Article Type : Original Articles
Authors : Saberian E, Jalili Sadrabad M, Petrasova A and Izadi A
Keywords : Dental Pulp; Stem Cells; Regeneration; Transplantation
Background: Dental loss and deformities caused by diseases and trauma are a major worry that have a negative impact on one's health and quality of life. Treatments for oral disorders now available can enhance clinical diagnostic criteria but do not restore damaged tissue. The pulp of the teeth is a unique source of stem cells. Human Dental Pulp Stem Cells (hDPSCs)-based regeneration techniques have showed considerable promise for treating dental abnormalities during the last few decades. The goal of this initiative is to use hDPSCs as a scaffold-base method for regenerative endodontics (hydroxyapatite: HA).
Methods: In this project, 8 incisor teeth of 4 rabbits were used that divided into 2 groups of case and control. In 2 cases hDPSCs injected (group A) and in 2 cases DPSC + hydroxyapatite (HA) injected (group B). All cases were checked in morphology of regenerated tissue (vascularization, innervation, intact odontoblastic layer, regenerative dentine) and side effects (degree of inflammation, necrosis) after 3 and 10 days.
Result: In 2 case groups(A, B) comparing with control group, the rate of inflammation was less and the morphology of regenerated pulp was improved, but no statistically significant difference between group A and control at the 3th and 10th day. Thus, in group B showed significant difference compare with control group in morphology and inflammation at 3 and 10 days (p=0.0132).
Conclusion: Our study suggests that the DPSCs+ HA transplanted to root canal decrease inflammation and promote cell proliferation and pulp regeneration. So, application of HA as a scaffold can significantly improve the findings rather than using DPSC alone.
Diseases and injuries cause bone and dental loss and deformities, which is a major problem with a high occurrence [1,2]. Autogenous bone transplantation is currently the gold standard treatment for bone abnormalities, however it is severely limited due to a lack of sources, difficulty harvesting grafts, and donor site morbidity. Other oral disease treatments currently available can only improve clinical diagnostic markers but not repair destroyed tissue [3,4]. In the nineteenth century, researchers focusing on embryonic development suggested the concept of stem cells. Following that, stem cells of various sorts were discovered in many tissues, including dental pulps. Dr. Irina Kerkis discovered dental pulp stem cells (DPSCs) as adult stem cells in 2005 . Because of their differentiation capacity and angiogenic capabilities, DPSCs have the potential to regenerate dentin and dental pulp tissue . Based on their characterization, such as expression of specific markers and multi-differentiation capacity, the separated cells may be called stem cells, which have shown to be promising for use in tissue regeneration . In vivo and in vitro models, DPSCs-HS (Human Serum) produced angiogenic growth factor concentrations comparable to DPSCs-FBS (Fetal Bovine Serum). DPSCs and stem cells from the apical papilla (SCAPs) were injected subcutaneously into immunocompromised mice in an in vivo model. All stem cell constructions showed osteogenic/odontogenic differentiation after 8 weeks, and regeneration of vascularized pulp-like tissue and mineralized tissue development after 12 weeks . For 4, 6, and 8 weeks, cultured DPSCs on human treated dentin (hTD), implanted in a mouse model. As a result, DPSCs combined with hTD in vivo, trigger the regeneration of dentin-like tissues such as dentin sialophosphoprotein and dentin matrix protein-1 . To use of dental pulp stem cells for pulp regeneration, in our experimental interventional study, 8 incisor teeth from 4 mature rabbits were used. Human DPSCs were cultured in proper culture medium and injected into the cleaned and washed canal area. In 2 case groups DPSCs injected (group A) and in 2 case groups DPSCs + Hydroxyapatite injected (group B). After 3 and 10 days, in 2 steps, each time, H&E staining was done to evaluate vital pulp tissue, inflammation, necrosis, vascularization, innervation, dentine quality (Osteodentine or Tubular dentine formation) and intact odontoblast layer.
