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Cell communication in a coculture system consisting of outgrowth endothelial cells and primary osteoblasts.

Herzog DP, Dohle E, Bischoff I, Kirkpatrick CJ - Biomed Res Int (2014)

Bottom Line: In addition, to gain a better understanding on the origin of different growth factors, both direct and indirect coculture strategies were employed.It could be shown that connexins, the gap junction proteins, were located around cell nuclei, where they await their transport to the cell membrane.In addition, areas in which two cells formed gap junctions were found.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathology, University Medical Center, Johannes Gutenberg University, Langenbeckstraße 1, 55101 Mainz, Germany.

ABSTRACT
Bone tissue is a highly vascularized and dynamic system with a complex construction. In order to develop a construct for implant purposes in bone tissue engineering, a proper understanding of the complex dependencies between different cells and cell types would provide further insight into the highly regulated processes during bone repair, namely, angiogenesis and osteogenesis, and might result in sufficiently equipped constructs to be beneficial to patients and thereby accomplish their task. This study is based on an in vitro coculture model consisting of outgrowth endothelial cells and primary osteoblasts and is currently being used in different studies of bone repair processes with special regard to angiogenesis and osteogenesis. Coculture systems of OECs and pOBs positively influence the angiogenic potential of endothelial cells by inducing the formation of angiogenic structures in long-term cultures. Although many studies have focused on cell communication, there are still numerous aspects which remain poorly understood. Therefore, the aim of this study is to investigate certain growth factors and cell communication molecules that are important during bone repair processes. Selected growth factors like VEGF, angiopoietins, BMPs, and IGFs were investigated during angiogenesis and osteogenesis and their expression in the cultures was observed and compared after one and four weeks of cultivation. In addition, to gain a better understanding on the origin of different growth factors, both direct and indirect coculture strategies were employed. Another important focus of this study was to investigate the role of "gap junctions," small protein pores which connect adjacent cells. With these bridges cells are able to exchange signal molecules, growth factors, and other important mediators. It could be shown that connexins, the gap junction proteins, were located around cell nuclei, where they await their transport to the cell membrane. In addition, areas in which two cells formed gap junctions were found.

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Gap junctional communication in the coculture model system. Several Cx43-positive areas could be detected in our coculture model system after a cultivation time of one week (a) and four weeks (b). Areas such as those shown were scaled up and presumptive gap junctions were found where cells were adjacent. A longer cultivation time led to an upregulation of relative connexin mRNA expression (c) and it seemed that pOBs expressed more connexin mRNA than did OECs (d). One week values (c) were set as control (=1.0) for cocultures and OEC values (d) were set as control (=1.0) to compare pOB and OEC monocultures. Scale bars: 75 μm. n = 3.
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fig4: Gap junctional communication in the coculture model system. Several Cx43-positive areas could be detected in our coculture model system after a cultivation time of one week (a) and four weeks (b). Areas such as those shown were scaled up and presumptive gap junctions were found where cells were adjacent. A longer cultivation time led to an upregulation of relative connexin mRNA expression (c) and it seemed that pOBs expressed more connexin mRNA than did OECs (d). One week values (c) were set as control (=1.0) for cocultures and OEC values (d) were set as control (=1.0) to compare pOB and OEC monocultures. Scale bars: 75 μm. n = 3.

Mentions: Connexins are either stored in vesicles around the cell nucleus near to the ER or in the plasma membrane of the cell, contributing to gap junctional intercellular communication. Cx43 expression was analysed in cocultures forming angiogenic structures and stained to give red fluorescence (Figures 4(a)–4(d)). CD-31 was used as endothelial marker and Hoechst dye for visualization of nuclei. A monoclonal Cx43 antibody was selected to reveal connexin localization. In cocultures consisting of pOBs and OECs, few perinuclear Cx43 spots were detectable after one week of coculturing (Figure 4(a)). After four weeks in coculture, when angiogenic structures have been established, many OECs showed typical perinuclear Cx43 conglomerates (Figure 4(b)). In addition, OECs showed Cx43-positive spots at locations where two cells are adjacent, seen between the nuclei of two cells. More mature and expanded microvessels appeared to contain even more gap junctional communication-positive areas in the plasma membrane of the cells. Large connexin conglomerates were located around several nuclei, especially in locations where OECs were merging to some kind of network. Interestingly, there were also a number of nonendothelial cells which contained Cx43-positive areas. The relative gene expression of the three important connexin isoforms during angiogenesis and osteogenesis, namely, Cx37, Cx40, and Cx43, was also assessed in this study. After 4 weeks of cocultivation, an upregulation of all three tested connexin isoforms could be detected. Cx37 expression rose to a 4-fold level and Cx40 and Cx43 to a 2.5-fold level (Figure 4(c)). Although statistical significance was not achieved, a clear trend could be detected. Indirect cocultures might provide the right platform to determine the origin of the connexin isoforms and to obtain some indication of the source of transcription. In general, it was established that connexin mRNA expression is higher in pOBs than in OECs (Figure 4(d)). This difference is relevant in all three connexin isoforms tested: Cx40 expression is 3-fold and Cx43 expression 3.5-fold higher in pOBs compared to OECs.


