Cartilage defects, which are caused by a variety of reasons such as traumatic injuries, osteoarthritis, or osteoporosis, represent common and severe clinical problems. Each year, over 6 million people visit hospitals in the U.S. for various knee, wrist, and ankle problems. As modern medicine advances, new and novel methodologies have been explored and developed in order to solve and improve current medical problems. One of the areas of investigation that has thus far proven to be very promising is tissue engineering [1, 2]. Since cartilage matrix is nanocomposite, the goal of the current work is to use nanomaterials and nanofabrication methods to create novel biologically inspired tissue engineered cartilage scaffolds for facilitating human bone marrow mesenchymal stem cell (MSC) chondrogenesis. For this purpose, through electrospinning techniques, we designed a series of novel 3D biomimetic nanostructured scaffolds based on carbon nanotubes and biocompatible poly(L-lactic acid) (PLLA) polymers. Specifically, a series of electrospun fibrous PLLA scaffolds with controlled fiber dimension were fabricated in this study. In vitro hMSC studies showed that stem cells prefer to attach in the scaffolds with smaller fiber diameter. More importantly, our in vitro differentiation results demonstrated that incorporation of the biomimetic carbon nanotubes and poly L-lysine coating can induce more chondrogenic differentiations of MSCs than controls, which make them promising for cartilage tissue engineering applications.

Ultrasound in the presence of microbubbles brings in transient increase in cell membrane permeability, which allows the entry of foreign molecules into cells. This platform has been applied in in vitro and in vivo gene delivery studies in recent years[1–2]. The frequently used microbubbles are air or inert gas encapsulated in a protein, lipid or polymer which is commonly used as FDA approved contrast agents in diagnostic ultrasound. On exposure to ultrasound the microbubbles lead to formation of small pores on the cell membrane. This work concentrates on application of this platform to enhance cellular uptake of nanoparticles and thereby achieve enhanced drug delivery. Nanoparticles can be manipulated at the nano level and have been applied in the realm of cancer detection and treatment for imaging, targeting tumors, and drug delivery purposes [2].

Nowadays, magnetic materials are receiving special attention due to their potential applications in different fields and in particular in medicine. Magnetic micro-nano-particles have been progressively employed as support materials for enzyme immobilization, and have been used as drug-delivery vehicles, contrast agents for magnetic resonance imaging as well as heat mediators for hyperthermia-based anti-cancer treatments and many other exciting biomedical applications. Magnetic materials have also attracted a big interest in the field of bone tissue regeneration because it has been demonstrated that magnetic nanoparticles have effect of osteoinduction even without external magnetic force. Therefore, one of the most big challenge in this field is the production of magnetic materials with good biocompatibility and biodegradability. In fact, the long-term effects in the human body of iron oxide (maghemite or magnetite), the most popular magnetic phase used in medicine and biotechnology, are not yet completely assessed. To this aim, in this work we developed an innovative biocompatible and bioresorbable superparamagnetic-like phase by doping nano-hydroxyapatite with Fe 2+ /Fe 3+ ions (FeHA). Moreover the same magnetic nanoparticles were used as nano-particulate emulsifier for the preparation of hollow hybrid Fe-HA-poly(L-lactic) acid (PLLA) micro-nano-spheres. PLLA has been used because poly(α-hydroxy-esters) are the most frequently used synthetic polymers for biomedical applications owing to their biocompatibility, hydrolytic degradation process and proper mechanical properties. These micro-nanospheres could be used as new type of scaffold for hard tissue regeneration. In fact, spherical scaffold display several advantages respect to the monolithic counterpart e.g., (i) improving control over sustained delivery of therapeutic agents, signalling biomolecules and even pluripotent stem cells, (ii) serving as stimulus-sensitive delivery vehicles for triggered release, (iii) introducing porosity and/or improve the mechanical properties of bulk scaffolds by acting as porogen or reinforcement phase, (iv) supplying compartmentalized micro-reactors for dedicated biochemical processes, (v) functioning as cell delivery vehicle, and, finally, (vi) giving possibility of preparing injectable and/or mouldable formulations to be applied by using minimally invasive surgery. Moreover, the same magnetic materials could find applications in nanomedicine as a multifunctional carrier. Their magnetic functionality could be utilized to move them into the body towards target organs by an external magnetic field. Furthermore, the superparamagnetic feature of the nanoparticles could allow to tailor the release of the therapeutic agent by switching (on-off) the external magnetic field and/or to treat cancer cells by hyperthermia.

