Deposition Of Hyaluronic Acid Research Articles

Stromal PDGFR-α Activation Enhances Matrix Stiffness, Impedes Mammary Ductal Development, and Accelerates Tumor Growth

The extracellular matrix (ECM) is critical for mammary ductal development and differentiation, but how mammary fibroblasts regulate ECM remodeling remains to be elucidated. Herein, we used a mouse genetic model to activate platelet derived growth factor receptor-alpha (PDGFRα) specifically in the stroma. Hyperactivation of PDGFRα in the mammary stroma severely hindered pubertal mammary ductal morphogenesis, but did not interrupt the lobuloalveolar differentiation program. Increased stromal PDGFRα signaling induced mammary fat pad fibrosis with a corresponding increase in interstitial hyaluronic acid (HA) and collagen deposition. Mammary fibroblasts with PDGFRα hyperactivation also decreased hydraulic permeability of a collagen substrate in an in vitro microfluidic device assay, which was mitigated by inhibition of either PDGFRα or HA. Fibrosis seen in this model significantly increased the overall stiffness of the mammary gland as measured by atomic force microscopy. Further, mammary tumor cells injected orthotopically in the fat pads of mice with stromal activation of PDGFRα grew larger tumors compared to controls. Taken together, our data establish that aberrant stromal PDGFRα signaling disrupts ECM homeostasis during mammary gland development, resulting in increased mammary stiffness and increased potential for tumor growth.

Loss of CD73 prevents accumulation of alternatively activated macrophages and the formation of prefibrotic macrophage clusters in irradiated lungs.

While radiotherapy is a mainstay for cancer therapy, pneumonitis and fibrosis constitute dose-limiting side effects of thorax and whole body irradiation. So far, the contribution of immune cells to disease progression is largely unknown. Here we studied the role of ecto-5'-nucelotidase (CD73)/adenosine-induced changes in the myeloid compartment in radiation-induced lung fibrosis. C57BL/6 wild-type or CD73-/- mice received a single dose of whole thorax irradiation (WTI, 15 Gy). Myeloid cells were characterized in flow cytometric, histologic, and immunohistochemical analyses as well as RNA analyses. WTI induced a pronounced reduction of alveolar macrophages in both strains that recovered within 6 wk. Fibrosis development in wild-type mice was associated with a time-dependent deposition of hyaluronic acid (HA) and increased expression of markers for alternative activation on alveolar macrophages. These include the antiinflammatory macrophage mannose receptor and arginase-1. Further, macrophages accumulated in organized clusters and expressed profibrotic mediators at ≥25 wk after irradiation (fibrotic phase). Irradiated CD73-/- mice showed an altered regulation of components of the HA system and no clusters of alternatively activated macrophages. We speculate that accumulation of alternatively activated macrophages in organized clusters represents the origins of fibrotic foci after WTI and is promoted by a cross-talk between HA, CD73/adenosine signaling, and other profibrotic mediators.-De Leve, S., Wirsdörfer, F., Cappuccini, F., Schütze, A., Meyer, A. V., Röck, K., Thompson, L. F., Fischer, J. W., Stuschke, M., Jendrossek, V. Loss of CD73 prevents accumulation of alternatively activated macrophages and the formation of prefibrotic macrophage clusters in irradiated lungs.

Abstract PR06: VEGF-targeted therapy induces extracellular matrix remodeling and increases mechanical barriers to therapy in colorectal cancer liver metastases

