Reduced Cell Adhesion on LightPLAS-Coated Implant Surfaces in a Three-Dimensional Bioreactor System.

Most implants used in trauma surgery are made of steel and remain inside the body only temporarily. The strong tissue interaction of such implants sometimes creates problems with their explantation. Modified implant surfaces, which decrease tissue attachment, might allow an easier removal and therefore a better outcome. Such a modification must retain the implant function, and needs to be biocompatible and cost-effective. Here, we used a novel VUV-light (Vacuum-Ultraviolett)-based coating technology (LightPLAS) to generate coated stainless-steel plates. The tested LightPLAS coating only had an average thickness of around 335 nm, making it unlikely to interfere with implant function. The coated plates showed good biocompatibility according to ISO 10993-5 and ISO 10993-12, and reduced cell adhesion after four different time points in a 2D cell culture system with osteoblast-like MG-63 cells. Furthermore, we could show decreased cell adhesion in our 3D cell culture system, which mimics the fluid flow above the implant materials as commonly present in the in vivo environment. This new method of surface coating could offer extended options to design implant surfaces for trauma surgery to reduce cell adhesion and implant ingrowth. This may allow for a faster removal time, resulting in shorter overall operation times, thereby reducing costs and complication rates and increasing patient wellbeing.

Osteogenic Effect of Pregabalin in Human Primary Mesenchymal Stem Cells, Osteoblasts, and Osteosarcoma Cells.

Seventy million patients worldwide are suffering from epilepsy. The long-term use of antiepileptic drugs causes the alteration of the bone tissue and its metabolism, thus increasing the risk of fractures. Clinical and pre-clinical studies have highlighted conflicting data on the influence of the relatively new antiepileptic drug pregabalin (Lyrica®). The objective of the present study was therefore to investigate its cytotoxicity in primary human osteoblasts (hOB). HOB and human mesenchymal stem cells (hMSC) were isolated from patients. The human osteosarcoma cells MG63 were included as established cell line. Cells were incubated with pregabalin at concentrations ranging from 0 to 40 µg/mL. Time-dependent cell proliferation was measured by automatic cell counting, and metabolism was determined by XTT assay and osseous differentiation by alkaline phosphatase (ALP) activity. Histological examinations of calcium deposit were performed with ALP, Alizarin Red, and von Kossa staining. A concentration-dependent increase in the proliferation of hOB and hMSC was observed after treatment with pregabalin. All cells showed a significant increase in cell metabolism. The osteogenic differentiation, confirmed by the increase of calcium deposit, was promoted by the administration of pregabalin. This effect was already significant at the therapeutic plasma concentration of pregabalin (10 µg/mL). In contrast to the other antiepileptic drugs, pregabalin showed no osteocatabolic effects. Conflicting in-vivo data must therefore be attributed to systemic effects of pregabalin.

The HIFα-Stabilizing Drug Roxadustat Increases the Number of Renal Epo-Producing Sca-1+ Cells.

Inhibition of the prolyl-4-hydroxylase domain (PHD) enzymes, leading to the stabilization of hypoxia-inducible factor (HIF) α as well as to the stimulation of erythropoietin (Epo) synthesis, is the functional mechanism of the new anti-anemia drug roxadustat. Little is known about the effects of roxadustat on the Epo-producing cell pool. To gain further insights into the function of PHD inhibitors, we characterized the abundance of mesenchymal stem cell (MSC)-like cells after roxadustat treatment of mice. The number of Sca-1+ mesenchymal cells following roxadustat treatment increased exclusively in the kidneys. Isolated Sca-1+ cells demonstrated typical features of MSC-like cells, including adherence to tissue culture plates, trilineage differentiation potential, and expression of MSC markers. Kidney-derived Sca-1+ MSC-like cells were cultured for up to 21 days. Within the first few days in culture, cells stabilized HIF-1α and HIF-2α and temporarily increased Epo production upon incubation in hypoxia. In summary, we have identified a Sca-1+ MSC-like cell population that is involved in renal Epo production and might contribute to the strong anti-anemic effect of the PHD inhibitor roxadustat.

Extracellular Vesicles Allow Epigenetic Mechanotransduction between Chondrocytes and Osteoblasts.

