Directing Stem Cell Commitment by Amorphous Calcium Phosphate Nanoparticles Incorporated in PLGA: Relevance of the Free Calcium Ion Concentration

The microenvironment of mesenchymal stem cells (MSCs) is responsible for the modulation in MSC commitment. Nanocomposites with an inorganic and an organic component have been investigated, and osteogenesis of MSCs has been attributed to inorganic phases such as calcium phosphate under several conditions. Here, electrospun meshes and two-dimensional films of poly(lactic-co-glycolic acid) (PLGA) or nanocomposites of PLGA and amorphous calcium phosphate nanoparticles (PLGA/aCaP) seeded with human adipose-derived stem cells (ASCs) were analyzed for the expression of selected marker genes. In a two-week in vitro experiment, osteogenic commitment was not found to be favored on PLGA/aCaP compared to pure PLGA. Analysis of the medium revealed a significant reduction of the Ca$^{2+}$ concentration when incubated with PLGA/aCaP, caused by chemical precipitation of hydroxyapatite (HAp) on aCaP seeds of PLGA/aCaP. Upon offering a constant Ca$^{2+}$ concentration, however, the previously observed anti-osteogenic effect was reversed: alkaline phosphatase, an early osteogenic marker gene, was upregulated on PLGA/aCaP compared to pristine PLGA. Hence, in addition to the cell-material interaction, the material-medium interaction was also important for the stem cell commitment here, affecting the cell-medium interaction. Complex in vitro models should therefore consider all factors, as coupled impacts might emerge

3D microtissue–derived human stem cells seeded on electrospun nanocomposites under shear stress: Modulation of gene expression

Objective Different microenvironments trigger distinct differentiation of stem cells. Even without chemical supplementation, mechanical stimulation by shear stress may help to induce the desired differentiation. The cell format, such as three-dimensional (3D) microtissues (MTs), MT-derived cells or single cells (SCs), may have a pivotal impact as well. Here, we studied modulation of gene expression in human adipose–derived stem cells (ASCs) exposed to shear stress and/or after MT formation. Materials and methods Electrospun meshes of poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles (PLGA/aCaP) at a weight ratio of 60:40 were seeded with human ASCs as MTs or as SCs and cultured in Dulbecco's modified Eagle's medium without chemical supplementation. After 2 weeks of static culture, the scaffolds were cultured statically for another 2 weeks or placed in a Bose® bioreactor with a flow rate per area of 0.16 mL cm−2 min−1. Stiffness of the scaffolds was assessed as a function of time. After 4 weeks, minimum stem cell criteria markers and selected markers of osteogenesis, endothelial cell differentiation, adipogenesis and chondrogenesis were analysed by quantitative real-time polymerase chain reaction. Additionally, cell distribution within the scaffolds and the allocation of the yes-associated protein (YAP) in the cells were assessed by immunohistochemistry. Results MTs decayed completely within 2 weeks after seeding on PLGA/aCaP. The osteogenic marker gene alkaline phosphatase and the endothelial cell marker gene CD31 were upregulated in MT-derived ASCs compared with SCs. Shear stress realised by fluid flow perfusion upregulated peroxisome proliferator–activated receptor gamma 2 expression in MT-derived ASCs and in SCs. The nuclear-to-cytoplasmic ratio of YAP expression was doubled under perfusion compared with that under static culture for MT-derived ASCs and SCs. Conclusions Osteogenic and angiogenic commitments were more pronounced in MT-derived ASCs seeded on bone biomimetic electrospun nanocomposite PLGA/aCaP than in SCs seeded without induction medium. Furthermore, the static culture was superior to the perfusion regimen used here, as shear stress resulted in adipogenic commitment for MT-derived ASCs and SCs, although the YAP nuclear-to-cytoplasmic ratio indicated higher cell tensions under perfusion, usually associated with preferred osteogenic differentiation

Cyclic uniaxial compression of human stem cells seeded on a bone biomimetic nanocomposite decreases anti-osteogenic commitment evoked by shear stress

