Inspired from biological systems, nanotechnology is beginning to revolutionize (and in many cases already has) revolutionized medicine including improved prevention, diagnosis, and treatment of numerous diseases. This talk will summarize efforts over the past decade that have synthesized novel nanoparticles, nanotubes, and other nanomaterials to improve medicine. Efforts focused on the use of nanomaterials to minimize immune cell interactions, inhibit infection, and increase tissue growth will be especially emphasized. Tissue systems covered will include the nervous system, orthopedics, bladder, cardiovascular, vascular, and the bladder. Due to complications translating in vitro to in vivo results, only in vivo studies will be emphasized here. Materials to be covered will include ceramics, metals, polymers, and composites thereof. Self-assembled nano-chemistries will also be emphasized. As the FDA has now approved several nanomaterials for medical applications, recent results from FDA trials will also be discussed. Importantly, this talk will also discuss what further advances we can make in medicine by using picotechnology compared to nanotechnology. In summary, this talk will provide the latest information concerning the design and use of numerous nanomaterials in regenerative medicine while highlighting what is necessary for this field to continue to grow through the exploration of picotechnology.

During the last decade, various functional nanostructured materials with interesting optical, magnetic, mechanical, and chemical properties have been extensively applied to biomedical areas including imaging, diagnosis, and therapy. In particular, interdisciplinary collaborative research between material science and biomedicine enabled nanomaterials to translate the medical issues and application to clinical trials. Cellular therapies by the administration of therapeutic cells such as stem cells or immune cells benefit greatly from the inclusion of nanomaterials to achieve high-resolution tracking of the cells. Another application of nanomaterials is as intrinsic chemotherapeutic agents. Long-term cell tracking can be realized by producing highly sensitive nanoparticles or by the control of particle-cell interactions. Engineering physical and chemical properties of nanoparticles - such as size, surface, and composition, enables to obtain optimized therapeutic potentials at target tissues with minimal toxicity. Mesoporous silica-coated hollow manganese oxide (HMnO@mSiO2) nanoparticles were fabricated for highly efficient T1 magnetic resonance imaging (MRI) contrast agent for labeling and MRI tracking of stem cells [1]. Computed tomograpy (CT) cell tracking methods with gold nanoparticles were developed [2]. The protective effects of ceria nanoparticles against ischemic stroke were studied, and demonstrated that intravenously administered ceria nanoparticles considerably reduced the brain infarct volume and nerve damage [3].
References
[1] T. Kim,dagger; T. Hyeon,* and Assaf A. Gilad,* "Mesoporous Silica-Coated Hollow Manganese Oxide Nanoparticles as Positive T1 Contrast Agents for Labeling and MRI Tracking of Adipose-Derived Mesenchymal Stem Cells" J. Am. Chem. Soc. 2011, 133, 2955-2961.
[2] T. Kim,dagger; T. Hyeon,* and Jeff W. M. Bulte,* “Micro-CT Imaging of Human Mesenchymal Stem Cells Labeled with Gold Nanoparticles” Manuscript in Preparations.
[3] C. K. Kim,dagger; T. Kim,dagger; S.-H. Lee,* and T. Hyeon,* "Ceria Nanoparticles that can Protect against Ischemic Stroke" Angew. Chem. Int. Ed. 2012, 51, 11039-11043.

Abstract: An in vitro blood-brain barrier (BBB) model was developed using murine brain endothelioma cells (b.End3 cells) and confirmed. Confirmation of the BBB model was completed by examining the permeability of FITC-Dextran at increasing exposure times in serum-free medium and comparing such values with values in the literature. After such confirmation, the permeability of five nanoparticles was determined by this model. Through such experiments, magnetic nanoparticles suitable for MRI use which would not pass through the BBB and magnetic nanoparticles suitable for drug delivery which would pass through the BBB to the were identified.
Materials and Methods: According to a patented process entitled “A biomimetic process for the synthesis of aqueous ferrofluids for biomedical applications”, five ferrofluids were synthesized by incubating a ferrous/ferric salt solution in phosphate-buffered saline supplemented with the additives of interest such as collagen, poly(vinyl) alcohol (PVA) and/or bovine serum albumin (BSA) using ammonium hydroxide under highly alkaline conditions. After synthesis, ferrofluids were centrifuged for a stability test so that the supernatant byproducts could be washed away. Dynamic light scattering and TEM were used to characterize their diameter and zeta potential was used to characterize the charge of those superparamagnetic iron oxide nanoparticles (SPIONs). An in vitro blood-brain barrier model based on b.End 3 cells was then used to test the permeability of the various nanoparticles which are 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). For this, nanoparticles were diluted 1:19 with HBSS and then inserts were exposed to them for 2 hours. After 2 hours, a 100 mu;L solution was taken from each well and full spectrum absorbance was used to determine the iron concentration that pass through the model. Each experiment was conducted in triplicate and repeated at least three times.
Results and Discussion: Results showed that the highest permeability was obtained from CPB and the lowest permeability was obtained from GGB. Also ferrofluids synthesized using collagen generally had higher permeability than those synthesized using glycine and glutamic acid. These results suggest that for nanoparticles that need to be delivered through BBB (i.e., for treating nuerological diseases), FF should be coated with collagen while, on the other hand, FF should be coated with glycine and glutamic acid to keep the nanoparticles from penetrating the BBB (i.e., for whole body MRI imaging to decrease brain toxicity).
Conclusions: An in vitro model of BBB was established using b.End3 cells. Results showed that in order to be delivered through BBB, ferrofluids should be coated with collagen and ferrofluids should be coated with glycine and glutamic acid to avoid penetration.

