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.

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.

Repairing Annulus Fibrosus Fissures Using Methacrylated Gellan Gum Combined with Novel Silk.

Intervertebral disc (IVD) herniation often causes severe pain and is frequently associated with the degeneration of the IVD. As the IVD degenerates, more fissures with increasing size appear within the outer region of the IVD, the annulus fibrosus (AF), favoring the initiation and progression of IVD herniation. For this reason, we propose an AF repair approach based on methacrylated gellan gum (GG-MA) and silk fibroin. Therefore, coccygeal bovine IVDs were injured using a biopsy puncher (⌀ 2 mm) and then repaired with 2% GG-MA as a filler material and sealed with an embroidered silk yarn fabric. Then, the IVDs were cultured for 14 days either without any load, static loading, or complex dynamic loading. After 14 days of culture, no significant differences were found between the damaged and repaired IVDs, except for a significant decrease in the IVDs' relative height under dynamic loading. Based on our findings combined with the current literature that focuses on ex vivo AF repair approaches, we conclude that it is likely that the repair approach did not fail but rather insufficient harm was done to the IVD.

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.

Biomedical applications of silk and its role for intervertebral disc repair.

Intervertebral disc (IVD) degeneration (IDD) is the main contributor to chronic low back pain. To date, the present therapies mainly focus on treating the symptoms caused by IDD rather than addressing the problem itself. For this reason, researchers have searched for a suitable biomaterial to repair and/or regenerate the IVD. A promising candidate to fill this gap is silk, which has already been used as a biomaterial for many years. Therefore, this review aims first to elaborate on the different origins from which silk is harvested, the individual composition, and the characteristics of each silk type. Another goal is to enlighten why silk is so suitable as a biomaterial, discuss its functionalization, and how it could be used for tissue engineering purposes. The second part of this review aims to provide an overview of preclinical studies using silk-based biomaterials to repair the inner region of the IVD, the nucleus pulposus (NP), and the IVD's outer area, the annulus fibrosus (AF). Since the NP and the AF differ fundamentally in their structure, different therapeutic approaches are required. Consequently, silk-containing hydrogels have been used mainly to repair the NP, and silk-based scaffolds have been used for the AF. Although most preclinical studies have shown promising results in IVD-related repair and regeneration, their clinical transition is yet to come.

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.

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.

Genipin-Enhanced Fibrin Hydrogel and Novel Silk for Intervertebral Disc Repair in a Loaded Bovine Organ Culture Model.

(1) Background: Intervertebral disc (IVD) repair represents a major challenge. Using functionalised biomaterials such as silk combined with enforced hydrogels might be a promising approach for disc repair. We aimed to test an IVD repair approach by combining a genipin-enhanced fibrin hydrogel with an engineered silk scaffold under complex load, after inducing an injury in a bovine whole organ IVD culture; (2) Methods: Bovine coccygeal IVDs were isolated from ~1-year-old animals within four hours post-mortem. Then, an injury in the annulus fibrosus was induced by a 2 mm biopsy punch. The repair approach consisted of genipin-enhanced fibrin hydrogel that was used to fill up the cavity. To seal the injury, a Good Manufacturing Practise (GMP)-compliant engineered silk fleece-membrane composite was applied and secured by the cross-linked hydrogel. Then, IVDs were exposed to one of three loading conditions: no load, static load and complex load in a two-degree-of-freedom bioreactor for 14 days. Followed by assessing DNA and matrix content, qPCR and histology, the injured discs were compared to an uninjured control IVD that underwent the same loading profiles. In addition, the genipin-enhanced fibrin hydrogel was further investigated with respect to cytotoxicity on human stem cells, annulus fibrosus, and nucleus pulposus cells; (3) Results: The repair was successful as no herniation could be detected for any of the three loading conditions. Disc height was not recovered by the repair DNA and matrix contents were comparable to a healthy, untreated control disc. Genipin resulted being cytotoxic in the in vitro test but did not show adverse effects when used for the organ culture model; (4) Conclusions: The current study indicated that the combination of the two biomaterials, i.e., genipin-enhanced fibrin hydrogel and an engineered silk scaffold, was a promising approach for IVD repair. Furthermore, genipin-enhanced fibrin hydrogel was not suitable for cell cultures; however, it was highly applicable as a filler material.

Functionalized silk fibers from transgenic silkworms for wound healing applications: Surface presentation of bioactive epidermal growth factor.

