Elsevier

Archives of Oral Biology

Volume 72, December 2016, Pages 138-145
Archives of Oral Biology

Maxillofacial bone regeneration with osteogenic matrix cell sheets: An experimental study in rats

https://doi.org/10.1016/j.archoralbio.2016.08.017Get rights and content

Highlights

  • We previously reported the method for creating cell sheets with osteogenic ability.

  • We make the sheets from mesodermal cells using a scraper.

  • It was unclear whether the sheets could repair the neuroectodermal bones.

  • Our study showed the sheets could regenerate the neuroectodermal bones.

  • The sheets can be applied for the alveolar cleft osteoplasty.

Abstract

Objective

Regeneration of maxillofacial bone defects, characterized by relatively small but complicated shapes, poses a significant clinical challenge. Osteogenic matrix cell sheets (OMCSs) have osteogenic ability and good shaping properties and may be ideal graft materials. Here, we assessed whether implantation of OMCSs could be used to repair maxillofacial bone defects.

Design

We adopted a rat mandibular symphysis model. The rat mandible is formed by a paired bone and the central portion consisting of fibrous tissue. There is no bone tissue at the site; accordingly, this site was interpreted as a physiological bone gap and was used for evaluation. Rat bone marrow cells were cultured in medium containing dexamethasone and ascorbic acid phosphate to create OMCSs. The OMCSs were implanted into the rat mandibular symphysis without a scaffold. Microcomputed tomography and histological analyses were conducted after 2, 4, and 8 weeks.

Results

Two weeks after implantation, microcomputed tomography images and histological sections showed some sparse granular calcification tissue within the bone gap at the mandibular symphysis. At 4 weeks, the calcification tissue spread, and the gap of the mandibles were continued. At 8 weeks, this continuous new bone tissue was matured. The experimental group showed abundant new bone tissue in the group with OMCS implantation, but not in the group with sham implantation.

Conclusions

Our present results indicated that use of OMCSs may be an optimal approach towards achieving maxillofacial regeneration.

Introduction

Maxillary alveolar cleft, facial trauma, bone resection due to cancer, periodontal disease, and bone atrophy after tooth extraction may result in non-healing maxillofacial bone defects. Autologous bone grafts are considered the gold standard for repairing such bone defects (Behnia et al., 2009; Liu, Tan, Luo, Hu, & Yue, 2014; Xie et al., 2007, Yoshioka et al., 2012). However, donor site morbidity is an important consideration. Maxillofacial bone defects are often smaller than those commonly encountered in orthopedic surgery, but have more complicated morphology (d’Aquino et al., 2009). Thus, the ability of the graft material to assume a complex shape is essential for maxillofacial bone regeneration.

Recently, researchers have been working to develop cell-based bone repair methods as a substitute for autologous bone grafts (Kawate et al., 2006, Morishita et al., 2006). We previously developed a cell transplantation method based on cell sheet technology with bone marrow-derived stromal cells (BMSCs), which were cultured in the presence of dexamethasone (Dex) and ascorbic acid phosphate (Akahane et al., 2008). These cells were lifted as cell sheets, termed osteogenic matrix cell sheets (OMCSs), with no special materials, such as thermosensitive polymers. OMCSs can be transplanted without a scaffold, resulting in bone formation (Inagaki et al., 2013, Nakamura et al., 2010). OMCSs are sufficiently malleable that they may represent optimal graft materials for maxillofacial bone regeneration. However, transplantation of OMCSs at the site of maxillofacial bone defects has not yet been attempted.

Recently, the rat mandibular symphysis, i.e., the central portion of the rat mandible, which consists of fibrous connective tissue and thus can be interpreted as a physiological bone gap, has been used to assess bone graft materials, particularly for the purpose of maxillofacial bone regeneration (Yagyuu, Kirita, Hattori, Tadokoro, & Ohgushi, 2015). Therefore, in this study, we adopted a rat mandibular symphysis model and examined whether implantation of OMCSs could fill the gap with new bone tissue, leading to bone union.

Section snippets

Animals

All animal studies were approved by the animal care and use committee of Nara Medical University before beginning the experiments. Fischer 344 (F344) rats were purchased from Japan SLC, Inc. (Hamamatsu, Japan). Seven-week-old male rats were used as donors for marrow cell preparation, and 15-week-old rats were used as recipients.

Cell culture and cell sheet preparation

OMCSs were used in this study and were prepared as previously reported (Akahane et al., 2008, Inagaki et al., 2013, Nakamura et al., 2010). In brief, rat bone marrow

In vitro evaluation of OMCSs

H&E-stained sections revealed that the OMCSs comprised several cell layers laminated along the sheet with abundant extracellular matrices (Fig. 2A). Immunohistochemical studies revealed that type I collagen was strongly expressed in the matrices secreted by the cultured BMSCs (Fig. 2B). Furthermore, OPN and OCN were expressed in the cultured cells (Fig. 2C and D).

Comparison of micro-CT images

Two weeks after implantation of OMCSs, the micro-CT scans exhibited sparse areas of calcification within the bone gap at the

Discussion

Cell sheet technology is a tissue engineering approach that does not require scaffolds (Matsuda, Shimizu, Yamato, & Okano, 2007; Matsuura, Utoh, Nagase, & Okano, 2014). Confluent cultures of cells can be harvested as a cell sheet without protease treatment. Avoiding protease treatment preserves complete cell–cell junctions, cell surface proteins, and the extracellular matrix in the cell sheet. Cell sheets are also soft, malleable, and easily molded. Cell sheet technology has been applied

Funding

This work was partially supported by JSPS KAKENHI Grant Number JP 24792245.

Competing interests

None of the authors have any conflicts of interest regarding this research.

Ethical approval

This study was approved by the animal care and use committee of Nara Medical University (protocol No. 10483).

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