INTRODUCTION:

The main goal of modern periodontal care is to preserve the teeth and their supporting structures. The regeneration technique of periodontal tissue aims to restore the supporting tissues of the teeth, such as bone, cementum, and periodontal ligament, so that they resemble periodontal tissue before any damage due to periodontal bacteria.^1,2

Bone grafting is a commonly used procedure to treat bone defects caused by various factors, including tumors, trauma, congenital bone defects, and degeneration due to pathological processes.

It is also utilized to reconstruct bone defects induced by periodontitis. Adding bone graft to the periodontal flap procedure is expected to accelerate the regeneration of lost periodontal tissue compared to bone grafting.^3–5

Human dentin from extracted teeth is currently the focus of most bone graft research as a bone graft material because the tooth's structure is similar to that of bone, making it a bio-inorganic material. Dentin is composed of the same inorganic and organic components as human bone, and it is believed that tooth graft transplants could promote the development of new bone.^6–8

Dentin and bone have distinct minor ion contents, such as Mg, N, CO3, and HPO4, and these minor ion variations impact dentin and bone crystal size and solubility. When Mg and CO3 ions react in biogenic apatite, they induce apatite crystals to grow smaller and more soluble.⁹

The xenografts that are often used in clinics are made from bovine bones. Bovine undergoes a deproteinization process so that what remains is the inorganic content. Bovine has perfect osteoconduction capabilities but has the disadvantage of being expensive. ¹⁰

Osteocalcin expression, a non-collagen protein produced by osteoblasts during the phase of bone mineralization, is thought to be an indicator of bone quality.¹¹ Bovine hydroxyapatite is known to stimulate osteocalcin expression in the same way and at the same time as autogenous bone transplant, the gold standard graft material.¹²

There is no research on the bone quality generated by the hydroxyapatite tooth graft content. Researchers are interested in comparing the potential of hydroxyapatite tooth transplant and hydroxyapatite xenograft to promote osteocalcin expression. This study compared osteocalcin expression in bone healing following socket grafting with a hydroxyapatite tooth graft and xenograft hydroxyapatite.

MATERIALS AND METHODS:

Study design:

This study was an experimental study conducted with an ethical clearance of 172/KKEPK.FKG/X/2014 from the Faculty of Dental Medicine Ethics Committee, Universitas Airlangga, Indonesia. Male guinea pigs (Cavia cobaya), aged 90-120 days and weighing 250-300 grams, were used. The sample size was determined using the Federer (1963) formula, resulting in 33 models, with each group comprising 11 guinea pigs.

They were randomly split into three groups and sedated with ketamine + xylazine 5-10 mg + 0.5 mg/head injections. The mandible's left incisor was removed, and sterile saline was used to irrigate the socket. For Group 1, the socket received a hydroxyapatite xenograft graft material made from cancellous bone taken from bovine (cow bone), a hydroxyapatite tooth graft transplant for Group 2 from teeth that had been extracted, and blood was used as a control for Group 3. Both xenograft graft material and tooth graft hydroxyapatite were obtained from Dr. Soetomo Tissue Bank, with particle sizes of 150 – 355 µm and 355 – 750 µm.

After 14 days, the guinea pig was sacrificed using 10% ether inhalation anesthesia, and a 70% buffered formalin solution was used to fix the preparation made from the bone tissue in the socket. After that, the tissue was calcified with EDTA solution and prepared with a thickness of 4 micrometers before staining with HE.

Immunohistochemistry method (IHC):

The tissue samples underwent immunohistochemical staining utilizing DAB (Deamino benzidine) and subsequently with an anti-osteocalcin monoclonal antibody (MoAb). The osteocalcin expression was calculated by assessing the color intensity within each group with an OLYMPUS microscope at 400x magnification.

Statistical analysis:

The non-parametric Kruskal-Wallis test was used to analyze study data, followed by the Mann-Whitney test with p<0.05 for statistical significance.

