Porous collagen scaffold reinforced with surfaced activated PLLA nanoparticles.

Porous collagen scaffold is integrated with surface activated PLLA nanoparticles fabricated by lyophilizing and crosslinking via EDC treatment. In order to prepare surface-modified PLLA nanoparticles, PLLA was firstly grafted with poly (acrylic acid) (PAA) through surface-initiated polymerization of acrylic acid. Nanoparticles of average diameter 316 nm and zeta potential -39.88 mV were obtained from the such-treated PLLA by dialysis method. Porous collagen scaffold were fabricated by mixing PLLA nanoparticles with collagen solution, freeze drying, and crosslinking with EDC. SEM observation revealed that nanoparticles were homogeneously dispersed in collagen matrix, forming interconnected porous structure with pore size ranging from 150 to 200 µm, irrespective of the amount of nanoparticles. The porosity of the scaffolds kept almost unchanged with the increment of the nanoparticles, whereas the mechanical property was obviously improved, and the degradation was effectively retarded. In vitro L929 mouse fibroblast cells seeding and culture studies revealed that cells infiltrated into the scaffolds and were distributed homogeneously. Compared with the pure collagen sponge, the number of cells in hybrid scaffolds greatly increased with the increment of incorporated nanoparticles. These results manifested that the surface-activated PLLA nanoparticles effectively reinforced the porous collagen scaffold and promoted the cells penetrating into the scaffold, and proliferation.

Multifunctional peptide-conjugated nanocarriers for pulp regeneration in a full-length human tooth root.

Dental pulp is a highly vascularized tissue, situated in an inextensible environment surrounded by rigid dentinal walls. The pulp receives its blood supply solely from the small apical foramen of a tooth root. Due to the unique anatomy that controls nutrition supply, regeneration of pulp tissue in a full-length tooth root has long been a challenge in regenerative endodontics. In this study, we designed and synthesized a multifunctional peptide-conjugated, pH-sensitive, non-viral gene vector for fast revascularization and pulp regeneration in a full-length human tooth root. The multifunctional peptide was designed to have distinctive features, including a cell-penetrating peptide to enhance cellular uptake, a nuclear localization signal peptide to assist in the translocation of an angiogenic gene into the nucleus, and a fluorescent tryptophan residue to visualize and quantify the transfection efficiency. Furthermore, a pH-sensitive dimethylmaleic anhydride (DMA) was integrated with the multifunctional peptide to enhance the transfected gene complex to escape from endosomes/lysosomes after internalization. In vitro experiments showed that the multifunctional non-viral gene vector significantly increased internalization and gene transfection efficiency as well as reduced cytotoxicity. After dental pulp stem cells (DPSCs) were transfected with the multifunctional gene vector/pVEGF complexes, the expression of VEGF from the DPSCs was upregulated for more than eight folds, which in turn greatly enhanced endothelial cell migration and vascular-like tube formation. Six weeks after implantation, the VEGF-transfected DPSCs accelerated new blood vessel formation and the regenerated pulp tissue occupied most of the area in the canal of a full-length human tooth root. The multifunctional peptide conjugated non-viral gene delivery is a safe and effective approach for regenerative endodontics. STATEMENT OF SIGNIFICANCE: Pulp regeneration in a full-length tooth root canal has long been a challenge in regenerative endodontics. This is due to the unique root anatomy that allows the blood supply of the tooth root only from a small apical foramen (< 1 mm), leading to a severe barrier for revascularization during pulp regeneration. In this work, we designed a multifunctional peptide-conjugated, pH-sensitive, non-viral gene vector to address this challenge. Our work shows that the peptide-conjugated system was an excellent carrier for fast revascularization and pulp tissue regeneration in a full-length toot root. This study will interest the multidisciplinary readership in gene delivery, biomaterials, and dental/craniofacial tissue engineering community.

Printability in extrusion bioprinting.

Extrusion bioprinting has been widely used to extrude continuous filaments of bioink (or the mixture of biomaterial and living cells), layer-by-layer, to build three-dimensional constructs for biomedical applications. In extrusion bioprinting, printability is an important parameter used to measure the difference between the designed construct and the one actually printed. This difference could be caused by the extrudability of printed bioink and/or the structural formability and stability of printed constructs. Although studies have reported in characterizing printability based on the bioink properties and printing process, the concept of printability is often confusingly and, sometimes, conflictingly used in the literature. The objective of this perspective is to define the printability for extrusion bioprinting in terms of extrudability, filament fidelity, and structural integrity, as well as to review the effect of bioink properties, bioprinting process, and construct design on the printability. Challenges related to the printability of extrusion bioprinting are also discussed, along with recommendations for improvements.

