Osteogenic Differentiation Protocols Research Articles

Co- and Triaxial Electrospinning for Stem Cell-based Bone Regeneration.

Bone tissue is composed of organic minerals and cells. It has the capacity to heal for certain minor damages, but when the bone defects surpass the critical threshold, they need fixing. Bone regeneration through natural and synthetic biodegradable materials requires various steps, such as manufacturing methods and materials selection. A successful biodegradable bone graft should have a high surface area/ volume ratio, strength, and a biocompatible, porous structure capable of promoting cell adhesion, proliferation, and differentiation. Considering these requirements, the electrospinning technique is promising for creating functional nano-sized scaffolds. The multi-axial methods, such as coaxial and triaxial electrospinning, are the most popular techniques to produce double or tri-layered scaffolds, respectively. Recently, stem cell culture on scaffolds and the application of osteogenic differentiation protocols on these scaffolds have opened new possibilities in the field of biomaterials research. This review discusses an overview of the progress in coaxial and triaxial technology through biodegradable composite bone materials. The review also carefully elaborates the osteogenic differentiation using stem cells and their performance with nano-sized scaffolds.

Regeneration of pulp-dentin complex using human stem cells of the apical papilla: in vivo interaction with two bioactive materials.

To compare the regenerative properties of human stem cells of the apical papilla (SCAPs) embedded in a platelet-rich plasma (PRP) scaffold, when implanted in vivo using an organotypic model composed of human root segments, with or without the presence of the bioactive cements - ProRoot MTA or Biodentine. SCAPs were isolated from third molars with incomplete rhizogenesis and expanded and characterized in vitro using stem cell and surface markers. The pluripotency of these cells was also assessed using adipogenic, chondrogenic, and osteogenic differentiation protocols. SCAPs together with a scaffold of PRP were added to the root segment lumen and the organotypic model implanted on the dorsal region of immunodeficient rats for a period of 4 months. Presence of SCAPs induced de novo formation of dentin-like and pulp-like tissue. A barrier of either ProRoot MTA or Biodentine did not significantly affect the fraction of sections from roots segments observed to contain deposition of hard material (P > 0.05). However, the area of newly deposited dentin was significantly greater in segments containing a barrier of Biodentine compared with ProRoot MTA (P < 0.001). SCAPs offer a viable alternative to other dental stem cells (DSCs) in their regenerative properties when enclosed in the microenvironment of human tooth roots. The present study also suggests that the presence of bioactive materials does not hinder or impede the formation of new hard tissues, but the presence of Biodentine may promote greater mineralized tissue deposition.

Osteogenic Differentiation of Human Mesenchymal Stem Cells by the Single Action of Luminescent Polyurea Oxide Biodendrimers.

Polyurea oxide (PURO) biodendrimers are a class of dendrimers that can trigger osteogenic differentiation of human mesenchymal stem cells (hMSCs). PURO biodendrimers are prepared by simple, solventless oxidation of polyurea dendrimers using hydrogen peroxide as the oxidant in quantitative yield, retaining both biocompatibility (up to 10 mg/mL for higher generations) and the non-traditional intrinsic luminescence. The effect of PURO biodendrimers in the differentiation of hMSCs was found by the single addition to a standard growth medium for MSCs differentiation (without differentiation inducers). After 21 days of incubation, the formation of osteoblasts was confirmed by the alizarin red staining assay and alkaline phosphatase activity. This is the first report of in vitro osteodifferentiation fully regulated by synthetic soft polymers such as dendrimers. Current osteogenic differentiation protocols rely on an in vitro inducing formulation (including dexamethasone, ascorbic acid, and β-glycerophosphate), which lacks therapeutic potential in vivo. The outstanding role of dendrimers in nanomedicine, under clinic translation, combined with this feature is envisaged to foster PURO dendrimers as an important strategy in cell therapy and regenerative medicine.

