Adipose stem cell microbeads as production sources for chondrogenic growth factors

Abstract

Microencapsulating stem cells in injectable microbeads can enhance delivery and localization, but their ability to act as growth factor production sources is still unknown. To address this concern, growth factor mRNA levels and production from alginate microbeads with encapsulated human adipose stem cells (ASC microbeads) cultured in both growth and chondrogenic media (GM and CM) were measured over a two week period. Human ASCs in microbeads were either commercially purchased (Lonza) or isolated from six human donors and compared to human ASCs on tissue culture polystyrene (TCPS). The effects of crosslinking and alginate compositions on growth factor mRNA levels and production were also determined. Secretion profiles of IGF-I, TGF-β3 and VEGF-A from commercial human ASC microbeads were linear and at a significantly higher rate than TCPS cultures over two weeks. For human ASCs derived from different donors, microencapsulation increased pthlh and both IGF-I and TGF-β3 secretion. CM decreased fgf2 and VEGF-A secretion from ASC microbeads derived from the same donor population. Crosslinking microbeads in BaCl2 instead of CaCl2 did not eliminate microencapsulation's beneficial effects, but did decrease IGF-I production. Increasing the guluronate content of the alginate microbead increased IGF-I retention. Decreasing alginate molecular weight eliminated the effects microencapsulation had on increasing IGF-I secretion. This study demonstrated that microencapsulation can enhance chondrogenic growth factor production and that chondrogenic medium treatment can decrease angiogenic growth factor production from ASCs, making these cells a potential source for paracrine factors that can stimulate cartilage regeneration.

Keywords: Adipose stem cells; Alginate; Cartilage; Growth factor delivery; Microencapsulation.

Figure 1.

Growth Factor Production and Secretion from ASC Microbeads over Time. (A) Linear regressions of cumulative growth factor secretion profiles from ASC cultures with measurements at 3, 5, 7, 10, 12, 14 and 15 days and normalized to DNA content at day 15. ASCs were cultured in growth medium (GM) and chondrogenic medium (CM) on tissue culture polystyrene (TCPS) or within alginate microbeads (μB). (B) Cumulative growth factor production/DNA content secreted and maintained within ASC microbeads after 7 and 14 days in GM and CM. (n = 6 samples, mean±SE, *p<0.05 vs. in microbead, #p<0.05 vs. wk1 GM, $p<0.05 vs. wk1 CM, ^p<0.05 vs. wk2 GM).

Figure 2.

Effect of Chondrogenic Medium on Growth Factor mRNA Levels and Secretion from ASCs Isolated from Different Donors. (A) mRNA levels and (B) growth factor secretion/DNA content on day 7 and day 14 from ASCs cultured in chondrogenic medium (CM) on tissue culture polystyrene (TCPS) and within alginate microbeads (μB) normalized to matching 2-D and 3-D cultures in growth medium (GM) (n = 6 donors, mean±SE, *p<0.05 CM vs. GM, #p<0.05 vs. wk1 TCPS, $p<0.05 vs. wk1 μB, ^p<0.05 vs. wk2 TCPS). mRNA and secretion levels of other genes and growth factors can be found in Table 2.

Figure 3.

Effect of Microencapsulation on Phenotypic mRNA Levels of ASCs Isolated from Different Donors. (A) mRNA levels on day 7 and day 14 of ASC microbeads cultured in growth medium or chondrogenic medium (CM) normalized to ASCs on tissue culture polystyrene (TCPS) cultured in matching media. (n = 6 donors, mean±SE, *p<0.05 μB vs. TCPS, #p<0.05 vs. wk1 GM, $p<0.05 vs. wk1 CM, ^p<0.05 vs. wk2 GM). mRNA levels of ASCs cultured in chondrogenic medium (CM) on tissue culture polystyrene (TCPS) and within alginate microbeads (μB) normalized to matching 2-D and 3-D cultures in growth medium can be found in Table 2.

Figure 4.

Effect of Microencapsulation on Growth Factor mRNA Levels and Secretion from ASCs Isolated from Different Donors. (A) mRNA levels and (B) growth factor secretion/DNA content on day 7 and day 14 from ASC microbeads cultured in growth medium or chondrogenic medium (CM) normalized to ASCs on tissue culture polystyrene (TCPS) cultured in matching media (n = 6 donors, mean±SE, *p<0.05 μB vs. TCPS, #p<0.05 vs. wk1 GM, $p<0.05 vs. wk1 CM, ^p<0.05 vs. wk2 GM). mRNA and secretion levels of other genes and growth factors can be found in Table 2.

Figure 5.

Effect of Divalent Crosslinks in ASC Microbead on Growth Factor mRNA Levels and Production. (A) mRNA levels and (B) growth factor secretion/DNA content on day 7 from ASC microbeads crosslinked in CaCl2 Ca++) or BaCl2 (Ba++), cultured in growth medium and normalized to ASCs on tissue culture polystyrene (TCPS). (C) Growth factor retained within microbeads normalized to DNA content (n = 6 samples, mean±SE, *p<0.05 μB vs. TCPS, #p<0.05 vs. Ca++).

Figure 6.

Effect of Alginate Molecular Weight and Chemistry in ASC Microbead on Growth Factor mRNA Levels and Production. (A) Growth factor mRNA and (B) growth factor secretion/DNA content on day 7 from ASC microbeads consisting of LVM, LVG, VLVM and MVM alginates; cultured in growth medium; and normalized to ASCs on tissue culture polystyrene (TCPS). (C) Growth factor retained in microbeads normalized to DNA content (n = 6 samples, mean±SE, *p<0.05 μB vs. TCPS, #p<0.05 vs. Ca++).

Figure 7.

Summative Affect of Chondrogenic Medium and Microencapsulation on ASCs