A morphological analysis of calcium hydroxylapatite and poly-l-lactic acid biostimulator particles.

Injectable fillers, pivotal in aesthetic medicine, have evolved significantly with recent trends favoring biostimulators like calcium hydroxylapatite (CaHA-CMC; Radiesse, Merz Aesthetics, Raleigh, NC) and poly-l-lactic acid (PLLA; Sculptra Aesthetics, Galderma, Dallas, TX). This study aims to compare the particle morphology of these two injectables and examine its potential clinical implications. Utilizing advanced light and scanning electron microscopy techniques, the physical characteristics of CaHA-CMC and PLLA particles were analyzed, including shape, size, circularity, roundness, aspect ratio, and quantity of phagocytosable particles. The findings reveal several morphological contrasts: CaHA-CMC particles exhibited a smooth, homogenous, spherical morphology with diameters predominantly ranging between 20 and 45 µm, while PLLA particles varied considerably in shape and size, appearing as micro flakes ranging from 2 to 150 µm in major axis length. The circularity and roundness of CaHA-CMC particles were significantly higher compared to PLLA, indicating a more uniform shape. Aspect ratio analysis further underscored these differences, with CaHA-CMC particles showing a closer resemblance to circles, unlike the more oblong PLLA particles. Quantification of the phagocytosable content of both injectables revealed a higher percentage of phagocytosable particles in PLLA. These morphological distinctions may influence the tissue response to each treatment. CaHA-CMC's uniform, spherical particles may result in reduced inflammatory cell recruitment, whereas PLLA's heterogeneous particle morphology may evoke a more pronounced inflammatory response.

Engineering Biomimetic Nanofiber Microspheres with Tailored Size, Predesigned Structure, and Desired Composition via Gas Bubble-Mediated Coaxial Electrospray.

Minimally invasive therapies avoiding surgical complexities evoke great interest in developing injectable biomedical devices. Herein, a versatile approach is reported for engineering injectable and biomimetic nanofiber microspheres (NMs) with tunable sizes, predesigned structures, and desired compositions via gas bubble-mediated coaxial electrospraying. The sizes and structures of NMs are controlled by adjusting processing parameters including air flow rate, applied voltage, distance, and spinneret configuration in the coaxial setup. Importantly, unlike the self-assembly method, this technique can be used to fabricate NMs from any material feasible for electrospinning or other nanofiber fabrication techniques. To demonstrate the versatility, open porous NMs are successfully fabricated that consist of various short nanofibers made of poly(ε-caprolactone), poly(lactic-co-glycolic acid), gelatin, methacrylated gelatin, bioglass, and magneto-responsive polymer composites. Open porous NMs support human neural progenitor cell growth in 3D with a larger number and more neurites than nonporous NMs. Additionally, highly open porous NMs show faster cell infiltration and host tissue integration than nonporous NMs after subcutaneous injection to rats. Such a novel class of NMs holds great potential for many biomedical applications such as tissue filling, cell and drug delivery, and minimally invasive tissue regeneration.

Three-Dimensional Objects Consisting of Hierarchically Assembled Nanofibers with Controlled Alignments for Regenerative Medicine.

Assembling electrospun nanofibers with controlled alignment into three-dimensional (3D), complex, and predesigned shapes has proven to be a difficult task for regenerative medicine. Herein, we report a novel approach inspired by solids of revolution that transforms two-dimensional (2D) nanofiber mats of a controlled thickness into once-inaccessible 3D objects with predesigned shapes. The 3D objects are highly porous, consisting of layers of aligned nanofibers separated by gaps ranging from several micrometers to several millimeters. Upon compression, the objects are able to recover their original shapes. The porous objects can serve as scaffolds, guiding the organization of cells and producing highly ordered 3D tissue constructs. Additionally, subcutaneous implantation in rats demonstrates that the 3D objects enable rapid cell penetration, new blood vessel formation, and collagen matrix deposition. This new class of 3D hierarchical nanofiber architectures offers promising advancements in both in vitro engineering of complex 3D tissue constructs/models or organs and in vivo tissue repair and regeneration.

Nanofiber Dressings Topically Delivering Molecularly Engineered Human Cathelicidin Peptides for the Treatment of Biofilms in Chronic Wounds.

