Sodium Alginate- and Cationic Cellulose-Functionalized Polycaprolactone Nanofibers for In Vitro and Antibacterial Applications.

The use of polyelectrolytes is emerging as a fascinating strategy for the functionalization of biomedical membranes, due to their ability to enhance biological responses using the interaction effect of charged groups on multiple interface properties. Herein, two different polyelectrolytes were used to improve the antibacterial properties of polycaprolactone (PCL) nanofibers fabricated via electrospinning. First, a new cationic cellulose derivative, cellulose-bearing imidazolium tosylate (CIMD), was prepared via the nucleophilic substitution of the tosyl group using 1-methylimidazole, as confirmed by NMR analyses, and loaded into the PCL nanofibers. Secondly, sodium alginate (SA) was used to uniformly coat the fibers' surface via self-assembly, as remarked through SEM-EDX analyses. Polyelectrolyte interactions between the CIMD and the SA, initially detected using a FTIR analysis, were confirmed via Z potential measurements: the formation of a CMID/SA complex promoted a substantial charge neutralization of the fibers' surfaces with effects on the physical properties of the membrane in terms of water adsorption and in vitro degradation. Moreover, the presence of SA contributed to the in vitro response of human mesenchymal stem cells (hMSCs), as confirmed by a significant increase in the cells' viability after 7 days in the case of the PCL/CMID/SA complex with respect to the PCL and PCL/CMID membranes. Contrariwise, SA did not nullify the antibacterial effect of CMID, as confirmed by the comparable resistance exhibited by S. mutans, S. aureus, and E. coli to the PCL/CIMD and PCL/CIMD/SA membranes. All the reported results corroborate the idea that the CIMD/SA functionalization of PCL nanofibers has a great potential for the fabrication of efficient antimicrobial membranes for wound healing.

Biomimetic Alginate/Gelatin Cross-Linked Hydrogels Supplemented with Polyphosphate for Wound Healing Applications.

In the present study, the fabrication of a biomimetic wound dressing that mimics the extracellular matrix, consisting of a hydrogel matrix composed of non-oxidized and periodate-oxidized marine alginate, was prepared to which gelatin was bound via Schiff base formation. Into this alginate/oxidized-alginate-gelatin hydrogel, polyP was stably but reversibly integrated by ionic cross-linking with Zn2+ ions. Thereby, a soft hybrid material is obtained, consisting of a more rigid alginate scaffold and porous structures formed by the oxidized-alginate-gelatin hydrogel with ionically cross-linked polyP. Two forms of the Zn-polyP-containing matrices were obtained based on the property of polyP to form, at neutral pH, a coacervate-the physiologically active form of the polymer. At alkaline conditions (pH 10), it will form nanoparticles, acting as a depot that is converted at pH 7 into the coacervate phase. Both polyP-containing hydrogels were biologically active and significantly enhanced cell growth/viability and attachment/spreading of human epidermal keratinocytes compared to control hydrogels without any adverse effect on reconstructed human epidermis samples in an in vitro skin irritation test system. From these data, we conclude that polyP-containing alginate/oxidized-alginate-gelatin hydrogels may provide a suitable regeneratively active matrix for wound healing for potential in vivo applications.

Self-Healing Properties of Bioinspired Amorphous CaCO3/Polyphosphate-Supplemented Cement.

There is a strong interest in cement additives that are able to prevent or mitigate the adverse effects of cracks in concrete that cause corrosion of the reinforcement. Inorganic polyphosphate (polyP), a natural polymer that is synthesized by bacteria, even those on cement/concrete, can increase the resistance of concrete to progressive damage from micro-cracking. Here we use a novel bioinspired strategy based on polyP-stabilized amorphous calcium carbonate (ACC) to give this material self-healing properties. Portland cement was supplemented with ACC nanoparticles which were stabilized with 10% (w/w) Na-polyP. Embedding these particles in the hydrated cement resulted in the formation of calcite crystals after a hardening time of 10 days, which were not seen in controls, indicating that the particles dissolve and then transform into calcite. While there was no significant repair in the controls without ACC, almost complete closure of the cracks was observed after a 10 days healing period in the ACC-supplemented samples. Nanoindentation measurements on the self-healed crack surfaces showed a similar or slightly higher elasticity at a lower hardness compared to non-cracked surfaces. Our results demonstrate that bioinspired approaches, like the use of polyP-stabilized ACC shown here, can significantly improve the repair capacity of Portland cement.

