Photocrosslinkable gelatin-treated dentin matrix hydrogel as a novel pulp capping agent for dentin regeneration: I. synthesis, characterizations and grafting optimization

Hydrogels are three-dimensional hydrophilic polymeric networks capable of swelling and retaining a large volume of water [1]. Lately, in situ hydrogel formation has been extensively employed as a carrier for biomedical applications because of its simple formation in any required shape, the lack of organic solvents or chemicals used, and the fact that the viscoelasticity of the in situ hydrogel is similar to the extracellular matrix (ECM), which can mimic the three-dimensional microenvironment of cells, support cell attachment, and induce cell proliferation and differentiation [2, 3]. Thus, hydrogels formed in situ met all typical physiological requirements that could meet the general requirements of a scaffold [1].

Surprisingly, in situ forming hydrogels were widely used for a variety of medical applications, including tissue engineering, medication delivery, wound healing, and tissue regeneration [4‐6]. Nowadays, gelatin-derived hydrogels are widely employed as interesting regenerative scaffolds for mimicking biotissues, creating beds for tissue healing and drug administration, etc. [7]. Gelatin (G) is a natural polymer and one of the derivatives of collagen that could be an appropriate component of scaffolds due to its low immunogenicity and similarity to the extracellular matrix (ECM). Moreover, it improves cell attachment and biodegradability of scaffolds and induces cell proliferation [8]. In recent years, gelatin has been used in interaction with human dental pulp stem cells for bone tissue engineering [9], dentin-pulp complex tissue engineering [10], dentin regeneration [11] , and Dental Follicle Stem Cells for tooth root regeneration [12].

Dentin matrix obtained from extracted teeth has been shown to act as a biocompatible scaffold for attachment, proliferation and differentiation of dental pulp stem cells into odontoblast-like cells [13]. The derived dentin matrix had no bearing on its infectivity because of the avascular and acellular nature of the dense collagenous matrix, thus rendering the allogenic human dentin matrix non-immunogenic [14]. Treated dentin matrix (TDM), as an acellular material, has been thought to be promise in dentin regeneration. Previous research suggested that treated dentin matrix (TDM) extracts contained a variety of extracellular matrix molecules, including dentin sialoprotein (DSP), transforming growth factor-β, decorin, biglycan, and dentin matrix protein 1 (DMP-1) that mediated cell proliferation and odontogenic differentiation [15, 16]. Furthermore, TDM was able to activate stem cells in pulp tissues to engage in pulp healing by promoting the production of restorative dentin, resulting in entire dentin regeneration (dentin, predentin, and the odontoblast layer) [6, 17, 18].

Photo-crosslinking to form hydrogels has attracted considerable interest in the field of tissue engineering. The reason for this interest is that gel constructs have similar water contents to the extracellular matrix and thus allow for efficient nutrient transport, which is important for maintaining cell viability, as well as contributing to biocompatibility by reducing mechanical irritation to the surrounding tissue [19‐21]. The photoinitiators used in visible light mediated hydrogelation have been somewhat limited due to cytotoxicity and solubility issues [22]. Potential sources of biocompatible photoinitiators include free-radical-generating molecules that are naturally found in biological systems. Riboflavin, also known as vitamin B2, is naturally occurring in the body, non-toxic and absorbs light strongly between 330–470 nm, making it an attractive alternative to the current synthetic photoinitiators. Riboflavin is a type II photoinitiator; hence, a coinitiator is required as a proton donor to start the polymerization reaction [23]. For the first time, glycine is combined with riboflavin as a safe and effective natural coinitiator rather than an amine initiator. Glycine is the simplest of amino acids and an integral component of critical biological molecules as well as a central component of many metabolic reactions [24].

The aim of this study is to synthesize gelatin- glycidyl methacrylate/treated dentin matrix hydrogel and optimize the grafting reaction through grafting conditions such as GMA concentration and reaction time. The efficiency of the photopolymerization system has been assessed in terms of its influence on the entire formed hydrogel properties e.g. swelling ratio, degradation degree, hydrogel morphology and thermal Stability. Moreover, the kinetic parameters of copolymers and cytotoxicity were tested.