Abstract Introduction: The anti-VEGF antibody bevacizumab in combination with chemotherapy is a standard treatment for metastatic colorectal cancer (mCRC) today, based on an overall survival improvement. However, this survival benefit is modest and mCRC ultimately progresses. The underlying mechanism of resistance to anti-angiogenic therapy remains unclear. Recent preclinical studies have shown that anti-angiogenic therapy increases collagen expression in tumors as a consequence of hypoxia. The extracellular matrix (ECM) play an important role in solid stress-induced blood vessel collapse as they transmit the mechanical stress created by proliferating cells within the confined space of a tumor. In this study, we investigated the effects of anti-angiogenic therapy on extracellular matrix expression, both collagenous and non-collagenous and perfusion as a novel mechanism of acquired resistance to anti-angiogenic therapy in liver metastases from CRC. Experimental Design: We used C57BL/6 and BALB/c, as well as CCR2-/- and ATR1-/- mice for this study. To generate liver metastasis, we exteriorized and transected spleen, inject syngenic CRC SL4 or CT26 cells (0.1 million cells) in one hemispleen and then, removed it. The other hemispleen remained intact. We monitored tumor burden by measuring blood Gaussia luciferase (Gluc) activity from Gluc transduced tumors and/or by high-frequency ultrasound imaging. Treatments include anti-VEGF monoclonal antibody B20.4-1.1 (5 mg/kg or 1 mg/kg i.p. 2x/week, Genentech), an anti-Ly6G antibody (5 mg/kg i.p. every 2days), pegylated hyaluronidase (PEGHAse, 4.5 mg/kg i.v. 2x/week 24 hour prior to administration of chemotherapy), 5-Fluorouracil (50 mg/kg i.v. 2x/week). We determined ECM–hyaluronic acid (HA), sulfated glycosaminoglycan (sGAG), and collagen contents—by ELISA, the Blyscan Proteoglycan and Glycosaminoglycan assay, and hydroxyproline assay, respectively. We further determined ECM, activated fibroblasts, vasculature, perfusion (Hechst33342), and hypoxia (pimonidazole) by immunohistochemistry, and immune cell profile using flow cytometry. We then, determined stiffness (Young's modulus) of tumors by unconfined compression tests (Cancer Res 60: 2497-503, 2000) and solid stress using a recently established method (PNAS 109: 15101-8, 2012). We obtained patient samples from the Heidelberg University Hospital and analyzed surgical specimens of patients who underwent liver resection for colorectal liver metastases without preoperative chemotherapy, after preoperative chemotherapy without bevacizumab or after preoperative chemotherapy with bevacizumab. Results: Here, we show that anti-VEGF therapy induces remodeling of the extracellular matrix with subsequent alteration of physical properties of liver metastases. We find that preoperative treatment with bevacizumab in patients with colorectal liver metastases resulted in a significant increase in HA deposition within the tumor. Moreover, in two syngeneic mouse models of mCRC, we show that anti-VEGF therapy markedly increased expression of HA and sGAG, without significant changes in collagen deposition. The density of these matrix components correlates with tumor stiffness; the latter was also significantly increased after anti-VEGF therapy. In time-course and immunofluorescence studies, treatment-induced hypoxia appeared as the driving force for enhanced extracellular matrix expression. Enzymatic depletion of HA partially reversed compromised perfusion in liver metastases after anti-VEGF therapy and prolonged survival in combination with anti-VEGF therapy and systemic chemotherapy. Conclusion: These findings suggest that extracellular matrix components such as HA could be a potential therapeutic target for reducing physical barriers to systemic treatments in patients with mCRC who receive anti-VEGF therapy. This abstract is also being presented as Poster B27. Citation Format: Nuh N. Rahbari, Dmitriy Kedrin, Joao Incio, Hao Liu, William T. Ho, Hadi T. Nia, Christina M. Edrich, Keehoon Jung, Julien Daubriac, Ivy Chen, Takahiro Heishi, John Martin, Yuhui Huang, Nir Maimon, Christoph Reissfelder, Juergen Weitz, Yves Boucher, Jeffrey W. Clark, Alan J. Grodzinsky, Dan G. Duda, Rakesh K. Jain, Dai Fukumura. VEGF-targeted therapy induces extracellular matrix remodeling and increases mechanical barriers to therapy in colorectal cancer liver metastases. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr PR06.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Anti-VEGF therapy induces ECM remodeling and mechanical barriers to therapy in colorectal cancer liver metastases.

The survival benefit of anti-vascular endothelial growth factor (VEGF) therapy in metastatic colorectal cancer (mCRC) patients is limited to a few months because of acquired resistance. We show that anti-VEGF therapy induced remodeling of the extracellular matrix with subsequent alteration of the physical properties of colorectal liver metastases. Preoperative treatment with bevacizumab in patients with colorectal liver metastases increased hyaluronic acid (HA) deposition within the tumors. Moreover, in two syngeneic mouse models of CRC metastasis in the liver, we show that anti-VEGF therapy markedly increased the expression of HA and sulfated glycosaminoglycans (sGAGs), without significantly changing collagen deposition. The density of these matrix components correlated with increased tumor stiffness after anti-VEGF therapy. Treatment-induced tumor hypoxia appeared to be the driving force for the remodeling of the extracellular matrix. In preclinical models, we show that enzymatic depletion of HA partially rescued the compromised perfusion in liver mCRCs after anti-VEGF therapy and prolonged survival in combination with anti-VEGF therapy and chemotherapy. These findings suggest that extracellular matrix components such as HA could be a potential therapeutic target for reducing physical barriers to systemic treatments in patients with mCRC who receive anti-VEGF therapy.

