Collagen Gel Research Articles

A micro-fragmented collagen gel as a cell-assembling platform for critical limb ischemia repair

Critical limb ischemia (CLI) is a devastating disease characterized by the progressive blockage of blood vessels. Although the paracrine effect of growth factors in stem cell therapy made it a promising angiogenic therapy for CLI, poor cell survival in the harsh ischemic microenvironment limited its efficacy. Thus, an imperative need exists for a stem-cell delivery method that enhances cell survival. Here, a collagen microgel (CMG) cell-delivery scaffold (40 × 20 μm) was fabricated via micro-fragmentation from collagen–hyaluronic acid polyionic complex to improve transplantation efficiency. Culturing human adipose-derived stem cells (hASCs) with CMG enabled integrin receptors to interact with CMG to form injectable 3-dimensional constructs (CMG-hASCs) with a microporous microarchitecture and enhanced mass transfer. CMG-hASCs exhibited higher cell survival (p < 0.0001) and angiogenic potential in tube formation and aortic ring angiogenesis assays than cell aggregates. Injection of CMG-hASCs intramuscularly into CLI mice increased blood perfusion and limb salvage ratios by 40 % and 60 %, respectively, compared to cell aggregate-treated mice. Further immunofluorescent analysis revealed that transplanted CMG-hASCs have greater muscle regenerative and angiogenic potential, with enhanced cell survival than cell aggregates (p < 0.05). Collectively, we propose CMG as a cell-assembling platform and CMG-hASCs as promising therapeutics to treat CLI.

Effect of Phytic Acid Addition on the Structure of Collagen-Hyaluronic Acid Composite Gel.

Composite collagen gels with hyaluronic acid are developed tissue-engineered structures for filling and regeneration of defects in various organs and tissues. For the first time, phytic acid was used to increase the stability and improve the mechanical properties of collagen gels with hyaluronic acid. Phytic acid is a promising cross-linker for collagen hydrogels and is a plant-derived antioxidant found in rich sources of beans, grains, and oilseeds. Phytic acid has several benefits due to its antioxidant, anticancer, and antitumor properties. In this work, studies were carried out on the kinetics of the self-assembly of collagen molecules in the presence of phytic and hyaluronic acids. It was shown that both of these acids do not lead to collagen self-assembly. Scanning electron microscopy showed that in the presence of phytic and hyaluronic acids, the collagen fibrils had a native structure, and the FTIR method confirmed the chemical cross-links between the collagen fibrils. DSC and rheological studies demonstrated that adding the phytic acid improved the stability and modulus of elasticity of the collagen gel. The presence of hyaluronic acid in the collagen gel slightly reduced the effect of phytic acid. The presence of phytic acid in the collagen gel improved the stability of the scaffold, but, after 1 week of cultivation, slightly reduced the viability of mesenchymal stromal cells cultured in the gel. The collagen type I gel with hyaluronic and phytic acids can be used to replace tissue defects, especially after the removal of cancerous tumors.

Extracellular matrix stiffness activates mechanosensitive signals but limits breast cancer cell spheroid proliferation and invasion.

Breast cancer is characterized by physical changes that occur in the tumor microenvironment throughout growth and metastasis of tumors. Extracellular matrix stiffness increases as tumors develop and spread, with stiffer environments thought to correlate with poorer disease prognosis. Changes in extracellular stiffness and other physical characteristics are sensed by integrins which integrate these extracellular cues to intracellular signaling, resulting in modulation of proliferation and invasion. However, the co-ordination of mechano-sensitive signaling with functional changes to groups of tumor cells within 3-dimensional environments remains poorly understood. Here we provide evidence that increasing the stiffness of collagen scaffolds results in increased activation of ERK1/2 and YAP in human breast cancer cell spheroids. We also show that ERK1/2 acts upstream of YAP activation in this context. We further demonstrate that YAP, matrix metalloproteinases and actomyosin contractility are required for collagen remodeling, proliferation and invasion in lower stiffness scaffolds. However, the increased activation of these proteins in higher stiffness 3-dimensional collagen gels is correlated with reduced proliferation and reduced invasion of cancer cell spheroids. Our data collectively provide evidence that higher stiffness 3-dimensional environments induce mechano-signaling but contrary to evidence from 2-dimensional studies, this is not sufficient to promote pro-tumorigenic effects in breast cancer cell spheroids.

