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ORIGINAL ARTICLE
Year : 2016  |  Volume : 34  |  Issue : 3  |  Page : 269-272
 

An in vitro evaluation of cytotoxicity of curcumin against human dental pulp fibroblasts


1 Department of Pedodontics and Preventive Dentistry, Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, Belagavi, Karnataka, India
2 Department of Microbiology and Molecular Biology, Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, Belagavi, Karnataka, India
3 Department of Pedodontics and Preventive Dentistry, Bapuji Dental College and Hospital, Davangere, Karnataka, India

Date of Web Publication25-Jul-2016

Correspondence Address:
Praveenkumar S Mandrol
Department of Pedodontics and Preventive Dentistry, Mandal's NGH Institute of Dental Sciences and Research Centre, Belagavi, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-4388.186757

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   Abstract 

Objective: The objective of this study was to evaluate the cytotoxicity of curcumin to primary dental pulp fibroblasts in vitro. Materials and Methods: Dental pulp fibroblasts from primary maxillary central incisors were cultured and used for cytotoxicity tests after the fourth passage. Ninety-five percent curcumin was diluted with dimethylsulfoxide to prepare 100%, 50%, and 25% concentrations. Each concentration of curcumin was added in triplicate into 96-well microtiter plate containing the fibroblast culture at 104/well. Cells without treatment served as a control group. The number of viable cells after 48 hrs incubation at 37°C in a humidified atmosphere of 5 % CO2 and 95 % air was determined by the 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay. The relative viability of pulp cells was expressed as color intensity of the number in the experimental wells relative to that of the control group. Absorbances were read at 492 nm on a microplate reader with a background subtraction at 620 nm. Results: Cell viability of primary dental pulp fibroblasts to 25%, 50%, and 100% curcumin concentration was 174%, 310%, and 317%, respectively. Conclusions: Curcumin promotes cell viability and induces proliferation of primary dental pulp fibroblasts and has the potential to be developed into an economical and reliable medicament for vital pulp therapy.


Keywords: Curcumin, cytotoxicity, pulp fibroblasts


How to cite this article:
Mandrol PS, Bhat K, Prabhakar A R. An in vitro evaluation of cytotoxicity of curcumin against human dental pulp fibroblasts. J Indian Soc Pedod Prev Dent 2016;34:269-72

How to cite this URL:
Mandrol PS, Bhat K, Prabhakar A R. An in vitro evaluation of cytotoxicity of curcumin against human dental pulp fibroblasts. J Indian Soc Pedod Prev Dent [serial online] 2016 [cited 2019 Mar 22];34:269-72. Available from: http://www.jisppd.com/text.asp?2016/34/3/269/186757



   Introduction Top


Management of the grossly carious primary molar is a common but sometimes challenging aspect of dental care for young children. Pulpotomy is performed in a primary tooth with extensive caries, but without evidence of radicular pathology when caries removal results in a carious or mechanical pulp exposure. The coronal pulp is amputated, and the remaining vital radicular pulp tissue surface is treated with a long-term clinically successful medicament.[1],[2] An optimum wound healing dressing or agent protects the wound tissue from bacterial infection, reduces inflammation, and induces cell proliferation to aid in the reconstruction of damaged tissue.[3] It would ideally also act as an anti-oxidant, as free radicals are considered the major cause of inflammation during wound healing process.[4] The objective is that radicular pulp should remain healthy without adverse clinical signs or symptoms such as sensitivity, pain or swelling, and also with no postoperative radiographic evidence of pathologic external or internal root resorption and no harm to succedaneous tooth [1],[5] so that the primary tooth fulfills its role in the dentition.

Traditional medicine is known to be fertile ground for the source of modern medicines. One medicine in that category is turmeric (Curcuma longa) and belongs to the ginger (Zingiberaceae) family. Components of turmeric are named curcuminoids, which include mainly curcumin (diferuloyl methane), demethoxycurcumin, and bisdemethoxycurcumin. Curcumin, a yellow phenolic pigment, is the most important fraction which is responsible for the biological activities of turmeric.[6] Extensive research on curcumin has demonstrated a wide spectrum of therapeutic actions such as anti-inflammatory, antibacterial, antiviral, antifungal, anti-diabetic, anti-coagulant, hepato-protective, anti-ulcer, hypo-tensive, and hypo-cholesteremic.[6],[7] How a single agent could exhibit all these effects is an enigma under intense analysis. Pharmacological safety and small cost make curcumin an attractive agent to investigate further.

