Ported tissue implant systems and methods of using same

Information

  • Patent Grant
  • 5733336
  • Patent Number
    5,733,336
  • Date Filed
    Thursday, March 30, 1995
    29 years ago
  • Date Issued
    Tuesday, March 31, 1998
    26 years ago
Abstract
A method and apparatus for implanting cells in a host is provided. In an embodiment, an implant assembly for a host tissue is provided comprising wall means defining a chamber for holding cells for implantation, the wall means including means for forming a porous boundary between the host tissue and the implanted cells in the chamber, the pore size of the boundary being sufficient to isolate the implanted cells from the immune response of the host tissue, and port means for providing selective access to the chamber.
Description

FIELD OF THE INVENTION
The inventions relate to systems and methods for implanting living cells within a host.
BACKGROUND OF THE INVENTIONS
For several years, researchers have been trying to surgically implant living cells in a host to treat various cell and molecular deficiency diseases. In theory, the implanted cells will generate biological products that the host, because of disease or injury, cannot produce for itself. For example, the implant assembly can contain pancreatic cells (clusters of which are called "islets"), which generate insulin that a diabetic host lacks.
Yet, in practice, conventional implant assemblies and methodologies usually fail to keep the implanted cells alive long enough to provide the intended therapeutic benefit. For example, pancreatic cells implanted for the treatment of diabetes usually die or become dysfunctional within a few days or weeks after implantation.
For a period after implantation, the region of the host tissue next to the implant assembly can be characterized as ischemic. "Ischemic" means that there is not a sufficient flow of blood in the tissue region closely surrounding the implant assembly. Usually, this ischemic condition exists during the first two weeks of implantation. Most implanted cells fail to live through this period.
During the ischemic period, a foreign body capsule forms around the implanted cells. The capsule consists of flattened macrophages, foreign body giant cells, and fibroblasts. Conventional hypotheses blame the foreign body capsule for causing implanted cells to die or become dysfunctional during the ischemic period.
The inventors have discovered that these widely held hypotheses are wrong. The inventors have discovered that the cells do not die because of the intervention of the foreign body capsule. Instead, the cells die because conventional implant assemblies and methodologies themselves lack the innate capacity to support the implanted cells' ongoing life processes during the critical ischemic period, when the host's vascular structures are not nearby. Because of this, the implanted cells perish before the host can grow new vascular structures close enough to sustain them.
When implanted cells die during the ischemic period, a classical foreign body capsule inevitably forms around the implant. The persistent presence of this capsule led previous researchers to the false conclusion that the host's foreign body reaction was the cause of implanted cell death, rather than its result.
The invention corrects these and other problems in existing implant assemblies and methodologies.
Many previous implant assemblies have also failed to be useful in a clinical setting, because they cannot be practically implanted and tolerated by the host without danger or discomfort.
For example, an implant assembly that housed cells within hollow fibers was recently used by CytoTherapeutics to successfully treat diabetes in rats. The assembly consisted of 7 fibers, each being 2 cm long and 0.073 cm in diameter. The pancreatic cells were present within the fibers at a density of about 25,000 cells per cm.sup.3. For this assembly to be clinically useful for the treatment of diabetes in humans, it would have to contain at least about 250,000 pancreatic islets (each islet contains about 1000 cells). This means that, to hold enough pancreatic cells to treat human diabetes, the assembly would have to be about 117 feet long. This makes the assembly unusable for clinical use in humans.
Recently, cells have also been encapsulated in tiny hydrogel vessels, called microcapsules. These tiny vessels cannot be implanted within the host's soft tissues, because they lack the physical strength to withstand the physiological stresses normally encountered close to the host tissue. Instead, the microcapsules are suspended in a free floating state within a solution that is infused into the host's peritoneal cavity.
In reality, the microcapsules have only limited clinical application. Not all persons can tolerate their injection free of danger or discomfort. Microcapsules are non-adhesive, and they do not stick to organs. Instead, they settle in large masses at the bottom of the peritoneal cavity. And, if implanted directly within the host's tissue, the microcapsules will rupture, and the contained cells would perish. For these reasons, microcapsules fail to provide a widely usable clinical solution to the problems surrounding the therapeutic implantation of cells.
Once implanted, a potential disadvantage of the known system is that once the cells die or are not longer sufficiently viable, a surgeon must implant a new device. The implantation of such a new device will typically require the patient to be placed under general anesthesia and entail the costs, risks, and discomforts inherent in any surgical procedure.
Additionally, many known devices seal the cells between the walls of the device when the device is created. Accordingly, it is not possible to test the devices to determine the integrity of the system prior to sealing the cells within the device.
SUMMARY OF THE INVENTION
To this end, in an embodiment, an implant assembly for a host tissue is provided comprising wall means defining a chamber for holding cells for implantation. The wall means includes means for forming a porous boundary between the host tissue and the implanted cells in the chamber, the pore size of the boundary being sufficient to isolate the implanted cells from the immune response of the host tissue. Port means are provided for selective access to the chamber.
In an embodiment, the port means is an elongated flexible tube.
In an embodiment, at least two port means are provided on opposite sides of the implant assembly.
In an embodiment, an implant assembly for a host tissue is provided comprising wall means having an interior surface defining a chamber for holding cells for implantation and an exterior surface defining an interface with the host tissue, the exterior surface having a conformation that results in growth of vascular structures by the host tissue close to the interface, the assembly including a port member for providing selective access to the chamber.
In another embodiment, an implant assembly for a host tissue is provided comprising a first device and a second device each having a chamber for holding cells defined by wall means including means for forming a porous boundary between the host tissue and the implanted cells in the chamber, the pore size of the boundary being sufficient to isolate the implanted cells from the immune response of the host tissue. The assembly further including a tube member having a first and a second end, the first end being coupled to the first device and a second end being coupled to the second device.
Preferably, the chamber of the first and second device are in fluid communication through the tube member.
In an embodiment, the tube includes means for allowing one to determine the location of the tube in the host without surgical intervention.
Additionally, the present invention provides a method for implanting cells in a host tissue comprising the steps of: creating an implant assembly for holding cells including wall means for forming a porous boundary between the host tissue and the implanted cells in the device, the pore size of the boundary being sufficient to isolate the implanted cells from the immune response, the device including a port; and adding cells to an interior of the implant assembly through the port.
In an embodiment, the method comprises the steps of: implanting the implant assembly in a host; and adding cells to the interior of the implant assembly after the implant assembly has been implanted.
In an embodiment, the cells are added to the implant assembly through the port prior to implantation.
In an embodiment, the method includes the steps of: providing two ports on opposite sides of the implant assembly; and flushing cells out of the implant assembly by exerting pressure through one port on the cells to cause them to flow through the second port.
In an embodiment, the method includes the step of removing cells from the implant assembly by aspirating the cells out of the port.
In an embodiment, the method includes the step of implanting a plurality of implant assemblies and coupling at least two tubes together with mechanical means. The mechanical means can include a clip.
In an embodiment, a method of implanting cells is provided comprising the steps of: providing a first implant assembly and a second implant assembly each having a chamber for holding cells defined by wall means including means for forming a porous boundary between the host tissue and the implanted cells in the chamber, the pore size of the boundary being sufficient to isolate the implanted cells from the immune response of the host tissue; providing a tube member including a first and a second end, the first end being coupled to the first implant assembly and a second end being coupled to the second implant assembly; severing the tube after the implant assembly is implanted; and adding cells through the severed tube to each chamber of each implant assembly.
Additionally, the present invention provides a method for implanting cells into a host tissue comprising the steps of: creating an implant assembly for holding cells including wall means for forming a porous boundary between the host tissue and the implanted cells in the implant assembly, the pore size of the boundary being sufficient to isolate the implanted cells from the immune response, the implant assembly including a port; adding cells to an interior of the implant assembly through the port; and testing the cells for therapeutic function prior to implanting the implant assembly.





Additional features and advantages of the present invention are described in, and will be apparent from, the detailed description of the presently preferred embodiments and from the drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a perspective view of an implant assembly that embodies the features of an embodiment of the invention being held in the hand of a practitioner;
FIG. 2 is an enlarged perspective view of the implant assembly shown in FIG. 1;
FIG. 3 is an enlarged and exploded perspective view of the implant assembly shown in FIG. 2;
FIG. 4 is a side section view of the implant assembly taken generally along line 4--4 in FIG. 2;
FIG. 5 is an enlarged and exploded perspective view of another implant assembly that embodies the features of the invention, showing the practitioner loading implanted cells into the assembly;
FIG. 6 is an enlarged assembled view of the assembly shown in FIG. 5, before the formation of a peripheral seal;
FIG. 7 is an enlarged view of the assembly shown in FIG. 6, partially peeled apart to show the interior;
FIG. 8 is an enlarged assembled view of the assembly shown in FIG. 5 after the formation of a peripheral seal;
FIG. 9 is a side section view of a portion of the sealed assembly taken generally along line 9--9 in FIG. 8;
FIG. 10 is a side section view of the assembly before sealing, taken generally along line 10--10 in FIG. 6;
FIG. 11 is a perspective view of a lamination slide holding the bottom layer of the laminated boundary structure that embodies the features of the invention;
FIG. 12 is a side section view of the lamination slide taken generally along line 12--12 in FIG. 11;
FIG. 13 is a perspective view of several lamination slides laid side by side for the application of adhesive filaments in the process of making the laminated boundary structure;
FIG. 14 is a side section view of the laminated boundary structure with its top layer laid over the cement filaments applied in FIG. 13;
FIG. 15 is a side section view of the laminated boundary structure clamped between two lamination slides while the cement filaments cure;
FIG. 16 is a perspective view of individual boundary wall elements being cut from the laminated structures made following the steps shown in FIGS. 11 to 15;
FIG. 17 is a diagrammatic depiction of an embodiment of the implant assembly that embodies the features of the invention after having been surgically implanted in host tissue;
FIG. 18 is a diagrammatic depiction of an embodiment of the implant assembly during the ischemic period, after about one or two days of implantation, showing the surrounding wound area filled with exudate;
FIG. 19 is a diagrammatic depiction of the implant assembly after about two weeks of implantation, showing the formation of vascular structures close to the boundary, ending the ischemic period;
FIG. 20 is a diagrammatic depiction of a section of the implant assembly in which the implanted cells have survived the ischemic period, showing the formation of vascular structures close to the boundary and the resulting alteration of the foreign body capsule;
FIG. 21 is a diagrammatic depiction of a section of the implant assembly in which the implanted cells have not survived the ischemic period, showing the lack of vascular structures close to the boundary and the resulting intervention of the foreign body capsule;
FIG. 22 is a graph showing the therapeutic loading curve for pancreatic cells derived in accordance with the invention.
