Proteoglycan Production Research Articles

Consensus Agreements on Regenerative Aesthetics: A Focus on Regenerative Biostimulation With Calcium Hydroxylapatite.

A growing population of patients is seeking treatments that not only affect their overlying features but also restore a more biologically youthful structure and function to the underlying tissue. These strategies are part of what is known as regenerative aesthetics (RA). As an emergent field, clarity regarding the precise definitions and aims of RA and methods to measure the regenerative capacity of RA treatments are lacking. A panel of 6 multidisciplinary experts discussed the foundational aspects of RA. Consensus statements covered aspects of RA including terminology, goals of treatment, treatment strategies, and biological benchmarks indicating regeneration. Consensus on a statement was defined as ≥75% agreement. Panelists emphasized the importance of natural, youthful tissue architecture and function including cellular and extracellular components. Replacement of a single biological component was not considered sufficient for an aesthetic treatment to be described as regenerative. Rather, the relative amounts, ratios, types, and organization are important to determine regenerative potential. Calcium hydroxylapatite is an example of an aesthetic injectable with evidence of regenerative capacity, as demonstrated by its ability to improve collagen type I/III ratios as well as induce the production of elastin and proteoglycans, which ultimately improve measures of skin quality.

Calcium Hydroxylapatite in Regenerative Aesthetics: Mechanistic Insights and Mode of Action.

Calcium Hydroxylapatite-Carboxymethylcellulose (CaHA-CMC) is a widely used soft-tissue filler known for its compatibility with human tissue and for its effective volumizing properties. The biodegradable CaHA microspheres function as a scaffold for the formation of new tissue by stimulating a variety of cellular responses leading to the production of collagen, elastin, vasculature and proteoglycans and thereby enhancing skin quality. Despite the promising regenerative effects of CaHA-CMC, substantial gaps remain in understanding its precise underlying mechanisms, and addressing these gaps is crucial for optimizing its clinical applications. A literature search was conducted using PubMed, google scholar and web of science for key words including "calcium hydroxylapatite", " tissue regeneration", "macrophages", "fibroblasts" and "calcium". This review elucidates the novel mechanisms of CaHA-CMC action within aesthetic medicine, with a focus on the dynamic interactions between macrophages and fibroblasts. The key role of calcium ions as critical biocues in directing skin regeneration pathways is explored alongside an examination of the distinct physicochemical properties of CaHA-CMC. CaHA-CMC helps in tissue regeneration by providing calcium ions that act as key biological cues, promoting fibroblast activity and collagen production.

Identification and culture of meniscons, meniscus cells with their pericellular matrix

Background aimsMeniscus injury is highly debilitating and often results in osteoarthritis. Treatment is generally symptomatic; no regenerative treatments are available. “Chondrons,” articular chondrocytes with preserved pericellular matrix, produce more hyaline cartilage extracellular matrix and improve cartilage repair. If meniscons exist in the meniscus and have similar therapeutic potential as chondrons, employing these cells has potential for meniscus cell therapy and tissue engineering. In this study, we isolated and cultured “meniscons,” meniscus cells surrounded by their native pericellular matrix, and investigated cell behavior in culture compared with chondrons. MethodsHuman meniscons were enzymatically isolated from osteoarthritic menisci and cultured up to 28 days in fibrin glue. Freshly isolated meniscons and chondrons were analyzed by histology and transmission electron microscopy. We used 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein hydrochloride labeling and type VI collagen immunohistochemistry to image pericellular matrix after 0 and 28 days of culture. Gene expression was quantified using real-time polymerase chain reaction and DNA content and proteoglycan production were analyzed using biochemical assays. ResultsMeniscons were successfully isolated from human meniscus tissue. The pericellular matrix of meniscons and chondrons was preserved during 28 days of culture. Meniscons and chondrons had similar cell proliferation and proteoglycan production. Meniscons and chondrons expressed similar levels of collagen type I alpha 1 chain, whereas collagen type II alpha 1 chain and aggrecan expression was lower in the meniscon population. ConclusionsFreshly isolated meniscons and meniscons cultured for 28 days share similarities with chondrons with regard to cell proliferation, morphology and biochemical activity. Rapid isolation of meniscons (45 min) demonstrates potential for one-stage meniscus regeneration and repair, which should be confirmed in vivo.

