Bone marrow-derived mesenchymal stromal cells (MSC) support bone regeneration by differentiating into osteogenic lineages in response to biochemical and mechanical cues such as fluid-flow and compression, processes replicable in vitro using bioreactors and 3D scaffolds.Human MSC were cultured on medium-stiffness and hard 3D polyurethane (PU) scaffolds, characterized by atomic force microscopy, scanning electron microscopy (SEM), and stiffness measurements, and subjected to fluid-flow and compression. Two-dimensional PU and polystyrene (PS) substrates served as controls. Cultures were maintained in expansion or osteogenic medium. Cell attachment and morphology were analyzed by SEM and F-actin staining; osteogenic differentiation was evaluated by qPCR. Alkaline phosphatase activity was quantified, and extracellular matrix (ECM) formation and mineralization were assessed using Picrosirius Red, Alizarin Red, and inductively coupled plasma mass spectrometry (ICP-MS). Mechanosensitivity was analyzed via FOS and PTGS2 expression, YAP/TAZ nuclear localization, and Wnt, Notch, and cAMP signaling.Cells adhered and spread throughout the scaffolds, exhibiting pore-spanning morphologies. Gene expression showed material- and time-dependent upregulation of osteoblastic and osteocytic markers, with PU scaffolds eliciting stronger responses than PS, even without osteogenic supplements. The scaffolds also supported ECM formation and mineralization. Mechanical stimulation via fluid-flow and compression induced time- and stimulus-dependent regulation of mechanoresponsive genes and activation of key mechanotransduction pathways, with distinct expression patterns for each loading mode.In this study, we developed a standardized, cost-effective, and easy-to-handle 3D PU scaffold that effectively supports hMSC adhesion and promotes osteogenic differentiation. The cells' mechanoresponse further highlights the scaffold's suitability as a versatile platform for in vitro studies under physiologically relevant conditions.

Multiple myeloma (MM) is a malignant plasma cell disease inducing osteolytic lesions by disrupting bone homeostasis, fostering catabolic and suppressing anabolic functions. While the impact on osteoblast generation and function is well documented, alterations of osteocyte function and extracellular matrix (ECM) are not yet fully understood. Thus, using a syngeneic mouse model of MM by injecting MOPC315.BM cells intratibially into BALB/c mice (n = 95), we performed transcriptomic profiling of an osteocyte-enriched population and identified a mechanosensitive matrisomal gene signature, which was disrupted by tumor engraftment. Non-invasive tibial loading restored the expression of 94 ECM-associated genes, including collagens, fibronectin, and aggrecan. Cross-species integration with RNA-seq data from 387 MM patients revealed eight ECM-related genes whose expression correlated with overall survival (VEGFA, BCAN, FGF13, TNFSF8, SDC1, LAMC1, SEMA3A, and CCL2). Four of these genes (Vegfa, Sdc1, Sema3a, Ccl2) were also load-responsive in a murine osteocyte (IDG-SW3 cells) bioreactor model. Our findings indicate that an existing mechanosensitive osteocytic repair program is suppressed by MM cells, which can be reinvigorated via a brief single loading session. It suggests that exercise-based interventions may be beneficial to restore bone mass through endochondral ossification programs in patients with MM.

The development of bone metastases poses a clinical challenge for patients with malignancies originating from diverse organs, including lungs, prostate, and breast. These metastatic events correlate with a poorer prognosis, and treatments are predominantly palliative. Furthermore, traditional in-vivo models for bone metastasis research have limitations, primarily due to species-specific differences. Therefore, we sought an alternative in-vitro model that is human cell based, robust and capable of a relatively quick establishment. Initially, we studied the potential of a decellularized porcine jejunum scaffold named Small Intestinal Submucosa with mucosa (SISmuc) and compared it to decellularized human bone and two synthetic scaffolds. We assessed various parameters related to osteoblast differentiation such as calcification, collagen I protein expression, and further differentiation to mature osteocytes indicated by sclerostin protein expression. The synthetic electrospun matrix demonstrated to be a good alternative to SISmuc, particularly when combined with bioactive glass 45S5, which enhanced calcification. We also incorporated osteoclasts into our model and demonstrated that monocytes could be more effectively differentiated into osteoclasts in a 3D environment compared to traditional 2D cultures. Within seven weeks, we successfully generate an in-vitro model incorporating osteoblasts, osteoclasts, and tumor cells. As a potential strategy for treating bone metastases, we found Chimeric Antigen Receptor T cells within lung and pancreatic bone metastasis models to be effective in reducing the number of tumor cells and inducing apoptosis. This innovative research establishes a foundation for more sophisticated 3D in-vitro models of bone metastasis, holding significant promise for advancing preclinical cancer research and therapeutic strategies.