Biofilms of multidrug-resistant bacteria in chronic wounds pose a great challenge in wound care. Herein, we report the topical delivery of molecularly engineered antimicrobial peptides using electrospun nanofiber dressings as a carrier for the treatment of biofilms of multidrug-resistant bacteria in diabetic wounds. Molecularly engineered human cathelicidin peptide 17BIPHE2 was successfully encapsulated in the core of pluronic F127/17BIPHE2-PCL core-shell nanofibers. The in vitro release profiles of 17BIPHE2 showed an in initial burst followed by a sustained release over 4 weeks. The peptide nanofiber formulations effectively killed methicillin-resistant Staphylococcus aureus (MRSA) USA300. Similarly, the 17BIPHE2 peptide containing nanofibers could also effectively kill other bacteria including Klebsiella pneumoniae (104 to 106 CFU) and Acinetobacter baumannii (104 to 107 CFU) clinical strains in vitro without showing evident cytotoxicity to skin cells and monocytes. Importantly, 17BIPHE2-containing nanofiber dressings without debridement caused five-magnitude decreases of the MRSA USA300 CFU in a biofilm-containing chronic wound model based on type II diabetic mice. In combination with debridement, 17BIPHE2-containing nanofiber dressings could completely eliminate the biofilms, providing one possible solution to chronic wound treatment. Taken together, the biodegradable nanofiber-based wound dressings developed in this study can be utilized to effectively deliver molecularly engineered peptides to treat biofilm-containing chronic wounds.

Tethering peptides onto biomimetic and injectable nanofiber microspheres to direct cellular response.

Biomimetic and injectable nanofiber microspheres (NMs) could be ideal candidate for minimally invasive tissue repair. Herein, we report a facile approach to fabricate peptide-tethered NMs by combining electrospinning, electrospraying, and surface conjugation techniques. The composition and size of NMs can be tuned by varying the processing parameters during the fabrication. Further, bone morphogenic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) mimicking peptides have been successfully tethered onto poly(ε-caprolactone) (PCL):gelatin:(gelatin-methacryloyl) (GelMA)(1:0.5:0.5) NMs through photocrosslinking of the methacrylic group in GelMA and octenyl alanine (OCTAL) in the modified peptides. The BMP-2-OCTAL peptide-tethered NMs significantly promote osteogenic differentiation of bone marrow-derived stem cells (BMSCs). Moreover, human umbilical vein endothelial cells (HUVECs) seeded on VEGF mimicking peptide QK-OCTAL-tethered NMs significantly up-regulated vascular-specific proteins, leading to microvascularization. The strategy developed in this work holds great potential in developing a biomimetic and injectable carrier to efficiently direct cellular response (Osteogenesis and Angiogenesis) for tissue repair.

Twisting electrospun nanofiber fine strips into functional sutures for sustained co-delivery of gentamicin and silver.

Surgical site infections (SSIs) represent the most common nosocomial infection among surgical patients. In order to prevent SSIs in a sustained manner and lessen side effects, we developed a twisting method for generation of nanofiber-based sutures capable of simultaneous delivery of silver and gentamicin. The prepared sutures are composed of core-sheath nanofibers with gentamicin/pluronic F127 in the core and silver/PCL in the sheath produced by co-axial electrospinning. The diameters of obtained sutures range from ~80 µm to ~1.2 mm. The in vitro release profiles of silver and gentamicin exhibit an initial burst followed by a sustained release over 5 weeks. The co-encapsulated sutures were able to kill bacteria much more effectively than gentamicin or silver alone loaded nanofiber sutures, without showing obvious impact on proliferation and migration of dermal fibroblasts and keratinocytes. The gentamicin and silver co-loaded PCL nanofiber sutures may hold great potential for prevention of SSIs.

Local Sustained Delivery of 25-Hydroxyvitamin D3 for Production of Antimicrobial Peptides.

