Design Of Biomaterial Surfaces Research Articles

Barrier Membrane with Janus Function and Structure for Guided Bone Regeneration.

Guided bone regeneration (GBR) technology has been demonstrated to be an effective method for reconstructing bone defects. A membrane is used to cover the bone defect to stop soft tissue from growing into it. The biosurface design of the barrier membrane is key to the technology. In this work, an asymmetric functional gradient Janus membrane was designed to address the bidirectional environment of the bone and soft tissue during bone reconstruction. The Janus membrane was simply and efficiently prepared by the multilayer self-assembly technique, and it was divided into the polycaprolactone isolation layer (PCL layer, GBR-A) and the nanohydroxyapatite/polycaprolactone/polyethylene glycol osteogenic layer (HAn/PCL/PEG layer, GBR-B). The morphology, composition, roughness, hydrophilicity, biocompatibility, cell attachment, and osteogenic mineralization ability of the double surfaces of the Janus membrane were systematically evaluated. The GBR-A layer was smooth, dense, and hydrophobic, which could inhibit cell adhesion and resist soft tissue invasion. The GBR-B layer was rough, porous, hydrophilic, and bioactive, promoting cell adhesion, proliferation, matrix mineralization, and expression of alkaline phosphatase and RUNX2. In vitro and in vivo results showed that the membrane could bind tightly to bone, maintain long-term space stability, and significantly promote new bone formation. Moreover, the membrane could fix the bone filling material in the defect for a better healing effect. This work presents a straightforward and viable methodology for the fabrication of GBR membranes with Janus-based bioactive surfaces. This work may provide insights for the design of biomaterial surfaces and treatment of bone defects.

Topological structure of electrospun membrane regulates immune response, angiogenesis and bone regeneration

The fate of biomaterials is orchestrated by biocompatibility and bioregulation characteristics, reported to be closely related to topographical structures. For the purpose to investigate the topography of fibrous membranes on the guided bone regeneration performance, we successfully fabricated poly (lactate-co-glycolate)/fish collagen/nano-hydroxyapatite (PFCH) fibrous membranes with random, aligned and latticed topography by electrospinning. The physical, chemical and biological properties of the three topographical PFCH membranes were systematically investigated by in vitro and in vivo experiments. The subcutaneous implantation of C57BL6 mice showed an acceptable mild foreign body reaction of all three topological membranes. Interestingly, the latticed PFCH membrane exhibited superior abilities to recruit macrophage/monocyte and induce angiogenesis. We further investigated the osteogenesis of the three topographical PFCH membranes via the critical-size calvarial bone defect model of rats and mice and the results suggested that latticed PFCH membrane manifested promising performance to promote angiogenesis through upregulation of the HIF-1α signaling pathway; thereby enhancing bone regeneration. Our research illustrated that the topological structure of fibrous membranes, as one of the characteristics of biomaterials, could regulate its biological functions, and the fibrous structure of latticed topography could serve as a favorable surface design of biomaterials for bone regeneration. Statement of significanceIn material-mediated regeneration medicine, the interaction between the biomaterial and the host is key to successful tissue regeneration. The micro-and nano-structure becomes one of the most critical physical clues for designing biomaterials. In this study, we fabricated three topological electrospun membranes (Random, Aligned and Latticed) to understand how topological structural clues mediate bone tissue regeneration. Interestingly, we found that the Latticed topographical PFCH membrane promotes macrophage recruitment, angiogenesis, and osteogenesis in vivo, indicating the fibrous structure of latticed topography could serve as a favorable surface design of biomaterials for bone regeneration.

High-Surface-Energy Nanostructured Surface on Low-Modulus Beta Titanium Alloy for Orthopedic Implant Applications

Nanostructured surfaces represent pertinent material technologies to improve the biocompatibility aspects of orthopedic implant surfaces. The present work reports the development of nanostructured titania surface on a low-modulus TNZT (Ti-35Nb-7Zr-5Ta) beta titanium alloy. The major focus is to gain more insights into the wettability, surface energy and corrosion resistance of the developed nanostructures. The developed nanomorphologies have been found to be nanopetal-like structures with nanotopographies and composed predominantly of anatase phase. Wettability studies revealed a highly hydrophilic surface with a concurrent enhancement in surface energy (68% increment in polar surface energy) advantageous for improved osseointegration. The developed layer has demonstrated comparable corrosion resistance with untreated surface. By incorporating the multiple aspects of low-modulus TNZT alloy, hydrophilic titania surface with elevated surface free energy and comparable corrosion resistance, the present work opens prospective avenues for effective biomaterial surface design paradigms.