Optimizing Anisotropic Polyurethane Scaffolds to Mechanically Match with Native Myocardium.

Biodegradable cardiac patch is desirable to possess mechanical properties mimicking native myocardium for heart infarction treatment. We fabricated a series of anisotropic and biodegradable polyurethane porous scaffolds via thermally induced phase separation (TIPS) and tailored their mechanical properties by using various polyurethanes with different soft segments and varying polymer concentrations. The uniaxial mechanical properties, suture retention strength, ball-burst strength, and biaxial mechanical properties of the anisotropic porous scaffolds were optimized to mechanically match native myocardium. The optimal anisotropic scaffold had a ball burst strength (20.7 ± 1.5 N) comparable to that of native porcine myocardium (20.4 ± 6.0 N) and showed anisotropic behavior close to biaxial stretching behavior of the native porcine myocardium. Furthermore, the optimized porous scaffold was combined with a porcine myocardium-derived hydrogel to form a biohybrid scaffold. The biohybrid scaffold showed morphologies similar to the decellularized porcine myocardial matrix. This combination did not affect the mechanical properties of the synthetic scaffold alone. After in vivo rat subcutaneous implantation, the biohybrid scaffolds showed minimal immune response and exhibited higher cell penetration than the polyurethane scaffold alone. This biohybrid scaffold with biomimetic mechanics and good tissue compatibility would have great potential to be applied as a biodegradable acellular cardiac patch for myocardial infarction treatment.

Glutathione-responsive biodegradable polyurethane nanoparticles for lung cancer treatment.

Lung cancer is one of the major causes of cancer-related deaths worldwide. Stimuli-responsive polymers and nanoparticles, which respond to exogenous or endogenous stimuli in the tumor microenvironment, have been widely investigated for spatiotemporal chemotherapeutic drug release applications for cancer chemotherapy. We developed glutathione (GSH)-responsive polyurethane nanoparticles (GPUs) using a GSH-cleavable disulfide bond containing polyurethane that responds to elevated levels of GSH within lung cancer cells. The polyurethane nanoparticles were fabricated using a single emulsion and mixed organic solvent method. Cisplatin-loaded GSH-sensitive nanoparticles (CGPU) displayed a GSH-dose dependent release of cisplatin. In addition, a significant reduction in in vitro survival fraction of A549 lung cancer cells was observed compared to free cisplatin of equivalent concentration (survival fraction of ~0.5 and ~0.7, respectively). The in vivo biodistribution studies showed localization of fluorescently labeled GPUs (~7% of total injected dose per gram tissue) in the lung tumor regions after mouse-tail IV injections in xenograft A549 lung tumor models. The animals exposed to CGPUs also exhibited the inhibition of lung tumor growth compared to animals administered with saline (tumor growth rate of 1.5 vs. 13 in saline) and free cisplatin (tumor growth rate of 5.9) in mouse xenograft A549 lung tumor models within 14 days. These nanoparticles have potential to be used for on-demand drug release for an enhanced chemotherapy to effectively treat lung cancer.

Recent advances in high-strength and elastic hydrogels for 3D printing in biomedical applications.

Three-dimensional (3D) printing enables the production of personalized tissue-engineered products with high tunability and complexity. It is thus an attractive and promising technology in the pharmaceutical and medical fields. Printable and biocompatible hydrogels are attractive materials for 3D printing applications because they offer favorable biomimetic environments for live cells, such as high water content, porous structure, bioactive molecule incorporation, and tunable mechanical properties and degradation rates. However, most conventional hydrogel materials are brittle and mechanically weak and hence cannot meet the mechanical needs for handling and soft and elastic tissue use. Thus, the development of printable, high-strength, and elastic hydrogel materials for 3D printing in tissue repair and regeneration is critical and interesting. In this review, we summarized the recent reports on high-strength and elastic hydrogels for printing use and categorized them into three groups, namely double-network hydrogels, nanocomposite hydrogels, and single-network hydrogels. The reinforcing mechanisms of these high-strength hydrogels and the strategies to improve their printability and biocompatibility were further discussed. These high-strength and elastic hydrogels may offer opportunities to accelerate the development of 3D printing technology and provide new insights for 3D-printed product design in biomedicine. STATEMENT OF SIGNIFICANCE: Biocompatible and biodegradable hydrogels are highly attractive in 3D printing because of their desirable printability and friendly environment for loading bioactive molecules and living cells. The development of high-strength and elastic hydrogels changes the conventional impression of weak and brittle hydrogels and provides new opportunities and inspirations for 3D printing and biomedical applications. In this review, we analyzed the hydrogel reinforcement mechanisms, summarized recent progresses in developing high-strength and elastic hydrogels for 3D printing, and discussed the strategies to improve the printability and biocompatibility of the hydrogel inks.

