The effects of intro-oral parathyroid hormone on the healing of tooth extraction socket: an experimental study on hyperglycemic rats

Abstract Objective To investigate the effects of intro-oral injection of parathyroid hormone (PTH) on tooth extraction wound healing in hyperglycemic rats. Methodology 60 male Sprague-Dawley rats were randomly divided into the normal group (n=30) and DM group (n=30). Type 1 diabetes mellitus (DM) was induced by streptozotocin. After extracting the left first molar of all rats, each group was further divided into 3 subgroups (n=10 per subgroup), receiving the administration of intermittent PTH, continuous PTH and saline (control), respectively. The intermittent-PTH group received intra-oral injection of PTH three times per week for two weeks. A thermosensitive controlled-release hydrogel was synthesized for continuous-PTH administration. The serum chemistry was determined to evaluate the systemic condition. All animals were sacrificed after 14 days. Micro-computed tomography (Micro-CT) and histological analyses were used to evaluate the healing of extraction sockets. Results The level of serum glucose in the DM groups was significantly higher than that in the non-DM groups (p<0.05); the level of serum calcium was similar in all groups (p>0.05). Micro-CT analysis showed that the DM group had a significantly lower alveolar bone trabecular number (Tb.N) and higher trabecular separation (Tb.Sp) than the normal group (p<0.05). The histological analyses showed that no significant difference in the amount of new bone (hard tissue) formation was found between the PTH and non-PTH groups (p>0.05). Conclusions Bone formation in the extraction socket of the type 1 diabetic rats was reduced. PTH did not improve the healing of hard and soft tissues. The different PTH administration regimes (continuous vs. intermittent) had similar effect on tissue healing. These results demonstrated that the metabolic characteristics of the hyperglycemic rats produced a condition that was unable to respond to PTH treatment.


Introduction
Diabetes mellitus (DM) has an increasingly higher occurrence of 463 million adults worldwide. 1 People with DM are more likely to suffer from earlier detrimental oral status and are prone to numerous oral diseases, including oral infection, periodontitis and difficulties in wound healing after tooth extraction. 2 It has been found that the prevalence of tooth extractions in DM patients is 1.88 times higher than the general population. 3 The high-glucose microenvironment would delay the healing of tooth extraction sockets. 4 High glucose could affect the osteoblastic function and matrix mineralization. 5 The soft tissue repair is associated with the defect in the formation of granulation tissue and collagen degradation induced by excessive matrix metalloproteinase. 6 The dysfunction of fibroblast could impair the collagen synthesis, resulting in decreased fiber accumulation. 7 Many studies have explored different approaches to accelerate the healing of tooth extraction sockets under DM conditions, such as the use of growth factors, 8 graft fillings, 9 ellagic acid, 10 and low-level laser therapy. 11 The effectiveness of these approaches, however, is unsatisfactory due to the complexity of DM and vulnerability of the oral environment. 12 Parathyroid hormone (PTH) is one of the important hormones to maintain the balance of the calcium and phosphorus metabolism. 13 PTH can regulate osteoclast activity and influence bone remodeling, and is currently the only clinical drug to promote osteogenesis for the treatment of osteoporosis. 14 The intermittent administration of PTH has been found to directly inhibit transcription of the sclerosis gene and stimulate bone formation; 15 the continuous administration of PTH could increase the receptor activator of the nuclear factor-kB ligand/osteoprotegerin ratio, resulting in an increased osteoclastogenesis. 16  The sol-gel-sol phase transition behavior was carried out as follows: PECE hydrogel was placed into 5 mL EP tube and incubated in a water bath at 0°C for ten min, and then was slowly heated at a rate of 0.5°C/min until 37°C. The sol-gel-sol phase transition diagram was recorded using the test tube-inverting method, which was visually observed by inverting the tube. The condition of sol and gel phase was defined as "flow liquid sol" and "no flow solid gel". Results are presented as mean±standard deviation.
The normality test was performed using the Shapiro-Wilk analysis; the statistical comparisons were analyzed by the Student's t-test, one-way ANOVA, and two-way ANOVA, with a significance level of 5%.
Statistical significance was defined as 0.05 α-level.

Characterization of PTH controlled-release hydrogel
The chemical structure of PECE gel was characterized by 1 H NMR (Figure 1). The peak of the caprolactone unit and the ethylene glycol unit were at 3.98 ppm and 3.57 ppm, respectively. The macromolecular weight estimated from 1 H NMR spectrum was 3246, which was consistent with theoretical estimation, indicating that the PECE gel was synthesized successfully.
The temperature-dependent sol-gel phase transition behavior was presented in Figure 2A. The hydrogel showed a sol state at lower temperature (0°C) and a gel state at body temperature (37°C).
The hydrogel underwent sol-gel-sol phase transition as the temperature increased and slowly released PTH at local part, which prolonged the therapeutic effect time and reduced the repeated injections.
The controlled-release ability of PECE hydrogel is shown in Figure 2B. The release rate of the drug was rapid in the first 168 hours, and then became comparatively slow and sustained in the next 168 hours. The cumulative release of protein reached 58.24% by the 14th day. Therefore, the experiment period for the PTH controlled-release hydrogel was set as 14 days in this study.

Serum chemistry
The level of serum glucose in the DM groups

Histological analysis of the extraction socket
The histological analysis of soft tissue (HE and Masson's trichrome staining) showed that more collagen in the extraction socket was observed in the groups treated with PTH (both iPTH and cPTH) than those not treated with PTH ( Figure 4A and B). The quantitative analysis of hard tissue (new alveolar bone formation) showed no statistical significance between DM and non-DM groups or between NS and PTH groups (p>0.05) ( Figure 4C).

Discussion
In this study, bone formation in the extraction socket in the DM rats was decreased when compared to  The effectiveness of PTH on promoting the healing of tooth extractions in the diabetic rats was not significant in the study. This may be due to the metabolic characteristics of hyperglycemic rats leading to a condition of ineffective PTH treatment. Some studies showed that PTH did not increase the insulinimmunoreactivity of β-cells of the pancreatic islets in diabetic rats. 24 The inflammation caused by diabetes would disrupt vascular endothelial cell function. 25 The expression of inflammatory cytokines such as TNF-α, IL-1 and IFN-γ in bone increased, 26,27  promote osteogenic differentiation. 34 The cPTH did not suppress osteogenesis, but the catabolic role on bone metabolism was greater than its anabolic role, resulting in bone loss. The cPTH increased slightly undifferentiated mesenchymal stem cell, osteoblast and osteocyte number at 14 days. 35 This may result in the non-significant difference between two regimes of PTH. Another possible reason might be that the rats used in the study were male. The cPTH was found to be not catabolic in male rats, as no change was found in BMD and cortical thickness in males. 36 The experiment period adopted in this study was two weeks. This was in consideration of the optimal slow-release effect of the PECE hydrogel. Furthermore, the epithelialization was generally complete and well-keratinized at approximately two weeks. 37,38 The maximum bone formation was achieved and the woven bone completely filled the extraction socket, 4 and almost replaced by substance with a radio-opacity during that timeframe, 37 so 14 days was appropriate for this study.