Osteoarthritis (OA) is a major global disease with increasing prevalence. It is one of the most significant causes of disability worldwide and represents a major burden in terms of healthcare delivery and impact on the quality of life of patients. It is a cause of severe chronic pain and has given rise to alarming levels of opioid use and addiction. Despite this prevalence, there are no disease-modifying treatments which delay or reverse the degrative changes within joints which are characteristics of the disease. All treatments are symptom-modifying with the exception of joint arthroplasty, which is currently the most common surgical procedure carried out in US hospitals. Several pharmaceutical and biological interventions have been tested in recent years, including metalloproteinase inhibitors, chondrogenic agents such as Kartogenin, IL-1 antagonists and monoclonal antibodies. So far, none of these has provided an effective disease-modifying treatment. Cellular therapies have a great deal of promise because of their anti-inflammatory and regenerative effects. Mesenchymal stromal cells (MSCs) have been widely studied as a treatment for OA in preclinical and clinical assessments with generally positive results. As the clinical testing of these cells proceeds serious questions emerge relating to the quality and consistency of the therapeutic product and the need for better standardisation with regard to, for example, the tissue source and expansion conditions. Of equal importance is the need for deeper insight into the therapeutic mechanism, specifically the activity and phenotype of cells transplanted to the OA environment, their fate and interaction with local cells.

Background

Mesenchymal stem cells (MSCs) are undergoing evaluation as a potential new therapy for immune and inflammatory-mediated conditions such as IVD degeneration (IDD). Both adipose (ASCs) and bone-marrow (BMSCs) derived MSCs have been widely used in this regard. The optimal tissue source and expansion conditions required to exploit the regenerative capacity of these cells are not yet fully elucidated. In addition the phenotypic response of transplanted cells to the disease environment is not well understood. In this study, ASCs and BMSCs were exposed to a combination of hypoxic conditioning and selected inflammatory mediators, conditions that mimic the microenvironment of the degenerate IVD, in an effort to understand their therapeutic potency for in vivo administration.

Familial osteochondritis dissecans (FOCD) is an inherited defect of cartilage and bone characterized by development of large cartilage lesions in multiple joints, short stature and early onset osteoarthritis. We have studied a family from Northern Sweden with FOCD over five generations. All affected family members have a heterozygous missense mutation on exon 17 of the aggrecan gene, resulting in a Val-Met amino acid replacement in the G3 aggrecan C-type lectin domain (CLD). Aggrecan, a major proteoglycan of articular cartilage produced by chondrocytes, has a large protein core richly substituted with sulfated glycosaminoglycan chains. The unique structure, its high concentration within the cartilage extracellular matrix and its ability to form a supermolecular complex with hyaluronan and bind to other matrix proteins all profoundly influence the biomechanical properties of the tissue. Deletion of CLD in a chick aggrecan construct was found to influence its secretion from chondrocytes and human aggrecan constructs carrying the V2303M mutation showed diminished interactions with the ECM proteins tenascin-R, fibulin-1 and fibulin-2. To investigate the pathogenesis of FOCD, we studied chondrogenic differentiation of patient bone marrow mesenchymal stem cells and induced pluripotent stem cells. We demonstrated that the mutation results in accumulation of unfolded or misfolded aggrecan within the lumen of the chondrocyte endoplasmic reticulum. Associated with this is the failure to assemble a normal extracellular matrix. This explains the susceptibility of these patients to cartilage injury and the degenerative changes that lead to early onset osteoarthritis.

