Induction of Intervertebral Disc–Like Cells From Adult Mesenchymal Stem Cells
http://www.100md.com
《干细胞学杂志》
Division of Experimental Orthopaedics, Orthopaedic Clinic, University of Heidelberg, Germany
Key Words. Intervertebral disc ? Articular cartilage ? Adult bone marrow stem cells ? Chondrogenic induction ? Gene expression
Correspondence: Dr. Wiltrud Richter, Division of Experimental Orthopaedics, Orthopaedic Clinic, University of Heidelberg, Schlierbacher Landstr. 200a, D-69118 Heidelberg, Germany. Telephone: 49-6221-969254; Fax: 49-6221-969288; e-mail: Wiltrud.Richter@ok.uni-heidelberg.de
ABSTRACT
Low back pain is one of the most common causes of disability, with 60% to 80% of people affected at some point during their lives. Several studies have shown that degeneration of intervertebral discs (IVDs) is one reason for a multiplicity of cases of low back pain , although the evidence for an indisputable link between clinical symptoms and IVD degeneration remains elusive. Current surgical treatments for IVD degeneration are disc excision or disc immobilization, procedures that do not repair the tissue.
One approach aiming to repair degenerated discs is tissue engineering of IVDs. Tissue engineering can be defined as the use of "living cells, manipulated through their extracellular environment or even genetically, to develop biological substitutes for implantation into the body and/or to foster the remodeling of tissue in some other active manner. The purpose is to either repair, replace, maintain, or enhance the function of a particular tissue or organ." . For tissue engineering of IVD, one major aim is the identification of suitable cell populations with the capacity to generate IVD tissue.
In several animal models, application of autologous cell sources was beneficial for the regeneration of degenerated discs . Although culture systems have been described that preserved the phenotype of native human IVD cells , expansion of cells is usually not possible under such conditions. However, high cell numbers are desired in cell therapy or tissue engineering approaches. Unfortunately, articular chondrocytes respond to monolayer expansion by dedifferentiation. They alter their cell morphology and matrix gene expression in comparison with native tissue , and the changes include the loss of the capacity to induce stable cartilage implants after intramuscular injection into nude mice . Mesenchymal stem cells (MSCs) isolated from bone marrow aspirates provide a nearly unlimited cell source with extremely high proliferation activity and the potential to differentiate into several mesenchymal cell lineages , including chondrogenic differentiation . Morphologically, articular cartilage and IVD tissue are clearly distinct, although both tissues have been described to harbor chondrocytes surrounded by extensive extracellular matrix . According to collagen type 2, sox 9, aggrecan, and proteoglycan expression , the differentiation status of IVD cells is believed to resemble that of articular chondrocytes. Intriguingly, the gene expression profile of both cell types, to our knowledge, has never been compared on the transcriptional level. Whether chondrogenic differentiation of MSCs may, thus, be suitable for generation of IVD-like or articular chondrocyte-like cells remained to be established.
In this study we aimed to identify an unlimited cell source with an expression profile resembling that of native IVD tissue. We hypothesized that extensive expansion of MSCs in monolayer followed by a TGF?-mediated induction protocol could yield such cells and be an attractive method for tissue engineering of IVD-like fibrocartilage. To our knowledge, this is the first study using cDNA array technology to characterize the gene expression profile of human IVD tissue and to compare it with expression levels in hyaline articular cartilage and MSCs after differentiation in 3D culture.
MATERIALS AND METHODS
Gene Expression Profiles in Mesenchymal Stem Cells After TGF?-Mediated Induction
The gene expression profile of undifferentiated in vitro expanded MSCs was characterized by expression of collagens types I and III, the small leucin-rich proteoglycans biglycan, lumican, and decorin, as well as alkaline phosphatase, osteonectin, endoglin, and chitinase 3-like 1 (Fig. 1B). Besides genes coding for the small leucin-rich proteoglycans and chitinase 3-like 1, none of these genes was described to be of relevance for chondrocytes.
