Ketul C. Popat

Bone tissue engineering: A review in bone biomimetics and drug delivery strategies

Critical‐sized defects in bone, whether induced by primary tumor resection, trauma, or selective surgery have in many cases presented insurmountable challenges to the current gold standard treatment for bone repair. The primary purpose of a tissue‐engineered scaffold is to use engineering principles to incite and promote the natural healing process of bone which does not occur in critical‐sized defects. A synthetic bone scaffold must be biocompatible, biodegradable to allow native tissue integration, and mimic the multidimensional hierarchical structure of native bone. In addition to being physically and chemically biomimetic, an ideal scaffold is capable of eluting bioactive molecules (e.g., BMPs, TGF‐βs, etc., to accelerate extracellular matrix production and tissue integration) or drugs (e.g., antibiotics, cisplatin, etc., to prevent undesired biological response such as sepsis or cancer recurrence) in a temporally and spatially controlled manner. Various biomaterials including ceramics, metals, polymers, and composites have been investigated for their potential as bone scaffold materials. However, due to their tunable physiochemical properties, biocompatibility, and controllable biodegradability, polymers have emerged as the principal material in bone tissue engineering. This article briefly reviews the physiological and anatomical characteristics of native bone, describes key technologies in mimicking the physical and chemical environment of bone using synthetic materials, and provides an overview of local drug delivery as it pertains to bone tissue engineering is included.

Optical sensing of biomolecules using microring resonators

A biosensor application of vertically coupled glass microring resonators with Q/spl sim/12 000 is introduced. Using balanced photodetection, very high signal to noise ratios, and thus high sensitivity to refractive index changes (limit of detection of 1.8/spl times/10/sup -5/ refractive index units), are achieved. Ellipsometry and X-ray photoelectron spectroscopy results indicate successful modification of biosensor surfaces. Experimental data obtained separately for a bulk change of refractive index of the medium and for avidin-biotin binding on the ring surface are reported. Excellent repeatability and close-to-complete surface regeneration after binding are experimentally demonstrated.

Biodegradable poly(ɛ-caprolactone) nanowires for bone tissue engineering applications

Critical-sized defects in bone, whether caused by cancer tumor resection, trauma, or selective surgery have in many cases presented insurmountable challenges to the current gold-standard treatment for bone repair. The primary purpose of a tissue-engineered scaffold is to incite and promote the natural healing process of bone, which does not occur in critical-sized defects. In this work, a solvent-free template synthesis technique was utilized to fabricate uniform arrays of substrate-bound poly(epsilon-caprolactone) (PCL) nanowires. Biodegradation of PCL nanowire surfaces was characterized using scanning electron microscopy (SEM) and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. Rat bone marrow-derived mesenchymal stem cells (MSCs) were employed to assess short-term biocompatibility and long-term bioactivity of nanowire surfaces. Short-term cell studies indicated that PCL nanowire surfaces supported enhanced cell adhesion and viability compared with control surfaces. MSCs seeded on nanowire surfaces also displayed increased levels of alkaline phosphatase (ALP) after 1, 2, and 3 weeks in culture. Calcium-phosphate mineralization was substantially accelerated on nanowire surfaces compared to control surfaces as indicated through calcium staining, von Kossa staining, SEM, and electron dispersive spectroscopy (EDS). Increased levels of inter- and extracellular levels of osteocalcin and osteopontin were observed on nanowire surfaces using immunofluorescence techniques after 3 weeks of culture. Considering the simplicity of the presented fabrication technique, capacity for solvent-free encapsulation of bioactive molecules or particles, and enhanced MSC performance on nanowire surfaces, this work presents an excellent foundation for the development of 3-D scaffolds for bone tissue regeneration.

Osteogenic differentiation of bone marrow stromal cells on poly(ε-caprolactone) nanofiber scaffolds

