Clumps of MSCs/ECM complexes generated with chondro-induction medium induces bone regeneration

1.

Pihlstrom, B.L., B.S. Michalowicz, and N.W. Johnson, Periodontal diseases. Lancet,

2005. 366(9499): p. 1809-1820.

2.

Kuo, L.C., A.M. Poison, and T. Kang, Associations between periodontal diseases and

systemic diseases: A review of the inter-relationships and interactions with

diabetes, respiratory diseases, cardiovascular diseases and osteoporosis. Public

Health, 2008. 122(4): p. 417-433.

3.

Bassani, D.G., M.T.A. Olinto, and N. Kreiger, Periodontal disease and perinatal

outcomes: a case-control study. Journal of Clinical Periodontology, 2007. 34(1): p. 3139.

4.

Bartold, P.M., R.I. Marshall, and D.R. Haynes, Periodontitis and rheumatoid

arthritis: A review. Journal of Periodontology, 2005. 76(11): p. 2066-2074.

5.

Scannapieco, F.A., R.B. Bush, and S. Paju, Associations between periodontal disease

and risk for atherosclerosis, cardiovascular disease, and stroke. A systematic

review. Ann Periodontol, 2003. 8(1): p. 38-53.

6.

Yoneda, M., et al., Involvement of a periodontal pathogen, Porphyromonas

gingivalis on the pathogenesis of non-alcoholic fatty liver disease. BMC

Gastroenterol, 2012. 12: p. 16.

7.

Langer, R. and J.P. Vacanti, TISSUE ENGINEERING. Science, 1993. 260(5110): p.

920-926.

8.

Christgau, M., et al., Guided tissue regeneration in intrabony defects using an

experimental bioresorbable polydioxanon (PDS) membrane. A 24-month split-mouth

study. J Clin Periodontol, 2002. 29(8): p. 710-23.

9.

Kitamura, M., et al., Periodontal Tissue Regeneration Using Fibroblast Growth

Factor-2: Randomized Controlled Phase II Clinical Trial. Plos One, 2008. 3(7). doi:

10.1371/journal.pone.0002611.

10.

Takayama, S., et al., Effects of basic fibroblast growth factor on human periodontal

ligament cells. Journal of Periodontal Research, 1997. 32(8): p. 667-675.

11.

Lynch, S.E., et al., A combination of platelet-derived and insulin-like growth factors

enhances periodontal regeneration. J Clin Periodontol, 1989. 16(8): p. 545-8.

12.

Mohammed, S., A.R.C. Pack, and T.B. Kardos, The effect of transforming growth

factor beta one (TGF-beta(1)) on wound healing, with or without barrier

membranes, in a Class II furcation defect in sheep. Journal of Periodontal

Research, 1998. 33(6): p. 335-344.

25

13.

Giannobile, W.V., et al., Recombinant human osteogenic protein-1 (OP-1) stimulates

periodontal wound healing in class III furcation defects. J Periodontol, 1998. 69(2):

p. 129-37.

14.

Wikesjö, U.M., et al., Periodontal repair in dogs: rhBMP-2 significantly enhances

bone formation under provisions for guided tissue regeneration. J Clin Periodontol,

2003. 30(8): p. 705-14.

15.

Blumenthal, N.M., et al., Effect of surgical implantation of recombinant human

bone morphogenetic protein-2 in a bioabsorbable collagen sponge or calcium

phosphate putty carrier in intrabony periodontal defects in the baboon. Journal of

Periodontology, 2002. 73(12): p. 1494-1506.

16.

Takeda, K., et al., Brain-derived neurotrophic factor enhances periodontal tissue

regeneration. Tissue Engineering, 2005. 11(9-10): p. 1618-1629.

17.

Bose, S., M. Roy, and A. Bandyopadhyay, Recent advances in bone tissue

engineering scaffolds. Trends in Biotechnology, 2012. 30(10): p. 546-554.

18.

