Patent Application
20100121446
The present invention generally relates to an implant assembly, and in particular such an implant assembly comprising a semipermeable membrane or barrier.
It is known in the organ transplantation technique that the human body opposes transplantation of an organ, tissue or even a cluster of cells by rejection phenomena caused by the immune defense of the body. In order to avoid immunosuppressive medicaments, bioartificial implants have been developed which comprise donor tissue or cells, which are to be implanted and a semipermeable barrier or filter, which allows diffusion of nutrients and oxygen from the donee's body to the implanted donor tissue or cell. This barrier should however not allow diffusion of the cells of the donee's immune defense mechanism. In addition and in particular in connection with xenotransplan, it could be advantageous to prevent diffusion of antibodies and components of the complement system of the recipient's body over the semipermeable barrier. The barrier should also of course allow diffusion of the desirable (therapeutic) substances produced by the cells in the implant into the donee's body.
One of the main obstacles in the art for obtaining successful usage of bioartificial implants is the growth of connective tissue in the donee's body around the implant. The connective tissue covers the pores of the semipermeable barrier, effectively inhibiting diffusion of nutrients and oxygen to the cells inside the implant. This will cause hypoxia and cell death.
The document WO 2005/030283 teaches a bioartifical implant, in which the outer surface of the semipermeable barrier is coated with a bioactive metal, such as titanium, in such a way that the coating does not impede the selective diffusion through the membrane. This coating has the advantage of inhibiting the growth of connective tissue (fibrosis) and promoting vessel formation.
There is though still a need for a bioartificial implant assembly having an implant construction that is highly safe, implying that cells present in the implant assembly cannot unintentionally leak out in the donee body after transplantation. This is particularly important for stem cells and genetically modified cells, which could be transformed into tumor-inducing cells if they escape the implant assembly. There is also a need for an implant assembly having an implant construction designed to minimize the growth of connective tissue.
The present invention overcomes these and other drawbacks of the prior art arrangements and meets the needs for an improved bioartificial implant.
It is a general object of the present invention to provide an improved bioartificial implant assembly.
Briefly, the present invention involves a bioartificial implant assembly comprising a frame structure and membranes. The frame structure comprises an inwardly protruding support flange and a respective frame portion extending above and below the support flange, respectively. The two membranes have outer circumferences matching the inner circumference of the frame structure. At least one the membranes, preferably both, is semipermeable. One of the membranes is positioned on a first side of the support flange with the other membrane positioned on the second opposite flange side. A sealing is formed at the first frame portion and at the second frame portion. This forms an implant with an enclosed, sealed inner chamber defined by the membranes and the frame structure.
The sealing can be effected by heat pressing the frame portions towards the support flange. Material of the frame portions will penetrate through the semipermeable membrane and attach to the flange material thereby forming effective seals.
Alternatively, foil rings having outer circumferences matching the outer circumference of the frame portions and inner circumferences that are smaller than the outer circumferences of the membranes are positioned on the respective frame portions. The foils rings are sealed, preferably through welding or using an adhesive, to the frame portions but also to the circumferential portions of the membranes.
Instead of utilizing single semipermeable membranes, membrane-comprising laminate structures can be used. In such a case, a sandwich structure comprising a semipermeable membrane, one or more nets of a bioactive metal and optionally one or more weldable rings can be fabricated and handled as a single unit.
The invention together with further objects and advantages thereof, may best be understood by making reference to the following description taken together with the accompanying drawings, in which:
Throughout the drawings, the same reference characters will be used for corresponding or similar elements.
The present invention relates to a bioartificial implant assembly based on a semipermeable barrier for allowing selective diffusion of substances over the barrier but also preventing diffusion of cells and possible soluble components, such as antibodies and complement components, of the immune defense. The implant assembly of the present invention has an implant design that reduces the risk for extensive growth of connective tissue around the membrane following implantation or at least reduces the amount of such connective tissue. The implant assembly also has a construction that achieves a highly safe sealing of the interior of the implant, thereby preventing unintentional leakage of, for instance, cells present in the implant interior to the donor body after implantation.
