Philipp Heher

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.

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.

Embryonic Stem Cells Facilitate the Isolation of Persistent Clonal Cardiovascular Progenitor Cell Lines and Leukemia Inhibitor Factor Maintains Their Self-Renewal and Myocardial Differentiation Potential in vitro

Compelling evidence for the existence of somatic stem cells in the heart of different mammalian species has been provided by numerous groups; however, so far it has not been possible to maintain these cells as self-renewing and phenotypically stable clonal cell lines in vitro. Thus, we sought to identify a surrogate stem cell niche for the isolation and persistent maintenance of stable clonal cardiovascular progenitor cell lines, enabling us to study the mechanism of self-renewal and differentiation in these cells. Using postnatal murine hearts with a selectable marker as the stem cell source and embryonic stem cells and leukemia inhibitory factor (LIF)-secreting fibroblasts as a surrogate niche, we succeeded in the isolation of stable clonal cardiovascular progenitor cell lines. These cell lines self-renew in an LIF-dependent manner. They express both stemness transcription factors Oct4, Sox2, and Nanog and early myocardial transcription factors Nkx2.5, GATA4, and Isl-1 at the same time. Upon LIF deprivation, they exclusively differentiate to functional cardiomyocytes and endothelial and smooth muscle cells, suggesting that these cells are mesodermal intermediates already committed to the cardiogenic lineage. Cardiovascular progenitor cell lines can be maintained for at least 149 passages over 7 years without phenotypic changes, in the presence of LIF-secreting fibroblasts. Isolation of wild-type cardiovascular progenitor cell lines from adolescent and old mice has finally demonstrated the general feasibility of this strategy for the isolation of phenotypically stable somatic stem cell lines.

Small Molecule Cardiogenol C Upregulates Cardiac Markers and Induces Cardiac Functional Properties in Lineage-Committed Progenitor Cells

Background/Aims: Cell transplantation into the heart is a new therapy after myocardial infarction. Its success, however, is impeded by poor donor cell survival and by limited transdifferentiation of the transplanted cells into functional cardiomyocytes. A promising strategy to overcome these problems is the induction of cardiomyogenic properties in donor cells by small molecules. Methods: Here we studied cardiomyogenic effects of the small molecule compound cardiogenol C (CgC), and structural derivatives thereof, on lineage-committed progenitor cells by various molecular biological, biochemical, and functional assays. Results: Treatment with CgC up-regulated cardiac marker expression in skeletal myoblasts. Importantly, the compound also induced cardiac functional properties: first, cardiac-like sodium currents in skeletal myoblasts, and secondly, spontaneous contractions in cardiovascular progenitor cell-derived cardiac bodies. Conclusion: CgC induces cardiomyogenic function in lineage-committed progenitor cells, and can thus be considered a promising tool to improve cardiac repair by cell therapy.

VUT-MK142 : a new cardiomyogenic small molecule promoting the differentiation of pre-cardiac mesoderm into cardiomyocytes

Intra-cardiac cell transplantation is a new therapy after myocardial infarction. Its success, however, is impeded by the limited capacity of donor cells to differentiate into functional cardiomyocytes in the heart. A strategy to overcome this problem is the induction of cardiomyogenic function in cells prior to transplantation. Among other approaches, recently, synthetic small molecules were identified, which promote differentiation of stem cells of various origins into cardiac-like cells or cardiomyocytes. The aim of this study was to develop and characterise new promising cardiomyogenic synthetic low-molecular weight compounds. Therefore, the structure of the known cardiomyogenic molecule cardiogenol C was selectively modified, and the effects of the resulting compounds were tested on various cell types. From this study, VUT-MK142 was identified as the most promising candidate with respect to cardiomyogenic activity. Treatment using this novel agent induced the strongest up-regulation of expression of the cardiac marker ANF in both P19 embryonic carcinoma cells and C2C12 skeletal myoblasts. The activity of VUT-MK142 on this marker superseded CgC; moreover, the novel compound significantly up-regulated the expression of other cardiac markers, and promoted the development of beating cardiomyocytes from cardiovascular progenitor cells. We conclude that VUT-MK142 is a potent new cardiomyogenic synthetic agent promoting the differentiation of pre-cardiac mesoderm into cardiomyocytes, which may be useful to differentiate stem cells into cardiomyocytes for cardiac repair. Additionally, an efficient synthesis of VUT-MK142 is reported taking advantage of continuous flow techniques superior to classical batch reactions both in yield and reaction time.

Desmin enters the nucleus of cardiac stem cells and modulates Nkx2.5 expression by participating in transcription factor complexes that interact with the nkx2.5 gene

ABSTRACT The transcription factor Nkx2.5 and the intermediate filament protein desmin are simultaneously expressed in cardiac progenitor cells during commitment of primitive mesoderm to the cardiomyogenic lineage. Up-regulation of Nkx2.5 expression by desmin suggests that desmin may contribute to cardiogenic commitment and myocardial differentiation by directly influencing the transcription of the nkx2.5 gene in cardiac progenitor cells. Here, we demonstrate that desmin activates transcription of nkx2.5 reporter genes, rescues nkx2.5 haploinsufficiency in cardiac progenitor cells, and is responsible for the proper expression of Nkx2.5 in adult cardiac side population stem cells. These effects are consistent with the temporary presence of desmin in the nuclei of differentiating cardiac progenitor cells and its physical interaction with transcription factor complexes bound to the enhancer and promoter elements of the nkx2.5 gene. These findings introduce desmin as a newly discovered and unexpected player in the regulatory network guiding cardiomyogenesis in cardiac stem cells. Summary: These data may be the first evidence for a nuclear function of an intermediate filament protein. In cardiac stem cells, desmin interacts with transcription factor complexes influencing Nkx 2.5 transcription.

