Induction Of Osteogenic Differentiation Research Articles

The effects of PPAR-γ inhibition on gene expression and the progression of induced osteogenic differentiation of human mesenchymal stem cells

Mesenchymal stem cells (MSCs) can differentiate into several cell types, such as osteoblasts and adipocytes, both in vitro and in vivo. Although these two differentiation pathways are distinct from each other, cross-communication between cells of the two lineages exists both systemically and peripherally in the tissue. The transcription factor PPAR-γ, the main switch in adipogenic differentiation of MSCs, has previously been described to have a negative effect on osteogenic differentiation. The aim of this study was to investigate the effect of PPAR-γ inhibition on osteogenic differentiation of human MSCs, in vitro. Extracellular matrix analysis and quantification of osteogenic markers, revealed how these cells respond when the adipogenic differentiation pathway is blocked during induction of osteogenic differentiation. The inhibition leads to a significant increase in mineralization of the extracellular matrix, as well as an increased activity or up-regulated gene expression of alkaline phosphatase, the key enzyme involved in matrix mineralization. Furthermore, it was also demonstrated by microarray analysis, that PPAR-γ inhibition during osteogenic induction leads to a significant up-regulation of a number of genes related to both osteogenesis and adipogenesis such as c10orf10, leptin, GDF5 and KLF15. In conclusion, inhibition of PPAR-γ during induction of osteogenesis leads to increased osteogenic differentiation of human MSCs.

A nanoscale ridge/groove pattern arrayed surface enhances adipogenic differentiation of human supernumerary tooth-derived dental pulp stem cells in vitro

ObjectiveThe aim of this study was to establish human dental pulp stem cells (hDPSCs) from supernumerary teeth and determine the effects of a 350-nm nano-patterned surface on adipogenic and osteogenic differentiation of hDPSCs. DesignSeveral surface markers were analysed by FACS to confirm the isolated cells as hDPSCs. To demonstrate the effects of a nano-patterned surface on the differentiation of hDPSCs, the cells were cultured on a nano-patterned surface with or without adipogenic or osteogenic induction factors. Cells were then stained with Oil red O or Alizarin red, and the lineage specific genes LPL and Runx-2 were analysed by real-time PCR at 3, 6 and 9 days after culture. ResultsThe hDPSCs on a nano-patterned surface showed a linear arrangement compared to irregular cells on a conventional surface. During adipogenic differentiation, more Oil red O stained cells were found in the nano-patterned group than in the conventional group. On the other hand, there was no significant difference in Alizarin red staining between the nano-pattern and conventional surface groups after induction of osteogenic differentiation. Gene expression analyses revealed significantly higher expression of LPL in the nano-patterned group than in the conventional group, whereas Runx-2 expression was higher in the conventional group than in the nano-patterned group. ConclusionThis study showed that a nano-patterned surface may be able to enhance adipogenic differentiation of hDPSCs by altering their morphology and gene expression patterns, whereas the same surface may inhibit or suppress osteogenic differentiation of the cells.

Induction of osteogenic differentiation by nanostructured alumina surfaces.

Permanent orthopedic implants are becoming increasingly important due to the demographic development. Their optimal osseointegration is key in obtaining good secondary stability. For anchorage dependent cells, topographic features of a surface play an essential role for cell adhesion, proliferation, differentiation and biomineralization. We studied the topographical effect of nanostructured alumina surfaces prepared by chemical vapor deposition on osteogenic differentiation and growth of human osteoblasts. Chemical vapor deposition of the single source precursor (tBuOAIH2)2 led to synthesis of one dimensional alumina nanostructures of high purity with a controlled stoichiometry. We fabricated different topographic features by altering the distribution density of deposited one dimensional nanostructures. Although the topography differed, all surfaces exhibited identical surface chemistry, which is the key requirement for systematically studying the effect of the topography on cells. Forty-eight hours after seeding, cell density and cell area were not affected by the nanotopography, whereas metabolic activity was reduced and formation of actin-fibres and focal adhesions was impaired compared to the uncoated control. Induction of osteogenic differentiation was demonstrated via up-regulation of alkaline phosphatase, bone sialoprotein, osteopontin and Runx2 at the mRNA level, demonstrating the potential of nanostructured surfaces to improve the osseointegration of permanent implants.

Aged human mesenchymal stem cells: the duration of bone morphogenetic protein-2 stimulation determines induction or inhibition of osteogenic differentiation.