Electrospinning of Bioactive Wound-Healing Nets.

The availability of appropriate dressings for treatment of wounds, in particular chronic wounds, is a task that still awaits better solutions than provided by currently applied materials. The method of electrospinning enables the fabrication of novel materials for wound dressings due to the high surface area and porosity of the electrospun meshes and the possibility to include bioactive ingredients. Recent results show that the incorporation of biologically active inorganic polyphosphate microparticles and microspheres and synergistically acting retinoids into electrospun polymer fibers yields biocompatible and antibacterial mats for potential dressings with improved wound-healing properties. The underlying principles and the mechanism of these new approaches in the therapy wounds, in particular wounds showing impaired healing, as well as for further applications in skin regeneration/repair, are summarized.

Amorphous Ca²âº polyphosphate nanoparticles regulate the ATP level in bone-like SaOS-2 cells.

Polyphosphate (polyP) is a physiologically occurring polyanion that is synthesized especially in bone-forming osteoblast cells and blood platelets. We used amorphous polyP nanoparticles, complexed with Ca(2+), that have a globular size of ∼100 nm. Because polyP comprises inorganic orthophosphate units that are linked together through high-energy phosphoanhydride bonds, we questioned whether the observed morphogenetic effect, elicited by polyP, is correlated with the energy-generating machinery within the cells. We show that exposure of SaOS-2 osteoblast-like cells to polyP results in a strong accumulation of mitochondria and a parallel translocation of the polyP-degrading enzyme alkaline phosphatase to the cell surface. If SaOS-2 cells are activated by the mineralization activation cocktail (comprising ß-glycerophosphate, ascorbic acid and dexamethasone) and additionally incubated with polyP, a tenfold intracellular increase of the ATP level occurs. Even more, in those cells, an intensified release of ATP into the extracellular space is also seen. We propose and conclude that polyP acts as metabolic fuel after the hydrolytic cleavage of the phosphoanhydride linkages, which contributes to hydroxyapatite formation on the plasma membranes of osteoblasts.

Development of collagen-chitosan dressing gel functionalized with propolis-zinc oxide nanoarchitectonics to accelerate wound healing.

Using an in situ sol-gel technique, new nanoarchitectonics of propolis loaded zinc oxide nanoarchitectonics (PP/ZnO-NPs) were developed in order to improve the in vivo outcomes of collagen-chitosan gel in wounded rats. The obtained nanoarchitectonics were fully characterized. The XRD results indicate the presence of a Zincite phase for ZnO-NPs and Zincite accompanied by a minor amount of zinc hydroxide for PP/ZnO-NPs samples. While the TEM findings illustrate the transfer of the ZnO-NPs from agglomerated spheres with an average particle size of 230 ± 29 nm to needle-like NPs of 323 ± 173 nm length (PP1/ZnO-NPs) and to a sheet-like NPs of 500 ± 173 nm diameter (PP2/ZnO-NPs). In addition, the incorporation of PP results in an increase in the surface negativity of ZnO-NPs to -31.4 ± 6.4 mV for PP2/ZnO-NPs. The antimicrobial activities of the nanocomposite gel loaded with 10%PP1/ZnO-NPs (G6) revealed the highest inhibition zone against E. coli (26 ± 2.31 mm). Remarkably, the in vivo outcomes showed that the nanocomposite gel (G6) has exceptional collagen deposition, quick wound closure rates, and re-epithelization. The outcomes demonstrate the nanocomposite gel encouraging biological properties for the treatment of damaged and infected wounds.

Egyptian propolis extract for functionalization of cellulose nanofiber/poly(vinyl alcohol) porous hydrogel along with characterization and biological applications.

