Arrangement Of Collagen Fibers Research Articles

Alginate-polylysine-alginate (APA) microencapsulated transgenic human amniotic epithelial cells ameliorate fibrosis in hypertrophic scars.

Hypertrophic scar (HS) is a severe skin fibrosis. Transplanting stem cells carrying anti-fibrotic cytokine genes, like interferon-gamma (IFN-γ), is a novel therapeutic strategy. Human amniotic epithelial cells (hAECs) are ideal seed cells and gene vectors. Microencapsulation creates a favorable environment for transplanted cells. This study investigates the effect of alginate-polylysine-alginate (APA)-microencapsulated hAECs modified with IFN-γ on HS fibrosis. hAECs were isolated from human placentas and characterized. The full-length IFN-γ gene was cloned into the pcDNA3.1 vector to create the recombinant plasmid IFN-γ-pcDNA3.1. This plasmid was then transfected into hAECs, resulting in the generation of IFN-γ-modified hAECs (IFN-γ-hAECs). Subsequently, these IFN-γ-hAECs were microencapsulated with APA to produce APA-IFN-γ-hAECs. In vitro, the release of IFN-γ, as well as the cellular and metabolic activities, growth, proliferation, migration, apoptosis, and trans-differentiation were assessed using HS-derived fibroblasts. In vivo, the weight loss of HS xenografts, collagen fiber arrangement, tissue oxidative stress, and inflammatory response were evaluated using a nude mouse model that had been transplanted with human HS tissues. In vitro, APA-IFN-γ-hAECs exhibited significantly sustained and enhanced IFN-γ release, increased cellular vitality, and inhibited fibroblast growth, proliferation, migration, and trans-differentiation into myofibroblasts. APA-IFN-γ-hAECs also remarkably downregulated extracellular matrix (ECM) components and promoted apoptosis. In vivo, they significantly accelerated the weight reduction of HS xenografts, improved collagen fiber arrangement, and mitigated oxidative stress and inflammation. This study suggests that APA-microencapsulated IFN-γ-hAECs may have potential in alleviating HS fibrosis, offering a new direction for exploring effective clinical HS management strategies.

SMILE-Derived Corneal Stromal Lenticule: Experimental Study as a Corneal Repair Material and Drug Carrier.

A detailed study of the physicochemical properties of SMILE-derived lenticules and evaluation of their drug delivery after loading with silver nanoparticles (AgNPs). The lenticules were decellularized and modified with crosslinking concentrations of 0.01 (0.01E/L), 0.05 (0.05E/L), and 0.25 (0.25E/L) mmol N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) per mg lenticule at 5:1 carbodiimide/N-hydroxysuccinimide (EDC/NHS) ratios. The degree of swelling, light transmittance, biomechanical properties, and stability of the non-crosslinked decellularized lenticules (DLs), 0.01E/L, 0.05E/L, and 0.25E/L were measured and characterized using Fourier transform infrared spectroscopy and transmission electron microscopy with non-crosslinked non-decellularized lenticules as controls. DLs, 0.01E/L, 0.05E/L, and 0.25E/L were soaked in AgNPs for 24 hours, and the concentration of the drug released was measured. There was no significant difference in the degree of swelling between the groups (P > 0.05). The light transmittance of the lenticules did not change after decellularization and crosslinking and decreased after loading with AgNPs. Non-decellularized lenticules biodegraded within 108 to 120 hours, and the other groups biodegraded within 96 to 108 hours in vitro. The 0.01E/L had the highest tensile strength. The absorption peak intensity ratio of the amide I band and the amide II band decreased, and the arrangement of collagen fibers was more compact in crosslinked decellularized lenticules. The 0.01E/L had the highest cumulative drug release (3.4 ± 0.91 μg). Crosslinking decellularization improved the biomechanical properties and resistance to water absorption of lenticules, increased covalent bonds between collagen fibers, and improved drug delivery. Crosslinked decellularized lenticules can be used as a new corneal patch material and drug delivery carrier for drug AgNPs.