Photopolymerizable materials are getting popular with extended use and applications with regard to the field of tissue engineering. The visible-light crosslinking method has been intensively utilized in dental clinics for in situ hydrogel material preparation. In the current study, a novel scaffold made from gelatin-treated dentin matrix hydrogel with the capability of being light-cured using a natural photoinitiating system composed of riboflavin and glycine was experimentally prepared and characterized for potential use in the regeneration of the dentin-pulp complex to maintain pulp vitality. Glycidyl methacrylate is a key ingredient in the production of light-cured hydrogels. Both its concentration and reaction time have a significant influence on the properties of prepared hydrogels, such as grafting optimization, swelling degree, degradation rate, biocompatibility, surface morphology, and thermal stability. As a result, the current study specifically investigated the effect of GMA concentration and reaction time on the various properties of gelatin-treated dentin matrix hydrogel in order to select the optimal ratio and reaction time for initiating the in vivo attempts of gelatin-treated dentin matrix hydrogel as a novel photocrosslinkable pulp capping agent for dentin regeneration in dog's teeth.

It was anticipated that increasing the final weight of the grafted hydrogel over the initial weight of the polymer component would demonstrate the grafting process. Evidently, the grafting percent and efficiency rise as the degree of substitution (DS) or grafting degree rises. As reported by Kim et al. [36], the DS of the prepared hydrogel by methacrylate increased with an increase in both GMA concentration and reaction time. This fact was linked to the rise in DS, which was also responsible for a greater degree of crosslinking in the hydrogel matrix. According to Crispim et al. [37] and Kamoun et al. [38], a higher DS denotes a significant amount of methacrylate groups connected to the polymer chains, which results in an increase in initiated sites on gelatin and a large number of monomer moieties attached. Furthermore, as the concentration of GMA and reaction time increased, the yield percent of grafted methacrylate on gelatin gradually increased. This effect might be caused by the gel phase's compactness, which might limit the mobility of polymeric chains and prevent further crosslinking reactions. This is comes in accordance with those of Pitarresi et al. [39] and Kim et al. [36].

One of the most crucial hydrogel features that have to be addressed is swelling, since it affects the surface morphology of hydrogels, the rate of releasing bioactive molecules and the hydrolytic degradation rate through the scaffold. It's interesting to note that the swelling degree (SD) values have drastically increased as a result of lower GMA ratios and they gradually decrease as the GMA content increases. This behaviour was attributed to hydrogels with low macromonomer contents (i.e., 0.048 M of GMA) possessing larger pore sizes than those with high GMA contents, as shown in Fig. 9. The hydrogel's 3D network with higher GMA contents became more compact and tight, which would limit the number of free hydroxyl groups available. As a result, it is much more difficult for water molecules to penetrate the hydrogel network, which results in a lower swelling degree. The increase in crosslinking degree restricts the polymeric matrix expansion, and subsequently less water is absorbed [22]. This finding is entirely consistent with the grafting and dextran-MA hydrogel formation results [36], showing dextran-methacrylate hydrogels displayed a wide range of swelling, ranging from 67 to 227%. As methacrylate substitutions in hydrogels decline, their swelling rises and the hydrogel matrices expand. Tiwari A et al. [40] studied the swelling properties of the photopolymerizable guar gum–methacrylate hydrogels (GG–MA), showed that the swelling ratio initially increased with decreasing methacrylate gel content, and that swelling was inhibited by the more rigid network at higher cross-linking density. This could be attributed to the increasing hydrophilicity of the GG–MA hydrogels resulting from a relatively lower number of hydrophobic poly(methacrylate) kinetic chains formed as a result of the lower degree of cross-linking. Furthermore, poly (γ-glutamic acid) hydrogel exhibited a lower swelling degree from 0.032 to 0.016 as the concentration of crosslinkers increased from 2 to 10% [41]. As the reaction time of prepared hydrogels increased, the degree of swelling significantly diminished. This implies that prolonging the reaction time increases the molecular weight [42], which improved the grafting optimization of G-GMA/TDM hydrogels as there is a linear relation between molecular weight and grafting optimization as mentioned by Yasuhiko Onishi et al. [43]. As grafting optimization increased, the substitution degree of G-GMA/TDM hydrogels and the crosslinking degree increased as well, and the swelling degree decreased [36, 44]. Additionally, this inverse relationship between the swelling degree, prolonging the reaction time and the substitution degree is consistent with the previous swelling results that were reported by Kamoun et al. [22].

