Collagen Matrices Research Articles

Impact of 3D collagen-based model and hydrostatic pressure on periodontal ligament fibroblast: A morpho-biochemical analysis

This study developed a customized hydrostatic pressure-based loading environment to investigate the effect of static hydrostatic pressure on the periodontal ligament fibroblasts (PDLf) in a three-dimensional (3D) collagen-based model. The cylindrical tissue constructs were comprised of PDL fibroblast cells seeded in type I collagen matrices and divided into three experimental groups: Control (no load), low-load (∼0.07 kPa), and high-load (∼60 kPa), all subjected to 24 h of experimental duration. Cells in the 3D construct were stained with fluorophore-conjugated antibodies for cytoskeletal protein F-actin and matricellular protein periostin. Cell culture supernatant was assessed for receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) expression. Transmission electron microscopy examined the contact between the cells and the collagen matrix. Ultrastructural changes in the 3D collagen matrix were also analyzed using scanning electron microscopy. Experiments were performed in triplicates, and data was analyzed using one-way ANOVA (p < 0.05). The 3D PDLf constructs from the low-load group demonstrated the highest levels of homogeneous cell distribution and higher expression of F-actin and periostin with enhanced interaction with the matrix. The collagen matrix in this group showed more closely packed fibers forming thicker bundles when compared to the control and the high-load 3D PDLf constructs. Nonuniform cell distribution with decreased expression of F-actin and periostin was observed in the control and high-load PDLf constructs. The high-load group showed the highest RANKL/OPG expression. This study demonstrated low-level hydrostatic pressure's role in regulating PDLf functions and extracellular matrix response, while excessive hydrostatic pressure may be detrimental to PDL fibroblast cell function.

Using x-ray micro computed tomography to quantify intracochlear fibrosis after cochlear implantation in a Guinea pig model

Cochlear implants (CIs) allow individuals with profound hearing loss to understand speech and perceive sounds. However, not all patients obtain the full benefits that CIs can provide and the cause of this disparity is not fully understood. One possible factor for the variability in outcomes after cochlear implantation, is the development of fibrotic scar tissue around the implanted electrode. It has been hypothesised that limiting the extent of fibrosis after implantation may improve overall CI function, and longevity of the device. Currently, histology is often used to quantify the extent of intracochlear tissue growth after implantation however this method is labour intensive, time-consuming, often involves significant user bias, and causes physical distortion of the fibrosis. Therefore, this study aimed to evaluate x-ray micro computed tomography (μCT) as a method to measure the amount and distribution of fibrosis in a guinea pig model of cochlear implantation. Adult guinea pigs were implanted with an inactive electrode, and cochleae harvested eight weeks later (n = 7) and analysed using μCT, to quantify the extent of tissue reaction, followed by histological analysis to confirm that the tissue was indeed fibrotic. Cochleae harvested from an additional six animals following implantation were analysed by μCT, before and after contrast staining with osmium tetroxide (OsO4), to enhance the visualisation of soft tissues within the cochlea, including the tissue reaction. Independent analysis by two observers showed that the quantification method was robust and provided additional information on the distribution of the response within the cochlea. Histological analysis revealed that μCT visualised dense collagenous material and new bone formation but did not capture loose, areolar fibrotic tissue. Treatment with OsO4 significantly enhanced the visible tissue reaction detected using μCT. Overall, μCT is an alternative and reliable method that can be used to quantify the extent of the CI-induced intracochlear tissue response and will be a useful tool for the in vivo assessment of novel anti-fibrotic treatments.

Role of Collagen in the Etiology of Inguinal Hernia Patients: A Case-Control Study.

