Prospects for microtechnology and nanotechnology in bioengineering of replacement microvessels. (Advances in the Science of Pathology).
Current understanding of cellular and molecular biology of organisms has progressed to the point where it is now possible to achieve de novo production of replacements or improvements for different tissues and organs. Many parts of the body have previously been considered as potential candidates in this regard. (1) The use of tissue-engineering technology will one day offer the possibility to regenerate damaged tissues with laboratory-grown specimens, such as bone, cartilage, skin, and blood vessels.
This review focuses on current and potential applications of tissue engineering in the replacement or functional improvement of microvascular structures. It is not exhaustive by any means and omits important aspects of microvessel replacement (such as stent microtechnology) that are considered too distantly related to our suggested approach. For the same reason, the angiogenic biomaterials are only briefly considered.
VASCULAR TISSUE ENGINEERING
With various degrees of success, the (almost) ex nihilo creation of new vessels has been reported recently in the literature as a promising novel option for the treatment of vasculopathies. This technology depends, however, on the caliber of the vessels being considered and on their desired destination. The most scientifically advanced experiments have already made attempts at reproducing the 3-tunic structure of large vessels. For example, in an early study conducted by Weinberg and Bell, (2) a model blood vessel was constructed in vitro. The vessel's multi-layered structure resembled that of an artery, and more importantly, it was able to resist the associated physiological pressure. The mechanical strength of this model vessel was directly dependent on its multiple layers of collagen, intermeshed with a sturdy Dacron network. Electron microscopy showed that the endothelial cells lining the lumen and the smooth muscle cells in the wall were functional and fairly well differentiated. The lining of the endothelial cells behaved as a permeability barrier and produced von Willebrand factor and prostacyclin.
Niklason et al (3) recently reported an improvement in the construction of replacement arteries on a scaffold of biodegradable polymer, using bovine aortic smooth muscle cells grown in vitro in a device providing a pulsatile perfusion. The associated fluid mechanic patterns profoundly influenced the distribution of cells in the device, inducing the relocalization of the seeded smooth muscle cells from an "adventitial" to an "intimal" location. These artificial vessels were further seeded luminally with autologous endothelial cells and grafted in the circulation of miniature swine, where they displayed angiographically documented patency for 24 days. Similar attempts for in vitro reconstruction of autologous ovine aorta on a polymer scaffold have been reported. (4)
Tissue-engineered blood vessels traditionally rely on synthetic or modified biological materials for structural support. A novel approach to tissue-engineered blood vessel production was recently reported, based exclusively on the use of cultured human cells. (5) In the study, human vascular smooth muscle cells exposed to ascorbic acid produced a cohesive cellular sheet. This sheet was placed around a tubular support to produce the media of the vessel. A similar sheet of human fibroblasts was wrapped around the media to provide the adventitia. After maturation, the tubular support was removed and endothelial cells were seeded in the lumen. This structure featured a well-defined, 3-layered organization and numerous extracellular matrix proteins, including elastin. In this environment, the smooth muscle cells reexpressed desmin, a differentiation marker known to be lost under standard culture conditions. The endothelium expressed von Willebrand factor, incorporated acetylated low-density lipoproteins, produced prostaglandin [I.sub.2], and strongly inhibited platelet adhesion in vitro. The complete vessel had a burst strength comparable to that of human vessels. A short-term grafting experiment conducted in a canine model demonstrated good handling and suturability characteristics.
In another innovative study, Campbell et al (6) suggested the development of an artificial blood conduit made fully from the cells of the host for autologous transplantation. (6) To this end, silastic tubings of variable lengths and diameters were inserted into the peritoneal cavity of rats or rabbits. Within 2 weeks, they were covered with several layers of myofibroblasts, collagen matrix, and a single layer of mesothelium. The tubing was then removed from the harvested implants, and the tube of living tissue was inverted such that it resembled a blood vessel with an inner lining of nonthrombotic mesothelial cells (the "intima") with a "media" of smooth muscle-like cells (myofibroblasts), collagen, and elastin, and an outer collagenous "adventitia." These tissue tubes, 10 to 20 mm long, were then successfully grafted by end-to-end anastomoses into the severed carotid artery or abdominal aorta of the same animal in which they were grown. The transplants remained patent for at least 4 months and developed structures resembling elastic lamellae. (6)
MICROVASCULAR TISSUE ENGINEERING
Because of their extremely small size and fragility, it is difficult to individually assemble very small vessels, such as capillaries, in vitro and then later implant them where they are needed. Instead, the approaches considered to date rely on bulk culturing of large quantities of capillary endothelial cells, as a homogenous population or combined with other cell types. For example, with the purpose of developing vascular-like networks inside tissue-engineered skins, a collagen biopolymer was fabricated in which 3 human cell types were cocultured (keratinocytes, dermal fibroblasts, and umbilical vein endothelial cells). (7,8) This "endothelialized" skin equivalent promoted spontaneous assembly of capillary-like structures in a differentiated extracellular matrix. Immunohistochemistry and transmission electron microscopy showed the characteristics of microvasculature in vivo, such as the expression of von Willebrand factor and the presence of Weibel-Palade bodies, basement membrane material, and intercellular junctions.
