Glycosaminoglycan Content Research Articles

Comparative Analysis of Decellularization Methods for the Production of Decellularized Umbilical Cord Matrix.

The importance of decellularized extracellular matrix (dECM) as a natural biomaterial in tissue engineering and regenerative medicine is rapidly growing. The core objective of the decellularization process is to eliminate cellular components while maximizing the preservation of the ECM's primary structure and components. Establishing a rapid, effective, and minimally destructive decellularization technique is essential for obtaining high-quality dECM to construct regenerative organs. This study focused on human umbilical cord tissue, designing different reagent combinations for decellularization protocols while maintaining a consistent processing time. The impact of these protocols on the decellularization efficiency of human umbilical cord tissue was evaluated. The results suggested that the composite decellularization strategy utilizing trypsin/EDTA + Triton X-100 + sodium deoxycholate was the optimal approach in this study for preparing decellularized human umbilical cord dECM. After 5 h of decellularization treatment, most cellular components were eliminated, confirmed through dsDNA quantitative detection, hematoxylin and eosin (HE) staining, and DAPI staining. Meanwhile, Masson staining, periodic acid-silver methenamine (PASM) staining, periodic acid-Schiff (PAS) staining, and immunofluorescent tissue section staining results revealed that the decellularized scaffold retained extracellular matrix components, including collagen and glycosaminoglycans (GAGs). Compared to native umbilical cord tissue, electron microscopy results demonstrated that the microstructure of the extracellular matrix was well preserved after decellularization. Furthermore, Fourier-transform infrared spectroscopy (FTIR) findings indicated that the decellularization process successfully retained the main functional group structures of extracellular matrix (ECM) components. The quantitative analysis of collagen, elastin, and GAG content validated the advantages of this decellularization process in preserving and purifying ECM components. Additionally, it was confirmed that this decellularized matrix exhibited no cytotoxicity in vitro. This study achieved short-term decellularization preparation for umbilical cord tissue through a combined decellularization strategy.

Poster 112: Toxicity of Chlorhexidine and Povidone-Iodine to Rotator Cuff Tendon

Objectives: Antimicrobial wound lavages containing chlorhexidine gluconate (CHG) or povidone-iodine (PI) are commonly used to prevent periprosthetic infection during shoulder arthroplasty. However, the potential toxicity of these solutions to the native surrounding rotator cuff tendon is not well understood. In total shoulder arthroplasty, the rotator cuff tendon is often concomitantly repaired, and any toxic effects from antimicrobial wound lavage during surgery may be detrimental to postoperative rotator cuff tendon healing. The purpose of this study was to determine the in vitro effects of a 1-minute CHG exposure and a 3-minute 0.35% PI exposure on the viability and biochemical content of rotator cuff tendon. Methods: Infraspinatus tendons (n=6 per group) were isolated from shoulders of Nubian goats (n=3; 2 female and 1 male). Tendons were submerged in Irrisept (0.05% CHG in sterile water) for 1 minute or 0.35% Betadine (PI) for 3 minutes per manufacturer guidelines, followed by phosphate-buffered saline wash and culture in tissue culture medium. Control tendon specimens were similarly exposed to tissue culture medium, followed by phosphate-buffered saline wash and culture in tissue culture medium. After seven days of culture, isolates from both the tendinous and enthesis portions of the rotator cuff were analyzed for viability, collagen content (hydroxyproline assay), and glycosaminoglycan (GAG) content (dimethylmethylene blue assay). Statistical analyses were performed via one-way ANOVA with post hoc Tukey’s test. Results: Within the tendinous portion of the rotator cuff, CHG-treated specimens demonstrated a decrease in mean tenocyte viability compared to controls (p=0.045) (Fig.1). While PI-treated specimens exhibited lower mean viability compared to controls, this difference was not statistically significant. Within the enthesis portion of the rotator cuff, tenocyte viability decreased in both CHG- and PI-treated groups; however, these decreases were not statistically significant (Fig. 2). There were no significant differences in mean collagen content/wet weight or GAG/wet weight among groups within the tendinous and enthesis portion of the rotator cuff (Fig. 3). Conclusions: Due to the potential catastrophic consequences associated with periprosthetic infection, CHG and PI antimicrobial lavages are commonly used to reduce the risk of periprosthetic infection during shoulder arthroplasty. However, in this study, brief 0.05% CHG and 0.35% PI exposures to rotator cuff tendon resulted in lower tenocyte viability 7 days after treatment. These results are consistent with the toxicity reported with exposure of CHG and PI to other musculoskeletal tissues, including articular cartilage. The toxicity of antimicrobial irrigation to the surrounding native rotator cuff tendon may be detrimental to the postoperative healing of concomitant rotator cuff repair in the setting of shoulder arthroplasty.

