Decrease In Glycosaminoglycans Research Articles

Age-Related Gene and Protein Expression in Mouse Mandibular Condyle Analyzed by Cap Analysis of Gene Expression and Immunohistochemistry

Introduction: Aging, an inevitable physiological process, leads to morphological and histological degenerative changes in the mandibular condylar cartilage (MCC); however, the molecular mechanism has not yet been elucidated, and little information is available on age-related factors. Therefore, this study was designed to identify age-related factors by investigating the age-related differentially expressed genes (DEGs) and localization of their translated protein expression in the mandibular condyle. Methods: Mandibular condyles were collected from 10- and 50-week-old mice. Total RNA was extracted from the samples and then analyzed using cap analysis of gene expression (CAGE) to identify age-related DEGs. Gene ontology (GO) enrichment analysis was performed to determine which biological processes were most affected by aging in terms of gene expression using Metascape. The mandibular condyle samples were processed for histology to investigate morphological changes caused by aging and for immunohistochemistry to localize the protein expression encoded by age-related genes identified with CAGE. Semi-quantitative immunohistochemistry was performed to assess age-related extracellular matrix (ECM) protein levels in the MCC. The histological sections were also used for Alcian blue histochemistry to detect glycosaminoglycans (GAGs). Results: GO enrichment analysis revealed that the genes related to “extracellular matrix organization,” including Acan, Col1a1, Col1a2, Col2a1, Mmp3, Mmp9, and Mmp13, were most differentially expressed in the aged mandibular condyle. Among these seven genes, Mmp3 was upregulated, and the others were downregulated with aging. Histological examination showed the age-related morphological and histological changes in the MCC. Immunohistochemical investigation showed the localization of matrix metalloproteinases (MMPs)-3, -9, and -13 and their substrate proteins, aggrecan, type I collagen, and type II collagen, in the mandibular condyle at 10 and 50 weeks, indicating different localizations between the young and the aged. In the aged MCC, semi-quantitative immunohistochemistry showed a significant decrease in the aggrecan protein level, and Alcian blue histochemistry showed a decrease in GAGs. Conclusion: MMP-3, MMP-9, and MMP-13 contribute to the remodeling of the ECM of the MCC and subchondral bone during aging by degrading ECM proteins at specific times and sites under the regulation of their production and secretion.

Biological Changes of Autologous Auricular Cartilage in Rhinoplasty.

Autologous auricular cartilage is used extensively as a good graft material in rhinoplasty. In this study, clinical specimens from patients who underwent revision rhinoplasty with auricular cartilage grafts were collected to compare the changes before and after auricular cartilage transplantation with the use of histologic, immunohistochemical, and quantitative assays. Patients who underwent revision rhinoplasty from 2018 to 2022 were analyzed. Fresh auricular cartilage left after surgery and auricular cartilage graft tissue were examined and compared. Compared with fresh auricular cartilage, local fibrosis was seen in the transplanted auricular cartilage with a slight decrease in elastic fibers, type II collagen, and extracellular matrix secretion. Quantitative assays showed a decrease in glycosaminoglycan, DNA, and total collagen content in the transplanted auricular cartilage tissue. The results of the study suggest that the histologic characteristics, cell functionality, and biochemical composition of the grafted cartilage changed to a certain extent after autologous auricular cartilage graft rhinoplasty. These results provide insights into the selection of graft/filler materials for rhinoplasty and what changes to expect.

Decrease in Glycosaminoglycan with Aging in Normal Rat Articular Cartilage Is Greater in Females than in Males.

ObjectiveOsteoarthritis (OA) is more prevalent in females. We hypothesized that changes in articular cartilage (AC) constituents with aging may cause differences. Herein, we aimed to compare the changes in AC constituents with aging in male and female normal rats.DesignThe glycosaminoglycan (GAG) and collagen (COL) contents of the AC in knee, hip, and shoulder joints of male and female rats were quantified and compared between age groups and sexes.ResultsThe amount of GAG was decreased in multiple joints in both males and females with aging. In females, it had a significant decrease in all joints measured. The decrease in GAG with aging was more severe in females than in males. Even in young rats, the amount of knee joint GAG was significantly less in females than in males. The amount of COL in the AC was unchanged with aging in both sexes.ConclusionsThe drastic GAG decrease with aging in female normal rats may explain the higher prevalence and more severe OA in females.

Small-diameter artery decellularization: Effects of anionic detergent concentration and treatment duration on porcine internal thoracic arteries.

