Proteoglycan Content Research Articles

Isolation of High-Quality Total RNA from Small Animal Articular Cartilage for Next-Generation Sequencing.

Articular cartilage is characterized by a low density of chondrocytes surrounded by an abundant extracellular matrix (ECM) consisting of a dense mixture of collagens, proteoglycans, and glycosaminoglycans. Due to its low cellularity and high proteoglycan content, it is particularly challenging to extract high-quality total RNA suitable for sensitive high-throughput downstream applications such as RNA sequencing (RNA-Seq). Available protocols for high-quality RNA isolation from articular chondrocytes are inconsistent, resulting in suboptimal yield and compromised quality. This poses a significant difficulty in the application of RNA-Seq to study the cartilage transcriptome. Current protocols rely either on dissociation of cartilage ECM by collagenase digestion or pulverizing cartilage using various methods prior to RNA extraction. However, protocols for cartilage processing vary significantly depending on the species and source of cartilage within the body. Protocols for isolating RNA from human or large mammal (e.g., horse or cattle) cartilage samples are available, but this is not the case for chicken cartilage, despite the species being extensively used in cartilage research. Here, we present two improved RNA isolation protocols based on pulverization of fresh articular cartilage using a cryogenic mill or on enzymatic digestion using 1.2% (w/v) collagenase II. Our protocols optimize the collection and tissue processing steps to minimize RNA degradation and enhance RNA purity. Our results show that RNA purified from chicken articular cartilage using these methods has appropriate quality for RNA-Seq experiments. The procedure is applicable for RNA extraction from cartilage from other species such as dog, cat, sheep, and goat. The workflow for RNA-Seq analysis is also described here. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Extraction of total RNA from pulverized chicken articular cartilage Alternate Protocol: Extraction of total RNA from collagen-digested articular cartilage Support Protocol: Dissection of chicken articular cartilage from the knee joint Basic Protocol 2: RNA sequencing of total RNA from chicken articular cartilage.

Histological Findings and T2 Relaxation Time in Canine Menisci of Elderly Dogs—An Ex Vivo Study in Stifle Joints

Osteoarthritis is a chronic disease that often affects the canine stifle joint. Due to their biomechanical function, the menisci in the canine stifle play an important role in osteoarthritis. They compensate for the incongruence in the joint and distribute and minimize compressive loads, protecting the hyaline articular cartilage from damage. Meniscal degeneration favors the development and progression of stifle joint osteoarthritis. Qualitative magnetic resonance imaging (MRI) is the current golden standard for detecting meniscal changes, but it has limitations in detecting early signs of meniscal degeneration. A quantitative MRI offers new options for detecting early structural changes. T2 mapping can especially visualize structural changes such as altered collagen structures and water content, as well as deviations in proteoglycan content. This study evaluated T2 mapping and performed a histological scoring of menisci in elderly dogs that had no or only low radiographic osteoarthritis grades. A total of 16 stifles from 8 older dogs of different sex and breed underwent ex vivo magnet resonance imaging, including a T2 mapping pulse sequence with multiple echoes. A histological analysis of corresponding menisci was performed using a modified scoring system. The mean T2 relaxation time was 18.2 ms and the mean histological score was 4.25. Descriptive statistics did not reveal a correlation between T2 relaxation time and histological score. Ex vivo T2 mapping of canine menisci did not demonstrate histological changes, suggesting that early meniscal degeneration can be present in the absence of radiological signs of osteoarthritis, including no significant changes in T2 relaxation time.

Hydroquinone, an Environmental Pollutant, Affects Cartilage Homeostasis through the Activation of the Aryl Hydrocarbon Receptor Pathway.

Exposure to environmental pollutants has a proven detrimental impact on different aspects of human health. Increasing evidence has linked pollution to the degeneration of tissues in the joints, although through vastly uncharacterised mechanisms. We have previously shown that exposure to hydroquinone (HQ), a benzene metabolite that can be found in motor fuels and cigarette smoke, exacerbates synovial hypertrophy and oxidative stress in the synovium. To further understand the impact of the pollutant on joint health, here we investigated the effect of HQ on the articular cartilage. HQ exposure aggravated cartilage damage in rats in which inflammatory arthritis was induced by injection of Collagen type II. Cell viability, cell phenotypic changes and oxidative stress were quantified in primary bovine articular chondrocytes exposed to HQ in the presence or absence of IL-1β. HQ stimulation downregulated phenotypic markers genes SOX-9 and Col2a1, whereas it upregulated the expression of the catabolic enzymes MMP-3 and ADAMTS5 at the mRNA level. HQ also reduced proteoglycan content and promoted oxidative stress alone and in synergy with IL-1β. Finally, we showed that HQ-degenerative effects were mediated by the activation of the Aryl Hydrocarbon Receptor. Together, our findings describe the harmful effects of HQ on articular cartilage health, providing novel evidence surrounding the toxic mechanisms of environmental pollutants underlying the onset of articular diseases.

