Traditional vital pulp treatments practiced in dentistry do not apply tissue engineering principles, that it relies on the formation of the dentin bridge on a tissue reaction to the alkaline substances applied at the exposure site. Since its use in dentistry in 1921, calcium hydroxide has been considered the gold standard for direct pulp capping(36). calcium hydroxide promotes hard bridge formation slowly, dissolves rapidly after marginal leakage and may dissolve during acid etching before the resin filling, and it does not chemically associate with the tooth or with the restored resin(37).
When calcium hydroxide applied directly to the pulp tissue, necrosis of 2mm depth occurs in the pulp tissue and inflammation in adjacent tissues due to the high pH of the calcium hydroxide(38). The formation of hard tissue occurs at the contact area of necrotic tissue and inflamed tissue(39). Under the necrosis layer, the pulpal stem cells differentiate into the odontoblast like cells, as a tissue reaction, and the dentin bridge matrix is placed(37).
For these reasons, the aim of this study was to try to regenerate dentin by a combination of bioactive and biodegradable polymers. A three-dimensional porous scaffold of a combination of biopolymers analogous to the extracellular matrix for dentin tissue engineering was fabricated. Tissue engineering is a multidisciplinary science that applies chemistry, materials engineering and medicine, aiming to repair and replace damaged or diseased tissues and organs(40). It requires three-dimensional, porous polymeric scaffolds that create the appropriate mechanical, structural and biological environment for tissue repair and regeneration(41, 42). Polymers used as a scaffold must be able to mimic the biological structure and functions of the ECM, which is a diverse composition of saccharides, proteins and signaling molecules(43), in terms of chemical and physical structure(44). Where ECM is responsible for cellular metabolism and forming new tissues(14, 44). Biopolymers were preferred in tissue engineering applications because of reduced inflammatory reactions, non-cellular toxicity, and biodegradation by blood enzymes in vivo(45).
In our study, we chose to crosslink chitosan and collagen as a result of the excellent properties of both polymers in tissue engineering and drug delivery. Because of its structural similarity to glycosaminoglycans in ECM and the possibility of forming it as porous scaffolds with morphological and mechanical properties similar to those of collagen scaffold, chitosan provides a good choice for tissue engineering applications(46, 47). Studies have shown that chitosan is a suitable candidate for tissue engineering due to non-toxicity, biocompatibility, and biodegradability(48). It also has the ability of histological regulation and displays the ability to stimulate cell proliferation(49). Chitosan has been shown to be highly compatible with osteoblast cells in vitro(50). This ability appeared in various formulations of chitosan(51). Good biological compatibility between neurons and chitosan has been reported, and it was found that chitosan is the best membrane for the proliferation of these cells(52). Chitosan has shown a characteristic enhancement of the vitality of neuronal cells and the results of in vitro cell culture indicate selective adhesion of Schwann cells(53).
Since collagen scaffolds are a distinctive template for renewable cell growth in vitro and in vivo, crosslinked collagen scaffolds have been used in regenerative medicine to promote the regeneration/repair of diseased and damaged tissues(54, 55). Extensive researches have been done on collagen scaffolds, these scaffolds have been proven to support cellular growth and researches have shown that collagen types-I can form a scaffold that resembles or even fully mimics the structural and biological properties of natural ECM collagen(56).
For our study, we selected high-deacetylated chitosan that was calculated by FT-IR spectroscopy to be 87% (data not shown). This implies that each 1mol of chitosan contains 0.87 mol of free positive amine groups allowing it to crosslink with molecules that have the opposite charge.
It has been found that a high degree of deacetylation enhances the activity of chitosan in each of its antimicrobial properties against a wide range of bacteria and fungi due to a strong positive charge that created by the protonation of free amino groups(57), positive charge adhering negatively bacteria and fungi to chitosan and prevents nutrients from reaching them. It was also found that chitosan with a high degree of deacetylation enhances the activity of fibroblast cells during wound healing processes for the same reason, high positive charge(58).
Since free radicals scavenging is one of the most useful properties to be achieved in biomedical compounds that used in regenerative medicine, it prevents the destruction of membrane fats, proteins, and DNA by radical oxygen reactive molecules(59). Chitosan has been preferred over other biopolymers in a tissue regeneration field due to its ability to dismantle active free radicals through fixation them by free amino and carboxyl groups in chitosan(60).
Dexamethasone, as known, is a synthetic glucocorticoid used clinically as an anti-inflammatory drug. It has been hypothesized also that dexamethasone increases the response of stem cells to materials used for differentiation61). Increased vitality and proliferation of stem cells derived from human bone marrow MSCs have been reported as a result of ongoing dexamethasone therapy(32).
