Collagenated heretologous cortico-cancelleus bone mix (CHCCBM) is largely employed in maxillary and dental surgery for regeneration procedures, and is similar to human bone from chemical and physical point of view and promotes osteogenesis. In order to get more inside how this biomaterial induces osteoblast gene expression to promote bone formation, the mRNA levels of bone related genes were compared in human osteoblasts and dental pulp stem cells, using real time RT-PCR. The obtained results demonstrated that CHCCBM enhance stem cells differentiation and deposition of matrix by the activation of osteoblast related genes SP7, FOSL1 and SPP1

A. Pellati

A. Piva

A. Russillo

C. Baserga

G. Beltramini

V. Candotto

English

AIR Universita degli studi di Milano

Collagenated heterologous cortico-cancelleus bone mix stimulated dental pulp derived stem cells

Beltramini, G

Russillo, A

Baserga, C

Pellati, Agnese

Piva, Alice

Candotto, Valentina

Archivio istituzionale della ricerca - Università di Ferrara

JOURNAL OF BIOLOGICAL REGULATORS & HOMEOSTATIC AGENTS0393-974X (2020)Copyright © by BIOLIFE, s.a.s.This publication and/or article is for individual use only and may not be furtherreproduced without written permission from the copyright holder.Unauthorized reproduction may result in financial and other penaltiesDISCLOSURE: ALL AUTHORS REPORT NO CONFLICTS OF INTEREST RELEVANT TO THIS ARTICLE.1(S2)Vol. 34, no. 1 (S2), 1-5 (2020)Collagenated heretologous cortico-cancelleus bone mix (CHCCBM) is a bovine bone derived employed in maxillary and oral surgery. Some reports have demonstrated its clinical efficacy to restore alveolar ridge in pre-prosthetic surgery (1-3).To investigate the molecular mechanism by which CHCCBM promotes osteoblast differentiation and proliferation, the quantitative expression of the mRNA of specific bone related genes, was examined in derived dental pulp stem cells treated CHCCBM. Dental pulp stem cells (DPSCs) represents an ideal source of stem cells because approachable niches containing a high number of stem cells compared to equal volumes of bone marrow (4-6). In this study the expression levels of specific genes were examined by means of real time RT-PCR in DPSCs after treatment with CHCCBM.Gene expression in DPSCs was then compared with the gene expression in treated Human Osteoblasts (HOb) to evaluate the potential effect of this material in osteoblasts differentiation.MATERIALS AND METHODSStem cells isolation from dental pulp, flow cytometric analyses and primary human osteoblasts cell culture were performed as previously described (4-6).Cell treatment DPSCs and HOb were seeded with CHCCBM (Gen-Os, Tecnoss srl, Giaveno, Torino, Italy) at the concentration of 10 mg/ml. Another set of wells containing untreated cells were used as control. The cells were maintained in a humidified atmosphere of 5% CO2 at 37°C. Cells were harvested at two time points, 15 and 30 days, for RNA extraction. RNA processing, RT-PCR and statistical analyses were conducted as previously described (4-6). Collagenated heretologous cortico-cancelleus bone mix (CHCCBM) is largely employed in maxillary and dental surgery for regeneration procedures, and  is similar to human bone from chemical and physical point of view and promotes osteogenesis. In order to get more inside how this biomaterial induces osteoblast gene expression to promote bone formation, the mRNA levels of bone related genes were compared in human osteoblasts and dental pulp stem cells, using real time RT-PCR. The obtained results demonstrated that CHCCBM enhance stem cells differentiation and deposition of matrix by the activation of osteoblast related genes SP7, FOSL1 and SPP1.Key words: bone, dental pulp, stem cells, gene expression, osteoblast differentiationCorresponding Author:Valentina Candotto, MD,Department of Biomedical, Surgical and Dental Sciences, University of Milan, Via Commenda 10, 20122 Milan, Italye-mail: candottovalentina@gmail.comCOLLAGENATED HETEROLOGOUS CORTICO-CANCELLEUS BONE MIX STIMULATED DENTAL PULP DERIVED STEM CELLSG. BELTRAMINI1, A. RUSSILLO1,2, C. BASERGA1,2, A. PELLATI3, A. PIVA4 and V. CANDOTTO1 1Department of Biomedical, Surgical and Dental Sciences, University of Milan, Milan, Italy; 2Maxillofacial and Dental Unit, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan, Italy; 3Department of Morphology, Surgery and Experimental medicine, University of Ferrara, 44121 Ferrara, Italy; 4Private practice, Ferrara, Italy2 (S1) F. GRAZIANI ET AL.of promotes osteogenesis. It is of paramount importance since several situation determine bone loss. Among them are socket preservation after tooth extraction, peri-implants bone maintenance (7-16), ridge reconstruction in syndromic patients (17-23) and oral rehabilitation after oncological treatments (24-35) In order to get more inside how CHCCBM acts on DPSCs, changes in expression of bone related marker genes (RUNX2, SPP1, SP7, COLIA1, COL3A1, ALPL and FOSL1) and mesenchymal stem cells marker (ENG) were investigated by real-time RT–PCR. DPSCs were isolated by enzymatic digestion and phenotipically characterized by flow cytometric analyses. Dental pulp derived cell were homogenously CD105+, CD90+, CD34-, CD45-, CD14-, which is a typical mesenchymal stem cells surface antigen profile.  