Neural Marker Expression in Adipose-Derived Stem Cells Grown in PEG-Based 3D Matrix Is Enhanced in the Presence of B27 and CultureOne Supplements.

Adipose-derived stem cells (ADSCs) have incredible potential as an avenue to better understand and treat neurological disorders. While they have been successfully differentiated into neural stem cells and neurons, most such protocols involve 2D environments, which are not representative of in vivo physiology. In this study, human ADSCs were cultured in 1.1 kPa polyethylene-glycol 3D hydrogels for 10 days with B27, CultureOne (C1), and N2 neural supplements to examine the neural differentiation potential of ADSCs using both chemical and mechanical cues. Following treatment, cell viability, proliferation, morphology, and proteome changes were assessed. Results showed that cell viability was maintained during treatments, and while cells continued to proliferate over time, proliferation slowed down. Morphological changes between 3D untreated cells and treated cells were not observed. However, they were observed among 2D treatments, which exhibited cellular elongation and co-alignment. Proteome analysis showed changes consistent with early neural differentiation for B27 and C1 but not N2. No significant changes were detected using immunocytochemistry, potentially indicating a greater differentiation period was required. In conclusion, treatment of 3D-cultured ADSCs in PEG-based hydrogels with B27 and C1 further enhances neural marker expression, however, this was not observed using supplementation with N2.

Adipose-Derived Stem Cells Spontaneously Express Neural Markers When Grown in a PEG-Based 3D Matrix.

Neurological diseases are among the leading causes of disability and death worldwide and remain difficult to treat. Tissue engineering offers avenues to test potential treatments; however, the development of biologically accurate models of brain tissues remains challenging. Given their neurogenic potential and availability, adipose-derived stem cells (ADSCs) are of interest for creating neural models. While progress has been made in differentiating ADSCs into neural cells, their differentiation in 3D environments, which are more representative of the in vivo physiological conditions of the nervous system, is crucial. This can be achieved by modulating the 3D matrix composition and stiffness. Human ADSCs were cultured for 14 days in a 1.1 kPa polyethylene glycol-based 3D hydrogel matrix to assess effects on cell morphology, cell viability, proteome changes and spontaneous neural differentiation. Results showed that cells continued to proliferate over the 14-day period and presented a different morphology to 2D cultures, with the cells elongating and aligning with one another. The proteome analysis revealed 439 proteins changed in abundance by >1.5 fold. Cyclic nucleotide 3'-phosphodiesterase (CNPase) markers were identified using immunocytochemistry and confirmed with proteomics. Findings indicate that ADSCs spontaneously increase neural marker expression when grown in an environment with similar mechanical properties to the central nervous system.

Bioresorbable zinc hydroxyapatite guided bone regeneration membrane for bone regeneration.

OBJECTIVES: The aim of this study was to investigate the bone regenerative properties of a heat treated cross-linked GBR membrane with zinc hydroxyapatite powders in the rat calvarial defect model over a 6-week period. MATERIAL AND METHODS: In vitro physio-chemical characterization involved X-ray diffraction analysis, surface topology by scanning electron microscopy, and zinc release studies in physiological buffers. Bilateral rat calvarial defects were used to compare the Zn-HAp membranes against the commercially available collagen membranes and the unfilled defect group through radiological and histological evaluation. RESULTS: The synthesized Zn-MEM (100 µm thick) showed no zinc ions released in the phosphate buffer solution (PBS) buffer, but zinc was observed under acidic conditions. At 6 weeks, both the micro-CT and histological analyses revealed that the Zn-MEM group yielded significantly greater bone formation with 80 ± 2% of bone filled, as compared with 60 ± 5% in the collagen membrane and 40 ± 2% in the unfilled control group. CONCLUSION: This study demonstrated the use of heat treatment as an alternative method to cross-linking the Zn-MEM to be applied as a GBR membrane. Its synthesis and production are relatively simple to fabricate, and the membrane had rough surface features on one side, which might be beneficial for cellular activities. In a rat calvarial defect model, it was shown that new bone formation was accelerated in comparison with the collagen membrane and the unfilled defect groups. These results would suggest that Zn-MEM has the potential for further development in dental applications.

Marine structure derived calcium phosphate-polymer biocomposites for local antibiotic delivery.