Many types of biomaterials have been utilized for the restorative and endodontic treatment procedures. The biological compatibility of dental materials is of vital importance to avoid or limit pulp tissue irritation or degeneration. Cytotoxicity screening assays provide a measure of cell death caused by materials or their extracts.[8] Primary cell cultures derived from dental pulp, i.e., pulp fibroblasts, may arguably be more relevant for testing the biocompatibility of materials for use in restorative dentistry and endodontics. Moreover, pulp fibroblast is highly sensitive to toxic substances, indicating that pulp cells could be a sensitive barometer to reveal the possible adverse effects of dental materials.[9],[10] As the rationale for the development of new therapeutic materials is to enhance successful clinical applications, trials must be carried out to evaluate their cytotoxicity. Hence, the aim of this study was to evaluate the cytotoxicity of curcumin on cultured human primary pulp fibroblasts.


   Materials and Methods Top


Source of explants

Human dental pulp fibroblasts were obtained from healthy patients presented to the Department of Pedodontics and Preventive Dentistry for orthodontic extractions. Included teeth were primary maxillary incisors with more than half root resorbed, which were devoid of any caries, restorations, and periodontal disease. Teeth were extracted aseptically after informed consent of parents. After extraction of teeth, pulp tissue was carefully extirpated from the root canal with sterile broach and sharp spoon excavator and was transported to the laboratory in Dulbecco's Modified Eagle's Medium (Hi-Media Laboratories, Mumbai, India) for fibroblast culture.

Fibroblast isolation and harvesting from dental pulp

Pulp tissue was placed in a sterile  Petri dish More Details containing a solution of 3 mg/ml collagenase and 4 mg/ml dispase for 1 h, till the tissue underwent enzymatic dissociation. Small pieces of pulp tissues were removed using a micropipette, re-suspended in 5–10 ml phosphate buffered saline, and centrifuged at 1800 rpm for 5 min to obtain pellet-containing cells. The supernatant was discarded and the pellet was re-suspended in 5 ml of Dulbecco's modified Eagle's medium. Single-cell suspensions of dental pulp were cultured in 24-well microtiter plate with Dulbecco's modified Eagle's medium and then incubated at 37°C in 5% CO2. The culture medium was changed for every 3 days until the cell confluence was achieved. Pulp tissue was minced into 1–2 mm fragments, and each piece was placed in 24-well microtiter plate with Dulbecco's Modified Eagle's Medium and then incubated at 37°C in 5% CO2. It should be considered that the total volume of the Dulbecco's Modified Eagle's Medium for outgrowth of cell must support the attachment of pulp pieces for further cell outgrowth (2–3 ml/well). Medium was changed after outgrowth was observed. The outgrown cells at confluence were sub-cultured at a ratio of 1:4. Cells were used for cytotoxic tests after the fourth passage.

Cytotoxicity assay

Ninety-five percent of curcumin (Hi-Media Laboratories, Mumbai, India) [Figure 1] was diluted with dimethylsulfoxide (Hi-Media Laboratories, Mumbai, India) in 100, 50, and 25 weight/volume %. Each concentration of curcumin was added in triplicate into 96-well microtiter plate containing the fibroblast culture. Cells without treatment served as a control group.
Figure 1: Crystalline curcumin (Hi-Media Laboratories, Mumbai, India)

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The number of viable cells after 48 h incubation at 37°C in a humidified atmosphere of 5% CO2 and 95% air was determined by the methyl-thiazol-diphenyl-tetrazolium (MTT) assay. For this, 20 µL of 5 mg/ml MTT was added to all the 12 wells (10 mg in 1000 µL, 5 mg in 1000 µL, 2.5 mg in 1000 µL, and control, in triplicate) in 96-well microtiter plate and incubated at 37°C, 5% CO2 with 98% humidity for 4 h. At the end of the incubation period, the medium with MTT was removed, and 100 μL of dimethylsulphoxide was added to each well. The plate was shaken on the microplate shaker to dissolve the purple MTT-formazan. The relative viability of dental pulp fibroblasts was expressed with color intensity of the number in the experimental wells relative to that of control. Absorbance was recorded at 492 nm on a microplate reader with background subtraction at 620 nm (Lisaplus, Aspen Diagnostics Pvt., Ltd., Mumbai, India). The percentage of viable cells was determined by using the following equation: cell viability % = (mean absorbance of experimental wells/mean absorbance of control wells) × 100%.


