Gregory H. Borschel

Processed allografts and type I collagen conduits for repair of peripheral nerve gaps

Autografting is the gold standard in the repair of peripheral nerve injuries that are not amenable to end‐to‐end coaptation. However, because autografts result in donor‐site defects and are a limited resource, an effective substitute would be valuable. In a rat model, we compared isografts with Integra NeuraGen® (NG) nerve guides, which are a commercially available type I collagen conduit, with processed rat allografts comparable to AxoGens Avance® human decellularized allograft product. In a 14‐mm sciatic nerve gap model, isograft was superior to processed allograft, which was in turn superior to NG conduit at 6 weeks postoperatively (P < 0.05 for number of myelinated fibers both at midgraft and distal to the graft). At 12 weeks, these differences were no longer apparent. In a 28‐mm graft model, isografts again performed better than processed allografts at both 6 and 22 weeks; regeneration through the NG conduit was often insufficient for analysis in this long graft model. Functional tests confirmed the superiority of isografts, although processed allografts permitted successful reinnervation of distal targets not seen in the NG conduit groups. Processed allografts were inherently non‐immunogenic and maintained some internal laminin structure. We conclude that, particularly in a long gap model, nerve graft alternatives fail to confer the regenerative advantages of an isograft. However, AxoGen processed allografts are superior to a currently available conduit‐style nerve guide, the Integra NeuraGen®. They provide an alternative for reconstruction of short nerve gaps where a conduit might otherwise be used. Muscle Nerve, 2008

Limitations of Conduits in Peripheral Nerve Repairs

Nerve conduits have emerged as alternatives to autologous nerve grafts, but their use in large-diameter nerve deficits remains untested. We report four patients who underwent repair of large-diameter nerves using absorbable nerve conduits and discuss the failed clinical outcomes. The reported cases demonstrate the importance of evaluating the length, diameter, and function of nerves undergoing conduit repair. In large-diameter nerves, the use of conduits should be carefully considered.

Mechanical properties of acellular peripheral nerve

BACKGROUND Acellular nerve has been used in experimental models as a peripheral nerve substitute. Our objective was to determine the difference in tensile strength between fresh and chemically treated acellularized peripheral nerve. MATERIALS AND METHODS F344 rat sciatic nerves were either fresh or acellularized and tested either whole (Part A) or transected and repaired (Part B). For all constructs, the mean ultimate stress, mean ultimate strain, Youngs modulus, and total mechanical work to fracture were calculated. The average ultimate strains for Groups A-1 and A-2 were 0.480 +/- 0.117 and 0.810 +/- 0.114, respectively. The Youngs moduli in Groups A-1 and A-2 were 576 +/- 160 and 580 +/- 150 kPa, respectively. In Groups A-1 and A-2, the normalized work to failure was 0.35 +/- 0.14 and 1.11 +/- 0.38 N. The specimens in Group B-1 withstood an average ultimate stress of 780 +/- 280 kPa. The specimens in Group B-2 withstood an average ultimate stress of 405 +/- 20 kPa. RESULTS The average ultimate strains for Groups B-1 and B-2 were 0.319 +/- 0.087 and 0.266 +/- 0.019, respectively. The Youngs moduli in Groups B-1 and B-2 were 4,030 +/- 1360 and 2,290 +/- 280 kPa, respectively. The normalized work to failure in Groups B-1 and B-2 was calculated as 0.22 +/- 0.04 and 0.11 +/- 0.02 N. CONCLUSIONS Although adequately robust for reconstructive procedures, the acellular peripheral nerve had decreased tensile strength compared with fresh nerve either when tested whole or when transected and repaired.

Contractile skeletal muscle tissue-engineered on an acellular scaffold.

For the reconstructive surgeon, tissue-engineered skeletal muscle may offer reduced donor-site morbidity and an unlimited supply of tissue. Using an acellularized mouse extensor digitorum longus muscle as a scaffold, the authors produced engineered skeletal muscle capable of generating longitudinal force. Eight extensor digitorum longus muscles from adult mice were made acellular using a protocol developed in the authors’ laboratory. The acellular muscles were then placed in a bath of 20% fetal bovine serum in Dulbecco’s modified Eagle’s medium and 100 U/ml penicillin for 1 week at room temperature. C2C12 myoblasts were injected into the acellular muscle matrix using a 26-gauge needle and a 100-&mgr;l syringe. The resulting constructs were placed in growth medium for 1 week at 37°C under 5% carbon dioxide, with media changes every 48 hours. The constructs were then placed in differentiation medium for 1 week, with media changes every 48 hours. Isometric contractile force testing of the constructs demonstrated production of longitudinal contractile force on electrical stimulation. A length-tension, or Starling, relationship was observed. Light and electron microscopy studies demonstrated recapitulation of some of the normal histologic features of developing skeletal muscle.

