Increasing the Number of Osteoblasts and Decreasing RANKL Expression on Diabetic Periodontitis in Rats Post-Administration of Nanoliposome Papaya Seed Extract

ABSTRACT

Objective:  To evaluate the impact of nanoliposome papaya seed extract on the number of osteoblasts and RANKL expression in a diabetic periodontitis rat model.

Material and Methods:  Thirty-six Sprague Dawley rats were randomly divided into three groups; each group was Group T₀ (rats induced to become diabetic periodontitis without treatment), Group T₁ (rats induced to become diabetic periodontitis and given 96% papaya seed extract) and Group T₂ (rats induced to become diabetic periodontitis and given 96% nanoliposome papaya seed extract). They were assessed at three points in time: day 3, day 7, and day 14. The extracts were given orally at 0.5 ml and drops as much as 0.03 ml on the gingival sulcus, and treatments were given once a day in each group at a predetermined time. Osteoblast cells were counted by hematoxylin-eosin (HE) staining, and RANKL expression was counted by immunohistochemistry (IHC) staining. The difference in osteoblast cells and RANKL expression between groups were analysed by one-way ANOVA and post hoc tests.

Results:  There was a significant difference in the number of osteoblasts on day 14 in the groups; a significant difference was also seen in RANKL expression on day 7 and day 14 in the groups.

Conclusion:  Administration of nanoliposome papaya seed extract affects the enhancement of the number of osteoblasts and reduction of RANKL expression in vivo.

Keywords:
Plant Extracts; Carica; Osteoblast; Diabetes Complications; Periodontics.

Introduction

Periodontal disease, an illness of supportive tissues of teeth, is a common oral infection suffered by humans [¹]. According to the Global Burden of Disease Study [²], periodontal disease was the 11^th most prevalent condition in the world. The type of periodontal disease that is known as a destructive and irreversible stage is periodontitis. Periodontitis causes destruction in the periodontal ligament, cementum, alveolar bone, and gingiva, and it influences quality of life. Periodontitis is a multifactorial and highly prevalent progressive disease; it was reported to affect 10.5%-12% of the world’s population. Chronic periodontitis is the most prevalent form of periodontitis, it is usually accompanied by the enhancement in the proportion of gram-negative organisms in subgingival biofilm like Porphromonas gingivalis, Tannerella forsythia, and Treponema denticola. The predominant bacterial pathogen in chronic periodontitis is Porphyromonas gingivalis, which has virulence factors that can invade periodontal tissue and also induce further immune responses with an increasing concentration of proinflammatory mediators that may increase periodontal breakdown [¹,³,⁴].

Epidemiological data proves that diabetes is a major risk factor for periodontitis, and periodontitis can cause various complications, one of which is worsening glycaemic control. People with diabetes mellitus have a high prevalence of periodontitis [⁵]. In Indonesia, the prevalence of periodontitis in diabetes mellitus patients reaches 75% [⁶]. Diabetic patients were three times more likely to develop alveolar bone loss and loss of attachment than patients who did not experience diabetes mellitus [⁷]. A previous study by Tse stated that diabetes mellitus has an interrelation with periodontitis; uncontrolled diabetes mellitus can increase the periodontal tissue inflammatory process, which aggravates alveolar bone destruction, and periodontitis can increase cytokine production, which worsens glycaemic control in diabetes mellitus [⁸]. Periodontal tissue condition may be exacerbated by impaired glycaemic control, immune dysfunction of bacterial invasion, oxidative stress, and increased inflammatory cytokine presence in diabetes. In the gingival sulcular fluid, several inflammatory cytokines can be triggered by hyperglycaemia. On the other hand, periodontitis enhances systemic oxidative stress and acute phase reactants such as C-reactive protein. So persistent chronic inflammation in periodontal tissue can affect the control of diabetes [⁹].

The main treatment in periodontitis is removing local factors as initial therapy with supporting therapy, such as using antibiotics and non-steroidal anti-inflammatory drugs if needed. Regenerative surgery is the gold standard in stimulating bone regeneration [¹⁰]. However, treatment with antibiotics may cause shortand long-term side effects, for example, bacterial resistance, infections, allergies, and diseases in the kidneys, heart, and liver [¹¹].