FIG. 23 illustrates schematically an embodiment of a ported implant assembly of the present invention.
FIG. 24 illustrates a cross-sectional view of the assembly of FIG. 23 along lines XXIV--XXIV thereof.
FIG. 25 illustrates another embodiment of a ported assembly of the present invention.
FIG. 26 illustrates an embodiment of the present invention that provides two ported implant assemblies coupled together.
FIG. 27a-27c illustrates schematically the use of an embodiment of the present invention including a number of ported implanted assemblies. FIG. 27(a) illustrates a plurality of assemblies 171-176 with respective tubes 181-186 of the assemblies clipped together with a location clip 187. FIG. 27(b) illustrates that after implantation into a body 191, clip 187 of FIG. 27(a) can be removed and a surgeon may fill each of the assemblies 171-176 with syringe 189. FIG. 27(c) illustrates that after assemblies 171-176 of FIGS. 27(b) have been filled, tubes 181-186 can be sealed with location clip 190.
FIG. 28 illustrates another embodiment of a ported assembly of the present invention.
FIG. 29 illustrates a further embodiment of the assembly of the present invention.
FIG. 30 illustrates a cross-sectional view of the assembly of FIG. 29 taken along lines XXX--XXX.
FIG. 31 illustrates a cross-sectional view of the assembly of FIG. 29 taken along lines XXXI--XXXI.
FIG. 32 illustrates a cross-sectional view of the assembly of FIG. 29 taken along lines XXXII--XXXII.





Before explaining the preferred embodiments, it is to be understood that the inventions are not limited in use to the details of construction or methodologies there set forth or as illustrated in the drawings. The inventions are capable of other embodiments and of being practiced and carried out in various ways.
DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS
FIGS. 1 to 4 show an implant assembly 10 that generally embodies features of the invention.
The assembly 10 can carry preselected types of living cells 12 for implanting within the soft tissue of a host. The implanted cells 12 generate biological products that the host, because of disease or injury, cannot produce for itself.
For example, the implant assembly 10 can carry clusters of pancreatic cells (called "islets"), which generate insulin for release into and use by a diabetic host.
The assembly 10 forms a porous, life sustaining boundary between the implanted cells 12 and the host. The porous boundary isolates the implanted cells 12 from attack and destruction by certain biological mechanisms of the host. At the same time, the porous boundary associates with the host's biological system closely enough to transfer nutrients and wastes in support of the biological processes of the implanted cells 12. The porous boundary also transfers the therapeutic products generated by the implanted cells 12 to the host.
In the embodiment shown in FIGS. 1 to 4, the assembly 10 includes a hoop-like housing 11. The housing 11 includes a first hoop element 14 and a second hoop element 16 that nests within the first hoop element 14. The assembly 10 also forms a cell chamber 18 within the hoop-like housing 11.
The first hoop element 14 has an upstanding cylindrical side wall 20 that peripherally defines an open area. First and second central openings 22 and 24 lead into the open area. The first central opening 22 is smaller than the second central opening 24. This forms an interior step or ledge 26 next to the first opening 22.
The second hoop element 16 also has a central opening 28. The second hoop element 16 has an outer diameter that is slightly greater than the inner diameter of the open area of the first hoop element 14. The peripheral edge of the second central opening 16 contains a slight chamfer 30 to receive the second hoop element 16. When assembled, the second hoop element 16 nests snugly in an interference press fit within the open area of the first hoop element 14 (see FIG. 2).
The first hoop element 14 and the second hoop element 16 are made of a durable biocompatible ceramic or metallic material, like titanium. Like titanium, the selected material should also preferably be subject to detection within the host tissue by fluoroscopy, x-ray, and the like.
The specific dimensions of the hoop-like housing 11 may vary according to its intended use and the volume of cells 12 it contains.
In one preferred embodiment, the side wall of the first hoop element 14 is about 0.055 inch in height and has an outer diameter of about 0.375 inch. The open area has an inner diameter of about 0.325 inch where it joins the inner edge of the chamber 30 of the second central opening 24. The second central opening 24 has an inner diameter of about 0.326 inch around the outer edge of the chamfer 30. The first central opening 14 has an inner diameter of about 0.275 inch and a depth of about 0.015 inch, where it joins the interior ledge 26.
In this embodiment, the associated second hoop element 16 has a height of about 0.025 inch; an outer diameter of about 0.326; and an inner diameter (for its central opening 28) of about 0.250 inch. The range of interference necessary to snugly join the second hoop element 16 within the first hoop element 14 will of course depend upon the nature of the materials selected.
The chamber includes a first porous wall element 32, a second porous wall element 34, and a sealing gasket or ring 36 that is sandwiched between them. The sealing ring 36 is made of a mesh polyester material.
The wall elements 32 and 34 and sealing ring 36 are sized to fit snugly within the confines of the hoop-like housing 11. And, as will be described in greater detail later, at least one (and preferably both) porous wall elements 32 and 34 have certain physical characteristics selected to protect and sustain the viability of the cells 12 within the host.
The ring 36 has a central open region 38. The open ring region 38, together with the overlying first and second porous wall elements 32 and 34, create the chamber 18 to hold the implanted cells 12 (see FIG. 4).
In making the assembly 10 shown in FIGS. 1 to 4, the practitioner lays one wall element 32 onto the ledge 26 formed in the first hoop element 14. The practitioner then lays the sealing ring 36 upon the wall element 32. Next, the practitioner inserts the desired amount of cells 12 to be implanted into the open region 38 of the ring 36. The amount of implanted cells 12 is sufficient to induce the expected therapeutic response in the host.
The practitioner next lays the other wall element 34 over the first wall element 32, sealing ring 36, and inserted cells 12. To complete the assembly 10, the practitioner presses the second hoop element 16 through the second central opening 24 into pressing engagement against the adjacent wall element 34. This seals the periphery of the cell holding chamber 18, which now snugly rests within the open area of the hoop-like housing 11.
Once assembled, one wall element 32 rests against the interior ledge 26 and is there exposed through the first central opening 22. The other wall element 34 rests against the second hoop element 16 and is there exposed through its central opening 28.
FIGS. 5 to 10 show another implant assembly 10' that embodies features of the invention. Like, the implant assembly 10 previously described, the assembly 10' includes a cell chamber 18' formed by first and second porous wall elements 32' and 34' and an intermediate sealing ring 36'.
Unlike the first described implant assembly 10, the assembly 10' does not rely upon the hoop-like housing 11 to hold and seal the chamber 18'. Instead, a preformed peripheral weld 40 bonds and seals the edges of the porous wall elements 32' and 34' to the interior ring 36'.
In making the assembly 10' shown in FIGS. 5 to 10, the practitioner lays the sealing ring 36' upon one wall element 32' and inserts the desired amount of cells 12 to be implanted into the open region 38' of the ring 36' (see FIG. 5). The practitioner overlays the other wall element 34' (as FIG. 6 shows). The practitioner then forms the weld 40 to seal the peripheral edges of the first and second wall elements 32' and 34' to the ring 36' (as FIG. 8 shows). The weld compresses the peripheral edge of the assembly 10' together, as FIG. 9 shows.
The practitioner selects a sealing technique that does not damage the cells 12 within the chamber 18'. For example, the inventors find that sonic welding can be used without damage to the inserted tissue cells.
In a preferred embodiment (using the laminated structure 72 made as shown in FIGS. 11 to 16, as will be described later), the practitioner uses a Branson sonic welder. The welder is operated at 40 Khz, with 941AES actuator, 947 m power supply, and 91C power controller. The horn amplitude is about 1.4 mils and is operated at a hold time of about 0.3 seconds; a weld time of about 0.20 seconds; a pressure of about 50 PSI; a trigger force of about 20 pounds; and a down speed of about 1.25 (machine setting).
These are typical operating ranges for making the sonic weld and can vary according to the materials used and degree of cell loading within the chamber.
The integral assembly 10' formed in this manner can be implanted directly within host tissue, without use of an exterior housing.
Preferably, as FIG. 8 shows, the assembly 10' includes an attached clip 42 made of a material that can be detected within the host tissue by fluoroscopy, x-ray, and the like. In this way, the practitioner can easily locate the assembly 10' within the host, if required.
