Enhanced osteogenic marker expression in alveolar bone via hydroxyapatite gypsum puger cassava starch scaffold: An in vivo study

Abstract

Aim:

To evaluate the effects of hydroxyapatite gypsum puger–cassava starch (HAGP-CS) scaffold on the number of osteoblasts and alkaline phosphatase (ALP) and osteocalcin (OCN) expression in the alveolar bone rat model.

Settings and Design:

In vivo study.

Materials and Methods:

Thirty-six Wistar rats were randomly divided into three groups: rat sockets were given a 1 mm × 1 mm × 1 mm HAGP scaffold, rat sockets were given a 1 mm × 1 mm × 1 mm HAGP-CS, and a control group (rat sockets were not given scaffold material). Following lower left molar extraction, scaffold materials were applied to the sockets. Assessments were conducted on days 7^th, 14^th, and 28^th, with osteoblast counts determined via hematoxylin-eosin staining, and ALP and OCN expressions were analyzed using immunohistochemistry (IHC) staining.

Statistical Analysis Used:

Two-way analysis of variance and Tukey’s test.

Results:

A significant increase in osteoblast count was observed on day 28 (P = 0.001). In addition, significant differences were noted in ALP expression on day 7 (P = 0.030) and day 28 (P = 0.001), as well as in OCN expression on days 7 and 28 (P = 0.001) across the groups.

Conclusions:

Administering a HAGP-CS scaffold significantly enhances osteoblast proliferation and increases ALP and OCN expression in the alveolar bone rat model.

INTRODUCTION

The recovery of alveolar bone following tooth extraction is critically important in the context of dental implant treatment within the field of prosthodontics. One of the main challenges in this recovery is alveolar bone regeneration, which can be disrupted due to the loss of natural bone structure. Optimal recovery depends on efforts to accelerate bone regeneration using materials or scaffolds that can support new bone formation. Scaffolds function as mechanical supports that allow cells to migrate, grow, and form new bone tissue.[1] Therefore, research on scaffold materials that can enhance osteogenesis, such as gypsum-cassava starch (CS) hydroxyapatite (HA), has excellent potential for dentistry.

HA is a biomaterial similar to bone’s mineral composition, often used in tissue engineering and bone regeneration applications. Previous studies have shown that HA can increase the number of osteoblasts and accelerate the mineralization of the extracellular matrix.[2,3,4]

Conversely, gypsum can form a porous structure that supports bone cell growth and tissue recovery. At the same time, CS, which is naturally occurring, can provide advantages in terms of biocompatibility and affordability.[5] Combining these two materials as a scaffold is expected to provide an environment that supports bone growth in the alveolar bone socket after tooth extraction.

The problem of bone regeneration in the alveolar bone socket after tooth extraction involves complex interactions between various types of cells, including osteoblasts, osteoclasts, and progenitor cells. Osteoblasts are cells that play a role in bone matrix formation and mineralization, while osteoclasts are responsible for bone resorption. An imbalance between osteoblast and osteoclast activity can disrupt optimal bone formation. Therefore, proper control of osteoblast proliferation and differentiation and control of osteoclast activity are essential in supporting healthy bone recovery.[6] One way to optimize bone regeneration is to use scaffold materials that provide a structural framework and modulate cellular activity in the bone socket. Scaffolds made of HA, gypsum, and CS can provide molecular signals that stimulate osteoblast proliferation and differentiation and reduce excessive osteoclast activity. In addition, this scaffold can help maintain the balance between bone formation and remodeling processes that are essential for successful recovery.[7]

HAGP-cassava starch (HAGP-CS) scaffold, HA from gypsum puger as the main ingredient in this scaffold, has a crystal structure similar to the mineral components in bones, so it is very effective in supporting the formation of new bone tissue. Previous studies have shown that HAGP can stimulate osteoblast proliferation and increase the expression of osteogenic markers, such as alkaline phosphatase (ALP) and osteocalcin (OCN).[4,8] HAGP is easy to form and biocompatible, noninflammatory, and nonimmunogenic and can also support bone healing by providing a stable framework for cell growth.[9,10,11] However, pure HAGP still has several weaknesses, namely low biomechanics, low porosity, and brittleness, so it needs to be combined with materials to improve the properties of this material, namely with biopolymer material (CS). CS is classified as a polysaccharide that contains amylopectin and amylose starch, which are beneficial for health and are a source of carbohydrates, proteins, vitamins, and minerals to build bone mass.[12] Its natural polysaccharide content in CS provides biodegradable properties that support the transition between the scaffold and the bone tissue that forms.[13] Combining these two materials is expected to create a scaffold that promotes bone growth structurally and functions biologically in stimulating bone formation. HAGP-CS scaffold can provide an environment that supports cellular interactions required for osteoblast formation and osteoclast inhibition. As a result, this scaffold has great potential to accelerate the bone regeneration process, especially in alveolar bone sockets that experience posttooth extraction deficiencies.[5]

