Symposium SM04—Translational Materials in Medicine---Prosthetics, Sensors and Smart Scaffolds

Tissue engineering has been with us for over 30 years and yet, I spite of some clinical successes, it has had little impact on the day-to-day practice of clinical medicine. This has been attributed to translational issues in scale-up and production, i.e., high costs; incompletely developed technologies and regulatory concerns. The concept ofin situtissue engineering is attractive in that it utilizes the patient’s own cells and also the patient’s own body as the bioreactor. Sphere templated porous polymers (STPP), where all interconnected pores are approximately 40 microns in diameter, have been found upon implantation to be rapidly infused with macrophages which then, in about 1 month, leads to vascularized, reconstructed tissue. Larger and smaller pore size materials do not demonstrate this phenomenon. Implantation of 40 micron pore STPP in bone sites yielded bone. Implantation in sclera yielded sclera tissue. Implantation in skin showed reconstruction of both the dermis and epidermis. Implantation in heart led to reconstruction of the heart stroma. Recent investigation of this in situtissue engineering reconstruction demonstrated that major changes are occurring in RNA signals from cells in 40 micron pores, compared to cells in 80 micron pores. Also, unique cells morphologies are seen in different pore sizes. Finally, there is evidence that the macrophages may be differentiating to an endothelial-like lineage contributing to the vigorous blood vessel ingrowth. (coauthors on this work are James Bryers, Neal Beeman and Le Zhen)

Silk proteins have emerged from the textile and suture worlds into a broader suite of medical utility over the past few decades. This progression started with new fundamental insights into the features of this unique protein polymer, and has subsequently evolved into new utility for these polymers in terms of clinical impact. We will review the historical, research and translational steps that have supported new medical materials and devices based on silks. We will also speculate on the future directions for this protein polymer based on the progress to date and the medical needs of the future.

Post-operative adhesions represent an important, unmet clinical need with costs to the U.S. healthcare system exceeding $2.5 billion annually. Adhesions form as a result of normal wound-healing processes following any type of surgery and develop after 95% of cases. In cardiac surgery, adhesions are particularly problematic during redo operations where surgeons must release pericardial adhesions from the surface of the heart before the intended procedure can begin. This process significantly lengthens operation times and introduces significant risks of hemorrhage and injury to the heart and lungs during sternal reentry and cardiac dissection. This presentation will discuss the use of a dynamically crosslinked polymer-nanoparticle (PNP) hydrogel adhesion barrier comprising hydrophobically-modified biopolymers (BPs) and biodegradable PEG-PLA nanoparticles (NPs). We demonstrate these materials to have desirable linear viscoelastic, yield-stress, and flow properties permitting simple spray delivery, robust tissue adherence, local tissue retention over the course of weeks and complete resorption within one month, as well as robust prevention of pericardial adhesion formation. We show that it is the distinct mechanical properties of these materials that dictate efficacy. Overall, this presentation will demonstrate the utility of a supramolecular hydrogel system as an effective solution for the prevention of post-operative pericardial adhesions.

We present here the development of a physical human eye model – based on hybrid soft/rigid materials - in order to create a test bench reproducing the optical eye properties.
We have developed phantom eye tissues in order to mimic the different parts like the sclera or the ciliary body as finely as possible regarding the optical properties. As a matter of fact, these parts are crucial in order to mimic human eye but often neglected in the literature. For the development of these models, we used the optical parameters (absorption and scattering coefficients and refractive index) extracted from an experimental study carried out on porcine eyes – which are close to human one. Moreover, we present a soft actuated model of the iris where the aperture ranges from 1 mm to 8 mm. Finally, all the different parts are put together to obtain a device mimicking exactly the optical properties of an eye.
This model could use to test medical imaging like diffuse optical tomography.

The use of non-covalent self-assembly to construct materials has become a prominent strategy in biomaterials science offering practical routes for the construction of increasingly functional materials. A variety of molecular building blocks can be used for this purpose, one such block that has attracted considerable attention in the last 20 years is de-novo designed peptides. Peptides offer a number of advantages to the biomaterial scientists. The library of 20 natural amino acids offers the ability to play with the intrinsic properties of the peptide such as structure, hydrophobicity, charge and functionality allowing the design of materials with a wide range of properties. Synthetic peptides are chemically fully defined and easy to purify through standard processes. Being build form natural amino acids they result usually in low toxicity and low immune response when used in-vivo and can be degraded and metabolised by the body. The β-sheet motif is of particular increasing interest as short peptides can be designed to form β-sheet rich fibres that entangle and consequently form very stable hydrogels. Through the fundamental understanding of the self-assembly and gelation processes of these peptides across length scales [Gao J. et al. Biomacromolecules 2017, 18, 826] we have been able to design hydrogels with tailored properties for a range of applications from tissue engineering [D. Kumar et al. Adv. Func. Mat. 2017, 27, 1702424] and cell culture [Castillo-Diaz L. et al. J. of Tissue Eng. 2016, 7, 2041731416649789] to drug delivery [Tang et al. Int J Pharm 2014, 465, 427], 3D bioprinting [Raphael, B. et al., Materials Letters 2017, 190, 103. and biosensing [King P. Et al. Chem. Comm. 2016, 52, 6697].

This talk will provide an overview of our recent developments in the design of smart materials for cell modulation. Bio-responsive nanomaterials are of growing importance with potential applications including drug delivery, diagnostics and tissue engineering (1) and recent examples in the design of nanostructured surfaces and elucidation of the cell-material interface will be presented [2, 3].

[1] Stevens MM, George JH, Exploring and engineering the cell surface interface, Science, 2005, 310, 1135-1138.
[2] Crowder S, Leonardo V, Whittaker T, Papathanasiou P, Stevens MM. Material cues as potent regulators of epigenetics and stem cell function. Cell Stem Cell. 2016. 18(1): 39-52.
[3] von Erlach TC, Bertazzo S, Wozniak MA, Horejs C, Maynard SA, Attwood S, Robinson BK, Autefage H, Kallepitis C, del Río Hernández A, Chen CS, Goldoni S, Stevens MM, Cell-geometry-dependent changes in plasma membrane order direct stem cell signalling and fate, Nature Materials. 2018. 17: 237–242.

Regenerative medicine has led to many advances in tissue replacement therapies for hard and soft tissues. Besides the numerous coatings and putty-like products, tissue engineered manufactured constructs are also making headways for healing large defects. In addition, these tissue engineered constructs have a new application as testbeds for evaluating diseases. For instance breast and prostate cancer have the propensity to metastasize to bone at which point the disease is incuravble. Lack of human samples at this stage of metastasis when patient is typically under hospice care and failure of animal models (since animals die before metastasis to bone) makes the humanoid test-bed a very advantageious drevice for studing cancer progression. We present a unique scaffold that uses biomimetic mineralization of hydroxyapatite inside nanoclays galleries to make the hierarchical structure of human bone with a stoichiometry characteristic of new bone; a niche to which cancer cells migrate. Prostate and breast cancer cell lines are seeded on to this tissue engineered bone scaffold leading to the creation of tumoroids of cancer. We also demonstrate that this engineered test-bed duplicates the last stage of cancer metastasis as indicated by the gene expression and immunocytochemistry analysis of the tumors generated in the testbed. Metastatic and non metastatic cell lines of both prostate and breast cancer are evaluated at metastasis site indicating different and unique behaviours at bone site. Nanomechanical evaluation in conjuction with gene and protein expressions are done for cancer progression at bone site. We demonstrate that the nanomechanical evaluation can describe the progression of the disease. Thus, unique biomimetic smart scaffolds provide new opportunities to evaluate cancer metastasis.

