Continuous Wet Spinning of Regenerated Silk Fibers from Spinning Dopes Containing 4% Fibroin Protein.

The wet spinning of fibers from regenerated silk fibroin has long been a research goal. Due to the degradation of the molecular structure of the fibroin protein during the preparation of the regenerated silk fibroin solution, fibroin concentrations with at least 10% protein content are required to achieve sufficient viscosity for wet spinning. In this study, a spinning dope formulation of regenerated silk fibroin is presented that shows a rheological behavior similar to that of native silk fibroin isolated from the glands of B. mori silkworm larvae. In addition, we present a wet-spinning process that enables, for the first time, the continuous wet spinning of regenerated silk fibroin with only 4% fibroin protein content into an endless fiber. Furthermore, the tensile strength of these wet-spun regenerated silk fibroin fibers per percentage of fibroin is higher than that of all continuous spinning approaches applied to regenerated and native silk fibroin published so far.

A Fast and Reliable Process to Fabricate Regenerated Silk Fibroin Solution from Degummed Silk in 4 Hours.

Silk fibroin has a high potential for use in several approaches for technological and biomedical applications. However, industrial production has been difficult to date due to the lengthy manufacturing process. Thus, this work investigates a novel procedure for the isolation of non-degraded regenerated silk fibroin that significantly reduces the processing time from 52 h for the standard methods to only 4 h. The replacement of the standard degumming protocol by repeated short-term microwave treatments enabled the generation of non-degraded degummed silk fibroin. Subsequently, a ZnCl2 solution was used to completely solubilize the degummed fibroin at only 45 °C with an incubation time of only 1 h. Desalting was performed by gel filtration. Based on these modifications, it was possible to generate a cytocompatible aqueous silk fibroin solution from degummed silk within only 4 h, thus shortening the total process time by 48 h without degrading the quality of the isolated silk fibroin solution.

Isotropic and Anisotropic Scaffolds for Tissue Engineering: Collagen, Conventional, and Textile Fabrication Technologies and Properties.

In this review article, tissue engineering and regenerative medicine are briefly explained and the importance of scaffolds is highlighted. Furthermore, the requirements of scaffolds and how they can be fulfilled by using specific biomaterials and fabrication methods are presented. Detailed insight is given into the two biopolymers chitosan and collagen. The fabrication methods are divided into two categories: isotropic and anisotropic scaffold fabrication methods. Processable biomaterials and achievable pore sizes are assigned to each method. In addition, fiber spinning methods and textile fabrication methods used to produce anisotropic scaffolds are described in detail and the advantages of anisotropic scaffolds for tissue engineering and regenerative medicine are highlighted.

Anisotropic Chitosan Scaffolds Generated by Electrostatic Flocking Combined with Alginate Hydrogel Support Chondrogenic Differentiation.

The replacement of damaged or degenerated articular cartilage tissue remains a challenge, as this non-vascularized tissue has a very limited self-healing capacity. Therefore, tissue engineering (TE) of cartilage is a promising treatment option. Although significant progress has been made in recent years, there is still a lack of scaffolds that ensure the formation of functional cartilage tissue while meeting the mechanical requirements for chondrogenic TE. In this article, we report the application of flock technology, a common process in the modern textile industry, to produce flock scaffolds made of chitosan (a biodegradable and biocompatible biopolymer) for chondrogenic TE. By combining an alginate hydrogel with a chitosan flock scaffold (CFS+ALG), a fiber-reinforced hydrogel with anisotropic properties was developed to support chondrogenic differentiation of embedded human chondrocytes. Pure alginate hydrogels (ALG) and pure chitosan flock scaffolds (CFS) were studied as controls. Morphology of primary human chondrocytes analyzed by cLSM and SEM showed a round, chondrogenic phenotype in CFS+ALG and ALG after 21 days of differentiation, whereas chondrocytes on CFS formed spheroids. The compressive strength of CFS+ALG was higher than the compressive strength of ALG and CFS alone. Chondrocytes embedded in CFS+ALG showed gene expression of chondrogenic markers (COL II, COMP, ACAN), the highest collagen II/I ratio, and production of the typical extracellular matrix such as sGAG and collagen II. The combination of alginate hydrogel with chitosan flock scaffolds resulted in a scaffold with anisotropic structure, good mechanical properties, elasticity, and porosity that supported chondrogenic differentiation of inserted human chondrocytes and expression of chondrogenic markers and typical extracellular matrix.

Silk Fiber-Reinforced Hyaluronic Acid-Based Hydrogel for Cartilage Tissue Engineering.

