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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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Immunohistochemistry staining and species specific genomic in situ hybridization of an implanted hydrogel upon harvest. A: Detection of collagen type I; B: collagen type II; C: aggrecan by immunohistochemistry. The blue dots are the DAPI stained nuclei. Note the even distribution of the nuclei across the samples, except for the edge in A. Species specific in situ hybridization discriminates between cells of human origin (D) and cells of murine origin (E). Mouse cells are predominantly found at the edge of the xenotransplant, human cells are distributed evenly within the transplant.
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Figure 4: Immunohistochemistry staining and species specific genomic in situ hybridization of an implanted hydrogel upon harvest. A: Detection of collagen type I; B: collagen type II; C: aggrecan by immunohistochemistry. The blue dots are the DAPI stained nuclei. Note the even distribution of the nuclei across the samples, except for the edge in A. Species specific in situ hybridization discriminates between cells of human origin (D) and cells of murine origin (E). Mouse cells are predominantly found at the edge of the xenotransplant, human cells are distributed evenly within the transplant.

Mentions: Concerning the hydrogel itself, histological inspection revealed a rather homogenous distribution of the disc cells within the hydrogel (Figure 4A-C). There were no obvious regions with particular features such as higher densities of viable cells at the edge or the centre. To verify the presence and the distribution of human cells in the transplants a species specific genomic hybridization was performed. It was possible to discriminate between cells of human origin and cells of murine origin. Mouse cells are predominantly found at the edge of the xenotransplant (Figure 4E) (corresponding with the collagen type I staining); human cells are distributed evenly within the transplant (Figure 4D), confirming the above notion of the initial rather homogenous cell distribution. It appears that few cells with positive mouse Alu hybridization are also present within the hydrogel (Figure 4E). These might be false positive signals, as the probes to mouse Alu sequences tend to weakly bind to human Alu sequences. Since there are fewer mouse sequences than human Alu, the detection reaction with NBT/BCIP has to be intensified in order to visualize bound probes per nucleus in mouse cells. In this case, the unspecific binding on human cells may also appear and cause the present false positive signals on some cells.


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)

Immunohistochemistry staining and species specific genomic in situ hybridization of an implanted hydrogel upon harvest. A: Detection of collagen type I; B: collagen type II; C: aggrecan by immunohistochemistry. The blue dots are the DAPI stained nuclei. Note the even distribution of the nuclei across the samples, except for the edge in A. Species specific in situ hybridization discriminates between cells of human origin (D) and cells of murine origin (E). Mouse cells are predominantly found at the edge of the xenotransplant, human cells are distributed evenly within the transplant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Immunohistochemistry staining and species specific genomic in situ hybridization of an implanted hydrogel upon harvest. A: Detection of collagen type I; B: collagen type II; C: aggrecan by immunohistochemistry. The blue dots are the DAPI stained nuclei. Note the even distribution of the nuclei across the samples, except for the edge in A. Species specific in situ hybridization discriminates between cells of human origin (D) and cells of murine origin (E). Mouse cells are predominantly found at the edge of the xenotransplant, human cells are distributed evenly within the transplant.
Mentions: Concerning the hydrogel itself, histological inspection revealed a rather homogenous distribution of the disc cells within the hydrogel (Figure 4A-C). There were no obvious regions with particular features such as higher densities of viable cells at the edge or the centre. To verify the presence and the distribution of human cells in the transplants a species specific genomic hybridization was performed. It was possible to discriminate between cells of human origin and cells of murine origin. Mouse cells are predominantly found at the edge of the xenotransplant (Figure 4E) (corresponding with the collagen type I staining); human cells are distributed evenly within the transplant (Figure 4D), confirming the above notion of the initial rather homogenous cell distribution. It appears that few cells with positive mouse Alu hybridization are also present within the hydrogel (Figure 4E). These might be false positive signals, as the probes to mouse Alu sequences tend to weakly bind to human Alu sequences. Since there are fewer mouse sequences than human Alu, the detection reaction with NBT/BCIP has to be intensified in order to visualize bound probes per nucleus in mouse cells. In this case, the unspecific binding on human cells may also appear and cause the present false positive signals on some cells.

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