Modeling Myeloma Dissemination In Vitro with hMSC-interacting Subpopulations of INA-6 Cells and Their Aggregation/Detachment Dynamics.

Multiple myeloma involves early dissemination of malignant plasma cells across the bone marrow; however, the initial steps of dissemination remain unclear. Human bone marrow-derived mesenchymal stromal cells (hMSC) stimulate myeloma cell expansion (e.g., IL6) and simultaneously retain myeloma cells via chemokines (e.g., CXCL12) and adhesion factors. Hence, we hypothesized that the imbalance between cell division and retention drives dissemination. We present an in vitro model using primary hMSCs cocultured with INA-6 myeloma cells. Time-lapse microscopy revealed proliferation and attachment/detachment dynamics. Separation techniques (V-well adhesion assay and well plate sandwich centrifugation) were established to isolate MSC-interacting myeloma subpopulations that were characterized by RNA sequencing, cell viability, and apoptosis. Results were correlated with gene expression data (n = 837) and survival of patients with myeloma (n = 536). On dispersed hMSCs, INA-6 saturate hMSC surface before proliferating into large homotypic aggregates, from which single cells detached completely. On confluent hMSCs, aggregates were replaced by strong heterotypic hMSC-INA-6 interactions, which modulated apoptosis time dependently. Only INA-6 daughter cells (nMA-INA6) detached from hMSCs by cell division but sustained adherence to hMSC-adhering mother cells (MA-INA6). Isolated nMA-INA6 indicated hMSC autonomy through superior viability after IL6 withdrawal and upregulation of proliferation-related genes. MA-INA6 upregulated adhesion and retention factors (CXCL12), that, intriguingly, were highly expressed in myeloma samples from patients with longer overall and progression-free survival, but their expression decreased in relapsed myeloma samples. Altogether, in vitro dissemination of INA-6 is driven by detaching daughter cells after a cycle of hMSC-(re)attachment and proliferation, involving adhesion factors that represent a bone marrow-retentive phenotype with potential clinical relevance. SIGNIFICANCE: Novel methods describe in vitro dissemination of myeloma cells as detachment of daughter cells after cell division. Myeloma adhesion genes were identified that counteract in vitro detachment with potential clinical relevance.

Liquid Overlay and Collagen-Based Three-Dimensional Models for In Vitro Investigation of Multiple Myeloma.

Multiple myeloma (MM) clones reside in the bone marrow (BM), which plays a role in its survival and development. The interactions between MM and their neighboring mesenchymal stromal cells (MSCs) have been shown to promote MM growth and drug resistance. However, those interactions are often missing or misrepresented in traditional two-dimensional (2D) culture models. Application of novel three-dimensional (3D) models might recapitulate the BM niche more precisely, which will offer new insights into MM progression and survival. Here, we aimed to establish two 3D models, based on MSC spheroids and collagen droplets incorporating both MM cells and MSCs with the goal of replicating the native myeloma context of the BM niche. This approach revealed that although MSCs can spontaneously assemble spheroids with altered metabolic traits, MSC spheroid culture does not support the integration of MM cells. On the contrary, collagen-droplet culture supported the growth of both cell types. In collagen, MSC proliferation was reduced, with the correlating decrease in ATP production and Ki-67 expression, which might resemble in vivo conditions, rather than 2D abundance of nutrients and space. MSCs and MMs were distributed homogenously throughout the collagen droplet, with an apparent CXCL12 expression in MSCs. In addition, the response of MM cells to bortezomib was substantially reduced in collagen, indicating the importance of 3D culture in the investigation of myeloma cell behavior, as drug resistance is one of the most pertinent issues in cancer therapy.

Enhancement of Immunosuppressive Activity of Mesenchymal Stromal Cells by Platelet-Derived Factors is Accompanied by Apoptotic Priming.