Introduction Technical faults are no longer accepted as the sole reason for recurrence following inguinal hernia (InH) repairs. Medical literature has been studied to find any contributing factors and collagen has emerged as a promising marker. Owing to their long half-lives, it has been found to best reflect the process of scarring, which is central to ensuring the formation of a proper fibrous tissue that incorporates the mesh with the abdominal wall. Methods Sixty participants were divided into two groups. The case group were patients diagnosed with InH and the control group had patients undergoing abdominal surgeries for indications other than abdominal wall hernias. A 0.5x0.5cm specimen of skin and transversalis fascia were biopsied and subsequently stained to determine the amount of collagen I and III. Results Collagen I, collagen III and the ratio of collagen I to III was measured. Collagen I was normal in the skin of both groups but decreased in transversalis fascia of cases. Collagen III was found to be normal in transversalis fascia of both cases and controls, but increased in the skin of cases. Ratio of collagen I to III was decreased in both skin and transversalis fascia of cases. Statistical analysis was carried out using an unpaired t-test, non-parametric Mann-Whitney test, ANOVA and chi-square test. Conclusions Our study has reported that in patients with inguinal hernia, collagen III or immature collagen is increased in skin and collagen I or mature collagen is decreased in the transversalis fascia. The ratio of collagen I/III is decreased in both skin and transversalis fascia.

Chitosan thread in the healing of the cecal wall of rabbits (Oryctolagus cuniculus) submitted to cecorrhaphy.

The development and evaluation of synthesis materials are crucial to reducing the morbidity and magnitude of post-enterorrhaphy surgical complications. Despite the possibility of production, chitosan thread has not yet been used in enterorrhaphy, and its effects on intestinal healing have not been evaluated. Therefore, this study aimed to evaluate the effects of chitosan thread on the intestinal wall repair of rabbits submitted to cecorrhaphy. For this, 42 rabbits were allocated into two groups with 21 animals. One group was submitted to cecorrhaphy with chitosan suture thread (CG) and the other with poliglecaprone suture thread (PG). The occurrence of postoperative complications, the intensity of edema, cellular response, formation of granulation tissue, as well as the deposition and maturation of collagen fibers, and the intensity of vascular endothelial growth factor (VEGF-α) expression, were evaluated during the intestinal wall repair process. The evaluations occurred on the 5th, 15th, and 25th postoperative (PO) days. The animals did not develop peritonitis, but adherence was observed in six animals from CG and seven from PG, with no difference between groups. The polymorphonuclear infiltrate showed higher intensity and higher amount of type III collagen fibers in CG on the 15th PO day. In contrast, a lower amount of type I collagen fibers was observed in CG samples on the 25th PO day. Therefore, the chitosan thread used for cecorrhaphy in rabbits results in minimal postoperative complications, presents biocompatibility, and bioactively assists the tissue repair process of the cecal wall, inducing minimal tissue reaction, stimulating the deposition of type III collagen fibers in the proliferative phase, with sustained VEGF-α expression, but with reduced deposition of type I fibers, indicating a delay in collagen maturation.

Biocompatibility Study with Baby Hamster Kidney Cells on Acetobacter Xylinum Pellicle for Diabetic Foot Laceration

Diabetes mellitus, commonly known as diabetes, is a persistent metabolic condition marked by elevated blood glucose levels. This condition's global prevalence has been consistently increasing, becoming a major cause for concern in public health on an international scale. This disease can lead to various complications, including impaired wound healing. The purpose of this study is to determine the cytotoxicity level of hydrogel membranes made from Acetobacter xylinum and whether the used method is appropriate for producing proper wound healing material. The findings of the study, employing BHK Kidney cells and the MTT Assay technique, revealed that the dehydrated hydrogel membrane derived from bacterial cellulose and processed through oven drying exhibited a toxicity level of less than 50% across all samples. Additionally, the swelling test indicated a rise in water absorption when the membrane was combined with collagen material

