Collagen Cross-linking Agents Research Articles

Improving bonding to eroded dentin by using collagen cross-linking agents: 2years of water storage.

The aim of this study was to investigate the effects of collagen cross-linking agents on nanomechanical and bonding properties of eroded dentin (ED), 24h and 2years after water storage. Human molar dentin surfaces, eroded by soft drinks or citric acid, were acid-etched and treated with primers containing proanthocyanidin (PA) and riboflavin (RI) or were untreated (control) and tested after 24h and 2years. After acid etching and adhesive application (Prime&Bond Elect (PBE); Scotchbond Universal (SBU); Tetric n-bond Universal (TEU)), specimens were sectioned into beams and tested for microtensile bond strength (μTBS) and silver nitrate deposition (NL) after 24h and 2years. The beams were used to evaluate the 24-h in situ conversion of degree (DC). Nanohardness (NH) and Young's modulus (YM) were evaluated via resin-bonded dentin slices after 24h and 2years. A three-way ANOVA and Tukey's test were used for statistical analysis (5%). For both storage times, ED with citric acid resulted in lower μTBS, NH, and YM and higher NL for each adhesive system than soft drink ED (p < 0.05). After 2years of water storage, cross-linking primers maintained the μTBS, NH, and YM (p < 0.05) when compared with the control group. Althougth, the NL values decreased for all groups after 2 years of water storage, PA and RI treatments showed NI values lower than control group (p < 0.001). No significant differences were observed between PA and RI treatments (p > 0.05). Cross-linking primers maintain or improve DC (p < 0.03). In general, TEU and SBU yielded higher μTBS, DC, NH, and YM and lower NL than PBE. Cross-linking agents improved the results and maintained the resin-ED interface bonding and nanomechanical properties, without jeopardizing adhesive polymerization. Cross-linking agents are a viable alternative for improving and maintaining resin-ED interface bonding and nanomechanical properties.

Influence of grape seed extract in adhesion on dentin surfaces conditioned with Er,Cr:YSGG laser.

The proanthocyanidin (PA)-rich grape seed extract (GSE) is a collagen cross-linking agent that can perform a chemical bond with the dentin's collagen. The objective of this study was to evaluate the influence on shear bond strength (SBS) of the pre-conditioning of GSE, on human dentin surfaces conditioned with Er,Cr:YSGG laser. The sample consisted of 64 non-carious human teeth, divided into eight groups, four groups conditioned with Er,Cr:YSGG laser (4.5W, 50Hz, 50μs, 70% air, 90% water) and four prepared with conventional methods (control). In both groups, a GSE solution was applied before using the two adhesives tested: Clearfil™ SE Bond (CSE) and Scotchbond™ Universal (SU). Subsequently, a SBS test, a scanning electron microscopy, and a statistical analysis were performed. In the laser groups, the best SBS mean (20.08 ± 4.01MPa) was achieved in the group treated with GSE and CSE. The control group with the application of CSE showed the highest SBS mean (24.27 ± 10.28MPa), and the group treated with laser and SU showed the lowest SBS mean (12.94 ± 6.51MPa). Between these two groups there was a statistically significant difference (p = 0.05). However, this was not observed among the laser or control groups. The type of dentin surface preparation can influence the SBS. The CSE showed better SBS in laser and control groups. The presence of GSE did not improve the adhesion on surfaces conditioned with laser, but more studies should be carried out in the future to confirm this conclusion.

