WO1999047047A1

WO1999047047A1 - Biological modification of vaso-occlusive devices

Info

Publication number: WO1999047047A1
Application number: PCT/US1999/005889
Authority: WO
Inventors: Gregory Anthony Helm; David Forest Kallmes; Harry J. Cloft
Original assignee: University Of Virginia Patent Foundation
Priority date: 1998-03-18
Filing date: 1999-03-18
Publication date: 1999-09-23
Also published as: AU3098099A

Abstract

Biologically modified vaso-occlusive devices which are seeded with a cell line and methods for the treatment of intracerebral aneurysms, arteriovenous malformations, and arteriovenous fistulae and for the local delivery of a cell line using such devices are provided. The vaso-occlusive devices are biologically modified to improve long term occlusion rates and increase intravascular scar formation. The cell line may be genetically modified to secrete desired proteins or growth factors such as transforming growth factors, fibroblast growth factors, platelet derived growth factors, epidermal growth factors, and mixtures thereof. The genetically modified cell line can also contain promoters which are inducible in order to control cell proliferation, gene expression, or combinations thereof.

Description

BIOLOGICAL MODIFICATION OF VASO-OCCLUSIVE DEVICES

FIELD OF THE INVENTION

The present invention relates to the fields of neurosurgery, endo vascular therapy, and interventional neuroradiology. In particular, it involves the biological modification of vaso-occlusive devices for the treatment of aneurysms, arteriovenous fistulae ("AVF") and arteriovenous malformations ("AVM"). as well as for the local delivery of a cell line to the vasculature of the patient to be treated.

BACKGROUND OF THE INVENTION Endovascular therapy has long been used in treating a variety of different conditions, including control of internal bleeding, occlusion of blood supply to tumors, and relief of vessel wall pressure in the region of an aneurysm. A variety of different embolic agents and vaso-occlusive devices are known as suitable for such therapy. One such class of embolic agents includes injectable fluids or suspensions, such as microfibrillar collagen, various polymeric beads, formable polymers, and polyvinyl alcohol foam. The polymeric agents additionally may be crosslinked, sometimes in vivo, to extend the persistence of the agent at the desired vascular site. These agents are often introduced into the vasculature through a catheter. After such introduction, the agents form a solid space-filling mass. Although they provide good short-term vaso-occlusion, they are ultimately reabsorbed in the process of vessel recanalization.

More common are vaso-occlusive devices. One such device is a balloon which may be carried to the vessel site at the end of the catheter, inflated with a suitable fluid, typically a polymerizable resin, and released from the end of the catheter. The balloon device has the advantage that it effectively fills the cross- section of the occluded vessel. However, when using intravascular balloon embolization of intracerebral aneurysms, inflation of a balloon into the aneurysm carries some risk of aneurysm rupture due to possible "overfilling" of portions of the aneurysm and due to the traction produced when detaching the balloon from the end of the catheter. Moreover, a vascular balloon can be difficult to retrieve after the resin within the balloon sets. Balloons have also been known to rupture during filling, to release prematurely during filling, or to leak monomeric resin into the vasculature during the period before the monomer sets into polymeric form.

Another type of vaso-occlusive device is a wire coil or braid which can be introduced through a catheter in stretched linear form and then assumes an irregular shape upon discharge of the device from the end of the catheter. For instance, U.S. Pat. No. 4.994,069, to Ritchart et al, describes a vaso- occlusive coil that assumes a linear helical configuration when stretched and a folded, convoluted configuration when relaxed. The stretched condition is used in placing the coil at the desired site (by its passage through the catheter) and the coil assumes a relaxed configuration (which is better suited to occlude the vessel) once the device is so placed. Ritchart et al. describe a variety of shapes. The secondary shapes of the disclosed coils include "flower" shapes and double vortices. A random secondary shape is described as well.

Vaso-occlusive coils having attached fibrous elements in a variety of secondary shapes are shown in U.S. Pat. No. 5,304,194, to Chee et al. Chee et al. describe a helically wound device having a secondary shape in which the fibrous elements extend in a sinusoidal fashion down the length of the coil. These coils, as with Ritchart et al., are produced in such a way that they will pass through the lumen of a catheter in a generally straight configuration and, when released from the catheter, form a relaxed or folded shape in the lumen or cavity chosen within the human body. The fibrous elements shown in Chee et al. enhance the ability of the coil to fill space within the vasculature and to facilitate formation of embolus and subsequent allied tissue.

A vaso-occlusive device with a stretch-resisting member contained therein is disclosed in U.S. Pat. No. 5,833,705 to Ken et al. The device comprises a helically wound coil which is formed by winding a wire into a first or primary helix to form an outer helical member having first and second ends. A stretch resistant member extends through the lumen and is fixedly attached to the coil in at least two locations. The primary helix, with its included stretch- resistant member, may be wound into a secondary form and heat-treated to preserve that form, desirably prior to the step of including the stretch-resisting member into the coil. The secondary form may be one which, when ejected from a delivery catheter, forms a specific shape. Such a shape might, e.g., fill a vascular cavity such as an aneurysm. The stiffness of the various parts of the coil may be tailored to enhance the utility of the device for specific applications. Fibrous materials may also be woven into the member or tied or wrapped onto it.

