Osteogenic differentiation of equine cord blood multipotent mesenchymal stromal cells within coralline hydroxyapatite scaffolds in vitro

2011 ◽  
Vol 24 (05) ◽  
pp. 354-362 ◽  
Author(s):  
R. J. Figueroa ◽  
T. G. Koch ◽  
D. H. Betts
Keyword(s):  
Alkaline Phosphatase ◽  
Cell Viability ◽  
Osteogenic Differentiation ◽  
Alkaline Phosphatase Activity ◽  
Stromal Cells ◽  
Culture Medium ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Coralline Hydroxyapatite ◽  
Osteogenic Induction

SummaryObjective: To investigate the osteogenic differentiation potential of equine umbilical cord blood-derived multipotent mesenchymal stromal cells (CB-MSC) within coralline hydro-xyapatite scaffolds cultured in osteogenic induction culture medium.Methods: Scaffolds seeded with equine CBMSC were cultured in cell expansion culture medium (control) or osteogenic induction medium (treatment). Cell viability and distribution were confirmed by the MTT cell viability assay and DAPI nuclear fluorescence staining, respectively. Osteogenic differentiation was evaluated after 10 days using reverse transcription polymerase chain reaction, alkaline phosphatase activity, and secreted osteocalcin concentration. Cell morphology and matrix deposition were assessed by scanning electron microscopy (SEM) after 14 days in culture.Results: Cells showed viability and adequate distribution within the scaffold. Successful osteogenic differentiation within the scaffolds was demonstrated by the increased expression of osteogenic markers such as Runx2, osteopontin, osteonectin, collagen IA increased levels of alkaline phosphatase activity increased osteocalcin protein secretion and bone-like matrix presence in the scaffold pores upon SEM evaluation.Clinical significance: These results demonstrate that equine CB-MSC maintain viability and exhibit osteogenic potential in coralline hydroxyapatite scaffolds when induced in vitro. Equine CB-MSC scaffold constructs deserve further investigation for their potential role as biologically active fillers to enhance bone-gap repair in the horse.

The inhibitory effects of vancomycin on rat bone marrow–derived mesenchymal stem cell differentiation

2021 ◽  
pp. 1-5
Author(s):  
Kari Hanson ◽  
Carly Isder ◽  
Kristen Shogren ◽  
Anthony L. Mikula ◽  
Lichun Lu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Osteogenic Differentiation ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
High Dose ◽  
Inhibitory Effects ◽  
Alizarin Red ◽  
Osteogenic Factors

OBJECTIVE The use of intrawound vancomycin powder in spine surgery has been shown to decrease the rate of surgical site infections; however, the optimal dose is unknown. High-dose vancomycin inhibits osteoblast proliferation in vitro and may decrease the rate of solid arthrodesis. Bone marrow–derived mesenchymal stem cells (BMSCs) are multipotent cells that are a source of osteogenesis in spine fusions. The purpose of this study was to determine the effects of vancomycin on rat BMSC viability and differentiation in vitro. METHODS BMSCs were isolated from the femurs of immature female rats, cultured, and then split into two equal groups; half were treated to stimulate osteoblastic differentiation and half were not. Osteogenesis was stimulated by the addition of 50 µg/mL l-ascorbic acid, 10 mM β-glycerol phosphate, and 0.1 µM dexamethasone. Vancomycin was added to cell culture medium at concentrations of 0, 0.04, 0.4, or 4 mg/mL. Early differentiation was determined by alkaline phosphatase activity (4 days posttreatment) and late differentiation by alizarin red staining for mineralization (9 days posttreatment). Cell viability was determined at both the early and late time points by measurement of formazan colorimetric product. RESULTS Viability within the first 4 days decreased with high-dose vancomycin treatment, with cells receiving 4 mg/mL vancomycin having 40%–60% viability compared to the control. A gradual decrease in alizarin red staining and nodule formation was observed with increasing vancomycin doses. In the presence of the osteogenic factors, vancomycin did not have deleterious effects on alkaline phosphatase activity, whereas a trend toward reduced activity was seen in the absence of osteogenic factors when compared to osteogenically treated cells. CONCLUSIONS Vancomycin reduced BMSC viability and impaired late osteogenic differentiation with high-dose treatment. Therefore, the inhibitory effects of high-dose vancomycin on spinal fusion may result from both reduced BMSC viability and some impairment of osteogenic differentiation.

