title: Transforming growth factor-beta 3 stimulates cartilage matrix elaboration by human marrow-derived stromal cells encapsulated in photocrosslinked carboxymethylcellulose hydrogels: potential for nucleus pulposus replacement.
abstract: Degeneration of the nucleus pulposus (NP) has been implicated as a major cause of low back pain. Tissue engineering strategies using marrow-derived stromal cells (MSCs) have been used to develop cartilaginous tissue constructs, which may serve as viable NP replacements. Supplementation with growth factors, such as transforming growth factor-beta 3 (TGF-β3), has been shown to enhance the differentiation of MSCs and promote functional tissue development of such constructs. A potential candidate material that may be useful as a scaffold for NP tissue engineering is carboxymethylcellulose (CMC), a biocompatible, cost-effective derivative of cellulose. Photocrosslinked CMC hydrogels have been shown to support NP cell viability and promote phenotypic matrix deposition capable of maintaining mechanical properties when cultured in serum-free, chemically defined medium (CDM) supplemented with TGF-β3. However, MSCs have not been characterized using this hydrogel system. In this study, human MSCs (hMSCs) were encapsulated in photocrosslinked CMC hydrogels and cultured in CDM with and without TGF-β3 to determine the effect of the growth factor on the differentiation of hMSCs toward an NP-like phenotype. Constructs were evaluated for matrix elaboration and functional properties consistent with native NP tissue. CDM supplemented with TGF-β3 resulted in significantly higher glycosaminoglycan content (762.69±220.79 ng/mg wet weight) and type II collagen (COL II) content (6.25±1.64 ng/mg wet weight) at day 21 compared with untreated samples. Immunohistochemical analyses revealed uniform, pericellular, and interterritorial staining for chondroitin sulfate proteoglycan and COL II in growth factor-supplemented constructs compared with faint, strictly pericellular staining in untreated constructs at 21 days. Consistent with matrix deposition, mechanical properties of hydrogels treated with TGF-β3 increased over time and exhibited the highest peak stress in stress-relaxation (σ(pk)=1.489±0.389 kPa) at day 21 among all groups. Taken together, these results demonstrate that hMSCs encapsulated in photocrosslinked CMC hydrogels supplemented with TGF-β3 are capable of elaborating functional extracellular matrix consistent with the NP phenotype. Such MSC-laden hydrogels may have application in NP replacement therapies.
authors.affiliation: Department of Biomedical Engineering, The City College of New York, New York, NY 10031, USA.
authors.forename: Michelle S
authors.initials: MS
authors.lastname: Gupta
authors.forename: Elana S
authors.initials: ES
authors.lastname: Cooper
authors.forename: Steven B
authors.initials: SB
authors.lastname: Nicoll
mesh_terms: D000328:Adult; D001696:Biomechanical Phenomena / Q000187:drug effects; D001854:Bone Marrow Cells / Q000166:cytology*; D002266:Carboxymethylcellulose Sodium / Q000494:pharmacology* / Q000528:radiation effects; D002356:Cartilage / Q000187:drug effects*; D018914:Cells, Immobilized / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D024043:Collagen Type II / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D020100:Hydrogels / Q000494:pharmacology; D007150:Immunohistochemistry; D007403:Intervertebral Disc / Q000187:drug effects*; D008027:Light; D008297:Male; D017154:Stromal Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D053782:Transforming Growth Factor beta3 / Q000494:pharmacology*
chemical_list: D024043:Collagen Type II; D003432:Cross-Linking Reagents; D006025:Glycosaminoglycans; D020100:Hydrogels; D053782:Transforming Growth Factor beta3; D004247:DNA; D002266:Carboxymethylcellulose Sodium