Meik Neufurth

A new printable and durable N,O-carboxymethyl chitosan–Ca2+–polyphosphate complex with morphogenetic activity

Biomimetic materials have been gaining increasing importance in tissue engineering since they may provide regenerative alternatives to the use of autologous tissues for transplantation. In the present study, we applied for bioprinting of a functionalized three-dimensional template, N,O-carboxymethyl chitosan (N,O-CMC), mimicking the physiological extracellular matrix. This polymer, widely used in tissue engineering, has been provided with functional activity by integration of polyphosphate (polyP), an osteogenically acting natural polymer. The two polymers, N,O-CMC and polyP, are linked together via Ca2+ bridges. This N,O-CMC + polyP material was proven to be printable and durable. The N,O-CMC + polyP printed layers and tissue units retain their properties to induce SaOS-2 bone-like cells to biomineralization. Subsequent in vivo experiments revealed a strong regeneration-inducing activity of the material in the rat calvarial defect model. In turn, N,O-CMC + polyP represents a promising hybrid material useful as a potential custom-designed scaffold for alternative tissue-engineering solutions.

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.

Modulation of the initial mineralization process of SaOS-2 cells by carbonic anhydrase activators and polyphosphate.

Ca-phosphate/hydroxyapatite (HA) crystals constitute the mineral matrix of vertebrate bones, while Ca-carbonate is the predominant mineral of many invertebrates, like mollusks. Recent results suggest that CaCO3 is also synthesized during early bone formation. We demonstrate that carbonic anhydrase-driven CaCO3 formation in vitro is activated by organic extracts from the demosponge Suberites domuncula as well as by quinolinic acid, one component isolated from these extracts. Further results revealed that the stimulatory effect of bicarbonate (HCO3−) ions on mineralization of osteoblast-like SaOS-2 cells is strongly enhanced if the cells are exposed to inorganic polyphosphate (polyP), a linear polymer of phosphate linked by energy-rich phosphodiester bonds. The effect of polyP, administered as polyP (Ca2+ salt), on HA formation was found to be amplified by addition of the carbonic anhydrase-activating sponge extract or quinolinic acid. Our results support the assumption that CaCO3 deposits, acting as bio-seeds for Ca-carbonated phosphate formation, are formed as an intermediate during HA mineralization and that the carbonic anhydrase-mediated formation of those deposits is under a positive–negative feedback control by bone alkaline phosphatase-dependent polyP metabolism, offering new targets for therapy of bone diseases/defects.

The enzyme carbonic anhydrase as an integral component of biogenic Ca‐carbonate formation in sponge spicules

The inorganic scaffold of the spicules, the skeletal elements of the calcareous sponges, is formed of calcium carbonate (CaCO3). The growth of the approximately 300‐μm large spicules, such as those of the calcareous sponge Sycon raphanus used in the present study, is a rapid process with a rate of about 65 μm/h. The formation of CaCO3 is predominantly carried out by the enzyme carbonic anhydrase (CA). The enzyme from the sponge S. raphanus was isolated and prepared by recombination. The CA‐driven deposition of CaCO3 crystallites is dependent on temperature (optimal at 52 °C), the pH value of the reaction assay (7.5/8.0), and the substrate concentration (CO2 and Ca2+). During the initial phase of crystallite formation, ≈40 μm large round‐shaped deposits are formed that remodel to larger prisms. These crystal‐like prisms associate to each other and form either rope‐/bundle‐like aggregates or arrange perfectly with their smaller planes along opposing surfaces of the sponge spicule rays. The CA‐dependent CaCO3 deposition can be inhibited by the CA‐specific inhibitor acetazolamide. The Michaelis–Menten constant for the CA‐driven mineralization has been determined to be around 8 mM with respect to CaCO3. The deposits formed have a Martens hardness of ≈5 GPa. The data presented here highlights for the first time that calcite deposition in the sponge system is decisively controlled enzymatically. This data will contribute to the development of new strategies applicable for the fabrication of novel biomaterials.