Elastic compliance of fibrillar assemblies in type I collagen

Fibrillary assemblies of Type I collagen find important applications in tissue engineering and as matrices for biophysical studies. The mechanical and structural properties of these structures are governed by factors such as protein concentration, temperature, pH and ionic strength. This study reports on an impedance based analysis of the elastic compliance of fibrillary assemblies of Type I collagen using quartz crystal microbalance with dissipation (QCM-D) at a fundamental frequency of 5 MHz and overtones (n = 3,5,7,9,11). Here, In situ partial fibrillation of the adsorbing collagen followed by its fibrillary assemblies on hydrophilic gold coated quartz surface have been crosslinked using Gallic acid (GA), Chromium (III) gallate (Cr-GA), Catechin (Cat), Tetrakis(hydroxymethyl)phosphonium sulfate (THPS) and Oxazolidine (Ox). This approach allows direct comparison of how viscoelastic properties track the structural evolution of the fiber and network length scales. The collagen crosslinking shows significant positive impact on the protein's mechanical behaviour and on the type of crosslinking agents used. The elastic modulus increases as collagen <GA < THPS < Cr-GA < Cat < Ox. Atomic force microscopic studies on the adsorbed collagen after cross linking confirmed the presence of fibrous assemblies.The results indicate stabilization and reinforcement through strong physical entanglement between the molecules of collagen as well as chemical interaction between collagen matrix and fibrils during cross linking. The elastic compliance evaluated from ΔDissipation/Δfreq. from QCM-D showed that cross linking with GA, Cr-GA and Ox resulted in flexible fibrillary network while agents like THPS and Cat showed elastic moduli similar to that of pure collagen. Results suggest that optimal collagen-crosslinking agent ratio and degree of crosslinking of collagen can help tailor the mechanical properties for specific applications in design of bio-materials of these composites.

Can Genipin-coated Sutures Deliver a Collagen Crosslinking Agent to Improve Suture Pullout in Degenerated Tendon? An Ex Vivo Animal Study.

BackgroundThe suture-tendon interface is often the weakest link in tendon-to-tendon or tendon-to-bone repair. Genipin is an exogenous collagen crosslink agent derived from the gardenia fruit that can enhance suture force to failure of the tendon-suture interface. Viable methods for intraoperative clinical delivery of genipin could be of clinical utility, but to our knowledge have not yet been extensively studied.Questions/purposesThe purposes of this study were (1) to evaluate whether sutures precoated with genipin can augment the suture-tendon interface to improve force to failure, stiffness, and work to failure in healthy and degenerated tendons; and (2) to determine the effect of genipin on the extent and distribution of crosslinking.MethodsSingle-stitch suture pullout tests were performed ex vivo on 25 bovine superficial digital flexor tendons. To assess effects on native tissue, one group of 12 tendons was cut in proximal and distal halves and randomized to treatment (n = 12) and control groups (n = 12) in a matched-pair design. One simple stitch with a loop with either a normal suture or genipin-coated suture was applied to tendons in both groups. To simulate a degenerative tendon condition, a second group of 13 tendons was cut in proximal and distal halves, injected with 0.2 mL of collagenase D (8 mg/mL) and incubated for 24 hours before suturing with either a genipin-coated suture (n = 13) or their matched controls (n = 13). Sutures from all groups then were loaded to failure on a universal materials testing machine 24 hours after suturing. Suture pullout force, stiffness, and work to failure were calculated from force-displacement data and compared between the groups. Additionally, fluorescence was measured to determine the degree of crosslinking quantitatively and a qualitative analysis of the distribution pattern was performed by microscopy.ResultsIn healthy tendon pairs, the median maximum pullout force was greater with genipin-coated sutures than with control sutures (median, 42 N [range, 24–73 N] versus 29 N [range, 13–48 N]; difference of medians, 13 N; p = 0.003) with corresponding increases in the required work to failure (median, 275 mJ [range, 48–369 mJ] versus 148 mJ [range, 83–369 mJ]; difference of medians, 127 mJ; p = 0.025) but not stiffness (median, 4.1 N/mm [range, 2.3–8.1 N/mm] versus 3.3 N/mm [range, 1.1–9.6 N/mm]; difference of medians, 0.8 N/mm; p = 0.052). In degenerated tendons, median maximum pullout force was greater with genipin-coated sutures than with control sutures (median, 16 N [range, 9-36 N] versus 13 N [range, 5-28 N]; difference of medians, 3 N; p = 0.034) with no differences in work to failure (median, 75 mJ [range, 11–249 mJ] versus 53 mJ [range, 14–143 mJ]; difference of medians, 22 mJ; p = 0.636) or stiffness (median, 1.9 N/mm [range, 0.7–13.4 N/mm] versus 1.6 N/mm [range, 0.5–5.6 N/mm]; difference of medians, 0.3 N/mm; p = 0.285). Fluorescence was higher in tendons treated with genipin-coated sutures compared with the control group, whereas higher fluorescence was observed in the treated healthy compared with the degenerated tendons (difference of means -3.16; standard error 1.08; 95% confidence interval [CI], 0.97–5.34; p = 0.006/healthy genipin: mean 13.04; standard error 0.78; 95% CI, 11.47-14.62; p < 0.001/degenerated genipin: mean 9.88; SD 0.75; 95% CI, 8.34-11.40; p < 0.001).ConclusionsGenipin-coated sutures improved force to failure of a simple stitch at the tendon-suture interface in healthy and degenerated tendons in an ex vivo animal model. Fluorescence was higher in tendons treated with genipin-coated sutures compared with the control group.Clinical RelevanceA genipin-coated suture represents a potential delivery vehicle for exogenous crosslink agents to augment suture retention properties. In vivo animal studies are the next logical step to assess safety and efficacy of the approach.