The implant assembly comprises three main constituents: a frame structure and two membranes, of which at least one is semi-permeable. The frame structure comprises an inwardly protruding support flange or shoulder employed as a support structure for the membranes or laminate structures comprising the membranes. A first frame portion of the frame structure extends above the support flange and a second opposite frame portion extends below the support flange as seen from a side view. The first membrane (laminate) is then positioned on a first side of the support flange, while the other membrane (laminate) is positioned on a second opposite side of the support flange. The inner chamber defined by the membranes and the frame structure is then sealed from the outside by a first seal formed at the first frame portion and a second seal formed at the second frame portion.
Herein different embodiments of the bioartificial implant assembly, will be described in more detail in connection with the figures.
The frame 10 is preferably made from a biocompatible material. More preferably, the frame 10 is made from a biocompatible polymer material, such as a plastic material. An example of such a material is polyethylene. The general form of the frame 10 could be circular as illustrated in the figure. However, this is merely an illustrative example and the invention is not limited thereto. The invention can thus be used in connection with any general frame form, including also elliptical, triangular, quadratic, or rectangular forms.
In a preferred implementation, the frame 10 is manufacture by molding, such as injection-molding. Other techniques known in the art can be selected to adopt for the particular frame material employed.
The frame thickness could be a few millimeters down to sub-millimeters, for example 1 mm or 0.5 mm. However, the present invention is not limited to L 0 any particular frame sizes.
A respective membrane 20, 30 is positioned on either side of and is supported by the flange 16, whereby at least one of the two membranes 20, 30, preferably both membranes 20, 30, constitutes a semipermeable barrier. The semipermeable membranes 20, 30 are designed to, from one side, allow diffusion or prevent diffusion of substances, materials, molecules or cells to the other opposite side. In a preferred implementation, the membranes 20, allow diffusion from one side to the other opposite side of nutrients and oxygen but prevent diffusion of cells and possibly soluble components, in particular cells and soluble components of the immune defense. Furthermore, the membranes 20, 30 are designed to, from the other opposite side, to allow diffusion or prevent diffusion of substances, materials, molecules or cells. In a preferred implementation, the membranes 20, 30 allow diffusion of substances present in the implant assembly 1 or produced by cells in the implant assembly 1 from the other opposite side. In the latter case, the membranes 20, 30 preferably prevent diffusion of any cells in the implant 1 to the outside of the implant assembly 1.
The semipermeable barriers 20, 30 can be made of a polymer material, such as polytetraflouroethylene (PTFE), GoreTex, alginated polymer crosslinked with Ca, carbohydrates, cellulose, plastics, polyvinylchloride (PVC), polycarbonate (PC), polyamides (PA), polyethylene (PE), polypropylene (PP), polyurethane (PU), polyvinylidene fluoride (PVDF), acrylic copolymers, such as polymethylmethacrylate (PMMA) or polyhydroxyethylmethacrylate (PHEMA), polyacetale (POM), polyethersulfone (PES), polysulfone (PS), polyesters, polyetherketone (PEEK), polyetherimide, polyester, polyacrylonitrile (PAN), polyvinylidendifluorid (PVDF), hydrogels. Such membranes 20, 30 are today available at the marked by for instance Millipore Inc, Baxter Inc and GE Healthcare.
Semipermeable membranes 20, 30 are available with different pore size distributions. As is well known in the art, pore sizes for semipermeable membranes are given as nominal pore sizes. In practice, there is a distribution around this average, nominal pore size. For example, typical nominal pore sizes for allotransplantation applications could be 0.45 or 0.22 μm. In the case of xenotransplantation applications, smaller pore size could be useful. The limiting pore size depend on the selective diffusion that is desired, i.e. allowing diffusion of therapeutic material present inside the assembly 1 or being produced inside the assembly 1 to the outside of the membranes 20, 30. Correspondingly, nutrients and oxygen present outside the membranes 20, 30 are preferably allowed to diffuse through the pores 25, 35 into any cells present in the implant assembly 1. However, the pore diameters are preferably not so large to allow cells to pass through the membranes 20, 30.
