Nanoparticles (NPs) loaded with human platelet-derived growth factor (h-PDGF) were prepared and characterized. These NPs were co-assembled with collagen to form highly aligned NP-collagen composite fibers by using an electrochemical process. PDGF can be released gradually from either NPs or aligned NP-collagen fibers. This investigation demonstrated a novel way to fabricate highly aligned composite fibers, which can locally release a growth factor in a controlled and gradual manner, potentially avoiding the fast clearance of the growth factor from the implantation site. Thus, the aligned NP-collagen fiber is a novel and promising implantation material for tendon/ligament repair and regeneration.

Structural polymers are susceptible to accumulated damage in the form of internal microcracks that propagate through the material, resulting in mechanical failure. Self- healing approaches offer a solution to repair these damages automatically. The first generation self-healing material system includes a microencapsulated healing agent within a catalyst-embedded matrix. Propagating microcracks rupture the microcapsules, releasing the liquid healing agent into the damaged region. Catalyst-triggered polymerization of the released healing agent repairs the damage. Our research focuses on a similar approach for addressing “damage accumulation failure” of poly(methyl methacrylate) (PMMA) bone cement caused by microcrack initiation and propagation. In this study, polyurethane (PU) microcapsules containing a tissue adhesive, 2-octylcyanoacrylate (OCA) were synthesized using in situ interfacial polymerization of toluene-2,4-diisocynate (TDI) and polyethylene glycol 200 (PEG 200) through an oil-in-oil-in-water microemulsion (o/o/w). The process was optimized by studying different combinations of organic solvents, surfactants, temperatures, agitation rates, pH, and reaction times and their effects on microencapsulation were observed. Microcapsule surface morphology, size, shell thickness, encapsulated OCA viability, thermal degradation, and chemical structure of the microcapsule shell were evaluated using a stereoscope, scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and fourier transform infrared spectroscopy (FT-IR).

Multipotential mesenchymal stromal stem cells (MMSSC) are an excellent model for testing of the toxicity and biocompatibility of natural-tissue-engineering scaffolds (extracellular matrix). Such studies allow prediction of the behavior of implanted materials in the human. In the present work, testing of a three-dimensional prototype of a smart material – nitinol (the intermetallic phase NiTi) – to evaluate chemotaxis and biocompatibility was conducted.

Porous samples were synthesized by the selective laser sintering (SLS) method, establishing different surface conditions in the samples. The surface microstructure and roughness were observed by scanning electron microscopy (SEM) and optical microscopy. The results revealed the clear influence of the surface roughness on stem cell proliferation, morphology, and adhesion. The NiTi samples were well tolerated by the cells but the number of focal contacts decreased with increasing porosity. The proliferation speed was 0.694 doubling/day in the control group and 0.532 doubling/day for the NiTi group. Whereas the control group showed immature and actively divided stem cells, cell growth to enormous sizes (i.e., rapid aging) and a fall in fission activity in the proximity of an external irritant (viz., the NiTi scaffold) was observed.

Yearly, there are over six million cataract surgeries worldwide that involve intraocular lenses (IOLs) [1]. However, preventing post-operative biofouling of these lenses remains a challenge. One major complication is post-operative bacterial infection [2]. Surface modification of IOLs may provide a solution. This study proposes the use of the anti-adhesive protein lubricin (LUB), a glycoprotein found in the synovial fluid, as a means to make polymer surfaces less prone to bacterial adhesion and proliferation, thus reducing the opportunity for post-operative infection [3]. This study used extended bacteria growth trials in the presence of lubricin, vitronectin, and mucin to investigate how lubricin and protein sub-regions of lubricin reduce bacterial functions. This study showed for the first time that polymer surface coatings of lubricin and vitronectin significantly reduce Staphylococcus aureus growth over the course of 15 hours, while mucin was only able to delay the start of the Staphylococcus aureus exponential growth phase and retard proliferation. In solution, both lubricin and mucin significantly reduce bacterial proliferation. Thus, the results of this study demonstrated that lubricin and its sub-regions mucin and vitronectin should be studied for a wide range of antibacterial applications.