Cell communication in a coculture system consisting of outgrowth endothelial cells and primary osteoblasts.

Herzog DP, Dohle E, Bischoff I, Kirkpatrick CJ - Biomed Res Int (2014)

Gap junctional communication in the coculture model system. Several Cx43-positive areas could be detected in our coculture model system after a cultivation time of one week (a) and four weeks (b). Areas such as those shown were scaled up and presumptive gap junctions were found where cells were adjacent. A longer cultivation time led to an upregulation of relative connexin mRNA expression (c) and it seemed that pOBs expressed more connexin mRNA than did OECs (d). One week values (c) were set as control (=1.0) for cocultures and OEC values (d) were set as control (=1.0) to compare pOB and OEC monocultures. Scale bars: 75 μm. n = 3.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016919&req=5

fig4: Gap junctional communication in the coculture model system. Several Cx43-positive areas could be detected in our coculture model system after a cultivation time of one week (a) and four weeks (b). Areas such as those shown were scaled up and presumptive gap junctions were found where cells were adjacent. A longer cultivation time led to an upregulation of relative connexin mRNA expression (c) and it seemed that pOBs expressed more connexin mRNA than did OECs (d). One week values (c) were set as control (=1.0) for cocultures and OEC values (d) were set as control (=1.0) to compare pOB and OEC monocultures. Scale bars: 75 μm. n = 3.
Mentions: Connexins are either stored in vesicles around the cell nucleus near to the ER or in the plasma membrane of the cell, contributing to gap junctional intercellular communication. Cx43 expression was analysed in cocultures forming angiogenic structures and stained to give red fluorescence (Figures 4(a)–4(d)). CD-31 was used as endothelial marker and Hoechst dye for visualization of nuclei. A monoclonal Cx43 antibody was selected to reveal connexin localization. In cocultures consisting of pOBs and OECs, few perinuclear Cx43 spots were detectable after one week of coculturing (Figure 4(a)). After four weeks in coculture, when angiogenic structures have been established, many OECs showed typical perinuclear Cx43 conglomerates (Figure 4(b)). In addition, OECs showed Cx43-positive spots at locations where two cells are adjacent, seen between the nuclei of two cells. More mature and expanded microvessels appeared to contain even more gap junctional communication-positive areas in the plasma membrane of the cells. Large connexin conglomerates were located around several nuclei, especially in locations where OECs were merging to some kind of network. Interestingly, there were also a number of nonendothelial cells which contained Cx43-positive areas. The relative gene expression of the three important connexin isoforms during angiogenesis and osteogenesis, namely, Cx37, Cx40, and Cx43, was also assessed in this study. After 4 weeks of cocultivation, an upregulation of all three tested connexin isoforms could be detected. Cx37 expression rose to a 4-fold level and Cx40 and Cx43 to a 2.5-fold level (Figure 4(c)). Although statistical significance was not achieved, a clear trend could be detected. Indirect cocultures might provide the right platform to determine the origin of the connexin isoforms and to obtain some indication of the source of transcription. In general, it was established that connexin mRNA expression is higher in pOBs than in OECs (Figure 4(d)). This difference is relevant in all three connexin isoforms tested: Cx40 expression is 3-fold and Cx43 expression 3.5-fold higher in pOBs compared to OECs.

Bottom Line: In addition, to gain a better understanding on the origin of different growth factors, both direct and indirect coculture strategies were employed.It could be shown that connexins, the gap junction proteins, were located around cell nuclei, where they await their transport to the cell membrane.In addition, areas in which two cells formed gap junctions were found.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathology, University Medical Center, Johannes Gutenberg University, Langenbeckstraße 1, 55101 Mainz, Germany.

ABSTRACT
Bone tissue is a highly vascularized and dynamic system with a complex construction. In order to develop a construct for implant purposes in bone tissue engineering, a proper understanding of the complex dependencies between different cells and cell types would provide further insight into the highly regulated processes during bone repair, namely, angiogenesis and osteogenesis, and might result in sufficiently equipped constructs to be beneficial to patients and thereby accomplish their task. This study is based on an in vitro coculture model consisting of outgrowth endothelial cells and primary osteoblasts and is currently being used in different studies of bone repair processes with special regard to angiogenesis and osteogenesis. Coculture systems of OECs and pOBs positively influence the angiogenic potential of endothelial cells by inducing the formation of angiogenic structures in long-term cultures. Although many studies have focused on cell communication, there are still numerous aspects which remain poorly understood. Therefore, the aim of this study is to investigate certain growth factors and cell communication molecules that are important during bone repair processes. Selected growth factors like VEGF, angiopoietins, BMPs, and IGFs were investigated during angiogenesis and osteogenesis and their expression in the cultures was observed and compared after one and four weeks of cultivation. In addition, to gain a better understanding on the origin of different growth factors, both direct and indirect coculture strategies were employed. Another important focus of this study was to investigate the role of "gap junctions," small protein pores which connect adjacent cells. With these bridges cells are able to exchange signal molecules, growth factors, and other important mediators. It could be shown that connexins, the gap junction proteins, were located around cell nuclei, where they await their transport to the cell membrane. In addition, areas in which two cells formed gap junctions were found.

Show MeSH
Related in: MedlinePlus