Biological evaluations of novel 2,3,3-Trisphosphonate in osteoclastic and osteoblastic activities

Bisphosphonates (BPs) are the first line treatment for many bone diseases including hypercalcimia associated with bone malignancies. In this paper, we introduce a new analogue of bisphosphonate called the 2,3,3-Trisphosphonate (2,3,3-TriPP) that was synthesised in a two steps reaction. In vitro investigations using a medically known bisphosphonate (Etidronate) and the 2,3,3-TrisPP were performed with an aim to evaluate biological effect of this novel compound in major bone cells. 2,3,3-TrisPP showed to have potential to supress the bone resorption process, as our data found that this novel compound exhibited cytotoxic effect in osteoclastic cells at a low concentration of 0.172 mg/mL (LC50). A molecular docking computational simulation calculated a high level of binding affinity between the human farnesyl pyrophosphate synthase (hFPPS) and 2,3,3-TrisPP. This calculation suggested 2,3,3TrisPP may have undergone the mevalonate pathway to prevent the prenylation step during biosynthesis and subsequently resulted in the deactivation of osteoclastic cells. Finally, high levels of osteoblast mineralisation potentials were recorded upon treatments with 2,3,3-TrisPP (0.01-0.1 mg/ml), which implied 2,3,3-TrsiPP may also facilitate bone regeneration.Peer reviewe

Biofabrication of 3D hard-soft and composite constructs for bone regeneration

Biofabrication encompasses the use of additive manufacturing techniques for fabricating complex constructs from a wide range of biomaterials, cells and bioactive substances as well as their maturation for the formation of tissue. The fabricated constructs should provide mechanical stability, porosity, and accurate positioning of cells. The aim of this work was the creation of hybrid constructs consisting of a combination of a thermoplastic hard polymer with and without addition of bioactive glass particles and a soft hydrogel matrix with immobilised cells. The hard phase should enhance the limited mechanical performance of the soft hydrogel phase. Moreover the addition of bioactive glass will enhance the local bioactivity of the scaffolds, of relevance for bone tissue engineering [1]. The hydrogel composition, based on alginate, was tailored to enable the proliferation, migration and differentiation of cells. The mechanical properties and the degradation kinetic of the constructs were investigated. Alginate-dialdehyde (ADA) gelatine (GEL) hydrogel (= ADA-GEL) containing murine bone marrow derived stroma cells (ST2) and polycaprolactone (PCL), polyethylene glycol (PEG) blends were used. Processing was done by additive manufacturing using a dispense plotter equipped with multiple cartridges. Process parameters like plotting speed, pressure and temperature were optimized for the two material systems. Porosity, degradation behaviour and mechanical stability of the PCL-PEG frame structure scaffolds were tested as well as the response of ST2 cells. The presence of bioactive glass leading to enhance local formation of hydroxyapatite was investigated. The cell behaviour and cell development were characterized by assessing the morphology and by measuring the viability of the immobilized cells in the ADA-GEL over an incubation period of 28 days. Both materials could be processed in a defined manner with optimized process parameters. The PEG phase could be dissolved and porous (bioactive) struts forming a framework structure were created. The viability of immobilized ST2 cells after hydrogel plotting was proven as well as their attachment, migration and proliferation by SEM and fluorescence microscopy images. Thus, two promising material systems for creating hybrid constructs were successfully evaluated. The two phase plotting approach enables the fabrication of hydrogel constructs with improved mechanical properties and bioactivity, which exhibit high potential for applications in bone regeneration. [1] A. J. Leite, et al., Bioplotting of a bioactive alginate dialdehyde-gelatin composite hydrogel containing bioactive glass nanoparticles, Biofabrication, 8, pp. 035005 (2016

Engineering of Metabolic Pathways by Artificial Enzyme Channels.

