Research Article
Volume 2 Issue 3 - 2015
Temporomandibular Joint Dysfunctions and Bio-Materials: Design, Free Radical Defense and Mechanism In Vitro
V Tamara Perchyonok1*, Tatiana Souza2, Desigar Moodley3, Nickolas Basson3, Shengmiao Zhang4 and Sias Grobler3
1VTPCHEM PTY LTD, Glenhuntly, Victoria, Australia
2Health Innovations Research Institute, RMIT University, Australia
3Oral and Dental Research Institute, School of Dentistry, The University of Western Cape, South Africa
4School of Material Science and Engineering, East China University of Science and Technology, China
*Corresponding Author: V Tamara Perchyonok, VTPCHEM PTY LTD, Glenhuntly, Victoria, 3163 Australia.
Received: August 01, 2015; Published: September 07, 2015
Citation: V Tamara Perchyonok., et al. “Temporomandibular Joint Dysfunctions and Bio-Materials: Design, Free Radical Defense and Mechanism In Vitro”. EC Dental Science 2.3 (2015): 307-316.
The aim of this investigation is to evaluate the suitability and flexibility of the bio-active containing designer materials to act as an “in vitro” probe to gain insights into molecular origin of TMJ. The hydroxyapatite/chitosan containing hydrogels represent a reliable biocompatible scaffold and allowed to evaluate the defense “build in” free radical defense mechanism of the functionalized bio-scaffolds on the molecular level, compare the effectiveness of the designer materials with the earlier reported capabilities of N-acetyl cysteine and apply the newly developed materials for the evaluation of new therapeutic treatment modalities in the TMJ therapy in vitro.
Materials and Methods: The hydrogels were prepared by dispersion of the corresponding component in glycerol and acetic acid with the addition of chitosan: hydroxyapatite as a molecular scaffold. The surface morphology (SEM), release behaviors (physiological pH and also in acidic conditions), stability of the antioxidant-chitosan were also evaluated. Structural investigations of the reactive surface of the hydrogel are reported. Bio-adhesive studies and mechanical properties of newly prepared materials were conducted to assess the suitability of these designer materials. Ability of the materials to release the phenolic components as potential therapeutic agents was assessed.
Results: The sustained release of total phenolic component as a potential therapeutic agent confirms the added benefit of synergistic action of a functional therapeutic delivery when comparing the newly designed chitosan/HA-based hydrogel molecular scaffold. The hydrogel formulations have a uniform distribution of drug content. The bio-adhesive capacity, tensile strength, compressive strength as well as modulus of elasticity of the hydroxyapatite containing materials in the “in vitro” systems was tested and quantified.
Conclusion: The added benefits of the chitosan hydroxyapatite treated hydrogels involved positive influence on the phenolic component release, sustainable bio-adhesion, tensile strength, compressive strength as well as modulus of elasticity of the hydroxyapatite containing materials in the “in vitro” systems was tested and demonstrated in vitro “build in” free radical defense mechanism.
Keywords: Hydroxyapatite; Drug release; Antimicrobial properties; Mechanical Properties; Biomolecular Scaffold
In the last few decades, tissue engineering has emerged as a promising multidisciplinary approach for the repair and regeneration of damaged bone tissue [1,2]. The molecular events that underline the degenerative temporomandibular joint (TMJ) diseases are poorly understood [3]. Mechanical stresses are generated during functional or para-functional movements of the jaw; adaptive mechanism of the TMJ may be exceeded by free radical accumulation leading to a dysfunctional state (i.e. disease state) [4]. Biologically relevant free radicals are very reactive and unstable molecular entities that have an unpaired electron and they can produce participate in the propagation chain reaction to form a new radical. Although oxygen free radicals participate in many physiological processes, they can be harmful to tissue, when either their action or their generation have been left uncontrolled. The most common source of free radicals in biological systems is oxygen. The elevation of reactive oxygen species (ROS) leads to oxidative stress that causes molecular damage to the vital structures and functions [5-7].
