Biopolymer Microparticles Prepared by Microfluidics for Biomedical Applications.

Biopolymers are macromolecules that are derived from natural sources and have attractive properties for a plethora of biomedical applications due to their biocompatibility, biodegradability, low antigenicity, and high bioactivity. Microfluidics has emerged as a powerful approach for fabricating polymeric microparticles (MPs) with designed structures and compositions through precise manipulation of multiphasic flows at the microscale. The synergistic combination of materials chemistry afforded by biopolymers and precision provided by microfluidic capabilities make it possible to design engineered biopolymer-based MPs with well-defined physicochemical properties that are capable of enabling an efficient delivery of therapeutics, 3D culture of cells, and sensing of biomolecules. Here, an overview of microfluidic approaches is provided for the design and fabrication of functional MPs from three classes of biopolymers including polysaccharides, proteins, and microbial polymers, and their advances for biomedical applications are highlighted. An outlook into the future research on microfluidically-produced biopolymer MPs for biomedical applications is also provided.

Highly purified mussel adhesive protein to secure biosafety for in vivo applications.

BACKGROUND: Unique adhesive and biocompatibility properties of mussel adhesive proteins (MAPs) are known for their great potential in many tissue engineering and biomedical applications. Previously, it was successfully demonstrated that redesigned hybrid type MAP, fp-151, mass-produced in Gram-negative bacterium Escherichia coli, could be utilized as a promising adhesive biomaterial. However, purification of recombinant fp-151 has been unsatisfactory due to its adhesive nature and polarity which make separation of contaminants (especially, lipopolysaccharide, a toxic Gram-negative cell membrane component) very difficult. RESULTS: In the present work, we devised a high resolution purification approach to secure safety standards of recombinant fp-151 for the successful use in in vivo applications. Undesirable impurities were remarkably eliminated as going through sequential steps including treatment with multivalent ion and chelating agent for cell membrane washing, mechanical cell disruption, non-ionic surfactant treatment for isolated inclusion body washing, acid extraction of washed inclusion body, and ion exchange chromatography purification of acid extracted sample. Through various analyses, such as high performance liquid chromatographic purity assay, limulus amoebocyte lysate endotoxin assay, and in vitro mouse macrophage cell tests on inflammation, viability, cytotoxicity, and apoptosis, we confirmed the biological safety of bacterial-derived purified recombinant fp-151. CONCLUSIONS: Through this purification design, recombinant fp-151 achieved 99.90% protein purity and 99.91% endotoxin reduction that nearly no inflammation response was observed in in vitro experiments. Thus, the highly purified recombinant MAP would be successfully used as a safety-secured in vivo bioadhesive for tissue engineering and biomedical applications.

Self-controllable proteinic antibacterial coating with bacteria-triggered antibiotic release for prevention of periprosthetic infection.

Periprosthetic infection is a devastating postimplantation complication in which a biofilm layer harboring invasive microorganisms forms around orthopedic implants, leading to severe implant failure and patient morbidity. Despite the development of several infection-triggered antibiotic release approaches, most current antibacterial coatings are susceptible to undesired antibiotic leakage or mechanical disintegration during prosthesis installation. Herein, we propose a self-controllable proteinic antibacterial coating capable of both long-lasting adherence onto titanium implant substrates over the implant fixation period and instantaneous bacterial eradication. Importantly, the pH-dependent reversible metal coordination of mussel adhesive protein (MAP) enabled bacterial concentration-dependent antibiotic delivery in response to infection-induced acidification. In addition, the MAP coating exhibited superior self-healable adhesive properties and scratch resistance, which enabled to avert issues associated with mechanical damages, including peeling and cracking, often occurring in conventional implant coating systems. The gentamicin-loaded MAP coating exhibited complete inhibition of bacterial growth in vivo against Staphylococcus aureus penetrations during implantation surgery (immediate infection) and even 4 weeks after implantation (delayed infection). Thus, our antibiotic-loaded MAP hydrogel coating can open new avenues for self-defensive antibiotic prophylaxis to achieve instant and sustainable bacteriocidal activity in orthopedic prostheses. © 2017 Elsevier Inc. All rights reserved.

