FLIM imaging revealed spontaneous osteogenic differentiation of stem cells on gradient pore size tissue-engineered constructs

Abstract

Background

There is an urgent clinical need for targeted strategies aimed at the treatment of bone defects resulting from fractures, infections or tumors. 3D scaffolds represent an alternative to allogeneic MSC transplantation, due to their mimicry of the cell niche and the preservation of tissue structure. The actual structure of the scaffold itself can affect both effective cell adhesion and its osteoinductive properties. Currently, the effects of the structural heterogeneity of scaffolds on the behavior of cells and tissues at the site of damage have not been extensively studied.

Methods

Both homogeneous and heterogeneous scaffolds were generated from poly(L-lactic acid) methacrylated in supercritical carbon dioxide medium and were fabricated by two-photon polymerization. The homogeneous scaffolds consist of three layers of cylinders of the same diameter, whereas the heterogeneous (gradient pore sizes) scaffolds contain the middle layer of cylinders of increased diameter, imitating the native structure of spongy bone. To evaluate the osteoinductive properties of both types of scaffold, we performed in vitro and in vivo experiments. Multiphoton microscopy with fluorescence lifetime imaging microscopy was used for determining the metabolic states of MSCs, as a sensitive marker of cell differentiation. The results obtained from this approach were verified using standard markers of osteogenic differentiation and based on data from morphological analysis.

Results

The heterogeneous scaffolds showed improved osteoinductive properties, accelerated the metabolic rearrangements associated with osteogenic differentiation, and enhanced the efficiency of bone tissue recovery, thereby providing for both the development of appropriate morphology and mineralization.

Conclusions

The authors suggest that the heterogeneous tissue constructs are a promising tool for the restoration of bone defects. And, furthermore, that our results demonstrate that the use of label-free bioimaging methods can be considered as an effective approach for intravital assessment of the efficiency of differentiation of MSCs on scaffolds.

Keywords

Heterogeneous scaffolds, MSC, Bone defects, Multiphoton microscopy & FLIM.