Hydrothermally converted coralline hydroxyapatite (HAp) particles loaded with medically active substances were used to develop polylactic acid (PLA) thin film composites for slow drug delivery systems. The effects of HAp particles within PLA matrix on the gentamicin (GM) release and release kinetics were studied. The gentamicin release kinetics seemed to follow Power law Korsmeyer Peppas model with mainly diffusional process with a number of different drug transport mechanisms. Statistical analysis shows very significant difference on the release of gentamicin between GM containing PLA (PLAGM) and GM containing HAp microspheres within PLA matrix (PLAHApGM) devices, which PLAHApGM displays lower release rates. The use of HAp particles improved drug stabilization and higher drug encapsulation efficiency of the carrier. HAp is also the source of Ca2+ for the regeneration and repair of diseased bone tissue. The release profiles, exhibited a steady state release rate with significant antimicrobial activity against Staphylococcus aureus (S. aureus) (SH1000) even at high concentration of bacteria. The devices also indicated significant ability to control the growth of bacterial even after four weeks of drug release. Clinical release profiles can be easily tuned from drug-HAp physicochemical interactions and degradation kinetics of polymer matrix. The developed systems could be applied to prevent microbial adhesion to medical implant surfaces and to treat infections mainly caused by S. aureus in surgery.

Coral exoskeletons as a precursor material for the development of a calcium phosphate drug delivery system for bone tissue engineering.

With the global rise in aging of populations, the occurrence of osteoporosis will continue to increase. Biomaterial and pharmaceutical scientists continue to develop innovative strategies and materials to address this disease. In this article, we describe a new perspective and approach into the use of coral exoskeletons as a precursor material to synthesize a calcium phosphate-based drug delivery system. Studies detailing the methodology of the conversion methods and the strategies and approach for the development of these novel drug delivery systems are described. Furthermore, in vivo studies in osteoporotic mice using a drug loaded and chemically modified version of the biomimetic delivery system showed significant cortical and cancellous bone increases. These studies support the notion and the rationale for future research and development of the use of coral exoskeletons as materials for drug delivery applications.

Bone regeneration of rat tibial defect by zinc-tricalcium phosphate (Zn-TCP) synthesized from porous Foraminifera carbonate macrospheres.

Foraminifera carbonate exoskeleton was hydrothermally converted to biocompatible and biodegradable zinc-tricalcium phosphate (Zn-TCP) as an alternative biomimetic material for bone fracture repair. Zn-TCP samples implanted in a rat tibial defect model for eight weeks were compared with unfilled defect and beta-tricalcium phosphate showing accelerated bone regeneration compared with the control groups, with statistically significant bone mineral density and bone mineral content growth. CT images of the defect showed restoration of cancellous bone in Zn-TCP and only minimal growth in control group. Histological slices reveal bone in-growth within the pores and porous chamber of the material detailing good bone-material integration with the presence of blood vessels. These results exhibit the future potential of biomimetic Zn-TCP as bone grafts for bone fracture repair.

Extracellular matrix remodelling during cell adhesion monitored by the quartz crystal microbalance.

A cell's ability to remodel adsorbed protein layers on surfaces is influenced by the nature of the protein layer itself. Remodelling is often required to accomplish cellular adhesion and extracellular matrix formation which forms the basis for cell spreading, increased adhesion and expression of different phenotypes. The adhesion of NIH3T3 (EGFP) fibroblasts to serum protein (albumin or fibronectin) precoated tantalum (Ta) and oxidised polystyrene (PS(ox)) surfaces was examined using the quartz crystal microbalance with dissipation (QCM-D) monitoring and fluorescence microscopy. The cells were either untreated or treated with cycloheximide to examine the contribution of endogenous protein production during cell adhesion to the QCM-D response over a period of 2h. Following adsorption of albumin onto Ta and PS(ox) there was no difference detected between the response to seeding untreated and cycloheximide treated cells. The QCM-D was able to detect differences in the untreated cellular responses to fibronectin versus serum precoated Ta and PS(ox) substrates, while cycloheximide treatment of the cells produced the same QCM-D response for fibronectin and serum precoatings on each of the materials. This confirmed that the process of matrix remodelling by the cells is dependent on the underlying substrate and the preadsorbed proteins and that the QCM-D response is dominated by changes in the underlying protein layer. Changes in dissipation correspond to the development of the actin cytoskeleton as visualised by actin staining.

Assessment of bone ingrowth into porous biomaterials using MICRO-CT.

