Effect of Poloxamer 407 as a carrier vehicle on rotator cuff healing in a rat model
© Kim et al.; licensee BioMed Central Ltd. 2014
Received: 6 September 2013
Accepted: 20 February 2014
Published: 1 March 2014
In vivo studies showing the effects of biologic healing-promoting factors on tendon-to-bone healing after rotator cuff repair have focused only on biologic healing-promoting factors and have not taken into consideration the effect of the carrier vehicle. Moreover, most studies have evaluated the healing process using different carrier vehicles, each of which may have specific effects on tendon healing. This may explain the large variability seen in outcomes in research studies. In this study, we investigated the effects of Poloxamer 407 as a carrier vehicle on rotator cuff healing at the repair site and compared it with those of a collagen sponge, which is a commonly used carrier vehicle.
Fifty-seven adult male Sprague–Dawley rats underwent detachment and immediate repair of the bilateral supraspinatus tendons. Rats were randomly assigned to three groups: repair only, repair with collagen sponge, and repair with Poloxamer 407. The repairs were evaluated at 1, 2, 4, and 8 weeks after surgery with histological analysis and biomechanical testing.
At 4 weeks, more cellular organization, a greater number of collagen fibers, and increased maturity of collagen fibers were observed in the repair with Poloxamer 407 group than in the other groups. The repair with collagen sponge group had delayed development and collagen fiber maturation. Significant differences in the biomechanical properties were found between groups at 4 weeks. Stiffness in the case of the repair with Poloxamer 407 group was significantly higher than that in the repair with collagen sponge group. The modulus was significantly lower in the repair with collagen sponge group than in the repair only group. However, the use of Poloxamer 407 versus the collagen sponge did not significantly affect the biomechanical properties of the repaired tendons at 8 weeks.
Carrier vehicles may have differing effects at the early stages of rotator cuff healing. The use of Poloxamer 407 as a carrier vehicle may be useful for promoting the early stages of healing and for maintaining the initial biomechanical properties of the repaired rotator cuff tendon.
Most rotator cuff tears are treated surgically; however, recent research has shown high re-rupture rates of 13%–94% [1–3]. Rotator cuff repair often requires tendon-to-bone healing, and previous studies have noted that failure of rotator cuff repair is a challenging clinical problem [3–5]. Several studies have focused on methods to enhance the healing process after rotator cuff repair, and biological approaches have improved tendon healing in animal models [6–10]. The healing capacity of various factors, including growth factors (i.e., vascular endothelial growth factor and platelet-derived growth factor), mesenchymal stem cells, platelet-rich plasma, and bone morphogenetic proteins (BMPs), has been tested in animals [11, 12].
In animal studies, these biologic healing-promoting factors have been delivered through various carrier vehicles such as type I collagen sponge, fibrin glue, and hyaluronan sponge to increase their retention at the repair site, localize tissue repair, and, in some instances, provide a scaffold for cellular ingrowth [10, 13]. However, most studies have focused only on the biologic healing-promoting factors and not taken into consideration the effect of the carrier vehicle. In addition, studies have evaluated the healing process using different carrier vehicles, each of which may have specific effects on tendon healing and lead to a large variability in outcomes.
Poloxamer 407 is a triblock polymer that exhibits concentration-dependent reverse thermal gelation, in which aqueous solutions are liquid at low temperatures (-10°C) and form semisolid gels at body temperature. This characteristic is potentially useful for sustained release of injectable drugs. Local injectable Poloxamer 407 formulations have been shown to promote slow and sustained drug release directly at the site of interest [14, 15]. Studies have also demonstrated that Poloxamer 407 can carry a sufficient amount of drug and shows good tolerability, biodegradability, non-toxicity, water solubility, and controlled release . Animal studies also support the applications of Poloxamer 407 as a reversible thermosensitive microvascular clamp for surgery  and as an antiadhesive material for laminectomy procedures .
