Osteoclastogenesis markers in craniofacial bone defects after demineralized dentin material membrane implantation as guided bone regeneration

Abstract

Guided bone regeneration (GBR) is an alternative treatment for craniofacial bone defects reconstruction through membrane barrier adaptation, such as demineralized dentin material membrane (DDMM). DDMM is used as a substitute for GBR material, which aligns with Green Economy principles, it has a good biological osteoinductive and osteoconductive effects, and its structure resembles bones. The balance of bone remodeling when experiencing craniofacial defects will be altered and allow changes to resorption activity, so the mechanisms of osteoclastogenesis and bone resorption are vital. Objective: this article aims to analyze the expression of TNF-α, RANKL, and osteoclast cells count after application of DDMM as GBR in mandibular bone defects. Methodology: this is an experimental study with a post-test only control group design, which began with the randomization of 120 rats into five groups: K(−), without membrane implantation; K(+), PPCM; P1, DDMM; P2, DDMM + bone graft; P3, PPCM + bone graft. The expression of TNF-α, RANKL, and osteoclast cells count were observed, followed by analysis using a one-way ANOVA and post hoc Tukey HSD comparison test. Results: there were significant differences in the expression of TNF-α, RANKL, and osteoclast cells count in all study groups (p=0.000). TNF-α showed a decreasing difference with the highest expression in the K(−) group on day 3 of 12.00±2.16. RANKL expression increased on day 14 and decreased on day 21 in all groups. The osteoclast cells count generally showed a critical period with the highest increase in the K(−) group on day 14 of 73.00±0.00. Conclusion: DDMM has the potential to be a superior membrane substitute compared to PPCM as GBR in alternative treatment for craniofacial bone defects reconstruction.

Keywords:
Demineralized dentin material membrane; Guided bone regeneration; TNF-α; RANKL; Osteoclastogenesis

Introduction

The craniofacial skeleton, comprising skull, face, and jaw bones, is a complex and vital structure for facial aesthetics, protection of vital organs, and masticatory function.¹ Pathological conditions such as congenital malformations, infections, trauma, tumor resection, or accidents can lead to craniofacial bone defects, causing significant challenges to patients’ quality of life and overall health.² In critical size defects where the body's natural regenerative capacity is insufficient, surgical intervention becomes necessary to restore its form and function.¹,³ Among the several surgical techniques available, guided bone regeneration (GBR) has been recognized as a promising approach for craniofacial bone defect reconstruction.⁴

GBR involves the application of barrier membranes to create an isolated space for bone regeneration, preventing soft tissue ingrowth and facilitating the migration of osteogenic cells to the defect site.⁴-⁶ Whereas GBR has demonstrated efficacy in promoting bone regeneration, challenges persist, particularly related to the choice of biomaterials. Porcine Pericardium Collagen Membrane (PPCM), a commonly used membrane in GBR procedures, presents drawbacks such as inflammation induction, immunogenicity, high costs, and non-permissible sourcing.⁷ Therefore, there is a demand for a permissible, cost-effective, and functionally analogous GBR materials.

Dentin, a mineralized tissue analogous to bones, emerges as a promising alternative. Demineralized dentin material membrane (DDMM) possesses excellent osteoinductive and osteoconductive properties, making it an attractive option for bone regeneration.⁸ DDMM contains growth factors and extracellular matrix proteins that promote cell proliferation and differentiation, enhancing bone formation and remodeling at the defect site.⁸,⁹

Understanding the cellular and molecular mechanisms underlying bone regeneration is essential for optimizing the efficacy of GBR procedures. Osteoclasts, specialized cells responsible for bone resorption, play a critical role in bone remodeling and repair.¹⁰,¹¹ The regulation of osteoclast activity is governed by several factors, including Receptor Activator of Nuclear Factor Kappa B Ligand (RANKL) and Tumor Necrosis Factor-alpha (TNF-α). Their levels fluctuate throughout bone healing, impairing the equilibrium between bone resorption and formation.¹²-¹⁴

