Development of an injectable, in situ crosslinkable, degradable polymeric carrier for osteogenic cell populations. Part 1. Encapsulation of marrow stromal osteoblasts in surface crosslinked gelatin microparticles

Richard G. Payne, Michael J Yaszemski, Alan W. Yasko, Antonios G. Mikos

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Abstract

This study investigated the temporary encapsulation of rat marrow stromal osteoblasts in surface crosslinked gelatin microparticles. Cells were encapsulated in uncrosslinked gelatin microparticles of average diameter of 630μm containing ∼53 cells. Gelatin microparticles were crosslinked to shell thicknesses of ∼75μm via exposure to 1mM dithiobis(succinimidylpropionate) (DSP) solution for 15min or 5mM DSP solution for 5min for the production of microparticles dispersing ∼60min after placement into a physiologic fluid at 37°C. Formed microparticles were placed into culture wells at a cell seeding density of 5.3×104 cells/cm2 and, following the degradation and/or dissolution of gelatin, the cells were cultured in the presence of osteogenic supplements for 28 days. Samples were taken at specified time points and analyzed by a DNA assay for cell number and a 3H-thymidine incorporation assay for proliferative potential. Samples were also obtained and analyzed at several time points by alkaline phosphatase, osteocalcin, and mineralization assays for early and late phenotypic expression markers of osteoblastic differentiation. The measurements from the different assays for encapsulated cells (EC) in uncrosslinked and crosslinked gelatin microparticles were normalized with the cell numbers from the DNA assay and compared with those for nonencapsulated control cells. The results demonstrated that the marrow stromal cells survived the encapsulation procedure in uncrosslinked gelatin microparticles and also retained their proliferative potential and osteoblastic phenotype over a 28 day period, although at a slightly lower level than the nonencapsulated cells. The results further showed that the marrow stromal cells survived the encapsulation in crosslinked gelatin microparticles prepared via exposure to 5mM DSP for 5min and also retained their proliferative potential and osteoblastic phenotype over a 28 day period, but at a slightly lower level than the EC in uncrosslinked gelatin microparticles. In contrast, exposure to 1mM DSP for 15min led to severely limited cell viability and phenotypic expression probably due to the increased crosslinking time. These results suggest that temporary encapsulation of cells in gelatin microparticles may protect cells from short-term environmental effects such as those associated with the crosslinking of an injectable polymeric carrier for bone tissue engineering.

Original languageEnglish (US)
Pages (from-to)4359-4371
Number of pages13
JournalBiomaterials
Volume23
Issue number22
DOIs
StatePublished - 2002

Fingerprint

Osteoblasts
Gelatin
Encapsulation
Assays
Bone Marrow
Cells
Injections
Population
Crosslinking
DNA
Cell Count
Stromal Cells
Phosphatases
Tissue engineering
Environmental impact
Rats
Bone
Dissolution
Phenotype
Degradation

Keywords

  • Bone tissue engineering
  • Cell transplantation
  • Gelatin microparticles
  • Injectable biomaterials
  • Marrow stromal osteoblasts
  • Poly(propylene fumarate)
  • Temporary cell encapsulation

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering

Cite this

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title = "Development of an injectable, in situ crosslinkable, degradable polymeric carrier for osteogenic cell populations. Part 1. Encapsulation of marrow stromal osteoblasts in surface crosslinked gelatin microparticles",
abstract = "This study investigated the temporary encapsulation of rat marrow stromal osteoblasts in surface crosslinked gelatin microparticles. Cells were encapsulated in uncrosslinked gelatin microparticles of average diameter of 630μm containing ∼53 cells. Gelatin microparticles were crosslinked to shell thicknesses of ∼75μm via exposure to 1mM dithiobis(succinimidylpropionate) (DSP) solution for 15min or 5mM DSP solution for 5min for the production of microparticles dispersing ∼60min after placement into a physiologic fluid at 37°C. Formed microparticles were placed into culture wells at a cell seeding density of 5.3×104 cells/cm2 and, following the degradation and/or dissolution of gelatin, the cells were cultured in the presence of osteogenic supplements for 28 days. Samples were taken at specified time points and analyzed by a DNA assay for cell number and a 3H-thymidine incorporation assay for proliferative potential. Samples were also obtained and analyzed at several time points by alkaline phosphatase, osteocalcin, and mineralization assays for early and late phenotypic expression markers of osteoblastic differentiation. The measurements from the different assays for encapsulated cells (EC) in uncrosslinked and crosslinked gelatin microparticles were normalized with the cell numbers from the DNA assay and compared with those for nonencapsulated control cells. The results demonstrated that the marrow stromal cells survived the encapsulation procedure in uncrosslinked gelatin microparticles and also retained their proliferative potential and osteoblastic phenotype over a 28 day period, although at a slightly lower level than the nonencapsulated cells. The results further showed that the marrow stromal cells survived the encapsulation in crosslinked gelatin microparticles prepared via exposure to 5mM DSP for 5min and also retained their proliferative potential and osteoblastic phenotype over a 28 day period, but at a slightly lower level than the EC in uncrosslinked gelatin microparticles. In contrast, exposure to 1mM DSP for 15min led to severely limited cell viability and phenotypic expression probably due to the increased crosslinking time. These results suggest that temporary encapsulation of cells in gelatin microparticles may protect cells from short-term environmental effects such as those associated with the crosslinking of an injectable polymeric carrier for bone tissue engineering.",
keywords = "Bone tissue engineering, Cell transplantation, Gelatin microparticles, Injectable biomaterials, Marrow stromal osteoblasts, Poly(propylene fumarate), Temporary cell encapsulation",
author = "Payne, {Richard G.} and Yaszemski, {Michael J} and Yasko, {Alan W.} and Mikos, {Antonios G.}",
year = "2002",
doi = "10.1016/S0142-9612(02)00184-9",
language = "English (US)",
volume = "23",
pages = "4359--4371",
journal = "Biomaterials",
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TY - JOUR