The TGF?-mediated induction of MSCs in 3D spheroid culture resulted in induction and further upregulation of many genes typical for articular cartilage, such as collagens type II, XI, and XII, biglycan, fibromodulin, cartilage oligomeric matrix protein (COMP), proline arginine-rich end leucine-rich repeat protein (PRELP), lumican, and decorin (Fig. 1C, closed circles). In parallel, collagen type X, bone sialoprotein, and osteopontin were induced, which are known to be expressed in hypertrophic chondrocytes or bone (Fig. 1C, closed boxes). In contrast, the dedifferentiation- and proliferation-associated markers chitinase 3-like 1 (cartilage glycoprotein 39, YKL40) and endoglin were downregulated after induction (Fig. 1C, dotted circles). Transcripts for the cartilage-relevant proteins collagen type II and COMP (Fig. 1D, dotted circles) and the hypertrophic markers collagen type X, bone sialoprotein, and osteopontin (Fig. 1D, dotted squares) were undetectable in cultured IVD cells. However, all of them except collagen type X were present in native IVD tissue (Fig. 1E, closed boxes and circles), which also, uniquely, expressed transcripts for cartilage intermediate layer protein (CILP), chondroadherin, and osteocalcin (Fig. 1E, dashed squares). In sum, the data demonstrated a highly similar although not identical gene expression profile between native IVD tissue and MSC spheroids after TGF?-mediated induction and indicated that IVD cells underwent dedifferentiation during monolayer expansion in culture.
Two weeks after TGF?-mediated induction, collagen type II protein expression commenced in most cases at the surface of the spheroids (Fig. 2B), whereas at 4 weeks, a ring-like structure with strong collagen type I and II content surrounded the stained centre of the spheroid (Figs. 2C, 2F). Collagen type I protein was detectable at all time points (Figs. 2D–2F). This was accompanied by metachromatic staining of Safranin-O (Fig. 2I). Figure 2A shows the morphology of expanded MSCs in monolayer before TGF?-mediated induction.
Figure 2. Morphological assessment of differentiated MSCs. MSCs were expanded in monolayer culture (A), and TGF?-mediated differentiation of spheroid cultures was induced for 2 weeks (B, D, E, I) or 4 weeks (C, F, G, H) before collagen type II (B, C, G) and collagen type I (E, F, H) protein was detected by immunhistochemistry. The negative control was obtained by omitting the primary antibody (D). Proteoglycan production was evident according to metachromatic staining by Safranin-O (I) 2 weeks after induction. Boxes in (C) and (F) represent areas shown at higher magnification in (G) and (H), respectively. Bars in (B) through (F) indicate 500 μm; in (G) through (I), 100 μm, respectively. These results are representative of three independent experiments. Abbreviations: MSC, mesenchymal stem cell; TGF?, transforming growth factor?.
TGF?-Mediated Differentiation of MSC Spheroids Yields Gene Expression Levels Closer to Native IVD Tissue Than to Hyaline Articular Cartilage
Quantitative evaluation of the gene expression profiles of native IVD tissue (n = 6) and MSC spheroids 2 weeks after TGF?-mediated induction (n = 7) revealed highly similar expression levels for most genes (Fig. 3A). Except for CILP, chondroadherin, and osteocalcin, which were not induced in the differentiated MSC spheroids, PRELP remained on a lower level (p < .05). On the other hand, collagen type X was unique for MSCs after induction, and lumican reached a significantly higher level (p < .05) compared with IVD tissue.
Figure 3. Quantitative analysis of gene expression levels of selected genes (signal intensity above 15% in IVD tissue, except collagen type X, which was negative). Spheroid cultures of MSCs 2 weeks after TGF?-mediated induction (n = 7) were compared with IVD tissue (n = 6) (A) and articular cartilage tissue (n = 5) (B). The signal intensities were normalized to the gene expression levels of the housekeeping genes on each filter. The medians of independent experiments are shown and expressed as relative values in percent of the housekeeping genes. Abbreviations: IVD, intervertebral disc; MSC, mesenchymal stem cell; nd, not determined; TGF?, transforming growth factor?.
When comparing native healthy articular cartilage (n = 5) with differentiated MSC spheroids, many cartilage differentiation markers, such as PRELP, COMP, decorin, biglycan, fibromodulin, aggrecan, collagen type XI, and melanoma inhibitory activity, were expressed at considerably higher levels in articular cartilage (Fig. 3B). Discrepant expression was evident for CILP, osteopontin, collagen type I, and collagen type X.
In conclusion, MSC spheroids attained a gene expression profile after TGF?-mediated induction, which was quantitatively closer to native IVD tissue than to articular cartilage.
