Novel acemannan-integrated bioceramic for enhancing human dental pulp stem cell viability and transforming growth factor beta-1 release in pulp regeneration

Abstract

Context:

Vital pulp therapy (VPT) preserves pulpal vitality by utilising bioactive materials that promote cell survival and regeneration. Calcium phosphate cement (CPC) and calcium sulfate hemihydrate (CSH) are biocompatible and osteoinductive, while acemannan, a polysaccharide from Aloe vera, exhibits immunomodulatory and regenerative properties. Their combination may synergistically enhance pulpal healing.

Aims:

This study evaluates the effects of a novel acemannan-integrated CPC–CSH (CPC–CSH–Ace) bioceramic on pH, human dental pulp stem cells (hDPSCs) viability, and transforming growth factor beta-1 (TGF-β1) release.

Settings and Design:

An in vitro experimental study conducted at ProSTEM (Prodia StemCell) Indonesia.

Subjects and Methods:

CPC and CSH were combined with 3%, 5%, and 10% acemannan to form test group with NeoPutty and EndoCem Mineral Trioxide Aggregate as controls. pH was measured at 3, 24, and 72 h. hDPSCs viability (%) was assessed using MTT assays at 24 and 72 h, and TGF-β1 levels (pg/mL) quantified by Enzyme-Linked Immunosorbent Assay after 7 days. Data were analysed using one-way analysis of variance followed by Tukey’s post hoc test (P < 0.05).

Results:

The CPC-CSH–Ace 3% group showed the highest cell viability at both 24 and 72 h (P < 0.05) and significantly increased TGF-β1 release (P < 0.05). This upregulation of TGF-β1 represents a key biological signal that promotes odontoblast proliferation and reparative odontogenesis – essential processes for successful pulp capping. All CPC-CSH-Ace groups displayed a slightly alkaline pH conducive to proliferation.

Conclusions:

The CPC–CSH–Ace 3% formulation showed superior bioactivity, indicating its potential as a novel material for VPT.

INTRODUCTION

Preserving pulpal vitality is a fundamental goal in conservative endodontics. Vital pulp therapy (VPT) allows the maintenance of functional pulp tissue following carious or traumatic exposure when a biologically active capping material is used to stimulate healing and regeneration.[1,2]

Calcium hydroxide has long been used as a pulp-capping agent; however, its solubility and weak sealing properties have led to the search for more advanced materials, such as Mineral Trioxide Aggregate (MTA) and Biodentine.[3,4] These materials offer excellent sealing and biocompatibility, but present drawbacks, including a prolonged setting time, high cost, nondegradable, poor adhesion with resin, and the potential to cause tooth discolouration.[2,4,5,6]

Current developments in regenerative endodontics emphasise biomaterials capable of not only releasing calcium ions but also modulating cellular signalling. Calcium phosphate cement (CPC) serves as a source of calcium ions, promoting the formation of hydroxyapatite, while calcium sulfate hemihydrate (CSH) enhances setting time, handling, and biodegradability.[7,8,9,10] Yet, these materials do not fully support cellular regeneration and the formation of a dentine bridge.[5]

Acemannan, a β-(1,4)-acetylated polymannose extracted from Aloe vera, has shown promise in regenerative and immunomodulatory properties, promoting fibroblast proliferation, anti-inflammatory properties, and enhancing the release of cytokines and growth factors contributing to dentine bridge formation.[11,12] Its integration into CPC–CSH (CPC–CSH–Ace) could provide synergistic benefits, resulting in enhanced bioactivity that improves the viability of human dental pulp stem cells (hDPSCs) and transforming growth factor-β1 (TGF-β1) secretion, which is crucial for odontoblastic differentiation and tertiary dentine formation.[13,14]

This study investigates the biological activity of CPC–CSH–Ace by evaluating pH, hDPSCs viability, and TGF-β1 secretion to identify the optimal acemannan concentration for VPT.