Effect of Baicalein on Matrix Metalloproteinases and Durability of Resin-Dentin Bonding.

In an attempt to increase resin-dentin bonding quality, this study used baicalein as a preconditioner in an etch-and-rinse adhesive to evaluate its effect on matrix metalloproteinases (MMPs) and adhesive durability. As a MMP inhibitor and potential collagen cross-linking agent, baicalein was used as a preconditioner in an etch-and-rinse adhesive system. The degree of conversion was evaluated by Fourier-transform infrared spectroscopy. EnzChek gelatinase/collagenase assay kits were then used to detect the MMP inhibitory effect of different concentrations of baicalein (0.1, 0.5, 2.5, and 5.0 μg/mL) on dentin powders. During in vitro bonding procedures, flat dentin surfaces on sound third molars were preconditioned with 2.5 μg/mL baicalein after being acid-etched; this step was followed by continuation of adhesive processes and build-up of resin composite. After resin-dentin stick preparation, bonding strength, failure mode, and interface nanoleakage were respectively evaluated via microtensile testing, stereomicroscopy, and field emission scanning electron microscopy either immediately or after storage in artificial saliva for three or six months. Data were analyzed by two-way analysis of variance and Tukey test (α=0.05). Baicalein at a concentration of 0-5.0 μg/mL did not influence the conversion of adhesives. However, it inhibited the activities of dentin-bond gelatinase and collagenase, especially at a concentration of 2.5 μg/mL, while effectively increasing microtensile bonding strength and decreasing nanoleakage in vitro, both immediately and after aging. Baicalein used as preconditioner in an etch-and-rinse adhesive system has an anti-MMP function and effectively improves resin-dentin bonding durability in vitro, which has potential value in clinical bonding procedures.

An ex vivo evaluation of effect of dentin pretreatment with various agents for varying time intervals on the shear bond strength of resin.

Background:This article highlights the contribution of collagen structure/stability to the bond strength. We hypothesize that induction of cross-linking in dentin collagen fibrils improves dentin collagen stability and thus bond strength with composite also improves.Aims:The aim of the study was to evaluate the effect of collagen cross-linking agents on the shear bond strength of composite resins.Subjects and Methods:One hundred human permanent teeth were randomly divided into three groups: Group I (n = 20) – no dentin pretreatment done, Group II – dentin pretreatment with 10% sodium ascorbate for 5 min (IIa) and 10 min (IIb), and Group III – dentin pretreatment with 6.5% proanthocyanidin (PA) for 5 min (IIIa) and 10 min (IIIb). A composite resin was bonded on prepared surfaces and cured. Thermocycling was done, and shear bond strength of the prepared samples was tested using a universal testing machine.Results:Values of Group I (control) were lowest whereas that of Group II (sodium ascorbate) were highest. The following order of shear bond strength was observed: IIb > IIa > IIIb ~ IIIa > I. Results for sodium ascorbate were found to be time dependent, whereas for PA, differences were nonsignificant.Conclusions:Treatment of dentinal surfaces with collagen cross-linking agent increases the shear bond strengths.

Correction to 'Quantification of the efficacy of collagen cross-linking agents to induce stiffening of rat sclera'.

[ J. R. Soc. Interface 14 , 20170014 (Published online 5 April 2017) ([doi:10.1098/rsif.2017.0014][2])][2] In our recent publication entitled, ‘Quantification of the efficacy of collagen cross-linking agents to induce stiffening of rat sclera’ published on 5 April 2017 (doi:10.1098/rsif.2017.

