Biomaterials (v.29, #23)

Characterization of microglial attachment and cytokine release on biomaterials of differing surface chemistry by Braden K. Leung; Roy Biran; Clay J. Underwood; Patrick A. Tresco (3289-3297).
The clinical usefulness of central nervous system recording electrodes is currently limited by inconsistent long-term performance that is believed to be governed by the brain tissue response to the implant. In this study, we observed persistent macrophage biomarker expression at the biotic–abiotic interface surrounding implanted electrodes over a 12-week indwelling period. Using the cell type-specific marker CD11b to examine the cells attached to electrodes retrieved over the indwelling period, we found that most of the cells were activated microglia, the resident macrophage of brain tissue, indicating that the implanted electrodes behave as a persistent inflammatory stimulus. To determine the potential usefulness of different materials as coatings for implanted electrodes, we examined brain-derived microglial cell attachment and cytokine release on a number of medically relevant materials. Our results suggest that activated microglia attach to many of the materials used as external coatings for electrode manufacture, and likely serve as a source of pro-inflammatory and neurotoxic cytokines that may be responsible for reducing the biocompatibility of such implants. Our results also indicate that low protein-binding coatings may be useful in reducing microglial attachment upon implantation in brain tissue and may provide a means of improving electrode biocompatibility.
Keywords: Cell attachment; Electrode; Foreign body response; Neural prosthesis; Protein adsorption; Surface treatment;

Peptide amphiphile nanostructure–heparin interactions and their relationship to bioactivity by Kanya Rajangam; Michael S. Arnold; Mark A. Rocco; Samuel I. Stupp (3298-3305).
Heparin–protein interactions are important in many physiological processes including angiogenesis, the growth of new blood vessels from existing ones. We have previously developed a highly angiogenic self-assembling gel, wherein the self-assembly process is triggered by the interactions between heparin and peptide amphiphiles (PAs) with a consensus heparin binding sequence. In this report, this consensus sequence was scrambled and incorporated into a new peptide amphiphile in order to study its importance in heparin interaction and bioactivity. Heparin was able to trigger gel formation of the scrambled peptide amphiphile (SPA). Furthermore, the affinity of the scrambled molecule for heparin was unchanged as shown by isothermal titration calorimetry and high Förster resonance emission transfer efficiency. However, both the mobile fraction and the dissociation rate constant of heparin, using fluorescence recovery after photobleaching, were markedly higher in its interaction with the scrambled molecule implying a weaker association. Importantly, the scrambled peptide amphiphile–heparin gel had significantly less angiogenic bioactivity as shown by decreased tubule formation of sandwiched endothelial cells. Hence, we believe that the presence of the consensus sequence stabilizes the interaction with heparin and is important for the bioactivity of these new materials.
Keywords: Nanoparticle; Angiogenesis; Heparin; Self-assembly;

The effect of calcium phosphate microstructure on bone-related cells in vitro by Xiaoming Li; Clemens A. van Blitterswijk; Qingling Feng; Fuzhai Cui; Fumio Watari (3306-3316).
Microstructure is essential for inductive bone formation in calcium phosphate materials after soft tissue implantation. We hereby evaluated activities (cell attachment, proliferation, ALP/DNA and protein/DNA) of three types of cells cultured on three kinds of calcium phosphate ceramic discs to study how microstructure takes its role in inductive bone formation. Three kinds of biphasic calcium phosphate (BCP) ceramic discs with the same chemistry and the same dimension of ∅10.0 × 1.0 mm3 (BCP1150-P, BCP1150-D and BCP1300), either having similar micropore sizes and surface roughness but different surface area (BCP1150-P vs BCP1150-D) or having similar surface area but different micropore sizes and different roughness (BCP1150-D vs BCP1300), were prepared. Conventionally Culturing C2C12, human bone marrow stromal cells (HBMSC) and MC3T3-E1 cells on BCP discs showed that, surface roughness did not affect cell attachment, cell proliferation and ALP expression of all cell types evaluated, while surface area did affect cell functions. ALP/DNA of C2C12 on BCP1150-P, having larger surface area, was significantly higher than on BCP1300 and BCP1150-D. Furthermore, all cells cultured on all of the three kinds of BCPs pre-soaked in culture medium having additional rhBMP-2 had a higher ALP expression than the conventional cell culture. Comparing with on BCP1300 and BCP1150-D, ALP/DNA of all cells tested increased more on BCP1150-P after the discs were pre-soaked in culture medium with rhBMP-2. The results indicated that increasing surface areas, microstructured calcium phosphate materials might concentrate more proteins (including bone-inducing proteins) that differentiate inducible cells to osteogenic cells that form inductive bone.
Keywords: Calcium phosphate; Osteoinduction; Microstructure; Alkaline phosphatase (ALP); Protein;

