Cell membrane-inspired phospholipid polymers for developing medical devices with excellent biointerfaces

Abstract

This review article describes fundamental aspects of cell membrane-inspired phospholipid polymers and their usefulness in the development of medical devices. Since the early 1990s, polymers composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) units have been considered in the preparation of biomaterials. MPC polymers can provide an artificial cell membrane structure at the surface and serve as excellent biointerfaces between artificial and biological systems. They have also been applied in the surface modification of some medical devices including long-term implantable artificial organs. An MPC polymer biointerface can suppress unfavorable biological reactions such as protein adsorption and cell adhesion – in other words, specific biomolecules immobilized on an MPC polymer surface retain their original functions. MPC polymers are also being increasingly used for creating biointerfaces with artificial cell membrane structures.     

Analysis of extracellular matrix production in artificial cartilage constructs by histology, immunocytochemistry, mass spectrometry, and NMR spectroscopy

Artificial cartilage constructs based on primary porcine chondrocytes embedded in agarose gel were cultivated for six weeks under static, free swelling conditions. Standard biochemical assays, immunocytochemical staining methods, MALDI-TOF mass spectrometry, and non-invasive 13C solid-state NMR spectroscopy were used to assess cell proliferation, chondrocyte metabolism, extracellular matrix composition, matrix production, and the nanoarchitecture of the macromolecules in the constructs. In particular the production of sulphated glycosaminoglycans such as chondroitin sulphate was investigated quantitatively. Standard methods such as histological and immunocytochemical tools as well as spectrophotometric assays indicated the production of extracellular matrix in the artificial cartilage constructs. In addition, MALDI-TOF mass spectrometric data allowed to clearly identify the production of chondroitin sulphate in the tissue engineered cartilage. While all these methods require invasive sample treatment, 13C NMR spectroscopy allows to study the composition of the artificial cartilage constructs without previous manipulations. Though lower in sensitivity, 13C NMR spectra clearly showed the presence of chondroitin sulphate in the constructs. To increase the sensitivity of the NMR method, a culture medium that contained uniformly 13C labelled glucose but no sodium pyruvate or L-glutamine was used. Thus, further insights into the chondrocyte metabolism ex vivo are possible. Therefore, MALDI-TOF mass spectrometry and 13C solid-state NMR are useful experimental techniques that can assist the quantitative evaluation and quality control of artificially engineered tissues.     

Thermally-reversible gel for 3-D cell culture of chondrocytes

Regeneration of destroyed articular cartilage can be induced by transplantation of cartilage cells into a defect. The best results are obtained by the use of autologus cells. However, obtaining large amounts of autologus cartilage cells causes a problem of creating a large cartilage defect in a donor site. Techniques are currently being developed to harvest a small number of cells and propagate them in vitro. It is a challenging task, however, due to the fact that ordinarily, in a cell culture on flat surfaces, chondrocytes do not maintain their in vivo phenotype and irreversibly diminish or cease the synthesis of the phenotypic markers for articular chondrocytes. Therefore, the research is continuing to develop culture conditions for chondrocytes with the preserved phenotype. We have investigated the use of thermoreversible gelling polymer based on N-isopropylacrylamide for the in vitro cell culture of chondrocytes.     

Gelatin/chitosan/hyaluronan ternary complex scaffold containing basic fibroblast growth factor for cartilage tissue engineering