Bone morphogenetic protein 2 (BMP-2) is a potent osteoinductive cytokine and a growing number of in vitro studies analyze its effects on human mesenchymal stem cells (hMSC) derived from aged or osteoporotic donors. In these studies the exact quantification of osteogenic differentiation capacity is of fundamental interest. Nevertheless, the experimental conditions for osteogenic differentiation of aged hMSC have not been evaluated systematically and vary to a considerable extend. Aim of the study was to assess the influence of cell density, osteogenic differentiation media (ODM) change intervals and duration of BMP-2 stimulation on osteoinduction. Furthermore, time series were carried out for osteogenic differentiation and BMP-2 concentration in ODM/BMP-2 cell culture supernatants. The experiments were performed using hMSC isolated from femoral heads of aged patients undergoing hip joint replacement. ODM change intervals of 96 hours resulted in significantly higher calcium deposition compared to shorter intervals. A cell density of 80% prior to stimulation led to stronger osteoinduction compared to higher cell densities. In ODM, aged hMSC showed a significant induction of calcium deposition after 9 days. Added to ODM, BMP-2 showed a stable concentration in the cell culture supernatants for at least 96 hours. Addition of BMP-2 to ODM for the initial 4 days led to a significantly higher induction of osteogenic differentiation compared to ODM alone. On the other hand, addition of BMP-2 for 21 days almost abrogated the osteoinductive effect of ODM. We could demonstrate that the factors investigated have a substantial impact on the extent of osteogenic differentiation of aged hMSC. Consequently, it is of upmost importance to standardize the experimental conditions in order to enable comparability between different studies. We here define standard conditions for osteogenic differentiation in regard to the specific features of aged hMSC. The finding that BMP-2 induces or inhibits osteogenic differentiation in a time dependent manner indicates an age related alteration in signal transduction of hMSC and requires further investigation.

Optimization of release pattern of FGF-2 and BMP-2 for osteogenic differentiation of low-population density hMSCs

In the modern design, most delivery systems for bone regeneration focus on a single growth factor (GF) or a simple mixture of multiple GFs, overlooking the coordination of proliferation and osteogenesis induced by various factors. In this study, core-shell microspheres with poly-l-lactide core-poly(lactic-co-glycolic acid) shell were fabricated, and two GFs, basic fibroblast growth factor 2 (FGF-2) and bone morphogenetic protein 2 (BMP-2) were encapsulated into the core or/and shell. The effects of different release patterns (parallel or sequential manners) of FGF-2 and BMP-2 from these core-shell microspheres on the osteogenic differentiation of low-population density human mesenchymal stem cells (hMSCs) were investigated and the temporal organization of GF release was optimized. In vitro experiments suggested that induction of osteogenic differentiation of low-population density hMSCs by the sequential delivery of FGF-2 followed by BMP-2 from the core-shell microspheres (group S2) was much more efficient than that by the parallel release of the two factors from uniform microspheres (group U). The osteogenic induction by the sequential delivery of BMP-2 followed by FGF-2 from core-shell microspheres (group S1) was even worse than that from microspheres loaded with BMP-2 in both core and shell (group B), although comparable to the cases of parallel delivery of dual GFs (group P). This study showed the advantages of group S2 microspheres in inducing osteogenic differentiation of low-population density hMSCs and the necessity of time sequence studies in tissue engineering while multiple GFs are involved.

Gangliosides as a potential new class of stem cell markers: the case of GD1a in human bone marrow mesenchymal stem cells

Owing to their exposure on the cell surface and the possibility of being directly recognized with specific antibodies, glycosphingolipids have aroused great interest in the field of stem cell biology. In the search for specific markers of the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) toward osteoblasts, we studied their glycosphingolipid pattern, with particular attention to gangliosides. After lipid extraction and fractionation, gangliosides, metabolically (3)H-labeled in the sphingosine moiety, were separated by high-performance TLC and chemically characterized by MALDI MS. Upon induction of osteogenic differentiation, a 3-fold increase of ganglioside GD1a was observed. Therefore, the hypothesis of GD1a involvement in hBMSCs commitment toward the osteogenic phenotype was tested by comparison of the osteogenic propensity of GD1a-highly expressing versus GD1a-low expressing hBMSCs and direct addition of GD1a in the differentiation medium. It was found that either the high expression of GD1a in hBMSCs or the addition of GD1a in the differentiation medium favored osteogenesis, providing a remarkable increase of alkaline phosphatase. It was also observed that ganglioside GD2, although detectable in hBMSCs by immunohistochemistry with an anti-GD2 antibody, could not be recognized by chemical analysis, likely reflecting a case, not uncommon, of molecular mimicry.