Removal of GAGs Regulates Mechanical Properties, Collagen Fiber Formation, and Alignment in Tissue Engineered Meniscus.

The complex fibrillar architecture of native meniscus is essential for proper function and difficult to recapitulate in vitro. In the native meniscus, proteoglycan content is low during the development of collagen fibers and progressively increases with aging. In vitro, fibrochondrocytes produce glycosaminoglycans (GAGs) early in culture, in contrast to native tissue, where they are deposited after collagen fibers have formed. This difference in the timing of GAG production hinders the formation of a mature fiber network in such in vitro models. In this study, we removed GAGs from collagen gel-based tissue engineered constructs using chondroitinase ABC (cABC) and evaluated the effect on the formation and alignment of collagen fibers and the subsequent effect on tensile and compressive mechanical properties. Removal of GAGs during maturation of in vitro constructs improved collagen fiber alignment in tissue engineered meniscus constructs. Additionally, removal of GAGs during maturation improved fiber alignment without compromising compressive strength, and this removal improved not only fiber alignment and formation but also tensile properties. The increased fiber organization in cABC-treated groups also appeared to influence the size, shape, and location of defects in these constructs, suggesting that treatment may prevent the propagation of large defects under loading. This data gives another method of modulating the ECM for improved collagen fiber formation and mechanical properties in tissue engineered constructs.

Differentiation of Sclera in Chick Embryo at Various Stages of its Development- A Histological Study

Background: The sclera is the outer strong opaque covering of the eye that functions as protective layer and provides site for attachment of extra ocular muscles. Aim: To study the microscopic anatomy of sclera in chick at various stages of embryological development keeping in mind the practical application of this information in understanding human development and disease processes. Methodology: It was an experimental study. The study was carried out at department of Anatomy, CPSP Regional Centre, Islamabad. Eyeballs were collected from group A, B and C (n=30 each) on day 10, 12 and 15 of incubation respectively. Dissected out tissues were processed and stained with Hematoxylin & Eosin and Alizarin red. The prepared specimens for group A and C were analysed under light microscope while the specimens from group B were observed via stereomicroscope. Data was evaluated by using SPSS version 23. Independent t-test was applied with p-value of less than 0.05 was considered significant. Results: As the chondrocytes in cartilaginous layer of sclera became mature, they change their shape from flattened to more rounded cells. The proteoglycan content in the cartilaginous matrix increased. The mean number of chondrocytes increased from 17.4 ±1.4/unit area on day 10 to 10.0 ±0.8/unit area on day 15. The mean thickness of cartilaginous layer increased from 44 ±2.4µm on day 10 to 77 ±8.3 µm on day 15 of development. Calcification of scleral ossicles became obvious at about 12 days of incubation when seen under stereomicroscope. Practical Implication: This study highlighted that understanding for the development of scleral elements (understudied tissue of the eye) can be critical in research involving autoimmune diseases and neural crest defects. Considering similar developmental stages among humans as in chick embryo. Conclusion: It was concluded that growth of sclera in eye of chick has been indicated by increase in thickness of its layers and increase in the number of chondrocytes/unit area and progressing calcification of ossicles in its bony part. Keywords: Chick Embryo, Chondrocytes, Hyaline Cartilage and Ossicular Ring.

