CORR® ORS Richard A. Brand Award for Outstanding Orthopaedic Research: Engineering Flexor Tendon Repair With Lubricant, Cells, and Cytokines in a Canine Model
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- Zhao, C., Ozasa, Y., Reisdorf, R.L. et al. Clin Orthop Relat Res (2014) 472: 2569. doi:10.1007/s11999-014-3690-y
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Adhesions and poor healing are complications of flexor tendon repair.
The purpose of this study was to investigate a tissue engineering approach to improve functional outcomes after flexor tendon repair in a canine model.
Flexor digitorum profundus tendons were lacerated and repaired in 60 dogs that were followed for 10, 21, or 42 days. One randomly selected repair from either the second or fifth digit in one paw in each dog was treated with carbodiimide-derivatized hyaluronic acid, gelatin, and lubricin plus autologous bone marrow stromal cells stimulated with growth and differentiation factor 5; control repair tendons were not treated. Digits were analyzed by adhesion score, work of flexion, tendon-pulley friction, failure force, and histology.
In the control group, 35 of 52 control tendons had adhesions, whereas 19 of 49 treated tendons had adhesions. The number of repaired tendons with adhesions in the control group was greater than the number in the treated group at all three times (p = 0.005). The normalized work of flexion in treated tendons was 0.28 (± 0.08), 0.29 (± 0.19), and 0.32 (± 0.22) N/mm/° at Day 10, Day 21, and Day 42 respectively, compared with the untreated tendons of 0.46 (± 0.19) at Day 10 (effect size, 1.5; p = 0.01), 0.77 (± 0.49) at Day 21 (effect size, 1.4; p < 0.001), and 1.17 (± 0.82) N/mm/° at Day 42 (effect size, 1.6; p < 0.001). The friction data were comparable to the work of flexion data at all times. The repaired tendon failure force in the untreated group at 42 days was 70.2 N (± 8.77), which was greater than the treated tendons 44.7 N (± 8.53) (effect size, 1.9; p < 0.001). Histologically, treated repairs had a smooth surface with intrinsic healing, whereas control repairs had surface adhesions and extrinsic healing.
Our study provides evidence that tissue engineering coupled with restoration of tendon gliding can improve the quality of tendon healing in a large animal in vivo model.
Tissue engineering may enhance intrinsic tendon healing and thus improve the functional outcomes of flexor tendon repair.
Flexor tendon injuries, one of the most common and difficult-to-treat hand injuries, can cause considerable functional loss and economic burden [2, 20, 24]. Primary surgical repair is the accepted treatment, but hand function may be compromised by postoperative complications such as poor intrinsic healing and adhesion formation [11, 24, 30]. One study showed that surface treatment with carbodiimide-derivatized gelatin, hyaluronic acid, and lubricin (cd-HA-lubricin) effectively decreased adhesions and improved digit function after flexor tendon repair in a canine model . However, this treatment also impaired tendon healing and caused another major complication: 6 weeks after repair, 35% of the tendons examined had ruptured (a significantly higher rate compared with the untreated control group) .
Cell-based therapy has been used to enhance tendon healing [8, 36]. Some studies have shown that in a tissue culture model, bone marrow stromal cells increased flexor tendon healing, especially if supplemented with growth differentiation factor 5 (GDF-5). GDF-5 is a growth factor that has been shown to differentiate stem cells toward a tendon phenotype [6, 13]. The purpose of our study was to test the hypothesis that delivery of stromal cells to the repair site through engineered cell patches could maintain the benefits of reduced adhesion from cd-HA-lubricin surface modification while maintaining tendon healing strength similar to that of repaired but otherwise untreated tendons.
Materials and Methods
Bone marrow was harvested 3 weeks before tendon surgery, and the bone marrow stromal cell-seeded patch was fabricated immediately before surgery. At surgery, the second and fifth flexor digitorum profundus tendons from one dog forepaw were transected and repaired. Cell patches were placed between lacerated tendon ends, and then the tendon surface was treated with the lubricating mixture. At postoperative Day 5, therapy was started on the surgically treated paw which continued until the designated survival times. After euthanasia of the animal and harvesting of the tendons, the repaired tendons were evaluated mechanically, biochemically, and histologically. The study was approved by our Institutional Animal Care and Use Committee.
