3D culture systems have the ability to mimic the natural microenvironment by allowing better cell-cell interactions. We have prepared an in vitro 3D osteogenic cell culture model using human adipose derived stem cells (hASCs) cultured atop recombinant elastin-like polypeptide (ELP) conjugated to a charged polyelectrolyte, polyethyleneimine (PEI). We demonstrate that hASCs cultured atop the ELP-PEI coated tissue culture polystyrene (TCPS) formed 3D spheroids and exhibited superior differentiation toward osteogenic lineage compared to the traditional two dimensional (2D) monolayer formed atop uncoated TCPS. Live/dead viability assay confirmed >90% live cells at the end of the 3-week culture period. Over the same culture period, higher protein content was observed in 2D monolayer than 3D spheroids, as the 2D environment allowed continued proliferation, while 3D spheroids underwent contact-inhibited growth arrest. The normalized alkaline phosphatase (ALP) activity, which is an indicator for early osteogenic differentiation was higher for 3D spheroids. The normalized osteocalcin (OCN) production, which is an indicator for osteogenic maturation was also higher for 3D spheroids while 2D monolayer had no noticeable OCN production. On day 22, increased Alizarin red uptake by 3D spheroids showed greater mineralization activity than 2D monolayer. Taken together, these results indicate a superior osteogenic differentiation of hASCs in 3D spheroid culture atop ELP-PEI coated TCPS surfaces than the 2D monolayer formed on uncoated TCPS surfaces. Such enhanced osteogenesis in 3D spheroid stem cell culture may serve as an alternative to 2D culture by providing a better microenvironment for the enhanced cellular functions and interactions in bone tissue engineering.

Objectives Collagen-based scaffolds for guided bone regeneration (GBR) are continuously improved to overcome the mechanical weaknesses of collagen. We have previously demonstrated superior mechanical characteristics of the elastin-like polypeptide (ELP) reinforced collagen composites. The objectives of this research were to evaluate the efficacy of ELP-collagen composites to culture human adipose-derived stem cells (hASCs) and allow them to undergo osteogenic differentiation. We hypothesized that hASCs would show a superior osteogenic differentiation in stiffer ELP-collagen composites compared to the neat collagen hydrogels. Methods Composite specimens were made by varying ELP (0–18 mg/mL) and collagen (2–6 mg/mL) in a 3:1 ratio. Tensile strength, elastic modulus, and toughness were determined by uniaxial tensile testing. hASCs cultured within the composites were characterized by biochemical assays to measure cell viability, protein content, and osteogenic differentiation (alkaline phosphatase activity, osteocalcin, and Alizarin red staining). Scanning electron microscopy and energy dispersive spectroscopy was used for morphological characterization of composites. Results All composites were suitable for hASCs culture with viable cells over the 22-day culture period. The ELP-collagen composite with 18 mg/mL of ELP and 6 mg/mL of collagen had greater tensile strength and elastic modulus combined with higher osteogenic activity in terms of differentiation and subsequent mineralization over a period of 3 weeks compared to other compositions. The extra-cellular matrix deposits composed of calcium and phosphorous were specifically seen in the 18:6 mg/mL ELP-collagen composite. Significance The success of the 18:6 mg/mL ELP-collagen composite to achieve long-term, 3-dimensional culture and osteogenic differentiation of indicates its potential as a GBR scaffold.