The role of osteoblasts in bone formation

Osteoblasts are the main cells involved in bone formation. They produce an extracellular matrix consisting of type I collagen and other proteins and carry out mineralization that produces hard bones. Osteoblasts are also expressed through osteogenic markers, such as ALP and OCN, which function as indicators of osteogenic activity.[5] Research shows that HA scaffolds can stimulate an increase in the number of osteoblasts and increase the expression of ALP and OCN, all of which contribute to new bone formation.[4,8] The administration of HAGP-CS scaffolds to posttooth extraction rats is expected to significantly increase osteoblast activity, thereby accelerating the healing process and regeneration of alveolar bone. Increased expression of osteogenic markers such as ALP and OCN will reflect the success of the osteogenesis process driven by this scaffold.[7] Thus, this study aims to evaluate how much the HAGP-CS scaffold can increase the number of osteoblasts and the expression of osteogenic markers – ALP and OCN in alveolar bone sockets.

MATERIALS AND METHODS

Making HAGP-CS scaffold

First, the hydroxyapatite gypsum puger (HAGP) material was made by dissolving 26.41 g of DHP powder into 400 ml of aquabidest and homogenizing it using a hotplate magnetic stirrer for 10 minutes, then 5 g of Puger gypsum powder was mixed into the DHP solution and then homogenized using a hotplate magnetic stirrer at a temperature of 40°C and a speed of 500 rpm for 15 minutes. After that, the hydrothermal process was carried out on the solution using an oven at a temperature of 100°C for 30 minutes. The solution was rinsed with aquadest and then filtered using filter paper until the pH was neutral. Then, the powder deposits on filter paper were dried in the oven at 50°C for 5 h. Then, it is sifted using a 100 mesh sieve so that HA powder from gypsum material is produced. Second, make CS by weighing 500 g of cassava grated then giving 1 l of water then filtering, the essence/solution is deposited for 12 h at room temperature, and then the sediment is dried at a temperature of 25°C then mashed and sifted with a 100 mesh sieve until CS powder is produced. Furthermore, the making of the HAGP CS scaffold by weighing 300 mg of solid gelatin is put in a glass tube 10 ml of water is heated to a temperature of 40°C 250 mg of HAGP is mixed until homogeneous then 250 mg of CS, and 10 ml of aquades, mixed using an ultrasonic homogenizer for 6 min, then the solution is put into an Eppendorf cylinder with a height of 30 mm and a diameter of 8 mm using a micropipette. It is then frozen at a temperature of −60°C for 24 h, and a freeze-drying process is carried out at a temperature of −80°C for 24 h. Then, the HAGP-CS scaffold material is formed and then cut into a size of 1 mm × 1 mm × 1 mm because the scaffold material will be applied to the rat socket with a very small space. Then, the scaffold material is sterilized with gamma radiation.

Experimental animals

This study has been evaluated and approved by the Ethical Committees of the Medical Research Faculty of Dentistry, University of Jember, with Certificate Number. 2616/UN25.8/KEPK/DL/2024.

Male Wistar rats, aged 8–12 weeks and healthy (±200–250 g), were acclimatized for 1 week in the experimental animal laboratory, Faculty of Dentistry, University of Jember. All animals were allowed free access to water and fed standard rat pellets. The number of samples in each group was 4 rats multiplied by 3 treatment groups, namely the HAGP group (rat sockets were given 1 mm × 1 mm × 1 mm HAGP scaffold), the HAGP-CS group (rat sockets were given HAGP-CS scaffold 1 mm × 1 mm × 1 mm), and the control group (rat sockets were not given scaffold) and multiplied by 3 observation groups, namely the 7^th, 14^th, and 28^th days so that there were a total sample of 36 rats, based on research Naini et al.[4]

