scholarly journals Revealing the influence of electron beam melted Ti-6Al-4V scaffolds on osteogenesis of human bone marrow-derived mesenchymal stromal cells

2021 ◽  
Vol 32 (9) ◽  
Author(s):  
Kristin S. Ødegaard ◽  
Lingzi Ouyang ◽  
Qianli Ma ◽  
Glenn Buene ◽  
Di Wan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Electron Beam ◽  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Bone Growth ◽  
Bone Mineralization ◽  
Bone Matrix ◽  
Porous Scaffolds ◽  
Alkaline Phosphatase Staining

AbstractPorous Titanium-6Aluminum-4Vanadium scaffolds made by electron beam-based additive manufacturing (AM) have emerged as state-of-the-art implant devices. However, there is still limited knowledge on how they influence the osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (BMSCs). In this study, BMSCs are cultured on such porous scaffolds to determine how the scaffolds influence the osteogenic differentiation of the cells. The scaffolds are biocompatible, as revealed by the increasing cell viability. Cells are evenly distributed on the scaffolds after 3 days of culturing followed by an increase in bone matrix development after 21 days of culturing. qPCR analysis provides insight into the cells’ osteogenic differentiation, where RUNX2 expression indicate the onset of differentiation towards osteoblasts. The COL1A1 expression suggests that the differentiated osteoblasts can produce the osteoid. Alkaline phosphatase staining indicates an onset of mineralization at day 7 in OM. The even deposits of calcium at day 21 further supports a successful bone mineralization. This work shines light on the interplay between AM Ti64 scaffolds and bone growth, which may ultimately lead to a new way of creating long lasting bone implants with fast recovery times.

Lenalidomide Modulates the Phenotype and Function of Human Mesenchymal Stromal Cells From Healthy Donors and MDS Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3816-3816
Author(s):  
Manja Wobus ◽  
Gwendolin Dünnebier ◽  
Silvia Feldmann ◽  
Gerhard Ehninger ◽  
Martin Bornhauser ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Clinical Efficacy ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Morphological Changes ◽  
Adipogenic Differentiation ◽  
Healthy Controls ◽  
Healthy Donors

Abstract Abstract 3816 Poster Board III-752 Introduction Recent studies in patients with MDS have clearly demonstrated the clinical efficacy of lenalidomide. However, its exact mechanisms of action have not been elucidated yet. Myelosuppression is the most common adverse event and seems to be dependent on dose as well MDS subtype, being rather infrequent in patients other than del5q. The aim of this study was to investigate whether lenalidomide affects the bone marrow microenvironment. Therefore, we analyzed in-vitro characteristics of isolated mesenchymal stromal cells (MSCs) from MDS patients and from healthy controls. Methods Bone marrow samples were collected from healthy donors (n=5) and patients with MDS (del5q MDS n=3, RA n=2, RAEB1/2 n=3). MSCs were isolated according to the standard adhesion protocol and cultured in the presence or absence of lenalidomide. Results Lenalidomide treatment of MSCs caused no morphological changes but proliferation was slightly increased. Typical surface molecules as CD73, CD90, CD105 and CD166 were expressed in MSCs from MDS patients at comparable levels to healthy controls. Lenalidomide treatment caused an upregulation of CD29 by 17.8 ± 4.4% and of CD73 by 24 ± 5.7% (mean fluorescence intensity). Investigating the cytokine production, we found lower IL-8 mRNA and protein levels in MSCs from MDS patients (mean in MDS MSC: 138.1 pg/ml vs. mean in healthy MSC: 1177 pg/ml). Interestingly, the IL-8 production can be increased by approximately 40% under lenalidomide treatment. MDS MSCs retained the capacity for adipogenic and osteogenic differentiation as well as their supportive function towards hematopoietic cells in long term culture-initiating assays (LTC-IC). However, the LTC-IC frequency was lower on MSC which had been preincubated with lenalidomide compared to controls. Lenalidomide also slightly accelerated osteogenic differentiation because mineralization started as early as on day 5 with lenalidomide whereas in the control cells first calcium deposits were visible after 7 days. Other samples showed augmented lipid vacuoles after adipogenic differentiation under lenalidomide treatment. Conclusion In conclusion, lenalidomide modulates the phenotype of MSC and leads to an increase of their IL-8 secretion by a yet unknown mechanism. Whether these in-vitro effects are associated with the clinical efficacy of this compound in patients with MDS remains to be investigated. Disclosures: Platzbecker: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

EphB4/ephrinB2 Interaction Mediate Chronic Myelogenous Leukemia Mesenchymal Stromal Cells Spreading and Osteogenic Differentiation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4772-4772
Author(s):  
Lin Li ◽  
Na Xu ◽  
Xuan Zhou ◽  
Jixian Huang ◽  
Yuling Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Direct Contact ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Qrt Pcr ◽  
Sh Group ◽  
Normal Human

