The regulation of nerve and blood vessel ingrowth in aneural and avascular intervertebral disc and articular cartilage

Introduction This review will discuss the regulatory mechanisms of both innervation and vascularisation within normally aneural and avascular tissues, and how they may become altered in degeneration enabling new nerve and blood vessel formation which is hypothesised to be a source of pain. Conclusion Normal intervertebral discs and articular cartilage are the largest aneural and avascular tissues in the human body yet during intervertebral disc degeneration and osteoarthritis these tissues become increasingly vascularised by small blood vessels and innervated by peptide containing sensory nerve fibres. The mechanism by which this process occurs remains largely unknown. Published data suggests that various factors present within the healthy tissues such as aggrecan, chondromodulin and semaphorins may act as repulsive barriers to neurite and endothelial cell invasion. During degeneration however, the synthesis of these molecules becomes disrupted, potentially leading to vascularisation and innervation of the tissue

Long-term in vitro 3D hydrogel co-culture model of inflammatory bowel disease

The in vitro study of the pathogenesis of inflammatory bowel disease (IBD) requires a cell model which closely reflects the characteristics of the in vivo intestinal epithelium. This study aimed to investigate the application of L-pNIPAM hydrogel as a scaffold to develop a long-term 3D co-culture model of Caco-2 and HT29-MTX cells under conditions analogous to inflammation, to determine its potential use in studying IBD. Monocultures and co-cultures were layered on L-pNIPAM hydrogel scaffolds and maintained under dynamic culture conditions for up to 12 weeks. Treatments with IL-1β, TNFα, and hypoxia for 1 week were used to create an inflammatory environment. Following prolonged culture, the metabolic activity of Caco-2 monoculture and 90% Caco-2/10% HT29-MTX co-cultures on L-pNIPAM hydrogels were increased, and finger-like structures, similar in appearance to villi were observed. Following treatment with IL-1β, TNFα and hypoxia, ALP and ZO-1 were decreased, MUC2 increased, and MUC5AC remained unchanged. ADAMTS1 was increased in response to hypoxia. Caspase 3 expression was increased in response to TNFα and hypoxic conditions. In conclusion, L-pNIPAM hydrogel supported long-term co-culture within a 3D model. Furthermore, stimulation with factors seen during inflammation recapitulated features seen during IBD

Immunohistochemical analysis of protein expression in formalin fixed paraffin embedded human intervertebral disc tissues

Immunohistochemistry (IHC) is a useful technique for the localization and semiquantification of protein expression within tissues. Adult human intervertebral disc (IVD) tissues contain a large amount of auto‐fluorescence which often makes immunofluorescence techniques inappropriate on tissue samples but can be applied to isolated cell samples. Thus, IHC remains one of, if not the most common application for protein detection within IVD tissue. Immunostaining localizes antigen expression through specific epitope‐antibody interactions. Within the field of IVD research, IHC is commonly used on fresh frozen and paraffin embedded tissues to elucidate the expression of antigens. Here, we discuss the principles of IHC applied to formalin fixed paraffin embedded IVD tissue and supply optimized protocols for antibodies used within our group to guide research within the IVD field

Assessment of the contribution of groundwater discharges to rivers using monthly flow statistics and flow seasonality

Groundwater discharge is believed to dominate dry season flows in perennial river systems and to sustain aquatic biodiversity. River flow statistics, extracted from the SPATSIM modelling system, were used to estimate the contribution of groundwater to river flow regimes. The flow statistics were compared for the principal aquifer types (based on major geological formations) in South Africa. This analysis focused on seasonal variation in flows rather than the annual totals or Baseflow Index. Groundwater discharge is expected to reduce flow variability and sustain flows, making flow concentrations lower than rainfall concentrations. Catchments dominated by carbonates have the greatest proportion of baseflow (37%), followed by basement complex (31%) and extrusive aquifer types (31%). The weak relationships between river flow indexes (particularly the Baseflow Index, Coefficient of Variation and Hydrological Index) and the seasonality or concentration statistics imply that catchment storage characteristics and other non-climatic factors play an important role in flow regulation. The geographic distribution of total flow concentrations differs markedly from rainfall concentrations, further evidence that non-climatic factors are important determinants of flow regimes. Karoo dykes and sills, extrusives and unconsolidated deposits are under-represented and the TMG sub-type, carbonates and basement complex and younger granites are over-represented among catchments with evenly distributed baseflows. The Baseflow Index and groundwater-fed baseflow are ecologically meaningful variables but lack clear thresholds that correspond with ecologically important changes in river flow regimes, for example perennial versus seasonal flow. Flow concentrations and percentage zero flows are useful and potentially ecologically important variables and should be tested as predictors of the aquatic and riparian biodiversity of river systems at a range of scales.Keywords: river flow statistics, baseflow, flow concentration, principal aquifer types, groundwater discharg

'Cell or Not to Cell' that is the question : for intervertebral disc regeneration?