T1 - Development of an injectable, in situ crosslinkable, degradable polymeric carrier for osteogenic cell populations. Part 1. Encapsulation of marrow stromal osteoblasts in surface crosslinked gelatin microparticles

AU - Payne, Richard G.

AU - Yaszemski, Michael J

AU - Yasko, Alan W.

AU - Mikos, Antonios G.

PY - 2002

Y1 - 2002

N2 - This study investigated the temporary encapsulation of rat marrow stromal osteoblasts in surface crosslinked gelatin microparticles. Cells were encapsulated in uncrosslinked gelatin microparticles of average diameter of 630μm containing ∼53 cells. Gelatin microparticles were crosslinked to shell thicknesses of ∼75μm via exposure to 1mM dithiobis(succinimidylpropionate) (DSP) solution for 15min or 5mM DSP solution for 5min for the production of microparticles dispersing ∼60min after placement into a physiologic fluid at 37°C. Formed microparticles were placed into culture wells at a cell seeding density of 5.3×104 cells/cm2 and, following the degradation and/or dissolution of gelatin, the cells were cultured in the presence of osteogenic supplements for 28 days. Samples were taken at specified time points and analyzed by a DNA assay for cell number and a 3H-thymidine incorporation assay for proliferative potential. Samples were also obtained and analyzed at several time points by alkaline phosphatase, osteocalcin, and mineralization assays for early and late phenotypic expression markers of osteoblastic differentiation. The measurements from the different assays for encapsulated cells (EC) in uncrosslinked and crosslinked gelatin microparticles were normalized with the cell numbers from the DNA assay and compared with those for nonencapsulated control cells. The results demonstrated that the marrow stromal cells survived the encapsulation procedure in uncrosslinked gelatin microparticles and also retained their proliferative potential and osteoblastic phenotype over a 28 day period, although at a slightly lower level than the nonencapsulated cells. The results further showed that the marrow stromal cells survived the encapsulation in crosslinked gelatin microparticles prepared via exposure to 5mM DSP for 5min and also retained their proliferative potential and osteoblastic phenotype over a 28 day period, but at a slightly lower level than the EC in uncrosslinked gelatin microparticles. In contrast, exposure to 1mM DSP for 15min led to severely limited cell viability and phenotypic expression probably due to the increased crosslinking time. These results suggest that temporary encapsulation of cells in gelatin microparticles may protect cells from short-term environmental effects such as those associated with the crosslinking of an injectable polymeric carrier for bone tissue engineering.

AB - This study investigated the temporary encapsulation of rat marrow stromal osteoblasts in surface crosslinked gelatin microparticles. Cells were encapsulated in uncrosslinked gelatin microparticles of average diameter of 630μm containing ∼53 cells. Gelatin microparticles were crosslinked to shell thicknesses of ∼75μm via exposure to 1mM dithiobis(succinimidylpropionate) (DSP) solution for 15min or 5mM DSP solution for 5min for the production of microparticles dispersing ∼60min after placement into a physiologic fluid at 37°C. Formed microparticles were placed into culture wells at a cell seeding density of 5.3×104 cells/cm2 and, following the degradation and/or dissolution of gelatin, the cells were cultured in the presence of osteogenic supplements for 28 days. Samples were taken at specified time points and analyzed by a DNA assay for cell number and a 3H-thymidine incorporation assay for proliferative potential. Samples were also obtained and analyzed at several time points by alkaline phosphatase, osteocalcin, and mineralization assays for early and late phenotypic expression markers of osteoblastic differentiation. The measurements from the different assays for encapsulated cells (EC) in uncrosslinked and crosslinked gelatin microparticles were normalized with the cell numbers from the DNA assay and compared with those for nonencapsulated control cells. The results demonstrated that the marrow stromal cells survived the encapsulation procedure in uncrosslinked gelatin microparticles and also retained their proliferative potential and osteoblastic phenotype over a 28 day period, although at a slightly lower level than the nonencapsulated cells. The results further showed that the marrow stromal cells survived the encapsulation in crosslinked gelatin microparticles prepared via exposure to 5mM DSP for 5min and also retained their proliferative potential and osteoblastic phenotype over a 28 day period, but at a slightly lower level than the EC in uncrosslinked gelatin microparticles. In contrast, exposure to 1mM DSP for 15min led to severely limited cell viability and phenotypic expression probably due to the increased crosslinking time. These results suggest that temporary encapsulation of cells in gelatin microparticles may protect cells from short-term environmental effects such as those associated with the crosslinking of an injectable polymeric carrier for bone tissue engineering.

KW - Bone tissue engineering

KW - Cell transplantation

KW - Gelatin microparticles

KW - Injectable biomaterials

KW - Marrow stromal osteoblasts

KW - Poly(propylene fumarate)

KW - Temporary cell encapsulation

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