Mechanical properties and in vitro behavior of nanofiber-hydrogel composites for tissue engineering applications

Hydrogel-based biomaterial systems have great potential for tissue reconstruction by serving as temporary scaffolds and cell delivery vehicles for tissue engineering (TE). Hydrogels have poor mechanical properties and their rapid degradation limits the development and application of hydrogels in TE. In this study, nanofiber reinforced composite hydrogels were fabricated by incorporating electrospun poly(ε-caprolactone) (PCL)/gelatin 'blend' or 'coaxial' nanofibers into gelatin hydrogels. The morphological, mechanical, swelling and biodegradation properties of the nanocomposite hydrogels were evaluated and the results indicated that the moduli and compressive strengths of the nanofiber reinforced hydrogels were remarkably higher than those of pure gelatin hydrogels. By increasing the amount of incorporated nanofibers into the hydrogel, the Young's modulus of the composite hydrogels increased from 3.29 ± 1.02 kPa to 20.30 ± 1.79 kPa, while the strain at break decreased from 66.0 ± 1.1% to 52.0 ± 3.0%. Compared to composite hydrogels with coaxial nanofibers, those with blend nanofibers showed higher compressive strength and strain at break, but with lower modulus and energy dissipation properties. Biocompatibility evaluations of the nanofiber reinforced hydrogels were carried out using bone marrow mesenchymal stem cells (BM-MSCs) by cell proliferation assay and immunostaining analysis. The nanocomposite hydrogel with 25 mg ml(-1) PCL/gelatin 'blend' nanofibers (PGB25) was found to enhance cell proliferation, indicating that the 'nanocomposite hydrogels' might provide the necessary mechanical support and could be promising cell delivery systems for tissue regeneration.     

In vitro evaluation of electrospun gelatin–glutaraldehyde nanofibers

The gelatin–glutaraldehyde (gelatin–GA) nanofibers were electrospun in order to overcome the defects of ex-situ crosslinking process such as complex process, destruction of fiber morphology and decrease of porosity. The morphological structure, porosity, thermal property, moisture absorption and moisture retention performance, hydrolytic resistance, mechanical property and biocompatibility of nanofiber scaffolds were tested and characterized. The gelatin–GA nanofiber has nice uniform diameter and more than 80% porosity. The hydrolytic resistance and mechanical property of the gelatin–GA nanofiber scaffolds are greatly improved compared with that of gelatin nanofibers. The contact angle, moisture absorption, hydrolysis resistance, thermal resistance and mechanical property of gelatin–GA nanofiber scaffolds could be adjustable by varying the gelatin solution concentration and GA content. The gelatin–GA nanofibers had excellent properties, which are expected to be an ideal scaffold for biomedical and tissue engineering applications.     

Design of porous polymeric scaffolds by gas foaming of heterogeneous blends

One of the challenges in tissue engineering scaffold design is the realization of structures with a pre-defined multi-scaled porous network. Along this line, this study aimed at the design of porous scaffolds with controlled porosity and pore size distribution from blends of poly(ε-caprolactone) (PCL) and thermoplastic gelatin (TG), a thermoplastic natural material obtained by de novo thermoplasticization of gelatin. PCL/TG blends with composition in the range from 40/60 to 60/40 (w/w) were prepared by melt mixing process. The multi-phase microstructures of these blends were analyzed by scanning electron microscopy and dynamic mechanical analysis. Furthermore, in order to prepare open porous scaffolds for cell culture and tissue replacement, the TG and PCL were selectively extracted from the blends by the appropriate combination of solvent and extraction parameters. Finally, with the proposed combination of gas foaming and selective polymer extraction technologies, PCL and TG porous materials with multi-scaled and highly interconnected porosities were designed as novel scaffolds for new-tissue regeneration.     

