Optimization Of Seed Cells Acquisition Derived From Cartilage For Tissue Engineering

BackgroundLacking self-healing capacity,cartilage were previously thought to be consist of single cellular component,which is chondrocyte.With the development of biotechnology,a number of researchers had isolated cells with the ability of self-renewal and multi-differentiation from articular cartilage,nasal septum cartilage,perichondrium,intervertebral discs and other tissues,called cartilage-derived stem/progenitor cells(CSPCS).CSPCS has advantages in chondrogenic differentiation,chondrogenic phenotype stabilization,resistance to cell hypertrophy and extracellular matrix deposition,thus became an ideal source of seed cells for cartilage tissue engineering,and gradually applied to cartilage tissue engineering and 3D bio-printing.The extraction and separation of seed cells are the basis of tissue engineering.The isolation methods and mechanisms of CSPCS are various,and there is no uniform standard,evaluation and optimization isolation method,which is important for the extensive application of CSPCS in cartilage tissue engineering.In addition,the sources of CSPCS are diverse.Currently,common sampling areas include joints,auricle,nasal septum and other parts.As a common source of autologous cartilage in clinic,costal cartilage has large amount of tissue,limited injury of donor site and mature harvest method.As an ideal source of CSPCS,costal cartilage has a good application prospect.Therefore,optimization of the sampling area is a problem need to be solved.Therefore,we believe that it is necessary for further study and optimize the isolation method and harvest site of costal cartilage for CSPCS,so as to provide experimental basis and reference for the construction of tissue-engineered cartilage by CSPCS as seed cells.Objective1.To compare and optimize methods of isolating CSPCs derived from rabbit cartilage.2.To clarify the influence of the source site on the cytological behavior of CSPCS and in vitro construction of cartilage tissue,and optimize the harvest site of costal cartilage for CSPCS.Methods and results1.Isolation and identification of CSPCS Methods:Three methods were used:fibronectin adhesion and low-density screening(FN+LD group),fibronectin adhesion alone(FN group),low-density screening(LD group)to isolate CSPCS and the expression of surface mesenchymal stem cell related marker proteins were assessed.Results:CSPCS isolated by all the three methods expressed CD44 and CD90 on a high level.2.Effects of different isolation methods on the biological characteristics of CSPCS cells Methods:the self-renewal ability of cells was detected by clone formation experiment.Flow cytometry was used to detect cell cycle.Cell migration ability was detected by cell scratch test.Induction of osteogenesis,lipogenesis and chondrogenesis was performed to detect the difference in the ability of multidirectional differentiation.Results:the CSPCS isolated by the combined method was superior to the low-density method in amplification and differentiation,and the fibronectin adhesion method was superior to the combined method.3.Influence of source site on the biological characteristics of CSPCS cells Methods:the whole segment of rabbit costal cartilage was divided into two groups:rib segment cartilage(R group)and free segment cartilage(F group).Stratification:CSPCS were extracted from superficial cartilage(group S)and deep cartilage(group D).Cell proliferation was determined by Cell Counting kit-8(cck-8)method.The self-renewal ability of cells was detected by clone formation experiment.Apoptosis was detected by TUNEL staining.Cell migration was measured by cell scratch test.Results:the CSPCS from the group R was superior to the CSPCS from the free end in cell proliferation,self-renewal and tolerance of nutrient deficiency environment,and the CSPCS from the surface source was superior to the deep ones,and self-renewal and tolerance of nutrient deficiency environment as well.4.Influence of source site on the construction of cartilage tissue of CSPCS in vitro Methods:cartilage tissue without scaffold was constructed by CSPCS from different sites in vitro.The histological features of the constructed cartilage tissues in each group were compared.Immunofluorescence staining and laser confocal microscopy were used to detect the cartilage related marker proteins and to evaluate the fine structure of the cartilage tissues.Results:the CSPCS derived from costal cartilage had good chondrogenic capacity,and the cartilage tissue constructed in vitro in each group was well deposited in the extracellular matrix.CD 146 was differentially expressed on the cell surface in new cartilage tissue.Conclusions1.CSPCS can be isolated by fibronectin adhesion combined with low-density screening,fibronectin adhesion alone and low-density screening.The difference in self-renewal and differentiation ability is related to the isolation method,and FN is better than FN+LD,both of which are better than LD.2.CSPCS derived from the surface layer and the proximal costal cartilage have a stronger ability in proliferate and self-renew,stronger ability to tolerate low nutritional conditions and better extracellular matrix deposition.3.When CSPCS are used as seed cells in tissue cartilage,the proximal costal cartilage segment and the superficial costal cartilage can be used as the selection of fine materials.