Collagen Fiber Alignment Research Articles

Abstract PO2-25-07: PEGylated Functional Upstream Domain (PEG-FUD): an anti-cancer therapy for breast Cancer

Abstract Background: Breast cancer (BC) is the most common cancer diagnosed in women. In breast cancers, the extracellular matrix (ECM) is known to play a key role in disease progression by mediating cell signaling processes that drive cancer cell proliferation, migration and invasion. Work by our group identified that aligned collagen fibers perpendicular to the tumor boundary are prognostic of poor patient prognosis. Further, through matrix targeted proteomics we determined that the ECM protein, Fibronectin (FN), organizes with aligned collagen fibers in BC tissues. FN is known as the master regulator of ECM assembly because FN deposition precedes and regulates the deposition of several other ECM proteins. Clinical evidence shows that high expression of FN levels in BC patients correlates with an increased mortality risk. Due to the abundant expression of FN in the tumor microenvironment, FN has been a useful biomarker for cancer imaging and therapy. Despite multiple efforts in developing therapies that target the ECM, currently there are no effective ECM treatments available due to toxicity and lack of specificity. Methods: To target abnormal FN deposition, we used a FN binding peptide, Functional Upstream Domain (FUD), derived from the F1 adhesin from Streptococcus pyogenes. PEGylated-FUD (PEG-FUD) is a potent inhibitor of FN assembly which binds the 70 kDa N-terminal region of FN with high affinity. We hypothesize that PEG-FUD will localize to the tumor site and that targeted disruption of FN assembly with PEG-FUD will reduce ECM deposition, thus block tumor progression in-vivo. In this study, we used an in vivo imaging system to assess the biodistribution of 20-kDa PEG-FUD following subcutaneous injection of Cy5 labeled peptide in 4T1 mammary tumor bearing mice. Additionally, in a second therapeutic experiment, we treated 4T1 mammary tumor bearing mice with 20-kDa PEG-FUD every 48 hr for a total of 10 treatments and measured tumor volume as an indicator of primary tumor burden. Results: As anticipated, we observed PEG-FUD’s accumulation and localization in 4T1 tumors. Additionally, PEG-FUD treatment significantly reduced tumor growth compared to saline treatment. There were no observed changes in standard toxicity assessments such as body weight and spleen weight among treatment groups. After PEG-FUD’s therapeutic treatment, we observed a significant reduction in the deposition of FN matrix and an increase in cleaved caspase-3, a known marker for apoptosis by western blotting providing a potential mechanism of action of PEG-FUD inducing changes in tumor growth. Conclusions: PEG-FUD’s localization in tumors suggests its utility as a solo cancer imaging agent while providing indications that PEG-FUD can be used to deliver other therapeutics to the tumor site as a conjugate in the future. In addition, therapeutic treatment of PEG-FUD inhibited tumor growth providing preliminary evidence for PEG-FUD’s use as a tumor targeting anti-cancer agent. Citation Format: Metti Gari. PEGylated Functional Upstream Domain (PEG-FUD): an anti-cancer therapy for 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 PO2-25-07.

Collagen fibers within skeletal muscle extracellular matrix dynamically change their alignment in a stretch-dependent fashion

