Thorpe Aa

Nucleus pulposus phenotypic markers to determine stem cell differentiation : fact or fiction?

Progress in mesenchymal stem cell (MSC) based therapies for nucleus pulposus (NP) regeneration are hampered by a lack of understanding and consensus of the normal NP cell phenotype. Despite the recent consensus paper on NP markers, there is still a need to further validate proposed markers. This study aimed to determine whether an NP phenotypic profile could be identified within a large population of mature NP samples. qRT-PCR was conducted to assess mRNA expression of 13 genes within human non-degenerate articular chondrocytes (AC) (n=10) and NP cells extracted from patients across a spectrum of histological degeneration grades (n=71). qRT-PCR results were used to select NP marker candidates for protein expression analysis. Differential expression at mRNA between AC and non-degenerate NP cells was only observed for Paired Box Protein 1 (PAX1) and Forkhead box F1 (FOXF1). In contrast no other previously suggested markers displayed differential expression between non-degenerate NP and AC at mRNA level. PAX1 and FOXF1 protein expression was significantly higher in the NP compared to annulus fibrosus (AF), cartilaginous endplate (CEP) and AC. In contrast Laminin-5 (LAM-332), Keratin-19 (KRT-19) and Hypoxia Inducible Factor 1 alpha (HIF1α) showed no differential expression in NP cells compared with AC cells. A marker which exclusively differentiates NP cells from AF and AC cells remains to be identified, raising the question: is the NP a heterogeneous population of cells? Or does the natural biological variation during IVD development, degeneration state and even the life cycle of cells make finding one definitive marker impossible?

Thermally triggered injectable hydrogel, which induces mesenchymal stem cell differentiation to nucleus pulposus cells: Potential for regeneration of the intervertebral disc

There is an urgent need for new therapeutic options for low back pain, which target degeneration of the intervertebral disc (IVD). Here, we investigated a pNIPAM hydrogel system, which is liquid at 39°C ex vivo, where following injection into the IVD, body temperature triggers gelation. The combined effects of hypoxia (5% O2) and the structural environment of the hydrogel delivery system on the differentiation of human mesenchymal stem cells (hMSCs), towards an NP cell phenotype was investigated. hMSCs were incorporated into the liquid hydrogel, the mixture solidified and cultured for up to 6weeks under 21% O2 or 5% O2 where viability was maintained. Immunohistochemistry revealed significant increases in NP matrix components: aggrecan; collagen type II and chondroitin sulphate after culture for 1week in 5% O2, accompanied by increased matrix staining for proteoglycans and collagen, observed histologically. NP markers HIF1α, PAX1 and FOXF1 were also significantly increased where hMSC were incorporated into hydrogels with accelerated expression observed when cultured in 5% O2. hMSCs cultured under hypoxic conditions, which mimic the native disc microenvironment, accelerate differentiation of hMSCs within the hydrogel system, towards the NP phenotype without the need for chondrogenic inducing medium or additional growth factors, thus simplifying the treatment strategy for the repair of 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 6weeks. 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. STATEMENT OF SIGNIFICANCE Low back pain (LBP) is associated with degeneration of the intervertebral disc (IVD). We have previously described development of a jelly delivery system (hydrogel). This has the potential to deliver adult stem cells to the centre of the IVD, known as the nucleus pulposus (NP). Here, we have demonstrated that adult stem cells can be safely injected into the NP using small bore needles, reducing damage to the disc. Following injection the hydrogel integrates with surrounding NP tissue, promotes differentiation of stem cells towards disc cells and restores IVD mechanical function. The hydrogel could be used to restore mechanical function to the IVD and deliver cells to promote regeneration of the disc as a minimally invasive treatment for LBP.

'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 acellular 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.

Leaping the hurdles in developing regenerative treatments for the intervertebral disc from preclinical to clinical

Chronic back and neck pain is a prevalent disability, often caused by degeneration of the intervertebral disc. Because current treatments for this condition are less than satisfactory, a great deal of effort is being applied to develop new solutions, including regenerative strategies. However, the path from initial promising idea to clinical use is fraught with many hurdles to overcome. Many of the keys to success are not necessarily linked to science or innovation. Successful translation to clinic will also rely on planning and awareness of the hurdles. It will be essential to plan your entire path to clinic from the outset and to do this with a multidisciplinary team. Take advice early on regulatory aspects and focus on generating the proof required to satisfy regulatory approval. Scientific demonstration and societal benefits are important, but translation cannot occur without involving commercial parties, which are instrumental to support expensive clinical trials. This will only be possible when intellectual property can be protected sufficiently to support a business model. In this manner, commercial, societal, medical, and scientific partners can work together to ultimately improve patient health. Based on literature surveys and experiences of the co‐authors, this opinion paper presents this pathway, highlights the most prominent issues and hopefully will aid in your own translational endeavors.

In vivo safety and efficacy testing of a thermally triggered injectable hydrogel scaffold for bone regeneration and augmentation in a rat model

Bone loss resulting from degenerative diseases and trauma is a significant clinical burden which is likely to grow exponentially with the aging population. In a number of conditions where pre-formed materials are clinically inappropriate an injectable bone forming hydrogel could be beneficial. The development of an injectable hydrogel to stimulate bone repair and regeneration would have broad clinical impact and economic benefit in a variety of orthopedic clinical applications. We have previously reported the development of a Laponite® crosslinked pNIPAM-co-DMAc (L-pNIPAM-co-DMAc) hydrogel delivery system, loaded with hydroxyapatite nanoparticles (HAPna), which was capable of inducing osteogenic differentiation of mesenchymal stem cells (MSCs) without the need for additional growth factors in vitro. However to enable progression towards clinical acceptability, biocompatibility and efficacy of the L-pNIPAM-co-DMAc hydrogel to induce bone repair in vivo must be determined. Biocompatibility was evaluated by subcutaneous implantation for 6 weeks in rats, and efficacy to augment bone repair was evaluated within a rat femur defect model for 4 weeks. No inflammatory reactions, organ toxicity or systemic toxicity were observed. In young male rats where hydrogel was injected, defect healing was less effective than sham operated controls when rat MSCs were incorporated. Enhanced bone healing was observed however, in aged exbreeder female rats where acellular hydrogel was injected, with increased deposition of collagen type I and Runx2. Integration of the hydrogel with surrounding bone was observed without the need for delivered MSCs; native cell infiltration was also seen and bone formation was observed within all hydrogel systems investigated. This hydrogel can be delivered directly into the target site, is biocompatible, promotes increased bone formation and facilitates migration of cells to promote integration with surrounding bone, for safe and efficacious bone repair.