A continuing challenge in cartilage tissue engineering for cartilage regeneration is the creation of a suitable synthetic microenvironment for chondrocytes and tissue regeneration. The aim of this study was to develop a highly tunable hybrid scaffold based on a silk fibroin matrix (SM) and a hyaluronic acid (HA) hydrogel. Human articular chondrocytes were embedded in a porous 3-dimensional SM, before infiltration with tyramine modified HA hydrogel. Scaffolds were cultured in chondropermissive medium with and without TGF-ß1. Cell viability and cell distribution were assessed using CellTiter-Blue assay and Live/Dead staining. Chondrogenic marker expression was detected using qPCR. Biosynthesis of matrix compounds was analyzed by dimethylmethylene blue assay and immuno-histology. Differences in biomaterial stiffness and stress relaxation were characterized using a one-step unconfined compression test. Cell morphology was investigated by scanning electron microscopy. Hybrid scaffold revealed superior chondro-inductive and biomechanical properties compared to sole SM. The presence of HA and TGF-ß1 increased chondrogenic marker gene expression and matrix deposition. Hybrid scaffolds offer cytocompatible and highly tunable properties as cell-carrier systems, as well as favorable biomechanical properties.

Textile cell-free scaffolds for in situ tissue engineering applications.

In this article, the benefits offered by micro-fibrous scaffold architectures fabricated by textile manufacturing techniques are discussed: How can established and novel fiber-processing techniques be exploited in order to generate templates matching the demands of the target cell niche? The problems related to the development of biomaterial fibers (especially from nature-derived materials) ready for textile manufacturing are addressed. Attention is also paid on how biological cues may be incorporated into micro-fibrous scaffold architectures by hybrid manufacturing approaches (e.g. nanofiber or hydrogel functionalization). After a critical review of exemplary recent research works on cell-free fiber based scaffolds for in situ TE, including clinical studies, we conclude that in order to make use of the whole range of favors which may be provided by engineered fibrous scaffold systems, there are four main issues which need to be addressed: (1) Logical combination of manufacturing techniques and materials. (2) Biomaterial fiber development. (3) Adaption of textile manufacturing techniques to the demands of scaffolds for regenerative medicine. (4) Incorporation of biological cues (e.g. stem cell homing factors).

Influence of Spinning Method on Shape Memory Effect of Thermoplastic Polyurethane Yarns.

Shape memory polymers are gaining increasing attention, especially in the medical field, due to their ability to recover high deformations, low activation temperatures, and relatively high actuation stress. Furthermore, shape memory polymers can be applied as fiber-based solutions for the development of smart devices used in many fields, e.g., industry 4.0, medicine, and skill learning. These kind of applications require sensors, actors, and conductive structures. Textile structures address these applications by meeting requirements such as being flexible, adaptable, and wearable. In this work, the influence of spinning methods and parameters on the effect of shape memory polymer yarns was investigated, comparing melt and wet spinning. It is shown that the spinning method can significantly influence the strain fixation and generated stress during the activation of the shape memory effect. Furthermore, for wet spinning, the draw ratio could affect the stress conversion, influencing its efficiency. Therefore, the selection of the spinning process is essential for the setting of application-specific shape-changing properties.

Polyphosphate-loaded silk fibroin membrane as hemostatic agent in oral surgery: a pilot study.

PURPOSE: Post-interventional hemorrhage can result in serious complications, especially in patients with hemostatic disorders. Identification of safe and efficient local hemostatic agents is important, particularly in the context of an ageing society and the emergence of new oral anticoagulants. The aim of this in vitro study was to investigate the potential of silk fibroin membranes coated with the inorganic polymer polyphosphate (polyP) as a novel hemostatic device in oral surgery. METHODS: Cocoons of the silkworm Bombyx mori were degummed and dissolved. Varying amounts of long-chain polyP (2-2000 µg/mm2) were adsorbed to the surface of silk fibroin membranes. Analysis of the procoagulant effect of polyP-coated silk membranes was performed using real-time thrombin generation assays in human plasma. Increasing concentrations of polyP (0.15-500 µg/ml) served as a positive control, while uncoated silk fibroin membranes were used as negative control. RESULTS: PolyP-coated silk fibroin membranes triggered coagulation when compared to plasma samples and pure silk fibroin membranes. A polyP-dose-dependent effect of thrombin generation could be found with a maximum (ETP = 1525.7 nM⋅min, peak thrombin = 310.1 nM, time to peak = 9.8 min, lag time = 7.6 min.) at 200 µg/mm2 of polymer loading on the silk fibroin membrane surface. CONCLUSIONS: In this study, it was demonstrated that silk fibroin membranes coated with polyP have the potential to act as a promising novel hemostatic device.

