Neutrophil-mediated enhancement of angiogenesis and osteogenesis in a novel triple cell co-culture model with endothelial cells and osteoblasts
Authors: Thanuja D K Herath
C James Kirkpatrick
Bee Tin Goh
Anis Larbi
Swee Hin Teoh
Journal: Journal of tissue engineering and regenerative medicine (2018)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: VEGF; angiogenesis; neutrophils; osteogenesis; tissue engineering
Affiliations: National Dental Centre Singapore, Singapore., Singapore Immunology Network, Agency for Science, Technology and Research, Singapore., Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore., REPAIR-Lab, Institute of Pathology, University Medical Centre, Johannes Gutenberg University, Mainz, Germany.

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. 

Retracted: No

PubPeer comments:
 Fig 4 A,B.  Influence of miR-424 on cell proliferation, migration and invasion when TNFAIP1 was silenced in A549 cells. A-C: TNFAIP1 silence significantly increased the number of migrated and invasive cells, as well as the number of colony for A549 cells. On the other side, the number of migrated cells, invasive cells and colony for A549 cells were significantly increased with miR-424 mimic addition compared to the control, however, the three kinds of cells were all declined approximately to that in control when cells were treated with miR-424 inhibitor . ![file] Unexpected similarities with Fig 3B from [Zhang et al (2016)] and Fig 3B from [Cao et al (2018)] . ![file] , 
 Fig. 1A,C.  Influence of miR-424 expression on A549 cells migration and invasion. A-B: Transwell assay showed that the migrated cells was drastically increased when cells were transfected with miR-424 than that in control, however, when cells were transfected with miR-424 inhibitor, there was no significant difference of migrated cells between control and Inhibitor group; C-D: miR424 overexpression significantly enhanced A549 cells invasion compared to the control, while this promoting effect was decreased in cells transfected with miR-424 inhibitor . ![file] Recognisably the same style as in [Zhang et al (2019)] , [Zhuang, Liu and Wu (2019)] and [Kong et al (2019)] . ![] The task of spotting matching panels is left as an exercise for the reader. , 
 Fig. 6B.  Influence of miR-424 on cell migration and invasion when TNFAIP1 was silenced in H1975 cells. ... B: qRT-PCR analysis and western blotting showed that TNFAIP1 level was negatively regulated by miR-424 . ![file] The tadpole blobs, and the presence of the same background in both bands at increased contrast, hint that this  Western blot may have been provided by an outside contractor. If I may plagiarise [a comment by Elisabeth Bik] : This paper belongs to a set of over 400 papers (as per February 2020) that share very similar Western blots with tadpole-like shaped bands, the same background pattern, and striking similarities in title structures, paper layout, bar graph design, and - in a subset - flow cytometry panels. Despite these similarities, these papers are authored by researchers from different departments and institutes, with almost no overlap in authors. The list of these papers is available here: https: docs.google.com spreadsheets d 1KXqTAyl4j-jVorFPMD2XRpr76LcIKJ0CVyIvRj0exYQ edit?usp=sharing A more detailed description of this set of papers, suspected to all be generated by a common source, a paper mill , can be found here: - https: forbetterscience.com 2020 01 24 the-full-service-paper-mill-and-its-chinese-customers (January 2020) - https: scienceintegritydigest.com 2020 02 21 the-tadpole-paper-mill (February 2020) The same paper mill has been described in these papers: - Jana Christopher - Systematic fabrication of scientific images revealed - FEBS Letters (2018) - https: febs.onlinelibrary.wiley.com doi full 10.1002 1873-3468.13201 - Jennifer A Byrne and Jana Christopher - Digital magic, or the dark arts of the 21st centuryu2014how can journals and peer reviewers detect manuscripts and publications from paper mills? - FEBS Letters (2020) - https: febs.onlinelibrary.wiley.com doi full 10.1002 1873-3468.13747 , 
 Fig 2D: ![file] Fig 2C from [Cao et al (2018)] : ![file] , 
 Recently retracted: https: www.cellphysiolbiochem.com Articles 000414 PDF 000414.pdf Kudos to the Editor for perseverance. --------------------------------- The article has been retracted by the current and former Publishers and the Editor. After the publication of this article, concerns were raised about the integrity of some of the data presented in figures 1, 4, and 6. The authors were unresponsive to our requests to provide the original data and respond to the concerns within the given timeframe. The matter was raised to the corresponding authoru2018s institution who did not respond to our request for an investigation. Given the serious nature of the concerns raised, the article has been retracted by the current and former publishers. The authors have not responded to our correspondence regarding this retraction despite multiple attempts of contact. ,