Sustained Delivery Of SDF-1and IGF-1Using A Thermosensitive Hydrogel For The Repair Of Chondrogenic Defect In Rabbit

IntroductionAs a basic pathological process, articular cartilage lesion involves in the early stage of pathologic changes of almost all the common joint diseases. The self-repair capacity of the cartilage tissue is very poor, even a tiny defect can’t be natural repaired due to the lack of blood vessels and lymphatic tissue in cartilage tissue, the content of chondrocytes is small, the small number of progenitor cells, which are essential for the cell differentiation, cannot move to the injury site and participate in the repair process because they are embedded in the thick extracellular matrix. Nowadays surgery is the main treatment method for articular cartilage injury clinically, the operative plan including:arthroscopic, micro-fracture surgery, autologous cartilage tissue transplantation and autologous chondrocyte transplantation. However, the above clinical treatment methods are not ideal, there are still many deficiencies exist. The development of tissue engineering technology provides a new potential way for the treatment of articular cartilage injury. Cartilage tissue engineering research mainly includes three aspects:the seed cells, growth factors and scaffolds. The scaffold provides a place for seed cells to obtain nutrition, exchange gas and fluids, evacuate waste, growth and metabolize. The seed cells attached to the scaffold and keep metabolize, differentiate into new cells. The growth factors involve in the regulation of metabolism of seed cells, promotion of cell proliferation and differentiation, and ultimately the formation of new cartilage tissue which is completely consistent with the normal cartilage tissue in both morphology and function.Adipose-derived stem cells(ADSCs) possess the following advantages which make it very suitable to be used as seed cells:1.easy to be got, less invasive, fewer complications and easily accepted by patients;2. easy to be isolated and cultured;3. high efficiency,5×103stem cells can be obtained from each1g fat tissue, which is300times as compared with those from1g bone marrow. Insulin-like growth factor-1(IGF-1) is closely related to the cartilage development and regeneration. It also plays a crucial role in the process of ADSCs differentiate into chondrocytes. It promotes the chondrocyte proliferation and re-differentiation. It also maintains the chondrocyte-specific phenotype and inhibits programmed apoptosis, and at the meantime, it promotes the synthesis and metabolism of cartilage matrix and inhibits the degradation of cartilage matrix. IGF-1is the most important growth factor that regulates the synthesis of cartilage aggrecan.Stromal cell-derived factor-1(SDF-1), also called "homing chemokine", plays a significant role in the launch and promotion of stem cell homing. Thermosensitive hydrogel possesses the following characteristics:injection with minimally invasive, non-toxic, excellent biocompatibility, high water content and high hydrophilicity. It can be rapidly transformed into a gel in situ after injected into the body at body temperature. It also provides a favorable environment for the growth of the seed cells.In the present study, we prepared IGF-1loaded sustained released PLGA microspheres and PLGA-PEG-PLGA thermosensitive hydrogel, studied the physical and chemical properties and the ability to promote the proliferation and differentiation of ADSCs in vitro. We also combined ADSCs and SDF-1with the PLGA-PEG-PLGA thermosensitive hydrogel to construct a hydrogel complex, and studied the repair effect of the hydrogel complex on chondrogenic defect in rabbit, thereby providing the experimental evidence for its clinical research in the future. This study contains three parts:(1) prepare the IGF-1loaded PLGA microspheres using the W/O/W emulsion-solvent evaporation method, and prepare the PLGA-PEG-PLGA hydrogel using the ring-opening polymerization method, study the morphology characteristic of the IGF-1loaded PLGA microspheres, the PLGA-PEG-PLGA hydrogel and the microspheres-hydrogel complex using the scanning electron microscopy(SEM), detect the drug loading and encapsulation efficiency, draw the drug release curve and observe the gelation property of the hydrogel;(2) isolate and culture rabbit ADSCs, study the effect of IGF-1loaded PLGA microspheres/SDF-1/PLGA-PEG-PLGA hydrogel complex on the ADSCs proliferation process and chondrogenic differentiation ability;(3) implant the ADSCs loaded IGF-1PLGA microspheres/SDF-1/PLGA-PEG-PLGA hydrogel complex into the femoral condyle cartilage defect in rabbit, study its effect on the repair of the chondrogenic defect.Part I Preparation of the IGF-1loaded sustained released PLGA microspheres and PLGA-PEG-PLGA thermosensitive hydrogelObjective:To investigate that whether the IGF-1loaded PLGA microspheres prepared using the W/O/W emulsion-solvent evaporation method and the PLGA-PEG-PLGA hydrogel prepared using the ring-opening polymerization method are suitable for cartilage tissue engineering research.Methods:Prepare the IGF-1loaded PLGA microspheres using the W/O/W emulsion-solvent evaporation method, prepare the PLGA-PEG-PLGA hydrogel using the ring-opening polymerization method and prepare the microspheres-hydrogel complex. Detect the drug loading and encapsulation efficiency of the IGF-1loaded PLGA microspheres, observe the temperature sensitive property of the PLGA-PEG-PLGA hydrogel, observe the release properties of IGF-1and SDF-1in vitro, study their morphology characteristics using both the transmission electron microscopy (TEM) and scanning electron microscopy (SEM), and observe the gelation property of the