Gene Expression Of Collagen Type Research Articles

Collagen peptides affect collagen synthesis and the expression of collagen, elastin, and versican genes in cultured human dermal fibroblasts.

Collagen is one of the major proteins of the skin and it is particularly important for its strength and resilience. Skin aging is a natural process that is characterized by the decrease and fragmentation of collagen in the dermis. Oral supplementation with collagen peptides has been clinically shown to have a positive effect on the skin condition. However, the mechanisms of aging-related changes synthesized by cells exposed to collagen are currently not well understood. Therefore, in this in vitro study, the mechanisms associated with collagen, elastin, and versican in human dermal fibroblasts were investigated after exposure to collagen peptides. The effects of different concentrations of collagen peptides on cell viability and metabolism were analyzed. For gene expression analysis, human dermal fibroblasts were treated with collagen peptides. This was then followed by RNA extraction and DNA synthesis. Gene expressions of collagen type 1 (COL1A1), elastin (ELN), and versican (VCAN) were quantified by quantitative reverse transcription polymerase chain reaction (RT-qPCR). In addition, collagen levels were analyzed by confocal scanning laser microscopy using immunostaining. Collagen peptides tested in the study increased the expression of the relevant COL1A1, ELN, and VCAN genes in human dermal fibroblasts (p < 0.005). Furthermore, confocal microscopy showed increased collagen expression in the dermal fibroblast culture after treatment with the collagen peptides (p < 0.005). These data provide cell-based evidence for the beneficial effects of exposure to collagen peptides on the skin's collagen content and on the molecules that provide firmness and elasticity. This may support the hypothesis that collagen peptides are important for maintaining extracellular matrix (ECM) structure and skin regeneration.

ARTICULAR CHONDROCYTES FROM DIABETIC AND NON-DIABETIC RATS EXPOSED TO NORMO- OR HYPERGLYCAEMIA RESPOND DIFFERENTIALLY TO THE COMPLEMENT SPLIT FRAGMENT ANAPHYLATOXIN C5A AND TNFA

Osteoarthritis (OA) and diabetis mellitus type 2 (DMT2) are pathogenetically linked. Complement dysregulation contributes to OA and could be involved in DMT2. The inflammatory anaphylatoxin C5a is released during complement activation. This study aims to understand the specific responses of chondrocytes isolated from diabetic and non-diabetic rats exposed to C5a and/or the proinflammatory cytokine TNFα in vitro dependent on the glucose supply. Articular chondrocytes of adult Zucker Diabetic Fatty (ZDF) rats (homozygous: fa/fa, diabetic, heterozygous: fa/+, lean controls) were exposed to 10 ng/mL TNFα and 25 ng/mL C5a alone or in combination, both, under normo- (NG, 1 g/L glucose) and hyperglycemic (HG, 4.5 g/L glucose) conditions (4 or 24 h). Chondrocyte survival, metabolic activity and gene expression of collagen type 2, suppressors of cytokine signaling (SOCS)1, −3 and anti-oxidative hemoxygenase-1 (HMOX1) were assessed. The complement regulatory protein CD46 and cell nuclei sizes were analyzed. Chondrocyte vitality remained unaffected by the treatment. Metabolic activity was impaired in chondrocytes of non-diabetic rats under HG conditions. Collagen type 2 transcription was suppressed by TNFα under HG condition in chondrocytes from nondiabetic donors and under both conditions in those of DMT2 rats (24 h)Except for DMT2 chondrocytes under HG (4 h), HMOX1 was generally induced by TNFα +/- C5a (NG, HG). C5a elevated HMOX1 only in chondrocytes of controls. The SOCS1/3 genes were increased by TNFα (NG, diabetic, non diabetic, 4 and 24 h). This could also be observed in chondrocytes of diabetic, but not of lean rats (24 h, HG). At 4 h, C5a induced SOCS1 only in non diabetic chondrocytes (NG, HG). Cytoprotective CD46 protein was suppressed by TNFα under NG condition. Nuclear volumes of chondrocyte were lower in chondrocytes from DMT2 rats compared to those from controls. The differential response suggests that chondrocytes are irreversibly compromised by DMT2.Achnowledgement: The authors are grateful for the support by the “Stiftung Edoprothetik (S 04/21)”

Comparison of Different Methods of Semiquantitative Assessment and Subjective Scores for Retropatellar Articular Cartilage Evaluation in Advancing Osteoarthritis.

