Three-dimensional Collagen Research Articles

Mueller matrix analysis of a biologically sourced engineered tissue construct as polarimetric phantom.

The polarimetric properties of biological tissues are often difficult to ascertain independent of their complex structural and organizational features. Conventional polarimetric tissue phantoms have well-characterized optical properties but are overly simplified. We demonstrate that an innovative, biologically sourced, engineered tissue construct better recapitulates the desired structural and polarimetric properties of native collagenous tissues, with the added benefit of potential tunability of the polarimetric response. We bridge the gap between non-biological polarimetric phantoms and native tissues. We aim to evaluate a synthesized tissue construct for its effectiveness as a phantom that mimics the polarimetric properties in typical collagenous tissues. We use a fibroblast-derived, ring-shaped engineered tissue construct as an innovative tissue phantom for polarimetric imaging. We perform polarimetry measurements and subsequent analysis using the Mueller matrix decomposition and Mueller matrix transformation methods. Scalar polarimetric parameters of the engineered tissue are analyzed at different time points for both a control group and for those treated with the transforming growth factor . Second-harmonic generation (SHG) imaging and three-dimensional collagen fiber organization analysis are also applied. We identify linear retardance and circular depolarization as the parameters that are most sensitive to the tissue culture time and the addition of . Aside from a statistically significant increase over time, the behavior of linear retardance and circular depolarization indicates that the addition of accelerates the growth of the engineered tissue, which is consistent with expectations. We also find through SHG images that collagen fiber organization becomes more aligned over time but is not susceptible to the addition of . The engineered tissue construct exhibits changes in polarimetric properties, especially linear retardance and circular depolarization, over culture time and under treatments. This tissue construct has the potential to act as a controlled modular optical phantom for polarimetric-based methods.

Matrix stiffening from collagen fibril density and alignment modulates YAP-mediated T-cell immune suppression

T-cells are essential components of the immune system, adapting their behavior in response to the mechanical environments they encounter within the body. In pathological conditions like cancer, the extracellular matrix (ECM) often becomes stiffer due to increased density and alignment of collagen fibrils, which can have a significant impact on T-cell function. In this study, we explored how these ECM properties—density and fibrillar alignment—affect T-cell behavior using three-dimensional (3D) collagen matrices that mimic these conditions. Our results show that increased matrix stiffness, whether due to higher density or alignment, significantly suppresses T-cell activation, reduces cytokine production, and limits proliferation, largely through enhanced YAP signaling. Individually, matrix alignment appears to lower actin levels in activated T-cells and changes migration behavior in both resting and activated T-cells, an effect not observed in matrices with randomly oriented fibrils. Notably, inhibiting YAP signaling was able to restore T-cell activation and improve immune responses, suggesting a potential strategy to boost the effectiveness of immunotherapy in stiff ECM environments. Overall, this study provides new insights into how ECM characteristics influence T-cell function, offering potential avenues for overcoming ECM-induced immunosuppression in diseases such as cancer.

Use of three-dimensional acellular collagen matrix in deep or tunnelling diabetic foot ulcers: a retrospective case series.

While most xenograft wound matrices are flat sheets not designed for deep or tunnelling wounds, three-dimensional acellular collagen matrices (3D-ACM) can fill deep wound beds and enable full wound wall apposition. This case series examines the use of 3D-ACM in treating diabetic foot ulcers (DFUs) that are deep, tunnelling, undermining, or irregularly shaped. We report outcomes of cases where 3D-ACM was applied to deep or tunnelling DFUs present for at least four weeks. In this retrospective case series, 3D-ACM was applied, healing was monitored and measurements were collected. Additional 3D-ACM was applied as needed. In total, 11 patients with 13 wounds were treated. Improved wound appearance and reduced size were observed at most follow-ups. Mean initial wound depth was 1.6cm, and several wounds showed significant depth reductions shortly after therapy initiation. In total, 62% of wounds (8/13) reached 50% closure by four weeks. Additionally, 54% (7/13) were fully closed by 12 weeks. The remaining 46% (6/13) took between 12-22.3 weeks to heal. Overall mean therapy time was 13.1 weeks (range: 2.0-22.3 weeks). Deeper wounds generally took longer to close. The findings of this case series showed that 3D-ACM could offer a protective microenvironment for wound management for deep or tunnelling DFUs. While some took >12 weeks to close, this may be attributable to large initial depths and volumes, rather than a failure to respond to the treatment modality. Other wounds that require a conforming 3D matrix, enabling full wound wall apposition, may benefit from 3D-ACM. Further investigations would be beneficial to understand the capabilities of this treatment modality.