Like the first described embodiment, the specific dimensions of the assembly 10' may vary according to its intended use. And, like the first described embodiment, at least one (and preferably both) porous wall elements 32' and 34' have certain physical characteristics selected to protect and sustain the viability of the cells within the host.
Regardless of the assembly used, the practitioner surgically implants it in the soft tissue 44 of the host (see FIG. 17). During surgery, the practitioner positions the assembly 10 so that the exposed first and second wall elements 32 and 34 rest close to the surrounding host tissue 44. In FIGS. 17 to 21, assembly 10 also encompasses assembly 10'.
The first and second wall elements 32 and 34 thereby together form the desired boundary 46 between the biological system of the host tissue 44 living outside the chamber 18 and the biological system of the implant tissue cells 12 living within the chamber 18.
For a period of time after implantation, the region of the host tissue 44 immediately surrounding the implant assembly 10 is ischemic (see FIG. 18). The region is ischemic, because the host treats the assembly 10 as a foreign body.
The host forms a wound area 48 around the assembly 10 (see FIG. 18). The wound area 48 has spaces that become filled with wound exudate 50. The wound exudate 50 keeps this area 48 ischemic.
Soon after implantation, host inflammatory cells enter and occupy the exudate area 48. "Inflammatory cells" include macrophages, foreign body giant cells, and fibroblasts.
The inflammatory cells try to remove the foreign implant assembly. Macrophages from the host try to ingest the foreign implant assembly 10. In some cases, the macrophages coalesce to form multinucleated giant cells. Fibroblast layers form to create a fibrous sac of cells and collagen around the foreign implant assembly 10, commonly called the foreign body capsule 52 (see FIG. 20).
The inventors have discovered that it is not the foreign body capsule 52 that most threatens the viability of the implanted cells during the ischemic period. Rather, the existence of the cells is most threatened during the ischemic period when the boundary 46 itself fails to allow enough extracellular nutrients like glucose and other metabolic support compounds present at the boundary 46 to pass to the cells. Without metabolic support, the implanted cells become dysfunctional or perish.
As FIG. 18 shows, the wound exudate 50 forms a fluid barrier between the vascular system of the host and the boundary 46. This barrier hinders the extracellular passage of nutrients from the host vascular system to the boundary 46. The concentrations of nutrients decrease as they transit the exudate barrier to reach the boundary 46.
The host's inflammatory cells that in time enter the wound exudate region 50 also create a metabolic sink. These cells compete for and further extract more of the host's extracellular nutrients before they reach the boundary.
If the host is stimulated to grow new vascular structures 54 close to the boundary 46, host endothelial cells will also enter the region 48. These cells begin the crucial process of forming the new vascular structures 54. Still, their presence further contributes to the metabolic sink effect. The host's endothelial cells further reduce the availability of nutrients for the implanted cells.
The ischemic period will end, if enough neovascular structures 54 from the host grow within the exudate region 50 close to the boundary 46 of the assembly 10 (as FIGS. 19 and 20 show). The close vascular structures 54 shorten the extracellular path that nutrients must travel to reach the boundary 46. The close vascular structures 54 provide nutrients in higher concentrations to the implanted cells. Close vascularization also transports the therapeutic products generated by the implanted cells 12 to the host.
However, all these desired benefits accrue only if the implanted cells 12 survive the critical ischemic period.
The inventors have discovered that the diminished concentrations of nutrients present at the boundary 46, although significantly reduced by the exudate barrier and metabolic sink effects, are still enough to sustain the implanted cells. This is true, even in the presence of a foreign body capsule.
Still, the cells will die, if the boundary 46 itself lacks the capacity to let enough of the remaining nutrients through to the cells at a sufficiently high rate. The inventors refer to this capacity as the metabolic transit value.
The inventors have discovered that the boundary 46 itself can also present another significant barrier to the passage of nutrients. The added barrier effect of the boundary 46 can further reduce the already diminished concentration of nutrients, until there is essentially nothing left to sustain the cells.
The series barriers to the extracellular passage of nutrients (the wound exudate 50, the boundary 46, and the metabolic sink effect) also inhibit the reverse passage metabolic wastes from the implanted cells.
The inventors have discovered that two principal factors threaten the survival of the implanted cells during the ischemic period. The first factor (which is conventionally recognized) is the failure to isolate the cells from the natural immune response of the host. The second factor (which is not conventionally recognized) is the undesirable additional barrier effect of the boundary 46 that impedes the essential flux of already scarce nutrients to the implanted cells before close vascularization fully develops. The same barrier effect impedes the flux of metabolic waste products away from the implanted cells to the host.
If the boundary 46 does not support the ongoing metabolic processes of the implanted cells while isolating them from the immune response of the host during the ischemic period, the implanted cells will not live long enough to derive the benefits of close vascularization, if it occurs.
According to this aspect of the invention, then, the porous boundary 46 is characterized in terms of its pore size; its ultimate physical strength; and its metabolic transit value. The first two characteristics serve to isolate the implant tissue cells from the immune response of the host. The last characteristic serves to transfer nutrients and waste products in support of the metabolic processes of implanted cells during the ischemic period, before close vascularization occurs. The last characteristic sustains the viability of the implanted cells during the ischemic period, even as a foreign body capsule forms.
According to another aspect of the invention, the assembly also includes an angiogenic material. The presence of an angiogenic material stimulates the neovascularization required close to the boundary 46 to bring an end to the ischemic period.
According to yet another aspect of the invention, the porous boundary 46 includes an interface 47 with the host tissue that is characterized by a conformation that supports and fosters the growth of vascular structures by the host close to the boundary 46.
Further details of the beneficial characteristics of the boundary 46 and its associated host interface 47 will now be individually described.
Boundary Pore Size
The boundary 46 has a pore size sufficient to isolate the implant tissue cells from the immune response of the host.
As used in this Specification, "pore size" refers to the maximum pore size of the material. The practitioner determines pore size using conventional bubble point methodology, as described in Pharmaceutical Technology, May 1983, pages 36 to 42.
As a threshold requirement, the pore size selected must make the boundary 46 impermeable to the vascular structure that forms close to the boundary 46. Penetration of the pores by the vascular structure breaches the integrity of the boundary 46, exposing the implanted cells to the complete immune response of the host. Generally speaking, pore sizes less than about 2 microns will block the ingress of vascular structures.
The ultimate pore size selected also depends upon the species of the host and the biologic relationship between the host and the donor of the implant tissue cells.
When the implanted cells are from another animal species (i.e., xenografts), the pore size must be sufficient to prevent the passage of both inflammatory cells and molecular immunogenic factors from the host into the implant tissue chamber. As used in this Specification, "molecular immunogenic factors" refers to molecules such as antibodies and complement.
Pore sizes sufficient to block passage of both inflammatory cells and molecular immunogenic factors in humans lie in the range of about 0.015 micron. Of course, these pore sizes are also impermeable to vascular structures.
When the implanted cells are from the same animal species but having a different genetic make up (i.e, allografts), the pore size usually must be sufficient to prevent the passage of only inflammatory cells from the host into the implant cell chamber. In allografts, molecular immunogenic factors do not seem to adversely affect the viability of the implanted cells. Still, some degree of tissue matching may be required for complete protection.
Pore sizes sufficient to block passage of inflammatory cells in humans lie in the range of below about 0.8 micron. These pore sizes, too, are impermeable to vascular structures.
When the implanted cells are isografts (autologous implants of genetically engineered cells), the pore size must be sufficient only to prevent the isografts from entering the host. Still, with isografts, the pore size selected must also prevent ingress of vascular structures.
Boundary Strength
The boundary 46 has an ultimate strength value that is sufficient to withstand, without rupture, the growth of new vascular structures, the growth of new cells within the chamber 18/18', and other physiological stresses close to the host tissue. Keeping the boundary 46 secure assures isolation of the implanted cells from both the immunogenic factors and inflammatory cells of the host.
These physiological stresses are caused when the host moves about in carrying out its normal life functions. The proliferation of implanted cells and the growth of vascular structures 54 also contributes to the physiological stresses close to the boundary 46. The stresses challenge the physical integrity of the boundary 46 by stretching or otherwise deforming it.
Absent a sufficient ultimate strength value, normal physiological stresses can rupture the boundary 46, exposing the implanted cells to the full effect of the host's immune and inflammatory systems.
The inventors presently believe that ultimate strength values sufficient to withstand physiological stresses close to the host tissue without rupture in animals lie above about 100 pounds per square inch (PSI). In comparison, the ultimate strength value for PVA hydrogel microcapsules is only about 2 to 2.5 PSI.
The ultimate strength values are determined by measuring the tensile strength of the material. Tensile strength is measured by ASTM D-412.
Metabolic Transit Value
The boundary 46 also has a metabolic transit value that sustains a flux of nutrients into the chamber 18 and waste products from the chamber 18 sufficient to sustain the viability of the implanted cells during the ischemic period.
The metabolic transit value takes into account the permeability value (P) and the porosity value (PORE) of the boundary 46.
The Permeability Value
The permeability value (P) is the measure of the amount of solute that travels through the boundary per unit time and unit surface area, given some fixed external solute concentration (measured in cm/sec in this specification). Example 1 sets forth a methodology for determining the permeability value according to this aspect of the invention.
The Porosity Value
The porosity value (PORE) represents the space in the boundary 46 that does not contain material, or is empty, or is composed of pores. Expressed as a percentage, the porosity value (PORE) measures the % volume of the boundary 46 that is not occupied by boundary material.