Engineered replacement materials have tremendous potential for vascular applications where over 400,000 damaged and diseased blood vessels are replaced annually in the United States alone. Unlike large diameter blood vessels, which are effectively replaced by synthetic materials, prosthetic small-diameter vessels are prone to early failure, restenosis, and reintervention surgery. We investigated the differential response of varying 0%-6% sodium dodecyl sulfate and sodium deoxycholate anionic detergent concentrations after 24 and 72h in the presence of DNase using biochemical, histological, and biaxial mechanical analyses to optimize the decellularization process for xenogeneic vascular tissue sources, specifically the porcine internal thoracic artery (ITA). Detergent concentrations greater than 1% were successful at removing cytoplasmic and cell surface proteins but not DNA content after 24 h. A progressive increase in porosity and decrease in glycosaminoglycan (GAG) content was observed with detergent concentration. Augmented porosity was likely due to the removal of both cells and GAGs and could influence recellularization strategies. The treatment duration on the other hand, significantly improved decellularization by reducing DNA content to trace amounts after 72 h. Prolonged treatment times reduced laminin content and influenced the vessel's mechanical behavior in terms of altered circumferential stress and stretch while further increasing porosity. Collectively, DNase with 1% detergent for 72 h provided an effective and efficient decellularization strategy to be employed in the preparation of porcine ITAs as bypass graft scaffolding materials with minor biomechanical and histological penalties.

4 The effects of clodronate disodium on normal and inflamed equine cartilage in vitro

Bisphosphonates (BP) are a group of drugs which modulate osteoclastic resorption and alter bone turnover. BP are only approved for use in navicular syndrome in adult horses, however, these drugs are being used off-label for their potential anti-inflammatory and pain-relieving effects. When the BP, clodronate disodium (CLO), is administered intramuscularly at the labeled dose, synovial fluid concentrations are detectable within 2 h of administration. There are currently no published studies evaluating the effects of CLO on equine joint tissues. This study evaluated the effects of CLO on normal and inflamed equine cartilage explants in vitro. Cartilage explants were collected from the femorotibial joints of 3 horses which underwent euthanasia for reasons other than stifle osteoarthritis. Only visually normal cartilage was selected for biopsy. The harvested cartilage from each femorotibial joint was subdivided into 24-well plates. Cartilage explants were subjected to 6 different treatments with 8 replicates each, after a 24 h equilibration period: Control (Con), CLO 5 ng/ml (C/lo), CLO 50 ng/ml (C/hi), rEqIL-1β 10 ng/ml (IL), C/lo plus IL (ILC/lo) and C/hi concentration plus IL (ILC/hi). Explant culture media was collected every 24 h and stored at −20°C until analysis of sulfated glycosaminoglycan (GAG) content and nitric oxide (NO) content using a Dimethylmethylene Blue (DMMB) assay and a Greiss reaction, respectively. At the conclusion of the experiment (96 h), cartilage explants were papain-digested and GAG were quantified using a DMMB assay. Data were analyzed using PROC MIXED procedure (SAS 9.4), where horses were considered a random effect, and treatment and day were fixed effects. For all analyses, values of P < 0.05 were considered significant. At d 1, d 2, and d 3, the IL treatment group had a significantly higher concentration of GAG within the media compared with the Con group ( P < 0.05), indicating the expected catabolic effect of IL-1β on the explants. Treatments with clodronate (C/lo, C/hi) alone did not result in an increase in GAG or NO within the media compared with the control indicating CLO did not have a cytotoxic effect. However, the addition of CLO to the IL-1β treated explants (ILC/lo, ILC/hi) did not result in a significant decrease in GAG or NO release into the media when compared with rEqIL-1β (IL) alone. No differences were observed between treatments when GAG content of cartilage explants was measured following papain-digestion. Our in vitro study suggests that CLO does not result in cartilage toxicity. However, no anti-inflammatory or chondroprotective effects were observed.

AGE-RELATED DETERIORATION OF BONE TOUGHNESS IS RELATED TO DIMINISHING AMOUNT OF MATRIX GLYCOSAMINOGLYCANS (GAGS).

ABSTRACTHydration status significantly affects the toughness of bone. In addition to the collagen phase, recent evidence shows that glycosaminoglycans (GAGs) of proteoglycans (PGs) in the extracellular matrix also play a pivotal role in regulating the tissue‐level hydration status of bone, thereby affecting the tissue‐level toughness of bone. In this study, we hypothesized that the amount of GAGs in bone matrix decreased with age and such changes would lead to reduction in bound water and subsequently result in a decrease in the tissue‐level toughness of bone. To test the hypothesis, nanoscratch tests were conducted to measure the tissue‐level toughness of human cadaveric bone specimens, which were procured only from male donors in three different age groups: young (aged 26 ± 6 years), mid‐aged (aged 52 ± 5 years), and elderly (aged 73 ± 5 years), with 6 donors in each group. Biochemical and histochemical assays were performed to determine the amount and major subtypes of GAGs and proteoglycans in bone matrix. In addition, low‐field nuclear magnetic resonance (NMR) measurements were implemented to determine bound water content in bone matrix. The results demonstrated that aging resulted in a statistically significant reduction (17%) of GAGs in bone matrix. Concurrently, a significant deterioration (20%) of tissue‐level toughness of bone with age was observed. Most importantly, the deteriorated tissue‐level toughness of bone was associated significantly with the age‐related reduction (40%) of bound water, which was partially induced by the decrease of GAGs in bone matrix. Furthermore, we identified that chondroitin sulfate (CS) was a major subtype of GAGs, and the amount of CS decreased with aging accompanied with a decrease of biglycan that is a major subtype of PGs in bone. The findings of this study suggest that reduction of GAGs in bone matrix is likely one of the molecular origins for age‐related deterioration of bone quality. © 2017 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Development of a Cytocompatible Scaffold from Pig Immature Testicular Tissue Allowing Human Sertoli Cell Attachment, Proliferation and Functionality.