Growth factors play a crucial role in wound healing in general and are promising tools for the treatment of chronic wounds as they can restore the physiological wound healing process. In growth factor-loaded wound dressings, human epidermal growth factor (EGF) is released in a burst and washed out quickly. The developed matrix consists of recombinant EGF produced in transgenic silkworms as a fusion protein with the fibroin light chain. The covalent linkage prevents EGF from draining into the surrounding tissue while presenting the growth factor on the surface. EGF-functionalized silk membranes and nonwovens lead to a 2.5-fold increase in the cell number of fibroblasts, while retaining full bioactivity even after e-beam sterilization. EGF is long-term presented without burst release and significantly reduces the wound area by 15% in an in vitro wound model. Hence, the cost-effective production of a biomaterial using transgenic silkworm larvae in combination with a growth factor paves the way for a promising new multifactorial wound cover for chronic wound healing. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2643-2652, 2018.

Differentiation of MSC and annulus fibrosus cells on genetically engineered silk fleece-membrane-composites enriched for GDF-6 or TGF-ß3.

Intervertebral disc (IVD) repair is a high-priority topic in our active and increasingly ageing society. Since a high number of people are affected by low back pain treatment options that are able to restore the biological function of the IVD are highly warranted. Here, we investigated whether the feasibility of genetically engineered (GE)-silk from Bombyx mori containing specific growth factors to precondition human bone-marrow derived mesenchymal stem cells (hMSC) or to activate differentiated human annulus fibrosus cells (hAFC) prior transplantation or for direct repair on the IVD. Here, we tested the hypothesis that GE-silk fleece can thrive human hMSC towards an IVD-like phenotype. We aimed to demonstrate a possible translational application of good manufacturing practice (GMP)-compliant GE-silk scaffolds in IVD repair and regeneration. GE-silk with growth and differentiation factor 6 (GDF-6-silk) or transforming growth factor ß3 (TGF-ß3, TGF-ß3-silk) and untreated silk (cSilk) were investigated by DNA content, cell activity assay and glycosaminoglycan (GAG) content and their differentiation potential by qPCR analysis. We found that all silk types demonstrated a very high biocompatibility for both cell types, that is, hMSC and hAFC, as revealed by cell activity, and DNA proliferation assay. Further, analyzing qPCR of marker genes revealed a trend to differentiation toward an NP-like phenotype looking at the Aggrecan/Collagen 2 ratio which was around 10:1. Our results support the conclusion that our GE-silk scaffold treatment approach can thrive hMSC towards a more IVD-like phenotype or can maintain the phenotype of native hAFC. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1324-1333, 2018.

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.

Selective laser-melted fully biodegradable scaffold composed of poly(d,l-lactide) and ß-tricalcium phosphate with potential as a biodegradable implant for complex maxillofacial reconstruction: In vitro and in vivo results.

OBJECTIVES: Scaffolds (SC) composed of poly(d,l-lactide) and ß-tricalcium phosphate of variable pore structures were manufactured by selective laser melting (SLM), which allowed the production of porous interconnected structures promoting cellular adhesion and vascular proliferation. Biocompatibility, rate of osseointegration and new bone formation (NB) were analyzed. MATERIAL AND METHODS: Powder based on the material composition was selective melted by a laser beam allowing layer-by-layer production. Pore size and biocompatibility were tested with mesenchymal stem cells (rMSC) and Saos 2 cells that were cultivated on SCs showing better proliferation, without toxicity, than controls. SCs with a 600- to 700-µm pore diameter proved ideal for fast and reliable cellular and vascular supply throughout the interconnecting pore system. Jaw and calvarial critical-size defects (CSD) with diameters of 5 or 16 mm were drilled in rats and either SLM test SCs (pore diameter 600 µm) or the previously removed autologs bone as controls were (re-) implanted. RESULTS: The SC in vivo led to complete bone ingrowth with minimal inflammatory reaction adjacent to and within the CSD as compared with controls. The SC promoted the differentiation of rMSC into osteoblasts, revealing osteoinductive properties. Promising NB ingrowth of the material was also obtained in the animal study. CONCLUSION: The SC showed complete bony replacement within 30 days in all rats; this ingrowth was significantly superior to that of controls and revealed no signs of significant foreign body reaction. Because of continuous replacement by bone this material composition is ideal for SCs fitting 3D bone defects. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1216-1231, 2017.

