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Conference Paper: Development of Three-Dimensional Culture System for Maintenance of Bovine Nucleus Pulposus Cells

TitleDevelopment of Three-Dimensional Culture System for Maintenance of Bovine Nucleus Pulposus Cells
Authors
Issue Date2014
PublisherGeorg Thieme Verlag. The Journal's web site is located at http://www.thieme.com/index.php?page=shop.product_details&flypage=flypage.tpl&product_id=1351&category_id=90&option=com_virtuemart&Itemid=53
Citation
World Forum for Spine Research (WFSR), Xi'an, China,15-17 May 2014. In Global Spine Journal, 2014, v. 4 n. Suppl. 1, p. S89, PO.085 How to Cite?
AbstractIntroduction Nucleus pulposus (NP) is located at the center of the intervertebral disc (IVD) and rich in collagen type II and proteoglycans. The glycosaminoglycans (GAGs) in the proteoglycans enable the retention of a high amount ofwater, which is important for the mechanical function of the IVD. In the pathology of degenerative disc disease (DDD), the loss of proteoglycan and water may be related to changes in NP cells (NPCs). To enable studies in disc cell biology, developing a 3D culture system for NPCs with maintained phenotype is critical. In this study, we compared two microencapsulation systems using collagen alone and collagen-glycosaminoglycan (GAG) coprecipitate, respectively, in maintaining NPC phenotype and used a micropatterning platform to screen for the type of extracellular matrix components. Materials and Methods NP from bovine caudal spines was digested to isolate bovine NPCs (bNPCs). In the first 3D culture system, the bNPCs were encapsulated in microspheres made of collagen and then cultured up to 2 weeks before evaluation. The second 3D culture system was based on collagen-GAG co-precipitate. In brief, it was fabricated by mixing chondroitin-6-sulfate, aminated type I collagen, and bNPCs, followed by centrifugation to collect the constructs, whichwere then cultured up to 2weeks followed by evaluation. Lastly, protein microstructures were fabricated using a laser-based crosslinking technique. Bovine serum albumin (BSA), together with extracellular matrix (ECM) proteins such as collagen, was crosslinked using laser and rose bengal. Cellswere seeded onto these microstructures and evaluated. Cell morphology and distribution were revealed by histology and actin staining. Viabilitywas examined by live/dead staining. Phenotype and components in the ECM were analyzed by immunohistochemistry and histology. Results Contraction of microspheres was monitored over time, especially from day 3 to day 7. Increase in cell densitywas observed over time, with a ring of dense cells at periphery starting to form since day 7. Some rounded cells with large vacuoles, simulating the morphology of bNPC in native NP tissue, were observed on day 7 in collagen microspheres. However, cells with such morphologywere not observed in later time points. Most cells were alive throughout the culture period. Alcian blue staining became stronger over time and was particularly strong in later time points at the periphery region. IHC of collagen type II was intense in both the cells and extracellular matrix except on day 3. The local and global mechanical properties of the microspheres may affect the cell morphology. Data on collagen-GAG co-precipitate and protein micropatterns will also be presented. Conclusion The collagen microspheres supported the survival and proliferation of bNPCs and partially maintained some phenotypic characteristics of bNPCs, including the production of collagen type II throughout the microspheres. The local and global mechanical properties of the microspheres may affect the distribution of actin and morphology of the cells. Disclosure of Interest None declared References 1. Chan BP, Ma JN, Xu JY, Li CW, Cheng JP, Cheng SH. Femtosecond laser-based free writing of 3D protein microstructures and micropatterns with sub-micrometer features: a study on voxels, porosity, and cytocompatibility. Adv Funct Mater 2014;24(3):277–294 2. Chan BP, Hui TY, Yeung CW, Li J, Mo I, Chan GC. Selfassembled collagen-human mesenchymal stem cell microspheres for regenerative medicine. Biomaterials 2007;28 (31):4652–4666 3. Choy AT, Leong KW, Chan BP. Chemical modification of collagen improves glycosaminoglycan retention of their coprecipitates. Acta Biomater 2013;9(1):4661–4672 4. Yuan M, Leong KW, Chan BP. Three-dimensional culture of rabbit nucleus pulposus cells in collagen microspheres. Spine J 2011;11(10):947–960
DescriptionConference theme: The Intervertebral Disc - from Degeneration to Therapeutic Motion Preservation
The abstract can be viewed at http://www.spineresearchforum.org/WFSR_2014_Thieme_AbstractBook_with_Cover.pdf
Persistent Identifierhttp://hdl.handle.net/10722/203893
ISSN
2015 SCImago Journal Rankings: 0.108

 