Despite the wealth of research focused on cancer, the testing ground for new drug delivery methods and novel drug candidates is a standard two-dimensional monolayer of cells. However, this method lacks the three-dimensional nature of a tumor, which has been shown to fundamentally alter gene expression. Furthermore, lack of diffusion within a tumor structure fosters necrosis and quiescence in the core. The three-dimensionality coupled with the tumor structure affords an increased resistance to chemotherapeutics. This project establishes a physiologically relevant in vitro model of a tumor that consists of a multicellular spheroid surrounded by a collagen matrix. Theo model is then used for subsequent testing with an expansile nanoparticle drug delivery system. Spheroids were made with a human breast cancer cell line (MDA-MB-231) and incorporated within a matrix to recapitulate stromal elements, whose interactions with tumors are fundamentally involved in the progression of cell phenotype from normal to malignant. Within in vitro models of cancer, both acellular and cellular stromal elements have afforded greater tolerance against chemotherapeutic agents. Cellular stromal elements are introduced via inclusion of fibroblast cells (murine fibroblast NIH3T3 cells) either as part of a spheroid or diffusely seeded within the collagen gel. In addition to the collagen matrix, Matrigel was included within the spheroid to mimic degradation of basement membrane associated with activated tumor stroma. Model plasticity allows for inclusion of any combination of these elements to analyze complex stroma/tumor interactions to evaluate nanoparticle drug delivery systems. Scanning electron and confocal microscopy indicate a well integrated tumor like structure with a necrotic core capable of ingrowth into the collagen matrix. Various spheroid constructs were treated against a polymeric expansile nanoparticle system releasing Paclitaxel (Pax-eNP) and Pax alone given as a bolus dose. Efficacy of a given treatment was judged by overall spheroid growth into the collagen. Nanoparticle and drug presence within the spheroid were measured by confocal microscopy and fluorescence activated cell sorting. Finally, cytotoxicity of the cells was quantified via the metabolic activity of disaggregated spheroids.

There are about 100,000 ACL reconstruction surgeries performed every year in the United States, with a failure rate ranging from 5-25%, depending on the criteria of the study [1]. It is believed that this high rate of failure is a result of insufficient healing at the tendon-to-bone insertion site (TBI). The TBI disperses critical stress concentrations that arise naturally between ligaments and bone by providing a compositional and mechanical transition from ligaments, through a fibrocartilaginous zone, to bone. However, this complex, inhomogeneous, and avascular tissue is incapable of regenerating following surgery. Therefore, there is considerable interest in the development of a nanostructured biomaterial that is capable of directing healthy regeneration of spatially controlled tissue across the TBI.
In this study, magnesium oxide (MgO) nanoparticles were used to mineralize poly(l-lactic acid) (PLLA) and tested for their ability to improve the attachment and growth of TBI-related orthopedic tissue. Magnesium is an essential mineral in bone which is thought to regulate the size and density of hydroxyapatite (HA) crystals, and further, Weng and Webster demonstrated that nano-rough MgO increased bone cell density three-fold compared to bulk MgO [2]. Presently, the ability of materials to promote tissue growth at the TBI was characterized via cell adhesion and proliferation experiments with fibroblasts and osteoblasts. Materials were also tested for their mechanical properties, and further characterization was performed using SEM, TEM, XRD, FTIR, EDS, and contact angle tests.
Results indicated for the first time that MgO nanoparticles in plain PLLA or PLLA/HA composites significantly increased osteoblast and fibroblast adhesion on PLLA. Interestingly, both cell lines followed the same general trend of adhesion on each sample, indicating that variations in only the secondary phase of a scaffold material will not be sufficient to direct the formation and maintenance of spatially controlled tissue heterogeneity at the TBI. However, nano-MgO can be used to mineralize different polymer phases to promote the formation of bone tissue at one end of the scaffold and fibrous tissue at the other end.
Mechanical tensile testing revealed that the addition of a secondary nano-phase to plain PLLA hardened the polymer, reducing the material elongation and increasing its elastic modulus. Moreover, the observed changes in mechanical strength of PLLA seemed to be dictated by the size and shape of its secondary nano-phase, indicating that the mechanical properties of PLLA composites can be tailored to align with the strength of bone or ligament tissue.
References
[1] Lui et al., Journal of Orthopaedic Surgery and Research, 5 (2010), 59.
[2] Weng L, Webster TJ, Nanotechnology. 23 (2012), 485105.

