Effect of Vitamin D Pretreatment of Human Mesenchymal Stem Cells on Ectopic Bone Formation

Abstract Adult mesenchymal stem cells (MSCs) are used in contemporary strategies for tissue engineering. The MSC is able to form bone following implantation as undifferentiated cells adherent to hydroxyapatite (HA)/tricalcium phosphate (TCP) scaffolds. Previous investigators have demonstrated that human MSCs (hMSCs) can be differentiated to osteoblasts in vitro by the inclusion of vitamin D and ascorbic acid. The aim of this study was to compare the osteogenic potential of predifferentiated and undifferentiated bone marrow–derived, culture-expanded hMSCs adherent to synthetic HA/TCP (60%/40%) following subcutaneous engraftment in severe combined immunodeficiency (SCID) mice. During the final 3 days of culture, cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and antibiotics or media containing 25-mM calcium supplementation with vitamin D and ascorbic acid. Four weeks following implantation in SCID mice, scoring analysis of bone formation within the cubes revealed the absence of bone formation in unloaded cubes. Bone formation compared by a qualitative bone index was 7.23% for undifferentiated cells compared to 5.20% for differentiated cells. Minimal resorption was observed at this early time point. In this ectopic model, predifferentiation using a combination of vitamin D and ascorbic acid failed to increase subsequent bone formation by implanted cells. Following implantation of hMSCs adherent to an osteoconductive scaffold, host factors may contribute dominant osteoinductive signals or impose inhibitory signals to control the fate of the implanted cell. Predifferentiation strategies require confirmation in vivo.

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Long-Term Quantitative Biodistribution and Side Effects of Human Mesenchymal Stem Cells (hMSCs) Engraftment in NOD/SCID Mice following Irradiation

Stem Cells International ◽

10.1155/2014/939275 ◽

2014 ◽

Vol 2014 ◽

pp. 1-13 ◽

Cited By ~ 7

Author(s):

Sabine François ◽

Benoit Usunier ◽

Luc Douay ◽

Marc Benderitter ◽

Alain Chapel

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Side Effects ◽

Total Body Irradiation ◽

Human Mesenchymal Stem Cells ◽

Scid Mice ◽

Human Mscs ◽

Long Term Treatment ◽

Total Body

There is little information on the fate of infused mesenchymal stem cells (MSCs) and long-term side effects after irradiation exposure. We addressed these questions using human MSCs (hMSCs) intravenously infused to nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice submitted to total body irradiation (TBI) or local irradiation (abdominal or leg irradiation). The animals were sacrificed 3 to 120 days after irradiation and the quantitative and spatial distribution of hMSCs were studied by polymerase chain reaction (PCR). Following their infusion into nonirradiated animals, hMSCs homed to various tissues. Engraftment depended on the dose of irradiation and the area exposed. Total body irradiation induced an increased hMSC engraftment level compared to nonirradiated mice, while local irradiations increased hMSC engraftment locally in the area of irradiation. Long-term engraftment of systemically administered hMSCs in NOD/SCID mice increased significantly in response to tissue injuries produced by local or total body irradiation until 2 weeks then slowly decreased depending on organs and the configuration of irradiation. In all cases, no tissue abnormality or abnormal hMSCs proliferation was observed at 120 days after irradiation. This work supports the safe and efficient use of MSCs by injection as an alternative approach in the short- and long-term treatment of severe complications after radiotherapy for patients refractory to conventional treatments.

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Investigation of early cell–surface interactions of human mesenchymal stem cells on nanopatterned β-type titanium–niobium alloy surfaces

Interface Focus ◽

10.1098/rsfs.2013.0046 ◽

2014 ◽

Vol 4 (1) ◽

pp. 20130046 ◽

Cited By ~ 15

Author(s):

Rebecca Medda ◽

Arne Helth ◽

Patrick Herre ◽

Darius Pohl ◽

Bernd Rellinghaus ◽

...

