The Choice Of Method For Seeding Chondrocytes Into Poly(Lactic-co-glycolic Acid) Scaffold With Small Pore Size

With the rapid development of our country’s transportation business and the process of aging society gradually, the orthopaedic trauma, infection, the incidence of degeneration increased year by year. On the pathological basis of articular cartilage injury of traumatic, degenerative osteoarthritis and other disorders, as well as joint pain and dysfunction caused by them has become clinical common diseases and frequently-occurring disease. The problem of repair for damage cartilage has been the hot and difficult issues for researchers of international orthopedic, sports medicine circles, and biological materials academic research.Due to the lack of the nutrition from blood vessels and nerves, the regeneration ability of damage cartilage is very low. The currently clinical method for repairing cartilage containing two categories:bone marrow stimulation and transplanted. The former, such as drilling technique, micro-fracture surgery; The latter such as periosteal grafting, mosaicplasty and autologous chondrocyte transplantation (ACI) surgery. Although these mature technologies could achieve certain clinical curative effect, but are difficult to obtain satisfactory repairing effect, for example, through stimulating the bone marrow method could repair the cartilage defects only by fibrocartilage; repair of cartilage defects by transplantation, inevitably lead to damage the donor site, if there was a more larger defect case, transplantation technology is difficult to meet clinical needs.The rapid development of tissue engineering technology provide new ideas and technology for cartilage repair, the U.S. Food and Drug Administration (FDA) approved tissue engineered products used in clinical treatment further confirmed the clinical effect of the tissue engineering products. Tissue engineering research mainly focus on seed cells, tissue engineering scaffolds, in vivo and in vitro building. Poly(lactic-co-glycolic acid)(PLGA) because have good biocompatibility, adjustable rate of degradation and good mechanical properties, had been widely applied in the field of tissue engineering research. The porosity tissue engineering scaffold is conducive to cell proliferation, migration, and nutrient substance exchange, simultaneously accelerate the degradation of scaffolds. The scaffolds of bone tissue engineering always have large pore size, this is good to exchange of substances and the ingrowth of blood vessels, promote matrix mineralization and bone formation; while the scaffolds of cartilage tissue engineering required smaller pore size, which can reduce the partial oxygen tension, increased the contact between cells, and were benefit of the regeneration of the cartilage. Threrfore, the pore size of scaffolds for bone tissue engineering have been reported mostly greater than100μm, while the pore size of scaffolds aimed at cartilage tissue engineering designed less than100μm that as the small pore size. Among them, seed cells into the pore of scaffolds are the first link to build a three-dimensional culture environment of tissue engineering. However, the pores of scaffolds exist a large number of gases, the surface tension between gas and liquid block nutrient substance exchange and migration of cells, at the same time, the hydrophobicity of scaffold affect cell adhesion, migration and proliferation to some degree. Studies have reported that the low-pressure seeding method could be seed cells within the pores of scaffolds by excluding gas exist in the scaffold porosity, enhanced the efficiency of the cells eeding efficiency, and confirmed the pressure of100mm Hg can obtain a better cell seeding effect. However, previous studies on the low-pressure seeding method mostly in the scaffold with the pore size greater than100μm for bone regeneration, and and pore size suitable for cartilage tissue engineering research, mostly less than100μm, the effect of low-pressure seeding method is applied to the small pore size scaffolds less than100μm is still unknown. Therefore, research on low-pressure seeding method applied to the small pore size scaffolds is very necessary. Literature reported that the cell adhered to the periphery of scaffolds during initial cells seeding affect the growth and migration of cells of into the scaffolds, and small pore size scaffold are more likely to produce the peripheral resistance than large pore size scaffold. For PLGA scaffolds with small pore size, which kind of seeding method is more suitable needs our further study.However, no matter what field of tissue engineering, the ultimate goal is to improve the effect of construcing engineering tissue. The verification in vivo of effects of new technology and product is an indispensable link, so the animal experiments in tissue engineering research is the key links of tissue engineering research. The role of animal experiments in cartilage tissue engineering:the verification of the feasibility of the the method, the evaluation of the safety of the products, best indicators forecast, as new technology and methods of preclinical studies, plays a vital role as a link. However, the selection of animal model during the experimental design is difficult and time-consuming, therefore, it is necessary to review that the principles of choosing animal models, the characteristics and application of animal models available in tissue engineering cartilage.In this paper, we mainly compared the effect of low-pressure seeding method when adopted to200-300μm and50-100μm pore size PLGA scaffolds and by comparing the seeding effect of low-pressure seeding method, injection, orbital shaker when adopted to seed chondrocytes into PLGA scaffolds with small pore size, to explore the optimal seeding method. At the end of this article, we reviewed the principle of selecting the animal model for research of osteochondral tissue engineering, animal models characteristics and situation of application.The choice of method for seeding chondrocytes into poly(lactic-co-glycolic acid) scaffold with small pore sizePart1.The isolation, expansion of and