Extracellular Matrix Of Many Tissues Research Articles

Active Media Perfusion in Bioprinted Highly Concentrated Collagen Bioink Enhances the Viability of Cell Culture and Substrate Remodeling.

The bioprinting of high-concentrated collagen bioinks is a promising technology for tissue engineering and regenerative medicine. Collagen is a widely used biomaterial for bioprinting because of its natural abundance in the extracellular matrix of many tissues and its biocompatibility. High-concentrated collagen hydrogels have shown great potential in tissue engineering due to their favorable mechanical and structural properties. However, achieving high cell proliferation rates within these hydrogels remains a challenge. In static cultivation, the volume of the culture medium is changed once every few days. Thus, perfect perfusion is not achieved due to the relative increase in metabolic concentration and no medium flow. Therefore, in our work, we developed a culture system in which printed collagen bioinks (collagen concentration in hydrogels of 20 and 30 mg/mL with a final concentration of 10 and 15 mg/mL in bioink) where samples flow freely in the culture medium, thus enhancing the elimination of nutrients and metabolites of cells. Cell viability, morphology, and metabolic activity (MTT tests) were analyzed on collagen hydrogels with a collagen concentration of 20 and 30 mg/mL in static culture groups without medium exchange and with active medium perfusion; the influence of pure growth culture medium and smooth muscle cells differentiation medium was next investigated. Collagen isolated from porcine skins was used; every batch was titrated to optimize the pH of the resulting collagen to minimize the difference in production batches and, therefore, the results. Active medium perfusion significantly improved cell viability and activity in the high-concentrated gel, which, to date, is the most limiting factor for using these hydrogels. In addition, based on SEM images and geometry analysis, the cells remodel collagen material to their extracellular matrix.

Eggshell Membrane Repurposing from the Perspective of Tissue Engineering: A Non-Systematic Review

Natural resources are rich in collagen and growth factors that can perfectly fit in tissue engineering and contribute to the extracellular matrix of many tissues. The thin membrane that coats the inner surface of the eggshell is one of these resources that meets the need of tissue engineering as it contains high collagen, growth factors, and glycosaminoglycans. With advanced technology, eggshell membrane (EgS-M) has been available in many forms like powder, solution, and thin sheets for broad applications. Furthermore, integrating nanoparticles has provided a broad-spectrum EgS-M biomaterial with antimicrobial, anti-inflammatory, biocompatible, and biodegradable properties. Herein, we discuss the current status of EgS-M's contribution to tissue engineering of skin, cartilage, bone, periodontal, myocardium, and nerve.

Peroxynitrous acid (ONOOH) modifies the structure of anastellin and influences its capacity to polymerize fibronectin

Anastellin (AN), a fragment of the first type III module in fibronectin (FN), initiates formation of superfibronectin, a polymer which resembles the native cell-derived fibrillar FN found in the extracellular matrix of many tissues, but which displays remarkably different functional properties. Here we demonstrate that exposure of AN to the biologically-important inflammatory oxidant, peroxynitrous acid (ONOOH), either as a bolus or formed at low levels in a time-dependent manner from SIN-1, impairs the capability of AN to polymerize FN. In contrast, exposure of FN to ONOOH does not seem to affect superfibronectin formation to the same extent. This oxidant-induced loss-of-function in AN occurs in a dose-dependent manner, and correlates with structural perturbations, loss of the amino acid tyrosine and tryptophan, and dose-dependent formation of modified amino acid side-chains (3-nitrotyrosine, di-tyrosine and 6-nitrotryptophan). Reagent ONOOH also induces formation of oligomeric species which decrease in the presence of bicarbonate, whereas SIN-1 mainly generates dimers. Modifications were detected at sub-stoichiometric (0.1-fold), or greater, molar excesses of oxidant compared to AN. These species have been localized to specific sites by peptide mass mapping. With high levels of oxidant (>100 times molar excess), ONOOH also induces unfolding of the beta-sheet structure of AN, thermal destabilization, and formation of high molecular mass aggregates. These results have important implications for the understanding of FN fibrillogenesis in vivo, and indicates that AN is highly sensitive to pathophysiological levels of oxidants such as ONOOH.

