Paracrine effects influenced by cell culture medium and consequences on microvessel-like structures in cocultures of mesenchymal stem cells and outgrowth endothelial cells

Mesenchymal stem cells (MSC) from bone marrow and outgrowth endothelial cells (OEC) from peripheral blood are considered as attractive cell types for applications in regenerative medicine aiming to build up complex vascularized tissue-engineered constructs. MSC provide several advantages such as the potential to differentiate to osteoblasts and to support the neovascularization process by release of proangiogenic factors. On the other hand, the neovascularization process can be actively supported by OEC forming perfused vascular structures after co-implantation with other cell types. In this study the formation of angiogenic structures in vitro was investigated in cocultures of MSC and OEC, cultured either in the medium for osteogenic differentiation of MSC (ODM) or in the medium for OEC cultivation endothelial cell growth medium-2 (EGM2 Bullet Kit). After 2 weeks, cocultures in EGM2 formed more microvessel-like structures compared to cocultures in ODM as demonstrated by immunofluorescence staining for the endothelial marker CD31. Increased expression of CD31 and CD146 in quantitative real-time polymerase chain reaction as well as a higher percentage of CD31- and CD146-positive cells in flow cytometry indicated a beneficial influence of EGM2 on endothelial cell growth and function. In addition, the improved formation of vascular structures in EGM2 correlates with higher levels of the proangiogenic factor vascular endothelial growth factor and platelet-derived growth factor in the supernatant of cocultures as well as in monocultures of MSC when cultivated in EGM-2. Nevertheless, ODM was more suitable for the differentiation of MSC to osteoblastic lineages in the cocultures, whereas EGM2 favored factors involved in vessel stabilization by pericytes. In conclusion, this study highlights the importance of medium components for cell interaction triggering the formation of angiogenic structures.     

Cellular reactions of osteoblasts to micron- and submicron-scale porous structures of titanium surfaces

Osteoblast reactions to topographic structures of titanium play a key role in host tissue responses and the final osseointegration. Since it is difficult to fabricate micro- and nano-scale structures on titanium surfaces, little is known about the mechanism whereby the topography of titanium surfaces exerts its effects on cell behavior at the cellular level. In the present study, the titanium surface was structured in micron- and submicron-scale ranges by anodic oxidation in either 0.2 M H3PO4 or 0.03 M calcium glycerophosphate with 0.15 calcium acetate. The average dimensions of pores in the structured surface were about 0.5 and 2 microm in diameter, with roughness averaging at 0.2 and 0.4 microm, respectively. Enhanced attachment of cells (SaOS-2) was shown on micron- and submicron-scale structures. Initial cell reactions to different titanium surfaces, e.g. the development of the actin-containing structures, are determined by the different morphology of the surfaces. It is demonstrated that on either micron- or submicron-structured surfaces, many well-developed filopodia were observed to be primary adhesion structures in cell-substrate interactions, and some of them entered pores using their distinct tips or points along their length for initial attachment. Therefore, porous structures at either micro- or submicrometre scale supply positive guidance cues for anchorage-dependent cells to attach, leading to enhanced cell attachment. In contrast, the cells attached to a smooth titanium surface by focal contacts around their periphery as predominant adhesion structures, since repulsive signals from the environment led to retraction of the filopodia back to the cell bodies. These cells showed well-organized stress fibres, which exert tension across the cell body, resulting in flattened cells.     

Osteogenic and angiogenic potentials of monocultured and co-cultured human-bone-marrow-derived mesenchymal stem cells and human-umbilical-vein endothelial cells on three-dimensional porous beta-tricalcium phosphate scaffold

The use of biodegradable beta-tricalcium phosphate (β-TCP) scaffolds holds great promise for bone tissue engineering. However, the effects of β-TCP on bone and endothelial cells are not fully understood. This study aimed to investigate cell proliferation and differentiation of mono- or co-cultured human-bone-marrow-derived mesenchymal stem cells (hBMSCs) and human-umbilical-vein endothelial cells (HUVECs) on a three-dimensional porous, biodegradable β-TCP scaffold. In co-culture studies, the ratios of hBMSCs:HUVECs were 5:1, 1:1 and 1:5. Cellular morphologies of HUVECs, hBMSCs and co-cultured HUVECs/hBMSCs on the β-TCP scaffolds were monitored using confocal and scanning electron microscopy. Cell proliferation was monitored by measuring the amount of double-stranded DNA (dsDNA) whereas hBMSC and HUVEC differentiation was assessed using the osteogenic and angiogenic markers, alkaline phosphatase (ALP) and PECAM-1 (CD31), respectively. Results show that HUVECs, hBMSCs and hBMSCs/HUVECs adhered to and proliferated well on the β-TCP scaffolds. In monoculture, hBMSCs grew faster than HUVECs on the β-TCP scaffolds after 7 days, but HUVECs reached similar levels of proliferation after 14 days. In monoculture, β-TCP scaffolds promoted ALP activities of both hBMSCs and HUVECs when compared to those grown on tissue culture well plates. ALP activity of cells in co-culture was higher than that of hBMSCs in monoculture. Real-time polymerase chain reaction results indicate that runx2 and alp gene expression in monocultured hBMSCs remained unchanged at days 7 and 14, but alp gene expression was significantly increased in hBMSC co-cultures when the contribution of individual cell types was not distinguished.     

