Andreas Herbert Teuschl

A novel bioreactor for the generation of highly aligned 3D skeletal muscle-like constructs through orientation of fibrin via application of static strain.

UNLABELLED The generation of functional biomimetic skeletal muscle constructs is still one of the fundamental challenges in skeletal muscle tissue engineering. With the notion that structure strongly dictates functional capabilities, a myriad of cell types, scaffold materials and stimulation strategies have been combined. To further optimize muscle engineered constructs, we have developed a novel bioreactor system (MagneTissue) for rapid engineering of skeletal muscle-like constructs with the aim to resemble native muscle in terms of structure, gene expression profile and maturity. Myoblasts embedded in fibrin, a natural hydrogel that serves as extracellular matrix, are subjected to mechanical stimulation via magnetic force transmission. We identify static mechanical strain as a trigger for cellular alignment concomitant with the orientation of the scaffold into highly organized fibrin fibrils. This ultimately yields myotubes with a more mature phenotype in terms of sarcomeric patterning, diameter and length. On the molecular level, a faster progression of the myogenic gene expression program is evident as myogenic determination markers MyoD and Myogenin as well as the Ca(2+) dependent contractile structural marker TnnT1 are significantly upregulated when strain is applied. The major advantage of the MagneTissue bioreactor system is that the generated tension is not exclusively relying on the strain generated by the cells themselves in response to scaffold anchoring but its ability to subject the constructs to individually adjustable strain protocols. In future work, this will allow applying mechanical stimulation with different strain regimes in the maturation process of tissue engineered constructs and elucidating the role of mechanotransduction in myogenesis. STATEMENT OF SIGNIFICANCE Mechanical stimulation of tissue engineered skeletal muscle constructs is a promising approach to increase tissue functionality. We have developed a novel bioreactor-based 3D culture system, giving the user the possibility to apply different strain regimes like static, cyclic or ramp strain to myogenic precursor cells embedded in a fibrin scaffold. Application of static mechanical strain leads to alignment of fibrin fibrils along the axis of strain and concomitantly to highly aligned myotube formation. Additionally, the pattern of myogenic gene expression follows the temporal progression observed in vivo with a more thorough induction of the myogenic program when static strain is applied. Ultimately, the strain protocol used in this study results in a higher degree of muscle maturity demonstrated by enhanced sarcomeric patterning and increased myotube diameter and length. The introduced bioreactor system enables new possibilities in muscle tissue engineering as longer cultivation periods and different strain applications will yield tissue engineered muscle-like constructs with improved characteristics in regard to functionality and biomimicry.

Shock wave treatment enhances cell proliferation and improves wound healing by ATP release-coupled extracellular signal-regulated kinase (ERK) activation.

Background: Signaling pathways underlying beneficial effects of extracorporeal shock wave treatment (ESWT) remain to be completely elucidated. Results: ESWT enhances cell proliferation in vitro and wound healing in vivo. Conclusion: ESWT-induced ATP release and subsequent extracellular signal-regulated kinase (ERK) activation are prerequisites for enhanced cell proliferation and wound healing. Significance: Deciphering the involved signaling cascades provides the basis for ESWT as clinical wound healing treatment. Shock wave treatment accelerates impaired wound healing in diverse clinical situations. However, the mechanisms underlying the beneficial effects of shock waves have not yet been fully revealed. Because cell proliferation is a major requirement in the wound healing cascade, we used in vitro studies and an in vivo wound healing model to study whether shock wave treatment influences proliferation by altering major extracellular factors and signaling pathways involved in cell proliferation. We identified extracellular ATP, released in an energy- and pulse number-dependent manner, as a trigger of the biological effects of shock wave treatment. Shock wave treatment induced ATP release, increased Erk1/2 and p38 MAPK activation, and enhanced proliferation in three different cell types (C3H10T1/2 murine mesenchymal progenitor cells, primary human adipose tissue-derived stem cells, and a human Jurkat T cell line) in vitro. Purinergic signaling-induced Erk1/2 activation was found to be essential for this proliferative effect, which was further confirmed by in vivo studies in a rat wound healing model where shock wave treatment induced proliferation and increased wound healing in an Erk1/2-dependent fashion. In summary, this report demonstrates that shock wave treatment triggers release of cellular ATP, which subsequently activates purinergic receptors and finally enhances proliferation in vitro and in vivo via downstream Erk1/2 signaling. In conclusion, our findings shed further light on the molecular mechanisms by which shock wave treatment exerts its beneficial effects. These findings could help to improve the clinical use of shock wave treatment for wound healing.

Decellularized human placenta chorion matrix as a favorable source of small-diameter vascular grafts.

