Osteogenic Potential and Histological Characteristics of Mesenchymal Stem Cell Sheet/Hydroxyapatite Constructs

Abstract

Abstract:We have developed a new cell transplantation technique, in which mesenchymal stem cells (MSCs) were cultured and lifted as a cell sheet structure. The sheet formed bone tissue after in vivo transplantation at a subcutaneous site, without the need for any scaffold. The sheet could also be transplanted with a scaffold, such as hydroxyapatite (HA). Transplantation of this sheet/HA construct resulted in extensive bone formation. The purpose of the present study was to analyze the spatial distribution and biological potential of the osteogenic sheet/HA constructs. Histological examination showed that the construct allowed bone formation inside the pores, as well as outside the HA at 2 weeks after transplantation. Interestingly, when two HAs were wrapped in one sheet, the bone on the outside of one HA could bond to the bone on the other HA, resulting in union of two HAs. Biochemical investigation showed that the sheet/HA constructs had higher osteogenic potential than previous methods, such as MSC/HA constructs. Our results indicate that this transplantation technique using MSC sheets has the potential to be a useful tool in bone tissue engineering.

Cell Sheet Injection as a Technique of Osteogenic Supply

“…Similar to Ma et al (17), we reported cell sheet fabrication using a scraping technique (8). Ma et al (17) reported that a scaffold-free transplanted cell sheet, which was created by a scraping technique, formed bone tissue within a subcutaneous site, consistent with our previous results (8,11,18). However, they used an open transplantation method through a skin incision.…”

“…Previously, we developed a method to fabricate cell sheets of cultured MSCs (8,11). The sheets demonstrated in vivo bone formation after subcutaneous transplantation, as well as following cell sheet injection without any scaffold.…”

Increased osteogenesis with hydroxyapatite constructs combined with serially-passaged bone marrow-derived mesenchymal stem cells

“…We have previously reported the preparation of HA constructs combined with BMSCs (BMSC/HA construct) [12,17]. Briefly, after release from the substratum, P3 cells were centrifuged and resuspended at a density of 1 × 10 6 cells/ml in standard medium.…”

“…RNA was isolated from four disks from each group using an Isogen RNA extraction kit (Nippon Gene Co. Ltd., Toyama, Japan). Briefly, each sample was placed in Isogen solution containing matrix beads and disrupted with a FastPrep FP24 Cell Disrupter (Qbiogene, Inc., Carlsbad, CA, USA) [12,17]. Subsequently, the remaining steps of RNA isolation were performed according to the manufacturer's instructions.…”

“…The combination of BMSCs with a scaffolding material, such as hydroxyapatite (HA), can promote the formation of new bone tissue in vivo after subcutaneous transplantation with freshly isolated bone marrow cells [13] or culture-expanded BMSCs (HA/ BMSCs construct) [2,[14][15][16][17], Recently, we reported a new cell transplantation technique in which BMSCs were cultured in culture medium containing Dex and ascorbic acid phosphate and transplanted as cell sheets [18]. These cell sheets can be combined with HA, in which they resulted in abundant bone formation compared with conventional techniques [17].…”