Secondary Structure Of Collagen Research Articles

The Complementary Use of Raman, ATR‐FTIR Spectroscopy, and Chemometrics for Investigating the Deterioration of Artificially Aged Parchment

ABSTRACTAmong the materials constituting our cultural heritage, parchment has a significant role as the substrate of many archival documents. The assessment of its state of preservation has an important benefit to conservation and delineates the adequate preventive measures that would lead to better‐controlled storage and exhibition conditions in museums and libraries worldwide. In the framework of this project, artificial aging of 48 new goat parchment samples was performed to induce deterioration similar to naturally aged parchment exposed to atmospheric pollutants. Five factors have been examined: relative humidity; NO2; SO2; exposure time, and the order of sequential exposure to NO2 and SO2. Temperature was kept constant at 25°C, while the effect of the above‐mentioned factors was examined at two levels (low and high). Statistics was involved in advance in the experimental design. Raman spectroscopy along with attenuated total reflectance‐Fourier transform infrared (ATR‐FTIR) spectroscopy were used to investigate the condition of collagen in parchment. The entire set of environmental factors along with detected changes in the spectra were investigated through a chemometrics scheme involving analysis of variance (ANOVA). According to the results, the onset of collagen's secondary structure decomposition was observed. Statistical elaboration of data reveals that the two analytical methods (Raman and ATR‐FTIR) function in a complementary manner related to the molecular changes and the chemical reactions that happen. In conclusion, both methods can increase their impact on the investigation of parchment degradation if they are combined with other analytical methods and chemometric applications.

Understanding the hierarchical structure of collagen fibers of the human periodontal ligament: Implications for biomechanical characteristics

The periodontal ligament (PDL) is a unique fibrous connective tissue that regulates periodontal homeostasis mechanisms. Its biomechanical properties primarily reside in the hierarchical and non-uniform collagenous network. This study aimed to investigate the region-specific structure and composition of collagen fibers in the PDL at various scales and to explore their relationship with mechanical properties in a split-mouth design. Fresh human cadaver transverse PDL specimens of maxillary anterior teeth were categorized into cervical, middle, and apical groups. These specimens were analyzed via Masson’s trichrome staining, scanning electron microscopy, picrosirius red (PSR) staining, three-dimensional (3D) reconstruction, Raman spectroscopy, and uniaxial tensile test. Statistical analyses were performed to compare the structural, compositional, and tensile properties among the groups. Notably, the middle PDL samples exhibited superior tensile strength and higher fiber area fraction than the other two transverse sections. Despite a higher mineral-to-matrix ratio and a different collagen secondary structure, the apical PDL demonstrated a relatively weaker tensile strength, possibly associated with its discovered sparser collagen fiber areal fraction. The cervical region, characterized by a mediocre fiber areal fraction, displayed diminished tensile strength. The 3D reconstructed collagenous network model and PSR staining exposed the fiber interaction and the micropores. Microscale porosity and variations in collagen secondary structure, particularly in the apical region, suggest adaptive mechanisms for accommodating compressive forces and maintaining functional integrity. Variance in the tensile properties of samples in different force directions indicated the significant influence of fiber orientation and root level on tissue mechanics. Statement of significanceThis study provides critical insights into the biomechanical and structural properties of the human periodontal ligament (PDL), particularly focusing on the underexplored anterior teeth. Through advanced techniques like SEM, histological staining, 3D reconstruction, Raman spectroscopy, and tensile testing, we reveal significant regional variations in PDL collagen organization, composition, and biomechanical properties. Our findings address a crucial knowledge gap concerning the material mechanics of the PDL, offering a foundational understanding for future periodontal tissue engineering and biomimetic material development. This multi-scale analysis underscores the importance of both mesoscale structural characteristics and nanoscale molecular structures in maintaining PDL mechanical integrity.

Collagen organization and structure in FBLN5-/- mice using label-free microscopy: implications for pelvic organ prolapse.

