Biointerface engineering through amalgamation of gene technology and site-specific growth factor conjugation for efficient osteodifferentiation.

The development of bone implants through bioinspired immobilization of growth factors remains a key issue in the generation of biological interfaces, especially in enhancing osteodifferentiation ability. In this study, we developed a strategy for surface functionalization of poly(lactide-glycolide) (PLGA) and hydroxyapatite (HA) composite substrates through site-specific conjugation of bone morphogenetic protein 2 containing 3,4-hydroxyphenalyalanine (DOPA-BMP2) mediated by tyrosinase and sortase A (SrtA). Firstly, the growth factor BMP2-LPETG containing LPETG motif was successfully expressed in Escherichia coli through recombinant DNA technology. The excellent binding affinity of binding growth factor (DOPA-BMP2) was achieved by converting the tyrosine residue (Y) of YKYKY-GGG peptide into DOPA (X) by tyrosinase, which bound to the substrates. Then its GGG motif was specifically bound to the end of BMP2-LPETG mediated by SrtA. Therefore, the generated bioactive DOPA-BMP2/PLGA/HA substrates significantly promoted the osteogenic differentiation of MC3T3-E1 cells. Thanks to this microbial-assisted engineering approach, our work presents a facile and highly site-specific strategy to engineer biomimetic materials for orthopedics and dentistry by effectively delivering growth factors, peptides, and other biomacromolecules.

Strategy to Immobilize Peptide Probe Selected through In Vitro Ribosome Display for Electrochemical Aptasensor Application.

In this study, we demonstrated an electrochemical aptasensor for calmodulin (CaM) detection and the peptide sequence (YWDKIKDFIGG) is obtained from in vitro ribosome display selection. To immobilize this peptide probe on the electrode surface, cystine was incorporated at the end of this peptide sequence. After a maleimide-functionalized poly(3,4-ethylenedioxythiophene), poly(EODT-MI), film was electropolymerized on the electrode, the peptide probe was immobilized through thiol-ene conjugation with the cystine end. Four peptides with different linkers were used for the binding test of bovine serum albumin and CaM using a quartz crystal microbalance. The zwitterionic linker EKEKEKEKEKEK provided good antifouling properties and the highest CaM binding. Furthermore, the immobilization of the peptide with this zwitterionic linker resulted in a minimal increase in the electrochemical impedance. By immobilizing the peptide with the selected zwitterionic linker, we successfully demonstrated an electrochemical aptasensor with a linear detection range for CaM from 0.01 to 10 mg/L and a detection limit of 0.001 mg/L.

Optimal therapeutic strategy using antigen-containing liposomes selectively delivered to antigen-presenting cells.

Recent immunotherapies have shown clinical success. In particular, vaccines based on particulate antigen (Ag) are expected to be implemented based on their efficacy. In the current study, we describe a strategy entailing Ag-encapsulating PEG-modified liposomes (PGL-Ag) as antigen protein delivery devices and show that the success of the liposome depends on the antigen-presenting cell (APC) capacity; after administration of PGL-Ag, dendritic cells (DCs) in particular take up the Ag and subsequently prime T cells. For the generation of antitumor T cell responses in the lymphoid tissues, the function of encapsulated Ag-capturing DCs in vivo could be a biomarker. We next designed a prime-boost strategy to enhance the antitumor effects of the PGL-Ag. In the tumor sites, we show that Ag retention in nanoparticle-capturing DCs promotes a robust antitumor response. Thus, this efficient particulate Ag-based host antigen-presenting cell delivery strategy provides a bridge between innate and adaptive immune response and offers a novel therapeutic option against tumor cells.

Tubular Network Formation by Mixing Amphiphilic Polypeptides with Differing Hydrophilic Blocks.