CNS/PNS proteoglycans functionalize neuronal and astrocyte niche microenvironments optimizing cellular activity by preserving membrane polarization dynamics, ionic microenvironments, ion fluxes, neuronal activation, and network neurotransductive capacity.

Central and peripheral nervous system (CNS/PNS) proteoglycans (PGs) have diverse functional roles, this study examined how these control cellular behavior and tissue function. The CNS/PNS extracellular matrix (ECM) is a dynamic, responsive, highly interactive, space-filling, cell supportive, stabilizing structure maintaining tissue compartments, ionic microenvironments, and microgradients that regulate neuronal activity and maintain the neuron in an optimal ionic microenvironment. The CNS/PNS contains a high glycosaminoglycan content (60% hyaluronan, HA) and a diverse range of stabilizing PGs. Immobilization of HA in brain tissues by HA interactive hyalectan PGs preserves tissue hydration and neuronal activity, a paucity of HA in brain tissues results in a pro-convulsant epileptic phenotype. Diverse CS, KS, and HSPGs stabilize the blood-brain barrier and neurovascular unit, provide smart gel neurotransmitter neuron vesicle storage and delivery, organize the neuromuscular junction basement membrane, and provide motor neuron synaptic plasticity, and photoreceptor and neuron synaptic functions. PG-HA networks maintain ionic fluxes and microgradients and tissue compartments that contribute to membrane polarization dynamics essential to neuronal activation and neurotransduction. Hyalectans form neuroprotective perineuronal nets contributing to synaptic plasticity, memory, and cognitive learning. Sialoglycoprotein associated with cones and rods (SPACRCAN), an HA binding CSPG, stabilizes the inter-photoreceptor ECM. HSPGs pikachurin and eyes shut stabilize the photoreceptor synapse aiding in phototransduction and neurotransduction with retinal bipolar neurons crucial to visual acuity. This is achieved through Laminin G motifs in pikachurin, eyes shut, and neurexins that interact with the dystroglycan-cytoskeleton-ECM-stabilizing synaptic interconnections, neuronal interactive specificity, and co-ordination of regulatory action potentials in neural networks.

Poster 352: Histopathological Etiology of Mucoid Degeneration of the Anterior Cruciate Ligament

Objectives: Mucoid degeneration of the anterior cruciate ligament (ACL) is an uncommon clinical condition with reported prevalence below 0.5%. There is a paucity of histopathological description in the literature, which has limited our understanding of the underlying pathophysiology. Such limited understanding has resulted in treatment options limited to mechanical debridement rather than biologic interventions to prevent development or progression of mucoid degeneration. The purpose of this study is to define the histopathology of mucoid degeneration of the ACL to better guide future biologic and surgical management options. Methods: This is a case control study where a retrospective series of 10 patients were identified to have mucoid degeneration of the ACL from 2002 to 2023. They were evaluated and treated by two senior attending orthopedic sports medicine surgeons. Magnetic resonance images (MRI) were evaluated for multiple anatomic and morphometric measures. Tissue samples were obtained at the time of arthroscopic partial debridement of the ACL for all patients. Intra-operative ACL samples and cadaveric control tissues were prepared for histologic examination of microstructure and composition, and gene expression using multiplex transcriptional profiling. For the gene expression analysis RNA was isolated from archived formalin-fixed and paraffin-embedded (FFPE) sections. Three consecutive 6mm sections were pooled to generate one RNA sample and total RNA was extracted using a High Pure FFPET RNA Isolation Kit per the manufacturer’s protocol and stored at -80°C until further analysis. RNA concentration and integrity was evaluated using an Agilent BioAnalyzer 2100 and a corrected input of 50ng of total RNA was used for NanoString analyses using the NanoString nCounter® Human Fibrosis V2 Panel for multiplex molecular analysis of 770 genes. Analysis was performed using nSolver 4.0 Software and only probes with counts above background were included following the manufacturer instructions. Results: All 10 patients reported pain and limited knee flexion. MRI exhibited a bulbous appearance of the ACL. There was an increased posterior tibial slope (PTS), narrow notch width index, and increased signal intensity of the ACL. Medial meniscus tear and cartilage degeneration were reported with high prevalence. The histological analysis showed significant differences in the architecture of the tissue compared with normal ACL controls, with disorganized collagen matrix and increased glycosaminoglycan content. NanoString analysis demonstrated a total of 155 differentially expressed genes (DEGs) between the mucoid degeneration and control groups after adjustments for multiple comparisons (p<0.05), with the top 20 DEGs summarized in Table 1. Among all the DEGs identified the 5 most upregulated ones include Fibronectin 1 (FN1), COL5A1, COL6A3, COL3A1 and COL1A2. There were also significant differences in the pathway scores for epithelial-to-mesenchymal transition (EMT), Extracellular matrix Degradation and Synthesis, Collagen Biosynthesis, Focal Adhesion Kinase, PDGF Signaling and PI3K-Akt (Fig. 1). The histology findings demonstrate distinct changes in the structure and composition of the ACL, and we hypothesize that repetitive microtrauma of the ACL may lead to cumulative damage that ultimately results in mucoid degeneration. Increased posterior tibial slope and narrow notch width index may contribute to cumulative loading of the ligament and subsequent mucoid degeneration. Identification of biomechanical risk factors may help to identify high risk patients. Associated injury of the medial meniscus and medial compartment cartilage also suggests that mucoid degeneration of the ACL may be part of an early degenerative process in the knee. Conclusions: Mucoid degeneration of the ACL is an uncommon condition that may represent the result of repetitive microtrauma to the ligament. Current interventions address the condition after it has progressed to an irreversible biological state. By defining the cellular and molecular mechanism(s) of the condition, we aim to broaden treatment options to potentially identify biologic interventions. The association of mucoid degeneration and medial compartment osteoarthritis suggests that patients may benefit from earlier interventions. [Figure: see text][Figure: see text]

