INTRODUCTION:

Tooth loss is a problem that is often encountered in the oral cavity. It can affect masticatory function, speech, aesthetics and even social relationships. Causes of tooth loss include dental caries, periodontal disease and trauma. Caries are the leading cause of tooth loss¹. Adults, children, and senior citizens can all experience tooth loss. According to data from the 2018 Basic Health Research (Riskesdas), 19% of Indonesians have tooth loss, with the most considerable percentage occurring at age 65(30.6%), followed by age 55–64 (29%)². Twenty teeth are needed to enable chewing. Malnutrition risk factors may rise in people who have lost 16 or more teeth³.

Treatment to replace missing teeth is by making artificial teeth. Artificial teeth can be removable (prostheses made of acrylic resin), bridge dentures (gold/porcelain/porcelain-fused-to-metal), and dental implants. An excellent dental implant must have good osseointegration, where osteoblasts can grow around the implant surface. Osseointegration is influenced by implant material, implant surface topography, bone quality/bone quantity, surgical technique, the healing process, and the impact of the prosthesis (loading, cleanliness)⁴.

The material often used for dental implants is titanium. Several studies have said that titanium can cause hypersensitivity reactions and grey colour in thin mucosa. Polymethylmethacrylate (PMMA) is a polymer material widely used in dentistry, including bone cement, denture base resins, fillers, and base materials (4). PMMA bone cement has good mechanical properties, often used as a bone repair or replacement material⁵. Apart from that, PMMA is also easy to manipulate. Therefore, PMMA can be a candidate implant material. However, PMMA has shortcomings in its osteoconductivity, so other materials need to be added to make it better⁶. Hydroxyapatite (HAp) is the main mineral found in bones and teeth. The arrangement of hydroxyapatite crystals has an identical appearance to hydroxyapatite in bone. This material is biocompatible, osteoconductive, and can blend with bone to increase the bone regeneration process⁷. PMMA combined with HAp can improve mechanical properties, biocompatibility, and osteoconductivity in both in vivo and in vitro studies⁸.

Osteoclast and osteoblast cells are involved in the regulation of the bone remodelling process following implantation. Osteoblasts express the receptor Activator of Nuclear Factor Kappa-β ligand (RANKL). To limit bone resorption and promote osteogenesis, osteoprotegerin (OPG) functions as a natural inhibitor of RANKL, binding to the Receptor Activator of Nuclear Factor Kappa-β (RANK). The biological effects of OPG are opposite to those mediated by RANKL and include inhibition of end stages of osteoclast differentiation, activation of matrix osteoclast suppression, and accelerating osteoclast apoptosis. Overall, the RANKL/OPG ratio determines the physiological balance of bone formation and turnover, with a higher ratio promoting increased bone resorption. Given the preceding context, studies on the ratio between OPG and RANKL following implantation using PMMA-HAp in Wistar rats' femurs (Rattus norvegicus) are required.

MATERIALS AND METHODS:

The white rats (Rattus norvegicus) used in this experimental in vivo study were male, 12–16 weeks old, and weighed 300±16grams. They were obtained from the Biochemistry Lab, Faculty of Medicine, Universitas Airlangga. The post-test-only control group is the study design that was employed. The Ethical Committee of Dental Research at Airlangga University 2022 gave this project ethical approval. Six groupings of animals were formed: Group control K7 (only drilling, n = 7) was sacrificed on day 7, K14 (only drilling, n = 7) was sacrificed on day 14, GMP7 (PMMA/HAp-GMP day 7) was sacrificed on day 7, GMP14 (PMMA/HAp-GMP day 14) was sacrificed on day 14, BBK-7 (PMMA/HAp BBK day 7) was sacrificed on day 7, and BBK-14 (PMMA/HAp BBK-14) was sacrificed on day 14.

Producing PMMA/HAp Implant Substances⁹

1. Polymethylmethacrylate (PMMA) is mixed with hydroxyapatite in a planetarium mixer with a ratio of 80: 20 for 3hours

2. For every 0.1gram PMMA/HAp mixture, 0.016ml of liquid monomer is added so that the PMMA: HAp ratio becomes 83.8:16.2

3. The ingredients are mixed using a cement spatula on a glass for 30-40 seconds until the dough reaches a dough state. Then, the dough is moulded using moulding made from polyvinyl chloride (PVC) with a diameter of 1mm and a height of 2mm

4. Set the time for 4-5minutes, then remove the implant material from the moulding

5. Sterilization

Sterilization of PMMA/Hap:

