To test and evaluate the effectiveness of local injection of autologous fat-derived mesenchymal stem cells (MSCs) into fracture site to prevent non-union in a clinically relevant model.

5 male Wistar rats underwent the same surgical procedure of inducing non-union. A mid-shaft tibial osteotomy was made with 1mm non-critical gap. Periosteum was stripped around the two fracture ends. Then, the fracture was fixed by ante-grade intramedullary nail. The non-critical gap was maintained by a spacer with minimal effect on the healing surface area. At the same surgical time, subcutaneous fat was collected from the ipsilateral inguinal region and stem cells were isolated and cultured in vitro. Within three weeks postoperatively, the number of expanded stem cells reached 5×10⁶ and were injected into the fracture site. Healing was followed up for 8 weeks and the quality was measured by serial x-rays, microCT, mechanical testing and histologically. Quality of healing was compared with that of previously published allogenic, xenogeneic MSCs and Purified Buffered Saline (PBS) controls.

All the five fractures united fully after 8 weeks. There was a progressive increase in the callus radiopacity during the eight-week duration, the average radiopacity in the autologous fat-MSC injected group was significantly higher than that of the allogeneic MSCs, xenogeneic MSCs and the control group, P < 0.0001 for treatment, time after injection, and treatment-time interaction (two-way repeated measure ANOVA). MicroCT, mechanical testing and histology confirmed radiological findings.

The autologous fat-MSCs are effective in prevention of atrophic non-union by stimulation of the healing process leading to a solid union. The quality and speed of repair are higher than those of the other types of cell transplantation tested.

The role of mesenchymal stem cells (MSCs) in enhancing healing process has been examined with allogeneic and xenogeneic cells in transplantation models. However, certain factors might limit the use of allogeneic cells in clinical practice, (e.g. disease transmission, ethical issues and patient acceptance). Adipose tissue represents an abundant source for autologous cells. The aim of this study was to evaluate adipose-derived autologous cells for preventing non-union.

Adults male Wistar rats (n=5) underwent a previously published surgical procedure known to result in non-union if no treatment is given. This consisted of a mid-shaft tibial osteotomy with peri/endosteal stripping stabilised by intramedullary nail fixation with a 1mm gap maintained by a spacer. During the same operation, ipsilateral inguinal subcutaneous fat was harvested and processed for cell isolation. After three weeks in culture, the cell number reached 5×106 and were injected into the fracture site.

At the end of the experiment, all tibias (injected with autologous fat-MSCs) developed union. These were compared with a control group injected with PBS (n=4) and with allogenic (n=5) and xenogeneic (n=6) cell transplantation groups. The amount of callus was noticeably large in the autologous cell group and the distal-callus index was significantly greater than that of the other groups, P-value =<0.05, unpaired t-test, corrected by Benjamini & Hochberg.

We report a novel method for autologous MSCs implantation to stimulate fracture healing. Local injection of autologous fat-MSCs into the fracture site resulted in a solid union in all the tibias with statistically significantly greater amounts of callus.

Abstract

Cutting rodent's bone ends and irrigation of the medullary canal is the common method used for cells collection in allogenic transplantation, however it does not yield sufficient cells for autologous transplantation. The aim of this experiment was to establish and validate a method for bone marrow collection for autologous MSCs transplantation. Two collection methods were examined: 1) Transection of the bone ends and irrigation of the medullary canal, 2) Trephining of the bone with a hypodermic needle without aspiration. Then cell harvesting was compared in the idealised laboratory situation and under simulated surgery.

First, two lower limbs were harvested from the same rat cadaver for comparison, bone marrow in one limb was collected by cutting the femoral head and the distal tibia and irrigation of the canal through drilled holes at the distal end of the femur and proximal end of the tibia. Other limb, hypodermic needle was used as a trephining tool into the medullary canal multiple times without applying negative pressure and rinsed from inside and outside. Second, bone marrow was harvested from another rat's cadaver in the surgery room to simulate the conditions needed for autologous transplantation.

The number of cells from irrigation method was 1.28*106 cells, whereas that from trephining method reached 17*106. The number cells from the bone marrow harvested in the surgery room was found 29.6*106. We report a novel technique for harvesting cells for autologous cell therapy from only one limb. A significantly larger number of cells from bone marrow could be collected using the needle trephining method. There is no negative effect on the viability of cells after bone marrow harvesting in the surgery room.

In atrophic non-union models, a minimally invasive technique is used to deliver stem cells into the fracture site via percutaneous injection. This technique is significantly affected by a backflow leakage and the net number of cells might be reduced. The Z-track method is a technique used in clinical practice for intramuscular injections to prevent backflow leakage.