Artificial tubular networks are promising structures for biomaterial applications because of their large surface areas. A tubular network was formed by co-assembling two different amphiphilic polypeptides, poly(ethylene glycol)-b-(l-Leu-Aib)6 (PL12) and polysarcosine-b-(l-Leu-Aib)6 (SL12). They both have the same hydrophobic 12-mer helical block (l-Leu-Aib)6 but different hydrophilic chains, poly(ethylene glycol) and polysarcosine. In water, both polypeptides self-assembled into a tubular structure having a uniform 80 nm diameter that was formed by packing among the hydrophobic L12 blocks. The SL12 nanotubes were short (200 nm), straight, and robust. PL12 formed long (>1 µm), bendable, and fusogenic nanotubes. The amphiphiles were then co-assembled with various mixing ratios to form tubular networks. Higher concentrations of PL12 made the nanotubes more bendable and fusogenic between open tube ends, which produced branching junctions under heat treatment.

Spontaneous Formation of Gating Lipid Domain in Uniform-Size Peptide Vesicles for Controlled Release.

Hybrid assemblies composed of phospholipids and amphiphilic polymers have been investigated previously as a biomimetic model of biological cells. However, these studies focused on the functions of polymers in a sea of membrane lipids. Here, we prepared a highly stable peptide-lipid hybrid vesicle from a combination of an amphiphilic polypeptide and the phospholipid, 1,2-dimyristoyl- sn-glycero-3-phosphocholine, with a mixing molar ratio of 1:1. The phase-separated structure of the hybrid vesicle was demonstrated by fluorescence resonance energy transfer analysis. The lipid domain of the hybrid vesicle had a phase-transition temperature of 38 °C and allowed the permeation of a hydrophilic molecule, fluorescein isothiocyanate-labeled polyethylene glycol ( Mw: 2000), above 38 °C. The designed peptide-lipid hybrid vesicle and a "lipidic gate" are a promising tool for smart drug delivery.

Thiophene-Conjugated Ligand Probe for Nonenzymatic Turn-On Electrochemical Protein Detection.

A new type of turn-on electrochemical protein detection is developed using an electropolymerizable molecular probe. To detect trypsin, a benzamidine ligand is conjugated with a thiophene moiety. Encapsulation of the probe in the trypsin pocket prevents electropolymerization, leading to efficient electron transfer from the electrolyte to the electrode. In contrast, unbound probes can become electropolymerized, yielding a polythiophene layer on the electrode. The polythiophene formed this way suppressed electron transfer. The detection limit of trypsin using this electrochemical strategy is 50 nM. The method is shown to be useful for nonenzymatic turn-on electrochemical detection.

Enhanced in Vitro Mineralization and in Vivo Osteogenesis of Composite Scaffolds through Controlled Surface Grafting of L-Lactic Acid Oligomer on Nanohydroxyapatite.

Nanocomposite of hydroxyapatite (HA) surface grafted with L-lactic acid oligomer (LAc oligomer) (op-HA) showed improved interface compatibility, mechanical property, and biocompatibility in our previous study. In this paper, composite scaffolds of op-HA with controlled grafting different amounts of LAc oligomer (1.1, 5.2, and 9.1 wt %) were fabricated and implanted to repair rabbit radius defects. The dispersion of op-HA nanoparticles was more uniform than n-HA in chloroform and nanocomposites scaffold. Calcium and phosphorus exposure, in vitro biomineralization ability, and cell proliferation were much higher in the op-HA1.1 wt %/PLGA scaffolds than the other groups. The osteodifferentiation and bone fusion in animal tests were significantly enhanced for op-HA5.2 wt %/PLGA scaffolds. The results indicated that the grafted LAc oligomer of 5.2 or 9.1 wt %, which formed a barrier layer on the HA surface, prevented the exposure of nucleation sites. The shielded nucleation sites of op-HA particles (5.2 wt %) might be easily exposed as the grafted LAc oligomer was decomposed easily by enzyme systems in vivo. Findings from this study have revealed that grafting 1.1 wt % amount of LAc oligomer on hydroxyapatite could improve in vitro mineralization, and 5.2 wt % could promote in vivo osteogenesis capacity of composite scaffolds.

Courses of change in knee adduction moment and lateral thrust differ up to 1 year after TKA.