This paper reports on the characterization of an integrated micro-fluidic platform for controlled electrical lysis of biological cells and subsequent extraction of intracellular biomolecules. The proposed methodology is capable of high throughput electrical cell lysis facilitated by nano-composite coated electrodes. The nano-composites are synthesized using Carbon Nanotube and ZnO nanorod dispersion in polymer. Bacterial cells are used to demonstrate the lysis performance of these nanocomposite electrodes. Investigation of electrical lysis in the microchannel is carried out under different parameters, one with continuous DC application and the other under DC biased AC electric field. Lysis in DC field is dependent on optimal field strength and governed by the cell type. By introducing the AC electrical field, the electrokinetics is controlled to prevent cell clogging in the micro-channel and ensure uniform cell dispersion and lysis. Lysis mechanism is analyzed with time-resolved fluorescence imaging which reveal the time scale of electrical lysis and explain the dynamic behavior of GFP-expressing E. coli cells under the electric field induced by nanocomposite electrodes. The DNA and protein samples extracted after lysis are compared with those obtained from a conventional chemical lysis method by using a UV–Visible spectroscopy and fluorimetry. The paper also focuses on the mechanistic understanding of the nano-composite coating material and the film thickness on the leakage charge densities which lead to differential lysis efficiency.

Achieving cell adhesion, growth and homeostasis on an underlying biomaterial surface may be a desirable feature in implant device design and tissue engineering. Insight has been gained from numerous cell patterning strategies where spatial cues and physical constraints have been shown to regulate the structure and function of cells. Despite significant advances in modifying substrates for cellular attachment, migration and proliferation, the achievement of confluent and aligned growth of functional endothelial cells on cardiovascular blood-contacting implants under physiologically significant wall shear stress has proven difficult. Recently we have reported on a method that enhances cellular adhesion under flow conditions on synthetic polymer surfaces, without reliance on pro-adhesive protein biomaterials, which are often thrombogenic. In this method we utilize electron beam lithography and size-dependent self-assembly to fabricate line arrays of nanowells allowing entrapment and retention of charged nanoparticles, covalently conjugated with a RGD adhesive ligand, GRGDSPK. This approach is an additive strategy of combining substrata topographic alteration, electrostatic charge and biochemical ligands, all uniquely incorporated as an ensemble of charged, ligand-bearing nanoparticles entrapped in arrays of nanowells. However, the modulation of endothelial cell physiologic mechanisms as a result of ensemble surface exposure remains to be characterized. In this report, we extend our studies and probe cell physiologic mechanisms altered as a result of nanofeatured surface exposure. We first examined the functional intactness or normalcy of endothelial cells adherent to the nanofeatured ensemble surface utilizing standard immunostaining and flow cytometry methods. We found β1-integrin expression dominated quiescent adherent endothelial cells while αVβ3-integrins expression was more common in migratory cells. Endothelial cells were noted to express high levels of PECAM-1 over time when exposed to nanofeatured surface and RGD peptides. For understanding the contribution of the nanofeatured surface (entrapped RGD conjugated nanoparticles) to cell adhesion, cytochalasin B was used to alter cell spreading. Confocal microscopy illustrated the uptake of nanoparticles in endothelial cells on composite surfaces, as well as the inhibition of this endocytosis by cytochalasin B. After prohibiting the cells from engulfing nanoparticles, we found an 80% reduction in cell adhesion; suggesting that an endocytic mechanism might play a role in maintaining cell adhesion. Nanofeatured ensemble surfaces appear to be good substrates for achieving a high level of EC adhesion, with maintained growth and stability.

Co-continuous phase structures of immiscible polymers can be developed under appropriate melt-blending conditions. Because of the presence of interfacial tension, such co-continuous structures start to coarsen when heated to a temperature higher than the melting/softening temperature of both phases. In this article, a systemic study of controllable growth of gradient porous structures utilizing variable coarsening rates in either a gradient temperature field or a gradient shear field is presented. Based on experimental results, the gradient of shear viscosity is identified as the mechanism for generating variable coarsening rates inside a co-continuous blend. By controllable variation of the shear viscosity distribution in a blend, a spatially varied and controllable gradient in phase structure is created. After dissolution of one of the two phases, the desired porous structure of the remaining polymer is obtained. A poly (lactic acid) (PLA)/polystyrene (PS) 50/50 wt% blend was used as a model system. By designing proper thermal and/or dynamic boundary conditions and introducing different thermal/shear rate gradients during annealing, several gradient porous structures of PLA were created.

