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Van de Voorde B, Benmeridja L, Giol ED, Van der Meeren L, Van Damme L, Liu Z, Toncheva A, Raquez JM, Van den Brande N, Skirtach A, Declercq H, Dubruel P, Van Vlierberghe S. Potential of poly(alkylene terephthalate)s to control endothelial cell adhesion and viability. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2021; 129:112378. [PMID: 34579897 DOI: 10.1016/j.msec.2021.112378] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/25/2021] [Revised: 08/10/2021] [Accepted: 08/17/2021] [Indexed: 10/20/2022]
Abstract
Poly(ethylene terephthalate) (PET) is known for its various useful characteristics, including its applicability in cardiovascular applications, more precisely as synthetic bypass grafts for large diameter (≥ 6 mm) blood vessels. Although it is widely used, PET is not an optimal material as it is not interactive with endothelial cells, which is required for bypasses to form a complete endothelium. Therefore, in this study, poly(alkylene terephthalate)s (PATs) have been studied. They were synthesized via a single-step solution polycondensation reaction, which requires mild reaction conditions and avoids the use of a catalyst or additives like heat stabilizers. A homologous series was realized in which the alkyl chain length varied from 5 to 12 methylene groups (n = 5-12). Molar masses up to 28,000 g/mol were obtained, while various odd-even trends were observed with modulated differential scanning calorimetry (mDSC) and rapid heat-cool calorimetry (RHC) to access the thermal properties within the homologous series. The synthesized PATs have been subjected to in vitro cell viability assays using Human Umbilical Vein Endothelial Cells (HUVECs) and Human Dermal Microvascular Endothelial Cells (HDMECs). The results showed that HUVECs adhere and proliferate most pronounced onto PAT(n=9) surfaces, which could be attributed to the surface roughness and morphology as determined by atomic force microscopy (AFM) (i.e. Rq = 204.7 nm). HDMECs were investigated in the context of small diameter vessels and showed superior adhesion and proliferation after seeding onto PAT(n=6) substrates. These preliminary results already pave the way towards the use of PAT materials as substrates to support endothelial cell adhesion and growth. Indeed, as superior endothelial cell interactivity compared to PET was observed, time-consuming and costly surface modifications of PET grafts could be avoided by exploiting this novel material class.
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Affiliation(s)
- Babs Van de Voorde
- Polymer Chemistry and Biomaterials Group (PBM), Centre of Macromolecular Chemistry (CMaC), Department of Organic and Macromolecular Chemistry, Ghent University, Krijgslaan 281 S4-bis, B-9000 Ghent, Belgium; SIM vzw, Technologiepark 48, B-9052 Zwijnaarde, Belgium
| | - Lara Benmeridja
- Department of Basic Medical Sciences, Tissue Engineering and Biomaterials Group, Ghent University, De Pintelaan 185, B3, B-9000 Ghent, Belgium
| | - Elena Diana Giol
- Polymer Chemistry and Biomaterials Group (PBM), Centre of Macromolecular Chemistry (CMaC), Department of Organic and Macromolecular Chemistry, Ghent University, Krijgslaan 281 S4-bis, B-9000 Ghent, Belgium
| | - Louis Van der Meeren
- Department of Biotechnology, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium
| | - Lana Van Damme
- Polymer Chemistry and Biomaterials Group (PBM), Centre of Macromolecular Chemistry (CMaC), Department of Organic and Macromolecular Chemistry, Ghent University, Krijgslaan 281 S4-bis, B-9000 Ghent, Belgium
| | - Zhen Liu
- Physical Chemistry and Polymer Science (FYSC), Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels, Belgium
| | - Antoniya Toncheva
- Laboratory of Polymeric and Composite Materials, University of Mons, 23 Place du Parc, B-7000 Mons, Belgium
| | - Jean-Marie Raquez
- Laboratory of Polymeric and Composite Materials, University of Mons, 23 Place du Parc, B-7000 Mons, Belgium
| | - Niko Van den Brande
- Physical Chemistry and Polymer Science (FYSC), Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels, Belgium
| | - André Skirtach
- Department of Biotechnology, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium
| | - Heidi Declercq
- Department of Basic Medical Sciences, Tissue Engineering and Biomaterials Group, Ghent University, De Pintelaan 185, B3, B-9000 Ghent, Belgium
| | - Peter Dubruel
- Polymer Chemistry and Biomaterials Group (PBM), Centre of Macromolecular Chemistry (CMaC), Department of Organic and Macromolecular Chemistry, Ghent University, Krijgslaan 281 S4-bis, B-9000 Ghent, Belgium
| | - Sandra Van Vlierberghe
- Polymer Chemistry and Biomaterials Group (PBM), Centre of Macromolecular Chemistry (CMaC), Department of Organic and Macromolecular Chemistry, Ghent University, Krijgslaan 281 S4-bis, B-9000 Ghent, Belgium.
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Anspach L, Tsaryk R, Seidmann L, Unger RE, Jayasinghe C, Simiantonaki N, Kirkpatrick CJ, Pröls F. Function and mutual interaction of BiP-, PERK-, and IRE1α-dependent signalling pathways in vascular tumours. J Pathol 2020; 251:123-134. [PMID: 32166747 DOI: 10.1002/path.5423] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2019] [Revised: 02/19/2020] [Accepted: 03/09/2020] [Indexed: 01/15/2023]
Abstract
Spontaneously regressing infantile haemangiomas and aggressive angiosarcomas are vascular tumours with excessive angiogenesis. When analysing haemangiomas and angiosarcomas immunohistochemically with respect to their chaperone profiles we found that angiosarcomas have significantly elevated protein levels of binding immunoglobulin protein (BIP) and PERK with concomitant attenuated IRE1α levels, whereas haemangioma tissue exhibits the same pattern as embryonal skin tissue. We show that BiP is essential for the maintenance of VEGFR2 protein, which is expressed in the endothelium of both tumour types. When studying the effects of BiP, the IRE1α/Xbp1 -, and PERK/ATF4-signalling pathways on the migration and tube-forming potential of endothelial cells, we show that downregulation of BiP, as well as inhibition of the kinase activity of IRE1α, inhibit in vitro angiogenesis. Downregulation of PERK (PKR-like kinase; PKR = protein kinase R) levels promotes Xbp1 splicing in endoplasmic reticulum (ER)-stressed cells, indicating that in angiosarcoma the elevated PERK levels might result in high levels of unspliced Xbp1, which have been reported to promote cell proliferation and increase tumour malignancy. The data presented in this study revealed that in addition to BiP or PERK, the kinase domains of IRE1α and Xbp1 could be potential targets for the development of novel therapeutic approaches for treating angiosarcomas and to control tumour angiogenesis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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Affiliation(s)
- Laura Anspach
- Institute of Pathology, University Medical Center Mainz, Mainz, Germany
| | - Roman Tsaryk
- Institute of Pathology, University Medical Center Mainz, Mainz, Germany
| | - Larissa Seidmann
- Institute of Pathology, University Medical Center Mainz, Mainz, Germany
| | - Ronald E Unger
- Institute of Pathology, University Medical Center Mainz, Mainz, Germany
| | - Caren Jayasinghe
- Department of Pathology, Laboratory Dr. Wisplinghoff, Cologne, Germany
| | | | | | - Felicitas Pröls
- Institute of Anatomy II, Medical Faculty, University of Cologne, Cologne, Germany
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Heller M, Bauer H, Schwab R, Blatt S, Peters K, Nezi‐Cahn S, Unger RE, Hasenburg A, Brenner W. The impact of intercellular communication for the generation of complex multicellular prevascularized tissue equivalents. J Biomed Mater Res A 2019; 108:734-748. [DOI: 10.1002/jbm.a.36853] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2019] [Revised: 11/21/2019] [Accepted: 11/22/2019] [Indexed: 01/04/2023]
Affiliation(s)
- Martin Heller
- Department of Gynecology University Medical Center of the Johannes Gutenberg University Mainz Mainz Germany
- BiomaTiCS—Biomaterials, Tissues and Cells in Science University Medical Center of the Johannes Gutenberg University Mainz Mainz Germany
| | - Heide‐Katharina Bauer
- Department of Gynecology University Medical Center of the Johannes Gutenberg University Mainz Mainz Germany
- BiomaTiCS—Biomaterials, Tissues and Cells in Science University Medical Center of the Johannes Gutenberg University Mainz Mainz Germany
| | - Roxana Schwab
- Department of Gynecology University Medical Center of the Johannes Gutenberg University Mainz Mainz Germany
- BiomaTiCS—Biomaterials, Tissues and Cells in Science University Medical Center of the Johannes Gutenberg University Mainz Mainz Germany
| | - Sebastian Blatt
- BiomaTiCS—Biomaterials, Tissues and Cells in Science University Medical Center of the Johannes Gutenberg University Mainz Mainz Germany
- Department of Maxillofacial Surgery University Medical Center of the Johannes Gutenberg University Mainz Mainz Germany
| | - Katharina Peters
- Department of Gynecology University Medical Center of the Johannes Gutenberg University Mainz Mainz Germany
- BiomaTiCS—Biomaterials, Tissues and Cells in Science University Medical Center of the Johannes Gutenberg University Mainz Mainz Germany
| | - Sandra Nezi‐Cahn
- Department of Gynecology University Medical Center of the Johannes Gutenberg University Mainz Mainz Germany
- BiomaTiCS—Biomaterials, Tissues and Cells in Science University Medical Center of the Johannes Gutenberg University Mainz Mainz Germany
| | - Ronald E. Unger
- BiomaTiCS—Biomaterials, Tissues and Cells in Science University Medical Center of the Johannes Gutenberg University Mainz Mainz Germany
- Institute for Pathology University Medical Center of the Johannes Gutenberg University Mainz Mainz Germany
| | - Annette Hasenburg