Abstract Background and Objective: Mesenchymal stromal cells (MSCs) are a major component of the leukemia bone marrow (BM) microenvironment.Recent studies have indicated interaction between acute leukemia cells and MSCs has a major role in cancer progression and resistance to treatment.Our previous study found that EphB4 receptor was over expressed in CML-Blast Crisis (BC) patients and resistant cell lines. Furthermore, we performed the experiment to prove that aberrant over expressed of EphB4 play an important role to change characterize of Imatinib-resistant in chronic myeloid leukemia cells. However,the contribution of over expressed of EphB4 molecules in leukemia cells to change MSCs function remains to be determined. Therefore,we hypothesis that the change of EphB4/ephrinB2 molecules on leukemia cells might play an important role to transform characterize of MSCs through direct contact, which finally support to leukemia progression and disruption of normal hematopoiesis in microenvironment of the bone marrow. Methods and Results: MSCs were prepared from bone marrow mononuclear cells isolated from normal human or patients' BM and cultures in CyagenBone marrow culture medium at 37 °C, 5% CO2 incubator. EphrinB2(2.628±0.2303 n=3; P<0.05), ALP\RUNX2 (early osteogenesis differentiation genes)(6.430±0.1343, n=3P<0.001; 4.948±0.1418,n=3P<0.001)were over expressed in MSCs (CML patients)in contrast to normal human MSC by QRT-PCR. After osteogenic induced for 2 weeks,MSCs from CML-initial patient showed significantly higher osteogenic differentiation (Osteogenesis Score 4.5P<0.01) and protein (later period osteogenesis differentiation)(2.1669%±0.1443, n=3P<0.001)was overexpressed in MSCs (CML patients) in contrast to normal human MSC(0.2993%±0.1612n=3) by western blot. In functional spreading assay, cultured MSC (CML patient) exposed to EphB4-Fc (5 μg/mL) were significantly rounder (p<0.001) and smaller (p<0.001) as demonstrated with F-actin staining, compared to control and human-Fc.No difference inmorphology was observed when MSC were cultured in the presence of ephrin-B2-Fc.Incubation of MSC with signaling pathway-specific inhibitors before the spreading assay, the PI3Kinase pathway (LY294002) (p<0.001),not the Src kinase pathway (PP2), inhibited MSCs (CML) attachment and spreading. The results revealed that the PI3Kinase pathway was activated upon ephrinB2 reverse signaling in response to EphB4-Fc to promote the contraction and rounding up of MSC. Activation of ephrin-B2 molecules expressed by MSCs (CML)by EphB4-Fc(5 μg/mL), human-Fc(5 μg/mL) or blank control, which cultured in osteogenic media for 2 weeks, more mineral in the presence of EphB4-Fc was visualized by Alizarin Red staining compared to the control human-Fc.Furthermore, after co-cultured respectively with K562-R or K562-R-EPHB4-SH for 72h,ALP\RUNX2 in MSCs (CML) were increased significantly in K562-R group(15.544±2.647; 6.378±2.0775 n=3)compared to K562-R-EPHB4-SH group (6.014±1.19273; 4.045±2.0273 n=3) and control group by QRT-PCR. However, ALP\RUNX2 in normal MSC was unaffected when respectively co-cultured with K562-R(0.741±2.2121; 0.129±0.9194 n=3) or K562-R-EPHB4-SH(0.171±2.062; 0.232±2.0474 n=3) for 72h. The co-cultured assays were also performed using transwells. ALP\RUNX2 in MSCs (CML) were no difference in K562-R group (6.28±1.7875; 4.232±2.4533 n=3) compared to K562-R-EPHB4-SH group (6.107±2.158; 4.139±1.9727 n=3). Conclusion: Our study illustrated that the change of EphB4/ephrinB2 molecules on leukemia cells may transform MSCs functional spreading and osteogenic differentiation through direct contact involved in PI3Kinase pathway. Disclosures No relevant conflicts of interest to declare.

scholarly journals Characterization of the Hematopoietic Supporting Activity of Osteoblasts Derived from Bone Marrow Mesenchymal Stromal Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4358-4358
Author(s):  
Manal Alsheikh ◽  
Roya Pasha ◽  
Nicolas Pineault
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Soluble Factors ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
The Impact ◽  
Myeloid Progenitors

