Effects of the Surface Characteristics of Nano-Crystalline and Micro-Particle Calcium Phosphate/Chitosan Composite Films on the Behavior of Human Mesenchymal Stem Cells In Vitro

Calcium phosphate (CaP) compounds, the main inorganic constituent of mammalian bone tissues, are believed to support bone precursor cell growth and osteogenic differentiation. Chitosan, a deacetylated derivative of chitin, is a versatile biopolymer to offer broad possibilities for cell-based tissue engineering. In the present study, different scales of CaP crystals on chitosan membranes were prepared for culture of human mesenchymal stem cells (hMSCs) in vitro. A series of aqueous CaP suspensions with different concentrations were mixed with chitosan solution and chitosan/calcium phosphate (C/CaP) films were fabricated by the solvent-casting method. With different weight ratios of CaP in chitosan solution, the various surface characteristics of nano-amorphous (C/CaP 0.1), nano-crystalline (C/CaP 0.5) and micro-particle (C/CaP 2) CaP compounds were examined by scanning electron microscopy and electron dispersion spectroscopy. X-ray diffraction on micro-particles of CaP indicated the formation of crystalline hydroxyapatite. The behavior of hMSCs, including proliferation, cell spreading and osteogenic differentiation, was studied on the C/CaP films. In basal culture medium, the incorporation of CaP into chitosan films could promote the proliferation of hMSCs. The C/CaP 0.5 film with connected CaP nano-crystals had better cellular viability. The fluorescence microscope images at 14 days of culture revealed extensive networks of F-actin filaments of hMSCs on chitosan, C/CaP 0.1 and C/CaP 0.5 films. The cellular morphology on C/CaP 2 film with discrete CaP micro-particles was partly restrained. In osteogenic medium, the alkaline phosphatase (ALP) activity of hMSCs increased and showed the process of osteogenic differentiation. The ALP levels on C/CaP 2 film were higher than those on C/CaP 0.1 and C/CaP 0.5 films. These results demonstrated that the crystallinity and topography of CaP on chitosan membranes could modulate the behaviors of cultured hMSCs in vitro.     

Overexpression of GDF5 through an adenovirus vector stimulates osteogenesis of human mesenchymal stem cells in vitro and in vivo

The use of stem cells combined with gene therapy could be an important way to facilitate bone regeneration. In this study, the aim was to investigate the potential of growth and differentiation factor-5 (GDF5) to genetically manipulate human mesenchymal stem cells (hMSCs) for bone regeneration. Recombinant adenovirus Ad-GDF5 and Ad-GFP were constructed and identified, and the titer of both were determined. Third-passage hMSCs were infected with adenovirus, and the expression of GDF5 was confirmed by detection of GFP-positive cells, GDF5 mRNA levels, Western blotting, and enzyme-linked immunosorbent assay (ELISA). hMSCs at passage 3 were divided into four groups: (1) an experimental group infected with Ad-GDF5, (2) a positive control group cultured with osteogenic differentiation medium, (3) a control group infected with Ad-GFP cultured with standard medium, and (4) a blank control group cultured with standard medium. Evaluation of cell morphology and proliferation, analysis of the expression of genes related to osteogenic differentiation, von Kossa staining, and immunofluorescent staining of collagen I were used to investigate the osteogenesis of cells among the groups. After culturing the cells for 2 days under each corresponding condition, the cells were detached and subcutaneously injected into the backs of nude mice to evaluate bone formation. Samples were collected for histological staining, protein Western blotting, and micro-computer tomography. When infected with Ad-GDF5, hMSCs could overexpress GDF5 for a prolonged period in vitro and reach a concentration of 160 ng/ml. Cells infected with Ad-GDF5 or cultured in osteogenic medium displayed osteogenic differentiation based on their histological and cellular properties and on their gene and protein expression patterns. Furthermore, Ad-GDF5 showed a better ability to upregulate the expression of collagen I, alkaline phosphatase, and osteocalcin mRNA than the osteogenic medium. Furthermore, Ad-GDF5 expression was associated with enhanced bone formation in vivo. Our findings suggest that hMSCs infected with Ad-GDF5 can differentiate in an osteogenic direction and may be a promising cell source for bone regeneration.     