Effect of polyelectrolyte size on multilayer conformation and dynamics at different temperatures and salt concentrations

Polyelectrolyte bilayers, which consist of poly-l-lysine (PLL) and hyaluronic acid (HA) were simulated with lipid membranes at different temperatures and ion concentrations. Starting with the sequential deposition of PLL and HA above the membrane surface, PLL and HA become completely mixed, leading to the formation of stable bilayers. PLL/HA bilayers are thicker at higher salt concentration because of weakened electrostatic interactions between PLLs and membrane lipids, in agreement with experiments. This salt effect decreases as PLL size increases. Also, bilayers become thinner at higher temperature because of the increased surface area of membrane. In particular, regardless of temperature and salt concentration, larger PLLs induce thicker bilayers, although larger PLLs have lower diffusivities than do smaller ones. Bilayers with larger PLLs show larger vacancy (more water) inside the bilayer, indicating that larger PLLs are less densely stacked on membrane surface than do smaller ones and thus form the thicker bilayer. These findings show the lower diffusivity of larger polyelectrolytes, which supports the experimental observation regarding the restricted diffusion of large polymers, and also imply the dependence of bilayer thickness on the polymer size.

Electrochemical Deposition of Materials for Biomedical Applications

Electrochemical methods have been developed for the deposition of biopolymer films containing hydroxyapatite, and other bioceramics, such as titania, silica and bioglass. Different electrochemical methods have been utilized for the deposition of natural biopolymers, such as chitosan, polyacrylic acid and hyaluronate. Chitosan was protonated and dissolved in acidic solutions and then deposited cathodically. In this approach the charge neutralization of cationic chitosan in the high pH region at the electrode surface resulted in the precipitation of insoluble chitosan. Electrodeposition of alginic acid was performed from sodium alginate solutions. In this method the pH decrease at the anode surface resulted in the charge neutralization of alginate macromolecules and the fabrication of alginic acid films on anodic substrates. A similar approach has been utilized for the deposition of hyaluronic acid from the sodium hyaluronate solutions. It was demonstrated that cathodic and anodic deposition methods can be used for the deposition of other biopolymers. Nanoparticles of hydroxyapatite were prepared by a chemical precipitation method. Precipitation was performed at 70 °C by the slow addition of a 0.6 M ammonium phosphate solution into a 1.0 M calcium nitrate solution. The pH of the solutions was adjusted to 11 by NH4OH. Stirring was performed during 8 h at 70 °C and then 24 h at room temperature. The precipitate was washed with water and finally with ethanol. The average length of the needle-shape HA crystals, prepared by this method, was about 150 nm and the average aspect ratio was 8. The composite films containing HA nanoparticles in chitosan, alginic acid and hyaluronic acid matrix were prepared on stainless steel, Ti and NiTi alloy substrates. The results of thermogravimetric analysis showed that hydroxyapatite content in the polymer matrix was varied in the range of 35-80 wt% by variation of hydroxyapatite or polymer concentration in the solutions. The deposition yield was studied at different deposition conditions. It was shown that film thickness can be varied in the range of 0.1-200 microns by variation of the deposition time in the range of 1-10 min and deposition voltage in the range of 10-60 V. The composite films were prepared as monolayers or multilayers containing hydroxyapatite-polymer layers, separated by the layers of pure polymer. The co-deposition of inorganic materials with biopolymers resulted in the fabrication of novel composite materials containing bioactive glass and other bioactive materials, such as silica and titania in a polymer matrix. Electrochemical techniques have been also applied for the fabrication of materials of graded composition (FGM) containing biopolymers, hydroxyapaite and other bioceramics and bioglass. Electrochemical impedance spectroscopy and potentiodynamic polarization studies showed that the composite coatings provided corrosion protection of the metallic substrates in simulated body fluid solutions. The use of biopolymers enabled the room temperature processing of the coatings, eliminating the problems related to the sintering of hydroxyapatite and other bioceramics. Moreover, the method enabled the incorporation of drugs and proteins in the composite material. Electrochemical strategies have been developed for the deposition of composite films containing heparin and albumin in a biopolymer matrix. The room temperature processing offers a possibility of co-deposition of other functional materials and fabrication of advanced nanocomposite coatings for biomedical applications. New deposition methods were developed for cathodic deposition of chiral biopolymers, such as poly-l-lysine and polyl-ornithine. As an extension of these investigations, the composite films, containing HA nanoparticles and other functional biomaterials in the matrix of chiral biopolymers were obtained and investigated. Further development of new deposition methods resulted in the development of electrophoretic strategies and deposition mechanisms for the deposition of linear polyethylenimine, polypyrrole, polyetheretherketone, polyacrylic acid. Fundamental mechanisms were developed for the co-deposition of enzymes and proteins with various biopolymers. We discovered a possibility of electrophoretic co-deposition of halloysite nanotubes with cationic and anionic biopolymers, such as chitosan, poly-l-lysine and polyl-ornithine, alginic acid, hyaluronic acid, polyacrylic acid. The halloysite nanotubes can be loaded with drugs and antimicrobial agents for the controlled release. The new composite materials are prtomising for applications in biomedical implants and biosensors. We investigated functional properties of new materials, fundamental deposition mechanisms and kinetics of deposition.

