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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.

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).

Since the days of ancient Greece, it has been known that articular cartilage has poor potential to repair. In April, 2013, autologous chondrocyte implantation (ACI) was approved by the Japanese ministry of Health, Labour and Welfare, as the first orthopedic cell therapy in Japan. Half a century has passed since Chesterman's report on rabbit allogeneic chondrocyte implantation, and approximately 20 years since the first human ACI report by Brittberg. In Japan, 24 years has passed since Wakitani's allogeneic chondrocyte implantation, and over 10 years since the human ACI report by Ochi. A long-term follow-up clinical trial with the high statistical power is needed to verify the safety and efficacy of new cartilage joint therapy. Moreover, we need the infrastructure, including drug and/or device regulation and timely approved by the government, to apply new therapies in similar time frames to other developed nations.

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., (Copyright © 2013. Published by Elsevier B.V.)

Background: Aiming to construct an osteochondral-like structure, the combination of a β-tricalcium phosphate (βTCP) block with a scaffold-free sheet formed using mesenchymal stem cells (MSCs) was investigated., Methods: Human bone marrow MSCs in a cell culture insert that was set in a 24-well plate were cultivated using a chondrogenic medium containing dexamethasone, IGF-1, and TGFβ3 for 3 weeks during which a cylindrical βTCP block was put on the sheet at day 1, and the cell sheet construct was harvested. In other experiments, at day 14, the construct was put on a cell sheet that was prepared the day before and cultivated for 3 weeks., Results: The addition of a βTCP block resulted in a combined osteochondral-like construct and comparable staining intensity by Alcian blue, while the expression levels of the aggrecan and type II collagen genes decreased a little. During the culture with the βTCP block, the expression levels of the aggrecan gene increased monotonically. The increase in the inoculum cell number from 1.86 to 3.72 × 10(6) cells resulted in marked increases in the thickness of cell sheet parts in the βTCP block and expression levels of the aggrecan and type II collagen genes, while the thickness of cell sheet parts on the βTCP block scarcely changed. On the other hand, the addition of a cell sheet that was prepared a day before to the construct at day 14 resulted in the marked increase in thickness of the cell sheet part on the βTCP block, while the thickness of that in the βTCP block did not increase., Conclusion: A combined osteochondral-like structure was produced by putting a βTCP block on the sheet of MSC. The thickness of the cell sheet parts in and on the βTCP block could be increased by the increase in inoculum cell number and by providing an additional cell sheet, respectively.

Study Design: Histopathological and immunohistochemical analysis., Objective: To investigate the histological changes and expression of bone morphogenetic protein (BMP) signaling component in hypertrophied ligamentum flavum (LF), and to clarify the effect of mechanical stress on them., Summary of Background Data: Hypertrophic changes of the LF are a major factor in degenerative lumbar canal stenosis (DLCS), but their mechanism remains unclear. BMPs are growth factors that regulate many cellular processes including proliferation, differentiation, and extracellular matrix synthesis. However, a few studies have investigated the expressions of BMP signaling in the hypertrophied LF., Methods: A total of 133 LF specimens from patients with DLCS and 17 control LF specimens from patients with lumbar disc herniation were analyzed histologically using hematoxylin and eosin, elastica van Gieson, and toluidine blue staining. To analyze the influence of mechanical stress, the DLCS specimens were divided into 2 groups: DLCS with and DLCS without hypermobility groups. The LF thickness was measured by magnetic resonance image, and the correlations between the thickness and the histological data were analyzed. Immunohistochemical analyses were carried out to confirm the expressions and localizations of BMP signaling components., Results: The cell number and cartilage matrix area were significantly increased in the hypertrophied LF, and those changes were more obvious in DLCS with hypermobility than in DLCS without hypermobility. The cellularity and percentage of cartilage matrix area had positive linear correlations with the LF thickness. BMP receptors and BMP ligands were both expressed by many cells of the hypertrophied LF, and some of these cells were positive for Sox9, CD105, and Msx2. The percentage of immunopositive cells for each BMP receptor type was significantly higher in DLCS with hypermobility than in DLCS without hypermobility., Conclusions: Higher cellularity and increased cartilage matrix area are important changes in LF hypertrophy. These results suggest that BMP signaling and mechanical stress may play a role in the hypertrophied LF.

MicroRNA-140 (miR-140) is specifically expressed in developing cartilage tissues. We have previously reported that miR-140 plays an important role during palatal cartilage development by modulating platelet-derived growth factor receptor alpha (pdgfra) in zebrafish. However, the regulatory mechanism of miR-140 in cartilage is still unknown. Using developing zebrafish, sox9a mutant (sox9a-/-) and sox9b mutant (sox9b-/-) zebrafish and SOX9 small interfering RNA in human chondrocytes, T/C-28 cells, we found that miR-140 is regulated by the cartilage master transcription regulator Sox9 in zebrafish and mammalian cells.

