Ann Thorac Surg 2000;70:824-828
© 2000 The Society of Thoracic Surgeons
Original articles: cardiovascular
Basic fibroblast growth factor may improve devascularized sternal healing
Atsushi Iwakura, MDa,
Yasuhiko Tabata, PhDb,
Kazunobu Nishimura, MDa,
Tatsuo Nakamura, MDb,
Yasuhiko Shimizu, MDb,
Masatoshi Fujita, MDc,
Masashi Komeda, MDa
a Department of Cardiovascular Surgery, Kyoto University Graduate School of Medicine, Kyoto, Japan
b Institute for Frontier Medical Sciences, Kyoto, Japan
c College of Medical Technology, Kyoto University, Kyoto, Japan
Address reprint requests to Dr Komeda, Department of Cardiovascular Surgery, Kyoto University, 54 Kawaharacho, Shogoin, Sakyo-ku, Kyoto, Japan 606–8507
e-mail: masakom{at}kuhp.kyoto-u.ac.jp
Presented at the Thirty-sixth Annual Meeting of The Society of Thoracic Surgeons, Fort Lauderdale, FL, Jan 31–Feb 2, 2000.
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Abstract
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Background. We have shown that a gelatin sheet incorporating basic fibroblast growth factor enhanced bone regeneration of the devascularized sternum. The purpose of this study was to determine if topical use of the gelatin sheet accelerated normal sternal regeneration and bone remodeling.
Methods. Thirty Wistar rats had median sternotomy and were divided into 3 groups: 10 had the bilateral internal thoracic arteries removed and basic fibroblast growth factor sheet applied on the sternum (group A), 10 had just the bilateral internal thoracic arteries removed (group B), and 10 had intact bilateral internal thoracic arteries (group C).
Results. Four weeks later the peristernal blood flow significantly increased and marked angiogenesis was seen around the sternum in group A. Histologically, the sternum was almost completely healed only in group A. In group A the bone mineral content was highest, but the bone mineral density was similar to that in other groups. The osteoclast index in group A was highest at the border zone of bone formation and remained high in regenerated bone.
Conclusions. The basic fibroblast growth factor sheet offset sternal ischemia and accelerated normal sternal bone regeneration and remodeling, not only by callus formation but also by callus resorption.
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Introduction
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Basic fibroblast growth factor (bFGF) is a potent mitogen regulating protein that induces proliferation of a variety of cells, including epithelial and mesenchymal, and that promotes the growth and regeneration of organs and tissues in vivo [1]. In osteogenic tissues, bFGF is produced by osteoblasts and stored in the bone matrix [2]. In experiments using bone and cartilage cells in culture, bFGF facilitated the proliferation of osteoblasts [3], chondrocytes [4], and periosteal cells [5]. In addition to these osteogenic potentials, bFGF also functions as an angiogenic factor [6].
Slow or poor healing of the sternum is one of the potential problems after sternotomy, and it is therefore a potential problem associated with heart surgery. Slow healing prolongs a patient’s hospital stay and increases health care costs considerably; it can also delay a patient’s return to work or social activities. Poor healing of the sternum often leads to deep sternal wound infection, a serious complication [7]. Previous studies [8, 9] have suggested the use of bilateral internal thoracic arteries (BITA) in sternal wound complications associated with coronary bypass surgery, because BITA provide the major blood supply to the sternum.
Recently, we have reported that the topical application of a gelatin sheet incorporating bFGF to the sternum after the removal of BITA accelerated sternal healing in rats [10]. In the present study, we examined whether topical use of a gelatin sheet incorporating bFGF accelerated normal sternal regeneration and bone remodeling.
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Material and methods
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Preparation of bFGF-incorporating gelatin hydrogel sheets
Gelatin with an isoelectric point of 4.9 was isolated from bovine bone collagen by an alkaline process using Ca(OH)2 (Nitta Gelatin Co, Osaka, Japan). Human recombinant bFGF with an isoelectric point of 9.6 was supplied from Kaken Pharmaceutical Co (Tokyo, Japan). Gelatin hydrogel sheets were made using the previously described process [11]. Sheets were freeze dried and then impregnated with an aqueous solution containing 100 µg of bFGF, to obtain gelatin hydrogels with incorporated bFGF. The prepared hydrogel sheets were rectangle-shaped (1 x 10 mm) and 0.7 mm thick. All experimental processes were conducted under sterile conditions.
