C. De Bari

A novel in vivo murine model of cartilage regeneration. Age and strain-dependent outcome after joint surface injury

Summary Objectives To generate and validate a murine model of joint surface repair following acute mechanical injury. Methods Full thickness defects were generated in the patellar groove of C57BL/6 and DBA/1 mice by microsurgery. Control knees were either sham-operated or non-operated. Outcome was evaluated by histological scoring systems. Apoptosis and proliferation were studied using TUNEL and Phospho-Histone H3 staining, respectively. Type II collagen neo-deposition and degradation were evaluated by immunostaining using antibodies to the CPII telopeptide and C1,2C (Col2-3/4Cshort), respectively. Aggrecanases and matrix metalloproteinases (MMPs) activity were assessed by immunostaining for TEGE373 and VDIPEN neo-epitopes. Results Young 8-week-old DBA/1 mice displayed consistent and superior healing of the articular cartilage defect. Age-matched C57BL/6 mice repaired poorly and developed features of osteoarthritis (OA). Compared to C57BL/6, DBA/1 mice displayed a progressive decline of chondrocyte apoptosis, cell proliferation within the repair tissue, persistent type II collagen neo-deposition, less type II collagen degradation, less aggrecanases and more MMP-induced aggrecan degradation. Eight-month-old DBA/1 mice failed to repair, but, in contrast to age-matched C57BL/6 mice, developed no signs of OA. Conclusion We have generated and validated a murine model of cartilage regeneration in which the outcome of joint surface injury is strain and age dependent. This model will allow, for the first time, the dissection of different pathways involved in joint surface regeneration in adult mammals using the powerful technology of mouse genetics.

Cell-based Approaches to Joint Surface Repair : A Research Perspective

Summary Repair of lesions of the articular cartilage lining the joints remains a major clinical challenge. Surgical interventions include osteochondral autograft transfer and microfracture. They can provide some relief of symptoms to patients, but generally fail to durably repair the cartilage. Autologous chondrocyte implantation has thus far shown the most promise for the durable repair of cartilage, with long-term follow-up studies indicating improved structural and functional outcomes. However, disadvantages of this technique include the need for additional surgery, availability of sufficient chondrocytes for implantation, and maintenance of their phenotype during culture-expansion. Mesenchymal stem cells offer an attractive alternative cell-source for cartilage repair, due to their ease of isolation and amenability to ex vivo expansion while retaining stem cell properties. Preclinical and clinical studies have demonstrated the potential of mesenchymal stem cells to promote articular cartilage repair, but have also highlighted several key challenges. Most notably, the quality and durability of the repair tissue, its resistance to endochondral ossification, and its effective integration with the surrounding host tissue. In addition, challenges exist related to the heterogeneity of mesenchymal stem cell preparations and their quality-control, as well as optimising the delivery method. Finally, as our knowledge of the cellular and molecular mechanisms underlying articular cartilage repair increases, promising studies are emerging employing bioactive scaffolds or therapeutics that elicit an effective tissue repair response through activation and mobilisation of endogenous stem and progenitor cells.

Bone marrow mesenchymal cells for haemophilia A gene therapy using retroviral vectors with modified long-terminal repeats