The size of the membranes 20, 30 is preferably selected so that their outer circumference matches the inner circumference of the frame 10, as is illustrated in
In this embodiment, the implant assembly 1 is closed and sealed by pressing down the extending portions 12, 14 of the frame 10 down towards the flange 16. By also applying heat during the pressing, the extending portions 12, 14 will melt together with the flange 16 to thereby form an effective seal.
This implant assembly exhibits several advantages over the prior art implants. Firstly, in the art, the membranes are typically directly welded together or fastened onto each other by applying an adhesive to the circumferential membrane portions. Such a closure may though not be effective enough in clinical applications. Thus, when the implant is transplanted into a host or donee body, the implant will be exposed to strain and external forces due to the movement of body portions. Depending on the actual implantation site, these forces can be large enough to actually break the weld or adhesion between the membranes, allowing content in the implant to leak into the donee body. If the content were cells, these cells will be targeted by the donee's immune defense, effectively killing the cells. The implant will therefore become ineffective. If the cells were, for example, stem cells or genetically modified cells producing a desired therapeutic agent, an undifferentiated form of the stem cells or a modified cell could become a cancer cell in the donee body.
The implant assembly of the invention having a dedicated frame structure supporting the membranes forms an effective seal that will withstand the strain and mechanical forces exerted following transplantation into a donee body, such as an animal body, preferably a mammalian body and more preferably a human body. The frame also provides a semi-rigid backbone that makes the implant less susceptible for the exerted forces.
Another disadvantage of the prior art implants is that, depending on the actual sealing technique employed, the circumferential end portion of the implant can be rough or even sharp. When transplanted, these rough portions will cause a local irritation in the body leading to growth of connective tissue around the implant to encapsulate the irritating implant portions. The connective tissue may, however, form a plug or cover preventing the diffusion of nutrients and oxygen into the inner chamber of the implant and the diffusion of therapeutic substances out from the implant. The cells present in those prior art implants may therefore starve after even a limited transplantation period in the host body due to this connective tissue.
The frame of the invention will after applying the welding (pressure and heat) have smooth, possibly rounded edges as illustrated in
The bioartificial implant assembly of the invention can be used for various therapeutic or diagnostic applications both in vitro and in vivo. Firstly, the internal space formed by the membranes and the sealed frame can house cells, such as xenogeneic cells, allogenic cells or autologous cells. These cells could be genetically modified for producing and releasing a substance or molecule exerting a therapeutic effect when reaching the donee body through the semipermeable membranes. The cells could instead be stem cells or cells derived from stem cells that have the capability of producing an agent of interest. This allows for a controlled and prolonged delivery of the therapeutic agent into the donee body without the risk of the immune defense cells killing the agent-producing cells.
The port 15 is preferably centered in the frame structure 10, in other words extends through the center of the frame 10 as illustrated in
The port 15 is in particular suitable for use in connection with a cell application. The implant assembly 10 can then first be manufactured and sealed and then cells, preferably a cell suspension of agent-producing cells and cell medium, are injected using a syringe through the port 15 into the internal space 18. Once the cell suspension has been added to the implant 1, the port 15 can be sealed by welding, by applying heat and pressure or by an adhesive to effectively close the channel. This sealing could be made close to the outer frame wall to form, after the sealing, a smooth outer frame surface. The remaining part of the port 15 is then discarded. However, if the sealing is performed a distance from the frame 10, the internal implant space 18 can be accessed after transplantation by cutting off the seal in the port 15. New cells can then be added to the internal chamber 18 and/or existing cells can be removed therefrom. The port 15 can then once again be sealed. This procedure can be repeated several times depending on the initial length of the port 15.
The implant assembly 1 of the present invention can be equipped with multiple ports 15 if advantageous.
The bioactive metal is a biocompatible metal that has the capability of attracting tissue and anchoring itself to the tissue. Examples of such materials include titanium, zirconium, tantalum, indium, nickel, aluminum and oxides and alloys thereof, such as titanium dioxide. Other bioactive metals include stainless steel and iridium. Also metals, oxides and alloys comprising chromium, molybdenum, cobalt, tungsten, magnesium, niobium, copper, bismuth, silver, gold, gallium, hafnium, rhenium and zinc could be used. A particular advantageous material is titanium, alloys and oxides thereof.