The impairment of the assembly of vimentin filaments leads to suppression of the formation and maturation of focal contacts and an alteration of the type of cellular protrusions

Cell migration is largely determined by the type of protrusions formed by the cell. Mesenchymal migration is accomplished by the formation of lamellipodia and/or filopodia, and membrane blebs lay at the basis of amoeboid migration. Changing the conditions of migration can lead to an alteration of the character of cell movement, for example, inhibition of Arp2/3-dependent actin polymerization by the CK-666 inhibitor leads to transition from mesenchymal to amoeboid motility type. The ability of cells to switch from one type of motility to another is called migration plasticity. The cellular mechanisms regulating migratory plasticity are poorly understood. One of the factors determining the possibility of migratory plasticity may be the presence and/or organization of vimentin intermediate filaments (VIFs). To investigate whether the organization of the VIF network affects the ability of fibroblasts to form membrane blebs, we used rat embryonic fibroblasts REF52 with normal VIF organization, with VIF knockout (REF–/–) and with a mutation inhibiting the assembly of full-length VIFs (REF117). Blebs formation was induced by treatment of cells with CK-666. Vimentin knockout did not lead to statistically significant increase of number of cells with blebs. In fibroblasts with short fragments of vimentin the number of cells forming blebs both spontaneously and in presence of CK-666 increased significantly. Disruption of the VIF organization did not lead to the significant changes in microtubules network or myosin light chain phosphorylation, but caused a significant reduction of the focal contact system. The most pronounced and statistically significant decrease in both the size and number of focal adhesions were observed in REF117. We believe that the regulation of membrane blebbing by VIFs is mediated by their effect on the focal adhesion system. Analysis of migration of fibroblasts with different organization of VIFs in a three-dimensional collagen gel showed that the organization of VIFs determines the type of cell protrusions, which in turn determines the character of cell movement. A novel role of VIF as a regulator of membrane blebbing, essential for the manifestation of migratory plasticity, is shown.

Selective mural cell recruitment of pericytes to networks of assembling endothelial cell-lined tubes.

Mural cells are critically important for the development, maturation, and maintenance of the blood vasculature. Pericytes are predominantly observed in capillaries and venules, while vascular smooth muscle cells (VSMCs) are found in arterioles, arteries, and veins. In this study, we have investigated functional differences between human pericytes and human coronary artery smooth muscle cells (CASMCs) as a model VSMC type. We compared the ability of these two mural cells to invade three-dimensional (3D) collagen matrices, recruit to developing human endothelial cell (EC)-lined tubes in 3D matrices and induce vascular basement membrane matrix assembly around these tubes. Here, we show that pericytes selectively invade, recruit, and induce basement membrane deposition on EC tubes under defined conditions, while CASMCs fail to respond equivalently. Pericytes dramatically invade 3D collagen matrices in response to the EC-derived factors, platelet-derived growth factor (PDGF)-BB, PDGF-DD, and endothelin-1, while minimal invasion occurs with CASMCs. Furthermore, pericytes recruit to EC tube networks, and induce basement membrane deposition around assembling EC tubes (narrow and elongated tubes) when these cells are co-cultured. In contrast, CASMCs are markedly less able to perform these functions showing minimal recruitment, little to no basement membrane deposition, with wider and shorter tubes. Our new findings suggest that pericytes demonstrate much greater functional ability to invade 3D matrix environments, recruit to EC-lined tubes and induce vascular basement membrane matrix deposition in response to and in conjunction with ECs.

