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Journal of Oral Biology and Craniofacial Research logoLink to Journal of Oral Biology and Craniofacial Research
. 2025 Jul 16;15(5):1021–1028. doi: 10.1016/j.jobcr.2025.07.007

Enhanced glass ionomer cement with bioactive additives for collagen synthesis and inflammation in pulp-rabbit teeth

Nattapon Rotpenpian a, Peerapon Sornying b, Sareepah Manmoo b, Krittee Dejyong b,⁎
PMCID: PMC12281183  PMID: 40697753

Abstract

Objective

This study aimed to enhance conventional glass ionomer cement (GIC) by incorporating 15 % chitosan, 5 % bovine serum albumin (BSA), 0.05 % tricalcium phosphate (TCP), and 1 μg translationally controlled tumor protein (TCTP), resulting in an enhanced-GIC formulation. The study evaluated its adhesion properties, biocompatibility, and ability to promote pulp tissue healing in rabbit anterior teeth.

Materials and methods

The enhanced-GIC was tested in a rabbit model to assess its physical adhesion and biological effects on pulp tissue. Following cavity preparation and material placement, the teeth were observed for 21 days. Histological evaluations focused on inflammation, toxicity, and collagen synthesis in pulp tissue.

Result

The enhanced-GIC showed comparable adhesion properties to conventional GIC. Histological analysis revealed no significant inflammation or toxicity in the pulp tissue of either group. The enhanced-GIC group exhibited superior biocompatibility, demonstrated by increased lymphocyte infiltration and enhanced collagen synthesis within the pulp tissue, suggesting its potential for promoting tissue regeneration.

Conclusion

The enhanced-GIC formulation shows promise as a sublining material in restorative dentistry, offering benefits in pulp tissue healing and collagen formation, while maintaining adhesion comparable to conventional GIC. This study highlights enhanced-GIC's potential for use in dental restorative procedures, particularly for cases requiring pulp regeneration.

Keywords: Glass ionomer cement, Biological property, Chitosan, Sub-lining, Dental material

1. Introduction

Dental restoration is a cornerstone of modern dentistry, focused on repairing damaged teeth by replacing compromised structures with suitable restorative materials. This procedure typically involves careful tooth preparation to ensure optimal outcomes, followed by the application of restorative materials specifically selected to meet the clinical needs of the patient.1 Tooth preparation involves mechanically refining a defective tooth to create an optimal surface for receiving restorative material, thereby ensuring the integrity and longevity of the restoration.

However, this process often requires the removal of infected carious tissues, which can introduce challenges such as heat generation, excessive pressure, and inadequate cooling during high-speed bur usage. These factors increase the risk of pulpal injury, particularly in cases of deep carious lesions. Such lesions often compromise the pulp cavity, with the risk of pulpal sensitivity being inversely proportional to the remaining dentin thickness—the distance between the pulp chamber and the cavity floor.2 Therefore, pulp protection is essential to minimize irritation from bacterial and chemical agents encountered during restorative procedures, safeguarding the vitality and function of the pulp tissue and ensuring the long-term health of the tooth.3

Pulp protective agents are broadly classified into dental liners and bases. Dental liners are thin, fluid materials applied over dentin to form a protective barrier against chemical irritants. They play a vital role in stimulating reparative dentin formation, promoting pulp healing, and enhancing the overall success of restorative procedures by safeguarding the pulp from potential harm during treatment.4 However, dental liners lack the strength and thickness required to withstand mechanical forces on their own, necessitating the use of complementary materials, such as dental bases, to provide the necessary structural support and ensure the long-term success of the restoration.5 Dental bases, typically applied over liners before the placement of the final restorative material, provide thermal insulation and protect the pulp from thermal, mechanical, and chemical trauma, including microleakage. An ideal base material should have adequate strength to withstand masticatory and condensation forces, ensuring the durability, stability, and integrity of the restoration throughout its lifespan.6