Textile Design of an Intervertebral Disc Replacement Device from Silk Yarn.

Low back pain is often due to degeneration of the intervertebral discs (IVD). It is one of the most common age- and work-related problems in today's society. Current treatments are not able to efficiently restore the full function of the IVD. Therefore, the aim of the present work was to reconstruct the two parts of the intervertebral disc-the annulus fibrosus (AF) and the nucleus pulposus (NP)-in such a way that the natural structural features were mimicked by a textile design. Silk was selected as the biomaterial for realization of a textile IVD because of its cytocompatibility, biodegradability, high strength, stiffness, and toughness, both in tension and compression. Therefore, an embroidered structure made of silk yarn was developed that reproduces the alternating fiber structure of +30° and -30° fiber orientation found in the AF and mimics its lamellar structure. The developed embroidered ribbons showed a tensile strength that corresponded to that of the natural AF. Fiber additive manufacturing with 1 mm silk staple fibers was used to replicate the fiber network of the NP and generate an open porous textile 3D structure that may serve as a reinforcement structure for the gel-like NP.

Development of electrospun, biomimetic tympanic membrane implants with tunable mechanical and oscillatory properties for myringoplasty.

Most commonly, autologous grafts are used in tympanic membrane (TM) reconstruction. However, apart from the limited availability and the increased surgical risk, they cannot replicate the full functionality of the human TM properly. Hence, biomimetic synthetic TM implants have been developed in our project to overcome these drawbacks. These innovative TM implants are made from synthetic biopolymer polycaprolactone (PCL) and silk fibroin (SF) by electrospinning technology. Static and dynamic experiments have shown that the mechanical and oscillatory behavior of the TM implants can be tuned by adjusting the solution concentration, the SF and PCL mixing ratio and the electrospinning parameters. In addition, candidates for TM implants could have comparable acousto-mechanical properties to human TMs. Finally, these candidates were further validated in in vitro experiments by performing TM reconstruction in human cadaver temporal bones. The reconstructed TM with SF-PCL blend membranes fully recovered the acoustic vibration when the perforation was smaller than 50%. Furthermore, the handling, medium adhesion and transparency of the developed TM implants were similar to those of human TMs.

Treatment of Critical-Size Femoral Bone Defects with Chitosan Scaffolds Produced by a Novel Process from Textile Engineering.

The purpose of this study was to investigate, in vitro and in vivo, the suitability of chitosan (CHS) scaffolds produced by the net-shape-nonwoven (NSN) technology, for use as bone graft substitutes in a critical-size femoral bone defect in rats. For in vitro investigations, scaffolds made of CHS, mineralized collagen (MCM), or human cancellous bone allograft (CBA) were seeded with human telomerase-immortalized mesenchymal stromal cells (hTERT-MSC), incubated for 14 days, and thereafter evaluated for proliferation and osteogenic differentiation. In vivo, CHS, MCM and CBA scaffolds were implanted into 5 mm critical-size femoral bone defects in rats. After 12 weeks, the volume of newly formed bone was determined by microcomputed tomography (µCT), while the degree of defect healing, as well as vascularization and the number of osteoblasts and osteoclasts, was evaluated histologically. In vitro, CHS scaffolds showed significantly higher osteogenic properties, whereas treatment with CHS, in vivo, led to a lower grade of bone-healing compared to CBA and MCM. While chitosan offers a completely new field of scaffold production by fibers, these scaffolds will have to be improved in the future, regarding mechanical stability and osteoconductivity.

Thermoresponsive Shape Memory Fibers for Compression Garments.

Their highly deformable properties make shape memory polymers (SMP) a promising component for the development of new compression garments. The shape memory effect (SME) can be observed when two polymers are combined. In here, polycaprolactone (PCL) and thermoplastic polyurethane (TPU) were melt spun in different arrangement types (blend, core-sheath, and island-in-sea), whereas the best SME was observed for the blend type. In order to trigger the SME, this yarn was stimulated at a temperature of 50 °C. It showed a strain fixation of 62%, a strain recovery of 99%, and a recovery stress of 2.7 MPa.

Collagen multifilament spinning.