The in vitro hydrolytic degradation or weight loss (%) of G-GMA/TDM hydrogels with different concentrations of macromonomers (GMA contents) and different reaction times was investigated in terms of weight loss for 3 weeks in PBS solution (pH 7.4 at 37 °C). Because gelatin is an excellent hydrophilic polymer and because of its porous structure after swelling Fig. 4C, it absorbs PBS buffer. Subsequently, the degradation of G-GMA/TDM hydrogels takes place through bulk erosion at the surface and interior, simultaneously. The weight loss rate of G-GMA/TDM hydrogels with low GMA contents was faster than that with high GMA contents. These results revealed that GMA contents have slowed down the hydrolytic degradation rate and weight loss (%) of hydrogels, which ranged from 41—11%, respectively, with increasing GMA contents. This is owing to high crosslinking density hydrogels related to high GMA contents, which increase the stability of the hydrogels and hinder the degradation rate. These outcomes are entirely consistent with the results of Kamoun et al. [26], who proved that the rate of degradation of PVA-g-GMA hydrogel became faster with decreasing GMA contents compared to the degraded hydrogels with high GMA contents. Moreover, Baier Leach et al. [21], who fabricated photocrosslinked GMA hyaluronic acid hydrogels (GMHA) found the degradation of 5% methacrylated GMHA gels (10.6 ± 2.0%) was significantly faster than that of 7% methacrylated GMHA gels (4.6 ± 0.8% p < 0.001), and the degradation of 7% was faster than that of 11% methacrylated GMHA gels (0.7 ± 0.3%; p < 0.0001). In terms of reaction time, the weight loss of G-GMA/TDM hydrogels with a short reaction time (6 h) is greater. The degree of crossing linking and the substitution degree of G-GMA/TDM hydrogels increase as reaction time increases, as was previously mentioned [36], and the stability of the hydrogels increases; therefore, the degradation rate will be low. It was concluded by Baghban et al. [7] that increasing the GMA concentrations and reaction time to the optimum values enhanced the physicochemical properties of the LED-curable methacrylated gelatin hydrogel.

Toxicology testing is thought to be an effective aspect of dental biomaterials. Ideal biomaterials shouldn't induce adverse reactions or release any hazardous compounds. According to ISO 10993–5 (E) [45], results revealed that the prepared scaffolds with 0.04 and 0.09 M GMA concentrations were biocompatible, as the cell viability (%) remained higher than 70%. The significant decrease in metabolic activity of cells treated with 0.195 and 0.391 M GMA could be attributed to high GMA content hydrogels exhibiting low protein adsorption and some toxic effects, whereas low GMA content hydrogels exhibited the highest safety on human fibroblast cells. This finding is consistent with the findings of Derya Sürmeliolu et al. and Beltrami R et al. [46, 47], who found that GMA has a concentration-dependent cytotoxic effect on fibroblast cells. Hong H et al. [48] showed that the fibroblast cell line NIH3T3 proliferation rate increased due to the elimination of residual unreacted GMA from modified silk fibroin with glycidyl methacrylate (Silk-GMA) by dialysis period. An optimal crosslinking reaction time produces a hydrogel with good physical properties via adequate crosslinking while ensuring optimum cell survival by lowering exposure to toxic reaction products [49]. All hydrogels with different reaction times (6 h, 12 h, and 24 h) were biocompatible except for the hydrogel with a 48 h reaction time, as gelatin is a cationic polymer that could have influenced the findings. According to Shuangcheng Tang et al. [42] and Bryn D. Monnery et al. [50], the molecular weight of cationic polymers will increase as a function of time, and materials with higher molecular weights are more toxic.