Pelvic organ prolapse (POP) is a gynecological disorder described by the descent of superior pelvic organs into or out of the vagina as a consequence of disrupted muscles and tissue. A thorough understanding of the etiology of POP is limited by the availability of clinically relevant samples, restricting longitudinal POP studies on soft-tissue biomechanics and structure to POP-induced models such as fibulin-5 knockout (FBLN5-/- ) mice. Despite being a principal constituent in the extracellular matrix, little is known about structural perturbations to collagen networks in the FBLN5-/- mouse cervix. We identify significantly different collagen network populations in normal and prolapsed cervical cross-sections using two label-free, nonlinear microscopy techniques. Collagen in the prolapsed mouse cervix tends to be more isotropic, and displays reduced alignment persistence via 2-D Fourier transform analysis of images acquired using second harmonic generation microscopy. Furthermore, coherent Raman hyperspectral imaging revealed elevated disorder in the secondary structure of collagen in prolapsed tissues. Our results underscore the need for in situ multimodal monitoring of collagen organization to improve POP predictive capabilities.

Spectral Features Differentiate Aging-Induced Changes in Parchment-A Combined Approach of UV/VIS, µ-ATR/FTIR and µ-Raman Spectroscopy with Multivariate Data Analysis.

From the moment of production, artworks are constantly exposed to changing environmental factors potentially inducing degradation. Therefore, detailed knowledge of natural degradation phenomena is essential for proper damage assessment and preservation. With special focus on written cultural heritage, we present a study on the degradation of sheep parchment employing accelerated aging with light (295-3000 nm) for one month, 30/50/80% relative humidity (RH) and 50 ppm sulfur dioxide with 30/50/80%RH for one week. UV/VIS spectroscopy detected changes in the sample surface appearance, showing browning after light-aging and increased brightness after SO2-aging. Band deconvolution of ATR/FTIR and Raman spectra and factor analysis of mixed data (FAMD) revealed characteristic changes of the main parchment components. Spectral features for degradation-induced structural changes of collagen and lipids turned out to be different for the employed aging parameters. All aging conditions induced denaturation (of different degrees) indicated by changes in the secondary structure of collagen. Light treatment resulted in the most pronounced changes for collagen fibrils in addition to backbone cleavage and side chain oxidations. Additional increased disorder for lipids was observed. Despite shorter exposure times, SO2-aging led to a weakening of protein structures induced by transitions of stabilizing disulfide bonds and side chain oxidations.

Analysis of riboflavin/ultraviolet a corneal cross-linking by molecular spectroscopy

Corneal cross-linking (CXL) with riboflavin and ultraviolet A light is a therapeutic procedure to restore the mechanical stability of corneal tissue. The treatment method is applied to pathological tissue, such as keratoconus and induces the formation of new cross-links. At present, the molecular mechanisms of induced cross-linking are still not known exactly. In this study, we investigated molecular alterations within porcine cornea tissue after treatment with riboflavin and ultraviolet A light by surface enhanced Raman spectroscopy (SERS). For that purpose, after CXL treatment a thin silver layer was vapor-deposited onto cornea flaps. To explore molecular alterations induced by the photochemical process hierarchical cluster analysis (HCA) was used. The detailed analysis of SERS spectra reveals that there is no general change in collagen secondary structure while modifications on amino acid side chains are the most dominant outcome. The formation of secondary and aromatic amine groups as well as methylene and carbonyl groups were observed. Even though successful cross-linking could not be registered in all treated samples, Raman signals of newly formed chemical groups are already present in riboflavin only treated corneas.

The Effect of Irradiation on the Skin upon Breast Cancer Radiotherapy Studied by FTIR Spectroscopy

Breast cancer affects the female population worldwide. Radiotherapy (RT) is part of the therapeutic modality in the management of breast cancer, after radical mastectomy or conserving surgery. The FTIR spectroscopic “marker bands” will lead us to approach the mechanism of skin damage due to the interaction of ionizing radiation and skin, on a molecular level at the very early stages. FT-IR spectroscopy, breast digital pictures, and ImageJ software were used in the study. Healthy breast skin was irradiated <em>ex-vivo</em> with a 4 Gy dose of a γ-<sup>60</sup>Co course Gammachamber 4000A. The FT-IR spectra showed that the low-dose irradiation induces skin dehydration, collagen secondary structure changes and advanced glycation end products (AGEs) as a result of free radicals as mediated products. The infrared “marker bands” at about 1743, 1160, and 870 cm<sup>-1</sup> are characteristic, indicating the development of inflammation, glycations, and peroxidations respectively, due to ionizing radiation-induced oxidative stress. ImageJ analysis provided the sharp surface of the skin after RT irradiation in contrast to the smooth surface of the non-irradiated healthy skin. The most important damages, induced by radiotherapy, were connective tissue lesions, glycosylation, and phosphorylation processes in the skin. The reactive oxygen species (ROS) free radicals prefer to abstract H atoms from lipids, sugar rings of glycoproteins, and base ribose of DNA. The produced intermediate free radicals, as a result of ROS reactions, led to the formation of AGEs and peroxides.