LY333531 attenuates contraction of tumor necrosis factor-α-sensitized human airway smooth muscle cells

Objective Protein kinase C (PKC) has been implicated in the increased contraction of human airway smooth muscle cells (HASMCs) in asthma. Using the three-dimensional collagen gel contraction system, the study aimed to determine the effects of LY333531, a specific inhibitor of the PKC-β isoform, on the contraction of tumor necrosis factor (TNF)-α-sensitized HASMCs. Methods Cultured HASMCs were divided into five groups: the control group received no treatment, and the cells in the TNF-α group were sensitized with 10 ng/mL TNF-α for 48 h, while TNF-α was administered to sensitize HASMCs in the presence of 0.1, 0.2, and 0.5 μM LY333531 for 48 h in the 0.1LY, 0.2LY, and 0.5LY groups, respectively. Following this, HASMCs contraction was stimulated with 1 mM acetylcholine (ACh) for up to 24 h in each group and assessed using a three-dimensional collagen gel contraction assay. Furthermore, western blot and immunofluorescence analysis were performed. Results The collagen gel contraction assay revealed that TNF-α increased the protein expression of phosphorylated PKC-β2, CPI-17, and MLC while exacerbating ACh-induced HASMCs contraction. LY333531 significantly attenuated HASMCs contraction and downregulated the protein expression of both p-CPI-17 and p-MLC. Conclusions At least in part by regulating CPI-17 and MLC phosphorylation, LY333531 attenuates augmented contraction of TNF-α-sensitized HASMCs in a collagen gel contraction system.

Cellular memory function from 3D to 2D: Three-dimensional high density collagen microfiber cultures induce their resistance to reactive oxygen species

Cell properties generally change when the culture condition is changed. However, mesenchymal stem cells cultured on a hard material surface maintain their differentiation characteristics even after being cultured on a soft material surface. This phenomenon suggests the possibility of a cell culture material to memorize stem cell function even in changing cell culture conditions. However, there are no reports about cell memory function in three-dimensional (3D) culture. In this study, colon cancer cells were cultured with collagen microfibers (CMF) in 3D to evaluate their resistance to reactive oxygen species (ROS) in comparison with a monolayer (2D) culture condition and to understand the effect of 3D-culture on cell memory function. The ratio of ROS-negative cancer cells in 3D culture increased with increasing amounts of CMF and the highest amount of CMF was revealed to be 35-fold higher than that of the 2D condition. The ROS-negative cells ratio was maintained for 7 days after re-seeding in a 2D culture condition, suggesting a 3D-memory function of ROS resistance. The findings of this study will open up new opportunities for 3D culture to induce cell memory function.

Deletion of Tgm2 suppresses BMP-mediated hepatocyte-to-cholangiocyte metaplasia in ductular reaction.

Transglutaminase 2 (Tgm2) plays an essential role in hepatic repair following prolonged toxic injury. During cholestatic liver injury, the intrahepatic cholangiocytes undergo dynamic tissue expansion and remodelling, referred to as ductular reaction (DR), which is crucial for liver regeneration. However, the molecular mechanisms governing the dynamics of active cells in DR are still largely unclear. Here, we generated Tgm2-knockout mice (Tgm2-/-) and Tgm2-CreERT2-Rosa26-mTmG flox/flox (Tgm2CreERT2-R26T/Gf/f) mice and performed a three-dimensional (3D) collagen gel culture of mouse hepatocytes to demonstrate how Tgm2 signalling is involved in DR to remodel intrahepatic cholangiocytes. Our results showed that the deletion of Tgm2 adversely affected the functionality and maturity of the proliferative cholangiocytes in DR, thus leading to more severe cholestasis during DDC-induced liver injury. Additionally, Tgm2 hepatocytes played a crucial role in the regulation of DR through metaplasia. We unveiled that Tgm2 regulated H3K4me3Q5ser via serotonin to promote BMP signalling activation to participate in DR. Besides, we revealed that the activation or inhibition of BMP signalling could promote or suppress the development and maturation of cholangiocytes in DDC-induced DR. Furthermore, our 3D collagen gel culture assay indicated that Tgm2 was vital for the development of cholangiocytes in vitro. Our results uncovered a considerable role of BMP signalling in controlling metaplasia of Tgm2 hepatocytes in DR and revealed the phenotypic plasticity of mature hepatocytes.