Mendelian randomization reveals interactions of the blood proteome and immunome in mitral valve prolapse

BackgroundMitral valve prolapse (MVP) is a common heart disorder characterized by an excessive production of proteoglycans and extracellular matrix in mitral valve leaflets. Large-scale genome-wide association study (GWAS) underlined that MVP is heritable. The molecular underpinnings of the disease remain largely unknown.MethodsWe interrogated cross-modality data totaling more than 500,000 subjects including GWAS, 4809 molecules of the blood proteome, and genome-wide expression of mitral valves to identify candidate drivers of MVP. Data were investigated through Mendelian randomization, network analysis, ligand-receptor inference and digital cell quantification.ResultsIn this study, Mendelian randomization identify that 33 blood proteins, enriched in networks for immunity, are associated with the risk of MVP. MVP- associated blood proteins are enriched in ligands for which their cognate receptors are differentially expressed in mitral valve leaflets during MVP and enriched in cardiac endothelial cells and macrophages. MVP-associated blood proteins are involved in the renewal-polarization of macrophages and regulation of adaptive immune response. Cytokine activity profiling and digital cell quantification show in MVP a shift toward cytokine signature promoting M2 macrophage polarization. Assessment of druggability identify CSF1R, CX3CR1, CCR6, IL33, MMP8, ENPEP and angiotensin receptors as actionable targets in MVP.ConclusionsHence, integrative analysis identifies networks of candidate molecules and cells involved in immune control and remodeling of the extracellular matrix, which drive the risk of MVP.

Myosin light chain phosphatase is a downstream target of Rho-kinase in endothelin-1-induced transactivation of the TGF-β receptor.

Rho-kinase (ROCK) regulates actomyosin contraction, coronary vasospasm, and cytoskeleton dynamics. ROCK and of NADPH oxidase (NOX) play an essential role in cardiovascular disease and proteoglycan synthesis, which promotes atherosclerosis by trapping low density lipoprotein. ROCK is activated by endothelin-1 (ET1) and transactivates the transforming growth factor beta receptor (TGFβR1), intensifying Smad signaling and proteoglycan production. This study aimed to identify the role of myosin light chain phosphatase (MLCP) as a downstream target of ROCK in TβR1 transactivation. Vascular smooth muscle cells were treated with ET1 and inhibitors of ROCK and MLCP were added. The phosphorylation levels of Smad2C, myosin light chain (MLC), and MLCP were monitored by western blot, and the mRNA expression of chondroitin 4-O-sulfotransferase 1 (C4ST1) was assessed by quantitative real-time PCR. We examined ROCK's role in ET1-induced TGFβR1 activation. ROCK phosphorylated MLCP at the MYPT1 T853 residue, blocked by the ROCK inhibitor Y27632. ROCK also increased MLC phosphorylation and actomyosin contraction in response to ET1, enhanced by the phosphatase inhibitor Calyculin A. Calyculin A also increased C4ST1 expression, GAG-chain synthesizing enzymes. This work suggests that ROCK is involved in ET1-mediated TβR1 activation through increased MLCP phosphorylation, which leads to Smad2C phosphorylation and stimulates C4ST1 expression.

Formulation and Characterization of Glucosamine Sulphate Potassium Chloride (GSPC) Loaded Emulgel for the Treatment of Osteoarthritis.

Osteoarthritis (OA) is becoming a major medical burden worldwide due to changing lifestyles and aging populations. Osteoarthritis is a disease characterized by a variety of anatomic and physiological changes to joints, including cartilage degradation, bone remodeling, and the formation of osteophytes. These changes cause pain, stiffness, swelling, and limitations in joint function. Glucosamine serves as a fundamental constituent for cartilage, the resilient connective tissue responsible for cushioning joints. Glucosamine Sulphate Potassium Chloride (GSPC) supplementation is widely employed to mitigate symptoms linked to osteoarthritis, a degenerative joint disorder hallmarked by cartilage degradation. Palliative care aims at minimizing pain and disability and improving function, performance, and quality of life. In this study, the emulgel formulation of GSPC was developed and checked for its potential. Currently, OA does not have a definitive treatment. Since conventional dosage forms cannot deliver the active drug content at a predefined target site in a predictable manner throughout the treatment period, a new carrier system is always required. Considering their reduced size, targeting potential, and site specificity, nanocarrier-based approaches could hold an answer to shortcomings associated with conventional routes. Thus, the objective of the current study was to formulate and characterize glucosamine sulphate potassium chloride-loaded emulgel for the treatment of osteoarthritis. Microemulsion of glucosamine sulphate potassium chloride was formulated using a spontaneous emulsification method comprising of oleic acid (oil phase), Tween 80, Tween 20 (surfactant) and PEG 400, Span 80 (co-surfactant), and distilled water (aqueous phase). The microemulsions were evaluated for surface morphology, globule size, poly-dispersibility index (PDI), zeta potential, and viscosity, and the final batch of microemulsions was selected. The optimized microemulsion contained 35% co-surfactant (propylene glycol), 20% surfactant (Tween 20), and 15% oil (oleic acid) and glucosamine sulphate potassium chloride in a dose of 60 mg, which has sufficient drug loading capacity with a droplet size of 182 nm for optimized formulation. The optimized microemulsion formulation was added to gel prepared by Carbopol 934 in a 1:1 (w/w) ratio, leading to the formulation of glucosamine sulphate potassium chloride- containing emulgel. The prepared emulgel was further evaluated for viscosity, drug content, pH, and in-vitro drug release. Emulgel formulation (F6) showed 88% drug release after 6 hours, and it followed the Higuchi model. Glucosamine Sulphate Potassium Chloride (GSPC) is used in the treatment of OA by increasing the production of proteoglycans, which can cause the cartilage to break down. Emulgel formulation (F3) showed 75.41% drug release, and formulation (F6) showed 88% drug release after 6 h. Therefore, it may be concluded that an emulgel of GSPC can be used as a controlled-release dosage form of the drug for local application in OA.