Summary. Bone marrow (BM) cells are attractive target cells for ex vivo gene therapy of genetic diseases, including haemophilia A. However, BM‐derived haematopoietic stem/progenitor cells (HSCs) transduced with factor VIII (FVIII) retroviral vectors, failed to express FVIII in vivo. To overcome the limitations of HSCs for haemophilia gene therapy, BM‐derived mesenchymal cells were explored as alternative target cells. The BM mesenchymal cell population contains self‐renewing mesenchymal stem/progenitor cells that give rise to different mesenchymal lineages and have been used safely in phase I gene‐marking trials. Human BM mesenchymal cells were transduced in vitro with an improved retroviral vector encoding a human B‐domain deleted FVIII (hFVIIIΔB) cDNA (MND‐MFG‐hFVIIIΔB). This vector contains multiple modifications in the cis‐acting elements within the MoMLV long‐terminal repeats (LTR) that prevent the binding of repressive transcription factors. These modifications were previously shown to increase and prolong gene expression in embryonic stem (ES) cells and HSCs. Transduction of BM mesenchymal cells with the MND‐MFG‐hFVIIIΔB retroviral vector resulted in high levels of functional human FVIII in vitro, ranging between 300 ± 50 SD and 700 ± 100 SD mU per 106 cells per 24 h. Following xenografting of the transduced human BM cells into immunodeficient NOD‐SCID mice, therapeutic hFVIII levels of 12 ± 10 ng mL−1 were detected in the plasma. Polymerase chain reaction analysis demonstrated long‐term engraftment (>3 months) of the human BM mesenchymal cells. The long‐term persistence of BM mesenchymal cells in the absence of myelo‐ablative conditioning and the therapeutic FVIII levels in vivo underscore the potential usefulness of BM‐derived mesenchymal cells for haemophilia gene therapy, as opposed to BM‐derived HSCs. Despite the modifications of the MoMLV LTR, FVIII expression declined, which coincided with a decrease in FVIII mRNA transcription levels, indicating that the salutary effect of the LTR modification on transgene expression is not universally applicable to all cell types.

In vitro growth rate of fibroblast-like synovial cells is reduced by methotrexate treatment

Background: Fibroblast-like synovial cells (FLS) can be cultured and expanded in vitro in monolayer. Little is known about the growth characteristics of FLS derived from different patients. Objective: To study FLS cultures, with particular attention to differences in growth rate of FLS from patients with rheumatoid arthritis (RA) and from other arthritic patients. Additionally, to analyse the influence of methotrexate (MTX) treatment, patient age, and disease duration on FLS growth characteristics. Materials and methods: FLS were isolated from needle arthroscopy biopsy specimens. Twenty four patients (11 RA, 8 spondyloarthropathy, 1 osteoarthritis, and 4 undifferentiated arthritis) were studied. FLS population doubling time was determined between passage 2 and passage 5. Differences in population doubling time between RA and non-RA FLS and between FLS from patients receiving MTX and those not receiving this drug were analysed. In addition, possible correlations between FLS population doubling time and patient age or disease duration were examined. Results: In vitro monolayer FLS cultures from needle arthroscopy biopsy specimens showed linear growth characteristics. Cell growth rate was not correlated with type of disease. Cells from patients undergoing treatment with MTX showed a longer population doubling time than FLS from patients not receiving this drug (Mann-Whitney test, p<0.05). No correlation was found with patient age or disease duration. Conclusion: The results suggest that FLS growth in monolayer is not dependent on the disease affecting the joint. MTX treatment, however, was more relevant in determining FLS growth rate.

Molecular basis of joint development

The introduction of the techniques of molecular biology has provided powerful tools in modern biomedical research including the field of developmental biology and new insights in the molecular pathways governing joint development. The critical signals involved in the joint formation process are also likely to be involved in the maintenance and repair of the joint structures following traumatic/degenerative or inflammatory injuries. Yet, the available data on the complex set of molecular players participating in joint specification and morphogenesis are often disconnected and sometimes controversial. In this review we try to depict a comprehensive state of the art in the field and to indicate connections that may link the different molecular pathways. The elucidation of these development cascades identifies new therapeutic targets and opportunities to enhance and control repair processes in joint disorders.

Mesenchymal differentiation propensity of a human embryonic stem cell line

Objectives:  To characterize basal differentiation tendencies of a human embryonic stem (hES) cell line, KCL‐002.