There are a variety of ways of discharging shaped coils and linear coils into the human vasculature. In addition to those patents which apparently describe the physical pushing of a coil out into the vasculature (e.g., Ritchart et al.), there are a number of other ways to release the coil at a specifically chosen time and site. U.S. Pat. No. 5,354,295 and its parent, U.S. Pat. No. 5,122,136, both to Guglielmi et al. describe an electrolytically detachable embolic device.

A variety of mechanically detachable devices are also known. For instance, U.S. Pat. No. 5,234,437, to Sepetka, shows a method of unscrewing a helically wound coil from a pusher having interlocking surfaces. U.S. Pat. No. 5,250,071, to Palermo, shows an embolic coil assembly using interlocking clasps mounted both on the pusher and on the embolic coil. U.S. Pat. No. 5,261,916, to Engelson, shows a detachable pusher- vaso-occlusive coil assembly having an interlocking ball and keyway-type coupling. U.S. Pat. No. 5,304,195, to Twyford et al., shows a pusher- vaso-occlusive coil assembly having an affixed, proximally extending wire carrying a ball on its proximal end and a pusher having a similar end. The two ends are interlocked and disengage when expelled from the distal tip of the catheter. U.S. Pat. No. 5,312,415, to Palermo, also shows a method for discharging numerous coils from a single pusher by use of a guidewire which has a section capable of interconnecting with the interior of the helically wound coil. U.S. Pat. No. 5,350,397, to Palermo et al., shows a pusher having a throat at its distal end and a pusher through its axis. The pusher sheath will hold onto the end of an embolic coil and will then be released upon pushing the axially placed pusher wire against the member found on the proximal end of the vaso-occlusive coil.

As discussed above, embolic coils have been used for purposes of vascular occlusion. A recent advance in coil technology is the development of electrolytically detachable platinum coils, marketed under the trade name Guglielmi Detachable Coil (GDC, Target Therapeutics, Fremont, CA). The GDC has gained widespread use in the treatment of intracranial aneurysms, with greater than 16,000 patients treated to date worldwide with the device. The design of the GDC allows safe coil delivery into aneurysms. The soft, pliable nature of the platinum used to construct the coil minimizes risk of vascular perforation, and the nonthrombogenic, inert nature of the platinum results in low rate of thromboembolic complications.

While embolic coils represent an important advance in the endovascular approach to cerebral aneurysm therapy, recent studies have shown disappointing results for large and giant aneurysms in both initial rates of aneurysm occlusion and in aneurysm recanalization. Aneurysm recanalization may result from either coil compaction or from growth of the aneurysm itself, but in most cases coil compaction is the primary mode of recanalization. While not being bound by theory, it is believed that coil compaction results from continuous pulsatile flow directed at the coil mass. The soft, pliable nature of the coil likely facilitates such compaction. In addition, the non-thrombogenic, inert nature of the coil results in a lack of organized thrombus formation within the aneurysm cavity. This lack of thrombus formation facilitates coil compaction in large and giant aneurysms, with resultant regrowth of the aneurysm lumen.

One proposed method, disclosed in U.S. Pat. No. 5,749,894 to Engelson, for reducing coil compaction includes the steps of sequentially or simultaneously at least partially filling the selected aneurysm with a coil and further with a formable polymeric composition. The coil device may be at least partially coated with the formable polymeric composition. The polymeric composition in-fills the interstices of the coil upon treatment with light or radio frequency radiation. The sequential or simultaneous step involves introduction of a polymeric material which can be coalesced, reformed, or solidified in the vasculature by use of heat applied with an amount of radiant energy, e.g., radio frequency (R.F.) or light. Additionally, various modifications aimed at increasing the "biological activity", or the activity relating to thrombus formation, collagen formation, or endothelialization within and adjacent to the aneurysm cavity, of a platinum coil have been proposed. These modifications have included additions of polyurethane coatings, collagen filaments, and basement membrane protein coatings. In vivo data suggests that these modifications may yield increases in the biologic activity of the coils.

Previous investigators have proposed the growth of cells onto endovascular devices before insertion. These techniques have included seeding of immortalized human endothelial cells. Others have suggested seeding the surface of metallic stents with cells that secrete tissue plasminogen activator, in hopes of improving short and long-term patency. However, none of these methodologies suggest the use of biologically modified vaso-occlusive devices in the treatment of malformations, such as intracerebral aneurysms, arteriovenous fistulae and arteriovenous malformations. None of the above mentioned modifications addresses the problems with vaso-occlusive devices, which are the subject of this invention .

It is therefore an object of the present invention to provide vaso- occlusive devices which lead to improved intravascular scar formation and improved device efficacy, as well as allow the local delivery of the 'biological modification' to selected sites within the vasculature of the patient to be treated. In particular, it is a further object of the present invention to provide an embolic coil with improved long term occlusion rates, increased intravascular scar formation, and reduced incidence of coil compaction.

These and other objects of the present invention will be apparent from the description of the invention which follows. SUMMARY OF THE INVENTION

The present invention addresses the foregoing problems and objects by providing a biologically modified vaso-occlusive device which is seeded with a desired cell line. The vaso-occlusive device improves long term occlusion rates and increases intravascular scar formation and allows for the local delivery of the desired cell line to a specific site within the vasculature of the patient to be treated.

Another aspect of the present invention is drawn to a method for at least partially filling an aneurysm comprising introducing into the aneurysm a biologically modified vaso-occlusive device which is seeded with a cell line. The method improves long term occlusion rates and increases intravascular scar formation.

