Increase In Collagen Type Research Articles

Intra-articular collagenase injection increases range of motion in a rat knee flexion contracture model

ObjectivesA knee joint contracture, a loss in passive range of motion (ROM), can be caused by prolonged immobility. In a rat knee immobilization flexion contracture model, the posterior capsule was shown to contribute to an irreversible limitation in ROM, and collagen pathways were identified as differentially expressed over the development of a contracture. Collagenases purified from Clostridium histolyticum are currently prescribed to treat Dupuytren’s and Peyronie’s contractures due to their ability to degrade collagen. The potential application of collagenases to target collagen in the posterior capsule was tested in this model.Materials and methodsRats had one hind leg immobilized, developing a knee flexion contracture. After 4 weeks, the immobilization device was removed, and the rats received one 50 µL intra-articular injection of 0.6 mg/mL purified collagenase. Control rats were injected with only the buffer. After 2 weeks of spontaneous remobilization following the injections, ROM was measured with a rat knee arthrometer, and histological sections were immunostained with antibodies against rat collagen types I and III.Results/conclusionCompared with buffer-injected control knees, collagenase-treated knees showed increased ROM in extension by 8.0°±3.8° (p-value <0.05). Immunohistochemical analysis revealed an increase in collagen type III staining (p<0.01) in the posterior capsule of collagenase-treated knees indicating an effect on the extracellular matrix due to the collagenase. Collagen I staining was unchanged (p>0.05). The current study provides experimental evidence for the pharmacological treatment of knee flexion contractures with intra-articular collagenase injection, improving the knee ROM.

Biomechanic and histologic analysis of fibroblastic effects of tendon-to-bone healing by transforming growth factor β1 (TGF-β1) in rotator cuff tears.

To evaluate the effect of transforming growth factor β1 (TGF-β1) on tendon-to-bone reconstruction of rotator cuff tears. Seventy-two rat supraspinatus tendons were transected and reconstructed in situ. At 8 and 16 weeks, specimens of three groups; that is control, L-dose (low dose), and H-dose (high dose) were harvested and underwent a biomechanical test to evaluate the maximum load and stiffness values. Histology sections of the tendon-to-bone interface were identified by hematoxylin-eosin or Masson trichrome stain. Collagen type III was observed by picric acid sirius red staining under polarized light. The level of insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF) was measured by the enzyme-linked immunosorbent assay (ELISA) method. Collagen type III of the H-dose group had a significant difference in histology structure compared with the L-dose group (P<0.05). The maximum load and stiffness decreased significantly in the control group compared with the values of the L-dose and H-dose groups. The stiffness among the three groups differed significantly at the same postoperative time (P<0.05). Interestingly, progressive reestablishment of collagen type III affected tendon-to-bone healing significantly in the later stages. The H-dose was associated with an increased collagen type III morphology stimulated by TGF-β1.

Autologous mesenchymal stem cells or meniscal cells: what is the best cell source for regenerative meniscus treatment in an early osteoarthritis situation?

BackgroundTreatment of meniscus tears within the avascular region represents a significant challenge, particularly in a situation of early osteoarthritis. Cell-based tissue engineering approaches have shown promising results. However, studies have not found a consensus on the appropriate autologous cell source in a clinical situation, specifically in a challenging degenerative environment. The present study sought to evaluate the appropriate cell source for autologous meniscal repair in a demanding setting of early osteoarthritis.MethodsA rabbit model was used to test autologous meniscal repair. Bone marrow and medial menisci were harvested 4 weeks prior to surgery. Bone marrow-derived mesenchymal stem cells (MSCs) and meniscal cells were isolated, expanded, and seeded onto collagen-hyaluronan scaffolds before implantation. A punch defect model was performed on the lateral meniscus and then a cell-seeded scaffold was press-fit into the defect. Following 6 or 12 weeks, gross joint morphology and OARSI grade were assessed, and menisci were harvested for macroscopic, histological, and immunohistochemical evaluation using a validated meniscus scoring system. In conjunction, human meniscal cells isolated from non-repairable bucket handle tears and human MSCs were expanded and, using the pellet culture model, assessed for their meniscus-like potential in a translational setting through collagen type I and II immunostaining, collagen type II enzyme-linked immunosorbent assay (ELISA), and gene expression analysis.ResultsAfter resections of the medial menisci, all knees showed early osteoarthritic changes (average OARSI grade 3.1). However, successful repair of meniscus punch defects was performed using either meniscal cells or MSCs. Gross joint assessment demonstrated donor site morbidity for meniscal cell treatment. Furthermore, human MSCs had significantly increased collagen type II gene expression and production compared to meniscal cells (p < 0.05).ConclusionsThe regenerative potential of the meniscus by an autologous cell-based tissue engineering approach was shown even in a challenging setting of early osteoarthritis. Autologous MSCs and meniscal cells were found to have improved meniscal healing in an animal model, thus demonstrating their feasibility in a clinical setting. However, donor site morbidity, reduced availability, and reduced chondrogenic differentiation of human meniscal cells from debris of meniscal tears favors autologous MSCs for clinical use for cell-based meniscus regeneration.

