Collagen Architecture Research Articles

Comparative Analysis of Commercially Available Extracellular Matrix Soft Tissue Bioscaffolds.

Decellularized extracellular matrix (dECM) products are widely established for soft tissue repair, reconstruction and reinforcement. These regenerative biomaterials mimic native tissue ECM with respect to structure and biology and are produced from a range of tissue sources and species. Optimal source tissue processing requires a balance between removal of cellular material and the preservation of structural and biological properties of tissue ECM. Despite the wide-spread clinical use of dECM products there is a lack of comparative information on these products Structurally, some dECM products showed a well-preserved collagen architecture with a broad porosity distribution, while others showed a significantly altered structure compared with native tissue. Decellularization varied across the products. Some materials surveyed (OFMm, PPN, PPC, OFMo, UBM, SISz, ADM, PADM and BADM) were essentially devoid of nuclear bodies (mean count of <5 cells per high powered field (HPF)), whereas others (SISu and SISb) demonstrated an abundance of nuclear bodies (>50 cells per HPF). Pathology assessment of the products demonstrated that OFMm, OFMo and PADM had the highest qualitative assessment score for collagen fiber orientation and arrangement, matrix porosity, decellularization efficiency, and residual vascular channels scoring 10.5±0.8, 12.8±1.0, and 9.7±0.7 out of a maximum total score of 16, respectively This analysis of commercially available dECM products in terms of their structure and cellularity includes 12 different commercial materials The findings highlight the variability of the products in terms of matrix structure and the efficacy of decellularization.

Type III Collagen Regulates Matrix Architecture and Mechanosensing during Wound Healing

Post-natal cutaneous wound healing is characterized by development of collagen-rich scar lacking the architecture and functional integrity of unwounded tissue. Directing cell behaviors to efficiently heal wounds while minimizing scar formation remains a major wound management goal. Herein, we demonstrate type III collagen (Col3) as a critical regulator of re-epithelialization and scar formation during healing of Col3-enriched, regenerative (Acomys), scar-permissive (CD-1 Mus and wild-type Col3B6/B6 mice), and Col3-deficient, scar-promoting (Col3F/F, a murine conditional knockdown model) cutaneous wound models. We define a scar-permissive fibrillar collagen architecture signature characterized by elongated and anisotropically-aligned collagen fibers that is dose-dependently suppressed by Col3. Further, loss of Col3 alters how cells interpret their microenvironment - their mechanoperception - such that Col3-deficient cells display mechanically-active phenotypes in the absence of increased microenvironmental stiffness via upregulation and engagement of the profibrotic integrin α11. Further understanding Col3's role in regulating matrix architecture and mechanoresponses may inform clinical strategies that harness pro-regenerative mechanisms.

Plasticity variable collagen-PEG interpenetrating networks modulate cell spreading

The extracellular matrix protein collagen I has been used extensively in the field of biomaterials due to its inherent biocompatibility and unique viscoelastic and mechanical properties. Collagen I self-assembly into fibers and networks is environmentally sensitive to gelation conditions such as temperature, resulting in gels with distinct network architectures and mechanical properties. Despite this, collagen gels are not suitable for many applications given their relatively low storage modulus. We have prepared collagen-poly(ethylene glycol) [PEG] interpenetrating network (IPN) hydrogels to reinforce the collagen network, which also induces changes to network plasticity, a recent focus of study in cell-matrix interactions. Here, we prepare collagen/PEG IPNs, varying collagen concentration and collagen gelation temperature to assess changes in microarchitecture and mechanical properties of these networks. By tuning these parameters, IPNs with a range of stiffness, plasticity and pore size are obtained. Cell studies suggest that matrix plasticity is a key determinant of cell behavior, including cell elongation, on these gels. This work presents a natural/synthetic biocompatible matrix that retains the unique structural properties of collagen networks with increased storage modulus and tunable plasticity. The described IPN materials will be of use for applications in which control of cell spreading is desirable, as only minimal changes in sample preparation lead to changes in cell spreading and circularity. Additionally, this study contributes to our understanding of the connection between collagen self-assembly conditions and matrix structural and mechanical properties and presents them as useful tools for the design of other collagen based biomaterials. Statement of significanceWe developed a collagen-poly(ethylene glycol) interpenetrating network (IPN) platform that retains native collagen architecture and biocompatibility but provides higher stiffness and tunable plasticity. With minor changes in collagen gelation temperature or concentration, IPN gels with a range of plasticity, storage modulus, and pore size can be obtained. The tunable plasticity of the gels is shown to modulate cell spreading, with a greater proportion of elongated cells on the most plastic of IPNs, supporting the assertion that matrix plasticity is a key determinant of cell spreading. The material can be of use for situations where control of cell spreading is desired with minimal intervention, and the findings herein may be used to develop similar collagen based IPN platforms.

