AMSCs Combined With TGF-β3-loaded PLGA Scaffolds For The Restoration Of Cartilage Defects

IntroductionCartilage injury is very common in clinical practice, all along, the treatment for it is a the hot and difficult problem in the field of orthopaedics. Self-healing cartilage tissue after injury in adult is fibrocartilage, but not hyaline cartilage which has function, thus inevitably appears progressive degeneration, eventually leading to the occurrence of osteoarthritis. The current treatments for cartilage defects are mainly focus on pain control, surgical intervention, and cartilage transplantation, but these can not yield satisfactory long-term efficacy. In this case, as an alternative treatment, mesenchymal stem cells based tissue engineering has emerged as a promising approach for cartilage defect repair. Three main conditions are needed for repair cartilage defect:First, sufficient number of properly functioning seed cells and chondrogenic capacity. Second, a three-dimensional scaffold with biocompatible, non-toxic degradation, suitable gap size and porosity. Third, cytokines which could adjust the seed cell differentiation and proliferation, and maintain their phenotypic. Adipose-derived mesenchymal stem cells (AMSCs) are adult stem cells originating from the mesoderm, because AMSCs are convenient of obtaining, adequate sources and small injury on body compared with others adult stem cells, similar biological characteristics and differentiation potential with bone-marrow mesenchymal stem cells (BMSCs), better proliferative capacity than BMSCs, AMSCs have become an ideal source of seed cells and research hotspot. As a three-dimensional scaffold which has been approved for human tissue engineering by the Food And Drug Administration (FDA) of American, poly-lactic-co-glycolic acid (PLGA) due to its advantages at good biocompatibility, wide source, easy to obtain and capable of mass production, more over, the space structure, general form, mechanical strength and degradation time of its could be design and control in advance, so it has become the most widely used in tissue engineering extracellular matrix material. Transforming growth factor-β (TGF-β) is a multifunctional regulator of a cell growth and extracellular matrix synthesis, it can promote the proliferation of chondrocytes increased proteoglycan and type Ⅱ collagen synthesis, but also to reduce the expression and secretion of the cartilage collagenase. The effect of TGF-β3inducing mesenchymal stem cells (mesenchymal stem cell, MSCs) differentiate to chondrocytes is better than TGF-β1, more over, TGF-β3could inhibit the inflammatory response and promote tissue inhibitor of metalloproteinase expression.Although AMSCs have been shown to have effects in the repair of cartilage defects, however, the studies which using non-induced AMSCs to repair full-thickness cartilage defects are still insufficient, whether non-induced AMSCs could be long-term survival after being implanted in the body and whether AMSCs repair cartilage defect by differentiating to chondrocytes derectly, these issues are unclear. Currently, no author has reported using AMSCs combined with TGF-β3-loaded PLGA scaffolds for the restoration of cartilage defects. In the present study, we based on the related research results of experiments in vitro, used superparamagnetic iron oxide (SPIO) labeled AMSCs combined with TGF-β3-loaded PLGA to design a AMSCs/TGF-β3/PLGA construct to repair the full-thickness cartilage defect model of rabbit and magnetic resonance imaging(MRI) to track the SPIO labeled AMSCs in vivo, observe their survival and distribution, at the same time, prussian blue staining to detect whether the new neo-chondrocytes were differentiated from implanted AMSCs directly.The study is mainly divided into three parts:(1) rabbit AMSCs isolation, culture, labeled by SPIO, then, to detect the sensitivity of MRI tracking SPIO-labeled AMSCs. Using a porogen leaching technology to fabricate bilayer PLGA scaffold. The results showed that AMSCs could be efficiently labeled by SPIO, MRI could track SPIO-labeled AMSCs sensitively when the concentration of cells was sufficient. The study confirmed the bilayer PLGA scaffold could be fabricate by porogen leaching technology. A bilayer PLGA scaffold which with suitable pore size for tissue engineering could be successfully produced by porogen leaching techniques.(2) investigation the effects of TGF-P3on the proliferation and differentiation of AMSCs in vitro. The results of present study showed that when the concentration of TGF-β3at lOng/ml, it had no effect on the proliferation of AMSCs in a short term. Treating AMSCs with TGF-β3in vitro could increase the espression of Collagen type II, Aggrecan and SRY-related of HMG box9(SOX-9) at gene and protein levels whether monolayer culture or three-dimensional culture in a time-dependent manner, but three-dimensional culture could promote the expression of them more efficient when compared to monolayer culture.