Regenerative engineering represents a new multidisciplinary paradigm to engineer complex tissues, organs, or organ systems through the integration of tissue engineering with advanced materials science, stem cell science and developmental biology. While possessing elements of tissue engineering, regenerative medicine, and morphogenesis, regenerative engineering is distinct from these individual disciplines since it specifically focuses on the integration and subsequent response of stem cells to biomaterials. One goal of regenerative engineering is the design of materials capable of inducing associated cells toward highly specialized functions. For example, the interaction of cells with calcium phosphate surfaces has proven to be an important signaling modality in promoting osteogenic differentiation. A biodegradable polymer-ceramic composite system has been developed from poly(lactide-co-glycolide) and in situ synthesized hydroxyapatite based on the three-dimensional sintered microsphere matrix platform. We have systematically optimized scaffold physico-chemical, mechanical, and structural properties for bone tissue regeneration applications by varying several parameters such as solution pH, polymer:ceramic ratio, sintering time and sintering temperature. The bioactivity of composite scaffolds is attributed to their ability to deliver calcium ions to surrounding medium and allow for reprecipitation of calcium phosphate on the scaffold surface. Furthermore, the composite scaffolds have demonstrated increased loading capacity of osteoinductive growth factor (BMP-2) and a more sustained release profile due to a greater number of adsorption sites provided by the ionic calcium and phosphate groups as well as a larger matrix surface area. In vitro cell studies were performed to investigate the efficacy of this composite system to induce osteogenic differentiation of human adipose-derived stem cells. Cells cultured on the ceramic containing scaffolds exhibited significantly higher expression of osteoblastic markers and greater extracellular matrix mineralization than non-ceramic containing scaffolds, indicating the potential for the ceramic phase to promote osteogenic differentiation. In addition, loaded BMP-2 retained its bioactivity as a mitogen and osteoinductive agent during the differentiation of adipose-derived stem cells into mature osteoblasts. In vivo evaluation using a critical-sized ulnar defect model in New Zealand white rabbits demonstrated the ability of composite scaffolds to support cellular infiltration throughout the scaffold pore structure and vascularization of new tissue, as well as facilitate formation of newly mineralized bone tissue. The work described herein provides strong evidence for the potential of polymer-ceramic composite scaffolds to function as osteoinductive bone graft substitutes, and paves the way for future development of advanced tissue-inducing materials.

A new family of A-B di-block copolymers based on the amino acid sequences of Nephila clavipes major ampulate dragline spider silk, which have a strong potential for applications in tissue regeneration and drug delivery, was synthesized and characterized. The morphology was assessed by SEM: HBA3 formed fibrillar structure and 2 μm diameter hollow micelles, while HBA4 and HBA6 formed hollow micelles in water solution. The secondary structures of water-cast spider silk-like block copolymer films were assessed by FTIR. The crystallinity was determined by Fourier self-deconvolution of the amide I spectra and confirmed by wide angle X-ray diffraction. Results indicate that the self-assembled morphology and the crystallinity can be varied by changing the length of A-block, and a minimum of 3 A-blocks are required to form β sheet crystalline regions in water-cast spider silk block copolymers. A theoretical model was used to predict the specific reversing heat capacity, Cp(T), which is crucial to the design of smart biomaterials. Excellent agreement was found between the theoretical value and the Cp(T) determined by temperature modulated differential scanning calorimetry. This method can serve as a standard by which to assess the thermal properties for other biologically inspired block copolymers, and then be further applied to control the biological interactions for use in drug delivery and smart biomaterials applications.

New materials are needed to replace degenerated intervertebral disc tissue and to provide longer-term solutions for chronic back-pain. Replacement tissue potentially could be engineered by seeding cells into a scaffold that mimics the architecture of natural tissue. Many natural tissues, including the nucleus pulposus (the central region of the intervertebral disc) consist of collagen nanofibers embedded in a gel-like matrix. Recently it was shown that electrospun micro- or nano-fiber structures of considerable thickness can be produced by collecting fibers in an ethanol bath. Here, randomly aligned polycaprolactone electrospun fiber structures up to 50 mm thick are backfilled with alginate hydrogels to form novel composite materials that mimic the fiber-reinforced structure of the nucleus pulposus. The composites are characterized using both indentation and tensile testing. The composites are mechanically robust, exhibiting substantial strain-to-failure. The method presented here provides a way to create large biomimetic scaffolds that more closely mimic the composite structure of natural tissue.

