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.

FIG. 1.

MSC contraction and metabolic activity as a function of time and scaffold type. (A) Pronounced cell-mediated contraction was observed for the low-density group with minor contractions in the high-density group and negligible contraction in the mineralized scaffold group. (B) Human MSCs remained metabolically active over extended culture times. ^aSignificantly different from other two experimental groups, ^bsignificantly different from the mineralized group, ^csignificantly different from the low-density group, ^∧significantly increased from previous time point, and ^∨significantly decreased from previous time point. MSC, mesenchymal stem cell.

FIG. 2.

Trends in chondrogenic gene expression. Expression profiles of chondrogenic genes (A) collagen type II alpha 1 (COL2A1), (B) aggrecan (ACAN), and (C) sex determining region Y box 9 (SOX9) were tracked for 21 days. After 21 days, expression levels of all three transcripts were depressed in the mineralized group with significantly higher expression of COL2A1 and ACAN observed in the high-density group. ^aSignificantly different from other two experimental groups, ^csignificantly different from the mineralized group, ^∧significantly increased from previous time point, and ^∨significantly decreased from previous time point.

FIG. 3.

Trends in osteogenic gene expression. Expression profiles of osteogenic genes (A) runt-related transcription factor 2 (RUNX2), (B) bone sialoprotein (BSP), (C) alkaline phosphatase (ALP), and (D) osteocalcin (OCN) were tracked for 21 days. After 21 days, BSP, ALP, and OCN were all significantly upregulated in the low-density nonmineralized group. ^aSignificantly different from other two experimental groups and ^∧significantly increased from previous time point.

FIG. 4.

Hematoxylin and eosin staining of scaffold sections. Histology sections show differences in MSC distribution and scaffold contraction. (A) Low density (growth), (B) low density (mixed), (C) high density (growth), (D) high density (mixed), (E) mineralized (growth), and (F) mineralized (mixed). Scale bars: 500 μm. Color images available online at www.liebertpub.com/tea

FIG. 5.

Alizarin red staining of scaffold sections. Histology sections reveal differential mineral content as a function of scaffold type and media treatment with the nonmineralized scaffold groups showing little mineralization at days 7 or 14, but robust mineralization in the mixed media groups at day 21. (A) Low density (growth), (B) low density (mixed), (C) high density (growth), (D) high density (mixed), (E) mineralized (growth), (F) mineralized (mixed). Scale bars: 500 μm. Color images available online at www.liebertpub.com/tea

FIG. 6.

Alcian blue staining of scaffold sections. Histology sections reveal differential GAG content as a function of scaffold type and media treatment with higher GAG levels initially in the lower density mixed groups, but higher levels in the high-density mixed group by day 21. (A) Low density (growth), (B) low density (mixed), (C) high density (growth), (D) high density (mixed), (E) mineralized (growth), (F) mineralized (mixed). Scale bars: 500 μm. Color images available online at www.liebertpub.com/tea