Increase In Glycosaminoglycans Research Articles

Abstract 3359: HEY1::NCOA2 fusion disturbs the transcriptional program of chondrogenic differentiation and activates oncogenes in mesenchymal chondrosarcoma tumorigenesis

Abstract Mesenchymal chondrosarcoma (MCS) is a rare high-grade mesenchymal neoplasm occurring in bone and soft tissue. It accounts for 2%~10% of all chondrosarcomas and affects mainly adolescents and young adults. Genetically, this tumor is characterized by the recurrent HEY1::NCOA2 fusion. To functionally characterize the fusion in MCS tumorigenesis, we generated stably transduced iPSC-derived mesenchymal stem cells (iPSC-MSC) with inducible expression of HEY1::NCOA2. We previously demonstrated that the expression of HEY1::NCOA2 significantly enhanced cell proliferation, upregulated PDGFB/PI3K/AKT signaling, and induced genes that are involved in chondrocyte differentiation. In this study, we evaluated the impact on chondrogenic lineage differentiation and dysregulated pathways resulting from HEY1::NCOA2 expression and further elaborated oncogenic pathways in MCS tumorigenesis. We first successfully established the differentiation culture of inducing iPSC-MSC on chondrogenic lineage differentiation, showing an increase in glycosaminoglycans and the expression of differentiation markers (COL1A1, COL2A1, ACAN, COMP, SOX9 and COL10A1) at 7-, 14-, and 21-days post treatment with differentiation medium. The iPSC-MSC-HEY1::NCOA2 cells and control cells were cultured for chondrogenic lineage differentiation. During differentiation culture, we collected cells from each condition at multiple time points to (1) monitor the expression of differentiation markers and (2) perform transcriptomic profiling. Principal component analysis showed that cells with the expression of HEY1::NCOA2 possessed a distinct feature from parental cells and control cells, however, HEY1::NCOA2 did not simply suppress chondrogenic differentiation. The differential gene expression indicated that HEY1::NCOA2 fusion disrupted genes related to fine tuning of chondrogenic differentiation (SOX9, COMP, and IGF1) and activated oncogenes (PDGFB, BCL2, CCND1, IGF2, etc.). Notably, we demonstrated that both BCL2 (an anti-apoptotic protein) and COMP (possess antiapoptotic function that protects chondrocytes against death and blocks activation of caspase 3) were directly targeted and consistently upregulated by HEY1::NCOA2 fusion. In addition, the functional classification of genes (n=86) consistently increased with HEY1:: NCOA2 expression through differentiation of the chondrogenic lineage demonstrated that the PI3K/AKT pathway was the most significantly enriched, which further supports targeting the PDGFB/PI3K/AKT axis in MCS therapy. Together, this study sheds light on how HEY1::NCOA2 fusion shapes key mechanisms of chondrogenic differentiation and exerts oncogenic activation. Our findings provide the rationale for anti-apoptosis along with anti-PDGFB/PI3K/AKT signaling in the treatment of mesenchymal chondrosarcoma. Citation Format: Wenqing Qi, Wojciech Rosikiewicz, Beisi Xu, Lu Wang. HEY1::NCOA2 fusion disturbs the transcriptional program of chondrogenic differentiation and activates oncogenes in mesenchymal chondrosarcoma tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3359.

Intracellular and Extracellular Water Balance in Patients with Lipedema.

Background: The presence of edema in patients with lipedema has been investigated in many studies. However, no study has been found that examines the amount of intracellular (ICF) and extracellular fluid (ECF) in these patients together. The aim of this study is to examine the amount of ICF and ECF in patients with stage 2 lipedema. Methods and Results: Twenty-four patients diagnosed with stage 2 lipedema were included in the study. Bioimpedance spectroscopy was applied to measure extracellular and intracellular water levels. The mean age of the participants was 44.58 ± 2.95 years. The mean body mass index (BMI) of the participants was 33.9 ± 1.84 kg/m2. It was observed that the amount of ECF in the patients was above the normal values (p < 0.001), whereas the amount of ICF was within the normal range (p = 0.801). In addition, it was observed that there was a moderate relationship between BMI and the amount of ECF. Conclusion: Although the amount of ICF is within the normal range in patients with lipedema, an increase in ECF is observed. The reason why edema is not observed in these patients despite the increase in ECF may be the increase in glycosaminoglycan and proteoglycan synthesis. In addition, the attention of these patients to weight control may contribute to slowing the course of the disease by preventing the increase in the amount of ECF. For a better diagnosis of lipedema, it is considered important to evaluate the amount of ECF in addition to routine evaluations.

Does a Temporary Increase in Glycosaminoglycan (GAG) Indicate Positive Intervertebral Disc Adaptation?