Objective assessment of corneal transparency using densitometry distribution analysis

Advancing the assessment of eye diseases necessitates novel biomarkers that surpass conventional morphological analysis. Historically, the exploration of microstructural details in corneal research has heavily relied on histological examination, a process necessitating post‐mortem tissue samples and inapplicable in vivo. The cornea comprises densely arranged collagen fibers and specialized cell types including keratocytes, endothelial cells, and epithelial cells. These intricate details are inaccessible through widely used clinical instrumentation, such as Scheimpflug or OCT imaging. However, it is possible to analyze in‐vivo microstructure indirectly using Densitometry Distribution Analysis (DDA). This method involves extracting microstructure‐related biomarkers, such as tissue transparency and homogeneity, from low‐resolution clinical corneal images using image processing supported by artificial intelligence. Indirect in‐vivo microstructure analysis requires evaluating the distribution of pixel intensities. In ophthalmic instruments, image formation is influenced by how light, after being emitted from the source, backscatters upon interacting with the cornea. Given the cornea's nonhomogeneous nature, its internal arrangement affects how light traverses through different layers and regions. This variation in light propagation leads to differing pixel intensities in the resultant images. By examining these intensity variations, we can derive microstructure‐related biomarkers, such as tissue transparency and homogeneity.In this talk, we will describe the DDA methodology and revisit its early successes in detecting eye diseases such as keratoconus before any clinical signs are apparent, with a 97% success rate, and up to 100% if clinical signs are observed. The DDA has also been used for early glaucoma detection, with a success rate of over 80%. Additionally, the DDA has been successfully applied to study the effects of contact lens wear on our eyes and to investigate certain rare genetic disorders affecting the cornea.

Kartogenin-Loaded Exosomes Derived From Bone Marrow Mesenchymal Stem Cells Enhance Chondrogenesis and Expedite Tendon Enthesis Healing in a Rat Model of Rotator Cuff Injury

Background: The insufficient regeneration of fibrocartilage at the tendon enthesis is the primary cause of retearing after surgical reattachment of the rotator cuff. Exosomes derived from bone marrow–derived mesenchymal stem cells (BMSC-Exos) and kartogenin (KGN) have been demonstrated to induce fibrocartilage formation. Loading drugs into exosomes may lead to a synergistic effect, significantly enhancing the inherent activity of both components. However, further investigation is necessary to determine whether loading KGN into BMSC-Exos could yield superior efficacy in promoting tendon enthesis healing. Purpose: To study the effect and mechanism of KGN-loaded BMSC-Exos (Kl-BMSC-Exos) on tendon enthesis repair and biomechanical properties in a rat rotator cuff injury (RCI) model. Study Design: Controlled laboratory study. Methods: The characteristics and in vivo retention of exosomes were demonstrated using nanoflow cytometry, transmission electron microscopy, and in vivo imaging of a small animal. The differentiation markers of BMSCs were assessed through quantitative polymerase chain reaction and immunofluorescence assays. Unilateral supraspinatus tenotomy and repair were performed in rats to establish the RCI model. Gelatin sponges were utilized to contain and deliver exosomes. In total, 44 rats were randomly assigned to 4 groups: sham, RCI, BMSC-Exos, and Kl-BMSC-Exos. Tendon enthesis regeneration and biomechanical properties were evaluated 8 weeks after surgery. RNA sequencing of BMSCs was performed to elucidate the underlying mechanism through which Kl-BMSC-Exos enhance tendon enthesis healing. Results: No discernible disparities in fundamental characteristics were evident between BMSC-Exos and Kl-BMSC-Exos. Incorporating exosomes into a gelatin sponge extended the in vivo retention time from 7 to 14 days. Kl-BMSC-Exos were more effective in inducing differentiation markers of BMSCs, improving fibrocartilage regeneration, organizing collagen fiber arrangement, and enhancing the biomechanical properties of tendon enthesis. Furthermore, transcriptomics suggested that Mospd1 was involved in Kl-BMSC-Exos–mediated tendon enthesis healing by enhancing fibrocartilage regeneration. Conclusion: The incorporation of exosomes into a gelatin sponge significantly enhances their in vivo retention time. Kl-BMSC-Exos can expedite the healing of RCI by enhancing chondrogenesis and fibrocartilage regeneration, providing more organized collagen fiber arrangement and superior biomechanical properties of the rotator cuff enthesis. The promotion of rotator cuff enthesis regeneration may contribute to enhancing the chondrogenic potential in BMSCs through Kl-BMSC-Exos–mediated upregulation of Mospd1. Clinical Relevance: As a cell-free therapeutic approach, Kl-BMSC-Exos displayed a better therapeutic effect on tendon enthesis healing than BMSC-Exos did, and these can be used as a biologic augmentation to enhance the healing of rotator cuff enthesis.

Quercetin Alleviates Scleral Remodeling Through Inhibiting the PERK-EIF2α Axis in Experiment Myopia.

