Differential effects of exogenous and endogenous hyaluronan on contraction and strength of collagen gels

The addition of exogenous hyaluronan to biomaterial scaffolds has been an important area of investigation for many decades. The ability to manipulate endogenous production of hyaluronan via the hyaluronan syntheses has offered another mechanism to study the effect of hyaluronan. While the literature suggests that exogenously added hyaluronan and endogenously produced hyaluronan will have varying impacts on extracellular matrix organization and function, no studies have directly shown this phenomenon. In this investigation, we demonstrate that the addition of exogenous high molecular weight (～1 MDa) hyaluronan and hyaluronan oligosaccharides have a distinct impact on both contraction and strength of smooth muscle cell-seeded collagen gels when compared to the effects of hyaluronan that is endogenously produced by the hyaluronan synthases. More specifically, the addition of exogenous high molecular weight hyaluronan resulted in more compact collagen gels with a higher ultimate tensile strength, whereas the endogenous overproduction of hyaluronan resulted in the opposite effect. We suggest that the addition of exogenous HA to collagen gels represents a model for the therapeutic administration of HA, whereas the addition of excess HA to a tissue via the endogenous overexpression of has represents a model for the pathological accumulation of HA.     

Tissue integrity signals communicated by high-molecular weight hyaluronan and the resolution of inflammation

The extracellular matrix polysaccharide hyaluronan (HA) exerts size-dependent effects on leukocyte behavior. Low-molecular weight HA is abundant at sites of active tissue catabolism and promotes inflammation via effects on Toll-like receptor signaling. Conversely, high-molecular weight HA is prevalent in uninjured tissues and is anti-inflammatory. We propose that the ability of high-molecular weight but not low-molecular weight HA to cross-link CD44 functions as a novel form of pattern recognition that recognizes intact tissues and communicates “tissue integrity signals” that promote resolution of local immune responses.     

Effect of unilateral labyrinthectomy on the molecular composition of perineuronal nets in the lateral vestibular nucleus of the rat

Disturbances in vestibular functions caused by unilateral labyrinthectomy (UL) are spontaneously restored during the process of vestibular compensation due to the plasticity of CNS. The underlying molecular background of vestibular compensation is not yet fully understood. Recent studies have shown that the extracellular matrix (ECM) molecules have either permissive or non-permissive effect on the neural plasticity. In our previous study we have demonstrated changes in the expression of hyaluronan (HA) in the vestibular nuclei (VN) of the frog following peripheral vestibular lesion. The present work was undertaken to examine the expression of the HA and chondroitin sulfate proteoglycans (CSPGs) in the lateral vestibular nucleus (LVN) of the rat following UL by using histochemical methods. On the first postoperative day, the condensation of the ECM around the neurons, the perineuronal net (PNN) was not distinguished from the surrounding neuropil on the side of UL indicating the desorganization of its molecular structure. At survival day 3, the PNN was recognizable with the HA probe, whereas its staining for the CSPGs was restored by the time of the seventh postoperative day. In the neuropil, the intensity of the HA increased on the operated side, while the CSPGs reaction almost completely disappeared. The present study have demonstrated for the first time that the UL is accompanied by the modification of the HA, and CSPG staining pattern in the PNN of the LVN in the rat. As the reorganization of the PNN corresponds to the restoration of spontaneous activity of vestibular neurons, our study implies the role of HA and CSPGs in the vestibular compensation.     

Hyaluronan fragments promote inflammation by down-regulating the anti-inflammatory A2a receptor