Keyword(s):

Stem Cells ◽

Gold Nanoparticles ◽

Mesenchymal Stem Cells ◽

Cell Surface ◽

Surface Interactions ◽

Bone Diseases ◽

Population Heterogeneity ◽

Human Mscs ◽

Adult Mesenchymal Stem Cells ◽

Cell Surface Interactions

Multi-potent adult mesenchymal stem cells (MSCs) derived from bone marrow have therapeutic potential for bone diseases and regenerative medicine. However, an intrinsic heterogeneity in their phenotype, which in turn results in various differentiation potentials, makes it difficult to predict the response of these cells. The aim of this study is to investigate initial cell–surface interactions of human MSCs on modified titanium alloys. Gold nanoparticles deposited on β-type Ti–40Nb alloys by block copolymer micelle nanolithography served as nanotopographical cues as well as specific binding sites for the immobilization of thiolated peptides present in several extracellular matrix proteins. MSC heterogeneity persists on polished and nanopatterned Ti–40Nb samples. However, cell heterogeneity and donor variability decreased upon functionalization of the gold nanoparticles with cyclic RGD peptides. In particular, the number of large cells significantly decreased after 24 h owing to the arrangement of cell anchorage sites, rather than peptide specificity. However, the size and number of integrin-mediated adhesion clusters increased in the presence of the integrin-binding peptide (cRGDfK) compared with the control peptide (cRADfK). These results suggest that the use of integrin ligands in defined patterns could improve MSC-material interactions, not only by regulating cell adhesion locally, but also by reducing population heterogeneity.

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Optimizing combination of vascular endothelial growth factor and mesenchymal stem cells on ectopic bone formation in SCID mice

Journal of Biomedical Materials Research Part A ◽

10.1002/jbm.a.36195 ◽

2017 ◽

Vol 105 (12) ◽

pp. 3326-3332 ◽

Cited By ~ 3

Author(s):

Chris H. Dreyer ◽

Kristian Kjaergaard ◽

Nicholas Ditzel ◽

Niklas R. Jørgensen ◽

Søren Overgaard ◽

...

Keyword(s):

Stem Cells ◽

Vascular Endothelial Growth Factor ◽

Mesenchymal Stem Cells ◽

Growth Factor ◽

Bone Formation ◽

Ectopic Bone Formation ◽

Scid Mice ◽

Vascular Endothelial ◽

Ectopic Bone ◽

Endothelial Growth Factor

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Four and a Half LIM Domains Protein 2 Mediates Bortezomib-Induced Osteogenic Differentiation of Mesenchymal Stem Cells in Multiple Myeloma Through p53 Signaling and β-Catenin Nuclear Enrichment

Frontiers in Oncology ◽

10.3389/fonc.2021.729799 ◽

2021 ◽

Vol 11 ◽

Author(s):

Zhenqing Xie ◽

Yan Xu ◽

Xiaojing Wei ◽

Gang An ◽

Mu Hao ◽

...

Keyword(s):