SUBJECTS AND METHODS

Study design

This in vitro study was performed at PT ProSTEM (Prodia StemCell) Indonesia with approval by the Institutional Ethical Committee, Universitas Trisakti (Ref. No. 903/S2-Sp/KEPK/FKG/5/2025) and adhered to the Declaration of Helsinki guidelines.

Materials

The materials used in this study included [Table 1]:

Table 1.

Materials used in the study

Materials	Composition	Manufacturer	Setting time
CPC-CSH- acemannan (%)
3	CPC, CSH, acemannan	-	Initial: 11 min
5
10
EndoCem MTA Premixed Regular®	Tricalcium silicate, dodecacalcium heptaaluminate, dimethyl sulfoxide, hydroxypropyl methylcellulose, calcium sulfate, zirconium oxide, lithium carbonate, silicon dioxide	Maruchi, Wonju, Korea	Initial: 4–5 min Final: 12 h
NeoPutty®	Tantalite, tricalcium silicate, calcium aluminate, dicalcium silicate, tricalcium aluminate, calcium sulfate, proprietary organic liquid, stabilizers	NuSmile Ltd (Avalon biomed), Houston, USA	Initial: 4 h Final: 24 h

CPC: Calcium phosphate cement, CSH: Calcium sulfate hemihydrate, MTA: Mineral trioxide aggregate

• CPC powder (Merck, Germany)

• CSH powder (Jinan Future Chemical Co., Ltd., China)

• Acemannan powder obtained from Xi’an Norson Biotech Co., Ltd., (China), with impurity content <0.3%.

Acemannan was supplied as a powder and used as received, without further purification. The CPC and CSH powders were mixed in a 7:3 weight ratio to prepare the base bioceramic composite (CPC–CSH). Acemannan powder was then incorporated into this blend to form three experimental groups containing 3%, 5%, and 10% acemannan (w/w), designated CPC–CSH–Ace 3%, CPC–CSH–Ace 5%, and CPC–CSH–Ace 10%, respectively.

Two commercially available premixed calcium silicate-based materials served as controls [Table 1]:

• NeoPutty (Avalon Biomed, USA) – premixed bioceramic putty

• EndoCem MTA Premixed Regular (Maruchi, Korea) – pozzolan-based premixed injectable bioceramic cement.

Preparation of extracts

Extracts of each material were prepared following ISO 10993-5 guidelines for cytotoxicity testing. Materials were incubated at 37°C, 5% CO₂, and 95% humidity for 48 h. After incubation, each material was finely ground in a sterile mortar and pestle to achieve uniform particle size and surface exposure. Subsequently, each material (50 mg) was suspended in 1 mL of sterile saline (OneMed Saline Wound Irrigation NaCl 0,9%, Indonesia). The suspensions were then centrifuged at ×4000 g at 25°C for 10 min, and the supernatant was collected and filtered through a 0.22 μm pore-size sterile filter (Sartorius AG, Goettingen, Germany).

pH measurement

Each material was immersed in 10 mL of distilled water, and pH was measured at 3, 24, and 72 h using a calibrated pH meter (Milwaukee MW150 Max, USA).

MTT assay for cell viability

hDPSCs were seeded in 96-well plates (4 × 10³ cells/cm³) and treated with material extracts for 24 and 72 h. Cells from the 7^th passage were used for all assays. 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) reagent (5 mg/mL) was added and incubated for 4 h at 37°C, 5% CO2, and 95% humidity, followed by solubilisation with 100 µL dimethyl sulfoxide. Absorbance was measured at 570 nm using a microplate reader (Stat Fax 2100, Palm City, FL). Cell viability (%) was calculated at 24 and 72 h relative to the control.

Enzyme-linked immunosorbent assay for transforming growth factor beta-1

After exposure to material extracts for 7 days, culture supernatants were collected, centrifuged, and analysed using a TGF-β1 Enzyme-Linked Immunosorbent Assay kit (Elabscience®, USA) following the manufacturer’s protocol.