Looking Inside the Matrix: Perineuronal Nets in Plasticity, Maladaptive Plasticity and Neurological Disorders.

The integrity of the central nervous system (CNS) matrix is crucial for its proper function. Loss of the lattice-like structure compromise synaptic stability and can lead to the disruption of the excitatory/inhibitory balance, astrocytosis, maladaptive plasticity and neuronal death. Perineuronal nets (PNNs) in the extracellular matrix (ECM) provide synaptic integration and control the functional wiring between neurons. These nets are significantly modified during CNS disorders, such as neurodegenerative, cerebrovascular and inflammatory diseases. The breakdown or the modification of PNNs could be due to the activity of matrix metalloproteinases (MMPs) or to the deposition of proteoglycans, glycoproteins, and hyaluronic acid. The expression and the activity of ECM-degrading enzymes can be regulated with tissue inhibitors of MMPs or via transcriptional and epigenetic silencing or enhancement (i.e. via histone deacetylases). The identification of molecules and mechanisms able to modify these processes will be essential for a new perspective on brain functioning in health and disease, leading to a target-directed approach with drugs directly interfering with the molecular mechanism underlying neurological disorders.

Study on biomimetic mineralization of lipopolysaccharide-amine nanopolymersomes/hyaluronic acid polyelectrolyte films on titanium surface

To explore biomimetic mineralization of polyelectrolyte multilayer films (PEM) of gene-loaded lipopolysaccharide-amine nanopolymersomes/hyaluronic acid self assembled on titanium surface. Via lay-by-layer self assembly technology, PEM were constructed on titanium or quartz surface using bone morphogenetic protein-2(BMP-2) plasmid-loaded lipopolysaccharide-amine nanopolymersomes(pLNP) as a polycation, and hyaluronic acid(HA) as a polyanion. The constructed PEM were defined as substrate-pLNP-(HA-pLNP)n, where a successive deposition of HA and pLNP on substrate surface was defined as one assembly cycle, and n was the cycle number. Biomimetic mineralization on surfaces of Ti-pLNP-(HA-pLNP)4(Group A, with outermost layer of pLNP), Ti-pLNP-(HA-pLNP)4.5(Group B, with outermost layer of HA), blank control(polished titanium, Ti) and alkaline-heat treated titanium(Ti-OH) were investigated. The biomimetic mineralization was analyzed by observing the topography under field-emisssion electron microscopy(FE-SEM), characterizing the surface chemical structure and components via X-ray diffractometer(XRD) and X-ray energy disperse spectroscopy(EDS). For experiment groups, XRD analysis showed that the diffraction peak of hydroxyapatite appeared, and its intensity was higher than that for Ti group. FE-SEM images showed that its surface was homogeneously covered by discrete agglomerate of big particles. EDS spectra showed that the percentage of Ca and P were 77.24% and 64.23%, and these were much higher than those in Ti group. The surface of Ti-pLNP-(HA-pLNP)n is favorable for in vitro biomimetic mineralization.

Dopamine-assisted deposition and zwitteration of hyaluronic acid for the nanoscale fabrication of low-fouling surfaces.

In this study, we proposed a bioinspired approach for the deposition and zwitteration of hyaluronic acid (HA) with a reduced glutathione (GSH) to form a composite layer that functions as a low fouling coating. A polyanion of the HA-dopamine conjugate (HADA) possessing catechol groups was synthesized by carbodiimide chemistry between HA and dopamine. Then, the dopamine conjugated biofunctional polymers (HADA) were grafted onto Au substrates via the transformation of catechol into a quinone group under mild oxidative conditions followed by a reaction with GSH to avoid undesired adhesion and also to shield the exposed Au substrate. Analysis of XPS spectra and wettability indicated that HADA and GSH were successfully grafted onto Au substrates. Surface plasmon resonance analysis showed that both HADA and further GSH modified surfaces exhibited reduced nonspecific adsorption. The attachment of GSH to HADA modified surfaces (HADA-G) resulted in better antifouling performance, with a low or ultralow protein adsorption of 0-7.51 ng cm-2 when exposed to single protein solutions, and a reduction in nonspecific adsorption from cow's milk to 10% compared to that of bare gold. The enhanced antifouling performance of HADA-G modified surfaces was likely due to the zwitterionic structure in GSH, which can induce stronger surface hydration through electrostatic interactions as well as the hydrogen bonding induced by HADA. Our results provide a facile and universal approach to surface modification and demonstrate the benefits of using a composite layer for the design of low fouling surfaces.