Cryopreservation of immature testicular tissue before chemo/radiotherapy is the only option to preserve fertility of cancer-affected prepubertal boys. To avoid reintroduction of malignant cells, development of a transplantable scaffold by decellularization of pig immature testicular tissue (ITT) able to support decontaminated testicular cells could be an option for fertility restoration in these patients. We, therefore, compared decellularization protocols to produce a cytocompatible scaffold. Fragments of ITT from 15 piglets were decellularized using three protocols: sodium dodecyl sulfate (SDS)-Triton (ST), Triton-SDS-Triton (TST) and trypsin 0.05%/ethylenediaminetetraacetic acid (EDTA) 0.02%-Triton (TET) with varying detergent concentrations. All protocols were able to lower DNA levels. Collagen retention was demonstrated in all groups except ST 1%, and a significant decrease in glycosaminoglycans was observed in the TST 1% and TET 1% groups. When Sertoli cells (SCs) were cultured with decellularized tissue, no signs of cytotoxicity were detected. A higher SC proliferation rate and greater stem cell factor secretion were observed than with SCs cultured without scaffold. ST 0.01% and TET 3% conditions offered the best compromise in terms of DNA elimination and extracellular matrix (ECM) preservation, while ensuring good attachment, proliferation and functionality of human SCs. This study demonstrates the potential of using decellularized pig ITT for human testicular tissue engineering purposes.

1p36 deletion results in a decrease in glycosaminoglycans which is associated with aggressiveness in neuroblastic tumors.

Despite our deep understanding of neuroblastic tumors, some patients still suffer treatment failure, so pre-treatment risk stratification still requires improvement and the search for new therapeutic targets must continue. Here we correlated prognostic clinical and biological features of neuroblastic tumors with the density of extracellular matrix glycosaminoglycans (the main components of the extracellular matrix 'ground substance'), in nearly 400 primary samples. We also studied the relationship between the density of extracellular matrix glycosaminoglycans and the expression of B3GALT6, an enzyme required for their synthesis. We associated a decrease in glycosaminoglycans with neuroblastomas that were histopathologically poorly-differentiated or undifferentiated, as well as with metastatic disease, and 1p36 deleted tumors. This decrease in glycosaminoglycans was also related to abnormal nuclear B3GALT6 expression in neuroblastic cells. These findings point towards the importance of the ground substance in the aggressiveness of neuroblastic tumors, which should therefore be considered when developing novel therapies for treating neuroblastomas.

Ageing affects chondroitin sulfates and their synthetic enzymes in the intervertebral disc

The depletion of chondroitin sulfates (CSs) within the intervertebral disc (IVD) during degenerative disc disease (DDD) results in a decrease in tissue hydration, a loss of fluid movement, cell apoptosis, a loss of nerve growth inhibition and ultimately, the loss of disc function. To date, little is known with regards to the structure and content of chondroitin sulfates (CSs) during IVD ageing. The behavior of glycosaminoglycans (GAGs), specifically CSs, as well as xylosyltransferase I (XT-I) and glucuronyltransferase I (GT-I), two key enzymes involved in CS synthesis as a primer of glycosaminoglycan (GAG) chain elongation and GAG synthesis in the nucleus pulposus (NP), respectively, were evaluated in a bovine ageing IVD model. Here, we showed significant changes in the composition of GAGs during the disc ageing process (6-month-old, 2-year-old and 8-year-old IVDs representing the immature to mature skeleton). The CS quantity and composition of annulus fibrosus (AF) and NP were determined. The expression of both XT-I and GT-I was detected using immunohistochemistry. A significant decrease in GAGs was observed during the ageing process. CSs are affected at both the structural and quantitative levels with important changes in sulfation observed upon maturity, which correlated with a decrease in the expression of both XT-I and GT-I. A progressive switch of the sulfation profile was noted in both NP and AF tissues from 6 months to 8 years. These changes give an appreciation of the potential impact of CSs on the disc biology and the development of therapeutic approaches for disc regeneration and repair.

Age-related histologic and biochemical changes in auricular and septal cartilage.