More than 100,000 total hip replacements surgeries are performed each year in France¹. Surgical site infections for these surgeries, mainly caused by the presence of microorganisms at the surface of the implant, are rare (0.5-5 % of the cases²) but can lead to serious damages such as implant failure and its removal. Currently, an efficient strategy for combating these infections is the use of systemic antibiotic prophylaxis. An alternative promising way to avoid its overuse is to use the implant as a support for the antimicrobial activity. Such a strategy allows the possibility of higher dosages near the affected site thereby improving efficiency and reducing the treatment duration and side effects³ . Titanium alloy is widely used as material for body implant for its biocompatibility; nevertheless it cannot induce bone regeneration. In order to combine osseoconduction and local drug delivery, implants can be coated with a hydroxyapatite layer, the constituting mineral of bones, known to improve the osseointegration of implants⁴.
The goal of this work was to develop an antibacterial coating on titanium alloy Ti6Al4V, associating a biomimetic hydroxyapatite and a natural antibacterial molecule, baicalein. This molecule is a flavonoid extracted from the root of the plant Scutellaria baicalensis, that has been used for a long time in the chinese traditional medicine for its antioxidant properties, and of which the efficiency has been proved against multi resistant bacteria⁵. Hydroxyapatite coating was synthesized by immersion of the metal in a mineralizing solution called Simulated Body Fluid (SBF), whose ionic composition is similar to blood plasma, at physiological pH and temperature⁶. Hybrid materials were obtained either in a one-pot synthesis, with incorporation of baicalein in SBF, or in a two steps process consisting in the adsorption of baicalein at the surface of a pre-formed hydroxyapatite layer. Hybrid coatings were characterized by scanning electron microscopy, X-ray electron spectrometry, and both Raman and Fourier transformed infrared spectroscopies for their physico-chemical properties and for their antimicrobial activity. Finally, release of baicalein from both coatings was investigated, as well as their cytotoxicity and osteoinductivity. For purpose of comparison and understanding, a study of the molecular interaction between calcium and baicalein in solution was also performed.

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Additive manufacturing (AM) or 3D printing (3DP) is becoming important for biomedical device fabrication, especially for patient matched implants due to lower cost and shorter lead time to manufacture. There are an estimated one million bone grafting procedures performed annually in the U.S. and a few million worldwide to repair bone defects, tumors, hip and knee replacements. Depending on the clinical need, different biomaterials are used for site specific or patient specific applications, for which different 3D printers are needed to create the biomedical device. Establishing process property relationships for different AM techniques are vital towards successful implementation of 3DP. However, additively manufactured components are still questioned for their reproducibility, machine to machine part quality variations and process specific material properties. Hard biomaterials, e.g., calcium phosphate (CaP) ceramics show significant promise towards bone implant applications, in both 3DP tissue engineering scaffolds and surface modified hip and knee replacement devices. We have used 3DP CaP scaffolds, for bone tissue engineering to control their degradation kinetics, mechanical strength, and biological properties with improved osteogenesis, angiogenesis and as drug delivery vehicle. An additional coating of polymer on both CaP scaffolds and hip / knee implant devices helped improve mechanical and biological properties while controlling the drug release kinetics. The presentation will include opportunities and challenges towards the use of 3DP or AM in developing biomedical devices.

Digital technology has provided the maxillofacial and craniofacial team with the ability to manipulate digital images using various 3D software’s to provide additional information which can be applied to improving patient treatment outcomes. CT and MRI data can be used in conjunction with 3D printers to produce anatomical models of the skull and provide the surgeon with the advantage of pre-planning the surgical procedure and preparing templates to act as guides during surgery to minimise tissue morbidity and reduce surgical time. Traditional methods of manufacturing maxillofacial prostheses are undertaken by hand carving the missing anatomical form in wax, and creating a mould into which pigmented silicone elastomer is placed. Over the last decade more modern technologies have been employed to manufacture anatomical face/body parts utilizing computed tomography (CT) data in conjunction with rapid prototyping (RP) techniques utilizing a hard plastic resin or thermoformed wax. However, these methods still require moulds into which a biocompatible pigmented silicone elastomer is placed.

The purpose of this presentation is to explore the development of direct printing of two component silicone elastomers in conjunction with a support structure to create complex shapes using a customized 3D printer. Ultimately, this would provide the maxillofacial prosthetist with a tool that manufactures prostheses reliably, with less emphasis placed on individual artistic interpretation. This technology has the potential to solve possible manufacturing solutions to complex shapes for both commercial and industry in addition to the current medical applications.
Method: The first step was to develop a two-component RTV silicone which was printable and had adequate properties suitable for fabrication of facial prostheses. The composition of silicone was varied to find the suitable combination with adequate strength. A 3D printer design had to be adapted to print the silicone.
Results: The base for two-component silicone was composed of 70% long, 20% medium, 10% short chain PDMS loaded with 30% filler showed optimal tear and tensile strength after printing. This newly developed two-component silicone also contained 5% cross-linker, 2.5% catalyst, 0.5% moderator and 3% thixotropic agent. The printer design and printing parameters were adapted to print the two-component silicone. Pigments were successfully printed through the printer and a hydrogel Pluronic F-127 was found to be suitable for use as support material for 3D printing of silicone prostheses.
Conclusions: The printing process and material have been developed to print silicone prostheses with suitable mechanical properties. The ways to incorporate colour data into printer and printing resolution need to research further.

3D Bioprinting has gained attention in tissue engineering due to its ability to spatially control the placement of cells, biomaterials and biological molecules. The development of new hydrogel bioinks with good printability and bioactive properties has made it possible to 3D bioprint and accelerate the maturation of complex 3D tissue-like models. In this talk, we present our recent work in bioink and material development for 3D bioprinting and culture of healthy tissues such as skin, bone and cartilage, as well as cancer tissues for disease modelling and studies of tumor cells interaction with their microenvironment.
In addition to the bioink development we will be talking about printing thermoplastics, 4D-printing and soft robotics.

Traumatic peripheral nerve injury occurs prevalently in the worldwide, which usually results in the disability of extremities. Grafting with a nerve guidance artificial conduit to guide neuron regrowth can enhance nerve regeneration.However, the commonly used nerve conduit cannot provide a spaciotemporal regulation and in-situ monitoring for localized neurons. Accordingly, in this study, neural implant device that is combined with regenerative tissue scaffolds and microelectrode arrays was developed to enhance cell growth through applying electrical stimulation and neural signals recording in a as-built tissues-mimicked micro environment ent. A new type of tissues scaffold, called Dopa-SFG in the composition of synthesized dopa-modified silk fibroin and gelatin was fabricated to exhibit tissue-mimic structural and mechanical peoperties, adhesive ability, and phase transition under a specific temperature. The aqueous Dopa-SFG precursors can be directly transformed to a gel bulk, in turn used to transfer print a complicated microelectrode structure that was pre-fabricated on PAA-Ca²⁺ sacrificial layer. Such transfer printed scaffold-based neural microelectrode array enabled conformable adhesion on a moisture-rich curvilinear surface and electrical signals transduction from the tiny microelectrode site, which is expected to provide more efficient peripheral nerve regeneration.
Results and discussion: After cooling transfer, Dopa-Silk fibroin gelatin(Dopa-SFG) is bonded to the underlying MEA. In the liquid state, Dopa-SFG dissolves the sacrificial layer of calcium ions above the MEA, and then transfers the MEA to the hydrogel through the adhesion of Dopa. Thus, an adhesive silk protein sleeve electrode having electrical stimulation and contact-guided growth and monitoring functions is produced.
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Regeneration of bone tissue in cases of non-union in fractures or spinal fusions present a significant medical and financial burden. Current approaches rely on scarcely available and undesirable allografts, new materials, or biologics, each presenting their own drawbacks and limiting efficacy. Electrical bone-growth stimulators have gained popularity with their promise of shorter recovery times; however, clinical meta analyses suggest mixed outcomes. Such invasive electrical approaches have relied on rigid wires inserted into the defect site. This approach is not ideal for regenerative engineering due to the remaining signaling and mechanical mismatch. To address this need, we turn to conducting polymers to develop a material that can fill the volume of a fracture defect site, allow for both passive and active stimulation of bone growth, and provide tissue-like characteristics. To this end, we demonstrate a thermoresponsive conducting hydrogel composite that shows differentiation of bone marrow stromal cells (BMSCs) into osteoblasts. The conductive hydrogel composite is based on a thermo-responsive hydrogel known as poly(polyethylene glycol citrate-co-N-isopropylacrylamide) (PPCN), and a conducting polymer alkoxysulfonate poly(3,4-ethylenedioxythiophene) (PEDOT-S). We explored PPCN/PEDOT-S composite ratios and characterized it to allow for maximal loading of the PEDOT-S, while still gelling at body temperature. Transport properties were characterized with two electrode impedance spectroscopy and 4-point probe. The composite’s osteoinductive properties were characterized through an in vitro 3D cell culture study using rat BMSCs in standard cell media and osteoinductive media. BMSC differentiation was determined based on Alkaline phosphatase (ALP) assay. The BMSCs with conducting hydrogel composite in standard cell media showed increased levels of ALP activity indicating differentiation of BMSCs into osteoblasts without the need of osteoinductive media. Given the potential for minimally invasive deployment of such thermo-responsive materials, these results show promise for this new class of conductive material for regenerative engineering. Furthermore, this study lays groundwork for external stimulation of this bulk material, which would re-imagine direct-current bone-growth stimulation.