The Effect of Halloysite Addition on the Material Properties of Chitosan-Halloysite Hydrogel Composites.

Chitosan-based hydrogels are being widely used in biomedical applications due to their eco-friendly, biodegradable, and biocompatible properties, and their ability to mimic the extracellular matrix of many tissues. However, the application of chitosan hydrogels has been limited due to their inherent mechanical weakness. Halloysite nanotubes (HNTs) are naturally occurring aluminosilicate clay minerals and are widely used as a bulk filler to improve the performance characteristics of many polymeric materials. HNTs have also been shown to be a viable nanocontainer able to provide the sustained release of antibiotics, chemicals, and growth factors. This study’s objective was to develop a stable drug delivery chitosan/HNT nanocomposite hydrogel that is biocompatible, biodegradable, and provides sustained drug release. In this study, chitosan/HNTs hydrogels containing undoped or gentamicin-doped HNTs were combined in different wt./wt. ratios and cross-linked with tripolyphosphate. The effects of chitosan and HNTs concentration and combination ratios on the hydrogel surface morphology, degradability, and mechanical properties, as well as its drug release capability, were analyzed. The results clearly showed that the addition of HNTs improved chitosan mechanical properties, but only within a narrow range. The nanocomposite hydrogels provided a sustained pattern of drug release and inhibited bacterial growth, and the live/dead assay showed excellent cytocompatibility.

X-ray Tomographic Imaging of Tensile Deformation Modes of Electrospun Biodegradable Polyester Fibers

Electrospinning allows the production of fibrous networks for tissue engineering, drug delivery and wound healing in healthcare. It enables the production of constructs with large surface area and a fibrous morphology that closely resembles the extracellular matrix of many tissues. A fibrous structure not only promotes cell attachment and tissue formation, but could also lead to very interesting mechanical properties. Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB)) is a biodegradable polyester that exhibits large (>400%) elongation before failure. In this study, synchrotron X-ray phase contrast imaging was performed during tensile deformation to failure on a non-woven fibre mat of P(3HB-co-4HB) fibres. Significant reorientation of the fibres in straining direction was observed, followed by localised necking and eventual failure. From an original average fibre diameter of 4.3 μm a bimodal distribution of fibre diameter (modal diameters of 1.9 and 3.7 μm) formed after tensile deformation. Extensive localised necking (thinning) of fibres between (thicker) fibre-fibre contacts was found to be the cause for non-uniform thinning of the fibres, a phenomenon that is expected, but has not been observed in 3D previously. The data presented here has implications not only in tissue regeneration but for fibrous materials in general.

Hyaluronan-based multifunctional hydrogels formed via enzyme-mediated crosslinking