Bee propolis is one of the most common natural extracts and has gained significant interest in biomedicine due to its high content of phenolic acids and flavonoids, which are responsible for the antioxidant activity of natural products. The present study report that the propolis extract (PE) was produced by ethanol in the surrounding environment. The obtained PE was added at different concentrations to cellulose nanofiber (CNF)/poly(vinyl alcohol) (PVA), and subjected to freezing thawing and freeze drying methods to develop porous bioactive matrices. Scanning electron microscope (SEM) observations displayed that the prepared samples had an interconnected porous structure with pore sizes in the range of 10-100 µm. The high performance liquid chromatography (HPLC) results of PE showed around 18 polyphenol compounds, with the highest amounts of hesperetin (183.7 µg/mL), chlorogenic acid (96.9 µg/mL) and caffeic acid (90.2 µg/mL). The antibacterial activity results indicated that both PE and PE-functionalized hydrogels exhibited a potential antimicrobial effects against Escherichia coli, Salmonella typhimurium, Streptococcus mutans, and Candida albicans. The in vitro test cell culture experiments indicated that the cells on the PE-functionalized hydrogels had the greatest viability, adhesion, and spreading of cells. Altogether, these data highlight the interesting effect of propolis bio-functionalization to enhance the biological features of CNF/PVA hydrogel as a functional matrix for biomedical applications.

A multicompartment vascular implant of electrospun wintergreen oil/ polycaprolactone fibers coated with poly(ethylene oxide).

BACKGROUND: The aim of the present study was to fabricate double layered scaffolds of electrospun polycaprolactone (PCL) and poly(ethylene oxide) (PEO). The electrospun PCL fibers were functionalized with wintergreen oil (WO) as a novel approach to prevent vascular grafts failure due to thrombosis by adjusting biomaterial-blood interactions. METHODS: PCL tubular scaffolds were prepared by electrospinning approach and coated with PEO as a hydrophilic polymer. The single and double layered scaffolds were characterized in terms of their morphological, chemical properties -as well as-hemocompatibility assays (i.e. prothrombin time, hemolysis percentage and platelets adhesion). Moreover, the antioxidant potential of WO-PCL samples were measured by 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) free radical assay. RESULTS: The results demonstrated that incorporation of WO during the electrospinning process decreased the PCL fiber diameter. In addition, the prothrombine time assay shows that WO could be used to lower the electrospun PCL fiber tendency to induce blood clotting. Moreover, SEM observations of platelets adhesion of both single and double layered PCL/PEO scaffolds fiber shows an increase of platelets number, compared with the scaffolds containing WO. CONCLUSIONS: The antioxidant potential and blood compatibility measurements of WO-PCL/PEO samples highlight the approach made so far as an ideal synthetic small size vascular grafts to overcome autogenous grafts shortages and drawbacks.

The biomaterial polyphosphate blocks stoichiometric binding of the SARS-CoV-2 S-protein to the cellular ACE2 receptor.

The effect of the polyanionic polymer of inorganic polyphosphate (polyP) involved in innate immunity on the binding of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein to the cellular ACE2 receptor was studied. The RBD surface comprises a basic amino acid stretch of four arginine residues which interact with the physiological polyP (polyP40) and polyP3. Subsequently, the interaction of RBD with ACE2 is sensitively inhibited. After the chemical modification of arginine, an increased inhibition by polyP, at a 1 : 1 molar ratio (polyP : RBP), is measured already at 0.1 µg mL-1. Heparin was ineffective. The results suggest a potential therapeutic benefit of polyP against SARS-CoV-2 infection.

Nanoparticle-directed and ionically forced polyphosphate coacervation: a versatile and reversible core-shell system for drug delivery.

