Liver fibrosis analysis using digital pathology.

Digital pathology has enabled the noninvasive quantification of pathological parameters. In addition, the combination of digital pathology and artificial intelligence has enabled the analysis of a vast amount of information, leading to the sharing of much information and the elimination of knowledge gaps. Fibrosis, which reflects chronic inflammation, is the most important pathological parameter in chronic liver diseases, such as viral hepatitis and metabolic dysfunction-associated steatotic liver disease. It has been reported that the quantitative evaluation of various fibrotic parameters by digital pathology can predict the prognosis of liver disease and hepatocarcinogenesis. Liver fibrosis evaluation methods include 1 fiber quantification, 2 elastin and collagen quantification, 3 s harmonic generation/two photon excitation fluorescence (SHG/TPE) microscopy, and 4 Fibronest™..　In this review, we provide an overview of role of digital pathology on the evaluation of fibrosis in liver disease and the characteristics of recent methods to assess liver fibrosis.

Brillouin spectroscopy of medically relevant samples of bovine jugular vein and pericardium.

There is the rapid growth in application of Brillouin scattering spectroscopy to biomedical objects in order to characterize their mechanoelastic properties in this way. However, the possibilities and limitations of the method when applied to tissues have not yet been clarified. Here, applicability of Brillouin spectroscopy for testing the elastic response of medically relevant tissues of bovine jugular vein and pericardium was considered. Parameters of the Brillouin peak were studied for samples untreated, diepoxide-fixed, and preserved after treatment in alcohol solutions. It was found that diepoxide cross-linking resulted to a slight tendency to increase the Brillouin position for hydrated tissues. The variations in the position and width of the Brillouin peaks, associated with local fluctuations in water concentration, were reduced after diepoxide treatment in the case of the pericardium, but not in the case of the vein wall. To obtain more information about the elastic response of the protein scaffold without the participation of water, dried samples were also studied. Brillouin spectra of the dried pericardium and vein wall revealed a significant increase in the Brillouin peak position (elastic modulus) after conservation in alcohol. In the case of the vein wall, this effect was found for both collagen and elastin-related peaks, which were identified in the Brillouin spectrum. This result corresponds to a denser packing of fibrous proteins after preservation in alcohol solutions. The ability of Brillouin spectroscopy to independently characterize the effect of treatment on the instantaneous elastic modulus of various tissue components is also attractive for its application in the development of new materials for bioimplants. A comparison of the Brillouin longitudinal and Young's elastic moduli determined for the hydrated samples of the vein and pericardium showed that there is no clear correspondence between these material parameters. The usefulness of using both experimental methods to obtain new information about the elastic response of the material is discussed.

Correction of age-related changes in the skin at the dermal and subdermal level using radiofrequency macroneedling therapy.

BACKGROUND: The negative effects of skin aging are primarily related to the destruction of dermal architectural structure. More specifically, this includes changes in the spatial arrangement of collagen, elastin fibers, mucopolysaccharides, proteoglycans, and ground substances. AIMS: The purpose of this study is to investigate the histologic effects of dermal and subdermal tissue after a controlled single treatment with radiofrequency (RF) macroneedling. This therapy provides a controlled, localized, thermal effect on the dermis whereby triggering the body's own healing processes of extracellular matrix remodeling. Clinically benefits include skin tightening. METHODS: Biopsies were obtained for histologic evaluation from four patients (n = 4), 4 weeks after completing a single RF macroneedling facial treatment. RESULTS: Age-related changes of the dermal and subdermal architecture were observed at baseline. After treatment, all biopsies demonstrated an increase in epidermal cells, collagen, elastin, fibroblasts, vasculature, and a decrease in inflammatory cells. CONCLUSIONS: The results of this histologic study confirm a significant "subsurfacing" thermal effect from the noncoagulative ascendant thermal injury. The obtained results characterize RF macroneedling therapy as an effective method for correcting age-related changes in facial skin.

Uterine Cesarean Scar Tissue-An Immunohistochemical Study.

