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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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Cumulated collagen production from cells of in vitro maintained and of in vivo implanted hydrogels. Note that most of the collagens from the in vitro cultures were found in the culture medium (middle bar). Mean values (n = 6) and standard deviations are shown. In vitro gel: collagen content of the in vitro cultured hydrogel; in vitro gel + medium: collagen content of the in vitro cultured hydrogel plus the collagen released into the combined medium supernatant from two weeks of culture; in vivo: collagen content of the in vivo implanted hydrogel. Statistic: paired t-test comparing the in vitro group (gel + medium) and the in vivo group, * = p < 0.05.
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Figure 6: Cumulated collagen production from cells of in vitro maintained and of in vivo implanted hydrogels. Note that most of the collagens from the in vitro cultures were found in the culture medium (middle bar). Mean values (n = 6) and standard deviations are shown. In vitro gel: collagen content of the in vitro cultured hydrogel; in vitro gel + medium: collagen content of the in vitro cultured hydrogel plus the collagen released into the combined medium supernatant from two weeks of culture; in vivo: collagen content of the in vivo implanted hydrogel. Statistic: paired t-test comparing the in vitro group (gel + medium) and the in vivo group, * = p < 0.05.

Mentions: In contrast to these staining patterns from the subcutaneous hydrogels, the in vitro cultured hydrogels displayed only very poor immunostaining for any antigen (data not shown, see also biochemical analysis in Figures 5 and 6). Upon macroscopic inspection, the hydrogels maintained in tissue culture for 14 days remained clear and soft with high plasticity, quite in contrast to the gels harvested from the mice. The results of the bulk biochemical analysis may explain the contrasting findings. Cultured hydrogels contained more than five times less glycosaminoglycans (GAG) than implanted gels (Figure 5). The collagen deposition in implanted gels was even 50-fold higher as in cultured gels (Figure 6). On the other hand, when the GAG and collagen content of the culture medium was taken into consideration, the total GAG and collagen output of the cultured cells was seemingly rather equal to that of the implanted cells (Figure 5 und6). In vitro cultured cells produced slightly but significantly more GAG than the subcutaneous cultured cells (1.2-fold), conversely collagen accumulation was significantly higher in in vivo hydrogels (1.4-fold). Taken together, the entire process of matrix deposition in vivo could be considered to be more efficient towards formation of a functional tissue whereas in vitro conditions seemingly delayed that process.


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)

Cumulated collagen production from cells of in vitro maintained and of in vivo implanted hydrogels. Note that most of the collagens from the in vitro cultures were found in the culture medium (middle bar). Mean values (n = 6) and standard deviations are shown. In vitro gel: collagen content of the in vitro cultured hydrogel; in vitro gel + medium: collagen content of the in vitro cultured hydrogel plus the collagen released into the combined medium supernatant from two weeks of culture; in vivo: collagen content of the in vivo implanted hydrogel. Statistic: paired t-test comparing the in vitro group (gel + medium) and the in vivo group, * = p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Cumulated collagen production from cells of in vitro maintained and of in vivo implanted hydrogels. Note that most of the collagens from the in vitro cultures were found in the culture medium (middle bar). Mean values (n = 6) and standard deviations are shown. In vitro gel: collagen content of the in vitro cultured hydrogel; in vitro gel + medium: collagen content of the in vitro cultured hydrogel plus the collagen released into the combined medium supernatant from two weeks of culture; in vivo: collagen content of the in vivo implanted hydrogel. Statistic: paired t-test comparing the in vitro group (gel + medium) and the in vivo group, * = p < 0.05.
Mentions: In contrast to these staining patterns from the subcutaneous hydrogels, the in vitro cultured hydrogels displayed only very poor immunostaining for any antigen (data not shown, see also biochemical analysis in Figures 5 and 6). Upon macroscopic inspection, the hydrogels maintained in tissue culture for 14 days remained clear and soft with high plasticity, quite in contrast to the gels harvested from the mice. The results of the bulk biochemical analysis may explain the contrasting findings. Cultured hydrogels contained more than five times less glycosaminoglycans (GAG) than implanted gels (Figure 5). The collagen deposition in implanted gels was even 50-fold higher as in cultured gels (Figure 6). On the other hand, when the GAG and collagen content of the culture medium was taken into consideration, the total GAG and collagen output of the cultured cells was seemingly rather equal to that of the implanted cells (Figure 5 und6). In vitro cultured cells produced slightly but significantly more GAG than the subcutaneous cultured cells (1.2-fold), conversely collagen accumulation was significantly higher in in vivo hydrogels (1.4-fold). Taken together, the entire process of matrix deposition in vivo could be considered to be more efficient towards formation of a functional tissue whereas in vitro conditions seemingly delayed that process.

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