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1.
J Biomed Mater Res B Appl Biomater ; 105(3): 568-574, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-26621079

RESUMEN

Neuroma management is an unresolved problem. Biomaterials to limit unwanted axonal growth could be a tool to manage neuroma. Hyaluronic acid/carboxymethyl cellulose (HA/CMC) is an antiadhesive, biodegradable material that is nontoxic to nerve. The purpose of this study was to evaluate the efficacy of this biomaterial to limit axonal growth. Rats received a sciatic nerve transection and repair with a short conduit (5 mm) containing HA/CMC, fibrin, or nothing (empty conduit). In another study, nerve was transected and either left undisturbed or wrapped with HA/CMC around the proximal and distal ends. In a final study, nerve was transected and repaired with an HA/CMC wrap. Four weeks following the procedures, nerves were harvested and assessed using histomorphometry to measure axonal regeneration. Axonal regeneration following transection was significantly inhibited by direct axonal contact with HA/CMC, whether within a conduit or wrapped around the transected proximal nerve end. Axonal regeneration following epineurial repair was not affected by HA/CMC wrapped around nerve, demonstrating axonal growth inhibition due to direct contact of regenerating axons with HA/CMC. These studies demonstrate the efficacy of HA/CMC to limit axonal outgrowth by contact with regenerating axons. HA/CMC barriers may prove to be a tool to prevent neuroma formation by inhibiting axonal growth. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 568-574, 2017.


Asunto(s)
Axones/fisiología , Carboximetilcelulosa de Sodio , Ácido Hialurónico , Regeneración/efectos de los fármacos , Nervio Ciático , Animales , Carboximetilcelulosa de Sodio/química , Carboximetilcelulosa de Sodio/farmacología , Ácido Hialurónico/química , Ácido Hialurónico/farmacología , Masculino , Neuroma/metabolismo , Neuroma/patología , Neuroma/prevención & control , Ratas , Ratas Endogámicas Lew , Nervio Ciático/lesiones , Nervio Ciático/metabolismo , Nervio Ciático/patología
2.
Hand (N Y) ; 11(4): 456-463, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-28149214

RESUMEN

Background: Nerve regeneration in vascularized composite allotransplantation (VCA) is not well understood. Allogeneic transplant models experience complete loss of nerve tissue and axonal regeneration without immunosuppressive therapy. The purpose of this study was to determine the impact of incomplete immunosuppression on nerve regeneration. Methods: In this study, transgenic mice (4 groups in total) with endogenous fluorescent protein expression in axons (Thy1-YFP) and Schwann cells (S100-GFP) were used to evaluate axonal regeneration and Schwann cell (SC) migration in orthotopic-limb VCA models with incomplete immunosuppression using Tacrolimus (FK506). Survival and complication rates were assessed to determine the extent of tissue rejection. Nerve regeneration was assessed using serial imaging of axonal progression and SC migration and viability. Histomorphometry quantified the extent of axonal regeneration. Results: Incomplete immunosuppression with FK506 resulted in delayed rejection of skin, muscle, tendon, and bone in the transplanted limb. In contrast, the nerve demonstrated robust axonal regeneration and SC viability based on strong fluorescent protein expression by SCs and axons in transgenic donors and recipients. Total myelinated axon numbers measured at 8 weeks were comparable in all VCA groups and not statistically different from the syngeneic donor control group. Conclusions: Our data suggest that nerve and SCs are much weaker antigens compared with skin, muscle, tendon, and bone in VCA. To our knowledge, this study is the first to prove the weak antigenicity of nerve tissue in the orthotopic VCA mouse model.


Asunto(s)
Axones/fisiología , Rechazo de Injerto/tratamiento farmacológico , Terapia de Inmunosupresión/métodos , Regeneración Nerviosa/fisiología , Neuronas/inmunología , Células de Schwann/inmunología , Alotrasplante Compuesto Vascularizado , Aloinjertos , Animales , Técnica del Anticuerpo Fluorescente , Rechazo de Injerto/fisiopatología , Inmunosupresores , Ratones , Ratones Transgénicos , Tacrolimus
3.
Hand (N Y) ; 10(3): 396-402, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26330769