In recent studies, polyurethane has shown multiple properties that make it an excellent candidate material in 4D printing. In this study, we present a simple and inexpensive additive method to print waterborne polyurethane paint based composites by adding carboxymethyl cellulose (CMC) and silicon oxide (SiO₂) nanoparticles to the paint. The first function of CMC and SiO₂ is to improve rheological properties of the polyurethane paint for making a printable precursor, which improves the printing resolution and enhances additive manufacturability. Second, the composite precursors improve the curing rate of the polyurethane paint without changing its inherited shape memory properties. Third, the printed composite parts shown enhanced mechanical strength compared with that of the parts printed with pure polyurethane. Finally, the 3D printed polyurethane-CMC and SiO₂ parts exhibit time-resolved shape transformation upon heat stimulation. To the best of our knowledge, this is the first study of using the polyurethane paint as the precursor for 4D printing, which would open new possibilities in future applications in biomedical engineering, soft robotics and so on.

Current blood diagnostics require ~2-10 milliliters (mL) of blood per test, taking hours, or days, for results. Drawing mLs of blood can induce hospital-acquired anemia (HAA) in premature infants, and in chronically, or critically ill patients. Reducing the volume drawn while increasing analysis speed and accuracy can improve healthcare, and decrease suffering and costs from infections and transfusions. Rapid results from µL blood samples can revolutionize quality and cost of care.
Theranos claimed it could analyze nanoliter (nL) blood droplets. FDA raids triggered by inaccuracies in tests, revealed Theranos diluted nL into mL-sized vials while using standard analysis methods with errors greater than the medically acceptable threshold of 10%.
However, HemaDropTM, [1-4] a “hyper-hydrophilic” surface coating, can solidify micro-liter (µL)-sized blood droplets within minutes microliter-sized droplets into Homogeneous Thin Solid Films (HTSFs), without phase separation or segregation. HTSFs can be analyzed within minutes using handheld or desktop analyzers, in air or in vacuo.
Blood HTSFs were tested for accuracy in measuring electrolytes (Na, K, Mg, Ca, Cl) and hematocrit (Fe) from single µL droplets. So-called “Lytes & Crit” (electrolytes & hematocrit) are often the first tests conducted in ER, ICU, NICU, OR, and hospitals, as general status depends critically on hydration (electrolytes concentration), and oxygenation (Fe hematocrit levels). These are repeated daily and sometimes every few hours.
Repeated measurements show that electrolytes and Fe in human blood are accurate within 10% using IBA, XRF, and XPS, from a few µL of blood solidified as HTSF when calibrated using commercial Balanced Saline Solution (BSS+) pre-solidified on strips coated with HemaDrop™, called DropFilmStrip™ [1-3]
XRF is of particular interest because it is a desktop/handheld tool which yields within minutes compositional analysis within the medically acceptable 10% for Na, K, Mg, Ca, Cl, Fe .
Prior to IBA, XPS, and XRF analysis, congealed, uniform HTSFs are compared to conventionally Dried Blood Spots (DBS). Solidification of droplets into HTSF is observed and timed via optical microscopy and video. In DBS, phase separation between platelets and serum yields rough, non-uniform thin solid films. But HTSFs solidified on HemaDropTM are smooth, and uniform without phase separation. Similar behavior can be seen with solidified Balanced Saline Solution (BSS) droplets. BSS congealed on HemaDropTM exhibits little crystallization and no phase separation[4] while simply dried BSS does.
HTSFs of human blood, canine blood and BSS solidified via HemaDropTM coated surfaces all underwent XRF in air, XPS in 10-10 Torr vacuo, and IBA in 10-7 Torr vacuo, performed on the same set of 5 and 10 µL-sized droplets congealed into HTSFs and compared to DBS.
HTSFs yield identical, reproducible 2 MeV RBS, XPS, and XRF spectra on different areas of HTSFs. C, N, O, Na, K, Ca, Mg, Cl, Fe are detected with leading edges falling within IBA resolution (< 15 keV, 2-3 channels), while DBS yield leading edges spread out over 100 keV.
The damage curve method extrapolates elemental composition while accounting for possible ion damage. Four consecutive spectra are taken on each analyzed area. RBS, XPS and XRF yields are interpolated to 0-analyzing dose to extract original concentrations.
IBA simulations with the software SIMNRA are fitted to the RBS spectra and matched to within 1%. Atomic compositions from sequential IBA, XPS and XRF spectra taken on different areas of HTSFs and on different samples are all within 5%.[5] Relative error analysis between multiple HTSFs establishes reproducibility within 10%. Comparative analysis from these results will be discussed to show how it guides optimization of the technology.
[1-3] Intern. /US Patents Pend. , Herbots N., et al (2016-2018)
[4-6] MRS Advances 2016, 2017, Bio-Interphases (2019)

Ti-50.3Ni-0.7Ag and Ti-18Zr-12.5Nb-2Sn (at.%) alloys scaffolds were fabricated by sintering the alloys fibers prepared by rapid solidification method. Secod phases such as Ti₂Ni and TiAg phases which have been observed in the bulk alloy prepared by conventional casting method were not observed in as-solidified Ti-Ni-Ag fibers. Ag content in the fibers was measured to be 0.57% much larger than that in the bulk alloy, which is beneficial to improve the antibacterial fuction. The two stage B2-R-B19' transformation occurred and good superelasticity with superelastic recovery ratio of 93% was obserevd in Ti-Ni-Ag fibers. As-spun Ti-Zr-Nb-Sn alloy fibers were composed of only the β phase. Tensile tests showed that Ti-Zr-Nb-Sn as-spun alloy fibers exhibited superelastic behavior over temperature ranges between 238 K and 318 K. A large recovery strain of 4.5% for 5% pre-strain was observed at room temperature.
In order to fabricate scaffolds, the fiber segments were put into the packing chamber of graphite mold and sintered at 1173 K for Ti-Ni-Ag alloy fibers and at 1573 K for Ti-Zr-Nb-Sn alloy fibers for 60 min in high vacuum condition. The alloy scaffolds prepared by sintering the fiber segments possessed three-dimensional and interconnected pores that were structured by the bonded small fiber segments with random directions. The porosity of the scaffolds were about 80%. The compressive strength of Ti-Ni-Ag alloy scaffold was about 5.6 MPa at the strain of 4% and that of Ti-Zr-Nb-Sn alloy scaffold was about 6 MPa at the strain of 7%, similar to that of cancellous bone ranging from 2 to 20 MPa. The elastic modulus of Ti-Ni-Ag and Ti-Zr-Nb-Sn alloy scaffolds was 0.67 GPa and 0.42 GPa, respectively, which were also similar to that of cancellous bone ranging from 0.1 to 2 GPa. Superelastic strain recovery of Ti-Ni-Ag and Ti-Zr-Nb-Sn alloy scaffolds was found to be 3% and 2.5%, respectively.

