Extracellular matrix protein mediated regulation of the osteoblast differentiation of bone marrow derived human mesenchymal stem cells

The biomimetic approach of tissue engineering exploits the favorable properties of the extracellular matrix (ECM), to achieve better scaffold performance and tissue regeneration. ECM proteins regulate cell adhesion and differentiation through integrin mediated signal transduction. In the present study, we have examined the role of ECM proteins such as collagen type I, fibronectin, laminin and vitronectin in regulating the proliferation and osteogenic differentiation of bone marrow derived human mesenchymal stem cells (hMSCs). hMSCs were grown on selected ECM protein treated tissue culture plates. The growth kinetics was assessed by calculating the doubling time of the cells on different ECM treated plates. The cells were directed to osteoblast lineage by growing them in osteogenic induction media for 21 day. Differentiation was evaluated at different time points by osteoblast differentiation associated gene expression, alkaline phosphatase (ALP) activity, histochemical staining for mineralized matrix and calcium quantification. The doubling time of hMSCs cultured on collagen type I was significantly low, which was followed by laminin and fibronectin treated plates. However, doubling time of hMSCs cultured on vitronectin treated plate was not significantly different than that of the untreated control. High ALP gene (ALPL) expression and associated enhancement of mineralization were observed on collagen type I, fibronectin and vitronectin treated plates. Collagen type I showed early onset of mineralization with high ALP activity and up-regulation of osteopontin, ALPL, bone sialoprotein and osteocalcin genes. Vitronectin also up-regulated these genes and showed the highest amount of calcium in the secreted mineral matrix. Therefore, we conclude that, ECM proteins indeed modified the growth patterns and induced the osteoblast differentiation of hMSCs. Our findings have significant implication for bone tissue engineering applications.     

Perfusion affects the tissue developmental patterns of human mesenchymal stem cells in 3D scaffolds

Human mesenchymal stem cells (hMSCs) developed in three‐dimensional (3D) scaffolds are significantly affected by culture conditions. We hypothesized that the hydrodynamic forces generated in perfusion bioreactors significantly affected hMSC functionality in 3D scaffolds by shaping the extracellular matrix (ECM) proteins. In this study, hMSCs were grown in 3D poly(ethylene terephthalate) (PET) scaffolds in static and a parallel perfusion system under similar initial conditions for up to 35 days. Results demonstrated that even at very low media velocities (O [10^?4 cm/sec]), perfusion cultures affected the ability of hMSCs to form an organized ECM network as illustrated by the immunostaining of collagen I and laminin fibrous structure. The change in the ECM microenvironment consequently influenced the nuclear shape. The hMSCs grown at the lower surface of static culture displayed a 15.2 times higher nuclear elongation than those at the upper surface, whereas cells grown in the perfusion bioreactor displayed uniform spherical nuclei on both surfaces. The difference in ECM organization and nuclear morphology associated with gene expression and differentiation characteristics of hMSCs. The cells exhibited lower CFU‐F colony forming ability and decreased expressions of stem‐cell genes of Rex‐1 and Oct‐4, implying a less primitive stem‐cell phenotype was maintained in the perfusion culture relative to the static culture conditions. The significantly higher expression level of osteonectin gene in the perfusion culture at day 28 indicated an upregulation of osteogenic ability of hMSCs. The study highlights the critical role of dynamic culture conditions on 3D hMSC construct development and properties. J. Cell. Physiol. 219: 421–429, 2009. © 2009 Wiley‐Liss, Inc.     