The benefits of fiber based implants and scaffolds for tissue engineering applications are their anisotropic, highly porous, and controllable macro-, micro-, and nanostructure. Collagen is one of the most commonly used material for the fabrication of scaffolds, as this biopolymer is present in the natural extracellular matrix. For textile processing and textile scaffold fabrication methods, multifilament yarns are required, however, only monofilaments can be generated by state-of-the-art collagen spinning. Hence, the research presented in here aimed at the development of a collagen multifilament wet-spinning process in reproducible quality as well as the characterization of non-crosslinked and crosslinked wet-spun multifilament yarns. Wet spun collagen yarns were comprised of 6 single filaments each having a fineness of 5 tex and a diameter of 80 µm. The tensile strength of the glutaraldehyde crosslinked yarns was 169 MPa (Young's modulus 3534 MPa) in the dry state and 40 MPa (Young's modulus 281 MPa) in the wet state. Furthermore, wet spun collagen filaments showed a characteristic fibrillar structure, which was similar the morphological structure of natural collagen fibers. The textile processing of collagen multifilament yarn was demonstrated by means of knitting technology.

Design of Complexly Graded Structures inside Three-Dimensional Surface Models by Assigning Volumetric Structures.

An innovative approach for designing complex structures from STL-datasets based on novel software for assigning volumetric data to surface models is reported. The software allows realizing unique complex structures using additive manufacturing technologies. Geometric data as obtained from imaging methods, computer-aided design, or reverse engineering that exist only in the form of surface data are converted into volumetric elements (voxels). Arbitrary machine data can be assigned to each voxel and thereby enable implementing different materials, material morphologies, colors, porosities, etc. within given geometries. The software features an easy-to-use graphical user interface and allows simple implementation of machine data libraries. To highlight the potential of the modular designed software, an extrusion-based process as well as a two-tier additive manufacturing approach for short fibers and binder process are combined to generate three-dimensional components with complex grading on the material and structural level from STL files.

Wet spinning and riboflavin crosslinking of collagen type I/III filaments.

Reconstituted fibrillary collagen is one of the most advantageous biomaterials for biomedical applications. The objective of the research project described in this paper was to evaluate whether riboflavin-induced photo-crosslinking could be used as a non-toxic alternative to glutaraldehyde (GA)-crosslinking for the preparation of wet spun collagen filaments. Collagen filaments were produced on a laboratory wet spinning line and crosslinked with GA or riboflavin with and without UV exposure. Based on mechanical and thermal analyses, it was concluded that the combination of riboflavin and UV light leads to crosslinked collagen filaments having improved mechanical and thermal properties. Furthermore, riboflavin-crosslinked filaments exhibited a higher cytocompatibility for human mesenchymal stem cells compared to GA-crosslinked filaments.

In silico modeling of structural and porosity properties of additive manufactured implants for regenerative medicine.

Additive manufacturing technologies are a promising technology towards patient-specific implants for applications in regenerative medicine. The Net-Shape-Nonwoven technology is used to manufacture structures from short fibers with interconnected pores and large functional surfaces that are predestined for cell adhesion and growth. The present study reports on a modeling approach with a particular focus on the specific structural properties. The overall porosities and mean pore-sizes of the digital models are simulated according to liquid-displacement porosity in a tool implemented in the modeling software. This allows adjusting the process parameters fiber length and fiber diameter to generate biomimetic structures with pore-sizes adapted to the requirements of the tissue that is to be replaced. Modeling the structural and porosity properties of scaffolds and implants leads to an efficient use of the processed biomaterials as the trial-and-error method is avoided.

Bulk collagen incorporation rates into knitted stiff fibre polymer in tissue-engineered scaffolds: the rate-limiting step.

Fabrication of tissue-engineered constructs in vitro relies on sufficient synthesis of extracellular matrix (ECM) by cells to form a material suitable for normal function in vivo. Collagen synthesis by human dermal fibroblasts grown in vitro on two polymers, polyethylene terephthalate (PET) and polyglycolic acid (PGA), was measured by high-performance liquid chromatography (HPLC). Cells were either cultured in a dynamic environment, where meshes were loaded onto a pulsing tube in a bioreactor, or in a static environment without pulsing. Collagen synthesis by cells cultured on a static mesh increased by six-fold compared to monolayer culture, and increased by up to a further 5.4-fold in a pulsed bioreactor. However, little of the collagen synthesized was deposited onto the meshes, almost all being lost to the medium. The amount of collagen deposited onto meshes was highest when cells were cultured dynamically on PET meshes (17.6 microg), but deposition still represented only 1.4% of the total synthesized. Although total collagen synthesis was increased by the use of 3D culture and the introduction of pulsing, the results suggest that the limiting factor for fabrication of a tissue-engineered construct within practical timescales is not the amount of collagen synthesized but the quantity retained (i.e. deposited) within the construct during culture. This may be enhanced by systems which promote or assemble true 3D multi-layers of cells.