Microcapsules are discussed in drug delivery, biomaterial and self-healing applications for their numerous advantages[1]–[3]. Plenty of stimuli are used to trigger the release of core contents[4]. However, any loss of core contents prior to their stimuli will lead to weakened function of encapsulated systems. In this research, materials of magnetic-sensitive microcapsules were successfully synthesized using Fe₃O₄/polycaprolactone (PCL) to construct the shell. In our previous work, pyrene based active Schiff base probe could be formed to complexes PE-M³⁺ (M = Fe/Cr/Al) to detect Fe³⁺, Cr³⁺ and Al³⁺ ions by its nano-aggregation induced emission enhancement (AIEE)[5]. As we known that Fe³⁺, Cr³⁺ and Al³⁺ ions are essential in the human body, Cr³⁺ plays an important role in the maintenance of an effective carbohydrates, lipid and protein metabolism[6]-[7]. In addition, Cr³⁺ ion deficiency leads to metabolic disorder inducing diabetes and cardiovascular disease risk[8]. Nevertheless, this probe is not able to perform its functions although it moves through digestive tracks. In order to protect the core contents and control release of encapsulated cargo that utilize microcapsule-based drug release system and high-frequency magnetic field (HFMF) to accelerate the rate of contents release. A pyrene based Schiff base derivative 2-((pyrene-1-ylmethylene)amino)ethanol (PE) was encapsulated for sensing Fe³⁺, Cr³⁺ and Al³⁺ ions to use in the fluorescence turn-on responses. Furthermore, the solvent evaporation method was used to fabricate the microcapsules using W/O/W emulsion. Experimental observations showed that the magnetic nanoparticles in the shell structure indicated that the shell structure was subjected to a sharp structural change when the magnetic NPs caused rapid heating when exposure to a HFMF. After the magnetic fields imposed, PCL shell was degraded in the surrounding, in the same time, schiff base probe PE was released into environmental solution also. In particular, the activity of this “OFF-ON” fluorescent sensor was not being obviously affected by encapsulated phenomenon. As shown in experimental observation, upon the increased addition of water fraction (fw) from 0% to 80% towards PE microcapsules in CH₃CN, the UV/PL peaks at 425/413 nm were red shifted to 463/470 nm, respectively.
This encapsulated fluorescent sensor exhibited a longer shelf life and better stability compared to the original sensor. The capsules’ shell improved the thermal stability of encapsuled PE, the thermal decomposition of these microcaspules around 350 deg. Likewise, the naked eye and fluorescence turn-on sensing responses were still performed well based on UV-vis absorption titrations and PL spectral investigations. PE sensor was well established by ¹H NMR titrations and its functionality was recognized during subsequent addition of M³⁺ ions, respectively. PCL was adopted as the shell material because of its good biocompatibility, biodegradability, and it can also be used as a cell growth support material. Moreover, the use of a HFMF stimulus can achieve an “instantaneous” burst release of drug by rapid temperature raise to induce a quick melting of the PE microcapsules. Therefore, the combination of magnetic and thermal properties would be a very attractive for detection of metal ions in organisms.
Keywords: Microcapsules; Magnetic-sensitive; Fe₃O₄ nanoparticles; PCL; Fluorescent sensor; Trivalent cations; High-frequency magnetic field (HFMF)

Bioprinted scaffolds with physico-mechanical properties mimicking the native extracellular matrix (ECM) environment holds great potential for successful regenerative medical treatments and/or 3D models of cellular behaviors. Recent advances highlight allylated gelatin (GelAGE) as a versatile hydrogel platform for both lithography-based and extrusion-based biofabrication with highly tailorable properties. This material is further known to be susceptible to photo-crosslinking with both UV- and Visible-light based photoinitiator systems. It is, however, unknown how these different photo-initiator systems affect long-term mechanical and rheological properties of GelAGE for the fabrication of tissue constructs with clinical relevant sized since UV light is known to be harmful for cells.
This study therefore aimed to study the polymerisation of GelAGE with UV (Irgacure 2959) and visible light (LAP, Ru/SPS) photoinitiators and concentrations suitable for lithography-based and extrusion-based printing applications based on resulting physico-mechanical and rheological properties.
Gelatin was reacted with 12 mmol of allyl glycidyl ether (AGE) and 2 mmol NaOH per gram of gelatin at 65 C for 1h followed by dialysis, lyophilisation and NMR characterisation. Hydrogel constructs were photo-polymerised (30mW/cm², 3min, 20wt.-% GelAGE) with linear dithiothreitol (DTT) at 1:1.5- 1:6 AGE:SH molar ratios and different photoinitiators using either visible-light (450nm, 0.05wt% Lithium phenyl-2,4,6-trimethylbenzoylphosphinate; LAP or 0.2-1mM ruthenium; Ru and 2-10mM sodium persulfate) or UV-light (365nm, 0.05wt% Irgacure 2959;Ig). Depth of cure, shape retention, viscosity, compressive modulus, mass loss, and mass swelling ratio were characterized. Mechanical properties was further studied as a function of time for hydrogels crosslinked with Ru/SPS and LAP.
GelAGE was successfully synthesized (DoM:42) and fabricated into 10mm large constructs. Compared with Ru/SPS and Irgacure 2959, hydrogels with LAP demonstrated a higher depth of cure, shape-fidelity and homogenous polymerisation. Depth of cure was independent of DTT concentration (10.58-10.84mm), unlike hydrogels with Ru/SPS or Irgacure 2959. Better shape retention was also observed for LAP samples post swelling, except the one with 1:1.5 AGE:SH molar ratio (partially dissolved, height drop to 6.32mm). GelAGE with UV photoinitiator (Irgacure 2959) resulted in lowest depth of cure and worst shape fidelity. The viscosity was observed to increase both with time of shearing and depended on the DTT concentration (0min = 10^-2 Pa s, 15min = 3x10^-2 Pa s for 1:1.5 AGE:SH molar ratio, 0min = 10^-2 Pa s, 15min =1.7x10^-1 Pa s for 1:6 AGE:SH molar ratio) which could be used to extend the fabrication window for extrusion-based bioprinting. The mechanical strength of Ru/SPS polymerised GelAGE constructs was further shown to be time-dependent (0min = 82 kPa, 10min = 34 kPa, 30min = no hydrogel formation) and tailorable with different ratio of Ru:SPS and weight percent of GelAGE. Further replenish of SPS resulted in stiffness close to original value at t=0. Mass loss and swelling ration were observed to be time-dependent as well. The time-dependent relationship of mechanical properties observed could be a powerful tool to achieve seamless mechanical gradients in biofabricated constructs by either pausing processing for a short term or replenish SPS at certain time point.
In conclusion, GelAGE with LAP initiated polymerization offers a thiol-ene clickable bioink formulation that can be applied to fabricate clinical relevant sized tissue constructs with high fidelity and shape retention. In addition, the Ru/SPS system can provide a wide range of stiffness and gradient stiffness formation while the viscosity can be adjusted by varying DTT concentration to fit extrusion-based printing. All these results support the use of visible light photoinitiators but rather than UV photoinitiator.

Recent advances in materials science open up new opportunities for integrating high performance electronics with the human body and other biological systems. This talk highlights progress in (1) large-area, wireless ‘epidermal electronic’ platforms for use in human-machine interfaces to robotic prosthetics and in sensors across the surfaces of residual limbs and prosthetic sockets and (2) multifunctional three dimensional mesosystems as active, electronic cellular scaffolds for study of proliferation, differentiation and communication across collections of muscle cells and neurons.

Access and adoption of advanced prosthetics for children has been limited due to a variety of factors including: high costs, limited insurance coverage options, challenges in adaptation and training leading to high rejection rates, and limitations in technology or performance. These factors have driven prescribers to limit prescriptions of advanced prosthetics for children with congenital amputations, which occurs in approximately 5 out of 10,000 live births. Significant progress is needed to advance the field not only in the technology be developed, but also in the overarching training and feedback provided to the patients. In this study we discuss new techniques that have been integrated to advance the design in wearable neuroprosthetics, and a gamified, visual approach to training in the pediatric environment.

This work has demonstrated the effectiveness of using electromyographic (EMG) actuation in gamified simulations to train the actuation of multiple hand position gestures in a virtual environment. This type of training leverages real time visual feedback to user inputs, in this case muscle flexure with varied magnitude and time dependent patterns. This provides a stronger link between the intentionality capture (input) and the assigned thresholds and patterns required to activate the multiple states (outputs). This is implemented as a game that simulates the control of the prosthetic and translates the EMG input into functional game mechanics, providing meaningful feedback in form of gameplay. Initial studies have focused on testing the signal actuation repeatability to target specific multi-point magnitude set thresholds, and have shown promising results.

The role of additive manufacturing in medical devices has been developing both in practice and in the regulatory framework. The FDA’s new non-binding best practices have been key towards standardizing process flow and ensuring repeatable. The device developed in this study has been produced using 3D printed acrylonitrile butadiene styrene (ABS) plastic, via fused deposition modeling (FDM), including a dissolvable acrylic copolymer to allow for complex internal channels. Interlocking parts are created to interface with mechanical components including: fasteners, springs, and cabling. The advantages of this type of manufacturing allow for rapid prototyping, custom sizing for short run manufacturing, and the ability to grow modular parts that can be used to resize the antebrachial and brachial segments. These parts have demonstrated exceptional strength to weight ratios and durability. Print orientation, orienting the fiber deposition direction in context with the part and applied mechanical loads, is critical to achieving durability.