Cell recognitive bioadhesive-based osteogenic barrier coating with localized delivery of bone morphogenetic protein-2 for accelerated guided bone regeneration.

Titanium mesh (Ti-mesh) for guided bone regeneration (GBR) approaches has been extensively considered to offer space maintenance in reconstructing the alveolar ridge within bone defects due to its superb mechanical properties and biocompatibility. However, soft tissue invasion across the pores of the Ti-mesh and intrinsically limited bioactivity of the titanium substrates often hinder satisfactory clinical outcomes in GBR treatments. Here, a cell recognitive osteogenic barrier coating was proposed using a bioengineered mussel adhesive protein (MAP) fused with Alg-Gly-Asp (RGD) peptide to achieve highly accelerated bone regeneration. The fusion bioadhesive MAP-RGD exhibited outstanding performance as a bioactive physical barrier that enabled effective cell occlusion and a prolonged, localized delivery of bone morphogenetic protein-2 (BMP-2). The MAP-RGD@BMP-2 coating promoted in vitro cellular behaviors and osteogenic commitments of mesenchymal stem cells (MSCs) via the synergistic crosstalk effects of the RGD peptide and BMP-2 in a surface-bound manner. The facile gluing of MAP-RGD@BMP-2 onto the Ti-mesh led to a distinguishable acceleration of the in vivo formation of new bone in terms of quantity and maturity in a rat calvarial defect. Hence, our protein-based cell recognitive osteogenic barrier coating can be an excellent therapeutic platform to improve the clinical predictability of GBR treatment.

Bioengineered Co-culture of organoids to recapitulate host-microbe interactions.

The recent spike in the instances of complex physiological host-microbe interactions has raised the demand for developing in vitro models that recapitulate the microbial microenvironment in the human body. Organoids are steadily emerging as an in vitro culture system that closely mimics the structural, functional, and genetic features of complex human organs, particularly for better understanding host-microbe interactions. Recent advances in organoid culture technology have become new avenues for assessing the pathogenesis of symbiotic interactions, pathogen-induced infectious diseases, and various other diseases. The co-cultures of organoids with microbes have shown great promise in simulating host-microbe interactions with a high level of complexity for further advancement in related fields. In this review, we provide an overview of bioengineering approaches for microbe-co-cultured organoids. Latest developments in the applications of microbe-co-cultured organoids to study human physiology and pathophysiology are also highlighted. Further, an outlook on future research on bioengineered organoid co-cultures for various applications is presented.

Tunicate-Inspired Photoactivatable Proteinic Nanobombs for Tumor-Adhesive Multimodal Therapy.

Near-IR (NIR) light-responsive multimodal nanotherapeutics have been proposed to achieve improved therapeutic efficacy and high specificity in cancer therapy. However, their clinical application is still elusive due to poor biometabolization and short retention at the target site. Here, innovative photoactivatable vanadium-doped adhesive proteinic nanoparticles (NPs) capable of allowing biological photoabsorption and NIR-responsive anticancer therapeutic effects to realize trimodal photothermal-gas-chemo-therapy treatments in a highly biocompatible, site-specific manner are proposed. The photoactivatable tumor-adhesive proteinic NPs can enable efficient photothermal conversion via tunicate-inspired catechol-vanadium complexes as well as prolonged tumor retention by virtue of mussel protein-driven distinctive adhesiveness. The incorporation of a thermo-sensitive nitric oxide donor and doxorubicin into the photoactivatable adhesive proteinic NPs leads to synergistic anticancer therapeutic effects as a result of photothermal-triggered "bomb-like" multimodal actions. Thus, this protein-based phototherapeutic tumor-adhesive NPs have great potential as a spatiotemporally controllable therapeutic system to accomplish effective therapeutic implications for the complete ablation of cancer.

Stretch-responsive adhesive microcapsules for strain-regulated antibiotic release from fabric wound dressings.