Collagen fiber architecture within the skeletal muscle extracellular matrix (ECM) contributes to muscle passive mechanical properties. There is evidence that collagen fibers change their alignment during muscle lengthening (stretching). Thus, the goal of this study was to characterize the dynamic changes of collagen fiber alignment in the muscle ECM during muscle stretching, and to assess the relationship between dynamic collagen re-alignment and muscle passive mechanics. We hypothesized that collagen fibers increase their alignment as the muscle is stretched, and that increased collagen alignment in stretched muscle is correlated to higher elastic stiffness. Extensor digitorum longus (EDL), soleus, and diaphragm muscles were collected from wildtype (WT) and D2.mdx (MDX) mice of 48-52 weeks of age. Muscles underwent a series of twitches to determine the optimum length (Lo) for isometric contraction, and then were decellularized to isolate the muscle ECM. Decellularized muscles (DCMs) were then mechanically tested using a series of stress-relaxation tests at 5% increments from 85% to 115% length relative to Lo. Elastic stiffness was measured as the slope of the tangent line on the stress-strain curve following stress-relaxation. Following mechanical testing, DCMs were imaged using second harmonic generation microscopy to visualize collagen alignment across the range of lengths used in mechanical testing. Collagen alignment was reported as an alignment index on a scale of 0 (perfect unalignment) to 1 (perfect alignment). We found that MDX diaphragm DCMs were less stiff than WT (WT: 910.2±586.7, MDX: 238±168.7, p=0.0041), but there were no significant differences in stiffness between WT and MDX EDL and soleus DCMs. We also observed a significant effect of stretch on collagen alignment in all DCMs tested (Diaphragm: p=0.0071, EDL: p=0.0004, soleus: p<0.0001). Interestingly, the collagen fibers of the epimysium, the outermost layer of the muscle ECM, were more aligned in WT diaphragm DCMs than in MDX (WT 85%: 0.672±.14, MDX 85%: 0.344±0.16, p=0.0027; WT 115%: 0.783±0.04, MDX 115%: 0.480±0.19, p=0.0036). Finally, we found a significant positive correlation between the alignment and elastic stiffness at 110% Lo across all DCMs (p=0.0038, R2=0.1713). These data indicate that collagen fibers dynamically re-align as muscle is stretched. Additionally, increased collagen fiber alignment is associated with higher passive stiffness in the decellularized muscle ECM. Overall, we find that dynamic architectural changes in the muscle ECM contribute to passive mechanical behavior during a muscle stretch. This work was supported by a grant from the NIH NIAMS (R01-AR079545). 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.

Multilayer woven scaffolds with gradient degradation and nano guidance properties for tendon reconstruction: Continuously promoting tissue infiltration and inducing tissue alignment

Limited tissue infiltration and disordered collagen alignment are primary factors in tendon repair failure. This study introduces a multilayer-induced scaffold (IS@DN) for tendon repair, combining gradient-degraded layers (DLs) and nano-guided layers (NLs). DL degradation creates 3-D pore channels for cell infiltration, while NLs offer continuous contact guidance for cell alignment. Enhanced cell penetration and alignment within the scaffold promote orderly collagen regeneration. IS@DN demonstrates suitable mechanical properties and degradation-enhanced porosity, promoting cell and collagen infiltration. Sequential exposure of NLs facilitates continuous alignment of cells and collagen fibers. At a macro level, IS@DN effectively regenerates the rat Achilles tendon, restoring 91.37 % of its native failure load. This integrative approach, incorporating gradient degradation and nano guidance, holds considerable promise for clinical tissue regeneration.

The prognostic and potentially immunomodulatory role of cartilage oligomeric matrix protein in patients with gastric and esophageal adenocarcinoma

BackgroundCartilage oligomeric matrix protein (COMP) is a novel regulator of the tumor microenvironment. Studies in colon cancer and pancreatobiliary adenocarcinoma have revealed COMP expression to be associated with decreased infiltration of immune cells in the tumor microenvironment. Herein, the expression of COMP was investigated in gastric and esophageal adenocarcinoma with particular reference to its the relationship with the immune microenvironment.MethodsCOMP expression was evaluated in tissue microarrays representing primary tumors from 159 patients with chemo- and radiotherapy naïve esophageal and gastric adenocarcinoma and 67 matched samples of lymph node metastases using immunohistochemistry. Additionally, collagen fibers were stained with Sirius Red and evaluated with the FIJI macro TWOMBLI algorithm.ResultsThe expression of COMP in cancer cells in the entire cohort was associated with shorter overall survival (OS) (p = 0.013) and recurrence-free survival (RFS) (p = 0.029), while COMP expression in the stroma was correlated with shorter RFS (p = 0.042). Similar correlations were found for patients with gastric adenocarcinoma, whereas COMP expression was not prognostic in esophageal adenocarcinoma. Further, in the entire cohort, the expression of COMP in the stroma was correlated with exclusion of different populations of immune cells (CD8+, CD3+, FoxP3+, CD20+) from the tumor microenvironment. Finally, higher density and alignment of collagen fibers were correlated with the expression of COMP in the stroma.ConclusionsExpression of COMP in gastric and esophageal adenocarcinoma was correlated with shorter OS and RFS. A reduced number of immune cells infiltrated the tumor microenvironment when COMP expression was detected. This phenomenon could be attributed to the denser collagen deposits, a hallmark of tumor fibrosis observed in COMP-expressing tumors.

