A Novel Bone Substitute Based on Recombinant Type I Collagen for Reconstruction of Alveolar Cleft.

This study aimed to examine the optimal cross-link density of recombinant peptide (RCP) particles, based on human collagen type I, for bone reconstruction in human alveolar cleft. Low- (group 1), medium- (group 2), and high- (group 3) cross-linked RCP particles were prepared by altering the duration of the heat-dependent dehydration reaction. Rat palatine fissures (n = 45), analogous to human congenital bone defects, were examined to evaluate the potential of bone formation by the three different RCP particles. Microcomputed tomography images were obtained to measure bone volume and bone mineral density at 4, 8, 12, and 16 weeks post grafting. Specimens were obtained for histological analysis at 16 weeks after grafting. Additionally, alkaline phosphatase and tartrate acid phosphatase staining were performed to visualize the presence of osteoblasts and osteoclasts. At 16 weeks, bone volume, bone mineral density, and new bone area measurements in group 2 were significantly higher than in any other group. In addition, the number of osteoblasts and osteoclasts on the new bone surface in group 2 was significantly higher than in any other group. Our results demonstrated that medium cross-linking was more suitable for bone formation-and could be useful in human alveolar cleft repairs as well.

Bone formation potential of collagen type I-based recombinant peptide particles in rat calvaria defects.

INTRODUCTION: This study aimed to examine the bone-forming ability of medium-cross-linked recombinant collagen peptide (mRCP) particles developedbased on human collagen type I, contains an arginyl-glycyl-aspartic acid-rich motif, fabricated as bone filling material, compared to that of the autologous bone graft. METHODS: Calvarial bone defects were created in immunodeficient rats though a surgical procedure. The rats were divided into 2 groups: mRCP graft and tibia bone graft (bone graft). The bone formation potential of mRCP was evaluated by micro-computed tomography and hematoxylin-eosin staining at 1, 2, 3, and 4 weeks after surgery, and the data were analyzed and compared to those of the bone graft. RESULTS: The axial volume-rendered images demonstrated considerable bony bridging with the mRCP graft, but there was no significant difference in the bone volume and bone mineral density between the mRCP graft and bone graft at 4 weeks. The peripheral new bone density was significantly higher than the central new bone density and the bottom side score was significantly higher than the top side score at early stage in the regenerated bone within the bone defects. CONCLUSION: These results indicate that mRCP has a high potential of recruiting osteogenic cells, comparable to that of autologous bone chips.

Extemporaneous Preparation of Injectable and Enzymatically Degradable 3D Cell Culture Matrices from an Animal-Component-Free Recombinant Protein Based on Human Collagen Type I.

Injectable hydrogels are considered important to realize safe and effective minimally invasive therapy. Although animal-derived natural polymers are well studied, they typically lack injectability and fail to eliminate the potential risks of immunogenic reactions or unknown pathogen contamination. Despite extensive research activities to explore ideal injectable hydrogels, such state-of-the-art technology remains inaccessible to non-specialists. In this article, the design of a new injectable hydrogel platform that can be extemporaneously prepared from commercially available animal-component-free materials is described. The hydrogels can be prepared simply by mixing mutually reactive aqueous solutions without necessitating specialized knowledge or equipment. Their solidification time can be adjusted by choosing proper buffer conditions from immediate to an extended period of time, that is, few or several tens of minutes depending on the concentration of polymeric components, which not only provides injectability, but enables 3D encapsulation of cells. Mesenchymal stromal/stem cells can be encapsulated and cultured in the hydrogels at least for 2 weeks by traditional cell culture techniques, and retrieved by collagenase digestion with cell viability of approximately 80%. This hydrogel platform accelerates future cell-related research activities.

An on-site preparable, novel bone-grafting complex consisting of human platelet-rich fibrin and porous particles made of a recombinant collagen-like protein.