Microvascular endothelial cells are under intense scrutiny by several biomedical engineering groups. As a matter of fact, in micropatterning experiments these cells seem to be the cells of choice. Chen et al (9) successfully demonstrated the controlled attachment of endothelium to predefined regions of various substrata through a combination of soft lithography and biochemical conjugation.
Soft lithography is a generic term for those techniques using stamps or channels fabricated in an elastomeric (ie, soft) material for pattern transfer (microcontact printing, patterning using microfluidic channels, laminar flow patterning, etc). Soft lithography is used for simple and inexpensive patterning of proteins and cells using nonphotolithographic microfabrication technologies. These techniques do not require stringent control of the laboratory environment and can be used to pattern surfaces with various ligands, offering good control over both the surface chemistry and the cellular environment. (10)
In a pioneering paper, Mrksich et al (11) described a technique for patterning substrates for endothelial cell culture that allowed the control of positions and sizes of attached cells. The proposed method used self-assembled monolayers (SAMs) of terminally substituted alkanethiolates adsorbed on optically transparent films of gold or silver. Self-assembled monolayers terminated in methyl groups were shown to adsorb proteins, while SAMs terminated in oligo(ethylene glycol) groups were resistant to the adsorption of protein. In the cited study, microcontact printing was used for the formation of SAMs at the micrometer scale. Patterned SAMs with hydrophobic, methyl-terminated lines were prepared and coated with fibronectin. Bovine capillary endothelial cells seeded in micropatterned dishes attached consequently only to the fibronectin-coated, methyl-terminated regions of the patterned SAMs and remained confined to the SAMs of the pattern for 5 to 7 days. Because these substrates were optically transparent, the cells could be visualized by inverted transmission and by fluorescence microscopy, after fixing and staining with fluorescein-labeled phalloidin. (11)
Progressive restriction of the available surface for adhesion of bovine and human endothelial cells, by culturing them on smaller and smaller micropatterned islands using the SAMs technology, was found to regulate the transition of cells from growth to apoptosis, thus confirming the important role played by a cell's shape in many of its functions. (9)
Moreover, the topographic features of the microscopic surface were shown to alter significantly the cell responses in vitro and the cellular reactions to implanted materials in vivo. (12) Careful observations revealed that topographic features of the attachment surface at nanoscale affect cellular attachment, alignment, cytoskeletal arrangement, direction of proliferation, growth rate, and metabolism (for a review, see Curtis and Wilkinson (13)).
More sophisticated, 3-dimensional microfluidic systems were generated as well, in order to produce micropatterns of proteins and cells on planar substrates. The spatial distribution of the microfluidic network in the stamp allowed the patterning of multiple types of proteins and cells in complex discontinuous structures on a surface. The channels formed by the stamp while in contact with the substrate limited the migration and growth of cells to the channels themselves. The cells stopped dividing once they formed a confluent layer, but the removal of the stamp allowed them to spread again and divide. (14)
These microvascular tissue-engineering studies, including the most recent, (15) focused primarily on the demonstration of the underlying technology. The full exploration of the cell physiology in those specific conditions, and of the benefit they may bring in practical applications, such as designing structures that are biologically and medically meaningful remains an aspiration for the future. Among the few exceptions, one study (16) showed that microvascular endothelial cells could choose between proliferation, apoptosis, or differentiation, when attached on adhesive domains of various well-defined sizes. In the latter instance, the cells tended to develop a small, tubular, intracellular and possibly extracellular empty space, distantly reminiscent of the capillary lumen.
