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Introduction: Three-dimensional bioprinting can be considered as an advancement of the classical tissue engineering concept. For bioprinting, cells have to be dispersed in hydrogels. Recently, a novel semi-synthetic thiolene hydrogel system based on norbornene-functionalized gelatin (GelNB) and thiolated gelatin (GelS) was described that resulted in the photoclick hydrogel GelNB/GelS. In this study, we evaluated the printability and biocompatibility of this hydrogel system towards adipose-tissue-derived mesenchymal stem cells (ASCs). Methods: GelNB/GelS was synthesized with three different crosslinking densities (low, medium and high), resulting in different mechanical properties with moduli of elasticity between 206 Pa and 1383 Pa. These hydrogels were tested for their biocompatibility towards ASCs in terms of their viability, proliferation and differentiation. The extrusion-based bioprinting of ASCs in GelNB/GelS-high was performed to manufacture three-dimensional cubic constructs. Results: All three hydrogels supported the viability, proliferation and chondrogenic differentiation of ASCs to a similar extent. The adipogenic differentiation of ASCs was better supported by the softer hydrogel (GelNB/GelS-low), whereas the osteogenic differentiation was more pronounced in the harder hydrogel (GelNB/GelS-high), indicating that the differentiation fate of ASCs can be influenced via the adaption of the mechanical properties of the GelNB/GelS system. After the ex vivo chondrogenic differentiation and subcutaneous implantation of the bioprinted construct into immunocompromised mice, the production of negatively charged sulfated proteoglycans could be observed with only minimal inflammatory signs in the implanted material. Conclusions: Our results indicate that the GelNB/GelS hydrogels are very well suited for the bioprinting of ASCs and may represent attractive hydrogels for subsequent in vivo tissue engineering applications. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) play an important role in tissue engineering applications aiming at the regeneration or substitution of damaged tissues. In this context, off-the-shelf allogeneic MSCs would represent an attractive universal cell source. However, immune rejection is a major limitation for the clinical use of allogeneic MSCs. Immune rejection is mediated by the expression of major histocompatibility complexes (MHC)-I and -II on the donor cells. In this study, we eliminated MHC-I and/or MHC-II expression in human MSCs by using the CRISPR/Cas9 technology and investigated the effect of the individual or combined knockout of MHC-I and MHC-II on MSC survival after transplantation into immunocompetent mice. Elimination of MHC-I and/or MHC-II expression did not affect mesenchymal marker gene expression, viability, proliferation and the differentiation potential of MSCs in vitro. However, cell survival of transplanted MSCs was significantly elevated in MHC-I and MHC-II deficient MSCs. A direct side-by-side comparison does not reveal any significant difference in the immunogenicity of MHC-I and MHC-II knockout MSCs. Moreover, double knockout of MHC-I and MHC-II did not further increase in vivo cell survival of transplanted MSCs. Our results demonstrate that knockout of MHC-I and/or MHC-II represents an effective strategy to prevent immune rejection of allogeneic MSCs. [ABSTRACT FROM AUTHOR]

The homogeneity of the genetically modified single-cells is a necessity for many applications such as cell line development, gene therapy, and tissue engineering and in particular for regenerative medical applications. The lack of tools to effectively isolate and characterize CRISPR/Cas9 engineered cells is considered as a significant bottleneck in these applications. Especially the incompatibility of protein detection technologies to confirm protein expression changes without a preconditional large-scale clonal expansion creates a gridlock in many applications. To ameliorate the characterization of engineered cells, we propose an improved workflow, including single-cell printing/isolation technology based on fluorescent properties with high yield, a genomic edit screen (Surveyor assay), mRNA RT-PCR assessing altered gene expression, and a versatile protein detection tool called emulsion-coupling to deliver a high-content, unified single-cell workflow. The workflow was exemplified by engineering and functionally validating RANKL knockout immortalized mesenchymal stem cells showing bone formation capacity of these cells. The resulting workflow is economical, without the requirement of large-scale clonal expansions of the cells with overall cloning efficiency above 30% of CRISPR/Cas9 edited cells. Nevertheless, as the single-cell clones are comprehensively characterized at an early, highly parallel phase of the development of cells including DNA, RNA, and protein levels, the workflow delivers a higher number of successfully edited cells for further characterization, lowering the chance of late failures in the development process. [ABSTRACT FROM AUTHOR]