Calcium phosphate (CaP) minerals have been widely studied as bone substitutes due to their excellent osteoconductivity and biocompatibility. However, their limited biodegradability remains a challenge. Magnesium phosphates (MgPs) are promising alternatives; offering similar properties to CaP while exhibiting enhanced bioresorption due to their higher solubility. To advance the relatively unexplored field of MgP research, this study investigated Cu‐doped MgP supraparticles with enhanced biodegradability as potential bone substitute materials. For this purpose, MgP nanoparticles (NPs) were synthesized using a modified sol–gel process with different parameter variations to tailor the crystal structure and particle size. The NPs were then spray‐dried with Cu ions (5.0 wt%) to produce antibacterial supraparticles whose unique properties were demonstrated in previous studies on CaP supraparticles. Subsequent calcination was used to study the impact of the crystal structure on Cu release experiments over 14 days as well as its efficacy against the gram‐positive bacterium Staphylococcus aureus . In particular, uncalcined, amorphous MgP supraparticles exhibited high Cu release in bi‐distilled water and antibacterial activity when tested as eluates. Interestingly, all MgP particle systems, with or without Cu, achieved a significant bactericidal effect against S. aureus at a particle concentration of 100 mg/mL in Mueller Hinton Broth, thereby demonstrating the antibacterial effect of Mg 2+ ions. The cell viability of the supraparticles was also investigated using human telomerase–immortalized mesenchymal stromal cells (hMSC‐TERT), which exhibited high biocompatibility at particle concentrations of 0.01–0.1 mg/L over 72 h. Uncalcined Cu‐doped MgP supraparticles induced the expression of proinflammatory markers (interleukin 1 beta, interleukin 6, interleukin 8, and serum amyloid A1), whereas the expression of osteogenic markers (alkaline phosphatase, bone gamma‐carboxyglutamate protein, and secreted phosphoprotein 1) was only slightly affected. However, the osteogenic differentiation of hMSC‐TERT showed a positive effect on mineralization when exposed to uncalcined MgP supraparticles. Overall, the results indicate that MgP supraparticles, both with and without Cu, exhibit high biocompatibility and hold promise for applications in bone tissue regeneration.

During three-dimensional (3D) bioprinting, the integration of living cells into hydrogel matrices results in complex biophysicochemical interactions between viscosity, shear stress, and temperature, critically influencing the structural and functional integrity of the resulting constructs. This study delves into the short-term biological ramifications of 3D extrusion printing of telomerase-immortalized human mesenchymal stromal cells (hMSC-TERT) embedded in bioinert hydrogels. Pluronic F127 and custom-synthesized poly(2-methyl-2-oxazoline)-block-poly(2-n-propyl-2-oxazine) (POx/POzi) are synthetic, block copolymers that create shear-thinning, physically crosslinked hydrogels that were used for this study. The rheological properties of the cell-free hydrogels and cell-laden bioinks were examined, revealing that they exhibited comparable behavior. Contrary to the original hypotheses, a key finding of this research is the reduction in cell viability (up to 50%) within 24 h post-printing, a trend consistently observed across varying initial conditions. The relative expression levels of the mechanoresponsive genes FOS and PTGS2 were increased, partly due to the suspension and incubation of the cells in both hydrogels. Only FOS was significantly upregulated in some cases because of the printing process after 2 and 4 h of incubation. These insights highlight the potential of using POx/POzi hydrogel as a matrix in 3D bioprinting, particularly for depositing hMSC-TERT into structures with vasculature-mimicking scaffolds or scaffolds designed for bone regeneration.