Studies show that DDMM modulates the expression of osteogenic and osteoclastic markers, promoting a favorable microenvironment for bone regeneration. Empirical evidence suggests that DDMM promotes osteoblast activity while mitigating undue osteoclast activity, facilitating the formation of new bone tissue and accelerating the healing process.¹⁵ Further research is warranted to reconcile the findings, as investigations about osteoclastogenesis in DDMM applications have yet to be conducted. Alterations in bone remodeling dynamics due to craniofacial defects need an examination of changes in resorption activity, highlighting the importance of understanding osteoclastogenesis and bone resorption mechanisms. This article aims to analyze the decrease in TNF-α expression and increase in RANKL expression and osteoclast cells count after applying DDMM as GBR in mandibular bone defects of Rattus norvegicus.

Methodology

Preparation and grouping of experimental animals

Surgical procedure on experimental animals was done in the laboratory at the Department of Pharmacology, Faculty of Medicine, Universitas Airlangga, Surabaya, approved by Universitas Airlangga Faculty of Dental Medicine Health Research Ethical Clearance Commission on letter number 0172/HRECC.FODM/III/2024. The experimental animals’ treatment has been thoroughly ensured to align with the ARRIVE protocol.¹⁶ One hundred twenty Wistar rats aged two to three months and weighing 250-300 grams were randomly divided into five groups. In the K(−) group, rats with mandibular bone defects were only given 10% povidone-iodine without implantation of GBR membrane, then suturing was performed, while in the K(+) group, PPCM (Jason®membrane, Botiss Dental, Germany) implantation was performed. In the treatment group, mandibular bone defects underwent membrane implantation, including DDMM (Tissue Bank, Dr. Soetomo General Hospital, Surabaya) in group P₁; DDMM and graft (Novocore Plus Graft, B&B Dental Implant, Italy) in group P₂; and PPCM and graft in the P₃ group. The graft used comprises 98% aragonitic calcium carbonate (CaCO3) or coralline hydroxyapatite in the form of granules or coral particles measuring 200-500 μm with a density of 1 g/cm³. Sixty mg of bone graft were implanted into the bone defect in groups P₂ and P₃. Subsequently, a 1×1 cm GBR membrane was applied over the bone graft or on top of the defect (Figure 1A). A marking line was made at the center of the defect using 3.0 silk thread to aid in cutting during paraffin block preparation.

Figure 1
(A) Preparation of the 1×1 cm GBR membrane; (B) retraction of incised tissue exposing the defect; (C) anesthesia with ketamine HCl in the femoral region; (D) suturing procedure using 3.0 silk thread.

Critical size defect induction and sample collection

A critical size defect with a 5 mm diameter was created in the posterior left mandibular area using a wheel bur and irrigated with 0.9% NaCl solution via a syringe. The soft tissue or skin was horizontally incised to expose the mandibular bone. The incised tissue was then retracted with arterial forceps to enhance the field of view (Figure 1B). The procedure began with an intramuscular injection of ketamine HCl in the femoral region at 20 mg/kg body weight (Figure 1C). The incision was then sutured with 3.0 silk thread after the membranes implantation (Figure 1D). On days 3, 7, 14, and 21 post-membrane implantation, six rats from each group were sacrificed for tissue specimen collection.

The rats’ euthanasia was performed using anesthetic gas administered by inhalation, where the rats were placed in a tube containing cotton soaked with ether. Tissue samples were excised from the posterior left ramus of the rat mandible using a scalpel, ensuring precision to avoid damage to surrounding tissues, and subsequently fixed in a 10% formalin solution for preservation and stabilization of cellular structures prior to further analysis (Figure 2A). Decalcification was then performed using a 10% EDTA solution at a neutral pH for approximately six weeks, with the solution being refreshed weekly until the mandibular bone tissue softened (Figure 2B). Following decalcification, tissue processing steps were conducted, including dehydration, clearing, impregnation, and embedding. Finally, the tissue was embedded in a paraffin block and sectioned vertically using a rotary microtome to a thickness of 3 μm. The sections were placed in water at 40-50°C to remove wrinkles and subsequently mounted onto glass slides.

