Tissue engineering approach to repair abdominal wall defects using cell-seeded bovine tunica vaginalis in a rabbit model

The aim of this study was to engineer skeletal muscle tissue for repair abdominal wall defects. Myoblast were seeded onto the scaffolds and cultivated in vitro for 5 days. Full thickness abdominal wall defects (3 × 4 cm) were created in 18 male New Zealand white rabbits and randomly divided into two equal groups. The defects of the first group were repaired with myoblast-seeded-bovine tunica vaginalis whereas the second group repaired with non-seeded-bovine tunica vaginalis and function as a control. Three animals were sacrificed at 7th, 14th, and 30th days of post-implantation from each group and the explanted specimens were subjected to macroscopic and microscopic analysis. In every case, seeded scaffolds have better deposition of newly formed collagen with neo-vascularisation than control group. Interestingly, multinucleated myotubes and myofibers were only detected in cell-seeded group. This study demonstrated that myoblast-seeded-bovine tunica vaginalis can be used as an effective scaffold to repair severe and large abdominal wall defects with regeneration of skeletal muscle tissue

Modulation of Differentiation and Bone Resorbing Activity of Human (Pre-) Osteoclasts After X-Ray Exposure

Low-dose radiotherapy (LD-RT) is a local treatment option for patients with chronic degenerative and inflammatory diseases, in particular musculoskeletal diseases. Despite reported analgesic and anti-inflammatory effects, cellular and molecular mechanisms related to osteoimmunological effects are still elusive. Here we test the hypothesis that X-irradiation inhibits the differentiation of precursor osteoclasts into mature osteoclasts (mOC) and their bone resorbing activity. Circulating monocytes from healthy donors were isolated and irradiated after attachment with single or fractionated X-ray doses, comparable to an LD-RT treatment scheme. Then monocytes underwent ex vivo differentiation into OC during cultivation up to 21 days, under conditions mimicking the physiological microenvironment of OC on bone. After irradiation, apoptotic frequencies were low, but the total number of OC precursors and mOC decreased up to the end of the cultivation period. On top, we observed an impairment of terminal differentiation, i.e. a smaller fraction of mOC, reduced resorbing activity on bone, and release of collagen fragments. We further analyzed the effect of X-irradiation on multinucleation, resulting from the fusion of precursor OC, which occurs late during OC differentiation. At 21 days after exposure, the observation of smaller cellular areas and a reduced number of nuclei per mOC suggest an impaired fusion of OC precursors to form mOC. Before, at 14 days, the nuclear translocation of Nuclear Factor Of Activated T Cells 1 (NFATc1), a master regulator of osteoclast differentiation and fusion, was decreased. In first results, obtained in the frame of a longitudinal LD-RT study, we previously reported a pain-relieving effect in patients. However, in a subgroup of patients suffering from Calcaneodynia or Achillodynia, we did not observe a consistent decrease of established blood markers for resorption and formation of bone, or modified T cell subtypes involved in regulating these processes. To assess the relevance of changes in bone metabolism for other diseases treated with LD-RT will be subject of further studies. Taken together, we observed that in vitro X-irradiation of monocytes results in an inhibition of the differentiation into bone-resorbing OC and a concomitant reduction of resorbing activity. The detected reduced NFATc1 signaling could be one underlying mechanism

Immature cardiomyocytes in the adult zebrafish heart and their role in cardiac regeneration