Transformation gélatineuse de la moelle osseuse : une série rétrospective monocentrique de 12 cas

PurposeGelatinous bone marrow transformation is a rare complication of unknown pathogenesis related to several underlying diseases. It is described as a focal loss of hematopoietic cells and a deposition of eosinophilic gelatinous substance rich in hyaluronic acid. We report a retrospective monocentric study followed by a literature review. MethodsWe have identified 12 patients with gelatinous transformation from 1999 to 2014. Clinical, biological and cytological data were collected. ResultsA bone marrow aspirate was performed for mild to severe pancytopenia (50%), less frequently for monocytopenia. Mean haemoglobin was 10.4g/dL, platelets 126.9G/L and leucocytes 3.82G/L. Gelatinous transformation was found in all patients. We have identified three hematologic malignancies, three cancers, three severe infections, two anorexia nervosa and one kidney injury. Ten patients had undernutrition, 9 out of the 12 patients died within a year due to advanced underlying disease and comorbidities. ConclusionGelatinous bone marrow transformation is found in various situations of severe undernutrition including anorexia nervosa, advanced cancers or severe sepsis, especially in HIV infection. The deposition of hyaluronic acid probably reduces haematopoiesis. Due to a complex inflammatory process, it alters the hematopoietic microenvironment and the bone marrow stroma. Severe undernutrition and other mechanisms are reviewed in this study. Gelatinous degeneration is still a rare disorder, indicative of an advanced underlying disease; its recognition could help guide investigations.

Construction of murine model of TAO by immunification with recombinant extracellular domain of human thyrotropin receptor in cationic liposomes

Background Thyroid-associated ophthalmopathy (TAO) is a kind of clinically common and incurable ocular disease, and its incidence is at top place.The etiology and pathologic mechanism of TAO are still unknown because of shortness of replicative animal models and difficulty to acquire the ocular tissues in the early stage of the disease.To better understand the pathogenesis of TAO and investigate effective treatable measures, an appropriate animal model should be developed. Objective This study was to immunize female BALB/c mice with the recombinant plasmid of human thyroid-stimulating hormone receptor (TSHR) extracellular domain in cationic liposomes for the establishement of TAO models. Methods Thirty-two 6- to 8-week-old female BALB/c mice were randomly assigned to four groups according to computer random allocation.pcDNA3.1+ /hTSHR289 of 100 μg in an adjuvant cationic liposomes was injected via anterior tibialis muscle and peritoneal cavity separately in the recombinant plasmid injection group in 0, 3, 6 weeks, and pcDNA3.1 or cationic liposomes was injected in the liposomes injection group or the blank plasmid group in the same way, respectively, and normal saline solution was injected in the blank control group.Body weight of the mice was measued before and 1 month, 2, 3 and 4 months after initial injection.The manifestations were observed after modeling.The mice were sacrificed 17 weeks after initial injection, and the histopathology examination was carried out on the thyroid gland and orbital tissue.The heart blood was collected from the mice, and serum contents of total thyroxin 4 (TT4) and thyroid-stimulating hormone (TSH) were assayed by ELISA. Results Protrusion, eyelid swell and keratitis occurred in 12 eyes of 6 mice in the recombinant plasmid injection group after immunization.A significant difference in the body weight of the mice was found among the blank control group, blank plasmid group, liposomes injection group and recombinant plasmid injection group (Fgroup=3.425, P=0.028), and the body weight was considerably reduced in the recombinant plasmid injection group in comparison with the blank control group, blank plasmid group, liposomes injection group (Ftime=0.838, P=0.023). The serum levels of TT4 were (7.75±1.00), (7.96±0.76), (6.76±1.10) and (4.43±2.88)μg/dl in the blank control group, liposomes injection group, blank plasmid group, and recombinant plasmid injection group, and those of TSH were (6.36±2.58), (4.83±3.96), (6.63±1.71) and (1.60±1.76)ng/ml, showing significant differences among the groups (F=7.150, P<0.001; F=5.521, P<0.01), and the serum levels of TT4 and TSH were remarkably lower in the recombinant plasmid injection group than those of the blank control group, liposomes injection group and blank plasmid group (all at P<0.05). Histopathology revealed the lymphocyte infiltration of thyroid gland in 6 mice and proliferation of orbital adipose tissue, infiltration of lymphocytes and mastocytes, deposition of hyaluronic acid as well as swell, breakage and inflammatory cell infiltration of extraocular muscle in 15 eyes of the recombinant plasmid injection group. Conclusions A murine model of TAO can be successfully induced by immunization with recombination plasmid pcDNA3.1+ /hTSHR289 and cationic liposomes.The histopathology characteristics and ocular findings of the animal models are similar to human TAO. Key words: Graves disease; Disease models, animal; Immunization/methods; Mice, inbred BALB/c; Receptors, thyrotropin/blood; Thyroxine/blood; Recombinant plasmid; Thyroid-associated ophthalmopathy