To derive the porosity value PORE (in %) for materials having a PORE equal to or greater than lot, the practitioner uses the following formula:
PORE=100(1-(.rho..sub.b /.rho..sub.m)
where:
.rho..sub.b is the density of the boundary as determined from its weight and volume, and
.rho..sub.m is the density of the boundary material.
To derive the porosity value PORE (in %) for materials having a PORE less than 10%, the practitioner uses using a scanning electron microscope to obtain the number of pores and their average diameter on the boundary. PORE is then derived according to the following formula:
PORE=N.pi.(d.sup.2 /4)
where:
N is the pore density and equals (p.sub.n /a),
p.sub.n is the number of pores in the boundary,
a is the total area of the boundary (in cm.sup.2), and
.pi. is the transcendental constant 3.1416 . . . ,
d is the average diameter of the pores (in (m).
The inventors have found that, above a threshold minimum porosity value, the permeability value is the principal influence upon the overall metabolic transit value. Still, below the threshold minimum porosity value, the metabolic transit value must also take into account the porosity value and the physical structure of the porous boundary 46. These considerations will be discussed later in greater detail.
To simplify the selection of a boundary 46, the inventors recommend the use of boundaries having a porosity value (PORE) greater than the observed minimum threshold value. Then, metabolic transit value and the permeability value can be treated as the same.
Referring now to FIGS. 23-24, a further embodiment of the assembly 100 is illustrated. In this embodiment, the assembly 100 includes a port member 102 that provides means for accessing the interior or cell chamber 104 of the assembly 100. In the embodiment illustrated, the port member 102 is an elongated flexible tube 106. The elongated flexible tube is in fluid communication with the cell chamber 104 that is defined by the wall elements of the assembly.
It should be noted, however, that the port member 102 does not have to be defined by an elongated flexible tube. Other structures can be used to provide access to the cell chamber of the assembly. For example, a resealable injection site can be provided on the assembly for providing access to the cell chamber.
By providing a port on the assembly, the port can be used to initially place cells 105 within the assembly 100. In this regard, a syringe or other device can be used to place the cells within the cell chamber 104 defined by the assembly 100.
For example, with respect to the embodiment illustrated in FIG. 23, the cannula of a syringe would be received by the flexible tube 106 allowing cells to be placed within the cell chamber 104. In filling the interior of the assembly, a syringe is utilized having a cannula that will be received within the full length of the port 102 and to an end 107 of the cell chamber 104 of the assembly. Using a syringe, as tissue is laid within the chamber 104 of the assembly 100, the syringe is slowly removed from the chamber. This methodology will insure that too great a pressure is not used that will damage the membrane as the tissue is laid. It is believed that the pressure used should be lower than syringe pressures. By using reduced pressure, additionally, this will insure that tissue is not damaged due to frictional forces that can shear the tissue if high pressures are utilized.
Of course, if desired, the cells can initially be sealed within the assembly 100 as set forth above with respect to the embodiments of the assembly 100 not including a port.
Because the cells can be placed within the assembly 100 through use of the port 102, the port allows the assembly to be implanted without cells for prevascularization of the assembly. Prevascularization of the assembly, and the benefits inherent therein, is disclosed in U.S. patent application Ser. No. 08/413,588 being filed on the same day herewith naming as inventors Steven Neuenfeldt, James Brauker, Robert Clarke, and Victoria Carr-Brandal. The disclosure of that application is incorporated herein by reference. Once the assembly 100 is vascularized within the host, cells can be added to the assembly 100 as described below.
Additionally, because the assembly 100 includes at least one port 102, the assembly can be "recharged" by placing new cells within the assembly after a fixed period of time. Heretofore, once the cells within the implanted assembly died or no longer were viable, it was necessary for a surgical intervention to remove the implanted assembly from the host and insert a new assembly including new cells. The present invention overcomes the disadvantages of the prior art by providing an assembly 100 that can be recharged with new cells.
To this end, in an embodiment, the assembly 100 is implanted with the flexible tube 106 being located near the outer epidermal layers of the patient. To recharge the assembly 100, a surgeon merely creates an incision in the skin exposing the end portion of the flexible tube 106. This allows the surgeon to thereby access the interior of the assembly 100 utilizing a syringe or other method for depositing new cells within the cell chamber 104 of the assembly 100 through the flexible tube 106.
After the cells have been placed within the assembly 100, the tube 106 can then be sealed using a heat seal, mechanical means, or another method. The tube 106 is then placed within the patient and the incision is closed.
Pursuant to the present invention, a port can be used on any of the embodiments of the implant assemblies set forth in this application, as well as other implant assemblies. For example, as illustrated in FIG. 25, the assembly 130 can comprise a disk-like device having an interior 136 with an O-ring or hoop that defines a cell chamber. The disk defines an aperture 138 for sealingly receiving the port 140. In the case of the illustrated embodiment, the port 140 is an elongated flexible tube.
In another embodiment of the invention illustrated in FIG. 26, an implant device 150 is provided including two assemblies 152 and 154. The two assemblies 152 and 154 share a flexible tube 156 therebetween. The tube 156 is in fluid communication with the cell chambers 155 and 157 of each assembly 152 and 154. In this regard, a first end 158 of the tube 156 is sealingly received by the assembly 152 and a second end 160 of the tube 156 is sealingly received by assembly 154.
Such an implant device 150 allows a variety of different applications and methods of use of the assemblies 152 and 154.
In an embodiment of the invention, the device 150 can be implanted and prevascularized in the host. After the device 150, and specifically assemblies 152 and 154 are vascularized, the cell chambers 155 and 157 of same can be filled with cells. To this end, a surgeon will create an incision in the patient and access the tube 156. To aid the surgeon in locating the tube 156 a location clip can be attached to the tube. However, other means can be used to allow the surgeon to identify the approximate location of the tube prior to creating an incision in the patient, e.g., the tube can include at least portions thereof that are radiopaque and can thereby be located by an x-ray of the patient.
Once the tube 156 is exposed, the surgeon will then sever the tube 156 in two. Each of the resultant tubes that are created will be in fluid communication with an interior of the respective assemblies 152 and 154. This will allow the surgeon to access the interior of the devices 152 and 154 and fill same with cells using a syringe or other means. The tubes are then sealed and replaced in the patient. The incision is then closed.
In another embodiment of the implant device 150, the cells can be sealed within the assemblies 152 and 154 of the device 150 by heat sealing or other method when the device is created. After the device 150 is implanted, when necessary, the tube 156 can then be accessed and severed allowing the surgeon to recharge the assemblies 152 and 154.
As illustrated in FIGS. 27a-c, the use of a ported device allows a plurality of implant assemblies 171-176 to be implanted in a patient and coupled together. Each of these ported implant assemblies can be charged either prior to being implanted into the patient or after implantation in the patient. Likewise, after a predetermined time, the assemblies can be charged to compensate for cells that are no longer viable or die.
As illustrated in FIG. 27a-27c, if a plurality of assemblies 171-176 are used, the respective tubes 181-186 of the assemblies can be clipped together with a location clip 187. This will allow the surgeon to easily locate and access the tubes 181-186.
As illustrated schematically in FIGS. 27a-27c, after the initial implantation, the clip 187 can act to seal the interiors of the tubes 181-186. When necessary, the surgeon will make an incision and access the clip 187. By removing the clip 187, the surgeon can access the interior of the tubes 181-186. This will allow the surgeon to fill each of the assemblies 171-176 with, for example, a syringe 189. After the assemblies 171-176 have been filled utilizing a syringe 189 or other device, the tubes 181-186 can be sealed again with the location clip 190. The tubes are then placed within the body 191 and the wound is closed. This allows the surgeon to easily and without a very invasive surgical procedure, to recharge the implanted assemblies 171-176.
In creating a ported assembly having a flexible tube, e.g., the assembly 100 of FIG. 23, the length of the tube 106 should be selected so that it allows for multiple recharges of the assembly. In this regard, upon each recharge of the assembly, the tube will be resealed. This sealing process can be accomplished by utilizing a heat seal. Therefore, in accessing the interior of the assembly through the tube, it will be necessary for the surgeon to remove a portion of the tube. Accordingly, the length of the tube should be selected so as to allow a sufficient number of recharges during the useful life of the implanted assembly.
As noted above, it is not necessary to heat seal the tube of the assembly, but rather, the tube can be sealed using mechanical means, e.g., the tube can be clipped. This will allow a reduced length of tubing to be initially utilized since in order to access the tube, the clip is merely removed rather than a portion of the tube being sliced off. Additionally, the tube can be sealed by injecting a curable substrate into the interior of the tube, such as silicon, and allowing the substrate to seal as a plug.
Depending on the implant assembly's construction, the port can be sealed within the assembly pursuant to a number of methods. For example, ultrasonic welding, over-molding, or crimping can be utilized to create an implant assembly having a port.
FIG. 28 illustrates another embodiment of the present invention. As illustrated, if desired, the assembly 192 can include a port at both ends of the assembly. This will allow, if desired, the assembly's cell chamber to be flushed after each recharge. In the illustrated embodiment, the assembly 192 thereby includes a first port 193 and a second port 194. Each port 193 and 194 is in fluid communication with the cell chamber 195. In use, either port 193 or 194 can be designed to receive cells to be laid in the cell chamber 195 of the assembly 192. To flush the cell chamber 195 prior to recharge, a fluid can be injected through port 193 by using a syringe. The original cells within the cell chamber 195 along with fluid would then exit the cell chamber through port 194.
