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Migration of intervertebral disc cells into dense collagen scaffolds intended for functional replacement.

Bron JL, Mulder HW, Vonk LA, Doulabi BZ, Oudhoff MJ, Smit TH - J Mater Sci Mater Med (2012)

Bottom Line: The aim of current study was to assess whether NP and surrounding annulus fibrosus (AF) cells are capable of migrating into dense collagen scaffolds.We seeded freshly harvested caprine NP and AF cells onto scaffolds consisting of 1.5 and 3.0% type I collagen matrices, prepared by plastic compression, to assess cell invasion.The migration distance appeared both time and density dependent and was higher for NP (25%) compared to AF (10%) cells after 4 weeks.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, VU University Medical Center, Amsterdam, The Netherlands. jl.bron@vumc.nl

ABSTRACT
Invasion of cells from surrounding tissues is a crucial step for regeneration when using a-cellular scaffolds as a replacement of the nucleus pulposus (NP). The aim of current study was to assess whether NP and surrounding annulus fibrosus (AF) cells are capable of migrating into dense collagen scaffolds. We seeded freshly harvested caprine NP and AF cells onto scaffolds consisting of 1.5 and 3.0% type I collagen matrices, prepared by plastic compression, to assess cell invasion. The migration distance appeared both time and density dependent and was higher for NP (25%) compared to AF (10%) cells after 4 weeks. Migration distance was not enhanced by Hst-2, a peptide derived from saliva known to enhance fibroblast migration, and this was confirmed in a scratch assay. In conclusion, we revealed invasion of cells into dense collagen scaffolds and therewith encouraging first steps towards the use of a-cellular scaffolds for NP replacement.

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Examples of 1.5% collagen scaffolds seeded with AF (a) and NP (b) cells after 28 days of migration (stain HE. Magnification ×20)
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Fig3: Examples of 1.5% collagen scaffolds seeded with AF (a) and NP (b) cells after 28 days of migration (stain HE. Magnification ×20)

Mentions: After the incubation period, the scaffolds were fixed with 4% formaldehyde and stored at 4 °C overnight. The scaffolds were cut in half and one half was dehydrated in a graded series of ethanol solutions, followed by xylene, and at last the scaffold was embedded in paraffin. Sections of 7 μm thickness were prepared orthogonal to the seeding plane. After deparaffinization of the sections with xylene substitute and hydration in a declining series of ethanol solutions, sections were stained with haematoxylin for 20 min, followed by eosin for 1 min. Sections were dehydrated in a series of ethanol solutions, transferred into xylene and coverslipped. A bright-field microscope (Leica Microsystems, Bannockburn, IL, USA) was used to visualize the sections. When the scaffold was too broad to be visualized in a single view, two pictures with sufficient overlap were taken. An overlay of these pictures was made using Photoshop (Adobe, San Jose, CA, USA). The pictures were read into Matlab (The MathWorks Inc., Eindhoven, The Netherlands). The level of migration M is defined by: where d is the distance travelled by the cells and w is the width of the scaffold (Fig. 3). Relative migration was used as a measure of migration because of deformations that where introduced in the scaffolds during the incubation period and fixation process. The migration in a scaffold is calculated as the mean of the four measures per scaffold. In turn, the migration for each different condition is the mean of the triple experiments. The standard deviation per condition is calculated as the root of the sum of the squared standard deviations of each individual scaffold. Only samples in which at least at two different positions migration could be determined are included in analysis.Fig. 3


Migration of intervertebral disc cells into dense collagen scaffolds intended for functional replacement.

Bron JL, Mulder HW, Vonk LA, Doulabi BZ, Oudhoff MJ, Smit TH - J Mater Sci Mater Med (2012)

Examples of 1.5% collagen scaffolds seeded with AF (a) and NP (b) cells after 28 days of migration (stain HE. Magnification ×20)
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3299969&req=5

Fig3: Examples of 1.5% collagen scaffolds seeded with AF (a) and NP (b) cells after 28 days of migration (stain HE. Magnification ×20)
Mentions: After the incubation period, the scaffolds were fixed with 4% formaldehyde and stored at 4 °C overnight. The scaffolds were cut in half and one half was dehydrated in a graded series of ethanol solutions, followed by xylene, and at last the scaffold was embedded in paraffin. Sections of 7 μm thickness were prepared orthogonal to the seeding plane. After deparaffinization of the sections with xylene substitute and hydration in a declining series of ethanol solutions, sections were stained with haematoxylin for 20 min, followed by eosin for 1 min. Sections were dehydrated in a series of ethanol solutions, transferred into xylene and coverslipped. A bright-field microscope (Leica Microsystems, Bannockburn, IL, USA) was used to visualize the sections. When the scaffold was too broad to be visualized in a single view, two pictures with sufficient overlap were taken. An overlay of these pictures was made using Photoshop (Adobe, San Jose, CA, USA). The pictures were read into Matlab (The MathWorks Inc., Eindhoven, The Netherlands). The level of migration M is defined by: where d is the distance travelled by the cells and w is the width of the scaffold (Fig. 3). Relative migration was used as a measure of migration because of deformations that where introduced in the scaffolds during the incubation period and fixation process. The migration in a scaffold is calculated as the mean of the four measures per scaffold. In turn, the migration for each different condition is the mean of the triple experiments. The standard deviation per condition is calculated as the root of the sum of the squared standard deviations of each individual scaffold. Only samples in which at least at two different positions migration could be determined are included in analysis.Fig. 3

Bottom Line: The aim of current study was to assess whether NP and surrounding annulus fibrosus (AF) cells are capable of migrating into dense collagen scaffolds.We seeded freshly harvested caprine NP and AF cells onto scaffolds consisting of 1.5 and 3.0% type I collagen matrices, prepared by plastic compression, to assess cell invasion.The migration distance appeared both time and density dependent and was higher for NP (25%) compared to AF (10%) cells after 4 weeks.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, VU University Medical Center, Amsterdam, The Netherlands. jl.bron@vumc.nl

ABSTRACT
Invasion of cells from surrounding tissues is a crucial step for regeneration when using a-cellular scaffolds as a replacement of the nucleus pulposus (NP). The aim of current study was to assess whether NP and surrounding annulus fibrosus (AF) cells are capable of migrating into dense collagen scaffolds. We seeded freshly harvested caprine NP and AF cells onto scaffolds consisting of 1.5 and 3.0% type I collagen matrices, prepared by plastic compression, to assess cell invasion. The migration distance appeared both time and density dependent and was higher for NP (25%) compared to AF (10%) cells after 4 weeks. Migration distance was not enhanced by Hst-2, a peptide derived from saliva known to enhance fibroblast migration, and this was confirmed in a scratch assay. In conclusion, we revealed invasion of cells into dense collagen scaffolds and therewith encouraging first steps towards the use of a-cellular scaffolds for NP replacement.

Show MeSH
Related in: MedlinePlus