Regenerative engineering was conceptualized by bridging the lessons learned in developmental biology and stem cell science with biomaterial constructs and engineering principles to ultimately generate de novo tissue. We seek to incorporate our understanding of natural tissue development to design tissue-inducing biomaterials, structures and composites than can stimulate the regeneration of complex tissues, organs, and organ systems through location-specific topographies and physico-chemical cues incorporated into a continuous phase. This combination of classical top-down tissue engineering approach with bottom-up strategies used in regenerative biology represents a new multidisciplinary paradigm. Advanced surface topographies and material scales are used to control cell fate and the consequent regenerative capacity.

Musculoskeletal tissues are critical to the normal functioning of an individual and following damage or degeneration they show extremely limited endogenous regenerative capacity. The increasing demand for biologically compatible donor tissue and organ transplants far outstrips the availability leading to an acute shortage. We have developed several biomimetic structures using various biomaterial platforms to combine optimal mechanical properties, porosity, bioactivity, and functionality to effect repair and regeneration of hard tissues such as bone, and soft tissues such as ligament and tendon. Starting with simple structures, we have developed composite and multi-scale systems that very closely mimic the native tissue architecture and material composition. Ultimately, we aim to modulate the regenerative potential, including proliferation, phenotype maturation, matrix production, and apoptosis through cell-scaffold and host –scaffold interactions developing complex tissues and organ systems.

Osteochondral (OC) tissue is comprised of articular cartilage, the subchondral bone and the central cartilage-bone interface. To facilitate proper regeneration, an equally complex and multiphasic matrix must be used. Although mono-phasic and bi-phasic matrices were previously applied, they failed to establish the OC interface upon regeneration. In this study, we designed and developed a novel matrix with increasing pore volume from one end to other, along the matrix length. For this matrix polylactide-co-glycolide (PLGA) 85:15 microspheres were combined with a water-soluble porogen in a layer-by-layer fashion and thermally sintered. The resulting matrix was then porogen-leached to form a gradiently-porous structured matrix. The formation of this gradient pore structure was established using Micro-Computed Tomography (μCT) scanning. A biodegradable hydrogel was infiltrated into the structure to form a unique OC matrix where the microsphere and hydrogel phases co-exist with opposing gradients. When the individual phases are associated with osteogenic and chondrogenic growth factors, the structureinduced factor delivery might provide the spatially controlled factor delivery necessary to regenerate osteochondral tissue structure. Overall, we designed a gradient matrix system that is expected to support osteochondral tissue engineering while forming a seamless interface between the cartilage and the bone matrix.

Ventilator associated pneumonia (VAP) is a serious and costly clinical problem. Specifically, receiving mechanical ventilation for over 24 hours increases the risk of VAP and is associated with high morbidity, mortality and medical costs. Cost effective endotracheal tubes (ETTs) that are resistant to bacterial infection could help prevent this problem. The objective of this study was to determine differences in the growth of Staphylococcus aureus (S. aureus) on nanomodified and unmodified polyvinyl chloride (PVC) ETTs under dynamic airway conditions. PVC ETTs were modified to have nanometer surface features by soaking them in Rhizopus arrhisus, a fungal lipase. Twenty-four hour experiments (supported by computational models) showed that air flow conditions within the ETT influenced both the location and concentration of bacterial growth on the ETTs especially within areas of tube curvature. More importantly, experiments revealed a 1.5 log reduction in the total number of S. aureus on the novel nanomodified ETTs compared to the conventional ETTs after 24 hours of air flow. This dynamic study showed that lipase etching can create nano-rough surface features on PVC ETTs that suppress S. aureus growth and, thus, may provide clinicians with an effective and inexpensive tool to combat VAP.

