Self-Assembling Peptide Hydrogels Modulate In Vitro Chondrogenesis of Bovine Bone Marrow Stromal Cells

Abstract

Our objective was to test the hypothesis that self-assembling peptide hydrogel scaffolds provide cues that enhance the chondrogenic differentiation of bone marrow stromal cells (BMSCs). BMSCs were encapsulated within two unique peptide hydrogel sequences, and chondrogenesis was compared with that in agarose hydrogels. BMSCs in all three hydrogels underwent transforming growth factor-b1-mediated chondrogenesis as demonstrated by comparable gene expression and biosynthesis of extracellular matrix molecules. Expression of an osteogenic marker was unchanged, and an adipogenic marker was suppressed by transforming growth factor-b1 in all hydrogels. Cell proliferation occurred only in the peptide hydrogels, not in agarose, resulting in higher glycosaminoglycan content and more spatially uniform proteoglycan and collagen type II deposition. The G1-positive aggrecan produced in peptide hydrogels was predominantly the full-length species, whereas that in agarose was predominantly the aggrecanase product G1-NITEGE. Unique cell morphologies were observed for BMSCs in each peptide hydrogel sequence, with extensive cell-cell contact present for both, whereas BMSCs in agarose remained rounded over 21 days in culture. Differences in cell morphology within the two peptide scaffolds may be related to sequence-specific cell adhesion. Taken together, this study demonstrates that selfassembling peptide hydrogels enhance chondrogenesis compared with agarose as shown by extracellular matrix production, DNA content, and aggrecan molecular structure.

Controlled Delivery of Transforming Growth Factor β1 by Self-Assembling Peptide Hydrogels Induces Chondrogenesis of Bone Marrow Stromal Cells and Modulates Smad2/3 Signaling

“…In (KLDL) 3 peptide, full-length aggrecan was the predominant species detected, consistent with the virtual absence of aggrecanase activity in peptide hydrogels. 30 In agarose with Med-TGF, a doublet band near 65 kDa was the major immunoreactive band detected, shown previously to be the G1-NITEGE species, which is generated by aggrecanase activity. 30 However, when TGF-b1 was adsorbed to the agarose, the intensity of this doublet band was decreased and fulllength aggrecan was the major product observed.…”

“…30 In agarose with Med-TGF, a doublet band near 65 kDa was the major immunoreactive band detected, shown previously to be the G1-NITEGE species, which is generated by aggrecanase activity. 30 However, when TGF-b1 was adsorbed to the agarose, the intensity of this doublet band was decreased and fulllength aggrecan was the major product observed. This suggests a reduced aggrecan fragment generation when TGF-b1 was adsorbed to agarose hydrogels compared to Med-TGF.…”

“…Increased DNA content for both Ads-TGF and Med-TGF relative to TGF-b1-free controls showed that the peptide hydrogel microenvironment stimulated cell proliferation for both conditions, similar to that found previously for Med-TGF stimulation using peptide gel scaffolds. 30 Adult equine BMSCs stimulated with Ads-TGF produced neotissue with statistically equivalent cartilage sGAG content and biosynthesis to equine BMSCs stimulated by Med-TGF.…”

“…30 The resulting 6.35-mm-diameter cell-seeded peptide disks, 50 mL initial volume, were cultured in high-glucose Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 1% ITSþ1 (Sigma-Aldrich, St. Louis, MO), 0.1 mM dexamethasone (Sigma-Aldrich), 37.5 mg/mL ascorbate-2-phosphate (Wako Chemicals, Richmond, VA), PSA, HEPES, L-proline, sodium pyruvate, and NEAA, with (medium-delivered TGF-b1 [Med-TGF]) or without (Control [Cntl]) 10 ng/mL recombinant human TGF-b1 (R&D Systems). In a similar manner, BMSCs were also mixed with 2% low-melting-point agarose (Invitrogen) and cast within similar acellular agarose rings, and the resulting cell-seeded disks were cultured as above.…”

“…[20][21][22][23][24] Benefits have been demonstrated for the use of these peptide hydrogels in cartilage, 25,26 liver, 27 and cardiovascular 28,29 tissues, and they have been shown to provide a microenvironment that enhances the chondrogenesis of BMSCs. 26,30 These peptides can also control the delivery and release of functional proteins, 21 therapeutic macromolecules, 22 and bioactive motifs. 20 Taken together, these studies suggest the potential to co-encapsulate growth factors and BMSCs in self-assembling peptide hydrogels to generate cartilage neotissue.…”