Effect of Vancomycin, Gentamicin and Clindamycin on Cartilage Cells In Vitro.

BACKGROUND: The treatment of grafts with vancomycin for ligament reconstruction in knee surgery is the current standard. However, high antibiotic concentrations have chondrotoxic effects. PURPOSE: To test the chondrotoxicity of clindamycin, gentamicin and vancomycin in comparable concentrations. In vitro and in vivo effective concentrations hugely vary from drug to drug. To allow for comparisons between these three commonly used antibiotics, the concentration ranges frequently used in orthopedic surgical settings were tested. STUDY DESIGN: Controlled laboratory study. METHODS: Human cartilage from 10 specimens was used to isolate chondrocytes. The chondrocytes were treated with clindamycin (1 mg/mL and 0.5 mg/mL), gentamicin (10 mg/mL and 5 mg/mL) or vancomycin (10 mg/mL and 5 mg/mL), at concentrations used for preoperative infection prophylaxis in ligament surgery. Observations were taken over a period of 7 days. A control of untreated chondrocytes was included. To test the chondrotoxicity, a lactate dehydrogenase (LDH) test and a water-soluble tetrazolium salt (WST-1) assay were performed on days 1, 3 and 7. In addition, microscopic examinations were performed after fluorescence staining of the cells at the same time intervals. RESULTS: All samples showed a reasonable vitality of the cartilage cells after 72 h. However, clindamycin and gentamicin both showed higher chondrotoxicity in all investigations compared to vancomycin. After a period of 7 days, only chondrocytes treated with vancomycin showed reasonable vitality. CONCLUSIONS: The preoperative treatment of ligament grafts with vancomycin is the most reasonable method for infection prophylaxis, in accordance with the current study results regarding chondrotoxicity; however, clindamycin and gentamicin cover a wider anti-bacterial spectrum. CLINICAL RELEVANCE: The prophylactic antibiotic treatment of ligament grafts at concentrations of 5 mg/mL or 10 mg/mL vancomycin is justifiable and reasonable. In specific cases, even the use of gentamicin and clindamycin is appropriate.

Articular Cartilage-From Basic Science Structural Imaging to Non-Invasive Clinical Quantitative Molecular Functional Information for AI Classification and Prediction.

This review presents the changes that the imaging of articular cartilage has undergone throughout the last decades. It highlights that the expectation is no longer to image the structure and associated functions of articular cartilage but, instead, to devise methods for generating non-invasive, function-depicting images with quantitative information that is useful for detecting the early, pre-clinical stage of diseases such as primary or post-traumatic osteoarthritis (OA/PTOA). In this context, this review summarizes (a) the structure and function of articular cartilage as a molecular imaging target, (b) quantitative MRI for non-invasive assessment of articular cartilage composition, microstructure, and function with the current state of medical diagnostic imaging, (c), non-destructive imaging methods, (c) non-destructive quantitative articular cartilage live-imaging methods, (d) artificial intelligence (AI) classification of degeneration and prediction of OA progression, and (e) our contribution to this field, which is an AI-supported, non-destructive quantitative optical biopsy for early disease detection that operates on a digital tissue architectural fingerprint. Collectively, this review shows that articular cartilage imaging has undergone profound changes in the purpose and expectations for which cartilage imaging is used; the image is becoming an AI-usable biomarker with non-invasive quantitative functional information. This may aid in the development of translational diagnostic applications and preventive or early therapeutic interventions that are yet beyond our reach.

Therapeutic Modulation of Cell Morphology and Phenotype of Diseased Human Cells towards a Healthier Cell State Using Lignin.

Despite lignin's global abundance and its use in biomedical studies, our understanding of how lignin regulates disease through modulation of cell morphology and associated phenotype of human cells is unknown. We combined an automated high-throughput image cell segmentation technique for quantitatively measuring a panel of cell shape descriptors, droplet digital Polymerase Chain Reaction for absolute quantification of gene expression and multivariate data analyses to determine whether lignin could therapeutically modulate the cell morphology and phenotype of inflamed, degenerating diseased human cells (osteoarthritic (OA) chondrocytes) towards a healthier cell morphology and phenotype. Lignin dose-dependently modified all aspects of cell morphology and ameliorated the diseased shape of OA chondrocytes by inducing a less fibroblastic healthier cell shape, which correlated with the downregulation of collagen 1A2 (COL1A2, a major fibrosis-inducing gene), upregulation of collagen 2A1 (COL2A1, a healthy extracellular matrix-inducing gene) and downregulation of interleukin-6 (IL-6, a chronic inflammatory cytokine). This is the first study to show that lignin can therapeutically target cell morphology and change a diseased cells' function towards a healthier cell shape and phenotype. This opens up novel opportunities for exploiting lignin in modulation of disease, tissue degeneration, fibrosis, inflammation and regenerative medical implants for therapeutically targeting cell function and outcome.