Silk ionomers for encapsulation and differentiation of human MSCs

The response of human bone marrow derived human mesenchymal stem cells (hMSCs) encapsulated in silk ionomer hydrogels was studied. Silk aqueous solutions with silk-poly-l-lysine or silk-poly-l-glutamate were formed into hydrogels via ultrasonication in situ with different net charges. hMSCs were encapsulated within the hydrogels and the impact of matrix charge was assessed over weeks in osteogenic, adipogenic and maintenance growth media. These modified silk charged polymers supported cell viability and proliferative potential, and the hMSCs were able to differentiate toward osteogenic or adipogenic lineages in the corresponding differentiation media. The silk/silk-poly-l-lysine hydrogels exhibited a positive effect on selective osteogenesis of hMSCs, inducing differentiation toward an osteogenic lineage even in the absence of osteogenic supplements, while also inhibiting adipogenesis. In contrast, silk/silk fibroin-poly-l-glutamate hydrogels supported both osteogenic and adipogenic differentiation of hMSCs when cultured under induction conditions. The results demonstrate the potential utility of silk-based ionomers in gel formats for hMSCs encapsulation and for directing hMSCs long term functional differentiation toward specific lineages.     

Core-shell PLGA/collagen nanofibers loaded with recombinant FN/CDHs as bone tissue engineering scaffolds

Poly(lactic-co-glycolic acid) (PLGA)/collagen nanofibrous scaffolds have been utilized in the tissue engineering field. It has been shown that both fibronectin (FN) and cadherin 11 (CDH) play important roles in the progress of osteogenesis and cell adhesion. The aim of this study was to fabricate recombinant FN/CDHs (rFN/CDHs)-loaded PLGA/collagen nanofibrous scaffolds and evaluate their effects on the adhesion and differentiation of human bone marrow mesenchymal stem cells (hMSCs). PLGA/collagen nanofibers were made by coaxial electrospinning. The morphology and mechanical properties of PLGA/collagen nanofibrous mats were analyzed by scanning electron microscopy and mechanical testing, respectively. The performance of scaffolds was evaluated in terms of the viability, morphology, and osteogenic gene expression levels of hMSCs. rFN/CDHs was successfully incorporated into the PLGA/collagen nanofibers. The release of rFN/CDHs from PLGA nanofibers was investigated by liquid chromatography–mass spectrometry. rFN/CDHs improved the mechanical properties of the PLGA/collagen nanofibers. The controlled release of rFN/CDHs can enhance the proliferation of hMSCs and induce osteogenic gene expression (alkaline phosphatase, RUNX2, and osteocalcin). Our data imply that rFN/CDHs may induce hMSCs differentiation into osteoblasts and PLGA/collagen nanofibers loaded with rFN/CDHs have potential in bone tissue engineering.     

人骨髓间充质干细胞的生物学特性及向神经前体细胞分化的实验研究

目的 观察体外培养的人骨髓间充质干细胞(hMSCs)的生物学特性,并探讨使其转分化为神经前体细胞(NPCs)的方法.方法以密度梯度离心和贴壁法相结合分离成人骨髓间充质干细胞,并观察细胞形态、生长、表面标记以及成骨和成软骨及成脂肪能力的情况.选用第3代细胞进行诱导,先经胚胎干细胞培养液扩增,再用加有5-氮胞苷和曲古菌素A的神经诱导液诱导,7d后,一部分样本进行Nestin、Sox2免疫荧光染色和RT-PCR检测;另一部分样本在含有B27的神经培养液中继续培养7d,然后进行NF-L的免疫荧光检测.结果分离培养的hMSCs纯度较高,CD29、CD44的阳性率均在90%以上;具有明显的成骨、成软骨和成脂肪能力;经5-氮杂胞苷和曲古菌素A作用后能向神经前体细胞分化,免疫荧光染色及RT-PCR结果显示,诱导后的细胞能特异性表达神经前体细胞标志物Nestin和Sox2;在神经培养液中继续培养后检测神经细胞标记物NF-L,可见较多阳性细胞.结论 hMSCs可在体外进行分离培养扩增,经药物修饰后具有向神经前体细胞分化的潜能.     

Response of Human Mesenchymal Stem Cells (hMSCs) to the Topographic Variation of Poly(3-Hydroxybutyrate-co-3-Hydroxyhexanoate) (PHBHHx) Films

The influence of the topographic morphology of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) films on human mesenchymal stem cells (hMSCs) was investigated in this study. PHBHHx films with various surface characteristics were prepared by compression-molding, solvent-casting and electrospinning. The adhesion, proliferation and differentiation behaviors of hMSCs were significantly modulated by the surface characteristics of these films. HMSCs could aggregate and form cellular clusters on the cast PHBHHx films, and the time to form cellular aggregates increased as the surface roughness increased. The aggregated hMSCs on the cast films kept their original surface markers and presented much higher viability during the regular culture and lower differentiation ability upon osteogenic induction than the spread cells on the compression-molded films and TCPS. HMSCs spread well and showed a specific orientation on the surface of the random electrospun fibrous films, they were not able to migrate into the interior of electrospun fibrous films, and they revealed the highest viability during the regular culture but a lower differentiation activity upon osteogenic induction. The electrospun fibrous PHBHHx films could serve as a suitable substrate for large quantity culturing of hMSCs when undifferentiated hMSCs are desired.     

