Experiment Of Repair Of Osteochondral Defect Via Bone Marrow Mesenchymal Stem Cells Composed Of β-TCP Bioceramic Scaffolds In Goat

BACKGROUNDArticular cartilage tissue lacks the blood supply to support self-repair and remolding.Because of this limited capacity for spontenous repair,minor articular cartilage injury can lead to irreversible degeneration of the joints.It is particularly prominent when the damage is involved the subchondral bone injury.The Articular subchondral bone cartilage plays an important role in the conduction of stress in the joint,and it acts as a strong support.Once osteochondral defect occurs in the jiont,the abnormal stress distribution and the collapse of articular suiface will be inevitable because the cartilage loses the solid suppot of subchondral bone,which can trigger a vicious circle that will result in the damage of normal articular cartilage.Eventually, it can lead to osteoarthritis or bony ankylosis.At present,many procedures have been proposed for the treatment of focal articular-cartilage injury,mainly including bone marrow stimulation and transplantation.Bone marrow stimulation includes subchondral drilling and microfracture techniques;the latter includes autogenous chondrocyte implantation (ACI) and mosaicplasty.However,all the technology above have their own limitations.The development of tissue engineering provides a new way for osteochondral defects repairing.Today complex organs and tissues construction are the direction for the development of tissue engineering.Ideal tissue-engineered repair materials should be able to provide a better intergration of the cartilage and subchondral bone of the host tissue,ACI technology can not reach this aim,and Mosaicplasty technology has many limitations.As we have known,bone-bone intergration model is much better than that of cartilage-cartilage and cartilage-bone model.The ideal tissue-engineered cartilage should meet the bone-bone intergration, which can support cartilage restoration and avoid the adverse combination of cartilage and bone problems.Studies have already reported numerous way of tissue engineering of bone and tissue engineering cartilage construct separately successful, and now composite osteochondral construction research have become the focus of attention in this field.At present,the bioreactor are the hot spots and important direction of development in the field of tissue engineering research,and its aim is to simulate the physiological environment of tissues and organs in vivo,which can promote the regeneration of them.For the normal bone and cartilage tissue,not only physical and chemical factors but also normal stress stimulation of articular cartilage also play an important role in maintain the physiological functions of cartilage.The appropriate mechanical load is a necessary physiological stimulus,which can overcome the deficiencies on the traditional cell culture.Mechanical factors affect the biological behavior of chondrocytes in an extremely complex process,involving cell types, morphology,growth cycle and distribution.Research has proved that external forces can affect the BMSCs differentiation towards osteoblasts and adipocyte,which suggest that stress factors may have multilineage differentiation potential for stem cells.Different forces in different ways of load,size and frequency of the role,can adjust and maintain normal chondrocyte biology through a mechanical signal transduction mechanism.Some studies show that fluid shear stress can increase the deposition of minerals,and enhance osteoblast phenotype expression and osteoblast secretion of extracellular matrix material in the stent with good distribution.Studies have found that a range of shear stress can promote the seed cells and vector-attached, and retain the osteoblast phenotype in vitro.In addition,stress load can adjust chondrocyte proliferation,matrix metabolism and synthesis in vitro.Although this mechanism of mechanical stimulation have not yet been fully clarified,it suggests that cells cultured in vitro,whether single-layer or three-dimensional culture,can improve their biological behavior of cultured cells through being exerted a certain dynamic mechanical stimulation,such as the promotion of cartilage cells extracellular matrix proteoglycan and collagen synthesis.By analoging the mechanical load in vivo, to study the seed cells for matrix synthesis,morphology,mechanical signal transduction mechanism and mechanical properties such as electricity,bioreactor has a great significance in the aspect of repairing osteochondral damage using tissue engineering methods.Calcium phosphate bioceramics has been widely accepted as an excellent scaffold for bone tissue engineering.The most widely used is hydroxyapatite(HA) andβ-tricalcium phosphate(β-TCP).