Novel silk protein barrier membranes for guided bone regeneration.

This study assesses the biocompatibility of novel silk protein membranes with and without modification, and evaluates their effect on facilitating bone formation and defect repair in guided bone regeneration. Two calvarian bone defects 12 mm in diameter were created in each of a total of 38 rabbits. Four different types of membranes, (silk-, hydroxyapatite-modified silk-, ß-TCP-modified silk- and commonly clinically used collagen-membranes) were implanted to cover one of the two defects in each animal. Histologic analysis did not show any adverse tissue reactions in any of the defect sites indicating good biocompatibility of all silk protein membranes. Histomorphometric and histologic evaluation revealed that collagen and ß-TCP modified silk membranes supported bone formation (collagen: bone area fraction p = 0.025; significant; ß-TCP modified silk membranes bone area fraction: p = 0.24, not significant), guided bone regeneration and defect bridging. The bone, which had formed in defects covered by ß-TCP modified silk membranes, displayed a more advanced stage of bone tissue maturation with restoration of the original calvarial bone microarchitecture when compared to the bone which had formed in defects, for which any of the other test membranes were used. Micro-CT analysis did not reveal any differences in the amount of bone formation between defects with and without membranes. In contrast to the collagen membranes, ß-TCP modified silk membranes were visible in all cases and may therefore be advantageous for further supporting bone formation beyond 10 weeks and preventing soft tissue ingrowth from the periphery. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2603-2611, 2017.

Microvascular stent anastomosis using N-fibroin stents: feasibility, ischemia time, and complications.

OBJECTIVE: To evaluate a novel microvascular anastomosis technique using N-fibroin stents. STUDY DESIGN: Cylinder stents of 1 mm diameter and 5 mm length were fabricated using N-fibroin from silkworms. In 22 rats, aortas were dissected, and the stent was inserted into the two ends of the aorta and fixed using methylmethacrylate. RESULTS: Stent anastomosis was successful in 21 (96%) rats. The mean ischemia time was 7.4 minutes, significantly shorter than the 15.9 minutes in the control group with conventional sutures (P < .0001). After 4 months, anastomosis was functionally patent in all cases. However, elastic fibers remained interrupted in all stent anastomosis cases, and marked host rejection was evident at the stent anastomosis sites. Around the stents, thrombi were frequent (52%). CONCLUSIONS: Our study demonstrated the basic feasibility of stent anastomosis using N-fibroin stents and reduced ischemia time. However, thrombus formation, frequent and severe abdominal infections, and heavy host rejection remain critical issues.

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).

Purmorphamine and oxysterols accelerate and promote osteogenic differentiation of mesenchymal stem cells in vitro.

AIM: The purpose of the present study was to find inexpensive and non-toxic additives enhancing and accelerating the osteogenesis of mesenchymal stem cells in vitro, which can be used for tissue engineering of bone material. MATERIALS AND METHODS: Osteogenic differentiation of rat mesenchymal stem cells was carried-out using classic differentiation medium containing or lacking purmorphamine, statins or oxysterols, respectively. Cell proliferation, alkaline phosphatase activity, calcium sedimentation and expression of bone matrix protein genes were measured to monitor differentiation. RESULTS: Purmorphamine substantially suppressed proliferation, enhanced and accelerated alkaline phosphatase activity and calcium sedimentation and increased the expression of osteopontin and osteocalcin in rat mesenchymal stem cells in vitro. A similar osteogenesis-promoting effect was observed for oxysterols but not for the two statins. CONCLUSION: Purmorphamine and oxysterols promote and accelerate osteogenesis of mesenchymal stem cells in vitro suggesting their potential application for tissue engineering of bone material.

Bone substitutes enhance osteogenic differentiation of mesenchymal stem cells in three-dimensional scaffolds.

AIM: The present study aimed to find bone substitutes to enhance osteogenic differentiation of mesenchymal stem cells in three-dimensional scaffolds in the absence of dexamethasone. MATERIALS AND METHODS: Seven commercial bone substitutes were added to a three-dimensional fibrin-matrix containing rat mesenchymal stem cells in a biocompatible poly-L-lactic-acid mesh. Cell viability, cytotoxicity and alkaline phosphatase activity were followed for three weeks. Expression of bone markers was examined by qualitative evaluation of corresponding transcripts. RESULTS: Six out of the seven bone derivatives exhibited an osteogenic-enhancing effect. CONCLUSION: The osteogenic-enhancing effect of the evaluated bone substitutes suggests their potential clinical application for preparation of autologous bone replacement material in three-dimensional carriers.