Many in the scientific community have been debating the extent to which the spine can adapt. While it is widely accepted that vertebral bones can adapt and become stronger/denser with appropriately dosed loading, many unanswered questions remain regarding intervertebral discs (IVDs). Recently, some claim that IVDs can adapt to stress under load and become stronger and more resilient over time based on the findings from a recent study “Imaging of exercise-induced spinal remodeling in elite rowers” by Frenken et al 2022 in the Journal of Science and Medicine in Sport. The Frenken paper investigated the IVD Glycosaminoglycan (GAG) increases in elite rowers over the course of a training period, which was to indicate a positive adaptation to training based on the assumption that GAG is decreased in discs with degeneration. A cause-and-effect relationship does not exist and in fact, is unlikely to exist particularly with a historically high prevalence of disc degeneration in rowers. The Frenken paper supports the notion that the GAG content alters in response to certain stimuli; however, we cannot conclude that a temporary increase in GAG content supports the hypothesis that the IVD adapts positively to load. An alternative postulate is that repetitive biomechanical loading of the intervertebral disc through intra-lumbar flexion (as observed in rowing) does stimulate nutrient transport across the vertebral endplates; however, this biochemical response is of limited benefit and should not be perceived as a positive adaptation to the overall longevity and health of the disc. In this context, the elevation in GAG may be more appropriately interpreted as a maladaptive indicator of undesirable biomechanical intra-lumbar flexion stress that progressively drives cartilaginous endplate sclerosis leading ultimately to the clinically observed accelerated disc degeneration, future back pain symptoms, and reduced GAG concentration in rowers. This contrasting interpretation is consistent with the clinical observation of intra-lumbar flexion biomechanical stress, the transient rise in GAG followed by a reduction that correlates with the radiological findings and back pain symptoms evolving over time in elite rowers.

Bioprinting of biomimetic self-organised cartilage with a supporting joint fixation device

Despite sustained efforts, engineering truly biomimetic articular cartilage (AC) via traditional top-down approaches remains challenging. Emerging biofabrication strategies, from 3D bioprinting to scaffold-free approaches that leverage principles of cellular self-organisation, are generating significant interest in the field of cartilage tissue engineering as a means of developing biomimetic tissue analogues in vitro. Although such strategies have advanced the quality of engineered cartilage, recapitulation of many key structural features of native AC, in particular a collagen network mimicking the tissue’s ‘Benninghoff arcade’, remains elusive. Additionally, a complete solution to fixating engineered cartilages in situ within damaged synovial joints has yet to be identified. This study sought to address both of these key challenges by engineering biomimetic AC within a device designed to anchor the tissue within a synovial joint defect. We first designed and fabricated a fixation device capable of anchoring engineered cartilage into the subchondral bone. Next, we developed a strategy for inkjet printing porcine mesenchymal stem/stromal cells (MSCs) into this supporting fixation device, which was also designed to provide instructive cues to direct the self-organisation of MSC condensations towards a stratified engineered AC. We found that a higher starting cell-density supported the development of a more zonally defined collagen network within the engineered tissue. Dynamic culture was implemented to further enhance the quality of this engineered tissue, resulting in an approximate 3 fold increase in glycosaminoglycan and collagen accumulation. Ultimately this strategy supported the development of AC that exhibited near-native levels of glycosaminoglycan accumulation (>5% WW), as well as a biomimetic collagen network organisation with a perpendicular to a parallel fibre arrangement (relative to the tissue surface) from the deep to superficial zones via arcading fibres within the middle zone of the engineered tissue. Collectively, this work demonstrates the successful convergence of novel biofabrication methods, bioprinting strategies and culture regimes to engineer a hybrid implant suited to resurfacing AC defects.

Altered presence of extra cellular matrix components in murine skin cancer: Modulation by Azadirachta indica leaf extract

Background and aimAlthough, the anticancer potential of Aqueous Azadirachta indica leaf extract (AAILE) has been robustly established against cutaneous squamous cell carcinoma (SCC) in mice, however, its ability in modulating tumor associated extra cellular matrix (ECM) is largely unknown. Therefore, the present study was conceived to explore changes in ECM during murine skin cancer and its chemoprevention by AAILE. Experimental procedureSkin tumors were induced using a two-stage model of carcinogenesis employing topical application of 7,12-Dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoyl phorbol-13-acetate (TPA) as carcinogen and promoter respectively. AAILE was administered orally to the animals. Male Laca mice were divided into four groups: control, AAILE, DMBA/TPA and AAILE + DMBA/TPA. ResultsThe tumors obtained in DMBA/TPA and AAILE + DMBA/TPA groups were histologically identified as SCC. Tumor induction in these groups was accompanied by raised serum carcinoembryonic antigen (CEA) levels when compared to control counterparts. Assessment of hydroxyproline levels and histochemical staining with sirius red and trichrome stain revealed an increase in collagen in tumors of DMBA/TPA group. An increase in glycosaminoglycans (GAGs) levels was also observed in DMBA/TPA group as made evident by biochemical studies and histochemical staining using mucicarmine and alcian blue-periodic acid schiff’s stain. Administration of AAILE to DMBA/TPA treated animals caused a decrease in collagen and GAG levels along with a decrease in serum CEA levels. ConclusionSkin tumors exhibited altered presence of ECM components which is indicative of a modified ECM. AAILE administration antagonised tumor associated ECM alterations which may be contributing to its chemopreventive activity as reported previously.

