A Soancă1, M Lupse2, M Moldovan3, E Pall4, M Cenariu4, A Roman5, O Tudoran6, P Surlin7, O Șorițău8. 1. Department of Periodontology, Faculty of Dental Medicine, Iuliu Haţieganu University of Medicine and Pharmacy, 15 V. Babeş St., 400012 Cluj-Napoca, Romania. 2. Department of Infectious Diseases, Faculty of Medicine, Iuliu Haţieganu University of Medicine and Pharmacy, 23 Iuliu Moldovan St., 400349 Cluj-Napoca, Romania. 3. Raluca Ripan Institute for Research in Chemistry, Babes-Bolyai University, 30 Fântânele St., 400294 Cluj-Napoca, Romania. 4. Department of Veterinary Reproduction, Obstetrics and Gynecology, Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine, 3-5 Mănăştur St., 400372 Cluj-Napoca, Romania. 5. Department of Periodontology, Faculty of Dental Medicine, Iuliu Haţieganu University of Medicine and Pharmacy, 15 V. Babeş St., 400012 Cluj-Napoca, Romania. Electronic address: Alexandra.Roman@umfcluj.ro. 6. Department of Functional Genomics and Experimental Pathology, Prof. Dr. Ion Chiricuţă Oncology Institute, 34-36 Republicii St., 400015 Cluj-Napoca, Romania. 7. Department of Periodontology, University of Medicine and Pharmacy, 2 Petru Rareş St., 200349 Craiova, Romania. 8. Laboratory of Radiotherapy, Tumor and Radiobiology, Prof. Dr. Ion Chiricuţă Oncology Institute, 34-36 Republicii St., 400015 Cluj-Napoca, Romania.
Abstract
BACKGROUND: Normal or inflamed gingival tissues are regarded as a source of mesenchymal stem cells (MSCs) abundant and easily accessible through minimally invasive dental procedures. Due to the proximity of dental resin composites to gingival tissues and to the possible local cytotoxic effect of the eluted components, gingiva-derived MSCs could be used to investigate the biocompatibility of dental biomaterials. PURPOSE: The present research aimed to isolate (MSCs) from inflamed and normal gingiva, to fully characterize them and to observe their behavior in relation with some commercial resin composite materials and one experimental material. MATERIAL AND METHODS: Following their isolation, putative MSCs from both gingival sources were grown under the same culture conditions and characterized by immunophenotyping of cell surface antigens by flow-cytometry and transcription factors by immunocytochemical staining. Moreover, stemness gene expression was evaluated by RT-PCR analysis. Multipotent mesenchymal differentiation potential was investigated. Osteogenic and neurogenic differentiated cells were highlighted by immunocytochemical staining, chondrogenic cells by cytochemical staining, and adipocytes by cytochemical staining and spectrophotometry, respectively. Resin composite cytotoxicity was evaluated by cell membrane fluorescent labeling with PKH 26 and MTT assay. The results of PKH labeling were statistically analysed using two-way RM ANOVA with Bonferroni post-tests. For MTT assay, two-way RM ANOVA with Bonferroni post-tests and unpaired t test with Welch's correction were used. RESULTS: A similar expression pattern of surface markers was observed. The cells were positive for CD105, CD73, CD90, CD49e, CD29, CD44 and CD166 and negative for CD45, CD34, CD14, CD79, HLA-DR and CD117 indicating a mesenchymal stem cell phenotype. The qRT-PCR analysis revealed a low gene expression for NOG, BMP4 and Oct3/4 and an increased expression for Nanog in both cells lines. Immunocytochemical analysis highlighted a more intense protein expression for Nanog, Oct3/4 and Sox-2 in MSCs derived from normal gingiva than from inflamed gingiva. Multipotent differentiation capacity of MSCs isolated from both sources was highlighted. The tested materials had no hazardous effect on MSCs as the two cell lines developed well onto resin composite substrates. Cell counting revealed some significant differences in the number of PKH-labeled MSCs at some experimental moments. Also, some differences in cell viability were recorded indicating better developmental conditions offered by some of the tested biomaterials. CONCLUSIONS: The experimental resin composite behaved like the most biocompatible commercial material. Inflamed gingiva-derived MSCs retain their stem cell properties and could be used as a valuable cell line for testing dental biomaterials.
BACKGROUND: Normal or inflamed gingival tissues are regarded as a source of mesenchymal stem cells (MSCs) abundant and easily accessible through minimally invasive dental procedures. Due to the proximity of dental resin composites to gingival tissues and to the possible local cytotoxic effect of the eluted components, gingiva-derived MSCs could be used to investigate the biocompatibility of dental biomaterials. PURPOSE: The present research aimed to isolate (MSCs) from inflamed and normal gingiva, to fully characterize them and to observe their behavior in relation with some commercial resin composite materials and one experimental material. MATERIAL AND METHODS: Following their isolation, putative MSCs from both gingival sources were grown under the same culture conditions and characterized by immunophenotyping of cell surface antigens by flow-cytometry and transcription factors by immunocytochemical staining. Moreover, stemness gene expression was evaluated by RT-PCR analysis. Multipotent mesenchymal differentiation potential was investigated. Osteogenic and neurogenic differentiated cells were highlighted by immunocytochemical staining, chondrogenic cells by cytochemical staining, and adipocytes by cytochemical staining and spectrophotometry, respectively. Resin composite cytotoxicity was evaluated by cell membrane fluorescent labeling with PKH 26 and MTT assay. The results of PKH labeling were statistically analysed using two-way RM ANOVA with Bonferroni post-tests. For MTT assay, two-way RM ANOVA with Bonferroni post-tests and unpaired t test with Welch's correction were used. RESULTS: A similar expression pattern of surface markers was observed. The cells were positive for CD105, CD73, CD90, CD49e, CD29, CD44 and CD166 and negative for CD45, CD34, CD14, CD79, HLA-DR and CD117 indicating a mesenchymal stem cell phenotype. The qRT-PCR analysis revealed a low gene expression for NOG, BMP4 and Oct3/4 and an increased expression for Nanog in both cells lines. Immunocytochemical analysis highlighted a more intense protein expression for Nanog, Oct3/4 and Sox-2 in MSCs derived from normal gingiva than from inflamed gingiva. Multipotent differentiation capacity of MSCs isolated from both sources was highlighted. The tested materials had no hazardous effect on MSCs as the two cell lines developed well onto resin composite substrates. Cell counting revealed some significant differences in the number of PKH-labeled MSCs at some experimental moments. Also, some differences in cell viability were recorded indicating better developmental conditions offered by some of the tested biomaterials. CONCLUSIONS: The experimental resin composite behaved like the most biocompatible commercial material. Inflamed gingiva-derived MSCs retain their stem cell properties and could be used as a valuable cell line for testing dental biomaterials.
Authors: Sofia Iozon; Gabriela Valentina Caracostea; Emőke Páll; Olga Şoriţău; Ionuţ Daniel Mănăloiu; Adriana Elena Bulboacă; Mihaela Lupşe; Carmen Mihaela Mihu; Alexandra Livia Roman Journal: Rom J Morphol Embryol Date: 2020 Impact factor: 1.033