The pro-inflammatory phase of bone healing, initiated by platelet activation and eventually hematoma formation, impacts bone marrow mesenchymal stromal cells (MSCs) in unknown ways. Here, we created platelet-rich plasma (PRP) hydrogels to study how platelet-derived factors modulate functional properties of encapsulated MSCs in comparison to a non-inflammatory fibrin (FBR) hydrogel environment. MSCs were isolated from human bone marrow, while PRP was collected from pooled apheresis thrombocyte concentrates and used for hydrogel preparation. After their encapsulation in hydrogels for 72 h, retrieved MSCs were analyzed for immunomodulatory activities, apoptosis, stem cell properties, senescence, CD9+, CD63+ and CD81+ extracellular vesicle (EV) release, and metabolism-related changes. PRP-hydrogels stimulated immunosuppressive functions of MSCs, along with their upregulated susceptibility to cell death in communication with PBMCs and augmented caspase 3/7 activity. We found impaired clonal growth and cell cycle progression, and more pronounced ß-galactosidase activity as well as accumulation of LC3-II-positive vacuoles in PRP-MSCs. Stimuli derived from PRP-hydrogels upregulated AKT and reduced mTOR phosphorylation in MSCs, which suggests an initiation of survival-related processes. Our results showed that PRP-hydrogels might represent a metabolically stressful environment, inducing acidification of MSCs, reducing polarization of the mitochondrial membrane and increasing lipid accumulation. These features were not detected in FBR-MSCs, which showed reduced CD63+ and CD81+ EV production and maintained clonogenicity. Our data revealed that PRP-derived hematoma components cause metabolic adaptation of MSCs followed by increased immune regulatory functions. For the first time, we showed that PRP stimuli represent a survival challenge and "apoptotic priming" that are detrimental for stem cell-like growth of MSCs and important for their therapeutic consideration.

Bone Metastases of Diverse Primary Origin Frequently Express the VDR (Vitamin D Receptor) and CYP24A1.

Active vitamin D (1,25(OH)2D3) is known to exert direct anti-cancer actions on various malignant tissues through binding to the vitamin D receptor (VDR). These effects have been demonstrated in breast, prostate, renal and thyroid cancers, which all have a high propensity to metastasise to bone. In addition, there is evidence that vitamin D catabolism via 24-hydroxylase (CYP24A1) is altered in tumour cells, thus, reducing local active vitamin D levels in cancer cells. The aim of this study was to assess VDR and CYP24A1 expression in various types of bone metastases by using immunohistochemistry. Overall, a high total VDR protein expression was detected in 59% of cases (39/66). There was a non-significant trend of high-grade tumours towards the low nuclear VDR expression (p = 0.07). Notably, patients with further distant metastases had a reduced nuclear VDR expression (p = 0.03). Furthermore, a high CYP24A1 expression was detected in 59% (39/66) of bone metastases. There was a significant positive correlation between nuclear VDR and CYP24A1 expression (p = 0.001). Collectively, the VDR and CYP24A1 were widely expressed in a multitude of bone metastases, pointing to a potential role of vitamin D signalling in cancer progression. This is of high clinical relevance, as vitamin D deficiency is frequent in patients with bone metastases.

Head and neck squamous cancer cells enhance the differentiation of human mesenchymal stem cells to adipogenic and osteogenic linages in vitro.

Human mesenchymal stem cells (hMSC) are multipotent cells with the ability to differentiate into a range of different cell types, including fat, bone, cartilage or muscle. A pro-tumorigenic effect of hMSC has been previously reported as part of the tumor stroma. In addition, studies have previously revealed the influence of hematopoietic and lymphoid tumors on hMSC differentiation to support their own growth. However, this possible phenomenon has not been explored in solid malignancies. Therefore, the aim of the present study was to investigate the effects of head and neck squamous cell carcinoma (HNSCC) lines Cal27 and HLaC78 on the induction of osteogenic and adipogenic differentiation in hMSCs. Native hMSCs were co-cultured with Cal27 and HLaC78 cells for 3 weeks. Subsequently, hMSC differentiation was assessed using reverse transcription-PCR and using Oil Red O and von Kossa staining. Furthermore, the effects of differentiated hMSCs on Cal27 and HLaC78 were examined. For this purpose, hMSCs differentiated into the adipogenic (adipo-hMSC) and osteogenic (osteo-hMSC) lineages were co-cultured with Cal27 and HLaC78. Cell viability, cytokine secretion and activation of STAT3 signaling were measured by cell counting, dot blot assay (42 cytokines with focus on IL-6) and western blotting (STAT3, phosphorylated STAT3, ß-actin), respectively. Co-culturing hMSCs with Cal27 and HLaC78 cells resulted in both adipogenic and osteogenic differentiation. In addition, the viability of Cal27 and HLaC78 cells was found to be increased after co-cultivation with adipo-hMSCs, compared with that of cells co-cultured with osteo-hMSC. According to western blotting results, Cal27 cells incubated with adipo-hMSCs exhibited increased STAT3 activation, compared with that in cells co-cultured with native hMSCs and osteo-hMSCs. IL-6 concentration in the media of Cal27 and HLaC78 after co-cultivation with respectively incubation with conditioned media of hMSCs, adipo-hMSCs and osteo-hMSCs were also found to be increased compared with that in the media of Cal27 and HLaC78 cells incubated with DMEM. To conclude, HNSCC cell lines Cal27 and HLaC78 induced hMSC differentiation towards the adipogenic and osteogenic lineages in vitro. Furthermore, a proliferative effect of adipo-hMSCs on Cal27 and HLaC78 cells was revealed with STAT3 activation as a possible mechanism. These results warrant further investigation of the interaction between HNSCC cells and hMSCs, with focus on the mechanism underlying the differentiation of hMSCs.