Fabrication of the Cell Patch
Dogs were sorted into three survival groups: 10 days, 21 days, or 42 days, with 20 dogs per group. The flexor digitorum profundus tendons of the second and fifth digits in one paw from each dog were sharply transected at the Zone II-D level  and repaired with a modified Pennington technique  using a 4-0 FiberWire® suture (Arthrex Inc, Naples, FL, USA) reinforced with a simple running suture of 6-0 Prolene™ (Ethicon Inc, Somerville, NJ, USA).
After tendon repair, the cell-augmented repaired tendon was treated with cd-HA-lubricin as previously described . Briefly, cd-HA-lubricin was formulated with 1% sodium HA (95%, molecular weight 1.5 × 106 Da; Acros Thermo Fisher Scientific, Rockford, IL, USA), 10% gelatin (Sigma, St Louis, MO, USA), 1% 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (Sigma), 1% N-hydroxysuccinimide (Pierce, Thermo Fisher Scientific, Rockford, IL USA), 0.1 mol/L 2-(N-morpholino) ethanesulfonic acid (MES) (pH 6.0), and 260 μg/mL bovine lubricin. After tendon repair, a radial neurectomy was performed through a lateral humeral incision to denervate the elbow and wrist extensors so that the dogs could not bear weight on the surgically treated limb . After surgery, the surgically treated paw was maintained in front of the chest with a custom sling for 5 days; then, synergistic motion rehabilitation was performed daily until euthanasia .
Less than 2 mm between tendon and surrounding tissue, easy to separate
3–4 mm can separate
More than 5 mm, difficult to separate
Sequences of polymerase chain reaction primers and the length of amplicons
Cell Tracking and Histology
Two repaired tendons from each group were used to track the implanted bone marrow stromal cells on postoperative Days 10, 21, and 42 to assess cell viability and distinguish bone marrow stromal cells from the host tenocytes, based on a previous report . Briefly, bone marrow stromal cells were labeled with PKH26 red fluorescent cell linker (Sigma) before seeding in the gel patch. Labeled cell patches then were transplanted into repaired tendon ends as described previously. After euthanasia of the dogs, tendons were harvested immediately, observed with a confocal microscope (LSM310; Zeiss, Oberkochen, Germany), fixed with 10% formalin, and embedded in paraffin. Tendon sections (7 µm thick) were cut and stained with hematoxylin and eosin. These sections were qualitatively evaluated by light microscopy.
The tendon rupture rate and adhesion score were analyzed with Fisher’s exact test. Since the mechanical testing and gene expression data were normally distributed, the data were reported as mean (SD); treated and untreated groups were compared with a paired t-test at each time because different digits in one dog were being compared. One-way ANOVA was used to test differences among the three times because comparisons were made among different dogs. Probability values less than 0.05 were considered statistically significant. JMP® software (SAS Institute Inc, Cary, NC, USA) was used for data analysis.
Numbers of adhesions in control and treated groups
Day 10 (n = 18)
Day 21 (n = 18)
Day 42 (n = 16)
Total (n = 52)
Day 10 (n = 16)
Day 21 (n = 17)
Day 42 (n = 16)
Total (n = 49)
Two major complications often are encountered after flexor tendon repair: adhesion formation and repair rupture [24, 30]. These complications are highly related to the flexor tendon’s low capacity for intrinsic healing because of its hypocellular and hypovascular nature [9, 32]. Delayed intrinsic healing not only increases the risk of repair rupture, but also provokes extrinsic healing, leading to adhesion formation. Although postoperative mobilization, especially using an active motion protocol, effectively decreases adhesions, it also may promote gap formation at the repair site, which further impairs tendon healing. We therefore sought to evaluate the effects of the combination of the surface treatment with lubricating molecules to prevent adhesion formation and stem cell-based therapy to enhance tendon intrinsic healing on functional outcomes after flexor tendon repair using a canine model.
