Introduction. Given the predominant functional role which aggrecan has in the intervertebral disc, particularly within the nucleus pulposus, it is necessary to evaluate the quality of aggrecan produced by cells within tissue engineered disc constructs. The aim here was to characterise the nanostructure of aggrecan synthesised by nucleus pulposus cells treated with growth differentiation factor [GDF]-6) seeded in hydrogels in comparison to aggrecan isolated from healthy disc. Methods. Aggrecan was isolated from bovine nucleus pulposus (NP) tissue (n=3 [<18 months old]) and primary bovine NP cells cultured with (+GDF6) or without GDF6 (−GDF6) for 28 days (n=2) in type I collagen hydrogels. Isolated aggrecan monomers were visualised by atomic force microscopy and categorised as either intact (globular domains visible at both the N and C termini) or non-intact. Core protein contour length (L. CP. ) was calculated for intact molecules. The proportion of non-intact/fragmented to intact aggrecan and the molecular area of all monomers was determined. Results. Very few aggrecan molecules were intact (1.3% in NP compared to 4.3% +GDF6 and 0% -GDF6). There was no significant difference in the mean L. CP. between NP (389 ± 37 nm) compared to +GDF6 (379.2 ± 26 nm) or the molecular area between NP (3560 ± 2179 nm. 2. ) and –GDF6 (3586 ± 2071 nm. 2. ). However, the molecular area in both cases was significantly lower than +GDF6 (4774 ± 3715 nm. 2. ) p≤0.0001. Discussion & conclusions. Aggrecan structure can be altered by culture conditions. GDF6 treatment promoted the synthesis of more intact monomers, with greater over all molecular area. Conflicts of interest: None. Funding: Impact Research Scholarship and the Presidents Doctoral Scholarship, provided by the University of Manchester

Aims. This study was performed to explore the effect of melatonin on pyroptosis in nucleus pulposus cells (NPCs) and the underlying mechanism of that effect. Methods. This experiment included three patients diagnosed with lumbar disc herniation who failed conservative treatment. Nucleus pulposus tissue was isolated from these patients when they underwent surgical intervention, and primary NPCs were isolated and cultured. Western blotting, reverse transcription polymerase chain reaction, fluorescence staining, and other methods were used to detect changes in related signalling pathways and the ability of cells to resist pyroptosis. Results. Western blot analysis confirmed the expression of cleaved CASP-1 and melatonin receptor (MT-1A-R) in NPCs. The cultured NPCs were identified by detecting the expression of CD24, collagen type II, and aggrecan. After treatment with hydrogen peroxide, the pyroptosis-related proteins NLR family pyrin domain containing 3 (NLRP3), cleaved CASP-1, N-terminal fragment of gasdermin D (GSDMD-N), interleukin (IL)-18, and IL-1β in NPCs were upregulated, and the number of propidium iodide (PI)-positive cells was also increased, which was able to be alleviated by pretreatment with melatonin. The protective effect of melatonin on pyroptosis was blunted by both the melatonin receptor antagonist luzindole and the nuclear factor erythroid 2–related factor 2 (Nrf2) inhibitor ML385. In addition, the expression of the transcription factor Nrf2 was up- or downregulated when the melatonin receptor was activated or blocked by melatonin or luzindole, respectively. Conclusion. Melatonin protects NPCs against reactive oxygen species-induced pyroptosis by upregulating the transcription factor Nrf2 via melatonin receptors. Cite this article: Bone Joint Res 2023;12(3):202–211

Background. Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP). We have developed an injectable hydrogel (NPgel), which following injection into bovine IVD explants, integrates with IVD tissue and promotes disc cell differentiation of delivered mesenchymal stem cells (MSCs) without growth factors. Here, we investigated the injection of NPgel+MSCs into IVD explants under degenerate culture conditions. Methods and Results. The NPgel integrated with bovine and human degenerate Nucleus Pulposus (NP) tissue and hMSCs produced matrix components: aggrecan, collagen type II and chondroitin sulphate in standard and degenerate culture conditions. Significantly increased cellular immunopositivty for aggrecan was observed within native NP cells surrounding the site where NPgel+MSCs were injected (P≤0.05). In NP explants a significant decrease in catabolic factors were observed where NPgel+MSCs was injected in comparison to controls. Conclusions. In agreement with our previous findings NPgel was sufficient alone to induce NP cell differentiation of MSCs following injection into NP tissue explants. Here, we have shown that viability is maintained even in degenerate conditions. Injection of NPgel with MSCs increased aggrecan expression and reduced MMP3 and IL-1R1 expression by native NP cells. The NPgel with incorporated MSCs has the potential to regenerate the NP and provide mechanical support, whilst reducing the catabolic phenotype of degenerate NP cells, as a treatment strategy for IVD degeneration. No conflicts of interest. Sources of funding: Funded by ARUK and MRC