Poster 367: Nano-Semiconductor Fibers in a Commercial Compression Sleeve Accelerates Healing of Achilles Tendon Injury in a Rat Model

Objectives: Commercial compression sleeves have been promoted to ameliorate pain secondary to arthritis and tendinopathy. The fabric of one particular sleeve is embedded with nano-semiconductor fibers that reflect thermal energy produced by the body at a photonic wavelength mimicking low-level laser therapy. The device is theorized to accelerate the inflammatory stage of tissue healing to reach the proliferation and remodeling stages at faster rates. Previous clinical studies have shown a significant improvement in patient-reported outcomes between patients who did and did not use this sleeve to treat osteoarthritis. The objective of this investigation was to determine if compression sleeves embedded with nano-semiconductor fibers can accelerate Achilles tendon healing in a rabbit model. Methods: Twelve New Zealand White rabbits—under IACUC approval—were administered intratendinous injections of 0.2% collagenase into the right Achilles tendon to simulate an acute tendon injury. Rabbits were divided into three groups: group I received the commercial compression sleeve without nano-semiconductors (n = 4), group II received the sleeve with nano-semiconductors (n = 4), and group III received an alternate sleeve design also with nano-semiconductors (n = 4). Calf circumference measurements and thermal radiation imaging of the right lower leg of each rabbit were collected days 7, 14, 21, and 28 post-injection. Two rabbits from each group were sacrificed at the 14-day and 28-day marks and the right Achilles tendons were harvested. Histological analysis using picrosirius red staining was performed to evaluate tissue organization. A custom MATLAB program (MathWorks, Natick, MA) was used to quantify the collagen fiber dispersion within the Achilles tendon samples. Results: A statistically significant difference in calf circumference was found at the 28-day mark between group I versus groups II and III: group I had an average calf circumference of 5.2 cm while groups II and III had an average calf circumference of 4.85 cm (p = 0.036) and 4.60 cm (p = 0.021) respectively. Additionally, after 28 days, group I demonstrated an average pixel density intensity on thermal radiation imaging of 1,531,192 while groups II and III yielded average pixel density intensities of 1,363,515 (p = 0.008) and 1,335,456 (p = 0.003) respectively. Histological staining showed observable alterations in collagen architecture, with group I displaying widespread disorganization and degradation of type I and type III collagen fibers near the injection site. Groups II and III displayed greater organization at the 28-day mark in comparison to group I, with signs of tissue proliferation and remodeling evident. No statistically significant differences were found between group I versus groups II and III regarding collagen fiber dispersion at both the 2-week mark (p = 0.145, p = 0.236) and the 4-week mark (p = 0.113, p = 0.268), respectively. Conclusions: The nano-semiconductor fibers embedded within a commercial sleeve significantly decreased swelling and temperature of the lower leg 28 days following acute Achilles tendon injury in a rabbit model. Furthermore, histologic analysis demonstrated greater collagen fiber and extracellular matrix organization as well as greater proliferation of type III collagen suggesting a faster transition to the proliferation stage of tissue healing.

A highly potent bi-thiazole inhibitor of LOX rewires collagen architecture and enhances chemoresponse in triple-negative breast cancer.

Lysyl oxidase (LOX) is upregulated in highly stiff aggressive tumors, correlating with metastasis, resistance, and worse survival; however, there are currently no potent, safe, and orally bioavailable small molecule LOX inhibitors to treat these aggressive desmoplastic solid tumors in clinics. Here we discovered bi-thiazole derivatives as potent LOX inhibitors by robust screening of drug-like molecules combined with cell/recombinant protein-based assays. Structure-activity relationship analysis identified a potent lead compound (LXG6403) with ∼3.5-fold specificity for LOX compared to LOXL2 while not inhibiting LOXL1 with a competitive, time- and concentration-dependent irreversible mode of inhibition. LXG6403 shows favorable pharmacokinetic properties, globally changes ECM/collagen architecture, and reduces tumor stiffness. This leads to better drug penetration, inhibits FAK signaling, and induces ROS/DNA damage, G1 arrest, and apoptosis in chemoresistant triple-negative breast cancer (TNBC) cell lines, PDX organoids, and invivo. Overall, our potent and tolerable bi-thiazole LOX inhibitor enhances chemoresponse in TNBC, the deadliest breast cancer subtype.

Image analysis Uncovers associations between immune landscape, collagen structure, and neoadjuvant chemotherapy in high-grade serous ovarian carcinomas

BackgroundThe changes in the tumor microenvironment of high-grade serous ovarian carcinomas following neoadjuvant chemotherapy are a complex area of study. Previous research underscores the importance of investigating the immune and collagen components within the tumor microenvironment for prognostic implications. MethodsIn this study, we utilized computational pathology techniques with Hematoxylin and Eosin-stained images to quantitatively characterize the immune and collagen architecture within the tumor microenvironment of patients with high-grade serous ovarian carcinoma. ResultsOur analysis of 12 pre- and post-neoadjuvant chemotherapy images revealed an increase in immune infiltrate, primarily within the epithelial region. Additionally, post-neoadjuvant chemotherapy images exhibited chaotic collagen architecture compared to pre-neoadjuvant chemotherapy images. Importantly, features extracted from post-neoadjuvant chemotherapy images showed associations with overall survival, potentially aiding in the selection of patients for immunotherapy trials. ConclusionsThese findings offer critical insights into the changes in the tumor microenvironment of high-grade serous ovarian carcinomas following neoadjuvant chemotherapy and their potential implications for clinical outcomes.