In preferred embodiments, the cell line may be genetically modified to secrete desired proteins or growth factors, such as transforming growth factors (TGF), fibroblast growth factors (FGF), platelet derived growth factors (PDGF), epidermal growth factors (EGF), and mixtures thereof.

In yet other embodiments, the genetically modified cell line can also include promoters which are inducible to control cell proliferation, gene expression, or combinations thereof. Yet another aspect of the present invention relates to methods for locally delivering a desired cell line preferably capable of secreting desired proteins and growth factors to selected sites within the vasculature of the patient to be treated.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 illustrates a photomicrograph of a sample taken at day 14 from a control animal (rat 2) using an unmodified control coil.

Figure 2 shows a photomicrograph of a sample taken at day 14 from a test animal (rat 4) using a biologically modified coil of the invention.

Figure 3 illustrates a photomicrograph of a sample taken at day 35 from a control animal (rat 5) using an unmodified control coil. 7

Figure 4 shows a photomicrograph of a sample taken at day 35 from a test animal (rat 7) using a biologically modified coil of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The biologically modified vaso-occlusive devices of the invention may include a wide variety of vaso-occlusive devices known in the art. For example, the device can be selected from the group consisting of a balloon, a coil, a braid, tufted or looped fibers, a ball or sphere of natural fibers, embolic fluids, biological matrices, non-cytotoxic polymers, and combinations thereof. The tufted or looped fibers can, for example, be formed of steel wool, and the ball or sphere of natural fibers can include a cotton ball or sphere or a wool ball or sphere. Embolic fluids can be any suitable agent known in the art which is capable of supporting cell growth. For instance, laminin, collagen, elastin, fibronectin, other extra-cellular matrix proteins and polypeptides, or combinations thereof can be used as embolic fluids. Additionally, the biological matrices can be any such matrices known in the art such as those formed from biological polymers, i.e. laminin, collagen, and fibronectin. Also, the non- cytotoxic polymers can be any polymer either naturally occurring or synthetic which is capable of supporting cell growth, such as fibrinogen (fibrin glue) or biocompatible polymer hydrogels. For example, the fibrinogen may be derived from a bovine source or be of human origin. Generally, embolic fluids, biological matrices, and non-cytotoxic polymers such as those disclosed in U.S. Patent No. 5,752,974 to Rhee et al., which is herein incorporated by reference for all purposes and in a manner consistent with this disclosure, that are capable of supporting cell growth can be used as a vaso-occlusive device of the invention.

Preferably, a coil, braid, or tufted or looped fibers can be used. The coil, braid, or tufted or looped fibers can be three-dimensional, helical, spherical, cylindrical, ovoid in shape, or may have other distorted spherical shapes and can contain both primary and secondary configurations, as well as stretch resistant components, such as disclosed in U.S. Patent Nos. 4,994,069 to Ritchart et al., 8

5,304,194 to Chee et al, and 5,833,705 to Ken et al., which are herein incorporated by reference in a manner consistent with our disclosure. Preferred materials of construction for the coil, braid, or tufted or looped fibers are platinum, tungsten, stainless steel, gold, palladium, nickel-titanium and their alloys, with platinum being the most preferred.

The vaso-occlusive device is biologically modified according to the present invention through seeding the device with a desired cell line. The desired cell line may be obtained from the patient to be treated, or from an established cell line. One possible source of a patient-derived cell line may be autologous dermal fibroblasts. The desired cell line used can include fibroblasts, endothelial cells, muscle cells, stem cells, and mixtures thereof. In preferred embodiments, the desired cell line may be genetically modified by methods generally known in the art, such as disclosed in Sambrook et. al., Molecular Cloning, A Laboratory Manual, Second Ed, 1989, pp. 16.30-16.67, which is herein incorporated by reference. The desired cell line can be genetically modified to secrete desired proteins or growth factors such as, but not limited to, transforming growth factors (TGF), fibroblast growth factors (FGF), preferably basic fibroblast growth factors (basic FGF or bFGF), platelet derived growth factors, epidermal growth factors, and mixtures thereof. In yet other embodiments, the genetically modified cell line can also contain promoters which are inducible in order to control cell proliferation, gene expression, or combinations thereof. For example, it is known that suicide genes, such as those encoding for thymidine kinase, allow selective cell killing under the control of acyclovir. See, for example, Oldfield et al., Gene therapy of the treatment of brain tumors using intratumolar transduction with the thymidine kinase gene and intravenous gangciclovir, Hum. Gene Ther. 1993; 4:39-69, which is incorporated herein by reference for all purposes and in a manner consistent with our disclosure. In addition, genes can be regulated under the control of inducible promoters, the activity of which depends on substances such as tetracycline. See Miller et al., Progress in transcriptionally targeted and regulatable vectors for gene therapy, Hum. Gene Ther. 1997; 8:803-815, also incorporated herein by reference for all purposes and in a manner consistent with the disclosure. These techniques are examples of inducible control mechanisms which can be used in the biologically modified vaso-occlusive device of this invention. The biological modification (or seeding) of the vaso-occlusive device with the desired cell line may be performed in any suitable manner, e.g., it may involve incubating the device with growth media including the desired cell line until the desired level of confluence on the surface of the vaso-occlusive device is obtained. The desired level of confluence means that the cells have grown on the vaso-occlusive device to such an extent as to provide for an adequate degree of cellular proliferation in vivo to improve the performance of the vaso- occlusive device as herein disclosed, e.g., to enhance thrombogenecity, to improve scar formation, and/or to enable the local delivery of a cell line to a site in the vasculature of a patient. The seeding of the vaso-occlusive device may also include sterilizing the device prior to incubation. The incubation can take place at conditions suitable for the growth of the desired cell line. Incubation conditions can be optimized by methods known in the art and preferably include temperature of 37 °C, 95% relative humidity and 5-10% by vol. CO₂.

