Potential Combination of Acemannan Sponge and Calcium Phosphate Cement-Calcium Sulfate Hemihydrate (CPC-CSH) as Direct Pulp Capping Alternative Materials for Reparative Dentin Formation

Abstract Background  Health problem that affects hard tissues of the teeth is dental caries, which is experienced by around 2.3 billion people in the world, with prevalence in Indonesia reaching 88.8. Direct pulp capping (DPC) is a vital pulp therapy used to maintain pulp vitality. Calcium hydroxide (Ca(OH) 2 ) is the gold standard pulp capping material, but has poor adhesion to dentin and mechanical properties, bacterial infiltration, formation of tunnel defects in the pulp resorption, and dentin bridge. Therefore, it is necessary to develop alternative therapy, namely, a combination of acemannan sponge and calcium phosphate cement (CPC)-calcium sulfate hemihydrate (CSH) for reparative dentin formation. Purpose  This article describes the potential combination of acemannan sponge and CPC-CSH as DPC alternative materials for reparative dentin formation. Reviews  Acemannan extracted from aloe vera exhibits anti-inflammatory, antimicrobial, and cytocompatibility properties. As DPC material, acemannan induces pulp proliferation and differentiation to osteoblast-like, growth factor synthesis, and promotes reparative dentin formation. However, acemannan sponge is radiolucent, allowing misinterpretation between pulp and acemannan sponge. CPC can be combined with CSH to shorten the setting time. CPC-CSH is radiopaque, has good compressive strength, and biocompatibility. CPC-CSH is a calcium-based material with neutral pH that can induce the dentin bridge formation. CPC-CSH showed good bioactivity because it induces the formation of apatite which plays a significant part in dentin and pulp regeneration. Conclusion  The combination of acemannan sponge and CPC-CSH has the potential as DPC alternative materials for reparative dentin formation.


Introduction
One of the main dental and oral health problems in many developing countries, such as Indonesia, is dental caries.The incidence of caries worldwide is 2.3 billion people with permanent teeth caries and 530 million children with primary teeth caries. 1 Caries prevalence in Indonesia is 88.8%. 2 Dental caries is a disease characterized by damage to the hard tissues of the teeth due to dissolution of organic components and demineralization of inorganic components caused by bacteria. 3Severe caries causes exposure of the dental pulp thereby affecting the vitality of the pulp.Vital pulp therapy has a purpose to maintain tooth vitality that is indicated for primary teeth with apical foramina that are still wide open. 4,5 dental treatment known as vital pulp therapy which removes infected pulp tissue and cleans the root canals, 6 based on case studies the success rate of this treatment is 73 to 99%. 7 Direct pulp capping (DPC) is a noninvasive vital pulp therapy that involves the application of drugs, dressings, or dental materials, used to support the formation of reparative dentin, maintain vitality, and seal exposed pulp. 8or decades, the gold standard for pulp capping materials has been calcium hydroxide [Ca(OH) 2 ].This material still has weaknesses such as adhesion to dentin restorations, bacterial infiltration, poor mechanical properties, formation of tunnel defects in the dentinal pulp resorption and bridge, and dissolution over time. 9This material still cannot prevent microleakage in the long term because it has poor adhesive qualities.Mineral trioxide aggregate (MTA) is a dental material that is also frequently used for pulp capping, but this material has the disadvantage of being easily broken during treatment and long setting time. 6herefore, it is necessary to develop alternative therapies to stimulate the reparative dentin formation to maintain the teeth vitality with exposed pulp.Currently, alternative pulp capping materials are being developed using natural ingredients, for example, acemannan.Acemannan is an active compound derived from aloe vera in the form of natural polysaccharides, which stimulates cell proliferation, osteogenic, immunomodulating, antimicrobial, synthesis of extracellular matrix, and increases growth factor expression.Acemannan material still has a weakness in the form of radiolucent properties, so it is necessary to combine it with radiopaque inorganic materials to prevent misinterpretation of radiographic images as empty cavities or secondary caries. 4Calcium phosphate cement (CPC) is a radiopaque material that is commonly used for hard tissue repair in the dental and medical fields because it is biocompatible, non-exothermic, and has a paste-like consistency so that it can enter various cavities or defects. 10he addition of calcium sulfate hemihydrate (CSH) material can shorten the setting time and is not toxic or inhibits cell proliferation, so it has the potential to be an alternative material of DPC therapy. 6

Methods and Materials
The search for literature reviews was conducted using the databases ScienceDirect, PubMed, and Google Scholar.The search was limited to articles focusing on acemannan and CPC-CSH in DPC for reparative dentine formation.The selected literature was published between 2016 and 2022 in English and Indonesian.The selected literature was accessed in full text by selecting the article type in the research and review form.Articles were selected and reviewed according to the inclusion criteria.Then, the selected articles were reviewed and summarized by the author from the relevant previous research results, so the author can make an article based on the study results.