Gelatin, chitosan and hyaluronan with a weight ratio of 82.6%, 16.5% and 0.1% were chosen as a scaffold material to mimic the composition of natural cartilage matrix for cartilage tissue engineering. Water soluble carbodiimide was added into the biomacromolecule solution with a concentration of 5% to crosslink the complex. Following a freeze-drying procedure, a porous scaffold (control) was then prepared. To enhance chondrogenesis, heparin was covalently immobilized onto the scaffold by carbodiimide chemistry, through which basic fibroblast growth factor (bFGF) was further incorporated by a bioaffinity force. Incubation in phosphate buffered saline (PBS, pH 7.4) at 37 °C caused the weight loss of all kinds of the scaffolds, which could be brought by both the degradation and dissolution of the biomacromolecules. Compared with the control, however, the heparinized scaffold showed stronger ability to resist the weight loss, implying that a higher crosslinking degree was achieved by incorporation of the heparin. Rabbit auricular chondrocytes were seeded onto the ternary complex scaffold containing bFGF to assess cell response. Chondrocytes could adhere and proliferate in all kinds of the scaffold, regardless of the existence of bFGF. No significant difference on glycosaminoglycan (GAG) secretion was recorded between these scaffolds after cultured for 7 and 21 days too, although the absolute value from the Scaffold-heparin-bFGF was somewhat higher. However, chondrocytes seeded in the Scaffold-heparin-bFGF indeed showed significant higher viability than that on the control scaffold. These results reveal that the ternary complex scaffolds, in particular the one containing bFGF, are a potential candidate for cartilage tissue engineering.     

Left ventricular assist device patient maintained on home hemodialysis: A novel class of patients to the home dialysis population

Severe heart failure is increasingly being managed by cardiac transplantation, and in some cases mechanical support devices serve as destination therapies. Left ventricular assist devices (LVADs) were approved for destination therapy for end stage heart failure patients before the more advanced total artificial heart modality became available. One common complication of mechanical assist device placement is acute kidney injury. Historically, patients with mechanical support devices have had to have inpatient hemodialysis until combined heart kidney transplant. Though, some units have started accepting LVAD patients in outpatient dialysis clinics. The cost of in center hemodialysis remains high and home dialysis modalities are becoming increasingly popular. We report the first patient with an LVAD to undergo training and successful home hemodialysis while awaiting combined heart kidney transplantation.     

A comparative study of the chondrogenic potential between synthetic and natural scaffolds in an in vivo bioreactor

Abstract

The clinical demand for cartilage tissue engineering is potentially large for reconstruction defects resulting from congenital deformities or degenerative disease due to limited donor sites for autologous tissue and donor site morbidities. Cartilage tissue engineering has been successfully applied to the medical field: a scaffold pre-cultured with chondrocytes was used prior to implantation in an animal model. We have developed a surgical approach in which tissues are engineered by implantation with a vascular pedicle as an in vivo bioreactor in bone and adipose tissue engineering. Collagen type II, chitosan, poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL) were four commonly applied scaffolds in cartilage tissue engineering. To expand the application of the same animal model in cartilage tissue engineering, these four scaffolds were selected and compared for their ability to generate cartilage with chondrocytes in the same model with an in vivo bioreactor. Gene expression and immunohistochemistry staining methods were used to evaluate the chondrogenesis and osteogenesis of specimens. The result showed that the PLGA and PCL scaffolds exhibited better chondrogenesis than chitosan and type II collagen in the in vivo bioreactor. Among these four scaffolds, the PCL scaffold presented the most significant result of chondrogenesis embedded around the vascular pedicle in the long-term culture incubation phase.     

A polylactide/fibrin gel composite scaffold for cartilage tissue engineering: fabrication and an in vitro evaluation

A composite scaffold for cartilage tissue engineering was fabricated by filling a porous poly (l-lactide) (PLLA) scaffold with fibrin gel. The porous PLLA scaffold prepared by a method of thermally induced phase separation has an average pore diameter of 200 μm and a porosity of 93%. Incorporation of fibrin gel into the scaffold was achieved by dropping a fibrinogen and thrombin mixture solution onto the scaffold. For a couple of minutes the fibrin gel was in situ formed within the scaffold. The filling efficiency was decreased along with the increase of the fibrinogen concentration. After fibrin gel filling, the compressive modulus and the yield stress increased from 5.94 MPa and 0.37 MPa (control PLLA scaffold in a hydrated state) to 7.21 MPa and 0.53 MPa, respectively. While the fibrin gel lost its weight in phosphate buffered saline up to ~50% within 3 days, 85% and 70% of the fibrin gel weight in the composite scaffold was remained within 3 and 35 days, respectively. A consistent significant higher level of rabbit auricular chondrocyte viability, cell number and glycosaminoglycan was measured in the composite scaffold than that in the control PLLA scaffold. Rabbit auricular chondrocytes with round morphology were also observed in the composite scaffold by confocal microscopy and scanning electron microscopy. Altogether with the features of better strength and cytocompatibility, this type of composite scaffold may have better performance as a matrix for cartilage tissue engineering.     

