RF plasma sputtering deposition of hydroxyapatite bioceramics : synthesis, performance, and biocompatibility

Hydroxyapatite (HA) coatings have numerous applications in orthopedics and dentistry, owing to their excellent ability to promote stronger implant fixation and faster bone tissue ingrowth and remodeling. Thermal plasma spray and other plasma-assisted techniques have recently been used to synthesize various calcium phosphate-based bioceramics. Despite notable recent achievements in the desired stoichiometry, phase composition, mechanical, structural, and bio-compatible properties, it is rather difficult to combine all of the above features in a single coating. For example, many existing plasma-sprayed HA coatings fall short in meeting the requirements of grain size and crystallinity, and as such are subject to enhanced resorption in body fluid. On the other hand, relatively poor interfacial bonding and stability is an obstacle to the application of the HA coatings in high load bearing Ti6Al4V knee joint implants. Here, we report on an alternative: a plasma-assisted, concurrent, sputtering deposition technique for high performance biocompatible HA coatings on Ti6Al4V implant alloy. The plasma-assisted RF magnetron co-sputtering deposition method allows one to simultaneously achieve most of the desired attributes of the biomimetic material and overcome the aforementioned problems. This article details the film synthesis process specifications, extensive analytical characterization of the material's properties, mechanical testing, simulated body fluid assessments, biocompatibility and cytocompatibility of the HA-coated Ti6Al4V orthopedic alloy. The means of optimization of the plasma and deposition process parameters to achieve the desired attributes and performance of the HA coating, as well as future challenges in clinical applications are also discussed.

Engineered microenvironments provide new insights into ovarian and prostate cancer progression and drug responses

Tissue engineering technologies, which have originally been designed to reconstitute damaged tissue structure and function, can mimic not only tissue regeneration processes but also cancer development and progression. Bioengineered approaches allow cell biologists to develop sophisticated experimentally and physiologically relevant cancer models to recapitulate the complexity of the disease seen in patients. Tissue engineering tools enable three-dimensionality based on the design of biomaterials and scaffolds that re-create the geometry, chemistry, function and signalling milieu of the native tumour microenvironment. Three-dimensional (3D) microenvironments, including cell-derived matrices, biomaterial-based cell culture models and integrated co-cultures with engineered stromal components, are powerful tools to study dynamic processes like proteolytic functions associated with cancer progression, metastasis and resistance to therapeutics. In this review, we discuss how biomimetic strategies can reproduce a humanised niche for human cancer cells, such as peritoneal or bone-like microenvironments, addressing specific aspects of ovarian and prostate cancer progression and therapy response.

Novel approaches for SER spectroscopic analysis of protein cofactors

Biomimetic systems employed for biotechnological applications i.e. as biosensors or bio fuel cells, require initial formation of conducting support/protein complexes with controlled properties. The specific interaction of the protein with the support determines important qualities of the device such as electrical communication, long-term stability and catalytic efficiency. In this respect the system parameters have to be chosen in a way that high protein loading on the support is achieved while protein denaturation upon adsorption is prevented. The conditions on the surface have to be adjusted in such a way that the desired surface reaction of the protein i.e. electron transfer to either the electrode or a second redox partner, is still guaranteed. Hence the choice of support, its functionlisation as well as the right adjustment of solution parameters play a crucial role in the rational design of these support/protein constructs.

The harnessing of peptide-monolith constructs for single step plasmid DNA purification

The availability of synthetic peptides has paved the way for their use in tailor-made interactions with biomolecules. In this study, a 16mer LacI-based peptide was used as an affinity ligand to examine the scale up feasibility for plasmid DNA purification. First, the peptide was designed and characterized for the affinity purification of lacO containing plasmid DNA, to be employed as a high affinity ligand for the potential capturing of plasmid DNA in a single unit operation. It was found there were no discernible interactions with a control plasmid that did not encode the lacO nucleotide sequence. The dissociation equilibrium constant of the binding between the 16mer peptide and target pUC19 was 5.0 ± 0.5 × 10-8 M as assessed by surface plasmon resonance. This selectivity and moderated affinity indicate that the 16mer is suitable for the adsorption and chromatographic purification of plasmid DNA. The suitability of this peptide was then evaluated using a chromatography system with the 16mer peptide immobilized to a customized monolith to purify plasmid DNA, obtaining preferential purification of supercoiled pUC19. The results demonstrate the applicability of peptide-monolith supports to scale up the purification process for plasmid DNA using designed ligands via a biomimetic approach.