Tissue engineering and regenerative medicine aim to produce tissue constructs in vitro which can subsequently be implanted in vivo to repair damaged or diseased tissues in a clinically relevant time scale. Coaxial electrospinning techniques are capable of producing 3D scaffolds with living cells, growth factors, and or time release drugs embedded within nano fibers. The fabrication of such electrospun mats requires a novel coaxial delivery system which provides encapsulation, protection and hydration of living cells. The electrospun fibers will form a mat adaptive to its collector that facilitates cell adhesion, differentiation, and proliferation. The embedded living cells may be employed to perform vital tasks such as secretion of matrix components and growth factors. These electrospun mats would improve viability of tissue engineered constructs, and may be made into biomedical devices. Cold water fish skin gelatin (high polarity natural polymer) is used to encapsulate cells while Poly(ethylene oxide) (PEO) serves as the electrospinning filler in the biosuspension. Glutaraldehyde (GTA) solution is used to preserve the 3D environment and structural integrity of the electrospun mats.

Current chemotherapeutic regimens involve the administration of a combination of agents with hopes of gaining synergistic cell-killing effects observed in vitro . However, drug synergy is rarely realized clinically given the different pharmacokinetic profiles of the drugs. Recent findings show that a combination of rapamycin and paclitaxel proves highly effective at hindering growth of tumors wherein the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. Our objective was to fabricate a micellar nanotherapeutic platform capable of delivering a multitude of agents shown to synergistically affect a specific pathway (PI3K/Akt/mTOR) in breast cancer. We hypothesized that this concomitant delivery strategy will result in increased antitumor efficacy, given the site-specific and controlled delivery of the two agents. Herein, we demonstrate the successful fabrication of a nanotherepeutic strategy for the treatment of breast tumors with aberrant PI3K/Akt/mTOR pathways. Resulting polymer micelles were small in size (∼30 nm) and showed high levels of drug incorporation efficiency of both rapamycin and paclitaxel. Current studies involve the examination of release kinetics and antitumor efficacy in in vitro and in vivo models.

Micro/nanometer protein fibers are fundamental building blocks of extra-/intracellular matrices, providing structural support, stability and protection to cells, tissue and organism [1]. Fabricating performance protein micro/nanofibers has been extensively pursued for a variety of biomedical application. In this study, silk-elastinlike protein (SELP) polymer will be electrospun into nanofibers as a biomaterial.

Several reports have described the relationship between size, aspect ratio, surface modification and internalization for a variety of nanoparticles (i.e. gold, polymer, carbon nanotubes). Nanodiamonds (NDs) in particular have recently been implicated in a variety of biomedical applications. One of the most promising is in utilizing NDs as drug delivery carriers where successful internalization is of utmost importance. A few reports recently have demonstrated the energy dependent internalization of bare NDs. In this report, we investigate the internalization mechanism and kinetics of functional ND-conjugate translocation.

The purpose of gene therapy is to introduce foreign genetic material into host cells to either supplement aberrant genes or to endow additional biological functions. To date, however, there has been only modest progress towards this goal, mainly due to the lack of safe, effective and broadly applicable delivery methods. Functional nanodiamonds (NDs) are rapidly emerging as promising platform carriers for next-generation therapeutics due to their innate biocompatibility, scalability, precise particle distribution, high surface area-to-volume ratio, near-spherical aspect ratio, and easily adaptable carbon surface for bioagent attachment. NDs have been functionalized with a range of therapeutics, proteins, antibodies, DNA, polymers, and other assorted biological agents. Furthermore, NDs are stable and dispersible in water, making them a promising and clinically important modality in improving the efficacy of the treatment of diseases and even some cancers at the molecular level. Mitochondrial function (MTT) and luminescent ATP production assays have demonstrated that NDs are not toxic to a wide variety of cell types. In this study, we functionalized NDs with amine groups via either covalent attachment of (3-aminopropyl) trimethoxysilane or surface immobilization of 800 Da low molecular weight polyethyleneimine (LMW PEI800) for plasmid DNA delivery. The latter delivery approach combines complementary characteristics of PEI800 and NDs to create a hybrid material that exhibits the high transfection efficiency of high molecular weight PEI, but without the inherent high cytotoxicity.