The Effects of ROCK Inhibitor on Prevention of Dexamethasone-Induced Glaucoma Phenotype in Human Trabecular Meshwork Cells.

This study investigated the effects of dexamethasone (Dex) on human trabecular meshwork (TM) cells, a model of glucocorticoid-induced glaucoma, and evaluated the impact of ripasudil (Rip) as a co-delivery or sequential dosing strategy. In vitro experiments were conducted to assess the effects of Dex and Rip on TM cells. Confocal microscopy was used to evaluate the impact of Dex and Rip on F-actin staining signals. Contractility of the TM cells upon Dex and Rip treatment mimicking co-delivery and sequential delivery was quantified using collagen gel contraction assay. Transepithelial electrical resistance (TEER) values and fluorescein isothiocyanate (FITC)-dextran permeability were also measured to assess the impact of Dex and Rip on TM cells. Dex and Rip did not exhibit cytotoxicity at the maximum tested concentration (20 µM). Dex-treated TM cells exhibited higher F-actin staining signals compared to controls, which were reduced when co-treated with Rip. Rip inhibited Dex-induced collagen gel contraction activity in both co-delivery and sequential treatments. Dex resulted in increased TEER values as the dose increased, whereas TEER values were maintained when co-treated with Rip. Co-delivery of Rip has the potential to prevent glaucoma symptoms when patients are treated with Dex. This study highlights the importance of identifying strategies to reduce the side effects of prolonged use of glucocorticoids, such as Dex, in the treatment of various diseases. This study demonstrates the potential of co-delivering ripasudil with dexamethasone to mitigate glucocorticoid-induced ocular hypertension and a secondary glaucoma that resembles primary open-angle glaucoma, providing insights for the development of novel preventive strategies in clinical care.

Hippo Signaling Mediates TGFβ-Dependent Transcriptional Inputs in Cardiac Cushion Mesenchymal Cells to Regulate Extracellular Matrix Remodeling.

The transforming growth factor beta (TGFβ) and Hippo signaling pathways are evolutionarily conserved pathways that play a critical role in cardiac fibroblasts during embryonic development, tissue repair, and fibrosis. TGFβ signaling and Hippo signaling are also important for cardiac cushion remodeling and septation during embryonic development. Loss of TGFβ2 in mice causes cardiac cushion remodeling defects resulting in congenital heart disease. In this study, we used in vitro molecular and pharmacologic approaches in the cushion mesenchymal cell line (tsA58-AVM) and investigated if the Hippo pathway acts as a mediator of TGFβ2 signaling. Immunofluorescence staining showed that TGFβ2 induced nuclear translocation of activated SMAD3 in the cushion mesenchymal cells. In addition, the results indicate increased nuclear localization of Yes-associated protein 1 (YAP1) following a similar treatment of TGFβ2. In collagen lattice formation assays, the TGFβ2 treatment of cushion cells resulted in an enhanced collagen contraction compared to the untreated cushion cells. Interestingly, verteporfin, a YAP1 inhibitor, significantly blocked the ability of cushion cells to contract collagen gel in the absence or presence of exogenously added TGFβ2. To confirm the molecular mechanisms of the verteporfin-induced inhibition of TGFβ2-dependent extracellular matrix (ECM) reorganization, we performed a gene expression analysis of key mesenchymal genes involved in ECM remodeling in heart development and disease. Our results confirm that verteporfin significantly decreased the expression of α-smooth muscle actin (Acta2), collagen 1a1 (Col1a1), Ccn1 (i.e., Cyr61), and Ccn2 (i.e., Ctgf). Western blot analysis indicated that verteporfin treatment significantly blocked the TGFβ2-induced activation of SMAD2/3 in cushion mesenchymal cells. Collectively, these results indicate that TGFβ2 regulation of cushion mesenchymal cell behavior and ECM remodeling is mediated by YAP1. Thus, the TGFβ2 and Hippo pathway integration represents an important step in understanding the etiology of congenital heart disease.

