Keywords: Free radicals; Dental restorative materials; Fucoidan; Chitosan; Bioactives
Functionalized biomaterials are ideal candidates to address problems associated with adhesion to biological tissues (soft and hard), due to the unique ability to build in suitable physical properties (elasticity, tensile and adhesive strength), biocompatibility and biodegradability in contact with physiological fluids [3,4].
The process of wound healing involves a series of cellular and cytokine-mediated events results in the generation of Reactive Oxygen Species (ROS), which have been found to play a deleterious role [5,6]. The surface application of substances with free radical scavenging properties has shown to significantly improve wound healing and protect tissues from oxidative damage [7,8].
Copaiba oleoresin is widely used as a popular medicine, through topical and oral administration [8,9]. It has various ethnopharmacological indications, including but not limited to wounds, asthma, as an antiseptic for wounds, skin ulcers, aching joints, general inflammations, as a tonic and to treat ulcers and other digestive diseases, and cancer [10,11].
Oblepicha oil has been used in traditional Chinese medicine since the Tang Dynasty, going back more than 1000 years [12,13]. This plant has been used extensively in the oriental traditional system of medicine for treatment of asthma, skin diseases, gastric ulcers and lung disorder [14,15]. A wide spectrum of pharmacological effects of oblepicha have been recently reported, including antioxidant, immunomodulatory, anti-atherogenic, anti-stress, hepatoprotective, radioprotective and tissue repair [16,17].
Chitosan has been used as a wound dressing material due to its superior tissue or mucoadhesive property hemostatic activity, low toxicity, relevant biodegradability and anti-infection activity [18-22]. Chitosan is a cationic polysaccharide and its adhesive properties are mainly based on ionic interactions with tissues or mucus layers. Low-molecular-weight chitosan is particularly known to facilitate closer interaction with the surface of the epithelial cells. Despite the advantages, the rigid crystalline structure of chitosan makes it hard to dissolve in water, and this has partially retarded its potential for such application. Modification of the chitosan with PEG can enhance the water solubility of chitosan and permit the formation of chitosan-based hydrogels by crosslinking of the PEG [23].
Fucoidan is a sulfated polysaccharide that contains L-fucose and sulfate. It is commonly found in marine brown seaweeds [24]. Fucoidan can increase the level of alkaline phosphatase (ALP), type-1 collagen expression, osteocalcin and BMP-2 and even helps in mineral deposition associated with bone mineralization [25].
As part of our continuous interest in developing functionalized biomaterials we report the synthesis, characterization and application of the newly developed chitosan-fucoidan biocomposites with additional bioactive components of oblepicha oil and copaiba oil as intra-dental and wound healing applications such as bio-adhesion, dentin bond strength and free radical defense mechanism for the compounds in the oral environment.
Gel formulation |
|
Bio-scaffold Chitosan or Fucoidan (% w/w) |
Oblepiha oil
|
Copaiba oil |
pH |
Chitosan |
Gel-1 |
5 |
0 |
0 |
6.14 |
chitosan/ oblepicha oil
|
Gel-2 |
5 |
1 |
0 |
6.35 |
Fucoidan |
Gel-3 |
5 |
0 |
0 |
6.01 |
fucoidan/oblepicha oil
|
Gel-4 |
5 |
1 |
0 |
6.15 |
chitosan/copaiba oil
|
Gel-5 |
5 |
0 |
1 |
6.23 |
fucoidan/ copaiba oil |
Gel-6 |
5 |
0 |
1 |
6.43 |
Further studies were conducted to evaluate and quantify the antioxidant potential of Oblepicha oil, Copaiba oil and combination with chitosan and fucoidan for the purpose of determining the stability of their activity and also correlating the micro-encapsulating influence of the chitosan on stability and efficacy of corresponding antioxidants.
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 [20]. 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 Oblepicha oil, Copaiba oil and corresponding chitosan and fucoidan complexes) tested to reach required concentration as shown in results, EDTA (0-4.8mM), Fe(NHs4)2(SO4)2 (0-4mM), ascorbate (4 mM) and H2O2 (0.2%). To chelate iron completely, 1.2 molar excess of EDTA was always used [21]. 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 [22]. The yield of OH radicals generated in the incubations was determined on the basis of degradation of deoxyribose [23]. Dityrosine formation was monitored by measuring fluorescence at 325 nm (excitation) and 415 nm (emission) according to the spectrophotometer [24].
Determination of gel pH: One gram of the prepared gels was accurately weighed and dispersed in 10 ml of purified water. The pH of the dispersions was measured using a pH meter (HANNA instruments, HI 8417,Portugal).
Morphology of the gels: The samples were prepared by freezing in liquid nitrogen for 10 min, and then were freeze-dried for 24 h. The prepared samples were fractured in liquid nitrogen using a razor blade. The fractured samples were attached to metal stubs, and sputter coated with gold under vacuum for SEM. The interior and the surface morphology were observed in scanning electron microscope (SEM, Hitachi S-4800, Japan).
Gel stability: Stability of the gel formulations was also investigated. The organoleptic properties (color, odor), pH, drug content, and release profiles of the gels stored at 20°C were examined on days (0, 15, 30 and 178).
