Effect of chitosan-oleuropein nanoparticles on dentin collagen cross-linking

Abstract

BACKGROUND:

The integrity and stability of collagen are crucial for the dentin structure and bonding strength at dentin-resin interface. Natural plant-derived polypehenols have been used as collagen crosslinkers.

OBJECTIVE:

The aims of the study were to develop novel chitosan oleuropein nanoparticles (CS-OL-NPs), and to investigate the CS-OL-NPs treated dentin’s the resistance to enzymatic degradation and mechanic property.

METHODS:

CS-OL-NPs were developed using the ionotropic gelation method. Release and biocompatibility of the CS-OL-NPs were tested. Twenty demineralized dentin collage specimens were randomized into four interventions groups: A, Deionized Water (DW); B, 5% glutaraldehyde solution (GA); C, 1 mg/ml chitosan (CS); and D, 100 mg/L CS-OL-NPs. After 1-min interventions, dentin matrix were evaluated by the micro-Raman spectroscopy for the modulus of elasticity test. Collagen degradation was assessed using hydroxyproline (HYP) assay.

RESULTS:

CS-OL-NPs were spherical core-shape with a size of 161.29 $\pm$ 8.19 nm and Zeta potential of 19.53 $\pm$ 0.26 mV. After a burst release of oleuropein in the initial 6 h, there was a long-lasting steady slow release. CS-OL-NPs showed a good biocompatibility for the hPDLSCs. The modulus of elasticity in the crosslinked groups were significantly higher than that in the control group ( $P<$ 0.05 for all). The specimens treated with CS-OL-NP showed a greater modulus of elasticity than those treated with GA and CS ( $P<$ 0.05 for both). The release of HYP in the crosslinked group was significantly lower than that in the non-crosslinked groups ( $P<$ 0.05 for all).

CONCLUSION:

CS-OL-NPs enhanced the dentin mechanical property and resistance to biodegradation, with biocompatibility and potential for clinical application.

Keywords

Dentin collagen oleuropein cross-linking nanoparticles

1. Introduction

Dentin is a complex tissue composed of minerals, water, and organic components such as collagen. Fibrillar type I collagen, the major organic component of dentin matrix, is of great importance for the functions of dentin by supporting the framework of mineralized tissue, hampering the lesion progression, and arresting biodegrade [1, 2, 3]. However, the collagen could collapse in some conditions, such as demeraliztion caused by acid attack of dental caries, or biochemically degradation by metalloproteinases (MMPs) [4, 5], or cysteine cathepsins [6], which has become the main cause of proteolytic activity of collagen.

Collagen degradation can significantly affect the structural integrity and biostability of dentin, and the bonding strength at dentin-resin interface [7, 8]. A number of studies have investigated a variety of methods to stabilize the collagen, including crosslinkers, remineralization agents, and collagenolytic enzyme inhibitors [9, 10]. In recent years, dentin crosslinking agents have gained great attention for their ability to stabilize collagen by inducing covalent bonds between collagen molecules and improving the biological and mechanical stability of collagen. Among them, some natural products (due to their superior biosafety and bio-compatibility) are more highly desirable than chemical agents for the further application. Polyphenols-a naturally occurring plant metabolite, such as epigallocatechin-3-gallate (EGCG) [11], proanthocyanidins (PA) [12] and hseperidine (HPN) [13], are widely available in peels, roots, seeds and leaves of plants. They have shown effects of facilitating the preservation of substrate shape in scaffolds, enhancing the mechanical property of collagen, and increasing the bond durability of dentin-resin adhesive [11, 12, 13].

Oleuropein (ole) extracted from olive, the most representative catecholic components of olives, contains phenolic acid with benzoyl structure. Its main components are ellagic acid and gallic acid. Oleuropein has demonstrated effects of anticancer, antibacterial, antihypertension, and antioxidation [14, 15] and an inhibitory activity against the oral microbial growth [16]. Oleuropein was also used as a cross-linking agent for bone engineering to enhance the HLC/n –Hap scaffolds [17]. The functional role of oleuropein as a cross-linker in dental tissue, however, is still unclear.

