Mesenchymal stem cells (MSCs) seeded on three-dimensional (3D) coralline (Porites lutea) biomatrices were irradiated with low-level laser irradiation (LLLI). The consequent phenotype modulation and development of MSCs towards ossified tissue was studied in this combined 3D biomatrix/LLLI system and in a control group, which was similarly grown, but was not treated by LLLI. The irradiated and non irradiated MSC were tested at 1-7, 10, 14, 21, 28 days of culturing via analysis of cellular distribution on matrices (trypan blue), calcium incorporation to newly formed tissue (alizarin red), bone nodule formation (von Kossa), fat aggregates formation (oil red O), alkaline phosphatase (ALP) activity, scanning electron microscopy (SEM) and electron dispersive spectrometry (EDS). The results obtained from the irradiated samples showed enhanced tissue formation, appearance of phosphorous peaks and calcium and phosphate incorporation to newly formed tissue. Moreover, in irradiated samples ALP activity was significantly enhanced in early stages and notably reduced in late stages of culturing. These findings of cell and tissue parameters up to 28 days of culture revealed higher ossification levels in irradiated samples compared with the control group. We suggest that both the surface properties of the 3D crystalline biomatrices and the LLLI have biostimulatory effects on the conversion of MSCs into bone-forming cells and on the induction of ex-vivo ossification.

This study introduces a novel three-dimensional biomatrix obtained from the marine hydrocoral Millepora dichotoma as a scaffold for hard tissue engineering. Millepora dichotoma was biofabricated under field and laboratory conditions. Three-dimensional biomatrices were made in order to convert mesenchymal stem cells (MSCs) to exemplify osteoblastic phenotype. We investigated the effect of the biomatrices on MSCs proliferation and differentiation at 2, 3, 4, 7, 10, 14, 21, 28, and 42 days. Different analyses were made: light microscopy, scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS), calcium incorporation to newly formed tissue (alizarin red), bone nodule formation (von Kossa), fat aggregate formation (oil red O), collagen type I immunofluorescence, DNA concentrations, alkaline phosphatase (ALP) activity, and osteocalcin concentrations. MSCs seeded on Millepora dichotoma biomatrices showed higher levels of calcium and phosphate incorporation and higher type I collagen levels than did control Porites lutea biomatrices. ALP activity revealed that MSCs seeded on M. dichotoma biomatrices are highly osteogenic compared to those on control biomatrices. The osteocalcin content of MSCs seeded on M. dichotoma remained constant up to 2 weeks before rising to surpass that of seeded P. lutea biomatrices after 28 days. Our study thus showed that M. dichotoma biomatrices enhance the differentiation of MSCs into osteoblast and hence have excellent potential as bioscaffold for hard tissue engineering.

Adipogenic and osteogenic cells share part of the early differentiation cascade of mesenchymal stem cells (MSCs). The choice of a mesenchymal precursor cell to differentiate into a particular cell type is dictated by many spatial and temporal cues, including growth factors, neighboring mature cells, and the extracellular matrix (ECM), which plays an important role in bone formation. Whether adipocytes that have initiated differentiation along one lineage can convert into osteogenic lineage by merely interacting with materials having specific surface parameters is unknown. Using crystalline three-dimensional (3D) biomatrices of marine origin (CaCO(3)), we explored whether preadipocytes can convert into osteoblasts. Cells (3T3F442A) were seeded on 3D biomatrices of marine origin (Porites lutea). Analyses were made at different time intervals-1, 2, 5, 7, 14, 21, and 28 days post-seeding. Cell characterizations were done using morphological (light microscopy and scanning electron microscopy), histological (Alizarin red, von Kossa and Oil red O staining), enzymatic (alkaline phosphatase activity, and quantitative PCR testing transcript levels of osteocalcin, alkaline phosphatase, core binding factor- 1 (Cbfa1), and fatty acid binding protein (aP2). We demonstrated 3T3F442A preadipocyte modulation and differentiation into bone-forming cells when grown on biomatrix of marine origin without addition of other bone morphogenesis inducers. We found an active ossification process typical of osteogenic phenotype as early as 2 days after seeding. It is suggested that this crystalline biomatrix having a particular 3D topology or surface parameters supports fast cellular adhesion, proliferation, and differentiation of preadipocytes to osteogenic phenotype.

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