Global fibroblast activation throughout the left ventricle but localized fibrosis after myocardial infarction

Fibroblast (Fb) differentiation and interstitial fibrosis contribute to cardiac remodeling and loss of function after myocardial infarction (MI). We investigated regional presence and regulation of fibrosis in a pig MI model. In vivo analysis of regional function and perfusion defined three regions: the scar, the myocardium adjacent to the scar (MIadjacent, reduced function, reduced perfusion reserve), and the remote myocardium (MIremote, minimal functional deficit, maintained perfusion). Interstitial and perivascular fibrosis, and increase of collagen type I, was only observed in the MIadjacent. Fb activated protein-alpha (FAP-α) was enriched in MIadjacent compared to MIremote. TGF-β1, which triggers Fb differentiation, was upregulated in both MIadjacent and MIremote, whereas lysyl oxidase, a regulator of collagen cross-linking, and the proteoglycans decorin and biglycan were only increased in the MIadjacent. Fb isolated and cultured for 4 days had myoFb characteristics with little difference between MIremote and MIadjacent, although RNA sequencing revealed differences in gene expression profiles. Fbs from all regions maintained proliferative capacity, and induced contraction of 3-D collagen matrices but scar myoFb was more effective. These data suggest that after MI, signaling through TGF-β1, possibly related to increased mechanical load, drives Fb activation throughout the left ventricle while regional signaling determines further maturation and extracellular matrix remodeling after MI.

Efficacy of thermoresponsive, photocrosslinkable hydrogels derived from decellularized tendon and cartilage extracellular matrix for cartilage tissue engineering.

Tissue engineering using adult mesenchymal stem cells (MSCs), a promising approach for cartilage repair, is highly dependent on the nature of the matrix scaffold. Thermoresponsive, photocrosslinkable hydrogels were fabricated by functionalizing pepsin-soluble decellularized tendon and cartilage extracellular matrices (ECM) with methacrylate groups. Methacrylated gelatin hydrogels served as controls. When seeded with human bone marrow MSCs and cultured in chondrogenic medium, methacrylated ECM hydrogels experienced less cell-mediated contraction, as compared against non-methacrylated ECM hydrogels. However, methacrylation slowed or diminished chondrogenic differentiation of seeded MSCs, as determined through analyses of gene expression, biochemical composition and histology. In particular, methacrylated cartilage hydrogels supported minimal due to chondrogenesis over 42 weeks, as hydrogel disintegration beginning at day 14 presumably compromised cell-matrix interactions. As compared against methacrylated gelatin hydrogels, MSCs cultured in non-methacrylated ECM hydrogels exhibited comparable expression of chondrogenic genes (Sox9, Aggrecan and collagen type II) but increased collagen type I expression. Non-methacrylated cartilage hydrogels did not promote chondrogenesis to a greater extent than either non-methacrylated or methacrylated tendon hydrogels. Whereas methacrylated gelatin hydrogels supported relatively homogeneous increases in proteoglycan and collagen type II deposition throughout the construct over 42 days, ECM hydrogels possessed greater heterogeneity of staining intensity and construct morphology. These results do not support the utility of pepsin-solubilized cartilage and tendon hydrogels for cartilage tissue engineering over methacrylated gelatin hydrogels. Methacrylation of tendon and cartilage ECM hydrogels permits thermal- and light-induced polymerization but compromises chondrogenic differentiation of seeded MSCs.