PURPOSE: This study seeks to develop fiber membranes for local sustained delivery of 25-hydroxyvitamin D3 to induce the expression and secretion of LL-37 at or near the surgical site, which provides a novel therapeutic approach to minimize the risk of infections. METHODS: 25-hydroxyvitamin D3 loaded poly(L-lactide) (PLA) and poly(ε-caprolactone) (PCL) fibers were produced by electrospinning. The morphology of obtained fibers was characterized using atomic force microscope (AFM) and scanning electron microscope (SEM). 25-hydroxyvitamin D3 releasing kinetics were quantified by enzyme-linked immunosorbent assay (ELISA) kit. The expression of cathelicidin (hCAP 18) and LL-37 was analyzed by immunofluorescence staining and ELISA kit. The antibacterial activity test was conducted by incubating pseudomonas aeruginosa in a monocytes' lysis solution. RESULTS: AFM images suggest that the surface of PCL fibers is smooth, however, the surface of PLA fibers is relatively rough, in particular, after encapsulation of 25-hydroxyvitamin D3. The duration of 25-hydroxyvitamin D3 release can last more than 4 weeks for all the tested samples. Plasma treatment can promote the release rate of 25-hydroxyvitamin D3. Human keratinocytes and monocytes express significantly higher levels of hCAP18/LL-37 after incubation with plasma treated and 25-hydroxyvitamin D3 loaded PCL fibers than the cells incubated with around ten times amount of free drug. After incubation with this fiber formulation for 5 days LL-37 in the lysis solutions of U937 cells can effectively kill the bacteria. CONCLUSIONS: Plasma treated and 25-hydroxyvitamin D3 loaded PCL fibers induce significantly higher levels of antimicrobial peptide production in human keratinocytes and monocytes without producing cytotoxicity.

Nitric Oxide: Physiological Functions, Delivery, and Biomedical Applications.

Nitric oxide (NO) is a gaseous molecule that has a central role in signaling pathways involved in numerous physiological processes (e.g., vasodilation, neurotransmission, inflammation, apoptosis, and tumor growth). Due to its gaseous form, NO has a short half-life, and its physiology role is concentration dependent, often restricting its function to a target site. Providing NO from an external source is beneficial in promoting cellular functions and treatment of different pathological conditions. Hence, the multifaceted role of NO in physiology and pathology has garnered massive interest in developing strategies to deliver exogenous NO for the treatment of various regenerative and biomedical complexities. NO-releasing platforms or donors capable of delivering NO in a controlled and sustained manner to target tissues or organs have advanced in the past few decades. This review article discusses in detail the generation of NO via the enzymatic functions of NO synthase as well as from NO donors and the multiple biological and pathological processes that NO modulates. The methods for incorporating of NO donors into diverse biomaterials including physical, chemical, or supramolecular techniques are summarized. Then, these NO-releasing platforms are highlighted in terms of advancing treatment strategies for various medical problems.

Extracellular Matrix Secretion Mechanically Reinforces Interlocking Interfaces.

Drawing inspiration for biomaterials from biological systems has led to many biomedical innovations. One notable bioinspired device, Velcro, consists of two substrates with interlocking ability. Generating reversibly interlocking biomaterials is an area of investigation, as such devices can allow for modular tissue engineering, reversibly interlocking biomaterial interfaces, or friction-based coupling devices. Here, a biaxially interlocking interface generated using electrostatic flocking is reported. Two electrostatically flocked substrates are mechanically and reversibly interlocked with the ability to resist shearing and compression forces. An initial high-throughput screen of polyamide flock fibers with varying diameters and fiber lengths is conducted to elucidate the roles of different fiber parameters on scaffold mechanical properties. After determining the most desirable parameters via weight scoring, polylactic acid (PLA) fibers are used to emulate the ideal scaffold for in vitro use. PLA flocked scaffolds are populated with osteoblasts and interlocked. Interlocked flocked scaffolds improved cell survivorship under mechanical compression and sustained cell viability and proliferation. Additionally, the compression and shearing resistance of cell-seeded interlocking interfaces increased with increasing extracellular matrix deposition. The introduction of extracellular matrix-reinforced interlocking interfaces may serve as binders for modular tissue engineering, act as scaffolds for engineering tissue interfaces, or enable friction-based couplers for biomedical applications.

Engineered Exosomes Containing Cathelicidin/LL-37 Exhibit Multiple Biological Functions.