The Actin Cytoskeleton as a Regulator of Proteoglycan 4.

The superficial zone (SZ) of articular cartilage is responsible for distributing shear forces for optimal cartilage loading and contributes to joint lubrication through the production of proteoglycan 4 (PRG4). PRG4 plays a critical role in joint homeostasis and is chondroprotective. Normal PRG4 production is affected by inflammation and irregular mechanical loading in post-traumatic osteoarthritis (PTOA). THe SZ chondrocyte (SZC) phenotype, including PRG4 expression, is regulated by the actin cytoskeleton in vitro. There remains a limited understanding of the regulation of PRG4 by the actin cytoskeleton in native articular chondrocytes. The filamentous (F)-actin cytoskeleton is a potential node in crosstalk between mechanical stimulation and cytokine activation and the regulation of PRG4 in SZCs, therefore developing insights in the regulation of PRG4 by actin may identify molecular targets for novel PTOA therapies. A comprehensive literature search on PRG4 and the regulation of the SZC phenotype by actin organization was performed. PRG4 is strongly regulated by the actin cytoskeleton in isolated SZCs in vitro. Biochemical and mechanical stimuli have been characterized to regulate PRG4 and may converge upon actin cytoskeleton signaling. Actin-based regulation of PRG4 in native SZCs is not fully understood and requires further elucidation. Understanding the regulation of PRG4 by actin in SZCs requires an in vivo context to further potential of leveraging actin arrangement to arthritic therapeutics.

FGF18 encoding circular mRNA-LNP based on glycerolipid engineering of mesenchymal stem cells for efficient amelioration of osteoarthritis.

Mesenchymal stem cells (MSCs) exhibit substantial potential for osteoarthritis (OA) therapy through cartilage regeneration, yet the realization of optimal therapeutic outcomes is hampered by their limited intrinsic reparative capacities. Herein, MSCs are engineered with circular mRNA (cmRNA) encoding fibroblast growth factor 18 (FGF18) encapsulated within lipid nanoparticles (LNP) derived from a glycerolipid to facilitate OA healing. A proprietary biodegradable and ionizable glycerolipid, TG6A, with branched tails and five ester bonds, forms LNP exhibiting above 9-fold and 41-fold higher EGFP protein expression in MSCs than commercial LNP from DLin-MC3-DMA and ALC-0315, respectively. The introduction of FGF18 not only augmented the proliferative capacity of MSCs but also upregulated the expression of chondrogenic genes and glycosaminoglycan (GAG) content. Additionally, FGF18 enhanced the production of proteoglycans and type II collagen in chondrocyte pellet cultures in a three-dimensional culture. In an OA rat model, transplantation with FGF18-engineered MSCs remarkably preserved cartilage integrity and facilitated functional repair of cartilage lesions, as evidenced by thicker cartilage layers, reduced histopathological scores, maintenance of zone structure, and incremental type II collagen and extracellular matrix (ECM) deposition. Taken together, our findings suggest that TG6A-based LNP loading with cmRNA for engineering MSCs present an innovative strategy to overcome the current limitations in OA treatment.

Positive Regulation of S-Adenosylmethionine on Chondrocytic Differentiation via Stimulation of Polyamine Production and the Gene Expression of Chondrogenic Differentiation Factors.