In this study, an in vitro blood-brain barrier model was developed using murine brain endothelioma cells (b.End3 cells). By comparing the permeability of FITC-Dextran at increasing exposure times in serum-free medium to such values in the literature, we confirm that the blood-brain barrier model was successfully established. After such confirmation, the permeability of five ferrofluid (FF) nanoparticle samples, GGB (ferrofluid synthesized using glycine, glutamic acid and BSA), GGC (glycine, glutamic acid and collagen), GGP (glycine, glutamic acid and PVA), BPC (BSA, PEG and collagen) and CPB (collagen, PVA and BSA), was determined using this model. In addition, all the five FF samples were characterized by zeta potential to determine their charge as well as TEM and dynamic light scattering for determining their hydrodynamic diameter. Results showed that FF coated with collagen had better permeability to the blood-brain barrier than FF coated with glycine and glutamic acid based on an increase of 4.5% in permeability. Through such experiments, magnetic nanomaterials, such as ferrofluids, that are less permeable to the blood brain barrier can be used to decrease neural tissue toxicity and magnetic nanomaterials with more permeable to the blood-brain barrier can be used for brain drug delivery.

Millions of medical implants and devices (e.g., screws, plates, and pins) are used each year worldwide in surgery, and traditionally the components have been limited to permanent metals (e.g., stainless steel, titanium alloys) and polyester-based absorbable polymers. Because of clinical problems associated with these traditional materials, a novel class of biodegradable metallic materials, i.e., magnesium-based alloys, attracted great attention and clinical interests. Magnesium (Mg) is particularly attractive for load-bearing orthopedic applications because it has comparable modulus and strength to cortical bone. Controlling the interface of Mg with the biological environment, however, is the key challenge that currently limits this biodegradable metal for broad applications in medical devices and implants. This paper will particularly focus on creating nanostructured interface between the biodegradable metallic implant and surrounding tissue for the dual purposes of (1) mediating the degradation of the metallic implants and (2) simultaneously enhancing bone tissue regeneration and integration. Nanophase hydroxyapatite (nHA) is an excellent candidate as a coating material due to its osteoconductivity that has been widely reported. Applying nHA coatings or nHA containing composite coatings on Mg alloys is therefore promising in serving the needed dual functions. The composite of nHA and poly(lactic-co-glycolic acid) (PLGA) as a dual functional interface provides additional benefits for medical implant applications. Specifically, the polymer phase promotes interfacial adhesion between the nHA and Mg, and the degradation products of PLGA and Mg neutralize each other. Our results indicate that nHA and nHA/PLGA coatings slow down Mg degradation rate and enhance adhesion of bone marrow stromal cells, thus promising as the next-generation multifunctional implant materials. Further optimization of the coatings and their interfacial properties are still needed to bring them into clinical applications.

A novel silicone-epoxy oligomer was synthesized and evaluated for its use as the polymer in photopolymerizable dental composites. This synthesized oligomer contained rigid and non-rigid groups with 1-8 epoxy functionalities as characterized using 1H NMR and 29Si NMR and MALDI-TOF analysis. In comparison to the traditional BisGMA/TEGDMA monomer system, the photo-polymerized silicone-epoxy demonstrated significantly improved material properties (148% greater elastic modulus, 12% greater ultimate strength, 48% greater fracture toughness), as well as 61% lower polymerization shrinkage and 58% lower polymerization stresses. Furthermore, the silicone-epoxy system demonstrated enhanced resistance to degradation of its material properties after accelerated (24hr) aging, i.e. exposure to severe hydrolytic (boiling in ethanol at 100 ᵒC), oxidative (exposed to 5% H2O2), and low pH (0.05M acetic acid) stress. Under these conditions, the properties of conventional BisGMA/TEGDMA systems deteriorated by 22-47%, while the properties of the silicone-epoxy composites decreased by 2-10%. Bisphosphonate additives enhanced the precipitation of mineral in a dose-dependent manner, but inhibited polymerization due to interactions with epoxy groups. Bisphosphonate additives also dose-dependently demonstrated anti-bacterial efficacy as demonstrated using live-dead, MTT and crystral violet assays. The silicone-epoxy polymer was demonstrated to be biocompatible when compared to tissue culture plastic. When calcium fluoride was incorporated into this system, fluoride was found to be released quantities significant enough to engender anti-bacterial effects. In summary, the designed multifunctional dental resin exhibits higher stability as demonstrated by lower chemical, mechanical and enzymatic degradation.