Nodule formation and mineralisation of human primary osteoblasts cultured on a porous bioactive glass scaffold

The aim of this study was to analyse human osteoblast responses to a porous bioactive glass scaffold. It was hypothesised that osteoblasts would attach, proliferate and form mineralised nodules in response to culture on the bioactive glass. As dissolution products are a key feature of bioactive glasses, this was measured by inductively coupled plasma optical emission spectroscopy to determine effects of both the glass surface and ion release. Osteoblasts attached and proliferated on the foams as demonstrated by scanning electron microscopy. Nodule formation was also observed in the pores of the glass and also in conditioned medium containing dissolution products at certain concentrations and these nodules were shown to be mineralised by alizarin red staining. Undiluted dissolution products from the foams however caused significant apoptosis suggesting an ion concentration dependent response.     

血管内皮细胞在生物材料引起血栓形成过程中的作用

血管内皮细胞——这一铺展于血管内腔、包绕着循环血的细胞,可合成和释放多种活性物质调节血液的凝血与抗凝平衡,在生物材料引起血栓形成的过程中起着重要的作用。同时它促栓和抗栓功能的异常改变也是衡量生物材料血液相容性优劣的灵敏指标。本文就近年来对内皮细胞对凝血—抗凝系统的调节及其在生物材料引起血栓形成中的作用等方面的研究以及尚待解决的一些问题作一综述。     

PEEK/SPEEK/HA生物材料对成骨细胞MG-63、成纤维细胞3T3的氧化应激作用*

目的对引入界面增强剂磺化聚醚醚酮的新型PEEK/HA生物材料进行生物相容性评价,研究该材料对骨环境细胞的氧化应激作用。方法选用成骨细胞MG-63、成纤维细胞3T3作为研究对象,比较材料对两种细胞产生ROS和MDA水平的影响,并观察两种细胞在HA质量占比30%的PEEK/SPEEK/HA材料上的生长形貌。结果在PEEK和SPEEK材料上培养导致成骨细胞MG-63和成纤维细胞3T3氧化应激水平上升,SPEEK材料相比PEEK材料能降低细胞氧化应激水平,PEEK/HA材料对成骨细胞MG-63和成纤维细胞3T3氧化应激作用相对PEEK和SPEEK材料减弱,与PEEK/HA生物材料相比,PEEK/SPEEK/HA生物材料使两种细胞氧化应激水平更低,当HA含量达到30%,成骨细胞MG-63氧化应激水平接近正常,并且两种细胞在该材料表面生长正常不受到限制。结论HA与SPEEK引入能降低细胞氧化作用。     

Polyurethane biomaterials for fabricating 3D porous scaffolds and supporting vascular cells

Successful tissue engineering of vascular grafts largely depends on synthetic scaffolds that support the survival, proliferation, and differentiation of seeded cells. To investigate the utility of polyurethanes for vascular tissue engineering, three-dimensional porous polyurethane scaffolds with highly interconnected pore structures were fabricated by a pressure differential/particulate leaching technique. Ammonium chloride and paraffin porogens were prepared to fabricate the scaffolds. Grinding of ammonium chloride resulted in particulates with uniform particle sizes but irregular shapes. Paraffin particulates made by a dispersion method, on the other hand, had spherical shapes and uniform particle sizes. Polyurethane scaffolds fabricated from these particulates had open faced, highly interconnected channels that could allow cellular infiltration and nutrient delivery. Human coronary artery smooth muscle and endothelial cell interactions with polyurethane surfaces revealed these biomaterials to maintain the contractile phenotype of human coronary artery smooth muscle cells and the formation of endothelial monolayers. During longer culture times, surface modification with cell adhesive extracellular matrix (ECM) protein promoted vascular cell proliferation, maintenance of the differentiated phenotype and endothelial monolayer integrity. Our results suggest that these polyurethanes, in conjunction with cell adhesive ECM proteins, could also support vascular cells in three-dimensional bioreactor-based culture conditions.     