Reviews Dental Caries
Dental caries is a pathological process that causes damage to the hard tissues of the teeth, as well as local and general complications. 11Caries is a multifactorial disease that is influenced by time, bacteria, and fermentable carbohydrates.Colonies of bacteria on teeth form biofilms through autoaggregation and coaggregation so that they have different morphological structures.The biofilm formed is supported by changes of microenvironment from aerobic to facultative anaerobic.In addition, the main cause of dental caries is a sticky substance that adheres to the tooth surface as a complex biofilm called dental plaque. 12The microorganism involved in caries initiation is Streptococcus mutans. 13aries is caused by the byproducts or acids of the fermentation of carbohydrates by bacteria in the mouth. 4S. mutans acts as enamel caries initiation through producing lactic acid as a result of fermentation, which then causes demineralization of the enamel. 12S. mutans initiates and sustains microbial growth, produces acid at low pH, metabolizes sugars into lactic acid, and also produces other organic acids that cause demineralization quickly than other plaque bacteria.S. mutans changes the environment into conditions rich in extracellular polysaccharides and low pH which creates a favorable acidogenic and aciduric environment bacteria so that it can assist other bacteria to establish dental caries. 14

Direct Pulp Capping
Maintaining pulp vitality and supporting the formation of dentinal bridges is the goal of vital pulp therapy. 15One of the alternative therapies to avoid root canal treatment by covering the exposed pulp with a dressing is DPC. 16DPC is a mechanical treatment using biomaterials that aims to maintain the exposed dental pulp to form reparative dentin. 17ndications for DPC treatment are: (1) pulp is still vital, (2) absence of clinical signs or symptoms indicating nonvital teeth such as abscess, spontaneous pain, and periodontal tissue swelling, (3) a deep carious lesion, (4) adequate structure of teeth for restoration, and (5) radiolucency absence in the periapical and furcation region, and root resorption. 18In addition, pulp capping material must be able to stimulate the formation of reparative dentin, maintain pulp vitality, has antibacterial properties agent, be adhesive between restorative materials and dentin, be sterile, be radiopaque, and be resistant to pressure during restoration placement and throughout the restoration period. 19a(OH) 2 has been the gold standard for pulp capping materials for decades. 20Widely, Ca(OH) 2 is used as an endodontic treatment because it has the ability to heal tissue.Ca(OH) 2 has the advantage of having excellent pH and antibacterial properties. 21The alkaline pH of Ca(OH) 2 is the reason for good antibacterial properties and plays a significant part in the initiation of tooth remineralization.High hydroxyl ions released from Ca(OH) 2 can kill microorganisms that cause inflammation by denaturing proteins and hydrolyzing lipopolysaccharides (LPS) so that bacterial cell walls are damaged and the bacteria die.The OH-ions in Ca(OH) 2 cause a low probability of bacteria survival, while Ca 2þ ions can stimulate the formation of dentine bridges and maintain pulp vitality.Ca(OH) 2 has several disadvantages, including weaknesses, formation of tunnel defects in the dentine bridge, having high solubility, the pulp surface becomes inflamed and necrotic after application, being degraded over time, and low mechanical resistance, which can lead to failed treatment and microfiltration. 15TA is a material that is often used in endodontic treatment.MTA is a powder containing trioxide and hydrophilic particles.MTA has a composition of 75% Portland cement, and 20% bismuth oxide, which provides radiopacity.MTA has conductive activity in calcified tissue and helps the mineralization process in dental pulp cells.That means MTA has potential as a material for pulp capping.MTA is proven to be biocompatible and has excellent potential when used in endodontic treatment.The advantages of MTA are that it has biocompatibility, bioactivity, and can promote the formation of mineralized tissue.MTA has many advantages over Ca (OH) 2 , among others, it can facilitate pulp cell proliferation, limited tissue necrosis, and stimulate the formation of a dentine bridge quickly. 5

