Proteoglycan Research Articles

Contrast enhanced imaging of human meniscus using cone beam CT

Meniscal injuries can lead to mechanical overloading of articular cartilage and eventually to knee osteoarthritis. The objective was to evaluate the potential of contrast enhanced computed tomography (CECT) to image contrast agent (CA) diffusion in human menisci with a clinical cone beam CT scanner. Isolated human menisci (n = 26) were imaged using magnetic resonance imaging (MRI) and CECT in situ. Diffusion of anionic CA into the meniscus was imaged for up to 30 h. The results of CECT were compared with water, collagen and proteoglycan (PG) contents, biomechanical properties, age and histological and MR images of the samples. Diffusion of CA required over 25 h to reach equilibrium. The CA partition (the CA concentration in the tissue divided by that in the bath) at the 40 min time point correlated significantly with that at the 30 h time point in both lateral (r = 0.706, P = 0.007) and medial (r = 0.669, P = 0.012) menisci. Furthermore, CA partition in meniscus after 30 h of diffusion agreed qualitatively with the distribution of PGs. The cross-sectional distribution of CA was consistent with that reported in a previous μCT study on bovine meniscus. The time required to reach diffusion equilibrium was found impractical for clinical applications. However, based on the present results, shorter delay between injection and imaging (e.g., 40 min) could be feasible in clinical diagnostics of meniscal pathologies.

Advances in the research of modulation of dermal collagen fibrin assembly by decorin

Formation of dermal collagen fiber is a complicated and sequential process with the progressive assembly of collagen. Collagen monomers form stepped and orderly protofibrils through longitudinal displacement. Subsequently, protofibrils or protofibrils and collagen are bonded by covalent bonds to form orderly lamellar structure of collagen fibers. Then collagen fibers are tightly wound into coarse collagen fiber bundles by covalent crosslinking. Decorin is a multifunctional small leucine-rich proteoglycan. It can prevent the aggregation of protofibrils by binding to the specific site of collagen with its core protein, and adjusting the spacing between the protofibrils with its glycosaminoglycan chain. Thus, by effecting the formation of collagen fibers with regulation of collagen assembly, decorin may help prevent scar formation and even promote regeneration.

Targeting PTEN with lentiviral shRNA promotes neurite outgrowth of cortical neurons and improves functional recovery in a rat spinal cord contusion model

Objective To observe the effects of PTEN silencing on axon regeneration and functional recovery after spinal cord injury (SCI). Methods Four groups were designed: DMEM、lenti-control、lenti-scramble、lenti-shRNA. Cortical neurons were seeded on or adjacent to chondroitin sulfate proteoglycans (CSPGs). Three days after lentivirus infection, the expression of PTEN were assessed by Western blot, and immunocytochemistry method were applied to examine the length, number and crossing behavior of neurites; Lentivirus was locally injected into the conical motor area of the rats with spinal cord injury. 1 week later, the expression of PTEN was assessed by Western blot. 6 weeks later, motor function of hind limbs was evaluated by Basso Beattie Bresnahan (BBB) score, while the expressions of green fluorescence in injured spinal cord were observed by fluorescence microscope, and immunohistochemical method was used to observe the expression of synaptophysin changes in spinal tissues. Results Less expression of PTEN was detected in lentivirus-shRNA, and neurites with PTEN silencing exhibited significant enhancements in elongation, initiation and crossing ability when they encountered CSPGs in vitro. Better hindlimb functional recovery, less expression of PTEN and more synaptophysin positive response area were detected in rats injected with lentivirus-shRNA compared with other groups. Moreover, green fluorescence was found in the caudal region of injured spinal cord of rats injected with lentivirus-shRNA. Conclusion Lentiviral-mediated PTEN-shRNA can significantly improve the axon regeneration and neurological functional recovery after spinal cord injury. Key words: Spinal cord injuries; RNA interference; Lentivirus; Axons; Regeneration

