Viability Test of Hydroxyapatite Tooth Graft on Osteoblast cell culture

 

Michael Ganda Wijaya1, Chiquita Prahasanti2*, Bambang Dwi Laksono2,

Westy Agrawanty1, Banun Kusumawardhani3, Maria Jessica Anggakusuma1

1Resident of Postgraduate Program in Periodontics, Faculty of Dental Medicine,

Universitas Airlangga, Surabaya, Indonesia.

2Department of Periodontology, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia.

3Department of Biomedical Sciences, Faculty of Dentistry, Universitas Jember, Jember, Indonesia.

*Corresponding Author E-mail: chiquita-p-s@fkg.unair.ac.id

 

ABSTRACT:

Surgery is required to restore bone loss brought on by regenerative periodontal diseases while retaining the patient's aesthetics. The bone deficits caused by periodontal disease have been repaired using a variety of transplant materials. One of the graft materials used is dentin since it resembles bone in terms of both organic and inorganic components. In order to evaluate the viability of dental grafts, this study intended to count the osteoblast cells that were still alive after a specific therapy. Osteoblast cell cultures in 42 well plates were employed in this work. The 42 well plate cell cultures were separated into seven groups for 24hour examinations and seven groups for 48 hour examinations in order to examine the cells using the MTT assay. Each group contained control cells, control media devoid of cells, and the treatment group, which received tooth transplant at doses of 8, 4, 2, 1, and 0.5mg/mL. Using an ELISA reader with a 595nm wavelength, the optical density of these cells was used to determine the viability of the cells. There are more than 50% of osteoblast cells in all concentrations, which is indicated by the number of these cells. The Shapiro-Wilk, Levene, and Oneway Anova tests were performed to assess the normality, uniformity, and degree of group differences in the data. This study demonstrates the biocompatibility of the tooth graft and the osteoblast cells.

 

KEYWORDS: Hydroxyapatite, Osteoblast cells, Tooth graft, Viability.

 

 


INTRODUCTION: 

The pattern of alveolar bone destruction that occurs in periodontal disorders varies greatly. The patient's aesthetics must be preserved while surgery is performed to replace bone loss brought on by regenerative periodontal diseases. Utilizing a range of transplant materials, the bone defects brought on by periodontal disease have been corrected. Two techniques—respective surgery and regenerative surgery—can be used to manage the treatment to remove and repair bone damage caused by periodontal tissue anomalies1–3.

 

Interest respective and regeneration surgery lessens the depth of the periodontal pocket and lessens the harm brought on by bacterial colonies that can be controlled by periodontal infection.

 

The second distinction is that surgical resection is not recommended in areas that require aesthetics since it simply removes the damaged tissue, increasing the likelihood of a recession in the treated area. Surgical regenerative beings potentially restore lost periodontal tissues and have an aesthetic value1,4. The tissue engineering triad, which describes the requirements for this regenerative paradigm, is necessary for treatment success. A scaffold, signaling, and cells are the three components that contribute to tissue engineering in this triangle. An extracellular matrix, which is formed from the materials utilized as graft material, serves as a framework to facilitate cell adhesion and movement in the tissue engineering triad idea5–8.

 

Non-toxic, non-irritating, and not causing an excessive inflammatory reaction are just a few of the terms used to describe the materials utilized as a graft9,10. The following categories of transplant materials are determined by where they came from: autograft, allograft, xenograft, and alloplast. The graft materials should be osteogenic, capable of osteoinduction and osteoconduction, and have a structure that is comparable to or similar to the tissue transplant recipient11,12.

 

By augmenting, it is possible to regenerate periodontal tissues for bone loss. An implanted tooth graft is one of the augmentation materials used to repair bone loss in the periodontal tissue (tooth-derived material bone graft or tooth graft). Currently, a wide range of graft materials are widely available for use as augmentation, but tooth graft material is regarded as the best (gold standard) and has the potential to be developed in the treatment of bone damage because it is derived from a gene or the same species (autogenous)13,14. The dentin and cementum of the tooth were used to harvest and prepare graft material for bone repair. Dentin's chemical and inorganic components resemble human bone. Bone morphogenetic proteins (BMPs), which promote the development of mesenchymal stem cells into chondrocytes, are also present in dentin15,16.