S-adenosylmethionine (SAM) is considered to be a useful therapeutic agent for degenerative cartilage diseases, although its mechanism is not clear. We previously found that polyamines stimulate the expression of differentiated phenotype of chondrocytes. We also found that the cellular communication network factor 2 (CCN2) played a huge role in the proliferation and differentiation of chondrocytes. Therefore, we hypothesized that polyamines and CCN2 could be involved in the chondroprotective action of SAM. In this study, we initially found that exogenous SAM enhanced proteoglycan production but not cell proliferation in human chondrocyte-like cell line-2/8 (HCS-2/8) cells. Moreover, SAM enhanced gene expression of cartilage-specific matrix (aggrecan and type II collagen), Sry-Box transcription factor 9 (SOX9), CCN2, and chondroitin sulfate biosynthetic enzymes. The blockade of the methionine adenosyltransferase 2A (MAT2A) enzyme catalyzing intracellular SAM biosynthesis restrained the effect of SAM on chondrocytes. The polyamine level in chondrocytes was higher in SAM-treated culture than control culture. Additionally, Alcian blue staining and RT-qPCR indicated that the effects of SAM on the production and gene expression of aggrecan were reduced by the inhibition of polyamine synthesis. These results suggest that the stimulation of polyamine synthesis and gene expression of chondrogenic differentiation factors, such as CCN2, account for the mechanism underlying the action of SAM on chondrocytes.

Recovery of Forearm and Fine Digit Function After Chronic Spinal Cord Injury by Simultaneous Blockade of Inhibitory Matrix Chondroitin Sulfate Proteoglycan Production and the Receptor PTPσ.

Spinal cord injuries (SCI), for which there are limited effective treatments, result in enduring paralysis and hypoesthesia, in part because of the inhibitory microenvironment that develops and limits regeneration/sprouting, especially during chronic stages. Recently, we discovered that targeted enzymatic removal of the inhibitory chondroitin sulfate proteoglycan (CSPG) component of the extracellular and perineuronal net (PNN) matrix via Chondroitinase ABC (ChABC) rapidly restored robust respiratory function to the previously paralyzed hemi-diaphragm after remarkably long times post-injury (up to 1.5 years) following a cervical level 2 lateral hemi-transection. Importantly, ChABC treatment at cervical level 4 in this chronic model also elicited improvements in gross upper arm function. In the present study, we focused on arm and hand function, seeking to highlight and optimize crude as well as fine motor control of the forearm and digits at lengthy chronic stages post-injury. However, instead of using ChABC, we utilized a novel and more clinically relevant systemic combinatorial treatment strategy designed to simultaneously reduce and overcome inhibitory CSPGs. Following a 3-month upper cervical spinal hemi-lesion using adult female Sprague Dawley rats, we show that the combined treatment had a profound effect on functional recovery of the chronically paralyzed forelimb and paw, as well as on precision movements of the digits. The regenerative and immune system related events that we describe deepen our basic understanding of the crucial role of CSPG-mediated inhibition via the PTPσ receptor in constraining functional synaptic plasticity at lengthy time points following SCI, hopefully leading to clinically relevant translational benefits.

Proteogenomic Analysis of Human Uterine Cervical Cancer (UCC) Reveals Treatment-Resistant Subtypes of UCC

Locally advanced uterine cervical cancer (UCC) is treated by radiotherapy with concurrent chemotherapy, but heterogenous treatment responses are frequently observed. To better optimize therapeutic options based on molecular signatures, we performed proteogenomic analysis of UCC. UCC tissue and blood samples were collected from patients who underwent primary radiotherapy ± chemotherapy at the National Cancer Center (NCC) in Korea from July 2004 to March 2020. Most samples were obtained via biopsy. Genomic DNA for WES was isolated from frozen biopsy tumor tissues and peripheral blood buffy-coat of patients. Both global proteome and phosphoproteome were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Whole exome sequencing, RNA sequencing, global proteomics and phosphoproteomics were performed using 253, 337, and 147 cervical cancer samples, respectively. Patient-derived xenograft were established using intratongue implantation using 2 primary cell lines from sub3 and sub5, and flow cytometric analysis was performed. Fourteen significantly mutated genes (SMG) were found in our study cohort which include 5 newly identified SMGs. Mutation-phosphorylation analysis revealed association with apoptosis and actin cytoskeleton pathway. Proteogenomic analysis defined 6 molecular subtypes of UCC. Of those, 3 subtypes (i.e., Sub3, Sub5 and Sub6) were associated with treatment-resistant phenotypes. The cell-type deconvolution analysis suggested activated stroma with activation of cancer-associated fibroblast in Sub 3, while Sub5 showed low levels of activated stroma and high levels of myeloid immune cells. FACS analysis of UCC mouse models established from these 2 radio-resistant primary cell lines showed high component of PDGFRA+CAF infiltration in Sub 3, and high level of PVR+CD45+ immune cells mainly composed of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) in Sub5. For Sub6, genes and/or protein signatures represented mucin-related processes (e.g., mucin glycosylation/sugar metabolism), which are linked to metastasis-associated Tn antigen production. The proteogenomic analysis thus suggests potential targets for radiotherapy-resistant subtypes of UCC; secretory factors from activated stroma and cancer-associated fibroblast (Sub3); RHOA signaling, PVR, and PMN-MDSCs (Sub5), and keratin/chondroitin sulfate proteoglycan and Tn antigen production (Sub6). Our study shows the importance of proteogenomic analysis in unveiling the subtype specific molecular pathways of UCC that are beyond reach by genomic data alone. The validity of our molecular pathway and cellular signatures linking these pathways should be further validated through detailed functional experiments and in larger UCC cohorts.

