Targeting gene expression during the early bone healing period in the mandible: A base for bone tissue engineeringJournal of Cranio-Maxillofacial Surgery

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Authors
Benedicta E. Beck-Broichsitter, Anneke N. Werk, Ralf Smeets, Alexander Gröbe, Max Heiland, Ingolf Cascorbi, Jörg Wiltfang, Robert Häsler, Stephan T. Becker
Year
2015
DOI
10.1016/j.jcms.2015.06.015
Subject
Surgery / Oral Surgery / Otorhinolaryngology

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Accepted Manuscript

Targeting gene expression during the early bone healing period in the mandible: a base for bone tissue engineering

Benedicta E. Beck-Broichsitter, MD, DMD, Research Assistant, Anneke N. Werk,

PhD, Ralf Smeets, MD, DMD, PhD, Alexander Gröbe, MD, DMD, PhD, Max Heiland,

MD, DMD, PhD, Ingolf Cascorbi, MD, PhD, Jörg Wiltfang, MD, DMD, PhD, Robert

Häsler, PhD, Stephan T. Becker, MD, DMD, PhD

PII: S1010-5182(15)00189-4

DOI: 10.1016/j.jcms.2015.06.015

Reference: YJCMS 2089

To appear in: Journal of Cranio-Maxillo-Facial Surgery

Received Date: 25 March 2015

Accepted Date: 16 June 2015

Please cite this article as: Beck-Broichsitter BE, Werk AN, Smeets R, Gröbe A, Heiland M, Cascorbi

I, Wiltfang J, Häsler R, Becker ST, Targeting gene expression during the early bone healing period in the mandible: a base for bone tissue engineering, Journal of Cranio-Maxillofacial Surgery (2015), doi: 10.1016/j.jcms.2015.06.015.

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ACCEPTED MANUSCRIPT

Targeting gene expression during the early bone healing period in the mandible: a base for bone tissue engineering

Benedicta E. Beck-Broichsitter1¶, MD, DMD; Anneke N. Werk2¶, PhD; Ralf Smeets1, MD, DMD, PhD;

Alexander Gröbe1, MD, DMD, PhD; Max Heiland1, MD, DMD, PhD; Ingolf Cascorbi2, MD, PhD; Jörg

Wiltfang3, MD, DMD, PhD; Robert Häsler4, PhD; Stephan T. Becker3, MD, DMD, PhD 1

University Medical Center Hamburg-Eppendorf, Department of Oral and Maxillofacial Surgery, Martinistraße 52, Campus Forschung Gebäude N27, 20246 Hamburg, Germany 2

Schleswig-Holstein University Hospital, Institute of Clinical and Experimental Pharmacology, Arnold-HellerStraße 3, Haus 30, 24105 Kiel, Germany 3

Schleswig-Holstein University Hospital, Department of Oral and Maxillofacial Surgery, Arnold-Heller-Straße 3, Haus 26, 24105 Kiel, Germany 4

Institute of Clinical Molecular Biology, Center for Molecular Biosciences, Christian Albrechts University of

Kiel, Am Botanischen Garten 11, 24118 Kiel, Germany ¶ contributed equally

This work is attributed to

Department of Oral and Maxillofacial Surgery, Schleswig-Holstein University Hospital

Head: Jörg Wiltfang, MD, DMD, PhD

Arnold-Heller-Straße 3, Haus 26, 24105 Kiel, Germany

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Corresponding Author:

Benedicta Elisabeth Beck-Broichsitter, MD, DMD, Research Assistant1,2

University Medical Center Hamburg-EppendorfAf

Martinistraße 52, Campus Forschung Gebäude N27 20246 Hamburg, Germany

Telephone: 0049-40-7410-53251

Fax: 0049-40-7410-55467

Email: b.beck-broichsitter@uke.de

Sources of Support (Grants)

This study was not financially supported by grants. 1

Permanent Address 2

Affiliation Address:

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SUMMARY

Purpose: Although bone tissue engineering techniques have become more and more sophisticated than in the past, natural bone healing mechanisms have not been sufficiently considered for further improvement of these techniques so far. We used an established animal model with transcriptome analysis to generate an unbiased picture of early bone healing to support tissue engineering concepts.

Material and Methods: In 30 Wistar rats, a 3-mm bone defect was created in the mandibular angle. Tissue was sampled at 5, 10, and 15 days, and the former defect area was excised to undergo transcriptome analysis after RNA extraction. Five differentially expressed genes were further evaluated with reverse transcription–polymerase chain reaction (rt-PCR).

Results: Transcriptome analysis revealed 2467 significantly over- and under-expressed transcripts after 5 days and 2265 after 15 days of bone healing, respectively. Validation via rtPCR confirmed overexpression of osteoactivin, angiopoietin-like factor–4, and metallomatrix proteinase–9 and underexpression of mastcellprotease-10 and proteoglycane-2 in comparison to values in the control group.

Conclusion: This systematic genome-wide transcriptome analysis helps to decipher the physiological mechanisms behind physiological bone healing. The exemplary depiction of 5 genes demonstrates the great complexity of metabolic processes during early bone healing. Here,

BMP-2 signaling pathways and local hypoxia play decisive roles in bone formation.

KEYWORDS

Bone tissue engineering, physiological bone healing, animal model, rat, transcriptome profiling, rt-PCR

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INTRODUCTION

Bone defects are a common challenge in every day clinical practice. Depending on the defect size, autologous bone grafts or bone replacement materials are widely used (Beck-Broichsitter et al., 2012a; Becker et al., 2012a; Becker et al., 2012b; Warnke et al., 2004). In this context, an autologous bone graft with its unique characteristic of osteoinductivity remains the gold standard, as bone replacement materials are not capable of inducing new bone formation at the transplantation site (Szabo et al., 2005). Studies have implied that these materials are highly biodegradable and biocompatible in order to serve as a guide track for cellular ingrowth (Arnold et al., 2002; Cai et al., 2009; Kotsakis et al., 2014), but histological evaluations have often revealed remaining particles of the bone replacement material within the regenerated bone tissue that was also surrounded by amounts of taut connective or scaring tissue (Aimetti et al., 2009;

Barone et al., 2008; Stavropoulos et al., 2010).

Bone defects below the species-specific critical size usually heal in the sense of a restitutio ad integrum without any scaring or residual tissue (Hoerth et al., 2014). These advantageous characteristics have inspired the formation of a new branch of science (Langer Vacanti, 1993) and industry over the last decades, which aimed to imitate natural bone regeneration in bone tissue engineering protocols using complex bioreactors or cell culture systems. Here, various scaffold materials and empiric or experimental dosages and timings of mainly one or two different growth factors were applied, which were previously determined to be involved in bone induction processes, such as bone morphogenic protein–2 (Beck-Broichsitter et al., 2012b;