Bone geometry of the hip is associated with obesity and early structural damage – A 3.0T MRI study of community-based adults

Bone geometry of the hip is associated with obesity and early structural damage – A 3.0T MRI study of community-based adults

Amino acid sequence surrounding the chondroitin sulfate attachment site of thrombomodulin regulates chondroitin polymerization

Thrombomodulin (TM) is a cell-surface glycoprotein and a critical mediator of endothelial anticoagulant function. TM exists as both a chondroitin sulfate (CS) proteoglycan (PG) form and a non-PG form lacking a CS chain (α-TM); therefore, TM can be described as a part-time PG. Previously, we reported that α-TM bears an immature, truncated linkage tetrasaccharide structure (GlcAβ1-3Galβ1-3Galβ1-4Xyl). However, the biosynthetic mechanism to generate part-time PGs remains unclear. In this study, we used several mutants to demonstrate that the amino acid sequence surrounding the CS attachment site influences the efficiency of chondroitin polymerization. In particular, the presence of acidic residues surrounding the CS attachment site was indispensable for the elongation of CS. In addition, mutants defective in CS elongation did not exhibit anti-coagulant activity, as in the case with α-TM. Together, these data support a model for CS chain assembly in which specific core protein determinants are recognized by a key biosynthetic enzyme involved in chondroitin polymerization.

Proteoglycans as potential microenvironmental biomarkers for colon cancer.

Glycosylation changes occur widely in colon tumours, suggesting glycosylated molecules as potential biomarkers for colon cancer diagnostics. In this study, proteoglycans (PGs) expression levels and their transcriptional patterns are investigated in human colon tumours in vivo and carcinoma cells in vitro. According to RT-PCR analysis, normal and cancer colon tissues expressed a specific set of PGs (syndecan-1, perlecan, decorin, biglycan, versican, NG2/CSPG4, serglycin, lumican, CD44), while the expression of glypican-1, brevican and aggrecan was almost undetectable. Overall transcriptional activity of the PGs in normal and cancer tissues was similar, although expression patterns were different. Expression of decorin and perlecan was down-regulated 2-fold in colon tumours, while biglycan and versican expression was significantly up-regulated (6-fold and 3-fold, respectively). Expression of collagen1A1 was also increased 6-fold in colon tumours. However, conventional HCT-116 colon carcinoma and AG2 colon cancer-initiating cells did not express biglycan and decorin and were versican-positive and -negative, respectively, demonstrating an extracellular origin of the PGs in cancer tissue. Selective expression of heparan sulfate (HS) proteoglycans syndecan-1 and perlecan in the AG2 colon cancer-initiating cell line suggests these PGs as potential biomarkers for cancer stem cells. Overall transcriptional activity of the HS biosynthetic system was similar in normal and cancer tissues, although significant up-regulation of extracellular sulfatases SULF1/2 argues for a possible distortion of HS sulfation patterns in colon tumours. Taken together, the obtained results suggest versican, biglycan, collagen1A1 and SULF1/2 expression as potential microenvironmental biomarkers and/or targets for colon cancer diagnostics and treatment.