Effect of Two Different Collagen Cross Linking Agents versus Topical Fluoride on Management of Root Caries (In Vitro Study)

Objectives: This study was designed to evaluate the effect of two different collagen cross linkers (GSE & SA) versus topical fluoride, and the effect of exposure time of these agents on management of root caries. Material and Method: 48 human premolar roots were used; the mesial or distal surface of each sample was ground flat to expose dentin surface, polished and then mounted on wax blocks. They were divided into 3 main groups (n=16); A1: received 6.5% GSE-treatment; A2: received 10% SA-treatment and A3: received topical 1.1%NaF gel. Each group was divided into 2 subgroups (n=8) according to treatment exposure time, where T1: was representing the short exposure time (10-min) and T2: was representing the long exposure time (30- min). Each group was pH-cycled for 3 constitutive days. Assessment was done using digital radiography (DR) and polarized light microscopy (PLM). Data was collected and statistically analyzed. Results: All agents used produced significant remineralization effect on the artificially demineralized root dentin following 30-min treatment, however the two collagen cross linkers produced significant mineralization effect after treatment for 10min. Conclusion: Using collagen cross linking agents could be of value

Quantification of the efficacy of collagen cross-linking agents to induce stiffening of rat sclera.

The concept of scleral stiffening therapies has emerged as a novel theoretical approach for treating the ocular disorders glaucoma and myopia. Deformation of specific regions of the posterior eye is innately involved in the pathophysiology of these diseases, and thus targeted scleral stiffening could resist these changes and slow or prevent progression of these diseases. Here, we present the first systematic screen and direct comparison of the stiffening effect of small molecule collagen cross-linking agents in the posterior globe, namely using glyceraldehyde, genipin and methylglyoxal (also called pyruvaldehyde). To establish a dose-response relationship, we used inflation testing to simulate the effects of increasing intraocular pressure in freshly harvested rat eyes stiffened with multiple concentrations of each agent. We used digital image correlation to compute the mechanical strain in the tissue as a metric of stiffness, using a novel treatment paradigm for screening relative stiffening by incubating half of each eye in cross-linker and using the opposite half as an internal control. We identified the doses necessary to increase stiffness by approximately 100%, namely 30 mM for glyceraldehyde, 1 mM for genipin and 7 mM for methylglyoxal, and we also identified the range of stiffening it was possible to achieve with such agents. Such findings will inform development of in vivo studies of scleral stiffening to treat glaucoma and myopia.

EFFECT OF NATURALLY DERIVED COLLAGEN CROSS-LINKERS ON DENTIN BONDING AT CLINICALLY RELEVANT EXPOSURE TIMES

Purpose: To investigate the effect of treatment the demineralized dentin with two naturally derived collagen cross-linking agents on resin-dentin shear bond strength using three clinically relevant treatment times.Materials and methods: 60 mid-coronal dentin specimens were randomly divided into two divisions (n=30) and either was treated with one of two cross-linking agents: 25% grape seed extract (GSE) and 25% ascorbic acid (ASA). The teeth of each division were further divided into three groups (n=10) according to the time of cross-linking agent application: 30 sec, 60 sec, and 90 sec. Additionally, 10 teeth were used as a control group (no treatment). After cross-linkers treatment and adhesive application, composite (Filtek Z250 XT) cylinders (3 mm diameter × 2 mm length) were built on all dentin surfaces. All specimens were stored in distilled water for 24 hours at 37˚C and then subjected to shear stress in a universal testing machine. Failure patterns were observed using a light microscope at 10X. The micromorphology of the fractured surfaces of selected specimens was evaluated using SEM. SBS data (MPa) were statistically analyzed by two-way ANOVA and t-test.Results: Both cross-linking agents resulted in a significant (P<0.0001) increase in resin-dentin SBS in comparison to control, regardless of application time. SBS values of both cross-linking agents were significantly increased by increasing their application time from 30 to 90 sec, while they were not affected, between other treatment times. There was no significant (P = 0.5) differences in SBS values between the two cross linkers at each corresponding treatment time.Conclusions: Resin-dentin SBS can be improved after treatment of demineralized dentin by GSE, and ASA cross-linking agents at a clinically relevant treatment times.