Reparative Dentin
Dentin is the hard tissue structure of the teeth that lies beneath the enamel, is slightly yellowish in color, and makes up the bulk of the tooth.Odontoblasts can produce dentin after going through the differentiation process of dental papillary cells.Meanwhile, the pulp is a tooth structure that includes soft connective tissue in the blood vessels and nerves in the dental cavity which is located under the dentin. 22Dentin chemically has a protein matrix composition and pathways that regulate the differentiation of odontoblasts and osteoblasts.Dentine morphologically does not have the ability to undergo remodeling, whereas dental pulp can differentiate into odontoblasts and osteoblasts, consisting of fibroblasts, endothelial cells, and progenitor cells.Damaged dentin and pulp due to injury induces an odontoblast and pulp response through the process of differentiating odontoblasts from progenitor cells in the dental pulp, and then secreting osteodentin reparative dentin matrix. 23parative dentin is a tertiary dentin matrix secreted by odontoblast-like cells to response a strong stimuli following the death of postmitotic odontoblasts responsible for primary or secondary dentin secretion due to trauma or caries. 23he incidence of caries has passed through the enamel layer and caused the exposed dentin to respond to the formation of reparative dentin on the pulp surface just below the exposed dentin.The reparative dentin formation aims to prevent the pulp from being exposed to harmful injury. 24The reparative dentin response occurs on the exposed side of the pulp due to missing odontoblasts, in this area the formation of dentin bridges also occurs.Formation of reparative dentin, which covers the pulp exposure, results in a bone-like structure made of collagen and noncollagenous proteins (osteocalcin and osteopontin).The composite structure of reparative dentine is called osteodentin. 5

Acemannan
Aloe vera is a herbal medicinal ingredient for the treatment or prevention of metabolic, skin, cancer, and cardiovascular diseases. 25Acemannan is a polymannose acetate molecule B-(1-4) produced from aloe vera gel extract. 26The main components of the acemannan molecule are mannose (57-77%), glucose (15-22%), and galactose (5-7%), forming a chain of repeating tetrasaccharide units o-(acetyl mannose)-o-(acetyl mannose)-o -(glucose)-o-(acetyl mannose) of four mannose residues in single-branched galactose on the second or fourth. 25cemannan material has been widely used for pharmacology from biology to medicine and industry, such as for the treatment of oral, metabolic, cardiovascular, and tumor diseases.Recent research on acemannan focused on dentistry, especially in wound healing in oral infections. 27Acemannan has good immunomodulating and antimicrobial properties as well as cytocompatibility and biocompatibility with various cell types. 28In vivo tests have shown that acemannan accelerates the formation of reparative dentin and oral wound healing.In addition, in vivo tests showed acemannan as a wound healing agent caused increased bone mineral density and faster wound healing compared with wounds that were not treated with acemannan.In vitro test showed acemannan caused bone marrow-derived mesenchymal stem/stromal cell (BMSC) proliferation, sialoprotein, vascular endothelial growth factor, alkaline phosphatase activity, BMP-2 and osteopontin expression, and increased mineralization. 29It is shown that acemannan stimulates the formation of dentin bridges and stimulates the formation of dental pulp tissue when DPC treatment was applied on rat molars. 30Based on these studies, the acemannan molecule functions as a bioactive molecule that stimulates differentiation into osteoblasts, extracellular matrix synthesis, and BMSC proliferation thereby inducing bone formation. 29

Calcium Phosphate Cement-Calcium Sulfate Hemihydrate
CPC is a bioactive material used in the medical and dental fields for hard tissue engineering because it has a chemical structure similar to bone and teeth. 31The main components of this material are dicalcium phosphate anhydrous (DCPA) and tetracalcium phosphate (TTCP). 9CPC material is biocompatible, non-exothermic, and has a paste-like consistency.The combination of calcium phosphate powder with water or solution will undergo a setting reaction through a hydrolysis process between components, followed by dissolution and re-precipitation at room temperature or body temperature. 10,31,32This process may benefit from CPC's molding ability, or it can enter cavities or defects with a fit. 33ydroxyapatite is the CPC application's final product, which is the main mineral in dentin that helps form the dentinal bridge barrier. 9SH is an inorganic component that is widely used in dentistry for repair of root perforations, treatment of periradicular lesions, repair of periodontal defects, and as a membrane barrier.This material is nontoxic, inexpensive, and biocompatible with bone and gingival tissue.This material is able to release calcium ions which can increase the activity of osteoblasts.Based on the research of Ulusoy et al, CSH as a material for DPC showed a fairly high success rate such as Ca(OH) 2 material. 34,35The combination of CPC-CSH can shorten setting time, maintain compressive strength, and has good handling properties. 6