Mechanical Articular Cartilage Injury Models and Their Relevance in Advancing Therapeutic Strategies.

This chapter details how Alan Grodzinsky and his team unraveled the complex electromechanobiological structure-function relationships of articular cartilage and used these insights to develop an impressively versatile shear and compression model. In this context, this chapter focuses (i) on the effects of mechanical compressive injury on multiple articular cartilage properties for (ii) better understanding the molecular concept of mechanical injury, by studying gene expression, signal transduction and the release of potential injury biomarkers. Furthermore, we detail how (iii) this was used to combine mechanical injury with cytokine exposure or co-culture systems for generating a more realistic trauma model to (iv) investigate the therapeutic modulation of the injurious response of articular cartilage. Impressively, Alan Grodzinsky's research has been and will remain to be instrumental in understanding the proinflammatory response to injury and in developing effective therapies that are based on an in-depth understanding of complex structure-function relationships that underlay articular cartilage function and degeneration.

Cell morphology as a biological fingerprint of chondrocyte phenotype in control and inflammatory conditions.

Introduction: Little is known how inflammatory processes quantitatively affect chondrocyte morphology and how single cell morphometric data could be used as a biological fingerprint of phenotype. Methods: We investigated whether trainable high-throughput quantitative single cell morphology profiling combined with population-based gene expression analysis can be used to identify biological fingerprints that are discriminatory of control vs. inflammatory phenotypes. The shape of a large number of chondrocytes isolated from bovine healthy and human osteoarthritic (OA) cartilages was quantified under control and inflammatory (IL-1ß) conditions using a trainable image analysis technique measuring a panel of cell shape descriptors (area, length, width, circularity, aspect ratio, roundness, solidity). The expression profiles of phenotypically relevant markers were quantified by ddPCR. Statistical analysis, multivariate data exploration, and projection-based modelling were used for identifying specific morphological fingerprints indicative of phenotype. Results: Cell morphology was sensitive to both cell density and IL-1ß. In both cell types, all shape descriptors correlated with expression of extracellular matrix (ECM)- and inflammatory-regulating genes. A hierarchical clustered image map revealed that individual samples sometimes responded differently in control or IL-1ß conditions than the overall population. Despite these variances, discriminative projection-based modeling revealed distinct morphological fingerprints that discriminated between control and inflammatory chondrocyte phenotypes: the most essential morphological characteristics attributable to non-treated control cells was a higher cell aspect ratio in healthy bovine chondrocytes and roundness in OA human chondrocytes. In contrast, a higher circularity and width in healthy bovine chondrocytes and length and area in OA human chondrocytes indicated an inflammatory (IL-1ß) phenotype. When comparing the two species/health conditions, bovine healthy and human OA chondrocytes exhibited comparable IL-1ß-induced morphologies in roundness, a widely recognized marker of chondrocyte phenotype, and aspect ratio. Discussion: Overall, cell morphology can be used as a biological fingerprint for describing chondrocyte phenotype. Quantitative single cell morphometry in conjunction with advanced methods for multivariate data analysis allows identifying morphological fingerprints that can discriminate between control and inflammatory chondrocyte phenotypes. This approach could be used to assess how culture conditions, inflammatory mediators, and therapeutic modulators regulate cell phenotype and function.

Prediction of six macrophage phenotypes and their IL-10 content based on single-cell morphology using artificial intelligence.

