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The Diabetic Dental Pulp Repair: Involvement of Vascular Endothelial Growth Factor and Bone Morphogenetic Protein 2

UMMARY Introduction/Objective We aimed to investigate the effects of diabetes mellitus (DM) on rat dental pulp repair by measuring time-dependent changes in expressions of vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP 2) following direct pulp capping. Methods Two groups, each of 20 Wistar rats, received either streptozotocin (for DM induction) or the same volume of sterile saline. One week later, the pulp of maxillary and mandibular right incisors in diabetic and nondiabetic groups were exposed and capped with calcium hydroxide in order to provoke reparative response. The levels of VEGF and BMP 2 were determined in the pulp tissue lysates one and seven days after the pulp capping, using enzyme-linked immunosorbent assays. Results Diabetic state per se increased VEGF level, with a peak at first day after the pulp capping (19.3 ± 0.9 pg/mg, p < 0.001), but did not affect BMP 2 levels. Significant increase of BMP 2 expression was noticed on seventh day in capped pulp, but only in diabetic rat (16.7 ± 1.0 pg/mg, p = 0.001). Positive correlation between VEGF and BMP 2 was found on seventh day following capping, only in diabetic pulp (r = 0.905, p = 0.003) . Conclusion Diabetes-induced increase in VEGF expression reflects changes in the inflammatory phase of pulp repair in DM. Increase in BMP 2 expression suggest that stimulating effect of calcium hydroxide appears seven days after diabetic pulp capping. Present results show that capping procedure induce VEGF expression being the prominent on the first day after the procedure while returning to control levels on the seventh day after the capping, suggesting significance of VEGF for the inflammatory phase of pulp repair after capping. Namely, previous studies showed an increase in capillary proliferation and inflammation being intensive one to three days after direct pulp capping of rat teeth with calcium hydroxide [12], suggesting time line for inflammatory phase following capping.


INTRODUCTION
Under pathological conditions, such as injury or infection, the dentine-pulp complex shows significant reparative response initiated by an inflammatory reaction, prerequisite for pulp healing and mediated principally by macrophages [1]. Besides clearing the injury site, macrophages are the main source of the growth factors, including vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP 2), required for tissue repair [2,3].
Dental pulp repair rely on dental pulp stem cells that migrate to the site of injury, differentiate and proliferate into endothelial cells, involved in angiogenesis, or into odontoblasts-for dentin generation, processes regulated by growth factors, among others VEGF and BMP 2 [1,4]. Recent study from Aksel et al. showed that in vitro delivery of VEGF and BMP 2 significantly enhanced the angiogenic and odontogenic potential of human dental pulp stem cells [4]. Noteworthy, the alteration in expression of VEGF and BMP 2 has been identified within human dental pulp cells in inflammation [5,6] as well as in human diabetic pulp tissue [7].
Diabetes mellitus (DM) impede healing of dental pulp resulting in inadequate reparative response [8], yet underlying molecular mechanisms are still not clarified. Recent study investigating diabetic wound healing point at alterations in the inflammatory phase due to macrophages dysfunction as a critical event in impaired tissue healing in DM [9]. Having in mind that dental pulp, due to its limited collateral circulation, is especially sensitive to diabetes-induced circulatory disorder and associated failure to deliver components of the immune system and growth factors [10], we aimed to investigate effects of diabetes on initial events of dental pulp repair by means of measuring time-dependent changes in VEGF and BMP 2 expression in rat dental pulp following direct capping.

Reagents
Streptozotocin (STZ) was purchased from Sigma-Aldrich (St. Louis, Mo, USA). The enzyme-linked immunosorbent assay (ELISA) kits for VEGF (Rat VEGF ELISA Kit) were purchased from RayBiotech Inc. (Norcross, GA, USA) and the ELISA kits for BMP 2 (Quantikine BMP-2 Immunoassay) were purchased from R&D Systems Inc. (Minneapolis, MN, USA). Other reagents, medicaments and dental materials were procured from standard local commercial suppliers.

Experimental animals
The study was conducted on 40 Wistar rats with a body weight between 250g and

Induction of diabetes
All the animals underwent overnight fasting prior to induction of hyperglycaemia by intraperitoneal injection of 60 mg/kg of STZ, ex tempore dissolved in sterile saline [11].
Animals in control group were injected with the same volume of sterile saline. Blood glucose levels were estimated on blood from tail vein using GlucoSure glucometer and Touch-In test strips (Apex Biotechnology Corp, Taiwan), 5 min. before, 24 h and 7 days after STZ or saline administration. Only the animals showing 200 mg/dl blood glucose level were considered as diabetic.