In vitro study of cartilage tissue engineering using human adipose-derived stem cells induced by platelet-rich plasma and cultured on silk fibroin scaffold

BackgroundCartilage tissue engineering is a promising technique for repairing cartilage defect. Due to the limitation of cell number and proliferation, mesenchymal stem cells (MSCs) have been developed as a substitute to chondrocytes as a cartilage cell-source. This study aimed to develop cartilage tissue from human adipose-derived stem cells (ADSCs) cultured on a Bombyx mori silk fibroin scaffold and supplemented with 10% platelet-rich plasma (PRP).MethodsHuman ADSCs and PRP were characterized. A silk fibroin scaffold with 500 μm pore size was fabricated through salt leaching. ADSCs were then cultured on the scaffold (ADSC-SS) and supplemented with 10% PRP for 21 days to examine cell proliferation, chondrogenesis, osteogenesis, and surface marker expression. The messenger ribonucleic acid (mRNA) expression of type 2 collagen, aggrecan, and type 1 collagen was analysed. The presence of type 2 collagen confirming chondrogenesis was validated using immunocytochemistry. The negative and positive controls were ADSC-SS supplemented with 10% foetal bovine serum (FBS) and ADSC-SS supplemented with commercial chondrogenesis medium, respectively.ResultsCells isolated from adipose tissue were characterized as ADSCs. Proliferation of the ADSC-SS PRP was significantly increased (p < 0.05) compared to that of controls. Chondrogenesis was observed in ADSC-SS PRP and was confirmed through the increase in glycosaminoglycans (GAG) and transforming growth factor-β1 (TGF-β1) secretion, the absence of mineral deposition, and increased surface marker proteins on chondrogenic progenitors. The mRNA expression of type 2 collagen in ADSC-SS PRP was significantly increased (p < 0.05) compared to that in the negative control on days 7 and 21; however, aggrecan was significantly increased on day 14 compared to the controls. ADSC-SS PRP showed stable mRNA expression of type 1 collagen up to 14 days and it was significantly decreased on day 21. Confocal analysis showed the presence of type 2 collagen in the ADSC-SS PRP and positive control groups, with high distribution outside the cells forming the extracellular matrix (ECM) on day 21.ConclusionOur study showed that ADSC-SS with supplemented 10% PRP medium can effectively support chondrogenesis of ADSCs in vitro and promising for further development as an alternative for cartilage tissue engineering in vivo.

Mast Cell/Proteinase Activated Receptor 2 (PAR2) Mediated Interactions in the Pathogenesis of Discogenic Back Pain.

Mast cells (MCs) are present in the painful degenerate human intervertebral disc (IVD) and are associated with disease pathogenesis. MCs release granules containing enzymatic and inflammatory factors in response to stimulants or allergens. The serine protease, tryptase, is unique to MCs and its activation of the G-protein coupled receptor, Protease Activated Receptor 2 (PAR2), induces inflammation and degradation in osteoarthritic cartilage. Our previously published work has demonstrated increased levels of MC marker tryptase in IVD samples from discogenic back pain patients compared to healthy control IVD samples including expression of chemotactic agents that may facilitate MC migration into the IVD. To further elucidate MCs’ role in the IVD and mechanisms underlying its effects, we investigated whether (1) human IVD cells can promote MC migration, (2) MC tryptase can mediate up-regulation of inflammatory/catabolic process in human IVD cells and tissue, and (3) the potential of PAR2 antagonist to function as a therapeutic drug in in vitro human and ex vivo bovine pilot models of disease. MC migration was quantitatively assessed using conditioned media from primary human IVD cells and MC migration examined through Matrigel. Exposure to soluble IVD factors significantly enhanced MC migration, suggesting IVD cells can recruit MCs. We also demonstrated significant upregulation of MC chemokine SCF and angiogenic factor VEGFA gene expression in human IVD cells in vitro in response to recombinant human tryptase, suggesting tryptase can enhance recruitment of MCs and promotion of angiogenesis into the usually avascular IVD. Furthermore, tryptase can degrade proteoglycans in IVD tissue as demonstrated by significant increases in glycosaminoglycans released into surrounding media. This can create a catabolic microenvironment compromising structural integrity and facilitating vascular migration usually inhibited by the anti-angiogenic IVD matrix. Finally, as a “proof of concept” study, we examined the therapeutic potential of PAR2 antagonist (PAR2A) on human IVD cells and bovine organ culture IVD model. While preliminary data shows promise and points toward structural restoration of the bovine IVD including down-regulation of VEGFA, effects of PAR2 antagonist on human IVD cells differ between gender and donors suggesting that further validation is required with larger cohorts of human specimens.