It should be noted that even in devices that include only one port, the cell chamber can be cleared prior to recharging. To this end, for example with the assembly of FIG. 23, the cell chamber 104 can be cleaned through aspiration of the cells through the port 102. For example, a syringe can be inserted into the cell chamber 104 through the port and a vacuum can be created to suction the cells present in the cell chamber into the syringe.
As illustrated in FIGS. 29-32, the assembly 200, in an embodiment, can include a spacer 202 for creating an area 204 within the interior of the assembly 200 for receiving the cells. In the illustrated embodiment, the spacer circumscribes a majority of the perimeter of the assembly 200. Such a structure would be desirable if the assembly 200 is to be implanted without cells and filled after vascularization in the host. The spacer or insert will maintain optimum cell depth, cell distribution, experimental pillow shape, and insure maximum chemical interactions. Cell space can be maintained in vivo prior to or following tissue loading using a filament or insert of approximate diameter equal to the desired tissue depth.
The assembly 200 including the spacer 202 is illustrated without a port. The port would be received and sealed, in the illustrated embodiment, within the opening 206 at an end 208 of the assembly 200.
Of course, the port can be used not only to deposit cells within the assembly, but to, if necessary, deliver nutrients or therapeutic agents to the cells within the assembly. Likewise, if necessary, the port can be used to aspirate a portion of, or all of, the implanted cells after the assembly has been implanted to modify the delivery of therapeutic agents by the assembly.
Additionally, due to use of a port on the present invention, one can test the assembly prior to inserting cells into the assembly or implanting same. For example, if desired, the cells can be inserted into the assembly and therapeutic function can be evaluated prior to implantation.
By way of example, and not limitation, examples evaluating the present invention will now be given. As the following Example 1 shows, the inventors have discovered that there is a direct correlation between the metabolic transit value and implanted cell survival during the ischemic period.
EXAMPLE 1
Embryonic lungs enclosed in membrane chambers having different permeability values were implanted in subcutaneous sites in rats.
1. Permeability
The permeability values for the membrane chambers were obtained for insulin diffusion in a conventional benchtop diffusion chamber, made by Crown Glass Company, Somerville, N.J. (Part Number DC-100), using radioactively labeled (.sup.125 I) insulin as the solute (obtained from ICN Biochemicals). The diffusion chamber had two chambers (which will be called Chambers A and B), each with a volume of 3 ml. The diffusion chamber presented a membrane surface area between the two chambers (where diffusion occurs) of 0.7 cm.sup.2.
The practitioner cuts the membrane material to be tested to a predetermined, known size.
If the membrane is hydrophobic, the practitioner wets the membrane before conducting the permeability test, using conventional wetting techniques.
The practitioner places the membrane in the diffusion chamber. The assembly of the diffusion chamber locates the membrane between the two chambers of equal volume, called Chamber A and Chamber B. In this way, the practitioner also fixes the cross sectional area (A) of the membrane. The diffusion chamber is uniformly heated to a temperature of about 37 degrees C during the test.
The practitioner loads equal amounts of buffer solution into Chamber A and Chamber B. The buffer solution can vary. In this Example, the practitioner can use phosphate buffered saline, 0.5% BSA as the buffer solution.
The practitioner then loads equal amounts of unlabeled (non-radioactive) insulin (about 3.4 micro units/ml) into Chamber A and Chamber B. Porcine pancreas insulin purchased from Sigma with an activity of 26.1 units/ml, or comparable material, can be used. The unlabeled insulin occupies any adsorption sites that may be present.
The practitioner uniformly stirs the fluids within the chamber at about 600 RPM, using a magnetic stir plate and magnetic stir rods (about 1 cm in length) placed in each Chamber A and B. The practitioner allows the system to equilibrate for about one hour.
The practitioner then removes a selected volume of buffer solution from Chamber A and adds back an equal volume of radioactive insulin. The radioactive insulin suspension is filtered before use to remove free .sup.125 I.
While stirring the fluids within Chamber A and Chamber B, the practitioner draws equal aliquots of fluid from each Chamber A and B (e.g. about 15 uL) at 2, 4, 6, 8, 10, 15, and 30 minute intervals.
The practitioner then counts the radioactivity levels in the samples using a gamma counter.
The practitioner determines the change in the counts (i.e., insulin concentration) in Chambers A and B per unit of time, suitably corrected for background noise.
The practitioner graphs the count and time pairs for each Chamber in terms of time versus the counts (with the counts being the Y-coordinates and time being the X-coordinates), restricting the analysis to points for which the counts in Chamber B are less than about 10% of the initial counts in Chamber A. The practitioner then derives a linear equation, fitting the range of counts (y) over the set of times (x) for each Chamber according to the following equations:
For Chamber A:
Y.sub.a =Y.sub.intercept -(N.sub.a *X)
where
Y.sub.intercept is the count value where the graph intersects the Y axis, and
N.sub.a is the slope of the Chamber A graph.
For Chamber B:
Y.sub.b Y.sub.intercept +(N.sub.b *X)
where
Y.sub.intercept is the count value where the graph intersects the Y axis, and
N.sub.b is the slope of the Chamber B graph.
The practitioner preferably uses a commercially available computer program to simplify the derivation process described above.
The practitioner then derives the permeability value (P) according to the general expression: ##EQU1## where V.sub.b is the volume of Chamber B
dM.sub.b /dT is the change in counts in Chamber B per unit time, which is the slope of the B graph derived above (N.sub.b),
P is the permeability value,
A is the area of the boundary tested, and
M.sub.a -M.sub.M.sub.b is the mass gradient of insulin across the membrane.
The practitioner knows V.sub.b and A, which remain constant throughout the test. The practitioner also knows dM.sub.b /dT, the slope of the g from the linear equation derive practitioner converts the unit min/min) into counts per minute/ (the number of seconds in a minute
The practitioner calculates M equation derived for Chamber A for (i.e., the mid point time for the mid point time for the test, the p average value for the period practitioner similarly calculates order linear equation derived for t=15 minutes. From these valu calculates M.sub.a -M.sub.b.
The practitioner can now der value (in cm/sec) as follows: ##EQU2##
Actually, the permeability includes the boundary layer effects with inevitable stagnate fluid lay surface in Chambers A and B during at the "true" intrinsic permeabil boundary, the practitioner would hav boundary layer effects. However, this invention, a knowledge of the permeability is not essential, b proportional to the experimental determined following the methodology
Yet, the practitioner can fo methodology to quantify the relative for selected boundaries, since boun will remain constant as long as the stirring method used remains the same.
The disclosed methodology can be used to assess whether a given boundary fits the criteria established for the permeability value according to this aspect of the invention.
2. Porosity
The porosity values (PORE) of the boundaries tested ranged from less than about 15% to greater than about 70%.
3. Determining Cell Survival
Embryonic lungs were removed from Lewis rat embryos between days 13.5 and 17.5 of development. The lungs were kept on ice in Dulbecco's Modified Eagle's Medium (DMEM), 20% fetal bovine serum. The lungs were minced until they were approximately 1 mm.sup.2. Minced lung tissue (5-10 .mu.l) was placed into implant assemblies like those shown in FIGS. 1 to 4. The lung tissue was encapsulated within test membranes having various permeabilities, porosities, and pore sizes. The implant assemblies were placed in DMEM (20% fetal bovine serum) at 37 degrees C until surgery, which occurred within 2 hours. The implant assemblies were implanted in subcutaneous or epididymal fat sites in male Lewis rats for 3 weeks.
After three weeks of implantation, the assemblies were explanted, trimmed of excess fat, and fixed with 2% glutaraldehyde in Sorensen's buffer. Sections of the assemblies were stained with hematoxylin and eosin.
Cell survival was scored based upon histological appearance of the implanted cells. Tissues were scored as "excellent" if they had normal characteristics of lung tissue, such as epithelial tubules, cilia, and formed cartilage. Tissues were scored as "good" if the tissue were still alive, but not well differentiated (for example, a high number of mesenchymal cells). The tissues were scored as "poor" if no or few cells remained alive.
In other histology studies using implanted pancreatic cells, survival assessment would involve analyzing the differentiated function of the pancreatic cells in terms of their insulin release in the response to a glucose challenge.
Table 1 shows the permeability value for those boundaries having a porosity value (PORE) greater than 70%, correlated with the survival of the implanted lung tissues.
TABLE 1______________________________________Membranes with PORE > 15% Pore Size orMembrane MW Cutoff Permeability Tissue Survival______________________________________cellulose acetate.sup.1 unknown 9 excellentcellulose acetate.sup.1 unknown 5.3 excellentBIOPORE .TM. .sup.2 0.45 .mu.m 2.6 excellentpolyvinyl difluoride.sup.1 unknown 2.5 goodcellulose mixed ester.sup.2 1.2 .mu.m 2.0 poorpolyvinyl difluoride.sup.1 unknown 1.7 goodpolypropylene.sup.3 0.075 .mu.m 1.4 poorcellulose acetate.sup.1 unknown 1.3 poorcellulose mixed ester.sup.2 0.45 .mu.m 0.9 poorpolyethylene.sup.3 0.08 .mu.m 0.9 poorcellulose.sup.4 300 kD 0.6 poorcellulose.sup.4 50 kD 0.2 poor______________________________________ *.times. 10.sup.-4 cm/s .sup.1 Baxter Healthcare Corporation (Deerfield, Il) .sup.2 Millipore Corporation (Bedford, Ma) .sup.3 Hoechst Celanese (Charlotte, NC) .sup.4 Spectrum Medical Instruments (Los Angeles, Ca)
Table 2 shows the permeability value of those boundaries having a porosity value (PORE) less than 15%, correlated with the survival of the implanted cells.
