Bmscs Co-cultured With Chondrocytes Were Induced To Chondrogenic Cells And Osteochondral Defct Repaired With Two Different Structure PLGA Scaffolds

The incidence of traumatic, infective and degeneraediseases which often caused cartilage defect of the articulation increased as the development of high speed traffic and the aging society of china. The pain and dysfunction of the joint which with pathological changes of cartilge have been a big problem, and the repair of cartilage was a challenge for specialist of orthopaedics and sport medicine, and researches on this issue is a high need.The rehealing ability of cartilage after injury is very limited, the reason for this may be the tissue construction is made of only particular high differented chondrocytes and the deficiency of vascular and lymphatictrophism.Subchondral bone injury was ofthen founded together with cartilage defect, which was disabled to support the superficial cartilage layerproperly and caused unnormal distribution of stress, afterthe osteochondral injury the articular cartilage degenerated inconvertibly, led to the pain, dysfunction and stiffness of the joints.The treatment strategy of osteochondral defect is mainly divied into two types: marrow stimulation method and transplantation method, the former include the technology of micro-drill of subchondral bone, micro-fracture of subchondral bone and so on; the later include Mosaicplastytechnology, osteochondral graft transplantation and autologous cartilage cell implantation. Both treatment methodhave badly satisfied repairing effect.problems such as limited cell source, high cell loss rate and unsure therapeutic effect exist in the conventional surgical treatment for cartilage lesion. The development of cartilage tissue engineering has led to the possibility for the repair of chondral damage.The problem we are faced with in the cartilage tissue engineering is the source of cells. The bone marrow mesenchymal stem cells (BMSCs) are considered to be one of the most important cell sources due to their easy availability and multilineage differentiation capacity. Many protocol has been developed to induce BMSCs to differentiate into chondroblast, like cytokine, biomechni stimulation, microenvironment inducing, as well as co-culture system, etc, of which BMSCs/chondrocytes co-culture is an economical.easy performing and theoretically reasonable stratagy.According to S. Miot et.al, goat is the most frequently used large animals in cartilage repair studies towing to its relatively large stifle size, ease of use, cost and availability. Besides, the goat knee’s intercondylar notch, cortical and trabecular bone structure are similar to those of humans. Yet to the best of our knowledge, rare reports have been published about the goat models in the co-culture system.The purpose of the present study was, firstly, to isolate and augment of goat BMSCs and goat rib cartilage cells. Secondly, to explore whether co-culture of goat chondrocytes and BMSCs in vitro entailing two populations culturing in monolayer, with one in a well plate and another upon the surface of a transwell membrane promotes the expression of extracellular cartilage matrix (ECM) and to compare chondrogenic effect with BMSCs induced by chondrogenic medium. Thirdly, to examine whether BMSCs seeded in3-dimesional PLGA sponge also gained increased differentiation potential by co-culturing with chondrocytes, the detail study are as follows: Part1.The isolation and expansion of goat BMSCs and goat rib cartilage cellsObjectiveTo isolate, expand and identify the goat BMSCs and cartilage cells needed in the co-culture experiment.method1. The isolation, expansion and identification of goat BMSCsThree goats were isolated BMSCs with the method of whole bone marrow adherence. Briefly, after anesthesia the goat iliac crest was punctured with a16-’gauge needle. About5-10mL of bone marrow was aspirated into a20mL syringe containing5000U of heparin and was mixed with15ml DMEM in50ml sterilized centrifuge tube. The diluted aspirate was centrifuged at1000r/min for5min to collect the precipitated cells, which were then cultured in a complete cell culture medium containing DMEM/10%v/v FBS,100mg/mL penicillin, and100U/mL streptomycin. After about12days a80%-90%confluent cell layer was formed, the cells were detached using0.25%trypsin adding0.01%EDTA and routinely passaged. The culture medium was changed every three days.The isolated cells were identified by the induction into osteogenic, chondrogenic and lipogenic cells and CD molecular marker detection with flow cytometry.2. The isolation, expansion and identification of goat rib cartilage cellsChondrocytes were isolated from goat rib free edge. In brief, goat was Anaesthed by injection with a Sumianxin(0.1ml/kg im) and atropine(0.1mg/kg im) as basic Anaesthesia, then further Anaesthed with butaylone(30-35mg/kg iv) and3cm free edge of the last rib were removed by sterile dissection. The soft tissue were removed under stereomicroscope and then rib were finely minced and washed with PBS and were then digested with0.25%trypsin (w/v) for30min, then0.1%type Ⅱ collagenase (Sigma,USA) in DMEM for8h at37℃, shaked every30min. The isolated chondrocytes were resuspended in DMEM (Gibco) supplemented with10%FBS (Gibco),100U/mL