Effect of dynamic 3‐D culture on proliferation, distribution, and osteogenic differentiation of human mesenchymal stem cells

Abstract

Ex vivo engineering of autologous bone tissue as an alternative to bone grafting is a major clinical need. In the present study, we evaluated the effect of 3-D dynamic spinner flask culture on the proliferation, distribution, and differentiation of human mesenchymal stem cells (MSCs). Immortalized human MSCs were cultured on porous 75:25 PLGA scaffolds for up to 3 weeks. Dynamically cultured cell/scaffold constructs demonstrated a 20% increase in DNA content (21 days), enhanced ALP specific activity (7 days and 21 days), a more than tenfold higher Ca2+ content (21 days), and significantly increased transcript levels of early osteogenesis markers (e.g., COL1A1, BMP2, RUNX-2) as compared with static culture. Despite the formation of a dense superficial cell layer, markedly increased cell ingrowth was observed by fluorescence microscopy on day 21. Furthermore, increased extracellular matrix deposition was visualized by scanning electron microscopy after 1 and 3 weeks of dynamic culture. The observed increased ingrowth and osteogenic differentiation of 3-D dynamically cultured human MSCs can be explained by generation of fluid shear stress and enhanced mass transport to the interior of the scaffold mimicking the native microenvironment of bone cells. This study provides evidence for the effectiveness of dynamic culture of human MSCs during the initial phase of ex vivo osteogenesis.

The effect of erythropoietin on bone

“…While conducting the experiments of Paper 2, the material safety classification of cetylpyridinium chloride was unfortunately altered and its use was prohibited at our institution. Therefore, before the altered version of the AZR protocol using 5% SDS in 0.5 M HCl for quantitative de-staining was tested and validated, an Arsenazo III assay was used in parts of Paper 2 as previously reported (Stiehler et al 2009). …”

A flow perfusion bioreactor with controlled mechanical stimulation: Application in cartilage tissue engineering and beyond

“…In another, adipose derived stem cells (ADSCs), inoculated into chitosan/gelatin hybrid hydrogels, and secreted signi icantly more proteoglycan in a spinner lask than in a static T-75 lask, as evidenced by stronger Alcian blue and Safranin O staining [16]. Spinner lasks also successfully induce both chondrogenesis and osteogenesis in bone-marrow-derived MSCs (BMSCs) [17][18][19]. Despite their bene its, spinner lasks can create turbulent low and high shear stresses at construct surfaces, leading to formation of ibrous capsules on the surface [20].…”

The effect of nano-scale topography on osteogenic differentiation of mesenchymal stem cells

“…There are a large number of reports on osteogenic differentiation that can result from mechanical stimuli [98][99][100] , including compression 84,101 and fluid shear stress 102 . Shear stress from the activation of mechanosensitive ion channels 103 , Ca 2+ channels 104,105 , heterotrimeric G-proteins, and protein kinases 102 enhances matrix mineralization and the expression of osteoblastic genes in human MSCs (ref. 99,100,106,107 ).…”

“…As signaling organelles, FA sites enable cells to touch their environment by transmitting mechanical and physical signals from the matrix to the cell 105,147,151 (Fig. 3).…”