Nanofiber poly(epsilon-caprolactone) (PCL) scaffolds were fabricated by electrospinning, and their ability to enhance the osteoblastic behavior of marrow stromal cells (MSCs) in osteogenic media was investigated. MSCs were isolated from Wistar rats and cultured on nanofiber scaffolds to assess short-term cytocompatibility and long-term phenotypic behavior. Smooth PCL substrates were used as control surfaces. The short-term cytocompatibility results indicated that nanofiber scaffolds supported greater cell adhesion and viability compared with control surfaces. In osteogenic conditions, MSCs cultured on nanofiber scaffolds also displayed increased levels of alkaline phosphatase activity for 3 weeks of culture. Calcium phosphate mineralization was substantially accelerated on nanofiber scaffolds compared to control surfaces as indicated through von Kossa and calcium staining, scanning electron microscopy and energy-dispersive X-ray spectroscopy. Increased levels of intra- and extracellular levels of osteocalcin and osteopontin were observed on nanofiber scaffolds using immunofluorescence techniques after 3 weeks of culture. These results demonstrate the enhanced tissue regeneration property of nanofiber scaffolds, which may be of potential use for engineering osteogenic scaffolds for orthopedic applications.

Preservation of FGF-2 bioactivity using heparin-based nanoparticles, and their delivery from electrospun chitosan fibers

Here we present a novel matrix-mimetic nanoassembly based on polysaccharides. Chitosan electrospun fiber networks are decorated with heparin-containing polyelectrolyte complex nanoparticles (PCNs) that present basic fibroblast growth factor (FGF-2), both stably adsorbed to the surfaces and released into solution. These FGF-2/PCN complexes can be released from the fibers with zero-order kinetics over a period of 30 days. Further modification of fibers with a single bilayer of polyelectrolyte multilayer (PEM) composed of N,N,N-trimethyl chitosan and heparin completely prevent release, and the FGF-2/PCN complexes are retained on the fibers for the duration of the release experiment (30 days). We also compare the mitogenic activity of these FGF-2/PCN complexes delivered in two different states: adsorbed to a surface and dissolved in solution. FGF-2/PCN complexes exhibit mitogenic activity with respect to ovine bone marrow-derived mesenchymal stem cells, even after being preconditioned by incubating for 27 days at 37°C in solution. However, when the FGF-2/PCN complexes are adsorbed to chitosan and coated with PEMs, the mitogenic activity of the FGF-2 steadily decreases with increasing preconditioning time. This work demonstrates a new system for stabilizing and controlling the delivery of heparin-binding growth factors, using polysaccharide-based matrix-mimetic nanomaterials. This work also contributes to our understanding of the preferred mode of growth factor delivery from porous scaffolds.

Hemocompatibility of titania nanotube arrays

Hemocompatibility is a key consideration for the long-term success of blood contacting biomaterials; hence, there is a critical need to understand the physiological response elicited from blood/nano-biomaterial interactions. In this study, we have investigated the adsorption of key blood serum proteins, in vitro adhesion and activation of platelets, and clotting kinetics of whole blood on titania nanotube arrays. Previous studies have demonstrated improved mesenchymal stem cell functionality, osteoblast phenotypic behavior, localized drug delivery, and the production of endothelial cell ECM on titania nanotube arrays. Furthermore, these titania nanotube arrays have elicited minimal levels of monocyte activation and cytokine secretion, thus exhibiting a very low degree of immunogenicity. Titania nanotube arrays were fabricated using anodization technique and the surface morphology was examined through scanning electron microscopy (SEM). The crystalline phases were identified using glancing angled X-ray diffraction (GAXRD). Nanoindentation and scratch tests were used to characterize the mechanical properties of titania nanotube arrays. The adsorption of key blood proteins (albumin, fibrinogen, and immunoglobulin-g) was evaluated using a micro-BCA assay and X-ray photoelectron spectroscopy (XPS). The adhesion and activation of platelets was investigated using live-cell staining, MTT assay, and SEM. Whole blood clotting kinetics was evaluated by measuring the free hemoglobin concentration, and SEM was used to visualize the clot formation. Our results indicate increased blood serum protein adsorption, platelet adhesion and activation, and whole blood clotting kinetics on titania nanotube arrays.

In vitro inflammatory response of nanostructured titania, silicon oxide, and polycaprolactone.