1. PBS (Phosphate Buffer Saline) is used thrice to wash each dish's implant sample.

2. After the implanted sample is dried, it is incubated in 70% ethanol for two hours.

3. Once the implant sample has dried, put it in a sealed sterile container.

4. The implant sample is exposed to UV-C radiation for two hours¹⁰.

Implantation PMAA/Hap¹¹

1. Animals that meet the inclusion criteria are placed in separate cages for one week for the acclimatization process to food. During treatment, the rats were anaesthetized using a combination of 10% ketamine and Xylazine in a ratio of 50:10, which was injected intramuscularly in the Semi Tendonosus muscle as an induction of anaesthesia in the gluteus.

2. The hair in the femur area where the implant will be installed is shaved and cleaned using 80% alcohol.

3. A 10mm long incision is made with a 15°C blade in the lateral area of the femur until it penetrates the bone surface. A drilling sequence is carried out according to the length and diameter of the implant using saline irrigation at a speed of no more than 1500rpm. Implant placement was carried out in the osteotomy area of the lateral surface of the femur until primary stability of the implant was obtained

4. Rats that were not given implant material in the control group only had drilling done to the same depth and size, and then the wound was closed using sewing thread.

5. Mice are marked with markers to differentiate groups

6. Implant osseointegration and markers are evaluated on days 7 and 14 after implant placement.

Immunohistochemical staining used in this study examined OPG and RANKL expression from femur rats (Rattus norvegicus). The primary antibodies OPG from Santacruz biotech US (anti-OPG/Osteoprotegerin (E-10): sc-390518) and RANKL from Santacruz biotech US (anti-RANKL Antibody (12A668): sc-52950) Histologic samples was performed immunohistochemical tests for OPG and RANKL. Statistical analyses were performed using SPSS software. All data were expressed as mean ± standard deviation (SD). Differences between experimental groups were analyzed with ANOVA. A p-value < 0.05 was considered statistically significant.

RESULTS:

OPG and RANKL expression were observed semi-quantitatively using immunohistochemical methods using indirect immunoperoxidase techniques. Observations were carried out on a light microscope (Olympus CX31) with 1000x magnification (for the calculation process). Image representation of observation results is presented as a photomicrograph (on the Panasonic Lumix GX-8).

Figure 1. Photomicrograft (1000x magnification) of OPG expression in Rattus norvegicus femur bone tissue observed in osteoblast cells (arrow). It can be seen that OPG expression is marked with a brown colour in the cytoplasm of osteoblast cells.

Figure 2. Photomicrograft (1000x magnification) of RANKL expression in Rattus norvegicus femur bone tissue, observed in osteoblast cells (arrow). It can be seen that OPG expression is marked with a brown colour in the cytoplasm of osteoblast cells.

The notation (*) indicates a significant difference (p<0.05)

Figure 3. Mean and various testing scores for the OPG expression groups on days 7 and 14.

Figure 3 displays the difference in OPG expression on days 7 and 14 between the GMP, BBK, and control groups. On days 7 and 14, the Figure 3 indicates that OPG expression was significantly higher in the BBK and GMP treatment groups than in the control group. The treatment groups did not differ significantly from one another.

The notation (*) indicates a significant difference (p<0.05)

Figure 4. Mean and various testing scores for the RANKL expression groups on days 7 and 14.

Figure 4 displays the difference in RANKL expression on days 7 and 14 between the GMP, BBK, and control groups. On days 7 and 14, the Figure 4 indicates that RANKL expression was significantly lower in the BBK and GMP treatment groups than in the control group. The treatment groups did not differ significantly from one another.

Description: the sign * indicates there is a significant difference (p <0.05). The sign ** indicates there is no significant difference (p>0.05)

Figure 5. Mean results of OPG/RANKL ratio analysis for the Control (K), PMMA/HAp-GMP (GMP) and PMMA/HAp-BBK (BBK) groups with Standard Deviation (SD) at lvvccong exposure times (7 and 14 days).

Results of the ANOVA comparison analysis of the OPG/RANKL CONTROL, BBK and GMP ratio groups on days 7 and 14. The OPG/RANKL ratio group on day 14 showed a significant difference (<0.05). Meanwhile, in the 7th-day group, the OPG/RANKL ratio showed no significant difference (>0.05).