Statistical analysis

Data normality and homogeneity were verified using Shapiro–Wilk and Levene’s tests. One-way analysis of variance followed by Tukey’s post-hoc test (P < 0.05). Statistical analysis was performed using SPSS v26.0 (IBM Corp., New York, USA).

RESULTS

Cell viability (%)

At 24 h, CPC–CSH–Ace 3% exhibited the highest viability (126.60% ±14%), followed by EndoCem MTA (88.23% ± 4%), CPC–CSH–Ace 5% (87.87% ±3%), and 10% (83.94% ± 3%). NeoPutty showed significantly lower cell viability (79.79% ± 5%) compared to other groups (P < 0.05). At 72 h, CPC–CSH–Ace 3% maintained the highest proliferation rate (258.6% ± 33%), remaining significantly higher than all other groups at both 24 and 72 h (P < 0.05) [Figure 1].

Figure 1.

Mean percentage of human dental pulp stem cells (hDPSCs) viability (%) after exposure to each material extract at 24 and 72 h, and quantitative analysis of transforming growth factor beta-1 (pg/mL) secreted by hDPSCs after 7 days of exposure to material extracts

Transforming growth factor beta-1 cytokine release

Seven days of exposure revealed that CPC–CSH–Ace 3% induced the highest TGF-β1 secretion (1.64 ± 0.16 pg/mL), followed by CPC–CSH–Ace 5% (0.30 ± 0.05 pg/mL), 10% (0.16 ± 0.03 pg/mL), EndoCem MTA (0.22 ± 0.03 pg/mL), and NeoPutty (0.17 ± 0.07 pg/mL). CPC–CSH–Ace 3% had significantly higher TGF-β1 cytokine release compared to all other groups (P < 0.05) [Figure 1].

pH profile

All CPC–CSH–Ace formulations demonstrated an alkaline pH conducive to pulp cell proliferation. The CPC–CSH–Ace 3% group showed stable alkalinity (7.47 at 3 h; 8.85 at 24 h; 7.05 at 72 h). The 5%, 10%, and EndoCem MTA groups displayed similar values (7–8.3), whereas NeoPutty recorded significantly higher pH (11.47 at 3 h and gradually stabilised at 72 h) [Figure 2].

Figure 2.

Comparative pH profiles of Calcium phosphate cement–calcium sulfate hemihydrate–Ace (3%, 5%, and 10%) formulations, NeoPutty, and EndoCem MTA at 3, 24, and 72 h

DISCUSSION

The findings of this study demonstrate that incorporating acemannan into calcium phosphate–calcium sulfate (CPC–CSH) bioceramic enhances its biological response on hDPSCs, with the 3% concentration exhibiting the most favourable outcomes in terms of cell viability, TGF-β1 release, and pH stability. Acemannan possesses immunomodulatory and regenerative properties by promoting fibroblast proliferation, macrophage activation, and the secretion of growth factors, including bone morphogenic protein (BMP)-2, BMP-4, vascular endothelial growth factors, alkaline phosphatase (ALP), and TGF-β1.[15] Its incorporation into CPC–CSH may enhance the biological potential of the material by modulating growth factor activity within the microenvironment, leading to improved odontoblastic activity and mineralisation.

The CPC–CSH–Ace 3% group demonstrated significantly higher viability of hDPSCs and TGF-β1 secretion than other formulations, indicating that 3% acemannan represents the optimal concentration for biological performance. This observation supports the biphasic dose–response concept, in which low concentrations of a bioactive compound stimulate cell proliferation and differentiation. In contrast, higher concentrations may induce oxidative stress, thus lowering cell viability and inhibiting cell function.[16] The CPC component provides a continuous calcium source that supports hydroxyapatite formation and odontoblastic differentiation, whereas CSH improves setting time, handling, biodegradation, and bioactivity of the cement.[7,8,9] The synergistic combination of CPC, CSH, and 3% acemannan therefore provides a balance between structural integrity and biofunctional stimulation.