Background: Three-dimensional (3D) scanning is an established method of breast volume estimation. However, this method can never be entirely precise, since the thoracic wall cannot be imaged by the surface scanner. Current methods rely on interpolation of the posterior breast border from the surrounding thoracic wall. Here, we present a novel method to calculate the posterior border and increase the accuracy of the measurement. Methods: Using principal component analysis, computed tomography images were used to build a statistical shape model (SSM) of the thoracic wall. The model was fitted to 3D images and the missing thoracic wall curvature interpolated (indirect volumetry). The calculations were evaluated by ordinary least squares regression between the preoperative and postoperative volume differences and the resection weights in breast reduction surgery (N = 36). Also, an SSM of the breast was developed, allowing direct volumetry. Magnetic-resonance images (MRI) and 3D scans were acquired from 5 patients in order to validate the direct 3D volumetry. Results: Volumetry based on a SSM exhibited a higher determination coefficient (R2 = 0,737) than the interpolation method (R2 = 0,404). The methods were not equivalent (p = 0.75), suggesting that the methods significantly differ. There was no influence of BMI on the correlation in either method. The MRI volumetry had a strong correlation with the 3D volumetry (R2 = 0,978). Conclusion: The SSM-based method of posterior breast border calculation is reliable and superior to the currently used method of interpolation. It should serve as a basis of software applications aiming at calculation of breast volume from 3D surface scanning data. [ABSTRACT FROM AUTHOR]

Introduction: Mesenchymal stem cells (MSCs) represent a very important cell source in the field of regenerative medicine and for bone and cartilage tissue engineering applications. Three-dimensional (3D) bioprinting has the potential to improve the classical tissue engineering concept as this technique allows the printing of cells with high spatial control of cell allocation within a 3D construct. In this study, we systematically compared different hydrogel blends for 3D bioprinting of MSCs by testing their cytocompatibility, ability to support osteogenic differentiation and their mechanical properties. In addition, we compared four different MSC populations isolated from different human tissues for their osteogenic differentiation capacity in combination with different hydrogels. The aim of this study was to identify the best MSC source and the most suitable hydrogel blend for extrusion-based bioprinting of 3D large-scaled osteogenic constructs. Materials and Methods: MSCs were isolated from different tissues (umbilical cord, adipose tissue, bone marrow). MSCs were seed onto or into different hydrogels and analyzed for cell viability, proliferation and osteogenic differentiation. In addition, viscoelastic properties of the hydrogels were determined. MSC-containing cubes with the size of 1 cm3 were printed by means of 3D extrusion-based bioprinting and analyzed by (immuno)histology for cell survival and production of a calcified extracellular matrix. Results: Adipose tissue derived MSCs (ASCs) showed the highest osteogenic differentiation potential. A complex hydrogel blend consisting of fibrin, gelatin, hyaluronic acid, glycerol (F/G/H/Gl), tuned with hydroxyapatite, showed the best viscoelastic properties in combination with an excellent biocompatibility towards ASCs. This cell/hydrogel combination was used to bioprint 3D cubes. The cubes showed good mechanical stability and the printed ASCs were viable and able to calcify the hydrogel after bioprinting. Conclusions: The combination of the HA-tuned F/G/H/Gl hydrogel blend along with ASCs can be considered as a very promising bioink for 3D bioprinting of artificial bone tissue equivalents for prospective applications in tissue engineering and regenerative medicine. [ABSTRACT FROM AUTHOR]

