Human Induced Pluripotent Stem Cell-Derived Neural Progenitor Cells Produce Distinct Neural 3D In Vitro Models Depending on Alginate/Gellan Gum/Laminin Hydrogel Blend Properties.

Stable and predictive neural cell culture models are a necessary premise for many research fields. However, conventional 2D models lack 3D cell-material/-cell interactions and hence do not reflect the complexity of the in vivo situation properly. Here two alginate/gellan gum/laminin (ALG/GG/LAM) hydrogel blends are presented for the fabrication of human induced pluripotent stem cell (hiPSC)-based 3D neural models. For hydrogel embedding, hiPSC-derived neural progenitor cells (hiNPCs) are used either directly or after 3D neural pre-differentiation. It is shown that stiffness and stress relaxation of the gel blends, as well as the cell differentiation strategy influence 3D model development. The embedded hiNPCs differentiate into neurons and astrocytes within the gel blends and display spontaneous intracellular calcium signals. Two fit-for-purpose models valuable for i) applications requiring a high degree of complexity, but less throughput, such as disease modeling and long-term exposure studies and ii) higher throughput applications, such as acute exposures or substance screenings are proposed. Due to their wide range of applications, adjustability, and printing capabilities, the ALG/GG/LAM based 3D neural models are of great potential for 3D neural modeling in the future.

Neuronal Differentiation from Induced Pluripotent Stem Cell-Derived Neurospheres by the Application of Oxidized Alginate-Gelatin-Laminin Hydrogels.

Biodegradable hydrogels that promote stem cell differentiation into neurons in three dimensions (3D) are highly desired in biomedical research to study drug neurotoxicity or to yield cell-containing biomaterials for neuronal tissue repair. Here, we demonstrate that oxidized alginate-gelatin-laminin (ADA-GEL-LAM) hydrogels facilitate neuronal differentiation and growth of embedded human induced pluripotent stem cell (hiPSC) derived neurospheres. ADA-GEL and ADA-GEL-LAM hydrogels exhibiting a stiffness close to ~5 kPa at initial cell culture conditions of 37 °C were prepared. Laminin supplemented ADA-GEL promoted an increase in neuronal differentiation in comparison to pristine ADA-GEL, with enhanced neuron migration from the neurospheres to the bulk 3D hydrogel matrix. The presence of laminin in ADA-GEL led to a more than two-fold increase in the number of neurospheres with migrated neurons. Our findings suggest that laminin addition to oxidized alginate-gelatin hydrogel matrices plays a crucial role to tailor oxidized alginate-gelatin hydrogels suitable for 3D neuronal cell culture applications.

Influence of nanoporous titanium niobium alloy surfaces produced via hydrogen peroxide oxidative etching on the osteogenic differentiation of human mesenchymal stromal cells.

Titanium niobium alloys exhibit a lower stiffness compared to Ti6Al4V, the 'gold standard' for load-bearing bone implants. Thus, the critical mismatch in stiffness between the implant and adjacent bone tissue could be addressed with TiNb alloys and thereby reduce stress shielding, which can result in bone resorption and subsequent implant loosening; however, the cellular response on the specific material is crucial for sufficient osseointegration. We therefore hypothesize that the response of human mesenchymal stromal cells (hMSC) and osteoblast-like cells on Ti45Nb surfaces can be improved by a novel nanoporous surface structure. For this purpose, an etching technique using hydrogen peroxide electrolyte solution was applied to Ti45Nb. The treated surfaces were characterized using SEM, LSM, AFM, nanoindentation, and contact angle measurements. Cell culture experiments using hMCS and MG-63 were conducted. The H2O2 treatment resulted in surface nanopores, an increase in surface wettability and a reduction in surface hardness. The proliferation of MG-63 was enhanced on TiNb45 compared to Ti6Al4V. MG-63 focal adhesion complexes were detected on all Ti45Nb surfaces, whereas the nanostructures notably increased the cell area and decreased cell solidity, indicating stimulated cell spreading and pseudopodia formation. Alizarin red stainings indicated that the nanoporous surfaces stimulated the osteogenic differentiation of hMSC. It can be concluded that the proposed surface treatment could potentially help to stimulate the osseointegration behaviour of the advantageous low stiff Ti45Nb alloy.

Biological Activation of Bioinert Medical High-Performance Oxide Ceramics by Hydrolytically Stable Immobilization of c(RGDyK) and BMP-2.

