We studied five conditions including autologous extrasynovial graft with and without surface treatment (n = 12), allograft intrasynovial tendon with and without surface treatment (n = 16), and normal surgical digits (n = 8) in 14 mixed breed dogs and compared adhesion scores and cell density between the conditions. Six dogs had autograft peroneus longus (PL) tendons as the donor grafting tendons and eight dogs had allograft flexor digitorum profundus (FDP) tendons as the graft donor tendons. Because this was a preliminary study, the sample size power analysis was not performed. In each dog, the second and fifth digits were randomly selected for a graft, treated either with saline solution as a control or cd-HA gelatin (ie, a total of 28 grafts). For the tendon cellularity study, the normal FDP tendon was used as the normal control group. Therefore, a total of five groups were designed: (1) autograft control (n = 6); (2) autograft treated (n = 6); (3) allograft control (n = 8); (4) allograft treated (n = 8); and (5) normal FDP tendon (n = 8). The cd-HA graft tendons were immersed in 1% sodium hyaluronate (Acros, 95%), 10% gelatin (from porcine skin; Sigma Chemical Co, St Louis, MO, USA), 1% 1-ethy1-3 [(3-dimethylaminopropyl) carbodiimide hydrochloride] [9
] (Sigma Chemical Co), and 1% N-hydroxysuccinimide (Sigma Chemical Co) in 0.1 M NaCl pH 6.0 and 0.9% phosphate-buffered saline for 5 minutes [27
]. We had prior approval of our Institutional Animal Care and Use Committee (IACUC).
The dogs were anesthetized using intravenous ketamine (10 mg/kg) and diazepam (0.6 mg/kg) and isoflurane inhalational during the surgical procedure. For the autograft dogs, both PL tendons from the hind paws of the same dog were harvested and immediately replaced the FDP tendons of the second and fifth digits of one paw in Zone II with one graft tendon treated with cd-HA and the other treated with saline as a control. For the allograft treated with cd-HA or saline control dog, however, a FDP tendon repair failure model was used to mimic the clinical scenario, ie, flexor tendon reconstruction in a scarred digit. In this clinically relevant model, the FDP tendon in Zone II was lacerated and repaired first. The dogs were immediately allowed free cage activity after surgery. All repaired FDP tendons ruptured within 1 week as a result of this unprotected postoperative ambulation, which we used as a mechanism to create a scarred tendon bed before reconstruction [29
]. Six weeks after primary repair, the digits underwent FDP tendon reconstruction. The allograft FDP tendons were harvested from dogs that were euthanized for other IACUC-approved studies not related to musculoskeletal disorders. The allograft tendons were immediately immersed in liquid nitrogen for 1 minute and then thawed for 5 minutes in warmed saline solution at 37°C. This procedure was repeated five times to induce tenocyte necrosis [23
]. The tendons were lyophilized with a custom-made lyophilizer and then gas-sterilized. The graft was rehydrated in a 0.9% NaCl bath in a closed, sterilized container for 24 hours in an incubator at 37°C before graft surgery.
The surgical procedures of the FDP reconstruction either in autograft or allograft followed a standard one-stage flexor tendon reconstruction as previously described for the canine model [28
]. Briefly, a space for the graft was created by removing the FDP tendon in Zone II. For both autograft and allograft procedures, one of the surgical digits was randomly selected for treatment with a graft coated with cd-HA gelatin, and the other digit was treated with a graft soaked in saline. A 3-mm drill was used to make a hole at the distal phalanx beneath the FDP tendon attachment. The graft distal end was sutured with 3/0 Ethibond suture (Ethicon, Inc, Somerville, NJ, USA) with modified Pennington locking loops [26
]. The suture was passed through the tunnel and out onto the dorsal aspect of the digit, and the distal graft was pulled into the bone tunnel. We used a dorsal button to fix the suture [28
]. The proximal end of the graft was sutured to the proximal recipient FDP tendon using a Pulvertaft weave suture. After the tendon graft, we performed a proximal radial neurectomy on the selected surgical forelimb to denervate the triceps muscle and prevent elbow extension and thus weightbearing [5
]. The operated paw was then immobilized. For the analgesics, a Buprenex intramuscular injection with a dose of 0.01 mg/kg every 8 hours for 2 days and then 4 mg/kg carprofen was given by subcutaneous injection for 1 week. On postoperative Day 5, rehabilitation was started with a synergistic wrist digit motion protocol (wrist flexion with the digit joints in extension and wrist extension with the digit joints in flexion) performed once daily for 6 weeks, after which the animal was euthanized with an overdose of pentobarbital.
During dissection, the adhesion status in Zone II was grossly assessed by two investigators (RLK, CZ) in a blinded fashion. The adhesion score system was modified based on previous flexor tendon repair grading criteria [26
]. The rating scale at each site ranged from 0 (no adhesion) to 4 (very severe) (Table ). The graft tendons were graded for adhesion formation at two sites: (1) between the tendon and sheath including the pulley and the synovial lining; and (2) between the tendon and tendon bed, including the flexor digitorum superficialis tendon and the surrounding soft tissues of the phalanx. Thus, the total of the scores at the two sites ranged from 0 to 8. Any disagreements in adhesion score were resolved by consensus.
Score for gross evaluation of the adhesion
After graft dissection, six autograft and eight allograft tendons were cut in 10 mm length at the proximal interphalangeal joint level. The graft tendons were then fixed in 10% neutral-buffered formalin, embedded in paraffin, and sectioned longitudinally in 5-μm thickness. Hematoxylin and eosin staining was performed. Two to three slides from each sample were examined in a high-resolution microscope linked to a digital image analysis system (microscope: Olympus BX51, Tokyo, Japan; camera: Sony DXC-970MD, 3CCD color camera, Tokyo, Japan) for gross observation of cell distribution, morphology, and appearance of foreign body giant and inflammatory cells. For the cellularity quantification, the cells in one slide from each tendon sample were counted by one investigator (FEK) in a blinded fashion from longitudinally sectional samples with the following protocol. The tendon slides were first observed under low-power magnification (20×), which included the entire tendon section (Fig. ). Using a stereological randomization technique, 10 areas of interest were randomly selected for cell counting. The scope was moved and focused on one circle (with the letter and number) centered in each grid (Fig. ) with medium magnification (100×). The microscope was switched to high-power magnification (400×) without any movement on the slide, and cells were counted. Then, the scope was switched to low magnification and moved to the other circle for cell counting. The cell number in 10 viewed areas in each slide was averaged for data analysis.
This is a typical slide showing a grid slide at 20× low magnification (A). For cell counting, the scope was moved and focused on the circle (with the letter and number) centered in each grid with a magnification of 100× (B).
Differences in cell count among five groups (autograft-cd-HA, autograft-saline, allograft-cd-HA, allograft-saline, and normal FDP tendon) and adhesion score in four groups (without normal FDP tendon group) were analyzed with analysis of variance and the post hoc Tukey’s honestly significantly different test was applied to assess differences among groups.