UNLABELLED Biomaterials based on decellularized tissues are increasingly attracting attention as functional alternatives to other natural or synthetic materials. However, a source of non-cadaver human allograft material would be favorable. Here we establish a decellularization method of vascular tissue from cryopreserved human placenta chorionic plate starting with an initial freeze-thaw step followed by a series of chemical treatments applied with a custom-made perfusion system. This novel pulsatile perfusion set-up enabled us to successfully decellularize the vascular tissue with lower concentrations of chemicals and shorter exposure times compared to a non-perfusion process. The decellularization procedure described here lead to the preservation of the native extracellular matrix architecture and the removal of cells. Quantitative analysis revealed no significant changes in collagen content and a retained glycosaminoglycan content of approximately 29%. In strain-to-failure tests, the decellularized grafts showed similar mechanical behavior compared to native controls. In addition, the mechanical values for ultimate tensile strength and stiffness were in an acceptable range for in vivo applications. Furthermore, biocompatibility of the decellularized tissue and its recellularizationability to serve as an adequate substratum for upcoming recellularization strategies using primary human umbilical vein endothelial cells (HUVECs) was demonstrated. HUVECs cultured on the decellularized placenta vessel matrix performed endothelialization and maintained phenotypical characteristics and cell specific expression patterns. Overall, the decellularized human placenta vessels can be a versatile tool for experimental studies on vascularization and as potent graft material for future in vivo applications. STATEMENT OF SIGNIFICANCE In the US alone more than 1million vascular grafts are needed in clinical practice every year. Despite severe disadvantages, such as donor site morbidity, autologous grafting from the patients own arteries or veins is regarded as the gold standard for vascular tissue repair. Besides, strategies based on synthetic or natural materials have shown limited success. Tissue engineering approaches based on decellularized tissues are regarded as a promising alternative to clinically used treatments to overcome the observed limitations. However, a source for supply of non-cadaver human allograft material would be favorable. Here, we established a decellularization method of vascular tissue from the human placenta chorionic plate, a suitable human tissue source of consistent quality. The decellularized human placenta vessels can be a potent graft material for future in vivo applications and furthermore might be a versatile tool for experimental studies on vascularization.

Regeneration of the anterior cruciate ligament: Current strategies in tissue engineering

Recent advancements in the field of musculoskeletal tissue engineering have raised an increasing interest in the regeneration of the anterior cruciate ligament (ACL). It is the aim of this article to review the current research efforts and highlight promising tissue engineering strategies. The four main components of tissue engineering also apply in several ACL regeneration research efforts. Scaffolds from biological materials, biodegradable polymers and composite materials are used. The main cell sources are mesenchymal stem cells and ACL fibroblasts. In addition, growth factors and mechanical stimuli are applied. So far, the regenerated ACL constructs have been tested in a few animal studies and the results are encouraging. The different strategies, from in vitro ACL regeneration in bioreactor systems to bio-enhanced repair and regeneration, are under constant development. We expect considerable progress in the near future that will result in a realistic option for ACL surgery soon.

Phototherapy With Led Light Modulates Healing Processes in an In Vitro Scratch-wound Model Using 3 Different Cell Types

BACKGROUND An effective way of modulating wound healing processes, including proliferation and apoptosis, is low-level light therapy. Because of several disadvantages of lasers, light-emitting diodes (LEDs) could be more feasible light sources. OBJECTIVE To evaluate and compare the effects of blue and red light from LEDs on different cell types in an in vitro scratch-wound model. METHODS Monolayers of C2C12 myoblasts, NIH/3T3 fibroblasts, and BICR10 keratinocytes were injured by mechanical scraping. Cells were illuminated on 5 consecutive days for 10 minutes by LED at 470 or 630 nm. Effects of light on in vitro wound healing were evaluated by analyzing time to closure, proliferation, apoptosis, and necrosis rates. RESULTS Illumination substantially affected cell viability and cell growth. Blue light strongly decreased proliferation and augmented apoptosis in all 3 cell types and increased necrosis rates in C2C12 and NIH/3T3 cells. In contrast, red light did not alter apoptosis in either cell type but promoted proliferation in all 3 cell types with significant effects in C2C12 and NIH/3T3 cells and shortened time to closure in all 3 cell types. CONCLUSION Light-emitting diode light illumination could be a therapeutic option and positively affect wound healing processes. By choosing appropriate wavelengths, variable effects can be achieved.

In vitro extracorporeal shock wave treatment enhances stemness and preserves multipotency of rat and human adipose-derived stem cells

BACKGROUND AIMS Adipose-derived progenitor/stem cells (ASCs) are discussed as a promising candidate for various tissue engineering approaches. However, its applicability for the clinic is still difficult due to intra- and inter-donor heterogeneity and limited life span in vitro, influencing differentiation capacity as a consequence to decreased multipotency. METHODS Extracorporeal shock wave treatment has been proven to be a suitable clinical tool to improve regeneration of a variety of tissues for several decades, whereas the mechanisms underlying these beneficial effects remain widely unknown. RESULTS In this study we show that human and rat adipose derived stem cells respond strongly to repetitive shock wave treatment in vitro, resulting not only in maintenance and significant elevation of mesenchymal markers (CD73, CD90, CD105), but also in significantly increased differentiation capacity towards the osteogenic and adipogenic lineage as well as toward Schwann-cell like cells even after extended time in vitro, preserving multipotency of ASCs. CONCLUSIONS ESWT might be a promising tool to improve ASC quality for cell therapy in various tissue engineering and regenerative medicine applications.

