Michael Wöltje

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).

Mesenchymal stem cells can be recruited to wounded tissue via hepatocyte growth factor-loaded biomaterials.

Mesenchymal stem cells (MSC) are precursor cells of mesodermal tissue and, because of their trophic phenotype, they are known to play beneficial roles in wound healing. In addition, various tissue engineering strategies are based on MSC/biomaterial constructs. As the isolation and expansion of MSCs is a long‐term process, a major goal is to develop an endogenous stem cell recruitment system that circumvents all ex vivo steps generally used for tissue engineering. Therefore collagen and silk fibroin were loaded with hepatocyte growth factor (HGF), a chemoattractant for MSCs. Collagen was mixed with HGF during polymerization, while silk fibroin and HGF were produced as fusion proteins by transgenic silkworms. To demonstrate release of active HGF, enzyme‐linked immunosorbent assay, in vitro migration assays and animal studies were performed to demonstrate MSC migration in vivo, followed by detailed examinations of the immunological effects of the biomaterials. Hepatocyte growth factor was released burst‐like, both from silk fibroin and collagen during the first 8 h and gradually for up to 168 h in vitro. Directed migration in vitro was demonstrated when MSCs were exposed to HGF. In vivo, HGF‐loaded collagen and silk fibroin were tolerated as subcutaneous implants. In addition, it was proved that endogenous MSCs were recruited from the local environment. These results show for the first time recruitment of endogenous MSCs to HGF‐loaded collagen (fast degradable) and silk fibroin scaffolds (long‐term degradable) in vitro and in vivo. This knowledge could be applied to make off‐the‐shelf, readily available constructs for use in patients with chronic wound or burns. Copyright

A review of the application of reinforced hydrogels and silk as biomaterials for intervertebral disc repair.

The degeneration of the intervertebral disc (IVD) within the spinal column represents a major pain source for many patients. Biological restoration or repair of the IVD using compressive-force-resistant and at the same time cytocompatible materials would be desirable over current purely mechanical solutions, such as spinal fusion or IVD implants. This review provides an overview of recent research on the repair of the inner (nucleus pulposus = NP) and the outer (annulus fibrous = AF) parts of the IVD tissue. Many studies have addressed NP repair using hydrogel-like materials. However, only a few studies have so far focused on AF repair. As the AF possesses an extremely low self-healing capacity and special attention to shear-force resistance is essential, special repair designs are required. In our review, we stated the challenges in IVD repair and highlighted the use of composite materials such as silk biomaterials and fibrin cross-linked reinforced hydrogels. We elaborated on the origin of silk and its many in tissue engineering. Furthermore, techniques such as electrospinning and 3D printing technologies allow the fabrication of versatile and functionalised 3D scaffolds. We summarised the research that has been conducted in the field of regenerative medicine over the recent years, with a special focus on the potential application and the potential of combining silk and reinforced - and thus mechanically tailored - hydrogels for IVD repair.

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

OBJECTIVESnScaffolds (SC) composed of poly(d,l-lactide) and β-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.nnnMATERIAL AND METHODSnPowder 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.nnnRESULTSnThe 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.nnnCONCLUSIONnThe 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.

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-, β-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 β-TCP modified silk membranes supported bone formation (collagen: bone area fraction pu2009=u20090.025; significant; β-TCP modified silk membranes bone area fraction: pu2009=u20090.24, not significant), guided bone regeneration and defect bridging. The bone, which had formed in defects covered by β-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, β-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.

Differentiation of MSC and annulus fibrosus cells on genetically-engineered silk fleece-membrane-composites enriched for GDF-6 or TGF-β3†

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 β3 (TGF‐β3, TGF‐β3‐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.

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.

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: FUNCTIONALIZED SILK FIBERS FROM TRANSGENIC SILKWORMS

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.

Intervertebral Disc Repair By A Combination Of Genipin-enhanced Fibrin Hydrogel And Growth Factor-enriched Silk-fleece