Effects of running exercise on cartilage in rats with an unstable knee joint

Objective To investigate the effect of running exercise on cartilage in rats with an unstable knee joint. Methods Twenty 8-week-old Sprague-Dawley rats had their left anterior cruciate ligament cut to model an unstable knee. They were randomly divided into a control group and an experimental group, 10 rats in each group. The control group was given no intervention, while the experimental group accepted running exercise training on na animal treadmill at a velocity of 15 m/min for an hour every day. After 3 and 6 weeks of training, 5 rats were sacrificed and cartilage from the medial condyle of the femur was sampled, decalcificated, embedded and sliced on the sagittal plane. After hematoxylin-eosin staining, toluidine blue staining and immunohistochemical staining, the cartilage thickness, Mankin' score, the content of matrix collagen and the proteoglycan content of the cartilage matrix were assessed, and the shape and structure of the unstable knee joints were observed under a transmission electron microscope. Results The cartilage thicknesses and Mankin's scores at 6 weeks were significantly different from those at 3 weeks in both groups. In the experimental group the average thickness of cartilage was 154±13 μm at 3 weeks and 131±15 μm at 6 weeks. The corresponding Mankin's scores were 9.93±1.36 and 11.23±1.57, respectively. Both were significantly different from the control group averages at the same time points. There was also a significant difference in the positive rate of toluidine blue and collagen typeⅡstaining between the experimental group and the control group at both time points, and in the experimental group between 3 and 6 weeks of training. After 3 weeks of training, fewer chondrocytes were observed under the transmission electron microscope in the experimental group, and fissures were seen on the surface of the cartilages. However, 3 weeks later, quite a few ruptures and a lot of necrotic cells could be seen. Conclusions Running exercise can damage the cartilage of unstable knee joints and speed up the development of osteoarthritis. Even moderate exercise could aggravate damage to unstable joints and the cartilage matrix, and accelerate chondrocyte degeneration. Key words: Anterior cruciate ligament; Rats; Knee joint; Joint cartilage; Exercise; Joint stability

참당귀 추출분말이 Monosodium Iodoacetate로 유발된 흰쥐의 골관절염에 대한 효과

참당귀 추출물의 골관절염에 대한 억제 또는 치료효과를 알고자 monosodium iodoacetate로 유발한 흰쥐의 골관절염 모델을 사용하여 30마리 6주령 SD흰쥐를 정상군(normal), 비처리군(untreated) 및 참당귀 처리군(treated)으로 각각 10마리씩 나누어 실험하였다. 실험기간 동안 실험 개시일부터 4일 간격으로 체중 변화를 측정하였다. 3주간 참당귀 추출물 처리 후 관절연골 내의 glycosaminoglycan (GAG)의 함량 및 proteoglycan (PG) 함량을 측정 비교하였다. 그 결과, 참당귀 추출물은 MIA로 증가한 GAG를 완화시켰으며, PG의 함량을 회복시켰다. 또한, 활액에서의 TNF-a함량은 미약하게 감소하였다. 한편, 경골 관절연골을 분리하여 Safranin-O 염색으로 관절연골 상태를 확인한 결과, MIA 처리로 유발된 관절연골 소실을 억제하였다. 이 참당귀 추출물을 HPLC로 분석한 결과, 주요 성분인 decursin 및 decursinol angelate의 총 함량은 <TEX>$10.5{\pm}0.2%$</TEX>이었다. To study the efficacy of extract powder of Angelica gigas in preventing and treating degeneration of the articular cartilage in rats with monosodium iodoacetate (MIA)-induced osteoarthritis, A total of 30 six-week-old Sprague-Dawley rats were randomly divided into normal control group, untreated group and Angelica gigas treated group, with 10 rats in each group. During the treatment period, body weight were measured in each four days interval from starting date. The rat were sacrificed at the end of 3rd week after daily administration of Angelica gigas and then rat tibia articular cartilage was removed. In articular cartilages, glycosaminoglycan (GAG) amount increased by MIA treatment were reduced while proteoglycan (PG) amount decreased by MIA treatment were fairly recovered by Angelica gigas treatment, respectively. The content of TNF-a was also slightly reduced sections of the cartilage were stained with safranin-0 were also partially recovered by Angelica gigas treatment. By HPLC analysis, the content of main compounds decursin and decursinol angelate was analyzed as <TEX>$10.5{\pm}0.2%$</TEX> of total extracts.

