Human Intervertebral Disc Cells from the Annulus: Three-Dimensional Culture in Agarose or Alginate and Responsiveness to TGF-β1

He, Guanghua; Ec, Fisher; Desai, Bhaloo J.; Aa, Stasky; Hoelscher, Gretchen L.; Hanley, EN

doi:10.1006/excr.1997.3647

Cited by 282 publications

(177 citation statements)

References 20 publications

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“…There was no evidence of an increase in either the compressive or shear moduli of the IVD cell-alginate constructs with time in culture. This was unexpected given previous findings that have demonstrated significant biosynthesis for IVD cells when cultured in alginate beads [7,9,10,16,30]. These results contrast with a previous study of articular chondrocytes in agarose culture [6], where the chondrocytes were able to synthesize a mechanically functional matrix.…”

Section: Discussioncontrasting

confidence: 70%

“…While there is ample evidence to show that these cells are able to synthesize significant quantities of ma-trix [7,9,10,16,30], these molecules do not appear to interact in a functional manner. This inability to synthesize a functional extracellular matrix in vitro may be caused by intrinsic behaviors of the IVD cells or by characteristics of the alginate culture system.…”

Section: Discussionmentioning

confidence: 99%

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Collagen gene expression and mechanical properties of intervertebral disc cell–alginate cultures

Baer

Wang

Kraus

et al. 2001

Journal Orthopaedic Research

132

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Cells of the intervertebral disc have a limited capacity for matrix repair that may contribute to the onset and progression of degenerative disc changes. In this study, the biosynthetic capacity of cells isolated from specific regions of the porcine intervertebral disc was evaluated in vitro. Using a competitive reverse transcription-polymerase chain reaction technique, gene expression levels for types I and I1 collagen were quantified in cells cultured for up to 21 d in a three-dimensional alginate culture system and compared to levels obtained for cells in vivo. The mechanical properties of cell-alginate constructs were measured in compression and shear after periods of culture up to 16 weeks. Cells from the anulus fibrosus expressed the most type I collagen mRNA in vivo and in vitro, while cells from the transition zone expressed the most type I1 collagen mRNA in vivo and in vitro. Mechanical testing results indicate that a mechanically functional matrix did not form at any time during the culture period; rather, decreases of up to 5o"h were observed in the compressive and shear moduli of the cell-alginate constructs compared to alginate with no cells. Together with results of prior studies, these results suggest that intervertebral disc cells maintain characteristics of their phenotype when cultured in alginate, but the molecules they synthesize are not able to form a mechanically functional matrix in vitro. 0 3001 Orthopaedic Research Society. Published by

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Section: Discussioncontrasting

confidence: 70%

Section: Discussionmentioning

confidence: 99%

Collagen gene expression and mechanical properties of intervertebral disc cell–alginate cultures

Baer

Wang

Kraus

et al. 2001

Journal Orthopaedic Research

132

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“…The utility of this culture system has been acknowledged by many authors (Guo et al 1989 ;Ha$ uselmann et al 1992Ha$ uselmann et al , 1994Maldonado & Oegema 1992 ;Grandolfo et al 1993 ;Chelburg et al 1995 ;Chiba et al 1997 ;Gruber et al 1997 1997 a, b, 1999 b). Earlier immunoblotting studies identified decorin and biglycan in media samples derived from ovine annulus fibrosus (AF) cells cultured in alginate beads , as well as the presence of catabolic fragments of the large high buoyant density aggrecan-like PGs (PGs) (Melrose et al 1997 b).…”

Section: mentioning

confidence: 97%

“…The alginate bead culture system has been shown to maintain chondrocyte (Guo et al 1989 ;Ha$ uselmann et al 1992Ha$ uselmann et al , 1994Grandolfo et al 1993) and intervertebral disc (IVD) cell viability for prolonged periods in culture and has been utilised to culture canine, ovine, lapine and human IVD cells (Maldonado & Oegema 1992 ;Chelburg et al 1995 ;Chiba et al 1997 ;Gruber et al 1997 ;Melrose et al 1997 a, b). The utility of this culture system has been acknowledged by many authors (Guo et al 1989 ;Ha$ uselmann et al 1992Ha$ uselmann et al , 1994Maldonado & Oegema 1992 ;Grandolfo et al 1993 ;Chelburg et al 1995 ;Chiba et al 1997 ;Gruber et al 1997 ;Melrose et al a, b, 1999.…”

Section: mentioning

confidence: 99%

“…The utility of the alginate bead culture system has been acknowledged by many authors (Guo et al 1989 ;Ha$ uselmann et al 1992Ha$ uselmann et al , 1994Maldonado & Oegema, 1992 ;Grandolfo et al 1993 ;Chelburg et al 1995 ;Chiba et al 1997 ;Gruber et al 1997 ;Melrose et al 1997 a, b). The methodology we have developed in the present study for the removal of sodium alginate from the de novo synthesised PGs should further improve the applicability of this culture system for studies of the PG metabolism and structure by cells cultured in this artificial matrix.…”

Section: mentioning

confidence: 99%

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Differential expression of proteoglycan epitopes by ovine intervertebral disc cells

