Cell isolation and culture
To isolate DPSC, healthy permanent teeth were collected from adult donors (under orthodontic treatment; 18–25 years old) at the Faculty of Dentistry, Universiti Kebangsaan Malaysia, Kuala Lumpur. Umbilical cord has been collected at the Universiti Kebangsaan Malaysia Medical Centre with informed consent from donors who deliver full term by elective caesarean section. The protocol of this study had been approved by Research Ethics Committee Faculty of Medicine Universiti Kebangsaan Malaysia (FF-2019-444).
Briefly, Tooth was cleansed with povidone-iodine and split at the cemento-enamel junction using sterilized dental burs to extract the dental pulp. Dental pulp tissue then minced into smaller fragments with sterile blade and digested using 4 mg/mL collagenase Type I enzyme (Gibco, Grand Island, NY, USA) for 40 min at 37 °C in water bath. The tissue fragments were resuspended in complete medium containing α-MEM supplemented with 10% fetal bovine serum (FBS), 1% GlutaMAX (Gibco, Grand Island, NY, USA) and 1% antibiotic–antimycotic. The cells were cultured in humidified incubator with 5% CO2 at 37 °C with medium change every 3 days until the cells reaches 80% confluence.
The umbilical cord was washed with Dulbecco’s phosphate-buffered saline and stripped off the umbilical arteries and vein. Wharton’s jelly was then minced into 2mm2 size and digested with 0.6% collagenase type II at 37 °C under gentle agitation. Resulting tissue fragments were suspended in DMEM low glucose supplemented with 1% GlutaMax™ (Gibco), 1% Antibiotic–Antimycotic (Gibco), with addition of 10% FBS, 10% hPL, or 10% human serum. The cells were cultured in humidified incubator with 5% CO2 at 37 °C with medium change every 3 days until the cells reaches 80% confluence and harvested using 1X TryPLE Select. The cells were expanded to passage 3 for the following experiments in this study.
DPSC conditioned medium collection
DPSCs were seeded at a density of 5000 cells/cm2 in culture flasks and grown until approximately 70–80% confluent. Cells then washed three times with PBS and incubated with freshly added serum-free DMEM containing penicillin–streptomycin at 24, 48 and 72 h, at 37 °C in 5% CO2. Supernatant then collected, centrifuged at 4 °C at 3000g for 3 min followed by 5 min at 1500g, filtered through 0.2-μm filters and stored in aliquots at − 80 °C as DPSC-CM.
Analysis of cell viability
To identify MSC viability when inducing differentiation with DPSC-CM, 3-(4,5-dimethylthiazoiyl-2)-2,5-diphenyltetrazolium bromide (MTT) coloration has been done. MSC were seeded at a density of 5000 cells/cm2 with the following media ratio: (a) Complete DMEM:CM 1:1; (b) Complete DMEM:CM 3:1; (c) Serum Free DMEM:CM 1:1; (d) Serum Free DMEM:CM 3:1. The cells were cultured in humidified incubator with 5% CO2 at 37 °C for 7 days with medium change every 3 days. MTT assay was done on day 1, 3, 5, and 7 to evaluate the effects of DPSC-CM towards MSC viability.
Differentiation of MSC into odontoblast
Four complete αMEM:CM combination aforementioned were used for MSC odontoblast differentiation induction, whereas StemPro™ Osteogenesis induction media (Gibco) was served as positive control treatment. The cells were differentiated for 7 and 14 days prior subsequent experiment.
Alizarin Red staining
At the end of each treatment, differentiated cells described above were stained with Alizarin Red to show positive calcium deposition. The staining dye was first observe under inverted microscope and then quantified using a mixture of 20% methanol and 10% acetic acid in distilled water for elution, the eluted dye was analyse using spectrophotometer at wavelength 450 nm.
Immunocytochemistry detection of odontogenic marker
Expression level of DMP-1 was evaluated using immunocytochemical analysis protocol reported by  with slight modification. In brief, cells were washed with DPBS and fixed with 4% paraformaldehyde (PFA) for 30 min, followed by cell permeabilization by 0.5% Triton X-100 solution for 20 min, and then blocked with 5% Bovine Serum Albumin for 1 h at 37 °C. 1:200 mouse anti DMP-1 antibody and 1:200 rabbit anti-GAPDH were incubated with the cells overnight. On the following day, the cells were washed before being incubated with 1:300 diluted Alexa Fluor 594 anti-rabbit IgG and Alexa Fluor 488 anti-mouse for 1 h at 37 °C. Nuclei were counterstained with DAPI. Fluorescence images were captured with a Nikon Eclipes Ti Fluorecence microscope.
Experiments were performed in triplicate and repeated on at least three biological samples (n = 3), and data are presented as mean ± SD. For statistical analysis, ANOVA was used. Statistical analysis was performed using Prism Version 7.0 software. Results were considered statistically significant at P < 0.05. All values are expressed as mean ± SD.