Effect of combined red and infrared wavelengths on inflammation, hemorrhage, and muscle damage caused by Bothrops leucurus snake venom.

To evaluate the effects of red and infrared wavelengths, separately and combined, on the inflammatory process and collagen deposition in muscle damage caused by B. leucurus venom. 112 mice were inoculated with diluted venom (0.6mg/kg) in the gastrocnemius muscle. The animals were divided into four groups: one control (CG) and three treatments, namely: 1) red laser (λ=660 nm) (RG), 2) infrared laser (λ=808 nm) (IG) and 3) red laser (λ=660 nm) + infrared (λ=808 nm) (RIG). Each group was subdivided into four subgroups, according to the duration of treatment application (applications every 24 hours over evaluation times of up to 144 hours). A diode laser was used (0.1 W, CW, 1J/point, ED: 10 J/cm2). Both wavelengths reduced the intensity of inflammation and the combination between them significantly intensified the anti-inflammatory response. Photobiomodulation also changed the type of inflammatory infiltrate observed and RIG had the highest percentage of mononuclear cells in relation to the other groups. Hemorrhage intensity was significantly lower in treated animals and RIG had the highest number of individuals in which this variable was classified as mild. As for collagen deposition, there was a significant increase in RG in relation to CG, in RIG in relation to CG and in RIG in relation to IG. Photobiomodulation proved to be effective in the treatment of inflammation and hemorrhage caused by B. leucurus venom and stimulated collagen deposition. Better results were obtained with the combined wavelengths.

Bioactive Peptide Profiling in Collagen Hydrolysates: Comparative Analysis Using Targeted and Untargeted Liquid Chromatography-Tandem Mass Spectrometry Quantification.

The investigation of collagen hydrolysates (CHs) is essential due to their widespread use in health, cosmetic, and therapeutic industries, attributing to the presence of bioactive dipeptides (DPs) and tripeptides (TPs). This study developed a novel targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with propyl chloroformate (PCF) derivatization to measure three bioactive peptides-Hydroxyprolyl-glycine (Hyp-Gly), Glycyl-prolyl-hydroxyproline (Gly-Pro-Hyp), and Prolyl-hydroxyproline (Pro-Hyp)-in CHs, with strong correlation coefficients (0.992, 1.000, and 0.995, respectively) and low limits of detection (LODs) of 1.40, 0.14, and 1.16 µM, respectively. Untargeted data-dependent acquisition (DDA) analyses measured peptide size distribution, while amino acid analysis assessed nutritional content. The analysis of ten commercial CHs revealed similar amino acid profiles but varied peptide lengths, indicating diverse hydrolysis conditions. Products with higher proportions of smaller peptides showed elevated levels of the targeted bioactive peptides, suggesting that a smaller peptide size may increase bioactivity. These findings can inform the optimization of CH supplements, providing consumers with detailed peptide content for more informed choices. Data are available via ProteomeXchange with the identifier PXD051699.

Fibrostricturing Crohn's Disease Is Marked by an Increase in Active Eosinophils in the Deeper Layers.

INTRODUCTION: Approximately 50% of patients with Crohn's disease (CD) develop intestinal strictures necessitating surgery. The immune cell distribution in these strictures remains uncharacterized. We aimed to identify the immune cells in intestinal strictures of patients with CD. METHODS: During ileocolonic resections, transmural sections of terminal ileum were sampled from 25 patients with CD and 10 non-inflammatory bowel disease controls. Macroscopically unaffected, fibrostenotic, and inflamed ileum was collected and analyzed for immune cell distribution (flow cytometry) and protein expression. Collagen deposition was assessed through a Masson Trichrome staining. Eosinophil and fibroblast colocalization was assessed through immunohistochemistry. RESULTS: The Masson Trichrome staining confirmed augmented collagen deposition in both the fibrotic and the inflamed regions, though with a significant increased collagen deposition in the fibrotic compared with inflamed tissue. Distinct Th1, Th2, regulatory T cells, dendritic cells, and monocytes were identified in fibrotic and inflamed CD ileum compared with unaffected ileum of patients with CD as non-inflammatory bowel disease controls. Only minor differences were observed between fibrotic and inflamed tissue, with more active eosinophils in fibrotic deeper layers and increased eosinophil cationic protein expression in inflamed deeper layers. Last, no differences in eosinophil and fibroblast colocalization were observed between the different regions. DISCUSSION: This study characterized immune cell distribution and protein expression in fibrotic and inflamed ileal tissue of patients with CD. Immunologic, proteomic, and histological data suggest inflammation and fibrosis are intertwined, with a large overlap between both tissue types. However strikingly, we did identify an increased presence of active eosinophils only in the fibrotic deeper layers, suggesting their potential role in fibrosis development.