Affinity-based release of glial-derived neurotrophic factor from fibrin matrices enhances sciatic nerve regeneration.

Glial-derived neurotrophic factor (GDNF) promotes both sensory and motor neuron survival. The delivery of GDNF to the peripheral nervous system has been shown to enhance regeneration following injury. In this study, we evaluated the effect of affinity-based delivery of GDNF from a fibrin matrix in a nerve guidance conduit on nerve regeneration in a 13 mm rat sciatic nerve defect. Seven experimental groups were evaluated which received GDNF or nerve growth factor (NGF) with the delivery system within the conduit, control groups excluding one or more components of the delivery system, and nerve isografts. Nerves were harvested 6 weeks after treatment for analysis by histomorphometry and electron microscopy. The use of the delivery system (DS) with either GDNF or NGF resulted in a higher frequency of nerve regeneration vs. control groups, as evidenced by a neural structure spanning the 13 mm gap. The GDNF DS and NGF DS groups were also similar to the nerve isograft group in measures of nerve fiber density, percent neural tissue and myelinated area measurements, but not in terms of total fiber counts. In addition, both groups contained a significantly greater percentage of larger diameter fibers, with GDNF DS having the largest in comparison to all groups, suggesting more mature neural content. The delivery of GDNF via the affinity-based delivery system can enhance peripheral nerve regeneration through a silicone conduit across a critical nerve gap and offers insight into potential future alternatives to the treatment of peripheral nerve injuries.

Outcome measures of peripheral nerve regeneration.

Animal models of nerve compression, crush, and transection injuries of peripheral nerves have been subject to extensive study in order to understand the mechanisms of injury and axon regeneration and to investigate methods to promote axon regeneration and improve functional outcomes following nerve injury. Six outcome measures of regenerative success including axon and neuron counts, muscle and motor unit contractile forces, and behavior are reviewed in the context of nerve injury types, crush, transection and nerve repair by direct coaptation, or transection and repair via a nerve graft or conduit. The measures are evaluated for sciatic, tibial, common peroneal, femoral, single nerve branches such as the soleus nerve, and facial nerves. Their validity is discussed in the context of study objectives and the nerve branch. The case is made that outcome measures of axon counts and muscle contractile forces may be valid during the early phases of axon regeneration when regenerating sprouts emerge asynchronously from the proximal nerve stump and regenerate towards their denervated targets. However, care must be taken especially when experimental interventions differentially affect how many neurons regenerate axons and the number of axons per neuron that sprout from the proximal nerve stumps. Examples of erroneous conclusions are given to illustrate the need for researchers to ensure that the appropriate outcome measures are used in the evaluation of the success of peripheral nerve regeneration.

Self-organization of rat cardiac cells into contractile 3-D cardiac tissue

The mammalian heart is not known to regenerate following injury. Therefore, there is great interest in developing viable tissue‐based models for cardiac assist. Recent years have brought numerous advances in the development of scaffold‐based models of cardiac tissue, but a self‐organizing model has yet to be described. Here, we report the development of an in vitro cardiac tissue without scaffolding materials in the contractile region. Using an optimal concentration of the adhesion molecule laminin, a confluent layer of neonatal rat cardiomyogenic cells can be induced to self‐organize into a cylindrical construct, resembling a papillary muscle, which we have termed a cardioid. Like endogenous heart tissue, cardioids contract spontaneously and can be electrically paced between 1 and 5 Hz indefinitely without fatigue. These engineered cardiac tissues also show an increased rate of spontaneous contraction (chronotropy), increased rate of relaxation (lusitropy), and increased force production (inotropy) in response to epinephrine. Cardioids have a developmental protein phenotype that expresses both α‐ and β‐tropomyosin, very low levels of SERCA2a, and very little of the mature isoform of cardiac troponin T.

The impact of motor and sensory nerve architecture on nerve regeneration.

