Table of Contents  
Year : 2022  |  Volume : 14  |  Issue : 1  |  Page : 3-11

Gaseous Ozone Treatment Augments Chondrogenic and Osteogenic Differentiation but Impairs Adipogenic Differentiation in Human Dental Pulp Stem Cells In Vitro

Department of Oral Medicine and Radiology, Dr. D. Y. Patil Dental College and Hospital, Pune, Maharashtra, India

Date of Submission10-Apr-2022
Date of Decision14-May-2022
Date of Acceptance23-May-2022
Date of Web Publication05-Aug-2022

Correspondence Address:
Lavanya Pasalkar
Dr. D. Y. Patil Vidyapeeth, Pimpri, Pune, Maharashtra 411018
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jofs.jofs_106_22

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Introduction: Stem cells have gotten a lot of attention because of their unique ability to differentiate and regenerate. Stem cells perform an important function in tissue regeneration and repair. Human dental pulp stem cells (hDPSCs) are a popular source of stem cells for accelerating wound healing. Over the last few decades, the use of ozone (O3) has progressed in medical specialties all over the world, resulting in significant clinical successes in the treatment of bone, vascular, and immunological ailments, as well as pain management. However, in the domain of tissue regeneration and differentiation, the effect of ozone on stem cells has received little attention. This is the first study to show that ozone therapy has an effect on hDPSCs. Materials and Methods: hDPSCs were isolated and grown in vitro from healthy extracted teeth. The culture media were allowed to absorb gaseous ozone. The ozone treatment was administered at concentrations of 0, 5, 10, 15, 20, 25, and 30 μg/mL, with a control concentration of 0 μg/mL. Proliferation was measured using the MTT reagent after 48 hours. The effect of ozone on the differentiation of DPSCs into osteoblasts, chondroblasts, and adipocytes was studied using the optimal ozone concentration. Results: One-way Analysis of variance (ANOVA) comparison between different concentrations of ozone showed statistically significant result (F = 23.895; P < 0.001). Maximum metabolic activity was observed with 10 μg/mL ozone. The proliferation increased up to 15 μg/mL; with further increase in O3 concentration, there was a marked reduction in proliferation. With 10 μg/mL, post-ozone treatment marked increase in osteogenic and chondrogenic. Chondrogenic differentiation was found to be statistically significant (P < 0.001) whereas marked decrease was seen with adipogenic differentiation. Conclusion: About 10 μg/mL ozonization slightly increased the proliferation in hDPSCs and distinctly increased the differentiation potential in chondrogenic and osteogenic lineages. But decreased adipogenic differentiation. With these findings, future studies will help to explain how ozonization affects hDPSCs to enhance their potency for clinical applications.

Keywords: Dental pulp stem cells, differentiation, ozone therapy, proliferation

How to cite this article:
Pasalkar L, Chavan M, Kharat A, Sanap A, Kheur S, Ramesh B. Gaseous Ozone Treatment Augments Chondrogenic and Osteogenic Differentiation but Impairs Adipogenic Differentiation in Human Dental Pulp Stem Cells In Vitro. J Orofac Sci 2022;14:3-11

How to cite this URL:
Pasalkar L, Chavan M, Kharat A, Sanap A, Kheur S, Ramesh B. Gaseous Ozone Treatment Augments Chondrogenic and Osteogenic Differentiation but Impairs Adipogenic Differentiation in Human Dental Pulp Stem Cells In Vitro. J Orofac Sci [serial online] 2022 [cited 2022 Dec 3];14:3-11. Available from:

  Introduction Top

Stem cell having the unique property of differentiation and regeneration has gained a huge attention in research field since its discovery. Stem cell plays an important role in the repair of injured tissues.[1] The three main varieties of stem cells are embryonic stem cells, adult stem cells, and induced pluripotent stem cells.[2] In human body, bone marrow, adipose tissue, amniotic fluid, umbilical cord, exfoliated deciduous teeth, dental pulp (DPSCs), and periodontal ligament are a few different sources of adult stem cells.[3] Specific cellular and molecular markers help to identify the unique phenotypes of stem cells. Human dental pulp stem cells (hDPSCs) possess mesenchymal (MSCs) phenotype which can evolve into diverse cell types such as osteoblasts, myoblasts, adipocytes, chondrocytes, liver cells, and β cells of islet of pancreas when subjected to preconditioning agents.[4] Dental stem cells are favorable for in vitro and in vivo experiments, and for therapy of immune-related diseases.

