Biodegradation of an injectable treated dentin matrix hydrogel as a novel pulp capping agent for dentin regeneration

Nowadays, regenerative dentistry is a promising area for dental tissue regeneration. Teeth affected by dental caries, traumatic dental injuries or even iatrogenesis could be benefited by this field advancing. Direct pulp capping (DPC), as an important treatment procedure for pulp exposure, requires adequate protection of the exposed pulp tissue without sacrificing its vitality and functions [1]. Regenerative dentistry based on tissue engineering allows to maintain not only structure of dentin-pulp complex but also its function, by stimulating dentinogenesis and restoring healthy tissues. The strategy focuses on creating scaffold-cell construct for inducing dental tissue regeneration [2, 3].

Many materials have been used to create scaffolds for dentin regeneration in DPC procedure. However, few have succeeded in obtaining complete dentin tissue [1]. It may be the case that while these materials support cell growth and mineralization, they are not capable of inducing differentiation towards an odontogenic specialization [4]. In order to achieve the perfect imitation for dentin regeneration, scaffold derived from dentin tissue is the best choice. A novel injectable mixture termed Treated Dentin Matrix Hydrogel (TDMH) combining alginate hydrogel as the matrix phase and TDM powder has been introduced as a promising material for restoring dentin defect in DPC [5]. It is not only a potential material satisfying the physical and chemical characteristics of a standard scaffold, but also the demand inductive factors for dentin-pulp complex regeneration [6‐9]. As a result, TDMH based scaffold facilitated dentinogenesis to reconstitute normal tissue continuum at the pulp-dentin border [5].

An ideal scaffold for tissue regeneration would be made of biodegradable material with good mechanical strength. Each material offers a unique structure, chemistry, composition, and degradation profile [10]. The optimal degradation rate of substrate is a crucial factor for superior tissue regeneration. The materials used for hydrogel formation must degrade by time to release the contents and create space for new tissue formation. Ideally, the degradation rate should coincide with the rate of new tissue formation. Rapid degradation will cause scaffolds to lose their carrier function for cell growth, whereas a slow degradation rate can decrease the available space and impede new tissue formation [11].

Alginate forms a hydrogel by the interaction between G-blocks that associate to form tightly held junctions in the presence of divalent cations such as Ca²⁺, Sr²⁺ and Ba²⁺. The ionic crosslinked alginate hydrogel is usually weak and loses its mechanical integrity over time due to the reversible crosslinking and the outward flux of ions from the hydrogel [12]. Alternatively, the balance between resorption of dentin matrix and new dentin formation on matrix is critical for optimal dentin regeneration, and partially demineralized dentin matrix used in this study is thought to have optimal conditions [13].

In our previous publications [5, 14], we demonstrated that TDMH contributed to dentin regeneration and vital pulp conservation. However, to date, no study evaluated the physiological biodegradation of this novel injectable material. Hereby, we present complimentary data specifically focused on assessing homogeneity and mechanical properties of this novel scaffold and its biodegradability in vitro and in vivo when used in restoring dentin defect in DPC procedure.

Regeneration of the dentin–pulp complex is of paramount importance to regain tooth vitality. The hybrid materials have undergone significant research to better mimic the physiological, biochemical, and physical cues of native tissues. At a minimum, the ideal biomaterial for tissue engineering needs to meet the following essential criteria: biodegradability, proper elastic modulus, and good biocompatibility [24, 25]. The present histological study was conducted under controlled conditions that used intact teeth in healthy patients to avoid the interference by confounding factors and by strict protocol for DPC procedure which included rubber dam isolation, disinfection of the operative field using 2% chlorhexidine-gluconate, and standardized pulp exposure size, as have been done in previous studies [5, 22].

The hydrogel scaffolds used for dentin–pulp complex regeneration should be degradable upon implantation to be replaced by newly formed tissues [26]. Ideally, the rate of scaffold degradation should be compatible with the rate of newly formed tissue. Too-rapid scaffold degradation can compromise its cell-supporting function, while a too slow degradation rate can hinder new tissue formation [11, 27]. Our previous study extensively focused on the dentinogenic properties of the novel injectable TDMH to restore normal tissues in dentin defect areas after DPC and revealed teeth treated with TDMH showed a positive trend to dentin regeneration [5]. However, the current study specifically investigated in vitro and in vivo degradation behavior of TDMH essential for new dentin regeneration and the various parameters involved in subsequent degradation of the hydrogels in the oral fluids, homogeneity, and the mechanical stability.

