Physical properties and cytotoxicity comparison of experimental gypsum-based biomaterials with two current dental cement materials on L929 fibroblast cells.

AIM: To evaluate physical properties and cytotoxicity of pure gypsum-based (pure-GYP) and experimental gypsum-based biomaterials mixed with polyacrylic acid (Gyp-PA). The results were compared with calcium hydroxide (CH) and glass ionomer cement (GIC) for application as base/liner materials.
MATERIALS AND METHODS: Vicat's needle was used to measure the setting time and solubility (%) was determined by percentage of weight loss of the materials following immersion in distilled water. For cytotoxicity test, eluates of different concentrations of materials were obtained and pipetted onto L-929 mouse fibroblast cultures and incubated for 3 days. Cellular viability was assessed using Dimethylthiazol diphenyltetrazolium bromide test to determine the cytotoxicity level. Statistical significance was determined by one-way analysis of variance followed by post hoc test (P < 0.05).
RESULTS: Setting time was significantly higher for pure-GYP and Gyp-PA; solubility test showed a similar tendency (pure-Gyp > Gyp-PA > CH = GIC). The pure-Gyp was found as the least cytotoxic materials at different concentrations. At 100 mg/mL dilutions of materials in growth medium highest cytotoxicity was observed with CH group.
CONCLUSION: Cytotoxic effect was not observed with pure-Gyp; application of this novel biomaterial on deeper dentin/an exposed pulp and possibility of gradual replacement of this biodegradable material by dentin like structure would be highly promising.

INTRODUCTION

In dentistry, pulp capping materials are used to protect exposed/near to be exposed vital pulp by capping the pulp tissue and it is expected that odontoblast cells would response to form tertiary/reparative dentin on the exposed site to protect the vitality of the pulp. It is needless to mention that the vital pulp tissue is the best root canal filling component; thus, protection and preservation of its vitality in different types of exposed condition is desirable but at the same time is very challenging. In this regard, calcium hydroxide (CH) and mineral trioxide aggregate (MTA) are widely used materials with distinct advantages and disadvantages.[12] CH has antibacterial property; however, it causes necrosis and inflammation when in contact with pulp tissue.[3] In addition, the handling properties are also less than ideal.[4] MTA has been reported to cause little inflammation and supports odontogenesis, resulting in more efficient pulp tissue regeneration.[3] Although MTA has superior biocompatibility when compared to the traditional materials used in root-end filling and root repair, it is a costly material and has poor handling characteristics.[5]

Gypsum (CaSO4.2H2O) is used in dentistry for decades. It is a cheap and easily available material which has been established as biomaterial for application at different sites of mineralized tissue and is being introduced in new fields of indications. Over the past 3 decades, the osteoconductive and biocompatible properties of the bioresorbable salt calcium sulfate (CaSO4) has been well-documented in the orthopedic literatures.[6789] Furthermore, the osteoconductive properties of CaSO4 -based materials have been successfully used in the augmentation of bone voids, resulting in complete cement resorption and replacement with host bone within 10 to 12 weeks.[10] Despite the advantage of bioresorption of gypsum that makes it an attractive candidate for certain applications, its relatively low mechanical properties have limited its scope of application as a bone replacement implant or even as bone cement. Different classes of materials were mixed with gypsum in order to improve its mechanical properties.[11] However, this potential gypsum-based biomaterials had never been evaluated/compared with commonly used dental lining and/base materials for application in conservative dentistry. In the present study, pure gypsum (pure-GYP) was mixed with liquid part of glass ionomer [polyacrylic acid (PA)] to improve its physical properties.

The aim of this preliminary study was to evaluate physical properties and cytotoxicity of gypsum-based biomaterials for application at deep cavity or on exposed pulp as base/liner materials. Physical properties and cytotoxic effects of the most commonly used CH for pulp capping materials and GIC indicated as base materials had also been compared.