Uterine leiomyoma as useful model to unveil morphometric and macromolecular collagen state and impairment in fibrotic diseases: An ex-vivo human study

Collagen is one of the main components of the extracellular matrix (ECM), involved, among all, in the maintenance of the structural support of tissues. In fibrotic diseases, collagen is overexpressed, and its production determines the formation of a significantly stiffer ECM. The cross-linking of high-resolution analytical tools, able to investigate both the tridimensional organization and the secondary structure of collagen in fibrotic diseases, could be useful to identify defined markers correlating the status of this protein with specific pathological conditions. To this purpose, an innovative multidisciplinary approach based on Phase-Contrast MicroComputed Tomography, Transmission Electron Microscopy, and Fourier Transform Infrared Imaging Spectroscopy was exploited on leiomyoma samples and adjacent myometrium to characterize microstructural collagen features. Uterine leiomyoma is a common gynecological disorder affecting women in fertile age. It is characterized by a massive collagen production due to the repairing processes occurring at myometrium level, and, hence, it represents a valuable model to investigate collagen self-organization in a pathological condition. Moreover, to evaluate the sensitivity of this multidisciplinary approach, the effects of eicosapentaenoic (EPA) and docosahexaenoic (DHA) omega-3 fatty acids in collagen reduction were also investigated.

Inhibition of Advanced Glycation End Product Formation in Rat Tail Tendons by Polydatin and p-Coumaric acid: an In Vitro Study.

Advanced glycation end products (AGEs) formed through non-enzymatic glycosylation between a protein and sugar molecule are highly harmful to the human body. In hyperglycemic patients, AGE formation is more due to high glucose circulating in the blood, causing inter and intra molecular cross-linking of collagen leading to reduction of collagen elasticity. This cross-linked collagen develops resistance to matrix metalloproteinases leading to impaired collagen turnover. The aim of this work is to determine the anti-glycation effects of polydatin and p-coumaric acid in preventing collagen cross-linking by incubating rat tail tendons (RTTs) as collagen source in high glucose concentration (50mM) for a week. The RTTs were then characterized for tensile strength, cross-linking efficiency, circular dichroism spectrometry, collagen, glucose, and aldehyde contents. Electrophoresis was carried out to evaluate the level of cross-linking in collagen and the results confirmed the ability of the drugs in preventing complex intermolecular cross-link formation induced by non-enzymatic glycosylation. CD data showed alteration in the secondary structure of collagen where AGE formation had occurred. More collagen was extracted by pepsin from RTTs treated with glucose alone (6.88mg/10mg tendon) when compared with drug-treated groups (4.25, 2.56mg/10mg tendon for polydatin and p-coumaric acid, respectively). Tensile strength (20.66% and 18.95%), cross-linking percentage (32.5% and 29.84%), and glucose content (2.3 and 1.8mg/100mg) of drug-treated groups were similar to the positive control (19.07%, 30.13%, and 2.61mg/100mg) thus proving the anti-glycation potential of the drugs. Hence, both polydatin and p-coumaric acid could play a pivotal role in preventing AGE formation.

Various Simulated Body Fluids Lead to Significant Differences in Collagen Tissue Engineering Scaffolds.