(3) on the basis of the previous two parts, the AMSCs/TGF-β3/PLGA constructs were used to repair the full-thickness cartilage defects of rabbit models, investigation the regeneration effects of them and the survival and distribution of implanted AMSCs as well as whether the neo-chondrocytes were differentiated from implanted AMSCs directly. The results showed that the AMSCs/TGF-β3/PLGA constructs could effective repair the articular cartilage defects. The AMSCs were still survival partly after12weeks being implanted in vivo, but the neo-chondrocytes were not differentiated from the AMSCs directly. Part1SPIO-labeling of AMSCs and fabrication of bilayer PLGA scaffoldObjective:To observe the sensitivity of MRI on SPIO-labeled AMSCs and whether the porogen leaching technology could produced a three-dimensional bilayer PLGA scaffold with suitable pore sizes for cartilage tissue engineering.Methods:Culturing AMSCs with medium containing25μg/ml SPIO for24hour, then, did prussian blue staining and observed the mark effect by light microscopy. The AMSCs were trypsinized, collected, resuspended in PBS, the sensitivity of MRI were observed when the concentrations SPIO-labeled AMSCs were5x104,1x105,5x105,1x106/ml. A bilayer PLGA scaffold with different pore size was fabricated by a porogen-leaching technique with gelatin particles as the porogen.Results:Culturing AMSCs with medium containing25μg/ml SPIO, the AMSCs could be marked efficiently, the rate of labeled AMSCs was nearly100%. When the cells concentration at1x105/ml, MRI could track the signal change, when the cells concentration at1x106/ml, presented a significant black signal on T2-weighted images. SEM photographs revealed that the bilayer PLGA scaffold possessed macropores and micropores. The average pore sizes of the micropores and macropores in the scaffold were about200μm and400μm, which are suitable for tissue engineering needs.Conclusions:The AMSCs could be marked efficiently by SPIO and tracked by MRI in vitro. A bilayer PLGA scaffold which is suitable for tissue engineering could be successfully produced using gelatin as porogen by porogen leaching technology. Part2The effects of TGF-β3on the proliferation and differentiation of AMSCs in vitroObjective:Observation of the effects of TGF-β3on the proliferation, differentiation of AMSCs in vitro.Methods:Adipose tissue was isolated from the groin area of adult New Zealand rabbits by enzyme digestion gradient centrifugation and adhesive culture methods. The third generation of AMSCs were used, the method of CCK-8was used to detect the effect of different concentrations (0,1,5,10,20,50ng/ml) of TGF-β3on the proliferation AMSCs at the1,3,7day respectively. lOng/ml TGF-β3was used to induce the differentiation of AMSCs, at the7and14days after induction, immunofluorescence staining was used to observe the expression of Collagen type Ⅱ. Real-time quantitative polymerase chain reaction (RT-qPCR) and western-blot methods were used to the expression of Aggrecan, Collagen type Ⅱ, SOX-9at gene and protein levels respectively.Results:After one or three days induced by different concentrations of TGF-β3, each concentration had no effect on the proliferation of AMSCs, but at7th day, all the concentrations of TGF-P3inhibited the proliferation of AMSCs, except lOng/ml. The immunofluorescence showed that after induction of TGF-β3seven days, the expression of Collagen type Ⅱ could be detected by immunofluorescence,14th day of the fluorescence signal intensity was significantly higher than7th day. Further, it can be seen that the AMSCs cultured by low serum culture medium also had a small amount of Collagen type II expression. RT-qPCR and western blot results showed that inducing by TGF-β3could increase the espression of Collagen type Ⅱ, Aggrecan and SOX-9at gene and protein levels in a time-dependent manner, the genes and proteins expression of the three were significantly higher in three-dimensional culture than that in monolayer culture.Conclusions:When the concentration of TGF-β3was10ng/ml, it had no effect on the proliferation of AMSCs in a short term. TGF-β3could promote AMSCs differentiating to chondrocytes in a time-dependent manner, three-dimensional culture was more efficient than monolayer culture.Part3The restore of articular cartilage defects using AMSCs/TGF-p3/PLGA constructs in animal modelsObjective:Observation the repair effects of AMSCs/TGF-β3/PLGA constructs on rabbit full-thickness articular cartilage defects and the effects and mechanisms of AMSCs in this process.Methods:Full-thickness defects were created surgically on the femoropatellar groove of the knee joints of64rabbits. The rabbits were randomly divided into four groups: Defect group, PLGA group, TGF-β3/PLGAgroup and AMSCs/TGF-β3/PLGA group.32rabbits took SEM scanning, then sacrificed at6or12weeks after surgery and the restored tissues were retrieved for morphology, histological observation. RT-qPCR and western-blot methods were used to the expression of Aggrecan, SOX-9, Collagen type I, Ⅱ and X at gene and protein levels respectively.Results:MRI confirmed that there still had survival AMSCs in the repair tissue at6weeks and12weeks after surgery, low signal change can be detected in T2-weight images. Prussian blue staining showed that the neo-cartilage layer and subchondral bone layer all had blue particles at6th week, but at12th week the blue particles existed only in the subchondral bone layer, while the neo-cartilage layer no blue particles. TGF-β3and AMSCs could promote the restore, these effects of them were exerted by regulating the expression of Aggrecan, SOX-9, Collagen type Ⅰ, Ⅱ and X at gene and protein levels in a time-dependent manner. The effect of PLGA itself in the process was no obvious.Conclusions:The AMSCs/TGF-β3/PLGA constructs could repair the full-thickness articular cartilage defects effectively. AMSCs could be survival after implanted in the defect by a long-term and promote cartilage repair effects, but the neo-chondrocytes were not differentiated from the AMSCs directly, AMSCs maybe promote cartilage defect repair by releasing some cell growth factors.