE staining and the isometric tetanic force measurement was 0.111. The correlation between the TAMI and the ChAT levels was 0.773. The correlation between the TAMI and the AChE-stained fibers was 0.640. Correlating AChE staining to the ChAT analysis produced a correlation of 0.712. Conclusions The great variability in all groups and weak correlations to the functional muscle assessments and the ChAT radiochemical assay made this technique an unreliable method of determining motor nerve viability.