Exosomes from embryonic mesenchymal stem cells alleviate osteoarthritis through balancing synthesis and degradation of cartilage extracellular matrix

BackgroundMesenchymal stem cell therapy for osteoarthritis (OA) has been widely investigated, but the mechanisms are still unclear. Exosomes that serve as carriers of genetic information have been implicated in many diseases and are known to participate in many physiological processes. Here, we investigate the therapeutic potential of exosomes from human embryonic stem cell-induced mesenchymal stem cells (ESC-MSCs) in alleviating osteoarthritis (OA).MethodsExosomes were harvested from conditioned culture media of ESC-MSCs by a sequential centrifugation process. Primary mouse chondrocytes treated with interleukin 1 beta (IL-1β) were used as an in vitro model to evaluate the effects of the conditioned medium with or without exosomes and titrated doses of isolated exosomes for 48 hours, prior to immunocytochemistry or western blot analysis. Destabilization of the medial meniscus (DMM) surgery was performed on the knee joints of C57BL/6 J mice as an OA model. This was followed by intra-articular injection of either ESC-MSCs or their exosomes. Cartilage destruction and matrix degradation were evaluated with histological staining and OARSI scores at the post-surgery 8 weeks.ResultsWe found that intra-articular injection of ESC-MSCs alleviated cartilage destruction and matrix degradation in the DMM model. Further in vitro studies illustrated that this effect was exerted through ESC-MSC-derived exosomes. These exosomes maintained the chondrocyte phenotype by increasing collagen type II synthesis and decreasing ADAMTS5 expression in the presence of IL-1β. Immunocytochemistry revealed colocalization of the exosomes and collagen type II-positive chondrocytes. Subsequent intra-articular injection of exosomes derived from ESC-MSCs successfully impeded cartilage destruction in the DMM model.ConclusionsThe exosomes from ESC-MSCs exert a beneficial therapeutic effect on OA by balancing the synthesis and degradation of chondrocyte extracellular matrix (ECM), which in turn provides a new target for OA drug and drug-delivery system development.

Thyroxine Increases Collagen Type II Expression and Accumulation in Scaffold-Free Tissue-Engineered Articular Cartilage.

Low collagen accumulation in the extracellular matrix is a pressing problem in cartilage tissue engineering, leading to a low collagen-to-glycosaminoglycan (GAG) ratio and poor mechanical properties in neocartilage. Soluble factors have been shown to increase collagen content, but may result in a more pronounced increase in GAG content. Thyroid hormones have been reported to stimulate collagen and GAG production, but reported outcomes, including which specific collagen types are affected, are variable throughout the literature. Here we investigated the ability of thyroxine (T4) to preferentially stimulate collagen production, as compared with GAG, in articular chondrocyte-derived scaffold-free engineered cartilage. Dose response curves for T4 in pellet cultures showed that 25 ng/mL T4 increased the total collagen content without increasing the GAG content, resulting in a statistically significant increase in the collagen-to-GAG ratio, a fold change of 2.3 ± 1.2, p < 0.05. In contrast, another growth factor, TGFβ1, increased the GAG content in excess of threefold more than the increase in collagen. In large scaffold-free neocartilage, T4 also increased the total collagen/DNA at 1 month and at 2 months (fold increases of 2.1 ± 0.8, p < 0.01 and 2.1 ± 0.4, p < 0.001, respectively). Increases in GAG content were not statistically significant. The effect on collagen was largely specific to collagen type II, which showed a 2.8 ± 1.6-fold increase of COL2A1 mRNA expression (p < 0.01). Western blots confirmed a statistically significant increase in type II collagen protein at 1 month (fold increase of 2.2 ± 1.8); at 2 months, the fold increase of 3.7 ± 3.3 approached significance (p = 0.059). Collagen type X protein was less than the 0.1 μg limit of detection. T4 did not affect COL10A1 and COL1A2 gene expression in a statistically significant manner. Biglycan mRNA expression increased 2.6 ± 1.6-fold, p < 0.05. Results of this study show that an optimized dosage of T4 is able to increase collagen type II content, and do so preferential to GAG. Moreover, the upregulation of COL2A1 gene expression and type II collagen protein accumulation, without a concomitant increase in collagens type I or type X, signifies a direct enhancement of chondrogenesis of hyaline articular cartilage without the induction of terminal differentiation.

Nicotine stimulates collagen type I expression in lung via \u03b17 nicotinic acetylcholine receptors