A drug encapsulation/delivery system using a novel principle is described that is based on an intra-particle migration of calcium ions between a central Ca2+-enriched nanoparticle core and the surrounding shell compartment. The supply of Ca2+ is needed for the formation of a coacervate shell around the nanoparticles, acting as the core of drug-loadable core-shell particles, using the physiological inorganic polymer polyphosphate (polyP). This polyanion has the unique property to form, at an alkaline pH and in the presence of a stoichiometric surplus of calcium ions, water-insoluble and stabile amorphous nanoparticles. At neutral pH a coacervate, the biologically active form of the polymer, is obtained that is composed of polyP and Ca2+. The drug-loaded core-shell particles, built from the Ca-polyP core and the surrounding Ca-polyP shell, were fabricated in two successive steps. First, the formation of the nanoparticle core at pH 10 and a superstoichiometric 2:1 molar ratio between CaCl2 and Na-polyP into which dexamethasone, as a phosphate derivative, was incorporated. Second, the preparation of the coacervate shell, loaded with ascorbic acid, by exposure of the Ca-polyP core to soluble Na-polyP and L-ascorbate (calcium salt). EDX analysis revealed that during this step the Ca2+ ions required for coacervate formation migrate from the Ca-polyP core (with a high Ca:P ratio) to the shell. Electron microscopy of the particles show an electron-dense 150-200 nm sized core surrounded by a less sharply delimited electron-sparse shell. The core-shell particles exhibited strong osteogenic activity in vitro, based on the combined action of polyP and of dexamethasone and ascorbic acid, which reversibly bind to the anionic polyP via ionic Ca2+ bonds. Drug release from the particles occurs after contact with a peptide/protein-containing serum, a process which is almost complete after 10 days and accompanied by the conversion of the nanoparticles into a coacervate. Human osteosarcoma SaOS-2 cells cultivated onto or within an alginate hydrogel matrix showed increased growth/viability and mineralization when the hybrid particles containing dexamethasone and ascorbic acid were embedded in the matrix. The polyP-based core-shell particles have the potential to become a suitable, pH-responsive drug encapsulation/release system, especially for bone, cartilage and wound healing.

Amorphous Polyphosphate and Ca-Carbonate Nanoparticles Improve the Self-Healing Properties of both Technical and Medical Cements.

Cement is used both as a construction material and for medical applications. Previously, it has been shown that the physiological polymer inorganic polyphosphate (polyP) is morphogenetically active in regeneration of skin, bone, and cartilage. The present study investigates the question if this polymer is also a suitable additive to improve the self-healing capacity not only of construction cement but also of inorganic bone void fillers. For the application in the cement, two different polyP-based amorphous nanoparticles (NP) are prepared, amorphous Ca-polyP NP and amorphous Ca-carbonate (ACC) NP. The particles are integrated into poly(methyl methacrylate) in a concentration ratio of 1:10. This material applied onto Portland cement blocks either by brush application or by blow spinning strongly accelerates the self-healing property of the cement after a 10 day incubation period. Most likely, this process depends on bacteria and their membrane-associated alkaline phosphatase, resulting in the formation of calcite from ACC. In a second approach, polyP is integrated into a calcium-silicate-based cement used in reconstitutive medicine. Subsequently, the cement becomes softer and more elastic. The data show that bioinspired polyP/ACC NP are suitable additives to improve the self-healing of construction cement and to biologize bone cement.

Biomimetic transformation of polyphosphate microparticles during restoration of damaged teeth.

OBJECTIVE: In the present study, we investigated the fusion process between amorphous microparticles of the calcium salt of the physiological polymer comprising orthophosphate units, of inorganic polyphosphate (polyP), and enamel. METHODS: This polymer was incorporated as an ingredient into toothpaste and the fusion process was studied by electron microscopy and by synchrotron-based X-ray tomography microscopy (SRXTM) techniques. RESULTS: The data showed that toothpaste, supplemented with the amorphous Ca-polyP microparticles (aCa-polyP-MP), not only reseals tooth defects on enamel, like carious lesions, and dentin, including exposed dentinal tubules, but also has the potential to induce re-mineralization in the enamel and dentin regions. The formation of a regeneration mineralic zone on the tooth surface induced by aCa-polyP-MP was enhanced upon exposure to artificial saliva, as demonstrated by SRXTM. Energy dispersive X-ray analysis revealed an increase in the calcium/phosphorus atomic ratio of the enamel deposits to values characteristic for the particles during the treatment with polyP applied in the toothpaste, indicating a fusion of the particles with the tooth mineral. SIGNIFICANCE: Our results suggest that toothpaste enriched with aCa-polyP-MP is a promising biomimetic material for accelerating enamel and dentin restoration.

