Three-dimensional visualization analysis of in vitro cultured bone fabricated by rat marrow mesenchymal stem cells

Marrow mesenchymal stem cells are well known for their differentiation into bone-forming osteoblasts and in vitro mineralized tissue formation. However, process details, including tissue structure and cellular environments, remain unclear. The present study demonstrates three-dimensional visualization of tissue fabricated by culturing MSCs in the presence of calcein, a fluorescent marker for bone mineralization. The 3D visualization was performed by computer-assisted confocal laser scanning microscopy and revealed that the in vitro tissue consisted of layers of a mineralized matrix with round cells in the matrix lacunae, an unmineralized matrix (osteoid), and osteoblastic cells on the osteoid surface. The findings show that the mineralization by cultured MSCs is an in vitro counterpart of in vivo bone formation and indicate that the novel technique of visualization without tissue fixation could be useful for continuous monitoring of tissue organization in an ongoing culture.     

Culture and neural differentiation of rat bone marrow mesenchymal stem cells in vitro

Adult bone marrow mesenchymal stem cells (MSCs) can differentiate into several types of mesenchymal cells, including osteocytes, chondrocytes, and adipocytes, but can also differentiate into non-mesenchymal cells, such as neural cells, under appropriate experimental conditions. Until now, many protocols for inducing neuro-differentiation in MSCs in vitro have been reported. But due to the differences in MSCs' isolation and culture conditions, the results of previous studies lacked consistency and comparability. In this study, we induced differentiation into neural phenotype in the same MSCs population by three different treatments: beta-mercaptoethanol, serum-free medium and co-cultivation with fetal mouse brain astrocytes. In all of the three treatments, MSCs could express neural markers such as NeuN or GFAP, associating with remarkable morphological modifications. But these treatments led to neural phenotype in a non-identical manner. In serum-free medium, MSCs mainly differentiated into neuron-like cells, expressing neuronal marker NeuN, and BME can promote this process. Differently, after co-culturing with astrocytes, MSCs leaned to differentiate into GFAP(+) cells. These data confirmed that MSCs can exhibit plastic neuro-differentiational potential in vitro, depending on the protocols of inducement.     

A novel tripolymer coating demonstrating the synergistic effect of chitosan, collagen type 1 and hyaluronic acid on osteogenic differentiation of human bone marrow derived mesenchymal stem cells

The biomimetic approach mimicking in vivo micro environment is the key for developing functional tissue engineered constructs. In this study, we used a tripolymer combination consisting of a natural polymer, chitosan and two extracellular matrix components; collagen type 1 and hyaluronic acid to coat tissue culture plate to evaluate their effect on osteogenic differentiation of human bone marrow derived mesenchymal stem cells (hMSCs). The polymers were blended at different mixing ratios and the tissue culture plates were coated either by polyblend method or by surface modification method. hMSCs isolated from adult bone marrow were directed to osteoblast differentiation on the coated plates. Our results showed that the tripolymer coating of the tissue culture plate enhanced mineralization as evidenced by calcium quantification exhibiting significantly higher amount of calcium compared to the untreated or individual polymer coated plates. We found that the tripolymer coated plates having a 1:1 mixing ratio of chitosan and collagen type 1, surface modified with hyaluronic acid is an ideal combination to achieve the synergistic effect of these polymers on in vitro osteogenic differentiation of hMSCs. These results thus, establish a novel biomimetic approach of surface modification to enhance osteoblast differentiation and mineralization. Our findings hold great promise in implementing a biomimetic surface coating to improve osteoconductivity of implants and scaffolds for various orthopaedic and bone tissue engineering applications.     