BackgroundTobacco-related chronic lung diseases are characterized by alterations in lung architecture leading to decreased lung function. Knowledge of the exact mechanisms involved in tobacco-induced tissue remodeling and inflammation remains incomplete. We hypothesize that nicotine stimulates the expression of extracellular matrix proteins, leading to relative changes in lung matrix composition, which may affect immune cells entering the lung after injury.MethodsPulmonary fibroblasts from wildtype and α7 nicotinic acetylcholine receptor knockout (α7KO) mice were exposed to nicotine and examined for collagen type 1 mRNA and protein expression. Testing the potential role on immune cell function, pulmonary fibroblasts were retained in culture for 120 h. The fibroblasts were eliminated by osmotic lysis and the remaining matrix-coated dishes were washed thoroughly. U937 cells were incubated on the matrix-coated dishes for 24 h followed by evaluation of IL-1β gene expression. Wildtype or α7KO C57BL/6 mice (female, 8–12 weeks) were fed normal diet and exposed to nicotine in their drinking water (100 μg/ml) for 8-12weeks. Lungs were processed for mRNA, protein, and histology. Statistical significance was determined at p ≤ .05 by two-tailed test or 2-way ANOVA with Bonferroni posttest.ResultsWe found that nicotine stimulated collagen type I mRNA and protein expression in a dose-dependent manner and up to 72 h in primary lung fibroblasts. The stimulatory effect of nicotine was inhibited in α7KO primary lung fibroblasts. Testing the potential role of these events on immune cell function, U937 monocytic cells were cultured atop matrices derived from nicotine-treated lung fibroblasts. These cells expressed more IL-1β than those cultured atop matrices derived from untreated fibroblasts, and antibodies against the α2β1 collagen integrin receptor inhibited the effect. Nicotine also stimulated fibroblast proliferation via MEK-1/ERK, unveiling a potentially amplifying pathway. In vivo, nicotine increased collagen type I expression was detected in wildtype, but not in α7KO mice. Wildtype mice showed increased collagen staining in lung, primarily around the airways.ConclusionsThese observations suggest that nicotine stimulates fibroblast proliferation and their expression of collagen type I through α7 nAChRs, thereby altering the relative composition of the lung matrix without impacting the overall lung architecture; this may influence inflammatory responses after injury.

Early Graft Tunnel Healing After Anterior Cruciate Ligament Reconstruction With Intratunnel Injection of Bone Marrow Mesenchymal Stem Cells and Vascular Endothelial Growth Factor.

Background:Bone marrow mesenchymal stem cells (BM-MSCs) are multipotent adult stem cells and have become an important source of cells for engineering tissue repair and cell therapy. Vascular endothelial growth factor (VEGF) promotes angiogenesis and contributes fibrous integration between tendon and bone during the early postoperative stage. Both MSCs and VEGF can stimulate cell proliferation, differentiation, and matrix deposition by enhancing angiogenesis and osteogenesis of the graft in the tunnel.Hypothesis:Injection of intratunnel BM-MSCs and VEGF enhances the early healing process of a tendon graft.Study Design:Controlled laboratory study.Methods:In this controlled animal laboratory study, each of 4 groups of rabbits underwent unilateral anterior cruciate ligament (ACL) reconstruction with use of the ipsilateral semitendinosus tendon. The rabbits received intratunnel injection of BM-MSCs and VEGF with a fibrin glue seal covering the distal tunnel at the articular site. Evaluation using magnetic resonance imaging (MRI), collagen type III expression, and biomechanical analyses were performed at 3- and 6-week intervals.Results:All parameters using MRI, collagen type III expression, and biomechanical analysis of pullout strength of the graft showed that application of intratunnel BM-MSCs and VEGF enhanced tendon-to-bone healing after ACL reconstruction.Conclusion:Intratunnel injections of BM-MSCs and VEGF after ACL reconstruction enhanced graft tunnel healing. Overall, the femoral tunnel that received BM-MSCs and VEGF had better advanced healing with increased collagen type III fibers and better outcomes on MRI and biomechanical analysis. MRI is the most reliable tool for clinical use in evaluating stages of ACL healing after reconstruction, since biopsy is an invasive procedure.

MK5 haplodeficiency attenuates hypertrophy and preserves diastolic function during remodeling induced by chronic pressure overload in the mouse heart.

