Polydopamine-Lysophosphatidate-Functionalised Titanium: A Novel Hybrid Surface Finish for Bone Regenerative Applications.

Aseptic loosening of total joint replacements (TJRs) continues to be the main cause of implant failures. The socioeconomic impact of surgical revisions is hugely significant; in the United Kingdom alone, it is estimated that £135m is spent annually on revision arthroplasties. Enhancing the longevity of titanium implants will help reduce the incidence and overall cost of failed devices. In realising the development of a superior titanium (Ti) technology, we took inspiration from the growing interest in reactive polydopamine thin films for biomaterial surface functionalisations. Adopting a "one-pot" approach, we exposed medical-grade titanium to a mildly alkaline solution of dopamine hydrochloride (DHC) supplemented with (3S)1-fluoro-3-hydroxy-4-(oleoyloxy)butyl-1-phosphonate (FHBP), a phosphatase-resistant analogue of lysophosphatidic acid (LPA). Importantly, LPA and selected LPA analogues like FHBP synergistically cooperate with calcitriol to promote human osteoblast formation and maturation. Herein, we provide evidence that simply immersing Ti in aqueous solutions of DHC-FHBP afforded a surface that was superior to FHBP-Ti at enhancing osteoblast maturation. The facile step we have taken to modify Ti and the biological performance of the final surface finish are appealing properties that may attract the attention of implant manufacturers in the future.

Development and biological evaluation of fluorophosphonate-modified hydroxyapatite for orthopaedic applications.

There is an incentive to functionalise hydroxyapatite (HA) for orthopaedic implant use with bioactive agents to encourage superior integration of the implants into host bone. One such agent is (3S) 1-fluoro-3-hydroxy-4-(oleoyloxy) butyl-1-phosphonate (FHBP), a phosphatase-resistant lysophosphatidic acid (LPA) analogue. We investigated the effect of an FHBP-HA coating on the maturation of human (MG63) osteoblast-like cells. Optimal coating conditions were identified and cell maturation on modified and unmodified, control HA surfaces was assessed. Stress tests were performed to evaluate coating survivorship after exposure to mechanical and thermal insults that are routinely encountered in the clinical environment. MG63 maturation was found to be three times greater on FHBP-modified HA compared to controls (p < 0.0001). There was no significant loss of coating bioactivity after autoclaving (P = 0.9813) although functionality declined by 67% after mechanical cleaning and reuse (p < 0.0001). The bioactivity of modified disks was significantly greater than that of controls following storage for up to six months (p < 0.001). Herein we demonstrate that HA can be functionalised with FHBP in a facile, scalable manner and that this novel surface has the capacity to enhance osteoblast maturation. Improving the biological performance of HA in a bone regenerative setting could be realised through the simple conjugation of bioactive LPA species in the future. Depicted is a stylised summary of hydroxyapatite (HA) surface modification using an analogue of lysophosphatidic acid, FHBP. a HA surfaces are simply steeped in an aqueous solution of 2 µM FHBP. b The polar head group of some FHBP molecules react with available hydroxyl residues at the mineral surfaces forming robust HA-O-P bonds leaving acyl chain extensions perpendicular to the HA surface. These fatty acyl chains provide points of integration for other FHBP molecules to facilitate their self-assembly. This final surface finish enhanced the human osteoblast maturation response to calcitriol, the active vitamin D3 metabolite.

Low pH and Anionic Lipid-dependent Fusion of Uukuniemi Phlebovirus to Liposomes.

Many phleboviruses (family Bunyaviridae) are emerging as medically important viruses. These viruses enter target cells by endocytosis and low pH-dependent membrane fusion in late endosomes. However, the necessary and sufficient factors for fusion have not been fully characterized. We have studied the minimal fusion requirements of a prototypic phlebovirus, Uukuniemi virus, in an in vitro virus-liposome assay. We show that efficient lipid mixing between viral and liposome membranes requires close to physiological temperatures and phospholipids with negatively charged headgroups, such as the late endosomal phospholipid bis(monoacylglycero)phosphate. We further demonstrate that bis(monoacylglycero)phosphate increases Uukuniemi virus fusion beyond the lipid mixing stage. By using electron cryotomography of viral particles in the presence or absence of liposomes, we observed that the conformation of phlebovirus glycoprotein capsomers changes from the native conformation toward a more elongated conformation at a fusion permissive pH. Our results suggest a rationale for phlebovirus entry in late endosomes.

