Preparation Of Growth Factor Modified PGCL Biomimetic Tissue Engineered Cornea And Its Study On Corneal Injury Repair

Purpose: To prepare stem cell biomimetic tissue engineering cornea with growth factor modified PGCL electrospun fiber membrane and to explore its application in corneal injury repair.Methods: 1.In this study,glycolide-caprolactone copolymer was synthesized by polymerization with glycolide and ε-caprolactone as monomers under the catalysis of initiator stannous octoate.By adjusting the blending ratio of glycolide and ε-caprolactone,biological polymeric materials with controllable mechanical and degradability can be synthesized.Based on electrospinning technology,electrospinning fiber membranes under different electrospinning parameters can be prepared by adjusting electrospinning process parameters such as voltage,solution concentration,liquid feeding speed,distance between spinneret and receiver,etc.Through the characterization of the surface morphology,hydrophilicity,mechanical properties,light transmittance and degradability of the electrospun fiber membrane,the electrospinning process parameters with the best performance were obtained at the same time,the toxicity test of the cells cultured with the material extract will provide a theoretical basis for the follow-up research.2.In order to further study the biocompatibility and degradability of PGCL,the model of subcutaneous material implantation on the back of rats was established,and the biocompatibility of the material in vivo was further evaluated by tissue section hematoxylin-eosin staining(HE)and Interleukin-6(IL-6)tissue immunofluorescence staining.And the macroscopic images of the materials were taken to evaluate the degradability of PGCL electrospinning membrane in vivo.3.The precursor sequence of DOPA adhesion group composed of tyrosine and lysine,namely YKTKY(Tyrosine-Lysine-Tyrosine-Lysine-Tyrosine,YKYKY),was added to the C-terminal of epidermal growth factor(EGF)and basic fibroblast growth factor(b FGF)by genetic engineering.The target proteins YKYKY-EGF and YKYKYb FGF were expressed and purified by E.coli,in which tyrosine and DOPA are similar in molecular structure.Furthermore,under the action of tyrosine hydroxylase,a dihydroxyl structure similar to DOPA was formed,that is,the adhesive recombinant growth factors DOPA-EGF and DOPA-b FGF were obtained.The amount of adhesion growth factor on the surface of spinning membrane and its release at different time points were studied by enzyme-linked immunosorbent assay(ELISA).The biological activity of recombinant growth factor was detected by mouse embryonic fibroblast cell line NIH 3T3.4.The primary rabbit limbal stem cells were cultured by tissue block method and enzyme digestion method,and the cell morphology was observed by inverted phase contrast microscope.The extracted cells were identified by cell immunofluorescence staining of LSC positive marker p63 and negative marker CK3.5.Different concentrations of target proteins YKYKY-EGF and YKYKY-b FGF were hydroxylated on the electrospinning membrane by tyrosine hydroxylase and cocultured with LSC.The optimum concentration of growth factors suitable for LSC proliferation was screened by CCK-8 method.According to the selected concentration,the in vitro cell experiment was carried out,and from the perspective of single application of growth factors and combined application of growth factors,the effect of growth factors on cell proliferation was evaluated by CCK-8 method.The biocompatibility of growth factors and materials to cells was observed indirectly by living/dead cell staining.The cytoskeleton structure at different time points was observed by fluorescence staining with phalloidin.The cell morphology and nucleus were stained by fluorescent dyes isothiocyanate(FITC)and 4’,6-diamido-2’-phenylindole(DAPI)respectively to observe the cell adhesion on the surface of electrospinning membranes.The expression of differentiation related genes such as CK3 and CK12 in vitro was analyzed by real-time quantitative PCR.6.The rabbit corneal mechanical injury model was constructed,and the repair of corneal injury by biomimetic tissue engineering cornea was evaluated by slit-lamp anterior segment photography,HE staining,Masson staining and immunohistochemical staining in vivo.Results: 1.According to the idea of orthogonal experimental design,the morphology of electrospun film was analyzed.The SEM results showed that the diameter of PGCL20 fiber was smooth and no beads were formed under the condition of Test 2 experiment.The contact angle results showed that the contact angles of each group were more than 120°,indicating that the electrospun membrane was hydrophobic.UV spectrophotometer test results show that the light transmittance of each group is less than 13%,and the transparency of the material is not good.The mechanical test results showed that the maximum tensile strength and elastic modulus of the spinning film in PGCL20 group were 12.8±0.42 Mpa and 43.22±7.98 MPa respectively.It is concluded that the electrospinning membrane with good mechanical properties and morphology can be prepared when the spinning solution concentration is 13wt%,the electrospinning distance is 20 cm and the static spinning voltage is 17 k V.The results of leaching experiment