Hydroxylapatite growth on single-crystal rutile substrates by Fredrik Lindberg; Jannica Heinrichs; Fredric Ericson; Peter Thomsen; Håkan Engqvist (3317-3323).
Titanium is widely used as an implant material. In addition to the bulk properties of titanium, the biological response is to a large degree controlled via the surface. The native amorphous titanium oxide that forms spontaneously on the surface gives a very good biological response. Lately it has been shown that crystalline titanium oxides (rutile and anatase) have in vitro bioactive properties. In addition to its potential for new materials development, this finding also opens up for the possibility of studying the mechanisms of bioactivity on materials with strictly controlled surfaces. In this paper the mechanisms behind the in vitro bioactivity are studied, using rutile single crystals. Three single-crystal rutile substrates: (100), (110), and (001), and a polycrystalline rutile substrate obtained by physical vapour deposition were soaked in a phosphate buffered saline solution for up to 4 weeks. The hydroxylapatite films that formed were analysed by X-ray diffraction, scanning electron microscopy, focused ion beam, and transmission electron microscopy. The hydroxylapatite grew faster on the (001) surface than on the other two. It was also found that on the (001) surface the direction of fast growth in hydroxylapatite was aligned parallel to the surface. This is in contrast to the (110) rutile surface where the fast growth of the hydroxylapatite crystal was directed outwards from the surface. The (100) face had poor adhesion at the interface. The orientations of the precipitated crystallites play a significant role in the faster coverage of the (001) rutile face. Based on the experimental results, a model for the hydroxylapatite growth process is given.
Keywords: Rutile; Titanium; Hydroxylapatite; Bioactivity; XRD; TEM;

In vivo study of anterior cruciate ligament regeneration using mesenchymal stem cells and silk scaffold by Hongbin Fan; Haifeng Liu; Eugene J.W. Wong; Siew L. Toh; James C.H. Goh (3324-3337).
Although most in vitro studies indicate that silk is a suitable biomaterial for ligament tissue engineering, in vivo studies of implanted silk scaffolds for ligament reconstruction are still lacking. The objective of this study is to investigate anterior cruciate ligament (ACL) regeneration using mesenchymal stem cells (MSCs) and silk scaffold. The scaffold was fabricated by incorporating microporous silk sponges into knitted silk mesh, which mimicked the structures of ligament extracellular matrix (ECM). In vitro culture demonstrated that MSCs on scaffolds proliferated vigorously and produced abundant collagen. The transcription levels of ligament-specific genes also increased with time. Then MSCs/scaffold was implanted to regenerate ACL in vivo. After 24 weeks, histology observation showed that MSCs were distributed throughout the regenerated ligament and exhibited fibroblast morphology. The key ligament ECM components including collagen I, collagen III, and tenascin-C were produced prominently. Furthermore, direct ligament–bone insertion with typical four zones (bone, mineralized fibrocartilage, fibrocartilage, ligament) was reconstructed, which resembled the native structure of ACL–bone insertion. The tensile strength of regenerated ligament also met the mechanical requirements. Moreover, its histological grading score was significantly higher than that of control. In conclusion, the results imply that silk scaffold has great potentials in future clinical applications.
Keywords: Tissue engineering; Ligament; Silk; Regeneration; Mesenchymal stem cells;

Injectable tissue-engineered bone composed of human adipose-derived stromal cells and platelet-rich plasma by Yunsong Liu; Yongsheng Zhou; Hailan Feng; Gui-e Ma; Yongwei Ni (3338-3345).
This study aimed to evaluate the effects of human platelet-rich plasma (hPRP) on the proliferation and osteogenic differentiation of human adipose-derived stromal cells (hADSCs) and to construct a novel injectable tissue-engineered bone (ITB) composed of hPRP and hADSCs. hADSCs were isolated from liposuction tissues of healthy patients. hPRP was obtained by traditional two-step centrifugation. MTT, alkaline phosphatase (ALP) activity and mineralization assays were used to evaluate the effects of different concentrations of hPRP on cell proliferation and osteogenic differentiation in vitro. hADSCs cultured in optimal concentration of activated hPRP were subcutaneously injected into the inguinal groove of nude mice with hPRP and thrombin. X-ray, H&E staining and immunohistochemical analysis were used to examine the bone formation. Studies in vitro revealed that cell proliferation, ALP activity and mineralization were induced by hPRP and 10–12.5% of hPRP seemed to be the optimal concentration. Studies in vivo showed that this ITB formed bone structure in heterotopic site of nude mice. These findings indicate that the ITB composed of hPRP and hADSCs may represent a prologue for the development of a novel biological solution to bone defect. However, further investigations should be done to fully reveal the characteristics of this ITB.
Keywords: Bone tissue engineering; Platelet-rich plasma; Adipose-derived stromal cells; Cell proliferation; Cell differentiation; Injectable bone;