Inhibition of Rho kinase (ROCK) impairs cytoskeletal contractility in human Müller glial cells without effects on cell viability, migration, and extracellular matrix production

The epiretinal membrane is a fibrocontractile tissue that forms on the inner surface of the retina, causing visual impairment ranging from mild to severe, and even retinal detachment. Müller glial cells actively participate in the formation of this membrane. Current research is constantly seeking for new therapeutic approaches that aim to prevent or treat cellular dysfunctions involved in the progression of this common fibrosis condition. The Rho GTPases signaling pathway regulates several processes associated with the epiretinal membrane, such as cell proliferation, migration, and contraction. Rho kinase (ROCK), an effector of the RhoA GTPase, is an interesting potential therapeutic target. This study aimed to evaluate the effects of a ROCK inhibitor (Y27632) on human Müller cells viability, growth, cytoskeletal organization, expression of extracellular matrix components, myofibroblast differentiation, migration, and contractility. Müller cells of the MIO-M1 lineage were cultured and treated for different periods with the inhibitor. Viability was evaluated by MTT assay and trypan blue exclusion method, and growth was evaluated by growth curve and BrdU incorporation assay. The actin cytoskeleton was stained with fluorescent phalloidin, intermediate filaments and microtubules were analyzed with immunofluorescence for vimentin and α-tubulin. Gene and protein expression of collagens I and V, laminin and fibronectin were evaluated by rt-PCR and immunofluorescence. Chemotactic and spontaneous cell migration were studied by transwell assay and time-lapse observation of live cells, respectively. Cell contractility was assessed by collagen gel contraction assay. The results showed that ROCK inhibition by Y27632 did not affect cell viability, but decreased cell growth and proliferation after 72 h. There was a change in cell morphology and organization of F-actin, with a reduction in the cell body, disappearance of stress fibers and formation of long, branched cell extensions. Microtubules and vimentin filaments were also affected, possibly because of F-actin alterations. The inhibitor also reduced gene expression and immunoreactivity of smooth muscle α-actin, a marker of myofibroblasts. The expression of extracellular matrix components was not affected by the inhibitor. Chemotactic cell migration showed no significant changes, while cell contractility was substantially reduced. No spontaneous migration of MIO-M1 cells was observed. In conclusion, pharmacological inhibition of ROCK in Müller cells could be a potentially promising approach to treat epiretinal membranes by preventing cell proliferation, contractility and transdifferentiation, without affecting cell viability.

The Rapid Generation of Cell-Laden, FACS-Compatible Collagen Gels

A three-dimensional cell culture in hydrogel beads can support cell growth and differentiation into multi-cellular structures, and these gel beads could be used as building blocks for more complex three-dimensional assemblies. This requires hydrogel beads that are robust enough to sort via FACS yet can be degraded by cell-secreted enzymes. Collagen polymers form hydrogels that are excellent cell growth substrates; however, collagen-containing hydrogel beads typically include additional polymers that limit their degradation. Here, we introduce a simple microfluidic method to generate robust, sortable, cell-laden collagen hydrogel beads. We use on-device pH control to trigger collagen gelation without exposing cells to low pH, ensuring high cell viability. We fabricate microfluidic devices to generate droplets with a wide size range, as demonstrated by production of both small (~55 µm diameter) and large (~300 µm diameter) collagen gels. All hydrogels are sufficiently robust to allow for sorting using FACS. Moreover, high cell viability is maintained throughout the process.

DYSREGULATION OF OSTEOCYTE SEMA3A BY MECHANICAL LOAD AND INFLAMMATION MAY DRIVE NEUROPLASTICITY AND PAIN IN OSTEOARTHRITIS