- Department of Gynecology University Medical Center of the Johannes Gutenberg University Mainz Mainz Germany
| | - Walburgis Brenner
- Department of Gynecology University Medical Center of the Johannes Gutenberg University Mainz Mainz Germany
- BiomaTiCS—Biomaterials, Tissues and Cells in Science University Medical Center of the Johannes Gutenberg University Mainz Mainz Germany
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Giol ED, Van Vlierberghe S, Unger RE, Schaubroeck D, Ottevaere H, Thienpont H, Kirkpatrick CJ, Dubruel P. Endothelialization and Anticoagulation Potential of Surface-Modified PET Intended for Vascular Applications. Macromol Biosci 2018; 18:e1800125. [DOI: 10.1002/mabi.201800125] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2018] [Revised: 05/07/2018] [Indexed: 01/17/2023]
Affiliation(s)
- Elena Diana Giol
- Polymer Chemistry and Biomaterials Research (PBM) Group; Centre of Macromolecular Chemistry; Ghent University; Krijgslaan 281, S4-bis B-9000 Belgium
| | - Sandra Van Vlierberghe
- Polymer Chemistry and Biomaterials Research (PBM) Group; Centre of Macromolecular Chemistry; Ghent University; Krijgslaan 281, S4-bis B-9000 Belgium
- Brussels Photonics (B-PHOT); Vrije Universiteit Brussel; Pleinlaan 2 B-1050 Belgium
| | - Ronald E. Unger
- REPAIR LAB; University Medical Center of the Johannes Gutenberg University Mainz; Langenbeckstraat 1 55131 Germany
| | - David Schaubroeck
- Centre of Microsystems Technology (CMST); imec and Ghent University; Technologiepark-Zwijnaarde15 B-9052 Belgium
| | - Heidi Ottevaere
- Brussels Photonics (B-PHOT); Vrije Universiteit Brussel; Pleinlaan 2 B-1050 Belgium
| | - Hugo Thienpont
- Brussels Photonics (B-PHOT); Vrije Universiteit Brussel; Pleinlaan 2 B-1050 Belgium
| | - Charles James Kirkpatrick
- REPAIR LAB; University Medical Center of the Johannes Gutenberg University Mainz; Langenbeckstraat 1 55131 Germany
| | - Peter Dubruel
- Polymer Chemistry and Biomaterials Research (PBM) Group; Centre of Macromolecular Chemistry; Ghent University; Krijgslaan 281, S4-bis B-9000 Belgium
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Freese C, Anspach L, Deller RC, Richards SJ, Gibson MI, Kirkpatrick CJ, Unger RE. Gold nanoparticle interactions with endothelial cells cultured under physiological conditions. Biomater Sci 2018; 5:707-717. [PMID: 28184390 DOI: 10.1039/c6bm00853d] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
PEGylated gold nanoparticles (AuNPs) have an extended circulation time after intravenous injection in vivo and exhibit favorable properties for biosensing, diagnostic imaging, and cancer treatment. No impact of PEGylated AuNPs on the barrier forming properties of endothelial cells (ECs) has been reported, but recent studies demonstrated that unexpected effects on erythrocytes are observed. Almost all studies to date have been with static-cultured ECs. Herein, ECs maintained under physiological cyclic stretch and flow conditions and used to generate a blood-brain barrier model were exposed to 20 nm PEGylated AuNPs. An evaluation of toxic effects, cell stress, the release profile of pro-inflammatory cytokines, and blood-brain barrier properties showed that even under physiological conditions no obvious effects of PEGylated AuNPs on ECs were observed. These findings suggest that 20 nm-sized, PEGylated AuNPs may be a useful tool for biomedical applications, as they do not affect the normal function of healthy ECs after entering the blood stream.
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Affiliation(s)
- C Freese
- REPAIR-lab, Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz and European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Langenbeckstr. 1, 55131 Mainz, Germany.
| | - L Anspach
- REPAIR-lab, Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz and European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Langenbeckstr. 1, 55131 Mainz, Germany.
| | - R C Deller
- University of Warwick, Department of Chemistry, Coventry, CV4 7AL, UK
| | - S-J Richards
- University of Warwick, Department of Chemistry, Coventry, CV4 7AL, UK
| | - M I Gibson
- University of Warwick, Department of Chemistry, Coventry, CV4 7AL, UK
| | - C J Kirkpatrick
- REPAIR-lab, Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz and European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Langenbeckstr. 1, 55131 Mainz, Germany.
| | - R E Unger
- REPAIR-lab, Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz and European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Langenbeckstr. 1, 55131 Mainz, Germany.
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Herath TDK, Larbi A, Teoh SH, Kirkpatrick CJ, Goh BT. Neutrophil-mediated enhancement of angiogenesis and osteogenesis in a novel triple cell co-culture model with endothelial cells and osteoblasts. J Tissue Eng Regen Med 2017; 12:e1221-e1236. [PMID: 28715156 DOI: 10.1002/term.2521] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2016] [Revised: 06/02/2017] [Accepted: 06/20/2017] [Indexed: 01/22/2023]
Abstract
Repair and regeneration of critical-sized bone defects remain a major challenge in orthopaedic and craniomaxillofacial surgery. Until now, attempts to bioengineer bone tissue have been hindered by the inability to establish proper angiogenesis and osteogenesis in the tissue construct. In the present study, we established a novel triple cell co-culture model consisting of osteoblasts, endothelial cells, and neutrophils and conducted a systematic investigation of the effects of neutrophils on angiogenesis and osteogenesis. Neutrophils significantly increased angiogenesis in the tissue construct, evidenced by the formation of microvessel-like structures with an extensive lattice-like, stable tubular network in the co-culture model. Moreover, neutrophils significantly induced the expression of pro-angiogenic markers, such as VEGF-A, CD34, EGF, and FGF-2 in a dose- and time-dependent manner. Subsequently, PCR arrays corroborated that neutrophils upregulate the important angiogenic markers and MMPs. Moreover, neutrophils also enhanced osteogenic markers, such as ALP, OCN, OPN, and COL-1 compared with the controls. As shown by the osteogenic gene arrays, neutrophils significantly regulated major osteogenic markers such as BMP2, BMP3, BMP4, BMP5, TGF-β2, RUNX2, and ECM proteins. Significantly higher mineralization was observed in triple cell co-culture compared with controls. Foregoing data indicate that the triple cell co-culture model can be used to stimulate the growth of microvasculature within a bone bioengineering construct to improve cell viability. Neutrophil-mediated enhancement of angiogenesis and osteogenesis could be a viable, clinically relevant tissue engineering strategy to obtain optimal bone growth in defect sites, in the field of oral and maxillofacial surgery.
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Affiliation(s)
| | - Anis Larbi
- Singapore Immunology Network, Agency for Science, Technology and Research, Singapore
| | - Swee Hin Teoh
- Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore
| | - C James Kirkpatrick
- REPAIR-Lab, Institute of Pathology, University Medical Centre, Johannes Gutenberg University, Mainz, Germany
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Zhang C, Hu K, Liu X, Reynolds MA, Bao C, Wang P, Zhao L, Xu HH. Novel hiPSC-based tri-culture for pre-vascularization of calcium phosphate scaffold to enhance bone and vessel formation. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2017. [DOI: 10.1016/j.msec.2017.05.035] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
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Hu C, Ni Z, Li BS, Yong X, Yang X, Zhang JW, Zhang D, Qin Y, Jie MM, Dong H, Li S, He F, Yang SM. hTERT promotes the invasion of gastric cancer cells by enhancing FOXO3a ubiquitination and subsequent ITGB1 upregulation. Gut 2017; 66:31-42. [PMID: 26370108 DOI: 10.1136/gutjnl-2015-309322] [Citation(s) in RCA: 98] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/04/2015] [Revised: 08/25/2015] [Accepted: 08/26/2015] [Indexed: 12/30/2022]
Abstract
BACKGROUND AND AIMS Human telomerase reverse transcriptase (hTERT) plays an important role in cancer invasion, but the relevant mechanism is not well known. This study aims to investigate the role and mechanism of hTERT in gastric cancer metastasis. DESIGN Proteomics analysis, qPCR and western blotting were used to screen for hTERT-regulated candidate molecules in gastric cancer invasion. Chromatin immunoprecipitation (ChIP) qPCR was performed to identify the binding sites of hTERT at the regulatory region of the integrin β1 (ITGB1) gene. ChIP assays were further applied to elucidate the transcription factors that bound to the regulatory region. The interactions between hTERT and the transcription factors were tested by co-immunoprecipitation (Co-IP) and glutathione S-transferase (GST) pull-down experiments. Moreover, the revealed pathway was verified in tumour-bearing nude mice and human gastric cancer tissues. RESULTS ITGB1 was identified as a downstream gene of hTERT, and there were two hTERT-binding regions within this gene. hTERT alleviated the binding of forkhead box O3 (FOXO3a) to FOXO3a binding element (+9972∼+9978), but it enhanced the binding of forkhead box M1 (FOXM1) to FOXM1 binding element (-1104∼-1109) in ITGB1 gene. Importantly, FOXO3a played a major role in hTERT-induced ITGB1 expression, and the hTERT/murine double minute 2 (MDM2) complex promoted the ubiquitin-mediated degradation of FOXO3a. Moreover, hTERT increased ITGB1 expression in xenograft gastric cancer, and the level of hTERT was positively correlated with that of ITGB1 in human gastric cancer tissues. CONCLUSIONS The hTERT/MDM2-FOXO3a-ITGB1 pathway markedly contributes to hTERT-promoted gastric cancer invasion, suggesting that this pathway might be a novel target for the prevention and treatment of gastric cancer metastasis.