The osteoinductive properties of CHCCBM were demonstrated by the up-regulation of SP7 during all the entire treatment both in treated DPSCs than in treated HOb. SP7 is a transcriptional factor involved in bone formation and osteoblast differentiation downstream of RUNX2 pathway. CHCCBM also modulate the expression of collagenic extracellular matrix genes, collagen type 1α1 (COL1A1) and collagen type 3α1 (COL3A1).  After 14 days, in treated DPSCs was observed the RESULTS Cell cultures were phenotipically characterized by flow cytometric analyses as previously reported (4-6). To study if CHCCBM stimulates osteoblasts differentiation and proliferation in DPSCs, several osteoblast genes and mesenchymal stem cells marker, were analyzed by quantitative real-time PCR after 14 days of treatment with CHCCBM. Treated DPSCs resulted in up-regulation of SP7, FOSL1 and SPP1 genes and down-regulation of ENG, RUNX2, COL3A1, COL1A1 and ALPL during the first 14 days of treatment. Analyzing the results obtained in HOb, we observed that after 14 days of treatment SP7, ENG, FOSL1, COL1A1, COL3A1 and SPP1 were up-regulated. The expression of RUNX2 and ALPL was down-regulated. “Two tailed ANOVA” comparison between DPSCs and HOb after the treatment showed that all genes were significantly differentially expressed after 14 days of treatment. (Table I). DISCUSSION CHCCBM (Gen-Os, Tecnoss srl, Giaveno, Torino, Italy) is widely used in several bone regeneration procedures in oral surgery due to its characteristics of resorption rate and capability Genes  DPSCs  HOb  Differentially expressed genes   Log10 RQ  Log10 RQ  p<0,005  SP7  1,22  0,52  0,001  ENG  -0,42  0,03  0,009  FOSL1  0,25  0,26  0,001  RUNX2  -0,42  -0,03  0,002  COL3A1  -1,46  0,02  0,001  COL1A1  -0,71  0,13  0,001  ALPL  -0,54  -0,23  0,001  SPP1  0,93  1,64  0,001   Table I. Differentially expressed genes between DPSCs and HOb after 14 days of treatment.(S1) 3Journal of Biological Regulators & Homeostatic Agentsfrom human dental pulp stem cells and osteosarcoma cell lines. Cell Biol Int 2008; 32(7):733-8. 5. Laino G, Carinci F, Graziano A, d’Aquino R, Lanza V, De Rosa A, Gombos F, Caruso F, Guida L, Rullo R, Menditti D, Papaccio G. In vitro bone production using stem cells derived from human dental pulp. J Craniofac Surg 2006; 17(3):511-5. 6. Laino G, d’Aquino R, Graziano A, Lanza V, Carinci F, Naro F, Pirozzi G, Papaccio G. A new population of human adult dental pulp stem cells: a useful source of living autologous fibrous bone tissue (LAB). J Bone Miner Res 2005; 20(8):1394-402. 7. Baj A, Bolzoni A, Russillo A, Lauritano D, Palmieri A, Cura F, Silvestre FJ, Giannì AB. Cone-morse implant connection system significantly reduces bacterial leakage between implant and abutment: an in vitro study. J Biol Regul Homeost Agents 2017; 31(2 Suppl 1):203-8.8. Baj A, Beltramini GA, Bolzoni A, Cura F, Palmieri A, Scarano A, Ottria L, Giannì AB. Bacterial colonization of the implant-abutment interface of conical connection with an internal octagon: an in vitro study using real-time PCR. J Biol Regul Homeost Agents 2017; 31(2 Suppl 1):163-8. 9. Candotto V, Lauritano D, Carinci F, Bignozzi CA, Pazzi D, Cura F, Severino M, Scarano A. Silver-Based Chemical Device as an Adjunct of Domestic Oral Hygiene: A Study on Periodontal Patients. Materials (Basel) 2018; 11(8). pii: E1483. 10. 1Lauritano D, Carinci F, Palmieri A, Cura F, Caruso S, Candotto V. Reuterinos(®) as adjuvant for peri-implant treatment: A pilot study. Int J Immunopathol Pharmacol 2019; 33:2058738419827745.11. Carinci F, Lauritano D, Bignozzi CA, Pazzi D, Candotto V, Santos de Oliveira P, Scarano A. A New Strategy Against Peri-Implantitis: Antibacterial Internal Coating. Int J Mol Sci 2019; 20(16): ii: E3897.12. Lauritano D, Boccalari E, Di Stasio D, Della Vella F, Carinci F, Lucchese A, Petruzzi M. Prevalence of Oral Lesions and Correlation with Intestinal Symptoms of Inflammatory Bowel Disease: A Systematic Review. Diagnostics (Basel) 2019; 9(3):pii: E77.13. Lauritano D, Moreo G, Della Vella F, Di Stasio D, Carinci F, Lucchese A, Petruzzi M. Oral Health Status and Need for Oral Care in an Aging Population: A down-regulation of COL1A1 and COL3A1. Instead, in HOb, the two collagens type were up-regulated. Osteopontine (SPP1) was up-regulated during all the treatment both in DPSCs than in HOb. The up-regulation of this gene suggests the differentiation effect of treatment.  ENG, also named CD105, a surface marker used to define a bone marrow stromal cell population capable of multilineage differentiation was down-regulated in DPSCs and up-regulated in HOb during the entire treatment.  Another gene involved in osteoblast differentiation and modulated by CHCCBM was FOSL1. Both in DPSCs than in HOb this gene was up-regulated. FOSL1 is a component of the dimeric transcription factor AP-1 involved in the transcription of bone related genes. 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Collagenated heterologous cortico-cancelleus bone mix stimulated dental pulp derived stem cells

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Archivio istituzionale della ricerca - Università di Ferrara