This study aims to investigate the effect of quercetin (QUE) on scleral remodeling by inhibiting the PERK-EIF2α signaling pathway and to evaluate its potential role in slowing myopia. Lens-induced myopia (LIM) guinea pigs were obtained and treated with QUE. After 4 and 6 weeks of treatments, ocular biological measurements were conducted. Hematoxylin and eosin (H&E) staining was used to observe the changes in scleral morphology and thickness, and Masson staining was used to examine scleral collagen fiber arrangement. Quantitative PCR (qPCR) and Western bolt were utilized to detect the mRNA and protein expression of PERK, EIF2α, MMP-2, TIMP-2, and collagen I in the scleral tissues. Calcium ion flow in each group was measured using noninvasive micro-test technology, and reactive oxygen species levels were detected by flow cytometry. Compared with the LIM group, the ocular measurements showed that the refractive errors and axial length of the eyes were significantly reduced in the LIM+QUE group (P < 0.01). H&E and Masson staining showed that sclera in the LIM+QUE group was thickened, collagen was dense, and the fiber gap was reduced. In the LIM+QUE group, the expression levels of PERK, EIF2α, and MMP-2 were decreased, whereas the expression levels of TIMP-2 and collagen I were increased. Calcium release and reactive oxygen species (ROS) in the LIM+QUE group were decreased. Quercetin ameliorates scleral remodeling in myopic guinea pigs by inhibiting the PERK-EIF2α signaling pathway, thereby alleviating the progression of myopia. These findings provide new experimental evidence for the potential application of quercetin in myopia prevention and treatment.

Allograft to bone-tunnel integration in a canine anterior cruciate ligament reconstruction model: a comparison study of allograft preparation methods

The procedure of anterior cruciate ligament (ACL) allograft preparation can be divided into fresh-frozen method (FF-allograft) or freeze-dried method (FD-allograft). This study aims to biomechanically and histologically compare the graft to bone tunnel integration between the two allografts. In-vitro results indicated that FF-allograft and FD-allograft showed excellent biocompatibility and biomechanics, while FD-allograft showed a denser collagen fiber arrangement than FF-allograft and autograft. Then, in-vivo preformation of the FF-allograft, FD-allograft, and autograft on bone tunnel integration was evaluated via a canine ACL reconstruction model. In-vivo results indicated that no signs of infection or osteoarthritis were shown in the femur-graft-tibia complexes, but more vascularity and synovitis formed around the implanted FF-allograft. Micro-computed tomography showed that peri-graft bone in the FF-allograft group was significantly increased and remodeled compared with the FD-allograft group; Histologically, the FF-allograft group exhibited similar graft-bone tunnel healing to the FD-allograft group. Tartrate-resistant acid phosphatase (TRAP) staining showed significantly more osteoclasts presented in the FD-allograft group compared to the FF-allograft group. Meanwhile, a significantly higher failure load was shown in the FF-allograft group when compared with the FD-allograft group (P < 0.05). In conclusion, the FF-allograft integrated more firmly into the bone tunnel than the FD-allograft when used in ACL reconstruction.

A Core of Keratocan-Negative Cells Survives in Old Corneal Scars

Corneal scars originate from keratocyte-derived fibroblasts and myofibroblasts that are ultimately cleared through apoptosis or revert to keratocytes. A mouse model expressing the keratocyte lineage-specific reporter KeraRT/tetO-Cre/mTmG (I-KeramTmG) was interrogated to elucidate cell phenotype dynamics during scar maturation. This model expresses tdTomato (red) in all keratocan-negative cells, while enhanced green fluorescent protein (green) is expressed only by keratocytes. A 1-mm full-thickness keratotomy was generated in adult I-KeramTmG mice. The presence of keratocytes was determined at 3, 6, and 10 months after injury. At 3 and 6 months, few green cells were visualized at the scar borders, while few or no green cells were seen in the central (core) scar. At 10 months, green cells were seen throughout the scar, but most cells were red. Proliferation of stromal cells after injury was studied by 5-ethynyl-2'-deoxyuridine labeling and Ki-67 staining; both assays showed proliferation only during the first 2 weeks after injury. Second harmonic generation microscopy showed thickened and irregularly arranged collagen fibers in scars, suggesting that neither extracellular matrix organization nor cell phenotype had changed significantly at 10 months after injury. Findings from invivo experiments suggest that in old corneal scars, a nonkeratocyte phenotype persists in an abnormal matrix with unique characteristics that probably prevent the regression of fibroblasts and myofibroblasts to keratocytes or invasion of surrounding keratocytes.

Quantitative structural organization of the sclera in chicks after deprivation myopia measured with second harmonic generation microscopy.