Objective: Gynecomastia is a common finding in the male population which is mostly idiopathic. The aim of our study was to analyze the histological differences in young and old patient groups and its association with recurrence rates. Methods: Three hundred and five gynecomastia patients (555 breasts) undergoing surgical treatment from 1997 to 2015 were divided into four groups: Group 1: 13-17 years, Group 2: 18-30 years, Group 3: 31-49 years and Group 4: 50-83 years. They were evaluated concerning clinical classification, histological differences and association with antiandrogen or steroids/immunosuppressive therapy. Results: We found that the rate of florid gynecomastia was higher in older patient groups, while fibrous gynecomastia was more common in adolescents and young adults (p = .0180). Glandular gynecomastia was more frequent in younger patients, while in the older patient groups, lipomatous gynecomastia was more common (p = .0006). Patients presenting with florid gynecomastia showed a higher rate of recurrence than patients with the fibrous type of gynecomastia (12.5 and 4.7%, respectively). Of note, 18.75% of florid gynecomastia was associated with antiandrogen agents or steroid/immunosuppressive therapy, while only 4.69% of fibrous gynecomastia was associated with antiandrogenic or immunosuppressive therapy. However, there was no increase of recurrence rates in patients using antiandrogen agents or undergoing steroid/immunosuppressive therapy. Conclusions: Fibrous gynecomastia was found to be more common in adolescents and young adults, while the florid type was more frequent in older patients. Patients presenting with florid gynecomastia showed a higher rate of recurrence than patients with the fibrous type of gynecomastia. [ABSTRACT FROM AUTHOR]

Introduction: C-reactive protein circulates as a pentameric protein (pCRP). pCRP is a well-established diagnostic marker as plasma levels rise in response to tissue injury and inflammation. We recently described pro-inflammatory properties of CRP, which are mediated by conformational changes from pCRP to bioactive isoforms expressing pro-inflammatory neo-epitopes [pCRP* and monomeric C-reactive protein (mCRP)]. Here, we investigate the role of CRP isoforms in renal ischemia/reperfusion injury (IRI). Methods: Rat kidneys in animals with and without intraperitoneally injected pCRP were subjected to IRI by the time of pCRP exposure and were subsequently analyzed for monocyte infiltration, caspase-3 expression, and tubular damage. Blood urea nitrogen (BUN) was analyzed pre-ischemia and post-reperfusion. CRP effects on leukocyte recruitment were investigated via intravital imaging of rat-striated muscle IRI. Localized conformational CRP changes were analyzed by immunohistochemistry using conformation specific antibodies. 1,6-bis(phosphocholine)-hexane (1,6-bisPC), which stabilizes CRP in its native pentameric form was used to validate CRP effects. Leukocyte activation was assessed by quantification of reactive oxygen species (ROS) induction by CRP isoforms ex vivo and in vitro through electron spin resonance spectroscopy. Signaling pathways were analyzed by disrupting lipid rafts with nystatin and subsequent ROS detection. In order to confirm the translational relevance of our findings, biopsies of microsurgical human free tissue transfers before and after IRI were examined by immunofluorescence for CRP deposition and co-localization of CD68+ leukocytes. Results: The application of pCRP aggravates tissue damage in renal IRI. 1,6-bisPC reverses these effects via inhibition of the conformational change that leads to exposure of pro-inflammatory epitopes in CRP (pCRP* and mCRP). Structurally altered CRP induces leukocyte-endothelial interaction and induces ROS formation in leukocytes, the latter can be abrogated by blocking lipid raft-dependent signaling pathways with Nystatin. Stabilizing pCRP in its native pentameric state abrogates these pro-inflammatory effects. Importantly, these findings are confirmed in human IRI challenged muscle tissue. Conclusion: These results suggest that CRP is a potent modulator of IRI. Stabilizing the native pCRP conformation represents a promising anti-inflammatory therapeutic strategy by attenuation of leukocyte recruitment and ROS formation, the primary pathomechanisms of IRI. [ABSTRACT FROM AUTHOR]