Collagen architecture and biomechanics of gracilis and adductor longus muscles from children with cerebral palsy.

Cerebral palsy (CP) describes some upper motoneuron disorders due to non-progressive disturbances occurring in the developing brain that cause progressive changes to muscle. While longer sarcomeres increase muscle stiffness in patients with CP compared to typically developing (TD) patients, changes in extracellular matrix (ECM) architecture can increase stiffness. Our goal was to investigate how changes in muscle and ECM architecture impact muscle stiffness, gait and joint function in CP. Gracilis and adductor longus biopsies were collected from children with CP undergoing tendon lengthening surgery for hamstring and hip adduction contractures, respectively. Gracilis biopsies were collected from TD patients undergoing anterior cruciate ligament reconstruction surgery with hamstring autograft. Muscle mechanical testing, two-photon imaging and hydroxyproline assay were performed on biopsies. Corresponding data were compared to radiographic hip displacement in CP adductors (CPA), gait kinematics in CP hamstrings (CPH), and joint range of motion in CPA and CPH. We found at matched sarcomere lengths muscle stiffness and collagen architecture were similar between TD and CP hamstrings. However, CPH stiffness (R2=0.1973), collagen content (R2=0.5099) and cross-linking (R2=0.3233) were correlated to decreased knee range of motion. Additionally, we observed collagen fibres within the muscle ECM increase alignment during muscular stretching. These data demonstrate that while ECM architecture is similar between TD and CP hamstrings, collagen fibres biomechanics are sensitive to muscle strain and may be altered at longer in vivo sarcomere lengths in CP muscle. Future studies could evaluate the impact of ECM architecture on TD and CP muscle stiffness across in vivo operating ranges. KEY POINTS: At matched sarcomere lengths, gracilis muscle mechanics and collagen architecture are similar in TD patients and patients with CP. In both TD and CP muscles, collagen fibres dynamically increase their alignment during muscle stretching. Aspects of muscle mechanics and collagen architecture are predictive of in vivo knee joint motion and radiographic hip displacement in patients with CP. Longer sarcomere lengths in CP muscle in vivo may alter collagen architecture and biomechanics to drive deficits in joint mobility and gait function.

A Comparative Study of the Effects of Different Crosslinking Methods on the Physicochemical Properties of Collagen Multifilament Bundles.

Crosslinking is usually required to improve the mechanical properties and stability of collagen-based scaffolds. Introducing exogenous crosslinks into collagen may however affect the collagen structure. Since the architecture of collagen is tied to its functionality, it is important to study the effect of crosslinking and to select a crosslinking method that preserves both the collagen structure and mechanical properties. The objective of this study is to compare the effect of various crosslinking methods on the structure and mechanical properties of bioartificial tendon-like materials (collagen multifilament bundles) fabricated by contact drawing. We examine both physical (ultraviolet light, UVC) and chemical (genipin, carbodiimide (EDC), and glutaraldehyde) crosslinking methods. The presence of collagen and the formation of well-ordered collagen structures are confirmed by attenuated total reflectance Fourier-transform infrared spectromicroscopy and wide-angle X-ray scattering for all crosslinking methods. The morphology of the collagen multifilament bundles is similar across crosslinking methods. Swelling of the multifilament bundles is dramatically reduced following crosslinking and varies by crosslinking method, with genipin- and carbodiimide-crosslinked specimens swelling the least. Ultimate tensile strength (UTS) and Young's modulus significantly improve for all crosslinked specimens compared to non-crosslinked specimens. Glutaraldehyde crosslinked collagen multifilament bundles display the highest UTS values ranging from 33.82±0.0 MPa to 45.59±0.76 MPa.

Primary Human Cell-Derived Extracellular Matrix from Decellularized Fibroblast Microtissues with Tissue-Dependent Composition and Microstructure.