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Affiliation(s)
- Changjiang Hu
- Department of Gastroenterology, Xinqiao Hospital, Third Military Medical University, Chongqing, China
| | - Zhenghong Ni
- Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Third Military Medical University, Chongqing, China
| | - Bo-Sheng Li
- Department of Gastroenterology, Xinqiao Hospital, Third Military Medical University, Chongqing, China
| | - Xin Yong
- Department of Gastroenterology, Xinqiao Hospital, Third Military Medical University, Chongqing, China
| | - Xin Yang
- Department of Gastroenterology, Xinqiao Hospital, Third Military Medical University, Chongqing, China
| | - Jian-Wei Zhang
- Department of Gastroenterology, Xinqiao Hospital, Third Military Medical University, Chongqing, China
| | - Dan Zhang
- Department of Gastroenterology, Xinqiao Hospital, Third Military Medical University, Chongqing, China
| | - Yong Qin
- Department of Gastroenterology, Xinqiao Hospital, Third Military Medical University, Chongqing, China
| | - Meng-Meng Jie
- Department of Gastroenterology, Xinqiao Hospital, Third Military Medical University, Chongqing, China
| | - Hui Dong
- Department of Gastroenterology, Xinqiao Hospital, Third Military Medical University, Chongqing, China.,Division of Gastroenterology, Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California, USA
| | - Song Li
- Department of Pharmaceutical Sciences, Center for Pharmacogenetics, School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Fengtian He
- Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Third Military Medical University, Chongqing, China
| | - Shi-Ming Yang
- Department of Gastroenterology, Xinqiao Hospital, Third Military Medical University, Chongqing, China
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Improving vascularization of engineered bone through the generation of pro-angiogenic effects in co-culture systems. Adv Drug Deliv Rev 2015; 94:116-25. [PMID: 25817732 DOI: 10.1016/j.addr.2015.03.012] [Citation(s) in RCA: 83] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2014] [Revised: 02/27/2015] [Accepted: 03/20/2015] [Indexed: 01/31/2023]
Abstract
One of the major problems with bone tissue engineering is the development of a rapid vascularization after implantation to supply the growing osteoblast cells with the nutrients to grow and survive as well as to remove waste products. It has been demonstrated that capillary-like structures produced in vitro will anastomose rapidly after implantation and become functioning blood vessels. For this reason, in recent years many studies have examined a variety of human osteoblast and endothelial cell co-culture systems in order to distribute osteoblasts on all parts of the bone scaffold and at the same time provide conditions for the endothelial cells to migrate to form a network of capillary-like structures throughout the osteoblast-colonized scaffold. The movement and proliferation of endothelial cells to form capillary-like structures is known as angiogenesis and is dependent on a variety of pro-angiogenic factors. This review summarizes human 2- and 3-D co-culture models to date, the types and origins of cells used in the co-cultures and the proangiogenic factors that have been identified in the co-culture models.
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Kirkpatrick CJ. Modelling the regenerative niche: a major challenge in biomaterials research. Regen Biomater 2015; 2:267-72. [PMID: 26816650 PMCID: PMC4676329 DOI: 10.1093/rb/rbv018] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2015] [Revised: 08/25/2015] [Accepted: 08/30/2015] [Indexed: 01/01/2023] Open
Abstract
By definition, biomaterials are developed for clinical application. In the field of regenerative medicine their principal function is to play a significant, and, if possible, an instructive role in tissue healing. In the last analysis the latter involves targeting the ‘regenerative niche’. The present paper will address the problem of simulating this niche in the laboratory and adopts a life science approach involving the harnessing of heterotypic cellular communication to achieve this, that is, the ability of cells of different types to mutually influence cellular functions. Thus, co-culture systems using human cells are the methodological focus and will concern four exemplary fields of regeneration, namely, bone, soft tissue, lower respiratory tract and airway regeneration. The working hypothesis underlying this approach is that in vitro models of higher complexity will be more clinically relevant than simple monolayer cultures of transformed cell lines in testing innovative strategies with biomaterials for regeneration.
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Affiliation(s)
- C James Kirkpatrick
- REPAIR-Lab, Institute of Pathology, University Medical Center, Johannes Gutenberg University Mainz, D-55101 Mainz, Germany;; Department of Biomaterials, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
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Tsaryk R, Gloria A, Russo T, Anspach L, De Santis R, Ghanaati S, Unger RE, Ambrosio L, Kirkpatrick CJ. Collagen-low molecular weight hyaluronic acid semi-interpenetrating network loaded with gelatin microspheres for cell and growth factor delivery for nucleus pulposus regeneration. Acta Biomater 2015; 20:10-21. [PMID: 25861947 DOI: 10.1016/j.actbio.2015.03.041] [Citation(s) in RCA: 86] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2014] [Revised: 03/07/2015] [Accepted: 03/18/2015] [Indexed: 12/19/2022]
Abstract
Intervertebral disc (IVD) degeneration is one of the main causes of low back pain. Current surgical treatments are complex and generally do not fully restore spine mobility. Development of injectable extracellular matrix-based hydrogels offers an opportunity for minimally invasive treatment of IVD degeneration. Here we analyze a specific formulation of collagen-low molecular weight hyaluronic acid (LMW HA) semi-interpenetrating network (semi-IPN) loaded with gelatin microspheres as a potential material for tissue engineering of the inner part of the IVD, the nucleus pulposus (NP). The material displayed a gel-like behavior, it was easily injectable as demonstrated by suitable tests and did not induce cytotoxicity or inflammation. Importantly, it supported the growth and chondrogenic differentiation potential of mesenchymal stem cells (MSC) and nasal chondrocytes (NC) in vitro and in vivo. These properties of the hydrogel were successfully combined with TGF-β3 delivery by gelatin microspheres, which promoted the chondrogenic phenotype. Altogether, collagen-LMW HA loaded with gelatin microspheres represents a good candidate material for NP tissue engineering as it combines important rheological, functional and biological features.
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Tsaryk R, Bartholomä NM, Simiantonaki N, Anspach L, Peters K, Heilmann C, Kirkpatrick CJ, Pröls F. Endoplasmic reticulum-resident chaperones modulate the inflammatory and angiogenic responses of endothelial cells. Br J Dermatol 2015; 173:416-27. [PMID: 25832777 DOI: 10.1111/bjd.13816] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/02/2015] [Indexed: 12/23/2022]
Abstract
BACKGROUND Wound healing depends on a well-balanced regulation of inflammation and angiogenesis. In chronic wounds the healing process is disturbed and inflammation persists. Regulation of wound closure is controlled by transmembrane and extracellular proteins, the folding and maturation of which occur in the endoplasmic reticulum (ER) by ER-resident chaperone machinery. OBJECTIVES To study the role of the ER-resident chaperones BiP/Grp78, its cochaperone Mdg1/ERdJ4, and Grp94 in chronic, nonhealing wounds. METHODS Immunohistochemical staining of these chaperones in individual human biopsies and investigation of the possible role of BiP and Mdg1 in endothelial cells, focusing on their inflammatory response and angiogenic potential. RESULTS In all chronic wounds investigated, the levels of these ER-resident chaperones were elevated in endothelial cells and leucocytes. The proangiogenic role of BiP has been shown in tumour growth studies before and was confirmed in this study. Proangiogenic activity of the cochaperone Mdg1 has been postulated before but could not be confirmed in this study. The chemokine tumour necrosis factor (TNF)-α was shown to trigger the presentation of proinflammatory adhesion molecules and the release of proinflammatory cytokines. Here we show that TNF-α does not affect endogenous chaperone levels, but that the ER-resident chaperones BiP and Mdg1 modulate the cellular TNF-α-induced proinflammatory response. CONCLUSIONS According to the presented data we assume that in chronic wounds upregulated levels of ER-resident chaperones might contribute to persistent inflammation in chronic wounds. Therapies to downregulate chaperone levels might provide a tool that switches the imbalanced chronic wound microenvironment from inflammation to healing.
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Affiliation(s)
- R Tsaryk
- Institute of Pathology, University Medical Center, Johannes Gutenberg University, Langenbeckstraße 1, 55131, Mainz, Germany
| | - N M Bartholomä
- Department of Rheumatology and Clinical Immunology, Medical University of Freiburg, Hugstetter Straße 55, 79106, Freiburg, Germany
| | - N Simiantonaki
- Zentrum für Pathologie Essen-Mitte, Am Deimelsberg 34a, 45276, Essen, Germany
| | - L Anspach
- Institute of Pathology, University Medical Center, Johannes Gutenberg University, Langenbeckstraße 1, 55131, Mainz, Germany
| | - K Peters
- Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, 18057, Rostock, Germany
| | - C Heilmann
- Department of Cardiovascular Surgery, Heart Center, University of Freiburg, Hugstetter Straße 55, 79106, Freiburg, Germany
| | - C J Kirkpatrick
- Institute of Pathology, University Medical Center, Johannes Gutenberg University, Langenbeckstraße 1, 55131, Mainz, Germany
| | - F Pröls
- Institute of Anatomy II, Medical University of Cologne, Joseph-Stelzmann-Straße 9, 50931, Cologne, Germany
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Freese C, Schreiner D, Anspach L, Bantz C, Maskos M, Unger RE, Kirkpatrick CJ. In vitro investigation of silica nanoparticle uptake into human endothelial cells under physiological cyclic stretch. Part Fibre Toxicol 2014; 11:68. [PMID: 25539809 PMCID: PMC4318365 DOI: 10.1186/s12989-014-0068-y] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2014] [Accepted: 11/14/2014] [Indexed: 12/18/2022] Open
Abstract
BACKGROUND In general the prediction of the toxicity and therapeutic efficacy of engineered nanoparticles in humans is initially determined using in vitro static cell culture assays. However, such test systems may not be sufficient for testing nanoparticles intended for intravenous application. Once injected, these nanoparticles are caught up in the blood stream in vivo and are therefore in continuous movement. Physical forces such as shear stress and cyclic stretch caused by the pulsatile blood flow are known to change the phenotype of endothelial cells which line the luminal side of the vasculature and thus may be able to affect cell-nanoparticle interactions. METHODS In this study we investigated the uptake of amorphous silica nanoparticles in primary endothelial cells (HUVEC) cultured under physiological cyclic stretch conditions (1 Hz, 5% stretch) and compared this to cells in a standard static cell culture system. The toxicity of varying concentrations was assessed using cell viability and cytotoxicity studies. Nanoparticles were also characterized for the induction of an inflammatory response. Changes to cell morphology was evaluated in cells by examining actin and PECAM staining patterns and the amounts of nanoparticles taken up under the different culture conditions by evaluation of intracellular fluorescence. The expression profile of 26 stress-related was determined by microarray analysis. RESULTS The results show that cytotoxicity to endothelial cells caused by silica nanoparticles is not significantly altered under stretch compared to static culture conditions. Nevertheless, cells cultured under stretch internalize fewer nanoparticles. The data indicate that the decrease of nanoparticle content in stretched cells was not due to the induction of cell stress, inflammation processes or an enhanced exocytosis but rather a result of decreased endocytosis. CONCLUSIONS In conclusion, this study shows that while the toxic impact of silica nanoparticles is not altered by stretch this dynamic model demonstrates altered cellular uptake of nanoparticles under physiologically relevant in vitro cell culture models. In particular for the development of nanoparticles for biomedical applications such improved in vitro cell culture models may play a pivotal role in the reduction of animal experiments and development costs.