β-TCP has a good biocompatibility, degradability and bone conduction capacity,moderate mechanical strength, microstructure and the degradation rate in vivo can be manipulated,and so on. Recently,scholars have found that the bioceramic aperture diameter of the inner connecting bone formation has an important impact,and it is necessary that the diameter must be larger than 50μm for the formation of mineralized bone.Calcium phosphate bioceramics has been used to construct tissue-engineered cartilage. Scholars have used the calcium phosphate as a scoffold to construct chondral tissue with chondrocyte and stem cells successful.In this study,the goat BMSCs was acted as seed cells,β-TCP bioceramics was acted as osteochondral scoffold for tissue engineering construction osteochondral graft. Firstly,the goat BMSCs were aspirated and differentiated into osteoblasts and chondrocyte induced by different induction medium and flow cytometry,which can identify its capacity to differentiation;then the goat BMSCs were labeled with bromodeoxyuridine,and explored the optimal concentration,time and cytotoxicity of BrdU labeling of goat BMSCs in vitro,and confirm its feasibility for stem cells labelling and tracer means for goat;Finally,we used the method of negative pressure suction in order to get the combination of goat BMSCs attached toβ-TCP ceramics, and we designed and producted the double-chamber stirring bioreactor,in which BMSCs could inducte and differentiate to osteoblasts and chondrocyte respectively; and the goats knee joint osteochondral defects model were created,and transplanted the engineered graft into the osteochondral defect site.Then,we observed the treatment effect of this tissue engineering mothod for repairing the osteochondral defect,which can provide a new method for clinical treatment of osteochondral defect using autologous BMSCs.MATERIAL AND METHODS1.Isolation,culture,osteogenic,identification,osteogenic and chondrogenic differentiation of goat BMSCs10-month-old goat was used in this experiment(The Southern Medical University institutional ethics committee approved all experiments in advance). 10ml bone marrow was aspirated;BMSCs were isolated and cultured using the whole bone marrow culture method in vitro.The 4rd passages of BMSCs(P4) were identified by flow cytometry.Add osteogenic and chondrogenic induction medium to the goat BMSCs.Osteogenic induction medium composition as follows:final concentration of 100nmol/L dexamethasone,10mmol/lβ- glycerophosphate,50ug/ml ascorbic acid of 10%FBS high glucose DMEM;chondrogenesis induction medium composition as follows:final concentration of 6.25μg/ml insulin,6.25μg/ml transferrin,50ug/ml ascorbic acid,100nmol/L dexamethasone and 10 ng/ml TGF-β1 of 10%FBS high glucose DMEM.Cytochemical staining, immunocytochemical staining and reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were performed to detect of the collagenâ… ,collagenâ…¡and Aggrecan expression at 0,1,2,4 weeks to identify the success of differentiation. The data was analyzed by SPSS 13.0 statistical software,p value＜0.05 was regarded significant difference.The doubling time of P4 and p8 goat BMSCs was analyzed with two-sample t test;the relative expression of mRNA and protein were analysised with One-way ANOVA,multiple comparisons between groups using LSD method.2.In vitro bromodeoxyuridine labeling of goat bone marrow mesenchymal stem cells. The 4^rd passage of goat BMSCs was incubated with BrdU at different concentrations(0,5,10,15 and 20μmol/L) for incubating time(12,24,48 and 72 h),to identify the optimal BrdU concentration and incubating time for goat BMSCs labeling.Immunofluorescence and trypanblau strain were performed respectively to calculate the labeling positive rate and the cells' survival rate for different time after BrdU labeling in vitro.Then we compared the difference of osteogencic and chondrogenic potentiality before and after BrdU labeling.MTT analysis was used to detect of the rate of cell proliferation and cytotoxicity of BrdU.Labeling rate and the survival rate before and after BrdU labeling was analyzed by factorial analysis of variance;P＜0.05 was regarded significant difference.The doubling time of P4 goat BMSCs of before and after BrdU labeling were analyzed with two-sample t test; P＜0.05 was regarded significant difference.3.The manufacture of double-chamber stirring bioreactor and tissue-engineered osteochondral graft repairing goat knee osteochondral defects in vivo.The double-chamber stirring bioreactor was designed and manufactured.Collect the 4rd-passage goat BMSCs and inoculate them to to theβ-TCP bioceramic composite at the density of 5×10⁷/ml.After induction in the double-chamber stirring bioreactor respectively for two weeks,the BMSCs-β-TCP bioceramic composites were transplanted into the defect area of goat knee joints.A defect of 6mm in diameter and 12mm in depth was created in the femoral medial condyle weight-bearing areas of both posterior limbs.Drilling at weight-bearing cartilage with the external diameter of 6mm trepan, bone and cartilage debris were removed.Autogenous goat BMSCs-β-TCP bioceramic composite was implanted into the defect area by press-fit method.After the implantation,the BMSCs-gel formed by 1.2%in algal and 102mmol/l sodium chloride crosslinked were covered on the surface of defect.According to whether the graft experienced mechanical stimulation during cultured and inducted in bioreactor, the goats were divided into three groups:group A:Mechanical stimulation+ two-direction induction in double-chamber bioreactor;group B:two-direction induction without mechanical stimulation;groups C:control group.Each group has four goats,totally 24 knees,12 goats.The goats were sacrificed at 12 weeks and 24 weeks after operation.Then detected the following items:general observation; histology and histochemical detection:HE staining,Toluidine blue staining,Masson staining,collagenâ…¡immunohistochemistry,Brdu immunofluorescence detection in the time point of 12 weeks.O'Driscoll,Keeley and Salter Histomorphology score. The data was showed as(?)