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Grigolo B.,Science Laboratorio Of Immunoreumatologia E Rigenerazione Tissutale
Journal of biological regulators and homeostatic agents | Year: 2011

The requirements for a successful regeneration of an osteo-chondral defect could effectively be met by using a bi-layered composite scaffold, able to support proliferation and differentiation of mesenchymal stem cells, while providing a biochemical environment promoting the formations of the two distinct tissues. The novel strategy here presented consists of developing a bio-mimetic scaffolds obtained by the combination of two integrated organic compounds (type I collagen and chitosan) with or without bioactive Mg-doped hydroxyapatite (Mg-HA) nanocrystals, depending on the specific layer, reproducing cartilaginous or subchondral bone tissue. An innovative patented methodology for scaffolds production, called - pH-dependent 3-phasic assembling -, allowed to development of a highly homogenous and chemically stable scaffold, presenting a very good integration among all three components, as confirmed by extensive SEM and thermogravimetric analyses. A preliminary in vitro evaluation was also carried out by seeding bi-layered scaffold with human bone marrow stromal cells (h-MSCs), by giving particular emphasis to cell viability and distribution at day 0, 7 and 14. Cells were viable and uniformly colonized the whole scaffold until day 14, indicating that the scaffold contributed to the maintenance of cell behaviour. Source

Grigolo B.,Science Laboratorio Of Immunoreumatologia E Rigenerazione Tissutale
Journal of biological regulators and homeostatic agents | Year: 2011

Association of biomaterials with autologous cells can provide a new generation of implantable devices for cartilage repair. An ideal scaffold should possess a preformed three-dimensional shape, fix the cells to the damaged area and prevent their migration into the articular cavity. Furthermore, the constructs should have sufficient mechanical strength to facilitate handling in a clinical setting and stimulate the uniform spreading of cells and a phenotype re-differentiation process. The aim of this study was to verify the ability of an equine collagen membrane to support the growth of human chondrocytes and to allow the re-expression of their original phenotype. This ability was assessed by the evaluation of collagen type I, II and aggrecan mRNA expression by Real-Time PCR. Immunohistochemical analyses were performed to evaluate collagen type I, II and proteoglycans synthesis. Electron microscopy was utilized to highlight the structure of the biomaterial and its interactions with the cells. Our data indicate that human chondrocytes seeded onto a collagen membrane express and produce collagen type II and aggrecan and downregulate the production of collagen type I during the experimental times analyzed. These results provide an in vitro demonstration for the therapeutic potential of autologous chondrocyte transplantation by an equine collagen membrane as a delivery vehicle in a tissue-engineered approach towards the repair of articular cartilage defects. Source

Manferdini C.,Science Laboratorio Of Immunoreumatologia E Rigenerazione Tissutale | Guarino V.,CNR Institute of Composite and Biomedical Materials | Zini N.,CNR Institute of Molecular Genetics | Raucci M.G.,CNR Institute of Composite and Biomedical Materials | And 8 more authors.
Biomaterials | Year: 2010

A biomimetic hyaluronic acid (HA)-based polymer scaffold was analysed in vitro for its characteristics and potential to support mineralization as carrier-vehicle. Biomimetic apatite crystal nucleation on the scaffold surface was obtained by a fine control of the pH level that increased ionic solubility thus controlling apatite formation kinetic. Different concentrations of human mesenchymal stromal cells (h-MSCs) were seeded on the scaffold, osteogenesis was induced in the presence or absence of fibroblast growth factor -2 and mineralization was analysed at different time points. We found that only at the highest h-MSCs concentration tested, the cells were uniformly distributed inside and outside the scaffold and proliferation started to decrease from day 7. Electron microscopy analysis evidenced that h-MSCs produced extracellular matrix but did not establish a direct contact with the scaffold. We found mineralized calcium-positive areas mainly present along the backbone of the scaffold starting from day 21 and increasing at day 35. FGF-2 treatment did not accelerate or increase mineralization. Non-biomimetic HA-based control scaffold showed immature mineralized areas only at day 35. Our data demonstrate that the biomimetic treatment of an HA-based scaffold promotes a faster mineralization process suggesting its possible use in clinics as a support for improving bone repair. © 2010 Elsevier Ltd. All rights reserved. Source