Abstract 2186: Incorporation of natural compounds into biomaterials to prevent breast cancer recurrence

Abstract Numerous studies have demonstrated that naturally occurring phytochemicals such as Tannic acid (TA) possess anti-cancer properties. Apoptotic activity is increased in breast cancer and prostate cancer cells in response to exposure to tannin extracts. Collagen type I is a biomaterial routinely used for cosmetic and reconstructive surgeries, including breast reconstruction following breast cancer mandated surgeries. TA functions as a collagen crosslinking agent through both hydrogen bonding and hydrophobic effects. Thus, if TA-crosslinked collagen type I is used for breast reconstruction procedures, the crosslinked collagen will be remodeled by growing breast epithelial cells and adipocytes where the incorporated TA will be released killing nearby residual cancer cells preventing recurrence. When adipocytes are grown on TA-crosslinked collagen beads the measureable released TA induces caspase-mediated apoptosis in ER+ and HER2+ breast cancer cells but has no effect on triple negative breast cancer cells and reduced apoptotic-inducing activity in normal breast epithelial cells. We are developing an injectable matrix comprised of TA-crosslinked collagen beads and adipocytes to serve as a tissue regeneration platform in patients post-lumpectomy. Variables such as collagen temperature, syringe pump rate, and drop distance have been studied to determine ideal method for producing beads of the desired size and shape. These promising results will lead to the construction of a matrix designed to not only promote tissue regeneration but also simultaneously help to prevent tumor recurrence. This matrix provides protection in breast cancer patients and holds promise for patients suffering from other soft tissue cancers. Citation Format: Lauren Jordan, Bailey-Jean Walker, Christopher Moody, Brian W. Booth. Incorporation of natural compounds into biomaterials to prevent breast cancer recurrence. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2186.

Degradation of dentin-bonded interfaces treated with collagen cross-linking agents in a cariogenic oral environment: An in situ study

ObjectivesTo evaluate the effect of treatment using collagen cross-linking agents as primer on resin–dentin bond interfaces subjected to cariogenic oral environment (COE). MethodsEach of forty human teeth had two cavities (4×4×1.5mm) prepared within enamel margins. These cavities were acid-etched and treated by the primers containing one of the following treatment agents (6.5% proanthocyanidins, 0.1% riboflavin-UVA activated light, 5% glutaraldehyde or distilled water as a control group). After that the cavities were bonded and restored with resin composite. One restoration for each tooth was tested immediately (IM) and another was included in an intra-oral palatal device that was placed in each mouth of ten adult volunteers for 14 days in COE. After 14 days, the teeth were removed and each restoration was sectioned to obtain a slice for Knoop microhardness (KHN) and resin–dentin bonded sticks for microtensile bond strength (μTBS) and nanoleakage (NL) evaluation. Data were evaluated by two-way ANOVA and Tukey’s tests (α=0.05). ResultsAfter 14days in a COE, the KHN was reduced for all groups, except for the glutaraldehyde group; however, the proanthocyanidins group retained the highest KHN in IM and after COE (p<0.05). The μTBS was not reduced after COE for the proanthocyanidins and glutaraldehyde groups, however only the proanthocyanidins treatment did not increase the NL after COE (p>0.05). ConclusionThe in situ study model seems to be a suitable short-term methodology to investigate the degradation of the bonding interfaces under a more realistic condition. Under COE, the proanthocyanidins and glutaraldehyde treatments produced stable interfaces that are worth further clinical investigation.

Proanthocyanidin: A natural dentin biomodifier in adhesive dentistry

Proanthocyanidin (PA), a plant flavonoid, has recently been used in adhesive and restorative dentistry as a natural collagen cross-linking agent. As the long-term stability of the resin-bonded dentin is still questionable due to hydrolysis of collagen by collagenolytic enzymes, the use of collagen cross-linking agents has been proposed to enhance mechanical properties of dentin matrix and reduce biodegradation rates of collagen. Therefore, this paper discusses the chemistry and properties of PA, its role in stabilizing the bonded interface and enhancing the clinical longevity of adhesive restorations, and also considers various factors related to its incorporation in the bonding protocol.

Occlusal loading and cross-linking effects on dentin collagen degradation in physiological conditions