RESULTS:

Overall, positive immunostaining for osteocalcin was observed in all groups. Still, the tooth graft group exhibited a higher expression level than the xenograft and control groups, as seen in Figure 1. This was indicated by a more intense brownish color in the immunostaining. Figure 2 summarizes the overall osteocalcin expression across the groups.

Figure 1. The average number of osteocalcin expressions among groups at 14 days postoperatively of Xenograft group (A), Tooth graft group (B), and Control group (C)

Figure 2. The average number of osteocalcin expressions among groups at 14 days postoperatively.

The Kruskal-Wallis test results, presented in Table 1, showed a significant difference in the mean values of the two treatment groups, with a significance value of p =0.001.

Table 1. Analysis of the Kruskal-Wallis test

Groups

Mean

Asymp. Sig.

Osteocalcin Xenograft

Tooth graft

Control

18.68

26.09

6.23

0.001

Tables 2 and 3 show the amounts of osteocalcin in the xenograft and tooth graft groups, respectively, compared to the control group. The xenograft group had a mean value of 11.82, significantly higher than the control group's mean value of 6.09 (p < 0.05). The tooth graft group had the highest mean value of 15.36, significantly higher than the control group's mean value of p =0.001.

Table 2. Mean, the minimum, and maximum number of osteocalcin expression in the xenograft group compared to the control group

Variable	Xenograft n = 11		Control n = 11		p Value
Variable	Mean ± SD	Min - Max	Mean ± SD	Min - Max	p Value
Osteocalcin	11.82 ± 2.040	8 – 15	6.09 ± 1.578	4 – 9	0.001

Table 3. Mean, the minimum, and maximum number of osteocalcin expression in the tooth graft group compared to the control group

Variable	Tooth graft n = 11		Control n = 11		p-value
Variable	Mean ± SD	Min - Max	Mean ± SD	Min - Max	p-value
Osteocalcin	15.36 ± 2.838	12 – 19	6.09 ± 1.578	4 – 9	0.001

Table 4 compares the amount of osteocalcin between the xenograft and tooth graft groups. The mean value of the xenograft group was 11.82, while the mean value of the tooth graft group was 15.36, indicating that the tooth graft group had a higher amount of osteocalcin. This difference was also statistically significant, p = 0.009.

Table 4. Mean, the minimum, and maximum number of osteocalcin expression in the xenograft group compared to the tooth graft group

Variable	Xenograft n = 11		Tooth graft n = 11		p-value
Variable	Mean ± SD	Min - Max	Mean ± SD	Min - Max	p-value
Osteocalcin	11.82 ± 2.040	8 – 15	15.36 ± 2.838	12 – 19	0.009

DISCUSSION:

Osteocalcin is one of the non-collagenous proteins expressed by osteoblasts in the last phase of the bone remodeling process, namely the bone mineralization phase, so osteocalcin can be considered a marker of mature osteoblasts.^13,14 Osteocalcin consists of 3 γ-carboxylated glutamic acid residues or bone Gla protein (BGP). This residue has an affinity for hydroxyapatite.^11,15 Research by Hoang (2003) found that osteocalcin requires free extracellular calcium (Ca²⁺) ions to bind hydroxyapatite. The calcium that interacts with osteocalcin will direct the surface of the Gla osteocalcin to the surface of hydroxyapatite so that osteocalcin can attach to hydroxyapatite.¹⁶

The primary hemostatic process begins with forming a blood clot that will contact the collagen connective tissue to create a platelet aggregation (blood clot). This blood clot will fill the socket cavity within a few minutes. Platelet activation will release several cytokines, such as IL-1, TNF-α, IL-6, and IL-8, stimulating inflammatory cells out of blood vessels to start the healing process. Inflammatory cells, predominantly macrophages and neutrophils, will produce pro-inflammatory growth factors and cytokines, such as VEGF, FGF, TGF-β1, and BMP, essential regulators of tissue repair. The fibrin clot will be replaced with fibrous tissue and blood vessels within four weeks. Various growth factors and cytokines released by platelets and inflammatory cells induce the differentiation of mesenchymal stem cells and bone marrow-derived cells into osteogenic cells locally in the socket. Growth factors and cytokines in the socket healing process can be derived from growth factors and cytokines released by pro-inflammatory cells, as well as cytokines and growth factors from mineralized bone deposits.^17–20

This study demonstrated that the socket healing process progressed to the bone mineralization phase at the end of the second week, as evidenced by the very high osteocalcin expression in the xenograft and tooth graft treatment groups compared to the control group.