The three-dimensional (3D) structure and architecture of biomaterial scaffolds play a critical role in bone formation as they affect the functionality of the tissue-engineered constructs. Assessment techniques for scaffold design and their efficacy in bone ingrowth studies require an ability to accurately quantify the 3D structure of the scaffold and an ability to visualize the bone regenerative processes within the scaffold structure. In this paper, a 3D micro-CT imaging and analysis study of bone ingrowth into tissue-engineered scaffold materials is described. Seven specimens are studied in this paper; a set of three specimens with a cellular structure, varying pore size and implant material, and a set of four scaffolds with two different scaffold designs investigated at early (4 weeks) and late (12 weeks) explantation times. The difficulty in accurately phase separating the multiple phases within a scaffold undergoing bone regeneration is first highlighted. A sophisticated three-phase segmentation approach is implemented to develop high-quality phase separation with minimal artifacts. A number of structural characteristics and bone ingrowth characteristics of the scaffolds are quantitatively measured on the phase separated images. Porosity, pore size distributions, pore constriction sizes, and pore topology are measured on the original pore phase of the scaffold volumes. The distribution of bone ingrowth into the scaffold pore volume is also measured. For early explanted specimens we observe that bone ingrowth occurs primarily at the periphery of the scaffold with a constant decrease in bone mineralization into the scaffold volume. Pore size distributions defined by both the local pore geometry and by the largest accessible pore show distinctly different behavior. The accessible pore size is strongly correlated to bone ingrowth. In the specimens studied a strong enhancement of bone ingrowth is observed for pore diameters>100 microm. Little difference in bone ingrowth is measured with different scaffold design. This result illustrates the benefits of microtomography for analyzing the 3D structure of scaffolds and the resultant bone ingrowth.

Engineering thick tissues--the vascularisation problem.

The ability to create thick tissues is a major tissue engineering challenge, requiring the development of a suitable vascular supply. Current trends are seeing the utilization of cells seeded into hybrid matrix/scaffold systems to create in vitro vascular analogues. Approaches that aim to create vasculature in vitro include the use of biological extracellular matrices such as collagen hydrogels, porous biodegradable polymeric scaffolds with macro- and micro-lumens and micro-channels, co-culture of cells, incorporation of growth factors, culture in dynamic bioreactor environments, and combinations of these. Of particular interest are those approaches that aim to create bioengineered tissues in vitro that can be readily connected to the host's vasculature following implantation in order to maintain cell viability.

Development and dissolution studies of bisphosphonate (clodronate)-containing hydroxyapatite-polylactic acid biocomposites for slow drug delivery.

An increase in clinical demand on the controlled release of bisphosphonates (BPs) due to complications associated with systemic administration, has been the current driving force on the development of BP drug-release systems. Bisphosphonates have the ability to bind to divalent metal ions, such as Ca2+ , in bone mineral and prevent bone resorption by influencing the apoptosis of osteoclasts. Localized delivery using biodegradable materials, such as polylactic acid (PLA) and hydroxyapatite (HAp), which are ideal in this approach, have been used in this study to investigate the dissolution of clodronate (non-nitrogen-containing bisphosphonate) in a new release system. The effects of coral structure-derived HAp and the release kinetics of the composites were evaluated. The release kinetics of clodronate from PLA-BP and PLA-HAp-BP systems seemed to follow the power law model described by Korsmeyer-Peppas. Drug release was quantified by 31 P-NMR with detection and quantification limits of 9.2 and 30.7 mM, respectively. The results suggest that these biocomposite systems could be tuned to release clodronate for both relatively short and prolonged period of time. In addition to drug delivery, the degradation of HAp supplies both Ca2+ and phosphate ions that can help in bone mineralization. Copyright © 2015 John Wiley & Sons, Ltd.

Natural and Synthetic Coral Biomineralization for Human Bone Revitalization.

Coral skeletons can regenerate replacement human bone in nonload-bearing excavated skeletal locations. A combination of multiscale, interconnected pores and channels and highly bioactive surface chemistry has established corals as an important alternative to using healthy host bone replacements. Here, we highlight how coral skeletal systems are being remolded into new calcified structures or synthetic corals by biomimetic processes, as places for the organized permeation of bone tissue cells and blood vessels. Progressive technologies in coral aquaculture and self-organization inorganic chemistry are helping to modify natural corals and create synthetic coral architectures able to accelerate bone regeneration with proper host integration at more skeletal locations, adapted to recent surgical techniques and used to treat intrinsic skeletal deformities and metabolic conditions.