COLLAGEN MATRIX MENISCAL WRAPPING REDUCES CONTACT PRESSURES IN MENISCAL REPAIR: A FINITE ELEMENT ANALYSIS STUDY

AbstractINTRODUCTIONTo preserve knee function and reduce degenerative, meniscal tears should be repaired where possible. Meniscal wrapping with collagen matrices has shown promising clinical outcome (AAOS meniscal algorithm), however there is limited basic science to support this.AIMto model the contact pressures on the human tibial plateau beneath a (1) a repaired radial meniscal tear and (2) a wrapped and repaired radial meniscal tear.METHODOLOGYComplete anterolateral radial tears were formed across 4 lateral human menisci, before repairing with ‘rip-stop’ H sutures using 2mm Arthrex Meniscal Suture tape. This was then repeated with the addition of a ChondroGide collagen matrix wrapping. From this experimental setup a finite element (FE) analysis model was construted.FE models of the two techniques (i) suture alone and (ii) suture and collagen-matrix wrap, were then modelled; bone was linear elastic, articular cartilage was a hyperelastic Yeoh model, and a linear elastic and transversely isotropic material model for the meniscus.The contact areas of the articulating surfaces, meniscus kinematics, and stress distribution around the repair were compared between the two systems.RESULTSMeniscal suture-tape repair had higher local stresses and strains (σ_max=51 MPa ε_max=25%) around the repair compared to with Collagen wrapping (σ_max=36.6MPa ε_max=15%). Radial displacement and pressure on the meniscal contact surfaces were higher in the suture only repair.CONCLUSIONCollagen-matrix wrapping strengthens the repair, reducing local peak stresses and strains around the suture-tape. This could reduce the chance of suture-tape pull-out and subsequent repair failure.

Design matters: A comparison of natural versus synthetic skin substitutes across benchtop and porcine wound healing metrics: An experimental study.

Skin substitutes, essential tools for helping close full thickness wounds with minimal scarring, are available in both collagen-based and synthetic polyurethane constructions. Here we explore fundamental differences between two frequently used skin substitutes and discuss how these differences may impact in vivo performance. Polyurethane- and collagen-based matrices were characterized in vitro for pore size via scanning electron microscopy, hydrophobicity via liquid contact angle, conformability via bending angle, and biocompatibility via fibroblast and keratinocyte adhesion and proliferation. These matrices were then evaluated in a full-thickness excisional pig wound study followed by histological analysis. Statistical analysis was performed using t-tests or one-way analysis of variances with Tukey's multiple post hoc comparisons, where appropriate. Average pore diameter in the tested polyurethane matrix was over four times larger than that of the collagen matrix (589 ± 297 µm vs. 132 ± 91 µm). Through liquid contact angle measurement, the collagen matrix (not measurable) was found to be hydrophilic compared to the hydrophobic polyurethane matrix (>90°). The collagen matrix was significantly more conformable than the polyurethane matrix (9 ± 2° vs. 84 ± 5° bending angle, respectively). Fibroblast and keratinocyte adhesion and proliferation assays elucidated a significantly greater ability of both cell types to attach and proliferate on collagen versus polyurethane. While the porcine study showed minimal contraction of either matrix material, histological findings between the two treatments were markedly different. Collagen matrices were associated with early fibroblast infiltration and fibroplasia, whereas polyurethane matrices elicited a strong multinucleated giant cell response and produced a network of comparatively aligned collagen fibrils. The more favorable in vitro properties of the collagen matrix led to less inflammation and better overall tissue response in vivo. Overall, our findings demonstrate how the choice of biomaterial and its design directly translate to differing in vivo mechanisms of action and overall tissue quality.

Dynamic Tensile Stress Promotes Regeneration of Achilles Tendon in a Panda Rope Bridge Technique Mice Model.

Regeneration of ruptured Achilles tendon remains a clinical challenge owing to its limited regenerative capacity. Dynamic tensile stress plays a positive role in the regeneration of tendon, although the specific underlying mechanisms remain unclear. In this study, the Achilles tendon defect-regeneration model was created in male C57BL/6 mice aged 8weeks. The animals were randomly assigned to four groups-repair, non-repair, repair with fixation, and non-repair with fixation. The repair groupand repair with fixation group adopted the panda rope bridge technique (PRBT) repair method. Our results demonstrated the presence of more densely aligned and mature collagen fibers, as well as more tendon-related makers, in the repair group at both 2- and 4-week post-surgery. Furthermore, the biomechanical strength of the regenerated tendon in the repair group was highly improved. Most importantly, the expressions of integrin αv and its downstream and the phosphorylation levels of FAK and ERK were remarkably higher in the repair group than in the other groups. Furthermore, blocking FAK or ERK with selective inhibitors PF573228 and U0126 resulted in obvious adverse effects on the histological structure of the regenerated Achilles tendon. In summary, this study demonstrated that dynamic tensile stress based on the PRBT could effectively promote the regeneration of the Achilles tendon, suggesting that dynamic tensile stress enhances the cell proliferation and tenogenic differentiation via the activation of the integrin/FAK/ERK signaling pathway.