Effect of lovastatin on shedding of heparan sulfate proteoglycan and syndecan-1 in lung tissues of rats with sepsis-induced acute lung injury

Objective To evaluate the effect of lovastatin on shedding of heparan sulfate proteoglycan (HSPG) and syndecan-1(SDC-1) in the lung tissues of rats with sepsis-induced acute lung injury. Methods One-hundred and twenty male Wistar rats aged 8-12 weeks, weighing 325-425 g, were randomly divided into 3 groups (n =40 each) using a random number table: sham operation group (group S), cecal ligation and puncture (CLP) group and lovastatin group (group L). Lovastatin 4 mg/kg was injected once a day for 5 consecutive days in S and L groups, while the equal volume of 0.5% CMC (the solvent) was given in CLP group. Sepsis was produced by CLP on 5th day of administration in CLP and L groups. The left lung was lavaged at 24 h after operation. The broncho-alveolar lavage fluid (BALF) was collected for determination of protein concentrations, white blood cell (WBC) count and percentage of neutrophils. Blood samples were collected for determination of the concentrations of HSPG and SDC-1 in serum (by ELISA). Evans blue was injected at 24 h after operation in the remaining 20 rats of each group. The lungs were removed for examination of the pathological changes and for measurement of HSPG and SDC-1 mRNA and protein expression (using Western blot and PCR), and Evans blue content (reflecting pulmonary capillary permeability) in the lung tissue. Results Compared with group S, the protein concentrations, WBC count and percentage of neutrophils in BALF, Evans blue content in lung tissues and the concentrations of HSPG and SDC-1 in serum were significantly increased, and HSPG and SDC-1 mRNA and protein expression was down-regulated in CLP and L groups. Compared with group CLP, the protein concentrations, WBC count and percentage of neutrophils in BALF, Evans blue content in lung tissues and the concentrations of HSPG and SDC-1 in serum were significantly decreased, and HSPG and SDC-1 mRNA and protein expression was up-regulated in group L. The pathological changes of lungs were significantly attenuated in group L as compared with group CLP. Conclusion The mechanism by which lovastatin attenuates acute lung injury induced by sepsis may be related to reduced shedding of HSPG and SDC-1 in lung tissues and improved function of pulmonary vascular endothelium in rats. Key words: Lovastatin; Sepsis; Respiratory distress syndrome, adult; Heparan sulfateproteoglycans; Syndecan-1

A4.2 IL4–10 synerkine for the treatment of osteoarthritis

Background and objectives Osteoarthritis (OA) is a degenerative joint disease, clinically characterised by joint pain and disability. Underlying structural changes are degeneration of articular cartilage, intra-articular inflammation with synovitis and changes in peri-articular and subchondral bone. The ideal drug to treat OA should have analgesic, anti-inflammatory and cartilage protective effects. To date, no such drug has been described. In human and animal in and ex-vivo models, the immunoregulatory cytokines interleukin 4 (IL-4) and interleukin 10 (IL-10), especially in combination therapy, can prevent joint degeneration effectively. To use the combined activities of both IL-4 and IL-10, we developed IL4–10 synerkine, preserving the functional activity of each cytokine. In several well-established animal models, we have shown the potential of IL4–10 synerkine to inhibit inflammatory pain in animal models. In this study, we evaluated the potential of the synerkine to reduce inflammation and to provide cartilage protection in an ex vivo model for human OA. Materials and methods Osteoarthritic cartilage (n = 8) and synovium (n = 6) was obtained at joint replacement surgery and was cultured for 72 h in conditioned medium, in the presence or absence of equimolar concentrations of IL-4 (10 ng/ml), IL-10 (10 ng/ml), the combination of IL-4 and IL-10 (10 ng/ml + 10 ng/ml) and IL4-10 synerkine (20 ng/ml). After culture, cartilage was selected to study changes in proteoglycan (PG) synthesis and release. Supernatants of both cartilage and synovium cultures were collected for measurement of a broad spectrum of inflammatory mediators. Results In both synovium and cartilage cultures, elevated levels of pro-inflammatory mediators like IL-6 (resp. 7.5 µg/mg and 531 µg/mg) and IL-8 (resp. 2.6 µg/mg and 142 µg/mg) were measured. These levels were strongly inhibited by the IL4-10 synerkine (about 80%; all p Conclusions IL4-10 synerkine directly affects proteoglycan turnover and cytokine production of OA cartilage, combined with reduced secretion of inflammatory cytokines and proteases by OA synovial tissue. The immunoregulatory and cartilage protective effects in combination with it’s analgesic effects, suggest that the synerkine is an effective disease modifying drug in the treatment of osteoarthritis.