Nanotechnology is a rapidly growing research fields with many applications in biology and medicine. At a heart of nanotechnology research is engineered nanostructures, which possess distinct optical, electronic, and magnetic properties based on their size, shape, and chemical composition. Researchers can now design their surface chemistry with small bi-functional organic molecules or amphiphillic polymers so that they are biocompatible and can be coated with bio-recognition molecules such as antibodies, aptamers, and peptides. Nanoparticles are used as a platform for drug delivery, as a physical trigger for controlling drug release, as a contrast agent for quantifying biological molecules. Thus, the applications of engineered nanostructures are diverse. In this presentation, an overview of the field of nanomedicine is described with an emphasis on results obtained from studying the in vivo interactions of nanostructures as it pertains to their applications in cancer.

Given their ability to dictate initial cell alignment and subsequent matrix organization, aligned electrospun scaffolds are a fitting means for engineering fiber-reinforced, anisotropic tissues such as tendon, ligament, the knee meniscus, and the annulus fibrosus [1–4]. However, one commonly observed limitation of such scaffolds is the relatively slow infiltration rates of surface-seeded cells, where the central thicknesses of constructs cultured for 10 weeks remain devoid of cells [3]. This limitation arises from the tight packing of fibers which yields small pore sizes, thereby hampering cell migration. Towards accelerating cell ingress, we have recently reported on two-polymer composite scaffolds containing both slow eroding poly(ε-caprolactone) (PCL) fibers as well as water-soluble poly(ethylene oxide) (PEO) fibers that serve as space holders during scaffold formation [5]. Removal of these PEO fibers prior to seeding resulted in improved cell infiltration after 3 weeks, but the long-term maturation of such constructs has yet to be characterized. To assess the effect of sacrificial PEO fiber content on construct growth, a triple-jet electrospinning device was employed to generate PCL/PEO scaffolds with PEO fiber fractions ranging from 0 to 60%. After seeding with human meniscus fibrochondrocytes (hMFCs), constructs were clamped in custom grips to maintain strip morphology. The mechanical and biochemical maturation of constructs was assessed over 12 weeks of free swelling culture in a chemically defined medium (CDM), along with cell infiltration and matrix distribution. We hypothesized that enhanced pore size in dual-fiber constructs would lead to not only to a better distribution of cells, but also to larger increases in stiffness resulting from enhanced matrix production and distribution.

Electro-active polymer working in aqueous medium has short response time and small power consumption. Polypyrrole is one of such electro-active polymers. Due to its stability, incorruptibility and micromachining compatibility, polypyrrole plays an important role in developing micro/nano-actuators for biological and chemical applications. This paper presents a flexible and reusable polypyrrole nano-valve array for biomedical applications. The nano-valves are formed by depositing polypyrrole on the anodic aluminum oxide (AAO) film by a novel method to ensure the integrity of the AAO film. The valves are actuated through the oxidation and reduction of the polypyrrole. The oxidation state of the polypyrrole makes sodium ions leaving the polypyrrole and hence contracts the polypyrrole. So the gold/polypyrrole bilayer was bended and the valves blocked. Conversely, the valve is opened as the reduction state. By controlling the open/close time with constant voltage, the diffusive quantity of the nano-valves can be easily changed.