Currently, tissue engineering approaches are exploring the different strategies for bone regeneration like mixture of osteoconductive substitutes, stem cells, growth factors with bone augmentation at planned site of dental implant and for periodontal defect repair. Which would in turn overcome the downside of the autologous bone graft which remains the gold standard treatment in large bony defects, as it comes with several drawbacks such as donor site morbidity and availability of sufficient graft volume.[5] hDPSCs are promising source of stem cells for the repair and reconstruction of large bony surgical defects such as reconstruction of cranial defects in craniofacial surgery.[5],[6] Studies are being carried out to evaluate the effect of various biochemical or physical agents on properties of stem cell to make it more potent for clinical application.

Ozone (O3) is found in nature as a triatomic molecule consisting of three oxygen atoms. The term ozone means odor in Greek language, and was first used in 1840 by Christian Friedrich Schonbein, a German chemist who is considered to be the father of ozone therapy.[7] It is thermodynamically highly unstable as it rapidly decomposes to oxygen. At 20°C it has a half-life of approximately 40 minutes. Its solubility in water is 10 times higher and it is 1.6-fold denser in comparison with oxygen. After fluorine and persulfate, it is the third most powerful oxidative agent.[8] Ozone has diverse properties such as: antimicrobial, analgesic, immunostimulant, detoxicating, antihypnotic, bioenergetics, and biosynthetic. O3 can be dispensed as (1) gaseous ozone, (2) ozonated water, and (3) ozonated oil. Ability of ozonated water and oil to entrap and substantially release ozone, makes them a good delivery system.[8],[9] In the field of medicine, ozone has been used for treatment for >100 years but its therapeutic application has been criticized due its possible toxicity.[9] However, since past few years the medical use of O3 has been gaining popularity worldwide as an adjuvant treatment modality for various diseases. Evidence for dose-dependent effects of O3 treatment has been recently demonstrated. Low O3 concentrations activate cell antioxidative response pathways by inducing the purported eustress in terms of cellular proliferation,[10] thus providing the therapeutic benefits required for the clinical practice.[10],[11] Accelerated wound healing process with increased amount of bone matrix formation have been noted in murine model post-O3 therapy.[12] Clinical use of ozone for wound healing and osteoradionecrosis of the jaw has shown promising results.[9] In tissue regeneration, ozone action has not been explored till now. Study done with adipose and neural-derived stem cells has shown enhanced proliferation and migration potential post mild ozonization.[10],[13] There are no substantial data in hDPSCs. In this study, we conditioned the hDPSCs with low O3 concentrations to shed light on the alteration in proliferation/differentiation of hDPSCs at molecular level and assess the effect of gaseous ozone on proliferation and differentiation potential of hDPSCs in vitro.

  Materials and Methods Top

Ethical approval for this study (protocol no. DPU/1184/46/2019) was provided by the Research and Recognition Committee under the Faculty of Dentistry, Dr. D. Y. Patil Vidyapeeth, Pune, on December 20, 2019. A total of five healthy teeth were extracted for periodontic reasons, orthodontic treatment, or third molars were taken. Signed informed consent forms were obtained in agreement to institutional ethics contemplations. Under aseptic conditions, vertical groove was created over the entire tooth surface using drills. The tooth was broken vertically in two halves with the help of elevator. The dental pulp was separated with a broach and transported to laboratory in Dulbecco’s modified Eagle medium (DMEM).