Procedure for tooth extraction of rats and application of scaffold material

Wistar rats were anesthetized intramuscularly using 100 mg/ml ketamine and 20 mg/ml xylazine base at a dose of 0.08–0.2 ml/kgBW; then, the lower left molar teeth were extracted using a needle holder. Then in the first treatment group, namely the HAGP scaffold was applied to the extraction socket measuring 1 mm×1 mm×1 mm, the second treatment group, namely the HAGP CS scaffold was applied to the extraction socket measuring 1 mm×1 mm×1 mm, in the third treatment group, namely the extraction socket was not given scaffold material but was directly sutured. All groups were sewed using DR SELLA Silk Braided USP 3/0 75 cm thread. Furthermore, observations were made on the 7^th, 14^th, and 28^th days. All rat samples were sacrificed; then, the left lower jaw was cut from anterior to posterior, cleaned with 0.9% NaCl, and then placed in a closed container containing 10% formalin buffer for fixation and sent to the anatomical pathology laboratory for histological preparation. Histopathological analysis used hematoxylin-eosin (HE) staining to count the number of osteoblast cells and immunohistochemistry (IHC) staining to count the amount of ALP and OCN expression. Identifying osteoblast cells in preparations with HE staining has a cuboidal or flat shape with one dark purple nucleus. Identification of ALP and OCN expression in preparations with IHC staining is determined through osteoblast cells; osteoblasts that express ALP and OCN positively are marked with dark brown cytoplasm. Counting of osteoblast cells, ALP, and OCN expression with quantitative image analysis by calculating the number of positive cells compared to total cells using morphology-based counters on a light microscope with a magnification of ×400 for five fields of view was performed on the alveolar edge of the mesiolabial region of molar teeth.

Statistical analysis

Data were analyzed using IBM SPSS Statistics 28 software (Armonk, New York, USA.)[14] with a significance level or probability value of 0.05 (P = 0.05) and a confidence level of 95% (α = 0.05). Data analysis was performed using the normality test with the Shapiro–Wilks test. A homogeneity test was performed using the Levene test (P > 0.05). One-way analysis of variance (ANOVA) test and Tukey test were performed to determine the significance of differences between groups (P < 0.05).

RESULTS

Histopathological observations of the alveolar bone around the lower left molar teeth were carried out on the 7^th, 14^th, and 28^th days. The average and standard deviation of osteoblasts, ALP, and OCN expression from each group are presented in Tables 1-3. There was a pattern of decreasing the number of osteoblasts in the control group and increasing the number of osteoblasts in the HAGP and HAGP-CS groups at three-time point evaluations. The average number of osteoblasts increased from day 7 with several cells of 23.3 per field of view on the 28^th day with the number of cells per field of view of 34 cells. Hence, the average highest number of osteoblasts was found in the HAGP-CS group on the 28^th day.

Table 1.

Differences in the number of osteoblasts on days 7, 14, and 28

Group	Number of osteoblast
	Day 7		Day 14		Day 28
	±SD	P	±SD	P	±SD	P
HAGP	16±1	0.020	26±1	0.020	30.3±1.52	0.001
HAGP-CS	23.3±1.52		30.3±2.08		34±3
Control	11.3±0.57		18.7±2.08		23.3±1.15

HAGP-CS: Hydroxyapatite gypsum puger–cassava starch, SD: Standard deviation

Table 3.

Differences in osteocalcin expression on days 7, 14, and 28

Group	OCN expression
	Day 7		Day 14		Day 28
	±SD	P	±SD	P	±SD	P
HAGP	5.22±0.83	0.001	6.11±0.38	0.001	7.22±0.19	0.001
HAGP-CS	4.66±0.57		8.66±0.57		12.66±0.57
Control	4.22±0.38		6.66±0.33		8.11±0.69

HAGP-CS: Hydroxyapatite gypsum puger–cassava starch, SD: Standard deviation, OCN: Osteocalcin

