Autologous Stem Cells in Achilles Tendinopathy (ASCAT): protocol for a phase IIA, single-centre, proof-of-concept study.

INTRODUCTION: Achilles tendinopathy (AT) is a cause of pain and disability affecting both athletes and sedentary individuals. More than 150 000 people in the UK every year suffer from AT.While there is much preclinical work on the use of stem cells in tendon pathology, there is a scarcity of clinical data looking at the use of mesenchymal stem cells to treat tendon disease and there does not appear to be any studies of the use of autologous cultured mesenchymal stem cells (MSCs) for AT. Our hypothesis is that autologous culture expanded MSCs implanted into an area of mid-portion AT will lead to improved pain-free mechanical function. The current paper presents the protocol for a phase IIa clinical study. METHODS AND ANALYSIS: The presented protocol is for a non-commercial, single-arm, open-label, phase IIa proof-of-concept study. The study will recruit 10 participants and will follow them up for 6 months. Included will be patients aged 18-70 years with chronic mid-portion AT who have failed at least 6 months of non-operative management. Participants will have a bone marrow aspirate collected from the posterior iliac crest under either local or general anaesthetic. MSCs will be isolated and expanded from the bone marrow. Four to 6 weeks after the harvest, participants will undergo implantation of the culture expanded MSCs under local anaesthetic and ultrasound guidance. The primary outcome will be safety as defined by the incidence rate of serious adverse reaction. The secondary outcomes will be efficacy as measured by patient-reported outcome measures and radiological outcome using ultrasound techniques. ETHICS AND DISSEMINATION: The protocol has been approved by the National Research Ethics Service Committee (London, Harrow; reference 13/LO/1670). Trial findings will be disseminated through peer-reviewed publications and conference presentations. TRIAL REGISTRATION NUMBER: NCT02064062.

The effect of temperature on the viability of human mesenchymal stem cells.

INTRODUCTION: Impaction allograft with cement is a common technique used in revision hip surgeries for the last 20 years. However, its clinical results are inconsistent. Recent studies have shown that mesenchymal stem cells (MSCs) seeded onto allograft can enhance bone formation. This in vitro study investigates whether the increase in temperature related to the polymerisation of bone cement will affect the viability of human MSCs. METHODS: The viability of human MSCs was measured after incubating them at temperatures of 38°C, 48°C and 58°C; durations 45 seconds, 80 seconds and 150 seconds. A control group was kept at 37°C and 5% carbon dioxide for the duration of the investigation (7 days). During the course of the study the human MSCs were analysed for cell metabolic activity using the alamarBlue™ assay, cell viability using both Trypan Blue dye exclusion and calcein staining under fluorescent microscopy, and necrosis and apoptosis using Annexin V and propidium iodide for flow cytometric analysis. A one-way analysis of variance with a priori Dunnett's test was used to indicate the differences between the treatment groups, when analysed against the control. This identified conditions with a significant difference in cell metabolic activity (alamarBlue™) and cell viability (Trypan Blue). RESULTS: Results showed that cell metabolism was not severely affected up to 48°C/150 seconds, while cells in the 58°C group died. Similar results were shown using Trypan Blue and calcein analysis for cell viability. No significant difference in apoptosis and necrosis of the cells was observed when human MSCs treated at 48°C/150 seconds were compared with the control group. CONCLUSIONS: The study suggests that human MSCs seeded onto allograft can be exposed to temperatures up to 48°C for 150 seconds. Exposure to this temperature for this time period is unlikely to occur during impaction allograft surgery when cement is used. Therefore, in many situations, the addition of human MSCs to cemented impaction grafting may be carried out without detrimental effects to the cells. Furthermore, previous studies have shown that this can enhance new bone formation and repair the defects in revision situations.

The effect of mesenchymal stromal cells on the osseoinduction of impaction grafts.