OBJECTIVE Chemical supplementation of culture media to induce differentiation of adult stem cells seeded on a scaffold may mask other differentiation triggers such as scaffold stiffness, chemical composition or mechanical stimulation. However, stem cells can be differentiated towards osteoblasts without any supplementation given an appropriate osteogenic scaffold and an adequate mechanical stimulation. MATERIALS AND METHODS Electrospun meshes of poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles (PLGA/aCaP) in a weight ratio of 60:40 were seeded with human adipose-derived stem cells (ASCs) and cultured in DMEM. After two weeks of static cultivation, they were either further cultivated statically for another two weeks (group 1), or placed in a Bose® bioreactor with a flow rate per area of 0.16 mL cm min (group 2). Furthermore, group 3 was also cultivated under perfusion, however, with an additional uniaxial cyclic compression. Stiffness of the scaffolds was assessed as a function of time. After a total of four weeks, minimum stem cell criteria markers as well as typical markers for osteogenesis, endothelial cell differentiation, adipogenesis and chondrogenesis were analyzed by quantitative real-time PCR, cell distribution within the scaffolds by histology and protein expression by immunohistochemistry. RESULTS Dynamic conditions (perfusion ± uniaxial cyclic compression) significantly upregulated gene and protein expression of PPAR-γ-2 compared to static cultivation, while osteogenic markers were slightly downregulated. However, the compression in the perfusion bioreactor favored osteogenesis compared to mere perfusion as indicated by upregulation of ALP, Runx2 and collagen I. This behavior was not only attributed to the compressive load, but also to the significant increase in stiffness of the scaffold. Furthermore, CD105 was significantly upregulated under compression. CONCLUSIONS Although an osteogenic electrospun composite material with an organic (PLGA) and an inorganic phase (aCaP nanoparticles) was used as scaffold, the dynamic cultivation as realized by either perfusion alone or an additional compression did not upregulate typical osteogenic genes when compared to static cultivation. In contrast, there was a significant upregulation of the adipogenic gene PPAR-γ-2. However, this anti-osteogenic starting point evoked by mere perfusion was partially reversed by an additional compression. Our findings exemplify that bone tissue engineering using adult stem cells should consider any other differentiations that may be triggered and overwhelm the desired differentiation, although experimental conditions theoretically provide cues to achieve it - like an osteogenic scaffold and mechanical stimulation

Effects of seeding adipose-derived stem cells on electrospun nanocomposite used as chest wall graft in a murine model

OBJECTIVES Malignant neoplasms infiltrating the chest wall often requires resection of the thoracic wall. To replace the defect, Gore-Tex(®) is usually employed as the gold standard material, however, Gore-Tex(®) is inert and not degradable. Novel materials are nowadays available which allow a full bio-integration due to their non-toxic degradability. Additionally, stem cell seeding has the capacity to reduce inflammatory response towards such grafts, thus integrating it better into the host organism

Cartilage/bone interface fabricated under perfusion: Spatially organized commitment of adipose-derived stem cells without medium supplementation

Tissue engineering of an osteochondral interface demands for a gradual transition of chondrocyte- to osteoblast-prevailing tissue. If stem cells are used as a single cell source, an appropriate cue to trigger the desired differentiation is the use of composite materials with different amounts of calcium phosphate. Electrospun meshes of poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles (PLGA/aCaP) in weight ratios of 100:0; 90:10, 80:20, and 70:30 were seeded with human adipose-derived stem cells (ASCs) and cultured in DMEM without chemical supplementation. After 2 weeks of static cultivation, they were either further cultivated statically for another 2 weeks (group 1), or placed in a Bose® bioreactor with a flow rate per area of 0.16 mL cm min (group 2). Markers for stem cell criteria, chondrogenesis, osteogenesis, adipogenesis and angiogenesis were analyzed by quantitative real-time PCR. Cell distribution, Sox9 protein expression and proteoglycans were assessed by histology. In group 2 (perfusion culture), chondrogenic Sox9 was upregulated toward the cartilage-mimicking side compared to pure PLGA. On the bone-mimicking side, Sox9 experienced a downregulation, which was confirmed on the protein level. Vice versa, expression of osteocalcin was upregulated on the bone-mimicking side, while it was unchanged on the cartilage-mimicking side. In group 1 (static culture), CD31 was upregulated in the presence of aCaP compared to pure PLGA, whereas Sox9 and osteocalcin expression were not affected. aCaP nanoparticles incorporated in electrospun PLGA drive the differentiation behavior of human ASCs in a dose-dependent manner. Discrete gradients of aCaP may act as promising osteochondral interfaces. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res B Part B: Appl Biomater, 2018

Directing Stem Cell Commitment by Amorphous Calcium Phosphate Nanoparticles Incorporated in PLGA: Relevance of the Free Calcium Ion Concentration

The microenvironment of mesenchymal stem cells (MSCs) is responsible for the modulation in MSC commitment. Nanocomposites with an inorganic and an organic component have been investigated, and osteogenesis of MSCs has been attributed to inorganic phases such as calcium phosphate under several conditions. Here, electrospun meshes and two-dimensional films of poly(lactic-co-glycolic acid) (PLGA) or nanocomposites of PLGA and amorphous calcium phosphate nanoparticles (PLGA/aCaP) seeded with human adipose-derived stem cells (ASCs) were analyzed for the expression of selected marker genes. In a two-week in vitro experiment, osteogenic commitment was not found to be favored on PLGA/aCaP compared to pure PLGA. Analysis of the medium revealed a significant reduction of the Ca2+ concentration when incubated with PLGA/aCaP, caused by chemical precipitation of hydroxyapatite (HAp) on aCaP seeds of PLGA/aCaP. Upon offering a constant Ca2+ concentration, however, the previously observed anti-osteogenic effect was reversed: alkaline phosphatase, an early osteogenic marker gene, was upregulated on PLGA/aCaP compared to pristine PLGA. Hence, in addition to the cell–material interaction, the material–medium interaction was also important for the stem cell commitment here, affecting the cell–medium interaction. Complex in vitro models should therefore consider all factors, as coupled impacts might emerge