Among modern restorative materials, glass ionomer cements (GICs) are extensively used as liners and bases due to their advantageous properties, including chemical bonding to tooth structures, fluoride release, and biocompatibility,7 GICs offer several advantages, including ease of use, caries protection, remineralization potential, and sustained moisture sensitivity. Recent advancements in GIC formulations have led to the development of resin-modified glass ionomer cements (RMGIC) and BIOGIC. RMGIC incorporates 2-hydroxyethyl methacrylate (HEMA), which improves aesthetics and reduces acidity, thereby enhancing its overall performance and versatility in clinical applications. These modifications aim to address some of the limitations of conventional GICs, offering improved mechanical properties and aesthetic outcomes for restorative dental procedures,8

However, despite these improvements, RMGIC remains sensitive to moisture and is generally unsuitable for use in pressure-bearing areas, which limits its application in certain restorative procedures, particularly in regions subject to heavy masticatory forces.9 BIOGIC represents a significant advancement in GIC formulations, enhanced with chitosan and bovine serum albumin (BSA). Chitosan, derived from the exoskeletons of crustaceans, offers antibacterial properties, promotes fluoride release, and supports dental pulp cell proliferation and differentiation. BSA contributes by releasing transforming growth factor beta-1 (TGF-β1), a crucial factor in dental pulp repair, which stimulates odontoblast activity and mineralisation. Additionally, the incorporation of tricalcium phosphate (TCP) into BIOGIC enhances calcium deposition, supporting the mineralisation process and further improving the material's ability to promote pulp healing and regeneration. This combination of bioactive components positions BIOGIC as a promising material for advanced dental restorative procedures.10

The latest advancement in BIOGIC involves the inclusion of translationally controlled tumour protein (TCTP), derived from Penaeus merguiensis shrimp. TCTP plays a crucial role in regulating cell growth, differentiation, and survival, and its incorporation into BIOGIC further enhances the material's capacity to promote pulp tissue healing and regeneration. This addition aims to improve the biocompatibility and regenerative potential of the cement, making it more effective in supporting dental pulp repair and enhancing the overall therapeutic outcomes in restorative dentistry.11

TCTP enhances cellular resilience to oxidative, thermal, and genetic stress, while promoting odontoblast differentiation and preventing apoptosis induced by bacterial by-products and HEMA. Furthermore, TCTP supports calcium deposition and stimulates reparative dentin mineralisation by upregulating osteopontin expression. These properties make TCTP a valuable component of BIOGIC, enhancing its potential for pulp tissue regeneration and strengthening the material's capacity to promote healing in dental restorations.12 However, the effects of TCTP are dose-dependent, with lower concentrations promoting odontoblastic activity, while higher concentrations may potentially induce tumourigenic effects. The incorporation of chitosan and BSA into GIC formulations extends the release of TCTP, thereby indirectly supporting odontoblast function and enhancing mineralisation. This controlled release mechanism optimises the regenerative benefits of TCTP, ensuring its therapeutic effects are maximised while minimizing the risks associated with excessive concentrations.

This study hypothesises that BIOGIC, enriched with TCP and TCTP, hereafter referred to as enhanced-GIC (enGIC), can promote odontoblast proliferation, differentiation, and mineralisation in vivo, while mitigating pulpal damage induced by bacterial by-products. Although previous studies have demonstrated the efficacy of enhanced-GIC (enGIC) in vitro, there is a notable lack of in vivo evidence to support its clinical application. To address this gap, animal studies are essential for assessing the biocompatibility, mineralisation potential, and biological responses of these advanced restorative materials under physiological conditions.

2. Materials and methods

2.1. Dental lining Materials

Two types of glass ionomer cement (GIC) were used in this study. The positive control group received a commercially available conventional GIC (batch no. 7609700), while the experimental group received an Enhanced-GIC, prepared by incorporating the following biomaterials into the GIC powder component (batch no. 7821600): 15 % (w/w) chitosan, 5 % (w/w) bovine serum albumin (BSA), 0.05 % tricalcium phosphate (TCP), and 1 μg of translationally controlled tumor protein (TCTP). All materials were sterilized and mixed thoroughly under aseptic conditions before application.