Human extracellular matrix (ECM) exhibits complex protein composition and architecture depending on tissue and disease state, which remains challenging to reverse engineer. One promising approach is based on cell-secreted ECM from primary human fibroblasts that can be decellularized into acellular biomaterials. However, fibroblasts cultured on rigid culture plastic or biomaterial scaffolds can experience aberrant mechanical cues that perturb the biochemical, mechanical, and the efficiency of ECM production. Here, we demonstrate a method for preparing decellularized ECM using primary human fibroblasts with tissue and disease-specific features with two case studies: (1) cardiac fibroblasts; (2) lung fibroblasts from healthy or diseased donors. Cells aggregate into engineered microtissues in low adhesion microwells that deposited ECM and can be decellularized. We systematically investigate microtissue morphology, matrix architecture, and mechanical properties, along with transcriptomic and proteomic analysis. Microtissues exhibited tissue-specific gene expression and proteomics profiling, with ECM complexity similar to native tissues. Healthy lung microtissues exhibited web-like fibrillar collagen compared to dense patches in healthy heart microtissues. Diseased lung exhibited more disrupted collagen architecture than healthy. Decellularized microtissues had tissue-specific mechanical stiffness that was physiologically relevant. Importantly, decellularized microtissues supported viability and proliferation of human cells. We show that engineered microtissues of primary human fibroblasts seeded in low-adhesion microwells can be decellularized to produce human, tissue and disease-specific ECM. This approach should be widely applicable for generating personalized matrix that recapitulate tissues and disease states, relevant for culturing patient cells ex vivo as well as implantation for therapeutic treatments. The online version contains supplementary material available at 10.1007/s12195-024-00809-y.

Biomimetic hydrogels with mesoscale collagen architecture for patient-derived tumor organoids culture

Patient-derived tumor organoids (PDTOs) shows great potential as a preclinical model. However, the current methods for establishing PDTOs primarily focus on modulating local properties, such as sub-micrometer topographies. Nevertheless, they neglect to capture the global millimeter or intermediate mesoscale architecture that have been demonstrated to influence tumor response to therapeutic treatment and tumor progression. In this study, we present a rapid technique for generating collagen bundles with an average length of 90 ± 27 μm and a mean diameter of 5 ± 1.5 μm from tumor tissue debris that underwent mechanical agitation following enzymatic digestion. The collagen bundles were subsequently utilized for the fabrication of biomimetic hydrogels, incorporating microbial transglutaminase (mTG) crosslinked gelatin. These biomimetic hydrogels, referred to as MC-gel, were specifically designed for patient-derived tumor organoids. The lung cancer organoids cultured in MC-gel exhibited larger diameters and higher cell viability compared to those cultured in gels lacking the mesoscale collagen bundle; moreover, their irregular morphology more closely resembled that observed in vivo. The MC-gel-based lung cancer organoids effectively replicated the histology and mutational landscapes observed in the original donor patient’s tumor tissue. Additionally, these lung cancer organoids showed a remarkable similarity in their gene expression and drug response across different matrices. This recently developed model holds great potential for investigating the occurrence, progression, metastasis, and management of tumors, thereby offering opportunities for personalized medicine and customized treatment options.

Abstract PO4-24-07: The Impact of GPER Modulation on Cancer Associated Fibroblasts on Extracellular Matrix Formation in Breast Cancer

Abstract Background: The G-protein coupled estrogen receptor (GPER) is a noncanonical estrogen receptor (ER) that is estimated to be present in roughly 50-60% of all breast cancer (BC) subtypes. Previous studies have suggested that the presence of GPER during primary disease aids in disease progression, metastasis, as well as chemotherapy resistance due to the agonistic activity of traditional chemotherapies for ER+ BC, tamoxifen and fulvestrant. It is also well established that the extracellular matrix (ECM), specifically collagen, contributes to the overall risk and progression of BC in patients. Importantly, fibroblasts are one of the primary cell types responsible for ECM deposition in the tumor microenvironment. We hypothesize that the modulation of cancer-associated fibroblasts (CAFs) via estrogen signaling through GPER regulates the deposition and structure of key ECM proteins, such as collagen, to aid in disease progression. Methods: CAFs, isolated from human BC surgical resections, were plated at 100% confluency, and incubated with either 10nM β-estradiol (E2), 50nM G1 (a GPER specific agonist) or a vehicle control and 50ug/mL ascorbic acid daily for 14 days to induce matrix deposition. Phenol-free, high glucose DMEM with 10% charcoal stripped FBS was used as media to reduce FBS sourced estrogen. Following the 14-day incubation, cell proliferation was quantified prior to extracting the cells to isolate the CAF-derived matrix (CDM). The organization and composition of ECM was analyzed in CDMs through immunofluorescence (IF) or western blot, respectively. An unpaired t-test was conducted on biological triplicates and between vehicle control and either G1 or E2. Significance was determined as p&amp;lt; 0.05. Results: Culturing CAFs with E2 resulted in a 10% increase in cell proliferation (n=3, p&amp;lt; 0.05) and a 1.6-fold (n=3, p&amp;lt; 0.05) increase in total ECM deposition by western blot. Key ECM proteins such as collagen, fibronectin, and periostin had a 1.8, 1.3, and 1.3-fold increase, respectively (n=3, p&amp;lt; 0.05), in protein deposition with E2 treatment when normalized to cell number. Collagen fiber analysis of IF stained CDM for collagen resulted in a coefficient of alignment with E2 of 0.76 compared to 0.51 without (n=3 for E2 and n=6 for vehicle, p&amp;lt; 0.05). To confirm E2 was acting through GPER, CDMs were subsequently generated with exposure to 50nM G1, a specific GPER agonist. Collagen fiber analysis of the G1 exposed CDMs resulted in a coefficient of alignment with G1 of 0.71 compared to 0.51 without (n=3 for G1 and n=6 for vehicle, p&amp;lt; 0.05). Conclusions: This study demonstrates that estrogen signaling through GPER modulates the deposition of fibronectin, periostin, and collagen as well as the organization of collagen architecture. These results highlight the need to further investigate how the interplay between the ECM, GPER activity, and fibroblasts impacts breast cancer progression. Citation Format: Shelby Fertal, Brian Burkel, Fern Murdoch, Kathy O'Leary, Linda Schuler, Suzanne Ponik. The Impact of GPER Modulation on Cancer Associated Fibroblasts on Extracellular Matrix Formation in Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-24-07.

