Shigeyuki Wakitani

Transplantation of cartilage-like tissue made by tissue engineering in the treatment of cartilage defects of the knee

We investigated the clinical, arthroscopic and biomechanical outcome of transplanting autologous chondrocytes, cultured in atelocollagen gel, for the treatment of full-thickness defects of cartilage in 28 knees (26 patients) over a minimum period of 25 months. Transplantation eliminated locking of the knee and reduced pain and swelling in all patients. The mean Lysholm score improved significantly. Arthroscopic assessment indicated that 26 knees (93%) had a good or excellent outcome. There were few adverse features, except for marked hypertrophy of the graft in three knees, partial detachment of the periosteum in three and partial ossification of the graft in one. Biomechanical tests revealed that the transplants had acquired a hardness similar to that of the surrounding cartilage. We conclude that transplanting chondrocytes in a newly-formed matrix of atelocollagen gel can promote restoration of the articular cartilage of the knee.

Cartilage Repair With Autologous Bone Marrow Mesenchymal Stem Cell Transplantation: Review of Preclinical and Clinical Studies

Clinical trials of various procedures, including bone marrow stimulation, mosaicplasty, and autologous chondrocyte implantation, have been explored to treat articular cartilage defects. However, all of them have some demerits. We focused on autologous culture-expanded bone marrow mesenchymal stem cells (BMSC), which can proliferate without losing their capacity for differentiation. First, we transplanted BMSC into the defective articular cartilage of rabbit and succeeded in regenerating osteochondral tissue. We then applied this transplantation in humans. Our previous reports showed that treatment with BMSC relieves the clinical symptoms of chondral defects in the knee and elbow joint. We investigated the efficacy of BMSC for osteoarthritic knee treated with high tibial osteotomy, by comparing 12 BMSC-transplanted patients with 12 cell-free patients. At 16-month follow-up, although the difference in clinical improvement between both groups was not significant, the arthroscopic and histological grading score was better in the cell-transplanted group. At the over 10-year follow-up, Hospital for Special Surgery knee scores improved to 76 and 73 in the BMSC-transplanted and cell-free groups, respectively, which were better than preoperative scores. Additionally, neither tumors nor infections were observed in all patients, and in the clinical study, we have never observed hypertrophy of repaired tissue, thereby guaranteeing the clinical safety of this therapy. Although we have never observed calcification above the tidemark in rabbit model and human histologically, the repair cartilage was not completely hyaline cartilage. To elucidate the optimum conditions for cell therapy, other stem cells, culture conditions, growth factors, and gene transfection methods should be explored.

Histone deacetylase-4 is required during early cranial neural crest development for generation of the zebrafish palatal skeleton

BackgroundHistone deacetylase-4 (Hdac4) is a class II histone deacetylase that inhibits the activity of transcription factors. In humans, HDAC4 deficiency is associated with non-syndromic oral clefts and brachydactyly mental retardation syndrome (BDMR) with craniofacial abnormalities.ResultsWe identify hdac4 in zebrafish and characterize its function in craniofacial morphogenesis. The gene is present as a single copy, and the deduced Hdac4 protein sequence shares all known functional domains with human HDAC4. The zebrafish hdac4 transcript is widely present in migratory cranial neural crest (CNC) cells of the embryo, including populations migrating around the eye, which previously have been shown to contribute to the formation of the palatal skeleton of the early larva. Embryos injected with hdac4 morpholinos (MO) have reduced or absent CNC populations that normally migrate medial to the eye. CNC-derived palatal precursor cells do not recover at the post-migratory stage, and subsequently we found that defects in the developing cartilaginous palatal skeleton correlate with reduction or absence of early CNC cells. Palatal skeletal defects prominently include a shortened, clefted, or missing ethmoid plate, and are associated with a shortening of the face of young larvae.ConclusionsOur results demonstrate that Hdac4 is a regulator of CNC-derived palatal skeletal precursors during early embryogenesis. Cleft palate resulting from HDAC4 mutations in human patients may result from defects in a homologous CNC progenitor cell population.

Articular cartilage endurance and resistance to osteoarthritic changes require transcription factor Erg.

To determine whether and how the transcription factor Erg participates in the genesis, establishment, and maintenance of articular cartilage.