Regulation of somatostatin expression by vitamin D3 and valproic acid in human adipose-derived mesenchymal stem cells

BackgroundAdipose-derived mesenchymal stem cells (ADMSC) are non-haematopoietic, fibroblast-like multipotent progenitor cells. They have the potential for trilineage (adipocyte, chondrocyte and osteocyte) differentiation as well as differentiation into endocrine pancreatic progenitors. In diabetic or cancer therapy, somatostatin (SST) expression plays a vital role. Small molecules such as valproic acid (VPA) and micronutrients like vitamin D3 have differentiation potential in ADMSC. Therefore, the aim of this study was to investigate the role of vitamin D3 machinery and its metabolic enzymes in ADMSC. Furthermore, the reprogramming effect of vitamin D3 and VPA was evaluated on somatostatin expression in pancreatic lineage differentiation.MethodsADMSC were characterised based on their cell surface marker profile using flow cytometry. Specific adipogenic and osteogenic differentiation protocols were used in this study. Gene expression of several pluripotent, endodermal, pancreatic progenitor and pancreatic endocrine lineage markers were investigated in native ADMSC and after stimulation with different concentration of vitamin D3 for five consecutive days (0, 50, 100, 150 nM) and VPA (0.5, 1, 1.5, 2 mM) by real-time PCR. Furthermore, somatostatin expression was confirmed with ELISA and immunocytochemistry.ResultsIn ADMSC, the expression of somatostatin mRNA, the vitamin D receptor (VDR) and its metabolising enzymes 1 α-Hydroxylase, 24-Hydroxylase and 25-Hydroxylase were detected. Upon stimulation with vitamin D3, nuclear translocation of vitamin D receptor (VDR) was observed. Interestingly, the presence of vitamin D3 reduced the transcription of the somatostatin gene. By contrast, VPA treatment of cultivated ADMSC showed enhancing effect on somatostatin gene expression. No other pluripotent, endodermal, pancreatic progenitor or pancreatic endocrine lineage mRNA expression was modulated under the influence of vitamin D3 and VPA.ConclusionHuman ADMSC carry the VDR. The vitamin D metabolising enzyme 25-Hydroxylase responded to the addition of vitamin D3. Moreover, our results demonstrate that somatostatin expression in ADMSC is constitutive, partially secreted and regulated by vitamin D3 and VPA.

Adenosine triphosphate enhances osteoblast differentiation of rat dental pulp stem cells via the PLC-IP3 pathway and intracellular Ca 2+ signaling.

Intracellular Ca2+ signals are essential for stem cell function and play a significant role in the differentiation process. Dental pulp stem cells (DPSCs) are a potential source of stem cells; however, the mechanisms controlling cell differentiation remain largely unknown. Utilizing rat DPSCs, we examined the effect of adenosine triphosphate (ATP) on osteoblast differentiation and characterized its mechanism of action using real-time Ca 2+ imaging analysis. Our results revealed that ATP enhanced osteogenesis as indicated by Ca 2+ deposition in the extracellular matrix via Alizarin Red S staining. This was consistent with upregulation of osteoblast genes BMP2, Mmp13, Col3a1, Ctsk, Flt1, and Bgn. Stimulation of DPSCs with ATP (1-300 µM) increased intracellular Ca 2+ signals in a concentration-dependent manner, whereas histamine, acetylcholine, arginine vasopressin, carbachol, and stromal-cell-derived factor-1α failed to do so. Depletion of intracellular Ca 2+ stores in the endoplasmic reticulum by thapsigargin abolished the ATP responses which, nevertheless, remained detectable under extracellular Ca 2+ free condition. Furthermore, the phospholipase C (PLC) inhibitor U73122 and the inositol triphosphate (IP 3 ) receptor inhibitor 2-aminoethoxydiphenyl borate inhibited the Ca 2+ signals. Our findings provide a better understanding of how ATP controls osteogenesis in DPSCs, which involves a Ca 2+ -dependent mechanism via the PLC-IP 3 pathway. This knowledge could help improve osteogenic differentiation protocols for tissue regeneration of bone structures.

