Search

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]

There is no consensus regarding follow-up after soft tissue sarcoma (STS) treatment. This study examines the efficacy and the cost-benefit of MRI imaging for discovering recurrence. A retrospective analysis was performed, collecting data on patient demography, tumor characteristics, treatment, and follow-up. Imaging was correlated to the clinical course, and sensitivity, specificity, and predictive values were calculated. The number needed to screen and costs of finding recurrence are reported. Amongst 216 sarcomas, 73 (35%) exhibited local recurrence during a follow-up of 5.3 ± 3.5 years. 173 entities had complete MRI follow-up with 58 (34%) local recurrences. Thirty-three (57%) were discovered by MRI, 8 (14%) by clinical presentation, and 17 (29%) simultaneously. There was a sensitivity of 100.00%, a specificity of 89%, a positive predictive value of 32%, and a negative predictive value of 100% for detecting local recurrence with MRI. Our data confirm the modalities and intervals proposed by the German guidelines for sarcoma care. The recommended MRI intervals should not be extended. MRI is more cost-effective than clinical examination; still, both modalities should be performed together to discover the maximum number of recurrences. [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]