Characterization of the soft-tissue wall lining residual periodontal pockets and implications in periodontal wound healing.

AIM: To characterize the soft-tissue wall of remaining periodontal pockets for wound healing-related parameters versus healthy gingival crevices in the same individuals. MATERIALS AND METHODS: Gingival tissues collected from the diseased interface of pockets (GT biopsies) and from healthy gingival crevices (G biopsies) were subjected to RT2-profiler PCR Array for wound healing-related markers and network analysis of differentially expressed genes. Lymphangiogenesis-related gene expression was determined by qRT-PCR. The migration potential of mesenchymal stem cells isolated from GT biopsies (GT-MSCs) and G biopsies (G-MSCs) was evaluated by the scratch- and the transwell migration assays. The total collagen protein content was determined in GT-MSCs and G-MSCs homogenates. RESULTS: Gene-ontology analysis on significantly upregulated genes expressed in GT biopsies revealed enrichment of several genes involved in processes related to matrix remodeling, collagen deposition, and integrin signaling. No significantly expressed genes were seen in G biopsies. Regarding lymphangiogenesis-related genes, GT biopsies demonstrated greater expression for PROX1 than G biopsies (p = 0.05). Lower migration potential (p < 0.001), yet greater production of collagen protein (p = 0.05), was found for GT-MSCs over G-MSCs. CONCLUSION: Differential expression patterns of various molecular pathways in biopsies and cell cultures of diseased versus healthy gingival tissues indicate a potential of the former for tissue remodeling and repair. CLINICAL RELEVANCE: In the course of periodontitis, granulation tissue is formed within a periodontal defect in an attempt to reconstruct the site. Following treatment procedures periodontal granulation tissue remains inflamed but appears to retain healing potential.

Minor salivary gland stem cells: a comparative study of the biological properties under clinical-grade culture conditions.

Development of clinical-grade, cell preparations is central to cGMP (good manufacturing practice compliant) conditions. This study aimed to investigate the potential of two serum/xeno-free, cGMP (StemPro, StemMacs) culture media to maintain "stemness" of human minor salivary gland stem cell (mSG-SC) cultures compared to a complete culture medium (CCM). Overall, StemMacs resulted in higher proliferation rates after p.6 compared to the conventional serum-based medium, while StemPro showed substantial delays in cell proliferation after p.9. The mSG-SCs cultures exhibited two distinct cell populations at early passages a mesenchymal subpopulation and an epithelial-like subpopulation. Expression of several markers (CD146, STRO-1, SSEA-4, CD105, CD106, CD34, K 7/8, K14, K18) variably decreased with prolonged passaging (all three media). The percentage of SA-ß-gal positive cells was initially higher for StemMacs compared to StemPro/CCM and increased with prolonged passaging in all cases. The telomere fragment length decreased with prolonged passaging in all three media but more pronouncedly for the CCM. Expansion under serum-free conditions caused pronounced upregulation of ALP and BMP-2, with parallel complete elimination of the baseline expressions of LPL (all three media) and ACAN (serum-free media), therefore, showing a preferential shift of the mSG-SCs towards osteogenic phenotypes. Finally, several markers (Nanog, SOX-2, PDX-1, OTX2, GSC, HCG) decreased with prolonged culture, indicating successive loss of "stemness". Based on the findings, it seems that StemPro preserve stemness of the mSG-SCs after prolonged culture. Nevertheless, there is still a vacant role for the ideal development of clinical-grade culture conditions.

Evaluation of stemness properties of cells derived from granulation tissue of peri-implantitis lesions.

OBJECTIVES: Peri-implantitis (PI) is an inflammatory disease associated with peri-implant bone loss and impaired healing potential. There is limited evidence about the presence of mesenchymal stromal cells (MSCs) and their regenerative properties within the granulation tissue (GT) of infrabony peri-implantitis defects. The aim of the present study was to characterize the cells derived from the GT of infrabony PI lesions (peri-implantitis derived mesenchymal stromal cells-PIMSCs). MATERIAL AND METHODS: PIMSC cultures were established from GT harvested from PI lesions with a pocket probing depth ≥6 mm, bleeding on probing/suppuration, and radiographic evidence of an infrabony component from four systemically healthy individuals. Cultures were analyzed for embryonic (SSEA4, NANOG, SOX2, OCT4A), mesenchymal (CD90, CD73, CD105, CD146, STRO1) and hematopoietic (CD34, CD45) stem cell markers using flow cytometry. PIMSC cultures were induced for neurogenic, angiogenic and osteogenic differentiation by respective media. Cultures were analyzed for morphological changes and mineralization potential (Alizarin Red S method). Gene expression of neurogenic (NEFL, NCAM1, TUBB3, ENO2), angiogenic (VEGFR1, VEGFR2, PECAM1) and osteogenic (ALPL, BGLAP, BMP2, RUNX2) markers was determined by quantitative RT-PCR. RESULTS: PIMSC cultures demonstrated high expression of embryonic and mesenchymal stem cell markers with inter-individual variability. After exposure to neurogenic, angiogenic and osteogenic conditions, PIMSCs showed pronounced tri-lineage differentiation potential, as evidenced by their morphology and expression of respective markers. High mineralization potential was observed. CONCLUSIONS: This study provides evidence that MSC-like populations reside within the GT of PI lesions and exhibit a multilineage differentiation potential. Further studies are needed to specify the biological role of these cells in the healing processes of inflamed PI tissues and to provide indications for their potential use in regenerative therapies.

Effects of orthodontic forces on bone turnover biomarkers in peri-miniscrew crevicular fluid: A systematic review.

OBJECTIVE: Peri-miniscrew crevicular fluid (PMCF) analysis of biomarkers representing bone formation or resorption could provide a non-invasive way to monitor bone turnover around miniscrews and the response to force loading. Our objective was to systematically investigate the relevant available evidence. MATERIALS AND METHODS: Search without restrictions in eight databases and hand searching until March 2020 took place. We searched for prospective human studies measuring the levels of markers of bone formation and resorption in PMCF under the effect of orthodontic forces. Following study retrieval and selection, relevant data was extracted and the risk of bias was assessed following the Cochrane Collaboration guidelines. RESULTS: Four studies, two randomized and two non-randomized, were finally identified, following miniscrews for a period up to 90 days. Loading of miniscrews led to a transient increase in C-telopeptide of type I collagen amounts. Temporary increases were also observed for the enzymes: alkaline phosphatase and aspartate aminotransferase. Under the effect of orthodontic loading the total amount of Receptor Activator of Nuclear Factor-κB Ligand (RANKL) in the PMCF consistently increased compared to the unloaded group, at all sampling points. These changes led to a stable decrease in the osteoprotegerin (OPG)/RANKL ratio under force application throughout the study period, as OPG in this group, together with OPG and RANKL in the unloaded group, remained mostly unchanged. No differences were detected for the total OPG quantity between the two loading groups. The levels of bone specific alkaline phosphatase and chondroitin sulfate did not change significantly during observation. All studies presented some issues of concern regarding the risk of bias. CONCLUSION: Biomarkers of bone turnover in PMCF showed variable responses following orthodontic loading. Overall, the findings were suggestive of adaptive bone alterations to physiologic force stimuli.

Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects "stemness" properties.

BACKGROUND: Development of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term "stemness" of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion. METHODS: DPSC and aBMMSC cultures (n = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (ß-galactosidase (SA-ß-gal) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR. RESULTS: Similar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6-7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in "osteogenic pre-disposition", evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6-7 under all expansion conditions. CONCLUSIONS: These findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics.