Aglinate coated, collagen matrix, cellular device, preparative methods, and uses thereof.

The present invention is directed to cellular devices comprising a collagen matrix, cell layer, and gelled alginate layer, processes for producing the devices, methods of implanting the devices, and methods of treatment therof

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

Ionically Gelled Alginate Foams: Physical Properties Controlled by Operational and Macromolecular Parameters

Alginates in the format of scaffolds provide important functions as materials for cell encapsulation, drug delivery, tissue engineering and wound healing among others. The method for preparation of alginate-based foams presented here is based on homogeneous, ionotropic gelation of aerated alginate solutions, followed by air drying. The method allows higher flexibility and better control of the pore structure, hydration properties and mechanical integrity compared to foams prepared by other techniques. The main variables for tailoring hydrogel properties include operational parameters such as degree of aeration and mixing times and concentration of alginate, as well as macromolecular properties such as the type of alginate (chemical composition and molecular weight distribution). Exposure of foams to γ-irradiation resulted in a dose-dependent (0–30 kGy) reduction in molecular weight of the alginate and a corresponding reduction in tensile strength of the foams

mRNA expression levels relative to GAPDH of selected genes relevant to chondrogenic differentiation, measured by RT qPCR.

<p>Structural ECM proteins (a–d) and enzymes involved in collagen processing and degradation (e–h).</p

Samples characterized in this study differed in cell seeding density (δ) and calcium concentration ([Ca²⁺]) in the differentiation medium.

<p>All samples were cultured with 1.05 mM calcium until day 10, and then switched to the given concentrations.</p

PCR primers and IHC antibodies used to probe respective proteins.

<p>PCR primers and IHC antibodies used to probe respective proteins.</p

TEM micrograph (combined from two tiles) of sample 10-B after three weeks of chondrogenic differentiation, showing distinct zones of matrix radially out from the cell surface.

<p>Scale bar is 1 µm.</p

SHG microscopy of selected samples identifying fibrillar collagens (white) counter stained with DAPI (blue).

<p> is shown to give weight to the darker end of the detector, as described in Materials and Methods. Scale bars are 20 µm.</p