scholarly journals Optimizing Osteogenic Differentiation of Ovine Adipose-Derived Stem Cells by Osteogenic Induction Medium and FGFb, BMP2, or NELL1 In Vitro

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Emil Østergaard Nielsen ◽  
Li Chen ◽  
Jonas Overgaard Hansen ◽  
Matilda Degn ◽  
Søren Overgaard ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Stromal Cells ◽  
Basic Medium ◽  
Induction Medium ◽  
Control Analysis ◽  
Alizarin Red ◽  
Osteogenic Induction Medium ◽  
Osteogenic Induction ◽  
Fibroblast Growth Factor Basic

Although adipose-derived stromal cells (ADSCs) have been a major focus as an alternative to autologous bone graft in orthopedic surgery, bone formation potential of ADSCs is not well known and cytokines as osteogenic inducers on ADSCs are being investigated. This study aimed at isolating ADSCs from ovine adipose tissue (AT) and optimizing osteogenic differentiation of ovine ADSCs (oADSC) by culture medium and growth factors. Four AT samples were harvested from two female ovine (Texel/Gotland breed), and oADSCs were isolated and analyzed by flow cytometry for surface markers CD29, CD44, CD31, and CD45. Osteogenic differentiation was made in vitro by seeding oADSCs in osteogenic induction medium (OIM) containing fibroblast growth factor basic (FGFb), bone morphogenetic protein 2 (BMP2), or NEL-like molecule 1 (NELL1) in 4 different dosages (1, 10, 50, and 100 ng/ml, respectively). Basic medium (DMEM) was used as control. Analysis was made after 14 days by Alizarin red staining (ARS) and quantification. This study successfully harvested AT from ovine and verified isolated cells for minimal criteria for adipose stromal cells which suggests a feasible method for isolation of oADSCs. OIM showed significantly higher ARS to basic medium, and FGFb 10 ng/ml revealed significantly higher ARS to OIM alone after 14 days.

OSTEOPLASTIC PROPERTIES OF MULTIPOTENT MESENCHYMAL STROMAL CELLS OF ADIPOSE TISSUE

2021 ◽  
Vol 74 (10) ◽  
pp. 2374-2378
Author(s):  
Andriy Bambuliak ◽  
Nataliia Kuzniak ◽  
Valentyna Honcharenko ◽  
Marianna Ostafiychuk ◽  
Alina Palamar
Keyword(s):  
Gene Expression ◽  
Alkaline Phosphatase ◽  
Adipose Tissue ◽  
Phosphatase Activity ◽  
Mesenchymal Stromal Cells ◽  
Alkaline Phosphatase Activity ◽  
Stromal Cells ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Tissue Samples ◽  
Medical University

The aim: Determining the ability of samples based on MMSC – AT differentiating in the osteogenic direction. Materials and methods: The study was conducting at Bukovinian State Medical University, Chernivtsi, Ukraine. Adipose tissue samples were obtaining from the neck of 60 experimental animals (white Wistar rats). Multipotent mesenchymal stromal cells of adipose tissue were obtained by grinding adipose tissue of rats in 0.1% collagenase 1A . Alkaline phosphatase activity was assessing by using the Alkaline Phosphatase Detection Kit (Sigma, USA) according to the manufacturer’s protocol. Osteopontin gene expression was determining by immunocytochemical method. To determine the mRNA used the PCR method, which is associated with reverse transcription (RT-PCR) in the area of quantification of gene expression to the marker BGP. Results: On the 21st day of observations, the expression of mRNA encoding the BGP gene decreased in samples № 1 and № 3 to 35,800 ± 420.0 copies and to 35,000 ± 400.0 copies, p1<0.01, p>0.05. Also was observing growth of copies of the BGP gene in samples № 2 and № 4 in 2.1, р<0.01 and 2.2 times, р-р2<0.05, relative to the data in sample № 1. Conclusions: Comparative study of osteoplastic properties samples MMSC-AT showed that a larger number of cells differentiate into the osteoblasts in samples containing MMSC-AT + PRP (№ 2) and MMSC-AT + PRP + «Kolapan» (№ 4). This has been proven higher alkaline phosphatase activity, higher levels osteopontin expression, and higher levels BGP gene expression.