Mechanistic details of the membrane perforation and passive translocation of TAT peptides

The trans-activator of transcription (TAT) peptide is regarded as the “gold standard” for cell-penetrating peptides, capable of traversing a mammalian membrane passively into the cytosolic space. This characteristic has been exploited through conjugation of TAT for applications such as drug delivery. However, the process by which TAT achieves membrane penetration remains ambiguous and unresolved. Mechanistic details of TAT peptide action are revealed herein by using three complementary methods: quartz crystal microbalance with dissipation (QCM-D), scanning electrochemical microscopy (SECM) and atomic force microscopy (AFM). When combined, these three scales of measurement define that the membrane uptake of the TAT peptide is by trans-membrane insertion using a “worm-hole” pore that leads to ion permeability across the membrane layer. AFM data provided nanometre-scale visualisation of TAT punctuation using a mammalian-mimetic membrane bilayer. The TAT peptide does not show the same specificity towards a bacterial mimetic membrane and QCM-D and SECM showed that the TAT peptide demonstrates a disruptive action towards these membranes. This investigation supports the energy-independent uptake of the cationic TAT peptide and provides empirical data that clarify the mechanism by which the TAT peptide achieves its membrane activity. The novel use of these three biophysical techniques provides valuable insight into the mechanism for TAT peptide translocation, which is essential for improvements in the cellular delivery of TAT-conjugated cargoes including therapeutic agents required to target specific intracellular locations.

Multi-parametric hydrogels support 3D in vitro bioengineered microenvironment models of tumour angiogenesis

Tumour microenvironment greatly influences the development and metastasis of cancer progression. The development of three dimensional (3D) culture models which mimic that displayed in vivo can improve cancer biology studies and accelerate novel anticancer drug screening. Inspired by a systems biology approach, we have formed 3D in vitro bioengineered tumour angiogenesis microenvironments within a glycosaminoglycan-based hydrogel culture system. This microenvironment model can routinely recreate breast and prostate tumour vascularisation. The multiple cell types cultured within this model were less sensitive to chemotherapy when compared with two dimensional (2D) cultures, and displayed comparative tumour regression to that displayed in vivo. These features highlight the use of our in vitro culture model as a complementary testing platform in conjunction with animal models, addressing key reduction and replacement goals of the future. We anticipate that this biomimetic model will provide a platform for the in-depth analysis of cancer development and the discovery of novel therapeutic targets.

Advanced tissue engineering scaffold design for regeneration of the complex hierarchical periodontal structure

AIM: This study investigated the ability of an osteoconductive biphasic scaffold to simultaneously regenerate alveolar bone, periodontal ligament and cementum. MATERIALS AND METHODS: A biphasic scaffold was built by attaching a fused deposition modelled bone compartment to a melt electrospun periodontal compartment. The bone compartment was coated with a calcium phosphate (CaP) layer for increasing osteoconductivity, seeded with osteoblasts and cultured in vitro for 6 weeks. The resulting constructs were then complemented with the placement of PDL cell sheets on the periodontal compartment, attached to a dentin block and subcutaneously implanted into athymic rats for 8 weeks. Scanning electron microscopy, X-ray diffraction, alkaline phosphatase and DNA content quantification, confocal laser microscopy, micro computerized tomography and histological analysis were employed to evaluate the scaffold's performance. RESULTS: The in vitro study showed that alkaline phosphatase activity was significantly increased in the CaP-coated samples and they also displayed enhanced mineralization. In the in vivo study, significantly more bone formation was observed in the coated scaffolds. Histological analysis revealed that the large pore size of the periodontal compartment permitted vascularization of the cell sheets, and periodontal attachment was achieved at the dentin interface. CONCLUSIONS: This work demonstrates that the combination of cell sheet technology together with an osteoconductive biphasic scaffold could be utilized to address the limitations of current periodontal regeneration techniques.

Magnetic and morphological properties of ferrofluid-impregnated hydroxyapatite/collagen scaffolds

In this article we present the morphological and magnetic characterization of ferrofluid-impregnated biomimetic scaffolds made of hydroxyapatite and collagen used for bone reconstruction. We describe an innovative and simple impregnation process by which the ferrofluid is firmly adsorbed onto the hydroxyapatite/collagen scaffolds. The process confers sufficient magnetization to attract potential magnetic carriers, which may be used to transport bioactive agents that favour bone regeneration. The crystalline structure of the magnetite contained in the ferrofluid is preserved and its quantity, estimated from the weight gain due to the impregnation process, is consistent with that obtained from energy dispersive X-ray spectroscopy. The magnetization, measured with a superconducting quantum interference device, is uniform throughout the scaffolds, demonstrating the efficiency of the impregnation process. The field emission gun scanning electron microscopy characterization demonstrates that the process does not alter the morphology of the hydroxyapatite/collagen scaffolds, which is essential for the preservation of their bioactivity and consequently for their effectiveness in promoting bone formation.