The effects of different cell-based therapies at a critical time point during the soft-tissue healing process

Background: Cell-based therapy for soft tissue injuries remains controversial. Adult mesenchymal stem cells (MSCs) are therapeutic candidates given their capacity for self-renewal, immunoprivilege, and differentiation capacity for chondrocyte and tenocyte lineages. Platelet rich plasma (PRP) has been reported to promote collagen synthesis and cell proliferation, influencing the healing of ligaments and cartilage. We hypothesize that allogeneic MSCs and PRP have additive effects on promoting ligament healing in an in-vivo rat medial collateral ligament (MCL) injury model. Methods: MCLs of 20 females Sprague rats were bilaterally transected and treated with either saline (controls) or 1 of 3 treatment groups; (1) allogeneic MSCs (105 cells), (2) PRP and (3) allogeneic MSCs &amp; PRP. In addition, five rats were used for the Sham group (surgery + no ligament injury). Rats were sacrificed two weeks post-surgery and the MCLs harvested for histological analysis by hematoxylin and eosin and alcian blue staining. Statistical analysis was performed using Fischer’s exact test with pair-wise comparisons and Bonferroni multiple comparison correction. Results: Histologically, differences across all injured groups (treatment groups and controls) were observed in cellularity (p &lt; 0.0185), regeneration of collagen fibers (p &lt; 0.0084), vascularity (p = 0.0129), inflammation (p = 0.0121) and glycosaminoglycan content (p = 0.0085). From pair-wise comparisons, only the combination allogeneic MSCs &amp; PRP group differed significantly from controls in increased cellularity (p = 9.04 x 10-4) and regeneration of collagen fibers (p = 6.58x10-4). In addition, the PRP group showed significant increase in glycosaminoglycan (p = 0.006) content when compared to the allogeneic MSCs group. Conclusion: The addition of allogeneic MSCs and PRP to an injured MCL show a significant histological increase in degree of cellularity, vascularity and the regeneration of collagen fibers when compared to controls. These data support a possible additive effect of combining allogeneic MSCs and PRP therapy to increase important repair factors during the proliferation/repair phase of post ligament injury. This preliminary study demonstrates that additional functional and biomechanical studies are warranted to determine the role that inflammatory responses versus tissue regeneration are contributing to this mechanism.

Short Link N promotes disc repair in a rabbit model of disc degeneration

BackgroundThe degeneration of the intervertebral disc (IVD) is characterized by proteolytic degradation of the extracellular matrix, and its repair requires the production of an extracellular matrix with a high proteoglycan-to-collagen ratio characteristic of a nucleus pulposus (NP)-like phenotype in vivo. At the moment, there is no medical treatment to reverse or even retard disc degeneration. The purpose of the present study was to determine if a low dose of short link N (sLN), a recently discovered fragment of the link N peptide, could behave in a manner similar to that of link N in restoring the proteoglycan content and proteoglycan-to-collagen ratio of the disc in a rabbit model of IVD degeneration, as an indication of its potential therapeutic benefit in reversing disc degeneration.MethodsAdolescent New Zealand white rabbits received an annular puncture with an 18-gauge needle into two noncontiguous discs to induce disc degeneration. Two weeks later, either saline (10 μL) or sLN (25 μg in 10 μL saline) was injected into the center of the NP. The sLN concentration was empirically chosen at a lower molar concentration equivalent to half that of link N (100 μg in 10 μL). The effect on radiographic, biochemical and histologic changes were evaluated.ResultsFollowing needle puncture, disc height decreased by about 25–30% within 2 weeks and maintained this loss for the duration of the 12-week study; a single 25-μg sLN injection at 2 weeks partially restored this loss in disc height. sLN injection led to an increase in glycosaminoglycans (GAG) content 12 weeks post-injection in both the NP and annulus fibrosus (AF). There was a trend towards maintaining control disc collagen-content with sLN supplementation and the GAG-to-collagen ratio in the NP was increased when compared to the saline group.ConclusionsWhen administered to the degenerative disc in vivo, sLN injection leads to an increase in proteoglycan content and a trend towards maintaining control disc collagen content in both the NP and AF. This is similar to link N when it is administered to the degenerative disc. Thus, pharmacologically, sLN supplementation could be a novel therapeutic approach for treating disc degeneration.

Differential Response of Bovine Mature Nucleus Pulposus and Notochordal Cells to Hydrostatic Pressure and Glucose Restriction.