Improvement of prognostic performance in severely injured patients by integrated clinico-transcriptomics: a translational approach

INTRODUCTION Severe trauma triggers a systemic inflammatory response that contributes to secondary complications, such as nosocomial infections, sepsis or multi-organ failure. The present study was aimed to identify markers predicting complications and an adverse outcome of severely injured patients by an integrated clinico-transcriptomic approach. METHODS In a prospective study, RNA samples from circulating leukocytes from severely injured patients (injury severity score ≥ 17 points; n = 104) admitted to a Level I Trauma Center were analyzed for dynamic changes in gene expression over a period of 21 days by quantitative RT-PCR. Transcriptomic candidates were selected based on whole genome screening of a representative discovery set (n = 10 patients) or known mechanisms of the immune response, including mediators of inflammation (IL-8, IL-10, TNF-α, MIF, C5, CD59, SPHK1), danger signaling (HMGB1, TLR2, CD14, IL-33, IL-1RL1), and components of the heme degradation pathway (HP, CD163, HMOX1, BLVRA, BLVRB). Clinical markers comprised standard physiological and laboratory parameters and scoring systems routinely determined in trauma patients. RESULTS Leukocytes, thrombocytes and the expression of sphingosine kinase-1 (SPHK1), complement C5, and haptoglobin (HP) have been identified as markers with the best performance. Leukocytes showed a biphasic course with peaks on day 0 and day 11 after trauma, and patients with sepsis exhibited significantly higher leukocyte levels. Thrombocyte numbers showed a typical profile with initial thrombopenia and robust thrombocytosis in week 3 after trauma, ranging 2- to 3-fold above the upper normal value. 'Relative thrombocytopenia' was associated with multi-organ dysfunction, the development of sepsis, and mortality, the latter of which could be predicted within 3 days prior to the time point of death. SPHK1 expression at the day of admission indicated mortality with excellent performance. C5-expression on day 1 after trauma correlated with an increased risk for the development of nosocomial infections during the later course, while HP was found to be a marker for the development of sepsis. CONCLUSIONS The combination of clinical and transcriptomic markers improves the prognostic performance and may represent a useful tool for individual risk stratification in trauma patients

Identification of ALP+/CD73+ defining markers for enhanced osteogenic potential in human adipose-derived mesenchymal stromal cells by mass cytometry

Background The impressive progress in the field of stem cell research in the past decades has provided the ground for the development of cell-based therapy. Mesenchymal stromal cells obtained from adipose tissue (AD-MSCs) represent a viable source for the development of cell-based therapies. However, the heterogeneity and variable differentiation ability of AD-MSCs depend on the cellular composition and represent a strong limitation for their use in therapeutic applications. In order to fully understand the cellular composition of MSC preparations, it would be essential to analyze AD-MSCs at single-cell level. Method Recent advances in single-cell technologies have opened the way for high-dimensional, high-throughput, and high-resolution measurements of biological systems. We made use of the cytometry by time-of-flight (CyTOF) technology to explore the cellular composition of 17 human AD-MSCs, interrogating 31 markers at single-cell level. Subcellular composition of the AD-MSCs was investigated in their naïve state as well as during osteogenic commitment, via unsupervised dimensionality reduction as well as supervised representation learning approaches. Result This study showed a high heterogeneity and variability in the subcellular composition of AD-MSCs upon isolation and prolonged culture. Algorithm-guided identification of emerging subpopulations during osteogenic differentiation of AD-MSCs allowed the identification of an ALP+/CD73+ subpopulation of cells with enhanced osteogenic differentiation potential. We could demonstrate in vitro that the sorted ALP+/CD73+ subpopulation exhibited enhanced osteogenic potential and is moreover fundamental for osteogenic lineage commitment. We finally showed that this subpopulation was present in freshly isolated human adipose-derived stromal vascular fractions (SVFs) and that could ultimately be used for cell therapies. Conclusion The data obtained reveal, at single-cell level, the heterogeneity of AD-MSCs from several donors and highlight how cellular composition impacts the osteogenic differentiation capacity. The marker combination (ALP/CD73) can not only be used to assess the differentiation potential of undifferentiated AD-MSC preparations, but also could be employed to prospectively enrich AD-MSCs from the stromal vascular fraction of human adipose tissue for therapeutic applications