Fluorophosphonate-functionalised titanium via a pre-adsorbed alkane phosphonic acid: a novel dual action surface finish for bone regenerative applications.

Enhancing vitamin D-induced human osteoblast (hOB) maturation at bone biomaterial surfaces is likely to improve prosthesis integration with resultant reductions in the need for revision arthroplasty consequent to aseptic loosening. Biomaterials that are less appealing to microorganisms implicated in implant failures through infection are also highly desirable. However, finding surfaces that enhance hOB maturation to active vitamin D yet deter bacteria remain elusive. In addressing this, we have sought to bio-functionalise titanium (Ti) with lysophosphatidic acid (LPA) and related, phosphatase-resistant, LPA analogues. The impetus for this follows our discovery that LPA co-operates with active vitamin D3 metabolites to secure hOB maturation in vitro including cells grown upon Ti. LPA has also been found, by others, to inhibit virulence factor production and biofilm formation of the human opportunistic pathogen Pseudomonas aeruginosa. Collectively, selected LPA species might offer potential dual-action surface finishes for contemporary bone biomaterials. In attaching a phosphatase-resistant LPA analogue to Ti we took advantage of the affinity of alkane phosphonic acids for TiO2. Herein, we provide evidence for the facile development of a dual-action Ti surface for potential orthopaedic and dental applications. Successful conjugation of an LPA analogue (3S)1-fluoro-3-hydroxy-4-(oleoyloxy)butyl-1-phosphonate (FHBP) to the Ti surface was supported through physiochemical characterisation using x-ray photoelectron spectroscopy and secondary ion mass spectrometry. hOB maturation to active vitamin D3 was enhanced for cells grown on FHBP-Ti whilst these same surfaces exhibited clear antiadherent properties towards a clinical isolate of Staphylococcus aureus.

HspA1A, a 70-kDa heat shock protein, differentially interacts with anionic lipids.

HspA1A, a 70-kDa heat shock protein, binds to specific lipids. This interaction allows HspA1A to associate with the plasma and other cellular membranes, where it regulates many vital functions like immunity, membrane stabilization, autophagy, and apoptosis. However, the molecular mechanism of the HspA1A-lipid interactions has yet to be fully characterized. Therefore, in this study, we characterized the interaction of HspA1A with three lipids, bis-(monoacylglycero)-phosphate, cardiolipin, and sulfatide. Our results revealed that, first, HspA1A embeds in membranes when bound to liposomes composed of cardiolipin and sulfatide. Second, the binding of HspA1A to lipids is complex and although important, electrostatic interactions alone cannot fully explain the observed binding. Third, the two HspA1A domains, the nucleotide-binding domain and the substrate-binding domain, differentially bind to lipids in a lipid-specific manner. Fourth, HspA1A lipid-binding is reduced by the presence of nucleotides, but it is unaffected by the presence of a peptide-substrate. These observations suggest that HspA1A binds to lipids via a multi-step mechanism and this interaction depends on the specific physicochemical properties of the lipid. We speculate that the association of HspA1A with lipids like the mitochondrial cardiolipin, which is an organelle marker, may facilitate the translocation and localized function of the molecular chaperone to particular sub-cellular compartments.

Differential effects of lipids and lyso-lipids on the mechanosensitivity of the mechanosensitive channels MscL and MscS.

