Evaluation of nanoarchitectured collagen type II molecules on cartilage engineering.

Scaffold architecture, including the geometry and dimension of scaffolds, is an important parameter in cell adhesion, migration, proliferation, and differentiation. Following the characterization of collagen type II nanoarchitectured molecules, collagen fibrils (CNFs) and collagen spheres (CNPs) prepared using a high-voltage electric field in our laboratory, we proposed to use these nanoarchitectured molecules to assess their influence on the culturing of chondrocytes in stirred bioreactors. The results demonstrate that chondrocytes rapidly formed more and larger chondrocyte pellets (spheroids) after the addition of nanoarchitectured molecules into the culture medium. The maintenance of chondrocytes with round morphology and increased glycosaminoglycan secretion indicated that these spheroids contained viable and un-dedifferentiated chondrocytes. No significant increases in DNA content were detected. These results show that the introduction of these molecules did not affect chondrocyte proliferation during a 3-day culture period. After the addition of CNPs and CNFs into the culture medium, the expression levels of collagen type II and aggrecan genes in chondrocytes increased significantly as demonstrated by real-time PCR analysis. Interestingly, chondrocytes exhibited distinct collagen type II and aggrecan gene expression profiles in culture with CNPs and CNFs. The aggrecan gene expression level of the chondrocytes was 2.5-fold greater following CFN addition than following the addition of CNPs. In contrast, the collagen type II expression level of the chondrocytes was 2.2-fold greater following the addition of CNPs than following the addition of CNFs. The chondrocyte pellets rapidly restored defects in articular cartilage during a 1-month implantation period in a rabbit model.

Studies on the microspheres comprised of reconstituted collagen and hydroxyapatite.

Microspheres comprised of hydroxyapatite particles, dispersed in reconstituted fibrous collagen, were prepared and characterized. The hydroxyapatite particles distributed evenly throughout the collagen matrix of the microsphere. Diameters of the reconstituted collagen fibers ranged from 30 to 90 nm, and exhibited a regular banding pattern with cross-striation of 50-60 nm under transmission electron microscope, suggesting that the reconstitution of collagen was not hindered by the hydroxyapatite particulates. When osteoblast cells isolated from newborn rat calvaria were seeded and cultured on the microspheres, the cell density increased from 2x10(4) to 3.2x10(4)cells/cm(2) in 8 days. Von Kossa staining exhibited spotty accumulation of mineral deposits on microspheres indicating matrix mineralization of the cultured cells. Analyses by electron microscopy and confocal microscopy showed that the osteoblast cells spread and attached to the microsphere via focal adhesion, while F-actin and DNA staining demonstrated the presence of stress fibers; moreover, mitotic cells could be observed. Together, these results indicate that osteoblast cells are capable of proliferating, differentiating and mineralizing in the matrix of the microspheres, and suggest that the microspheric composite is a potential grafting material for future clinical applications.

Osteogenic enrichment of bone-marrow stromal cells with the use of flow chamber and type I collagen-coated surface.

The stromal cells of the bone marrow are able to attach to the surface and differentiate into cells with bone-forming capability when stimulated with osteogenic supplements. In this study, we have employed a flow-chamber device containing a collagen-coated surface to enrich the potential osteoprogenitor cells from bone marrow stromal cells (BMSCs). The population of the cells attached to the collagen-coated substratum is about twice that attached to the uncoated surface. In the flow chamber, almost all marrow cells attached on the untreated glass were flushed out at the shear stress of 1.10 dyne/cm(2). On the other hand, 25% of the marrow cells remained attached to the collagen-coated glass, even under the shear stress of 1.30 dyne/cm(2). The collagen-attached marrow cells exhibited similar, specific alkaline phosphatase activity compared with that of the cells attached to the uncoated dish in the early stage of culturing. Nevertheless, only the collagen-attached marrow cells later expressed significant amounts of osteocalcin, which is a specific marker for osteoblast cells. Thus, we have successfully developed a protocol that uses a collagen-coated surface efficiently in a flow chamber to enrich the osteogenic cells from the BMSCs. This provides a useful tool to obtain osteogenic cells from bone marrow for biologic and clinical applications.

Collagen as an immobilization vehicle for bone marrow stromal cells enriched with osteogenic potential.

The bone marrow contains mesenchymal cells that can be divided into two categories: cells of hemopoietic lineage and stromal cells. The stromal cells are adhesive to the surface of culture dish, and could be differentiated into cells with bone-forming capability when stimulated with osteogenic supplements. In this study, we have employed collagen to immobilize cells with osteogenic potential from bone marrow. A more than two-fold increase in cell density was obtained on the collagen-coated substratum as compared to the uncoated ones. The selected marrow cells exhibited elevated alkaline phosphatase activity in parallel with the proliferation of the cells attached to the collagen surface. The osteoblastic expression of the selected cells was further confirmed by the histological stains of alkaline phosphatase and mineral deposit. This method provides a simple and fast screening technique to isolate osteoprogenitor-enriched population from the bone marrow stromal cells. It has a great potential for future biological and clinical applications.