DPSC isolation and culture

Isolation and in vitro expansion of hDPSCs was performed using the explant culture process defined beforehand.[14] Dental pulp tissue was crushed into minute pieces, and the fragments were positioned in a plastic culture dish. Necessary volume of fetal bovine serum was supplemented to the tissues to dip them entirely. Afterward, 24 hours incubation at 37°C and 5% CO2 was complete for explant tissue culture; the entire hDPSCs explant culture arrangement was additionally continued in DMEM supplemented with 20% Fetal Bovine Serum (FBS) and a small amount of antibiotic–antimycotic solution at the same temperature and CO2 settings. The complete cell culture/cell growth medium was replaced every alternate day, and the growth of hDPSCs and healthy morphological characteristics were observed frequently with an inverted phase-contrast microscope. About 80% confluent hDPSCs were detached using 0.25% trypsin and transferred to a bigger T-25 polystyrene culture flask. Confluent dental pulp stem cells (DPSCs) were detached using 0.25% trypsin and then uninterruptedly passaged in for growth and further experiments.

Flow cytometry for the characterization of the DPSCs for MSC-specific cell surface markers

Flow cytometry was used to assess cell with phenotypic MSCs characteristics. Cells were trypsinized followed by washing with Phosphate Buffer Solution (PBS). The concentration of cell was adjusted to 1 × 107 cells/mL in suspension and were transferred to a fresh Eppendorf tube (Cat no: z336769, sigma, India). Further, the cells were fixed by addition of 500 μL fixative solutions. At room temperature, 50 μL goat serum were used to block the cells for 15 to 20 minutes. The cells were incubated at 4°C with mouse anti-human fluorescein isothiocyanate-labeled monoclonal anti-CD90 and CD105, mouse anti-human phycoerythrin (PE)-labeled monoclonal anti-CD73 and CD45, mouse anti-human PE-labeled monoclonal anti-CD34 and Human Leuckocyte Antigen- DR isotype (HLA-DR) with dilution of 1:50 for 20 minutes. Twice washing with PBS was done to remove excess antibodies. Cells were acquired by using a BD FACSCaliber (BD biosciences, India) and analyzed by using Quest software (version 3.3; BD biosciences, India). To check the negative control, immunoglobulin G-stained cells having Fluroscein isothiocynate (FITC) and PE label were used.

Assessment of proliferation (MTT assay)

Proliferation of hDPSCs after preconditioning with different concentrations of ozone (5, 10, 15, 20, 25, and 30 μg/mL) was assessed by MTT assay. Ozone gas was induced in the culture media by attaching thin silicon tube to ozonator and by dispensing the ozone gas in the culture media present in the closed beaker. hDPSCs at the density of 1 × 104 cells per well were allowed to seed in 96 well plates. The cells after adherence (for minimum 12 hours) were treated with different concentrations of ozone, at 37°C for 48 hours. The proliferation rate of cells was determined after incubation by MTT assay on second day. To the each well, 10 μL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) at the concentration of 5 mg/mL was added and incubation was done at 37°C for 4 hours. Then MTT was taken off from the wells and blue formazan crystals were allowed to dissolve in 100 μL of Dimethyl Sulfoxide (DMSO). Reading of the absorbance was done at 570 nm using a microplate reader. Optimum concentrations showing proliferation effects (if any) was used with standard differentiation cocktails to study additive effects on differentiation.

Differentiation assay (osteogenic, chondrogenic, and adipogenic differentiation)

The cells were treated with standard differentiation cocktails + ozone and standard differentiation cocktails alone. The osteogenic, chondrogenic, and adipogenic effect was evaluated in both groups. The cells were treated with optimum concentration of ozone for 0, 8, and 16 days. Ozone gas was induced in the culture media by attaching thin silicon tube to ozonator and by dispensing the ozone gas in the culture media present in the closed beaker.

Osteogenic differentiation: A 24-well plate used to seed the cells at 2500/cm2 density with growth medium. Cells were exposed to osteogenic induction medium (DMEM containing 10% FBS, 0.1 mM dexamethasone, 1% antibiotic – antimycotic, 50 mM ascorbate-2-phosphate, and 10 mM β-glycerophosphate + 10 μg/mL O3) after 24 hours. The medium was changed weekly. After that, 2% alizarin red S was used to fix the pellet and stained the cells on 21st day in order to obtain the osteogenic differentiation and mineralization.