Research conducted by Araújo et al. (2010) on extraction sockets treated with hydroxyapatite graft material showed that in the second week, the osteoclasts appeared to have been replaced by osteoblasts, and the remineralization process of trabecular bone (mature bone) began.²¹ It follows the research conducted by Chaves et al. (2012).¹²

The osteocalcin expression in the xenograft and tooth graft groups was higher than in the control group, with a significant difference in amount (p < 0.05). The hydroxyapatite graft material will stimulate the remodeling process more quickly than the normal healing process without the graft because hydroxyapatite can osteoinduction and osteoconduction. The osteoconductive knowledge of hydroxyapatite derived from the hydroxyapatite porous structure will facilitate angiogenesis so that osteogenic cells can attach and migrate to the graft, which induces new bone growth.^1,22,23

The actual osteoinduction ability of hydroxyapatite is still under debate until now. Research conducted by Prahasantiet et al. (2019) showed that hydroxyapatite particles cultured in human exfoliated deciduous teeth (SHED) could regulate the four osteoblast markers, namely type 1 collagen, osteocalcin, alkaline phosphatase (ALP), and osteopontin.¹ Sun et al. (2008) also proved the osteoinduction ability of hydroxyapatite. They found the expression of osteoblast marker genes, namely Runx2, osteopontin, collagen type 1, and osteocalcin, in human mesenchymal stem cells (hMSC) cultured on hydroxyapatite particles.²⁴ Meanwhile, Sotto-Maior et al. (2011), who researched the femurs of Wistar rats, found the presence of collagen fiber deposits around the hydroxyapatite particles.²²

Osteoinduction of hydroxyapatite originates from the microporous surface. The presence of microporous provides interconnections between macroporous grafts so that interstitial fluid circulation occurs along the matrix. After the graft is placed on the bone defect, the hydroxyapatite granules will dissolve into a layer of biological apatite (calcium phosphate) on the surface of the hydroxyapatite. Calcium phosphate has a high affinity for endogenous proteins and growth factors, including BMP, so it can induce the differentiation of pluripotent stem cells into cells that play a role in osteogenesis.^1,25 Extracellular calcium (Ca²⁺) will increase the production of autocrine growth factors such as IGF-I, IGF-II, BMP-1, and BMP-2, which are effective stimulators of osteoblast differentiation. Calcium ions are essential in bone mineralization because osteocalcin requires free calcium ions to bind hydroxyapatite. Phosphate ion ((PO₄)³) will enter the cell and then induce the process of bone mineralization and the expression of osteocalcin as a marker of mature osteoblasts.⁴

The osteocalcin expression in the tooth graft treatment group was higher than in the xenograft group, with a significant difference in amount (p<0.05). The tooth's structure is similar to the bone, so the tooth can also be considered a bio-inorganic material. Dentin contains the same inorganic and organic components as those in human bone. A comparison of inorganic and organic ingredients has a ratio of 96%: 4% enamel, 70-75%: 30% dentin, and 45-50% cementum: 50-55%. In the alveolar bone, the inorganic content is about 65%, and the organic is 25%.^4,26,27

Dentin and bone have different minor ion, such as Mg, N, CO₃, and HPO₄. The concentration of Mg ions, in dentin is 1.23, and in bone, is 0.72. For CO₃ ions, the attention in dentin is 5.6, and in bone, it is 7.4. The difference in these ions, especially Mg and CO₃, causes differences in crystal size and solubility (solubility) between dentin apatite and bone apatite, where CO₃ ions have a more significant influence than Mg ions. The higher the Mg and CO₃ ions, the smaller the apatite crystal size formed and the higher the particle solubility. The crystal size of bone apatite ±2.5x0.3 nm is almost the same as that of dentin apatite, which is ±2x0.4 nm, but the solubility of bone apatite is higher than that of dentin apatite.⁹