Treatment with synchronized radiofrequency and facial muscle stimulation: Histologic analysis of human skin for changes in collagen and elastin fibers.

BACKGROUND: Skin's exposure to intrinsic and extrinsic factors causes age-related changes, leading to a lower amount of dermal collagen and elastin. AIM: This study investigated the effects of a novel facial muscle stimulation technology combined with radiofrequency (RF) heating on dermal collagen and elastin content for the treatment of facial wrinkles and skin laxity. METHODS: The active group subjects (N = 6) received four 20-min facial treatments with simultaneous RF and facial muscle stimulation, once weekly. The control subject (N = 1) was untreated. Skin biopsies obtained at baseline, 1-month and 3-month follow-up were evaluated histologically to determine collagen and elastin fibers content. A group of independent aestheticians evaluated facial skin appearance and wrinkle severity. Patient safety was followed. RESULTS: In the active group, collagen-occupied area reached 11.91 ± 1.80 × 106 µm2 (+25.32%, p < 0.05) and 12.35 ± 1.44 × 105 µm2 (+30.00%, p < 0.05) at 1-month and 3-month follow-up visits. Elastin-occupied area at 1-month and 3-month follow-up was 1.64 ± 0.14 × 105 µm2 (+67.23%, p < 0.05), and 1.99 ± 0.21 × 105 µm2 (+102.80%, p < 0.05). In the control group, there was no significant difference (p > 0.05) in collagen and elastin fibers. Active group wrinkle scores decreased from 5 (moderate, class II) to 3 (mild, class I). All subjects, except the control, improved in appearance posttreatment. No adverse events or side effects occurred. CONCLUSION: Decreased dermal collagen and elastin levels contributes to a gradual decline in skin elasticity, leading to facial wrinkles and unfirm skin. Study results showed noticeable improvement in facial appearance and increased dermal collagen and elastin content subsequent to simultaneous, noninvasive RF, and facial muscle stimulation treatments.

Detrimental Effects of Chlorhexidine on Articular Cartilage Viability, Matrix, and Mechanics.

BACKGROUND: Chlorhexidine gluconate (CHG) solution is commonly used as an antiseptic irrigation for bacterial decontamination during orthopaedic surgery. Although the chondrotoxicity of CHG on articular cartilage has been reported, the full extent of CHG-related chondrotoxicity and its effects on the extracellular matrix and mechanical properties are unknown. PURPOSE: To investigate the in vitro effects of a single 1-minute CHG exposure on the viability, biochemical content, and mechanics of native articular cartilage explants. STUDY DESIGN: Controlled laboratory study. METHODS: Articular cartilage explants (6 per group) were harvested from femoral condyles of the porcine stifle and sectioned at tidemark. Explants were bathed in CHG solution (0.05% CHG in sterile water) at varying concentrations (0% control, 0.01% CHG, and 0.05% CHG) for 1 minute, followed by complete phosphate-buffered saline wash and culture in chondrogenic medium. At 7 days after CHG exposure, cell viability, matrix content (collagen and glycosaminoglycan [GAG]), and compressive mechanical properties (creep indentation testing) were assessed. RESULTS: One-minute CHG exposure was chondrotoxic to explants, with both 0.05% CHG (2.6% ± 4.1%) and 0.01% CHG (76.3% ± 8.6%) causing a decrease in chondrocyte viability compared with controls (97.5% ± 0.6%; P < .001 for both). CHG exposure at either concentration had no significant effect on collagen content, while 0.05% CHG exposure led to a significant decrease in mean GAG per wet weight compared with the control group (2.6% ± 1.7% vs 5.2% ± 1.9%; P = .029). There was a corresponding weakening of mechanical properties in explants treated with 0.05% CHG compared with controls, with decreases in mean aggregate modulus (177.8 ± 90.1 kPa vs 280.8 ± 19.8 kPa; P < .029) and shear modulus (102.6 ± 56.5 kPa vs 167.9 ± 16.2 kPa; P < .020). CONCLUSION: One-minute exposure to CHG for articular cartilage explants led to dose-dependent decreases in chondrocyte viability, GAG content, and compressive mechanical properties. This raises concern for the risk of mechanical failure of the cartilage tissue after CHG exposure. CLINICAL RELEVANCE: Clinicians should be judicious regarding the use of CHG irrigation at these concentrations in the presence of native articular cartilage.