Additionally, the invention titled "Material for the Release of Substances from Glass Ionomer Cement Modified with Chitosan" was granted a patent on January 31, 2023, under patent number 0501002755 in Thailand. Another related invention, titled "Resin Composition for Application in Glass Ionomer Cement and Its Manufacturing Method," was granted a patent on March 13, 2023, under patent number 1001001095 in Thailand. These patents contribute to advancements in the field of dental materials, specifically enhancing the properties of glass ionomer cement for improved therapeutic outcomes in restorative dentistry.

2.2. Animal experiment

This in vivo study was approved by the Institutional Animal Care and Use Committee at Prince of Songkla University (Approval No. MHESI 68014/1785) and adhered to the ARRIVE 2.0 guidelines for animal experimentation.

Six New Zealand White male rabbits, each weighing approximately 1 kg, were randomly assigned to three experimental groups. Procedures were performed on both the upper and lower incisor teeth of each rabbit, with a total of four teeth per rabbit. Excluding the negative control group, the remaining groups were further randomised into Pattern A and Pattern B (Fig. 1).

Fig. 1.

Fig. 1

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The group randomization pattern (a) and the negative control group (b).

The first group, the Negative Control, received no restorative material (n = 4 teeth, Rabbit 1). The second group, the Positive Control, was restored with conventional GIC (n = 20 teeth, Rabbits 1–6), while the third group, the Experimental Group, received restoration with enhanced-GIC (n = 20 teeth, Rabbits 1–6). Teeth were randomly assigned to the Positive and Experimental groups using a computer-generated random number sequence, stratified by rabbit, to ensure balanced allocation across animals and reduce potential confounding. Furthermore, evaluators responsible for histological examination and scoring were blinded to group assignments to minimize assessment bias.

The rabbits were anaesthetised using 0.2 mg/kg of Dexmedetomidine (Dextometor®, Zoetis, USA) and 20 mg/kg of Tiletamine/Zolazepam (Zoletil®, Virbac Corporation, FR) were administered intramuscularly, followed by subcutaneously administering an analgesia; 5 mg/kg of Tramadol HCl (TRAMADOL®, T.P. Corporation, Thailand) prior to the procedure.13Anaesthesia depth was monitored by assessing the toe/tail pinch reflex every 5 min. If necessary, 1 mg/kg of Atipamezole (Atipam®, Zoetis, US) was administered intraperitoneally to reverse the effects of alpha-2 adrenergic receptor agonism and shorten the recovery time.

A standardized 1 mm cavity was created in each upper and lower incisor using a low-speed dental bur under sterile saline irrigation to expose the dental pulp.14 Following tooth preparation, the cavities were restored using either GIC or enhanced-GIC and subsequently sealed with a resin composite. Post-operative pain and discomfort were assessed through monitoring feed intake and evaluating facial expressions using the Rabbit Facial Grimace Scale.15 Post-operative analgesia was provided through a subcutaneous injection of Tramadol HCl at a dose of 15 mg/kg. Right lateral dental radiographs were obtained on days 0, 3, 7, 14, and 21 under anaesthesia with 1.5–2 % isoflurane in oxygen (1–1.5 L/min) to evaluate the adhesion and the interface distance between the tooth and the restorative material. The rabbits were euthanised on day 21 for histopathological analysis of their incisor teeth using Thiopental Na 100 mg/kg intravenously.

2.3. Histological analysis

The samples were carefully dissected along with the surrounding maxillary and mandibular tissues. Blood was cleaned off under running water. Each specimen was then fixed in 10 % neutral-buffered formalin for 48 h, followed by decalcification, dehydration, and embedding in paraffin wax using standard histological techniques. Serial longitudinal sections (5 μm thick) were cut in the labiolingual direction to include the right lower incisors and their supporting structures. The sections were then mounted on glass slides, dried at 37–40 °C overnight or at 60 °C for 1–2 h, and deparaffinised in xylene. Rehydration was performed using a graded ethanol series (100 %, 95 %, 70 %, and 50 % v/v) for 1–2 min each, followed by a rinse in distilled water.16

The tissue slides were stained with hematoxylin for 5–10 min and then rinsed in tap water. To differentiate the staining, the slides were briefly exposed to 1 % acid alcohol (1 % HCl in 70 % ethanol), followed by rinsing in tap water. The slides were then immersed in a bluing solution, such as ammonia water, for 1–2 min to enhance the hematoxylin staining. Subsequently, the slides were stained with eosin for 1–3 min, rinsed in distilled water, and dehydrated through a graded series of ethanol solutions. The tissue sections were cleared in xylene and mounted with a resin-based mounting medium under coverslips.