In this interventional experimental study, 8 incisors teeth of upper jaw as clinical cases, from 4 male mature albino rabbits were used. The rabbits were 1-2 years old with 2.5 kg average weight. All the procedures and materials were performed at Pasargad Tissue and Gene Knowledge Company (Histogenotech, Tehran, Iran).
1-Culture of DPSCs
DPSCs line was obtained from the National Center for Genetic Resources of Iran. Cells were cultured in DMEM medium containing penicillin / streptomycin antibiotics and Fetal Bovine Serum (FBS) and under a luminar hood. The cell culture medium was changed every two days. Once a week, by briefly trypsinizing for 5 minutes with trypsin 0.25%, the cells were separated and cultured at a density of 1× 105 cell per square centimeter in a single flask. The new flasks were stored in 37 °C and 5% CO2 (Figure 1).
Figure 1: Cells cultured in two different groups with or without hydroxyapatite.
Determining the percentage of living cells
Trypan blue dye was used to determine the percentage of living cells. Trypan blue is a vital dye, so living cells do not allow it to pass through and repel it if it enters. But the membranes of dead cells are not able to remove vital dye from the cytoplasm. Therefore, dead cells turn blue and are easily recognizable from living cells that do not stain. For this purpose, first, 10 ?l of trypan blue solution of 0.4 wt% by volume was added to 90 ?l of cell suspension (trypsinized cells suspended in 1 ml of culture medium) and pipetted several times. Then, 10 microliters of the above solution was poured under a neobar slide and the percentage of stained (blue) cells was determined as the percentage of dead cells by light microscopy.
To perform most of the daily cell culture processes such as freezing, passage, etc., it is necessary to know the number of cells. Using the right number of cells and constant improves cell growth and helps to standardize and repeat the tests in the cell culture process. One of the methods of cell count in a cell solution (suspension) is the use of a neobar slide (homocytometer). After trispinizing and separation of both cell lines from the bottom of the culture dishes, the cell suspension was centrifuged at 1000 rpm at 4 ° C for five minutes. The supernatant was then discarded and the cells were homogenized in one milliliter of culture medium. For cell count, 10 ?l of the cell suspension was mixed with 90 ?l of methyl green solution and counted under a neobar slide (homocytometer) under a microscope. Before pouring the cells onto the slide, the cell suspension was homogenized several times with a sampler to prevent the formation of cell clamps and cell deposition and to increase the accuracy of the work.
Figure 2: Cell count using neobar slide.
When filling the counting chamber, the cells were randomly distributed in the counting chamber, and to determine the total number of cells, the cells in the 4 squared corners of the scaled slide of the homocytometer were counted and the number of living cells was calculated according to the following equation:
Number of viable cells per milliliter of cell solution = Mean of cells counted in 4 neobar slide areas × Percentage of viable cells ×Slice dilution coefficient (10) × Slide number (104) (Figure 2).
General anesthesia was done with 100mg/kg ketamine and 5mg/kg Xylazine IP (Intra Pritoneal) injection. Then the access hole was made on palatal surface of each tooth with round carbide bur #2 and high speed hand piece, to appear the pulp. For initial access to dental apex, K-file#6 was used. After complete cleansing of the pulp tissue from the roots and crown (pulpectomy) with barbed broach and K-file, number 6-30, and washing with normal saline and sodium hypochlorite, we passed the K-file#30 through the canal apex to induce bleeding. Then the canal cleaning would continue with K-file#30 to dilate the apical foramen to 0.5 mm diameter. During preparation steps, the tooth canal was washed with sodium hypochlorite 5.25% and was dried with sterile paper points. 0.06 gr hydroxyapatite as a scaffold was dissolved in 200 landa PBS, and finally 10 landa in 105 cells is obtained. In each rabbit, one upper incisor tooth was in an interventional (case) group. In 2 case groups DPSCs injected (group A) and in 2 case groups DPSCs + final obtained hydroxyapatite injected (group B). Another upper incisor tooth was in control group that after canal cleaning, we didn’t put anything inside it. Then all these incisors were sealed with Self-cured Glass Ionomer (GC Company) (Figure 3).