Stem Cells ◽

Multiple Myeloma ◽

Mesenchymal Stem Cells ◽

Bone Formation ◽

Osteogenic Differentiation ◽

Bone Disease ◽

Marker Genes ◽

Human Mscs ◽

P53 Signaling ◽

Lim Domains

Myeloma bone disease (MBD), caused by the inhibition of osteoblast activity and the activation of osteoclast in the bone marrow environment, is the most frequent and life-threatening complication in multiple myeloma (MM) patients. Bortezomib (Bzb) was shown to promote MM-derived mesenchymal stem cells (MM-MSCs) differentiation to osteoblast in vitro and in animal models, promoting the bone formation and regeneration, may be mediated via β-catenin/T-cell factor (TCF) pathway. Further defining molecular mechanism of Bzb-enhanced bone formation in MM will be beneficial for the treatment of myeloma patients. The present study has identified for the first time four and a half LIM domains protein 2 (FHL2), a tissue-specific coregulator that interacts with many osteogenic marker molecules, as a therapeutic target to ameliorate MM bone disease. First, increased messenger RNA (mRNA) and protein levels of FHL2, and the mRNA level of main osteoblast markers (including Runx2, ALP, and Col1A1), were found in MM-patients-derived MSCs after Bzb treatment. FHL2 KD with short hairpin RNA (shRNA) reduced the expression of osteoblast marker genes and blocked the osteogenic differentiation of MM-MSCs regardless of the presence or absence of Bzb, implying that FHL2 is an important activator of the osteogenic differentiation of human MSCs under a proteasome inhibition condition. Molecular analysis showed that the enhanced expression of FHL2 was associated with the Bzb-induced upregulation of p53. No significant change at protein level of total β-catenin was observed with or without Bzb treatment. However, it was mostly enriched to nuclei in MSCs after Bzb treatment. Moreover, β-catenin was restricted to the perinuclear region in FHL2 KD cells. These data provide evidence that FHL2 is essential for promoting β-catenin nuclear enrichment in MM-MSCs. In conclusion, FHL2 is critical for Bzb-induced osteoblast differentiation of MM-MSCs and promotes the osteogenesis, through p53 signaling and β-catenin activation. Targeting FHL2 in MM may provide a new therapeutic strategy for treating MBD.

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Mesenchymal stem cells overexpressing BMP-9 by CRISPR-Cas9 present high in vitro osteogenic potential and enhance in vivo bone formation

Gene Therapy ◽

10.1038/s41434-021-00248-8 ◽

2021 ◽

Author(s):

Gileade P. Freitas ◽

Helena B. Lopes ◽

Alann T. P. Souza ◽

Maria Paula O. Gomes ◽

Georgia K. Quiles ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Bone Formation ◽

Osteogenic Potential ◽

In Vivo Bone Formation

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Recapitulation of endochondral bone formation using human adult mesenchymal stem cells as a paradigm for developmental engineering

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1000302107 ◽

2010 ◽

Vol 107 (16) ◽

pp. 7251-7256 ◽

Cited By ~ 302

Author(s):

C. Scotti ◽

B. Tonnarelli ◽

A. Papadimitropoulos ◽

A. Scherberich ◽

S. Schaeren ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Bone Formation ◽

Endochondral Bone Formation ◽

Endochondral Bone ◽

Human Adult ◽

Adult Mesenchymal Stem Cells ◽

Developmental Engineering

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Bone Regeneration from Frozen Marrow Mesenchymal Cells/Recombinant Human Bone Morphogenetic Protein/Hydroxyapatite Transplantation

Key Engineering Materials ◽

10.4028/www.scientific.net/kem.309-311.1009 ◽

2006 ◽

Vol 309-311 ◽

pp. 1009-1012

Author(s):