Finally, the slides were examined under a light microscope (Bright field Polarization Microscope; Carl Zeiss Axioskop 40) in 6 sections per group, using image analysis software for evaluation of inflammatory cell infiltration. Inflammatory cell infiltration was assessed in 20 fields of view at 40x magnification for each group. The degree of inflammation was graded as shown in Table 1.17

Table 1.

Degree of inflammation and Collagen deposition grading.

Grade Degree of inflammation Collagen deposition
0 No inflammatory infiltrates; normal tissue. No collagen fibers
I 1–10 % of vessels infiltrated by small lymphocytes. Small amount of collagen with coarse arrangement and pale blue stain
II 11–50 % of vessels infiltrated by small lymphocytes, with mild primary dentin inflammation. Short to medium continuous arrangement of collagen bands with blue stain
III More than 51 % of vessels infiltrated by small lymphocytes, with mild secondary dentin inflammation. Long collagen fibers with dark blue stain
IV Extensive inflammation, with infiltrates extending into the base of the predentin.
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For collagen analysis, the slides were stained with 0.1 % Sirius Red in saturated aqueous picric acid for 60 min at room temperature. Excess stain was removed by rinsing the slides in 0.5 % acetic acid, followed by dehydration and clearing in xylene. The sections were then mounted with a resin-based medium. Collagen fibers were assessed under a polarized light microscope (Brightfield Polarization Microscope; Carl Zeiss Axioskop 40) in six sections per group, using software for analysis. Collagen deposition was graded based on color intensity and arrangement in 12 areas of surrounding tissue adjacent to the filling material, with evaluation performed in 20 fields of view at 40x magnification in each group as shown in Table 1.18

2.4. Statistical analysis

Data are presented as means ± standard deviation (SD). Normality was assessed using the Shapiro-Wilk test. The Mann–Whitney U test was used to compare histological criteria and the means between groups on days 21. A p-value of ≤0.05 was considered statistically significant.

3. Results

3.1. Gross anatomy and radiographic evaluation after GIC and Enhanced-GIC treatment

The study results revealed no statistically significant differences in the gross anatomy or radiographic images of the teeth between the GIC and enhanced-GIC groups at any time point. Evaluations were conducted on pre-treatment drilled incisors (D0 Pre) and subsequent stages, including immediately after treatment (D0 Tx), as well as on days 3, 7, 14, and 21 post-operations in six rabbits. Unfortunately, rabbits E and F died on days 14 and 7, respectively, before reaching the experimental endpoint due to heatstroke caused by a malfunction in the air conditioning system. Representative images of the gross anatomy and radiographic evaluations are presented in Fig. 2, Fig. 3, Fig. 4.

Fig. 2.

Fig. 2

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Gross anatomy of teeth from pre-treatment drilled incisors (D0 Pre) and subsequent stages: GIC and enhanced-GIC embedded (D0 Tx), Day 3, Day 7, Day 14, and Day 21 post-operation in six rabbits. The gross scores were evaluated at three levels based on the extent of cavity filling compared to pre-treatment drilled incisors: 0 = no filling of the drilled incisor cavities, 0.5 = partial cavity filling, and 1 = complete cavity filling.

Fig. 3.

Fig. 3

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Comparison of teeth anatomy during pulp and dentine regeneration: (a) radiolucent appearance of the lower incisor cavity, (b) radiopaque appearance of GIC, and (c) the disappearance of the radiolucent cavity corresponding with pulp and dentine regeneration. These images illustrate the progressive changes in tooth anatomy as the restorative materials interact with the pulp and dentine, highlighting the regenerative process over time.