Heavy Chain Transfer by Tumor Necrosis Factor-stimulated Gene 6 to the Bikunin Proteoglycan

We present data that hyaluronan (HA) polysaccharides, about 14-86 monosaccharides in length, are capable of accepting only a single heavy chain (HC) from inter-α-inhibitor via transfer by tumor necrosis factor-stimulated gene 6 (TSG-6) and that this transfer is irreversible. We propose that either the sulfate groups (or the sulfation pattern) at the reducing end of the chondroitin sulfate (CS) chain of bikunin, or the core protein itself, enables the bikunin proteoglycan (PG) to accept more than a single HC and permits TSG-6 to transfer these HCs from its relatively small CS chain to HA. To test these hypotheses, we investigated HC transfer to the intact CS chain of the bikunin PG, and to the free chain of bikunin. We observed that both the free CS chain and the intact bikunin PG were only able to accept a single HC from inter-α-inhibitor via transfer by TSG-6 and that HCs could be swapped from the bikunin PG and its free CS chain to HA. Furthermore, a significant portion of the bikunin PG was unable to accept a single heavy chain. We discuss explanations for these observations, including the intracellular assembly of inter-α-inhibitor. In summary, these data demonstrate that the sulfation of the CS chain of bikunin and/or its core protein promote HC transfer by TSG-6 to its relatively short CS chain, although they are insufficient to enable the CS chain of bikunin to accept more than one HC in the absence of other cofactors.

Bimolecular based heparin and self-assembling hydrogel for tissue engineering applications

One major goal of tissue engineering is to develop new biomaterials that are similar structurally and functionally to the extracellular matrix (ECM) to mimic natural cell environments. Recently, different types of biomaterials have been developed for tissue engineering applications. Among them, self-assembling peptides are attractive candidates to create artificial cellular niches, because their nanoscale network and biomechanical properties are similar to those of the natural ECM. Here, we describe the development of a new biomaterial for tissue engineering composed by a simple combination of the self-assembling peptide RAD16-I and heparin sodium salt. As a consequence of the presence of heparin moieties the material acquired enhances the capacity of specific binding and release of growth factors (GFs) with heparin binding affinity such as VEGF165. Promising results were obtained in the vascular tissue engineering area, where the new composite material supported the development of tubular-like structures within a three dimensional (3D) culture model. Moreover, the new scaffold enhances the cell survival and chondrogenic commitment of adipose-derived stem cells (ADSC). Interestingly, the expression of specific markers of mature cartilage tissue including collagen type II was confirmed by western blot and real-time PCR. Furthermore, positive staining for proteoglycans (PGs) indicated the synthesis of cartilage tissue ECM components. Finally, the constructs did not mineralize and exhibited mechanical properties of a tissue undergoing chondrogenesis. Altogether, these results suggest that the new composite is a promising “easy to prepare” material for different reparative and regenerative applications.

The structural and compositional transition of the meniscal roots into the fibrocartilage of the menisci.