cartilage layer thickness, composition of the extracellular matrix (ECM), geometry and configuration of retropatellar cartilage partially differ significantly from those found at other locations and are essential for patellofemoral biomechanics. 119 serial medial and lateral patella facet samples of patients undergoing implantation of a total knee endoprosthesis of areas showing mild (Group A, macroscopically ICRS Grade 1b) respectively advanced (Group B, macroscopically ICRS Grade 3a/3b) (38 each) osteoarthritis according to the histological-histochemical grading system (HHGS) were compared with 22 healthy biopsies by means of immunohistochemistry and histology investigations. We quantified our results on the gene expression of collagen type I and II and aggrecan with real-time (RT)-PCR rsp using colourimetry for proteoglycan content. The digitized images of histology and immunohistochemistry staining with Safranin O, Alcian blue, PAS, Masson Goldner, immunostaining, e.g. for collagen I and II were also analyzed with Photoshop software. The most significant decrease in staining intensity was revealed for Safranin-O staining in Group B at the lateral patellar facet, and the most relevant increase was for Col I staining at the same site. The lateral patella site in Group B also showed the highest increase in the ratio of expression indices for the genes Col1A1 and the reference gene following the equation 2-ΔCt with a quotient of 29.6. 1. Comparisons of our retropatellar cartilage analysis with femoral and tibial studies utilizing similar techniques show significant differences. 2. Cartilage layer thickness, ECM composition, geometry and configuration are essential for patellofemoral biomechanics. 3. Consequently, there is a need for diversified approaches towards retropatellar surface during TKA as well as for advanced cartilage restoration techniques.

In Vitro Investigation of Gelatin/Polycaprolactone Nanofibers in Modulating Human Gingival Mesenchymal Stromal Cells.

The aesthetic constancy and functional stability of periodontium largely depend on the presence of healthy mucogingival tissue. Soft tissue management is crucial to the success of periodontal surgery. Recently, synthetic substitute materials have been proposed to be used for soft tissue augmentation, but the tissue compatibility of these materials needs to be further investigated. This study aims to assess the in vitro responses of human gingival mesenchymal stromal cells (hG-MSCs) cultured on a Gelatin/Polycaprolactone prototype (GPP) and volume-stable collagen matrix (VSCM). hG-MSCs were cultured onto the GPP, VSCM, or plastic for 3, 7, and 14 days. The proliferation and/or viability were measured by cell counting kit-8 assay and resazurin-based toxicity assay. Cell morphology and adhesion were evaluated by microscopy. The gene expression of collagen type I, alpha1 (COL1A1), α-smooth muscle actin (α-SMA), fibroblast growth factor (FGF-2), vascular endothelial growth factor A (VEGF-A), transforming growth factor beta-1 (TGF-β1), focal adhesion kinase (FAK), integrin beta-1 (ITG-β1), and interleukin 8 (IL-8) was investigated by RT-qPCR. The levels of VEGF-A, TGF-β1, and IL-8 proteins in conditioned media were tested by ELISA. GPP improved both cell proliferation and viability compared to VSCM. The cells grown on GPP exhibited a distinct morphology and attachment performance. COL1A1, α-SMA, VEGF-A, FGF-2, and FAK were positively modulated in hG-MSCs on GPP at different investigation times. GPP increased the gene expression of TGF-β1 but had no effect on protein production. The level of ITG-β1 had no significant changes in cells seeded on GPP at 7 days. At 3 days, notable differences in VEGF-A, TGF-β1, and α-SMA expression levels were observed between cells seeded on GPP and those on VSCM. Meanwhile, GPP showed higher COL1A1 expression compared to VSCM after 14 days, whereas VSCM demonstrated a more significant upregulation in the production of IL-8. Taken together, our data suggest that GPP electrospun nanofibers have great potential as substitutes for soft tissue regeneration in successful periodontal surgery.

Inflammatory liver tissue formation using oxygen permeable membrane based culture platform

During chronic liver injury, inflammation leads to liver fibrosis, particularly due to the activation of hepatic stellate cells (HSCs). The involvement of inflammatory cytokines in HSC activation and the interplay among different liver cells are elaborated. To examine their interactions invitro, many cultured liver tissue models are performed in organoid or spheroid culture with random 3D structure. Herein, we demonstrated the hierarchical coculture of primary rat hepatocytes with non-parenchymal cells such as the human-derived HSC line (LX-2) and liver sinusoidal endothelial cell line (TMNK-1). The cocultured tissue had high usability with simple operation of separating solid and liquid phases with improved liver functions such as albumin production and hepatic cytochrome P450 3A4 activity. We also studied the effects of stimulation by both oxygen tension and the key pro-fibrogenic cytokine, transforming growth factor beta (TGF-β), on HSC activation. Gene expression of collagen type I and alpha-smooth muscle actin were enhanced in the hierarchical coculture under lower oxygen tension and TGF-β1 stimulation. Therefore, this hierarchical invitro cocultured liver tissue could provide a useful platform as a disease model for elucidating the interactions of various liver cell types and biochemical signals in future liver fibrogenesis studies.

Considering the Value of 3D Cultures for Enhancing the Understanding of Adhesion, Proliferation, and Osteogenesis on Titanium Dental Implants.

