Limits...
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

(a–c) Histology of CPSs (hematoxylin and eosin staining). CPSs expanded with 1% HS-STK1 and 1% HS-STK1+3 exhibiting thicker outgrowth of the peripheral region formed by extracellular matrix–rich multilayered cells than 10% FBS-M199. (d–f) Immunohistochemical observations of PCNA-positive cells. (g) PCNA-positive cells were notably increased at the peripheral region of the CPSs expanded with 1% HS-STK1 and 1% HS-STK1+3. (h) Measurement of CPS thickness. (a–c) bar, 200 µm and (d–f) bar, 50 µm.CPS: cultured periosteal sheet; HS: human serum supplemented; FBS: fetal bovine serum; PCNA: proliferating cell nuclear antigen.n = 3, *p < 0.05, **p < 0.01, compared with controls.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2 - License 3
getmorefigures.php?uid=PMC3927863&req=5

fig2-2041731413509646: (a–c) Histology of CPSs (hematoxylin and eosin staining). CPSs expanded with 1% HS-STK1 and 1% HS-STK1+3 exhibiting thicker outgrowth of the peripheral region formed by extracellular matrix–rich multilayered cells than 10% FBS-M199. (d–f) Immunohistochemical observations of PCNA-positive cells. (g) PCNA-positive cells were notably increased at the peripheral region of the CPSs expanded with 1% HS-STK1 and 1% HS-STK1+3. (h) Measurement of CPS thickness. (a–c) bar, 200 µm and (d–f) bar, 50 µm.CPS: cultured periosteal sheet; HS: human serum supplemented; FBS: fetal bovine serum; PCNA: proliferating cell nuclear antigen.n = 3, *p < 0.05, **p < 0.01, compared with controls.

Mentions: Periosteal cells were developed as outgrowths from periosteal tissue fragments to form CPSs on culture plates regardless of the type of culture media used. Cell migration was observed earlier in the 1% HS-STK1 and 1% HS-STK1+3 groups than in the control group: the diameter of the CPSs and total DNA content recovered from the CPSs were significantly higher in the 1% HS-STK1 and 1% HS-STK1+3 groups than in the controls (p < 0.05; Figure 1(a) and (b)). The formation of a multilayer structure was observed only in the area adjacent to the primary periosteal segment, and the peripheral outgrowth presented a single-layer structure with a sparse cell distribution in the control group (Figure 1(e) and (h)). In contrast, the formation of a dense extracellular matrix and a multilayer structure was characteristic in the peripheral region in the 1% HS-STK1 and 1% HS-STK1+3 groups (Figure 1(c), (d), (f), and (g)). The thickness of the CPSs in the peripheral region was approximately 50 µm in the control group and approximately 90 µm in both the 1% HS-STK1 and 1% HS-STK1+3 groups (Figure 2(a)–(c) and (h)). In good agreement with the increase in CPS thickness, the percentage of PCNA-positive cells was also significantly higher in stem-cell culture media than in the control medium (p < 0.05; Figure 2(d)–(g)). No significant differences were noted in the expansion and formation of the multilayer structure between the 1% HS-STK1 and 1% HS-STK1+3 groups.


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)

(a–c) Histology of CPSs (hematoxylin and eosin staining). CPSs expanded with 1% HS-STK1 and 1% HS-STK1+3 exhibiting thicker outgrowth of the peripheral region formed by extracellular matrix–rich multilayered cells than 10% FBS-M199. (d–f) Immunohistochemical observations of PCNA-positive cells. (g) PCNA-positive cells were notably increased at the peripheral region of the CPSs expanded with 1% HS-STK1 and 1% HS-STK1+3. (h) Measurement of CPS thickness. (a–c) bar, 200 µm and (d–f) bar, 50 µm.CPS: cultured periosteal sheet; HS: human serum supplemented; FBS: fetal bovine serum; PCNA: proliferating cell nuclear antigen.n = 3, *p < 0.05, **p < 0.01, compared with controls.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2 - License 3
Show All Figures
getmorefigures.php?uid=PMC3927863&req=5

fig2-2041731413509646: (a–c) Histology of CPSs (hematoxylin and eosin staining). CPSs expanded with 1% HS-STK1 and 1% HS-STK1+3 exhibiting thicker outgrowth of the peripheral region formed by extracellular matrix–rich multilayered cells than 10% FBS-M199. (d–f) Immunohistochemical observations of PCNA-positive cells. (g) PCNA-positive cells were notably increased at the peripheral region of the CPSs expanded with 1% HS-STK1 and 1% HS-STK1+3. (h) Measurement of CPS thickness. (a–c) bar, 200 µm and (d–f) bar, 50 µm.CPS: cultured periosteal sheet; HS: human serum supplemented; FBS: fetal bovine serum; PCNA: proliferating cell nuclear antigen.n = 3, *p < 0.05, **p < 0.01, compared with controls.
Mentions: Periosteal cells were developed as outgrowths from periosteal tissue fragments to form CPSs on culture plates regardless of the type of culture media used. Cell migration was observed earlier in the 1% HS-STK1 and 1% HS-STK1+3 groups than in the control group: the diameter of the CPSs and total DNA content recovered from the CPSs were significantly higher in the 1% HS-STK1 and 1% HS-STK1+3 groups than in the controls (p < 0.05; Figure 1(a) and (b)). The formation of a multilayer structure was observed only in the area adjacent to the primary periosteal segment, and the peripheral outgrowth presented a single-layer structure with a sparse cell distribution in the control group (Figure 1(e) and (h)). In contrast, the formation of a dense extracellular matrix and a multilayer structure was characteristic in the peripheral region in the 1% HS-STK1 and 1% HS-STK1+3 groups (Figure 1(c), (d), (f), and (g)). The thickness of the CPSs in the peripheral region was approximately 50 µm in the control group and approximately 90 µm in both the 1% HS-STK1 and 1% HS-STK1+3 groups (Figure 2(a)–(c) and (h)). In good agreement with the increase in CPS thickness, the percentage of PCNA-positive cells was also significantly higher in stem-cell culture media than in the control medium (p < 0.05; Figure 2(d)–(g)). No significant differences were noted in the expansion and formation of the multilayer structure between the 1% HS-STK1 and 1% HS-STK1+3 groups.

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