Background and Objectives: Wound healing encompasses a multitude of factors and entails the establishment of interactions among components of the basement membrane. The quantification of particle concentrations can serve as valuable biomarkers for assessing biomechanical muscle properties. The objective of this study was to examine the immunoexpression and immunoconcentration of myometrial collagen type VI, elastin, alpha-smooth muscle actin, and smooth muscle myosin heavy chain, as well as the expression of platelets and clusters of differentiation 31 in the uterine scar following a cesarean section (CS). Materials and Methods: A total of 177 biopsies were procured from a cohort of pregnant women who were healthy, specifically during the surgical procedure of CS. The participants were categorized into seven distinct groups. Group 1 consisted of primiparas, with a total of 52 individuals. The subsequent groups were organized based on the duration of time that had elapsed since their previous CS. The analysis focused on the immunoexpression and immunoconcentration of the particles listed. Results: No significant variations were observed in the myometrial immunoconcentration of collagen type VI, elastin, smooth muscle myosin, and endothelial cell cluster of differentiation 31 among the analyzed groups. The concentration of alpha-smooth muscle actin in the myometrium was found to be significantly higher in patients who underwent CS within a period of less than 2 years since their previous CS, compared to those with a longer interval between procedures. Conclusions: Our findings indicate that the immunoconcentration of uterine myometrial scar collagen type VI, elastin, smooth muscle myosin heavy chain, alpha-smooth muscle actin, and endothelial cell marker cluster of differentiation 31 remains consistent regardless of the duration elapsed since the previous CS. The findings indicate that there are no significant alterations in the biomechanical properties of the uterine muscle beyond a period of 13 months following a CS.

Investigating the relationship between residual stress and micromechanical properties of blood vessels using atomic force microscopy.

The magnitude of vascular residual stress, an inherent characteristic exclusive to the vasculature, exhibits a strong correlation with vascular compliance, tensile resistance, vascular rigidity, and vascular remodeling subsequent to vascular transplantation. Vascular residual stress can be quantified by evaluating the magnitude of the opening angle within the vascular ring. For decellularized vessels, the vascular ring's opening angle diminishes, consequently reducing residual stress. The decellularization process induces a laxity in the vascular fiber structure within decellularized vessels. To investigate the interrelation between the magnitude of residual stress and the microstructure as well as mechanical properties of elastin and collagen within blood vessels, this study employed fresh blood vessels, stress-relieved vessels, and sections of decellularized blood vessels. Structural scanning and force map experiments on the surface of the sections were conducted using atomic force microscopy (AFM). The findings revealed well-organized arrangements of elastin and collagen within fresh vessels, wherein the regularity of collagen and elastin exhibited variability as residual stress declined. Furthermore, both stress-relieved and decellularized vessel sections exhibited a reduction in the mean Young's modulus to varying extents in comparison to fresh vessels. The validity of our experimental results was further corroborated through finite element simulations. Hence, residual stress assumes a crucial role in upholding the structural stability of blood vessels, and the intricate association between residual stress and the microstructural and micromechanical properties of blood vessels holds significant implications for comprehending the impact of vascular diseases on vascular structure and advancing the development of biomimetic artificial blood vessels that replicate residual stress. RESEARCH HIGHLIGHTS: In this inquiry, we scrutinized the interconnection amid vascular residual stress and the microscale and nanoscale aspects of vascular structure and mechanical function, employing AFM. We ascertained that residual stress assumes a pivotal role in upholding vascular microstructure and mechanical attributes. The experimental outcomes were subsequently validated through finite element simulation.

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.

Comapping Cellular Content and Extracellular Matrix with Hemodynamics in Intact Arterial Tissues Using Scanning Immunofluorescent Multiphoton Microscopy.

Deviation of blood flow from an optimal range is known to be associated with the initiation and progression of vascular pathologies. Important open questions remain about how the abnormal flow drives specific wall changes in pathologies such as cerebral aneurysms where the flow is highly heterogeneous and complex. This knowledge gap precludes the clinical use of readily available flow data to predict outcomes and improve treatment of these diseases. As both flow and the pathological wall changes are spatially heterogeneous, a crucial requirement for progress in this area is a methodology for acquiring and comapping local vascular wall biology data with local hemodynamic data. Here, we developed an imaging pipeline to address this pressing need. A protocol that employs scanning multiphoton microscopy was developed to obtain three-dimensional (3D) datasets for smooth muscle actin, collagen, and elastin in intact vascular specimens. A cluster analysis was introduced to objectively categorize the smooth muscle cells (SMC) across the vascular specimen based on SMC actin density. Finally, direct quantitative comparison of local flow and wall biology in 3D intact specimens was achieved by comapping both heterogeneous SMC data and wall thickness to patient-specific hemodynamic results.