In biogenic apatite, CO₃ ions can replace PO₄ or OH^- ions so that they can change the hydroxyapatite formula to Ca10-2X/3(PO₄)6-X(CO₃) X (OH)2-X/3, which causes calcium deficiency. Pan et al. (2009) found that replacing PO₄ ions with CO₃ ions causes a shift in the balance of hydroxyapatite so that it is more soluble.²⁸ Due to too many inflammatory cells, the high solubility of graft material can affect the remodeling and bone mineralization process, damaging the healing process. The graft material loses its osteoconductive capability rapidly as it is absorbed quickly by the body, and the disintegration of the graft material causes bone apposition.^29,30

Bone and dentin have different Ca/P ratios, which are the ratios of calcium ion content to phosphate ions in hydroxyapatite. The higher the Ca/P ratio, the higher the calcium ion content in the material. The Ca/P ratio of biogenic apatites such as bone and teeth differ from the Ca/P ratio of geological apatite or pure hydroxyapatite due to the presence of ions or minor minerals (trace minerals) in biogenic apatite. Pure hydroxyapatite has a Ca/P ratio of 1.67; bone hydroxyapatite has a Ca/P ratio of 1.71, dentin hydroxyapatite has a Ca/P ratio of 1.63, while bovine hydroxyapatite heated to 1000^oC has a Ca/P ratio of 1,85.4,33,34 Ca/P ratio hydroxyapatite is essential to improve the mechanical ability of the graft.^31,32 Chan (2010) states that mechanical stability to endure shear stresses, such as those caused by chewing pressure, is one need for the optimum graft material. A robust graft surface that can be loaded bearing is crucial for bone remodeling.²⁵ Ramesh et al. (2007), in their study comparing the mechanical strength of hydroxyapatite with different Ca/P ratios heated at temperatures of 1000^oC-1350^oC, found that pure hydroxyapatite (Ca/P ratio: 1.67) had the best and most stable mechanical strength, followed by calcium-deficient hydroxyapatite (Ca/P ratio: 1.57), and calcium-rich hydroxyapatite (Ca/P ratio: 1.87) has the lowest mechanical strength.³¹

The Ca/P ratio of hydroxyapatite tooth graft (1.63) is close to the value of the Ca/P ratio of pure hydroxyapatite (1.67), so the mechanical strength of tooth graft is better than xenograft with a Ca/P ratio of 1.85. It has an influence on the ability of the graft to induce new bone growth, so the results of this study showed that the expression of osteocalcin was higher in the group of hydroxyapatite tooth graft than in the group of hydroxyapatite xenograft (p < 0.05) but contradicted the research conducted by Suzuki et al. (2000), showed that osteoblast activity was higher in hydroxyapatite with a higher Ca/P ratio.³³

This study used a hydroxyapatite graft with particle sizes of 150 – 355 µm and 355 – 750 µm. If the particle size of the graft is too tiny, the graft will be rapidly absorbed by macrophages so that the ability to induce bone is not maximized. On the other hand, in grafts with particle size, resorption by macrophages is too slow, which can hinder the process of new bone formation.⁶ Therefore, this study used particle sizes of 150 – 355 µm and 355 – 750 µm, which were mixed manually to obtain the maximum effect of the graft material.

CONCLUSION:

This study revealed that osteocalcin expression was higher in hydroxyapatite tooth grafts than in hydroxyapatite xenografts. This indicates that tooth grafts have the potential to serve as a promising alternative material in the field of dentistry, specifically in the treatment of periodontal diseases. However, additional research is required to examine hydroxyapatite tooth grafts' physical and chemical properties before they can be utilized for human applications.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 15.04.2024 Revised on 07.02.2025

Accepted on 22.06.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):263-268.

DOI: 10.52711/0974-360X.2026.00037

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.