MicroRNAs (miRNAs) can be transported in extracellular vesicles (EVs) and are qualified as possible messengers for cell-cell communication. In the context of osteoarthritis (OA), miR-221-3p has been shown to have a mechanosensitive and a paracrine function inside cartilage. However, the question remains if EVs with miR-221-3p can act as molecular mechanotransducers between cells of different tissues. Here, we studied the effect of EV-mediated transport in the communication between chondrocytes and osteoblasts in vitro in a rat model. In silico analysis (Targetscan, miRWalk, miRDB) revealed putative targets of miRNA-221-3p (CDKN1B/p27, TIMP-3, Tcf7l2/TCF4, ARNT). Indeed, transfection of miRNA-221-3p in chondrocytes and osteoblasts resulted in regulation of these targets. Coculture experiments of transfected chondrocytes with untransfected osteoblasts not only showed regulation of these target genes in osteoblasts but also inhibition of their bone formation capacity. Direct treatment with chondrocyte-derived EVs validated that chondrocyte-produced extracellular miR-221-3p was responsible for this effect. Altogether, our study provides a novel perspective on a possible communication pathway of a mechanically induced epigenetic signal through EVs. This may be important for processes at the interface of bone and cartilage, such as OA development, physiologic joint homeostasis, growth or fracture healing, as well as for other tissue interfaces with differing biomechanical properties.

The Interaction between microRNAs and the Wnt/ß-Catenin Signaling Pathway in Osteoarthritis.

Osteoarthritis (OA) is a chronic disease affecting the whole joint, which still lacks a disease-modifying treatment. This suggests an incomplete understanding of underlying molecular mechanisms. The Wnt/ß-catenin pathway is involved in different pathophysiological processes of OA. Interestingly, both excessive stimulation and suppression of this pathway can contribute to the pathogenesis of OA. microRNAs have been shown to regulate different cellular processes in different diseases, including the metabolic activity of chondrocytes and osteocytes. To bridge these findings, here we attempt to give a conclusive overview of microRNA regulation of the Wnt/ß-catenin pathway in bone and cartilage, which may provide insights to advance the development of miRNA-based therapeutics for OA treatment.

Laser Ablated Periodic Nanostructures on Titanium and Steel Implants Influence Adhesion and Osteogenic Differentiation of Mesenchymal Stem Cells.

Metal implants used in trauma surgeries are sometimes difficult to remove after the completion of the healing process due to the strong integration with the bone tissue. Periodic surface micro- and nanostructures can directly influence cell adhesion and differentiation on metallic implant materials. However, the fabrication of such structures with classical lithographic methods is too slow and cost-intensive to be of practical relevance. Therefore, we used laser beam interference ablation structuring to systematically generate periodic nanostructures on titanium and steel plates. The newly developed laser process uses a special grating interferometer in combination with an industrial laser scanner and ultrashort pulse laser source, allowing for fast, precise, and cost-effective modification of metal surfaces in a single step process. A total of 30 different periodic topologies reaching from linear over crossed to complex crossed nanostructures with varying depths were generated on steel and titanium plates and tested in bone cell culture. Reduced cell adhesion was found for four different structure types, while cell morphology was influenced by two different structures. Furthermore, we observed impaired osteogenic differentiation for three structures, indicating reduced bone formation around the implant. This efficient way of surface structuring in combination with new insights about its influence on bone cells could lead to newly designed implant surfaces for trauma surgeries with reduced adhesion, resulting in faster removal times, reduced operation times, and reduced complication rates.

Current Methods for Skeletal Muscle Tissue Repair and Regeneration.

Skeletal muscle has the capacity of regeneration after injury. However, for large volumes of muscle loss, this regeneration needs interventional support. Consequently, muscle injury provides an ongoing reconstructive and regenerative challenge in clinical work. To promote muscle repair and regeneration, different strategies have been developed within the last century and especially during the last few decades, including surgical techniques, physical therapy, biomaterials, and muscular tissue engineering as well as cell therapy. Still, there is a great need to develop new methods and materials, which promote skeletal muscle repair and functional regeneration. In this review, we give a comprehensive overview over the epidemiology of muscle tissue loss, highlight current strategies in clinical treatment, and discuss novel methods for muscle regeneration and challenges for their future clinical translation.

Enhanced Production of Exosome-Associated AAV by Overexpression of the Tetraspanin CD9.

