How to Select Cases and Utilize Stem Cells in Tendon Injury

1. Introduction

The use of stem cells for the treatment of tendon injuries has gained acceptance over the past decade. While there has been no randomized controlled trials for the use of mesenchymal stem cells (MSCs) in the treatment of tendon overstrain injuries, an increasing amount of experimental^1–3 and clinical data⁴ have mostly demonstrated improved quality of equine tendon healing with cellular treatments.

2. Case Selection

The cells are most commonly delivered by intratendinous injection and therefore require a defect within the tendon that is surrounded by an intact paratenon to retain the cells after implantation. The size of the lesion is not critical, although small lesions are more difficult to inject and generally carry a better prognosis and therefore cellular therapies are less indicated for the smaller lesions. Cell retention is very poor if the paratenon is not intact (unpublished data). Hence, percutaneous lesions, where this is not the case, are not appropriate for treatment unless at the stage when a granulation bed is present that can act as this retaining layer. 

Analysis of the clinical data shows improved outcome with earlier implantation although not always significantly different statistically. For racehorses, there was an average of 46 days between injury and implantation for successful cases compared to 54 days for cases that subsequently re-injured⁴; for sports horses, the re-injury rate was lower for horses treated within 1 month of the injury compared to those treated after a month (unpublished data). Autologous treatments are a two-step procedure requiring first the aspiration of bone marrow (or other tissue) to obtain the MSCs and second, after a variable time depending on the preparation used, to implant the cells. In the intervening time, the horse is either maintained on box-rest (if lame or severely injured) or given up to 10 minutes hand-walking per day. For bone marrow, a period of approximately 3 weeks for culture is needed, which limits any earlier implantation, and so bone marrow should be aspirated as soon as is possible after injury. The use of banked or allogenic cells allows even earlier implantation although it is logical to target the cell implantation to when there is a vascular supply established to support the cells after implantation—normally at least 1 to 2 weeks after injury.

3. Cell Choice

There are a number of options with respect to cell choice. The most commonly used preparations are MSCs recovered from bone marrow or fat, with more recent additions of umbilical cord cells⁵ and fetal tendon cells.⁶ The major differences between these cell products are more related to the preparation methods rather than the source. Of the autologous treatments, cells can either be ‘minimally manipulated,’ when cells are released from tissue by enzymes and a cell mixture returned to the veterinarian for implantation (often the case when fat is chosen as the tissue source), or MSC populations are expanded by culture in a laboratory. Culture and expansion is necessary if it is deemed important to produce an abundant and homogenous stem cell preparation because of the low numbers of MSCs in the source material (e.g., only 1 in 100,000 of the nucleated cells in bone marrow is an MSC). Allogenic cells simplify the process and may become more commonly used, providing legislation allows, as it avoids the first step and is easier and cheaper. Allogenic cells do not appear to cause immunological reactions because of the immunosuppressive effects and appear to be well tolerated (unpublished data). However, such products have not been extensively researched or well characterized to date to ensure quality and effectiveness and, therefore, should be used with caution.

4. Dose

There have been no good studies documenting a dose response. Initially, 10 million cells (suspended in bone marrow supernatant at a concentration of 5 million cells/mL) were used for an ‘average-sized’ lesion occupying up to 30% of the cross-sectional area of the superficial digital flexor tendon. More recent studies have shown that there is limited retention of cells after implantation^7–11 and an analysis of treated horses showed a reduced re-injury rate in horses treated with 20 million or more cells when compared to the original empirical does of 10 million cells/lesion. Therefore this ‘routine’ dose has been increased to 20 million cells (at the same concentration of 5–10 million cells/mL) with the option to increase this twofold for larger lesions.

Step 1–How to Obtain a Bone Marrow Aspirate

Equipment Needed

• Ultrasound machine with 7.5 MHz or above linear transducer
• Sedative
• 2 mL syringe (for local analgesia)
• Local anaesthetic (mepivicaine)
• Heparin (stock solution concentration: 5000 IU/mL)
• 11 scalpel blade
• 11 G 10 cm Jamshidi needle
• Two 10 mL or one 20 mL syringe
• At least two 5 mL syringes
• Sterile swabs
• Sterile containers and transport pack (to ship the bone marrow to the laboratory for culture).

Technique: Sternum

As the bone marrow has to be transported to the laboratory after it is obtained, aspirations should only be done on a Monday to Thursday to avoid prolonged delivery over weekends.

The horse is first sedated with a combination of an α2 agonist and opiate (e.g., detomidine HCl and butorphanol tartate). A 10 cm wide band overlying the sternum is clipped and scrubbed with surgical scrub (e.g., chlorhexidine) and surgical spirit.

While some don’t advocate ultrasound, ultrasound is advised to ensure accurate aspiration and therefore minimize the risks of inadvertent penetration of the heart. The sternum is examined ultrasonographically to identify the three most caudal sternebrae by the appearance of their intersternebral spaces (Fig. 1). The most caudal one is avoided as it is thinner and hence penetration of its deeper surface is easier.
 

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