Graphene Oxide/Polymer‐Based Biomaterials

Since its discovery in 2004, derivatives of graphene have been developed and heavily investigated in the field of tissue engineering. Among the most extensively studied forms of graphene, graphene oxide (GO), and GO/polymer‐based nanocomposites have attracted great attention in various forms such as films, 3D porous scaffolds, electrospun mats, hydrogels, and nacre‐like structures. In this review, the most actively investigated GO/polymer nanocomposites are presented and discussed, these nanocomposites are based on chitosan, cellulose, starch, alginate, gellan gum, poly(vinyl alcohol) (PVA), poly(acrylamide), poly(?‐caprolactone) (PCL), poly(lactic acid) (PLLA), poly(lactide‐co‐glycolide) (PLGA), gelatin, collagen, and silk fibroin (SF). The biological and mechanical performance of such nanocomposites are comprehensively scrutinized and ongoing research questions are addressed. The analysis of the literature reveals overall the great potential of GO/polymer nanocomposites in tissue engineering strategies and indicates also a series of challenges requiring further research efforts.

     

PLLA–collagen and PLLA–gelatin hybrid scaffolds with funnel-like porous structure for skin tissue engineering

In skin tissue engineering, a three-dimensional porous scaffold is necessary to support cell adhesion and proliferation and to guide cells moving into the repair area in the wound healing process. Structurally, the porous scaffold should have an open and interconnected porous architecture to facilitate homogenous cell distribution. Moreover, the scaffolds should be mechanically strong to protect deformation during the formation of new skin. In this study, the hybrid scaffolds were prepared by forming funnel-like collagen or gelatin sponge on a woven poly(l-lactic acid) (PLLA) mesh. The hybrid scaffolds combined the advantages of both collagen or gelatin (good cell-interactions) and PLLA mesh (high mechanical strength). The hybrid scaffolds were used to culture dermal fibroblasts for dermal tissue engineering. The funnel-like porous structure promoted homogeneous cell distribution and extracellular matrix production. The PLLA mesh reinforced the scaffold to avoid deformation. Subcutaneous implantation showed that the PLLA–collagen and PLLA–gelatin scaffolds promoted the regeneration of dermal tissue and epidermis and reduced contraction during the formation of new tissue. These results indicate that funnel-like hybrid scaffolds can be used for skin tissue regeneration.     

PLLA–collagen and PLLA–gelatin hybrid scaffolds with funnel-like porous structure for skin tissue engineering

Abstract

In skin tissue engineering, a three-dimensional porous scaffold is necessary to support cell adhesion and proliferation and to guide cells moving into the repair area in the wound healing process. Structurally, the porous scaffold should have an open and interconnected porous architecture to facilitate homogenous cell distribution. Moreover, the scaffolds should be mechanically strong to protect deformation during the formation of new skin. In this study, the hybrid scaffolds were prepared by forming funnel-like collagen or gelatin sponge on a woven poly(l-lactic acid) (PLLA) mesh. The hybrid scaffolds combined the advantages of both collagen or gelatin (good cell-interactions) and PLLA mesh (high mechanical strength). The hybrid scaffolds were used to culture dermal fibroblasts for dermal tissue engineering. The funnel-like porous structure promoted homogeneous cell distribution and extracellular matrix production. The PLLA mesh reinforced the scaffold to avoid deformation. Subcutaneous implantation showed that the PLLA–collagen and PLLA–gelatin scaffolds promoted the regeneration of dermal tissue and epidermis and reduced contraction during the formation of new tissue. These results indicate that funnel-like hybrid scaffolds can be used for skin tissue regeneration.     

Rapid Production of Cell‐Laden Microspheres Using a Flexible Microfluidic Encapsulation Platform

This study establishes a novel microfluidic platform for rapid encapsulation of cells at high densities in photocrosslinkable microspherical hydrogels including poly(ethylene glycol)‐diacrylate, poly(ethylene glycol)‐fibrinogen, and gelatin methacrylate. Cell‐laden hydrogel microspheres are advantageous for many applications from drug screening to regenerative medicine. Employing microfluidic systems is considered the most efficient method for scale‐up production of uniform microspheres. However, existing platforms have been constrained by traditional microfabrication techniques for device fabrication, restricting microsphere diameter to below 200 µm and making iterative design changes time‐consuming and costly. Using a new molding technique, the microfluidic device employs a modified T‐junction design with readily adjustable channel sizes, enabling production of highly uniform microspheres with cell densities (10–60 million cells mL^?1) and a wide range of diameters (300–1100 µm), which are critical for realizing downstream applications, through rapid photocrosslinking (≈1 s per microsphere). Multiple cell types are encapsulated at rates of up to 1 million cells per min, are evenly distributed throughout the microspheres, and maintain high viability and appropriate cellular activities in long‐term culture. This microfluidic encapsulation platform is a valuable and readily adoptable tool for numerous applications, including supporting injectable cell therapy, bioreactor‐based cell expansion and differentiation, and high throughput tissue sphere‐based drug testing assays.     