Recently, people focused on traditional herbs with natural compounds as an alternative treatment. It showed fewer side effects, low cost, high availability, and ability as a treatment and therapy agent. One of the potential traditional herbs is papaya seeds (Carica papaya Linn); it contains various bioactive compounds, namely alkaloids, flavonoids, saponins and phenols, which act as antibacterial and anti-inflammatory agents that promote wound healing [¹²]. The active compounds found in papaya seed extract that play a role in wound healing, especially flavonoids, have secondary compounds, such as quercetin, that have anti-inflammatory properties capable of increasing the production of osteoblasts in the osteogenesis process, and phenols, which have bioactive components, such as chlorogenic acid, that are capable of inhibiting osteoclast production [¹³].

Remodelling alveolar bone depends on the balance of bone deposition by osteoblasts and bone resorption by osteoclasts. Osteoblasts are a type of mesenchymal cell that play a role in producing the organic matrix of bone in the process of bone formation and development [¹⁴]. Receptor activator of nuclear factor-kappa B ligand (RANKL), its receptor activator of nuclear factor-kappa B (RANK) and osteoprotegerin (OPG) are key molecules in regulating osteoclast differentiation, recruitment and function. RANKL is essential for the complete differentiation of osteoclast precursor cells, so it plays an important role in periodontal bone resorption [¹⁵].

Although natural compounds showed high potency as treatment and therapy agents, they generally have a large molecular size and low water solubility leading to poor absorption and bioavailability [¹⁶,¹⁷]. Nanoliposomes are one way to modify drug delivery systems. They can optimise the therapeutic effect by providing specific targets in action. Liposomes are artificial vesicles made of phospholipids and cholesterol. They have been widely used as a carrier of active substances in medicine because of their ability to increase drug stability and are easily absorbed by the membrane [¹⁸]. This study aimed to evaluate the effectiveness of nanoliposome papaya seed extract on the number of osteoblasts and RANKL expression in a diabetic periodontitis rat model.

Material and Methods

Ethical Clearance

This research was evaluated and approved by the Ethical Committee of The Institute of Bioscience, Universitas Brawijaya (Certificate number 1198-KEP-UB). This was true experimental design research and randomised post-test-only method.

Papaya Seeds Extract Nanoliposome Synthesis

Extraction of papaya seeds with 96% ethanol solvent and synthesis of nanoliposomes was accomplished using the same method as previous research done by Pusporini et al. [¹³,¹⁹]. Papaya seeds were extracted using the maceration method and then synthesised into nanoliposome by combining Mozafari and sonication methods to reduce the particle size by 1-1000 nm [²⁰,²¹].

Experimental Animals

Young, male and healthy Sprague Dawley rats (± 250-300 g) were acclimatised for one week at Animal House, Faculty of Dentistry, Universitas Brawijaya. All animals were allowed free access to water and fed with standard rat pellets. Thirty-six Sprague Dawley rats were randomly divided into three groups: group T₀ (rats induced to become diabetic periodontitis without treatment), group T₁ (rats induced to become diabetic periodontitis and given 96% papaya seed extract), and group T₂ (rats induced to become diabetic periodontitis and given 96% nanoliposome papaya seed extract). Observations were assessed at three points in time: day 3, day 7, and day 14, so each group consisted of four rats.

After the rats had fasted for at least eight hours, they were induced to be in a diabetic condition by injection of nicotinamide 150 mg/kgBW dissolved with normal saline intraperitoneally, followed 15 minutes later by an injection of STZ 50 mg/kgBW dissolved with a citrate buffer (pH 4.5). This induction was repeated 24 hours later with the same dose. After induction, blood was collected from rats, and glucose levels were checked using a commercial glucometer. Rats reached diabetes mellitus if fasting blood glucose > 126 mg/dL [²²,²³]. For the induction of periodontitis, previously rats were anaesthetised by injecting ketamine HCL intramuscularly into the hamstring at a dose of 0.2ml/200grBW; further induction was done by tying a silk ligature of 3.0 size to sub gingiva around central incisor of lower jaw and dropping 5 µg LPS P. gingivalis in 0.05 ml of PBS once a day on the central labial incisors of the lower jaw for seven days [¹⁹,²⁴,²⁵]. The examination results of periodontitis condition are the gingiva looks redness and swelling, a periodontal pocket exists, and BOP (+) [²⁶]. Both the papaya seed extract and nanoliposomes papaya seed extract were administered orally at 0.5 ml and locally at 0.03 ml on the pocket. The treatments were given once daily in each group at a predetermined time [¹⁹,²⁷]. All samples were sacrificed with the cervical dislocation method under anaesthesia on days 4, 8, and 15. After being sacrificed, the lower jaw was cleaned with NaCl 0.9%, then placed in a closed container containing a 10% formalin buffer for fixation and sent to the anatomical pathology laboratory for histological preparation. Rats were properly buried.

