title: Ingrowth of human mesenchymal stem cells into porous silk particle reinforced silk composite scaffolds: An in vitro study.
abstract: Silk fibroin protein is biodegradable and biocompatible, exhibiting excellent mechanical properties for various biomedical applications. However, porous three-dimensional (3-D) silk fibroin scaffolds, or silk sponges, usually fall short in matching the initial mechanical requirements for bone tissue engineering. In the present study, silk sponge matrices were reinforced with silk microparticles to generate protein-protein composite scaffolds with desirable mechanical properties for in vitro osteogenic tissue formation. It was found that increasing the silk microparticle loading led to a substantial increase in the scaffold compressive modulus from 0.3 MPa (non-reinforced) to 1.9 MPa for 1:2 (matrix:particle) reinforcement loading by dry mass. Biochemical, gene expression, and histological assays were employed to study the possible effects of increasing composite scaffold stiffness, due to microparticle reinforcement, on in vitro osteogenic differentiation of human mesenchymal stem cells (hMSCs). Increasing silk microparticle loading increased the osteogenic capability of hMSCs in the presence of bone morphogenic protein-2 (BMP-2) and other osteogenic factors in static culture for up to 6 weeks. The calcium adsorption increased dramatically with increasing loading, as observed from biochemical assays, histological staining, and microcomputer tomography (μCT) analysis. Specifically, calcium content in the scaffolds increased by 0.57, 0.71, and 1.27 mg (per μg of DNA) from 3 to 6 weeks for matrix to particle dry mass loading ratios of 1:0, 1:1, and 1:2, respectively. In addition, μCT imaging revealed that at 6 weeks, bone volume fraction increased from 0.78% for non-reinforced to 7.1% and 6.7% for 1:1 and 1:2 loading, respectively. Our results support the hypothesis that scaffold stiffness may strongly influence the 3-D in vitro differentiation capabilities of hMSCs, providing a means to improve osteogenic outcomes.
authors.affiliation: Department of Biomedical Engineering, Tufts University, Medford, MA, USA.
authors.forename: Danielle N
authors.initials: DN
authors.lastname: Rockwood
authors.forename: Eun Seok
authors.initials: ES
authors.lastname: Gil
authors.forename: Sang-Hyug
authors.initials: SH
authors.lastname: Park
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Kluge
authors.forename: Warren
authors.initials: W
authors.lastname: Grayson
authors.forename: Sarindr
authors.initials: S
authors.lastname: Bhumiratana
authors.forename: Rangam
authors.initials: R
authors.lastname: Rajkhowa
authors.forename: Xungai
authors.initials: X
authors.lastname: Wang
authors.forename: Sung Jun
authors.initials: SJ
authors.lastname: Kim
authors.forename: Gordana
authors.initials: G
authors.lastname: Vunjak-Novakovic
authors.forename: David L
authors.initials: DL
authors.lastname: Kaplan
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D001696:Biomechanical Phenomena / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D003094:Collagen / Q000378:metabolism; D006801:Humans; D008422:Materials Testing / Q000379:methods*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology; D008903:Minerals / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects / Q000235:genetics; D016062:Porosity / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D047011:Silk / Q000494:pharmacology*; D012995:Solubility / Q000187:drug effects; D054457:Tissue Scaffolds / Q000737:chemistry*; D055114:X-Ray Microtomography
chemical_list: D015415:Biomarkers; D008903:Minerals; D012333:RNA, Messenger; D047011:Silk; D003094:Collagen; D000469:Alkaline Phosphatase; D002118:Calcium