Implantable zoledronate-PLGA microcapsules ameliorate alveolar bone loss,gingival inflammation and oxidative stress in an experimental periodontitis rat model

Abstract

The effect of implantable Zoledronate-PLGA microcapsules (PLGA-ZOL) in periodontitis remains unclear. In this study, we aimed to explore the potential role of PLGA-ZOL in protecting periodontitis and elucidate the underlying mechanism. A rat model of periodontitis was established by ligation the mandibular first molars, then PLGA-ZOL was implanted. The healing volume was scanned by cone-beam computed tomography. Cytokine levels in the gingival tissues were determined by ELISA and RT-PCR. Oxidative stress was indicated by detecting superoxide dismutase concentration and catalase activity. After periodontitis model was successfully established in rats, PLGA-ZOL treatment significantly attenuated alveolar bone loss, as indicated by the increased total healing volume, bone volume/tissue volume and osteoprotegerin level, as well as decreased sRANKL level. PLGA-ZOL treatment also suppressed the inflammatory activities by inhibiting pro-inflammatory cytokine production (TNF-α, IL-1β) but increasing anti-inflammatory cytokine secretion (IL-10). Furthermore, PLGA-ZOL was found to ameliorate oxidative stress in gingival tissues. In conclusion, PLGA-ZOL microcapsules ameliorate alveolar bone loss, gingival inflammation and oxidative stress in an experimental rat model of periodontitis.

Keywords

PLGA-ZOL alveolar bone loss inflammation oxidative stress periodontitis

Introduction

Periodontitis is a common infectious and inflammatory oral disease, and will finally lead to loose teeth and teeth loss.¹ Periodontitis is characterized by continuous alveolar bone loss and the destruction of supporting periodontal tissues.^1–3 Initially, periodontitis is the inflammatory response to protect against periodontal pathogens, and the production of the pro-inflammatory cytokines further results in periodontal tissue destruction.² Inflammation was commonly believed to be involved in periodontitis progress. During periodontitis development, hydrolytic enzymes, inflammatory cytokines and oxygen radicals were released by resident cells, such as tumor necrosis factor (TNF)-α, interleukin 1β (IL-1β) and IL-8 promoting the alveolar bone loss and periodontal tissue destruction, while IL-10 protecting against periodontitis.^4–6 Clinically, surgery and mechanical therapy were used for periodontitis treatment.⁷ However, the effects are limited and unsatisfactory. Moreover, it is hard to avoid periodontitis-induced alveolar bone loss and periodontal tissue destruction.⁶ Therefore, it is critical to find out a new therapy for periodontitis treatment.

Periodontitis was commonly induced by placing a bacterial plaque retentive ligature in the gingival sulcus around the molar teeth. In the rat periodontitis model, microbial dysbiosis was necessary for the development of periodontitis. Studies indicated that oral microbial dysbiosis contributed to periodontitis. During the development of periodontitis, microbial complexes formed, such as the orange complex (consists of gram-negative anaerobic species and red complex (consists of the periodontal pathogens).⁸ Clinically, healthy periodontal tissue maintains mild state of inflammation, while diseased periodontal tissues exhibits severe inflammation.^9–12 It is suggested that the conversion from the symbiotic microflora to dysbiotic pathogenic microflora leads to severe inflammation in host and thus contributes to the tissue destruction and alveolar bone loss in periodontitis.⁹ When analyzing the clinical samples from periodontitis patients and healthy control, the significant clustering of periodontitis-associated microbiota was observed.¹³ Zoledronate (ZOL), also known as zoledronic acid, is a new bisphosphonate, and is widely used for hypercalcemia treatment.^14,15 Recently, the study of the effect of ZOL on older women with osteopenia demonstrated that the older women receiving ZOL treatment showed a lower risk of vertebral fractures, symptomatic fractures, height loss and nonvertebral fragility fractures,¹⁶ indicating the protective role of ZOL in osteopenia. However, the normal route of ZOL is oral administration, while the absorption efficiency is relatively low from the gastrointestinal tract,¹⁷ and thus limited the application of ZOL clinically. Interestingly, there is evidence that bisphosphonates including ZOL could be used as an adjunctive local drug.¹⁸ Polylactide-co-glycolide (PLGA), a biodegradable material approved by the Food and Drug Administration, was widely used for implanted medical devices.^19–21 PLGA prolonged the bioactivity of implanted drugs via regulating degradation time.^22,23 Therefore, PLGA was commonly used for promoting therapy efficiency in tissue repair by controlling drug release.

