Mesenchymal stem cell therapies for intervertebral disc degeneration: consideration of the degenerate niche

We have previously reported a synthetic Laponite® crosslinked pNIPAM‐co‐DMAc (NPgel) hydrogel, which induces nucleus pulposus (NP) cell differentiation of human MSCs (hMSCs) without the need for additional growth factors. Furthermore NP gel supports integration following injection into the disc and restores mechanical function to the disc. However, translation of this treatment strategy into clinical application is dependent on the survival and differentiation of hMSC to the correct cell phenotype within the degenerate IVD. Here, we investigated the viability and differentiation of hMSCs within NP gel within a catabolic microenvironment. Human MSCs were encapsulated in NPgel and cultured for 4 weeks under hypoxia (5% O2) with ± calcium, IL‐1β and TNFα either individually or in combination to mimic the degenerate environment. Cell viability, and cellular phenotype was investigated. Stem cell viability was maintained within hydrogel systems for the 4 weeks investigated under all degenerate conditions. NP matrix markers: Agg and Col II and NP phenotypic markers: HIF‐1α, FOXF1 and PAX1 were expressed within the NPgel cultures and expression was not affected by culture within degenerate conditions. Alizarin red staining demonstrated increased calcium deposition under cultures containing CaCl2 indicating calcification of the matrix. Interestingly MMP's, ADAMTS 4 and Col I expression by hMSCs cultured in NPgel was upregulated by calcium but not by pro‐inflammatory cytokines IL‐1β and TNFα. Importantly IL‐1β and TNFα, regarded as key contributors to disc degeneration, were not shown to affect the NP cell differentiation of MSCs in the NPgel. In agreement with our previous findings, NPgel alone was sufficient to induce NP cell differentiation of MSCs, with expression of both aggrecan and collagen type II, under both standard and degenerate culture conditions; thus could provide a therapeutic option for the repair of the NP during IVD degeneration

Thermally Triggered Hydrogel Injection Into Bovine Intervertebral Disc Tissue Explants Induces Differentiation Of Mesenchymal Stem Cells And Restores Mechanical Function.

We previously reported a synthetic Laponite® crosslinked pNIPAM-co-DMAc (L-pNIPAM-co-DMAc) hydrogel which promotes differentiation of mesenchymal stem cells (MSCs) to nucleus pulposus (NP) cells without additional growth factors. The clinical success of this hydrogel is dependent on: integration with surrounding tissue; the capacity to restore mechanical function; as well as supporting the viability and differentiation of delivered MSCs. Bovine NP tissue explants were injected with media (control), human MSCs (hMSCs) alone, acellular L-pNIPAM-co-DMAc hydrogel or hMSCs incorporated within the L-pNIPAM-co-DMAc hydrogel and maintained at 5% O2 for 6 weeks. Viability of native NP cells and delivered MSCs was maintained. Furthermore hMSCs delivered via the L-pNIPAM-co-DMAc hydrogel differentiated and produced NP matrix components: aggrecan, collagen type II and chondroitin sulphate, with integration of the hydrogel with native NP tissue. In addition L-pNIPAM-co-DMAc hydrogel injected into collagenase digested bovine discs filled micro and macro fissures, were maintained within the disc during loading and restored IVD stiffness. The mechanical support of the L-pNIPAM-co-DMAc hydrogel, to restore disc height, could provide immediate symptomatic pain relief, whilst the delivery of MSCs over time regenerates the NP extracellular matrix; thus the L-pNIPAM-co-DMAc hydrogel could provide a combined cellular and mechanical repair approach

Cutibacterium acnes is present in non-herniated human discs; its positivity rate correlates with the patients age

