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Experimental Example 1
Compressive loading culture experiment of human synovium derived cells using MIGHTY (in vitro arthritis model).
Suspension of human derived synovium cells was mixed with atelocollagen and seeded to MIGHTY. After 3 days of culture, cyclic compressive loading of 40kPa with CLS (Fig1-1) was applied to the sponge for 1 hour and then elevation of expression levels was observed for arthritis related genes PGE2, IL-6 and IL-8. Under the same condition, addition of dexamethasone, works as anti-inflammatory drugs, gave suppressive effects to up-regulated protein expressions of PGE2, IL-6 and IL-8 in dose-dependent manner(Fig1-2). These results show usefulness of MIGHTY in an in vitro arthritis model(8)
(K. Shimomura and K. Nakata, Osaka University, Department of Health and Sport Sciences)
Experimental Example 2
Culture of fibroblast cells under mechanical stimuli using MIGHTY (in vitro glanulation tissue compression model).
Suspension of rat fibroblasts was mixed with atelocollagen and seeded to MIGHTY. The sponge was cultured under mechanical stimulus with compression load device uniquely prepared and increase of apoptosis was observed after 1 day from seeding in the group of stimulated (Fig2-1). Increases of gene expression levels are also observed for HSP90α (relating to process of protein repair or enhance of cell survival), Hyaluronic acid (HA; relating to load durability of extracellular matrix) and COX-2 (its expression level is enhanced by interaction of HA and CD44) (Fig2-2). Concentrations in culture supernatant of HSP90, HA and PGE2 (known as downstream molecule of COX-2) were found to be elevated by mechanical stimulus (Fig2-3) and the result suggested that these molecules can be biomarkers for delayed wound healing(7)
(G. Nakagami and H. Sanada, The University of Tokyo, Department of gerontological Nursing/Wound care Management)
Experimental Example 3
Transplantation of human umbilical cord perivascular cells (HUPVC) to rat calvarial defect using MIGHTY and observation of osteogenesis.
Bone mesenchymal stem cells (BMSC)derived from human bone marrow, conditioned medium for BMSC (BMSC-CM) , human umbilical cord perivascular cells (HUCPVC) and HUCPVC+BMSC-CM are added to MIGHTY and transplanted to the rat calvarial defect. In the result of bone mass determination among each group, both groups of HUCPVC (E) and HUCPVC+BMSC-CM (F) showed remarkable increase of bone mass amount compared to sham control (A) or CSM (Alone) group (B), although no significant difference was considered between them (Fig3-1) . After treatment of hematoxylin and eosin staining (H&E staining), generation of soft tissue was remarkably observed in group of CSM (Alone), while amount of neonatal bone in CSM was found be increased in groups of HUCPVC or HUCPVC+BMSC-CM (Fig3-2) . Furthermore, survival of transplanted HUCPVC and bone differentiation are observed from immunofluorescence staining results(2)
(S. Kajiyama, Tsurumi University, School of Dental Medicine)
Experimental Example 4
Induction of chondrocyte differentiation from dedifferentiated fat cells and adipose derived stem cells with MIGHTY.
Suspension of Adipose derived Stem Cells (ASC) from human buccal fat pad or Dedifferentiated Fat Cell (DAFT) was mixed with atelocollagen and seeded to MIGHTY. It was observed that cells were seeded equally to MIGHTY, not only the area of upper or lower area but also of the middle (Fig4-1). They were cultured for 3 weeks, expression levels of markers of chondrocyte such as aggrecan, collagen type 2 (col2) and Sox2 are found to be increased in the group of DAFT compared to these of group of ASC (Fig4-2). Production of functional extracellular matrix was observed from the result of Alcian Blue staining or immunostaining with anti aggrecan (3)
(A. Nishio and Y. Hashimoto, Osaka Dental University, Second Department of Oral and Maxillofacial Surgery and Department of Biomaterials)
Experimennat Example 5
Sustained released of rhBMP-9 with MIGHTY and observation of osteogenesis.
1µg of rhBMP-9 (L-BMP-9) or 5µg of rhBMP-9 (H-BMP-9) was added to MIGHY(CSM) and transplanted to the rat calvarial defect
. After 8 weeks from transplantation process, both of rhBMP-9 /CSM groups showed remarkable increase of bone mass amount compared to that of control or group of CSM (Alone). (Fig5-1).And in the evaluation of histology, both groups of MMP-9/CSM showed significant difference from control in defect closure rate while most of contents consist of connective tissues in the group of CSM (Alone)(1)
(T. Nakamura and K. Noguchi, Kagoshima University, Deapartment of Periodontology)