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Affiliation(s)
- Christian Freese
- REPAIR-lab, Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz, European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Mainz, Germany.
| | - Daniel Schreiner
- REPAIR-lab, Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz, European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Mainz, Germany.
| | - Laura Anspach
- REPAIR-lab, Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz, European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Mainz, Germany.
| | | | | | - Ronald E Unger
- REPAIR-lab, Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz, European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Mainz, Germany.
| | - C James Kirkpatrick
- REPAIR-lab, Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz, European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Mainz, Germany.
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15
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Tsaryk R, Silva-Correia J, Oliveira JM, Unger RE, Landes C, Brochhausen C, Ghanaati S, Reis RL, Kirkpatrick CJ. Biological performance of cell-encapsulated methacrylated gellan gum-based hydrogels for nucleus pulposus regeneration. J Tissue Eng Regen Med 2014; 11:637-648. [DOI: 10.1002/term.1959] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2014] [Accepted: 09/17/2014] [Indexed: 01/08/2023]
Affiliation(s)
- Roman Tsaryk
- REPAIR Lab, Institute of Pathology, University Medical Center; Johannes Gutenberg University; Mainz Germany
| | - Joana Silva-Correia
- 3Bs Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho; Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine; Guimarães Portugal
- ICVS/3Bs-PT Government Associate Laboratory; Braga/Guimarães Portugal
| | - Joaquim Miguel Oliveira
- 3Bs Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho; Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine; Guimarães Portugal
- ICVS/3Bs-PT Government Associate Laboratory; Braga/Guimarães Portugal
| | - Ronald E. Unger
- REPAIR Lab, Institute of Pathology, University Medical Center; Johannes Gutenberg University; Mainz Germany
| | - Constantin Landes
- Department for Oral, Cranio-maxillofacial and Facial Plastic Surgery, Medical Center; Goethe University; Frankfurt Germany
| | - Christoph Brochhausen
- REPAIR Lab, Institute of Pathology, University Medical Center; Johannes Gutenberg University; Mainz Germany
| | - Shahram Ghanaati
- REPAIR Lab, Institute of Pathology, University Medical Center; Johannes Gutenberg University; Mainz Germany
- Department for Oral, Cranio-maxillofacial and Facial Plastic Surgery, Medical Center; Goethe University; Frankfurt Germany
| | - Rui L. Reis
- 3Bs Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho; Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine; Guimarães Portugal
- ICVS/3Bs-PT Government Associate Laboratory; Braga/Guimarães Portugal
| | - C. James Kirkpatrick
- REPAIR Lab, Institute of Pathology, University Medical Center; Johannes Gutenberg University; Mainz Germany
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Unger RE, Peters K, Sartoris A, Freese C, Kirkpatrick CJ. Human endothelial cell-based assay for endotoxin as sensitive as the conventional Limulus Amebocyte Lysate assay. Biomaterials 2014; 35:3180-7. [DOI: 10.1016/j.biomaterials.2013.12.059] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2013] [Accepted: 12/19/2013] [Indexed: 01/04/2023]
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Marei MK, Nagy NB, Saad MS, Zaky SH, Elbackly RM, Eweida AM, Alkhodary MA. Strategy for a Biomimetic Paradigm in Dental and Craniofacial Tissue Engineering. Biomimetics (Basel) 2013. [DOI: 10.1002/9781118810408.ch6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
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18
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Tsaryk R, Peters K, Unger RE, Feldmann M, Hoffmann B, Heidenau F, Kirkpatrick CJ. Improving cytocompatibility of Co28Cr6Mo by TiO2 coating: gene expression study in human endothelial cells. J R Soc Interface 2013; 10:20130428. [PMID: 23825117 DOI: 10.1098/rsif.2013.0428] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023] Open
Abstract
Cobalt-based materials are widely used for coronary stents, as well as bone and joint implants. However, their use is associated with high corrosion incidence. Titanium alloys, by contrast, are more biocompatible owing to the formation of a relatively inactive titanium oxide (TiO2) layer on their surface. This study was aimed at improving Co28Cr6Mo alloy cytocompatibility via sol-gel TiO2 coating to reduce metal corrosion and metal ion release. Owing to their role in inflammation and tissue remodelling around an implant, endothelial cells present a suitable in vitro model for testing the biological response to metallic materials. Primary human endothelial cells seeded on Co28Cr6Mo showed a stress phenotype with numerous F-actin fibres absent on TiO2-coated material. To investigate this effect at the gene expression level, cDNA microarray analysis of in total 1301 genes was performed. Compared with control cells, 247 genes were expressed differentially in the cells grown on Co28Cr6Mo, among them genes involved in proliferation, oxidative stress response and inflammation. TiO2 coating reduced the effects of Co28Cr6Mo on gene expression in endothelial cells, with only 34 genes being differentially expressed. Quantitative real-time polymerase chain reaction and protein analysis confirmed microarray data for selected genes. The effect of TiO2 coating can be, in part, attributed to the reduced release of Co(2+), because addition of CoCl2 resulted in similar cellular responses. TiO2 coating of cobalt-based materials, therefore, could be used in the production of cobalt-based devices for cardiovascular and skeletal applications to reduce the adverse effects of metal corrosion products and to improve the response of endothelial and other cell types.
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Affiliation(s)
- R Tsaryk
- Institute of Pathology, University Medical Center, Johannes Gutenberg University of Mainz, Mainz, Germany.
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The Role of Oxidative Stress in the Response of Endothelial Cells to Metals. SPRINGER SERIES IN BIOMATERIALS SCIENCE AND ENGINEERING 2013. [DOI: 10.1007/978-1-4614-4328-5_4] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
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Bulnheim U, Müller P, Neumann HG, Peters K, Unger RE, Kirkpatrick CJ, Rychly J. Endothelial cells stimulate osteogenic differentiation of mesenchymal stem cells on calcium phosphate scaffolds. J Tissue Eng Regen Med 2012; 8:831-40. [PMID: 23038605 DOI: 10.1002/term.1590] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2011] [Revised: 01/25/2012] [Accepted: 07/07/2012] [Indexed: 12/14/2022]
Abstract
The interaction of mesenchymal stem cells (MSCs) with endothelium in vivo is significant for regenerative processes in organisms. To design concepts for tissue engineering for bone regeneration based on this interaction, the osteogenic differentiation of human bone marrow-derived MSCs in a co-culture with human dermal microvascular endothelial cells (HDMECs) was studied. The experiments were focussed on the regulation of MSCs in a co-culture with HDMECs on different calcium phosphate scaffolds. Alkaline phosphatase (ALP) activity and mRNA expression of various osteogenic markers increased significantly when cells were co-cultured on materials with calcium phosphate scaffolds compared to tissue culture polystyrene or when MSCs were cultured alone. In addition, it was observed that the expression of osteopontin and osteocalcin was highly sensitive to the substrate for cell adhesion. Whereas these late osteogenic markers were down-regulated in co-cultures on polystyrene, they were up-regulated on calcium phosphate and moreover, were differentially expressed on the three calcium phosphate scaffolds tested. To enhance the osteogenic differentiation of MSCs in a co-culture, direct cell-cell interactions were required. Concerning molecular mechanisms in the interactions between both cell types, it was found that connexin 43 was expressed in contact sites and more apparently, endothelial cells grew over the MSCs, which facilitated direct cellular interactions mediated by various adhesion receptors. This study revealed significant findings for the design of implant materials suitable for regeneration of bone by stimulating the functional interaction of MSCs with endothelial cells.
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Affiliation(s)
- Ulrike Bulnheim
- Laboratory of Cell Biology, Medical Faculty, University of Rostock, Schillingallee 69, 18057, Rostock, Germany
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Silicon nitride films for the protective functional coating: blood compatibility and biomechanical property study. J Mech Behav Biomed Mater 2012; 16:9-20. [PMID: 23137618 DOI: 10.1016/j.jmbbm.2012.06.020] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2012] [Revised: 06/17/2012] [Accepted: 06/26/2012] [Indexed: 11/20/2022]
Abstract
Behaviors of silicon nitride films and their relation to blood compatibility and biomechanical have been interesting subjects to researchers. A systematic blood compatibility and biomechanical property investigation on the deposition of silicon-nitride films under varying N₂ and CF₄ flows was carried out by direct current unbalanced magnetron sputtering techniques. Significant role of surface property, chemical bonding state of silicon nitride film and blood compatibility, mechanical properties for the films were observed. The chemical bonding configurations, surface topography, contact angle and mechanical properties were characterized by means of X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and nano-indentation technique and CSEM pin-on-disk tribometer. Blood compatibility of the films was evaluated by platelet adhesion investigation. The results of the platelet adhesion tests shown that the effect of fluorine and nitrogen-doped revealed an intimate relationship between the ratio of polar component and dispersion component of the surface energy and its hemocompatibility. Si-N-O coating can be a great candidate for developing antithrombogenic surfaces in blood contacting materials. The chemical bonding state made an adjustment in microstructured surfaces, once in the totally wettable configuration, may improve the initial contact between platelet and biomedical materials, due to the appropriate ratio of dispersion component and polar component. To resist wear, biomedical components require coatings that are tough and hard, have low friction, and are bio-inert. The study suggests that by Si-N coating the metal surfaces could be a choice to prolong the life of the sliding pair Co-Cr-Mo alloy/UHMWPE implants.