There was a pattern of decreasing the number of ALP expressions in the control group and increasing the number of osteoblasts in the HAGP and HAGP-CS groups at three-time point evaluations. The average number of ALP increased from day 7 with a number of cells of 7.77 per field of view to day 28 with a number of cells of 11 per field of view. Hence, the average highest number of ALP expressions was found in the HAGP-CS group on day 28. There was a pattern of decreasing the number of OCN expressions in the control group and increasing the number of OCN expressions in the HAGP and HAGP-CS groups at three-time point evaluations. The average number of OCN expressions increased from day 7 with several cells of 4.66 per field of view to day 28 with several cells of 12.66 per field of view. Hence, the average number of OCN expressions was found in the HAGP-CS group on the 28^th day. The fluctuations are explained in Tables 1-3, while the histopathology of osteoblasts, ALP, and OCN expression is described in Figures 1-3. Based on the normality and homogeneity tests, show that the data is normal and homogeneous. The one-way ANOVA analysis was conducted to compare three treatment groups on days 7, 14, and 28; in this analysis, a significant difference was found in the number of osteoblasts on day 7^th (P = 0.020), day 14^th (P = 0.020), and day 28^th (P = 0.001); a significant difference was also seen in ALP expression on day 7^th (P = 0.030), day 14^th (P = 0.001), and day 28^th (P = 0.001); a significant difference was also seen in OCN expression on day 7^th (P = 0.001), day 14^th (P = 0.001), and day 28^th (P = 0.001) [Tables 1-3].

Figure 1.

Microscopic image of osteoblast cells with hematoxylin-eosin staining at ×400 magnification, the image is indicated by the yellow arrow. (a) Hydroxyapatite gypsum puger (HAGP) scaffold group, (b) HAGP-cassava starch (HAGP-CS) scaffold group, (c) control group

Figure 3.

Microscopic image of osteocalcin with immunohistochemistry staining at ×400, the image is indicated by the yellow arrow. (a) Hydroxyapatite gypsum puger (HAGP) scaffold group, (b) HAGP-cassava starch (HAGP-CS) scaffold group, (c) control group

Figure 2.

Microscopic image of alkaline phosphatase with immunohistochemistry staining at ×400, the image is indicated by the yellow arrow. (a) Hydroxyapatite gypsum puger (HAGP) scaffold group, (b) HAGP-cassava starch (HAGP-CS) scaffold group, (c) control group

The post hoc Tukey HSD assay in osteoblasts showed significant differences between the control group and HAGP (P = 0.007) as well as the control group and HAGP-CS (P = 0.000) on day 7. There were significant differences between the control group and HAGP (P = 0.000) and the control group and HAGP-CS (P = 0.000) on day 14. There was a significant difference between the control group and HAGP (P = 0.000) as well as the control group and HAGP-CS (P = 0.000) on day 28.

The post hoc Tukey HSD test on ALP showed significant differences between the control group and HAGP (P = 0.000) as well as the control group and HAGP-CS (P = 0.000) on day 7. There was a significant difference between the control group and HAGP (P = 0.000) as well as the control group and HAGP-CS (P = 0.044) on day 14. There was a significant difference between the control group and HAGP (P = 0.000) as well as the control group and HAGP-CS (P = 0.000) on day 28. No significant differences were found in the HAGP and HAGP-CS groups on day 28 (P = 0.189).

The post hoc Tukey HSD assay on OCN showed a significant difference between the control group and HAGP (P = 0.044) and no significant difference between the control group and HAGP-CS (P = 0.334) on day 7. There was a significant difference between the control group and HAGP-CS (P = 0.000) and no significant difference between the control group and HAGP (P = 0.220) on day 14. There were significant differences between the control group and HAGP-CS (P = 0.000) as well as the HAGP and HAGP-CS groups (P = 0.000); no significant differences were found in the control group and HAGP (P = 0.059) on day 28.

DISCUSSION

Posttooth extraction alveolar bone regeneration refers to the process of restoring or repairing alveolar bone tissue after tooth extraction. After tooth extraction, the empty tooth socket often experiences changes in the shape and volume of the alveolar bone. This process is essential to prepare the area to accommodate dental implants or other restorations.[15,16] In this study, scaffolds or supporting matrices, such as HAGP-CS, can support bone regeneration by providing a framework for osteogenic cells to grow. Osteogenic cells include osteoblasts, ALP expression, and OCN.[17,18]

This article aims to evaluate the increase in the number of osteoblasts, ALP expression, and OCN in the alveolar bone socket of rats after administration of the HAGP scaffold combined with CS through the freeze-drying method. The findings in this study were an increase in the number of osteoblasts in the alveolar bone sockets of rats given HAGP-CS scaffolds. The results of the examination of osteoblast cells in the HAGP-CS scaffold group on day 28^th showed a higher number of osteoblast cells, 1.45 times higher than the control group [Table 1]. They were significantly different compared to the control, so administering HAGP-CS scaffolds to the alveolar bone sockets could potentially increase the number of osteoblasts. Osteoblasts are cells responsible for bone formation, and an increase in their number indicates positive stimulation of the bone regeneration process.[19,20]

