Periodontitis is a chronic inflammatory disease affecting almost half of the adult US population. Gingiva is an integral part of the periodontium and has recently been identified as a source of adult gingiva-derived mesenchymal stem cells (GMSCs). Given the prevalence of periodontitis, the purpose of this study is to evaluate differences between GMSCs derived from healthy and diseased gingival tissues and explore their potential in bone engineering. Primary clonal cell lines were established from harvested healthy and diseased gingival and characterized for expression of known stem-cell markers and multi-lineage differentiation potential. Finally, they were cultured on electrospun polycaprolactone (PCL) scaffolds and evaluated for attachment, proliferation, and differentiation. Flow cytometry demonstrated cells isolated from healthy and diseased gingiva met the criteria defining mesenchymal stem cells (MSCs). However, GMSCs from diseased tissue showed decreased colony-forming unit efficiency, decreased alkaline phosphatase activity, weaker osteoblast mineralization, and greater propensity to differentiate into adipocytes than their healthy counterparts. When cultured on electrospun PCL scaffolds, GMSCs from both sources showed robust attachment and proliferation over a 7-day period; they exhibited high mineralization as well as strong expression of alkaline phosphatase. Our results show preservation of ‘stemness’ and osteogenic potential of GMSC even in the presence of disease, opening up the possibility of using routinely discarded, diseased gingival tissue as an alternate source of adult MSCs.

Guided tissue regeneration (GTR) aims to regenerate the lost attachment apparatus caused by periodontal disease through the use of a membrane. The goal of this study is to create and characterize a novel hybrid membrane that contains biologically active fibrin matrix within a synthetic polycaprolactone (PCL) electrospun membrane. Three-dimensional fibrin matrices and fibrin-incorporated electrospun membrane were created from fresh frozen plasma by centrifugation in glass vials under three different conditions: 400 g for 12 min, 1450 g for 15 min and 3000 g for 60 min. Half the membranes were crosslinked with 1% genipin. Degradation against trypsin indicated biologic stability while uniaxial tensile testing characterized mechanical properties. Continuous data was analyzed by ANOVA to detect differences between groups (p = 0.05). Fibrin-incorporated electrospun membranes showed statistically significant increase in mechanical properties (elastic modulus, strain at break and energy to break) compared to fibrin matrices. While crosslinking had marginal effects on mechanical properties, it did significantly increase biologic stability against trypsin (p < 0.0001). Lastly, membranes generated at 400 g and 1450 g were superior in mechanical properties and biologic stability compared to those generated at 3000 g. Fibrin-incorporated, crosslinked electrospun PCL membranes generated at lower centrifugation forces offers a novel strategy to generate a potentially superior membrane for GTR procedures.

Synthetic polymers offer control over composition, architecture, mechanical properties and degradation kinetics. Predictable sterilization of synthetic polymeric scaffolds made from low temperature melting polymers, remains a challenge to clinical translation. We previously demonstrated successful room temperature sterilization of electrospun polycaprolactone scaffolds (ePCL) using peracetic acid (PA). The current paper investigates the effects of PA sterilization on two different scaffolds types—ePCL and commercially available porous polystyrene (Alvetex^®) scaffolds using mouse calvarial osteoblasts cell line (MC3T3) and Live-Dead Assay. We report cytotoxicity in PA-treated ePCL scaffolds (PA-ePCL), while control scaffolds strongly supported cell survival. Treatment of PA-ePCL scaffolds with known methods of PA residual elimination (sodium thiosulfate, catalase, washing and aeration) had minimal effect on MC3T3 survival. However, incubation in 80% ethanol for 30 min successfully eliminated the toxic PA residuals and restored scaffold cytocompatibility. On the other hand, PA treatment of Alvetex^® scaffolds induced diametrically opposite effects: cell survival and proliferation was enhanced after PA exposure and these responses were reversed following ethanol wash. These results suggest that PA treatment can induce different biological effects based on polymer chemistry and scaffold architecture and presents interesting opportunities to modulate biological properties of tissue engineering scaffolds.