Polyphenol, a chemical active agent with phenolic hydroxyls in structure, has been found to provide a good modification effect on collagen, especially under acidic nonoxidized conditions [18]. However, polyphenol is prone to be oxidized into quinones, in turn, affecting its biological properties and application. In recent years, chitosan has received more attention as a delivery of therapeutic agent for its ideal availability, biocompatibility and biodegradability. The interaction between biodegradable cationic and anionic biopolymers lead to the formation of polyionic hydrogels, with a desired property of agent delivery [19, 20]. The polyphenols-chitosan nanoparticles have demonstrated sustained release of encapsulants and biodegradable characteristics [21, 22].

Chitosan is a hydrophilic biopolymers with free hydroxyl and amino groups that are prone to interact with collagen molecules to form crosslinks and inhibit the collagenase activity [23]. The Type I collagen fiber in demineralization dentin after crosslinked by chitosan nanoparticles showed a significant increase in the resistance to bacterial-mediated enzymatic degradation and hydrolysis [7, 24]. There has been reported a stabilizing effect of chitosan nanoparticles in addition to crosslinker (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide) EDC on the adhesive interface [25]. A combination of chitosan nanoparticles with crosslinkers has exhibited superior mechanical and biological properties of collagen substrates compared with the crosslinkers alone [26]. The effect of chitosan-loaded oleuropein nanoparticles, as a crosslinker on the mechanical and biological properties of Type I collagen, is still poorly understood.

The aims of this study were to develop a novel chitosan-loaded oleuropein nanoparticle (CS-OL-NP) by ionotropic gelation method, and to evaluate the mechanical variation and collagen degradation resistance associated with the crosslinking of the dentin collagen matrix with the CS-OL-NP. The hypothesis of the study was that the CS-OL-NP incorporated in dentin collagen has dual advantages of stabilizing collagen with chitosan and oleuropein and playing a protective role of chitosan on oleuropein to maintain the stable biological activity of oleuropein, thus resulting in an improved structural stability of the collagen matrix.

2. Materials and methods

2.1 Preparation of the chitosan-loaded oleuropein nanoparticles (CS-OL-NP)

The nanoparticles were synthesized by ionotropic gelation method as described in the literature [19]. Briefly, chitosan was dissolved in the solution of hydrochloric acid to obtain the CS solution at a concentration of 1 mg/ml, and the pH was adjusted to 5.5 using NaOH. Then, 1 mL 0.2w/v oleuropein solution was added into the chitosan solution, subsequently 0.25% sodium tripolyphosphate solution was slowly added into oleuropein and chitosan mixture solution with the Volumatic ration of 1:5 under continuous stirring at a speed of 1000 rpm for 30 minutes at room temperature.

2.2 Character of chitosan-loaded oleuropein nanoparticles (CS-OL-NP)

The size and zeta potential of the CS-OL-NP were measured on a Zeta sizer Nano-ZS (Malvern Instruments, Malvern, UK) based on the Dynamic light scattering (DLS) techniques. All measurements were performed in triplicate to calculate the average. The morphology of the CS-OL-NP was observed using the transmission electron microscopy (TEM) (JEM 2100F, JEOL, Japan).

2.3 Measurement of the oleuropein release

Release of CS-OL-NPs in vitro was assessed according to the literature [27]. 5 mL of CS-OL-NP solution was added in 20 mL of phosphate-buffered saline solution (PBS) (pH $=$ 7.4, 37 ${{}^{\circ}}$ C), and then incubated in a shaker at 120 rpm at 37 ${{}^{\circ}}$ C. The concentration of released oleuropein was measured at different time points of 1, 2, 4, 6, 12, 24, 36, 48ï¼Œ60, and 70 hours; aliquots of 2 mL medium was removed and replaced by 2 mL fresh PBS. The amount of oleuropein in the solution was determined by the UV spectrometry at 540 nm. The incubation medium was used to form a calibration curve. All measurements were performed in triplicate.

2.4 In vitro biocompatibility test

Human PDL stem cells (hPDLSCs) were used for the biocompatibility test according to the literature [28]. Periodontal ligament tissues were scraped from the middle of the root surfaces and cultured in minimum essential alpha medium ( $\alpha$ -MEM), containing 10% fetal bovine serum (FBS) and 1% penicillin streptomycin. The cytotoxicity of the CS-OL-NP was evaluated by the Cell Counting Kit-8 (CCK-8) assay. The cells were cultured with different treatments for 1, 3, and 5 days; 10 $\mu$ L of CCK-8 reagent was added into 100 $\mu$ L medium each well for incubation, and the absorbance of the solution was then tested by a microplate reader (Molecular Devices, San Jose, CA, USA) at 450 nm. All experiments were conducted in triplicate.