Pathological Eye Growth in Myopia: a Limited Review on Endocrine Physiology for Future Research

Purpose: Myopia or short-sightedness: first manifests in school-children (early-onset), and also in young adults (late-onset). Pathological myopia can lead to stretching of the eyeball to the point of blindness from a detached retina. This title presents a preliminary understanding of endocrine, juxtacrine, and paracrine factors: hoping to stimulate further research and enable early therapeutic intervention. Background: The optical stimulus for eye growth is a blurred image on the retina that results from under-focused accommodation of the internal crystalline lens. Neural signals and biochemical messaging from the retina and vascular choroid provide local impetus to alter collagen structure in the outer white sclera of the eye. In persons that develop highly myopic refractive error, additional factors from blood and tissue chemistry, might influence intra-ocular pressure (IOP), posited to influence eyeball elongation. New research shows that GABA and dopamine are involved, but other factors remain unexplored. Eye Physiology: Human sclera has developmental origination same as any other connective tissue derived from the neural crest and mesoderm. Bundles of collagen fibers exist in the white opaque sclera and are more stringently oriented in the dome of the clear, transparent cornea. “Deformation compliance” of the sclera is governed by complexity of the collagen architecture, and “reduced scleral rigidity” is a known factor for myopic elongation, but developmental physiology is not well understood. Muscular contraction is required for sustained eye focusing to objects located at normal working distances. Short-term contractions can reduce IOP but increased muscle density from sustained near accommodation can promote progression of myopia. This implicates nitric oxide and calcium channels, and a role for associated biochemical and nutritional agonists and antagonists. Endocrine Influences: In his 1954 lecture to the Royal College of Surgeons, Dr. Howard C. Naffziger pointed to all forms of “ocular protrusion,” not just “thyroid exophthalmos,” as being physiologically related. A 2016 review paper on autonomic nervous physiology, aside from sympathetic (adrenergic) and parasympathic (collinergic) control, also implicates Substance P (SP), Vasoactive Intestinal Peptide (VIP), and Phospholipase C (PC). These topics are further described in limited detail. SP is a neuropeptide of the tachykinin family, is released from the terminals of sensory nerves, and regulates emotion (hypothalamus, amygdala, and the periaqueductal gray). It is also a vasodilator dependent on release of nitric oxide. Experimental eye physiology and clinical research pertaining to these topics appears warranted. VIP regulates cardiac contractions, relaxes smooth muscle of the trachea, and affects prolactin secretion by the pituitary gland. Cardiac contractions lead to pulsations of eye pressure about three times in two seconds, and pulmonary effects on IOP occur once every five seconds. Research into these mechanisms requires cross-disciplinary collaborations. PC is a membrane-associated enzyme that cleaves dietary phospholipids and has downstream effects on calcium channels. Binding to blood platelets, of agonists such as thrombin, epinephrine, or collagen: activates PC to release arachidonic acid from cleavage of phosphatidylinositol and phosphatidyl choline. Behavioral effects include visual attention-span and memory: both of which also concern education of children, and effectiveness of adults at work. Conclusions: The author is pleased to report growing interest and evidence for endocrine influences upon the outer coat of the eye (cornea and sclera). A 2022 study on “keratoconus” from Texas and Oklahoma provides in-vitro as well as in-vivo data, suggesting the existence of what they call, the Hypothalamic-Pituitary-Adrenal-Corneal axis. Effective communication across university disciplines and clinic doctors appears necessary for further progress. Funding: The author received no funds from Federal or State granting mechanisms, and no wages from corporate employers. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The phenomenon of skin contraction in CO2 LASER surgical incisions using superpulse and continuous emission mode - preliminary study.