Chondrogenic differentiation: Centrifuging of 2.5 × 105 cells from each group were carried out in a 15-mL polypropylene tube at 1000 rpm for 5 minutes followed by DMEM wash. Then cultured DMEM containing of 1X-ITS, 100 nM dexamethasone, 1 mM sodium pyruvate, 50 mg/mL ascorbate-2-phosphate, 40 mg/mL L-proline, and 10 ng/mL Transforming Growth Factor (TGF)-β3 with 10 μg/mL O3. Followed by incubation for 4 weeks at 37°C in an atmosphere containing 5% CO2; the medium was changed weekly. Then, 4% paraformaldehyde was used to fix the pellet on 28th overnight. And 0.1% alcian blue was used to stain the paraffin-embedded sections to demonstrate chondrogenic differentiation.

Adipogenic differentiation: A 24-well plate (2500/cm2) used for seeding the hDPSCs. After 24 hours incubation, adipogenic induction medium was introduced to the cells and changed weekly (DMEM supplemented with 10% FBS, 1% antibiotic-antimycotic, 1 mM dexamethasone, 200 mM indomethacin, 0.5 mM isobutyl-methylxanthine, and 10 mM insulin + 10 μg/mL O3). Differentiated adipocytes were confirmed by 0.3% oil red O stain.

Quantification of the stained cells was carried out by measuring intensities from the images with Image J software.

Statistical analysis

The outcomes were displayed as the mean ± standard deviation of the experimental values. For proliferation assay, one-way Analysis of variance (ANOVA) test was used for comparing the difference among all groups together followed by post hoc test to compare each group with all the other groups. For differentiation assay, unpaired t test was used to compare the values obtained from ozone exposed group with its respective control group for osteoblast, adipocytes, and chondroblast differentiation.

  Results Top

The present study was carried out to evaluate the proliferation potential of hDPSCs after conditioning with different concentrations of ozone. Primarily the isolated cells from dental pulp were analyzed by flow cytometry for stem cell surface markers. After characterization of DPSCs, the ozone gas was allowed to flow in the conditioning media in concentration of 0, 5, 10, 15, 20, 25, and 30 μg/mL, respectively, at 37°C. Proliferation potential was obtained by MTT assay after 48 hours of treatment. The optimum concentration found to stimulate the proliferation of hDPSCs were further used to demonstrate the effect of ozone treatment on the trilineage differentiation potential of hDPSCs into osteoblasts, chondroblasts, and adipocytes.

hDPSCs were isolated by explant culture method. The cells were showing MSC-like morphology [Figure 1]A. Further to characterize the cells, MSC-specific cell surface markers were utilized to define cultured cells. Obtained data indicate that hDPSCs expressed CD73, CD90, and CD105. These findings are consistent with their undifferentiated state; hDPSCs did not express other surface markers such as HLA-DR, CD34, or CD45 (hematopoietic markers) [Figure 1]B.
Figure 1 (A) Morphological features of DPSCs (scale bar, 100 μm). (B) Flow cytometry analysis of cell surface markers in hDPSCs. Cells were labelled with antibodies against MSC markers (CD90, CD105, and CD73) and hematopoietic antigens (CD34, CD45, and HLA-DR). DPSCs, dental pulp stem cells.

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To evaluate the further effect of ozone on the proliferation of DPSCs, the DPSCs were exposed to various concentrations of ozone. The experiment was performed in triplicates to avoid an error and precise readings were obtained. [Table 1] shows the reading obtained from the MTT assay after 48 hours. One-way ANOVA comparison between different concentrations of ozone for amount of proliferation showed statistically significant result (F = 23.895; P < 0.001). The proliferation potential mildly increased at ozone concentration of 10 μg/mL after which there is a marked decrease in proliferation potential. The post hoc test used to compare each concentration group with all the other concentration groups showed that there is statistically significant decrease in hDPSCs proliferation rate at 25 and 30 μg/mL O3. It can be concluded that there is a marginal difference among the readings of optimum density obtained with control 5, 10, and 15 μg/mL O3 concentration groups. However, optimum density obtained with 10 μg/mL O3 conditioned media was the highest. Further increase in O3 concentration decreases the optimum density significantly compared to the control and lower concentration groups [Figure 2].
Table 1 MTT assay to assess the proliferation in DPSCs treated with various concentrations of ozone

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Figure 2 DPSCs were treated with various concentrations of ozone (0 μg/mL, 5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL, 25 μg/mL, and 30 μg/mL) for 48 hours and comparative analysis was done to check the proliferative activity of ozone to DPSCs. ns, not significant. **P < 0.01.