Sodium alginate (SA) is a common term used for a family of unbranched polymers composed of 1,4-linked β-d-mannuronic and α-l-guluronic acid residues in varying proportions, sequence, and molecular weight. Its gelation takes place when divalent cations (usually Ca²⁺), interact ionically with blocks of guluronic acid residues, resulting in formation of the three-dimensional network which is usually described by ‘egg-box’ model [28]. As the encapsulation method is mild, and done at room temperature in aqueous medium, several sensitive drugs, proteins, living cell, enzymes have been successfully released through SA hydrogels [29, 30]. Previous studies have confirmed TDM could release dentinogenesis related proteins that not only play vital role in the proliferation and differentiation of DPSCs into odontoblasts, but also form a network to form new dentin and regulate mineralization during dentin development and regeneration [31, 32]. As a result, TDMH based scaffold induced a natural biological regeneration to reconstitute normal tissue continuum at the pulp-dentin border.

Tissue engineering scaffolds require structural uniformity not just for uniform cell distribution but also for well-controlled material degradation. The diffusion of nutrients into all parts of the gel and the elimination of metabolic wastes are ensured by uniform pore size and distribution. If the structure is homogeneous, mechanical properties are more consistent across the hydrogel and between samples as concluded by Espona et al. [15]. The findings of the present study showed that TDMH was homogenous ensuring degradation behavior equal all over the hydrogel mass.

Moreover, adequate mechanical properties are essential for hydrogel scaffolds to provide support for the cells and withstand mechanical loading [33, 34]. The reduction in the mechanical strength and degree of crosslinking of the hydrogels may lead to a faster degradation rate after arriving at exposure site. So, they cannot reside for a sufficiently long time in the defect site. Matrix stiffness also affects the phenotype and differentiation pathway of mesenchymal stem cells (MSCs) [35]. Hydrogel mechanical properties are influenced by hydrogel composition, concentration, method of fabrication in addition to crosslinking density, porosity and hydrogel modification [36‐38]. Decreased porosity and increased crosslinking density can be used to increase the material's mechanical properties, but these changes may compromise cellular response and degradability [33, 39]. Therefore, a balance should exist between hydrogel mechanical properties and degradability [40]. Alternatively, the current study revealed that TDMH ranged within the described native hard tissues elastic moduli values (25–40 kPa) [41, 42], validating this novel injectable scaffold as 3D matrix able to mimic the characteristics of native hard tissues that encouraged odontogenic/ osteogenic differentiation as increasing the calcium content and crosslinking density could allow tailoring the mechanical properties.

Regarding the in vitro degradation behavior of TDMH, after the hydrogels were immersed in PBS for 4 weeks, pores of the hydrogels swelled and became larger than those of the as-obtained hydrogels (week 0). After fourth week the hydrogels lost its porous structure and at the end of the degradation experiment (week 8), TDMH became not porous. While after 8 weeks, it was completely degraded. Shahriari et al. [43] studied the in vitro degradation of alginate scaffolds and concluded that the scaffolds generally maintained their channels and bulk geometry for at least 28 days. Moshaverinia et al. [18] investigated the degradation behavior of hydrogel based on oxidized sodium alginate with different degrees of oxidation in PBS at 37 °C showing more degradability of oxidized alginate hydrogels and after 4 weeks of storage in PBS, almost 50% of the initial weight of the alginate hydrogels has been lost. Other studies have performed cell attachment studies on alginate for up to 10 weeks and suggested techniques such as adding NaCl to preserve material integrity [44, 45]. Moreover, the hydrogel properties can be further regulated by multifunctional crosslinking molecules, which provide a wider range and tighter control over degradation rates, as demonstrated by Lee et al. [46]. In the current study, such degradation rate of TDMH may be attributed to the combined crosslinking of calcium ions to SA and the presence of TDM powder which acting as an inorganic crosslinker that could restrict the movability of the SA polymer chains and therefore slow down their degradation rates [47].