In tissue engineering applications, vascularization can be accomplished by coimplantation of tissue forming cells and endothelial cells (ECs), whereby the latter are able to form functional blood vessels. The use of three-dimensional (3D) bioprinting technologies has the potential to improve the classical tissue engineering approach because these will allow the generation of scaffolds with high spatial control of endothelial cell allocation. This study focuses on a side by side comparison of popular commercially available bioprinting hydrogels (Matrigel, fibrin, collagen, gelatin, agarose, Pluronic F-127, alginate, and alginate/gelatin) in the context of their physicochemical parameters, their swelling/degradation characteristics, their biological effects on vasculogenesis-related EC parameters and their printability. The aim of this study was to identify the most suitable hydrogel or hydrogel combination for inkjet printing of ECs to build prevascularized tissue constructs. Most tested hydrogels displayed physicochemical characteristics suitable for inkjet printing. However, Pluronic F-127 and the alginate/gelatin blend were rapidly degraded when incubated in cell culture medium. Agarose, Pluronic F- 127, alginate and alginate/gelatin hydrogels turned out to be unsuitable for bioprinting of ECs because of their nonadherent properties and/or their incapability to support EC proliferation. Gelatin was able to support EC proliferation and viability but was unable to support endothelial cell sprouting. Our experiments revealed fibrin and collagen to be most suitable for bioprinting of ECs, because these hydrogels showed acceptable swelling/degradation characteristics, supported vasculogenesis-related EC parameters and showed good printability. Moreover, ECs in constructs of preformed spheroids survived the printing process and formed capillary-like cords. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) represent a very attractive cell source for tissue engineering applications aiming at the generation of artificial bone substitutes. The use of three-dimensional bioprinting technologies has the potential to improve the classical tissue engineering approach because bioprinting will allow the generation of hydrogel scaffolds with high spatial control of MSC allocation within the bioprinted construct. In this study, we have performed direct comparisons between commercially available hydrogels in the context of their cytocompatibility toward MSCs and their physicochemical parameters with the aim to identify the most suitable hydrogel for drop-on-demand (DoD) printing of MSCs. In this context, we examined matrigel, fibrin, collagen, gelatin, and gelatin/alginate at various hydrogel concentrations. Matrigel, fibrin, collagen, and gelatin were able to support cell viability, but the latter showed a limited potential to promote MSC proliferation. We concentrated our study on fibrin and collagen hydrogels and investigated the effect of hydroxyapatite (HA) inclusion. The inclusion of HA enhanced proliferation and osteogenic differentiation of MSCs and prevented degradation of fibrin in vitro. According to viscosity and storage moduli measurements, HA-blends displayed physicochemical characteristics suitable for DoD printing. In bioprinting experiments, we confirmed that fibrin and collagen and their respective HA-blends represent excellent hydrogels for DoD-based printing as evidenced by high survival rates of printed MSCs. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3231-3241, 2017. [ABSTRACT FROM AUTHOR]

Background: Synovial sarcoma account for approximately 10 % of all soft-tissue tumors and occur most frequently in young adults. A specific translocation in this sarcoma induces fusion of the SYT gene on chromosome 18 to the SSX genes on chromosome X, leading to proliferation of the tumor cells. The need for non-invasive biomarkers indicating recurrence and activity of this disease has sparked research into short non-coding RNA known as microRNA (miRNA). Methods: Blood samples of patients with active synovial sarcoma and of synovial sarcoma patients in complete remission as well as of healthy donors and patients with active leiomyosarcoma, MPNST, Ewing sarcoma and liposarcoma were collected. Whole blood RNA was extracted and samples of patients with active synovial sarcoma and of healthy donors were analyzed using an Affymetrix GeneChip miRNA Array v. 4.0. qRT-PCR was carried out to confirm a panel of miRNAs which where differentially expressed in the miRNA array. This miRNA-panel was further evaluated in patients with synovial sarcoma in complete remission and patients with active leiomyosarcoma, MPNST, Ewing sarcoma and liposarcoma as well as in an independent cohort of synovial sarcoma patients. Results: Unsupervised hierarchical clustering of the miRNA arrays separated patients with active synovial sarcoma from healthy controls. A panel of seven miRNAs (miR-99a-5p, miR-146b-5p, miR-148b-3p, miR-195-5p, miR-223-3p, miR-500b-3p and miR-505-3p) was further validated by qRT-PCR to be significantly upregulated in synovial sarcoma patients. Moreover, most of the analyzed miRNAs were shown to be significantly upregulated in synovial sarcoma patients compared to leiomyosarcoma, MPNST, Ewing sarcoma and liposarcoma patients. Validation of the miRNA panel in an independent cohort of synovial sarcoma patients confirmed higher expression levels compared to healthy controls and patients in complete remission. Conclusion: Our results have identified a specific whole blood miRNA signature that may serve as an independent biomarker for the diagnosis of local recurrence or distant metastasis of synovial sarcoma. It even distinguishes synovial sarcoma from other sarcoma subtypes, thus potentially serving as a specific biomarker for synovial sarcoma. [ABSTRACT FROM AUTHOR]