The unsaturated fatty acids of cell membrane lipid are susceptible to peroxidative reaction. Lipid peroxidation of cell membranes has been implicated in the wide range of tissue injuries and diseases [8]. Accumulation of lipid hydro-peroxides in a membrane disrupts its function and causes it to collapse and have range of cytotoxic radicals. The most serious one is byproducts aldehydes. They may also react with transition metals like iron or cop-per to form stable aldehyde such as Malondialdehyde for example, which will damage the cell membrane. Because hemoglobin constitutes the largest iron store in the body, it is speculated to be a potential source of redox activity iron, which can catalyze the formation of free radicals that might be damaging to the joint [8]. Antioxidants can be classified as enzymatic or non-enzymatic [9]. The non-enzymatic antioxidants are further subdivided as nutrient based (such as alpha tocopherol, β- Carotene ascorbate, Glutathione, Selenium, Proanthocyanidin, Lycopene, Green tea) and non-nutrient based (such as Ceruloplasmin, Transferrin, uric acid, Peptides Camosine Anserine [9].
Antioxidants end the electron stealing reaction of free radicals by donating one of their electrons. The antioxidant does not become a free radical by donating an electron because they are stable in either form. Antioxidants act by scavenging or chain breaking, such as Vitamin E (alpha tocopherol), Vitamin C (ascorbic acid), or Vitamin A (beta carotene) and preventative antioxidants that function basically by sequestering transition metal ions and preventing fenton reactions and are therefore predominantly proteins by nature (Eg., albumin, transferring, or lactoferrin)[9].
The a etiological factors of TMJ disorders are as follows: systemic diseases (rheumatoid arthritis, psoriasis, pseudo gout, ankylosing spondylitis, etc.), secondary inflammatory component from the neighboring regions (otitis, maxillary sinusitis, tonsillitis), trauma (chronical), prevalence of dental arch defects e.g. missing of molar teeth [10], malocclusion, endocrinological disturbances, odontogenic infections (impacted third molars) [11]. Specific bacterial organisms such as S. aureus, S. mitis, M. fermentas, and Actinobacillus actinomycetemcomitans have been found in the synovial fluid [12]. Serum antibodies against Chlamydia species in patients with mono arthritis of the TMJ have also been reported [13].
Chitosan (CS), obtained through the alkaline deacetylation of chitin, is a copolymer of N-acetyl-D-glucosamine and D-glucosamine. CS stands out by a unique combination of favorable biological properties such as nontoxicity, biocompatibility and biodegradability, along with mucoadhesive, bacteriostatic, and wound-healing properties [14,15].
The aim of this investigation is to evaluate the suitability and flexibility of the designer materials to act as an “in vitro” probe to gain insights into molecular origin of TMJ and more specifically excessive damage caused by oxygen centered radicals and the defense “build in” free radical defense mechanism of the functionalized bio-scaffolds on the molecular level, compare the effectiveness of the designer materials with the earlier reported capabilities of N-acetyl cysteine and apply the newly developed materials for the evaluation of new therapeutic treatment modalities in the TMJ therapy in vitro.
Preparation of various hydrogels: general protocol
The bio-active containing gel was prepared by dispersion of 0.2 gm in glycerol (5% w/w) (1 ml) using a mortar and a pestle following the earlier reported generic protocol [14,15]. Ten milliliters of glacial acetic acid (3% w/w) was then added with continuous mixing and finally chitosan/hydroxyapaptite (10% chitosan w/w) polymer was mixed well to form the required gel. The strength of the prepared gel (10 gm) is 0.02g of bio-additive in each gram of the base.
Swelling/Weight Loss Tests and Bioactive Release
The swelling/weight loss tests were performed when triplicates of each samples composition (approximately 2 cm2, weight normalized) were immersed in 2 mL of different fluids at 37oC for each time interval studied (1, 2, 4, 24, and 96h). Two different media were used in accordance with the ISO 10993-9 standard. The first media was Phosphate Buffered Saline (PBS, Sigma Aldrich), intended to mimic the inorganic phase of human plasma [16]. The other media was PBS with a reduced pH which was intended to simulate the local inflammatory environment of the wounds [16,17]. This is termed Solution pH 4.0. The pH was lowered using Lactic Acid (Sigma Aldrich). The fluid absorption of each sample was calculated according to Equation 2 to obtain their swelling degree (SD). WS is the weight of the sample at each time interval (swollen weight) and WD is the dry weight before swelling [18]. After 4 days of immersion, the samples were dried and weighed in order to calculate their weight loss (WL) [Equation 3], where WD and WDS are the weight of the dried samples before and after swelling tests, respectively