Animal experiments
Thirty male Wister rats weighing between 300 and 400 g were orally intubated after receiving anesthesia with 99.5% ether and ventilated on a small volume-cycled animal ventilator (rodent ventilator model 683, Harvard Apparatus, Holliston, MA, USA). Anesthesia was maintained during the operation with 1% to 2% isoflurane. After a midline skin incision, with the animal in the supine position, the bilateral major pectoral muscles were divided from the junction of the sternum, and intercostal muscles on both sides of the sternum were exposed. A median sternotomy was carefully performed using a rotating saw (D-7200, AESCULAP, Tuttlingen, Germany) leaving part of the narrow sternum on both sides. The bleeding from the bone marrow was stopped with bone wax. The BITA were ligated using 6–0 polypropylene sutures near the take off and at the distal bifurcation, and the BITA with their beds were destroyed using an electrical coagulator. The 30 rats were randomly divided into three groups: group A (n = 10) had the BITA removed and a gelatin hydrogel sheet with incorporated bFGF was placed on the posterior table of the sternum before closing the sternum, group B (n = 10) had the BITA removed and the sternum was closed without using the sheet, and group C (n = 10) had the BITA left intact and sternal closure without the sheet. When placing the sheets that incorporated bFGF (100 µg/sheet) in the animals in Group A, the destroyed ITA beds were also covered by the sheet completely from the inside of the chest wall and it was stabilized with 6-0 polypropylene sutures on all sides to avoid movement behind the sternum. After applying positive end-expiratory pressure to fully inflate the lung, the sternum was parasternally closed with four interrupted braided polyester sutures. The muscle layer and skin were then carefully sutured with 4-0 nylon monofilaments. Streptomycin was administered intramuscularly just after skin closure (50 mg/rat). Rats were sacrificed by intravenous administration of a lethal dose of sodium pentobarbital 4 weeks (10 animals in each group) after surgery. The sternum was excised and fixed in 10% formaldehyde solution in PBS for 4 days to assess the extent of bone regeneration. All of the animal experiments were performed according to the institutional guidelines on animal experimentation of Kyoto University.
Measurement of peristernal blood flow
Peristernal blood flow (PBF, mL/min/100 g) at the capillary blood perfusion level was measured using a noncontact laser flowmeter (ALF21N, Advance Co, Tokyo, Japan) before median sternotomy, after closure of the sternum and 4 weeks after surgery. A beam of He-Ne laser light was directed through an optic fiber to a measuring probe with a diameter of 3.0 mm. The probe was placed over the intercostal muscles near the sternum, separated by 10 mm in a straight line, so that the area of measurement was about 5 mm in diameter and 1 mm in depth. The He-Ne beam was then changed to a diode laser (2 mW, 780 nm) to measure PBF, which was calculated on the basis of the Doppler shift. The probe included two optic fibers: one for laser illumination and the other for receiving reflected and dispersed light. Three readings for each measurement were recorded after a stable baseline had been obtained, and the three values were averaged.
Qualitative and quantitative analysis of the regenerated sternal formation
Bone regeneration of the sternum was assessed by dual energy roentgenogram absorptometry (DEXA) and histologic examinations. The bone mineral density (BMD) and the bone mineral content (BMC) of each sternum was measured with DEXA utilizing a bone mineral analyzer (Dichroma scan 600, Aloka Co, Tokyo, Japan) 4 weeks after surgery. The instrument was calibrated with a phantom of known mineral content. Each scan was performed at a speed of 20 mm/s, and the scanning length was 1 mm.
Analysis of periosteal sternal osteoclasts
Bone specimens were demineralized in 10 wt% EDTA solution at 4°C for 3 days, embedded in paraffin and sectioned at 10 µm thickness. The sections were obtained at the third, fourth and fifth intercostal spaces of the sternum and stained with hematoxylin-eosin 4 weeks after surgery. The histologic sections were viewed at a magnification of 40x with a light microscope, and the number of osteoclasts were counted to evaluate bone remodeling after surgery. Measurements were performed in ten squared-shaped areas (0.3 x 0.3 mm) including each completely regenerated bone tissue and the border zone between regenerated bone tissue and cartilage in each histologic section. Completely regenerated bone tissue was defined as new bone with bone marrow.
Five different sections were measured for each experimental group. The number of osteoclasts, characterized by basophilic cuboided cytoplasm and plural nuclei located adjacent to the bone surface, was counted in each area. The bone perimeter in each area was measured using an image analyzer (SP-1000, Olympus, Tokyo, Japan) and National Institutes of Health Image version 1.56 software under a light microscope, and the osteoclast index (the number of osteoclast/sternum perimeter in mm) was calculated [12]. The cell number per perimeter was calculated from the data of 15 independent areas.