Bioactive membranes for bone regeneration applications: Effect of physical and biomolecular signals on mesenchymal stem cell behavior

This study focuses on the in vitro characterization of bioactive elastin-like recombinamer (ELR) membranes for bone regeneration applications. Four bioactive ELRs exhibiting epitopes designed to promote mesenchymal stem cell adhesion (RGDS), endothelial cell adhesion (REDV), mineralization (HAP), and both cell adhesion and mineralization (HAP-RGDS) were synthesized using standard recombinant protein techniques. The materials were then used to fabricate ELR membranes incorporating a variety of topographical micropatterns including channels, holes and posts. Primary rat mesenchymal stem cells (rMSCs) were cultured on the different membranes and the effects of biomolecular and physical signals on cell adhesion, morphology, proliferation, and differentiation were evaluated. All results were analyzed using a custom-made MATLAB program for high throughput image analysis. Effects on cell morphology were mostly dependent on surface topography, while cell proliferation and cell differentiation were largely dependent on the biomolecular signaling from the ELR membranes. In particular, osteogenic differentiation (evaluated by staining for the osteoblastic marker osterix) was significantly enhanced on cells cultured on HAP membranes. Remarkably, cells growing on membranes containing the HAP sequence in non-osteogenic differentiation media exhibited significant up-regulation of the osteogenic marker as early as day 5, while those growing on fibronectin-coated glass in osteogenic differentiation media did not. These results are part of our ongoing effort to develop an optimized molecularly designed periosteal graft.     

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.     

Regulation of the fate of human mesenchymal stem cells by mechanical and stereo-topographical cues provided by silicon nanowires

Extracellular stimuli imposed on stem cells enable efficient initiation of mechanotransductive signaling to regulate stem cell fates; however, how such physical cues conferred by the stereo-topographical matrix govern the fate of stem cells still remains unknown. The purpose of this study is to delineate the effects of stereotopography and its various relevant physical properties on the fate regulation of human mesenchymal stem cells (hMSCs). Stereo-topographical silicon nanowires (SiNWs) that were precisely controlled with respect to their various dimensions and their growth orientation were used in this study. hMSCs cultured on stereo SiNWs of different lengths in the absence of biochemical osteogenic induction cues displayed a spherical and less-elongated morphology and showed an approximately 10% loss of cell viability compared to those grown on two-dimensional (2-D) flat Si. Moreover, osteogenic gene expression of COL1A1 and Runx2 in hMSCs cultured on the shortest SiNWs was significantly higher than those grown on the longer SiNWs and 2-D flat Si. hMSCs grown on shorter SiNWs also demonstrated higher expression levels for F-actin, phosphorylated focal adhesion kinase (pFAK), vinculin and alpha 2 integrin. Stereo-topographical cues provided by SiNWs are able to regulate osteogenic differentiation of hMSCs via cytoskeleton remodeling and this is correlated with the differential expression of alpha 2/beta 1 integrin heterodimers and the focal adhesion molecules pFAK and vinculin. The findings in this study provide insights in terms of the design of stereo-topographical structures for use in tissue engineering, bone regeneration and relevant medical applications.     

Pro-osteogenic trophic effects by PKA activation in human mesenchymal stromal cells

Human mesenchymal stromal cells (hMSCs) are able to differentiate into a wide variety of cell types, which makes them an interesting source for tissue engineering applications. On the other hand, these cells also secrete a broad panel of growth factors and cytokines that can exert trophic effects on surrounding tissues. In bone tissue engineering applications, the general assumption is that direct differentiation of hMSCs into osteoblasts accounts for newly observed bone formation in vivo. However, the secretion of bone-specific growth factors, but also pro-angiogenic factors, could also contribute to this process. We recently demonstrated that secretion of bone specific growth factors can be enhanced by treatment of hMSCs with the small molecule db-cAMP (cAMP) and here we investigate the biological activity of these secreted factors. We demonstrate that conditioned medium contains a variety of secreted growth factors, with differences between medium from basic-treated and cAMP-treated hMSCs. We show that conditioned medium from cAMP-treated hMSCs increases proliferation of various cell types and also induces osteogenic differentiation, whereas it has differential effects on migration. Microarray analysis on hMSCs exposed to conditioned medium confirmed upregulation of pathways involved in proliferation as well as osteogenic differentiation. Our data suggests that trophic factors secreted by hMSCs can be tuned for specific applications and that a good balance between differentiation on the one hand and secretion of bone trophic factors on the other, could potentially enhance bone formation for bone tissue engineering applications.     