Tissue engineering is an interdisciplinary field, aimed at maintaining, restoring and enhancing normal tissue and organ functions by merging of engineering, biological sciences and medicine. To date, tissue engineering has been successfully applied to engineer many types of tissues including bone, cartilage and cardiovascular. One of the central themes in the field of tissue engineering is the development of proper biomaterials with tunable characteristics to mimic the body&’s architectural and geometrical intricacies and induce proper cell-cell and cell-matrix interactions. In the past few years, we have been actively involved in the development of innovative biomaterials for tissue engineering and stem cell bioengineering applications. We have demonstrated that that hydrogels are excellent scaffolding materials mainly due to their 3D microenvironment, homogenous cell distribution, and tunable mechanical and chemical properties. For instance, we have used ‘top-down&’ microfabrication techniques (i.e. micropatterning) to develop cell-laden gelatin methacrylate (GelMA) hydrogels with precise geometrical features for bone and cardiovascular tissue engineering applications. Recently, we demonstrated microfabrication of elastin-based hydrogels (methacrylated torpoelastin, MeTro) with remarkable properties for engineering of various types of tissues. We have shown that the fabricated MeTro hydrogels exhibit excellent mechanical characteristics such as high resilience upon stretching and reversible deformation with low energy loss. In addition, we have demonstrated that the mechanical properties of fabricated MeTro gels can be finely tuned to select desirable stiffness for a specific application (i.e. cardiovascular) depending on the methacrylation degree and concentration of the hydrogels. In this talk, I will present recent findings in our lab covering fabrication of novel biomaterials for engineering 3D tissue constructs.

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 talk will particularly focus on how to create 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 addressing the challenges in commercializing biodegradable metallic implants. 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.

For the last two decades, the advance of material sciences and biotechnology enabled the development of numerous types of scaffolds for regenerative medicine. These scaffolds aim at tissue regeneration involving minimally invasive surgical procedures.
In this work we report the development of injectable scaffold prepared with chitosan and collagen-coated gold nanoparticles (NPs). Chitosan is a hydrogel known for its porous structure, biocompatibility and biodegradation. However, its low mechanical resistance and the requirement of toxic crosslinkers in its preparation, like glutaraldehyde, have hampered its application in biomedical devices. The incorporation of gold NPs aims at improving cell proliferation through mechano-transduction events due to the nanotopography. Besides, gold NPs provide the in situ delivery of bioactive molecules, as for example, collagen type I, which is a bioactive molecule reported to stimulate chondrocytes growth. Finally, since gold is an antibacterial material, the presence of gold NPs may reduce the risk of infection normally caused by scaffold implants.
Gold NPs were prepared by reduction of gold (III) chloride hydrate using sodium citrate. Collagen was linked to gold NPs by EDC/NHS coupling reaction. NPs size distribution and zeta;-potential were measured by dynamic light scattering. The mean diameter was measured as 52.82nm and the zeta;-potential was of -7.6mV. After NPs coating the mean diameter and zeta;-potential were 300nm and -7,3mV, respectively. NPs were also characterized by UV-Vis spectrometry. The maximum of absorption was at 522nm for bare NPs, which is characteristic of gold NPs of 20nm of diameter. Absorbance spectrum of collagen-coated NPs presented two additional bands at 280 and 220nm. These bands are characteristic of proteins, proving that the collagen was linked to the NPs. The concentration of collagen in NPs suspension was determined by Bradford method as being 161mg.mL-1. The hydrogel was obtained by preparing chitosan solution 2% (w/v) in acetic acid 2%. Following, collagen-coated gold NPs were dispersed into the gel and 20mu;L of glutaraldehyde 25% was added. After 12h the hydrogel was acetylated by treatment with acetic anhydride.
Our results demonstrated that the incorporation of gold NPs into chitosan provided chitosan crosslinking at glutaraldehyde concentration 10 times lower than conventional procedures. Additionally, the chitosan collagen-coated gold NPs scaffold presented better mechanical resistance compared to the scaffold of chitosan. Scaffold morphology was characterized by SEM. Compared to the scaffold composed of chitosan only, chitosan-gold NPs scaffold presented a structure with reduced porosity and higher topographical disorder. Studies of cellular proliferation and bacterial test are being performed.
This work has been sponsored by FAPESP.