Osteoarthritis (OA) of the spine and diarthrodial joints is by far the most common cause of chronic disability in people over 50 years of age. The disease has a striking impact on quality of life and represents an enormous societal and economic cost, a burden that will increase greatly as populations age. OA is a complex condition with broad pathology. Damage to the articular cartilage is a consistent feature, accompanied by changes to the subchondral bone and synovium. Progression of the disease involves further degeneration of the articular cartilage, damage to the underlying bone and morphological changes that include subchondral bone thickening, development of cysts, osteophytes and inflammation of the synovium. Enhanced production of proinflammatory cytokines and matrix metalloproteinases accelerates degradation of the articular cartilage. It is striking that no approved pharmacological intervention, biological therapy or procedure prevents the progressive destruction of the OA joint. All current treatments, without exception, produce symptomatic rather than regenerative results. While there have been some exciting developments in the search for OA treatments in the last decade, including matrix metalloproteinase inhibitors, anti-TNF and anti-IL1 drugs for example, none of these has to date emerged as an effective medicinal product. There is thus an urgent and compelling need to identify, validate and test new biological therapeutics. Stromal cell therapy represents one such compelling approach. The results from several early clinical studies have indicated that this approach holds a great deal of promise for the treatment of OA. Most studies have involved direct intraarticular injection of a suspension of mesenchymal stromal cells (MSCs) for treatment of knee OA. Results from a number of controlled patient studies have suggested that this treatment results in an effective repair response. Although data regarding mechanism of action are limited, it appears that the cells have an anti-inflammatory effect, possibly targeting cells within the synovium, rather than a direct cartilage repair effect. Several recent reports have highlighted a dramatic and sustained response in patients receiving MSC treatment. For example, allogeneic expanded adipose-derived MSCs have been shown to be safe and effective in the treatment of complex perianal fistulas in Crohn's disease. Also, allogeneic bone marrow-derived MSCs has a been shown to have a positive effect in pediatric acute graft versus host disease. These observations point to a mechanism of action that involves host immunomodulation, but this needs further examination. Within the field of musculoskeletal disease effective translation of MSC technology has been hindered by a lack of randomized controlled patient studies, severe inconsistencies regarding the preparation and characterization of the cell product, and an incomplete understanding of the therapeutic mechanism. Direct to consumer clinics have flourished in some countries, providing cell treatments to OA patients. Most or all of these utilize unexpanded cell fractions from marrow or fat without even rudimentary product characterization and may report an exaggerated clinical outcome. Data from these clinics is not likely to yield information that will be useful. In fact, a recent systemic review of clinical trials involving MSC treatment in OA indicated that only a limited number of studies provided high quality evidence and long term follow up. Many suffered from a lack of consistency, including a diversity of methods for MSC preparation, and thus did not contribute to a supporting evidence base. There is a compelling need to provide clear and unambiguous clinical proof of concept relating to MSC treatment for OA. The ADIPOA2 study, currently active in Europe, will go some way towards achieving this. This is a 150 patient, phase 2b study designed to to assess the efficacy of a single injection of autologous adipose-derived MSCs in the treatment of mild to moderate OA of the knee, active and unresponsive to conservative therapy for at least 12 months.

Clinical translation of MSC therapies in orthopaedics has been hampered by heterogeneity and a lack of standardised and validated testing protocols for quality assurance. Although minimal criteria have been proposed¹, it is apparent that these do not predict performance in vivo. We used a combinatorial antibody profiling tool to probe the surface immunophenotype of human bone marrow derived MSCs and used this to define new marker panels. Cells were cultured from three marrow donors using specified expansion conditions and probed by high throughput flow cytometry using a panel of 230 antibodies. Analysis of expression of the surface proteins revealed significant variation in response to culture conditions and considerably less variation between donors. Of the panel of 230 markers 107 were negative, 24 had high expression in all samples, 1 had low expression and 98 displayed significant differences between cell preparations. Cluster analyses revealed that marker expression in one culture condition varied considerably from the other two. Phenotypic characterization of the cell preparations, assessed by analysis of differentiation propensity, showed similar patterns of variability between these samples. This suggests that the selected panel may be used as phenotypic MSC markers. Ongoing work involves the generation of novel antibody arrays which will be used as quality tests in a manufacturing environment. These tests will be used for in-process and product release applications for enhanced cell manufacturing and improved clinical outcomes.