Research on cell-free vesicles revealed a multitude of characteristics, in particular of microvesicles and exosomes, that range from their potential as biomarkers to a function in horizontal transfer of genetic information from cell to cell and also include supportive functions in viral infection. Exosome-associated adeno-associated viruses (exo-AAVs) are of particular interest for the past couple of years, because they introduced a new source of highly potent recombinant AAVs with improved features, including accelerated transduction rates and more efficient immune escape. However, key factors like the mode of action, efficiency of production, or engineering of exo-AAVs remain elusive to a large extent. Here, we used the established system of CD9 overexpression to boost the exosome output of AAV producing HEK-AAV cells. The CD9-powered high-exosome environment was established during exo-AAV1 production, and we could demonstrate that the yield of exo-AAVs dramatically increased when compared to standard exo-AAVs. Furthermore, we report that exo-AAV-CD9GFP was more efficient in transduction of cells in the same titer ranges as standard exo-AAVs. Our results provide a technological approach for the generation of exo-AAVs with superior performance.

Identification and characterization of human xylosyltransferase II promoter single nucleotide variants.

The human isoenzymes xylosyltransferase-I and -II (XT-I, XT-II) catalyze the rate-limiting step in proteoglycan biosynthesis. Therefore, serum XT activity, mainly representing XT-II activity, displays a powerful biomarker to quantify the actual proteoglycan synthesis rate. Serum XT activity is increased up to 44% in disorders which are characterized by an altered proteoglycan metabolism, whereby underlying regulatory mechanisms remain unclear. The aim of this study was to investigate new regulatory pathways by identifying and characterizing naturally occurring XYLT2 promoter sequence variants as well as their potential influence on promoter activity and serum XT activity. XYLT2 promoter single nucleotide variants (SNVs) were identified and genotyped in the genomic DNA of 100 healthy blood donors by promoter amplification and sequencing or restriction fragment length polymorphism analysis. The SNVs were characterized by an in silico analysis considering genetic linkage and transcription factor binding sites (TBSs). The influence of SNVs on promoter activity and serum XT activity was determined by dual luciferase reporter assay and HPLC-ESI mass spectrometry. Allele frequencies of seven XYLT2 promoter sequence variants identified were investigated. In silico analyses revealed a strong genetic linkage of SNVs c.-80delG and c.-188G > A, c.-80delG and c.-1443G > A, as well as c.-188G > A and c.-1443G > A. However, despite the generation of several SNV-associated changes in TBSs in silico, XYLT2 promoter SNVs did not significantly affect promoter activity. Serum XT activities of SNV carriers deviated up to 8% from the wild-type, whereby the differences were also not statistically significant. This is the first study which identifies, genotypes and characterizes XYLT2 promoter SNVs. Our results reveal a weak genetic heterogeneity and a strong conservation of the human XYLT2 promoter region. Since the SNVs detected could be excluded as causatives for strong interindividual variabilities in serum XT activity, our data provide increasing evidence that XT-II activity is obviously regulated by hitherto unknown complex genetic pathways, such as cis- or trans-acting enhancers, silencers or miRNAs.

First description of the complete human xylosyltransferase-I promoter region.

BACKGROUND: Human xylosyltransferase-I (XT-I) catalyzes the rate-limiting step in proteoglycan glycosylation. An increase in XYLT1 mRNA expression and serum XT activity is associated with diseases characterized by abnormal extracellular matrix accumulation like, for instance, fibrosis. Nevertheless, physiological and pathological mechanisms of transcriptional XT regulation remain elusive. RESULTS: To elucidate whether promoter variations might affect the naturally occurring variability in serum XT activity, a complete sequence analysis of the XYLT1 promoter was performed in genomic DNA of healthy blood donors. Based on promoter amplification by a specialized PCR technique, sequence analysis revealed a fragment of 238 bp, termed XYLT1 238*, which has never been described in the human XYLT1 reference sequence so far. In silico characterization of this unconsidered fragment depicted an evolutionary conservation between sequences of Homo sapiens and Pan troglodytes (chimpanzee) or Mus musculus (mouse), respectively. Promoter activity studies indicated that XYLT1 238* harbors various transcription factor binding sites affecting basal XYLT1 expression and inducibility by transforming growth factor-ß1, the key fibrotic mediator. A microsatellite and two single nucleotide variants (SNV), c.-403C>T and c.-1088C>A, were identified and genotyped in 100 healthy blood donors. Construct associated changes in XYLT1 promoter activity were detected for several sequence variants, whereas serum XT activity was only marginally affected. CONCLUSIONS: Our findings describe for the first time the entire XYLT1 promoter sequence and provide new insights into transcriptional regulation of XT-I. Future studies should analyze the impact of regulatory XYLT1 promoter variations on XT-associated diseases.