Individuals with pathologic conditions and restorative deficiencies might benefit from a combinatorial approach encompassing stem cells and dental implants; however, due to the various surface textures and coatings, the influence of titanium dental implants on cells exhibits extensive, wide variations. Three-dimensional (3D) cultures of stem cells on whole dental implants are superior in testing implant properties and were used to examine their capabilities thoroughly. The surface micro-topography of five titanium dental implants manufactured by sandblasting with titanium, aluminum, corundum, or laser sintered and laser machined was compared in this study. After characterization, including particle size distribution and roughness, the adhesion, proliferation, and viability of adipose-derived stem cells (ADSCs) cultured on the whole-body implants were tested at three time points (one to seven days). Finally, the capacity of the implant to induce ADSCs' spontaneous osteoblastic differentiation was examined at the same time points, assessing the gene expression of collagen type 1 (coll-I), osteonectin (osn), alkaline phosphatase (alp), and osteocalcin (osc). Laser-treated (Laser Mach and Laser Sint) implants exhibited the highest adhesion degree; however, limited proliferation was observed, except for Laser Sint implants, while viability differences were seen throughout the three time points, except for Ti Blast implants. Sandblasted surfaces (Al Blast, Cor Blast, and Ti Blast) outpaced the laser-treated ones, inducing higher amounts of coll-I, osn, and alp, but not osc. Among the sandblasted surfaces, Ti Blast showed moderate roughness and the highest superficial texture density, favoring the most significant spontaneous differentiation relative to all the other implant surfaces. The results indicate that 3D cultures of stem cells on whole-body titanium dental implants is a practical and physiologically appropriate way to test the biological characteristics of the implants, revealing peculiar differences in ADSCs' adhesion, proliferation, and activity toward osteogenic commitment in the absence of specific osteoinductive cues. In addition, the 3D method would allow researchers to test various implant surfaces more thoroughly. Integrating with preconditioned stem cells would inspire a more substantial combinatorial approach to promote a quicker recovery for patients with restorative impairments.

IRISIN STIMULATES ANABOLISM AND MATRIX SYNTHESIS IN HUMAN NUCLEUS PULPOSUS CELLS IN VITRO: NEW INSIGHTS INTO A CROSS-TALK BETWEEN THE MUSCLE AND THE INTERVERTEBRAL DISC

This study aimed to investigate the effect of irisin on human nucleus pulposus cells (hNPCs) in vitro. Our hypothesis was that irisin would improve hNPC metabolism and proliferation.hNPCs were isolated from intervertebral discs and cultured in alginate beads. hNPCs were exposed to phosphate-buffered saline (PBS) or recombinant irisin (r-irisin) at 5, 10 and 25 ng/mL (n=4). Each experiment was performed in triplicate. Cell proliferation was assessed with trypan blue staining-automated cell counting and PicoGreen assay. Glycosaminoglycan (GAG) content was measured using the DMMB assay. Metabolic activity was assessed with the MTT assay and the Griess Reagent System. Gene expression of collagen type II (COL2), matrix metalloproteinase (MMP)-13, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and −3, aggrecan, interleukin (IL)-1β, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 was measured by RT-PCR. MTT assay and ADAMTS-5, COL2, TIMP-1 and IL-1β gene expression were evaluated following incubation with 5, 10 and 25 ng/mL r-irisin for 24 hours and subsequent culture with 10 ng/ml IL-1β and vice versa (incubation for 24 hours with IL-1β and subsequent culture with r-irisin).Irisin increased hNPC proliferation (p&lt;0.001), metabolic activity (p&lt;0.05), GAG content (p&lt;0.01), as well as COL2 (p&lt;0.01), aggrecan (p&lt;0.05), TIMP-1 and −3 (p&lt;0.01) gene expression, while decreasing MMP-13 (p&lt;0.05) and IL-1β (p&lt;0.001) mRNA levels. r-irisin pretreatment of hNPCs cultured in pro-inflammatory conditions resulted in a rescue of metabolic activity (p&lt;0.001) and a decrease of IL-1β (p&lt;0.05) levels. Similarly, incubation of hNPCs with IL-1β and subsequent exposure to r-irisin increased hNPC metabolic activity (p&lt;0.001), COL2 gene expression (p&lt;0.05) and decreased IL-1β (p&lt;0.05) and ADAMTS-5 levels (p&lt;0.01).Irisin stimulates hNPC proliferation, metabolic activity, and anabolism by reducing IL-1β and catabolic enzyme expression while promoting matrix synthesis.

Aging Fibroblasts Adversely Affect Extracellular Matrix Formation via the Senescent Humoral Factor Ependymin-Related Protein 1

Skin senescence is characterized by a decrease in extracellular matrix and the accumulation of senescent fibroblasts in the dermis, and their secretion of humoral factors. Ependymin-related protein 1 (EPDR1) is involved in abnormal fibroblast metabolism and collagen deposition, however, its relation to skin aging is unclear. We investigated whether and how EPDR1 is involved in age-related dermal deterioration. When young dermal fibroblasts and senescent cells were co-cultured in a semipermeable membrane separation system, the young fibroblasts showed decreased gene expression of collagen type I α1 chain (COL1A1) and elastin, and increased expression of matrix metalloproteinase (MMP)1 and MMP3. Senescence marker expression and EPDR1 production were increased in the culture medium of senescent cells. Treatment of young fibroblasts with recombinant EPDR1, enhanced matrix-related gene expression and suppressed COL1A1 expression, whereas EPDR1 knockdown had the opposite effects. EPDR1 gene and protein expression were increased in aged skin, compared to young skin. These results suggest that senescent cells affect nearby fibroblasts, in part through EPDR1 secretion, and exert negative effects on matrix production in the dermis. These results may lead to the discovery of potential candidate targets in the development of skin anti-aging therapies.