Figure 2
(A) Harvesting the mandibular ramus of experimental animals; (B) decalcified specimen.

Osteoclastogenesis markers examination

The parameters were TNF-α expression and osteoclast cells count on days 3, 7, 14, and 21, with the expression of RANKL on days 7, 14, and 21 post-membrane implantations. The data for all parameters were derived from observations of specimens subjected to immunohistochemical (IHC) staining for TNF-α and RANKL expression using monoclonal antibody: anti-rat TNF-α (52B83: sc-52746, Santa Cruz Biotechnology, Inc., United States); and anti-rat RANKL (12A668: sc-52950, Santa Cruz Biotechnology, Inc., United States), while samples for osteoclast cells count were stained using Mallory-Azan (Mallory-Azan stain kit, Sigma-Aldrich, United States).

The IHC procedure involves deparaffinization with xylene, followed by rehydration through graded alcohols and washing with deionized water. Endogenous peroxidase activity is quenched with hydrogen peroxide and methanol. The samples are treated with primary and secondary antibodies, and chromogen is applied. Counter-staining is done with hematoxylin-eosin, followed by dehydration with alcohol solutions and clearing with xylene.¹⁷ Finally, the samples are mounted with a cover glass. Mallory-Azan staining follows the same deparaffinization and rehydration process as the IHC procedure, but continues with azocarmine staining, differentiation, and subsequent staining with Mallory solution, which contains orange G, aniline blue, and acetic acid. The specimen is then cleared with xylene and mounted with a cover glass through additional steps of incubation, washing, and alcohol dips.

These assessments were performed on six slides per group during each day of observation using a light microscope (SMZ-1, Nikon, Japan) at 400x magnification. Each sample slide received a score based on the cumulative count of cells across nine observed fields of view.

Statistical analysis

The research data analysis was conducted using IBM SPSS Statistics v25 software, including a normality test with the Kolmogorov-Smirnov and a homogeneity test using Levene's test to observe the data distribution, followed by a comparison test. All variables show normality test results with a significance value of p>0.05, indicating that the data are normally distributed. All variables also show homogeneous data variations with p-value>0.05, so the assumptions of normality and homogeneity of the data are met. Normally distributed and homogeneous data were subjected to a one-way parametric analysis of variance (ANOVA) test to analyze the differences between groups P₁, P₂, P₃, K(+), and K(−) on days 3, 7, 14, and 21, with a significance level of p<0.05. Post-hoc Tukey HSD was also performed to compare significant differences between pair of groups on each observation day with the same significance value as one-way ANOVA test.

Results

The immunohistochemical staining results showed differences in TNF-α expression for each group on day 3 after treatment (Figure 3). The TNF-α expression was the highest on day 3 in all groups, especially in the negative control group without GBR membrane implantation group at 12.00 ± 2.16, as shown in Table 1. Furthermore, all groups showed a gradual decrease in TNF-α expression from days 3, 7, 14, and 21, and there was a significant decrease in the negative control group compared to the other groups.

Figure 3
TNF-α expression on day 3 (A) negative control; (B) positive control; (C) DDMM group; (D) DDMM + bone graft group; (E) PPCM + bone graft group [IHC; 400x].

Table 1
Mean expression of TNF-α in all study groups and one-way ANOVA test results.