With great structure comes great functionality: Understanding and emulating spider silk

The overarching aim of biomimetic approaches to materials synthesis is to mimic simultaneously the structure and function of a natural material, in such a way that these functional properties can be systematically tailored and optimized. In the case of synthetic spider silk fibers, to date functionalities have largely focused on mechanical properties. A rapidly expanding body of literature documents this work, building on the emerging knowledge of structure–function relationships in native spider silks, and the spinning processes used to create them. Here, we describe some of the benchmark achievements reported until now, with a focus on the last five years. Progress in protein synthesis, notably the expression on full-size spidroins, has driven substantial improvements in synthetic spider silk performance. Spinning technology, however, lags behind and is a major limiting factor in biomimetic production. We also discuss applications for synthetic silk that primarily capitalize on its nonmechanical attributes, and that exploit the remarkable range of structures that can be formed from a synthetic silk feedstock.

A multiscale study on the structural and mechanical properties of the luffa sponge from Luffa cylindrica plant

Cellular materials that are often observed in biological systems exhibit excellent mechanical properties at remarkably low densities. Luffa sponge is one of such materials with a complex interconnecting porous structure. In this paper, we studied the relationship between its structural and mechanical properties at different levels of its hierarchical organization from a single fiber to a segment of whole sponge. The tensile mechanical behaviors of three single fibers were examined by an Instron testing machine and the ultrastructure of a fractured single fiber was observed in a scanning electronic microscope. Moreover, the compressive mechanical behaviors of the foam-like blocks from different locations of the sponge were examined. The difference of the compressive stress-strain responses of four sets of segmental samples were also compared. The result shows that the single fiber is a porous composite material mainly consisting of cellulose fibrils and lignin/hemicellulose matrix, and its Young's modulus and strength are comparable to wood. The mechanical behavior of the block samples from the hoop wall is superior to that from the core part. Furthermore, it shows that the influence of the inner surface on the mechanical property of the segmental sample is stronger than that of the core part; in particular, the former's Young's modulus, strength and strain energy absorbed are about 1.6 times higher. The present work can improve our understanding of the structure-function relationship of the natural material, which may inspire fabrication of new biomimetic foams with desirable mechanical efficiency for further applications in anti-crushing devices and super-light sandwich panels.

Biophysical Characterization of Oxidized Phospholipids : Implications for Altered Membrane Structure and Function

The present study aims to elucidate the modifications in the structure and functionality of the phospholipid matrix of biological membranes brought about by free radical-mediated oxidative damage of its molecular constituents. To this end, the surface properties of two oxidatively modified phospholipids bearing an aldehyde or carboxyl function at the end of truncated sn-2 acyl chain were studied using a Langmuir balance. The results obtained reveal both oxidized species to have a significant impact on the structural dynamics of phospholipid monolayers, as illustrated by the progressive changes in force-area isotherms with increasing mole fraction of the oxidized lipid component. Moreover, surface potential measurements revealed considerable modifications in the electric properties of oxidized phospholipid containing monolayers during film compression, suggesting a packing state-controlled reorientation of the intramolecular electric dipoles of the lipid headgroups and acyl chains. Based on the above findings, a model describing the conformational state of oxidized phospholipid molecules in biological membranes is proposed, involving the protrusion of the acyl chains bearing the polar functional groups out from the hydrocarbon phase to the surrounding aqueous medium. Oxidative modifications alter profoundly the physicochemical properties of unsaturated phospholipids and are therefore readily anticipated to have important implications for their interactions with membrane-associating molecules. Along these lines, the carboxyl group bearing lipid was observed to bind avidly the peripheral membrane protein cytochrome c. The binding was reversed following increase in ionic strength or addition of polyanionic ATP, thus suggesting it to be driven by electrostatic interactions between cationic residues of the protein and the deprotonated lipid carboxyl exposed to the aqueous phase. The presence of aldehyde function bearing oxidized phospholipid was observed to enhance the intercalation of four antimicrobial peptides into phospholipid monolayers and liposomal bilayers. Partitioning of the peptides to monolayers was markedly attenuated by the aldehyde scavenger methoxyamine, revealing it to be mediated by the carbonyl moiety possibly through efficient hydrogen bonding or, alternatively, formation of covalent adduct in form of a Schiff base between the lipid aldehydes and primary amine groups of the peptide molecules. Lastly, both oxidized phospholipid species were observed to bind with high affinity three small membrane-partitioning therapeutic agents, viz. chlorpromazine, haloperidol, and doxorubicin. In conclusion, the results of studies conducted using biomimetic model systems support the notion that oxidative damage influences the molecular architecture as well as the bulk physicochemical properties of phospholipid membranes. Further, common polar functional groups carried by phospholipids subjected to oxidation were observed to act as molecular binding sites at the lipid-water interface. It is thus plausible that oxidized phospholipid species may elicit cellular level effects by modulating integration of various membrane-embedded and surface-associated proteins and peptides, whose conformational state, oligomerization, and functionality is known to be controlled by highly specific lipid-protein interactions and proper physical state of the membrane environment.