Highly Porous Microcarriers for Minimally Invasive In Situ Skeletal Muscle Cell Delivery

Microscale cell carriers have recently garnered enormous interest in repairing tissue defects by avoiding substantial open surgeries using implants for tissue regeneration. In this study, the highly open porous microspheres (HOPMs) are fabricated using a microfluidic technique for harboring proliferating skeletal myoblasts and evaluating their feasibility toward cell delivery application in situ. These biocompatible HOPMs with particle sizes of 280–370 µm possess open pores of 10–80 µm and interconnected paths. Such structure of the HOPMs conveniently provide a favorable microenvironment, where the cells are closely arranged in elongated shapes with the deposited extracellular matrix, facilitating cell adhesion and proliferation, as well as augmented myogenic differentiation. Furthermore, in vivo results in mice confirm improved cell retention and vascularization, as well as partial myoblast differentiation. These modular cell‐laden microcarriers potentially allow for in situ tissue construction after minimally invasive delivery providing a convenient means for regeneration medicine.     

Poly(L-lactic acid) nanocylinders as nanofibrous structures for macroporous gelatin scaffolds

Electrospun Nanofiber sheets have been shown to mimic the structure of extracellular matrix (ECM). Although these nanofibers have shown great potential for use as tissue engineering scaffolds, it is difficult for the electrospun nanofiber based sheets to be shaped into the desired three-dimensional structure. In this study, poly(L-lactic acid) (PLLA), a biodegradable and biocompatible polyester, was electrospun to produce nanofibers that were treated with an amino group containing base in order to fabricate polymeric nanocylinders. The aspect ratio of the PLLA nanocylinders was tunable by varying the aminolysis time and density of the amino group containing base. The effects of changes in nanofibrous morphology of the PLLA nanocylinders/macro-porous gelatin scaffolds on cell adhesion and proliferation were evaluated. The results revealed different cell morphology, adhesion, and proliferation in the nanocylinders composite gelatin scaffold versus gelatin scaffold alone. Confocal laser scanning microscopy observation showed more spreading and a more flattened cell morphology after NIH3T3 cells were cultured on PLLA nanocylinders/gelatin scaffolds for 10 hours and 4 days. These results indicate that the gelatin/PLLA nanocylinder composite is a promising way to fabricate 3D nanofibrous scaffolds that accelerates cell adhesion and proliferation for tissue engineering.     

Fabrication of Nanofiber Reinforced Protein Structures For Tissue Engineering

Extracellular matrices and degradable nanofibers are two very promising materials in the field of tissue engineering; however both of these structures face limitations as tissue engineering scaffolds. Extracellular matrices, such as collagen, gelatin, and laminin, have excellent biocompatibility and allow cell in growth and survival, but structural weakness makes them difficult to handle and greatly limits their uses. Degradable nanofibers support cell attachment and can provide structural support and directional guidance, but individual degradable nanofibers are fragile and have a tendency to form dense fiber bundles which limit cell penetration into the spaces between the nanofibers, especially in the case of aligned nanofibers. To overcome these difficulties, degradable loose nanofibers were embedded in protein matrix in an attempt to fabricate a hybrid scaffold with improved properties, such as improved strength, guidance, spacing among nanofibers, etc. Polycaprolactone (PCL) was used as a model material for degradable nanofibers. Gelatin was employed as a model protein for matrix structure formation. Thin hybrid films (average thickness = 2.78 um) were fabricated by wetting the loose aligned undirectional nanofiber arrays or loose aligned bi-directional nanofiber grids with a gelatin aqueous solution, which also allows for live cell loading into the nanofiber-protein composite if cell are premixed with protein solution or on the surface of the films. Gelatin film alone without nanofiber reinforcement is difficult to handle due to the weakness of the thin membrane. Gelatin films with a fiber density as low as 3% v/v were structurally robust enough for handling, and manipulation into complex shapes. Mechanical testing confirmed that the addition of nanofibers enhanced the strength of gelatin films, in both dry and hydrated state. In vitro testing confirmed that nanofiber reinforced films were biocompatible and provided cells with directional guidance. Results demonstrate the promise of gelatin/PCL nanofiber composites as a tissue scaffolding material.     