Manferdini C.,Science Laboratorio Of Immunoreumatologia E Rigenerazione Tissutale | Gabusi E.,Science Laboratorio Of Immunoreumatologia E Rigenerazione Tissutale | Grassi F.,Science Laboratorio Of Immunoreumatologia E Rigenerazione Tissutale | Piacentini A.,Science Laboratorio Of Immunoreumatologia E Rigenerazione Tissutale | And 6 more authors.
Journal of Cellular Physiology | Year: 2011

Human bone cells used for in vitro studies are mainly derived from bone marrow (BM) or trabecular bone (TB). There are no specific markers or procedures for isolation and growth of these cells. To validate the potentiality of these cells, we isolated human mesenchymal stromal cells (MSCs) and osteoblasts (OBs) from the tibial plateau of the same subject, grown in two different media (α-MEM and DMEM/F12) and analyzed for cell growth, proliferation, phenotype and osteogenic potential. We found that OBs grew well in both media tested, but MSCs were able to grow only in α-MEM medium. OBs in DMEM/F12 showed reduced proliferation capability and expressed a low level of alkaline phosphatase (AP), RUNX-2, osteocalcin (OC), bone sialoprotein (BSP), collagen type I (Col.I) compared with OBs in α-MEM but high level of collagen type XV (Col.XV). Compared with MSCs in α-MEM, OBs have an increased ability to proliferate and express more OC and BSP at molecular level but less AP, RUNX-2 and Col.I than MSCs. Time-course experiments to analyze the osteogenic potential of these cells showed that OBs were more efficient than MSCs. However, these cells obtained from tibial plateau showed a different trend of AP, OC and Col.I osteogenic markers compared to control MSCs from the iliac crest. This study shows that bone-adherent OBs grown in α-MEM medium are more efficient for osteogenic differentiation than BM MSCs and contribute to defining their phenotypic and functional characteristics, so providing a rationale for their use in bone tissue engineering or therapeutic purposes. © 2011 Wiley-Liss, Inc. Source

Gabusi E.,Laboratorio RAMSES | Gabusi E.,Science Laboratorio Of Immunoreumatologia E Rigenerazione Tissutale | Manferdini C.,Laboratorio RAMSES | Manferdini C.,Science Laboratorio Of Immunoreumatologia E Rigenerazione Tissutale | And 15 more authors.
Journal of Cellular Physiology | Year: 2012

Fluctuation in extracellular calcium (Ca2+) concentration occurs during bone remodeling. Free ionized Ca2+ plays a critical role in regulating osteoblast functions. We analyzed the effects of different concentrations of free ionized Ca2+ (0.5, 1.3, and 2.6mM) on human osteoblasts and we evaluated osteoblastic phenotype (marker expression and cell morphology) and functions (osteogenic differentiation, cell proliferation, and cell signaling). Our data show human osteoblasts that chronically stimulated with 0.5, 1.3, or 2.6mM Ca2+ significantly increase intracellular content of alkaline phosphatase, collagen type I, osteocalcin, and bone sialoprotein, whereas collagen type XV was down-modulated and RUNX2 expression was not affected. We also found a Ca2+ concentration-dependent increase in osteogenic differentiation and cell proliferation, associated to an increase of signaling protein PLCβ1 and p-ERK. Human osteoblast morphology was affected by Ca2+ as seen by the presence of numerous nucleoli, cells in mitosis, cell junctions, and an increased number of vacuoles. In conclusion, our data show a clear phenotypical and functional effect of extracellular Ca2+ on human osteoblasts and support the hypothesis of a direct role of this cation in the bone remodeling processes. © 2011 Wiley Periodicals, Inc. Source

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