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Angiogenic response induced by acellular brain scaffolds grafted onto the chick embryo chorioallantoic membrane
Brain Research 989 (2003) 9–15 www.elsevier.com / locate / brainres
Research report
Angiogenic response induced by acellular brain scaffolds grafted onto the chick embryo chorioallantoic membrane Domenico Ribatti a , *, Maria Teresa Conconi b , Beatrice Nico a , Silvia Baiguera b , Patrizia Corsi c , Pier Paolo Parnigotto b , Gastone G. Nussdorfer b a
Department of Human Anatomy and Histology, University of Bari Medical School, Policlinico, Piazza Giulio Cesare 11, I-70124 Bari, Italy b Department of Human Anatomy and Physiology, Section of Anatomy, University of Padua Medical School, Padua, Italy c Department of Physiology and Pharmacology, University of Bari Medical School, Policlinico, Piazza Giulio Cesare 11, I-70124 Bari, Italy Accepted 27 June 2003
Abstract The repair and regeneration of injured tissues and organs depend on the re-establishment of the blood flow needed for cellular infiltration and metabolic support. Among the various materials used in tissue reconstruction, acellular scaffolds have recently been utilized. In this study, we investigated the angiogenic response induced by acellular brain scaffolds implanted in vivo onto the chick embryo chorioallantoic membrane (CAM), a useful model for such investigations. The results show that acellular brain scaffolds are able to induce a strong angiogenic response, comparable to that of fibroblast growth factor-2 (FGF-2), a well known angiogenic cytokine. The response may be considered dependent on a direct angiogenic effect exerted by the scaffold, because no inflammatory infiltrate was detectable in CAM’s mesenchyme beneath the implant. Acellular brain scaffolds might induce the release of endogenous angiogenic factors, such as FGF-2 and vascular endothelial growth factor (VEGF) released from the extracellular matrix of the developing CAM. In addition, the angiogenic response may depend, in part, also on the presence in the acellular matrix of transforming growth factor beta 1 (TGFb1). 2003 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Cell differentiation and migration Keywords: Angiogenesis; Brain; Chorioallantoic membrane; Regeneration; Scaffold
1. Introduction The repair and regeneration of injured tissues and organs depend on the re-establishment of the blood flow needed for cellular infiltration and metabolic support. Among the various materials used in tissue reconstruction, acellular scaffolds have recently been utilized [7]. Acellular scaffolds are the noncellular part of a tissue and consist of proteins such as collagen and carbohydrate structures
*Corresponding author. Tel.: 139-080-547-8240; fax: 139-080-5478310. E-mail address: [email protected] (D. Ribatti). 0006-8993 / 03 / $ – see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0006-8993(03)03225-6
secreted by the resident cells. They can be transplanted without rejection and provide a conductive environment for normal cellular growth, differentiation and angiogenesis and a framework for tissue regeneration, because they are completely replaced by the host tissue [7]. In the last few years, acellular matrices have been successfully used to substitute and repair skin [23], bladder [21], urethra [13], small bowel [14], and skeletal muscle [11] defects. Angiogenesis, the process by which new blood vessels arise from pre-existing ones, is a crucial event in the repair of lesions of nervous tissue [5]. In this study, we investigated the angiogenic response induced by acellular brain scaffolds implanted in vivo onto the chick embryo chorioallantoic membrane (CAM), a useful model for such investigations [19].
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D. Ribatti et al. / Brain Research 989 (2003) 9–15
2. Materials and methods
2.3. CAM assay
2.1. Acellular scaffold preparation
Fifty fertilized White Leghorn chicken eggs were incubated at 37 8C at constant humidity. On day 3 of incubation a square window was opened in the egg shell after removal of 2–3 ml of albumen so as to detach the developing CAM from the shell. The window was sealed with glass and the eggs were returned to the incubator. On day 8, eggs were treated with 1 mm 3 acellular brain scaffolds implanted on the top of growing CAMs under sterile conditions. In some experiments, brain scaffolds were mixed with 400 ng / embryo of anti-FGF-2 antibody (Santa Cruz Biotechnology) before implantation or with 400 ng / embryo of anti-VEGF antibody against N-terminal peptide 1–140 (Santa Cruz) or with a mixture of the two antibodies. Moreover, 1 mm 3 sterilized gelatin sponges (Gelfoam, Upjohn, Kalamazoo, MI, USA) adsorbed with fibroblast growth factor-2 (FGF-2) (500 ng / embryo) dissolved in 2 ml PBS or with PBS alone, used as positive and negative control, respectively, were implanted on day 8 on the top of some CAMs, as previously reported [16]. CAMs were examined daily until day 12 and photographed in ovo with a stereomicroscope equipped with a Camera System MC 63 (Zeiss, Oberkochen, Germany). Blood vessels entering the implants or the sponges within the focal plane of the CAM were counted by two observers in a double-blind fashion at 503 magnification [2]. On day 12, CAMs were processed for light microscopy. Eight micrometer serial sections were cut in a plane parallel to the surface of the CAM, stained with a 0.5% aqueous
Whole brains, obtained from adult Sprague–Dawley rats, were rinsed two times in phosphate-buffered saline (PBS) containing 1% antibiotic and antimycotic solution (Sigma, St. Louis, MO, USA), and treated according to Meezan et al. [12]. Samples were frozen and thawed four times and were then processed twice as follows: distilled water for 18 h at 4 8C, 4% sodium deoxycholate (Sigma) for 1 h, and 2000 kU Dnase I (Sigma) in 1 M NaCl for 15 min. The absence of cellular elements was confirmed histologically (hematoxylin–eosin staining) (Merck, Darmstadt, Germany), and acellular matrices were stored in PBS at 4 8C.