Effects of Extracellular Vesicles from Osteogenic Differentiated Human BMSCs on Osteogenic and Adipogenic Differentiation Capacity of Naïve Human BMSCs.

Osteoporosis, or steroid-induced osteonecrosis of the hip, is accompanied by increased bone marrow adipogenesis. Such a disorder of adipogenic/osteogenic differentiation, affecting bone-marrow-derived mesenchymal stem cells (BMSCs), contributes to bone loss during aging. Here, we investigated the effects of extracellular vesicles (EVs) isolated from human (h)BMSCs during different stages of osteogenic differentiation on the osteogenic and adipogenic differentiation capacity of naïve (undifferentiated) hBMSCs. We observed that all EV groups increased viability and proliferation capacity and suppressed the apoptosis of naïve hBMSCs. In particular, EVs derived from hBMSCs at late-stage osteogenic differentiation promoted the osteogenic potential of naïve hBMSCs more effectively than EVs derived from naïve hBMSCs (naïve EVs), as indicated by the increased gene expression of COL1A1 and OPN. In contrast, the adipogenic differentiation capacity of naïve hBMSCs was inhibited by treatment with EVs from osteogenic differentiated hBMSCs. Proteomic analysis revealed that osteogenic EVs and naïve EVs contained distinct protein profiles, with pro-osteogenic and anti-adipogenic proteins encapsulated in osteogenic EVs. We speculate that osteogenic EVs could serve as an intercellular communication system between bone- and bone-marrow adipose tissue, for transporting osteogenic factors and thus favoring pro-osteogenic processes. Our data may support the theory of an endocrine circuit with the skeleton functioning as a ductless gland.

Preservation of the naïve features of mesenchymal stromal cells in vitro: Comparison of cell- and bone-derived decellularized extracellular matrix.

The fate and behavior of bone marrow mesenchymal stem/stromal cells (BM-MSC) is bidirectionally influenced by their microenvironment, the stem cell niche, where a magnitude of biochemical and physical cues communicate in an extremely orchestrated way. It is known that simplified 2D in vitro systems for BM-MSC culture do not represent their naïve physiological environment. Here, we developed four different 2D cell-based decellularized matrices (dECM) and a 3D decellularized human trabecular-bone scaffold (dBone) to evaluate BM-MSC behavior. The obtained cell-derived matrices provided a reliable tool for cell shape-based analyses of typical features associated with osteogenic differentiation at high-throughput level. On the other hand, exploratory proteomics analysis identified native bone-specific proteins selectively expressed in dBone but not in dECM models. Together with its architectural complexity, the physico-chemical properties of dBone triggered the upregulation of stemness associated genes and niche-related protein expression, proving in vitro conservation of the naïve features of BM-MSC.

Impaired regenerative capacity and senescence-associated secretory phenotype in mesenchymal stromal cells from samples of patients with aseptic joint arthroplasty loosening.