The nets 40, 50 have several advantageous effects to the implant 1. Firstly, it increases the mechanical strength of the implant 1 and provides stability. This is especially important if the implant 1 is equipped with a port as illustrated in
Most importantly, the bioactive metal, preferably titanium or an oxide of titanium, of the nets 40, 50 significantly eliminates the fibrosis problem of prior art implants. A possible theory for the prevention of growth of connective tissue around the implant in connection with transplantation into the donee, is that the bioactive metal structure will tightly adhere to and become fixed to surrounding tissue. This adherence reduces the movement of the implant inside the body and thereby reduces the risk of local tissue irritation and fibrosis. In addition, experiments performed indicate that the bioactive metal seems to attract blood vessels in the donee body. The growth of connective tissue close to the net surface will be blocked, and such growth of connective tissue will not have time to start again before the blood vessels grow close to the surface of the bioactive nets 40, 50. This has the further advantage that the blood vessels and growth of blood vessels close to the implant 1 allows nutrients and oxygen from the blood vessels to be effectively transported (diffuse) through the implant unimpeded by connective tissue. The nets 40, 50 therefore lower the risk of hypoxia to cells provided in the internal implant space.
The metal nets 40, 50 are permeable to the substances being able to diffuse through the membranes 20, 30. This means that the openings through the nets 40, 50 have a diameter (size) larger than the pore size of the membranes 20, 30. An example of such a metal net is Tigran titanium net with a pore size of 0.12 mm.
In order to achieve close contact between the nets 40, 50 and the underlying semipermeable membranes 20, 30, a support matrix, such as cell growth support matrix can be provided in the inner chamber enclosed by the membranes 20, 30, which is further discussed herein. The support matrix then pushes the membranes 20, 30 close to the nets 40, 50 and prevents the membranes 20, 30 from bend towards the inner chamber space. If no such support is provided, close contact between net 40, 50 and membrane 20, 30 can be achieved using an adhesive that is provided between the net 40, 50 and the membrane 20, 30. If the adhesive is provided as a layer over the whole membrane surface, it is important that it does not plug the pores in the semipermeable membrane 20, 30. Alternatively, the adhesive could be provided at only a limited portion of the plan membrane surface, thereby leaving the vast majority of membrane pores unaffected by the net-membrane adhesive.
In
In a first approach, a ring 60 is first punched from the carrier foil and the membrane 20, net 40 and punched foil 60 are placed onto each other and aligned as illustrated in
Alternatively, a hole is first punched through the cover foil 60 where this hole has a circumference slightly less than the circumference of the final laminate structure 100. The net sheet is then placed on the membrane sheet and the punched foil sheet is placed on the net. A welding is applied, causing the protective foil to melt through the net and attach to the membrane. The resulting sandwich structure 100 is punched with a punch having a slightly larger circumference than the punch employed for forming the hole in the cover foil 60. The resulting laminate structure 100 can then be handled in one piece.
Instead of using the net of bioactive metal, a permeable coating of the bioactive metal could be formed from small grains and granulates of the bioactive metal deposited on the semipermeable membranes using some well-known technique, such as atomizing process or thin-film technique, including evaporation or sputtering. Even smaller metal particles, such as metal powder or dust can be “painted” in dry form or in a suspension onto the semipermeable membrane. The coating is preferably substantially continuous to prevent connective tissue form growing on the underlying semipermeable barrier. The coating should also be free from significant knots that could cause growth of connective tissue. It has been found that excellent results (in terms of no clogging of membrane pores, free diffusion of therapeutic substances, nutrients and oxygen through the coating and fibrosis reduction) are achieved using a metal coating thickness of from about 5 nm, preferably about 50 to 250 nm, though also thicker coating such as about or over 1 μm can also be used. For more reference of coating a semipermeable membrane with a bioactive metal reference is made to the patent application WO 2005/030283, the teaching of which is hereby incorporated by reference.