The tissue microenvironment plays a critical role in regulating inflammation. Chronic inflammation leads to an influx of inflammatory cells and mediators, extracellular matrix turnover, and increased extracellular adenosine. Low molecular weight (LMW) fragments of hyaluronan (HA), a matrix component, play a critical role in lung inflammation and fibrosis by inducing inflammatory gene expression at the injury site. Adenosine, a crucial negative regulator of inflammation, protects tissues from immune destruction via the adenosine A2a receptor (A2aR). Therefore, these two extracellular products of inflammation play opposing roles in regulating immune responses. As such, we wanted to determine the effect of LMW HA on A2aR function. In this article, we demonstrate that LMW HA causes a rapid, significant, and sustained down-regulation of the A2aR. CD44 was found to be necessary for LMW HA to down-modulate the A2aR as was protein kinase C signaling. We also demonstrate that LMW HA induces A2aR down-regulation during inflammation in vivo, and that this down-regulation can be blocked by treatment with an HA-blocking peptide. Because adenosine plays a critical role in limiting inflammation, our data provide a novel mechanism whereby LMW HA itself may further augment inflammation. By defining the pro- and anti-inflammatory properties of extracellular matrix components, we will be better able to identify specific pharmacologic targets as potential therapies.     

Differences in the expression of glycosaminoglycans in human fibroblasts derived from gingival overgrowths is related to TGF-beta up-regulation

Glycosaminoglycans (GAGs) play important roles in cell behavior and have the ability to bind and modulate cytokines. Using primary cultured fibroblasts from hereditary gingival fibromatosis (HGF), normal gingiva (NG), and NG treated with cyclosporin-A (NGc) we show changes in the expression and structural characteristics of GAGs as well as in the expression of enzymes involved in their biosynthesis and degradation. In addition, we show the over-expression of TGF-β1 and TGF-β type II receptor in HGF and NGc. There is an increase in the GAGs retained in the cellular fraction, and the fine structure of galactosaminoglycans show a decrease in α-l-iduronic acid content in HGF and NGc. Elevated extracellular levels of low molecular weight hyaluronan (HA) are found in HGF due to increase in the expression of HA synthase 3 and hyaluronidases 1 and 2. The results bring new insights to the accumulation of extracellular matrix related to TGF-β over-expression.     

Low molecular weight hyaluronan from stretched lung enhances interleukin-8 expression

Mechanical ventilation has been shown to cause ventilator-induced lung injury (VILI), probably by overdistending or stretching the lung. Hyaluronan (HA), a component of the extracellular matrix, in low molecular weight (LMW) forms has been shown to induce cytokine production. LMW HA is produced by hyaluronan synthase 3 (HAS 3). We found that HAS 3 mRNA expression was upregulated and that LMW HA accumulated in an animal model of VILI. We hypothesized that stretch-induced LMW HA production that causes cytokine release in VILI was dependent on HAS 3 mRNA expression. We explored this hypothesis with in vitro lung cell stretch. Cell stretch induced HAS 3 mRNA expression and LMW HA in fibroblasts. Nonspecific inhibitors of HAS 3 (cyclohexamide and dexamethasone), a nonspecific inhibitor of protein tyrosine kinases (genistein), and a janus kinase 2 inhibitor (AG490) blocked stretch-induced HAS 3 expression and synthesis of LMW HA. Stretch-induced LMW HA from fibroblasts caused a significant dose-dependent increase in interleukin-8 production both in static and stretched epithelial cells. These results indicated that de novo synthesis of LMW HA was induced in lung fibroblasts by stretch via tyrosine kinase signaling pathways, and may play a role in augmenting induction of proinflammatory cytokines in VILI.     

Potential use of collagen-chitosan-hyaluronan tri-copolymer scaffold for cartilage tissue engineering

A three-dimensional biodegradable porous scaffold plays a vital role in a tissue engineering approach. Collagen, chitosan and hyaluronan (HA) are natural extracellular matrix (ECM) or similarity, and may provide appropriate environment for the generation of cartilage-like tissue. In this study, we prepared a collagen/chitosan/HA tri-copolymer porous scaffold by freezing and lyophilization to evaluate physico-chemical properties of the tri-copolymer scaffold and its capacity to sustain chondrocytes proliferation and differentiation in vitro. The results show that the mechanical strength, the resistance to enzymatic degradation, and the waterblinding capacity were improved when chitosan and hyaluronan were incorporated into a collagen scaffold. After 21 days of culture, the porous scaffold had been surfaced with cartilaginous tissue. DNA and glycosaminoglycan (GAG) contents were significantly higher during culture periods in collagen/ chitosan/hyaluronan matrix compared to collagen alone matrix, and most seeded cells preserved the chondrocytic phenotype during culture within the scaffold. The collagen/chitosan/hyaluronan tri-copolymer scaffold has potential applications in a cartilage tissue engineering scaffold field.     