Fig. 4.

Fig. 4

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Radiographic anatomy of teeth from pre-treatment drilled incisors (D0 Pre) and subsequent stages: GIC and Enhanced-GIC embedded (D0 Tx), Day 3, Day 7, Day 14, and Day 21 post-operation in six rabbits. The radiographs show the radiopaque shadow of GIC and pulp cavities, providing visual assessment of the restorative material's integrity and the pulp’s condition at different time points.

The remaining GIC and enhanced-GIC after treatment were scored as follows: 1 for complete retention of material, 0.5 for partial retention, and 0 for total loss of material. These scores were then analysed using the Wilcoxon signed-rank test in GraphPad Prism 10.1.1 (GraphPad, USA). On Day 0, both groups had comparable scores close to 1. Over time, a gradual reduction in scores was observed in both groups. By Day 3, the scores had significantly decreased, indicating early changes in material or enamel performance. From Day 7 to Day 21, the scores fluctuated but remained relatively stable, suggesting that the performance of both materials had reached a steady state after the initial adjustment phase. While the Enhanced-GIC group demonstrated slightly higher scores than the GIC group on Days 14 and 21, these differences were within the error margins, indicating no statistically significant disparity in performance as shown in Fig. 5.

Fig. 5.

Fig. 5

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There was no statistically significant difference in gross scores over time between GIC and enhanced-GIC groups (enGIC) (p≥0.05). The bar graph illustrates the mean scores with standard deviation from Day 0 to Day 21. Both groups show a decline in scores from baseline (Day 0), with fluctuations observed over the study period.

3.2. Histological assessment

After 21 days of restoration with GIC and Enhanced-GIC, all restorations remained within the cavity of the incisor. H&E staining revealed normally structured pulp tissue adjacent to the dentin, in tubular contact with the cavity floor (score 0). Histological analysis demonstrated that both the GIC and Enhanced-GIC liners provided effective protection to the pulp-dentine complex and did not induce inflammatory reactions in the pulp tissue, as shown in Fig. 6A and Table 2. Furthermore, the Enhanced-GIC group exhibited a small amount of collagen with a coarse arrangement and pale blue staining (grade I), while the GIC group showed no collagen synthesis, as illustrated in Fig. 6B and Table 2.

Fig. 6.

Fig. 6

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Histological analysis of pulpal tissue on day 21 following treatment with GIC and enhanced-GIC was performed using Haematoxylin and Eosin (H&E) and Picrosirius Red staining. H&E staining revealed pulpal responses in GIC-treated samples observed at 40× magnification (Aa), and in enhanced-GIC-treated samples observed at both 4× and 40× magnifications (Ab), with a scale bar of 5 mm. Picrosirius Red staining was similarly employed to assess collagen deposition, with GIC-treated samples examined at 40× magnification (Ba) and enhanced-GIC-treated samples at 40× magnification (Bb), also using a scale bar of 5 mm.

Table 2.

Summary of the statistical analysis of the histology criteria compared between the control and enhanced-GIC.

Histology criteria Day21
GIC Enhanced-GIC p-value
Number of new microvessels (Mann–Whitney U test) 0.43 ± 0.13 0.52 ± 0.21 0.432
Number of fibroblast or collagen (Mann–Whitney U test) 7.83 ± 3.28 8.43 ± 2.58 0.356
Inflammation intensity (Mann–Whitney U test) 0.13 ± 0.04 0.10 ± 0.07 0.768
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4. Discussion

This study evaluates Enhanced-GIC, a glass ionomer cement modified with biomaterials, for its potential as a sublining material in dental restorations. The enhancement, achieved by incorporating chitosan, bovine serum albumin (BSA), tricalcium phosphate (TCP), and translationally controlled tumor protein (TCTP), aims to optimize the biocompatibility, mechanical properties, and regenerative potential of the material. Biocompatibility is crucial for its success as a restorative material, and the study confirmed the non-toxic nature of Enhanced-GIC, along with its ability to support cellular activity, particularly in pulp tissue. Chitosan, a key component, exhibited antibacterial properties against oral pathogens such as Streptococcus mutans, which helped reduce bacterial colonization near the pulp, further supporting the material's potential for use in dental applications.19

Additionally, chitosan enhances adhesion between the cement and dentin, forming polyelectrolyte complexes that improve structural stability.20 Its ability to promote pulp cell proliferation and differentiation into osteogenic lineages21 aligns with existing research suggesting its role in wound healing and tissue regeneration.