To our knowledge, there have been few attempts at bioengineering microvascular networks in vitro. Among them, micromachining was used to create grooves on silicon wafers, where endothelial and hepatocyte cords were assembled, without any specific emphasis on lumen formation. These cellular cords were subsequently detached from their scaffold for further intermixing, with the intention to produce an "artificial liver." (17)
Therapeutic angiogenesis is an emerging strategy for treating diseases by inducing new blood vessel formation in ischemic tissues. Therapeutic angiogenesis may therefore become an important medical tool, helping to preserve the integrity of tissues subjected to ischemia. In this respect, it may be seen as an example of microvascular tissue engineering, consisting of induction of new capillaries from preexisting vessels by a sprouting mechanism (18) via biochemical and/or genetic means.
In studies aimed at inducing therapeutic angiogenesis, a variety of growth factors, such as vascular endothelial growth factor, fibroblast growth factor, or insulin-like growth factor, were perfused in the blood, injected locally, or administered by slow release. (19) For example, fibroblast growth factor was adsorbed to microspheres of precapillary size that were injected via a coronary catheter. Fibroblast growth factor was released from these microspheres and taken up by endothelial cells, which started to proliferate. In the future, this method for application of growth factors would allow for the delivery of angiogenic substances to selected parts of the heart or other organs, apparently without causing inflammation or ischemia. (20)
Use of angiogenic biomaterials, obtained by the embedding of angiogenic factors in biogels, is another method for local stimulation of angiogenesis. For example, vascular endothelial growth factor embedded in fibrin matrices (21) or covalently conjugated with fibrin (22) were reported to be effective stimulants of endothelialization.
Alternatively, the genes encoding angiogenic factors have been introduced at sites where angiogenesis is needed, either by viral or liposomal vectors, or as naked plasmids. (23)
As it was aptly stated, however, therapeutic angiogenesis is "not yet for prescription." (24) Several clinical phase I trials suggested the feasibility and short-term safety of treatment with angiogenic factors or with their genes. The VIVA trial, a phase II trial described recently, did not demonstrate any benefit (such as the increase of exercise time or reduction of angina) for the patients treated with vascular endothelial growth factor over a strong placebo effect. Moreover, there are concerns that pathologic processes dependent on angiogenesis, for instance tumor development, atherosclerosis, and proliferative retinopathies, may be exacerbated, requiring further careful monitoring. Another problem is related to the direct effect of the locally administered growth factors, recently shown to induce nonfunctional capillary tubes, and/or the occurrence of angioma (25) rather than functional capillaries. Consequently, proangiogenic and proarteriogenic therapies will need further investigation before they enter common clinical practice.
A NONSPROUTING MECHANISM FOR MICROVASCULAR LUMEN FORMATION
We recently discovered the importance of a preformed appropriate local topology for the development of capillaries in vivo. (26) In this respect, we described the role played by monocytes/macrophages in preparing the extracellular matrix bed for the development of new capillaries and/or for tissular insemination of circulating precursor endothelial cells.
In the cardiac tissue of a transgenic mouse in which monocytes were attracted to the myocardium by the targeted overexpression of monocyte chemoattractant protein 1 (MCP-1), we found tunnels that were endothelial nitric oxide synthase-, and platelet endothelial cell adhesion molecule 1-negative, and that occasionally contained blood-derived cells. Immunohistochemical data showed that monocytes/macrophages drilled these tunnels using the proteolytic enzyme mouse macrophage metalloelastase. DNA synthesis and the neoendothelial markers present in the microvasculature of MCP-1 mouse hearts indicated an active angiogenic process. Based on these data, we suggested that the macrophage-drilled tunnels might evolve to become "true" capillaries, when connected to the existing vessels, and might possibly be colonized by circulating endothelial cell progenitors. This phenomenon might represent another mechanism, in addition to the secretion of the angiogenic factors, by which monocytes/ macrophages could participate in the elaboration of new blood vessels in adult tissues.
Angiogenesis appears, therefore, to be a cooperative process, seldom involving a single cell type. In particular, cells with high penetration capabilities are likely to assist the progression of fragile capillary sprouts or, alternatively, of endothelial precursors. This assertion is strongly supported by in vitro data as well, in which either candidate pericytes (27) or a subpopulation of endothelial cells (28) were shown to play a role in the processing of extracellular matrix to the benefit of actual capillaries.
The mechanism of capillary lumen formation described in the context of a sprouting paradigm is the transcellular fusion of vacuoles occurring within neighboring endothelial cells. (29) The lesson derived from our observations, however, is that (in some instances at least) the capillary lumen is apparently formed before the cellular structure itself. If so, this principle may be applied for the design of replacement microvessels.
