19 research outputs found
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, labâtoâlab variability jeopardizes the muchâneeded 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 reâdifferentiation 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 reâdifferentiation media and techniques were also investigated. Results: Recommended protocols are provided for extraction, expansion, and reâdifferentiation 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 speciesâspecific pronase usage, 60â100 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 crossâlab comparisons on NP cells worldwide
Cell therapy for intervertebral disc repair: Clinical perspective
Low back pain is the main cause of disability and is associated with intervertebral disc degeneration. Contemporary treatments are limited to palliative therapeutics or aggressive surgical interventions; however, current advancements in cell therapy offer to fill this breach. Clinical data suggest that cell transplantation can accomplish pain relief without any observed adverse effects. Despite a large variety of preclinical studies and preliminary clinical investigations, controversy remains on the optimal cell type and transplantation strategies. The translational potential of this article lies in the aim to update on the current state of intervertebral disc cell therapy and to identify current obstacles
Clinical Development of Regenerative Medicine Targeted for Intervertebral Disc Disease
Low back pain is critical health, social, and economic issue in modern societies. This disease is often associated with intervertebral disc degeneration; however, contemporary treatments are unable to target this underlying pathology to alleviate the pain symptoms. Cell therapy offers a promising novel therapeutic that, in theory, should be able to reduce low back pain through mitigating the degenerative disc environment. With the clinical development of cell therapeutics ongoing, this review aims to summarize reporting on the different clinical trials and assess the different regenerative strategies being undertaken to collectively obtain an impression on the potential safety and effectiveness of cell therapeutics against intervertebral disc-related diseases
Regenerative technologies to bed side: Evolving the regulatory framework
There are high expectations for the clinical application of regenerative medicine technologies to treat musculoskeletal disorders. However, there are still big hurdles in bringing cell-based products to the market, mainly due to strict regulatory frameworks to approve these. Recently, the Japanese Pharmaceuticals and Medical Devices Agency adopted new regulations under legislature. The translational potential of this article is to inform on the regulations to bring experimental phase regenerative concepts to market approval in the United States and Europe, and highlight the opportunities granted by Japanese regulatory framework. Furthermore, we discuss the perspectives on the quickly evolving regulatory environment
Corrigendum to âRegenerative technologies to bed side: Evolving the regulatoryâ [J Orthop Translat 9 (2017) 1â7]
Wnt3a and wnt5a as Potential Chondrogenic Stimulators for Nucleus Pulposus Cell Induction: A Comprehensive Review
Low back pain remains a highly prevalent pathology engendering a tremendous socioeconomic burden. Low back pain is generally associated with intervertebral disc (IVD) degeneration, a process involving the deterioration of nucleus pulpous (NP) cells and IVD matrix. Scientific interest has directed efforts to restoring cell numbers as a strategy to enable IVD regeneration. Currently, mesenchymal stromal cells (MSCs) are being explored as cell therapy agents, due to their easy accessibility and differentiation potential. For enhancement of MSCs, growth factor supplementation is commonly applied to induce differentiation towards a chondrogenic (NP) cell phenotype. The wnt signaling pathways play a crucial role in chondrogenesis, nonetheless, literature appears to present controversies with regard to wnt3a and wnt5a for the induction of NP cells, chondrocytes, and MSCs. This review aims to summarize the reporting on wnt3a/wnt5a mediated NP cell differentiation, and to elucidate the mechanisms involved in wnt3a and wnt5a mediated chondrogenesis for potential application as cell therapy supplements for IVD regeneration. Our review suggests that wnt3a, subsequently replaced with a chondrogenic stimulating growth factor, can enhance the chondrogenic potential of MSCs in vitro. Contrariwise, wnt5a is suggested to play a role in maintaining cell potency of differentiated NP or chondrogenic cells
Homing of vertebralâdelivered mesenchymal stromal cells for degenerative intervertebral discs repair â an in vivo proofâofâconcept study