penicillin G (Gibco),and100μg/mL streptomycin (Gibco). The cells were then plated at a density of1x105cells/mL and placed in a5%CO2incubator at37℃. The culture medium was changed every3days.The isolated cells were identified by Toluidine blue staining.ResultWe isolated and expanded cells successfully from the aspirates of bone marrow, and were identified as BMSCs by the three differentiationability and CD molecular identification. And we successfully obtained cartilage cells identified by Toluidine blue staining.ConclusionThe BMSCs could be isolated, expanded conveniently with the method of whole bone marrow adherence, and rib cartilage could be obtained by the digestion with trypsin and type Ⅱ collagenase.Part2.To induce BMSCs into chondrogenic cells by rib cartilage cells in Transwell co-culture systemObjectiveTo evaluate the BMSCs’ability of secreting chondrocytes ECM under the Tranwell co-culture system.MethodIn Transwell coculture system Chondrocytes(passage3) and BMSCs (passage4) were co-cultured to induce the BMSCs to chondrogenic cells. Three experimental groups were established as follows:in group A, chondrocytes and BMSCs were co-cultured at ratios of1:2in DMEM (Gibco) supplemented with10%FBS (Gibco),100U/mL penicillin G (Gibco), and100μg/mL streptomycin (Gibco),seeding chondrocytes in the insert chamber and BMSCs in the bottom of six-well plate behind the insert chamber. in groups B, BMSCs was induced in chondrogenic medium (cyagen) in six-well palte, kit component including DMEM supplemented with10%FBS,100U/mL penicillin G,100μg/mL streptomycin,40μg/mL ITS+(Sigma),10ng/mL TGF-β3(Invitrogen, USA),10-7M dexamethasone (Sigma), and50μg/mL ascorbate2-phosphate (Sigma), in group C, BMSCs were cultured in six-well plate in DMEM (Gibco) supplemented with10%FBS (Gibco),100U/mL penicillin G (Gibco) and100μg/mL streptomycin (Gibco). Groups C served as the control groups, and groups A and B as the experimental groups. All groups initially contained1x105cells plated at a density of1x104cells per cm2. The medium was changed every3days, the discard medium were frozen at-80°or GAG content assay and the cells were harvested on days14for Q-PCR detection and21for histological assess.7500software for7500and7500fast real-time-PCR software were employed to analyze the gene expression in the tanswell co-culture system. We utilized GAPDH as the house-keeping gene and controlled group C as the reference group.ResultIn transwell co-culture system metachromatic toluidine blue staining as well as positive collagen Ⅱ immunofluorescence in group A and group B indicated the successful secretion of GAG and collagen Ⅱ while group C failed to do so. The amount of GAG was assessed at7days and21days culture. The GAG content in group A and group B were96.04±1.04μg/ml and116.20±26.45μg/ml at the7th day culture, which were significantly different from that in group C (P<0.05)(Fig.4A).At the point of21th day culture, group A obtained243.92±5.50μg/ml GAG, which was significantly lower than that in group B (299.85±3.79μg/ml) and higher than that in group C (92.58±6.80μg/ml). During the2weeks culture, up-regulated expression of collagen Ⅱ gene, COL2a and osteopontin were found in group A and in group B, of which2.17times and2.94times more collagen Ⅱ gene.2.23times and5.50times more COL2a,4.81times and3.65times more osteopontin were obtained in group A and group B respectively. The expression of collagen I in group A was down-regulated as0.18times as much as that in group C, while group B achieved1.68times collagen I as much as that in group C (Fig.5A-D). However, no significant change was observed in the expression of COL2a in group A and group C (p>0.05)(Fig.5A). Besides, group B obtained significantly higher expression of collagen I and collagen Ⅱ than group A did.ConculsionGoat chondrocytes and BMSCs co-culture system can serve to be a proper microenvironment to support the differentiation of BMSCs and passage3chondrocytes had chondro-inductive affection on BMSCs. Part3.To induce BMSCs to chondrogenic cells in3D PLGA scaffold.ObjectiveTo evaluate the BMSCs’secretingcartilage ECM ability in3D scaffolds while culturedfloating on the six-well plate with or without cartilage cell seeded in the bottom.MethodIn PLGA sponge co-culture system,chondrocytes were seeded in24-well plate and the constructs of BMSCs seeding into PLGA sponge were suspended in the medium in24-well plate, and the constructs suspending in24-well plate without chondrocytes seeding in the bottom served as control group. The culture medium comprise DMEM (Gibco) supplemented with10%FBS (Gibco),100U/mL penicillin G (Gibco) and100μg/mL streptomycin (Gibco). The culture medium was changed every other day and in days21the sponges were sectioned for histologically assess.Result In PLGA scaffolds Results from HE staining, toluidine blue staining and collagen Ⅱ immunol histochemistry demonstrated intensive proliferation of BMSCs and secretion of extracellular matrix (ECM) including abundant collagen Ⅱ and GAG in the experimental group. Negative collagen Ⅱ immunol histochemistry and negative GAG toluidine blue staining as well as light staining ECM were observed in the control group.ConclusionThe BMSCs were induced to chondrogenic cells evidenced by the enhanced secreting cartilage ECM ability.