The prognosis of patients with emphysema is extremely poor.1,2 The surgical treatments for this disease include lung volume reduction surgery (LVRS) and lung transplantation. The indications for LVRS are limited, and lung function generally declines slowly after surgery.3,4 With lung transplantation, there are many difficulties related to graft donation, such as immunosuppression and bronchiolitis obliterans syndrome.5 However, if reconstruction of the emphysematous lung tissue toward a normal situation were possible, it could provide a promising new avenue for the treatment for emphysema.
Basic fibroblast growth factor (bFGF) was originally discovered as a growth factor in fibroblast cells. It has a variety of activities, and its specificity is low. During the fetal stage, bFGF promotes the induction of alveoli and vasculature in the lung.6,7 Furthermore, it has been reported that multi-potential mesenchymal cells exist in many adult tissues.8 A recent study has demonstrated that tracheal cartilage is regenerated by slow release of bone morphogenetic protein or bFGF from bioabsorbable gelatin sponge.9–12 In the present experiment, emphysematous lung tissue was reconstructed using bFGF slowly released from bioabsorbable gelatin microspheres (MS) delivered into the pulmonary microvasculature.
Materials and Methods
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, and published by the National Academy Press, revised 1996 (http://nap.edu/catalog/5140.html) and the “Guide for the Care and Use of Experimental Animals” prepared by Kagawa University (1999). The present study design was approved by the “Conference for animal experiments at Kagawa University.”
Development of a Canine Pulmonary Emphysema Model
Twenty hybrid beagle dogs (body weight 10–11 kg, mean body weight 10.3 kg) were used. The animals were divided into four groups: control group (n = 5), emphysema group (n = 5), bFGF-releasing microsphere group (FGF-MS, n = 5), and microsphere group (MS, n = 5). A canine pulmonary emphysema model was developed as described previously.13 Briefly, porcine pancreatic elastase [(PPE), 350 U/kg body weight; Elastin Products Co., Owensville, MO] was dissolved in double-distilled water (1 ml/kg body weight). The fifteen dogs in the experimental groups were anesthetized by intramuscular injection of xylazine (Selactal, 5.0 mg/kg; Byer Ltd., Tokyo, Japan) and ketamine (Ketalar, 125 mg/kg; Sankyo Co. Ltd., Tokyo, Japan). After introducing a bronchoscope (Olympus: 1T40, Tokyo, Japan) into the bronchi, a catheter was inserted into the left lingular bronchus through the channel of the bronchoscope under mechanical ventilation. The PPE solution was then injected selectively into the lingual segment.
Preparation of bFGF-incorporated Gelatin Hydrogel Microspheres
Gelatin microspheres were prepared by glutaraldehyde cross-linking as reported previously.14 Briefly, gelatin with an isoelectric point of 4.9 was isolated from bovine bone collagen by an alkaline process (Nitta Gelatin Inc., Osaka, Japan). An aqueous solution of human recombinant bFGF with an isoelectric point of 9.6 was obtained from Kaken Pharmaceutical Co. Ltd., Tokyo, Japan. Ten microliters of a 10% aqueous solution of gelatin preheated to 40°C was dribbled into 350 ml of olive oil with stirring at 420 rpm for 10 min at 40°C. The emulsion was then cooled in crushed ice and stirred at 420 rpm for 30 min. Acetone (100 ml, 4°C) was then added to the emulsion and stirring was continued for 60 min. The resulting microspheres were washed three times with acetone and recovered by centrifugation (5,000 rpm, at 4°C, 5 min). The microspheres were filtered through sieves with a pore size of 125 and 75 μm, and dried in air at 4°C to obtain noncross-linked gelatin microspheres 75–125 μm in diameter. The noncross-linked gelatin microspheres (20 mg) were placed in 0.1% Tween 80 aqueous solution containing 0.13% glutaraldehyde, and cross-linking was allowed to proceed at 4°C for 24 h. After collection by centrifugation (5,000 rpm, 5 min), the microspheres were stirred in 20 ml of 100 mM glycine solution for 1 h. The resulting microspheres were washed with double-distilled water, recovered by centrifugation (5,000 rpm, 4°C, 5 min) and freeze-dried (Figure 1). bFGF was incorporated into the gelatin microspheres by dropping 1 mg/ml bFGF solution (100 μl) on 10 mg of freeze-dried gelatin microspheres. The bFGF-incorporated gelatin microspheres were suspended in saline (5 ml) before injection (Figure 2). In accordance with a previous study, it was recognized that the bFGF-incorporating gelatin microspheres had slowly released bFGF for about 2 weeks during the process of microsphere degradation in vivo.14,15
All the dogs were anesthetized by inhalation of sevoflurane, oxygen and dinitrogen monoxide, and then intubated for mechanical ventilation after intramuscular injection of xylazine and ketamine.
