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

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

3D Printing of Piezoelectric Barium Titanate-Hydroxyapatite Scaffolds with Interconnected Porosity for Bone Tissue Engineering

The prevalence of large bone defects is still a major problem in surgical clinics. It is, thus, not a surprise that bone-related research, especially in the field of bone tissue engineering, is a major issue in medical research. Researchers worldwide are searching for the missing link in engineering bone graft materials that mimic bones, and foster osteogenesis and bone remodeling. One approach is the combination of additive manufacturing technology with smart and additionally electrically active biomaterials. In this study, we performed a three-dimensional (3D) printing process to fabricate piezoelectric, porous barium titanate (BaTiO3) and hydroxyapatite (HA) composite scaffolds. The printed scaffolds indicate good cytocompatibility and cell attachment as well as bone mimicking piezoelectric properties with a piezoelectric constant of 3 pC/N. This work represents a promising first approach to creating an implant material with improved bone regenerating potential, in combination with an interconnected porous network and a microporosity, known to enhance bone growth and vascularization

Highly Porous Polymer-Derived Bioceramics Based on a Complex Hardystonite Solid Solution

Highly porous bioceramics, based on a complex hardystonite solid solution, were developed from silicone resins and micro-sized oxide fillers fired in air at 950 °C. Besides CaO, SrO, MgO, and ZnO precursors, and the commercial embedded silicone resins, calcium borate was essential in providing the liquid phase upon firing and favouring the formation of an unprecedented hardystonite solid solution, corresponding to the formula (Ca0.70Sr0.30)2(Zn0.72Mg0.15Si0.13) (Si0.85B0.15)2O7. Silicone-filler mixtures could be used in the form of thick pastes for direct ink writing of reticulated scaffolds or for direct foaming. The latter shaping option benefited from the use of hydrated calcium borate, which underwent dehydration, with water vapour release, at a low temperature (420 °C). Both scaffolds and foams confirmed the already-obtained phase assemblage, after firing, and exhibited remarkable strength-to-density ratios. Finally, preliminary cell tests excluded any cytotoxicity that could be derived from the formation of a boro-silicate glassy phase

In-vitro study of the bioactivity and cytotoxicity response of Ti surfaces modified by Nb and Mo diffusion treatments

This work focuses on the bioactivity and biological response of modified Ti surfaces produced by powder metallurgy. They are processed by diffusion of two beta-stabilizing elements, Nb and Mo, deposited onto the surface of PM Ti substrates. Moreover, the addition of an activating agent, NH4Cl, to the suspension has been carried out by thermo-reactive diffusion process. The surface modification led to a gradient in composition (Ti-Nb or Ti-Mo) and microstructure (beta / alpha + beta / alpha phases). This work presents the bioactivity results of these Ti-Mo and Ti-Nb surfaces as well as the cell-material response of the Ti-Nb surfaces. The reactivity of the materials was tested through immersion in simulated body fluid considering Ca and P precipitation in order to assess the ability of the materials to induce hydroxyapatite formation. The in-vitro cell response was evaluated by human osteoblast-like cells incubation on the different surfaces for 48 h. The investigation led to positive results in terms of surface bioactivity and an improved cell-material interaction of the PM modified Ti-Nb surfaces compared to the reference Ti material.The authors would like to thank the funding provided for this research by the Regional Government of Madrid (program MULTIMAT-CHALLENGE-CM, ref. S2013/MIT-2862), and by the University Carlos III of Madrid for the research stay of three months in the Institute of Biomaterials (University of Erlangen-Nurnberg)

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

Cellular behaviour of bone marrow stromal cells on modified Ti-Nb surfaces

The cellular behaviour of bone marrow stromal cells on titanium surfaces modified by niobium diffusion is presented in order to test their osteogenic differentiation response after culturing for 21 days. The surface modification of Ti substrates produced by powder metallurgy was performed through niobium diffusion treatments. Ti-Nb exhibited a beta-Ti surface together with a microstructural (beta / alpha + beta / alpha) and compositional (Ti-Nb) gradient which enhances hardness, wear resistance and lowers the elastic modulus making it more similar to the human bone. Furthermore, the Ti-Nb-NH4Cl by means of the activating agent achieved three times the hardness of Ti together with a porous surface. The in vitro osteogenic differentiation response of bonemarrowstromal cells on both Ti-Nb surfaces indicated the positive cell-material interaction. The osteogenic differentiation of cells was successful after 21 days, considering the positive response in terms of increased cell viability, lactate dehydrogenase-(LDH) activity, alkaline phosphatase-(ALP) activity expression (osteogenicmarker) and bone-like nodules deposition by ST-2 cells as a bone mineralization cue. Therefore, the positive effect of a low elastic modulus Ti-Nb surface and a porous nitride TiNbNH4Cl with suitable wettability and average roughness values on the osteogenic differentiation response of bone marrow stromal cells is demonstrated. (C) 2017 Elsevier Ltd. All rights reserved.The authors would like to thank the funding provided for this research by the Regional Government of Madrid (program MULTIMAT-CHALLENGE-CM, ref. S2013/MIT-2862)

Evaluation of Electrospun Poly(ε-Caprolactone)/Gelatin Nanofiber Mats Containing Clove Essential Oil for Antibacterial Wound Dressing

The objective of this study was to produce antibacterial poly(ε-caprolactone) (PCL)-gelatin (GEL) electrospun nanofiber mats containing clove essential oil (CLV) using glacial acetic acid (GAA) as a “benign” (non-toxic) solvent. The addition of CLV increased the fiber diameter from 241 ± 96 to 305 ± 82 nm. Aside from this, the wettability of PCL-GEL nanofiber mats was increased by the addition of CLV. Fourier-transform infrared spectroscopy (FTIR) analysis confirmed the presence of CLV, and the actual content of CLV was determined by gas chromatography–mass spectrometry (GC-MS). Our investigations showed that CLV-loaded PCL-GEL nanofiber mats did not have cytotoxic effects on normal human dermal fibroblast (NHDF) cells. On the other hand, the fibers exhibited antibacterial activity against Staphylococcus aureus and Escherichia coli. Consequently, PCL-GEL/CLV nanofiber mats are potential candidates for antibiotic-free wound healing applications