The enzymatic catalyzed synthesis and gelation of an ionic peptide and its use to create hydrogels for 3D cell culture is discussed. Time resolved small angle scattering in conjunction with imaging technique allowed the structural changes occurring through this enzymatic reaction to be assessed. In turn, the structural information about the fibrillar network and its local density proved key in facilitating the understanding of the relationships between self-assembly behavior, local nanostructure and final physical properties of the materials. The understanding of the gelation process of these materials allowed the design of a simple and efficient methodology to prepare gels for cell culture. Tetrapeptide/enzyme solution containing cells could be injected into cell culture plate with subsequent gelation of the materials leading to encapsulation of the cells into a 3D network. This system was evaluated for the 3D cell culture of human dermal fibroblasts (HDF). Microscopy showed that cells were uniformly distributed within the gel matrix. Cell counting and live/dead staining showed proliferation of HDF with limited cell death over 10 days.

The aim of this work was to develop a new Ag-doped bioactive material with antibacterial behavior, optimizing the properties of the new fabricated composite material in the system SiO2 58.6 -P2O5 7.2 -Al2O3 4.2 -CaO 24.9 -Na2O 2.1 -K2O 3 (wt%). Two systems with different concentrations in Ag2O (Ba with 2.1 and Bb with 4.2 wt%) were prepared by the sol-gel method and compared to the respective silver-free control composite (CONTROL). The microstructural characteristics of the developed compositions were characterized by different techniques as UV/VIS spectroscopy, X-ray diffraction analysis (XRD) and Scanning Electron Microscopy with Energy Dispersive Spectroscopy (SEM-EDS). The antibacterial properties of the Ag-doped glass-ceramics were tested against the bacterial colony Staphylococcus aureus (S. aureus) which is very characteristic oral bacteria and the material-cell interaction was monitored in a primary culture of Human Gingival Fibroblasts (HGFs). Our study shows the successful incorporation of the silver ions in the ceramic structure and the preparation of new Ag-doped composite materials with cell-proliferation-inductive, as well as antibacterial properties indicating their potential application dental tissue restoration strategies.

With multifunctionality and nanoscale dimensions, self-assembled rosette nanotubes (RNTs) exhibit unique biological and mechanical properties, making them promising to serve as a new generation of implants. Synthetic twin G^C base features the hydrogen bonding arrays of both guanine and cytosine and has the ability to self-organize spontaneously into nanotubes with a 3.5 nm outer diameter, a 1.1 nm inner channel running the length of the nanotube which can reach several micrometers in length. In this study, a twin G^C motif functionalized with an aminobutyl side chain (referred to as TBL) was synthesized, assembled into bioactive RNTs and used along with poly(2-hydroxyethyl methacrylate) (pHEMA) and hydroxyapatite (HA) nanoparticles to prepare RNTs/HA/pHEMA composites for orthopedic applications. The properties of these composites was investigated, notably the solidification process, surface morphology, mechanical properties, and cytocompatibility properties. The RNTs assembled from TBL and HA nanoparticles were found to be effective towards increasing the bioactivity of the composites thus establishing the potential of TBL/HA/pHEMA composites as very promising injectable orthopedic implant materials.