2.5 Sample preparation

Ethics of the study was approved by the Ethics Committee of Nangjing Stomatological Hospital, China. Written informed consent was obtained from each individual participant. Thirty healthy human third molars, without any caries or crack (age 20–40 years old), were collected and stored in 0.1% thymol solution, and used within one week. The study flowchart is shown in Fig. 1.

Figure 1.

Illustration of the specimen preparation process.

Dentin stubs were prepared by cutting parallel 2 mm below the cement-enamel junction and followed additional cuts in the occlusal-apical direction to remove side walls of the enamel. Then the dentin stubs were sectioned in the mesial-distal direction to obtain the slabs with a dimension of 0.5 mm $\times$ 0.5 mm $\times$ 1.0 mm using a low speed cutting saw (IsoMet, Buehler, Lake Bluff, IL, USA) under constant water coolant.

The dentin slabs were randomly divided into four interventions groups ( $N=$ 15 per group): A, Deionized Water (DW); B, 5% glutaraldehyde solution (GA); C, 1 mg/ml chitosan (CS); and D, 100 mg/L CS-OL-NP.

Specimens were demineralized according to the literature [29] using 1% citric acid (pH 3.8) for 1 min to expose type I collagen fibrils of dentin, and thoroughly rinsed with distilled water, then stored in sterile de-ionized water at 4 ${}^{\circ}$ C. The etched dentin specimens were immersed in respective solution for 1 minute to mimic the clinical situation.

2.6 Raman investigation of the dentin disks

Raman spectra analysis of the demineralized dentin specimens after different interventions were conducted using the Raman spectrophotometer (LabRam HR Evolution, Paris, France). The configuration of the equipment was He-Ne laser with 50 mW power, 785 nm laser wavelength, 10 s acquisition time, 2.0 cm ${}^{-1}$ spectral resolution, and 5 consecutive scans with an accumulation time of 10 s each at each site. The laser beam was focused using an optical lens with a magnification factor of 100.

2.7 Mechanic property test

Ten specimens of each group were fixed on a universal testing machine (Tapezium X type tester, Shimadzu Corporation, Japan) to evaluate the apparent modulus elasticity. A tensile load was applied at a crosshead speed of 0.1 mm/min until failure occurred. A 100-N-load cell with sensitivity of $\pm$ 0.5% was used in this experiment. The stress-strain curves of all specimens were plotted from the load-displacement curves, and data was presented in MPa.

2.8 Collagenase degradation and hydroxyproline assay

Demineralized dentin beams were randomly allocated to the four groups as described above ( $n=$ 5 per group). For the collagenase solution, 5 mg I collagenase with a molecular weight of 110 kDa (Collagenase Type I, Sigma-Aldrich, St. Louis, USA) was dissolved in 50 ml 0.2 M ammonium bicarbonate buffer (pH $=$ 7.5) to a final concentration of 100 ug/ml.

The specimens in each group were treated with the collagenase solution for 48 hours. All treated dentin beams were individually incubated in plastic vials with 2.5 ml collagenase solution per well and shaken at 37 ${}^{\circ}$ C. The collagenase solution was renewed every 12 h during the incubation. The HYP concentration in supernatant was estimated by an Assay kit (Jiancheng, Nanjing, China) according to the manufacturer’s instructions. The amount of HYP was measured using a spectrophotometer (Lambda 365, PerkinElmer, USA) with the absorbance detected at a wavelength of 560 nm. The measurement of the HYP content of each dentin beam was performed in triplicate.

2.9 Statistical analysis

The Kolmogorov-Smirnov and Levene’s tests were used to assess the data normality and homogeneity of variance assumptions. The one-way ANOVA and the Tukey post hoc test were used to analyze the cell viability, apparent elastic modulus, HYP content, and pairwise comparison between groups. The SPSS software (version 17.0) was used for the statistical analyses. A $P$ -value of $<$ 0.05 was considered statistically significant.

3. Results

3.1 Characteristic of the nanoparticles (CS-OL-NPs)

The CS-OL-NPs were spherical core-shape with an average particle size of 161.29 $\pm$ 8.19 nm (Figs 2 and 3). Zeta potential for the CS-OL-NPs was 19.53 $\pm$ 0.26 mV.

Figure 2.

Transmission electron micrograph of the CS-OL-NPs (chitosan oleuropein nanoparticles).