To characterize the histologic and biochemical properties of auricular and septal cartilage and analyze age-related changes in middle-aged to older adults. Cross-sectional study of auricular and septal cartilage from 33 fresh cadavers. Auricular and septal cartilage specimens were stained using Safranin O for glycosaminoglycans, Verhoeff's stain for elastin, and Masson's trichrome for collagen. Percentage of tissue stained, cell density and size were quantified. Relationships between donor characteristics and histologic properties were evaluated using mixed model analyses. The average donor age was 75 years (standard deviation = 11 years; range, 55-93 years). In auricular cartilage, each 1-year increase in age was associated with a 0.97% decrease in glycosaminoglycans (P < .001) and a 0.98% decrease in elastin (P < .001). In septal cartilage, glycosaminoglycans decreased 2.4% per year (P < .001). Age did not affect collagen content significantly in auricular (P = .417) or septal cartilage (P = .284). Cell density and cell size declined with age in auricular (both P < .001) and septal cartilage (P = .044, P = .032, respectively). Compared to septal cartilage in patients of all ages, auricular cartilage had more glycosaminoglycans, less collagen, higher cell density, and smaller cells. In auricular and septal cartilage, glycosaminoglycans, elastin, cell density, and cell size decrease significantly with age in patients over 55 years of age. Glycosaminoglycan content declines faster with age in septal cartilage than auricular cartilage. These age-related changes may affect biomechanical properties and tissue viability, and thereby have implications for graft choice in functional, aesthetic, and reconstructive nasal surgery. NA. Laryngoscope, 127:E399-E407, 2017.

Diet-induced hypercholesterolemia alters liver glycosaminoglycans and associated-lipoprotein receptors in rats.

Glycosaminoglycans (GAGs) play an important role in lipoprotein metabolism. In liver, it facilitates the uptake of remnants through receptor-independent endocytosis. However, changes in liver GAGs during diet-induced hypercholesterolemia with normal levels of fat feeding are unknown. Present paper highlights the effect of diet-induced hypercholesterolemia with normal levels (5%) of fat on liver GAGs and other associated lipoprotein receptors. Hypercholesterolemia was induced in rats by feeding diet supplemented with 0.5% cholesterol and 0.125% bile salts. Hypercholesterolemia showed significantly decreased GAGs of both heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (CS/DS) classes of molecules. Quantitative real-time polymerase chain reaction analysis of GAG biosynthetic enzymes and other genes revealed significant changes in expression profile. The decrease in GAGs was prevented by simvastatin treatment; a drug that inhibits endogenous cholesterol synthesis that was used as a positive control in our study. Furthermore, there was a comparatively decreased binding of GAGs from hypercholesterolemic rats to lipoprotein lipase. LRP1 which plays a major role in lipoprotein uptake was also significantly decreased, and it was attenuated in simvastatin-treated hypercholesterolemic rats. Furthermore, LDLR and ApoE were also decreased significantly in liver of hypercholesterolemic rats. Thus, diet-induced hypercholesterolemia results in dysregulation of cholesterol homeostasis apparently through changes in GAGs in conjunction with other associated players.

Non-invasive assessments of cartilage macromolecular composition in patients with acute cartilage injury

Purpose: Cartilage degeneration or defective repair after an injury is typically associated with changes in the macromolecular composition of cartilage. The major function of large aggregating glycosaminoglycans (GAGs) is hydration of the cellular matrix, ensuring cartilage resiliency through electrostatic repulsion. A decrease in GAGs is likely to expose cartilage to dehydration, predisposing the joint to further degeneration. In parallel, roughening of the articular surface may also occur as a result of cartilage fibrillation, a process involving loosening of the collagen network, denaturation of the collagen and loss of the collagen fiber organization. Such a complex matrix makes any progression or repair of the defect challenging to monitor. New MRI developments allowing for a combined evaluation of both macromolecules are considered as a non-invasive alternative to cartilage biopsies. Thus, recent reports suggest that GAG chemical exchange saturation transfer (gagCEST) and T2 mapping MRI sequences are well suited for the detection of change in GAGs and collagen content and structure, respectively. However, clinical proof is still required to monitor cartilage degeneration after injury using biochemical MRI methods. Therefore, the purpose of this study is to establish sensitivity and reproducibility of quantitative imaging biomarkers, such as gagCEST asymmetry values and T2 relaxation times values of the superficial and deep layers of cartilage. Combined MRI measurements were performed in patients with an acute cartilage injury likely to worsen during the observational period due to several risk factors. Methods: Imaging sessions were performed on 7 patients with cartilage defect(s) in the knee joint (ICRS grade 1 or 2) as well as high risk factors (concomitant meniscus damage and/or ligament instability) for further progression of defect. Cartilage gagCEST and T2 images (Fig.1) were obtained for all patients at 7T using following MRI sequences and acquisition parameters: a 3D RF-spoiled GRE sequence with a resolution of 0.9x0.9x2.2 mm3 for gagCEST imaging, and a 3D-TESS sequence with a resolution of 0.25x0.25x3 mm3 for T2 mapping. For image analysis, regions-of-interest were defined on 2D-TSE morphological images in the suspicious regions for defective and normal appearing cartilage, and transferred to gagCEST and T2 images for quantitative analysis. All MRI measurements were repeated 8 days later for reproducibility assessment Results: The gagCEST asymmetry values (N=5 , preliminary data) were 28% lower in damaged areas as compared to adjacent healthy regions in cartilage (3.06±2.18% vs 4.27±1.61%, p=0.17). Disruption in the zonal organization of collagen fibrils was also observed in the damaged areas. T2 relaxation values becoming more uniform between the superficial and deep cartilage layers in defect region ([superficial/deep]T2 ratio in the defective region: 0.81±0.22 vs in the healthy region: 0.68±0.10, p=0.08). Both, gagCEST and T2 measurements showed high correlation between baseline and day-8 values (r=0.78 and r=0.86 , both p<0.0005, respectively). Conclusions: Both MRI methods, as applied here in combination to patients with low-grade cartilage injury, appear to be reproducible and might be able to detect differences between a defective and healthy region of cartilage. These preliminary results support the use of a comprehensive MRI approach in observational and therapeutic trials to differentiate early formation of hyaline from fibrotic cartilage without resorting to serial biopsies.