Bifunctional dentifrice: Amorphous polyphosphate a regeneratively active sealant with potent anti-Streptococcus mutans activity.

OBJECTIVE: In this study we demonstrate that inorganic polyphosphate (polyP) exhibits a dual protective effect on teeth: it elicits a strong antibacterial effect against the cariogenic bacterium Streptococcus mutans and, in form of amorphous calcium polyP microparticles (size of 100-400nm), it efficiently reseals cracks/fissures in the tooth enamel and dentin. METHODS: Three different formulations of amorphous polyP microparticles (Ca-polyP, Zn-polyP and Sr-polyP) were prepared. RESULTS: Among the different polyP microparticles tested, the Ca-polyP microparticles, as a component of a newly developed formulation of a dentifrice, turned out to be most effective in inhibiting growth of S. mutans. Further studies have shown that it is mainly the soluble polyP, which has a strong antibacterial activity, either given as sodium salt of polyP or formed by partial disintegration of the microparticles via the alkaline phosphatase present in the oropharyngeal cavity. In addition, we demonstrate that the developed toothpaste containing incorporated amorphous polyP microparticles, efficiently reduces dental biofilm formation. SIGNIFICANCE: From our results we conclude that polyP microparticles, if added to toothpaste in an amorphous state, might be beneficial not only for restoring tooth damages but also because they provide a suitable depot of functionally/antibacterially active soluble polyP.

A Novel Biomimetic Approach to Repair Enamel Cracks/Carious Damages and to Reseal Dentinal Tubules by Amorphous Polyphosphate.

Based on natural principles, we developed a novel toothpaste, containing morphogenetically active amorphous calcium polyphosphate (polyP) microparticles which are enriched with retinyl acetate ("a-polyP/RA-MP"). The spherical microparticles (average size, 550 ± 120 nm), prepared by co-precipitating soluble Na-polyP with calcium chloride and supplemented with retinyl acetate, were incorporated into a base toothpaste at a final concentration of 1% or 10%. The "a-polyP/RA-MP" ingredient significantly enhanced the stimulatory effect of the toothpaste on the growth of human mesenchymal stem cells (MSC). This increase was paralleled by an upregulation of the MSC marker genes for osteoblast differentiation, collagen type I and alkaline phosphatase. In addition, polyP, applied as Zn-polyP microparticles ("Zn-a-polyP-MP"), showed a distinct inhibitory effect on growth of Streptococcus mutans, in contrast to a toothpaste containing the broad-spectrum antibiotic triclosan that only marginally inhibits this cariogenic bacterium. Moreover, we demonstrate that the "a-polyP/RA-MP"-containing toothpaste efficiently repairs cracks/fissures in the enamel and dental regions and reseals dentinal tubules, already after a five-day treatment (brushing) of teeth as examined by SEM (scanning electron microscopy) and semi-quantitative EDX (energy-dispersive X-ray spectroscopy). The occlusion of the dentin cracks by the microparticles turned out to be stable and resistant against short-time high power sonication. Our results demonstrate that the novel toothpaste prepared here, containing amorphous polyP microparticles enriched with retinyl acetate, is particularly suitable for prevention/repair of (cariogenic) damages of tooth enamel/dentin and for treatment of dental hypersensitivity. While the polyP microparticles function as a sealant for dentinal damages and inducer of remineralization processes, the retinyl acetate acts as a regenerative stimulus for collagen gene expression in cells of the surrounding tissue, the periodontium.

Amorphous polyphosphate-hydroxyapatite: A morphogenetically active substrate for bone-related SaOS-2 cells in vitro.