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Hoppe A, Güldal NS, Boccaccini AR. A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics. Biomaterials 2011; 32:2757-74. [PMID: 21292319 DOI: 10.1016/j.biomaterials.2011.01.004] [Citation(s) in RCA: 1365] [Impact Index Per Article: 97.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2010] [Accepted: 01/04/2011] [Indexed: 01/08/2023]
Abstract
Several inorganic materials such as special compositions of silicate glasses, glass-ceramics and calcium phosphates have been shown to be bioactive and resorbable and to exhibit appropriate mechanical properties which make them suitable for bone tissue engineering applications. However, the exact mechanism of interaction between the ionic dissolution products of such inorganic materials and human cells are not fully understood, which has prompted considerable research work in the biomaterials community during the last decade. This review comprehensively covers literature reports which have investigated specifically the effect of dissolution products of silicate bioactive glasses and glass-ceramics in relation to osteogenesis and angiogenesis. Particularly, recent advances made in fabricating dense biomaterials and scaffolds doped with trace elements (e.g. Zn, Sr, Mg, and Cu) and investigations on the effect of these elements on the scaffold biological performance are summarized and discussed in detail. Clearly, the biological response to artificial materials depends on many parameters such as chemical composition, topography, porosity and grain size. This review, however, focuses only on the ion release kinetics of the materials and the specific effect of the released ionic dissolution products on human cell behaviour, providing also a scope for future investigations and identifying specific research needs to advance the field. The biological performance of pure and doped silicate glasses, phosphate based glasses with novel specific compositions as well as several other silicate based compounds are discussed in detail. Cells investigated in the reviewed articles include human osteoblastic and osteoclastic cells as well as endothelial cells and stem cells.
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Affiliation(s)
- Alexander Hoppe
- Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, Cauerstrasse 6, 91058 Erlangen, Germany
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23
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“In vivo” evaluation of the vascular pattern in oral peri-implant tissues. Arch Oral Biol 2011; 56:148-52. [DOI: 10.1016/j.archoralbio.2010.08.020] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2010] [Revised: 08/06/2010] [Accepted: 08/31/2010] [Indexed: 11/20/2022]
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Abstract
Blood vessels have a fundamental role both in skeletal homeostasis and in bone repair. Angiogenesis is also important for a successful bone engineering. Therefore, scaffolds should be tested for their ability to favour endothelial cell adhesion, proliferation and functions. The type of endothelial cell to use for in vitro assays should be carefully considered, because the properties of these cells may depend on their source. Morphological and functional relationships between endothelial cells and osteoblasts are evaluated with co-cultures, but this model should still be standardized, particularly for distinguishing the two cell types. Platelet-rich plasma and recombinant growth factors may be useful for stimulating angiogenesis.
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25
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The rapid anastomosis between prevascularized networks on silk fibroin scaffolds generated in vitro with cocultures of human microvascular endothelial and osteoblast cells and the host vasculature. Biomaterials 2010; 31:6959-67. [PMID: 20619788 DOI: 10.1016/j.biomaterials.2010.05.057] [Citation(s) in RCA: 157] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2010] [Accepted: 05/23/2010] [Indexed: 11/24/2022]
Abstract
The survival and functioning of a bone biomaterial upon implantation requires a rapidly forming and stably functioning vascularization that connects the implant to the recipient. We have previously shown that human microcapillary endothelial cells (HDMEC) and primary human osteoblast cells (HOS) in coculture on various 3-D bone biomaterial scaffolds rapidly distribute and self-assemble into a morphological structure resembling bone tissue. Endothelial cells form microcapillary-like structures containing a lumen and these were intertwined between the osteoblast cells and the biomaterial. This tissue-like self-assembly occurred in the absence of exogenously added angiogenic stimuli or artificial matrices. The purpose of this study was to determine whether this in vitro pre-formed microvasculature persists and functions in vivo and to determine how the host responds to the cell-containing scaffolds. The scaffolds with cocultures were implanted into immune-deficient mice and compared to scaffolds without cells or with HDMEC alone. Histological evaluation and immunohistochemical staining with human-specific antibodies of materials removed 14 days after implantation demonstrated that the in vitro pre-formed microcapillary structures were present on the silk fibroin scaffolds and showed a perfused lumen that contained red blood cells. This proved anastomosis with the host vasculature. Chimeric vessels in which HDMEC were integrated with the host's ingrowing (murine) capillaries were also observed. No HDMEC-derived microvessel structures or chimeric vessels were observed on implanted silk fibroin when precultured with HDMEC alone. In addition, there was migration of the host (murine) vasculature into the silk fibroin scaffolds implanted with cocultures, whereas silk fibroin alone or silk fibroin precultured only with HDMEC were nearly devoid of ingrowing host microcapillaries. Therefore, not only do the in vitro pre-formed microcapillaries in a coculture survive and anastomose with the host vasculature to become functioning microcapillaries after implantation, the coculture also stimulates the host capillaries to rapidly grow into the scaffold to vascularize the implanted material. Thus, this coculture-based pre-vascularization of a biomaterial implant may have great potential in the clinical setting to treat large bone defects.
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Sprindzuk M, Dmitruk A, Kovalev V, Bogush A, Tuzikov A, Liakhovski V, Fridman M. Computer-aided Image Processing of Angiogenic Histological. J Clin Med Res 2009; 1:249-61. [PMID: 22481986 PMCID: PMC3311439 DOI: 10.4021/jocmr2009.12.1274] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/27/2009] [Indexed: 12/11/2022] Open
Abstract
UNLABELLED This article reviews the questions regarding the image evaluation of angiogeneic histological samples, particularly the ovarian epithelial cancer. Review is focused on the principles of image analysis in the field of histology and pathology. The definition, classification, pathogenesis and angiogenesis regulation in the ovaries are also briefly discussed. It is hoped that the complex image analysis together with the patient's clinical parameters will allow an acquiring of a clear pathogenic picture of the disease, extension of the differential diagnosis and become a useful tool for the evaluation of drug effects. The challenge of the assessment of angiogenesis activity is the heterogeneity of several objects: parameters derived from patient's anamnesis as well as of pathology samples. The other unresolved problems are the subjectivity of the region of interest selection and performance of the whole slide scanning. KEYWORDS Angiogenesis; Image processing; Microvessel density; Cancer; Pathology.
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Affiliation(s)
- Matvey Sprindzuk
- United Institute of Informatics Problems, National Academy of Sciences of Belarus, Belarus
| | - Alexander Dmitruk
- United Institute of Informatics Problems, National Academy of Sciences of Belarus, Belarus
| | - Vassili Kovalev
- United Institute of Informatics Problems, National Academy of Sciences of Belarus, Belarus
| | - Armen Bogush
- United Institute of Informatics Problems, National Academy of Sciences of Belarus, Belarus
| | - Alexander Tuzikov
- United Institute of Informatics Problems, National Academy of Sciences of Belarus, Belarus
| | - Victor Liakhovski
- United Institute of Informatics Problems, National Academy of Sciences of Belarus, Belarus
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Campillo-Fernández AJ, Unger RE, Peters K, Halstenberg S, Santos M, Sánchez MS, Dueñas JMM, Pradas MM, Ribelles JLG, Kirkpatrick CJ. Analysis of the Biological Response of Endothelial and Fibroblast Cells Cultured on Synthetic Scaffolds with Various Hydrophilic/Hydrophobic Ratios: Influence of Fibronectin Adsorption and Conformation. Tissue Eng Part A 2009; 15:1331-41. [DOI: 10.1089/ten.tea.2008.0146] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Affiliation(s)
| | - Ronald E. Unger
- Institute of Pathology, Johannes Gutenberg University, Mainz, Germany
| | - Kirsten Peters
- Institute of Pathology, Johannes Gutenberg University, Mainz, Germany
| | - Sven Halstenberg
- Institute of Pathology, Johannes Gutenberg University, Mainz, Germany
| | - Marina Santos
- 3B's Research Group—Biomaterials, Biodegradables and Biomimetics, University of Minho, Braga, Portugal
| | - Manuel Salmerón Sánchez
- Center for Biomaterials, Universidad Politécnica de Valencia, Valencia, Spain
- Centro de Investigación Príncipe Felipe, Avda. Autopista del Saler 16, Valencia, Spain
| | | | - Manuel Monleón Pradas
- Center for Biomaterials, Universidad Politécnica de Valencia, Valencia, Spain
- Centro de Investigación Príncipe Felipe, Avda. Autopista del Saler 16, Valencia, Spain
| | - Jose Luis Gómez Ribelles
- Center for Biomaterials, Universidad Politécnica de Valencia, Valencia, Spain
- Centro de Investigación Príncipe Felipe, Avda. Autopista del Saler 16, Valencia, Spain
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Crosstalk between osteoblasts and endothelial cells co-cultured on a polycaprolactone-starch scaffold and the in vitro development of vascularization. Biomaterials 2009; 30:4407-15. [PMID: 19487022 DOI: 10.1016/j.biomaterials.2009.05.004] [Citation(s) in RCA: 155] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2009] [Accepted: 05/05/2009] [Indexed: 11/23/2022]
Abstract
The reconstruction of bone defects based on cell-seeded constructs requires a functional microvasculature that meets the metabolic demands of the engineered tissue. Therefore, strategies that augment neovascularization need to be identified. We propose an in vitro strategy consisting of the simultaneous culture of osteoblasts and endothelial cells on a starch-based scaffold for the formation of pre-vascular structures, with the final aim of accelerating the establishment of a vascular bed in the implanted construct. Human dermal microvascular endothelial cells (HDMECs) were co-cultured with human osteoblasts (hOBs) on a 3D starch-based scaffold and after 21 days of culture HDMEC aligned and organized into microcapillary-like structures. These vascular-like structures evolved from a cord-like configuration to a more complex branched morphology, had a lumen and stained in the perivascular region for type IV collagen. Genetic profiling of 84 osteogenesis-related genes was performed on co-culture vs. monoculture. Osteoblasts in co-culture showed a significant up-regulation of type I collagen and immunohistochemistry revealed that the scaffold was filled with a dense matrix stained for type I collagen. In direct contact with HDMEC hOBs secreted higher amounts of VEGF in relation to monoculture and the highest peak in the release profile correlated with the formation of microcapillary-like structures. The heterotypic communication between the two cell types was also assured by direct cell-cell contact as shown by the expression of the gap junction connexin 43. In summary, by making use of heterotypic cellular crosstalk this co-culture system is a strategy to form vascular-like structures in vitro on a 3D scaffold.