Human fascia lata ECM scaffold augmented with immobilized hyaluronan: inflammatory response and remodeling in the canine body wall and shoulder implantation sites

We postulate that immobilization of tyramine-substituted hyaluronan (THA) into an extracellular matrix (ECM) scaffold may be a strategy to promote an anti-inflammatory response to the ECM. Further, we posit that the implantation site could influence the inflammatory response and remodeling of an ECM scaffold. Eight beagles underwent implantation of fascia ECM grafts, treated with either immobilized low molecular weight (57 kDa) THA or water only, in both the shoulder injury and body wall sites. Dogs were euthanized at 12 weeks and fascia grafts harvested en bloc for histology. Grafts implanted at the body wall had significantly higher inflammatory cell infiltrate and vascularity, and significantly lower retardance (collagen density), than grafts at the shoulder, suggestive of a more intense, persistent, and perhaps degradative inflammatory and remodeling response at the body wall than shoulder injury site in the canine model. However, the presence of immobilized low MW THA had no effect on the inflammation response or remodeling of fascia ECM compared to water-treated controls. Importantly, these results suggest that the inflammatory response and remodeling of biomaterial implants depends on the location of implantation and therefore our animal models need to be carefully chosen. Further, the potential anti-inflammatory advantages of hyaluronan (HA) in wound healing do not appear to be realized when presenting it to the host as non-degradable hydrogel even if its capacity for binding HA binding protein is maintained. Further study treating ECM with uncross-linked (free) HA or immobilized low MW THA as a means to deliver free HA or other biomolecules to a surgical repair site is warranted.     

Structural and functional insights into sclerostin-glycosaminoglycan interactions in bone

In order to improve bone defect regeneration, the development of new adaptive biomaterials and their functional and biological validation is warranted. Glycosaminoglycans (GAGs) are important extracellular matrix (ECM) components in bone and may display osteogenic properties that are potentially useful for biomaterial coatings. Using hyaluronan (HA), chondroitin sulfate (CS) and chemically modified highly sulfated HA and CS derivatives (sHA3 and sCS3; degree of sulfation ∼3), we evaluated how GAG sulfation modulates Wnt signaling, a major regulator of osteoblast, osteoclast and osteocyte biology. GAGs were tested for their capability to bind to sclerostin, an inhibitor of Wnt signaling, using surface plasmon resonance and molecular modeling to characterize their interactions. GAGs bound sclerostin in a concentration- and sulfate-dependent manner at a common binding region. These findings were confirmed in an LRP5/sclerostin interaction study and an in vitro model of Wnt activation. Here, pre-incubation of sclerostin with different GAGs led to a sulfate- and dose-dependent loss of its bioactivity. Using GAG-biotin derivatives in a competitive ELISA approach sclerostin was shown to be the preferred binding partner over Wnt3a. In conclusion, highly sulfated GAGs might control bone homeostasis via interference with sclerostin/LRP5/6 complex formation. Whether these properties can be utilized to improve bone regeneration needs to be validated in vivo.     

Fragment size- and dose-specific effects of hyaluronan on matrix synthesis by vascular smooth muscle cells