Visual deprivation causes enhanced eye growth and the development of myopia, which is associated with a change in the arrangement of collagen fibers within the sclera. A second harmonic generation (SHG) microscope has been used to image the collagen fibers of unstained scleral punches from the posterior part of chicken eyes. We aimed to analyze the fibrous scleral tissue and quantify the changes in collagen organization in relation to the extent of induced deprivation myopia. The scleral architecture was assessed with the Radon transform (RT) through the parameter called structural dispersion (SD) that provides an objective tool to quantify the level of organization of the collagen network. We found that final refraction and axial length changes were linearly correlated. However, no significant differences in scleral thickness were found for different amounts of induced myopia. In contrast, a significant correlation between SD and refraction was demonstrated, ranging from a non-organized (in the control sclerae) to a quasi-aligned distribution (with a dominant direction of the fibers, in the sclera of myopic chicks). These findings demonstrate a remodeling process of the scleral collagen associated with myopia progression that can be measured accurately combining SHG imaging microscopy and RT algorithms.

Validation of Fixed Ultrasonography for Achilles Tendon Assessment: A Reliability Study

Background: It is important to highlight the advantages of ultrasound in assessing muscular and tendinous behavior due to its non-invasive nature and capacity for dynamic studies. However, evaluating tendons via ultrasound can be challenging given the complexity of anisotropic phenomena related to collagen fiber arrangement. This study aims to validate the reliability of fixed ultrasound compared to manual acquisition in measuring Achilles tendon thickness. Method: Twenty participants, six men and fourteen women, were recruited. Ultrasound was used to measure the Achilles tendon’s thickness at two specific points (4 and 6 cm from the calcaneal insertion of the Achilles tendon). The measurements were conducted by two examiners, one with previous experience and another without. Results: The measurements at 6 cm from the calcaneal insertion showed α = 0.996, α = 0.998 for measurements at 4 cm using manual acquisition, and α = 0.997 for measurements with fixed ultrasound at rest. For the weight-bearing and ankle dorsiflexion measurements, the reliability was excellent (α = 0.999 and α = 1.000). Conclusions: The findings demonstrated excellent reliability in the ultrasound measurements of the Achilles tendon’s thickness, even when performed by different evaluators and under load-bearing conditions. This study suggests the clinical utility of assessing anatomical structures under load, enhancing ultrasound’s applicability beyond the examination table. It is concluded that fixed ultrasound acquisition exhibits excellent reliability in measuring the Achilles tendon’s thickness, offering potential benefits for precise diagnosis of pathologies, planning surgical interventions, and reducing possible errors related to operator variability.

Soluble allogenic telocollagen as a direct protein therapeutic: Results of serial injections in a rodent rotator cuff tear model

HypothesisDelivery of soluble allogeneic type I telocollagen (allo-telocollagen) will accelerate and improve the healing of damaged tendons. Our hypothesis draws from known mechanochemical properties of type I collagen that direct its incorporation into damaged connective tissue. We further suggest that allo-telocollagen will raise a minimal immunogenic reaction due to homology within species. MethodsSeventy-eight shoulders (39 Sprague-Dawley rats) had their supraspinatus tendon surgically detached from its footprint on the humerus and repaired (72 shoulders) or left uninjured (6 shoulders). The repaired tissue was treated with an injection of 100 μl of saline, 10 mg/ml allogeneic atelocollagen (allo-atelocollagen), or 10 mg/ml allo-telocollagen at 0-, 1-, and 2-weeks post-surgery. At 30- and 60-days post-surgery, the tendons were assessed by mechanical testing (failure load, failure stress, stiffness, and relaxation) and by semiquantitative histological scoring. ResultsAt 30-days post-surgery, the mechanical and histological outcomes were not statistically different. However, at day 60, allo-telocollagen improved the failure strength of the supraspinatus (29.9 ± 4.7 N) relative to saline (20.0 ± 3.5 N; P value <= 0.001) or allo-atelocollagen (23.2 ± 1.5 N; P value = 0.025) treated tendons, and it approached that of uninjured controls (36.9 ± 5.0 N; P value = 0.021). Allo-telocollagen improved the failure stress of the supraspinatus (34.1 ± 9.3 MPa) relative to the saline treated tendons (21.4 ± 6.0 MPa; P value = 0.031; 160% improvement) and was no different than uninjured controls (33.4 ± 9.9 MPa; P value = 0.999) or allo-atelocollagen (32.3 ± 7.4 MPa; P value = 0.977). The stiffness of uninjured controls was far greater than any of injured/treated tendons (>200% stiffer). Histological scoring showed that the allo-telocollagen treated tendons produced better collagen fiber arrangement (1.55 ± 0.17) than saline (2.50 ± 0.29; P value = 0.001) or allo-atelocollagen (2.23 ± 0.28; P value = 0.042) treated tendons and that it did not increase markers of immunogenesis (1.10 ± 0.42) relative to either saline (1.44 ± 0.20; P value = 0.369) or allo-atelocollagen (0.68 ± 0.41; P value = 0.1058). ConclusionsWhile all three treatments produced similar results at 30 days, by 60 days, soluble allo-telocollagen clearly separated from the other interventions, yielding better mechanical and histological outcomes in a torn/repaired rotator cuff rat model. Allo-telocollagen treated tendons also approached the failure strength and matched the failure stresses of uninjured control tendons. The data suggest a new use for allo-telocollagen as a deliverable direct protein mechanotherapeutic that can improve both healing quality and speed.