Background. Intervertebral disc (IVD) degeneration is a major cause of Low back pain (LBP). We have reported an injectable hydrogel (NPgel), which following injection into bovine NP explants, integrates with NP tissue and promotes NP cell differentiation of delivered mesenchymal stem cells (MSCs) without growth factors. Here we investigated the injection of NPgel+MSCs into bovine NP explants under degenerate culture conditions to mimic the in vivo environment of the degenerate IVD. Methods. hMSCs were incorporated within liquid NPgel and injected into bovine NP explants alongside controls. Explants were cultured for 6 weeks under hypoxia (5%) with ± calcium 5.0mM CaCl. 2. or IL-1β individually or in combination to mimic the degenerate microenvironment. Cell viability was assessed by caspase 3 immunohistochemistry. Histological and immunohistochemical analysis was performed to investigate altered matrix synthesis and matrix degrading enzyme expression. Results. CFSe positive hMSCs were identified in all NPgel injected explants and cell viability was maintained. The NPgel integrated with NP tissue and hMSCs produced matrix components: aggrecan, collagen type II and chondroitin sulphate in standard and degenerate culture conditions. Increased cellular immunopositivty for aggrecan and collagen type II as well as decreased cellular immunopositivity for degrading enzyme expression was observed within NP tissue removed from the injection site. Conclusion. MSCs incorporated within NPgel could be used to regenerate the NP and restore the healthy NP phenotype of degenerate NP cells as a treatment strategy for LBP. We are currently investigating the survival and differentiation capacity of hMSCs delivered via the NPgel into degenerate human NP explants and thus ascertain the future clinical success of this therapy. Conflicts of Interest: None. Funding: BMRC, MERI Sheffield Hallam University

Purpose of study and background. We have previously reported the development of injectable hydrogels for potential disc regeneration (NPgel) or bone formation which could be utilized in spinal fusion (Bgel). As there are multiple sources of mesenchymal stem cells (MSCs), this study investigated the incorporation of patient matched hMSCs derived from adipose tissue (AD) and bone marrow (BM) to determine their ability to differentiate within both hydrogel systems under different culture conditions. Methods and Results. Human fat pad and bone marrow derived MSCs were isolated from femoral heads of patients undergoing hip replacement surgery for osteoarthritis with informed consent. MSCs were encapsulated into either NPgel or Bgel and cultured for up to 6 weeks in 5% (NPgel) or 21% (Bgel) O. 2. Histology and immunohistochemistry was utilized to determine phenotype. Both fat and bone marrow derived MSCs, were able to differentiate into both cell lineages. NPgel culture conditions increased expression of matrix components such as collagen II and aggrecan and NP phenotypic markers FOXF1 and PAX1, whereas Bgel induced expression of collagen I and osteopontin, indicative of osteogenic differentiation. Conclusion. NPgel and Bgel were able to differentiate patient derived MSCs from different sources into both NP and osteogenic lineages, which may give rise to novel treatment strategies for IVD degeneration and spinal fusion, enabling choice for cell source according to patients' circumstances and needs. C Le Maitre and C Sammon hold a patent for the hydrogel described. Funded by MRC and Versus Arthritis

Aims

Lumbar spinal stenosis (LSS) is a common skeletal system disease that has been partly attributed to genetic variation. However, the correlation between genetic variation and pathological changes in LSS is insufficient, and it is difficult to provide a reference for the early diagnosis and treatment of the disease.

Background. Degeneration of the intervertebral disc (IVD) is a major cause of Low back pain. We have recently reported a novel, injectable liquid L-pNIPAM-co-DMAc hydrogel (NPgel), which promote differentiation of MSCs to nucleus pulposus (NP) cells without the need for additional growth factors. Here, we investigated the behaviour of hMSCs incorporated within the hydrogel injected into NP tissue. Methods. hMSCs were injected either alone or within NPgel, into bovine NP tissue explants and maintained at 5% O. 2. for up to 6wks. Media alone and acellular NPgel were also injected into NP explants to serve as controls. Cell viability was assessed by Caspase 3 immunohistochemistry and the phenotype of injected hMSC was assessed by histology and immunohistochemistry. Mechanical properties were also assessed via dynamic mechanical analysis (DMA). Results. No significant difference in the elastic modulus was observed between NPgel injected NP tissue and media injected controls. CFSe positive hMSCs were identified in all injected tissue samples and cell viability was maintained. Where hMSCs were delivered via NPgel, the hydrogel integrated with native NP tissue and cells producing NP matrix components: aggrecan; collagen type II and chondroitin sulphate. Conclusion. hMSC incorporated within L-pNIPAM-co-DMAc hydrogel and injected into NP explants, integrate with native NP tissue and promote differentiation towards the NP phenotype; thus potentially could be used to regenerate the NP as a treatment strategy for LBP. No conflict of interest. Funding: BMRC, MERI Sheffield Hallam University