The growth media can be developed by methods well known in the art. For example, one suitable growth medium includes Dulbecco Modified Eagle Medium with 5% fetal bovine serum. The concentration of cells of the desired cell line originally added to the tissue culture dish ("seeding density") can be optimized by methods known in the art. In one embodiment, the seeding density is about 7.5 X 10⁵ to about 10 X 10⁵ cells per ml of the growth medium. During incubation, the cells migrate onto the device and proceed to grow onto the inner and outer surface of the vaso-occlusive device until the desired level of confluence is obtained. Preliminarily, it may be desirable to coat some types of vaso-occlusive devices, such as a coil, a balloon, a braid or non-cytotoxic polymers, with a coating, such as collagen, extracellular matrix proteins, laminin, fibronectin, elastin, or combinations thereof to enhance cellular adhesion and growth. The coating can be applied to the vaso-occlusive device 10

initially, and then the desired cell line can be grown on the coating. The coating may have any desirable thickness, so long as it does not impede the growth of the cells and it does not impede the insertion of the vaso-occlusive device into the patient's body. The biologically modified vaso-occlusive devices of the invention can be used for the treatment of aneurysms. arteriovenus fistulae ("AVF"), and arteriovenus malformations ("AVMs"), as well as for the local delivery of a desired cell line to specific sites within the vasculature of a patient to be treated. Once the vaso-occlusive device has been biologically modified and the desired cell line has reached the desired level of confluence, the biologically modified vaso-occlusive device can be inserted into the patient^'s vasculature and guided to the desired location within the patient, such as to an aneurysm, an AVF, or AVM, in a usual fashion known in the art. Suitable methods are disclosed, for example, in U.S. Pat. Nos. 4,994,069 to Ritchart et al.; 5,234,437 to Sepetka; 5,250,071 to Palermo; 5,261,916 to Engelson; and 5.354,295 and 5,122,136, both to Guglielmi et al., all of which are herein incorporated by reference for all purposes and in a manner that is consistent with our disclosure. Additionally, embolic fluids, biological matrices, and non-cytotoxic polymers seeded with a cell line can be introduced into the patients vasculature through the use of a delivery balloon or a catheter , and thereafter the balloon or catheter is removed from the patient's vasculature. Such delivery methods are well known in the art. While not wishing to be bound by any theory of operability, it is believed that within the patient's vasculature the cells divide and multiply on the surface of the biologically modified vaso-occlusive device. It is believed that the cells increase thrombus organization, improve endothelialization, and produce collagen and other matrix proteins, as well as any proteins and growth factors which the cells have been genetically engineered to produce, thereby leading to increased scar formation and providing for improved occlusion and local delivery of the desired cell line. Certain biologically modified vaso-occlusive devices, such as coils or braids, may further include fibrous adjuncts, e.g., loops, tufts, braided coverings, 11

or a combination thereof, to enhance the thrombogenicity. The fibrous adjuncts can be attached to the outside of the appropriate vaso-occlusive device in any suitable manner and can have shapes, sizes, and configurations known in the art, as disclosed for example in U.S. Pat. No. 5,304,194 to Chee et al. which is incorporated herein by reference for all purposes and in a manner consistent with our disclosure.

Also, cellular adhesion to a vaso-occlusive device may be improved with surface modification of the vaso-occlusive device with ion implantation, performed in a manner known in the art. One suitable method is disclosed, for example, in Murayama et al., Ion implantation and protein coating of detachable coils for endovascular treatment of cerebral aneurysms: concepts and preliminary results in swine models, Neurosergery 1997; 40:1233-1244, incorporated herein by reference for all purposes and in a manner consistent with our disclosure. Further, more than one of the biologically modified vaso-occlusive devices of the invention can be introduced into the patient's vasculature at the desired location. For instance, biologically modified vaso-occlusive devices such as coils, balloons, tufted or looped fibers, balls or spheres of natural fibers, non-cytotoxic polymers, and biological matrices can be introduced into the patient's vasculature in conjunction with an embolic fluid, such as fibrillar collagen, non-fibrillar collagen, laminin, or mixtures thereof. In particular, suitable embolic fluids and methods for their introduction into the patient's vasculature are disclosed, for example, in Rhee et al., U.S. Pat. No. 5,752,974, which is herein incorporated by reference for all purposes and in a manner consistent with our disclosure. The embolic fluid itself can also be biologically modified if desired. Additionally, the biologically modified vaso-occlusive device may be introduced into the patient's vasculature in conjunction with a formable polymeric composition (or "formable polymer") which may be reformed , solidified, cured, or crosslinked in situ. The terms "reformed", "solidified", "cured", or "cross-linked" when applied to the formable polymeric composition are used interchangeably herein. The polymeric composition may 12

be reformed by any suitable method, such as via light, heat, or R.F. The formable polymeric composition may be introduced into the patient's body separately from the vaso-occlusive device, substantially simultaneously with, or subsequently to, the introduction of the vaso-occlusive device. The formable polymeric composition can be introduced into the patient's body in any suitable manner, such as those in U.S. Pat. No. 5,749,894 to Engelson, which is herein incorporated by reference for all purposes and in a manner that is consistent with our disclosure. Formable polymers such as those disclosed in Engelson can be used. As used herein, the term "collagen" is intended to encompass collagen of any type, from any source, including, but not limited to, collagen extracted from tissue or produced recombinantly, collagen analogs, collagen derivatives, modified collagens, and denatured collagens.

