Isolation of high‐quality RNA from intervertebral disc tissue via pronase predigestion and tissue pulverization

Caprez, Stephanie; Menzel, Ursula; Li, Zhen; Grad, Sibylle; Alini, Mauro; Peroglio, Marianna

doi:10.1002/jsp2.1017

Cited by 22 publications

(30 citation statements)

References 25 publications

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“…Recently, AngII was also demonstrated to have haemodynamic-independent effects, such as contribution to inflammation, hypersensitivity and fibrosis in many organs, including liver, kidney and skin (Kranzhofer et al, 1999;Paul et al, 2006;Ruiz-Ortega et al, 2001). Previous studies have also shown that inflammatory cells expressed RAS components and AngII contributed to axon sprouting and regeneration (Chakrabarty et al, 2008;Hoch et al, 2009). Therefore, the definition of "local" or "tissue" renin-angiotensin system (tRAS) was introduced (Paul et al, 2006).…”

mentioning

confidence: 99%

The tissue-renin-angiotensin-system of the human intervertebral disc

Li¹,

Wystrach²,

Bernstein³

et al. 2020

eCM

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Symptomatic intervertebral disc (IVD) degeneration accounts for significant socioeconomic burden. Recently, the expression of the tissue renin-angiotensin system (tRAS) in rat and bovine IVD was demonstrated. The major effector of tRAS is angiotensin II (AngII), which participates in proinflammatory pathways. The present study investigated the expression of tRAS in human IVDs, and the correlation between tRAS, inflammation and IVD degeneration. Human IVD tissue was collected during spine surgery and distributed according to principal diagnosis. Gene expression of tRAS components, proinflammatory and catabolic markers in the IVD tissue was assessed. Hydroxyproline (OHP) and glycosaminoglycan (GAG) content in the IVD tissue were determined. Tissue distribution of tRAS components was investigated by immunohistochemistry. Gene expression of tRAS components such as angiotensin-converting enzyme (ACE), Ang II receptor type 2 (AGTR2), angiotensinogen (AGT) and cathepsin D (CTSD) was confirmed in human IVDs. IVD samples that expressed tRAS components (n = 21) revealed significantly higher expression levels of interleukin 6 (IL-6), tumour necrosis factor α (TNF-α), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 4 and 5 compared to tRAS-negative samples (n = 37). Within tRAS-positive samples, AGT, matrix-metalloproteinases 13 and 3, IL-1, IL-6 and IL-8 were more highly expressed in traumatic compared to degenerated IVDs. Total GAG/DNA content of non-tRAS expressing IVD tissue was significantly higher compared to tRAS positive tissue. Immunohistochemistry confirmed the presence of AngII in the human IVD. The present study identified the existence of tRAS in the human IVD and suggested a correlation between tRAS expression, inflammation and ultimately IVD degeneration.

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mentioning

confidence: 99%

The tissue-renin-angiotensin-system of the human intervertebral disc

Li¹,

Wystrach²,

Bernstein³

et al. 2020

eCM

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“…1). Thus, www.ecmjournal.org they were defined as markers of functional AF cells (Bron et al, 2009;Costi et al, 2007;Nakamichi et al, 2016;Schmidt et al, 2007). Each of these markers had previously been shown to be related to AF tissue function.…”

Section: Discussionmentioning

confidence: 99%

“…Each of these markers had previously been shown to be related to AF tissue function. Collagen type I expression at both gene and protein level is a well-known marker of AF cells in all species related with the tissue ECM property, since the proportion of collagen type I increases from the NP towards the outer AF (Bron et al, 2009). MKX expression at both gene and protein level has recently been identified as a transcription factor for AF cell differentiation, where MKX knock-out mice showed a much smaller collagen fibril diameter and a more rapid IVD degeneration as compared with the wild type mice (Nakamichi et al, 2016).…”

Section: Discussionmentioning

confidence: 99%

“…The acute symptoms are relieved by removing herniated tissue. However, AF defects remain unrepaired, which leads to a 10-30 % re-herniation rate resulting in recurrent pain and continuing disc degeneration (Ambrossi et al, 2009;Carragee et al, 2003;Smith et al, 2010). Aggressive discectomy with removal of all NP material reduces the risk of re-herniation but will lead to a post-discectomy syndrome with rapid disc degeneration and pain in nearly one third of the patients (Watters and McGirt, 2009).…”