Fibrotic Diseases of the Human Urinary and Genital Tract: Current Understanding and Potential Strategies for Treatment.

Fibrosis is a disease condition characterized by abnormalities of the extracellular matrix, such as accumulation of the transforming growth factor β, infiltration by myofibroblasts, deposition of collagen, and a generalized dysregulation of collagen maturation. It can severely impair the function of organs by replacing normal tissue with a highly collagenized matrix, thereby reducing the elasticity and compliance of tissues. Fibrotic diseases of the genitourinary tract present relevant problems in healthcare, and their principles of pathophysiology remain unclarified; hence, the armamentarium for prevention and treatment is limited. These diseases include renal fibrosis, Peyronie's disease and ureteral and urethral strictures due to perturbations in the process of wound healing in response to injuries. Such deteriorations may contribute to obstructive uropathies or sexual dysfunction. This review provides a brief overview of the most frequent fibrotic diseases of the genitourinary system and of how the pathophysiology is related to symptoms, and also highlights potential therapeutic strategies to address the abnormal deposition of collagen. Although the understanding of factors associated with fibrotic conditions of the urinary and genital tract is still limited, some beneficial advances have been made. Further research will serve to provide a more comprehensive insight into factors responsible for the development of fibrotic tissue deposition.

Biological investigation of resinous endodontic sealers containing calcium hydroxide.

The purpose of this study was to evaluate, in vivo, the biocompatibility, biomineralization, collagen maturation and the in vitro antibacterial and cytotoxicity of resinous endodontic sealers containing calcium hydroxide. Forty rats were implanted with polyethylene tubes containing Sealer 26, Sealer Plus, Dia-ProSeal and an empty tube, examined after 7, 15, 30 and 60 days. Antimicrobial activity was evaluated against Enterococcus faecalis by Agar Diffusion Test (ADT) through inhibition zones. For cytotoxicity, undifferentiated pulp cells (OD-21) were cultured and assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, exposed to dilution of serial extracts at 6, 24, 48h. Cytotoxicity was analyzed by two-way ANOVA and Bonferroni correction. Kruskal-Wallis test followed by Dunn test was performed for nonparametric data (p<0.05). MTT assay revealed cell proliferation affected by sealers extract in all periods (p<0.0001), except for Dia-Proseal and Sealer Plus ⅛ dilution. Subcutaneous analysis showed at day 7th moderate inflammatory infiltration. After 30 days, Sealer 26 still showed moderate inflammatory infiltrate compared to mild inflammation from control and Dia-ProSeal (p = 0.006). At day 60th, all groups showed similar mild inflammatory infiltrate (p>0.05). Sealer 26 induced more biomineralization than other sealers in all periods. At 7 and 15 days, all sealers had significant percentage of immature collagen fibers. After 60 days Sealer 26 showed more mature fibers compared to other sealers (p<0.001). All sealers had a smaller zone of inhibition than chlorhexidine, but with no significant difference among any group (p>0.05). All sealers showed satisfactory biological responses with in vitro/in vivo biocompatibility and antimicrobial activity against planktonic bacteria. Sealer 26 induced more biomineralization than Sealer Plus and Dia-ProSeal.

Histological assessment and gene expression analysis of intra-oral soft tissue graft donor sites.

To determine the structural and gene expression features of different intra-oral soft tissue donor sites (i.e., anterior palate, posterior palate, maxillary tuberosity and retromolar pad). Standardized mucosal tissue punch biopsies were collected from at least one donor site per subject. Histological processing was performed to determine tissue morphometry and quantify collagen composition. Site-specific gene distribution was mapped using targeted gene expression analysis and validated using real time polymerase chain reaction (qPCR). A total of 50 samples from 37 subjects were harvested. Epithelial thickness did not differ between sites. However, lamina propria was thicker in the maxillary tuberosity (2.55 ± 0.92 mm) and retromolar pad (1.98 ± 0.71 mm) than in the lateral palate. Type I collagen was the predominant structural protein in the lamina propria (75.06%-80.21%). Genes involving collagen maturation and extracellular matrix regulation were highly expressed in the maxillary tuberosity and retromolar pad, while lipogenesis-associated genes were markedly expressed in the lateral palate. The retromolar pad showed the most distinct gene expression profile, and the anterior and posterior palate displayed similar transcription profiles. Tissue samples harvested from the anterior and posterior palate differed morphologically from those from the maxillary tuberosity and retromolar pad. Each intra-oral site showed a unique gene expression profile, which might impact their biological behaviour and outcomes of soft tissue augmentation procedures.

