Platelet-rich Plasma Combined With Collagen Hydrogel Induces Chondrogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells

Articular cartilage defects are relatively common in clinic.Because articular cartilage lacks neurovascular distribution and poor regenerative ability,it is extremely difficult to repair itself after articular cartilage damage.Therefore,complete treatment of cartilage defects has become a difficult and hot topic in clinical treatment and basic research.With the deepening of research on cartilage defects with tissue engineering and regenerative medicine,it provides a more appropriate treatment plan for the repair of cartilage defects.In this study,rabbit bone marrow mesenchymal stem cells were used as seed cells for repair of cartilage defects.Collagen hydrogel was used as a scaffold for cartilage tissue engineering.Autologous platelet-rich plasma was used as a cell growth factor.The three were used to construct tissue engineering cartilage.Its effect of cartilage and cartilage repair in vivo and in vitro.Part I Role of PRP-complexed BMSCs/collagen hydrogel(COL)in cartilage induction in vitroObjective: To construct tissue-engineered cartilage with PRP-complexed BMSCs/COL and observe the effects of PRP-activated growth factors on cell proliferation,differentiation,and extracellular matrix secretion in tissue engineered cartilage.Methods: Collagen hydrogels were prepared using type I collagen.Three-dimensional mesenchymal stem cells and collagen hydrogels were mixed to construct a three-dimensional structure.Rabbit peripheral blood was extracted by second centrifugation to extract platelet-rich plasma and activated by CaCl2.Experimental group: BC group was collagenous group,third generation BMSCs and COL constructed tissue engineered cartilage,BCT group was third generation BMSCs and COL constructed tissue engineered cartilage,cartilage inducing solution containing TGF-? was added for culture;BCP The group constructed tissue-engineered cartilage for third-generation BMSCs and COLs and added PRP extract to culture.Each group was cultured for 7 days,14 days,and 21 days.Samples were taken for each day and the following correlation tests were performed: 1.Detection of DNA content contrasted the proliferation of cells in the tissue-engineered cartilage of each group;2.Performed HE staining and observed each The morphological changes of cells in the tissue-engineered cartilage were observed.3.The calcein-PI fluorescence staining was performed to compare the survival status of the cells in the tissue-engineered cartilage of each group.4.Phalloidin-hoechst33258 staining was performed to compare the tissue engineering of each group.The distribution of fibrial actin(F-actin)in cartilage;5.Safranin O staining and detection of DMMB reagents,comparing the different GAG content of cartilage-specific matrix secreted by cells in tissue-engineered cartilage of each group;6.Using qRT-PCR technique was used to detect the changes of cartilage-specific genes(ColII,Acan,ColI,SOX9)in each group during tissue-engineered cartilage formation.7.Immunohistochemical staining was used to detect type II tissue-engineered cartilage in each group.Collagen secretion to detect the degree of cartilage.Results: 1.Histological staining(HE staining,calcein-PI fluorescence staining,safranin O staining,and phalloidin-hoechst 33258 fluorescence staining)revealed that cartilage differentiation was observed in the BC group,the BCP group,and the BCT group,and the induction effect was observed in the BCP group.It is better than BC group,which shows that the induction effect is more obvious after adding platelet-rich plasma.Both PRP and collagen hydrogel can play a better role in the cartilage induction process.2,through biochemical detection(DNA content and GAG content detection),BCP group cell proliferation than BCT group and BC group is high,indicating that platelet-rich plasma released by the activation of growth factors have the role of promoting BMSCs proliferation.3.Immunohistochemistry COLII staining and RT-PCR gene detection showed that cartilage-specific matrix proteoglycans and COLII expression in BCP group was superior to BC group,indicating that platelet-rich plasma combined with collagen material can increase mesenchymal stem cells The effect of cartilage differentiation.Compared with BCT group,the expression of type I collagen was lower in BCP group,indicating that platelet rich plasma can maintain the cartilage phenotype and prevent cartilage dedifferentiation.Conclusion: 1.Autologous PRP can promote the proliferation of BMSCs and increase the activity of cells.2.Autologous PRP promotes the expression of cartilage-specific genes and extracellular matrix secretion of BMSCs in vitro and induces cartilage formation.3.Autologous PRP promotes cartilage formation.Similar to TGF-?1,in addition,its type I collagen expression is lower than TGF-?1,indicating that it can better maintain the tissue engineering cartilage phenotype.Part? The role of PRP-complexed BMSCs/collagen in the repair of knee cartilage defectsObjective:Feasibility of Constructing Tissue-engineered Cartilage to Repair Articular Cartilage Defects with PRP and BMSCs/collagen Probes.Methods: Firstly,PRP was extracted by second centrifugation.BMSCs were extracted by gradient density centrifugation.After three generations of culture,tissue-engineered cartilage was constructed with collagen hydrogel to create a rabbit knee cartilage defect model.Experimental group: BC group implanted with BMSCs/collagen tissue engineered cartilage;BCT group implanted with BMSCs/collagen tissue engineered cartilage,intra-articularly Injection of autologous TGF-?;BCP group implanted with BMSCs/collagen tissue engineered cartilage,intraarticular injection of autologous PRP.After 8 weeks in each group,the following materials were taken for detection: 1.Specimen observation: Observe the repair of cartilage defect,and roughly score the site of cartilage repair;2.Safranin-solid green staining to observe the cell morphology and extracellular matrix secretion of the repaired site,examine the condition of new cartilage and the surrounding The interface of the tissue was used.3.The repair of cartilage defects was compared by gross scores and histological scores.4.Western Blot was used to detect the expression of tissue-engineered cartilage-specific protein COL2A1 in the defect repair site.Results:By constructing cartilage defect models in animals,combined with gross specimen observation,histological staining(safranine-solid green staining),and gross and histological scores,we found that BC group(control group)and BCT group(positive control group)BCP group(experimental group)had repair of articular cartilage.Compared with BC group,BCP group had better repair effect.Western Blot detection by immunoblot showed that BCP group(experimental group)showed significantly better expression of cartilage-specific type ? collagen than BC group(control group),indicating that materials,seed cells,and growth factors have a common advantage in repairing articular cartilage.Conclusion: 1.Autologous PRP composite BMSCs and Col can effectively promote the repair of articular cartilage defects and extracellular matrix secretion of new chondrocytes;2.autologous sources of PRP promote articular cartilage repair with the same time as TGF-?1.