Collagen-GAG Scaffold Biophysical Properties Bias MSC Lineage Choice in the Presence of Mixed Soluble Signals

Abstract

Biomaterial strategies for regenerating multitissue structures require unique approaches. One strategy is to design scaffolds so that their local biophysical properties can enhance site-specific effects of an otherwise heterogeneous biomolecular environment. This investigation examined the role of biomaterial physical properties (relative density, mineral content) on the human mesenchymal stem cell phenotype in the presence of mixed soluble signals to drive osteogenesis or chondrogenesis. We tested a series of three-dimensional collagen-glycosaminoglycan scaffolds with properties inspired by extracellular matrix characteristics across the osteotendinous interface (tendon, cartilage, and bone). We found that selective scaffold mineralization induced a depressed chondrogenic response compared with nonmineralized groups as demonstrated by gene expression and histological analyses. Interestingly, the greatest chondrogenic response was found in a higher density, nonmineralized scaffold variant despite increased contraction and cellular condensation in lower density nonmineralized scaffolds. In fact, the lower density scaffolds demonstrated a significantly higher expression of osteogenic transcripts as well as ample mineralization after 21 days of culture. This effect may be due to local stiffening of the scaffold microenvironment as the scaffold contracts, leading to increased cell density, accelerated differentiation, and possible endochondral ossification as evidenced by a transition from a glycosaminoglycan (GAG)-rich milieu to higher mineralization at later culture times. These findings will help shape the design rules for graded biomaterials to regenerate distinct fibrillar, fibrocartilagenous, and mineralized regions of orthopedic interfaces.

Mineralized collagen scaffolds induce hMSC osteogenesis and matrix remodeling

“…Measures of metabolic activity give a good representation of the overall metabolic health of the cell-seeded biomaterials, 32, 34, 61 they are not exact measures of total cell number. Given previous studies favorably comparing explicit measures of cell number versus metabolic activity of cell-seeded CG scaffolds 62, 63 as well as the large number of scaffold groups and experimental end-points in this study, we chose not to explicitly determine cell number via destructive assays, but rather tracked construct metabolic activity via non-destructive AlamarBlue.…”

The Effect of Gradations in Mineral Content, Matrix Alignment, and Applied Strain on Human Mesenchymal Stem Cell Morphology within Collagen Biomaterials

“…We showed anisotropic scaffolds containing structural alignment cues can enhance alignment, proliferation and transcriptomic stability of equine tenocytes, [15, 16] while also selectively activate mechanotransduction paths and MSC tenogenic differentiation in the absence of growth factors supplementation. [9, 17, 18] We have separately demonstrated a hydroxyapatite mineralized CG scaffold that enhanced MSC differentiation towards an osteogenic lineage, again in the absence of conventional osteogenic supplements. [18, 19] We have recently described a method to generate multi-compartment scaffolds that contain discrete scaffold regions connected by a continuous interface.…”

Strategies to balance covalent and non-covalent biomolecule attachment within collagen-GAG biomaterials

“…Previous work with CG scaffolds has shown that the incorporation of soluble and covalent biomolecules into CG scaffolds impacts cellular behavior. 20, 26, 29, 53 However, these studies have focused on contributions of the total factor present without distinguishing the impacts of specific attachment and fouling individually. For this study we examined how the manipulation of treatment parameters for two covalent immobilization strategies impacted both covalent attachment and fouling.…”

“…However, like with many biomaterial platforms, it has become apparent that biomolecular signals added to culture media can enhance or help direct cellular responses. 26–29 …”