We aimed to investigate the effects of a carrier vehicle on rotator cuff healing at the repair site by using Poloxamer 407 (polymer) and a collagen sponge, both of which are commonly used as carrier vehicles. We evaluated rotator cuff healing rates at the tendon-to-bone repair site after surgical repair with either collagen sponge or Poloxamer 407 and examined whether tendon-to-bone remodeling after surgical repair of the supraspinatus tendon was affected by local application of a collagen sponge or Poloxamer 407. We hypothesized that each would have different effects on tendon-to-bone healing, biomechanical strength, and remodeling of the tendon repair. We also hypothesized that Poloxamer 407 (polymer) would have a better effect on early tendon-to-bone healing process than collagen sponge.
Bilateral shoulders of 57 adult male Sprague–Dawley rats (mean body weight, 457 g; range, 410–500 g) were randomly allocated to three groups: 19 rats (38 shoulders) underwent tendon-to-bone repair only, 19 (38 shoulders) underwent tendon-to-bone repair with application of the collagen sponge carrier vehicle to the repair site (repair + CS), and 19 (38 shoulders) underwent tendon-to-bone repair with application of the Poloxamer 407 carrier vehicle to the repair site (repair + P407). Principles of laboratory animal care (NIH publication no. 86–23, revised 1985) were followed throughout the study, and approval for the study was obtained by the institutional animal care and use committee (#KIACUC-09-0016).
Specimens were fixed overnight in 4% paraformaldehyde and decalcified in 14% ethylenediamine tetraacetic acid. Specimens were embedded in paraffin, sectioned at 5 μm, and dried for 1 h at 60°C, after which they were stained with hematoxylin and eosin (cell morphology, inflammation, cellularity, vascular proliferation, and fibroblast proliferation), toluidine blue (fibrocartilage formation and tidemark), and Picrosirius red (collagen organization). Picrosirius red staining and examination with monochromatic polarized microscopy was used for analyzing collagen organization at the tendon-to-bone repair site . We evaluated these histologic parameters with the grading criteria used for the tendon-to-bone maturation scoring system described by Ide et al. . Immunohistochemical analysis of angiogenesis and capillary formation was performed on 5-μm sections cut from formalin-fixed paraffin-embedded tissue that was stained with rabbit monoclonal anti-CD34 (EP373Y; 1:50, Abcam, Cambridge, MA, USA). The visualization system used was the BenchMark XT (Ventana, Tucson, AZ, USA) with heat-induced epitope retrieval (CC1 solution, Ventana, Tucson, AZ, USA). Sections were incubated with primary antibodies for 32 min at 37°C. Staining was detected with the ultraView Universal DAB detection kit (Ventana). Microvessels were counted in 10 separate × 400 magnification fields. The three fields with the highest angiogenesis out of the 10 areas were taken as the microvessel density (MVD). The lower third MVD was considered mild, the middle third as moderate, and the highest as severe according to the relative comparison of all specimens. Three to five sections per group were evaluated based on section quality. The sections were evaluated by an independent experienced blinded pathologist.
Biomechanical results were compared using a one-way ANOVA for groups (repair only, repair + CS, and repair + P407) and time (2, 4, and 8 weeks) followed by Tukey's multiple comparison test. Primary endpoints included maximum load, maximum stress, modulus, and stiffness in each group at each time point. Data are presented as means ± standard deviations. p < 0.05 was considered statistically significant. Histology-based results were qualitative in nature and were not statistically compared.
Clinical studies on rotator cuff repair have demonstrated a high rate of incomplete healing and gap formation between tendon and bone [1, 2, 23, 24]. Re-tears and failure with continuity after rotator cuff repair occur in the early postoperative period [25, 26], and the tendon may re-rupture from the repair site because of its inability to control the loads placed on it during the early postoperative period . Therefore, enhancing tissue regeneration and maintaining repair strength during the early period after rotator cuff repair may decrease the re-tear or failure rates.