Effect of Irisin on Human Nucleus Pulposus Cells: New Insights into the Biological Cross-talk Between Muscle and Intervertebral Disk.

In vitro study. To investigate the effect of irisin on human nucleus pulposus cells (hNPCs) in vitro. Physical exercise (PE) favours weight loss and ameliorates function in patients with low back pain. Although there is no biological evidence that the intervertebral disk (IVD) can respond to PE, recent studies have shown that running is associated with increased IVD hydration and hypertrophy. Irisin, a myokine released upon muscle contraction, has demonstrated anabolic effects on different cell types, including chondrocytes. hNPCs were exposed to 5, 10, and 25ng/mL irisin. Cell proliferation, glycosaminoglycan (GAG) content, metabolic activity, gene expression of collagen type II (COL2), matrix metalloproteinase (MMP)-13, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and TIMP-3, aggrecan (ACAN), interleukin (IL)-1β, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 were assessed. In addition, MTT assay and ADAMTS-5, COL2, TIMP-1, and IL-1β gene expression were evaluated following incubation with irisin for 24 hours and subsequent culture with 10ng/mL IL-1β and vice versa (incubation for 24 hours with IL-1β and subsequent culture with irisin). Irisin increased hNPC proliferation, metabolic activity, and GAG content, as well as COL2, ACAN, TIMP-1 and TIMP-3 gene expression, while decreasing MMP-13 and IL-1β mRNA levels. Irisin pretreatment of hNPCs cultured in proinflammatory conditions resulted in a rescue of metabolic activity and a decrease of IL-1β levels. Similarly, incubation of hNPCs with IL-1β and subsequent exposure to irisin led to an increment of metabolic activity, COL2 gene expression, and a reduction of IL-1β and ADAMTS-5 levels. Irisin increases hNPC proliferation, GAG content, metabolic activity, and promotes anabolic gene expression while reducing catabolic markers. Irisin may be one of the mediators by which PE and muscle tissues modulate IVD metabolism, suggesting the existence of a biological cross-talk between the muscle and IVD.

Extracorporeal shockwave therapy in an immobilized knee model in rats prevents progression of joint contracture.

Joint immobilization, which ensures rest and accelerates tissue recovery in musculoskeletal disorders, often causes joint contracture, for which there is still no effective prevention. To address this, we investigated the effects of extracorporeal shockwave therapy (ESWT) in preventing joint contracture, in a unilaterally immobilized knee rat model. Under general anesthesia, ESWT (0.25 mJ/mm2 , 3000 shot, 4 Hz, 3 days/week) was administered from 1 day after immobilization up to 2, 4, and 6 weeks. The immobilized control group received general anesthesia without ESWT. We evaluated joint angle, tissue elasticity, and gene and protein expression related to fibrosis, inflammation, and angiogenesis in the joint capsule. Relative to the control, the ESWT group had greater joint angle at 4 and 6 weeks, and lower posterior-capsule elasticity at 6 weeks. In the ESWT group, at 6 weeks, gene expression of collagen type I (col1α1), connective tissue growth factor (CTGF), and α-smooth muscle actin (α-SMA) was significantly downregulated, whereas interleukin-6 (IL-6) and hypoxia-inducible factor-1α (HIF-1α) gene expression was upregulated, relative to that in the control. Compared with that in the control, at 4 and 6 weeks, the ratio of CTGF+ cells was significantly lower in the ESWT group; at 4 weeks, the ESWT group had significantly fewer CD68+ cells in the adhesion area, and at 6 weeks, significantly more blood vessels. Statement of Clinical Significance: In a rat model, ESWT counteracted fibrosis, suppressed macrophage infiltration, and promoted neovascularization, reducing elasticity, and increasing joint range-ofmotion. ESWT offers a potential new strategy to prevent progression in joint contracture.