This study aims to point out the main drawback with respect to the design of simulated body environments. Three media commonly used for the simulation of the identical body environment were selected, i.e., Kokubo’s simulated body fluid that simulates the inorganic component of human blood plasma, human blood plasma, and phosphate buffer saline. A comparison was performed of the effects of the media on collagen scaffolds. The mechanical and structural effects of the media were determined via the application of compression mechanical tests, the determination of mass loss, and image and micro-CT analyses. The adsorption of various components from the media was characterized employing energy-dispersive spectrometry. The phase composition of the materials before and after exposure was determined using X-ray diffraction. Infrared spectroscopy was employed for the interpretation of changes in the collagen secondary structure. Major differences in terms of the mechanical properties and mass loss were observed between the three media. Conversely, only minor structural changes were detected. Since no general recommendation exists for selecting the simulated body environment, it is necessary to avoid the simplification of the results and, ideally, to utilize alternative methods to describe the various aspects of degradation processes that occur in the media.

Bone biomechanical properties and tissue-scale bone quality in a genetic mouse model of familial dysautonomia.

Familial dysautonomia (FD) is associated with a high prevalence of bone fractures, but the impacts of the disease on bone mass and quality are unclear. The purpose of this study was to evaluate tissue through whole-bone scale bone quality in a mouse model of FD. Femurs from mature adult Tuba1a-Cre; Elp1LoxP/LoxP conditional knockouts (CKO) (F = 7, M = 4) and controls (F = 5, M = 6) were evaluated for whole-bone flexural material properties, trabecular microarchitecture and cortical geometry, and areal bone mineral density (BMD). Adjacent maps spanning the thickness of femur midshaft cortical bone assessed tissue-scale modulus (nanoindentation), bone mineralization, mineral maturity, and collagen secondary structure (Raman spectroscopy). Consistent with prior studies on this mouse model, the Elp1 CKO mouse model recapitulated several key hallmarks of human FD, with one difference being the male mice tended to have a more severe phenotype than females. Deletion of Elp1 in neurons (using the neuronal-specific Tuba1a-cre) led to a significantly reduced whole-bone toughness but not strength or modulus. Elp1 CKO female mice had reduced trabecular microarchitecture (BV/TV, Tb.Th, Conn.D.) but not cortical geometry. The mutant mice also had a small but significant reduction in cortical bone nanoindentation modulus. While bone tissue mineralization and mineral maturity were not impaired, FD mice may have altered collagen secondary structure. Changes in collagen secondary structure were inversely correlated with bone toughness. BMD from dual-energy x-ray absorptiometry (DXA) was unchanged with FD. The deletion of Elp1 in neurons is sufficient to generate a mouse line which demonstrates loss of whole-bone toughness, consistent with the poor bone quality suspected in the clinical setting. The Elp1 CKO model, as with human FD, impacts the nervous system, gut, kidney function, mobility, gait, and posture. The bone quality phenotype of Elp1 CKO mice, which includes altered microarchitecture and tissue-scale material properties, is complex and likely influenced by these multisystemic changes. This mouse model may provide a useful platform to not only investigate the mechanisms responsible for bone fragility in FD, but also a powerful model system with which to evaluate potential therapeutic interventions for bone fragility in FD patients.

Comparison of different protocols for demineralization of cortical bone

Bone is a biological composite material consisting of two main components: collagen and mineral. Collagen is the most abundant protein in vertebrates, which makes it of high clinical and scientific interest. In this paper, we compare the composition and structure of cortical bone demineralized using several protocols: ethylene-diamine-tetraacetic acid (EDTA), formic acid (CH2O2), hydrochloric acid (HCl), and HCl/EDTA mixture. The efficiencies of these four agents were investigated by assessing the remaining mineral quantities and collagen integrity with various experimental techniques. Raman spectroscopy results show that the bone demineralized by the CH2O2 agent has highest collagen quality parameter. The HCl/EDTA mixture removes the most mineral, but it affects the collagen secondary structure as amide II bands are shifted as observed by Fourier transform infrared spectroscopy. Thermogravimetric analysis reveals that HCl and EDTA are most effective in removing the mineral with bulk measurements. In summary, we conclude that HCl best demineralizes bone, leaving the well-preserved collagen structure in the shortest time. These findings guide on the best demineralization protocol to obtain high-quality collagen from bone for clinical and scientific applications.