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.

Polyhydroxybutyrate-based osteoinductive mineralized electrospun structures that mimic components and tissue interfaces of the osteon for bone tissue engineering.

Scaffolds for bone tissue engineering should enable regeneration of bone tissues with its native hierarchically organized ECM and multiple tissue interfaces. To achieve this, inspired by the structure and properties of bone osteon, we fabricated polyhydroxybutyrate-based mineralized electrospun fibrous scaffolds. After studying multiple polyhydroxybutyrate (PHB)-based fibers, we chose 7%PHB/1%Gelatin fibers (PG) to fabricate mineralized fibers that mimic mineralized collagen fibers in bone. The mineralized PG (mPG) surface had a rough, hydrophilic layer of low crystalline calcium phosphate which was biocompatible to BMSCs, induced their proliferation and was osteoinductive. Subsequently, by modulating the electrospinning process, we fabricated mPG-based novel higher order fibrous scaffolds that mimic the macroscale geometries of osteons of bone ECM. Inspired by the aligned collagen fibers in bone lamellae, we fabricated mPG scaffolds with aligned fibers that could direct anisotropic elongation of mBMSCs. Further, we fabricated electrospun mPG-based osteoinductive tubular constructs which can mimic cylindrical bone components like osteons or lamellae or be used as long bone analogues based on their dimensions. Finally, to regenerate tissue interfaces in bone, we introduced a novel bi-layered scaffold-based approach. An electrospun bi-layered tubular construct that had PG in the outer layer and 7%PHB/0.5%Polypyrrole fibers (PPy) in the inner layer was fabricated. The bi-layered tubular construct underwent preferential surface mineralization only on its outer layer. This outer mineralized layer supported osteogenesis while the inner PPy layer could support neural cell growth. Thus, the bi-layered tubular construct may be used to regenerate haversian canal in the osteons which hosts nerve fibers. Overall, the study introduced novel techniques to fabricate biomimetic structures that can regenerate components of bone osteon and its multiple tissue interfaces. The study lays foundation for the fabrication of a modular scaffold that can regenerate bone with its hierarchical structure and complex tissue interfaces.

Abstract 4271: The ITGA2 (CD49b) collagen receptor excludes the CD8+ T cell infiltration in pancreatic ductal adenocarcinoma