The invention will be further described with reference to the following illustrative examples utilizing an embolic coil, the Guglielmi detachable coil (GDC), as the vaso-occlusive device and fibroblasts as the cell line.

Example 1 : Characterization of Growth Pattern of Fibroblasts on GDC Coil in vitro A. Procedure Modified fibroblasts were co-cultured with short segments of platinum embolic coils. The coil segments 10mm in length were cut from 8-mm x 30-cm GDC devices (GDC-10, GDC-18; Target Therapeutics, Fremont, Calif). These coils are constructed from platinum filaments with a diameter of 0.002 inch (0.05 mm) and 0.004 inch (0.1mm) for the GDC-10 and the GDC-18, respectively. These 10-mm-long segments of coil were placed in the well plates immediately after the suspended cells had been added to the walls. Prior to placing the coil segments in the tissue culture plates, the coil segments were immersed in a solution containing either murine laminin (10 μg/ml) (Sigma Chemical), human type I collagen (100 μg/ml) (Gibco/Life Technologies), murine type IV collagen (100 μg/ml) (Gibco/Life Technologies), or human fibronectin (100 μg/ml) (Gibco/Life Technologies) for 45 minutes and were 13

then air dried Uncoated coil segment controls containing a strand of bovine type I collagen (ReGen Biologies, Franklin Lakes, NJ) within the lumen were also studied. The cultures of fibroblasts and coil segments were viewed daily using an inverted microscope to assess the extent of cellular proliferation on the coil surfaces. In addition, selected samples were processed for scanning electron microscopy (SEM).

The fibroblast cell line used in this example was a cell line of genetically modified NIH 33 fibroblasts. These cells had been transfected with a Zip-neo vector that contained a 1.1 kilobase insert of copy DNA that coded four separate clones of basis Fibroblast Growth Factor (basic FGF or bFGF). These cells constitutively produce bFGF. Cells were grown in Dulbecco modified Eagle medium (Gibco/Life Technologies, Gaithersburg, MD) with 5% fetal bovine serum (Hyclone Laboratories, Logan, Utah), penicillin (100 U/mL) (Sigma Chemical, St. Louis, MO), streptomycin (0.1 mg/mL) (Sigma Chemical), and amphotericin-B (25 μg/mL) (Sigma Chemical). Suspensions with 75,000 - 100,000 cells were added to 12-well plates (Falcon Products, Franklin Lakes, NJ).

Basic FGF serves to promote proliferation and migration of endothelial cells and fibroblasts, and serves to increase the rate of collagen synthesis by fibroblasts. These mechanisms of action tend to improve the efficacy of coil immobilization by forming a stable, fibrous matrix covered by intact endothelium.

The assessment of cell density was performed by two investigators, blinded to the type of coating at the time of viewing, who arrived at a consensus opinion with regard to the predominant pattern of cell growth along the coils. To that end, a grading system was developed for the semiquantitative measurement of cell density along the coils. The grading system included a five-point scale, as follows: 1 = no cells, 2 = scattered single cells, 3 = near confluence, 4 = single-layer confluence, 5 = multilayer confluence. (Kallmes et al., In Vitro Proliferation and Adhesion of Basic Fibroblast Growth Factor- Producing Fibroblasts on Platinum Coils, 206 Radiology 237 (1998), 14

incorporated herein by reference, includes Figures illustrating coil samples with cell density corresponding to the five-point scale.) To assess the influence of various coil coatings on the rapidity of cell proliferation on the coils, multiple samples of GDC- 10s and GDC- 18s were coated as described earlier. Each sample was evaluated on days 1, 2 and 3 of culture.

Prompt cell proliferation over the surface of the coil segments such that confluence was attained by day 3 was observed for coated and uncoated GDC- 10s and DC-18s coils. In addition, the cells had a propensity to grow between the primary winds of the coils into the central lumen of the coil segments. The coating of the coil segments with fibronectin, laminin, and type I collagen slightly increased the rate of cell proliferation, while there appeared to be an inhibitory effect of type IV collagen on the rate of cell growth, as compared to all other samples. While not being bound by theory, it is believed that the form of type IV collagen used may have contained other biologically active molecules, such as transforming growth factor β, that could inhibit the in vitro proliferation of fibroblasts.

The coil samples that housed the collagen filament demonstrated growth patterns similar to those of other samples, except that the filament appeared to inhibit the ingrowth of cells into the coil lumen, probably because of swelling of the collagen filament in vitro to occupy the entire lumen of the coil.

B. Secretion of Growth Factor by Modified Fibroblasts Measurement of bFGF concentrations in the media after cells attained confluence was performed with the cell supernatant by using an enzyme-linked immunosorbent assay (ELISA) (Quantikine Human FGF basic immunoassay; R&D Systems, Minneapolis, Minn).