Treiser, M.D., et al., Cytoskeleton-based forecasting of stem cell lineage fates.

Proceedings of the National Academy of Sciences of the United States of America,

2010. 107(2): p. 610-615.

19.

Thomson, J.A. and V.S. Marshall, Primate embryonic stem cells. Current Topics in

Developmental Biology, Vol 38, 1998. 38: p. 133-165.

20.

Takahashi, K., et al., Induction of pluripotent stem cells from adult human

fibroblasts by defined factors. Cell, 2007. 131(5): p. 861-872.

21.

Takahashi, K. and S. Yamanaka, Induction of pluripotent stem cells from mouse

embryonic and adult fibroblast cultures by defined factors. Cell, 2006. 126(4): p.

663-676.

22.

Pittenger, M.F., et al., Multilineage potential of adult human mesenchymal stem

cells. Science, 1999. 284(5411): p. 143-147.

23.

Colnot, C., Cell Sources for Bone Tissue Engineering: Insights from Basic Science.

Tissue Engineering Part B-Reviews, 2011. 17(6): p. 449-457.

24.

Kawaguchi, H., et al., Enhancement of periodontal tissue regeneration by

transplantation of bone marrow mesenchymal stem cells. Journal of Periodontology,

2004. 75(9): p. 1281-1287.

25.

Hasegawa, N., et al., Behavior of transplanted bone marrow-derived mesenchymal

stem cells in periodontal defects. Journal of Periodontology, 2006. 77(6): p. 10031007.

26.

Nagahara, T., et al., Introduction of a Mixture of beta-Tricalcium Phosphate Into a

Complex of Bone Marrow Mesenchymal Stem Cells and Type I Collagen Can

26

Augment the Volume of Alveolar Bone Without Impairing Cementum Regeneration.

Journal of Periodontology, 2015. 86(3): p. 456-464.

27.

Liechty, K.W., et al., Human mesenchymal stem cells engraft and demonstrate site-

specific differentiation after in utero transplantation in sheep. Nature Medicine,

2000. 6(11): p. 1282-1286.

28.

Hoogendoorn, H.A., et al., LONG-TERM STUDY OF LARGE CERAMIC

IMPLANTS (POROUS HYDROXYAPATITE) IN DOG FEMORA. Clinical

Orthopaedics and Related Research, 1984(187): p. 281-288.

29.

Oreffo, R.O.C. and J.T. Triffitt, Future potentials for using osteogenic stem cells and

biomaterials in orthopedics. Bone, 1999. 25(2): p. 5S-9S.

30.

Kittaka, M., et al., Clumps of a mesenchymal stromal cell/extracellular matrix

complex can be a novel tissue engineering therapy for bone regeneration.

Cytotherapy, 2015. 17(7): p. 860-873.

31.

Takewaki, M., et al., MSC/ECM Cellular Complexes Induce Periodontal Tissue

Regeneration. Journal of Dental Research, 2017. 96(9): p. 984-991.

32.

Takeshita, K., et al., Xenotransplantation of interferon-gamma-pretreated clumps of

a human mesenchymal stem cell/extracellular matrix complex induces mouse

calvarial bone regeneration. Stem Cell Research & Therapy, 2017. 8(1). doi:

10.1186/s13287-017-0550-1.

33.

Motoike, S., et al., Clumps of mesenchymal stem cell/extracellular matrix complexes

generated with xeno-free conditions facilitate bone regeneration via direct and

indirect osteogenesis. International Journal of Molecular Sciences, 2019. 20(16). doi:

10.3390/ijms20163970.

34.

Ernestina, S, et al., Hypoxia in cartilage: HIF-1α is essential for chondrocyte growth

arrest and survival. Genes & Development, 2001. 15(21): p. 2865-2876.

35.

Steck, E., et al., Mesenchymal stem cell differentiation in an experimental cartilage

defect: restriction of hypertrophy to boneclose neocartilage. Stem Cells and

Development, 2009. 18: p. 969-978.