Mechanosensitive (MS) channels of small (MscS) and large (MscL) conductance are the major players in the protection of bacterial cells against hypoosmotic shock. Although a great deal is known about structure and function of these channels, much less is known about how membrane lipids may influence their mechanosensitivity and function. In this study, we use liposome coreconstitution to examine the effects of different types of lipids on MscS and MscL mechanosensitivity simultaneously using the patch-clamp technique and confocal microscopy. Fluorescence lifetime imaging (FLIM)-FRET microscopy demonstrated that coreconstitution of MscS and MscL led to clustering of these channels causing a significant increase in the MscS activation threshold. Furthermore, the MscL/MscS threshold ratio dramatically decreased in thinner compared with thicker bilayers and upon addition of cholesterol, known to affect the bilayer thickness, stiffness and pressure profile. In contrast, application of micromolar concentrations of lysophosphatidylcholine (LPC) led to an increase of the MscL/MscS threshold ratio. These data suggest that differences in hydrophobic mismatch and bilayer stiffness, change in transbilayer pressure profile, and close proximity of MscL and MscS affect the structural dynamics of both channels to a different extent. Our findings may have far-reaching implications for other types of ion channels and membrane proteins that, like MscL and MscS, may coexist in multiple molecular complexes and, consequently, have their activation characteristics significantly affected by changes in the lipid environment and their proximity to each other.

Antagonism and synergy of single chain sphingolipids sphingosine and sphingosine-1-phosphate toward lipid bilayer properties. Consequences for their role as cell fate regulators.

A recurring question in membrane biological chemistry is whether bioactive signaling lipids act only as second messenger ligands or also through an effect on bilayer physical properties. Sphingosine (Sph) and sphingosine-1-phosphate (S1P) are single-chained charged sphingolipids that have antagonistic functions in the "sphingolipid rheostat" which determines cell fate. Sph and S1P respectively promote apoptosis and cell growth. In the present study, potential effects of these bioactive lipids on physicochemical properties of the lipid bilayer of cell membranes were evaluated. We have investigated the effect of both sphingolipids, incorporated separately or, for the first time, together, in large or giant phosphadidylcholine (PC) unilamellar vesicles. Three bilayer properties were examined: membrane surface charge, lipid packing, and formation of membrane microdomains. Sph and S1P appear to have distinct, when not inverse, effects on all three properties. Besides, when both sphingolipids are mixed together, their effects on lipid packing are synergistic, whereas their effects on microdomain formation and zeta-potential are mostly antagonistic. These results are interpreted as arising from different electrostatic interactions between lipid headgroups. In particular, Sph and S1P may interact together electrostatically and form a complex. These mostly inverse and opposing effects of both single-chain phospholipids on membrane physical properties might be involved in their antagonistic role in regulating cell fate. Particularly, the mutual interaction between Sph and S1P as a complex might be able to sequester both molecules in a biologically inactive form and therefore to promote a mutual regulation of their biological activities, depending on their ratio, consistent with the sphingolipid rheostat.

Bis (monoacylglycero) phosphate interfacial properties and lipolysis by pancreatic lipase-related protein 2, an enzyme present in THP-1 human monocytes.

The interfacial physical properties of bis(monoacylglycero)phosphate (BMP) and its derivatives with three oleoyl chains (hemi-BDP) and four oleoyl chains (bis(diacylglycero)phosphate, BDP) were investigated using Langmuir monomolecular films. The mean molecular area of BMP at the collapse surface pressure (45mN m(-1)) was similar to those measured with other phospholipids bearing two acyl chains (66 and 59.6Å(2) molecule(-1) at pH 5.5 and 8.0, respectively). In Hemi-BDP and BDP, the mean molecular area increased by 26 and 35Å(2) molecule(-1) per additional acyl chain at pH 5.5 and 8.0, respectively. When BMP was added to a phospholipid mixture mimicking late endosome membrane composition at pH 8.0, the mean phospholipid molecular area increased by 7% regardless of the surface pressure. In contrast, the variation in molecular area was surface pressure-dependent at pH 5.5, a pH value close to that of intra-endosomal content. BMP and hemi-BDP, but not BDP, were hydrolyzed by pancreatic lipase-related protein 2 (PLRP2), which exhibits phospholipase A(1) activity. At pH 5.5, the maximum activities of PLRP2 on BMP were recorded at high surface pressures (25-35mN/m). At pH 8.0, the PLRP2 activity vs. surface pressure showed a bell-shaped curve with maximum activities at 15mN/m for both BMP and hemi-BDP. This is a new activity for this enzyme which could degrade cellular BMP since both human PLRP2 (HPLRP2) and BMP were localized in human monocytic THP-1 cells. This is the first report on the cellular localization of HPLRP2 in human monocytes.