Histopathological analysis used HE staining to calculate the number of osteoblast cells and IHC staining to calculate the RANKL expression number. Identification of osteoblast cells on preparation with HE staining have the form; cuboidal or flat with a single dark purple nucleus [²⁸]. Identification of RANKL expression on preparation with IHC staining was determined through osteoblast cells; osteoblasts that positively expressed RANKL were marked by dark brown cytoplasms [²⁹]. Calculation of osteoblast cells and RANKL expression used a light microscope with 400x magnification for five fields of view; it was done in the alveolar margin of the mesiolabial region of the incisors.

Statistical Analysis

The data was analysed using IBM SPSS statistics 22 with a significance level or probability value of 0.05 (p=0.05) and a confidence level of 95% (α=0.05). The data analysis was performed using a normality test with the Shapiro-Wilks test. The homogeneity test was done using the Levene test (p>0.05). One-way ANOVA tests and post hoc tests were performed to determine the significance of differences between groups (p<0.05).

Results

The clinical presentation of periodontitis condition and characteristics of animal model of diabetic periodontitis can be seen in Figure 1.

Figure 1
Periodontitis model in rat. A. Normal; B. Periodontitis; C. Characteristics of diabetic periodontitis model.

Histopathological observation on the alveolar bone around the central incisor of the lower jaw was carried out on days 3, 7, and 14. The mean and standard deviation of osteoblasts and RANKL expression from each group were presented in Figures 2 and 3. There were patterns of decreased numbers of osteoblasts in group T₀ and an enhanced number of osteoblasts in groups T₁ and T₂ at three time-point evaluations. The highest average number of osteoblasts was found in the nanoliposome papaya seed extract group (group T₂) on day 14. There were patterns of enhanced numbers of RANKL expression in group T₀ and decreased numbers of RANKL expression in groups T₁ and T₂ at three time-point evaluations. The lowest average number of RANKL expression is found in the nanoliposome papaya seed extract (group T₂) on day 14. The fluctuation is described in Figures 2 and 3, and the histopathological image of osteoblasts and RANKL expression is described in Figures 4 and 5.

Figure 2
Average number of osteoblast at different times of evaluation.

Figure 3
Average number of RANKL expression at different times of evaluation.

Figure 4
Overview of osteoblast cells using HE staining. The red round mark shows osteoblast cells in the alveolar bone that were observed using a 400x magnification Olympus digital microscope. A) T₀-3; B) T₁-3; C) T₂-3; D) T₀-7; E) T₁-7; F) T₂-7; G) T₀-14; H) T₁-14; I) T₂-14.

Figure 5
Overview of RANKL expression using IHC staining. Arrows and inserts indicate RANKL expression in the alveolar bone that was observed using a 400x magnification Olympus digital microscope. A) T₀-3; B) T₁-3; C) T₂-3; D) T₀-7; E) T₁-7; F) T₂-7; G) T₀-14; H) T₁-14; I) T₂-14.

According to the normality and homogeneity test, it showed that the data is normal and homogenous. The one-way ANOVA test showed a significant difference in the number of osteoblasts on day 14 (p=0.002); a significant difference was also seen in RANKL expression on day 7 (p=0.002) and day 14 (p=0.000). Post hoc tests showed a significant difference in the number of osteoblasts in group T₀ compared with group T₂ and group T₁ compared with group T₂ on day 14. Post hoc tests showed a significant difference in the number of RANKL expression in each group on day 7, also group T₀ compared with group T₁, and group T₀ compared with group T₂ on day 14 (Table 1).