Conjugation of Hyaluronic Acid onto Surfaces via the Interfacial Polymerization of Dopamine to Prevent Protein Adsorption.

A versatile, convenient, and cost-effective method that can be used for grafting antifouling materials onto different surfaces is highly desirable in many applications. Here, we report the one-step fabrication of antifouling surfaces via the polymerization of dopamine and the simultaneous deposition of anionic hyaluronic acid (HA) on Au substrates. The water contact angle of the Au surfaces decreased from 84.9° to 24.8° after the attachment of a highly uniform polydopamine (PDA)/HA hybrid film. The results of surface plasmon resonance analysis showed that the Au-PDA/HA surfaces adsorbed proteins from solutions of bovine serum albumin, lysozyme, β-lactoglobulin, fibrinogen, and soybean milk in ultralow or low amounts (4.8-31.7 ng/cm(2)). The hydrophilicity and good antifouling performance of the PDA/HA surfaces is attributable to the HA chains that probably attached onto their upper surface via hydrogen bonding between PDA and HA. At the same time, the electrostatic repulsion between PDA and HA probably prevents the aggregation of PDA, resulting in the formation of a highly uniform PDA/HA hybrid film with the HA chains (with a stretched structure) on the upper surface. We also developed a simple method for removing this PDA/HA film and recycling the Au substrates by using an aqueous solution of NaOH as the hydrolyzing agent. The Au surface remained undamaged, and a PDA/HA film could be redeposited on the surface, with the surface exhibiting good antifouling performance even after 10 such cycles. Finally, it was found that this grafting method is applicable to other substrates, including epoxy resins, polystyrene, glass, and steel, owing to the strong adhesion of PDA with these substrates.

Hyaluronic Acid Filler in HIV-Associated Facial Lipoatrophy: Evaluation of Tissue Distribution and Morphology with MRI

Objective: This prospective observational study evaluated magnetic resonance imaging (MRI) findings of hyaluronic acid (HA) injections used for the correction of HIV-associated facial lipoatrophy. Methods: Ten consecutive males underwent subdermal HA injection (mean 1.3 ± 0.6 ml per side) and MRI examinations prior to and then 1, 6 and 12 months after injection. Two radiologists blinded to the clinical data assessed morphologic and quantitative changes. Results: MRI revealed HA deposition in the subdermal and deep fat compartments. A significant HA volume increase was observed 1 month after injection (mean increase 331%, p < 0.0001) as compared to the injected amount. No volume reduction occurred at 12 months (p = 0.9961). The measured bound water content did not change (p > 0.9991), whereas skin thickness and tissue vascularization increased during the first 6 months (p = 0.01). Conclusion: Our data show that the cosmetic results of HA injections are caused by water binding in the deep facial fat and by a transient increase in vascularization and skin thickness.

Nitric Oxide Stimulates Matrix Synthesis and Deposition by Adult Human Aortic Smooth Muscle Cells Within Three-Dimensional Cocultures