To achieve quantifiable outcomes, including arm system performance and the influence on patient’s quality of life, a clinical trial protocol for assessment has been developed in conjunction with Oregon Health & Science University (OHSU) and the University of Central Florida (UCF). This protocol includes assessment data points four times across the course of the one year study. Occupational therapists from OHSU will support the evaluation and provide valuable insight as to when multi-gesture hand states are appropriate to “unlock” for the patient. This novel development of technology and clinical translation is critical to advancing the accessibility of advances prosthetics for pediatrics. The use of additive manufacturing has enabled significantly lower costs, rapid prototyping, and advanced customization critical to improving clinical outcomes and experience.

Implantable devices and scaffolds with new functions interfacing with neural systems enables new techniques to fire and reconnect neurons. Specifically, we will report our recent progresses on a fiber based photoacoustic convertors for succesful in vitro and in vivo neuro stimulation and a biocompatible silk-based nanoladder scaffolds for facilitating neuron functional reconnection after injuries. In the first application, we design and develop a miniaturized fiber with diameters ranging between 500 micons to 1 milimeter composed of two layers: a diffusion layer and a expansion layer with nanocomposites to produce high intensity and controlled frequency of localized ultrasound when excited with nanosecond laser light. In vitro and in vivo neuron stimulation was successfully achieved with a laser duration of 20 milisecond. Neural stimulation in motor cortex of intact awaken mouse brains confirmed the stimulation was localized with a high spatial resolution of 500 microns and not through direct auditory stimuation. In the second application, we design and demonstrate a silk based scaffold composed of micron size fibers and nanoscale protrusions to direct axon growth along a predefined direction and facilitate neuron functional reconnection in vitra and in vivo.

Perineural invasion (PNI) is the neoplastic invasion of nerves. However, the mechanisms underlying its pathogenesis remain largely unknown. In this study, we created a hydrogel biochip to mimic a nerve tissue microenvironment for investigating the interaction between schwann cells and cancer cells. A new type of conductive tissue scaffold was developed via conjugating gelatin with poly(3,4-ethylenedioxythiophene (PEDOT), within which a naturally occurring polymer, melanin was incorporated to improve electrical properties. The resultant composite exhibited porous 3D structure with elastic conductivity and high chemical stability. The existence of Melanin and PEDOT was analyzed by FTIR confirming the bonding. Through SEM image we observed 300 nm spherical Melanin nanoparticles and PEDOT polymer both embedded in the hydrogel pore walls. The analysis of physicochemical characteristics indicates the electrical and mechanical properties increases as melanin and PEDOT incorporate with hydrogel. Furthermore, the in vitro biodegradation of compound showed a decreasing current as the conductive materials increases. At last we use the compound to create a hydrogel-based Electrode texture by transferprinting technique. As a result, a degradable nerve-mimicking biochip was formed to investigate Perineural invasion.

Laser-based additive manufacturing techniques such as two photon polymerization, matrix assisted pulsed laser evaporation-direct write, and laser induced forward transfer have been used over the past two decades to create surfaces with small-scale features for medical applications. For example, the two photon polymerization technique involves use of femtosecond laser (e.g., a titanium:sapphire laser) pulses for selective polymerization within highly localized regions of a photosensitive resin. Polymerization and hardening occur at sites within the photosensitive resin where the excitation threshold of the photoinitiator is exceeded. Polymerization of microscale and sub-microscale features is possible since the two photon absorption exhibits a nonlinear relationship with the incident light intensity. Two photon polymerization has been used to prepare structures with sub-microscale features out of photosensitive acrylate-based polymers, zirconium oxide hybrid materials, and organically-modified ceramic materials. Several types of medically-relevant structures, such as scaffolds for tissue engineering and medical devices, have been prepared using two photon polymerization. For example, we have used two photon polymerization to prepare small-scale lancet-shaped devices known as microneedles for transdermal delivery of pharmacologic agents or transdermal sampling of body fluids. Efforts to optimize the two photon polymerization processing procedures and postprocessing procedures for medical applications will be considered. In addition, selection of an appropriate photoinitiator for use in medical applications will be considered. The biological and functional evaluation of two photon polymerization-created medial devices and scaffolds for tissue engineering will be described. Necessary steps in the development of two photon polymerization as a commercially viable manufacturing method will be described.

Orthopedic surgeons need devices that can replace the articular surface of cartilage, then regenerate the cartilage to replace the device, all while recruiting cells from the underlying bone marrow. Such devices do not yet available, but they could be, using our new hybrid biomaterials. Hybrids have nanoscale co-networks of inorganic glass and organic components, e.g. sol-gel silica and biodegradable polymers. We now have hybrids that can “bounce” and self-heal. The hybrids are ideal for 3-D printing inks, which can yield bespoke scaffold architectures. Osteochondral devices can now be 3D printed that stimulate articular cartilage production and also provide the bearing surface with tribology similar to native cartilage.

Bone implants are frequently used to treat osteoarthritis and reconstruct large bony defects. The aging population as well as increasing numbers of obese patients will enhance the future need for orthopedic implants. Although the use of these implants in total joint replacements has become one of the most successful procedures in medical practice, two major challenges remain: implant-associated infection and implant longevity. As a result, the bar for proper functioning implants has been raised: implants should ideally prevent infection by bacteria that are increasingly resistant to antibiotics as well as induce and support sustained tissue integration between implant and surrounding bone tissue to secure implant longevity. Both requirements can be fulfilled by bone implants with multifunctional surfaces that we have synthesized through a combination of rational design, selective laser melting (SLM) and plasma electrolytic oxidation (PEO) in the presence of metallic nanoparticles (NPs).
Additive manufacturing has boosted new research areas in which the functionalities of biomaterials do not depend on the material properties but are direct consequences of the topology and can therefore be rationally designed. This has enabled the synthesis of highly porous biomaterials that closely mimic the mechanical properties of native bone. Furthermore, geometrical features such as porosity, pore size and curvature can be rationally designed to promote tissue integration. However, porous biomaterials are at risk for infection as they may have surface areas up to several orders of magnitude larger than solid biomaterials. Therefore, it is essential to biofunctionalize the surface such that not only bone regeneration is improved yet simultaneously infection is prevented.
Here, we present the synthesis and characterization of multifunctional titanium bone implants. We designed long porous implants that were synthesized from medical grade Ti-6Al-4V powder by SLM resulting in porous implants with a 4 times enlarged surface area compared to solid implants. Subsequently, the implant surface was biofunctionalized by PEO using an electrolyte consisting of Ca/P species and Ag, Cu and/or Zn NPs. PEO is very suitable for highly porous structures as it generates a homogeneous oxide layer in which the NPs become entrapped, thereby preventing nanotoxicity. Following biofunctionalization, biomaterial surface morphology was analyzed by scanning electron microscopy, chemical composition by energy dispersive X-ray spectroscopy, phase composition by X-ray diffraction and ion release kinetics by inductively coupled plasma optical emission spectroscopy. Antibacterial testing was performed against methicillin-resistant Staphylococcus aureus (MRSA), a pathogen commonly involved in implant-associated infection. We determined the antibacterial leaching activity in a Kirby-Bauer assay and quantified bactericidal activity in vitro as well as in an ex vivo murine infection model. Subsequently, through live/dead staining, metabolic assays and measurement of alkaline phosphatase activity the cytotoxicity and osteogenic differentiation of human mesenchymal stem cells (hMSC) were analyzed.
PEO biofunctionalization of the porous titanium implants resulted in a micro/nano-porous TiO₂ surface layer that contained hydroxyapatite. The immobilized NPs in the surface generated the release of Ag, Cu and/or Zn ions for at least 28 days resulting in strong antibacterial leaching activity and prevention of bacterial adhesion in vitro and ex vivo, with AgNPs demonstrating the largest antibacterial activity. The biofunctionalized surfaces did not induce cytotoxicity and improved the metabolic activity of hMSC. Altogether, the biofunctionalized porous titanium implants presented here are suitable candidates for further preclinical development to prevent implant-associated infection and improve implant longevity.