High-performance oxide ceramics (HPOC), such as alumina, zirconia, and dispersion ceramics thereof are successfully used as articulating components in joint arthroplasty. HPOC exhibit excellent wear resistance, high strength, and cytocompatible behavior; however, they lack sufficient tissue bonding capability. Thus, they are primarily deployed as low-wear-bearing articulating components in arthroplasty without direct tissue contact, although proper cellular stimulation would hold significant advantages. Here, we describe a surface modification approach for HPOC, enabling hydrolytically stable interfacial binding of c(RGDyK) peptides and BMP-2 proteins to significantly improve the adhesion and osteogenic differentiation of human mesenchymal stem cells (hMSCs) without altering the mechanical properties of the underlying ceramic substrates. Analyses of cellular attachment of murine fibroblasts (L929), human alveolar basal epithelial cells (A549), hMSCs on c(RGDyK), and osteogenic differentiation of hMSCs on BMP-2-coated interfaces demonstrate significant improvements of cell adhesion and an enhanced osteogenic differentiation potential in vitro. The presented approach provides a strategy for the development of a novel class of bioactive HPOC with osseointegration potential that could lead to novel therapeutic solutions for biomedical applications. Furthermore, the developed surface modification is designed in a way to be readily translated to other medically employed bioinert materials in the future.

Periodic microstructures on bioactive glass surfaces enhance osteogenic differentiation of human mesenchymal stromal cells and promote osteoclastogenesis in vitro.

Bioactive glasses (BG) are known for their ability to bond to hard and soft tissues. We hypothesized that the stimulation of bone remodeling, including cellular bone forming and bone resorbing processes, can be increased by applying periodic microstructures on the glass surfaces in vitro. To test our hypothesis, two different BG (45S5 and 13-93) were microstructured in a groove-and-ridge pattern of different sizes by a novel casting process and tested in cell culture experiments using human mesenchymal stromal cells (hMSCs) and RAW 264.7 cells. The microstructures induced contact guidance of hMSCs and increased osteogenic marker gene expression of the stem cells, compared to non-structured glass surfaces as verified by ELISA and quantitative real-time PCR (qPCR) analyses. Furthermore, the structures stimulated the differentiation of RAW cells to osteoclast-like cells confirmed by TRAP gene expression and their resorption activity causing visible resorption lacunae. Our results demonstrate that periodically microstructured BG (especially 45S5) might improve the osteogenic differentiation of hMSCs and influence the activity of material resorbing cells in vitro. Hence, microstructuring of BG could enhance the remodeling process of bone substitutes critical for the formation of new bone tissue in vivo and thus be used to trigger bone remodeling kinetics in vivo. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1965-1978, 2018.

Response of umbilical cord mesenchymal stromal cells to varying titanium topographical signals.

A wide variety of titanium implant modifications have been developed to improve tissue- or cell-material interactions including bone bonding, implant failure, and contact osteogenesis. Osteogenesis can be stimulated by mechanobiological signals such as topography though translation of in vivo reactions to in vitro bioactivity and stem cell culture data, and vice versa, is challenging. We hypothesized that a systematic in vitro approach comparing clinically well-accepted implant surface topographical modifications could shed light on potential cell biological mechanisms provoked by submicron-, micron- or macrostructured surfaces. In this study, we investigated the response of umbilical cord derived mesenchymal stromal cells (UC-MSCs) to anodized, particle blasted, and plasma sprayed highly porous Plasmapore surfaces, which is known to promote bony ingrowth in vivo. After 21 days, UC-MSCs undergo a morphological shift from a 2D to 3D behavior on micro- or macrostructures visualized by actin-vinculin fluorescence and are able to fill the porous surfaces completely. Cell viability after 7 days was significantly decreased on the micro- and macrostructured surfaces particle blasted and Plasmapore, compared to polished controls. The analysis of osteogenic differentiation under noninduced conditions revealed a significantly elevated ALP activity on Plasmapore, indicating a beneficial effect of this macrostructured surface toward osteogenic differentiation supported by late elevated gene expression of osteopontin evaluated by qPCR. Mineralization as well as in vitro bioactivity was pronounced on anodized surfaces. Our findings point to synergistic implant modification strategies allowing early contact osteogenesis and bone ingrowth for future implant designs. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 180-191, 2018.

Engineered Potato virus X nanoparticles support hydroxyapatite nucleation for improved bone tissue replacement.

