Growth and Osteogenic Differentiation of Discarded Gingiva-Derived Mesenchymal Stem Cells on a Commercial Scaffold

Abstract

Background: In periodontal patients with jawbone resorption, the autologous bone graft is considered a "gold standard" procedure for the placing of dental prosthesis; however, this procedure is a costly intervention and poses the risk of clinical complications. Thanks to the use of adult mesenchymal stem cells, smart biomaterials, and active biomolecules, regenerative medicine and bone tissue engineering represent a valid alternative to the traditional procedures.

Aims: In the past, mesenchymal stem cells isolated from periodontally compromised gingiva were considered a biological waste and discarded during surgical procedures. This study aims to test the osteoconductive activity of FISIOGRAFT Bone Granular^® and Matriderm^® collagen scaffolds on mesenchymal stem cells isolated from periodontally compromised gingiva as a low-cost and painless strategy of autologous bone tissue regeneration.

Materials and methods: We isolated human mesenchymal stem cells from 22 healthy and 26 periodontally compromised gingival biopsy tissues and confirmed the stem cell phenotype by doubling time assay, colony-forming unit assay, and expression of surface and nuclear mesenchymal stem cell markers, respectively by cytofluorimetry and real-time quantitative PCR. Healthy and periodontally compromised gingival mesenchymal stem cells were seeded on FISIOGRAFT Bone Granular^® and Matriderm^® scaffolds, and in vitro cell viability and bone differentiation were then evaluated.

Results: Even though preliminary, the results demonstrate that FISIOGRAFT Bone Granular^® is not suitable for in vitro growth and osteogenic differentiation of healthy and periodontally compromised mesenchymal stem cells, which, instead, are able to grow, homogeneously distribute, and bone differentiate in the Matriderm^® collagen scaffold.

Conclusion: Matriderm^® represents a biocompatible scaffold able to support the in vitro cell growth and osteodifferentiation ability of gingival mesenchymal stem cells isolated from waste gingiva, and could be employed to develop low-cost and painless strategy of autologous bone tissue regeneration.

Keywords: FISIOGRAFT Bone Granular®; Matriderm®; autologous bone tissue regeneration; bone resorption; oral MSCs; periodontal disease; periodontally compromised GMSCs; waste gingival tissue.

FIGURE 1

GMSC cultures (P0). Representative image of (A) healthy and (B) periodontally compromised GMSCs immediately after mechanical and enzymatic digestion, showing a rounded morphology (10×); representative image of (C) healthy and (D) periodontally compromised GMSCs at 7th day from digestion, with the typical fibroblast-like morphology (10×).

FIGURE 2

Cell growth analysis and colony-forming unit assay. Panels (A) and (B) respectively show the cell growth curve of H-GMSCs and P-GMSCs (P2) evaluated by Trypan blue viability assay and the doubling time of H-GMSCs and P-GMSCs calculated according to the literature data (http://www.doublingtime.com/compute.php); Panels (C) and (D) respectively show the colonies (<50 cells) (left) and the monolayer subculture (right) of H-GMSCs and P-GMSCs (P1) stained with Crystal Violet, and the quantification histogram of the colony-forming unit assay (CFU-F); data are reported as mean values ± SD of three independent experiments. P-value *P ≤ 0.05; **P ≤ 0.01.

FIGURE 3

Mesenchymal stem cell feature analysis. Representative fields of flow-cytometric analysis of (A) hematopoietic stem cell markers CD45 and HLA-DR and (B) MSC markers CD29, CD73, CD90, and CD105 in H-GMSCs and P-GMSCs (P5) (control: isotype anti-IgG1 for CD45, CD29, CD90, CD73, and CD105; isotype anti-IgG2 for HLA-DR); (C) the histogram shows the expression of nuclear MSC markers NANOG, Oct4, and SOX-2 in H-GMSCs and P-GMSCs (P3). Data are reported as mean values ± SD of three independent experiments. Actin-β was used as the housekeeping gene; FC = fold change; the mRNA expression of analyzed genes was normalized against BM-MSCs (positive control); P-value *P ≤ 0.05.

FIGURE 4

Cell viability analysis. (A) WST1 viability values of H-GMSCs and P-GMSCs (P3) grown in the FISIOGRAFT Bone Granular^® scaffold for 24, 48, and 72 h; (B) MTT viability values of H-GMSCs and P-GMSCs (P3) grown in the Matriderm^® collagen scaffold for 24, 48, and 72 h; data are reported as mean values ± SD of three independent experiments; P-values *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

FIGURE 5

Cell distribution analysis. (A) Fluorescent representative images of a Live/Dead assay of H-GMSCs and P-GMSCs (P5) grown for 24, 48, and 72 h in the Matriderm^® collagen scaffold (4X); (B) (left) MaxI P and (right) volumetric images of DAPI/Actin Green confocal microscopy assay of H-GMSCs and P-GMSCs (P5) grown for 2, 7, and 10 days in the Matriderm^® collagen scaffold (4X); scale bars = 100 μm; depth = 190,336 μm for H-GMSCs; depth = 182,80 μm for P-GMSCs.

FIGURE 6

Osteoblastic differentiation assay. (A) Representative images of control H-GMSCs and P-GMSCs (P3) grown in osteogenic differentiation medium (ODM), with or without Biochanin A 300 nM and 1 μM, and stained with Red S Alizarin (4×); (B) histogram representing the quantitative analysis of Red S Alizarin by spectrophotometry (550 nm OD), of H-GMSCs and P-GMSCs (P3) grown in ODM, in presence or non-presence of the Matriderm^® collagen scaffold, with or without Biochanin A 300 nM and 1 μM; (C) histogram showing the relative mRNA expression of the osteoblastic markers Runx2, OPN, and OCN in H-GMSCs and P-GMSCs (P3) grown in ODM, in presence or non-presence of the Matriderm^® collagen scaffold, with or without Biochanin A 300 nM and 1 μM. Actin-β was used as the housekeeping gene; FC = fold change.