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Application of stem-cell media to explant culture of human periosteum: An optimal approach for preparing osteogenic cell material.

Uematsu K, Nagata M, Kawase T, Suzuki K, Takagi R - J Tissue Eng (2013)

Bottom Line: Cultured periosteal sheet diameter and DNA content were significantly higher, and the multilayer structure was prominent in 1% human serum-supplemented STK1 and 1% human serum-supplemented STK1+3.The messenger RNA of osteoblastic markers was significantly upregulated in 1% human serum-supplemented STK1+3.Expansion in 1% human serum-supplemented STK1+3 successfully induced cultured periosteal sheet growth while retaining osteogenic potential, and subsequent osteoblastic induction promoted the production of homogeneous cell material.

View Article: PubMed Central - PubMed

Affiliation: Division of Oral and Maxillofacial Surgery, Department of Oral Health Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan ; Division of Oral Bioengineering, Department of Tissue Regeneration and Reconstitution, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.

ABSTRACT
As part of our clinical tests on bone regeneration using cultured periosteal sheets, here, we prepared cultured periosteal sheets in two types of stem-cell culture media, STK1 and STK3. Human periosteum was expanded either in 1% human serum-supplemented STK1 for 28 days, in 1% human serum-supplemented STK1 for 14 days followed by 1% human serum-supplemented STK3 for 14 days (1% human serum-supplemented STK1+3), or in 10% fetal bovine serum-supplemented Medium 199 for 28 days (control). Cultured periosteal sheet diameter and DNA content were significantly higher, and the multilayer structure was prominent in 1% human serum-supplemented STK1 and 1% human serum-supplemented STK1+3. The messenger RNA of osteoblastic markers was significantly upregulated in 1% human serum-supplemented STK1+3. Osteopontin-immunopositive staining and mineralization were evident across a wide area of the cultured periosteal sheet in 1% human serum-supplemented STK1+3. Subcutaneous implantation in nude mice following expansion in 1% human serum-supplemented STK1+3 produced the highest cultured periosteal sheet osteogenic activity. Expansion in 1% human serum-supplemented STK1+3 successfully induced cultured periosteal sheet growth while retaining osteogenic potential, and subsequent osteoblastic induction promoted the production of homogeneous cell material.

No MeSH data available.


Related in: MedlinePlus

Gene expression analysis of osteoblastic markers and bone-related growth factors of periosteal sheets expanded with 1% HS-STK1, 1% HS-STK1+3, and 10% FBS-M199 by qRT-PCR. (a) Runx2, (b) ALP, (c) col1a1, (d) OPN, (e) BMP-2, (f) RANKL, (g) VEGF-A, and (h) FGFR1.HS: human serum supplemented; FBS: fetal bovine serum; Runx2: runt-related transcription factor 2; qRT-PCR: quantitative reverse transcriptase polymerase chain reaction; ALP: alkaline phosphatase; OPN: osteopontin; RANKL: receptor activator of nuclear factor kappa-B ligand; col1a1: collagen, type I, alpha 1; BMP: bone morphogenetic protein; VEGF-A: vascular endothelial growth factor-A; FGFR1: fibroblast growth factor receptor 1.n = 4, *p < 0.05, **p < 0.01, compared with controls.
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fig4-2041731413509646: Gene expression analysis of osteoblastic markers and bone-related growth factors of periosteal sheets expanded with 1% HS-STK1, 1% HS-STK1+3, and 10% FBS-M199 by qRT-PCR. (a) Runx2, (b) ALP, (c) col1a1, (d) OPN, (e) BMP-2, (f) RANKL, (g) VEGF-A, and (h) FGFR1.HS: human serum supplemented; FBS: fetal bovine serum; Runx2: runt-related transcription factor 2; qRT-PCR: quantitative reverse transcriptase polymerase chain reaction; ALP: alkaline phosphatase; OPN: osteopontin; RANKL: receptor activator of nuclear factor kappa-B ligand; col1a1: collagen, type I, alpha 1; BMP: bone morphogenetic protein; VEGF-A: vascular endothelial growth factor-A; FGFR1: fibroblast growth factor receptor 1.n = 4, *p < 0.05, **p < 0.01, compared with controls.

Mentions: In good agreement with our previous findings,16 expression of mRNA encoding osteoblastic markers (Runx2, ALP, col1a1, and OPN) was observed in CPSs cultured in the 10% FBS-M199 medium without specific osteoblastic induction (Figure 4(a)–(d)). When the mRNA levels in the 1% HS-STK1 group were compared with those in the control group, the ALP mRNA level was upregulated, while the OPN mRNA (a late-stage differentiation marker) was downregulated, suggesting suppression of final differentiation into osteoblasts in the 1% HS-STK1 group (Figure 4(a)–(d)). On the other hand, the mRNA levels of all osteoblastic markers were upregulated in the 1% HS-STK1+3 group (Figure 4(a)–(d)). This suggests that switching the medium from the STK1 medium designed for primary stem-cell culture to the STK3 medium designed for differentiation of progenitors into osteoblasts at the midpoint of the 28-day culture resulted in the production of CPSs expressing high levels of osteoblastic markers. The 1% HS-STK1+3 group also exhibited remarkably high mRNA expression of local bone-regeneration factors BMP-2, RANKL, and VEGF-A, which mediate cell differentiation into osteoblasts, recruitment of osteoclasts, and angiogenesis, respectively (Figure 4(e)–(g)).