For the preliminary experiment, we developed the MS- control group (n = 3) to investigate the effect of microvessel embolization due to MS incorporation. In this group, a suspension of 100 mg gelatin microspheres without bFGF was injected into the right atrium with a Swan-Ganz catheter (TC-504, Nihon Koden, Toyko, Japan) to reach the total lung micro-arteries via the right femoral vein. Total lung administration was done to allow recognition of the systemic effects of gelatin microsphere as well as localized effects. Injection was performed with continuous monitoring of vital signs for immediate effects after injection. Additionally, arterial blood gas analysis was performed 1, 2, and 4 h later under mechanical ventilation with the breathing frequency set at 10 breath/min, tidal volume at 20 ml/kg, and fraction of inspired oxygen 1.0. At 1, 2, and 4 weeks after injection in each dog, histological evaluation was done by biopsy of the treated lung, and functional evaluation by arterial blood gas analysis.
Administration of bFGF-incorporated Gelatin Microsphere Suspension
In the control group (n = 5), untreated model dogs underwent lung biopsy of the lingual segment via a fourth intercostal thoracotomy. In the emphysema group (n = 5), PPE-induced emphysema model dogs underwent lung biopsy for confirmation of the emphysematous change.
In the experimental groups, 10 PPE-induced emphysema model dogs were used. The left inferior truncus of the pulmonary artery was clamped through a left thoracotomy, and the suspension of bFGF-incorporated gelatin microspheres (n = 5) or a suspension of gelatin microspheres alone (n = 5) was injected into the left lingual segmental artery with a 27G needle (Nipro Co., Osaka, Japan). In the previous examination, embolization of small arterioles by MS was confirmed histologically by biopsy just after infusion of MS (Figure 3). In the FGF-MS group, bFGF-positive cells were observed around small arterioles 1 week after injection by immunohistochemical examination to examine the cellular uptake of bFGF (Figure 4).
Four weeks after injection, the treated lung and normal lung were observed histologically. The tissue specimens were stained with hematoxylin-eosin (HE), and for immunohistological analysis with a polyclonal rabbit antihuman antibody against factor VIII-related antigen, which is specific for the endothelial cells of blood vessels.16 The mean linear intercept (Lm), the standard technique for measuring alveolar surface area, was calculated for normal lungs, emphysematous lungs, and treated lungs to assess the degree of emphysematous change.17 In addition, the tissue vascularity was assessed by counting each individual microvessel identified by the HE staining and immunohistochemical staining for factor VIII, as the average counts of 20 areas under light microscopy at ×200 magnification. The microvessel count was performed by a single investigator under the supervision of the senior author.
Results of Preliminary Experiment
No immediate effect was observed after injection of 100 mg microspheres by continuous monitoring of vital signs and blood gas analysis. Injected gelatin microsphere remained in the small arterioles at 1 wk. Proliferation of fibroblasts was observed around the microspheres, without evidence of necrosis, bleeding or edema. At 2 and 4 wk, the injected microspheres were not detectable, and only fibroblast proliferation was observed. Arterial blood gas analysis at any time point showed no worsening of parameters in comparison with the preinjection status.
Results of Main Experiment
An open lung biopsy of the left lingula was done 4 wk after injection of the PPE, and this confirmed uniform development of emphysematous changes in the emphysema group (Figure 5A). The normal lung architecture was not evident in any field. In the control group, open lung biopsy of the left lingual segment was performed under general anesthesia, and this confirmed that the normal lung architecture was completely maintained (Figure 5B). The Lm for the bFGF-treated lungs and MS-treated lungs was each significantly smaller than in the emphysema group (p < 0.0001) (Table 1). The dilated alveoli were similar in size to those of the control group in the FGF-MS group and MS group. These changes were more evident in the FGF-MS group, where alveolus-like architecture with type-1 pneumocytes (100–200 μm in diameter) and dense microvascularization were observed around small pulmonary arteries. Although slight proliferation of fibroblasts was observed around the arteries, parenchymal thickening or fibrosis of alveoli was not detected (Figure 6).