   Results Top


All the tests were done in triplicate. The mean absorbance (optical density [OD]) and percentage values of cell viability at various concentrations of curcumin to human primary dental pulp fibroblasts are shown in [Table 1].
Table 1: Mean survival of human primary dental pulp fibroblasts as measured by MTT assay after exposure to various dilutions of curcumin for 48 h

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   Discussion Top


The biologic and toxicologic properties of biomaterials are important for their clinical usage.In vitro cytotoxic screening as a primary factor of biocompatibility is determined by cell culture. In addition, in vitro tests are simple, reproducible, cost-effective, relevant, and suitable for evaluating the basic biological properties of dental materials.[10] Data on cell viability have long been obtained from in vitro cytotoxicity assays, and the MTT cell survival assay is widely used for measuring the cytotoxic potential of a compound.[11] The present study investigated the in vitro cytotoxicity of curcumin against primary dental pulp fibroblasts by MTT assay. No cytotoxicity was detected for curcumin at any of the concentrations used (25%, 50%, and 100%). The results revealed that the viability of primary dental pulp fibroblasts increased with an increasing concentration of curcumin [Figure 2]. It was observed that curcumin increased the cell proliferation of primary dental pulp fibroblasts, resulting in almost three times more cell viability than control in 50% and 100% groups.
Figure 2: Viability of primary dental pulp fibroblast at 25%, 50%, and 100% concentrations of curcumin

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The choice of cell line for in vitro cytotoxicity screening assays remains debatable, as the apparent cytotoxicity of a material can be significantly affected by the cell line selected for the test. Nevertheless, primary cell cultures derived from the tissues of ultimate concern may arguably be more relevant to the clinical situation.[10]

The removal of the coronal portion of the pulp is an accepted procedure for treating both primary and permanent teeth with carious pulp exposures. The justification for this procedure is that the coronal pulp tissue, which is adjacent to the carious exposure, usually contains microorganisms and shows evidence of inflammation and degenerative change. The abnormal tissue can be removed, and the healing can be allowed to take place at the entrance of the pulp canal in an area of essentially normal pulp.[5] Placement of a medicament directly over the exposed pulp tissue has been suggested as a way to promote pulp healing and generate reparative dentin. If successful, this procedure precludes the need for more invasive, more extensive, and more expensive treatment.[12]

A therapeutic agent selected for the treatment of wounds should ideally improve one or more phases of healing without producing deleterious side effects.[7] Curcumin has been shown to have significant wound healing properties. Because of its low water-solubility, curcumin makes a suitable candidate for topical applications.[4] The wound healing potential of curcumin is attributed to its biochemical effects such as its anti-inflammatory,[13] anti-infectious,[14],[15] and antioxidant [16],[17] activities. The effects of curcumin topical treatment on different stages of cutaneous wound healing can be summarized as follows: (1) Stage of inflammation - inflammation is the crucial phase of the wound healing process. Uncontrolled inflammatory responses may lead to undesirable effects and subsequently tissue damage. Considering that tissue injury causes almost an immediate onset of acute inflammation, controlling inflammation is, therefore, desirable and can optimize the wound repair process. Curcumin has been shown to inhibit the production of tumor necrosis factor alpha and interleukin-1, two main cytokines released from monocytes and macrophages that play important roles in the regulation of inflammatory responses. Of equal importance is curcumin's ability to inhibit the activity of nuclear factor kappa-light-chain-enhancer of activated B-cells, a transcription factor that regulates many genes implicated in the initiation of inflammatory responses. (2) Stage of proliferation - the infiltration of fibroblasts into wound site is essential for granulation tissue formation/tissue remodeling, collagen production, and deposition of curcumin enhances fibroblast migration, granulation tissue formation, collagen deposition, and in general, re-epithelialization. Being apoptotic in the early phase of wound healing, curcumin eliminates unwanted inflammatory cells from the wound site. (3) Stage of remodeling - the re-organization and remodeling of the extracellular matrix are a prerequisite for wounds to heal completely. The extracellular matrix provides support to cells and it is composed of various proteins and polysaccharides including granulation tissue and collagen. Transforming growth factor beta (TGF-β) is an important cytokine that is involved in the repair, chemotaxis, and deposition of collagen in a wound site. Curcumin improves wound contraction by increasing the production of TGF-β and therefore increasing fibroblast proliferation.[7]

In this study, the cell viability values were consistently above 100% for all the test concentrations of curcumin. Apart from the action of curcumin, this could also be because the color of curcumin will add on to the OD values obtained.