RESUMEN

BACKGROUND: Individual contributions of exogenous Schwann cells (SCs) and vascular endothelial growth factor (VEGF) were evaluated in acellular nerve allografts (ANAs). ANA processing removes SCs and vasculature, likely contributing to reduced regeneration compared to autografts. Exogenous SCs may improve the regenerative microenvironment, and VEGF has been shown to stimulate angiogenesis. Replacing these components in ANAs may improve regeneration. METHODS: A rat sciatic nerve transection model was used to study 20-mm grafts. Four graft types were studied: (1) isograft, (2) ANA, (3) ANA-SCs, and (4) ANA-VEGF. After 10 weeks in vivo, the midgraft and distal nerve to the grafts were analyzed for axonal regeneration using histomorphometry to assess total myelinated axon counts, density, width, and percent neural tissue. RESULTS: The most axons in the distal nerve were regenerated in the isograft followed by the ANA- SC group, with 9171 ± 1822 and 7103 ± 1576 regenerated axons respectively. Both the ANA and ANA-VEGF groups had significantly fewer regenerated axons compared to the isograft (p < 0.05) with 5225 ± 2994 and 5709 ± 2657 regenerated axons, respectively. The ANA and ANA-VEGF groups also had significantly reduced fiber density and percent nerve compared to the isograft; the isograft and ANA-SC groups were not significantly different (p < 0.05). CONCLUSIONS: These results show that SCs improve axonal regeneration in a 20 mm ANA to a greater extent than VEGF. VEGF treatment showed a trend toward increased axonal regeneration but was not significantly different compared to the untreated ANA. The role of VEGF may be clearer in longer grafts where ischemia is a greater factor.

4.
Biotechnol Bioeng ; 111(9): 1886-94, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24728940

RESUMEN

Glial cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor known to enhance motor nerve regeneration following its delivery. However, recent studies have determined that extended GDNF delivery to regenerating axons can entrap motor axons at the site of GDNF delivery. This entrapment leads to reduced motor axons available to reinnervate muscle. To address this issue, we designed a cell-based GDNF expression system that can temporally regulate protein expression using an inducible gene excision mechanism to prevent entrapment at the site of expression. To design this system for regulation of GDNF expression, we transduced two lentiviral vectors, one containing a constitutively active GDNF transgene flanked by two loxP sites, and the other containing a tetracycline-inducible cre transgene along with its constitutively active transactivator, into Schwann cells (SCs). These SCs over-express GDNF, but expression can be suppressed through the administration of tetracycline family antibiotics, such as doxycycline. The engineered SCs produced significantly more GDNF as compared to untransduced controls, as measured by enzyme-linked immunosorbent assay (ELISA). Following doxycycline treatment, these SCs produced significantly lower levels of GDNF and induced less neurite extension as compared to untreated SCs. Engineered SCs treated with doxycycline showed a marked increase in Cre recombinase expression, as visualized by immunohistochemistry (IHC), providing evidence of a mechanism for the observed changes in GDNF expression levels and biological activity. This cell-based GDNF expression system could have potential for future in vivo studies to provide a temporally controlled GDNF source to promote axon growth.


Asunto(s)
Factor Neurotrófico Derivado de la Línea Celular Glial/biosíntesis , Células de Schwann/metabolismo , Transducción Genética , Animales , Células Cultivadas , Regulación de la Expresión Génica , Vectores Genéticos , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Lentivirus/genética , Ratas Endogámicas Lew , Recombinación Genética
8.
Exp Neurol ; 247: 165-77, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23644284

RESUMEN

Repair of large nerve defects with acellular nerve allografts (ANAs) is an appealing alternative to autografting and allotransplantation. ANAs have been shown to be similar to autografts in supporting axonal regeneration across short gaps, but fail in larger defects due to a poorly-understood mechanism. ANAs depend on proliferating Schwann cells (SCs) from host tissue to support axonal regeneration. Populating longer ANAs places a greater proliferative demand on host SCs that may stress host SCs, resulting in senescence. In this study, we investigated axonal regeneration across increasing isograft and ANA lengths. We also evaluated the presence of senescent SCs within both graft types. A sciatic nerve graft model in rats was used to evaluate regeneration across increasing isograft (~autograft) and ANA lengths (20, 40, and 60 mm). Axonal regeneration and functional recovery decreased with increased graft length and the performance of the isograft was superior to ANAs at all lengths. Transgenic Thy1-GFP rats and qRT-PCR demonstrated that failure of the regenerating axonal front in ANAs was associated with increased levels of senescence related markers in the graft (senescence associated ß-galactosidase, p16(INK4A), and IL6). Lastly, electron microscopy (EM) was used to qualitatively assess senescence-associated changes in chromatin of SCs in each graft type. EM demonstrated an increase in the presence of SCs with abnormal chromatin in isografts and ANAs of increasing graft length. These results are the first to suggest that SC senescence plays a role in limited axonal regeneration across nerve grafts of increasing gap lengths.