Fibronectin-coated polyurethane meniscal scaffolding supplemented with MSCs improves scaffold integration and proteoglycan production in a rabbit model.

The role of mesenchymal stem cells (MSC) in supporting the formation of new meniscal tissue in a meniscal scaffold is not well understood. The objective of this study was to assess the quality of the meniscal tissue produced in a fibronectin (FN)-coated polyurethane (PU) meniscal scaffold after a meniscal injury was made in an experimental rabbit model. Twelve New Zealand white rabbits were divided in two groups after performing a medial meniscectomy of the anterior horn. In group 1, the meniscal defect was reconstructed with a non-MSC supplemented FN-coated PU scaffold. On the other hand, the same scaffold supplemented with MSCs was used in group 2. The animals were sacrificed at 12week after index surgery. A modified scoring system was used for histological assessment. This new scoring (ranging from 0 to 15) includes a structural evaluation (meniscal scaffold interface and extracellular matrix production) and tissue quality evaluation (proteoglycan and type I-collagen content). The meniscal scaffold was found loose in the joint in three cases, corresponding to two cases in group 1 and 1 case in group 2. No differences were observed between the groups in terms of the total score (7.0 ± 0.9 vs. 9.4 ± 2.6, p = 0.09). However, differences were observed in group 2 in which 2 out of the 5 scored items, scaffold integration (1 ± 0.0 vs. 1.9 ± 0.6, p = 0.03) and proteoglycan production (1.2 ± 0.3 vs. 2.4 ± 0.2, p = 0.001). A trend to a higher production of Type I-Collagen production was also observed in group 2 (1.1 ± 0.4 vs. 1.4 ± 0.7, p = 0.05). In a rabbit model at 12weeks, the adhesion of MSCs to a FN-coated PU scaffold improves scaffold integration, proteoglycan production and the characteristics of the new meniscal-like tissue obtained when compared to a non-supplemented scaffold. This fact could be a major step toward improving the adhesion of the MSCs to meniscal scaffolds and, consequently, the obtention of better quality meniscal tissue.

Proton and Carbon Ion Irradiation Changes the Process of Endochondral Ossification in an Ex Vivo Femur Organotypic Culture Model.

Particle therapy (PT) that utilizes protons and carbon ions offers a promising way to reduce the side effects of radiation oncology, especially in pediatric patients. To investigate the influence of PT on growing bone, we exposed an organotypic rat ex vivo femur culture model to PT. After irradiation, histological staining, immunohistochemical staining, and gene expression analysis were conducted following 1 or 14 days of in vitro culture (DIV). Our data indicated a significant loss of proliferating chondrocytes at 1 DIV, which was followed by regeneration attempts through chondrocytic cluster formation at 14 DIV. Accelerated levels of mineralization were observed, which correlated with increased proteoglycan production and secretion into the pericellular matrix. Col2α1 expression, which increased during the cultivation period, was significantly inhibited by PT. Additionally, the decrease in ColX expression over time was more pronounced compared to the non-IR control. The chondrogenic markers BMP2, RUNX2, OPG, and the osteogenic marker ALPL, showed a significant reduction in the increase in expression after 14 DIV due to PT treatment. It was noted that carbon ions had a stronger influence than protons. Our bone model demonstrated the occurrence of pathological and regenerative processes induced by PT, thus building on the current understanding of the biological mechanisms of bone.

Fibronectin sensitizes activation of contractility, YAP, and NF-κB in nucleus pulposus cells.

