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3D porous calcium-alginate scaffolds cell culture system improved human osteoblast cell clusters for cell therapy.

Chen CY, Ke CJ, Yen KC, Hsieh HC, Sun JS, Lin FH - Theranostics (2015)

Bottom Line: The Ca-Alginate scaffold facilitated the growth and differentiation of human bone cell clusters, and the functionally-closed process bioreactor system supplied the soluble nutrients and osteogenic signals required to maintain the cell viability.This system preserved the proliferative ability of cells and cell viability and up-regulated bone-related gene expression and biological apatite crystals formation.The described strategy could be used in therapeutic application and opens new avenues for surgical interventions to correct skeletal defects.

View Article: PubMed Central - PubMed

Affiliation: 1. Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei, Taiwan (R.O.C.).

ABSTRACT
Age-related orthopedic disorders and bone defects have become a critical public health issue, and cell-based therapy is potentially a novel solution for issues surrounding bone tissue engineering and regenerative medicine. Long-term cultures of primary bone cells exhibit phenotypic and functional degeneration; therefore, culturing cells or tissues suitable for clinical use remain a challenge. A platform consisting of human osteoblasts (hOBs), calcium-alginate (Ca-Alginate) scaffolds, and a self-made bioreactor system was established for autologous transplantation of human osteoblast cell clusters. The Ca-Alginate scaffold facilitated the growth and differentiation of human bone cell clusters, and the functionally-closed process bioreactor system supplied the soluble nutrients and osteogenic signals required to maintain the cell viability. This system preserved the proliferative ability of cells and cell viability and up-regulated bone-related gene expression and biological apatite crystals formation. The bone-like tissue generated could be extracted by removal of calcium ions via ethylenediaminetetraacetic acid (EDTA) chelation, and exhibited a size suitable for injection. The described strategy could be used in therapeutic application and opens new avenues for surgical interventions to correct skeletal defects.

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The morphology and size distribution of bone-like tissues. (A to C) The morphology of hOBs @ Ca-Alginate scaffolds were examined by SEM under 350x observation; through the EDX determination, the calcium and phosphorous ions increase over time; (A1 to C1) the images were under 1000x observation; (D) showed the size distribution of bone-like tissues after 7 and 14 days perfusion. The third part was confocal images of the bone-like tissue after 14 days perfusion. (E1) in green indicated the cell bodies of bone-like tissue with calcein AM dye; (E2) in blue with Hoechst 33342 revealed the nucleus location; (E3) was the fluorescent merge image; (E4) showed the z-axis; (E5) displayed the bone-like tissue within Ca-Alginate scaffold in bright field; (E6) was the merge image of the bone-like tissue within Ca-Alginate scaffold.
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Figure 5: The morphology and size distribution of bone-like tissues. (A to C) The morphology of hOBs @ Ca-Alginate scaffolds were examined by SEM under 350x observation; through the EDX determination, the calcium and phosphorous ions increase over time; (A1 to C1) the images were under 1000x observation; (D) showed the size distribution of bone-like tissues after 7 and 14 days perfusion. The third part was confocal images of the bone-like tissue after 14 days perfusion. (E1) in green indicated the cell bodies of bone-like tissue with calcein AM dye; (E2) in blue with Hoechst 33342 revealed the nucleus location; (E3) was the fluorescent merge image; (E4) showed the z-axis; (E5) displayed the bone-like tissue within Ca-Alginate scaffold in bright field; (E6) was the merge image of the bone-like tissue within Ca-Alginate scaffold.

Mentions: The morphology of hOBs in Ca-Alginate scaffolds was revealed by scanning electron microscopy (SEM), and the calcium/phosphorous signals were detected by SEM with energy dispersive X-ray spectroscopy (SEM/EDX, Fig. 5). The SEM images showed that individual cells were embedded within the Ca-Alginate scaffolds on day 1, and only calcium signal from scaffolds were detected through the SEM/EDX measurement (Fig. 5A1 and 5A2). Under dynamic perfusion, hOBs proliferated and by day 7 aggregated into cell clusters that could secrete and amplify large quantity of ECM (Fig. 5B1 and 5B2). At day 14, large cell clusters were surrounded by ECM, and the EDX data indicated that a layer of apatite of biological origin was formed at the surface (Fig. 5C1 and 5C2). These hOBs cell clusters presented 3D structures and exhibited osteogenic functions, such as mineralization, suggesting that Ca-Alginate scaffolds integrated with the perfusion system provide a suitable environment for bone-like tissue formation.


