Combination of heterologous fibrin sealant and bioengineered human embryonic stem cells to improve regeneration following autogenous sciatic nerve grafting repair

Peripheral nerve injury is a worldwide clinical problem, and the preferred surgical method for treating it is the end-to-end neurorrhaphy. When it is not possible due to a large nerve gap, autologous nerve grafting is used. However, these surgical techniques result in nerve regeneration at highly variable degrees. It is thus very important to seek complementary techniques to improve motor and sensory recovery. One promising approach could be cell therapy. Transplantation therapy with human embryonic stem cells (hESCs) is appealing because these cells are pluripotent and can differentiate into specialized cell types and have self-renewal ability. Therefore, the main objective of this study was to find conditions under which functional recovery is improved after sciatic nerve neurorrhaphy. We assumed that hESC, either alone or in combination with heterologous fibrin sealant scaffold, could be used to support regeneration in a mouse model of sciatic nerve injury and repair via autografting with end-to-end neurorrhaphy. Methods Five millimeters of the sciatic nerve of C57BL/6 J mice were transected off and rotated 180 degrees to simulate an injury, and then stumps were sutured. Next, we applied heterologous fibrin sealant and/or human embryonic stem cells genetically altered to overexpress fibroblast growth factor 2 (FGF2) at the site of the injury. The study was designed to include six experimental groups comprising neurorrhaphy (N), neurorrhaphy + heterologous fibrin sealant (N + F), neurorrhaphy + heterologous fibrin sealant + doxycycline (N + F + D), neurorrhaphy + heterologous fibrin sealant + wild-type hESC (N + F + W), neurorrhaphy + heterologous fibrin sealant + hESC off (N + F +T), and neurorrhaphy + heterologous fibrin sealant + hESC on via doxycycline (N + F + D + T). We evaluated the recovery rate using Catwalk and von Frey functional recovery tests, as well as immunohistochemistry analysis. Results The experiments indicated that sensory function improved when transgenic hESCs were used. The regeneration of sensory fibers indeed led to increased reflexes, upon stimulation of the paw ipsilateral to the lesion, as seen by von-Frey evaluation, which was supported by immunohistochemistry. Conclusions Overall, the present data demonstrated that transgenic embryonic stem cells, engineered to overexpress FGF-2 in an inducible fashion, could be employed to support regeneration aiming at the recovery of both motor and sensory functions.(AU)

Combined polyglycolic acid tube and autografting versus autografting or polyglycolic acid tube alone. A comparative study of peripheral nerve regeneration in rats

PURPOSE: To compare sciatic nerve regeneration in rats using three different techniques of repair. METHODS: Fifteen isogonics rats were divided into three groups according to the method used to repair a 5-mm long defect created in the sciatic nerve: autogenous graft (Group A), polyglycolic acid tube (PGAt) (Group B), and of the association of PGAt with the graft (Group C). Histological analysis, regenerated myelinated axon number count and functional analysis were used to compare after six weeks. RESULTS: There was no difference in fiber diameter and degree of myelinization presented by Groups A, B and C. Group B presented the lowest number of regenerated axons. The groups did not display any significant functional difference after walking track analysis (p<0.05). CONCLUSION: No differences between the three groups in terms of functional recovery, although there were histological differences among them. .

Effect of amnion-wrapped allogenic nerve bridging on peripheral nerve injury / Efecto del puente alogénico de nervio envuelto en membrana amniótica sobre la lesión de nervio periférico

This work aims to investigate the effect of fetal amnion-wrapped acellular allogenic nerve transplantation on peripheral nerve injury (PNI) in dogs and to explore its advantages and feasibility in PNI repair. A total of 15 dogs were divided into three groups: the allogenic nerve transplantation (A), amnion-wrapped allogenic nerve transplantation (B), and allogenic nerve donor (C) groups. Neurite counts after myelin and H-E stainings, soleus muscle action potentials, and sciatic nerve conductive velocities were compared between the A and B groups at 16 w after operation. The B group showed better nerve regeneration than the A group at 16 w. Compared with the A group, the B group showed a better growth continuity of the transplanted nerve and milder inflammatory reactions around the nerve. The B group presented much more proliferated Schwannocytes and regenerated nerve fibers than the A group. The neurite density and the amplitude of the soleus muscle action potentials in the B group were significantly higher than those in the A group (P < 0.05). The two groups did not show significant differences in nerve conductive velocities (P > 0.05). Amnion-wrapped acellular allogenic nerve transplantation can improve defected nerve morphology and the quality of transplanted nerve regeneration.