Abstract Introduction Cell transplantation shows promising results for intervertebral disc (IVD) repair, however, contemporary strategies present concerns regarding needle puncture damage, cell retention, and straining the limited nutrient availability. Mesenchymal stromal cell (MSC) homing is a natural mechanism of longâdistance cellular migration to sites of damage and regeneration. Previous ex vivo studies have confirmed the potential of MSC to migrate over the endplate and enhance IVDâmatrix production. In this study, we aimed to exploit this mechanism to engender IVD repair in a rat disc degeneration model. Methods Female Sprague Dawley rats were subjected to coccygeal disc degeneration through nucleus pulposus (NP) aspiration. In part 1; MSC or saline was transplanted into the vertebrae neighboring healthy or degenerative IVD subjected to irradiation or left untouched, and the ability to maintain the IVD integrity for 2 and 4âweeks was assessed by disc height index (DHI) and histology. For part 2, ubiquitously GFP expressing MSC were transplanted either intradiscally or vertebrally, and regenerative outcomes were compared at days 1, 5, and 14 postâtransplantation. Moreover, the homing potential from vertebrae to IVD of the GFP+ MSC was assessed through cryosection mediated immunohistochemistry. Results Part 1 of the study revealed significantly improved maintenance of DHI for IVD vertebrally receiving MSC. Moreover, histological observations revealed a trend of IVD integrity maintenance. Part 2 of the study highlighted the enhanced DHI and matrix integrity for discs receiving MSC vertebrally compared with intradiscal injection. Moreover, GFP rates highlighted MSC migration and integration in the IVD at similar rates as the intradiscally treated cohort. Conclusion Vertebrally transplanted MSC had a beneficial effect on the degenerative cascade in their neighboring IVD, and thus potentially present an alternative administration strategy. Further investigation will be needed to determine the longâterm effects, elucidate the role of cellular homing versus paracrine signaling, and validate our observations on a large animal model
Nâacetylcysteine attenuates oxidative stressâmediated cell viability loss induced by dimethyl sulfoxide in cryopreservation of human nucleus pulposus cells: A potential solution for mass production
Abstract Background Cell therapy is considered a promising strategy for intervertebral disc (IVD) regeneration. However, cell products often require longâterm cryopreservation, which compromises cell viability and potency, thus potentially hindering commercialization and offâtheâshelf availability. Dimethyl sulfoxide (DMSO) is a commonly used cryoprotectant, however, DMSO is associated with cytotoxicity and cell viability loss. This study aimed to investigate the effects of DMSO on human nucleus pulposus cells (NPC) and the role of oxidative stress in DMSOâinduced cytotoxicity. Furthermore, we examined the potential of antioxidant Nâacetylcysteine (NAC) supplementation to mitigate the negative effects of DMSO. Methods NPC were exposed to various concentrations of DMSO with or without a freezing cycle. Cell viability, cell apoptosis and necrosis rates, intracellular reactive oxygen species (ROS) levels, and gene expression of major antioxidant enzymes were evaluated. In addition, NAC was added to cryopreservation medium containing 10% DMSO and its effects on ROS levels and cell viability were assessed. Results DMSO concentrations â€1% for 24âh did not significantly affect the NPC viability, whereas exposure to 5 and 10% DMSO (most commonly used concentration) caused cell viability loss (loss of 57% and 68% respectively after 24âh) and cell death in a doseâ and timeâdependent manner. DMSO increased intracellular and mitochondrial ROS (1.9âfold and 3.6âfold respectively after 12âh exposure to 10% DMSO) and downregulated gene expression levels of antioxidant enzymes in a doseâdependent manner. Tempering ROS through NAC treatment significantly attenuated DMSOâinduced oxidative stress and supported maintenance of cell viability. Conclusions This study demonstrated doseâ and timeâdependent cytotoxic effects of DMSO on human NPC. The addition of NAC to the cryopreservation medium ameliorated cell viability loss by reducing DMSOâinduced oxidative stress in the freezeâthawing cycle. These findings may be useful for future clinical applications of whole cells and cellular products
Different isolation methods for nucleus pulposus progenitor cells
Introduction: Nucleus Pulposus Progenitor Cells (NPPCs), positive for the angiopoietin-1 receptor (Tie2), were demonstrated in human, mouse, canine and bovine NP tissue. Tie2+ NPPCs possess a multi-lineage differentiation potential, and regeneration potential is attributed to them. However, the isolation of Tie2+ NPPCs can be cumbersome. Hence, three isolation methods were compared.
Methods: Bovine NP cells were isolated from 10-14-month-old animals. Cell sorting was performed with an antibody against Tie2 (bs-1300R, Bioss) using FACS, magnetic-activated cell sorting (MACS) and pluriSelect, a size-based sorting method. Outcomes were evaluated by cell yield of Tie2+ cells, the ability of sorted cells to form colonies and tri-lineage differentiation assays.
Results: FACS resulted in the highest Tie2+ cell yield (5.0 ± 4.0%) followed by MACS (1.6 ± 2.9%) and pluriSelect (1.1 ± 1.4%). Colony forming ability did not differ between Tie2+ and Tie2- cells for any isolation method. However, Tie2+ cells obtained by MACS resulted in more colonies than pluriSelect (p < 0.05). Osteogenic and adipogenic differentiation of Tie2+ and Tie2- cells did not result in a clear distinction for MACS and pluriSelect; Tie2+ FACS-sorted cells demonstrated superior osteogenic and adipogenic differentiation over Tie2- cells. Also for chondrogenesis, the Tie2+ FACS-sorted NPPCs tended to produce more proteoglycan vs Tie2- NPPCs, whereas for MACS and pluriSelect no difference was found.
Conclusion: Isolation of Tie2+ NPPC is possible with all three methods tested. However, FACS resulted in the highest cell yield and a clearer separation after differentiation making it the method of choice for Tie2+ NPPC isolation