MATERIALS AND METHODS

Preparation of materials and setting time measurement

Pure α-hemihydrate gypsum [(pure-GYP), Noritake, Thailand], CH, Dycal, Dentsply, USA) and GIC (GC, JaChemical">pan) materials were mixed according to the manufacturers instruction. Gypsum-based polyacrylic material (Gyp-PA) were prepared by mixing pure-GYP and PA liquid to a proportion of 5:1 (by weight), respectively, using a weighing machine (E. Mettler, Zurich, Switzerland) of an accuracy of 0.1 g. The above ratio was chosen after a pilot study revealed that it had better handling properties. The list of materials used and their mixing ratios are listed in Table 1. Mixing of each specimen was carried at an ambient room temperature of 23°C and 60 ± 5% relative humidity.

Table 1

Materials used in the study

The Vicat's needle penetration test according to ISO-9694: 1996[12] was performed to measure the setting time; the diameter and load of the Vicat's needle were 2 mm and 3 N. The freshly mixed cement material was poured in to a plastic mould (diameter 15 mm, height 10 mm). Setting time was determined as the total time from the start of mixing to the time when the needle failed to make a depth of more than 1 mm (marked at the indenter) on the surface of the cement. Five samples of each test group were measured.

Solubility/disintegration

A thin layer of Vaseline was coated in to the inner side of the moulds (similar dimension for setting time measurements) to facilitate removal of specimens. The mixed cement materials were loaded into the plastic mould; before that a dental floss was placed in such a way so that approximately 6 cm of the floss past through the mould after being attached with the poured material on the other side. The mold assembly was then placed in an incubator at 37 ± 2°C and 90 ± 5% relative humidity for 1 h. Two specimens were suspended in 50 mL of distilled water container ensuring they were not in contact with each other. The container was then placed in a humidified incubator at 37 ± 2°C and 90 ± 5% relative humidity for 24 h. At 1 day following soaking of the specimens, cement discs were rinsed briefly with deionized water, gently blotted dry, weighed to an accuracy of ±0.1 mg, then placed in a desiccator with silica gel and weighed daily. This procedure was repeated until the weight change from one measurement to the next was less than 0.001 g. The percentage weight loss or solubility was calculated as below:

M1 = weight of disc 1 h after the start of mixing

M2 = weight of disc after soaking in water for 24 h and desiccation

The measurements were repeated for 5 times for each of the four material groups.

Cytotoxicity test

Cell cultures preparation

In this study, the cryopreserved L929 cell had been thawed and centrifuged. The dimethyl sulphoxide (DMSO) and fetal bovine serum was evacuated and 1 mL of α modification essential medium was added. Cell culture was immediately introduced into tissue culture flask containing 5 mL of the culture medium. When the flask became confluent, the cells were detached using 0.25% trypsin, centrifuged and added to tissue culture flask.

Preparation of the materials and their extracts

All the four groups of materials were introduced into paraffin wax mould, supported by sterile stainless steel. After setting, they were subjected to ultraviolet radiation for 30 min. The cements were weighed and then were introduced into sterile glass bottles with α modification medium (200 mg/mL). Finally, materials were incubated for 72 h at 37°C. After incubation, the materials extract were filtered into another sterile glass bottle and were added to the culture medium with a series of concentrations labeled as 100, 50, 25, 12.5, 6.25, 3.12, and 1.56 mg/mL in five replicates.

Procedure of MTT assay

L929 mouse fibroblast cell culture was applied into the wells containing full and diluted concentrations of the material extract and then incubated for 48 h at 37°C and 5% CO2. MTT solution was added to each plate and was incubated to be solubilized with DMSO. After 48 h, the effects of the materials on mitochondrial function were measured using an enzyme-linked immunosorbent assay reader (at wave length of 570 nm).

Statistical analysis

One-way analysis of variances (ANOVAs) followed by multiple comparison tests were performed to determine the significant differences of setting time and solubility (%) of the materials. For cell cytotoxicity, the data were presented as mean values. This is a descriptive analysis using IC50 [IC50 = concentration when only 50% of cells proliferate]. Material concentration more than IC50 is cytotoxic to the cells. Five replicates of each concentration were performed for each material. Differences in mean cell viability values at each concentrations of materials extract of different materials were assessed using one-way ANOVAs followed by multiple comparison test (P < 0.05).

RESULTS

Setting time

The setting time of Gyp-PA was 13.10 min, while with pure-GYP, the mean setting time was 9.85 min. The mean setting time for CH and GIC were 0.97 and 3.62 min, respectively. The result of setting time with significant differences among the groups is shown in Figure 1.

