Clumps of a mesenchymal stromal cell/extracellular matrix complex can be a novel tissue engineering therapy for bone regeneration.
OBJECTIVE
The transplantation of mesenchymal stromal cells (MSCs) to damaged tissue has attracted attention in scientific and medical fields as an effective regenerative therapy. Nevertheless, additional studies are required to develop an MSC transplant method for bone regeneration because the use of an artificial scaffold restricts the number of transplanted cells and their function. Furthermore, regulating the degree of cell differentiation in vitro is desirable for a more effective regenerative therapy. To address these unresolved issues, with the use of a self-produced extracellular matrix (ECM), we developed clumps of an MSC/ECM complex (C-MSCs).


METHODS
MSCs isolated from rat femur were cultured in growth medium supplemented with 50 μg/mL of ascorbic acid for 7 days. To obtain C-MSCs, confluent cells were scratched with the use of a micropipette tip to roll up the cellular sheet, which consisted of ECM produced by the MSCs. The biological properties of C-MSCs were assessed in vitro and their bone regenerative activity was tested by use of a rat calvarial defect model.


RESULTS
Immunofluorescent confocal microscopic analysis revealed that type I collagen formed C-MSCs. Osteopontin messenger RNA expression and amount of calcium content were higher in C-MSCs cultured in osteo-inductive medium than those of untreated C-MSCs. The transplantation of osteogenic-differentiated C-MSCs led to rapid bone regeneration in the rat calvarial defect model.


CONCLUSIONS
These results suggest that the use of C-MSCs refined by self-produced ECM, which contain no artificial scaffold and can be processed in vitro, may represent a novel tissue engineering therapy.
Department of Periodontal Medicine, Applied Life Sciences, Institute of Biomedical and Health Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Mizuho
M
Kittaka
Department of Periodontal Medicine, Applied Life Sciences, Institute of Biomedical and Health Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Mikihito
M
Kajiya
Department of Periodontal Medicine, Applied Life Sciences, Institute of Biomedical and Health Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan. Electronic address: bashihi@hiroshima-u.ac.jp.
Hideki
H
Shiba
Department of Periodontal Medicine, Applied Life Sciences, Institute of Biomedical and Health Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Manabu
M
Takewaki
Department of Periodontal Medicine, Applied Life Sciences, Institute of Biomedical and Health Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Kei
K
Takeshita
Department of Periodontal Medicine, Applied Life Sciences, Institute of Biomedical and Health Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Rathvisal
R
Khung
Department of Periodontal Medicine, Applied Life Sciences, Institute of Biomedical and Health Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Takako
T
Fujita
Department of Periodontal Medicine, Applied Life Sciences, Institute of Biomedical and Health Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Tomoyuki
T
Iwata
Department of Periodontal Medicine, Applied Life Sciences, Institute of Biomedical and Health Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Truong Quoc
TQ
Nguyen
Department of Periodontal Medicine, Applied Life Sciences, Institute of Biomedical and Health Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Kazuhisa
K
Ouhara
Department of Periodontal Medicine, Applied Life Sciences, Institute of Biomedical and Health Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Katsuhiro
K
Takeda
Department of Periodontal Medicine, Applied Life Sciences, Institute of Biomedical and Health Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Tsuyoshi
T
Fujita
Department of Periodontal Medicine, Applied Life Sciences, Institute of Biomedical and Health Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Hidemi
H
Kurihara
D000818:Animals; D001861:Bone Regeneration / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D064987:Cell- and Tissue-Based Therapy / Q000379:methods*; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D003470:Culture Media / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005269:Femur / Q000166:cytology; D008297:Male; D045164:Mesenchymal Stem Cell Transplantation / Q000379:methods*; D059630:Mesenchymal Stem Cells / Q000166:cytology; D018919:Neovascularization, Physiologic / Q000502:physiology; D010012:Osteogenesis / Q000502:physiology; D053495:Osteopontin / Q000096:biosynthesis / Q000235:genetics; D010294:Parietal Bone / Q000293:injuries / Q000601:surgery*; D051381:Rats; D011916:Rats, Inbred F344; D023822:Tissue Engineering / Q000379:methods*
D024042:Collagen Type I; D003470:Culture Media; C506897:Spp1 protein, rat; D053495:Osteopontin; D002118:Calcium
bone regeneration; complex mesenchymal stromal cells; extracellular matrix; mesenchymal stromal cells