Among biopolymers, collagen and polysaccharide, chitosan, are suitable for topical drug delivery systems, providing the advantage of using them as a natural biological material with tissue healing properties(62, 63). A dug can be linked to polymer matrix by physical methods or by chemical reagents due to the availability of functional groups that is able to interact with the drug in various ways(64). Whereas glutaraldehyde is one of the most important chemical reagents in the field of drug binding to chitosan and polymers containing amine groups(65-67). The physical crosslinking between the drug and the collagen matrix plays an important role in loading and releasing the drug from collagen without adding any crosslinking agents that may be cytotoxic when applying this drug-loaded matrix topically(68). Physical methods of linking a drug to chitosan by electrostatic or hydrogen bonds have been also shown to be effective in controlled drug release(69, 70). This interaction between the polymer and a drug leads to stable production and loading of the drug with great efficacy and prolonged release of the drug; this has been reported in other research(71).
In the present study, we have used freeze-drying technique to fabricate bio-sponge consisting of chitosan and collagen, so as to benefit from the properties of collagen which promotes cellular adhesion, differentiation as well as its hemostatic properties(18, 72, 73), also to benefit from the adhesive, antibacterial, antifungal and hemostatic properties of chitosan(74-76), that justified our selection of both polymers for crosslinking and histologically study.
Since most studies about biopolymer matrix for medical applications used a different ways to crosslinking polymers to form scaffolds by utilizing functional groups in each component within the mixture, crosslinking treatment is one of the most important issues for bio-scaffolds, consideration should be given to the intensity of the crosslinking and the preservation of the biological activity and biological properties provided by each component before crosslinking. There are two types of crosslinking methods; Physical and chemical methods. In the chemical methods group, glutaraldehyde is the most comfortable traditional agent used in the treatment of porous collagen scaffolds, and amino biopolymers(77), which achieves a high and undesirable crosslinking degree and potential cytotoxicity of this agents(67, 78).
Physical methods are an attempt to establish a binding without the introduction of cytotoxic chemical reagents and maintain excellent biocompatibility of tissue engineering materials(79).
Here we used the physical linking method by creating ionic bonds between amino groups of chitosan and carboxyl groups of the glutamic and aspartic residues of collagen.
Fig. 1, 2, 3, 4 show FT-IR results of characterization of the polymer blend and dexamethasone-loaded polymers films synthesized by the solvent cast method showed the forming of electrostatic and H-bonding between collagen and chitosan and the forming of H-bonding between dexamethasone and studied homopolymers and polymer blend. Fig. 5 shows SEM images for sponges also showed a porous structure for homopolymer and hybrid sponges, collagen –chitosan sponge at 1:1 weight ratio showed an average pore size of 100 um that is suitable for cell growth(80), dexamethasone was clearly shown immobilized on the surface and embedded in polymer sponges (Fig. 6).
Figure 1. FT-IR for a: dexamethasone, b: collagen, c: chitosan film.
Figure 2. FT-IR for, a: dexamethasone, b dexamethasone-loaded chitosan film, c: chitosan film.
Fig. 3. FT-IR for, a: dexamethasone, b: dexamethasone-loaded collagen, c: collagen film.
Fig. 4. FT-IR for, a: dexamethasone, b: dexamethasone-loaded chitosan:collagen (1:1) film, c: chitosan:collagen (1:1) film.
Fig. 5. SEM for chitosans:collagen (1:1) sponge.
Fig. 6. SEM for dexamethasone loaded chitosan: collagen (1:1) sponge.
The drugs can be loaded into the polymer matrix or condensed on the surface of the matrix, the maximum loading in the drug formulation can be achieved by incorporating the drug during the formation of pharmaceutical molecules(81). Here we used the physical method of linking the drug to the polymer sponge by hydrogen bonding between dexamethasone and the polymer mixture before lyophilized.
Drug release study from the sponge shown that the release of drug from the hybrid sponge was faster than the chitosan sponge, which ended in 16h, and was slower than the collagen sponge which completed within 5h. The formulation based on collagen-chitosan (1:1 wt %) blend was able to control drug release within 10h(82) . Thus, the dexamethasone loaded -blend of high molecular weight chitosan and collagen, to form a bio-sponge hydrogel for DPC, will have advantages of both collagen and chitosan properties, in addition to the properties of prolonging released dexamethasone, which is about 30 times more effective than cortisone(83).
Our results revealed that the combination of bio-polymers that act as a natural extracellular matrix loaded with a steroidal anti-inflammatory drug, that acts as an osteoconductivity agent, can be considered as an effective alternative to high-alkaline mineral oxides in order to avoid its side effects occurring in a teeth pulp, such as pulp calcifications, tissue burns, and pulp stones. Bio-polymer sponge with a neutral PH activates stem cells to differentiate into odontoplast cells and form the dentin bridge.
The results revealed that the dentin bridge was formed without any inflammatory response in the pulp tissues, maybe due to the release of dexamethasone, and faster than that in the Ca(OH)2 group, with a shorter duration. The dentin bridge was regular and thick under the bio-scaffold, and The odontoblasts layer appeared to exist under the formed dentin bridge, which indicates that the formation of the hard tissue was as a renewed dentin layer and not as a tissue reaction towards Ca(OH)2 alkalinity.