 

In this investigation, the extracted tooth's root dentin was used to make the graft material hydroxyapatite. It is vital to examine the biocompatibility of the graft material in order to achieve the best results and to treat bone loss safely. The capacity of a material to avoid producing a negative biological reaction when it is ingested is known as biocompatibility. Materials' cytotoxicity, mutagenicity, and cancer-causing potential can all be considered as aspects of biocompatibility17–21.

 

One of the first testing stages, cytotoxicity essay, is crucial for a material that will be employed in dentistry22,23. A chemical's level of toxicity must be determined in order for the substance to be absorbed by the host, not to irritate, not to cause cancer, and not to trigger allergic reactions. Toxicity tests can be scored in the viability category18. A dental hydroxyapatite graft's potential to survive in the presence of osteoblast cell cultures was investigated in this study.

 

MATERIALS AND METHODS:

Setting and design study:

The research involves osteoblast cell cultures in an experimental setting using a post-only control group design24. The Laboratory of Parasitology, Faculty of Medicine, Universitas Gajah Mada, carried out a viability test using the MTT assay. The equipment utilized in this investigation includes digital scales, conventional incubators, an incubator with CO2, Roux bottles, Petri dishes, multi-channel pippets, microplates, shakers, and Elisa readers. While the active ingredients were osteoblast cell cultures, tooth graft, DMEM, 10% bovine serum, PBS, trypsin-versene, hydroxyapatite powder from tooth graft, MTT, and DMSO.

 

 

Samples size:

Samples from the extracted root dentin tooth used to create the dental graft were obtained, sanitized, and formed into particles measuring 355-710 μm18,25,26. Federer(1977) provided the following formula to calculate the number of samples or replications for this study: (t-1) (r-1)> 15 18. There were t treatments, and there were r replications. In this research, there were 12 treatments total—6 for 24 hours and 6 for 48 hours. Calculation of replication as follows:

 

(12-1) (r-1) > 15

11 (r-1) > 15

r >15+11

       11

r > 2.37 ≈  r = 3

Replication in this study consisted of three replications.

 

Research variable:

The number and size of the tooth grafts served as the study's independent variables. Osteoblast cell culture viability was the dependent variable. The laboratory techniques, the quantity of cells in the culture, and the duration of interaction with the cell culture test materials were all simultaneously controlled variables (24 and 48 hours).

 

Operational definition:

1.   A tooth graft was a graft made from the extracted root dentin of a tooth. This material is then deproteinized, resulting in crystals of 100% hydroxyapatite with a particle size of 355-710 µm.

2.   The cultivation of osteoblasts in Dulbecco's Modified Eagle's Medium was the source of osteoblast cell culture (DMEM).

3.   Osteoblast cell cultures were monitored for viability using the MTT assay method, which was read by an ELISA reader with an absorbance wavelength of 595 nm. Purplish blue formazan was viewed from living cells.

 

Preparation of tooth graft:

Graft material made from teeth that had been removed. After separating the tooth's partial crown and root, the powdery dentin of the tooth root was obtained. A 3% H2O2 solution was used to cleanse the dentin until all of the fat has been removed. Heat treatment was used in the deproteinization procedure, leaving crystalline hydroxyapatite at its purest state. Particles between 355-710µm in size were used in the Bank Network Dr. Soetomo's tooth graft. After that, the tooth transplant material was dissolved in DMSO liquid to create a white suspension at five concentrations: 8mg/mL, 4mg/mL, 2 mg/mL, 1mg/mL, and 0.5mg/mL.