Enhanced cell adhesion on silk fibroin via lectin surface modification.

Various tissue engineering (TE) approaches are based on silk fibroin (SF) as scaffold material because of its superior mechanical and biological properties compared to other materials. The translation of one-step TE approaches to clinical application has generally failed so far due to the requirement of a prolonged cell seeding step before implantation. Here, we propose that the plant lectin WGA (wheat germ agglutinin), covalently bound to SF, will mediate cell adhesion in a time frame acceptable to be part of a one-step surgical intervention. After the establishment of a modification protocol utilizing carbodiimide chemistry, we examined the attachment of cells, with a special focus on adipose-derived stromal cells (ASC), on WGA-SF compared to pure native SF. After a limited time frame of 20min the attachment of ASCs to WGA-SF showed an increase of about 17-fold, as compared to pure native SF. The lectin-mediated cell adhesion further showed an enhanced resistance to trypsin (as a protease model) and to applied fluid shear stress (mechanical stability). Moreover, we could demonstrate that the adhesion of ASCs on the WGA-SF does not negatively influence proliferation or differentiation potential into the osteogenic lineage. To test for in vitro immune response, the proliferation of peripheral blood mononuclear cells in contact with the WGA-SF was determined, showing no alterations compared to plain SF. All these findings suggest that the WGA modification of SF offers important benefits for translation of SF scaffolds into clinical applications.

Emerging Trends in Abdominal Wall Reinforcement: Bringing Bio‐Functionality to Meshes

Abdominal wall hernia is a recurrent issue world-wide and requires the implantation of over 1 million meshes per year. Because permanent meshes such as polypropylene and polyester are not free of complications after implantation, many mesh modifications and new functionalities have been investigated over the last decade. Indeed, mesh optimization is the focus of intense development and the biomaterials utilized are now envisioned as being bioactive substrates that trigger various physiological processes in order to prevent complications and to promote tissue integration. In this context, it is of paramount interest to review the most relevant bio-functionalities being brought to new meshes and to open new avenues for the innovative development of the next generation of meshes with enhanced properties for functional abdominal wall hernia repair.

Extracorporeal shockwave treatment: A novel tool to improve Schwann cell isolation and culture

BACKGROUND AIMS As new approaches for peripheral nerve regeneration are sought, there is an increasing demand for native Schwann cells for in vitro testing and/or reimplantation. Extracorporeal shockwave treatment (ESWT) is an emergent technology in the field of regenerative medicine that has also recently been shown to improve peripheral nerve regeneration. METHODS In this study, we elucidate the effects of ESWT on Schwann cell isolation and culture. Rat sciatic nerves were dissected and treated with ESWT, and Schwann cells were isolated and cultured for 15 passages. RESULTS Single treatment of the whole nerve ex vivo led to significantly increased extracellular adenosinetriphosphate as an immediate consequence, and subsequently a number of effects on the culture were observed, starting with a significantly increased Schwann cell yield after isolation. In the ESWT group, the quality of culture, reflected in consistently higher purity (S100b, morphology), proliferation rate (5-bromo-2-deoxyuridine, population doublings per passage) and expression of regenerative phenotype-associated markers (P75, glial fibrillary acidic protein, c-Jun), was significantly improved. In contrast, the control group exhibited progressively senescent behavior, reflected in a decrease of proliferation, loss of specific markers and increase in P16(INK4A) expression. CONCLUSIONS ESWT has beneficial effects on Schwann cell isolation and culture.

Sericin removal from raw Bombyx mori silk scaffolds of high hierarchical order.

Silk fibroin has previously been described as a promising candidate for ligament tissue engineering (TE) approaches. For biocompatibility reasons, silkworm silk requires removal of sericin, which can elicit adverse immune responses in the human body. One disadvantage of the required degumming process is the alteration of the silk fiber structural properties, which can hinder textile engineering of high order hierarchical structures. Therefore, the aim of this study was to find a way to remove sericin from a compact and highly ordered raw silk fiber matrix. The wire rope design of the test model scaffold comprises several levels of geometric hierarchy. Commonly used degumming solutions fail in removing sericin in this wire rope design. Weight loss measurements, picric acid and carmine staining as well as scanning electron microscopy demonstrated that the removal of sericin from the model scaffold of a wire rope design can be achieved through a borate buffer-based system. Furthermore, the borate buffer degummed silks were shown to be nontoxic and did not alter cell proliferation behavior. The possibility to remove sericin after the textile engineering process has taken place eases the production of highly ordered scaffold structures and may expand the use of silk as scaffold material in further TE and regenerative medicine applications.