Guilherme Giusti

Comparison and characterization of multiple biomaterial conduits for peripheral nerve repair

Four biomaterial tubes, poly(lactic-co-glycolic acid) (PLGA), poly(caprolactone fumarate) (PCLF), a neutral oligo[(polyethylene glycol) fumarate] (OPF) hydrogel or a positively charged oligo[(polyethylene glycol) fumarate] (OPF(+)) hydrogel with a PCLF sleeve, have previously been shown to have benefits for nerve repair. However, no direct comparison to identify the optimal material have been made. Herein, these nerve tubes were implanted in a rat sciatic nerve model and nerve regeneration was quantified and compared by using accepted nerve assessment techniques. Using standard statistical methods, no significant differences of individual parameters were apparent between groups despite PCLF showing a tendency to perform better than the others. Using a mean-variance based ranking system of multiple independent parameters, statistical differences became apparent. It was clear that the PLCF tube supported significantly improved nerve regeneration and recovery compared to the other three biomaterial conduits. The ability to simultaneously compare a number of regenerative parameters and elucidate the best material from the combination of these individual parameters is of importance to the nerve regeneration area and has implications for the tissue engineering field. By using this method of comparison, a number of biomaterial constructs may be compared under similar conditions and the optimal construct elucidated using the minimal number of animals and materials.

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.

The effect of collagen nerve conduits filled with collagen- glycosaminoglycan matrix on peripheral motor nerve regeneration in a rat model

BACKGROUND Bioabsorbable unfilled synthetic nerve conduits have been used in the reconstruction of small segmental nerve defects with variable results, especially in motor nerves. We hypothesized that providing a synthetic mimic of the Schwann cell basal lamina in the form of a collagen-glycosaminoglycan (GAG) matrix would improve the bridging of the nerve gap and functional motor recovery. METHODS A unilateral 10-mm sciatic nerve defect was created in eighty-eight male Lewis rats. Animals were randomly divided into four experimental groups: repair with reversed autograft, reconstruction with collagen nerve conduit (1.5-mm NeuraGen, Integra LifeSciences), reconstruction with collagen nerve conduit filled with collagen matrix, and reconstruction with collagen nerve conduit filled with collagen-GAG (chondroitin-6-sulfate) matrix. Nerve regeneration was evaluated at twelve weeks on the basis of the compound muscle action potential, maximum isometric tetanic force, and wet muscle weight of the tibialis anterior muscle, the ankle contracture angle, and nerve histomorphometry. RESULTS The use of autograft resulted in significantly better motor recovery compared with the other experimental methods. Conduit filled with collagen-GAG matrix demonstrated superior results compared with empty conduit or conduit filled with collagen matrix with respect to all experimental parameters. Axon counts in the conduit filled with collagen-GAG matrix were not significantly different from those in the reversed autograft at twelve weeks after repair. CONCLUSIONS The addition of the synthetic collagen basal-lamina matrix with chondroitin-6-sulfate into the lumen of an entubulation repair significantly improved bridging of the nerve gap and functional motor recovery in a rat model. CLINICAL RELEVANCE Use of a nerve conduit filled with collagen-GAG matrix to bridge a motor or mixed nerve defect may result in superior functional motor recovery compared with commercially available empty collagen conduit. However, nerve autograft remains the gold standard for reconstruction of a segmental motor nerve defect.

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.

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.

Effect of Vascular Endothelial Growth Factor Administration on Nerve Regeneration after Autologous Nerve Grafting

BACKGROUND The aim of this study was to evaluate the effect of vascular endothelial growth factor (VEGF) administration around the autologous nerve graft on nerve recovery in a rat model. METHODS A total of 69 rats were randomly divided into three experimental groups. A 10-mm sciatic nerve defect was made and reconstructed with the reversed nerve segment. Group I received an osmotic pump with saline, group II received an osmotic pump with VEGF, and group III added a silicone tube around the nerve graft to decrease the surrounding blood supply. Nine animals in each group were sacrificed on day 3 to evaluate improvement in new vessel formation. In each group 14 animals were sacrificed at 16 weeks after the initial procedure to evaluate the functional motor nerve regeneration using compound muscle action potential, isometric tetanic force, wet muscle weight, and nerve histomorphometry. RESULTS The average vascular density on day 3 was 10.7% in group I, 21.4% in group II, and 0.9% in group III. These differences were significant. However, the average maximum isometric tetanic force at 16 weeks was 54.4% in group I, 57.5% in group II, and 47.6% in group III. No difference was found with or without VEGF administration. Histomorphometric analysis was also not significantly different between the groups. CONCLUSIONS New vessel formation on autologous nerve graft was enhanced by VEGF administration. However, the neovascularization effect of VEGF administration did not translate into better motor nerve recovery.

Validation of Isometric Tetanic Force as a Measure of Muscle Recovery After Nerve Injury in the Rabbit Biceps

PURPOSE The purpose of this study was to describe and validate a technique for measurement of isometric tetanic force (ITF) in the rabbit biceps muscle. MATERIALS AND METHODS Eighteen New Zealand White rabbits were randomized to test either the right side or the left side first. Under propofol anesthesia, the brachial plexus and biceps brachii were exposed. The middle trunk (C6, C7) was secured in a bipolar electrode. Compound muscle action potential (CMAP) was measured. The proximal, tendinous portion of the biceps was severed at the shoulder and clamped in a custom-made force transducer. Muscle preload and electrical stimulation variables were optimized to obtain the highest tetanic muscle contraction. Wet muscle weight (WMW) and nerve histomorphometry were analyzed. Statistical analysis was performed to determine side-to-side equivalence. RESULTS The rabbit biceps muscle force demonstrated side-to-side equivalence with overlapping 95% confidence intervals (95% CI). The right side, expressed as a percentage of the left, averaged 99.69% (95% CI, 88.89%-110.5%). The WMW of the right expressed as a percentage of the left was 98.9% (95% CI, 95.8%-102%). CONCLUSIONS The ITF is equivalent from side to side in the rabbit as demonstrated by the high degree of overlap in the 95% CIs for each side. The width of the 95% CI implies that there is more variability in the rabbit upper extremity than for the lower extremity of the rabbit or rat models, and researchers should take this into account when performing sample size estimates in pre-experimental planning. CLINICAL RELEVANCE The rabbit biceps muscle ITF measurements can be used to measure motor recovery in a rabbit model of brachial plexus injury and compared with the contralateral uninjured side.