Exosomes show great potential in diagnostic and therapeutic applications. Inspired by the human innate immune defense, herein, we report engineered exosomes derived from monocytic cells treated with immunomodulating compounds 1α,25-dihydroxyvitamin D3, and CYP24A1 inhibitor VID400 which are slowly released from electrospun nanofiber matrices. These engineered exosomes contain significantly more cathelicidin/LL-37 when compared with exosomes derived from either untreated cells or Cathelicidin Human Tagged ORF Clone transfected cells. In addition, such exosomes exhibit multiple biological functions evidenced by killing bacteria, facilitating human umbilical vein endothelial cell tube formation, and enhancing skin cell proliferation and migration. Taken together, the engineered exosomes developed in this study can be used as therapeutics alone or in combination with other biomaterials for effective infection management, wound healing, and tissue regeneration.

Bioinspired elastomer composites with programmed mechanical and electrical anisotropies.

Concepts that draw inspiration from soft biological tissues have enabled significant advances in creating artificial materials for a range of applications, such as dry adhesives, tissue engineering, biointegrated electronics, artificial muscles, and soft robots. Many biological tissues, represented by muscles, exhibit directionally dependent mechanical and electrical properties. However, equipping synthetic materials with tissue-like mechanical and electrical anisotropies remains challenging. Here, we present the bioinspired concepts, design principles, numerical modeling, and experimental demonstrations of soft elastomer composites with programmed mechanical and electrical anisotropies, as well as their integrations with active functionalities. Mechanically assembled, 3D structures of polyimide serve as skeletons to offer anisotropic, nonlinear mechanical properties, and crumpled conductive surfaces provide anisotropic electrical properties, which can be used to construct bioelectronic devices. Finite element analyses quantitatively capture the key aspects that govern mechanical anisotropies of elastomer composites, providing a powerful design tool. Incorporation of 3D skeletons of thermally responsive polycaprolactone into elastomer composites allows development of an active artificial material that can mimic adaptive mechanical behaviors of skeleton muscles at relaxation and contraction states. Furthermore, the fabrication process of anisotropic elastomer composites is compatible with dielectric elastomer actuators, indicating potential applications in humanoid artificial muscles and soft robots.

Enhancing the stiffness of electrospun nanofiber scaffolds with a controlled surface coating and mineralization.

A new method was developed to coat hydroxyapatite (HAp) onto electrospun poly(lactic-co-glycolic acid) (PLGA) nanofibers for tendon-to-bone insertion site repair applications. Prior to mineralization, chitosan and heparin were covalently immobilized onto the surface of the fibers to accelerate the nucleation of bone-like HAp crystals. Uniform coatings of HAp were obtained by immersing the nanofiber scaffolds into a modified, 10-fold-concentrated simulated body fluid (m10SBF) for different periods of time. The new method resulted in thicker and denser coatings of mineral on the fibers compared to those produced by previously reported methods. Scanning electron microscopy measurements confirmed the formation of nanoscale HAp particles on the fibers. A mechanical property assessment demonstrated a higher stiffness with respect to previous coating methods. A combination of the nanoscale fibrous structure and bonelike mineral coating could mimic the structure, composition, and function of mineralized tissues.

Freeze-Casting with 3D-Printed Templates Creates Anisotropic Microchannels and Patterned Macrochannels within Biomimetic Nanofiber Aerogels for Rapid Cellular Infiltration.

A new approach is described for fabricating 3D poly(ε-caprolactone) (PCL)/gelatin (1:1) nanofiber aerogels with patterned macrochannels and anisotropic microchannels by freeze-casting with 3D-printed sacrificial templates. Single layer or multiple layers of macrochannels are formed through an inverse replica of 3D-printed templates. Aligned microchannels formed by partially anisotropic freezing act as interconnected pores between templated macrochannels. The resulting macro-/microchannels within nanofiber aerogels significantly increase preosteoblast infiltration in vitro. The conjugation of vascular endothelial growth factor (VEGF)-mimicking QK peptide to PCL/gelatin/gelatin methacryloyl (1:0.5:0.5) nanofiber aerogels with patterned macrochannels promotes the formation of a microvascular network of seeded human microvascular endothelial cells. Moreover, nanofiber aerogels with patterned macrochannels and anisotropic microchannels show significantly enhanced cellular infiltration rates and host tissue integration compared to aerogels without macrochannels following subcutaneous implantation in rats. Taken together, this novel class of nanofiber aerogels holds great potential in biomedical applications including tissue repair and regeneration, wound healing, and 3D tissue/disease modeling.