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Semenoff TADV, Semenoff Segundo A, de Figueiredo JAP. Biocompatibility of different intracanal medications in rat bucal submucosa tissue. J Appl Oral Sci 2009; 16:12-7. [PMID: 19089283 PMCID: PMC4327274 DOI: 10.1590/s1678-77572008000100004] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2007] [Accepted: 10/24/2007] [Indexed: 11/22/2022] Open
Abstract
The aim of this study was to analyze the buccal tissue responses of Wistar rats to 2% chlorhexidine solution, calcium hydroxide and the association of both products. For this purpose, 30 specimens were randomly implanted in the filtrum of the four upper and lower hemiarches with a polyethylene tube containing one of the following substances: 2% chlorhexidine solution, calcium hydroxide and 2% chlorhexidine solution (test groups); calcium hydroxide and distilled water and distilled water (control groups). Ten rats each were distributed according to time interval of evaluation at 7, 15 and 30 days. The histological sections were stained with Harris hematoxylin and eosin. Analysis was performed with an optical microscope at x100, x200 and x400 magnifications by an expert examiner blinded to the materials. The sections were classified by scores attributed to inflammatory events and by a ranking determined according to the severity of the inflammation. The results of the inflammatory events and severity ranking were submitted to the Kruskal-Wallis test at a 0.05 level of significance. No statistically significant difference occurred among the tested materials; however, all materials showed a decreased of severity with respect to longer time intervals.
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30
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Santos MI, Tuzlakoglu K, Fuchs S, Gomes ME, Peters K, Unger RE, Piskin E, Reis RL, Kirkpatrick CJ. Endothelial cell colonization and angiogenic potential of combined nano- and micro-fibrous scaffolds for bone tissue engineering. Biomaterials 2008; 29:4306-13. [DOI: 10.1016/j.biomaterials.2008.07.033] [Citation(s) in RCA: 152] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2008] [Accepted: 07/23/2008] [Indexed: 11/29/2022]
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Peters K, Unger RE, Stumpf S, Schäfer J, Tsaryk R, Hoffmann B, Eisenbarth E, Breme J, Ziegler G, Kirkpatrick CJ. Cell type-specific aspects in biocompatibility testing: the intercellular contact in vitro as an indicator for endothelial cell compatibility. JOURNAL OF MATERIALS SCIENCE. MATERIALS IN MEDICINE 2008; 19:1637-44. [PMID: 17914634 DOI: 10.1007/s10856-007-3227-y] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/18/2006] [Accepted: 10/27/2006] [Indexed: 05/17/2023]
Abstract
Endothelial cells cover the inner surface of blood vessels and form the interface between the blood and the tissues. Endothelial cells are involved in regulating barrier function, which is maintained by the interendothelial cell contacts. These interendothelial cell contacts are established by the interaction of different molecules. The maintenance of the barrier requires an appropriate signalling between these molecules. Thus, a number of different signalling pathways are integrated within interendothelial contacts. Since endothelial cells are important in tissue-implant interactions (especially for stent materials) this study examines the expression pattern of different interendothelial contact molecules to determine the usefulness in the analysis of biocompatibility in vitro. The effects of different pro-inflammatory and toxic stimuli and contact of human microvascular endothelial cells to metallic surfaces were examined for their impact on the pattern of interendothelial contact molecules. Striking modifications in the arrangement of these molecules were induced and the mode of modification was dependent on the tested compound. Thus, examining the pattern of expression of specific interendothelial contact molecules in vitro may be useful for testing the endothelial cell compatibility of biomaterials and their corrosion products.
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Affiliation(s)
- Kirsten Peters
- Institute of Pathology, Johannes Gutenberg-University, Langenbeckstr. 1, 55101 Mainz, Germany.
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Peters K, Unger RE, Gatti AM, Sabbioni E, Tsaryk R, Kirkpatrick CJ. Metallic nanoparticles exhibit paradoxical effects on oxidative stress and pro-inflammatory response in endothelial cells in vitro. Int J Immunopathol Pharmacol 2008; 20:685-95. [PMID: 18179741 DOI: 10.1177/039463200702000404] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
Particulate matter is associated with different human diseases affecting organs such as the respiratory and cardiovascular systems. Very small particles (nanoparticles) have been shown to be rapidly internalized into the body. Since the sites of internalization and the location of the detected particles are often far apart, a distribution via the blood stream must have occurred. Thus, endothelial cells, which line the inner surface of blood vessels, must have had direct contact with the particles. In this study we tested the effects of metallic nanoparticles (Co and Ni) on oxidative stress and pro-inflammatory response in human endothelial cells in vitro. Exposure to both nanoparticle types led to a concentration-dependent cytotoxic effect. However, the effects on oxidative stress and pro-inflammatory response differed dramatically. Due to the nanoparticle-induced effects, a comparison between metallic nanoparticle- and metal ion-treatment with the corresponding ions was made. Again, divergent effects of nanoparticles compared with the ions were observed, thus indicating differences in the signaling pathways induced by these compounds. These paradoxical responses to different metallic nanoparticles and ions demonstrate the complexity of nanoparticle-induced effects and suggest the need to design new strategies for nanoparticle toxicology.
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Affiliation(s)
- K Peters
- Institute of Pathology, Johannes Gutenberg University, Mainz, Germany.
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Thimm BW, Unger RE, Neumann HG, Kirkpatrick CJ. Biocompatibility studies of endothelial cells on a novel calcium phosphate/SiO
2
-xerogel composite for bone tissue engineering. Biomed Mater 2008; 3:015007. [DOI: 10.1088/1748-6041/3/1/015007] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
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James Kirkpatrick C, Fuchs S, Iris Hermanns M, Peters K, Unger RE. Cell culture models of higher complexity in tissue engineering and regenerative medicine. Biomaterials 2007; 28:5193-8. [PMID: 17761278 DOI: 10.1016/j.biomaterials.2007.08.012] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2007] [Accepted: 08/07/2007] [Indexed: 11/17/2022]
Abstract
Cell culture techniques have tended to be used in biomaterial research as a screening method prior to embarking on specific in vivo experimentation. This presentation aims at showing that it is possible to develop more sophisticated in vitro systems using primary human cells in co-culture with other cell types and biomaterials in a three-dimensional setting. While the predictive value of such systems is still not proven these models can be employed to unravel the complexity of biological systems in order to understand molecular mechanisms of cell-cell and cell-material interactions. The brief overview is under the headings of basic principles of relevant culture systems, the study of inflammation and the healing response, scenarios for specific biomaterial applications and future directions. How human endothelial cells can be usefully incorporated into more complex cell culture models is presented as an example of how relevant questions in tissue engineering and regenerative medicine can be addressed. The central tenet of this paper is that it is possible to refine in vitro methodology using cells of human origin to establish relevant assay systems that more closely simulate the cellular and molecular microenvironment encountered in a specific situation of regeneration using biomaterials.
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Peters K, Unger RE, Kamp G, Rychly J, Kirkpatrick CJ. In vitro methodologies to evaluate biocompatibility: Status quo and perspectives. Toxicology 2007. [DOI: 10.1016/j.tox.2007.06.016] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
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Unger RE, Sartoris A, Peters K, Motta A, Migliaresi C, Kunkel M, Bulnheim U, Rychly J, Kirkpatrick CJ. Tissue-like self-assembly in cocultures of endothelial cells and osteoblasts and the formation of microcapillary-like structures on three-dimensional porous biomaterials. Biomaterials 2007; 28:3965-76. [PMID: 17582491 DOI: 10.1016/j.biomaterials.2007.05.032] [Citation(s) in RCA: 320] [Impact Index Per Article: 17.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2007] [Accepted: 05/30/2007] [Indexed: 11/18/2022]
Abstract
The survival and functioning of a bone biomaterial requires a rapid and stable vascularization after implantation. However, the mechanisms involved in the context of the complex healing microenvironment are poorly understood. To evaluate the vascularization potential of bone biomaterials, angiogenic stimuli were added to human dermal microvascular endothelial cells (HDMEC) growing on three-dimensional (3-D) bone biomaterials consisting of porous hydroxyapatite, porous calcium phosphate, porous nickel-titanium, successfully being used in humans, and also silk fibroin nets. HDMEC did not migrate to form microcapillary-like structures as they did on cell culture plastic. In cocultures of HDMEC and primary human osteoblast cells (HOS) or the human osteoblast-like cell line MG-63 on these biomaterials, a tissue-like self-assembly of cells occurred with time, with endothelial cells forming microcapillary-like structures containing a lumen and giving a strong PECAM-1 expression at cell interfaces. These microcapillary-like structures were intertwined between cell layers of osteoblasts and did not form when exogenous angiogenic stimuli were added to these cocultures. The life span of HDMEC was also significantly enhanced by coculture; with HDMEC being present for up to at least 42 days, compared to the monoculture where cells began to die rapidly after 1 week without passage. This coculture system may be applicable to a prevascularization strategy for biomaterials prior to implantation. Irrespective of this, the coculture model holds promise for studies to deepen our understanding of bone regeneration on 3-D substrates. Most importantly, these data raise important questions concerning the exact nature of pro-angiogenic drug- or gene-delivery systems to be incorporated into scaffolds. Our results underline the necessity to take into account the in situ production of growth factors by invading mesenchymal cells in the regenerative niche.
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Affiliation(s)
- Ronald E Unger
- Institute of Pathology, REPAIR Lab, Johannes Gutenberg University, Mainz 55101, Germany.