Introduction: The last decade has led to rapid developments and increased usage of computational tools at the single-cell level. However, our knowledge remains limited in how extracellular cues alter quantitative macrophage morphology and how such morphological changes can be used to predict macrophage phenotype as well as cytokine content at the single-cell level. Methods: Using an artificial intelligence (AI) based approach, this study determined whether (i) accurate macrophage classification and (ii) prediction of intracellular IL-10 at the single-cell level was possible, using only morphological features as predictors for AI. Using a quantitative panel of shape descriptors, our study assessed image-based original and synthetic single-cell data in two different datasets in which CD14+ monocyte-derived macrophages generated from human peripheral blood monocytes were initially primed with GM-CSF or M-CSF followed by polarization with specific stimuli in the presence/absence of continuous GM-CSF or M-CSF. Specifically, M0, M1 (GM-CSF-M1, TNFα/IFNγ-M1, GM-CSF/TNFα/IFNγ-M1) and M2 (M-CSF-M2, IL-4-M2a, M-CSF/IL-4-M2a, IL-10-M2c, M-CSF/IL-10-M2c) macrophages were examined. Results: Phenotypes were confirmed by ELISA and immunostaining of CD markers. Variations of polarization techniques significantly changed multiple macrophage morphological features, demonstrating that macrophage morphology is a highly sensitive, dynamic marker of phenotype. Using original and synthetic single-cell data, cell morphology alone yielded an accuracy of 93% for the classification of 6 different human macrophage phenotypes (with continuous GM-CSF or M-CSF). A similarly high phenotype classification accuracy of 95% was reached with data generated with different stimuli (discontinuous GM-CSF or M-CSF) and measured at a different time point. These comparably high accuracies clearly validated the here chosen AI-based approach. Quantitative morphology also allowed prediction of intracellular IL-10 with 95% accuracy using only original data. Discussion: Thus, image-based machine learning using morphology-based features not only (i) classified M0, M1 and M2 macrophages but also (ii) classified M2a and M2c subtypes and (iii) predicted intracellular IL-10 at the single-cell level among six phenotypes. This simple approach can be used as a general strategy not only for macrophage phenotyping but also for prediction of IL-10 content of any IL-10 producing cell, which can help improve our understanding of cytokine biology at the single-cell level.

Characterization and In Vitro Cytotoxicity Safety Screening of Fractionated Organosolv Lignin on Diverse Primary Human Cell Types Commonly Used in Tissue Engineering.

There is limited data assessing the cytotoxic effects of organosolv lignin with cells commonly used in tissue engineering. Structural and physico-chemical characterization of fractionated organosolv lignin showed that a decrease of the molecular weight (MW) is accompanied by a less branched conformation of the phenolic biopolymer (higher S/G ratio) and an increased number of aliphatic hydroxyl functionalities. Enabling stronger polymer-solvent interactions, as proven by the Hansen solubility parameter analysis, low MW organosolv lignin (2543 g/mol) is considered to be compatible with common biomaterials. Using low MW lignin, high cell viability (70-100%) was achieved after 2 h, 24 h and 7 days using the following lignin concentrations: MSCs and osteoblasts (0.02 mg/mL), gingival fibroblasts and keratinocytes (0.02 to 0.04 mg/mL), periodontal ligament fibroblasts and chondrocytes (0.02 to 0.08 mg/mL). Cell viability was reduced at higher concentrations, indicating that high concentrations are cytotoxic. Higher cell viability was attained using 30/70 (w/v) NaOH vs. 40/60 (w/v) EtOH as the initial lignin solvent. Hydrogels containing low MW lignin (0.02 to 0.3 mg/mL) in agarose dose-dependently increased chondrocyte attachment (cell viability 84-100%) and hydrogel viscosity and stiffness to 3-11 kPa, similar to the pericellular matrix of chondrocytes. This suggests that low MW organosolv lignin may be used in many tissue engineering fields.

Controlled Growth Factor Delivery and Cyclic Stretch Induces a Smooth Muscle Cell-like Phenotype in Adipose-Derived Stem Cells.

Adipose-derived stem cells (ASCs) are an abundant and easily accessible multipotent stem cell source with potential application in smooth muscle regeneration strategies. In 3D collagen hydrogels, we investigated whether sustained release of growth factors (GF) PDGF-AB and TGF-ß1 from GF-loaded microspheres could induce a smooth muscle cell (SMC) phenotype in ASCs, and if the addition of uniaxial cyclic stretch could enhance the differentiation level. This study demonstrated that the combination of cyclic stretch and GF release over time from loaded microspheres potentiated the differentiation of ASCs, as quantified by protein expression of early to late SMC differentiation markers (SMA, TGLN and smooth muscle MHC). The delivery of GFs via microspheres produced large ASCs with a spindle-shaped, elongated SMC-like morphology. Cyclic strain produced the largest, longest, and most spindle-shaped cells regardless of the presence or absence of growth factors or the growth factor delivery method. Protein expression and cell morphology data confirmed that the sustained release of GFs from GF-loaded microspheres can be used to promote the differentiation of ASCs into SMCs and that the addition of uniaxial cyclic stretch significantly enhances the differentiation level, as quantified by intermediate and late SMC markers and a SMC-like elongated cell morphology.