Figure 3.

Particle size distribution of the CS-OL-NPs (chitosan oleuropein nanoparticles).

Figure 4.

The release profile of Oleuropein from CS-OL-NPs.

3.2 In vitro release experiment the nanoparticles (CS-OL-NPs)

The release profile of CS-OL-NPs (Fig. 4, the cumulative percentage release of polyphenols from CS-OL-NPs) demonstrated a rapid burst in initial period, followed by a relative slow release. In the initial 6 h nearly 48% of oleuropein was released from CS-OL-NPs, and thereafter followed by a long-lasting slow release. The cumulative release of oleuropein was about 80% within 70 h.

3.3 In vitro biocompatibility experiment

The GA group showed a significant decrease in the viability of hPDLSCs, indicating a time-dependent cytotoxicity; the other three groups showed a good viability of hPDLSCs (Fig. 5). No significant difference of cytotoxicity (or viability of hPDLSCs) was found between CS and CS-OL-NP groups; and both showed a steady proliferation of hPDLSCs with the culture time.

Figure 5.

Cell viability was determined as using a CCK-8 kit. ${}^{***}p<$ 0.01.

Figure 6.

Representative Raman spectra of specimens after different biomodification treatments.

3.4 Raman analysis

Raman spectra of the dentin collagen in the control and biomodification groups are shown in Fig. 6. Compared with the unmodified collagen, the major bands of type I collagen in the control group did not change significantly (i.e. amide I at 1652 cm ${}^{-1}$ , CH ${}_{2}$ bending at 1450 cm ${}^{-1}$ and amide III at 1250 cm ${}^{-1}$ ), indicating that the backbone structure of collagen was preserved. Treatments with the GA and CS-OL-NPs lead to a significant change in the collagen structure, including the widened amide I ( $-$ 1650 cm ${}^{-1}$ ) and shoulder formation of amide II ( $-$ 1540 cm ${}^{-1}$ ). Collagen treated with GA showed an increased intensity of the Raman peak at 1035 cm ${}^{-1}$ , indicating the pyridinium ring vibration induced by the interaction between GA and collagen.

3.5 Mechanical property of the CS-OL-NPs

Figure 7.

Apparent elastic modulus values of dentin samples in different groups ( ${}^{**}p<$ 0.05, ${}^{***}p<$ 0.01).

Figure 8.

Hydroxyproline concentration of the dentin samples in different groups ( ${}^{**}p<$ 0.05, ${}^{***}p<$ 0.01).

Figure 9.

Chemical structure of the Oleuropein.

The apparent elastic modulus values of each different treatment were illustrated in Fig. 7. After crosslinking, the apparent elastic modulus in group B, C, and D (8.94 $\pm$ 0.44 MPa, 5.99 $\pm$ 0.34 MPa, and 11.74 $\pm$ 0.64 MPa, respectively) were significantly higher than that in group A ( $P<$ 0.05). The multiple comparisons among different groups revealed that the apparent elastic modulus values were significantly higher in group B than that in group D, and group C ( $P<$ 0.05 for all).

3.6 Hydroxyproline assay

The mean HYP concentrations in the chemically crosslinked groups (1.58 $\pm$ 0.04, 2.94 $\pm$ 0.11, and 1.08 $\pm$ 0.13 um/mL) were significantly lower than that in the control group (6.36 $\pm$ 0.53 um/mL) ( $p<$ 0.05) (Fig. 8). The specimens treated with CS-OL-NPs demonstrated a significant lower HYP release than the other crosslinked groups ( $P<$ 0.05). Group B showed a significant higher resistance against biodegradation than group C.

4. Discussion

To the best of our knowledge, this is the first research to investigate the crosslinking effect of CS-OL-NPs on dentin collagen. The findings of the study showed that the CS-OL-NPs could enhance the mechanical property of the dentin. Among various crosslinking agents, glutaraldehyde is widely used as a gold-standard crosslinker to interact with collagen because of its economy, effectiveness, and fast response speed [30, 31, 32, 33]. However, glutaraldehyde has been found to be cytotoxic and calcification of implants, with a limited clinical application [34]. An alternative collagen crosslinker with low cytotoxicity and effectiveness of stabilizing collagen will be beneficial.