An abnormality in glucocorticoid receptor expression differentiates steroid responders from nonresponders in keloid disease.

SummaryBackgroundGlucocorticoids (GCs) are first‐line treatment for keloid disease (KD) but are limited by high incidence of resistance, recurrence and undesirable side‐effects. Identifying patient responsiveness early could guide therapy.MethodsNineteen patients with KD were recruited at week 0 (before treatment) and received intralesional steroids. At weeks 0, 2 and 4, noninvasive imaging and biopsies were performed. Responsiveness was determined by clinical response and a significant reduction in vascular perfusion following steroid treatment, using full‐field laser perfusion imaging (FLPI). Responsiveness was also evaluated using (i) spectrophotometric intracutaneous analysis to quantify changes in collagen and melanin and (ii) histology to identify changes in epidermal thickness and glycosaminoglycan (GAG) expression. Biopsies were used to quantify changes in glucocorticoid receptor (GR) expression using quantitative reverse transcriptase polymerase chain reaction, immunoblotting and immunohistochemistry.ResultsAt week 2, the FLPI was used to separate patients into steroid responsive (n = 12) and nonresponsive groups (n = 7). All patients demonstrated a significant decrease in GAG at week 2 (P < 0·05). At week 4, responsive patients exhibited significant reduction in melanin, GAG, epidermal thickness (all P < 0·05) and a continued reduction in perfusion (P < 0·001) compared with nonresponders. Steroid‐responsive patients had increased GR expression at baseline and showed autoregulation of GR compared with nonresponders, who showed no change in GR transcription or protein.ConclusionsThis is the first demonstration that keloid response to steroids can be measured objectively using noninvasive imaging. FLPI is a potentially reliable tool to stratify KD responsiveness. Altered GR expression may be the mechanism gating therapeutic response.

Upstream Upregulation of Glycosaminoglycans Production by Targeting Synthesizing Enzymes Using A Nonviral Gene Delivery Method for Intervertebral Disk Regeneration

IntroductionProteoglycans, described as ground substance of the intervertebral disk tissue, are proteins rich in glycosaminoglycans (GAGs) providing stiffness and elasticity to the IVD. During IVD degeneration, a decrease of GAG is observed. This decrease results in loss of water content which alters its mechanical properties1. Glucuronyltransferase I (GT-I) and Xylosyltransferase I (XT-I) are two key enzymes involved in the synthesis of GAGs. Both enzymes have been shown to be downregulated in degenerated cartilage2,3. While GT-I has been reported to have a crucial role in GAG synthesis in the IVD, the implication of XT-I has not yet been demonstrated. Nonviral-based gene therapy is an attractive therapeutic approach because of its relative safety compared to viral therapies. The hypothesis of this study is that nonviral mediated delivery of GT-I and XT-I plasmids will increase the GAG production in IVD cells. The specific objectives of this study were1 to investigate the expression of GT-I and XT-I in relation to aging within the IVD tissue and2 to upregulate GT-I and XT-I expression using a polymeric transfecting agent for gene delivery of p GT-I and p XT-I.Materials and Methods(1)Expression of GAGs, GT-I and XT-I in relation to aging IVD tissues were harvested from 6-month-old, 1-, 2-, and 8-year-old bovine tails. One half of the IVD tissue was fixed by 4% paraformaldehyde. After sucrose treatment and sectioning, safranin O/Fast green staining and immunostaining of XT-I and GT-I were carried out. The other half was digested by 0.5 mg/mL of proteinase K for biochemical (DMMB assay) and HPLC analyses.(2)Polymeric transfection-mediated gene delivery of pGT-I and pXT-I Nucleus pulposus and annulus fibrosus cells (passage 1) and adipose derived stem cells (ADSCs) (passage 3), extracted from 6-month-old bovine caudal disks and rabbit adipose tissue, respectively, were seeded at a density of 50,000 cells/cm2. After 24 hours, cells were treated with XT-I and/or GT-I TOPO TA plasmids DNA complexed with commercial partial degraded polyamidoamine dendrimer (SuperFect, Qiagen) for 30 minutes before addition to cells (1 µg of plasmid DNA final concentration). p Luc was used as the nontargeting plasmid and control for transfection (luciferase activity) (empty vector (EV)). Medium was then changed daily until the completion of the study. Cellular metabolic activity (AlamarBlue) and proliferation (Picogreen), GAG quantification (DMMB), gene expression (XT-I, GT-I and aggrecan), and enzyme activity analyses were conducted on day 2 and 4 posttransfection.Results(1)Expression of GAGs, XT-I, and GT-I with aging A decrease of the intensity of GAG staining associated with a disorganization of the tissue was observed with aging. GAG downregulation was confirmed by a significant decrease of sulfated GAGs (6092 ± 393 µg GAG/µg DNA for 6-month-old IVD to 1033 ± 530 µg GAG/µg DNA for 8-year-old IVD) within the tissue (Fig. 1). Associated with this phenomenon, a decrease of XT-I and GT-I expression was noted by immunohistochemistry. Indeed, the high expression of XT-I and GT-I observed within 6-month-old bovine IVD decreased with aging to become nondetectable within 8-year-old bovine IVD.(2)Nonviral mediated pXT-I and pGT-I delivery After 2 and 4 days posttreatment, using polymer and pDNA complexes (polyplexes), over 80% cell viability was seen regardless of the plasmid and cell types. ADSCs, NP, and AF cells showed luciferase activity 2 and 4 days posttransfection. The expression of both plasmids GT-I and XT-I was observed in the three cell types.DownloadOpen in new tabDownload in PowerPointFigure 1 I-sGAG content within NP and AF tissues with aging. p < 0.05, Anova one-way test. II-GAG distribution in 6-month-old, 1-, 2-, and 8-year-old bovine IVD tissues (Safranin O (GAG, orange), Fast Green (collagen fibers, green) staining) (A-D) NP tissue (E-H) AF tissue.ConclusionA downregulation of GAGs was observed within bovine IVDs with aging. This decrease is associated with a downregulation of XT-I and GT-I expression known to be implicated in the GAG production. The use of a nonviral system shows promising results for gene transfer by their ability to transfect IVD cells.Acknowledgments Science Foundation Ireland, Research Frontiers Programme (07/RFP/ENMF482), Science Foundation Ireland, 07/SRC/B1163, AO Foundation (S-09-7P), 07/RFP/ENMF482 STTF 09.I confirm having declared any potential conflict of interest for all authors listed on this abstractNoDisclosure of InterestNone declaredHalloran D, et al. Tissue Engineering 2007;13:1927–1954Venkatesan N, et al. Proc Natl Acad Sci USA 2004;101(52):18087–18092Venkatesan N, et al. FASEB J 2009;23(3):813–822