The effect of medium composition on deposition of collagen type 1 and expression of osteogenic genes in mesenchymal stem cells derived from human adipose tissue and bone marrow

The two mesenchymal stem cell (MSC) populations that have gained most attention in relation to bone tissue engineering are adipose tissue (AT) MSCs and bone marrow (BM) MSCs. The purpose of this study was to investigate the ability of human BM-MSCs and AT-MSCs to survive, proliferate and deposit collagen type 1 when cultured on polycaprolactone nanofiber scaffolds and to ascertain the effect of medium composition on collagen type 1 formation and expression of osteogenic genes. The cells were seeded on polycaprolactone nanofiber scaffolds and cultured in three different types of media that differed by the presence of ascorbic acid, β-glycerophosphate and dexamethasone, that are typical components used for osteogenic differentiation of MSCs in vitro.In summary, AT-MSCs were proliferating significantly faster than BM-MSCs. AT-MSCs also showed better ability to deposit collagen type 1 and had a higher expression of early osteogenic markers, whereas BM-MSCs had higher expression of late osteogenic markers. This suggests that MSCs from diverse sources have different attributes and with respect to osteogenic differentiation, AT-MSCs are more immature compared to BM-MSCs. Collagen formation was depending on medium composition and the organization of collagen type 1 appeared to be influenced by the presence of dexamethasone.     

Stimulating factors for regulation of osteogenic and chondrogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs), distributed in many tissues in the human body, are multipotent cells capable of differentiating in specific directions. It is usually considered that the differentiation process of MSCs depends on specialized external stimulating factors, including cell signaling pathways, cytokines, and other physical stimuli. Recent findings have revealed other underrated roles in the differentiation process of MSCs, such as material morphology and exosomes. Although relevant achievements have substantially advanced the applicability of MSCs, some of these regulatory mechanisms still need to be better understood. Moreover, limitations such as long-term survival in vivo hinder the clinical application of MSCs therapy. This review article summarizes current knowledge regarding the differentiation patterns of MSCs under specific stimulating factors.     

A scaffold-free in vitro model for osteogenesis of human mesenchymal stem cells

For studying cellular processes three-dimensional (3D) in vitro models are of a high importance. For tissue engineering approaches osseous differentiation is performed on 3D scaffolds, but material depending influences promote cellular processes like adhesion, proliferation and differentiation. To investigate developmental processes of mesenchymal stem cells without cell-substrate interactions, self-contained in vitro models mimicking physiological condition are required. However, with respect to scientific investigations and pharmaceutical tests, it is essential that these tissue models are well characterised and are of a high reproducibility. In order to establish an appropriate in vitro model for bone formation, different protocols are compared and optimised regarding their aggregate formation efficiency, homogeneity of the aggregates, the viability and their ability to induce differentiation into the osteogenic lineage. The protocols for the generation of 3D cell models are based on rotation culture, hanging drop technique, and the cultivation in non adhesive culture vessels (single vessels as well as 96 well plates). To conclude, the cultivation of hMSCs in 96 well non adhesive plates facilitates an easy way to cultivate homogenous cellular aggregates with high performance efficiency in parallel. The size can be controlled by the initial cell density per well and within this spheroids, bone formation has been induced.     

Activation of JNKs is essential for BMP9-induced osteogenic differentiation of mesenchymal stem cells

Although BMP9 is highly capable of promoting osteogenic differentiation of mesenchymal stem cell (MSCs), the molecular mechanism involved remains to be fully elucidated. Here, we explore the possible involvement and detail role of JNKs (c-Jun N-terminal kinases) in BMP9-induced osteogenic differentiation of MSCs. It was found that BMP9 stimulated the activation of JNKs in MSCs. BMP9-induced osteogenic differentiation of MSCs was dramatically inhibited by JNKs inhibitor SP600125. Moreover, BMP9-activated Smads signaling was decreased by SP600125 treatment in MSCs. The effects of inhibitor are reproduced with adenoviruses expressing siRNA targeted JNKs. Taken together, our results revealed that JNKs was activated in BMP9-induced osteogenic differentiation of MSCs. What is most noteworthy, however, is that inhibition of JNKs activity resulted in reduction of BMP9-induced osteogenic differentiation of MSCs, implying that activation of JNKs is essential for BMP9 osteoinductive activity. [BMB Reports 2013; 46(8):422-427]     

Rice bran extract affects differentiation of mesenchymal stem cells potency into osteogenic cells