Intervertebral disc degeneration involves the breakdown of the discs of the spine due to genetics, aging, or faulty mechanical loading. As part of the progression of the disease, nucleus pulposus cells lose their phenotypic characteristics, inducing inflammation and extracellular matrix (ECM) alterations that result in a loss of disc mechanical homeostasis. Fibronectin is one ECM molecule that has been shown to be upregulated in disc degeneration and plays an important role in the progression of a wide variety of fibrotic diseases. Fragments of fibronectin have also long been associated with both osteoarthritis and disc degeneration. The goal of this work isto test the effects of fibronectin on disc cell phenotype, mechanosensing, and inflammatory signaling. We identify that fibronectin increases the activation of cellular contractility, the mechanosensitive transcription factor Yes-associated protein, and the inflammatory transcription factor nuclear factor-κB. This results in decreased production and expression of proteoglycans, which are required to maintain healthy disc function. Thus, fibronectin is a potential regulator of phenotypic changes in disc degeneration, and a potential target for treating disc degeneration at the cellular level. Understanding the role of fibronectin, and its potential as a therapeutic target, could provide new approaches for preventing or reversing disc degeneration.

Optimization of treatment of vulvar scleroatrophic lichen with using modern laser technologies

Background. Vulvar lichen sclerosus (VLS) is a chronic inflammatory dermatosis, which is characterized by focal skin atrophy of the vulva and mucous membranes. It leads to deformation of the perineum. The prevalence of VLS in children is up to 0.1%, among the adults — 3%. The average age of the first symptoms appearance in girls is 5.4 years, in women — 55.1 years. The diagnosis of VLS is made on the base of anamnesis, assessment of pubertal development, physical examination of the external genitalia, advanced vulvoscopy and vaginoscopy, bacteriological, bacterioscopic and cytological studies of vaginal wet mount, assessment of the hormonal profile. The treatment methods include high-potency steroids as the initial treatment, usage of diode, fractional CO2 and erbium lasers with different wavelengths and injections of platelet-rich plasma to improve the regeneration processes of the affected tissue by improving collagenogenesis, vasculogenesis, production of proteoglycans and glycoproteins.
 The aim of the study is to compare the effectiveness of photodestruction with a manual diode laser of the Dornier Medilas D LiteBeam, wavelength of 940 nm, minimum power in pulsed mode on the girls’ vulva mucosa with various types of VLS during neutral and preadolescent age. To obtain the results there were analyzed criteria including the size of the lesion after photodestruction therapy, the disappearance of cracks and angiomatosis in the LS area. The objectives of the complex therapy of VLS in girls were elimination of itching, decrease of the inflammatory changes, improvement of the skin and mucosa blood supply, and stimulation of local immunological mechanisms.
 Materials and methods. The study was conducted on the base of the gynecological department of Russian Children’s Clinical Hospital. The effectiveness of the laser-induced thermotherapy with a wavelength of 940 nm, minimum power in pulsed mode on the vulva of VLS girls of neutral and preadolescent periods as a part of the complex therapy was evaluated. Ninety 5 to 13 year VLS patients took part in the trial. They were divided into 3 groups. The first group included 30 patients (average age 7.5 ± 1.1) with atrophic form of lichen treated with a laser with a wavelength of 940 nm. The second group consisted of 30 patients (average age 6.8 ± 1.3) with erythematous-edematous lichen, who were treated with a laser with a wavelength of 635 nm. The third group included 30 patients (average age 7.3 ± 1.2) with erosive and ulcerative form of VLS, who received high-potency corticosteroids therapy. 
 Results. The treatment results in the three subgroups of VLS patients show a diode laser with a wavelength of 940 nm therapy to be the most effective. After the first treatment course 27 (90%) patients in the first group showed a positive trend with a decrease in itching and burning, pink-color presence in vulva mucousа, a decrease of the skin pigmentation area and an increase in the elasticity of the skin. After 2 courses, 29 (96.7%) patients had positive trend. The second most effective method of treatment is photodestruction with a 635 nm wavelength laser. After the 4 courses (two hospitalisations), 25 (83,3%) patients noted a decrease in itching and in a “tightening” feeling. In 24 (80%) patients there was an improvement in the condition of the mucous membranes of the vulva. Reduction of edema was observed in 23 (76.7%) patients by the time of an examination in 3 weeks after the last therapy course. However, it was not possible to completely restore the mucous membranes of the vulva in patients of this group. The least effective — drug therapy with high-potency steroids. 8 (26.7%) patients noted positive dynamics only by the 4th day therapy, 11 (36,7%) by the time of the 3 weeks after the initial application of the ointment, 12 (40%) by the 2nd month. In 5 (16.7%) patients, the visible condition of the mucous membranes did not change, the number of bleeding ulcers decreased minimally.
 Conclusions. The results of the trial indicate the high efficiency of laser therapy of VLS in children compared with the topical glucocorticoids treatment. The duration of the therapy and condition of the external genitalia after treatment depends on the histological form and stage of VLS. According to our study, laser therapy contributes to the remission onset in pediatric patients with different types of VLS in the shortest possible time after the photodestruction procedure.