Aim of the study: We developed an experimental rat model to explore the possibility of enhancing the healing of critical-size bone defects. The aim of this study was to demonstrate the feasibility of this concept by achieving high local BMP-2 expression via a transduced muscle flap that would facilitate bony union while minimizing systemic sequelae. Methods: The transduction potential of the adenoviral vector encoding for BMP-2 was tested in different cell lines in vitro. In vivo experiments consisted of harvesting a pedicled quadriceps femoris muscle flap with subsequent creation of a critical-size defect in the left femur in Sprague-Dawley rats. Next, the pedicled muscle flap was perfused with high titers of Ad.BMP-2 and Ad.GFP virus, respectively. Twelve animals were divided into three groups comparing the effects of Ad.BMP-2 transduction to Ad.GFP and placebo. Bone healing was monitored radiologically with subsequent histological analysis post-mortem. Results: The feasibility of this concept was demonstrated by successful transduction in vitro and in vivo as evidenced by a marked increase of BMP-2 expression. The three examined groups only showed minor difference regarding bone regeneration; however, one complete bridging of the defect was observed in the Ad.BMP-2 group. No evidence of systemic viral contamination was noted. Conclusions: A marked increase of local BMP-2 expression (without untoward systemic sequelae) was detected. However, bone healing was not found to be significantly enhanced, possibly due to the small sample size of the study. [ABSTRACT FROM AUTHOR]

Vascularization is essential in bone tissue engineering and recent research has focused on interactions between osteoblasts (hOBs) and endothelial cells (ECs). It was shown that cocultivation increases the stability of osteoblastic alkaline phosphatase (ALP) mRNA. We investigated the mechanisms behind this observation, focusing on mRNA binding proteins. Using a luciferase reporter assay, we found that the 3'-untranslated region (UTR) of ALP mRNA is necessary for human umbilical vein endothelial cells (HUVEC)-mediated stabilization of osteoblastic ALP mRNA. Using pulldown experiments and nanoflow-HPLC mass spectrometry, vimentin was identified to bind to the 3'-UTR of ALP mRNA. Validation was performed by Western blotting. Functional experiments inhibiting intermediate filaments with iminodipropionitrile and specific inhibition of vimentin by siRNA transfection showed reduced levels of ALP mRNA and protein. Therefore, ALP mRNA binds to and is stabilized by vimentin. This data add to the understanding of intracellular trafficking of ALP mRNA, its function, and have possible implications in tissue engineering applications. [ABSTRACT FROM AUTHOR]

Vascularization plays an important role in tissue engineering applications. It is known that implantation of differentiated endothelial cells or endothelial progenitor cells (EPCs) from cord blood (cbEPCs) gives rise to the formation of a complex functional neovasculature, whereas EPCs isolated from peripheral blood (pbEPCs) have a limited capability to form blood vessels upon implantation. MicroRNA-126 (miR-126) has been shown to have pro-angiogenic effects in vivo. In this study, we investigated whether modulation of miR-126 expression in pbEPCs may alter their angiogenic properties. Gain of function and loss of function experiments revealed that miR-126 has anti-angiogenic effects in pbEPCs. Overexpression of miR-126 resulted in decreased proliferation, migration, invasion and tube formation, while inhibition of miR-126 induced the opposite effects. However, modulation of miR-126 expression did not influence apoptotic susceptibility of pbEPCs. This study provides evidence that inhibition of miR-126 improves angiogenesis-related growth parameters in pbEPCs and may represent a therapeutic option to ameliorate the angiogenic and vasculogenic properties of pbEPCs. [ABSTRACT FROM AUTHOR]