AbstractOBJECTIVEChanges in subchondral bone are one of few disease characteristics to correlate with pain in OA1. Profound neuroplasticity and nociceptor sprouting is displayed within osteoarthritic (OA) subchondral bone and is associated with pain and pathology2. The cause of these neural changes remains unestablished. Correct innervation patterns are indispensable for bone growth, homeostasis, and repair. Axon guidance signalling factor, Sema3A is essential for the correct innervation patterning of bony tissues3, expressed in osteocytes4 and known to be downregulated in bone OA mechanical loading5. Bioinformatic analysis has also shown Sema3a as a differentially expressed pathway by bone in human OA patients6.HYPOTHESIS: Pathological mechanical load and inflammation of bone causes dysregulation of Sema3A signalling leading to perturbed sensory nerve plasticity and pain.METHODSHuman KOLF2-C1 iPSC derived nociceptors were generated by TALEN-mediated insertion of transcription factors NGN2+Brn3A and modified chambers differentiation protocol to produce nociceptor-like cells. Nociceptor phenotype was confirmed by immunocytochemistry. Human Y201-MSC cells were embedded in 3D type-I collagen gels (0.05 × 106 cell/gel), in 48-well plates and silicone plates, were differentiated to osteocytes for 7 days before stimulation with IL-6 (5ng/ml) and soluble IL-6 receptor (sIL-6r (40ng/ml), IL6/sIL6r and mechanical load mimetic Yoda1 (5μM) or unstimulated (n=5/group) (48-well plates) or were mechanically loaded in silicone plates (5000μstrain, 10Hz, 3000 cycles) or not loaded (n=5/group). Conditioned media transfer was performed from osteocyte to nociceptor cultures assessed by continuous 24-hour phase contrast confocal microscopy. 24-hours after stimulation RNA was quantified by RT-qPCR (IL6) or RNAseq whole transcriptome analysis/DEseq2 analysis (Load). Protein release was quantified by ELISA. Normally distributed data with homogenous variances was analysed by two-tailed t test.RESULTSIPSC-derived nociceptor-like cells display elongated (&gt;5mm) dendritic projections and nociceptive molecular markers such as TUJ1, PrPH and Neun and TrkA. Sema3A signalling ligands were expressed in 100% of osteocyte cultures. Mechanical loading regulated the Sema3 pathway; Sema3A (0.4-fold, p&lt;0.001), Sema3B (13-fold, p&lt;0.001), Sema3C (0.4-fold, p&lt;0.001). Under inflammatory stimulation by IL6/IL6sR, SEMA3A (7-fold, p=0.01) and receptor Plexin1 (3-fold, p=0.03) show significant regulation. Sema3A protein release showed a significant downregulation of Sema3A release by IL6/sIL6r+Yoda1 (2-fold, p=0.02). Continuous 24-hour phase contrast confocal microscopy measuring the number of extending/retreating dendritic projections revealed that sensory nerve cultures exposed to media from osteocytes stimulated with IL-6/sIL-6R+Yoda1 displayed significantly more invading dendritic projections (p=0.0175, 12-fold±SEM 3.5) across 3 random fields of view within a single stimulated neural culture and significantly fewer retracting dendritic projections (p=0.0075, 2-fold±SEM 0.33) compared to controls.CONCLUSIONSHere we show osteocytic regulation of Sema3A under pathological mechanical loading and the ability of media pathologically loaded osteocyte cultures to induce the branching and invasion of cultured nociceptor-like cells as displayed in OA subchondral bone.Declaration of Interest(b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

Polymeric reinforcements for cellularized collagen-based vascular wall models: influence of the scaffold architecture on the mechanical and biological properties.

A previously developed cellularized collagen-based vascular wall model showed promising results in mimicking the biological properties of a native vessel but lacked appropriate mechanical properties. In this work, we aim to improve this collagen-based model by reinforcing it using a tubular polymeric (reinforcement) scaffold. The polymeric reinforcements were fabricated exploiting commercial poly (ε-caprolactone) (PCL), a polymer already used to fabricate other FDA-approved and commercially available devices serving medical applications, through 1) solution electrospinning (SES), 2) 3D printing (3DP) and 3) melt electrowriting (MEW). The non-reinforced cellularized collagen-based model was used as a reference (COL). The effect of the scaffold's architecture on the resulting mechanical and biological properties of the reinforced collagen-based model were evaluated. SEM imaging showed the differences in scaffolds' architecture (fiber alignment, fiber diameter and pore size) at both the micro- and the macrolevel. The polymeric scaffold led to significantly improved mechanical properties for the reinforced collagen-based model (initial elastic moduli of 382.05 ± 132.01kPa, 100.59 ± 31.15kPa and 245.78 ± 33.54kPa, respectively for SES, 3DP and MEW at day 7 of maturation) compared to the non-reinforced collagen-based model (16.63 ± 5.69kPa). Moreover, on day 7, the developed collagen gels showed stresses (for strains between 20% and 55%) in the range of [5-15] kPa for COL, [80-350] kPa for SES, [20-70] kPa for 3DP and [100-190] kPa for MEW. In addition to the effect on the resulting mechanical properties, the polymeric tubes' architecture influenced cell behavior, in terms of proliferation and attachment, along with collagen gel compaction and extracellular matrix protein expression. The MEW reinforcement resulted in a collagen gel compaction similar to the COL reference, whereas 3DP and SES led to thinner and longer collagen gels. Overall, it can be concluded that 1) the selected processing technique influences the scaffolds' architecture, which in turn influences the resulting mechanical and biological properties, and 2) the incorporation of a polymeric reinforcement leads to mechanical properties closely matching those of native arteries.