Many indwelling medical devices that are critical to patient care (e.g., catheters, vascular grafts, extracorporeal circuits) suffer from major clinical problems related to clotting, inflammation, and infection. One approach to improving the biocompatibility of medical devices is to develop materials that release nitric oxide (NO), a known potent inhibitor of platelet adhesion/activation and also an endogenous antimicrobial agent. Healthy endothelial cells exhibit a NO flux of 0.5-4 ×10^-10 mol cm^-2 min^-1, and materials that mimic this range of NO release rates are expected to have similar anti-thrombotic and antimicrobial properties. Incorporation of NO donor molecules such as S-nitrosothiols (RSNOs) into polymers, either non-covalently dispersed or covalently bound, are studied that mimic the endogenous NO release levels. The therapeutic NO release from these RSNO-based materials can be initiated via heat, metal ions, or light. Novel solvent impregnation methods have been studied that incorporate NO donor molecules, such as S-nitroso-N-acetylpenicillamine (SNAP), into biomedical grade polymers or polymeric devices. The NO releasing polymers are characterized in vitro for their NO release kinetics, NO donor leaching, physical/material properties, and antimicrobial properties. Nitric oxide release kinetics are measured under physiological conditions using a chemiluminescence NO analyzer. Antimicrobial effects of the NO vs. control polymers are evaluated using in vitro using common pathogens related to medical device infections (P. aeruginosa, S. aureus) and quantitated by plate counting. The potential clinical applications of these NO releasing polymers to improve the biocompatibility of devices such as catheters, insulin cannulas, and extracorporeal life support are evaluated via short-term and long-term preclinical animal models to evaluate the efficacy on prevention of thrombosis and infection. The NO releasing polymers exhibit promising biocompatibility properties as compared to controls by significantly reducing platelet adhesion/activation, and reducing viable bacteria on the device surfaces. The NO-releasing polymers developed have the potential to be applied to and improve the biocompatibility of a wide range of medical device applications.

One of the most important scientific and technological frontiers of our time is the interfacing of electronics with the human nervous system. This endeavour promises to deliver new tools for diagnosis and treatment of disease. Current solutions, however, are limited by the materials that are brought in contact with the tissue and transduce signals across the biotic/abiotic interface. Recent advances in organic electronics have made available materials with a unique combination of attractive properties, including mechanical flexibility, mixed ionic/electronic conduction, enhanced biocompatibility, and capability for drug delivery. I will present examples of novel devices for recording and stimulation of neurons and show that organic electronic materials offer tremendous opportunities to study the nervous system and treat its pathologies.

Cellular adhesion and orientation plays a large role in many cellular processes such as migration, angiogenesis, wound healing and proliferation. Such functionality in a biomaterial at the interface due solely to the physical characteristics opens the possibility of modulating the biological response to implanted biomaterials in a dynamic and endogenous manner. In this study, a unique corrugated surface architecture with two distinct microstructures of regularly spaced curved microchannels and flat plateau-like areas was created by cutting capillary alginate hydrogel (Capgel^TM) along the capillary long-axis with a compression-assisted vibratome (Compresstome, Precisionary LLC, Boston, MA, USA). Capgel^TM is an alginate hydrogel containing patent, parallel capillaries created during the ionic crosslinking of a precursor alginate polymer solution into a 3D polymer network. The balancing of fluid dynamic forces, molecular dynamics and electrostatic interactions during the gel synthesis yields an ordered array of patent capillaries running in parallel as the diffusion front of cations (in this case Cu²⁺) progresses through an alginate polymer solution. Cultured human foreskin fibroblasts atop of the films of Capgel^TM (~200 micrometer thickness) showed a dual orientation of fibroblasts on this tissue scaffold material due to the physical features inherent to the hydrogel. Fluorescent staining with Hoecht 33342 (Thermofisher Scientific, Waltham, MA, USA) and ActinGreen^TM (Thermofisher Scientific, Waltham, MA, USA) further support this assertion along with the quantification of nuclear and cellular alignment achieved by ImageJ analysis that showed distinct populations of oriented cells. These results suggest that the microcapillaries induced one particular cellular orientation as the fibroblasts followed and oriented along the capillary long-axis while the flat plateau-like regions created by the Compresstome cutting between the capillary channels yielded an additional distinct direction of cellular alignment orthogonal to the capillary channel-induced alignment. Future studies exploring the surface preparation parameters (i.e. oscillation frequency during Compresstome cutting, chemical composition of the hydrogel) may further modulate the potential cellular responses to this unique biomaterial. Additionally, the highly preferential orientation observed for the fibroblasts in two distinct directions as well as the highly polarized nucleus can have implications on cellular proliferation, motility and extracellular matrix deposition. For instance, deposition and orientation of extracellular matrix components such as collagen fibrils has been associated with fibroblast morphology. Capgel^TM biomaterials are therefore a unique scaffold material for engineering anisotropic tissue microstructures and organs.

With the aging population growing and the prevalence of osteoarthritis rising, the need to develop life-lasting implants is bigger than ever. Total joint replacements are often referred to as one of the most successful surgical interventions, but in the light of current developments, young and active patients will most certainly outlive their implants. Aseptic loosening, the mechanical failure of the bone-implant interface, is one of the main reasons. It is often caused by the particulate wear debris coming from the articulating surfaces of the prosthesis, which initiates the foreign body response of the patient’s immune system leading to inflammatory bone loss. Bone loss may also be the result of stress shielding, caused by the mechanical mismatch of the bone-implant interface. With the introduction of a metallic implant, the physiological loading condition of the bone changes, since most of the load is carried by the implant. According to Wolff’s Law, this will cause bone to adapt itself by reducing its volume in places it is no longer needed. Biomaterial optimization is therefore inevitable, when working towards the next generation of life-lasting implants.

The emerging concept of metamaterials offers a promising route to the development of such implant biomaterials with unique combinations of mechanical (e.g. Negative Poisson’s Ratio (NPR)), mass-and fluid-transport (e.g. diffusivity and permeability) and biological properties (e.g. tissue regeneration performance). The topology of these so-called meta-biomaterials, may be rationally designed to exhibit unprecedented properties for tissue regeneration and sustained mechanical support. The tremendous developments in metal additive manufacturing have allowed us to fabricate these highly complex micro-architectures, including triply periodic minimal surfaces and hybrid combinations of auxetic and conventional meta-biomaterials.

Unlike conventional meta-biomaterials, auxetic meta-biomaterials have a negative Poisson’s ratio and expand laterally in response to axial stretch. A recent study has proven their importance within the field of orthopaedics, in which a rational distribution of negative (auxetic) and positive Poisson’s ratios was used to improve implant-bone contact and potentially implant longevity in the femoral component (i.e.hip stem) of a Total Hip Replacement (THR). Since the hip stem is repeatedly loaded under bending, the lateral side of a conventional implant will be retracting from the bone under tensile loading. The bone-implant interface is not only more susceptible to failure when subjected to tension, but the implant’s retraction also reduces bone-implant contact and allows wear particles to enter the bone-implant interface space. Laterally applying an auxetic meta-biomaterial therefore resulted in compression on both of the implant’s contact lines with the surrounding bone, decreasing the chance of bone-implant interface failure and stimulating osseointegration.

In this work, we characterize the mechanical properties of additively manufactured, titanium auxetic lattices, based on the re-entrant hexagonal honeycomb. The mechanical properties of this unit cell can be tuned through slight alterations in its geometry, which has been done to obtain a variety of Poisson’s ratios. The limits of the Selective Laser Melting (SLM) process were explored to synthesize structures with optimal bone-ingrowth properties. Their architecture was therefore evaluated using micro-CT, while its quasi-static and fatigue properties were assessed in a compression test. The Poisson’s ratio was experimentally determined using Digital Image Correlation (DIC), based on the movement of a randomly applied speckle pattern. With this comprehensive library of mechanical and morphological properties, we hope to contribute to the adoption of auxetic lattices within the field of orthopaedics, as an ideal substitute for bone in life-lasting implants.

Orthopedic implants are used routinely worldwide for many applications and due to our ageing population, the practice of inserting implants into younger patients, a rising prevalence of risk factors such as obesity and the necessity to maintain active lifestyles means that the number who receive an implant is rising significantly. Orthopedic implant survivorship increases following osteointegration where bone grows adjacent to and in direct contact with the implant surface. However, conditions for spontaneous bone healing and implant integration are not always ideal and pathological conditions, infection, tissue degeneration and bone loss as a result of age, disease or implant failure cause a cell regulatory imbalance in the activities of osteoblasts and osteoclasts as well as proliferation and differentiation of their stem cell progenitors, thus limiting bone regeneration and repair. Fixation of massive bone tumor endoprosthetic replacements is not as successful as conventional joint replacement surgery and aseptic loosening is the major cause of implant failure. This presentation will focus on translational research and will describe how increased bone growth and direct contact to the implant shaft is vital in reducing failure. The use of surface modification techniques, bioactive coatings and 3D printed technology is shown to significantly improve integration and implant survival. Additionally, stem cell therapy is a method used to augment the regeneration of damaged tissues and over recent years, we have investigated bone marrow derived autologous and allogeneic stem cells as a method to enhance osteointegration and bone repair. A novel technique where stem cells are sprayed onto the implant surface will be presented as well as techniques that use cells to enhance the repair and reconstruction of bone tissue. Infection is a growing global concern and a devastating implant complication that is rising in incidence and severity and this presentation will also focus on methods targeted at preventing and treating implant infection.