Aiming for the clinical application of cartilage regeneration, a culture method for mesenchymal stem cells (MSCs) derived from human bone marrow to obtain scaffold-free cartilage-like disk-shaped sheet of uniform sizes without the shrinkage was investigated. A disk-shaped cell sheet having the same diameter as that of the membrane without the shrinkage was formed after the cultivation of MSCs (18.6 × 10(5)cells/well) for 3 weeks in a cell culture insert (CCI) containing a flat membrane whose porosity was 12%, while 6.2 and 31.0 × 10(5)MSCs/well, respectively, resulted in the shrinkage of the aggregate and the hole formation in the center part of the sheet. Cell aggregates shrunk also in a 96-well plate and CCIs having lower porosity. The disk-shaped cell sheet showed the comparable thickness (1.2mm) and sulfated glycosaminoglycan (sGAG) density to those of the pellet formed in a pellet culture. The gene expression levels of aggrecan and type II collagen in the disk-shaped cell sheet were not lower than those in the pellet. In conclusion, the usage of CCI having 12% porosity and 18.6 × 10(5)MSCs/well could avoid the shrinkage from the formation of the scaffold-free cartilage-like disk-shaped cell sheet., (Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

An ex vivo cartilage defect model for the evaluation of cartilage regeneration using mesenchymal stem cells (MSCs) was developed. Porcine chondrocytes and human MSCs were transplanted into cartilage defects created on the porcine osteochondral and chondral discs and cultivated for 3 weeks. Although the regeneration of cartilage-like tissues was observed after the transplantation of chondrocytes to defects on both of the osteochondral and chondral discs, the transplanted MSCs formed cartilage-like tissues only in the defect on the chondral disc, in which an in vivo cartilage-like structure was partly observed, and a degraded tissue was observed in the defect on the osteochondral disc. The effects of medium additives such as serum, transforming growth factor-β3 (TGF-β3), and fibroblast growth factor-2, and the transfection of TGF-β3 gene to MSCs could be clearly estimated using the cartilage defect model. In conclusion, a chondral disc with defects is useful for evaluating cartilage regeneration using MSCs., (Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Porcine articular cartilage tissue was crushed using Multi-Beads-Shocker() and digested with trypsin and collagenase type II. Chondrocyte cell yield was maximized by automatic crushing for 10 s, which was 4 times that by manual dicing with a surgical blade and might be useful for the automation of the cell processing., (2009 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The objective of this study was to evaluate the effects of the mechanical environment on the formation of cartilage tissue in transplanted embryonic stem (ES) cells. Full-thickness osteochondral defects were created on the patella groove of SD rats, and ES cells (CCE ES cells obtained from 129/Sv/Ev mice and Green ES FM260 ES cells obtained from 129SV [D3] - Tg [NCAG-EGFP] CZ-001-FM260Osb mice) were transplanted into the defects embedded in collagen gel. The animals were randomly divided into either the joint-free group (JF group) or the joint-immobilized group (JI group) for 3 weeks after a week postoperatively. The results showed that cartilage-like tissue formed in the defects of the JF group whereas large teratomatous masses developed in the defects of the JI group. Some parts of the cartilage-like tissue and the teratomatous masses were positively stained with immunostain for GFP when the Green ES FM260 ES cells were transplanted. It is suggested that the environment plays an important role for ES cells in the process of repairing cartilage tissue in vivo., ((c) 2007 Orthopaedic Research Society.)

This study assessed the effectiveness of autologous bone marrow stromal cell transplantation for the repair of full-thickness articular cartilage defects in the patellae of a 26-year-old female and a 44-year-old male. These two patients presented in our clinic because their knee pain prevented them from walking normally. After thorough examination, we concluded that the knee pain was due to the injured articular cartilage and decided to repair the defect with bone marrow stromal cell transplantation. Three weeks before transplantation, bone marrow was aspirated from the iliac crest of each patient. After erythrocytes had been removed by use of dextran, the remaining nucleated cells were placed in culture. When the attached cells reached subconfluence, they were passaged to expand in culture. Adherent cells were subsequently collected, embedded in a collagen gel, transplanted into the articular cartilage defect in the patellae, and covered with autologous periosteum. Six months after transplantation, clinical symptoms (pain and walking ability) had improved significantly and the improvement has remained in effect (5 years and 9 months posttransplantation in one case, and 4 years in the other), and both patients have been satisfied with the outcome. As early as 2 months after transplantation, the defects were covered with tissue that showed slight metachromatic staining. Two years after the first and 1 year after the second transplantation, arthroscopy was performed and the defects were repaired with fibrocartilage. Results indicate autologous bone marrow stromal cell transplantation is an effective approach in promoting the repair of articular cartilage defects.

A micromachine was used to study the response of mesenchymal tissue to mechanically controlled motion in vivo. The middle portion of the coccygeal vertebra of Fischer 344 rats was osteotomized, and continuous bending motion was applied for 4 weeks. The experimental groups were divided into two groups with higher sliding displacement applied at the osteotomized gap of group II. Hyaline cartilage tissue was generated at the osteotomized ends, and was predominantly formed on the side that extended during the bending motion. These newly formed tissues stained intensively with safranin O and toluidine blue, positively with immunostain for type II collagen, but negatively with immunostain for type I collagen. Articular cartilage-like tissues with a surface and a layer structure were obtained in group II, in which higher sliding motion was applied. Light and electron microscopy revealed morphological features similar to those of normal articular cartilage tissue in the superficial and middle zones of the tissues obtained in group II. Collagen fibrils in the superficial zone were found aligned parallel to the smooth surface. Although tidemark formation was not observed in the deep zone, the structure was much more natural than that of any other tissue-engineered cartilage reported to date. These results suggest that controlled sliding stimulation can elicit the generation of articular cartilage structure in vivo.

