Osteoblastic Differentiation of HMSCs on 3D-Scaffolds is Reduced without Microtopography

Objective: Mesenchymal stem cells (MSCs) provide a multipotent cell source for tissue engineering, particularly MSCs seeded in 3D structures or scaffolds. Bone tissue engineering requires osteoblastic differentiation of progenitor cells. This differentiation is generally obtained by osteogenic induction media in vitro before subsequent implantation in vivo. Several biomaterial surface modifications have been applied to induce MSC differentiation. In vitro, surface roughness induces osteoblastic differentiation of MSCs, increasing osteogenic gene expression and creating an osteogenic microenvironment. However, it is not clear whether 3D scaffolds have the potential to induce MSCs osteoblastic differentiation in the absence of exogenous growth factors.

Method: Human MSCs were cultured for 7 days in MSC growth medium on 2D substrates [tissue culture polystyrene (TCPS), hydroxyapatite (HA), or sandblasted/acid-etched titanium (SLA)] or 3D [TCPS, polycaprolactone (PCL)] scaffolds. Expression of integrins, BMPs, Wnts, and osteogenic markers were measured by real-time qPCR. In a second set of samples, DNA, alkaline phosphatase, osteocalcin, osteoprotegerin, BMP-2, BMP-4, VEGF, and FGF were measured (n=6/condition, ANOVA/Bonferroni’s modified Student’s t-test).

Result: Cells on SLA expressed more RUNX2, BGLAP, BMP2, BMP4, WNT5A, ITGA1, ITGA2, ITGAV, and ITGB1 in comparison to all materials tested. 3D scaffolds increased ITGA5 and WNT3A in comparison with HA and SLA. 3D-TCPS and PCL showed the highest DNA content. SLA and HA increased alkaline phosphatase, osteocalcin, osteoprotegerin, BMP-2, BMP-4, VEGF, and FGF in comparison to 2D and 3D-TCPS and PCL. The most robust effect was observed in SLA surfaces. Interestingly, protein levels on 3D-TCPS and PCL were similar to 2D-TCPS.

Conclusion: The data indicate that microtexture induces osteogenic differentiation, increasing osteogenic gene expression and producing an osteogenic niche. Our study demonstrates that a 3D environment in the absence of microstructure and exogenous factors is not sufficient to induce osteogenic differentiation and suggest that surface modification of 3D structures can control MSC fate.