PURPOSE: In total knee arthroplasty (TKA), dynamic knee loading may loosen the artificial joint and bone or cause polyethylene wear after prolonged use. TKA decreases knee adduction moment at 6 months, but this effect is lost by 1 year post-operatively. However, lateral thrust after TKA has not been clarified. We hypothesized that like knee adduction moment, lateral thrust would return to baseline levels by 1 year post-operatively. METHODS: Participants were 15 patients who underwent TKA for medial knee OA. Japanese Orthopaedic Association (JOA) score, numeric rating scale, and gait analysis (measurement of peak knee adduction moment, knee varus angle at peak knee adduction moment, lateral thrust, and gait speed) were performed preoperatively (baseline) and 3 weeks, 3 and 6 months, and 1 year post-operatively. RESULTS: JOA score improved from 55 ± 9.8 to 78 ± 12.1 at 1 year post-operatively, and pain decreased significantly from baseline at each follow-up (p < 0.001). Significant increases in gait speed were observed at 6 months and 1 year (p < 0.001). Peak knee adduction moment during stance phase was significantly lower at 3 weeks, 3 months, and 6 months compared to baseline (p < 0.05), but no significant changes were seen at 1 year. Knee varus at peak knee adduction moment did not differ significantly between any measurement points, while lateral thrust was decreased at 6 months and 1 year compared to baseline (p < 0.05). CONCLUSIONS: Temporal courses of changes up to 1 year after TKA differed between knee adduction moment and lateral thrust, so our hypothesis was rejected. LEVEL OF EVIDENCE: IV.

A Bioorthogonal Approach for the Preparation of a Titanium-Binding Insulin-like Growth-Factor-1 Derivative by Using Tyrosinase.

The generation of metal surfaces with biological properties, such as cell-growth-enhancing and differentiation-inducing abilities, could be potentially exciting for the development of functional materials for use in humans, including artificial dental implants and joint replacements. However, currently the immobilization of proteins on the surfaces of the metals are limited. In this study, we have used a mussel-inspired bioorthogonal approach to design a 3,4-hydroxyphenalyalanine-containing recombinant insulin-like growth-factor-1 using a combination of recombinant DNA technology and tyrosinase treatment for the surface modification of titanium. The modified growth factor prepared in this study exhibited strong binding affinity to titanium, and significantly enhanced the growth of NIH3T3 cells on the surface of titanium.

Polydopamine-modified konjac glucomannan scaffold with sustained release of vascular endothelial growth factor to promote angiogenesis.

The fabrication of scaffolds capable of the sustained release of the vascular endothelial growth factor (VEGF) to promote angiogenesis for a long time remains a challenge in tissue engineering. Here, we report a facile approach for effectively fabricating a bioactive scaffold that gradually releases VEGF to promote angiogenesis. The scaffold was fabricated by coating polydopamine (PDA) on a konjac glucomannan (KGM) scaffold, followed by the surface immobilization of VEGF with PDA. The resulting VEGF-PDA/KGM scaffold, with a porous and interconnected microstructure (392 µm pore size with 84.80 porosity), combined the features of long-term biodegradability (10 weeks with 51 % degradation rate), excellent biocompatibility, and sustained VEGF release for up to 21 days. The bioactive VEGF-PDA/KGM scaffold exhibited multiple angiogenic activities over time, as confirmed by in vivo and in vitro experiments. For example, the scaffold significantly promoted the attachment and proliferation of human umbilical vein endothelial cells and the formation of vascular tubes in vitro. Moreover, the in vivo results demonstrated the formation and maturation of blood vessels after subcutaneous implantation in rats for four weeks. This promising strategy is a feasible approach for producing bioactive materials that can induce angiogenesis in vivo. These findings provide a new avenue for designing and fabricating biocompatible and long-term biodegradable scaffolds for sustained VEGF release to facilitate angiogenesis.

An osteoinductive surface by adhesive bone morphogenetic protein-2 prepared using the bioorthogonal approach for tight binding of titanium with bone.