Platelet-rich fibrin (PRF) is widely used in regenerative medicine. Nonetheless, major issues include its controversial effects on bone regeneration and a lack of quality-assured glass tubes required for coagulation. We used porous particles (FBG) comprising a recombinant RGD motif-enriched collagen I-like protein to activate the coagulation pathway and examined the effects of the resulting PRF-FBG complex on bone regeneration. Human whole-blood samples were mixed with FBG in plastic tubes and centrifuged to prepare a PRF-FBG complex. Platelet-derived growth factor-BB (PDGF-BB) levels and cell growth activity were determined by ELISA and a bioassay using osteoblasts. Bone regenerative activity was assessed using a mouse model of calvarial bone defect. FBG facilitated PRF-like matrix formation during centrifugation. In this PRF-FBG complex, the microstructure of fibrin fibers was similar to that of PRF prepared conventionally in glass tubes. PDGF-BB levels and mitogenic action were not significantly influenced by FBG. In the bone defect model, although PRF did not exert any significant positive effects on its own, in combination with FBG, it synergistically stimulated new bone formation. This study demonstrated that incorporation of FBG into whole-blood samples induces PRF formation without the aid of glass tubes. The resulting PRF-FBG complex could be a promising bone grafting material in clinical settings. © 2018 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1420-1430, 2019.

Human immunodeficiency virus type 1 Vpr induces cell cycle arrest at the G(1) phase and apoptosis via disruption of mitochondrial function in rodent cells.

Vpr of human immunodeficiency virus type 1 causes cell cycle arrest at the G(2)/M phase and induces apoptosis after G(2)/M arrest in primate cells. We have reported previously that Vpr also induces apoptosis independently of G(2)/M arrest in human HeLa cells. By contrast, Vpr does not induce G(2)/M arrest in rodent cells, but it retards cell growth. To clarify the relationship between cell cycle arrest and apoptosis, we expressed Vpr endogenously in rodent cells and investigated cell cycle profiles and apoptosis. We show here that Vpr induces cell cycle arrest at the G(1) phase and apoptosis in rodent cells. Vpr increased the activity of caspase-3 and caspase-9, but not of caspase-8. Moreover, Vpr-induced apoptosis could be inhibited by inhibitors of caspase-3 and caspase-9, but not by inhibitor of caspase-8. We also showed that Vpr induces the release of cytochrome c from mitochondria into the cytosol and disrupts the mitochondrial transmembrane potential. Finally, we showed that apoptosis occurred in HeLa cells through an identical pathway. These results suggest that disruption of mitochondrial functions by Vpr induces apoptosis via cell cycle arrest at G(1), but that apoptosis is independent of G(2)/M arrest. Furthermore, it appears that Vpr acts species-specifically with respect to induction of cell cycle arrest but not of apoptosis.

A novel role for Vpr of human immunodeficiency virus type 1 as a regulator of the splicing of cellular pre-mRNA.

Vpr, one of the accessory gene products of human immunodeficiency virus type 1 (HIV-1), affects aspects of both viral and cellular proliferation, being involved in long terminal repeat (LTR) activation, arrest of the cell cycle at the G2 phase, and apoptosis. We have discovered a novel role for Vpr as a regulator of the splicing of pre-mRNA both in vivo and in vitro. We found, by RT-PCR and RNase protection analysis, that Vpr caused the accumulation of incompletely spliced forms of alpha-globin 2 and beta-globin pre-mRNAs in cells that had been transiently transfected with a Vpr expression vector. We postulated that this novel effect of Vpr might occur via a pathway that is distinct from arrest of the cell cycle at G2. By analyzing splicing reactions in vitro, we showed that Vpr inhibited the splicing of beta-globin pre-mRNA in vitro. The splicing of intron 1 of alpha-globin 2 pre-mRNA was modestly inhibited by Vpr but the splicing of intron 2 was unaffected. Interestingly, an experimental infection system which utilizes high-titered HIV-1/vesticular stomatitis virus G protein showed that Vpr expressed from an HIV-1 provirus was sufficient to accumulate endogenous alpha-globin 2 pre-mRNA. Thus, it is likely that Vpr contributes to selective inhibition of the splicing of cellular pre-mRNA.