Adequate vascularization is an essential requirement for bone development, fracture healing and bone tissue engineering. We have previously described the coculture of primary human osteoblasts (hOBs) and human endothelial cells (HUVECs), designed to investigate the interactions between these cells. In this system, we showed that cocultivation of these two cell types leads to a downregulation of platelet-derived growth factor receptor-α (PDGFR-α) in hOBs, which was a consequence of reduced mRNA stability. In the current study we investigated the possible involvement of microRNAs in this process. Firstly, we performed a microarray analysis of osteoblastic miRNAs following cocultivation with HUVECs, revealing an upregulation of miR-126. This result was confirmed by RT-qPCR, and we observed that the increase is dependent on direct cell-to-cell contacts. Gain-of-function and loss-of-function experiments showed that miR-126 is a negative regulator of PDGFR-α mRNA. Additionally, migration of hOBs was inhibited by miR-126 overexpression and stimulated by miR-126 inhibition. Addition of PDGFR-α blocking antibody to hOB culture also inhibited hOB migration. There was no effect of miR-126 modulation on osteoblast proliferation, apoptosis rate or differentiation. In conclusion, we report that the miR-126/PDGFR-α system regulates the migratory behavior of human osteoblasts, without exerting effects on cell survival and differentiation. [ABSTRACT FROM AUTHOR]

Bone tissue is dependent on an efficient blood supply to ensure delivery of nutrients and oxygen. One method to acquire a vascular-engineered bone tissue could be the use of an angiogenic gene-activated scaffold. In the current study, porous chitosan/hydroxyapatite (C/HA) scaffolds were fabricated via freeze-drying with desired pore size, and then combined with the adenoviral vector encoding vascular endothelial growth factor and green fluorescence protein (Ad-VEGF). Human osteoblasts were cultured and seeded on characterized scaffolds. The attachment, proliferation, and differentiation of cells on gene-activated and unactivated C/HA scaffolds were evaluated in vitro and in vivo by histo- and immunohistochemistry. Findings confirmed that human osteoblasts cultured on gene-activated C/HA scaffold secreted vascular endothelial growth factor, besides maintaining its characteristic phenotype with specific extracellular matrix production. In vivo experiments indicated that scaffolds were tissue biocompatible, and that gene-activated scaffold provided a suitable environment for neovessel formation by recruiting host endothelial cells into the newly forming ectopic bone-like tissue. This study revealed that the Ad-VEGF-activated C/HA composite scaffold has potential for vascular bone regeneration applications. [ABSTRACT FROM AUTHOR]

Angiogenesis is a fundamental process in bone formation, remodeling, and regeneration. Moreover, for the regeneration of bone in tissue engineering applications, it is essential to support neovascularization. This can be achieved by cell-based therapies using primary endothelial cells, which are able to form functional blood vessels upon implantation. In bone composite grafts, coimplanted endothelial cells do not only support neovascularization but also support osteogenic differentiation of osteoblasts and osteoprogenitor cells. In this study, we investigated the effect of endothelial cells on proliferation and cell survival of human primary osteoblasts (hOBs) and human mesenchymal stem cells (MSCs). Human umbilical vein endothelial cells (HUVECs) stimulated hOB and MSC proliferation, whereas proliferation of HUVECs was unaffected by cocultured hOBs or MSCs. The effect of HUVEC cocultivation on hOB and MSC proliferation was more pronounced in direct cocultures than in indirect cocultures, indicating that this effect is at least partially dependent on the formation of heterotypic cell contacts between HUVECs and hOBs or MSCs. Furthermore, HUVEC cocultivation reduced low-serum induced apotosis of hOBs and MSCs by a mechanism involving increased phosphorylation and inactivation of the proapoptotic protein Bad. In summary, our experiments have shown that cocultured HUVECs increase the proliferation and reduce low-serum induced apoptosis of hOBs and MSCs. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1682-1689, 2012 [ABSTRACT FROM AUTHOR]

Background: The diagnosis of soft-tissue sarcomas of the limbs is often delayed, sometimes markedly so, even though prompt and appropriate treatment improves survival and lowers the amputation rate. [ABSTRACT FROM AUTHOR]

