Dissertation
Dissertation > Medicine, health > Basic Medical > Medical science in general > Biomedical Engineering > General issues > Biomaterial

The Restoration of Osteochondral Defects Using Non-connective Bilayered Poly(Lactic-co-glycolic Acid) Scaffolds Loaded with Autologous BMSCs and Platelet-rich-plasma in Rabbits

Author ZhangYongTao
Tutor JinDan
School Southern Medical University,
Course Surgery
Keywords platelet-rich plasma poly(lactide-co-glycolide) bilayered non-connective osteochondral defect
CLC R318.08
Type Master's thesis
Year 2013
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Orthopedic surgeons generally believe that the large cartilage defect is difficult to heal. Currently, conventional surgery such as intra-articular injections, microfracture of the subchondral plate, implantation of autologous chondrocytes, osteochondral grafting, joint replacement surgery and other techniques have their own advantages and limitations. There are some difference in the structure and function between the reconstruct and repaired articular cartilage and normal cartilage. The reconstruction of large osteochondral defects is still a challenge for the basic clinical research in orthopedic. Tissue engineering and regenerative medicine provides a new way of reconstructing articular cartilage defect and improving the structure and function of repaired articular cartilage.Application of extracellular matrix materials, biomimetic materials and synthetic materials to contruct scaffolds loads cells and cytokines in vitro and in vivo is one of the important content in the tissue engineering research. The application of autologous or allogeneic derived extracellular matrix scaffold is limit for reasons of the relative shortage of sources, risk of the spread of disease and poor biomechanical properties. Synthetic biodegradable material has the advantage of easy access and can be plastic to match defect, it is subjected to the attention of researchers. PLGA scaffolds have been used to repair the tendon, ligament, cartilage and bone defects. Researchers believe that the application of a two layer scaffold is necessary to contruct the articular carilage and subchondral bone for the reconstruction of articular osteochondral defects. Therefore, it is a good choice to construct the osteochondral defect using a bilayered PLGA scaffold.Considering that a number of biological factors act in a highly coordinated manner during native tissue development, the use a single factor to stimulate and regulate process of chondrogenic differentiation may be limited. Forther more, the price of recombinant growth factor is relatively expensive and only a small number of growth factor available in clinic. Recently, studies found that PRP can release a variety of growth factors, such as platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor-β(TGF-β), insulin-like growth factor (IGF) and fibroblast growth factor (FGF). As the important source of autologous growth factors, it has many advantanges, such as avoiding the risk of disease, low prices and containing more types of growth factor. The results indicate that PRP can improve mesenchymal stem cells (MSCs) proliferate and differentiate into chondrogenic, other studies show that PRP can promote the progenitor cells migrate from the subchondral bone. The authors confirmed that PRP composite the MSC can promote the formation of bone in vivo and its positive role in the repair of osteochondral defect. MSC has advantage of easy access and differentiation potential. Therefore, BMSCs can be seeded on the scaffold to contruct the composite of tissue engineered osteochondral. Composite of engineered osteochondral can be contruct using the autologous PRP and BMSCs of New Zealand rabbit in the present study.Application of PRP in the construction of osteochondral defect in the area of weight-bearing is limited for its low mechanical properties. In the present study, we design a non-connective bilayered PLGA scaffold. It was used as a carrier for autologous BMSCs and PRP and was implanted to the osteochondral defect in rabbits. The aim of this study is to discus the feasibility of bilayed PLGA scaffold for the reconstruction of osteochondral defect and to observe the effect of autologous BMSCs and PRP on the reconstruction of osteochondral defect in rabbit. The study materials, methods, results and conclusions are described as follows:Part1.The restoration of osteochondral defects using bilayered poly(lactide-co-glycolide) scaffolds loaded with autologous platelet-rich plasma in rabbitsObjectiveTo discuss the feasibility of non-connective bilayered PLGA scaffold for the restoration reconstruction of osteochondral defect in rabbitsMethods1. Fabrication of porous non-connective bilayered PLGA scaffoldsThe "room-temperature" compression molding/particulate leaching technique was used to generate a porous PLGA. The PLGA scaffold is composed of cartilage layer (92%porosity,50-100μm pore size,0.3mm thickness), intermediate adhesive layer (PLGA film,0.3mm thickness) and subchondral layer (92%porosity,300-450μm pore size,3.4mm thickness). To fabricate the nonconnective bilayed scaffold, cylinder mixtures with different pore size porogen and the PLGA film were stuck by dichloromethane under pressure, and cut into4mm in diameter and4mm in thickness。After leaching porogen by water, we obtained the non-connective bilayered PLGA scaffold. Non-connective bilayered PLGA scaffolds were obtained from the Department of Macromolecular Science of Fudan University.Bilayered PLGA scaffolds were sterilized by immersion into75%ethanol for30min, followed by rinsing three times with phosphate-buffered saline (PBS). 