Alterations in the Structure, Composition, and Organization of Galactosaminoglycan-Containing Proteoglycans and Collagen Correspond to the Progressive Stages of Dupuytren's Disease.

Dupuytren's disease (DD) is a prevalent fibroproliferative disorder of the hand, shaped by genetic, epigenetic, and environmental influences. The extracellular matrix (ECM) is a complex assembly of diverse macromolecules. Alterations in the ECM's content, structure and organization can impact both normal physiological functions and pathological conditions. This study explored the content and organization of glycosaminoglycans, proteoglycans, and collagen in the ECM of patients at various stages of DD, assessing their potential as prognostic indicators. This research reveals, for the first time, relevant changes in the complexity of chondroitin/dermatan sulfate structures, specifically an increase of disaccharides containing iduronic acid residues covalently linked to either N-acetylgalactosamine 6-O-sulfated or N-acetylgalactosamine 4-O-sulfated, correlating with the disease's severity. Additionally, we noted an increase in versican expression, a high molecular weight proteoglycan, across stages I to IV, while decorin, a small leucine-rich proteoglycan, significantly diminishes as DD progresses, both confirmed by mRNA analysis and protein detection via confocal microscopy. Coherent anti-Stokes Raman scattering (CARS) microscopy further demonstrated that collagen fibril architecture in DD varies importantly with disease stages. Moreover, the urinary excretion of both hyaluronic and sulfated glycosaminoglycans markedly decreased among DD patients.Our findings indicate that specific proteoglycans with galactosaminoglycan chains and collagen arrangements could serve as biomarkers for DD progression. The reduction in glycosaminoglycan excretion suggests a systemic manifestation of the disease.

A feasibility study of in vivo quantitative ultra-short echo time-MRI for detecting early cartilage degeneration

ObjectivesTo explore the feasibility of Ultra-short echo time (UTE) – MRI quantitative imaging in detecting early cartilage degeneration in vivo and underlying pathological and biochemical basis.MethodsTwenty volunteers with osteoarthritis (OA) planning for total knee arthroplasty (TKA) were prospectively recruited. UTE-MRI sequences and conventional sequences were performed preoperatively. Regions of interests (ROIs) were manually drawn on the tibial plateau and lateral femoral condyle images to calculate MRI values. Cartilage samples were collected during TKA according to the preset positions corresponding to MR images. Pathological and biochemical components of the corresponding ROI, including histological grading, glycosaminoglycan (GAG) content, collagen integrity, and water content were obtained.Results91 ROIs from volunteers of 7 males (age range: 68 to 78 years; 74 ± 3 years) and 13 females (age range: 57 to 79 years; 67 ± 6 years) were evaluated. UTE-MTR (r = −0.619, p < 0.001), UTE-AdiabT1ρ (r = 0.568, p < 0.001), and UTE-T2* values (r = −0.495, p < 0.001) showed higher correlation with Mankin scores than T2 (r = 0.287, p = 0.006) and T1ρ (r = 0.435, p < 0.001) values. Of them, UTE-MTR had the highest diagnostic performance (AUC = 0.824, p < 0.001). UTE-MTR, UTE-AdiabT1ρ and UTE-T2* value was mainly related to collagen structural integrity, PG content and water content, respectively (r = 0.536, −0.652, −0.518, p < 0.001, respectively).ConclusionUTE-MRI have shown greater in vivo diagnostic value for early cartilage degeneration compared to conventional T2 and T1ρ values. Of them, UTE-MTR has the highest diagnostic efficiency. UTE-MTR, UTE-AdiabT1ρ, and UTE-T2* value mainly reflect different aspects of cartilage degeneration--integrity of collagen structure, PG content, and water content, respectively.Critical relevance statementUltra-short echo time (UTE)-MRI has the potential to be a novel image biomarkers for detecting early cartilage degeneration in vivo and was correlated with biochemical changes of early cartilage degeneration.Key PointsConventional MR may miss some early cartilage changes due to relatively long echo times.Ultra-short echo time (UTE)-MRI showed the ability in identifying early cartilage degeneration in vivo.UTE-MT, UTE-AdiabT1ρ, and UTE-T2* mapping mainly reflect different aspects of cartilage degeneration.Graphical