Inorganic biomaterials are used in various orthopedic and dental implants. Nevertheless, they cause clinical issues such as loosening of implants and patient morbidity. Therefore, inspired by mussel adhesive proteins, we aimed to design an adhesive and dimer-forming highly active bone morphogenetic protein-2 (BMP-2) using bioorthogonal chemistry, in which recombinant DNA technology was combined with enzymatic modifications, to achieve long-term osseointegration with titanium. The prepared BMP-2 exhibited substantially higher binding activity than wild-type BMP-2, while the adhered BMP-2 was more active than soluble BMP-2. Therefore, the adhesive BMP-2 was immobilized onto titanium wires and screws and implanted into rat bones, and long-term osteogenesis was evaluated. Adhesive BMP-2 promoted the mechanical binding of titanium to bones, enabling efficient bone regeneration and effective stabilization of implants. Thus, such adhesive biosignaling proteins can be used in regenerative medicine.

Mussel inspired sequential protein delivery based on self-healing injectable nanocomposite hydrogel.

Polysaccharide based self-healing and injectable hydrogels with reversible characteristics have widespread potential in protein drug delivery. However, it is a challenge to design the dynamic hydrogel for sequential release of protein drugs. Herein, we developed a novel mussel inspired sequential protein delivery dynamic polysaccharide hydrogel. The nanocomposite hydrogel can be fabricated through doping polydopamine nanoparticles (PDA NPs) into reversible covalent bond (imine bonds) crosslinked polymer networks of oxidized hyaluronic acid (OHA) and carboxymethyl chitosan (CEC), named PDA NPs@OHA-l-CEC. Besides multiple capabilities (i.e., injection, self-healing, and biodegradability), the nanocomposite hydrogel can achieve sustained and sequential protein delivery of vascular endothelial growth factor (VEGF) and bovine serum albumin (BSA). PDA NPs doped in hydrogel matrix serve dual roles, acting as secondary protein release structures and form dynamic non-covalent interactions (i.e., hydrogen bonds) with polysaccharides. Moreover, by adjusting the oxidation degree of OHA, the hydrogels with different crosslinking density could control overall protein release rate. Analysis of different release kinetic models revealed that Fickian diffusion drove rapid VEGF release, while the slower BSA release followed a Super Case II transport mechanism. The novel biocompatible system achieved sequential release of protein drugs has potentials in multi-stage synergistic drug deliver based on dynamic hydrogel.

Tough, self-healing and injectable dynamic nanocomposite hydrogel based on gelatin and sodium alginate.

Biomacromolecules based injectable and self-healing hydrogels possessing high mechanical properties have widespread potential in biomedical field. However, dynamic features are usually inversely proportional to toughness. It is challenging to simultaneously endow these properties to the dynamic hydrogels. Here, we fabricated an injectable nanocomposite hydrogel (CS-NPs@OSA-l-Gtn) stimultaneously possessing excellent autonomous self-healing performance and high mechanical strength by doping chitosan nanoparticles (CS-NPs) into dynamic polymer networks of oxidized sodium alginate (OSA) and gelatin (Gtn) in the presence of borax. The synergistic effect of the multiple reversible interactions combining dynamic covalent bonds (i.e., imine bond and borate ester bond) and noncovalent interactions (i.e., electrostatic interaction and hydrogen bond) provide effective energy dissipation to endure high fatigue resistance and cyclic loading. The dynamic hydrogel exhibited excellent mechanical properties like maximum 2.43 MPa compressive strength, 493.91 % fracture strain, and 89.54 kJ/m3 toughness. Moreover, the integrated hydrogel after injection and self-healing could withstand 150 successive compressive cycles. Besides, the bovine serum albumin embedded in CS-NPs could be sustainably released from the nanocomposite hydrogel for 12 days. This study proposes a novel strategy to synthesize an injectable and self-healing hydrogel combined with excellent mechanical properties for designing high-strength natural carriers with sustained protein delivery.

A novel red fluorescent and dynamic nanocomposite hydrogel based on chitosan and alginate doped with inclusion complex of carbon dots.