2. Preparation of autologous PRPBlood samples (10ml peripheral blood containing1ml3.8%of citrate as an anticoagulant) were obtained from New Zealand rabbits. Autologous PRP was prepared using two centrifugation techniques at room temperature. The tubs were spun at400g for15min and were centrifuged at800g for10min at the second time. The supernatant plasma was discarded and remaining0.8ml of plasma containing precipitated platelets was blend evenly and was designated as PRP. Autologous PRP was prepared before the surgery. The platelets in the PRP and whole blood samples were counted manually. Bone marrow samples were obtained using bone marrow aspirate techniques, and PRP was prepaired using the method above.3. Construction of PLGA/PRP compositeTwelve sterile bilayered PLGAs were carefully placed into a6-well plates. The PRP prepared before surgery and10%calcium chloride (0.05ml/ml of PRP) were added to the6-well plates.4. Surgical implantationEighteen healthy adult New Zealand white rabbit were randomly divided into three equal groups. An osteochondral defect (diameter4mm, thickness4mm) was created through the articular cartilage and subchondral bone of each medial femoral condyle in the two knees using a drill equipped with a4mm diameter drill bit. PLGA/PRP composites and PLGA scaffolds were implanted to the defects in PLGA/PRP group and PLGA group. Osteochondral defects were left untreated in the untreated group. The rabbits were returned to their cages and allowed to move freely without joint immobilization. The rabbits (6knees/group) were euthanized at4and12weeks after surgery. Gross morphology, histology, and expression of collagen type Ⅱ were assessed at4and12weeks after surgery. Micro-CT imaging observation and relative expression level of specific genes were assessed at12weeks after surgery.Result The non-connective bialayered scaffolds were gained as designed. The platelet concentrations in PRP samples were found to be4.9-fold that of the whole blood samples. The number of platelets increased1.41-fold in samples of bone marrow derived PRP compared to peripheral blood derived PRP. The total score with respect to gross appearance and total histological score in the PLGA/PRP group was higher than in other groups at4weeks and12weeks after surgery (P<0.05). Positive immunohistochemical staining of collagen II was observed at4weeks and12weeks. It was highest in the PLGA/PRP group. At12weeks after surgery, the relative expression level of collagen type Ⅱ and aggrecan of the samples in the PLGA/PRP group was significantly higher than that of the PLGA and untreated groups (P<0.05). More subchondral bone formed in the PLGA/PRP than in the PLGA group or untreated group at12weeks after surgery, as indicated by Micro-CT imaging.ConclusionNon-connective bilayered PLGA scaffolds loaded with autologous PRP were found to improve the restoration of osteochondral defects in rabbit model This is an effective way of reconstructing osteochondral defects in rabbits.Part2.Addition of autologous bone mesenchymal stem cells and platelet-rich plasma to the bilayered PLGA scaffolds is beneficial to the restoration of osteochondral defects in rabbitsObjectiveTo observe the effect of non-connective bilayered scaffold load autologous BMSCs and PRP on the restoration and repair of defects in rabbitsMethod1. Isolation and expansion of BMSCsBMSCs were aspirated from the bone marrow(3-4ml), gradient centrifuged, and plated into flasks to be curtured. BMSCs at passage3were used for experimentation.2. Cell Seedding1ml of4.0x106/ml and4x105/ml BMSCs suspension were prepaired separatly. The centrifuge tube was used seeding the BMSCs onto the cartilage layer and subchondral layer. The number of BMSCs in the suspension was counted manually and the seeding efficiency was calculated. PLGA/BMSCs composite were cultured into6-well plates. The cell adhesion on scaffolds were observed by electron microscope scanning3. PRP loadingAutologous PRP was prepaired using the method above. The PLGA/BMSCs composite was transferred to a6-well plate (2/well), then the autologous PRP was added to the6-well plates.4. Surgical implantationSixteen New Zealand rabbits were divided four groups. The PLGA/BMSCs/PRP composite, PLGA/PRP composite and PLGA scaffolds were implanted to the deffects (4mm diameter,4mm thickness) in the experimental and control groups, defects was not implanted to any material in the untreated group. At6months after surgery, gross appearance, histological evaluation, immunohistochemical straining, Q-PCR and Micro-CT scanning were performed. The score of BV/TV in the area of subchondral were analyzed.ResultsAutologous BMSCs can be obtained by bone marrow puncture technique and was expanded in vitro. BMSCs can be seeded to bilayered PLGA scaffolds using two centrifugation seeding techniques. The efficiency was (81.47±2.53)%in bone layer and (85.27±1.79)%in cartilage layer. Histological evaluation and evaluation of the expression of aggrecan and collagen type II on repaired cartilage revealed no significant differences between the PLGA/BMSCs/PRP group and the PLGA/PRP group, it is significantly higher in the PLGA/BMSCs/PRP group than that of in PLGA group and untreated group. The ratio of newly formed subchondral bone to tissue volume in the defects (BV/TV) was (57±14)%in PLGA/BMSCs/PRP group. This was significantly higher than that of the PLGA/PRP group ((42.3±2.6)%), PLGA group ((28.8±5.9)%), or untreated group ((34.8±5.7)%).ConclusionsThe implantation of composite of PLGA/BMSCsPRP was found to improve the restoration of cartilage and subchondral bone. It is a cheap and effective method of reconstructing osteochondral defects using composites of bilayered non-connective PLGA loaded with autologous BMSCs and PRP in rabbits

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