DISCUSSION
The authors wish to thank Stephanie Kadel, Katrin G?tzke, and Thea Hennig for excellent technical assistance and Dr. Sven Schneider for statistical support. This work was supported by a grant from the research fund of the Stiftung Orthop?dische Universit?tsklinik Heidelberg and by Cytonet, Weinheim.
REFERENCES
Buckwalter JA. Aging and degeneration of the human intervertebral disc. Spine 1995;20:1307–1314.
Cats-Baril WL, Frymoyer JW. Identifying patients at risk of becoming disabled because of low-back pain: the Vermont Rehabilitation Engineering Center predictive model. Spine 1991;16:605–607.
Humzah MD, Soames RW. Human intervertebral disc: structure and function. Anat Rec 1988;220:337–356.
Kranzler LI, Schaffer L, Siqueira EB. Recent advances in the treatment of ruptured lumbar intervertebral disks. Neurol Clin 1985;3:405–416.
Hunter CJ, Matyas JR, Duncan NA. The notochordal cell in the nucleus pulposus: a review in the context of tissue engineering. Tissue Eng 2003;9:667–677.
Sato M, Asazuma T, Ishihara M et al. An experimental study of the regeneration of the intervertebral disc with an allograft of cultured annulus fibrosus cells using a tissue-engineering method. Spine 2003;28:548–553.
Sakai D, Mochida J, Yamamoto Y et al. Transplantation of mesenchymal stem cells embedded in Atelocollagen gel to the intervertebral disc: a potential therapeutic model for disc degeneration. Biomaterials 2003;24:3531–3541.
Gruber HE, Johnson TL, Leslie K et al. Autologous intervertebral disc cell implantation: a model using Psammomys obesus, the sand rat. Spine 2002;27:1626–1633.
Okuma M, Mochida J, Nishimura K et al. Reinsertion of stimulated nucleus pulposus cells retards intervertebral disc degeneration: an in vitro and in vivo experimental study. J Orthop Res 2000;18:988–997.
Yung LJ, Hall R, Pelinkovic D et al. New use of a three-dimensional pellet culture system for human intervertebral disc cells: initial characterization and potential use for tissue engineering. Spine 2001;26:2316–2322.
Manning WK, Bonner WM Jr. Isolation and culture of chondrocytes from human adult articular cartilage. Arthritis Rheum 1967;10:235–239.
Benya PD, Shaffer JD. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 1982;30:215–224.
Aulthouse AL, Beck M, Griffey E et al. Expression of the human chondrocyte phenotype in vitro. In Vitro Cell Dev Biol 1989;25:659–668.
Benz K, Breit S, Lukoschek M et al. Molecular analysis of expansion, differentiation, and growth factor treatment of human chondrocytes identifies differentiation markers and growth-related genes. Biochem Biophys Res Commun 2002;293:284–292.
Archer CW, McDowell J, Bayliss MT et al. Phenotypic modulation in sub-populations of human articular chondrocytes in vitro. J Cell Sci 1990;97(pt 2):361–371.
Dell’Accio F, De Bari C, Luyten FP. Molecular markers predictive of the capacity of expanded human articular chondrocytes to form stable cartilage in vivo. Arthritis Rheum 2001;44:1608–1619.
Sekiya I, Vuoristo JT, Larson BL et al. In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis. Proc Natl Acad Sci U S A 2002;99:4397–4402.
Caplan AI. Mesenchymal stem cells. J Orthop Res 1991;9:641–650.
Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.
Winter A, Breit S, Parsch D et al. Cartilage-like gene expression in differentiated human stem cell spheroids: a comparison of bone marrow-derived and adipose tissue-derived stromal cells. Arthritis Rheum 2003;48:418–429.
Buckwalter JA, Pedrini-Mille A, Pedrini V et al. Proteoglycans of human infant intervertebral disc: electron microscopic and biochemical studies. J Bone Joint Surg Am 1985;67:284–294.
Cs-Szabo G, Ragasa-San Juan D, Turumella V et al. Changes in mRNA and protein levels of proteoglycans of the anulus fibrosus and nucleus pulposus during intervertebral disc degeneration. Spine 2002;27:2212–2219.
Sive JI, Baird P, Jeziorsk M et al. Expression of chondrocyte markers by cells of normal and degenerate intervertebral discs. Mol Pathol 2002;55:91–97.