Nonenzymatic Transformation of Amorphous CaCO3 into Calcium Phosphate Mineral after Exposure to Sodium Phosphate in Vitro: Implications for in Vivo Hydroxyapatite Bone Formation

Studies indicate that mammalian bone formation is initiated at calcium carbonate bioseeds, a process that is driven enzymatically by carbonic anhydrase (CA). We show that amorphous calcium carbonate (ACC) and bicarbonate (HCO3−) cause induction of expression of the CA in human osteogenic SaOS‐2 cells. The mineral deposits formed on the surface of the cells are rich in C, Ca and P. FTIR analysis revealed that ACC, vaterite, and aragonite, after exposure to phosphate, undergo transformation into calcium phosphate. This exchange was not seen for calcite. The changes to ACC, vaterite, and aragonite depended on the concentration of phosphate. The rate of incorporation of phosphate into ACC, vaterite, and aragonite, is significantly accelerated in the presence of a peptide rich in aspartic acid and glutamic acid. We propose that the initial CaCO3 bioseed formation is driven by CA, and that the subsequent conversion to calcium phosphate/calcium hydroxyapatite (exchange of carbonate by phosphate) is a non‐enzymatic exchange process.

The sponge silicatein-interacting protein silintaphin-2 blocks calcite formation of calcareous sponge spicules at the vaterite stage

Ca-carbonate, the inorganic matrix of the spicules from the calcareous sponges, is formed as the result of an enzyme-catalyzed reaction with the carbonic anhydrase [CA] as a decisive component. The growth and the morphology of the spicules are genetically controlled, and are taxon-specific. In the present study it is shown that the silicatein-interacting protein silintaphin-2 is present at the surface of the siliceous spicules of the demosponge Suberites domuncula and prevents the association of calcareous crystals synthesized in vitro to these skeletal elements. Silintaphin-2 comprises a Ca2+-binding domain that is formed by a 22 amino acid-long peptide, N-DDDSQGEIQSDMAEEEDDDNVD-C. This very acidic 22-meric peptide, termed D/E-peptide, is shown to decelerate the in vitro synthesis of calcite through blocking the transformation of amorphous Ca-carbonate to the crystalline morph calcite at the level of vaterite. This effect is seen at a molar ratio of D/E-peptide : Ca2+ of 1 : 5000. During the deposition of Ca-carbonate the peptide becomes incorporated into the crystallites. Determinations of the mechanical characteristics of the formed Ca-carbonate deposits revealed a hardness of 1.98 ± 0.31 GPa (calcite) and 1.38 ± 0.39 GPa (vaterite), an elastic modulus of 72.83 ± 11.68 GPa (calcite) and 39.13 ± 8.04 GPa (vaterite) and a creep of 5.44 ± 1.15 (calcite) and 9.95 ± 1.60 (vaterite) per maximal depth (%). It is concluded that the D/E-peptide interferes with the Ca2+ ions within the growing vaterite crystals and freezes this unstable phase formed during calcite formation. It is postulated that proteins, like silintaphin-2 with its Ca2+-binding domain D/E-peptide, are involved in the taxon-specific control of the synthesis of the inorganic matrix of the sponge spicules.

Enzyme-accelerated and structure-guided crystallization of calcium carbonate: Role of the carbonic anhydrase in the homologous system

The calcareous spicules from sponges, e.g. from Sycon raphanus, are composed of almost pure calcium carbonate. In order to elucidate the formation of those structural skeletal elements, the function of the enzyme carbonic anhydrase (CA), isolated from this species, during the in vitro calcium carbonate-based spicule formation, was investigated. It is shown that the recombinant sponge CA substantially accelerates calcium carbonate formation in the in vitro diffusion assay. A stoichiometric calculation revealed that the turnover rate of the sponge CA during the calcification process amounts to 25 CO2s(-1) × molecule CA(-1). During this enzymatically driven process, initially pat-like particles are formed that are subsequently transformed to rhomboid/rhombohedroid crystals with a dimension of ~50 μm. The CA-catalyzed particles are smaller than those which are formed in the absence of the enzyme. The Martens hardness of the particles formed is ~4 GPa, a value which had been determined for other biogenic calcites. This conclusion is corroborated by energy-dispersive X-ray spectroscopy, which revealed that the particles synthesized are composed predominantly of the elements calcium, oxygen and carbon. Surprising was the finding, obtained by light and scanning electron microscopy, that the newly formed calcitic crystals associate with the calcareous spicules from S. raphanus in a highly ordered manner; the calcitic crystals almost perfectly arrange in an array orientation along the two opposing planes of the spicules, leaving the other two plane arrays uncovered. It is concluded that the CA is a key enzyme controlling the calcium carbonate biomineralization process, which directs the newly formed particles to existing calcareous spicular structures. It is expected that with the given tools new bioinspired materials can be fabricated.