Exosomes derived from macrophages upon cobalt ion stimulation promote angiogenesis

Angiogenesis is critical for tissue repair and regeneration, including implant osseointegration. It is well known that macrophages exert immunomodulatory functions in angiogenesis. However, whether macrophage-derived exosomes participate in the process is still unclear. Cobalt (Co) ions are frequently used as implant additives to mimic hypoxic microenvironment, which can induce angiogenesis through stabilizing hypoxia inducible factor-1α (HIF-1α) of macrophages and endothelial cells (ECs). The present work attempts to investigate whether exosomes derived from macrophages upon Co ion stimulation can mediate angiogenesis and the possible mechanism. The results show that the exosomes promote endothelial migration and angiogenesis in vitro and in vivo, particularly when Co ion concentration is 200 μM. Further studies reveal that the exosomes upregulating nitric oxide (NO), vascular endothelial growth factor (VEGF), and integrin β1 expression may be the underlying mechanism of the observations. Our findings provide new insights for Co ion mediated macrophage-EC communication and surface design of biomaterials from the perspective of pro-angiogenesis.

Synergistic effects of immunoregulation and osteoinduction of ds-block elements on titanium surface

Ds-block elements have been gaining increasing attention in the field of biomaterials modification, owing to their excellent biological properties, such as antibiosis, osteogenesis, etc. However, their function mechanisms are not well understood and conflicting conclusions were drawn by previous studies on this issue, which are mainly resulted from the inconsistent experimental conditions. In this work, three most widely used ds-block elements, copper, zinc, and silver were introduced on titanium substrate by plasma immersion ion implantation method to investigate the rule of ds-block elements in the immune responses. Results showed that the implanted samples could decrease the inflammatory responses compared with Ti sample. The trend of anti-inflammatory effects of macrophages on samples was in correlation with cellular ROS levels, which was induced by the implanted biomaterials and positively correlated with the number of valence electrons of ds-block elements. The co-culture experiments of macrophages and bone marrow mesenchymal stem cells showed that these two kinds of cells could enhance the anti-inflammation and osteogenesis of samples by the paracrine manner of PGE2. In general, in their steady states on titanium substrate (Cu2+, Zn2+, Ag), the ds-block elements with more valence electrons exhibit better anti-inflammatory and osteogenic effects. Moreover, molecular biology experiments indicate that the PGE2-related signaling pathway may contribute to the desired immunoregulation and osteoinduction capability of ds-block elements. These findings suggest a correlation between the number of valence electrons of ds-block elements and the relevant biological responses, which provides new insight into the selection of implanted ions and surface design of biomaterials.

Controlling Cell Behavior through the Design of Biomaterial Surfaces: A Focus on Surface Modification Techniques

AbstractSurface interaction at the biomaterial–cell interface is essential for a variety of cellular functions, such as adhesion, proliferation, and differentiation. Nevertheless, changes in the biointerface enable to trigger specific cell signaling and result in different cellular responses. In order to manufacture biomaterials with higher functionality, biomaterials containing immobilized bioactive ligands have been widely introduced and employed for tissue engineering and regenerative medicine applications. Moreover, a number of physical and chemical strategies have been used to improve the functionality of biomaterials and specifically at the material interface. Here, the interactions between materials and cells at the interface levels are described. Then, the importance of surface properties in cell function is discussed and recent methods for surface modifications are systematically highlighted. Additionally, the impact of bulk material properties on the cellular responses is briefly reviewed.

Nanostructural Surfaces with Different Elastic Moduli Regulate the Immune Response by Stretching Macrophages.