Upstream Upregulation of Glycosaminoglycans Production by Targeting Synthesizing Enzymes Using a Nonviral Gene Delivery Method for Intervertebral Disk Regeneration

Introduction Proteoglycans, described as ground substance of the intervertebral disk tissue, are proteins rich in glycosaminoglycans (GAGs) providing stiffness and elasticity to the IVD. During IVD degeneration, a decrease of GAG is observed. This decrease results in loss of water content which alters its mechanical properties1. Glucuronyltransferase I (GT-I) and Xylosyltransferase I (XT-I) are two key enzymes involved in the synthesis of GAGs. Both enzymes have been shown to be downregulated in degenerated cartilage2,3. While GT-I has been reported to have a crucial role in GAG synthesis in the IVD, the implication of XT-I has not yet been demonstrated. Nonviral-based gene therapy is an attractive therapeutic approach because of its relative safety compared to viral therapies. The hypothesis of this study is that nonviral mediated delivery of GT-I and XT-I plasmids will increase the GAG production in IVD cells. The specific objectives of this study were1 to investigate the expression of GT-I and XT-I in relation to aging within the IVD tissue and2 to upregulate GT-I and XT-I expression using a polymeric transfecting agent for gene delivery of p GT-I and p XT-I. Materials and Methods (1)Expression of GAGs, GT-I and XT-I in relation to aging IVD tissues were harvested from 6-month-old, 1-, 2-, and 8-year-old bovine tails. One half of the IVD tissue was fixed by 4% paraformaldehyde. After sucrose treatment and sectioning, safranin O/Fast green staining and immunostaining of XT-I and GT-I were carried out. The other half was digested by 0.5 mg/mL of proteinase K for biochemical (DMMB assay) and HPLC analyses. (2)Polymeric transfection-mediated gene delivery of pGT-I and pXT-I Nucleus pulposus and annulus fibrosus cells (passage 1) and adipose derived stem cells (ADSCs) (passage 3), extracted from 6-month-old bovine caudal disks and rabbit adipose tissue, respectively, were seeded at a density of 50,000 cells/cm2. After 24 hours, cells were treated with XT-I and/or GT-I TOPO TA plasmids DNA complexed with commercial partial degraded polyamidoamine dendrimer (SuperFect, Qiagen) for 30 minutes before addition to cells (1 µg of plasmid DNA final concentration). p Luc was used as the nontargeting plasmid and control for transfection (luciferase activity) (empty vector (EV)). Medium was then changed daily until the completion of the study. Cellular metabolic activity (AlamarBlue) and proliferation (Picogreen), GAG quantification (DMMB), gene expression (XT-I, GT-I and aggrecan), and enzyme activity analyses were conducted on day 2 and 4 posttransfection. Results (1)Expression of GAGs, XT-I, and GT-I with aging A decrease of the intensity of GAG staining associated with a disorganization of the tissue was observed with aging. GAG downregulation was confirmed by a significant decrease of sulfated GAGs (6092 ± 393 µg GAG/µg DNA for 6-month-old IVD to 1033 ± 530 µg GAG/µg DNA for 8-year-old IVD) within the tissue ( Fig. 1 ). Associated with this phenomenon, a decrease of XT-I and GT-I expression was noted by immunohistochemistry. Indeed, the high expression of XT-I and GT-I observed within 6-month-old bovine IVD decreased with aging to become nondetectable within 8-year-old bovine IVD. (2)Nonviral mediated pXT-I and pGT-I delivery After 2 and 4 days posttreatment, using polymer and pDNA complexes (polyplexes), over 80% cell viability was seen regardless of the plasmid and cell types. ADSCs, NP, and AF cells showed luciferase activity 2 and 4 days posttransfection. The expression of both plasmids GT-I and XT-I was observed in the three cell types. [Figure: see text] Conclusion A downregulation of GAGs was observed within bovine IVDs with aging. This decrease is associated with a downregulation of XT-I and GT-I expression known to be implicated in the GAG production. The use of a nonviral system shows promising results for gene transfer by their ability to transfect IVD cells. Acknowledgments Science Foundation Ireland, Research Frontiers Programme (07/RFP/ENMF482), Science Foundation Ireland, 07/SRC/B1163, AO Foundation (S-09-7P), 07/RFP/ENMF482 STTF 09. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared Halloran D, et al. Tissue Engineering 2007;13:1927–1954 Venkatesan N, et al. Proc Natl Acad Sci USA 2004;101(52):18087–18092 Venkatesan N, et al. FASEB J 2009;23(3):813–822