Distinct spectral signatures unfold ECM stiffness-triggered biochemical changes in breast cancer cells.

Cancer progression often accompanies the stiffening of extracellular matrix (ECM) in and around the tumor, owing to extra deposition and cross-linking of collagen. Stiff ECM has been linked with poor prognosis and is known to fuel invasion and metastasis, notably in breast cancer. However, the underlying biochemical or metabolic changes and the cognate molecular signatures remain elusive. Here, we explored Raman spectroscopy to unveil the spectral fingerprints of breast cancer cells in response to extracellular mechanical cues. Using stiffness-tuneable hydrogels, we showed that cells grown on stiff ECM displayed morphological changes with high proliferation. We further demonstrated that Raman Spectroscopy, a label-free and non-invasive technique, could provide comprehensive information about the biochemical environment of breast cancer cells in response to varying ECM stiffness. Raman spectroscopic analysis classified the cells into distinct clusters based on principal component-based linear discriminant analysis (PC-LDA). Multivariate curve resolution-alternating least squares (MCR-ALS) analysis indicated that cells cultured on stiff ECM exhibited elevated nucleic acid content and lesser lipids. Interestingly, increased intensity of Raman bands corresponding to cytochrome-c was also observed in stiff ECM conditions, suggesting mitochondrial modulation. The key findings harboured by spectral profiles were also corroborated by transmission electron microscopy, confirming altered metabolic status as reflected by increased mitochondria number and decreased lipid droplets in response to ECM stiffening. Collectively, these findings not only give the spectral signatures for mechanoresponse but also provide the landscape of biochemical changes in response to ECM stiffening.

Structure and properties of the acellular porcine cornea irradiated with 60Co-γ and electron beam and its histocompatibility.

Acellular porcine cornea (APC) has been used in corneal transplantation and treatment of the corneal diseases. Sterilization is a key step before the application of graft, and irradiation is one of the most commonly used methods. In this paper, APC was prepared by the physical freeze-thawing combined with biological enzymes, and the effects of the electron beam (E-beam) and cobalt 60 (60Co-γ) at the dose of 15 kGy on the physicochemical properties, structure, immunogenicity, and biocompatibility of the APC were investigated. After decellularization, the residual DNA was 20.86 ± 1.02 ng/mg, and the α-Gal clearance rate was more than 99%. Irradiation, especially the 60Co-γ, reduced the cornea's transmittance, elastic modulus, enzymatic hydrolysis rate, swelling ratio, and cross-linking degree. Meanwhile, the diameter and spacing of the collagen fibers increased. In the rat subcutaneous implantation, many inflammatory cells appeared in the unirradiated APC, while the irradiated had good histocompatibility, but the degradation was faster. The lamellar keratoplasty in rabbits indicated that compared to the E-beam, the 60Co-γ damaged the chemical bond of collagen to a larger extent, reduced the content of GAGs, and prolonged the complete epithelization of the grafts. The corneal edema was more serious within 1 month after the surgery. After 2 months, the thickness of the APC with the two irradiation methods tended to be stable, but that in the 60Co-γ group became thinner. The pathological results showed that the collagen structure was looser and the pores were larger, indicating the 60Co-γ had a more extensive effect on the APC than the E-beam at 15 kGy.