The Effect of Exosomes from Adipose derived Mesenchymal Stem Cells in Photoaged Rat Skin versus Topically Applied Growth Factors. A Histological Study

Abstract Introduction Skin photoaging is characterized by coarse wrinkles, loss of elasticity and dryness. The worst scenario of ultraviolet (UV) ray exposure can reach up to skin malignant changes. The topical application of growth factors has proven to be effective in ameliorating the photoaging. Among the paracrine products of stem cells are exosomes which also play an important role. Aim of the Work To investigate the effect of exosomes derived from adipose mesenchymal stem cells on the histological structure of photoaged rat thin skin versus topically applied growth factors. Materials and Methods Forty-five young adult male albino rats aged between 6 - 8 weeks were used in this study. Animals were subjected to gentle shaving of the hair over the center of their backs and they were randomly classified into four groups. Control group (I), 20 rats were divided into four subgroups (five animals each). Subgroup Ia, animals were left without treatment, Subgroup Ib, animals were injected subcutaneously with 1 ml of Phosphate Buffer Saline (PBS) at the shaved areas. Subgroup Ic, shaved areas were topically treated with growth factors gel after micro-needling procedure (Three topical treatments were repeated each one performed every 10 days). Subgroup Id, rats were subcutaneously injected with one dose of purified exosome concentrate containing 300 µg of total protein. Group (II)– (photoaged untreated group), five rats, each was exposed to UVA + UVB irradiation for two weeks (five days per week) on the shaved areas using UV lamp. Group (III)- (photoaged exosome-treated group), five rats, each was exposed to UVA + UVB irradiation similar to group II. The animals were injected with one dose of purified exosome concentrate containing 300 µg of total protein subcutaneously at the shaved areas. Group (IV)- (Photoaged and topically treated with growth factors), five rats each was exposed to UVA + UVB irradiation similar to group II. Then, the animals were treated topically as in subgroup C. The back skin was dissected and processed to prepare sections for light microscopic examination with H&amp;E, Masson’s trichrome, Orcein and PAS stains. Immunostaining for KI67and CD31 were done. Morphometric and statistical analysis were performed. Results Group II revealed significant decrease in the epidermal thickness, hair follicles were scanty and disorganized. Orcein sections showed short fragmented elastic fibers and irregular arrangement of collagen fibers with Masson’s trichrome stain. KI67 positive cells was higher than other groups and CD31 was the opposite. Groups III and IV showed marked decrease in the previously mentioned microscopic changes denoting amelioration of the recorded photoaged epidermal and dermal changes. Conclusion The current study concluded that both exosomes and topically applied Growth factors over three divided doses accompanied with micro-needling have resulted in a significant amelioration of the altered microscopic photoaging changes observed after exposure of the rat thin skin to the UV rays. Furthermore, the improvement was more obvious with the topically applied growth factors compared to one dose of exosomes concentrate. Further studies are recommended using repeated application of exosomes and also in combination with micro- needling to stabilize the best protocol for treatment.

Revealing chemistry-structure-function relationships in shark vertebrae across length scales

Shark cartilage presents a complex material composed of collagen, proteoglycans, and bioapatite. In the present study, we explored the link between microstructure, chemical composition, and biomechanical function of shark vertebral cartilage using Polarized Light Microscopy (PLM), Atomic Force Microscopy (AFM), Confocal Raman Microspectroscopy, and Nanoindentation. Our investigation focused on vertebrae from Blacktip and Shortfin Mako sharks. As typical representatives of the orders Carcharhiniformes and Lamniformes, these species differ in preferred habitat, ecological role, and swimming style. We observed structural variations in mineral organization and collagen fiber arrangement using PLM and AFM. In both sharks, the highly calcified corpus calcarea shows a ridged morphology, while a chain-like network is present in the less mineralized intermedialia. Raman spectromicroscopy demonstrates a relative increase of glucosaminocycans (GAGs) with respect to collagen and a decrease in mineral-rich zones, underlining the role of GAGs in modulating bioapatite mineralization. Region-specific testing confirmed that intravertebral variations in mineral content and arrangement result in distinct nanomechanical properties. Local Young's moduli from mineralized regions exceeded bulk values by a factor of 10. Overall, this work provides profound insights into a flexible yet strong biocomposite, which is crucial for the extraordinary speed of cartilaginous fish in the worlds’ oceans. Statement of significanceShark cartilage is a morphologically complex material composed of collagen, sulfated proteoglycans, and calcium phosphate minerals. This study explores the link between microstructure, chemical composition, and biological mechanical function of shark vertebral cartilage at the micro- and nanometer scale in typical Carcharhiniform and Lamniform shark species, which represent different vertebral mineralization morphologies, swimming styles and speeds. By studying the intricacies of shark vertebrae, we hope to lay the foundation for biomimetic composite materials that harness lamellar reinforcement and tailored stiffness gradients, capable of dynamic and localized adjustments during movement.