Hydroxyapatite (HAP) is a biocompatible bio-ceramic whose structure and composition is similar to bone. However, its lack of strength and toughness have seriously hampered its applications as a bone graft substitute material. Attempts have been made to overcome these mechanical properties deficiencies by combining HAP bioceramic material with absorbable polymers in order to improve its mechanical properties. However, poor interfacial bonding between the HAP and the polymers has limited the benefits of such biocomposite structures. At the other end of the biomaterials spectrum is collagen, which constitutes the most abundant proteins in the body and exhibits properties such as biodegradability, bioadsorbability with low antigenicity, high affinity to water, and the ability to interact with cells through integrin recognition. These favorable properties renders collagen as a natural candidate for the modification and compatibilization of the polymer-HAP biocomposite. In this study, we developed a novel approach to the synthesis of a potential bone graft material, where the HAP moiety acts not only as a bioceramic filler, but also constitutes the initiator surface that promotes the in-situ polymerization of the adsorbable polymer of choice. The synthesis of poly(D,L-lactide-co-glycolide) (PLGA) polymer was catalyzed by nano-hydroxyapatite (nHAP) particles and upon reaction completion, the biocomposite material was tethered with collagen. The synthesis was monitored by 1H NMR and FTIR spectroscopies and the products after each step were characterized by thermal analysis to probe both thermal stability, morphological integrity and mechanical properties.

Pioneer works on nanocomposites were focused in carbon nanofibers or nanotubes dispersed in epoxy matrix, a viscous liquid facilitating the compounding stage. The interest in developing new composites aimed for biomedical applications led us to design new nanocomposites based in biodegradable polymers with demonstrated biological performance.

We report herein the development of micro-nano composites by extruding poly(butylene succinate) (PBS) microfibers with two different diameters, 200 and 500 µm, reinforced with electrospun chitosan nanofibers. Analysis of the microfibers showed high levels of alignment of the reinforcing phase and excellent distribution of the nanofibers in the composite. Its geometry facilitates the development of orthotropy, maximizing the reinforcement in the axial fiber main axis.

The biodegradable microfiber composites show an outstanding improvement of mechanical properties and of the kinetics of biodegradation, with very small fractions (0.05 and 0.1 wt.%) of electrospun chitosan nanofibers reinforcement. The high surface area-to-volume ratio of electrospun nanofibers combined with the increased water uptake capability of chitosan justify the accelerated kinetics of biodegradation of the composite as compared with the unfilled synthetic polymer.

Advanced biomaterials that mimic the structure and function of native tissues and permit stem cells to adhere and differentiate is of paramount importance in the development of stem cell therapies for bone defects. Successful bone repair approaches may include an osteoconductive scaffold that permits excellent cell adhesion and proliferation, and cells with an osteogenic potential. The objective of this study was to evaluate the cell proliferation, viability and osteocyte differentiation of equine-derived bone marrow mesenchymal stem cells (EqMSCs) when seeded onto biocompatible and biodegradable calcium-deficient hydroxyapatite (CdHA) tubular-shaped bacterial cellulose scaffolds (BC-TS) of various sizes. The biocompatible gel-like BC-TS was synthesized using the bacterium Gluconacetobacter sucrofermentans under static culture in oxygen-permeable silicone tubes. The BC-TS scaffolds were modified using a periodate oxidation to yield biodegradable scaffolds. Additionally, CdHA was deposited in the scaffolds to mimic native bone tissues. The morphological properties of the resulting BC-TS and its composites were characterized using scanning electron microscopy. The ability of the BC-TS and its composites to support and maintain EqMSCs growth, proliferation and osteogenic differentiation in vitro was also assessed. BC-TS and its composites exhibited aligned nanofibril structures. MTS assay demonstrated increasing proliferation and viability with time (days 1, 2 and 3). Cell-scaffold constructs were cultured for 8 days under osteogenic conditions and the resulting osteocytes were positive for alizarin red. In summary, biocompatible and biodegradable CdHA BC-TS composites support the proliferation, viability and osteogenic differentiation of EqMSCs cultured onto its surface in vitro, allowing for future potential use for tissue engineering therapies.

