Bone regeneration utilizing a newly developed octacalcium phosphate/weakly denatured collagen scaffold.

INTRODUCTION: Autologous bone grafting or various bone-regenerating materials are used to treat bone defects caused by tumor resection and accident trauma. Octacalcium phosphate, a reasonable bone regenerative material, activates osteoblasts. We fabricated a composite material, octacalcium phosphate/weakly denatured collagen, as a new scaffold. We aimed to investigate the osteoregenerative effect of the octacalcium phosphate/weakly denatured collagen scaffold (compared with that of weakly denatured collagen) in skull defects in a canine model. METHODS: Atelocollagen was extracted from porcine skin via pepsin treatment. The weakly denatured collagen scaffold was fabricated with a freeze-dried and thermally crosslinked atelocollagen suspension at pH 7.4. Octacalcium phosphate was synthesized using Ca-acetate and NaH2PO4. Octacalcium phosphate particles (diameter, 199-298 µm) were mixed with a collagen matrix to fabricate an octacalcium phosphate/weakly denatured collagen scaffold. Bilateral defects (diameter, 10 mm; full-thickness) were induced in dog skulls, and the octacalcium phosphate/weakly denatured collagen and weakly denatured collagen scaffolds were implanted into the defects. RESULTS: Eight weeks after implantations, bone regeneration was evaluated via histopathological analysis. It revealed osteoblast infiltration and osteoregeneration in all defects treated with the octacalcium phosphate/weakly denatured collagen scaffold. The defects treated with weakly denatured collagen scaffold or without any scaffold mostly contained connective tissue, with no neo-osteogenesis. DISCUSSION: The novel octacalcium phosphate/weakly denatured collagen scaffold better promotes osteoregeneration than the weakly denatured collagen scaffold; this "in situ tissue engineering" approach is potentially clinically applicable for bone reconstruction.

Optimal dehydrothermal processing conditions to improve biocompatibility and durability of a weakly denatured collagen scaffold.

Collagen scaffolds are essential for tissue regeneration; however, preprocessing of these scaffolds is necessary because of their poor mechanical properties. The aim of this study was to determine the optimal condition for preparing a collagen scaffold with biocompatibility and durability. An atelocollagen fiber suspension was made and stored at -10°C in a container that could be cooled from the bottom to provide an orientation perpendicular to the collagen fiber and facilitate cell infiltration into the scaffold. After freeze-drying the frozen suspension, various collagen scaffolds were made by dehydrothermal (DHT) treatment under different conditions (processing temperature: 120-160°C for 0-28 h). Sections of the obtained materials were embedded under the back skin of rats, and the thickness and biocompatibility of the residual scaffold were evaluated after 2 weeks. The number of foreign body giant cells was counted to evaluate biocompatibility. Although the residual scaffold was thick, excessive DHT treatment caused a strong foreign body reaction. Weak DHT treatment resulted in a collagen scaffold with good biocompatibility but with reduced thickness. Overall, these results showed the restricted optimal conditions to make a collagen scaffold with good biocompatibility and ability to maintain sufficient space for tissue regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2301-2307, 2017.

Dominant trait linked to chromosome 1 in DBA/2 mice for the resistance to autoimmune gastritis appears in bone marrow cells.

Neonatal thymectomy (NTx) induces autoimmune gastritis (AIG) in BALB/c mice, a model for human type A chronic atrophic gastritis, but not in DBA/2 mice and rarely in CDF1 mice (a hybrid of BALB/c and DBA/2 mice). The aim of this study was to clarify the mechanisms of AIG-resistance in mice bearing the dominant trait of DBA/2. Linkage groups associated with, and cells related to AIG resistance were examined with CDF1-BALB/c backcrosses. Intracellular staining and flow-cytometric bead array for several cytokines were performed on NTx BALB/c mice and NTx DBA/2-chimeric BALB/c mice receiving DBA/2-bone marrow cells. In NTx BALB/c mice, IFN-γ-secreting CD4(+) T cells were increased, but not in NTx DBA/2 mice. Because Vß6(+) T cell-bearing mice of half of their backcrosses developed AIG, but the other half of Vß6(+) T cell-negative mice developed scarcely, resistance for AIG generation is associated with the presence of the Mls-1a locus on chromosome 1 in DBA/2 mice, which deletes Vß6(+) T cells. NTx DBA/2-chimera BALB/c mice showed dominant production of IL-10 and resistance for AIG, although the deletion of Vß6(+) T cells was found not to be a cause of AIG-resistance from Mls-1a locus segregation experiments. Although NTx DBA/2-chimeric BALB/c mice did not suffer from AIG, they brought immediate precursors of T cells for AIG. It is concluded that DBA/2 mice generate bone marrow-derived cells that produce anti-inflammatory cytokines to prevent the activation of AIG-T cells.

Direct generation of induced pluripotent stem cells from human nonmobilized blood.

The use of induced pluripotent stem cells (iPSCs) is an exciting frontier in the study and treatment of human diseases through the generation of specific cell types. Here we show the derivation of iPSCs from human nonmobilized peripheral blood (PB) and bone marrow (BM) mononuclear cells (MNCs) by retroviral transduction of OCT3/4, SOX2, KLF4, and c-MYC. The PB- and BM-derived iPSCs were quite similar to human embryonic stem cells with regard to morphology, expression of surface antigens and pluripotency-associated transcription factors, global gene expression profiles, and differentiation potential in vitro and in vivo. Infected PB and BM MNCs gave rise to iPSCs in the presence of several cytokines, although transduction efficiencies were not high. We found that 5 × 10(5) PB MNCs, which corresponds to less than 1 mL of PB, was enough for the generation of several iPSC colonies. Generation of iPSCs from MNCs of nonmobilized PB, with its relative efficiency and ease of harvesting, could enable the therapeutic use of patient-specific pluripotent stem cells.

Generation of induced pluripotent stem cells by efficient reprogramming of adult bone marrow cells.

Reprogramming of somatic cells provides potential for the generation of specific cell types, which could be a key step in the study and treatment of human diseases. In vitro reprogramming of somatic cells into a pluripotent embryonic stem (ES) cell-like state has been reported by retroviral transduction of murine fibroblasts using four embryonic transcription factors or through cell fusion of somatic and pluripotent stem cells. Here we show that mouse adult bone marrow mononuclear cells (BM MNCs) are competent as donor cells and can be reprogrammed into pluripotent ES cell-like cells. We isolated BM MNCs and mouse embryonic fibroblasts (MEFs) from Oct4-GFP transgenic mice, fused them with ES cells, or infected them with retroviruses expressing Oct4, Sox2, Klf4, and c-Myc. Fused BM MNCs formed more ES-like colonies than did MEFs. Infected BM MNCs gave rise to induced pluripotent stem (iPS) cells, although transduction efficiencies were not high. It was more efficient to pick up iPS colonies as compared with MEFs. BM-derived iPS (BM iPS) cells expressed ES cell markers, formed teratomas, and contributed to chimera mice with germ line development. Clonal analysis revealed that BM iPS clones had diversity, although some clones were found to be genetically identical with different phenotypes. Our findings imply that BM MNCs have potential advantages to generate iPS cells for the clinical application.