The ELISA demonstrated concentrations of bFGF on the order of 800- 1200 pg/mL, which is greater than the concentration typically required to exert biological activity in vivo.

C. Retention of Viable Cells after Passage Through Microcatheter After the cells had attained confluence over the surface of the coil segment, the coil segment was passed through a Tracker 18 microcatheter 15

(Target Therapeutics) and then placed back into growth medium. The coils were viewed through an inverted microscope immediately before and immediately after passage through the microcatheter.

The vast majority of the cells had been removed from the surface of the coil segment, but the cells within the inner lumen of the coil segment remained in place. Furthermore after placing the coil segment back into the growth medium, there was prompt regrowth of the cells, indicating that the cells retained inside the lumen of the coils were viable. D. Discussion of Results The results demonstrate that genetically modified NIH 3T3 cells grow readily on GDCs in vitro. Coating of coils with fibronectin, laminin, and type 1 collagen slightly increases the rate of cell proliferation, but even uncoated GDC- 18 coils reached confluence in less than 48 hours. Uncoated GDC- 10s showed slightly less rapid cell proliferation, but near confluence was reached by day 3. The reason for slight differences between findings from uncoated GDC-10 and GDC-18 samples is unclear. However, near confluence of cells along the surface of the coil is considered to be adequate and it is not believed that the slight differences between uncoated GDC-10 and GDC-18 coils are important. With both GDC-10 and GDC-18 coils we noted rapid growth of cells between the primary coil windings into the lumen of the coils. This occurred even though the diameter of a typical fibroblast is several times wider than the stated tolerance of the interwinding distance of less than 0.001 -inch (0.025 mm). The viability of the cells that reside in the lumen of the coils was shown by means of the rapid proliferation of cells after the coils were reimmersed in the medium after embolization through the microcatheter. As such, even though cells are sheared from the outer surface of the coil during passage through the micro-catheter, our system allowed reliable deposition of cells.

Example 2: In Vivo Growth of Modified Fibroblasts In vivo Experiments were performed to demonstrate that platinum embolic coils can be used to deliver genetically modified, growth factor- 16

secreting fibroblast grafts into the endovascular space. See Kallmes et al., Platinum Coil-mediated Implantation of Growth Factor-secreting Endovascular Tissue Grafts: An In Vivo Study, 207 Radiology 519 (1998), incorporated herein by reference. A. Procedure

As outlined in Example 1 , genetically modified fibroblasts were grown onto GDCs in the same manner as in Example 1. Ten-millimeter-long segments were cut from an 8-mm x 30-mm GDC-18 device (Target Therapeutics, Fremont, CA). These coil segments were placed into the tissue culture well plates immediately after the cells were plated. The coils were viewed daily with an inverted microscope until the cells reached confluence along the outer surface of the coil. Once confluence was attained, the biologically modified GDCs were implanted into a given animal.

All animal experimentation was performed under an approved protocol reviewed by the Animal Research Committee at applicant's institution.

Because a murine fibroblast cell line was used, nude rats were needed to reduce the difficulties related to xenograft rejection. The rats (weight, 150-200 g) (Harlan Sprague Dawley, Indianapolis, IN) were anesthetized with intramuscular administration of ketamine hydrochloride (80 mg/kg) and xylazine hydrochloride (8 mg/kg). A midline incision was performed over the neck. The left carotid sheath was exposed and carefully incised, and the common carotid artery was isolated. Proximal and distal control of the vessel was achieved with 4-0 silk suture. A small incision was made in the middle portion of the artery; a beveled 24-guage Teflon sheath was inserted into the incision in retrograde fashion. The tip of the sheath was placed approximately 10 mm cephalic to the origin of the left common carotid artery.

One-centimeter segments of a GDC-18 were prepared, as in Example 1, each of which was placed in a given animal. These GDC segments comprised unmodified coils (n = 4) to serve as control samples and test coils coated with genetically modified fibroblasts (n = 4). Coil segments were loaded manually into the plastic introducer provided in the standard packaging material for the 17

GDC. The introducer was opposed to the hub of the indwelling arterial sheath. The coil segments were introduced into the artery of the given animal with a 0.018-inch-diameter coil pusher (Target Therapeutics) under direct visualization. The caudal end of the coil segment was positioned approximately 3 mm cephalic to the origin of the left common carotid artery. The sheath was then removed, and the distal aspect of the artery was ligated with 4-0 silk suture. The wound was closed with running suture. The incubation time was 14 days (control coils, n = 2; test coils, n = 2) or 35 days (control coils, n = 2; test coils n = 2). At the end of the respective incubation times, the animals were anesthetized with ketamine and xylazine, and transcardiac perfusion was performed with 4% paraformaldehyde. The coil segments were carefully harvested, along with surrounding soft tissues, and placed in 4% formaldehyde for at least 72 hours. The coil segments were than carefully extracted from the carotid artery, after which the tissues were mounted in paraffin. The samples were sectioned with a rotary microtome and stained with Hematoxylin-eosin and Trichrome stains. Histologic sections were viewed by a pathologist (M.B.S.L.), who was blinded to the treatment group. Hematoxylin-eosin stain was used primarily to assess cellular content, whereas Trichrome stain was used to evaluate cellular and collagen content. B. Results