Interfering Hsa_circRNA_0060640 Suppresses TGF-β2-Induced Proliferation, Motility and EMT in Human Lens Epithelium Cells by Targeting miR-214-3p and Collagen Type I alpha2 Chain

BackgroundCircular RNA (circRNA) is a novel star factor in the research of ocular diseases including cataract and the most common postoperative complication posterior capsule opacification (PCO). Hsa_circRNA_0060640 (circ_0060640) is an age-related cataract-related circRNA. However, its role in cataractogenesis is unrevealed yet.MethodsPCO in vitro model was established in human lens epithelium cells (hLECs) induced by transforming growth factor-beta2 (TGF-β2). RNA and protein expressions were respectively detected by quantitative PCR and western blotting. Direct interaction between two RNAs was predicted by Starbase tool and confirmed by dual-luciferase reporter assay. MTS and EdU assays measured cell proliferation; Transwell, starch wound and western blotting assays evaluated cell motility and epithelial-mesenchymal transition (EMT).ResultsCirc_0060640 expression is higher in anterior lens capsule tissues from human cataractous eyes and TGF-β2-stimulated hLECs cells line SRA01/04. RNA interference of circ_0060640 could prevent SRA01/04 cells from TGF-β2-induced cell proliferation, migration and invasion, accompanied with decreased N-cadherin and α-smooth muscle actin and increased E-cadherin. Mechanistically, circ_0060640 directly controls microRNA (miR)-214-3p expression and then regulates gene expression of collagen type I alpha2 chain (COL1A2). Notably, COL1A2 inhibition is underlying the protective role of circ_0060640 silencing and miR-214-3p ectopic expression in TGF-β2-stimulated SRA01/04 cells.ConclusionCirc_0060640 is a novel cataract-related gene and its silencing could block TGF-β2-evoked hLECs proliferation, motility and EMT in vitro via targeting miR-214-3p-COL1A2 axis. Therefore, targeting circ_0060640 via RNA interference might be a treatment strategy for PCO development.

MTA-Based Cements: Biocompatibility and Effects on the Gene Expression of Collagen Type 1 and TGF-β1.

Objective This study sought to evaluate the biocompatibility of Neomineral Trioxide Aggregate (Neo-MTA), MTA Repair High Plasticity (MTA-HP), and Mineral Trioxide Aggregate-Angelus white (MTA-Ang) in fibroblasts of human dental pulp. Materials and Methods Morphology was evaluated after 24 h of incubation. LIVE/DEAD assay and cell adhesion tests were performed at 24 h of treatment. Cell proliferation assays (MTSs) and Annexin V were performed at 48 h incubation with different treatments. The expression of Col-1 and TGF-β1 was tested by endpoint PCR at 5 days of treatment. Results Morphological changes were observed in all groups. Neo-MTA and MTA-Ang were associated with increased cell viability, and all materials induced apoptosis, with a higher percentage in the MTA-HP group than in the other groups. In the LIVE/DEAD assay, there was more damage to the cell membrane in the group of cells treated with MTA-HP than in the other groups. Conclusion Neo-MTA and MTA-Ang presented similar biocompatibility, and both showed greater biocompatibility than MTA-HP. MTA-HP and MTA-Ang increased Col-1A gene expression, and Neo-MTA and MTA-Ang increased TGF-β1 gene expression in a similar way.

Mesenchymal Stromal Cells (MSCs) Isolated from Various Tissues of the Human Arthritic Knee Joint Possess Similar Multipotent Differentiation Potential

(1) Background: The mesenchymal stromal cells (MSCs) of different tissue origins are applied in cell-based chondrogenic regeneration. However, there is a lack of comparability determining the most suitable cell source for the tissue engineering (TE) of cartilage. The purpose of this study was to compare the in vitro chondrogenic potential of MSC-like cells from different tissue sources (bone marrow, meniscus, anterior cruciate ligament, synovial membrane, and the infrapatellar fat pad removed during total knee arthroplasty (TKA)) and define which cell source is best suited for cartilage regeneration. (2) Methods: MSC-like cells were isolated from five donors and expanded using adherent monolayer cultures. Differentiation was induced by culture media containing specific growth factors. Transforming growth factor (TGF)-ß1 was used as the growth factor for chondrogenic differentiation. Osteogenesis and adipogenesis were induced in monolayer cultures for 27 days, while pellet cell cultures were used for chondrogenesis for 21 days. Control cultures were maintained under the same conditions. After, the differentiation period samples were analyzed, using histological and immunohistochemical staining, as well as molecularbiological analysis by RT-PCR, to assess the expression of specific marker genes. (3) Results: Plastic-adherent growth and in vitro trilineage differentiation capacity of all isolated cells were proven. Flow cytometry revealed the clear co-expression of surface markers CD44, CD73, CD90, and CD105 on all isolated cells. Adipogenesis was validated through the formation of lipid droplets, while osteogenesis was proven by the formation of calcium deposits within differentiated cell cultures. The formation of proteoglycans was observed during chondrogenesis in pellet cultures, with immunohistochemical staining revealing an increased relative gene expression of collagen type II. RT-PCR proved an elevated expression of specific marker genes after successful differentiation, with no significant differences regarding different cell source of native tissue. (4) Conclusions: Irrespective of the cell source of native tissue, all MSC-like cells showed multipotent differentiation potential in vitro. The multipotent differentiation capacity did not differ significantly, and chondrogenic differentiation was proven in all pellet cultures. Therefore, cell suitability for cell-based cartilage therapies and tissue engineering is given for various tissue origins that are routinely removed during total knee arthroplasty (TKA). This study might provide essential information for the clinical tool of cell harvesting, leading to more flexibility in cell availability.