Background: Biomaterials&’ physical shape and structural feature size on the micro and nano scales are increasingly recognized to play important roles in their function as scaffolds for tissue engineering and as vehicles for controlled or targeted therapeutic delivery. Recently, our laboratory developed injectable polymeric nanofibrous hollow microspheres and demonstrated their advantages over traditional cell carriers for knee cartilage regeneration in rabbits. However, the nanofibrous hollow microspheres could only be self-assembled from a star-shaped poly(L-lactic acid) (PLLA) with unclear mechanisms. Objective: To investigate the scientific mechanism of the nanofibrous hollow microsphere formation, to develop facile techniques to generate nanofibrous hollow objects from a variety of polymers, and to employ these vehicles to carry cells for regeneration. Methods: A non-aqueous emulsion system was developed to emulsify polymer solutions into microspheres. A thermally-induced phase separation technique was integrated in the process to generate nanofibrous structure. In one approach, a mixed solvent system was employed to increase the affinity of a polymer solution (such as a linear PLLA) to the emulsion medium to initiate and stabilize double emulsion formation, leading to hollow object formation. In a different approach, appropriate emulsifiers were incorporated into different emulsion systems (including a PLLA solution emulsified in glycerol, a Nylon6 solution emulsified in olive oil, and a polyacrylonitrile (PAN) solution emulsified in silicone oil), which also led to the formation of nanofibrous hollow microspheres. In addition, nanofibrous hollow microspheres with controllable open hole size, nanofibrous hollow discs and nanofibrous shells could be fabricated through adjusting the emulsification process. We seeded pre-adipocytes inside PLLA nanofibrous hollow microspheres, where the cells differentiated into mature adipocytes with rich lipid droplets formation. Conclusion: We formed a generalized theory of nanofibrous hollow structure formation and developed techniques to fabricate nanofibrous hollow microspheres using linear PLLA, PAN and Nylon 6 for the first time. In addition, we developed techniques to control the open hole size on the nanofibrous hollow microspheres. Furthermore, we developed new and facile fabrication techniques to generate nanofibrous hollow discs and nanofibrous shells for the first time. These nanofibrous hollow objects are promising injectable cell carriers for tissue regeneration.

Nanoporous silica has been widely investigated as a novel material for biomedical applications. We have shown that coatings of nanoporous silica on implants have a favourable biocompatibility [1] and offer the possibility to locally deliver bioactive molecules (e.g. growth factors) [2] and small-molecule drugs [3]. Animal experiments have shown that healing reactions after implantation operations could successfully be improved [4], as the interaction between the surface (coating) of an implant and the surrounding tissue is a crucial factor for the efficiency of a prosthesis.
Nanoporous silica nanoparticles (NPSNPs) offer similar possibilities with regard to growth factor and drug delivery. Often, these nanoparticles are used as suspensions, which are added to cell culture media or injected into animals for testing their efficacy. However, they can also be integrated into tissue engineering scaffolds. Here, we report on the decoration of nanoporous silica nanoparticles with polysialic acid, a polysaccharide involved in neural development, on the NPSNP-based delivery of BMP2, a growth factor, and of dexamethasone, a small-molecule drug. Both are involved in the differentiation of mesenchymal stem cells. Due to their rich surface chemistry, NPSNPs are highly versatile with regard to different surface modifications which allows to adapt their chemical interactions in a way which facilitates their integration into various scaffold materials. As an example, the integration of functionalized NPSNPs into collagen-based scaffolds will be presented. Periodic mesoporous organosilicas (PMOs) offer an interesting alternative to nanoporous silica with a more hydrophobic character.
[1] C. Turck, G. Brandes, I.Krueger, P. Behrens, H. Mojallal, T. Lenarz, M. Stieve. Acta Oto-Laryngol. 2007, 127, 801-808.
[2] N. Ehlert, A. Hoffmann, T. Luessenhop, G. Gross, P.P. Mueller, M. Stieve, T. Lenarz, P. Behrens. Acta Biomater., 2011, 7, 1772-79.
[3] N. Ehlert, M. Badar, A. Christel, S.J. Lohmeier, T. Luessenhop, M. Stieve, T. Lenarz, P.P. Mueller, P. Behrens. J. Mater. Chem. 2011, 21, 752-60.
[4] N. Ehlert, P.P. Mueller, M. Stieve, T. Lenarz, P. Behrens. Chem. Soc. Rev. 2013, 42, 3847-3861.