Biomimetic approach for an articular cartilage patch: Combination of decellularized cartilage matrix and silk-elastin-like-protein (SELP) hydrogel.

Articular cartilage degradation due to injury, disease and aging is a common clinical issue as current regenerative therapies are unable to fully replicate the complex microenvironment of the native tissue which, being avascular, is featured by very low ability to self-regenerate. The extracellular matrix (ECM), constituting almost 90% of the entire tissue, plays a critical role in its function and resistance to compressive forces. In this context, the current tissue engineering strategies are only partially effective in restoring the biology and function of the native tissue. A main issue in tissue regeneration is treatment failure due to scarce integration of the engineered construct, often following a gradual detachment of the graft. In this scenario, we aimed to create an adhesive patch able to adequately support cartilage regeneration as a promising tool for the treatment of cartilage injuries and diseases. For this, we produced an engineered construct composed of decellularized ECM (dECM) obtained from horse joint cartilage, to support tissue regeneration, coupled with a Silk-Elastin-Like Proteins (SELP) hydrogel, which acts as a biological glue, to guarantee an adequate adherence to the host tissue. Following the production of the two biomaterials we characterized them by assessing: 1) dECM morphological, chemical, and ultrastructural features along with its capability to support chondrocyte proliferation, specific marker expression and ECM synthesis; 2) SELP microarchitecture, cytocompatibility and mechanical properties. Our results demonstrated that both materials hold unique properties suitable to be exploited to produce a tailored microenvironment to support cell growth and differentiation providing a proof of concept concerning the in vitro biological and mechanical efficacy of the construct. The SELP hydrogel displayed a very interesting physical behavior due to its high degree of resistance to mechanical stress, which is generally associated with physiological mechanical load during locomotion. Intriguingly, the shear-thinning behavior of the hydrogel may also make it suitable to be applied and spread over non-homogeneous surfaces, therefore, we hypothesize that the hybrid biomaterial proposed may be a real asset in the treatment of cartilage defects and injuries.

LC-MS/MS analysis of elastin crosslinker desmosines and microscopic evaluation in clinical samples of patients with hypertrophy of ligamentum flavum.

Ligamentum flavum (LF) pathologies often lead to severe myelopathy or radiculopathy characterized by reduced elasticity, obvious thickening, or worsened ossification. Elastin endows critical mechanical properties to tissues and organs such as vertebrae and ligaments. Desmosine (DES) and isodesmosine (IDES) are crosslinkers of elastin monomers called tropoelastin. These crosslinkers are potential biomarkers of chronic obstructive pulmonary disease. As a biological diagnostic tool that supplements existing symptomatic, magnetic resonance imaging scanning or radiological imaging diagnostic measures for LF hypertrophy and associated pathologies, an isotope-dilution liquid chromatography-tandem mass spectrometry method with selected reaction monitoring mode for the quantitation of DESs in human plasma, urine, cerebrospinal fluid (CSF), and yellow ligamentum was investigated. Isotopically labeled IDES-13C3,15N1 was used as an internal standard (ISTD) for DES quantitation for the first time. The samples plus ISTD were hydrolyzed with 6 N hydrochloric acid. Analytes and ISTD were extracted using a solid phase extraction cellulose cartridge column. The assays were repeatable, reproducible, and accurate with % CV ≤ 7.7, ISTD area % RSD of 7.6, and % AC ≤ (101.2 ± 3.90) of the calibrations. The ligamentum samples gave the highest average DES/IDES content (2.38 µg/mg) on a dry-weight basis. A high percentage of the CSF samples showed almost no DESs. Urine and plasma samples of patients showed no significant difference from the control (p-value = 0.0519 and 0.5707, respectively). Microscopy of the yellow ligamentum samples revealed dark or blue-colored zones of elastin fibers that retained the hematoxylin dye and highly red-colored zones of collagen after counterstaining with van Gieson solution. Thus, we successfully developed a method for DES/IDES quantitation in clinical samples.

Reduced fibrous capsule elastic fibers from biologic ECM-enveloped CIEDs in minipigs, supported with a novel compression mechanics model.