The Influence of Arthroscopic Shaver Mincing and Platelet-Rich Plasma on Chondrocytes of Intraoperatively Harvested Human Cartilage.

Minced cartilage implantation (MCI) has seen a renaissance in recent years. In this evolved technique, human articular cartilage is harvested with an arthroscopic shaver, augmented with platelet-rich plasma (PRP), and implanted with autologous thrombin. This modified technique combines the possibility of cell-based surgical cartilage repair with a minimally invasive autologous 1-step procedure. However, evidence on cell survival and preserved function after shaver-based mincing and PRP supplementation is limited. To evaluate the effects of arthroscopic shaver mincing and augmentation with PRP on human cartilage tissue. Controlled laboratory study. Standardized samples were taken from 12 donors during autologous MCI. A comparison of cell outgrowth, cell viability, proliferation capacity, and ability to produce extracellular matrix-specific proteoglycans after chondrogenic redifferentiation was made between cartilage taken by curettage from the border of the cartilage defect, cartilage tissue minced by an arthroscopic shaver, and cartilage tissue minced by an arthroscopic shaver that was additionally augmented with autologous PRP. There was no difference between all 3 groups in terms of cell outgrowth or proliferation capacity. Metabolic activity relative to the cell number of chondrocytes isolated from shaver-minced cartilage was higher compared with chondrocytes isolated from cartilage that was derived by curettage or shaver-minced cartilage that was augmented with PRP. After chondrogenic stimulation, the normalized proteoglycan content was higher in spheroids of cells derived from shaver-minced cartilage augmented with PRP than in spheroids of cells derived from curettage. A high correlation of cell outgrowth, proliferation capacity, and viability between isolated cells from all 3 groups taken from an individual donor was observed. Chondrocytes isolated from human cartilage tissue that was harvested and minced with an arthroscopic shaver remained viable and proliferative. The augmentation of shaver-minced cartilage with PRP led to the enhanced proteoglycan production of chondrogenic spheroids in vitro, pointing toward the development of a cartilage-specific extracellular matrix. This in vitro study yields promising results regarding the use of an arthroscopic shaver and augmentation with PRP in the context of MCI. Knowledge that shaver mincing and augmentation with PRP are feasible for processing articular cartilage during MCI is highly relevant for surgical cartilage repair.

Understanding Snail Mucus Biosynthesis and Shell Biomineralisation through Genomic Data Mining of the Reconstructed Carbohydrate and Glycan Metabolic Pathways of the Giant African Snail (Achatina fulica).

The giant African snail (Order Stylommatophora: Family Achatinidae), Achatina fulica (Bowdich, 1822), is the most significant and invasive land snail pest. The ecological adaptability of this snail involves high growth rate, reproductive capacity, and shell and mucus production, driven by several biochemical processes and metabolism. The available genomic information for A. fulica provides excellent opportunities to hinder the underlying processes of adaptation, mainly carbohydrate and glycan metabolic pathways toward the shell and mucus formation. The authors analysed the 1.78 Gb draft genomic contigs of A. fulica to identify enzyme-coding genes and reconstruct biochemical pathways related to the carbohydrate and glycan metabolism using a designed bioinformatic workflow. Three hundred and seventy-seven enzymes involved in the carbohydrate and glycan metabolic pathways were identified based on the KEGG pathway reference in combination with protein sequence comparison, structural analysis, and manual curation. Fourteen complete pathways of carbohydrate metabolism and seven complete pathways of glycan metabolism supported the nutrient acquisition and production of the mucus proteoglycans. Increased copy numbers of amylases, cellulases, and chitinases highlighted the snail advantage in food consumption and fast growth rate. The ascorbate biosynthesis pathway identified from the carbohydrate metabolic pathways of A. fulica was involved in the shell biomineralisation process in association with the collagen protein network, carbonic anhydrases, tyrosinases, and several ion transporters. Thus, our bioinformatic workflow was able to reconstruct carbohydrate metabolism, mucus biosynthesis, and shell biomineralisation pathways from the A. fulica genome and transcriptome data. These findings could reveal several evolutionary advantages of the A. fulica snail, and will benefit the discovery of valuable enzymes for industrial and medical applications.