Figure 1

Setting time of the materials tested; same superscript letter indicates no significant differences among the groups (P > 0.05)

For Gyp-PA, the mean percentage of weight loss following immersion in deionized water resulted in a 14.66%. This weight loss was increased up to 26.36% with pure-GYP which was significantly higher than Gyp-PA [Figure 2]; significantly reduced solubility of 4.51% was observed with CH. For GIC, the mean solubility or weight loss was 2.32% and was not significantly different than the CH group (P > 0.05).

Figure 2

Solubility (%) of materials; same superscript letter indicates no significant differences among the groups (P > 0.05)

The results of cytotoxicity are shown in Figure 3. The results showed that at all concentration cell viability of pure-GYP was above 50%. While Gyp-PA and CH showed toxicity effect at concentration around 25 mg/mL. Table 2 shows the significant differences in mean cell viability (%) values at each concentrations of materials extract of tested groups.

Figure 3

Cell viability (%) at different concentration of the materials extract

Table 2

Mean values of cell viability (%) at each concentration with different materials; groups from the same column that are identified with the same superscript letter are not significantly different (P>0.05)

DISCUSSION

The focus of the present study was to evaluate gypsum-based materials in search of an ideal biomaterials for application at different sites in conservative dentistry mainly at traumatic exposure of pulp and/in deep dentin. Cytotoxicity test with L929 fibroblast cell lines obviously showed a favorable result for pure-GYP materials as maximum cell viability was observed even with the highest concentration of material extract. Mainly, PA and water are present in the liquid component of GIC material. Gypsum was mixed with PA to increase the adhesive and cohesive properties of the mixed product. Although solubility rate was decreased when PA was mixed with gypsum compared with pure-GYP, setting time was increased; the presence of PA in the mixing liquid probably interfered with the formation of gypsum crystals during the setting procedures; thus, setting time was increased with gypsum-PA. The use of modifiers such as K2SO4 to reduce the setting time[13] to an acceptable limit of the gypsum-based materials might be effective and will be considered in future study. Moreover, mixing of biocompatible polymer with gypsum materials would be less cytotoxic and would reduce the solubility (%) of gypsum materials and will be considered in further studies. Considering the cytotoxicity results, significantly higher level of toxicity was observed with CH at 50 and at 100 mg/mL concentration level. The above finding is in line with previous studies on Dycal which had found strong cytotoxic effect as well.[1415] Further study on pH of the gypsum-based products will be helpful to consider this prospective material for successful application especially on sites of pulp exposure, as the differences on partial hard tissue formation by pulpal tissue in response to different dental materials with varying of pH were reported.[16]

According to the manufacturer's information the α hemidydrate gypsum used in this study was processed from 96.99% of calcium dihydrate gypsum which was further heat processed for more purification. The additional materials which present in the composition are CaSO4 anhydrite gypsum and CaO of about 1.48% and 1.53%, respectively. Thus, the abundance presence of Ca+2 in this material which is also the main component of dentin might be beneficiary in formation of intermingling structure with dentin. From molecular point of view, the presence of Ca+2 was found to be advantageous as application of pure gypsum on bone suggested that extracellular calcium plays an important role in the regulations of bone cells.[17] It has been suggested that moderate high extracellular Ca+ is a chemotactic and proliferating signal for osteoblast and stimulates the differentiation of MC3T3-E1 preosteoblast.[18] Considering the above facts, application of biocompatible gypsum materials on exposed pulp/at deep dentin might has a stimulatory effect on preodontoblast cells or stem cells in the pulp tissue; thus, probable formation of dentin like structure in response of differentiated preodontoblast cells or stem cells would be a great success in the field of conservative dentistry. The higher solubility of gypsum might not be harmful as the material has a unique property of biodegradability; thus, partially soluble material is unlikely to produce any toxic effect as previously resin-based lining material had been found cytotoxic to surrounding tissues.[19]

Pure gypsum showed the least cytotoxic properties. Application of this novel biomaterial on deeper dentin/an exposed pulp and possibility of gradual replacement of this biodegradable material by dentin-like structure in response of underlying cells would be highly promising. Further studies to improve the physical properties of experimental gypsum-based biomaterial and its effects on dental pulp cells are under considerations.