Application Of Sericin In Tissue Engineering Of Peripheral Nerve And 3D Microvascular Model

Part One Sustained Local Release of NGF from a Chitosan/Sericin Composite Scaffold for Treating Chronic Nerve Compression ObjectiveTo explore the efficacy of NGF-loaded chitosan/sericin composite scaffold in treating chronic nerve compression.MethodsSericin was isolated from Bombyx mori,140 Nd-s cocoons by Li Br extraction method.Then the chitosan(CS)and sericin(SS)solutions were mixed at five predetermined volume ratios(CS100/SS0,CS80/SS20,CS50/SS50,CS20/SS80,CS0/SS100).The composite scaffolds were fabricated through crosslinking chitosan and sericin via genipin.The physical characterizations of CS/SS composite scaffolds,including swelling ratio,porosity,microstructure and mechanical property,were determined by water absorption test,ethanol replacement method,scanning electron microscopy and compression test,respectively.Next,the biocompatibility of CS/SS scaffolds was analyzed by culturing RSC96 cells on the surface of composite scaffolds.Subsequently,degradation products of composite scaffolds were obtained.The effects of the degradation products on(1)the transcription of glialcellderived neurotrophic factor(GDNF),early growth response 2(EGR2)and neural cell adhesion molecule(NCAM)in RSC96 cells;(2)the transcription of tumor necrosis factor alpha(TNF-?)and interleukin-1 beta(IL-1?)in RAW 264.7 cells;and(3)the differentiation rate of undifferentiated PC-12 cells were tested,respectively.Finally,a rat model of chronic sciatic nerve compression was established.The tissue-engineered scaffold encapsulating NGF(NGF/scaffold)was applied for the treatment of chronically compressed nerves in a rat model.The functional recovery was evaluated by thermal withdrawal latency test,mechanical withdrawal threshold test,nerve conduction study and analysis of gastrocnemius muscles.The microstructural restoration was determined by myelin basic protein(MBP),?-3 tubulin immunofluorescence staining and transmission electron microscopy.Results(1)CS/SS composite scaffolds possessed high porosity(reaching 80%),suitable pore size(ranging from 130 ?m to 146 ?m),adjustable mechanical properties(ranging from 25.4 k Pa to 47.8 k Pa)and swelling ratios(ranging from 113.5% to 165.3%).(2)These composite scaffolds were able to support the growth of Schwann cells for 9 days.(3)By increasing sericin within the scaffolds,the treatment using their degradation products gradually upregulated the m RNA levels of GDNF(CS0/SS100 group was 5.24-fold higher than CS100/SS0 group),EGR2(CS0/SS100 group was 6.01-fold higher than CS100/SS0 group)and NCAM(CS0/SS100 group was 2.31-fold higher than CS100/SS0 group)in RSC96 cells,while gradually down-regulated the m RNA levels of TNF-?(CS0/SS100 group was 0.74-fold lower than CS100/SS0 group)and IL-1?(CS0/SS100 group was 0.34-fold lower than CS100/SS0 group)in RAW 264.7 cells.(4)The composite scaffold with 1:1 chitosan-tosericin possessed optimal neurotropic properties.(5)At Week 4 after treatment,the tissueengineered strategy achieved significant nerve recovery in a CNC animal model by decreasing neuralgia,improving nerve conduction velocity(blank control group was 46.3 m/s,while NGF/scaffold group was 63.4 m/s),accelerating microstructure restoration(the thickness of myelin sheath in blank control group was 0.77 ?m,while NGF/scaffold group was 1.44 ?m),and attenuating gastrocnemius muscles dystrophy(the relative wet weight of gastrocnemius muscle in blank control group was 84.39%,while NGF/scaffold group was 93.76%).ConclusionThis novel tissue-engineered strategy achieved significant functional and histological recovery when compared to traditional surgical decompression,suggesting a promising alternative for clinically treating peripheral nerve compression.Part Two Sericin Nerve Guidance Conduit Delivering Clobetasol for Functional and Structural Regeneration of Transected Peripheral NervesObjectiveTo explore the efficacy of clobetasol-loaded sericin nerve guidance conduit in treating large nerve gap.MethodsSericin was extracted from the cocoons of Bombyx mori(140 Nd-s)silkworm strain via the Li Br extraction method.Sericin nerve guidance conduit(GSC)and sericin film(GSF)were fabricated using glutaraldehyde as the crosslinker.The physical characterizations of GSC,including swelling ratio,porosity,microstructure and mechanical property,were determined by water absorption test,ethanol replacement method,scanning electron microscopy and compression test,respectively.Next,the effects of GSF on(1)the growth of RSC96 cells;(2)the transcription and translation of neurotrophin-3(NT-3)and neurotrophin-4(NT-4)in RSC96 cells were tested.The effects of clobetasol on(1)the proliferation of RSC96 cells;(2)the transcription and translation of neuregulin 1(NRG1)and early growth response 2(EGR2)in RSC96 cells were tested.Finally,clobetasol-loaded GSC(Clobetasol/GSC)was utilized in bridging a 10 mm long sciatic nerve gap in a rat model.The functional recovery was evaluated by calculation of static sciatic index,thermal withdrawal latency test,nerve conduction study and analysis of gastrocnemius muscles.The microstructural regeneration was determined by S100,myelin basic protein(MBP),?