Delayed Dissolution and Small Molecule Release from Atomic Layer Deposition Coated Electrospun Nanofibers

Electrospun poly (vinyl alcohol) nanofibers are coated with aluminum oxide using atomic layer deposition (ALD) to control the dissolution rate of the nanofiber mats in high‐humidity and aqueous environments. In this regard, ALD offers an effective method to provide a robust, conformal coating to the entire nanofiber surface without modifying the core material. The thickness of the coating, controlled by varying the number of ALD cycles from 2 to 200, enables tuning of the nanofiber stability in water from a few seconds for an uncoated sample to over 5 weeks for a 200 cycle coated sample. Changing the rate of nanofiber dissolution modulates the release of embedded small molecules within the polymer matrix from minutes to weeks while minimizing the “burst” effect typically associated with nanoscale systems. This simple nanofiber coating technique shows great potential as a method to tune shelf‐life, mat degradation, and small molecule release from highly water‐soluble polymers, hitherto unexplored, in a wide range of fields, including biomedical, agricultural, and packaging.     

A Micro‐Ark for Cells: Highly Open Porous Polyhydroxyalkanoate Microspheres as Injectable Scaffolds for Tissue Regeneration

To avoid large open surgery using scaffold transplants, small‐sized cell carriers are employed to repair complexly shaped tissue defects. However, most cell carriers show poor cell adherences and viability. Therefore, polyhydroxyalkanoate (PHA), a natural biopolymer, is used to prepare highly open porous microspheres (OPMs) of 300–360 µm in diameter, combining the advantages of microspheres and scaffolds to serve as injectable carriers harboring proliferating stem cells. In addition to the convenient injection to a defected tissue, and in contrast to poor performances of OPMs made of polylactides (PLA OPMs) and traditional less porous hollow microspheres (PHA HMs), PHA OPMs present suitable surface pores of 10–60 µm and interconnected passages with an average size of 8.8 µm, leading to a high in vitro cell adhesion of 93.4%, continuous proliferation for 10 d and improved differentiation of human bone marrow mesenchymal stem cells (hMSCs). PHA OPMs also support stronger osteoblast‐regeneration compared with traditional PHA HMs, PLA OPMs, commercial hyaluronic acid hydrogels, and carrier‐free hMSCs in an ectopic bone‐formation mouse model. PHA OPMs protect cells against stresses during injection, allowing more living cells to proliferate and migrate to damaged tissues. They function like a micro‐Noah's Ark to safely transport cells to a defect tissue.     

Room-Temperature-Formed PEDOT:PSS Hydrogels Enable Injectable,Soft, and Healable Organic Bioelectronics

There is an increasing need to develop conducting hydrogels for bioelectronic applications. In particular, poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) hydrogels have become a research hotspot due to their excellent biocompatibility and stability. However, injectable PEDOT:PSS hydrogels have been rarely reported. Such syringe-injectable hydrogels are highly desirable for minimally invasive biomedical therapeutics. Here, an approach is demonstrated to develop injectable PEDOT:PSS hydrogels by taking advantage of the room-temperature gelation property of PEDOT:PSS. These PEDOT:PSS hydrogels form spontaneously after syringe injection of the PEDOT:PSS suspension into the desired location, without the need of any additional treatments. A facile strategy is also presented for large-scale production of injectable PEDOT:PSS hydrogel fibers at room temperature. Finally, it is demonstrated that these room-temperature-formed PEDOT:PSS hydrogels (RT-PEDOT:PSS hydrogel) and hydrogel fibers can be used for the development of soft and self-healable hydrogel bioelectronic devices.     