Abstract BACKGROUND: Pancreatic ductal adenocarcinoma cancer (PDAC) is characterized by an enriched extracellular matrix and limited T cell infiltration, which poses challenges for immunotherapy. The significant accumulation of collagen type I (COL1) in PDAC tumor microenvironment raises questions about its functional role in PDAC progression and its interactions with the cellular compartments. OBJECTIVE: The objective of this study was to investigate whether the collagen receptors expressed by cancer cells modulate the anti-tumor activity of CD8+ tumor-infiltrated lymphocytes (CD8+ TILs).METHODS: Collagen receptor expression patterns in PDAC were examined using flow cytometry, western blot, multiplex immunofluorescence, RNA seq, and scRNA seq databases in KPC and treatment-naïve PDAC that were resected from patients. The KPC cell line, known as Hy15549, was established from KPC mouse model (Ptf1-Cre; LSL-KRAS-G12D; Trp53 Lox/+). Using CRISPR-Cas9, we conducted targeted deletions for either ITGA2(CD49b) or ITGB1(CD29), as well as generating double knockouts for both CD49b and CD29. Growth of these cell lines implanted into the flanks of immunocompetent, immunodeficient mice was measured, as well as after CD8+ T cell depletion in immunocompetent mice. We analyzed TILs using FACs. We used multiplex immunofluorescence to analyze tumors microenvironment including collagen fiber alignment. Downstream signaling pathways were assessed using RNA seq and western blot. RESULTS: CD49b is highly expressed specifically in PDAC cancer cells, as assessed in both KPC cell flank tumors and resected human PDAC. Using adhesion assay, we found that CD49b specifically binds to Col1 rather than ColIV. KPC CD29b KO exhibited a complete loss of adhesion to both Col1 and ColIV. Tumor growth of the KPC CD49b KO, KPC CD29 KO, KPC CD49b CD29 KO was observed to be significantly slower than KPC WT despite identical in vitro growth kinetics. Knockout of CD49b in tumors promoted intratumoral infiltration of CD8+ T cells and significantly impeded tumor growth. Tumor growth by KPCCD49bKO was rescued by depletion of CD8+ T cells. In contrast to observations in immune-competent mice, KPCCD49bKO tumor growth was similar to KPCwt in immunodeficient nude mice. Analysis of downstream signaling in KPCCD49bKO tumors revealed a decrease of FAK as well as pFAk expression pathway. The proteomic profiling of KPCCD49bKO revealed an increase of CXCL10 chemokine. Moreover, In the absence of CD49b, the orientation of collagen fiber alignment appeared more scattered, with thinner fibers. This altered alignment could potentially contribute to immune cell infiltration.Conclusion:These findings reveal a previously unreported function of CD49b in PDAC cancer progression and the immune response within the TME. Together, our study reveals a mechanism underlying immune exclusion and suggests a novel immunotherapeutic target in PDAC. Citation Format: Ibrahim Ragab Eissa, Eric D. Abston, Darshini Kuruppu, Yongtao Wang, Guoliang Qiao, Motaz Qadan, Michael Lanuti, Kenneth K. Tanabe. The ITGA2 (CD49b) collagen receptor excludes the CD8+ T cell infiltration in pancreatic ductal adenocarcinoma [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 4271.

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.

Fractal analysis of extracellular matrix for observer-independent quantification of intestinal fibrosis in Crohn’s disease

Prevention of intestinal fibrosis remains an unresolved problem in the treatment of Crohn’s disease (CD), as specific antifibrotic therapies are not yet available. Appropriate analysis of fibrosis severity is essential for assessing the therapeutic efficacy of potential antifibrotic drugs. The aim of this study was to develop an observer-independent method to quantify intestinal fibrosis in surgical specimens from patients with CD using structural analysis of the extracellular matrix (ECM). We performed fractal analysis in fibrotic and control histological sections of patients with surgery for CD (n = 28). To specifically assess the structure of the collagen matrix, polarized light microscopy was used. A score to quantify collagen fiber alignment and the color of the polarized light was established. Fractal dimension as a measure for the structural complexity correlated significantly with the histological fibrosis score whereas lacunarity as a measure for the compactness of the ECM showed a negative correlation. Polarized light microscopy to visualize the collagen network underlined the structural changes in the ECM network in advanced fibrosis. In conclusion, observer-independent quantification of the structural complexity of the ECM by fractal analysis is a suitable method to quantify the degree of intestinal fibrosis in histological samples from patients with CD.

