ARTICLE IN PRESS doi:10.1510/icvts.2008.197012
Interactive CardioVascular and Thoracic Surgery 8 (2009) 610–614 www.icvts.org
Institutional report - Experimental
Morphologic features of biocompatibility and neoangiogenesis onto a biodegradable tracheal prosthesis in an animal model Stefano Brizzolaa, Magda de Eguileorb, Tiziana Brevinic, Annalisa Grimaldib, Terenzio Congiub, Peter Neuenschwanderd , Fabio Acocellaa,* Department of Veterinary Clinical Science, Faculty of Veterinary Medicine, University of Milan, Via Celoria 10, 20133 Milano, Italy b Department of Structural and Functional Biology, University of Insubria, Varese, Italy c Laboratory of Biomedical Embryology, Center for Stem Cell Research, University of Milan, Italy d Department of Materials, Institute of Polymers, ETH, Zurich, Switzerland
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Received 23 October 2008; received in revised form 25 February 2009; accepted 27 February 2009
Abstract We evaluated a newly designed bioresorbable polymer (Degrapol䊛 ) tracheal prosthesis in an in-vivo angiogenesis-inducing animal model focusing on the specific tissue reaction, the neo-angiogenesis and also the eventual cathepsin B role during the polymer degradation. Fifteen rabbits were divided into three groups (2, 6 and 8 weeks) and our tube-shaped porous prosthesis was implanted using the common carotid artery and the internal jugular vein as vascular pedicle. Optical and electron microscopy, immunohistochemistry and immunocytochemistry were performed at the end of each period, showing cells and fibrils, in direct contact with the Degrapol䊛 scaffold, strongly increased with time. Blood vessel neoformation was visible with CD31 expression localized at the endothelial cells forming the neovascular walls. Over time many of them differentiate in muscle fibers as validated by the expression of a-smooth muscle actin (SMA). Few inflammatory cells, expressing CD14, were visible while most cells adopting a pronounced spreading phenotype showed a strong positivity for cathepsin B. We concluded that this bioresorbable polymer provided a good substrate for fibrous tissue deposition with an excellent degree of neo-angiogenesis. Also, cathepsin B seems to contribute to the polymer degradation and particularly to neovascularization by stimulating capillary-like tubular structures and cell proliferation. 䊚 2009 Published by European Association for Cardio-Thoracic Surgery. All rights reserved. Keywords: Tissue engineering; Neovascularization; Animal model; Degrapol䊛; Cathepsin B
1. Introduction Tracheal circumferential defects involving more than half of the tracheal wall still represent an unsolved problem w1x. Several studies have developed different methods to help repair cartilage and improve healing w2x but a suitable tracheal reconstruction or replacement has not been achieved yet w3–5x. Recently, the tissue engineering seems to be promising to generate scaffolds in a controlled fashion and predetermined shape that can mimic the organ in all its anatomical part w6–9x. We performed a preliminary morphologic study as described because, to our knowledge, there is no study or data available on the in vivo tubular implant made by Degrapol䊛 w10x. We therefore evaluated the behavior of a newly designed electrospun bioresorbable polymer (Degrapol䊛) tracheal prosthesis in an in-vivo angiogenesis-inducing animal model. This study has sought to characterize the cells invading the engineered scaffold, to determine the presence of neo-formed tissues basing it on a well-defined method of induced angiogenesis through a vascular flap w11x. Herein, we evaluate the presence and *Corresponding author. Tel.yfax: q39 02 50317809. E-mail address:
[email protected] (F. Acocella). 䊚 2009 Published by European Association for Cardio-Thoracic Surgery
the eventual action of cathepsin B during the process of polymer degradation. This cysteine protease has been found in different processes of angiogenesis and cell invasion either in physiological rather than pathological state. Optical and electron microscopy, and immunohistochemistry and immunocytochemistry have been used for this purpose. 2. Material and methods 2.1. Tracheal prosthesis The biodegradable prosthesis was made by three 3608 rings of 2.0 mm in length interposed by 0.5 mm of interring space. The tubular skeleton measured 6 mm outside and 5 mm inside diameter, respectively, and the entire length was 1 cm of electrospun Degrapol䊛, with 6y25 mm ⭋ fibers and 30y80 mm inter-fiber space. The molecular weight of the electrospun polymer used was of 60.765 daltons, measured by means of gel permeation chromatography (GPC). A cylinder made of Teflon䊛 was introduced inside the lumen to prevent tissue ingrowth. All the prostheses were submitted to low-temperature plasma sterilizers (STERRAD䊛).