Event Abstract Back to Event Hyaluronan-based multifunctional hydrogels formed via enzyme-mediated crosslinking Daniela S. Ferreira1, 2, Dominic W. Collis1, 2 and Helena S. Azevedo1, 2 1 Queen Mary, University of London, School of Engineering and Materials Science, United Kingdom 2 Queen Mary University of London, Institute of Bioengineering, United Kingdom Introduction: Hydrogels have generated great interest to develop 3D artificial matrices that recapitulate features of the natural extracellular matrix (ECM) for applications in tissue regeneration or cell expansion[1],[2]. Covalent crosslinking is a common method to obtain stable hydrogels with precise control over crosslinking density. However, many of the crosslinking reagents used are toxic, precluding their use for cell encapsulation applications. Enzyme-mediated crosslinking reactions are finding increasing applications for developing hydrogels due to their mild conditions and enzyme specificity[3]. FXIIIa is a transglutaminase that catalyses the reaction between an ε-amine of lysine (K) onto a γ-carboxamide residue of glutamine (Q), resulting in the covalent bond formation of an ε-(γ-glutamyl)lysine isopeptide which is highly resistant to proteolytic degradation[4]. This crosslinking mechanism naturally occurs in vivo during the processes of wound healing. Hyaluronan (HA), a linear polysaccharide present in the ECM of many tissues, has been chemically modified to develop hydrogels[5]-[7]. In this work, HA was functionalized with two different transglutaminase peptide substrates to obtain HA hydrogels by enzyme-mediated crosslinking. Materials and Methods: Peptides were synthesized manually using standard Fmoc based solid phase chemistry and purified, as described elsewhere[8]. Hyaluronan (60 kDa) was functionalized with acrylate groups using a two-step synthesis (Fig 1-A, as described elsewhere[9]. Peptides were coupled to acrylated HA via Michael-type addition by reacting 1.2-fold molar excess of peptide over acrylate groups in 0.3 M triethanolamine (pH 8.0) overnight at 37°C. Results and Discussion: Peptides were designed as factor XIIIa substrates, containing either a lysine donor peptide CH3CONH-GFKGG-ERCG-CONH2 (TG-K, Mw = 950.08 g/mol) or a glutamine acceptor peptide - CH3CONH-GNQEQVSPL-ERCG-CONH2 (TG-Q, Mw = 1457.58 g/mol). In addition, a glutamine acceptor containing a cell adhesion ligand RGDS, which is recognized by several integrin receptors, was also synthesized, CH3CONH--GNQEQVSPLRGDSPG-CONH2 (TG-Q-RGDSPG, Mw = 1581.67 g/mol). To render hydrogels susceptible to degradation by proteolytic enzymes, like natural fibrin, a lysine donor peptide was synthesized bearing a substrate for matrix metalloproteases (MMPs) Ac-GFKGGGPQGIWGQ-ERCG-CONH2 (TG-MMP-K, Mw = 1581.67 g/mol). Acrylated HA was obtained by a two-step reaction and the degree of acrylation was found to be 16%, as determined by NMR (Fig. 1-B). Transglutaminase peptide substrates were grafted onto acrylate groups of HA backbone via Michael-type addition as confirmed by NMR analysis (Fig. 2). Conclusion: HA macromers functionalized with transglutaminase substrate peptides were successfully obtained. These HA-peptide macromers will be used as precursors to develop hydrogels that can be formed in situ (e.g. wound site), where FXIIIa is produced. The incorporation of integrin-specific adhesive ligands and cell-sensitive proteolytic domains in these hydrogels will aid in guiding and enhancing local tissue regeneration. The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007‐2013) under REA grant agreement n° 608765.

Material-based strategies to engineer fibronectin matrices for regenerative medicine

Engineering biomaterials for regenerative medicine involves a myriad of aspects to be considered for the successful design, interaction with cells and integration with living tissues (i.e. pore size, mechanical properties, degradation rate, biological activity). Among different technologies used to functionalise synthetic biomaterials and promote cell adhesion, cell growth and cell differentiation, this review focuses on strategies to organise extracellular matrix (ECM) proteins in a biomimetic way, as cells do in natural tissues in vivo (the ECM is a mesh of proteins that surrounds cells, and therefore, constitutes the scaffolding of a tissue), but using functional materials instead of living cells. The authors critically review material-based strategies to organise fibronectin (FN), a core component in the ECM of many tissues, and engineer microenvironments that recapitulate the structure and properties of the ECM. Material-driven organisation of FN in analogy with their natural cell-mediated assembly is a powerful route to engineer the network structure and biological activity of FN fibrillar matrices, seeking to develop biomimetic scaffolds for regenerative medicine. Here, the authors discuss different routes to promote the cell-free formation of FN fibrils as well as the biological impact of these engineered cellular microenvironments.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

1H, 13C and 15N resonance assignments for the fibrillin-1 EGF2-EGF3-hybrid1-cbEGF1 four-domain fragment

Fibrillins are large extracellular glycoproteins that form the principal component of microfibrils. These perform a vital structural function in the extracellular matrix of many tissues. Fibrillins have also been implicated in mediating a number of protein–protein interactions, some of which may be significant in regulating growth factors such as transforming growth factor β. Here we present the backbone and side-chain 1H, 13C and 15N assignments for a 19 kDa protein fragment derived from the N-terminus of human fibrillin-1, encompassing four domains in total. These domains include the second and third epidermal growth factor-like (EGF) domains, the first hybrid domain (hyb1), and the first calcium-binding EGF domain of fibrillin-1. This region of fibrillin-1 is of particular interest as the hyb1 domain has been suggested to play a role in microfibril assembly, as well as several other protein–protein interactions.