Adipose- and bone marrow-derived mesenchymal stem cells display different osteogenic differentiation patterns in 3D bioactive glass-based scaffolds

Mesenchymal stem cells can be isolated from a variety of different sources, each having their own peculiar merits and drawbacks. Although a number of studies have been conducted comparing these stem cells for their osteo-differentiation ability, these are mostly done in culture plastics. We have selected stem cells from either adipose tissue (ADSCs) or bone marrow (BMSCs) and studied their differentiation ability in highly porous three-dimensional (3D) 45S5 Bioglass®-based scaffolds. Equal numbers of cells were seeded onto 5 × 5 × 4 mm3 scaffolds and cultured in vitro, with or without osteo-induction medium. After 2 and 4 weeks, the cell-scaffold constructs were analysed for cell number, cell spreading, viability, alkaline phosphatase activity and osteogenic gene expression. The scaffolds with ADSCs displayed osteo-differentiation even without osteo-induction medium; however, with osteo-induction medium osteogenic differentiation was further increased. In contrast, the scaffolds with BMSCs showed no osteo-differentiation without osteo-induction medium; after application of osteo-induction medium, osteo-differentiation was confirmed, although lower than in scaffolds with ADSCs. In general, stem cells in 3D bioactive glass scaffolds differentiated better than cells in culture plastics with respect to their ALP content and osteogenic gene expression. In summary, 45S5 Bioglass-based scaffolds seeded with ADSCs are well-suited for possible bone tissue-engineering applications. Induction of osteogenic differentiation appears unnecessary prior to implantation in this specific setting. Copyright © 2013 John Wiley & Sons, Ltd.

The Distinct Stem Cell Population Derived From Chorionic Plate Of Human Placenta Which Has Differentiation Potential Into Erythroid Lineage Cells

Background/AimsPlacenta is a specialized organ which has a property of fetal and adult tissues simultaneously. Mesenchymal stem cell and hematopoietic stem cells have been founded in the parenchyma of placenta tissue contributed by fetal side. Recently, it was reported that placenta is a major hematopoietic organ contributing to both generation and expansion of multipotent hematopoietic stem and progenitor cells. We hypothesized that hematopoietic stem cells in placenta might be originated from another placental stem cells which has been so-called “placenta mesenchymal stem cells”. To prove this hypothesis, this study is designed to induce in vitro differentiation of placental stem cells into the hematopoietic stem cells without genetic transduction. MethodsPlacental stem cells was obtained from surgically isolated placental chorionic plates from healthy women who had undergone abortion at 6-8 weeks of gestation. To prevent the contamination of hematopoietic stem cells, CD34-CD44+ cells were purified by flowcytometric isolation. After mesenchymal differentiation potential was identified in these cells by induction of adipogenic and osteogenic differentiation, they were cultured in vitro with serum-free media including cytokine cocktail which was known to induce differentiation of embryonic stem cells into hematopoietic lineage (erythropoietin, Flt-3/Flk-2 ligand, G-CSF, GM-CSF, IL-6/7/15, stem cell factor, and thrombopoietin). In vitro differentiation was performed in conventional cell culture dish (group 1) and Ultra-low attachment dish (group2) for 3 weeks, respectively (Figure 1). Expression of hematopoietic stem and progenitor cell markers were analyzed at every 1 week. [Display omitted] ResultsIn group 1 (conventional cell culture dish), no hematopoietic differentiation was identified regardless of the concentrations of cytokine cocktail. In group 2 (Ultra-low attachment dish), expression of pan-hematopoietic markers, CD34 and CD45 was also not observed. However, after 2 weeks of differentiation induction, red cells aggregates and expression of erythroid-lineage markers (CD235a and CD38) were detected in cells cultured using high concentration cytokines (Media 3 in Figure 1). The proportion of cells expressing erythroid-lineage markers became increased during in vitro differentiation (Figure 2). [Display omitted] ConclusionAlthough this study failed to show the differentiation of placenta-derived stem cells into hematopoietic stem cells, the results demonstrated that placenta-derived stem cells could be differentiated into the erythroid lineage under certain condition (i.e. liquid culture + high concentration cytokines). Therefore, placenta-derived stem cells which have been so-called “placenta mesenchymal stem cells” is considered to be a distinct stem cell population including property of erythroid progenitor cells. Disclosures:No relevant conflicts of interest to declare.

