Human Hair Keratin Promotes Nerve Repair After Crush Injury Of Sciatic Nerve In Rats

Background: Axon regeneration and functional recovery after peripheral nerve damage remains a clinical challenge.Injury leads to axonal disintegration in which Schwann cells(SCs)and macrophages re-engage in the process of regeneration.With the development of tissue regeneration engineering,more and more biomaterials have been applied to peripheral nerve injury.Keratin as a natural substance is widely found in hairs,nails and feathers,which has the advantages of stable amino acid composition,strong plasticity,good biocompatibility and biodegradability.It has been reported that keratin extracted from wool or feathers can support cell growth and tissue repair.The current study performed a method of reduction to extract keratin from human hair.In this study,a human hair keratin(HHK)extracted by reducing method and a human hair keratin sponge(HHK sponge)we obtained by changing the preparation conditions were used for in vitro and in vivo experiments,respectively.The results suggested that HHK can promote cell adhesion,proliferation,migration as well as the secretion of neurotrophic factors by SCs and the regulation of the expression of macrophage inflammatory cytokines,extension of dorsal root ganglion(DRG)neurons in vitro.The motor deficits caused by a sciatic nerve crush injury were alleviated by HHK sponge dressing.Moreover,HHK sponge can be decomposed and absorbed by the body within 5 weeks,creating a suitable microenvironment for the early repair while not affecting the extension of axons during the later stages.Thus,HHK has been identified as a promising biomaterial that can enhancing nerve regeneration.Objectives: To investigate the role of HHK as a new type of tissue engineering biomaterial in regulating the biological activity of SCs,macrophages and DRG neurons in vitro,and promoting the regeneration of axons after peripheral nerve injury in vivo.Methods:(1)Extraction of HHK and preparation of HHK sponge.(2)The effects of HHK on SCs adhesion,proliferation,migration and nutritional factor expression were observed by counting analysis,CCK-8 test,Transwell chamber,scratch test and RT-q PCR.(3)Immunofluorescence was used to detect the effect of HHK on the axon extension of DRG neurons.(4)The effect of HHK on the expression of macrophage inflammatory factors was detected by RT-q PCR.(5)The model of sciatic nerve crush injury in SD rats was established in vivo.(6)The effects of HHK sponge on functional recovery after sciatic nerve injury were observed by sciatic nerve function index(SFI),footprint analysis,wet weight of gastrocnemius muscle and muscle fiber morphology.(7)The regulation of myelin regeneration by HHK sponge was analyzed by transmission electron microscope(TEM)analysis.Results:(1)HHK was extracted by reducing method and HHK sponge was prepared.(2)HHK could promote SCs adhesion,proliferation,migration and expression of neurotrophic factor.(3)HHK can promote the axon growth of DRG neurons.(4)HHK could inhibit the expression of pro-inflammatory factor IL-6,TNF-?,i-NOS and IL-1?,and increase the expression of anti-inflammatory factor IL-10.(5)HHK sponge accelerated the recovery of nerve and muscle after sciatic nerve crush injury in SD rats.Conclusions:(1)Successfully extract keratin from human hair and prepare HHK sponge.(2)HHK can regulate the biological activity of a variety of cells involved in nerve damage repair in vitro.(3)The HHK sponge accelerates the regeneration of axon after injury in vivo,which provides valuable time for the connection between axon and target organ.