Co-Culture Of Adipose-Derived Stem Cells And Chondrocytes With TGFβ3 Promote Chondrogenic Differentiation And Construction

In repair and reconstruction surgery,cartilage defects caused by various reasons,such as congenital heredity,postnatal trauma,tumor and inflammatory reaction,are very common.Due to the limited repair potential of cartilage,the damaged cartilage is general replaced by fibrous tissue or scarred cartilage,which is far from the shape and function of primary body cartilage.At present,artificial substitutes or autogenous cartilages have many disadvantages in clinical cartilage defect repair.At the same time,autogenous cartilage transplantation also has some disadvantages,such as insufficient donor site,long cell cycle of digestion and culture,prone to pathological changes in donor site,and difficult to maintain chondrocyte phenotype and function.Therefore,it is an urgent problem to find cartilage source that is easy to obtain,minimally invasive,good in shape and function.In recent years,the rise and development of biological tissue engineering in the field of repair and reconstruction surgery provides new ideas and methods for the treatment of cartilage defects.Tissue engineering is a new discipline established and developed by the combination of seed cells and scaffold materials.The core and key of tissue engineering is appropriate seed cells and construction mode.Therefore,the primary basis of tissue engineering cartilage construction is seed cells.Ideal cartilage seed cells should have the following characteristics:1)easy to obtain,less damage;2)strong in vitro expansion ability,short-term access to a certain number of seed cells;3)directional differentiation to chondrocytes,stable phenotype and so on.How to select and obtain suitable cartilage seed cells has become the primary task of tissue engineering cartilage construction.Adipose derived stem cells(ADSCs/ASCs)are a kind of adult stem cells existing in adipose tissue.Because of their wide distribution in human and animal bodies,abundant sources and easy access,ADSCs/ASCs have become one of the seed cells for tissue engineering in recent years.Zuk et al.Extracted adipose derived stem cells from aspirated adipose tissue for the first time and made a more in-depth study,which proved that they could differentiate into osteocytes,chondrocytes and adipocytes under specific induction conditions.Previous studies in our laboratory have proved that the combination of TGF β 1,IGFI and dexamethasone can induce adipose derived stem cells to differentiate into chondrocytes,construct tissue-engineered cartilage,artificially create knee cartilage defects,and transplant the induced cell material complex into pig knee joint,It is proved that TGF-1 can improve the potential of repairing articular cartilage defects.As an essential inducer,TGF-β1 and TGF-β3 have the ability to induce adipose derived stem cells to differentiate into chondrocytes in the TGF superfamily.Recent studies have found that TGF-β3 has more advantages in adipose derived stem cells to differentiate into chondrocytes.TGF-β3 is an important promoter of chondrocyte lineage differentiation.The addition of TGF-β3 can rapidly initiate chondrocyte lineage differentiation,significantly enhance the expression of col Ⅱ and AGG in cell population,promoting cartilage repair and accelerate cartilage differentiation through Smad pathway,and also control matrix metalloproteinase(MMP),The expression of matrix metalloproteinase(TIMP)was stimulated to promote the formation of cartilage associated collagen.Currently,for TGFβ1 there have been many studies on the cartilage differentiation of adipose derived stem cells,and it has been verified that TGF-β1 has a certain cartilage construction potential for adipose derived stem cells.However,there are few studies on the differentiation of adipose derived stem cells into chondrocytes induced by TGF-β3,especially the construction of tissue-engineered cartilage in vitro and in vivo,which is worthy of further study in order to obtain a more optimized tissue-engineered cartilage Bone induction protocol.In addition,the literature shows that the co culture of adipose derived stem cells can not only promote the differentiation of seed cells,improve the induction efficiency,but also reduce the influence of interference source cells on seed cells.When adipose derived stem cells and chondrocytes are co cultured,not only the differentiation efficiency of adipose derived stem cells to chondrocytes is accelerating,but also the proliferation rate and extracellular matrix surface area of chondrocytes are increased both of them had an increasing effect.Huang et al.Found that after chondrocytes and mesenchymal stem cells were co cultured,mesenchymal stem cells continuously expressed cartilage phenotype and differentiated into chondrocytes.Cai et al.Confirmed that in the three-dimensional environment,adipose derived stem cells and chondrocytes co cultured to form macromass,adipose derived stem cells were induce to differentiate into chondrocytes.After implanted into nude mice,the expression of cartilage in the co-cultured macromass was better than that in the single culture group.Although we have a