The skin contraction phenomenon occurs due to the energy emitted by the surgical CO2 LASER affecting the collagen architecture and intracellular water content in tissues. The study aimed to assess how gender, age, breed, body-weight, CO2 LASER emission mode, and potency influence skin contraction following the incision. The study involved 80 dogs (N = 80) of both genders, multiple breeds, undergoing major surgery with CO2 LASER. Subjects were grouped based on LASER potency (12 or 15 Watts) and emission mode (Superpulse-SP or Continuous-CT): GSP12, GSP15, GCT12, and GCT15. A 10mm incision was performed using the surgical CO2 LASER beam, consistently employing a focal point of 0.4mm, positioned at a distance of 1mm from the skin surface, and always maintained perpendicular to it, and resulting lengths measured with a digital caliper. Results were considered significant for p-value < 0.05. GSP12 showed minimal contraction, while GCT15 exhibited the most significant. Male subjects in GCT12, GCT15, and GSP12 experienced less contraction than females. Purebred dogs had greater contraction than mixed breeds. GSP12 individuals showed age-related contraction decrease (p < 0.01), with skin contracting by 0.09mm per year. Weight and skin contraction trended towards significance (p = 0.06), with a 0.02mm increase per unit weight. For a constant power of 12 W, the analysis of the relationship between the emission mode of the LASER beam and the final skin contraction (GSP12 vs. GCT12) revealed statistically significant differences (p < 0.01). This study suggests that the use of the Continuous mode of LASER emission, regardless of the power used, is associated with a higher level of final skin contraction. TRIAL REGISTRATION NUMBER AND DATE OF REGISTRATION FOR PROSPECTIVELY REGISTERED TRIALS: Project approval registration number by the Research and Teaching Ethics Committee (CEIE),Faculty of Veterinary Medicine-University of Lisbon (FMV_ULisboa), Lisboa-Portugal, N/Refª 015/2022.

Deletion of mouse lysyl oxidase in megakaryocytes affects bone properties in a sex-dependent manner

AbstractLysyl oxidase (LOX) is a facilitator of extracellular matrix cross-linking. Using newly developed megakaryocyte-specific LOX knockout mice, we show that LOX expressed in these scarce bone marrow cells affects bone volume and collagen architecture in a sex-dependent manner.

High angular resolution diffusion imaging (HARDI) of porcine menisci: a comparison of diffusion tensor imaging and generalized q-sampling imaging.

Diffusion magnetic resonance imaging (MRI) allows for the quantification of water diffusion properties in soft tissues. The goal of this study was to characterize the 3D collagen fiber network in the porcine meniscus using high angular resolution diffusion imaging (HARDI) acquisition with both diffusion tensor imaging (DTI) and generalized q-sampling imaging (GQI). Porcine menisci (n=7) were scanned ex vivo using a three-dimensional (3D) HARDI spin-echo pulse sequence with an isotropic resolution of 500 µm at 7.0 Tesla. Both DTI and GQI reconstruction techniques were used to quantify the collagen fiber alignment and visualize the complex collagen network of the meniscus. The MRI findings were validated with conventional histology. DTI and GQI exhibited distinct fiber orientation maps in the meniscus using the same HARDI acquisition. We found that crossing fibers were only resolved with GQI, demonstrating the advantage of GQI over DTI to visualize the complex collagen fiber orientation in the meniscus. Furthermore, the MRI findings were consistent with conventional histology. HARDI acquisition with GQI reconstruction more accurately resolves the complex 3D collagen architecture of the meniscus compared to DTI reconstruction. In the future, these technologies have the potential to nondestructively assess both normal and abnormal meniscal structure.

Abstract 681: Rewiring collagen architecture using a highly potent bi-thiazole inhibitor of LOX mediates chemosensitization in triple-negative breast cancer

Abstract Lysyl oxidase (LOX) is the founding member of the LOX family proteins and mediates the conversion of lysine residues in collagen I and elastin precursors into highly reactive aldehydes, leading to crosslinking of extracellular matrix (ECM). LOX is upregulated in aggressive tumors with high stiffness, and its higher expression correlates with metastasis, therapy resistance, and worse clinical outcome; however, relatively LOX-specific, orally bioavailable inhibitors are still lacking. Here we discovered bi-thiazole derivatives as potent LOX inhibitors by combining a robust screening platform for drug-like molecules, cell- and recombinant protein-based assays. Structure-activity relationship analysis further identified a potent and relatively LOX-specific lead compound (LXG6403) that reduces collagen content and crosslinking, and fibronectin assembly, leading to increased chemoresponse in triple-negative breast cancer (TNBC) cell lines and PDX organoids in 3D collagen. LXG6403 shows favorable pharmacokinetic properties, reduces collagen content and crosslinking, as demonstrated by the state-of-the-art MALDI-MSI and MP-SHG analyses, respectively. This leads to better drug penetration, inhibition of FAK signaling, and induction of ROS generation/DNA damage, leading to G1 arrest and apoptosis in chemoresistant TNBC PDXs. Overall, our novel, potent, tolerable bi-thiazole LOX inhibitor enhances chemoresponse in TNBC, the deadliest breast cancer subtype. Citation Format: Metin Cetin, Ozge Saatci, Abdol-Hossein Rezaeian, Chintada N. Rao, Chad Beneker, Harrison Taylor, Breanna Pederson, Ioulia Chatzistamou, Brian Buckley, Susan Lessner, Peggi Angel, Campbell McInnes, Ozgur Sahin. Rewiring collagen architecture using a highly potent bi-thiazole inhibitor of LOX mediates chemosensitization in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 681.

