Bone graft materials used for bone regeneration are categorized into four groups of autografts, allografts, xenografts, and alloplastic materials. They can lead to bone regeneration by inducing osteogenesis, osteoconduction, and osteoinduction. Autografts were long regarded as the gold standard graft materials, but due to the specific nature and morphology of the alveolar ridge defects, and the need to modify the bone grafts into small bone chips to match the defect size, autogenous bone graft materials are subject to early resorption. According to a review study, xenografts have superior performance compared with allografts and alloplasts. Alloplastic materials can serve as osteoconductive scaffolds, and their prolonged and even permanent presence ensures that the space is protected. To overcome the existing limitations, allogenic, xenogeneic, and synthetic bone materials are used. However, synthetic bone substitutes cannot stimulate osteogenesis and do not possess osteoinductive properties.
Stem cells provide a good source for tissue engineering because of their easy use, high division rate, and optimal regeneration capacity. Stem cells can be set apart from oral tissues, for example, the periodontal ligament and dental pulp, and their bone regenerative potential have been studied. Mesenchymal stem cells are among the most commonly used stem cells for bone regeneration since they are the precursors of osteoblasts and osteocytes. Bone marrow mesenchymal stem cells are a suitable origin for cell-based treatments due to their easy availability, differentiation to bone, cartilage, and muscle tissue, and immunosuppressive effects. Due to the difficult isolation of bone marrow stem cells (and their inadequate number), researchers have attempted to isolate stem cells from other origins such as dental tissues. Dental pulp stem cells (DPSCs) have the potential to be discriminated into osteogenic cells and make a mineralized matrix. In addition, they have the potential to proliferate and can be used with different bone scaffolds. Isolation of DPSCs does not require a surgical procedure and is not associated with postoperative complications. Also, their osteogenic property is comparable to that of bone marrow stem cells. Thus, DPSCs are often preferred for bone regeneration.
In vitro studies can greatly help to enhance basic knowledge about the biological reactions of different cells and scaffolds. Bio-Oss is commonly used for the purpose of comparison in the assessment of new xenograft scaffolds. Camel scaffold is a recently introduced scaffold which has not been comprehensively studied. Thus, this study aimed to compare the biological reactions of DPSCs cultured on new xenograft bone substitutes derived from camel and bovine bones.
MATERIALS AND METHODS
Cell preparation and materials
DPSCs (IBRC C10896) were acquired from the Iranian Cell Bank Biological Resource Center. following defrosting, the cells were plated in a 75-cc culture flask, which included Dulbecco's modified Eagles medium (Gibco, Grand Island, NY, USA), 10% fetal bovine serum (Gibco, USA), 100 IU/mL penicillin (Gibco, USA), and 100 mg/mL streptomycin (Gibco, USA). Then, the cells were incubated in a humidified chamber at 37°C and 5% CO2 under sterile conditions. After 24 h, the culture medium was substituted with 1% fetal bovine serum. Following 24 h of incubation, the cells were exposed to biomaterials and incubated in a humidified chamber at 37°C, 5% CO2, and 95% H2O under sterile conditions (a cell-culture hood). When the cells achieved 80% confluence (passaged with 0.25% trypsin and 0.2% EDTA), they were utilized in their log phase to provide a cellular suspension, and the rest was kept in a nitrogen storage tank. A cell suspension with a density of 10000 cells/mL as seeded on a 96-well plate for the methyl thiazolyl tetrazolium (MTT) assay. A cell suspension with 30000 cells/mL density was used in two 24-well plates for the alkaline phosphatase (ALP) test and Alizarin red staining. The plates were incubated at 37°C and 95% H2O for 24 h. Scaffolds were first cultivated in 6-well plates at 16 μg/mL concentration, and Eagle's culture medium was added to the scaffolds. Then, they were incubated for 24 h. The cells were added to the scaffolds at a concentration of 16 μg/mL with a culture medium in each well. After 24 h, an osteogenic culture medium was used for osteogenic differentiation.