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Bahramsoltani M, Plendl J. Different ways to antiangiogenesis by angiostatin and suramin, and quantitation of angiostatin-induced antiangiogenesis. APMIS 2007; 115:30-46. [PMID: 17223849 DOI: 10.1111/j.1600-0463.2007.apm_405.x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Angiogenesis, i.e. sprouting of new vessels, their remodelling and regression, is a prerequisite for growth and differentiation of organs and tissues. It is involved in many pathological processes, particularly growth and metastasis of tumours. Angiostatic therapy is a promising new strategy in the treatment of cancer. Angiogenesis inhibitors could intervene in the different phases of the angiogenic cascade, i.e. migration, proliferation, differentiation and three-dimensional organisation of endothelial cells, to inhibit the generation of tumour vessels. The aim of this study was to explore whether in a previously validated in vitro model for quantitation of angiogenesis the effects of the angiostatic factors angiostatin and suramin can be investigated and quantified. Examination of angiostatin and suramin showed that angiostatin-induced antiangiogenesis resulted in inverse angiogenesis. The addition of suramin initially resulted in increased angiogenesis. However, long-term incubation ultimately led to disintegration of endothelial structures, thus establishing the angiostatic effects of suramin. Antiangiogenesis was not only quantified using the previously validated method. It also lent itself to assessment of the extent of antiangiogenesis within the various phases of the angiogenic cascade. This method may therefore be employed in trial studies of potential angiostatic substances and related cellular mechanisms.
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Kirkpatrick CJ, Fuchs S, Peters K, Brochhausen C, Hermanns MI, Unger RE. Visions for Regenerative Medicine: Interface Between Scientific Fact and Science Fiction. Artif Organs 2006; 30:822-7. [PMID: 17026583 DOI: 10.1111/j.1525-1594.2006.00306.x] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
This article gives a brief overview of the authors' views on the future development of tissue engineering with respect to the challenges both to the materials and life sciences. Emphasis will be placed on the advantages of three-dimensional bioresorbable polymers in combination with relevant molecular cues and the application of autologous stem or progenitor cells. There is a requirement for much more diversity in the synthesis of so-called "intelligent" materials, which respond to external stimuli, as well as the development of novel drug and gene delivery systems. In addition, much more basic research is necessary in developmental biology and the application of modern cell and molecular biology to biomaterial questions.
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Affiliation(s)
- C James Kirkpatrick
- Laboratory for REgenerative PAthology & Interface Research (REPAIR-lab), Institute of Pathology, Johannes Gutenberg University Mainz, Germany.
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Laschke MW, Harder Y, Amon M, Martin I, Farhadi J, Ring A, Torio-Padron N, Schramm R, Rücker M, Junker D, Häufel JM, Carvalho C, Heberer M, Germann G, Vollmar B, Menger MD. Angiogenesis in Tissue Engineering: Breathing Life into Constructed Tissue Substitutes. ACTA ACUST UNITED AC 2006; 12:2093-104. [PMID: 16968151 DOI: 10.1089/ten.2006.12.2093] [Citation(s) in RCA: 380] [Impact Index Per Article: 20.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Long-term function of three-dimensional (3D) tissue constructs depends on adequate vascularization after implantation. Accordingly, research in tissue engineering has focused on the analysis of angiogenesis. For this purpose, 2 sophisticated in vivo models (the chorioallantoic membrane and the dorsal skinfold chamber) have recently been introduced in tissue engineering research, allowing a more detailed analysis of angiogenic dysfunction and engraftment failure. To achieve vascularization of tissue constructs, several approaches are currently under investigation. These include the modification of biomaterial properties of scaffolds and the stimulation of blood vessel development and maturation by different growth factors using slow-release devices through pre-encapsulated microspheres. Moreover, new microvascular networks in tissue substitutes can be engineered by using endothelial cells and stem cells or by creating arteriovenous shunt loops. Nonetheless, the currently used techniques are not sufficient to induce the rapid vascularization necessary for an adequate cellular oxygen supply. Thus, future directions of research should focus on the creation of microvascular networks within 3D tissue constructs in vitro before implantation or by co-stimulation of angiogenesis and parenchymal cell proliferation to engineer the vascularized tissue substitute in situ.
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Affiliation(s)
- Matthias W Laschke
- Institute for Clinical and Experimental Surgery, University of Saarland, Homburg, Germany.
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Laschke MW, Harder Y, Amon M, Martin I, Farhadi J, Ring A, Torio-Padron N, Schramm R, Rücker M, Junker D, Hä JM, Carvalho C, Heberer M, Germann G, Vollmar B, Menger MD. Angiogenesis in Tissue Engineering: Breathing Life into Constructed Tissue Substitutes. ACTA ACUST UNITED AC 2006. [DOI: 10.1089/ten.2006.12.ft-130] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
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Kirkpatrick CJ, Peters K, Hermanns MI, Bittinger F, Krump-Konvalinkova V, Fuchs S, Unger RE. In vitro methodologies to evaluate biocompatibility: status quo and perspective. ACTA ACUST UNITED AC 2005. [DOI: 10.1016/j.rbmret.2005.04.008] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
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Unger RE, Peters K, Huang Q, Funk A, Paul D, Kirkpatrick CJ. Vascularization and gene regulation of human endothelial cells growing on porous polyethersulfone (PES) hollow fiber membranes. Biomaterials 2005; 26:3461-9. [PMID: 15621235 DOI: 10.1016/j.biomaterials.2004.09.047] [Citation(s) in RCA: 66] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2004] [Accepted: 09/08/2004] [Indexed: 01/04/2023]
Abstract
Open-cell hollow fibers made of polyethersulfone (PES) manufactured in the absence of solvents with pore diameters smaller than 100 microm were examined for vascularization by human endothelial cells. The goal of this study was to determine whether the 3-D porous character of the PES surface affected human endothelial cell morphology and functions. Freshly isolated human endothelial cells from the skin (HDMEC), from the lung (HPMEC) and from umbilical cords (HUVEC) and two human endothelial cell lines, HPMEC-ST1.6R and ISO-HAS.c1 were added to PES fibers and cell adherence and growth was followed by confocal laser scanning microscopy. Prior coating of PES with gelatin or fibronectin was necessary for adhesion and spreading of cells over the uneven porous surface with time. Confluent cells exhibited typical strong PECAM-1 expression at cell-cell borders. Little expression of the activation markers E-selectin, ICAM-1, and VCAM-1 was observed by RT-PCR of endothelial cells growing on PES. However, after stimulation for 4h by LPS, activation of these markers was observed and it was shown by immunofluorescent staining that induction occurred in most of the cells, thus confirming an intact functionality. Finally, cells growing as a monolayer on PES migrated to form microvessel-like structures when placed under conditions that stimulated angiogenesis. Thus, human endothelial cells grown on fibronectin-coated PES fibers retain important endothelial-cell specific morphological and functional properties and PES may serve as a useful biomaterial in tissue engineering and biotechnology applications.
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Affiliation(s)
- Ronald E Unger
- Institute of Pathology, Johannes Gutenberg University, Langenbeckstr. 1, 55101 Mainz, Germany.
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Unger RE, Huang Q, Peters K, Protzer D, Paul D, Kirkpatrick CJ. Growth of human cells on polyethersulfone (PES) hollow fiber membranes. Biomaterials 2005; 26:1877-84. [PMID: 15576161 DOI: 10.1016/j.biomaterials.2004.05.032] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2004] [Accepted: 05/27/2004] [Indexed: 11/28/2022]
Abstract
A novel material of porous hollow fibers made of polyethersulfone (PES) was examined for its ability to support the growth of human cells. This material was made in the absence of solvents and had pore diameters smaller than 100 microm. Human cell lines of different tissue and cell types (endothelial, epithelial, fibroblast, glial, keratinocyte, osteoblast) were investigated for adherence, growth, spread and survival on PES by confocal laser microscopy after staining of the cells with Calcein-AM. Endothelial cell attachment and growth required pre-coating PES with either fibronectin or gelatin. The other cell types exhibited little difference in growth, spread or survival on coated or uncoated PES. All the cells readily adhered and spread on the outer, inner and cut surfaces of PES. With time confluent monolayers of cells covered the available surface area of PES and in some cases cells grew as multilayers. Many of the cells were able to survive on the PES for up to 7 weeks and in some cases growth was so extensive that the underlying PES was no longer visible. Scanning electron microscope observations of cells on the materials correlated with the confocal morphometric data. Thus, PES is a substrate for the growth of many different types of human cells and may be a useful scaffolding material for tissue engineering.
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Affiliation(s)
- R E Unger
- Institute of Pathology, Johannes Gutenberg University, Mainz, Langenbeckstr. 1, 55101, Mainz, Germany.
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Sun XT, Zhang MY, Shu C, Li Q, Yan XG, Cheng N, Qiu YD, Ding YT. Differential gene expression during capillary morphogenesis in a microcarrier-based three-dimensional in vitro model of angiogenesis with focus on chemokines and chemokine receptors. World J Gastroenterol 2005; 11:2283-90. [PMID: 15818740 PMCID: PMC4305813 DOI: 10.3748/wjg.v11.i15.2283] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/13/2004] [Revised: 03/14/2004] [Accepted: 04/13/2004] [Indexed: 02/06/2023] Open
Abstract
AIM To globally compare the gene expression profiles during the capillary morphogenesis of human microvascular endothelial cells (HMVECs) in an in vitro angiogenesis system with affymetrix oligonucleotide array. METHODS A microcarrier-based in vitro angiogenesis system was developed, in which ECs migrated into the matrix, proliferated, and formed capillary sprouts. The sprouts elongated, branched and formed networks. The total RNA samples from the HMVECs at the selected time points (0.5, 24, and 72 h) during the capillary morphogenesis were used for microarray analyses, and the data were processed with the softwares provided by the manufacturers. The expression patterns of some genes were validated and confirmed by semi-quantitative RT-PCR. The regulated genes were grouped based on their molecular functions and expression patterns, and among them the expression of chemokines and chemokine receptors was specially examined and their functional implications were analyzed. RESULTS A total of 1 961 genes were up- or down-regulated two-folds or above, and among them, 468 genes were up- or down-regulated three-folds or above. The regulated genes could be grouped into categories based on their molecular functions, and were also clustered into six groups based on their patterns of expression. As for chemokines and chemokine receptors, CXCL1/GRO-alpha, CXCL2/GRO-beta, CXCL5/ENA-78, CXCL6/GCP2, IL-8/CXCL8, CXCL12/SDF-1, CXCL9/Mig, CXC11/ITAC, CX3CL1/fractalkine, CCL2/MCP-1, CCL3, CCL5/RANTES, CCL7, CCL15, CCL21, CCL23, CCL28, and CCR1, CCR9, CXCR4 were identified. Moreover, these genes demonstrated different changing patterns during the capillary morphogenesis, which implied that they might have different roles in the sequential process. Among the chemokines identified, CCL2/MCP-1, CCL5/RANTES and CX3CL1 were specially up-regulated at the 24-h time point when the sprouting characterized the morphological change. It was thus suggested that they might exert crucial roles at the early stage of angiogenesis. CONCLUSION The present study demonstrates a global profile of gene expression during endothelial capillary morphogenesis, and the results provide us much information about the molecular mechanisms of angiogenesis, with which further evaluation of individual genes can be conducted.