Micropatterning the organization of multicellular structures in 3D biological hydrogels; insights into collective cellular mechanical interactions

Control over the organization of cells at the microscale level within supporting biomaterials can push forward the construction of complex tissue architectures for tissue engineering applications and enable fundamental studies of how tissue structure relates to its function. While cells patterning on 2D substrates is a relatively established and available procedure, micropatterning cells in biomimetic 3D hydrogels has been more challenging, especially with micro-scale resolution, and currently relies on sophisticated tools and protocols. We present a robust and accessible ‘peel-off’ method to micropattern large arrays of individual cells or cell-clusters of precise sizes in biological 3D hydrogels, such as fibrin and collagen gels, with control over cell–cell separation distance and neighboring cells position. We further demonstrate partial control over cell position in the z-dimension by stacking two layers in varying distances between the layers. To demonstrate the potential of the micropatterning gel platform, we study the matrix-mediated mechanical interaction between array of cells that are accurately separated in defined distances. A collective process of intense cell-generated densified bands emerging in the gel between near neighbors was identified, along which cells preferentially migrate, a process relevant to tissue morphogenesis. The presented 3D gel micropatterning method can be used to reveal fundamental morphogenetic processes, and to reconstruct any tissue geometry with micrometer resolution in 3D biomimetic gel environments, leveraging the engineering of tissues in complex architectures.

Design and evaluation of new wound dressings based on collagen-cellulose derivatives

The purpose of this research was to design and characterize new hydrogels, respectively, their lyophilized forms, spongious matrices, based on collagen and methylcellulose or hydroxyethylcellulose to be used as wound dressings. 1.1% collagen gel was blended with 3% cellulose ethers gels in different ratios. To design the new potential dressings, we evaluated the compatibility of biopolymers through rheological and spectroscopic analyses for hydrogels, respectively spectral, morphological, goniometric, enzymatic, and thermal stability. Rheological measurements indicated that all hydrogels were pseudoplastic, a required property for their manipulation and application. Spectroscopic and spectral analyses showed the conservation of the specific triple helix structure of collagen for samples with 30% cellulose ethers. The morphological evaluation demonstrated that matrices presented hydrophilic and porous structures with proper swelling ability. The thermal and biodegradation study illustrated the increase of matrices’ stability with the addition of cellulose ethers gels. Biocompatibility tests showed that none of the tested matrices had any cytotoxic effect and some compositions of collagen and methylcellulose or hydroxyethylcellulose release factors that might promote fibroblasts’ proliferation and had no inhibitor effect on fibroblasts migration ability; all these results guarantee the development of a promising wound dressing based on 70% collagen gel and 30% cellulose ethers gels.

Protein tyrosine phosphatase sigma and zeta1 regulate cardiac fibroblast contractile function through TGF-beta dependent and independent pathways