Abstract Osteoblasts (OST) found within the endosteal niche are important regulators of Hematopoietic Stem and Progenitor Cells (HSPC) under steady state and during hematopoietic reconstitution. OST are derived from mesenchymal stromal cells (MSC) following osteogenic differentiation. MSC and OST secrete a wide array of soluble factors that sustain hematopoiesis. Recently, we showed that media conditioned with OST derived from MSC (referred as M-OST) after 6 days of osteogenic differentiation were superior to MSC conditioned media (CM) for the expansion of cord blood (CB) progenitors, and CB cells expanded with M-OST CM supported a more robust engraftment of platelets in NSG mice after transplantation. These findings raised the possibility that M-OST could be superior to MSC for the ex vivoexpansion HSPC. In this study, we set out to test the hypothesis that the growth modulatory activity of M-OST would vary as a function of their maturation status. The objectives were to first monitor the impact of M-OST differentiation and maturation status on the expression of soluble factors that promote HSPC expansion and in second, to investigate the capacity of M-OST CMs prepared from M-OST at distinct stages of differentiation to support the expansion and differentiation of HSPCs in culture. M-OST at distinct stages of differentiation were derived by culturing bone marrow MSC in osteogenic medium for various length of time (3 to 21 days). All CB CD34+ enriched (92±7% purity) cell cultures were done with serum free media conditioned or not with MSC or M-OST and supplemented with cytokines SCF, TPO and FL. We first confirmed the progressive differentiation and maturation of M-OST as a function of osteogenic culture length, which was evident by the induction of the osteogenic transcription factors Osterix, Msx2 and Runx2 mRNAs, the gradual increase in osteopontin and alkaline phosphatase positive cells and quantitative increases in calcium deposit. Next, we investigated the expression in MSC and M-OSTs of genes known to collaborate for the expansion of HSPCs by Q-PCR. Transcript copy numbers for IGFBP-2 increased swiftly during osteogenic differentiation, peaking at day-3 (˃100-fold vs MSC, n=2) and returning below MSC level by day-21. In contrast, ANGPTL members (ANGPTL-1, -2, -3 and -5) remained superior in M-OSTs throughout osteogenic differentiation with expression levels peaking around day 6 (n=2). Next, we tested the capacity of media conditioned with primitive (day-3, -6), semi-mature (day-10, -14) and mature M-OST (day-21) to support the growth of CB cells. All M-OST CMs increased (p˂0.03) the growth of total nucleated cells (TNC) after 6 days of culture compared to non-conditioned medium used as control (mean 2.0-fold, n=4). Moreover, there was a positive correlation between cell growth and M-OST maturation status though differences between the different M-OST CMs tested were not significant. The capacity of M-OST CMs to increase (mean 2-fold, n=4) the expansion of CD34+ cells was also shared by all M-OST CMs (p˂0.05), as supported by significant increases with immature day-3 (mean ± SD of 18 ± 6, p˂0.02) and mature day-21 M-OST CMs (14 ± 5, p˂0.05) vs. control (8 ± 3, n=4). Conversely, expansions of TNC and CD34+ cells in MSC CM cultures were in-between that of control and M-OST CMs cultures. Interestingly, M-OST CMs also modulated the expansion of the HSPC compartment. Indeed, while the expansion of multipotent progenitors defined as CD34+CD45RA+ was promoted in control culture (ratio of 4.5 for CD34+CD45RA+/CD34+CD45RA- cells), M-OST CMs supported greater expansion of the more primitive CD34+CD45RA- HSPC subpopulation reducing the ratio to 3.3±0.4 for M-OST cultures (cumulative mean of 10 cultures, n=2). Moreover, the expansions of CD34+CD38- cells and of the long term HSC-enriched subpopulation (CD34+CD38-CD45RA-Thy1+) in M-OST CM cultures were respectively 2.7- and 2.8-fold greater than those measured in control cultures (n=2-4). Finally, the impact of M-OST CMs on the expansion of myeloid progenitors was investigated using a colony forming assay; expansion of myeloid progenitors were superior in all M-OST CM cultures (1.6±0.2 fold, n=2). In conclusion, our results demonstrate that M-OST rapidly acquire the expression of growth factors known to promote HSPC expansion. Moreover, the capacity of M-OST CMs to support the expansion of HSPCs appears to be a property shared by M-OST at various stages of maturation. Disclosures No relevant conflicts of interest to declare.

A calcium-induced signaling cascade leading to osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells

Biomaterials ◽  
2012 ◽  
Vol 33 (11) ◽  
pp. 3205-3215 ◽  
Author(s):  
Ana M.C. Barradas ◽  
Hugo A.M. Fernandes ◽  
Nathalie Groen ◽  
Yoke Chin Chai ◽  
Jan Schrooten ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Signaling Cascade
Sign in / Sign up

Export Citation Format

Share Document