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2.2. Animals Fifteen New Zealand white rabbits (4–4.5 kg) were divided into three groups of five animals each and were sacrificed after two (Group 1), six (Group 2) and eight (Group 3) weeks post-op, respectively. All animals were treated in accordance with the European Communities Council directive (86y609yEEC), to the laws and regulations on animal welfare enclosed in D.L.G.S. 116y92 and approved by the Italian Health Department. 2.3. Surgical procedure The scaffold was placed in the neck visceral space as previously described w11x. Briefly, during general anesthesia, the prosthesis was placed laterally to the isolated vascular structures in the jugular space (common carotid trunk and internal jugular vein). The vessels and the prosthesis were wrapped with a 2=3 cm latex sheet to avoid the surrounding tissue invasion. The envelope was closed using a 7-0 polypropylene suture (Prolene䊛, ETHICON). Antibiotic prophylaxis was instituted using Enrofloxacin (Baytril䊛, Bayer), 10 mgykg intramuscularly for 5 days. 2.4. Gross examination Immediately after euthanasia, the prosthesis and surrounding tissues were examined and gross findings recorded. 2.5. Hematoxylin and eosin, Mallory’s trichrome, sirius red staining Two transverse sections were collected at 1 cm from each extremity of the prosthesis. Serial sections (5 mm) were stained with hematoxylin and eosin (HE) and Mallory’s trichrome for general morphology. Sirius red staining (Dystar, Leverkusen) has been used for selective light microscopic demonstration of collagenic material. 2.6. Light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) Samples were fixed for 2 h in 0.1 M cacodylate buffer pH 7.2, containing 3% glutaraldehyde. After standard serial ethanol dehydration, specimens were embedded in an Epon-Araldite 812 mixture. Sections were obtained with a Reichert Ultracut S ultratome (Leica, Wien, Austria). Semithin sections were stained by conventional methods (crystal violet and basic fuchsin) according to Moore et al. (1960), and observed under a light microscope (Olympus, Tokyo, Japan). Images were acquired with a DS-5M-L1 digital camera system (Nikon, Tokyo, Japan). Thin sections were stained by uranyl acetate and lead citrate and observed with a Jeol 1010 EX electron microscope (Jeol, Tokyo, Japan). Samples for SEM were fixed and dehydrated as described above, treated with hexamethyldisilazane and mounted on polylysinated slides. Subsequently, the samples were air-dried and covered with a 9-nm gold film by flash evaporation of carbon in an Emitech K 250 sputter coater (Emitech, Baltimore, MD, USA). The samples were exam-
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ined with an SEM-FEG Philips XL-30 microscope (Philips, Eindhoven, Netherlands). 2.7. Indirect immunofluorescence staining Samples were embedded in polyfreeze tissue freezing medium (Polysciences, Eppelheim, Germany) and immediately frozen in liquid nitrogen. Cryosections (7 mm) were obtained with a Leica CM 1850 cryotome. The samples were incubated with primary antibodies to either anti-CD31 (early indicator during the endothelial differentiation), CD14 (specific marker for macrophage), a-smooth muscle actin (SMA) and cathepsin B (dilution 1:20) for 30 min. Then the sections were incubated with secondary antibodies for 30 min. The slides were mounted in Citifluor (Citifluor Ltd, London, UK) with coverslips and examined with an Olympus BH2 microscope (Olympus). In control samples, antibodies were omitted and sections were treated with BSA-containing PBS. 2.8. Enzymatic activity Unstained cryosections (7 mm), were incubated for 1 h at 37 8C in cathepsin B solution (Sigma) 10 Uyml in PBS to test the active biopolymer degradation due to the enzyme action. In control samples, cathepsin B solution was omitted and sections were treated only with PBS. Experiments were performed in triplicate. Cryosections were mounted in Citifluor (Citifluor Ltd) and slides were examined with an Olympus BH2 microscope (Olympus). Data were recorded with a DS-5M-L1 digital camera system (Nikon). 3. Results 3.1. Gross findings There were no complications or deaths during the operative procedures and in the post-op period. The prefabricated flaps were excised en-bloc with the vascular carrier and examined after opening the latex sheet immediately before being fixed. A thin fibro-connective tissue was clearly present around the tracheal scaffold in all the subjects (Fig. 1). The tissue invasion starts from the vascular axis and proceed time depending (G1™G3) in the anti-pedicle direction. We did not find a complete polymer resorption during the 8 weeks of implantation. 3.2. Light and electron microscopical observations 3.2.1. SEM analysis In all samples examined, cells and fibrils, in direct contact with the Degrapol䊛 scaffold, were visible (Fig. 2a–h). Initially (Group 1) few fibrillar material and few cells adhered on biomaterial and spanned within it (Fig. 2a–d). While fibrils bridging the construct surface reduced the interconnected pore network, cells, showing a migratory phenotype, glued on surfaces of polymer filaments hugging and pressing them (Fig. 2b, d). Cells and fibrillar material strongly increased with time and after 8 weeks (Group 3) the scaffold’s external surface was completely concealed (Fig. 2e) while the polymer
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Fig. 1. Macroscopic evaluation of the complex vascular pedicle-prosthesis for angiogenesis and tissue neoformation. The images clearly show a progressive time-dependent neo-vascular tissue growth onto and through the scaffold starting from the vascular axis.