Flexor Tendon Tissue Engineering: Acellularization of Human Flexor Tendons with Preservation of Biomechanical Properties and Biocompatibility

Acellular human tendons are a candidate scaffold for tissue engineering flexor tendons of the hand. This study compared acellularization methods and their compatibility with allogeneic human cells. Human flexor tendons were pretreated with 0.1% ethylenediaminetetracetic acid (EDTA) for 4 h followed by 24 h treatments of 1% Triton X-100, 1% tri(n-butyl)phosphate, or 0.1% or 1% sodium dodecyl sulfate (SDS) in 0.1% EDTA. Outcomes were assessed histologically by hematoxylin and eosin and SYTO green fluorescent nucleic acid stains and biochemically by a QIAGEN DNeasy kit, Sircol collagen assay, and 1,9 dimethylmethylene blue glycosaminoglycan assay. Mechanical data were collected using a Materials Testing System to pull to failure tendons acellularized with 0.1% SDS. Acellularized tendons were re-seeded in a suspension of human dermal fibroblasts. Attachment of viable cells to acellularized tendon was assessed biochemically by a cell viability assay and histologically by a live/dead stain. Data are reported as mean±standard deviation. Compared with the DNA content of fresh tendons (551±212 ng DNA/mg tendon), only SDS treatments significantly decreased DNA content (1% SDS [202.8±37.4 ng DNA/mg dry weight tendon]; 0.1% SDS [189±104 ng DNA/mg tendon]). These findings were confirmed by histology. There was no decrease in glycosaminoglycans or collagen following acellularization with SDS. There was no difference in the ultimate tensile stress (55.3±19.2 [fresh] vs. 51.5±6.9 [0.1% SDS] MPa). Re-seeded tendons demonstrated attachment of viable cells to the tendon surface using a viability assay and histology. Human flexor tendons were acellularized with 0.1% SDS in 0.1% EDTA for 24 h with preservation of mechanical properties. Preservation of collagen and glycoaminoglycans and re-seeding with human cells suggest that this scaffold is biocompatible. This will provide a promising scaffold for future human flexor tendon tissue engineering studies to further assess biocompatibility through cell proliferation and in vivo studies.

Time dependence of changes of two cartilage layers in anterior cruciate ligament insertion after resection on chondrocyte apoptosis and decrease in glycosaminoglycan

BackgroundThe purpose of this study is to clarify the differences in time-dependent histological changes (chondrocyte apoptosis and glycosaminoglycan (GAG) layer thickness decrease) between uncalcified fibrocartilage (UF) and calcified fibrocartilage (CF) layers at the anterior cruciate ligament (ACL) insertion after ACL resection of rabbits.MethodsForty male Japanese white rabbits underwent ACL substance resection in the right knee (resection group) and same operation without resection in the left knee (sham group). Animals were sacrificed 1, 2, 4 and 6 weeks after surgery.ResultsIn the UF layer, the apoptosis rate in the resection group was significantly higher than that in the sham group at 1 and 2 weeks. The GAG layer thicknesses of the UF layer in the resection group at 1, 2, 4 and 6 weeks were lower than those in the sham group. In the CF layer, the apoptosis rate in the resection group was significantly higher than that in the sham group at 2 and 4 weeks. The GAG layer thickness of the CF layer in the resection group was lower than that in the sham group only at 6 weeks.ConclusionThe increase in chondrocyte apoptosis rate preceded the decrease in GAG layer thickness in both layers. In the UF layer, the increase in chondrocyte apoptosis rate and the decrease in GAG layer thickness preceded those in the CF layer. Using a surviving ligament and minimizing a debridement of ACL remnant during ACL reconstruction may be important to maintain cartilage layers of ACL insertion. An injured ACL should be repaired before degenerative changes of the insertion occur.