Comparison of Gene Expression of Inflammation- and Fibrosis-Related Factors Between the Anterior and Posterior Capsule in Patients With Rotator Cuff Tear and Shoulder Stiffness.

Background:Arthroscopic capsular release is an effective treatment for refractory shoulder stiffness, yet there are no basic studies that can explain the extent of the release.Purpose:This study aimed to compare the genetic expression of inflammation- and fibrosis-related factors between the anterior and posterior capsules in patients with shoulder stiffness and rotator cuff tear.Study Design:Descriptive laboratory study.Methods:Enrolled in this study were 35 patients who underwent arthroscopic capsular release for shoulder stiffness along with the rotator cuff repair. Anterior and posterior glenohumeral joint capsular tissues were obtained during the capsular release. For the control tissue, anterior capsule was obtained from 40 patients without stiffness who underwent arthroscopic rotator cuff repair. The gene expression of collagen types I and III, fibronectin, extracellular matrix, basic fibroblast growth factor, transforming growth factor–β, connective tissue growth factor, matrix metalloproteinase (MMP)-1, MMP-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, intercellular adhesion molecule 1, interleukin 1, and tumor necrotizing factor–α were analyzed using real-time reverse transcription polymerase chain reaction. Differences in gene expression between the anterior capsule, the posterior capsule, and the control tissue were compared with the Kruskal-Wallis test.Results:The expression levels of collagen types I and III were significantly higher in the anterior capsule with stiffness com (pared with both the posterior capsule with stiffness (P = .010 for both) and the control (P = .038 and .010, respectively). The levels of fibronectin, MMP-2, and MMP-9 in the anterior capsule were significantly higher than in both the posterior capsule (P = .013, .003, and .006, respectively) and the control (P = .014, .003, and .005, respectively).Conclusion:Genetic analysis of the shoulder capsule revealed that more fibrogenic processes occur in the anterior capsule compared with the posterior capsule in patients with shoulder stiffness.Clinical Relevance:Capsular release for shoulder stiffness should be more focused on the anterior capsule than on the posterior capsule.

A Prototype Skin Substitute, Made of Recycled Marine Collagen, Improves the Skin Regeneration of Sheep.

Simple SummaryMarine ecosystems are a huge source of unexplored “blue” materials for different applications. The edible part of sea urchin is limited, and the vast majority of the product ends up as waste. Our studies intend to fully recycle wastes from the food industry and reconvert them in high added-value products, as innovative biocompatible skin substitutes for tissue regeneration. The aim of the present work is to apply the pioneering skin substitute in in vivo experimental wounds to test its regenerative potential and compare it, in a future study, to the available commercial membranes produced with collagen of bovine, porcine, and equine origin. Results are encouraging since the skin substitute made with marine collagen reduced inflammation, promoted the deposition of granulation tissue, and enhanced a proper re-epithelialization with the adequate development of skin appendages. In summary, our findings might be of great interest for processing industries and biotech companies which transform waste materials in high-valuable and innovative products for Veterinary advanced applications.Skin wound healing is a complex and dynamic process that aims to restore lesioned tissues. Collagen-based skin substitutes are a promising treatment to promote wound healing by mimicking the native skin structure. Recently, collagen from marine organisms has gained interest as a source for producing biomaterials for skin regenerative strategies. This preliminary study aimed to describe the application of a collagen-based skin-like scaffold (CBSS), manufactured with collagen extracted from sea urchin food waste, to treat experimental skin wounds in a large animal. The wound-healing process was assessed over different time points by the means of clinical, histopathological, and molecular analysis. The CBSS treatment improved wound re-epithelialization along with cell proliferation, gene expression of growth factors (VEGF-A), and development of skin adnexa throughout the healing process. Furthermore, it regulated the gene expression of collagen type I and III, thus enhancing the maturation of the granulation tissue into a mature dermis without any signs of scarring as observed in untreated wounds. The observed results (reduced inflammation, better re-epithelialization, proper development of mature dermis and skin adnexa) suggest that sea urchin-derived CBSS is a promising biomaterial for skin wound healing in a “blue biotechnologies” perspective for animals of Veterinary interest.

Morpho-functional effects of different universal dental adhesives on human gingival fibroblasts: an in vitro study

To analyze the effects of four universal adhesives (Optibond Solo Plus—OB, Universal Bond—UB, Prime&Bond Active—PBA, FuturaBond M + —FB) on human gingival fibroblasts in terms of cytotoxicity, morphology and function. After in vitro exposure for up to 48 h, fibroblast viability was determined by the MTT assay determined, morphology by phase-contrast microscopy and migration by the scratch wound assay. Expression levels of IL1β, IL6, IL8, IL10, TNFα and VEGF genes were assessed by RT-PCR and their protein production by Western blot analysis. Apoptosis and cell cycle were analyzed by flow cytometry. OB and UB induced early morphological changes on fibroblasts (3 h) with extended cell death at 24 h/48 h. Gene expression of collagen type I and fibronectin increased fivefold compared with controls, elastin disappeared and elastase increased threefold, indicating gingival tissue tended to become fibrotic. Only UB and OB increased gene expression of inflammatory markers: IL1β at 3 and 48 h (up to about three times), IL6 and IL8 at 3 h (up to almost four times) which corresponded to the increase of the activated form NF-kB. All adhesives showed an effect on the functionality of fibroblasts with cytotoxic effect time and concentration dependent. Among all the OB and UB adhesives, they showed the greatest cell damage. The in-depth analysis of the effects of universal adhesives and possible functional effects represents an important information for the clinician towards choosing the most suitable adhesive system.