2000

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The alginate bead culture system has been utilised by several groups to examine the in vitro proteoglycan (PG) metabolism of chondrocytes and intervertebral disc cells, but the nature of the PGs produced has not been examined in detail. This is largely due to the difficulty of separating the anionically charged sodium alginate support matrix from PGs which are similarly charged. In the present study ovine annulus fibrosus, transitional zone and nucleus pulposus cells were dissociated enzymatically from their respective matrices by sequential digestion with pronase\clostridial collagenase and DNAase and then cultured in alginate beads for 10 d. The beads were solubilised and subjected to DEAE Sepharose CL6B anion exchange chromatography to separate the sodium alginate bead support matrix material quantitatively from the disc cell PGs. The alginate free bead PGs were then subjected to composite agarose polyacrylamide gel electrophoresis to resolve PG populations and the PGs were transferred to nitrocellulose membranes by semidry electroblotting. The PGs were identified by probing the blots with a panel of antibodies to defined PG core protein and glycosaminoglycan side chain epitopes. Alginate beads of disc cells were also embedded in paraffin wax and 4 µm sections cut to immunolocalise decorin, biglycan, versican, and the 7-D-4 PG epitope within the beads. Decorin and biglycan had similar distributions in the beads, being localised on the cell surface whereas versican and the 7-D-4 PG epitope were immunolocalised interterritoriarly. This study is the first to demonstrate that ovine disc cells synthesise versican in alginate bead culture. Furthermore the immunoblotting studies also showed that a proportion of the 7-D-4 PG epitope was colocalised with versican.Key words : Alginate beads ; disc cells ; versican ; 7-D-4 epitope ; biglycan\decorin. The alginate bead culture system has been shown to maintain chondrocyte (Guo et al. 1989 ;Ha$ uselmann et al. 1992Ha$ uselmann et al. , 1994Grandolfo et al. 1993) and intervertebral disc (IVD) cell viability for prolonged periods in culture and has been utilised to culture canine, ovine, lapine and human IVD cells (Maldonado & Oegema 1992 ;Chelburg et al. 1995 ;Chiba et al. 1997 ;Gruber et al. 1997 ; Melrose et al. 1997 a, b). The utility of this culture system has been acknowledged by many authors (Guo et al. 1989 ;Ha$ uselmann et al. 1992Ha$ uselmann et al. , 1994Maldonado & Oegema 1992 ;Grandolfo et al. 1993 ;Chelburg et al. 1995 ;Chiba et al. 1997 ;Gruber et al. 1997 1997 a, b, 1999 b). Earlier immunoblotting studies identified decorin and biglycan in media samples derived from ovine annulus fibrosus (AF) cells cultured in alginate beads , as well as the presence of catabolic fragments of the large high buoyant density aggrecan-like PGs (PGs) (Melrose et al. 1997 b). The PGs which were incorporated into the bead extracellular matrix in these earlier studies were not characterised due to the difficulty of separating them from the polyuronic acid alg...

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Three-dimensional cartilage formation by bone marrow-derived cells seeded in polylactide/alginate amalgam

Caterson

Nesti

et al. 2001

J. Biomed. Mater. Res.

179

116

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Bone marrow-derived cells are considered as candidate cells for cartilage tissue engineering by virtue of their ability to undergo chondrogenesis in vitro when cultured in high density or when embedded within a three-dimensional matrix in the presence of growth factors. This study evaluated the potential of human bone marrow-derived cells for cartilage tissue engineering by examining their chondrogenic properties within a three-dimensional amalgam scaffold consisting of the biodegradable polymer, poly-L-lactic acid (PLA) alone, and with the polysaccharide gel, alginate. Cells were suspended either in alginate or medium and loaded into porous PLA blocks. Alginate was used to improve cell loading and retention within the construct, whereas the PLA polymeric scaffold provided appropriate mechanical support and stability to the composite culture. Cells seeded in the PLA/alginate amalgams and the plain PLA constructs were treated with different concentrations of recombinant human transforming growth factor-beta1 (TGF-beta 1) either continuously (10 ng/mL) or only for the initial 3 days of culture (50 ng/mL). Chondrogenesis was assessed at weekly intervals with cultures maintained for up to 3 weeks. Histological and immunohistochemical analysis of the TGF-beta 1-treated PLA/alginate amalgam and PLA constructs showed development of a cartilaginous phenotype from day 7 to day 21 as demonstrated by colocalization of Alcian blue staining with collagen type II and cartilage proteoglycan link protein. Expression of cartilage specific genes, including collagen types II and IX, and aggrecan, was detected in TGF-beta 1-treated cultures by reverse transcription-polymerase chain reaction analysis. The initiation and progression of chondrogenic differentiation within the polymeric macrostructure occurred with both continuous and the initial 3-day TGF-beta 1 treatment regimens, suggesting that key regulatory events of chondrogenesis take place during the early period of cell growth and proliferation. Scanning electron microscopy revealed abundant cells with a rounded morphology in the PLA/alginate amalgam. These findings suggest that the three-dimensional PLA/alginate amalgam is a potential candidate bioactive scaffold for cartilage tissue engineering applications.

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Human Intervertebral Disc Cells from the Annulus: Three-Dimensional Culture in Agarose or Alginate and Responsiveness to TGF-β1

Cited by 282 publications

References 20 publications

Collagen gene expression and mechanical properties of intervertebral disc cell–alginate cultures

Collagen gene expression and mechanical properties of intervertebral disc cell–alginate cultures

Differential expression of proteoglycan epitopes by ovine intervertebral disc cells

Three-dimensional cartilage formation by bone marrow-derived cells seeded in polylactide/alginate amalgam

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