Figure 3: Cell transplant into the opened dental canal.
So, 4 teeth were in case group and 4 teeth in control group. After passing 3 and 10 days, in two steps, each time 2 rabbits were taken from the samples. By observing ethical standards, general anesthesia was done with 100mg/kg ketamine and 5mg/kg Xylazine IP (Intra Pritoneal) injection. Then the infiltrated injection was done in the depth of incisor tooth vestibule. The tooth with elevator and dicidious teeth forceps was extracted. The extracted teeth were put in formalin solution 10% immediately for 24-72 hours for fixing the tissue. Then dehydration with ethyl alcohol was done. This helps paraffin to penetrate into the tissue for cutting easily. Then put in xylol solution for removing extra alcohol and paraffin. Then samples were colored with Hematoxylin and Eosin (H&E) staining. The samples were observed with light microscope to compare and check in inflammation, necrosis, vascularization, innervation, dentine quality (osteodentine or tubular dentine formation) and intact odontoblast layer (Figure 4).
Figure 4: Tooth extraction.
In H&E staining, the degree of inflammation was classified according to the following score:
· There are one or more scattered inflammatory cells in the pulp below the injection site.
· Polymorphonuclear leukocytes (acute) or mononuclear lymphocytes (chronic) in an inflammatory lesion
· Severe inflammatory lesion that appears as an abscess or dense infiltration in one-third or more of the crown pulp
· The pulp is completely necrotic
The pulp soft tissue morphology was classified according to the following score:
· Morphology of normal or relatively natural tissue below the injection site and throughout the pulp, with vascularization, innervation, tubular dentine formation and intact odontoblast layer
· Lack of morphology of normal pulp tissue below the injection site, with a relatively normal appearance in deeper areas
· Loss of general pulp morphology and cellular organization below the injection site
· Without vascularization, innervation and intact odontoblast layer, with osteodentine dentine formation
Comparison of the group A and the control group according to the inflammation degree after 3 days
Based on the inflammation scoring that mentioned in method, in both groups, the rate of inflammation (as an abscess in one third of the cronal pulp area) was almost high (Mean degree in the group A was 3.000±0.5774 and mean degree in the control group was 3.667±0.3333). So there was no statistically significant differences between control group and the group A with DPSCs transplantation at 3 day (P value = 0.3739) (Figure 5).
Figure 5: Histological view of the inflammation degree of the group A and the control group after 3 days.
Comparison of the group A and the control group according to the inflammation degree after 10 days
Histological examination showed that the inflammation degree was higher in the control group (Mean = 3.333±0.3333) than the group A (Mean = 2.333±0.3333). In the control group, severe inflammatory lesion was observed as an abscess in one third of the coronary pulp area, while in the 10-day cell group, polymorphonuclear leukocytes (acute) or mononuclear lymphocytes (chronic) were observed at the transplant site. So, there was no significant difference between two groups in terms of inflammation (P value = 0. 1012). According to comparison result in each group (Table 1), with time passing, there was no significant difference in inflammation rate in the control group (P value = 0.5185). In the group A, the inflammation rate decreased with time passing but it wasn’t statistically significant differences (P value=0.3739). In the 3rd day, both groups had no significant difference in inflammation (P value = 0.3739). In the 10th day the inflammation rate in group A was less than control group but there is no statistically significant differences (P value=0.1012).
Comparison of the group B and the control group according to the inflammation degree after 3 days
Based on the inflammation scoring, the rate of inflammation in the case group B with DPSCs+HA transplantation at 3 days (Mean = 1.667±0.3333) was less than the control group (Mean = 3.667±0.3333) and in the control group the pulp was completely necrosed. So the difference was statistically significant (P value = 0.0132) (Table 1). In the group B, it was observed some polymorphonuclear leukocytes (acute) or mononuclear lymphocytes (chronic) at the surgical sites (Figure 6).