Osteoarthritis (OA) is a degenerative disease with a strong inflammatory component. Intra-articular (IA) injections of mesenchymal stem cells (MSCs) modulate local inflammation, although the lack of engraftment suggests that they undergo apoptosis. The aim of this study is to investigate the fate of IA-delivered MSCs in an animal model of OA and to assess the role of apoptosis in vitro. Collagenase-induced OA (CIOA) was performed on C57BL/6 mice and 2×10∧5 GFP+ MSCs were IA-injected in the animals. 3 days later, knee joints were digested into a single-cell suspension and MSCs retrieved by cell sorting. Conditioned medium (CM) of retrieved cells was tested on murine macrophages and cytokine secretion was measured. Apoptosis of MSCs was induced in vitro with staurosporine (STS) and evaluated by Annexin V/Sytox Blue staining; activation of caspases was measured by FLICA assays. Murine lymphocytes were cocultured with apoptotic MSCs and their proliferation measured by quantification of Cell Trace Violet. 1.63% of injected cells were retrieved and proliferated in culture. Their CM significantly modulated activation of macrophages, with greater effects from OA-induced MSCs. STS induced apoptosis with activation of Caspase 3/7. Apoptotic MSCs significantly prevented the proliferation of murine lymphocytes. MSCs can be administered and retrieved from murine knees. Retrieval yield is low, consistent with previous studies. MSCs were licensed from the OA joint to produce an immunosuppressive milieu that modulated macrophages ex vivo. In vitro, apoptosis increased the immunomodulatory potential of MSCs. This suggests that apoptosis may contribute to the therapeutic effects of MSCs in OA.

Early clinical studies investigating the effects of delivery of mesenchymal stromal cells (MSCs) to degenerated intervertebral discs have shown promising results, but with an incomplete understanding of the therapeutic mechanism(s) of action. To address this, we have developed a 3D co-culture system to unravel the biological interaction between MSCs and nucleus pulposus (NP) cells. Alginate constructs were created using a biphasic configuration consisting of a cylindrical shell with an encapsulated bead. Human NP cells were seeded in monolayer or encapsulated within alginate and cultured in hypoxia with variations of pH, osmolarity and growth factors (n = 6) to replicate healthy or degenerative conditions. Wells and gels were fixed and stained for ECM content, and retrieved cells and media were analysed for ECM and inflammatory factor expression. Encapsulated hNPCs showed no migration from either alginate structure and full bead separation was achieved over 14 days, reinforcing the construct as a separable 3D co-culture method. Addition of the degenerative growth factors TNFα and IL-1β as well as the adjustment of media pH to degenerative levels (pH 6.8) caused the hNPCs to decrease in size and proliferate significantly higher than control levels. TGF-β3 addition showed higher incidence of aggrecan deposition over addition of IL-1β. Addition of FGF2 altered cell morphology and ECM deposition including formation of pseudo lamellae, indicating a phenotype shift toward annulus fibrosis cells as shown in late-stage degenerative disc disease. The data from this study will be used in future MSC:NPC co-cultures to determine immunoregulatory interactions in a degenerative environment.

Clinical trials are underway to elucidate a successful MSC-based therapy for the repair and regeneration of intervertebral disc (IVD) tissue. Currently, there is a lack of knowledge surrounding the relationship between naïve MSCs and the inflammatory microenvironment of the degenerate disc. To inform a phase II clinical trial, this study tests the hypothesis that cytokines, IL-1ß and TNFα regulate the expression of neuropeptides and neurotrophic factors from MSCs, thus exacerbating pain in those patients that have the presence of sensory nerve fibres within the IVD. Patient-matched MSCs derived from bone marrow (BM) or adipose (AD) tissue were stimulated with IL-1β (10ng/ml) or TNFα (10ng/ml) for 48 hours in either 21% or 5% O2. qRT-PCR was performed to assess expression of trophic factors involved in the survival or nerves (NGF & BDNF), blood vessels (VEGF) as well as pain related peptides (SP & CGRP) and inflammatory factors. Conditioned culture medium was analysed using ELISAs to identify secretion of soluble factors. IL-1β did not regulate neurotrophic factor expression from BM-MSCs under normoxic or hypoxic conditions. However, TNFα increased NGF, BDNF, SP and CGRP under normoxic conditions. In ADMSCs, VEGF was increased following IL-1β and TNFα stimulation; with TNFα also increasing NGF and CGRP under normoxic conditions. When exposed to hypoxia, the trophic effect of TNFα on human BM-MSCs was reduced. Overall this data suggests a role for priming or pre-stimulation of naïve MSCs prior to implantation to prevent exacerbation of pain from sensory nerve fibres.

