Hisashi Mera

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.

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.

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.

Effect of epigallocatechin-3-gallate on the increase in type II collagen accumulation in cartilage-like MSC sheets

With the aim to increase type II collagen content in the scaffold-free cartilage-like cell sheet using human bone marrow mesenchymal stem cells, we examined the effect of epigallocatechin-3-gallate (EGCG) addition to the chondrogenic medium for the cell sheet culture. The addition of EGCG (10 μM) increased the content of type II collagen 2-fold, while the addition did not markedly change the expression level of the genes encoding type II collagen and Sox 9. The reactive oxygen species level in the cells in cell sheets was thought to be too low to suppress the accumulation of type II collagen. On the other hand, the addition of EGCG markedly decreased both the matrix metalloproteinase-13 concentration in the supernatant of cell sheet culture and the type II collagen degradation activity in that supernatant. Taken together, EGCG may enhance the accumulation of type II collagen by suppressing type II collagen degradation. Addition of EGCG to the sheet culture of MSC markedly increased not only wet weight (A) and sGAG amount (B) but also type II collagen amount (C) in the cartilage-like cell sheet.

Effect of epigallocatechin-3-o-gallate and quercetin on the cryopreservation of cartilage tissue.

The effects of epigallocatechin-3-o-gallate (EGCG) and quercetin on the contents of extracellular matrix (ECM) in porcine cartilage at 4 °C were investigated. The addition of quercetin at 0.01 mM for the incubation of porcine cartilage disks at 4 °C for 2 week could suppress the decrease in ECM and the compliance of the disks, markedly greater than those of EGCG (1.0 mM).

Irx3 and Bmp2 Regulate Mouse Mesenchymal Cell Chondrogenic Differentiation in Both a Sox9-Dependent and -Independent Manner.

Sox9, a master regulator of cartilage development, controls the cell fate decision to differentiate from mesenchymal to chondrogenic cells. In addition, Sox9 regulates the proliferation and differentiation of chondrocytes, as well as the production of cartilage‐specific proteoglycans. The existence of Sox9‐independent mechanisms in cartilage development remains to be determined. Here, we attempted to identify genes involved in such putative mechanisms via microarray analysis using a mouse chondrogenic cell line, N1511. We first focused on transcription factors that exhibited upregulated expression following Bmp2 treatment, which was not altered by subsequent treatment with Sox9 siRNA. Among these, we selected positive regulators for chondrogenesis and identified Iroquois‐related homeobox 3 (Irx3) as one of the candidate genes. Irx3 expression gradually increased with chondrocyte terminal differentiation in a reciprocal manner to Sox9 expression, and promoted the chondrogenic differentiation of mesenchymal cells upon Bmp2 treatment. Furthermore, Irx3 partially rescued impaired chondrogenesis by upregulating the expression of epiphycan and lumican under reduced Sox9 expression. Finally, Irx3 was shown to act in concert with Bmp2 signaling to activate the p38 MAPK pathway, which in turn stimulated Sox9 expression, as well as the expression of epiphycan and lumican in a Sox9‐independent manner. These results indicate that Irx3 represents a novel chondrogenic factor of mesenchymal cells, acts synergistically with Bmp2‐mediated signaling, and regulates chondrogenesis independent of the transcriptional machinery associated with Sox9‐mediated regulation.

Effect of the direct injection of bone marrow mesenchymal stem cells in hyaluronic acid and bone marrow stimulation to treat chondral defects in the canine model

Introduction The purpose of this study was to assess the direct injection of bone marrow-derived mesenchymal stem cells (BMSCs) suspended in hyaluronic acid (HA) combined with drilling as a treatment for chondral defects in a canine model. Methods Tibial bone marrow was aspirated, and BMSCs were isolated and cultured. One 8.0-mm diameter chondral defect was created in the femoral groove, and nine 0.9-mm diameter holes were drilled into the defect. BMSCs (2.14 × 107 cells) suspended in HA were injected into the defect. HA alone was injected into a similar defect on the contralateral knee as a control. Animals were sacrificed at 3 and 6 months. Results Although the percentage of coverage assessed macroscopically was significantly better at 6 months than at 3 months in both the BMSC (p = 0.02) and control (p = 0.001) groups, there were no significant differences in the International Cartilage Repair Society grades. The Wakitani histological score was significantly better at 6 months than at 3 months in the BMSC and control groups. While the control defects were mostly filled with fibrocartilage, several of the defects in the BMSC group contained hyaline-like cartilage. The mean Wakitani scores of the BMSC group improved from 7.0 ± 1.0 at 3 months to 4.6 ± 0.9 at 6 months, and those of the control group improved from 9.4 ± 1.2 to 6.0 ± 0.6. The BMSC group showed significantly better regeneration than the control group at 3 months (p = 0.04), but the difference at 6 months was not significant (p = 0.06). Conclusions The direct injection of BMSCs in HA combined with drilling enhanced cartilage regeneration.