Study On Treatment Of Osteochondral Defect With Collagen-hybridized Antigen-extracted Cancellous Bone Supplemented With Mesenchymal Stem Cell

Partâ… Impact of microenvironment on differentiation of fibrous callus in joint cavity compared with bone marrow cavityObjective: To evaluate compositional variation of fibrous callus under the environments of bone marrow cavity and joint cavity. Methods: Metaphysis bone marrow and synovial fluid were harvested from ten adult rabbits by aspiration. Their pH value and oxygen partial pressure were determined by means of blood-gas analysis. Paired-T test was performed to find out any statistical difference. For another 15 adult rabbits, closed radius fracture models were made under anaesthesia for callus transplantation,and the autologous fibrous callus was transplanted in the right knee joint cavity at 12 d postoperatively. All of the animals were sacrificed by overdose of anesthetic 3 w after transplantation, and the transplanted fibrous calluses, the healed bones of radius fractures were procured for the assessment of compositional variation. Results: Significant statistical difference was found between synovial fluid and bone marrow with respect to pH value and oxygen pressure. For pH value, synovial fluid was 6.213±0.097,and bone marrow was 7.223±0.0677(P<0.05);for oxygen partial pressure, synovial fluid was 7.62±0.466mmHg , bone marrow was 50.50±2.47mmHg(P<0.05). Pure fibrous composition was found in the callus at fracture sites 12 d postoperatively, and for 11 out of the 15 animals, the fibrous callus was transformed into cartilaginous tissue after 3 w of transplantation, and some progenitor cells took on a transitional form to chondrocytes at peripheral site. But the fibrous callus was absent in the other 4 animals. The fibrous callus at the original site, i.e., the fracture locus, had differentiated into bony tissue. Conclusion: The bone marrow cavity and joint cavity are contrastingly different microenvironments. Joint cavity may facilitate the differentiation of the callus in chondrogenetic pathway, and bone marrow cavity encourages osteogenesis.Partâ…¡Impact of antigen extraction on microstructure of cancellous boneObjective: To evaluate microstructure alteration of cancellous bone by two approaches of antigen extraction during degreasing, deproteinization and decalcification. Methods: Fresh cancellous bone of young cattle was harvested and sawed in to blocks 15mm×10mm×10mm in size. They were subsequently treated to extract antigen by two approaches(5 samples for each): one was degreased with methanol:chloroform=1:1(ml:ml) for 24 h→deproteinization with 30% H₂O₂ for 24 h→decalcification with 0.6mol/L HCl for 6 min;the other was degreased with methanol:chloroform=1:1(ml:ml)→sequential deproteinization by 2mol/L CaCl₂ 24h, 8mol/L LiCl 24h, 10 % H₂O₂ 12h→decalcification by 0.5mol/L Na4EDTA for 15 d. The samples were then fixed, dehydrated and embedded. TEM was applied to observe compositional variation of microstructure of the antigen-extracted cancellous bone. Results: There was no evident difference of the cancellous bone in gross observation between two approaches of antigen extraction, namely, taking on a white, semitransparent appearance and a elastic, hydrophilic nature. Microstructure of cancellous bone treated with first method was impaired with collagen in the matrix dissolved to a homogeneous form; the collagen deposition, however, could be observed in the cancellous bone treated by the second method. Conclusion: The microstructure can be well preserved by the second method compared with the first method despite the comparable gross appearance.Partâ…¢Characterization of compatibility and chondrogenic ability after being loaded with chondrocytesObjective: To evaluate biocompatibility and subcutaneous chondrogenic capability of antigen-extracted cancellous bone in nude mice after being loaded with chondrocytes. Methods: Antigen-extracted cancellous bone was prepared by the second method in part II. Bone marrow was harvested by bone aspiration and mesenchymal stem cells were procured by cultruring and proliferation; articular cartilage from biopsy was diced and digested, and chondrocytes were harvested after expansion. Mesenchymal stem cells and chondrocytes were seeded in the antigen-extracted cancellous bone at 1×10⁵/ml, the growth kinetics were observed with inverted biological microscope and SEM. Antigen-extracted cancellous bone was also seeded with chondrocytes at 1×10⁶/ml and implanted subcutaneously in nude mice, and the profile of chondrogenesis was observed after 6 w. Results: The cell suspension was easy to be absorbed by antigen-extracted cancellous bone. Mesenchymal stem cells and chondrocytes could grow and proliferate immediately next to the antigen-extracted cancellous bone as seen by inverted biological microscopy observation and were found adhering on the cancellous bone and secreting matrix by SEM observation. However, satisfactory chondrogenesis was not found in nude mice subcutis and much fibrous tissue infiltration was observed