The nucleus pulposus of the human intervertebral disc contains 2 cell types: notochordal (NC) and mature nucleus pulposus (MNP) cells. NC cell loss is associated with disc degeneration and this process may be initiated by mechanical stress and/or nutrient deprivation. This study aimed to investigate the functional responses of NC and MNP cells to hydrostatic pressures and glucose restriction. Bovine MNP and NC cells were cultured in 3-dimensional alginate beads under low (0.4-0.8 MPa) and high (1.6-2.4 MPa) dynamic pressure for 24 hours. Cells were cultured in either physiological (5.5 mM) glucose media or glucose-restriction (0.55 mM) media. Finally, the combined effect of glucose restriction and high pressure was examined. Cell viability and notochordal phenotypic markers were not significantly altered in response to pressure or glucose restriction. MNP cells responded to low pressure with an increase in glycosaminoglycan (GAG) production while high pressure significantly decreased ACAN gene expression compared with atmospheric controls. NC cells showed no response in matrix gene expression or GAG production with either loading regime. Glucose restriction decreased NC cell TIMP-1 expression but had no effect on MNP cells. The combination of glucose restriction and high pressure only affected MNP cell gene expression, with decreased ACAN, Col2α1, and ADAMTS-5 expression. This study shows that NC cells are more resistant to acute mechanical stresses than MNP cells and provides a strong rationale for future studies to further our understanding the role of NC cells within the disc, and the effects of long-term exposure to physical stresses.

Compromised mechanical homeostasis in arterial aging and associated cardiovascular consequences.

Aging leads to central artery stiffening and associated hemodynamic sequelae. Because healthy arteries exhibit differential geometry, composition, and mechanical behaviors along the central vasculature, we sought to determine whether wall structure and mechanical function differ across five vascular regions-the ascending and descending thoracic aorta, suprarenal and infrarenal abdominal aorta, and common carotid artery-in 20 versus 100-week-old male wild-type mice. Notwithstanding generally consistent changes across these regions, including a marked thickening of the arterial wall, diminished in vivo axial stretch, and loss of elastic energy storage capacity, the degree of changes tended to be slightly greater in abdominal than in thoracic or carotid vessels. Likely due to the long half-life of vascular elastin, most mechanical changes in the arterial wall resulted largely from a distributed increase in collagen, including thicker fibers in the media, and localized increases in glycosaminoglycans. Changes within the central arteries associated with significant increases in central pulse pressure and adverse changes in the left ventricle, including increased cardiac mass and decreased diastolic function. Given the similar half-life of vascular elastin in mice and humans but very different life-spans, there are important differences in the aging of central vessels across these species. Nevertheless, the common finding of aberrant matrix remodeling contributing to a compromised mechanical homeostasis suggests that studies of central artery aging in the mouse can provide insight into mechanisms and treatment strategies for the many adverse effects of vascular aging in humans.

Platelet‐rich plasma enhances the integration of bioengineered cartilage with native tissue in anin vitromodel

Current therapies for cartilage repair can be limited by an inability of the repair tissue to integrate with host tissue. Thus, there is interest in developing approaches to enhance integration. We have previously shown that platelet-rich plasma (PRP) improves cartilage tissue formation. This raised the question as to whether PRP could promote cartilage integration. Chondrocytes were isolated from cartilage harvested from bovine joints, seeded on a porous bone substitute and grown in vitro to form an osteochondral-like implant. After 7 days, the biphasic construct was soaked in PRP for 30 min before implantation into the core of a donut-shaped biphasic explant of native cartilage and bone. Controls were not soaked in PRP. The implant-explant construct was cultured for 2-4 weeks. PRP-soaked bioengineered implants integrated with host tissue in 73% of samples, whereas controls only integrated in 19% of samples. The integration strength, as determined by a push-out test, was significantly increased in the PRP-soaked implant group (219 ± 35.4 kPa) compared with controls (72.0 ± 28.5 kPa). This correlated with an increase in glycosaminoglycan and collagen accumulation in the region of integration in the PRP-treated implant group, compared with untreated controls. Immunohistochemical studies revealed that the integration zone contained collagen type II and aggrecan. The cells at the zone of integration in the PRP-soaked group had a 3.5-fold increase in matrix metalloproteinase-13 gene expression compared with controls. These results suggest that PRP-soaked bioengineered cartilage implants may be a better approach for cartilage repair due to enhanced integration.