Vascular diseases are characterized by the over-proliferation and migration of aortic smooth muscle cells (SMCs), and degradation of extracellular matrix (ECM) within the vessel wall, leading to compromise in cell-cell and cell-matrix signaling pathways. Tissue engineering approaches to regulate SMC over-proliferation and enhance healthy ECM synthesis showed promise, but resulted in low crosslinking efficiency. Here, we report the benefits of exogenous nitric oxide (NO) cues, delivered from S-Nitrosoglutathione (GSNO), to cell proliferation and matrix deposition by adult human aortic SMCs (HA-SMCs) within three-dimensional (3D) biomimetic cocultures. A coculture platform with two adjacent, permeable 3D culture chambers was developed to enable paracrine signaling between vascular cells. HA-SMCs were cultured in these chambers within collagen hydrogels, either alone or in the presence of human aortic endothelial cells (HA-ECs) cocultures, and exogenously supplemented with varying GSNO dosages (0-100 nM) for 21 days. Results showed that EC cocultures stimulated SMC proliferation within GSNO-free cultures. With increasing GSNO concentration, HA-SMC proliferation decreased in the presence or absence of EC cocultures, while HA-EC proliferation increased. GSNO (100 nM) significantly enhanced the protein amounts synthesized by HA-SMCs, in the presence or absence of EC cocultures, while lower dosages (1-10 nM) offered marginal benefits. Multi-fold increases in the synthesis and deposition of elastin, glycosaminoglycans, hyaluronic acid, and lysyl oxidase crosslinking enzyme (LOX) were noted at higher GSNO dosages, and coculturing with ECs significantly furthered these trends. Similar increases in TIMP-1 and MMP-9 levels were noted within cocultures with increasing GSNO dosages. Such increases in matrix synthesis correlated with NO-stimulated increases in endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) expression within EC and SMC cultures, respectively. Results attest to the benefits of delivering NO cues to suppress SMC proliferation and promote robust ECM synthesis and deposition by adult human SMCs, with significant applications in tissue engineering, biomaterial scaffold development, and drug delivery.

Polysaccharide-coated PCL nanofibers for wound dressing applications.

Polysaccharide-based nanofibers with a multilayered structure are prepared by combining electrospinning (ESP) and layer-by-layer (LBL) deposition techniques. Charged nanofibers are firstly prepared by electrospinning poly(ε-caprolactone) (PCL) with a block-copolymer bearing carboxylic acid functions. After deprotonation of the acid groups, the layer-by-layer deposition of polyelectrolyte polysaccharides, notably chitosan and hyaluronic acid, is used to coat the electrospun fibers. A multilayered structure is achieved by alternating the deposition of the positively charged chitosan with the deposition of a negatively charged polyelectrolyte. The construction of this multilayered structure is followed by Zeta potential measurements, and confirmed by observation of hollow nanofibers resulting from the dissolution of the PCL core in a selective solvent. These novel polysaccharide-coated PCL fiber mats remarkably combine the mechanical resistance typical of the core material (PCL)-particularly in the hydrated state-with the surface properties of chitosan. The control of the nanofiber structure offered by the electrospinning technology, makes the developed process very promising to precisely design biomaterials for tissue engineering. Preliminary cell culture tests corroborate the potential use of such system in wound healing applications.

Using hyaluronidase medication to correct adverse events post soft tissue augmentation

As hyaluronic acid (HA) has become one of the most common facial aesthetic augmentation filler agents, the need to effectively deal with procedure-related complications is much greater. Adverse events such as overcorrection and asymmetries owing to product misplacement through poor injection technique can undermine positive treatment outcomes because, although they are classed as temporary, HA-based dermal fillers can last up to 12 months or longer. Hyaluronidase treatment fortunately offers aesthetic practitioners a potential solution to multiple HA-filler based adverse effects. When used correctly, hyaluronidase is also an effective treatment for some serious adverse reactions to HA and can help to remove unwanted HA deposits. It has also been shown to be useful in managing rare granulomatous foreign-body reactions to HA and delayed onset nodules, once infection or inflammation have been treated or ruled out.

MP17-03 HYALURONAN AND ITS HYALURONAN RECEPTORS IN RAT BLADDER REGENERATION AND THEIR ROLE IN ANGIOGENESIS

INTRODUCTION AND OBJECTIVES: Hyaluronic acid (HA), a non-sulfated glycosaminoglycan and an essential component of the extracellular matrix (ECM), plays critical roles in phases of wound healing and tissue regeneration. This study was designed to characterize temporal and anatomical HA deposition and its major receptors (CD44, LYVE-1, RHAMM, and HARE) expression in a rat bladder regeneration model and to understand the effectiveness of HA in promoting angiogenesis. METHODS: Fifteen Sprague-Dawley rats were subjected to partial cystectomy with interposition of distal sections of porcine small intestinal submucosa (SIS). SIS-augmented bladders were harvested on post-operative days 2, 7, 14, 28, and 56 for histological studies. Effectiveness of HA in stimulating endothelial cell proliferation, tube formation, and angiogenic activity were determined in vitro cell and ex ovo chick chorioallantoic membrane (CAM) models. RESULTS: Bladder regeneration proceeded without complications; and all regenerated bladders had complete urothelial lining and smooth muscle bundle formation by day 56 post-augmentation. As determined by immunohistochemistry, strong positive HA staining was observed on days 28 and 56 post-operation in the ECM of the stroma. CD44 immunoreactivity was detected on days 28 and 56 in the cytoplasm of urothelial cells and LYVE-1 immunoreactivity was detected exclusively in lymphatic vessels at days 28 and 56. RHAMM mRNA increased within 2 days post-operation, whereas HARE mRNA level did not change during the course of bladder regeneration. Exogenous HA significantly stimulated growth and tube formation of cultured endothelial cells, and enhanced angiogenesis in CAM assay. CONCLUSIONS: During bladder regeneration HA is synthesized throughout the course of regeneration processes. HA deposition coincides with urothelial differentiation. Temporal regulation and spatial distribution of two major HARs (CD44 and LYVE-1) follow the same temporal pattern as HA deposition. Therapeutic modalities through local delivery of exogenous HA can be viable to significantly improve angiogenesis and enhance the outcome and completeness of bladder regeneration.

