| BACKGROUNDSince the autogenous rib cartilage have the features as abundant volume,viability,good biomechanical property,non-immunogenicity,et al.,it is always taken as one of the most common material for repairing,padding,or supporting in plastic surgery.The most common way of cartilage transplantation is implanting it in the form of huge block directly,or in the form of a framework pieced together.However,this way brings many problems,such as waste,inflexibility,poor efficiency,complicated process and so on.In order to achieve more elaborate outcomes,some plastic surgeons advocate that dice the cartilage into small block before the implantation.Moreover,researches and clinical applications in joint surgery showed that diced cartilage transplantation not only brought the flexibility,but the promotion of the property of the chondrocytic proliferation.This makes this way of transplantation achieve some result of tissue engineering without the drawbacks of it.In order to make clear the mechanism of chondrocytic proliferation and historical fate of the transplanted cartilage micro-tissue,an inquiry experiment include in-vitro culturing of cartilage micro-tissue,in-vitro culturing of cartilage micro-tissue&biomimetic matrix complex and in-vivo culturing of cartilage micro-tissue&biomimetic matrix complex was designed.OBJECTIVE1.To observe the migration and the proliferation of chondrocyte in cartilage micro-tissue when cultured in vitro,and to observe the influence from the diameter of micro-tissue on the migration and the proliferation.2.To confect biomimetic matrix for cartilage micro-tissue,to fabricate the complex of cartilage micro-tissue&biomimetic matrix,and to observe the influence of the biomimetic matrix on migration and the proliferation of chondrocyte in the complex.3.To culture the cartilage micro-tissue&biomimetic matrix complex in vivo,and to observe the historical fate of the implanted complex and migration and the proliferation of chondrocyte in the complex when cultured in vivo.METHODS2.The cartilage harvested from experimental mini-porcine was diced into small blocks,namely micro-tissues.And all of the micro-tissues,according to their diameters,were divided into 3 groups as<200um,200um?400um,400um-800um,with the help of experimental cell strainer.And all groups of the mirco-tissues were cultured in vitro,the migration and the proliferation of chondrocyte in cartilage micro-tissue were observed during this process,and the contrast among the groups was observed.3.Liquid biomimetic matrix was confected with chondroitin sulfate,sodium hyaluronate(HA)and type ? collagen(COL-?)extracted from allogeneic rib cartilage in proportion as COLII/HA/CS=8/0.5/1.5mg/ml.10 X DMEM was added into the biomimetic matrix in a volume ratio of 1:10,to establish the biomimetic matrix with DMEM for in-vitro culturing.Mix the cartilage micro-tissues and equal volume of the biomimetic matrix to fabricate the complex of cartilage micro-tissue&biomimetic matrix.And the complexes were cultured in vitro after being solidified.14 days later all the complexes underwent HE stain.And observe the migration and the proliferation of chondrocyte histologically.3.The cartilage harvested from experimental mini-porcine was diced into micro-tissues.And the complexes of cartilage micro-tissue&biomimetic matrix were fabricated and autologous subcutaneous implantation was undergone within 4 hours since the cartilages was harvested.After a 2 months or 4 months in-vivo culture,the complexes was harvested,and all the complexes underwent HE stain.The historical fate of the implanted complex and migration and the proliferation of chondrocyte were observed.RESULTS:1.Formation of the cartilage micro-tissues and research of the influence from the diameter of micro-tissue on the migration and the proliferation(1)The micro-tissues formed with the thin blade knife and cell strainer were well survived,and some cartilage lacuna were split.(2)The micro-tissues and the chondrocytes in all the groups of<200um,200um?400um,400um?800um survived on the 3rd,7th,14th day of the in vitro culturing.(3)The migration and the proliferation of chondrocyte could not be observed on the3rd,7th,14th day of the in vitro culturing in the 3 group of micro-tissues under the microscope.2.Confecting of the biomimetic matrix and the influence of the biomimetic matrix on migration and the proliferation of chondrocyte in the complex cultured in vitro(1)The solidified biomimetic matrix turned to be white,and had certain hardness and elasticity.When mixed with DMEM,good biocompatibility was achieved.(2)No significant change of the form of the complex was seen during the in-vitro culturing.And no significant change of the volume of the complex when compared with its counterpart before culturing.(3)Under the microscope,the histological images showed the micro-tissues and the chondrocytes survived,and the migration and proliferation of chondrocyte could not be observed.3.Inquiry experiment of in vivo culturing of the complex of cartilage micro-tissue&biomimetic matrix(1)A irregular form was achieved when the cartilage micro-tissue&biomimetic matrix complex was cultured in vivo for 2 or 4 months.And the volume of the complex was decreased increasingly.(2)Under general observation,after 2 months in-vivo culture,irregular cartilage-like masses and fibrous tissues could be seen on the profile of complex.The hardness is lower than normal cartilage.The condition of complex cultured for 4 months was similar with 2 months except the cartilage-like hardness.(3)Under the microscope,the histological images of complex cultured for 2 months showed the micro-tissues and the chondrocytes survived well survived.And absorption of the biomimetic matrix could be seen in the histological image.However,there was no significant migration and proliferation of chondrocyte.(4)Under the microscope,the histological images of complex cultured for 4 months showed the micro-tissues and the chondrocytes survived well survived.And abundant fibrous tissues winded around the cartilage micro-tissues and made the gap between the micro-tissues shoter than cultured for 2 months.Little biomimetic matrix was left.And under 100×scope,two kind formation of chondrocytic migration and the proliferation can be seen in local:cell group around the micro-tissue without obvious secretion and scattered proliferation with matrix secretion.CONCLUSION:1.Formation of the cartilage micro-tissues and research of the influence from the diameter of micro-tissue on the migration and the proliferation(1)The method of forming micro-tissues with the thin blade knife and cell strainer can split the cartilage lacuna and assure the liveness of the cartilage micro-tissues.The micro-tissues and the chondrocytes survived can survived when cultured in vitro.(2)The migration and proliferation of chondrocyte can not occur during 14 days in-vitro culture.(3)There is no significant relationship between the diameter of the mirco-tissue and the migration and proliferation of chondrocyte.2.Confecting of the biomimetic matrix and the influence of the biomimetic matrix on migration and the proliferation of chondrocyte in the complex cultured in vitro(1)The solidified biomimetic matrix confected with COL-II,CS and HA achieves good biocompatibility,micro-tissues and the chondrocytes survived during the in-vitro culturing.(2)There is no significant change of the volume of the complex during the 14 days in-vitro culture.(3)In vitro,no significant migration and proliferation of chondrocyte occur,and the biomimetic matrix can not induce the chondrocyte to migrate or proliferate.3.Inquiry experiment of in vivo culturing of the complex of cartilage micro-tissue&biomimetic matrix(1)The complex of cartilage micro-tissue&biomimetic matrix had good biocompatibility in vivo and biomimetic matrix can be absorbed.(2)The complex survived in vivo and the micro-tissues were packaged as a whole by the fibrous tissue when culture in vivo.(3)A small number of migration and the proliferation of chondrocyte occurred in the autologous subcutaneous implantation.And the migration and the proliferation formed some cell groups around the micro-tissue or some cartilage-like mass. |
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