To analyze the bio-active release (propolis (Brazilian or Uruguan), Copaiba oil, Oblepicha Oil) based on the total phenolic concentration, the swelling media was analyzed after 1, 2, 24, and 96h of immersion via UV-Vis spectrometer, from 300 to 800 nm, using polystyrene cuvettes [19]. For quantification of the amount of propolis released, a standard curve was created by diluting the original propolis in isopropanol resulting in several aliquots of known concentration, which were then analyzed in the same wavelength range. The area of the peak of these aliquots (of known concentration of bio-additive) was calculated and used to compare with those of the bio-additive released by the samples.
Total phenol concentration in Propolis (Brazilian and Uruguyan), Copaiba oil and Oblepicha Oil
Total phenols assay Total phenols content was estimated by a colorimetric assay based on the same procedure realized by Rocha., et al. [20] and described by Waterman & Mole [21] with some modifications. The samples were diluted in distilled water to obtain a concentration of 5 µg/mL of total phenols. The concentration of 5% v/v of Folin-Ciocalteu reagent and 10% v/v sodium carbonate (35% w/v) reagents were added to the samples. After the addition of the reagents, the solutions were kept in the dark at room temperature for 30 min and the absorbance was read at the wavelength of 760 nm in a spectrophotometer UV mini-1240 (Shimadzu Co., Kyoto, Japan). Gallic acid (Aldrich, Australia) was used as a standard. The analyses were performed in triplicate.
Statistical analysis
The total flavonoids, total phenolics, quercetrin and afzelin contents as well as the antioxidant activity results were statistically analyzed by the variance analysis (ANOVA) by the Prism 6 software. In all analyses, a 5% significance level was considered.
Protein cross-linking as a model for detection of free radical activity and activation of “molecular defense forces”
Bovine serum albumin (BSA), a completely water-soluble protein, was polymerized by hydroxyl radicals generated by the Fenton reaction system of Fe2+/EDTA/ H2O2/ascorbate [22]. As a result, the protein loses its water-solubility and the polymerized product precipitates. The decrease in the concentration of the water-soluble protein can easily be detected.
The in vitro incubation mixtures contained reagents, added in the sequence as follows, at the final concentrations: bovine serum albumin (0.8 mg/ml), phosphate buffer, pH 7.4 (10 mM), water to reach 2.5 ml total volume, antioxidant (such as Oblepiha oil, Copaiba oil and corresponding chitosan) tested to reach required concentration as shown in results, EDTA (0-4.8 mM), Fe(NH4)2(SO4)2 (0-4 mM), ascorbate (4 mM) and H2O2 (0.2%). To chelate iron completely, 1.2 molar excess of EDTA was always use [23]. The reaction mixture was incubated for 20 min at ambient temperature. The supernatant was precipitated with an equal volume of trichloroacetic acid (10%) at 0 degrees Celsius. The precipitate thus obtained was re-dissolved in 1 ml of Na2CO3 (10%) in NaOH (0.5 M) and the final volume made up to 2.5 ml by water. An aliquot of the solution was used for protein determination [24]. The yield of OH radicals generated in the incubations was determined on the basis of degradation of deoxyribose [25]. Bityrosine formation was monitored by measuring fluorescence at 325 nm (excitation) and 415 nm (emission) according spectrophotometer [26].
Tensile strength testing of the material
Tensile testing was conducted using Instron 5565. Following American Standardized Testing Materials Standard D3039, rectangular samples were approximately 6-8 mm in length, 1 mm in width a 1 mm in thickness, and tested with a gauge length of 3.5 ± 0.4 mm. Samples were elongated at a rate of 1% of gauge length per second. The cross-sectional area of samples was evaluated using Image J image analysis software. Stress-strain curve were developed from the load-displacement curve. Young’s modulus was quantified by finding the slope of the stress strain curve, and ultimate tensile strength was calculated.
Microbiology and TMJ