A proper immune response is key for the successful implantation of biomaterials, and designing and fabricating biomaterials to regulate immune responses is the future trend. In this work, three different nanostructures were constructed on the surface of titanium using a hydrothermal method, and through a series of in vitro and in vivo experiments, we found that the aspect ratio of nanostructures can affect the elastic modulus of a material surface and further regulate immune cell behaviors. This work demonstrates that nanostructures with a higher aspect ratio can endow a material surface with a lower elastic modulus, which was confirmed by experiments and theoretical analyses. The deflection of nanostructures under the cell adsorption force is a substantial factor in stretching macrophages to enhance cell adhesion and spreading, further inducing macrophage polarization toward the M1 phenotype and leading to intense immune responses. In contrast, a nanostructure with a lower aspect ratio on a material surface leads to a higher surface elastic modulus, making deflection of the material difficult and creating a surface that is not conducive to macrophage adhesion and spreading, thus reducing the immune response. Moreover, molecular biology experiments indicated that regulation of the immune response by the elastic modulus is primarily related to the NF-κB signaling pathway. These findings suggest that the immune response can be regulated by constructing nanostructural surfaces with the proper elastic modulus through their influence on cell adhesion and spreading, which provides new insights into the surface design of biomaterials.

Adsorption-associated orientational changes of immunoglobulin G and regulated phagocytosis of Staphylococcus epidermidis.

Understanding the adsorption of immunoglobulin G (IgG) on biomaterials surfaces is crucial for design and modification of the surfaces to alleviate inflammatory responses after implantation. Here, we report direct visualization and two-dimensional (2D) image interpretation of the IgG molecule adsorbed on simplified surfaces by single particle electron microscopy and atomic force microscopy. Influence of the orientational changes in adsorbed IgG on phagocytosis of macrophages against Staphylococcus epidermidis is further examined. Untreated amorphous carbon film and -COOH and -NH2 grafted carbon films are employed as the model surfaces for the adsorption testing. Results show that IgG displays flat orientation lying on the untreated surface, while forms vertical orientations standing on the functionalized surfaces. These specific spatial alignments are associated with altered unfolding extent and structure rearrangement of IgG domains, which are influenced synergistically by surface charge and wettability of the substrata. The changes in interdomain distance of IgG molecules subsequently regulate immune behaviors of macrophages and phagocytosis of S. epidermidis. The results would give insight into appropriate design of biomaterial surfaces in nanoscales for desired inflammatory responses. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2838-2849, 2018.

Measuring the Interactions between Protein-Coated Microspheres and Polymer Brushes in Aqueous Solutions.

Hydrophilic or zwitterionic polymer-functionalized surfaces have become attractive biomaterials in bioscience and technology due to their excellent protein-resistant ability. Understanding the fundamental interactions between proteins and polymers plays an essential role in the surface design of biomaterials. In this work, we studied the interactions between bovine serum albumin (BSA) and two sorts of polymer brushes including zwitterionic poly(carboxybetaine methacrylate) (PCBMA) and hydrophilic poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA) in NaCl aqueous solutions directly with a self-established total internal reflection microscope (TIRM) to provide a better understanding of the underlying nonfouling mechanism of polymers. Our results indicate that both the surface charge and brushes length can affect protein adsorption through electrostatic and steric repulsions, respectively. Both PCBMA- and POEGMA-coated surfaces display negative charge properties due to incomplete coverage and ionic adsorption. As a result, strong electrostatic repulsions between proteins and negatively charged polymer-coated surfaces could contribute to the resistance of protein-coated particles in solutions with low ionic strength (0.1, 0.5, and 1 mM) and disappear in solutions with high ionic strength (10 mM). The measured interaction profiles demonstrate that PCBMA brushes could provide apparent steric forces only at high ionic strength (10 mM), where zwitterionic brushes exhibit a relatively extended conformation with a lack of electrostatic forces between intra- and interpolymers. In contrast, the steric repulsion between proteins and POEGMA brushes appears when particles diffuse at low positions in all salt concentrations (0.1-10 mM) with similar steric decay lengths, which results from the unresponsiveness of POEGMA brushes to the salt stimulus.

Host Responses to Biomaterials and Anti-Inflammatory Design-a Brief Review.

Host responses toward foreign implants that lead to chronic inflammation and fibrosis may result in failure of the biomedical device. To solve these problems, first a better understanding of the biomaterial-induced host reactions including protein adsorption, leukocyte activation, inflammatory and fibrotic responses to biomaterials is required; second an improved design of biomaterial surfaces is needed that results in an appropriate host response, causing less inflammatory response, and supporting tissue regeneration. Hence, this review provides a brief overview on the host response to implants, as well as in vitro models to study inflammatory and fibrotic responses to biomaterials to predict the clinical outcome of implantation. Moreover, the review highlights anti-inflammatory strategies to improve the biocompatibility of implants, which contain the modification of physicochemical surface properties of materials as well as the immobilization of anti-inflammatory reagents and bioactive molecules on biomaterials.