Molecular MR imaging for the evaluation of the effect of dynamic stabilization on lumbar intervertebral discs

The dynamic stabilization of lumbar spine is a non-fusion stabilization system that unloads the disc without the complete loss of motion at the treated motion segment. Clinical outcomes are promising but still not definitive, and the long-term effect on instrumented and adjacent levels is still a matter of discussion. Several experiments have been devised in order to gain a better understanding of the effect of the device on the intervertebral disc. One of the hypotheses was that while instrumented levels are partially relieved from loading, adjacent levels suffer from the increased stress. But this has not been proved yet. The aim of this study was to investigate the long-term effect of dynamic stabilization in vivo, through the quantification of glycosaminoglycans (GAG) concentration within instrumented and adjacent levels by means of the delayed Gadolinium-Enhanced Magnetic Resonance Imaging of Cartilage (dGEMRIC) protocol. Ten patients with low back pain, unresponsive to conservative treatment and scheduled for Dynesys implantation at one to three lumbar spine levels, underwent the dGEMRIC protocol to quantify GAG concentration before and 6 months after surgery. Each patient was also evaluated with visual analog scale (VAS), Oswestry, Prolo, Modic and Pfirrmann scales, both at pre-surgery and at follow-up. Six months after implantation, VAS, Prolo and Oswestry scales had improved in all patients. Pfirrmann scale could not detect any change, while dGEMRIC data already showed a general improvement in the instrumented levels: GAG was increased in 61% of the instrumented levels, while 68% of the non-instrumented levels showed a decrease in GAG, mainly in the posterior disc portion. In particular, seriously GAG-depleted discs seemed to have the greatest benefit from the Dynesys implantation, whereas less degenerated discs underwent a GAG depletion. dGEMRIC was able to visualize changes in both instrumented and non-instrumented levels. Our results suggest that the dynamic stabilization of lumbar spine is able to stop and partially reverse the disc degeneration, especially in seriously degenerated discs, while incrementing the stress on the adjacent levels, where it induces a matrix suffering and an early degeneration.

Targeting of the CNS in MPS-IH Using a Nonviral Transferrin-α-l-iduronidase Fusion Gene Product

Mucopolysaccharidosis type I (Hurler syndrome) is caused by a deficiency of the enzyme α-l-iduronidase (IDUA), and is characterized by widespread lysosomal glycosaminoglycan (GAG) accumulation. Successful treatment of central nervous system (CNS) diseases is limited by the presence of the blood-brain barrier, which prevents penetration of the therapeutic enzyme. Given that the brain capillary endothelial cells that form this barrier express high levels of the transferrin receptor (TfR), we hypothesized that the coupling of IDUA to transferrin (Tf) would facilitate IDUA delivery to the CNS. A plasmid bearing a fusion gene consisting of Tf and IDUA was constructed which, when delivered in vivo, resulted in the production of high levels of an enzymatically active protein that was transported into the CNS by TfR-mediated endocytosis. Short-term treatment resulted in a decrease in GAGs in the cerebellum of mucopolysaccharidosis type I (MPS I) mice. This approach, therefore, represents a potential strategy for the delivery of therapeutic enzyme to the CNS.

Biochemical, Pathological, and Skeletal Improvement of Mucopolysaccharidosis VI After Gene Transfer to Liver but Not to Muscle

Mucopolysaccharidosis VI (MPS VI) is caused by deficient activity of arylsulfatase B (ARSB), resulting in intralysosomal storage of dermatan sulfate (DS) and multisystem disease without central nervous system involvement. After gene transfer, muscle or liver can theoretically be converted into factories for systemic ARSB secretion, leading to uptake by non-transduced cells. We have injected newborn MPS VI rats and cats with adeno-associated viral (AAV) vectors expressing ARSB under the control of liver-specific, muscle-specific, or universally active promoters. After systemic or intramuscular (IM) administration of AAV, therapeutic levels of circulating ARSB are achieved, resulting in skeletal improvements and significant decrease in glycosaminoglycan (GAG) storage, inflammation and apoptosis (despite a neutralizing immune response to ARSB in MPS VI rats). In addition, we have observed wide-spread dissemination of vector after IM AAV administration. This results in secretion of therapeutic levels of ARSB when the universally active cytomegalovirus (CMV) but not the muscle-specific muscle creatine kinase (MCK) promoter is used, suggesting that transduction of extramuscular sites rather than enzyme secretion from muscle occurs after muscle ARSB gene transfer. We conclude that AAV-mediated expression of ARSB from liver represents a feasible therapeutic strategy for MPS VI, potentially avoiding multiple infusions of costly recombinant enzyme associated with enzyme replacement therapy.