36.

Phuong, N.D., et al., Endochondral Ossification in Critical-Sized Bone Defects via

Readily Implantable Scaffold-Free Stem Cell Constructs. Stem Cells Translational

Medicine, 2017. 6(7): p. 1644-1659.

37.

Bouxsein, M.L., et al., Guidelines for Assessment of Bone Microstructure in Rodents

Using Micro-Computed Tomography. Journal of Bone and Mineral Research, 2010.

25(7): p. 1468-1486.

27

38.

Xin, Z., et al., Chondrocytes transdifferentiate into osteoblasts in endchondral bone

during development, postnatalgrowth and fracture healing in mice. PLOS Genetics,

2014. 10(12). doi: 10.1371/journal.pgen.1004820.

39.

Y Jing., et al.,

Chondrocytes Directly Transform into Bone Cells in Mandibular

Condyle Growth. Journal of Dental Research, 2015. 94(12): p. 1668-1675.

40.

Adam, J.E., et al., Matrix elasticity directs stem cell lineage specification. Cell,

2006. 126(4): p. 677-689.

41.

Komatsu, N., et al., Type I collagen deposition via osteoinduction ameliorates

YAP/TAZ activity in 3D floating culture clumps of mesenchymal stem

cell/extracellular matrix complexes. Stem Cell Research & Therapy, 2018. 9(1). doi:

10.1186/s13287-018-1085-9.

42.

Li, F., X. Wang, and C. Niyibizi, Bone marrow stromal cells contribute to bone

formation following infusion into femoral cavities of a mouse model of osteogenesis

imperfecta. Bone, 2010. 47(3): p. 546-555.

43.

Zhou, Y., et al., Implantation of osteogenic differentiated donor mesenchymal stem

cells causes recruitment of host cells. Journal of Tissue Engineering and

Regenerative Medicine, 2015. 9(2): p. 118-126.

44.

Zhou, Y., et al., Mesenchymal stromal cells regulate the cell mobility and the

immune response during osteogenesis through secretion of vascular endothelial

growth factor A. Journal of Tissue Engineering and Regenerative Medicine, 2018.

12(1): p. E566-E578.

45.

Chelsea, S. B., et al., Stem cell derived endochondral cartilage stimulates bone

healing by tissue transformation. Journal of Bone and Mineral Research, 2014.

29(5): p. 1269-1282.

46.

Motoike, S., et al., Cryopreserved clumps of mesenchymal stem cell/extracellular

matrix complexes retain osteogenic capacity and induce bone regeneration. Stem

Cell Research & Therapy, 2018. 9(1). doi: 10.1186/s13287-018-0826-0.

28

図. 1

Xeno-free/Serum-free の軟骨誘導培地による C-MSCs 作製

(A&B) 5、10、15 日目でサンプルの回収を行い、HE 染色(A)およびサフラニン O 染色(B)