Asunto(s)
Regeneración Nerviosa/fisiología , Recuperación de la Función/fisiología , Células de Schwann/fisiología , Células de Schwann/trasplante , Nervio Ciático/trasplante , Animales , Caspasa 3/metabolismo , Cromatina/patología , Cromatina/ultraestructura , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes , Masculino , Microscopía Electrónica de Transmisión , Unión Neuromuscular/fisiología , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Ratas Transgénicas , Proteínas S100/metabolismo , Células de Schwann/ultraestructura , Nervio Ciático/patología , Nervio Ciático/ultraestructura , Neuropatía Ciática/cirugía , Trasplante Homólogo/métodos , beta-Galactosidasa/metabolismo
9.
Plast Reconstr Surg ; 131(4): 499e-511e, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23542267

RESUMEN

BACKGROUND: Successful nerve regeneration is critical to the functional success of composite tissue allografts. The present study was designed to characterize the effect of acute rejection on nerve regeneration and functional recovery in the setting of orthotopic limb transplantation. METHODS: A rat orthotopic limb transplantation model was used to evaluate the effects of acute rejection on nerve regeneration and motor recovery. Continuous administration of FK506 (full suppression), administration of FK506 for the first 8 of 12 weeks (late rejection), or delayed administration of FK506/dexamethasone following noticeable rejection (early rejection) was used to preclude or induce rejection following limb transplantation. Twelve weeks postoperatively, nerve regeneration was assessed by means of histomorphometric analysis of explanted sciatic nerve, and motor recovery was assessed by means of evoked muscle force measurement in extensor digitorum longus muscle. RESULTS: A single episode of acute rejection that occurs immediately or late after reconstruction does not significantly alter the number of regenerating axonal fibers. Acute rejection occurring late after reconstruction adversely affects extensor digitorum longus muscle function in composite tissue allografts. CONCLUSIONS: Collected data reinforce that adequate immunosuppressant administration in cases of allogeneic limb transplantation ensures levels of nerve regeneration and motor functional recovery equivalent to that of syngeneic transplants. Prompt rescue following acute rejection was further demonstrated not to significantly affect nerve regeneration and functional recovery postoperatively. However, instances of acute rejection that occur late after reconstruction affect graft function. In total, the present study begins to characterize the effect of immunosuppression regimens on nerve regeneration and motor recovery in the setting of composite tissue allografts.


Asunto(s)
Extremidades/inervación , Extremidades/trasplante , Rechazo de Injerto , Regeneración Nerviosa , Enfermedad Aguda , Animales , Rechazo de Injerto/tratamiento farmacológico , Masculino , Ratas , Ratas Endogámicas Lew
10.
Muscle Nerve ; 47(2): 213-23, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23169341

RESUMEN

INTRODUCTION: We sought to determine whether supplementation of acellular nerve allografts (ANAs) with Schwann cells overexpressing GDNF (G-SCs) would enhance functional recovery after peripheral nerve injury. METHODS: SCs expanded in vitro were infected with a lentiviral vector to induce GDNF overexpression. Wild-type SCs (WT-SCs) and G-SCs were seeded into ANAs used to repair a 14-mm nerve gap defect. Animals were harvested after 6 and 12 weeks for histomorphometric and muscle force analysis. RESULTS: At 6 weeks, histomorphometry revealed that ANAs supplemented with G-SCs promoted similar regeneration compared with isograft at midgraft. However, G-SCs failed to promote regeneration into the distal stump. At 12 weeks, ANAs with G-SCs had lower maximum and specific force production compared with controls. CONCLUSIONS: The combined results suggest that consistent overexpression of GDNF by G-SCs trapped axons in the graft and prevented functional regeneration.


Asunto(s)
Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Tejido Nervioso/trasplante , Traumatismos de los Nervios Periféricos/cirugía , Recuperación de la Función/fisiología , Células de Schwann/trasplante , Animales , Masculino , Regeneración Nerviosa/fisiología , Tejido Nervioso/metabolismo , Traumatismos de los Nervios Periféricos/metabolismo , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Células de Schwann/metabolismo
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