Xeno-free and shrinkage-free preparation of scaffold-free cartilage-like disc-shaped cell sheet using human bone marrow mesenchymal stem cells

Aiming for the clinical application of cartilage regeneration, the xeno-free cultivation method to obtain a scaffold-free cartilage-like disc-shaped cell sheet using mesenchymal stem cells (MSCs) derived from human bone marrow without the shrinkage of the sheet was investigated. MSCs were inoculated into Cell Culture Insert (0.3 cm(2), pore size; 0.4 μm, pore density; 1.0 × 10(8)/cm(2)) using serum-free chondrogenic differentiation medium containing TGF-β3, IGF-1 and dexamethasone or other modified media, and cultured at 37 °C in 5% CO2 for 3 weeks. Sheet thickness, cartilage specific genes expression, ECM accumulation were determined, and the sections of sheets were stained with alcian blue. A novel mixed medium consisting of a growth medium (10% FCS) with a serum-free chondrogenic differentiation medium could prevent the shrinkage of the sheet and produced a disc-shaped cell sheet. The depth of the sheet was approximately 0.7 mm and the gene expression levels were higher than those in cells in normal human cartilage. The use of human serum instead of FCS did not cause shrinkage and did not decrease the accumulation levels of sGAG and type 2 collagen in the sheet. The cultivation of MSCs grown with completely xeno-free materials using the mixed medium containing human serum in a cell culture insert showed a sheet depth of 1.0 mm and gene expression levels higher than those in normal cartilage. The scaffold-free and xeno-free cartilage-like cell sheet was successfully formed without shrinkage using human bone marrow MSCs and the chondrogenic differentiation medium containing human serum.

Transplantation of Scaffold-Free Cartilage-Like Cell-Sheets Made from Human Bone Marrow Mesenchymal Stem Cells for Cartilage Repair A Preclinical Study

Objective The object of this study was to determine culture conditions that create stable scaffold-free cartilage-like cell-sheets from human bone marrow–derived mesenchymal stem cells (hBMSCs) and to assess their effects after transplantation into osteochondral defects in nude rats. Design (Experiment 1) The hBMSCs were harvested from 3 males, the proliferative and chondrogenic capacities were assessed at passage 1, and the cells were expanded in 3 different culture conditions: (1) 5% fetal bovine serum (FBS), (2) 10% FBS, and (3) 5% FBS with fibroblast growth factor 2 (FGF-2). The cells were harvested and made chondrogenic pellet culture. The cell proliferation rate, glycosaminoglycan/DNA ratio, and safranin-O staining intensity of pellets cultured condition 3 were higher than those of conditions 1 and 2. (Experiment 2) The hBMSCs were expanded and passaged 3 times under culture condition 3, and fabricate the cell-sheets in chondrogenic medium either with or without FBS. The cell-sheets fabricated with FBS maintained their size with flat edges. (Experiment 3) The cell-sheets were transplanted into osteochondral defects in nude rats. Histological analysis was performed at 2, 4, and 12 weeks after surgery. Results The osteochondral repair was better after sheet transplantation than in the control group and significantly improved Wakitani score. Immunostaining with human-specific vimentin antibody showed that the transplanted cells became fewer and disappeared at 12 weeks. Conclusions These results indicate that culture with FGF-2 may help to quickly generate sufficient numbers of cells to create stable and reliable scaffold-free cartilage-like cell-sheets, which contribute to the regeneration of osteochondral defects.

Quality Evaluation of Human Bone Marrow Mesenchymal Stem Cells for Cartilage Repair

Quality evaluation of mesenchymal stem cells (MSCs) based on efficacy would be helpful for their clinical application. In this study, we aimed to find the factors of human bone marrow MSCs relating to cartilage repair. The expression profiles of humoral factors, messenger RNAs (mRNAs), and microRNAs (miRNAs) were analyzed in human bone marrow MSCs from five different donors. We investigated the correlations of these expression profiles with the capacity of the MSCs for proliferation, chondrogenic differentiation, and cartilage repair in vivo. The mRNA expression of MYBL1 was positively correlated with proliferation and cartilage differentiation. By contrast, the mRNA expression of RCAN2 and the protein expression of TIMP-1 and VEGF were negatively correlated with proliferation and cartilage differentiation. However, MSCs from all five donors had the capacity to promote cartilage repair in vivo regardless of their capacity for proliferation and cartilage differentiation. The mRNA expression of HLA-DRB1 was positively correlated with cartilage repair in vivo. Meanwhile, the mRNA expression of TMEM155 and expression of miR-486-3p, miR-148b, miR-93, and miR-320B were negatively correlated with cartilage repair. The expression analysis of these factors might help to predict the ability of bone marrow MSCs to promote cartilage repair.