2.2. Immunohistochemistry Five micrometer thick sections of whole brain and acellular matrix were incubated at 37 8C with primary polyclonal antibody anti-transforming growth factor beta-1 (TGFb1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:200 in 1% BSA for 60 min. Immunostaining was performed using a large volume Dako LSAB (labelled streptavidin–biotin)1Kit / HPR (horseradish peroxidase) (Dako, Carpinteria, CA, USA). A preimmune rabbit serum (Dako) replacing the primary antibody served as negative control.
Fig. 1. Histological sections of a rat brain after (A) and before (B) detergent–enzymatic treatment. Note in (A) the less compact structure and the absence of cells. Original magnification: 3100.
D. Ribatti et al. / Brain Research 989 (2003) 9–15
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solution of toluidine blue (Merck) and with naphthol-ASD-chloroacetate esterase to highlight mononuclear cells and observed under a Leitz-Dialux 20 light photomicroscope (Leitz, Wetzlar, Germany). The angiogenic response was assessed histologically by a planimetric method of ‘point counting’ [16]. Briefly, every third section within 30 serial slides from each specimen was analyzed under a 144-point mesh inserted in the eyepiece of the photomicroscope. The total number of intersection points occupied by transversally cut vessels (3–10 mm diameter) was counted at 3250 at the boundary between the implants and the surrounding CAM mesenchyme in six randomly chosen microscopic fields for each section. Mean values6standard deviation (S.D.) were determined for each analysis. The vascular density was indicated by the final mean number of occupied intersection points. Statistical analysis was performed using Student’s t-test for unpaired data. The same experimental procedure was utilized to quantify the mononuclear cell infiltrate.
3. Results
3.1. Acellular scaffold Sections of the matrix obtained following the detergent– enzymatic treatment of rat brain are shown in Fig. 1A. Histological examination revealed the absence of cells; moreover, the acellular matrix presented a less compact structure compared to that observed in untreated tissue (Fig. 1B). The detergent–enzymatic treatment preserved the presence of TGFb1 from brain tissues, as demonstrated by the diffuse strong immunoreactivity to TGFb1 in both whole brain and acellular matrix (Fig. 2A and B).
Fig. 2. Sections of a rat brain (A) and of an acellular matrix (B) immunoreactive to TGFb1. Original magnification: 3250.
3.2. Macroscopic examination Macroscopic observations on day 12 of incubation showed that the implants of the acellular brain scaffolds were surrounded by allantoic vessels that developed radially towards the implant in a ‘spoked-wheel’ pattern (Fig. 3A). As shown in Table 1, the angiogenic response induced by the acellular brain scaffolds was comparable to that exerted by FGF-2, a well-known angiogenic cytokine. No vascular reaction was detectable around the sponge treated with vehicle alone. To assess whether the angiogenic response elicited by the brain scaffolds was due to an increased mobilization of endogenous FGF-2 or VEGF, brain scaffolds were added to the CAM in the presence of anti-FGF-2 or anti-VEGF antibodies. Under these experimental conditions, anti-FGF-2 reduced the angiogenic response by 55% and anti-VEGF by 30%. When the two antibodies were added together, they acted in a synergistic way, reducing the angiogenic response by 80%.
3.3. Microscopic examination On day 12 of incubation, in the area away from the implant, the CAM was composed of a surface epithelium arising from the ectoderm (chorion), an intermediate mesenchyme, containing arterial and venous vessels merging with a capillary meshwork running under the chorion, and a deep epithelium arising from the endoderm (allantois). Acellular brain scaffolds were adherent to the chorion without invading the mesenchyme (Fig. 4A). Numerous newly formed blood vessels were recognizable arranged radially under the implants and around large blood vessels of the CAM (Fig. 4B). At some points, blood vessels of the CAM invaded the chorionic epithelium (under normal conditions, on day 12 of incubation, blood vessels of the CAM are located beneath the chorionic epithelium) and merged inside the brain scaffolds over the epithelium (Fig.