The Effect of Crosslinking Agents on the Properties of Type II Collagen Biomaterials

Type II collagen has been perceived as the indispensable element and plays a crucial role in cartilage tissue engineering. Thus, materials based on type II collagen have drawn farther attention in both academic and research for developing new systems for the cartilage regeneration. The disadvantage of using type II collagen as a biomaterial for tissue repairing is its reduced biomechanical properties. This can be solved by physical, enzymatic or chemical cross-linking processes, which provide biomaterials with the required mechanical properties for medical applications. To enhance type II collagen properties, crosslinked collagen scaffolds with different cross-linking agents were prepared by freeze-drying technique. The present research work studied the synthesis of type II collagen biomaterials with and without crosslinking agents. Scaffolds morphology was observed by MicroCT, showing in all cases an appropriate microstructure for biological applications, and the mechanical studies were performed using compressive tests. DSC showed an increase in denaturation temperature with an increase in cross-linking agent concentration. FTIR suggested that the secondary structure of collagen is not affected after the cross-linking; supplementary, to confirm the characteristic triple-helix conformation of collagen, the CD investigation was performed. The results showed that the physical-chemical properties of type II collagen were improved by cross-linking treatments.

Raman spectroscopy reveals age- and sex-related differences in cortical bone from people with osteoarthritis

Bone strength in human cortical bone is determined by the composition and structure of both the mineral and collagen matrices and influenced by factors such as age, gender, health, lifestyle and genetic factors. Age-related changes in the bone matrix are known to result in loss of mechanical strength and increased fragility. In this study we show how Raman spectroscopy, with its exquisite sensitivity to the molecular structure of bone, reveals new insights into age- and sex-related differences. Raman analysis of 18 samples of cortical hip bone obtained from people aged between 47–82 years with osteoarthritis (OA) found subtle changes in the lipid and collagen secondary structure, and the carbonate (CO32−) and phosphate (PO43−) mineral ratios in the bone matrix. Significant differences were observed between older and younger bones, and between older female and older male bones; no significant differences were observed between younger male and female bones. Older female bones presented the lowest mineral to matrix ratios (MMR) and highest CO32−/PO43− ratios, and relative to lipid/collagen –CH2 deformation modes at 1450 cm−1 they had lowest overall mineral content, higher collagen cross linking and lipid content but lower levels of α-helix collagen structures than older male and younger male and female bones. These observations provided further insight on bone composition changes observed in the bone volume fraction (BV/TV) for the older female bones from microCT measurements on the same samples, while tissue mineral density (TMD) measurements had shown no significant differences between the samples.

Effect of magnesium ascorbyl phosphate on collagen stabilization for wound healing application

Wound healing is a complex process which requires appropriate structural support for restoration of tissue continuity and function. Collagen can act as a template for cellular activities but poor physico-chemical properties necessitates the stabilization of collagen without impairing its structure and function. This study investigates the effect of magnesium ascorbyl phosphate (MAP) on collagen with reference to physico-chemical properties. Incorporation of MAP enhanced the rate of collagen fibrillation signifying increased interaction at reduced time interval. MAP did not induce any changes in the secondary structure of collagen while there was an increase in shear viscosity with increase in shear stress at different shear rate. MAP stabilized collagen film exhibited higher denaturation temperature and showed an increase in Young's Modulus when compared with that of collagen film. In vivo studies showed complete wound closure on day 16 in case of stabilized collagen film. Mechanical properties of healed skin revealed that MAP collagen film treated rat skin completely regained its properties similar to that of normal skin thereby making them a potential candidate for wound healing application.

Increased Fibroblast Metabolic Activity of Collagen Scaffolds via the Addition of Propolis Nanoparticles.

Propolis natural extracts have been used since ancient times due to their antioxidant, anti-inflammatory, antiviral, and antimicrobial activities. In this study, we produced scaffolds of type I collagen, extracted from Wistar Hanover rat tail tendons, and impregnated them with propolis nanoparticles (NPs) for applications in regenerative medicine. Our results show that the impregnation of propolis NPs to collagen scaffolds affected the collagen denaturation temperature and tensile strength. The changes in structural collagen self-assembly due to contact with organic nanoparticles were shown for the first time. The fibril collagen secondary structure was preserved, and the D-pattern gap increased to 135 ± 28 nm, without losing the microfiber structure. We also show that the properties of the collagen scaffolds depended on the concentration of propolis NPs. A concentration of 100 μg/mL of propolis NPs with 1 mg of collagen, with a hydrodynamic diameter of 173 nm, was found to be an optimal concentration to enhance 3T3 fibroblast cell metabolic activity and cell proliferation. The expected outcome from this research is both scientifically and socially relevant since the home scaffold using natural nanoparticles can be produced using a simple method and could be widely used for local medical care in developing communities.