The combination of HA scaffolds from puger gypsum with natural materials such as CS has a three-dimensional porous structure with a pore diameter size of 112.42 μm and a pore area of 10,168.69 μm² which serves as a framework for cells to attach, grow, multiply, and differentiate, and it has high mechanical strength and can withstand pressure without cracking so that it can provide support for osteoblasts to develop and function optimally.[18,21] These results align with research showing that combining HA scaffolds and CS can increase osteoblast proliferation and differentiation and accelerate the healing of damaged alveolar bones.[4,8,21] A precisely designed scaffold not only becomes a mechanical support but also serves as a bioactive system that promotes osteogenesis and inhibits osteoclasty. These effects occur through molecular pathways such as BMP/SMAD, Wnt/β-catenin, and RANKL/OPG regulation, which can be confirmed through histological analyses such as ALP staining, TRAP, and bone mineralization.[22]

The HAGP CS scaffold has biocompatible properties that do not trigger tissue inflammatory responses through TNF alpha mediators, do not cause immunogenic reactions through IgG mediators, and are biodegradable, thus creating a microenvironment suitable for cell growth, such as osteoblasts.[23,24] The use of HAGP for wound healing has an effect, namely with its osteoconductive properties, so that it can facilitate osteoblast adhesion originating from the differentiation of fibroblast cells by fibroblast growth factor in the periosteum. Osteoblast cells will carry out the process of bone formation (osteogenesis) with the mechanisms of intramembranous ossification and endochondral ossification. Osteoblast cells will develop into osteocytes, which play a role in bone remodeling to maintain bone integrity and vitality. In line with other studies, inorganic materials from bone substitutes can create adhesion and increase osteoblastic cells.[18]

The results of the ALP examination in the HAGP-CS scaffold group on the 28^th day showed a significantly higher ALP value, 1.34 times higher than the control group [Table 2]. This indicates that administering HAGP scaffolds after tooth extraction in Wistar rats can increase the amount of ALP. In line with the results of osteoblast examination, the increasing number of osteoblasts also increases ALP protein, because osteoblasts will secrete bone matrix protein, namely ALP. ALP is an enzyme expressed by osteoblasts and is vital in bone mineralization. ALP activity is related to the formation of hard teeth and bone tissue, so ALP is widely used as a marker of bone formation. ALP prepares an alkaline environment in the osteoid tissue so that calcium can easily precipitate in the tissue. In addition, ALP in bones can increase phosphate concentration so that calcium–phosphate bonds are formed in the form of HA crystals. Based on the law of mass action, these crystals eventually precipitate in the bone.[8,25] This is consistent with other studies showing that chitosan application can increase ALP expression in osteoblast cells planted in chitosan–carbonate apatite nano scaffolds.[20] This study showed that the activity of ALP in human osteoblast cells increased significantly between days 7 and 21, reaching a peak around day 7, and then showing a plateau until day 35 [Table 3]. This indicates an intensive phase of matrix synthesis at the beginning of the osteogenesis process, followed by the subsequent phases of mineralization and differentiation.[26]

Table 2.

Differences in alkaline phosphatase expression on days 7, 14, and 28

Group	ALP expression
	Day 7		Day 14		Day 28
	±SD	P	±SD	P	±SD	P
HAGP	9.33±0.57	0.030	9.77±0.19	0.001	10.55±0.5	0.001
HAGP-CS	7.77±0.38		8.66±0.57		11±1
Control	6.88±0.38		7.55±0.38		8.22±0.19

HAGP-CS: Hydroxyapatite gypsum puger–cassava starch, SD: Standard deviation, ALP: Alkaline phosphatase

In the OCN examination, the HAGP-CS scaffold group on day 28^th had a higher OCN cell count, 1.56 times higher than the control group. This shows that administering the HAGP scaffold after tooth extraction in Wistar rats can increase the number of OCN cells. In line with the osteoblast examination, the increasing number of osteoblasts also increases the OCN protein, because osteoblasts will secrete bone matrix proteins, namely OCN. OCN is one of the biological markers used to assess the process of mineralization and formation of the extracellular matrix in bones.[27] OCN is one of the most commonly used markers for mature osteoblasts. Increased OCN expression indicates that this scaffold can stimulate the formation of the extracellular matrix needed for better bone mineralization and integration.[28] HA can increase the expression of proteins such as osteopontin and OCN in mesenchymal stem cells grown on HA-collagen scaffolds. This study showed that HA interacts with cells through integrin β1, triggers the activation of the Wnt/β-catenin pathway, and increases the expression of osteogenic genes such as Runx2 and OCN.[29]