Plunge-Freezing Cryopreservation of Tendons.

Allograft transplantation is an important method for tendon reconstruction after injury, and its clinical success highly relies on the storage and transportation of the grafts. Cryopreservation is a promising strategy for tendon storage. In this study, we report a novel cryopreservation agent (CPA) formulation with a high biocompatibility for tendon cryopreservation. Mainly composed of natural zwitterionic betaine and the biocompatible polymer poly(vinylpyrrolidone) (PVP), it exhibited ideal abilities to depress the freezing point and inhibit ice growth and recrystallization. Notably, after cryopreservation via plunge-freezing for 1 month, Young's modulus (144 MPa, 98% of fresh tendons) and ultimate stress (46.7 MPa, 99% of fresh tendons) remained stable, and the cross-linking of collagen microfibers, protein structures, and glycosaminoglycan (GAG) contents changed slightly. These results indicate that the formulation (5 wt % betaine and 5 wt % PVP in phosphate-buffered saline, PBS solution) effectively maintains the biomechanical properties and tissue structure. This work offers a novel cryopreservation method for tendons and may also provide insights into the long-term preservation of various other tissues.

Fisetin Protects the Wistar Rats from the Oxidative and Inflammatory Damage Caused by Ethelene Glycol Induced Urolithiasis

Urolithiasis, commonly referred to as urinary stones, is the third most prevalent condition affecting the urinary system and affects 12% of people worldwide. In this investigation, we assessed the prospective flavonoid fisetin's anti-urolithiasis activity. Fisetin reduced the nucleation, aggregation, and crystallization of kidney stones in in vitro tests at dosages of 62.5, 125, 250, 500, and 1,000 ug/mL in a dose-dependent manner. Twenty-four male Wistar rats (n = 6) were induced with urolithiasis using ethylene glycol (0.75% v/v) with ammonium chloride (1% w/v) in drinking water for seven days. Except for the animals in the vehicle control and disease control groups, the remaining animals were supplemented with either Fisetin 15 mg/kg or 30 mg/kg body weight from days 7 to 21. On day 22, a urine evaluation showed that fisetin significantly increased the glycosaminoglycan (GAG) content, corrected urine pH, increased creatinine clearance, and increased urine volume. Additionally, fisetin decreased the levels of calcium, creatinine, uric acid, and N-acetyl glucouronidase (NAG) activity. Serum biochemical analysis revealed that fisetin reduced the excretion of creatinine, uric acid, and blood urea nitrogen (BUN). Further, fisetin reduced the content of total protein, calcium, oxalate, protein carbonyl content (PCC), malondialdehyde (MDA), tumor necrosis factor (TNF-α), interlukin-6 (IL-6), and increased the content of reduced glutathione (GSH), the activities of superoxide dismutase (SOD), and catalase (CAT). Histological findings also support our findings. Based on the above observations, fisetin can be a potential candidate for the treatment of urolithiasis.

Widespread Vascularization and Correlation of Glycosaminoglycan Accumulation to Tendon Pain in Human Plantar Fascia Tendinopathy