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Fig. 3. In ovo picture on day 12 of incubation showing an implant of an acellular brain scaffold surrounded by allantoic vessels developing radially towards it (asterisk) in a ‘spoked-wheel’ pattern (A). The vasoproliferative response is comparable to that induced by a gelatin sponge (asterisk) soaked with FGF-2 (B). No vascular reaction is detectable around the vehicle-treated sponge (asterisk) (C). Original magnification: (A)–(C) 350.
Table 1 Macroscopic evaluation of the angiogenic activity of acellular brain scaffolds (ABSs) in the chick embryo CAM Treatment
No. of blood vessels (mean6S.D.)
PBS FGF-2 ABS ABS1anti-FGF-2 antibody ABS1anti-VEGF antibody ABS1anti-FGF-21anti-VEGF antibodies
862 3465* 3064* 1465** 2164*** 662**
*Statistically different from PBS (P,0.001). **Statistically different from ABS (P,0.001). ***Statistically different from ABS (P,0.01).
4C and D). In the mesenchyme of the CAM a scarce mononuclear cell inflammatory infiltrate expressing naphthol-AS-D-chloroacetate esterase activity was detectable (Fig. 4E). In agreement with the macroscopic and microscopic observations, a higher microvessel density was detectable within the CAMs implanted with brain scaffolds than in those treated with vehicle alone (and comparable to the microvessel density of the CAMs treated with FGF-2) when the angiogenic response was quantified by a morphometric method of ‘point counting’ (Table 2). Moreover, no significant increase of the mononuclear cell infiltrate was observed in the CAMs treated with the brain scaffolds, with respect to vehicle-treated samples, ruling out the possibility that the angiogenic activity
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Fig. 4. On a microscopic level, scaffolds are adherent to the chorion without invading the mesenchyme (asterisk in A). At higher magnification, numerous newly formed blood vessels are recognizable arranged radially under the chorion (CH) and around large blood vessels of the CAM (B). At some points, bloods vessels (arrows) invade the chorion (CH) (C) or are recognizable upon it (D). A few naphthol-AS-D-chloroacetate esterase-positive mononuclear cells can be recognized in a perivascular position (E). Original magnifications: (A) 3100; (C)–(E) 3400.
exerted by the brain scaffolds was the consequence of the triggering of an inflammatory response (Table 2).
4. Discussion It is commonly believed that adult neurons contain the capacity to regrow if provided with an appropriate substrate, including a peripheral nerve graft, injection of neurotrophin-secreting cells, or artificial matrices sealed with growth-promoting molecules [8]. Using the de-
tergent–enzymatic method, highly antigenic proteins are extracted [24], so that acellular scaffolds can be transplanted without rejection and provide a conductive environment for normal cellular growth, differentiation and angiogenesis and a framework for tissue regeneration [7]. Acellular matrices are remodeled in vivo acting as a temporary scaffold that is colonized by host cells generating new tissue [9]. In this study we have demonstrated, for the first time, that acellular brain scaffolds are able to induce a strong angiogenic response, comparable to that of FGF-2, a well
Table 2 Microscopic evaluation of the angiogenic response and of mononuclear cell infiltrate induced by acellular brain scaffolds (ABSs) in the chick embryo CAM Treatment
PBS FGF-2 ABS ABS1anti-FGF-2 ab ABS1anti-VEGF ab ABS1anti-FGF-21anti-VEGF ab
Angiogenic response
Mononuclear cell infiltrate
No. of intersection points
Microvessel density (%)
No. of intersection points
Cell density (%)
361 3065* 2764* 1363** 1964*** 561**
2.1 20.8 18.8 9.0 13.0 3.4
462 663 562 462 561 562
2.8 4.1 3.4 2.8 3.4 3.4
*Statistically different from PBS (P,0.001). **Statistically different from ABS (P,0.001). ***Statistically different from ABS (P,0.01).