Engineering the molecular design of intelligent biomaterials by controlling recognition and specificity is the first step in coordinating and duplicating complex biological and physiological processes. Recent developments in protein delivery have been directed towards the preparation of targeted formulations for protein delivery to specific sites, use of environmentally-responsive polymers to achieve pH- or temperature-triggered delivery, usually in modulated mode, and improvement of the behavior of their mucoadhesive behavior and cell recognition. We address design and synthesis characteristics of novel crosslinked networks capable of protein release as well as artificial molecular structures capable of specific molecular recognition of biological molecules. Molecular imprinting and microimprinting techniques, which create stereo-specific three-dimensional binding cavities based on a biological compound of interest can lead to preparation of biomimetic materials for intelligent drug delivery, drug targeting, and tissue engineering. We have been successful in synthesizing novel glucose- and protein-binding molecules based on non-covalent directed interactions formed via molecular imprinting techniques within aqueous media.

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.

Moldable, settable bone grafts that possess mechanical strength exceeding that of host bone and maintain strength while remodeling could improve the clinical management of numerous orthopaedic conditions. Polyurethane (PUR) composites are an attractive alternative to calcium phosphate cements due to their tough mechanical properties and active remodeling. 45S5 bioactive glass (BG) particles have widely been used for bone regeneration purposes due to its osteoconductivity. Although physiological loads are cyclic, fatigue properties of biomaterials utilized in load-bearing applications are rarely reported. We investigated the quasi-static and dynamic compressive mechanical properties of BG/PUR composites. We hypothesized that a BG/PUR composite comprised of surface-modified BG would improve the mechanical properties compared to one made with cleaned-BG, and that this composite would maintain its strength throughout in vivo remodeling. BG particles were grafted with 3-aminopropyl-trietoxysilane (APTES) and surface-polymerized polycaprolactone (PCL). Composites were made from a lysine triisocyanate- poly(ethylene glycol) prepolymer, polyester triol (70% caprolactone, 20% glycolide, 10% lactide polyol, Mn ~300 g mol-1), triethylene diamine catalyst in dipropyl glycol, and BG (56.7 volume %). Under quasi-static compression, cleaned and modified BG/PUR composites had strengths of 7.9 ± 3.2 and 53.8 ± 6.5 MPa, respectively. Fatigue testing was completed in cyclic sinusoidal (5 Hz) compression, reaching maximum stress levels of 5 to 15 MPa. Under a maximum stress level of 5 MPa, the mean fatigue life of the cleaned- and modified-BG composites was 197 ± 257 and 904,172 ± 130,054 cycles, respectively. The compressive modulus of the modified-BG composite gradually decreased until failure. Its residual strain increased throughout the testing due to plastic deformation. The compressive mechanical properties of the composites were dependent on the interfacial bonding between the surface-polymerized PCL chains and the PUR network, formed in situ. Results related to the relative average length of surface chains, with respect to PUR network mesh size, support the hypothesis that the observed increase mechanical strength and fatigue life is due to chain entanglements and physical crosslinks. For the in vivo study, a 11mm diam X 18mm length defect was created in the diaphysis of sheep femur and BG composites were implanted/cured. After 8 and 16 weeks, the defect site was explanted and quasi-static compression mechanical testing was conducted. The mechanical properties of explanted BG/PUR composite from the sheep femoral plug defect maintained strength above the local native bone control. By comparing these compressive mechanical properties of cleaned- versus modified-BG/PUR composites, we conclude that surface modification significantly extends the BG/PUR composite&’s ability to withstand physiologically relevant dynamic stresses, while actively remodeling in vivo.

Osteochondral (OC) tissue is a complex structure comprised of an upper layer of articular cartilage, the subchondral bone and the central cartilage-bone interface. In order to facilitate proper regeneration of the tissue it is essential to devise a matrix that ensures cartilage-bone interface formation along with the regeneration of the individual tissue layers. Although mono-phasic and bi-phasic matrices were previously applied to OC defect repair, they failed to establish the proper osteochondral interface upon regeneration. In this study, we design and develop a gradiently porous matrix with increasing pore volume from one end to other, along the scaffold length. We hypothesize that such a design will enable OC tissue regeneration, including the cartilage-bone interface. For this matrix polylactide-co-glycolide or PLGA 85:15 microspheres were combined with a water-soluble porogen in a layer-by-layer fashion, increasing the porogen content (from bottom to top), and thermally sintered. The resulting matrix was then porogen-leached to form a gradiently porous structure. Micro-CT scanning was performed to establish the gradient pore structure with pore volume continuously increasing from 30-60%. A biodegradable hydrogel was infused into the gradient pore structure to form a unique OC graft where the microsphere and hydrogel phases co-exist with opposing gradients. When the individual phases are loaded with osteogenic and chondrogenic growth factors, the structure would ensure the spatial control of growth factor delivery necessary to regenerate osteochondral tissue structure. The uniqueness of this approach is to design a matrix system where bone- and cartilage-forming phases are unified in a way that supports the complexity of OC tissue regeneration. As determined by immunostaining, the gradient matrix system seeded with human bone marrow stromal cells show osteogenic as well as chondrogenic differentiation. Overall, through this study we designed a gradient matrix system that would support osteochondral tissue engineering while forming a seamless interface between the articular cartilage and the underlying bone matrix.