Several studies have suggested that biologic healing-promoting factors improve healing after rotator cuff tendon-to-bone repair, such as cartilage-derived morphogenetic protein 2 , osteoinductive growth factors (BMPs 2–7), transforming growth factor-β 1–3, and fibroblast growth factor (FGF)  and FGF-2 . Different carrier vehicles have been used in these studies, including collagen sponge, fibrin glue/sealant, and hyaluronan sponge/paste, to increase local retention and slow the release of healing-promoting factors at the repair site. However, the influence of the various carrier vehicles on the rotator cuff tendon healing has not been reported, and little is known about the effect of the carrier vehicles used for delivery of healing-promoting factors on rotator cuff healing.
We evaluated the effect of two carrier vehicles on rotator cuff healing at the tendon-to-bone repair site: Poloxamer 407, the use of which has been extended to in vivo studies in various surgical specialties [14, 16, 18], and collagen sponge, which is a commonly used carrier vehicle. To assess the direct and measurable effect of these two carrier vehicles on rotator cuff tendon-to-bone healing, we applied a collagen sponge or Poloxamer 407 to the interface of the detached supraspinatus tendon and footprint in the bone rather than applying these carrier vehicles over the rotator cuff tendon-to-bone repair site.
Our histological and biomechanical results show that the initial tendon-to-bone healing process was affected by local administration of the carrier vehicle and that the healing process differed depending on the carrier vehicle used.
Among the biomechanical properties, stiffness of the repair + P407 group was significantly higher than that of the repair + CS group at 4 weeks. However, modulus and stiffness in the repair + CS group were significantly lower than those in the repair only or repair + P407 groups. These biomechanical results were consistent with histological findings. Development and maturity of collagen fibers were higher in the repair + P407 group than in the repair only and repair + CS groups at 2 and 4 weeks. The amount and maturity of collagen fibers were more distinct at 4 weeks in the repair + P407 group, and this was reflected by improved biomechanical properties. In the repair + CS group, fewer and less mature collagen fibers were observed than in the other groups even at 8 weeks. These data suggest that Poloxamer 407 exhibited an early healing process with respect to collagen fiber maturation compared with the collagen sponge. Based on our findings, we postulate that Poloxamer 407 as a carrier vehicle may be more useful to maintain the repair strength, particularly during the early stages of the healing period, than a collagen sponge. These initial increased material properties and healing process may decrease the risk of early re-tear failure after rotator cuff repair.
Our results confirmed our hypothesis that different carrier vehicles have different effects on the biomechanical properties and healing process of repaired rotator cuff tendons. Based on our histological and biomechanical findings, we suggest that the use of a carrier vehicle may delay or promote the early stage of tendon-to-bone healing and also affect the repair strength. The early stage of tendon-to-bone healing may be a critical period when re-tear might occur after rotator cuff repair.
The present study has some limitations. First, the surgical repair was performed for an acute rotator cuff tear rather than for degenerated and retracted tendon tears, which would have been more clinically relevant. Second, the use of a quadruped animal associated with a weight-bearing forelimb to model the human shoulder may be a limitation . Third, this study was limited to 8 weeks postoperatively; therefore, the durability of these results cannot be evaluated and should be addressed. Additionally, we evaluated only the carrier vehicles and did not evaluate their optimal amounts, interactions, or effects when used with biologic healing-promoting agents such as growth factors, cytokines, and stem cells.
The carrier vehicles used for healing-promoting agent retention have different effects on rat rotator cuff tendon-to-bone healing depending on the carrier vehicle used, suggesting that the use of a carrier vehicle alters tendon biology and mechanical strength, particularly during the early stage of healing. The use of Poloxamer 407 as a carrier vehicle may be useful for promoting the early stages of the healing process and for maintaining the initial biomechanical properties of repaired rotator cuff tendons. In the early healing stage, the biomechanical properties of the repaired tendon-to-bone insertion in the regenerated tissue of an in vivo rotator cuff were better with Poloxamer 407 compared to the collagen sponge, suggesting the probable feasibility of regenerating rotator cuffs using current tissue engineering techniques with this polymer.
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (2009–0065193).
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