Rajendra S. Bhatnagar

Modulation of proteoglycan and collagen profiles in human dermal fibroblasts by high density micromass culture and treatment with lactic acid suggests change to a chondrogenic phenotype.

Cartilage formation during embryonic development and in fracture healing in adult animals involves chondrogenic differentiation of mesenchymal precursors. Here we describe an in vitro model whereby human dermal fibroblasts, considered to be restricted to a fibroblast lineage, are apparently redirected toward a chondrogenic phenotype by high density micromass culture in the presence of lactic acid. Micromass cultures treated with 40 mM lactate exhibited increased levels of Alcian blue staining and sulfate incorporation, indicative of elevated sulfated glycosaminoglycan synthesis. Northern analysis revealed an up-regulation of chondroitin sulfate proteoglycan 1 (aggrecan) and transforming growth factor-β1 mRNA and a decrease in type I collagen expression. Type II collagen was detected by reverse transcription-PCR only in experimental cultures. Although the observed changes in biosynthesis and gene expression were consistent with differentiating chondrocytes. the cells displayed an elongated. fibroblast-like morphology. These findings suggest that dermal fibroblasts may be committed to differentiate along a chondrogenic paths as by to in vitro culture under specific forcing conditions.

Biomimetic scaffolds for tissue engineering

A major goal of tissue engineering is to generate living cellular constructs with 3-D, tissue-like organization of cells and matrices. The generation of mechanical forces by the cellular cytoskeleton plays a critical role in the organization of matrix and of cellular colonies. The anchorage of the cytoskeleton to a substrate is essential for cellular tractional processes. We show that placing cells in scaffolds which contain a collagen-related synthetic peptide P-15 allows them to generate highly organized 3-D colonies and matrices. These observations suggest that P-15 may replicate cells physiological collagen anchorage.

Synthetic peptides cytomodulin-1 (CM-1) and cytomodulin-2 (CM-2) promote collagen synthesis and wound healing in vitro

Transforming growth factor-/spl beta/1 (TGF-/spl beta/1) is believed to be a key player in wound healing, promoting cell proliferation, migration, and matrix synthesis in a variety of cell types. We have designed two peptides, i.e., cytomodulin-1 (CM-1) and cytomodulin-2 (CM-2), to simulate the binding domain of TGF-/spl beta/1. In this study we examined the bioactivity of the two synthetic peptides CM-1 and CM-2 on human foreskin fibroblasts (HFF). Synthetic peptides CM-1 and CM-2 in culture media increased wound healing of fibroblasts in an injury model in vitro. In addition, CM-1 and CM-2 enhanced the gene expression of collagen I and increased the production of pro-collagen I peptide in HFF. These results suggest that CM-1 and CM-2 have potentially useful clinical applications in wound healing.

Fine structure of collagen: Molecular mechanisms of the interactions of collagen

Collagen is a multifunctional protein which enters into highly specific interactions with many proteins including cell surface receptors. The physiological solid state of collagen complicates explanations of its behavior as a bioactive material, since conformational parameters for collagen molecules are deduced either from X-ray diffraction of fibers, or derived from properties of individual molecules in solution. Physiological interactions of collagen involve molecules on the fiber surface. The anisotropic environment of these molecules can be expected to induce local conformational fluctuations relevant to intermolecular recognition and allosteric binding. We have examined conformational heterogeneity within the triple helix using stereochemical mapping, synthetic polypeptide models, and cryogenic atomic force microscopy. We have identified a 15-residue domain inα1(I) chain residues766GTPGPQGIAGQRGVV780 which shows equal preference for a strand-bend-strand conformation and for the collagen fold. A synthetic peptide analogous to this sequence generates aβ-rich structure in mixed solvents with the central —IA— residues forming the core of aβ-bend. This peptide is a potent force-conducting ligand for collagen receptors, serving as an anchorage for cells for tissue engineering. Our studies provide a rational mechanism for intermolecular recognition based on local conformational tautomerism of a helix-domain sequence.