Bacterial infection of a wound is a major complication that can significantly delay proper healing and even necessitate surgical debridement. Conventional non-woven fabric dressings, including gauzes, bandages and cotton wools, often fail in treating wound infections in a timely manner due to their passive release mechanism of antibiotics. Here, we propose adhesive mechanically-activated microcapsules (MAMCs) capable of strongly adhering to a fibrous matrix to achieve a self-regulated release of antibiotics upon uniaxial stretching of non-woven fabric dressings. To achieve this, a uniform population of polydopamine (PDA)-coated MAMCs (PDA-MAMCs) are prepared using a microfluidics technique and subsequent oxidative dopamine polymerization. The PDA-MAMC allows for robust mechano-activation within the fibrous network through high retention and effective transmission of mechanical force under stretching. By validating the potential of a PDA-MAMCs-laden gauze to release antibiotics in a tensile strain-dependent manner, we demonstrate that PDA-MAMCs can be successfully incorporated into a woven material and create a smart wound dressing for control of bacterial infections. This new mechano-activatable delivery approach will open up a new avenue for a stretch-triggered, on-demand release of therapeutic cargos in skin-mountable or wearable biomedical devices.

Mechano-activated biomolecule release in regenerating load-bearing tissue microenvironments.

Although mechanical loads are integral for musculoskeletal tissue homeostasis, overloading and traumatic events can result in tissue injury. Conventional drug delivery approaches for musculoskeletal tissue repair employ localized drug injections. However, rapid drug clearance and inadequate synchronization of molecule provision with healing progression render these methods ineffective. To overcome this, a programmable mechanoresponsive drug delivery system was developed that utilizes the mechanical environment of the tissue during rehabilitation (for example, during cartilage repair) to trigger biomolecule provision. For this, a suite of mechanically-activated microcapsules (MAMCs) with different rupture profiles was generated in a single fabrication batch via osmotic annealing of double emulsions. MAMC physical dimensions were found to dictate mechano-activation in 2D and 3D environments and their stability in vitro and in vivo, demonstrating the tunability of this system. In models of cartilage regeneration, MAMCs did not interfere with tissue growth and activated depending on the mechanical properties of the regenerating tissue. Finally, biologically active anti-inflammatory agents were encapsulated and released from MAMCs, which counteracted degradative cues and prevented the loss of matrix in living tissue environments. This unique technology has tremendous potential for implementation across a wide array of musculoskeletal conditions for enhanced repair of load-bearing tissues.

Bone Graft Biomineral Complex Coderived from Marine Biocalcification and Biosilicification.

Bone graft materials have been mainly developed based on inorganic materials, including calcium phosphate. However, these graft materials usually act as osteoconductive rather than osteoinductive scaffolds. To improve bone reconstruction, a combination of several materials has been proposed. However, there are still no alternatives that can completely replace the existing animal-derived bone graft materials. In this work, a marine-inspired biomineral complex was suggested as a potential bone graft material. The proposed biosilicified coccolithophore-derived coccoliths using bioengineered mussel adhesive proteins show osteopromotive ability through the synergistic effects of osteoconductivity from calcium carbonate and osteoinductivity from silica. Its possibility of use as a bone substitute was determined by evaluating the in vitro osteogenic behaviors of multipotent mesenchymal stem cells and in vivo bone regeneration in a rat calvarial defect model. Therefore, the marine-inspired biomineral complex developed in this study could be successfully used for bone tissue engineering.

Biosilicated collagen/ß-tricalcium phosphate composites as a BMP-2-delivering bone-graft substitute for accelerated craniofacial bone regeneration.

BACKGROUND: Bioceramic ß-tricalcium phosphate (ß-TCP) is used as a bone-grafting material and a therapeutic drug carrier for treatment of bone defects in the oral and maxillofacial regions due to the osteoconductivity and biocompatibility. However, the low mechanical strength and limited osteoinductivity of ß-TCP agglomerate restrict bone regenerating performance in clinical settings. METHODS: Herein, a biomimetic composite is proposed as a bone morphogenetic protein-2 (BMP-2)-delivering bone graft substitute to achieve a robust bone grafting and augmented bone regeneration. RESULTS: The sequential processes of brown algae-inspired biosilicification and collagen coating on the surface of ß-TCP enable the effective incorporation of BMP-2 into the coating layer without losing its bioactivity. The sustained delivery of BMP-2 from the biosilicated collagen and ß-TCP composites promoted in vitro osteogenic behaviors of pre-osteoblasts and remarkedly accelerated in vivo bone regeneration within a rat calvarial bone defect. CONCLUSIONS: Our multicomposite bone substitutes can be practically applied to improve bone tissue growth in bone grafting applications with further expansion to general bone tissue engineering.