Abstract 7382: Artificial intelligence derived features of collagen architecture identify high risk HPV associated oropharyngeal squamous cell carcinoma patients

Abstract Introduction: Human papilloma virus (HPV) induced oropharyngeal squamous cell carcinoma (OPSCC) is a common subtype of head and neck carcinoma. Dysregulation of the extracellular matrix (ECM), an important part of the tumor microenvironment (TME), is shown in HPV related cancers. We studied collagen fibers (CF), the amplest component of the ECM, and evaluated whether their characteristics within the TME can offer a prognostic value in OPSCC. Methods: Whole slide images (WSIs) of OPSCC patients were obtained from Washington University (WU, n=107), Houston Veterans Administration Hospital (HV, n=94), Cleveland Clinic (CC, n=336), John Hopkins University (JH, n=121) and Vanderbilt Medical Centre (VM, n=158). For the identified CF in tumor-stroma regions of WSIs, the extracted CF features included: CF fragmentation measure, CF bundling percentage, CF rigidity measure, CF anisotropy index, and CF density index. For survival analysis using overall survival (OS) and Disease-Free Survival (DFS) as endpoints, the median risk score in the training set (WU) was applied for risk stratification in the validation sets (HV, CC, JH, VM) by means of a Least Absolute Shrinkage and Selection Operator-Cox regression model. Multivariable Cox Proportional Hazards Regression was done for both OS and DFS. Results: The patients defined as “high risk” had worse OS than those identified as “low risk” (HV(p=0.03), CC(p=0.03), JH(p=0.02), VM(p=0.04)). Multivariable analysis showed that CF features were prognostic independent of T/N stages, age, sex, and smoking status with HR=1.99 (95% CI=1.46-2.72, p=0.00001) for OS and HR=1.73 (95% CI=1.21-247, p=0.002) for DFS (Table 1). Table 1: Multivariable Survival Analysis for OS and DFS, HR=Hazard Ratio, CI=Confidence Interval Variable Overall Survival Disease Free Survival HR (95% CI) P value HR (95% CI) P value Age (&amp;lt;65 years vs. &amp;gt;=65 years) 0.99 (0.93-1.06) 0.78444 0.96 (0.82-1.12) 0.62864 Sex (Female vs. Male) 0.56 (0.29-1.10) 0.09042 0.50 (0.22-1.13) 0.09385 Smoking status (Yes vs. No) 1.85 (1.31-2.62) 0.00051 1.03 (0.72-1.47) 0.86871 T-stage (T3/4 vs T1/2) 1.86 (1.38-2.50) 0.00004 1.91 (1.36-2.69) 0.00018 N-stage (N+ vs N0) 1.76 (1.31-2.37) 0.00016 2.04 (1.46-2.86) 0.00003 Collagen fiber features (High risk vs low risk) 1.99 (1.46-2.72) 0.00001 1.73 (1.21-2.47) 0.00286 Conclusion: Artificial intelligence derived features of CF architecture can help identify high vs low risk patients in HPV related OPSCC. These findings deserve further confirmation in larger prospectively collected OPSCC data sets and could impact future treatment paradigms. Citation Format: Reetoja Nag, Haojia Li, Germán Corredor, Pingfu Fu, Nabil F. Saba, Mehmet A. Bilen, Tilak Pathak, Paula Toro, Mojgan Mokhtari, Krunal Pandav, Michael Gilkey, Shlomo Koyfman, Deborah Chute, Wade L. Thorstad, Mitra Mehrad, Patricia D. Castro, Andrew G. Sikora, Rebecca D. Chernock, Jingqin Luo, Mitchell Frederick, Vlad Sandulache, David J. Adelstein, James Lewis Jr., Anant Madabhushi. Artificial intelligence derived features of collagen architecture identify high risk HPV associated oropharyngeal squamous cell carcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7382.

Avenanthramide and β-Glucan Therapeutics Accelerate Wound Healing Via Distinct and Nonoverlapping Mechanisms.