Operative procedures
One week after the STZ or sterile saline injection, all animals underwent cavity preparation procedures on distal surfaces of right mandibular and maxillary incisors in order to provoke pulp reparative response. Left incisors remained intact and served as controls. The rats were anaesthetized with an intramuscular injection of 20 mg/kg tiletamin-zolazepam combination (Zoletil 100, Virbac, Carros, France). Before cavity preparation, oral cavity was disinfected with 0,2% chlorhexidine digluconate (Curasept 220, Curaden International AG, Kriens, Switzerland) and teeth additionally scrubbed with cotton pellet soaked with 70% ethanol. Aided with magnifying glasses (magnification 4,5x; Zeiss, Aalen, Germany), cavities were prepared with a micro motor handpiece and a carbide round burs (ISO 006; NTI, Kahla, Germany) until the pulp was visible. The cavities were prepared under constant water cooling. Pulp exposure was subsequently created with a sterile sharp probe (Ref. 27-3; HLW, Wernberg-Köblitz, Germany). The cavities were rinsed with saline solution, and haemostasis obtained with sterile, saline soaked paper points. After the careful air-drying of the cavities, pulp tissue was directly capped with calcium hydroxide (Ca(OH) 2 ) paste (Life; Kerr Corp., Orange, CA, USA) in order to induce reparative response, and cavities were restored using a self-etch, flowable composite restoration material (Vertise Flow; Kerr Corp., Orange, CA, USA).

Sample collection and preparation
Sacrifices of randomly chosen 10 animals in both diabetic and non-diabetic group were done one day after the capping procedure, using an overdose of thiopental-Na (Trapanal, Nycomed, Konstanz, Germany). The sacrifices of remaining 10 animals in diabetic and nondiabetic groups were done in the same way 7 days after the induction of pulp reparation. The incisor teeth were extracted and split using excavators and pliers, and pulp tissue evacuated with sterile probes and tweezers. The samples of pulp in reparation were formed as pools of pulp tissue from right mandibular and maxillary incisors of a single animal. Similarly, pools of intact pulp tissue were obtained from contra lateral intact incisors.
Specimens were transferred directly to previously weighed Eppendorf tubes. Tubes with pulp tissue were then measured for total weight and stored at -70°C until further use. All weight measurements were conducted using high precision (readability: 10 -4 g) Adventurer TM digital balance (OHAUS, Corp., Pine Brook, NJ, USA). The samples weights were calculated by subtracting total and empty tubes weights.
After homogenization, the pulp tissue lysates were centrifuged at 5000g for 10 min in micro-centrifuge (Heraeus* Biofuge Primo R, Thermo Fisher Scientific, Waltham, MA, USA), the supernatants were collected, divided into two aliquots (for VEGF and BMP 2 concentration measurement) and stored at -70°C until further analysis.

VEGF and BMP 2 quantification
Concentrations of VEGF and BMP 2 were measured in supernatants of rat pulp tissue lysates using ELISA kits according to the manufacturer's instructions. The absorbencies of microplate wells at 450 nm were recorded using Multiskan EX microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Each standard and sample was run in duplicate for both growth factors (GF) and the value used for statistical analysis was the average of two readings. The final GF concentrations of each pulp sample were normalized to its total weight. Results were expressed as pg/mg of pulp tissue.

Statistical analysis
Mean values and standard error of mean (SEM) were used for descriptive statistics of the sample. Parametric statistic analysis was used since the obtained concentrations of GF, blood glucose levels and body weight were normally distributed (Shapiro-Wilk test, P>0.05) and variance of observed groups were homogenous (P>0.05). Data were analyzed using one way analysis of variance (ANOVA) with post hoc Holm-Sidak method for pair-wise comparisons, and Pearson correlation coefficient. Analyses were computed with the statistical software (SPSS 18.0, IBM Statistics, USA). A P value less than 0.05 was considered to be statistically significant.

Variables of diabetic state
Diabetic animals expressed hyperglycaemia and significant loss of body weight (Table 1).

VEGF protein expression in dental pulp
Diabetic rats showed significant increase of the VEGF pulp levels (P<0.001) compared to nondiabetic in both intact and capped pulp, regardless of examined capping time duration (Fig 1). Diabetes per se increased VEGF levels at both time points: 19.   (Fig 2).

VEGF-BMP 2 correlations
In nondiabetic animals, there was a significant negative correlation between pulp VEGF and BMP 2 levels on the first day after capping. In diabetic pulp samples, VEGF and BMP 2 were positevely correlated on the seventh day after capping ( Table 2).