In the present study, we aimed to develop the PLGA-ZOL microcapsules and to analyze their characterization, such as the chemical structure, morphology, size distribution and cumulative release. In addition, we determined the effect of PLGA-ZOL microcapsules on alveolar bone loss, inflammatory cytokines release, superoxide dismutase and catalase concentrations in established periodontitis rat model.

Methods

PLGA-ZOL microcapsules

The oil phase was prepared by dissolving 0.68 g PLGA (Jinan Daigang Bioengineering Co., Ltd, Jinan, China) into 1.2 ml solution that was prepared by mixing dichloromethane (Titan Technology Co., Ltd., Shanghai, China) and ethyl acetate (Titan Technology Co., Ltd., Shanghai, China) at the ratio of 2:3. The 0.2 ml ZOL (Jizhi Technology Co., Ltd., Shanghai, China) solution in water with a concentration of 20 mg/mL²⁴ (and following our unpublished study) was served as internal water phase. Then, colostrum was prepared by slowly adding the internal water phase in the oil phase, followed by mixing 3000 rpm for 6 min using a magnetic mixer. Then, the double emulsion was prepared by slowly dropping the colostrum into 10 ml water solution with 4% polyvinyl alcohol (Titan Technology Co., Ltd., Shanghai, China) and 3% NaCl (Titan Technology Co., Ltd., Shanghai, China), followed by mixing 3000 rpm for 10 min using a magnetic mixer. Then, stirred at very low speed until completely volatilizing the oil phase. Collecting the microcapsules at the bottom of the tube by washing with ultrapure water repeatedly after centrifugation for 10 min at 4000 rpm. Then, freeze-dry the microcapsules after another centrifugation. Store the microcapsules at –20°C until use.

PLGA-ZOL microcapsules release assay

PLGA-ZOL microcapsules release assay was performed at 37 °C using phosphate-buffered saline (PBS) or normal saline (NS) media. The method followed our unpublished study and a previously published study.²⁴ Briefly, a total of 20 mg PLGA-ZOL microcapsules were added into 10 mL media and were oscillated at 37 °C on 100 rpm. On different time points, 2 ml samples were collected for ZOL amounts detection by Ion chromatography. Cumulative release of ZOL on different time points (Qn) (0, 10, 20, 30, 40, 50 and 60 hours for short time, 0, 5, 10, 20, 25 and 30 days for long time) was calculated as:

Q n = CnV 0 + (C 1 + C 2 + C 3 + \dots + C n - 1) \times V

Cn is the ZOL concentration on different time points, V0 is the total release medium volume, V is sample volume at different time points. The cumulative release rate (Q) was calculated as: Q = (Qn/W) × 100%.

W is encapsulated ZOL content in PLGA-ZOL microcapsules.

Periodontitis rat model

The SD rats with the body weight around 220 g were purchased from Laboratory Animal Center of Jiangsu University and were kept into animal facility following the instructions. The study was supported by the ethics committee of The Affiliated Stomatological Hospital of Nanjing Medical University (2018–035-d2). The rats were randomly divided into 5 groups with 8 rats in each group: Control, Ligation, Ligation + PLGA, Ligation + ZOL, Ligation + PLGA-ZOL. To establish the periodontitis rat model, the rats were first anesthetized by intraperitoneal injection of 10% chloral hydrate at the dose of 300 µL/100 g body weight. Then, the ligation was performed on the mandibular first molars using the 0.2 mm orthodontic steel wire. In the following 4 weeks, the ligation was determined weekly, and replaced it once it was loosened. After the periodontitis model was established, 4 mg PLGA, ZOL or PLGA-ZOL was implanted on the site of ligation. Four weeks later, the rats were sacrificed for further detections.

In vivo imaging

Four weeks after ligation, the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate at the dose of 300 µL/100 g body weight. Then, the cone-beam computed tomography (CB-CT) was used for scanning. The repair in each rat was scanned continuously. The software Mimics Research 20.0 was used for analyzing CT images and calculating the healing volume.

Bone analysis

Maxillae from different groups were collected after the rats were sacrificed and were fixed in formalin solution, followed by analysis using micro-CT. The ratio of bone volume in tissue volume (BV/TV) was calculated using SCANCO software.