With the explosion of the field of nanobiotechnology, questions have arisen whether the use of new nanoscale materials (including nanotubes, nanowires, nanowhiskers, fullerenes or buckyballs, and quantum dots) might have unintended human health hazards and environmental consequences. The quantum size effect, altered physicochemical characteristics due to surface-induced effects, resulted from the nanoparticles “minute size” and may trigger injurous responses in biological tissue. Toxicity characterization of nanomaterials is more complex than that of conventional chemicals (such as drug molecules) because it involves, in addition to elemental composition, aspects of size, shape and surface properties. To date, there is a lack of consensus in the published literature on nanoparticle toxicity due to the variability of methods, materials, and cell lines used

We will report a throughput electrical biosensing system to rapid assess the human health and environmental toxicity of nanoscale materials to find the kinetics of nanotoxic at cellular level. In this project, we developed a unique biosensing system for analyzing nanotoxicity assays of gold, silver, cadmium oxide and carbon nanotubes nanomaterials. An array formatted electrical impedance sensing system was utilized to kinetically analyze the cytotoxicity of different sizes as well as different concentrations of the materials by measuring the resistance of each electrode as the cells attach simultaneously and in real-time, a critical feature in the monitoring of cytotoxicity. To further enhance our results, each electrode was deposited with a gold salt in order to improve the reproducibility of each electrode in monitoring the cell attachment. An electrical impedance measuring biosensor to perform cytotoxicity assays on human lung fibroblasts and rainbow trout gill epithelial cells is made. Several metallic nanoparticles, carbon nanotubes and grapheme nanosheets are tested. This method provides shorter run times, easier performance and more precise results. By measuring the impedance change when cells attach to the electrodes on the biosensing chip, real time kinetic effects of toxicity of the various materials are recorded. The biological and physical characterization of nanotoxic kinetics of engineered nanomaterials using the biosensing chip will also provide an alternative approach for setting standards for assessing toxicities of other nanomaterials. This system was able to provide a more rapid, efficient and straightforward method of measurement compared to the current method of incorporating the nanoparticles into a cell culture directly.

Surface plasmon resonance (SPR) is a quantum electromagnetic phenomenon arising from the interaction of light with free electrons at a metal-dielectric interface. At a specific angle or wavelength of light, the photons energy is transferred to the excitation of the free electrons oscillation on the surface. The change in the refractive index (RI) is influenced by the analyte concentration in the medium in contact with the metal surface. SPR has been widely used for the detection of gaseous, liquid, or solid samples. In this study, the use of a novel SPR module (PhotonicSys SPR H5) was tested for the detection of the stroke biomarker N-terminal pro-brain natriuretic peptide (NT-proBNP). NT-proBNP is secreted from the heart ventricles in response to excessive stretching of cardiomyocytes. NT-proBNP is proven to be a good biomarker for stroke diagnosis, with sensitivity >90% and specificity >80%. However, there is still a need for new diagnostics techniques, mainly addressing assay specificity and set up. The PhotonicSys SPR H5 system, is a miniature fast and portable system, which can be easily integrated with other instruments and used in the field; therefore, it is attractive as a point-of-care sensor. The novelty in the technology is in the SPR substrates and the reading methodology. The system provides self-referenced measurement to compensate for drifts and provide a stable measurement. NT-proBNP SPR detection was tested with two different types of SPR substrates having different top interacting surfaces (Au and SiO₂), in water, buffer and plasma samples. In addition, specific detection was done by bonded anti-NT-proBNP antibody onto the SPR substrate. NT-proBNP was detected in a range of clinically relevant concentrations for stroke, from 0.1ng/ml to 80ng/ml. The sensor demonstrated a clinically relevant limit-of-detection (LOD) of 0.1ng/ml. In the case of silicon oxide substrate, the RI showed an increasing pattern with increasing concentrations. However, in the case of the metal substrate (Au-Ag), the RI showed a decreasing pattern with increasing concentrations. More detailed explanation for the difference in the two patterns will be given by examining further the interaction of NT-proBNP with each surface.

We will report how porous, elastic, and biocompatible membranes can be made by combining sheets from soft dendritic colloids (SDCs) with a magnetically-responsive, 3D printed silicone scaffold. The resulting multilayer structures could be used as tissue matrix surrogate scaffolds in magnetically-actuated cyclic strain bioreactors. SDCs are a new class of polymeric material characterized by a branched corona of nanofibers spread out in all directions. They are produced in a scalable polymer precipitation process under intensive shear. Their fractal, hierarchical structure enables remarkable adhesion and networking properties, mimicking the contact splitting effect seen in gecko leg adhesion. We show that SDC nonwovens composed of a thermoplastic polyurethane can be fabricated into porous, nonwoven membranes with morphological features similar to that of the branched, fibrous architectures comprising physiological tissues such as lung tissue. The SDC membranes are ultrasoft, with controllable moduli of physiological softness that can mimic both soft, healthy tissue and stiffened, diseased tissues characterized by stiffened fibers resulting from pathologies such as cystic fibrosis. These SDC membranes, combined with a new method of 3D printing of magnetically-responsive silicone mesh scaffolds, allow modulated, cyclic actuation of the material to predetermined strain values. The inclusion of these ultrasoft, porous membranes in a magnetically-actuated bioreactor setup will allow their facile, untethered, long-term actuation during cell growth to determine the effects of the scaffold’s mechanical properties on cell proliferation and viability for in vitro toxicological and human performance evaluations.

Paraquat is a commonly used pesticide in many parts of Asia, Pacific nations, and the Americas in the past few decades. However, numerous clinical cases of paraquat poisoning have been reported, mostly originated from suicide and accidental exposure. To perform suitable treatment for paraquat poisoning patients, a rapid, accurate diagnosis method is necessary. In general, Urine sodium dithionite colorimetric assay is used for detecting paraquat in urine within 1-2 hours, but it cannot accurately reflect paraquat poisoning prognosis in patients. On the other hand, HPLC analysis provides sensitive determination of paraquat in blood although it is time-consuming (> 24 hours) and costly. Over the past few years, surface-enhanced Raman spectroscopy (SERS) has been widely applied in health and environment monitoring, especially for detecting extremely small amounts of molecules. The important advance is attributed to the delicate fabrication of SERS substrates providing strong Raman signal enhancement. Recently, some studies demonstrated that three-dimensional substrates perform better than two-dimensional planer substrates. The main reason is that three-dimensional substrates extend the hotspots distribution into the third dimension along the z-axis, accordingly enhancing the Raman signals within the depth of focus (DOF) of the optical system. Among three dimensional substrates, paper composed of cellulose nanofibers and microfibers is very attractive due to its low-lost and flexibility. Therefore, this inspired us to develop a cost-effective paper-based SERS substrate as a rapid detection platform for the diagnosis of paraquat poisoning patients.

Herein, we fabricated the paper-based SERS substrates via direct sputtering deposition of gold on a filter paper. By finely controlling the deposition rate, deposition time and vacuum pressure during the process, we successfully created non-continuous gold nanoislands (AuNPs) on the paper surface. The gaps between the AuNPs would generate extremely high electric fields to enhance the Raman signals of target molecules. Besides, we modified the paper with a hydrophobic layer to inhibit the diffusion of analytes into the deep location of papers. Therefore, the analytes in a sample droplet could be condensed on the paper surface and consequently raising their effective concentration within the hotspots supported by the AuNPs. Then, we observed the morphologies of the AuNPs on paper by SEM and used a three-dimensional finite-difference time–domain (3D-FDTD) simulation to analyze their near-field electromagnetic responses. Moreover, we evaluated the as-fabricated SERS papers by using methylene blue as analyte. The optimized AuNPs-deposited papers exhibited a significant Raman enhancement factor up to 10⁷, even by using a portable 785-nm Raman spectrometer. More importantly, the SERS papers demonstrated a sensitive detection toward paraquat with the concentration from 10^-2 to 10⁴ ppm in DI water. Note that the blood paraquat level of poisoned patients is typically higher than 1 ppm, indicating our SERS papers have a great potential to be used as a rapid clinical diagnosis tool for paraquat poisoning. Further parallel analyses with other commonly used pesticides (e.g., dimethoate, methamidophos and chlorpyrifos) and the determination of serum paraquat level of paraquat-poisoned patients (9 patients) are undergoing. Further results will be presented at the conference.