Background: Plantar fasciitis is a painful tendinous condition (tendinopathy) with a high prevalence in athletes. While a healthy tendon has limited blood flow, ultrasound has indicated elevated blood flow in tendinopathy, but it is unknown if this is related to a de facto increase in the tendon vasculature. Likewise, an accumulation of glycosaminoglycans (GAGs) is observed in tendinopathy, but its relationship to clinical pain is unknown. Purpose: To explore to what extent vascularization, inflammation, and fat infiltration were present in patients with plantar fasciitis and if they were related to clinical symptoms. Study Design: Descriptive laboratory study. Methods: Biopsy specimens from tendinopathic plantar fascia tissue were obtained per-operatively from both the primary site of tendon pain and tissue swelling (“proximal”) and a region that appeared macroscopically healthy at 1 to 2 cm away from the primary site (“distal”) in 22 patients. Biopsy specimens were examined with immunofluorescence for markers of blood vessels, tissue cell density, fat infiltration, and macrophage level. In addition, pain during the first step in the morning (registered during an earlier study) was correlated with the content of collagen and GAGs in tissue. Results: High vascularization (and cellularity) was present in both the proximal (0.89%) and the distal (0.96%) plantar fascia samples, whereas inconsistent but not significantly different fat infiltration and macrophage levels were observed. The collagen content was similar in the 2 plantar fascia regions, whereas the GAG content was higher in the proximal region (3.2% in proximal and 2.8% in distal; P = .027). The GAG content in the proximal region was positively correlated with the subjective morning pain score in the patients with tendinopathy (n = 17). Conclusion: In patients with plantar fasciitis, marked tissue vascularization was present in both the painful focal region and a neighboring nonsymptomatic area. In contrast, the accumulation of hydrophilic GAGs was greater in the symptomatic region and was positively correlated with increased clinical pain levels in daily life. Clinical Relevance: The accumulation of GAGs in tissue rather than the extent of vascularization appears to be linked with the clinical degree of pain symptoms of the disease.

Influence of feeding a soft diet on proteoglycan expression in rat temporomandibular joint discs

ObjectivesExtracellular matrix components play a significant role in maintaining tissue integrity and pathological processes of the temporomandibular joint (TMJ). This study aimed to evaluate the influence of a soft diet on the mRNA expression of proteoglycans and glycosaminoglycans (GAGs) linked to proteoglycan core proteins in rat TMJ discs. MethodsThirty 4-week-old male Wistar rats were assigned to one of two groups: a control group fed a regular pellet diet and a soft diet group fed a powdered diet for 4 weeks. The mRNA expression levels of 12 proteoglycans in TMJ discs were evaluated using real-time polymerase chain reaction (PCR). In addition, histomorphometric and biochemical analyses were performed to evaluate the thickness and deoxyribonucleic acid (DNA), GAG, and water content of the TMJ discs. ResultsThe TMJ disc thickness in the anterior, intermediate, and posterior bands decreased significantly in the soft diet group. The GAG content decreased significantly in the soft-diet group, whereas no significant differences in DNA content or water content ratio were observed between the groups. Real-time PCR indicated that the expression levels of aggrecan, versican, biglycan, decorin, fibromodulin, lumican, and chondroadherin decreased in the soft diet group. The expression levels of all versican isoforms decreased in the soft diet group. ConclusionsThese results indicate that the biomechanical environment of the TMJ caused by a soft diet is closely related to the expression of proteoglycans in TMJ discs, which may eventually increase the fragility of the TMJ discs.

Decellularized skin pretreatment by monophosphoryl lipid A and lactobacillus casei supernatant accelerate skin recellularization.

Bioscaffolds and cells are two main components in the regeneration of damaged tissues via cell therapy. Umbilical cord stem cells are among the most well-known cell types for this purpose. The main objective of the present study was to evaluate the effect of the pretreatment of the foreskin acellular matrix (FAM) by monophosphoryl lipid A (MPLA) and Lactobacillus casei supernatant (LCS) on the attraction of human umbilical cord mesenchymal stem cells (hucMSC). The expression of certain cell migration genes was studied using qRT-PCR. In addition to cell migration, transdifferentiation of these cells to the epidermal-like cells was evaluated via immunohistochemistry (IHC) and immunocytochemistry (ICC) of cytokeratin 19 (CK19). The hucMSC showed more tissue tropism in the presence of MPLA and LCS pretreated FAM compared to the untreated control group. We confirmed this result by scanning electron microscopy (SEM) analysis, glycosaminoglycan (GAG), collagen, and DNA content. Furthermore, IHC and ICC data demonstrated that both treatments increase the protein expression level of CK19. Pretreatment of acellular bioscaffolds by MPLA or LCS can increase the migration rate of cells and also transdifferentiation of hucMSC to epidermal-like cells without growth factors. This strategy suggests a new approach in regenerative medicine.