Statistical analysis
All data were analyzed by one-way ANOVA to assess statistical significance among experimental groups. Experimental results were expressed as mean ± standard deviation. Results of the statistical analyses were regarded as significant when the p value was less than 0.05.
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Results
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Assessment of sternal angiogenesis
Figure 1 shows results of PBF just after removal of the BITA and 4 weeks after surgery in each group. Although PBF did not change after the median sternotomy alone (ie, intact BITA), PBF after ITA removal fell to 50.3% ± 6.7% of the preoperative level. The PBF 4 weeks after the surgery in group A, group B, and group C were 114.0% ± 12.3%, 77.5% ± 8.8%, and 98.8% ± 7.7% of the preoperative level, respectively. Significant differences were noted among the three groups (p < 0.01).
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Fig 1. The peristernal blood flow was measured by means of a noncontact laser Doppler flowmeter after removal of the bilateral internal thoracic arteries (BITA) and 4 weeks after surgical treatment as follows: In group A, a gelatin sheet containing 100 µg of basic fibroblast growth factor was applied after removal of the bilateral internal thoracic arteries (BITA). In group B, the BITA were removed, but no gelatin sheet was applied. In group C, the BITA were left intact..
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Histologic sections of the tissue around the sternum confirmed this increase in vascular number. There were more capillaries and arterioles (10 to 50 µm in diameter) around the sternum in group A than in groups B and C (Fig 2).
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Fig 2. Micrographs (hematoxylin-eosin stained, 20 x magnification) showing new vessels in the connective tissue around the sternum after surgical treatment in three study groups. (A) A gelatin sheet containing 100 µg of basic fibroblast growth factor was applied after removal of the bilateral internal thoracic arteries (BITA). (B) The BITA were removed, but no gelatin sheet was applied. (C)The BITA were left intact.
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Histologic assessment of bone formation
Figure 3 shows histologic sternum sections 4 weeks after surgery with the different procedures. Four weeks after surgery groups B and C had partial enchondral ossification around the original sternum (Figures 3B and 3C). Group A subjects, on the other hand, had almost completely healed sternums filled with regenerated bone tissue and bone marrow (Fig 3A). Each section of the regenerated sternum was much larger than that of the native sternum, because of enchondral ossification of the large costal cartilage next to the native sternum. Many osteoblasts in the active form were seen at the border zone between the regenerated cartilage and cancellous bone.
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Fig 3. Histologic cross-sections of regenerated sternum were obtained 4 weeks after surgical treatment as follows: (A) A gelatin sheet containing 100 µg of basic fibroblast growth factor was applied after removal of the bilateral internal thoracic arteries (BITA). (B) The BITA were removed, but no gelatin sheet was applied. (C)The BITA were left intact.
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Qualitative and quantitative analysis of regenerated sternal formation
The bone mineral content, as assessed by DEXA, was greatest in group A (70.1 ± 9.2 mg), less in group C (46.4 ± 8.9 mg) and slightly less in group B (42.7 ± 8.6 mg); differences were statistically significant (p < 0.01). These results are in good agreement with the photographs in Figure 3. However, the bone mineral density of the regenerated sternum was similar among 3 groups (group A: 50.0 ± 9.0 mg/mm2; group B, 49.0 ± 8.0 mg/mm2; group C, 42.0 ± 9.0 mg/mm2), suggesting normal bone histology in all groups (Fig 4).
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Fig 4. The bone mineral content and density was assessed by dual energy roentgenogram absorptometry after surgical treatment. In group A, a gelatin sheet containing 100 µg of basic fibroblast growth factor was applied after removal of the bilateral internal thoracic arteries (BITA). In group B, the BITA were removed, but no gelatin sheet was applied. In group C, the BITA were left intact.
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Periosteal sternal osteoclasts
At the border zone between the new bone and cartilage of the sternum during early stages of enchondral ossification, the osteoclast index in group A increased to more than twofold that in groups B and C (Fig 5). Contrary to the above findings, the osteoclast index in all groups was greater at the regenerated bone site than that at the border zone. The osteoclast index in group A, which was significantly higher than in group C (p < 0.05), tended to be larger among the three groups in the latter stages of enchondral ossification.
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Fig 5. The osteoclast index was calculated as the number of osteoclast/sternum perimeter in mm after surgical treatment. In group A, a gelatin sheet containing 100 µg of basic fibroblast growth factor was applied after removal of the bilateral internal thoracic arteries (BITA). In group B, the BITA were removed, but no gelatin sheet was applied. In group C, the BITA were left intact.