Biocompatibility markers for the study of interactions between osteoblasts and composite biomaterials

Biphasic calcium phosphate (BCP), a mixture of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP), has attracted attention as an excellent bone graft substitute. Mixtures of ceramics with agarose, as natural biodegradable binder, have been recently performed in order to increase the flexibility of the ceramic component and to facilitate the biomaterial preparation. In previous studies we have evaluated the response of both L929 fibroblasts and Saos-2 osteoblasts to hydroxyapatite-betaTCP/agarose disks observing a higher sensitivity of osteoblasts to this biomaterial. In the present study, the use of specific fluorescent probes and antibodies has allowed to evaluate different cell function parameters as biocompatibility markers for the cell/biomaterial interaction of Saos-2 osteoblasts cultured for 7 days on hydroxyapatite-betaTCP/agarose disks. The cell cycle subG(1) fraction, the exposition of phosphatidylserine on the outside surface of the plasma membrane and the analysis of plasma membrane integrity versus cell size, indicate that the interaction with the biomaterial induces a light increase of apoptosis in osteoblasts without producing cell necrosis. The high percentage of viable cells on the biomaterial and the preservation of endothelial nitric oxide synthase (eNOS) expression, eNOS activity and mitochondrial membrane potential (Deltapsi(m)), demonstrate the good biocompatibility of hydroxyapatite-betaTCP/agarose disks and its potential utility for bone substitution and repair.     

Formation of focal contacts by osteoblasts cultured on orthopedic biomaterials.

The nature of the contact sites formed during the adhesion of osteoblasts to orthopedic implant materials was investigated by fluorescence microscopy. More specifically, the cytoskeletal organization of and the focal contact formation by neonatal rat calvarial osteoblasts attaching to and spreading on 316L stainless steel, Ti-6Al-4V, Co-Cr-Mo, Synamel (hydroxyapatite), alumina, and borosilicate glass were examined. Focal contacts are regions where the plasma membrane approaches the substrate to within 10-15 nm and where bundles of cytoskeletal microfilaments terminate. Fluorescent-labeling of F-actin-containing microfilaments demonstrated a typical sequence of events as rounded, suspended osteoblasts spread onto the substrates. Immunofluorescent-labeling of the protein vinculin, which is found at the cytoplasmic face of focal contacts, initially showed the formation of streak-like focal patches. On the biomaterials, the vinculin staining subsequently extended up and along, but ventral to, the microfilament bundles. The fibrillar patterns observed at later times may evidence the formation of extracellular matrix contacts.     

The effect of human osteoblasts on proliferation and neo-vessel formation of human umbilical vein endothelial cells in a long-term 3D co-culture on polyurethane scaffolds

Angiogenesis is a key element in early wound healing and is considered important for tissue regeneration and for directing inflammatory cells to the wound site. The improvement of vascularization by implementation of endothelial cells or angiogenic growth factors may represent a key solution for engineering bone constructs of large size. In this study, we describe a long-term culture environment that supports the survival, proliferation, and in vitro vasculogenesis of human umbilical vein endothelial cells (HUVEC). This condition can be achieved in a co-culture model of HUVEC and primary human osteoblasts (hOB) employing polyurethane scaffolds and platelet-rich plasma in a static microenvironment. We clearly show that hOB support cell proliferation and spontaneous formation of multiple tube-like structures by HUVEC that were positive for the endothelial cell markers CD31 and vWF. In contrast, in a monoculture, most HUVEC neither proliferated nor formed any apparent vessel-like structures. Immunohistochemistry and quantitative PCR analyses of gene expression revealed that cell differentiation of hOB and HUVEC was stable in long-term co-culture. The three-dimensional, FCS-free co-culture system could provide a new basis for the development of complex tissue engineered constructs with a high regeneration and vascularization capacity.     