The role of cell-matrix adhesion and cell migration in breast tumor growth and progression.

During breast cancer progression, there is typically increased collagen deposition resulting in elevated extracellular matrix rigidity. This results in changes to cell-matrix adhesion and cell migration, impacting processes such as the epithelial-mesenchymal transition (EMT) and metastasis. We aim to investigate the roles of cell-matrix adhesion and cell migration on breast tumor growth and progression by studying the impacts of different types of extracellular matrices and their rigidities. We embedded MCF7 spheroids within three-dimensional (3D) collagen matrices and agarose matrices. MCF7 cells adhere to collagen but not agarose. Contrasting the results between these two matrices allows us to infer the role of cell-matrix adhesion. We found that MCF7 spheroids exhibited the fastest growth rate when embedded in a collagen matrix with a rigidity of 5.1kPa (0.5mg/mL collagen), whereas, for the agarose matrix, the rigidity for the fastest growth rate is 15kPa (1.0% agarose) instead. This discrepancy is attributable to the presence of cell adhesion molecules in the collagen matrix, which initiates collagen matrix remodeling and facilitates cell migration from the tumor through the EMT. As breast tumors do not adhere to agarose matrices, it is suitable to simulate the cell-cell interactions during the early stage of breast tumor growth. We conducted further analysis to characterize the stresses exerted by the expanding spheroid on the agarose matrix. We identified two distinct MCF7 cell populations, namely, those that are non-dividing and those that are dividing, which exerted low and high expansion stresses on the agarose matrix, respectively. We confirmed this using Western blot which showed the upregulation of proliferating cell nuclear antigen, a proliferation marker, in spheroids grown in the 1.0% agarose (≈13kPa). By treating the embedded MCF7 spheroids with an inhibitor or activator of myosin contractility, we showed that the optimum spheroids' growth can be increased or decreased, respectively. This finding suggests that tumor growth in the early stage, where cell-cell interaction is more prominent, is determined by actomyosin tension, which alters cell rounding pressure during cell division. However, when breast tumors begin generating collagen into the surrounding matrix, collagen remodeling triggers EMT to promote cell migration and invasion, ultimately leading to metastasis.

Invasion/chemotaxis- and extravasation-chip models for breast cancer bone metastasis.

Bone is one of the most frequently targeted organs in metastatic cancers including the breast. Breast cancer bone metastasis often results in devastating outcomes as limited treatment options are currently available. Therefore, innovative methods are needed to provide earlier detection and thus better treatment and prognosis. Here, we present a new approach to model bone-like microenvironments to detect invasion and extravasation of breast cancer cells using invasion/chemotaxis (IC-) and extravasation (EX-) chips, respectively. Our results show that the behaviors of MDA-MB-231 breast cancer cells on IC- and EX-chip models correlate with their in vivo metastatic potential. Our culture model constitutes cell lines representing osteoblasts, bone marrow stromal cells, and monocytes embedded in three-dimensional (3D) collagen I-based extracellular matrices of varying composition and stiffness. We show that collagen I offers a better bone-like environment for bone cells and matrix composition and stiffness regulate the invasion of breast cancer cells. Using in situ contactless rheological measurements under cell culture conditions, we show that the presence of cells increased the stiffness values of the matrices up to 1200 Pa when monitored for five days. This suggests that the cellular composition has a significant effect on regulating matrix mechanical properties, which in turn contribute to the invasiveness. The platforms we present here enable the investigation of the underlying molecular mechanisms in breast cancer bone metastasis and provide the groundwork of developing preclinical tools for the prediction of bone metastasis risk.

Phosphorylation of collagen fibrils enhances intrafibrillar mineralization and dentin remineralization.