Influence of Staphylococcus epidermidis on Collagen Crimp Patterns of Soft Tissue Allograft.

BACKGROUND: Postoperative infections, commonly from Staphylococcus epidermidis, may result in anterior cruciate ligament graft failure and necessitate revision surgery. In biomechanical studies, S. epidermidis has been shown to establish biofilms on tendons and reduce graft strength. PURPOSE/HYPOTHESIS: The goal of this study was to determine the effect of bacterial bioburden on the collagen structure of tendon. It was hypothesized that an increase in S. epidermidis biofilm would compromise tendon crimp, a pattern necessary for mechanical integrity, of soft tissue allografts. STUDY DESIGN: Controlled laboratory study. METHODS: Cultures of S. epidermidis were used to inoculate tibialis anterior cadaveric tendons. Conditions assessed included 5 × 105 colony-forming units or concentrated spent media from culture (no living bacteria). Incubation times of 30 minutes, 3 hours, 6 hours, and 24 hours were utilized. Second-harmonic generation imaging allowed for visualization of collagen autofluorescence. Crimp lengths were determined using ImageJ and compared based on incubation time. RESULTS: Incubation time positively correlated with increasing S. epidermidis bioburden. Both fine and coarse crimp patterns lengthened with increasing incubation time. Significant coarse crimp changes were observed after only 30-minute incubations (P < .029), whereas significant fine crimp lengthening occurred after 6 hours (P < .0001). No changes in crimp length were identified after incubation in media lacking living bacteria. CONCLUSION: The results of this study demonstrate that exposure to S. epidermidis negatively affects collagen crimp structure. Structural alterations at the collagen fiber level occur within 30 minutes of exposure to media containing S. epidermidis. CLINICAL RELEVANCE: Our study highlights the need for antimicrobial precautions to prevent graft colonization and maximize graft mechanical strength.

Skeletal calcification patterns of batoid, teleost, and mammalian models: Calcified cartilage versus bone matrix.

This study compares the skeletal calcification pattern of batoid Raja asterias with the endochondral ossification model of mammalians Homo sapiens and teleost Xiphias gladius. Skeletal mineralization serves to stiffen the mobile elements for locomotion. Histology, histochemistry, heat deproteination, scanning electron microscopy (SEM)/EDAX analysis, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and Fourier transform infrared spectrometry (FTIR) have been applied in the study. H. sapiens and X. gladius bone specimens showed similar profiles, R. asterias calcified cartilage diverges for higher water release and more amorphous bioapatite. In endochondral ossification, fetal calcified cartilage is progressively replaced by bone matrix, while R. asterias calcified cartilage remains un-remodeled throughout the life span. Ca2+ and PO4 3- concentration in extracellular matrix is suggested to reach the critical salts precipitation point through H2 O recall from extracellular matrix into both chondroblasts or osteoblasts. Cartilage organic phase layout and incomplete mineralization allow interstitial fluids diffusion, chondrocytes survival, and growth in a calcified tissue lacking of a vascular and canalicular system. HIGHLIGHTS: Comparative physico-chemical characterization (TGA, DTG and DSC) testifies the mass loss due to water release, collagen and carbonate decomposition of the three tested matrices. R. asterias calcified cartilage water content is higher than that of H. sapiens and X. gladius, as shown by the respectively highest dehydration enthalpy values. Lower crystallinity degree of R. asterias calcified cartilage can be related to the higher amount of collagen in amorphous form than in bone matrix. These data can be discussed in terms of the mechanostat theory (Frost, 1966) or by organic/inorganic phase transformation in the course evolution from fin to limbs. Mineral analysis documented different charactersof R. asterias vs H. sapiens and X. gladius calcified matrix.

Crystal structure and induced stability of trimeric BxpB: implications for the assembly of BxpB-BclA complexes in the exosporium of Bacillus anthracis.