scholarly journals Combined Fluorescence-Based in Vitro Assay for the Simultaneous Detection of Cell Viability and Alkaline Phosphatase Activity during Osteogenic Differentiation of Osteoblast Precursor Cells

2020 ◽  
Vol 3 (2) ◽  
pp. 30 ◽  
Author(s):  
Sebastian Wilkesmann ◽  
Fabian Westhauser ◽  
Joerg Fellenberg
Keyword(s):  
Alkaline Phosphatase ◽  
Cell Viability ◽  
Osteogenic Differentiation ◽  
Simultaneous Detection ◽  
Cell Number ◽  
Precursor Cells ◽  
Vitro Assay ◽  
Alp Activity ◽  
Bone Substitute Materials

Novel bone substitute materials need to be evaluated in terms of their osteogenic differentiation capacity and possible unwanted cytotoxic effects in order to identify promising candidates for the therapy of bone defects. The activity of alkaline phosphatase (ALP) is frequently quantified as an osteogenic marker, while various colorimetric assays, like MTT assay, are used to monitor cell viability. In addition, the DNA or protein content of the samples needs to be quantified for normalization purposes. As this approach is time consuming and often requires the analysis of multiple samples, we aimed to simplify this process and established a protocol for the combined fluorescence-based quantification of ALP activity and cell viability within one single measurement. We demonstrate that the fluorogenic substrate 4-methylumbelliferone-phosphate (4-MUP) and the commonly used para-nitrophenylphosphate (p-NPP) produce comparable and highly correlating results. We further show that fluorescein–diacetate (FDA) can be used to quantify both cell viability and cell number without interfering with the quantification of ALP activity. The measurement of additional normalization parameters is, therefore, unnecessary. Therefore, the presented assay allows for a time-efficient, simple and reliable analysis of both ALP activity and cell viability from one sample and might facilitate experiments evaluating the osteogenic differentiation of osteoblast precursor cells.

scholarly journals In vitro modulation of alkaline phosphatase activity in neutrophils from patients with chronic myelogenous leukemia by monocyte-derived activity

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 492-497 ◽  
Author(s):  
T Matsuo
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Chronic Myelogenous Leukemia ◽  
Alkaline Phosphatase Activity ◽  
Culture Medium ◽  
Underlying Mechanism ◽  
Myelogenous Leukemia ◽  
Neutrophil Alkaline Phosphatase

Abstract To clarify the underlying mechanism of low neutrophil alkaline phosphatase (NAP) activity in chronic myelogenous leukemia (CML), CML neutrophils were cultured in liquid medium with different numbers of monocytes. Alkaline phosphatase activity in CML neutrophils, assessed cytochemically, increased with the numbers of monocytes. NAP activity was not induced by the interaction between neutrophils and monocytes, but by the presence of a monocyte-derived soluble activity. NAP activity in normal neutrophils was also lowered by depletion of monocytes from culture medium. Under such monocyte-depleted conditions, both CML and normal neutrophils proliferated and differentiated to produce mature neutrophils. Thus induction of NAP activity can be modified in vitro by changing the amount of NAP-inducing activity released from monocytes. However, whether a reduction of NAP-inducing activity in CML neutrophil is the cause of low NAP activity in vivo remains uncertain.

scholarly journals Clonal analysis in vitro of osteogenic differentiation of marrow CFU-F

1987 ◽  
Vol 87 (5) ◽  
pp. 731-738 ◽  
Author(s):  
M.E. Owen ◽  
J. Cave ◽  
C.J. Joyner
Keyword(s):  
Alkaline Phosphatase ◽  
Osteogenic Differentiation ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Wide Spectrum ◽  
Epidermal Growth ◽  
Single Precursor ◽  
Low Levels ◽  
Marrow Cells

Fibroblastic colonies, each of which is derived from a single precursor cell (CFU-F), are formed when suspensions of marrow cells are cultured in vitro. The ability of marrow CFU-F to differentiate in vitro was investigated using the expression of alkaline phosphatase activity as a marker for osteogenic differentiation. In cultures of rabbit marrow cells the colonies formed varied in size, morphology and expression of enzyme activity, indicating that marrow stromal CFU-F are a heterogeneous population. Growth and differentiation of marrow CFU-F can be modified in vitro. Epidermal growth factor increased average colony size and reduced clonal expression of alkaline phosphatase activity to very low levels. Hydrocortisone activated the osteogenic differentiation programme within the cellular progeny of a wide spectrum of CFU-F. The results support the possible development of in vitro clonal methods for the study of differentiation and regulation of the osteogenic and other fibroblastic cell lines of the marrow stromal system.