The influence of biomaterials on endothelial cell thrombogenicity

Driven by tissue engineering and regenerative medicine, endothelial cells are being used in combination with biomaterials in a number of applications for the purpose of improving blood compatibility and host integration. Endothelialized vascular grafts are beginning to be used clinically with some success in some centers, while endothelial seeding is being explored as a means of creating a vasculature within engineered tissues. The underlying assumption of this strategy is that when cultured on artificial biomaterials, a confluent layer of endothelial cells maintain their non-thrombogenic phenotype. In this review the existing knowledge base of endothelial cell thrombogenicity cultured on a number of different biomaterials is summarized. The importance of selecting appropriate endpoint measures that are most reflective of overall surface thrombogenicity is the focus of this review. Endothelial cells inhibit thrombosis through three interconnected regulatory systems (1) the coagulation cascade, (2) the cellular components of the blood such as leukocytes and platelets and (3) the complement cascade, and also through effects on fibrinolysis and vascular tone, the latter which influences blood flow. Thus, in order to demonstrate the thrombogenic benefit of seeding a biomaterial with EC, the conditions under which EC surfaces are more likely to exhibit lower thrombogenicity than unseeded biomaterial surfaces need to be consistent with the experimental context. The endpoints selected should be appropriate for the dominant thrombotic process that occurs under the given experimental conditions.     

不同比例共培养内皮细胞和成骨细胞的成骨及成血管效应*☆

背景：近年来研究显示内皮细胞和成骨细胞共培养修复骨组织的过程中存在相互促进的作用。 目的：观察不同比例下内皮细胞和成骨细胞共培养对其成血管和成骨作用的影响。 方法：分离培养人脐静脉内皮细胞和成骨细胞MG63,内皮细胞和成骨细胞按照1∶0,8∶1,4∶1,1∶1,1∶4,1∶8,      0∶1在24孔板中进行共培养,内皮细胞和成骨细胞分别单纯单层培养作对照。在第7,14,21天时观察细胞生长状态、血管内皮生长因子表达水平及碱性磷酸酶活性。 结果与结论：各组细胞在21 d中生长良好,第14天1∶8组可见成骨细胞开始集落式生长,第21天1∶4组钙化染色结节要明显强于1∶8组,而单纯成骨细胞组并未出现钙化结节。1∶4组碱性磷酸酶含量在3个时间点均明显增高(P < 0.05)且第7天时血管内皮生长因子开始增高,直到第21天时达到高峰。说明内皮细胞和成骨细胞共培养比例为4∶1时,成血管作用较强,而比例为1∶4时成骨作用更强。      

Differentiation of osteoblasts and in vitro bone formation from murine embryonic stem cells

Pluripotent embryonic stem (ES) cells have the potential to differentiate to all fetal and adult cell types and might represent a useful cell source for tissue engineering and repair. Here we show that differentiation of ES cells toward the osteoblast lineage can be enhanced by supplementing serum-containing media with ascorbic acid, beta-glycerophosphate, and/or dexamethasone/retinoic acid or by co-culture with fetal murine osteoblasts. ES cell differentiation into osteoblasts was characterized by the formation of discrete mineralized bone nodules that consisted of 50-100 cells within an extracellular matrix of collagen-1 and osteocalcin. Dexamethasone in combination with ascorbic acid and beta-glycerophosphate induced the greatest number of bone nodules and was dependent on time of stimulation with a sevenfold increase when added to ES cultures after, but not before, 14 days. Co-culture with fetal osteoblasts also provided a potent stimulus for osteogenic differentiation inducing a fivefold increase in nodule number relative to ES cells cultured alone. These data demonstrate the application of a quantitative assay for the derivation of osteoblast lineage progenitors from pluripotent ES cells. This could be applied to obtain purified osteoblasts to analyze mechanisms of osteogenesis and for use of ES cells in skeletal tissue repair.     

Tissue engineering by self-assembly of cells printed into topologically defined structures

Understanding the principles of biological self-assembly is indispensable for developing efficient strategies to build living tissues and organs. We exploit the self-organizing capacity of cells and tissues to construct functional living structures of prescribed shape. In our technology, multicellular spheroids (bio-ink particles) are placed into biocompatible environment (bio-paper) by the use of a three-dimensional delivery device (bio-printer). Our approach mimics early morphogenesis and is based on the realization that the genetic control of developmental patterning through self-assembly involves physical mechanisms. Three-dimensional tissue structures are formed through the postprinting fusion of the bio-ink particles, in analogy with early structure-forming processes in the embryo that utilize the apparent liquid-like behavior of tissues composed of motile and adhesive cells. We modeled the process of self-assembly by fusion of bio-ink particles, and employed this novel technology to print extended cellular structures of various shapes. Functionality was tested on cardiac constructs built from embryonic cardiac and endothelial cells. The postprinting self-assembly of bio-ink particles resulted in synchronously beating solid tissue blocks, showing signs of early vascularization, with the endothelial cells organized into vessel-like conduits.     

