Cell sheet transplantation of cultured mesenchymal stem cells enhances bone formation in a rat nonunion model
Introduction
We have developed a cell transplantation method in which bone marrow-derived mesenchymal stem cells (MSCs) are cultured and lifted as a cell sheet structure [1]. No special equipment, such as temperature-based culture dishes and thermoresponsive films [2], [3], is necessary with our method, because MSCs are cultured only in medium containing dexamethasone (Dex) and ascorbic acid phosphate (vitamin C), and lifted by a scraper as a single cell sheet structure. The cell sheet made by our method is easily detached from the culture substrate, so that adhesion molecules on the cell surface and cell–cell interactions remain intact because no enzymatic treatments, such as trypsin digestion, are necessary to harvest the cell sheet. In an earlier report, we showed that such sheets could form bone tissue after transplantation into a subcutaneous site without a scaffold [1].
Delay and failure of bone union are common clinical problems confronting orthopedic surgeons. It has been reported that 5–10% of fractures result in either delayed union or nonunion, depending on the duration of incomplete healing [4], [5]. Fracture repair and nonunion treatments have been widely investigated in experimental research and at the clinical level [6], [7], [8], [9]. However, treatments for nonunion remain difficult in some clinical cases. There are two main types of nonunion, namely hypertrophic and atrophic types [10]. The hypertrophic type has the ability of osteogenesis, whereas the atrophic type is incapable of bone formation. Therefore, hypertrophic nonunions can often be treated by stable fixation of the fragments, while atrophic nonunions require bone grafting for healing. Operations for nonunions are relatively extensive and should be recommended only when bone union is improbable or obviously impossible without a change in treatment. For many years, the most frequently used method of nonunion treatment has been grafting of autogenous bone obtained from the ilium and tibia [10]. Free vascularized bone graft has also been used for bridging long bony defects in nonunions [11], [12]. These seem to be ideal for skeletal reconstruction, because of the lower possibility of disease transmission and immunological rejection compared with allografts. However, these surgical treatments require harvest of autogenous bones from an intact site, and surgeons sometimes face complications of pain and nerve palsy after bone harvesting.
Recently, cell-based treatments based on tissue engineering techniques have advanced, and thus, are considered a promising therapy for certain diseases [13], [14]. In the treatment of bone and joints, MSCs have been applied to total joint arthroplasty, osteonecrosis treatment and repair of cartilage defects [15], [16], [17]. These reports used scaffolds, such as hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP), to maintain the cells for transplantation. However, scaffolds are not generally used for surgical treatment of nonunions. If transplantation of MSCs without scaffold could enhance bone union at the fracture site, scaffold-free cell-based treatments could be employed, representing an ideal approach to the treatment of fracture, nonunion and osteonecrosis.
In the present study, cell sheets of MSCs were transplanted onto fractured femurs without scaffolds to enhance bone formation in a rat nonunion model. The results of the present study indicate that scaffold-free cell sheet transplantation can be applied as a treatment for fracture and nonunion.
Section snippets
Animals
Fischer 344 (F344) rats were purchased from Japan SLC, Inc. Seven-week-old male rats were used as donors for marrow cell preparation. Male rats of 300–350 g body weight were also used as recipients in the bone repair model except in experiments to verify the origin of newly formed bone. We used male rats as donors and females as recipients to verify the origin by detecting the sex-determining region Y (Sry) gene on the Y-chromosome, which was used as a marker for donor cells. The animal
Biochemical analysis
Fig. 2A, B, C shows the mRNA expression levels of cultured cells in MEM, osteoinductive and sheet groups in vitro. The expression levels of alkaline phosphatase (ALP) and osteocalcin (OC) in the sheet group (P = 0.004 and 0.003 vs. MEM group, respectively) as well as those in the osteoinductive group (P = 0.000 and 0.000 vs. MEM group, respectively) were significantly higher than those in the MEM group. No significant difference was observed between the osteoinductive group and the sheet group. We
Discussion
It has been reported that there is a population of undifferentiated cells in bone marrow, referred to as MSCs, which can differentiate into osteoblasts, adipocytes, chondrocytes, neurons and myogenic cells [22], [23], [24], [25], [26]. Differentiation into an osteoblastic lineage is induced by their in vitro culture in osteoinductive medium including Dex [18], [22], [27]. The in vitro differentiated osteoblasts are useful for regenerating bone tissue and have already been used in combination
Conflict of interest statement
The author state that they have no conflicts of interest.
Acknowledgments
We thank Dr. K. Hattori (National Institute of Advanced Industrial Science and Technology, Japan) for his work on biomechanical analysis. We also thank Ms. M. Yoshimura for her technical assistance (Nara Medical University School of Medicine, Japan).
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