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Research regarding tissue-engineered bone rapidly develops in recent years. How to construct tissue-engineered bone with structure similar to natural bone remains unclear, which has become a rapidly growing area of research. Cell sheet engineering technology enables novel approaches to tissue engineering and has attracted increasing attention. Strong evidence exists that cell sheet engineering technology has been used to successfully construct single cell sheets of cornea, periodontal ligament, skin, and urothelium and multiple cell sheets of myocardium, smooth muscle renal glomerulus, and hepatic lobule[1-4]. The present study used decalcified bone matrix (DBM) as scaffold to compound prepared cell sheets and observed the biocompatibility of cell sheets to DBM, as well as cell sheet growth on DBM.

With the progress in research of tissue-engineered bone, how to construct large-scale tissue-engineered bone with structure similar to natural bone and stable blood supply has become a hot spot. The basic requirements are to construct clinically useful, large-scale, functional tissue-engineered bone with structure similar to natural bone.
Cell sheet engineering technology enables novel approaches to tissue-engineered bone with natural bone-like structure. This technology utilizes a theory, in which temperature-responsive medium is used, that temperature change makes          poly(N-isopropylacrylamide) (PIPAAm) from hydrophobic into hydrophilic, thus cells cultured would separate from medium surface and form cell sheets and attach onto extracellular matrix[8-9]. Such cell sheets do not require proteolytic enzyme and cells would not be injured. The extracellular matrix at the bottom can directly form basal lamina to construct three-dimensional tissue structure. This cell inoculation method provides cells for bone formation and these cells exhibit the arrangement of osteoblasts during natural bone formation. Zhou et al[10] used this technology to successfully construct tissue-engineered bone with cortical bone- and spongy bone-like structure, which suggests the feasibility of this technology. Cell sheet engineering promotes the development of tissue-engineered bone, but there are some problems to be solved, for example, source of seed cells and control of cell sheet thickness[11]. 
Allogenic DBM has been shown to be a good biological scaffold material owing to its excellent biocompatibility and degradability, satisfactory mechanical strength, and easy sterilization[12]. Following defatting, decalcification, and noncollagen protein removal procedures, DBM exhibits low immunogenicity, reserves the growth factors and collagen for inducing bone formation. Its three-dimensional latticed structure provides good place for the attachment, proliferation, differentiation, osteogenesis, and vascularization of seed cells[13].
The present study constructed DBM/cell sheets complex. Results showed that cell sheets well attached to DBM, without obscission. During the process of construction, cell sheets were gently placed on DBM and 2.0-3.0 mL of culture medium was dropped. Thereafter, there was a 4.0-5.0 hours of culture procedure, which provides sufficient time for cell attachment. Finally, medium was slowly dropped along the culture plate wall, which contributes to good attachment. Scanning electron microscope results showed that at 3 days after inoculation, cell sheets well attached to DBM, showing satisfactory activity; at 5 days, cell sheets began to grow towards adjacent scaffolds; at 7 days, cell sheets rapidly grew towards adjacent scaffolds, and scaffold had be a part covered by cell sheets. These results demonstrate that DBM shows good biocompatibility to cell sheets and promotes cell proliferation and differentiation. DBM/cell sheet complex extremely promotes construction of tissue-engineered bone with structure similar to natural bone.

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