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known angiogenic cytokine, when implanted onto the chick CAM. The newly formed blood vessels grow radially around the scaffold and invade it at some points. The angiogenic response may be considered dependent on the direct angiogenic activity exerted by the scaffold, because no inflammatory infiltrate is detectable in the CAM’s mesenchyme beneath the implant, as demonstrated by histochemical detection of mononuclear cells and by microscopic counts. Using the CAM assay under other experimental conditions, the angiogenic response may be attributable to the angiogenic cytokines released by the inflammatory cells, localized in the CAM mesenchyme around the newly formed vessels [10,17,18]. For example, when bioptic specimens obtained from rheumatoid arthritis tissues were implanted on the CAM, morphometric quantitation showed that microvessels and mononuclear cell density were highly correlated [18]. In contrast, under our experimental conditions, acellular brain scaffolds might induce the release of endogenous angiogenic factors, such as FGF-2 and VEGF [15,20], stored in the extracellular matrix of the developing CAM. This finding is also confirmed by the fact that application of anti-FGF-2 or anti-VEGF antibody reduced the angiogenic response by 55 and 30%, respectively, and when the two antibodies were added together, they acted synergistically, reducing the angiogenic response by 80%. Although there are no data that suggest that the mechanism of growth factor release from the extracellular matrix is via the release of proteolytic enzymes, it is conceivable to hypothesize this mechanism of action. The angiogenic response may depend, in part, also on the presence in the acellular matrix of TGFb1, as already demonstrated [22]. The main limitation of the CAM assay is its non-specific inflammatory reactions to grafts, resulting in a secondary vasoproliferative reaction, making it difficult to quantify the primary response being investigated. In fact, inflammatory angiogenesis per se, in which infiltrating inflammatory cells may be the source of angiogenic factors, cannot be distinguished from the direct angiogenic activity of the test material. In this study we have clearly demonstrated that no inflammatory infiltrate is detectable in the CAM’s mesenchyme beneath the implant, as evidenced by histochemical detection of mononuclear cells and by microscopic counts. Another important drawback of the CAM assay is represented by the timing of the CAM angiogenic response. Many studies determine angiogenesis after 24 h, a time at which there is only vasodilation. In this study we evaluated the angiogenic response 96 h after grafting and it is worth pointing out that measurements of vessel density are really measurements of newly formed vessels. In this regard, morphometric evaluation at the macroscopic and microscopic levels is important. It is also conceivable to hypothesize that the angiogenic activity may be dependent, in part, on the inherent angiogenic activity of the matrix and / or its chemoattractant activity. Other angiogenic factors, besides TGF-b,
might be retained in the matrix. In fact, it is conceivable that brain extraction techniques may not remove all the factors that impact on angiogenesis. Under our experimental conditions, the implants remain avascular. Addition to the brain acellular scaffolds of exogenous angiogenic cytokines, such as VEGF [1,4], FGF-2 or neurotrophic factors with angiogenic capability, such as nerve growth factor (NGF) [3], might induce the ingrowth of adjacent newly formed blood vessels of the CAM into the implants. For example, it has been demonstrated that VEGF significantly increases blood vessel penetration within silicone nerve regeneration chambers [6]. Further experiments using our in vivo experimental model are required to further elucidate this matter.
Acknowledgements This work was supported, in part, by a grant from the Ministero dell’Istruzione, dell’Universita` e della Ricerca (FIRB ‘‘Interuniversity Funds for Basic Research’’), Rome, Italy, to DR and GGN.
References [1] D. Bartholid, B.P. Rubin, M.E. Schwab, VEGF mRNA induction correlates with changes in the vasculature upon spinal cord damage in the rat, Eur. J. Neurosci. 9 (1997) 2549–2560. [2] P.C. Brooks, R.A.F. Clark, D.A. Cheresh, Requirement of vascular integrin a v b 3 for angiogenesis, Science 264 (1994) 569–571. [3] G. Cantarella, L. Lempereur, M. Presta, D. Ribatti, G. Lombardo, P. ` C. Pafumi, R. Bernardini, Nerve growth Lazarovici, G. Zappala, factor–endothelial cell interaction leads to angiogenesis in vitro and in vivo, FASEB J. 16 (2002) 1307–1309. [4] P. Carmeliet, E. Storkebaum, Vascular and neuronal effects of VEGF in the nervous system: implications for neurological disorders, Semin. Cell Dev. Biol. 13 (2002) 39–53. [5] M.I. Hobson, R. Brown, C.J. Green, G. Terenghi, Interrelationships between angiogenesis and nerve regeneration: a histochemical study, Br. J. Plast. Surg. 50 (1997) 125–131. [6] M.I. Hobson, C.J. Green, G. Terenghi, VEGF enhances intraneuronal angiogenesis and improves nerve regeneration after axotomy, J. Anat. 197 (2000) 591–605. [7] J. Hodde, Naturally occurring scaffolds for soft tissue repair and regeneration, Tissue Eng. 8 (2002) 295–308. [8] P.J. Horner, F.H. Gage, Regenerating the damaged central nervous system, Nature 407 (2000) 963–970. [9] S.A. Livesey, D.N. Herndon, Transplanted acellular allograft dermal matrix. Potential as a template for the reconstruction of viable dermis, Transplantation 60 (1995) 1–9. [10] E. Lucarelli, L. Sangiorgi, S. Benassi, D. Donati, G.A. Gobbi, P. Picci, A. Vacca, D. Ribatti, Angiogenesis in lipoma: an experimental study in the chick embryo chorioallantoic membrane, Int. J. Mol. Med. 4 (1999) 593–596. [11] M. Marzaro, M.T. Conconi, L. Perin, S. Giuliani, P.G. Gamba, P. De Coppi, G.P. Perrino, P.P. Parnigotto, G.G. Nussdorfer, Autologous satellite cell seeding improves in vivo biocompatibility of homologous muscle acellular matrix implants, Int. J. Mol. Med. 10 (2002) 177–182. [12] E. Meezan, J.T. Hjelle, K. Brendel, A simple, versatile, non
