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Rheological and biological properties of a hydrogel support for cells intended for intervertebral disc repair.

Benz K, Stippich C, Osswald C, Gaissmaier C, Lembert N, Badke A, Steck E, Aicher WK, Mollenhauer JA - BMC Musculoskelet Disord (2012)

Bottom Line: The expression of cartilage- and disc-specific mRNAs was maintained in hydrogels in vitro and in vivo, demonstrating the maintenance of a stable specific cellular phenotype, compared to monolayer cells.Matrix deposition could be specified by immunohistology for collagen types I and II, and aggrecan and was found only in areas where predominantly cells of human origin were detected by species specific in situ hybridization.The data demonstrate that the hydrogels form stable implants capable to contain a specifically functional cell population within a physiological environment.

View Article: PubMed Central - HTML - PubMed

Affiliation: NMI Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen, Germany.

ABSTRACT

Background: Cell-based approaches towards restoration of prolapsed or degenerated intervertebral discs are hampered by a lack of measures for safe administration and placement of cell suspensions within a treated disc. In order to overcome these risks, a serum albumin-based hydrogel has been developed that polymerizes after injection and anchors the administered cell suspension within the tissue.

Methods: A hydrogel composed of chemically activated albumin crosslinked by polyethylene glycol spacers was produced. The visco-elastic gel properties were determined by rheological measurement. Human intervertebral disc cells were cultured in vitro and in vivo in the hydrogel and their phenotype was tested by reverse-transcriptase polymerase chain reaction. Matrix production and deposition was monitored by immuno-histology and by biochemical analysis of collagen and glycosaminoglycan deposition. Species specific in situ hybridization was performed to discriminate between cells of human and murine origin in xenotransplants.

Results: The reproducibility of the gel formation process could be demonstrated. The visco-elastic properties were not influenced by storage of gel components. In vitro and in vivo (subcutaneous implants in mice) evidence is presented for cellular differentiation and matrix deposition within the hydrogel for human intervertebral disc cells even for donor cells that have been expanded in primary monolayer culture, stored in liquid nitrogen and re-activated in secondary monolayer culture. Upon injection into the animals, gels formed spheres that lasted for the duration of the experiments (14 days). The expression of cartilage- and disc-specific mRNAs was maintained in hydrogels in vitro and in vivo, demonstrating the maintenance of a stable specific cellular phenotype, compared to monolayer cells. Significantly higher levels of hyaluronan synthase isozymes-2 and -3 mRNA suggest cell functionalities towards those needed for the support of the regeneration of the intervertebral disc. Moreover, mouse implanted hydrogels accumulated 5 times more glycosaminoglycans and 50 times more collagen than the in vitro cultured gels, the latter instead releasing equivalent quantities of glycosaminoglycans and collagen into the culture medium. Matrix deposition could be specified by immunohistology for collagen types I and II, and aggrecan and was found only in areas where predominantly cells of human origin were detected by species specific in situ hybridization.

Conclusions: The data demonstrate that the hydrogels form stable implants capable to contain a specifically functional cell population within a physiological environment.

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mRNA expression profiles of human disc cell preparations cultured in vitro in monolayer or hydrogel. P1 cells were harvested at days 4, 7, and 14 and the expression of collagen type I (COL1), collagen type II (COL2), and aggrecan (ACAN) was analyzed. Expression values of the P1 cultures are expressed relative to the median expression of the cells at the end of the expansion phase (P0) (median because the data are not normally distributed). Boxes represent 25%/75% percentiles, mean values (dotted line) and median values (solid line).
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Figure 2: mRNA expression profiles of human disc cell preparations cultured in vitro in monolayer or hydrogel. P1 cells were harvested at days 4, 7, and 14 and the expression of collagen type I (COL1), collagen type II (COL2), and aggrecan (ACAN) was analyzed. Expression values of the P1 cultures are expressed relative to the median expression of the cells at the end of the expansion phase (P0) (median because the data are not normally distributed). Boxes represent 25%/75% percentiles, mean values (dotted line) and median values (solid line).

Mentions: In an in vitro reference culture the alterations in gene expression of the matrix genes collagen type I, type II and aggrecan were analyzed at different time points during the culture of disc cells in monolayer and hydrogel cultures. This data set expression was compared to the expression data of the initial cell population (P0) used for the subculture (Figure 2). Collagen type I expression was almost unchanged over time and independent from culture type. Collagen type II expression decreased in monolayer cultures, at the end of the 14 days monolayer culture the median expression was 4-fold less (mean 18-fold) than the expression in the P0 cells. In contrast, in hydrogels the expression at all three time points was higher than in the P0 cells (median: 2 - 5-fold; mean: 1,7 - 11-fold). Although these differences were not statistically significant due to restricted number of patients (n = 4) and high individual variations a clear tendency was visible: the hydrogel culture improved the collagen type II expression compared to the monolayer (median: 10-fold, mean: 200-fold higher at day 14). Aggrecan expression was less effected by culture type. Nonetheless the same tendency for improved expression in hydrogels can be seen as for collagen type II mRNA expression.


