Daniel Eberli

The influence of extracellular matrix derived from skeletal muscle tissue on the proliferation and differentiation of myogenic progenitor cells ex vivo

Skeletal muscle relies upon regeneration to maintain homeostasis and repair injury. This process involves the recruitment of the tissues resident stem cell, the muscle progenitor cell, and a subsequent proliferative response by newly generated myoblasts, which must then align and fuse to generate new muscle fibers. During regeneration, cells rely on environmental input for direction. Extracellular matrix (ECM) represents a crucial component of a cells microenvironment that aids in guiding muscle regeneration. We hypothesized that ECM extracted from skeletal muscle would provide muscle progenitor cells and myoblasts with an ideal substrate for growth and differentiation ex vivo. To test this hypothesis, we developed a method to extract ECM from the large thigh muscles of adult rats and present it to cells as a surface coating. Myogenic cells cultured on ECM extract experienced enhanced proliferation and differentiation relative to standard growth surfaces. As the methodology can be applied to any size muscle, these results demonstrate that bioactive ECM can be readily obtained from skeletal muscle and used to develop biomaterials that enhance muscle regeneration. Furthermore, the model system demonstrated here can be applied to the study of interactions between the ECM of a particular tissue and a cell population of interest.

Influence of the fiber diameter and surface roughness of electrospun vascular grafts on blood activation.

Electrospun grafts have been widely investigated for vascular graft replacement due to their ease and compatibility with many natural and synthetic polymers. Here, the effect of the processing parameters on the scaffolds architecture and subsequent reactions of partially heparinized blood triggered by contacting these topographies were studied. Degrapol® (DP) and poly(lactic-co-glycolic acid) (PLGA) electrospun fibrous scaffolds were characterized with regard to fiber diameter, pore area and scaffold roughness. The study showed that electrospinning parameters greatly affect fiber diameter together with pore dimension and overall scaffold roughness. Coagulation cascade activation, early platelet adhesion and activation were analyzed after 2h of exposure of blood to the biomaterials. While no differences were found between DP and PLGA with similar topographies, the blood reactions were observed to be dependent on the fiber diameter and scaffold roughness. Scaffolds composed of thin fibers (diameter <1μm) triggered very low coagulation and almost no platelets adhered. On the other hand, scaffolds with a bigger fiber diameter (2-3μm) triggered higher thrombin formation and more platelets adhered. The highest platelet adhesion and activations rates as well as coagulation cascade activation were found in blood incubated in contact with the scaffolds produced with the biggest fiber diameter (5μm). These findings indicate that electrospun grafts with small fiber diameter (<1μm) could perform better with reduced early thrombogenicity due to lower platelet adhesion and lower activation of platelets and coagulation cascade.

Bioengineered corporal tissue for structural and functional restoration of the penis

Various reconstructive procedures have been attempted to restore a cosmetically acceptable phallus that would allow normal reproductive, sexual, and urinary function in patients requiring penile reconstruction. However, these procedures are limited by a shortage of native penile tissue. We previously demonstrated that a short segment of the penile corporal body can be replaced using naturally derived collagen matrices with autologous cells. In the current study, we examined the feasibility of engineering the entire pendular penile corporal bodies in a rabbit model. Neocorpora were engineered from cavernosal collagen matrices seeded with autologous cells using a multistep static/dynamic procedure, and these were implanted to replace the excised corpora. The bioengineered corpora demonstrated structural and functional parameters similar to native tissue and male rabbits receiving the bilateral implants were able to successfully impregnate females. This study demonstrates that neocorpora can be engineered for total pendular penile corporal body replacement. This technology has considerable potential for patients requiring penile reconstruction.

Urological surgery and antiplatelet drugs after cardiac and cerebrovascular accidents.

PURPOSE The perioperative treatment of patients on dual antiplatelet therapy after myocardial infarction, cerebrovascular event or coronary stent implantation represents an increasingly frequent issue for urologists and anesthesiologists. We assess the current scientific evidence and propose strategies concerning treatment of these patients. MATERIALS AND METHODS A MEDLINE and PubMed search was conducted for articles related to antiplatelet therapy after myocardial infarction, coronary stents and cerebrovascular events, as well as the use of aspirin and/or clopidogrel in the context of surgery. RESULTS Early discontinuation of antiplatelet therapy for secondary prevention is associated with a high risk of coronary thrombosis, which is further increased by the hypercoagulable state induced by surgery. Aspirin has recently been recommended as a lifelong therapy. Clopidogrel is mandatory for 6 weeks after myocardial infarction and bare metal stents, and for 12 months after drug-eluting stents. Surgery must be postponed beyond these waiting periods or performed with patients receiving dual antiplatelet therapy because withdrawal therapy increases 5 to 10 times the risk of postoperative myocardial infarction, stent thrombosis or death. The shorter the waiting period between revascularization and surgery the greater the risk of adverse cardiac events. The risk of surgical hemorrhage is increased approximately 20% by aspirin and 50% by clopidogrel. CONCLUSIONS The risk of coronary thrombosis when antiplatelet agents are withdrawn before surgery is generally higher than the risk of surgical hemorrhage when antiplatelet agents are maintained. However, this issue has not yet been sufficiently evaluated in urological patients and in many instances during urological surgery the risk of bleeding can be dangerous. A thorough dialogue among surgeon, cardiologist and anesthesiologist is essential to determine all risk factors and define the best possible strategy for each patient.