A type strain of Staphylococcus aureus (ATCC 12600), obtained from the American Type Culture Collection (Manassas, USA) was used as test bacterium for estimating the antibacterial activity of the hydrogels. The antibacterial activity of the prepared chitosan hydrogels were tested using the standard Kirby-Bauer agar disc diffusion method [27]. Five to 6 mm deep Muller-Hinton agar (Oxoid, Basingstoke, UK) plates were inoculated by streaking a standardized inoculum suspension that match a 0.5 McFarland standard and containing 107- 108 colony forming units/ml with a throat cotton swab. For each test sample 500 µg of hydrogel was applied to a 6 mm diameter paper disc. The paper discs were placed on the inoculated Muller-Hinton agar medium and incubated at 37ºC for 24 hours. The diameter of the zones of growth inhibition was measured with a caliper. Each measurement was done in triplicate and the testing of each sample was repeated 3 times. The antibacterial efficacy of the prepared gels were compared to antibiotic sensitivity discs (Mast Laboratories, Merseyside UL) containing 30 µg of tetracycline per disc.
Scanning electron microscope characterization of the bio-active restorative materials
The SEM images were obtained to characterize the microstructure of the freeze-dried gels and are presented in Figure 1. The ‘skin’ of the prepared modified flow able composites can be seen, and the collapse of the surface pores may be due to artifacts (freeze-drying process).
Figure 1: SEM of the surfaces of the materials after polymerization where
a. Hydroxyapatite/Copaiba Oil/Chitosan
b. Hydroxyapatite/Brazilian Propolis/Chitosan
c. Hydroxyapatite/Brazilian Propolis/Chitosan and
d. Hydroxyapatite/Brazilian Propolis/Chitosan.
Free radical defense and bio-functional hydrogels
Inflammation of the temporomandibular joint (TMJ) is often treated with arthrocentesis, which is thought to have an anti-inflammatory effect [28,29]. Several mechanisms elucidating the antimicrobial activity of chitosan have been postulated. The most acceptable mechanism is the interaction between positively charged chitosan molecules and negatively charged microbial cell membranes. The interaction is mediated by the electrostatic forces between the protonated NH3+ groups of chitosan and the electronegative charges of the microbial cell surfaces. The electrostatic interaction results in the following types of interference: a) by promoting changes in the properties of the membrane wall permeability, thus provoking internal osmotic imbalance and therefore inhibiting the growth of the microorganisms and b) by the hydrolysis of the peptidoglycans in the microorganism wall, leading to the leakage of the intracellular electrolytes such as potassium ions and the low molecular weight protenaceous constituents (Eg. proteins, nucleic acid, glucose and lactate dehydrogenase) [30]. Since such a mechanism is based on electrostatic interactions, it also suggests that the greater the number of cationized groups, the higher the antimicrobial activity.
Figure 2: Free radical defense and biomaterials as a function of the BSA solubility (%) at pH 6.7.
One of the advantages of the bioactives over the conventional antibiotic based therapy is the attempt to address an emergence of resistant strains [31], being a challenge to treat fungal infections [32].
Tensile strength of the modified biomaterials
The mean and standard deviation values (MPa) for the tensile strength are summarized in Figure 3.
Figure 3: Tensile strength of the modified biomaterials.
Swelling and bio-additive release for the newly prepared hydrogels
The swelling tests in PBS and in Solution pH 4.0 for 4 days revealed that when reaching the equilibrium swelling degree (ESD), all samples swelled in accordance with (Figure 4a and 4b). Based on the ESD values, the gels can be considered as superabsorbent [33]. A peak of media uptake was observed at the beginning of all curves, and after 1 day of swelling there was a plateau-the ESD. The ESD occurs when the hydration forces (the network stretching by the initial fluid uptake) and the elastic force of the cross-linkages reach the equilibrium [34,35].
As discussed before, high amounts of bioactive led to low degree of crystallinity and low percentage of crystalline phase. When the samples swell, the amorphous chains could have more freedom to move and, if they were not cross-linked, with the help of the media, they could detach from the network to the media, increasing the weight loss [36].