Diagnosis of Osteoarthritis at an Early Stage via Infrared Spectroscopy Combined Chemometrics in Human Serum: A Pilot Study

Methods applied for early diagnosis of osteoarthritis (OA) are limited. Early prevention and treatment can effectively reduce the pain of OA patients and save costs. The present study aimed to develop a rapid non-destructive detection method for early diagnosis of OA by evaluating infrared (IR) spectroscopy combined chemometrics. Our cohort consisted of (a) 15 patients with osteoarthritis (OA) and (b) 10 without clinical signs of the disease and they were used as controls. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was used to investigate serum samples (50 µL) collected from these patients. A supervised classification algorithm namely discriminant analysis (DA) was applied to evaluate the diagnostic accuracy spectral processing and chemometrics analysis allowed for detecting spectral biomarkers that discriminated the two cohorts. About 250 infrared spectra were statistically important for separating the groups. Peaks at 1000 cm−1 in OA serum were associated mainly with C–O stretching vibration associated with the changes in the proteoglycan contents previously reported in OA. A good overall classification accuracy of 74.47% was obtained from the DA model. Our findings indicated that this discriminating model, which incorporated the ATR-FTIR spectrum, could provide a rapid and cost-effective blood test, thus facilitating the early diagnosis of human OA.

Injury-related cell death and proteoglycan loss in articular cartilage: Numerical model combining necrosis, reactive oxygen species, and inflammatory cytokines.

Osteoarthritis (OA) is a common musculoskeletal disease that leads to deterioration of articular cartilage, joint pain, and decreased quality of life. When OA develops after a joint injury, it is designated as post-traumatic OA (PTOA). The etiology of PTOA remains poorly understood, but it is known that proteoglycan (PG) loss, cell dysfunction, and cell death in cartilage are among the first signs of the disease. These processes, influenced by biomechanical and inflammatory stimuli, disturb the normal cell-regulated balance between tissue synthesis and degeneration. Previous computational mechanobiological models have not explicitly incorporated the cell-mediated degradation mechanisms triggered by an injury that eventually can lead to tissue-level compositional changes. Here, we developed a 2-D mechanobiological finite element model to predict necrosis, apoptosis following excessive production of reactive oxygen species (ROS), and inflammatory cytokine (interleukin-1)-driven apoptosis in cartilage explant. The resulting PG loss over 30 days was simulated. Biomechanically triggered PG degeneration, associated with cell necrosis, excessive ROS production, and cell apoptosis, was predicted to be localized near a lesion, while interleukin-1 diffusion-driven PG degeneration was manifested more globally. Interestingly, the model also showed proteolytic activity and PG biosynthesis closer to the levels of healthy tissue when pro-inflammatory cytokines were rapidly inhibited or cleared from the culture medium, leading to partial recovery of PG content. The numerical predictions of cell death and PG loss were supported by previous experimental findings. Furthermore, the simulated ROS and inflammation mechanisms had longer-lasting effects (over 3 days) on the PG content than localized necrosis. The mechanobiological model presented here may serve as a numerical tool for assessing early cartilage degeneration mechanisms and the efficacy of interventions to mitigate PTOA progression.

Anti-inflammatory and pro-anabolic effects of 5-aminosalicylic acid on human inflammatory osteoarthritis models.

Osteoarthritis (OA) is the most common degenerative joint disease, mainly affecting the elderly worldwide, for which the drug treatment remains a major challenge. Low-grade inflammation plays a pivotal role in OA onset and progression. Exploration of notable anti-inflammatory and disease-modifying drugs on human samples could facilitate the evaluation of therapeutic strategies for OA. The anti-inflammatory drug 5-aminosalicylic acid (5-ASA) is a first-line drug for ulcerative colitis (UC), however no study has explored the effects of 5-ASA on articular chondrocytes. In this work, both in vitro (chondrocyte pellets) and ex vivo (osteochondral explants) human inflammatory OA models were applied to evaluate the effects of 5-ASA. In the inflammatory pellet model, 5-ASA remarkably downregulated the gene expression of interleukin-6 (IL-6), and cyclooxygenase-2 (COX-2) while upregulating proteoglycan 4 (PRG4) and cartilage oligomeric matrix protein (COMP) gene expression. Total glycosaminoglycan (GAG) synthesis by pellets was markedly increased in 5-ASA-treated groups compared with the inflammatory group. In conditioned medium, inflammatory mediators (IL-8, nitric oxide) were markedly inhibited upon 5-ASA treatment. Moreover, histological staining showed 5-ASA retained proteoglycan content and inhibited degradation of extracellular matrix (ECM) core components, aggrecan (ACAN) and collagen type II (COL2). In the inflammatory explant model, 5-ASA mitigated signs of OA development by reducing inflammatory mediators and GAG loss. These findings suggest that 5-ASA has anti-inflammatory and pro-anabolic effects on human chondrocyte pellet and osteochondral explant inflammatory OA models. Disease-modifying OA drugs are an unmet clinical need for the treatment of OA. Our study explored and demonstrated the anti-inflammatory and protective effects of 5-ASA on in vitro and ex vivo human inflammatory OA models, showing its translational potential for OA treatment.