Nanofibrous hollow microspheres self-assembled from star-shaped polymers as injectable cell carriers for knee repair

To repair complexly shaped tissue defects, an injectable cell carrier is desirable to achieve an accurate fit and to minimize surgical intervention. However, the injectable carriers available at present have limitations, and are not used clinically for cartilage regeneration. Here, we report nanofibrous hollow microspheres self-assembled from star-shaped biodegradable polymers as an injectable cell carrier. The nanofibrous hollow microspheres, integrating the extracellular-matrix-mimicking architecture with a highly porous injectable form, were shown to efficiently accommodate cells and enhance cartilage regeneration, compared with control microspheres. The nanofibrous hollow microspheres also supported a significantly larger amount of, and higher-quality, cartilage regeneration than the chondrocytes-alone group in an ectopic implantation model. In a critical-size rabbit osteochondral defect-repair model, the nanofibrous hollow microspheres/chondrocytes group achieved substantially better cartilage repair than the chondrocytes-alone group that simulates the clinically available autologous chondrocyte implantation procedure. These results indicate that the nanofibrous hollow microspheres are an excellent injectable cell carrier for cartilage regeneration.     

Designing artificial cells to harness the biological ion concentration gradient

Cell membranes contain numerous nanoscale conductors in the form of ion channels and ion pumps that work together to form ion concentration gradients across the membrane to trigger the release of an action potential. It seems natural to ask if artificial cells can be built to use ion transport as effectively as natural cells. Here we report a mathematical calculation of the conversion of ion concentration gradients into action potentials across different nanoscale conductors in a model electrogenic cell (electrocyte) of an electric eel. Using the parameters extracted from the numerical model, we designed an artificial cell based on an optimized selection of conductors. The resulting cell is similar to the electrocyte but has higher power output density and greater energy conversion efficiency. We suggest methods for producing these artificial cells that could potentially be used to power medical implants and other tiny devices.     

Controlling Cell Behavior Through the Design of Polymer Surfaces

Polymers have gained a remarkable place in the biomedical field as materials for the fabrication of various devices and for tissue engineering applications. The initial acceptance or rejection of an implantable device is dictated by the crosstalk of the material surface with the bioentities present in the physiological environment. Advances in microfabrication and nanotechnology offer new tools to investigate the complex signaling cascade induced by the components of the extracellular matrix and consequently allow cellular responses to be tailored through the mimicking of some elements of the signaling paths. Patterning methods and selective chemical modification schemes at different length scales can provide biocompatible surfaces that control cellular interactions on the micrometer and sub‐micrometer scales on which cells are organized. In this review, the potential of chemically and topographically structured micro‐ and nanopolymer surfaces are discussed in hopes of a better understanding of cell–biomaterial interactions, including the recent use of biomimetic approaches or stimuli‐responsive macromolecules. Additionally, the focus will be on how the knowledge obtained using these surfaces can be incorporated to design biocompatible materials for various biomedical applications, such as tissue engineering, implants, cell‐based biosensors, diagnostic systems, and basic cell biology. The review focusses on the research carried out during the last decade.     