Anti-Inflammatory Therapeutic Approaches to Prevent or Delay Post-Traumatic Osteoarthritis (PTOA) of the Knee Joint with a Focus on Sustained Delivery Approaches.

Inflammation plays a central role in the pathogenesis of knee PTOA after knee trauma. While a comprehensive therapy capable of preventing or delaying post-traumatic osteoarthritis (PTOA) progression after knee joint injury does not yet clinically exist, current literature suggests that certain aspects of early post-traumatic pathology of the knee joint may be prevented or delayed by anti-inflammatory therapeutic interventions. We discuss multifaceted therapeutic approaches that may be capable of effectively reducing the continuous cycle of inflammation and concomitant processes that lead to cartilage degradation as well as those that can simultaneously promote intrinsic repair processes. Within this context, we focus on early disease prevention, the optimal timeframe of treatment and possible long-lasting sustained delivery local modes of treatments that could prevent knee joint-associated PTOA symptoms. Specifically, we identify anti-inflammatory candidates that are not only anti-inflammatory but also anti-degenerative, anti-apoptotic and pro-regenerative.

Articular Chondrocyte Phenotype Regulation through the Cytoskeleton and the Signaling Processes That Originate from or Converge on the Cytoskeleton: Towards a Novel Understanding of the Intersection between Actin Dynamics and Chondrogenic Function.

Numerous studies have assembled a complex picture, in which extracellular stimuli and intracellular signaling pathways modulate the chondrocyte phenotype. Because many diseases are mechanobiology-related, this review asked to what extent phenotype regulators control chondrocyte function through the cytoskeleton and cytoskeleton-regulating signaling processes. Such information would generate leverage for advanced articular cartilage repair. Serial passaging, pro-inflammatory cytokine signaling (TNF-α, IL-1α, IL-1ß, IL-6, and IL-8), growth factors (TGF-α), and osteoarthritis not only induce dedifferentiation but also converge on RhoA/ROCK/Rac1/mDia1/mDia2/Cdc42 to promote actin polymerization/crosslinking for stress fiber (SF) formation. SF formation takes center stage in phenotype control, as both SF formation and SOX9 phosphorylation for COL2 expression are ROCK activity-dependent. Explaining how it is molecularly possible that dedifferentiation induces low COL2 expression but high SF formation, this review theorized that, in chondrocyte SOX9, phosphorylation by ROCK might effectively be sidelined in favor of other SF-promoting ROCK substrates, based on a differential ROCK affinity. In turn, actin depolymerization for redifferentiation would "free-up" ROCK to increase COL2 expression. Moreover, the actin cytoskeleton regulates COL1 expression, modulates COL2/aggrecan fragment generation, and mediates a fibrogenic/catabolic expression profile, highlighting that actin dynamics-regulating processes decisively control the chondrocyte phenotype. This suggests modulating the balance between actin polymerization/depolymerization for therapeutically controlling the chondrocyte phenotype.

An Evidence-Based Systematic Review of Human Knee Post-Traumatic Osteoarthritis (PTOA): Timeline of Clinical Presentation and Disease Markers, Comparison of Knee Joint PTOA Models and Early Disease Implications.