Abstract 426: The molecular microenvironment critically affects the formation of isoDGR-dependent integrin binding sites in fibronectin

Abstract Fibronectin (FN) is an adhesive protein present in most body fluids and in the extracellular matrix of many tissues. This protein mediates a variety of cellular interactions with extracellular matrix and play important roles in haemostasis, thrombosis, inflammation, wound repair and angiogenesis. FN is constituted of three types of repeating homologous modules termed FN-I, FN-II and FN-III. Each module contains binding sites for molecules of the extracellular matrix and for cell adhesion receptors, such as integrins. Moreover, FN contains four NGR sites located in the: 5th type I, 2nd type II, 7th type I repeat, 9th type III repeats. We have previously shown that the NGR site of the 5th type I repeat (FN-I5) rapidly undergoes deamidation leading to formation of isoDGR. A deamidated FN fragment corresponding to this domain binds αvβ3, inhibits endothelial cell adhesion and proliferation and inhibits tumor growth in animal models. In the present study we have investigated the molecular mechanisms that regulate isoDGR formation in FN in vivo. We provide evidence to suggest that FN purified from plasma contains very low amounts of isoDGR (<3%) despite its long half life (48h). In contrast, using anti-isoDGR antibodies, we observed that isoDGR is formed in significant amounts in small FN fragments injected in mice and left to circulate for 4 or 6 h (>17% in 4 h). In vitro cell adhesion assays, antibody binding assays and integrin binding studies showed that FN deamidation can rapidly occur when FN is bound to microtiter plates, a condition known to cause conformational changes in this protein, but not with FN in solution. Rapid deamidation was observed to occur also with short fragments (FN-I5), either adsorbed on microtiter plates or in solution. These results suggest that the kinetics of NGR-to-isoDGR transition in plasma FN is slower than its clearance rate. Furthermore, the finding that conformational changes and/or protein fragmentation increase the kinetics of NGR-to-isoDGR transition suggest that changes in the molecular microenvironment of NGR sites, potentially occurring after FN deposition in tissues, could be critical for the formation of isoDGR integrin binding sites. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 426. doi:10.1158/1538-7445.AM2011-426

Characterization of bionanocomposite scaffolds comprised of amine‐functionalized gold nanoparticles and silicon carbide nanowires crosslinked to an acellular porcine tendon

As one of the most common proteins found in the human body, collagen is regarded as biocompatible and has many properties making it ideal for soft-tissue repair applications. However, collagen matrices fabricated from purified forms of collagen are notoriously weak and easily degraded by the body. The extracellular matrix of many tissues including human dermis, porcine dermis, and porcine small intestine submucosa are often utilized instead, and several of these scaffolds are crosslinked. Crosslinking has been shown to improve the mechanical properties of collagenous tissues and increase their resistance to degradation. In this study we investigated two novel "bionanocomposite" materials in which either gold nanoparticles or silicon carbide nanowires were crosslinked to a porcine tendon. Scanning electron micrographs confirmed that the nanomaterials were successfully crosslinked to the tissues. A collagenase assay, tensile testing, flow cytometry, and bioreactor studies were also performed to further characterize the properties of these novel materials. The results of these studies indicated that crosslinking porcine diaphragm tissues with nanomaterials resulted in scaffolds with improved resistance to enzymatic degradation and appropriate biocompatibility characteristics, thus warranting further study of these materials for soft tissue repair and tissue engineering applications.

