Bringing osteogenic activity to MC3T3-E1-loaded microscaffolds

Abstract

Bone-related pathologies and injuries represent a complex matter of study due to the major limitations of tissue self-healing and the diverse factors into play. When treating large defects, above the critical size, the native repair process results to be insufficient and the incorporation of engineered materials and devices is fundamental to achieve a full recovery. In this context, regenerative medicine plays a pivotal role in the development and evaluation of new solutions, allowing to address a wider range of cases and conditions. Novel approaches aim to exploit and merge stem cell differentiation with diverse stimulating agents, whether synthetically or naturally derived. Autografts are currently considered the gold standard procedure in this field The graft provides osteogenic and osteoinductive cues alongside stem cells while restoring the mechanical and biological properties of the damaged tissue However, limiting factors in their application are high surgical costs and times, and an increased risk of comorbidities at the donor site. Scaffold-based strategies are preferred as alternative option due to the possibility to tune the device characteristics to the intended use and gain better control on different aspects of the procedure . A broad range of biocompatible materials can be examined to support cell expansion and differentiation, also allowing to incorporate further factors in the final construct. BMP 2 is one of the most frequently examined osteogenic factors in bone regeneration, alongside ceramic compounds resembling the native tissue composition and properties. In the current study, a recently developed microscaffold was utilised to guide and sustain cell culture. The main purpose was to gain better insight into the achievable differentiation with the combination of diverse factors. MC3T3 E1 cells have been selected for their capability to differentiate into osteoblasts when exposed to BMP 2. This latter aspect has been tested and confirmed in 2D culture first, measuring the promoted extracellular mineralization via Alizarin Red S ARS ) staining. Afterwards, a three-dimensional setup was designed with the involvement of a fullerene-shaped microscaffold, named buckyball (BB), able to shield cellular aggregates from mechanical damages and allow for the integration of other cues, such as calcium microparticles and BMP 2. Their inclusion was expected to promote major osteodifferentiation in respect of standard culture conditions. Samples were analysed with ARS and Calcein Green staining, and qPCR over a 14 days culture period BMP 2 was proven to trigger a higher calcium deposition, similarly to the 2D case. Furthermore, the combination of BMP 2 and calcium microparticles seemed to accelerate the differentiation process, with osteogenic markers' overexpression and matrix remodelling taking place after only one week of culture. However, the control groups also showed minor mineral content and gene upregulation, suggesting that the passage to three-dimensional conditions could have been sufficient to trigger some differentiation. The MC3T3 E1 cell line was thus considered suitable for testing growth factors-functionalized microscaffolds for regenerative solutions in bone repair, opening up to further applications to find the most effective combination of osteogenic factors to achieve a greater regenerative potential.