Objective: Given the significant economic, health care, and personal burden of acute and chronic wounds, we investigated the dose dependent wound healing mechanisms of two Avena sativa derived compounds: avenanthramide (AVN) and β-Glucan. Approach: We utilized a splinted excisional wound model that mimics human-like wound healing and performed subcutaneous AVN and β-Glucan injections in 15-week-old C57BL/6 mice. Histologic and immunohistochemical analysis was performed on the explanted scar tissue to assess changes in collagen architecture and cellular responses. Results: AVN and β-Glucan treatment provided therapeutic benefits at a 1% dose by weight in a phosphate-buffered saline vehicle, including accelerated healing time, beneficial cellular recruitment, and improved tissue architecture of healed scars. One percent AVN treatment promoted an extracellular matrix (ECM) architecture similar to unwounded skin, with shorter, more randomly aligned collagen fibers and reduced inflammatory cell presence in the healed tissue. One percent β-Glucan treatment promoted a tissue architecture characterized by long, thick bundles of collagen with increased blood vessel density. Innovation: AVN and β-Glucan have previously shown promise in promoting wound healing, although the therapeutic efficacies and mechanisms of these bioactive compounds remain incompletely understood. Furthermore, the healed ECM architecture of these wounds has not been characterized. Conclusions: AVN and β-Glucan accelerated wound closure compared to controls through distinct mechanisms. AVN-treated scars displayed a more regenerative tissue architecture with reduced inflammatory cell recruitment, while β-Glucan demonstrated increased angiogenesis with more highly aligned tissue architecture more indicative of fibrosis. A deeper understanding of the mechanisms driving healing in these two naturally derived therapeutics will be important for translation to human use.

Matrix viscoelasticity promotes liver cancer progression in the pre-cirrhotic liver.

Type 2 diabetes mellitus is a major risk factor for hepatocellular carcinoma (HCC). Changes in extracellular matrix (ECM) mechanics contribute to cancer development1,2, and increased stiffness is known to promote HCC progression in cirrhotic conditions3,4. Type 2 diabetes mellitus is characterized by an accumulation of advanced glycation end-products (AGEs) in the ECM; however, how this affects HCC in non-cirrhotic conditions is unclear. Here we find that, in patients and animal models, AGEs promote changes in collagen architecture and enhance ECM viscoelasticity, with greater viscous dissipation and faster stress relaxation, but not changes in stiffness. High AGEs and viscoelasticity combined with oncogenic β-catenin signalling promote HCC induction, whereas inhibiting AGE production, reconstituting the AGE clearance receptor AGER1 or breaking AGE-mediated collagen cross-links reduces viscoelasticity and HCC growth. Matrix analysis and computational modelling demonstrate that lower interconnectivity of AGE-bundled collagen matrix, marked by shorter fibre length and greater heterogeneity, enhances viscoelasticity. Mechanistically, animal studies and 3D cell cultures show that enhanced viscoelasticity promotes HCC cell proliferation and invasion through an integrin-β1-tensin-1-YAP mechanotransductive pathway. These results reveal that AGE-mediated structural changes enhance ECM viscoelasticity, and that viscoelasticity can promote cancer progression in vivo, independent of stiffness.

Mesothelioma of Uncertain Malignant Potential (MUMP) of the Tunica Vaginalis: Proposal for Reclassification as "Complex Mesothelial Tumor of the Tunica Vaginalis".

A well-differentiated papillary mesothelial tumor (WDPMT) and malignant mesothelioma are 2 well-recognized entities arising from the testis tunica vaginalis. Another mesothelial lesion exclusively seen at this site is mesothelioma of uncertain malignant potential (MUMP)-a lesion reminiscent of WDPMT yet demonstrating variable proportions of more complex architectural patterns that could be confused with invasion. MUMP was first described in 2010 with a total of 11 cases reported to date. Herein, we describe 19 additional patients who underwent hydrocelectomy, excision, and/or orchiectomy. Novel morphologic patterns found in addition to the 2010 series include spindle cells, keloidal-type collagen, and multicystic architecture lined by bland mesothelial cells. Clinical follow-up in 9 patients for more than 1 year (1.5 to 22.5y, median 4.5y) revealed no evidence of disease recurrence or metastases. Despite greater architectural complexity, MUMP has (1) bland cytology; (2) merging in with WDPMT areas; (3) low mitotic rate and Ki-67 nuclear labeling index; (4) retention of MTAP and BAP1 expression; and (5) benign clinical follow-up. If these cases were malignant mesotheliomas, one would have expected at least some of the patients to demonstrate disease recurrence/progression without adjuvant therapy within the available follow-up time, particularly with limited resection in most patients. Thus, we propose that "mesothelioma of uncertain malignant potential" be renamed as "complex mesothelial tumor of the tunica vaginalis." Using the term "complex" draws a contrast with the simple cuboidal lining and simple papillary architecture seen in WDPMT. Also, labeling the lesion as "tumor" removes the stigmata of "uncertain malignant potential" and "mesothelioma" that are alarming to patients and clinicians, and potentially could unduly lead to more extensive surgery in an attempt at "complete" resection. At the same time, not definitively labeling the lesion as benign allows recommendations for follow-up.