On the electropolishing of NiTi braided stents – challenges and solutions

Nickel Titanium (NiTi) alloys possess special mechanical properties and good biocompatibility hence used as base material for the production of vascular stents. Normally, vascular stents are machined from NiTi tubes, using laser cutting processes. Braiding is a promising alternative for the machining of certain NiTi stents. However, a surface finish treatment, such as electropolishing of the braided stents, is still required in order to achieve a medical‐grade surface finish. The thermally‐grown oxide resulting from the shape‐setting heat treatment, following the braiding must be removed. Moreover, electropolishing is required to achieve optimum corrosion resistance. Therefore, the aim of this study is to find suitable parameters for the effective electropolishing of NiTi textile stents. Electropolishing of a device with such a complex geometry is challenging, hence a custom‐designed electrolytic cell was constructed and used in this study. We examined the stent surfaces before and after electropolishing, using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Potentiodynamic tests were performed in NaCl 0.9% solution for as‐received and electropolished samples. The results from the present study indicate an improvement in surface quality of the braided stents after electropolishing. Potentiodynamic tests revealed that electropolishing improves the corrosion resistance of the NiTi stents.     

Autologous urothelial cells transplantation onto a prefabricated capsular stent for tissue engineered ureteral reconstruction

In this study, we have fabricated an artificial ureter by transplantation of in vitro-expanded urothelial cells onto an in vivo-prefabricated capsular stent using tissue engineering methods. Spiral poly (l-lactic acid) (PLLA) stents were transplanted into the subcutaneous of Wistar rats for a period of 1, 2 or 3 weeks to induce the formation of connective tissue capsules on their surfaces. The capsular PLLA stents were then decellularized and further recellularized with bladder epithelial cells to fabricate artificial ureters. The results showed that the entrapped cells in all capsules remained continuously proliferation and lined up in continuous layers. In addition, the urothelial cells on the capsular stents with an embedding period of 2 or 3 weeks showed higher proliferative viability compared with the cells on the stents with an embedding time of 1 week (P < 0.05). The results of the study indicated that the prefabricated capsular stents could serve as alternative cell carriers for tissue engineered ureters, especially with embedding time from 2 to 3 weeks.     

Electrochemically Powered,Energy‐Conserving Carbon Nanotube Artificial Muscles

While artificial muscle yarns and fibers are potentially important for many applications, the combination of large strokes, high gravimetric work capacities, short cycle times, and high efficiencies are not realized for these fibers. This paper demonstrates here electrochemically powered carbon nanotube yarn muscles that provide tensile contraction as high as 16.5%, which is 12.7 times higher than previously obtained. These electrochemical muscles can deliver a contractile energy conversion efficiency of 5.4%, which is 4.1 times higher than reported for any organic‐material‐based artificial muscle. All‐solid‐state parallel muscles and braided muscles, which do not require a liquid electrolyte, provide tensile contractions of 11.6% and 5%, respectively. These artificial muscles might eventually be deployed for a host of applications, from robotics to perhaps even implantable medical devices.     

Artificial Muscles Powered by Glucose

Untethered actuation is important for robotic devices to achieve autonomous motion, which is typically enabled by using batteries. Using enzymes to provide the required electrical charge is particularly interesting as it will enable direct harvesting of fuel components from a surrounding fluid. Here, a soft artificial muscle is presented, which uses the biofuel glucose in the presence of oxygen. Glucose oxidase and laccase enzymes integrated in the actuator catalytically convert glucose and oxygen into electrical power that in turn is converted into movement by the electroactive polymer polypyrrole causing the actuator to bend. The integrated bioelectrode pair shows a maximum open‐circuit voltage of 0.70 ± 0.04 V at room temperature and a maximum power density of 0.27 µW cm^?2 at 0.50 V, sufficient to drive an external polypyrrole‐based trilayer artificial muscle. Next, the enzymes are fully integrated into the artificial muscle, resulting in an autonomously powered actuator that can bend reversibly in both directions driven by glucose and O₂ only. This autonomously powered artificial muscle can be of great interest for soft (micro‐)robotics and implantable or ingestible medical devices manoeuvring throughout the body, for devices in regenerative medicine, wearables, and environmental monitoring devices operating autonomously in aqueous environments.     