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chorioallantoic membrane of the anti-angiogenic activity of cyclosporine in rheumatoid arthritis versus osteoarthritis, Inflamm. Res. 49 (2000) 418–423. D. Ribatti, B. Nico, A. Vacca, L. Roncali, P.H. Burri, V. Djonov, Chorioallantoic membrane capillary bed: a useful target for studying angiogenesis and anti-angiogenesis in vivo, Anat. Rec. 264 (2001) 317–324. D. Ribatti, B. Nico, L. Morbidelli, S. Donnini, M. Ziche, A. Vacca, L. Roncali, M. Presta, Cell-mediated delivery of fibroblast growth factor-2 and vascular endothelial growth factor onto the chick embryo chorioallantoic membrane: endothelial fenestrations and angiogenesis, J. Vasc. Res. 38 (2001) 389–397. R.S. Sutherland, L.S. Baskin, S.W. Hayward, G.R. Cunha, Regeneration of bladder urothelium, smooth muscle, blood vessels and nerves into an acellular tissue matrix, J. Urol. 156 (1996) 571–577. J. Taipale, J. Saharinen, K. Hedman, J.J. Keski-Oja, Latent transforming growth factor-beta 1 and its binding protein are components of extracellular matrix microfibrils, J. Histochem. Cytochem. 44 (1996) 875–879. Y. Takami, T. Matsuda, M. Yoshitake, M. Hanumadass, R.J. Walter, Dispase / detergent treatment dermal matrix as a dermal substitute, Burns 22 (1996) 182–190. G.J. Wilson, H. Yeger, Acellular matrix allograft small caliber vascular prostheses, Trans. Am. Soc. Artif. Intern. Organs 36 (1990) 340–343.
Research report
Angiogenic response induced by acellular brain scaffolds grafted onto the chick embryo chorioallantoic membrane Domenico Ribatti a , *, Maria Teresa Conconi b , Beatrice Nico a , Silvia Baiguera b , Patrizia Corsi c , Pier Paolo Parnigotto b , Gastone G. Nussdorfer b a
Department of Human Anatomy and Histology, University of Bari Medical School, Policlinico, Piazza Giulio Cesare 11, I-70124 Bari, Italy b Department of Human Anatomy and Physiology, Section of Anatomy, University of Padua Medical School, Padua, Italy c Department of Physiology and Pharmacology, University of Bari Medical School, Policlinico, Piazza Giulio Cesare 11, I-70124 Bari, Italy Accepted 27 June 2003
Abstract The repair and regeneration of injured tissues and organs depend on the re-establishment of the blood flow needed for cellular infiltration and metabolic support. Among the various materials used in tissue reconstruction, acellular scaffolds have recently been utilized. In this study, we investigated the angiogenic response induced by acellular brain scaffolds implanted in vivo onto the chick embryo chorioallantoic membrane (CAM), a useful model for such investigations. The results show that acellular brain scaffolds are able to induce a strong angiogenic response, comparable to that of fibroblast growth factor-2 (FGF-2), a well known angiogenic cytokine. The response may be considered dependent on a direct angiogenic effect exerted by the scaffold, because no inflammatory infiltrate was detectable in CAM’s mesenchyme beneath the implant. Acellular brain scaffolds might induce the release of endogenous angiogenic factors, such as FGF-2 and vascular endothelial growth factor (VEGF) released from the extracellular matrix of the developing CAM. In addition, the angiogenic response may depend, in part, also on the presence in the acellular matrix of transforming growth factor beta 1 (TGFb1). 2003 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Cell differentiation and migration Keywords: Angiogenesis; Brain; Chorioallantoic membrane; Regeneration; Scaffold
1. Introduction The repair and regeneration of injured tissues and organs depend on the re-establishment of the blood flow needed for cellular infiltration and metabolic support. Among the various materials used in tissue reconstruction, acellular scaffolds have recently been utilized [7]. Acellular scaffolds are the noncellular part of a tissue and consist of proteins such as collagen and carbohydrate structures
*Corresponding author. Tel.: 139-080-547-8240; fax: 139-080-5478310. E-mail address: [email protected] (D. Ribatti). 0006-8993 / 03 / $ – see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0006-8993(03)03225-6
secreted by the resident cells. They can be transplanted without rejection and provide a conductive environment for normal cellular growth, differentiation and angiogenesis and a framework for tissue regeneration, because they are completely replaced by the host tissue [7]. In the last few years, acellular matrices have been successfully used to substitute and repair skin [23], bladder [21], urethra [13], small bowel [14], and skeletal muscle [11] defects. Angiogenesis, the process by which new blood vessels arise from pre-existing ones, is a crucial event in the repair of lesions of nervous tissue [5]. In this study, we investigated the angiogenic response induced by acellular brain scaffolds implanted in vivo onto the chick embryo chorioallantoic membrane (CAM), a useful model for such investigations [19].