Osteogenic differentiation of mesenchymal stem cells in fibrin-hydroxyapatite matrix in a 3-dimensional mesh scaffold.

AIM: To explore the feasibility of culturing mesenchymal stem cells in an hydroxyapatite-fibrin matrix held by a mesh scaffold and inducing osteogenic differentiation of these cells. The aim was to obtain bone-material in vitro in a desired form. MATERIALS AND METHODS: Rat mesenchymal stem cells were mixed with fibrin and nanocrystalline hydroxyapatite in tubular scaffolds constructed from a poly(L-lactic acid) mesh, and cultured under standard and osteogenic differentiating conditions. Cell viability, cytotoxicity and alkaline phosphatase activity were followed for 3 weeks. Living cells and the expression of bone markers were visualized by fluorescence staining and immunofluorescence staining, respectively. Attachment of cells to the scaffold mesh surface was examined by scanning electron microscopy. RESULTS: Cell viability decreased and cytotoxicity increased rapidly during the first day of culture but stabilized gradually afterwards, indicating fast adaptation of the cells in the matrix-scaffold environment. From day 17, cytotoxicity started to decrease, paralleled by an increase in alkaline phosphatase activity, indicating osteogenic differentiation. A large number of living cells were visible in the matrix and on the mesh scaffold. Expression of collagen type I, osteoponin, osteocalcin and core binding factor 1 were evident under osteogenic differentiation conditions. CONCLUSION: The three-dimensional construction of a fibrin-hydroxyapatite matrix in a biocompatible poly(L-lactic acid) as mesh-scaffold provides a promising carrier for producing bone-material in vitro in a desired form for applications in regenerative medicine.

Transgenic protein production in silkworm silk glands requires cathepsin and chitinase of Autographa californica multicapsid nucleopolyhedrovirus.

The silkworm Bombyx mori represents an established in vivo system for the production of recombinant proteins. Baculoviruses have been extensively investigated and optimised for the expression of high protein levels inside the haemolymph of larvae and pupae of this lepidopteran insect. Current technology includes deletion of genes responsible for the activity of virus-borne proteases, which in wild-type viruses, cause liquefaction of the host insect and enhance horizontal transmission of newly synthesised virus particles. Besides the haemolymph, the silk gland of B. mori provides an additional expression system for recombinant proteins. In this paper, we investigated how silk gland can be efficiently infected by a Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). We demonstrated that the viral chitinase and the cysteine protease cathepsin are necessary to permit viral entry into the silk gland cells of intrahaemocoelically infected B. mori larvae. Moreover, for the first time, we showed AcMNPV crossing the basal lamina of silk glands in B. mori larvae, and we assessed a new path of infection of silk gland cells that can be exploited for protein production.

Transcriptome analysis of MSC and MSC-derived osteoblasts on Resomer® LT706 and PCL: impact of biomaterial substrate on osteogenic differentiation.

BACKGROUND: Mesenchymal stem cells (MSC) represent a particularly attractive cell type for bone tissue engineering because of their ex vivo expansion potential and multipotent differentiation capacity. MSC are readily differentiated towards mature osteoblasts with well-established protocols. However, tissue engineering frequently involves three-dimensional scaffolds which (i) allow for cell adhesion in a spatial environment and (ii) meet application-specific criteria, such as stiffness, degradability and biocompatibility. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we analysed two synthetic, long-term degradable polymers for their impact on MSC-based bone tissue engineering: PLLA-co-TMC (Resomer® LT706) and poly(ε-caprolactone) (PCL). Both polymers enhance the osteogenic differentiation compared to tissue culture polystyrene (TCPS) as determined by Alizarin red stainings, scanning electron microscopy, PCR and whole genome expression analysis. Resomer® LT706 and PCL differ in their influence on gene expression, with Resomer® LT706 being more potent in supporting osteogenic differentiation of MSC. The major trigger on the osteogenic fate, however, is from osteogenic induction medium. CONCLUSION: This study demonstrates an enhanced osteogenic differentiation of MSC on Resomer® LT706 and PCL compared to TCPS. MSC cultured on Resomer® LT706 showed higher numbers of genes involved in skeletal development and bone formation. This identifies Resomer® LT706 as particularly attractive scaffold material for bone tissue engineering.