The hierarchical assembly of nanoapatite within a type I collagen matrix was achieved through biomimetic mineralization in vitro, cooperatively regulated by non-collagenous proteins and small biomolecules. Here, we demonstrated that IP6 could significantly promote intrafibrillar mineralization in two- and three-dimensional collagen models through binding to collagen fibrils via hydrogen bonds (the interaction energy ∼10.21 kJ mol-1), as confirmed by the FTIR spectra and isothermal experimental results. In addition, we find that IP6 associated with dental collagen fibrils can also enhance the remineralization of calcium-depleted dentin and restore its mechanical properties similar to the natural dentin within 4 days. The promoting effect is mainly due to the chemical modification of IP6, which alters the interfacial physicochemical properties of collagen fibrils, strengthening the interaction of calcium phosphate minerals and mineral ions with collagen fibrils. This strategy of interfacial regulation to accelerate the mineralization of collagen fibrils is essential for dental repair and the development of a clinical product for the remineralization of hard tissue.

Zirconium ion ligand Cross-Linked carbon nanotubes and leather collagen fibers for Flexible, Stable, and highly efficient underwater sensors

Flexible sensing materials have recently found widespread applications in integrating various underwater sensors. In this study, we adopted oil-tanned sheepskin as a “structural template” and harnessed its distinctive three-dimensional collagen fiber network. By leveraging the coordination complexation of zirconium ions (Zr4+), we brided collagen fibers with carbon nanotubes (CNTs) to enhance the bonding fastness of the two. We finally constructed a double cross-linked network to produce a new flexible conductive leather called CNTs@Zr@OTL. This material is the basis for preparing leather bio-based piezoresistive sensors （LBPS） and further developing leather biosensing systems (LBS). Experimental outcomes revealed that when CNTs were employed at a concentration of 8.5 %, CNTs@Zr@OTL exhibited an electrical resistivity of approximately 7.4 Ω·cm and a shrinkage temperature of around 74.5 °C. Furthermore, this material demonstrated remarkable characteristics, including 24-hour biodegradability, a broad sensing range (20 %-120 %), and high sensitivity (with response and recovery times of 300 ms and 100 ms, respectively). Based on multiple cross-linked networks, we propose for the first time the underwater sensing mechanism of “Submarine Tunnel” to achieve strong stability in the water environment. Finally, we simulated the LBPS's response to minor external stimuli, including rainwater exposure, multiangle reactions, and water depth sensitivity. By interfacing with wireless modules and mobile phones, the LBS enabled the execution of emergency SOS signals, highlighting its potential applications in underwater motion monitoring and urgent rescue operations.

ALA-PDT promotes the death and contractile capacity of hypertrophic scar fibroblasts through inhibiting the TGF-β1/Smad2/3/4 signaling pathway

BackgroundHypertrophic scars, an abnormal wound-healing response to burn injuries, are characterized by massive fibroblast proliferation and excessive deposition of extracellular matrix and collagen. 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) is a promising therapy for hypertrophic scar, details of the mechanisms remain to be elucidated. In this study, we aimed to investigate the molecular mechanisms involved in ALA-PDT against hypertrophic scar fibroblasts. MethodsThe morphologies of hypertrophic scar fibroblasts (HSFs) treated with ALA-PDT were observed under a light microscopy. The viability of HSFs was detected using the CCK-8 assay. HSFs-populated collagen gel contraction assays were conducted to examine the fibroblast contractility and the cytotoxicity of HSFs in 3D collagen tissues were observed using confocal microscopy. The effect of ALA-PDT on TGF-β1/Smad2/3/4 signaling pathway activation and effector gene expression were verified by immunoprecipitation, western blot and real-time quantitative PCR analysis. ResultsWe observed significant changes in cell morphology after ALA-PDT treatment of HSFs. As ALA concentration and light dose increased, the viability of HSFs significantly decreased. ALA-PDT can significantly alleviate the contractile capacity and promote the death of HSFs induced by TGF-β1 treatment in a three-dimensional collagen culture model. TGF-β1 treatment of HSFs can significantly induce phosphorylation of Smad2/3 (p-Smad2/3) in whole cells, as well as p-Smad2/3 and Smad4 proteins into the nucleus and increase the mRNA levels of collagen 1/3 and α-SMA. ALA-PDT hampers the TGF-β1-Smad2/3/4 signaling pathway activation by inducing K48-linked ubiquitination and degradation of Smad4. ConclusionsOur results provide evidence that ALA-PDT can inhibit fibroblast contraction and promote cell death by inhibiting the activation of the TGF-β1 signaling pathway that mediates hypertrophic scar formation, which may be the basis for the efficacy of ALA-PDT in the treatment of hypertrophic scars.