Highly Elastic Biodegradable Single-Network Hydrogel for Cell Printing.

Cell printing is becoming a common technique to fabricate cellularized printed scaffold for biomedical application. There are still significant challenges in soft tissue bioprinting using hydrogels, which requires live cells inside the hydrogels. Moreover, the resilient mechanical properties from hydrogels are also required to mechanically mimic the native soft tissues. Herein, we developed a visible-light cross-linked, single-network, biodegradable hydrogel with high elasticity and flexibility for cell printing, which is different from previous highly elastic hydrogel with double-network and two components. The single-network hydrogel using only one stimulus (visible light) to trigger gelation can greatly simplify the cell printing process. The obtained hydrogels possessed high elasticity, and their mechanical properties can be tuned to match various native soft tissues. The hydrogels had good cell compatibility to support fibroblast growth in vitro. Various human cells were bioprinted with the hydrogels to form cell-gel constructs, in which the cells exhibited high viability after 7 days of culture. Complex patterns were printed by the hydrogels, suggesting the hydrogel feasibility for cell printing. We believe that this highly elastic, single-network hydrogel can be simply printed with different cell types, and it may provide a new material platform and a new way of thinking for hydrogel-based bioprinting research.

Biodegradable Nanoparticles Enhanced Adhesiveness of Mussel-Like Hydrogels at Tissue Interface.

Popular bioadhesives, such as fibrin, cyanoacrylate, and albumin-glutaraldehyde based materials, have been applied for clinical applications in wound healing, drug delivery, and bone and soft tissue engineering; however, their performances are limited by weak adhesion strength and rapid degradation. In this study a mussel-inspired, nanocomposite-based, biodegradable tissue adhesive is developed by blending poly(lactic-co-glycolic acid) (PLGA) or N-hydroxysuccinimide modified PLGA nanoparticles (PLGA-NHS) with mussel-inspired alginate-dopamine polymer (Alg-Dopa). Adhesive strength measurement of the nanocomposites on porcine skin-muscle constructs reveals that the incorporation of nanoparticles in Alg-Dopa significantly enhances the tissue adhesive strength compared to the mussel-inspired adhesive alone. The nanocomposite formed by PLGA-NHS nanoparticles shows higher lap shear strength of 33 ± 3 kPa, compared to that of Alg-Dopa hydrogel alone (14 ± 2 kPa). In addition, these nanocomposites are degradable and cytocompatible in vitro, and elicit in vivo minimal inflammatory responses in a rat model, suggesting clinical potential of these nanocomposites as bioadhesives.

Low-Initial-Modulus Biodegradable Polyurethane Elastomers for Soft Tissue Regeneration.

The mechanical match between synthetic scaffold and host tissue remains challenging in tissue regeneration. The elastic soft tissues exhibit low initial moduli with a J-shaped tensile curve. Suitable synthetic polymer scaffolds require low initial modulus and elasticity. To achieve these requirements, random copolymers poly(δ-valerolactone-co-ε-caprolactone) (PVCL) and hydrophilic poly(ethylene glycol) (PEG) were combined into a triblock copolymer, PVCL-PEG-PVCL, which was used as a soft segment to synthesize a family of biodegradable elastomeric polyurethanes (PU) with low initial moduli. The triblock copolymers were varied in chemical components, molecular weights, and hydrophilicities. The mechanical properties of polyurethanes in dry and wet states can be tuned by altering the molecular weights and hydrophilicities of the soft segments. Increasing the length of either PVCL or PEG in the soft segments reduced initial moduli of the polyurethane films and scaffolds in dry and wet states. The polymer films are found to have good cell compatibility and to support fibroblast growth in vitro. Selected polyurethanes were processed into porous scaffolds by a thermally induced phase-separation technique. The scaffold from PU-PEG1K-PVCL6K had an initial modulus of 0.60 ± 0.14 MPa, which is comparable with the initial modulus of human myocardium (0.02-0.50 MPa). In vivo mouse subcutaneous implantation of the porous scaffolds showed minimal chronic inflammatory response and intensive cell infiltration, which indicated good tissue compatibility of the scaffolds. Biodegradable polyurethane elastomers with low initial modulus and good biocompatibility and processability would be an attractive alternative scaffold material for soft tissue regeneration, especially for heart muscle.

