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Perlecan, a heparan sulfate proteoglycan, is widely distributed in developing and adult tissues and plays multiple, important physiological roles. Studies with knockout mouse models indicate that expression of perlecan and heparan sulfate is critical for proper skeletal morphogenesis. Heparan sulfate chains bind and potentiate the activities of various growth factors such as fibroblast growth factor 2 (FGF-2). Previous studies indicate that important biological activities are associated with the heparan sulfate-bearing domain I of perlecan (PlnDI; French et al. J. Bone Miner. Res. 17 , 48, 2002). In the present study, we have used recombinant, glycosaminoglycan-bearing PlnDI to reconstitute three-dimensional scaffolds of collagen I. Collagen I fibrils bound PlnDI much better than native collagen I monomers or heat-denatured collagen I preparations. Heparitinase digestion demonstrated that recombinant PlnDI was substituted with heparan sulfate and that these heparan sulfate chains were critically important not only for efficient integration of PlnDI into scaffolds, but also for FGF-2 binding and retention. PlnDI-containing collagen I scaffolds to which FGF-2 was bound sustained growth of both MG63, an osteoblastic cell line, and human bone marrow stromal cells (hBMSCs) significantly better than scaffolds lacking either PlnDI or FGF-2. Collectively, these studies demonstrate the utility of PlnDI in creating scaffolds that better mimic natural extracellular matrices and better support key biological activities.

Extracellular matrix (ECM) molecules in cartilage cooperate with growth factors to regulate chondrogenic differentiation and cartilage development. Domain I of perlecan (Pln) bears heparan sulfate chains that bind and release heparin binding growth factors (HBGFs). We hypothesized that Pln domain I (PlnDI) might be complexed with collagen II (P-C) fibrils to improve binding of bone morphogenetic protein-2 (BMP-2) and better support chondrogenesis and cartilage-like tissue formation in vitro. Our results showed that P-C fibrils bound more BMP-2 than collagen II fibrils alone, and better sustained BMP-2 release. Polylactic acid (PLA)-based scaffolds coated with P-C fibrils immobilized more BMP-2 than either PLA scaffolds or PLA scaffolds coated with collagen II fibrils alone. Multipotential mouse embryonic mesenchymal cells, C3H10T1/2, were cultured on 2-dimensional P-C fibrils or 3-dimensional P-C/BMP-2-coated (P-C-B) PLA scaffolds. Chondrogenic differentiation was indexed by glycosaminoglycan (GAG) production, and expression of the pro-chondrogenic transcription factor, Sox9, as well as cartilaginous ECM proteins, collagen II, and aggrecan. Immunostaining for aggrecan, perlecan, tenascin, and collagen X revealed that both C3H10T1/2 cells and primary mouse embryonic fibroblasts cultured on P-C-B fibrils showed the highest expression of chondrogenic markers among all treatment groups. Safranin O-Fast Green staining indicated that cartilage-like tissue was formed in the P-C-B scaffolds, while no obvious cartilage-like tissue formed in other scaffolds. We conclude that P-C fibrils provide an improved biomimetic material for the binding and retention of BMP-2 and support chondrogenic differentiation.