At 14 days, animals with control coils showed early unorganized thrombus organization within the vessel lumen, while test animals showed extensive fibroblast proliferation within the vessel lumens. In the animals with control coils (see Figure 1), the common carotid artery was filled with blood elements, primarily red blood cells and fibrin. In most samples, there was early thrombus organization along the vessel wall. This finding was characterized by focal accumulation of fibrin, cellular elements, and early neovascularization and fibroblast proliferation, as shown in Figure 1. In Figure 1 , the coil was removed before embedding and C indicates the area formerly filled with the coil. The photomicrograph (axial orientation at approximately the center of the vessel 18

segment that contained the coil) shows that the vast majority of tissue in the vessel lumen is involuting red blood cells, with only a small amount of collagen formation and few fibroblasts (straight arrows). Note the vessel wall (curved arrows). (Note also, Trichrome stain used; original magnification xlOO). At 14 days in the animals with biologically modified test coils (see

Figure 2), the carotid artery was filled with cellular elements, primarily fibroblasts. Trichrome staining showed early collagen deposition. Small blood vessels (which indicate neovascularity) were also present. In Figure 2, the coil was removed before embedding. The photomicrograph (axial orientation at approximately the center of the vessel segment that contained the coil) shows that the vessel lumen is essentially occluded by the proliferation of fibroblasts and deposition of collagen (straight arrows). Note the vessel wall (curved arrows). (Note also, Trichrome stain used; original magnification xlOO).

At 35 days the difference between test and control samples was even more marked, with abundant collagen present within test samples compared to mild collagen formation in control samples. In the animals with control coils, there was a small amount of cellular proliferation and collagen formation along the periphery of the vessel lumen, as shown in Figure 3. In Figure 3, the coil and tissue within the lumen of the coil were removed before embedding. C indicates the area formerly filled with the coil. The photomicrograph (axial orientation at approximately the center of the vessel segment that contained the coil) shows the periphery of the vascular lumen only focally filled with fibroblast proliferation and a small degree of collagen deposition (short arrow). Note the vessel wall (long arrow). (Note also, Trichrome stain used; original magnification x 100).

At 35 days, in the animals with the biologically modified test coils of the invention (see Figure 4), there was a marked increase in the degree of cellularity within the vessel lumen compared with that in the 14-day samples, along with marked collagen production. Scattered areas of neovascularization were also seen. In Figure 4, the coil was removed before embedding and C indicates the area formerly filled with the coil. Photomicrograph (axial orientation at 19

approximately the center of the vessel segment that contained the coil) shows the vessel lumen filled completely with proliferating fibroblasts, with some expansion of the original vessel diameter. Note the vessel wall (arrow). (Note also, Trichrome stain used; original magnification xlOO). C. Discussion of Results

This study demonstrates the feasibility of endovascular cell delivery with the GDC. Marked proliferation of fibroblasts was noted at both 14 and 35 days after implantation in vessels with modified coils, whereas arteries with control coils demonstrated minimal fibroblast proliferation. Collagen formation was extensive in arteries with the biologically modified coils, whereas minimal collagen formation was noted in the control samples. The results suggest that long-term occlusion rates for large and giant cerebral aneurysms treated with embolic coils could be improved with coil-mediated implantation of growth factor-secreting fibroblasts using the biologically modified vaso-occlusive device of the invention.

Example 3 : In Vivo Aneurysm Model

Further in vivo testing of biologically modified embolic coils according to the invention requires a specialized model of aneurysms. Most previous animal models of human aneurysms have relied on surgical aneurysm creation, with arteriotomy and suturing of vein patch grafts. This procedure causes extensive local damage to the vessel wall with resultant release of growth factors and recruitment of macrophages and fibroblasts, thereby influencing any experimental results. Therefore, a non-surgical method for bifurcation aneurysm creation in a rabbit was developed. Briefly, a natural arterial trifurcation (the rabbit brachiocephalic artery, left common carotid artery, and aortic arch) are converted into a bifurcation aneurysm model. Initially, the left common carotid artery is occluded approximately 15 mm beyond its origin using a detachable latex balloon. The stump of the carotid artery is then incubated with intraluminal, concentrated porcine elastase. The result is an arterial bifurcation 20

aneurysm of approximately 4-7 mm in diameter, which is created non-surgically and has been validated to remain patent for at least four months. The lumen of the non-surgically created aneurysm is lined by intact endothelium, and demonstrates the attenuation of the elastic lamina typical of human saccular aneurysms. Notably, there is no propensity for fibroblast recruitment or proliferation and the endovascularly-created rabbit aneurysm model is valid for testing of fibroblast implantation.

The invention has been described in connection with the preferred embodiments. These embodiments, however, are merely illustrative and the invention is not restricted thereto. It will be understood by those skilled in the art that other variations and modifications can easily be made which are within the scope of the invention.

Claims

21What is claimed is:

1. A method for at least partially filling an aneurysm comprising the step of: introducing at least one biologically modified vaso-occlusive device which has been seeded with a cell line into an aneurysm.

2. The method of claim 1 wherein said cell line is selected from the group consisting of fibroblasts, endothelial cells, muscle cells, stem cells, and mixtures thereof.

3. The method of claim 2 wherein said cell line is genetically modified to secrete proteins, growth factors, or a combination thereof.

4. The method of claim 3 wherein said proteins or growth factors are selected from the group consisting of transforming growth factors, fibroblast growth factors, platelet derived growth factors, epidermal growth factors, and mixtures thereof.

