Patent Publication Number: US-2010121446-A1

Title: Implant assembly

Description:
TECHNICAL FIELD 
     The present invention generally relates to an implant assembly, and in particular such an implant assembly comprising a semipermeable membrane or barrier. 
     BACKGROUND 
     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&#39;s body to the implanted donor tissue or cell. This barrier should however not allow diffusion of the cells of the donee&#39;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&#39;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&#39;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&#39;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. 
     SUMMARY 
     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. 
    
    
     
       SHORT DESCRIPTION OF THE DRAWINGS 
       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: 
         FIG. 1A  is an exploded view of an embodiment of a bioartificial implant assembly according to the present invention; 
         FIG. 1B  illustrates the implant assembly of  FIG. 1A  before sealing the implant assembly; 
         FIG. 1C  is a cross sectional view of the implant assembly of  FIG. 1B ; 
         FIG. 2A  illustrates the implant assembly of  FIG. 1B  after sealing; 
         FIG. 2B  is a cross sectional view of the implant assembly of  FIG. 2A ; 
         FIG. 3  is a cross sectional view of another embodiment of a bioartificial implant assembly according to the present invention; 
         FIG. 4  is an exploded view of a further embodiment of a bioartificial implant assembly according to the present invention; 
         FIG. 5  is an exploded view of a portion of an embodiment of laminate structure that can be used in the implant assembly according to the present invention; 
         FIG. 6  is an exploded view of still another embodiment of a bioartificial implant assembly according to the present invention; 
         FIG. 7A  is an exploded view of a further embodiment of a bioartificial implant assembly according to the present invention; 
         FIG. 7B  illustrates the implant assembly of  FIG. 7A  after sealing the implant assembly; 
         FIG. 7C  is a cross sectional view of the implant assembly of  FIG. 7B . 
         FIG. 8A  is a cross sectional view of another laminate structure that can be used in the implant assembly according to the present invention; 
         FIG. 8B  is a cross sectional view of a further laminate structure that can be used in the implant assembly according to the present invention; 
         FIG. 9A  is a cross sectional view of an embodiment of a bioartifical implant assembly according to the present invention housing granulates in its inner chamber; 
         FIG. 9B  is a cross sectional view of an embodiment of a bioartifical implant assembly according to the present invention housing a net in its inner chamber; 
         FIG. 9C  is a cross sectional view of an embodiment of a bioartifical implant assembly according to the present invention housing cells in its inner chamber; 
         FIG. 10  is a flow diagram illustrating a method of producing a bioartificial implant assembly according to the present invention; 
         FIG. 11  is a flow diagram illustrating an embodiment of the sealing step of the method illustrated in  FIG. 10 ; 
         FIG. 12  is a flow diagram illustrating another embodiment of the sealing step of the method illustrated in  FIG. 10 ; 
         FIG. 13  is a graph illustrating increase in hemoglobin values in mice having received an implant assembly containing EPO-producing cells or control cells; 
         FIG. 14  is a graph showing percentage of starting value hemoglobin in mice receiving implant assemblies containing EPO-producing cells and having a Biopore membrane or a laminate of a Biopore membrane and titanium net; 
         FIG. 15  illustrates an implant assembly explanted from a mouse after 24 days. Left panel: removal of the skin tissue, middle panel: vessel formation in the skin tissue facing the implant, and right panel: microscopic view of the skin side of the implant; 
         FIG. 16  illustrates photographs showing cross-sections of the implant assembly in 100× magnification with implant assemblies without titanium net (upper panels) and with titanium nets (lower panels); 
         FIG. 17  is a view of the implant assembly with a 0.12 mm net thread size showing the implant wall facing the skin in the left panel and the wall facing the abdomen on the right panel; 
         FIG. 18  is a view of the implant assembly with a 0.10 mm net thread size showing the implant wall facing the skin in the left panel and the wall facing the abdomen on the right panel; 
         FIG. 19  is a view of the implant assembly with a 0.10 mm net thread size plus titanium powder coating showing the implant wall facing the skin in the left panel and the wall facing the abdomen on the right panel; 
         FIG. 20  illustrates a cross section of the wall facing the abdomen of the implant assembly with a 0.12 mm net thread size at 100× magnification; and 
         FIG. 21  illustrates cross sections of titanium powder coating of the implant assembly with a 0.10 mm net thread size plus titanium coating at 200× magnification (left panel) and 400× magnification (right panel). 
     