MAPK-activated protein kinase-5 (MK5) is a protein serine/threonine kinase that is activated by p38 MAPK and the atypical MAPKs ERK3 and ERK4. The physiological function(s) of MK5 remains unknown. Here, we examined the effect of MK5 haplodeficiency on cardiac function and myocardial remodeling. At 12 wk of age, MK5 haplodeficient mice (MK5+/-) were smaller than age-matched wild-type littermates (MK5+/+), with similar diastolic function but reduced systolic function. Transverse aortic constriction (TAC) was used to induce chronic pressure overload in 12-wk-old male MK5+/- and MK5+/+ mice. Two weeks post-TAC, heart weight-to-tibia length ratios were similarly increased in MK5+/- and MK5+/+ hearts, as was the abundance of B-type natriuretic peptide and β-myosin heavy chain mRNA. Left ventricular ejection fraction was reduced in both MK5+/+ and MK5+/- mice, whereas regional peak systolic tissue velocities were reduced and isovolumetric relaxation time was prolonged in MK5+/+ hearts but not in MK5+/- hearts. The TAC-induced increase in collagen type 1-α1 mRNA observed in MK5+/+ hearts was markedly attenuated in MK5+/- hearts. Eight weeks post-TAC, systolic function was equally impaired in MK5+/+ and MK5+/- mice. In contrast, the increase in E wave deceleration rate and progression of hypertrophy observed in TAC MK5+/+ mice were attenuated in TAC MK5+/- mice. MK5 immunoreactivity was detected in adult fibroblasts but not in myocytes. MK5+/+, MK5+/-, and MK5-/- fibroblasts all expressed α-smooth muscle actin in culture. Hence, reduced MK5 expression in cardiac fibroblasts was associated with the attenuation of both hypertrophy and development of a restrictive filling pattern during myocardial remodeling in response to chronic pressure overload.NEW & NOTEWORTHY MAPK-activated protein kinase-5 (MK5)/p38-regulated/activated protein kinase is a protein serine/threonine kinase activated by p38 MAPK and/or the atypical MAPKs ERK3 and ERK4. MK5 immunoreactivity was detected in adult ventricular fibroblasts but not in myocytes. MK5 haplodeficiency attenuated the progression of hypertrophy, reduced collagen type 1 mRNA, and protected diastolic function in response to chronic pressure overload.

Collagen analysis of the Achilles tendon injured and treated with F1 protein. A study using transmission electron, polarized light microscopy and western blot.

IntroductionIt is estimated that $30 billion are spent on musculoskeletal injuries in the United States each year and tendon/ligament injuries represent ~45% of these cases. Incidence of rupture of the Achilles tendon in North America is 5.5 to 9.9/100,000 habitants. After surgery, the patients return to their activity on approximately 85 days, but may range up to 270 days.AimThe effect of F1 protein extracted from Hevea brasiliensis in Achilles tendon repair in rats subjected to tenotomy.MethodsThree months‐old male Sprague Dawley rats were divided into 3 experimental groups (90 total n): control (C), Kessler‐Tajima suture (K), Kessler‐Tajima plus F1 protein (KP). Catgut suture 5‐0 was used to surgery. Transmission electron microscopy was used to analyze collagen morphology and ultrastructural aspects; western blotting was used to quantify collagen type I and III and light microscopy under polarized light to demonstrates the collagen distribution. All groups were analyzed 2 and 4 weeks after surgery.ResultsTransmission electron microscopy analysis revealed ultrastructural characteristics, in which the presence of macrophages and mast cell were noted in K and KP groups in both analyzed times. Blood vessels among collagen fibrils were noted especially in the groups analyzed after 4 weeks of injury and surgery independent of the treatment. After 2 weeks of surgery in the treated (KP) and non‐treated (K) groups the tendons' weight was increased ~7 times and decreased to ~5.6 after 4 weeks in both groups. Tendon of K group (2 weeks after injury) presented a higher ratio of collagen type III than type I when compared to all other groups, however treated groups analyzed after 2 and 4 weeks presented more collagen type I than non‐treated groups, as well as a higher density of the fibrils collagen. Polarized light microscopy shows collagen fibers arrangement for all tissue. K and KP groups (2 weeks) presented more collagen type III near muscle, and type I near of calcaneus bone; both groups after 4 weeks presented the two types of collagen along the tissue with predominance of type I. C group had only collagen type I in this type of analyze.ConclusionF1 protein increases collagen type I and density of collagen fibrils when compared to non‐treated group, therefore apparently may improve tendon repair.Support or Funding InformationFAPESP #2015/24087‐3

Inhibition of inducible nitric oxide synthase (iNOS) by simvastatin attenuates cardiac hypertrophy in rats.