Table 1
Post hoc test result of osteoblasts and RANKL expressions.

Groups	Osteoblasts			RANKL Expressions
	T0-14	T1-14	T2-14	T0-7	T1-7	T2-7	T0-14	T1-14	T2-14
T0-7				-	0.038*	0.002*
T1-7				-	-	0.049*
T2-7				-	-	-
T0-14	-	0.052	0.002*				-	0.000*	0.000*
T1-14	-	-	0.036*				-	-	0.165
T2-14	-	-	-				-	-	-

• ^*
  Statistically Significant.

Discussion

There is a reciprocal relationship between diabetes mellitus and periodontitis. Periodontitis can increase the production of cytokines that worsens glycaemic control; conversely, high blood sugar levels can increase the inflammatory process of the periodontal tissue [³⁰]. The bone remodelling cycle operates continually as osteoclasts constantly remove mature bone with new bone simultaneously formed by osteoblasts. A balance between bone resorption by osteoclast and bone formation by osteoblast determines the level of bone mass. Osteoblast cells influence the regulation of osteoclastogenesis through the regulatory system of OPG/RANKL/RANK. RANKL/OPG ratio is a determinant of the bone resorption process. RANKL is a member of TNF, it will bind to its receptor RANK to stimulate osteoclast differentiation and activation, they play a role in the formation and function of osteoclasts. OPG is a membrane that surrounds and secretes proteins attached to RANKL to inhibit its role on RANK receptors [¹⁵]. Hyperglycaemia, a condition related to diabetes mellitus, can increase the production of advanced glycation end products (AGEs), receptor of AGEs (RAGE), and reactive oxygen species (ROS). Interaction between AGEs and RAGE causes immune dysfunction and enhancement of proinflammatory cytokine expression. ROS can stimulate the enhancement of proinflammatory cytokine by activating intercellular signalling such as mitogen-activated protein kinase (MAPKs) and nuclear factor kappa B (NF-kB); it can damage tissue repair [³¹].

In this study, the condition of the experimental animals was made periodontitis condition accompanied by diabetes mellitus; in addition, they received different treatments as already mentioned in the method. The clinical signs that were seen in the rat models were bleeding on probing, redness, swelling, periodontal pocket, and fasting blood glucose >126 mg/dl. In group T₀, which did not receive any treatment, there was a pattern of decreased number of osteoblasts and an increased number of RANKL expression on the predetermined time series, namely days 3, 7, and 14. In each time series, the lowest number of osteoblasts and the highest number of RANKL expression were found in group T₀. This shows there is an inflammatory process in the group. Statistically, on day three, there were no significant differences in osteoblast number and RANKL expression between groups. The reduction of osteoblasts in periodontitis rats in this research was caused by inflammation triggered by LPS P. gingivalis that can activate multiple cell types via the production of proinflammatory cytokines [²⁶,³²].

Histopathologically periodontitis was signalled by the increase of polymorphonuclear leukocyte infiltration, production of proinflammatory cytokines and prostaglandins, amplification of lytic enzyme, and activation of osteoclasts along with a high level of ROS [¹⁹,³³]. This condition is exacerbated by the presence of high blood sugar levels; hyperglycaemia can lead to the regulation of the immune response through direct influence on the function of immune cells, and it can worsen inflammation. Some research show there is an increase of inflammatory mediators such as expression of TNF-α, IL-β, IL-6, and PGE₂ in chronic periodontitis with diabetes mellitus. Enhancing inflammatory mediators can increase the expression of RANKL, which will bind to its receptor RANK as the initial process of alveolar bone destruction. This condition will stimulate the osteoclast precursor to differentiate and induce TNF receptor-associated factor 6 (TRAF 6), which activates NF-kB and MAPKs, which are key to osteoclast formation; it will be in line with the reduction of the number of osteoblast cells. It leads to the destruction of inorganic minerals from the alveolar bone; then, bone resorption could occur. The uncontrollable destruction will cause high severity [³¹,³⁴].