Therapeutic Interventions to Reduce Radiation Induced Dermal Injury in a Murine Model of Tissue Expander Based Breast Reconstruction.

Radiation therapy (XRT) induced dermal injury disrupts type I collagen architecture. This impairs cutaneous viscoelasticity, which may contribute to the high rate of complications in expander-based breast reconstruction with adjuvant XRT. The objective of this study was to further elucidate the mechanism of radiation-induced dermal injury and to determine if amifostine (AMF) or deferoxamine (DFO) mitigates type I collagen injury in an irradiated murine model of expander-based breast reconstruction. Female Lewis rats (n = 20) were grouped: expander (control), expander-XRT (XRT), expander-XRT-AMF (AMF), and expander-XRT-DFO (DFO). Expanders were surgically placed. All XRT groups received 28 Gy of XRT. The AMF group received AMF 30 minutes before XRT, and the DFO group used a patch for delivery 5 days post-XRT. After a 20-day recovery period, skin was harvested. Atomic force microscopy and Raman spectroscopy were performed to evaluate type I collagen sheet organization and tissue compositional properties, respectively. Type I collagen fibril disorganization was significantly increased in the XRT group compared with the control (83.8% vs 22.4%; P = 0.001). Collagen/matrix ratios were greatly reduced in the XRT group compared with the control group (0.49 ± 0.09 vs 0.66 ± 0.09; P = 0.017). Prophylactic AMF demonstrated a marked reduction in type I collagen fibril disorganization on atomic force microscopy (15.9% vs 83.8%; P = 0.001). In fact, AMF normalized type I collagen organization in irradiated tissues to the level of the nonirradiated control (P = 0.122). Based on Raman spectroscopy, both AMF and DFO demonstrated significant differential protective effects on expanded-irradiated tissues. Collagen/matrix ratios were significantly preserved in the AMF group compared with the XRT group (0.49 ± 0.09 vs 0.69 ± 0.10; P = 0.010). β-Sheet/α-helix ratios were significantly increased in the DFO group compared with the XRT group (1.76 ± 0.03 vs 1.86 ± 0.06; P = 0.038). Amifostine resulted in a significant improvement in type I collagen fibril organization and collagen synthesis, whereas DFO mitigated abnormal changes in collagen secondary structure in an irradiated murine model of expander-based breast reconstruction. These therapeutics offer the ability to retain the native microarchitecture of type I collagen after radiation. Amifostine and DFO may offer clinical utility to reduce radiation induced dermal injury, potentially decreasing the high complication rate of expander-based breast reconstruction with adjuvant XRT and improving surgical outcomes.

Collagen stabilization using ionic liquid functionalised cerium oxide nanoparticle

Nanoparticles owing to their size have a substantial influence on the biological behavior of collagen, thereby opening new channels to unfold the propensity of nanoparticles in terms of collagen stabilization. The present study aims to synthesize and characterize cerium oxide nanoparticles and to investigate their crosslinking efficiency on collagen. Cerium oxide nanoparticles, known biocatalysts, form an effective oxidation system due to their variable oxidation state. Ionic liquid functionalizes cerium oxide nanoparticles (IL-CONP) have been synthesis by the sonication method and characterizes using techniques such as Dynamic Light Scattering, and X-ray Diffraction. The hydrodynamic diameter, Zeta potential and polydispersity index of nanoparticles is 192.3 ± 2.14 nm, −13.76 ± 1.5 mV and 0.387, respectively. Changes in the secondary structure of collagen upon treatment with increasing concentration of IL-CONP indicate conformational modifications at the molecular level. Differential scanning calorimetry studies on rat tail tendon collagen fibers with IL-CONP indicate an increase in thermal stability of collagen from 61 to 87 °C. Thus, cerium oxide-based nanoparticle crosslinking invokes a considerable array of interest as a potential crosslinking agent for collagen.

