Heparan Sulfate Proteoglycan-Mimetic Hydrogels
Growth factors (GFs) are retained within the Extracel® hydrogel and are slowly released from it over several weeks. The hydrogel protects the GFs from proteolysis and allows them to remain bioactive. When thiol-modified heparin is added to Extracel®, as it is with Extracel-HP® hydrogel kits, the avidity of GF binding to the polymer network increases. The thiol-modified heparin further protects the GFs from proteolysis while sequestering and slowly releasing them from the hydrogel. The immobilized heparin in Extracel-HP® in the form of Heprasil® (thiol-modified hyaluronan blended with thiol-modified heparin) appears to mimic heparan sulfate proteoglycans normally present in the extracellular matrix.
Six GFs have been studied for in vitro release from Heprasil®-only and Extracel-HP® hydrogels:
- basic fibroblast growth factor (bFGF)2
- vascular endothelial growth factor (VEGF)2
- angiopoietin-1 (Ang-1)3
- keratinocyte growth factor (KGF)3
- platelet-derived growth factor-AA (PDGF)3
- transforming growth factor β1 (TGF-β1)3
The GFs are released over several weeks with varying release-rate kinetics. The release rates for bFGF and VEGF have been published2. Those for Ang-1, KGF, PDGF, and TGF- β are summarized below, along with the bFGF and VGF data.
Growth-Factor Release Rates
Growth factors were incorporated non-covalently into Extracel-HP® hydrogels prior to crosslinking to determine their release rate, and the time course of their appearance outside the gel was quantified by ELISA. Human recombinant Ang-1, KGF, PDGF, and TGF- β1 and their respective ELISA kits were purchased from R & D Systems of Minneapolis. The final GF amounts were 1 ng/μL for PDGF, KGF, and Ang-1 and 0.2 ng/μL for TGF- β1. The same methods were used for GF release quantification as described in Pike, et al2. Release profiles for the GFs were sustained over thirty-five days and were curve fit with first-order exponential kinetics (R2 > 0.9 for all cases). The most remarkable result of these experiments was a dramatic variation in the total mass released, which varied by as much as 54.6% of the initial load for bFGF and as little as 1.4% for PDGF in fourteen days (see Table 1 and Table 2).
Table 1. GF Release over Seven Days
| Heprasil® | Extracel-HP® | |
| bFGF | 13.0 | 13.0 |
| VEGF | 8.0 | 9.6 |
| Ang-1 | 1.3 | 2.4 |
| KGF | 13.6 | 2.7 |
| PDGF | 1.6 | 1.2 |
| TGF-β1 | 6.6 | 31.6 |
Table 2. GF Release over Fourteen Days
| Heprasil® | Extracel-HP® | |
| bFGF | 23.5 | 54.6 |
| VEGF | 9.1 | 11.7 |
| Ang-1 | 2.9 | 5.3 |
| KGF | 16.1 | 17.4 |
| PDGF | 1.9 | 1.4 |
| TGF-β1 | 20.4 | 45.1 |
Hydrogel Composition
The Extracel-HP® Hydrogel Kit contains Heprasil® (thiol-modified hyaluronan with thiol-modified heparin), Gelin-S® (thiol-modified gelatin, denatured collagen), and Extralink® (thiol-reactive crosslinking agent). It gels at temperatures from ambient to 37°C at physiological pH, with no low-temperature or low-pH steps in its preparation. The researcher has complete control over gelation time (as short as twenty minutes or as long as several hours), hydrogel stiffness, and hydrogel composition. Extracel-HP® is tested for bacteria growth, lactate dehydrogenase-elevating virus (LDEV), and endotoxins.
References
- S. Cai, Y. Liu, X. Z. Shu, G. D. Prestwich, “Injectable glycosaminoglycan hydrogels for controlled release of human basic fibroblast growth factor,”Biomaterials, 26, 6054-6067 (2005).
- D. B. Pike, S. Cai, K. R. Pomraning, M. A. Firpo, R. J. Fisher, X. Z. Shu, G. D. Prestwich, R. A. Peattie, “Heparin-regulated release of growth factors in vitro and angiogenic response in vivo to implanted hyaluronan hydrogels containing VEGF and bFGF”, Biomaterials, 27, 5242–5251 (2006).
- Unpublished data from the Rob Peattie lab (Oregon State University) and S. Cai and B. Yu of the Glenn Prestwich (University of Utah) lab.