Microporogen-Structured Collagen Matrices for Embedded Bioprinting of Tumor Models for Immuno-Oncology.

Embedded bioprinting enables the rapid design and fabrication of complex tissues that recapitulate in vivo microenvironments. However, few biological matrices enable good print fidelity, while simultaneously facilitate cell viability, proliferation, and migration. Here, we introduce a new microporogen-structured (μPOROS) matrix for embedded bioprinting, in which matrix rheology, printing behavior, and porosity are tailored by adding sacrificial microparticles composed of a gelatin-chitosan complex to a prepolymer collagen solution. To demonstrate its utility, we created a three-dimensional tumor model via embedded printing of a murine melanoma cell ink within the μPOROS collagen matrix at 4°C. The collagen matrix is subsequently crosslinked around the microparticles upon warming to 21°C, followed by their melting and removal at 37°C. This process results in a μPOROS matrix with a fibrillar collagen type-I network akin to that observed in vivo. Printed tumor cells remain viable and proliferate, while antigen-specific cytotoxic T cells incorporated in the matrix migrate to the tumor site, where they induce cell death. The integration of our μPOROS matrix with embedded bioprinting opens new avenues for creating complex tissue microenvironments in vitro that may find widespread use in drug discovery, disease modeling, and tissue engineering for therapeutic use. This article is protected by copyright. All rights reserved.

Efficacy of a collagen matrix for soft tissue augmentation after implant placement compared to connective tissue grafts: A multicenter, noninferiority, randomized controlled trial.

To test whether soft tissue volume augmentation using a collagen matrix (VCMX) leads to noninferior results in terms of gain of mucosal thickness at single implant sites, compared to connective tissue grafts (SCTG). The study was designed as a multi-center randomized controlled clinical trial. Subjects in need of soft tissue volume augmentation at single tooth implant sites were consecutively recruited at nine centers. The deficient mucosal thickness at the implant sites (one per patient) was augmented by applying either a VCMX or a SCTG. Patients were examined at 120 days (abutment connection = primary endpoint), 180 days (final restoration), and 360 days (1-year after insertion of the final restoration). Outcome measures included: transmucosal probing of the mucosal thickness (crestal = primary outcome), profilometric measurements of the tissue volume, and patient-reported outcome measures (PROMs). Out of the 88 patients, 79 attended the one-year follow-up. The median increase of the crestal mucosal thickness between pre-augmentation and 120 days was 0.3 ± 2.1 mm in the VCMX group and 0.8 ± 1.6 mm in the SCTG group (p = .455). Non-inferiority of the VCMX compared to the SCTG was not observed. The respective numbers at the buccal aspect amounted to 0.9 ± 2.0 mm (VCMX) and 1.1 ± 1.4 mm (SCTG; p = .431). PROMs including pain perception favored the VCMX group. It remains inconclusive whether soft tissue augmentation using a VCMX is noninferior to SCTG in terms of crestal mucosal thickening at single implant sites. However, the use of collagen matrices favors PROMs especially pain perception, while achieving similar buccal volume gains along with comparable clinical and aesthetic parameters to SCTG.

Porcine Resorbable Collagen Matrix Shows Good Incorporation of Liquid Platelet-Rich Fibrin In Vitro.