    
    
     DETAILED DESCRIPTION 
     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. 
       FIG. 1A  is a schematic overview of the including elements in an embodiment of the implant assembly  1 . The implant assembly  1  comprises a frame structure  10  and at least one semipermeable membrane, barrier or filter  20 ,  30 . The other membrane or barrier  30  is preferably also semipermeable but could alternative be impermeable or have another lower permeability than the first membrane  20 . The frame structure  10  comprises an inwardly protruding or extending support flange  16 . The support flange  16  is preferably protruding from a central position of the frame backbone  10 . This means that a first or upper portion  12  of the frame  10  extends above the flange  16  and a second or lower portion  14  extends below the flange  16 . 
     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  FIG. 1B , where one of the membranes  20  has been positioned inside the frame  10  onto the flange  16 . The other membrane  30  is likewise positioned on the other side of the flange  16 .  FIG. 1C  is a cross-sectional view of the implant assembly  1  of  FIG. 1B . This figure clearly illustrates how the two membranes  20 ,  30  rest inside the frame  10  on the inwardly protruding flange  16 . At this position, the frame  10  and the two membranes  20 ,  30  define an internal space or chamber  18  that is isolated from the external environment and only accessibly through the pores  25 ,  35  of the membranes  20 ,  30 . This height of this inner chamber  18  is dictated by the thickness of the support flange  16 . 
     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.  FIG. 2A  schematically illustrates the bioartificial implant  1  after sealing the frame portions  12 ,  14  onto the flange  16 .  FIG. 2B  is a cross-sectional illustration of  FIG. 2A . The seals  2 ,  3  will lock the membranes  20 ,  30  to the frame structure  10  as material of the frame  10  will, when applying the heat, flow through the pores of the membrane portions positioned on the flange  16  and contact and melt together with the flange  16 . The seals  2 ,  3  will in this manner be totally secure and prevent any unintentionally leakage of substance between the frame inside and the membranes  20 ,  30 . As is seen best in  FIG. 2B , the circumferential portions of the membranes  20 ,  30  arranged connected to the flange  16  will become embedded in the frame  10 . 
     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&#39;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  FIGS. 2A and 2B . There will therefore be no sharp edges that cause local irritation and connective tissue ingrowth. 
     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. 
       FIG. 3  is a cross-sectional view of another embodiment of an implant assembly  1  of the invention. This implant  1  is equipped with a port  15 , illustrated in the form of a tube with an inner channel. This port  15  is arranged in the central portion of the frame  10  and provides a channel into the inner chamber  18 . The port  15  can be provided in the frame  10  by inserting the tube  15  into the injection-mold used for forming the frame  10 . In an alternative approach, the tube  15  and frame  10  are injection-molded together in a single piece. A further alternative is to first manufacture the frame  10  with a dedicated orifice in its frame backbone and then inserting the tube  15  therein. The tube  15  is then attached to the frame  10  and the orifice is sealed by welding around the lateral tube area in connection with the outer frame wall. However, from a sealing point of view, manufacturing the tube  15  and frame  10  together as a single piece is most preferred, followed by manufacturing the tube  15  separately and inserting the tube  15  into the injection-mold for the frame  10 . 
     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  FIG. 3 . This means that the port  15  typically also extends through the support flange  16  in addition to the frame backbone  10 . The end of the port  15  positioned in the inner chamber  18  can be aligned with or end closely outside of the inner frame wall or the short end of the support flange  16 . Alternatively, the port end can extend some distance into the inner chamber  18  possibly even close up to the opposite inner frame wall or opposite flange end. 
     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. 
       FIG. 4  is a schematic overview of the elements of a further embodiment of an implant assembly  1  of the present invention. In this embodiment, the implant  1  comprises two nets  40 ,  50  of bioactive metal. These nets  40 ,  50  have a size similar to the semipermeable membranes  20 ,  30  and are positioned onto these membranes  20 ,  30 . When sealing the implant  1 , both the membranes  20 ,  30  and the nets  40 ,  50  will becomes embedded into the frame structure  10 . 
     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  FIG. 3 . In such a case, the injection of a cell suspension will exert a pressure towards the membranes  20 ,  30  causing them to bulge outwards until enough cell medium has diffused through the membrane pores  25 ,  35 . The nets  40 ,  50  will provide a counter-pressure to this bulging force, preventing the membranes  20 ,  30  from bulging too much, which may cause damage and even rapture of the membranes  20 ,  30 . The nets  40 ,  50  also function as a protection to the sensitive semipermeable membranes  20 ,  30  thereby preventing the rapture of the membranes  20 ,  30  upon contact with external objects. 
     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  FIG. 4 , the semipermeable membranes  20 ,  30  and the nets  40 ,  50  are punched separately from a respective membrane sheet and net sheet. The membranes  20 ,  30  are first positioned onto the flange  16  followed by the nets  40 ,  50 . An alternatively approach forms a sandwich or laminate structure  100 , in which the net  40  is first adhered to the membrane  20  and then the whole structure  100  is positioned on the implant flange. This approach is illustrated in  FIG. 5 . A carrier foil  60  is first welded to the membrane  20  with the net  40  positioned between the carrier foil  60  and the membrane  20 . 
