Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Orthop. Oct 18, 2017; 8(10): 754-760
Published online Oct 18, 2017. doi: 10.5312/wjo.v8.i10.754
Bone regeneration with osteogenic matrix cell sheet and tricalcium phosphate: An experimental study in sheep
Tsutomu Kira, Manabu Akahane, Shohei Omokawa, Takamasa Shimizu, Kenji Kawate, Tadanobu Onishi, Yasuhito Tanaka
Tsutomu Kira, Takamasa Shimizu, Tadanobu Onishi, Yasuhito Tanaka, Department of Orthopedic Surgery, Nara Medical University, Nara 634-8522, Japan
Manabu Akahane, Department of Public Health, Health Management and Policy, Nara Medical University School of Medicine, Nara 634-8522, Japan
Shohei Omokawa, Department of Hand Surgery, Nara Medical University, Nara 634-8522, Japan
Kenji Kawate, Department of Artificial Joint and Regenerative Medicine for Bone and Cartilage, Nara Medical University, Nara 634-8522, Japan
Author contributions: Kira T designed the study and conducted the animal experiments; Akahane M and Shimizu T conducted biological analysis; Omokawa S designed the study and interpreted the results; Kawate K and Onishi T conducted the data analysis; Tanaka Y interpreted the results.
Supported by Grant-in-Aid for scientific research from the Ministry of Health, Labour and Welfare, Japan.
Institutional animal care and use committee statement: All experimental protocols using animals were approved by the institutional animal care and use committee of Nara Medical University before experiments commenced.
Conflict-of-interest statement: There is no conflict of interest related to this study.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Tsutomu Kira, MD, PhD, Department of Orthopedic Surgery, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan. gs212005@naramed-u.ac.jp
Telephone: +81-744-223051 Fax: +81-744-256449
Received: February 12, 2017
Peer-review started: February 15, 2017
First decision: March 27, 2017
Revised: June 13, 2017
Accepted: July 7, 2017
Article in press: July 10, 2017
Published online: October 18, 2017
Processing time: 127 Days and 9.9 Hours
Abstract
AIM

To determine the effects of a cell sheet created from sheep bone marrow and tricalcium phosphate (TCP) on osteogenesis.

METHODS

Bone marrow cells were harvested from a sheep and cultured in a minimal essential medium (MEM) containing ascorbic acid phosphate (AscP) and dexamethasone (Dex). After 2 wk, the formed osteogenic matrix cell sheet was lifted from the culture dish using a scraper. Additionally, harvested bone marrow cells were cultured in MEM only as a negative control group, and in MEM with AscP, Dex, and β-glycerophosphate as a positive control group. For in vitro evaluation, we measured the alkaline phosphatase (ALP) activity and osteocalcin (OC) content in the media of the cultured cells from each group. For in vivo analysis, a porous TCP ceramic was used as a scaffold. We prepared an experimental group comprising TCP scaffolds wrapped with the osteogenic matrix cell sheets and a control group consisting of the TCP scaffold only. The constructs were implanted subcutaneously into athymic rats and the cell donor sheep, and bone formation was confirmed by histology after 4 wk.

RESULTS

In the in vitro part, the mean ALP activity was 0.39 ± 0.03 mg/well in the negative control group, 0.67 ± 0.04 mg/well in the sheet group, and 0.65 ± 0.07 mg/well in the positive control group. The mean OC levels were 1.46 ± 0.33 ng/well in the negative control group, 3.92 ± 0.16 ng/well in the sheet group, and 4.4 ± 0.47 ng/well in the positive control group, respectively. The ALP activity and OC levels were significantly higher in the cell sheet and positive control groups than in the negative control group (P < 0.05). There was no significant difference in ALP activity or OC levels between the cell sheet group and the positive control group (P > 0.05). TCP constructs wrapped with cell sheets prior to implantation showed bone formation, in contrast to TCP scaffolds alone, which exhibited poor bone formation when implanted, in the subcutaneous layer both in athymic rats and in the sheep.

CONCLUSION

This technique for preparing highly osteoinductive TCP may promote regeneration in large bone defects.

Keywords: Cell sheet; Osteogenesis; Sheep; Bone marrow; Mesenchymal stromal cell

Core tip: An osteogenic matrix cell sheet derived from sheep bone marrow enhances osteogenic differentiation. We found that the osteogenic matrix cell sheets on tricalcium phosphate discs efficiently promotes bone formation.