Abstract Background Heart failure is a frequently fatal cardiovascular disease with limited therapeutic options. To improve our understanding of signaling pathways in affected cells is therefore important. Protein tyrosine phosphatase sigma (PTPRσ) and zeta 1 (PTPRz1) have been shown to be involved in cardiac development and disease [1,2]. Both phosphatases are upregulated in patients with end stage heart failure. We demonstrated that PTPRz1 inhibits while PTPRσ is required for migration of neonatal cardiac fibroblasts (nCF) [3]. Purpose To further evaluate the influence of PTPRσ and PTPRz1 on fibroblast phenotype with regard to stress fiber formation and collagen contraction and their interaction with the TGFβ pathway. Methods nCF were transfected with either scrambled-, PTPRσ-, PTPRz1-, or both siRNAs. mRNA lysates were harvested 24h after transfection with subsequent rt-PCR analysis for the phosphatases. Transfected cells were seeded and stained with labeled phalloidin to measure the formation of stress fibers or in collagen gel matrices to evaluate their contractile capacities. To evaluate the nuclear translocation of SMAD3 immunohistochemical colocalization of SMAD3 and DAPI was quantified. Data were analyzed by Kruskal-Wallis and Mann-Whitney-U or ANOVA followed by t-test with Welsh correction using GraphPad prism. Results PTPRz1 and -σ knockdown (kd) was highly efficient (&amp;gt;85%). PTPRz1 mRNA was upregulated by 48% after PTPRσ kd (p &amp;lt; 0.05, n=4) but not vice versa and not after combined kd. Compared to control, nCF showed a 26% increase in the number of stress fibers in response to the PTPRz1 kd (p = 0.057, n=3-4) and an increase of 29% after PTPRσ/-z1 kd (p &amp;lt; 0.05, n=4). Collagen gels were contracted to about 70% of their surface by TGFβ in control (p&amp;lt;0.05, n=4) or any kd condition. They were also contracted by 24,5% after PTPRz1 knockdown (p &amp;lt; 0.01, n=4), whereas the additional kd of PTPRσ obliterated the effect. Nuclear translocation of SMAD3 was roughly doubled upon TGFβ stimulation in controls and all kd conditions. In unstimulated nCF, PTPRσ kd induced a 28%, PTPRz1 kd a 45%, and their combination a 114% increase of nuclei positive for SMAD3 (p &amp;lt; 0.05). Conclusion SMAD3 nuclear translocation is additively induced by PTPRz1 and PTPRσ kd independent of TGFβ. Stress fiber formation, however, seems to be inhibited by PTPRz1 independent of PTPRσ, while PTPRz1-kd-induced collagen contraction does depend on the presence of PTPRσ. PTPRσ and PTPRz1 thus have separate, overlapping and antagonistic effects that are TGFβ dependent and independent. The elucidation of the involvement of the phosphatases in fibroblast signaling and function could improve our understanding of adverse remodeling during heart failure and deliver novel options for therapeutic interventions.

Development of a New Collagen Gel Product for Leather Finishing.

Leather finishing is a critical process in the leather industry, as it significantly influences the final appearance, durability, and quality of leather products. Traditional leather finishing techniques often involve the use of synthetic chemicals, which may lead to environmental concerns and potential health hazards. In this study, we investigate the feasibility and effectiveness of a new collagen-based product for leather finishing. Collagen, a natural protein found abundantly in animals, has shown promise as an environmentally friendly and sustainable alternative for leather finishing. The new collagen gel product obtained from bovine hide waste by using an alkaline extraction method with lime was functionalized through an enzymatic treatment that allows to achieve a finishing product suitable for coating formulations, and at the same time, a biodegradable finishing. The collagen gel product was optimized by varying parameters, such as temperature, pH, and enzyme quantity. The optimized collagen gel product exhibits a wide particle size range and retains the triple-helical structure of collagen. The leather samples treated with the collagen gel product show enhanced properties compared to those with conventional finishes. The results show that the collagen gel product enhances water vapor permeability, color stability, and touch in the finishes. However, a low resistance to wet rubbing is obtained; therefore, it is necessary to study how to improve this parameter.

Bevacizumab promotes tenogenic differentiation and maturation of rat tendon-derived cells in vitro.