Engineering Anisotropic Cell Models: Development of Collagen Hydrogel Scaffolds with Magneto‐Responsive PEG Microgels for Tissue Engineering Applications

AbstractMimicking tissue‐oriented organization in vitro has been extensively studied in recent years, using both natural and synthetic materials in combination with external magnetic fields to establish anisotropic conditions. Here, a new combination between magneto‐responsive anisometric PEG microgels and collagen hydrogels is explored to establish anisotropic in vitro models. Different sizes of PEG microgels are tested to assess the impact of both width and aspect ratio on the formation and alignment of collagen hydrogels. Results show that the key properties of collagen hydrogels, regarding fibrillogenesis, rheological properties, and fiber diameter are kept consistent upon the combination with PEG microgels. Furthermore, partial collagen fiber alignment is observed when larger (width 10 µm) PEG microgels are employed and magnetically aligned. In vitro studies show cell alignment within the anisotropic collagen hydrogels from the first day in culture. Interestingly, PEG microgels with higher width and length tend to induce less hydrogel contraction even after 7 days in culture. The results demonstrate the ability to establish a 3D unidirectional collagen hydrogel by magnetically aligning anisometric microgels during the gelation process, which can be promising for different tissue engineering applications.

Bioprinting Cell-Laden Hydrogels for Studies of Epithelial Tissue Morphogenesis.

The extracellular matrix (ECM) provides dynamic structural and molecular signals that affect the form and function of developing tissues. In order to parse how the individual features of the ECM impact cell- and tissue-level behavior during development, engineered culture models should reproduce key structural and molecular features of native ECM. Here, we describe a protocol for bioprinting epithelial cell aggregates embedded within a collagen-Matrigel ink in order to study the dynamic interplay between epithelial tissues and aligned networks of type I collagen fibers. Collagen fiber alignment and geometry can be spatially controlled by modulating the printing speed, nozzle geometry, surface chemistry, and degree of molecular crowding in the printing ink. We provide detailed procedures for generating epithelial cell aggregates, microextrusion printing collagen-Matrigel bioinks, culturing the three-dimensional (3D)-printed tissues, and imaging 3D-printed collagen-Matrigel constructs.

Evaluating wound-healing effect of silicon-zinc-boron-containing glycerohydrogel and its effect on mammary glands of high producing dairy cows

Cow’s milk quality, which may deteriorate due to inflammatory processes in the mammary glands, remains one of the important problems of dairy farming and requires effective, safe and affordable therapeutic agents. Nanocomposite silicon-zinc-boron-containing glycerohydrogel (Si-Zn-B-gel) may become a very good solution to the issue. The paper demonstrates wound-healing effect of the glycerohydrogel and confirms its effectiveness for teat hyperkeratosis treatment indairy cows. Results of a rat burn model – based experiment suggest that Si-Zn-B-gel is a promising wound healing agent for topical use. Thus, on Day 9 complete re-epithelialization of the burn surface was observed, with fibrous structures prevailing in the granulation tissue of the dermal layer, on Day 19 a mature scar was formed with a longitudinal alignment of collagen fibers. The production tests conducted inhigh producing dairy cows have demonstrated good therapeutic effect of the Si-Zn-B gel for teat-end hyperkeratosis and confirmed its long-term effect that helps to longer maintain the results achieved during treatment. After a 7-day treatment physiological structure of up to 27.8% teats improved, on Day 14 of the experiment no severe hyperkeratotic lesions were observed and the number of teats that correspond to the physiological norm was 72.2%. Analysis of the data collected shows that the Si-Zn-B-gel is effective for teat-end hyperkeratosis treatment, thus, it prevents mastitis in animals and improves the milk quality.

Mimicked Spinal Cord Fibers Trigger Axonal Regeneration and Remyelination after Injury.