The xenografts used in this study had a particle size of 1000–2000 μm and included Bone Plus (natural bovine bone substitute, NovaTeb Co., Iran), Camel Bone (natural Camel bone substitute; NovaTeb Co., Iran), and Bio-Oss (Geistlich Biomaterials, Geistlich AG, Wolhusen, Switzerland). The prepared xenografts were incubated with the cell culture at 37°C for 1 h. After 1 h, the culture medium was pulled out. In order to investigate the adhesion of cells to xenografts and their possible application as scaffolds in tissue engineering, the bone substitutes were covered with 10,896 DPSCs in 200 μL of culture medium. Next, half of the samples received 500 μL of standard culture medium (Dulbecco's modified Eagles' medium, 10% fetal bovine serum, 100 IU/mL of 1% penicillin, 100 mg/mL streptomycin, and 2 mmol/L glutamine) and the other half received the same osteogenic medium (standard culture medium plus 10 μM dexamethasone, 10 μM beta-glycerophosphate, and 50 μg/mL ascorbic acid). The cell cultures were then incubated at 37°C and 5% CO2. Twenty-four hours after cell culture, scanning electron microscopy (Iran's Sharif Solar [IRASOL] Co.) was performed. For this purpose, the scaffolds were washed twice with phosphate-buffered saline (PBS) and also by 2.5% glutaraldehyde 2 h later. They were rinsed with 1% osmium 1 h after fixation. They were then dehydrated with ascending concentrations of ethanol (30%, 70%, 90%, 95%, and 100%). Finally, the specimens were observed under an electron microscope.
Methyl thiazolyl tetrazolium assay
The 96-well plates were pulled out from the incubator and studied after 24, 48, and 72 h below a light microscope at ×4 and ×10 magnifications. To ensure the proliferation and soundness of cells, the culture medium of the wells was removed and they were washed with PBS (IRBC, Iran) for several times. The MTT (Sigma, USA) solution was diluted by the medium at 1:10 ratio and 10 μL of it was added to each well. The cells were then incubated at 37°C, 100% H2O, and 5% CO2 for 4 h. The culture medium was removed from the wells following observation of purple color change below a light microscope, and then 100 μL of dimethyl sulfoxide was poured to each well and it was slightly vibrate in order to melt the formazan crystals. Capped plates stayed in the dark for 2–4 h. Then, they were uncapped and the optical density of the wells was read at 570 nm wavelength using an ELISA-reader to evaluate the metabolic activity of the cells. Cell viability was determined and reported as the percentage of viable cells compared with the negative control group (undifferentiated DPSCs in culture medium). The positive control group included undifferentiated DPSCs without scaffolds in an osteogenic medium.
Alkaline phosphatase assay
The ALP activity test was performed to assess the osteoblastic activity 30 days following the exposure of cells to the bone substitutes. The cells were washed with PBS (IRBC, Iran) and then 500 μL of the assay buffer solution available in the kit (ParsAzmoon, Iran) was added to each well in accordance with the manufacturer's instructions. After centrifugation (13000 rpm/min for 10 min), the supernatant was conveyed to 96-well plates. Next, a certain amount of P-nitro phenyl phosphate solution available in the kit (ParsAzmoon, Iran) was poured to each well in accordance with the manufacturer's instructions. The specimens were placed in the dark for 1 h and the stop solution was added to all specimens. The optical density of the wells was read at 450 nm wavelength by an ELISA-reader and reported. The same steps were repeated at 7 days.
Alizarin red staining
Alizarin red staining was done in order to verify mineralization and formation of calcified nodules after 7 and 21 days. The culture medium was pulled out from each well, and the cells were gently washed with PBS (IRBC, Iran) three times. The cells were fixed in 100% methanol for 15 min at room temperature. Then, the fixative was pulled out, and the cells were cleansed with PBS (IRBC, Iran) three times for 5 min. After removing the standard dilution solution, 40 mM of Alizarin red dye (pH of 2.4–4.4) was applied over the cells for 2 min at room temperature. Then, the dye was taked out and the cells were cleansed with distilled water. Each well was deliberated below a light microscope at ×10 and ×20 magnifications in order to assess the morphology of the cells and the formation of calcified nodules (orange-red color).