Recombinant human bone morphogenetic protein (rhBMP-2) plays a major role in differentiation of marrow stromal cells (MSCs). Peptides based on the active domains of rhBMP- 2, like the LYLTSIASLETPVSSAKPIK (BMP peptide), have been proposed as an alternative to reduce the side effects associated with high doses of rhBMP-2. The objective of this work was to determine the osteogenic activity of the BMP peptide grafted to poly(lactide fumarate) nanoparticles (PLAF NPs). A cysteine-terminated BMP peptide was grafted to PLAF NPs by linking the cysteine residue to the fumarate groups. Groups included blank NPs, free BMP peptide, free BMP-2 protein, and BMP-grafted NPs. The decrease in cell numbers after 21 days was consistent with an increase in mineral content and decrease in proliferation with osteogenic differentiation of MSCs. Alkaline phosphatase (ALPase) activity peaked at 14 days, consistent with the start of the osteogenic cascade. A slightly higher mineral content was observed in the BMP-grafted NP group after 14 days. m-RNA and immunostaining showed that cells in the BMP-grafted NP group had a higher content of osteocalcin protein after 21 days. Results suggest that the BMP-grafted NPs have a greater affinity to BMP cell surface receptors, leading to a stronger activation of the pathways leading to osteogenesis.

The objective of the present in vitro research was to determine cardiomyocyte functions on poly-lactic-co-glycolic acid (50:50 (PLA:PGA); PLGA) with greater amounts of carbon nanofibers (CNFs) using an in vitro electrical stimulation system for myocardial tissue engineering applications. The addition of CNFs can increase the conductivity and strength of pure PLGA. For this reason, different PLGA: CNF ratios (100:0, 75:25, 50:50, 25:75, 0:100 wt%) were created where conductivity and cytocompatibility properties under electrical stimulation with human cardiomyocytes were determined. Results showed that PLGA:CNF materials were conductive and that conductivity increased with greater amounts of PLGA added, from 0 S.m-1 for 100:0 wt% (pure PLGA) to 6.5x10-3 S.m-1 for 0:100 wt% (pure CNFs) materials. Furthermore, results indicated that cardiomyocyte cell density increased with continuous electrical stimulation (rectangular, 2 nm, 5 V/cm, 1 Hz) after 1, 3, and 5 days as well as a slight increase in Troponin I excretion compared to non-electrically stimulated normal cardiomyocyte cell functions. This study, thus, provides an alternative conductive scaffold using nanotechnology which should be further explored for numerous cardiovascular applications.

The nanoscale physical properties of newly electrospun polyamide nanofibrillar matrices < 1 year old versus those that were > 3 year old were investigated with transmission electron microscopy, selected area electron diffraction, contact angle measurements, and Raman spectroscopy. Significant differences in crystallinity, hydrophobicity, and chemistry were found and correspondingly different cell responses by cerebellar granular neurons were observed. The properties of the aged nanofibrillar scaffolds evoked a response for neuron burrowing into a more 3-dimensional environment in addition to better facilitation of neurite outgrowth. The nanophysical properties of tissue scaffolds have been recently shown to directly and indirectly regulate cellular responses. As physical properties can evolve over time, the present investigation addresses the issue of tissue scaffold shelf life, with possible changes in directive signals to cells.

Cylindrical porous polycaprolactone (PCL) scaffolds containing 25, 35, and 50 wt% demineralized bone matrix (DBM) were fabricated using a salt-leaching method for application in bone engineering. In the present work, PCL-DBM scaffolds were monitored for calcium and phosphorus deposition in both deionized (DI) water and simulated body fluid (SBF) for time periods of 5, 10, 15, and 20 days at 37°C under constant rotation. An in vitro assessment of the bioactivity of synthetic materials using SBF under physiological conditions can be used as a barometer of scaffold behavior in vivo. DBM, an osteoinductive material, was used to gauge if there was a correlation between the concentration of DBM within a scaffold and the apatite formation on its surface. Biochemical assays, alizarin red S staining, and scanning electron microscopy (SEM) with elemental analysis of calcium and phosphorus were consistent in that they confirmed that PCL scaffolds containing 35 wt% DBM in SBF at 14 days post-immersion showed signs of early apatite formation.

Polyurethane (PU) materials are used in a wide variety of implantable devices and technologies, e.g. stents, breast augmentation, nose surgery and bladder reconstruction. Despite the excellent chemical control for manufacturing bulk materials and the good biocompatibility, a major challenge remains interfacing of PU with biological environments. A chemically controlled surface engineering approach could improve desired protein adsorption processes and cellular interactions within different tissues, preventing uncontrolled events of the implant especially in early stages shortly after surgical procedures.