Physical Therapy Diagnosis of Lumbar Disc Herniation: Role and Function - an Overview

Background: Discus hernia represents an intervertebral disk prolapse. The intervertebral disk provides stability in rest state and distributes the load of the spinal column on the move. Changes that lead to the occurrence of disc prolapse are desiccation, reduction of proteoglycan content, mucoid degeneration, and fibrosing tissue uptake. Predisposing factors of lumbar pain are heavy physical tasks with lifting loads over twenty pounds, especially professional use of heavy tools. Objective: The aims of this article is the review of diagnostics and physical therapy of lumbar disc herniation at levels L4/L5 and L5/S1. Methods: Authors used descriptive method for review lumbar disc herniation including diagnostic procedures and physical therapx based on scientific literature stored in medical databses PubMed, Scopus, Hinari, etc.. Results: The most common area of the spine to experience a herniated disk is the low back, just below the waist level. Herniated disks also commonly occur in the neck.. Conclusion: Rehabilitation is a delicate problem and requires long-term treatment and multidisciplinary cooperation, and is carried out in accordance with the principles of diagnostic and physical therapy of lumbar dics herniation depending of level of demaged and level of causes of lumbar disc herniation. The majority of herniated disks do not require surgery, and respond best to physical therapy. Physical therapists design personalized treatment programs to help people with herniated disks regain normal movement, reduce pain, and get back to their regular activities.

Early removal of the infrapatellar fat pad/synovium complex beneficially alters the pathogenesis of moderate stage idiopathic knee osteoarthritis in male Dunkin Hartley guinea pigs

BackgroundThe infrapatellar fat pad (IFP) is the largest adipose deposit in the knee; however, its contributions to the homeostasis of this organ remain undefined. To determine the influence of the IFP and its associated synovium (IFP/synovium complex or IFP/SC) on joint health, this study evaluated the progression of osteoarthritis (OA) following excision of this unit in a rodent model of naturally-occurring disease.MethodsMale Dunkin-Hartley guinea pigs (n=18) received surgical removal of the IFP in one knee at 3 months of age; contralateral knees received sham surgery as matched internal controls. Mobility and gait assessments were performed prior to IFP/SC removal and monthly thereafter. Animals were harvested at 7 months of age. Ten set of these knees were processed for microcomputed tomography (microCT), histopathology, transcript expression analyses, and immunohistochemistry (IHC); 8 sets of knees were dedicated to microCT and biomechanical testing (material properties of knee joints tissues and anterior drawer laxity).ResultsFibrous connective tissue (FCT) developed in place of the native adipose depot. Gait demonstrated no significant differences between IFP/SC removal and contralateral hindlimbs. MicroCT OA scores were improved in knees containing the FCT. Quantitatively, IFP/SC-containing knees had more osteophyte development and increased trabecular volume bone mineral density (vBMD) in femora and tibiae. Histopathology confirmed maintenance of articular cartilage structure, proteoglycan content, and chondrocyte cellularity in FCT-containing knees. Transcript analyses revealed decreased expression of adipose-related molecules and select inflammatory mediators in FCTs compared to IFP/SCs. This was verified via IHC for two key inflammatory agents. The medial articular cartilage in knees with native IFP/SCs showed an increase in equilibrium modulus, which correlated with increased amounts of magnesium and phosphorus.Discussion/conclusionFormation of the FCT resulted in reduced OA-associated changes in both bone and cartilage. This benefit may be associated with: a decrease in inflammatory mediators at transcript and protein levels; and/or improved biomechanical properties. Thus, the IFP/SC may play a role in the pathogenesis of knee OA in this strain, with removal prior to disease onset appearing to have short-term benefits.