scholarly journals In vitro modulation of alkaline phosphatase activity in neutrophils from patients with chronic myelogenous leukemia by monocyte-derived activity

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 492-497
Author(s):  
T Matsuo
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Chronic Myelogenous Leukemia ◽  
Alkaline Phosphatase Activity ◽  
Culture Medium ◽  
Underlying Mechanism ◽  
Myelogenous Leukemia ◽  
Neutrophil Alkaline Phosphatase

To clarify the underlying mechanism of low neutrophil alkaline phosphatase (NAP) activity in chronic myelogenous leukemia (CML), CML neutrophils were cultured in liquid medium with different numbers of monocytes. Alkaline phosphatase activity in CML neutrophils, assessed cytochemically, increased with the numbers of monocytes. NAP activity was not induced by the interaction between neutrophils and monocytes, but by the presence of a monocyte-derived soluble activity. NAP activity in normal neutrophils was also lowered by depletion of monocytes from culture medium. Under such monocyte-depleted conditions, both CML and normal neutrophils proliferated and differentiated to produce mature neutrophils. Thus induction of NAP activity can be modified in vitro by changing the amount of NAP-inducing activity released from monocytes. However, whether a reduction of NAP-inducing activity in CML neutrophil is the cause of low NAP activity in vivo remains uncertain.

scholarly journals Secretion of niche signal molecules in conditions of osteogenic differentiation of multipotent mesenchymal stromal cells induced by textured calcium phosphate coating

2019 ◽  
Vol 65 (4) ◽  
pp. 339-346
Author(s):  
L.S. Litvinova ◽  
V.V. Shupletsova ◽  
K.A. Yurova ◽  
O.G. Khaziakhmatova ◽  
N.M. Todosenko ◽  
...  
Keyword(s):  
Calcium Phosphate ◽  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Calcium Phosphate Coating ◽  
Signal Molecules ◽  
Humoral Factors ◽  
Alizarin Red

Secretion of 21 cytokines, chemokines and growth factors (LIF, SCF, SDF-1a, SCGF-b, M-CSF, MCP-3, MIF, MIG, TRAIL, GRO-a; IL-1a, IL-2ra, IL-3, IL-12(p40), IL-16, IL-18, HGF, TNF-b, b-NGF, IFN-a2, CTACK) has been studied in vitro in the culture of human adipose-derived multipotent mesenchymal stromal cells (hAMMSCs) in conditions of its osteogenic differentiation caused by 14-day contact with calcium phosphate (CP) surface with different roughness. Bilateral X-ray amorphous CP coatings were prepared on the samples of commercially pure titanium in the anodal regime using a micro-arc method. An aqueous solution prepared from 20 wt% phosphoric acid, 6 wt% dissolved hydrohyapatite nanopowder (particle diameter 10-30 nm with single agglomerates up to 100 nm), and 9 wt% dissolved calcium carbonate was used to obtain CP coating. hAMMSCs isolated from lipoaspirate were co-cultured after 4 passages with the CP-coated samples at final concentration of 1.5´105 viable karyocytes per 1.5 mL of standard nutrition medium (without osteogenic stimulators) for 14 days (a determination of [CD45,34,14,20], CD73, CD90 и CD105 cell immunophenotype; an analysis of secretory activity) and 21 days (alizarin red S staining of culture) with medium replacement every 3-4 days. Under conditions of in vitro contact with rough CP coating hAMMSCs differentiated into osteoblasts synthesizing the mineralized bone matrix; this was accompanied by 2-3-fold increasing ratio of [CD45,34,14,20]+ hemopoietic cells. The following humoral factors of hemopoietic niches acted as the signal molecules escalating in vitro the hemopoietic base in 14 days of differentiating three-dimensional culture of hAMMSCs: either leukemia inhibitory factor (LIF) and stem cell factor (SCF) cytokines under mean index of CP roughness Ra=2.4-2.6 mm or stromal derived factor-1 (SDF-1a, CXCL12 chemokine) under Ra=3.1-4.4 mm.

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