Effect of different solutions in reversing the damage caused by radiotherapy in dentin structure.

Background Previous studies have shown that radiotherapy of the head and neck region can cause direct changes in dental structure. This study evaluated the effect of different solutions on the dentin chemical composition and collagen structure of irradiated dentin. Material and Methods Sixty maxillary canines were distributed in 2 groups (n=30): non-irradiated and irradiated (radiotherapy: X-rays of 6 MV in 30 cycles of 2 Gy to 60 Gy). The teeth were sectioned, sanded, and polished to obtain 3x3x2 mm fragments, which were redistributed in 3 subgroups (n=10) according to the treatment employed: chlorhexidine 2% (CL), chitosan 0.2% (QT), and 0.5 M carbodiimide (EDC). The samples were analyzed in FTIR at time zero (T0-control) and after 1 (T1), 3 (T3), and 5 (T5) minutes of immersion in the tested solutions. The data for the areas of the carbonate (C), amide I (AI) bands, and the ratio between the areas of the amide III/proline and hydroxyproline (AIII/PH) bands were analyzed using ANOVA and Tukey test (α=5%). Results QT showed lower C values at T1, T3, and T5 (P<0.0001), presenting lower values when compared to CL and EDC subgroups (P<0.05). AI values at T3 and T5 were higher than T0-control and T1, independently of the radiotherapy and dentin treatment factors (P<0.05). At T0-control, the AIII/PH ratio was lower in the irradiated group (P<0.05), whereas the EDC treatment at T1, T3, and T5 and QT at T3 and T5 increased these values (P<0.05), making them similar to non-irradiated subgroups (P>0.05). Conclusions Radiotherapy changes the secondary structure of collagen, and EDC was able to restore collagen integrity after 1 minute of immersion, without changing dentin inorganic composition. Key words:Radiotherapy, collagen, dentin, FTIR, chemical composition.

Vancomycin-releasing cross-linked collagen sponges as wound dressings.

The study presents a novel vancomycin-releasing collagen wound dressing derived from Cyprinus carpio collagen type I cross-linked with carbodiimide which retarded the degradation rate and increased the stability of the sponge. Following lyophilization, the dressings were subjected to gamma sterilization. The structure was evaluated via scanning electron microscopy images, micro-computed tomography, and infrared spectrometry. The structural stability and vancomycin release properties were evaluated in phosphate buffered saline. Microbiological testing and a rat model of a wound infected with methicillin-resistant Staphylococcus aureus (MRSA) were then employed to test the efficacy of the treatment of the infected wound. Following an initial mass loss due to the release of vancomycin, the sponges remained stable. After 7 days of exposure in phosphate buffered saline (37°C), 60% of the material remained with a preserved collagen secondary structure together with a high degree of open porosity (over 80%). The analysis of the release of vancomycin revealed homogeneous distribution of the antibiotic both across and between the sponges. The release of vancomycin was retarded as proved by in vitro testing and further confirmed by the animal model from which measurable concentrations were observed in blood samples 24 hours after the subcutaneous implantation of the sponge, which was more than observed following intraperitoneal administration. The sponge was also highly effective in terms of reducing the number of colony-forming units in biopsies extracted from the infected wounds 4 days following the inoculation of the wounds with the MRSA solution. The presented sponges have ideal properties to serve as wound dressing for prevention of surgical site infection or treatment of already infected wounds.

Characterization of the Nanostructure of Collagen Fibers Following the Application of Dilute Hydrogen Peroxide used in Dental Whitening Treatments

This work aims to understand the effect of dilute hydrogen peroxide (H2O2) solutions, used in dental whitening treatments, on the nanostructure of type I collagen fibers. Using this approach, collagen fibers were exposed to 1%, 3% and 5% H2O2 solutions, and the changes in their nanostructure were analyzed by combining topographical and phase-shift atomic force microscopy (T-AFM and PS-AFM) results with circular dichroism (CD) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The combination of these techniques demonstrates that after exposure to 1 to 5% peroxide solutions, collagen fibers form agglomerates while the rupture of intramolecular bonds occurs, leading to collagen secondary structure loss and degradation. These results show for the first time that even dilute H2O2 solutions have a significant effect on collagen at the nanostructural level.