Influence of mechanical fluid shear stress on the osteogenic differentiation protocols for Equine adipose tissue-derived mesenchymal stem cells

Cell-based therapies have become a promising approach to promote tissue regeneration and the treatment of musculoskeletal disorders. Bone regeneration maintains bone homeostasis, mechanical stability and physical performance. Mechanical stimulation showed to induce stem cell differentiation into the osteogenic fate. However, the effect of various osteogenic protocols on the osteogenic commitment of equine adipose-derived stem cells is not fully elucidated. Here we examined the influence of fluid-based shear stress (FSS) via mechanical rocking to assess whether mechanical stimulation promotes osteogenic differentiation of equine adipose-derived stem cells (ASCs). ASCs were induced into osteogenic fate using osteogenic differentiation medium (ODM) protocol or additional supplementation of 5 mM CaCl2 and 7.5 mM CaCl2 protocol compared to cells cultivated in basal medium (BM) up to 21 day. The ASCs proliferation pattern was evaluated using the sulforhodamine B (SRB) protein assay. Osteogenic differentiation examined via semi-quantification of alizarin red staining (ARS) and alkaline phosphatase activity (ALP) as well as, via quantification of osteocalcin (OC), alkaline phosphatase (ALP), osteopontin (OP), and collagen type-1 (COL1) gene expression using RT-qPCR. We show that mechanical FSS increased the proliferation pattern of ASCs compared to the static conditions. Mechanical FSS together with 5 mM CaCl2 and 7.5 mM CaCl2 promoted osteogenic nodule formation and increased ARS intensity compared to the standard osteogenic protocols. We observed that combined mechanical FSS with ODM protocol increase ALP activity compared to static culture conditions. We report that ALP and OC osteogenic markers expression were upregulated under mechanical FSS culture condition particularly with the ODM protocol. Taken together, it can be assumed that mechanical stress using FSS promotes the efficiency of the osteogenic differentiation protocols of ASCs through independent mechanisms.

Optimized osteogenic differentiation protocol from R1 mouse embryonic stem cells in vitro

Embryonic stem cells (ESCs) are a unique model that allows the study of molecular pathways underlying commitment and differentiation. One such lineage is osteoblasts, which are responsible for forming bone tissue in the body. There are many osteogenic differentiation protocols in the literature utilizing different soluble factors. The goal of the present study was to increase the efficacy of our osteogenic differentiation protocol from R1 cells. We have studied the effects of the addition of the following factors: dexamethasone, retinoic acid, and peroxisome-proliferator-activated receptor-gamma inhibitor, which have been reported to enhance osteogenesis. We found that among the 6 different protocols that were tested, the addition of retinoic acid with later addition of dexamethasone gives the most enrichment of osteogenic lineage cells. Thus, our findings provide valuable guidelines for culture condition to differentiate mouse R1 ESCs to osteoblastic cells in vitro.

A Site-Specific Integrated Col2.3GFP Reporter Identifies Osteoblasts Within Mineralized Tissue Formed In Vivo by Human Embryonic Stem Cells.

The use of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) for study and treatment of bone diseases or traumatic bone injuries requires efficient protocols to differentiate hESCs/iPSCs into cells with osteogenic potential and the ability to isolate differentiated osteoblasts for analysis. We have used zinc finger nuclease technology to deliver a construct containing the Col2.3 promoter driving GFPemerald to the AAVS1 site (referred to as a "safe harbor" site), in human embryonic stem cells (H9Zn2.3GFP), with the goal of marking the cells that have become differentiated osteoblasts. In teratomas formed using these cells, we identified green fluorescent protein (GFP)-positive cells specifically associated with in vivo bone formation. We also differentiated the cells into a mesenchymal stem cell population with osteogenic potential and implanted them into a mouse calvarial defect model. We observed GFP-positive cells associated with alizarin complexone-labeled newly formed bone surfaces. The cells were alkaline phosphatase-positive, and immunohistochemistry with human specific bone sialoprotein (BSP) antibody indicates that the GFP-positive cells are also associated with the human BSP-containing matrix, demonstrating that the Col2.3GFP construct marks cells in the osteoblast lineage. Single-cell cloning generated a 100% Col2.3GFP-positive cell population, as demonstrated by fluorescence in situ hybridization using a GFP probe. The karyotype was normal, and pluripotency was demonstrated by Tra1-60 immunostaining, pluripotent low density reverse transcription-polymerase chain reaction array and embryoid body formation. These cells will be useful to develop optimal osteogenic differentiation protocols and to isolate osteoblasts from normal and diseased iPSCs for analysis.