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After choosing the appropriate concentration of ozone, the effect of ozone on the differentiation potential of hDPSCs was assessed. The optimum O3 concentration of 10 μg/mL was considered to evaluate the change in trilineage differentiation potential of hDPSCs. Induction of O3 was given on days 0, 8, and 16 followed by reading which was taken on 21st day using Alizarin red stain for osteoblasts, alcian blue stain for chondroblasts, and oil red O stain for adipocytes.

Paired t test used for comparison of hDPSCs differentiation into osteoblasts, adipocytes, and chondroblasts with and without O3 exposure. In [Table 2] and [Table 3], comparison of mean score between osteogenic control and osteogenic ozone exposed group (t = 0.649; P = 0.552) [Figure 3] and adipogenic control and adipogenic ozone exposed group showed statistically insignificant result (t =0.803; P = 0.467) [Figure 4].
Figure 3 Quantification of functional staining (alizarin red S) in osteogenic differentiation of DPSCs treated with 10 μg/mL ozone and comparative analysis of staining intensities (scale bar, 100 μm). ns, not significant.

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Figure 4 Quantification of functional staining (oil red O) in adipogenic differentiation of DPSCs treated with 10 μg/mL ozone and comparative analysis of staining intensities (scale bar, 100 μm). ns, not significant.

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Table 2 Quantification of osteogenic differentiation

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Table 3 Quantification of adipogenic differentiation

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Whereas, in [Table 4], the comparison of mean score between chondrogenic control and chondrogenic ozone exposed with respective cocktails showed statistically significant result (t = 11.761; P < 0.001) [Figure 5]. It can be concluded that only chondrogenic differentiation was statistically significant in ozone-treated DPSCs. Whereas, decrease in the adipogenic differentiation was observed when exposed to O3 along with adipogenic induction compared to only adipogenic induction. Osteogenic differentiation showed slight increase in mineralization post ozone exposure.
Table 4 Quantification of chondrogenic differentiation

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Figure 5 Quantification of functional staining (alcian blue) in chondrogenic differentiation of DPSCs treated with 10 μg/mL ozone and comparative analysis of staining intensities (scale bar, 100 μm). **P < 0.01.

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  Discussion Top

Regenerative medicine and tissue engineering have given opportunity to modify, regenerate, and reestablish the functioning of various human parts. Stem cells is a fundamental tool in regenerative medicine which is well-defined as cells proficient of both self-renewal and multilineage differentiation potential.[1],[2] Tooth is found to be a substantial source of adult stem cells and the cells derived are called as hDPSCs. hDPSCs possess properties like ability to self-renew and multilineage differentiation.[15],[16] hDPSCs are easiest to harvest from exfoliated primary teeth and extracted wisdom teeth obtained from young adults, which are otherwise discarded as medical waste. Easy accessibility and capability of differentiation into several phenotypes has made it well-liked throughout the last decade in the discipline of regenerative medicine.[16]

Bone reconstruction with MSCs is an actively growing field which facilitates tissue healing even in cases of unfavorable circumstances.[6],[17] Chamieh et al.,[5] in their experiment treated two symmetrical full-thickness defects with a rat DPSCs having dense collagen gel scaffold and acellular scaffold. de Mendonca Costa et al.[6] treated eight rats having cranial defects with collagen membrane and combination of collagen membrane and hDPSC. In comparison to only collagen, the mixture used has shown to form more mature bone with no graft rejection. Thus, hDPSC is considered a promising tool for repair of large bony defects in craniofacial surgery. Martínez-Sarrà et al.[18] have shown wound healing in mouse model having muscular dystrophy with the help of therapeutic potential of hDPSCs. hDPSCs has improved angiogenesis by secreting several growth factors, matrix deposition, and enriched vascularization with all models; thus concluding that hDPSCs accelerate the wound healing process with reduced rate of dystrophic muscle degeneration.[6]