Natural products have been found to be promising therapeutic agents in dentistry due to their availability, favorable biosafety, and cost-effectiveness. Oleuropein consists of three structural subunits: a polyphenol (hydroxytyrosol), a secoiridoid (elenolic acid), and a glucose molecule [35]. The deglucosidation and oxidation of oleuropein engenders a poly $\alpha$ , $\beta$ -unsaturated aldehyde which is similar in structure to polymerised glutaraldehyde. Aldehydes have a firm affinity for nucleophilic binding sites such as amino and thiol functional groups of collagen to create intramolecular and intermolecular crosslinks [36].

Although there is a remarkable time-dependent effect of PA-base extracts on the mechanical property of the demineralized dentin [37], the application time is crucial for clinically acceptable. Long time treatment not only influences the clinical relevance but also gives rise to some uncertainties related to the self-polymerization of polyphenolic compound. It has been found that one minute application time of cross linker as a primer of dentin with the use of either self-etch or etch&rinse was an effective time to improve dentin-resin bonding over time [38]. In light of this, in the present study, demineralized dentins were treated by different modifications for one minute to mimic the clinical situation, and the modulus of elasticity and hydroxyproline release demonstrated a positive effect of CS-OL-NPs on the mechanical and biological stability of the dentin collagen. This is in agreement with the literature in which the improved effect of polyohenoles on dentin collagen was found after one minute application [39].

Hydroxyproline is considered as one of the specific amino acids of collagen, which accounts for about 13.4% of collagen protein and is very rare in other proteins [40]. Therefore, hydroxyproline as a characteristic amino acid in collagen molecule can be determined by measuring its content in enzymatic hydrolysate to evaluate the degradation of collagen. In the present study, the glutaraldehyde and CS-OL-NPs groups showed a lower HYP release than the control, indicating a protective effect on the collagen; and the collagen crosslinked by CS-OL-NPs showed the highest resistance to degradation. This may be due to the synergistic effect of chitosan and oleuropein on the collagen biostability. This is also consistent with the previous studies in which the chitosan displayed enhanced mechanical and biostability properties induced by crosslinking [26, 41]. The oleuropein contains multiples glucose and phenol structural units, which is the structural basis of the polyphenols to interact with proteins. The hydrophilic phenolic hydroxyl groups can bond with hydroxyl, carboxyl, amino and amide groups in the protein side chain to form hydrogen [42]. At the same time, the ortho hydroxyl structure of oleuropein makes it prone to form hydrogen bond, which plays a role in improving thermal stability and maintaining the triple helix configuration of collagen [40]; the more phenolic hydroxyl groups, the stronger the hydrogen bonding.

The cross-linking effect of oleuropein is also related to its deglycosylation by glycoside hydrolases (GHs) [43]. GHs is a group of enzymes which have the ability to degrade gycosidic bond between carbohydrates or between carbohydrates and non-carbohydrates that were mainly involved in the degradation of carbohydrate main chains [44]. Previous researches have shown GHs play an important role in the development of supergingival and subgingival biofilm [45]. The increase in activity of several GHs in plaque may serve as a potential biomarker for phenotypic changes associated with dental plaque formation [46]. It has been speculated that when the oleuropein contact with salivary or plaque, the glucose molecule of oleuropein is cleaved by the GHs in the saliva or dental plaque to produce an unsaturated aglycone, the functional unsaturated aglycone react with the amino groups of lysine and the hydroxylysine residues of type I collagen to form the irreversible covalent crosslinking between oleuropein and collagen.

Chitosan contains a large number of free amino groups, providing a number of sites for the formation of complex with collagen to form cross-linking by non-specific interaction and enhance the stability of collagen fiber network. Treating dentin collagen with chitosan nanoparticles could improve the resistance of dentin surface collagen to bacterial degradation [23]. Fang et al. found the photodynamic-activated (PDA) crosslinker functioned chitosan nanoparticles could increase mechanical properties of root dentin by the crosslink effects [41]. Compared with their study, in which the dentin surface was treated by CS and PDA in turn, the dentin samples only need to be processed by CS-OL-NPs in one step in the current research, thus simplifying the operation procedure. In this study, although the exogenous collagenase was used, the release of hydroxyproline was partly due to the effect of crosslinkers on endogenous MMPs and cysteine cathepsins. Further investigation into the inhibitory effect of cross linkers on endogenous MMPs and cysteine cathepsins in dentin is still required. Moreover, chitosan is insoluble in neutral or alkaline environment, and is soluble only under acidic conditions. In the clinical situations such as caries and acid etched dentin, the local pH is acidic which is helpful to the dissolution of chitosan. Therefore this is also a favorable condition for CS-OL-NPs to play a role in acid attack.