Boos N, Weissbach S, Rohrbach H et al. Classification of age-related changes in lumbar intervertebral discs: 2002 Volvo Award in basic science. Spine 2002;27:2631–2644.
Haynesworth SE, Baber MA, Caplan AI. Cell surface antigens on human marrow-derived mesenchymal cells are detected by monoclonal antibodies. Bone 1992;13:69–80.
Reyes M, Lund T, Lenvik T et al. Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 2001;98:2615–2625.
Cancedda R, Descalzi CF, Castagnola P. Chondrocyte differentiation. Int Rev Cytol 1995;159:265–358.
Silva WA Jr, Covas DT, Panepucci RA et al. The profile of gene expression of human marrow mesenchymal stem cells. Stem Cells 2003;21:661–669.
Aigner T, Gresk-Otter KR, Fairbank JC et al. Variation with age in the pattern of type X collagen expression in normal and scoliotic human intervertebral discs. Calcif Tissue Int 1998;63:263–268.
Gebhard PM, Gehrsitz A, Bau B et al. Quantification of expression levels of cellular differentiation markers does not support a general shift in the cellular phenotype of osteoarthritic chondrocytes. J Orthop Res 2003;21:96–101.
Stevens RL, Ewins RJ, Revell PA et al. Proteoglycans of the intervertebral disc: homology of structure with laryngeal proteoglycans. Biochem J 1979;179:561–572.
Pearce RH, Grimmer BJ, Adams ME. Degeneration and the chemical composition of the human lumbar intervertebral disc. J Orthop Res 1987;5:198–205.
Gruber HE, Fisher EC Jr, Desai B et al. Human intervertebral disc cells from the annulus: three-dimensional culture in agarose or alginate and responsiveness to TGF-beta1. Exp Cell Res 1997;235:13–21.
Wang JY, Baer AE, Kraus VB et al. Intervertebral disc cells exhibit differences in gene expression in alginate and monolayer culture. Spine 2001;26:1747–1751.
Alini M, Li W, Markovic P et al. The potential and limitations of a cell-seeded collagen/hyaluronan scaffold to engineer an intervertebral disc-like matrix. Spine 2003;28:446–454.(Eric Steck, Helge Bertram)
Key Words. Intervertebral disc ? Articular cartilage ? Adult bone marrow stem cells ? Chondrogenic induction ? Gene expression
Correspondence: Dr. Wiltrud Richter, Division of Experimental Orthopaedics, Orthopaedic Clinic, University of Heidelberg, Schlierbacher Landstr. 200a, D-69118 Heidelberg, Germany. Telephone: 49-6221-969254; Fax: 49-6221-969288; e-mail: Wiltrud.Richter@ok.uni-heidelberg.de
ABSTRACT
Low back pain is one of the most common causes of disability, with 60% to 80% of people affected at some point during their lives. Several studies have shown that degeneration of intervertebral discs (IVDs) is one reason for a multiplicity of cases of low back pain , although the evidence for an indisputable link between clinical symptoms and IVD degeneration remains elusive. Current surgical treatments for IVD degeneration are disc excision or disc immobilization, procedures that do not repair the tissue.
One approach aiming to repair degenerated discs is tissue engineering of IVDs. Tissue engineering can be defined as the use of "living cells, manipulated through their extracellular environment or even genetically, to develop biological substitutes for implantation into the body and/or to foster the remodeling of tissue in some other active manner. The purpose is to either repair, replace, maintain, or enhance the function of a particular tissue or organ." . For tissue engineering of IVD, one major aim is the identification of suitable cell populations with the capacity to generate IVD tissue.