The bioactive coating will react, at transplantation into the donee body, with blood to form a blood clot. This clot works as an adhesive effectively attaching the implant to the surrounding tissue and thereby reducing the risk of inflammation and growth of connective tissue. The porosity of the coat material will have a capillary effect, providing a suction effect towards external tissue. This will enhance the attachment to the tissue.
In an embodiment, a protective membrane can be positioned on the outside of the semipermeable membrane. This protective membrane protects the semipermeable membrane and function as a coating bearing membrane. This means that the coating of the bioactive metal is provided on a first side of the protective membrane, while the second opposite side lies on the membrane. The protective membrane should then be permeability that does not impede with the semipermeability of the semipermeable membrane.
It is actually possible to combine the usage of a net 40, 50 of bioactive metal with a bioactive metal coating 55 on the semipermeable membrane 20, 30, which is illustrated in
The bioactive metal (powder or granulate) coating can alternatively, or preferably in addition, be provided on at least a portion of the outer surface of the frame structure 10. It is indeed possible, during manufacture of the frame 10 to attach titanium oxide powder particles or granulates (or indeed some other bioactive metal) into the outer frame surfaces. This further increases the percentage of the outer implant surface that comprises a bioactive metal. As a consequence, the secure anchoring of the implant into surrounding tissue, the promotion of growth of blood vessels close up to the implant and the suppression of growth of connective tissue are even further boosted by also presenting bioactive metal on the frame surfaces.
As an alternative, or a combination, the final implant assembly of the present invention could, after manufacture, be introduced into a net pouch made of a bioactive metal, such as an oxide of titanium.
Other forms of tissue anchoring structure could alternatively be employed, including different hooks, barbs and suture sleeves.
In this case the sealing 2, 3 of the implants are made between the rings 80, 90 and the frame portions 12, 14 and between the rings 80, 90 and the membranes 20, 30 or membrane-containing laminate structures positioned on the flange 16.
A preferred implementation applies a weld between the rings 80, 90 and the frame portions 12, 14 and between the rings 80, 90 and the membranes 20, 30. This weld then forms an effective seal 2, 3 between the frame 10 and the rings 80, 90 and between the rings 80, 90 and the membranes 20, 30. The enclosed interior chamber 18 is fully isolated from the exterior of the implant 1 except through the pores 25, 35 in the membranes 20, 30. An alternative to using welds between ring and frame and ring and membrane is to use and adhesive or a combination of adhesive and welding.
As has been described in the foregoing, the semipermeable membranes of the invention can form part of a laminate structure. Such a structure 100 is illustrated in
The laminate structure 100 is positioned on the support flange of the frame structure. With this laminate embodiment, the net 40 can be extended away from the enclosed inner chamber, i.e. facing the exterior of the implant. The net then have the previously described advantageous effects in tissue anchoring, reduction of connective tissue and promoting angiogenesis. Alternatively, the net 40 of the laminate structure 100 faces towards the inner chamber when placed on the support flange. In such a case, the net provides a good cell growth matrix or surface, onto which cells present in the enclosed inner chamber can anchor. This significantly promotes cell survival as the cells then have a favorable surface to attach to and grow on. The net 40 also provides mechanical support and protection to the more sensitive semipermeable membrane 20, in particular when a cell suspension is introduced, such as through the port.
An alternative laminate structure 100 is illustrated in cross sectional view in
It is anticipated by the present invention that the bioartificial implant assembly of the present invention can have at least one of the semipermeable membranes exchanged for a membrane-containing laminate structure, such as illustrated in
If the laminate structure illustrated in
The weldable rings 60, 70 can be made of weldable materials selected from the ones given for the semipermeable membrane above. A typical example is polyethylene (PE).
In a particular embodiment of the invention, the internal space of the implant can be filled with or partly filled with a cell matrix providing a support for the growth of cells added to the internal chamber. Any such cell matrix available in the art could be used according to the invention. An example is the use of porous titanium grains or granulates 110 or such granulates 110 made of an oxide of titanium as illustrated in
The granulates 110 have a further advantageous effect in that the hold the semipermeable membranes 20, 30 in close contact with any net of bioreactive metal that may be provided onto the membranes 20, 30. This in turn reduces the distance between any blood vessels and soft tissue attached to or provided close to the metal net and thereby improves the transport of oxygen and nutrients from the outside into cells present in the cell chamber 18.