The adult mammalian heart is a non-regenerative organ that fails to recover neither functionally nor structurally after insults. Although, reports show that the presences of mitotic nuclei after pathological or physiological cardiac stress in humans, it is widely accepted that the regenerative capacity of the human heart is immensely inadequate to restore the loss of cardiomyocytes (CMs) (Beltrami et al., 2001; Kajstura et al., 1998). Consequently, myocardial infarctions (MIs) are the primary cause of cardiovascular morbidity and mortality. MIs is the irreversible loss of cardiac myocytes due to prolonged myocardial ischemia caused by an imbalance of the metabolic demand of the myocardium and myocardial blood flow (Whelan et al., 2010). Patients with MIs often die prematurely because of heart failure, resulting from irreversible scar formation on the ventricular wall and undermined heart function (Jessup and Brozena, 2003). Despite early intervention and advancements of medical devices for prevention, MIs are still untreatable, unless the heart transplantation approach considered, which is very limited by heart donation (Augoustides and Riha, 2009). Therefore, there is a high demand for standard therapy for heart failure that can restore the loss of CMs, prompt myocardial regeneration, and eventually, reduce morbidity and mortality rate of the disease. Contrary to the adult mammalian heart, zebrafish display an extraordinary capacity for heart regeneration after the cardiac insult (Poss et al., 2002). This regenerative response relies on the ability of CMs to proliferate and replenish the lost tissue. Zebrafish is indeed one of the most commonly used experimental models for developmental and regenerative biology studies (Gemberling et al., 2013; Gonzalez-Rosa et al., 2017). For decades, the process of cardiac regeneration has been investigated using various cardiac injury models. The most commonly used and well-established injury methods are ventricular apical resection (Poss et al., 2002; Raya et al., 2003), cryoinjury (Chablais et al., 2011; Schnabel et al., 2011), as well as genetic and chemical ablation of heart cells (Curado et al., 2007; Wang et al., 2011). The origin of new cells is one of the most fundamental questions to be addressed during organ regeneration in any regenerative organism, and understanding of such phenomenon is crucial to design effective therapeutic strategies for non-regenerative organisms (Gonzalez-Rosa et al., 2017; Tanaka and Reddien, 2011). Despite the robust cardiac regenerative potential, to date, only a handful of lineage tracing experiments have been reported in zebrafish heart regeneration. It was proposed that the cellular source of the renewed cardiac tissue might arise from progenitor or stem cells (Lepilina et al., 2006), through CMs dedifferentiation (Jopling et al., 2010; Kikuchi et al., 2010), transdifferentiation from other cell types in the heart tissue, and/or direct proliferation of the existing CMs (Kikuchi and Poss, 2012). Fate-mapping studies using transgenic lines driven by the myl7 promoter have shown that pre-existing CMs contribute to myocardial regeneration. However, myl7 expression is activated at early developmental stages in cardiac progenitor cells and hence precluding the identification of genuinely mature CMs in adult stages. Therefore, the cellular origin of the regenerating CMs remains elusive. Moreover, CM heterogeneity in the developing and adult zebrafish heart has never been explored to provide full insight into the process of regeneration. Therefore, I set out to identify genes exclusively expressed by either immature or mature CMs, generate promoter-driven reporter and CreERT2 lines to characterize the reporters during zebrafish heart development, and regeneration, and eventually to determine the contribution of the immature CMs to the regenerating CMs....Das Herz eines erwachsenen Säugetiers ist ein nicht regenerative Organ, das sich nach Verletzungen weder funktionell noch strukturell erholt. Obwohl Berichte zeigen, dass das Vorhandensein von mitotischen Kernen nach pathologischem oder physiologischem Herzstress beim Menschen besteht, wird allgemein anerkannt, dass die Regenerationsfähigkeit des menschlichen Herzens vollkommen unzureichend ist, um den Verlust von Kardiomyozyten (KMs) wiederherzustellen (Beltrami et al., 2001) Kajstura et al., 1998). Folglich sind Herzinfarkte (Myokardinfarkte, MIs) die Hauptursache für Herz-Kreislauf-Morbidität und Mortalität. MI ist der irreversible Verlust von Herzmuskelzellen aufgrund einer verlängerten myokardialen Ischämie, verursacht durch ein Ungleichgewicht des metabolischen Bedarfs des Myokards und des myokardialen Blutflusses (Whelan et al., 2010). Patienten mit MI sterben häufig vorzeitig an einer Herzinsuffizienz, die auf irreversible Narbenbildung an der Ventrikelwand und unzureichender Herzfunktion zurückzuführen ist (Jessup und Brozena, 2003). Trotz frühzeitiger Interventionen und Weiterentwicklungen von medizinischen Geräten zur Vorbeugung sind MIs immer noch nicht behandelbar, sofern nicht der Ansatz der Herztransplantation in Betracht gezogen wird, der durch die Zahl der Herzspenden sehr begrenzt ist (Augoustides und Riha, 2009). Daher besteht ein hoher Bedarf an Standardtherapien für Herzinsuffizienz, die den Verlust von KMs wiederherstellen, die Myokardregeneration auslösen und schließlich die Morbidität und Mortalitätsrate der Erkrankung senken können. Im Gegensatz zum Herz eines erwachsenen Säugetiers weisen Zebrafische eine außergewöhnliche Fähigkeit zur Herzregeneration nach Herzinsuffizienz auf (Poss et al., 2002). Diese regenerative Antwort beruht auf der Fähigkeit der KMs, zu proliferieren und das verlorene Gewebe zu ersetzen. Zebrafisch ist in der Tat eines der am häufigsten verwendeten experimentellen Modelle für Entwicklungs und Regenerationsbiologie-Studien (Gemberling et al., 2013; Gonzalez-Rosa et al., 2017)..