Nucleated cells circulating in the peripheral blood contribute to the repair of osteochondral defects only in the early phase of healing

The role of cells circulating in the peripheral blood to participate in the natural repair process of osteochondral defects was evaluated in a green fluorescent protein (GFP) transgenic and wild rat parabiosis model. Two weeks after the parabiosis operation, vascular communication between the conjoined rats was confirmed by flow‐cytometry analysis. A 1.5 mm diameter and 1.0 mm depth osteochondral defect was made in the patellar groove of each rat femoral bone. Histological examination was performed at 1, 2, 4 and 24 weeks following surgery. In the early postoperative phase (1–4 weeks) there were GFP‐negative and ‐positive cells in the defects of both parabiotic rats. GFP‐positive chondrocytes were confirmed partly in the repair tissue of the wild parabiotic rat. In the late postoperative phase (24 weeks), the repaired defects were occupied by cells originating from the adjacent tissue and not from the peripheral blood. The ratio of cells originating from the peripheral blood was approximately 30–40% in the repair tissue at 1 week after surgery, reduced to 0–7% at 24 weeks. From these results it is confirmed that cells circulating in the peripheral blood contributed to the repair of the osteochondral defects, particularly in the early phase of healing. Thus, peripheral blood not only supplies the factors needed for repair but also provides a cell population involved in the wound‐healing process. Copyright

Systemic Administration of Granulocyte Colony-Stimulating Factor for Osteochondral Defect Repair in a Rat Experimental Model

Objective: The objective of this study was to assess the effect of granulocyte colony-stimulating factor (G-CSF) on osteochondral defect repair in the rat knee. Design: Twenty-six 12-week-old male Lewis rats were randomly divided into 2 groups. From day 0 to day 4, the G-CSF group received glycosylated G-CSF, and the control group received phosphate-buffered saline. A 1.5-mm diameter and 1.0-mm deep osteochondral defect was introduced in the patellar groove of the bilateral femur in all rats on day 4. The peripheral blood nucleated cells were counted for 14 days from the first day of injection, the appearance of the cartilage repair was observed histologically and macroscopically for 2, 4, 8, 12, and 24 weeks after surgery. Results: The number of peripheral blood leukocytes increased 3 days and returned to normal levels 7 days after the first injection. Compared with the control group, the G-CSF group had more fibrous and/or bony tissue at earlier points in time. The tissue repair rate, which is defined as the percentage of repaired osteochondral defects, was significantly higher in the G-CSF group 4 weeks after surgery. However, there were no significant differences in the cartilage repair rate and the modified Wakitani score between the 2 groups at each time point. Conclusions: The defect filling was significantly better in the G-CSF group in the early phases. Our findings suggest that G-CSF may promote the repair of osteochondral defects by mediating an increase in the number of peripheral blood nucleated cells.

Loss of lean body mass affects low bone mineral density in patients with rheumatoid arthritis – results from the TOMORROW study

Abstract Objectives: Osteoporosis is one of the complications for patients with rheumatoid arthritis (RA). Rheumatoid cachexia, the loss of lean body mass, is another. However, the relationship between decreased lean body mass and reduced bone mineral density (BMD) in patients with RA has not been well studied. Methods: This study included 413 participants, comprising 208 patients with RA and 205 age- and sex-matched healthy volunteers. Clinical data, BMD, bone metabolic markers (BMM) and body composition, such as lean body mass and percent fat, were collected. Risk factors for osteoporosis in patients with RA including the relationship BMD and body composition were analyzed. Results: Patients with RA showed low BMD and high BMM compared with controls. Moreover, lean body mass was lower and percent fat was higher in patients with RA. Lean body mass correlated positively and percent fat negatively with BMD. Lean body mass was a positive and disease duration was a negative independent factor for BMD in multivariate statistical analysis. Conclusion: BMD and lean body mass were significantly lower in patients with RA compared to healthy controls. Lean body mass correlated positively with BMD and decreased lean body mass and disease duration affected low BMD in patients with RA. Trial Registration: [UMIN Clinical Trials Registry, http://www.umin.ac.jp/ctr/, UMIN000003876].