Functional analysis of MSC-interacting subpopulations (A–C): Functional enrichment analysis of differentially expressed genes (from RNA-seq) using Metascape. A, Gene ontology (GO) cluster analysis of gene lists that are unique for MA (left) or nMA (right) INA-6. Circle nodes represent subsets of input genes falling into similar GO term. Node size grows with the number of input genes. Node color defines a shared parent GO term. Two nodes with a similarity score > 0.3 are linked. B, Enrichment analysis of pairwise comparisons between MA subpopulations and their overlaps (arranged in columns). GO terms were manually picked and categorized (arranged in rows). Raw Metascape results are shown in Supplementary Fig. S6. For each GO term, the P values (x axis) and the counts of matching input genes (circle size) were plotted. The lowest row shows enrichment of gene lists from the TRRUST database. C, Circos plots by Metascape. Sections of a circle represent lists of differentially expressed genes. Purple lines connect same genes appearing in two gene lists. ∩: Overlapping groups, MA: MSC-adhering, nMA: non–MSC-adhering, CM: MSC-conditioned medium. D, INA-6 were cocultured on confluent hMSC for 24 or 48 hours, separated by WPSC and subcultured for 48 hours under IL6 withdrawal (n = 6), except the control (IL6 + INA-6; n = 3). Signals were normalized (red line) to INA-6 cells grown without hMSCs and IL6 (n = 3). Statistics (D): Paired t test, two-factor RM-ANOVA. Datapoints represent the mean of four technical replicates. INA-6 were isolated from independent cocultures with hMSCs from 6 unique donors.

Abstract 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.

Separation and gene expression of INA-6 subpopulations. A, Schematic of WPSC separating nMA-INA6 from MA-INA6. A coculture 96-well plate is turned upside down and attached on top of a “catching plate,” forming a “well-plate sandwich.” nMA-INA6 cells are collected in the catching plate by subsequent rounds of centrifugation and gentle washing. MA-INA6 are enzymatically dissociated from hMSCs or by rough pipetting. Subsequent RNA-seq of MSC-interacting subpopulations reveals distinct expression clusters [right, multidimensional scaling plot (n = 5)]. B, Separation was microscopically tracked after each centrifugation step. C–E, qRT-PCR of genes derived from RNA-seq results. Expression was normalized to the median of CM-INA6. Samples include those used for RNA-seq and six further cocultures (n = 11; non-detects were discarded). C, Adhesion factors, ECM proteins and matrix metalloproteinases. D, Factors involved in bone remodeling and bone homing chemokines. E, Factors involved in (immune) signaling. Statistics: (C–E): Paired t-test. Datapoints represent the mean of three (B–E) technical replicates. INA-6 were isolated from independent cocultures with hMSCs from 5 (A, B), 9 (C--E) unique donors.

Supplementary Figure 1. Principle and quantification of the V-well adhesion assay of fluorescently labeled myeloma cells adapted by Weetall et al. 2001. Supplementary Figure 2. Validation of image cytometric analysis of cell cycle in four INA-6 cultures. Supplementary Figure 3. Cell cycle analysis of INA-6 pellets gained from V-Well Adhesion assay (Fig. 3). Supplementary Figure 4. Representative (one of the four independent sample sets as seen in Supplementary Figure 3) curve fitting analysis of cell cycle profiles generated by Image Cytometry. Supplementary Figure 5. Correlation of RNAseq with qPCR Left: Validation of RNAseq results (Fig. 4) with qPCR showing the log2(foldchange expression) of 18 genes. Supplementary Figure 6. Functional enrichment analysis by Metascape using genes that are differentially expressed between MSC-interacting subpopulations. Supplementary Figure 7. Expression levels of adhesion genes that are downregulated and associated with survival (p < 0.01). Bone Marrow Plasma Cell (BMPC), Monoclonal Gammopathy of Undetermined Significance (MGUS), Smoldering Multiple Myeloma (sMM), Multiple Myeloma (MM), Multiple Myeloma Relapse (MMR). Supplementary Figure 8. Expression levels of adhesion genes that are not downregulated and associated with survival (p < 0.01).