Tissue engineering of vascular elastin matrices disrupted by mechanical injury, disease, or congenitally absent, is among other factors, limited by the lack of suitable cell scaffolds to up-regulate and guide innately poor elastin synthesis by adult vascular smooth muscle cells (SMCs). Evidence suggests that scaffolds based on hyaluronan (HA), a glycosaminoglycan, may be useful to elicit elastogenic cell responses, although these effects appear to be dictated by HA fragment size and/or dose. This study investigates the efficacy of a simple, frequently adopted exogenous HA supplementation model to test this hypothesis. Rat aortic SMCs were cultured with HA (2 x 10(6) Da (HMW) > or = MW < or = 2.2 x 10(4) Da) supplemented at doses between 0.2 and 200 microg/ml. Cell layers were biochemically assayed for DNA, elastin and collagen content. Fragmented, but not high molecular weight (HMW) HA, stimulated cell proliferation in inverse correlation fragment size while the opposite effect was observed for synthesis of soluble and matrix elastin; almost no dose effects were observed within any group. SDS-Page/Western Blot and a desmosine assay semi-quantitatively confirmed the observed biochemical trends for tropoelastin and matrix elastin, respectively. Quantitative differences in elastin deposition were mirrored in TEM micrographs. Elastin was mostly deposited in the form of amorphous clumps but fibers were increasingly present in cell layers cultured with HMW HA. HA and its fragments did not disrupt normal fibrillin-mediated mechanisms of elastin matrix deposition. While the current outcomes confirm that the effects of HA on elastin synthesis are fragment size-specific, this study shows that an exogenous supplementation model does not necessarily simulate cellular matrix synthesis responses to HA-based biomaterial scaffolds.     

Differential expression of CD44 in canine melanocytic tumours

CD44, the main cell surface receptor for hyaluronan (HA), is often overexpressed in tumour cells, and its presence has been related to cell proliferation and migration. Many of the functions of CD44 are mediated through its interaction with hyaluronan. This study investigated the expression of CD44 in CML-1 and CML-10c2 canine melanoma cell lines and melanoma biopsies, and the production of hyaluronan and versican by the canine melanoma cell lines. Versican is an extracellular proteoglycan that binds hyaluronan, forming a tridimensional pericellular coat surrounding the cells. Both canine melanoma cell lines expressed CD44 and produced HA, but only CML-1 produced versican. Cells expressing all three components (CD44, HA and versican) formed abundant extracellular matrices as demonstrated by a particle exclusion assay. CD44 was present within benign and malignant melanomas, but its expression was more intense in malignant melanomas (P < 0.01). In high CD44-expressing tumours, CD44 tended to be present in the periphery of malignant melanomas, whereas its expression was homogeneous in benign melanomas.     

Hyaluronic acid and periodontitis

Hyaluronic acid (HA; synonyms--Hyaluronan, Hyaluronate) is a glycosaminoglycan found in the connective tissue of vertebrates. It is the most abundant glycosaminoglycan of higher molecular weight in the extracellular matrix of soft periodontal tissues. The use of HA in the treatment of inflammatory process is established in medical areas such as orthopedics, dermatology and ophthalmology. In the field of dentistry, it has shown anti-inflammatory and anti-bacterial effects in gingivitis and periodontitis therapy. Due to its tissue healing properties, it could be used as an adjunct to mechanical therapy in the treatment of periodontitis.     

The effect of hyaluronic acid with different molecular weights on collagen crosslink synthesis in cultured chondrocytes embedded in collagen gels

Hyaluronic acid (HA) is a component of the extracellular matrix of cartilage and has various effects on three-dimensional cultured chondrocytes. We measured Pyridinoline (Pyr), which is a crosslink of collagen in cultured chondrocyte-collagen composites treated with HA of different molecular weights to investigate the effects of the various molecular weights on collagen crosslink synthesis. The control group was collagen gel without cells; group N was treated without HA; and the others were treated with HA with an average molecular weight of 2.3 x10(6) Da (group H), 8.0 x10(5) Da (group M), and 2.3 x10(4) Da (group L). In the control group, the Pyr content decreased, at week 4, being one-tenth that of preculture levels. In groups H and M, it was significantly greater than that in groups L and N at week 4. Pyr/hydroxyproline, which indicates the concentration of Pyr per collagen, decreased greatly in the control group at week 3. In groups H and M, it was significantly higher than that in groups L and N at week 4 and increased to 80 and 76% of normal rabbit articular cartilage, respectively. The concentration of Pyr per collagen in cultured chondrocyte-collagen composites was similar to that of normal articular cartilage in vivo, and higher molecular weight HA may have a greater effect on the maturation of collagen in the composite.     