Tannic acid-inspired, self-healing, and dual stimuli responsive dynamic hydrogel with potent antibacterial and anti-oxidative properties.

Due to their intrinsic injectable and self-healing characteristics, dynamic hydrogels, based on dynamic covalent bonds, have gained a great attention. In this study, a novel dynamic hydrogel based on the boronic ester dynamic covalent bond is facilely developed using phenylboronic acid-modified hyaluronic acid (HA-PBA) and plant-derived polyphenol-tannic acid (TA). The dynamic hydrogel gelated quickly under mild conditions and had favorable viscoelastic properties with good self-healing and shear-thinning capabilities. Moreover, the simultaneous utilization of TA as a reductant for the green synthesis of silver nanoparticles (AgNP) inspired the preparation of a TA-reduced AgNP hybrid dynamic hydrogel with potent and broad-spectrum antibacterial activities. The dynamic hydrogels could also be applied for pH- and reactive oxygen species (ROS)-responsive release of loaded protein molecules without showing evident cytotoxicity and hemolysis in vitro. In addition, the dynamic hydrogels showed the anti-oxidative properties of high free radical and ROS scavenging capacity, which was verified by the DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical assay and ROS fluorescence staining. Overall, this novel class of cytocompatible, self-healing, dual stimuli responsive, antibacterial, anti-oxidative, and injectable hydrogels could be promising as a wound dressing for chronic wound healing.

Electrostatic Flocking of Insulative and Biodegradable Polymer Microfibers for Biomedical Applications.

Electrostatic flocking, a textile engineering technique, uses Coulombic driving forces to propel conductive microfibers toward an adhesive-coated substrate, leaving a forest of aligned fibers. Though an easy way to induce anisotropy along a surface, this technique is limited to microfibers capable of accumulating charge. This study reports a novel method, utilizing principles from the percolation theory to make electrically insulative polymeric microfibers flockable. A variety of well-mixed, conductive materials are added to multiple insulative and biodegradable polymer microfibers during wet spinning, which enables nearly all types of polymer microfibers to accumulate sufficient charges required for flocking. Biphasic, biodegradable scaffolds are fabricated by flocking silver nanoparticle (AgNP)-filled poly(ε-caprolactone) (PCL) microfibers onto substrates made from 3D printing, electrospinning, and thin-film casting. The incorporation of AgNP into PCL fibers and use of chitosan-based adhesive enables antimicrobial activity against methicillin-resistant Staphylococcus aureus. The fabricated scaffolds demonstrate both favorable in vitro cell response and new tissue formation after subcutaneous implantation in rats, as evident by newly formed blood vessels and infiltrated cells. This technology opens the door for using previously unflockable polymer microfibers as surface modifiers or standalone structures in various engineering fields.

Gold nanocages covered by smart polymers for controlled release with near-infrared light.

Photosensitive caged compounds have enhanced our ability to address the complexity of biological systems by generating effectors with remarkable spatial/temporal resolutions. The caging effect is typically removed by photolysis with ultraviolet light to liberate the bioactive species. Although this technique has been successfully applied to many biological problems, it suffers from a number of intrinsic drawbacks. For example, it requires dedicated efforts to design and synthesize a precursor compound for each effector. The ultraviolet light may cause damage to biological samples and is suitable only for in vitro studies because of its quick attenuation in tissue. Here we address these issues by developing a platform based on the photothermal effect of gold nanocages. Gold nanocages represent a class of nanostructures with hollow interiors and porous walls. They can have strong absorption (for the photothermal effect) in the near-infrared while maintaining a compact size. When the surface of a gold nanocage is covered with a smart polymer, the pre-loaded effector can be released in a controllable fashion using a near-infrared laser. This system works well with various effectors without involving sophisticated syntheses, and is well suited for in vivo studies owing to the high transparency of soft tissue in the near-infrared region.

New forms of electrospun nanofiber materials for biomedical applications.