showed that the cell viability was good and the material was non-toxic.The results of degradation experiments in vitro showed that the material was basically degraded at 12 weeks of degradation,and the fiber morphology was lost by SEM,and the mass loss percentage,p H value of degradation solution and molecular weight gradually decreased with the extension of degradation time.In vivo degradation experiment showed that the material was basically degraded at the 10 th week of degradation.The results of subcutaneous implantation on the back of rats showed that there was inflammatory cell infiltration in the tissue around the material on the 1st and 7th day after operation,and the IL-6 staining was positive,and the inflammatory cell infiltration basically disappeared on the 28 th day after operation,and no toxicity was found in the HE results of the five internal organs of rats,suggesting that the material had good biocompatibility.2.Y-EGF and Y-b FGF were successfully expressed and prepared by genetic engineering technology,and the biological activity of the recombinant growth factor was verified.DOPA-EGF and DOPA-b FGF with strong adhesion can be obtained by tyrosinase hydroxylation.The fitting curve of the amount of adhesion protein was obtained by ELISA.With the increase of the concentration of growth factor,the quality of the combination of growth factor and material increased.And with the extension of release time,the high concentration of growth factor is closely combined with the material,which is not easy to release,and the release rate of low concentration growth factor increases.The contact angle of electrospun film modified by DOPA-EGF and DOPA-b FGF is about 30 degree,which indicates that the material has changed from hydrophobic to hydrophilic.The light transmittance is increased from less than 13% before modification to more than 50% after modification,and the light transmittance is obviously improved.3.LSC was successfully extracted by enzyme digestion method and tissue block method.The electrospinning membrane adhered to growth factor was co-cultured with it,and the amount of adherent growth factor per unit area was calculated.The CCK-8 method was used to screen the optimal concentration of growth factors to promote cell proliferation.The results showed that the optimal concentrations of DOPA-EGF and DOPA-b FGF were 5 ng/cm~2 and 10 ng/cm~2,respectively.Furthermore,the OD value of the combined application of the two growth factors was 1.65±0.07,which was significantly higher than that of the growth factor group and the control group alone(P<0.001).There was almost no red fluorescence in the staining results of living/dead cells,and the green fluorescence range of DOPA-EGF&b FGF group was significantly higher than that of other groups on the 7th day.Through FITC staining and phalloidin staining,it was found that the cells in each group adhered well,the spreading area of cells was well,the number of cells was large,and the microfilament structure in cells was clearly visible,especially in the group of combined application of growth factors.The results of gene level detection showed that after 7 days of cell culture,the expression of CK3 and CK12 in the adhesion growth factor group increased,while the expression of p63 and PAX6 in the control group increased.4.In the in vivo experiment in the form of combined application of growth factors,slit lamp examination showed that the central corneal epithelial turbidity was obvious in the PGCL group,and the turbidity area accounted for about 90% of the corneal defect area,forming pannus,and it was not easy to observe the posterior anatomical structure through the cornea;the corneal epithelium in the Control group and the GF@PGCL group were slightly turbid,and the central cornea could be seen with clouds;the corneal transparency in the GF&Cell@PGCL group was the highest and the repair effect of the injury was good.The results of immunohistochemical staining showed that CK3+CK12 and Vimentin were positive.The results of HE staining showed that the epithelial thickness in GF@PGCL and GF&Cell@PGCL groups was significantly higher than that in Control and PGCL groups.The results of Masson staining showed that the deposition of matrix collagen was orderly in GF&Cell@PGCL group.Conclusions: 1.PGCL electrospun fiber membrane with controllable mechanical properties and degradability was successfully synthesized and prepared by chemical synthesis and electrospinning technology,and it has good biological safety and biocompatibility.2.The recombinant growth factors YKYKY-EGF and YKYKY-b FGF with strong adhesion and biological activity were successfully synthesized by genetic engineering technology,and they also had a certain sustained-release ability.3.The electrospinning membrane modified by growth factors have good hydrophilicity and good light transmittance in wet state.4.The cells were co-cultured with the electrospun membrane attached with growth factors,and the biomimetic basement membrane of tissue engineering cornea was constructed in vitro.It was confirmed that the combined application of DOPAEGF&b FGF and growth factors had better cell viability,better promotion of cell adhesion and proliferation.5.GF&Cell@PGCL can better promote the repair of corneal injury in vivo.