Section: Introductionmentioning

confidence: 99%

“…Several studies have shown that TGF-β1 enhances ECM production and cell proliferation of human AF cells in both 2D and 3D in vitro cultures (Chou et al, 2016;Gruber et al, 2004). TGF-β1 increases myofibroblast contractility in wound healing, which plays a pivotal role in physiological tissue remodelling (Tomasek et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Functional cell phenotype induction with TGF-β1 and collagen-polyurethane scaffold for annulus fibrosus rupture repair

Du¹,

Long²,

Nakai³

et al. 2020

eCM

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Appropriate cell sources, bioactive factors and biomaterials for generation of functional and integrated annulus fibrosus (AF) tissue analogues are still an unmet need. In the present study, the AF cell markers, collagen type I, cluster of differentiation 146 (CD146), mohawk (MKX) and smooth muscle protein 22α (SM22α) were found to be suitable indicators of functional AF cell induction. In vitro 2D culture of human AF cells showed that transforming growth factor β1 (TGF-β1) upregulated the expression of the functional AF markers and increased cell contractility, indicating that TGF-β1-pre-treated AF cells were an appropriate cell source for AF tissue regeneration. Furthermore, a tissue engineered construct, composed of polyurethane (PU) scaffold with a TGF-β1-supplemented collagen type I hydrogel and human AF cells, was evaluated with in vitro 3D culture and ex vivo preclinical bioreactor-loaded organ culture models. The collagen type I hydrogel helped maintaining the AF functional phenotype. TGF-β1 supplement within the collagen I hydrogel further promoted cell proliferation and matrix production of AF cells within in vitro 3D culture. In the ex vivo IVD organ culture model with physiologically relevant mechanical loading, TGF-β1 supplement in the transplanted constructs induced the functional AF cell phenotype and enhanced collagen matrix synthesis. In conclusion, TGF-β1containing collagen-PU constructs could induce the functional cell phenotype of human AF cells in vitro and in situ. This combined cellular, biomaterial and bioactive agent therapy has a great potential for AF tissue regeneration and rupture repair.

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Proinflammatory intervertebral disc cell and organ culture models induced by tumor necrosis factor alpha

Pfannkuche

Lang

et al. 2020

JOR Spine

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Inflammation plays an important role in the pathogenesis of intervertebral disc (IVD) degeneration. The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) has shown markedly higher expression in degenerated human disc tissue compared with healthy controls. Anti-inflammatory treatment targeting TNF-α has shown to alleviate discogenic pain in patients with low back pain. Therefore, in vitro and ex vivo inflammatory models utilizing TNF-α provide relevant experimental conditions for drug development in disc degeneration research. The current method article addressed several specific questions related to the model establishment. (a) The effects of bovine and human recombinant TNF-α on bovine nucleus pulposus (NP) cells were compared. (b) The required dose for an inflammatory IVD organ culture model with intradiscal TNF-α injection was studied. (c) The effect of TNF-α blocking at different stages of inflammation was evaluated. Outcomes revealed that bovine and human recombinant TNF-α induced equivalent inflammatory effects in bovine NP cells. A bovine whole IVD inflammatory model was established by intradiscal injection of 100 ng TNF-α/ cm 3 disc volume, as indicated by increased nitric oxide, glycosaminoglycan, interleukin 6 (IL-6), and interleukin 8 (IL-8) release in culture media, and upregulation of MMP3, ADAMTS4, IL-8, IL-6, and cyclooxygenase (COX)-2 expression in NP tissue. However, results in human NP cells showed that the time point of anti-inflammatory treatment was crucial to achieve significant effects. Furthermore, anticatabolic therapy in conjunction with TNF-α inhibition would be required to slow down the pathologic cascade of disc degeneration.

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Isolation of high‐quality RNA from intervertebral disc tissue via pronase predigestion and tissue pulverization

Cited by 22 publications

References 25 publications

The tissue-renin-angiotensin-system of the human intervertebral disc

The tissue-renin-angiotensin-system of the human intervertebral disc

Functional cell phenotype induction with TGF-β1 and collagen-polyurethane scaffold for annulus fibrosus rupture repair

Proinflammatory intervertebral disc cell and organ culture models induced by tumor necrosis factor alpha

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