BSA contributes significantly to biocompatibility through its release of transforming growth factor-beta 1 (TGF-β1).22 This cytokine is pivotal for odontoblast function, type I collagen synthesis, and mineralisation. Previous studies corroborate its role in modulating inflammatory responses and enhancing cellular repair mechanisms,23 further substantiating its inclusion in Enhanced-GIC.

The inclusion of tricalcium phosphate (TCP) addresses the critical need for enhanced mineralisation, a key factor in the success of dental restorations. TCP provides essential calcium and phosphate ions, which mimic the natural composition of tooth structure and promote the formation of reparative dentin. Previous studies have demonstrated that TCP-based materials facilitate mineral deposition both in vitro and in vivo, highlighting their significant role in improving the efficacy of dental restorations.11 In this study, Enhanced-GIC with TCP demonstrated increased calcium deposition in the treated pulp tissue, further supporting the findings that TCP enhances mineralisation and contributes to the regenerative potential of restorative materials. This enhanced calcium deposition may facilitate the formation of reparative dentin, thus promoting improved dental restoration outcomes.

TCTP, derived from Penaeus merguiensis, plays a multifaceted role in cellular protection and repair.10 By preventing apoptosis and oxidative stress-induced damage, TCTP ensures the survival of odontoblasts and other pulp cells.11 Additionally, its promotion of mineralisation through upregulation of osteogenic markers, such as osteopontin, emphasizes its importance in reparative dentinogenesis. However, TCTP's dose-dependent effects necessitate careful optimization to avoid potential adverse effects.

Histological analysis revealed the formation of a new collagen matrix and an absence of necrosis or severe inflammatory response in tissues treated with Enhanced-GIC. These findings underscore its excellent biocompatibility and regenerative potential. The observed collagen synthesis aligns with previous studies that emphasize the role of components such as chitosan and BSA in promoting extracellular matrix formation and facilitating tissue healing. This further supports the potential of Enhanced-GIC as an effective restorative material in dental applications.24 The comparable adhesive performance between Enhanced-GIC and conventional GIC further establishes its viability as a restorative material. This similarity in adhesion strength suggests that Enhanced-GIC can offer reliable bonding to dental tissues, making it a promising alternative to traditional materials in clinical applications. The ability to maintain effective adhesion while providing enhanced biological and mechanical properties positions Enhanced-GIC as a competitive option in restorative dentistry.

The integration of biomaterials such as chitosan, BSA, TCP, and TCTP into GIC enhances its biological performance by promoting cell adhesion, proliferation, and tissue regeneration. Chitosan, a natural polysaccharide, exhibits antimicrobial properties and improves mechanical strength and biocompatibility. BSA serves as a protein carrier and supports cellular functions and matrix interactions. TCP contributes to remineralization and osteoconductivity by releasing calcium and phosphate ions, essential for hard tissue repair. TCTP plays a role in cell survival and proliferation, potentially enhancing wound healing and anti-inflammatory responses. Together, these additives may synergistically improve the therapeutic potential of GIC in dental and biomedical applications.20, 21, 22, 23, 24, 25

After the in vivo implantation of the biomaterial, it was expected to initiate a normal wound-healing mechanism, a complex process involving dynamic interactions among various cell types.25 This study evaluated these responses by quantifying and correlating the cell populations involved in the healing process around the test specimens. Fibroblasts, which play a pivotal role in tissue regeneration, were highlighted for their ability to produce extracellular matrix components, such as collagen, and release growth factors critical for maintaining tissue homeostasis. Previous studies demonstrated that GIC particles observed in histological sections did not induce foreign body giant cell formation, granulomatous inflammation, necrosis, or significant chronic inflammation around the implanted material.26 Similarly, the current experiment confirmed the biocompatibility of GIC by showing no adverse inflammatory responses in the test specimens.