Study of bilineage differentiation of human-bone-marrow-derived mesenchymal stem cells in oxidized sodium alginate/N-succinyl chitosan hydrogels and synergistic effects of RGD modification and low-intensity pulsed ultrasound

The level of formation of new bone and vascularization in bone tissue engineering scaffold implants is considered as a critical factor for clinical application. In this study, an approach using an RGD-grafted oxidized sodium alginate/N-succinyl chitosan (RGD–OSA/NSC) hydrogel as a scaffold and low-intensity pulsed ultrasound (LIPUS) as mechanical stimulation was proposed to achieve a high level of formation of new bone and vascularization. An in vitro study of endothelial and osteogenic differentiations of human-bone-marrow-derived mesenchymal stem cells (hMSCs) was conducted to evaluate it. The results showed that RGD–OSA/NSC composite hydrogels presented good biological properties in attachment, proliferation and differentiation of cells. The MTT cell viability assay showed that the total number of cells increased more significantly in the LIPUS-stimulated groups with RGD than that in the control ones; similar results were obtained for alkaline phosphatase activity/staining and mineralized nodule formation assay of osteogenic induction and immunohistochemical test of endothelial induction. The positive synergistic effect of LIPUS and RGD on the enhancement of proliferation and differentiation of hMSCs was observed. These findings suggest that the hybrid use of RGD modification and LIPUS might provide one approach to achieve a high level of formation of new bone and vascularization in bone tissue engineering scaffold implants.     

Flow perfusion culture of human mesenchymal stem cells on coralline hydroxyapatite scaffolds with various pore sizes

Bone grafts are widely used in orthopaedic reconstructive surgery, but harvesting of autologous grafts is limited due to donor site complications. Bone tissue engineering is a possible alternative source for substitutes, and to date, mainly small scaffold sizes have been evaluated. The aim of this study was to obtain a clinically relevant substitute size using a direct perfusion culture system. Human bone marrowderived mesenchymal stem cells were seeded on coralline hydroxyapatite scaffolds with 200 μm or 500 μm pores, and resulting constructs were cultured in a perfusion bioreactor or in static culture for up to 21 days and analysed for cell distribution and osteogenic differentiation using histological stainings, alkaline phosphatase activity assay, and real-time RT-PCR on bone markers. We found that the number of cells was higher during static culture at most time points and that the final number of cells was higher in 500 μm constructs as compared with 200 μm constructs. Alkaline phosphatase enzyme activity assays and real time RT-PCR on seven osteogenic markers showed that differentiation occurred primarily and earlier in statically cultured constructs with 200 μm pores compared with 500 μm ones. Adhesion and proliferation of the cells was seen on both scaffold sizes, but the vitality and morphology of cells changed unfavorably during perfusion culture. In contrast to previous studies using spinner flask that show increased cellularity and osteogenic properties of cells when cultured dynamically, the perfusion culture in our study did not enhance the osteogenic properties of cell/scaffold constructs. The statically cultured constructs showed increasing cell numbers and abundant osteogenic differentiation probably because of weak initial cell adhesion due to the surface morphology of scaffolds. Our conclusion is that the specific scaffold surface microstructure and culturing system flow dynamics has a great impact on cell distribution and proliferation and on osteogenic differentiation, and the data presented warrant careful selection of in vitro culture settings to meet the specific requirements of the scaffolds and cells, especially when natural biomaterials with varying morphology are used.     

The effect of heparin-functionalized PEG hydrogels on three-dimensional human mesenchymal stem cell osteogenic differentiation

Poly(ethylene glycol) (PEG) hydrogels functionalized with heparin were utilized as a three-dimensional culture system for human mesenchymal stem cells (hMSCs). Heparin-functionalized hydrogels supported hMSC viability, as quantified through live/dead imaging, and induced osteogenic differentiation, as measured by increased alkaline phosphatase (ALP) production and osteopontin (OPN) and collagen I (COL I) gene expression over the 5-week study. Further exploration of the potential mechanism of heparin-induced osteogenic differentiation was performed. Specifically, the availability of bone morphogenetic protein 2 (BMP2) and fibronectin (FN) in the culture system was controlled and hMSC osteogenic differentiation was evaluated as a function of the microenvironment. BMP2 availability increased both ALP production and OPN gene expression, while FN increased ALP production, but not OPN gene expression. Furthermore, immunostaining of integrin expression revealed that viability and differentiation were differentially affected by integrin production, where both alpha5beta1 and alphavbeta3 integrin-ligand interactions supported viability, while only the alpha5beta1 integrin played a role in hMSC osteogenic differentiation.     