Single-molecule imaging of hyaluronan in human synovial fluid

Human synovial fluid contains a high concentration of hyaluronan, a high molecular weight glycosaminoglycan that provides viscoelasticity and contributes to joint lubrication. In osteoarthritis synovial fluid, the concentration and molecular weight of hyaluronan decrease, thus impairing shock absorption and lubrication. Consistently, substitution of hyaluronan (viscosupplementation) is a widely used treatment for osteoarthritis. So far, the organization and dynamics of hyaluronan in native human synovial fluid and its action mechanism in viscosupplementation are poorly characterized at the molecular level. Here, we introduce highly sensitive single molecule microscopy to analyze the conformation and interactions of fluorescently labeled hyaluronan molecules in native human synovial fluid. Our findings are consistent with a random coil conformation of hyaluronan in human synovial fluid, and point to specific interactions of hyaluronan molecules with the synovial fluid matrix. Furthermore, single molecule microscopy is capable of detecting the breakdown of the synovial fluid matrix in osteoarthritis. Thus, single molecule microscopy is a useful new method to probe the structure of human synovial fluid and its changes in disease states like osteoarthritis.     

Hyaluronan oligosaccharides promote functional recovery after spinal cord injury in rats

Hyaluronan is a component of the extracellular matrix of the central nervous system, and forms perineuronal nets around neurons. It has been recently reported that the hyaluronan-degrading enzyme hyaluronidase promotes lateral mobility of AMPA-type glutamate receptors and enhances synaptic plasticity. However, the biological significance of hyaluronan-degrading products (oligosaccharides) has not been studied in depth. Here we investigated the effects of hyaluronan oligosaccharides on motor function recovery after spinal cord injury in rats. The disaccharide HA2 and especially the tetrasaccharide HA4, significantly improved motor function, unlike the case with oligosaccharides composed of 6-12 saccharides. Consistent with this finding, HA4 treatment enhanced axonal regeneration/sprouting, as assessed by corticospinal tract tracer fiber counts. HA4 treatment also significantly suppressed accumulation of Iba-1-positive cells in a lesion two weeks after injury. In vitro experiments demonstrated that NMDA-induced neuronal cell death was partly blocked by HA4, but not by other oligosaccharides, whereas proteoglycan-mediated inhibition of neurite outgrowth was not affected by treatment with any oligosaccharide examined. Taken together, the present results revealed that due in part to its neuroprotective activity, HA4 promotes motor function recovery after spinal cord injury.     

Activation and transforming growth factor-beta production in eosinophils by hyaluronan

To investigate whether extracellular matrix glycosaminoglycan hyaluronan (HA) modulates eosinophil activation and transforming growth factor (TGF)-beta production by eosinophils, human peripheral blood eosinophils (purity > 99%) from 12 patients with mild to moderate asthma or six healthy subjects were isolated and incubated with increasing concentrations of low molecular weight (mol wt) HA ( approximately 0.2 x 10(6) D) or high mol wt HA (3.0 to approximately 5.8 x 10(6) D). We found that the low mol wt HA has a pronounced effect on eosinophil survival in both patients with asthma and healthy subjects in a dose-dependent fashion on Days 2 and 4. Whereas the high mol wt HA had a smaller effect on eosinophil survival than did the low mol wt HA. The HA-mediated eosinophil survival was partially but significantly inhibited ( approximately 50% inhibition) by a blocking monoclonal antibody for CD44, a specific receptor of HA, and largely inhibited by an anti-granulocyte macrophage colony-stimulating factor (GM-CSF) neutralizing antibody but not by an anti-interleukin (IL)-3 or anti-IL-5 neutralizing antibody. In addition, the low mol wt HA increased GM-CSF messenger RNA (mRNA) expression and protein secretion by eosinophils in a dose-dependent fashion, suggesting that the HA-mediated eosinophil survival is due mainly to induction of GM-CSF release through partial CD44 signaling. Furthermore, we demonstrated that the low mol wt HA results in morphologic changes in eosinophils such as transforming from a round to a spindle shape and in homotypic aggregation, upregulates intercellular adhesion molecule-1 expression, and increases TGF-beta mRNA expression and protein secretion by eosinophils. These observations suggest previously unforeseen interactions between eosinophils and low mol wt extracellular matrix and, thus, novel pathways by which eosinophils may contribute to the regulation of airway inflammation and airway remodeling.     