Osteogenic Differentiation from Embryonic Stem Cells.

Embryonic stem (ES) cells have been widely studied due to their pluripotency and their potential of self-renewal. Murine ES cells are useful in investigating the molecular pathways underlying their differentiation to various mature cell types in the body. This chapter describes the maintenance of murine ES cells in culture and a routine ES cell osteogenic differentiation protocol utilized in our laboratory.

FGF‐2 addition during expansion of human bone marrow‐derived stromal cells alters MSC surface marker distribution and chondrogenic differentiation potential

Although clinical applications using mesenchymal stromal cells (MSCs) are becoming more frequent, procedures for their in vitro culture are far from standardized. Growth factors such as FGF-2 are frequently added during expansion to improve population growth and differentiation characteristics. However, up to now its influence on surface marker distribution of MSCs has been close to unknown. The purpose of this study was therefore to analyse effects of FGF-2 supplementation on pre-selection of MSC subpopulations. Mesenchymal stromal cells were harvested from bone marrow of six patients and expanded in alpha-MEM or DMEM-LG. Starting in passage 2, 10ng/ml FGF-2 was administered and non-supplemented media were used as controls. Growth indices were calculated from P0 to P4. After P4, fluorescence cytometry for common MSC surface markers was performed and standard chondrogenic, adipogenic and osteogenic differentiation protocols were applied. Cell population growth indices were higher for those in FGF-2 supplemented media. Significant differences in surface marker distribution were observed for CD13, CD14, CD49, CD90, CD340 and STRO-1 depending on respective culture conditions. FGF-2 suppressed CD146 expression in both alpha-MEM and DMEM-LG. No differences in adipogenic and osteogenic differentiation potential could be observed, while FGF-2 significantly improved chondrogenic differentiation in DMEM-LG. While holding the benefit of improving MSC chondrogenic differentiation potential, FGF-2 pre-selects certain MSC subtypes. Our data clearly show that expansion culture conditions have a significant effect on distribution of a number of MSC surface markers.

Abstract 4913: Investigation of the in vitro differentiation ability of hybrid cell clones derived from spontaneous cell fusion events between murine breast cancer cells and murine mesenchymal stem cells.

Abstract We have previously shown that murine breast cancer cell lines are capable of acquiring malignant attributes through cell fusion with stem cell lines in vitro. Hybrid cell clones emerged from spontaneous fusion events in a co-culture model showed an increased resistance against chemotherapeutic substances together with a higher proliferation rate and genetic alterations compared to their parental cell lines. Since solid tumors, including breast cancer, have pronounced heterogeneity of cell populations, cell fusion between cancer cells and tumor infiltrating immune cells as well as stem cells is one mechanism how tumor cells could acquire new properties. With relation to the importance of cell fusion in tumor heterogeneity and progression the objective of this study was to characterize and investigate if hybrid cell clones arised from spontaneous fusion events between hygromycin resistant murine 67NR-Hyg mammary carcinoma cells and puromycin resistant murine bone marrow derived stem cells (BMDCs) from Tg(GFPU)5Nagy/J mice to maintain their ability to differentiate into the neural and osteogenic lineage. Dual hygromycin/puromycin resistant mBMDC/67NRHyg cells originated by cell fusion were confirmed by a dual drug selection procedure and PCR analysis to examine an overlap of parental markers. BMDCs are generally defined as self-renewable, multipotent progenitor cells with the ability to differentiate into several mesenchymal lineages. Differentiation was performed by cultivating the cell clones and parental cell lines under appropriate conditions to an established neural and osteogenic differentiation protocol, respectively. Our results revealed through molecular and biochemical analyses that hybrid cell clones and parental cell lines were positive for neuronal as well as osteogenic markers. Differentiation of hybrid cell clones into the osteogenic lineage was associated with expression of osteocalcin, bone sialoprotein II and collagen type I alpha 1 and increasing alkaline phosphatase activity, while differentiation into neuronal lineage was concomitant with expression of neurofilament M, GAD67 and class III beta-tubulin. In conclusion, our data show that hybrid cell clones are able to differentiate into specialized cells from both osteogenic and neuronal lineages. These stem cell properties indicate that cell clones derived from cell fusion events maintain abilities of the parental cell lines. Citation Format: Marieke Mohr, Kurt S. Zaenker, Thomas Dittmar. Investigation of the in vitro differentiation ability of hybrid cell clones derived from spontaneous cell fusion events between murine breast cancer cells and murine mesenchymal stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4913. doi:10.1158/1538-7445.AM2013-4913

