Kosuke Tateishi

In Vitro Generation of a Scaffold-Free Tissue-Engineered Construct (TEC) Derived from Human Synovial Mesenchymal Stem Cells: Biological and Mechanical Properties and Further Chondrogenic Potential

The purpose of this study was to characterize a tissue-engineered construct (TEC) generated with human synovial mesenchymal stem cells (MSCs). MSCs were cultured in medium with ascorbic acid 2-phosphate (Asc-2P) and were subsequently detached from the substratum. The detached cell/matrix complex spontaneously contracted to develop a basic TEC. The volume of the TEC assessed by varying initial cell density showed that it was proportional to initial cell densities up to 4 x 10(5) cells/cm(2). Assessment of the mechanical properties of TEC using a custom device showed that the load at failure and stiffness of the constructs significantly increased with time of culture in the presence of Asc-2P, while in the absence of Asc-2P, the constructs were mechanically weak. Thus, the basic TEC possesses sufficiently self-supporting mechanical properties in spite of not containing artificial scaffolding. TEC further cultured in chondrogenic media exhibited positive alcian blue staining with elevated expression of chondrogenic marker genes. Based on these findings, such human TEC may be a promising method to promote cartilage repair for future clinical application.

The influence of skeletal maturity on allogenic synovial mesenchymal stem cell-based repair of cartilage in a large animal model.

One of the potential factors that may affect the results of mesenchymal stem cell (MSC)-based therapy is the age of donors and recipients. However, there have been no controlled studies to investigate the influence of skeletal maturity on the MSC-based repair of cartilage. The purpose of this study was to compare the repair quality of damaged articular cartilage treated by a scaffold-free three-dimensional tissue-engineered construct (TEC) derived from synovial MSCs between immature and mature pigs. Synovial MSCs were isolated from immature and mature pigs and the proliferation and chondrogenic differentiation capacities were compared. The TEC derived from the synovial MSCs were then implanted into equivalent chondral defects in the medial femoral condyle of both immature and mature pigs, respectively. The implanted defects were morphologically and biomechanically evaluated at 6 months postoperatively. There was no skeletal maturity-dependent difference in proliferation or chondrogenic differentiation capacity of the porcine synovial MSCs. The TEC derived from synovial MSCs promoted the repair of chondral lesion in both immature and mature pigs without the evidence of immune reaction. The repaired tissue by the TEC also exhibited similar viscoelastic properties to normal cartilage regardless of the skeletal maturity. The results of the present study not only suggest the feasibility of allogenic MSC-based cartilage repair over generations but also may validate the use of immature porcine model as clinically relevant to test the feasibility of synovial MSC-based therapies in chondral lesions.

Comparison of Human Serum with Fetal Bovine Serum for Expansion and Differentiation of Human Synovial MSC: Potential Feasibility for Clinical Applications:

The aim of this study was to evaluate the effect of human serum (HS) on growth and differentiation capacity of human synovium-derived mesenchymal stem cells (MSC) in comparison to cells grown in fetal bovine serum (FBS). Human MSCs were isolated from the synovium of knee joints of three donors and the cells were cultured individually in varying concentrations of allogenic HS or FBS. Bovine MSCs were isolated from synovium and cultured in the same manner. Cell proliferation was assessed by the tetrazolium assay after passage 3. The capacity for chondrogenic and osteogenic differentiation was investigated in specific media followed by 1,9-dimethylmethylene blue assay and alcian blue staining, or by alizarin red staining, respectively. Human MSCs proliferated significantly more rapidly in the presence of HS than with equivalent levels of FBS. Chondrogenic or osteogenic differentiation occurred to nearly identical levels in HS or FBS. The results of this study indicate that HS is superior for the culture of human MSCs compared with FBS in terms of cellular expandability, without losing chondrogenic or osteogenic differentiation capacity. Coupled with the advantage in eliminating the potential risk accompanied with the use of xeno-derived materials, pooled, well-characterized HS could be a useful reagent to promote cellular expansion for clinical synovial stem cell-based therapy.