Aseptic loosening of total hip and knee joint replacements is the most common indication for revision surgery after primary hip and knee arthroplasty. Research suggests that exposure and uptake of wear by mesenchymal stromal cells (MSC) and macrophages results in the secretion of proinflammatory cytokines and local osteolysis, but also impaired cell viability and regenerative capacity of MSC. Therefore, this in vitro study compared the regenerative and differentiation capacity of MSC derived from patients undergoing primary total hip arthroplasty (MSCprim) to MSC derived from patients undergoing revision surgery after aseptic loosening of total hip and knee joint implants (MSCrev). Regenerative capacity was examined by measuring the cumulative population doubling (CPD) in addition to the number of passages until cells stopped proliferating. Osteogenesis and adipogenesis in monolayer cultures were assessed using histological stainings. Furthermore, RT-PCR was performed to evaluate the relative expression of osteogenic and adipogenic marker genes as well as the expression of markers for a senescence-associated secretory phenotype (SASP). MSCrev possessed a limited regenerative capacity in comparison to MSCprim. Interestingly, MSCrev also showed an impaired osteogenic and adipogenic differentiation capacity compared to MSCprim and displayed a SASP early after isolation. Whether this is the cause or the consequence of the aseptic loosening of total joint implants remains unclear. Future research should focus on the identification of specific cell markers on MSCprim, which may influence complication rates such as aseptic loosening of total joint arthroplasty to further individualize and optimize total joint arthroplasty.

Modeling of the Human Bone Environment: Mechanical Stimuli Guide Mesenchymal Stem Cell-Extracellular Matrix Interactions.

In bone tissue engineering, the design of in vitro models able to recreate both the chemical composition, the structural architecture, and the overall mechanical environment of the native tissue is still often neglected. In this study, we apply a bioreactor system where human bone-marrow hMSCs are seeded in human femoral head-derived decellularized bone scaffolds and subjected to dynamic culture, i.e., shear stress induced by continuous cell culture medium perfusion at 1.7 mL/min flow rate and compressive stress by 10% uniaxial load at 1 Hz for 1 h per day. In silico modeling revealed that continuous medium flow generates a mean shear stress of 8.5 mPa sensed by hMSCs seeded on 3D bone scaffolds. Experimentally, both dynamic conditions improved cell repopulation within the scaffold and boosted ECM production compared with static controls. Early response of hMSCs to mechanical stimuli comprises evident cell shape changes and stronger integrin-mediated adhesion to the matrix. Stress-induced Col6 and SPP1 gene expression suggests an early hMSC commitment towards osteogenic lineage independent of Runx2 signaling. This study provides a foundation for exploring the early effects of external mechanical stimuli on hMSC behavior in a biologically meaningful in vitro environment, opening new opportunities to study bone development, remodeling, and pathologies.

Curcumin-primed human BMSC-derived extracellular vesicles reverse IL-1ß-induced catabolic responses of OA chondrocytes by upregulating miR-126-3p.

BACKGROUND: Curcumin has anti-inflammatory effects and qualifies as a potential candidate for the treatment of osteoarthritis (OA). However, curcumin has limited bioavailability. Extracellular vesicles (EVs) are released by multiple cell types and act as molecule carrier during intercellular communication. We assume that EVs can maintain bioavailability and stability of curcumin after encapsulation. Here, we evaluated modulatory effects of curcumin-primed human (h)BMSC-derived EVs (Cur-EVs) on IL-1ß stimulated human osteoarthritic chondrocytes (OA-CH). METHODS: CellTiter-Blue Viability- (CTB), Caspase 3/7-, and live/dead assays were used to determine range of cytotoxic curcumin concentrations for hBMSC and OA-CH. Cur-EVs and control EVs were harvested from cell culture supernatants of hBMSC by ultracentrifugation. Western blotting (WB), transmission electron microscopy, and nanoparticle tracking analysis were performed to characterize the EVs. The intracellular incorporation of EVs derived from PHK26 labeled and curcumin-primed or control hBMSC was tested by adding the labeled EVs to OA-CH cultures. OA-CH were pre-stimulated with IL-1ß, followed by Cur-EV and control EV treatment for 24 h and subsequent analysis of viability, apoptosis, and migration (scratch assay). Relative expression of selected anabolic and catabolic genes was assessed with qRT-PCR. Furthermore, WB was performed to evaluate phosphorylation of Erk1/2, PI3K/Akt, and p38MAPK in OA-CH. The effect of hsa-miR-126-3p expression on IL-1ß-induced OA-CH was determined using CTB-, Caspase 3/7-, live/dead assays, and WB. RESULTS: Cur-EVs promoted viability and reduced apoptosis of IL-1ß-stimulated OA-CH and attenuated IL-1ß-induced inhibition of migration. Furthermore, Cur-EVs increased gene expression of BCL2, ACAN, SOX9, and COL2A1 and decreased gene expression of IL1B, IL6, MMP13, and COL10A1 in IL-1ß-stimulated OA-CH. In addition, phosphorylation of Erk1/2, PI3K/Akt, and p38 MAPK, induced by IL-1ß, is prevented by Cur-EVs. Cur-EVs increased IL-1ß-reduced expression of hsa-miR-126-3p and hsa-miR-126-3p mimic reversed the effects of IL-1ß. CONCLUSION: Cur-EVs alleviated IL-1ß-induced catabolic effects on OA-CH by promoting viability and migration, reducing apoptosis and phosphorylation of Erk1/2, PI3K/Akt, and p38 MAPK thereby modulating pro-inflammatory signaling pathways. Treatment of OA-CH with Cur-EVs is followed by upregulation of expression of hsa-miR-126-3p which is involved in modulation of anabolic response of OA-CH. EVs may be considered as promising drug delivery vehicles of curcumin helping to alleviate OA.