This finding is consistent with previous research demonstrating that robusta coffee extract enhances OCN expression in osteoblasts during orthodontic tooth movement.[30] The HAGP-CS scaffold evaluated in this study exhibits potential as a biomaterial suitable for bone tissue engineering. HA is a biomaterial similar to the mineral composition of natural bones, thus providing an environment that supports the growth of osteogenic cells such as osteoblasts. On the other hand, gypsum puger and CS starch, as natural-based materials, provide good biocompatibility and biodegradability properties. This combination, through the freeze-drying process, produces a scaffold with a porous structure that supports the migration and proliferation of osteogenic cells and facilitates the transfer of oxygen and nutrients needed in the bone healing process.[8,21] This is supported by research showing that the combination of HA-Gelatin-Propolis scaffold can increase bone regeneration activity as seen from the levels of ALP and OCN secreted by osteoblasts.[31] The results of this study showed that the 28-day HAGP-CS scaffold can increase the number of osteoblasts, ALP expression, and OCN, which all contribute to early bone healing. Day 28 is the initial final phase of healing or the initial phase of remodeling. Therefore, this scaffold has the potential to be an alternative therapy in the early regeneration of alveolar bones, especially in patients who have experienced tooth loss or other alveolar bone damage. More research is needed to explore longer research and the long-term effects of this scaffold, including clinical trials in humans, as well as to assess its mechanical stability and biodegradability in biological environments so that it can provide additional insights into the development of more effective scaffolds.[32,33,34]

This research material is different from other synthetic materials, HA in this study was obtained from natural materials, namely local gypsum from the Puger limestone mountains of Indonesia which was combined with cassava starch. This material is easier to get and cheaper than synthetic scaffold materials on the market and has characteristics according to the Japanese HA 200 and HA bovine scaffold standards.[3,9] Bone scaffolding made from HA, gypsum puger, and CS contributes synergistically to supporting osteoblast proliferation and osteogenic protein expression. HA improves cell adhesion and differentiation through its similarity to bone minerals as well as activation of certain molecular pathways. Gypsum acts as a source of calcium that supports mineralization and strengthens scaffolding structures, while CS provides a biocompatible matrix that supports the microenvironment for cell growth. The combination of all three results in an ideal scaffolding for bone regeneration biologically and mechanically.[20,35]

In other studies as a comparison to contextualize the novelty of HAGP CS is a study on similar scaffolds such as Hydroxyapatite chitosan and tapioca starch with the freeze dry manufacturing method has the advantage of smaller pores, better interconnection but has the disadvantage of small pores that can inhibit vascularization and cell growth, as well as other studies on Hydroxyapatite, chitosan, PCL scaffolds have higher mechanical strength, better degradation control but have disadvantages in the manufacturing process is more complex, higher costs. Degradation control is better but has the disadvantage of a more complex manufacturing process, higher cost.[36]

HAGP-CS scaffolding shows excellence in terms of sustainability, with optimal pore size (75–100 μm), porosity structure that supports bone regeneration, and potential for functional modification. Biodegradability of about 39% on the 1^st day and 55% on the 21^st day, despite having a mechanical strength (2.07 MPa) that needs to be improved, HAGP-CS offers a promising alternative in bone tissue engineering, especially in areas with limited access to imported raw materials.[21,24]

Research limitations in animal response variability and histology/immunohistochemical techniques are small sample sizes and short and limited observation times. Therefore, increasing sample sizes, using appropriate controls, antibody validation, and combinations with other methods, biochemical or molecular, can help improve the reliability and reproducibility of the data. Further studies are needed to optimize scaffold design, evaluate long-term biocompatibility, and explore its potential application in clinical practice to guide future research.

CONCLUSIONS

The administration of HAGP-CS scaffold in a rat alveolar bone model can increase the number of osteoblasts as well as the expression levels of ALP and OCN in vivo, indicating its potential as an effective strategy to support alveolar bone regeneration after tooth extraction in preparation for dental implant treatment in the field of prosthodontics.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

This research is financially supported by LPPM University of Jember, Indonesia (Grant Number: 7554/UN25/KP/2024).