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Affiliation(s)
- Xi-Tai Sun
- Department of Hepatobiliary Surgery, Affiliated Drum Tower Hospital of Medical College, Hepatobiliary Research Institute, Nanjing University, Nanjing 210008, Jiangsu Province, China
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Peters K, Schmidt H, Unger RE, Kamp G, Pröls F, Berger BJ, Kirkpatrick CJ. Paradoxical effects of hypoxia-mimicking divalent cobalt ions in human endothelial cells in vitro. Mol Cell Biochem 2005; 270:157-66. [PMID: 15792365 DOI: 10.1007/s11010-005-4504-z] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Divalent cobalt ions (Co2+) induce the expression of hypoxia responsive genes and are often used in cell biology to mimic hypoxia. In this in vitro study we compared the effects of hypoxia and Co2+ on human endothelial cells and examined processes that are stimulated in hypoxia in vivo (proliferation and angiogenesis). We analyzed the expression of the hypoxia-inducible factor-1alpha (HIF-1alpha) under different hypoxic conditions (3% and nearly 0% O2) and Co2+ -concentrations (0.01-0.7 mM). As in hypoxia, the amount of HIF-1alpha protein was enhanced by exposure to Co2+ (did not correlate with mRNA amount). however, contrary to the results of hypoxia, in vitro-angiogenesis was inhibited after exposure to even low Co2+-concentrations (> or =0.01 mM). This led to the conclusion that although hypoxia signaling after Co2+ -exposure took place, further yet unknown Co2+ -induced event(s) must have occurred.
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Affiliation(s)
- Kirsten Peters
- Institute of Pathology, Johannes Gutenberg-University, Langenbeckstr. 1, 55101 Mainz, Germany.
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Hoffmann B, Kokott A, Shafranska O, Detsch R, Winter S, Eisenbarth E, Peters K, Breme J, Kirkpatrick CJ, Ziegler G. Einfluss einer TiO2-Beschichtung auf Biokompatibilität, Korrosionsund Auslaugverhalten verschiedener Implantatlegierungen / Corrosion behaviour, metal release and biocompatibility of implant materials coated by TiO2-sol gel chemistry. BIOMED ENG-BIOMED TE 2005; 50:320-9. [PMID: 16300047 DOI: 10.1515/bmt.2005.045] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Alloys based on titanium or cobalt have been used as implant materials for decades with good success. Because of their natural oxide layer these alloys reveal good corrosion behaviour. In contact with physiological solution metal release takes place, which can cause inflammation. Coatings can improve the corrosion behaviour. In this study Ti6Al4V and Co28Cr6Mo alloys, which are frequently used as implant materials, were tested. Polished discs of these alloys and polished discs, which were coated with TiO2-layers by sol-gel chemistry, were compared regarding their corrosion behaviour and metal ion releasing. The releasing of Al, V, Ti, Co, Cr and Mo was quantified by ICP-MS analysis. The TiO2-coating reduced the release of all ions except of the Al-ion. Both alloys showed a deviating kinetic of ion releasing. In addition, cell response (cell vitality, cell proliferation, endothelial marker CD31 and actin allocation) of osteoblasts and endothelial cells were investigated.
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Affiliation(s)
- B Hoffmann
- Friedrich-Baur-Forschungsinstitut für Biomaterialien, Universität Bayreuth, Ludwig-Thoma-Str. 36c, 95440 Bayreuth.
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Unger RE, Peters K, Wolf M, Motta A, Migliaresi C, Kirkpatrick CJ. Endothelialization of a non-woven silk fibroin net for use in tissue engineering: growth and gene regulation of human endothelial cells. Biomaterials 2004; 25:5137-46. [PMID: 15109837 DOI: 10.1016/j.biomaterials.2003.12.040] [Citation(s) in RCA: 144] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2003] [Accepted: 12/08/2003] [Indexed: 02/08/2023]
Abstract
We have previously shown that a biomaterial consisting of a non-woven fibroin net produced from silk (Bombyx mori) cocoons is an excellent scaffolding material for a wide variety of human cells of different tissue types. Endothelialization must take place for a biomaterial to be successful after implantation. Therefore, primary human endothelial cells and the human endothelial cell lines, HPMEC-ST1.6R and ISO-HAS-1, were examined for adherence and growth patterns on the fibroin nets by confocal laser scanning microscopy after vital staining of the cells and by electron microscopy. Endothelial cells adhered and spread along individual fibers of the nets and did not fill the gaps between individual fibers. Higher attachment and growth coverage was obtained if nets were first coated with gelatin, fibronectin or collagen type I. Proinflammatory markers of endothelial cells on the fibers exhibited a non-activated state and LPS-stimulated cells exhibited activation of these markers. Furthermore, a typical PECAM-1 localization at cell-cell contacts was observed. Scanning electron microscopic examination of fibroin nets after removal of cells did not demonstrate any changes to the fibroin structure. HUVEC and HDMEC on fibroin nets embedded in collagen type I gels formed microvessel-like structures. Thus, silk fibroin nets are a highly endothelial cell-compatible scaffolding material that support the growth, normal and inducible cell functions and angiogenesis potential of human endothelial cells in vitro similar to that observed in vivo.
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Affiliation(s)
- R E Unger
- Institute of Pathology, Johannes Gutenberg University, Langenbeckstr. 1, Mainz 55101, Germany.
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Sun XT, Ding YT, Yan XG, Wu LY, Li Q, Cheng N, Qiu YD, Zhang MY. Angiogenic synergistic effect of basic fibroblast growth factor and vascular endothelial growth factor in an in vitro quantitative microcarrier-based three-dimensional fibrin angiogenesis system. World J Gastroenterol 2004; 10:2524-8. [PMID: 15300897 PMCID: PMC4572154 DOI: 10.3748/wjg.v10.i17.2524] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To develop an in vitro three-dimensional (3-D) angiogenesis system to analyse the capillary sprouts induced in response to the concentration ranges of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) and to quantify their synergistic activity.
METHODS: Microcarriers (MCs) coated with human microvascular endothelial cells (HMVECs) were embedded in fibrin gel and cultured in 24-well plates with assay media. The growth factors bFGF, or VEGF, or both were added to the system. The wells (n = 8/group) were digitally photographed and the average length of capillary-like sprouts (ALS) from each microcarrier was quantitated.
RESULTS: In aprotinin-stabilized fibrin matrix, human microvascular endothelial cells on the MCs invaded fibrin, forming sprouts and capillary networks with lumina. The angiogenic effects of bFGF or VEGF were dose-dependent in the range from 10 to 40 ng/mL. At d 1, 10 ng/mL of bFGF and VEGF induced angiogenesis with an ALS of 32.13 ± 16.6 μm and 43.75 ± 27.92 μm, respectively, which were significantly higher than that of the control (5.88 ± 4.45 μm, P < 0.01), and the differences became more significant as the time increased. In addition, the combination of 10 ng/mL of bFGF and VEGF each induced a more significant effect than the summed effects of bFGF (10 ng/mL) alone and VEGF (10 ng/mL) alone when analyzed using SPSS system for general linear model (GLM) (P = 0.011), and that also exceeded the effects by 20 ng/mL of either bFGF or VEGF.
CONCLUSION: A microcarrier-based in vitro three-dimensional angiogenesis model can be developed in fibrin. It offers a unique system for quantitative analysis of angiogenesis. Both bFGF and VEGF exert their angiogenic effects on HMVECs synergistically and in a dose-dependent manner.
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Affiliation(s)
- Xi-Tai Sun
- Department of Hepatobiliary Surgery, Affiliated Gu Lou Hospital of Medical College, Hepatobiliary Research Institute, Nanjing, Jiangsu Province, China
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Berger BJ, Müller TS, Buschmann IR, Peters K, Kirsch M, Christ B, Pröls F. High levels of the molecular chaperone Mdg1/ERdj4 reflect the activation state of endothelial cells. Exp Cell Res 2003; 290:82-92. [PMID: 14516790 DOI: 10.1016/s0014-4827(03)00316-1] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Mdg1/ERdj4, a mammalian chaperone that belongs to the HSP40 protein family, has been reported to be located in the endoplasmic reticulum (ER), is induced by ER stress, and protects ER stressed cells from apoptosis. Here we show that under normal physiological conditions, Mdg1/ERdj4 is expressed at various levels in the vasculature due to different activation states of the endothelium. To elucidate the stimuli that induce ER stress and thus upregulate Mdg1/ERdj4, we investigated the effect of several endothelium specific stressors on its expression. Mdg1/ERdj4 mRNA is induced by activated macrophages, by nitric oxide (NO) and heat shock, and during terminal cell differentiation, whereas shear stress does not affect Mdg1/ERdj4 expression levels. While the mRNA stability of BiP/GRP78 is unaffected in ER stressed cells, the stability of Mdg1/ERdj4 mRNA is prolonged during ER stress resulting in rapid increases and high levels of Mdg1/ERdj4 mRNA. Mdg1/ERdj4 protein is localized in the ER under control conditions. While heat shock induces a rapid translocation of Mdg1/ERdj4 to the nucleoli, no translocation could be observed during ER stress. This indicates that Mdg1/ERdj4 protein has diverse mechanisms to protect stressed cells from apoptosis.
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Affiliation(s)
- Bernhard J Berger
- Institute of Anatomy and Cell Biology II, Albert Ludwigs-University, 79104 Freiburg, Germany
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