Insulin Enhances the In Vitro Osteogenic Capacity of Flexor Tendon-Derived Progenitor Cells

Abstract

There is increased incidence of tendon disorders and decreased tendon healing capacity in people with diabetes mellitus (DM). Recent studies have also suggested pathological ossification in repair tendon of people with DM. Therefore, the objective of this study is to investigate the effects of insulin supplementation, an important pathophysiologic stimulus of DM, on tendon progenitor cell (TPC) proliferation and in vitro osteogenic capacity. Passage 3 TPCs were isolated from collagenase-digested, equine superficial digital flexor tendons. TPC proliferation was measured via MTT assay after 3 days of monolayer culture in medium supplemented with 0, 0.007, 0.07, and 0.7 nM insulin. In vitro osteogenic capacity of TPCs (Alizarin Red staining, osteogenic mRNA expression, and alkaline phosphatase bioactivity) was assessed with 0, 0.07, and 0.7 nM insulin-supplemented osteogenic induction medium. Insulin (0.7 nM) significantly increased TPC proliferation after 3 days of monolayer culture. Alizarin Red staining of insulin-treated TPC osteogenic cultures demonstrated robust cell aggregation and mineralized matrix secretion, in a dose-dependent manner. Runx2, alkaline phosphatase, and Osteonectin mRNA and alkaline phosphatase bioactivity of insulin-treated TPC cultures were significantly higher at day 14 of osteogenesis compared to untreated controls. Addition of picropodophyllin (PPP), a selective IGF-I receptor inhibitor, mitigated the increased osteogenic capacity of TPCs, indicating that IGF-I signaling plays an important role. Our findings indicate that hyperinsulinemia may alter TPC phenotype and subsequently impact the quality of repair tendon tissue.

Figure 1

(a) TPC proliferation measured via MTT assay after 3 days of monolayer culture in basal medium supplemented with 0, 0.007, 0.07, and 0.7 nM/L insulin. ∗ represents a significant difference between 0 and 0.7 nM/L insulin (p ≤ 0.05). (b) Representative phase-contrast photomicrographs of TPC monolayer cultures maintained in basal medium and basal medium with 0.7 nM insulin for 3 days. A significant increase in TPCs is seen in insulin-supplemented medium. Scale bar = 100 microns.

Figure 2

(a) Alizarin Red-stained TPC osteogenic cultures supplemented with 0, 0.07, and 0.7 nM/L insulin. Insulin promoted cell aggregation and mineralized matrix secretion as evidenced by intensity of Alizarin Red stain uptake. Scale bar = 500 microns. (b) mRNA levels (normalized to EF1a) of RUNX2, ALP, and Osteonectin (OSN) in TPC osteogenic cultures supplemented with 0, 0.07, and 0.7 nM/L insulin. Insulin treatment significantly increased osteogenic gene expression. ∗ represents a significant difference (p ≤ 0.05) between treatment groups. (c) Alkaline phosphatase bioactivity (normalized to total DNA content) of TPC osteogenic cultures supplemented with 0, 0.07, and 0.7 nM/L insulin. ∗ represents a significant increase in alkaline phosphatase activity (p ≤ 0.05).

Figure 3

mRNA levels (normalized to EF1a) of IGF-I receptor and insulin receptor of TPC control monolayer cultures maintained in basal medium (Ctrl) and TPC osteogenic cultures supplemented with 0, 0.07, and 0.7 nM/L insulin. ∗ represents a significant increase (p ≤ 0.05).

Figure 4

(a) Alizarin Red-stained TPC osteogenic cultures with 0, 0.07, and 0.7 nM/L insulin and 100 nM of picropodophyllin (PPP). PPP mitigated aggregation and mineralized matrix secretion of TPCs seen with insulin treatment. Scale bar = 500 microns. (b) mRNA levels (normalized to EF1a) of RUNX2, ALP, and Osteonectin (OSN) in TPC osteogenic cultures without insulin (Ctrl), supplemented with 0.7 nM/L insulin (INS) and with 0.7 nM/L insulin and 100 mM picropodophyllin (INS+PPP). PPP returned the increased osteogenic gene expression seen in insulin-treated TPCs back to baseline. ∗ represents a significant increase between treatment groups (p ≤ 0.05). (c) Alkaline phosphatase bioactivity (normalized to DNA) of TPC osteogenic cultures without insulin (Ctrl), supplemented with 0.7 nM/L insulin (INS) and with 0.7 nM/L insulin and 100 mM picropodophyllin (INS+PPP). ∗ represents a significant increase between treatment groups (p ≤ 0.05).