Understanding the causality of the post-traumatic osteoarthritis (PTOA) disease process of the knee joint is important for diagnosing early disease and developing new and effective preventions or treatments. The aim of this review was to provide detailed clinical data on inflammatory and other biomarkers obtained from patients after acute knee trauma in order to (i) present a timeline of events that occur in the acute, subacute, and chronic post-traumatic phases and in PTOA, and (ii) to identify key factors present in the synovial fluid, serum/plasma and urine, leading to PTOA of the knee in 23-50% of individuals who had acute knee trauma. In this context, we additionally discuss methods of simulating knee trauma and inflammation in in vivo, ex vivo articular cartilage explant and in vitro chondrocyte models, and answer whether these models are representative of the clinical inflammatory stages following knee trauma. Moreover, we compare the pro-inflammatory cytokine concentrations used in such models and demonstrate that, compared to concentrations in the synovial fluid after knee trauma, they are exceedingly high. We then used the Bradford Hill Framework to present evidence that TNF-α and IL-6 cytokines are causal factors, while IL-1ß and IL-17 are credible factors in inducing knee PTOA disease progresssion. Lastly, we discuss beneficial infrastructure for future studies to dissect the role of local vs. systemic inflammation in PTOA progression with an emphasis on early disease.

Mechanotransduction and Stiffness-Sensing: Mechanisms and Opportunities to Control Multiple Molecular Aspects of Cell Phenotype as a Design Cornerstone of Cell-Instructive Biomaterials for Articular Cartilage Repair.

Since material stiffness controls many cell functions, we reviewed the currently available knowledge on stiffness sensing and elucidated what is known in the context of clinical and experimental articular cartilage (AC) repair. Remarkably, no stiffness information on the various biomaterials for clinical AC repair was accessible. Using mRNA expression profiles and morphology as surrogate markers of stiffness-related effects, we deduced that the various clinically available biomaterials control chondrocyte (CH) phenotype well, but not to equal extents, and only in non-degenerative settings. Ample evidence demonstrates that multiple molecular aspects of CH and mesenchymal stromal cell (MSC) phenotype are susceptible to material stiffness, because proliferation, migration, lineage determination, shape, cytoskeletal properties, expression profiles, cell surface receptor composition, integrin subunit expression, and nuclear shape and composition of CHs and/or MSCs are stiffness-regulated. Moreover, material stiffness modulates MSC immuno-modulatory and angiogenic properties, transforming growth factor beta 1 (TGF-ß1)-induced lineage determination, and CH re-differentiation/de-differentiation, collagen type II fragment production, and TGF-ß1- and interleukin 1 beta (IL-1ß)-induced changes in cell stiffness and traction force. We then integrated the available molecular signaling data into a stiffness-regulated CH phenotype model. Overall, we recommend using material stiffness for controlling cell phenotype, as this would be a promising design cornerstone for novel future-oriented, cell-instructive biomaterials for clinical high-quality AC repair tissue.

Lack of a skeletal muscle phenotype in adult human bone marrow stromal cells following xenogeneic-free expansion.

BACKGROUND: Many studies have elegantly shown that murine and rat bone marrow-derived mesenchymal stromal cells (bmMSCs) contribute to muscle regeneration and improve muscle function. Yet, the ability of transplanted human bmMSCs to manifest myogenic potential shows conflicting results. While human adipose- and umbilical cord-derived MSCs can be differentiated into a skeletal muscle phenotype using horse serum (HS), bmMSCs have only been shown to differentiate towards the skeletal muscle lineage using a complex mixture of cytokines followed by transfection with notch intracellular domain. METHODS: Since xenogeneic-free growth supplements are increasingly being used in the expansion of bmMSCs in clinical trials, we investigated the effects of human plasma and platelet lysate (P/PL) on the expression of neuromuscular markers and whether P/PL-expanded human bmMSCs could be differentiated towards a skeletal myogenic phenotype. Neuromuscular markers were measured using the highly sensitive droplet digital polymerase chain reaction for measuring the expression of Myf5, MyoD, MyoG, ACTA1, Desmin, GAP-43, and Coronin 1b transcripts, by performing immunofluorescence for the expression of Desmin, GAP-43, and MEF2, and flow cytometry for the expression of CD56/neural cell adhesion molecule (NCAM). RESULTS: Despite that bmMSCs expressed the myogenic regulatory factor (MRF) MEF2 after expansion in P/PL, bmMSCs cultured under such conditions did not express other essential MRFs including Myf5, MyoD, MyoG, or ACTA1 needed for myogenesis. Moreover, HS did not induce myogenesis of bmMSCs and hence did not induce the expression of any of these myogenic markers. P/PL, however, did lead to a significant increase in neurogenic GAP-43, as well as Desmin expression, and resulted in a high baseline expression of the neurogenic gene Coronin 1b which was sustained under further P/PL or HS culture conditions. Fetal bovine serum resulted in equally high levels of GAP-43 and Coronin 1b. Moreover, the proportion of CD56/NCAM-positive bmMSCs cultured in P/PL was 5.9 ± 2.1. CONCLUSIONS: These data suggest that P/PL may prime a small portion of bmMSCs towards an early neural precursor cell type. Collectively, this shows that P/PL partially primes the cells towards a neurogenic phenotype, but does not prime adult human bmMSCs towards the skeletal muscle lineage.