 

Preparation of Cell Culture Osteoblasts:

Osteoblast cell culture roux kept in bottles containing 10% bovine serum and Dulbecco's Modified Eagle's Medium (DMEM) until confluent (evenly when viewed with a microscope). Roux vials with osteoblast cell cultures were incubated in a typical incubator at 37°C for 24hours. The growth media, Dulbecco's Modified Eagle's Medium (DMEM) with 10% bovine serum, was taken out of the bottle. PBS should be used to clean the osteoblast cell cultures in Roux bottles. Trypsin-versene 0.5% was released for 20minutes on osteoblast cell cultures that adhere to the base of the container. Following release, a growth media containing 10% FBS was supplied to the osteoblast cell culture. After centrifuging the cell suspension, DMEM +10% FBS was added to the precipitate cells. Cells were placed in 36 healthy plates with cell culture growth medium and six-well plates with growth media only, each containing 100 mL of replication as needed, and the plates were cultured for 24hours at 37°C in a 5% CO2 incubator14.

 

MTT Test Preparation:

Each group received treatments of 8mg/mL, 4mg/mL, 2mg/mL, 1mg/mL, and 0.5mg/mL as the control group, media control. Prior to treatment, osteoblast cell cultures were taken out of the incubator and examined under an inverted microscope. By washing with PBS solution and drying with sterile tissue, discard the growing media. 100mL of the tooth graft suspension should be added to the replication plate with the appropriate concentration. Each group was incubated for 24 and 48hours at a temperature of 37°C in an incubator with 5% CO2. Removing the tooth graft suspension from the appropriate well plates in the incubator, washing with a PBS solution, and patting dry were all necessary steps. Each healthy plate should have 100mL of MTT reagent added before being incubated for 2 to 4hours. When adding the stopper SDS 10% to 100mL of N.HCl, the sample checked under a microscope to see if formazan has developed. 24hour incubation in the dark at room temperature with the plate wrapped in aluminum foil. Elisa reader read well plates using a 595nm wavelength after being examined27.

 

Data analysis:

Data were statistically analyzed by Oneway ANOVA test at the significance level (α = 0.05) with SPSS 20 (UBM, USA).

 

RESULT:

The results of the study on the viability of the graft tooth that was conducted at the Laboratory of Parasitology, Universitas Gajah Mada, were divided into the control group and the treatment group 8mg/mL, 4mg/mL, 2 mg/mL, 1mg/mL and 0.5mg/mL at osteoblast cell cultures using the method of MTT assay was obtained optical density (OD) and do readings with Elisa Reader, were as follows:

 

Table 1: Optical density control group and the treatment group after the exposure of the tooth hydroxyapatite graft in cultured osteoblast cells (24 hours).

Treatment (mg/mL)

Mean+SD

Sig.

Viabilitassel (%)

Control

0.501 + 0.043267

0.537

100

8

0.482 + 0.042462

0.067

97

4

0.502 + 0.008185

0.593

100

2

0.508 + 0.006110

0.637

101

1

0.502 + 0.023438

0.328

100

0.5

0.475 + 0.009504

0.942

96

 

Table 2: The optical Density control group and the treatment group after the exposure of the tooth hydroxyapatite graft in cultured osteoblast cells (48 hours).

Treatment (mg/mL)

Mean + SD

Sig.

Cell viability (%)

Control

0.570 + 0.019502

0.246

100

8

0.564 + 0.014000

0.274

99

4

0.535 + 0.025239

0.189

95

2

0.515 + 0.004726

0.407

92

1

0.520 + 0.015373

0.187

93

0.5

0.527 + 0.020306

0.188

94

 

Data analysis:

Based on the research done on osteoblast cells exposed to tooth graft, a value of absorbance (Optical Density) was high, both in the MTT test 24 hours and 48 hours. The highest absorbance produced in the control group 48 h = 0.57+0.019502 being produced the lowest absorbance in the treatment group 0.5mg/mL 24hours = 0.475+0.009504.

 

As a result of the sample size being less than50, the data was then examined for Shapiro-Wilk normality. All of the data's p-values were greater than 0.05 as a result of the normality test. It denotes a normal distribution in the collected data. Levene homogeneity test results were used to examine the data, and they revealed p=0.03 (p<0.05). It demonstrated that the data acquired are not all equal. As a result of the non-homogeneous nature of the data obtained, a one-way Anova test was then conducted to compare the means of each group and determine the difference across groups. At p=0.01 (p<0.05), the Oneway Anova test revealed a significant difference in absorbance. Post hoc Games-Howell was used to compare the data for each group when there were no significant differences between them. Unless the comparison between groups of 0.5mg/mL 24 hours with a group of 8mg/mL 48hours, p=0.015, and the groups of 0.5mg/mL 24 hours with a group of 0.5 mg/mL 48 hours, p=0.044, showed a significant difference in the data obtained.