The meniscal roots, or insertional ligaments, firmly attach the menisci to tibial plateau. These strong attachments anchor the menisci and allow for the generation of hoop stress in the tissue. The meniscal roots have a ligament-like structure that transitions into the fibrocartilagenous structure of the meniscal body. The purpose of this study was to carry out a complete analysis of the structure and tissue organization from the body of the meniscus through the transition region and into the insertional roots. Serial sections were obtained from the meniscal roots into the meniscal body in fixed juvenile bovine menisci. Sections were stained for collagen and proteoglycans (PG) using fast green and safranin-o staining protocols. Unstained sections were imaged used a backlit stereo microscope. Optical projection tomography (OPT) was employed to evaluate the three-dimensional collagen architecture of the root-meniscus transition in lapine menisci. Tie-fibres were observed in the sections of the ligaments furthest from the bovine meniscal body. Blood vessels were observed to be surrounded by these tie-fibres and a PG-rich region within the ligaments. Near the tibial insertion, the roots contained large ligament-like collagen fascicles. In sections approaching the meniscus, there was an increase in tie-fibre size and density. Small tie-fibres extended into the ligament from the epiligamentous structure in the outermost sections of the meniscal roots, while large tie-fibre bundles were apparent at the meniscus transition. The staining pattern indicates that the root may continue into the outer portion of the meniscus where it then blends with the more fibrocartilage-like inner portions of the tissue. In unstained sections it was observed that the femoral side of the epiligamentous structure surrounding the root becomes more fibrous and thickens in the inferior inner portion of the posterior medial root. This thickening changes the shape of the root to more closely resemble the meniscus wedge shape. These observations support the concept of root continuity with the outer portion of the meniscus, thereby connecting with the hoop-like structure of the peripheral meniscus. OPT identified continuous collagen organization from the root into the meniscal body in longitudinal sections. In the radial direction, the morphology of the root continues into the meniscal body consistent with the serially sectioned bovine menisci. Blood vessels were prevalent on the periphery of the root. These blood vessels then arborized to cover the anterior femoral surface of the meniscus. This is the first study of the structural transition between the insertional ligaments (roots) and the fibrocartilagenous body of the menisci. These new structural details are important to understanding the meniscal load-bearing mechanism in the knee.

Ultrastructure Organization of Collagen Fibrils and Proteoglycans of Stingray and Shark Corneal Stroma.

We report here the ultrastructural organization of collagen fibrils (CF) and proteoglycans (PGs) of the corneal stroma of both the stingray and the shark. Three corneas from three stingrays and three corneas from three sharks were processed for electron microscopy. Tissues were embedded in TAAB 031 resin. The corneal stroma of both the stingray and shark consisted of parallel running lamellae of CFs which were decorated with PGs. In the stingray, the mean area of PGs in the posterior stroma was significantly larger than the PGs of the anterior and middle stroma, whereas, in the shark, the mean area of PGs was similar throughout the stroma. The mean area of PGs of the stingray was significantly larger compared to the PGs, mean area of the shark corneal stroma. The CF diameter of the stingray was significantly smaller compared to the CF diameter in the shark. The ultrastructural features of the corneal stroma of both the stingray and the shark were similar to each other except for the CFs and PGs. The PGs in the stingray and shark might be composed of chondroitin sulfate (CS)/dermatan sulfate (DS) PGs and these PGs with sutures might contribute to the nonswelling properties of the cornea of the stingray and shark.