Furthermore, earlier research indicated that stimulating Wnt/β-catenin signaling in animal models with small cavities lacking pulp exposure could enhance reactionary dentin formation.27 For example, when a collagen sponge soaked with Tideglusib, a GSK3 antagonist, was placed in a tooth cavity, it activated Wnt signaling in odontoblasts and dental pulp cells through the remaining dentin. However, challenges such as collagen sponge degradation led to space formation beneath the capping material, resulting in cracks and restoration failures.28 GIC has consistently shown cellular biocompatibility across various studies, including applications in human gingival fibroblast cultures, subcutaneous tissue, rat alveoli, and deep cavities in human teeth.29,30,31,32

This study demonstrates that Enhanced-GIC maintains the biocompatibility of traditional glass ionomer cement and shows promise as a sublining material for promoting dentin regeneration. However, further optimization is needed to enhance its mechanical stability. The inclusion of additives like chitosan and BSA can affect the setting time and structural integrity, requiring further investigation to refine these interactions. Additionally, ensuring the scalability and cost-effectiveness of Enhanced-GIC with biomaterials such as TCTP is crucial for its broader clinical application. Addressing these issues will be key to enhancing the viability of Enhanced-GIC for modern restorative dentistry.

5. Conclusion

Enhanced-GIC represents a notable advancement in dental materials, integrating biomaterials such as chitosan, BSA, TCP, and TCTP to achieve an optimal balance of biocompatibility, antibacterial properties, and mineralisation capacity. This study highlights its potential as a sublining material that supports early healing responses. However, while its biological benefits are evident, further investigation is required to enhance its mechanical properties and validate its clinical efficacy. The findings contribute to the growing body of evidence supporting the integration of biologically active components in restorative dentistry, yet do not explicitly address the novelty or robustness of the study design within the discussion or conclusion.

6. Strengths and limitations of the study

This study has several strengths that enhance its reliability and relevance. The randomized design, stratified by animal, with both positive and negative controls, reduces bias and confounding. Using a bilateral incisor model allows each rabbit to serve as its own control, improving consistency and minimizing animal use. Validated tools like the Rabbit Facial Grimace Scale and established histological criteria, combined with blinded evaluators, ensure objective and reliable assessments.

Ethical rigor was maintained through adherence to ARRIVE 2.0 guidelines and institutional approval, reflecting high standards of animal welfare. The use of a patented Enhanced-GIC adds clinical relevance and potential for future application. Finally, the integration of anatomical, radiographic, histological, and collagen analyses provides a comprehensive evaluation of material performance and biological response.

However, the limitations of the study was the small sample size, including the loss of two rabbits, reduced statistical power. The semi-quantitative scoring system may have limited sensitivity, and the 21-day duration was insufficient to assess long-term outcomes. Additionally, collagen assessment was qualitative, lacking molecular or quantitative analysis for tissue response.

Patient's/Guardian's consent

No.

Human ethics and consent to participate declarations

Not applicable.

Ethical clearance

This animal study was approved by the Institutional Animal Care and Use Committee at Prince of Songkla University (MHESI 68014/1785).

Funding

This research was supported by the National Science, Research and Innovation Fund (NSRF) and Prince of Songkla University (Ref. No. DEN6801013S).

Declaration of competing interest

The authors declare that there are no conflicts of interest regarding the publication of this article.

Acknowledgments

The authors would like to express their gratitude to the Department of Oral Biology and Occlusion, Faculty of Dentistry, Prince of Songkla University, and the Faculty of Veterinary Science, Prince of Songkla University, for their valuable support. This research was funded by the National Science, Research and Innovation Fund (NSRF) and Prince of Songkla University (Ref. No. DEN6801013S).

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