There is increasing evidence that inorganic calcium-polyphosphates (polyP) are involved in human bone hydroxyapatite (HA) formation. Here we investigated the morphology of the particles, containing calcium phosphate (CaP) with different concentrations of various Na-polyP concentrations, as well as their effects in cell culture. We used both SaOS-2 cells and human mesenchymal stem cells. The polymeric phosphate readily binds calcium ions under formation of insoluble precipitates. We found that addition of low concentrations of polyP (<10wt.%, referred to the CaP deposits) results in an increased size of the HA crystals. Surprisingly, at higher polyP concentrations (>10wt.%) the formation of crystalline HA is prevented and amorphous polyP/HA hybrid particles with a size of ≈50nm are formed, most likely consisting of polyP molecules linked via Ca(2+) bridges to the surface of the CaP deposits. Further studies revealed that the polyP-CaP particles cause a strong upregulation of the expression of the genes encoding for two marker proteins of bone formation, collagen type I and alkaline phosphatase. Based on their morphogenetic activity the amorphous polyP-CaP particles offer a promising material for the development of bone implants, formed from physiological inorganic precursors/polymers. STATEMENT OF SIGNIFICANCE: Hydroxyapatite (HA) is a naturally occurring mineral of vertebrate bone. Natural HA, a bio-ceramic material which is crystalline to different scale, has been used as a biomaterial to fabricate scaffolds for in situ bone regeneration and other tissue engineering purposes. In contrast to natural HA, synthetic apatite is much less effective. In general, while HA is bioactive, its interaction and biocompatibility with existing bone tissue is low. These properties have been attributed to a minimal degradability in the physiological environment. In the present study we introduce a new Ca-phosphate (CaP) fabrication technology, starting from calcium chloride and dibasic ammonium phosphate with the HA characteristic Ca/P molar ratio of 10:6 and report that after addition >10% (by weight) of polyphosphate (polyP) amorphous CaP/HA samples were obtained. Those samples elicits strong morphogenetic activity let us to conclude that polyP/HA-based material might be beneficial for application as bone substitute implant.

A biomimetic approach to ameliorate dental hypersensitivity by amorphous polyphosphate microparticles.

OBJECTIVE: Dental hypersensitivity has become one of the most common and most costly diseases in the world, even though those maladies are very rarely life threatening. Using amorphous microparticles, fabricated from the natural polymer (polyphosphate), we intend to reseal the dentinal tubules exposed and reduce by that the hypersensitivity. METHODS: Amorphous microparticles (termed aCa-polyP-MP) were prepared from Na-polyphosphate (polyP) and CaCl2, then incubated with human teeth. The potential of the microparticles to plug the dentinal tubules was determined by microscopic and spectroscopic techniques. RESULTS: We demonstrate that, in contrast to polyP, the aCa-polyP-MP efficiently reseal dentinal tubules exposed at the tooth surface. Scanning electron microscopical (SEM) and energy dispersive X-ray spectroscopic (EDX) studies showed that the tooth cement and dentin surfaces, incubated with aCa-polyP-MP, form a nearly homogenous, approximately 50-µm thick solid polyP layer on the tooth cement and dentin surfaces, while no coating on the tooth surface, incubated with Na-polyP [Ca(2+)], was observed. Determination of the mechanical properties of the polyP coating revealed a Martens hardness of 3.85±0.64GPa and a reduced elastic modulus of 94.72±8.54GPa already after a 3h exposure to the aCa-polyP-MP, which become close to those of the natural enamel (4.33±0.69GPa and 101.61±8.52GPa, respectively) after prolonged incubation periods. In addition, aCa-polyP-MP turned out to display morphogenetic activity. Incubation of precursor odontoblasts cultures in the presence of aCa-polyP-MP resulted in a 7-fold increase of the steady-state-expression level of the gene encoding for the alkaline phosphatase (ALP) during a 7 d incubation period. SIGNIFICANCE: Ca-polyP microparticles, consisting of the biocompatible natural polymer polyP, provide a potential sealing material for dentinal tubules on the tooth surface.