In several animal models, application of autologous cell sources was beneficial for the regeneration of degenerated discs . Although culture systems have been described that preserved the phenotype of native human IVD cells , expansion of cells is usually not possible under such conditions. However, high cell numbers are desired in cell therapy or tissue engineering approaches. Unfortunately, articular chondrocytes respond to monolayer expansion by dedifferentiation. They alter their cell morphology and matrix gene expression in comparison with native tissue , and the changes include the loss of the capacity to induce stable cartilage implants after intramuscular injection into nude mice . Mesenchymal stem cells (MSCs) isolated from bone marrow aspirates provide a nearly unlimited cell source with extremely high proliferation activity and the potential to differentiate into several mesenchymal cell lineages , including chondrogenic differentiation . Morphologically, articular cartilage and IVD tissue are clearly distinct, although both tissues have been described to harbor chondrocytes surrounded by extensive extracellular matrix . According to collagen type 2, sox 9, aggrecan, and proteoglycan expression , the differentiation status of IVD cells is believed to resemble that of articular chondrocytes. Intriguingly, the gene expression profile of both cell types, to our knowledge, has never been compared on the transcriptional level. Whether chondrogenic differentiation of MSCs may, thus, be suitable for generation of IVD-like or articular chondrocyte-like cells remained to be established.
In this study we aimed to identify an unlimited cell source with an expression profile resembling that of native IVD tissue. We hypothesized that extensive expansion of MSCs in monolayer followed by a TGF?-mediated induction protocol could yield such cells and be an attractive method for tissue engineering of IVD-like fibrocartilage. To our knowledge, this is the first study using cDNA array technology to characterize the gene expression profile of human IVD tissue and to compare it with expression levels in hyaline articular cartilage and MSCs after differentiation in 3D culture.
MATERIALS AND METHODS
Gene Expression Profiles in Mesenchymal Stem Cells After TGF?-Mediated Induction
The gene expression profile of undifferentiated in vitro expanded MSCs was characterized by expression of collagens types I and III, the small leucin-rich proteoglycans biglycan, lumican, and decorin, as well as alkaline phosphatase, osteonectin, endoglin, and chitinase 3-like 1 (Fig. 1B). Besides genes coding for the small leucin-rich proteoglycans and chitinase 3-like 1, none of these genes was described to be of relevance for chondrocytes.
The TGF?-mediated induction of MSCs in 3D spheroid culture resulted in induction and further upregulation of many genes typical for articular cartilage, such as collagens type II, XI, and XII, biglycan, fibromodulin, cartilage oligomeric matrix protein (COMP), proline arginine-rich end leucine-rich repeat protein (PRELP), lumican, and decorin (Fig. 1C, closed circles). In parallel, collagen type X, bone sialoprotein, and osteopontin were induced, which are known to be expressed in hypertrophic chondrocytes or bone (Fig. 1C, closed boxes). In contrast, the dedifferentiation- and proliferation-associated markers chitinase 3-like 1 (cartilage glycoprotein 39, YKL40) and endoglin were downregulated after induction (Fig. 1C, dotted circles). Transcripts for the cartilage-relevant proteins collagen type II and COMP (Fig. 1D, dotted circles) and the hypertrophic markers collagen type X, bone sialoprotein, and osteopontin (Fig. 1D, dotted squares) were undetectable in cultured IVD cells. However, all of them except collagen type X were present in native IVD tissue (Fig. 1E, closed boxes and circles), which also, uniquely, expressed transcripts for cartilage intermediate layer protein (CILP), chondroadherin, and osteocalcin (Fig. 1E, dashed squares). In sum, the data demonstrated a highly similar although not identical gene expression profile between native IVD tissue and MSC spheroids after TGF?-mediated induction and indicated that IVD cells underwent dedifferentiation during monolayer expansion in culture.
Two weeks after TGF?-mediated induction, collagen type II protein expression commenced in most cases at the surface of the spheroids (Fig. 2B), whereas at 4 weeks, a ring-like structure with strong collagen type I and II content surrounded the stained centre of the spheroid (Figs. 2C, 2F). Collagen type I protein was detectable at all time points (Figs. 2D–2F). This was accompanied by metachromatic staining of Safranin-O (Fig. 2I). Figure 2A shows the morphology of expanded MSCs in monolayer before TGF?-mediated induction.
Figure 2. Morphological assessment of differentiated MSCs. MSCs were expanded in monolayer culture (A), and TGF?-mediated differentiation of spheroid cultures was induced for 2 weeks (B, D, E, I) or 4 weeks (C, F, G, H) before collagen type II (B, C, G) and collagen type I (E, F, H) protein was detected by immunhistochemistry. The negative control was obtained by omitting the primary antibody (D). Proteoglycan production was evident according to metachromatic staining by Safranin-O (I) 2 weeks after induction. Boxes in (C) and (F) represent areas shown at higher magnification in (G) and (H), respectively. Bars in (B) through (F) indicate 500 μm; in (G) through (I), 100 μm, respectively. These results are representative of three independent experiments. Abbreviations: MSC, mesenchymal stem cell; TGF?, transforming growth factor?.
