High-strength silk protein scaffolds for bone repair

Abstract

Biomaterials for bone tissue regeneration represent a major focus of orthopedic research. However, only a handful of polymeric biomaterials are utilized today because of their failure to address critical issues like compressive strength for load-bearing bone grafts. In this study development of a high compressive strength (~13 MPa hydrated state) polymeric bone composite materials is reported, based on silk protein-protein interfacial bonding. Micron-sized silk fibers (10-600 µm) obtained utilizing alkali hydrolysis were used as reinforcement in a compact fiber composite with tunable compressive strength, surface roughness, and porosity based on the fiber length included. A combination of surface roughness, porosity, and scaffold stiffness favored human bone marrow-derived mesenchymal stem cell differentiation toward bone-like tissue in vitro based on biochemical and gene expression for bone markers. Further, minimal in vivo immunomodulatory responses suggested compatibility of the fabricated silk-fiber-reinforced composite matrices for bone engineering applications.

Fig. 1.

(A) Schematic representation of silk-fiber fabrication steps, (B) SEM image showing degummed silk-fiber morphology and possible arrangements of crystalline and less crystalline domains, and (C) hydrolyzed silk microfibers of varied lengths used as fillers for fabricating reinforced scaffolds. (Scale bar, 400 µm.)

Fig. 2.

(A) Varied lengths of silk microfibers obtained after alkali hydrolysis, (B) compressive modulus of silk microfiber-reinforced scaffolds of ratios 1∶1 and 1∶3, before and after cell culture (28 d), (C) ALP activity of seeded hMSCs under differentiating conditions on silk microfiber-reinforced scaffolds, and (D) cell proliferation showing normalized values of cell growth within silk scaffolds over a period of 4 wk. (Scale bar, 200 µm.) Data represents mean ± standard deviation (n = 5), where **P ≤ 0.01 and *P ≤ 0.05.

Fig. 3.

SEM images showing silk scaffold characteristics including pore size, microfiber bonding, porosity, and surface roughness. Inset shows fabricated scaffold used for cell culture. (Scale bar, 200 µm.)

Fig. 4.

Real-time gene expression conducted on silk microfiber-reinforced scaffolds seeded with hMSCs under differentiating conditions showing fold expression of osteogenic genes: (A) ALP, (B) Collagen 1a1, (C) OP, and (D) BSP. (Scale bar, 200 µm.) Data represents mean ± SD (n = 4), where **P ≤ 0.01 and *P ≤ 0.05.

Fig. 5.

Histological images showing in vivo immunological response of fabricated silk microfiber scaffolds in mice. Sample sections were stained with H&E. Microscopic images (Left) show scaffolds implanted subcutaneously in mice and a harvested highly vascularized implant after 4 wk.