Tailoring Adipose Stem Cell Trophic Factor Production with Differentiation Medium Components to Regenerate Chondral Defects

Abstract

Recent endeavors to use stem cells as trophic factor production sources have the potential to translate into viable therapies for damaged or diseased musculoskeletal tissues. Adipose stem cells (ASCs) can be differentiated into chondrocytes using the chondrogenic medium (CM), but it is unknown if this approach can optimize ASC growth factor secretion for cartilage regeneration by increasing the chondrogenic factor production, while decreasing angiogenic and hypertrophic factor production. The objective of this study was to determine the effects the CM and its components have on growth factor production from ASCs to promote cartilage regeneration. ASCs isolated from male Sprague-Dawley rats and cultured in monolayer or alginate microbeads were treated with either the growth medium (GM) or the CM for 5 days. In subsequent studies, ASC monolayers were treated with either the GM supplemented with different combinations of 50 μg/mL ascorbic acid-2-phosphate (AA2P), 100 nM dexamethasone (Dex), 10 ng/mL transforming growth factor (TGF)-β1, and 100 ng/mL bone morphogenetic protein (BMP)-6 or with the CM excluding different combinations of AA2P, Dex, TGF-β1, and BMP-6. mRNA levels and growth factor production were quantified at 8 and 24 h after the last media change, respectively. The CM increased chondrogenic factor secretion (TGF-β2, TGF-β3, and insulin-like growth factor [IGF]-I) and decreased angiogenic factor production (the vascular endothelial growth factor [VEGF]-A, the fibroblast growth factor [FGF]-2). Microencapsulation in the GM increased production of the chondrogenic (IGF-I, TGF-β2) and angiogenic (VEGF-A) factors. AA2P increased secretion of chondrogenic factors (IGF-I, TGF-β2), and decreased angiogenic factor (VEGF-A) secretion, in addition to decreasing mRNA levels for factors associated with chondrocyte hypertrophy (FGF-18). Dex increased mRNA levels for hypertrophic factors (BMP-2, FGF-18) and decreased angiogenic factor secretion (VEGF-A). TGF-β1 increased angiogenic factor production (FGF-2, VEGF-A) and decreased chondrogenic factor mRNA levels (IGF-I, PTHrP). BMP-6 increased hypertrophic mRNA levels (FGF-18) and chondrogenic factor production (TGF-β2). When ASC microbeads preconditioned with the CM were implanted in a focal cartilage defect and immobilized within an RGD-conjugated hydrogel, tissue infiltration from the edges of the defect and perichondrium was observed. These results show that differentiation media components have distinct effects on ASC's production of angiogenic, chondrogenic, and hypertrophic factors and that AA2P may be the most beneficial CM component for preconditioning ASCs to stimulate cartilage regeneration.

Enhancing adipose stem cell chondrogenesis: A study on the roles of dexamethasone, transforming growth factor β3 and ascorbate supplements and their combination

“…VEGF-A, the major angiogenic factor [78], induces MMP expression in arthritic cartilage and OA [79,80]. Our results for hADSCs in micromass at day 7 are consistent with results obtained by Lee et al [81] on a 5-day monolayer of rat ADSCs which showed Dex, Asc and TGF-β supplementation down-regulate VEGF-A expression compared to NC. Both studies indicate Dex supplementation results in the least expression of VEGF-A.…”

Growth factor gene expression profiles of bone morphogenetic protein-2-treated human adipose stem cells seeded on calcium phosphate scaffolds in vitro

“…The environment in which the tissue regeneration takes place needs to be cleaned of damaged tissue and remodeled, inflammation needs to be allowed and suppressed at the proper stages of healing, and blood vessel formation has to keep up with the regeneration process to allow oxygen and nutrients to reach the metabolically very active bone forming osteoblasts. ASCs can both differentiate towards osteoblasts [22], and form trophic factors that influence the before mentioned processes [19]. As it would make sense that ASCs produce increasingly more trophic factors while healing propagates, we hypothesized that as the differentiation of ASCs progresses hASCs produce more factors associated with processes such as angiogenesis and bone remodeling.…”

“…We found that these factors were expressed at moderate levels when compared to other factors (Table 1). It is possible that the presence of ascorbic acid in our culture medium somewhat diminished VEGFA expression, since it has been shown that ascorbic acid-2 phosphate reduces VEGFA secretion in rat ASCs [19]. Of course Shown are the average BMP-2/control ratios (AESD), of those trophic factors who's expression by ASCs is affected by both substrate type (Fig.…”

“…VEGF promotes the growth of vascular endothelial cells in vitro and induces vascular leakage in vivo, which is important in inflammation control and de novo blood vessel formation and sprouting [18]. ASCs also produce members of the fibroblast growth factor and transforming growth factor-b family [19]. Thus, ASCs produce factors that potentially support bone regeneration in vivo.…”

Evaluation of electrospun PLLA-ECM scaffolds as biomaterials for bone regeneration

“…For example, hMSCs are able to secrete growth factors depending on the stem cell fate; fibroblast growth factor (FGF) during proliferation (Kim & Ma et al 2013), BMP--2 during osteogenic differentiation (Lee et al 2009;Huang et al 2007) and TGF--β during chondrogenic differentiation (Lee et al 2013;). …”