The effect of charged groups on protein interactions with poly(HEMA) hydrogels.

Proteins, lipids and other biomolecules interact strongly with the acrylic-based biomaterials used for contact lenses. Although hydrogels are nominally resistant to protein fouling, many studies have reported considerable amounts of protein bound to poly(2-hydroxyethylmethacrylate) (PHEMA) lenses. This study examined the binding of a series of biomolecules (tear protein analogues, mucin and cholesterol) to poly(methylmethacrylate) (PMMA) and three HEMA-based hydrogels (PHEMA, HEMA plus methacrylic acid (P(HEMA-MAA)), HEMA plus methacrylic acid plus N-vinylpyrrolidone (P(HEMA-MAA-NVP))) by use of a quartz crystal microbalance with dissipation (QCM-D) monitoring. The QCM-D estimates changes in the mass and viscous constant for the adsorbed layer through measurements of frequency and dissipation. Protein interaction with each of the test materials caused a net increase in mass of the material indicating protein binding except for lysozyme interacting with P(HEMA-MAA). A net decrease in mass was observed for lysozyme interacting with P(HEMA-MAA) which may be ascribed to lysozyme collapsing the hydrogel by expelling water. A net mass decrease was observed for cholesterol interacting with each of the hydrogel materials, while a mass increase was observed on PMMA.

Lysozyme interaction with poly(HEMA)-based hydrogel.

Lysozyme interaction with an acrylic-based hydrogel, poly(2-hydroxyethyl methacrylate) co-methacrylic acid (P(HEMA-MAA)), was investigated using a combination of quartz crystal microbalance with dissipation (QCM-D), surface plasmon resonance (SPR) and dual polarisation interferometry (DPI). This combination of techniques demonstrated that lysozyme initially absorbed into the hydrogel matrix and displaced water from the hydrogel while subsequent lysozyme additions were adsorbed onto the surface of the hydrogel material. QCM-D, being sensitive to bound water, showed an overall decrease in mass and stiffening of the layer after lysozyme addition. SPR, a water insensitive technique, showed a net mass increase after addition of lysozyme and buffer rinses. DPI showed that the first exposure of lysozyme to P(HEMA-MAA) was consistent with lysozyme absorption while subsequent lysozyme exposures were consistent with lysozyme adsorption.

Monitoring cell adhesion on tantalum and oxidised polystyrene using a quartz crystal microbalance with dissipation.

The quartz crystal microbalance with dissipation (QCM-D) (Q-Sense AB, Sweden) has been established as a useful tool for evaluating interactions between various biological and non-biological systems, and there has been increasing interest in using the QCM-D technique for cell monitoring applications. This study investigated the potential of the QCM-D to characterise the initial adhesion and spreading of cells in contact with protein precoated biocompatible surfaces. The QCM-D technique is attractive for monitoring cell adhesion and spreading as it allows in situ real-time measurements. The adhesion of NIH3T3 (EGFP) fibroblasts to tantalum (Ta) and oxidised polystyrene (PS(ox)) surfaces precoated with serum proteins was examined using the QCM-D for a period of either 2 or 4 h. Time-lapse photography was performed at 30 min intervals to visually examine cell adhesion and spreading in order to relate cell morphology to the QCM-D response. Following adsorption of albumin, fibronectin or newborn calf serum onto the surfaces, QCM-D measurements showed that cells adhered and spread on the fibronectin and serum coated surfaces, while few cells adhered to the albumin coated surfaces. Cells adhered to albumin coated surfaces had a rounded morphology. The responses to fibronectin and serum precoated surfaces were quite different for each of the underlying substrates indicating that the process of cell adhesion and spreading elicits different responses depending on both the protein coating composition and the influence of the underlying substrate. The different response may be due to extracellular matrix remodelling as well as cytoskeletal changes. Frequency (f) and dissipation (D) changes associated with cell adhesion were less than would be expected from the Sauerbrey relation due to the viscoelastic properties of the cells.

Analysis of 3D bone ingrowth into polymer scaffolds via micro-computed tomography imaging.