Bioresponsive microspheres for on-demand delivery of anti-inflammatory cytokines for articular cartilage repair.

Despite innovations in surgical interventions, treatment of cartilage injury in osteoarthritic joints remains a challenge due to concomitant inflammation. Obstructing a single dominant inflammatory cytokine has shown remarkable clinical benefits in rheumatoid arthritis, and similar strategies are being suggested to target inflammatory pathways in osteoarthritis (OA). Here, we describe the utility of gelatin microspheres that are responsive to proteolytic enzymes typically expressed in arthritic flares, resulting in on-demand and spatiotemporally controlled release of anti-inflammatory cytokines for cartilage preservation and repair. These microspheres were designed with a net negative charge to sequester cationic anti-inflammatory cytokines, and the magnitude of the negative charge potential increased with an increase in crosslinking density. Collagenase-mediated degradation of the microspheres was dependent on the concentration of the enzyme. Release of anti-inflammatory cytokines from the loaded microspheres directly correlated with the degradation of the gelatin matrix. Exposure of the IL-4 and IL-13 loaded microspheres reduced the inflammation of chondrocytes up to 80%. Hence, the delivery of these microspheres in an OA joint can attenuate the stimulation of chondrocytes and the resulting secretion of catabolic factors such as proteinases and nitric oxide. The microsphere format also allows for minimally invasive delivery and is less susceptible to mechanically induced drug release. Consequently, bioresponsive microspheres can be an effective tool for cartilage preservation and arthritis treatment.

Engineering the geometrical shape of mesenchymal stromal cells through defined cyclic stretch regimens.

Stem cells have been predicted to improve disease outcomes and patient lives. Steering stem cell fate - through controlling cell shape - may substantially accelerate progress towards this goal. As mesenchymal stromal cells (MSCs) are continuously exposed in vivo to a dynamically changing biomechanical environment, we hypothesized that exogenous forces can be applied for engineering a variety of significantly different MSC shapes. We applied specific cyclic stretch regimens to human MSCs and quantitatively measured the resulting cell shape, alignment, and expression of smooth muscle (SMC) differentiation markers, as those have been associated with elongated morphology. As proof of principle, a range of different shapes, alignments, and correlating SMC marker levels were generated by varying strain, length, and repetition of stretch. However, the major determinant of biomechanically engineering cellular shape was the repetition of a chosen stretch regimen, indicating that the engineered shape and associated differentiation were complex non-linear processes relying on sustained biomechanical stimulation. Thus, forces are key regulators of stem cell shape and the targeted engineering of specific MSC shapes through biomechanical forces represents a novel mechanobiology concept that could exploit naturally occurring in vivo forces for improving stem cell fate in clinical regenerative therapies.

The geometrical shape of mesenchymal stromal cells measured by quantitative shape descriptors is determined by the stiffness of the biomaterial and by cyclic tensile forces.

Controlling mesenchymal stromal cell (MSC) shape is a novel method for investigating and directing MSC behaviour in vitro. it was hypothesized that specifigc MSC shapes can be generated by using stiffness-defined biomaterial surfaces and by applying cyclic tensile forces. Biomaterials used were thin and thick silicone sheets, fibronectin coating, and compacted collagen type I sheets. The MSC morphology was quantified by shape descriptors describing dimensions and membrane protrusions. Nanoscale stiffness was measured by atomic force microscopy and the expression of smooth muscle cell (SMC) marker genes (ACTA2, TAGLN, CNN1) by quantitative reverse-transcription polymerase chain reaction. Cyclic stretch was applied with 2.5% or 5% amplitudes. Attachment to biomaterials with a higher stiffness yielded more elongated MSCs with fewer membrane protrusions compared with biomaterials with a lower stiffness. For cyclic stretch, compacted collagen sheets were selected, which were associated with the most elongated MSC shape across all investigated biomaterials. As expected, cyclic stretch elongated MSCs during stretch. One hour after cessation of stretch, however, MSC shape was rounder again, suggesting loss of stretch-induced shape. Different shape descriptor values obtained by different stretch regimes correlated significantly with the expression levels of SMC marker genes. Values of approximately 0.4 for roundness and 3.4 for aspect ratio were critical for the highest expression levels of ACTA2 and CNN1. Thus, specific shape descriptor values, which can be generated using biomaterial-associated stiffness and tensile forces, can serve as a template for the induction of specific gene expression levels in MSC. Copyright © 2017 John Wiley & Sons, Ltd.