289-POS

Objectives Pre-eclampsia (PE) is a serious pregnancy complication which affects 3–5% of the pregnant population. To date there is no ‘cure’ for PE. However, the placenta seems to play an important role in the pathogenesis of this disease. Human placenta is an abundant source of proteoglycans to which glycosaminoglycan (GAG) side chains are attached. Heparan sulphate proteoglycans (HSPGs) have been implicated in many biological processes including, anticoagulation, angiogenesis and inflammation. However, their role in human placentae is largely unknown. The aim of this study was to determine the mRNA expression and protein abundance of HSPGs as well as the structure and relative abundance of GAGs in PE-affected placentae compared to gestation-matched controls. Methods The mRNA expression of HSPGs was determined using real-time PCR. Proteoglycans (PGs) were isolated from placental tissue by anion-exchange chromatography. PG enriched samples were investigated using ELISA with antibodies against the protein core of a range of PGs, as well as GAG chains, and Heparan Sulphate (HS) linkage regions following 0.01 U/mL heparinase III digestion. Results The mRNA expression of HSPGS, syndecan 1 & 2, glypican 1 & 3 and perlecan was significantly reduced in PE-affected placentae compared to controls ( p n = 40 each, Mann–Whitney U test). Syndecans1-4, glypicans1 & 3, biglycan, decorin and perlecan were identified in placental tissues by ELISA following anion exchange chromatography. HS abundance was further investigated using ELISA following digestion with heparinase III to reveal the HS linkage region common to all HS and heparin chains. In PE-affected placentae there was significantly less HS linkage regions compared to controls (1.53 ± 0.0.19 vs 1.03 ± 0.11, n = 20 each, p = 0.0391, Mann–Whitney U test). This correlated with the reduction in HSPG mRNA expression. Conclusions It is plausible that the reduction in PG expression and HS abundance may contribute to the pathogenesis of PE by altering thrombin management or angiogenic and inflammatory processes within the placenta. Disclosures T. Gunatillake: None. A. Chui: None. M. Lord: None. J. Whitelock: None. P. Murthi: None. V. Ignatovic: None. P. Monagle: None. S. Brennecke: None. J. Said: None.

Immunolocalization of CCL2-expressing cells in EAE and EAE-MSC cerebral cortex

The chemokine CCL2 has been considered as a mediator of inflammation in different diseases of the central nervous system, including experimental autoimmune encephalomyelitis (EAE), where the chemokine mediates extravasation of mononuclear leukocytes and loss of microvessel barrier function [1]. Previous studies have demonstrated that cellular sources of CCL2 during both EAE and multiple sclerosis (MS) are astrocytes and microvessel endothelial cells (ECs). Initially, we have demonstrated that in a MOG-induced model of EAE in C57BL/6 mice, 6 hrs after the intravenous treatment with bone marrow derived mesenchymal stem cells (MSCs) [2], the junctional staining patter of blood-brain barrier (BBB) microvessels and their functional effectiveness to permeability tracers seem to be restored. We have subsequently analysed, in the same experimental models, EAE and EAE-MSC mice, expression and immunolocalization of chemokine CCL2 by double immmunolabelling with cell-specific markers: endothelial PECAM-1 (CD31), OPCs (oligodendrocyte precursor cells) proteoglycan NG2, astrocytic GFAP, and Iba1 for microglia cells. Surprisingly, in the adopted model of cerebral cortex EAE, astrocytes and ECs do not show any detectable CCL2 expression, instead a strong staining is observed on activated parenchymal and perivascular microglia. Astrogliosis, microglia activation, and CCL2 overexpression appearing strongly reduced in EAE mice after MSC treatment. These observations identify microglia cells as the major source of CCL2 in EAE mice, whose barrier is damaged, and suggest the downregulation of the chemokine in perivascular microglia as a possible mechanism involved in BBB protection after MSC administration.

Human Cartilage Tissue Engineering Using Type I Collagen/Heparan Sulfate Scaffolds

Human Cartilage Tissue Engineering Using Type I Collagen/Heparan Sulfate Scaffolds Introduction: Cartilage engineering may provide a promising alternative solution to current methods of cartilage repair. The aim of this study was to evaluate the suitability of type I collagen (Col I) scaffolds with and without heparan sulfate (HS) to support attachment, proliferation and chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Materials and Methods: hBMSCs were cultured in Col I, Col I+1%HS, Col I+2%HS and Col I+3%HS scaffolds in both chondrogenic and non-differentiation media for 15 and 30 days. The resulting neo-tissues were analyzed using histochemistry, immunohistochemistry, electron microscopy (EM) and molecular biology. Collagen released into the media by the constructs was also measured by dye-binding assays. Results: Our results showed successful growth and proliferation of hBMSCs on all scaffolds analyzed. Better results were obtained in chondrogenic cultures after 30 days, in which we observed oval/ rounded cells along the scaffolds, and extracellular matrix (ECM) by EM; this ECM was strongly positive for proteoglycan (PG) safranin O staining and staining for type II (Col II). Greater total collagen release was found in supernatants of chondrogenic cultures than in controls. The best results for all analyses were found in scaffolds including HS. Conclusions: We conclude that the Col I and HS scaffolds used in this study are suitable supports for hBMSCs to differentiate toward chondrocyte-like cells in chondrogenic medium. We observe that the addition of HS to Col I scaffolds improves the chondrogenic phenotype of the cells, with Col I+3%HS being the best scaffold.