TABLE 2______________________________________Membranes with PORE < 15% Perme- TissueMembrane* Pore Size ability.Yen. survival______________________________________NUCLEPORE.sup.1 0.8 4.4 FairNUCLEPORE 0.4 3.1 PoorNUCLEPORE 0.22 2.3 PoorPORETICS.sup.2 0.1 2.2 PoorPORETICS 0.08 0.5 PoorPORETICS 0.05 1.2 PoorPORETICS 0.03 0.9 PoorPORETICS 0.01 0.2 Poor______________________________________ *polycarbonate .Yen..times. 10.sup.-4 Cm/s .sup.1 Nuclepore Corporation (Pleasanton, Ca) .sup.2 Poretic Corporation (Livermore, Ca)
Tables 1 and 2 demonstrate the direct relationship between the metabolic transit value of the boundary and implanted cell survival. More particularly, the Tables show that implanted cell survival significantly improves when the permeability value of the boundary increases.
For the type of cells studied in Example 1, boundaries having a permeability value for insulin less than about 1.5.times.10.sup.-4 cm/sec, as determined using the described methodology, consistently did not support cell survival, regardless of the porosity value. Yet, boundaries having a permeability value for insulin greater than about 1.5 .times.10.sup.-4 cm/sec and a porosity value greater than about 15% uniformly supported vigorous cell survival.
Boundaries having a lower porosity value (less than about 15%) also supported cell survival (see Table 2). Still, the metabolic transit value for these less porous boundaries requires a higher relative permeability value. For the type of cells studied in Example 1, boundaries having a lower porosity value (less than about 15%) supported cell survival when the permeability value for insulin was greater than about 4.0.times.10.sup.-4 cm/sec.
The inventors believe that, when considering less porous boundaries, their specific physical structure must also be taken into account. The less porous interfaces used in Example 1 were track-etched membranes. These membranes have uniform cylindrical pores separated by relatively large, nonporous regions.
The poor tissue survival using the low porosity boundaries could be due to uneven localization of areas of high permeability, or due to constraints produced by cells on the particular physical properties of the track-etched membranes. For example, the cells may be more efficient at plugging up the cylindrical pores of the track-etched membranes either with cell extensions or cell secretions. Thus, although the track-etched membranes have high permeability values in vitro, the response of the cells in vivo may prevent the attainment of sufficient metabolic transit to support the graft cells.
Example 1 demonstrates a methodology that can be followed to identify for other cell types the applicable metabolic transit value that assures cell survival during the ischemic period after implantation.
The absolute permeability and porosity values that constitute a given metabolic transport value will depend upon the type of cell and the methodologies of determining permeability and porosity. Different conditions will give different absolute values. Still, regardless of the test conditions, the relative differences in permeability and porosity values derived under constant, stated conditions will serve as an indicator of the relative capabilities of the boundaries to support implanted cell viability.
Tables 1 and 2 also show that good tissue survival occurs even with membrane materials that are subject to the formation of an avascular fibrotic response (the so-called "foreign body capsule"). The fact that these membrane materials create this response has, in the past, led to the widely held view that the formation of the foreign body capsule caused poor diffusion of nutrients. Example 1 shows the error of this conventional wisdom.
As Table 1 shows, the use of relative thicker cellulose acetate membranes with 0.45 micron pore size (130 microns thick) having an insulin permeability of 0.9.times.10.sup.-4 cm/sec results in poor tissue survival. On the other hand. the use of relatively thinner cellulose acetate membranes with the same approximate pore size (10 microns thick) and having a greater permeability of 5.3.times.10.sup.-4 cm/sec results in excellent tissue survival.
The thickness of the membrane does not alter the foreign body response; a foreign body capsule will form whether the membrane is relatively thick or thin. However, membrane thickness does alter the permeability value.
Thus, the cells died when the thicker boundary was used, not because of the formation of the foreign body capsule, but because of poor nutrition and poor waste removal due to the low permeability of the thicker boundary. The tissue survived when the thinner boundary is used, because the higher permeability provided improved cell nutrition and improved waste removal to support cell metabolism, even when the same foreign body capsule forms.
EXAMPLE 2
In an experiment, the practitioner grew RAT-2 fibroblasts (ATCC CRL 1764) in 20% Fetal Bovine Serum, 2 mM 1-glutamine, and DMEM (Sigma) (high glucose) until 100% confluent. The RAT-2 cells were split 1:2 in the above media, 16 to 24 hours before surgery.
On the day of surgery, the cells were washed with 15 ml of HBSS (no ions) and trypsinized off the culture flask. The practitioner neutralized the trypsin by adding 5 ml of the above media. The practitioner pelleted the cells by centrifugation (1000 rpm, 10 minutes, at 22 degrees C).
The pelleted cells were counted and resuspended in media in three concentrations: 5.3.times.10.sup.3 cells/10 .mu.l; 5.8.times.10.sup.5 cells/10 .mu.l; and 5.8.times.10.sup.6 cells/10 .mu.l.
Implant assemblies like that shown in FIGS. 1 to 4 having boundaries of differing permeability values were made. The permeability values ranged from 0.2.times.10.sup.-4 cm/sec to 9.times.10.sup.-4 cm/sec (see Tables 1 and 2 to follow). The total boundary area for each assembly was about 0.77 cm.sup.2.
The various cell concentrations were loaded into the assemblies. The practitioner implanted the assemblies both subcutaneously and within the epididymal fatpad of host rats.
After 3 weeks, the assemblies were explanted and examined histologically, as described previously.
The inventors observed that assemblies loaded with 5.8.times.10.sup.3 cells and 5.8.times.10.sup.5 cells displayed excellent results, given sufficient boundary permeability values. After 3 weeks of implantation, the initial load of 5.8.times.10.sup.5 cells proliferated to approximately 2.0.times.10.sup.7 cells. The inventors observed that assemblies having higher initial loads of 5.8.times.10.sup.6 cells displayed poorer results.
Lower initial loads (less than 5.times.10.sup.6) were able to survive the ischemic period and even proliferate 30 to 3000 fold. The final cell counts in the assemblies with lower initial loads were three times higher than the initial load of the assemblies that failed because of higher initial loads. Thus, high loads of cells (greater than 5.times.10.sup.6) are unable to survive during the ischemic period, yet the same cell loads are able to survive after the ischemic period as progeny of the cells from lower initial loads.
Close Vascularization at the Boundary
(1) presence of Angiogenic Material
Neovascularization close to the boundary is essential to the long tern survival of the implanted cells within the host. The inventors have found that the host will not grow new vascular structures 54 close to the boundary (as FIGS. 24 and 25 show), unless it is stimulated to do so. Without proper stimulation, the ischemic period never ends, because a classical foreign body reaction occurs.
The assembly 10 therefore includes an angiogenic material 56 for stimulating neovascularization close to the boundary.
The specific identity of the angiogenic material 56 is not known. Still, the inventors have determined that the presence of certain cells stimulate neovascularization, while others do not.
For example, the presence of lung tissues; pancreatic islets; adult pancreatic ducts; and cultured cell lines of fibroblasts, mammary gland, and smooth muscle cells induces or stimulates neovascularization, when compared to the vascularization on control grafts where these cell types were not present.
On the other hand, the presence of primary skin fibroblasts and microvascular endothelial cells do not induce neovascularization.
The inventors believe that certain cells induce or stimulate neovascularization by secreting angiogenic factors. Because the stimulus crosses membranes that are impermeable to cells, it must be a molecular signal that the living cell generates. This further underscores the need to support the implanted cells during the ischemic period. If angiogenic source cells perish, the molecular signal stops, and the neovascularization process comes to a halt.
According to this aspect of the invention, when cells are implanted that have a desired therapeutic effect, but do not secrete angiogenic material, the assembly 10 includes a separate angiogenic source cell or material 56.
Following the invention, the practitioner selects an boundary 46 having a sufficient metabolic transit value to support the viability of the implanted cells, i.e., the angiogenic source cells and other non-angiogenic, therapeutic cells (when present) implanted with them. The practitioner also selects a pore size and ultimate physical strength to make the boundary 46 impermeable to the neovascular growth that the angiogenic source cells stimulate.
Alternatively, the practitioner may coat the exterior of the boundary 46 itself with an angiogenic material 56. Of course, the coated boundary 46 still must have sufficient pore size, ultimate strength, and metabolic transit value to sustain the cells 12 isolated behind the boundary 46.
Because the new vascular structures 54 cannot penetrate the boundary 46, and because the angiogenic signal to the host continues, the new vasculature proliferates close to the boundary 46.
As FIG. 21 shows, when the cells 12 die during the ischemic period, and close vascularization is not stimulated, the fibroblasts of the foreign body capsule 52 become closely packed and dense. However, as FIG. 20 shows, when the cells 12 survive the ischemic period, and the process of close vascularization is stimulated, the fibroblasts of the foreign body capsule 52 is altered to form a less dense and more dispersed structure.
(2) Conformation for Close Vascularization
In the preferred embodiment, the porous boundary 46 includes an interface 47 with the host tissue that is characterized by a structural conformation that further enhances the growth of vascular structures by the host close to the boundary.
To achieve this result, each wall element 32/32' and 34/34' of the assemblies 10/10' includes a first porous region 58 and a different second porous region 60. The first porous region 58 comprises the boundary 46 previously described. The second porous region 60 comprises the interface 47.
