Study On The Effect Of LIPU To Tissue-engineered Cartilage By CFDBM Co-cultured With Chondrocytes And BMSCs In Vitro

In the field of joint surgery, articular cartilage injury had been a focus, and there was currently no effective treatment. Since the articular cartilage had no nerves, lack of blood supply, the lower ability to proliferate and migrate, self-repair capacity was limited. Therefor it was easily lead to irreversible damage and dysfunction. Through clinically subchondral bone plate, microfracture drilling or other technologies to migrate undifferentiated medullary cavity of bone marrow mesenchymal stem cell (Bone marrow Mesenchymal Stem Cells, BMSCs) to the injury site, then proliferating and differentiatiing to form hyaline cartilage type organization, the injury of cartilage could be cured. Despite the long-term effect was not yet conformed, it suggested that prompted by a certain method articular cartilage had a certain capacity to be repaired. Thereby increasing BMSCs to differentiate into chondrocytes would be helpful to repair articular cartilage injury. Recent studies indicated that low-intensity pulsed ultrasound (Low Intensity Pulsed Ultrasound, LIPU) could promote healing fracture and nonunion and also accelerating differentiating BMSCs to the articular cartilage cells and secreting the cartilage-specific extracellular matrix. Through the molecular and genetic studies, it was suggested that LIPU's effects might relate to the mechanical and thermal. But under different culturement and different environment conditions the conclusions were also different. The repairing after injury and the specific mechanism of LIPU in articular cartilage injuries is not yet totally disclosed, and there was still a long way to go before effective clinical application.Objective:1. To obtain a large number of New Zealand rabbit articular cartilage cells by isolating, cultureing and proliferating;2. To obtain a large number of New Zealand rabbit BMSCs by isolating, cultureing and proliferating;3. To build a new type of demineralized bone matrix (Cell-Free Deminerized Bone Matrix, CFDBM), and used it as three-dimensional tissue-engineered cartilage cell stents to explore its feasibility;4. To unclose the ability of rabbit articular cartilage cells and BMSCs in the CFDBM to proliferating, differentiating and secreting extracellular matrix;5. By giving the experimental group a certain intensity LIPU stimulus to explore LIPU's effect on rabbit articular cartilage cells and the BMSCs,it maight be helpful to the animal experimental study and to provide some experimental reference for the LIPU finally and reasonably applied to the treatment of articular cartilage injury;Methods:1. By mechanical crushing combination of enzyme digesting articular cartilage, rabbit articular cartilage cells could be obtained in vitro, then through plane-amplificatiing, passageing and applicating cell morphology, II collagen immunohistochemical staining method,cell morphology and identification of cell phenotype could be observed;2. By the Whole bone marrow rinsing method and Adherent screening method, BMSCs of bone marrow tissue couled be separated and purified, then proliferated through monolayer culture in vitro. Cell morphology and identification of cell phenotype could be observed by cell morphology and immunohistochemical staining method;3. CFDBM was made by the new method, and using CFDBM composite allogenic rabbit articular cartilage cells and BMSCs to construct three-dimensional tissue-engineered cartilage co-culture system, then observe cell proliferation and matrix synthesis secretion in phase contrast microscopy, HE staining and collagen type II immunohistochemical staining methods was used to observe the CFDBM's biological characteristics;4. Constructing of BMSCs-catilage cells-CFDBM co-culture system, then stimulating it by 1.0MHz,10mW/cm2,20min per day of LIPU, the cells morphology in the CFDBM, growth and proliferation situation and the type II collagen synthesis and secretion was detected paraffin section and HE staining of collagen type II immunohistochemical staining methods;Results:1. 1st-4rd generation chondrocytes and BMSCs of biological characteristics was similar to primary cells;2. Chondrocytes and BMSCs could maintain a high proliferation capacity in CFDBM; 1st-3rd generation chondrocytes and BMSCs maintained their phenotype stability; BMSCs and articular chondrocytes co-culturing could induce BMSCs differentiating to chondrocytes and secreting the specific extracellular matrix;3. Planting BMSCs alone in CFDBM there was no specific extracellular matrix synthesised and secreted;4. Planting chondrocytes alone in CFDBM, it was gradually reduced for extracellular matrix synthesised and secreted;5. Planting both chondrocytes and BMSCs alone in CFDBM,the chondrocyte-specific extracellular matrix was synthesised and secreted more than planted chondrocytes alone;6.1.0MHz,10mW/cm2,20min per day of LIPU stimulation does not affect the activity of BMSCs;7. Planting both chondrocytes and BMSCs alone in CFDB, then giving 1.0MHz, 10mW/cm2,20min per day of LIPU stimulation, the chondrocyte-specific extracellular matrix was synthesised and secreted more than without LIPU, but no significantly promoting cell growth and proliferation.Conclusion:1.CFDBM could be used as tissue-engineered cartilage cells stent in vitro;2.LIPU could promote differentiating BMSCs to the articular cartilage cells and keeping chondrocytes phenotype stability, stimulating the synthesis and secretion of extracellular matrix, but having significantly promoting cells proliferation.