The Role of Platelet-Rich Plasma in Promoting Cartilage Integration and Chondrocyte Migration

Objectives:Current therapies for cartilage repair either do not result in regeneration of articular cartilage, or there is inadequate integration with the host tissue leading to failure of the repair. Thus, there is an interest in developing alternative approaches. The mechanisms of cartilage integration remain relatively unknown, however it is believed that chondrocyte migration is crucial to this process. Previously, we showed that platelet rich plasma (PRP) enhances in vitro cartilage tissue formation. We hypothesized that PRP will enhance the integration of bioengineered cartilage with native cartilage due to increased matrix accumulation at the interface and that PRP could promote chondrocyte migration.Methods:Isolated bovine chondrocytes were seeded on a porous bone substitute and grown in vitro to form osteochondral-like tissue. After 7 days the biphasic constructs were soaked in PRP for 30 minutes prior to implantation into the core of a ring-shaped osteochondral explant. Controls were not soaked in PRP. The resulting implant-explant construct was cultured in a stirring bioreactor for 2 weeks (contact model). Alternatively, the PRP soaked biphasic construct was placed 2mm away from a native cartilage/bone plug of equal dimensions to assess chondrocyte migration between the two tissues (non-contact model). The integration zone was visualized histologically. A push-out test was performed to assess the strength of integration. Matrix accumulation at the zone of integration was assessed biochemically and the gene expression of the cells in this region was assessed by RT-PCR. Cell migration was evaluated by video microscopy over 8 days. Significance (p<0.05) was determined by a χ2 test, a student’s t-test or one-way ANOVA with tukey’s post hoc.Results:PRP soaked implants (contact model) integrated with host tissue in 73% of samples, whereas control implants integrated in 19% of samples (p<0.05). The integration strength was significantly increased in the PRP soaked implant group compared to controls (219 ± 35.4 kPa and 72.0 ± 28.5 kPa, respectively, p<0.05). This correlated with an increase in glycosaminoglycan and collagen accumulation in the region of integration in the PRP treated implant group (p<0.05). Immunohistochemical studies revealed that the integration zone was rich in collagen type II and aggrecan. The cells at the zone of integration in the PRP soaked group had a 3.5 fold increase in matrix metalloproteinase 13 (MMP13) gene expression (p<0.05) compared to controls. In the non-contact cultures, a network of fibers developed to connect the PRP soaked construct to the native plug. No fibers formed when the constructs were not soaked in PRP. Time-lapse videos showed chondrocytes with a round phenotype migrating along the fibers and undergoing cell division within 24 hours. These cells came from the bioengineered cartilage whereas migrating cells from native cartilage were only seen after 5 days in select cultures. After 2 weeks, the cells deposited cartilage-like matrix around the fibers as seen histologically. A single layer of cells expressed MMP13 on the outer aspects of the tissue.Conclusion:PRP soaked bioengineered cartilage implants showed improved integration with native cartilage compared to non-soaked implants perhaps due to increased matrix accumulation. Chondrocytes grew out from the in vitro formed tissue and migrated along fibers after PRP soaking. The contribution of these cells to integration requires further study.

Role of RHEB in Regulating Differentiation Fate of Mesenchymal Stem Cells for Cartilage and Bone Regeneration.

Advances in mesenchymal stem cells (MSCs) and cell replacement therapies are promising approaches to treat cartilage and bone defects since substantial differentiation capacities of MSCs match the demands of tissue regeneration. Our understanding of the dynamic process requiring indispensable differentiation of MSCs remains limited. Herein, we describe the role of RHEB (Ras homolog enriched in brain) regulating gene signature for differentiation of human adipose derived mesenchymal stem cells (ASCs) into chondrogenic, osteogenic, and adipogenic lineages. RHEB-overexpression increases the proliferation of the ASCs. RHEB enhances the chondrogenic differentiation of ASCs in 3D culture via upregulation of SOX9 with concomitant increase in glycosaminoglycans (GAGs), and type II collagen (COL2). RHEB increases the osteogenesis via upregulation of runt related transcription factor 2 (RUNX2) with an increase in the calcium and phosphate contents. RHEB also increases the expression of osteogenic markers, osteonectin and osteopontin. RHEB knockdown ASCs were incapable of expressing sufficient SRY (Sex determining region Y)-box 9 (SOX9) and RUNX2, and therefore had decreased chondrogenic and osteogenic differentiation. RHEB-overexpression impaired ASCs differentiation into adipogenic lineage, through downregulation of CCAAT/enhancer binding protein beta (C/EBPβ). Conversely, RHEB knockdown abolished the negative regulation of adipogenesis. We demonstrate that RHEB is a novel regulator, with a critical role in ASCs lineage determination, and RHEB-modulated ASCs may be useful as a cell therapy for cartilage and bone defect treatments.

* Thermosensitive Poly(N-vinylcaprolactam) Injectable Hydrogels for Cartilage Tissue Engineering.