Detachment of INA-6 daughter cells after cell division. A–D, INA-6 divisions in interaction with confluent hMSCs. Seeding ratio INA-6:MSC = 4:20. A, Three examples of dividing INA-6 cells generating either two MA, or one MA and one nMA daughter cells as described in G. Dashed circles mark mother cells (white), MA cell (blue), and first position of nMA cell (green). Scale bar: 20 µm. B, Cell division of MSC-adhering (MA) mother cell can yield one mobile non–MSC-adhering (nMA) daughter cell. C, Frequencies of INA-6 pairs defined in A and B per observed cell division. A total of 65 divisions were evaluated for each of three independent time-lapse recordings. D, Rolling duration of nMA cells after division did not depend on hMSC donor [H(2) = 5.250, P-unc = 0y.072]. Datapoints represent single nMA cells after division. E–G, Adhesive and cell cycle assessment of MSC-interacting INA-6 subpopulations using the V-Well assay. E, Schematic of V-Well Assay (see Supplementary Fig. S1 for detailed analysis). MSC-interacting subpopulations were separated by subsequent centrifugation and removal of the pellet. The pellet size was quantified by its total fluorescence brightness. Adhering subpopulations were resuspended by rough pipetting. F, Relative cell pellet sizes of adhesive INA-6 subpopulations that cycle either asynchronously or were synchronized at mitosis. Gray lines in-between points connect dependent measurements of cocultures (n = 9) that shared the same hMSC-donor and INA-6 culture. Cocultures were incubated for three different durations (1, 2, and 3 hours after INA-6 addition). Timepoints were pooled, since time did not show an effect on cell adhesion [F(2,4) = 1.414, P-unc = 0.343]. Factorial RM-ANOVA shows an interaction between cell cycle and the kind of adhesive subpopulation [F(1, 8) = 42.67, P-unc = 1.82e-04]. Technical replicates = 4 per datapoint. G, Cell cycles were profiled in cells gathered from the pellets of four independent cocultures (n = 4) and the frequency of G0–G1 cells are displayed depending on coculture duration (see Supplementary Fig. S3 for cell cycle profiles). Four technical replicates were pooled after pelleting. Statistics: D: Kruskal–Wallis H-test. F: Paired t test. G: Paired t test, two-factor RM-ANOVA. Datapoints represent INA-6 from independent cocultures with hMSCs from 3 unique donors.

Tumor cells are decorated with aberrant glycan structures on cell surfaces. It is well known that the glycocalyx serves as a main cellular regulator, although its role in cancer is still not completely understood. Over recent decades, several non-natural monosaccharides carrying clickable groups have been introduced in melanoma cells. This technique, called Metabolic Glycoengineering (MGE), opens up the possibility of altering the cell’s glycocalyx via click chemistry using a two-step approach. This study expands the field of MGE by showing the successful metabolic incorporation of novel alternative artificial glucosamine derivatives. The latter were either deoxygenated or blocked by methyl ether in position 4 to generate deficient glycosylation patterns, while being extended by an alkyne to enable click chemistry as a one-step approach. As a result, we observed a reduced proliferation rate of melanoma cells. Furthermore, using a lectin array, the decrease in high mannose epitopes was observed. In summary, the successful use of alternative artificial glucosamine derivatives enabled a significant alteration in the glycocalyx, consequently influencing cell behavior.