Laminin regulates the osteogenic differentiation of dental follicle cells via integrin-α2/-β1 and the activation of the FAK/ERK signaling pathway

Dental follicle cells (DFCs) are ideal for studies concerning the differentiation of dental precursor cells into alveolar osteoblasts and cementoblasts. Previous investigations have suggested that the extracellular matrix (ECM) protein laminin and the ECM receptor integrin-α2/-β1 play regulatory roles during the osteogenic differentiation of DFCs. Our present data indicate that laminin impairs alkaline phosphatase (ALP) activity following osteogenic induction while inducing integrin-α2/-β1 expression, osteogenic differentiation marker elevation, and DFC biomineralization. Integrin-α2/-β1 facilitates the laminin-dependent expression of osteogenic differentiation markers and the laminin-dependent inhibition of ALP activity. Moreover, these laminin-dependent effects on the osteogenic differentiation of DFCs can be reversed by the inhibition of the FAK/ERK signaling pathway. Thus, laminin regulates the inhibition of early osteogenic differentiation markers and the induction of late osteogenic differentiation markers via integrin-α2/-β1 and the activation of the FAK/ERK signaling pathway.     

Autocrine fibroblast growth factor 2‐mediated interactions between human mesenchymal stem cells and the extracellular matrix under varying oxygen tension

Human mesenchymal stromal or stem cells (hMSCs) are being investigated for cell therapy in a wide range of diseases. MSCs are a potent source of trophic factors and actively remodel their immediate microenvironment through the secretion of bioactive factors in response to external stimuli such as oxygen tension. In this study, we examined the hypothesis that hypoxia influences hMSC properties in part through the regulation of extracellular milieu characterized by the extracellular matrix (ECM) matrices and the associated fibroblast growth factor‐2 (FGF‐2). The decellularized ECM matrices derived from hMSC culture under both hypoxic (e.g., 2% O₂) and the standard culture (e.g., 20% O₂) conditions have different binding capacities to the cell‐secreted and exogenenous FGF‐2. The reduced hMSC proliferation in the presence of FGF‐2 inhibitor and the differential capacity of the decellularized ECM matrices in regulating hMSC osteogeneic and adipogenic differentiation suggest an important role of the endogenous FGF‐2 in sustaining hMSC proliferation and regulating hMSC fate. Additionally, the combination of the ECM adhesion and hypoxic culture preserved hMSC viability under serum withdrawal. Together, the results suggest the synergistic effect of hypoxia and the ECM matrices in sustaining hMSC ex vivo expansion and preserving their multi‐potentiality and viability under nutrient depletion. The results have important implication in optimizing hMSC expansion and delivery strategies to obtain hMSCs in sufficient quantity with required potency and to enhance survival and function upon transplantation. J. Cell. Biochem. 114: 716–727, 2013. © 2012 Wiley Periodicals, Inc.     

Development of decellularized amniotic membrane as a bioscaffold for bone marrow-derived mesenchymal stem cells: ultrastructural study

Developing effective stem cell-based therapies requires the design of complex in vitro culture systems for accurate representation of the physiological stem cell niche. Human amniotic membrane (hAM) has been successfully used in clinical grafting applications due to its unique biological and regenerative properties. Decellularized hAM (d-hAM) has been previously applied to the culture of human bone marrow mesenchymal stem cells (hMSCs), promoting their expansion and differentiation into adipogenic and osteogenic lineages. In the present study, hAM was decellularized by NaOH-treatment, to provide the three-dimensional (3D) bioscaffold for culturing hMSCs. The ultrastructural differences between intact hAM and decellularized hAM were characterized using the transmission electron microscope (TEM), as well as the 3D interaction between d-hAM and hMSCs cultured on the membrane. TEM examination of the intact hAM showed many microvilli on the epithelial layer cells, active Golgi apparatus, smooth endolplasmic reticulum and the characteristic pinocytic vesicles. The epithelial layer with its structures was absent in the d-hAM. However, no observable difference was detected in the ultrastructural characteristics of the compact stromal layer of d-hAM compared to intact hAM. Both contained bundles of extra cellular matrix (ECM) proteins, and scattered elastic fibres. Cultured human mesenchymal stem cells (hMSCs) examined by TEM appeared oval to spherical in shape and had a rough and non-uniform surface with distinct protrusions or irregular fillopodia. Their diameter ranged from 20.49 to 21.6 µm. Most of the cellular organelles were also noticed. SEM examination of the prepared samples revealed unique 3D interaction between the hMSC and d-hAM, where the latter seems to envelop the segments of the hMSCs lying on the surrounding membrane. This study shows that the decellularization process affected the epithelial layer only of hAM and had no effect on altering the presence of ECM components present in the stromal layer of the d-hAM. The interaction between hMSCs and d-hAM maybe mediated by hAM components other than human amniotic epithelial cells, such as ECM components or MSCs present in the deeper spongy layer of the membrane or/and the adhesive components of the basement membrane of the removed epithelial layer.     