INTRODUCTION: It is well accepted that two incidents are causing discogenic back pain: trauma or disc degeneration. In the cases of disc material protrusion of the inner annulus fibrosus (AF) and/or injuries of the outer AF, we aimed to repair the intervertebral disc (IVD) from an “inside-out” approach. Therefore, we propose using hydrogel in combination with a genetically modified but GMP-compliant silk. The silk’s fibrinogen contains the human growth and differentiation factor 6 (GDF6), directly produced by the baculovirus transduced Bombyx mori larvae in culture. GDF6 was shown to drive stem cells towards an IVD-like phenotype. Within this study, we investigated the feasibility of a genipin cross-linked fibrin hydrogel as a filling material for the IVD as well as a glue for the silk membrane-fleece using an ex vivo organ culture approach. Additionally, different physiological loading regimes were applied to investigate the IVDs cellular response in situ. Furthermore, cytotoxicity and proliferation potential of human mesenchymal stem cells (MSC) within the silk material was assessed. METHODS: Bovine IVDs of 10-14 month old animals were harvested under aseptic conditions. After inducing an IVD injury by a circular 2 mm biopsy punch, the cavity was filled with an FDA-approved human based fibrin hydrogel (Baxter Tisseel) enhanced with 4.2 mg/ml of a cross-linker; genipin (Wako Chemicals GmbH). The defect was closed with a GMP-compliant silk membrane-fleece composite (Spintec Engineering GmbH) that was placed on the hydrogel. Subsequently, the IVDs were subjected to in vitro organ culture for 14 days using three different and independent loading regimes: 1) complex loading of 0.2MPa compression and 0 ±2° torsion at 0.2Hz for 8h/day, 2) static diurnal loading of 0.2MPa and 3) no loading (free swelling control). For complex loading a custom built two-degree of freedom bioreactor was used. At the end of culture, the discs were harvested and controlled for seal failure, disc height, metabolic activity (alamar blue), cell death by necrosis (LDH assay) and apoptosis (Caspase 3/7), DNA, GAG and collagen (via hydroxyl proline = HYP) contents and qPCR of ECM production and inflammation was performed. Histologies for collagen (Picrosirius red), proteoglycan (SafraninO/Fast Green) and cytoplasm and nuclei (H&E) were performed on plastic embedded sagittal cuts and the latter two also on transversal cryo-sections. Proliferation potential of GDF6-silk was investigated by seeding MSC (P 2) at a density of 12’000 cells per 5x5mm silk fleece-membrane composite for 21 days. As controls, silk without growth factors (cSilk), silk with transforming growth factor (TGFβ1) and silk with exogenous GDF6 were used. Metabolic activity, DNA and GAG content as well as qPCR [aggrecan (ACAN), collagen 2 (COL2), and others] were measured on day 0, 7, 14 and 21. All experiments were performed with five bovine and five human donors, respectively. Two-way ANOVA followed by Bonferroni’s multiple comparisons test was performed using GraphPad Prism. RESULTS SECTION: Macroscopic inspection revealed that the silk seal was not displaced throughout the culture period. Further, cellular metabolic activity (data not shown), DNA and GAG content and disc height of the repaired discs did not differ significantly from the injured IVDs. Except for a higher DNA content under static loading for the repaired discs compared to the injured IVDs (p-value £ 0.004, Fig. 1). Examination of the histological sections indicated that the injury created a cavity in the injured discs. Whereas in the repaired discs the induced injury was closed and the cavity was filled with tissue (Fig. 2). In vitro experiments on the cellular level attributed a good cell compatibility within the silk and GDF6 silk. Also proliferation, DNA and GAG content did not reveal significant differences among the different silks (data not shown). qPCR of MSC revealed a trend towards a higher ACAN to COL2 ratio. This indicated the differentiation of the MSC towards a nucleus pulposus phenotype (Fig. 3). DISCUSSION: Strikingly, the discs responded to the injury on the opposite sides equally, suggesting exchange of cytokines either throughout the disc or the culture media. The in vitro silk experiments attributes the silk a good biocompatibility. Further, GDF6 silk thrives MSC towards a NP-like phenotype. The silk and the hydrogel offer a promising approach to repair and regenerate the IVD after nucleotomy upon disc herniation. SIGNIFICANCE: Exploring the possibilities of combining natural biomaterials with growth factors might lead towards new treatment approaches in the field of IVD regeneration. Which is of importance due to the lack of satisfying options and a high incidence rate. REFERENCES: 1. L.E. Clarke et al. (2014), Arthritis Res. Ther. 16(2): R67, 2. M Likhitpanichkul et al. (2014), Eur Cell Mater 28, 25-38, 3. S.C. Chan, B. Gantenbein-Ritter (2012), J Vis Exp 60: 3490, 4. J. Walser et al. (2012), John Wiley & Sons, Ltd. ACKNOWLEDGEMENTS: We thank Eva Roth for her help in IVD isolation and biomechanical assays. Microscopy was performed on equipment supported by the Microscopy Imaging Center (MIC), University of Bern, Switzerland. This project was supported by the Gebert Rüf Foundation project # GRS-X028/13. ORS 2017 Annual Meeting Poster No.0833