-3 tubulin,CD34 immunofluorescence staining and Luxol fast blue(LFB)staining.Results(1)The GSC possessed high porosity(82.56%)and suitable pore size(36.73 ?m).(2)The GSF exhibited excellent biocompatibility and promoted RSC96 cells to produce selected neurotrophic factors,NT-3(GSF group was 3.48 fold-higher than Control group at Day 7)and NT-4(GSF group was 3.75 fold-higher than Control group at Day 7).(3)Clobetasol significantly enhanced the proliferation of RSC96 cells and promoted the transcription and translation of two myelin-critical genes,NRG1(the m RNA level of NRG1 in Clo group was 2.84 fold-higher than DMSO group)and EGR2(the m RNA level of EGR2 in Clo group was 15.83 fold-higher than DMSO group),in RSC96 cells.(4)The implantation of clobetasolloaded GSC in vivo led to successful regeneration of the transected sciatic nerve.The GSC alone led to a moderate functional and histological recovery,which was further improved by the inclusion of clobetasol into the GSC.(5)The regeneration outcome of Clobetasol/GSC group was functionally comparable to autologous nerve grafting(evidenced by three parameters,i.e.static sciatic index,relative reaction time and nerve conduction velocity).Specifically,the static sciatic index(SSI),relative reaction time(RRT)and nerve conduction velocity(NCV)in Clobetasol/GSC group were-74.55,1.30,and 46.4 m/s at Week 12,respectively,while these parameters were-64.53,1.23,and 49.8 m/s in Autograft group.ConclusionClobetasol could become a therapeutic agent in peripheral nerve regeneration due to its effects on enhancing the proliferation of RSC96 cells and promoting the transcription and translation of NRG1 and EGR2 in RSC96 cells.The inclusion of clobetasol into the GSC effectively promoted functional and structural regeneration in sciatic nerve,suggesting its clinical translational potential.Part Three Sericin Applied in Developing a Microfluidic 3D Microvascular ModelObjectiveTo explore the effect of sericin in developing a microfluidic 3D microvascular model.MethodsSericin was extracted from the cocoons of Bombyx mori(140 Nd-s)silkworm strain via the heat and alkaline degumming method.The sericin powder was obtained by lyophilization and dissolved to reach a concentration of 0.1%(w/v).The effect of 0.1% sericin on the growth and proliferation of human umbilical vein endothelial cell(HUVEC)was tested.The poly(dimethylsiloxane)(PDMS)surface was chemically modified with PLASMA,(3-aminopropyl)triethoxy silane(APTES)and glutaraldehyde(GA).Next,the efficiency of chemical modification in immobilizing sericin was determined by BCA assays.After that,water contact-angle of the functionalized(chemical modification + sericin coating)surface was measured.The number of residual cells after rinsing with PBS and cell spread area on the functionalized surface were calculated.Finally,the capillary-like structures were formed in microfluidic chips by using soft lithography.HUVECs were subsequently cultured on the functionalized surface of the micro-channels and the cell viability was determined.Results(1)Sericin was naturally biocompatible with HUVEC and possessed nutritive function.(2)Chemical modification significantly enhanced the immobilization of sericin on PMDS surface(PDMS group was 0.01 ?g/mm2,while PDMS+APTES+GA group was 0.25 ?g/mm2).(3)The contact angle of the native PDMS(115.8o)decreased significantly with the surface functionalization(57.1o),suggesting an enhanced surface wettability.(4)After rinsing with PBS,the number of residual cells on the functionalized surface(219/mm2)was significantly higher than that on the unmodified surface(9/mm2)and the surface with sericin adsorption only(23/mm2).(5)There was a significant increase in cell spreading area on the functionalized surface(1004 ?m2)as compared to the unmodified surface(660 ?m2).Furthermore,the HUVEC spreading area on the functionalized surface was comparable to that on the surface of tissue culture plate(1378 ?m2).(6)HUVECs were able to adhere to the functionalized surface of the micro-channels and keep alive in the microfluidic chip for more than 7 days.ConclusionChemical modification effectively improved the immobilization of protein on PMDS surface.Sericin was an ideal biomaterial which could be applied in surface coating of PDMS.A microfluidic 3D microvascular model was successfully established,it could be further applied in exploring the process of metastasis of different tumors by using different experimental conditions.