Table 2 shows data for the microvessel count. The microvessel count in the bFGF-treated lungs was significantly higher than in the emphysema group (p = 0.0003). The count was higher in the FGF-MS group than in the MS group, but not to a significant degree. These effects were observed sporadically, and their distribution paralleled that of the small pulmonary arteries. Six to seven foci of reconstructed tissue were observed in low-power fields (×2). In the MS group, slight fibrotic change was observed around small pulmonary arteries, but no alveolus architecture was evident (Figure 7).
bFGF was originally discovered as a growth factor in fibroblast cells. It has a variety of activities, and its specificity is low. In this study, focusing on the induction of lung differentiation during the fetal stage and neovascularization,6,7 bFGF was used in an attempt to reconstruct lung tissue. Since cells do not exist independently in the body but interact with the surrounding milieu to proliferate and differentiate, it is necessary to create an environment in which they are likely to do so.18 It is difficult to reconstruct tissue using in vitro cell culture techniques, and therefore an in vivo environment is needed to achieve tissue reconstruction. A variety of growth factors such as fibroblast growth factor, hepatocyte growth factor and transforming growth factor are involved in differentiation of the lungs.19 During the fetal stage, bFGF is expressed in both epithelial and mesenchymal cells, and plays a crucial role in lung development through yet unknown mechanisms.
As growth factors are labile and their in vivo half-life is short, it is impossible to expect tissue reconstruction simply by administering them to a specific site.18 To maintain effective concentrations of growth factors for the required period, gelatin microspheres absorbable by the body were used to release bFGF in a sustained manner. For nearly a decade, bFGF-releasing gelatin microspheres have been applied in various fields of regenerative research,20–22 and their effect has been generally accepted. A previous study has clearly indicated that bFGF is released as a result of microsphere degradation in vivo,23 and can be controlled by changing the water content of the gelatin hydrogel. Moreover, the microspheres are completely degraded in the body, and thus do not elicit inflammatory or pharmacological responses in vivo. In fact, the results of the preliminary experiment in this study confirmed the tolerability of a 100-mg gelatin microsphere bolus injection via the pulmonary artery. In this manner, regeneration of tracheal cartilage has been achieved by slow release of bFGF from gelatin sponge.9–12 To allow bFGF to act on the emphysematous lung, gelatin microspheres must remain in the lung tissue and release bFGF in a sustained manner. To achieve this, embolization of vessels with gelatin microspheres is required. Since the target arterioles were 100–200 μm in diameter, gelatin microspheres 100 μm in diameter were used.
It is apparent that alveoli and micro-circulation are essential for pulmonary function. In this study, the FGF-MS embolized small-caliber pulmonary arteries, and might have released bFGF at the embolized site. In only the FGF-MS group, bFGF-positive cells were observed around small pulmonary arteries, so it is conceivable that bFGF was present for at least 1 week after embolization. Finally, an alveolus-like architecture, including type I pneumocytes and dense microvascularization, was observed after embolization with bFGF-MS. The microvessel count data corroborated the neovascularization effect of the bFGF-incorporating microspheres. An important finding was that addition of bFGF elicited the appearance of type I pneumocytes and induction of neovascularization.
Massaro and Massaro have reported that exogenous retinoic acid can induce alveolar regeneration in an experimental emphysema model using adult mice.24 A subsequent clinical trial of retinoic acid for the treatment of chronic obstructive pulmonary disease (COPD) demonstrated no improvement.25 Morino et al. reported that trans-bronchial application of bFGF induced an increase of pulmonary blood flow and led to recovery of pulmonary function in dogs with PPE-induced emphysema.26,27 In the present study, we found for the first time that transarterial injection of bFGF induced a reduction of alveolar space and neovascularization. The advantage of intra-arterial administration compared with other methods is its ease of administration and uniform delivery of the materials. However, emphysematous change was introduced in only a limited area, and no functional study was attempted. The effects of our method on pulmonary function remain to be determined in future studies.
Patients with diffuse emphysema are usually not good candidates for LVRS because there is no specific “target area” where emphysematous changes are more severe. If it becomes possible to induce micro-circulation and alveoli in such patients, then the use of this technique in combination with conventional LVRS may become a potentially effective novel treatment.
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