An adaptable hydrogel array format for 3-dimensional cell culture and analysis by Leenaporn Jongpaiboonkit; William J. King; Gary E. Lyons; Amy L. Paguirigan; Jay W. Warrick; David J. Beebe; William L. Murphy (3346-3356).
Hydrogels have been commonly used as model systems for 3-dimensional (3-D) cell biology, as they have material properties that resemble natural extracellular matrices (ECMs), and their cell-interactive properties can be readily adapted in order to address a particular hypothesis. Natural and synthetic hydrogels have been used to gain fundamental insights into virtually all aspects of cell behavior, including cell adhesion, migration, and differentiated function. However, cell responses to complex 3-D environments are difficult to adequately explore due to the large number of variables that must be controlled simultaneously. Here we describe an adaptable, automated approach for 3-D cell culture within hydrogel arrays. Our initial results demonstrate that the hydrogel network chemistry (both natural and synthetic), cell type, cell density, cell adhesion ligand density, and degradability within each array spot can be systematically varied to screen for environments that promote cell viability in a 3-D context. In a test-bed application we then demonstrate that a hydrogel array format can be used to identify environments that promote viability of HL-1 cardiomyocytes, a cell line that has not been cultured previously in 3-D hydrogel matrices. Results demonstrate that the fibronectin-derived cell adhesion ligand RGDSP improves HL-1 viability in a dose-dependent manner, and that the effect of RGDSP is particularly pronounced in degrading hydrogel arrays. Importantly, in the presence of 70 μm RGDSP, HL-1 cardiomyocyte viability does not decrease even after 7 days of culture in PEG hydrogels. Taken together, our results indicate that the adaptable, array-based format developed in this study may be useful as an enhanced throughput platform for 3-D culture of a variety of cell types.
Keywords: Extracellular matrix; Degradation; RGD peptide; Tissue engineering; Cardiomyocyte;

Towards control of smooth muscle cell differentiation in synthetic 3D scaffolds by Simon C. Baker; Jennifer Southgate (3357-3366).
A central tenant of tissue engineering is that cells should be able to recapitulate full functional tissue capability when placed within an appropriate architecture or scaffold. The aim of this study was to examine the effect of three-dimensional (3D) architecture on the differentiated phenotype of human smooth muscle cells derived from the stroma of the lower urinary tract. Stromal cell cultures were established from surgical specimens and the differentiated smooth muscle cell phenotype was monitored by gene expression, immunofluorescence and immunoblotting. Expression of contractile proteins, including smooth muscle myosin and smoothelin, was lost by cultures grown on two-dimensional (2D) tissue culture polystyrene, but was regained to some extent by the removal of serum and by the addition of TGFβ1. Stromal cells were seeded onto plasma-coated electrospun polystyrene scaffolds to examine the influence of 3D architecture on smooth muscle cell phenotype, but differentiation was inhibited by serum proteins that adsorbed non-specifically onto the large surface area of the scaffold. Stromal cells failed to adhere to the scaffold in serum-free conditions, but laminin pre-coating of the scaffold prevented serum adsorption and promoted cell attachment and differentiation. The study highlights how non-specific factors, such as serum adsorption, may confound the development of materials for tissue engineering.
Keywords: Bladder; Tissue culture; Smooth muscle cell; Differentiation; Electrospinning; Polystyrene; Scaffold;