Dielectrophoresis (DEP) approach was employed to achieve highly aligned multi-walled carbon nanotubes (MWCNTs) within the gelatin methacrylate (GelMA) hydrogels in a facile, rapid, inexpensive, and reproducible manner. This approach enabled us to make different CNTs alignments (e.g., vertical or horizontal alignments) within the GelMA hydrogel using different electrode designs or configurations. Anisotropically aligned GelMA-CNTs hydrogels showed considerably higher conductivity compared to randomly distributed CNTs dispersed in the GelMA hydrogel and the pristine and non-conductive GelMA hydrogel. Adding 0.3 mg/mL CNTs to the GelMA hydrogel led to a slight increase in the mechanical properties of the GelMA and made it to behave as a viscoelastic material. Therefore, it can be used as a suitable scaffold for soft tissues, such as skeletal muscle tissue. 3D microarrays of skeletal muscle myofibers were then fabricated based on the GelMA and GelMA-CNTs hydrogels and they were characterized in terms of gene expressions related to the muscle cell differentiation and contraction. Owing to high electrical conductivity of aligned GelMA-CNTs hydrogels, the engineered muscle tissues cultivated on these materials demonstrated superior maturation and functionality particularly after applying the electrical stimulation (voltage 8 V, frequency 1 Hz, and duration 10 ms for 2 days) compared to the corresponding tissues obtained on the pristine GelMA and randomly distributed CNTs within the GelMA hydrogel.

Poly(glycerol sebacate) (PGS) is a biodegradable and biocompatible elastomer that has been used in a wide range of biomedical applications. While a porous format is common for tissue engineering scaffolds, to allow cell ingrowth, PGS degradation has been primarily studied in a nonporous format. The purpose of this research was to investigate the degradation of porous PGS at three frequently used cure temperatures: 120°C, 140°C, and 165°C. The thermal, chemical, mechanical, and morphological changes were examined using thermogravimetric analysis, differential scanning calorimetry, Fourier transform infrared spectroscopy, compression testing, and scanning electron microscopy. Over the course of the 16-week degradation study, the samples’ pores collapsed. The specimens cured at 120°C demonstrated the most degradation and became gel-like after 16 weeks. Thermal changes were most evident in the 120°C and 140°C cure PGS specimens, as shifts in the melting and recrystallization temperatures occurred. Porous samples cured at all three temperatures displayed a decrease in compressive modulus after 16 weeks. This in vitro study helped to elucidate the effects of porosity and cure temperature on the biodegradation of PGS and will be valuable for the design of future PGS scaffolds.

This study reports a novel class of biodegradable polyurethane biomaterials and three-dimensional scaffolds for tissue engineering. Solvent casted polyurethane films were studied for biocompatibility by seeding with human bone marrow derived stromal cells. In order to develop a three-dimensional and porous structure, a dynamic solvent sintering method was applied to the polyurethanes for the first time. Microstructural studies on the sintered scaffolds reveal porous structure formation with bonding between the adjacent microspheres. In conclusion, this study establishes new polyurethane biomaterials that are fully absorbable for tissue engineering applications.

This study used Langmuir-Schaefer (LS) method to produce thermo-responsive poly(N-isopropylacrylamide) (PIPAAm) modified surface. Block copolymer composed of polystyrene (PSt) and PIPAAm was synthesized by RAFT polymerization. PSt-block-PIPAAm (St-IP) with various chemical compositions was dropped on an air-water interface and formed Langmuir film by compression. Then, the Langmuir film changing a density was transferred on a hydrophobic modified glass substrate to produce St-IP transferred surface (St-IP LS surface). From the observation of atomic force microscope images, a nanostructure was observed on the transference of Langmuir films. Cell adhesion and detachment were also evaluated on the LS surfaces in response to temperature. Cell adhesion on LS surfaces at 37 °C was controlled by changing the chemical compositions and densities. After reducing temperature to 20 °C, adhering cells rapidly detached themselves with lower Am and higher composition of PIPAAm. Our method should be proved novel insights for investigating cell adhesion and detachment on thermo-responsive surfaces.