Nanostructured Multilayer Films Assembled from Poly(dopamine)‐Coated Carbon Nanotubes for Controlling Cell Behavior

AbstractNano‐topographic surfaces have been used as an effective tool to control cell behavior such as adhesion and proliferation. In this study, multilayer films with nano‐topographic features were fabricated by alternatively assembling poly(l‐lysine) (PLL) and poly(dopamine)‐coated carbon nanotubes (CNTs@PDA) layers. The growth of PLL/CNTs@PDA film presented a perfect linear relationship with the number of bilayers. A nanostructured morphology with interpenetrating CNT networks was observed by scanning electron microscopy (SEM). Adhesion and proliferation of endothelial cells (ECs) and smooth muscle cells (SMCs) on the PLL/CNTs@PDA multilayer films have been evaluated. The films support initial adhesion of both ECs and SMCs. Interestingly, the PLL/CNTs@PDA multilayer films were found to promote proliferation of SMCs and inhibited proliferation of ECs. Further, pheochromocytoma (PC12) cells were employed to evaluate the influence of PLL/CNTs@PDA multilayer films on the outgrowth of synapses. We found that the nanostructured surface significantly promoted the synapses of PC12 cell growth and formation. Our findings suggest that cytophilic surfaces with the nanostructured morphology have diverse effects on different cells, which sheds light on new design of biomaterial surfaces in cell‐based applications.

Surface chemistry from wettability and charge for the control of mesenchymal stem cell fate through self-assembled monolayers

Self-assembled monolayers (SAMs) of alkanethiols on gold are highly controllable model substrates and have been employed to mimic the extracellular matrix for cell-related studies. This study aims to systematically explore how surface chemistry influences the adhesion, morphology, proliferation and osteogenic differentiation of mouse mesenchymal stem cells (mMSCs) using various functional groups (-OEG, -CH3, -PO3H2, -OH, -NH2 and -COOH). Surface analysis demonstrated that these functional groups produced a wide range of wettability and charge: -OEG (hydrophilic and moderate iso-electric point (IEP)), -CH3 (strongly hydrophobic and low IEP), -PO3H2 (moderate wettability and low IEP), -OH (hydrophilic and moderate IEP), -NH2 (moderate wettability and high IEP) and -COOH (hydrophilic and low IEP). In terms of cell responses, the effect of wettability may be more influential than charge for these groups. Moreover, compared to -OEG and -CH3 groups, -PO3H2, -OH, -NH2 and -COOH functionalities tended to promote not only cell adhesion, proliferation and osteogenic differentiation but also the expression of αv and β1 integrins. This finding indicates that the surface chemistry may guide mMSC activities through αv and β1 integrin signaling pathways. Model surfaces with controllable chemistry may provide insight into biological responses to substrate surfaces that would be useful for the design of biomaterial surfaces.

Multifunctional and stable bone mimic proteinaceous matrix for bone tissue engineering

Biomaterial surface design with biomimetic proteins holds great promise for successful regeneration of tissues including bone. Here we report a novel proteinaceous hybrid matrix mimicking bone extracellular matrix that has multifunctional capacity to promote stem cell adhesion and osteogenesis with excellent stability. Osteocalcin-fibronectin fusion protein holding collagen binding domain was networked with fibrillar collagen, featuring bone extracellular matrix mimic, to provide multifunctional and structurally-stable biomatrices. The hybrid protein, integrated homogeneously with collagen fibrillar networks, preserved structural stability over a month. Biological efficacy of the hybrid matrix was proven onto tethered surface of biopolymer porous scaffolds. Mesenchymal stem cells quickly anchored to the hybrid matrix, forming focal adhesions, and substantially conformed to cytoskeletal extensions, benefited from the fibronectin adhesive domains. Cells achieved high proliferative capacity to reach confluence rapidly and switched to a mature and osteogenic phenotype more effectively, resulting in greater osteogenic matrix syntheses and mineralization, driven by the engineered osteocalcin. The hybrid biomimetic matrix significantly improved in vivo bone formation in calvarial defects over 6 weeks. Based on the series of stimulated biological responses in vitro and in vivo the novel hybrid proteinaceous composition will be potentially useful as stem cell interfacing matrices for osteogenesis and bone regeneration.

