Patricia F. Friedrich

Treatment of a Segmental Nerve Defect in the Rat with Use of Bioabsorbable Synthetic Nerve Conduits: A Comparison of Commercially Available Conduits

BACKGROUND The use of biodegradable synthetic nerve conduits for the reconstruction of segmental nerve defects has been extensively reported in both animal and human studies, with a majority of studies evaluating sensory nerve recovery. However, few studies have compared these nerve conduits for functional motor recovery. The purpose of this study was to compare three commercially available, synthetic, bioabsorbable nerve conduits and autograft with respect to compound muscle action potentials, maximum isometric tetanic force, wet muscle weight, and nerve histomorphometry. METHODS Eighty Lewis rats were divided into four groups according to the type of repair of a 10-mm excision of the sciatic nerve: group I had a reversed autograft; group II, a poly-DL-lactide-epsilon-caprolactone conduit; group III, a type-I collagen conduit; and group IV, a polyglycolic acid conduit. All results were compared with the contralateral side. At twelve weeks, the rats underwent bilateral measurements of the compound muscle action potentials of the tibialis anterior and flexor digiti quinti brevis muscles, isometric tetanic force and muscle weight of the tibialis anterior, and peroneal nerve histomorphometry. RESULTS At twelve weeks, no difference in the percentage of recovery between the autograft and the poly-DL-lactide-epsilon-caprolactone conduit was observed with respect to compound muscle action potentials, isometric muscle force, muscle weight, and axon count measurements. The poly-DL-lactide-epsilon-caprolactone and collagen conduits remained structurally stable at twelve weeks, while the polyglycolic acid conduits had completely collapsed. The polyglycolic acid conduit had the poorest results, with a recovery rate of 15% for compound muscle action potentials and 29% for muscle force. CONCLUSIONS The functional outcome in this rat model was similar for the autograft and the poly-DL-lactide-epsilon-caprolactone conduits when they were used to reconstruct a 10-mm sciatic nerve defect. Functional recovery following the use of the polyglycolic acid conduit was the poorest.

Isometric tetanic force measurement method of the tibialis anterior in the rat

Tetanic force measurements of rat tibialis anterior (TA) muscles have been described, but with a variety of stimulation parameters. This study presents a novel functional method of force measurement of the rat TA muscle and describes the optimization of stimulation parameters. Bilateral TA muscles in 10 male Lewis rats were attached to a force transducer after the corresponding hindlimb was fixed. Preload, stimulus intensity, duration, and frequency were optimized for each individual muscle and the isometric maximal tetanic muscle force was measured. The mean left side tetanic force as a percentage of the right was 100.0 ± 4.4% and was statistically equivalent. Large standard deviations between sides (35–50%) were observed in the optimized parameters (preload, stimulus intensity, duration, and frequency). Optimization of the variables affecting isometric tetanic force resulted in reproducible and reliable side‐to‐side measurements of the TA muscle in the rat model.

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.

Measurement of bone blood flow using the hydrogen washout Technique—Part I: Quantitative evaluation of tissue perfusion in the laboratory rat

The measurement of blood flow in bone is of interest in both clinical and experimental settings. Such determinations would ideally provide accurate, quantitative, and reproducible data with relatively simple and safe technology, even in the small bones of experimental animals. Methods that provide absolute or quantitative measurements include positron emission tomography, “isotope fractioning” using radioactive or fluorescent‐labeled microspheres, and measurement of the rate of washout of diffusible tracers delivered either by injection or inhalation. In this article, we describe in detail a modification of the original Whiteside hydrogen washout technique, using modern hydrogen sensors, a micromanipulator for probe placement, and custom software that greatly simplifies blood flow determination and is effective in the small bones of experimental animals.