TGF?-Mediated Differentiation of MSC Spheroids Yields Gene Expression Levels Closer to Native IVD Tissue Than to Hyaline Articular Cartilage
Quantitative evaluation of the gene expression profiles of native IVD tissue (n = 6) and MSC spheroids 2 weeks after TGF?-mediated induction (n = 7) revealed highly similar expression levels for most genes (Fig. 3A). Except for CILP, chondroadherin, and osteocalcin, which were not induced in the differentiated MSC spheroids, PRELP remained on a lower level (p < .05). On the other hand, collagen type X was unique for MSCs after induction, and lumican reached a significantly higher level (p < .05) compared with IVD tissue.
Figure 3. Quantitative analysis of gene expression levels of selected genes (signal intensity above 15% in IVD tissue, except collagen type X, which was negative). Spheroid cultures of MSCs 2 weeks after TGF?-mediated induction (n = 7) were compared with IVD tissue (n = 6) (A) and articular cartilage tissue (n = 5) (B). The signal intensities were normalized to the gene expression levels of the housekeeping genes on each filter. The medians of independent experiments are shown and expressed as relative values in percent of the housekeeping genes. Abbreviations: IVD, intervertebral disc; MSC, mesenchymal stem cell; nd, not determined; TGF?, transforming growth factor?.
When comparing native healthy articular cartilage (n = 5) with differentiated MSC spheroids, many cartilage differentiation markers, such as PRELP, COMP, decorin, biglycan, fibromodulin, aggrecan, collagen type XI, and melanoma inhibitory activity, were expressed at considerably higher levels in articular cartilage (Fig. 3B). Discrepant expression was evident for CILP, osteopontin, collagen type I, and collagen type X.
In conclusion, MSC spheroids attained a gene expression profile after TGF?-mediated induction, which was quantitatively closer to native IVD tissue than to articular cartilage.
DISCUSSION
The authors wish to thank Stephanie Kadel, Katrin G?tzke, and Thea Hennig for excellent technical assistance and Dr. Sven Schneider for statistical support. This work was supported by a grant from the research fund of the Stiftung Orthop?dische Universit?tsklinik Heidelberg and by Cytonet, Weinheim.
REFERENCES
Buckwalter JA. Aging and degeneration of the human intervertebral disc. Spine 1995;20:1307–1314.
Cats-Baril WL, Frymoyer JW. Identifying patients at risk of becoming disabled because of low-back pain: the Vermont Rehabilitation Engineering Center predictive model. Spine 1991;16:605–607.
Humzah MD, Soames RW. Human intervertebral disc: structure and function. Anat Rec 1988;220:337–356.
Kranzler LI, Schaffer L, Siqueira EB. Recent advances in the treatment of ruptured lumbar intervertebral disks. Neurol Clin 1985;3:405–416.
Hunter CJ, Matyas JR, Duncan NA. The notochordal cell in the nucleus pulposus: a review in the context of tissue engineering. Tissue Eng 2003;9:667–677.
Sato M, Asazuma T, Ishihara M et al. An experimental study of the regeneration of the intervertebral disc with an allograft of cultured annulus fibrosus cells using a tissue-engineering method. Spine 2003;28:548–553.
Sakai D, Mochida J, Yamamoto Y et al. Transplantation of mesenchymal stem cells embedded in Atelocollagen gel to the intervertebral disc: a potential therapeutic model for disc degeneration. Biomaterials 2003;24:3531–3541.
Gruber HE, Johnson TL, Leslie K et al. Autologous intervertebral disc cell implantation: a model using Psammomys obesus, the sand rat. Spine 2002;27:1626–1633.
Okuma M, Mochida J, Nishimura K et al. Reinsertion of stimulated nucleus pulposus cells retards intervertebral disc degeneration: an in vitro and in vivo experimental study. J Orthop Res 2000;18:988–997.
Yung LJ, Hall R, Pelinkovic D et al. New use of a three-dimensional pellet culture system for human intervertebral disc cells: initial characterization and potential use for tissue engineering. Spine 2001;26:2316–2322.