Amorphous polyphosphate/amorphous calcium carbonate implant material with enhanced bone healing efficacy in a critical-size defect in rats.

In this study the effect of amorphous calcium carbonate (ACC) microparticles and amorphous calcium polyphosphate (polyP) microparticles (termed aCa-polyP-MP) on bone mineral forming cells/tissue was investigated in vitro and in vivo. The ACC particles (termed ACC-P10-MP) were prepared in the presence of Na-polyP. Only the combinations of polyP and ACC microparticles enhanced the proliferation rate of human mesenchymal stem cells (MSCs). Gene expression studies revealed that ACC causes an upregulation of the expression of the cell membrane-associated carbonic anhydrase IX (CA IX; formation of ACC), while the transcript level of the alkaline phosphatase (ALP; liberation of orthophosphate from polyP) changes only relatively little. In contrast, aCa-polyP-MP primarily induces ALP expression. If both components are applied together a strong stimulation of expression of both marker genes is observed. In order to investigate whether ACC also enhances bone regeneration induced by polyP in vivo, the particles were encapsulated into PLGA (poly(d,l-lactide-co-glycolide)) microspheres (diameter ~800 µm) and implanted into rat critical-size calvarial defects. The studies revealed that animals that received aCa-polyP-MP microspheres showed an increased rate of regeneration compared to ß-tri-calcium phosphate (ß-TCP) controls. This effect is even accelerated if microspheres with both aCa-polyP-MP and ACC-P10-MP (1 : 1 weight ratio) are applied, resulting in an almost complete restoration of the defect area after 12 weeks. qRT-PCR analyses of tissue sections through the regeneration zone with microspheres containing both aCa-polyP-MP and ACC-P10-MP revealed a significantly higher upregulation of expression of the marker genes compared to each of the components alone. The Young's moduli for microspheres containing aCa-polyP-MP (1.74 MPa) and aCa-polyP-MP/ACC-P10-MP (2.38 MPa) were markedly higher compared to ß-TCP-controls (0.63 mPa). Our results show that the combined application of ACC and Ca-polyP (both in the amorphous state) opens new strategies for the development of regenerative implants for the reconstruction of bone defects.

Modular Small Diameter Vascular Grafts with Bioactive Functionalities.

We report the fabrication of a novel type of artificial small diameter blood vessels, termed biomimetic tissue-engineered blood vessels (bTEBV), with a modular composition. They are composed of a hydrogel scaffold consisting of two negatively charged natural polymers, alginate and a modified chitosan, N,O-carboxymethyl chitosan (N,O-CMC). Into this biologically inert scaffold two biofunctionally active biopolymers are embedded, inorganic polyphosphate (polyP) and silica, as well as gelatin which exposes the cell recognition signal, Arg-Gly-Asp (RGD). These materials can be hardened by exposure to Ca(2+) through formation of Ca(2+) bridges between the polyanions, alginate, N,O-CMC, and polyP (alginate-Ca(2+)-N,O-CMC-polyP). The bTEBV are formed by pressing the hydrogel through an extruder into a hardening solution, containing Ca(2+). In this universal scaffold of the bTEBV biomaterial, polycations such as poly(L-Lys), poly(D-Lys) or a His/Gly-tagged RGD peptide (three RGD units) were incorporated, which promote the adhesion of endothelial cells to the vessel surface. The mechanical properties of the biopolymer material (alginate-Ca(2+)-N,O-CMC-polyP-silica) revealed a hardness (elastic modulus) of 475 kPa even after a short incubation period in CaCl2 solution. The material of the artificial vascular grafts (bTEBVs with an outer size 6 mm and 1.8 mm, and an inner diameter 4 mm and 0.8 mm, respectively) turned out to be durable in 4-week pulsatile flow experiments at an alternating pressure between 25 and 100 mbar (18.7 and 75.0 mm Hg). The burst pressure of the larger (smaller) vessels was 850 mbar (145 mbar). Incorporation of polycationic poly(L-Lys), poly(D-Lys), and especially the His/Gly-tagged RGD peptide, markedly increased the adhesion of human, umbilical vein/vascular endothelial cells, EA.HY926 cells, to the surface of the hydrogel. No significant effect of the polyP samples on the clotting of human plasma is measured. We propose that the metabolically degradable polymeric scaffold bTEBV is a promising biomaterial for future prosthetic vascular grafts.