Cytotoxic Effect of Niobium Phosphate Glass–based Gutta-Percha Points on Periodontal Ligament Fibroblasts In Vitro

IntroductionThe outcome of root canal obturation might be affected by the chemical components of the chosen filling materials. Niobium phosphate glass–based gutta-percha (GNB) was proposed as a biomaterial-based obturation point. This study aimed to investigate the cytotoxic and cell modulation effects of GNB points on human periodontal ligament fibroblasts (PDLFs) in vitro. MethodsHuman PDLFs were cultured for the assays. Extracts of regular gutta-percha (GP) points and GNB were obtained, serially diluted (1:5, 1:10, and 1:25), and used to stimulate PDLFs. A cell viability assay was performed using alamarBlue reagent (Molecular Probes, Waltham, MA), and reverse transcription quantitative polymerase chain reaction was used to assess the gene expression for collagen type I and cementum protein 1. One-way analysis of variance followed by the Tukey post hoc test was performed (P < .05). ResultsRegular GP reduced cell viability only in pure extracts, whereas GNB exhibited cytotoxicity to PDLFs in pure extracts as well as 1/5 and 1/10 dilutions. The gene expression of collagen type I was down-regulated only in the GNB group (P < .05). The expression of cementum protein 1 remained unaltered by both tested materials. ConclusionsThe addition of niobium phosphate glass to GP points increased cytotoxicity, affecting PDLF viability and partially disturbing physiological cell function.

Stereolithography 3D Bioprinting Method for Fabrication of Human Corneal Stroma Equivalent.

3D bioprinting technology is a promising approach for corneal stromal tissue regeneration. In this study, gelatin methacrylate (GelMA) mixed with corneal stromal cells was used as a bioink. The visible light-based stereolithography (SLA) 3D bioprinting method was utilized to print the anatomically similar dome-shaped structure of the human corneal stroma. Two different concentrations of GelMA macromer (7.5 and 12.5%) were tested for corneal stroma bioprinting. Due to high macromer concentrations, 12.5% GelMA was stiffer than 7.5% GelMA, which made it easier to handle. In terms of water content and optical transmittance of the bioprinted scaffolds, we observed that scaffold with 12.5% GelMA concentration was closer to the native corneal stroma tissue. Subsequently, cell proliferation, gene and protein expression of human corneal stromal cells encapsulated in the bioprinted scaffolds were investigated. Cytocompatibility in 12.5% GelMA scaffolds was observed to be 81.86 and 156.11% at day 1 and 7, respectively, which were significantly higher than those in 7.5% GelMA scaffolds. Elongated corneal stromal cells were observed in 12.5% GelMA samples after 7days, indicating the cell attachment, growth, and integration within the scaffold. The gene expression of collagen type I, lumican and keratan sulfate increased over time for the cells cultured in 12.5% GelMA scaffolds as compared to those cultured on the plastic tissue culture plate. The expression of collagen type I and lumican were also visualized using immunohistochemistry after 28days. These findings imply that the SLA 3D bioprinting method with GelMA hydrogel bioinks is a promising approach for corneal stroma tissue biofabrication.

Cell-Laden Agarose-Collagen Composite Hydrogels for Mechanotransduction Studies.

The increasing investigation of cellular mechanotransduction mechanisms requires biomaterials combining biofunctionality and suitable mechanical properties. Agarose is a standard biomaterial for cartilage and intervertebral disc mechanobiology studies, but lacks adhesion motifs and the necessary cell-matrix interaction for mechanotransduction. Here, collagen type I was blended at two concentrations (2 and 4.5 mg/mL) with agarose 2% wt/vol. The composite hydrogels were characterized in terms of structural homogeneity, rheological properties and size stability. Nucleus pulposus (NP) cell viability, proliferation, morphology, gene expression, GAG production, adhesion and mechanotransduction ability were further tested. Blended hydrogels presented a homogenous network of the two polymers. While the addition of 4.5 mg/mL collagen significantly decreased the storage modulus and increased the loss modulus of the gels, blended gels containing 2 mg/mL collagen displayed similar mechanical properties to agarose. Hydrogel size was conserved over 21 days for all agarose-based gels. Embedded cells were viable (>80%) and presented reduced proliferation and a round morphology typical of NP cells in vivo. Gene expression of collagen types I and II and aggrecan significantly increased in blended hydrogels from day 1 to 7, further resulting in a significantly superior GAG/DNA ratio compared to agarose gels at day 7. Agarose-collagen hydrogels not only promoted cell adhesion, contrary to agarose gels, but also showed a 5.36-fold higher focal adhesion kinase phosphorylation (pFAK/β-tubulin) when not compressed, and increased pFAK/FAK values 10 min after compression. Agarose-collagen thus outperforms agarose, mimics native tissues constituted of non-fibrillar matrix and collagens, and allows exploring complex loading in a highly reproducible system.