Fabrication and mechanical characterization of 3D electrospun scaffolds for tissue engineering

Electrospinning is a polymer processing technique that produces fibrous structures comparable to the extracellular matrix of many tissues. Electrospinning, however, has been severely limited in its tissue engineering capabilities because this technique has produced few three-dimensional structures. Sintering of electrospun materials provides a method to fabricate unique architectures and allow much larger structures to be made. Electrospun mats were sintered into strips and cylinders, and their tensile and compressive mechanical properties were measured. In addition, electrospun materials with salt pores (salt embedded within the material and then leached out) were fabricated to improve porosity of the electrospun materials for tissue engineering scaffolds. Sintered electrospun poly(d,l-lactide) and poly(l-lactide) (PDLA/PLLA) materials have higher tensile mechanical properties (modulus: 72.3 MPa, yield: 960 kPa) compared to unsintered PLLA (modulus: 40.36 MPa, yield: 675.5 kPa). Electrospun PDLA/PLLA cylinders with and without salt-leached pores had compressive moduli of 6.69 and 26.86 MPa, respectively, and compressive yields of 1.36 and 0.56 MPa, respectively. Sintering of electrospun materials is a novel technique that improves electrospinning application in tissue engineering by increasing the size and types of electrospun structures that can be fabricated.

Injectable, Biodegradable Hydrogels for Tissue Engineering Applications

Hydrogels have many different applications in the field of regenerative medicine. Biodegradable, injectable hydrogels could be utilized as delivery systems, cell carriers, and scaffolds for tissue engineering. Injectable hydrogels are an appealing scaffold because they are structurally similar to the extracellular matrix of many tissues, can often be processed under relatively mild conditions, and may be delivered in a minimally invasive manner. This review will discuss recent advances in the field of injectable hydrogels, including both synthetic and native polymeric materials, which can be potentially used in cartilage and soft tissue engineering applications.

HDL as a biomarker, potential therapeutic target, and therapy.

Emerging evidence suggests that HDL function is not always accurately predicted by HDL cholesterol levels. The functions of HDL include reverse cholesterol transport and modulation of inflammation. These functions appear to have evolved as part of the innate immune system. In healthy individuals, in the absence of systemic oxidative stress and inflammation, HDL is anti-inflammatory. However, in those with chronic illnesses such as diabetes that are characterized by systemic oxidative stress and inflammation, HDL may actually promote the inflammatory response (i.e., it may become proinflammatory). HDL may be thought of as a shuttle. The size of the shuttle can be estimated by HDL cholesterol levels. The shuttle's cargo can change dramatically from one that efficiently promotes reverse cholesterol transport and is anti-inflammatory to one that is less effective in promoting reverse cholesterol transport and is also proinflammatory without any change in the size of the shuttle (i.e., these changes in HDL cargo can occur without any change in HDL cholesterol levels). Understanding these issues may lead to improved use of HDL as a biomarker and may also lead to new therapeutic targets and therapies. ### HDL can modulate LDL oxidation. Lipoproteins evolved to facilitate the extracellular transport of lipids in multicellular organisms. The major protein in LDL is apolipoprotein (apo)-B. This protein contains a binding domain that causes LDL to be deposited in the extracellular matrix of many tissues, particularly in arteries that are predisposed to atherosclerosis. As a result of the binding of apoB-containing proteins to extracellular matrix molecules, the concentration of apoB in the subendothelial space of even normal arteries is twofold higher than in plasma (1). The deposition of LDL in the extracellular matrix of the subendothelial space predisposes it to oxidation. The oxidized lipids that result evoke a tissue response similar to that which occurs in response to a Mycobacterium (2, …

Microporous collagen spheres produced via thermally induced phase separation for tissue regeneration

Collagen is an abundant protein found in the extracellular matrix of many tissues. Due to its biocompatibility, it is a potentially ideal biomaterial for many tissue engineering applications. However, harvested collagen often requires restructuring into a three-dimensional matrix to facilitate applications such as implantation into poorly accessible tissue cavities. The aim of the current study was to produce a conformable collagen-based scaffold material capable of supporting tissue regeneration for use in wound repair applications. Microporous collagen spheres were prepared using a thermally induced phase separation (TIPS) technique and their biocompatibility was assessed. The collagen spheres were successfully cross-linked with glutaraldehyde vapour, rendering them mechanically more stable. When cultured with myofibroblasts the collagen spheres stimulated a prolonged significant increase in secretion of the angiogenic growth factor, vascular endothelial growth factor (VEGF), compared with cells alone. Control polycaprolactone (PCL) spheres failed to stimulate a similar prolonged increase in VEGF secretion. An enhanced angiogenic effect was also seen in vivo using the chick embryo chorioallantoic membrane assay, where a significant increase in the number of blood vessels converging towards collagen spheres was observed compared with control PCL spheres. The results from this study indicate that microporous collagen spheres produced using TIPS are biologically active and could offer a novel conformable scaffold for tissue regeneration in poorly accessible wounds.