The pH profile of the CPC–CSH–Ace formulations remained within a mildly alkaline range (7.05–8.85) throughout the observation period, creating an environment favourable for mitochondrial activity and cell function.[17] Such alkalinity also provides antibacterial effects while avoiding cytotoxic stress.[17,18] In contrast, NeoPutty showed the highest initial pH (11.4), which contributed to reduced cell proliferation, consistent with prior reports linking extreme alkalinity to inhibition of mitochondrial respiration and reduced cell viability.[17] EndoCem MTA, along with CPC–CSH–Ace 5% and 10%, demonstrated moderate biological activity and maintained pH values between 7.0 and 8.3, indicating that overly alkaline and overly modified matrices may compromise pulpal cell responses.

TGF-β1 is a crucial cytokine involved in odontoblastic differentiation and reparative dentine formation.[13,14] Its upregulation in the CPC–CSH–Ace 3% group indicates effective stimulation of cellular differentiation and regenerative signalling. Previous studies have shown that 1 ng/mL of TGF-β1 can increase ALP for 14 days, contributing to matrix secretion and maturation. However, excessively elevated TGF-β1 levels (>5 ng/mL) have been reported to inhibit mineralisation and excessive suppression of odontogenic markers such as ALP, dentine sialophosphoprotein, and dentine matrix protein-1 during odontoblast differentiation.[19] The moderate yet significantly increased TGF-β1 expression observed in the 3% group may therefore represent an ideal biological range that promotes controlled regeneration rather than pathological overexpression.

Another important advantage of CPC-CSH-Ace lies in its favourable setting characteristics. The incorporation of CSH notably accelerates the initial setting reaction of CPC, resulting in an ideal working and setting time for clinical procedures such as direct pulp capping. The setting time is a critical factor in endodontic procedures, as materials with prolonged setting durations are more susceptible to washout.[20,21] Excessively soluble materials that disintegrate easily may expose the pulp to bacterial contamination, intensify inflammatory reactions, and negatively influence pulpal healing.[22] Furthermore, environmental conditions can significantly affect the performance of pulp-capping materials. Traditional materials, such as MTA, are more susceptible to contamination by heavy metals due to their prolonged setting time. Studies have also reported that MTA may release trace amounts of lead, arsenic, and chromium ions when immersed in deionised water or physiological solutions.[20] In this study, the CPC–CSH–Ace material exhibited an initial setting time of approximately 11 min, which is considerably faster than conventional silicate-based materials such as NeoPutty and comparable to EndoCem MTA. A setting duration within the range of 10–15 min is regarded as ideal for pulp-capping applications, providing sufficient working time while ensuring early material stability.[23] This setting behaviour allows sufficient working time for manipulation while ensuring early material hardening, thereby reducing the risk of microleakage or material displacement under clinical conditions.

Collectively, the results suggest that CPC–CSH–Ace 3% provides an optimal physicochemical and biological balance for VPT by maintaining a stable alkaline pH, great setting time, supporting the proliferation of hDPSCs, and inducing the regulated release of the cytokine TGF-β1. Previous studies have also reported that 3% and 5% concentrations of Aloe vera significantly increased the bond strength of dual-cure resin cement to root dentine, owing to their ability to rehydrate the dentine substrate, prevent collagen fibre collapse, and promote hybrid layer formation.[24] This formulation could therefore be a promising candidate for regenerative endodontic use. Nevertheless, as this study was conducted under in vitro conditions, further in vivo and clinical studies are needed to confirm its long-term effects on dentine bridge formation, inflammatory modulation, pulpal healing under physiological conditions, and provide a favourable substrate for restorative adhesion following VPT.

CONCLUSIONS

CPC–CSH–Ace 3% demonstrated the most favourable biological characteristics, achieving the highest hDPSC viability, stable alkaline pH, and enhanced TGF-β1 secretion compared with higher acemannan concentrations and commercial bioceramics. The formulation provides a promising bioactive material for VPT and regenerative endodontic applications.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.