Sustained release of TGFbeta3 from PLGA microspheres and its effect on early osteogenic differentiation of human mesenchymal stem cells

Abstract

Despite the widespread role of transforming growth factor-beta3 (TGFbeta3) in wound healing and tissue regeneration, its long-term controlled release has not been demonstrated. Here, we report microencapsulation of TGFbeta3 in poly-d-l-lactic-co-glycolic acid (PLGA) microspheres and determine its bioactivity. The release profiles of PLGA-encapsulated TGFbeta3 with 50:50 and 75:25 PLA:PGA ratios differed throughout the experimental period. To compare sterilization modalities of microspheres, bFGF was encapsulated in 50:50 PLGA microspheres and subjected to ethylene oxide (EO) gas, radio-frequency glow discharge (RFGD), or ultraviolet (UV) light. The release of bFGF was significantly attenuated by UV light, but not significantly altered by either EO or RFGD. To verify its bioactivity, TGFbeta3 (1.35 ng/mL) was control-released to the culture of human mesenchymal stem cells (hMSC) under induced osteogenic differentiation. Alkaline phosphatase staining intensity was markedly reduced 1 week after exposing hMSC-derived osteogenic cells to TGFbeta3. This was confirmed by lower alkaline phosphatase activity (2.25 +/- 0.57 mU/mL/ng DNA) than controls (TGFbeta3- free) at 5.8 +/- 0.9 mU/mL/ng DNA (p < 0.05). Control-released TGFbeta3 bioactivity was further confirmed by lack of significant differences in alkaline phosphatase upon direct addition of 1.35 ng/mL TGFbeta3 to cell culture (p > 0.05). These findings provide baseline data for potential uses of microencapsulated TGFbeta3 in wound healing and tissue-engineering applications.

FIG. 1

Fabrication and degradation of PLGA microspheres. (A) Representative SEM image of microspheres fabricated from poly-d-l-lactic-co-glycolic acid (PLGA) with 50:50 PLA/PGA ratio with encapsulated TGFβ3. The average diameter of TGFβ3-encapsulating PLGA microspheres was 108 ± 62 μm. (B) Representative SEM image of anticipated degradation of TGFβ3-encapsulating PLGA microspheres in PBS solution after 4 days.

FIG. 2

Morphological changes of sterilized PLGA microspheres under SEM. (A) Unsterilized PLGA microspheres. (B) Ultraviolet (UV) light-sterilized PLGA microspheres (30 min) showing severe detrimental effect of UV sterilization. (C) Ethylene oxide (EO) gas-sterilized PLGA microspheres for 24 h. (D) Radiofrequency glow discharge (RFGD)-sterilized PLGA microspheres (4 min, 100 W). In contrast to severe surface degradation changes induced by UV light, EO gas and RFGD did not yield marked surface degradation of PLGA microspheres.

FIG. 3

Release kinetics of bFGF from PLGA microspheres. UV light significantly altered the release rate of bFGF from PLGA microspheres up to 21 days (n = 3, *p < 0.05). No significant changes in release kinetics were observed after EO gas or RFGD sterilizations. Ethylene oxide seemed to be the most economically efficient and safe sterilization method for cytokine-encapsulating PLGA microspheres.

FIG. 4

Release kinetics of TGFβ3 from PLGA microspheres in 1% BSA solution. TGFβ3 was released in a sustained fashion up to 36 and 42 days from 50:50 or 75:25 co-polymer ratios of PLGA microspheres, respectively, as detected by ELISA. Initial burst-like release was observed for both co-polymer ratios, although the 50:50 PLA/PGA ratio yielded a more rapid release rate than the 75:25 PLA/PGA ratio did.

FIG. 5

Alkaline phosphatase (ALP) activity of human mesenchymal stem cells (hMSCs) cultured with osteogenic-supplemented medium for 7 days. (A) ALP staining (red) upon exposure to TGFβ3-free PLGA microspheres. (B) ALP staining (red) upon exposure to TGFβ3 released from PLGA microspheres. Red stain was limited to isolated regions, as shown by white arrowhead. (C) ALP activity of hMSCs cultured in osteogenic-supplemented medium quantified by ALP reagent. Significant decrease in staining was observed for hMSCs cultured in osteogenic-supplemented medium in PLGA microsphere-delivered TGFβ3, suggesting that TGFβ3 at 1.35 ng/mL inhibits early osteogenic differentiation of hMSCs in vitro (n = 3, p < 0.05). magnification × 10 (Color images available online at <www.liebertpub.com/ten>.)