SP0168 The Synovium as a Stem Cell Niche for Joint Repair

The synovium has remarkable self-renewal ability and self-regenerates in patients with inflammatory arthritis undergoing synovectomy. In addition, during osteoarthritis the joint demonstrates apparent repair mechanisms that can result in aberrant formation of cartilage and bone in the form of chondro-osteophytes. Altogether, this presumes existence of endogenous stem cells in the joint. More than a decade ago, we isolated from the adult human synovium mesenchymal stem cells (MSCs) with multipotency inherent at the single cell level (De Bari et al., Arthritis Rheum 2001). In a comparative study of MSCs from multiple tissue sources including bone marrow, the synovial MSCs displayed superiority in cartilage formation (Sakaguchi et al., Arthritis Rheum 2005), pointing to synovial MSCs as possible ideal chondroprogenitors for articular cartilage repair. We have postulated that postnatally the synovium may function as a reservoir of stem cells for the regeneration / repair of joint tissues such as articular cartilage and menisci that are known to have poor intrinsic regeneration potential. In this regard, the synovium shares with the joint surface a common embryonic derivation from the developmental joint interzone (Koyama et al., 2008; Rountree et al., 2004), while bone marrow and periosteum which, like synovium, contain MSCs in adult life, appear to have distinct developmental origins. In adult life, stem cells are considered as quiescent slow-cycling cells which, following injury, become activated and undergo proliferation and differentiation into mature cell type(s) in order to safeguard tissue anatomy and function. On the basis of this notion, we recently provided data on the identification and characterization of endogenous resident MSCs in the adult mouse knee joint synovium in vivo. We used a double nucleoside labelling scheme in a mouse model of knee joint surface injury (Eltawil et al., Osteoarthritis Cartilage 2009) and identified in the adult knee synovium long-term label-retaining slow-cycling cells that, following injury to the articular cartilage, proliferated and differentiated into chondrocytes, hence displaying typical features of adult stem cells (Kurth et al., Arthritis Rheum 2011). These slow-cycling cells were non-haematopoietic, non-endothelial stromal cells with a phenotype compatible with MSCs, and were located in the lining layer and in perivascular areas. Notably, these cells were distinct from pericytes. The existence of functional MSC niches in the adult joint opens up unprecedented opportunities for pharmacological interventions by using medications that would target MSC niches and related reparative signalling pathways to activate and modulate intrinsic joint tissue regeneration and, in a broader picture, to influence outcomes of joint disorders and restore joint homeostasis. Disclosure of Interest None Declared

54 A NOVEL AGE- AND STRAIN-DEPENDENT IN VIVO MODEL OF ARTICULAR CARTILAGE HEALING IN MICE

(lengthwise and crosswise) at 5-mm intervals, and the number of infrared rays blocked by the animal was counted. The total locomotor activity in every 30min was shown graphically and used as a daily behavioral pattern. The total locomotor activity over 24 hours was measured as daily locomotor activity. Measurement of tail surface temperature before and after moxibustion in each group: The tail surface temperature was measured using a thermograph in group I and II. To avoid the influence of haircoat, the tail temperature which is used as an indicator for peripheral circulation was measured. Before and after, after 15min of acclimation to the environment, the tail surface temperature was measured in conscious animals in a windless room maintained at a temperature of 15±1oC and a relative humidity of 60±10%. Temperature of the rat tails was measured at a distance of 1m from the thermgraphy device. Rats were acclimated before the experiment. Results: Changes in locomotor activity before and after moxibustion stimulation: Before moxibustion stimulation, in group I, the pattern of locomotor activity was regular. In the group I, there was no clear diference in the pattern of locomotor activity between the active and resting phases and the pattern of locomotor activity was irregular. The daily level of locomotor activity at 24 hours was significantly higher in the group I than in group II. After moxibustion stimulation of the group I, the pattern of locomotor activity became diphasic with clear active and resting phases, similar to that observed in group II. Moreover, the daily level of locomotor activity at 24 hours in group I was significantly higher than that in group II. Changes in tail surface temperature before and after before moxibustion stimulation: Before moxibustion stimulation, the tail surface temperature was significantly lower in group I than in group II. After moxibustion stimulation, there was no significant difference in the tail surface temperature between group I and group II. Conclusions: The increased locomotor activity of rat AA was presumably ascribable to the removal of blood circulation reduced blood flow in the peripheral circulation rather than the induction of stress by moxibustion. These results demonstrate that moxibustion stimulation is effective for the treatment of pain relief.