The first porous region 58 faces the implanted cells 12 (see FIG. 20). The first porous region 58 has the boundary characteristics, above described, of pore size; ultimate physical strength; and metabolic transit value. It is this region 58 that isolates the implanted cells from the immune mechanisms of the host, while sustaining their viability through the flux of nutrients and wastes during the ischemic period.
The second porous region 60 faces the host tissue 44 and forms the interface 47 with it (see FIG. 20). The second porous region 60 has an architecture that enhances the formation of vascular structures 54 close to the boundary 46. The formation of these vascular structures 54 within the second region 60 mark the end of the ischemic period. Vascularization in the second region 60 sustains the viability of the implanted cells 12 after the ischemic period ends.
A foreign body capsule 52 still forms about the implanted assembly 10. However, close vascularization within the second porous region 60 can alter the normal configuration of the foreign body capsule 52. As FIG. 20 shows, a life sustaining vascular bed forms within the capsule 52 close to the boundary 46, keeping flattened macrophages, foreign body giant cells, and fibroblasts from pressing against and blocking the boundary 46.
Because of the pore size, strength, and permeability characteristics of the porous first region 58, it is impermeable to the neovasculature 54 formed in the second region 60.
The inventors believe that close vascularization occurs if the three dimensional conformation of second region 60 creates certain host inflammatory cell behavior.
The inventors have observed by light and electron microscopy that close vascularization occurs if, in the initial period of implantation, at least some macrophages entering the material are not activated. Activated macrophage are characterized by cell flattening.
The inventors observe close vascularization in regions of an implant where the macrophages that have entered the cavities of the material retain a rounded appearance when viewed through light microscopy (-400.times.). At 3000.times. (TEM) the rounded macrophage is observed to have substantially conformed to the contours of the material. Although there is a correlation with macrophage shape, it is not clear that macrophages control the observed, response. However, it is clear that invasion of the structure by host cells is required. Although the bulk of the cells appear to be macrophages, it is possible that other inflammatory cells control the response, therefore the inventors refer to the invading cells as "inflammatory cells," which include but are not limited to macrophages.
On the other hand, foreign body capsule formation occurs when, in the initial period of implantation, inflammatory cells in contact with the implant material flatten against those portions of the material which present an area amenable to such flattening behavior by an inflammatory cell.
The material for the second region 60 that results in formation of close vascular structures is a polymer membrane having an average nominal pore size of approximately 0.6 to about 20 .mu.m, using conventional methods for determination of pore size in the trade. Preferably, at least approximately 50% of the pores of the membrane have an average size of approximately 0.6 to about 20 .mu.m.
The structural elements which provide the three dimensional conformation may include fibers, strands, globules, cones or rods of amorphous or uniform geometry which are smooth or rough. These elements, referred to generally as "strands," have in general one dimension larger than the other two and the smaller dimensions do not exceed five microns.
In one arrangement, the material consists of strands that define "apertures" formed by a frame of the interconnected strands. The apertures have an average size of no more than about 20 Am in any but the longest dimension. The apertures of the material form a framework of interconnected apertures, defining "cavities" that are no greater than an average of about 20 pn in any but the longest dimension.
In this arrangement, the material for the second region has at least some apertures having a sufficient size to allow at least some vascular structures to be created within the cavities. At least some of these apertures, while allowing vascular structures to form within the cavities, prevent connective tissue from forming therein because of size restrictions.
Further details of the material are set forth in copending U.S. application Ser. No. 735,401 entitled "Close Vascularization Implant Material" filed Jul. 24, 1991, which is incorporated into this Specification by reference.
Making a Boundary
FIGS. 11 to 16 show a method of making a preferred embodiment of the wall elements 32 and 34 that forms the boundary. The method integrally joins material selected for the first region 58 to another material selected for the second region 60. The two joined materials form the composite, or laminated, structure 72 shared by both wall elements 32 and 34. The laminated structure 72 joins the interface 47 to the boundary 46.
In the illustrated embodiment, a porous PTFE membrane material having a thickness of about 35 microns and a pore size of about 0.4 micron is selected for the first region 58. This material is commercially available from Millipore Corporation under the tradename Biopore.TM..
The porous material selected for the first region 58 has a thickness of about 30 microns and an ultimate (tensile) strength value of at least 3700 PSI, which is well above the desired minimum value. The selected material has pore size of 0.35 microns, which blocks the passage of inflammatory cells. The selected material has a permeability value for insulin of 2.6.times.10.sup.-4 cm/sec and a porosity value of greater than 70%. The membrane therefore meets the metabolic transit value requirements.
It should be appreciated that other, comparable materials can meet the stated requirements for the first region 58. For example, polyethylene, polypropylene, cellulose acetate, cellulose nitrate, polycarbonate, polyester, nylon, and polysulfone materials can be used. Mixed esters of cellulose, polyvinylidene, difluoride, silicone, and polyacrylonitrile can also be used.
In the illustrated embodiment, a membrane material made by W. L. Gore and Associates (Elkton, Md.) under the tradename GORE-TEX.TM. is selected for the second region 60. The GORE-TEX.TM. material comprises a microporous membrane made from PTFE. The membrane is 15 microns thick and has a pore size of 5 microns. Polyester strands 61 join the PTFE membrane to form a backing for it. The backing has a depth of about 120 microns.
The GORE-TEX.TM. material also has an ultimate strength value well above the desired minimum value. The conformation of the polyester strands 61 also meets the criteria, set forth earlier, for promoting the growth of neovascular structures.
In Step 1 (see FIGS. 10 and 11), the practitioner secures the edges of a strip of the GORE-TEX.TM. material (second region 60) to a lamination slide 62, with the polyester backing 61 facing the slide 62.
In Step 2 (see FIG. 13), the practitioner places 2 or 3 lamination slides 62 side-by-side on a work surface. Using a syringe 64, the practitioner applies cement or adhesive in continuous filaments 66 in a back and forth pattern across the lamination slides 62. The practitioner touches the syringe tip 64 to the work surface at the end of each filament 66 to begin a new filament 66.
Step 2 forms a criss-crossing pattern of cement filaments 66 across the secured strips of the second region material, as FIG. 13 shows.
The cement selected can vary. For example, the cement can be cellulose acetate or similar epoxy material. In the illustrated embodiment, the cement comprises a mixture of VYNATHENE EY 90500 EVA resin and toluene (made by Mallinckrodt).
In forming the EVA cement mixture, the practitioner adds about 30 grams of resin and an equal amount of toluene to a bottle. The practitioner seals the bottle to allow the resin to dissolve. The bottle may be periodically shaken to accelerate this process.
The relative amounts of resin and toluene may have to be slightly adjusted to arrive at the right consistency for the cement. If the cement is too thin to form continuous filaments when applied, use less toluene. If the cement is to viscous to be expressed from the syringe, use more toluene. Small changes in the amount of toluene added result is significant changes in the viscosity of the cement.
In Step 3 (as FIG. 14 shows), the practitioner places preformed strips of the BIOPORE.TM. membrane material (first region 58) upon the cement filaments 66 applied in Step 2. In the illustrated embodiment, the practitioner precuts the Biopore.sup.TM membrane material into disks having the diameter desired for the wall elements 32 and 34.
In Step 4 (as FIG. 15 shows), the practitioner lays a strip of release material 68 (like PATAPAR) over the first region material 58 and covers the layered structure with another lamination slide 70. The practitioner clamps the lamination slides 62 and 70 together, bringing the membrane layers into intimate contact.
In Step 5, the practitioner places the clamped lamination slides 62 and 70 in an oven for about 5 to 10 minutes at a temperature of about 80 degrees C. The heat melts the EVA cement.
In Step 6, the heated lamination slides 62 and 70 are allowed to cool to room temperature. Upon cooling and solidification, the filaments 66 securely join the BIOPORE.TM. membrane material to the GORE-TEX.TM. membrane material. The practitioner then unclamps the lamination slides 62 and 70 and removes the finished composite structure 72 (in strips).
In Step 7 (as FIG. 16 shows), the practitioner lays the composite structure 72 strips on a polypropylene cutting slab 74. The practitioner aligns a presized punch 76 over each precut disk, striking the punch with a hammer. The practitioner thereby frees the wall elements 32 or 34 formed of the composite structure of the desired dimensions. Small scissors may be used to snip any adherent polyester strands not cut by the die.
Implant assemblies 10/10' are made using the wall elements in the manner previously described.
It should be appreciated that the first region material 58 can be applied to the second region material 60 by various alternative means to form the laminated structure 72. For example, the first region material 58 can be extruded in place upon the second region material 60.
EXAMPLE 3
Assemblies like that shown in FIGS. 1 to 4 and constructed according to the foregoing process have been successfully used to accomplish complete correction of diabetes in partially pancreatectomized and streptozotocin-treated rat hosts. The animals were corrected up to 293 days. Upon removal of the implants, the animals reverted to a diabetic state. Histology of the implants revealed the presence of vascular structures close to the boundary.
These assemblies presented a boundary area of about 0.77 cm.sup.2. Each assembly sustained an initial cell load of about 600 pancreatic islets (or about 600,000 pancreatic cells).
When implanted, the assemblies sustained cell densities of about 200,000 islets/cm.sup.3. These assemblies, made and used in accordance with the invention, supported 8 times more pancreatic islets in a given volume than the CytoTherapeutics assemblies (having cell densities of only 25,000 islets/cm.sup.3).