Kazuhide Miyazaki ◽

Takafumi Yoshikawa ◽

Jin Iida ◽

Y. Ueda ◽

M. Koizumi ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Bone Formation ◽

Bone Morphogenetic Protein ◽

Mesenchymal Cells ◽

Human Bone ◽

Osteogenic Potential ◽

Morphogenetic Protein ◽

Human Bone Morphogenetic Protein ◽

Recombinant Human Bone

Introduction: Marrow mesenchymal cells contain stem cells and can regenerate tissues. We previously reported the clinical application of autologous cultured bone to regeneration therapy. However, in cases with low numbers of active cells, culture is often unsatisfactory. If frozen marrow cells retain their osteogenic potential, we could clinically use them in regeneration therapy as alternatives to high active cells obtained from youngsters. Here, we examined osteogenic potential of frozen human mesenchymal stem cells in combination with recombinant human bone morphogenetic protein (rhBMP) using biochemical and histological analyses. Method: Marrow fluid was aspirated from the human iliac bone of a 46-year-old man with lumbar canal stenosis during surgery. Two weeks after primary culture in standard medium, bone marrow mesenchymal stem cells (BMSCs) were trypsinized for the preparation of a cell suspension, and cells were concentrated to 106 cells/ml by centrifugation. Cells were kept at – 80 °C until use. To impregnate porous hydroxyapatite (HA) with rhBMP, 1 3g rhBMP/20 3l 0.1 % trifluoroacetic acid was applied on HA, and then desiccated under vacuum. In the present study, we used 4 subgroups: BMSC/rhBMP/HA, BMSC/HA, rhBMP/HA, and HA only. HA constructs from the 4 subgroups were implanted at subcutaneous sites on the back of 5-week-old nude mice (BALB/cA Jcl-nu). Eight weeks after implantation, implanted HA constructs were harvested, and biochemical and histological analyses were performed. Alkaline phosphatase activity (ALP) and human osteocalcin (hOs) levels were measured. Results and Discussion: ALP activity and hOs in the BMSC/BMP/HA subgroup were 2 or 3 times that in the BMSC/HA subgroup. Histological analysis showed that significant bone formation was observed in these two subgroups, and supported biochemical data. However, in the BMP/HA and HA only subgroups, significant bone formation could not be detected histologically nor biochemically. These results indicated that a combination of rhBMP and BMSCs, and only with a minimal amount of 1 3g rhBMP, allowed successful generation of human bone. In the human body, rhBMP in the order of milligrams is necessary for bone formation. However, by combining BMSCs, HA and rhBMP, only a small amount of rhBMP was needed to dramatically enhance osteogenic potential. As we reported here, cryopreserved BMSCs also showed high osteoblastic activity. In conclusion, this study provided histological and biochemical evidence that combination of cryopreserved BMSCs, BMP, and porous HA could enhance osteogenic potential.

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Mesenchymal Stem Cells from Reaming Material Possess High Osteogenic Potential and React Sensitively to Bone Morphogenetic Protein 7

Journal of Applied Biomaterials & Functional Materials ◽

10.5301/jabfm.5000333 ◽

2017 ◽

Vol 15 (1) ◽

pp. 54-62 ◽

Cited By ~ 1

Author(s):

Melanie Hoellig ◽

Fabian Westhauser ◽

Kira Kornienko ◽

Kai Xiao ◽

Gerhard Schmidmaier ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Bone Formation ◽

Bone Morphogenetic Proteins ◽

Osteogenic Potential ◽

Autogenous Bone ◽

Alternative Source ◽

Morphogenetic Protein

Background Femoral material harvested using the Reamer-Irrigator-Aspirator (RIA) system is an alternative source for autogenous bone material in the treatment of non-unions, especially in combination with bone morphogenetic proteins (BMP). So far, there is no in vivo evidence of BMP-7 interacting with mesenchymal stem cells (MSCs) from RIA material (RIA-MSCs) and iliac crest autogenous bone (BMSCs). The aim of this study was to compare their osteogenic potential when stimulated with BMP-7 in vivo. Methods RIA-MSC and BMSC from 11 donors were isolated and the character of MSCs was investigated in vitro. Constructs consisting of MSC, β-tricalcium phosphate and 2 concentrations of BMP-7 (0.1 µg/mL and 1 µg/mL) were implanted in mice for 8 weeks. Bone formation in the constructs was analyzed quantitatively and qualitatively. Results All cell populations used were determined to be MSCs. The qualitative in vivo analysis showed formation of bone tissue. With regard to quantity, bone formation was significantly higher in RIA constructs without or with stimulation with 0.1 µg/mL BMP-7, compared with BMSC constructs. We found no significant differences between constructs stimulated with 1 µg/mL BMP-7. In the RIA group, we observed a significant increase in bone formation after stimulation with 0.1 µg/mL BMP-7. No significant change could be found using a higher concentration. In the BMSC group, we detected a significant increase when using 0.1 µg/mL and 1 µg/mL BMP-7. Conclusions RIA material is a source of MSCs with high osteogenic potential. Our results showed that stimulation by BMP-7 leads to an increased osteogenic potential of MSCs. In this respect, RIA-MSCs reacted more sensitively than BMSCs.

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