Low back pain, strongly associated with intervertebral disc degeneration, is one of the most prevalent health problems in the western world today. Current treatments have been directed toward alleviating patient symptoms but have been shown to accelerate degenerative changes in adjacent discs. New approaches in tissue engineering have provided a variety of treatment options including the delivery of regenerative cells, either alone or together with hydrogel scaffolds in order to restore/maintain disc biomechanics whilst simultaneously regenerating the matrix. This review paper discusses the use of cellular and a cellular therapeutic strategies for IVD degeneration with an emphasis on the importance of tailoring the treatment strategy with stage of degeneration, thus offering insight into the future clinical options for IVD regeneration

Hydroxyapatite nanoparticle injectable hydrogel scaffold to support osteogenic differentiation of human mesenchymal stem cells

Bone loss associated with degenerative disease and trauma is a clinical problem increasing with the aging population. Thus, effective bone augmentation strategies are required; however, many have the disadvantages that they require invasive surgery and often the addition of expensive growth factors to induce osteoblast differentiation. Here, we investigated a Laponite crosslinked, pNIPAMDMAc copolymer (L-pNIPAM-co-DMAc) hydrogel with hydroxyapatite nanoparticles (HAPna), which can be maintained as a liquid ex vivo, injected via narrowgauge needle into affected bone, followed by in situ gelation to deliver and induce osteogenic differentiation of human mesenchymal stem cells (hMSC). L-pNIPAMco-DMAc hydrogels were synthesised and HAPna added post polymerisation. Commercial hMSCs from one donor (Lonza) were incorporated in liquid hydrogel, the mixture solidified and cultured for up to 6 weeks. Viability of hMSCs was maintained within hydrogel constructs containing 0.5 mg/mL HAPna. SEM analysis demonstrated matrix deposition in cellular hydrogels which were absent in acellular controls. A significant increase in storage modulus (G’) was observed in cellular hydrogels with 0.5 mg/mL HAPna. Semi-quantitative immunohistochemistry and histological analysis demonstrated that bone differentiation markers and collagen deposition was induced within 48 h, with increased calcium deposition with time. The thermally triggered hydrogel system, described here, was sufficient without the need of additional growth factors or osteogenic media to induce osteogenic differentiation of commercial hMSCs. Preliminary data presented here will be expanded on multiple patient samples to ensure differentiation is seen in these samples. This system could potentially reduce treatment costs and simplify the tre

Physical disruption of intervertebral disc promotes cell clustering and a degenerative phenotype

© 2019, The Author(s). To test the hypothesis that physical disruption of an intervertebral disc disturbs cell-matrix binding, leading to cell clustering and increased expression of matrix degrading enzymes that contribute towards degenerative disc cell phenotype. Lumbar disc tissue was removed at surgery from 21 patients with disc herniation, 11 with disc degeneration, and 8 with adolescent scoliosis. 5 μm sections were examined with histology, and 30-µm sections by confocal microscopy. Antibodies were used against integrin α5beta1, matrix metalloproteinases (MMP) 1, MMP-3, caspase 3, and denatured collagen types I and II. Spatial associations were sought between cell clustering and various degenerative features. An additional, 11 non-herniated human discs were used to examine causality: half of each specimen was cultured in a manner that allowed free ‘unconstrained’ swelling (similar to a herniated disc in vivo), while the other half was cultured within a perspex ring that allowed ‘constrained’ swelling. Changes were monitored over 36 h using live-cell imaging. 1,9-Di-methyl methylene blue (DMMB) assay for glycosaminoglycan loss was carried out from tissue medium. Partially constrained specimens showed little swelling or cell movement in vitro. In contrast, unconstrained swelling significantly increased matrix distortion, glycosaminoglycan loss, exposure of integrin binding sites, expression of MMPs 1 and 3, and collagen denaturation. In the association studies, herniated disc specimens showed changes that resembled unconstrained swelling in vitro. In addition, they exhibited increased cell clustering, apoptosis, MMP expression, and collagen denaturation compared to ‘control’ discs. Results support our hypothesis. Further confirmation will require longitudinal animal experiments

Tissue engineering laboratory models of the small intestine.

In recent years, three-dimensional (3D) cell culture models of the small intestine have gained much attention. These models support cell proliferation, migration, and differentiation, and encourage tissue organization which is not possible in two-dimensional (2D) culture systems. Furthermore, the use of a wide variety of cell culture scaffolds and support substrates have revealed considerable differences in cell behavior and tissue organization. These systems have been used in combination with intestinal stem cells, organoid units or human colonic adenocarcinoma cell lines such as Caco-2 and HT29-MTX to generate a number of in vitro and in vivo models of the intestine. Here, we review the current 2D and 3D tissue engineering models of the intestine to determine the most effective sources of intestinal cells and current research on support scaffolds capable of inducing the morphological architecture and function of the intestinal mucosa