Role of bis(monoacylglycero)phosphate in propranolol binding to phospholipid membranes under acidic conditions as measured by high-performance frontal analysis/capillary electrophoresis.

Bis(monoacylglycero)phosphate (BMP) is localized in acidic organelles such as late endosomes or lysosomes. It has been reported that BMP levels increase under phospholipidosis induced by cationic amphiphilic drugs. In the present study, the effect of BMP on the binding of propranolol (PRO) to phospholipid liposomes under acidic conditions was investigated. Binding experiments were conducted by high-performance frontal analysis/capillary electrophoresis. PRO showed nonspecific binding to BMP-containing liposomes (BMP:phosphatidylcholine = 1:4), when numbers of bound drug molecules per lipid molecule (r) ranged 0.01-0.06. Total binding affinity increased depending on the BMP content. Binding affinity was decreased by low ionic strength, or by substitution of BMP with diacylglycerol, suggesting that electrostatic interactions were involved. The binding-enhancement effect of BMP was almost equivalent to that of phosphatidylglycerol, and slightly larger than that of phosphatidylserine. An acidic environment (pH 5.0) decreased total binding affinity to BMP-containing liposomes. This could be explained by the pH-partition theory (i.e., the loss in affinity was caused by a decrease in the neutral form of the drug accessible to the membrane core). These results suggest that PRO binding is enhanced by BMP in late endosomes or lysosomes, whereas an acidic environment weakens such binding.

Partially polymerized liposomes: stable against leakage yet capable of instantaneous release for remote controlled drug delivery.

A critical issue for current liposomal carriers in clinical applications is their leakage of the encapsulated drugs that are cytotoxic to non-target tissues. We have developed partially polymerized liposomes composed of polydiacetylene lipids and saturated lipids. Cross-linking of the diacetylene lipids prevents the drug leakage even at 40 °C for days. These inactivated drug carriers are non-cytotoxic. Significantly, more than 70% of the encapsulated drug can be instantaneously released by a laser that matches the plasmon resonance of the tethered gold nanoparticles on the liposomes, and the therapeutic effect was observed in cancer cells. The remote activation feature of this novel drug delivery system allows for precise temporal and spatial control of drug release.

Sphingosine 1-Phosphate Liposomes for Targeted Nitric Oxide Delivery to Mediate Anticancer Effects against Brain Glioma Tumors.

Specifically targeting glioblastoma multiforme (GBM) blood vessels and actively enhancing the permeability of the brain-blood-tumor barrier (BBTB) are two extremely difficult challenges currently hindering the development of effective therapies against GBM. Herein, a liposome drug delivery system (S1P/JS-K/Lipo) is described, which delivers the nitric oxide (NO) prodrug JS-K, O2 -(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl) piperazin-1-yl] diazen-1-ium-1,2-diolate, to GBM tumors using sphingosine-1-phosphate (S1P)-signaling molecules as active targeting lipid ligands. It is revealed that S1P/JS-K/Lipo actively penetrates the BBTB, aided by caveolin-1-mediated transcytosis, and it is demonstrated that the system specifically interacts with S1P receptors (S1PRs), which are highly expressed on GBM cells. Nondestructive ultrasound imaging in GBM mouse models is also utilized to observe microsized NO bubble production from JS-K, as catalyzed by the glutathione S-transferases (GSTs) resident in GBM cells. Given that these NO bubbles strongly promote GBM cell death in vivo, the S1PR-targeted liposome delivery system-which successfully achieves BBTB penetration and tumor targeted delivery of a complex multicomponent drug regimen-represents a promising approach for targeted therapies against GBM and other carcinomas characterized by elevated S1PR expression.

Ligand extraction properties of the GM2 activator protein and its interactions with lipid vesicles.

