Perlecan Domain I Promotes Fibroblast Growth Factor 2 Delivery in Collagen I Fibril Scaffolds

Abstract

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.

Potential Role for Heparan Sulfate Proteoglycans in Regulation of Transforming Growth Factor-β (TGF-β) by Modulating Assembly of Latent TGF-β-binding Protein-1

“…4C shows a schematic diagram of these fibronectin fragments). HSPG (Perlecan) was purchased from Sigma, and purified perlecan domain I was produced as described elsewhere (37).…”

Perlecan, the “jack of all trades” proteoglycan of cartilaginous weight‐bearing connective tissues

“…(95) The importance of the HS chains of domain-I of perlecan for skeletogenesis is inferred from in vitro experiments conducted using recombinant perlecan domain-I, which induced C3H10T1/2 cells to enter a chondrogenic differentiation pathway (96,97) and promoted FGF-2 delivery to collagen fibril-I scaffolds enhancing the growth of an osteoblastic cell line, MG63, as well as human bone marrow stromal cells in vitro. (98) Thus the HS-deficient perlecan of hspg2 D3À/D3À mice is apparently able to sustain normal skeletal growth and stabilise the ECM through interactions involving its core protein. However, when the hspg2 D3À/D3À mice are challenged, damage to the ECM is less able to undergo repair due to deficient FGF-2 cell signalling since this is normally mediated through the HS chains of perlecan domain I. Perlecan isolated from fibroblast and smooth muscle cells of hspg2 D3À/D3À mice, however, is still substituted with HS; thus HS substitution sites outside domain-I do exist and are probably located in domain V. (95,96) This could compensate for the HS deficiency due to deletion of domain-I in hspg2 D3À/D3À mice, while functional redundancy by other cell surface HS-proteoglycans may also alleviate this HS deficiency.…”

Growth Factor Delivery Through Self-assembling Peptide Scaffolds

“…A single dose of HB-IGF-1 resulted in sustained delivery and stimulation of proteoglycan synthesis for at least 8 days. The use of HB-IGF-1 with the KLD peptide system could be enabled by mixing in heparin, heparan sulfate, or the heparan sulfate proteoglycan, perlecan, with KLD [18,19,51]. Although adsorption of TGF-b1 to KLD effectively increased proteoglycan production in our study and a previous study [26], changing the degradation rates of KLD by changing the concentration or adding crosslinks may offer a way of improving delivery.…”