Abstract
IntroductionAdipose stem cells (ASCs) secrete many trophic factors that can stimulate tissue repair, including angiogenic factors, but little is known about how ASCs and their secreted factors influence cartilage regeneration. Therefore, the aim of this study was to determine the effects ASC-secreted factors have in repairing chondral defects.MethodsASCs isolated from male Sprague Dawley rats were cultured in monolayer or alginate microbeads supplemented with growth (GM) or chondrogenic medium (CM). Subsequent co-culture, conditioned media, and in vivo cartilage defect studies were performed.ResultsASC monolayers and microbeads cultured in CM had decreased FGF-2 gene expression and VEGF-A secretion compared to ASCs cultured in GM. Chondrocytes co-cultured with GM-cultured ASCs for 7 days had decreased mRNAs for col2, comp, and runx2. Chondrocytes treated for 12 or 24 hours with conditioned medium from GM-cultured ASCs had reduced sox9, acan, and col2 mRNAs; reduced proliferation and proteoglycan synthesis; and increased apoptosis. ASC-conditioned medium also increased endothelial cell tube lengthening whereas conditioned medium from CM-cultured ASCs had no effect. Treating ASCs with CM reduced or abolished these deleterious effects while adding a neutralizing antibody for VEGF-A eliminated ASC-conditioned medium induced chondrocyte apoptosis and restored proteoglycan synthesis. FGF-2 also mitigated the deleterious effects VEGF-A had on chondrocyte apoptosis and phenotype. When GM-grown ASC pellets were implanted in 1 mm non-critical hyaline cartilage defects in vivo, cartilage regeneration was inhibited as evaluated by radiographic and equilibrium partitioning of an ionic contrast agent via microCT imaging. Histology revealed that defects with GM-cultured ASCs had no tissue ingrowth from the edges of the defect whereas empty defects and defects with CM-grown ASCs had similar amounts of neocartilage formation.ConclusionsASCs must be treated to reduce the secretion of VEGF-A and other factors that inhibit cartilage regeneration, which can significantly influence how ASCs are used for repairing hyaline cartilage.
Highlights
Adipose stem cells (ASCs) secrete many trophic factors that can stimulate tissue repair, including angiogenic factors, but little is known about how ASCs and their secreted factors influence cartilage regeneration
Angiogenic growth factor production from ASCs ASC monolayers and microbeads cultured in growth medium (GM) had 1.5 to 3.0 times higher levels of fgf2 and vegfa compared to chondrocytes while chondrogenic medium (CM) reduced both vegfa and fgf2 to levels similar to that of chondrocytes (Figure 1B)
Effect of ASC paracrine signaling and secreted factors on chondrocyte gene expression Chondrocytes co-cultured with ASC monolayers cultured in GM experienced 3.5- to 4.0-fold reductions in col2, comp, and runx2 compared to chondrocytes with no co-culture (Figure 2B)
Summary
Adipose stem cells (ASCs) secrete many trophic factors that can stimulate tissue repair, including angiogenic factors, but little is known about how ASCs and their secreted factors influence cartilage regeneration. The secretory profile of ASCs for cartilage regeneration has been investigated [9], an in-depth study determining the role ASC-secreted factors have in chondrocyte proliferation, phenotype, and cartilage regeneration has yet to be conducted Techniques such as microencapsulation and differentiation medium treatments have been used to improve the regenerative capacity for mesenchymal stem cells [10,11,12], but no study has investigated the effects these two parameters have on the secretion of trophic factors from ASCs. the overall objective of this study was to determine the role ASC-secreted trophic factors have in cartilage regeneration. ASCs were implanted into a chondral defect in the xiphoid to determine the effect ASCs have on hyaline cartilage regeneration
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