A concise synthesis of the bioactive meroterpenoid natural product (+/-)-liphagal, a potent PI3K inhibitor

A short, diversity-oriented synthesis that follows a biomimetic route to the marine natural product liphagal, from a commercially available building block, is delineated.

Additively manufactured device for dynamic culture of large arrays of 3D tissue engineered constructs

The ability to test large arrays of cell and biomaterial combinations in 3D environments is still rather limited in the context of tissue engineering and regenerative medicine. This limitation can be generally addressed by employing highly automated and reproducible methodologies. This study reports on the development of a highly versatile and upscalable method based on additive manufacturing for the fabrication of arrays of scaffolds, which are enclosed into individualized perfusion chambers. Devices containing eight scaffolds and their corresponding bioreactor chambers are simultaneously fabricated utilizing a dual extrusion additive manufacturing system. To demonstrate the versatility of the concept, the scaffolds, while enclosed into the device, are subsequently surface-coated with a biomimetic calcium phosphate layer by perfusion with simulated body fluid solution. 96 scaffolds are simultaneously seeded and cultured with human osteoblasts under highly controlled bidirectional perfusion dynamic conditions over 4 weeks. Both coated and noncoated resulting scaffolds show homogeneous cell distribution and high cell viability throughout the 4 weeks culture period and CaP-coated scaffolds result in a significantly increased cell number. The methodology developed in this work exemplifies the applicability of additive manufacturing as a tool for further automation of studies in the field of tissue engineering and regenerative medicine.

Polymer assisted hydroxyapatite microspheres suitable for biomedical application

Hollow Microspheres of hydroxyapatite-polymer composite can be used as carriers in drug delivery and fillers in tissue engineering. Based on the concept of soft chemistry, a battery of technique is available in the literature to synthesize hollow microspheres, however, an economically viable synthesis route, having good control over the microarchitect and easy to be scaled up, is yet to be developed. Polymer matrix mediated synthesis of inorganic nanoparticles is known to synthesize nanoparticles with controlled morphology and dimensions. It is termed as biomimetic synthesis. Integrating the biomimetic synthesis of nano-particles and spray drying techniques, a novel process of producing hydroxyapatite-polymer composite hollow microspheres is briefly discussed here.

An Experimental and Numerical Study of Calliphora Wing Structure

Experiments are performed to determine the mass and stiffness variations along the wing of the blowfly Calliphora. The results are obtained for a pairs of wings of 10 male flies and fresh wings are used. The wing is divided into nine locations along the span and seven locations along the chord based on venation patterns. The length and mass of the sections is measured and the mass per unit length is calculated. The bending stiffness measurements are taken at three locations, basal (near root), medial and distal (near tip) of the fly wing. Torsional stiffness measurements are also made and the elastic axis of the wing is approximately located. The experimental data is then used for structural modeling of the wing as a stepped cantilever beam with nine spanwise sections of varying mass per unit lengths, flexural rigidity (EI) and torsional rigidity (GJ) values. Inertial values of nine sections are found to approximately vary according to an exponentially decreasing law over the nine sections from root to tip and it is used to calculate an approximate value of Young's modulus of the wing biomaterial. Shear modulus is obtained assuming the wing biomaterial to be isotropic. Natural frequencies, both in bending and torsion, are obtained by solving the homogeneous part of the respective governing differential equations using the finite element method. The results provide a complete analysis of Calliphora wing structure and also provide guidelines for the biomimetic structural design of insect-scale flapping wings.