The outermost exosporium layer of Bacillus anthracis spores, the causative agents of anthrax, is comprised of a basal layer and an external hair-like nap. The nap includes filaments composed of trimers of the collagen-like glycoprotein BclA. Essentially all BclA trimers are attached to the spore in a process in which part of the 38-residue amino-terminal domain (NTD) of BclA forms an extremely stable interaction with the basal layer protein BxpB. Evidence indicates that the BclA-BxpB interaction is direct and requires trimeric BxpB. To further investigate the nature of the BclA-BxpB interaction, we determined the crystal structure of BxpB. The structure was trimeric with each monomer consisting of 11 ß strands with connecting loops. The structure did not include apparently disordered amino acids 1-19, which contain the only two cysteine residues of the 167-residue BxpB. The orientation of the structure reveals regions of BxpB that could be involved in interacting with the BclA NTD and with adjacent cysteine-rich proteins in the basal layer. Furthermore, the BxpB structure closely resembles that of the 134-residue carboxyl-terminal domain of BclA, which forms trimers that are highly resistant to heat and detergent. We demonstrated that BxpB trimers do not share this resistance. However, when BxpB trimers are mixed with a peptide containing residues 20-38 of BclA, they form a complex that is as stable as BclA-BxpB complexes extracted from spores. Together, our results provide new insights into the mechanism of BclA-BxpB attachment and incorporation into the exosporium. IMPORTANCE The B. anthracis exosporium plays major roles in spore survival and infectivity, but the complex mechanism of its assembly is poorly understood. Key steps in this process are the stable attachment of collagen-like BclA filaments to the major basal layer structural protein BxpB and the insertion of BxpB into an underlying basal layer scaffold. The goal of this study is to further elucidate these interactions thereby advancing our understanding of exosporium assembly, a process shared by many spore-forming bacteria including important human pathogens.

Fast detection of liver fibrosis with collagen-binding single-nanometer iron oxide nanoparticles via T1-weighted MRI.

SNIO-CBP, a single-nanometer iron oxide (SNIO) nanoparticle functionalized with a type I collagen-binding peptide (CBP), was developed as a T1-weighted MRI contrast agent with only endogenous elements for fast and noninvasive detection of liver fibrosis. SNIO-CBP exhibits 6.7-fold higher relaxivity compared to a molecular gadolinium-based collagen-binding contrast agent CM-101 on a per CBP basis at 4.7 T. Unlike most iron oxide nanoparticles, SNIO-CBP exhibits fast elimination from the bloodstream with a 5.7 min half-life, high renal clearance, and low, transient liver enhancement in healthy mice. We show that a dose of SNIO-CBP that is 2.5-fold lower than that for CM-101 has comparable imaging efficacy in rapid (within 15 min following intravenous injection) detection of hepatotoxin-induced liver fibrosis using T1-weighted MRI in a carbon tetrachloride-induced mouse liver injury model. We further demonstrate the applicability of SNIO-CBP in detecting liver fibrosis in choline-deficient L-amino acid-defined high-fat diet mouse model of nonalcoholic steatohepatitis. These results provide a platform with potential for the development of high relaxivity, gadolinium-free molecular MRI probes for characterizing chronic liver disease.

Micro-Raman spectroscopy analysis of dentin remineralization using eggshell derived nanohydroxyapatite combined with phytosphingosine.