Elisa

The concentrations of sRANKL, osteoprotegerin (OPG), TNF-α, IL-1β, CXCL1/CINC-1 and IL-10 in gingival tissues were determined by commercial ELISA kits following to manufacturer’s instructions. The ELISA kits are purchased from MyBioSource (San Diego, CA).

Superoxide dismutase (SOD) concentration and catalase (CAT) activity

For SOD detection, the gingival tissues were homogenized, followed by mixing supernatant with a solution in pH = 7.8 with 50 nM phosphate buffer, 100 nM EDTA, 19.5 mM L-methionine. Then, added a 150 ml solution with 10 nM riboflavin and 750 nM NBT, followed by exposure to 20 W light for 15 mins. SOD was valued as the absorbance at 560 nm.²⁵ For CAT detection, 20 µl homogenate was mixed with 5 nM Tris-HCl EDTA buffer and 3% H₂O₂. The absorbance at 230 nm was detected immediately and 6 mins after mixing samples.²⁵

RT-PCR

RNA from gingival tissues was isolated using TRIzol reagent, followed by detecting RNA concentrations. Then, the reverse transcription was performed using the cDNA Synthesis Kit (Qiagen, Valencia, CA). RT-qPCR which was performed by using the SYBR TaqTM kit. The primers used in this study are: TNF-α: Forward: 5’-CGG GGT GAT CGG TCC CAA CAA G-3’, Reverse: 5’-GTG GTT TGC TAC GAC GTG GGC-3’, IL-1β: Forward: 5’-TGC TGT CTG ACC CAT GTG AG-3’, Reverse: 5’- CCA AGG CCA CAG GGA TTT TG-3’, iNOS: Forward: 5’-AGG CAC AAG ACT CTG ACA CC-3’, Reverse: 5’-GGT AGG GTA GAG GAG GGG AG-3’, IL-6: Forward: 5’-GGA TAC CAC CCA CAA CAG AC-3’ and Reverse: 5’-TTG CCG AGT AGA CCT CAT AG-3’; GAPDH: Forward: 5’-GGA CCA GGT TGT CTC CTG TG-3’, Reverse: 5’-CAT TGA GAG CAA TGC CAG CC-3’. GAPDH expression was used as a control. 2^−△△Ct method was used to detect the relative expression levels of genes.

Statistical analysis

All data were presented as mean ± standard deviation (SD). The data were analyzed by one or two-way ANOVA analysis followed with a Tukey or Bonferroni post hoc test, respectively. P < 0.05 was believed as statistically significant.

Results

Characterization of PLGA-ZOL microcapsules

The chemical structure of ZOL was shown in Figure 1(a). To explore the effect of ZOL in the periodontitis rat model, we first implanted ZOL to PLGA, which is a biodegradable material approved by FDA, and was widely used for implanted medical devices.^19–21 Figure 1(b) showed the PLGA-ZOL microcapsules under a microscope. Further, under a scanning electron microscope, the PLGA-ZOL surface was smooth without any adhesion (Figure 1(c)). The microcapsules size distribution of PLGA-ZOL was further verified. As shown in Figure 1(d), the size of microcapsules is 16–20 µm and the average Zeta potential of the microcapsules is below −36 mV, suggesting that the property of PLGA-ZOL microcapsules is stable.

ZOL release in different medias

ZOL release from PLGA-ZOL microcapsules was determined at 1,02,03,04,05,060 hours (Figure 2(a)). At the beginning, the release of PLGA-ZOL microcapsules was very slow both in PBS and normal saline (NS) media. Twenty-four hours later, about 40% of PLGA-ZOL microcapsules were released into media, suggesting a burst release effect. Moreover, the long-term release was also determined at 51,01, 52,02,530 days. Cumulative release rate at 10 days was around 70%, and 80% was released in 20 days, and 90% was released in 30 days (Figure 2(b)).