Spatial mapping of proteoglycan content in articular cartilage using near-infrared (NIR) spectroscopy.

Diagnosis of articular cartilage pathology in the early disease stages using current clinical diagnostic imaging modalities is challenging, particularly because there is often no visible change in the tissue surface and matrix content, such as proteoglycans (PG). In this study, we propose the use of near infrared (NIR) spectroscopy to spatially map PG content in articular cartilage. The relationship between NIR spectra and reference data (PG content) obtained from histology of normal and artificially induced PG-depleted cartilage samples was investigated using principal component (PC) and partial least squares (PLS) regression analyses. Significant correlation was obtained between both data (R(2) = 91.40%, p<0.0001). The resulting correlation was used to predict PG content from spectra acquired from whole joint sample, this was then employed to spatially map this component of cartilage across the intact sample. We conclude that NIR spectroscopy is a feasible tool for evaluating cartilage contents and mapping their distribution across mammalian joint.

Proteoglycan from salmon nasal cartridge promotes in vitro wound healing of fibroblast monolayers via the CD44 receptor

Proteoglycans (PGs) are involved in various cellular functions including cell growth, adhesion, and differentiation; however, their physiological roles are not fully understood. In this study, we examined the effect of PG purified from salmon nasal cartilage (SNC-PG) on wound closure using tissue-cultured cell monolayers, an in vitro wound-healing assay. The results indicated that SNC-PG significantly promoted wound closure in NIH/3T3 cell monolayers by stimulating both cell proliferation and cell migration. SNC-PG was effective in concentrations from 0.1 to 10μg/ml, but showed much less effect at higher concentrations (100–1000μg/ml). The effect of SNC-PG was abolished by chondroitinase ABC, indicating that chondroitin sulfates (CSs), a major component of glycosaminoglycans (GAGs) in SNC-PG, are crucial for the SNC-PG effect. Furthermore, chondroitin 6-sulfate (C-6-S), a major CS of SNC-PG GAGs, could partially reproduce the SNC-PG effect and partially inhibit the binding of SNC-PG to cells, suggesting that SNC-PG exerts its effect through an interaction between the GAGs in SNC-PG and the cell surface. Neutralization by anti-CD44 antibodies or CD44 knockdown abolished SNC-PG binding to the cells and the SNC-PG effect on wound closure. These results suggest that interactions between CS-rich GAG-chains of SNC-PG and CD44 on the cell surface are responsible for the SNC-PG effect on wound closure.

Rat Articular Cartilages Change Their Tissue and Protein Compositions During Perinatal Period.

Articular cartilage (AC) covers the surface of bones in joints and functions as a cushion against mechanical loading. The tissue contains abundant extracellular matrix (ECM), which mainly consists of proteoglycans (PG) and collagen (COL) fibres. The property of AC is gradually changing by ageing with gravity loading. To know the property change of AC by initial gravity loading during short period after birth, we performed histological assays and proteomics assay on the AC of the femoral condyle in knee joints of perinatal rats. The water content (%) was significantly decreased in neonate AC compared with fetal AC. During the perinatal stages (E19 and P0), the localizations of glycosaminoglycan (GAG) and type I and II COLs were homogeneous. The density of chondrocytes was significantly decreased in the deeper layers comparing with the surface layer in neonate AC. In addition, we found a drastic change in the protein expression pattern on proteomic analysis. The expressions of ECM components were relatively increased in neonate AC compared with fetal AC.