ObjectiveThis study evaluated the ability of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) to improve the stability of demineralized dentin collagen matrices when subjected to mechanical cycling by means of Chewing Simulation (CS). MethodsDemineralized dentin disks were randomly assigned to four groups (N=4): (1) immersion in artificial saliva at 37°C for 30 days; (2) pre-treatment with 0.5M EDC for 60s, then stored as in Group 1; (3) CS challenge (50N occlusal load, 30s occlusal time plus 30s with no load, for 30 days); (4) pre-treatment with 0.5M EDC as in Group 2 and CS challenge as in Group 3. Collagen degradation was evaluated by sampling storage media for ICTP and CTX telopeptides. ResultsEDC treated specimens showed no significant telopeptides release, irrespective of the aging method. Cyclic stressing of EDC-untreated specimens caused significantly higher ICTP release at day 1, compared to static storage, while by days 3 and 4, the ICTP release in the cyclic group fell significantly below the static group, and then remained undetectable from 5 to 30 days. CTX release in the cyclic groups, on EDC-untreated control specimens was always lower than in the static group in days 1–4, and then fell to undetectable for 30 days. SignificanceThis study showed that chewing stresses applied to control untreated demineralized dentin increased degradation of collagen in terms of CTX release, while collagen crosslinking agents may prevent dentin collagen degradation, irrespective of simulated occlusal function.

Genipin, a Cross-linking Agent, Promotes Odontogenic Differentiation of Human Dental Pulp Cells

IntroductionThe aim of this study was to investigate the effects of genipin, a natural collagen cross-linking agent, on odontogenic differentiation of human dental pulp cells (hDPCs) because the mechanical properties of collagen allow it to serve as a scaffold for engineering of pulp-dentin complex. Furthermore, the role of extracellular signal–regulated kinase (ERK) was investigated as a mediator of the differentiation. MethodsThe odontogenic differentiation was analyzed by alkaline phosphatase activity, real time-polymerase chain reaction, Western blotting, and alizarin red S staining. The morphologic features of hDPCs cultured in genipin-treated collagen were evaluated by scanning electron microscopy. For the assessment of mechanical properties of collagen treated with genipin, the surface roughness and compressive strength were measured. ResultsAlkaline phosphatase activity, the expression of odontogenic markers, and mineralized nodule formation increased in the genipin-treated group. Genipin also activated ERK, and treatment with ERK inhibitor blocked the expression of the markers. The cells cultured in genipin-treated collagen spread across the substrate and attached in close proximity to one another. The proliferation and differentiation of hDPCs cultured in genipin-treated collagen were facilitated. The mechanical properties of collagen, such as surface roughness and compressive strength, were increased by treatment with genipin. ConclusionsOur results show that genipin promotes odontogenic differentiation of hDPCs via the ERK signaling pathway. Furthermore, the enhanced mechanical properties of the collagen scaffold induced by genipin may play important roles in cell fate. Consequently, the application of genipin might be a new strategy for dentin-pulp complex regeneration.

Effect of different collagen cross-linking agents on shear bond strength of composite to dentin – An in vitro study

Effect of different collagen cross-linking agents on shear bond strength of composite to dentin – An in vitro study

Combined use of chondroitinase-ABC, TGF-β1, and collagen crosslinking agent lysyl oxidase to engineer functional neotissues for fibrocartilage repair

Patients suffering from damaged or diseased fibrocartilages currently have no effective long-term treatment options. Despite their potential, engineered tissues suffer from inferior biomechanical integrity and an inability to integrate in vivo. The present study identifies a treatment regimen (including the biophysical agent chondroitinase-ABC, the biochemical agent TGF-β1, and the collagen crosslinking agent lysyl oxidase) to prime highly cellularized, scaffold-free neofibrocartilage implants, effecting continued improvement in vivo. We show these agents drive in vitro neofibrocartilage matrix maturation toward synergistically enhanced Young's modulus and ultimate tensile strength values, which were increased 245% and 186%, respectively, over controls. Furthermore, an in vitro fibrocartilage defect model found this treatment regimen to significantly increase the integration tensile properties between treated neofibrocartilage and native tissue. Through translating this technology to an in vivo fibrocartilage defect model, our results indicate, for the first time, that a pre-treatment can prime neofibrocartilage for significantly enhanced integration potential in vivo, with interfacial tensile stiffness and strength increasing by 730% and 745%, respectively, compared to integration values achieved in vitro. Our results suggest that specifically targeting collagen assembly and organization is a powerful means to augment overall neotissue mechanics and integration potential toward improved clinical feasibility.

Characterization of genipin-modified dentin collagen.