Curcumin Involve the Myofibrosis Process of Rabbit Valve Interstitial Cells based on Expression Alpha- Smooth Muscle Actin: Experimental Posttest-only Control Group Design

Introduction: Following an acute rheumatic fever infection brought on by an inflammatory reaction to streptococcal bacteria, rheumatic heart valve disease results in valve degeneration. Elements of the immune system play an important role, in facilitating myofibroblast transdifferentiation and clearing damaged tissue of apoptotic cells. Myelofibrosis arising in valve components is the main cause of valve dysfunction. Valve myelofibrosis showed a significantly increased collagen, proteoglycan, and elastin content in myofibrotic valves compared to healthy valves. Objective: To establish curcumin’s capacity to prevent rabbit valve interstitial cells (VIC) from differentiating into myofibroblasts after being stimulated by TGF-1 by comparing the results to controls. Method: This study uses a posttest-only control group design for an in-vitro laboratory experiment. Heart VIC of a New Zealand rabbit were isolated (Oryctolagus cuniculus), and induced fibrosis by administration of TGF-β1 5 ng/mL. VIC pretreated with TGF-β1 were treated with low-dose curcumin (20 nanoM/L) and high-dose curcumin (50 nanoM/L). The immunocytochemical method based on SMA expression observed the inhibition of myofibroblastic differentiation. Statistical significance was analyzed by Kruskal-Wallis statistical test with a p-value < 0.05 as the limit of significance. Result: Administration of curcumin with low doses (20 nanoM/L) and high doses (50 nanoM/L), significantly decreased TGF-β1-induced myofibroblastic differentiation of VIC, which was characterized by decreased SMA expression in all low-dose curcumin treatment groups (20 nanoM/L) (1.40 ± 6.30) and high-dose curcumin (50 nanoM/L) (1.40 ± 8.30). Conclusion: Curcumin potentially prevent the development of SMA-expressing VIC into myofibroblasts.

Computed Tomography of Cartilage: An Exploration of Novel Cationic Bismuth Contrast Agent.

Accurate diagnosis of minor cartilage injuries with delayed contrast-enhanced computed tomography (CECT) is challenging as poor diffusion and toxicity issues limit the usage of common CT contrast agents. Hence, the design of safe contrast agents with physiochemical properties suitable for fast, deep cartilage imaging is imminent. Herein, a novel cationic bismuth contrast agent (Bi-DOTAPXD) based on dodecane tetraacetic acid (DOTA) was synthesized and examined for CECT of cartilage. The complex was designed to improve diagnosis by utilising a net-positive charge for enhanced permeability through cartilage, inherent low-toxicity and high X-ray attenuation of bismuth. Osteochondral plugs (n = 12), excised from visually intact porcine articular cartilage were immersed in Bi-DOTAPXD (8mg/mL) and Gd-DOTAPXD (10mg/mL) contrast agents and scanned with a high-resolution microcomputed tomography scanner at multiple time-points. The mean Bi-DOTAPXD and Gd-DOTAPXD partitions at 45-min time-point were 85.7 ± 35.1 and 69.8 ± 30.2%, and the partitions correlated with the histopathological analysis of cartilage proteoglycan (PG) content (r) at 0.657 and 0.632, respectively. The time diffusion constants (τ) for Bi-DOTAPXD and Gd-DOTA were 121 and 159min, respectively. Diffusion Bi-DOTAPXD and Gd-DOTAPXD reflected inter-sample variation in cartilage PG content. Cationic Bi-DOTAPXD may have the potential as a CT agent for the diagnosis of cartilage.

Toughness Variations among Natural Casings: An Exploration on Their Biochemical and Histological Characteristics.

We investigated the mechanical, biochemical, and histological properties of hog and sheep casings produced in different countries to elucidate the responsible factors for the toughness quality of natural casings. The toughness and collagen characteristics of sheep and lamb casings were also investigated to elucidate the effect of animal slaughter age on the relationships between connective tissue and the mechanical properties of natural casings. The results showed that the main component of hog and sheep casings was collagen with many layers of sheets. The contents of collagen, elastin, and proteoglycan in hog and sheep casings were similar. The toughest Chinese casings (p < 0.01) possessed a significantly lower heat solubility of collagen (p < 0.01), and a different size and arrangement of collagen fibers. Sheep casings were significantly tougher than lamb casings (p < 0.01). Compared with lamb casings, sheep casings had a significantly low heat-labile collagen content, a low heat solubility of collagen, a large size of collagen fibers, and a high pyridinoline concentration (p < 0.01). Therefore, the high thermal and structural stability of collagen in aged animals may contribute to the enhanced mechanical properties of casings.