Introduction: The presence of bacteria in the intervertebral discs (IVDs) and their role in disc degeneration is an area of controversy. Numerous studies have detected Cutibacterium acnes and other microbes with 16S DNA Sequencing and microbial cultures. However, those studies fail to determine whether the bacteria are in-vivo disc bacteria or perioperative contamination. Capoor et al. 1 performed confocal scanning laser microscopy for a limited number of herniated IVDs and detected C. acnes biofilms within the human specimens. Our study investigated the presence of Gram-positive bacteria C. acnes and Staphylococcus aureus in non-herniated human IVDs. Furthermore, expression of cellular recognition receptors Toll-like receptor (TLR) 2, TLR4 and NLR family pyrin domain containing 3 (NLRP3) and the pyroptosis marker Gasdermin D were investigated. Methods: Immunohistochemical staining for Gram-positive bacteria, S. aureus, C.acnes TLR2, TLR4, NLRP3 and Gasdermin D was performed on 75 non-herniated human IVD samples. Cell detection and classification was performed using QuPath. Fluorescently labelled S. aureus cells were co-cultured with human NP cells in monolayer across multiplicity of Infection (MOI) range (1:10- 1:100), and analysed by confocal imaging. Furthermore, human nucleus pulposus (NP) cells in monolayer were treated with Lipopolysaccharide (LPS) (5-50μg/ml) and Peptidoglycan (PGN) (5-50 μg/ml) for 48h, and cells in 3D alginate with PGN for up to 72h. Secretome analysis was performed using Luminex for cytokines, chemokines, matrix degrading enzymes and other secreted factors. Statistical analysis was performed using Kruskal-Wallis, Dunn’s multiple comparison test and Pearson correlation. Results Co-culture of S. aureus with NP cells showed internalisation of bacteria. Immunohistochemical staining demonstrated gram positive bacteria was solely detected within cells and not as biofilm within the tissue. The positivity rate of C. acnes ranged between 5-99%. The number of C.acnes positive cells showed a correlation with the age of the patients (r=0.41, p= 0.007). However, it did not correlate with grade of degeneration. The positivity rate of TLR2 ranged between 5-99% and TLR4 from 3-72%. TLR2 and TLR4 showed a strong correlation (r= 0.62, p= 1.5e-006). A significant decrease in TLR2 was observed in females showing a mid-degenerative grade compared to females showing no signs of degeneration. Investigation of the presence and the correlation between NLRP3, GasderminD, S. aureus and the above-mentioned factors is undergoing. Treatment of NP cells with LPS and PGN resulted in an increase of several catabolic cytokines such as IL-1, TNF, IL-6 and IFN-γ alongside increased production of chemokines, neurotrophic and angiogenic factors associated with IVD degeneration. Conclusion This study demonstrated the presence of Gram-positive bacteria such as C. acnes in non-herniated and cadaveric human disc samples. The internalisation of bacteria by human NP cells was demonstrated and aligns with previous publications. Furthermore, this shows a correlation between age and the presence of C. acnes as well as a strong correlation between the two TLRs. Moreover, bacterial cell membrane components triggered a catabolic response in human disc cells. Ongoing interaction studies between bacteria and NP cells will give us insight it to the potential role of bacteria in disc degeneration

A proposed classification of invasive alien plant species in South Africa: Towards prioritising species and areas for management action

Many invasive alien plant species in South Africa are already well-established and cause substantial damage, while scores of others are at the early stages of invasion (only recently introduced and/or entering a phase of rapid population growth). Management programmes must target well-established invaders, but must also give appropriate attention to emerging problems. Protocols for objectively prioritizing species in the two groups for management action are lacking. To this end, we describe the objective derivation of two lists of invasive alien plants in South Africa, using available quantitative data and expert knowledge on current patterns of distribution and abundance, life-history traits, and (for emerging invaders) estimates of potential habitat. ‘Major invaders’ are those invasive alien species that are well-established, and which already have a substantial impact on natural and semi-natural ecosystems. ‘Emerging invaders’ currently have less influence, but have attributes and potentially suitable habitat that could result in increased range and consequences in the next few decades. We describe the derivation of lists that contain 117 major invaders (categorized into groups based on geographical range and abundance) and 84 emerging invaders (categorized into groups based on current propagule-pool size and potentially invasible habitat). The main lists, and groupings within them, provide a useful means for prioritizing species for a range of management interventions at national, regional and local scale

Aquaporin expression in the human and canine intervertebral disc during maturation and degeneration