PLGA-ZOL ameliorates alveolar bone loss

To detect the effect of PLGA-ZOL in alveolar bone loss, we first established periodontitis rat model, and performed PLGA-ZOL treatment, while PLGA treatment and ZOL treatment alone was used as a control for PLGA-ZOL treatment. Cone-beam computed tomography (CB-CT) images of alveolar bone defects (ABD) was illustrated in Figure 3(a). PLGA treatment (Ligation + PLGA) had no influence on total healing volume as compared with the Ligation group. ZOL (Ligation + ZOL) and PLGA-ZOL (Ligation + PLGA-ZOL) treatment significantly increased the total healing volume when compared with the Ligation group. In addition, the total healing volume in the Ligation + PLGA-ZOL group is much higher than Ligation + ZOL group (Figure 3(b)). Then percentage of bone volume/tissue volume (BV/TV) was significantly increased in Ligation + ZOL group, and PLGA-ZOL treatment further enhanced the BV/TV percentage in the established rat periodontitis model (Figure 3(c)). Furthermore, the sRANKL and OPG concentrations in gingival tissues were detected. As expected, periodontitis development significantly enhanced the sRANKL level, while ZOL, especially PLGA-ZOL treatment dramatically suppressed the sRANKL level (Figure 3(d)). OPG level significantly decreased in periodontitis rat (Ligation), while ZOL treatment dramatically enhanced OPG level in gingival tissues. More importantly, OPG level in Ligation + PLGA-ZOL group is much higher than the Ligation + ZOL group (Figure 3(e)). These results indicated that ZOL ameliorates alveolar bone loss, while ZOL implantation into PLGA significantly enhanced the effect of ZOL in ameliorating alveolar bone loss.

PLGA-ZOL altered inflammatory cytokines

Inflammation was crucial for periodontitis development.²⁶ Accordingly, we detected the effect of PLGA-ZOL treatment on inflammatory cytokines in gingival tissues of the established periodontitis model. As shown in Figure 4(a) to (c), the concentrations of TNF-α, IL-1β and CINC-1 significantly increased after periodontitis rat (Ligation) when compared with normal rat (Control). ZOL treatment, especially PLGA-ZOL treatment significantly suppressed these cytokines concentrations. IL-10 concentration dramatically decreased in periodontitis rats, while PLGA-ZOL treatment significantly enhanced IL-10 concentration in gingival tissues of periodontitis rats (Figure 4(d)).

PLGA-ZOL suppressed pro-inflammatory cytokine expression

We further determined the effect of PLGA-ZOL treatment on pro-inflammatory cytokine expression. Periodontitis development (Ligation) significantly enhanced the mRNA expression levels of TNF-α (Figure 5(a)), IL-1β (Figure 5(b)), inducible nitric oxide synthase (iNOS) (Figure 5(c)) and IL-6 (Figure 5(d)). ZOL treatment dramatically suppressed the mRNA levels of these cytokines. More importantly, the levels of these cytokines in the Ligation + PLGA-ZOL group were much lower than in Ligation + ZOL group. These results suggested that ZOL treatment suppressed these pro-inflammatory cytokines, while ZOL implantation into PLGA (PLGA+ZOL) significantly enhanced the effect of ZOL.

PLGA-ZOL enhanced SOD concentration and CAT activity

Finally, we explored the effect of PLGA-ZOL on superoxide dismutase (SOD) concentration and catalase (CAT) activation in gingival tissues. Periodontitis development (Ligation) significantly suppressed SOD concentration and CAT activity as compared with normal rat (Control). ZOL treatment, especially PLGA-ZOL treatment dramatically enhanced SOD concentration (Figure 6(a)) and CAT activity (Figure 6(b)) in gingival tissues of periodontitis rats. These results indicated that PLGA-ZOL enhanced SOD concentration and CAT activity.

Discussion

Periodontitis still remains a threat to many people worldwide as it is a chronic inflammation-related oral disease. Currently, the efficiency of periodontitis treatment is limited partly due to the fast release of drugs, which leads to lower absorption efficiency. We therefore explored how to prolong the drug release by implanting ZOL onto PLGA and detected the effect of PLGA-ZOL in protecting periodontitis using an established rat model.

Animal models were widely used to study periodontitis. Numerous animal models in different species such as rats, hamsters, rabbits, ferrets, pigs, dogs, and primates have been used for modeling human periodontal diseases and treatments.^27,28 Rats were most widely used for the experiential periodontal animal model. In this study, the periodontitis model was established using rats by ligating the mandibular first molars. The successfully established rat periodontitis model was evidenced by reduction of healing area, increase of pro-inflammatory cytokine expression levels, and decrease of SOD level and CAT activity in gingival tissues. In this study, we prepared the PLGA-ZOL microcapsules as described in methods, detected the size distribution of PLGA-ZOL, and determined the cumulative release of ZOL from PLGA-ZOL microcapsules in different media. To this end, the prolonged and stable release of ZOL from PLGA-ZOL microcapsules was confirmed. PLGA-ZOL treatment dramatically enhanced the healing volume, the bone volume/tissue volume percentage, OPG concentration in gingival tissues, SOD level and CAT activity, while suppressed pro-inflammatory cytokine expression levels and concentrations in gingival tissues. All the data from this study demonstrated that PLGA-ZOL ameliorates alveolar bone loss, gingival inflammation and oxidative stress in an experimental periodontitis rat model.