Day	Mean ± SD TNF-α (cells/HPF)					p-value*
	K (−)	K (+)	P1	P2	P3
3	12.00±2.16	9.50±1.29	8.00±1.63	6.50±0.57	7.00±1.63	0.000
7	8.00±0.81	8.00±1.63	7.00±0.81	4.00±0.81	5.50±1.29
14	6.00±0.81	7.00±1.63	5.50±0.57	2.50±1.29	4.50±1.29
21	2.00±0.81	4.50±0.57	3.50±1.29	1.00±0.00	2.50±0.57

• ^*
  Significance: p<0.05

The result of immunohistochemical staining also revealed that there were differences in the increase of RANKL expression on day 14 in all groups, as in Figure 4. It was followed by a decrease in RANKL expression on day 21. The highest average RANKL expression value was in the negative control group on day 14, 529.0 ± 32.51, while the lowest average RANKL expression was in the positive control group implanted using PPCM on day 7, as many as 36.0 ± 0.00 (Table 2).

Figure 4
RANKL expression on day 14 (A) positive control; (B) DDMM group; (C) DDMM + bone graft group; (D) PPCM + bone graft group [IHC; 400x].

Table 2
Mean expression of RANKL in all study groups and one-way ANOVA test results.

Day	Mean ± SD RANKL (cells/HPF)					p-value*
	K (−)	K (+)	P1	P2	P3
7	273.0±66.05	36.0±0.00	262.0±77.50	125.0±102.0	127.0±95.00	0.000
14	529.0±32.51	177.0±62.50	525.0±1.73	446.0±212.5	150.0±40.00
21	222.0±26.05	161.0±2.64	214.0±155.5	74.0±23.00	114.0±38.00

• ^*
  Significance: p<0.05

Figure 5 shows a difference in the increase of osteoclast cells count with Mallory-Azan staining for each group on days 3, 7, and 14, followed by a decrease in the number of osteoclast cells on day 21. There was a significant increase in the negative control group on day 14, making this group the highest average number of osteoclast cells, which is 73.00 ± 0.00, while the lowest average number of osteoclast cells was in the negative control group and group P₁ implanted using DDMM on day 3 where no osteoclast cells was detected (Table 3).

Figure 5
HPA image of osteoclast cells on day 14 (A) negative control; (B) positive control; (C) DDMM group; (D) DDMM + bone graft group; (E) PPCM + bone graft group [Mallory Azan; 400x].

Table 3
Mean quantity of osteoclast samples in all study groups and one-way ANOVA test results.

Day	Mean ± SD Osteoclast Cell (cells/HPF)					p-value*
	K (−)	K (+)	P1	P2	P3
3	0.00±0.00	2.00±2.00	0.00±0.00	3.00±2.64	1.33±1.52	0.000
7	6.00±1.00	41.67±60.96	1.00±1.73	6.33±9.29	2.67±3.05
14	73.00±0.00	58.67±31.21	38.00±14.52	30.00±12.00	41.67±18.77
21	10.00±0.00	24.00±18.52	46.00±16.09	26.33±24.33	8.00±5.29

• ^*
  Significance: p<0.05

For the comparison test group, the one-way ANOVA parametric test revealed a significant value of p<0.05 for the expression of TNF-α, RANKL, and osteoclast cell count, indicating significant differences among the research groups. Post hoc analysis was performed, showing significant differences in TNF-α expression across several time points. On day 3, the K(−) group differed significantly from the P₁, P₂, and P₃ groups. On day 7, the P₂ group exhibited a significant difference compared to the K(−) and K(+) groups. This pattern continued on day 14, where the P₂ group again differed significantly from the K(−) and K(+) groups. On day 21, a significant difference was observed between the K(+) group and the P₂ group.

The post hoc test for RANKL expression showed significant differences (p<0.05) only on day 14, where RANKL expression in the K(+) group displayed significant differences compared to the K(−), P₁, and P₂ groups, and in the P₃ group differed from the K(−), P₁, and P₂ groups. However, no significant differences (p>0.05) were observed in the number of osteoclast cells across all group comparisons on days 3, 7, 14, and 21.

