Cell-Secreted Matrices Enhance Osteogenesis in Adipose-Derived Stem Cells Undergoing Lineage Specification

Abstract

Osteogenic differentiation of mesenchymal stem cells (MSC) is important in the field of bone tissue engineering. The identification of biological factors that influence osteogenesis is vital for developing a broader understanding of how complex microenvironments play a role in differentiation. The aim of this study was to demonstrate that adipose-derived stem cell (ADSC) osteogenesis is enhanced through interaction with extracellular matrices (ECM) secreted by ADSC undergoing osteogenesis. ADSC were obtained from human patients following elective abdominoplasty. Cells were selected for plastic adherence, characterized, and induced to differentiate using osteogenic supplements (OS; dexamethasone, ascorbic acid, and beta-glycerol phosphate). Cells were removed at several time points during osteogenesis and the secreted ECM was isolated. Undifferentiated cells were re-seeded onto the cell secreted ECMs and induced to differentiate with OS. At several time points, cells cultured on ECMs or tissue culture plastic controls (i.e. uncoated surface) were collected and RNA isolated. QPCR and gene array analysis revealed enrichment of osteogenic markers and more rapid progression through osteogenic maturational phases in cells seeded onto ECM secreted at the midpoint in differentiation (ca. 15 days). Our results demonstrate that the cumulative deposition of ECM reaches a critical point at approximately 15 days, before which there appear to be no definitive osteogenic cues from the matrix, and after which, strong drivers of osteogenesis are present. The creation of microenvironments that contain essential morphogenic matrix signals is an important step towards methods of growing and differentiating MSC in a rapid effective manner, particularly for bone-related clinical applications.

Dermatopontin in the extracellular matrix enhances osteogenic differentiation of adipose-derived mesenchymal stem cells

“…Western blot confirmation of higher DPT protein expression in ADSC at the midpoint in differentiation versus ADSC at earlier in differentiation Our previous work demonstrates that ADSC at the midpoint in osteogenesis secrete osteogenesis-enhancing ECM [10]. Alternatively, ADSC at an earlier time point in differentiation secrete osteogenesis-inhibiting ECM.…”

“…However, small, non-collagenous, matricellular ECM components may have equally or more important roles in modulating the differentiation process and are much less studied. For this reason, we selected DPT, a small extracellular protein highly expressed at the gene and protein levels at the midpoint in ADSC osteogenesis [10], for overexpression studies to determine its effect on osteogenesis. Our previous work [10] suggests that ADSC secrete osteogenic matrices at day 16 during differentiation.…”

“…For this reason, we selected DPT, a small extracellular protein highly expressed at the gene and protein levels at the midpoint in ADSC osteogenesis [10], for overexpression studies to determine its effect on osteogenesis. Our previous work [10] suggests that ADSC secrete osteogenic matrices at day 16 during differentiation. To determine potential proteins responsible for this enhanced activity, we identified genes that code for ECM components that were upregulated at day 16 in ADSC differentiation -one of which was DPT.…”

“…Human ADSC were obtained, isolated, and characterized as previously described [10] under a protocol approved by the Wake Forest School of Medicine Institutional Review Board (no. IRB00007586).…”

“…In a previous study, we identified the DPT gene as being differentially expressed during adipose-derived stem cell (ADSC) osteogenic differentiation [10]. We demonstrated that matrices secreted by ADSC at the midpoint in a 30-day cell culture differentiation protocol enhanced osteogenesis.…”