Biomechanical properties of single chondrocytes and chondrons determined by micromanipulation and finite-element modelling

A chondrocyte and its surrounding pericellular matrix (PCM) are defined as a chondron. Single chondrocytes and chondrons isolated from bovine articular cartilage were compressed by micromanipulation between two parallel surfaces in order to investigate their biomechanical properties and to discover the mechanical significance of the PCM. The force imposed on the cells was measured directly during compression to various deformations and then holding. When the nominal strain at the end of compression was 50 per cent, force relaxation showed that the cells were viscoelastic, but this viscoelasticity was generally insignificant when the nominal strain was 30 per cent or lower. The viscoelastic behaviour might be due to the mechanical response of the cell cytoskeleton and/or nucleus at higher deformations. A finite-element analysis was applied to simulate the experimental force-displacement/time data and to obtain mechanical property parameters of the chondrocytes and chondrons. Because of the large strains in the cells, a nonlinear elastic model was used for simulations of compression to 30 per cent nominal strain and a nonlinear viscoelastic model for 50 per cent. The elastic model yielded a Young''s modulus of 14 ± 1 kPa (mean ± s.e.) for chondrocytes and 19 ± 2 kPa for chondrons, respectively. The viscoelastic model generated an instantaneous elastic modulus of 21 ± 3 and 27 ± 4 kPa, a long-term modulus of 9.3 ± 0.8 and 12 ± 1 kPa and an apparent viscosity of 2.8 ± 0.5 and 3.4 ± 0.6 kPa s for chondrocytes and chondrons, respectively. It was concluded that chondrons were generally stiffer and showed less viscoelastic behaviour than chondrocytes, and that the PCM significantly influenced the mechanical properties of the cells.     

Engineered articular cartilage: influence of the scaffold on cell phenotype and proliferation

Articular cartilage defects do not heal. Biodegradable scaffolds have been studied for cartilage engineering in order to implant autologous chondrocytes and help cartilage repair. We tested some new collagen matrices differing in collagen type, origin, structure and methods of extraction and purification, and compared the behavior of human chondrocytes cultured on them. Human chondrocytes were grown for three weeks on four different equine type I collagen matrices, one type I, III porcine collagen matrix and one porcine type II collagen matrix. After 21 days, samples were subjected to histochemical, immunohistochemical and histomorphometric analysis to study phenotype expression and cell adhesion. At 7, 14 and 21 days cell proliferation was studied by incorporation of [3H]-thymidine. Our data evidence that the collagen type influences cell morphology, adhesion and growth; indeed, cellularity and rate of proliferation were significantly higher and cells were rounder on the collagen II matrix than on either of the collagen I matrices. Among the collagen I matrices, we observed a great variability in terms of cell adhesion and proliferation. The present study allowed us to identify one type I collagen matrix and one type II collagen matrix that could be usefully employed as a scaffold for chondrocyte transplantation.     

Tetrahedral DNA Nanostructure: A Potential Promoter for Cartilage Tissue Regeneration via Regulating Chondrocyte Phenotype and Proliferation

Utilizing biomaterials to regulate the phenotype and proliferation of chondrocytes is a promising approach for effective cartilage tissue regeneration. Recently, a significant amount of effort has been invested into directing chondrocytes toward a desired location and function by utilizing biomaterials to control the dedifferentiation and phenotypic loss of chondrocytes during in vitro monolayer culture. Here, the transmission signals resulting from tetrahedral DNA nanostructures (TDNs) in the regulation of chondrocyte phenotype and proliferation are exploited. TDNs, new DNA nanomaterials, have been considered as promising materials in biomedical fields. Upon exposure to TDNs, chondrocyte phenotype is significantly enhanced, accompanied by lower gene expression related to Notch signaling pathway and higher expression of type II collagen. In addition, the cell proliferation and morphology of chondrocytes are changed after exposure to TDNs. In conclusion, this work demonstrates that TDNs are potentially useful mechanism in cartilage tissue regeneration from chondrocytes, whereby chondrocyte phenotype and proliferation can be retained.     