To gain better control over the PU surfaces we polymerized different bulk PU materials and developed a transfer-nanolithography technique to deposit inorganic Au-nanoparticles with defined structural features on the PU surface. Different nanoparticle patterns were transferred and analyzed by scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (HR-TEM). Topographical features of PU substrates were investigated by atomic force microscopy (AFM). Transferred Au-nanoparticles showed high stability on PU substrates even under extreme sonication conditions. In a final step, those nanoparticles were functionalized with peptides to facilitate cellular adhesion under physiologically relevant conditions. As proof of concept, rat embryonic fibroblast cells were cultured on a peptide functionalized PU interface and investigated by SEM.

In conclusion, we developed a versatile method to prepare nanostructured and biofunctionalized PUs. These PUs showed good stability characteristics and in vitro biocompatibility in cell culture assays.

Astrocytes are cellular bridges between the neurons and capillaries in the blood brain barrier. It was recently suggested that the nanophysical properties of the basement membrane of the blood brain barrier can influence astrocyte and neuron responses. In this work, cerebral cortical astrocytes were cultured on standard poly-L-Lysine coated glass substrates, Aclar substrates, and electrospun polyamide nanofibers whose properties may recapitulate those of the basement membrane. The nanoscale elasticity of each culture environment was investigated by force curve analysis and compared. The elasticity of the individual nanofibers on nanofibrillar surfaces was also investigated. Finally, variations in elasticity of scaffolds were correlated with astrocyte responses.

The importance of the extracellular mechanical environment in stem cell differentiation has been extensively studied over the last decade. In neuronal cell differentiation, matrix stiffness and neurite outgrowth have been correlated, highlighting the impact of matrix effects on neuronal cell morphology. In addition, on materials that approach the physiological mechanical properties of brain tissue, neurons from mixed phenotype primary cultures will prevail. However, if the same mixed culture is grown on polystyrene, glial populations are more prevalent. Enhancing the understanding of these differentiation processes will further expand the ability to design materials for neuronal implants that are conducive to neuronal survival, resist glial scarring and promote neurite outgrowth and cell connectivity. Specifically, elastomers such as poly(glycerol sebacate) (PGS) hold promise in neuronal tissue engineering, due to their mechanical tunability. PGS is biocompatible, biodegradable and possesses mechanical properties similar to that of living tissue. Neuronal cell differentiation was studied on PGS, using P19 embryonic carcinoma cells, which can be differentiated into a neuronal phenotype using retinoic acid. Varying cure temperatures of PGS including 120°, 140° and 165°C were selected, which equate to an elastic modulus of 0.07, 0.43 and 2.30 MPa respectively. Cells were characterized via immunocytochemistry. A primarily astrocytic population, with limited neuronal differentiation and neurite outgrowth were observed on the PGS 120°C. Cells grown on PGS 140°C demonstrated marked neurite outgrowth, with an increase in neuronal cells. Cells grown on the PGS 165°C exhibited the largest population of neurons, with significant neurite outgrowth. These results indicate that substrate mechanical properties do impact neuronal differentiation, but that a material with a Young’s modulus similar to that of neuronal tissue (PGS 120°C) may not necessarily be the most conducive to in vitro differentiation.

The aim of the current work was to examine the human monocyte response to 444 ferritic stainless steel fibre networks. 316L austenitic fibre networks, of the same fibre volume fraction, were used as control surfaces. Fluorescence and scanning electron microscopies suggest that the cells exhibited a good degree of attachment and penetration throughout both networks. Lactate Dehydrogenase (LDH) and TNF-α releases were used as indicators of cytotoxicity and inflammatory responses respectively. LDH release indicated similar levels of monocyte viability when in contact with the 444 and 316L fibre networks. Both networks elicited a low level secretion of TNF-α, which was significantly lower than that of the positive control wells containing zymosan. Collectively, the results suggest that 444 ferritic and 316L austenitic networks induced similar cytotoxic and inflammatory responses from human monocytes.