Manning WK, Bonner WM Jr. Isolation and culture of chondrocytes from human adult articular cartilage. Arthritis Rheum 1967;10:235–239.
Benya PD, Shaffer JD. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 1982;30:215–224.
Aulthouse AL, Beck M, Griffey E et al. Expression of the human chondrocyte phenotype in vitro. In Vitro Cell Dev Biol 1989;25:659–668.
Benz K, Breit S, Lukoschek M et al. Molecular analysis of expansion, differentiation, and growth factor treatment of human chondrocytes identifies differentiation markers and growth-related genes. Biochem Biophys Res Commun 2002;293:284–292.
Archer CW, McDowell J, Bayliss MT et al. Phenotypic modulation in sub-populations of human articular chondrocytes in vitro. J Cell Sci 1990;97(pt 2):361–371.
Dell’Accio F, De Bari C, Luyten FP. Molecular markers predictive of the capacity of expanded human articular chondrocytes to form stable cartilage in vivo. Arthritis Rheum 2001;44:1608–1619.
Sekiya I, Vuoristo JT, Larson BL et al. In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis. Proc Natl Acad Sci U S A 2002;99:4397–4402.
Caplan AI. Mesenchymal stem cells. J Orthop Res 1991;9:641–650.
Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.
Winter A, Breit S, Parsch D et al. Cartilage-like gene expression in differentiated human stem cell spheroids: a comparison of bone marrow-derived and adipose tissue-derived stromal cells. Arthritis Rheum 2003;48:418–429.
Buckwalter JA, Pedrini-Mille A, Pedrini V et al. Proteoglycans of human infant intervertebral disc: electron microscopic and biochemical studies. J Bone Joint Surg Am 1985;67:284–294.
Cs-Szabo G, Ragasa-San Juan D, Turumella V et al. Changes in mRNA and protein levels of proteoglycans of the anulus fibrosus and nucleus pulposus during intervertebral disc degeneration. Spine 2002;27:2212–2219.
Sive JI, Baird P, Jeziorsk M et al. Expression of chondrocyte markers by cells of normal and degenerate intervertebral discs. Mol Pathol 2002;55:91–97.
Boos N, Weissbach S, Rohrbach H et al. Classification of age-related changes in lumbar intervertebral discs: 2002 Volvo Award in basic science. Spine 2002;27:2631–2644.
Haynesworth SE, Baber MA, Caplan AI. Cell surface antigens on human marrow-derived mesenchymal cells are detected by monoclonal antibodies. Bone 1992;13:69–80.
Reyes M, Lund T, Lenvik T et al. Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 2001;98:2615–2625.
Cancedda R, Descalzi CF, Castagnola P. Chondrocyte differentiation. Int Rev Cytol 1995;159:265–358.
Silva WA Jr, Covas DT, Panepucci RA et al. The profile of gene expression of human marrow mesenchymal stem cells. Stem Cells 2003;21:661–669.
Aigner T, Gresk-Otter KR, Fairbank JC et al. Variation with age in the pattern of type X collagen expression in normal and scoliotic human intervertebral discs. Calcif Tissue Int 1998;63:263–268.
Gebhard PM, Gehrsitz A, Bau B et al. Quantification of expression levels of cellular differentiation markers does not support a general shift in the cellular phenotype of osteoarthritic chondrocytes. J Orthop Res 2003;21:96–101.
Stevens RL, Ewins RJ, Revell PA et al. Proteoglycans of the intervertebral disc: homology of structure with laryngeal proteoglycans. Biochem J 1979;179:561–572.
Pearce RH, Grimmer BJ, Adams ME. Degeneration and the chemical composition of the human lumbar intervertebral disc. J Orthop Res 1987;5:198–205.
Gruber HE, Fisher EC Jr, Desai B et al. Human intervertebral disc cells from the annulus: three-dimensional culture in agarose or alginate and responsiveness to TGF-beta1. Exp Cell Res 1997;235:13–21.
Wang JY, Baer AE, Kraus VB et al. Intervertebral disc cells exhibit differences in gene expression in alginate and monolayer culture. Spine 2001;26:1747–1751.
Alini M, Li W, Markovic P et al. The potential and limitations of a cell-seeded collagen/hyaluronan scaffold to engineer an intervertebral disc-like matrix. Spine 2003;28:446–454.(Eric Steck, Helge Bertram)