Osteogenic Differentiation and Biocompatibility of Bovine Teeth Scaffold with Rat Adipose-derived Mesenchymal Stem Cells

Abstract

Objective Adipose-derived mesenchymal stem cells (ADMSCs) have great potential for regenerative medicine. These have been combined with biomaterials such as Bovine teeth that are preferred as a periodontal regeneration material. The main purpose of this study is to evaluate and analyze a biocompatibility test and osteogenic differentiation of bovine teeth scaffold seeded with ADMSCs in vitro. Materials and Methods A true experimental study with post-test only group design was conducted. Random sampling and Lameshow’s formula were used to determine the sample. The scaffold, obtained from bovine teeth as the bone graft material, was analyzed using 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and its attachment was evaluated by scanning electron microcopy (SEM) and micro-computed tomography with ADMSCs. ADMDSCs attachment present in the bovine teeth scaffold was assessed using SEM at 1-hour, 12-hour, and 24-hour intervals. Statistical Analysis Analysis of variance was used to analyze the MTT assay results (p < 0.05) based on normality and homogeneity test (p > 0.05). Results The highest viability of cells (97.08%) was found at a concentration of 10% by means of an MTT test (p < 0.05). The results of three-dimensional bovine teeth Abstract Keywords ► adipose-derived mesenchymal stem cells ► bovine teeth ► scaffold ► tissue engineering

Osteogenic Differentiation Bovine Teeth Scaffold Combined Adipose Mesenchymal Stem Cells Sari et al. 207 European Journal of Dentistry Vol. 13 No. 2/2019 Introduction The tissue engineering technique incorporating the use of cells and growth factors combined with scaffold for periodontal tissue regeneration is currently gaining popularity. This technique utilizes biocompatible scaffold seeds with growth factor, stem cells, or both that are implanted into a site to stimulate the reformation or repair of the missing tissues.1,2 Bovine teeth have been the most widely employed substitutes for the human variety in dental research, with their use increasing over the past 30 years.3,4 As bone graft material, bovine teeth possess osteoinductive and osteoconductive properties responsible for the construction of new bones. The term “osteoinduction” signifies that the grafted material is chemotactic to undifferentiated osteoprogenitor cells in the host and induces differentiation into osteoblasts. Osteoconduction is defined as a process that permits the growth of osteogenic cells from exposed bone surface into the adjacent graft material.5,6 Bovine teeth are predominantly composed of inorganic material (70%), organic material (20%), and water (10%). The inorganic component is largely hydroxyapatite and organic content consisting of collagen type I and growth factor.7,8,9 The purpose of scaffolds is to support cell attachment and migration, while also providing growth factors to support tissue and bone formation. The combination of scaffold and stem cells for bone growth is synergic in character. The self-renewal abilities of stem cells and their capability to differentiate into multiple cell lineages render them promising candidates for cell-based tissue engineering. Adipose-tissue derived from adult stem cells is most commonly used for periodontal regeneration.10 Adipose-derived mesenchymal stem cells (ADMSCs) can be easily isolated, providing an enormous number of stem cells that are vital for tissue engineering and stem cells-based therapies.11,12 The International Society for Cellular Therapy set three minimum criteria for the definition of MSCs: plastic-adherence; expression of CD73, CD90, and CD105; and the absence of CD45, CD14, CD19, human leukocyte antigen - DR isotype (HLA-DR) expressions and their trilineage differentiation potential into osteoblasts, chondrocytes, adipocytes.13,14 Research into bovine teeth implantation in the calvarial defects of rats showed an increase in bone density after 6 weeks. Other research conducted by George et al state that umbilical mesenchymal stem cells demonstrate a tendency to differentiate and proliferate after binding to the tooth surface in vitro.15,16 The purpose of this study is to analyze the osteogenic potential and biocompatibility test of bovine teeth scaffold seeded with ADMSCs in vitro. Materials and Methods This study received ethical clearance (number 637-KE) from the Animal Care and Use Committee Faculty, Veterinary University of Airlangga, Surabaya, Indonesia. Three, 4-weekold, male Wistar rat subjects were sacrificed by euthanasia. Isolation of the ADMSCs of these subjects was performed by washing adipose tissue with saline phosphates containing 10% antimycotic–antibiotic agent. The adipose tissue was cut into pieces and immersed in a 0.2% collagenase type I (Worthington, Lakewood, New Jersey, United States) solution with the addition of Dulbecco’s phosphate buffer saline (STEMCELL Technologies, Nucleos, Singapore) and agitated slowly for 40 minutes at 37°C. The tissue was filtered using a 10 µm mesh filter (Pluriselect; Leipzig, DE) before being centrifuged at 1,250 rpm for 4 minutes, with the supernatant subsequently discarded.17 Isolation and Culture of Adipose-Derived Mesenchymal Stem Cells ADMSCs MSCs were cultured with α-modified minimum essential medium eagle (αMEM) (Gibco, Roskilde Denmark) plus 15% fetal bovine serum (Biowest; Missouri, United States), 2 mM of L-glutamine) (Gibco), 100 mg/mL streptomycin (Gibco), 2.5 μg/mL fungizone) (Gibco), and 100 IU/mL penicillin (Gibco) before being incubated at 37°C with 5% CO2 . The cells were grown in six wells on a tissue culture plate at a concentration of 107 in each well. The medium was changed on the 7th day and every 3 days thereafter. Observation of the cells was performed using an inverted microscope (80× magnification).13-18 Characterization ADMSCs by Immunocytochemical Staining and Flow Cytometry A single cell subjected to a process of trypsinization was centrifuged, fixed with formaldehyde (TCI, Chuo-ku, Tokyo), and washed with PBS. FITC anti-rat CD105 monoclonal antibody (BioLegend, San Diego, California, United States) and FITC anti-rat CD45 monoclonal antibody (BioLegend) were mixed into the sample and incubated at 37°C for 45 minutes. Several drops of 50% glycerin (TCI, Chuo-ku, Tokyo) were then applied to glass objects and observed under a fluorescent microscope. During the trypsinization process, the single scaffold showed the average particle size to be 500 µm. ADMSCs cell attachment to the scaffold bovine teeth showed a significant increase in the number of cells attached after 24 hours compared with those at 1 and 12 hours. Alizarin red staining showed an increase in ADMSC osteogenic differentiation after it was combined with bovine teeth scaffold.