The left ventricular hypertrophy (LVH) occurs in response to the haemodynamic overload in some physiological and pathological conditions. This study was designed to investigate the possible cardioprotective effect of simvastatin (SIM) treatment against isoproterenol (ISO)-induced LVH and the probable underlying mechanism in adult male Wistar rats. Animals were allocated into four groups. Rats of control group received normal saline orally for 30 days and intraperitoneally for the last 7 days. Rats of SIM group received SIM orally (10 mg/kg/day in saline) for 30 days. Rats of ISO group received normal saline orally for 30 days and ISO intraperitoneally (5 mg/kg) for the last 7 days to induce LVH. Rats of ISO/SIM group received SIM for 30 days and ISO intraperitoneally for the last 7 days. At the end of the experiment, all animals were sacrificed by cervical decapitation under anaesthesia. Truncal blood was collected and serum was separated and used for biochemical assay. The heart was dissected and processed for histological and immunohistochemical studies. The results of the present study confirmed the ISO-induced myocardial lesions including significant increase of heart weight (HW), heart weight/body weight (HW/BW) ratio, LVH, interstitial myocardial fibrosis (increased collagen types I and III), inflammatory cellular infiltration, necrosis of cardiomyocytes, and increased expression of inducible nitric oxide synthase (iNOS) and thioredoxin in cardiomyocytes. These changes were accompanied by significant increase in serum levels of troponin-T, creatine phosphokinase-MB (CPK-MB), tumour necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). Co-administration of SIM to ISO-injected rats significantly reduced all these cardiac changes and serum biochemical markers in addition to marked depletion of iNOS and thioredoxin expression in cardiomyocytes. It is concluded that SIM co-administration attenuated ISO-induced cardiac lesions including LVH by inhibiting iNOS expression in cardiomyocytes.

Transforming growth factor beta-1 (TGF-β1) stimulates collagen synthesis in cultured rainbow trout cardiac fibroblasts.

Cold acclimation of rainbow trout, Oncorhynchus mykiss, causes collagen to increase within the extracellular matrix (ECM) of the myocardium, while warm acclimation has the opposite effect. The mechanism responsible for this remodelling response is not known. In mammals, transforming growth factor beta-1 (TGF-β1) stimulates collagen deposition within the myocardial ECM. Therefore, we hypothesized that TGF-β1 regulates trout myocardial ECM turnover and predicted that TGF-β1 would induce collagen deposition in cultured rainbow trout cardiac fibroblasts. We found that treatment of trout cardiac fibroblasts with 15 ng ml-1 human recombinant TGF-β1 caused an increase in total collagen at 48 and 72 h and an increase in collagen type I protein after 7 days. We also found that TGF-β1 treatment caused an increase in the transcript abundance of tissue inhibitor of metalloproteinase 2 (timp-2) and matrix metalloproteinase 9 (mmp-9) at 24 h. Cells treated with TGF-β1 also had lower levels of the gene transcript for mmp-2 after 48 h and higher levels of the gene transcript for collagen type I α1 (col1a1) after 72 h. These changes in gene expression suggest that the increase in collagen deposition is due to a decrease in the activity of matrix metalloproteinases and an increase in collagen synthesis. Together, these results indicate that TGF-β1 is a regulator of ECM composition in cultured trout cardiac fibroblasts and suggest that this cytokine may play a role in regulating collagen content in the trout heart during thermal acclimation.

Evaluation effect of low level Helium-Neon laser and Iranian propolis extract on Collagen Type I gene expression by human gingival fibroblasts: an &lt;i&gt;in vitro&lt;/i&gt; study

production of collagen by fibroblast cells is a key component in wound healing. Several studies have shown that low level laser therapy (LLLT) and propolis extract stimulate collagen Type I production. The aim of this study is to evaluation the combined effect of LLL helium neon (632.8 nm) and Iranian propolis extract on collagen Type I gene expression by human gingival fibroblasts (HGF3-PI 53). Human gingival fibroblasts after culturing divided into six experimental groups: G1-control group, which received no irradiation and propolis extract, G2-irradiated at1.5 J/cm2, G3-irradiated at 0.15 J/cm2, G4-recived extract of propolis, G5- combined extract of propolis and 1.5 J/cm2 laser irradiation and G6- combined extract of propolis and 0.15 J/cm2 laser irradiation. The experiments were conducted in triplicate. After 24 hour, the total RNA was extracted and cDNA synthesis was performed. Type I collagen mRNA expression was determined with real time PCR. The obtained results illustrated a statistically significant difference between G3 (0.15 J/cm2) and G1 (control group) in levels of collagen Type I messenger RNA (mRNA) expression (p<0.05). The irradiated cells showed a 1.4 times increase in mRNA expression of the collagen Type I gene. Expression of this gene decreases in other groups that this difference was statistically significant. LLLT in different dosage and propolis extract may result in decreased or increased collagen type I gene expression. However this effect should be investigated in clinical studies.