DESIGN OF AN ANGIOGENESIS ASSIST DEVICE
The success of therapeutic angiogenesis relies on the assumption of preexistence of intact microvessels, containing viable endothelial cells, in tissues or in proximity to the sites where angiogenesis is expected to be induced. The sprouting mechanism suggests that new functional capillaries then develop from these mature sources, as an effect of stimulation by angiogenic factors. Obviously, in many instances in which the local microcirculation is compromised, this condition is not met. In severe cases of tissue ischemia and/or necrosis, no viable microvessels exist to sprout new capillaries.
Taking this fact into consideration, we suggested a different approach, which combines the current developments in cellular biology of endothelium with the advantages of several recently developed technologies. In particular, we propose the implementation of a more complex protocol, in which both the cellular "seeds" and the stimuli for their growth are provided through the use of a silicon-based "angiogenesis assist device," or "angiochip."
The platform for the creation of the angiochip would be the nanofilter-based, drug-delivery silicon capsules developed by our group. (30) By surface micromachining and sacrificial layer technology, our team succeeded in creating molecular filters with pores of controlled size in the 10- to 200-nm range ("nanofilters"). These filters, incorporated into millimeter-sized polyhedral silicon frames, already have multiple applications, ranging from molecular filtering (31) to controlled drug-delivery systems. (32,33)
Other groups have recently shown that silicon microchips have the ability of storage and controlled release of multiple chemicals (eg, by electrochemical dissolution of thin anode membranes covering microreservoirs filled with chemicals in solid, liquid, or gel form). (34) Future integration of electronics, such as microprocessors, remote control units, or biosensors, may lead to the development of "smart" microchip implants or tablets that release drugs into the body either automatically or when needed. (35)
Angiogenic or angiostatic substances may be placed inside these nanofilter-based capsules. To function as an endothelial carrier, the surface of the silicon capsule can be covered with endothelial cells. As a refinement, our suggested tunneling mechanism of angiogenesis sets the background for the use of capsules with a micromachined surface provided with capillary-sized grooves, in which the endothelial cells could be assembled in microvascular-like channels. (36) On implantation along with their carrier (the grooved capsule), these capillary primordia would develop under the effect of angiogenic factors released from inside and become connected to the blood supply. This capillary-like network would fuse with the patient's own microvasculature in a stage of capsule-driven angiogenic activity, resembling autograph revascularization. (7)
The angiochip, as a hybrid bioinorganic device, would have several advantages: (a) controlled delivery of angiogenic factors useful for the development of both the exogenous and the endogenous microvasculature, (b) the ability to provide the implanted tissue with a fresh endothelial network from which further sprouting can proceed, (c) mechanical robustness required for implantation and retrieval using surgical means, (d) repeated use of the same device, (e) inclusion in the capsule's wall of miniature electronic components for remotely activated release of capsule content, and (f) compatibility with further incorporation of electronic biosensors for in situ diagnostics and for the individualized administration of angiogenic therapy.
This work was supported in part by the Shannon Award (RR13370) from the National Institutes of Health (NIH), Bethesda, Md (Dr Ferrari), and by NIH grant RO1 HL 65983 (Dr Moldovan). The authors are grateful to Pascal Goldschmidt-Clermont, MD, and Derek Hansford, PhD, for useful suggestions, and to Christine Roos for critical reading of the manuscript.
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(36.) Moldovan NI. Assisting the birth of new blood vessels: from cells to the silicon chip, and retour. Paper presented at: BioMEMS and Biomedical Nanotechnology World 2000 Congress; September 23-26, 2000; Columbus, Ohio. Available at: http://www.healthtech.com/conference/00mem/abstracts/moldvan.htm.
Accepted for publication September 20, 2001.
From the Biomedical Engineering Center and Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio. Dr Ferrari is the scientific founder of iMEDD, Inc, dedicated to the exploitation of some applications of the nanopore-based capsule briefly described in the text, and he owns stock options in the company. Dr Moldovan has no material interests related to this work.
Presented at the 10th Annual William Beaumont Hospital Seminar on Molecular Pathology, DNA Technology in the Clinical Laboratory, Royal Oak, Mich, March 8-10, 2001.
Reprints: Nicanor I. Moldovan, PhD, Heart and Lung Research Institute, Room 305A, 473 W 12th Ave, Columbus, OH 43210 (e-mail: email@example.com).