The action of curcumin has never been reported for therapeutic uses in pediatric endodontic therapies. Possession of desired biological actions, pharmacological safety, and negligible cost makes curcumin an attractive agent to explore further for its potential uses in vital pulp therapy procedures in primary and permanent teeth and also for regenerative endodontic procedures in permanent teeth.


   Conclusion Top


Curcumin promotes cell viability and induces proliferation of primary dental pulp fibroblasts and has the potential to be developed into an economical and reliable medicament for vital pulp therapy, and it needs to be evaluated further in animal models for usage tests.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

1.
American Academy of Pediatric Dentistry. Clinical guidelines on pulp therapy for primary and young permanent teeth: Reference manual 2006-07. Pediatr Dent 2006;28:144-8.  Back to cited text no. 1
    
2.
Fuks AB. Vital pulp therapy with new materials for primary teeth: New directions and treatment perspectives. J Endod 2008;34 7 Suppl: S18-24.  Back to cited text no. 2
    
3.
Kulac M, Aktas C, Tulubas F, Uygur R, Kanter M, Erboga M, et al. The effects of topical treatment with curcumin on burn wound healing in rats. J Mol Histol 2013;44:83-90.  Back to cited text no. 3
    
4.
Mohanty C, Das M, Sahoo SK. Sustained wound healing activity of curcumin loaded oleic acid based polymeric bandage in a rat model. Mol Pharm 2012;9:2801-11.  Back to cited text no. 4
    
5.
McDonald RE, Avery DR, Jeffrey AD. Treatment of deep caries, vital pulp exposure, and pulpless teeth. In: McDonald RE, Avery DR, editors, Dentistry for the Child and Adolescent. 9th ed. Missouri, UK: Mosby, Inc. (an affiliate of Elsevier Inc.); 2011. p. 350.  Back to cited text no. 5
    
6.
Chattopadhyay I, Biswas K, Bandyopadhyay U, Banerjee RK. Turmeric and curcumin: Biological actions and medicinal applications. Curr Sci 2004;87:44-53.  Back to cited text no. 6
    
7.
Akbik D, Ghadiri M, Chrzanowski W, Rohanizadeh R. Curcumin as a wound healing agent. Life Sci 2014;116:1-7.  Back to cited text no. 7
    
8.
Murray PE, García Godoy C, García Godoy F. How is the biocompatibilty of dental biomaterials evaluated? Med Oral Patol Oral Cir Bucal 2007;12:E258-66.  Back to cited text no. 8
    
9.
van Wyk CW, Olivier A, Maritz JS. Cultured pulp fibroblasts: Are they suitable for in vitro cytotoxicity testing? J Oral Pathol Med 2001;30:168-77.  Back to cited text no. 9
    
10.
Odabas ME, Ertürk M, Çinar Ç, Tüzüner T, Tulunoglu Ö. Cytotoxicity of a new hemostatic agent on human pulp fibroblasts in vitro. Med Oral Patol Oral Cir Bucal 2011;16:e584-7.  Back to cited text no. 10
    
11.
Sumantran VN. Cellular chemosensitivity assays: An overview. Methods Mol Biol 2011;731:219-36.  Back to cited text no. 11
    
12.
Hilton TJ. Keys to clinical success with pulp capping: A review of the literature. Oper Dent 2009;34:615-25.  Back to cited text no. 12
    
13.
Liang G, Yang S, Zhou H, Shao L, Huang K, Xiao J, et al. Synthesis, crystal structure and anti-inflammatory properties of curcumin analogues. Eur J Med Chem 2009;44:915-9.  Back to cited text no. 13
    
14.
Mun SH, Joung DK, Kim YS, Kang OH, Kim SB, Seo YS, et al. Synergistic antibacterial effect of curcumin against methicillin-resistant Staphylococcus aureus. Phytomedicine 2013;20:714-8.  Back to cited text no. 14
    
15.
Singh RK, Rai D, Yadav D, Bhargava A, Balzarini J, De Clercq E. Synthesis, antibacterial and antiviral properties of curcumin bioconjugates bearing dipeptide, fatty acids and folic acid. Eur J Med Chem 2010;45:1078-86.  Back to cited text no. 15
    
16.
Ak T, Gülçin I. Antioxidant and radical scavenging properties of curcumin. Chem Biol Interact 2008;174:27-37.  Back to cited text no. 16
    
17.
Meng B, Li J, Cao H. Antioxidant and antiinflammatory activities of curcumin on diabetes mellitus and its complications. Curr Pharm Des 2013;19:2101-13.  Back to cited text no. 17
    


    Figures

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    Tables

  [Table 1]



 

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