The decrease of mechanical properties of the demineralized dentin matrix makes it susceptible to be destroyed by the mechanic damage and collapse by chemical degradation. The mechanic damage helps to remove the protective effect of carboxyl terminal peptide on collagen specific enzymolysis site, and promotes the combination of collagenase and specific enzymolysis site. On the other hand, mechanical damage causes breakage of the crosslinking at the carboxyl end of collagen molecule, which makes the $\alpha$ chain move and destroys the triple helix structure of collagen [36]. Previous studies showed higher fatigue resistance, elastic modulus or other mechanical properties of dentin with crosslinking treatments [39, 41]. The findings from the current study showed the apparent elastic modulus of the CS-OL-NPs group in the study was significantly higher than that of the other groups. This confirms the role of CS-OL-NPs in stabilizing dentin collagen.

Biocompability is a basic requirement for a biomaterial to be used in the clinic. The new CS-OL-NPs in the current study also showed a good biocompability for hPDLSCs. In contrast, an obvious decrease of cell viability was observed in the glutaraldehyde group, which was consistent with a previous study [47]. The cytotoxicity of glutaraldehyde might be attributed to the residues from depolymerization along with uncured molecules [48].

The enhanced stability of collagen is closely related with the duration of crosslinking agents. The accumulated release curve of CS-OL-NPs showed a burst release of oleuropein in the first 6 hour and a slow release thereafter. The initial burst release may be due to the oleuropein adsorbed on the shell of the nanoparticle; the subsequent slow release may be due to the hard hydrophobic shell of chitosan nanoparticles, which limited the rapid release of oleuropein and became a long-last steady release.

The structural changes of demineralized dentin collagen after different treatments showed the incorporation of chitosan did not result in any obvious additional bands in the spectrum, this may be attributed to the spectra of collagen overlap with that of chitosan due to the presence of amid bands in both [49]. In the current study, the CS-OL-NPs modified dentin matrix showed broadening of amide I (1650 cm ${}^{-1}$ ) and emergence of a shoulder at amide II (1540 cm ${}^{-1}$ ). This has been established that the phenolic hydroxyl groups of oleuropein had interaction with amide groups of collagen to form hydrogen bonding, an important evidence of collagen crosslinking according to previous researches [21, 50]. Future studies could consider evaluating whether CS-OL-NPs can be served as the primer of the adhesive or added into acid etchant to promote the dentin-resin bond.

5. Conclusion

CS-OL-NPs enhanced the dentin mechanical property and resistance to biodegradation, with biocompatibility and potential for clinical application.

Footnotes

Acknowledgments

The authors have no acknowledgments.

Conflict of interest

The authors declare that they have no conflict of interest.

Funding

The authors report no funding.

References

Ortiz

Boyce

. Materials science. Bioinspired structural materials. Science. 2008; 319: 1053-54.

Bedran-Russo

Pauli

Chen

S-N

. Dentin biomodification: Strategies, renewable resources and clinical applications. Dent Mater. 2014; 30: 62-76.

Kishen

. Mechanisms and risk factors for fracture predilection in endodontically treated teeth. Endodont Top. 2006; 13: 57-83.

Tezvergil-Mutluay

Mutluay

Agee

, et al. Carbodiimide cross-linking inactivates soluble and matrix-bound MMPs in vitro . J Dent Res. 2012; 91: 192-196.

Mazzoni

Mannello

Tay

, et al. Zymographic analysis and characterization of MMP-2 and -9 forms in human sound dentin. J Dent Res. 2007; 86: 436-40.

Scaffa

Breschi

Mazzoni

, et al. Co-distribution of cysteine cathepsins and matrix metalloproteases in human dentin. Arch Oral Biol. 2017; 74: 101-7.

Kishen

Shrestha

, et al. Characterizing the collagen stabilizing effect of crosslinked chitosan nanoparticles against collagenase degradation. Dent Mater. 2016; 32: 968-77.

Liu

Dusevich

Wang

. Proanthocyanidins rapidly stabilize the demineralized dentin layer. J Dent Res. 2013; 92: 746-52.