Polymerase chain reaction
The expression of osteocalcin (OCN) gene was evaluated qualitatively at 3, 7, and 14 days after the test in all groups. In order to analyze its expression, the cells were placed in 60-mm plates at a concentration of 2 × 200000 cells/well. Total RNA was extracted using RNeasy kit (PrimeScriptTM RT reagent Kit#RR037A, v. 0610, Japan). In accordance with the manufacturer's order, 1 μg of total RNA was extracted, and the cDNA was synthesized using the cDNA synthesis kit (PrimeScriptTM RT reagent Kit#RR037A, v. 0610, Japan). The expression of OCN mRNA was characterized by certain primers using reverse-transcription polymerase chain reaction (PCR). PCR was done in the following order: 95°C for 2 min, 35 cycles of denaturation at 95°C for 30 s, annealing for 30 s, and extension at 72°C for 30s [Table 1]. The final extension was carried out at 72°C for 5 min. The B-actin gene was utilized as the housekeeping gene for internal control. The process was conducted on 1.8% agarose gel and evaluated by ultraviolet light. Each test was repeated twice with different random emulsions of xenografts. In order to obtain semi-quantitative results, data were imported to ImageJ software (ImageJ bundled with Java 1.8.0). A definite range for each gene was defined, which was twice as long as the color change in the transverse dimension. Then, a numerical output for each gene was obtained by ImageJ software (ImageJ bundled with Java 1.8.0). All steps were performed by one examiner and repeated three times.
Normal distribution of the data was evaluated by the Kolmogorov-Smirnov test. A comparison between groups and different time points was carried out using two-way ANOVA. Because the interaction result of group and time was found to be notable, pairwise comparisons were done using one-way ANOVA and Tukey's honestly significant difference (HSD) test. P < 0.05 was considered statistically significant. The data were analyzed by SPSS version 20 International Business Machines Corporation (IBM), Armonk, New York. Two-way ANOVA was applied in order to analyze the impact of time on optical density. By reasons of the significant impact of time, subgroup analysis was carried out. One-way ANOVA was applied to assess the impact of assessment time on each group.
Cell proliferation and viability assay
The results manifested an obvious difference in optical density at 24, 48, and 72 h (P < 0.001). The Tukey's test showed meaningful dissimilarity in optical density at different time points in each of the Camel Bone, Bone Plus, and Bio-Oss groups (P < 0.001). The Tukey's HSD test showed that the optical density was obviously different at 24, 48, and 72 h in all three groups (P < 0.001). Pairwise comparisons showed the lowest optical density in the Bio-Oss group after 24 h. The optical density of the Camel Bone and the negative control group was significantly higher than that of the Bio-Oss group at 24 h (P < 0.001). The mean optical density of the Camel, Bio-Oss, and Bone Plus groups was greater than that of the positive and negative control groups, indicating that DPSCs proliferated in all experimental groups. However, greater mean optical density was noted in the Camel Bone group at 24, 48, and 72 h, which indicates higher cell proliferation in this group compared with other groups (P < 0.001). Comparison of viability at different time points showed significantly different viability at 24 and 48 h. Also, the viability at 72 h was greater than that after 48 and 24 h in all three groups [Figure 1].
Alkaline phosphatase activity test
The results showed that in the Bio-Oss group, the mean optical density was greater than the mean optical density of the control group at all three-time points (1–3 min). This finding indicates higher proliferation and differentiation of DPSC into osteoblastic cells and expression of OCN in the Bio-Oss group than in other groups (Camel Bone and Bone Plus). The ALP activity was higher in the Bio-Oss group in comparison with the control, Camel Bone, and Bone Plus groups. However, in quality assessment by Alizarin red staining under a light microscope, red color was observed indicating extracellular calcium deposition, confirming cell differentiation into osteoblastic cells in all three groups of camel bone, Bio-Oss, and bone plus [Figure 2] and [Figure 3]. According to the results, there was a significant correlation between cell proliferation and ALP activity in all three groups.
