Shinichi Tsutsumi

Evaluation of posterolateral spinal fusion using mesenchymal stem cells : Differences with or without osteogenic differentiation

Study Design. An animal study to achieve posterolateral intertransverse process spine fusion using mesenchymal stem cell (MSC). Objective. We investigated the effectiveness of graft material for spinal fusion using a rabbit model by examining the MSC with or without osteogenic differentiation. Summary of Background Data. Posterolateral spinal fusion is commonly performed. Autogenous bone graft is the gold standard, although various problems are reported. Recently, MSCs from bone marrow have been studied in various fields. Thus, we supposed that MSCs have the ability to spinal fusion. Methods. Twenty-four mature male Japanese white rabbits (weight, 3.0–4.0 kg) were divided into 4 groups: 1) autologous bone (AG), 2) hydroxyapatite (HA), 3) MSC, and 4) osteogenic MSC (OMSC). Each group underwent fusion of the intertransverse processes. The lumbar spine was harvested en bloc, and the fusion mass was evaluated radiographically, by manual palpation test, and by histologic analysis at 6 weeks after surgery. Results. Fusion success or failure was assumed based on the results from manual palpation of the harvested spine. Four of 5 rabbits in the OMSC group, 4 of 6 rabbits in the AB group, 2 of 6 rabbits in the MSC group, and none of 6 rabbits in the HA group achieved fusion. In the OMSC group and AG group, new bone formation was observed histologically. In the HA group, fibrous tissue and cartilage were observed and there was no new bone. In the MSC group, less mature bone formation was present in the grafted fragments. Conclusion. The present study suggested that MSCs that have been cultured with osteogenic differentiation medium may induce the formation of new bone in experimental spinal fusion. Further studies are needed to determine the suitable level of osteogenic differentiate of MSC as well as the most appropriate carrier for MSC.

Rhythmic expression of DEC1 and DEC2 in peripheral tissues: DEC2 is a potent suppressor for hepatic cytochrome P450s opposing DBP

The mammalian master molecular clock consisting of several clock gene products in the suprachiasmatic nucleus (SCN) drives circadian rhythms in behaviour and physiology. Molecular clocks consisting of the same components also exist in various peripheral organs. DEC1 and DEC2, basic helix-loop-helix transcription factors, were recently reported to be involved in the central clock in the SCN. We examined the expression profile of DEC1 and DEC2 in the periphery and their roles in the regulation of oscillating target genes in the liver. Levels of DEC1 and DEC2 mRNA exhibited a day-night variation in various peripheral tissues of rats. In the liver, their expression was high during the subjective night. Transfection assays showed that DEC2, but not DEC1, suppressed the transcription of the cholesterol 7alpha-hydroxylase gene (CYP7A), overwhelming the potent enhancement by D-site binding protein (DBP). Electrophoretic mobility shift assays indicated that DEC2 binds to the E-box (CACATG) at the -219/-214 region of CYP7A. The transcriptional activities of the other sterol metabolizing cytochrome P450s (Cyps), CYP8B and CYP51, were also suppressed by DEC2 but not DEC1. DEC2, but not DEC1, works as a direct output mediator that transmits the circadian signals to the hepatic functions, including the CYP7A, CYP8B, and CYP51 expression.

Transplantation of autologous rabbit BM-derived mesenchymal stromal cells embedded in hyaluronic acid gel sponge into osteochondral defects of the knee

BACKGROUND Mesenchymal stromal cells (MSC) have the potential to differentiate into distinct mesenchymal tissues including cartilage, suggesting that these cells are an attractive cell source for cartilage tissue engineering approaches. Various methods, such as using hyaluronan-based materials, have been employed to improve transplantation for repair. Our objective was to study the effects of autologous transplantation of rabbit MSC with hyaluronic acid gel sponges into full-thickness osteochondral defects of the knee. METHODS Rabbit BM-derived MSC were cultured and expanded with fibroblast growth factor (FGF). Specimens were harvested at 4 and 12 weeks after implantation, examined histologically for morphologic features, and stained immunohistochemically for type II collagen and CD44. RESULTS The regenerated area after autologous transplantation of hyaluronic acid gel sponge loaded with MSC into the osteochondral defect at 12 weeks after surgery showed well-repaired cartilage tissue, resembling the articular cartilage of the surrounding structure, of which the histologic score was significantly better than that of the untreated osteochondral defect. In the regenerated cartilage, type II collagen was found in the pericellular matrix of regenerative chondrocytes, while CD44 expression in the regenerative tissue could not be revealed. DISCUSSION These data suggest that the autologous transplantation of MSC embedded in hyaluronan-based material may support chondrogenic differentiation and be useful for osteochondral defect repair.

RGD-CAP (βig-h3) is expressed in precartilage condensation and in prehypertrophic chondrocytes during cartilage development

RGD-CAP ((beta)ig-h3), isolated from cartilage as a collagen-associated protein, was demonstrated to have a binding ability to collagen and to enhance the adhesion of chondrocytes via integrin alpha(1)beta(1). However, the role of this protein in cartilage development remains unclear. In this study, we investigated the expression of RGD-CAP ((beta)ig-h3) in chick embryos and cultured mesenchymal stem cells (MSCs) during the differentiation to chondrocytes. The effects of recombinant RGD-CAP on adhesion and DNA synthesis of MSCs and mineralization were also examined. Tissue sections from chick embryos at Hamburger-Hamilton (HH) stages 19-37 were immunostained with anti-chick RGD-CAP antibodies. The expression of RGD-CAP was slightest in chick embryos at HH stage 19, whereas a considerable expression of RGD-CAP was observed in the developing vertebrae and precartilage aggregate in the limb bud of chick embryos at HH stage 26. The expression of RGD-CAP was significantly reduced in vertebrae of chick embryo at HH stage 32. Reverse transcriptional polymerase chain reaction (RT-PCR) analysis showed that RGD-CAP was highly expressed in cultured MSCs and decreased by 4-day treatment with 10(-8) M dexamethasone when MSCs proliferated to adipocyte-like cells, whereas it was recovered by co-treatment with 3 ng/ml TGF-beta for 8-12 days when MSCs proliferated to hypertrophic chondrocyte-like cells. The adhesion and DNA synthesis of MSCs cultured on RGD-CAP-coated dishes increased significantly compared with the controls. RGD-CAP was distributed in the prehypertrophic zone in matured cartilage of the vertebrae of chick embryos at HH stage 37. Recombinant RGD-CAP inhibited the mineralization of hypertrophic chondrocytes. These results suggest that RGD-CAP ((beta)ig-h3) exerts an essential role in the early cartilage development by enhancing the adhesion and growth of the pre-chondrogenic cells, and functions as a negative regulator for mineralization at the terminal stage of the chondrogenic differentiation.