The amount of bioactives (such as Brazilian propolis, Uruguyan propolis, Copaiba oil and Oblepicha oil) release in swelling media was analyzed after 1, 2, 24, and 96h of immersion (Figure 5a and 5b). The propolis release by polymeric systems usually occurs in two steps: the release of certain amounts of propolis in the first day of swelling as well as a prolonged release in some cases [37]. A trend could be observed in all curves after 4 days of immersion: there was a high bioactive release in the initial hours and the cumulative release reached constant values up to 1 day of immersion. No prolonged release was observed.
The amount of bioactive release in swelling media was analyzed after 1, 2, 24, and 96h of immersion (Figure 4b). The bioactive release by polymeric systems usually occurs in two steps: the release of certain amounts of propolis in the first day of swelling as well as a prolonged release in some cases [38]. Nonetheless, the samples released more bioactive to PBS than to Solution pH 4.0, probably indicating that the propolis release can be influenced by the media pH.
Figure 4: Swelling degree (SD) results of Ch/HA and of Ch/HA–bioactive samples, after regular time intervals (1, 2, 4, 24, and 96 h), when immersed in (a) PBS and (b) Solution pH 4.0 for 4 days.
Figure 5: Bioactive cumulative release profile of Ch/HA-bioactive samples. The Ch/HA-bioactive samples were immersed in (a) PBS and (b) Solution pH 4.0 and the propolis delivered was quantified after regular intervals of time for 4 days.
Sample Weight loss in PBS Solution pH 4
CH/HA hydrogel 12.6 ± 5.1 12.5 ± 5.8
CH/HA/Brazilian 15.5 ± 4.8 6.78 ± 2.4
CH/HA/Uruguyan 14.3 ± 4.1 5.9 ± 2.8
CH/HA/Oblepicha 16.4 ± 5.6 6.9 ± 3.1
CH/HA/Copaiba 15.4 ± 5.4 5.9 ± 2.4
Table 1: Total Weight Loss of the CH/HA and the CH/HA–Bioactive Samples After 4 days of Immersion in PBS and in Solution pH 4.0.
Inhibition Zone against Staphylococcus aureous and bioactive hydrogels
All the test samples gave an average inhibition zone larger than the tetracycline control disc, thereby confirming the antibacterial activity of the different nano-diamond combinations against Stapylococcus aureous (Figure 6). Using the Student's T-test (p < 0.01), there was a significant difference between the rest of the samples when compared to each other and the positive control. The hydrogel with the highest antibacterial activity contained bioactive and hydroxyapatite/chitosan and the antibacterial activity was still higher than the tetracycline control disc (Figure 6).
Figure 6: Antibacterial inhibition zones produced by the different bio-active containing hydrogels against Staphylococus aureous.
The process of inflammation plays a crucial role in wound healing. In the event of an injury, inflammatory cells such as the granulocytes, monocytes, and macrophages migrate to the injury site. Fibroblasts are activated by the growth factor that is released, and they produce matrix and new collagen fibrils [39]. But these collagen fibrils do not align with the original connective tissue. Instead, they grow at right angles to the plane of the injury. The integrity of the zone of healing is further compromised as the elastin fibers do not heal at all. Anti-inflammatory medications usually prescribed after injury allays pain and swelling but unfortunately they diminish the healing response [40]. As a result of this incomplete healing, the joint remains painful with normal or even sub normal physical activity. Such a joint may become hyper mobile and prone to re-injury due to incomplete ligament support. Newly designed hydrogels may also be beneficial in TMD management owing to its anti-microbial effect. Chlamydia, Mycoplasma genitalium, Staphylococcus aureus, Mycoplasma fermentans, Actinobacillus actinomycetemcomitans, and Streptococcus mitis have been cultured from the TMJ. The presence of S. aureus in the TMJ synovial fluid has been related to TMD [41].
In this investigation we prepared and evaluated bioactive materials containing Chitosan/Hydroxyapatite, which have proven to be suitability and flexibility of the designer materials to act as an “in vitro” probe to gain insights into molecular origin of TMJ and more specifically excessive damage caused by oxygen centered radicals and the defense “build in” free radical defense mechanism of the functionalized bio-scaffolds on the molecular level and apply the newly developed materials for the development of new therapeutic treatment modalities in the TMJ therapy.
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Copyright: © 2015 V Tamara Perchyonok., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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PMID: 29333536 [PubMed]