Application of industrial enzymes for production of valuable chemical compounds has greatly benefited from recent developments in Systems and Synthetic Biology. Both, in vivo and in vitro systems have been established, allowing conversion of simple into complex compounds. Metabolic engineering in living cells needs to be balanced which is achieved by controlling gene expression levels, translation, scaffolding, compartmentation, and flux control. In vitro applications are often hampered by limited protein stability/half-life and insufficient rates of substrate conversion. To improve stability and catalytic activity, proteins are post-translationally modified and arranged in artificial metabolic channels. Within the review article, we will first discuss the supramolecular organization of enzymes in living systems and second summarize current and future approaches to design artificial metabolic channels by additive manufacturing for the efficient production of desired products

Fabrication and characterization of alginate-keratin based composite microspheres containing bioactive glass for tissue engineering applications

3D cell encapsulation within hydrogels has attracted more and more attention in tissue engineering applications because hydrogels provide a hydrated environment closely mimicking the in vivo environment for cell and tissue growth1. This present study considers the fabrication of alginate-keratin based composite microspheres containing bioactive glass (BG) of 45S5 composition for cell encapsulation. We propose the use of alginate di-aldehyde (ADA) synthesized via periodate oxidation of alginate to enhance the biodegradability of alginate, and the incorporation of keratin into the alginate based hydrogel to improve cellular interaction of the hydrogel. Keratins extracted from wool contain cell adhesive peptide sequences including RGD (arginine-glycine-aspartic acid), and LDV (leucine-aspartic acid-valine)2. BG particles, well known for promoting calcium phosphate deposition, were incorporated into the microspheres to enhance osseointegration3. The microspheres were prepared via a pressure-driven extrusion technique. Weight loss, protein release measurements, and FTIR spectroscopy of the fabricated microspheres were carried out. The morphology and microstructure of the microspheres were investigated by light microscopy and scanning electron microscopy (SEM), respectively. The results demonstrated that the composition of the hydrogels had a significant effect on their physical properties. Biological properties of ADA-keratin based microspheres were evaluated by encapsulating MG-63 osteosarcoma cells into the microspheres. Cell viability of MG-63 cells in ADA-keratin-1%BG hydrogels was found to be comparable to that of alginate-keratin and ADA-keratin after culturing for 21 days. The results proved that such novel composite hydrogel might be a promising material for biofabrication in bone healing approaches. Please click Additional Files below to see the full abstract

Cell adhesion evaluation of laser-sintered HAp and 45S5 bioactive glass coatings on micro-textured zirconia surfaces using MC3T3-E1 osteoblast-like cells

Laser texturing is a technique that has been increasingly explored for the surface modification of several materials on different applications. Laser texturing can be combined with conventional coating techniques to functionalize surfaces with bioactive properties, stimulating cell differentiation and adhesion. This study focuses on the cell adhesion of laser-sintered coatings of hydroxyapatite (HAp) and 45S5 bioactive glass (45S5 BG) on zirconia textured surfaces using MC3T3-E1 cells. For this purpose, zirconia surfaces were micro-textured via laser and then coated with HAp and 45S5 BG glass via dip coating. Afterwards, the bioactive coatings were laser sintered, and a reference group of samples was conventionally sintering. The cell adhesion characterisation was achieved by cell viability performing live/dead analysis using fluorescence stains and by SEM observations for a qualitative analysis of cell adhesion. The in vitro results showed that a squared textured pattern with 100μm width grooves functionalized with a bioactive coating presented an increase of 90% of cell viability compared to flat surfaces after 48h of incubation. The functionalized laser sintered coatings do not present significant differences in cell viability when compared to conventionally sintered coatings. Therefore, the results reveal that laser sintering of HAp and 45S5 BG coatings is a fast and attractive coating technique.publishe

Structure optimisation and biological evaluation of bone scaffolds prepared by co-sintering of silicate and phosphate glasses

A degradable phosphate glass (ICEL) and a bioactive silicate glass (CEL2) were mixed in different ratios (wt-%: 100%ICEL, 70%ICEL-30%CEL2, 30%ICEL-70%CEL2, 100%CEL2; codes 100-0, 70-30, 30-70, 0-100) and then co-sintered to obtain three-dimensional porous scaffolds by gel casting foaming. Thermal analyses were carried out on the glass mixtures and were used as a starting point for the optimisation of the scaffold sintering treatment. The microcomputed tomography and field emission scanning electron microscope analyses allowed the selection of the optimal sintering temperature to obtain an adequate structure in terms of total and open porosity. The scaffolds showed an increasing solubility with increasing ICEL glass content, and for 30-70 and 0-100, the precipitation of hydroxyapatite in simulated body fluid was observed. In vitro tests indicated that all the scaffolds showed no cytotoxic effect. The co-sintering of silicate and phosphate glasses showed to be a promising strategy to tailor the scaffold osteoconductivity, degradation and bioactivit