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D. Ribatti et al. / Brain Research 989 (2003) 9–15
2. Materials and methods
2.3. CAM assay
2.1. Acellular scaffold preparation
Fifty fertilized White Leghorn chicken eggs were incubated at 37 8C at constant humidity. On day 3 of incubation a square window was opened in the egg shell after removal of 2–3 ml of albumen so as to detach the developing CAM from the shell. The window was sealed with glass and the eggs were returned to the incubator. On day 8, eggs were treated with 1 mm 3 acellular brain scaffolds implanted on the top of growing CAMs under sterile conditions. In some experiments, brain scaffolds were mixed with 400 ng / embryo of anti-FGF-2 antibody (Santa Cruz Biotechnology) before implantation or with 400 ng / embryo of anti-VEGF antibody against N-terminal peptide 1–140 (Santa Cruz) or with a mixture of the two antibodies. Moreover, 1 mm 3 sterilized gelatin sponges (Gelfoam, Upjohn, Kalamazoo, MI, USA) adsorbed with fibroblast growth factor-2 (FGF-2) (500 ng / embryo) dissolved in 2 ml PBS or with PBS alone, used as positive and negative control, respectively, were implanted on day 8 on the top of some CAMs, as previously reported [16]. CAMs were examined daily until day 12 and photographed in ovo with a stereomicroscope equipped with a Camera System MC 63 (Zeiss, Oberkochen, Germany). Blood vessels entering the implants or the sponges within the focal plane of the CAM were counted by two observers in a double-blind fashion at 503 magnification [2]. On day 12, CAMs were processed for light microscopy. Eight micrometer serial sections were cut in a plane parallel to the surface of the CAM, stained with a 0.5% aqueous
Whole brains, obtained from adult Sprague–Dawley rats, were rinsed two times in phosphate-buffered saline (PBS) containing 1% antibiotic and antimycotic solution (Sigma, St. Louis, MO, USA), and treated according to Meezan et al. [12]. Samples were frozen and thawed four times and were then processed twice as follows: distilled water for 18 h at 4 8C, 4% sodium deoxycholate (Sigma) for 1 h, and 2000 kU Dnase I (Sigma) in 1 M NaCl for 15 min. The absence of cellular elements was confirmed histologically (hematoxylin–eosin staining) (Merck, Darmstadt, Germany), and acellular matrices were stored in PBS at 4 8C.
2.2. Immunohistochemistry Five micrometer thick sections of whole brain and acellular matrix were incubated at 37 8C with primary polyclonal antibody anti-transforming growth factor beta-1 (TGFb1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:200 in 1% BSA for 60 min. Immunostaining was performed using a large volume Dako LSAB (labelled streptavidin–biotin)1Kit / HPR (horseradish peroxidase) (Dako, Carpinteria, CA, USA). A preimmune rabbit serum (Dako) replacing the primary antibody served as negative control.
Fig. 1. Histological sections of a rat brain after (A) and before (B) detergent–enzymatic treatment. Note in (A) the less compact structure and the absence of cells. Original magnification: 3100.
D. Ribatti et al. / Brain Research 989 (2003) 9–15
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solution of toluidine blue (Merck) and with naphthol-ASD-chloroacetate esterase to highlight mononuclear cells and observed under a Leitz-Dialux 20 light photomicroscope (Leitz, Wetzlar, Germany). The angiogenic response was assessed histologically by a planimetric method of ‘point counting’ [16]. Briefly, every third section within 30 serial slides from each specimen was analyzed under a 144-point mesh inserted in the eyepiece of the photomicroscope. The total number of intersection points occupied by transversally cut vessels (3–10 mm diameter) was counted at 3250 at the boundary between the implants and the surrounding CAM mesenchyme in six randomly chosen microscopic fields for each section. Mean values6standard deviation (S.D.) were determined for each analysis. The vascular density was indicated by the final mean number of occupied intersection points. Statistical analysis was performed using Student’s t-test for unpaired data. The same experimental procedure was utilized to quantify the mononuclear cell infiltrate.