Discussion

Guided bone regeneration (GBR) is a clinically proven method for efficient bone healing, often using bioresorbable barrier membranes like collagen membranes derived from porcine pericardium.¹⁸,¹⁹ Whereas these membranes have shown efficacy in promoting tissue integration, challenges such as delayed blood vessel invasion associated with collagen fibril cross-linking and membrane collapse persist.¹⁹ Previous research indicates that combining GBR with particulate bone graft can prevent membrane collapse, maintain space, and immobilize bone grafts, effectively supporting bone regeneration.²⁰ To address these issues, this study compares the effectiveness of PPCM with a newly developed material, demineralized dentin material membrane (DDMM), both with and without bone graft.⁶,¹⁵

Tumor Necrosis Factor-alpha (TNF-α), a multifunctional cytokine, plays a pivotal role in inflammation and bone resorption of diseases related to bone destruction. It stimulates the proliferation of osteoclast precursors and collaborates with several cytokines and growth factors, including RANKL, IL-6, and TGF-β, to facilitate osteoclast formation and bone resorption in vivo.²¹ Observations from the current study reveal that TNF-αα expression peaks on day 3 post-implantation with the highest value in the K(−) group of 12.00 ± 2.16, followed by a gradual decrease until day 21. This temporal pattern aligns with previous findings by Mulyawan, et al.⁷ (2021), indicating a similar peak of TNF-α expression around day 5 post-implantation, coinciding with the heightened activity of macrophages during the acute inflammatory phase. During this phase, macrophage en masse enters the damaged area by releasing several mediators and enzymes. The subsequent decline in TNF-α expression is attributed to the resolution of inflammation and the transition to the early repair phase, in which macrophages will continue to produce cytokines that stimulate fibroblasts to proliferate and secrete bone growth factors such as BMP-2, ALP, and OSX so that bone formation increases and proinflammatory cytokines gradually decrease.³,⁷

A previous study by Lou, et al.²² (2018) stated that TNF-α cannot induce osteoclastogenesis without RANKL. However, it can enhance osteoclastogenesis along with RANKL, as confirmed by the increase in the number of tartrate-resistant acid phosphate (TRAP)-positive multinucleated osteoclasts and increased TRAP activity. TRAP-6 and 2 are enzymes in the RANKL and TNF-α receptors; they bind to each other to activate several transcription factors that work in the NF-κB signaling pathway.²¹ The NF-κB signaling pathway, which is activated during inflammation, assumes a critical role in osteoclastogenesis. Toll-like receptors (TLRs) are triggered in response to tissue injury or infection, leading to NF-κB activation and the production of inflammatory mediators by M1 macrophages.²³ The balance between M1 and M2 macrophages significantly influences the resolution of inflammation and subsequent bone regeneration. M1 macrophages primarily produce proinflammatory cytokines such as TNF-α. Conversely, M2 macrophages are associated with anti-inflammatory responses and tissue repair processes.²³ Out of all the TNF-α expression reductions seen here, the P₂ group's expression on day 21 was the lowest, at 1.00 ± 0.00. This indirectly points to the superior ability of DDMM implanted with bone graft to dampen inflammation compared to other treatment groups because a variety of endogenous growth hormones, including VEGF, FGF-2, IGF-1, and most frequently, TGF-β, are found in the dentin matrix. These factors play a variety of roles in the wound-healing process, including fibroblast proliferation, angiogenesis, inflammation, and collagen production.²⁴ The results above are corroborated by recent studies employing a bilayer membrane incorporating a demineralized dentin matrix, which has been proven to suppress inflammatory responses or create an immunosuppressive environment by polarizing M1 to M2 macrophages.²⁵