Purpose: To evaluate the incorporation of liquid platelet-rich fibrin in different collagen matrices in vitro. Materials and Methods: Collagen matrices with liquid platelet-rich fibrin were used and divided into the following test groups (n = 5): Mucoderm (MD), Mucograft (MG), and Fibro-Gide (FG). After incubating the collagen matrices in liquid platelet-rich fibrin, histologicl and fluid absorption capacity analysis were performed. Intergroup comparisons of cell count, blood plasma penetration area, and fluid absorption capacity were performed using one-way ANOVA and Tukey tests. Intragroup comparisons of fluid absorption capacity were made using the independent t test with a 5% significance level. Results: Descriptive qualitative analysis showed total incorporation of liquid platelet-rich fibrin in the FG group, while the MG and MD groups showed only partial and shallow incorporation, respectively. There was a statistically significant difference among the three groups regarding inflammatory cell infiltration (P = .000), with the FG group presenting the highest number of cells in the matrices (577.15 ± 54.88). The FG group showed an area of total blood plasma penetration into the matrix, followed by the MG group with partial penetration, and the MD group with minor penetration area (P = .000). Considering the fluid absorption capacity analysis, only groups FG and MG were statistically different when comparing the liquid platelet-rich fibrin absorption coefficient (P = .017), with higher absorption in group FG (14.30 ± 3.35). Conclusions: The FG collagen matrix showed a good capacity for liquid platelet-rich fibrin incorporation in vitro.

A Comparative Evaluation of Collagen in Ameloblastoma and Oral Squamous Cell Carcinoma using Picrosirius Red Staining with Polarizing Microscopy and CD44v6 Immunoreactivity.

Solid multicystic ameloblastoma (SMA) is a locally aggressive, benign odontogenic tumor of odontogenic origin with greater rate of recurrence. Epithelial-mesenchymal interaction plays an important role in tooth morphogenesis that shows complete differentiation of epithelial and ectomesenchymal components to the level of tooth formation. Tumor stroma in ameloblastoma is normal mature collagen that prevents differentiation to the level of tooth formation. Current study evaluates the role of stromal elements in aggressive behavior of SMA using picrosirius red staining with polarizing microscopy and CD44v6 immunohistochemistry (IHC). To compare nature of collagen using picrosirius red staining under polarized microscope and IHC expression of CD44v6 marker in SMA and oral squamous cell carcinoma (OSCC). Thirty blocks were retrieved from departmental archives and subjected to picrosirius red staining and CD44v6 IHC staining. Slides stained with picrosirius red were observed under polarized microscope to report the birefringence pattern. IHC slides were annotated for intensity of staining of tumor cells. In contrast to OSCC's 40% red, 40% yellowish-red, and 20% greenish-yellow birefringence, SMA displayed 87% red, 13% yellowish-red, and 0% greenish-yellow. Compared to OSCC, which had tumor cells stained 9% strongly, 64% moderately, 27% mildly, and 0% negatively, SMA revealed 0% strong, 10% moderate, 60% weak, and 30% negative staining. As opposed to OSCC, which exhibited a greater quantity of greenish-yellow birefringence of immature collagen, SMA showed predominantly red birefringence, which is suggestive of mature collagen with a lack of metastasis. Comparing SMA to OSCC, the lack of significant CD44v6 positivity suggests that there has not been perineural invasion or regional metastases in SMA.

Poster 136: Augmented Meniscus Radial Tear Healing via Local Transforming Growth Factor-beta 1 (TGF- β1) Gene Therapy in a Rat Model