Analysis of high-throughput screening reveals the effect of surface topographies on cellular morphology

Surface topographies of materials considerably impact cellular behavior as they have been shown to affect cell growth, provide cell guidance, and even induce cell differentiation. Consequently, for successful application in tissue engineering, the contact interface of biomaterials needs to be optimized to induce the required cell behavior. However, a rational design of biomaterial surfaces is severely hampered because knowledge is lacking on the underlying biological mechanisms. Therefore, we previously developed a high-throughput screening device (TopoChip) that measures cell responses to large libraries of parameterized topographical material surfaces. Here, we introduce a computational analysis of high-throughput materiome data to capture the relationship between the surface topographies of materials and cellular morphology. We apply robust statistical techniques to find surface topographies that best promote a certain specified cellular response. By augmenting surface screening with data-driven modeling, we determine which properties of the surface topographies influence the morphological properties of the cells. With this information, we build models that predict the cellular response to surface topographies that have not yet been measured. We analyze cellular morphology on 2176 surfaces, and find that the surface topography significantly affects various cellular properties, including the roundness and size of the nucleus, as well as the perimeter and orientation of the cells. Our learned models capture and accurately predict these relationships and reveal a spectrum of topographies that induce various levels of cellular morphologies. Taken together, this novel approach of high-throughput screening of materials and subsequent analysis opens up possibilities for a rational design of biomaterial surfaces.

Three-dimensional reconstruction of surface nanoarchitecture from two-dimensional datasets

The design of biomaterial surfaces relies heavily on the ability to accurately measure and visualize the three-dimensional surface nanoarchitecture of substrata. Here, we present a technique for producing three-dimensional surface models using displacement maps that are based on the data obtained from two-dimensional analyses. This technique is particularly useful when applied to scanning electron micrographs that have been calibrated using atomic force microscopy (AFM) roughness data. The evaluation of four different surface types, including thin titanium films, silicon wafers, polystyrene cell culture dishes and dragonfly wings confirmed that this technique is particularly effective for the visualization of conductive surfaces such as metallic titanium. The technique is particularly useful for visualizing surfaces that cannot be easily analyzed using AFM. The speed and ease with which electron micrographs can be recorded, combined with a relatively simple process for generating displacement maps, make this technique useful for the assessment of the surface topography of biomaterials.

Cell adhesion on phase-separated surface of block copolymer composed of poly(2-methacryloyloxyethyl phosphorylcholine) and poly(dimethylsiloxane)

We investigated the morphological effect of phase-separated block copolymer surfaces composed of poly(2-methacryloyloxyethyl phosphorylcholine (MPC)) (PMPC) and poly(dimethylsiloxane) (PDMS) on protein adsorption and cell adhesion behavior. We observed three different types of phase-separated surface morphologies by TEM and AFM. The elemental composition of phosphorus on the surface increases with the PMPC composition. Furthermore, the polymer surface formed by a block copolymer-containing a higher MPC unit composition shows a slightly lower static water contact angle. This result indicates that the elemental surface ratio of the surface depends on the MPC composition in the block copolymer. Protein adsorption tests revealed that only hydrophobic PDMS domains showed selective protein adsorption. Cell adhesion tests revealed that the number of adhered cells increased with increasing hydrophobic PDMS domain size of block copolymers in serum-containing media. In contrast, no cells adhered onto block copolymer surfaces in serum-free media, whereas a large amount of adhered cells were observed on the hydrophobic PDMS surface. This result indicates that segregated hydrophobic domains on a biocompatible PMPC surface strongly affect serum protein adsorption, thereby promoting considerable cell adhesion, although the surface is hydrophilic. Thus, both the composition of MPC units and the segregated hydrophobic surface morphology are important considerations in biomaterial surface design.