SR = (WS - Wd) / Wd * 100%
In this way were 90 teeth samples (each containing 2 studs) prepared and divided into 18 groups of 8 each, A-T (Table 2) and stored in a solution of artificial saliva. These groups was then
Group A |
37% of phosphoric acid +primer+ Bonding immediately (negative control) |
Group B |
Self-etching primer + Bonding immediately (positive control) |
Group C |
Oblepicha oil (0.005g)+ primer+ Bonding immediately |
Group D |
Copaiba oil (0.005g)+ primer+ Bonding immediately |
Group E |
Gel1+primer+ Bonding immediately |
Group F |
Gel2+primer+ Bonding immediately |
Group J |
Gel3+primer+ Bonding immediately |
Group K |
Gel 4 + primer +Bonding immediately |
Group L |
Gel 5 + primer +Bonding immediately |
Group M |
Gel 6 + primer +Bonding immediately |
Group L |
Oblepicha oil (0.005g)+ Bonding immediately |
Group M |
Copaiba oil (0.005g)+ Bonding immediately |
Group N |
Gel1+ Bonding immediately |
Group O |
Gel2+ Bonding immediately |
Group P |
Gel3+ Bonding immediately |
Group R |
Gel4 + Bonding immediately |
Group S |
Gel5 + Bonding immediately |
Group T |
Gel6 + Bonding immediately |
Hydrogel |
Adhesive Force(N) ± SD (Dentin) |
Work of Adhesion(N.cm) ±SD (Dentin) |
chitosan/oblepicha |
1.78± 0.41 |
6.01 ± 0.25 |
Chitosan |
1.12± 0.19 |
3.45± 0.15 |
Fucoidan |
1.18± 0.12 |
3.54± 0.18 |
Oblepicha oil |
1.61± 0.11 |
2.95± 0.16 |
Copaiba oil |
1.58± 0.16 |
2.45± 0.14 |
Fucoidan/oblepicha |
1.85± 0.41 |
6.25 ± 0.30 |
chitosan/copaiba |
1.92± 0.32 |
6.75 ± 0.32 |
Fucoidan/Copaiba |
2.10± 0.41 |
6.98 ± 0.31 |
Hydrogel |
Adhesive Force(N) ± SD (Skin) |
Work of Adhesion(N.cm) ± SD (Skin) |
chitosan/oblepicha |
3.78± 0.41 |
16.01 ± 0.45 |
Func/oblepicha |
2.85± 0.41 |
18.25 ± 0.50 |
Chitosan |
1.22± 0.13 |
3.75± 0.15 |
Fucoidan |
1.28± 0.12 |
3.64± 0.16 |
Oblepicha oil |
1.81± 0.14 |
3.25± 0.14 |
Copaiba oil |
1.58± 0.16 |
2.65± 0.16 |
chitosan/copaiba |
3.92± 0.32 |
15.75 ± 0.62 |
Fucoidan/Copaiba |
4.10± 0.41 |
18.98 ± 0.61 |
Although the correlation between the force and work of adhesion was noticeable for all samples this performance can be expected because of the well-known intrinsic bio-adhesive properties of this material. Also ionic vs covalent bonding of the chitosan: therapeutic agent complex may depend on the pH of the environment as the -COOH groups in substituents of oblepicha or copaiba oils ionize at alkaline pH and form covalent "amide" linkage at low pH. The adequate water absorption capacity, together with its cationic nature, which promotes binding to the negative surface of skin or dentin structure, can also explain these results. Hydration of the polymer causes mobilization of the polymer chains and hence influences polymeric adhesion [18-22]. Appropriate swelling is important to guarantee bio-adhesion; however, over-hydration can form slippery nonadhesive hydrogels [18-22].
Chitosan and fucoidan are potent antioxidants with multiple free hydroxyl groups [18-24]. These hydroxyl groups can form bridge-type hydrogen bonds within the side chains of hydroxyl, carboxyl, amino or amide groups of the collagen molecules [18- 24]. The formation of these hydrogen bonds is the reason of the stability of chitosan-collagen or fucoidan: collagen interaction [24,25]. By positioning itself between collagen molecules, host: guest complex formed by chitosan: bioactive agent (oblepicha or copaiba oils and its active in) can potentially also form ionic bonds, as well as covalent bonds with collagen fibrils [24,25]. Furthermore, in the process of formation of hydrogen bonds host: guest complex, molecules can replace the water molecules bound to collagen in the extra-complex compartments [24,25]. This study has shown that newly developed hydrogels are capable of improving the modulus of elasticity of demineralised dentine.
SEM images of the dentin surface exposed to BIOF-IPNs Gels 1-4 for 2 weeks in artificial saliva. The surface of the dentin has been significantly influenced by the treatment with bioactive containing gels, illustrating previously reported unique properties of the chitosan and fucoidan in promoting the formation of the hydroxyapatite crystals as demonstrated in figures 4A-4D.
Interestingly, the surface of the dentin has been significantly influenced by the treatment with bio-active containing gels, confirming previously reported unique properties of the chitosan in promoting a formation of the hydroxyapatite crystals, as well as prevent demineralization of enamel and dentin, which has been reported previously (Figure 4) [18-22]. The potential applications of chitosan and fucoidan in dentistry include strengthening of the collagen matrix, increasing resin–dentine bond strength, inactivation of collagen-bound proteases and remineralization of root caries [18-22]. The detailed investigation of the potential mechanism is currently being researched in our laboratory.
The stability of bio-active oil-loaded chitosan complexes has been measured during storage and no significant decomposition observed after 6 month storage at room temperature (24°C).
We reported earlier that 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
We adopted the method for recording changes in water solubility of the model protein Bovine Serum Albumin (BSA) exposed to free radicals generated by an inorganic chemical system. As clearly demonstrated by the Figure 6, upon exposure to standard H2O2 in the form of Fe2+/EDTA/ H2O2/ascorbate used as a control, upon incorporation of the chitosan substituted
The present results demonstrate the capability of the newly designed hydrogel system to play an important role as a "build in" free radical defense mechanism, and acting as a "proof of concept" for the functional multi-dimensional restorative repair materials. Further biological evaluations of the above materials such as cytotoxicity and antimicrobial properties are currently being evaluated in our laboratories.
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