 

Calculating the percentage of cell viability of osteoblasts following exposure to the tooth graft requires data on optical density treatment of a group 24 and 48 hours later compared to corresponding control groups. Cell viability (%) = (OD treated/OD control) x 100% is the formula used. The treatment group 2 mg/mL 24 hours had the maximum viability (101%) and the treatment group 2 mg/mL 48 hours had the lowest viability (92%).

 

DISCUSSION:

The Laboratory of Parasitology, Faculty of medicine, Universitas of Gajah Mada, processed hydroxyapatite from a tooth graft with a particle size of 355-710m and tested the survival of osteoblast cells in this experiment. Dentistry treatments always involve replacing damaged oral cavity components with new ones using materials. Each material must adhere to a number of specifications, one of which is that it be made of a substance that is biocompatible and won't harm or be poisonous to the surrounding biological environment both locally and systemically. A toxicity viability test is conducted in clinical studies to assess a material's level. A viability test is frequently used to evaluate a material's biocompatibility28–31. Enzymatic testing using MTT assay reagent is one way to determine a material's viability32.

 

Hydroxyapatite graft testing on osteoblast cell culture is used to determine the vitality of teeth. It is based on the idea that osteoblast cells play a key role in bone remodeling by forming the bone matrix and participating in osteogenesis33–36. In regenerative periodontal surgery, hydroxyapatite will be applied clinically to the tooth transplant before being implanted in the injured bone to hasten bone tissue regeneration or prossesus alveolar19,37,38.

 

The MTT assay uses colorimetry to analyze the metabolic activities of live cells to evaluate a cell's viability. By examining the proportion of the total number of living cells in the culture, it is possible to acquire quantitative information from experimental research on the survivability of hydroxyapatite tooth grafts in cultivated osteoblast cells. The optical densities of each group can be used to calculate the proportion of live cells39.

 

There is a variation in the survivability of each treatment group when calculating the outcomes of research on osteoblast cells. Tellis standard, which states that a substance is deemed dangerous when more than 50% of live cells are present after exposure to the substance, was utilized as a reference for the toxicity characteristics of the material18. The viability of cells exposed to tooth graft hydroxyapatite for 24 and 48 hours with certain concentration showed excellent results; compared to the control group, cell viability varied from 92 to 101 percent, indicating that the substance is not harmful.

 

The average viability treatment group did not differ substantially from the control group between the hours of 24 and 48. The chemical composition of the tooth graft's hydroxyapatite is expected to have an impact on viability, proliferation, and differentiation, as are other elements like its porosity and roughness, which are influenced by the media's pH. Graft porosity will impact the blood vessel-forming cells. Surface roughness of a graft material influences osteoblast cell attachment33,40.

 

Due to the presence of the tissue-regenerating mineral hydroxyapatite in tooth graft, it does not produce harmful effects. The inorganic substance hydroxyapatite, also known as Ca10(PO4)6(OH)2, has a hexagonal structure with the formula Ca5(PO4)3(OH). The matrix of the bones and teeth contains hydroxyapatite, which gives the structure rigidity. The bioactive mineral hydroxyapatite can directly connect with bone. The human body contains these substances. There are no harmful effects of the hydroxyapatite used in tooth grafts37,40–42.

 

CONCLUSION:

The high osteoblast cell viability of hydroxyapatite in dental grafts indicates that the material is not harmful to osteoblast cells. After being subjected to the hydroxyapatite of a tooth graft, differentiation culture proliferation, and osteoblast cells must be tested.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

ACKNOWLEDGMENTS:

There are no acknowledgements to declare.

 

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Received on 17.11.2022            Modified on 28.07.2023

Accepted on 11.12.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(2):855-859.

DOI: 10.52711/0974-360X.2024.00132