fibers were incorporated in a dense fibrous tissue with interposed cells (Fig. 2f, h). 3.2.2. Optical analysis Histological examination showed an increased amount of cells and extracellular fibrillary matrix, starting from the external side of the samples toward the lumen (Fig. 3). The cellular invasion proceeded throughout the scaffold thickness (Fig. 4a–c) and in correspondence of the maximum tissue deposition blood vessel neoformation was visible (Fig. 4c). 3.2.3. TEM analysis In all samples examined, cells were wrapped up collagen fibrils as validated by ultrastructural evidence (Fig. 4d, e). The newly-synthesized and secreted collagen was organized into tightly packed fibrils regularly and parallely arranged, forming bundles (Fig. 4d, e). Fibroblast-like cells, characterized by a spreading phenotype, dug into the surrounding extracellular matrix that showed a clear sign of degradation (Fig. 4e). As cells proliferated and migrated centripetally filling pores of the scaffold, small muscle fibers (Fig. 4f) and
Fig. 2. Scanning electron microscope images showing (panels a–d) the Degrapol䊛 scaffold (d) partially covered by cells and fibrillar material. After 2 weeks the cells by their pseudopodia are in close contact with the biomaterial structures (arrowheads) bridging them (arrow). After 8 weeks (panels e–h) cells and fibrillar material increased in number and quantity up to conceal the Degrapol scaffold (d). Bars: a – 50 mm; b – 5 mm; c–h – 20 mm.
Fig. 3. Histological analysis. Scattered early evidence of fibrovascular invasion (left). Presence of large vascular spaces, providing evidence for a neoangiogenetic process, filled with a variable amount of erythrocytes (middle, right) (Hematoxylin–Eosin).
endothelial cells defining capillary structure were visible (Fig. 4f, g). 3.3. Sirius red staining and trichrome These sections showed a regular and increased deposition of collagen starting from the surface and filling the biopolymer network (Fig. 4h). At the end of the invasion process the collagen was also localized around the neovessels (Fig. 4i).
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expression of a-SMA (Fig. 4l). In all samples few inflammatory cells, expressing CD14, were visible (Fig. 4m) while most cells adopting a pronounced migratory and spreading phenotype showed a strong positivity for cathepsin B (Fig. 4n). 3.5. Enzymatic activity The degradation of Degrapol scaffold, presumably due to cathepsin B action, was tested using the enzyme on prosthesis cryosections. After incubation for 1 h at 37 8C the fibers of biopolymer were broken and dimensionally reduced in comparison with the fibers of biopolymer treated only with PBS buffer (Fig. 4o, p). 4. Discussion
Fig. 4. Semithin cross section of excised tracheal scaffold (panels a-c). Progressively, cells and fibrillar material invade centripetally the Degrapol scaffold. Transmission electron microscope images showing a detail of the Degrapol䊛 scaffold (d) covered by cells and fibrillar material. Large amount of collagen bundles longitudinally sectioned (d), fibroblast-like cells (e) surrounded by collagen bundles cross-sectioned (arrowheads), small muscle fibers (f) and minute blood vessels (g) were visible. Prosthesis cross-sectioned (h, i) showing an increased deposition of collagen (in red color with sirius red staining and in gray-blue with Masson trichrome) filling the biopolymer network (h) and surrounding the neovessels (i, v). Immunofluorescence ( j, k) with monoclonal anti-CD31, followed by detection with fluoresceine isothiocyanate-conjugated secondary antibodies. Immunofluorescence with antibodies anti-SMA (l), anti-CD14 (m), and anti-cathepsin B (n) followed by detection with tetramethylrhodamine isothiocyanate-conjugated secondary antibodies. Shown in red, respectively: muscle fibers (l), macrophages (m), and cells characterized by cathepsin B production (n) are visible. In blue are nuclei stained with DAPI. Cryosections of Degrapol scaffold before (o) and after (p) incubation with cathepsin B enzyme for 1 h at 37 8C. The fibers of biopolymer were broken and reduced in dimension (p) due to the enzyme degradation. Bars: a–c, i, o, p – 100 mm; d–f – 2 mm; g – 4 mm; h – 50 mm.