As rice bran contains various nutrients and other proteins of which a part has biological effects on animal cells, we tested the effect of rice bran extract on rat mesenchymal stem cells (rMSCs) obtained from bone marrow. These rMSCs are pluripotent and can be readily induced to differentiate into a number of cell types, including bone and cartilage. rMSC was aggregated by culturing in serum-free condition with rice bran extract, but was not aggregated by culturing in serum-free condition or in serum-containing medium. Moreover, the longer aggregates of rMSCs were cultured in serum-free condition with rice bran extract, the more the aggregates grew. After two passages in serum-free conditions, rMSCs lost their potency for differentiation into osteogenic cells; however, the addition of rice bran extract to serum-free medium successfully prevented the loss of this ability for differentiation. In addition, MSC makers CD105 and CD166 gene expression in serum-free condition with rice barn extract corresponded to these expressions in serum-containing medium. This result suggests that certain factors in rice bran could be bioactive and contribute toward retaining the ability of MSCs to differentiate into osteogenic cells after passaging.     

NT-3 promotes osteogenic differentiation of mouse bone marrow mesenchymal stem cells by regulating the Akt pathway

Objectives:To investigate the effect of neurotrophin-3 (NT-3) on osteogenic/adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).Methods:Osteogenic differentiation was detected by alkaline phosphatase (ALP) staining and alizarin red staining (ARS). Adipogenic differentiation was detected by oil red O (ORO) staining. The expression of bone-related genes (Runx2, Osterix, OCN, ALP) and lipogenic genes (FABP4, PPAR, CEBP, LPL) was detected by real-time quantitative polymerase chain reaction (real-time qPCR). The expression of p-Akt and Akt protein was detected by Western blot assay.Results:ALP staining and ARS staining showed that the overexpression of NT-3 could promote the differentiation into osteoblasts, while knockdown of NT-3 could inhibit that. Real-time qPCR showed that the overexpression of NT-3 could increase the expression of osteoblast genes, while knockdown of NT-3 could inhibit that. ORO staining showed that the overexpression of NT-3 could inhibit the differentiation into adipogenesis, while knockdown of NT-3 can promote that. Real-time qPCR showed that the overexpression of NT-3 could reduce the expression of lipogenic genes. while knockdown NT-3 could increase that. In addition, the overexpression of NT-3 increased p-Akt/Akt levels significantly, while knockdown NT-3 reduced that significantly.Conclusion:NT-3 could promote the differentiation of mouse BMSCs into osteoblasts and inhibit their differentiation into adipogenesis.     

Basement membrane collagen type IV expression by human mesenchymal stem cells during adipogenic differentiation

During adipogenic differentiation human mesenchymal stem cells (hMSC) produce collagen type IV. In immunofluorescence staining differentiating hMSCs started to express collagen type IV when Oil Red O-positive fat droplets appeared intracellularly. Quantitative real time-polymerase chain reaction confirmed progressive increase of collagen type IV α1 and α2 mRNA levels over time, 18.6- and 12.2-fold by day 28, respectively, whereas the copy numbers of α3-α6 mRNAs remained rather stable and low. Type IV collagen was in confocal laser scanning microscopy seen around adipocytes, where also laminins and nidogen were found, suggesting pericellular deposition of all key components of the fully developed basement membrane. Immunofluorescence staining of matrix metalloproteinase-2 (MMP-2, 72 kD type IV collagenase, gelatinase A) and MMP-9 (92 kD type IV collagenase, gelatinase B) disclosed only faint staining of MSCs, but MMP-9 was strongly induced during adipogenesis, whereas MSC supernatants disclosed in zymography pro-MMP-2 and faint pro-MMP-9 bands, which increased over time, with partial conversion of pro-MMP-2 to its active 62 kD form. Differentiation was associated with increasing membrane type 1-MMP/MMP-14 and tissue inhibitor of metalloproteinase-2 (TIMP-2) staining, which may enable participation of type IV collagenases in basement membrane remodelling via ternary MT1-MMP/TIMP-2/MMP-2 or -9 complexes, focalizing the fully active enzyme to the cell surface. MMP-9, which increased more in immunofluorescence staining, was perhaps preferentially bound to cell surface and/or remodelling adipocyte basement membrane. These results suggest that upon MSC-adipocyte differentiation collagen type IV synthesis and remodelling become necessary when intracellular accumulation of fat necessitates a dynamically supporting and instructive, partly denatured adipogenic pericellular type IV collagen scaffold.     