5. The method of claim 3 wherein said cell line is further genetically modified to include inducible promoters which control cell proliferation, gene expression, or combinations thereof.

6. The method of claim 1 wherein said vaso-occlusive device is selected from the group consisting of a balloon, a coil, a braid, tufted or looped fibers, balls or spheres of natural fibers, embolic fluids, biological matrices, and a non-cytotoxic polymer.

7. The method of claim 1 wherein said balloon, coil, braid or non-cytotoxic polymer has been further modified to include a collagen coating, a polymeric coating, collagen filaments, laminin, fibronectin, other extracellular matrix proteins, or combinations thereof.

8. The method of claim 1 wherein said vaso-occlusive device is a coil or braid. 22

9. The method of claim 8 further comprises the step of:

introducing an embolic agent into said aneurysm in conjunction with said coil or braid.

10. The method of claim 9 wherein said embolic agent is selected from the group consisting of fibrillar collagen, non-fibrillar collagen, laminin, polymers, and mixtures thereof.

11. The method of claim 8 wherein said coil or braid is helical, spherical, or cylindrical in shape.

12. The method of claim 8 wherein said coil or braid is made of a material selected from the group consisting of platinum, tungsten, stainless steel, gold, palladium, nickel-titanium and their alloys.

13. The method of claim 8 wherein said coil or braid is made of platinum.

14. The method of claim 8 wherein said coil or braid is augmented with external fibrous adjuncts selected from the group consisting of loops, tufts, and braided coverings.

15. A method for introducing a cell line into a site in the vasculature of a patient comprising the step of: introducing at least one biologically modified vaso-occlusive device which has been seeded with said cell line into said site in the vasculature of a patient.

16. The method of claim 15 wherein said site in the vasculature of a patient is an aneurysm.

17. The method of claim 15 wherein said cell line is selected from the group consisting of fibroblasts, endothelial cells, muscle cells, stem cells, and mixtures thereof.

18. The method of claim 17 wherein said cell line is genetically modified to secrete proteins, growth factors, or a combination thereof.

19. The method of claim 18 wherein said proteins or growth factors are selected from the group consisting of transforming growth factors, fibroblast growth factors, platelet derived growth factors, epidermal growth factors, and mixtures thereof.

20. The method of claim 18 wherein said cell line is further genetically modified to include inducible promoters which control cell proliferation, gene expression, or combinations thereof.

21. The method of claim 15 wherein said vaso-occlusive device is selected from the group consisting of a balloon, a coil, a braid, tufted or looped fibers, balls or spheres of natural fibers, embolic fluids, biological matrices, and non-cytotoxic polymers.

22. The method of claim 15 wherein said vaso-occlusive device is a coil or a braid.

23. The method of claim 22 wherein said coil or braid is helical, spherical, or cylindrical in shape.

24. The method of claim 22 wherein said coil or braid is made of a material selected from the group consisting of platinum, tungsten, stainless steel, gold, palladium, nickel-titanium and their alloys.

25. The method of claim 22 wherein said coil or braid is made of platinum.

26. The method of claim 22 wherein said coil or braid is augmented with external fibrous adjuncts selected from the group consisting of loops, tufts, and braided coverings.

27. A biologically modified vaso-occlusive device which has been seeded with a cell line. 24

28. The biologically modified vaso-occlusive device of claim 27 wherein said cell line is selected from the group consisting of fibroblasts, endothelial cells, muscle cells, stem cells, and mixtures thereof.

29. The biologically modified vaso-occlusive device of claim 28 wherein said cell line is genetically modified to secrete proteins, growth factors, or a combination thereof.

30. The biologically modified vaso-occlusive device of claim 29 wherein said proteins or growth factors are selected from the group consisting of transforming growth factors, fibroblast growth factors, platelet derived growth factors, epidermal growth factors, and mixtures thereof.

31. The biologically modified vaso-occlusive device of claim 24 wherein said cell line is further genetically modified to include inducible promoters which control cell proliferation, gene expression, or a combination thereof.

32. The biologically modified vaso-occlusive device of claim 29 wherein said cell line is further genetically modified to include inducible promoters which control cell proliferation, gene expression, or a combination thereof.

33. The biologically modified vaso-occlusive device of claim 27 wherein said vaso-occlusive device is selected from the group consisting of a balloon, a coil, a braid, tufted or looped fibers, balls or spheres of natural fibers, embolic fluids, biological matrices, and non-cytotoxic polymers.

34. The biologically modified vaso-occlusive device of claim 27 wherein said vaso-occlusive device is a balloon, a coil or a braid.

35. The biologically modified vaso-occlusive device of claim 34 has been further modified to include a collagen coating, a polymeric coating, 25

collagen filaments, laminin, fibronectin, other extracellular matrix proteins, or combinations thereof.

36. The biologically modified vaso-occlusive device of claim 35 wherein said coil or braid is helical, spherical, or cylindrical in shape.

37. The biologically modified vaso-occlusive device of claim 36 wherein said coil or braid is made of a material selected from the group consisting of platinum, tungsten, stainless steel, gold, palladium, nickel- titanium and their alloys.

38. The biologically modified vaso-occlusive device of claim 36 wherein said coil or braid is made of platinum.

39. The biologically modified vaso-occlusive device of claim 36 wherein said coil or braid is augmented with external fibrous adjuncts selected from the group consisting of loops, tufts, and braided coverings.