Exploring the Role of Desmoplastic Physical Stroma in Pancreatic Cancer Progression Using a Three-Dimensional Collagen Matrix Model.

Pancreatic ductal adenocarcinoma (PDAC) is a refractory tumor with a poor prognosis, and its complex microenvironment is characterized by a fibrous interstitial matrix surrounding PDAC cells. Type I collagen is a major component of this interstitial matrix. Abundant type I collagen promotes its deposition and cross-linking to form a rigid and dense physical barrier, which limits drug penetration and immune cell infiltration and provides drug resistance and metabolic adaptations. In this study, to identify the physical effect of the stroma, type I collagen was used as a 3D matrix to culture Capan-1 cells and generate a 3D PDAC model. Using transcriptome analysis, a link between type I collagen-induced physical effects and the promotion of Capan-1 cell proliferation and migration was determined. Moreover, metabolomic analysis revealed that the physical effect caused a shift in metabolism toward a glycolytic phenotype. In particular, the high expression of proline in the metabolites suggests the ability to maintain Capan-1 cell proliferation under hypoxic and nutrient-depleted conditions. In conclusion, we identified type I collagen-induced physical effects in promoting Capan-1 cells, which cause PDAC progression, providing support for the role of dense stroma in the PDAC microenvironment and identifying a fundamental method for modeling the complex PDAC microenvironment.

Morpho-mechanical mapping of human dura mater microstructure

The human dura mater is known to impact vastly traumatic brain injury mechanopathology. In spite of this involvement, dura mater is typically neglected in computational and physical human head models. The lack of location-dependent microstructural and related mechanical data of dura mater may be considered a rationale behind this simplification. The anisotropic nature of dura mater under various loading conditions so far remains unelucidated. Furthermore, principal collagen fiber orientation is yet to be quantified for a morpho-mechanically-informed material model on the dura mater. This study aims to assess how location-dependent mechanical anisotropy is linked to principal collagen fiber orientation. Uniaxial extension tests were performed in a heated tissue bath for 60 samples from six individuals and correlated to the three-dimensional collagen structure in four individuals using second-harmonic generation (SHG) imaging. Failure stress and stretch at failure, elastic modulus, and a microstructurally motivated material model were integrated to examine local differences in dura mater morpho-mechanics. The quantitative observation of collagen fiber orientation and dispersion confirmed that collagen is highly aligned in the human dura mater and that both fiber orientation and dispersion differ depending on the location investigated. This observation provides a possible explanation for the previously observed isotropic mechanical behavior, as the main collagen fiber direction is not oriented along the anterior-posterior or medial-lateral direction at most of the mapped locations. Additionally, these site-dependent structural properties have implications for the mechanical load response and therefore potentially for the regional functions dura mater has to fulfill. The here chosen non-symmetrical fiber dispersion material model fits the data well and provides a comprehensive parameter base for further studies and future finite element models. Statement of significanceThe human dura mater greatly affects traumatic brain injury mechanisms, but it is often ignored in computational and physical head models. This is because there is a lack of detailed microstructural and mechanical data specific to the dura mater. Its anisotropic nature and collagen fiber orientation have not been fully understood, hindering the development of an accurate material model. Hence, this study combines morphological data on collagen fiber orientation and dispersion at multiple locations of human cranial dura mater, and links microstructure to location-specific load-displacement behavior. It provides microstructurally informed mechanical information towards realistic head models for predicting location-dependent tissue behavior and failure for assessing brain injury and graft material development.