The aim of this study was to assess the remineralization efficacy of chicken eggshell-derived nano-hydroxyapatite (CEnHAp) combined with phytosphingosine (PHS) on artificially induced dentinal lesions. PHS was commercially procured whereas CEnHAp was synthesized using microwave-irradiation method and characterized using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), high-resolution scanning electron microscopy-energy-dispersive X-ray spectroscopy (HRSEM-EDX), and transmission electron microscopy (TEM). A total of 75 pre-demineralized coronal dentin specimens were randomly treated with one of the following test agents (n = 15 each): artificial saliva (AS), casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), CEnHAp, PHS, and CEnHAp-PHS under pH cycling for 7, 14, and 28 days. Vickers microhardness indenter, HRSEM-EDX, and micro-Raman spectroscopy methods were used to assess the mineral changes in the treated dentin samples. Data were submitted to Kruskal-Wallis and Friedman's two-way analyses of variance (p < 0.05). HRSEM and TEM analysis depicted irregular spherical structure of the prepared CEnHAp with a particle size of 20-50 nm. The EDX analysis confirmed the presence of Ca, P, Na and Mg ions. The XRD pattern showed the characteristic crystalline peaks for hydroxyapatite and calcium carbonate that are present in the prepared CEnHAp. Dentin treated with CEnHAp-PHS revealed highest microhardness values along with complete tubular occlusion compared to other groups at all test time intervals (p < 0.05). Specimens treated with CEnHAp showed increased remineralization than those treated with CPP-ACP followed by PHS and AS groups. The intensity of mineral peaks, as observed in the EDX and micro-Raman spectra, confirmed these findings. Further, the molecular conformation of the collagen's polypeptide chains, and amide-I and CH2 peaks attained peak intensities in dentin treated with CEnHAp-PHS and PHS whereas other groups revealed poor stability of collagen bands. Microhardness, surface topography, and micro-Raman spectroscopy analyses revealed that dentin treated with CEnHAp-PHS have an improved collagen structure and stability as well as highest mineralization and crystallinity.

Histological Skin Assessment of Patients Submitted to Bariatric Surgery: A Prospective Longitudinal Cohort Study.

BACKGROUND: Obesity is a stigmatizing disease that can cause dermatological aberrations, such as sagging after rapid weight loss. OBJECTIVE: This study is to evaluate the effects of obesity and massive weight loss following bariatric surgery on collagen and elastic fibers of the extracellular matrix of the skin. METHODS: Thirty-three skin biopsies were collected from patients prior to bariatric surgery and one year after surgery. Histological analyses were performed using hematoxylin-eosin and Weigert's resorcin-fuchsin staining for collagen and elastic and elaunin fibers, respectively. Differences between means were submitted to the Student's t-test or Mann-Whitney U test, with p < 0.05 significant. RESULTS: The study demonstrated an architectural alteration of the skin 1 year after bariatric surgery. In the histological analysis of the skin samples, a significant difference in the thickness of the epidermis was found 1 year after surgery in all age groups as well as in the 38-to-68-kg weight loss group (p < 0.0001). In addition to laxity, disorganization of collagen was found, with an apparent decrease in quantity and an increase in elastic fibers, although fragmented (p < 0.0001). CONCLUSION: Obesity and massive weight loss following bariatric surgery cause the disorganization of collagen fibers and the fragmentation of elastic fibers of the extracellular matrix of the skin.

Regional and disease specific human lung extracellular matrix composition.

Chronic lung diseases, such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), are characterized by regional extracellular matrix (ECM) remodeling which contributes to disease progression. Previous proteomic studies on whole decellularized lungs have provided detailed characterization on the impact of COPD and IPF on total lung ECM composition. However, such studies are unable to determine the differences in ECM composition between individual anatomical regions of the lung. Here, we employ a post-decellularization dissection method to compare the ECM composition of whole decellularized lungs (wECM) and specific anatomical lung regions, including alveolar-enriched ECM (aECM), airway ECM (airECM), and vasculature ECM (vECM), between non-diseased (ND), COPD, and IPF human lungs. We demonstrate, using mass spectrometry, that individual regions possess a unique ECM signature characterized primarily by differences in collagen composition and basement-membrane associated proteins, including ECM glycoproteins. We further demonstrate that both COPD and IPF lead to alterations in lung ECM composition in a region-specific manner, including enrichment of type-III collagen and fibulin in IPF aECM. Taken together, this study provides methodology for future studies, including isolation of region-specific lung biomaterials, as well as a dataset that may be applied for the identification of novel ECM targets for therapeutics.

Phosphotungstic Acid-treated Picrosirius Red Staining Improves Whole-slide Quantitative Analysis of Collagen in Histological Specimens.