Injectable hydrogels have gained prominence in the field of tissue engineering for minimally invasive delivery of cells for tissue repair and in the filling of irregular defects. However, many injectable hydrogels exhibit long gelation times or are not stable for long periods after injection. To address these concerns, we used thermosensitive poly(N-vinylcaprolactam) (PNVCL) hydrogels due to their cytocompatibility and fast response to temperature stimuli. Changes in the PNVCL molecular weight and concentration enabled the development of hydrogels with tunable mechanical properties and fast gelation times (<60 s when the temperature was raised from room temperature to physiologic temperature). Chondrocytes (CHs) and mesenchymal stem cells were encapsulated in PNVCL hydrogels and exhibited high viability (∼90%), as monitored by Live/Dead staining and Alamar Blue assays. Three-dimensional constructs of CH-laden PNVCL hydrogels supported cartilage-specific extracellular matrix production both in vitro and after subcutaneous injection in nude rats for up to 8 weeks. Moreover, biochemical analyses of constructs demonstrated a time-dependent increase in glycosaminoglycans (GAGs) and collagen, which were significantly augmented in the implants cultured in vivo. Histological analyses also demonstrated regular distribution of synthesized cartilage components, including abundant GAGs and type II collagen. The findings from this study demonstrate thermosensitive PNVCL as a candidate injectable biomaterial to deliver cells for cartilage tissue engineering.

Increase of glycosaminoglycans and metalloproteinases 2 and 9 in liver extracellular matrix on early stages of extrahepatic cholestasis.

Cholestasis produces hepatocellular injury, leukocyte infiltration, ductular cells proliferation and fibrosis of liver parenchyma by extracellular matrix replacement. Analyze bile duct ligation effect upon glycosaminoglycans content and matrix metalloproteinase (MMPs) activities. Animals (6-8 weeks; n = 40) were euthanized 2, 7 or 14 days after bile duct ligation or Sham-surgery. Disease evolution was analyzed by body and liver weight, seric direct bilirubin, globulins, gamma glutamyl transpeptidase (GGT), alkaline phosphatase (Alk-P), alanine and aspartate aminotransferases (ALT and AST), tissue myeloperoxidase and MMP-9, pro MMP-2 and MMP-2 activities, histopathology and glycosaminoglycans content. Cholestasis caused cellular damage with elevation of globulins, GGT, Alk-P, ALT, AST. There was neutrophil infiltration observed by the increasing of myeloperoxidase activity on 7 (P = 0.0064) and 14 (P = 0.0002) groups which leads to the magnification of tissue injuries. Bile duct ligation increased pro-MMP-2 (P = 0.0667), MMP-2 (P = 0.0003) and MMP-9 (P<0.0001) activities on 14 days indicating matrix remodeling and establishment of inflammatory process. Bile duct ligation animals showed an increasing on dermatan sulfate and/or heparan sulfate content reflecting extracellular matrix production and growing mitosis due to parenchyma depletion. Cholestasis led to many changes on rats' liver parenchyma, as so as on its extracellular matrix, with major alterations on MMPs activities and glycosaminoglycans content.

Assessment of lumbar intervertebral disc glycosaminoglycan content by gadolinium-enhanced MRI before and after 21-days of head-down-tilt bedrest.

During spaceflight, it has been shown that intervertebral discs (IVDs) increase in height, causing elongation of the spine up to several centimeters. Astronauts frequently report dull lower back pain that is most likely of discogenic origin and may result from IVD expansion. It is unknown whether disc volume solely increases by water influx, or if the content of glycosaminoglycans also changes in microgravity. Aim of this pilot study was to investigate effects of the spaceflight analog of bedrest on the glycosaminoglycan content of human lumbar IVDs. Five healthy, non-smoking, male human subjects of European descent were immobilized in 6° head-down-tilt bedrest for 21 days. Subjects remained in bed 24 h a day with at least one shoulder on the mattress. Magnetic Resonance Imaging (MRI) scans were taken according to the delayed gadolinium-enhanced magnetic resonance imaging (dGEMRIC) protocol before and after bedrest. The outcome measures were T1 and ΔT1. Scans were performed before and after administration of the contrast agent Gd-DOTA, and differences between T1-values of both scans (ΔT1) were computed. ΔT1 is the longitudinal relaxation time in the tissue and inversely related to the glycosaminoglycan-content. For data analysis, IVDs L1/2 to L4/5 were semi-automatically segmented. Zones were defined and analyzed separately. Results show a highly significant decrease in ΔT1 (p<0.001) after bedrest in all IVDs, and in all areas of the IVDs. The ΔT1-decrease was most prominent in the nucleus pulposus and in L4/5, and was expressed slightly more in the posterior than anterior IVD. Unexpected negative ΔT1-values were found in Pfirrmann-grade 2-discs after bedrest. Significantly lower T1 before contrast agent application was found after bedrest compared to before bedrest. According to the dGEMRIC-literature, the decrease in ΔT1 may be interpreted as an increase in glycosaminoglycans in healthy IVDs during bedrest. This interpretation seems contradictory to previous findings in IVD unloading.