Application of biomodification techniques to dentin can improve its biochemical and biomechanical properties. Several collagen cross-linking agents have been reported to strengthen the mechanical properties of dentin. However, the characteristics of collagen that has undergone agent-induced biomodification are not well understood. The objective of this study was to analyze the effects of a natural cross-linking agent, genipin (GE), on dentin discoloration, collagen stability, and changes in amino acid composition and lysyl oxidase mediated natural collagen cross-links. Dentin collagen obtained from extracted bovine teeth was treated with three different concentrations of GE (0.01%, 0.1%, and 0.5%) for several treatment times (0–24 h). Changes in biochemical properties of NaB3H4-reduced collagen were characterized by amino acid and cross-link analyses. The treatment of dentin collagen with GE resulted in a concentration- and time-dependent pigmentation and stability against bacterial collagenase. The lysyl oxidase-mediated trivalent mature cross-link, pyridinoline, showed no difference among all groups while the major divalent immature cross-link, dehydro-dihydroxylysinonorleucine/its ketoamine in collagen treated with 0.5% GE for 24 h, significantly decreased compared to control (P < 0.05). The newly formed GE-induced cross-links most likely involve lysine and hydroxylysine residues of collagen in a concentration-dependent manner. Some of these cross-links appear to be reducible and stabilized with NaB3H4.

Tannic Acid Preferentially Targets Estrogen Receptor-Positive Breast Cancer

Research efforts investigating the potential of natural compounds in the fight against cancer are growing. Tannic acid (TA) belongs to the class of hydrolysable tannins and is found in numerous plants and foods. TA is a potent collagen cross-linking agent; the purpose of this study was to generate TA-cross-linked beads and assess the effects on breast cancer cell growth. Collagen beads were stable at body temperature following crosslinking. Exposure to collagen beads with higher levels of TA inhibited proliferation and induced apoptosis in normal and cancer cells. TA-induced apoptosis involved activation of caspase 3/7 and caspase 9 but not caspase 8. Breast cancer cells expressing the estrogen receptor were more susceptible to the effects of TA. Taken together the results suggest that TA has the potential to become an anti-ER+ breast cancer treatment or preventative agent.

Whole globe inflation testing of exogenously crosslinked sclera using genipin and methylglyoxal

Exogenous collagen cross-linking has been investigated as method of reinforcing scleral biomechanics, with the goal of counteracting scleral weakening that occurs at the onset of myopia. This study uses whole globe inflation testing to investigate the biomechanical effect of treating posterior sclera with the collagen cross-linking agents methylglyoxal and genipin. Pairs of porcine eyes were treated in four ways. Three groups involved 1% methylglyoxal: two-hour (Group I) or thirty-minute (Group II) incubation of the whole globe, and thirty-minute incubation of only the posterior sclera of the intact eye (Group III). Group IV consisted of a thirty-minute incubation of the posterior sclera in 1% genipin. Following treatment, each eye was subjected to inflation testing under physiological pressure levels (0–150 mmHg); four strain markers on the posterior pole were tracked, providing displacement measurements in two directions. Results were used to derive load versus deformation behavior and to calculate stiffness at 0.25% strain (toe stiffness) and at peak strain (peak stiffness). Toe stiffness of Group I was 4.8 and 1.3 times greater than controls (sagittal and transverse directions, respectively: 5.23 ± 0.39 vs. 0.90 ± 0.08 mHg, P < 0.001; and 3.41 ± 0.19 vs. 1.51 ± 0.22 mHg, P < 0.01; values in mean ± SE). Group II was 7.4 and 4.3 times stiffer than controls (sagittal and transverse directions, respectively: 5.26 ± 0.49 vs. 0.63 ± 0.10 mHg, P < 0.02; and 3.44 ± 0.44 vs. 0.65 ± 0.07 mHg, P < 0.003). Group III was 3.6 and 3.4 times stiffer than controls (sagittal and transverse directions, respectively: 5.21 ± 0.39 vs. 1.13 ± 0.31 mHg, P < 0.01; and 4.94 ± 1.48 vs. 1.13 ± 0.25, P < 0.01), while Group IV was 8.2 and 2.8 times stiffer than controls (sagittal and transverse: 12.36 ± 1.96 vs. 1.35 ± 0.14 mHg, P < 0.01; and 12.45 ± 1.34 vs. 3.27 ± 0.50 mHg, P < 0.05). In all groups, there was no significant difference in peak stiffness after scleral cross-linking (SXL). At low strain, the posterior sclera was stiffer in both measured directions following methylglyoxal and genipin treatments, however at peak strain the treated sclera was not stiffer. Additionally, the saturation level of scleral stiffening by methylglyoxal can be reached within thirty minutes of treatment.