Musculoskeletal joint motion is facilitated by synchronized interactions between multiple tissue types and the seamless integration of bone with soft tissues such as ligaments, tendons or cartilage. Many of these soft tissues transit into bone through a multi-region fibrocartilaginous interface, which serves to minimize the formation of stress concentrations while enabling load transfer between soft and hard tissues. With its functional significance, re-establishment of the soft tissue-to-bone interface is thus critical for promoting the integration of biological as well as synthetic soft tissue grafts. A pressing challenge for interface tissue engineering is how to harness the repair potential of mesenchymal stem cells for the simultaneous regeneration of more than one type of tissue, which is essential for multi-tissue integration. To this end, our approach centers on identifying and optimizing composite scaffold design parameters such as scaffold composition and fiber organization, especially when coupled with mechanical loading in directing stem cell differentiation into the ligament fibroblasts, fibrochondroytes or osteoblasts. This lecture will describe our work with nanofiber-based scaffolds and how controlling scaffold diameter, alignment, composition and/or mechanical loading can direct MSC differentiation into interface-relevant cell populations, without concurrent stimulation with growth factor or inductive media. These insights provide new understandings of stem cell-biomaterials interactions, and will lead to a new generation fixation devices for integrative connective tissue repair and regeneration.

Synthetic biodegradable polymers have been widely investigated as scaffolds for tissue engineering applications. For certain applications (e.g. loading bearing hard tissues), the polymeric scaffolds need to possess sufficient mechanical strength. Reinforcing agents possessing high intrinsic mechanical property allow efficient load transfer, increasing the mechanical properties of the polymeric implants. These reinforcing agents should not only improve the bulk properties of the polymer, but also not elicit any adverse effects on cells and tissues. In this study, we report the efficacy of several organic and inorganic one- and two- dimensional nanomaterials (single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs), single-walled graphene oxide nanoribbons (SWGONRs), multi-walled graphene oxide nanoribbons (MWGONRs), graphene oxide nanoplatelets (GONPs), tungsten sulfide nanotubes (WSNTs) and molybdenum sulfide nanoplatelets (MSNPs) as reinforcing agents to enhance mechanical properties of polypropylene fumarate (PPF) for tissue engineering applications. We also report the in vitro biocompatibility of the PPF nanocomposites at nanomaterials loading concentrations that provide maximum mechanical reinforcement. PPF nanocomposites were prepared by dispersing SWCNTs, MWCNTs, SWGONRs, MWGONRs, GONPs, WSNTs and MSNPs in PPF [1,2]. Nanocomposites were used for compression and flexural testing. Subsequently, cytotoxicity of nanocomposites (yielding maximum mechanical reinforcement) against MC3T3 pre-osteoblasts was evaluated. Confocal and scanning electron microscopies (SEM) were performed to study cell attachment.
All PPF nanocomposites showed statistically significant enhancement in the mechanical properties with 0.2 wt% MoS2 nanoplatelets showing the highest Young&’s modulus of asymp; 10GPa; a 100% increase compared to PPF (control). Inorganic nanoparticles showed equivalent or better mechanical reinforcement compared to carbon nanoparticles. Cytotoxicity study showed more than 78% viability for cells in contact with crosslinked samples, which increases to ~100% with media dilution, as expected for a dose dependent cytotoxicity. Confocal and SEM imaging analysis showed about 40-49% cell attachment.
1Dand 2D carbon and inorganic- nanostructures as reinforcing agents significantly improve the mechanical properties of PPF. PPF nanocomposites exhibit low cytotoxicity, good cell attachment and spreading. The results taken together indicate that 1Dand 2D carbon and inorganic- nanostructures are promising materials as reinforcing agents to improve the bulk and functional properties of polymeric implants for tissue engineering applications.
Acknowledgements:
This work was sponsored by National Institutes of Health (grants No. 1DP2OD007394-01).
References
[1]. Lalwani, G., et al., Biomacromolecules, 2013. 14(3): p. 900-90.
[2]. Lalwani, G., et al., Acta Biomaterialia (In Press)