Bionanoparticles based on filamentous phages or flexuous viruses are interesting candidates for meeting the challenges of tailoring biomineralization in hydrogel-based bone tissue substitutes. We hypothesized that hydroxyapatite crystal nucleation and matrix mineralization can be significantly increased by mineralization-inducing (MIP) and integrin binding motif (RGD) peptides presented on biomimetic nanoparticles. In this study, Potato virus X (PVX), a flexible rod-shaped plant virus was genetically engineered to present these functional peptides on its particle surface. Recombinant PVX-MIP/RGD particles were isolated from infected Nicotiana benthamiana plants and characterized by western blot, SEM, TEM, and TPLSM in MSC cultures. The presence of RGD was proven by cell attachment, spreading, and vinculin cluster analysis, and MIP by in vitro mineralization and osteogenic differentiation assays. Thus the tailored surface of genetically engineered PVX forms fibril-like nanostructures which enables enhanced focal adhesion-dependent cell adhesion, and matrix mineralization verified by Alizarin. Hydroxyapatite crystal nucleation is supported on recombinant PVX particles leading to a biomimetic network and bundle-like structures similar to mineralized collagen fibrils. In conclusion, the recombinant flexuous PVX nanoparticles exhibit properties with great potential for bone tissue substitutes. STATEMENT OF SIGNIFICANCE: A suitable biomaterial for tissue engineering should be able to mimic the endogenous extracellular matrix by presenting biochemical and biophysical cues. Novel hydrogel-based materials seek to meet the criteria of cytocompatibility, biodegradability, printability, and crosslinkability under mild conditions. However, a majority of existing hydrogels lack cell-interactive motifs, which are crucial to modulate cellular responses. The incorporation of the plant virus PVX to the hydrogel could improve functions like integrin-binding and mineralization due to peptide-presentation on the particle surface. The tailored surface of genetically engineered PVX forms fibril-like nanostructures which enables enhanced focal adhesion-dependent cell adhesion and matrix mineralization and offers great potential for the development of new hydrogel compositions for bone tissue substitutes.

The m-AAA Protease Associated with Neurodegeneration Limits MCU Activity in Mitochondria.

Mutations in subunits of mitochondrial m-AAA proteases in the inner membrane cause neurodegeneration in spinocerebellar ataxia (SCA28) and hereditary spastic paraplegia (HSP7). m-AAA proteases preserve mitochondrial proteostasis, mitochondrial morphology, and efficient OXPHOS activity, but the cause for neuronal loss in disease is unknown. We have determined the neuronal interactome of m-AAA proteases in mice and identified a complex with C2ORF47 (termed MAIP1), which counteracts cell death by regulating the assembly of the mitochondrial Ca2+ uniporter MCU. While MAIP1 assists biogenesis of the MCU subunit EMRE, the m-AAA protease degrades non-assembled EMRE and ensures efficient assembly of gatekeeper subunits with MCU. Loss of the m-AAA protease results in accumulation of constitutively active MCU-EMRE channels lacking gatekeeper subunits in neuronal mitochondria and facilitates mitochondrial Ca2+ overload, mitochondrial permeability transition pore opening, and neuronal death. Together, our results explain neuronal loss in m-AAA protease deficiency by deregulated mitochondrial Ca2+ homeostasis.

Inkjet printed periodical micropatterns made of inert alumina ceramics induce contact guidance and stimulate osteogenic differentiation of mesenchymal stromal cells.

Bioinert high performance ceramics exhibit detrimental features for implant components with direct bone contact because of their low osseointegrating capability. We hypothesized that periodical microstructures made of inert alumina ceramics can influence the osteogenic differentiation of human mesenchymal stromal cells (hMSC). In this study, we manufactured pillared arrays made of alumina ceramics with periodicities as low as 100µm and pillar heights of 40µm employing direct inkjet printing (DIP) technique. The response of hMSC to the microstructured surfaces was monitored by measuring cell morphology, viability and formation of focal adhesion complexes. Osteogenic differentiation of hMSCs was investigated by alkaline phosphatase activity, mineralization assays and expression analysis of respective markers. We demonstrated that MSCs react to the pillars with contact guidance. Subsequently, cells grow onto and form connections between the microstructures, and at the same time are directly attached to the pillars as shown by focal adhesion stainings. Cells build up tissue-like constructs with heights up to the micropillars resulting in increased cell viability and osteogenic differentiating properties. We conclude that periodical micropatterns on the micrometer scale made of inert alumina ceramics can mediate focal adhesion dependent cell adhesion and stimulate osteogenic differentiation of hMSCs.

Low-aspect ratio nanopatterns on bioinert alumina influence the response and morphology of osteoblast-like cells.

Topographical features on the nanometer scale are known to influence cellular behavior. The response of specific cell types to various types of surface structures is currently still being investigated. Alumina ceramics play an important role as biomaterials, e.g., in medical and dental applications. In this study, we investigated the influence of nanoscale surface features with low aspect ratio (< 0.1) on the response of osteoblast-like MG-63 cells. To this end, low-energy ion irradiation was employed to produce shallow nanoscale ripple patterns on Al2O3(0001) surfaces with lateral periodicities of 24 nm and 179 nm and heights of only 0.7 and 11.5 nm, respectively. The nanopatterning was found to increase the proliferation of MG-63 cells and may lead to pseudopodia alignment along the ripples. Furthermore, focal adhesion behavior and cell morphology were analyzed. We found that MG-63 cells are able to recognize surface nanopatterns with extremely low vertical variations of less than 1 nm. In conclusion, it is shown that surface topography in the sub-nm range significantly influences the response of osteoblast-like cells.