Prakki

Xiong

Bortolatto

, et al. Functionalized epigallocatechin gallate copolymer inhibit dentin matrices degradation: Mechanical, solubilized telopeptide and proteomic assays. Dent Mater. 2018; 34: 1625-33.

10.

Epasinghe

Yiu

CKY

Burrow

, et al. The inhibitory effect of proanthocyanidin on soluble and collagen-bound proteases. J Dent. 2013; 41: 832-39.

11.

Islam

Hiraishi

Nassar

, et al. In vitro effect of hesperidin on root dentin collagen and de/re-mineralization. Dent Mater J. 2012; 31: 362-67.

12.

Alkhateeb

Al-Duais

Qnais

. Beneficial effects of oleuropein on glucose uptake and on parameters relevant to the normal homeostatic mechanisms of glucose regulation in rat skeletal muscle. Phytother Res. 2018; 32: 651-56.

13.

Kucukgul

Isgor

Duzguner

, et al. Antioxidant effects of oleuropein on hydrogen peroxide-induced neuronal stress- an in vitro study. Anti inflamm Antiallergy Agents Med Chem. 2020; 19: 74-84.

14.

Karygianni

Cecere

Argyropoulou

, et al. Compounds from Olea europaea and Pistacia lentiscus inhibit oral microbial growth. BMC Complement Altern Med. 2019; 19: 51.

15.

Fan

Hui

Duan

Fan

Deng

. Novel scaffolds fabricated using oleuropein for bone tissue engineering. Biomed Res Int. 2014; 2014: 652432.

16.

Muzzalupo

Badolati

Chiappetta

, et al. In vitro antifungal activity of olive (olea europaea) leaf extracts loaded in chitosan nanoparticles. Front Bioeng Biotechnol. 2020; 8: 151.

17.

Wang

, et al. Crosslinked collagen/chitosan matrix for artificial livers. Biomaterials. 2003; 24: 3213-20.

18.

Shao

Fang

Zhao

Cao

. Mechanism and effects of polyphenol derivatives for modifying collagen. ACS Biomater Sci Eng. 2019; 5: 4272-4284.

19.

Liang

Fang

, et al. Synthesis, characterization and cytotoxicity studies of chitosan-coated tea polyphenols nanoparticles. Colloids Surf B Biointerfaces. 2011; 82: 297-301.

20.

Luo

. Chitosan-based nanocarriers for encapsulation and delivery of curcumin: A review. Int J Biol Macromol. 2021 May 15; 179: 125-135.

21.

Shi

, et al. Modification of collagen with a natural cross-linker, procyanidin. Int J Biol Macromol. 2011; 48: 354-59.

22.

Kumar

Meena

Rajamani

. Fabrication of BSA-Green Tea Polyphenols-Chitosan Nanoparticles and Their Role in Radioprotection: A Molecular and Biochemical Approach. J Agric Food Chem. 2016; 64: 6024-34.

23.

Taravel

Domard

. Relation between the physicochemical characteristics of collagen and its interactions with chitosan: I. Biomaterials. 1993; 14: 930-938.

24.

Shrestha

Friedman

Kishen

. Photodynamically crosslinked and chitosan-incorporated dentin collagen. J. Dent Res. 2011; 90: 1346-1351.

25.

Xiong

Shen

Jiang

Kishen

. Effect of crosslinked chitosan nanoparticles on the bonding quality of fiber posts in root canals. J Adhes Dent. 2020; 22: 321-330.

26.

Shrestha

Friedman

Kishen

. Photodynamically crosslinked and chitosan-incorporated dentin collagen. J Dent Res. 2011; 90: 1346-51.

27.

Liang

Fang

, et al. Synthesis, characterization and cytotoxicity studies of chitosan-coated tea polyphenols nanoparticles. Colloids Surf B Biointerfaces. 2011; 82: 297-301.

28.

Ren

Yao

Zhang

Fan

Yang

Zhang

Liu

Sun

Miao

. Aligned fibers fabricated by near-field electrospinning influence the orientation and differentiation of hPDLSCs for periodontal regeneration. J. Biomed. Nanotechnol. 2017; 13: 1725-1734.

29.

Osorio

Aguilera

Medina-Castillo

Toledano

Toledano-Osorio

. Silver improves collagen structure and stability at demineralized dentin: A dynamic-mechanical and Raman analysis. J Dent. 2018 Dec; 79: 61-67.

30.

