DONATED CARTILAGE STEM CELLS FOR CARTILAGE DEFECT REPAIR – ALLOGENEIC CHONDROPROGENITOR THERAPY PHASE II TRIAL (ACT2)

Jade Perry, Duncan Carroll, Charlotte Hulme, Larissa Rix, Tian Lan, Sally Roberts, Martyn Snow and Karina Wright

Funded by the Medical Research Council

Currently, the most cost-effective treatment for cartilage defects is autologous chondrocyte implantation (ACI) according to the National Institute for Clinical Excellence. ACI involves two surgeries: the first to collect a biopsy of healthy cartilage, which is then used to grow the patients own chondrocytes (cartilage cells) which are then implanted in a second surgery around 4 weeks later. Unfortunately, for many reasons, there has been limited uptake by NHS centres across the UK to adopt the ACI procedure, and the RJAH Orthopaedic Hospital remains one of the few centres to perform this procedure.

After multiplying chondrocytes in the lab, they can lose their ability to produce cartilage, as they usually do not multiply in the body, so attention has turned to a subpopulation of cells in the cartilage called Chondroprogenitors (CPs). CPs are a stem-like cell, meaning they have some properties similar to stem-cells, such as the ability to multiply and not lose function. The aim of the ACT2 trial is to obtain CPs from cartilage donated through the NHS Blood and Transplant (NHSBT) organ donation service, these will then be multiplied and stored cryogenically until they are needed by patients, where they will be thawed and implanted into the cartilage defect (on a patch made from collagen or hyaluronic acid) in a single surgery. This could result in a higher uptake of ACT over ACI as a single surgery would cost less for the NHS, and multiple patients could be treated from a single donor.

At the RJAH we are optimising the process of harvesting, cryogenically storing and transferring the CPs to the patch (scaffold). This has involved receiving donated cartilage (see picture insert of Prof Snow’s dissection) from NHSBT, or US commercial sources (JRF Ortho and RTI Surgical) and processing it to isolate the CPs and testing different variables throughout this process.

In brief, CPs were isolated from full depth human articular cartilage from healthy cadaveric osteochondral allografts from the knee (n=6, aged 24- 42 yrs) and talus (n=6; aged 15- 32 yrs) using selective adhesion to vitronectin. Cell viability across the donors varied from 24—100% and live chondrocyte prep yields varied considerably. Chondroprogenitors isolated from all donors were sterile and were successfully culture expanded. Furthermore, all donors achieved the minimum functionality criteria set for the cells (a measure of their ability to produce cartilage repair tissue components). All chondroprogenitors were immunopositive (>95%) for chondropotency markers, mesenchymal stem cell markers and integrin markers. To date, cryogenic stability testing at 6-months confirmed an acceptable viability value of more than 50% for the 5 grafts to have reached this time point. For the 3 grafts where functionality data is available, the minimum functionality criteria was passed and was above the target value of 90% of pre-cryopreservation values.

In additional work we wanted to assess how the CPs would adhere to the chosen biological scaffolds, and whether the solution they were stored (cryopreservant) in would affect their adherent properties. To do this we seeded 1 cm squares of the Chondro-Gide® collagen scaffold with varying cell numbers in 100 uL of different cryopreservant mixes. Results showed that a cryopreservant with 5% DMSO (an antifreeze used to stop cells from bursting as they’re frozen) performed best for cell attachment, and that a one-centimetre square of scaffold could hold up to 4 million cells.

We further assessed cell attachment by isolating seeded scaffolds in their own nutrient media and measured a metabolite of the cells to determine an accurate cell number, as well as visualising the live cells attached (stained green) using confocal microscopy, see picture insert). In the future we will look at differences between two biological scaffolds: Chondro-Gide® (collagen-based) and Hyalofast® (hyaluronan[1]based), and whether cells perform differently on these scaffolds in terms of cell attachment and cell metabolism.