Osteogenic differentiation of human mesenchymal stem cells in mineralized alginate matrices.

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 (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), 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.

Alterations in mucus barrier function and matrix structure induced by guluronate oligomers.

The effect of guluronate oligomers on the barrier properties of mucous matrices was investigated in terms of the mobility of nanoparticles in mucous matrices by fluorescence recovery after photobleaching (FRAP), cellular uptake of nanoparticles in mucus secreting cells (HT29-MTX), and mucin matrix architecture by scanning electron microscopy (SEM). Guluronate oligomers improved nanoparticle mobility in both native and highly purified mucus matrices and improved cellular uptake of nanoparticles through a mucus layer. Addition of guluronate oligomers to mucin matrices resulted in a decrease in the density of network cross-links and an increase in matrix pore size. Based on these data, we conclude that guluronate oligomers are able to improve nanoparticle mobility in several mucus matrices and alter network architecture in mucin matrices in a manner that suggests a reduction in barrier function. As such, there may be a potential application for guluronate oligomers in mucosal delivery of nanomedicines.

Biochemical and structural characterization of neocartilage formed by mesenchymal stem cells in alginate hydrogels.

A popular approach to make neocartilage in vitro is to immobilize cells with chondrogenic potential in hydrogels. However, functional cartilage cannot be obtained by control of cells only, as function of cartilage is largely dictated by architecture of extracellular matrix (ECM). Therefore, characterization of the cells, coupled with structural and biochemical characterization of ECM, is essential in understanding neocartilage assembly to create functional implants in vitro. We focused on mesenchymal stem cells (MSC) immobilized in alginate hydrogels, and used immunohistochemistry (IHC) and gene expression analysis combined with advanced microscopy techniques to describe properties of cells and distribution and organization of the forming ECM. In particular, we used second harmonic generation (SHG) microscopy and focused ion beam/scanning electron microscopy (FIB/SEM) to study distribution and assembly of collagen. Samples with low cell seeding density (1e7 MSC/ml) showed type II collagen molecules distributed evenly through the hydrogel. However, SHG microscopy clearly indicated only pericellular localization of assembled fibrils. Their distribution was improved in hydrogels seeded with 5e7 MSC/ml. In those samples, FIB/SEM with nm resolution was used to visualize distribution of collagen fibrils in a three dimensional network extending from the pericellular region into the ECM. In addition, distribution of enzymes involved in procollagen processing were investigated in the alginate hydrogel by IHC. It was discovered that, at high cell seeding density, procollagen processing and fibril assembly was also occurring far away from the cell surface, indicating sufficient transport of procollagen and enzymes in the intercellular space. At lower cell seeding density, the concentration of enzymes involved in procollagen processing was presumably too low. FIB/SEM and SHG microscopy combined with IHC localization of specific proteins were shown to provide meaningful insight into ECM assembly of neocartilage, which will lead to better understanding of cartilage formation and development of new tissue engineering strategies.

Viscoelastic properties of mineralized alginate hydrogel beads.

Alginate hydrogels have applications in biomedicine, ranging from delivery of cells and growth factors to wound management aids. However, they are mechanically soft and have shown little potential for the use in bone tissue engineering. Here, the viscoelastic properties of alginate hydrogel beads mineralized with calcium phosphate, both by a counter-diffusion (CD) and an enzymatic approach, are characterized by a micro-manipulation technique and mathematical modeling. Fabricated hydrogel materials have low mineral content (below 3 % of the total hydrogel mass, which corresponds to mineral content of up to 60 % of the dry mass) and low dry mass content (<5 %). For all samples compression and hold (relaxation after compression) data was collected and analyzed. The apparent Young's modulus of the mineralized beads was estimated by the Hertz model (compression data) and was shown to increase up to threefold upon mineralization. The enzymatically mineralized beads showed higher apparent Young's modulus compared to the ones mineralized by CD, even though the mineral content of the former was lower. Full compression-relaxation force-time profiles were analyzed using viscoelastic model. From this analysis, infinite and instantaneous Young's moduli were determined. Similarly, enzymatic mineralized beads, showed higher instantaneous and infinite Young's modulus, even if the degree of mineralization is lower then that achieved for CD method. This leads to the conclusion that both the degree of mineralization and the spatial distribution of mineral are important for the mechanical performance of the composite beads, which is in analogy to highly structured mineralized tissues found in many organisms.

Alginate-controlled formation of nanoscale calcium carbonate and hydroxyapatite mineral phase within hydrogel networks.

A one-step method was used to make nanostructured composites from alginate and calcium carbonate or calcium phosphate. Nanometer-scale mineral phase was successfully formed within the gel network of alginate gel beads, and the composites were characterized. It was found that calcite was the dominating polymorph in the calcium carbonate mineralized beads, while stoichiometric hydroxyapatite was formed in the calcium phosphate mineralized beads. A combination of electron microscopy, Fourier-transform infrared spectroscopy, thermogravimetric analysis and powder X-ray diffraction showed that alginate played an active role in controlling mineral size, morphology and polymorphy. For the calcium phosphate mineralized beads, alginate was shown to modulate stoichiometric hydroxyapatite with low crystallinity at room temperature, which may have important applications in tissue engineering. The results presented in this work demonstrate important aspects of alginate-controlled crystallization, which contributes to the understanding of composite material design.