Polyphosphate as a donor of high-energy phosphate for the synthesis of ADP and ATP

ABSTRACT Here, we studied the potential role of inorganic polyphosphate (polyP) as an energy source for ADP and ATP formation in the extracellular space. In SaOS-2 cells, we show that matrix vesicles are released into the extracellular space after incubation with polyP. These vesicles contain both alkaline phosphatase (ALP) and adenylate kinase (AK) activities (mediated by ALPL and AK1 enzymes). Both enzymes translocate to the cell membrane in response to polyP. To distinguish the process(es) of AMP and ADP formation during ALP hydrolysis from the ATP generated via the AK reaction, inhibition studies with the AK inhibitor A(5′)P5(5′)A were performed. We found that ADP formation in the extracellular space occurs after enzymatic ATP synthesis. After exposure to polyP, a significant increase of the ADP level was observed, which is likely to be been catalyzed by ALP. This increase is not due to an intensified ATP release via exocytosis. The ATP level in the extracellular space of SaOS-2 cells is strongly increased in response to polyP, very likely mediated by the AK. We propose that the ALP and AK enzymes are involved in the extracellular ADP and ATP synthesis. Summary: Polyphosphate is a prime candidate for the compound that serves as a donor for the formation of extracellular ‘high-energy’ bonds during the formation of extracellular ATP and ADP, and hence can act as a metabolic fuel.

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 β-tri-calcium phosphate (β-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 Youngs 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 β-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.

3D printing of hybrid biomaterials for bone tissue engineering: Calcium-polyphosphate microparticles encapsulated by polycaprolactone

Here we describe the formulation of a morphogenetically active bio-ink consisting of amorphous microparticles (MP) prepared from Ca2+ and the physiological inorganic polymer, polyphosphate (polyP). Those MP had been fortified by mixing with poly-ε-caprolactone (PCL) to allow 3D-bioprinting. The resulting granular PCL/Ca-polyP-MP hybrid material, liquefied by short-time heating to 100 °C, was used for the 3D-printing of tissue-like scaffolds formed by strands with a thickness of 400 µm and a stacked architecture leaving ≈0.5 mm2-sized open holes enabling cell migration. The printed composite scaffold turned out to combine suitable biomechanical properties (Youngs modulus of 1.60 ± 0.1 GPa; Martens hardness of 153 ± 28 MPa), matching those of cortical and trabecular bone, with morphogenetic activity. This scaffold was capable of attracting and promoting the growth of human bone-related SaOS-2 cells as demonstrated by staining for cell viability (Calcein AM), cell density (DRAQ5) and SEM studies. Furthermore, the hybrid material was demonstrated to upregulate the steady-state-expression of the cell migration-inducing chemokine SDF-1α. EDX analysis and FTIR measurements revealed the presence of hydroxyapatite in the mineral deposits formed on the scaffold surface. Based on the results we conclude that granular PCL/Ca-polyP-MP hybrid material is suitable for the fabrication of bioprintable scaffold which comprises not only biomechanical stability but also morphogenetic potential. STATEMENT OF SIGNIFICANCE In present-day regenerative engineering efforts, biomaterial- and cell-based strategies are proposed that meet the required functional and spatial characteristics and variations, especially in the transition regions between soft (cartilage, tendon or ligament) and hard (bone) tissues. In a biomimetic approach we succeeded to fabricate amorphous Ca-polyP nanoparticles/microparticles which are highly biocompatible. Together with polycaprolactone (PCL), polyP can be bio-printed. This hybrid material attracts the cells, as documented optically as well as by a gene-expression studies. Since PCL is already a FDA-approved organic and inert polymer and polyP a physiological biologically active component this new bio-hybrid material has the potential to restore physiological functions, including bone remodelling and regeneration if used as implant.