趋化因子受体CX3CR1调控人主动脉瓣膜间质细胞成骨分化的作用研究

目的:探究趋化因子受体CX3CR1调控人主动脉瓣膜间质细胞成骨分化的作用和机制,为钙化性主动脉瓣膜疾病的早期干预和治疗提供新思路。方法:取非钙化主动脉瓣(3例)和钙化主动脉瓣(5例),免疫组织化学染色检测成骨相关转录因子Runx2、骨桥蛋白OPN和骨钙蛋白OCN的表达;取3例非钙化的主动脉瓣,采用胶原酶连续消化法分离人主动脉瓣膜间质细胞,观察细胞形态及生长状态,并采用细胞免疫荧光进行表型鉴定。对成骨诱导培养的人主动脉瓣膜间质细胞分别过表达和干扰趋化因子受体CX3CR1,平行设置CM组、OM组和negative control+OM组,采用qPCR和Western blot检测Runx2、OPN和OCN的表达,Western blot检测AKT和p-AKT的表达。茜素红S染色评价晚期钙结节形成情况。结果:临床标本显示钙化的主动脉瓣较非钙化的主动脉瓣高表达CX3CR1(P 0. 05);成功分离人主动脉瓣膜间质细胞,α-SMA和Vimentin阳性,vWF阴性。与CM、OM、negative control组比较,CX3CR1+OM组Runx2、OPN和p-AKT表达上调(P 0. 05),且茜素红S染色可见明显钙结节;与CM、OM、negative siRNA control+OM组比较,si CX3CR1+OM组Runx2、OPN和p-AKT表达下调(P 0. 05),且茜素红S染色可见钙结节减少。结论:趋化因子受体CX3CR1可能通过AKT信号通路促进人主动脉瓣膜间质细胞成骨分化。     

FGF-2 increases osteogenic and chondrogenic differentiation potentials of human mesenchymal stem cells by inactivation of TGF-β signaling

Human mesenchymal stem cells (hMSCs) are able to self-replicate and differentiate into a variety of cell types including osteoblasts, chondrocytes, adipocytes, endothelial cells, and muscle cells. It was reported that fibroblast growth factor-2 (FGF-2) increased the growth rate and multidifferentiation potentials of hMSCs. In this study, we investigated the genes involved in the promotion of osteogenic and chondrogenic differentiation potentials of hMSCs in the presence of FGF-2. hMSCs were maintained in the medium with FGF-2. hMSCs were harvested for the study of osteogenic or chondrogenic differentiation potential after 15 days’ culture. To investigate osteogenic differentiation, the protein levels of alkaline phosphatase (ALP) and the mRNA expression levels of osteocalcin were measured after the induction of osteogenic differentiation. Moreover, the investigation for chondrogenic differentiation was performed by measuring the mRNA expression levels of type II and type X collagens after the induction of chondrogenic differentiation. The expression levels of ALP, type II collagen, and type X collagen of hMSCs cultured with FGF-2 were significantly higher than control. These results suggested that FGF-2 increased osteogenic and chondrogenic differentiation potentials of hMSCs. Furthermore, microarray analysis was performed after 15 days’ culture in the medium with FGF-2. We found that the overall insulin-like growth factor-I (IGF-I) and transforming growth factor-β (TGF-β) signaling pathways were inactivated by FGF-2. These results suggested that the inactivation of IGF-I and TGF-β signaling promotes osteogenic and chondrogenic differentiation potential of hMSCs in the presence of FGF-2.     