Fracture toughness has occasionally been neglected in the development of tissue engineering scaffolds. In fact, almost all recent developments aim to achieve transparent scaffolds with the tensile strength and elastic modulus closely-matched to those of native cornea despite the fact that cornea is normally subjected to below-ultimate-strength cyclic tensile loadings due to intraocular pressure, ocular muscle contractions and eye blink. Similarly to other soft collagenous tissues, toughening mechanisms in cornea are not well understood, but the lamellar structure of orthogonally aligned collagen fibrils in corneal stroma is thought to account for its toughness. To examine this, transparent laminates of gelatin nanofibers in chitosan-alginate gel, mimicking the corneal lamellar structure, were created in a three-step process. First, stacks of orthogonally aligned gelatin nanofibers were created by electrospinning followed by chemical cross-linking. Next, dehydrated cross-linked gelatin fibers were swollen in chitosan-alginate solution, forming fiber-reinforced hydrogel composites. Finally, the resulting structures were subjected to cycles of dehydration and chemical cross-linking to increase their mechanical properties and optical transparency. Fracture toughness and time-independent tensile behaviors of the orthogonally-aligned fiber-reinforced hydrogels were characterized using trouser tearing and uniaxial tensile tests. Their behaviors were compared to those of pure hydrogels and hydrogels reinforced with randomly-oriented or parallel gelatin fibers. Relative orientation of fibers in adjacent layers was found to significantly affect the overall fracture toughness and time-independent tensile behaviors of the fiber-reinforced hydrogels and is therefore a key to achieve tough biomimetic scaffolds for corneal tissue engineering.

Advanced composites comprise a growing component of research and development of medical products. Composites range in complexity from heterophase single materials to multi-component constructs of combination medical products. The diversity of requirements leading to regulatory approval and successful marketing of medical products based on the range of composites is enormous. For all regulated medical products, there are criteria which must be met in order to achieve a commercially successful outcome. Technical, regulatory, economic and societal considerations can be subdivided into several essential components each. Careful consideration of each of these criteria should be given before committing substantial resources to product development. A good point at which to make this evaluation is often just following proof of concept. Numerous examples exist of successful and unsuccessful medical product development efforts. The failure to develop a successful medical product may well be determined by even one or a few of the critical criteria not being met.

Natural origin biodegradable polymers have outstanding properties for many biomedical applications including as scaffolds for tissue engineering. The cell recognition sites, the biodegradability and the cytocompatibility of many natural origin biomaterials are particularly appealing in those applications. However, the limitations in its processing and the narrower spectra properties when compared to its synthetic counterparts, frequently hinder its application in more demanding biomedical devices. The composition of those materials with synthetic biodegradable polymers allows conferring them easier processability, which is a key issue for many applications but in particular for tissue engineering scaffolding. The effective reinforcement of biodegradable biomaterials with fibres or with particulates enable tailoring its properties for defined applications and provide enhanced opportunities for using biomimetic approaches in the biomaterial development for advanced therapies.
A critical ingredient of many strategies aiming the development of Advanced Therapies and in particular using Tissue Engineering concepts is the need for scaffolds providing temporary structure to assist the cells in the regeneration of tissue defects. The scaffolds should be specifically designed to create environments that promote tissue development and not merely to support the maintenance of communities of cells. To achieve that goal, highly functional and porous scaffolds may combine specific morphologies and surface chemistry with the local release of bioactive agents.
Many composites were already proposed in scaffolds aiming the regeneration of a wealth of tissues. We have a particular interest in developing systems based in biodegradable polymers for the regeneration of bone and articular cartilage. Those mechanically demanding applications require a combination of mechanical properties, processability, cell-friendly surfaces and tuneable biodegradability that need to be adjusted for the specific application envisioned (the properties of both bone and cartilage are highly dependent of the anatomical site considered). Those biomaterials may be processed by different routes into devices with wide range of morphologies such as fibers and meshes, films or particles with outstanding biological and structural performance for biomedical applications. We will review herein our latest developments in this fascinating research area.

Cell culture scaffolds act as a template for tissue regeneration, and encourages cells to form healthy and functional tissues. Cell behaviors, including cellular adhesion, proliferation, migration, and differentiation, are regulated by the interactions between cells and the microenvironment of the cells; therefore, the chemical, topological, and mechanical properties of scaffold surfaces are significant for regulating the cell behavior. We have reported that honeycomb-patterned porous polymer films can be prepared by casting a polymer solution under humid conditions and using condensed water droplets as templates of pores. In this report, we show the preparation of honeycomb scaffolds for cell culturing by using above-mentioned “breath figure” method, and we found that their mechanical and topographical properties strongly affect the adhesion of fibroblasts. By photo-crosslinking of the poly(1,2-butadiene), the hardness of the honeycomb scaffold can be successfully controlled without any surface chemical changes, and detail modulus values of scaffolds were measured by atomic force microscopy. We found that only small numbers of the cells adhered on the softer honeycomb scaffolds, which has even higher modulus value than conventional gels, comparing with flat films and a hard honeycomb scaffold. These results indicate that the elastomeric honeycomb substrates are useful for evaluating the effect of the mechanical signal-derived geometry on the transduction system of cells.
We also found that honeycomb scaffolds led human mesenchymal stem cells (hMSCs) to osteospecific and myospecific differentiations depending on the size of pores without any hazardous chemicals and supplements. Polystyrene honeycomb scaffolds with different pore sizes were successfully fabricated by casting a polymer solution under humid conditions in order to investigate the effect of porous microtopography on hMSC differentiation. We have used honeycomb scaffolds to achieve the microtopography-induced differentiation of hMSCs without any hazardous chemicals. Honeycomb scaffolds led hMSCs to osteospecific and myospecific differentiations depending on the size of pores, which identified by immunofluorescent microscopy. This selective differentiation suggested that surface microtopography may be effective for using hMSCs in regenerative medicine and tissue engineering.