神经干细胞与透明质酸水凝胶材料生物相容性的研究

目的:评价神经干细胞与改性透明质酸水凝胶支架新材料的生物相容性,研究该透明质酸水凝胶支架作为中枢神经组织工程载体材料的可行性,为用组织工程及干细胞技术治疗中枢神经系统损伤提供基础。方法:以冷冻干燥法制备透明质酸水凝胶材料支架,通过化学接枝法将抗Nogo受体抗体(Anti-NogoR)和多聚赖氨酸(poly-l-lysine,PLL)分子接枝到水凝胶上对其改性,制成新的支架材料。体外培养胚胎13.5d大鼠前脑泡神经干细胞.将神经干细胞与生物支架共培养,通过扫描电镜观察透明质酸水凝胶的内部结构及神经干细胞在支架材料上的粘附与生长情况,通过细胞免疫组织化学技术观察神经干细胞在透明质酸水凝胶材料上的存活与分化情况。结果:制备的透明质酸水凝胶材料具有疏松的三维多孔结构,神经干细胞在支架材料上能够粘附并且有突起长出,生长良好。神经干细胞在支架材料上能够分化。结论:神经干细胞与经过改性的透明质酸水凝胶新材料有很好的生物相容性,能够在材料上存活分化。该新透明质酸水凝胶材料有望作为修复中枢神经损伤的组织工程载体。     

流体剪切力下 CD44-HA 介导的 MDA-MB-231细胞及 HL60 细胞的滚动黏附

目的 探究胞外基质中的透明质酸(hyaluronic acid, HA)如何调控血流中CD44⁺肿瘤细胞的黏附滚动行为。方法 采用平行平板流动腔装置,观察记录流场中MDA-MB-231细胞及HL60细胞在固定HA上的运动,提取细胞滚动黏附特征参数。结果 MDA-MB-231细胞在HA底板上的黏附受到HA浓度的正向调控,但不受HA分子量影响;与物理吸附相比,生物素-亲和素固定的HA可显著提高细胞的黏附比率。在30～50 mPa剪切力范围内,剪切力的增加加快了细胞的滚动速度,降低了细胞的黏附比率,但对细胞的栓缚时间影响不大。同样流场中,与MDA-MB-231细胞比较,CD44表达水平较低的HL60细胞在HA底板上的栓缚时间短、滚动速度快、黏附比率低(<1.5%)。结论 流体剪切力可能通过调节CD44-HA的结合速率而非解离速率来调控MDA-MB-231细胞的滚动速度;CD44-HA相互作用参与HL60细胞的初始黏附,但不起主要作用。研究结果为抗肿瘤药物的开发提供参考。     

Hyaluronan enhances cartilage repair through low grade tissue remodeling involving cytokines and matrix metalloproteinases

OBJECTIVE AND DESIGN: To determine whether the ability of high molecular weight hyaluronan (HA) to reverse cartilage damage caused by specific catabolic mediators of cartilage damage, fibronectin fragments (Fn-fs), occurs through a low grade of enhanced catabolic events such as enhanced matrix metalloproteinase (MMP) expression or cytokine activities. MATERIAL: HA from 6.8-kDa to 2 million daltons was studied. TREATMENT: The ability of HA to enhance matrix metalloproteinase-3 (MMP-3) epitopes and cartilage proteoglycan (PG) degradation neoepitopes was tested in bovine cartilage, as well as the ability of recombinant human interleukin-1 receptor antagonist protein (rhIRAP) to reverse PG depletion in cartilage first exposed to Fn-f. RESULTS: All HA forms enhanced MMP-3 epitopes and PG degradation in normal undamaged cartilage and in the case of HA800, the degradation was not sufficient to decrease steady state levels of cartilage PG. When HA800 was added to Fn-f damaged cartilage, restoration of PG occurred, but this was blocked by rhIRAP. CONCLUSIONS: These results collectively suggest that some of the repair activity of HA800 is through proteolytic activity which is not sufficient to decrease matrix PG content, but is nonetheless elevated above levels in cartilage not treated with HA800.