Nanostructured materials are ubiquitous in tissue engineering, drug delivery, and biosensing applications. Nonetheless, little is known about the inflammatory response of materials differing in surface nanoarchitecture. Here we report human monocyte viability and morphology, in addition to inflammatory cytokines (IL-1alpha and B, IL-6, IL-10, IFN-alpha and gamma, TNF-alpha, IL-12, MIP-1alpha and beta), and reactive oxygen species production on several nanostructured surfaces, compared to flat surfaces of the same material. The surfaces studied were titiania nanotubes, short and long silicon oxide, and polycaprolactone nanowires. The results indicate that inflammation on titanium, polycaprolactone, and silicon oxide materials can be reduced by restructuring the surface with nanoarchitecture. Nanostructured surfaces display a reduced inflammation response compared to a respective flat control, with significant differences between titanium and nanotubular titanium. Little difference is observed in the inflammatory response between short and long nanowires of PCL and silicon oxide. All surfaces are significantly less inflammatory than the positive control, lipopolysaccharide. Additionally, we show that flat titanium is more inflammatory than silicon oxide and polycaprolactone. This study shows that nanoarchitecture can be used to reduce the inflammatory response of human monocytes in vitro.

Surface Modification of SU-8 for Enhanced Biofunctionality and Nonfouling Properties

SU-8 is a chemically amplified, epoxy-based negative photoresist typically used for producing ultrathick resist layers during device manufacturing in the semiconductor industry. As a simple resist, SU-8 has garnered attention as a possible material for a variety of biomedical applications, including tissue engineering, drug delivery, as well as cell-based screening and sensing. However, as a hydrophobic material, the use of SU-8 is limited due to a high degree of nonspecific adsorption of biomolecules, as well as limited cell attachment. In this work, surface chemistry is utilized to modify the SU-8 surface by covalently attaching poly(ethylene glycol) (PEG) to increase biofunctionality and improve its nonfouling properties. Different molecular weights and concentrations of PEG were used to form films of various grafting densities on SU-8 surfaces. X-ray photoelectron spectroscopy (XPS) was used to verify the presence of PEG moieties on the SU-8 surface. High-resolution C1s spectra show that, with an increase in concentration and immobilization time, the grafting density of PEG also increases. Further, a standard overlayer model was used to calculate the thickness of the PEG films formed. The effect of PEG-modified SU-8 was examined in terms of protein adsorption on the surface and fibroblast-surface interactions.

Template synthesized poly(ɛ-caprolactone) nanowire surfaces for neural tissue engineering

Tissue engineering therapies targeted at nerve regeneration in spinal cord injuries (SCI) have broad social and economic benefits to the American population. Due to the complicated pathophysiology of SCI, there are very few options available for functional regeneration of the spinal column. Nanotechnology offers interesting avenues to explore tissue engineering in SCI. In this study, we have developed a novel solvent free nanotemplating technique for fabricating poly(epsilon-caprolactone) (PCL) surfaces with controlled arrays of high aspect ratio substrate-bound nanowires for the growth and maintenance of differentiated states of neuronal cells. PC12 cells were used to evaluate the ability of nanowire surfaces to promote neuronal phenotypic behavior. Cell adhesion, proliferation and viability were investigated for up to 4 days of culture using fluorescence microscopy, scanning electron microscopy (SEM) and MTT activity. Our results indicate significantly higher cell adhesion and subsequent proliferation and viability of PC12 cells cultured on nanowire surfaces as compared to control surfaces without any nanoarchitecture. Further, the adhered cells were maintained in a differentiated state for 7 days and neuronal network formation and expression of neuronal markers were investigated using fluorescence microscopy, SEM and immunofluorescence. Cells on nanowire surfaces expressed key neuronal markers and demonstrated neuronal phenotypic behavior as compared to the cells on control surfaces.

In vitro immunogenicity of silicon-based micro- and nanostructured surfaces.

The increasing use of micro- and nanostructured silicon-based devices for in vivo therapeutic or sensing applications highlights the importance of understanding the immunogenicity of these surfaces. Four silicon surfaces (nanoporous, microstructured, nanochanneled, and flat) were studied for their ability to provoke an immune response in human blood derived monocytes. The monocytes were incubated with the surfaces for 48 h and the immunogenicity was evaluated based on the viability, shape factors, and cytokine expression. Free radical oxygen formation was measured at 18 h to elicit a possible mechanism invoking immunogenicity. Although no cytokines were significantly different comparing the response of monocytes on the tissue culture polystyrene surfaces to those on the micropeaked surfaces, on average all cytokines were elevated on the micropeaked surface. The monocytes on the nanoporous surface also displayed an elevated cytokine response, overall, but not to the degree of those on the micropeaked surface. The nanochanneled surface response was similar to that of flat silicon. Overall, the immunogenicity and biocompatibility of flat, nanochanneled, and nanoporous silicon toward human monocytes are approximately equivalent to tissue culture polystyrene.