Spinal cord injury (SCI) causes tissue structure damage and composition changes of the neural parenchyma, resulting in severe consequences for spinal cord function. Mimicking the components and microstructure of spinal cord tissues holds promise for restoring the regenerative microenvironment after SCI. Here, we have utilized electrospinning technology to develop aligned decellularized spinal cord fibers (A-DSCF) without requiring synthetic polymers or organic solvents. A-DSCF preserves multiple types of spinal cord extracellular matrix proteins and forms a parallel-oriented structure. Compared to aligned collagen fibers (A-CF), A-DSCF exhibits stronger mechanical properties, improved enzymatic stability, and superior functionality in the adhesion, proliferation, axonal extension, and myelination of differentiated neural progenitor cells (NPCs). Notably, axon extension or myelination has been primarily linked to Agrin (AGRN), Laminin (LN), or Collagen type IV (COL IV) proteins in A-DSCF. When transplanted into rats with complete SCI, A-DSCF loaded with NPCs improves the survival, maturation, axon regeneration, and motor function of the SCI rats. These findings highlight the potential of structurally and compositionally biomimetic scaffolds to promote axonal extension and remyelination after SCI.

Microfluidically Aligned Collagen to Maintain the Phenotype of Tenocytes In Vitro.

Tendon is a highly organized tissue that transmits forces between muscle and bone. The architecture of the extracellular matrix of tendon, predominantly from collagen type I, is important for maintaining tenocyte phenotype and function. Therefore, in repair and regeneration of damaged and diseased tendon tissue, it is crucial to restore the aligned arrangement of the collagen type I fibers of the original matrix. To this end, a novel, user-friendly microfluidic piggyback platform is developed allowing the controlled patterned formation and alignment of collagen fibers simply on the bottom of culture dishes. Rat tenocytes cultured on the micropatterns of aligned fibrous collagen exhibit a more elongated morphology. The cells also show an increased expression of tenogenic markers at the gene and protein level compared to tenocytes cultured on tissue culture plastic or non-fibrillar collagen coatings. Moreover, using imprinted polystyrene replicas of aligned collagen fibers, this work shows that the fibrillar structure of collagen per se affects the tenocyte morphology, whereas the biochemical nature of collagen plays a prominent role in the expression of tenogenic markers. Beyond the controlled provision of aligned collagen, the microfluidic platform can aid in developing more physiologically relevant in vitro models of tendon and its regeneration.

Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement.

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a localized fibrocartilage phenotype characterized by accumulation of glycosaminoglycan (GAG)-rich matrix and remodeling of the collagen network. While fibrocartilage is a normal feature in impinged regions of healthy tendons, excess GAG deposition and disorganization of the collagen network are hallmark features of tendinopathy. Accordingly, impingement is clinically recognized as an important extrinsic factor in the initiation and progression of tendinopathy. Nevertheless, the mechanobiology underlying tendon impingement remains understudied. Prior efforts to elucidate the cellular response to tendon impingement have applied uniaxial compression to cells and excised tendon explants in vitro. However, isolated cells lack a three-dimensional extracellular environment crucial to mechanoresponse, and both in vitro and excised explant studies fail to recapitulate the multiaxial strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region. Moreover, in vivo models of tendon impingement lack control over the mechanical strain environment. To overcome these limitations, we present a novel murine hind limb explant model suitable for studying the mechanobiology of Achilles tendon impingement. This model maintains the Achilles tendon in situ to preserve local anatomy and reproduces the multiaxial strain environment generated by impingement of the Achilles tendon insertion upon the calcaneus during passively applied ankle dorsiflexion while retaining cells within their native environment. We describe a tissue culture protocol integral to this model and present data establishing sustained explant viability over 7 days. The representative results demonstrate enhanced histological GAG staining and decreased collagen fiber alignment secondary to impingement, suggesting elevated fibrocartilage formation. This model can easily be adapted to investigate different mechanical loading regimens and allows for the manipulation of molecular pathways of interest to identify mechanisms mediating phenotypic change in the Achilles tendon in response to impingement.