Sortilin is upregulated during osteoblastic differentiation of mesenchymal stem cells and promotes extracellular matrix mineralization

Osteoblasts and adipocytes are derived from a common precursor in bone marrow, the mesenchymal stem cell (MSC). Factors driving human MSCs (hMSCs) to differentiate down the two lineages play important roles in determining bone density because it has been shown that bone volume loss associated with osteoporosis and aging is accompanied by reduced osteoblastic bone formation and increased marrow adipose tissue. The genes upregulated in hMSCs during osteogenic differentiation were screened using cDNA microarrays and were semi-quantitated by real-time RT-PCR. One of the genes identified was sortilin, which was upregulated one day after osteogenic induction and remained upregulated for a week. The overexpression of sortilin in hMSCs using an adenovirus vector resulted in the acceleration of mineralization during osteogenic differentiation without affecting alkaline phosphatase activity. Lipoprotein lipase (LPL), produced by adipocytes, is bound by sortilin, which may mediate its endocytosis. By adding LPL to osteogenic induction medium, osteoblastic mineralization was inhibited in a dose-dependent manner. Interestingly, sortilin overexpression abolished the LPL-mediated suppression of osteogenic differentiation. hMSCs exist in marrow where LPL-producing adipose cells are abundant and where osteogenesis is negatively regulated by LPL. Sortilin has a counter effect of promoting osteogenesis by acting as a scavenger of LPL.     

Chitosan enhances mineralization during osteoblast differentiation of human bone marrow-derived mesenchymal stem cells, by upregulating the associated genes

Objectives: Chitosan is widely used as a scaffold for bone tissue engineering. However, up‐to‐date, no previous detailed study has been conducted to elucidate any mechanism of osteogenesis by chitosan itself. Here, we have evaluated effects of chitosan‐coated tissue culture plates on adhesion and osteoblast differentiation processes of human mesenchymal stem cells (hMSCs), isolated from adult bone marrow. Materials and methods: Tissue culture plates coated with chitosan at different coating densities were used to evaluate the effects on hMSC adhesion and osteoblast differentiation. hMSCs were induced to differentiate into osteoblasts on the chitosan‐coated plates and were evaluated using established techniques: alkaline phosphatase assay, demonstration of presence of calcium and real time PCR. Results: The cells adhered to plates of lower coating density of chitosan, but formed viable cell aggregates at higher coating density (100 μg/sq.cm). Coating density of 25 μg/sq.cm, supporting cell adhesion was chosen for osteoblast differentiation experiments. Differentiating hMSCs showed higher mineral deposition and calcium content on chitosan‐coated plates. Chitosan upregulated genes associated with calcium binding and mineralization such as collagen type 1 alpha 1, integrin‐binding sialoprotein, osteopontin, osteonectin and osteocalcin, significantly. Conclusions: We demonstrate for the first time that chitosan enhanced mineralization by upregulating the associated genes. Thus, the study may help clinical situations promoting use of chitosan in bone mineralization, necessary for healing non‐union fractures and more.     

Mineralization by mesenchymal stromal cells is variously modulated depending on commercial platelet lysate preparations

Background aims

Numerous cellular models have been developed to investigate calcification for regenerative medicine applications and for the identification of therapeutic targets in various complications associated with age-related diseases. However, results have often been contradictory due to specific culture conditions, cell type ontogeny and aging status. Human platelet lysate (hPL) has been recently investigated as valuable alternative to fetal bovine serum (FBS) in cell culture and bone regeneration. A parallel comparison of how all these multiple factors may converge to influence mineralization has yet to be reported.