を行った。左は増殖培地で作製した C-MSCs、右は軟骨誘導培地で作製した C-MSCs を示

す。それぞれ左図が弱拡大、右図が強拡大像 (スケールバー=100 µm) を示す。

29

図. 1

Xeno-free/Serum-free の軟骨誘導培地による C-MSCs 作製

(C) 3、5、7、10、13、15 日目で回収を行い、real-time PCR を行った。使用したマーカ

ーは、Sox9, Aggrecan (ACAN), Col 2, Col 10, IHH である。ハウスキーピング遺伝子とし

て 18s を用いて、ΔΔCT 法により目的遺伝子の相対比を産出した。黒線が増殖培地で作製

した C-MSCs、赤線が軟骨誘導培地で作製した C-MSCs を示す。

30

図. 2

Xeno-free/Serum-free の軟骨誘導培地で作製された C-MSCs の SCID マウスの頭

蓋冠骨欠損への移植

(A) C-MSCs 移植時の肉眼所見を示す。上段が移植前、下段が C-MSCs 移植後。写真は軟

骨誘導培地で 10 日間培養した C-MSCs 移植時のもの。(B) 移植 4、8、12 週後の micro

CT 画像。比較として非移植群および増殖培地で 5 日間培養した C-MSCs を用いた。(スケ

ールバー=250 µm) (C) 移植 4、8、12 週後の骨欠損部における再生骨量 (BV) /全体組織量

(TV)。グラフはそれぞれ、左が 4 週、中央が 8 週、右が 12 週目のもの。エラーバーは

S.D.値を示す。**P < 0.01、*P < 0.05

31

32

図. 3

Xeno-free/Serum-free の軟骨誘導培地で培養した C-MSCs の頭蓋冠骨欠損への移

植時の組織像

(A&B) 移植 12 週後の組織像を示す。(A) 厚さ 8 µm で切片を作製し、HE 染色を行っ

た。(スケールバー=250 µm) (B) 厚さ 20 µm で切片を作製し、抗ヒト Vimentin 抗体で蛍

光免疫染色を行った。青色が細胞核、緑色がヒト Vimentin の分布を示す。上段が弱拡大

(スケールバー=250 µm)、下段が強拡大像 (スケールバー=50 µm)。左が非移植群、中央が

通常培地 5 日培養群、右が軟骨誘導培地 5 日培養群。

(C) 移植 4、8、12 週後の組織像を示す。厚さ 8 µm で切片を作製し、HE 染色およびサフ

ラニ O 染色を行った。上図が軟骨誘導培地 10 日培養群、下図が軟骨誘導培地 15 日培養

群を示す。それぞれ上段が HE 染色弱拡大像、下段左図が HE 染色強拡大像、下段右図が

サフラニン O 染色像を示す (スケールバー=250 µm)。左が移植 4 週後、中央移植 8 週

後、右が移植 12 週後。

(D&E) 移植 4、8、12 週後の組織像を示す。 厚さ 20 µm で切片を作製し、抗ヒト

Vimentin 抗体で蛍光免疫染色を行った。軟骨誘導培地 10 日培養群 (D)、軟骨誘導培地 15

日培養群(E)。青色が細胞核、緑色がヒト Vimentin の分布を示す。上段が弱拡大 (スケー

ルバー=250 µm)、下段が強拡大像 (スケールバー=50 µm)。それぞれ左が移植 4 週後、中

央移植 8 週後、右が移植 12 週後。

33

図. 4

Xeno-free/Serum-free の軟骨誘導培地で 10 日間培養した C-MSCs の頭蓋冠骨欠

損への移植時の短期的な観察

移植 3、7、14 日後に観察した。(A) micro CT 画像を示す。(スケールバー=250 µm) (B)上

段が HE 染色像、下段がサフラニン O 染色像を示す。左図が弱拡大、右図が強拡大像。

(スケールバー=500 µm) (C) 抗ヒト Vimentin 抗体で蛍光免疫染色を行った。それぞれ左

図が弱拡大(スケールバー=500 µm)、右図が強拡大像(スケールバー=50 µm)。青色が細胞

核、緑色がヒト Vimentin の分布を示す。

34

表. 1 real-time PCR プライマー

18s

Sox9

ACAN

Col 2

IHH

Col 10

F: GTAACCCGTTGAACCCCATT

R: CCATCCAATCGGTAGTAGCG

F: CATGAGCGAGGTGCACTCC

R: TCGCTTCAGGTCAGCCTTG

F: TGAGGAGGGCTGGAACAAGTACC

R: GGAGGTGGTAATTGCAGGGAACA

F: TTTCCCAGGTCAAGATGGTC

R: CTTCAGCACCTGTCTCACCA

F: AACTCGCTGGCTATCTCGGT

R: GCCCTCATAATGCAGGGACT

F: CCCTTTTTGCTGCTAGTATCC

R: CTGTTGTCCAGGTTTTCCTGGCAC

35

...