Exogenous interleukin-1 beta stimulation regulates equine tenocyte function and gene expression in three-dimensional culture which can be rescued by pharmacological inhibition of interleukin 1 receptor, but not nuclear factor kappa B, signaling

We investigated how Interleukin 1 beta (IL-1β) impacts equine tenocyte function and global gene expression in vitro and determined if these effects could be rescued by pharmacologically inhibiting nuclear factor-κB (NF-KB) or interleukin 1 signalling. Equine superficial digital flexor tenocytes were cultured in three-dimensional (3D) collagen gels and stimulated with IL-1β for two-weeks, with gel contraction and interleukin 6 (IL6) measured throughout and transcriptomic analysis performed at day 14. The impact of three NF-KB inhibitors on gel contraction and IL6 secretion were measured in 3D culture, with NF-KB-P65 nuclear translocation by immunofluorescence and gene expression by qPCR measured in two-dimensional (2D) monolayer culture. In addition, daily 3D gel contraction and transcriptomic analysis was performed on interleukin 1 receptor antagonist-treated 3D gels at day 14. IL-1β increased NF-KB-P65 nuclear translocation in 2D culture and IL6 secretion in 3D culture, but reduced daily tenocyte 3D gel contraction and impacted > 2500 genes at day 14, with enrichment for NF-KB signaling. Administering direct pharmacological inhibitors of NF-KB did reduce NF-KB-P65 nuclear translocation, but had no effect on 3D gel contraction or IL6 secretion in the presence of IL-1β. However, IL1Ra restored 3D gel contraction and partially rescued global gene expression. Tenocyte 3D gel contraction and gene expression is adversely impacted by IL-1β which can only be rescued by blockade of interleukin 1 receptor, but not NF-KB, signalling.

Increased Matrix Metalloproteinase-1 Activation Enhances Disruption and Regression of k-RasV12–Expressing Arteriovenous Malformation-Like Vessels

This study sought to identify potential mechanisms by which k-RasV12-expressing endothelial cell (EC) tubes demonstrate an increased propensity to regress compared with controls. Activated k-Ras mutations play a role in a variety of pathological conditions, including arteriovenous malformations, which are prone to bleed, causing serious hemorrhagic complications. ECs expressing active k-RasV12 demonstrate markedly excessive lumen formation with widened and shortened tubes accompanied by reduced pericyte recruitment and basement membrane deposition, leading to deficient capillary network assembly. The current study showed that active k-Ras-expressing ECs secreted greater amounts of MMP-1 proenzyme compared with control ECs, and readily converted it to increased active MMP-1 levels through the action of plasmin or plasma kallikrein (generated from their added zymogens). Active MMP-1 degraded three-dimensional collagen matrices, leading to more rapid and extensive regression of the active k-Ras-expressing EC tubes, in conjunction with matrix contraction, compared with control ECs. Under conditions where pericytes protect control EC tubes from plasminogen- and MMP-1-dependent tube regression, this failed to occur with k-RasV12 ECs, due to reduced pericyte interactions. In summary, k-RasV12-expressing EC vessels showed an increased propensity to regress in response to serine proteinases through accentuated levels of active MMP-1, a novel pathogenic mechanism that may underlie hemorrhagic events associated with arteriovenous malformation lesions.

ALK5 Inhibition of Subconjunctival Scarring From Glaucoma Surgery: Effects of SB-431542 Compared to Mitomycin C in Human Tenon's Capsule Fibroblasts.