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications.

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

Synthesis and characterization of conductive, biodegradable, elastomeric polyurethanes for biomedical applications.

Biodegradable conductive polymers are currently of significant interest in tissue repair and regeneration, drug delivery, and bioelectronics. However, biodegradable materials exhibiting both conductive and elastic properties have rarely been reported to date. To that end, an electrically conductive polyurethane (CPU) was synthesized from polycaprolactone diol, hexadiisocyanate, and aniline trimer and subsequently doped with (1S)-(+)-10-camphorsulfonic acid (CSA). All CPU films showed good elasticity within a 30% strain range. The electrical conductivity of the CPU films, as enhanced with increasing amounts of CSA, ranged from 2.7 ± 0.9 × 10(-10) to 4.4 ± 0.6 × 10(-7) S/cm in a dry state and 4.2 ± 0.5 × 10(-8) to 7.3 ± 1.5 × 10(-5) S/cm in a wet state. The redox peaks of a CPU1.5 film (molar ratio CSA:aniline trimer = 1.5:1) in the cyclic voltammogram confirmed the desired good electroactivity. The doped CPU film exhibited good electrical stability (87% of initial conductivity after 150 hours charge) as measured in a cell culture medium. The degradation rates of CPU films increased with increasing CSA content in both phosphate-buffered solution (PBS) and lipase/PBS solutions. After 7 days of enzymatic degradation, the conductivity of all CSA-doped CPU films had decreased to that of the undoped CPU film. Mouse 3T3 fibroblasts proliferated and spread on all CPU films. This developed biodegradable CPU with good elasticity, electrical stability, and biocompatibility may find potential applications in tissue engineering, smart drug release, and electronics. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2305-2314, 2016.

New generation ICG-based contrast agents for ultrasound-switchable fluorescence imaging.

Recently, we developed a new technology, ultrasound-switchable fluorescence (USF), for high-resolution imaging in centimeter-deep tissues via fluorescence contrast. The success of USF imaging highly relies on excellent contrast agents. ICG-encapsulated poly(N-isopropylacrylamide) nanoparticles (ICG-NPs) are one of the families of the most successful near-infrared (NIR) USF contrast agents. However, the first-generation ICG-NPs have a short shelf life (<1 month). This work significantly increases the shelf life of the new-generation ICG-NPs (>6 months). In addition, we have conjugated hydroxyl or carboxyl function groups on the ICG-NPs for future molecular targeting. Finally, we have demonstrated the effect of temperature-switching threshold (Tth) and the background temperature (TBG) on the quality of USF images. We estimated that the Tth of the ICG-NPs should be controlled at ~38-40 °C (slightly above the body temperature of 37 °C) for future in vivo USF imaging. Addressing these challenges further reduces the application barriers of USF imaging.

Triggerable Degradation of Polyurethanes for Tissue Engineering Applications.

Tissue engineered and bioactive scaffolds with different degradation rates are required for the regeneration of diverse tissues/organs. To optimize tissue regeneration in different tissues, it is desirable that the degradation rate of scaffolds can be manipulated to comply with various stages of tissue regeneration. Unfortunately, the degradation of most degradable polymers relies solely on passive controlled degradation mechanisms. To overcome this challenge, we report a new family of reduction-sensitive biodegradable elastomeric polyurethanes containing various amounts of disulfide bonds (PU-SS), in which degradation can be initiated and accelerated with the supplement of a biological product: antioxidant-glutathione (GSH). The polyurethanes can be processed into films and electrospun fibrous scaffolds. Synthesized materials exhibited robust mechanical properties and high elasticity. Accelerated degradation of the materials was observed in the presence of GSH, and the rate of such degradation depends on the amount of disulfide present in the polymer backbone. The polymers and their degradation products exhibited no apparent cell toxicity while the electrospun scaffolds supported fibroblast growth in vitro. The in vivo subcutaneous implantation model showed that the polymers prompt minimal inflammatory responses, and as anticipated, the polymer with the higher disulfide bond amount had faster degradation in vivo. This new family of polyurethanes offers tremendous potential for directed scaffold degradation to promote maximal tissue regeneration.