We tried to prevent nonspecific nuclear staining (NS-NS) of picrosirius red (PSR) staining by treating the specimens with one of the heteropoly acids phosphotungstic acid (PTA). We analyzed a total of 35 cases of non-cancerous liver tissue for fibrosis and NS-NS under PSR-alone, phosphomolybdic acid (PMA)-pretreated PSR (PMA + PSR), or PTA-pretreated PSR (PTA + PSR) condition. In addition, we analyzed the photosensitivity of PMA or PTA single stain specimens. PTA + PSR significantly suppressed NS-NS compared with PSR. The color of the specimens did not change into blue by 30 times the exposure to whole slide scanner (WSS) light. The PTA + PSR condition showed the highest correlation with the Ishak score (pathological evaluation of liver fibrosis) compared with other conditions. Furthermore, Sirius Red-positive percentage (SRP%) in PSR was increased in the NS-NS observed cases. SRP% in PMA + PSR was significantly affected by WSS light exposure time. Moreover, the deposition of non-polarized PSR-stained substances (NP-PSR+S) clinging to the collagen fibers potentially explains why SRP% seemed bigger under PSR than PTA + PSR. Our protocol enabled us to analyze the whole slide image of PSR staining by high magnification, which would contribute to the accurate analysis of collagen amount in the tissue sections.

Biochemical Compositional Analysis of Cadaveric Rib Cartilage.

Background: Cadaveric rib is used as a cartilage source for reconstructive rhinoplasty in patients who lack sufficient native septal cartilage; however, these grafts are known to warp. Objective: To measure and compare the biochemical properties of cadaveric rib as related to age, gender, and cortical versus core location. Methods: Seven cadaveric rib cartilage specimens were obtained and sectioned into cortical and core segments. Biochemical assays were used to determine total collagen and sulfated glycosaminoglycan (sGAG) content. Results: Collagen was present in higher amounts in cortical segments than core samples (72.8 ± 35.14 vs. 37.3 ± 16.99 µg/mgww, p = 0.0005). sGAG was also shown to be more prevalent in cortical segments (25.47 ± 11.59 vs. 12.17 ± 7.15 µg/mgww, p < 0.0001). The concentrations of collagen and sGAG demonstrated a positive correlation (R2 = 0.44, p = 0.0004). Collagen and sGAG content decreased with the age of the donor (p = 0.001 and p < 0.0001, respectively), but donor gender did not appear to affect collagen or sGAG content (p = 0.62 and p = 0.43, respectively). Conclusion: Collagen and sGAG content was higher in cortical segments of cadaveric rib cartilage than in core segments, and higher in samples from younger cadavers as well.

Development and characterisation of 3D collagen-gelatin based scaffolds for breast cancer research.

While 2D culture presents a useful tool for cancer research, it fails to replicate the tumor microenvironment as it lacks proper three-dimensional cell-cell/cell-matrix interactions, often resulting in exaggerated responses to therapeutic agents. 3D models that aim to overcome the issues associated with 2D culture research offer a new frontier for cancer research with cell growth, morphology and genetic properties that more closely match in vivo cancers. Herein, we aim to develop a collagen-based scaffold that supports the attachment and proliferation of breast cancer (BC) cells as a 3D culture model. Scaffolds were produced on a repeatable basis using a freeze-drying procedure. The constructs were highly porous (>99%) with homogenous pore sizes (150-300 µm) and an interconnected structure. The application of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) crosslinking resulted in scaffolds with elastic moduli in the range of 1-2 kPa, mimicking cancerous breast tissue stiffness. Furthermore, the incorporation of gelatin into the scaffolds enabled the porosity, pore size and mechanical properties to be tailored, resulting in scaffolds with stiffness values that accurately replicate the stiffness of human BC extracellular matrix (ECM) (1.3-1.7 kPa). Scaffolds displayed high in vitro stability with 90% of mass remaining after 14 days of culture. The scaffolds were shown to be highly biocompatible, and capable of supporting the attachment, infiltration and proliferation of MCF7 breast cancer (BC) cells over +14 days. These results confirm the suitability of these scaffolds as culture models for BC cells. These collagen-based scaffolds offer significant potential for the exploration of aspects of BC, such as gene expression profiles and patterns, and for the assessment of the efficacy of therapeutic agents in treating BC.