BACKGROUND: Fibrous capsules (Fb) in response to cardiovascular implantable electronic devices (CIEDs), including a pacemaker (P) system, can produce patient discomfort and difficulties in revision surgery due partially to their increased compressive strength, previously linked to elevated tissue fibers. OBJECTIVE: A preliminary study to quantify structural proteins, determine if biologic extracellular matrix-enveloped CIEDs (PECM) caused differential Fb properties, and to implement a realistic mechanical model. METHODS: Retrieved Fb (-P and -PECM) from minipigs were subjected to biomechanical (shear oscillation and uniaxial compression) and histological (collagen I and elastin) analyses. RESULTS: Fb-PECM showed significant decreases compared to Fb-P in: low strain-loss modulus (390 vs. 541 Pa) across angular frequencies, high strain-compressive elastic modulus (1043 vs. 2042 kPa), and elastic fiber content (1.92 vs. 3.15 µg/mg tissue). Decreases in elastin were particularly noted closer to the implant's surface (Fb-PECM = 71% vs. Fb-P = 143% relative to dermal elastin at mid-tangential sections) and verified with a solid mechanics hyperelasticity with direction-dependent fiber viscoelasticity compression simulation (r2 ≥ 98.9%). CONCLUSIONS: The biologic envelope composed of decellularized porcine small intestine submucosa ECM for CIEDs promoted fibrous tissues with less elastic fibers. Novel compression modeling analyses directly correlated this singular reduction to more desirable subcutaneous tissue mechanics.

Characterization of a decellularized rat larynx: comparison between microscopy techniques.

BACKGROUND: No effective method has yet been developed to efficiently reconstruct the larynx and restore its function. Decellularization has recently been tested for this purpose with very promising results. The goal of decellularization is to remove cells leaving an intact scaffold made of an extracellular matrix (ECM). Although the use of hematoxylin/eosin and Masson trichrome stains is widely accepted to highlight tissue structure, the methods based on evaluation of collagen and elastin are considered highly variable. The aim of this study was to develop a whole organ decellularization protocol and compare the qualitative and quantitative efficiency of some microscopy techniques for collagen and elastin detection in paraffin-embedded tissues. METHODS: H&E, Masson Trichrome and DAPI staining as well as DNA quantification were used to evaluate decellularization efficiency. Van Gieson stain, Picrosirius Red stain (PRS) and multiphoton laser scanning microscopy (MPM) were carried out for collagen detection and quantitative assessment. Polarized PRS was used to investigate collagen network, and Weigert stain and MPM were used to detect and estimate elastin content. RESULTS: The decellularization process removed the cellular components without affecting glycosaminoglycan, collagen and elastin content. Concerning collagen quantification, Van Gieson stain underestimated collagen content, while PRS, apparently less fading, did not reach reliable results when used as quantitative method. MPM effectively quantified collagen content. Collagen fibers were visualized much better under polarized light microscopy, allowing to underline that decellularization process affects the homogeneity of 3D collagen network. Concerning elastin detection, Weigert stain and MPM produced overlapping results. CONCLUSIONS: An efficient protocol to decellularize the whole larynx was developed, allowing the removal of cells without affecting ECM integrity. The results supported the use of non-polarized PRS to highlight collagen, even the thin fibers, second harmonic generation for major fibrillar collagens and polarized PRS for 3D collagen network. Concerning elastin, Weigert stain and MPM showed similar results, thus the use of MPM, rather than that of the Weigert stain, may be suitable to avoid the additional time and costs of a histological staining.

Routine Elastin Staining in Surgically Resected Colorectal Cancer: Impact on Venous Invasion Detection and its Association With Oncologic Outcomes.