El objetivo fue investigar el efecto del trasplante alogénico de nervio acelular envuelto en membrana amniótica fetal sobre la lesión del nervio periférico (LNP) en perros, y explorar sus ventajas y viabilidad en la reparación de LNP. Quince 15 perros se dividieron en tres grupos: grupo trasplante alogénico de nervio (A), grupo trasplante alogénico de nervio envuelto en membrana amniótica (B), y grupo donante alogénico de nervio (C). Se compararon el recuento de neuritas posterior a la tinción de hematoxilina-eosina (HE) y para mielina, potenciales de acción del músculo sóleo, y velocidades conductoras nerviosas del nervio ciático entre los grupos A y B, 16 semanas después de la operación. El grupo B mostró una mejor regeneración de los nervios que el grupo A a las 16 semanas. En comparación con el grupo A, el grupo B mostró una mejor continuidad del crecimiento del nervio trasplantado con reacciones inflamatorias leves alrededor del nervio. El grupo B presentó fibras nerviosas donde proliferaron más los Schwannocitos y regeneración que el grupo A. La densidad de las neuritas y la amplitud de los potenciales de acción del músculo sóleo en el grupo B fueron significativamente más altos (p <0,05). Ambos grupos no mostraron diferencias significativas en las velocidades conductoras nerviosas (P> 0,05). El trasplante alogénico de nervio acelular envuelto en membrana amniótica puede mejorar la morfología del nervio lesionado y la calidad de regeneración del nervio trasplantado.

Non-neuronal cells are not the limiting factor for the low axonal regeneration in C57BL/6J mice

Peripheral axonal regeneration was investigated in adult male mice of the C57BL/6J (C), BALB/cJ (B) and A/J (A) strains and in their F1 descendants using a predegenerated nerve transplantation model. Four types of transplants were performed: 1) isotransplants between animals of the C, B and A strains; 2) donors of the C strain and recipients of the C x B and C x A breeding; 3) donors of the B strain and recipients of the C x B breeding, and 4) donors of the A strain and recipients of the C x A breeding. Donors had the left sciatic nerve transected and two weeks later a segment of the distal stump was transplanted into the recipient. Four weeks after transplantation the regenerated nerves were used to determine the total number of regenerated myelinated fibers (TMF), diameter of myelinated fibers (FD) and myelin thickness (MT). The highest TMF values were obtained in the groups where C57BL/6J mice were the donors (C to F1 (C x B) = 4658 + OR - 304; C to F1 (C x A) = 3899 + OR - 198). Also, A/J grafts led to a significantly higher TMF (A to F1 (C x A) = 3933 + OR - 565). Additionally, isotransplant experiments showed that when the nerve is previously degenerated, C57BL/6J mice display the largest number of myelinated fibers (C to C = 3136 + OR - 287; B to B = 2759 + OR - 170, and A to A = 2835 + OR - 239). We also observed that when C57BL/6J was the graft donor, FD was the highest and MT did not differ significantly when compared with the other groups. These morphometric results reinforce the idea that Schwann cells and the nerve environment of C57BL/6J provide enough support to the regenerative process. In this respect, the present results support the hypothesis that the non-neuronal cells, mainly Schwann cells, present in the sciatic nerve of C57BL/6J mice are not the main limiting factor responsible for low axonal regeneration

Estudo sobre a influência da sutura distal em enxertos longos de nervo com emprego de técnica microcirúrgica / Study on the influence of distal suture in long nerve grafts using a microsurgery technique

Foram operados 14 coelhos nos quais se realizaram enxertos longos de nervo fibular do tipo autólogo, usando-se técnica microcirúrgica. Num grupo (sete coelhos), as suturas proximal e distal foram feitas no mesmo estágio cirúrgico e, no outro (sete coelhos), a sutura distal foi realizada 60 dias após a sutura proximal. A análise dos resultados näo demonstrou haver diferença estatisticamente significante no número de fibras mielinizadas no segmento proximal, no enxerto propriamente dito e no segmento distal em ambos os grupos

Injertos nerviosos vascularizados: estudio experimental / Vascularized nerve grafts: experimental study

La vascularizacion de los injertos nerviosos es de primordial importancia para la supervivencia del injerto. Preservamos el pediculo proximal en el nervio ciatico del conejo y reparamos un defecto de 4.5 cm. Un modelo experimental de injertos nerviosos vascularizados es presentado como una alternativa para la recuperacion mas rapida de la funcion. Utilizamos 18 conejos, en el lado derecho se efectuo un injerto de 4.5 cm preservando el pediculo vascular, en el lado contralateral se efectuo un injerto de la misma longitud sin preservar ningun pediculo. La neurorrafia se efectuo con nylon 10/0 y microscopio quirurgico. Posteriormente se sacrificaron los animales entre la 5 y la 15 semana del postoperatorio y los nervios se estudiaron con microscopio de luz. El analisis cuantitativo se efectuo en un computador teniendo en cuenta principalmente el espesor de la vaina de mielina. Este experimento demostro que cuando se efectua un injertpo nervioso vascularizado la rata de la maduracion axonal se aumenta hasta 2 veces la rata observada en injertos convencionales. La maduracion de las fibras ocurre de una manera mas rapida y aun utilizando la misma longitud del nervio no se presento ningun caso de necrosis central. En conclusion los resultados son mas favorables en cuanto a regeneracion cuando se utiliza un injerto nervioso vascularizado