In groups T₁ (given 96% papaya seed extract) and T₂ (given 96% nanoliposome papaya seed extract), there was a pattern of an increased number of osteoblasts and a decreased number of RANKL expression on the predetermined time series, namely days 3, 7 and 14. In each time series, the highest number of osteoblasts and the lowest number of RANKL expression was found in group T₂. Statistically, there is a significant difference between groups in RANKL expression on days 7 and 14 and the number of osteoblasts on day 14. It indicated that treatment with papaya seed extract has the potential to reduce periodontitis via an anti-inflammatory mechanism. Based on the previous findings, papaya seed extract contains active compounds such as flavonoids and phenols that promote alveolar bone recovery. Flavonoids in papaya seed have antibacterial, antifungal, and antioxidant properties that are able to restore alveolar bone density by preventing bone resorption. Flavonoids have a secondary flavonol compound, namely quercetin which has antioxidant and anti-inflammatory activity that can reduce the inflammatory production of IL-1, IL-6 and, TNF-α; quercetin is also capable of inhibiting intracellular signalling pathways such as MAPKs and NF-kB resulting in reduced inflammation and oxidative stress [¹⁹]. Chlorogenic acid is a phenolic compound in papaya seed that acts as an antioxidant and anti-inflammatory agent that could reduce RANKL production, inhibit osteoclast activity and bone resorption resulting in increasing the number of osteoblast cells [³⁵].

Based on a post hoc test on day 14, there is no significant difference between groups T₀ and T₁ in the number of osteoblasts; however, in RANKL expression, that significant difference exists. There is no significant difference between groups T₁ and T₂ in the number of RANKL expression; however, that significant difference exists in the number of osteoblasts. This condition can be caused by several possibilities. The use of herbal medicine has several disadvantages: low solubility, lack of permeability to penetrate the absorption barrier, low bioavailability, and unpredictable toxicity [¹⁶,¹⁷]. Efforts to optimise the active compounds of papaya seed extract can be carried out in the form of liposomes. In this research, the treatment with papaya seed extract administered with nanoliposome showed the best result.

Various types of nanoliposomes are currently used clinically as delivery systems for drugs. The nanoliposomal system is a potential drug delivery system because nanoliposomes incorporate hydrophilic and lipophilic therapeutic agents relatively easily and can be delivered directly to an accessible body site such as the periodontium. With relative non-immunogenicity and low toxicity, nanoliposomes also naturally target macrophages [³⁶]. The process of alveolar bone repair includes two stages of cellular activity, i.e. the resorption of old bone by osteoclasts through the process of acidification and proteolytic digestion and the formation of new bone by osteoblasts; osteoblast cells will invade the area, and formation occurs by secreting osteoid (collagen and protein matrix) which then mineralises. This phase lasts for 7 to 14 days, and after that, there is suppression of osteoclast formation and stimulation of osteoblastogenesis. During the repair process, differentiation occurs, including chemotaxis, cell attachment, mitosis, and osteoblast differentiation leading to deposition of new alveolar bone. The limitation of this study is that observations were made only up to day 14; more time is necessary to observe the process of osteoblastogenesis in more detail [³⁷]. In addition, problems often arise in the preparation of nanoparticles, the occurrence of rapid aggregation, and uneven particle sizes, so the stability of the dispersion system becomes difficult to control. It is important to examine particle size, particle morphological characters, and potential zeta values as a solution to these problems [³⁸].

Conclusion

There is an effect of the administration of nanoliposome papaya seed extract on a diabetic periodontitis rat model; it can increase the number of osteoblasts and decrease the number of RANKL expression in vivo. Comparing the different doses and observation times, as well as toxicity and material characterisation tests, is necessary to do for further research.

Data Availability

The data used to support the findings of this study can be made available upon request to the corresponding author.

• Financial Support
  This research is financially supported by Faculty of Dentistry, Univesrsitas Brawijaya (Grant Number: 042.01.2.400919/2017), Indonesia.

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Edited by

• Academic Editor:
  Myroslav Goncharuk-Khomyn

Publication Dates

• Publication in this collection
  20 Dec 2024
• Date of issue
  2025

History

• Received
  09 Jan 2023
• Reviewed
  29 Aug 2023
• Accepted
  08 May 2024