3. Results
3.1. Acellular scaffold Sections of the matrix obtained following the detergent– enzymatic treatment of rat brain are shown in Fig. 1A. Histological examination revealed the absence of cells; moreover, the acellular matrix presented a less compact structure compared to that observed in untreated tissue (Fig. 1B). The detergent–enzymatic treatment preserved the presence of TGFb1 from brain tissues, as demonstrated by the diffuse strong immunoreactivity to TGFb1 in both whole brain and acellular matrix (Fig. 2A and B).
Fig. 2. Sections of a rat brain (A) and of an acellular matrix (B) immunoreactive to TGFb1. Original magnification: 3250.
3.2. Macroscopic examination Macroscopic observations on day 12 of incubation showed that the implants of the acellular brain scaffolds were surrounded by allantoic vessels that developed radially towards the implant in a ‘spoked-wheel’ pattern (Fig. 3A). As shown in Table 1, the angiogenic response induced by the acellular brain scaffolds was comparable to that exerted by FGF-2, a well-known angiogenic cytokine. No vascular reaction was detectable around the sponge treated with vehicle alone. To assess whether the angiogenic response elicited by the brain scaffolds was due to an increased mobilization of endogenous FGF-2 or VEGF, brain scaffolds were added to the CAM in the presence of anti-FGF-2 or anti-VEGF antibodies. Under these experimental conditions, anti-FGF-2 reduced the angiogenic response by 55% and anti-VEGF by 30%. When the two antibodies were added together, they acted in a synergistic way, reducing the angiogenic response by 80%.
3.3. Microscopic examination On day 12 of incubation, in the area away from the implant, the CAM was composed of a surface epithelium arising from the ectoderm (chorion), an intermediate mesenchyme, containing arterial and venous vessels merging with a capillary meshwork running under the chorion, and a deep epithelium arising from the endoderm (allantois). Acellular brain scaffolds were adherent to the chorion without invading the mesenchyme (Fig. 4A). Numerous newly formed blood vessels were recognizable arranged radially under the implants and around large blood vessels of the CAM (Fig. 4B). At some points, blood vessels of the CAM invaded the chorionic epithelium (under normal conditions, on day 12 of incubation, blood vessels of the CAM are located beneath the chorionic epithelium) and merged inside the brain scaffolds over the epithelium (Fig.
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Fig. 3. In ovo picture on day 12 of incubation showing an implant of an acellular brain scaffold surrounded by allantoic vessels developing radially towards it (asterisk) in a ‘spoked-wheel’ pattern (A). The vasoproliferative response is comparable to that induced by a gelatin sponge (asterisk) soaked with FGF-2 (B). No vascular reaction is detectable around the vehicle-treated sponge (asterisk) (C). Original magnification: (A)–(C) 350.
Table 1 Macroscopic evaluation of the angiogenic activity of acellular brain scaffolds (ABSs) in the chick embryo CAM Treatment
No. of blood vessels (mean6S.D.)
PBS FGF-2 ABS ABS1anti-FGF-2 antibody ABS1anti-VEGF antibody ABS1anti-FGF-21anti-VEGF antibodies
862 3465* 3064* 1465** 2164*** 662**
*Statistically different from PBS (P,0.001). **Statistically different from ABS (P,0.001). ***Statistically different from ABS (P,0.01).
4C and D). In the mesenchyme of the CAM a scarce mononuclear cell inflammatory infiltrate expressing naphthol-AS-D-chloroacetate esterase activity was detectable (Fig. 4E). In agreement with the macroscopic and microscopic observations, a higher microvessel density was detectable within the CAMs implanted with brain scaffolds than in those treated with vehicle alone (and comparable to the microvessel density of the CAMs treated with FGF-2) when the angiogenic response was quantified by a morphometric method of ‘point counting’ (Table 2). Moreover, no significant increase of the mononuclear cell infiltrate was observed in the CAMs treated with the brain scaffolds, with respect to vehicle-treated samples, ruling out the possibility that the angiogenic activity
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Fig. 4. On a microscopic level, scaffolds are adherent to the chorion without invading the mesenchyme (asterisk in A). At higher magnification, numerous newly formed blood vessels are recognizable arranged radially under the chorion (CH) and around large blood vessels of the CAM (B). At some points, bloods vessels (arrows) invade the chorion (CH) (C) or are recognizable upon it (D). A few naphthol-AS-D-chloroacetate esterase-positive mononuclear cells can be recognized in a perivascular position (E). Original magnifications: (A) 3100; (C)–(E) 3400.
exerted by the brain scaffolds was the consequence of the triggering of an inflammatory response (Table 2).
