Osteogenic Differentiation of Human Mesenchymal Stem Cells in Mineralized Alginate Matrices

<div><p>Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (<i>RUNX2</i>, <i>COL1AI</i>, <i>SP7</i>, <i>BGLAP</i>) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (<i>SOST</i>) and dental matrix protein-1 (<i>DMP1</i>), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.</p></div

SEM micrographs of collagen fibrils in beads cultured in osteogenic medium.

<p>(A, C) low and high magnification of the space close to a cell producing collagen in ALP modified beads (ALP+). Mineral crystals similar to those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120374#pone.0120374.g002" target="_blank">Fig. 2</a> are indicated by *; (B, D) low and high magnification micrographs of the space close to a cell producing collagen in unmineralized beads. Images were collected at 21 days post encapsulation.</p

SEM micrographs of the alginate network in ALP-modified (A, C, E) or unmodified (B, D, F) beads taken at day 21 post encapsulation.

<p>Cells were cultured in either growth medium (A, B) or osteogenic medium (C-F). Mineral particles with spherical morphology are clearly visible for heavily mineralized samples shown at high and low magnification in panel C and E.</p

Mineralization of alginate beads.

<p>Light microscopy of unmodified (A) or ALP-modified (B) beads taken at day 2 and day 21 post encapsulation. Cells were cultured in either growth medium (GM) or osteogenic medium (OM). Mineralized beads appear dark whereas unmineralized beads appear transparent. Scale bar 500μm.</p

Viability of hMSCs in alginate beads cultured in osteogenic medium.

<p><b>(A)</b> Live/dead fluorescent staining of cells cultured in osteogenic medium was visualized using confocal microscopy (LSM 510 META FCS, Zeiss). Left images: Confocal cross sections through overlaid transmitted light of hMSCs in alginate beads; Right images: three dimensional reconstructions of cross sections through the beads. Live cells appear green, dead cells appear red. Scale bar 500 μm. <b>(B)</b> Metabolic activity of MSC in alginate beads cultured in growth medium (GM) or osteogenic medium (OM) at day 2, 7, 14 and 21 post encapsulation measured by Alamar Blue assay. Metabolic activity was significantly different in ALP modified beads cultured in OM compared with unmodified beads cultured in OM at day 14, p <0.001, day 17, p< = 0.01 and day 21, p< = 0.001, Sidak’s multiple comparison test. The difference in metabolic activity between unmodified and ALP-modified beads cultured in GM was not statistically significant. The data presented are mean values +/- SD, n = 3. ALP: Containing 0.25mg/mL ALP.</p

Alkaline phosphatase activity in cells cultured in ALP-modified or unmodified beads for 9 days as indicated.

<p>p values indicate statistical significant differences by Sidak’s multiple comparison test.</p

Relative mRNA expression of osteoblast/ osteocyte markers.

<p>MSCs were cultured in unmodified (no ALP), ALP-modified (ALP) alginate beads or on traditional culture plates (2D). Samples were cultured in either growth medium (GM) or osteogenic medium (OM) for 21 days post encapsulation. RUNX2 (A), Osterix (SP7) (B), COL1A1 (C), and sclerostin (SOST) (E) mRNA expression are relative to mRNA expression in cells cultured on traditional culture plates in osteogenic medium for 7 days. Osteocalcin (BGLAP) (D) and DMP1 (F) mRNA expressions are relative to mRNA expression in cells in unmodified alginate beads cultured in growth medium for 7 days post encapsulation. ND = not detected.</p

Additional file 1: of Chemerin is elevated in multiple myeloma patients and is expressed by stromal cells and pre-adipocytes

Figure S1. Expression of CCRL2 and CMKLR1 in primary myeloma cells (pMM, n = 24) and cell lines (n = 9) analyzed by qPCR. GAPDH was used as an endogenous control. (DOCX 51 kb