Venous invasion (VI) is a powerful yet underreported prognostic factor in colorectal cancer (CRC). Its detection can be improved with an elastin stain. We evaluated the impact of routine elastin staining on VI detection in resected CRC and its relationship with oncologic outcomes. Pathology reports from the year before (n=145) and the year following (n=128) the implementation of routine elastin staining at our institution were reviewed for established prognostic factors, including VI. A second review, using elastin stains, documented the presence/absence, location, number, and size of VI foci. The relationship between VI and oncologic outcomes was evaluated for original and review assessments. VI detection rates increased from 21% to 45% following implementation of routine elastin staining (odds ratio [OR]=3.1; 95% confidence interval [CI]: 1.8-5.3; P<0.0001). The second review revealed a lower VI miss rate postimplementation than preimplementation (22% vs. 48%, respectively; P=0.007); this difference was even greater for extramural VI-positive cases (9% vs. 38%, respectively; P=0.0003). Missed VI cases postimplementation had fewer VI foci per missed case (P=0.02) and a trend towards less extramural VI than those missed preimplementation. VI assessed with an elastin stain was significantly associated with recurrence-free survival (P=0.003), and cancer-specific survival (P=0.01) in contrast to VI assessed on hematoxylin and eosin alone (P=0.053 and 0.1, respectively). The association between VI and hematogenous metastasis was far stronger for elastin-detected VI (OR=11.5; 95% CI: 3.4-37.1; P<0.0001) than for hematoxylin and eosin-detected VI (OR=3.7; 95% CI: 1.4-9.9; P=0.01). Routine elastin staining enhances VI detection and its ability to stratify risk in CRC and should be considered for evaluation of CRC resection specimens.

Percolation of collagen stress in a random network model of the alveolar wall.

Fibrotic diseases are characterized by progressive and often irreversible scarring of connective tissue in various organs, leading to substantial changes in tissue mechanics largely as a result of alterations in collagen structure. This is particularly important in the lung because its bulk modulus is so critical to the volume changes that take place during breathing. Nevertheless, it remains unclear how fibrotic abnormalities in the mechanical properties of pulmonary connective tissue can be linked to the stiffening of its individual collagen fibers. To address this question, we developed a network model of randomly oriented collagen and elastin fibers to represent pulmonary alveolar wall tissue. We show that the stress-strain behavior of this model arises via the interactions of collagen and elastin fiber networks and is critically dependent on the relative fiber stiffnesses of the individual collagen and elastin fibers themselves. We also show that the progression from linear to nonlinear stress-strain behavior of the model is associated with the percolation of stress across the collagen fiber network, but that the location of the percolation threshold is influenced by the waviness of collagen fibers.

Quantitative not qualitative histology differentiates aneurysmal from nondilated ascending aortas and reveals a net gain of medial components.

Medial degeneration is a common histopathological finding in aortopathy and is considered a mechanism for dilatation. We investigated if medial degeneration is specific for sporadic thoracic aortic aneurysms versus nondilated aortas. Specimens were graded by pathologists, blinded to the clinical diagnosis, according to consensus histopathological criteria. The extent of medial degeneration by qualitative (semi-quantitative) assessment was not specific for aneurysmal compared to nondilated aortas. In contrast, blinded quantitative assessment of elastin amount and medial cell number distinguished aortic aneurysms and referent specimens, albeit with marked overlap in results. Specifically, the medial fraction of elastin decreased from dilution rather than loss of protein as cross-sectional amount was maintained while the cross-sectional number, though not density, of smooth muscle cells increased in proportion to expansion of the media. Furthermore, elastic lamellae did not thin and interlamellar distance did not diminish as expected for lumen dilatation, implying a net gain of lamellar elastin and intralamellar cells or extracellular matrix during aneurysmal wall remodeling. These findings support the concepts that: (1) medial degeneration need not induce aortic aneurysms, (2) adaptive responses to altered mechanical stresses increase medial tissue, and (3) greater turnover, not loss, of mural cells and extracellular matrix associates with aortic dilatation.

Feasibility and Performance of Elastin Trichrome as a Primary Stain in Colorectal Cancer Resection Specimens: Results of an Interobserver Variability Study.