Objectives: Meniscus tears cause significant musculoskeletal impairment and are among the most common knee injuries. Tears in the inner third of the meniscus have intrinsically poor healing capacity due to insufficient blood supply. In an effort to improve healing, use of biologic augments is a promising area of investigation. Tissue engineering strategies, such as growth factor gene therapy, have emerged as encouraging approaches to improving meniscal healing. Transforming Growth Factor-Beta 1 (TGF-β1) gene therapy has shown promise in vitro and ex vivo as a therapeutic strategy, but its effects on meniscus healing in vivo remain unknown. This study aimed to determine whether local delivery of viral vectors carrying the TGF-β1 gene could improve the healing of a meniscus tear involving the avascular zone in vivo in a rat model. Methods: Recombinant adenoviruses were generated using the AdEasy system. Briefly, the coding regions of mouse TGFβ1 were PCR amplified and cloned into an adenoviral shuttle vector and subsequently used to generate recombinant adenoviruses in HEK293 cells. The resulting adenoviruses were designated as Adv-TGF-β1. An analogous adenovirus expressing only GFP (Adv-GFP) was used as mock virus controls. To achieve local delivery of viral vectors to the injury site, we utilized a thermoresponsive biodegradable polydiolcitrate-gelatin delivery scaffold (PPCNg). A full-thickness radial tear was made in the medial meniscus of the left hind leg of twelve Wistar rats. Rats were divided into two groups: one receiving a mixture of PPCNg+TGF-β1 (n = 3) and the other receiving PPCNg+GFP (n = 3). A 4ºC pre-chilled solution of PPCNg seeded with Adv-TGF-β1 or Adv-GFP at 1011 infectious viral particles/injection was administered at the site of the induced meniscus tear. After 3 weeks, rats were euthanized, and the menisci were histologically evaluated by hematoxylin and eosin (H&amp;E) staining. The histologic assessment and scoring of meniscus tissue wound healing were done by one blinded and one unblinded observer, each using selected wound healing parameters as described in Table 1, and the results were averaged. The intra-class correlation coefficient (ICC) was computed based on a mean rating (k=2), absolute agreement, and two-way random effects model to assess the agreement between the wound healing score rating of the two observers. Statistical significance between groups was determined using the Mann-Whitney Wilcoxon rank sum test. A value of p &lt; 0.05 was considered statistically significant. All animal procedures were performed according to an Institutional Animal Care and Use Committee protocol at the home institution. Results: Immediately after and 3 weeks post-surgery, there was no evidence of infection, dehiscence, or functional deficit. Histologic evaluation of the sections (n = 8/group) of harvested rat menisci (n = 2/group) revealed improved healing of tears in the PPCNg+TGF-β1 group compared to the control group (5.7 ± 0.48 vs. 5.0 ± 0.89, p = 0.011) (Figure 1B). The lesion area demonstrated significantly greater neovascularization (2.75 ± 0.45 vs. 2.06 ± 0.68, p = 0.004), better collagen organization (1.94 ± 0.25 vs. 1.62 ± 0.50, p = 0.038), and increased hypercellularity (1.0 ± 0.00 vs. 1.3 ± 0.48, p = 0.018) in the PPCNg+TGF-β1 group compared to the controls (Figure 1C). Moderate absolute agreement between the two raters was observed in their wound healing score ratings (ICC = 0.615). Conclusions: Our results indicate that PPCNg-mediated delivery of Adv-TGF-β1 enhanced the early (3 weeks) healing of the meniscus lesion in rats compared with control (Adv-GFP) gene therapy. Further, localized administration of Adv-TGF-β1 induced greater neovascularization, better organization of collagen fibers, and increased hypercellularity at the injury site in the Adv-TGFβ-1 rat group compared with controls at 3 weeks post-injury. These results together suggest that TGF-β1 gene therapy may enhance meniscus healing by inducing more cellular proliferation and infiltration and greater neovascularization at the injury site to serve as nourishment for the establishment of new wound matrix, leading to more significant deposition and remodeling of mature collagen matrix. Further study that includes larger sample sizes, confirmation of successful gene transfer to meniscal cells around the tear site, and investigations of both later time points of wound healing and the mechanisms by which local TGF-β1 gene therapy may modulate augmented meniscus healing in this rat model of meniscal defect is necessary to better determine the potential future clinical utility of local TGF-β1 gene therapy to treat of meniscus tears with poor healing capacity. [Table: see text]

Extracellular matrix-derived mechanical force governs breast cancer cell stemness and quiescence transition through integrin-DDR signaling