Biophysical properties of hydrogels for mimicking tumor extracellular matrix.

The extracellular matrix (ECM) is an essential component of the tumor microenvironment. It plays a critical role in regulating cell-cell and cell-matrix interactions. However, there is lack of systematic and comparative studies on different widely-used ECM mimicking hydrogels and their properties, making the selection of suitable hydrogels for mimicking different in vivo conditions quite random. This study systematically evaluates the biophysical attributes of three widely used natural hydrogels (Matrigel, collagen gel and agarose gel) including complex modulus, loss tangent, diffusive permeability and pore size. A new and facile method was developed combining Critical Point Drying, Scanning Electron Microscopy imaging and a MATLAB image processing program (CSM method) for the characterization of hydrogel microstructures. This CSM method allows accurate measurement of the hydrogel pore size down to nanometer resolution. Furthermore, a microfluidic device was implemented to measure the hydrogel permeability (Pd) as a function of particle size and gel concentration. Among the three gels, collagen gel has the lowest complex modulus, medium pore size, and the highest loss tangent. Agarose gel exhibits the highest complex modulus, the lowest loss tangent and the smallest pore size. Collagen gel and Matrigel produced complex moduli close to that estimated for cancer ECM. The Pd of these hydrogels decreases significantly with the increase of particle size. By assessing different hydrogels' biophysical characteristics, this study provides valuable insights for tailoring their properties for various three-dimensional cancer models.

The intriguing role of collagen on the rheology of cancer cell spheroids.

Spheroids are multicellular systems with an interesting rheology giving rise to elasto-visco-plastic properties. They are good tumor models, but the role of the extracellular matrix (ECM) is not fully understood. ECM is an important link between cells and may have a significant impact on tissue organization. Here we determine viscoelastic properties of spheroids including different collagen I amounts using AFM and predict new frequency-dependent properties leading to soft glassy rheology behavior. A unified model - similar to single cell behavior - is proposed and discussed, while complementary confocal experiments reveal the microstructure of spheroids, with collagen I fibers serving as a skeleton for cells, thus reinforcing the spheroid viscoelastic behavior.

The influence of glutaraldehyde on the microscopic structure of human pericardium.

OBJECTIVES: In our study, we investigate the collagen structure of human pericardium microscopically in dependence of glutaraldehyde (GA) concentration and fixation time. METHODS: Pericardial samples were taken from 9 patients aged 40+ years who underwent cardiac surgery, either coronary artery bypass surgery or valve implantation/reconstruction. Specimens were cut in 5 equal pieces and treated with GA at fixed concentrations (0.3125%, 0.625%, or 1.25%) but different exposer times (5 min, 10 min, 20 min, 30 min, and 60 min). Elastica van Gieson (EvG) staining was used for microscopic examination of pericardial collagen structure. RESULTS: The collagen structure studied microscopically depended on both GA incubation time and GA concentration. At low GA concentrations (0.3125%, 0.625%) and short incubation times, individual collagen fibers appeared separately. After one hour incubation period, single collagen fibers could not be distinguished at any GA concentration. For fixed incubation times no differences were seen in the collagen structure when 0.3125% and 0.625% GA were used. However, at a concentration of 1.25% GA fusion of individual collagen fibers was already observed at low incubation times. CONCLUSION: Pericardial collagen structure changes with increasing incubation time and increasing GA concentration by raising fusion of single fibers. For GA concentrations of ≤0.625%, fiber fusion depends plainly on incubation time. That is relevant as this concentration is used in cardiac surgery. At a concentration of 1.25% GA, single collagen fibers could not be separated, even at short incubation times. Fusion of individual collagen fibers and changes in appearance (less undulating) were assumed to be responsible for stiffening of GA-fixed pericardium.