The gold standard for managing postoperative ocular fibrosis in glaucoma surgery is the chemotherapeutic mitomycin C (MMC) despite its association with significant adverse effects. This study compares in vitro the antifibrotic efficacy and cytotoxicity of the small-molecule TGFβ1 inhibitor SB-431542 (SB) to MMC. To measure collagen contraction, human Tenon's capsule fibroblasts (HTCFs) embedded in a three-dimensional collagen lattice were exposed to 0.2 mg/mL MMC or 20 µM SB followed by incubation with 2 ng/mL TGFβ1. Total protein extracted from experimentally treated HTCFs underwent immunoblotting for α-smooth muscle actin (α-SMA), matrix metallopeptidase 9 (MMP-9), and EDA splice-variant fibronectin (EDA-FN) expression. Cytotoxicity and cell metabolism were assessed using LIVE/DEAD staining, lactate dehydrogenase (LDH) assay, and methylthiazole tetrazolium (MTT) assay. Collagen lattice contraction in TGFβ1-induced HTCFs was significantly lowered by SB and MMC. Pretreatment with SB and MMC significantly lowered protein expression of α-SMA, MMP-9, and EDA-FN in HTCFs relative to TGFβ1 alone. HTCF viability in collagen lattices was significantly reduced with MMC pretreatment but not SB pretreatment. MMC-pretreated HTCFs had a significant increase in LDH release after 3 hours and a decrease in MTT activity after 20 minutes, while SB-pretreated HTCFs showed no significant changes via MTT or LDH assay during the same treatment period. SB shows comparable efficacy to MMC in reducing expression of fibrosis-promoting proteins in HTCFs and in vitro scarring activity. SB distinguishes itself from MMC by exhibiting less cytotoxicity in both two-dimensional and three-dimensional in vitro assays. This study demonstrates in vitro the potential of SB as a safer alternative ocular antifibrotic agent.

Biomimetic Remineralized Three-Dimensional Collagen Bone Matrices with an Enhanced Osteostimulating Effect.

Bone grafts with a high potential for osseointegration, capable of providing a complete and effective regeneration of bone tissue, remain an urgent and unresolved issue. The presented work proposes an approach to develop composite biomimetic bone material for reconstructive surgery by deposition (remineralization) on the surface of high-purity, demineralized bone collagen matrix calcium phosphate layers. Histological and elemental analysis have shown reproduction of the bone tissue matrix architectonics, and a high-purity degree of the obtained collagen scaffolds; the cell culture and confocal microscopy have demonstrated a high biocompatibility of the materials obtained. Adsorption spectroscopy, scanning electron microscopy, microcomputed tomography (microCT) and infrared spectroscopy, and X-ray diffraction have proven the efficiency of the deposition of calcium phosphates on the surface of bone collagen scaffolds. Cell culture and confocal microscopy methods have shown high biocompatibility of both demineralized and remineralized bone matrices. In the model of heterotopic implantation in rats, at the term of seven weeks, an intensive intratrabecular infiltration of calcium phosphate precipitates, and a pronounced synthetic activity of osteoblast remodeling and rebuilding implanted materials, were revealed in remineralized bone collagen matrices in contrast to demineralized ones. Thus, remineralization of highly purified demineralized bone matrices significantly enhanced their osteostimulating ability. The data obtained are of interest for the creation of new highly effective osteoplastic materials for bone tissue regeneration and augmentation.

Met-Signaling Controls Dendritic Cell Migration in Skin by Regulating Podosome Formation and Function

Signaling through the HGF receptor/Met in skin-resident Langerhans cells (LCs) and dermal dendritic cells (DCs) is essential for their emigration toward draining lymph nodes upon inflammation-induced activation. In this study, we addressed the role of Met signaling in distinct steps of LC/dermal DC emigration from the skin by employing a conditionally Met-deficient mouse model (Metflox/flox). We found that Met deficiency severely impaired podosome formation in DCs and concomitantly decreased the proteolytic degradation of gelatin. Accordingly, Met-deficient LCs failed to efficiently cross the extracellular matrix-rich basement membrane between the epidermis and the dermis. We further observed that HGF-dependent Met activation reduced the adhesion of bone marrow-derived LCs to various extracellular matrix factors and enhanced the motility of DCs in three-dimensional collagen matrices, which was not the case for Met-deficient LCs/DCs. We found no impact of Met signaling on the integrin-independent amoeboid migration of DCs in response to the CCR7 ligand CCL19. Collectively, our data show that the Met-signaling pathway regulates the migratory properties of DC in HGF-dependent and HGF-independent manners.