Extracellular matrix remodeling, integrin expression, and downstream signaling pathways influence the osteogenic differentiation of mesenchymal stem cells on poly(lactide-co-glycolide) substrates

Abstract

The possibility of using multipotent adult bone marrow-derived mesenchymal stem cells (MSCs) for tissue-engineering applications hinges on the ability to predictably control their differentiation. Previously, we showed the osteogenic potential of adult bone marrow-derived MSCs cultured on thin films of poly(lactide-co-glycolide) (PLGA) depends in part on the identity of extracellular matrix (ECM) ligands initially deposited onto the material from serum in the culture medium. Here we have addressed the hypothesis that remodeling of the PLGA surface via the de novo synthesis of ECM proteins by the MSCs may also play an important role in governing their osteogenic differentiation. Supporting this hypothesis, increasing amounts of fibronectin and type-I collagen were synthesized and deposited onto thin-film PLGA substrates, whereas vitronectin levels diminished over a 28-day time course. Integrin expression profiles changed accordingly, with higher levels of alpha2beta1 and alpha5beta1 than alphavbeta3 at three different time points. The mitogen-activated protein kinase (MAPK) and phosphatidyl inositol-3-kinase (PI3K) pathways were also activated in MSCs cultured on these substrates, and their inhibition significantly inhibited osteogenic differentiation as assessed according to alkaline phosphatase activity and mineral deposition. These data indicate that initial ECM deposition, subsequent matrix remodeling, and corresponding integrin expression profiles influence osteogenesis in MSCs cultured on PLGA in part by engaging MAPK and PI3K signaling pathways. Understanding the mechanisms by which stem cells respond to different polymers will be critical in their eventual therapeutic use.

FIG. 1.

Extracellular matrix (ECM) and integrin expression in mesenchymal stem cells (MSCs) cultured on poly(lactide-co-glycolide) (PLGA) substrates. (A) MSCs were cultured on non-tissue culture dishes coated with 85:15 PLGA in the presence of normal growth medium (serum medium) or medium containing osteoinductive supplements (OS medium) for 1, 7, 14, or 28 days. At each time point, cell lysates were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot for levels of fibronectin (FN) and vitronectin (VN). Results shown are from a representative blot, similar to those obtained from multiple experiments. (B) MSCs cultured on thin PLGA films in OS medium were also fixed and fluorescently stained for extracellular type I ollagen, VN, or FN. Images shown are representative of one of the intermediate time points (7 days). Scale bar represents 10 μm. (C) Quantitative flow cytometry was used to assess the relative expression of the α2β1, α5β1, and αvβ3 integrins in MSCs, with mean fluorescent intensities used to determine the relative expression of each integrin.

FIG. 2.

Extracellular matrix (ECM) and osteogenic gene expression levels in mesenchymal stem cells (MSCs) cultured on poly(lactide-co-glycolide) (PLGA). (A) MSCs were cultured on spin-coated PLGA substrates for 1, 7, 14 and 28 days in normal growth medium or osteoinductive medium (OS medium). Type I collagen, fibronectin, runt-related transcription factor 2 (Runx2), bone sialoprotein, and osteocalcin gene expression levels were assessed by RT-PCR at each timepoint. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA expression was also examined as an internal control. Representative images of ethidium bromide–stained agarose gels are shown. (B–E) After scanning densitometry, type I collagen (B), fibronectin (C), Runx2 (D), and bone sialoprotein (E) gene expression levels were normalized to corresponding GAPDH expression levels, and the ratios were compared to OS medium day 28 condition. Statistical comparisons were made at each time point between the two different media conditions (*p < 0.05, ***p < 0.001).

FIG. 2.

Extracellular matrix (ECM) and osteogenic gene expression levels in mesenchymal stem cells (MSCs) cultured on poly(lactide-co-glycolide) (PLGA). (A) MSCs were cultured on spin-coated PLGA substrates for 1, 7, 14 and 28 days in normal growth medium or osteoinductive medium (OS medium). Type I collagen, fibronectin, runt-related transcription factor 2 (Runx2), bone sialoprotein, and osteocalcin gene expression levels were assessed by RT-PCR at each timepoint. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA expression was also examined as an internal control. Representative images of ethidium bromide–stained agarose gels are shown. (B–E) After scanning densitometry, type I collagen (B), fibronectin (C), Runx2 (D), and bone sialoprotein (E) gene expression levels were normalized to corresponding GAPDH expression levels, and the ratios were compared to OS medium day 28 condition. Statistical comparisons were made at each time point between the two different media conditions (*p < 0.05, ***p < 0.001).

FIG. 3.

Coating poly(lactide-co-glycolide) (PLGA) substrates with purified extracellular matrix (ECM) proteins alters alkaline phosphatase (ALP) activity. Non-tissue culture dishes spin-coated with a 1% solution of 85:15 PLGA were incubated with a purified ECM protein solution [type I collagen (100 μg/mL), fibronectin (5 μg/mL), or vitronectin (2 μg/mL)] or kept uncoated overnight at 4°C. Mesenchymal stem cells were cultured on these substrates, and ALP activity was assessed after 14 and 21 days of culture. The ALP values for all of the ECM-coated PLGA substrates were significantly higher than the uncoated control surface (***p < 0.001).

FIG. 4.

Mitogen-activated protein kinase (MAPK) and phosphatidyl inositol-3-kinase (PI3K) pathways are activated in mesenchymal stem cells (MSCs) cultured on poly(lactide-co-glycolide) (PLGA). (A) MSCs were cultured on spin-coated PLGA substrates for 1, 7, 14, and 28 days in normal growth medium (labeled FBS) or osteoinductive medium (labeled OS). Cell lysates at each time point were analyzed using electrophoresis and Western blot by probing for phosphorylated MAPK, phosphorylated Akt, osteopontin, and β-actin. Membranes were stripped and reprobed for the total levels of extracellular signal–regulated kinase and Akt-1. Western blots representative of multiple experiments are shown. (B) MSCs were cultured on substrates coated with fibronectin, type I collagen, and vitronectin in basal medium lacking serum (labeled DMEM), normal growth medium (labeled FBS), or osteoinductive medium (labeled OS). Shown are representative Western blots from lysates generated after 7 days.

FIG. 5.

Mitogen-activated protein kinase (MAPK) and phosphatidyl inositol-3-kinase (PI3K) pathways are required for the osteogenic differentiation of mesenchymal stem cells (MSCs) on poly(lactide-co-glycolide) (PLGA) substrates. (A) MSCs were cultured on spin-coated PLGA substrates in OS medium with or without the MAPK pathway inhibitor PD98059 (50 μM) for 1, 7, or 14 days, with the inhibitor replenished at every medium change. Cell lysates analyzed using Western blot for p-MAPK confirmed the pharmacologic inhibition of the MAPK pathway via PD98059. (B) Inhibition of the PI3K pathway via LY294002 (20 μM) generated similar inhibition of that pathway. (C) MSCs cultured on PLGA substrates with or without PD98059 or LY294002 (20 μM) in OS medium show significant reductions in alkaline phosphatase activity. (D) Pharmacologic inhibition of these pathways also induced qualitative reductions in mineral deposition at 14 days, as revealed by von Kossa staining (mineral deposits denoted by black arrows).