Effects of Osteogenic Differentiation Inducers on in Vitro Expanded Adult Mesenchymal Stromal Cells

For bone regeneration therapy using stem cells, well-defined ex vivo protocols to expand mesenchymal stromal cells (MSC), as well as assays to show their potential differentiation into the osteogenic lineage, are needed. Aim of this study was to analyze the role of the biochemical osteogenic inducers, i.e. ascorbic acid, dexamethasone, and ß-glycerophosphate, employed in the current protocols for osteogenic differentiation of MSC in vitro, to address the requirements for reliable differentiation systems. MSC were isolated from the bone marrow of donors (46-73 years of age) undergoing total hip replacement, and expanded in vitro. At confluence, MSC were cultured under four different conditions: α-MEM plus serum (basal medium or C1), basal medium plus ascorbate (C2), basal medium plus ascorbate and dexamethasone (C3), or basal medium plus ascorbate, dexamethasone and ß-glycerophosphate (C4). Morphology, proliferation, mineralization, alkaline phosphatase, collagen and expression of bone-related genes of MSC under the different media were analyzed at fixed time points. MSC proliferation and the number of colony forming units were increased by ascorbic acid, whereas dexamethasone enhanced the proportion of ALP-positive CFU and was critical for mineral deposition. Runx-2 and type I collagen gene expression decreased along with additive-induced MSC differentiation, i.e. from C1 to C4, while ALP and osteocalcin were differently regulated. Our findings support the role of different inducers on the sequential stages of MSC expansion and osteogenic differentiation in vitro, suggesting the addition of DEX following proliferation to ensure mineralization, as an index of in vivo osteogenic potency of human mesenchymal cells.

Differential bone-forming capacity of osteogenic cells from either embryonic stem cells or bone marrow-derived mesenchymal stem cells

For more than a decade, human mesenchymal stem cells (hMSCs) have been used in bone tissue-engineering research. More recently some of the focus in this field has shifted towards the use of embryonic stem cells. While it is well known that hMSCs are able to form bone when implanted subcutaneously in immune-deficient mice, the osteogenic potential of embryonic stem cells has been mainly assessed in vitro. Therefore, we performed a series of studies to compare the in vitro and in vivo osteogenic capacities of human and mouse embryonic stem cells to those of hMSCs. Embryonic and mesenchymal stem cells showed all characteristic signs of osteogenic differentiation in vitro when cultured in osteogenic medium, including the deposition of a mineralized matrix and expression of genes involved in osteogenic differentiation. As such, based on the in vitro results, osteogenic ES cells could not be discriminated from osteogenic hMSCs. Nevertheless, although osteogenic hMSCs formed bone upon implantation, osteogenic cells derived from both human and mouse embryonic stem cells did not form functional bone, indicated by absence of osteocytes, bone marrow and lamellar bone. Although embryonic stem cells show all signs of osteogenic differentiation in vitro, it appears that, in contrast to mesenchymal stem cells, they do not possess the ability to form bone in vivo when a similar culture method and osteogenic differentiation protocol was applied.

Ethanol alters the osteogenic differentiation of amniotic fluid-derived stem cells.

