Enhanced Differentiation of Human Embryonic Stem Cells on Extracellular Matrix-Containing Osteomimetic Scaffolds for Bone Tissue Engineering

Abstract

Current methods of treating critical size bone defects include autografts and allografts, however, both present major limitations including donor-site morbidity, risk of disease transmission, and immune rejection. Tissue engineering provides a promising alternative to circumvent these shortcomings through the use of autologous cells, three-dimensional scaffolds, and growth factors. We investigated the development of a scaffold with native bone extracellular matrix (ECM) components for directing the osteogenic differentiation of human embryonic stem cells (hESCs). Toward this goal, a microsphere-sintering technique was used to fabricate poly(lactic-co-glycolic acid) (PLGA) scaffolds with optimum mechanical and structural properties. Human osteoblasts (hOBs) were seeded on these scaffolds to deposit bone ECM for 14 days. This was followed by a decellularization step leaving the mineralized matrix intact. Characterization of the decellularized PLGA scaffolds confirmed the deposition of calcium, collagen II, and alkaline phosphatase by osteoblasts. hESCs were seeded on the osteomimetic substrates in the presence of osteogenic growth medium, and osteogenicity was determined according to calcium content, osteocalcin expression, and bone marker gene regulation. Cell proliferation studies showed a constant increase in number for hESCs seeded on both PLGA and ECM-coated PLGA scaffolds. Calcium deposition by hESCs was significantly higher on the osteomimetic scaffolds compared with the control groups. Consistently, immunofluorescence staining demonstrated an increased expression of osteocalcin in hESCs seeded on ECM-coated osteomimetic PLGA scaffolds. Gene expression analysis of RUNX2 and osteocalcin further confirmed osteogenic differentiation of hESCs at the highest expression level on osteomimetic PLGA. These results together demonstrate the potential of PLGA scaffolds with native bone ECM components to direct osteogenic differentiation of hESCs and induce bone formation.

Optimization of Polymer-ECM Composite Scaffolds for Tissue Engineering: Effect of Cells and Culture Conditions on Polymeric Nanofiber Mats

“…This requires the polymer to have functionalizable pendant chains or end groups [29]. A less common, but increasingly investigated approach is to use cells to deposit their native ECM onto the scaffold, followed by decellularization and the use of this ECM-polymer composite scaffold as a bioactive support for subsequent recellularization [33,34,35]. This approach (which we refer to as the cellularization-decell-recell cycle) harnesses the naturally-occurring bioactive signals from a selected cell source to modify the biological responses of subsequently seeded cell populations.…”

“…This approach (which we refer to as the cellularization-decell-recell cycle) harnesses the naturally-occurring bioactive signals from a selected cell source to modify the biological responses of subsequently seeded cell populations. For instance, scaffolds containing human osteoblast-derived ECM were shown to enhance osteogenic differentiation of human embryonic stem cells [35]. In order for cells to deposit their native ECM, the scaffold should allow cellular attachment and proliferation, which requires basic cell-adhesiveness of the scaffold.…”

Biomaterials control of pluripotent stem cell fate for regenerative therapy

“…Although the uncoated microspheres also induced mouse ESCs toward an osteogenic lineage with the supply of differentiating soluble factors, the ECM-coated microspheres significantly enhanced the osteogenesis and maturation of cells with higher levels of osteocalcin expression and calcium deposition than the uncoated microspheres (Fig. 5.4) [211]. The ECM of osteoblasts, holding sufficient level of biochemical molecules and biophysical properties mimicking native bone tissue, may properly guide PSCs to change their fate into an osteogenic lineage.…”

Pluripotent Stem Cells and Skeletal Regeneration—Promise and Potential

“…Jukes et al differentiated ES cells into a cartilage matrix to recapitulate endochondral ossification, and when these matrices were transplanted into critical-size cranial defects in rats, they underwent cartilage hypertrophy, calcification, and ultimately replacement by bone (25). Other scaffolds that have been tested include decellularized osteoblast-derived extracellular matrix alone (26, 27) or on a PLGA scaffold (28), microcarriers (29), type I collagen gel (30), and hydroxyapatite/tricalcium phosphate (HA/TCP) (24, 31). Furthermore, investigators have tried to simulate mechanical loading in culture with approaches such as cyclic loading in a compression chamber (32) or culture on BioFlex plates (33).…”