Elsevier

Bone

Volume 46, Issue 2, February 2010, Pages 418-424
Bone

Cell sheet transplantation of cultured mesenchymal stem cells enhances bone formation in a rat nonunion model

https://doi.org/10.1016/j.bone.2009.08.048Get rights and content

Abstract

Orthopedic surgeons have long been troubled by cases involving nonunion of fractured bones. This study aimed to enhance bone union by cell sheet transplantation of mesenchymal stem cells. A nonunion model was made in rat femur, and rat bone marrow cells were cultured in medium containing dexamethasone and ascorbic acid phosphate to create a cell sheet that could be scraped off as a single sheet. Cell sheets were transplanted onto fractured femurs without a scaffold in the model. X-ray and histological analysis were performed at 2, 4 and 8 weeks. Ultrasonography and biomechanical analysis were performed at 8 weeks. X-ray photographs and histological sections showed callus formation around the fracture site in the cell sheet-transplanted group (sheet group). Bone union was obtained in the sheet group at 8 weeks. By contrast, the control group (without sheet transplantation) showed nonunion of the femur. The results of pullout evaluation in the vertical direction of the femur in the sheet group were significantly better than that of the control group. Analysis of the origin of de novo formed bone using the Sry gene, which was used as a marker for donor cells, showed that transplanted cells without scaffolds could survive and differentiate into osteogenic lineage cells in vivo. These results showed that the femoral fracture in our model was completely cured by the transplantation of a cell sheet created by tissue engineering techniques. Thus, we think that cell sheet transplantation can contribute to hard tissue reconstruction in cases involving nonunion, bone defects and osteonecrosis.

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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