PMCID: PMC5766278

EC Neurology
Longer Duration of Downslope Treadmill Walking Induces Depression of H-Reflexes Measured during Standing and Walking.

PMID: 31032493 [PubMed]

PMCID: PMC6483108

EC Microbiology
Onchocerciasis in Mozambique: An Unknown Condition for Health Professionals.

PMID: 30957099 [PubMed]

PMCID: PMC6448571

EC Nutrition
Food Insecurity among Households with and without Podoconiosis in East and West Gojjam, Ethiopia.

PMID: 30101228 [PubMed]

PMCID: PMC6086333

EC Ophthalmology
REVIEW. +2 to +3 D. Reading Glasses to Prevent Myopia.

PMID: 31080964 [PubMed]

PMCID: PMC6508883

EC Gynaecology
Biomechanical Mapping of the Female Pelvic Floor: Uterine Prolapse Versus Normal Conditions.

PMID: 31093608 [PubMed]

PMCID: PMC6513001

EC Dental Science
Fiber-Reinforced Composites: A Breakthrough in Practical Clinical Applications with Advanced Wear Resistance for Dental Materials.

PMID: 31552397 [PubMed]

PMCID: PMC6758937

EC Microbiology
Neurocysticercosis in Child Bearing Women: An Overlooked Condition in Mozambique and a Potentially Missed Diagnosis in Women Presenting with Eclampsia.

PMID: 31681909 [PubMed]

PMCID: PMC6824723

EC Microbiology
Molecular Detection of Leptospira spp. in Rodents Trapped in the Mozambique Island City, Nampula Province, Mozambique.

PMID: 31681910 [PubMed]

PMCID: PMC6824726

EC Neurology
Endoplasmic Reticulum-Mitochondrial Cross-Talk in Neurodegenerative and Eye Diseases.

PMID: 31528859 [PubMed]

PMCID: PMC6746603

EC Psychology and Psychiatry
Can Chronic Consumption of Caffeine by Increasing D2/D3 Receptors Offer Benefit to Carriers of the DRD2 A1 Allele in Cocaine Abuse?

PMID: 31276119 [PubMed]

PMCID: PMC6604646

EC Anaesthesia
Real Time Locating Systems and sustainability of Perioperative Efficiency of Anesthesiologists.

PMID: 31406965 [PubMed]

PMCID: PMC6690616

EC Pharmacology and Toxicology
A Pilot STEM Curriculum Designed to Teach High School Students Concepts in Biochemical Engineering and Pharmacology.

PMID: 31517314 [PubMed]

PMCID: PMC6741290

EC Pharmacology and Toxicology
Toxic Mechanisms Underlying Motor Activity Changes Induced by a Mixture of Lead, Arsenic and Manganese.

PMID: 31633124 [PubMed]

PMCID: PMC6800226

EC Neurology
Research Volunteers' Attitudes Toward Chronic Fatigue Syndrome and Myalgic Encephalomyelitis.

PMID: 29662969 [PubMed]

PMCID: PMC5898812

EC Pharmacology and Toxicology
Hyperbaric Oxygen Therapy for Alzheimer's Disease.

PMID: 30215058 [PubMed]

PMCID: PMC6133268

News and Events

August Issue Release

We always feel pleasure to share our updates with you all. Here, notifying you that we have successfully released the August issue of respective journals and can be viewed in the current issue pages.

Submission Deadline for September Issue

Ecronicon delightfully welcomes all the authors around the globe for effective collaboration with an article submission for the September issue of respective journals. Submissions are accepted on/before August 15, 2020.

Certificate of Publication

Ecronicon honors with a "Publication Certificate" to the corresponding author by including the names of co-authors as a token of appreciation for publishing the work with our respective journals.

Best Article of the Issue

Editors of respective journals will always be very much interested in electing one Best Article after each issue release. The authors of the selected article will be honored with a "Best Article of the Issue" certificate.

Certifying for Review

Ecronicon certifies the Editors for their first review done towards the assigned article of the respective journals.

Latest Articles

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