Collagen fiber arrangement in the normal bladder lamina propria and their potential impact on the pathological substaging of bladder cancer stage T1.

The lamina propria (LP) of the urinary bladder lies between the urothelial mucosa and the muscularis propria. This complex stratum is composed of extracellular matrix, several cell types, and collagen types I and III fibers. LP invasion by urothelial carcinoma (progression from stage Ta to T1) is a determinant of bladder cancer advancement. We attempted to characterize collagen fiber arrangement in the LP. This could enrich our understanding of this important layer and potentially provide clues for sub-staging of the T1 bladder cancer. A total of 24 Masson trichrome-stained images of normal bladder, including 12,530 collagen fibers were quantitatively analyzed using the Dragonfly software. The LP was divided according to fiber orientation into superficial LP (SLP, 15% of the thickness) and the deep LP (DLP, 85% of the thickness). Collagen fiber geometry analysis demonstrated that the SLP fibers are more parallel to the urothelium with an average angle of 260±230 compared to 400±260 in the DLP (p=3.4X10-144), more packed (average distance to the closest fiber of 0.61±0.67 compared to 0.66±0.77, p=0.0001), and their aspect ratio is considerably longer (average of 1.93±0.12 compared to 0.20±0.11, p=2.84x10-8). No difference was found in fiber perimeter or Feret diameter. Thus, we conclude that bladder collagen fibers are arranged in two distinct layers: a dense-ordered SLP and a loose disorder DLP. This indicates that the physical barrier to cancer cell invasion probably lies in the SLP, immediately underneath the urothelium. Once this barrier is breached, the looser and disorganized DLP poses no remarkable obstacle. Thus, we believe that histology-based subdivisions of stage T1 are expected to fail in providing clinically meaningful prognostic information.

Comparative Analysis of Commercially Available Extracellular Matrix Soft Tissue Bioscaffolds.

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

Impact of cathepsin K-induced proteoglycans degradation on dentin collagen

ObjectivesThis study aimed to investigate the effects of cathepsin K (catK) on proteoglycans (PGs) and the subsequent impacts on dentin collagen degradation. Materials and MethodsDemineralized dentin samples were prepared and divided into the following groups: deionized water (DW), 0.1 U/mL chondroitinase ABC (C-ABC), and 1 μM odanacatib (ODN). Then, they were immersed for 48 h and then incubated in 1 mL of PBS (pH=5.5) at 37 °C for 5 d. Glycosaminoglycan (GAG) were examined to explore the degradation of PGs by catK. To determine the effect of catK-mediated PGs on dentin collagen degradation, hydroxyproline (HYP) assays, assessment of the degree of dentin crosslinking, and scanning electron microscopy (SEM) were assessed. Statistical analysis was conducted using one-way ANOVA followed by Tukey’s tests or Welch's ANOVA followed by Dunnett's tests at a significance level of 0.05. ResultsThe production of GAG was significantly lower in the ODN group than in the DW group (P < 0.05), revealing that PG degradation was reduced in dentin after ODN treatment. Additionally, ODN treatment minimized the gaps in collagen fibers, improved fiber arrangement, and significantly increased the degree of collagen crosslinking, subsequently reducing the total amount of collagen fiber degradation in the dentin (P < 0.05). ConclusionsCatK-mediated degradation of PGs negatively impacted the stability of collagen fibers, promoted gaps, led to a less organized arrangement of dentin collagen fibers, ultimately increasing collagen degradation.

Comparison of Normal Human Skin and Hypertrophic Scar Tissue Samples of Different Ages, Locations, and Stages of Maturity.