Time-lapse analysis of INA-6 detachment from INA-6 aggregates and hMSCs. A, Frequency of observed INA-6 aggregates that did or did not lose INA-6 cell(s). A total of 87 aggregates were evaluated per datapoint. B, Example of a “disseminating” INA-6 aggregate growing on fluorescently (PKH26) stained hMSC (from A–D). Dashed green lines are trajectories of detached INA-6 cells. Scale bar = 50 µm. C–E, Quantitative assessment of INA-6 detachments. A total of 45 detachment events were evaluated per datapoint. Seeding ratio INA-6:MSC = 4:1. C, Most INA-6 cells dissociated from another INA-6 cell and not from an hMSC [F(1, 3) = 298, P-unc = 4.2e-4]. D, Detachment frequency of aggregate size categories. E, Detachment frequency of INA-6 cells detaching as single, pairs or more than three cells. Statistics: (A): Paired t test; (C--E): Paired t test, two-factor RM-ANOVA; Datapoints represent three (A) or four (C–E) independent time-lapse recordings of cocultures with hMSCs from 2 (A) or 3 (C–E) unique donors.

Adhesion and ECM genes (shown in Fig. 6A) were filtered by their association with patient survival (P-adj. < 0.01) and were categorized as continuously downregulated during disease progression

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.

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, self-assembling MSC spheroid culture does not support the integration of MM cells. On the other hand, 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. Additionally, 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. Impact statement: The application of 3D models in the investigation of multiple myeloma will provide better insight into their behaviour and drug resistance, allowing us to develop better treatment strategies. Here, we optimized a collagen-based approach which has shown to be reproducible, cost-effective and already providing an altered feedback in therapy response.

Multiple myeloma (MM) is a hematological malignancy whose curability is greatly challenged by recurrent patient relapses and therapy resistance. We have previously proposed the high expression of ADAM8, ADAM9 and ADAM15 (A Disintegrin And Metalloproteinase 8/9/15) as adverse prognostic markers in MM. This study focused on the so far scarcely researched role of ADAM8/9/15 in MM using two patient cohorts and seven human MM cell lines (HMCL). High ADAM8/9/15 expression was associated with high-risk cytogenetic abnormalities and extramedullary disease. Furthermore, ADAM8/15 expression increased with MM progression and in relapsed/refractory MM compared to untreated patient samples. RNA sequencing and gene set enrichment analysis comparing ADAM8/9/15high/low patient samples revealed an upregulation of proliferation markers and proliferation-associated gene sets in ADAM8/9/15high patient samples. High ADAM8/9/15 expression correlated with high Ki67 and high ADAM8/15 expression with high MYC protein expression in immunohistochemical stainings of patient tissue. Conversely, siRNA-mediated knockdown of ADAM8/9/15 in HMCL downregulated proliferation-related gene sets. Western blotting revealed that ADAM8 knockdown regulated IGF1R/AKT signaling and ADAM9 knockdown decreased mTOR activation. Lastly, high ADAM8/9/15 expression levels were verified as prognostic markers independent of Ki67/MYC expression and/or high-risk abnormalities. Overall, these findings suggest that ADAM8/9/15 play a role in MM progression and proliferation signaling.

Survival of patients with multiple myeloma regarding the expression levels of adhesion and bone retention genes. A,P-value distribution of genes associated with patient survival (n = 535) depending on high or low expression levels. Red dashed line marks the significance threshold of P-adj = 0.05. Histogram of P values was plotted using a bin width of −log10(0.05)/2. Patients with high and low gene expression were delineated using maximally selected rank statistics (maxstat). B, Survival curves for three genes taken from the list of adhesion genes shown in A, maxstat thresholds defining high and low expression were: CXCL12: 81.08; DCN: 0.75; TGM2: 0.66 normalized counts. C, Gene expression (RNA-seq, n = 873) measured in normalized counts (edgeR) of CXCL12, DCN in BMPC, MGUS, smoldering multiple myeloma (sMM), multiple myeloma (MM), multiple myeloma relapse (MMR), human myeloma cell lines (HMCL). The red dashed line marks one normalized read count. Statistics (A, B): log-rank test; (C): Kruskal–Wallis, Mann–Whitney U test. All P values were corrected using the Benjamini–Hochberg procedure.