Paracrine effects of mesenchymal stem cells enhance vascular regeneration in ischemic murine skin

New theories on the regeneration of ischemic vasculature have emerged indicating a pivotal role of adult stem cells. The aim of this study was to investigate homing and hemodynamic effects of circulating bone marrow-derived mesenchymal stem cells (MSCs) in a critically ischemic murine skin flap model. Bone marrow-derived mesenchymal stem cells (Lin(-)CD105(+)) were harvested from GFP(+)-donor mice and transferred to wildtype C57BL/6 mice. Animals receiving GFP(+)-fibroblasts served as a control group. Laser scanning confocal microscopy and intravital fluorescence microscopy were used for morphological analysis, monitoring and quantitative assessment of the stem cell homing and microhemodynamics over two weeks. Immunohistochemical staining was performed for GFP, eNOS, iNOS, VEGF. Tissue viability was analyzed by TUNEL-assay. We were able to visualize perivascular homing of MSCs in vivo. After 4 days, MSCs aligned along the vascular wall without undergoing endothelial or smooth muscle cell differentiation during the observation period. The gradual increase in arterial vascular resistance observed in the control group was abolished after MSC administration (P<0.01). At capillary level, a strong angiogenic response was found from day 7 onwards. Functional capillary density was raised in the MSC group to 197% compared to 132% in the control group (P<0.01). Paracrine expression of VEGF and iNOS, but not eNOS could be shown in the MSC group but not in the controls. In conclusion, we demonstrated that circulating bone marrow-derived MSCs home to perivascular sites in critically ischemic tissue, exhibits paracrine function and augment microhemodynamics. These effects were mediated through arteriogenesis and angiogenesis, which contributed to vascular regeneration.

Engineering functional bladder tissues

Purpose: End stage bladder disease can seriously affect patient quality of life and often requires surgical reconstruction with bowel tissue, which is associated with numerous complications. Bioengineering of functional bladder tissue using tissue‐engineering techniques could provide new functional tissues for reconstruction. In this review, we discuss the current state of this field and address different approaches to enable physiologic voiding in engineered bladder tissues in the near future. Materials and Methods: In a collaborative effort, we gathered researchers from four institutions to discuss the current state of functional bladder engineering. A MEDLINE® and PubMed® search was conducted for articles related to tissue engineering of the bladder, with special focus on the cells and biomaterials employed as well as the microenvironment, vascularisation and innervation strategies used. Results: Over the last decade, advances in tissue engineering technology have laid the groundwork for the development of a biological substitute for bladder tissue that can support storage of urine and restore physiologic voiding. Although many researchers have been able to demonstrate the formation of engineered tissue with a structure similar to that of native bladder tissue, restoration of physiologic voiding using these constructs has never been demonstrated. The main issues hindering the development of larger contractile tissues that allow physiologic voiding include the development of correct muscle alignment, proper innervation and vascularization. Conclusion: Tissue engineering of a construct that will support the contractile properties that allow physiologic voiding is a complex process. The combination of smart scaffolds with controlled topography, the ability to deliver multiple trophic factors and an optimal cell source will allow for the engineering of functional bladder tissues in the near future. Copyright

Optimization of human skeletal muscle precursor cell culture and myofiber formation in vitro

Muscle bioengineering is proposed as a treatment option for various conditions requiring restoration of muscle function. In order to allow for rapid clinical translation culture conditions have to be optimized for human application. The optimal isolation and culture technique should be able to support cell growth and differentiation using defined media only. Therefore, we have evaluated alternative culture conditions to determine the optimal growth condition for the engineering of human skeletal muscle. In this research, we present protocols for consistent isolation and growth of human muscle precursor cells (MPCs). MPCs were grown from human biopsies and expanded in culture using defined media and collagen coated dishes only. The best results were achieved using a one-step pre-plating and by supplementing the growth medium with insulin, dexamethasone, human basic fibroblast growth factor (hFGF) and human epithelial growth factor (hEGF). Detailed cell characterization using fluorescence-activated cell-sorting analysis and morphological analysis at different passages were performed. Further, the applicability of these cells for tissue engineering purposes was assessed by measuring expansion potential, formation of myofibers and fused myotubes. We have established a culture technique for human MPCs that allows for reliable cell growth and expansion using collagen coated dishes and defined media only. Cell characterization demonstrated a muscle phenotype and the ability to form myofibers in vitro.