MK-801 Attenuates Capillary Bed Compression and Hypoperfusion following Incomplete Focal Cerebral Ischemia

The effects of the N-methyl-d-aspartate (NMDA) antagonist MK-801 on capillary beds and CBF following 1 h of transient incomplete focal cerebral ischemia were studied by examining 133Xe CBF, capillary diameter, and area of perfused vasculature. Capillary diameter increased from a control of 5.24 ± 0.37 to 8.62 ± 0.57 μm (p < 0.001) and area of perfused vasculature from 20,943 ± 1,151 to 30,442 ± 1,691 μm2/×10 magnification field (p < 0.001) with MK-801 1.0 mg/kg administered 30 min prior to ischemia. After flow restoration in control animals, there was a relative hypoperfusion with eventual normalization of CBF over 60 min. Alternatively, in MK-801 1.0 mg/kg animals, there was rapid normalization of CBF upon flow restoration without the postischemic hypoperfusion observed in controls. On histological analysis, there was consistently less neuronal edema in MK-801-treated animals. These results support the hypothesis that hypoperfusion following incomplete focal cerebral ischemia may be due in part to NMDA-mediated cellular edema with subsequent extravascular capillary bed compression.

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.

Measurement of bone blood flow using the hydrogen washout technique—part II: Validation by comparison to microsphere entrapment

Accurate and reproducible measurement of bone blood flow has important clinical and experimental applications. Hydrogen washout is simple, safe, and widely used, but its use in bone tissue has not been validated. To this end, we have compared cortical bone blood flow measurements obtained by radioactive‐labeled microsphere entrapment with those from hydrogen washout. Blood flow was measured in tibial cortical bone of 12 New Zealand White rabbits by radioactive microsphere entrapment and by hydrogen washout. Besides a control group (n = 6), four animals were treated with systemic epinephrine (0.8 µg/kg/min) (group 2) and two with nitroprusside (100 µg/kg/min) (group 3). Furthermore, nine femora from seven rats were isolated on their vascular pedicles and repeated bone blood flow measurements were made at each location with the hydrogen washout method to confirm reproducibility of blood flow determinations by hydrogen washout. An average flow of 2.3 ± 2.0 mL/min/100 g was obtained with the microsphere method and 2.0 ± 0.5 mL/min/100 g with the hydrogen washout method. There was a significant correlation and agreement: R2 = 0.97 (p < 0.01). No consistent flow variations were found with systemic vasoactive drug administration. Hydrogen washout provided reproducible results and showed high sensitivity to flow changes. Hydrogen washout is both sensitive and reproducible in measuring bone blood flow. Results agree well with flow values obtained by labeled microsphere entrapment.

Transplantation of a vascularized rabbit femoral diaphyseal segment: Mechanical and histologic properties of a new living bone transplantation model

A new vascularized bone transplantation model is described, including the anatomy and surgical technique of isolating a rabbit femoral diaphyseal segment on its nutrient vascular pedicle. The histologic and biomechanical parameters of pedicled vascularized femoral autotransplants were studied following orthotopic reimplantation in the resulting mid‐diaphyseal defect. Vascularized femur segments were isolated in 10 rabbits on their nutrient pedicle, and then replaced orthotopically with appropriate internal fixation. Postoperative weightbearing and mobility were unrestricted, and the contralateral femora served as no‐treatment controls. After 16 weeks, the bone flaps were evaluated by x‐ray (bone healing), mechanical testing (material properties), microangiography (quantification of intraosseous vasculature), histology (bone viability), and histomorphometry (bone remodeling). Bone healing occurred by 2 weeks, with further callus remodeling throughout the survival period. Eight transplants healed completely, while two had a distal pseudarthrosis. Microangiography demonstrated patent pedicles in all transplants. Intraosseous vessel densities were comparable to nonoperated (control) femora. We found ultimate strength and elastic modulus to be significantly reduced when compared to normal controls. Viable bone, increased mineral apposition rate, and bone turnover were demonstrated in all transplants. The method described, and the data provided will be of value for the further study of isolated segments of living bone, and in particular, for investigations of reconstruction of segmental bone loss in weight‐bearing animal models. This study also provides important normative data on living autologous bone flap material properties, vascularity, and bone remodeling. We intend to use this method and data for comparison in subsequent studies of large bone vascularized allotransplantation.