The intervertebral disc (IVD) is a highly hydrated tissue, the rich proteoglycan matrix imbibes water, enabling the disc to withstand compressive loads. During ageing and degeneration increased matrix degradation leads to dehydration and loss of function. Aquaporins (AQP) are a family of transmembrane channel proteins that selectively allow the passage of water in and out of cells and are responsible for maintaining water homeostasis in many tissues. Here, the expression of all 13 AQPs at gene and protein level was investigated in human and canine non‐degenerate and degenerate IVDs to develop an understanding of the role of AQPs during degeneration. Furthermore, in order to explore the transition of notochordal cells (NCs) towards nucleus pulposus (NP) cells, AQP expression was investigated in canine IVDs enriched in NCs to understand the role of AQPs in IVD maturation. AQP0, 1, 2, 3, 4, 5, 6, 7 and 9 were expressed at gene and protein level in both non‐degenerate and degenerate human NP tissue. AQP2 and 7 immunopositivity increased with degeneration in human NP tissue, whereas AQP4 expression decreased with degeneration in a similar way to AQP 1 and 5 shown previously. All AQP proteins that were identified in human NP tissue were also expressed in canine NP tissue. AQP2, 5, 6 and 9 were found to localise to vacuole‐like membranes and cell membranes in NC cells. In conclusion, AQPs were abundantly expressed in human and canine IVDs. The expression of many AQP isotypes potentially alludes to multi‐faceted functions related to adaption of NP cells to the conditions they encounter within their microenvironment in health and degeneration. The presence of AQPs within the IVD may suggest an adaptive role for these water channels during the development and maintenance of the healthy, mature IVD

A proposed prioritization system for the management of invasive alien plants in South Africa

Every country has weed species whose presence conflicts in some way with human management objectives and needs. Resources for research and control are limited, so priority should be given to species that are the biggest problem. The prioritization system described in this article was designed to assess objectively research and control priorities of invasive alien plants at a national scale in South Africa. The evaluation consists of seventeen criteria, grouped into five modules, that assess invasiveness, spatial characteristics, potential impact, potential for control, and conflicts of interest for each plant species under consideration. Total prioritization scores, calculated from criterion and module scores, were used to assess a species' priority. Prioritization scores were calculated by combining independent assessments provided by several experts, thus increasing the reliability of the rankings. The total confidence score, a separate index, indicates the reliability and availability of data used to make an assessment. Candidate species for evaluation were identified and assessed by several experts using the prioritization system. The final ranking was made by combining two separate indices, the total prioritization score and the total confidence score. This approach integrates the plant's perceived priority with an index of data reliability. Of the 61 species assessed, those with the highest ranks (Lantana camara, Chromolaena odorata and Opuntia ficus-indica) had high prioritization and high confidence scores, and are thus of most concern. Those species with the lowest ranks, for example, Harrisia martinii, Opuntia spinulifera and Opuntia exaltata, had low prioritization scores and high confidence scores, and thus are of least concern. Our approach to ranking weeds offers several advantages over existing systems because it is designed for multiple assessors based on the Delphi decision-making technique, the criteria contribute equally to the total score, and the system can accommodate incomplete data on a species. Although the choice of criteria may be criticized and the system has certain limitations, it appears to have delivered credible results

A proposed prioritization system for the management of invasive alien plants in South Africa

Every country has weed species whose presence conflicts in some way with human management objectives and needs. Resources for research and control are limited, so priority should be given to species that are the biggest problem. The prioritization system described in this article was designed to assess objectively research and control priorities of invasive alien plants at a national scale in South Africa. The evaluation consists of seventeen criteria, grouped into five modules, that assess invasiveness, spatial characteristics, potential impact, potential for control, and conflicts of interest for each plant species under consideration. Total prioritization scores, calculated from criterion and module scores, were used to assess a species' priority. Prioritization scores were calculated by combining independent assessments provided by several experts, thus increasing the reliability of the rankings. The total confidence score, a separate index, indicates the reliability and availability of data used to make an assessment. Candidate species for evaluation were identified and assessed by several experts using the prioritization system. The final ranking was made by combining two separate indices, the total prioritization score and the total confidence score. This approach integrates the plant's perceived priority with an index of data reliability. Of the 61 species assessed, those with the highest ranks (Lantana camara, Chromolaena odorata and Opuntia ficus-indica) had high prioritization and high confidence scores, and are thus of most concern. Those species with the lowest ranks, for example, Harrisia martinii, Opuntia spinulifera and Opuntia exaltata, had low prioritization scores and high confidence scores, and thus are of least concern. Our approach to ranking weeds offers several advantages over existing systems because it is designed for multiple assessors based on the Delphi decision-making technique, the criteria contribute equally to the total score, and the system can accommodate incomplete data on a species. Although the choice of criteria may be criticized and the system has certain limitations, it appears to have delivered credible results