Another possible cell matrix providing cell growth support is to use a net 120. This net 120 can, but does not have to be, made of a bioactive metal, preferably a titanium oxide. Other examples include nets made of polymers and plastics. Instead of utilizing nets, different forms of membranes, typically having larger nominal pore size as compared to the semipermeable membranes could be employed. Actually any cell matrix material and design known in the art can be used in connection with the implant assembly 1, including matrices pre-mixed with cells.
It could be possible to use protective membranes in connection with a cell matrix. The protective membranes will then protect the semipermeable membranes from any mechanical ware caused by the cell matrix. In such a case, during manufacture of the implant, a respective protective membrane is first placed onto either side of the flange and then the semipermeable membranes are positioned on the protective membranes. Alternatively, the protective membranes form part of a laminate structure with the membranes and optionally at least one metal net.
The protective membranes should not prevent or negatively effect the diffusion through the semipermeable membranes too much. Thus, their only function is to provide protection to the more sensitive semipermeable membranes.
The cross sectional views of
The teachings of the different embodiment described above and illustrated in the drawings may be combined. For example, the implant assemblies illustrated in
The implant assembly of the present invention is particularly suitable as a cell implant, in which the cells are provided in the sealed, enclosed inner chamber. The cells are natural or are genetically engineered for producing and releasing one or more substances that are of medical interest to the exterior of the implant. The substance could be a medical agent that the donee body lacks or has a reduced level of. The implant assembly is though not limited to usage in connection with deficiency diseases. In clear contrast, the cells can produce different types of inhibitors, antagonists, antibodies or agonists that interact with different receptors, proteins and other molecules and cells in the donee body.
Thus, the implant assembly can be used, when filled with one or more cell types, for treating or preventing a disease or medical disorder in an animal body, preferably mammalian body and more preferably human body, where the at least one cell type produces and releases an agent having the capability of treating or preventing the disease or disorder.
In another approach, the bioartificial implant assembly of the invention does not comprise cells as a source of the therapeutic agent. In clear contrast, the agent is provided directly inside the implant chamber, possibly with a suitable excipient, carrier, etc. The agent will then diffuse in a controlled and prolonged way through the pores of the semipermeable membranes. Also a continuous pumping of medicament from an implanted source or external (non-implanted) source through a port into the implant assembly and then out into the recipient body can be used.
Another use of the implant assembly of the invention is a protector or chamber for a diagnostic appliance, such as a sensor head. The sensor is then able to detect the presence of or measure the concentration of a defined substance present in the donee body. This substance is then able to pass through the semipermeable membranes. The implant assembly will protect the sensitive sensor head from mechanical strains. The sensor could in turn be connected to a medical pump, delivering a therapeutic agent to the subject's body in response to the sensor measurements.
A next step S2 positions a first membrane, preferably semipermeable membrane, on a first side of the support flange. For this purpose, the outer circumference (diameter) of the membrane matches (is equal to or close to) the inner circumference (diameter) of the frame structure (10). A second membrane, preferably semipermeable membrane, is likewise positioned on a second opposite side of the support flange. The inner chamber enclosed by the membranes and the frame is sealed in step S4 by forming a first seal in connection with the first frame portion and a second seal in connection with the second frame portion. The method then ends.
The order at which the steps S2 to S4 are performed can differ from what is illustrated in
Any material to be provided in the interior of the chamber, such as cell matrix, medicament agent source, including agent-producing cells, can be added prior positioning one of the membranes on the support flange or even after sealing utilizing one or more frame ports.
The correct shape of the semipermeable membrane and optionally the net, protective membrane and rings can be individually formed, such as by punching from larger sheets. The formed membranes, rings and nets can then be placed on the flange or each other to form an optional sandwich structure. Alternatively, sheets are handled together in the form of a pre-fabricated laminate structure that is positioned onto the flange.