Sensory nerve autografting is the standard of care for injuries resulting in a nerve gap. Recent work demonstrates superior regeneration with motor nerve grafts. Improved regeneration with motor grafting may be a result of the nerves Schwann cell basal lamina tube size. Motor nerves have larger SC basal lamina tubes, which may allow more nerve fibers to cross a nerve graft repair. Architecture may partially explain the suboptimal clinical results seen with sensory nerve grafting techniques. To define the role of nerve architecture, we evaluated regeneration through acellular motor and sensory nerve grafts. Thirty-six Lewis rats underwent tibial nerve repairs with 5 mm double-cable motor or triple-cable sensory nerve isografts. Grafts were harvested and acellularized in University of Wisconsin solution. Control animals received fresh motor or sensory cable isografts. Nerves were harvested after 4 weeks and histomorphometry was performed. In 6 animals per group from the fresh motor and sensory cable graft groups, weekly walking tracks and wet muscle mass ratios were performed at 7 weeks. Histomorphometry revealed more robust nerve regeneration in both acellular and cellular motor grafts. Sensory groups showed poor regeneration with significantly decreased percent nerve, fiber count, and density (p<0.05). Walking tracks revealed a trend toward improved functional recovery in the motor group. Gastrocnemius wet muscle mass ratios show a significantly greater muscle mass recovery in the motor group (p<0.05). Nerve architecture (size of SC basal lamina tubes) plays an important role in nerve regeneration in a mixed nerve gap model.

Fibrin Matrices With Affinity-Based Delivery Systems and Neurotrophic Factors Promote Functional Nerve Regeneration

Glial‐derived neurotrophic factor (GDNF) and nerve growth factor (NGF) have both been shown to enhance peripheral nerve regeneration following injury and target different neuronal populations. The delivery of either growth factor at the site of injury may, therefore, result in quantitative differences in motor nerve regeneration and functional recovery. In this study we evaluated the effect of affinity‐based delivery of GDNF or NGF from fibrin‐filled nerve guidance conduits (NGCs) on motor nerve regeneration and functional recovery in a 13 mm rat sciatic nerve defect. Seven experimental groups were evaluated consisting of GDNF or NGF and the affinity‐based delivery system (DS) within NGCs, control groups excluding the DS and/or growth factor, and nerve isografts. Groups with growth factor in the conduit demonstrated equivalent or superior performance in behavioral tests and relative muscle mass measurements compared to isografts at 12 weeks. Additionally, groups with GDNF demonstrated greater specific twitch and tetanic force production in extensor digitorum longus (EDL) muscle than the isograft control, while groups with NGF produced demonstrated similar force production compared to the isograft control. Assessment of motor axon regeneration by retrograde labeling further revealed that the number of ventral horn neurons regenerating across NGCs containing GDNF and NGF DS was similar to the isograft group and these counts were greater than the groups without growth factor. Overall, the GDNF DS group demonstrated superior functional recovery and equivalent motor nerve regeneration compared to the isograft control, suggesting it has potential as a treatment for motor nerve injury. Biotechnol. Bioeng. 2010;106: 970–979.

Tissue-engineered axially vascularized contractile skeletal muscle

Background: As tissue-engineered muscle constructs increase in scale, their size is limited by the need for a vascular supply. In this work, the authors demonstrate a method of producing three-dimensional contractile skeletal muscles in vivo by incorporating an axial vascular pedicle. Methods: Primary myoblast cultures were generated from adult F344 rat soleus muscle. The cells were suspended in a fibrinogen hydrogel contained within cylindrical silicone chambers, and situated around the femoral vessels in isogeneic adult recipient rats. The constructs were allowed to incubate in vivo for 3 weeks, at which point they were explanted and subjected to isometric force measurements and histologic evaluation. Results: The resulting three-dimensional engineered skeletal muscle constructs produced longitudinal contractile force when electrically stimulated. Length-tension, force-voltage, and force-frequency relationships were similar to those found in developing skeletal muscle. Desmin staining demonstrated that individual myoblasts had undergone fusion to form multinucleated myotubes. Von Willebrand staining showed that the local environment within the chamber was richly angiogenic, and capillaries had grown into and throughout the constructs from the femoral artery and vein. Conclusions: Three-dimensional, vascularized skeletal muscle can be engineered in vivo. The resulting tissues have histologic and functional properties consistent with native skeletal muscle.