We have been investigating the use of artificial organs made of biodegradable materials to induce regeneration of defective or missing host tissues.1–6 In a previous study, we focused on inducing regeneration of the trachea to produce an artificial prosthesis.4 The normal trachea is composed of a mucosal layer, a submucosal layer, tracheal glands, a smooth muscle layer, and the C-letter–shaped tracheal cartilage. The cartilage is one of the most important structures, because it maintains the integrity of the tracheal lumen. The first step in our attempt to produce an artificial trachea was to induce regeneration of the tracheal cartilage.
In our previous studies, we have reported that slow release of bone morphogenetic protein 2 from a gelatin sponge implanted in a gap in the tracheal cartilage induced regeneration of the cartilage.5,6 However, regenerated cartilage was observed only at the edges of the stumps of each cartilage ring, and the gap was not filled with regenerated cartilage. According to the literature, basic fibroblast growth factor (b-FGF) promotes the regeneration of chondrocytes in articular cartilage defects in vivo.7–10 We therefore examined whether slow release of b-FGF from a gelatin sponge could induce the regeneration of tracheal cartilage.
Materials and Methods
Preparation of Gelatin Sponge Containing b-FGF
A 5% (by weight) aqueous solution of gelatin with an isoelectric point of 5.0 (Nitta Gelatin Co., Osaka, Japan), containing 0.05% by weight glutaraldehyde (Wako Pure Chemical Industries, Osaka, Japan), was cast into a Teflon mold, then stored at 4°C for 12 hours for completion of cross-linking reaction. The material was then immersed in glycine solution at 37°C for 1 hour to block any residual glutaraldehyde, washed with distilled water, freeze-dried, and finally sterilized by exposure to ethylene oxide gas. The gelatin sponge was trimmed into pieces measuring approximately 10 × 50 × 2 mm3 (Figure 1). Just before implantation, 100 μg aqueous b-FGF solution was dissolved in 0.3 ml saline, and the resulting solution was applied dropwise to the gelatin sponge. The sponge was then left to stand for 15 minutes to allow the b-FGF solution to soak in completely.
Fifteen hybrid beagle dogs aged 2 to 3 years and weighing 10 to 13 kg were used. After general anesthesia with 10 mg/kg ketamine and 5 mg/kg xylazine, with 2 μg/kg atropine sulfate added to control secretions intramuscularly, all of the dogs were intubated via the trachea, and their breathing was maintained by mechanical ventilation with a mixture of nitrous oxide, halothane, and oxygen. A 1-cm gap was made in the midventral portion of each of 10 consecutive cervical tracheal cartilages from rings 4 to 13 by resecting a portion of the rings. The tracheal mucosa was carefully preserved throughout the procedure (Figure 2). The gelatin sponge fit exactly into each gap. In the five dogs acting as the control group, the gap was left unfilled. In the five dogs acting as the gelatin group, a gelatin sponge with no added solution was implanted in the gap. In the five remaining dogs acting as the b-FGF group, a gelatin sponge slowly releasing 100 μg of b-FGF was implanted in the gap. The implanted gelatin sponge was fixed by 1-0 silk sutures to prevent dislodgement (Figure 3).
We killed one of the five dogs in each group by bolus injection of a fatal dose of pentobarbital at 1 month after implantation, and another one at 3 months (Table 1). The trachea was removed and examined macroscopically and microscopically. We first observed an overview of the resected trachea. We then made cross-sections of the middle portions of the implant sites to observe the internal lumen of the trachea. Then we made thin slice sections and stained them with hematoxylin and eosin (HE) and alcian blue (AB) for light microscopic examination. Immunohistological evaluation was performed as required using proliferating cell nuclear antigen (PCNA) for detection of proliferating cells and alpha-smooth muscle actin (SMA) for detection of myofibroblasts.
This experiment was carried out in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council (published by the National Academy Press, revised 1996).
The nine dogs allowed to survive are still alive with no problems, and the remainder survived with no problems until they were euthanatized as planned (Table 1).
In the control and gelatin groups, each tracheal cartilage gap was filled with soft granulation tissue at 1 and 3 months. In contrast, in the b-FGF group, the gap was filled with elastic hard tissue at 1 month. This elastic hard tissue connected the stumps of each cartilage ring (Figure 4a), but at this stage no cartilage regeneration was evident in the gap. At 3 months in the b-FGF group, however, the gap was filled with regenerated cartilage, and the regenerated cartilage connected the stumps of each cartilage ring (Figure 4b).
In the control group, the shortest linear distances between host cartilage stumps were 5.0 mm and 3.0 mm at 1 and 3 months, respectively, after implantation (Figure 5). The gap had shrunk in comparison with the original length of 10 mm. At both 1 and 3 months, the submucosal layer in the gap was approximately 1.3 mm thick and thicker than that of normal portions. The submucosal layer consisted of dense collagen bundles and fibroblasts, and connected the host cartilage stumps at 1 and 3 months. No regenerated cartilage was observed at the implant site, and the cutting surfaces of the host cartilage stumps were covered with perichondrium at 1 and 3 months (Figure 5). PCNA-positive myofibroblasts were not observed.
In the gelatin group, the shortest linear distance between host cartilage stumps at 1 month was 1.0 mm, and it had shrunk compared with the original length of 10 mm (Figure 6). No regenerated cartilage was observed, and the submucosal layer, which contained dense collagen bundles, was 2.2 mm thick. No PCNA-positive myofibroblasts were observed in the gap.