Exploiting lipid raft transport with membrane targeted nanoparticles: A strategy for cytosolic drug delivery by Kathryn C. Partlow; Gregory M. Lanza; Samuel A. Wickline (3367-3375).
The ability to specifically deliver therapeutic agents to selected cell types while minimizing systemic toxicity is a principal goal of nanoparticle-based drug delivery approaches. Numerous cellular portals exist for cargo uptake and transport, but after targeting, intact nanoparticles typically are internalized via endocytosis prior to drug release. However, in this work, we show that certain classes of nanoparticles, namely lipid-coated liquid perfluorocarbon emulsions, undergo unique interactions with cells to deliver lipophilic substances to target cells without the need for entire nanoparticle internalization. To define the delivery mechanisms, fluorescently-labeled nanoparticles complexed with αvβ3-integrin targeting ligands were incubated with αvβ3-integrin expressing cells (C32 melanoma) under selected inhibitory conditions that revealed specific nanoparticle-to-cell interactions. We observed that the predominant mechanism of lipophilic delivery entailed direct delivery of lipophilic substances to the target cell plasma membrane via lipid mixing and subsequent intracellular trafficking through lipid raft-dependent processes. We suggest that local drug delivery to selected cell types could be facilitated by employing targeted nanoparticles designed specifically to utilize alternative membrane transport mechanisms.
Keywords: Drug delivery; Nanoparticle; Confocal microscopy; Molecular imaging; Membrane fusion; Lipid exchange;

Reducible heparin nanogels cross-linked with disulfide linkages were developed for efficient cellular uptake of therapeutic heparin to induce apoptotic cell death. The heparin nanogels were synthesized by forming nanocomplexes between thiolated heparin and poly(ethylene glycol) in a selected organic solvent, and subsequently producing intermolecular disulfide bonds between thiolated heparin molecules by ultrasonication. The resultant heparin nanogels had a stable structure with an average diameter of 248.7 ± 26.8 nm in aqueous solution. However, they rapidly disintegrated and released free heparin molecules under reductive environments, such as intracellular cytosol, through the cleavage of disulfide cross-links within their network structure. Confocal laser scanning microscopy and flow cytometric analysis revealed that these heparin nanogels significantly inhibited proliferation of mouse melanoma cells by inducing caspase-mediated apoptotic cell death. The present study suggested that the reducible heparin nanogels exhibiting a remarkable apoptotic activity could be potentially applied for cancer cell targeted delivery when combined with various therapeutic and diagnostic agents.
Keywords: Heparin; Nanogel; Disulfide; Apoptosis; Drug delivery system;

The importance of particle size and DNA condensation salt for calcium phosphate nanoparticle transfection by Claudio E. Pedraza; David C. Bassett; Marc D. McKee; Valentin Nelea; Uwe Gbureck; Jake E. Barralet (3384-3392).
Calcium phosphate has been used for over 30 years to deliver genetic material to mammalian cells. This vector has proven advantages over other transfection species such as viruses and dendrimers in terms of superior biocompatibility and reduced immune response. However, clinical application of calcium phosphate based transfection techniques is hampered by poor understanding of the key factors underlying its action. Despite widespread in vitro use, little attention has been given to the physico-chemical characteristics of the calcium phosphate particles mediating transfection. In this study parameters were optimised to produce calcium phosphate nanoparticles onto which plasmid DNA (pDNA) was adsorbed that were more effective than a commercial dendrimer vector in delivering pDNA to an osteoblastic cell line and compared favourably in a fibroblastic cell line without the need for special culture conditions such as cell cycle synchronization or glycerol shock treatment. Addition of the pDNA after nanoparticle synthesis allowed for characterisation of particle morphology, size, surface charge and composition. We found that the key parameters for effective calcium phosphate nanoparticle transfection were an optimal concentration of calcium and chloride ions and a nanosized non-agglomerated precipitate.
Keywords: Calcium phosphate; Nanoparticles; Non-viral; Transfection; Green fluorescent protein; Gene therapy;

A phenomenological model for the degradation of biodegradable polymers by Ying Wang; Jingzhe Pan; Xiaoxiao Han; Csaba Sinka; Lifeng Ding (3393-3401).
This paper presents a phenomenological diffusion–reaction model for the biodegradation of biodegradable polymers. The biodegradation process is modelled using a set of simplified reaction–diffusion equations. These partial differential equations are non-dimensionalised giving two normalised parameters which control the interplay between the hydrolysis reaction and the monomer diffusion. The equations are firstly solved for simple cases of plates and pins. The numerical results are presented in the form of biodegradation maps which show the conditions where the biodegradation is controlled by auto-catalysed hydrolysis, non-catalysed hydrolysis, a combination of auto-catalysed and non-catalysed hydrolyses, or a combination of hydrolysis and monomer diffusion, respectively. The degradation maps provide a clear guide for the design of biodegradable fixation devices used in orthopaedic surgeries. Finally the diffusion–reaction equations are solved using the finite element method for strip and square meshes, showing how the model can be used to assist the design of sophisticated fixation devices.
Keywords: Biodegradable polymers; Biodegradation; Modelling; Finite element analysis;