A study of crystalline biomaterials for articular cartilage bioengineering

This study examines the suitability of marine origin coral species, Porites lutea (POR) and the hydrozoan Millepora dichotoma (MIL), for use as novel three dimensional growth matrices in the field of articular cartilage tissue engineering. Therefore, mesenchymal stem cells (MSCs) and chondrocytes were grown on the skeletal material obtained from each of these two organisms to investigate their potential use as three dimensional scaffolding for cartilage tissue growth. Chondrogenic induction of MSCs was achieved by addition of transforming growth factor-β1 (TGF-β1) and insulin growth factor-I (IGF-I). Cell adherence, proliferation, differentiation and tissue development were investigated through six weeks of culture. Cartilage tissue growth and chondrocytic phenotype maintenance of each cell type were examined by cell morphology, histochemical analyses, expression of collagen type II and quantitative measures of glycosaminoglycan (GAG) content. The MSCs and the chondrocytes were shown good adherence to the scaffolds and maintenance of the chondrocytic phenotype in the initial stages of culture. However after two weeks of culture on MIL and three weeks on POR these cultures began to exhibit signs of further differentiation and phenotypic loss. The shown results indicated that POR was a better substrate for chondrocytes phenotype maintenance than MIL. We believe that surface modification of POR combined with mechanical stimuli will provide a suitable environment for chondrogenic phenotype maintenance. Further investigation of POR and other novel coralline biomatrices is indicated and warranted in the field of cartilage tissue engineering applications.     

Fibrin-mediated endothelial cell adhesion to vascular biomaterials resists shear stress due to flow

In vitro endothelial cell (EC) seeding onto biomaterials for blood-contacting applications can improve the blood compatibility of materials. Adhesive proteins adsorbed from serum that is supplemented with the culture medium intercede the initial cell adhesion and subsequent spreading on material surface during culture. Nevertheless, physical and chemical properties of vascular biomaterial surface fluctuate widely between materials resulting in dissimilarity in protein adsorption characteristics. Thus, a variation is expected in cell adhesion, growth and the ability of cell to resist shear stress when tissue engineering on to vascular biomaterials is attempted. This study was carried out with an objective to determine the significance of a matrix coating on cell adhesion and shear stress resistance when cells are cultured on materials such as polytetrafluoroethylene (PTFE, Teflon) and polyethyleneterephthalate (Dacron), ultra high molecular weight polyethylene (UHMWPE) and titanium (Ti), that are used for prosthetic devices. The study illustrates the distinction of EC attachment and proliferation between uncoated and matrix-coated surfaces. The cell attachment and proliferation on uncoated UHMWPE and titanium surfaces were not significantly different from matrix-coated surfaces. However, shear stress resistance of the cells grown on composite coated surfaces appeared superior compared to the cells grown on uncoated surface. On uncoated vascular graft materials, the cell adhesion was not supported by serum alone and proliferation was scanty as compared to matrix-coated surface. Therefore, coating of implant devices with a composite of adhesive proteins and growth factors can improve EC attachment and resistance of the cells to the forces of flow.     

Cytocompatibility of titanium metal injection molding with various anodic oxidation post-treatments

Metal injection molding (MIM) is a near net shape manufacturing method that allows for the production of components of small to moderate size and complex shape. MIM is a cost-effective and flexible manufacturing technique that provides a large innovative potential over existing methods for the industry of implantable devices. Commercially pure titanium (CP-Ti) samples were machined to the same shape as a composite feedstock with titanium and polyoxymethylene, and these metals were injected, debinded and sintered to assess comparative biological properties. Moreover, we treated MIM-Ti parts with BIOCOAT®, BIODIZE® and BIOCER®, three different anodic oxidation techniques that treat titanium using acid, alkaline and anion enriched electrolytes, respectively. Cytocompatibility as well as morphological and chemical features of surfaces was comparatively assessed on each sample, and the results revealed that MIM-Ti compared to CP-Ti demonstrated a specific surface topography with a higher roughness. MIM-Ti and BIOCER® samples significantly enhanced cell proliferation, cell adhesion and cell differentiation compared to CP-Ti. Interestingly, in the anodization post-treatment established in this study, we demonstrated the ability to improve osseointegration through anionic modification treatment. The excellent biological response we observed with MIM parts using the injection molding process represents a promising manufacturing method for the future implantable devices in direct contact with bones.