Broad Bandwidth,Self‐Powered Acoustic Sensor Created by Dynamic Near‐Field Electrospinning of Suspended,Transparent Piezoelectric Nanofiber Mesh

Freely suspended nanofibers, such as spider silk, harnessing their small diameter (sub‐micrometer) and spanning fiber morphology, behave as a nonresonating acoustic sensor. The associated sensing characteristics, departing from conventional resonant acoustic sensors, could be of tremendous interest for the development of high sensitivity, broadband audible sensors for applications in environmental monitoring, biomedical diagnostics, and internet‐of‐things. Herein, a low packing density, freely suspended nanofiber mesh with a piezoelectric active polymer is fabricated, demonstrating a self‐powered acoustic sensing platform with broad sensitivity bandwidth covering 200–5000 Hz at hearing‐safe sound pressure levels. Dynamic near‐field electrospinning is developed to fabricate in situ poled poly(vinylidene fluoride‐co‐trifluoroethylene) (P(VDF‐TrFE)) nanofiber mesh (average fiber diameter ≈307 nm), exhibiting visible light transparency greater than 97%. With the ability to span the nanomesh across a suspension distance of 3 mm with minimized fiber stacking (≈18% fiber packing density), individual nanofibers can freely imitate the acoustic‐driven fluctuation of airflow in a collective manner, where piezoelectricity is harvested at two‐terminal electrodes for direct signal collection. Applications of the nanofiber mesh in music recording with good signal fidelity are demonstrated.     

Electrospun poly(l-lactide)/zein nanofiber mats loaded with <Emphasis Type="Italic">Rana chensinensis</Emphasis> skin peptides for wound dressing

Electrospun nanofiber mats can display impressive performance as an ideal wound dressing. In this study, poly(l-lactide)(PLLA)/zein nanofiber mats loaded with Rana chensinensis skin peptides (RCSPs) were successfully produced by two different electrospinning techniques, blend and coaxial, with the goal of developing a wound dressing material. The nanofiber mats were investigated by environmental scanning electron microscope (ESEM), transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FTIR), differential scanning calorimeter (DSC), water contact angle, mechanical tests and cell viability. The resulting nanofiber mats exhibited smooth surfaces, tiny diameters and different cross-sectional shapes from pure PLLA and zein nanofibers. The FTIR result showed that PLLA, zein and RCSPs were well dispersed, without chemical interactions. Compared with coaxial nanofiber mats, blending zein-RCSPs with PLLA enhanced hydrophilicity but decreased mechanical properties. Adding RCSPs into the electrospun nanofibers significantly improved the mechanical properties of the mats. Cell viability studies with human foreskin fibroblasts demonstrated that cell growth on PLLA/zein-RCSPs nanofiber mats was significantly higher than that on PLLA/zein nanofiber mats. The results indicate that nanofiber mats containing RCSPs are potential candidates for wound dressing.     

Advanced Nanotechnology Leading the Way to Multimodal Imaging‐Guided Precision Surgical Therapy

Surgical resection is the primary and most effective treatment for most patients with solid tumors. However, patients suffer from postoperative recurrence and metastasis. In the past years, emerging nanotechnology has led the way to minimally invasive, precision and intelligent oncological surgery after the rapid development of minimally invasive surgical technology. Advanced nanotechnology in the construction of nanomaterials (NMs) for precision imaging‐guided surgery (IGS) as well as surgery‐assisted synergistic therapy is summarized, thereby unlocking the advantages of nanotechnology in multimodal IGS‐assisted precision synergistic cancer therapy. First, mechanisms and principles of NMs to surgical targets are briefly introduced. Multimodal imaging based on molecular imaging technologies provides a practical method to achieve intraoperative visualization with high resolution and deep tissue penetration. Moreover, multifunctional NMs synergize surgery with adjuvant therapy (e.g., chemotherapy, immunotherapy, phototherapy) to eliminate residual lesions. Finally, key issues in the development of ideal theranostic NMs associated with surgical applications and challenges of clinical transformation are discussed to push forward further development of NMs for multimodal IGS‐assisted precision synergistic cancer therapy.     

Herstellung von hochporösen,endkonturnahen Titan‐Formkörpern für biomedizinische Anwendungen

Near‐net‐shape manufacturing of highly porous titanium parts for biomedical applications The production of highly porous titanium parts is attractive for biomedical applications. Preferrentially, these parts are produced by powdermetallurgical means using suitable spacer materials. Porosities up to 75 % and well defined pore sizes in the range of 0.1 to 2.0 mm are achieved adjusting the amount and the particle size of the spacer material. Up to now, near‐net‐shape manufacturing of highly porous parts was hindered by the plastic deformation of the sintered network during machining leading to a partial or complete closing of the open porosity. A new manufacturing route is presented, where the shaping is already done in the unsintered state starting from pressed compacts. The stability of the compacts was found to be sufficient to machine the compacts without additional binders. The manufacturing route was successfully applied to the prototype of an acetabular cup. Additionally, some investigations are presented characterizing the highly porous titanium.     

