A Hybrid Silk/RADA-Based Fibrous Scaffold with Triple Hierarchy for Ligament Regeneration

Abstract

While silk-based microfibrous scaffolds possess excellent mechanical properties and have been used for ligament tissue-engineering applications, the microenvironment in these scaffolds is not biomimetic. We hypothesized that coating a hybrid silk scaffold with an extracellular matrix (ECM)-like network of self-assembling peptide nanofibers would provide a biomimetic three-dimensional nanofibrous microenvironment and enhance ligament tissue regeneration after bone marrow-derived mesenchymal stem cell (BMSC)-seeding. A novel scaffold possessing a triple structural hierarchy comprising macrofibrous knitted silk fibers, a silk microsponge, and a peptide nanofiber mesh was developed by coating self-assembled RADA16 peptide nanofibers on a silk microfiber-reinforced-sponge scaffold. Compared with the uncoated control, RADA-coated scaffolds showed enhanced BMSC proliferation, metabolism, and fibroblastic differentiation during the 3 weeks of culture. BMSC-seeded RADA-coated scaffolds showed an increasing temporal expression of key fibroblastic ECM proteins (collagen type I and III, tenascin-C), with a significantly higher tenascin-C expression compared with the controls. BMSC-seeded RADA-coated scaffolds also showed a temporal increase in total collagen and glycosaminoglycan production (the amount produced being higher than in control scaffolds) during 3 weeks of culture, and possessed 7% higher maximum tensile load compared with the BMSC-seeded control scaffolds. The results indicate that the BMSC-seeded RADA-coated hybrid silk scaffold system has the potential for use in ligament tissue-engineering applications.

Time dependence of material properties of polyethylene glycol hydrogels chain extended with short hydroxy acid segments

“…The double-stranded DNA content of the samples was determined using a Quant-it PicoGreen assay (Invitrogen, Carlsbad, CA) at emission and excitation wavelengths of 485 and 528 nm, respectively, according to the manufacturer's instructions as described [10]. Total collagen content was measured with a Sircol collagen assay based on selective binding of G-X-Y amino acid sequence of collagen to Sircol dye at absorbance wavelength of 555 nm, according to manufacturer's instructions as described [16, 31]. For RNA analysis, at each time point, total cellular RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA) plus RNeasy Mini-Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions as described [16].…”

Ligament regeneration using an absorbable stent-shaped poly-l-lactic acid scaffold in a rabbit model

“…For ligament tissue engineering, usage of various materials including silk, polytetrafluoroethylene, poly(lactic-coglycolic acid) and PLLA has been reported [18][19][20][21][22]. Ideally, scaffolds should have good mechanical strength during the early stages, which it can adequately maintain during tissue regeneration.…”

Self‐assembling peptide nanofiber hydrogels in tissue engineering and regenerative medicine: Progress, design guidelines, and applications

“…Ligament injuries affect millions worldwide and current treatment often involves the use of allografts which have been associated with serious complications such as ligament laxity, donor site morbidity, and inflammatory response. Chen et al fabricated a composite material which consisted of a silk‐based microfibrous scaffold, which possesses good mechanical properties, coated with the Ac‐(RADA) 4 ‐CONH 2 self‐assembling peptide hydrogel which provides an ECM‐like network of peptide nanofibers . The authors seeded this composite material with BMSCs and observed that compared with the uncoated control, the peptide‐coated scaffolds showed enhanced BMSC proliferation, metabolism, and fibroblastic differentiation during 3 weeks of culture.…”

“…Chen et al fabricated a composite material which consisted of a silk-based microfibrous scaffold, which possesses good mechanical properties, coated with the Ac-(RADA) 4 -CONH 2 self-assembling peptide hydrogel which provides an ECMlike network of peptide nanofibers. 48 The authors seeded this composite material with BMSCs and observed that compared with the uncoated control, the peptide-coated scaffolds showed enhanced BMSC proliferation, metabolism, and fibroblastic differentiation during 3 weeks of culture. Furthermore, BMSC-seeded, peptide-coated scaffolds showed increased temporal expression of key fibroblastic ECM proteins including collagen type I and III, tenascin-C, and glycosaminoglycan, and possessed 7% higher maximum tensile load compared with the controls.…”