Ozone being a potent oxidant, leading to a dose-dependent oxidative stress due to its ability to generate the free radicals and reactive oxygen species resulting from the cell wall lipoperoxidation, protein oxidation, DNA damage, enzymatic inactivation, and cell apoptosis. Although, in the western countries, ozone therapy has been widely used for medicinal practice over the last few years thus giving significant clinical results in the treatment of bony, vascular, and immune disorders, and pain management.[9] At the same time, there are initial experiments coming which show direct effects of ozone on different adult stem cells. Costanzo et al.[10] investigated the effects of ozonization on MSCs obtained from adipose tissues, it has been noticed that mild ozone treatment enhances adipogenesis in stem cells without causing deleterious effects. Controlled reactive oxygen species are known to initiate the required signaling associated with cell proliferation, viability, and differentiation. Tricarico et al.[13] studied effect of ozone on neural stem cells and stated that based on concentrations used, ozone significantly alter the cytokine release,distance of cell migration with mild changes in cell proliferation. It also increased proliferation and cytokine production. Dental stem cells were still lagging behind in this process as no study had shown direct effect of O3 on hDPSCs. This is the very first research which shows the influence of ozone on the hDPSCs and aimed to put forth, hypothetically, the change in molecular mechanisms associated with the ozone treatment.

In our study, ozonization showed significant difference in proliferation of various groups. A 10 μg/mL of O3 has shown to increase hDPSCs proliferation potential. Maximum optimum density was obtained at 10 μg O3/mL. The further increase in O3 concentration markedly raised the cell death rate. There found to be marginal difference among the optimum densities of hDPSCs between the 10 μg O3/mL and control group. hDPSCs on exposure to optimum O3 concentration of 10 μg O3/mL with respective differentiating media showed significantly increased chondrogenic lineage differentiation as compared to its untreated counterpart. With marginal increase in case of osteogenic differentiation. Whereas, negative impact seen on adipogenic differentiation; contrary to the previous report,[10] adipogenic potential was found to be decreased post O3 exposure.

Still, further studies are necessary to give extensive understanding for the mechanisms of action of ozone on dental tissues. Main hurdles faced in this investigation were difficult to get exact concentration of gaseous ozone and reproducible results. Nevertheless, we have evidently demonstrated that majority of effects obtained after ozonization to the various other stem cell types were partly comparable to those of with hDPSCs. On the basis of these contemporary findings, further research is obligatory to elucidate the effects of ozone therapy on hDPSC, in order to formulate the targeted protocols of ozone treatment for its use in regenerative dentistry and clinical dental practices.

  Conclusion Top

Within the scope of this research, it can be concluded that hDPSCs are a good choice for investigating the effects of ozonization on cell proliferation and differentiation. At varied concentrations, ozonization caused a statistically significant (P < 0.001) shift in hDPSC proliferation. On hDPSCs, 10 μg/mL can moderately augment proliferation. Proliferation was dramatically inhibited at increased O3 concentrations. When cells were allowed to differentiate into the osteogenic lineage at the optimum concentration of 10 μg/mL of O3, the mineralization increased noticeably but not significantly. In the O3-treated hDPSCs, chondrogenesis was considerably greater (P < 0.001). Following O3 treatment, adipogenic differentiation was found to be reduced. Based on the current preliminary finding, additional molecular research can be conducted to elucidate the action of low ozonization on hDPSCs, with the goal of developing systematic ozone treatment regimens for hDPSC-based tissue regeneration and clinical translation.

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Conflicts of interest

Authors declare no conflicts of interest.

  References Top

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Chamieh F, Collignon AM, Coyac BR et al. Accelerated craniofacial bone regeneration through dense collagen gel scaffolds seeded with dental pulp stem cells. Sci Rep 2016;6:38814.  Back to cited text no. 5
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2], [Table 3], [Table 4]


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