THE USE OF CONFOCAL LASER SCANNING MICROSCOPY TO ASSESS THE POTENTIAL SUITABILITY OF 3D SCAFFOLDS FOR TISSUE REGENERATION, BY MONITORING EXTRA‐CELLULAR MATRIX DEPOSITION AND BY QUANTIFYING CELLULAR INFILTRATION AND PROLIFERATION

Abstract

The great many varieties of dermal regeneration materials currently being developed requires that sound in vitro assessments of their potential to be infiltrated with host cells are needed prior to any in vivo studies. The biocompatibility of scaffolds is conventionally determined by indirect biochemical assays as measures of cell number/viability within these materials. A disadvantage of these methods is the failure to provide information pertaining to the spatial distribution of cells in situ. In the case of dermal regeneration, if cells are only present in appreciable numbers on the outer portion of a scaffold, then critical aspects such as extra‐cellular matrix (ECM) production and vascularization will be limited, and the likelihood of successful tissue regeneration low. Thus, direct enumeration of cells within scaffolds, with respect to spatial distribution and time, is required to adequately assess the potential biocompatibility. In this article we demonstrate a systematic approach to the quantification of infiltration and proliferation of primary human dermal fibroblasts into the dermal regeneration scaffold, Integra^® (the most frequently used dermal regeneration template in burns patients), using confocal laser scanning microscopy (CLSM). Scaffold samples were placed on top of confluent cell layers and infiltrated by cells migrating from the culture dish against gravity. Cells were then enumerated (by nuclear staining) with respect to their location within the scaffold and over a time period of up to 28 days. Large increases in cell numbers were observed on the surface of the scaffolds, together with measurable increases in cell numbers within their interiors. We also describe the immunofluorescent staining of fibroblasts and ECM components as well as the subsequent use of CLSM to qualitatively assess the potential for scaffold re‐modeling. Of particular note was the cellular deposition of a “front” of fibronectin matching the maximum extent of cell infiltration at each time point. We suggest that this key ECM component is a useful indicator of the extent of cellular re‐modeling of a biomaterial.

Keywords:

• α‐smooth muscle actin
• Alexafluor
• Biocompatibility
• Collagen I
• Fibronectin
• Image analysis

ACKNOWLEDGMENTS

This work was funded by The Technology Strategy Board (TSB) (project number: TP/4/BIO/6/1/22353). TSB is a business‐led executive non‐departmental public body, established by the government. Its mission is to promote and support research into, and development and exploitation of, technology and innovation for the benefit of U.K. business, in order to increase economic growth and improve the quality of life. It is sponsored by the Department for Innovation, Universities and Skills (DIUS). Please visit www.innovateuk.org for further information.

The primary human fibroblast cell strain SKF371 was produced in‐house as part of a cell bank developed during a DTI‐funded project (MMP4.5).