Cultivation of Head and Neck Squamous Cell Carcinoma Cells with Wound Fluid Leads to Cisplatin Resistance via Epithelial-Mesenchymal Transition Induction.

Locoregional recurrence is a major reason for therapy failure after surgical resection of head and neck squamous cell carcinoma (HNSCC). The physiological process of postoperative wound healing could potentially support the proliferation of remaining tumor cells. The aim of this study was to evaluate the influence of wound fluid (WF) on the cell cycle distribution and a potential induction of epithelial-mesenchymal transition (EMT). To verify this hypothesis, we incubated FaDu and HLaC78 cells with postoperative WF from patients after neck dissection. Cell viability in dependence of WF concentration and cisplatin was measured by flow cytometry. Cell cycle analysis was performed by flow cytometry and EMT-marker expression by rtPCR. WF showed high concentrations of interleukin (IL)-6, IL-8, IL-10, CCL2, MCP-1, EGF, angiogenin, and leptin. The cultivation of tumor cells with WF resulted in a significant increase in cell proliferation without affecting the cell cycle. In addition, there was a significant enhancement of the mesenchymal markers Snail 2 and vimentin, while the expression of the epithelial marker E-cadherin was significantly decreased. After cisplatin treatment, tumor cells incubated with WF showed a significantly higher resistance compared with the control group. The effect of cisplatin-resistance was dependent on the WF concentration. In summary, proinflammatory cytokines are predominantly found in WF. Furthermore, the results suggest that EMT can be induced by WF, which could be a possible mechanism for cisplatin resistance.

Biomimetic Mineralization Promotes Viability and Differentiation of Human Mesenchymal Stem Cells in a Perfusion Bioreactor.

In bone tissue engineering, the design of 3D systems capable of recreating composition, architecture and micromechanical environment of the native extracellular matrix (ECM) is still a challenge. While perfusion bioreactors have been proposed as potential tool to apply biomechanical stimuli, its use has been limited to a low number of biomaterials. In this work, we propose the culture of human mesenchymal stem cells (hMSC) in biomimetic mineralized recombinant collagen scaffolds with a perfusion bioreactor to simultaneously provide biochemical and biophysical cues guiding stem cell fate. The scaffolds were fabricated by mineralization of recombinant collagen in the presence of magnesium (RCP.MgAp). The organic matrix was homogeneously mineralized with apatite nanocrystals, similar in composition to those found in bone. X-Ray microtomography images revealed isotropic porous structure with optimum porosity for cell ingrowth. In fact, an optimal cell repopulation through the entire scaffolds was obtained after 1 day of dynamic seeding in the bioreactor. Remarkably, RCP.MgAp scaffolds exhibited higher cell viability and a clear trend of up-regulation of osteogenic genes than control (non-mineralized) scaffolds. Results demonstrate the potential of the combination of biomimetic mineralization of recombinant collagen in presence of magnesium and dynamic culture of hMSC as a promising strategy to closely mimic bone ECM.

A novel proangiogenic B cell subset is increased in cancer and chronic inflammation.