Chondrogenic Differentiation of Human Chondrocytes and Stem Cells in Different Cell Culture Systems Using IGF-1-Coupled Particles

Various cell-based therapies use the transplantation of ex vivo cultured chondrocytes or stem cells to support repair of cartilage defects. Cell expansion in vitro is required prior to transplantation accompanied by cell dedifferentiation, resulting in unwanted fibrocartilage formation in vivo. Targeted application of growth factors during in vitro cultivation is intended to enhance chondrogenic differentiation of cells. In previous studies, collagen-based scaffolds enriched with silica particles coupled with the insulin-like growth factor (IGF) 1 were tested, concerning their suitability to increase the in vitro redifferentiation of human chondrocytes. Accordingly, in the present study chondrogenic differentiation potential of IGF-1-coupled particles was investigated using human chondrocytes cultured in scaffold-free spheroid pellet culture. Further, influence of IGF-1-coupled particles on mesenchymal stem cells derived from bone marrow (BM-MSCs) cultured onto collagen–based scaffold or in pellet culture was examined as well pellet culture was examined. Chondrogenic differentiation was induced by the growth factor IGF-1 applied as I) soluble IGF-1 or II) conjugated to red fluorescent silica particles. In addition, control silica particles conjugated with NH2 were used to exclude adverse side effects. Besides cell proliferation, collagen type II and glycosaminoglycan synthesis was quantified and histological staining performed to investigate the chondrogenic differentiation. In pellet culture, IGF-1-coupled particles were applied during the pellet formation only. Traceable red fluorescent particles showed homogenous distribution within the pellets. Adverse effects were not detected. Human chondrocyte pellets displayed significantly increased collagen type II synthesis using IGF-1-coupled particles, compared to soluble IGF-1. Independent of the application mode, induction of chondrogenic differentiation of BM-MSCs cultured in pellets was not suitable with the addition of IGF-1 only. However, BM-MSCs cultivation onto collagen-based scaffold enriched with IGF-1-coupled particle showed superior glycosaminoglycan synthesis, compared to soluble IGF-1 application. Using IGF-1 coupled to particles within a three-dimensional matrix resulted in an increased stimulatory chondrogenic effect, indicating a promising tool for controlled growth factor delivery during treatment of cartilage lesion.

Impaired contraction and decreased detrusor innervation in a female rat model of pelvic neuropraxia

Bilateral pelvic nerve injury (BPNI) is a model of post-radical hysterectomy neuropraxia, a common sequela. This study assessed the time course of changes to detrusor autonomic innervation, smooth muscle (SM) content and cholinergic-mediated contraction post-BPNI. Female Sprague-Dawley rats underwent BPNI or sham surgery and were evaluated 3, 7, 14, and 30days post-BPNI (n = 8/group). Electrical field-stimulated (EFS) and carbachol-induced contractions were measured. Gene expression was assessed by qPCR for muscarinic receptor types 2 (M2) and 3 (M3), collagen type 1α1 and 3α1, and SM actin. Western blots measured M2 and M3 protein expression. Bladder sections were stained with Masson's trichrome for SM content and immunofluorescence staining for nerve terminals expressing vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH), and neuronal nitric oxide synthase (nNOS). Bilateral pelvic nerve injury caused larger bladders with less SM content and increased collagen type 1α1 and 3α1 gene expression. At early time points, cholinergic-mediated contraction increased, whereas EFS-mediated contraction decreased and returned to baseline by 30days. Protein and gene expression of M3 was decreased 3 and 7days post-BPNI, whereas M2 was unchanged. TH nerve terminals surrounding the detrusor decreased in all BPNI groups, whereas VAChT and nNOS terminals decreased 14 and 30days post-BPNI. Bilateral pelvic nerve injury increased bladder size, impaired contractility, and decreased SM and autonomic innervation. Therapeutic strategies preventing nerve injury-mediated decline in neuronal input and SM content may prevent the development of a neurogenic bladder and improve quality of life after invasive pelvic surgery.

Knockdown of TREM-1 suppresses IL-1β-induced chondrocyte injury via inhibiting the NF-κB pathway

Triggering receptor expressed on myeloid cells 1 (TREM-1) is a recently discovered molecule that modulates inflammatory responses. This study aimed to investigate the specific function of TREM-1 in chondrocytes and its association with the pathophysiology of osteoarthritis (OA). We observed upregulation of TREM-1 in OA cartilage compared to normal tissues. Knockdown of TREM-1 suppressed interleukin 1 beta (IL-1β)-induced extracellular matrix (ECM) metabolic imbalance, pro-inflammatory cytokine production, decrease in cell viability and apoptosis. Mechanistic analyses further revealed that IL-1β-induced activation of the NF-κB pathway is suppressed upon TREM-1 knockdown, similar to the effect of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB. TREM-1 expression was consistently increased in a mouse OA model in vivo, and its silencing led to inhibition of matrix metallopeptidase-13 (MMP-13) production, increased collagen type II synthesis and decreased NF-κB signaling. Our data collectively suggest that TREM-1 plays a critical in OA development through regulation of NF-κB signaling. Pharmacological inhibition of TREM-1 may therefore present an effective novel therapeutic approach for OA.