identification of the costal chondrocytes of the New Zealand white rabbitObjectiveTo isolate, expand and identify the costal chondrocytes of the New Zealand white rabbit which were used as seed cells.Methods1. The isolation, primary culture and identification of rabbit costal chondrocytesObtain bilateral costal cartilage under sterile conditions, cut it into pieces, digested with0.25%trypsin under37℃for30min, centrifuged and abandoned supernatant,0.2%collagenase digested for6-8h, centrifuged and rinsed precipitation with DMEM. Seeded chondrocytes in flasks, add cell culture medium, incubated in37℃and5%of CO2, the first medium change after3d, cells were observed and identified the ability of secreting matrix with alcian blue staining.2. The expansion and identification of rabbit primary culture costal chondrocytesWhen primary cultured chondrocytes reached80%-90%confluence, digested cells with0.25%-0.01%trypsin-EDTA mixture for passaged expanding culture, until second generation, took collagen type Ⅱ immunohistochemical staining, identificated the chondrocyte phenotype and the ability of secreting of type Ⅱ collagen.ResultsNew Zealand white rabbits costal chondrocytes were isolated, primary cultured chondrocytes stained with alcian blue was positive, subculture to the passage2did collagen type Ⅱ immunohistochemical staining, showed positive expression, were identified correspond to the rib chondrocytes characteristics.ConclusionThe two-step enzymatic digestion method could obtain the experimental requirements of costal chondrocytes, subcultured to second generation still maintained the phenotype of hyaline cartilage cell.Part2. The influence of pore size of scaffolds on the effect of low-pressure seeding method using for chondrocytesObjectiveTo compare the effect of low-pressure seeding method using for seeding chondrocytes into200-300μm and50-100μm poly (lactic-co-glycolic acid)(PLGA) scaffolds.MethodsThe second generation of the chondrocytes were seeded by low-pressure seeding method into the different pore size PLGA scaffolds, respectively.. Set200-300μm group for group A,50-100μm group for group B (n=9).48h later, The DNA content of per construct was analyzed using Hoechst33285fluorometric detection and standardized by the dry weight of construct, to detected the efficiency of low-pressure seeding method; we seed fibrin gel contains FITC(Fluorescein Isothiocyanate) without chondrocytes to observed gel distribution to confirm that the gel would not block the pore size of scaffolds; analyzed the cell distribution in the scaffold through hard tissue slice and DAPI (4’,6-diamidino-2-phenylindole) nuclei staining;7d after seed chondrocytes, the morphology of cells in the outer periphery and the interior of the scaffold was observed by scanning electron microscope(SEM); evaluated cell proliferation trend in scaffolds by alamarblue assay, set1d,3d,5d,7d,9d after seed as point-in-time.ResultsDNA quantitative results showed that the DNA content of group A was498.51±40.18ng/mg, group B was657.32±89.68ng/mg, group A<group B (t=2.799, P=0.049); fibrin gel of both groups can realize uniform distribution in PLGA scaffolds; the results of DAPI staining showed that the distribution of cells in group A were more uniform, while cells more distributed in the outer periphery in group B; SEM indicated that within pores of group A have more chondrocytes adhesion, but group B showed none of chondrocyte adhesion; the result of alamarblue assay showed a consistent trend of proliferation of group A and group B, group A was faster than group B in late proliferation, no statistical differences in the amount of cells in the two groups to9d.(t=2.246, P=0.088).ConclusionLow-pressure seeding method applied to200-300μm PLGA scaffolds can obtain better effect, but it is not suitable for seeding chondrocytes into PLGA scaffolds with50-100μm pore size.Part3. The choice of method for seeding chondrocytes into PLGA scaffold with small pore sizeObjectiveCompare the chondrocytes seeding effect of low-pressure method, injection and orbital shaker method for50-100μm PLGA scaffold, to explore the best seeding method.MethodsIsolated and cultured of chondrocytes from rib cartilage of New Zealand white rabbits, sub-cultured to the second generation, seed fibrin gels containing chondrocytes into50-100μm PLGA scaffolds (porosity92%) by low-pressure method, injection and orbital shaker,respectively(n=9).48h later, the DNA content of per construct was analyzed by Hoechst33285fluorometric detection standardized by the dry weight of scaffold; assessed the cell distribution in the scaffold through hard tissue slice and DAPI (4’,6-diamidino-2-phenylindole) nuclei staining. Observed gel distribution by seeding fibrin gel contains FITC (fluorescein isothiocyanate) without chondrocytes using all above methods, respectively.7d after seed cells, observed the morphology of chondrocytes in the outer periphery and the interior of the scaffold by scanning electron microscope(SEM); some constructs implanted subcutaneously into nude mice(n=3), after eight weeks, took out them did toluidine blue and collagen type II immunohistochemistry staining, used the Image-Pro Plus6.0software analyzed the images to assessed the in vivo chondrogenic effect.ResultsDNA quantitative results showed that the DNA content of low-pressure group was657.32±89.68ng/mg, injection group was755.79±80.50ng/mg, orbital shaker was650.18±106.33ng/mg, there was no significant difference between groups (F=1.214, P=0.361); the results of DAPI staining indicated that the distribution of cells in injection group was more uniform than the other two groups; the fibrin gel of all groups could be uniformly distributed in the scaffolds; though SEM we discovered cells distributed in the outer periphery in low-pressure group and orbital shaker group, only the inside pores of injection group had visibly cell adhesion; the staining results of paraffin section display the extent of matrix and collagen type Ⅱ of injection group was more wide than low-pressure group and orbital shaker group (P=0.000).ConclusionFor50-100μm PLGA scaffold, injection is a quick, efficient cell seeding method.