hydrogel complex.Results:The IGF-1loaded PLGA microspheres, the PLGA-PEG-PLGA hydrogel and their complex are prepared successfully. The average value of drug loading and encapsulation efficiency of the IGF-1loaded PLGA microspheres are0.0622%and81.8%, respectively. The gelation temperature of the20wt%PLGA-PEG-PLGA hydrogel is33.2±0.1℃. The burst release rates of IGF-1and SDF-1in the microspheres-hydrogel complex are14.4%and27.7%, respectively. TEM and SEM show that the diameter range of the PLGA microspheres is from50to80μm, and the PLGA microspheres maintain a good shape in the hydrogel. The gelation time of the hydrogel is3minutes.Conclusions:The prepared IGF-1loaded PLGA microspheres/SDF-1/PLGA-PEG-PLGA hydrogel complex possess good gelation property and can sustained release the SDF-1and IGF-1. It’s suitable for cartilage tissue engineering research.Part II The effect of IGF-1loaded PLGA microspheres/SDF-1/PLGA-PEG-PLGA hydrogel complex on the proliferation and differentiation of rabbit ADSCsObjective:To study the effect of IGF-1loaded PLGA microspheres/SDF-1/PLGA-PEG-PLGA hydrogel complex on the rabbit ADSCs proliferation process and chondrocyte differentiation ability in vitro.Methods:ADSCs were isolated from the groin adipose tissue in rabbit and cultured in vitro, draw the cell growth curve. Observe the osteogenic, chondrogenic and adipogenic differentiation ability of the third generation ADSCs. Study the effect of the IGF-1loaded PLGA microspheres, the SDF-1loaded PLGA-PEG-PLGA hydrogel and the microspheres-hydrogel complex on the proliferation of rabbit ADSCs using MTT assay. Detect their impact on SOX-9, Collagen Ⅱ and Aggrecan gene expression of ADSCs using Real-time quantitative PCR. Detect their impact on SOX-9, Collagen Ⅱ and Aggrecan protein expression of ADSCs using Western Blot.Results:The osteogenic, chondrogenic and adipogenic differentiation of the cultured ADSCs are all successful. There are no adverse effect of the PLGA microspheres and PLGA-PEG-PLGA hydrogel on cell viability and proliferation of rabbit ADSCs. The IGF-1loaded PLGA microspheres and the microspheres-hydrogel complex obviously promote the proliferation of ADSCs at3and7days. The SOX-9, Collagen Ⅱ and Aggrecan gene expression level of the ADSCs co-cultured with the the IGF-1loaded PLGA microspheres and that with the microspheres-hydrogel complex are all creased significantly at7and14days. The results of protein expression in each group were consistent with the results of PCR.Conclusions:The prepared IGF-1loaded PLGA microspheres/SDF-1/PLGA-PEG-PLGA hydrogel complex is not only a good drug loading system, its three-dimensional structure and high water content property can provide an excellent environment for the growth of seed cells. It can obviously promote the induced chondrogenic differentiation of ADSCs. The microspheres-hydrogel complex has broad application prospects in the field of cartilage tissue engineering.Part Ⅲ The effect of ADSCs/IGF-1loaded PLGA microspheres/SDF-1/PLGA-PEG-PLGA hydrogel complex on the chondrogenic defect repair in rabbitObjective:Using the IGF-1loaded PLGA microspheres/SDF-1/PLGA-PEG-PLGA hydrogel complex as a carrier to load the ADSCs and implant the complex into the femoral condyle cartilage defect in rabbit and study its effect on the repair of the chondrogenic defect in rabbit.Methods:50New Zealand white rabbits were randomly divided into five groups: control group:nothing was implanted into the femoral condyle cartilage defect in rabbit; hydrogel+MS group:implant the10mg/ml IGF-1loaded PLGA microspheres encapsulated PLGA-PEG-PLGA hydrogel gelation complex into the femoral condyle cartilage defect in rabbit without SDF-1; hydrogel+SDF-1group:implant the0.5μg/ml SDF-1encapsulated PLGA-PEG-PLGA hydrogel gelation complex into the femoral condyle cartilage defect in rabbit without IGF-1loaded PLGA microspheres; hydrogel+MS+SDF-1group:implant the10mg/ml IGF-1loaded PLGA microspheres and0.5μg/ml SDF-1encapsulated PLGA-PEG-PLGA hydrogel gelation complex into the femoral condyle cartilage defect in rabbit; hydrogel+MS+SDF-1+ADSCs group: implant the10mg/ml IGF-1loaded PLGA microspheres,0.5μg/ml SDF-1and1×106/ml ADSCs encapsulated PLGA-PEG-PLGA hydrogel gelation complex into the femoral condyle cartilage defect in rabbit. Specimens were collected at6weeks and12weeks after surgery, the morphology characteristics and scores, the histology and scores and the immunohistochemical staining of Collagen Ⅰ, Collagen Ⅱ and Aggrecan were studied, the SOX-9, Collagen Ⅰ、Collagen Ⅱ、Collagen X and Aggrecan gene expression of the specimens of each group were studied using Real-time quantitative PCR.Results:The ICRS score and the MODS score of the hydrogel+MS+SDF-1group and the hydrogel+MS+SDF-1+ADSCs group are significantly better than those of the other three groups at6weeks and12weeks after surgery, the immunohistochemical staining results show the significant repair effect of these two groups. The results of Real-time quantitative PCR show the significant promotion ability of these two groups on SOX-9, Collagen II and Aggrecan gene expression.Conclusions:The PLGA-PEG-PLGA thermosensitive hydrogel can be rapidly transformed into a gel in situ and sustained release growth factors after injected into the body. It also provides a favorable micro-environment for the growth and differentiation of ADSCs. A complex prepared by using the hydrogel as a carrier to load the ADSCs, SDF-1and IGF-1loaded sustained released PLGA microspheres can effectively promote the cartilage regeneration, so it is very suitable for cartilage tissue engineering research. It also has broad application prospects in the clinical research of cartilage tissue repair.