Fetal alcohol spectrum disorder (FASD) is a set of developmental defects caused by prenatal alcohol exposure. Clinical manifestations of FASD are highly variable and include mental retardation and developmental defects of the heart, kidney, muscle, skeleton, and craniofacial structures. Specific effects of ethanol on fetal cells include induction of apoptosis as well as inhibition of proliferation, differentiation, and migration. This complex set of responses suggests that a bioinformatics approach could clarify some of the pathways involved in these responses. In this study, the responses of fetal stem cells derived from the amniotic fluid (AFSCs) to treatment with ethanol have been examined. Large-scale transcriptome analysis of ethanol-treated AFSCs indicates that genes involved in skeletal development and ossification are up-regulated in these cells. Therefore, the effect of ethanol on osteogenic differentiation of AFSCs was studied. Exposure to ethanol during the first 48 hours of an osteogenic differentiation protocol increased in vitro calcium deposition by AFSCs and increased alkaline phosphatase activity. In contrast, ethanol treatment later in the differentiation protocol (day 8) had no significant effect on the activity of alkaline phosphatase. These results suggest that transient exposure of AFSCs to ethanol during early differentiation enhances osteogenic differentiation of the cells.

Spontaneous immortalization of neural crest-derived corneal progenitor cells after chromosomal aberration

In a previous study, we have reported the existence of neural crest-derived stem cell-like cells originating from the corneal limbus of juvenile mice (termed murine corneal cells, MCCs). To yield a sufficient number of MCCs, for example, for cell-therapy approaches, here we have investigated MCCs' ability for extensive proliferation, and we have evaluated their stem cell qualities and genetic stability after large-scale culture. MCCs were established from corneal limbal tissue of juvenile mice. To determine their cell proliferation and self-renewing potential, MTT tests and an estimation of colony forming unit efficiency were carried out. Multipotency of cell differentiation was examined by applying adipogenic and osteogenic differentiation protocols. Moreover, karyotyping was performed and expression of stem cell markers and cell cycle-associated genes was analysed. MCCs, as primary cells, could be cultured for more than 60 passages. We observed increased cell proliferation and high number of colony forming units (CFUs) after extensive culture. Interestingly, there were no changes in expression of MCC markers. Furthermore, cell differentiation potentials remained comparable with MCCs at early passages. However, karyotyping revealed numeric chromosomal aberrations at higher passages. Moreover, tumour suppressor genes such as p16 and p21 were found to be down-regulated after large-scale cell culture. MCCs immortalize spontaneously after extensive cell culture, but still demonstrate stem cell-like qualities.

Inhibition of Histone Acetylation as a Tool in Bone Tissue Engineering

Our approach to bone tissue engineering is the in vitro expansion and osteogenic differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) and their subsequent implantation on porous ceramic materials. Current osteogenic differentiation protocols use dexamethasone to initiate the osteogenic process, thus ignoring the multiple signaling pathways that control osteogenesis in vivo. Supporting osteogenesis at multiple stages might further enhance the bone-forming capacity of hMSCs. As reported previously, inhibition of so-called histone deacetylases (HDACs) stimulates osteoblast maturation, and in this report, we investigated whether trichostatin A (TSA), a widely used HDAC inhibitor, can be implemented in bone tissue engineering. We confirmed that TSA treatment of hMSCs results in increased expression of alkaline phosphatase (ALP) with concomitant increase in mineralization. Flow cytometry demonstrated that TSA increases the percentage of ALP-positive hMSCs as well as their average ALP expression level, but the robustness of the response differs between donors. Unfortunately, TSA has a profound negative effect on cell proliferation, so we investigated whether hMSCs respond to TSA after reaching confluence. Confluent hMSCs on tissue culture plastic displayed enhanced ALP expression. Therefore, we seeded TSA-treated hMSCs onto ceramic particles and analyzed ectopic bone formation upon implantation in immune-deficient mice. Unfortunately, TSA-treated hMSCs did not display better bone formation in vivo than control cells. Finally, we observed that TSA treatment strongly enhanced bone formation of ex vivo cultured mouse calvaria, which warrants further exploration of TSA in bone tissue engineering.