Transcriptional activation of LON Gene by a new form of mitochondrial stress: A role for the nuclear respiratory factor 2 in StAR overload response (SOR).

High output of steroid hormone synthesis in steroidogenic cells of the adrenal cortex and the gonads requires the expression of the steroidogenic acute regulatory protein (StAR) that facilitates cholesterol mobilization to the mitochondrial inner membrane where the CYP11A1/P450scc enzyme complex converts the sterol to the first steroid. Earlier studies have shown that StAR is active while pausing on the cytosolic face of the outer mitochondrial membrane while subsequent import of the protein into the matrix terminates the cholesterol mobilization activity. Consequently, during repeated activity cycles, high level of post-active StAR accumulates in the mitochondrial matrix. To prevent functional damage due to such protein overload effect, StAR is degraded by a sequence of three to four ATP-dependent proteases of the mitochondria protein quality control system, including LON and the m-AAA membranous proteases AFG3L2 and SPG7/paraplegin. Furthermore, StAR expression in both peri-ovulatory ovarian cells, or under ectopic expression in cell line models, results in up to 3-fold enrichment of the mitochondrial proteases and their transcripts. We named this novel form of mitochondrial stress as StAR overload response (SOR). To better understand the SOR mechanism at the transcriptional level we analyzed first the unexplored properties of the proximal promoter of the LON gene. Our findings suggest that the human nuclear respiratory factor 2 (NRF-2), also known as GA binding protein (GABP), is responsible for 88% of the proximal promoter activity, including the observed increase of transcription in the presence of StAR. Further studies are expected to reveal if common transcriptional determinants coordinate the SOR induced transcription of all the genes encoding the SOR proteases.

StAR enhances transcription of genes encoding the mitochondrial proteases involved in its own degradation.

Steroidogenic acute regulatory protein (StAR) is essential for steroid hormone synthesis in the adrenal cortex and the gonads. StAR activity facilitates the supply of cholesterol substrate into the inner mitochondrial membranes where conversion of the sterol to a steroid is catalyzed. Mitochondrial import terminates the cholesterol mobilization activity of StAR and leads to mounting accumulation of StAR in the mitochondrial matrix. Our studies suggest that to prevent mitochondrial impairment, StAR proteolysis is executed by at least 2 mitochondrial proteases, ie, the matrix LON protease and the inner membrane complexes of the metalloproteases AFG3L2 and AFG3L2:SPG7/paraplegin. Gonadotropin administration to prepubertal rats stimulated ovarian follicular development associated with increased expression of the mitochondrial protein quality control system. In addition, enrichment of LON and AFG3L2 is evident in StAR-expressing ovarian cells examined by confocal microscopy. Furthermore, reporter studies of the protease promoters examined in the heterologous cell model suggest that StAR expression stimulates up to a 3.5-fold increase in the protease gene transcription. Such effects are StAR-specific, are independent of StAR activity, and failed to occur upon expression of StAR mutants that do not enter the matrix. Taken together, the results of this study suggest the presence of a novel regulatory loop, whereby acute accumulation of an apparent nuisance protein in the matrix provokes a mitochondria to nucleus signaling that, in turn, activates selected transcription of genes encoding the enrichment of mitochondrial proteases relevant for enhanced clearance of StAR.

GLTP mediated non-vesicular GM1 transport between native membranes.

Lipid transfer proteins (LTPs) are emerging as key players in lipid homeostasis by mediating non-vesicular transport steps between two membrane surfaces. Little is known about the driving force that governs the direction of transport in cells. Using the soluble LTP glycolipid transfer protein (GLTP), we examined GM1 (monosialotetrahexosyl-ganglioside) transfer to native membrane surfaces. With artificial GM1 donor liposomes, GLTP can be used to increase glycolipid levels over natural levels in either side of the membrane leaflet, i.e., external or cytosolic. In a system with native donor- and acceptor-membranes, we find that GLTP balances highly variable GM1 concentrations in a population of membranes from one cell type, and in addition, transfers lipids between membranes from different cell types. Glycolipid transport is highly efficient, independent of cofactors, solely driven by the chemical potential of GM1 and not discriminating between the extra- and intracellular membrane leaflet. We conclude that GLTP mediated non-vesicular lipid trafficking between native membranes is driven by simple thermodynamic principles and that for intracellular transport less than 1 µM GLTP would be required in the cytosol. Furthermore, the data demonstrates the suitability of GLTP as a tool for artificially increasing glycolipid levels in cellular membranes.