Cartilage regeneration treatments using stem cells are associated with problems due to the cell source and the difficulty of delivering the cells to the cartilage defect. We consider labeled induced pluripotent stem (iPS) cells to be an ideal source of cells for tissue regeneration, and if iPS cells could be delivered only into cartilage defects, it would be possible to repair articular cartilage. Consequently, we investigated the effect of magnetically labeled iPS (m-iPS) cells delivered into an osteochondral defect by magnetic field on the repair of articular cartilage. iPS cells were labeled magnetically and assessed for maintenance of pluripotency by their ability to form embryoid bodies in vitro and to form teratomas when injected subcutaneously into nude rats. These cells were delivered specifically into cartilage defects in nude rats using a magnetic field. The samples were graded according to the histologic grading score for cartilage regeneration. m-iPS cells differentiated into three embryonic germ layers and formed teratomas in the subcutaneous tissue. The histologic grading score was significantly better in the treatment group compared to the control group. m-iPS cells maintained pluripotency, and the magnetic delivery system proved useful and safe for cartilage repair using iPS cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Articular cartilage defects that do not repair spontaneously induce osteoarthritic changes in joints over a long period of observation. In this study, we examined the usefulness of transplanting culture-expanded bone marrow mesenchymal cells into osteochondral defects of joints with cartilage defects. First, we performed experiments on rabbits and up on obtaining good results proceeded to perform the experiments on humans. Macroscopic and histological repair with this method was good, and good clinical results were obtained although there was no significant difference with the control group. Recent reports have indicated that this procedure is comparable to autologous chondrocyte implantation, and concluded that it was a good procedure because it required one step less than that required by surgery, reduced costs for patients, and minimized donor site morbidity. Although some reports have previously shown that progenitor cells formed a tumor when implanted into immune-deficient mice after long term in vitro culture, the safety of the cell transplantation was confirmed by our clinical experience. Thus, this procedure is useful, effective, and safe, but the repaired tissues were not always hyaline cartilage. To obtain better repair with this procedure, treatment approaches using some growth factors during in vitro culture or gene transfection are being explored. J. Cell. Physiol. 225: 291–295, 2010. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Abstract: The effect of the addition of monosaccharides composing glycosaminoglycans (GAGs) on the accumulation of type 2 collagen (COL(II)) in a three-dimensional (3D) culture of porcine chondrocyte cells was investigated for possible application to cartilage regenerative medicine. Primary chondrocytes from porcine cartilage were cultivated in three-dimension employing atelo collagen gel for 3 weeks with the addition of several saccharides. The addition of d-glucuronic acid (d-GlcA), N-acetyl-d-galactosamine (d-GalNAc), chondroitin sulfate C (CSC), d-galactose, N-acetyl-d-glucosamine, and l-iduronic acid increased markedly not aggrecan but COL(II) accumulation although the addition of d-fructose and d-mannose not composing GAGs did not show such an effect. The addition of d-GlcA and d-GalNAc had no synergistic effect. The addition of CSC, d-GlcA, and d-GalNAc also increased COL(II) mRNA expression while aggrecan mRNA expression was not increased by these compounds. In conclusion, the addition of monosaccharides composing GAGs might be valuable for increasing COL(II) accumulation in the 3D culture of chondrocytes. [Copyright &y& Elsevier]

The effect of glycosaminoglycan addition on a three-dimensional (3D) culture of porcine chondrocyte cells was investigated with a view to use in cartilage regenerative medicine. Chondroitin sulfate C increased the mRNA expression of type 2 collagen, while chondroitin sulfate A did not. Hyaluronic acid of high molecular weight markedly decreased the mRNA expression of both aggrecan and type 2 collagen, although hyaluronic acid of low molecular weight showed no apparent effect. [Copyright &y& Elsevier]

Abstract: Although tribological function is the most important mechanical property of articular cartilage, few studies have examined this function in tissue-engineered cartilage. We investigated changes in the frictional properties of cartilage regenerated from the inoculation of rabbit chondrocytes into fibroin sponge. A reciprocating friction-testing apparatus was used to measure the friction coefficient of the regenerated cartilage under a small load. The specimen was slid against a stainless steel plate in a water vessel filled with physiological saline. The applied load was 0.03N, the stroke length was 20mm, and the mean sliding velocity was 0.8mm/s. The friction coefficient of the regenerated cartilage decreased with increasing cultivation time, because a hydrophilic layer of synthesized extracellular matrix was formed on the fibroin sponge surface. The friction coefficient of the regenerated cartilage was as low as that of natural cartilage in the early stages of the sliding tests, but it increased with increasing duration of sliding owing to exudation of interstitial water from the surface layer. [Copyright &y& Elsevier]