Mineralized synthetic matrices as an instructive microenvironment for osteogenic differentiation of human mesenchymal stem cells

The effect of substrate-mediated signals on osteogenic differentiation of hMSCs is studied using a synthetic bone-like material comprising both organic and inorganic components that supports adhesion, spreading, and proliferation of hMSCs. hMSCs undergo osteogenic differentiation even in the absence of osteogenesis-inducing supplements. They exhibit higher expressions of Runx2, BSP, and OCN compared to their matrix-rigidity-matched, non-mineralized hydrogel counterparts. The mineralized-hydrogel-assisted osteogenic differentiation of hMSCs could be attributed to their exposure to high local concentrations of calcium and phosphate ions in conjunction with chemical and topological cues arising from the hydrogel-bound calcium phosphate mineral layer.     

FGF-2对人骨髓间充质干细胞增殖和向成骨细胞分化的影响

探讨体外培养条件下,成纤维细胞生长因子-2（FGF-2）和地塞米松（Dex）对第7代人骨髓间充质干细胞（MSCs）增殖和向成骨细胞分化的作用以及两者联合使用的效应。MSCs经含FGF-2或／和Dex的培养液作用后,于不同时间采用MTT法测定细胞增殖情况;对硝基苯磷酸（pNPP）法测定碱性磷酸酶（ALP）活性;ELISA法测定骨钙蛋白（OC）含量;茜素红S染色法对沉积的钙盐进行染色。发现：（1）FGF-2组细胞的生长速度为对照组的1．31倍,Dex／FGF-2组细胞的生长速度为FGF-2组的1．12倍。（2）Dex组的ALP活性、OC含量和细胞外基质钙盐沉积分别为对照组的17．0倍、2．12倍和10．56倍,并能形成成熟的羟基磷灰石（HA）结晶和骨结节;FGF-2组的ALP活性比对照组降低了76．7％,虽然OC含量、钙盐沉积增加,但不能形成成熟的HA结晶和骨结节;FGF-2对Dex诱导的ALP活性增加和HA结晶形成有拮抗作用。由此证明：（1）FGF-2可促进MSCs的增殖,Dex对MSCs的增殖无明显作用;Dex能增强FGF-2对MSCs的促增殖效应。（2）Dex可使MSCs分化为成熟的成骨细胞,是一个有效的成骨细胞分化诱导剂;FGF-2可使MSCs分化为未成熟的成骨细胞;FGF-2拮抗Dex诱导MSCs分化为成熟的成骨细胞。     

Osteostimulation scaffolds of stem cells: BMP‐7‐derived peptide‐decorated alginate porous scaffolds promote the aggregation and osteo‐differentiation of human mesenchymal stem cells

The scaffolds for stem cell‐based bone tissue engineering should hold the ability to guide stem cells osteo‐differentiating. Otherwise, stem cells will differentiate into unwanted cell types or will form tumors in vivo. Alginate, a natural polysaccharide with great biocompatibility, was widely used in biomedical applications. However, the limited bioactivity and poor osteogenesis capability of pristine alginate hampered its further application in tissue engineering. In this work, a bone forming peptide‐1 (BFP‐1), derived from bone morphogenetic protein‐7, was grafted to alginate polymer chains to prepare peptide‐decorated alginate porous scaffolds (pep‐APS) for promoting osteo‐differentiation of human mesenchymal stem cells (hMSCs). SEM images of pep‐APS exhibited porous structure with about 90% porosity (pore size 100–300 μm), which was appropriate for hMSCs ingrowth. The adhesion, proliferation and aggregation of hMSCs grown on pep‐APS were enhanced in vitro. Moreover, pep‐APS promoted the alkaline phosphatase (ALP) activity of hMSCs, and the osteo‐related genes expression was obviously up‐regulated. The immunochemical staining and western blot analysis results showed high expression level of OCN and Col1a1 in the hMSCs grown on pep‐APS. This work provided a facile and valid strategy to endow the alginate polymers themselves with specific bioactivity and prepare osteopromoting scaffold with enhanced osteogenesis ability, possessing potential applications in stem cell therapy and regenerative medicine.