Scars disrupt the normal structure and function of the skin. The primary goal of plastic surgery is to prevent and reduce scarring. Therefore, we aimed to establish a comparison scheme between normal skin (NS) tissues of different ages and locations; hypertrophic scars (HTS) of different ages, locations, and maturities; and NS and HTS tissues to provide evidence on scar severity for improving treatment evaluation. Various methods including histology, immunohistochemistry, and immunofluorescence were employed to compare the general appearance, macrophage infiltration, fibroblast activity, degree of angiogenesis, and collagen fiber type and arrangement in human-sourced NS and HTS tissues of different ages, locations, and maturities in seven patients (three with NS and four with HTS) from the Department of Burn and Plastic Surgery of the Shandong Provincial Hospital from January 2019 to December 2020. The thicknesses of the epidermis and dermis of NS tissues varied with age and location. The epidermis of the upper arms, face, and upper eyelids of NS tissues sequentially thickened, whereas the dermis was sequentially thinner. Several glandular structures were identified in the upper eyelids but rarely in the face and upper arms. Histological changes in HTS tissue of different ages, locations, and maturity occur as scar formation time is prolonged, accompanied by increased CD86 levels and fibrosis. As the scar matured, connexin and VEGFR2 expression decreased, indicating reduced inflammation, fibroblast activity, and angiogenesis. The comparison between NS and HTS tissue also revealed significant differences; the positive expression of VEGFR2 and total collagen in HTS tissue was higher than that in NS tissue. We discovered significant differences among NS, HTS, and NS and HTS tissues of different ages, locations, and maturities. Further, this study may provide a basis for clarifying the treatment effect of different methods for HTS compared with those for NS, efficiently individualizing patients' treatment plans and ultimately shortening the scar treatment process.

Silk fibroin microgrooved zirconia surfaces improve connective tissue sealing through mediating glycolysis of fibroblasts

The use of zirconia has significantly enhanced the aesthetic outcomes of implant restorations. However, peri-implantitis remains a challenge to long-term functionality of implants. Unlike the perpendicularly arranged collagen fibers in periodontal tissue, those in peri-implant tissue lie parallel to the abutment surface and contain fewer fibroblasts, making them more prone to inflammation. Studies have shown that microgroove structures on implant abutments could improve surrounding soft tissue structure. However, creating precise microgrooves on zirconia without compromising its mechanical integrity is technically challenging. In this study, we applied inkjet printing, an additive manufacturing technique, to create stable silk fibroin microgroove (SFMG) coatings of various dimensions on zirconia substrates. SFMG significantly improved the hydrophilicity of zirconia and showed good physical and chemical stability. The SFMG with 90 μm interval and 10 μm depth was optimal in promoting the proliferation, alignment, and extracellular matrix production of human gingival fibroblasts (HGFs). Moreover, the in vitro results revealed that SFMG stimulated key glycolytic enzyme gene expression in HGFs via the PI3K-AKT-mTOR pathway. Additionally, the in vivo results of histological staining of peri-abutments soft tissue showed that SFMG promoted the vertical alignment of collagen fibers relative to the abutment surface, improving connective tissue sealing around the zirconia abutment. Our results indicated that SFMG on zirconia can enhance HGF proliferation, migration and collagen synthesis by regulating glycolysis though PI3K-AKT-mTor pathway, thereby improving connective tissue sealing.

Oriented cellulose hydrogel: Directed tissue regeneration for reducing corneal leukoplakia and managing fungal corneal ulcers

Fungal corneal ulcer is one of the leading causes of corneal blindness in developing countries. Corneal scars such as leukoplakia are formed due to inflammation, oxidative stress and non-directed repair, which seriously affect the patients' subsequent visual and life quality. In this study, drawing inspiration from the oriented structure of collagen fibers within the corneal stroma, we first proposed the directional arrangement of CuTA-CMHT hydrogel system at micro and macro scales based on the 3D printing extrusion method combined with secondary patterning. It played an antifungal role and induced oriented repair in therapy of fungal corneal ulcer. The results showed that it effectively inhibited Candida albicans, Aspergillus Niger, Fusarium sapropelum, which mainly affects TNF, NF-kappa B, and HIF-1 signaling pathways, achieving effective antifungal functions. More importantly, the fibroblasts interacted with extracellular matrix (ECM) of corneal stroma through formation of focal adhesions, promoted the proliferation and directional migration of cells in vitro, induced the directional alignment of collagen fibers and corneal stromal orthogonally oriented repair in vivo. This process is mainly associated with MYLK, MYL9, and ITGA3 molecules. Furthermore, the downregulation the growth factors TGF-β and PDGF-β inhibits myofibroblast development and reduces scar-type ECM production, thereby reducing corneal leukoplakia. It also activates the PI3K-AKT signaling pathway, promoting corneal healing. In conclusion, the oriented CuTA-CMHT hydrogel system mimics the orthogonal arrangement of collagen fibers, inhibits inflammation, eliminates reactive oxygen species, and reduces corneal leukoplakia, which is of great significance in the treatment of fungal corneal ulcer and is expected to write a new chapter in corneal tissue engineering.