certain understanding of the induction mode of cell level co culture,there is still a lack of three-dimensional research on co culture,especially how to use co culture to construct high-quality three-dimensional tissueengineered cartilage for the repair of craniomaxillofacial cartilage defects,which needs further research and discussion.Therefore based on the research of adipose derived stem cells in the literature and previous experiments,we propose to use TGFβ3 as the inducing factor,and adopt the coculture mode of adipose derived stem cells and chondrocytes to evaluate the feasibility of adipose derived stem cells to construct tissue-engineered cartilage.Hoping to obtain a more optimized cartilage induction and construction scheme,which can provide reference for clinical cartilage engineering Repair of the defect to provide help.[Purpose]Objective to investigate the feasibility of adipose derived stem cells(ADSCs)differentiating into chondrocytes under the biphasic induction mode of TGF-β 3 and cell co culture,and the ability of PGA/PLA composite to construct tissue-engineered cartilage in vitro and in vivo.[Methods]1 Adipose derived stem cells differentiate into chondrocytes:adipose derived stem cells(ADSCs)and chondrocytes were obtained from porcine abdominal adipose tissue and ear cartilage tissue by enzymatic digestion.The proliferation ability in vitro was observing and the P2 adipose derived cells were collecting and identified by flow cytometry.After eliminating the cell interference,the chondrogenic differentiation potential was identifying by osteogenic induction,chondrogenic induction and adipogenic induction.The second generation ADSCs were collected and cultured with TGF-β 3(10 ng/ml tgf-b3,10 ng/ml BMP6,50 ng/ml IGF-1,0.1 um dexamethasone,50 mg/ml VC,40 mg/ml proline).The experiment was divided into four groups:ADSCs+ACS+TGF-β 3 group,ADSCs+ACS group,ADSCs+TGF-β3 group,ACS+TGF-β3 group.Each group was inducing and identified from two-dimensional cell level and macromass.After cultured in TGF-β3 medium for 3-4 weeks,the ability of adipose derived stem cells to differentiate into chondrocytes was evaluating by morphological observation,cell proliferation sequencing,quantitative reverse transcription polymerase chain reaction,he staining and immunohistochemical staining.2 Adipose derived stem cells were used to construct cartilage tissue in vitro:adipose derived stem cells and chondrocytes were used as seed cells,and polyglycolic acid/polylactic acid(PGA/PLA)was used as scaffold material to construct ADSCs+ACS+material complex,and the ability of adipose derived stem cells to construct cartilage tissue in vitro was analyzed.The P2 cells were collecting according to the culture method of adipose derived stem cells and chondrocytes in the first part,and then combined with PGA/PLA scaffolds to construct cell material composites.ADSCs+ACS+TGF-β 3 group,ADSCs+ACS group,ADSCs+ TGF-β 3 group and ACS+TGF-β 3 group were set up respectively.The cells were culturing in vitro with TGFβ3 induction medium for 4 weeks.After 4 weeks,the ability of adipose derived stem cells to construct cartilage tissue in vitro and the biphasic induction scheme of TGF-β 3 co culture were evaluated by gross observation,wet weight test,biomechanical test,GAG content test,col Ⅱ content test,immunohistochemical staining and safranine-o staining.3 Adipose derived stem cells were used to construct cartilage tissue in vivo:according to the second part of the experiment,the cell material complex was constructed,includingADSCs+ACS+TGFβ 3、ADSCs+ACS group、ADSCs+TGFβ3 group、ACS+TGFβ 3 group and other four groups were treated with TGF-β 3 induction medium for 2 weeks in vitro,and then the composite was transferred to nude mice for 4 weeks in vivo.The ability of adipose derived stem cells to construct cartilage tissue in vivo and the biphasic induction scheme of TGF-β 3 co culture were evaluated by gross observation,wet weight test,biomechanical test,GAG content test,col II content test,immunohistochemical staining and safranine-o staining.[Results]1 At the cellular level,porcine adipose derived stem cells can differentiate into chondrocytes through the dual induction mode of TGFβ3 and co-culture,and the dual induction mode of TGF-β 3 and co culture has a synergistic effect.The combined effect is greater than the single induction mode.2 In vitro,through the two-way induction mode of TGF-β 3 and co culture,porcine adipose derived stem cells can form tissue-engineered cartilage like tissue on PGA/PLA scaffolds,which has a certain ability to construct cartilage in vitro.Moreover,the dual induction mode of TGF-β 3 and co culture has a synergistic effect,and the co induction effect of TGF-β 3 and co culture is better than that of single-phase induction mode.3 In vivo,through the two-way induction mode of TGF-β 3 and co culture,porcine adipose derived stem cells can form tissue-engineered cartilage like tissue on PGA/PLA scaffolds,which has a certain ability of cartilage construction in vivo.Moreover,the dual induction mode of TGF-β3 and co culture has a synergistic effect,and the co induction effect of TGF-β 3 and co culture is better than that of single-phase induction mode.