Composite scaffolds for the engineering of hollow organs and tissues

Several types of synthetic and naturally derived biomaterials have been used for augmenting hollow organs and tissues. However, each has desirable traits which were exclusive of the other. We fabricated a composite scaffold and tested its potential for the engineering of hollow organs in a bladder tissue model. The composite scaffolds were configured to accommodate a large number of cells on one side and were designed to serve as a barrier on the opposite side. The scaffolds were fabricated by bonding a collagen matrix to PGA polymers with threaded collagen fiber stitches. Urothelial and bladder smooth muscle cells were seeded on the composite scaffolds, and implanted in mice for up to 4 weeks and analyzed. Both cell types readily attached and proliferated on the scaffolds and formed bladder tissue-like structures in vivo. These structures consisted of a luminal urothelial layer, a collagen rich compartment and a peripheral smooth muscle layer. Biomechanical studies demonstrated that the tissues were readily elastic while maintaining their pre-configured structures. This study demonstrates that a composite scaffold can be fabricated with two completely different polymer systems for the engineering of hollow organs. The composite scaffolds are biocompatible, possess adequate physical and structural characteristics for bladder tissue engineering, and are able to form tissues in vivo. This scaffold system may be useful in patients requiring hollow organ replacement.

Computed tomography perfusion imaging of renal cell carcinoma: systematic comparison with histopathological angiogenic and prognostic markers.

PurposeThe aim of this study was to systematically analyze the correlation between computed tomography (CT) perfusion and histopathological angiogenic and prognostic markers in patients with renal cell carcinoma (RCC). Material and MethodsFifteen patients (12 men; mean age, 64.5 ± 9.4 years) with RCC underwent contrast-enhanced CT perfusion imaging (scan range, 10 cm; scan time, 40 seconds; dual-source 128-section CT) 1 day before surgery. The procedure for surgical specimen processing was modified to obtain an exact match with CT images. Microvessel density (MVD) was quantified by CD34 staining, and lymphatic vessel density (LVD) was stained with D2-40 antibodies. The CT perfusion values blood flow (BF), blood volume (BV), and flow extraction product (KTrans) were calculated using the maximum-slope and a delay-corrected modified Patlak approach and were correlated to MVD and LVD. The relationship between CT perfusion and the prognostic markers pT stage, Fuhrman grade, and tumor necrosis was evaluated. ResultsHistopathology revealed varying high MVD but low or absent intratumoral LVD. The BF and BV of RCC, both including and excluding necrotic regions, showed significant correlations with MVD (r = 0.600–0.829, P < 0.05 each). Significant correlations between MVD and KTrans were found only in small tumor areas exhibiting no necrosis (r = 0.550, P < 0.05). No significant correlation was found between BF, BV, and KTrans with intratumoral LVD (P = 0.35–0.82). With higher pT stage and Fuhrman grade, BF, BV, and KTrans were lower, similar to the MVD, but without reaching statistical significance. Blood flow, BV, and KTrans were significantly higher in RCCs with less than 50% necrosis than in those with 50% or grater necrosis (P < 0.05 each). ConclusionOur study indicates that BF and BV from CT perfusion reflect blood vessels of RCC. Computed tompgraphic perfusion parameters differ significantly depending upon the degree of tumor necrosis.

Hemostatic properties and the role of cell receptor recognition in human hair keratin protein hydrogels

Driven by new discoveries in stem-cell biology and regenerative medicine, there is broad interest in biomaterials that go beyond basic interactions with cells and tissues to actively direct and sustain cellular behavior. Keratin biomaterials have the potential to achieve these goals but have been inadequately described in terms of composition, structure, and cell-instructive characteristics. In this manuscript we describe and characterize a keratin-based biomaterial, demonstrate self-assembly of cross-linked hydrogels, investigate a cell-specific interaction that is dependent on the hydrogel structure and mediated by specific biomaterial-receptor interactions, and show one potential medical application that relies on receptor binding - the ability to achieve hemostasis in a lethal liver injury model. Keratin biomaterials represent a significant advance in biotechnology as they combine the compatibility of natural materials with the chemical flexibility of synthetic materials. These characteristics allow for a system that can be formulated into several varieties of cell-instructive biomaterials with potential uses in tissue engineering, regenerative medicine, drug and cell delivery, and trauma.