Various approaches to endoscopic carpal tunnel release have been described, including the advantages of the open compared with the endoscopic technique. However recent results suggest that both are equal in terms of outcome and morbidity. The misconception about the potential morbidity and the hope of successful conservative treatment can sometimes delay operative release of the carpal tunnel. The aim of this study was to evaluate whether the preoperative duration of symptoms influences outcome and recovery. Patients who had endoscopic release of the carpal tunnel using a modified one-port method were included in this retrospective study. Patients' satisfaction and general outcome were analysed with a questionnaire. A total of 242 patients were included in the study, and the data of 170 endoscopic decompression operations were analysed (70%). There were no major operative complications, except the conversion to open release in one case. There was a significant association between the preoperative interval of symptoms and the return to everyday activities and normal function ( p < 0.001). Patients with longer-lasting symptoms also had reduced recovery of postoperative strength, which was negatively associated with the duration of preoperative symptoms ( p < 0.001). Operative decompression by the endoscopic one-port method is a low risk procedure with a low morbidity. The decision for operative decompression should be made as early as possible to avoid complications seen in patients with long-lasting symptoms and permanent nerve damage. [ABSTRACT FROM AUTHOR]

Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in osteoblast function. Inhibition of PDGFR activity leads to a suppression of osteoblast proliferation, whereas mineralized matrix production is enhanced. In previous experiments, we showed that co-cultivation of human primary endothelial cells and human primary osteoblasts (hOBs) leads to a cell contact-dependent downregulation of PDGFR-α expression in the osteoblasts. In this study, we investigated this effect in more detail, revealing that human umbilical vein endothelial cell (HUVEC)-mediated PDGFR-α downregulation is dependent on time and cell number. This effect was specific to endothelial cells and was not observed when hOBs were co-cultured with human primary chondrocytes or fibroblasts. Likewise, HUVEC-mediated suppression of PDGFR-α expression was only seen in hOBs and mesenchymal stem cells but not in immortalized osteoblastic cell lines. Functional inhibition of gap junctional communication between HUVECs and hOBs by 18α-glycyrrhetinic acid had no effect on HUVEC-mediated PDGFR-α downregulation, whereas inhibition of p38 mitogen-activated protein kinase (MAPK) prevented the HUVEC-mediated reduction in osteoblastic PDGFR-α expression. To delineate the molecular mechanism underlying the PDGFR-α downregulation, we examined the effect of HUVEC co-cultivation on osteoblastic PDGFR-α promoter activity as well as mRNA stability. Co-cultivation of HUVECs with hOBs significantly shortened the half-life of osteoblastic PDGFR-α mRNA, but did not decrease its promoter activity. In summary, our data show that PDGFR-α is downregulated in hOBs by co-cultivation with human primary endothelial cells through a p38 MAPK-dependent post-transcriptional mechanism. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The neovascularization of three-dimensional voluminous tissues, such as bone, represents an important challenge in tissue engineering applications. The formation of a preformed vascular plexus could maintain cell viability and promote vascularization after transplantation. We have developed a three-dimensional spheroidal coculture system consisting of human primary endothelial cells and human primary osteoblasts (hOBs) to improve angiogenesis in bone tissue engineering applications. In this study, we investigated the survival and vascularization of the engineered implants in vivo. Endothelial cell spheroids were cocultured with hOBs in fibrin and seeded into scaffolds consisting of processed bovine cancellous bone (PBCB). The cell-seeded scaffolds were evaluated for their angiogenic potential in two different in vivo assays: the chick embryo chorioallantoic membrane (CAM) model and the severe combined immunodeficiency disorder (SCID) mouse model. In both assays, the development of a complex three-dimensional network of perfused human neovessels could be detected. After subcutaneous implantation into immunodeficient mice, the newly formed human vasculature was stabilized by the recruitment of murine smooth muscle α-actin-positive mural cells and anastomoses with the mouse vasculature. We conclude that this endothelial cell spheroid system can be used to create a network of functional perfused blood vessels in vivo. The finding that this process takes place with high efficacy in the presence of co-implanted primary osteoblasts and in an osteoconductive environment provided by the PBCB scaffold, suggests that this system may be suitable for improving vascularization in bone tissue engineering. [ABSTRACT FROM AUTHOR]