TMIC-03. TARGETING THE COLLAGEN RECEPTOR DDR1 PROMOTES ANTI-GLIOMA IMMUNITY BY REMODELING COLLAGEN FIBER ARCHITECTURE

Abstract Glioblastoma (GBM), characterized by its aggressive nature and profound immunosuppression, poses a significant therapeutic challenge with limited treatment options. We previously demonstrated that targeting Col1α1 eradicates oncostreams and improves survival in murine models. However, the underlying mechanisms by which collagen reshapes the glioma tumor microenvironment (TME) remain unknown. Through RNA-seq analysis we identified that DDR1, a collagen receptor, is overexpressed in genetically engineered glioma mouse models (GEMMs) including NPA (NRAS/shp53/shATRX), and NPD (NRAS/shp53/PDGFβ), compared to healthy brain tissue. Pharmacological inhibition of DDR1 enhanced radiosensitivity, disrupted oncostreams in vitro, and ex vivo, imaged with time-lapse confocal microscopy. GEMMs of DDR1 knockdown (NRAS/shp53/shATRX/shDDR1) using the Sleeping Beauty transposase system significantly increased median survival by over 50%. Gene Ontology analysis of differentially expressed genes between wild-type and DDR1 knockdown gliomas revealed enrichment in immune reactivity, cytokine-chemokine activity, and extracellular matrix. Inhibition of DDR1 enhanced intratumoral infiltration of CD45+, CD8+, and CD3+ immune cells, accompanied by a decrease in the abundance of immune-suppressive cells, suggesting that gliomas exploit DDR1-mediated mechanisms to promote immunosuppression and facilitate tumor growth. We hypothesize that DDR1 in glioma impedes immune surveillance by promoting collagen fiber alignment, as evaluated using collagen-specific second-harmonic generation microscopy. Furthermore, analysis of human datasets demonstrated a negative correlation between DDR1 expression and CD8, suggesting DDR1’s role as a key regulator of immune surveillance in gliomas. Interestingly, when DDR1 knockdown gliomas were implanted in immune deficient mouse models, NSG (NOD-scid-IL2Rγnull) or CD8−/− immune-deficient mice, no significant differences in survival were observed, indicating DDR1 expression in glioma cells dampens anti-glioma immunity. In conclusion, we propose that DDR1 within glioma cells induces an immune-inhibitory TME, reprograms cytokine and chemokine profiles, and promotes collagen fiber alignment, thus inhibiting anti-glioma immunity. Our data highlight DDR1 as a promising therapeutic target, providing a novel avenue for GBM treatment with significant potential.

Neuro-regenerative behavior of adipose-derived stem cells in aligned collagen I hydrogels.

Peripheral nerve injuries persist as a major clinical issue facing the US population and can be caused by stretch, laceration, or crush injuries. Small nerve gaps are simple to treat, and the nerve stumps can be reattached with sutures. In longer nerve gaps, traditional treatment options consist of autografts, hollow nerve guidance conduits, and, more recently, manufactured fibrous scaffolds. These manufactured scaffolds often incorporate stem cells, growth factors, and/or extracellular matrix (ECM) proteins to better mimic the native environment but can have issues with homogenous cell distribution or uniformly oriented neurite outgrowth in scaffolds without fibrous alignment. Here, we utilize a custom device to fabricate collagen I hydrogels with aligned fibers and encapsulated adipose-derived mesenchymal stem cells (ASCs) for potential use as a peripheral nerve repair graft. Initial results of our scaffold system revealed significantly less cell viability in higher collagen gel concentrations; 3mg/mL gels showed 84.8±7.3% viable cells, compared to 6mg/mL gels viability of 76.7±9.5%. Mechanical testing of the 3mg/mL gels showed a Young's modulus of 6.5±0.8kPa nearly matching 7.45kPa known to support Schwann cell migration. Further analysis of scaffolds coupled with stretching in vitro revealed heightened angiogenic and factor secretion, ECM deposition, fiber alignment, and dorsal root ganglia (DRG) neurite outgrowth along the axis of fiber alignment. Our platform serves as an in vitro testbed to assess neuro-regenerative potential of ASCs in aligned collagen fiber scaffolds and may provide guidance on next-generation nerve repair scaffold design.