Furthermore, the histological findings in this study showed SA hydrogel was totally degraded after 8 weeks and replaced with newly formed dentin, leaving partial degradable TDM at the exposure site, after that it was significantly replaced by newly formed dentin. These findings correlate well with its in vitro degradation behavior for the first 8 weeks of degradation, in which SA hydrogel totally degrades after 8 weeks. This comes in accordance with Lee et al. [48] who concluded that ionically crosslinked alginate hydrogels disintegrate progressively in vivo due to the release of the divalent cations crosslinking the hydrogel into the surrounding media in exchange with the monovalent cations, such as sodium ions.

Higher dentin area induced by TDMH was observed at 16 weeks compared with 2 weeks after DPC. Moreover, histomorphometric measurements revealed a significant reduction in TDMH area fraction with a simultaneous increase in new dentin area with a significant negative correlation. Until now, the exact mechanisms of demineralized dentin in vivo degradation have not been fully clarified. Both enzymatic digestion and cellular phagocytosis are the dynamic processes involved in the organ absorption [49, 50]. Partially demineralized TDM used in this study is thought to have optimal conditions as biodegradable scaffold for dentin regeneration as reported by Koga et al. [13]. This could be explained by the partial demineralization of TDM leads to superficial decalcified dentin that expose the organic matrix with an inner core of mineralized dentin that could be enzymatically degraded. The exposed collagen matrices were found to be degraded by enzymes under different physiological and pathological conditions [50]. In vitro dentin bio-absorption by collagenase digestion was also successfully evaluated [10].

Additionally, mineralized tissues are known to be degraded by multinucleated giant cells via phagocytosis [51]. The cellular phagocytosis of TDM by giant cells was revealed in some histological sections in the present study. Thus, we hypothesize that TDMH in our study was degraded by cellular and enzymatic digestion altogether. This comes in accordance with those of Kabir et al. [10] who concluded that gradual absorption of the DDM by multinucleated giant cells and the presence of osteoblasts suggests active bone remodeling at the grafted site.

Nevertheless, the size and shape of dentin matrix particles are also important parameters that can be altered in order to tailor the degradation profile of the composite for a specific application. The current study showed that TDM with large dentin particles approximately 500 μm particles sized powder accommodating the defect size, successfully acted as a bio-absorbable scaffold. The histological findings clearly demonstrated significant bio-absorption of the TDMH at 16 weeks compared to 2 weeks. The loss of the structural integrity of the scaffold confirmed the physiological absorption. This comes in agreement with Koga et al. [13] and Togari et al. [52] who demonstrated that the larger the particle size of TDM, the more prominent the bone regeneration as smaller particles implanted in bone defects had more resorbability, and they may have been resorbed in vivo before the initiation of new bone formation. In contrast, Chen et al. [53] used hTDM as a paste for DPC with smaller particle size < 76 μm and concluded that TDM paste could achieve the dental pulp reparative procedure but with short follow up period and no emphasis on its degradability.

Newly formed dentin in the defect site was directly connected with TDM and native dentin. Altogether, the results proved that TDMH acted as a biodegradable scaffold, and absorption of TDM provided sufficient spaces for the newly generated dentin into defect areas after DPC procedure with degradation rate matched the rate of new dentin formation. This study highlights TDMH might contribute as a novel scaffold for dentin engineering. However, the use of different teeth for analysis (not a repeated measure), and the presence of a RMGIC liner that may influence cell fate and tissue formation could be addressed as limitations to the present study. Therefore, further research is recommended at earlier and later endpoints, and the applicability of the scaffold for large defects should be further studied. Cryopreservation of TDM scaffold is also recommended to overcome time-consuming preparation technique.

TDMH was homogenous and exhibited significant stability and almost completely recovered after excessive compression. TDMH generally maintained their bulk geometry throughout 7 weeks. The in vivo response to TDMH was characterized by extensive degradation of the hydrogel and dentin matrix particles and abundant formation of new dentin. Higher dentin area induced by TDMH was observed at 16 weeks compared with 2 weeks after DPC. The degradation rate of TDMH matched the rate of new dentin formation.