Anterior cruciate ligament (ACL) reconstructive surgery is a major health concern world-wide because of a large aging population and increased occurrence of sport-related injuries. Tissue engineering is a rapidly growing interdisciplinary field that offers a promising new approach for ACL repair. The aim of this project is to explore novel “smart” surgical fixation devices that not only secure a graft in place without strength failure, but also incorporate and release bioactive materials, intended to promote bone tissue growth. In order to facilitate bioactive reagent release, biopolymeric scaffolds with continuous micro-porous structure were developed. The morphology of the porous structures in the poly-L-lactic acid (PLLA) matrix reflects the differential influence of the concentration of sacrificial material (PS-polystyrene), phase separation levels of the immiscible polymers (PLLA and PS), and melt-blending conditions (Fig. 1) [1]. During removal of the sacrificial material, the chemical solvent might introduce some chemical reactant into the scaffolds. In order to assess the feasibility of using the micro-porous structures for medical applications, 7F2 osteoblasts were cultured on these scaffolds for 7 days. The attachment and proliferation of 7F2 cells on all scaffolds were assessed by fluorescent nuclear staining with Hoechst 33258 and phalloidin. The morphology of 7F2 osteoblasts on solid PLLA and PLLA/HA with 40% porous structure scaffolds till the 7 days pos-seeding was observed under confocal microscopy (Fig. 2A and B). The results showed that removal of the sacrificial material does not influence cell growth and the composites are biocompatible. Besides in vitro cytotoxicity test, in vivo test of all the micro-porous structural scaffolds was performed through rat subcutaneous surgery. Histological analysis (H&E staining) of the porous PLLA/HA with 40% pores retrieved from rat subcutaneous tissue 4 weeks postimplantation show that cells start to grow inside the porous scaffold (Fig. 3A). The morphology of surrounding extracellular matrix (ECM) growing on the scaffolds was observed under SEM. Figure 3B shows soft tissue attached onto PLLA/HA porous scaffold after 1 month post implantation time point, which indicates the good biocompatibility of the scaffolds. Based on these data we predict that these scaffolds will be suitable for inducing and sustaining bone tissue regeneration, and will be feasible for ACL repair.

A massively-parallel molecular delivery system for mammalian cells is demonstrated by pulsed-laser irradiation of a gold nanoparticle array situated below a cell monolayer. This system is capable of high throughput and spatially-targeted delivery into desired areas of a cell culture by designing the laser irradiation pattern. Rapid fabrication of gold nanoparticle arrays over large areas (>1 mm 2 ) is achieved by polymer mold guided near-field photothermal annealing.

Indentation, in addition to the traditional tensile testing, has been widely used for evaluating mechanical properties of hard materials such as metals and bone as well as soft materials like polymer and soft tissues. However, it is difficult to measure the contact area and surface deformation in conventional indentation tests of soft tissue which will bring large errors to the evaluation of the material properties. Also the assumption of isotropic property limited the usage of indentation test in characterizing the nonlinear, anisotropic properties of soft tissue thin film. In this project, 2D and 3D finite element analyses has been carried out to predict hyperelastic material response under indentation and punch tests. A novel indentation test system was developed, which made the direct measurement of local deformation and contact area possible. The apparatus consists of a transparent indenter, a digital microscope, and a computer based control and data acquisition system. The proposed testing system and associated finite element analysis are used to characterize the mechanical properties of multiscale (bulk and thin film) biological tissues.

A drug delivery system utilizing nanodiamonds has been proposed for the controlled release of the chemotherapeutic doxorubicin (DOX) [1,2]. The drug delivery system consists of a patch which is envisioned to be implanted immediately after a tumor removal surgery to target residual cancerous cells such that tumor reoccurrence can be effectively prevented. Therefore, long release times are desired for the patch to prevent any new cancerous tissue from forming. The patch is to be assembled in three different layers beginning with a bottom layer comprised of nonporous parylene. A layer of DOX loaded nanodimonds is deposited onto the base layer. Then a porous layer of parylene is accumulated on top of the nanodimonds. Nanodimonds are utilized because their release rates are based on pH, which changes near cancerous cells. Due to the complexity of the drug delivery system, models are created at several different scales.

Here, we incorporate the results of our new “altered phase theory” (Sikdar et al. 2008a) into design of new polymer clay nanocomposites (PCNs) for bone biomaterials applications. Montmorillonite (MMT) clay was modified using unnatural amino acids as potentially new biocompatible modifiers. The longer carbon chain structures of the unnatural amino acids are expected to enhance non bonded interactions with clay as well as maintaining the usefulness of functional groups of natural amino acids. The specific choice of amino acids is based on both the antibacterial activity reported in literature and also our previous studies on role of chain length, functional groups etc of modifiers in influencing mechanical behavior in PCNs. Biocompatibility studies using cell culture experiments as well as mechanical behavior is evaluated for the PCNs. FTIR spectroscopy is used to compare changes to molecular structure. The increase in d 001 spacing of modified clay compared to pure clay obtained from XRD experiments confirms successful intercalation of modifier. The osteoblast cells were found to grow and proliferate over the substrates. The major contribution of this work is the design of novel amino acid biopolymer-clay nanocomposites for biomaterials applications. Porous scaffold structures were also designed and fabricated.