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