Immunomodulation Using BMP-7 and IL-10 to Enhance the Mineralization Capacity of Bone Progenitor Cells in a Fracture Hematoma-Like Environment.

Following biomaterial implantation, a failure to resolve inflammation during the formation of a fracture hematoma can significantly limit the biomaterial's ability to facilitate bone regeneration. This study aims to combine the immunomodulatory and osteogenic effects of BMP-7 and IL-10 with the regenerative capacity of collagen-hydroxyapatite (CHA) scaffolds to enhance in vitro mineralization in a hematoma-like environment. Incubation of CHA scaffolds with human whole blood leads to rapid adsorption of fibrinogen, significant stiffening of the scaffold, and the formation of a hematoma-like environment characterized by a limited capacity to support the infiltration of human bone progenitor cells, a significant upregulation of inflammatory cytokines and acute phase proteins, and significantly reduced osteoconductivity. CHA scaffolds functionalized with BMP-7 and IL-10 significantly downregulate the production of key inflammatory cytokines, including IL-6, IL-8, and leptin, creating a more permissive environment for mineralization, ultimately enhancing the biomaterial's osteoconductivity. In conclusion, targeting the onset of inflammation in the early phase of bone healing using BMP-7 and IL-10 functionalized CHA scaffolds is a promising approach to effectively downregulate inflammatory processes, while fostering a more permissive environment for bone regeneration.

Selective CNS Targeting and Distribution with a Refined Region-Specific Intranasal Delivery Technique via the Olfactory Mucosa.

Intranasal drug delivery is a promising approach for the delivery of drugs to the CNS, but too heterogenous, unprecise delivery methods without standardization decrease the quality of many studies in rodents. Thus, the lack of a precise and region-specific application technique for mice is a major drawback. In this study, a previously developed catheter-based refined technique was validated against the conventional pipette-based method and used to specifically reach the olfactory or the respiratory nasal regions. This study successfully demonstrated region-specific administration at the olfactory mucosa resulting in over 20% of the administered fluorescein dose in the olfactory bulbs, and no peripheral bioactivity of insulin detemir and Fc-dependent uptake of two murine IgG1 (11C7 and P3X) along the olfactory pathway to cortex and hippocampus. An scFv of 11C7 showed hardly any uptake to the CNS. Elimination was dependent on the presence of the IgG's antigen. In summary, it was successfully demonstrated that region-specific intranasal administration via the olfactory region resulted in improved brain targeting and reduced peripheral targeting in mice. The data are discussed with regard to their clinical potential.

Basement membrane stiffness determines metastases formation.

The basement membrane (BM) is a special type of extracellular matrix and presents the major barrier cancer cells have to overcome multiple times to form metastases. Here we show that BM stiffness is a major determinant of metastases formation in several tissues and identify netrin-4 (Net4) as a key regulator of BM stiffness. Mechanistically, our biophysical and functional analyses in combination with mathematical simulations show that Net4 softens the mechanical properties of native BMs by opening laminin node complexes, decreasing cancer cell potential to transmigrate this barrier despite creating bigger pores. Our results therefore reveal that BM stiffness is dominant over pore size, and that the mechanical properties of 'normal' BMs determine metastases formation and patient survival independent of cancer-mediated alterations. Thus, identifying individual Net4 protein levels within native BMs in major metastatic organs may have the potential to define patient survival even before tumour formation. The ratio of Net4 to laminin molecules determines BM stiffness, such that the more Net4, the softer the BM, thereby decreasing cancer cell invasion activity.

Adhesive Properties of the Hyaluronan Pericellular Coat in Hyaluronan Synthases Overexpressing Mesenchymal Stem Cells.

Hyaluronan (HA), a natural component of the extracellular matrix, is supposed to have a regulatory function in the stem cell niche. Bone marrow-derived human mesenchymal stem cells (hMSCs) are known to express all three hyaluronan synthases (HASes), which are responsible for HA production. HA is extruded into the extracellular matrix, but also stays bound to the plasma membrane forming a pericellular coat, which plays a key role during early cell adhesion. Since HAS isoenzymes, HAS1, HAS2 and HAS3, produce HA with different molecular weights, a difference in their role for cell adhesion is expected. Here, we transduced the immortalized hMSC cell line SCP1 to constitutively express eGFP-tagged HASes (SCP1-HAS-eGFP) by lentiviral gene transfer. The overexpression of the HAS-eGFP was shown on RNA and protein levels, HA was determined by ELISA and the stained HA-coat was analyzed using confocal microscopy. Time-lapse microscopy, spreading assay and single cell force spectroscopy using atomic force microscopy were applied to characterize adhesion of the different HAS transduced SCP1 cells. We showed in this study that HAS3 overexpressing cells formed the thickest pericellular coat compared with control or HAS1 and HAS2 transduced cells. Furthermore, SCP1-HAS3-eGFP displayed faster and stronger adhesion compared to cells overexpressing the other synthases or control cells. We conclude that overexpression of HASes in hMSCs differentially modulates their initial adhesive interactions with the substrate. This observation might be helpful in regenerative medicine goals.

Three-dimensional self-assembling nanofiber matrix rejuvenates aged/degenerative human tendon stem/progenitor cells.

The poor healing capacity of tendons is known to worsen in the elderly. During tendon aging and degeneration, endogenous human tendon stem/progenitor cells (hTSPCs) experience profound pathological changes. Here, we explored a rejuvenation strategy for hTSPCs derived from aged/degenerated Achilles tendons (A-TSPCs) by providing three-dimensional (3D) nanofiber hydrogels and comparing them to young/healthy TSPCs (Y-TSPCs). RADA peptide hydrogel has a self-assembling ability, forms a nanofibrous 3D niche and can be further functionalized by adding RGD motifs. Cell survival, apoptosis, and proliferation assays demonstrated that RADA and RADA/RGD hydrogels support A-TSPCs in a comparable manner to Y-TSPCs. Moreover, they rejuvenated A-TSPCs to a phenotype similar to that of Y-TSPCs, as evidenced by restored cell morphology and cytoskeletal architecture. Transmission electron, confocal laser scanning and atomic force microscopies demonstrated comparable ultrastructure, surface roughness and elastic modulus of A- and Y-TSPC-loaded hydrogels. Lastly, quantitative PCR revealed similar expression profiles, as well a significant upregulation of genes related to tenogenesis and multipotency. Taken together, the RADA-based hydrogels exert a rejuvenating effect by recapitulating in vitro specific features of the natural microenvironment of human TSPCs, which strongly indicates their potential to direct cell behaviour and overcome the challenge of cell aging and degeneration in tendon repair.

Age related changes in cell stiffness of tendon stem/progenitor cells and a rejuvenating effect of ROCK-inhibition.

Tendon stem/progenitor cells (TSPC) are potential targets for regenerative medicine and the treatment of tendon injuries. The frequency of such injuries increases in elderly patients while the proportion of functional TSPCs in tendon tissue decreases, protracting tendon repair. Using atomic force microscopy (AFM), we show that cell stiffness and size increase in TSPCs isolated from elderly patients (A-TSPC) compared to TSPCs from younger patients (Y-TSPC). Additionally, two-photon excited fluorescence (TPEF) microscopy revealed a denser, well-structured actin cytoskeleton in A-TSPC, which correlates with the augmented cell stiffness. Treating A-TSPC with ROCK-inhibitor, reverses these age-related changes, and has rejuvenating effect on cell morphology and stiffness. We assume that cellular stiffness is a suitable marker for cell aging and ROCK a potential target for therapeutic applications of cell rejuvenation.