Polyblend Nanofibers to Regenerate Gingival Tissue: A Preliminary In Vitro Study

Abstract

Aim: The regeneration of small periodontal defects has been considered an important divide and challenging issue for dental practitioners. The aim of this preliminary in vitro study was to analyze the effects of polycaprolactone (PCL) nanofibers enriched with hyaluronic acid and vitamin E vs. nude nanofibers on gingival fibroblasts activity, an innovative graft for periodontal soft tissue regeneration purposes.Methods: Nanofibers were produced in PCL (NF) or PCL enriched with hyaluronic acid and vitamin E (NFE) by electrospinning technique. NF and NFE were stereologically and morphologically characterized by scanning electron microscope (SEM), and composition was analyzed by infrared spectroscopy. Human fibroblasts were obtained from one gingival tissue fragment (HGF) and then seeded on NF, NFE, and plastic (CT). Cell adhesion and morphology were evaluated using SEM at 24 h and cell viability after 24, 48, and 72 h by alamarBlue® assay. Gene expression for COL-I, LH2b, TIMP-1, PAX, and VNC was analyzed by real-time RT-PCR in samples run in triplicate and GAPDH was used as housekeeping gene. Slot blot analysis was performed and immunoreactive bands were revealed for MMP-1 and COL-I. YAP and p-YAP were analyzed by Western blot and membranes were reprobed by α-tubulin. Statistical analysis was performed.Results: IR spectrum revealed the presence of PCL in NF and PCL and vitamin E and hyaluronic acid in NFE. At 24 h, HGF adhered on NF and NFE conserving fibroblast like morphology. At 72 h from seeding, statistically significant differences were found in proliferation of HGF cultured on NF compared to NFE. Expression of genes (LH2b, TIMP-1, and MMP-1) and proteins (COL-I) related to collagen turnover revealed a reduction of COL-1 secretion in cells cultured on NF and NFE compared to CT; however, NFE stimulated cross-linked collagen deposition. Mechanosensor genes (PAX, VNC, and YAP) were upregulated in HGF on NF while they were decreased in cells grown on NFE.Conclusion: Preliminary data suggest that PCL-enriched nanofibers could represent a support to induce HGF proliferation, adhesion, collagen cross-linking, and to reduce collagen degradation, therefore favoring collagen deposition in gingival connective tissue.

Clinical, histological, immunohistochemical, and biomolecular analysis of hyaluronic acid in early wound healing of human gingival tissues: A randomized, split‐mouth trial

“…A customized digital counting grid was employed to evaluate the MVD of each tissue slide through histomorphometric analysis. The intersection points of the grid that fell on the vessels were manually counted, and the ratio between test points and total points of the grid that fell on the overall connective tissue was calculated and expressed as a percentage value 28 …”

“…Considering the absence of previous studies providing data on the in vivo effect of HA in G early wound healing, the sample size was calculated based on data from a study of Canullo et al., 31 in which the MVD average was from 8% to 12% with a standard deviation of 3%. Therefore, the population of the study was calculated to be n > 7.35 28,31 . Considering a dropout of 15%, eight patients were required.…”

“…The intersection points of the grid that fell on the vessels were manually counted, and the ratio between test points and total points of the grid that fell on the overall connective tissue was calculated and expressed as a percentage value. 28…”

“…Sirius red staining ‡ was performed to stain and analyze interstitial fibrillary collagen organization and content. 28 Slides were deparaffinized and immersed for 30 min in saturated aqueous picric acid containing 0.1% sirius red F3BA § and were observed under polarized light ¶ in order to distinguish the newly deposited collagen, describe collagen maturation, and evaluate collagen fiber arrangement.…”

“…Therefore, the population of the study was calculated to be n > 7.35. 28,31 Considering a dropout of 15%, eight patients were required.…”