Treating long-bone defects still to date is a major challenge in orthopedic surgery. Current treatment modalities such as autografts and allografts have many limitations and often fail. Tissue Engineering has emerged as a promising and more fitting alternative. Despite the tremendous efforts by researchers, the traditional scaffold designs support intramembranous ossification through the direct differentiation of mesenchymal stem cells into bone forming cells (osteoblasts). Bone formation via this route does not involve vascularization process and therefore relies solely on diffusion for nutrient and waste transport; this results in extremely hypoxic conditions in the central regions of scaffolds and leads to limited amount of bone regeneration. On the other hand, long-bones development, naturally, occurs through cartilage mediated ossification (endochondral); where bone formation occurs through cartilage matrix formation, matrix vascularization and finally ossification [1]. As such, in order to address the unique challenges of long-bone regeneration, an advanced polymer-hydrogel scaffold is proposed. Our system is composed of a poly(85 lactide-co-15 glycolide) acid microsphere scaffold as a load-bearing phase. Thermal sintering and porogen leaching technique was used to develop PLGA microsphere scaffolds with tunable pore volume [2].The second component of our system is a hydrogel matrix that will closely mimic the natural three dimensional extra cellular matrix (ECM) network required for stem cells differentiation into chondrocytes and their subsequent maturation into hypertrophic chondrocyte. In addition, the hydrogel will serve to encapsulate cells rapidly and efficiently, as well as providing a venue for drug and agent delivery. Through this study, we successfully developed a hydrogel-polymer scaffold system where the hydrogel phase supports cartilage-mediated bone formation, while the stable polymer phase acts as a load-bearing graft essential for long-bone repair. Together, the polymer-hydrogel matrix design is proposed as a novel matrix system for bone regeneration via endochondral ossification.
[1] Amini A R, Laurencin C T and Nukavarapu S P 2012 Bone tissue engineering: recent advances and challenges Critical reviews in biomedical engineering 40 363-408
[2] Amini A R, Adams D J, Laurencin C T and Nukavarapu S P 2012 Optimally porous and biomechanically compatible scaffolds for large-area bone regeneration Tissue engineering. Part A 18 1376-88

Existing strategies for bone regeneration based on tissue engineering principles are driven by challenge of developing scaffolds capable of eliciting cell response favorable for tissue formation and providing mechanical support until the regenerating tissue has enough strength to support itself or restore function at defective sites. The effect of mechanical stimuli on cellular processes has broadened the scope of mechanical properties with the need to consider the mechanical properties at nanoscale in addition to the macroscale. Biodegradable composites prepared by incorporating inorganic fillers in biodegradable polymers have great potential for fabricating scaffolds that can meet the challenges of bone tissue engineering. Biomimetic nanoclay-hydroxyapatite (HAP) hybrid was prepared using sodium montmorillonite (Na-MMT) clay modified with an unnatural amino acid and for preparing biodegradable polymer (both natural and synthetic) composite films and scaffolds in our studies. The choice of MMT clay was based on insights gained through “altered phase theory” for polymer-clay nanocomposites (PCNs), reinforcing capability of MMT clay, medicinal properties of MMT clay and its reported use for pharmaceutical applications. A biomimetic approach was used to mineralize HAP in modified MMT clay galleries to obtain the nanoclay-HAP hybrid and it was found through infrared spectroscopy studies that carboxylic groups of unnatural amino acid in modifed clay were involved in HAP mineralization. Nanoindentation tests performed on biodegradable polymer (synthetic) composite films containing nanoclay-HAP hybrid showed significant improvement in nanomechanical properties thus emphasizing the reinforcing effect of nanoclay-HAP hybrid at nanoscale. Biocompatibility and osteoconductive potential of the biodegradable polymer composite films and scaffolds was studied through appropriate assays involving human mesenchymal stem cells (hMSCs). In addition to studies related to