The complexity of the angiogenic cascade limits cellular approaches to studying angiogenic endothelial cells (ECs). In turn, in vivo assays do not allow the analysis of the distinct cellular behavior of ECs during angiogenesis. Here we show that ECs can be grafted as spheroids into a matrix to give rise to a complex three-dimensional network of human neovessels in mice. The grafted vasculature matures and is connected to the mouse circulation. The assay is highly versatile and facilitates numerous applications including studies of the effects of different cytokines on angiogenesis. Modifications make it possible to study human lymphangiogenic processes in vivo. EC spheroids can also be coimplanted with other cell types for tissue engineering purposes. [ABSTRACT FROM AUTHOR]

Background: RhoA and Rho kinase inhibitors overcome the inhibition of axonal regeneration posed by central nervous system (CNS) substrates. Methods: To investigate if inhibition of the Rho pathway augments the neurite extension that naturally occurs in the peripheral nervous system (PNS) following nerve damage, dorsal root ganglion neurons and Schwann cell co-cultures were incubated with culture medium, C3 fusion toxin, and the Rho kinase (ROCK) inhibitors Y27632 and H1152. The longest neurite per neuron were measured and compared. Incubation with Y27632 and H1152 resulted in significantly longer neurites than controls when the neurons were in contact with Schwann cells. When separated by a porous P.E.T. membrane, only the group incubated with H1152 developed significantly longer neurites. This work demonstrates that Rho kinase inhibition augments neurite elongation in the presence of contact with a PNS-like substrate. [ABSTRACT FROM AUTHOR]

Neovascularization is a critical step in tissue engineering applications since implantation of voluminous grafts without sufficient vascularity results in hypoxic cell death of central tissues. We have developed a three-dimensional spheroidal coculture system consisting of human umbilical vein endothelial cells (HUVECs) and human primary fibroblasts (hFBs) to improve angiogenesis in tissue engineering applications. Morphological analysis of cryosections from HUVEC/hFB cospheroids revealed a characteristic temporal and spatial organization with HUVECs located in the center of the cospheroid and a peripheral localization of fibroblasts. In coculture spheroids, the level of apoptosis of endothelial cells was strongly decreased upon cocultivation with fibroblasts. Collagen-embedded HUVEC spheroids develop numerous lumenized capillary-like sprouts. This was also apparent for HUVEC/hFB cospheroids, albeit to a lesser extent. Quantification of cumulative sprout length revealed an approximately 35% reduction in endothelial cell sprouting upon cocultivation with fibroblasts in cospheroids. The slight reduction in endothelial cell sprouting was not mediated by a paracrine mechanism but is most likely due to the formation of heterogenic cell contacts between HUVECs and hFBs within the cospheroid. The model system introduced in this study is suitable for the development of a preformed lumenized capillary-like network ex vivo and may therefore be useful for improving angiogenesis in in vivo tissue engineering applications. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

As the use of ultrasound-assisted liposuction (UAL) increases, the technique grows more popular in breast surgery, especially in reduction mammaplasty and treatment of gynecomastia. The aim of our study was to investigate the effect of UAL on breast tissue using histological examinations, and analyze the effect of this technique on a cellular level. Biopsies from 10 patients undergoing ultrasonically assisted lipectomy prior to classic reduction mammaplasty were taken from the treated areas of the breast. Biopsies were fixed in formalin and embedded in paraffin. Sections were stained with hematoxilin-eosin, and analyzed for defective adipocytes, and the effects of UAL on breast tissue. Untreated breast tissue and breast tissue that had been treated only with conventional aspiration lipectomy served as controls. Sections were analyzed using light microscopy. Compared to the breast tissue treated only with conventional lipectomy, a stronger destruction of the cellular structure of adipocytes could be detected. The destruction was visible even in areas more distant from the aspiration channel. In contrast, the breast tissue was mostly intact, no signs of ultrasonic-induced cellular destruction were visible. The glandular structure was kept intact. Beside the direct mechanical destruction by the probe and the canula, no further alterations of the cellular integrity of the glandular parts were visible. In conclusion our results indicates that UAL is also a safe technique for use in breast surgery. Besides easy handling and improved modelling, the destructive effect of the ultrasound does not include the glandular breast tissue. [ABSTRACT FROM AUTHOR]