Previous work suggested that tenogenic differentiation of tendon stem/progenitor cells (TSPCs) was suppressed by upregulated expression of the angiogenic marker vascular endothelial growth factor (VEGF). The purpose of this study was to test the hypothesis that anti-VEGF antibody, bevacizumab, promotes in vitro tenogenic differentiation and maturation of two distinct types of TSPCs, tendon proper-derived cells (TDCs), and paratenon-derived cells (PDCs) originating from rat Achilles tendon. TDCs and PDCs were isolated from the tendon proper and the paratenon of rat Achilles tendons. TDCs and PDCs were cultured for 3 days on plates with or without VEGF. TDCs and PDCs were also cultured in collagen gel matrix, and the blocking effect of VEGF was examined by the addition of 100 ng/mL of bevacizumab. Effects of bevacizumab on tenogenic differentiation were assessed using real-time PCR, immunofluorescent staining, and western blotting. VEGF significantly attenuated expression of the Tnmd gene in both PDCs and TDCs (P<0.05). Expressions of the Scx, Tnmd, and Col1a1 genes were significantly upregulated by the addition of bevacizumab (P<0.05). Immunofluorescent staining showed that the percentage of tenomodulin-positive PDCs and TDCs was significantly higher with bevacizumab treatment than in control cultures (P<0.05). Western blotting showed that bevacizumab suppressed pVEGFR-2 protein expression in both PDCs and TDCs. Bevacizumab promoted the in vitro tenogenic differentiation and maturation of two distinct TSPCs derived from rat Achilles tendon. Since the previous studies demonstrated that TSPCs have a potential to contribute to tendon repair, attenuating VEGF levels in TSPCs by administration of bevacizumab is a novel candidate therapeutic option for promoting tendon repair.

Spatial Configurations of 3D Extracellular Matrix Collagen Density and Anisotropy Simultaneously Guide Angiogenesis.

Extracellular matrix (ECM) collagen density and fibril anisotropy are thought to affect the development of new vasculatures during pathologic and homeostatic angiogenesis. Computational simulation is emerging as a tool to investigate the role of matrix structural configurations on cell guidance. However, prior computational models have only considered the orientation of collagen as a model input. Recent experimental evidence indicates that cell guidance is simultaneously influenced by the direction and intensity of alignment (i.e., degree of anisotropy) as well as the local collagen density. The objective of this study was to explore the role of ECM collagen anisotropy and density during sprouting angiogenesis through simulation in the AngioFE and FEBio modeling frameworks. AngioFE is a plugin for FEBio (Finite Elements for Biomechanics) that simulates cell-matrix interactions during sprouting angiogenesis. We extended AngioFE to represent ECM collagen as deformable 3D ellipsoidal fibril distributions (EFDs). The rate and direction of microvessel growth were modified to depend simultaneously on the ECM collagen anisotropy (orientation and degree of anisotropy) and density. The sensitivity of growing neovessels to these stimuli was adjusted so that AngioFE could reproduce the growth and guidance observed in experiments where microvessels were cultured in collagen gels of varying anisotropy and density. We then compared outcomes from simulations using EFDs to simulations that used AngioFE's prior vector field representation of collagen anisotropy. We found that EFD simulations were more accurate than vector field simulations in predicting experimentally observed microvessel guidance. Predictive simulations demonstrated the ability of anisotropy gradients to recruit microvessels across short and long distances relevant to wound healing. Further, simulations predicted that collagen alignment could enable microvessels to overcome dense tissue interfaces such as tumor-associated collagen structures (TACS) found in desmoplasia and tumor-stroma interfaces. This approach can be generalized to other mechanobiological relationships during cell guidance phenomena in computational settings.

The electron beam induced cross-linking of bovine collagen gels with various concentrations: The mechanical properties and secondary structure

This study considered the examination of the influence of electron beam irradiation (2 and 100 kGy) on bovine collagen gels with various concentrations (3, 6 and 8 wt%). The impact of irradiation was verified via the analysis of the mechanical properties and the secondary structure of the collagen. The irradiated collagen gels behaved non-linearly and stiffened with increasing strain in contrast to the non-irradiated state in which they softened with increasing strain. The impact of irradiation was reflected by an increase in the tensile modulus and strain energy density. The observed increase was greater the higher the water content of the collagen gels. The impact of irradiation on the secondary structure was less clear; infrared spectrometry revealed that the events that take place are complex, with the occurrence of cross-linking, partial denaturation and other structural changes. The electron beam irradiated collagen gels continued to exhibit collagenous polymeric structures and integrity.