Thready stripe-patterned thermo-responsive surfaces were prepared and their surface properties were characterized. Prepared 3 μm wide stripe-patterned surfaces were evaluated by observing the adhesions and detachments of three types of cells: HeLa cells (HeLas), human umbilical vein endothelial cells (HUVECs), and NIH-3T3 cells (3T3s). Although cell adhesion and detachment in response to temperature were observed on all cells on a conventional thermo-responsive surface without patterns, the thermo-responsive surface with a 3 μm striped-pattern exhibited various cell adhesion properties. HeLas hardly adhered to the patterned surface even at 37 °C. On the other hand, although HUVECs adhered on the patterned surface at 12 h after incubation at 37 °C, the adhered HUVECs detached themselves after another 12 h incubation at 37 °C. 3T3s adhered to the patterned surface at 37 °C and detached themselves after reducing temperature to 20 °C. A mixture of HeLa, HUVEC and 3T3 was separated using their different specific cell-adhesion properties, and the composition of cells was analyzed by a flow-cytometry. As a result, the conventional thermo-responsive surface with a stripe-pattern was found to function as a cell-separating interface by using specific cell adhesion properties.

Here we present a single mask sacrificial molding process that allows ultrathin 2-dimensional membranes to be fabricated using biocompatible polymeric materials. For initial investigations, polycaprolactone (PCL) was chosen as a model material. The process is capable of creating 250-500 nm thin, through-hole PCL membranes with various geometries, pore-sizes and spatial features approaching 2.5 micrometers using contact photolithography. The technique uses a mold created from two layers of lift-off resist (LOR). The upper layer is patterned, while the lower layer acts as a sacrificial release layer for the polymer membrane. For mold fabrication, photoresist on top of the layers of lift-off resist is patterned using conventional photolithography. During development the mask pattern is transferred onto the first LOR layer and the photoresist is removed using acetone, leaving behind a thin mold. The mold is filled with a solution of the desired polymer. Subsequently, both the patterned and lower LOR layers are dissolved by immersion in an alkaline solution. The membrane can be mounted onto support structures pre-release to facilitate handling.

Multi-walled nanofibers with their outstanding properties have found expanding applications on drug delivery systems, biosensors, self-healing materials and many other state-of-the-art technologies. This work investigates the fabrication and morphological control of multi-walled structured electrospun polymeric nanofibers by multi-axial electrospinning system. This process is based on a nozzle allowing multi-axial extrusion of different fluids with concentric orders. Two spinnable polymers of poly(methyl methacrylate) and polyacrylamide are chosen for the fabrication of middle and outer walls of co-axial hollow nanofibers, respectively. Hansen’s solubility parameters are used to systematically optimize the solvent selection for each layer and control the degree of miscibility of layers with the purpose of tailoring the final wall morphology of nanofibers. Characterization studies are performed by Scanning Electron Microscopy, Energy-Dispersive X-ray Spectroscopy, Fourier Transform Infrared Spectroscopy, and Thermal Gravimetric Analyzer.

We developed a rapidly-gelling chitosan sponge crosslinked with Guanosine 5'-Diphosphate (GDP). GDP has not been previously explored as an anionic crosslinker, and it was used in this application since the nucleoside guanosine has been shown to improve remyelination in situ, and thus its presence in the sponge composition was hypothesized to induce Oligodendrocyte Progenitor Cells' (OPC) differentiation. In addition to the chemical composition tailored to target OPCs, the developed chitosan sponge possesses a wide range of desirable physicochemical properties such as: rapid gelation, high porosity with interconnected pores, moduli of elasticity resembling that of soft tissue and cytocompatibility with many cell types. Moreover, protein encapsulation into the sponges was possible with high encapsulation efficiencies (e.g. BMP-7 and NT-3). In this study, BDNF was encapsulated in the chitosan sponges with an encapsulation efficiency greater than 80% and a sustained release over a 16-day period was achieved. We demonstrate here for the first time, the attachment of human fetal OPCs to the sponges and their differentiation after 12 days of culture. Overall, this newly-introduced injectable sponge is a promising therapeutic modality that can be used to enhance remyelination post-spinal cord injuries.