The GM2 activator protein (GM2AP) is an accessory protein required for the enzymatic conversion of GM2 to GM3 by hydrolases in the lysosomal compartments of cells. Here, GM2AP interactions with lipid vesicles are investigated by sucrose-loaded vesicle sedimentation and gel filtration assays, and the effects of pH and lipid composition on membrane binding and lipid extraction are characterized. The sedimentation experiments allow for facile quantification of the percentage of protein in solution and on the bilayer surface, with detailed analysis of the protein:lipid complex that remains in solution. Optimum binding and ligand extraction is found for pH 4.8 where <15% of the protein remains surface associated regardless of the lipid composition. In addition to extracting GM2, we find that GM2AP readily extracts dansyl-headgroup-labeled lipids as well as other phospholipids from vesicles. The ability of GM2AP to extract dansyl-DHPE from vesicles is altered by pH and the specific ligand GM2. Although the unique endosomal lipid, bis(monoacylglycero)phosphate, is not required for ligand extraction, it does enhance the extraction efficiency of GM2 when cholesterol is present in the vesicles.

Endothelial cell migration on RGD-peptide-containing PEG hydrogels in the presence of sphingosine 1-phosphate.

Sphingosine 1-phosphate (S1P) is a potent chemokinetic agent for endothelial cells that is released by activated platelets. We previously developed Arg-Gly-Asp (RGD)-containing polyethylene glycol biomaterials for the controlled delivery of S1P to promote endothelialization. Here, we studied the effects of cell adhesion strength on S1P-stimulated endothelial cell migration in the presence of arterial levels of fluid shear stress, since an upward shift in optimal cell adhesion strengths may be beneficial for promoting long-term cell adhesion to materials. Two RGD peptides with different integrin-binding specificities were added to the polyethylene glycol hydrogels. A linear RGD bound primarily to beta(3) integrins, whereas a cyclic RGD bound through both beta(1) and beta(3) integrins. We observed increased focal adhesion formation and better long-term adhesion in flow with endothelial cells on linear RGD peptide, versus cyclic RGD, even though initial adhesion strengths were higher for cells on cyclic RGD. Addition of 100 nM S1P increased cell speed and random motility coefficients on both RGD peptides, with the largest increases found on cyclic RGD. For both peptides, much of the increase in cell migration speed was found for smaller cells (<1522 microm(2) projected area), although the large increases on cyclic RGD were also due to medium-sized cells (2288-3519 microm(2)). Overall, a compromise between high cell migration rates and long-term adhesion will be important in the design of materials that endothelialize after implantation.

The phenyltetraene lysophospholipid analog PTE-ET-18-OMe as a fluorescent anisotropy probe of liquid ordered membrane domains (lipid rafts) and ceramide-rich membrane domains.

The conjugated phenyltetraene PTE-ET-18-OMe (all-(E)-1-O-(15'-phenylpentadeca-8',10',12',14'-tetraenyl)-2-O-methyl-rac-glycero-3-phosphocholine) is a recently developed fluorescent lysophospholipid analog of edelfosine, (Quesada et al. (2004) J. Med. Chem. 47, 5333-5335). We investigated the use of this analog as a probe of membrane structure. PTE-ET-18-OMe was found to have several properties that are favorable for fluorescence anisotropy (polarization) experiments in membranes, including low fluorescence in water and moderately strong association with lipid bilayers. PTE-ET-18-OMe has absorbance and fluorescence properties similar to those of diphenylhexatriene (DPH) probes, with about as large a difference between its fluorescence anisotropy in liquid disordered (Ld) and ordered states (gel and Lo) as observed for DPH. Also like DPH, PTE-ET-18-OMe has a moderate affinity for both gel state ordered domains and Lo state ordered domains (rafts). However, unlike fluorescent sterols or DPH (Megha and London (2004) J. Biol. Chem. 279, 9997-10004), PTE-ET-18-OMe is not displaced from ordered domains by ceramide. Also unlike DPH, PTE-ET-18-OMe shows only slow exchange between the inner and outer leaflets of membrane bilayers, and can thus be used to examine anisotropy of an individual leaflet of a lipid bilayer. Since PTE-ET-18-OMe is a zwitterionic molecule, it should not be as influenced by electrostatic interactions as are other probes that do not cross the lipid bilayer but have a net charge. We conclude that PTE-ET-18-OMe has some unique properties that should make it a useful fluorescence probe of membrane structure.

Analyzing and modeling the inhibitory effect of phosphatidic acid on the GTP-gamma-S binding activity of Goalpha.