Chondrogenic differentiation and immunological properties of mesenchymal stem cells in collagen type I hydrogel

Allogeneic mesenchymal stem cells (MSCs) are regarded as promising seed cells for engineering cartilage. However, few researches have covered the immune properties of seeded MSCs. Collagen has been considered as good scaffold, whether it has inherent chondrogenic inducibility for MSCs is still in debate. In this study, engineering grafts are constructed by neonatal rabbit MSCs and collagen Type I hydrogel. After periods of culture, the appearance of chondroid tissue in the grafts and the cartilage matrix‐specific genes expressions of seeded cells prove the inducibility of collagen hydrogel, even if the growth factors are absence. With the differentiation, immunological properties of MSCs are changing. The expressions of main histocompatibility complex (MHC) molecules increase and the ability to inhibit the proliferation of activated lymphocytes may be declined. But to a large extent, it keeps the low stimulating to allogeneic lymphocytes and the small absolute value of MHCs. The changes are adverse for avoiding inflammation and rejection. Therefore, suitable scaffold and engineering strategies should be selected. For the grafts based on Collagen I hydrogel and MSCs, a longer culture period might not be necessary. To maintain the immune regulation, a higher initial MSCs density in engineering grafts may be more meaningful. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Role of human amnion-derived mesenchymal stem cells in promoting osteogenic differentiation by influencing p38 MAPK signaling in lipopolysaccharide -induced human bone marrow mesenchymal stem cells

Periodontitis is a chronic inflammatory disease induced by bacterial pathogens, which not only affect connective tissue attachments but also cause alveolar bone loss. In this study, we investigated the anti-inflammatory effects of Human amnion-derived mesenchymal stem cells (HAMSCs) on human bone marrow mesenchymal stem cells (HBMSCs) under lipopolysaccharide (LPS)-induced inflammatory conditions. Proliferation levels were measured by flow cytometry and immunofluorescence staining of 5-ethynyl-2′-deoxyuridine (EdU). Osteoblastic differentiation and mineralization were investigated using chromogenic alkaline phosphatase activity (ALP) activity substrate assays, Alizarin red S staining, and RT-PCR analysis of HBMSCs osteogenic marker expression. Oxidative stress induced by LPS was investigated by assaying reactive oxygen species (ROS) level and superoxide dismutase (SOD) activity. Here, we demonstrated that HAMSCs increased the proliferation, osteoblastic differentiation, and SOD activity of LPS-induced HBMSCs, and down-regulated the ROS level. Moreover, our results suggested that the activation of p38 MAPK signal transduction pathway is essential for reversing the LPS-induced bone-destructive processes. SB203580, a selective inhibitor of p38 MAPK signaling, significantly suppressed the anti-inflammatory effects in HAMSCs. In conclusion, HAMSCs show a strong potential in treating inflammation-induced bone loss by influencing p38 MAPK signaling.     

Donepezil hydrochloride as a novel inducer for osteogenic differentiation of mesenchymal stem cells on PLLA scaffolds in vitro

Over the past decades, bone defects caused by illness or trauma have been the most common traumatic injuries in humans and treatment of orthopedic infections has always been a serious challenge to experts in the world. In this project, poly L-lactic acid (PLLA) nanofibrous scaffolds were synthesized as a nontoxic, eco-friendly, and cost-effective scaffold by the electrospinning technique. Then, the impact of PLLA on the cell proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs) was assayed in the presence and absence of donepezil hydrochloride (DH) which was prescribed in patients with Alzheimer's disease. Also, hMSCs were seeded on PLLA scaffold in the presence (PLLA-DH) and absence of 1 μg mL^-1 of DH under osteogenic induction media. Osteogenic differentiation of hMSCs was assessed by specific bone-related tests including alkaline phosphatase (ALP) activity, Alizarin red and von Kossa staining, calcium content assay. Also, Osteocalcin and osteopontin were evaluated as osteogenic proteins as well as ALP, osteonectin, osteocalcin, collagen type I (Col-I) and Runx2 as osteogenic genes via immunocytochemistry (ICC) and Real-time PCR analyses. The obtained data showed the higher ALP enzyme activity and biomineralization, more intensity during von Kossa staining as well as the increase in the expression rate of osteogenic related gene and protein markers in differentiated hMSCs on PLLA-DH. In conclusion, the present study revealed that the combination of PLLA scaffold with DH provides a scope to develop a suitable matrix in bone tissue engineering applications.     

