| OBJECTIVE: A group of PLLA/Lignin nanocomposite fiber membrane were prepared with electrospinning techniques,using them to study the differences ability of bone marrow mesenchymal stem cells differentiate into chondrocytes induced by fibrin membranes with different proportions,filter out the best ratio for effect of stem cell growth and induction in vitro.This group of fiber membrane would implanted into the model of articular cartilage defects surgical operation by Sprague-Dawley(SD)rats to verify the effect of nanocomposite fiber membrane could repairing a large area of superficial cartilage defects.Methods: First part,the synthesis of nanofiber membranes.LP copolymer were synthesized with Lignin and PLA,then,mixed with PLLA in different ratios and The nano-composite fiber membrane was made through electrospinning technology.Characterization of electrospun nanofibers with Aseries of detection: Scanning electron micrographs,fiber morphology and fiber diameter were measured;The mechanical properties of the PLLA/LP nanofibers were characterized by Tensile strength and Young's modulus;The viscoelastic behavior of the nanofibers was investigated using a dynamic mechanical analysis.In the second part,we investigated the chondrogenic differentiation of bone marrow mesenchymal stem cells into nanocomposite fibrin membranes in vitro.Different proportions of nano-composite fiber membranes were used as research objects and slide-graft group was established as control,bone marrow mesenchymal stem cells was extracted from SD rats,expanded and cultured in vitro,then,inoculated into each fiber membrane group and control group,cultured after 7 days,the adhesion morphology of the cells and the scaffold was observed with a scanning electron microscope(SEM);after 21 days,calcein/propidium iodide staining and DNA proliferation were examined for the ability of the fibrous membrane to support growth of the cells;The secretion glycosaminoglycans(GAG),phalloidin/33258 fluorescence staining and immunofluorescence staining of collagen II were used to detect the characteristics of chondrogenic differentiation;7-,14-,and 21-day cultured samples were used to detect the expression of cartilage-related characteristic genes(fluorescence quantitative PCR),verification the ability of chondrogenic differentiation.In third part,the nanocomposite fibrous membrane was used to repair the superficial cartilage defect of rat joint in vivo.The SD rat's joint cavity was opened and a cartilage notch was made by electric drill.The model of superficial cartilage defect was prepared with rat.The cells-nano-composite fiber membranes which has Selected were Implanted in vivo and the control group was established at the same time.Samples were obtained and collected at2,4 and 6 weeks after operation.Gross observation scores and histologicalscores were performed.After fixed with Paraformaldehyde,the joints were used to HE staining,Safranin O staining and immunohistochemical staining to observe the repair of articular cartilage defects.RESULTS: In this paper,LP copolymer was successfully synthesized and a series of PLLA/Lignin nanocomposite fiber membranes were made by electrospinning.Through material characterization,the fiber diameter ranges from 712 ± 63 nm to 350 ± 80 nm,the tensile strength of the nanofibers from3.49 ± 0.20 MPa for PLLA to 2.49 ± 0.14 MPa,the Young's modulus from 56.8± 1.6 to 66.8 ± 2.8 MPa and so on.In the experiment of induction of bone marrow mesenchymal stem cells into cartilage in vitro,cell proliferation or activity was assayed by cell proliferation assay and Calcein-AM/PI fluorescence staining,which significant of PLLA-Ligp30% group;Scanning electron microscopy observed cells adhesion and growth conditions In the material and cells with each group of fiber membrane growth better than control,furthermore,the change of shape are most obvious in PLLA-Ligp 30% group,a certain differentiation trend can be observed;The results of GAG secretion assay,collagen II immunofluorescence staining and PCR shows that the chondrogenic differentiation of mesenchymal stem cells has some trends can be observed in each fiber membrane group compared to control group,the expression of COL II,SOX9,and ACAN which were characteristic genes of cartilage was significantly higher.Overall,the cells of PLLA-Ligp30% group had cartilage feature markers obviously;The interfilation of the actin cytoskeleton around the nucleus was observed by fibrous actin secretion assay.The actin skeleton of PLLA-Ligp30%group was fully intertwined with the material fibers,which was consistent with the above detection.Through in vivo experiments of SD rats,the joints which were implanted with cell-nano composite fiber membrane had a better repairedeffects of cartilage defects compared to the control significantly.In addition,inhibition of osteoarthritis and protection effect can be observed;The results of histological staining also consistent to the gross observation.The Implant of mesenchymal stem cells with nanofiber membranes can chondrogenic differentiation and repair of superficial cartilage defects of joints in vivo.Conclusion: The nano-composite fiber membranes in different proportions can be prepared by electrospinning with lignin-PLA copolymers successfully.The nano-composite fiber membranes can induce mesenchymal stem cells into chondrogenic differentiation in vitro and showed a good curative effect on the articular superficial cartilage repairing in vivo.This study can promote direction for innovative composite biomaterials and provide theoretical basis for the design of cartilage-inducing materials. |
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