Biosilica-loaded poly(ϵ-caprolactone) nanofibers mats provide a morphogenetically active surface scaffold for the growth and mineralization of the osteoclast-related SaOS-2 cells.

Bioprinting/3D cell printing procedures for the preparation of scaffolds/implants have the potential to revolutionize regenerative medicine. Besides biocompatibility and biodegradability, the hardness of the scaffold material is of critical importance to allow sufficient mechanical protection and, to the same extent, allow migration, cell-cell, and cell-substrate contact formation of the matrix-embedded cells. In the present study, we present a strategy to encase a bioprinted, cell-containing, and soft scaffold with an electrospun mat. The electrospun poly(ϵ-caprolactone) (PCL) nanofibers mats, containing tetraethyl orthosilicate (TEOS), were subsequently incubated with silicatein. Silicatein synthesizes polymeric biosilica by polycondensation of ortho-silicate that is formed from prehydrolyzed TEOS. Biosilica provides a morphogenetically active matrix for the growth and mineralization of osteoblast-related SaOS-2 cells in vitro. Analysis of the microstructure of the 300-700 nm thick PCL/TEOS nanofibers, incubated with silicatein and prehydrolyzed TEOS, displayed biosilica deposits on the mats formed by the nanofibers. We conclude and propose that electrospun PCL nanofibers mats, coated with biosilica, may represent a morphogenetically active and protective cover for bioprinted cell/tissue-like units with a suitable mechanical stability, even if the cells are embedded in a softer matrix.

Effect of bioglass on growth and biomineralization of SaOS-2 cells in hydrogel after 3D cell bioprinting.

We investigated the effect of bioglass (bioactive glass) on growth and mineralization of bone-related SaOS-2 cells, encapsulated into a printable and biodegradable alginate/gelatine hydrogel. The hydrogel was supplemented either with polyphosphate (polyP), administered as polyP • Ca2+-complex, or silica, or as biosilica that had been enzymatically prepared from ortho-silicate by silicatein. These hydrogels, together with SaOS-2 cells, were bioprinted to computer-designed scaffolds. The results revealed that bioglass (nano)particles, with a size of 55 nm and a molar ratio of SiO2 : CaO : P2O5 of 55 : 40 : 5, did not affect the growth of the encapsulated cells. If silica, biosilica, or polyP • Ca2+-complex is co-added to the cell-containing alginate/gelatin hydrogel the growth behavior of the cells is not changed. Addition of 5 mg/ml of bioglass particles to this hydrogel significantly enhanced the potency of the entrapped SaOS-2 cells to mineralize. If compared with the extent of the cells to form mineral deposits in the absence of bioglass, the cells exposed to bioglass together with 100 µmoles/L polyP • Ca2+-complex increased their mineralization activity from 2.1- to 3.9-fold, or with 50 µmoles/L silica from 1.8- to 2.9-fold, or with 50 µmoles/L biosilica from 2.7- to 4.8-fold or with the two components together (100 µmoles/L polyP • Ca2+-complex and 50 µmoles/L biosilica) from 4.1- to 6.8-fold. Element analysis by EDX spectrometry of the mineral nodules formed by SaOS-2 revealed an accumulation of O, P, Ca and C, indicating that the mineral deposits contain, besides Ca-phosphate also Ca-carbonate. The results show that bioglass added to alginate/gelatin hydrogel increases the proliferation and mineralization of bioprinted SaOS-2 cells. We conclude that the development of cell-containing scaffolds consisting of a bioprintable, solid and cell-compatible inner matrix surrounded by a printable hard and flexible outer matrix containing bioglass, provide a suitable strategy for the fabrication of morphogenetically active and biodegradable implants.