Deformation-Dependent Enzyme Mechanokinetic Cleavage of Type I Collagen

Collagen is a key structural protein in the extracellular matrix of many tissues. It provides biological tissues with tensile mechanical strength and is enzymatically cleaved by a class of matrix metalloproteinases known as collagenases. Collagen enzymatic kinetics has been well characterized in solubilized, gel, and reconstituted forms. However, limited information exists on enzyme degradation of structurally intact collagen fibers and, more importantly, on the effect of mechanical deformation on collagen cleavage. We studied the degradation of native rat tail tendon fibers by collagenase after the fibers were mechanically elongated to strains of epsilon=1-10%. After the fibers were elongated and the stress was allowed to relax, the fiber was immersed in Clostridium histolyticum collagenase and the decrease in stress (sigma) was monitored as a means of calculating the rate of enzyme cleavage of the fiber. An enzyme mechanokinetic (EMK) relaxation function T(E)(epsilon) in s(-1) was calculated from the linear stress-time response during fiber cleavage, where T(E)(epsilon) corresponds to the zero order Michaelis-Menten enzyme-substrate kinetic response. The EMK relaxation function T(E)(epsilon) was found to decrease with applied strain at a rate of approximately 9% per percent strain, with complete inhibition of collagen cleavage predicted to occur at a strain of approximately 11%. However, comparison of the EMK response (T(E) versus epsilon) to collagen's stress-strain response (sigma versus epsilon) suggested the possibility of three different EMK responses: (1) constant T(E)(epsilon) within the toe region (epsilon<3%), (2) a rapid decrease ( approximately 50%) in the transition of the toe-to-heel region (epsilon congruent with3%) followed by (3) a constant value throughout the heel (epsilon=3-5%) and linear (epsilon=5-10%) regions. This observation suggests that the mechanism for the strain-dependent inhibition of enzyme cleavage of the collagen triple helix may be by a conformational change in the triple helix since the decrease in T(E)(epsilon) appeared concomitant with stretching of the collagen molecule.

Sulfated Polysaccharides Promote the Assembly of Amyloid β1–42 Peptide into Stable Fibrils of Reduced Cytotoxicity

The histopathological hallmarks of Alzheimer disease are the self-aggregation of the amyloid beta peptide (Abeta) in extracellular amyloid fibrils and the formation of intraneuronal Tau filaments, but a convincing mechanism connecting both processes has yet to be provided. Here we show that the endogenous polysaccharide chondroitin sulfate B (CSB) promotes the formation of fibrillar structures of the 42-residue fragment, Abeta(1-42). Atomic force microscopy visualization, thioflavin T fluorescence, CD measurements, and cell viability assays indicate that CSB-induced fibrils are highly stable entities with abundant beta-sheet structure that have little toxicity for neuroblastoma cells. We propose a wedged cylinder model for Abeta(1-42) fibrils that is consistent with the majority of available data, it is an energetically favorable assembly that minimizes the exposure of hydrophobic areas, and it explains why fibrils do not grow in thickness. Fluorescence measurements of the effect of different Abeta(1-42) species on Ca(2+) homeostasis show that weakly structured nodular fibrils, but not CSB-induced smooth fibrils, trigger a rise in cytosolic Ca(2+) that depends on the presence of both extracellular and intracellular stocks. In vitro assays indicate that such transient, local Ca(2+) increases can have a direct effect in promoting the formation of Tau filaments similar to those isolated from Alzheimer disease brains.