Polymerase chain reaction test
The results of the qualitative assessment showed expression of osteopontin (OPN), OCN, and osteonectin (OSN) genes in DPSCs + Camel Bone, DPSCs + Bone Plus and DPSC + Bio-Oss groups. Also, the expression of OPN and OCN in DPSC + Camel Bone and DPSCs + Bone Plus groups was greater than that in DPSCs + Bio-Oss group. However, the expression of OSN gene in DPSCs + Camel Bone and DPSCs + Bio-Oss groups was greater than that in DPSCs + Bone Plus group [Figure 4].
In quantitative assessment, expression of OPN, OCN, and OSN was noted in all three groups of DPSCs + Camel Bone, DPSCs + Bone Plus, and DPSCs + Bio-Oss. Analysis shows the comparison of groups, indicating that the OPN gene was expressed in all three groups (P < 0.05). Nevertheless, the pronouncement of OPN gene in the DPSCs + Bio-Oss group was not notably different from that in the control group (P > 0.05). Also, the OSN gene expression in DPSCs + Bone Plus and DPSCs + Bio-Oss groups had a significant difference with its expression in DPSCs + Camel Bone and control groups (P < 0.05). OCN gene was also expressed in all three groups (P < 0.05).
Cell adhesion assessment
As shown in [Figure 5], cell adhesion to all three scaffolds (Camel Bone, Bio-Oss, Bone Plus) was noted on micrographs [Figure 5].
Xenograft bone substitutes have been widely used for bone regenerative procedures. However, the processing method of xenografts can affect the results with respect to cell proliferation. In this study, the bone regeneration ability of DPSCs on three xenograft bone substitutes from three different sources was compared, and cell proliferation and viability were studied by the MTT assay at 24, 48, and 72 h. The ALP activity, Alizarin red staining, and PCR tests were done to assess osteogenic differentiation and mineralization at 30 days. Cell adhesion to the scaffolds was assessed using scanning electron microscopy (IRASOL Co). According to the results of the MTT assay, the interaction effect of time and group on cell proliferation was significant (P < 0.05). The proliferation rate was in the following order: Negative control > camel bone > bone plus > Bio-Oss > positive control. The differences among the test groups at 24, 48, and 72 h were statistically significant (P < 0.05).
Kubler et al., in 2004 compared the effects of different bone substitutes on the proliferation of human iliac bone marrow stem cells. The results showed that human osteoblasts exhibited different growth patterns on different bone substitutes. However, Bio-Oss xenograft showed the lowest cell proliferation and differentiation rate among the five tested bone substitutes. It was concluded that bone graft material had a different effect on the pattern of proliferation of human osteoblasts in vitro. Ayobian et al., in 2012 added four types of Tutodent, Osteon, Bio-Oss, and Cerasorb bone substitutes to SaOS2 cell culture. Cell differentiation rate, cell viability, and ALP activity in SaOS2 cell culture were evaluated by using electron microscopy, cell viability assay, and phase-contrast microscopy. The results showed that the control group (SaOS2 cells cultured in the Dulbecco's modified Eagle's medium without bone substitute) indicated the highest cell viability. All four bone substitutes showed significant ALP activity (P < 0.001). The present results demonstrated optimal proliferation and differentiation of DPSCs in presence of three types of xenograft bone substitutes from three different sources.
Lafzi et al., in 2016 compared the effects of freeze-dried bone allograft and demineralized freeze-dried bone allograft scaffolds on proliferation rate and differentiation of MG-63 cell line. They concluded that the cell proliferation rate increased from 24 to 72 h on freeze-dried bone allograft scaffold (P < 0.05). Due to the use of different types of cell lines and bone substitutes, their results cannot be compared with the present findings.
