Study On The Characterization Of Nerve Scaffold Fabricated With NGF,PLGA And Acellular Pigskin

Peripheral nerve defects caused by various kinds of trauma can result in motor and sensory disabilities. Direct end-to-end suturing is suggested for a short nerve injury. For larger nerve defects or gaps, implantation of a nerve graft is often necessary to bridge the proximal and distal nerve stumps for facilitating nerve regeneration and functional recovery. At present, to recover the disrupted nerve is clinically carried out by autograft, but there still exist certain limits and disadvantages: the donator source limitation for autograft, and the accompanying risks in harvesting autograft. To overcome those disadvantages, artificial nerve scaffold has been developed and showed great potentiality. In recent research, biodegradation never conduits have attracted much more attention. By the end of 2006, there are some conduits that were FDA approved for clinic, including SaluBridge Nerve cuff, Neurolac nerve conduit, Neuro Matrix, Neuroflex and Neurotube. In our country, a few of institutions are applying for national medical equipment certificates. However, different kinds of conduits have different effectiveness on nerve regeneration. In addition, these present treatments are served for small defects (several millimeters) and do not address larger peripheral nerve injuries. Thus, it was deserved to further develop new nerve scaffolds to satisfy the requirement of clinical using.Polylactic acid-polyglycolic acid (PLGA) has been widely utilized in tissu engineering, due to its good biocompatibility, biodegradation and easy fabrication. But the scaffold fabricated by PLGA is fragile and hard. So it is essential to compounded PLGA with other kinds of materials in order to improve its biomechanics. Meanwhile, acidity from PLGA degradation in vivo could result in local acid collection, which would change the pH stabilization of tissue. Thus how to overcome this problem is should be taken consideration.Moreover, studies confirm the addition of nerve growth factors [NGFs] into artificial nerve scaffold can promote its regeneration and repair, however, due to the very short half-life time of NGFs in vivo, thus sustained release of NGFs is required for them to function better.It is well known that porcine-derived extracellular matrix (ECM) has good biocompability and will not cause the risk that sufferer is infected with porcine endogenous retrovirus. ECM, which is composed with collagen and a little laminin and fibronectin, has been shown to play a significant role in axonal regeneration. However it is difficult to fabricate a nerve scaffold with ECM molecules directly. As porcine-derived ECM has favourable hydrophilicity and water insoluble, we mixed ECM with PLGA to fabricate a compound conduit, which could keep its shape in vivo for a long time.In this study, we manufactured a compound nerve scaffold with porcine-derived ECM and PLGA, which could release NGF sustainly. The compound scaffold was evaluated by vitro testing and animal experiments. The concrete contents are as follows:①Porcine-derived extracellular matrix was prepared by enzymolysis. The matrix was underwent H-E Staining, SEM observation and DNA remains assay to ensure the pigskin free of cell. It was seen cells have been completely removed by H.E staining and scanning electron macrograph (SEM) observation. The result of DNA remains assay showed that there is no DNA band in acellular matrix.②Gelatin microballoon with bioactive NGF was fabricated. By laser particle diameter distribution device testing, it was found that the particle diameter was range from 1μm to 20μm and the mean diameter from 8μm to12μm. The encanpsulation ratio of NGF is 80.50% by capillary electrophoresis.③Alkaline substance added into the scaffold could neutralize acidoid from degradation of PLGA to maintain tissue pH. As for pure PLGA, the pH of degradation solution will decrease gradually as it was degradated in purified water. Variety kinds of weak base moderators, which were combinated with PLGA, could regulate the pH of degradation solution. But the regulated effectiveness vary with the kinds of moderator and the percentage of moderator added to the PLGA. According to our experiment, all the basic additives investigated in the experiment showed good potential to relieve the level of acid collection in the course of PLGA degradation. Among those, the relieving ability of NaHCO3 was in the top, basic amino acid in the moderate, while Algin and chitosan ranked lower. 5% addition of Lysine produced a best outcome that is the pH remainning at 7.28~6.62.④Comparing the compound scaffolds with different mixture ratio of PLGA and ECM, the results were as follows : (1) The pore diamater and the appearance were vary with the increasement of the content of ECM in the scaffold; (2)the breaking strength increased up to the peak with the extent of ECM in the scaffold increasing to 20%~30%; (4) the breaking extensibility decreased gradually with increment of ECM in scaffold; (5) the max water-uptake and mass losing rate of conduits increased with the increment of ECM in the scaffold during the degradation period. By comprehend evaluation; it is considered that the most optimization content of ECM in scaffold is about 20%~30%.⑤Target scaffold has been fabricated.⑥According to test in vitro, the result displayed as follows: (1)Mechanical testing showed that the breaking strength was about 8.308MPa, the breaking extensibility was about 38.98 % and the elastic modulus was about 77.27 MPa. (2) SEM result showed PLGA has mixed with ECM to form cellular structure. (3) During 8 weeks of degradation in vitro, and the mass loss ratio was about 15.6%, the accumulative releas rate of hydroxyproline is 62.8%. (4) The release of NGF could be still detected on the 30th day. (5) Cells could adhere to the scaffold and extend on it.⑦The results of animal experiment suggested that the scaffold implanted into rat's sciatic nerve has good biocompatibility, no inflammation and is free of hypersensitive response.To sum up, the scaffold could release NGF sustainedly and protect NGF from denaturation; it was free of cytotoxicity and could promote Schwann cells'growth; it's three dimensional structure is the prerequisite of SCs extended on it; it has appropriate mechanics property and good bio-degradablity; Addition alkaline substance is an effective method for neutralization acidoid from PLGA. So the compound scaffold may be a potential artificial nerve scaffold.