Epidemiological studies have shown that accumulated mechanical stress is a risk factor for the development of osteoarthritis (OA). This debilitating progressive clinical condition affects a broad spectrum of patients and will ultimately lead to definitive arthroplasty surgery as the endpoint treatment option in many cases. The aim of this study is to establish a graded murine model of OA by medial meniscotibial destabilisation of the knee joint and in phase two, to investigate the migration and engraftment of radioisotope labeled mesenchymal stem cells (MSCs) at varying points of disease progression. The first phase of the study was to establish the murine model, an Irish first. All procedures were performed aseptically under general anaesthesia via a midline medial parapatellar approach on a murine fracture table. Microsurgical dissection was performed through necropsy analysed layers to the joint space and the meniscotibial ligament identified and transected. Validated histopathological analysis was performed at two, four, eight and twelve weeks postoperatively. The results showed a gradation of OA changes from mild unicondylar changes at two weeks, moderate unicompartmental change at four, severe unicompartmental change at eight and severe bicompartmental change at twelve weeks post-operatively. In vivo Bazooka-Single Photon Emission Computed Tomography (SPECT) (Phase 2) imaging studies are currently ongoing following the model establishment.

Background

70% of Breast Cancer patients develop metastatic bone deposits, predominantly spinal metasases. Adult Mesenchymal Stem Cells (MSCs) are multiprogenitor stem cells found within the bone marow which have the ability to self renew and differentiate into multiple cell types. MSCs home specifically to tumour sites, highlighting their potential as delivery vehicles for therapeutic agents. However studies show they may also increase tumour metastatic potential.

Background: 70% of Breast Cancer patients develop metastatic bone deposits, predominantly spinal metasases. Adult Mesenchymal Stem Cells (MSCs) are multiprogenitor stem cells found within the bone marow which have the ability to self renew and differentiate into multiple cell types. MSCs home specifically to tumour sites, highlighting their potential as delivery vehicles for therapeutic agents. However studies show they may also increase tumour metastatic potential.

Aims: The aim of this study was to investigate interactions between MSCs and breast cancer cells to further elucidate their role in the tumour microenvironment and hence understand factors involved in stimulating the formation of bone metastases.

Methods: MSCs harvested from the iliac crest of healthy volunteers were grown for collection of conditioned medium (CM), containing all factors secreted by the cells. Breast cancer cell lines (T47D, SK-BR3) were then cultured in MSC CM +/− antibodies to TGFβ, VEGF, MCP-1 and CCL5 for 72hrs. Cell proliferation was assessed using an Apoglow^® assay and RNA harvested for analysis of changes in Epithelial Mesenchymal Transition specific gene expression: N-Cadherin, E-Cadherin, Vimentin, Twist, Snail.

Results: A significant down regulation of breast cancer cell proliferation in the presence of MSC secreted factors was observed (p< 0.05). There was a dramatic increase in expression of EMT specific genes in both cell lines following exposure to MSC-secreted factors. Inclusion of antibodies to TGF, VEGF, MCP-1 and CCL5 inhibited the effect seen, suggesting these paracrine factors played a role in the elevated expression levels.

Conclusion: MSCs clearly have a distinct paracrine effect on breast cancer epithelial cells, mediated at least in part through secretion of growth factors and chemokines. These factors play an important role in the metastatic cascade and may represent potential therapeutic targets to inhibit MSC-breast cancer interactions, helping to prevent the formation of bone metastases in cancer.