Osteogenic potential of rat mesenchymal stem cells after several passages

Osteogenic potential of serially passaged rat bone marrow derived mesenchymal stem cells (BMCs) was evaluated for clinical feasibility. Osteogenic differentiation in vitro was evaluated by means of the concentration and mRNA expression of alkaline phosphatase and osteocalcin. For in vivo osteogenesis, BMCs in various degrees of differentiation were implanted into the athymic mice. Although elevated levels of osteogenic markers were prominent in the less passaged BMCs continuously cultured with osteogenic supplements (OS group), they decreased with passaging. Similar to the in vitro experiments, abundant bone and cartilage formations inside the membrane were observed in the P0 through P2 cells of the OS group. In the P3 cells, however, the chambers were filled with fibrous tissues showing the failure of osteogenesis. Establishment of the culture conditions that permit the rapid expansion of BMCs while retaining their potential for differentiation will be required for future clinical applications.     

Growth factors and chondrogenic differentiation of mesenchymal stem cells

The main purpose of the article is to review recent knowledge about growth factors and their effect on the chondrogenic differentiation of mesenchymal stem cells under in vitro conditions. Damaged or lost articular cartilage leads to progressive debilitation, which have major impact on the life quality of the affected individuals of both sexes in all age groups. Mature hyaline cartilage has a very low self-repair potential due to intrinsic properties - lack of innervation and vascular supply. Another limiting factor is low mitotic potential of chondrocytes. Small defects are healed by migration of chondrocytes, while large ones are healed by formation of inferior fibrocartilage. However, in many cases osteoarthritis develops. Recently, cellular therapy combining mesenchymal stem cells and proper differentiation factors seems to be promising tool for hyaline cartilage defects healing.     

Changes in cell migration of mesenchymal cells during osteogenic differentiation

We showed that the migration, morphology and adhesiveness of undifferentiated mesenchymal cells dramatically changed during osteogenic differentiation. The migration of these cells was transiently upregulated early in osteogenic differentiation. At a later stage, migration was decreased but adhesiveness was increased. Furthermore, Cdc42 and Rac1 Rho-family small GTPases were activated at early stages of differentiation and the phosphorylation level of FAK decreased as differentiation progressed. We also showed cell migration was promoted by inhibition of the Rho-ROCK-myosin signaling. Finally, using a mouse model of ectopic bone formation, we confirmed that treatment with ROCK inhibitor, Y-27632 increased cell movement into bone formation sites, resulting in enhanced osteogenesis. These results provide a new insight into the link between cell migration and osteogenic differentiation.     

Primary bone-derived cells induce osteogenic differentiation without exogenous factors in human embryonic stem cells

We developed a new and efficient method for osteoblastic differentiation of human embryonic stem cells (hESCs) using primary bone-derived cells (PBDs). Three days after embryoid body (hEB) formation, cells were allowed to adhere to culture surface where PBDs were pre-plated and mitomycin C-treated in DMEM/F12 medium supplemented with 5% knockout serum replacement. As early as 14 days, mineralization and formation of nodule-like structures in cocultured hEBs were prominent by von Kossa and Alizarin S staining, and expressions of osteoblast-specific markers including bone sialoprotein, alkaline phosphates, osteocalcin, collagen 1, and core binding factor alpha1 by RT-PCR. In addition, FACS analysis revealed that over 19% of the differentiated cells expressed osteocalcin. These results suggest that PBDs not only have osteogenic effects releasing osteogenic factors as bone morphogenic protein (BMP) 2 and BMP 4 but also have exerted other effects, whether chemical or physical, for the differentiation of hESCs.