Artificial Periosteum with Oriented Surface Nanotopography and High Tissue Adherent Property.

Massive periosteal defects often significantly impair bone regeneration and repair, which have become a major clinical challenge. Unfortunately, current engineered periosteal materials can hardly currently focus on achieving high tissue adhesion property, being suitable for cell growth, and inducing cell orientation concurrently to meet the properties of nature periosteum. Additionally, the preparation of oriented surface nanotopography often relies on professional equipment. In this study, inspired by the oriented collagen structure of nature periosteum, we present a composite artificial periosteum with a layer of oriented nanotopography surface containing carbon nanotubes (CNTs), cross-linked with adhesive polydopamine (PDA) hydrogel on both terminals. An oriented surface structure that can simulate the oriented alignment of periosteal collagen fibers can be quickly and conveniently obtained via a simple stretching of the membrane in a water bath. With the help of CNTs, our artificial periosteum exhibits sufficient mechanical strength and desired oriented nanotopological structure surface, which further induces the directional arrangement of human bone marrow mesenchymal stem cells (hBMSCs) on the membrane. These oriented hBMSCs express significantly higher levels of osteogenic genes and proteins, while the resultant composite periosteum can be stably immobilized in vivo in the rat model of massive calvarial defect through the PDA hydrogel, which finally shows promising bone regeneration ability. We anticipate that the developed functional artificial periosteum has great potential in biomedical applications for the treatment of composite defects of the bone and periosteum.

Cyclic Stretching Triggers Cell Orientation and Extracellular Matrix Remodeling in a Periodontal Ligament 3D In Vitro Model.

During orthodontic tooth movement (OTM), the periodontal ligament (PDL) plays a crucial role in regulating the tissue remodeling process. To decipher the cellular and molecular mechanisms underlying this process in vitro, suitable 3D models are needed that more closely approximate the situation in vivo. Here, a customized bioreactor is developed that allows dynamic loading of PDL-derived fibroblasts (PDLF). A collagen-based hydrogel mixture is optimized to maintain structural integrity and constant cell growth during stretching. Numerical simulations show a uniform stress distribution in the hydrogel construct under stretching. Compared to static conditions, controlled cyclic stretching results in directional alignment of collagen fibers and enhances proliferation and spreading ability of the embedded PDLF cells. Effective force transmission to the embedded cells is demonstrated by a more than threefold increase in Periostin protein expression. The cyclic stretch conditions also promote extensive remodeling of the extracellular matrix, as confirmed by increased glycosaminoglycan production. These results highlight the importance of dynamic loading over an extended period of time to determine the behavior of PDLF and to identify in vitro mechanobiological cues triggered during OTM-like stimulus. The introduced dynamic bioreactor is therefore a useful in vitro tool to study these mechanisms.

Super-Resolution Second-Harmonic Generation Imaging with Multifocal Structured Illumination Microscopy.

Second-harmonic generation (SHG) is a noninvasive imaging technique that enables the exploration of physiological structures without the use of an exogenous label. However, traditional SHG imaging is limited by optical diffraction, which restricts the spatial resolution. To break this limitation, we developed a novel approach called multifocal structured illumination microscopy-SHG (MSIM-SHG). By combination of SHG with MSIM, SHG-based super-resolution imaging of material molecules can be achieved, and this SHG super-resolution imaging has a wide range of applications for biological tissues and cells. MSIM-SHG achieved a lateral full width at half-maximum (fwhm) of 147 ± 13 nm and an axial fwhm of 493 ± 47 nm by imaging zinc oxide (ZnO) particles. Furthermore, MSIM-SHG was utilized to quantify collagen fiber alignment in various tissues such as the ovary, muscle, heart, kidney, and cartilage, demonstrating its feasibility for identifying collagen characteristics. MSIM-SHG has potential as a powerful tool for clinical diagnosis and biological research.