Rheological and biological properties of a hydrogel support for cells intended for intervertebral disc repair.

Benz K, Stippich C, Osswald C, Gaissmaier C, Lembert N, Badke A, Steck E, Aicher WK, Mollenhauer JA - BMC Musculoskelet Disord (2012)

mRNA expression profiles of human disc cell preparations cultured in vitro in monolayer or hydrogel. P1 cells were harvested at days 4, 7, and 14 and the expression of collagen type I (COL1), collagen type II (COL2), and aggrecan (ACAN) was analyzed. Expression values of the P1 cultures are expressed relative to the median expression of the cells at the end of the expansion phase (P0) (median because the data are not normally distributed). Boxes represent 25%/75% percentiles, mean values (dotted line) and median values (solid line).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3375205&req=5

Figure 2: mRNA expression profiles of human disc cell preparations cultured in vitro in monolayer or hydrogel. P1 cells were harvested at days 4, 7, and 14 and the expression of collagen type I (COL1), collagen type II (COL2), and aggrecan (ACAN) was analyzed. Expression values of the P1 cultures are expressed relative to the median expression of the cells at the end of the expansion phase (P0) (median because the data are not normally distributed). Boxes represent 25%/75% percentiles, mean values (dotted line) and median values (solid line).
Mentions: In an in vitro reference culture the alterations in gene expression of the matrix genes collagen type I, type II and aggrecan were analyzed at different time points during the culture of disc cells in monolayer and hydrogel cultures. This data set expression was compared to the expression data of the initial cell population (P0) used for the subculture (Figure 2). Collagen type I expression was almost unchanged over time and independent from culture type. Collagen type II expression decreased in monolayer cultures, at the end of the 14 days monolayer culture the median expression was 4-fold less (mean 18-fold) than the expression in the P0 cells. In contrast, in hydrogels the expression at all three time points was higher than in the P0 cells (median: 2 - 5-fold; mean: 1,7 - 11-fold). Although these differences were not statistically significant due to restricted number of patients (n = 4) and high individual variations a clear tendency was visible: the hydrogel culture improved the collagen type II expression compared to the monolayer (median: 10-fold, mean: 200-fold higher at day 14). Aggrecan expression was less effected by culture type. Nonetheless the same tendency for improved expression in hydrogels can be seen as for collagen type II mRNA expression.

Bottom Line: The expression of cartilage- and disc-specific mRNAs was maintained in hydrogels in vitro and in vivo, demonstrating the maintenance of a stable specific cellular phenotype, compared to monolayer cells.Matrix deposition could be specified by immunohistology for collagen types I and II, and aggrecan and was found only in areas where predominantly cells of human origin were detected by species specific in situ hybridization.The data demonstrate that the hydrogels form stable implants capable to contain a specifically functional cell population within a physiological environment.

View Article: PubMed Central - HTML - PubMed

Affiliation: NMI Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen, Germany.

ABSTRACT

Background: Cell-based approaches towards restoration of prolapsed or degenerated intervertebral discs are hampered by a lack of measures for safe administration and placement of cell suspensions within a treated disc. In order to overcome these risks, a serum albumin-based hydrogel has been developed that polymerizes after injection and anchors the administered cell suspension within the tissue.

Methods: A hydrogel composed of chemically activated albumin crosslinked by polyethylene glycol spacers was produced. The visco-elastic gel properties were determined by rheological measurement. Human intervertebral disc cells were cultured in vitro and in vivo in the hydrogel and their phenotype was tested by reverse-transcriptase polymerase chain reaction. Matrix production and deposition was monitored by immuno-histology and by biochemical analysis of collagen and glycosaminoglycan deposition. Species specific in situ hybridization was performed to discriminate between cells of human and murine origin in xenotransplants.

Results: The reproducibility of the gel formation process could be demonstrated. The visco-elastic properties were not influenced by storage of gel components. In vitro and in vivo (subcutaneous implants in mice) evidence is presented for cellular differentiation and matrix deposition within the hydrogel for human intervertebral disc cells even for donor cells that have been expanded in primary monolayer culture, stored in liquid nitrogen and re-activated in secondary monolayer culture. Upon injection into the animals, gels formed spheres that lasted for the duration of the experiments (14 days). The expression of cartilage- and disc-specific mRNAs was maintained in hydrogels in vitro and in vivo, demonstrating the maintenance of a stable specific cellular phenotype, compared to monolayer cells. Significantly higher levels of hyaluronan synthase isozymes-2 and -3 mRNA suggest cell functionalities towards those needed for the support of the regeneration of the intervertebral disc. Moreover, mouse implanted hydrogels accumulated 5 times more glycosaminoglycans and 50 times more collagen than the in vitro cultured gels, the latter instead releasing equivalent quantities of glycosaminoglycans and collagen into the culture medium. Matrix deposition could be specified by immunohistology for collagen types I and II, and aggrecan and was found only in areas where predominantly cells of human origin were detected by species specific in situ hybridization.

Conclusions: The data demonstrate that the hydrogels form stable implants capable to contain a specifically functional cell population within a physiological environment.

Show MeSH
Related in: MedlinePlus