Sprayable Adhesive Nanotherapeutics: Mussel-Protein-Based Nanoparticles for Highly Efficient Locoregional Cancer Therapy.

Following surgical resection for primary treatment of solid tumors, systemic chemotherapy is commonly used to eliminate residual cancer cells to prevent tumor recurrence. However, its clinical outcome is often limited due to insufficient local accumulation and the systemic toxicity of anticancer drugs. Here, we propose a sprayable adhesive nanoparticle (NP)-based drug delivery system using a bioengineered mussel adhesive protein (MAP) for effective locoregional cancer therapy. The MAP NPs could be administered to target surfaces in a surface-independent manner through a simple and easy spray process by virtue of their unique adhesion ability and sufficient dispersion property. Doxorubicin (DOX)-loaded MAP NPs (MAP@DOX NPs) exhibited efficient cellular uptake, endolysosomal trafficking, and subsequent low pH microenvironment-induced DOX release in cancer cells. The locally sprayed MAP@DOX NPs showed a significant inhibition of tumor growth in vivo, resulting from the prolonged retention of the MAP@DOX NPs on the tumor surface. Thus, this adhesive MAP NP-based spray therapeutic system provides a promising approach for topical drug delivery in adjuvant cancer therapy.

Antibacterial efficacy of poly(vinyl alcohol) composite nanofibers embedded with silver-anchored silica nanoparticles.

Silver has been widely used as an effective antibacterial agent especially for treating burns and wounds. However, release of silver from materials often arouse side effects due to toxicity of silver towards mammalian cells. Argyria and argyrosis are well known problems of acute toxicity of silver towards human body. Immobilization of silver is an effective approach to reduce silver release. Herein, we present poly(vinyl alcohol) (PVA) composite nanofibers embedded with silver-anchored silica nanoparticles (SSNs) as a novel antibacterial material. Silver nanoparticles anchored on silica nanoparticles were prepared and incorporated into PVA nanofibers to fabricate silver-silica embedded PVA nanofibers (SSN-PVA) by electrospinning. Incorporation of SSNs into PVA was confirmed by TEM and SEM results revealed regular nanofibers whose diameter increased with successive addition of SSNs. The SSN-PVA nanofibers showed significant antibacterial efficacy against both Gram-negative and Gram-positive bacteria. Our research results demonstrated SSN-embedded polymeric nanofibers can open up a promising prospect for the prevention of bacterial infection in diverse biomedical fields including wound dressing. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1121-1128, 2018.

Diatom-Inspired Silica Nanostructure Coatings with Controllable Microroughness Using an Engineered Mussel Protein Glue to Accelerate Bone Growth on Titanium-Based Implants.

Silica nanoparticles (SiNPs) have been utilized to construct bioactive nanostructures comprising surface topographic features and bioactivity that enhances the activity of bone cells onto titanium-based implants. However, there have been no previous attempts to create microrough surfaces based on SiNP nanostructures even though microroughness is established as a characteristic that provides beneficial effects in improving the biomechanical interlocking of titanium implants. Herein, a protein-based SiNP coating is proposed as an osteopromotive surface functionalization approach to create microroughness on titanium implant surfaces. A bioengineered recombinant mussel adhesive protein fused with a silica-precipitating R5 peptide (R5-MAP) enables direct control of the microroughness of the surface through the multilayer assembly of SiNP nanostructures under mild conditions. The assembled SiNP nanostructure significantly enhances the in vitro osteogenic cellular behaviors of preosteoblasts in a roughness-dependent manner and promotes the in vivo bone tissue formation on a titanium implant within a calvarial defect site. Thus, the R5-MAP-based SiNP nanostructure assembly could be practically applied to accelerate bone-tissue growth to improve the stability and prolong the lifetime of medical implantable devices.

Mussel-Inspired Anisotropic Nanocellulose and Silver Nanoparticle Composite with Improved Mechanical Properties, Electrical Conductivity and Antibacterial Activity.