Expression of Desmoglein 2, Desmocollin 3 and Plakophilin 2 in Placenta and Bone Marrow-Derived Mesenchymal Stromal Cells.

Many controversial results exist when comparing mesenchymal stromal cells (MSCs) derived from different sources. Reasons include not only variables in tissue origin, but also methods of cell preparation or choice of expansion media which can strongly influence the expression and hence, function of the cells. In this short report we aimed to investigate the expression of the cell anchoring proteins desmoglein 2, desmocollin 3 and plakophilin 2 in early passage placenta-derived MSCs of fetal (fetal pMSCs) and maternal (maternal pMSCs) origins versus adult bone marrow-derived MSCs (bmMSCs) that were expanded and cultured under the same good manufacturing practice (GMP) conditions. Comprehensive gene expression microarray analysis profiling indicated differential expression of these genes in the different MSC-derived types with fetal pMSCs expressing the highest levels of PKP2, DSC3 and DSG2, followed by maternal pMSCs, while bmMSCs expressed the lowest levels. A higher expression of PKP2 and DSC3 genes in fetal pMSCs was confirmed by qRT-PCR suggesting neonatal increases in the expression of these desmosomal genes vs. adult MSCs. Intracellular desmocollin 3 and desmoglein 2 expression was observed by flow cytometry and cytoplasmic plakophilin 2 by immunofluorescence in all three MSC sources. These data suggest that fetal pMSCs, maternal pMSCs and bmMSCs may anchor intermediate filaments to the plasma membrane via desmocollin 3, desmoglein 2 and plakophilin 2.

Comparative phenotypic transcriptional characterization of human full-term placenta-derived mesenchymal stromal cells compared to bone marrow-derived mesenchymal stromal cells after differentiation in myogenic medium.

Placenta-derived mesenchymal stromal cells (pMSCs) are a very attractive source of MSCs. In this short report we evaluated the expression of phenotypic markers from fetal and maternal pMSCs after exposure to myogenic medium commonly used to differentiate bone marrow MSCs (bmMSCs) to smooth muscle-like cells (SMCs). In order to reveal differences between these different MSC sources, cells were expanded and differentiated to elucidate whether this differentiation protocol facilitated efficient differentiation of SMCs from human pMSCs. We report that TGF-ß1, PDGF and ascorbic acid is not sufficient to produce SMCs from pMSCs.

Stretching human mesenchymal stromal cells on stiffness-customized collagen type I generates a smooth muscle marker profile without growth factor addition.

Using matrix elasticity and cyclic stretch have been investigated for inducing mesenchymal stromal cell (MSC) differentiation towards the smooth muscle cell (SMC) lineage but not in combination. We hypothesized that combining lineage-specific stiffness with cyclic stretch would result in a significantly increased expression of SMC markers, compared to non-stretched controls. First, we generated dense collagen type I sheets by mechanically compressing collagen hydrogels. Atomic force microscopy revealed a nanoscale stiffness range known to support myogenic differentiation. Further characterization revealed viscoelasticity and stable biomechanical properties under cyclic stretch with >99% viable adherent human MSC. MSCs on collagen sheets demonstrated a significantly increased mRNA but not protein expression of SMC markers, compared to on culture flasks. However, cyclic stretch of MSCs on collagen sheets significantly increased both mRNA and protein expression of α-smooth muscle actin, transgelin, and calponin versus plastic and non-stretched sheets. Thus, lineage-specific stiffness and cyclic stretch can be applied together for inducing MSC differentiation towards SMCs without the addition of recombinant growth factors or other soluble factors. This represents a novel stimulation method for modulating the phenotype of MSCs towards SMCs that could easily be incorporated into currently available methodologies to obtain a more targeted control of MSC phenotype.