Red fluorescent hydrogels possessing injectable and self-healing properties have widespread potential in biomedical field. It is still a challenge to achieve a biomacromolecules based dynamic hydrogels simultaneously combining with excellent red fluorescence, good mechanical properties, and biocompatibility. Here we first explore hydrophilic inclusion complex of (R-CDs@α-CD) derived from hydrophobic red fluorescent carbon dots (R-CDs) and α-cyclodextrin (α-CD), and then achieved a red fluorescent and dynamic polysaccharide R-CDs@α-CD/CEC-l-OSA hydrogel. The nanocomposite hydrogel can be fabricated through controlled doping of red fluorescent R-CDs@α-CD into dynamic polymer networks, taking reversibly crosslinked N-carboxyethyl chitosan (CEC) and oxidized sodium alginate (OSA) as an example. The versatile red fluorescent hydrogel simultaneously combines the features of injection, biocompatibility, and augmented mechanical properties and self-healing behavior, especially in rapid self-recovery even after integration. The R-CDs@α-CD uniformly dispersed into dynamic hydrogel played the role of killing two birds with one stone, that is, endowing red emission of a hydrophilic fluorescent substance, and improving mechanical and self-healing properties as a dynamic nano-crosslinker, via forming hydrogen bonds as reversible crosslinkings. The novel red fluorescent and dynamic hydrogel based on polysaccharides is promising for using as biomaterials in biomedical field.

Polysarcosine-Coated liposomes attenuating immune response induction and prolonging blood circulation.

HYPOTHESIS: Liposomes coated with long polysarcosine (PSar) chains at a high density might enable long blood circulation and attenuate accelerated blood clearance (ABC) phenomenon. EXPERIMENTS: In this study, we controlled the length (23, 45, 68 mers) and density (5, 10, 15 mol%) of PSar on liposomal coatings and, furthermore, investigated the effects of PSar length and density on the blood circulation time, biodistribution, immune response, and ABC phenomenon induction. Length-controlled PSar-bound lipids (PSar-PEs) were synthesized using a click reaction and inserted into bare liposomes at different combinations of chain lengths and proportions. FINDINGS: Although all PSar-coated liposomes (PSar-lipos) had similar morphological, physical, and chemical properties, they had different blood circulation times and biodistribution, and exerted varied effects on the immune system. All PSar-lipos with different PSar length and density showed a similar anti-PSar IgM response. Liposomes modified with the longest PSar chain (68 mers) at a high density (15 mol%) showed the longest blood circulation time and, additionally, attenuated ABC phenomenon compared with PEG-lipo. The ex vivo analysis of the biodistribution of liposomes revealed that a thick PSar layer enhanced the blood circulation time of liposomes due to the reduction of the accumulation of liposomes in the liver and spleen. These findings provide new insights into the relationship between IgM expression and ABC phenomenon inhibition.

Photo-reactive polymers for the immobilisation of epidermal growth factors.

Photo-reactive polymers are important for biomaterials, including devices with a 3D-structure. Here, different types of photo-reactive polymers were prepared and utilised for immobilisation of growth factors. They were synthesised by conjugation of gelatin with the azidophenyl group or by copolymerisation of the azidophenyl group-coupled methacrylate with poly(ethylene glycol) methacrylate. The azidophenyl content and the zeta potential of the prepared polymers were measured. After spin coating of polymers, the thickness and the water contact angle of coated layers were measured. The amount of the immobilised epidermal growth factor (EGF) was determined using fluorescence labelling. Cell adhesion responded to the nature of photo-reactive polymers but did not depend on the immobilised EGF. However, cell growth was dependent on the amount of immobilised EGF and was significantly affected by the nature of photo-reactive polymers. The study shows that the properties of the photo-immobilisation matrix significantly influence the biological activity.

Natural polyphenol tannin-immobilized composites: rational design and versatile applications.