Materials for wearable devices, tissue engineering and bio-sensing applications require both antibacterial activity to prevent bacterial infection and biofilm formation, and electrical conductivity to electric signals inside and outside of the human body. Recently, cellulose nanofibers have been utilized for various applications but cellulose itself has neither antibacterial activity nor conductivity. Here, an antibacterial and electrically conductive composite was formed by generating catechol mediated silver nanoparticles (AgNPs) on the surface of cellulose nanofibers. The chemically immobilized catechol moiety on the nanofibrous cellulose network reduced Ag⁺ to form AgNPs on the cellulose nanofiber. The AgNPs cellulose composite showed excellent antibacterial efficacy against both Gram-positive and Gram-negative bacteria. In addition, the catechol conjugation and the addition of AgNP induced anisotropic self-alignment of the cellulose nanofibers which enhances electrical and mechanical properties of the composite. Therefore, the composite containing AgNPs and anisotropic aligned the cellulose nanofiber may be useful for biomedical applications.

Bioengineered mussel glue incorporated with a cell recognition motif as an osteostimulating bone adhesive for titanium implants.

Successful titanium implantation strongly depends on early fixation through an osseointegration between the titanium fixture and adjacent bone tissue. From a clinical perspective, rapid recruitment of functional biomolecules from the blood and osteogenic cell binding is critical for osseointegration immediately after implant insertion. Thus, surface modifications aiming to improve the interactions between the blood and implant and to enhance the binding of osteogenic cells onto the implant surface can contribute to successful osseointegration. Mussel adhesive proteins (MAPs) derived from marine mussels have been considered as promising bioadhesives that have strong adhesion and coating abilities onto organic and inorganic surfaces, even in wet environments. Here, we investigated the in vitro and in vivo osteostimulating ability of the bioengineered mussel glue MAP-RGD, which is a recombinant MAP fused with an Arg-Gly-Asp (RGD) peptide, an effective cell recognition motif for activating intracellular signaling pathways, using a titanium mesh (Ti-mesh) as a model titanium implant. We found that the in vitro cell behaviors of pre-osteoblast cells, such as attachment, proliferation, spreading, and osteogenic differentiation, increased significantly on the MAP-RGD-coated Ti-mesh surface. In vitro blood responses including blood wetting, blood clotting, and platelet adhesion were also highly enhanced on the MAP-RGD-coated surface. Importantly, implantation of the MAP-RGD-coated Ti-mesh resulted in a remarkable acceleration of in vivo bone regeneration and maturation of a new bone in a rat calvarial defect. Consequently, the bioengineered mussel glue can be successfully utilized as an osteostimulating bone bioadhesive for titanium implant applications with further expansion to general bone tissue engineering.

Surface-independent antibacterial coating using silver nanoparticle-generating engineered mussel glue.

During implant surgeries, antibacterial agents are needed to prevent bacterial infections, which can cause the formation of biofilms between implanted materials and tissue. Mussel adhesive proteins (MAPs) derived from marine mussels are bioadhesives that show strong adhesion and coating ability on various surfaces even in wet environment. Here, we proposed a novel surface-independent antibacterial coating strategy based on the fusion of MAP to a silver-binding peptide, which can synthesize silver nanoparticles having broad antibacterial activity. This sticky recombinant fusion protein enabled the efficient coating on target surface and the easy generation of silver nanoparticles on the coated-surface under mild condition. The biosynthesized silver nanoparticles showed excellent antibacterial efficacy against both Gram-positive and Gram-negative bacteria and also revealed good cytocompatibility with mammalian cells. In this coating strategy, MAP-silver binding peptide fusion proteins provide hybrid environment incorporating inorganic silver nanoparticle and simultaneously mediate the interaction of silver nanoparticle with surroundings. Moreover, the silver nanoparticles were fully synthesized on various surfaces including metal, plastic, and glass by a simple, surface-independent coating manner, and they were also successfully synthesized on a nanofiber surface fabricated by electrospinning of the fusion protein. Thus, this facile surface-independent silver nanoparticle-generating antibacterial coating has great potential to be used for the prevention of bacterial infection in diverse biomedical fields.