Assessment of cell proliferation rate in each group at different time points (indirect MTT) showed that the cell proliferation rate increased from 24 to 72 h in all groups; this increase was significant in the three xenograft groups and not significant in the positive and negative control groups. Cell proliferation rate in the Camel group after 72 h was significantly greater than that in Bio-Oss and Bone Plus groups. Thus, Camel Bone may also have osteoinductive properties. Given the novelty of this material and the absence of any studies on it, it is recommended to interpret the results with caution. Further in vitro and clinical studies are imperative on this topic.
The ALP activity test can be used to assess both osteogenic differentiation of stem cells and osteoblast maturation. In order to assess the osteogenic differentiation, Alizarin red staining is also recommended. Moreover, OCN gene expression was evaluated in this study both quantitatively and qualitatively by PCR and Alizarin red staining. The qualitative assessment showed that the ALP activity was in the following order: Bio-Oss < camel bone < bone plus. The dissimilarity among the test groups was statistically significant, which indicates optimal osteogenic differentiation of DPSCs confirmed by ALP activity. In a qualitative assessment by Alizarin red staining, extracellular calcium nodules were noted in all three groups. In addition, the red color is an indicator confirming the differentiation of DPSCs to osteoblastic cells. The quantitative assessment by Alizarin red staining showed the following order for cell activity: Bio-Oss < camel < bone plus < control group.
Wang et al., Akkouch et al. (2014) and El-Gendy et al. (2013) assessed the osteogenic differentiation of DPSCs in another culture media by Alizarin red staining. The present results regarding the ability of DPSCs for mineralization and formation of calcified nodules on different scaffolds with three different sources in osteogenic culture medium were similar to the findings of Wang et al., Akkouch et al., and El-Gendy et al. According to the results, the presence of a scaffold causes a remarkable difference in the rate of mineralization; Nevertheless, there was a remarkable difference in the rate of mineralization between different scaffolds. In addition, according to the scanning electron microscopic images, DPSCs can adhere to the scaffolds. Despite the fact that xenografts were not floating in the plate because of their weight, the cells adhered to them and were not separated.
In a study by Vaziri et al., conducted in 2012, three commercially available demineralized dried bone allografts (Cenebone, Osseo + Alloss) with 8 mg/mL and 16 mg/mL concentrations were added to SaOS2 cell culture medium. The cell proliferation rate, osteogenic activity, and osteoinductive ability in SaOS2 cell culture medium were evaluated by the MTT assay and Alizarin red staining. Gene declaration was also assessed at 3 and 5 days. They reported an increase in cell proliferation rate in presence of 8 mg/mL concentration of Cenebone and Osseo + after 48 h; however, cell proliferation rate decreased in presence of 16 mg/mL concentration of Cenebone, Osseo +, and Alloss. The study results showed that all bone substitutes improved cell differentiation. In study of Hu et al., in 2011; the osteogenic activity of bovine bone and EMUS is ostrich (Dromaius novaehollandiae) derived from natural bone marrow proteins was evaluated and compared. The results showed that ALP activity in bovine bone marrow proteins culture (C2C12 culture) increased from 2 to 10 times during 3–14 days (P < 0.05–0.001). Also, ALP activity in bovine bone marrow proteins and EMUS cultures was not significantly different at 14 days (P > 0.05). However, the osteogenic activity of cells in both bone marrow proteins and EMUS cultures increased (P < 0.05). Bone marrow proteins and EMUS cultures can have osteogenic activity in vitro.
Considering the in vitro design of this study and the differences of DPSCs and osteoblasts and preosteoblasts in the clinical setting, the generalizability of the results should be done with caution. This study could suggest a hypothesis for future studies, and further studies are recommended on this topic.
Considering the present results, it may be concluded that DPSCs in presence of the tested scaffolds can show a significant proliferation rate. The cells adhered to all three types of xenografts as scaffolds. According to the consequence of the PCR test, the Camel Bone group revealed a high rate of cell proliferation and osteogenic differentiation in both qualitative and quantitative assessments in vitro. Since the Camel Bone and bovine Bone Plus xenografts are new bone substitutes, further in vitro and in vivo studies on them are recommended.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
The authors would like to thank the Dental Research Center, Research Institute of Dental Sciences, Dental School, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
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