Harmonization and standardization of nucleus pulposus cell extraction and culture methods

Background: In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab‐to‐lab variability jeopardizes the much‐needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. Methods: The most commonly applied methods for NP cell extraction, expansion, and re‐differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re‐differentiation media and techniques were also investigated. Results: Recommended protocols are provided for extraction, expansion, and re‐differentiation of NP cells from common species utilized for NP cell culture. Conclusions: This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species‐specific pronase usage, 60–100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross‐lab comparisons on NP cells worldwide

Network Analysis Identifies Gene Regulatory Network Indicating the Role of RUNX1 in Human Intervertebral Disc Degeneration

Intervertebral disc (IVD) degeneration (IDD) is a multifactorial physiological process which is often associated with lower back pain. Previous studies have identified some molecular markers associated with disc degeneration, which despite their significant contributions, have provided limited insight into the etiology of IDD. In this study, we utilized a network medicine approach to uncover potential molecular mediators of IDD. Our systematic analyses of IDD associated with 284 genes included functional annotation clustering, interaction networks, network cluster analysis and Transcription factors (TFs)-target gene network analysis. The functional enrichment and protein–protein interaction network analysis highlighted the role of inflammatory genes and cytokine/chemokine signaling in IDD. Moreover, sub-network analysis identified significant clusters possessing organized networks of 24 cytokine and chemokine genes, which may be considered as key modulators for IDD. The expression of these genes was validated in independent microarray datasets. In addition, the regulatory network analysis identified the role of multiple transcription factors, with RUNX1 being a master regulator in the pathogenesis of IDD. Our analyses highlighted the role of cytokine genes and interacting pathways in IDD and further improved our understanding of the genetic mechanisms underlying IDD

Correction: Sangadala, S. et al. FK506 Induces Ligand-Independent Activation of the Bone Morphogenetic Protein Pathway and Osteogenesis. Int. J. Mol. Sci. 2019, 20, 1900

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Sclerostin small-molecule inhibitors promote osteogenesis by activating canonical Wnt and BMP pathways

Background: The clinical healing environment after a posterior spinal arthrodesis surgery is one of the most clinically challenging bone-healing environments across all orthopedic interventions due to the absence of a contained space and the need to form de novo bone. Our group has previously reported that sclerostin in expressed locally at high levels throughout a developing spinal fusion. However, the role of sclerostin in controlling bone fusion remains to be established. Methods: We computationally identified two FDA-approved drugs, as well as a single novel small-molecule drug, for their ability to disrupt the interaction between sclerostin and its receptor, LRP5/6. The drugs were tested in several in vitro biochemical assays using murine MC3T3 and MSCs, assessing their ability to (1) enhance canonical Wnt signaling, (2) promote the accumulation of the active (non-phosphorylated) form of β-catenin, and (3) enhance the intensity and signaling duration of BMP signaling. These drugs were then tested subcutaneously in rats as standalone osteoinductive agents on plain collagen sponges. Finally, the top drug candidates (called VA1 and C07) were tested in a rabbit posterolateral spine fusion model for their ability to achieve a successful fusion at 6 wk. Results: We show that by controlling GSK3b phosphorylation our three small-molecule inhibitors (SMIs) simultaneously enhance canonical Wnt signaling and potentiate canonical BMP signaling intensity and duration. We also demonstrate that the SMIs produce dose-dependent ectopic mineralization in vivo in rats as well as significantly increase posterolateral spine fusion rates in rabbits in vivo, both as standalone osteogenic drugs and in combination with autologous iliac crest bone graft. Conclusions: Few if any osteogenic small molecules possess the osteoinductive potency of BMP itself – that is, the ability to form de novo ectopic bone as a standalone agent. Herein, we describe two such SMIs that have this unique ability and were shown to induce de novo bone in a stringent in vivo environment. These SMIs may have the potential to be used in novel, cost-effective bone graft substitutes for either achieving spinal fusion or in the healing of critical-sized fracture defects. Funding: This work was supported by a Veteran Affairs Career Development Award (IK2-BX003845)

Harmonization and standardization of nucleus pulposus cell extraction and culture methods

Abstract Background In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab‐to‐lab variability jeopardizes the much‐needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. Methods The most commonly applied methods for NP cell extraction, expansion, and re‐differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re‐differentiation media and techniques were also investigated. Results Recommended protocols are provided for extraction, expansion, and re‐differentiation of NP cells from common species utilized for NP cell culture. Conclusions This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species‐specific pronase usage, 60–100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross‐lab comparisons on NP cells worldwide