A high-throughput model of post-traumatic osteoarthritis using engineered cartilage tissue analogs

A number of in vitro models of post-traumatic osteoarthritis (PTOA) have been developed to study the effect of mechanical overload on the processes that regulate cartilage degeneration. While such frameworks are critical for the identification therapeutic targets, existing technologies are limited in their throughput capacity. Here, we validate a test platform for high-throughput mechanical injury incorporating engineered cartilage. We utilized a high-throughput mechanical testing platform to apply injurious compression to engineered cartilage and determined their strain and strain rate dependent responses to injury. Next, we validated this response by applying the same injury conditions to cartilage explants. Finally, we conducted a pilot screen of putative PTOA therapeutic compounds. Engineered cartilage response to injury was strain dependent, with a 2-fold increase in glycosaminoglycan (GAG) loss at 75% compared to 50% strain. Extensive cell death was observed adjacent to fissures, with membrane rupture corroborated by marked increases in lactate dehydrogenase (LDH) release. Testing of established PTOA therapeutics showed that pan-caspase inhibitor [Z-VAD-FMK (ZVF)] was effective at reducing cell death, while the amphiphilic polymer [Poloxamer 188 (P188)] and the free-radical scavenger [N-Acetyl-L-cysteine (NAC)] reduced GAG loss as compared to injury alone. The injury response in this engineered cartilage model replicated key features of the response of cartilage explants, validating this system for application of physiologically relevant injurious compression. This study establishes a novel tool for the discovery of mechanisms governing cartilage injury, as well as a screening platform for the identification of new molecules for the treatment of PTOA.

ЦИСТИТ КАК ВЕРОЯТНЫЙ ЭТИОЛОГИЧЕСКИЙ ФАКТОР СИНДРОМА НЕДЕРЖАНИЯ МОЧИ У СОБОЛЕЙ

«Wet belly» disease of fur animals, the sables particularly, has little been studied, and the data have been mostly limited to publications in 1960-1970. In our examinations carried out earlier on the sables with urinary incontinence, the incidence of anemia, anorexia, the inflammation in gastrointestinal tract, and the weight loss were also found, indicating the urinary incontinence not to be the only symptom of the disease. This article gives new information for histological changes in the urinary bladder of 7-month black sables, injured by «wet belly» (a «syndrome of urinary incontinence»). The catharal and haemorrhagical cystitis were indicated as possible etiological factors of the disease. Post-mortem examination of 76 sables with «wet belly», or «syndrome of urinary incontinence», showed that 28 % of the males, and 41 % of the females have various types of inflammation in the urinary bladder. Using morphometric analysis, a loss of the urinary bladder mucosa and muscle layer was demonstrated in the urinary bladder of sick animals if compared to healthy sables. Authors suggest that the phenomenon is probably related to high epithelium exfoliation and bladder dysfunction, such as atony and dilatation in the muscle layer of urinary bladder. In the young sables, injured by haemorrhagic cystitis with the symptoms of «wet belly» disease, an increase of glycosaminoglycans in the urinary bladder tissue was detected by hystochemical methods, indicating activation of local defense mechanisms due to the inflammation. These facts support a hypothesis, whereby a various form of cystitis may account for the development of «wet belly» disease in young sables.

The Effect of IGF-I on Anatomically Shaped Tissue-Engineered Menisci

This study investigates the effect of insulin-like growth factor (IGF)-I on the development of anatomically-shaped alginate menisci seeded with meniscal fibrochondrocytes. To accomplish this, bovine meniscal fibrochondrocytes were seeded into 2% w/v alginate, crosslinked with calcium sulfate, and injected into anatomical molds derived from microcomputed tomography scans. The meniscal constructs were then cultured for up to 4 weeks with or without 100 ng/mL IGF-I supplemented in the media. Histological, immunohistological, biochemical, and mechanical analyses were performed to characterize tissue development, accumulation and localization of extracellular matrix, and mechanical properties. After 4 weeks of culture, IGF-I treatment significantly improved mechanical and biochemical properties, while maintaining DNA content, with a 26-fold increase in glycosaminoglycan (GAG) content and 10-fold increase in collagen content compared to 0-week controls, and a 3-fold increase in the equilibrium modulus at 2 weeks compared to controls. IGF-I-treated menisci had ∼60% of the GAG content of native tissue and the compressive equilibrium modulus matched native properties by 2 weeks of culture. Further, IGF-I-treated menisci developed a distinct surface layer similar to native tissue with elongated cells and collagen fibers aligned parallel to the surface, the presence of types I and II collagen, and accumulation of lubricin. This study demonstrates that IGF-I treatment can greatly increase the mechanical and biochemical properties of engineered tissues and aid in the development of a distinct surface zone similar to the superficial zone of native menisci.