Additionally, the surface morphology of the G-GMA/TDM cryogels with different GMA concentrations and different reaction times was prepared by cryo-fixation and cryo-fracturing techniques [51]. The improvement of the morphological surface of hydrogels with high GMA concentration and prolonged reaction time was attributed to the fact that the higher the crosslinking degree, the higher the substitution degree copolymer contains high levels of photocurable methacrylate groups, allowing the formation of hydrogels with high crosslinking density and uniform surface structure. These results are fully consistent with those obtained by Kamoun and Menzel et al. [22], who revealed that the high substitution degree of pullulan-HEMA hydrogels crosslinked by carboxylated camphorquinone folic acid coinitiator under visible light exhibited dense and compacted surface hydrogel morphology compared to the low DS copolymer. Further, Tiwari A et al. [40] examined the morphology of the photopolymerizable guar gum–methacrylate hydrogels (GG–MA), which exhibited a 3-D interconnected open pore microstructure. As the porosity increased with decreasing methacrylate hydrogel content. Thus, guar gum–methacrylate.hydrogels with a higher methacrylate gel content resulting from the higher methacrylation degree percent of the corresponding GG–MA macromonomers had a smaller pore size and a higher pore density.

The thermal gravimetric analyses of pure gelatin and G-GMA/TDM dried hydrogels have been described according to the thermal weight loss (%) and T_onset values. The overall TGA parameters have improved with increasing GMA content and reaction time until 24 h, and afterwards they returned to a lower level with prolonging the reaction time until 48 h. This behavior could be attributed to the grafting of GMA onto gelatin and the prolonged reaction time, which enhanced the thermal stability of the formed hydrogel due to the high crosslinking density and compactness of hydrogels, as the enhanced thermal properties of the G-GMA/TDM with high GMA concentration and 24 h reaction time synthesized hydrogels suggested an effective methacryloyl conjugation leading to a greater amount of covalent crosslinking density [7, 33, 35]. These result are consistent with Kamoun et al. [38] who studied PVA-g-GMA hydrogels with different GMA concentrations (0, 0.025, 0.05, 0.07, 0.09, 0.15 and 0.25 M), all TGA determined parameters of PVA-g-GMA hydrogels have thermally improved due to the increase of the ratio of GMA until 0.09 M later, they return to reduce again with high ratios of GMA at 0.125 and 0.25 M.

According to our research results, the ratio and reaction time of GMA (0.097 M ~ 2 mL for 24 h) have been selected as the optimal ratio and optimal reaction time for initiating the in vivo attempts of G-GMA/TDM as a novel photocrosslinkable pulp capping agent for dentin regeneration in dog's teeth, which will be the second part of this research. As G-GMA/TDM (0.097 M ~ 2 mL for 24 h) synthesized hydrogel, a biocompatible scaffold, this is a key consideration for selecting pulp capping material that is not cytotoxic on dental pulp cells, and another significant concern is an improved swelling degree, as it is a crucial point in preparing hydrogel for dentin regeneration that allows the feasible release of signaling molecules along with calcium and phosphate from the scaffold for tissue mineralization,, as well as offer required features for improving surface morphology, ideal grafting reaction conditions with a convenient degradation rate with better thermal stability.

The novel gelatin-GMA/treated dentin matrix hydrogels are based on a riboflavin visible-light induced system and glycine as a coinitiator for the first time with riboflavin. The synthesis procedure of an uncrosslinked Gelatin- GMA copolymer was described in detail and confirmed via NMR spectra, while the crosslinking of G-GMA/TDM hydrogel was determined by IR spectroscopy. The concentration of GMA and its reaction time were utilized to adjust the properties of the resultant crosslinked hydrogels. As a result, using 0.097 M of GMA content and prolonging its reaction time for 24 h resulted in high crosslinked density hydrogels, high thermal stability, compacted surface morphology hydrogels, and biocompatible hydrogels. Thus, visible-light photocrosslinked G-GMA/TDM hydrogels could be employed as novel biomaterial and scaffold in dentin engineering.