Self-assembled composite matrix in a hierarchical 3-D scaffold for bone tissue engineering

It is of high clinical relevance in bone tissue engineering that scaffolds promote a high seeding efficiency of cells capable of osteogenic differentiation, such as human bone marrow-derived mesenchymal stem cells (hMSCs). We evaluated the effects of a novel polycaprolactone (PCL) scaffold on hMSC seeding efficiency, proliferation, distribution and differentiation. Porous PCL meshes prepared by fused deposition modeling (FDM) were embedded in matrix of hyaluronic acid, methylated collagen and terpolymer via polyelectrolyte complex coacervation. Scaffolds were cultured statically and dynamically in osteogenic stimulation medium for up to 28 days. Compared to naked PCL scaffolds, embedded scaffolds provided a higher cell seeding efficiency (t-test, P<0.05), a more homogeneous cell distribution and more osteogenically differentiated cells, verified by a more pronounced gene expression of the bone markers alkaline phosphatase, osteocalcin, bone sialoprotein I and bone sialoprotein II. Dynamic culture resulted in higher amounts of DNA (day 14 and day 21) and calcium (day 21 and day 28), compared to static culture. Dynamic culture and the embedding synergistically enhanced the calcium deposition of hMSC on day 21 and day 28. This in vitro study provides evidence that hybrid scaffolds made from natural and synthetic polymers improve cellular seeding efficiency, proliferation, distribution and osteogenic differentiation.     

Differentiation of mesenchymal stem cells onto highly adherent radio frequency-sputtered carbonated hydroxylapatite thin films

In this work, an improved version of the radio frequency magnetron sputtering (RF-MS) technique was used to prepare highly adherent B-type carbonated hydroxylapatite (B-CHA) thin films. Fourier transform infrared spectroscopy (FTIR) and grazing incidence X-ray diffraction studies proved that the coatings maintained the composition and revealed the polycrystalline structure of HA. Scanning electron microscopy analysis showed that the CHA films are rough and exhibit a homogeneous microstructure. Energy-dispersive X-ray spectroscopy (EDX) mapping demonstrated a uniform distribution of the Ca and P cations while a Ca/P ratio of 1.8 was found. In addition, the FTIR experiments showed a remarkable reproducibility of the nanostructures. Human mesenchymal stem cells (hMSCs), in vitro differentiated osteoblasts, and explanted bone cells were grown over the surface of CHA coatings for periods between a few hours and 21 days. Osteoprogenitor cells maintained viability and characteristic morphology after adhesion on CHA coatings. Ki67-positive osteoblasts were the evidence of cell proliferation events. Cells showed positive staining for markers of osteoblast phenotype such as collagen type I, bone sialoprotein and osteonectin. Our data showed the formation of mineralized foci by differentiation of hMSCs to human primary osteoblasts after cultivation in osteogenic media on RF-sputtered films. The results demonstrate the capacity of B-type CHA coating to support MSCs adhesion and osteogenic differentiation ability.     

TAK-778 enhances osteoblast differentiation of human bone marrow cells cultured on titanium

TAK-778 induces bone growth in in vitro and in vivo models. The aim of this study was to evaluate the osteogenic potential of TAK-778 on human bone marrow cells cultured on commercially pure titanium (cpTi). Cells were cultured either in absence or in presence of TAK-778 (10(-5)M) on cpTi in supplemented alpha-MEM. For attachment evaluation, cells were cultured for 4 and 24h. After 7, 14, and 21 days, cell proliferation, cell viability, total protein content, alkaline phosphatase (ALP) activity, and bone-like formation were evaluated. TAK-778 did not affect cell attachment and viability. Cell number was reduced by TAK-778. ALP activity, total protein content, and bone-like formation were increased by TAK-778. These results suggest that initial cell events such as cell attachment are not affected by TAK-778 while events that indicate osteoblast differentiation including reduced cell proliferation, and increased both ALP activity and bone-like formation are enhanced by TAK-778 in presence of cpTi. It means that TAK-778 could be a useful drug to improve the osseointegration of implants by both enhancing and accelerating bone formation on Ti surface.