B cells contribute to immune responses through the production of immunoglobulins, antigen presentation, and cytokine production. Several B cell subsets with distinct functions and polarized cytokine profiles have been reported. In this study, we used transcriptomics analysis of immortalized B cell clones to identify an IgG4+ B cell subset with a unique function. These B cells are characterized by simultaneous expression of proangiogenic cytokines including VEGF, CYR61, ADM, FGF2, PDGFA, and MDK. Consequently, supernatants from these clones efficiently promote endothelial cell tube formation. We identified CD49b and CD73 as surface markers identifying proangiogenic B cells. Circulating CD49b+CD73+ B cells showed significantly increased frequency in patients with melanoma and eosinophilic esophagitis (EoE), two diseases associated with angiogenesis. In addition, tissue-infiltrating IgG4+CD49b+CD73+ B cells expressing proangiogenic cytokines were detected in patients with EoE and melanoma. Our results demonstrate a previously unidentified proangiogenic B cell subset characterized by expression of CD49b, CD73, and proangiogenic cytokines.

The human arthritic hip joint is a source of mesenchymal stromal cells (MSCs) with extensive multipotent differentiation potential.

BACKGROUND: While multiple in vitro studies examined mesenchymal stromal cells (MSCs) derived from bone marrow or hyaline cartilage, there is little to no data about the presence of MSCs in the joint capsule or the ligamentum capitis femoris (LCF) of the hip joint. Therefore, this in vitro study examined the presence and differentiation potential of MSCs isolated from the bone marrow, arthritic hyaline cartilage, the LCF and full-thickness samples of the anterior joint capsule of the hip joint. METHODS: MSCs were isolated and multiplied in adherent monolayer cell cultures. Osteogenesis and adipogenesis were induced in monolayer cell cultures for 21 days using a differentiation medium containing specific growth factors, while chondrogenesis in the presence of TGF-ß1 was performed using pellet-culture for 27 days. Control cultures were maintained for comparison over the same duration of time. The differentiation process was analyzed using histological and immunohistochemical stainings as well as semiquantitative RT-PCR for measuring the mean expression levels of tissue-specific genes. RESULTS: This in vitro research showed that the isolated cells from all four donor tissues grew plastic-adherent and showed similar adipogenic and osteogenic differentiation capacity as proven by the histological detection of lipid droplets or deposits of extracellular calcium and collagen type I. After 27 days of chondrogenesis proteoglycans accumulated in the differentiated MSC-pellets from all donor tissues. Immunohistochemical staining revealed vast amounts of collagen type II in all differentiated MSC-pellets, except for those from the LCF. Interestingly, all differentiated MSCs still showed a clear increase in mean expression of adipogenic, osteogenic and chondrogenic marker genes. In addition, the examination of an exemplary selected donor sample revealed that cells from all four donor tissues were clearly positive for the surface markers CD44, CD73, CD90 and CD105 by flow cytometric analysis. CONCLUSIONS: This study proved the presence of MSC-like cells in all four examined donor tissues of the hip joint. No significant differences were observed during osteogenic or adipogenic differentiation depending on the source of MSCs used. Further research is necessary to fully determine the tripotent differentiation potential of cells isolated from the LCF and capsule tissue of the hip joint.

Decellularized human bone as a 3D model to study skeletal progenitor cells in a natural environment.

There has been an increasing interest in exploring naturally derived extracellular matrices as an material mimicking the complexity of the cell microenvironment in vivo. Bone tissue-derived decellularized constructs are able to preserve native structural, biochemical, and biomechanical cues of the tissue, therefore providing a suitable environment to study skeletal progenitor cells. Particularly for bone decellularization, different methods have been reported in the literature. However, the used methods critically affect the final ultrastructure and surface chemistry as well as the decellularization efficiency, consequently causing complications to draw conclusions and compare results in between studies. In this chapter, an optimized protocol for the preparation of human bone derived scaffolds is described, including processing techniques and further characterization methods, which allow the final construct to be recognized as a major platform for bone therapeutic and/or diagnostic applications.

Novel molecular cues for dental defects in hypophosphatasia.