Lee

Sabatini

. Glutaraldehyde collagen cross-linking stabilizes resin-dentin interfaces and reduces bond degradation. Eur J Oral Sci. 2017; 125: 63-71.

31.

Hass

Wang

Nguyen

Peng

Wang

. Methacrylate-functionalized proanthocyanidins as novel polymerizable collagen cross-linkers – Part 1: Efficacy in dentin collagen bio-stabilization and cross-linking. Dent Mater. 2021; 37: 1183-1192.

32.

Mathapati

Bishi

Guhathakurta

Cherian

Venugopal

Ramakrishna

Verma

. Biomimetic acellular detoxified glutaraldehyde cross-linked bovine pericardium for tissue engineering. Mater Sci Eng C Mater Biol Appl. 2013; 33: 1561-72.

33.

Constable

Burton

Lawless

Gramigna

Buchan

Espino

. Effect of glutaraldehyde based cross-linking on the viscoelasticity of mitral valve basal chordae tendineae. Biomed Eng Online. 2018; 171: 93.

34.

Oryan

Kamali

Moshiri

, et al. Chemical crosslinking of biopolymeric scaffolds: Current knowledge and future directions of crosslinked engineered bone scaffolds. Int J Biol Macromol. 2018; 107: 678-88.

35.

Ahamad

Toufeeq

Khan

, et al. Oleuropein: A natural antioxidant molecule in the treatment of metabolic syndrome. Phytother Res. 2019; 33: 3112-28.

36.

Matyašovský

Jurkovič

Duchovič

, et al. Utilisation of collagen biopolymers and amaranth in polycondensation adhesives. 2009.

37.

Han

Jaurequi

Tang

, et al. Proanthocyanidin: A natural crosslinking reagent for stabilizing collagen matrices. J Biomed Mater Res A. 2003; 65: 118-124.

38.

Parise Gré

Pedrollo Lise

Ayres

, et al. Do collagen cross-linkers improve dentin’s bonding receptiveness? Dent Mater. 2018; 34: 1679-89.

39.

Moreira

Souza

Sousa

, et al. Efficacy of new natural biomodification agents from Anacardiaceae extracts on dentin collagen cross-linking. Dent Mater. 2017; 33: 1103-1109.

40.

Frazier

Deaville

Green

, et al. Interactions of tea tannins and condensed tannins with proteins. Journal of Pharmaceutical and Biomedical Analysis. 2010; 51: 490-5.

41.

Nicholson

Singh

Kishen

. Microtissue engineering root dentin with photodynamically cross-linked nanoparticles improves fatigue resistance of endodontically treated teeth. J Endod. 2020; 46: 668-674.

42.

Vidal

Zhu

Manohar

Aydin

Keiderling

Messersmith

Bedran-Russo

. Collagen-collagen interactions mediated by plant-derived proanthocyanidins: A spectroscopic and atomic force microscopy study. Acta Biomater. 2016; 41: 110-8.

43.

Frazier

Deaville

Green

, et al. Interactions of tea tannins and condensed tannins with proteins. J Pharm Biomed Anal. 2010; 51: 490-495.

44.

White

Lamed

Bayer

Flint

. Biomass utilization by gut microbiomes. Annu Rev Microbiol. 2014; 68: 279-296.

45.

Cantarel

Lombard

Henrissat

. Complex carbohydrate utilization by the healthy human microbiome. PLoS One. 2012; 7(6): e28742.

46.

Kishen

Shrestha

Cheng

Goh

. Characterizing the collagen stabilizing effect of crosslinked chitosan nanoparticles against collagenase degradation. Dent Mater. 2016; 32(8): 968-77.

47.

Hass

Luque-Martinez

Gutierrez

, et al. Collagen cross-linkers on dentin bonding: Stability of the adhesive interfaces, degree of conversion of the adhesive, cytotoxicity and in situ MMP inhibition. Dent Mater. 2016; 32(6): 732-741.

48.

Khor

. Methods for the treatment of collagenous tissues for bioprostheses. Biomaterials. 1997; 18(2): 95-105.

49.

Sathishkumar

Mun

. Binding affinity of proanthocyanidin from waste Pinus radiata bark onto proline-rich bovine achilles tendon collagen type I. Chemosphere. 2007; 67: 1618-27.

50.

Shi

Zhang

Shi

Lin

. Modification of collagen with a natural cross-linker, procyanidin. Int J Biol Macromol. 2010; 48: 354-9.