Microfluidic Generation of Nanomaterials for Biomedical Applications

As nanomaterials (NMs) possess attractive physicochemical properties that are strongly related to their specific sizes and morphologies, they are becoming one of the most desirable components in the fields of drug delivery, biosensing, bioimaging, and tissue engineering. By choosing an appropriate methodology that allows for accurate control over the reaction conditions, not only can NMs with high quality and rapid production rate be generated, but also designing composite and efficient products for therapy and diagnosis in nanomedicine can be realized. Recent evidence implies that microfluidic technology offers a promising platform for the synthesis of NMs by easy manipulation of fluids in microscale channels. In this Review, a comprehensive set of developments in the field of microfluidics for generating two main classes of NMs, including nanoparticles and nanofibers, and their various potentials in biomedical applications are summarized. Furthermore, the major challenges in this area and opinions on its future developments are proposed.     

Injectable and Crosslinkable PLGA‐Based Microribbons as 3D Macroporous Stem Cell Niche

Poly(lactide‐co‐glycolide) (PLGA) has been widely used as a tissue engineering scaffold. However, conventional PLGA scaffolds are not injectable, and do not support direct cell encapsulation, leading to poor cell distribution in 3D. Here, a method for fabricating injectable and intercrosslinkable PLGA microribbon‐based macroporous scaffolds as 3D stem cell niche is reported. PLGA is first fabricated into microribbon‐shape building blocks with tunable width using microcontact printing, then coated with fibrinogen to enhance solubility and injectability using aqueous solution. Upon mixing with thrombin, firbornogen‐coated PLGA microribbons can intercrosslink into 3D scaffolds. When subject to cyclic compression, PLGA microribbon scaffolds exhibit great shock‐absorbing capacity and return to their original shape, while conventional PLGA scaffolds exhibit permanent deformation after one cycle. Using human mesenchymal stem cells (hMSCs) as a model cell type, it is demonstrated that PLGA μRB scaffolds support homogeneous cell encapsulation, and robust cell spreading and proliferation in 3D. After 28 days of culture in osteogenic medium, hMSC‐seeded PLGA μRB scaffolds exhibit an increase in compressive modulus and robust bone formation as shown by staining of alkaline phosphatase, mineralization, and collagen. Together, the results validate PLGA μRBs as a promising injectable, macroporous, non‐hydrogel‐based scaffold for cell delivery and tissue regeneration applications.     

3-D Nanofibrous electrospun multilayered construct is an alternative ECM mimicking scaffold

Extra cellular matrix (ECM) is a natural cell environment, possesses complicated nano- and macro- architecture. Mimicking this three-dimensional (3-D) web is a challenge in the modern tissue engineering. This study examined the application of a novel 3-D construct, produced by multilayered organization of electrospun nanofiber membranes, for human bone marrow-derived mesenchymal stem cells (hMSCs) support. The hMSCs were seeded on an electrospun scaffold composed of poly ε-caproloactone (PCL) and collagen (COL) (1:1), and cultured in a dynamic flow bioreactor prior to in vivo implantation. Cell viability after seeding was analyzed by AlamarBlue™ Assay. At the various stages of experiment, cell morphology was examined by histology, scanning electron microscopy (SEM) and confocal microscopy. Results: A porous 3-D network of randomly oriented nanofibers appeared to support cell attachment in a way similar to traditionally used tissue culture polysterene plate. The following 6 week culture process of the tested construct in the dynamic flow system led to massive cell proliferation with even distribution inside the scaffold. Subcutaneous implantation of the cultured construct into nude mice demonstrated good integration with the surrounding tissues and neovascularization. Conclusion: The combination of electrospinning technology with multilayer technique resulted in the novel 3-D nanofiber multilayered construct, able to contain efficient cell mass necessary for a successful in vivo grafting. The success of this approach with undifferentiated cells implies the possibility of its application as a platform for development of constructs with cells directed into various tissue types.