Discussion
One of the treatments for exposed pulp due to caries, mechanical and chemical trauma, or mechanical excavation is root canal treatment or vital pulp therapy.However, for teeth condition with a wide-open apical foramen (immature permanent teeth) due to the incomplete root formation process, it is not appropriate to treat them using root canal treatment.Root formation does not occur without vital pulp because tooth eruption stops, the roots are thin and not strong enough to withstand masticatory forces, so the tooth will break easily.Therefore, one of the treatments to maintain pulp vitality as a substitute for root canal treatment is DPC. 4 Invasive complex treatments such as endodontics or extractions can be prevented if DPC is successful. 36DPC is indicated for teeth with vital pulp tissue without residual pain due to pulp exposure or trauma with a width of less than 1.5 mm, deep dental caries lesion, carious lesions located near the pulp to the lamina dura, and the furcation and periphery areas with no radiolucency, pulp obliteration chamber and root canals, or the occurrence of internal or external root resorption. 17,18he ideal material for DPC should have bactericidal properties, be nonhazardous to the pulp and its structure, stimulate formation of reparative dentine, and does not interfere with the physiological process of root resorption by osteoclasts. 5The process by which dental pulp stem cells (DPSCs) differentiate into odontoblast-like cells will be the beginning of the formation of reparative dentin structures when the pulp is exposed and the existing odontoblast layer is damaged.DPSCs migrate and proliferate in the materio-pulpal complex, this event undergoes a process of reparative dentin formation resulting from odontogenic differentiation. 37 recent decades, the popular material for pulp capping treatment in dental restoration is Ca(OH) 2 .Despite being the gold standard, Ca(OH) 2 has several disadvantages especially the formation of tunnel defects in the dentinal bridge.This can lead to failure of microfiltration and restoration treatment.MTA is a widely used pulp capping material and provides excellent result in in vitro and clinical tests. 15In a systematic review, MTA had higher efficacy than Ca(OH) 2 , showed more predictable dentine bridge formation than Ca (OH) 2 , and less pulp inflammation. 38In a previous studies, MTA as dentin regeneration material for dentin regeneration as a treatment material for primary and permanent teeth whose pulp chambers have not been completely closed.However, this material still has some drawbacks that need to be considered, such as being expensive, there is a discoloration of the teeth after treatment, and a long setting time of approximately 2 to 3 hours 6 (►Fig.1).
Because of its anti-inflammatory and antibacterial qualities, acemannan is recommended as an efficient and effective DPC agent.Acemannan exhibits good biocompatibility and induces reparative dentin formation.In a study by Elhag et al, acemannan as a material for direct pup capping the success rate on mandibular permanent first molars with carious lesions was 77.8%, while in human primary teeth it was 72.73% after 6 months of observation.When applied as a DPC agent, acemannan material will induce the growth of dental pulp cells to stimulate dental pulp cells differentiation into growth factor synthesis and osteoblast-like cells of the extracellular matrix, as well as the formation of new dentin and mineral deposition, according to Elhag et al 28 in vitro studies. 28Gonna et al evaluated the histological impacts of acemannan and formocresol as a material for pulp-dressing treatment in primary teeth undergoing pulpotomy treatment.The acemannan and formocresol groups had effectiveness of 75 and 25%. 39In vivo investigation showed acemannan induces pulp repair and reparative dentin formation in caries-exposed canines with LPS-induced reversible pulpitis, as well as reversible pulpitis in human primary teeth.Acemannan application, like MTA application, produced a dentine bridge with normal supporting pulp tissue and no pulp inflammation or necrosis 4 (►Fig.2).