In vivo differentiation of human periodontal ligament cells leads to formation of dental hard tissue

Following trauma, periodontal disease, or orthodontic tooth movement, residual periodontal ligament (PDL) cells at the defect site are considered mandatory for successful regeneration of the injured structures. Recent developments in tissue engineering focus, as one pillar, on the transplantation of PDL cells to support periodontal regeneration processes. Here, we examined the ability of osteogenically predifferentiated PDL cells to undergo further osteoblastic or cementoblastic differentiation and to mineralize their extracellular matrix when transplanted in an in vivo microenvironment. Using collagen sponges as carriers, osteogenically predifferentiated human PDL cells were transplanted subcutaneously into six immunocompromised CD-1® nude mice. Following explantation after 28 days, osteogenic and cementogenic marker protein expression was visualized immunohistochemically. After 28 days, transplanted PDL cells revealed both cellular, cytoplasmatic and extracellular immunoreactivity for the chosen markers alkaline phosphatase, osteopontin, PTH-receptor 1, and osteocalcin. Specific osteogenic and cementoblastic differentiation was demonstrated by RUNX2 and CEMP1 immunoreactivity. Early stages of mineralization were demonstrated by calcium and phosphate staining. Our results reinforce the previously published reports of PDL cell mineralization in vivo and further demonstrate the successful induction of specific osteogenic and cementogenic differentiation of transplanted human PDL cells in vivo. These findings reveal promising possibilities for supporting periodontal remodeling and regeneration processes with PDL cells being potential target cells with which to influence the process of orthodontically induced root resorption.

Mesenchymal stem cells from osteoporotic patients feature impaired signal transduction but sustained osteoinduction in response to BMP-2 stimulation

Osteoporotic fractures show reduced callus formation and delayed bone healing. Cellular sources of fracture healing are mesenchymal stem cells (MSC) that differentiate into osteoblasts by stimulation with osteoinductive cytokines, such as BMP-2. We hypothesized that impaired signal transduction and reduced osteogenic differentiation capacity in response to BMP-2 may underlie the delayed fracture healing.Therefore, MSC were isolated from femoral heads of healthy and osteoporotic patients. Grouping was carried out by bone mineral densitometry in an age-matched manner. MSC were stimulated with BMP-2. Signal transduction was assessed by western blotting of pSMAD1/5/8 and pERK1/2 as well as by quantitative RT-PCR of Runx-2, Dlx5, and Osteocalcin. Osteogenic differentiation was assessed by quantifying Alizarin Red staining.Osteoporotic MSC featured an accurate phosphorylation pattern of SMAD1/5/8 but a significantly reduced activation of ERK1/2 by BMP-2 stimulation. Furthermore, osteoporotic MSC showed significantly reduced basal expression levels of Runx-2 and Dlx5. However, Runx-2, Dlx5, and Osteocalcin expression showed adequate up-regulation due to BMP-2 stimulation. The global osteogenic differentiation in standard osteogenic differentiation media was reduced in osteoporotic MSC. Nevertheless, osteoporotic MSC were shown to feature an adequate induction of osteogenic differentiation due to BMP-2 stimulation.Taken together, we here demonstrate osteoporosis associated alterations in BMP-2 signaling but sustained specific osteogenic differentiation capacity in response to BMP-2. Therefore, BMP-2 may represent a promising therapeutic agent for the treatment of fractures in osteoporotic patients.

Functional analysis of Bombyx mori Decapentaplegic gene for bone differentiation in a mammalian cell