Molecular Simulation To Characterize the Adsorption Behavior of a Fibrinogen γ-Chain Fragment

Implants invoke inflammatory responses from the body even if they are chemically inert and nontoxic. It has been shown that a crucial precedent event in the inflammatory process is the spontaneous adsorption of fibrinogen (Fg) on implant surfaces, which is typically followed by the presence of phagocytic cells. Interactions between the phagocyte integrin Mac-1 and two short sequences within the fibrinogen gamma chain, gamma190-202 and gamma377-395, may partially explain phagocyte accumulation at implant surfaces. These two sequences are believed to form an integrin binding site that is inaccessible when Fg is in its soluble-state structure but then becomes available for Mac-1 binding following adsorption, presumably due to adsorption-induced conformational changes. The objective of this research was to theoretically investigate this possibility by using molecular dynamics simulations of the gamma-chain fragment of Fg over self-assembled monolayer (SAM) surfaces presenting different types of surface chemistry. The GROMACS software package was used to carry out the molecular simulations in an explicit solvation environment over a 5 ns period of time. The adsorption of the gamma-chain of fibrinogen was simulated on five types of SAM surfaces. The simulations showed that this protein fragment exhibits distinctly different adsorption behavior on the different surface chemistries. Although the trajectory files showed that significant conformational changes did not occur in this protein fragment over the time frame of the simulations, it was predicted that the protein does undergo substantial rotational and translational motions over the surface prior to stabilizing in various preferred orientations. This suggests that the kinetics of surface-induced conformational changes in a protein's structure might be much slower than the kinetics of orientational changes, thus enabling the principles of adsorption thermodynamics to be used to guide adsorbing proteins into defined orientations on surfaces before large conformational changes can occur. This finding may be very important for biomaterial surface design as it suggests that surface chemistry can potentially be used to directly control the orientation of adsorbing proteins in a manner that either presents or hides specific bioactive sites contained within a protein's structure, thereby providing a mechanism to control cellular responses to the adsorbed protein layer.

Confocal imaging of biofilm formation process using fluoroprobed Escherichia coli and fluoro-stained exopolysaccharide.

We developed a novel method of evaluating biofilm architecture on a synthetic material using green fluorescent protein-expressing Escherichia coli and red fluorescence staining of exopolysaccharides. Confocal laser scanning microscopy observation revealed the time course of the change in the in situ three-dimensional structural features of biofilm on a polyurethane film without structural destruction: initially adhered cells are grown to form cellular aggregates and secrete exopolysaccharides. These cells were spottily distributed on the surface at an early incubation time but fused to form a vertically grown biofilm with incubation time. Fluorescence intensity, which is a measure of the number of cells, determined using a fluorometer and biofilm thickness determined from confocal laser scanning microscopy vertical images were found to be effective for quantification of time-dependent growth of biofilms. The curli (surface-located fibers specifically binding to fibronectin and laminin)-producing Escherichia coli strain, YMel, significantly proliferated on fibronectin-coated polyurethane, whereas the curli-deficient isogenic mutant, YMel-1, did not. The understanding of biofilm architecture in molecular and morphological events and new fluorescence microscopic techniques may help in the logical surface design of biomaterials with a high antibacterial potential.

A molecular modeling study of the effect of surface chemistry on the adsorption of a fibronectin fragment spanning the 7-10th type III repeats.

Although it is well documented that proteins adsorb onto biomaterial surfaces, relatively little is quantitatively understood about the effects of adsorption on protein orientation and conformation. Because this is the primary determining factor of protein bioactivity, the ability to accurately predict a protein's orientation and conformation following adsorption will be essential for the rational design of biomaterial surfaces to control biological responses. Force field-based computational chemistry methods provide an excellent means to theoretically address this issue, with the nontrivial requirement that the force field must be tailored to appropriately represent protein adsorption behavior. Accordingly, we have modified an existing force field (CHARMm) based on semiempirical quantum-mechanical peptide adsorption data to enable it to simulate protein adsorption behavior in an implicit aqueous environment. This modified force field was then applied to predict the adsorption behavior of the 7-10 type III repeats of fibronectin on functionalized surfaces. Predicted changes in adsorption energy and adsorption-induced conformation as a function of surface chemistry were found to correlate well with experimentally observed trends for these same systems. This work represents a first attempt towards the development of a molecular mechanics force field that is specifically parameterized to accurately simulate protein adsorption to biomaterial surfaces.