because of the large pore size and limited proliferation capability of chondrocytes. Conclusion: Antigen-extracted cancellous bone has good biocompatibility and hydrophilic nature . However, the large pore size and limited proliferation rate of seeded chondrocytes allow for infiltration of fibrous tissue after subcutaneous implantation.Partâ…£Hybridization of antigen-extracted cancellous bone with collagen and evaluation of biocompatibilityObjective: To prepare rat tail collagen and apply it to hybridize antigen-extracted cancellous bone to reduce the pore size, thus making an even distribution of seeded chondrocytes and well structured chondrogenesis. Methods: Collagen was extracted from rat tail by means of acidolysis and enzymolysis and used to hybridize the antigen-extracted cancellous bone. Quantitative and qualitative analysis was performed as follows: molecular weight was estimated by SDS-PAGE, and absorbtion peak of UV was measured by spectrophotometric analysis and nitrogen composition was determined by micro Kjeldahl method. 50% collagen solution was then made by being dissolved in 0.05mol/L acetic acid and used to hybridize the antigen-extracted cancellous bone. Mesenchymal stem cells and chondrocytes were harvested as with test II and III and seeded in the collagen-hybridized antigen-extracted cancellous bone at 1×105/ml. Biocompatibility was observed. Chondrocytes were seeded in the collagen-hybridized antigen-extracted cancellous bone at 1×106/ml and resulting composite was implanted subcutaneously in nude mice, and chondrogenesis was observed after 6 w. Results: The Collagen was readily available by means of acidolysis and enzymolysis from rat tail. SDS-PAGE revealed the molecular weight was arounc 97KD, and aborbsion of UV at 230nm wavelength, which was typical of type I collagen. Micro Kjeldahl method demonstrated the concentration of collagen was 4.0mg/ml,purity was 96%. Smaller pores formed by collagen were observed in the original large pores of cancellous bone. Image analysis showed pore size was reduced from 400-1000um to 50-80um after collagen hybridization. Mesenchymal stem cells and chondrocytes could adhere and proliferate well on the collagen. Subcutaneous implantation of chondrocytes in the collagen-hybridized antigen-extracted cancellous bone could lead to abundant chondrogenesis. Conclusion: Rat tail collagen is type I collagen with high purity and good biocompatibility. Mesenchymal stem cells and chondrocytes can grow and proliferate on collagen-hybridized antigen-extracted cancellous bone. The reduced pore size after collagen hybridization make seeded chondrocytes evenly distributed, and can lead to chondrogenesis with good structure subcutaneously. Partâ…¤Collagen hybridized antigen-extracted cancellous bone scaffold supplemented with mesenchymal stem cells to treat osteochondral defect in rabbit modelObjective To prepare the collagen hybridized antigen-extracted cancellous bone supplemented with autologous mesenchymal stem cells and apply it as a single unit to treat osteochondral defect. Methods:Collagen hybridized antigen-extracted cancellous bone was prepared according to test IV. Autologous mesenchymal stem cells were harvested by bone aspiration of bone marrow from rabbit model, cultured and expanded in vitro and then seeded in the collagen hybridized antigen-extracted cancellous bone scaffold to repair osteochondral defect of the left knee in the rabbit model; and as an auto-control, the right knee of the same rabbit was implanted with a same scaffold that was cell-free. Reparative profile of the defect was assessed by histological examination and Pineda scoring system at regular intervals of 6th,12th, 24th and 48th week. Results: At 6th week, the defects from both knees were filled with fibrous like tissue. There was no evidence of chondrogenesis and subchondral osteogenesis indicated by H&E and saffron"O"staining; at 12th week, the tissues newly generated in the defects of the experimental side were stained slightly positive for chondrogenesis, and subchondral bone was partially generated; while on the control side, both the cartilage and the subchondral bone were not regenerated; at 24th week, the cartilage and subchondral bone of the defects on the experimental side were fully regenerated, but on the control side, the defects were filled with fibrous tissue that was apparently depressed and subchondral bone was partially regenerated; at 48th week, the defects on the experimental side were still filled with cartilage, but a little bit depressed, and the subchondral bone was well structured; on the control side, the defects were significantly depressed. Pineda score revealed statistical difference between the experimental and the control sides at each time point, but more significant at 12th,24th and 48th week. Conclusion: The collagen hybridized antigen-extracted cancellous bone scaffold supplemented with autologous mesenchymal stem cells can be used to treat osteochondral defect as a single unit eliminating the need of osteogenic and chondrogenic induction in vitro.