Venous invasion (VI) is a powerful prognostic factor in colorectal cancer (CRC) that is widely underreported. The ability of elastin stains to improve VI detection is now recognized in several international CRC pathology protocols. However, concerns related to the cost and time required to perform and evaluate these stains in addition to routine hematoxylin and eosin (H&E) stains remains a barrier to their wider use. We therefore sought to determine whether an elastin trichrome (ET) stain could be used as a "stand-alone" stain in CRC resections, by comparing the sensitivity, accuracy, and reproducibility of detection of CAP-mandated prognostic factors using ET and H&E stains. Representative H&E- and ET-stained slides from 50 CRC resections, including a representative mix of stages and prognostic factors, were used to generate 2 study sets. Each case was represented by H&E slides in 1 study set and by corresponding ET slides from the same blocks in the other study set. Ten observers (3 academic gastrointestinal [GI] pathologists, 4 community pathologists, 3 fellows) evaluated each study set for CAP-mandated prognostic factors. ET outperformed H&E in the assessment of VI with respect to detection rates (50% vs. 28.6%; P<0.0001), accuracy (82% vs. 59%, P<0.0001), and reproducibility (k=0.554 vs. 0.394). No significant differences between ET and H&E were observed for other features evaluated. In a poststudy survey, most observers considered the ease and speed of assessment at least equivalent for ET and H&E for most prognostic factors, and felt that ET would be feasible as a stand-alone stain in practice. If validated by others, our findings support the use of ET, rather than H&E, as the primary stain for the evaluation of CRC resections.

Relationship between calcification, atherosclerosis and matrix proteins in the human aorta.

INTRODUCTION: Extracellular matrix (ECM) proteins have been associated with atherosclerotic complications, such as plaque rupture, calcification and aneurysm. It is not clear what role different types of collagen play in the pathomechanism of atherosclerosis. The aim of the study was to analyze the content of elastin and major types of collagen in the aortic wall and how they associated are with course of atherosclerosis. MATERIAL AND METHODS: In this work we present six biochemical parameters related to ECM proteins and collagen-specific amino acids (collagen type I, III, and IV, elastin, proline and hydroxyproline) analyzed in 106 patients' aortic wall specimens characterized by different degree of atherosclerosis. Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC/ESI-MS/MS), ELISA and immunohistochemical methods were used. The severity of atherosclerosis was assessed on the six-point scale of the American Heart Association, taking into account the number and location of foam cells, the presence of a fatty core, calcium deposits and other characteristic atherosclerotic features. RESULTS: The results show that there is a relationship between the content of collagen-specific amino acids and development of atherosclerosis. The degree of atherosclerotic lesions was negatively correlated with the content of proline, hydroxyproline and the ratio of these two amino acids. Calcium deposits and surrounding tissue were compared and it was demonstrated that the ratio of type I collagen to type III collagen was higher in the aortic tissue than in aortic calcification areas, while the ratio of collagen type III to elastin was smaller in the artery than in the calcium deposits. CONCLUSIONS: We suggest that increase in collagen type III presence in the calcification matrix may stem from disorders in the structure of the type I and III collagen fibers. These anomalous fibers are likely to favor accumulation of the calcium salts, an important feature of the process of atheromatosis.

Assessment of Transdermal Delivery of Topical Compounds in Skin Scarring Using a Novel Combined Approach of Raman Spectroscopy and High-Performance Liquid Chromatography.

Objective: The goal of any topical formulation is efficient transdermal delivery of its active components. However, delivery of compounds can be problematic with penetration through tough layers of fibrotic dermal scar tissue. Approach: We propose a new approach combining high-performance liquid chromatography (HPLC) and Raman spectroscopy (RS) using a topical of unknown composition against a well-known antiscar topical (as control). Results: Positive detection of compounds within the treatment topical using both techniques was validated with mass spectrometry. RS detected conformational structural changes; the 1,655/1,446 cm-1 ratio estimating collagen content significantly decreased (p < 0.05) over weeks 4, 12, and 16 compared with day 0. The amide I band, known to represent collagen and protein in skin, shifted from 1,667 to 1,656 cm-1, which may represent a change from ß-sheets in elastin to α-helices in collagen. Confirmatory elastin immunohistochemistry decreased compared with day 0, conversely the collagen I/III ratio increased in the same samples by week 12 (p < 0.05, and p < 0.0001, respectively), in keeping with normal scar formation. Optical coherence tomography attenuation coefficient representing collagen deposition was significantly decreased at week 4 compared with day 0 and increased at week 16 (p < 0.05). Innovation: This study provides a platform for further research on the simultaneous evaluation of the effects of compounds in cutaneous scarring by RS and HPLC, and identifies a role for RS in the therapeutic evaluation and theranostic management of skin scarring. Conclusions: RS can provide noninvasive information on the effects of topicals on scar pathogenesis and structural composition, validated by other analytical techniques.

Multiplexed imaging mass spectrometry of the extracellular matrix using serial enzyme digests from formalin-fixed paraffin-embedded tissue sections.