Pharmacologic iron chelation reduces markers of chondrocyte hypertrophy and osteoarthritis-associated cartilage lesions in an animal model of idiopathic disease

Purpose: It has previously been documented that chondrocytes from joints afflicted with osteoarthritis (OA) undergo improper dedifferentiation into hypertrophic chondrocytes; thus, it is postulated that this process may contribute to the progression of OA. Relative to unaffected adult chondrocytes, hypertrophic chondrocytes mimic cellular processes for endochondral ossification observed during initial development of long bones. In particular, they express markers of hypertrophic differentiation such as runx family transcription factor 2 (runx2), collagen type x, and the matrix degrading enzyme matrix metalloproteinase-13 (MMP-13). In addition to these markers, hypertrophic chondrocytes exhibit increased apoptosis, decreased autophagy, and promote cartilage vascularization and mineralization of the extracellular matrix. One theory for why this hypertrophic state occurs is that oxidative stress from reactive oxygen species (ROS) contributes to this shift in chondrocyte phenotype. We had previously demonstrated the ability of systemic iron reduction, achieved by an iron deficient diet, to reduce ROS production and thereby decrease structural changes of OA in an animal model of idiopathic/spontaneous disease. Given the success of this study, we hypothesized that systemic iron chelation - achieved by administration of the pharmacologic iron chelator deferoxamine (DFO) - would reduce markers of chondrocyte hypertrophy and decrease the development of knee OA in this animal model. Methods: All procedures received University approval and were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Three-month-old male Dunkin-Hartley guinea pigs were randomly assigned to receive either DFO or vehicle control (n=8 per group). Animals within the DFO group received 46 mg/kg of DFO injected subcutaneously twice daily while animals within the control group received an equivalent dose of saline. Animals were subjected to 10-minute sessions of open field behavior monitoring once per month for the duration of the study, with baseline activity levels collected prior to initiating treatment. The study was terminated when animals were 8-months-old, at which time body weights were recorded. Hind limbs were removed and the right tibia was measured using calipers. Articular cartilage was collected from the right knee joint for gene expression analysis using Nanostring nCounter technology. Femoral head cartilage and liver tissue were collected for iron quantification by atomic absorption spectroscopy. Left knee joints were formalin-fixed, paraffin-embedded, and stained with toluidine blue. For histologic analysis, all joint compartments were blindly scored by two assessors using OARSI published guidelines. Data were compared using non-parametric Mann-Whitney U tests, Welch’s t-tests, or unpaired student’s t-tests as appropriate given the normality and heteroscedasticity of the data. All statistical analyses were performed with Prism 8.0; statistical significance was set at P≤0.05. Results: No significant differences in body weight (p=0.576) or tibia length (p=0.628) were present between groups. As anticipated, animals treated with DFO had lower levels of iron within the liver (p=0.0218) and articular cartilage (p=0.0005) tissue than control animals. Histologic grading of knee joints confirmed that animals treated with DFO had lower total joint OA scores than control animals (p<0.0001). Gene transcript analysis demonstrated that, relative to controls, animals treated with DFO had lower expression of runx2 (p=0.0010), MMP-13 (p=0.0151), vascular endothelial growth factor (VEGF; p=0.0140), β- catenin (p<0.0001), wnt (p=0.0008), mammalian target of rapamycin (mTOR; p=0.0018), and the serine/threonine protein kinase AKT (p=0.0041). Animals treated with DFO also had increased expression of b cell lymphoma 2 (BCL-2; p<0.0001), a prototypical anti-apoptotic marker. There was no significant difference in the gene expression of collagen type x between groups (p=0.3163). Open field behavior monitoring indicated that DFO treated animals maintained their baseline activity level throughout the study, while the movement of control animals declined throughout the study. Conclusions: Administration of a pharmacologic iron chelator was successful in reducing iron levels both systemically and within articular cartilage tissue, which was associated with a reduced development of OA-associated cartilage lesions and maintained mobility in these animals. Gene transcript analysis of cartilage tissue revealed that DFO treated animals had a decreased expression of: select markers of chondrocyte hypertrophy (runx2 and MMP-13), negative regulators of autophagy (AKT and mTOR), the angiogenesis promoter VEGF, and genes associated with chondrocyte dedifferentiation (β- catenin and wnt); while displaying increased expression of the anti-apoptotic protein BCL-2. These results indicate that removal of excess iron may help prevent chondrocyte hypertrophy and development of OA-related changes within the cartilage of knee joints.