We studied the structure and mechanical properties of DNA i-motif nanowires by means of molecular dynamics computer simulations. We built up to 230 nm-long nanowires, based on a repeated TC5 sequence from NMR crystallographic data, fully relaxed and equilibrated in water. The unusual C●C+ stacked structure, formed by four ssDNA strands arranged in an intercalated tetramer, is here fully characterized both statically and dynamically. By applying stretching, compression and bending deformations with the steered molecular dynamics and umbrella sampling methods, we extract the apparent Young’s and bending moduli of the nanowire, as well as estimates for the tensile strength and persistence length. According to our results, i-motif nanowires share similarities with structural proteins, as far as their tensile stiffness, but are closer to nucleic acids and flexible proteins, as far as their bending rigidity is concerned. Curiously enough, their tensile strength makes such DNA fragments tough as mild steel or a nickel alloy. Besides their yet to be clarified biological significance, i-motif nanowires may qualify as interesting candidates for nanotechnology templates, due to such outstanding mechanical properties.

Scanning acoustic microscopy (SAM), when applied to biological samples has the potential to resolve the longitudinal acoustic wave speed and hence stiffness of discrete tissue components. The heterogeneity of biological materials combined with the action of cryosectioning and rehydrating can, however, create variations in section topography. Here, we set out to determine how variations in specimen thickness influence apparent acoustic wave speed measurements

Cryosections (5μm nominal thickness) of human skin biopsies were adhered to glass slides before washing and rehydrating in water. Multiple regions (200x200 μm; n = 3) were imaged by SAM to generate acoustic wave speed maps. Subsequently co-localised 30x30 μm sub-regions were imaged by atomic force microscopy (AFM) in fluid. The images were then registered using Image J. Each pixel was allocated both a height and wave speed value before their relationship was then plotted on a scattergram. The mean section thickness measured by AFM was 3.48 ± 1.12 (SD) μm. Regional height variations influenced apparent wave speed measurements. A 3.5 μm height difference was associated with a 400 ms-1 increase in wave speed. In the present study we show that local variations in specimen thickness influence apparent wave speed. We also show that a true measure of wave speed can be calculated if the thickness of the specimen is known at each sampling point.

Cellulose nanofibers (Cel-F) were extracted by a simple and harmless Star Burst (SB) method, which produced aqueous cellulose-nanofiber solution just by running original cellulose beads under a high pressure of water in the synthetic SB chamber. By optimizing the SB process conditions, the cellulose nanofibers with high aspect ratios and the small diameter of ∼23 nm were obtained, which was confirmed by transmission electron microscopy (TEM). From the structural analysis of the Cel-F/PVA composite by the scanning electron microscopy (SEM), it was found that the Cel-F were homogeneously dispersed in the PVA matrix. Considering the high molecular compatibility of the cellulose and PVA due to the hydrogen bonding, a good adhesive interface could be expected for the Cel-F and the PVA matrix. The influences of the morphological change in Cel-F on the mechanical properties of the composites were analysed. The Young’s modulus rapidly increased from 2.2 GPa to 2.9 GPa up to 40 SB treatments (represented by the unit Pass), whereas the Young’s modulus remained virtually constant above 40 Pass. Due to the uniform dispersibility of the Cel-F, the Young’s modulus of the 100 Pass composite at the concentration of 5 wt% increased up to 3.2 GPa. The experimental results corresponded well with the general theory of the composites with dispersed short-fiber fillers, which clearly indicated that the potential of the cellulose nanofibers as reinforcement materials for hydrophilic polymers was sufficiently confirmed.