Skeletal muscle tissue engineering using biological scaffolds for repair of abdominal wall defects in a rabbit model

Abdominal wall defects caused by trauma, tumour ablation, muscle deficiency or postsurgical loss of muscle mass may lead to the need for restoration of damaged muscle tissues as loss of functional muscle tissue could cause severe impairments of the functionality of skeletal muscle. Hence, the present study was aimed mainly to engineer skeletal muscle tissue using myoblast seeded bovine pericardium (BP) and bovine tunica vaginalis (BTV) scaffolds in a rabbit model. Myoblast were harvested successfully from soleus muscles of 5-day-old male White New Zealand rabbit and based on the purity test using immunocytochemistry (desmin staining) and flow cytometric analysis, more than 97% of the isolated skeletal myoblast have got myogenic phenotype. Myoblast were labelled with PKH26-fluorescent dye and seeded onto the scaffolds and incubated in vitro for 5 days. The in vitro findings of myoblast-seeded BP and BTV scaffolds suggest that myoblast harvested from primary culture are able to form myotube on both types of scaffolds and these naturally derived collagenbased scaffolds showed a tremendous potential for in vitro cultivation of skeletal muscle that can be used as substrate for filling of wound bed or for the delivery of cells. A total of thirty-six male New Zealand white rabbits which were divided into two groups (BP and BTV groups) of eighteen rabbits each were used in this study. The rabbits in each group were further subdivided into two groups of nine rabbits each: the treatment groups (I and II) and control groups (III and IV). Myoblast seeded-BP and myoblast seeded-BTV scaffolds were implanted on the artificially created 3 x 4 cm2 defects at mid-ventral abdominal wall on nine rabbits of the treatment groups I and II, respectively. Whereas, control groups III and IV were repaired with non-seeded BP and BTV scaffolds, respectively. Three rabbits from each group were euthanized at 7th, 14th and 30th days of postimplantation and their ex-implanted specimens were examined macroscopically and microscopically. Macroscopic examination of the abdominal wall post-implantation showed no evidence of herniation, signs of rejection and infection in both treatment and control groups of both type of scaffolds. However, 33.33% and 22.22% mild type of adhesion were found in the control groups III and IV, respectively. Whereas, 11.11% mild type of adhesion and absence of adhesion were found in the treatment groups I and II, respectively. At 7th day of post-implantation, microscopic examinations revealed more intense infiltration of granulocytes and macrophages in the treatment and control groups of both types of scaffolds. Whereas, on 14th and 30th days of post-implantation, the fibroblast migration, deposition of newlyformed collagen, neovascularisation and skeletal muscle cells ingrowths were detected in the treatment groups: I and II. However, not a single of skeletal-muscle cell were found in the control groups III and IV. In conclusion, this study demonstrated that myoblast seeded BP and BTV can be successfully transplanted into abdominal wall defects and resulted in the regeneration of skeletal muscle tissue

Myoblast seeded bovine tunica vaginalis for repair abdominal wall defects and method for preparing the same

The present invention relates to a biomaterial for repair of abdominal wall defects comprising a sterile processed bovine tunica vaginalis and myoblast cells and a method for making the same. The preferred embodiment, collagen based biomaterials, which can be used for reconstruction of a abdominal wall defects for regeneration of skeletal muscle tissue in human and veterinary medicine. The present invention also provides a novel method for making/ preparing which increased the biocompatibility and shef life of the invented scaffolds, thereby following of seeding of myoblast on the bovine tunica vaginalis to improve its mechanical performances through skeletal muscle tissue engineering

Myoblast seeded bovine pericardium for repair of abdominal wall defect

The present invention comprises myoblast seeded bovine pericardium and a method of making the same. The preferred embodiment, biological biomaterials which can be used for reconstruction of abdominal wall defects for regeneration o skeletal muscle both in human medicine and veterinary medicine. The present invention also provides a novel method of making/ preparation which possesses an excellent biocompatibility as well as an increased stability of the grant thereby following of seeding of myoblast on the bovine pericardium

Modulation of Differentiation and Bone Resorbing Activity of Human (Pre-) Osteoclasts After X-Ray Exposure

Grant: BMBF; GREWIS-alph

Focal adhesions are essential to drive zebrafish heart valve morphogenesis

Elucidating the morphogenetic events that shape vertebrate heart valves, complex structures that prevent retrograde blood flow, is critical to understanding valvular development and aberrations. Here, we used the zebrafish atrioventricular (AV) valve to investigate these events in real time and at single-cell resolution. We report the initial events of collective migration of AV endocardial cells (ECs) into the extracellular matrix (ECM), and their subsequent rearrangements to form the leaflets. We functionally characterize integrin-based focal adhesions (FAs), critical mediators of cell–ECM interactions, during valve morphogenesis. Using transgenes to block FA signaling specifically in AV ECs as well as loss-of-function approaches, we show that FA signaling mediated by Integrin α5β1 and Talin1 promotes AV EC migration and overall shaping of the valve leaflets. Altogether, our investigation reveals the critical processes driving cardiac valve morphogenesis in vivo and establishes the zebrafish AV valve as a vertebrate model to study FA-regulated tissue morphogenesis