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Management of mesenchymal stem cell enhances the efficacy of microfracture in cartilage repair

Management of mesenchymal stem cell enhances the efficacy of microfracture in cartilage repair

초록/요약

The limited healing potential of articular cartilage is a well-known problem in orthopedic surgery. Thus, a variety of surgical techniques has been developed to treat the joint pain and improve the joint function. Microfracture (MFX) as a bone marrow stimulation technique is the most common applied cartilage repair procedure. Unfortunately, the deficiencies of fibrocartilaginous repair tissue lead to breakdown under normal joint loading and clinical results become worse with time after treatment. This study also showed the histomorphochemical comparison of MFX as a first-line and a salvage procedure. To overcome the shortcomings of MFX, an enhanced MFX technique was developed with an additional stem cell mobilization factor as rhGM-CSF (recombinant human granulocyte-macrophage colony-stimulating factor). Finally, our results suggest that MFX as a salvage procedure resulted in overall inferior cartilage repair histomorphochemically compared with MFX as a first-line procedure. And, rhGM-CSF administration enhanced the healing of MFX treated cartilage defect. In chapter I, MFX is considered as the first-line procedure for knee cartilage repair, but the results of MFX seem less predictable and rather controversial in a salvage situation. Thus, the purpose of the study was to histomorphochemically compare MFX as a salvage procedure with MFX as a first-line procedure in a rabbit model. We hypothesized that MFX in a salvage situation would result in histomorphochemically inferior cartilage repair compared to MFX as a first-line procedure, and the inferiority would be attributed to less migration of reparable marrow cells to the defect due to the the destruction of the microarchitecture of the subchondral bone. Thirty-six New Zealand white rabbits were divided into three groups: (1) untreated full-thickness chondral defect, (2) single MFX treatment (1st MFX group) and (3) repeated MFX in 8 weeks after the first procedure (2nd MFX group). In each group, rabbits were sacrificed at the end of 8 weeks, and osteochondral specimens at the repair sites were obtained for histomorphochemical analysis. Results showed that MFX as a salvage procedure resulted in overall inferior cartilage repair histomorphochemically compared with MFX as a first-line procedure and that the inferiority was due to deteriorative changes in the quality of underlying subchondral bone rather than intrinsic incapability to recruit the reparative cells in the defect area. In conclusion, although a comparable number of repairable cells are anticipated, MFX should not be attempted in a salvage situation because of mechanically weakened subchondral bone. In chapter II, The MFX technique is widely used to treat articular cartilage defect. However, it has a problem that the result may be suboptimal because of the fibrocartilaginous repair which is possibly due to the paucity of bone marrow mesenchymal stem cells. This study investigated the effects of rhGM-CSF, a hematopoietic cytokine that mobilizes stem cells, on MFX-treated cartilage defect in a rabbit model. Forty New Zealand white rabbits were subjected to full-thickness chondral defects on their knees and divided into 4 groups: (1) Defect, (2) Defect + intravenous injection of rhGM-CSF (i.v.GM-CSF), (3) Defect + MFX, (4) Defect + i.v.GM-CSF + MFX. The repaired cartilage tissues were retrieved at 4, 8 and 12 weeks after the treatment and examined by the histological observation, the quantitative assessment, and the biochemical assays. With the bone marrow from the defect area, a number of mesenchymal stem cells were measured by colony forming unit-fibroblast (CFU-F) test. And the distribution of rhGM-CSF in the bone marrow was also evaluated by the NIR fluorescence imaging and ELISA. In histological observations and chemical assay, MFX after rhGM-CSF administration showed slightly better cartilage repair than when it had been done alone without rhGM-CSF at 4, 8 and 12 weeks. MFX after administration of rhGM-CSF showed the significantly higher amount of bone marrow mesenchymal stem cells than the MFX without rhGM-CSF treatment. In conclusion, this study showed that intravenous injection of rhGM-CSF could improve the therapeutic effect of MFX on the articular defect probably by increasing the amount of bone marrow mesenchymal stem cells coming out from the cartilage defect after MFX.

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목차

Background 1
1.1. Articular cartilage 2
1.2. Articular cartilage lesion 4
1.3. Microfracture methods 7
1.4. Current study in MFX 7
1.5. The aim of this study 9
CHAPTER I: Histomorphochemical comparison of microfracture as a first-line and a salvage procedure: Is microfracture still a viable option for knee cartilage repair in a salvage situation? 10
2.1. Introduction 11
2.2. Material and Methods 14
2.2.1.Surgical procedures and determination of the depth of microfracture 14
2.2.2.Cell Culture and Assay of Fibroblast CFU 19
2.2.3.Histological Evaluation 21
2.2.4.Biochemical Assay of Repaired Cartilage 25
2.2.5.Radiological Evaluation 25
2.2.6.Statistics 25
2.3. Results 27
2.3.1.Fibroblast CFU assay 27
2.3.2.Histological Evaluation 29
2.3.3.Biochemical Assay of Repaired Cartilage 32
2.3.4.Radiological Evaluation 34
2.4. Discussion 38
CHAPTER II: Granulocyte macrophage-colony stimulating factor significantly enhances articular cartilage repair by microfracture 42
3.1. Introduction 43
3.2. Methods 46
3.2.1.Experimental design 46
3.2.2.Surgical procedures 46
3.2.3.Histology and immunohistology analysis 47
3.2.4.Biochemical analysis 47
3.2.5.Collection of bone marrow exudate and assay of fibroblast CFU 48
3.2.6.Chondrogenic differentiation of human chondrocytes, human MSCs,
osteoarthritis (OA) human chondrocytes in pellet culture 48
3.2.7.Measurement GM-CSF concentration 48
3.2.8.In vivo animal imaging 49
3.2.9.Statistics 49
3.3. Results 50
3.3.1.Histological evaluation 50
3.3.2.Biochemical analysis 55
3.3.3.Collection of bone marrow exudate and assay of fibroblast CFU 55
3.3.4.Chondrogenic differentiation of human chondrocytes, human MSCs,
osteoarthritis (OA) human chondrocytes in pellet culture 57
3.3.5.Measurement GM-CSF concentration 59
3.3.6.In vivo animal imaging 60
3.4. Discussion 62
Conclusions 68
Reference 70

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