±s,and two-way ANOVA and LSD was used for stastistcs analysis,P＜0.05 was regarded significant differenceRESULTS1.The goat BMSCs of primary culture were cambiform or spindle-like,and it has the character of good refractive and adherent growth.With the extension of culture time,the growth of BMSCs was colony-like and typical spindle-like of cells can be observed in the colony.The cells would reach 80%of fusion after 7～8d cultivation,90%of fusion after 9～10d cultivation.Most of the cells were long spindle-like;cell growth rate was faster than the primary cells.After cultured for 2～3d,the cells began to enter the logarithmic-like growth phase,and 7～8d into the plateau phase.The result of flow cytometry showed:cell surface antigen expression of MSCs markers CD29 can reach 86.4%,and the expression of hemopoietic stem cell marker CD34 was only 0.4%.Under the induction of osteogenic induction medium and the chondrogenic induction medium,the goat BMSCs could be differentiated toward osteoblasts and chondrocytes respectively;Identified osteoblast formed from BMSCs with alkaline phosphatase stain and alizarin red staining,the result showed positive in 2 weeks.Identified chondrocyte formed from BMSCs with toluidine blue and Alican blue staining,the result showed positive in 2 weeks.RT-PCR,Western blot and immunohistochemical staining results were confirmed osteoblastic and chondrogenic cell differentiation and,after the BMSCs were induced towards osteogenesis and chondrogenesis for 2 weeks,The expression Collagenâ… ,â…¡and Aggrecan mRNA and protein had no significance stastistic difference with that of induction 4 weeks.and it has significance stastistic difference with induction 1 week and non-induction. 2.We performed immunofluorescence staining after labeled by the BrdU.The goat BMSCs' nucleus show green fluor under fluorescence microscope.The labeling positive rate increased gradually with the increase of incubating time and concentration of BrdU.More than 90%goat BMSCs could be labeled when the cells were incubated for 48h and the concentration of BrdU maintaining 15μmol/L at the same time,and the survival rate of labeled cells were more than 98%.but the labeling rate did not increase with prolong of time and the increase of concentration of BrdU.It indicated that the optimal time for labeling goat BMSCs is 48h and the optimal concentration is 15μmol/L of BrdU.The morphous,growth,proliferation and differentiation of the labeled BMSCs were not influenced compare to the unlabelled BMSCs.3.After 12 weeks,the result showed the operation area of group A articular surface was smooth,with the surrounding normal cartilage naturally straight flush, transparent form new cartilage tissue;Group B restoration surgery the basic integrity of the cartilage tissue,but the center of many not fully integrated,there is slight depression,surface wear,good subchondral bone formation;control group, depression joint operation areas,non-articular cartilage formation,such as lack of bone joints.BrdU immunofluorescence confirmed that the new cartilage cells from some of the goat implanted autologous BMSCs.Histological examination showed thatβ-TCP bioceramic degradation has been absorbed.After 24 weeks,the operation area of group A was more smooth,and the surrounding normal cartilage naturally straight flush,transparent form new cartilage,subchondral bone formation in good condition;Group B restoration surgery the basic integrity of the cartilage tissue, center is not yet fully integrated,there was slight depression;Collagenâ…¡immunohistochemistry of cartilage that was new brown area.Group C has no formation of articular cartilage.The growth and the intergration of subchondral bone of group A and B were better.The result of histological test showed that:the quality of goat cartilage formed in group A in vivo is superior to that of group B.O'Driscoll Keeley and Salter histomorphology score:group A of 12 weeks(n＝4) 16.00±0.82,24 weeks(n＝4) 18.75±0.96;group B(n＝4) 12 weeks 11.00±0.82,24 weeks(n＝4) 14.75±0.96; group C did not form cartilage,12 weeks and 24 weeks in each group,the difference was statistically significant(P＜0.05).CONCLUSIONS1,We isolated the goat BMSCs from iliac of adult Chinese goats.And we established a method about isolation,culture and identification for the goat BMSCs.The 4rd passage BMSCs cultured by the whole bone marrow culture method was of higher purity and thriving.This experiment confirmed that the goat BMSCs had a great potentiality of proliferation and osteogenic and chondrogenic differentiation in vitro.When cultured in osteogenic and chondrogenic induction medium for 2 weeks,the cells could express the characteristic of osteocyte and chondrocyte.Goat BMSCs are an ideal seed cell for tissue engineering and it has broad application prospects,which provide an experimental foundation for goat BMSCs being used in bone and cartilage tissue engineering in vivo.2,BrdU can be used as a labeling marker for goat BMSCs.When the concentration is 15μmol/L and the incubation time is 48 hours,the optimal labeling effect can be gained.Goat BMSCs labeled with BrdU is of high efficiency.BrdU has no obvious cytotoxicity and influence on the growth, proliferation and differentiation to the cells.3,We can construct tissue engineering osteochondral graft simultaneously at the same piece ofβ-TCP bioceramics with goat BMSCs act as seed cells and cultured and inducted in double-chamber stirring bioreactor.The experiment in vivo of goat proved that mechanical stimulation could improve the quality of goat cartilage formation.