Immobilization strategy for optimizing VEGF's concurrent bioactivity towards endothelial cells and osteoblasts on implant surfaces

Orthopedic implant failure is mainly due to defective osseointegration and bacterial infection. Hence, a promising strategy to overcome these two problems is to functionalize the implant surface with both growth factors (GFs) and anti-infective agents. Covalent immobilization is widely used for such functionalization, but few studies have investigated the possible decrease in the GF's bioactivity as a result of conformational changes upon immobilization. In our study, vascular endothelial growth factor (VEGF) was immobilized on titanium surface via either covalent binding or heparin-VEGF interaction, and its bioactivity on endothelial cells (ECs) was compared. Although a similar surface density of immobilized VEGF was achieved by these two strategies, the bioactivity of the covalently immobilized VEGF on EC functions is significantly lower than that of the heparin-bound VEGF. The heparin-bound VEGF also enhanced mineralization in an osteoblast/endothelial cell co-culture to a much greater extent than in an osteoblast monoculture, illustrating the importance of crosstalk between osteoblasts and endothelial cells. In addition, the surface of the substrates with heparin-bound VEGF is highly hydrophilic and negatively-charged, which significantly inhibits Staphylococcus aureus adhesion. These results suggest that our strategy of immobilizing VEGF on titanium via heparin-VEGF interaction can preserve the GF's bioactivity on both osseous and vascular components and concomitantly reduce bacterial infection, which is promising to enhance the long-term stability of implants.     

Regenerative potential of human periodontal ligament derived stem cells on three-dimensional biomaterials: a morphological report

Recent studies have shown that mesenchymal stem cells obtained from periodontal ligament (PDL-MSCs) are multipotent cells that have similar features of the bone marrow and dental pulp MSCs and are capable of proliferating and producing different types of tissue such as bone and tooth associated-tissues. Human PDL-MSCs expanded ex vivo were induced to osteogenesis, seeded in three-dimensional biocompatible scaffolds (fibrin sponge, bovine-derived substitutes) and examined using light, scanning and transmission electron microscopy. Morphological observations showed extensive growth of cellular biomass partially covering the scaffolds after 4 weeks of incubation in mineralization medium. These findings indicate that periodontal ligament can be an easily and efficient autologous source of stem cells with a high expansion capacity and ability to differentiate in osteogenic cells that can colonize and grow connected to bio-compatible scaffold. It can be suggested that the use of PDL-MSCs for generating graft biomaterials is advantageous for bone tissue engineering in regenerative dentistry.     

Coculture of osteoblasts and endothelial cells: optimization of culture medium and cell ratio

Vascularization strategies in cell-based bone tissue engineering depend on optimal culture conditions. The present study aimed to determine optimal cell culture medium and cell ratio for cocultures of human marrow stromal cells (HMSCs) and human umbilical vein endothelial cells (HUVECs) in view of both osteogenic and angiogenic outcome parameters upon two-dimensional and three-dimensional culture conditions. Cultures were performed in four different media: osteoblastic cell proliferation medium, osteogenic medium (OM), endothelial medium, and a 1:1 mixture of the latter two media. Mineralization within the cocultures was observed only in OM. Subsequent experiments in OM showed that alkaline phosphatase activity, mineralization, and CD31(+) staining were highest for cocultures at a 50:50 HMSC/HUVEC ratio. Therefore, the results from the present study show that a HMSC/HUVEC coculture ratio of 50:50 in OM is the best combination to obtain both osteogenic and angiogenic differentiation.     

The effect of three-dimensional matrix-embedding of endothelial cells on the humoral and cellular immune response

The endothelium is a unique immunologic target. The first host-donor reaction in any cell, tissue or organ transplant occurs at the blood-tissue interface, the endothelium. When endothelial cells are themselves the primary component of the implant a second set of immunologic reactions arises. Injections of free endothelial cell implants elicit a profound major histocompatibility complex (MHC) II dominated immune response with significant sensitivity, cascade enhancement and immune memory. Endothelial cells embedded within three-dimensional matrices retain all the biosecretory capacity of quiescent endothelial cells. Perivascular implants of such cells are the most potent inhibitor of intimal hyperplasia and thrombosis following controlled vascular injury, but without any immune reactivity. Allo- and even xenogeneic endothelial cells evoke no significant humoral or cellular immune response in immunocompetent hosts when embedded within matrices. Moreover, endothelial implants are immunomodulatory, reducing the extent of the memory response to previous free cell implants. Attenuated immunogenicity results in muted activation of adaptive and innate immune cells. These findings point toward a pivotal role of matrix-cell-interconnectivity for the cellular immune phenotype and might therefore assist in the design of extracellular matrix components for successful tissue engineering.