This paper illustrates the utility of micro-computed tomography (micro-CT) to study the process of tissue engineered bone growth. A micro-CT facility for imaging and visualising biomaterials in three dimensions (3D) is described. The facility is capable of acquiring 3D images made up of 2000(3) voxels on specimens up to 60mm in extent with resolutions down to 2 microm. This allows the 3D structure of tissue engineered materials to be imaged across three orders of magnitude of detail. The capabilities of micro-CT are demonstrated by imaging the Haversian network within human femoral cortical bone (distal diaphysis) and bone ingrowth into a porous scaffold at varying resolutions. Phase identification combined with 3D visualisation enables one to observe the complex topology of the canalicular system of the cortical bone. Imaging of the tissue engineered bone at a scale of 1cm and resolutions of 10 microm allows visualisation of the complex ingrowth of bone into the polymer scaffold. Further imaging at 2 microm resolution allows observation of bone ultra-structure. These observations illustrate the benefits of tomography over traditional techniques for the characterisation of bone morphology and interconnectivity and performs a complimentary role to current histomorphometric techniques.

Protein adsorption on derivatives of hyaluronic acid and subsequent cellular response.

The modulation of biological interactions with artificial surfaces is a vital aspect of biomaterials research. Serum protein adsorption onto photoreactive hyaluronic acid (Hyal-N(3)) and its sulfated derivative (HyalS-N(3)) was analyzed to determine extent of protein interaction and protein conformation as well as subsequent cell adhesion. There were no significant (p < 0.01) differences in the amount of protein adsorbed to the two polymers; however, proteins were found to be more loosely bound on HyalS-N(3) compared with Hyal-N(3). Fibronectin was adsorbed onto HyalS-N(3) in such an orientation as to allow the availability of the cell binding region, while there was more restricted access to this region on fibronectin adsorbed onto Hyal-N(3). This was confirmed by reduced cell adhesion on fibronectin precoated Hyal-N(3) compared with fibronectin precoated HyalS-N(3). Minimal cell adhesion was observed on albumin and serum precoated Hyal-N(3). The quartz crystal microbalance confirmed that specific cell-surface interactions were experienced by cells interacting with the fibronectin precoated polymers and serum precoated HyalS-N(3).

The correlation of pore morphology, interconnectivity and physical properties of 3D ceramic scaffolds with bone ingrowth.

In the design of tissue engineering scaffolds, design parameters including pore size, shape and interconnectivity, mechanical properties and transport properties should be optimized to maximize successful inducement of bone ingrowth. In this paper we describe a 3D micro-CT and pore partitioning study to derive pore scale parameters including pore radius distribution, accessible radius, throat radius, and connectivity over the pore space of the tissue engineered constructs. These pore scale descriptors are correlated to bone ingrowth into the scaffolds. Quantitative and visual comparisons show a strong correlation between the local accessible pore radius and bone ingrowth; for well connected samples a cutoff accessible pore radius of approximately 100 microM is observed for ingrowth. The elastic properties of different types of scaffolds are simulated and can be described by standard cellular solids theory: (E/E(0))=(rho/rho(s))(n). Hydraulic conductance and diffusive properties are calculated; results are consistent with the concept of a threshold conductance for bone ingrowth. Simple simulations of local flow velocity and local shear stress show no correlation to in vivo bone ingrowth patterns. These results demonstrate a potential for 3D imaging and analysis to define relevant pore scale morphological and physical properties within scaffolds and to provide evidence for correlations between pore scale descriptors, physical properties and bone ingrowth.

Hollow-fiber assay for ligand-mediated cell adhesion.

BACKGROUND: The investigation of receptor-ligand interactions in the cellular context presents significant technical challenges, first, to immobilize the ligand in a manner that preserves functional properties and, second, to relate ligand properties to cell adhesion and other cellular processes. METHODS: Ligand-mediated cell adhesion was characterized by the development of a cellulose hollow-fiber adhesion assay in which ligand (protein A) was immobilized onto the cellulose membrane as a recombinant fusion protein containing a cellulose-binding domain affinity tag. Modules containing single cellulose hollow fibers were connected to a micro-flow system for cell deposition and detachment with fluid shear stress. The cell adhesion process that occurred inside a segment of hollow fiber was observed in real time by using an inverted microscope equipped with a CCD camera and digital frame grabber. Image analysis software was developed to count cells and record digital images. RESULTS: Cell adhesion strength was characterized by counting the number of cells that were detached by application of fluid shear stress with values that ranged from 2.3 to 185 dyne/cm2. The median shear stress of detachment of KG1a cells was directly related to the duration of membrane contact and the amount of immobilized monoclonal antibody (anti-CD34). CONCLUSIONS: The hollow-fiber assay provides a general method to determine functional properties of molecular domains that interact with cell surface receptors and markers.