Application of stem-cell media to explant culture of human periosteum: An optimal approach for preparing osteogenic cell material.

Uematsu K, Nagata M, Kawase T, Suzuki K, Takagi R - J Tissue Eng (2013)

Gene expression analysis of osteoblastic markers and bone-related growth factors of periosteal sheets expanded with 1% HS-STK1, 1% HS-STK1+3, and 10% FBS-M199 by qRT-PCR. (a) Runx2, (b) ALP, (c) col1a1, (d) OPN, (e) BMP-2, (f) RANKL, (g) VEGF-A, and (h) FGFR1.HS: human serum supplemented; FBS: fetal bovine serum; Runx2: runt-related transcription factor 2; qRT-PCR: quantitative reverse transcriptase polymerase chain reaction; ALP: alkaline phosphatase; OPN: osteopontin; RANKL: receptor activator of nuclear factor kappa-B ligand; col1a1: collagen, type I, alpha 1; BMP: bone morphogenetic protein; VEGF-A: vascular endothelial growth factor-A; FGFR1: fibroblast growth factor receptor 1.n = 4, *p < 0.05, **p < 0.01, compared with controls.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3927863&req=5

fig4-2041731413509646: Gene expression analysis of osteoblastic markers and bone-related growth factors of periosteal sheets expanded with 1% HS-STK1, 1% HS-STK1+3, and 10% FBS-M199 by qRT-PCR. (a) Runx2, (b) ALP, (c) col1a1, (d) OPN, (e) BMP-2, (f) RANKL, (g) VEGF-A, and (h) FGFR1.HS: human serum supplemented; FBS: fetal bovine serum; Runx2: runt-related transcription factor 2; qRT-PCR: quantitative reverse transcriptase polymerase chain reaction; ALP: alkaline phosphatase; OPN: osteopontin; RANKL: receptor activator of nuclear factor kappa-B ligand; col1a1: collagen, type I, alpha 1; BMP: bone morphogenetic protein; VEGF-A: vascular endothelial growth factor-A; FGFR1: fibroblast growth factor receptor 1.n = 4, *p < 0.05, **p < 0.01, compared with controls.
Mentions: In good agreement with our previous findings,16 expression of mRNA encoding osteoblastic markers (Runx2, ALP, col1a1, and OPN) was observed in CPSs cultured in the 10% FBS-M199 medium without specific osteoblastic induction (Figure 4(a)–(d)). When the mRNA levels in the 1% HS-STK1 group were compared with those in the control group, the ALP mRNA level was upregulated, while the OPN mRNA (a late-stage differentiation marker) was downregulated, suggesting suppression of final differentiation into osteoblasts in the 1% HS-STK1 group (Figure 4(a)–(d)). On the other hand, the mRNA levels of all osteoblastic markers were upregulated in the 1% HS-STK1+3 group (Figure 4(a)–(d)). This suggests that switching the medium from the STK1 medium designed for primary stem-cell culture to the STK3 medium designed for differentiation of progenitors into osteoblasts at the midpoint of the 28-day culture resulted in the production of CPSs expressing high levels of osteoblastic markers. The 1% HS-STK1+3 group also exhibited remarkably high mRNA expression of local bone-regeneration factors BMP-2, RANKL, and VEGF-A, which mediate cell differentiation into osteoblasts, recruitment of osteoclasts, and angiogenesis, respectively (Figure 4(e)–(g)).

Bottom Line: Cultured periosteal sheet diameter and DNA content were significantly higher, and the multilayer structure was prominent in 1% human serum-supplemented STK1 and 1% human serum-supplemented STK1+3.The messenger RNA of osteoblastic markers was significantly upregulated in 1% human serum-supplemented STK1+3.Expansion in 1% human serum-supplemented STK1+3 successfully induced cultured periosteal sheet growth while retaining osteogenic potential, and subsequent osteoblastic induction promoted the production of homogeneous cell material.

View Article: PubMed Central - PubMed

Affiliation: Division of Oral and Maxillofacial Surgery, Department of Oral Health Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan ; Division of Oral Bioengineering, Department of Tissue Regeneration and Reconstitution, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.

ABSTRACT
As part of our clinical tests on bone regeneration using cultured periosteal sheets, here, we prepared cultured periosteal sheets in two types of stem-cell culture media, STK1 and STK3. Human periosteum was expanded either in 1% human serum-supplemented STK1 for 28 days, in 1% human serum-supplemented STK1 for 14 days followed by 1% human serum-supplemented STK3 for 14 days (1% human serum-supplemented STK1+3), or in 10% fetal bovine serum-supplemented Medium 199 for 28 days (control). Cultured periosteal sheet diameter and DNA content were significantly higher, and the multilayer structure was prominent in 1% human serum-supplemented STK1 and 1% human serum-supplemented STK1+3. The messenger RNA of osteoblastic markers was significantly upregulated in 1% human serum-supplemented STK1+3. Osteopontin-immunopositive staining and mineralization were evident across a wide area of the cultured periosteal sheet in 1% human serum-supplemented STK1+3. Subcutaneous implantation in nude mice following expansion in 1% human serum-supplemented STK1+3 produced the highest cultured periosteal sheet osteogenic activity. Expansion in 1% human serum-supplemented STK1+3 successfully induced cultured periosteal sheet growth while retaining osteogenic potential, and subsequent osteoblastic induction promoted the production of homogeneous cell material.

No MeSH data available.


Related in: MedlinePlus