MP17-04 NOVEL INSIGHTS INTO HUMAN DETRUSOR SMOOTH MUSCLE MUSCARINIC RECEPTOR PHYSIOLOGY: A ROLE FOR THE LARGE CONDUCTANCE VOLTAGE- AND CALCIUM-ACTIVATED POTASSIUM CHANNELS

INTRODUCTION AND OBJECTIVES: Hyaluronic acid (HA), a non-sulfated glycosaminoglycan and an essential component of the extracellular matrix (ECM), plays critical roles in phases of wound healing and tissue regeneration. This study was designed to characterize temporal and anatomical HA deposition and its major receptors (CD44, LYVE-1, RHAMM, and HARE) expression in a rat bladder regeneration model and to understand the effectiveness of HA in promoting angiogenesis. METHODS: Fifteen Sprague-Dawley rats were subjected to partial cystectomy with interposition of distal sections of porcine small intestinal submucosa (SIS). SIS-augmented bladders were harvested on post-operative days 2, 7, 14, 28, and 56 for histological studies. Effectiveness of HA in stimulating endothelial cell proliferation, tube formation, and angiogenic activity were determined in vitro cell and ex ovo chick chorioallantoic membrane (CAM) models. RESULTS: Bladder regeneration proceeded without complications; and all regenerated bladders had complete urothelial lining and smooth muscle bundle formation by day 56 post-augmentation. As determined by immunohistochemistry, strong positive HA staining was observed on days 28 and 56 post-operation in the ECM of the stroma. CD44 immunoreactivity was detected on days 28 and 56 in the cytoplasm of urothelial cells and LYVE-1 immunoreactivity was detected exclusively in lymphatic vessels at days 28 and 56. RHAMM mRNA increased within 2 days post-operation, whereas HARE mRNA level did not change during the course of bladder regeneration. Exogenous HA significantly stimulated growth and tube formation of cultured endothelial cells, and enhanced angiogenesis in CAM assay. CONCLUSIONS: During bladder regeneration HA is synthesized throughout the course of regeneration processes. HA deposition coincides with urothelial differentiation. Temporal regulation and spatial distribution of two major HARs (CD44 and LYVE-1) follow the same temporal pattern as HA deposition. Therapeutic modalities through local delivery of exogenous HA can be viable to significantly improve angiogenesis and enhance the outcome and completeness of bladder regeneration.

Effect of astaxanthin on vocal fold wound healing

Our previous study demonstrated that a large amount of reactive oxygen species (ROS) is produced during the early phase of vocal fold wound healing. In the current study, we investigated the effect of astaxanthin, which is a strong antioxidant, on the regulation of oxidative stress and scarring during vocal fold wound healing. Prospective animal experiment with control. Sprague-Dawley rats were dosed with astaxanthin (Ast-treated group, 100 mg/kg/day) or olive oil (sham-treated group) by oral gavage daily from preinjury day 1 to postinjury day 4. After vocal folds were injured under the endoscope, larynges were harvested for histological and immunohistochemical examinations on postinjury days 1, 3, 5, and 56, and quantitative real time polymerase chain reaction (PCR) on postinjury days 1 and 3. The expression of 4-hydroxy-2-nonenal, which is an oxidative stress marker, was reduced significantly in the lamina propria of the Ast-treated group as compared to the sham-treated group. Histological examination showed significantly less tissue contraction with favorable deposition of hyaluronic acid in the lamina propria of the Ast-treated group compared to the sham-treated group. Real time PCR revealed significantly upregulated mRNA expression of basic fibroblast growth factor on postinjury day 1 and procollagen type I in the Ast-treated group compared to the sham-treated group. These findings suggest that astaxanthin has the potential to prevent vocal fold scarring by regulating oxidative stress during the early phase of vocal fold wound healing.