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Comment
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Sternal wound complications after a median sternotomy are uncommon, but once they develop, they are often grave and increase mortality and morbidity. Such complications have been associated with the patient’s preoperative state, including obesity, diabetes, and cardiac cachexia or with a history of various extended surgical procedures [13, 14]. The use of BITA has especially been believed to increase the chance of sternal complications. The removal of BITA after median sternotomy decreases sternal blood supply considerably. It has recently been reported that the harvesting of BITA in a skeletonized fashion may decrease the chance of sternal complications. Cohen and colleagues [15] showed that a skeletonized ITA did not reduce blood flow to the left side of the sternum, compared with a pedicled ITA as assessment with single emission computed tomography. However, there are wide variations of the anatomy of collateral vessels around the ITA. Jesus and colleagues [16] classified the collaterals in six types in a human anatomical study. In some types in which collaterals from the lateral chest wall to the ITA connect to the anterior wall of the ITA through a vertical common channel, the collaterals from the chest wall to sternum can be potentially preserved by careful skeletonization of ITA (ie, if the vertical common channel alone is served). However, if the collaterals connect to the side wall of the ITA (ie, no vertical common channel exists) even a careful skeletonization method can destroy the collaterals from the lateral chest wall to the sternum.
Thus, the skeletonization may be useful for some but not all patients who need or may potentially benefit from extended use of BITA for coronary revascularization. Our method, which uses bFGF for sternal healing, can increase the blood supply of the devascularized sternum and enhance the bone regeneration. As for the sternal blood supply, the PBF in group A, as measured with a noncontact laser Doppler flowmeter, significantly increased 4 weeks after the surgery compared with that in groups B and C. Our histologic study of blood vessels around the sternum showed an increase in vascular number, which suggested that the increased PBF in group A was associated with the angiogenic effect of bFGF. However, our models in which BITA were destroyed using an electrical coagulator may not be analogous to a patient with BITA harvest. Because BITA beds were destroyed posteriorly, branch vessels coursing from the artery anteriorly towards the chest wall probably survived. This may support our results that PBF after ITA removal decreased to about 50% of the preoperative level. In addition, the enhancement of bone regeneration that occurred only in group A produced almost completely healed sternums 4 weeks after the surgery. As shown in the results, however, the histologic features of a regenerated sternum in rats after surgery were much larger than those of the native sternum. To assess the bone quality of the large regenerated sternum, we measured the bone mineral density using DEXA. There were no significant differences among the 3 groups. This suggested that enhanced new bone regeneration had normal bone histologic quality, and the new bone would be healthy and strong enough to be clinically helpful. Furthermore, our method may have a significant long-term effect on bone strength and stability, because we have confirmed that the histologic features in group B were the same as those in group A 3 months after surgery.
It has been reported that bFGF has accelerated bone remodeling of a tibial fracture model in dogs not only by callus formation, as a result of mitogenic effects on periosteal cells, but also by osteoclastic callus resorption [12]. Recent in vitro studies have shown that differentiation of osteoclast progenitors into osteoclasts is required for the cell-to-cell contact of osteoblastic cells and osteoclast progenitors and that osteoclast function was activated by osteoblastic cells [18, 19]. Moreover, bFGF has been shown to develop expression of transforming growth factor-ß, which has been shown to increase the number of osteoclasts after repeated injection into the periosteum of the neonatal rat parietal bone [20]. In our results, the osteoclast index at the border zone between the new bone and the cartilage in group A was higher than in groups B and C, suggesting that group A sternums started osteoclastic resorption in the middle of active osteoblastic callus formation. Although the osteoclast index at the regenerated bone site markedly increased in groups B and C, the index in Group A also remained at a high level. We believe that our findings confirm the acceleration of bone remodeling due to enchondral ossification of the sternum. However, the present experimental study may be insufficient to elucidate the involvement of bFGF in bone remodeling, because bone turnover in rats is considerably different from that in humans. Further investigation using a larger animal model that has similar anatomical and physiologic features to humans is necessary.
In conclusion, use of a gelatin hydrogel sheet incorporating bFGF offset sternal ischemia and facilitated healing despite BITA removal after the sternotomy in our rat models, probably due to angiogenic and osteogenic effects of bFGF. Moreover, the acceleration of normal sternal healing was caused not only by osteoblastic callus formation but also by osteoclastic callus resorption. This method may help decrease the chance of sternal necrosis in high-risk patients and can potentially help extended use of BITA in coronary bypass surgery for patients in that category. [17]
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Abstract
Introduction