Mineralization disorders with a broad range of etiological factors represent a huge challenge in dental diagnosis and therapy. Hypophosphatasia (HPP) belongs to the rare diseases affecting predominantly mineralized tissues, bones and teeth, and occurs due to mutations in the ALPL gene, which encodes tissue-nonspecific alkaline phosphatase (TNAP). Here we analyzed stem cells from bone marrow (BMSCs), dental pulp (DPSCs) and periodontal ligament (PDLSCs) in the absence and presence of efficient TNAP inhibitors. The differentiation capacity, expression of surface markers, and gene expression patterns of donor-matched dental cells were compared during this in vitro study. Differentiation assays showed efficient osteogenic but low adipogenic differentiation (aD) capacity of PDLSCs and DPSCs. TNAP inhibitor treatment completely abolished the mineralization process during osteogenic differentiation (oD). RNA-seq analysis in PDLSCs, comparing oD with and without TNAP inhibitor levamisole, showed clustered regulation of candidate molecular mechanisms that putatively impaired osteogenesis and mineralization, disequilibrated ECM production and turnover, and propagated inflammation. Combined alteration of cementum formation, mineralization, and elastic attachment of teeth to cementum via elastic fibers may explain dental key problems in HPP. Using this in vitro model of TNAP deficiency in DPSCs and PDLSCs, we provide novel putative target areas for research on molecular cues for specific dental problems in HPP.

Interactions between Muscle and Bone-Where Physics Meets Biology.

Muscle and bone interact via physical forces and secreted osteokines and myokines. Physical forces are generated through gravity, locomotion, exercise, and external devices. Cells sense mechanical strain via adhesion molecules and translate it into biochemical responses, modulating the basic mechanisms of cellular biology such as lineage commitment, tissue formation, and maturation. This may result in the initiation of bone formation, muscle hypertrophy, and the enhanced production of extracellular matrix constituents, adhesion molecules, and cytoskeletal elements. Bone and muscle mass, resistance to strain, and the stiffness of matrix, cells, and tissues are enhanced, influencing fracture resistance and muscle power. This propagates a dynamic and continuous reciprocity of physicochemical interaction. Secreted growth and differentiation factors are important effectors of mutual interaction. The acute effects of exercise induce the secretion of exosomes with cargo molecules that are capable of mediating the endocrine effects between muscle, bone, and the organism. Long-term changes induce adaptations of the respective tissue secretome that maintain adequate homeostatic conditions. Lessons from unloading, microgravity, and disuse teach us that gratuitous tissue is removed or reorganized while immobility and inflammation trigger muscle and bone marrow fatty infiltration and propagate degenerative diseases such as sarcopenia and osteoporosis. Ongoing research will certainly find new therapeutic targets for prevention and treatment.

Lumenal calcification and microvasculopathy in fetuin-A-deficient mice lead to multiple organ morbidity.

The plasma protein fetuin-A mediates the formation of protein-mineral colloids known as calciprotein particles (CPP)-rapid clearance of these CPP by the reticuloendothelial system prevents errant mineral precipitation and therefore pathological mineralization (calcification). The mutant mouse strain D2,Ahsg-/- combines fetuin-A deficiency with the calcification-prone DBA/2 genetic background, having a particularly severe compound phenotype of microvascular and soft tissue calcification. Here we studied mechanisms leading to soft tissue calcification, organ damage and death in these mice. We analyzed mice longitudinally by echocardiography, X-ray-computed tomography, analytical electron microscopy, histology, mass spectrometry proteomics, and genome-wide microarray-based expression analyses of D2 wildtype and Ahsg-/- mice. Fetuin-A-deficient mice had calcified lesions in myocardium, lung, brown adipose tissue, reproductive organs, spleen, pancreas, kidney and the skin, associated with reduced growth, cardiac output and premature death. Importantly, early-stage calcified lesions presented in the lumen of the microvasculature suggesting precipitation of mineral containing complexes from the fluid phase of blood. Genome-wide expression analysis of calcified lesions and surrounding (not calcified) tissue, together with morphological observations, indicated that the calcification was not associated with osteochondrogenic cell differentiation, but rather with thrombosis and fibrosis. Collectively, these results demonstrate that soft tissue calcification can start by intravascular mineral deposition causing microvasculopathy, which impacts on growth, organ function and survival. Our study underscores the importance of fetuin-A and related systemic regulators of calcified matrix metabolism to prevent cardiovascular disease, especially in dysregulated mineral homeostasis.