3.4. Indirect immunofluorescence staining The monoclonal CD31 antibody analysis showed that positive cells were visibly crowded especially on the external surface (Fig. 4j) while after a short time from the cell invasion, the expression of CD31 was localized at the endothelial cells forming the blood vessel walls (Fig. 4k). Cells increased over time (G1–G3) in number and many of them differentiate in muscle fibers as validated by the
These in vivo studies corroborate previous data w11x about the animal model inducing angiogenesis through a vascular flap providing a suitable substrate for the development of tracheal structure. The early observation (Group 1) suggested that tissue formation proceeded from the vascular pedicle toward the prosthetic material. As predictable, the zones closer to the vascular carrier were reached by extracellular fibrillary matrix-synthesizing fibroblasts before the zones located at a greater distance w11x. The pedicle region showed a scattered early obviousness of neoangiogenesis by means of tissue deposition accompanied by new formation of blood vessels. In all samples large vascular spaces filled with erythrocytes were observed in close proximity to the vascular pedicle. A few macrophages were the inflammatory cells involved in the reactive process mainly localized in proximity to the blood vessels. On the other hand, there was no evidence of cells involved in the immune process, confirming the good biocompatibility of the polymer. Our analysis shows that over time there is a migration of cells CD31 positive generally expressed by endothelial progenitor cells derived by earlier common myeloid progenitor or as hematopoietic stem cell. Based on the hypothesis that CD31 is an early indicator during the endothelial differentiation, CD31-bright cells are precursor cells moving from the vascular pedicle, and colonizing the thickness of Degrapol䊛 prosthesis. The prostheses revealed a conspicuous amount of neo-formed tissues that progressively substitute the biopolymer fibers filling the spaces with cells and extracellular fibrillar material made of collagen that, as a structural protein, provides mechanical support to the tissues. Morphological and immunocytochemical characterization is consistent with endothelial cells migration, spatial disposition and differentiation of migrating stem cells in various cell types. The positivity of cathepsin B in the present cells advocates its properties to promote angiogenesis through a process of migration and invasion, already described by others either in vitro than in vivo studies w12, 13x. Its lysosomial activity is only one of the characteristics of this endocellular protease that has been found even over the cell membrane and in the pericellular space. These peculiarities enable it to have a paramount role in destruction and remodeling of the matrix, and in turn in cell adhesion and migration. Cathepsin B seems to be implicated in different pathological
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processes such as tumor angiogenesis, neurodegeneration or abdominal aorta aneurysm w14x, but how it regulates angiogenesis, cell proliferation, invasion and apoptosis is still poorly understood. Moreover, our simple enzymatic activity analysis shows that the cathepsin B could be liable to the polymer degradation such as for biological matrix. Our data show the expression of cathepsin B in progenitor cells (ECS) expressing CD31 and SMA, and are consistent with a migratory and angiogenetic cellular phase. To our knowledge, it is the first time that cathepsin has been found during an in-vivo evaluation of a scaffold polymer degradation w15x. Further and more ad hoc studies on the interaction between cathepsin and Degrapol䊛 chemical structure would improve our knowledge on which part of the polymer is subjected to the degradation process. We can also speculate to design a scaffold made up of distinct components characterized by a different degradability when in contact with cellular enzyme (viz. cathepsins), to allow a polymer fibers substitution with timing and predetermined direction for an ad hoc tissue regeneration. Thus the 3-D scaffold realized, exploiting the physiological cell proteolytic pathways of degradation, could be more suitable for tissue replacement.
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