Imatinib mesylate inhibits osteoclastogenesis and joint destruction in rats with collagen-induced arthritis (CIA).

Macrophage colony-stimulating factor (M-CSF) is a key factor for osteoclastogenesis at the bone–pannus interface in patients with rheumatoid arthritis as well as a receptor activator of NF-κB ligand (RANKL). Imatinib mesylate inhibits the phosphorylation of c-fms, a receptor for M-CSF. The present study investigates the effect of imatinib mesylate on joint destruction in rats with collagen-induced arthritis (CIA) and on osteoclastogenesis in vitro. Imatinib mesylate (50 or 150 mg/kg), dexamethasone, or vehicle was administered daily to CIA rats for 4 weeks from the onset of arthritis. Hind-paw swelling and body weight were measured weekly. At weeks 2 and 4, the metatarsophalangeal (MTP) joints and the ankle and subtalar joints were radiographically and histologically assessed. The effect of imatinib mesylate on osteoclast formation from rat bone marrow cells with M-CSF and soluble RANKL (sRANKL) in vitro was also examined. Radiographic assessment showed that 150 mg/kg imatinib mesylate suppressed the destruction of the MTP and the ankle and subtalar joints at week 2, and MTP joint destruction at week 4 in CIA rats, although hind-paw swelling was not suppressed. The number of TRAP-positive cells at the bone–pannus interface was significantly reduced in the group administered with 150 mg/kg imatinib mesylate compared with that given vehicle at week 4. Imatinib mesylate dose-dependently inhibited the proliferation of M-CSF-dependent osteoclast precursor cells in vitro as well as osteoclast formation induced by M-CSF and sRANKL. These findings suggest that imatinib mesylate could prevent joint destruction in patients with rheumatoid arthritis.

Repair of meniscal lesions using a scaffold-free tissue-engineered construct derived from allogenic synovial MSCs in a miniature swine model

The menisci of the knee are fibro-cartilaginous tissues and play important roles in the joint, and the loss of the meniscus predisposes the knee to degenerative changes. However, the menisci have limited healing potential due to the paucity of vascularity. The purpose of the present study was to test the feasibility of a scaffold-free tissue-engineered construct (TEC) derived from synovial mesenchymal stem cells (MSCs) to repair incurable meniscal lesions. Porcine synovial MSCs were cultured in monolayers at high density in the presence of ascorbic acid followed by the suspension culture to develop a three-dimensional cell/matrix construct (TEC). A 4-mm cylindrical defect was created bilaterally in the medial meniscus of skeletally mature miniature pigs. The defects were implanted with an allogenic TEC or were left empty. After 6 months, the TEC-treated defects were consistently repaired by a fibro-cartilaginous tissue with good tissue integration to the adjacent host meniscal tissue, while the untreated were either partially or not repaired. The ratio of Safranin O positive area within the central body of the meniscus adjacent to the original defect was significantly higher in the TEC-treated group than in the control group. Moreover, TEC treatment significantly reduced the size and severity of post-traumatic chondral lesions on the tibial plateau. These results suggest that the TEC could be a promising stem cell-based implant to repair meniscal lesions with preventive effects from meniscal body degeneration and the development of post-traumatic arthritis.