Aims

Inflammatory response plays a pivotal role in the pathophysiological process of intervertebral disc degeneration (IDD). A20 (also known as tumour necrosis factor alpha-induced protein 3 (TNFAIP3)) is a ubiquitin-editing enzyme that restricts nuclear factor-kappa B (NF-κB) signalling. A20 prevents the occurrence of multiple inflammatory diseases. However, the role of A20 in the initiation of IDD has not been elucidated. The aim of the study was to investigate the effect of A20 in senescence of TNF alpha (TNF-α)-induced nucleus pulposus cells (NPCs).

Introduction. Type 1 neurofibromatosis is a serious hereditary disease in which mainly skin, nervous, muscular, and bone systems are damaged. In bone systems the most common deformities are thoracic kyphosis and scoliosis. Data for morphological changes in the structural components of spine in neurofibromatosis are scarce. Thus our study aimed to investigate morphological changes in structural components of the spine in NF1 neurofibromatosis. Methods. Growth plates, intervertebral discs, and fragments of vertebral bodies from deformed and adjacent segments of the spine were obtained from 15 patients aged 10–14 years with scoliosis (Cobb angle 90–120°) caused by neurofibromatosis. Preoperative examination included MRI study of the spine and brain to exclude intracanal masses, and radiographic study of the spine. Patients did not present any neurological symptoms. All children underwent anterior release and interbody fusion. Structural spinal components from children aged 12–14 years collected at forensic autopsy were used as controls. Tissues were investigated by conventional histochemical and ultrastructural methods. The levels of aggrecan and NF1 gene expression were studied with the PCR method. Results. The study of growth plate and intervertebral disc specimens removed during surgery for scoliosis in neurofibromatosis showed a clear boundary between their convex and concave sides. Both growth plate and intervertebral disc in convex side retain their architectonic and histochemical characteristics. The concave side of the growth plate is presented by small chondroblasts densely spaced without a definite orientation and surrounded by homogeneous matrix, which is made up of chondroitin sulphates. These embryonic-type chondroblasts are poorly differentiated. Chondroblasts proliferate beyond the growth plate. Proliferating cells invade into vertebral body and are bordered by thin bone lamellae, causing the scalloping of vertebral body as a radiological symptom of the pathology. Changes occurring in the intervertebral disc are of considerable interest. Concave-side disc is characterised by isolated proliferation zones containing poorly differentiated chondroblasts and fibroblasts, and neurinoma-like masses. Bone trabeculae inside a concave-side vertebra are passing the stage of osteogenesis imperfecta. Detected morphological changes in spinal structures are consistent with findings of Stevenson, who registered cartilage and bone deficiencies in animal model (mice with NF1 genemutation). Thus, morphological studies testify to structural disorder in concave side of the growth plate, but unchanged regularities and stages of chondroblast differentiation and adequate osteogenesis in the convex side. NF1 gene regulates the growth, differentiation, and proliferation of chondroblasts at the early stage of embryogenesis. Gene inactivation at a somite stage results in altered development of definitive spinal structures. Continued growth with adequate proliferation, differentiation, osteogenesis, and topochemical characteristics occurs in the convex-side growth plate, and growth disorder in the concave-side part with continued load cause growth asymmetry and development of spinal deformity. Scoliosis associated with neurofibromatosis is notable for deformity progression and pseudoarthrosis development after surgery. Deformity progression (modulation) should be considered in connection with disorder in osteogenic potency of osteoblasts. Conclusions. The causal factor of spinal deformity development in NF1 neurofibromatosis is NF1 gene mutation. Inactivation of NF1 gene results in disorder in chondrogenesis and osteogenesis within structurally altered zones. A continued load causes development of scoliotic spinal deformity

Mesenchymal stem-cell based therapies have been proposed as novel treatments for intervertebral disc degeneration, a prevalent and disabling condition associated with back pain. The development of these treatment strategies, however, has been hindered by the incomplete understanding of the human nucleus pulposus phenotype and by an inaccurate interpretation and translation of animal to human research. This review summarises recent work characterising the nucleus pulposus phenotype in different animal models and in humans and integrates their findings with the anatomical and physiological differences between these species. Understanding this phenotype is paramount to guarantee that implanted cells restore the native functions of the intervertebral disc.

Cite this article: Bone Joint Res 2013;2:169–78.