Revision total hip replacement has a failure rate of up to 25%. Part of the reason for this high failure is the lack of bone stock. In this study, we investigated whether mesenchymal stromal cells (MSCs) or osteoprogenitors (OPs) contribute to bone formation in impacted allograft or an allograft and hydroxyapatite (HA) combination. Eight samples were inserted in the paraspinal muscles of six sheep, which were allograft or an HA/allograft mix, seeded with autologous MSCs, autologous OPs, or no cells (control), and impacted with a clinically relevant force at 3 kN preimplantation. Other groups were HA blocks seeded with or without MSCs. Samples were retrieved at 12 weeks. MSCs incorporated into allograft, HA/allograft, and HA blocks displayed higher mean values of new bone formation compared with the controls, but there was no statistical difference between allograft groups. However, in HA/allograft groups, significantly more new bone was found with MSCs than without cells (p = 0.046) or with OPs (p = 0.028). In the HA blocks, more new bone was found in the MSC group than in the HA block without cells (p = 0.028). In conclusion, MSCs used with combinations of allograft and HA enhance new bone formation in an ovine ectopic site after being subjected to realistic forces that are used during impaction grafting.

Use of mesenchymal stem cells to enhance bone formation around revision hip replacements.

Tissue engineering approaches to regenerate bone stock in revision total hip replacements could enhance the longevity of the implant and benefit the quality of the patient's life. This study investigated the impaction of allograft with mesenchymal stem cells in an ovine hip hemiarthroplasty model. In total, 10 sheep were divided into two groups with 5 sheep in each group. The groups were: 1) mesenchymal stem cells mixed with allograft; 2) allograft only as a control. Ground reaction force was assessed for limb function and showed that there was no significant difference in the recovery for animals in different groups. The amount of bone regenerated around the hip replacement was assessed using un-decalcified histology. The results showed that the stem cell group generated significantly more new bone at the implant-allograft interface and within the graft than the control group. The results from this study indicate that the use of stem cells on an allograft scaffold increases bone formation indicating that the use of stem cells for revision hip arthroplasty may be beneficial for patients undergoing revision surgery where the bone stock is compromised.

Can mesenchymal stem cells survive under normal impaction force in revision total hip replacements?

Impaction allograft as a scaffold for bone-forming cells is a tissue-engineering approach for filling bone defects that are commonly encountered during revision total joint replacement (THR). The purpose of this in vitro study is to assess the viability of mesenchymal stem cells (MSC) grown on allograft following impaction using forces similar to those measured during revision total hip replacements. Impaction forces of 0, 3, 6, and 9 kN were used representing normal and high impact. The results showed that the viability in the 3 and 6 kN groups was not significantly reduced compared with that of the 0 kN group, while the survival of the MSCs was significantly reduced after 9 kN impaction force. This study suggests that the addition of MSCs to the allograft scaffold will survive normal impaction force in revision THR.

A novel gain-of-function mutation of the integrin alpha2 VWFA domain.

Integrin alpha2beta1 is the major receptor for collagens in human tissues, being involved in cell adhesion and the control of collagen and collagenase gene expression. The collagen binding site of alpha2beta1 has been localized to the alpha2 von Willebrand Factor type A (VWFA) domain (A-domain or I-domain) and the residues responsible for the interaction with collagen have been mapped. We report a study of alpha2 VWFA domain in which residue E318, which lies outside the collagen binding site, is mutated to tryptophan, showing that this is a gain-of-function mutation. Recombinant alpha2-E318W VWFA domain showed elevated and specific binding to collagen I compared with the wild-type. Side chain hydrophobicity was important for the gain-of-function as elevated binding was seen with E318I and E318Y, but not with E318R. The E318W mutation had additional effects on VWFA domain properties as alpha2-E318W VWFA domain differed from the wild-type in its cation preferences for ligand binding and in binding to monoclonal antibody JA203, which bound at a site distal to E318. The gain-of-function effect was not restricted to binding to collagen I as alpha2-E318W also showed elevated binding to collagen IV, collagen I C-propeptide, laminin and E-cadherin. Binding to these ligands was inhibited by collagen peptide containing the GFOGER motif, indicating that these bound to the VWFA domain by a similar mechanism to collagen I. These data indicate that residue E318 plays a novel and important role in modulating alpha2 VWFA domain--ligand binding and may be involved in the conformational changes associated with its regulation.