The extracellular matrix (ECM) serves as signals that regulate specific cell states in tumor tissues. Increasing evidence suggests that extracellular biomechanical force signals are critical in tumor progression. In this study, we aimed to explore the influence of ECM-derived biomechanical force on breast cancer cell status. Experiments were conducted using 3D collagen, fibrinogen, and Matrigel matrices to investigate the role of mechanical force in tumor development. Integrin-cytoskeleton-AIRE and DDR-STAT signals were examined using RNA sequencing and western blotting. Data from 1358 patients and 86 clinical specimens were used for ECM signature-prognosis analysis. Our findings revealed that ECM-derived mechanical force regulated tumor stemness and cell quiescence in breast cancer cells. A mechanical force of ~45 Pa derived from the extracellular substrate activated integrin β1/3 receptors, stimulating stem cell signaling pathways through the cytoskeleton/AIRE axis and promoting tumorigenic potential and stem-like phenotypes. However, excessive mechanical force (450 Pa) could drive stem-like cancer cells into a quiescent state, with the removal of mechanical forces leading to vigorous proliferation in quiescent cancer stem cells. Mechanical force facilitated cell cycle arrest to induce quiescence, dependent on DDR2/STAT1/P27 signaling. Therefore, ECM-derived mechanical force governs breast cancer cell status and proliferative characteristics through stiffness alterations. We further established an ECM signature based on the fibrinogen/fibronectin/vitronectin/elastin axis, which efficiently predicts patient prognosis in breast cancer. Our findings highlight the vital role of ECM-derived mechanical force in governing breast cancer cell stemness/quiescence transition and suggest the novel use of ECM signature in predicting the clinical prognosis of breast cancer.

Cells immersed in collagen matrices show a decrease in plasma membrane fluidity as the matrix stiffness increases

Cells are constantly adapting to maintain their identity in response to the surrounding media's temporal and spatial heterogeneity. The plasma membrane, which participates in the transduction of external signals, plays a crucial role in this adaptation. Studies suggest that nano and micrometer areas with different fluidities at the plasma membrane change their distribution in response to external mechanical signals. However, investigations linking fluidity domains with mechanical stimuli, specifically matrix stiffness, are still in progress. This report tests the hypothesis that the stiffness of the extracellular matrix can modify the equilibrium of areas with different order in the plasma membrane, resulting in changes in overall membrane fluidity distribution. We studied the effect of matrix stiffness on the distribution of membrane lipid domains in NIH-3 T3 cells immersed in matrices of varying concentrations of collagen type I, for 24 or 72 h. The stiffness and viscoelastic properties of the collagen matrices were characterized by rheometry, fiber sizes were measured by Scanning Electron Microscopy (SEM) and the volume occupied by the fibers by second harmonic generation imaging (SHG). Membrane fluidity was measured using the fluorescent dye LAURDAN and spectral phasor analysis. The results demonstrate that an increase in collagen stiffness alters the distribution of membrane fluidity, leading to an increasing amount of the LAURDAN fraction with a high degree of packing. These findings suggest that changes in the equilibrium of fluidity domains could represent a versatile and refined component of the signal transduction mechanism for cells to respond to the highly heterogeneous matrix structural composition. Overall, this study sheds light on the importance of the plasma membrane's role in adapting to the extracellular matrix's mechanical cues.

Extracellular matrix composition affects outgrowth of dendrites and dendritic spines on cortical neurons.

The composition of the extracellular matrix (ECM) in nervous tissue plays an important role in controlling neuronal outgrowth and synapse development. Changes in both protein and glycosaminoglycan components of the ECM occur with tissue injury and may affect neuron growth. To investigate neuron responses to alterations in fibronectin (FN), a major component of the wound ECM, we grew cortical neurons on cell-derived decellularized matrices composed of wild type FN (FN+/+) or of a mutant form of FN (FNΔ/+) from which the III13 heparin-binding site had been deleted by CRISPR-Cas 9 gene editing. The most significant effect of the mutant FN was a reduction in dendrite outgrowth. Not only were dendrites shorter on mutant FNΔ/+-collagen (COL) matrix than on wild type (FN+/+-COL) matrix, but the number of dendrites and dendritic spines per neuron and the spine densities were also dramatically reduced on FNΔ/+-COL matrices. Mass spectrometry and immunostaining identified a reduction in tenascin-C (TN-C) levels in the mutant matrix. TN-C is an ECM protein that binds to the III13 site of FN and modulates cell-matrix interactions and has been linked to dendrite development. We propose that TN-C binding to FN in the wound matrix supports dendrite and spine development during repair of damaged neural tissue. Overall, these results show that changes in ECM composition can dramatically affect elaboration of neurites and support the idea that the ECM microenvironment controls neuron morphology and connectivity.

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.