Enthesis maturation in engineered ligaments is differentially driven by loads that mimic slow growth elongation and rapid cyclic muscle movement

Entheses are complex attachments that translate load between elastic-ligaments and stiff-bone via organizational and compositional gradients. Neither natural healing, repair, nor engineered replacements restore these gradients, contributing to high re-tear rates. Previously, we developed a culture system which guides ligament fibroblasts in high-density collagen gels to develop early postnatal-like entheses, however further maturation is needed. Mechanical cues, including slow growth elongation and cyclic muscle activity, are critical to enthesis development in vivo but these cues have not been widely explored in engineered entheses and their individual contribution to maturation is largely unknown. Our objective here was to investigate how slow stretch, mimicking ACL growth rates, and intermittent cyclic loading, mimicking muscle activity, individually drive enthesis maturation in our system so to shed light on the cues governing enthesis development, while further developing our tissue engineered replacements. Interestingly, we found these loads differentially drive organizational maturation, with slow stretch driving improvements in the interface/enthesis region, and cyclic load improving the ligament region. However, despite differentially affecting organization, both loads produced improvements to interface mechanics and zonal composition. This study provides insight into how mechanical cues differentially affect enthesis development, while producing some of the most organized engineered enthesis to date. Statement of significanceEntheses attach ligaments to bone and are critical to load transfer; however, entheses do not regenerate with repair or replacement, contributing to high re-tear rates. Mechanical cues are critical to enthesis development in vivo but their individual contribution to maturation is largely unknown and they have not been widely explored in engineered replacements. Here, using a novel culture system, we provide new insight into how slow stretch, mimicking ACL growth rates, and intermittent cyclic loading, mimicking muscle activity, differentially affect enthesis maturation in engineered ligament-to-bone tissues, ultimately producing some of the most organized entheses to date. This system is a promising platform to explore cues regulating enthesis formation so to produce functional engineered replacements and better drive regeneration following repair.

Stromal cells regulate mechanics of tumour spheroid

The remarkable contractility and force generation ability exhibited by cancer cells empower them to overcome the resistance and steric hindrance presented by a three-dimensional, interconnected matrix. Cancer cells disseminate by actively remodelling and deforming their extracellular matrix (ECM). The process of tumour growth and its ECM remodelling have been extensively studied, but the effect of the cellular tumour microenvironment (TME) has been ignored in most studies that investigated tumour-cell-mediated ECM deformations and realignment. This study reports the integration of stromal cells in spheroid contractility assays that impacts the ECM remodelling and invasion abilities of cancer spheroids. To investigate this, we developed a novel multilayer in vitro assay that incorporates stromal cells and quantifies the contractile deformations that tumour spheroids exert on the ECM. We observed a negative correlation between the spheroid invasion potential and the levels of collagen deformation. The presence of stromal cells significantly increased cancer cell invasiveness and altered the cancer cells' ability to deform and realign collagen gel, due to upregulation of proinflammatory cytokines. Interestingly, this was observed consistently in both metastatic and non-metastatic cancer cells. Our findings contribute to a better understanding of the vital role played by the cellular TME in regulating the invasive outgrowth of cancer cells and underscore the potential of utilising matrix deformation measurements as a biophysical marker for evaluating invasiveness and informing targeted therapeutic opportunities.

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.

Investigating the Combined Effects of Mechanical Stress and Nutrition on Muscle Hypertrophic Signals Using Contractile 3D-Engineered Muscle (3D-EM).

Since 3D-EM closely resembles in vivo muscles, the aim of this study was to investigate the effects of exercise (electrical pulse stimulation (EPS)) and nutrition (maca), which contains triterpenes, on muscle hypertrophy by using 3D-EM for the first time. The 3D-EM was composed of C2C12 cells and type 1 collagen gel, was differentiated for 14 days, and was divided into four groups: control, maca, EPS, and maca + EPS. The medium was replaced every two days before each EPS intervention, and the concentration of maca in the culture solution was 1 mg/mL. The intervention conditions of the EPS were 30 V, 1 Hz, and 2 ms (24 h on, 24 h off, for one week). The expression levels of proteins were examined by Western blotting. The intervention of maca and EPS upregulated the expression of MHC-fast/slow (both p < 0.05) compared with the control group, and the addition of maca had no effect on the phosphorylation of mTOR (p = 0.287) but increased the AMPK phosphorylation (p = 0.001). These findings suggest that intervention with maca and EPS has a positive effect on muscle hypertrophy, which has a positive impact on sarcopenia. However, the underlying mechanisms remain to be further explored.