In the b-FGF group, the shortest linear distances between host cartilage stumps were 7.0 and 9.0 mm at 1 and 3 months, respectively, and the gap had therefore been maintained at close to its original length of 10 mm. The regenerated fibrous cartilage contained abundant collagen fibers but little hyaluronic acid-positive matrix, unlike the hyaline cartilage of native tracheal cartilage. At 1 month, it stained positively with AB, had scant collagen fibers, and was observed at the edges of the host cartilage stumps (Figures 7a and 7b). The regenerated cartilage showed overgrowth with a concentric pattern from the perichondrium of the host cartilage stumps and was covered with perichondrium (Figure 7c). However, the gap was not totally filled with regenerated cartilage. The residual gap in the middle portion of the implant site was filled with granulation tissue and collagen bundles. PCNA-positive myofibroblasts were observed in the granulation tissue in the gap between the regenerated fibrous cartilage (Figure 8; Top, Bottom). At 3 months, fibrous cartilage had regenerated in the total length of the gap and had bridged the host cartilage stumps (Figure 9; Top). The regenerated cartilage in the gap was covered with perichondrium, which was continuous from one side to the other of the perichondrium of the host cartilage. The regenerated cartilage at 3 months was defined as not hyaline cartilage but fibrous cartilage (Figure 9; Bottom). No PCNA- positive myofibroblasts were detected at the implant site.
By using the slow release of b-FGF from a gelatin sponge, we succeeded in inducing the regeneration of cartilage that filled the gap in canine tracheal cartilage defects and bridged the cartilage stumps firmly at 3 months after implantation. Even macroscopically, it was evident that regenerated cartilage connected the stumps of each cartilage ring at 3 months. Although not as straight as the host cartilage, the regenerated cartilage bridged the gap between the host cartilage stumps in a slightly meandering manner. However, no cartilage regeneration was evident at 1 month because the regenerated cartilage was present only at the edges of the host cartilage stumps. The regenerated cartilage was covered with perichondrium, which grew from the perichondrium of the host cartilage. Perichondrium is known to have chondrogenic potential11 and to be the only possible source that can regenerate cartilage at defect sites in adults.12 It is well known that b-FGF is expressed in the process of cartilage regeneration.12 Therefore, we can speculate that the cartilage regenerated in our study originated from perichondrium, the regeneration of which was stimulated by the slow release of b-FGF. Moreover, even at 1 month in the b-FGF group, regenerated cartilage was observed at the edges of the host cartilage stumps, and this regenerated cartilage was also covered with perichondrium. Therefore, we speculated that b-FGF, which was released slowly from the gelatin sponge, recruited the chondrogenic potential of the perichondrium to stimulate the regeneration of chondrocytes.
Moreover, the regeneration of cartilage and the generation of granulation tissue containing proliferating myofibroblasts in dense collagen bundles maintained the distance between host cartilage stumps, and this aided the process of cartilage regeneration. Maintenance of the distance between the stumps at 9.0 mm at 3 months in the b-FGF group meant that the rate of cartilage regeneration was exceeded the rate of shrinkage of the cartilage gap. Therefore, the distances between the stumps were maintained at 7.0 to 9.0 mm in the b-FGF group, compared with 1.0 to 5.0 mm in the other groups. Especially in the gelatin group, the distance between the stumps had shrunk severely (to 1 mm) at 1 month. In addition, the submucosal layer in the gelatin group was thicker than that in the control group at 1 month. This result suggests that implantation of gelatin alone can accelerate the shrinkage process in the gap and trigger a foreign body reaction.5,13,14 Gelatin is biodegradable, and in this study, it was used for slow release of b-FGF. Because of the short half-life of b-FGF in the body, it cannot be expected to efficiently induce tracheal cartilage regeneration if administered in solution form. b-FGF was ionically combined with the gelatin and then released as a result of degradation of the gelatin. It has been reported that b-FGF combined with gelatin is released slowly for 15 days.15 Therefore, b-FGF was not released from the implanted gelatin which had been absorbed at 1 and 3 months after the implantation. We speculate that b-FGF released for the initial 15 days acted on the host perichondrium, which was stimulated to regenerate chondrocytes gradually 1 to 3 months after implantation of the gelatin.
In the b-FGF group, PCNA-positive myofibroblasts were observed at 1 month but not at 3 months. Myofibroblasts appear during the process of wound healing and play an important role in wound contraction.16 They have the morphologic characteristics of both smooth muscle and fibroblasts. Generally, myofibroblasts disappear selectively through apoptosis as the regeneration process progresses.17 The reason for the absence of myofibroblasts in the control and gelatin groups under microscopic examination using SMA staining is not clearly understood. However, we speculated that the wound repair process would have been finished by 1 month after implantation in these groups and that the myofibroblasts would therefore have disappeared within 1 month. However, myofibroblasts were clearly observed and were proliferating actively at 1 month in the b-FGF group, as shown by positive staining for PCNA. We speculated that this result was caused by the effect of the slow release of b-FGF from the gelatin sponge.
The regenerated cartilage was not hyaline but fibrous cartilage. This inconsistency would cause serious problems in articular cartilage, but not in tracheal cartilage. In general, fibrous cartilage has less mechanical strength than hyaline cartilage in its response to loading. However, unlike articular cartilage (for example, in the knee joint), tracheal cartilage does not need to endure continuous loading. Tracheal cartilage needs only to endure short-duration loading during coughing, or static loading during normal respiration. Therefore, we consider that although regeneration of hyaline cartilage would be ideal, regeneration of fibrous cartilage is enough to maintain the internal lumen of the trachea.
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