DISCUSSION:

Polymethylmethacrylate (PMMA) and hydroxyapatite (HAp) are combined to produce materials with better mechanical and biological characteristics¹². PMMA and HAp material can impact the transmembrane bone morphogenic protein receptor (BMPR). The amount of calcium in HA can cause BMP receptors to respond. This transmembrane receptor transmits data from the extracellular matrix to the intracellular matrix through the SMAD pathway. The SMAD pathway enables and subsequently stimulates osterix (Osx) transcription. Receptor activators of nuclear factor kappa b ligand (RANKL) and osteoprotegerin (OPG) expression are crucial for osteoblastogenesis.

According to the study's observations of OPG expression, the BBK and GMP groups showed an increase compared to the control group on days 7 and 14. It might happen due to the HAp content's biomaterial characteristics, like roughness and nano- or microstructure. These HAp characteristics boost BMP receptor expression, affecting the rise in BMP sensitivity¹³.

OPG expression in the GMP group increased slightly compared to the BBK group on days seven and 14. The physical properties of HAp are determined by the extraction source and synthesis method. BBK HAp synthesis uses wet precipitation, while GMP HAp synthesis uses freeze-dried or demineralization and deproteinase methods. In this process, bone mineral is removed so that bone protein becomes exposed. Exposed bovine protein has been proven to have osteoinductive properties so that it can recruit recipient stem cells to differentiate into osteoblasts and stimulate recipient osteoblasts to produce new bone¹⁴. Thus, the GMP HAp material is slightly better than the BBK Hap¹⁵. These two methods can produce different HAp sizes, morphologies, and Ca/P ratios. All three are essential in determining the compatibility and osteoinduction of HAp as a biomaterial. HAp should have a Ca/P ratio similar to bone, namely 1.67¹⁶. Research by Manalu et al. (2015) indicated that the Ca/P ratio of bovine bone was 1.91¹⁷. However, research by Pridanti et al. (2020) found that the Ca/P ratio of BBK from the results of energy dispersive X-ray (EDX) was 1.64¹⁸. It could account for the GMP group's slightly higher rise in OPG and RANKL expression than the BBK group's^17,18. However, more research is required since the Ca/P ratio was not studied in this study.

On day 14, the study's control group had the highest RANKL expression values. According to Hassumi's (2017) research, RANKL expression peaked on day 14 and, after that, declined¹⁹. This result is consistent with that finding. Osteoblast and osteoclast cells are involved in bone remodelling. A process known as bone resorption precedes the rebuilding of bone. When equilibrium is reached, osteoblasts create new bone while osteoclasts absorb broken or unnecessary bone²⁰.

In this study, the OPG/RANKL ratio on day 7 increased in the GMP and BBK groups compared to the control group on day 7. The shape and size of HA activate the Wnt/β catenin signalling pathway, which is essential in cell determination, proliferation and differentiation, and bone mass maintenance by regulating the activity of bone-forming osteoblast cells and, indirectly, osteoclast cells. In several studies in transgenic mice, if there was an increase in Wnt signalling, there was an increase in OPG production and a decrease in osteoclasts. Activation of the Wnt/β catenin signalling pathway by binding to the FZD receptor and co-receptor LRP 5/6 induces phosphorylation of the degradation complex (axin, DVL) to liberate β catenin. β catenin accumulates in the cytoplasm and then translocates to the nucleus, which induces RUNX2 and Osx and regulates RANKL and OPG expression²¹.

In the GMP-7 group, the OPG/RANKL ratio was slightly lower than in the BBK-7 group. It may have occurred because the bone remodelling process had not occurred entirely. On the 14th day, both treatment groups experienced significant increases. The results of this study are supported by studies by Hassumi et al. and Lenneras et al., which state that the OPG/RANKL ratio influences the level of bone remodelling. On the 7th and 14^th days, the OPG/RANKL ratio will increase and then decrease on the following day^19,22. This study demonstrates the suitability of BBK and GMP materials for bone remodelling. However, further investigation is required compared to the gold standard dental implant, which is titanium.

CONCLUSION:

There was an increase in OPG and a decrease in RANKL after implantation of Polymethylmethacrylate (PMMA) – Hydroxyapatite (HAp) in the femur of Wistar rats (Rattus norvegicus).

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENT:

This study is funded by Directorate General of Higher Education, Research, and Technology, Ministry of Education and Culture, Republic of Indonesia 2022.

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Received on 28.02.2024 Revised on 22.08.2024

Accepted on 06.12.2024 Published on 27.03.2025

Available online from March 27, 2025

Research J. Pharmacy and Technology. 2025;18(3):1258-1262.

DOI: 10.52711/0974-360X.2025.00182

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