A non-invasive in vitro monitoring system reporting skeletal muscle differentiation.

Monitoring of cell differentiation is a crucial aspect of cell-based therapeutic strategies depending on tissue maturation. In this study, we have developed a noninvasive reporter system to trace murine skeletal muscle differentiation. Either a secreted bioluminescent reporter (Metridia luciferase) or a fluorescent reporter (green fluorescent protein [GFP]) was placed under the control of the truncated muscle creatine kinase (MCK) basal promoter enhanced by variable numbers of upstream MCK E-boxes. The engineered pE3MCK vector, coding a triple tandem of E-Boxes and the truncated MCK promoter, showed twentyfold higher levels of luciferase activation compared with a Cytomegalovirus (CMV) promoter. This newly developed reporter system allowed noninvasive monitoring of myogenic differentiation in a straining bioreactor. Additionally, binding sequences of endogenous microRNAs (miRNAs; seed sequences) that are known to be downregulated in myogenesis were ligated as complementary seed sequences into the reporter vector to reduce nonspecific signal background. The insertion of seed sequences improved the signal-to-noise ratio up to 25% compared with pE3MCK. Due to the highly specific, fast, and convenient expression analysis for cells undergoing myogenic differentiation, this reporter system provides a powerful tool for application in skeletal muscle tissue engineering.

Efficacy of Topical Hemostatic Agents: A Comparative Evaluation of Two Gelatin/Thrombin-Based Hemostatic Matrices in a Porcine Kidney Surgical Model

Abstract Purpose: Topical hemostatic agents are an important means of controlling or preventing bleeding. This study was performed to compare gelatin–thrombin matrix with smooth particles (SmGM) versus gelatin–thrombin matrix with stellate particles (StGM) in a porcine kidney bleeding model. Materials and methods: In male pigs, reproducible lesions (diameter and depth ∼10 mm) were created in the renal cortex. Each lesion was treated topically using either SmGM or StGM. Blood loss was quantified before and 2, 5 and 10 minutes after treatment. Dry mass, ultrastructural and histologic analyses were also performed. Results: Thirty-two lesions were treated with SmGM and 32 with StGM; median initial bleeding rates were 27.6 and 29.1 mL/min, respectively. Two minutes post-application, SmGM was associated with significantly less bleeding than StGM (0.574 vs 0.920 mL/min; p < .0001). This difference stemmed principally from lesions with initial blood loss >29 mL/min, where bleeding rates at 2 minutes were ∼3-fold higher with StGM (1.636 vs 0.567 mL/min; p ≥ 0.040). Dry mass per unit volume of hemostatic agent was significantly higher with SmGM versus StGM. SmGM formed discrete, smooth particles, while StGM particles were stellate and tended to coalesce. Histologic analysis showed more solid mass, larger particles and less intervening space with SmGM versus StGM. Conclusions: In a severe, high-volume bleeding model, residual bleeding at 2 minutes was significantly lower with SmGM versus StGM, and SmGM showed greater consistency across bleeding intensities. These findings may be attributable to dry mass per unit volume and/or ultrastructural differences between the two agents.

The Importance of Biophysical and Biochemical Stimuli in Dynamic Skeletal Muscle Models

Classical approaches to engineer skeletal muscle tissue based on current regenerative and surgical procedures still do not meet the desired outcome for patient applications. Besides the evident need to create functional skeletal muscle tissue for the repair of volumetric muscle defects, there is also growing demand for platforms to study muscle-related diseases, such as muscular dystrophies or sarcopenia. Currently, numerous studies exist that have employed a variety of biomaterials, cell types and strategies for maturation of skeletal muscle tissue in 2D and 3D environments. However, researchers are just at the beginning of understanding the impact of different culture settings and their biochemical (growth factors and chemical changes) and biophysical cues (mechanical properties) on myogenesis. With this review we intend to emphasize the need for new in vitro skeletal muscle (disease) models to better recapitulate important structural and functional aspects of muscle development. We highlight the importance of choosing appropriate system components, e.g., cell and biomaterial type, structural and mechanical matrix properties or culture format, and how understanding their interplay will enable researchers to create optimized platforms to investigate myogenesis in healthy and diseased tissue. Thus, we aim to deliver guidelines for experimental designs to allow estimation of the potential influence of the selected skeletal muscle tissue engineering setup on the myogenic outcome prior to their implementation. Moreover, we offer a workflow to facilitate identifying and selecting different analytical tools to demonstrate the successful creation of functional skeletal muscle tissue. Ultimately, a refinement of existing strategies will lead to further progression in understanding important aspects of muscle diseases, muscle aging and muscle regeneration to improve quality of life of patients and enable the establishment of new treatment options.

An overview of surgical sealant devices: current approaches and future trends

ABSTRACT Introduction: Wound leakage is a common complication after surgical incision or resection. In the past, a variety of clinically-approved surgical sealant devices have been used as an adjunct or alternative to conventional means of tissue sealing. However, there is still an unmet need for a sealant that can be universally applied over a wide range of clinical applications. This has further driven the emergence of both novel liquid surgical sealant devices and active hemostatic sealant patches to deal with the increasing complexity of surgical interventions in the field of hemorrhage control and wound leakage. Areas covered: Emphasizing the literature from the past 5 years, this review covers the current offering of natural, synthetic or composite surgical sealant devices in liquid or patch form, their appropriate clinical indications as well as emerging technologies, strategies and products. Expert commentary: Recent years have been marked by dramatic and continuous progress towards the development of novel surgical sealant devices and their rigorous clinical testing. In addition, next-generation sealant formulations such as active hemostatic sealant patches and bio-inspired or nanoparticle-based sealant concepts have been developed and are constantly being evaluated and refined.