title: Adipose-derived stromal cells protect intervertebral disc cells in compression: implications for stem cell regenerative disc therapy.
abstract: BACKGROUND
  Abnormal biomechanics plays a role in intervertebral disc degeneration. Adipose-derived stromal cells (ADSCs) have been implicated in disc integrity; however, their role in the setting of mechanical stimuli upon the disc's nucleus pulposus (NP) remains unknown. As such, the present study aimed to evaluate the influence of ADSCs upon NP cells in compressive load culture.
  
  
  METHODS
  Human NP cells were cultured in compressive load at 3.0MPa for 48 hours with or without ADSCs co-culture (the ratio was 50:50). We used flow cytometry, live/dead staining and scanning electron microscopy (SEM) to evaluate cell death, and determined the expression of specific apoptotic pathways by characterizing the expression of activated caspases-3, -8 and -9. We further used real-time (RT-) PCR and immunostaining to determine the expression of the extracellular matrix (ECM), mediators of matrix degradation (e.g. MMPs, TIMPs and ADAMTSs), pro-inflammatory factors and NP cell phenotype markers.
  
  
  RESULTS
  ADSCs inhibited human NP cell apoptosis via suppression of activated caspase-9 and caspase-3. Furthermore, ADSCs protected NP cells from the degradative effects of compressive load by significantly up-regulating the expression of ECM genes (SOX9, COL2A1 and ACAN), tissue inhibitors of metalloproteinases (TIMPs) genes (TIMP-1 and TIMP-2) and cytokeratin 8 (CK8) protein expression. Alternatively, ADSCs showed protective effect by inhibiting compressive load mediated increase of matrix metalloproteinases (MMPs; MMP-3 and MMP-13), disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs; ADAMTS-1 and 5), and pro-inflammatory factors (IL-1beta, IL-6, TGF-beta1 and TNF-alpha).
  
  
  CONCLUSIONS
  Our study is the first in vitro study assessing the impact of ADSCs on NP cells in an un-physiological mechanical stimulation culture environment. Our study noted that ADSCs protect compressive load induced NP cell death and degradation by inhibition of activated caspase-9 and -3 activity; regulating ECM and modulator genes, suppressing pro-inflammatory factors and preserving CK8. Consequently, the protective impact of ADSCs found in this study provides an essential understanding and expands our knowledge as to the utility of ADSCs therapy for intervertebral disc regeneration.
authors.affiliation: 1. Department of Orthopedics, Xijing Hospital, Fourth Military Medical University, West Changle Road, Xi'an, 710032, China;
authors.forename: Zhen
authors.initials: Z
authors.lastname: Sun
authors.affiliation: 2. Department of Orthopedics, Changhai Hospital, The Second Military Medical University, Shanghai, 200433, China;
authors.forename: Beier
authors.initials: B
authors.lastname: Luo
authors.affiliation: 3. Department of Orthopedics, Air Force Hospital, Youyi Road 269, Xi'an, China;
authors.forename: Zhi-Heng
authors.initials: ZH
authors.lastname: Liu
authors.affiliation: 4. Department of Orthopaedics and Traumatology, University of Hong Kong, Pokfulam, Hong Kong, SAR, China.
authors.forename: Dino
authors.initials: D
authors.lastname: Samartzis
authors.affiliation: 1. Department of Orthopedics, Xijing Hospital, Fourth Military Medical University, West Changle Road, Xi'an, 710032, China;
authors.forename: Zhongyang
authors.initials: Z
authors.lastname: Liu
authors.affiliation: 1. Department of Orthopedics, Xijing Hospital, Fourth Military Medical University, West Changle Road, Xi'an, 710032, China;
authors.forename: Bo
authors.initials: B
authors.lastname: Gao
authors.affiliation: 1. Department of Orthopedics, Xijing Hospital, Fourth Military Medical University, West Changle Road, Xi'an, 710032, China;
authors.forename: Liangliang
authors.initials: L
authors.lastname: Huang
authors.affiliation: 1. Department of Orthopedics, Xijing Hospital, Fourth Military Medical University, West Changle Road, Xi'an, 710032, China;
authors.forename: Zhuo-Jing
authors.initials: ZJ
authors.lastname: Luo
mesh_terms: D017667:Adipocytes / Q000166:cytology*; D000293:Adolescent; D000328:Adult; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002478:Cells, Cultured; D019483:Disintegrins / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007403:Intervertebral Disc / Q000473:pathology*; D055959:Intervertebral Disc Degeneration / Q000628:therapy*; D053533:Keratin-8 / Q000378:metabolism; D008297:Male; D053509:Matrix Metalloproteinase 13 / Q000378:metabolism; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D060888:Real-Time Polymerase Chain Reaction; D017154:Stromal Cells / Q000166:cytology* / Q000502:physiology*; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism; D055815:Young Adult
chemical_list: D019483:Disintegrins; D053533:Keratin-8; C560822:TIMP1 protein, human; C560823:TIMP2 protein, human; D019715:Tissue Inhibitor of Metalloproteinase-1; D019716:Tissue Inhibitor of Metalloproteinase-2; D053148:Caspase 3; D053453:Caspase 9; D053509:Matrix Metalloproteinase 13; D019278:Matrix Metalloproteinase 3
keywords: adipose-derived stromal cells; compressive load.; intervertebral disc; nucleus pulposus