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor (TGF-<TEX>${\beta}$</TEX>) superfamily and are involved in osteoblastic differentiation. The largest TGF-<TEX>${\beta}$</TEX> superfamily subgroup shares genetic homology with human BMPs (hBMPs) and silkworm decapentaplegic (dpp). In addition, hBMPs are functionally interchangeable with Drosophila dpp. Bombyx mori dpp may induce bone formation in mammalian cells. To test this hypothesis, we synthesized the 1,285-base pairs cDNA of full-length B. mori dpp using total RNAs obtained from the fat body of 3-day-old of the <TEX>$5^{th}$</TEX> instar larvae and cloned the cDNA into the pCEP4 mammalian expression vector. Next, B. mori dpp was expressed in C3H10T1/2 cells. The target cells transfected with the pCEP4-Bm dpp plasmid showed biological functions similar to those of osteogenic differentiation induction growth factors such as hBMPs. We determined the relative mRNA expression rates of Runt-related transcription factor 2 (RUNX2), osterix, osteocalcin, and alkaline phosphatase (ALP) to validate the osteoblast-specific differentiation effects of B. mori dpp by performing quantitative real-time RT-PCR. Interestingly, mRNA expression levels of the 3 marker genes except RUNX2, in cells expressing B. mori dpp were much higher than those in control cells and C3H10T1/2 cells transfected with pCEP4. These results suggested that B. mori dpp signaling regulates osterix expression during osteogenic differentiation via RUNX2-independent mechanisms.

Bisphosphonate-Linked Hyaluronic Acid Hydrogel Sequesters and Enzymatically Releases Active Bone Morphogenetic Protein-2 for Induction of Osteogenic Differentiation

Regeneration of bone by delivery of bone morphogenetic proteins (BMPs) from implantable scaffolds is a promising alternative to the existing autologous bone grafting procedures. Hydrogels are used extensively in biomaterials as delivery systems for different growth factors. However, a controlled release of the growth factors is necessary to induce bone formation, which can be accomplished by various chemical functionalities. Herein we demonstrate that functionalization of a hyaluronan (HA) hydrogel with covalently linked bisphosphonate (BP) ligands provides efficient sequestering of BMP-2 in the resulting HA-BP hydrogel. The HA-BP hydrogel was investigated in comparison with its analogue lacking BP groups (HA hydrogel). While HA hydrogel released 100% of BMP-2 over two weeks, less than 10% of BMP-2 was released from the HA-BP hydrogel for the same time. We demonstrate that the sequestered growth factor can still be released by enzymatic degradation of the HA-BP hydrogel. Most importantly, entrapment of BMP-2 in HA-BP hydrogel preserves the growth factor bioactivity, which was confirmed by induction of osteogenic differentiation of mesenchymal stem cells (MSCs) after the cells incubation with the enzymatic digest of the hydrogel. At the same time, the hydrogels degradation products were not toxic to MSCs and osteoblasts. Furthermore, BP-functionalization of HA hydrogels promotes adhesion of the cells to the surface of HA hydrogel. Altogether, the present findings indicate that covalent grafting of HA hydrogel with BP groups can alter the clinical effects of BMPs in bone tissue regeneration.

Induction of Osteogenic Differentiation of Multipotent Mesenchymal Stromal Cells from Human Adipose Tissue

We studied of osteogenic differentiation of multipotent mesenchymal stromal cells from human adipose tissue. Experiments showed that 1α,25-dihydroxycalciferol is a more effective inductor of osteogenesis than dexamethasone. Comparative analysis revealed activation of gene expression for the major osteogenic markers on day 7 of culturing in a medium containing 1α,25-dihydroxycalciferol. It was found that transcription of genes encoding type 1 collagen proteins, osteopontin, osteocalcin, and bone sialoprotein peaked on day 14 in culture, while the expression of alkaline phosphatase and bone morphogenetic protein-2 genes increased over 21 days. Intensive mineralization of the extracellular matrix was observed starting from day 14 in culture. On the basis of the analysis of these data, optimal terms for osteogenic induction (day 14) and an optimal inductor (1α,25-dihydroxycalciferol) were chosen and the protocol of effective osteogenic differentiation of multipotent mesenchymal stromal cells from human adipose tissue was developed for creation of tissue-engineered bone equivalents.

Effect of the HDAC inhibitor vorinostat on the osteogenic differentiation of mesenchymal stem cells in vitro and bone formation in vivo