Associations of human femoral condyle cartilage structure and composition with viscoelastic and constituent-specific material properties at different stages of osteoarthritis

The relationships between structure and function in human knee femoral cartilage are not well-known at different stages of osteoarthritis. Thus, our aim was to characterize the depth-dependent composition and structure (proteoglycan content, collagen network organization and collagen content) of normal and osteoarthritic human femoral condyle cartilage (n = 47) and relate them to their viscoelastic and constituent-specific mechanical properties that are obtained through dynamic sinusoidal testing and fibril-reinforced poroelastic material modeling of stress-relaxation testing, respectively. We characterized the proteoglycan content using digital densitometry, collagen network organization (orientation angle and anisotropy) using polarized light microscopy and collagen content using Fourier transform infrared spectroscopy. In the superficial cartilage (0–10 % of thickness), the collagen network disorganization and proteoglycan loss were associated with the smaller initial fibril network modulus - a parameter representing the pretension of the collagen network. Furthermore, the proteoglycan loss was associated with the greater strain-dependent fibril network modulus - a measure of nonlinear mechanical behavior. The proteoglycan loss was also associated with greater cartilage viscosity at a low loading frequency (0.005 Hz), while the collagen network disorganization was associated with greater cartilage viscosity at a high loading frequency (1 Hz). Our results suggest that proteoglycan loss and collagen network disorganization reduce the pretension of the collagen network while proteoglycan degradation also increases the nonlinear mechanical behavior of the collagen network. Further, the results also highlight that proteoglycan loss and collagen disorganization increase the viscosity of femoral cartilage, but their contribution to increased viscosity occurs in completely different loading frequencies.

Unloader bracing in osteoarthritis of the knee – Is there a direct effect on the damaged cartilage?

BackgroundUnloading knee braces represent a conservative treatment option for non-pharmalogical management of unicompartmental osteoarthritis of the knee. Though there is consensus on the clinical effectiveness of unloading, the effect mechanism of bracing remains part of a debate. Our study was designed to assess the effect of unloader bracing on damaged cartilage via MRI cartilage mappings. MethodsFourteen patients (7 female, 7 male, mean age 43.1 ± 9.4 years) with unicompartmental cartilage wear in knees with varus or valgus malalignment were enrolled. Clinical scores, radiographs and MR-graphic properties (T2/T2* mapping, T1 Delayed Gadolinium Enhanced MRI of the cartilage (dGEMRIC) mapping, high-resolution PDw sequences) of knee cartilage were recorded before and three months after brace use. ResultsBracing the knees for a mean of 14.4 ± 2.0 weeks (range 11 to 18 weeks) resulted in significant pain reduction (VAS changed from 5.9 ± 2.0 to 2.0 ± 1.3, p < 0.001) and improvement in knee function (KOOS increased from 42.1 ± 22.7 to 64.8 ± 18.7, p < 0.001). In the affected cartilage regions T2 relaxation times significantly decreased from 56.1 ± 11.4 ms to 46.5 ± 11.2 ms (p < 0.05). No changes in T1-dGEMRIC and T2* relaxation times, thickness or the extent of the damaged cartilage area could be detected. ConclusionsOur results suggest, that unloader bracing improves the biochemical properties of the damaged cartilage by increasing collagen and proteoglycan concentration as well as decreasing the cartilage edema.

Proteoglycans play a role in the viscoelastic behaviour of the canine cranial cruciate ligament.