DC FieldValueLanguage
dc.contributor.authorLi, HYen_US
dc.contributor.authorMa, JNen_US
dc.contributor.authorChooi, WHen_US
dc.contributor.authorChan, Den_US
dc.contributor.authorCheah, KSEen_US
dc.contributor.authorChan, BPen_US
dc.date.accessioned2014-09-19T16:43:15Z-
dc.date.available2014-09-19T16:43:15Z-
dc.date.issued2014en_US
dc.identifier.citationWorld Forum for Spine Research (WFSR), Xi'an, China,15-17 May 2014. In Global Spine Journal, 2014, v. 4 n. Suppl. 1, p. S89, PO.085en_US
dc.identifier.issn2192-5682-
dc.identifier.urihttp://hdl.handle.net/10722/203893-
dc.descriptionConference theme: The Intervertebral Disc - from Degeneration to Therapeutic Motion Preservation-
dc.descriptionThe abstract can be viewed at http://www.spineresearchforum.org/WFSR_2014_Thieme_AbstractBook_with_Cover.pdf-
dc.description.abstractIntroduction Nucleus pulposus (NP) is located at the center of the intervertebral disc (IVD) and rich in collagen type II and proteoglycans. The glycosaminoglycans (GAGs) in the proteoglycans enable the retention of a high amount ofwater, which is important for the mechanical function of the IVD. In the pathology of degenerative disc disease (DDD), the loss of proteoglycan and water may be related to changes in NP cells (NPCs). To enable studies in disc cell biology, developing a 3D culture system for NPCs with maintained phenotype is critical. In this study, we compared two microencapsulation systems using collagen alone and collagen-glycosaminoglycan (GAG) coprecipitate, respectively, in maintaining NPC phenotype and used a micropatterning platform to screen for the type of extracellular matrix components. Materials and Methods NP from bovine caudal spines was digested to isolate bovine NPCs (bNPCs). In the first 3D culture system, the bNPCs were encapsulated in microspheres made of collagen and then cultured up to 2 weeks before evaluation. The second 3D culture system was based on collagen-GAG co-precipitate. In brief, it was fabricated by mixing chondroitin-6-sulfate, aminated type I collagen, and bNPCs, followed by centrifugation to collect the constructs, whichwere then cultured up to 2weeks followed by evaluation. Lastly, protein microstructures were fabricated using a laser-based crosslinking technique. Bovine serum albumin (BSA), together with extracellular matrix (ECM) proteins such as collagen, was crosslinked using laser and rose bengal. Cellswere seeded onto these microstructures and evaluated. Cell morphology and distribution were revealed by histology and actin staining. Viabilitywas examined by live/dead staining. Phenotype and components in the ECM were analyzed by immunohistochemistry and histology. Results Contraction of microspheres was monitored over time, especially from day 3 to day 7. Increase in cell densitywas observed over time, with a ring of dense cells at periphery starting to form since day 7. Some rounded cells with large vacuoles, simulating the morphology of bNPC in native NP tissue, were observed on day 7 in collagen microspheres. However, cells with such morphologywere not observed in later time points. Most cells were alive throughout the culture period. Alcian blue staining became stronger over time and was particularly strong in later time points at the periphery region. IHC of collagen type II was intense in both the cells and extracellular matrix except on day 3. The local and global mechanical properties of the microspheres may affect the cell morphology. Data on collagen-GAG co-precipitate and protein micropatterns will also be presented. Conclusion The collagen microspheres supported the survival and proliferation of bNPCs and partially maintained some phenotypic characteristics of bNPCs, including the production of collagen type II throughout the microspheres. The local and global mechanical properties of the microspheres may affect the distribution of actin and morphology of the cells. Disclosure of Interest None declared References 1. Chan BP, Ma JN, Xu JY, Li CW, Cheng JP, Cheng SH. Femtosecond laser-based free writing of 3D protein microstructures and micropatterns with sub-micrometer features: a study on voxels, porosity, and cytocompatibility. Adv Funct Mater 2014;24(3):277–294 2. Chan BP, Hui TY, Yeung CW, Li J, Mo I, Chan GC. Selfassembled collagen-human mesenchymal stem cell microspheres for regenerative medicine. Biomaterials 2007;28 (31):4652–4666 3. Choy AT, Leong KW, Chan BP. Chemical modification of collagen improves glycosaminoglycan retention of their coprecipitates. Acta Biomater 2013;9(1):4661–4672 4. Yuan M, Leong KW, Chan BP. Three-dimensional culture of rabbit nucleus pulposus cells in collagen microspheres. Spine J 2011;11(10):947–960-
dc.languageengen_US
dc.publisherGeorg Thieme Verlag. The Journal's web site is located at http://www.thieme.com/index.php?page=shop.product_details&flypage=flypage.tpl&product_id=1351&category_id=90&option=com_virtuemart&Itemid=53-
dc.relation.ispartofGlobal Spine Journalen_US
dc.rightsGlobal Spine Journal. Copyright © Georg Thieme Verlag.-
dc.titleDevelopment of Three-Dimensional Culture System for Maintenance of Bovine Nucleus Pulposus Cellsen_US
dc.typeConference_Paperen_US
dc.identifier.emailLi, HY: olili@hku.hken_US
dc.identifier.emailChan, D: chand@hku.hken_US
dc.identifier.emailCheah, KSE: hrmbdkc@hku.hken_US
dc.identifier.emailChan, BP: bpchan@hkucc.hku.hken_US
dc.identifier.authorityChan, D=rp00540en_US
dc.identifier.authorityCheah, KSE=rp00342en_US
dc.identifier.authorityChan, BP=rp00087en_US
dc.identifier.hkuros240462en_US
dc.identifier.hkuros239674-
dc.identifier.volume4-
dc.identifier.issueSuppl. 1-
dc.identifier.spageS89, PO.085-
dc.identifier.epageS89, PO.085-
dc.publisher.placeGermany-

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