Exploring the in vivo wound healing effects of a recombinant hemolin from the caterpillar Lonomia obliqua.

BackgroundHemolin proteins are cell adhesion molecules from lepidopterans involved in a wide range of cell interactions concerning their adhesion properties. However, hemolin’s roles in cell proliferation and wound healing are not fully elucidated. It has been recently reported that rLosac, a recombinant hemolin from the caterpillar Lonomia obliqua, presents antiapoptotic activity and is capable of improving in vitro wound healing. Therefore, this study aimed to explore rLosac’s in vivo effects using a skin wound healing model in rats.MethodsCircular full-thickness wounds in the rat dorsum skin were treated either with rLosac, or with saline (control), allowing healing by keeping the wounds occluded and moist. During the wound healing, the following tissue regeneration parameters were evaluated: wound closure and collagen content. Furthermore, tissue sections were subjected to histological and immunohistochemical analyses.ResultsThe rLosac treatment has demonstrated its capacity to improve wound healing, as reflected in findings of a larger number of activated fibroblasts, proliferation of epithelial cells, increase of collagen type 1, and decrease of inflammatory infiltrate.ConclusionThe findings have indicated the rLosac protein as a very promising molecule for the development of new wound-healing formulations.

Extracellular matrix remodeling derangement in ex-obese patients.

A known consequence of the large weight loss after bariatric surgery is the appearance of large skinfolds, particularly in the abdomen region of the patients. The balance between the synthesis of extracellular matrix (ECM) components and their proteolysis, mainly by fibrinolytic systems and matrix metalloproteases (MMPs), may be disturbed in these patients. The causes underlying the deregulation of ECM remodeling that occurs in these patients are not, however, clear. We investigated molecular mechanisms responsible for this dysfunction of ECM remodeling process, comparing it to normal skin. Collagen types, MMP2 and MMP9 expression and activity, interleukins 1β (IL1β) and 6 (IL6), and transcription coactivator PGC-1β expression were analyzed in 16 patients. Ex-obese patients presented increased expression of collagen types III and IV mRNA, increased expression of MMP2, decreased expression and activity of MMP9, and increased expression of PGC-1β in the skin. Inflammation markers IL1β and IL6 mRNA were not different. We have demonstrated that obese patients with extensive weight loss after bariatric surgery have increased expression of PGC-1β in the skin, which can result in a decreased expression and activity of MMP9 and increased collagen types III and IV deposition. These molecular changes may contribute for the formation of saggy skinfolds observed in these patients and impair wound healing.

Differential regulation of angiotensin II-induced extracellular signal regulated kinase-1/2 and -5 in progressive glomerulonephritis.

Extracellular signal regulated kinase (ERK)1/2 and ERK5 are key kinases of the signalling pathways involved in various cellular responses to kidney injury; however, the mechanistic links between those kinase and renin-angiotensin system (RAS) activations in glomerulonephritis (GN) have not been fully elucidated. In this study, we sought to clarify the potential roles of ERK1/2 and ERK5 via RAS activation in the pathogenesis of GN. A rat model of progressive GN was induced by anti-glomerular basement membrane (GBM) injection and the signal transduction pathway in angiotensin II (Ang II)-induced glomerular pathologic alterations were investigated in primary cultured mesangial cells (MCs). Rats developed typical cellular crescents in glomeruli on day 7 that progressed to severe fibrocellular crescents and glomerulosclerosis on day 28. Strong expression of phospho-ERK1/2 was observed on day 7 and phospho-ERK5 expression was markedly increased on day 28 of GN. An angiotensin II type 1 receptor blocker (ARB) suppressed those augmentations. Moreover, ARB treatment attenuated the increases in macrophage infiltration and PCNA-positive cells observed on day 7 in GN rats, as well as the increase in collagen type 1 expression on day 28. Consistently, MCs stimulated by Ang II showed significant increases in proliferation and the expression of MCP-1 and collagen type 1. Interestingly, while the ERK1/2 inhibitor PD98059 abolished the elevations in MCP-1 expression and cell proliferation, the ERK5 inhibitor BIX02189 abrogated the elevation in collagen type 1 expression. Altogether, these data suggest that ERK1/2 regulates acute inflammatory reactions, while ERK5 promotes the development of RAS-induced chronic glomerular fibrosis activation in GN.