Age and oxidative stress regulate Nrf2 homeostasis in human articular chondrocytes

The purpose of this study was to investigate the effect of age and oxidative stress on regulation of nuclear factor erythroid-2-related factor 2 (Nrf2) in young, old, and osteoarthritic (OA) human articular chondrocytes. Levels of Nrf2 in primary human chondrocytes isolated from young, old, and OA donors were measured by immunoblotting, qPCR, and immunohistochemistry. Effects on levels of Nrf2, antioxidant proteins regulated by Nrf2, as well as p65, and the anabolic response to insulin-like growth factor-1 (IGF-1) were evaluated after induction of oxidative stress with menadione, Nrf2 knockdown with siRNA, and/or Nrf2 activation with RTA-408. Nrf2 protein levels were significantly lower in older adult chondrocytes (∼0.59 fold; p=0.034) and OA chondrocytes (∼0.50 fold; p=0.016) compared to younger cells. Menadione significantly increased Nrf2 protein levels in young chondrocytes by just under four-fold without changes in old chondrocytes. Nrf2 knockdown and activation differentially regulated levels of anti-oxidant proteins including sulfiredoxin and NAD(P)H quinone dehydrogenase 1. Nrf2 activation with RTA-408 also decreased basal p65 phosphorylation, increased aggrecan and type II collagen gene expression, and increased production of proteoglycans in OA chondrocytes treated with IGF-1. Targeted therapeutic strategies aimed at maintaining Nrf2 activity could be useful in maintaining chondrocyte homeostasis through maintenance of intracellular antioxidant function and redox balance.

Glucosamine Sulphate Potassium Chloride in the Management of Osteoarthritis- Considering Emulgel Dosage Form: A Review

Background: In addition to lifestyle, diet, environmental, and physiological factors, genetics also contributes to an increase in mitochondrial vulnerability. With depression, the hypothalamic, pituitary, and adrenal (HPA) axis are overactive, which is linked to higher CNS glucocorticoid levels. In recent years, topical drug delivery has been used on a priority basis compared to other drug delivery systems because of its specific site action and sustained release capacity. An emulsion gel is prepared by an emulsion incorporated into a gel by a gelling agent. Glucosamine Sulphate Potassium Chloride (GSPC) is used to treat osteoarthritis. It does this by increasing the production of proteoglycans, which can cause the cartilage to break down. Objectives: The aim behind choosing the glucosamine sulphate potassium chloride for the management of Osteoarthritis (OA) is that it has many clinical investigations and has topical properties which are effective against osteoarthritis. Method: Osteoarthritis is a prevalent rheumatic musculoskeletal disorder. It is a pervasive disorder affecting people worldwide and can affect any joint, mainly the knees, hips, spine, and hand joints. The review is based on many studies extracted from Google Scholar, Google Patents, etc. Data from other sources were gathered to do further research on osteoarthritis. Result: Extensive literature was studied about Glucosamine Sulphate Potassium Chloride. It has been shown that GSPC is effective against osteoarthritis, and also, some patents regarding GSPC have a therapeutic effect on making our joints healthy. Conclusion: Topical formulations are widely used for better patient compliance, and emulgel is a good choice regarding viscosity and spreadability. GSPC is effective against osteoarthritis; many clinical studies have evidence of this.

E3 Ubiquitin Ligase Constitutive Photomorphogenic 1 Regulates Differentiation and Inflammation via MAPK Signaling Pathway in Rabbit Articular Chondrocytes

Constitutive photomorphogenic 1 (COP1), is an E3 ubiquitin ligase that plays a role in the regulation of various cellular processes including cell growth, differentiation, and survival in mammals. In certain conditions such as overexpression or loss of function, COP1 acts either as an oncogenic protein or as a tumor suppressor by targeting specific proteins for ubiquitination-mediated degradation. However, the precise role of COP1 has not been well studied in primary articular chondrocytes. In this study, we investigated the role of COP1 in chondrocyte differentiation. Western blotting and reverse transcription-polymerase chain reaction analysis demonstrated that COP1 overexpression reduced type II collagen expression, promoted cyclooxygenase 2 (COX-2) expression, and reduced sulfated proteoglycan synthesis, as detected by Alcian blue staining. Upon siRNA treatment, revived type II collagen, sulfated proteoglycan production, and decreased COX-2 expression. Phosphorylation of p38 kinase and ERK-1/-2 signaling pathways was regulated by COP1 upon cDNA and siRNA transfection in chondrocytes. The inhibition of the p38 kinase and ERK-1/-2 signaling pathways with SB203580 and PD98059 ameliorated the expression of type II collagen and COX-2 in transfected chondrocytes, thus suggesting that COP1 regulates differentiation and inflammation in rabbit articular chondrocytes via the p38 kinase and ERK-1/-2 signaling pathway.