Shedding light on the effects of blood on meniscus tissue: the role of mononuclear leukocytes in mediating meniscus catabolism

Traumatic meniscal injuries can cause acute pain, hemarthrosis (bleeding into the joint), joint immobility, and post-traumatic osteoarthritis (PTOA). However, the exact mechanism(s) by which PTOA develops following meniscal injuries is unknown. Since meniscus tears commonly coincide with hemarthrosis, investigating the direct effects of blood and its constituents on meniscus tissue is warranted. The goal of this study was to determine the direct effects of blood and blood components on meniscus tissue catabolism. Porcine meniscus explants or primary meniscus cells were exposed to whole blood or various fractions of blood for 3days to simulate blood exposure following injury. Explants were then washed and cultured for an additional 3days prior to collection for biochemical analyses. Whole blood increased matrix metalloproteinase (MMP) activity. Fractionation experiments revealed blood-derived red blood cells did not affect meniscus catabolism. Conversely, viable mononuclear leukocytes induced MMP activity, nitric oxide (NO) production, and loss of tissue sulfated glycosaminoglycan (sGAG) content, suggesting that these cells are mediating meniscus catabolism. These findings highlight the potential challenges of meniscus healing in the presence of hemarthrosis and the need for further research to elucidate the in vivo effects of blood and blood-derived mononuclear leukocytes due to both hemarthrosis and blood-derived therapeutics.

ROLE OF NF-κB TRANSCRIPTION FACTOR ACTIVATION IN THE PROCESSES OF CONNECTIVE TISSUE COMPONENTS DEGRADATION IN SKELETAL MUSCLES UNDER METABOLIC SYNDROME

Connective tissue, encompassing both cellular and non-cellular elements, plays an important role in the progression of numerous pathological processes across various organs and tissues. Within skeletal muscle tissue, the extracellular matrix not only plays a structural and supporting function, but it is a complex multicomponent system that performs a number of regulatory functions. At present, the effect of activation of the transcription factor NF-κB on the quantitative and qualitative composition of the components of the extracellular matrix in skeletal muscles under metabolic syndrome development is insufficiently studied. The aim of this work is to study the effect of ammonium pyrrolidinedithiocarbamate on the concentration of glycosaminoglycans, the concentration of individual fractions of glycosaminoglycans, the content of free L-oxyproline and sialic acids in the biceps femoris muscle of rats under metabolic syndrome. The study was conducted on 24 male Wistar rats weighing 200-260 g, which were randomly divided into 4 groups of 6 animals each. The first was control; the second made up the metabolic syndrome modeling group. Metabolic syndrome was reproduced by adding a 20% fructose solution to the standard vivarium diet as the only source of drinking water. Metabolic syndrome was modelled for 60 days. The third group received ammonium pyrrolidinedithiocarbamate administration at a dose of 76 mg/kg intraperitoneally (i/p) three times a week for 60 days. The fourth group underwent combined treatment involving the administration of both ammonium pyrrolidinedithiocarbamate and metabolic syndrome modeling. In a 10% homogenate of the biceps femoris muscle, the total concentration of glycosaminoglycans, the concentration of the heparin-heparan fraction of glycosaminoglycans, the keratan-dermatan fraction of glycosaminoglycans, the chondroitin fraction of glycosaminoglycans, and the content of free L-oxyproline and sialic acids were assessed. The introduction of ammonium pyrrolidinedithiocarbamate under metabolic syndrome modelling led to a decrease in the total concentration of glycosaminoglycans by 9.2% compared to the metabolic syndrome group. Under these conditions, the concentration of the heparin-heparan fraction increased by 121.1%, the keratan-dermatan fraction decreased by 32.8%, and the concentration of the chondroitin fraction decreased by 38.7% compared to the metabolic syndrome group. The concentration of free L-oxyproline and sialic acids in the biceps femoris muscle decreased by 19.8% and 24.4%, respectively, compared to the metabolic syndrome group. Blockade of activation of the transcription factor NF-κB by intraperitoneal administration of ammonium pyrrolidinedithiocarbamate against the background of metabolic syndrome modelling leads to a decrease in the depolymerization of glycoproteins and proteoglycans, reduces the intensity of collagenolysis and leads to a redistribution of concentrations of individual fractions of glycosaminoglycans, characterized by an increase in the content of the heparin-heparan fraction and a decrease in chondroitin and keratan-dermatan fractions in the biceps femoris muscle of rats.

Biochemical Variability of Nasal Cartilages: Implications in Functional Rhinoplasty.

Nasal valve compromise is a common issue for patients presenting to an otolaryngologist. Anatomically, the dorsal septal cartilage (DSA) articulates with the upper lateral nasal cartilage (ULC). When the ULC weakens, it is prone to collapse and may result in nasal obstruction. Our objectives were to analyze histological differences between quadrangular and DSA nasal cartilages and compare histological composition of these tissues among patients with diagnosis of nasal valve compromise (NVC) versus those without NVC (no NVC). Prospective cross-sectional study of nasal cartilages from seventy-three (73) live donors. Quadrangular cartilage (QC), and DSA from patients undergoing septorhinoplasty were collected. Safranin O histochemical staining was used to observe glycosaminoglycans (GAGs) content and cell count. Masson's Trichrome staining was used to assess collagen content in these nasal cartilages. Mean GAG content was lower in DSA compared with QUAD cartilage (68.18% vs. 87.22%, p < 0.0001), and was lowest in DSA of patients with NVC (59.07%). Mean collagen content was higher in DSA compared with QUAD cartilage, (65.43% vs. 46.65%, p = 0.0002), and was highest in DSA of patients with NVC (74.86%). The DSA showed decreased GAG and increased collagen content compared with quadrangular cartilage. Within the DSA subsite, patients with presurgical NVC had the lowest GAG and highest collagen levels. This observation suggests that the biochemical composition of the DSA may contribute to its weakening, potentially resulting in its inability to withstand minor trauma and/or the effects of aging, ultimately leading to NVC. N/A Laryngoscope, 134:4252-4258, 2024.