Scaffold-Free Tissue-Engineered Construct–Hydroxyapatite Composites Generated by an Alternate Soaking Process : Potential for Repair of Bone Defects

Mesenchymal stem cell (MSC)-based tissue-engineered construct (TEC)-hydroxyapatite (HAp) composites were developed by an alternate soaking process. The TEC derived from cultured synovial MSCs was alternately immersed in varying concentrations of CaCl(2)/Tris-HCl and Na(2)HPO(4)/Tris-HCl buffers, and HAp formation was analyzed by Fourier transform infrared spectroscopy (FT-IR), wide-angle X-ray diffraction, and scanning electron microscopy (SEM). These analyses clearly demonstrated HAp formation in the TEC. Specifically, SEM assessments showed that spherical HAp crystals of approximately 1 mum were directly formed on the surfaces of the cells and extracellular matrix (ECM) fibers. Cytotoxicity from exposure to calcium or phosphate buffers of >100 mM concentrations as assessed by LIVE/DEAD staining and total DNA assays was detected, but such cytotoxicity was not detected following exposure to concentrations of <50 mM. The HAp nanocrystals (ca. approximately 500 nm) were formed after 20 cycles in 10 mM calcium or phosphate buffers, and cell survival in the composites was confirmed. Moreover, preliminary implantation of TEC-HAp composites derived from rabbit synovial MSCs to rabbit osteochondral defects exhibited accelerated osteoinduction. These composites may be the first example of a hybrid material that consists of ECM, HAp nanocrystals, and living MSCs, and the TEC-HAp composite could be a unique and useful material for bone tissue engineering.

Compressive properties of cartilage-like tissues repaired in vivo with scaffold-free, tissue engineered constructs

BACKGROUND It is crucial to develop an effective methodology for restoring adequate compressive properties to osteoarthritic cartilage. We have developed a scaffold-free tissue engineered construct cultured from synovium-derived mesenchymal stem cells. However, the compressive properties of cartilage-like tissues repaired with the construct have not been fully determined. METHODS Synovium-derived mesenchymal stem cells were cultured in Dulbeccos modified Eagles medium to produce the tissue engineered construct. Implantation of the construct into cylindrically-shaped partial defects in femoral cartilage in an experimental porcine model was performed. Six months after implantation, cartilage-like tissues repaired with the construct were subjected to static and cyclic compression tests using a micro-unconfined compression test apparatus developed in our laboratory. FINDINGS The developed apparatus was validated in preliminary examinations. The repaired tissues exhibited rate-dependent viscoelastic properties; the compressive modulus was slightly lower than that of normal cartilage at a rate of 4 microm/s, while no difference was observed at a rate of 100 microm/s. In contrast, the repaired tissue without the construct exhibited rate-independent, non-viscoelastic properties. In the cyclic compression test, however, the compressive strain was significantly larger in both repaired tissues as compared with normal cartilage. INTERPRETATION Although the quasi-static compressive properties of the repaired tissue with the construct, indicating rate-dependent and viscoelastic behaviors, are comparable to normal cartilage, the cyclic compressive strain increases more rapidly than in normal cartilage. It is suggested that the differences between the tissues and normal cartilage are attributable to the increased permeability of the extracellular matrix.

Scaffold-Free Tissue Engineered Construct (TEC) Derived from Synovial Mesenchymal Stem Cells: Characterization and Demonstration of Efficacy to Cartilage Repair in a Large Animal Model

This chapter describes in vitro generation of a scaffold-free mesenchymal stem cell (MSC)-based tissue-engineered construct (TEC) to facilitate cartilage repair and regeneration. Synovial MSCs were cultured in monolayer at high density and were subsequently detached from the substratum. The cell/matrix complex spontaneously contracted to develop a basic TEC. Immunohistochemical analysis showed that the TEC was rich in collagen I and III, fibronectin, and vitronectin. The TEC exhibited stable adhesion to the surface of a porcine cartilage matrix in an explant culture system. The TEC cultured in chondrogenic media exhibited elevated expression of glycosaminoglycan and chondrogenic marker genes. Implantation of a TEC into chondral defects initiated repair with a chondrogenic-like tissue, as well as secure biological integration to the adjacent cartilage. Histologically, the repair tissue stained positively with Safranin O and for collagen II. Biomechanical evaluation revealed that repair tissue exhibited mechanical properties similar to those of normal porcine cartilage in static compression and friction tests. The TEC technology could be a unique and promising method for stem-cell based cartilage repair.