Lack of effect on bone healing of injectable BMP-2 augmented hyaluronic acid

Autologous bone graft is the gold standard for the filling of large osseous defects. Because of its limited supply and complications such as pain, bleeding or infection, the development of alternative bone substitutes has been the subject of several studies. In clinical practice, the most commonly used bone substitutes are calcium phosphates like hydroxyapatite or tricalcium phosphate. With the aim to improve the osseointegration of these materials, growth factors such as bone morphogenetic protein-2 (BMP-2) have been added. Preferably, an injectable bone substitute should be made available. Hyaluronic acid is a component of the extracellular matrix of many tissues, including bone. We examined the bone regenerative effect of commercially available, injectable hyaluronic acid (Hyalart) with and without addition of bone morphogenetic protein-2 (BMP-2). Trepanation defects of 9.4 mm diameter in the intercondylar groove of sheep femora were filled with pure and augmented (200 microg BMP-2) hyaluronic acid. As controls, empty defects and defects treated with autologous bone graft harvested from the contralateral side were used. After 3 months, the defects were analysed by fluorescence microscopy after intravital fluorescence staining, contact microradiography, histology and histomorphometry. Treatment of the defects with loaded and unloaded hyaluronic acid resulted in a significant lack of bone formation inside the defects. Untreated defects showed an amount of 5.1% newly formed bone, and defects treated with autologous bone graft revealed a bone content of 20%. The difference between both groups was statistically significant (P < 0.05). Furthermore, there was neither a remarkable effect in the periphery of the defects nor ectopic bone formation. The application of the used injectable hyaluronic acid (Hyalart) with and without BMP-2 is not advantageous as sole bone substitute for the filling of osseous defects.

Imaging and tracking of single hyaluronan molecules diffusing in solution

Hyaluronan is an important soluble component of the extracellular matrix of many tissues with well known space-filling, lubricating and signaling functions. As such, hyaluronan can regulate cell adhesion, migration, differentiation and proliferation. Ultrastructural studies showed the existence of fibers and networks of hyaluronan molecules at surfaces, while bulk studies of hyaluronan in solution indicated that the polymer forms random coils. Here, we show that single hyaluronan molecules can be visualized and tracked in three-dimensional samples at room temperature in aqueous buffer. Using a wide-field fluorescence microscope equipped with laser excitation and an sensitive and fast EMCCD camera for fluorescence detection, single FITC-labeled hyaluronan molecules from rooster comb were detected in aqueous solutions. Freely moving hyaluronan-FITC could be tracked over up to 20 images acquired at a frame rate of 98 Hz. Analysis of the trajectories revealed Brownian motion of hyaluronan in tris-buffered saline with an average diffusion coefficient D=3.0+/-0.2 microm(2)/s. These observations confirm the concept that hyaluronan molecules form random coils in solution. The possibility of following the tracks of single hyaluronan molecules in solution facilitates the analysis of processes that lead to the formation of more organized forms of hyaluronan and its interactions with cells with very high spatial and temporal accuracy.

Identification of heparin affin regulatory peptide domains with potential role on angiogenesis

Heparin affin regulatory peptide (HARP) is a growth factor displaying high affinity for heparin. It is present in the extracellular matrix of many tissues, interacting with heparan sulfate and dermatan/chondroitin sulfate glycosaminoglycans. We have previously shown that HARP is implicated in the control of angiogenesis and its effects are mimicked, at least in part, by synthetic peptides that correspond to its N and C termini. In the present work, we show that HARP is cleaved by plasmin, leading to the production of five peptides that correspond to distinct domains of the molecule. Heparin, heparan sulfate and dermatan sulfate, at various HARP to glycosaminoglycan ratios, partially protect HARP from plasmin degradation. The molecules with higher affinity to HARP are the more protective, heparin being the most efficient. The peptides that are produced from cleavage of HARP by plasmin, affect in vivo and in vitro angiogenesis and modulate the angiogenic activity of vascular endothelial growth factor on human umbilical vein endothelial cells. Similar results were obtained in vitro with recombinant HARP peptides, identical to the peptides generated after treatment of HARP with plasmin. These results suggest that different regions of HARP may induce or inhibit angiogenesis.