A hybrid construct of decellularized matrix and fibrin for differentiating adipose stem cells into insulin-producing cells, an optimized in vitro assessment.

The generation of insulin-producing cells (IPCs) is an attractive approach for replacing damaged β cells in diabetic patients. In the present work, we introduced a hybrid platform of decellularized amniotic membrane (dAM) and fibrin encapsulation for differentiating adipose tissue-derived stem cells (ASCs) into IPCs. ASCs were isolated from healthy donors and characterized. Human AM was decellularized, and its morphology, DNA, collagen, glycosaminoglycan (GAG) contents, and biocompatibility were evaluated. ASCs were subjected to four IPC differentiation methods, and the most efficient method was selected for the experiment. ASCs were seeded onto dAM, alone or encapsulated in fibrin gel with various thrombin concentrations, and differentiated into IPCs according to a method applying serum-free media containing 2-mercaptoethanol, nicotinamide, and exendin-4. PDX-1, GLUT-2 and insulin expression were evaluated in differentiated cells using real-time PCR. Structural integrity and collagen and GAG contents of AM were preserved after decellularization, while DNA content was minimized. Cultivating ASCs on dAM augmented their attachment, proliferation, and viability and enhanced the expression of PDX-1, GLUT-2, and insulin in differentiated cells. Encapsulating ASCs in fibrin gel containing 2 mg/ml fibrinogen and 10 units/ml thrombin increased their differentiation into IPCs. dAM and fibrin gel synergistically enhanced the differentiation of ASCs into IPCs, which could be considered an appropriate strategy for replacing damaged β cells.

Mechanical properties, collagen and glycosaminoglycan content of equine superficial digital flexor tendons are not affected by training.

Physical activity can activate extracellular matrix (ECM) protein synthesis and influence the size and mechanical properties of tendon. In this study, we aimed to investigate whether different training histories of horses would influence the synthesis of collagen and other matrix proteins and alter the mechanical properties of tendon. Samples from superficial digital flexor tendon (SDFT) from horses that were either (a) currently race trained (n = 5), (b) previously race trained (n = 5) or (c) untrained (n = 4) were analysed for matrix protein abundance (mass spectrometry), collagen and glycosaminoglycan (GAG) content, ECM gene expression and mechanical properties. It was found that ECM synthesis by tendon fibroblasts invitro varied depending upon the previous training history. In contrast, fascicle morphology, collagen and GAG content, mechanical properties and ECM gene expression of the tendon did not reveal any significant differences between groups. In conclusion, although we could not identify any direct impact of the physical training history on the mechanical properties or major ECM components of the tendon, it is evident that horse tendon cells are responsive to loading invivo, and the training background may lead to a modification in the composition of newly synthesised matrix.

Elderberry (Sambucus nigra) and hawthorn (Crataegus oxyacantha) extract additives in carboxymethyl chitosan scaffolds for osteochondral tissue engineering applications

Elderberry (<i>Sambucus nigra</i>) and hawthorn (<i>Crataegus oxyacantha</i>) extract additives in carboxymethyl chitosan scaffolds for osteochondral tissue engineering applications