Over the past two decades, electrospinning has emerged as an enabling nanotechnology to produce nanofiber materials for various biomedical applications. In particular, therapeutic/cellloaded nanofiber scaffolds have been widely examined in drug delivery, wound healing, and tissue repair and regeneration. However, due to the insufficient porosity, small pore size, noninjectability, and inaccurate spatial control in nanofibers of scaffolds, many efforts have been devoted to exploring new forms of nanofiber materials including expanded nanofiber scaffolds, nanofiber aerogels, short nanofibers, and nanofiber microspheres. This short review discusses the preparation and potential biomedical applications of new forms of nanofiber materials.

Converting 2D Nanofiber Membranes to 3D Hierarchical Assemblies with Structural and Compositional Gradients Regulates Cell Behavior.

New methods are described for converting 2D electrospun nanofiber membranes to 3D hierarchical assemblies with structural and compositional gradients. Pore-size gradients are generated by tuning the expansion of 2D membranes in different layers with incorporation of various amounts of a surfactant during the gas-foaming process. The gradient in fiber organizations is formed by expanding 2D nanofiber membranes composed of multiple regions collected by varying rotating speeds of mandrel. A compositional gradient on 3D assemblies consisting of radially aligned nanofibers is prepared by dripping, diffusion, and crosslinking. Bone mesenchymal stem cells (BMSCs) on the 3D nanofiber assemblies with smaller pore size show significantly higher expression of hypoxia-related markers and enhanced chondrogenic differentiation compared to BMSCs cultured on the assemblies with larger pore size. The basic fibroblast growth factor gradient can accelerate fibroblast migration from the surrounding area to the center in an in vitro wound healing model. Taken together, 3D nanofiber assemblies with gradients in pore sizes, fiber organizations, and contents of signaling molecules can be used to engineer tissue constructs for tissue repair and build biomimetic disease models for studying disease biology and screening drugs, in particular, for interface tissue engineering and modeling.

Dual Delivery of Alendronate and E7-BMP-2 Peptide via Calcium Chelation to Mineralized Nanofiber Fragments for Alveolar Bone Regeneration.

The fixation and stability of dental implants is governed by the quality of the underlying alveolar bone. The current study investigates if the dual delivery of calcium chelating bone therapeutics from mineralized nanofiber fragments can help regenerate alveolar bone in vivo. Alendronate (ALN) or/and bone morphogenetic protein-2-mimicking peptide conjugated to a heptaglutamate moiety (E7-BMP-2) were incorporated onto mineralized nanofiber fragments of polylactide-co-glycolide-collagen-gelatin (PCG in 2:1:1 weight ratios) via calcium coupling/chelation. Two mg of the single-loaded (ALN) and coloaded (ALN + E7-BMP-2) mineralized nanofiber PCG grafts was filled into critical-sized (2 mm diameter × 2 mm depth) alveolar bone defects in rat maxillae and let heal for 4 weeks. X-ray microcomputed tomography analysis of the retrieved maxillae revealed significantly elevated new bone formation parameters for the ALN and ALN + E7-BMP-2 groups compared with the unfilled defect controls. However, no significant differences between the single and coloaded nanofiber grafts were noted. Furthermore, the histopathological analysis of the tissue sections divulged islands of new bone tissue in the ALN and ALN + E7-BMP-2 groups, whereas the control defect was covered with gingival tissue. Together, the presented strategy using mineralized nanofiber fragments in the sustained delivery of dual calcium chelating therapeutics could have potential applications in enhancing bone regeneration.

Complexation-induced resolution enhancement of 3D-printed hydrogel constructs.

Three-dimensional (3D) hydrogel printing enables production of volumetric architectures containing desired structures using programmed automation processes. Our study reports a unique method of resolution enhancement purely relying on post-printing treatment of hydrogel constructs. By immersing a 3D-printed patterned hydrogel consisting of a hydrophilic polyionic polymer network in a solution of polyions of the opposite net charge, shrinking can rapidly occur resulting in various degrees of reduced dimensions comparing to the original pattern. This phenomenon, caused by complex coacervation and water expulsion, enables us to reduce linear dimensions of printed constructs while maintaining cytocompatible conditions in a cell type-dependent manner. We anticipate our shrinking printing technology to find widespread applications in promoting the current 3D printing capacities for generating higher-resolution hydrogel-based structures without necessarily having to involve complex hardware upgrades or other printing parameter alterations.