3D porous calcium-alginate scaffolds cell culture system improved human osteoblast cell clusters for cell therapy.

Chen CY, Ke CJ, Yen KC, Hsieh HC, Sun JS, Lin FH - Theranostics (2015)

The morphology and size distribution of bone-like tissues. (A to C) The morphology of hOBs @ Ca-Alginate scaffolds were examined by SEM under 350x observation; through the EDX determination, the calcium and phosphorous ions increase over time; (A1 to C1) the images were under 1000x observation; (D) showed the size distribution of bone-like tissues after 7 and 14 days perfusion. The third part was confocal images of the bone-like tissue after 14 days perfusion. (E1) in green indicated the cell bodies of bone-like tissue with calcein AM dye; (E2) in blue with Hoechst 33342 revealed the nucleus location; (E3) was the fluorescent merge image; (E4) showed the z-axis; (E5) displayed the bone-like tissue within Ca-Alginate scaffold in bright field; (E6) was the merge image of the bone-like tissue within Ca-Alginate scaffold.
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Related In: Results  -  Collection

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Figure 5: The morphology and size distribution of bone-like tissues. (A to C) The morphology of hOBs @ Ca-Alginate scaffolds were examined by SEM under 350x observation; through the EDX determination, the calcium and phosphorous ions increase over time; (A1 to C1) the images were under 1000x observation; (D) showed the size distribution of bone-like tissues after 7 and 14 days perfusion. The third part was confocal images of the bone-like tissue after 14 days perfusion. (E1) in green indicated the cell bodies of bone-like tissue with calcein AM dye; (E2) in blue with Hoechst 33342 revealed the nucleus location; (E3) was the fluorescent merge image; (E4) showed the z-axis; (E5) displayed the bone-like tissue within Ca-Alginate scaffold in bright field; (E6) was the merge image of the bone-like tissue within Ca-Alginate scaffold.
Mentions: The morphology of hOBs in Ca-Alginate scaffolds was revealed by scanning electron microscopy (SEM), and the calcium/phosphorous signals were detected by SEM with energy dispersive X-ray spectroscopy (SEM/EDX, Fig. 5). The SEM images showed that individual cells were embedded within the Ca-Alginate scaffolds on day 1, and only calcium signal from scaffolds were detected through the SEM/EDX measurement (Fig. 5A1 and 5A2). Under dynamic perfusion, hOBs proliferated and by day 7 aggregated into cell clusters that could secrete and amplify large quantity of ECM (Fig. 5B1 and 5B2). At day 14, large cell clusters were surrounded by ECM, and the EDX data indicated that a layer of apatite of biological origin was formed at the surface (Fig. 5C1 and 5C2). These hOBs cell clusters presented 3D structures and exhibited osteogenic functions, such as mineralization, suggesting that Ca-Alginate scaffolds integrated with the perfusion system provide a suitable environment for bone-like tissue formation.

Bottom Line: The Ca-Alginate scaffold facilitated the growth and differentiation of human bone cell clusters, and the functionally-closed process bioreactor system supplied the soluble nutrients and osteogenic signals required to maintain the cell viability.This system preserved the proliferative ability of cells and cell viability and up-regulated bone-related gene expression and biological apatite crystals formation.The described strategy could be used in therapeutic application and opens new avenues for surgical interventions to correct skeletal defects.

View Article: PubMed Central - PubMed

Affiliation: 1. Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei, Taiwan (R.O.C.).

ABSTRACT
Age-related orthopedic disorders and bone defects have become a critical public health issue, and cell-based therapy is potentially a novel solution for issues surrounding bone tissue engineering and regenerative medicine. Long-term cultures of primary bone cells exhibit phenotypic and functional degeneration; therefore, culturing cells or tissues suitable for clinical use remain a challenge. A platform consisting of human osteoblasts (hOBs), calcium-alginate (Ca-Alginate) scaffolds, and a self-made bioreactor system was established for autologous transplantation of human osteoblast cell clusters. The Ca-Alginate scaffold facilitated the growth and differentiation of human bone cell clusters, and the functionally-closed process bioreactor system supplied the soluble nutrients and osteogenic signals required to maintain the cell viability. This system preserved the proliferative ability of cells and cell viability and up-regulated bone-related gene expression and biological apatite crystals formation. The bone-like tissue generated could be extracted by removal of calcium ions via ethylenediaminetetraacetic acid (EDTA) chelation, and exhibited a size suitable for injection. The described strategy could be used in therapeutic application and opens new avenues for surgical interventions to correct skeletal defects.

Show MeSH
Related in: MedlinePlus