Isorhamnetin inhibits hypertrophic scar formation through TGF-β1/Smad and TGF-β1/CREB3L1 signaling pathways

BackgroundHypertrophic scar (HS) is a common fibrotic skin disease that occurs secondary to burns or injuries. The activation of the TGF-β signaling pathway contributes immensely to HS formation. Isorhamnetin (ISO) is a type of flavonoid compound that exerts an antifibrotic effect via TGF-β signaling suppression. However, whether ISO can inhibit HS formation via TGF-β signaling is yet to be elucidated. This study aimed to examine the influence of ISO on HS pathogenesis and TGF-β signaling, especially the downstream molecules and networks of TGF-β signaling that facilitate HS formation. MethodsHypertrophic scar fibroblasts (HSFBs) were isolated from human HS tissues. The in vitro proliferation, migration, contractile ability, cell cycle, and apoptosis of HSFBs after ISO treatment were determined using cell viability assay, EdU staining, wound healing assay, collagen gel contraction assay, and flow cytometry. The expressions of genes and proteins involved in TGF-β signaling and its downstream molecules in ISO-treated HSFBs were determined using quantitative PCR (qPCR), immunofluorescence, and western blotting. In vivo, a rabbit HS model was established, and the effects of ISO on rabbit HS formation were investigated using histological analysis, immunohistochemical staining, and qPCR. ResultsIn vitro studies indicated that ISO treatment suppressed the proliferation, migration, and contractile ability of HSFBs; attenuated the expressions of COL Ⅰ, COL Ⅲ, and α-SMA; and inhibited TGF-β1 signaling-induced activation of HSFBs by decreasing the levels of phosphorylated Smad2/3 and cleaved CREB3L1 in a dose-dependent manner. Furthermore, ISO augmented apoptosis and G2 phase cell cycle arrest of HSFBs by upregulating the expressions of the proapoptotic proteins Bax and cleaved caspase-3 and downregulating the expression of the antiapoptotic protein Bcl-2. In vivo studies revealed that ISO ameliorated HS formation in the rabbit ear by lowering the scar elevation index, attenuating the collagen density, facilitating the regular arrangement of collagen fibers, and downregulating the expressions of TGF-β1, CREB3L1, COL Ⅰ, COL Ⅲ, and α-SMA. ConclusionsISO suppressed HS pathogenesis by dampening TGF-β1/Smad and TGF-β1/CREB3L1 signaling pathways, which suggests that it may serve as a candidate inhibitor of TGF-β1 signaling and a promising anti-HS drug with a high therapeutic potential.

Harnessing oriented arrangement of collagen fibers by 3D printing for enhancing mechanical and osteogenic properties of mineralized collagen scaffolds

As the structural basis of connective and load-bearing tissues, collagen fibers with orientation play an important role in the mechanical properties and physiological and biochemical functions of the tissues, but viable methods for preparing scaffolds with highly oriented collagenous structure still need to be further studied. In this study, pure collagen was used as printing ink to 3D printing. Harnessing oriented collagen fiber structure by 3D printing for promoting mechanical and osteogenic properties of scaffolds. The scaffolds with different printed angles and thicknesses were prepared to fit the bone defect site and realize personalized customization. The orientation assembly of collagen fibers was promoted by shear force action of 3D printing, the regular arrangement of collagen fibers and stabilization of fiber structure were promoted by pH adjustment and glutaraldehyde cross-linking, and the collagen fibers were mineralized by cyclic mineralization method. The microscopic morphology of fiber arrangement in the scaffolds were investigated by scanning electron microscopy. Results demonstrated that collagen fibers were changed from non-oriented to oriented after 3D printing. And the tensile modulus of the scaffolds with oriented collagen fibers was nine times higher than that of the scaffolds with non-oriented fibers. Moreover, the effects of oriented collagen fibers on the proliferation, differentiation and mineralization of MC3T3-E1 cells were studied by CCK-8 assay, live/dead cell staining, alkaline phosphatase activity test, and Alizarin red staining. The results indicated that cell proliferation, differentiation and mineralization were significantly promoted by oriented collagen fibers, and the cells proliferated directionally in the direction of the fibers. Taken together, mineralized collagen fiber scaffolds with oriented collagen fibers have great potential in bone tissue engineering applications.