As a result, acemannan has the potential to be a useful capping material to stimulate the formation of economical and efficient reparative dentin.On the other hand, acemannan sponges are radiolucent due to their monosaccharide composition.According to cone beam computed tomography pictures, the dentine bridge covering the dental pulp was found in the radiolucent zone between the acemannan sponge material and the dental pulp.To avoid any misunderstanding as empty cavities or secondary caries, acemannan must be combined with other materials, namely calcium phosphate in an acemannan sponge, to create a radiopaque hybrid organic-inorganic picture that cannot be misinterpreted. 4PC is an inorganic material that is radiopaque and biocompatible because its chemical structure is similar to that of teeth. 10The addition of CSH aims to shorten cement setting time.Initial setting time of cement consisting of CPC-CSH is short, approximately 15 to 20 minutes, sufficient time for the doctor to apply the material over the exposed pulp without applying pressure. 6CPC-CSH biphasic cement had a pH of 6.5 to 6.9, which was near to neutral.Several investigations have shown that significant alkaline nature of a material is not necessary to promote dentine bridge formation, and that neutral calcium-based materials can induce dentine bridge formation in non-necrotic locations. 9n addition to a pH that supports CPC-CSH as a pulp capping material, CPC-CSH also shows satisfactory bioactivity in simulated body fluid (SBF).The CPC-CSH cement releases calcium ions into the SBF, which causes the concentration of calcium ions to become saturated.Calcium ions react with phosphate ions at higher concentrations, causing apatite to develop on the cement surface.After 28 days of hydration, morphology of the cement submerged in SBF revealed that CPC-CSH formed a significant amount of hydroxyapatite.Hydroxyapatite plays a vital function in dentin production and pulp regeneration due to the formation of a bioactive surface.Hydroxyapatite supports the differentiation and growth of odontoblastic process in human dental pulp cells (HDPCs). 9Based on previous studies, CPC-CSH cement showed minimal toxicity to HDPC. 6,9cemannan was isolated from a homogenized, centrifuged, and alcohol-precipitated aloe vera gel.To remove protein and monosaccharide molecules, acemannan was inserted into a 10,000 MWCO (Thermo Scientific-Pierce Biotechnology) semipermeable dialysis tube for 24 hours and then lyophilized.Gas chromatography-mass spectrometry and 13C nuclear magnetic resonance were used to examine monosaccharide composition and polysaccharide structure, with acemannan being the polysaccharide isolated from aloe vera gel.The extraction yield of acemannan is approximately 0.2%, then the acemannan is dissolved and sterilized by autoclaving.Thereafter, an acemannan sponge was made by freezing and lyophilizing the acemannan solution aseptically. 29CPA and TTCP are the components of CPC.TTCP powder [Ca 4 (PO 4 ) 2 O] is made by heating 2 moles of calcium carbonate (CaCO 3 ) and 1 mol of calcium pyrophosphate (Ca 2 P 2 O 7 ) to 1400°C for 3 hours in a solid-state process.The product was allowed to cool to room temperature before being pulverized in a mortar and sieved to obtain TTCP powder, which was then combined with DCPA powder (CaHPO 4 ) to form CPC. CPC has a low viscosity and handling properties increase when CSH is added.Mixing between CPC-CSH is done by adding deionized water with a liquid-to-powder (L/P) ratio of 0.35 mL/g to form a paste-shaped mixture. 9The combination of CPC and CSH was made with a ratio of 70% CPC and 30% CSH.The addition of CSH can shorten setting time and improve handling properties, but reduce compressive strength.This comparison is used to get the right setting Fig. 1 Radiographic features before and after direct pulp capping with mineral trioxide aggregate (MTA). 4ime and optimal compressive strength for clinical use. 6herefore, CPC-CSH has the potential to have a synergistic effect with the addition of acemannan sponge as a DPC material in the production of reparative dentin and the preservation of pulp vitality.

Conclusion
The combination of acemannan sponge and CPC-CSH has the potential as DPC alternative materials for reparative dentin formation.

Clinical Relevance
The use of alternative material on direct pulp capping treatment is recommended to improve the performance of dentin reparative formation.The use of combination between acemannan sponge and calcium phosphate cementcalcium sulfate hemihydrate (CPC-CSH) is effective for dentine reparative formation to maintain pulp vitality.Fig. 2 Radiographic appearance of dentine bridge formation and continued root formation after application of acemannan to teeth. 4