Proteoglycans (PGs) are minor extracellular matrix proteins, and their contributions to the mechanobiology of complex ligaments such as the cranial cruciate ligament (CCL) have not been determined to date. The CCLs are highly susceptible to injuries, and their extracellular matrix comprises higher PGs content than the other major knee ligaments. Hence these characteristics make CCLs an ideal specimen to use as a model in this study. This study addressed the hypothesis that PGs play a vital role in CCL mechanobiology by determining the biomechanical behaviour at low strain rates before and after altering PGs content. For the first time, this study qualitatively investigated the contribution of PGs to key viscoelastic characteristics, including strain rate dependency, hysteresis, creep and stress relaxation, in canine CCLs. Femur-CCL-tibia specimens (n = 6 pairs) were harvested from canine knee joints and categorised into a control group, where PGs were not depleted, and a treated group, where PGs were depleted. Specimens were preconditioned and cyclically loaded to 9.9N at 0.1, 1 and 10%/min strain rates, followed by creep and stress relaxation tests. Low tensile loads were applied to focus on the toe-region of the stress-strain curves where the non-collagenous extracellular matrix components take significant effect. Biochemical assays were performed on the CCLs to determine PGs and water content. The PG content was ∼19% less in the treated group than in the control group. The qualitative study showed that the stress-strain curves in the treated group were strain rate dependent, similar to the control group. The CCLs in the treated group showed stiffer characteristics than the control group. Hysteresis, creep characteristics (creep strain, creep rate and creep compliance), and stress relaxation values were reduced in the treated group compared to the control group. This study suggests that altering PGs content changes the microstructural organisation of the CCLs, including water molecule contents which can lead to changes in CCL viscoelasticity. The change in mechanical properties of the CCLs may predispose to injury and lead to knee joint osteoarthritis. Future studies should focus on quantitatively identifying the effect of PG on the mechanics of intact knee ligaments across broader demography.

Effects of Different Types of Electric Fields on Mechanical Properties and Microstructure of Ex Vivo Porcine Brain Tissues.

Electrotherapy plays a crucial role in regulating neuronal activity. Nevertheless, the relevant therapeutic mechanisms are still unclear; thus, the effects of electric fields on brain tissue's mechanical properties and microstructure need to be explored. In this study, focusing on the changes in mechanical properties and microstructure of ex vivo porcine brain tissues under different types of electric fields, directional and alternating electric fields (frequencies of 5, 20, 50, and 80 Hz, respectively) integrate with a custom-designed indentation device. The experimental results showed that for the ex vivo brain tissue, the directional electric field (DEF) can reduce the elastic properties of brain tissue. Simultaneously, the DEF can increase the cell spacing and reduce the proteoglycan content. The transmission electron microscope (TEM) analysis observed that the DEF can reduce the integrity of the plasma membrane, the endoplasmic reticulum's stress response, and the myelin lamella's separation. The alternating electric field (AEF) can accelerate the stress relaxation process of brain tissue and change the time-dependent mechanical properties of brain tissue. Meanwhile, with the increase in frequency, the cell spacing decreased, and the proteoglycan content gradually approached the control group without electric fields. TEM analysis observed that with the increase in frequency, the integrity of the plasma membrane increases, and the separation of the myelin lamella gradually disappears. Understanding the changes in the mechanical properties and microstructure of brain tissue under AEF and DEF enables a preliminary exploration of the therapeutic mechanism of electrotherapy. Simultaneously, the essential data was provided to support the development of embedded electrodes. In addition, the ex vivo experiments build a solid foundation for future in vivo experiments.

Alleviation of cartilage degeneration by laser acupuncture: An ultrasound biomicroscopic study of early postmenopausal osteoarthritis in rats

This study aims to evaluate the therapeutic effects of laser acupuncture (LA) on articular cartilage degradation at the early stage of postmenopausal osteoarthritis (PMOA) by an ultrasound biomicroscopic technology. Twenty-one healthy female Sprague-Dawley rats were randomly and evenly divided into three groups - sham, ovariectomized (OVX) and OVX ​+ ​LA groups. Only the peri-ovarian fatty tissue of the sham rats was exteriorized. The animals in the OVX and OVX ​+ ​LA groups underwent bilateral ovariectomy to create a menopause model. On day 8 after the ovariectomy, the 2-week LA therapy at acupoints Guanyuan (CV-4), Sanyinjiao (SP-6), and Zusanli (ST-36) was performed in the OVX ​+ ​LA group, while the sham and OVX groups had a sham treatment. All the animals were sacrificed on day 22. The cartilage tissues in the left knee joint were examined by ultrasound and histology. Compared with the sham group, the significantly degenerate alterations (p ​< ​0.01) in ultrasound roughness index (URI), reflection coefficient (RC1) and cartilage thickness were found in the OVX group, while the values of these parameters were significantly recovered (p ​< ​0.01) in the OVX ​+ ​LA group. The LA treatment enhanced proteoglycans (PGs) content with a significant reduce of the modified Mankin score (p ​< ​0.01). The acoustic assessment strongly correlated with the histologic evaluation. The results showed that LA treatment could alleviate the postmenopause-induced cartilage degradation and promote tissue repair. LA has potential to be a simple and safe non-pharmacological countermeasure against PMOA for geriatric patient population. The ultrasound biomicroscopic technology could be a quantitative evaluation tool of the structural and morphological responses of articular cartilage to LA intervention.