Hyperosmolar expansion medium improves nucleus pulposus cell phenotype

Background: Repopulating the degenerated intervertebral disc (IVD) with tissue-specific nucleus pulposus cells (NPCs) has already been shown to promote regeneration in various species. Yet the applicability of NPCs as cell-based therapy has been hampered by the low cell numbers that can be extracted from donor IVDs and their potentially limited regenerative capacity due to their degenerated phenotype. To optimize the expansion conditions, we investigated the effects of increasing culture medium osmolarity during expansion on the phenotype of dog NPCs and their ability to produce a healthy extracellular matrix (ECM) in a 3D culture model. Methods: Dog NPCs were expanded in expansion medium with a standard osmolarity of 300 mOsm/L or adjusted to 400 or 500 mOsm/L in both normoxic and hypoxic conditions. Following expansion, NPCs were cultured in a 3D culture model in chondrogenic culture medium with a standard osmolarity. Read-out parameters included cell proliferaton rate, morphology, phenotype and healthy ECM production. Results: Increasing the expansion medium osmolarity from 300 to 500 mOsm/L resulted in NPCs with a more rounded morphology and a lower cell proliferation rate accompanied by the expression of several healthy NPC and progenitor markers at gene ( KRT18, ACAN, COL2, CD73, CD90) and protein (ACAN, PAX1, CD24, TEK, CD73) level. The NPCs expanded at 500 mOsm/L were able to retain most of their phenotypic markers and produce healthy ECM during 3D culture independent of the oxygen level used during expansion. Conclusions: Altogether, our findings show that increasing medium osmolarity during expansion results in an NPC population with improved phenotype, which could enhance the potential of cell-based therapies for IVD regeneration

Hyperosmolar expansion medium improves nucleus pulposus cell phenotype

Background:Repopulating the degenerated intervertebral disc (IVD) with tissue-spe-cific nucleus pulposus cells (NPCs) has already been shown to promote regenerationin various species. Yet the applicability of NPCs as cell-based therapy has been ham-pered by the low cell numbers that can be extracted from donor IVDs and theirpotentially limited regenerative capacity due to their degenerated phenotype. Tooptimize the expansion conditions, we investigated the effects of increasing culturemedium osmolarity during expansion on the phenotype of dog NPCs and their abilityto produce a healthy extracellular matrix (ECM) in a 3D culture model.Methods:Dog NPCs were expanded in expansion medium with a standard osmolar-ity of 300 mOsm/L or adjusted to 400 or 500 mOsm/L in both normoxic and hypoxicconditions. Following expansion, NPCs were cultured in a 3D culture model in chon-drogenic culture medium with a standard osmolarity. Read-out parameters includedcell proliferaton rate, morphology, phenotype and healthy ECM production.Results:Increasing the expansion medium osmolarity from 300 to 500 mOsm/Lresulted in NPCs with a more rounded morphology and a lower cell proliferation rateaccompanied by the expression of several healthy NPC and progenitor markers atgene (KRT18, ACAN, COL2, CD73, CD90) and protein (ACAN, PAX1, CD24, TEK,CD73) level. The NPCs expanded at 500 mOsm/L were able to retain most of theirphenotypic markers and produce healthy ECM during 3D culture independent of theoxygen level used during expansion. Conclusions:Altogether, our findings show that increasing medium osmolarity duringexpansion results in an NPC population with improved phenotype, which couldenhance the potential of cell-based therapies for IVD regeneration