We report a multiplexed imaging mass spectrometry method which spatially localizes and selectively accesses the extracellular matrix on formalin-fixed paraffin-embedded tissue sections. The extracellular matrix (ECM) consists of (1) fibrous proteins, post-translationally modified (PTM) via N- and O-linked glycosylation, as well as hydroxylation on prolines and lysines, and (2) glycosaminoglycan-decorated proteoglycans. Accessing all these components poses a unique analytical challenge. Conventional peptide analysis via trypsin inefficiently captures ECM peptides due to their low abundance, intra- and intermolecular cross-linking, and PTMs. In previous studies, we have developed matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) techniques to capture collagen peptides via collagenase type III digestion, both alone and after N-glycan removal via PNGaseF digest. However, in fibrotic tissues, the buildup of ECM components other than collagen-type proteins, including elastin and glycosaminoglycans, limits efficacy of any single enzyme to access the complex ECM. Here, we have developed a novel serial enzyme strategy to define the extracellular matrix, including PTMs, from a single tissue section for MALDI-IMS applications. Graphical Abstract.

Novel Recombinant Tropoelastin Implants Restore Skin Extracellular Matrix.

BACKGROUND: Elastin is an essential component of the dermis, providing skin with elasticity and integrity. Elastin and other dermal components are gradually lost through aging, sun damage, and following injury, highlighting a need to replace these components to repair the skin. Tropoelastin (TE) in monomeric form was previously shown to be utilized as a substrate by dermal fibroblasts during the production of elastin fibers in vitro. OBJECTIVE: To analyze coaccumulation of elastin and collagen and gene expression of biomarkers associated with elastin production, examine the ex vivo effects of recombinant human TE (rhTE) and hyaluronic acid (HA) on epidermal and dermal structures, and evaluate the in vivo response following intradermal injections of rhTE and HA. METHODS: Human dermal fibroblasts and 3-D skin patch models were cultured for in vitro analysis. Ex vivo analysis was performed using skin explants. In vivo studies were done in 6-week-old male CD Hairless rats. Different formulations of rhTE, soluble or crosslinked using derivatized HA (dHA), were tested and analyzed. RESULTS: rhTE in monomeric form was utilized as a substrate by dermal fibroblasts during the production of branched elastin and fibrous collagen networks in vitro. Formulations of rhTE crosslinked with dHA demonstrated increased expression of hyaluronic acid synthase 1 and ex vivo results revealed increased moisture content and glycosaminoglycan (GAG) deposition versus dermal filler control. Intradermal rhTE‒dHA injection produced colocalized human‒rat elastin fibers in vivo. CONCLUSIONS: These results suggest that the novel rhTE‒dHA matrix is an attractive material to support skin tissue repair.J Drugs Dermatol. 2020;19(12): doi:10.36849/JDD.2020.5375.

Lifelong SIRT-1 overexpression attenuates large artery stiffening with advancing age.

Advanced age is accompanied by aortic stiffening that is associated with decreased vascular expression of sirtuin-1 (SIRT-1). Interventions that increase SIRT-1 expression also lower age-related aortic stiffness. Therefore, we sought to determine if lifelong SIRT-1 overexpression would attenuate age-related aortic stiffening. Aortic pulse wave velocity (PWV) was assessed from 3-24 months in SIRT-1 transgenic overexpressing (SIRTTG) and wild-type (WT) mice. To determine the role of aortic structural changes on aortic stiffening, histological assessment of aortic wall characteristics was performed. Across the age range (3-24 mo), PWV was 8-17% lower in SIRTTG vs. WT (P<0.05). Moreover, the slope of age-related aortic stiffening was lower in SIRTTG vs. WT (2.1±0.2 vs. 3.8±0.3 cm/sec/mo, respectively). Aortic elastin decreased with advancing age in WT (P<0.05 old vs. young WT), but was maintained in SIRTTG mice (P>0.05). There was an age-related increase in aortic collagen, advanced glycation end products, and calcification in WT (P<0.05 old vs. young WT). However, this did not occur in SIRTTG (P>0.05). These findings indicate that lifelong SIRT-1 overexpression attenuates age-related aortic stiffening. These functional data are complemented by histological assessment, demonstrating that the deleterious changes to the aortic wall that normally occur with advancing age are prevented in SIRTTG mice.