Tannins, which are natural plant polyphenols, are widely used in different fields, especially in biomedical applications due to their unique properties, including high abundance, low cost, structural diversity, protein precipitation, biocompatibility, and biodegradability. However, they fail to satisfy the requirements in some specific applications (e.g., environmental remediation) on account of their water solubility, making their separation and regeneration difficult. Inspired by the design of composite materials, tannin-immobilized composites have emerged as promising and novel materials and combine or even surpass the advantages of each of their components. This strategy can endow tannin-immobilized composites with efficient manufacturing properties, high strength, good stability, easy chelating/coordinating ability, excellent antibacterial property, biological compatibility, bioactivity, chemical/corrosion resistance, and strong adhesive performance, which significantly expand their application in various fields. In this review, initially we summarize the design strategy of tannin-immobilized composites, mainly concentrating on the choice of immobilized substrate (e.g., natural polymers, synthetic polymers, and inorganic materials) as well as the binding interaction (e.g., Mannich reaction, Schiff base reaction, graft copolymerization, oxidation coupling, electrostatic interaction, and hydrogen bonding) between them. Further, the application of tannin-immobilized composites in the biomedical (tissue engineering, wound healing, cancer therapy, and biosensors) and other (leather materials, environmental remediation, and functional food packaging) fields is highlighted. Finally, we conclude with some thoughts on the open challenges and future perspectives of tannin composites. It can be anticipated that tannin-immobilized composites will continuously draw attention from more and more researchers, and further promising applications of tannin composites will be explored.

Elongated polyproline motifs facilitate enamel evolution through matrix subunit compaction.

Vertebrate body designs rely on hydroxyapatite as the principal mineral component of relatively light-weight, articulated endoskeletons and sophisticated tooth-bearing jaws, facilitating rapid movement and efficient predation. Biological mineralization and skeletal growth are frequently accomplished through proteins containing polyproline repeat elements. Through their well-defined yet mobile and flexible structure polyproline-rich proteins control mineral shape and contribute many other biological functions including Alzheimer's amyloid aggregation and prolamine plant storage. In the present study we have hypothesized that polyproline repeat proteins exert their control over biological events such as mineral growth, plaque aggregation, or viscous adhesion by altering the length of their central repeat domain, resulting in dramatic changes in supramolecular assembly dimensions. In order to test our hypothesis, we have used the vertebrate mineralization protein amelogenin as an exemplar and determined the biological effect of the four-fold increased polyproline tandem repeat length in the amphibian/mammalian transition. To study the effect of polyproline repeat length on matrix assembly, protein structure, and apatite crystal growth, we have measured supramolecular assembly dimensions in various vertebrates using atomic force microscopy, tested the effect of protein assemblies on crystal growth by electron microscopy, generated a transgenic mouse model to examine the effect of an abbreviated polyproline sequence on crystal growth, and determined the structure of polyproline repeat elements using 3D NMR. Our study shows that an increase in PXX/PXQ tandem repeat motif length results (i) in a compaction of protein matrix subunit dimensions, (ii) reduced conformational variability, (iii) an increase in polyproline II helices, and (iv) promotion of apatite crystal length. Together, these findings establish a direct relationship between polyproline tandem repeat fragment assemblies and the evolution and the design of vertebrate mineralized tissue microstructures. Our findings reveal that in the greater context of chordate evolution, the biological control of apatite growth by polyproline-based matrix assemblies provides a molecular basis for the evolution of the vertebrate body plan.

Design and synthesis of binding growth factors.

Growth factors play important roles in tissue regeneration. However, because of their instability and diffusible nature, improvements in their performance would be desirable for therapeutic applications. Conferring binding affinities would be one way to improve their applicability. Here we review techniques for conjugating growth factors to polypeptides with particular affinities. Conjugation has been designed at the level of gene fusion and of polypeptide ligation. We summarize and discuss the designs and applications of binding growth factors prepared by such conjugation approaches.

Structure formation and catalytic activity of DNA dissolved in organic solvents.

Equal-opportunity dissolver: By attaching polyethylene glycol at its 5' end, DNA (PEG-DNA) can be solubilized in various organic solvents and was shown to form G-quadruplexes by CD spectroscopy. A complex containing iron(III) protoporphyrin IX (hemin) and G-quadruplex-forming PEG-DNA catalyzed an oxidative reaction in methanol (see scheme).