4. Discussion It is commonly believed that adult neurons contain the capacity to regrow if provided with an appropriate substrate, including a peripheral nerve graft, injection of neurotrophin-secreting cells, or artificial matrices sealed with growth-promoting molecules [8]. Using the de-
tergent–enzymatic method, highly antigenic proteins are extracted [24], so that acellular scaffolds can be transplanted without rejection and provide a conductive environment for normal cellular growth, differentiation and angiogenesis and a framework for tissue regeneration [7]. Acellular matrices are remodeled in vivo acting as a temporary scaffold that is colonized by host cells generating new tissue [9]. In this study we have demonstrated, for the first time, that acellular brain scaffolds are able to induce a strong angiogenic response, comparable to that of FGF-2, a well
Table 2 Microscopic evaluation of the angiogenic response and of mononuclear cell infiltrate induced by acellular brain scaffolds (ABSs) in the chick embryo CAM Treatment
PBS FGF-2 ABS ABS1anti-FGF-2 ab ABS1anti-VEGF ab ABS1anti-FGF-21anti-VEGF ab
Angiogenic response
Mononuclear cell infiltrate
No. of intersection points
Microvessel density (%)
No. of intersection points
Cell density (%)
361 3065* 2764* 1363** 1964*** 561**
2.1 20.8 18.8 9.0 13.0 3.4
462 663 562 462 561 562
2.8 4.1 3.4 2.8 3.4 3.4
*Statistically different from PBS (P,0.001). **Statistically different from ABS (P,0.001). ***Statistically different from ABS (P,0.01).
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known angiogenic cytokine, when implanted onto the chick CAM. The newly formed blood vessels grow radially around the scaffold and invade it at some points. The angiogenic response may be considered dependent on the direct angiogenic activity exerted by the scaffold, because no inflammatory infiltrate is detectable in the CAM’s mesenchyme beneath the implant, as demonstrated by histochemical detection of mononuclear cells and by microscopic counts. Using the CAM assay under other experimental conditions, the angiogenic response may be attributable to the angiogenic cytokines released by the inflammatory cells, localized in the CAM mesenchyme around the newly formed vessels [10,17,18]. For example, when bioptic specimens obtained from rheumatoid arthritis tissues were implanted on the CAM, morphometric quantitation showed that microvessels and mononuclear cell density were highly correlated [18]. In contrast, under our experimental conditions, acellular brain scaffolds might induce the release of endogenous angiogenic factors, such as FGF-2 and VEGF [15,20], stored in the extracellular matrix of the developing CAM. This finding is also confirmed by the fact that application of anti-FGF-2 or anti-VEGF antibody reduced the angiogenic response by 55 and 30%, respectively, and when the two antibodies were added together, they acted synergistically, reducing the angiogenic response by 80%. Although there are no data that suggest that the mechanism of growth factor release from the extracellular matrix is via the release of proteolytic enzymes, it is conceivable to hypothesize this mechanism of action. The angiogenic response may depend, in part, also on the presence in the acellular matrix of TGFb1, as already demonstrated [22]. The main limitation of the CAM assay is its non-specific inflammatory reactions to grafts, resulting in a secondary vasoproliferative reaction, making it difficult to quantify the primary response being investigated. In fact, inflammatory angiogenesis per se, in which infiltrating inflammatory cells may be the source of angiogenic factors, cannot be distinguished from the direct angiogenic activity of the test material. In this study we have clearly demonstrated that no inflammatory infiltrate is detectable in the CAM’s mesenchyme beneath the implant, as evidenced by histochemical detection of mononuclear cells and by microscopic counts. Another important drawback of the CAM assay is represented by the timing of the CAM angiogenic response. Many studies determine angiogenesis after 24 h, a time at which there is only vasodilation. In this study we evaluated the angiogenic response 96 h after grafting and it is worth pointing out that measurements of vessel density are really measurements of newly formed vessels. In this regard, morphometric evaluation at the macroscopic and microscopic levels is important. It is also conceivable to hypothesize that the angiogenic activity may be dependent, in part, on the inherent angiogenic activity of the matrix and / or its chemoattractant activity. Other angiogenic factors, besides TGF-b,
might be retained in the matrix. In fact, it is conceivable that brain extraction techniques may not remove all the factors that impact on angiogenesis. Under our experimental conditions, the implants remain avascular. Addition to the brain acellular scaffolds of exogenous angiogenic cytokines, such as VEGF [1,4], FGF-2 or neurotrophic factors with angiogenic capability, such as nerve growth factor (NGF) [3], might induce the ingrowth of adjacent newly formed blood vessels of the CAM into the implants. For example, it has been demonstrated that VEGF significantly increases blood vessel penetration within silicone nerve regeneration chambers [6]. Further experiments using our in vivo experimental model are required to further elucidate this matter.
Acknowledgements This work was supported, in part, by a grant from the Ministero dell’Istruzione, dell’Universita` e della Ricerca (FIRB ‘‘Interuniversity Funds for Basic Research’’), Rome, Italy, to DR and GGN.
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