Vorinostat, a histone deacetylase (HDAC) inhibitor currently in a clinical phase III trial for multiple myeloma (MM) patients, has been reported to cause bone loss. The purpose of this study was to test whether, and to what extent, vorinostat influences the osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro and bone formation in vivo. Bone marrow-derived MSCs were prepared from both normal donors and MM patients. The MSCs were cultured in an osteogenic differentiation induction medium to induce osteogenic differentiation, which was evaluated by alkaline phosphatase (ALP) staining, Alizarin Red S staining and the mRNA expression of osteogenic markers. Naïve mice were administered vorinostat (100 mg/kg, ip) every other day for 3 weeks. After the mice were sacrificed, bone formation was assessed based on serum osteocalcin level and histomorphometric analysis. Vorinostat inhibited the viability of hMSCs in a concentration-dependent manner (the IC50 value was 15.57 μmol/L). The low concentration of vorinostat (1 μmol/L) did not significantly increase apoptosis in hMSCs, whereas pronounced apoptosis was observed following exposure to higher concentrations of vorinostat (10 and 50 μmol/L). In bone marrow-derived hMSCs from both normal donors and MM patients, vorinostat (1 μmol/L) significantly increased ALP activity, mRNA expression of osteogenic markers, and matrix mineralization. These effects were associated with upregulation of the bone-specifying transcription factor Runx2 and with the epigenetic alterations during normal hMSCs osteogenic differentiation. Importantly, the mice treated with vorinostat did not show any bone loss in response to the optimized treatment regimen. Vorinostat, known as a potent anti-myeloma drug, stimulates MSC osteogenesis in vitro. With the optimized treatment regimen, any decrease in bone formation was not observed in vivo.

MiR-125b regulates osteogenic differentiation of human bone marrow mesenchymal stem cells by targeting Smad4

To investigate whether miR-125b regulates the osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs) by modulating Smad4, a predicted target in silicon. Smad4 3'-UTR-luciferase vector was constructed and dual-luciferase reporter gene assay was employed to examine the effect of miR-125b on luciferase activity. MSCs were isolated and cultured from human bone marrow, and then transfected with miR-125b mimics followed by induction of osteogenic differentiation. qRT-PCR and Western blot were used to detect the expressions of Smad4 mRNA and protein. MSCs were induced into the osteoblasts after transfecting with Smad4 siRNA, and the effect of Smad4 downregulation on osteogenic differentiation was observed by AKP activity and RUNX2 mRNA levels. miR-216b bound Smad4 3'-UTR and inhibited the luciferase activity (P<0.05). Smad4 mRNA and protein expressions were significantly down-regulated in the MSCs induced into osteogenic differentiation when miR-125b was overexpressed. Downregulation of Smad4 suppressed the AKP activity and RUNX2 mRNA expression, indicating that Smad4 siRNA simulated at least in part the function of miR-125b as the regulator of MSCs osteogenic differentiation. miR-125b can suppress MSCs osteogenic differentiation by directly targeting Smad4.

In vitro osteogenic differentiation of human amniotic fluid-derived stem cells on a poly(lactide-co-glycolide) (PLGA)–bladder submucosa matrix (BSM) composite scaffold for bone tissue engineering

Stem cells have become an important component of tissue regeneration, as they are able to differentiate into various cell types if guided appropriately. It is well known that cellular differentiation is greatly influenced by the surrounding microenvironment. We have developed a composite scaffold system using a collagen matrix derived from porcine bladder submucosa matrix (BSM) and poly(lactide-co-glycolide) (PLGA). In this study, we investigated whether a composite scaffold composed of naturally derived matrix combined with synthetic polymers would provide a microenvironment to facilitate the induction of osteogenic differentiation. We first showed that human amniotic fluid-derived stem cells (hAFSCs) adhered to the composite scaffolds and proliferated over time. We also showed that the composite scaffolds facilitated the differentiation of hAFSCs into an osteogenic lineage. The expression of osteogenic genes, including RUNX2, osteopontin (OPN) and osteocalcin (OCN) was upregulated in cells cultured on the composite scaffolds incubated in the osteogenic medium compared with ones without. Increased alkaline phosphatase (ALP) activity and calcium content indicates that hAFSCs seeded on 3D porous BSM–PLGA composite scaffolds resulted in higher mineralization rates as the duration of induction increased. This was also evidenced by the mineralized matrix within the scaffolds. The composite scaffold system provides a proper microenvironment that can facilitate osteogenic differentiation of AFSCs. This scaffold system may be a good candidate material for bone tissue engineering.