Regional structure-function relationships of lumbar cartilage endplates

Cartilage endplates (CEPs) act as protective mechanical barriers for intervertebral discs (IVDs), yet their heterogeneous structure–function relationships are poorly understood. This study addressed this gap by characterizing and correlating the regional biphasic mechanical properties and biochemical composition of human lumbar CEPs. Samples from central, lateral, anterior, and posterior portions of the disc (n = 8/region) were mechanically tested under confined compression to quantify swelling pressure, equilibrium aggregate modulus, and hydraulic permeability. These properties were correlated with CEP porosity and glycosaminoglycan (s-GAG) content, which were obtained by biochemical assays of the same specimens. Both swelling pressure (142.79 ± 85.89 kPa) and aggregate modulus (1864.10 ± 1240.99 kPa) were found to be regionally dependent (p = 0.0001 and p = 0.0067, respectively) in the CEP and trended lowest in the central location. No significant regional dependence was observed for CEP permeability (1.35 ± 0.97 * 10−16 m4/Ns). Porosity measurements correlated significantly with swelling pressure (r = −0.40, p = 0.0227), aggregate modulus (r = −0.49, p = 0.0046), and permeability (r = 0.36, p = 0.0421), and appeared to be the primary indicator of CEP biphasic mechanical properties. Second harmonic generation microscopy also revealed regional patterns of collagen fiber anchoring, with fibers inserting the CEP perpendicularly in the central region and at off-axial directions in peripheral regions. These results suggest that CEP tissue has regionally dependent mechanical properties which are likely due to the regional variation in porosity and matrix structure. This work advances our understanding of healthy baseline endplate biomechanics and lays a groundwork for further understanding the role of CEPs in IVD degeneration.

Structural Elucidation of Glycosaminoglycans in the Tissue of Flounder and Isolation of Chondroitin Sulfate C.

Glycosaminoglycans (GAGs) are valuable bioactive polysaccharides with promising biomedical and pharmaceutical applications. In this study, we analyzed GAGs using HPLC-MS/MS from the bone (B), muscle (M), skin (S), and viscera (V) of Scophthalmus maximus (SM), Paralichthysi (P), Limanda ferruginea (LF), Cleisthenes herzensteini (G), Platichthys bicoloratus (PB), Pleuronichthys cornutus (PC), and Cleisthenes herzensteini (CH). Unsaturated disaccharide products were obtained by enzymatic hydrolysis of the GAGs and subjected to compositional analysis of chondroitin sulfate (CS), heparin sulfate (HS), and hyaluronic acid (HA), including the sulfation degree of CS and HS, as well as the content of each GAG. The contents of GAGs in the tissues and the sulfation degree differed significantly among the fish. The bone of S. maximus contained more than 12 μg of CS per mg of dry tissue. Although the fish typically contained high levels of CSA (CS-4S), some fish bone tissue exhibited elevated levels of CSC (CS-6S). The HS content was found to range from 10-150 ug/g, primarily distributed in viscera, with a predominant non-sulfated structure (HS-0S). The structure of HA is well-defined without sulfation modification. These analytical results are independent of biological classification. We provide a high-throughput rapid detection method for tissue samples using HPLC-MS/MS to rapidly screen ideal sources of GAG. On this basis, four kinds of CS were prepared and purified from flounder bone, and their molecular weight was determined to be 23-28 kDa by HPGPC-MALLS, and the disaccharide component unit was dominated by CS-6S, which is a potential substitute for CSC derived from shark cartilage.

Characterization and modulation of the pro-inflammatory effects of immune cells in the canine intervertebral disk.

Intervertebral disk (IVD) degeneration affects both humans and canines and is a major cause of low back pain (LBP). Mast cell (MC) and macrophage (MØ) infiltration has been identified in the pathogenesis of IVD degeneration (IVDD) in the human and rodent model but remains understudied in the canine. MC degranulation in the IVD leads to a pro-inflammatory cascade and activates protease activated receptor 2 (PAR2) on IVD cells. The objectives of the present study are to: (1) highlight the pathophysiological changes observed in the degenerate canine IVD, (2) further characterize the inflammatory effect of MCs co-cultured with canine nucleus pulposus (NP) cells, (3) evaluate the effect of construct stiffness on NP and MCs, and (4) identify potential therapeutics to mitigate pathologic changes in the IVD microenvironment. Canine IVD tissue was isolated from healthy autopsy research dogs (beagle) and pet dogs undergoing laminectomy for IVD herniation. Morphology, protein content, and inflammatory markers were assessed. NP cells isolated from healthy autopsy (Mongrel hounds) tissue were co-cultured with canine MCs within agarose constructs and treated with cromolyn sodium (CS) and PAR2 antagonist (PAR2A). Gene expression, sulfated glycosaminoglycan content, and stiffness of constructs were assessed. CD 31+ blood vessels, mast cell tryptase, and macrophage CD 163+ were increased in the degenerate surgical canine tissue compared to healthy autopsy. Pro-inflammatory genes were upregulated when canine NP cells were co-cultured with MCs and the stiffer microenvironment enhanced these effects. Treatment with CS and PAR2 inhibitors mediated key pro-inflammatory markers in canine NP cells. There is increased MC, MØs, and vascular ingrowth in the degenerate canine IVD tissue, similar to observations in the clinical population with IVDD and LBP. MCs co-cultured with canine NP cells drive inflammation, and CS and PAR2A are potential therapeutics that may mitigate the pathophysiology of IVDD in vitro.