Our study has several limitations. First, we used female dogs only to minimize the confounding factors caused by sex difference. Therefore, the findings might not generalize to both genders, and future studies may evaluate the gender effect of the treatments. Second, we did not test either the cell-based therapy or surface modification alone. However, some studies have shown that bone marrow stromal cells do increase tendon healing capacity [8, 10, 15], and others have shown that surface modification decreases tendon adhesions [14, 40]. Therefore, the purposes of our study were to investigate the effects of combining these treatments on postoperative adhesion formation and tendon healing. The surface modification procedures were not blinded because a placebo substance was not used. Third, the viable transplanted cells were not counted. Although qualitative images showed cell density decreasing with time, we do not know the survival rate of implanted cells. Fourth, because of difficulty with accurate measurement of the cross-sectional area of the repaired tendon with adhesions, the breaking strength values could not be normalized. Fifth, the endpoint was 42 days. Longer followup may be needed. Finally, 2 to 3 weeks are needed for the bone marrow stromal cell preparation, which limits clinical applicability for acute tendon injuries. However, delayed primary repairs are commonly performed, and a delay of as much as 3 weeks is not associated with worse results .
Cell-based therapy has been investigated as a means to accelerate tendon healing. Embryonic stem cells, induced pluripotent stem cells, and adult mesenchymal stem cells [4, 16, 34] have been studied for their differentiation potential, function, regenerative capacity, and side effects. Adult mesenchymal stem cells most commonly are used clinically and experimentally. Although the adult mesenchymal stem cells can be derived from bone marrow, adipose, muscle, and other tissue, the most common use in tendon healing to date is the use of bone marrow stromal cells to treat equine flexor tendon injures [3, 5, 19, 22, 23]. Several studies have shown that bone marrow stromal cells increase repaired flexor tendon strength and stiffness in a canine ex vivo model [6, 13, 36]. Manning et al.  used adipose mesenchymal stem cells to treat flexor tendon lacerations using a canine in vivo model, and cells remained viable at the repair site for 9 days. However, no mechanical data such as digit function or repair strength were reported in that study. They did not find any difference in gene expression comparing treated and untreated repairs, which was similar to our findings. The lack of difference in gene expression may relate to the fact that the sample they used and the sample that we used for analysis was a segment of the tendon in which transplanted cells and local resident cells were admixed.
Several growth factors have been used to enhance cell-based therapy for tendon regeneration, including GDF-5, GDF-6, platelet-derived growth factor, and platelet-rich plasma [1, 6, 7]. Tan et al.  used GDF-5 to treat bone marrow stromal cells and reported that a 100-ng/mL treatment caused cells to increase collagen expression, upregulate tenogenic marker expression (including scleraxis and tenascin-C), and downregulate nontenogenic marker expression (including runt-related transcription factor 2 [Runx2] and sex-determining region Y-box 9 [Sox 9]). They concluded that GDF-5 induced bone marrow stromal cell tenogenic differentiation . Hayashi et al.  examined tendon healing in a tissue culture model and found that GDF-5 (100 ng/mL) promoted bone marrow stromal cell production and increased tendon intrinsic healing. These studies provided a strong rationale for using 100 ng/mL GDF-5 for stimulation of bone marrow stromal cells in the current in vivo flexor tendon healing model.
Augmentation of a cd-HA-lubricin-treated tendon repair with GDF-5 stimulated bone marrow stromal cells in a collagen patch delivered to the repair site at the time of surgery improved but did not completely correct the impaired healing previously noted with use of cd-HA-lubricin alone . The augmentation did not appear to result in any additional adhesions. Considering these results, we believe that cell-based repair therapy, coupled with methods to reduce tendon surface friction, has the potential to enhance tendon intrinsic healing. However, further studies are warranted to assess the use of different cell sources, different cell doses, different carrier vehicles, and different cytokines, in various combinations, to achieve the ultimate goal of strong tendon healing without adhesions.
We thank Masanori Hayashi MD, PhD and Kumar Chokalingam PhD, Department of Orthopedic Surgery, Mayo Clinic for their assistance with surgical procedures and RT-PCR analysis.