Deriving a Therapeutic Loading Factor
As earlier described, one aspect of the invention provides the ability to identify a metabolic transit value associated with a given cell type. Knowing the required metabolic transit value, in turn, makes it possible to identify the clinically practical region of operation, where compact implant assemblies can sustain therapeutically large volumes of cells.
This aspect of the invention provides the methodology to derive and use a therapeutic loading factor (L) to characterize and predict the clinical effectiveness of a given implant assembly for a given cell type.
The therapeutic loading factor (L) takes into account the number of cells (N) that are required to be implanted to achieve the desired therapeutic effect; the effective area (A) of the boundary between the implanted cells and host that the host can be reasonably expected to tolerate; and the metabolic transit value (T) needed to sustain cell viability.
The therapeutic loading factor for a given implant assembly and given implanted cell type can be expressed as follows:
L.sub.c =(A/N.sub.c)*T.sub.min
where
c is the given cell type,
L.sub.c is the therapeutic loading factor for the given cell type,
A is the area of boundary between the implanted cells and the host offered by the given implant assembly,
N.sub.c is the number of cells supported by the boundary area (A), and
T.sub.min is the minimum metabolic transit value that will support cell survival during the ischemic period, determined according the methodology set forth in Example 1.
If the practitioner selects boundaries having a porosity value of greater than 15%, then the permeability value (P) alone can be used to express the metabolic transit value (T). The therapeutic load factor can then be expressed:
L.sub.c =(A/N.sub.c)*P.sub.min
where P.sub.min is the minimum permeability value that will support cell survival during the ischemic period.
In the assemblies described in Example 3, the observed ratio between the boundary area and the number of implanted cells (A/N.sub.c) for the successful implantation of pancreatic cells was 128 .mu.m.sup.2 /pancreatic cell. The inventors believe that a somewhat larger ratio of about 150 .mu.m.sup.2 /pancreatic cell will provide a satisfactory margin for variance among different hosts.
As earlier discussed, given a boundary porosity value of greater than 15%, a permeability value (P) greater than about 1.5.times.10.sup.-4 cm/sec for insulin should be provided a metabolic transit value that will sustain cell survival during the ischemic period and afterward.
FIG. 22 shows the therapeutic loading curve for pancreatic cells generated based upon the above considerations. The curve displays the predicted region of cell survival in terms of the boundary area-to-cell number ratio A/N (x-coordinate) and permeability value P (y-coordinate) (given a porosity value of greater than about 15%).
FIG. 22 predicts that assemblies operating to the right of the therapeutic loading curve will sustain implanted pancreatic cells. FIG. 22 predicts that assemblies operating to the left of the therapeutic loading curve will not.
The inventors believe that a human diabetic will require the transplantation of about 250,000 pancreatic islets (or about 250 million pancreatic cells) to derive a therapeutic benefit. With this in mind, one can calculate a range of sizes for an implant assembly based upon the A/N ratio.
The equation for calculating the side dimension (L) in cm of a square implant assembly based upon the A/N ratio is as follows: ##EQU3## where: the factor 10.sup.-8 converts micron.sup.2 to cm.sup.2.
The equation for calculating the diameter (D) in cm of a round implant assembly based upon the A/N ratio is as follows: ##EQU4## where: the factor 10.sup.-8 converts micron.sup.2 to cm.sup.2.
Table 3 lists a range of L's and D's at different A/N ratios for an implant assembly holding 250,000 pancreatic islets
______________________________________A/N A (cm.sup.2)/side L(cm) D(cm)______________________________________128 160 12.6 14.3150 188 13.7 15.5200 250 15.8 17.8328 410 20.2 22.8463 579 24.0 24.1______________________________________
Based upon the foregoing considerations, the inventors believe that A/N ratios less than about 200 .mu.m.sup.2 /pancreatic cell define the operating region of implant assemblies that offer compact, clinically practical implant boundary areas. FIG. 22 shows this preferred region.
As FIG. 22 also shows, a practitioner can provide an implant assembly that combines the benefits of compact size with the ability to sustain the requisite therapeutical number of cells, by selecting a permeability value for the boundary that achieves a region of operation to the right of the therapeutic loading curve. The practitioner also selects the prescribed pore size and ultimate physical strength determined in accordance with the invention.
FIG. 22 shows that the prior art hollow fiber implant assembly made by CytoTherapeutics (described in the Background section of this Specification) falls well outside the preferred region of clinically practical operation. This assembly offers an A/N ratio of about 328 .mu.m.sup.2 /pancreatic cell, about 1.5 times the A/N ratio of the invention.
FIG. 22 also shows a prior art hollow fiber implant assembly made by W. R. Grace and Co. (Lexington, Mass.), as reported by Proc. Natl. Acad. Sci. U.S.A., Vol. 88, pp. 11100-11104 (December 1991). Each hollow fiber had a length of 2-3 cm, and an inside diameter of 0.177 cm. There were 200 to 400 pancreatic islets loaded into each fiber for implantation. Taking an average length of 2.5 cm and an average cell load of 300 islets, the associated A/N ratio is 463, more than twice the A/N ratio of the invention.
The foregoing establishes a methodology to derive and use a therapeutic loading factor for pancreatic islets. This methodology can be followed to identify a therapeutic loading factor for other cell types and other ranges of metabolic transit values. The absolute value of the therapeutic loading factor derived will of course depend upon the type of cell and the methodologies used to determine permeability and porosity. Different conditions will give different absolute values for the therapeutic loading factor.
Still, regardless of the test conditions, the relative differences in the A/N ratios, permeability values, and porosity values derived under constant, stated conditions will serve as a means to characterize and predict the clinical effectiveness of a given implant assembly for a given cell type.
It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications be covered by the appended claims.
Claims
  • 1. A method for implanting cells into a host tissue comprising the steps of:
  • providing an implant assembly for holding cells including wall means having an interior surface defining a chamber for holding cells for implantation and exterior surface defining an interface with the host tissue, the wall means having a pore size sufficient to isolate the implanted cells from the immune response of the host tissue and having a metabolic transit value sufficient to sustain viability of implanted cells during ischemia, the exterior surface having a conformation which allows growth of vascular structures by the host tissue close to but not substantially into the interface, the implant assembly including a port;
  • adding cells into the chamber of the implant assembly through the port; and
  • implanting the implant assembly and port within a host.
  • 2. The method of claim 1, wherein the cells are added to the implant assembly by using a syringe.
  • 3. A method for implanting cells into a host tissue comprising the steps of:
  • providing an implant assembly for holding cells including wall means having an interior surface defining a chamber for holding cells for implantation and exterior surface defining an interface with the host tissue, the wall means having a pore size sufficient to isolate the implanted cells from the immune response of the host tissue and having a metabolic transit value sufficient to sustain viability of implanted cells during ischemia, the exterior surface having a conformation which allows growth of vascular structures by the host tissue close to but not substantially into the exterior surface, the implant assembly including a port;
  • implanting the implant assembly and port within a host; and
  • accessing the port and adding cells to the chamber of the implant assembly through the port after the implant assembly has been implanted.
  • 4. The method of claims 1 or 3, including the step of sealing the port after an addition of the cells to the implant assembly.
  • 5. The method of claim 4, wherein the port is sealed by a heat seal.
  • 6. The method of claim 4, wherein the port is sealed by mechanical means.
  • 7. The method of claim 4, wherein the port is sealed by a plug created by a curable substrate.
  • 8. The method of claims 1 or 3, including the steps of:
  • providing two ports on opposite sides of the implant assembly; and
  • flushing cells out of the implant assembly by exerting pressure through one port on the cells within the assembly to cause them to flow through the second port.
  • 9. The method of claim 8, including the step of adding new cells through one of the ports into the implant assembly after cells have been flushed out of the implant assembly.
  • 10. The method of claims 1 or 3, including the step of removing cells from the implant assembly by aspirating the cells out through the port.
  • 11. The method of claim 10, including the step of adding new cells through the port into the implant assembly.
  • 12. The method of claims 1 or 3, wherein the port is a tube.
  • 13. The method of claim 12, including the step of implanting a plurality of implant assemblies and coupling at least two tubes of the plurality of implant assemblies together with mechanical means.
  • 14. The method of claim 13, wherein the mechanical means is a clip.
  • 15. A method for implanting cells into a host tissue comprising the steps of:
  • providing a plurality of implant assemblies for holding cells, each implant assembly including wall means for forming a porous boundary between the host tissue and the implanted cells in the assembly, the pore size of the boundary being sufficient to isolate the implanted cells from the immune response, the assembly including a tube;
  • adding cells to an interior of the implant assembly through the tube; and
  • coupling at least two tubes of the plurality of implant assemblies together with mechanical means.
  • 16. The method of claim 15, wherein the mechanical means is a clip.
RELATED APPLICATION

This is a division of application Ser. No. 08/179,860, filed on Jan. 11, 1994, which is a continuation-in-part of U.S. application Ser. No. 07/933,871 entitled: "Close Vascularization Implant Material" filed Aug. 21, 1992, abandoned, which is a continuation-in-part of U.S. application Ser. No. 07/735,401 filed Jul. 24, 1991, abandoned, which is a continuation-in-part of U.S. application Ser. No. 07/606,791 entitled: "Membrane for Vascularization of Foreign Body Capsule" filed Oct. 3, 1990, abandoned.

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Divisions (1)
Number Date Country
Parent 179860 Jan 1994
Continuation in Parts (3)
Number Date Country
Parent 933871 Aug 1992
Parent 735401 Jul 1991
Parent 606791 Oct 1990