Bone mesenchymal stem cells deliver exogenous lncRNA CAHM via exosomes to regulate macrophage polarization and ameliorate intervertebral disc degeneration

Exosomes derived from bone marrow mesenchymal stem cells (BMSC-Exos) were reported to have therapeutic potential in degenerative diseases. This study aimed to explore the effects of BMSC-Exos on inhibiting M1 macrophage polarization, reducing excessive nucleus pulposus cells (NPCs) apoptosis, and inhibiting ECM degradation during intervertebral disc degeneration (IDD). Rat IDD models were established by acupuncture. For the co-culture experiment, we used BMSC-Exo or human monocyte leukemia (THP-1) medium to incubate THP-1 or NPCs, respectively. BMSC-Exo was isolated from the BMSC medium, identified by TEM and NTA, and injected into the intervertebral discs of IDD rats. The macrophage infiltration in intervertebral disc tissue was evaluated by immunohistochemistry and immunofluorescence. ELISA was used to measure the levels of TNF-α, IL-6 and IL-10. The ECM degradation was analyzed by Western blot. The cell proportion and apoptosis were measured by flow cytometry. The morphological change of the intervertebral disc was analyzed by HE and safranin O fixation staining. In intervertebral disc tissues of IDD rats, we found the increased infiltration of M1 macrophages, with upregulated iNOS, TNF-α and IL-6 levels. Compared with BMSCs, the expression of CAHM in BMSC-Exo was significantly higher. Using co-cultured experiments, we proved that BMSC-Exo reduced apoptosis and ECM degradation of NPCs by inhibiting M1-type macrophage polarization by delivering CAHM. In addition, BMSC-Exo could improve IDD in vivo, including increased proteoglycan content, reduced macrophage infiltration and ECM degradation, and decrease expression of inflammatory factors by delivering CAHM. In conclusion, BMSC-Exo delivered exogenous CAHM via exosomes to regulate macrophage polarization and ameliorate IDD.

Subacromial bursa increases the failure force in a mouse model of supraspinatus detachment and repair.

It has been shown that subacromial bursa (SAB) harbors connective tissue progenitor cells. The purpose of this study was to evaluate the effects of implantation of SAB-derived cells (SBCs) suspended in a fibrin sealant bead and implantation of SAB tissue at rotator cuff repair site on biomechanical properties of the repair in a mouse (C57Bl/6) model of supraspinatus tendon (ST) detachment and repair. Part 1: Murine SAB tissue was harvested and cultured. Viability of SBCs suspended in 10 μL of fibrin sealant beads was confirmed invitro and invivo. Eighty mice underwent right ST detachment and repair augmented with either fibrin sealant bead (control group) or fibrin sealant bead with 100,000 SBCs (study group) applied at the repair site. Part 2: 120 mice underwent right ST detachment and repair and were randomized equally into 4 groups: (1) a tissue group, which received a piece of freshly harvested SAB tissue; (2) a cell group, which received SBCs suspended in fibrin sealant bead; (3) a fibrin sealant group, which received plain fibrin sealant bead without cells; and (4) a control group, which received nothing at the ST repair site. An equal number of mice in each group were killed at 2 and 4 weeks. Specimens underwent biomechanical testing to evaluate failure force (part 1 and 2) and histologic analysis of the repair site (part 1 only). Part 1: The mean failure force in the study group was significantly higher than controls at 2 and 4 weeks (3.25±1.03 N vs. 2.43±0.56 N, P=.01, and 4.08±0.99 N vs. 3.02±0.8 N, P=.004, respectively). Mean cell density of the ST at the repair site was significantly lower in the study group at 2 weeks than in controls (18,292.13±1706.41 vs. 29,501.90±3627.49, P=.001). Study group specimens had lower proteoglycan contents than controls, but this difference was not statistically significant. Part 2: There was no difference in failure force between cell and tissue groups at the 2- and 4-week time points (P=.994 and P=.603, respectively). There was no difference in failure force between fibrin sealant bead and control groups at the 2- and 4-week time points (P=.978 and P=.752, respectively). This study shows that the application of SBCs and SAB tissue at the rotator cuff repair site increases the strength of repair in a murine model of rotator cuff detachment and repair.