G proteins are the molecular switches of G-protein-coupled signal transmembrane transduction, which plays a pivotal role in diverse cellular processes. The guanine nucleotide binding states of Galpha-subunits are considered key factors for their functions. We report here that phosphatidic acid (PA) inhibits the [(35)S]-GTPgammaS binding activity of Goalpha. To elucidate this inhibitory effect, biochemical analyses are carried out and a structure-based model is proposed. The experimental results show that PA particularly inhibits the activity of the Goalpha in a dose-dependent manner, whereas other lipids tested do not. Further analysis on the effects of PA analogs demonstrate that a phosphate head group together with at least one fatty acid chain is necessary for the inhibition. Using a lipid-protein binding assay, it is shown that Goalpha specifically and directly interacts with PA. In addition to these experimental studies, a 3D structure of Goalpha is constructed, based on sequence homology greater than 70% to E. coli Gialpha(1). Molecular docking is performed with PA and PA analogs, and the results are compared and analyzed. Collectively, the results of this investigation provide direct experimental evidence for an inhibitory effect of PA on GTP binding activity of Goalpha, and also suggest a structural model for the inhibitory mechanism. The lipid-protein model suggests that PA may occupy the channel for exchanging guanine nucleotides, thus leading to the inhibition. These findings reveal a potential new drug target for the diseases caused by genetic G-protein abnormalities.

Determining the effects of PEI adsorption on the permeability of 1,2-dipalmitoylphosphatidylcholine/bis(monoacylglycero)phosphate membranes under osmotic stress.

Polycations are used for a number of biological applications, including antibiotics and gene therapy. One aspect of the use of polycation gene carriers such as polyethylenemine (PEI) in gene therapy that is not well understood is their ability to escape from the vesicles they are internalized in. Here, in an attempt to gain a better understanding of PEI interaction with endosomal lipids under osmotic stress, we performed investigations using monolayers and vesicles derived from a mixture of neutral and negative lipids (1,2-dipalmitoylphosphatidylcholine (DPPC) and bis(monoacylglycero)phosphate (BMP), respectively). X-ray reflectivity (XR) and Langmuir trough measurements confirmed PEI adsorption to the negatively charged membrane. Confocal microscopy imaging indicated that PEI adsorption actually increases the overall integrity of the DPPC/BMP vesicle against osmotic stresses while also causing overall deformation and permeabilization of the lipid membrane, thus leading to leakage of contents from the interior of the vesicle. These confocal microscopy observations were also supported by data gathered by dynamic light scattering (DLS). STATEMENT OF SIGNIFICANCE: In recent decades, researchers have investigated polyamine-based gene delivery systems as useful alternatives to viral gene carriers. One step that is crucial to the performance of polyamine gene carriers such as polyethylenemine (PEI) is escape from late endosomal vesicles during intracellular delivery. However, the ability of polyamine/DNA polyplexes to effectively escape from endosomes is a little-understood part of the gene therapy techniques that use these polyplexes. Here, we performed investigations using monolayers and vesicles derived from a mixture of neutral and negative lipids (1,2-dipalmitoylphosphatidylcholine (DPPC) and bis(monoacylglycero)phosphate (BMP), respectively) as model systems for late endosomes in order to examine the interactions of PEI with the DPPC/BMP membranes and study the subsequent effects on the stability and permeability of these membranes.

Examining the effect of regioisomerism on the physico-chemical properties of lysophosphatidylethanolamine-containing liposomes using fluoro probes.

Lysophospholipids (LysoPLs) receive steadily increasing attention in the area of lipid chemistry and biology. However, the physico-chemical properties of individual LysoPL regioisomers have not yet been investigated. Herein, we report the synthesis of fluoro analogues of lysophosphatidylethanolamines (LPEs) and examine the physico-chemical properties of the LPE regioisomers using chemically synthesized fluoro probes.

Alginate hydrogels of varied molecular weight distribution enable sustained release of sphingosine-1-phosphate and promote angiogenesis.