It is a high challenge to use PLGA-based microcapsules for chronic inflammatory periodontitis treatment since the absorption efficiency is relatively low and the prolonged release of drugs was needed. Summarizing the published papers, we prepared the PLGA-ZOL microcapsules using the double emulsion-solvent evaporation methods as described in methods. When detecting the cumulative release in different media, we confirmed the stable and prolonged release of ZOL from PLGA-ZOL microcapsules. This is the basis of our study to explore the enhanced effect of PLGA-ZOL in periodontitis treatment.

Inflammation was commonly believed to be associated with the development of periodontitis. Various anti-inflammation agents were evaluated to regulate the host response to decrease cytokine release or activation.^29,30 In periodontitis patients, the pro-inflammatory cytokines (IFN‐γ, IL‐2, TNF‐α and IL‐1β) in serum and gingival tissue biopsies significantly increased when compared with healthy controls. Interestingly, the ratio of TNF‐α/IL‐4 and IL‐1β/IL‐10 are both corrected with periodontitis severity.³¹ Similarly, 5 mg/kg S. maritima administration in rat periodontitis model attenuated alveolar bone loss and inhibited the production of pro-inflammatory cytokine,²⁵ indicating that the effect to attenuate periodontitis in animal models was partially via suppressing inflammation. The short-term inflammation induced by hypoxic pretreatment improved cellular behavior in vitro and enhanced regenerative potential in vivo of periodontal ligament stem cells.³² In an animal model of periodontitis, treatment with resolvin-E1 completely eliminated the signs of chronic inflammation, which was reported to be associated with the regeneration of lost tissues.³³ In human gingival fibroblasts and primary human mesenchymal stem cells, reduction of inflammatory mediator release re-established the impaired collagen metabolism, increased hMSCs differentiation towards the osteoblastic lineage, and promoted hard tissue regeneration of the periodontium.³⁴ We found that PLGA-ZOL treatment significantly suppressed the pro-inflammatory cytokine production and expression levels (TNF-α, IL-1β, CINC-1, iNOS and IL-6), and promoted the SOD levels and CAT activity in gingival tissues of experimental rat periodontitis model.

It is very important to point out the limitations of the present study. First, the effect of PLGA-ZOL in attenuating periodontitis is only detected in the rat model. The mouse periodontitis model would be helpful to identify the common effect of PLGA-ZOL in periodontitis. Second, the periodontitis associated cell line should be used to detect the direct effect of PLGA-ZOL in attenuating periodontitis. Third, the mechanism is not determined. The biological effector of PLGA-ZOL and the signaling pathways should be detected to clarify the potential mechanism. Fourth, microbial dysbiosis was necessary for periodontitis development. Oral microbial dysbiosis was observed in the animal models and human periodontitis patients.^8,13 The microbial dysbiosis should be detected. Finally, clinical studies should be performed to further verify the results from animal models. However, these are all we will do in future studies.

In summary, this study provided evidence that PLGA-ZOL treatment significantly attenuated alveolar bone loss, suppressed pro-inflammatory cytokine expression levels and concentration, and ameliorated oxidative stress in gingival tissues of established rat periodontitis model. This study supplied theoretical support for the potential use of PLGA-ZOL for periodontitis treatment in the clinic. After clinical studies, PLGA-ZOL may be used for periodontitis treatment.

Conclusions

PLGA-ZOL microcapsules ameliorate alveolar bone loss, gingival inflammation and oxidative stress in an experimental periodontitis rat model.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by Construction project of advantageous disciplines in Colleges and universities of Jiangsu Province (2018-87); No. 20 project supported by scientific research of the fifth “169 project” of Zhenjiang City; Qingdao Municipal Medical Research Guidance Plan 2019 (2019-WJZD078, 2019-WJZD060).

ORCID iDs

Chun Yao

Feimin Zhang

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