Large, bulky, power-hungry traditional mechanical actuators are poorly suited for small, biological applications such as medical devices. Shape changing polymers are an emerging class of actuators which can utilize environmental conditions to undergo large, complex shape changes. Liquid crystalline self-assembly is one promising strategy to program structural orientation and resulting actuation in polymeric materials. This molecular ordering can be spatially patterned, resulting in monolithic materials that undergo complex shape change. However, liquid crystal polymer networks are typically hydrophobic and only respond to stimuli that would be incompatible with biological environments, such as high temperatures and organic solvents. We have used two strategies to overcome these limitations: 1) engineering liquid crystal elastomers chemistry to respond near body temperature and 2) building gels from water-soluble, chromonic liquid crystals to respond to aqueous stimuli.

Thermotropic, hydrophobic liquid crystal elastomers have significant advantages of facile patterning, a variety of available monomers, and well-studied elastomer chemistries. These patternable elastomers can undergo high amounts of actuation strain, but these strains are typically not realized below 100-200 C, as determined by the liquid crystalline phase transition. To lower this actuation temperature to near body temperature, we use a 2-click thiol-acrylate/thiol-ene chemistry to copolymerize mesogens with isotropic monomers, which can shift the transition temperature and resulting actuation temperature from 105 C to 28 C. This control of phase behavior enables liquid crystal elastomers which can morph in response to contact with skin or warm tap water. Additionally, these low-temperature elastomers can be 3D printed, allowing structural freedom which is unfeasible in many other liquid crystal elastomer chemistries.

Alternatively, we can synthesize gels which respond to aqueous stimuli, such as pH, by building hydrogels from chromonic liquid crystals. We have synthesized the first hydrogel from chromonic liquid crystals which can be ordered through surface alignment and copolymerized with responsive monomers. By controlling the crosslink density of the gel with non-polymerizable chromonic precursors, we can polymerize gels ranging from ~10 kPa to ~300 kPa. The modulus of the gel is ~2x stiffer along the axis of molecular alignment, which guides anisotropic actuation in response to temperature and pH. This control over crosslink density not only allows for control over the magnitude of shape change but also allows us to synthesize gels similar to soft tissues with anisotropic mechanical properties.

With these strategies, we have taken steps towards the use of liquid crystalline polymers in biological applications. Both liquid crystal elastomers and liquid crystal gels have been used as substrates for mammalian cell culture to confirm the biocompatibility of these materials. By controlling the molecular orientation of these ordered gels and elastomers, we can potentially affect cellular behavior both statically by anisotropic modulus and dynamically by temperature-based or pH-based actuation.

Nanotechnology has enabled unprecedented control of the interactions between materials and biological entities, from the microscale down to the molecular level. For example, nanosurfaces and nanostructures have been used to mimic or interact with biological microenvironments, to support specific biological functions such as cell adhesion, mobility and differentiation, as well as tissue healing.
Recently, a new paradigm has been proposed for nanomedicine to exploit the intrinsic properties of nanomaterials as active devices rather than as passive structural units or carriers for medications. In this view, the nanomaterial itself is the active device that responds to external stimuli by modifying its intrinsic chemical and/or physical characteristics, so as to provide useful bio-stimulation and/or bio-signaling.¹
Our research approach falls into this latter, “active” category: we develop “smart” nanomaterials that change their structural/functional properties in response to specific external stimuli (electric or magnetic fields, electromagnetic radiation, ultrasound, etc.). Specifically, we develop multifunctional nanostructured materials that are pharmacologically active and that can be actuated by virtue of their magnetic, dielectric, optically active, or piezoelectric properties.²
More in details, in this talk I will approach applications of smart nanomaterials such as piezoelectric nanotransducers³, nanotechnological antioxidants⁴, and hybrid lipid/magnetic nanovectors for advanced theranostic applications⁵.
This presentation will summarize our main results to date, and highlights the most promising examples that could have a practical translation into the clinical or technological realms.

[1] Genchi G.G., […], Ciofani G. Frontiers in Bioengineering and Biotechnology, 5: 80 (2017)
[2] Genchi G.G., […], Ciofani G. Advanced Healthcare Materials, 6(9): 1700002 (2017)
[3] Marino A., […], Ciofani G. Nano Today, 14: 9-12 (2017)
[4] Tapeinos C., […], Ciofani G. ACS Omega, 3(8): 8952-8962 (2018)
[5] Tapeinos C., […], Ciofani G. Nanoscale, 10.1039/C8NR05520C

Because of their excellent biocompatibility and osteoconductivity, ceramic biomaterials are clinically utilised as bone grafts for the reconstruction of injured bone tissues. The fundamental concept for bone tissue engineering is to use the natural biological responses in the host body. Here, we explored the biophysical and material properties of bone tissue in order to develop a rationale for improved ceramic bone grafts. Especially we focused on the pcarbonate substitutions in apatite minerals of bone matrix and bone responses to mechanical strain. Material characterization of bone matrix shows that bone mineral includes carbonate ions in their crystal lattice. Mechanical loading in bone tissue induces an electrical potential generated by piezoelectricity arising from displacement of collagen fibrils. The electrical potential is stored in collagen fibrils as well as apatite minerals. The stable electrical potentials stored in apatite minerals are enough to stimulate the osteogenic cells during bone remodeling. Using these material properties, we modify the surface characteristics of bone graft materials in order to improve the osteoconductivity. The surface modification of ceramic biomaterials used for medical devices is expected to improve the osteoconductivity through control of the bio-interfaces between the materials and bone tissues.
The purpose of this study was to investigate a mechanism through which the surface wettability of biomaterials can be improved and determine the effects of biomaterial surface characteristics on cellular behaviors. Polarization treatment induced surface charges on hydroxyapatite, b-tricalcium phosphate, carbonate-substituted hydroxyapatite and yttria-stabilized zirconia regardless of the differences in the carrier ions participating in the polarization. Characterization of the surfaces revealed that the wettability of the polarized ceramic biomaterials was improved through the increase in the surface free energies compared with conventional ceramic surfaces. In addition, sintering atmosphere affects the polarization capacity of hydroxyapatite by changing hydroxide ion content and grain size. Compared with hydroxyapatite sintered in air, hydroxyapatite sintered in saturated water vapor had a higher polarization capacity that increased surface free energy and improved wettability, which in turn accelerated cell adhesion. We determined the optimal conditions of hydroxyapatite polarization for the improvement of surface wettability and acceleration of cell adhesion. Understanding the osteoconduction mechanism may allow us to design new biomaterials and select biomaterials for implantation that will last the lifetime of the recipient.

The use of 3D bioprinting techniques to creats 3D cell cultures offers new opportunities in the creation of disease models, the assessment of new drugs, and regenerative medicine. However, clinical application of 3D bioprinting requires that some outstanding challenges are overcome, primarily related to productivity and quality. This presentation will describe a new bioprinting system called Reactive Jet Impingement (ReJI), which prints cell-laden hydrogels. Droplets of gel precursor solutions are jetted at one another such that they meet and react in mid-air before the gel droplets fall to the substrate. This technique works effectively with a range of hydrogels, including alginate, fibrin and collagen hybrid gels. The ReJI process gives a high deposition rate, and allows high cell density gels to be printed with a high viability. Cell density plays a major role in defining the maturation rate of micro-tissues, and we have demonstrated the value of high cell density in the development of bone microtissues from immortalized human bone marrow stem cells (hMSCs). hMSCs were printed with high viability within a collagen-alginate-fibrin gel, and bone tissue specific proteomic and genetic markers indicated significantly higher tissue maturation rates in cell filled gels deposited with higher cell densities. Further work is exploring use of the technique for musculoskeletal and cancer co-cultures with multiple bio-ink deposition.