We evaluated the potential of the Z-track injection technique for preventing cell loss in non-union models by determining the behaviour of observable marker fluids. Firstly, toluene blue stain was used as an injection material to allow visual detection of its distribution. Rat's cadaver legs were used and tibias were kept unbroken to ensure intact skin and overlying soft tissue. Technique includes pulling the skin over the shin of tibia towards the ankle and injection of the dye around the mid-shaft. The needle was then partially pulled back, the skin was returned to its normal position and a complete extraction of the needle was followed. Secondly, a mixture of contrast material and toluene blue was used to allow direct visual and radiological detection of the injected material into the fracture site. Ante-grade nailing of tibia via tibial tuberosity was carried out followed by a 3 point closed fracture. Injection was performed into the fracture gap similarly to the steps above. X-rays were taken to visualise the location and distribution of the injected material.

Observation revealed no blue stain could be detected over the skin, X -rays revealed that the radiopaque dye remained around the tibia with no escape of the material into the superficial layers or onto the skin surface. Therefore, the number of cells delivered and maintained at a target site could be increased by the Z-track method and therefore, the therapeutic benefit of stem cell injections could be optimised with this simple technique.

Background

There are currently no effective treatments for skeletal muscle fibrosis. Myofibroblasts are the major cellular effectors of fibrosis but their origin in muscle is unknown. We report that PDGFRβ (platelet derived growth factor receptor beta) Cre inactivates genes in murine PDGFRβ+ cells and myofibroblasts in muscle with high efficiency. We used this system to delete the integrin αv subunit because of the suggested role of multiple αv integrins as central mediators of fibrosis in multiple organs.

Scar tissue formation secondary to acute muscle injury, surgical wounding and compartment syndrome can result in significant functional impairment and predispose to further injury. The source of fibroblasts, and the molecular mechanisms driving their activation and persistence in skeletal muscle fibrosis are not known. We hypothesized that cells expressing PDGFRβ become fibroblasts in response to injury and that targeting αv integrins in these cells reduces skeletal muscle fibrosis.

We used double-fluorescent reporter mice to demonstrate that cells expressing PDGFRβ become activated myofibroblasts in response to cardiotoxin (CTX) induced skeletal muscle injury. Following injury, PDGFRβ+ cells moved from perivascular locations into the interstitium in a distribution characteristic of fibroblasts, and showed marked induction of fibroblastic genes including αSMA and collagen1 (all p<0.0001). To confirm that αv integrins present on PDGFRβ cells critically regulate skeletal muscle fibrosis we used Itgavflox/flox;PDGFRβ-Cre mice (transgenic mice in which αv integrins are ‘knocked-down’ in PDGFRβ+ cells). These mice were significantly protected from CTX induced fibrosis (p<0.01). To demonstrate potential clinical utility of targeting αv integrins, we used a small molecule inhibitor of αv integrins (CWHM12). Treatment with CWHM12 significantly reduced fibrosis when delivered from the time of injury (p<0.01) and when delivered after the fibrotic response had become established (p<0.01).

We have identified a core pathway regulating fibrosis in skeletal muscle. Pharmacologic inhibition of αv integrins has potential clinical utility in the treatment and prevention of skeletal muscle fibrosis.

Adipose tissue is an attractive source of mesenchymal stem cells (MSCs) as it is largely dispensable and readily accessible through minimally invasive procedures such as lipoaspiration. Until recently MSCs could only be isolated in a process involving ex-vivo culture. Pericytes (CD45−, CD146+, and CD34−) and adventitial cells (CD45−, CD146−, CD34+) represent two populations of MSCs (collectively termed perivascular stem cells or PSCs) that can be prospectively purified using fluorescence activated cell sorting (FACS). We performed FACS on lipoaspirate samples from n=129 donors to determine the frequency and yield of PSCs and to establish patient and processing factors that influence yield.

The mean number of stromal vascular fraction (SVF) cells from 100ml of lipoaspirate was 37.8×106. Within the SVF, mean cell viability was 82%, with 31.6% of cells being heamatopoietic (CD45+). Adventitial cells and pericytes represented 31.6% and 7.9% of SVF cells respectively. As such, 200ml of lipoaspirate would theoretically yield 24.5 million MSCs –a sufficient number to enable point-of-care delivery for use in several orthopaedic applications. The yield and prevalence of PSCs were minimally affected by donor age, sex and BMI. Storing lipoaspirate samples for up to 72 hours prior to processing had no significant deleterious effects on MSC yield or viability.

Our study confirms that pure populations of MSC-precursors (PSCs) can be prospectively isolated from adipose tissue, in sufficient quantities to negate the necessity for culture expansion while widening possible applications to include trauma, where a time delay between extraction and implantation excludes their use.