Mechanical properties of cell- and microgel bead-laden oxidized alginate-gelatin hydrogels

3D-printing technologies, such as biofabrication, capitalize on the homogeneous distribution and growth of cells inside biomaterial hydrogels, ultimately aiming to allow for cell differentiation, matrix remodeling, and functional tissue analogues. However, commonly, only the mechanical properties of the bioinks or matrix materials are assessed, while the detailed influence of cells on the resulting mechanical properties of hydrogels remains insufficiently understood. Here, we investigate the properties of hydrogels containing cells and spherical PAAm microgel beads through multi-modal complex mechanical analyses in the small- and large-strain regimes. We evaluate the individual contributions of different filler concentrations and a non-fibrous oxidized alginate-gelatin hydrogel matrix on the overall mechanical behavior in compression, tension, and shear. Through material modeling, we quantify parameters that describe the highly nonlinear mechanical response of soft composite materials. Our results show that the stiffness significantly drops for cell- and bead concentrations exceeding four million per milliliter hydrogel. In addition, hydrogels with high cell concentrations (≥6 mio ml−1) show more pronounced material nonlinearity for larger strains and faster stress relaxation. Our findings highlight cell concentration as a crucial parameter influencing the final hydrogel mechanics, with implications for microgel bead drug carrier-laden hydrogels, biofabrication, and tissue engineering

Surface modifcation of SPIONs in PHBV microspheres for biomedical applications

Surface modification of superparamagnetic iron oxide nanoparticles (SPIONs) has been introduced with lauric acid and oleic acid via co-precipitation and thermal decomposition methods, respectively. This modification is required to increase the stability of SPIONs when incorporated in hydrophobic, biodegradable and biocompatible polymers such as poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). In this work, the solid-in-oil-in-water (S/O/W) emulsion-solvent extraction/evaporation method was utilized to fabricate magnetic polymer microspheres incorporating SPIONs in PHBV. The prepared magnetic PHBV microspheres exhibited particle sizes <1 µm. The presence of functional groups of lauric acid, oleic acid and iron oxide in the PHBV microspheres was confirmed by Fourier Transform Infrared spectroscopy (FTIR). X-ray diffraction (XRD) analysis was performed to further confirm the success of the combination of modified SPIONs and PHBV. Thermogravimetric analysis (TGA) indicated that PHBV microspheres were incorporated with SPIONsLauric as compared with SPIONsOleic. This was also proven via magnetic susceptibility measurement as a higher value of this magnetic property was detected for PHBV/SPIONsLauric microspheres. It was revealed that the magnetic PHBV microspheres were non-toxic when assessed with mouse embryotic fibroblast cells (MEF) at different concentrations of microspheres. These results confirmed that the fabricated magnetic PHBV microspheres are potential candidates for use in biomedical applications

Antibacterial 45S5 Bioglass®-based scaffolds reinforced with genipin cross-linked gelatin for bone tissue engineering

45S5 Bioglass® (BG) scaffolds with high porosity (>90%) were coated with genipin cross-linked gelatin (GCG) and further incorporated with poly(p-xylyleneguanidine) hydrochloride (PPXG). The obtained GCG coated scaffolds maintained the high porosity and well interconnected pore structure. A 26-fold higher compressive strength was provided to 45S5 BG scaffolds by GCG coating, which slightly retarded but did not inhibit the in vitro bioactivity of 45S5 BG scaffolds in SBF. Moreover, the scaffolds were made antibacterial against both Gram-positive and Gram-negative bacteria by using polyguanidine, i.e. PPXG, in this study. Osteoblast-like cells (MG-63) were seeded onto PPXG and GCG coated scaffolds. PPXG was biocompatible with MG-63 cells at a low concentration (10 μg mL−1). MG-63 cells were shown to attach and spread on both uncoated and GCG coated scaffolds, and the mitochondrial activity measurement indicated that GCG coating had no negative influence on the cell proliferation behavior of MG-63 cells. The developed novel antibacterial bioactive 45S5 BG-based composite scaffolds with improved mechanical properties are promising candidates for bone tissue engineering