Alginate hydrogels have been widely validated for controlled release of growth factors and cytokines, but studies exploring sustained release of small hydrophobic lipids are lacking. Sphingosine-1-phosphate (S1P), a bioactive lipid, is an appealing small molecule for inducing blood vessel formation in the context of ischemic conditions. However, there are numerous biological and engineering challenges associated with designing biomaterial systems for controlled release of this lipid. Thus, the objective of this study was to design an injectable, alginate hydrogel formulation that provides controlled release of S1P to establish locally sustained concentration gradients that promote neovascularization. Herein, we varied the molecular weight distribution of alginate polymers within the hydrogel to alter the resultant mechanical properties in a manner that provides control over S1P release. With increasing high molecular weight (HMW) content, the hydrogels exhibited stiffer material properties and released S1P at slower rates. Accordingly, S1P released from hydrogels with 100% HMW content led to enhanced directed migration of outgrowth endothelial cells and blood vessel development assessed using a chick chorioallantoic membrane assay as compared to hydrogels with less HMW content. Overall, this study describes how alginate hydrogels of varied molecular weight may be used to control S1P release kinetics for therapeutic applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 138-146, 2018.

Saposin B mobilizes lipids from cholesterol-poor and bis(monoacylglycero)phosphate-rich membranes at acidic pH. Unglycosylated patient variant saposin B lacks lipid-extraction capacity.

Sphingolipid activator proteins (SAPs), GM2 activator protein (GM2AP) and saposins (Saps) A-D are small, enzymatically inactive glycoproteins of the lysosome. Despite of their sequence homology, these lipid-binding and -transfer proteins show different specificities and varying modes of action. Water-soluble SAPs facilitate the degradation of membrane-bound glycosphingolipids with short oligosaccharide chains by exohydrolases at the membrane-water interface. There is strong evidence that degradation of endocytosed components of the cell membrane takes place at intraendosomal and intralysosomal membranes. The inner membranes of the lysosome differ from the limiting membrane of the organelle in some typical ways: the inner vesicular membranes lack a protecting glycocalix, and they are almost free of cholesterol, but rich in bis(monoacylglycero)phosphate (BMP), the anionic marker lipid of lysosomes. In this study, we prepared glycosylated Sap-B free of other Saps by taking advantage of the Pichia pastoris expression system. We used immobilized liposomes as a model for intralysosomal vesicular membranes to probe their interaction with recombinantly expressed Sap-B. We monitored this interaction using SPR spectroscopy and an independent method based on the release of radioactively labelled lipids from liposomal membranes. We show that, after initial binding, Sap-B disturbs the membrane structure and mobilizes the lipids from it. Lipid mobilization is dependent on an acidic pH and the presence of anionic lipids, whereas cholesterol is able to stabilize the liposomes. We also show for the first time that glycosylation of Sap-B is essential to achieve its full lipid-extraction activity. Removal of the carbohydrate moiety of Sap-B reduces its membrane-destabilizing quality. An unglycosylated Sap-B variant, Asn215His, which causes a fatal sphingolipid storage disease, lost the ability to extract membrane lipids at acidic pH in the presence of BMP.

* Injectable Graft Substitute Active on Bone Tissue Regeneration.

With the aim to obtain an injectable bioactive scaffold that can accelerate bone formation in sinus lift augmentation, in bony void and fracture repair, we have developed a three-dimensional (3D) jelly collagen containing lysophosphatidic acid (LPA) and 1α,25-dihydroxyvitamin D3 (1,25D3). Using an in vitro 3D culture model of bone fracture, we show that the contraction of the collagen gel is mediated by Rho-kinase activation in osteoblasts. The gel contraction showed dependence on cell concentration and was increased by LPA, which favored apposition and fastening of bone fragments approach. LPA was shown to act through actin cytoskeleton reorganization and myosin light chain phosphorylation of human primary osteoblasts (hOB). Moreover, LPA conferred osteoconductive properties as evidenced by the induction of proliferation, differentiation, and migration of hOB. The addition of 1,25D3 did not enhance cell-mediated gel contraction, but stimulated the maturation of hOB in vitro through the production of extracellular matrix of higher quality. On the basis of these observations, the collagen gel enriched with LPA and 1,25D3 described herein can be considered an injectable natural scaffold that allows the migration of cells from the side of bone defect and a promising candidate to accelerate bone growth and fracture healing.