This study also evaluates the expression of RANKL, an essential osteoclastogenesis regulator. RANKL expression displays an increase on day 14 post-implantation in all study groups, followed by a subsequent decrease on day 21. It happens because, on day 14, RANKL has progressed from the activation phase to the resorption phase, causing the endochondral tissue to start actively resorbing. As RANKL increases, more osteoclast cells grow.¹³ Notably, the combination of PPCM or DDMM with bone grafts in this study showed a lower RANKL expression compared to membranes alone because the collagen membrane attached to the bone graft diminishes the osteoclastic resorption activity of the bone graft due to soft tissue growth, so RANKL, a crucial protein for osteoclast activation, also experiences a decrease in its expression.²⁶ These results align with a similar study by Kresnoadi, Sari and Laksono²⁷ (2023) that observed RANKL expression in tooth extraction sockets, showing the group given polyethylene glycol (PEG) as control had higher value compared to the group given a combination of PEG and bone graft. The RANKL/OPG ratio influences osteoclastogenesis. Previous studies have observed the simultaneous expression of RANKL and OPG, finding a significant negative correlation between those two.²⁸ The explanations above indicate that after trabecular bone formation, marked by decreased RANKL on day 21, the group implanted with DDMM and bone graft showed superior results compared to all treatment groups because it showed the lowest RANKL expression on that observation day, indirectly proving that the OPG ratio increased, thus inhibiting bone resorption activity.

The results showed the lowest number of osteoclasts in all groups on day 3, especially in the K(−) group and the group implanted with DDMM, where osteoclasts were undetected (0.00 ± 0.00). Perhaps because, on day 3, post-bone defect, the activation phase has not yet begun.²⁹ A similar study by Yang, et al.²⁶ (2014) supports this finding, where no resorption activity by osteoclasts towards the graft was found in the control group and treatment groups using collagen membranes even up to two weeks post-implantation. The critical period of osteoclastogenesis occurs on day 14, where the number of osteoclasts increases and is followed by a decrease on day 21. The decrease indicates the beginning of the formation phase, performed by osteoblasts depositing the resorbed area with mineralized tissue.³⁰ The highest number of osteoclasts on day 14 was observed in the K(−) group at 73.00 ± 0.00. This is because in groups without membrane implantation, osteoclast formation occurs more extensively.

The findings indicate that the osteoclastogenesis markers observed, TNF-α, RANKL, and osteoclasts, are interrelated and act on several common signaling pathways, especially the Nf-κB pathway. The use of membranes, whether PPCM or DDMM, aims to reduce inflammation and inhibit osteoclast formation. The findings also suggest DDMM could be a more effective alternative for GBR in clinical settings, potentially leading to improved outcomes in craniofacial bone defect reconstruction. Adding bone grafts jointly with DDMM further supports the regenerative process by maintaining space and providing a scaffold for osteoblasts, which are crucial for healing. Stem cells and growth factors, along with scaffold, are key elements for tissue regeneration. DDMM supplies important growth factors that support regenerative processes, and its superior efficacy in decreasing osteoclast activity and enhancing bone formation may translate into better healing and more successful long-term outcomes for patients.

Conclusion

This research has concluded that there is a significant difference in the expression of TNF-α, RANKL, and osteoclast cells count after the application of DDMM and PPCM as GBR in mandibular bone defects in Rattus norvegicus rats. Hence, DDMM has the potential to be a superior membrane substitute to PPCM as GBR in the alternative treatment for craniofacial bone defects reconstruction.

Acknowledgements

The study was funded by Universitas Airlangga International Research Network Grant Scheme Number 1655/UN3.LPPM/PT.01.03/2023. We thank Universitas Airlangga for the financial aid.

Data availability statement

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Edited by

• Editor:
  Ana Carolina Magalhães

• Associate Editor:
  Mariana Schutzer Ragghianti Zangrando

Publication Dates

• Publication in this collection
  10 Jan 2025
• Date of issue
  2025

History

• Received
  13 June 2024
• Reviewed
  15 Oct 2024
• Accepted
  04 Nov 2024