Induction of osteogenic differentiation of stem cells via a lyophilized microRNA reverse transfection formulation on a tissue culture plate

MicroRNA (miRNA) regulation is a novel approach to manipulating the fate of mesenchymal stem cells, but an easy, safe, and highly efficient method of transfection is required. In this study, we developed an miRNA reverse transfection formulation by lyophilizing Lipofectamine 2000-miRNA lipoplexes on a tissue culture plate. The lipoplexes can be immobilized on a tissue culture plate with an intact pseudospherical structure and lyophilization without any lyoprotectant. In this study, reverse transfection resulted in highly efficient cellular uptake of miRNA and enabled significant manipulation of the intracellular target miRNA level. Reverse transfection formulations containing Lipofectamine 2000 1 μL per well generated much higher transfection efficiency without obvious cytotoxicity compared with conventional and other transfection methods. Further, the transfection efficiency of the reverse transfection formulations did not deteriorate during 90 days of storage at 4°C and −20°C. We then assessed the efficiency of the miRNA reverse transfection formulation in promoting osteogenic differentiation of mesenchymal stem cells. We found that transfection with anti-miR-138 and miR-148b was efficient for enhancing osteogenic differentiation, as indicated by enhanced osteogenesis-related gene expression, amount of alkaline phosphatase present, production of collagen, and matrix mineralization. Overall, the miRNA reverse transfection formulation developed in this study is a promising approach for miRNA transfection which can control stem cell fate and is suitable for loading miRNAs onto various biomaterials.

Induction of Osteogenic Differentiation of Adipose Derived Stem Cells by Microstructured Nitinol Actuator-Mediated Mechanical Stress

The development of large tissue engineered bone remains a challenge in vitro, therefore the use of hybrid-implants might offer a bridge between tissue engineering and dense metal or ceramic implants. Especially the combination of the pseudoelastic implant material Nitinol (NiTi) with adipose derived stem cells (ASCs) opens new opportunities, as ASCs are able to differentiate osteogenically and therefore enhance osseointegration of implants. Due to limited knowledge about the effects of NiTi-structures manufactured by selective laser melting (SLM) on ASCs the study started with an evaluation of cytocompatibility followed by the investigation of the use of SLM-generated 3-dimensional NiTi-structures preseeded with ASCs as osteoimplant model. In this study we could demonstrate for the first time that osteogenic differentiation of ASCs can be induced by implant-mediated mechanical stimulation without support of osteogenic cell culture media. By use of an innovative implant design and synthesis via SLM-technique we achieved high rates of vital cells, proper osteogenic differentiation and mechanically loadable NiTi-scaffolds could be achieved.

Rigid matrix supports osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED)

Stem cell fate can be induced by the grade of stiffness of the extracellular matrix, depending on the developed tissue or complex tissues. For example, a rigid extracellular matrix induces the osteogenic differentiation in bone marrow derived mesenchymal stem cells (MSCs), while a softer surface induces the osteogenic differentiation in dental follicle cells (DFCs). To determine whether differentiation of ectomesenchymal dental precursor cells is supported by similar grades of extracellular matrices (ECMs) stiffness, we examined the influence of the surface stiffness on the proliferation and osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED). Cell proliferation of SHED was significantly decreased on cell culture surfaces with a muscle-like stiffness. A dexamethasone-based differentiation medium induced the osteogenic differentiation of SHED on substrates of varying mechanical stiffness. Here, the hardest surface improved the induction of osteogenic differentiation in comparison to that with the softest stiffness. In conclusion, our study showed that the osteogenic differentiation of ectomesenchymal dental precursor cells SHED and DFCs are not supported by similar grades of ECM stiffness.

DNA Methylation Plasticity of Human Adipose-Derived Stem Cells in Lineage Commitment

Adult stem cells have an enormous potential for clinical use in regenerative medicine that avoids many of the drawbacks characteristic of embryonic stem cells and induced pluripotent stem cells. In this context, easily obtainable human adipose-derived stem cells offer an interesting option for future strategies in regenerative medicine. However, little is known about their repertoire of differentiation capacities, how closely they resemble the target primary tissues, and the potential safety issues associated with their use. DNA methylation is one of the most widely recognized epigenetic factors involved in cellular identity, prompting us to consider how the analyses of 27,578 CpG sites in the genome of these cells under different conditions reflect their different natural history. We show that human adipose-derived stem cells generate myogenic and osteogenic lineages that share much of the DNA methylation landscape characteristic of primary myocytes and osteocytes. Most important, adult stem cells and in vitro-generated myocytes and osteocytes display a significantly different DNA methylome from that observed in transformed cells from these tissue types, such as rhabdomyosarcoma and osteosarcoma. These results suggest that the plasticity of the DNA methylation patterns plays an important role in lineage commitment of adult stem cells and that it could be used for clinical purposes as a biomarker of efficient and safely differentiated cells.