DISCUSSION
Pulp healing in rats shows histological similarity to pulp healing in humans after direct pulp capping with different dentinogenetic-stimulating agents [12]. Although rat molar teeth are more frequently used as model for human teeth for histological analysis of dentin repair [13], in the present study, due to voluminous pulp, incisor teeth were used in order to provide the required amount of pulp tissue necessary for VEGF and BMP 2 quantification.
Rodent incisor teeth, although differing from human for their constant growth, were proposed as a useful model for evaluating potential human dental pulp reactions to pulp capping agents and were used previously to evaluate several aspects of pulp repair and inflammation [14,15]. The repair of dental pulp after direct capping by Ca(OH) 2 implies following sequential steps: a moderate inflammation, migration of dental pulp stem cells, their proliferation and differentiation resulting in reparatory dentin formation [1]. Inflammation is prerequisite for pulp repair as a first step, resolved during the first week and is characterized by infiltration of macrophages starting from the first day after trauma or capping [1,16]. Spiller et al. showed that M1 macrophages, predominant in initial inflammatory phase, express VEGF gene in order to support angiogenesis [17] but also, VEGF is necessary for M1 to shift to M2 macrophage phenotype and to resolve inflammation [18].
Present results show that capping procedure induce VEGF expression being the prominent on the first day after the procedure while returning to control levels on the seventh day after the capping, suggesting significance of VEGF for the inflammatory phase of pulp repair after capping. Namely, previous studies showed an increase in capillary proliferation and inflammation being intensive one to three days after direct pulp capping of rat teeth with calcium hydroxide [12], suggesting time line for inflammatory phase following capping.
The effects of Ca(OH) 2 as capping material rely on its dissociation into calcium and hydroxyl ions. Hydroxyl ions exhibit antibacterial properties due to alcaline reaction and stimulates reparative dentin formation [19]. Oxidation of hydroxyl ions results in formation of hydroxyl radicals, which is able, as reactive oxygen species (ROS), to induce VEGF expression [20]. Beside ROS, another potent stimulus for VEGF induction is hypoxia. In line with this, our results show that experimentally induced diabetes caused a significant increase of VEGF levels in intact and capped dental pulp. This is probably induced by both hyperglycaemia and hypoxia, strong stimuli for VEGF induction, effects potentiated by the fact that dental pulp has limited or no collateral circulation [21], therefore is more prone to hypoxia-induced VEGF induction. Furthermore, intensive ROS generation and resulting oxidative stress are hallmark of diabetes and induced VEGF [22] and, accordingly, our previous studies showed oxidative stress in dental pulp tissue of patients with type 2 DM [23]. In line with the proposed mechanism of redox-mediated VEGF induction by Ca(OH) 2 and DM is the fact that present results show the greatest VEGF induction in both capped and diabetic dental pulp. Having in mind that diabetic state is characterized by an imbalance in the ratio of M2-"anti-inflammatory" and M1-"pro-inflammatory" macrophages in the favor of the latter [24], present results of enhanced VEGF expression in DM could be reflecting proinflammatory state induced by M1 macrophages prevalance but also, VEGF overexpression could contribute to resolving inflammation by activation of M2 "antiinflammatory macrophages".
Regarding BMP-2 levels, present results show significant increase in BMP 2 expression only in diabetic rats, 7 days after pulp capping, suggesting that BMP-2 induction depends on both Ca(OH) 2 and diabetic state. It is well known that BMP 2 promotes the differentiation of pulp stem cells into odontoblasts and production of reparative dentin, phases following inflammation resolution [25] which is in line with presently observed negative correlation between VEGF and BMP-2 observed on the first day after capping in nondiabetic pulps. Regarding mechanisms underlying stimulatory effects of calcium hydroxide on BMP 2 expression under diabetic state, it is noteworthy that in addition to ROS-stimulated BMP 2 expression [26], increased availability of Ca +2 ions is also associated with an increase in cellular BMP 2 expression [27]. These effects are potentiated in the diabetic state-state of oxidative stress and acidic environment which enhance BMP 2 expression and stability [28] but also, BMP 2 -VEGF mutual association, suggested by observed positive correlation between BMP-2 and VEGF in DM. Having in mind that dental pulp stem cells isolated from diabetic patients show diminished capacity for proliferation and differentiation and that BMP 2 supplementation enhanced differentiation of pulp stem cells into odontoblasts in vitro [29], present results point at the beneficial effects of calcium hydroxide as the direct capping agent in the DM for pulp repair.

CONCLUSION
Studies of dental pulp repair processes in vivo in humans are ethically and practically limited and, therefore, present results obtained in diabetic rat represent biologic background for consideration of therapies directed toward maintaining pulp vitality in diabetic dental   Figure 1. VEGF levels (pg/mg of tissue) in intact and capped dental pulp in diabetic and nondiabetic rat. Bars represent mean ± SEM of pulp VEGF levels on first (A) and seventh day (B) after pulp capping in diabetic and nondiabetic rat a-p < 0.001 diabetic compared to nondiabetic rats b-p < 0.001 capped compared to intact pulp