The order of the operation steps S20 to S23 can be different from what is illustrated in the figure. For instance, the welding of step S22 can be performed prior to the positioning step S21. Alternatively, steps S20 and S21 are performed in parallel and steps S22 and S23 are performed in parallel.
If a laminate structure is employed instead of single membranes, such a laminate structure can be performed by position a net of a bioactive metal, metal alloy or metal oxide on a first die of the membrane. A weldable ring is positioned on the net and is welded to the membrane to form the laminate structure. In another embodiment, a second net and second weldable ring are welded to the second opposite side of the membrane.
Tissue integration was studied using the implant assembly of the present invention containing erythropoietin (EPO) producing cells implanted into mice.
The implant assembly of the present invention was used in connection with Biopore membrane (Millipore, 0.4 μm average pore size) with or without the usage of an outer titanium net (0.12 mm thickness of titanium wire).
These two different types of implant assemblies (with or without net) were filled with allogenic mouse myoblast cells modified to produce EPO. The cells were introduced as a suspension in culture medium through a port of the implant. Thereafter the port was closed by heat-welding. Before implantation, EPO production was tested in culture to assure that the cells were viable.
Male Balb/C mice were anaesthetized using isofluorane and one filled implant assembly was implanted in each mouse. After implantation hemoglobin values were collected and the mice were sacrificed after 24 days. EPO is a hormone that is known to stimulate the production of erythrocytes. Erythrocytes contain hemoglobin (Hgb), which therefore can be used as good indicator on the production of EPO. Vessel formation around the implanted devices was photo documented. One implant assembly per group was cultured after explantation and the release of EPO was followed. Remaining implant assemblies were cross-sectioned and stained using hematoxylin-eosin.
Extensive vessel observation could be seen, see
Soft-tissue integration of the implant assembly, of the invention with different forms of titanium applied to the membrane surface has also been investigated.
Two female Lewis rats received three implant assemblies each. The implant assembly had a laminate structure consisting of an inner non-coated Biopore membrane (Millipore, average pore size of 0.4 μm) onto which a titanium net was positioned, either with a thread size of 0.10 mm or 0.12 mm. The implant assembly with the 0.12 mm thread size had a frame with a thickness of the support flange of about 1.0 mm and was filled with titanium granules (average diameter<0.75 mm) as cell matrix. The implant assembly with thread size of 0.10 mm had a frame with flange thickness of about 0.5 mm. The third assembly type was identical to the implant assembly with thread size of 0.10 mm but was also coated with a layer of titanium oxide powder.
The rats were anaesthetized using isofluorane, approximately 3% in 400 ml air and 150 ml oxygen per minute. The abdominal skin was opened and devices placed under the fascia connected to the subcutis. The fascia was then closed with a 7-0 suture and the skin was closed with wound clips. After closure of the skin, the animals received Temgesic to achieve pain relief.
The rats were anaesthetized using isofluorane during retrieval. The implant assemblies from one animal were retrieved with as little tissue as possible to visualize vessels on the device. The retrieved implants from the second rat were collected together with surrounding tissue and were placed in 4% buffered formalin. After retrieval the rats were sacrificed by cutting the heart open.
The retrieved materials were incubated in 4% formalin for approximately 24 hours before transfer to 70% ethanol. Plastic embedding, sectioning and staining were performed by HistoCenter AB. The sections were prepared 20 μm thick to avoid loosing metal material. They were then placed on glass slides and stained with hematoxylin and eosin. Digital images of the areas of interest were taken in a light microscope.
The inclusion of the granulate-based cell matrix improved the close contact between the implant and the tissue, possibly due to that the granulates press the semipermeable membrane close onto the net. Lack of the granulates may cause the membranes to collapse into the enclosed space, thereby obtaining a greater distance between the nets and the membranes.
The application of the titanium powder coating provides a good integration in tissue and also promotes the formation of blood vessels.
It will be understood by a person skilled in the art that various modifications and changes may be made to the present invention without departure from the scope thereof, which is defined by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0700460-9 | Feb 2007 | SE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/SE08/00126 | 2/18/2008 | WO | 00 | 8/19/2009 |