     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  FIG. 5 . A welding is then applied to the circumferential part of the foil  60 , melting the foil  60  through the net  40  and adhering it to the membrane  20 . 
     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  FIG. 6 . In such a case, the implant  1  has the tissue integrating and blood vessel attracting functions of the net  40 ,  50  and coating  60  and the stabilizing function of the net  40 ,  50 . 
     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. 
       FIG. 6  also illustrates another useful element of the frame structure  10 , which can be used for securely anchoring the implant assembly  1  to a structure, typically bone structure in the host body. The frame  10  comprises a tissue anchoring structure  13  illustrated in the form of a protruding frame element  13  extending beyond the circumference of the frame  10 . This anchoring structure  13  comprises, in this embodiment, a bore through which a bone screw can be entered. Such a screw is then secured (screwed into) a bone of the host body to thereby securely attach the implant  1  to the bone and prevent any significant movement thereof following implantation. 
     Other forms of tissue anchoring structure could alternatively be employed, including different hooks, barbs and suture sleeves. 
       FIG. 7A  is an exploded view of another embodiment of the bioartificial implant assembly  1  according to the present invention. As described above, two membranes  20 ,  30  of which at least one preferably both are semipermeable are positioned on either side of the central support flange  16 . In this case, the sealing is not realized by heat pressing the frame portions  12 ,  14  down towards the flange  16 . Instead the frame portions  12 ,  14  form a sealing surface onto which sealing foil rings  80 ,  90  are positioned. The foil rings  80 ,  90  have a respective outer circumference matching the circumference of the frame portions  12 ,  14 . The inner circumference of the rings  80 ,  90  is though smaller than the outer circumference of the membranes  20 ,  30 . As a consequence, when the rings  80 ,  90  are positioned on the frame portions  12 ,  14 , the rings  80 ,  90  will extend slightly over the circumferential portions of the membranes. This is more clearly seen in  FIG. 7B  and in the cross sectional view of  FIG. 7C . 
     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  FIG. 8A . In this laminate embodiment, the structure comprises, in sequential order, a semipermable membrane  20 , a net of bioactive metal or metal alloy positioned on the membrane  20  and a cover or weldable ring  60  positioned on the net  40 . By welding the ring  60  to the membrane  20 , a portion of the ring material will move through the net  40  and attach to the membrane  20 . The net  40  therefore becomes fixed to the membrane  20  and the laminate structure  100  can be handled as a single unit. 
     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  FIG. 8B . This structure  100  comprises two nets  40 ,  50  of bioactive metal or metal alloy positioned on either side of the semipermeable membrane  20 . A respective weldable ring  60 ,  70  is positioned outside of the nets  40 ,  50  and is welded to the membrane  20 , thereby anchoring the nets  40 ,  50  to the membrane  20 . This laminate structure  100  presents the bioactive metal nets  40 ,  50  to both the inner chamber and the implant exterior. 
     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  FIG. 8A  or  8 B. This means that the implant can have two single semipermeable membranes, one semipermeable membrane and one membrane-containing laminate structure or two, similar or different, membrane-containing laminate structures. 
     If the laminate structure illustrated in  FIG. 8B  is employed or the one in  FIG. 8A  with the net faced towards the inner chamber, the sealing between the foil ring and the laminate structure is mainly effected between the foil ring and the weldable ring. As a consequence, the respective inner circumferences (diameters in the case of circular implant) of the weldable rings  60 ,  70  and the foil rings  80 ,  90  then preferably matches, i.e. being substantially the same. 
     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  FIG. 9A . Titanium granulates  110  produced by the well-known Hunter process or Kroll process are potentially good candidates for cell support. The resulting titanium granulates  110  are highly porous and have a large specific surface area. This specific surface area of the porous titanium granulates  110  is preferably 0.05 m 2 /g or at least larger than 0.005 m 2 /g. Suitable titanium granulates  110  for use as cell matrix according to the invention is marked as “Alfa Aesar” (Johnson Matthey), USA, Product No. 042459. An example of granulate size could be 0.1 mm to 0.7 mm. The granulates  110  are preferably entered in the enclosed inner chamber  18  before adding one of the membranes  20 ,  30  or laminate structures to the flange  16 . Alternatively and depending on the granulate diameter versus the inner diameter of the port channel, the granulates  110  could be entered in the chamber  18  after sealing and by injection through the port. 
     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.  FIG. 9B  illustrates a net  120  provided as cell matrix in the inner chamber  18 . The net  120  could free, i.e. not anchored to the frame structure  10  or the membranes  20 ,  30 , or be attached to the implant assembly  1 , such as through the frame structure  10 . The net  120  could occupy a substantial area of the inner chamber  18  as illustrated in the figure. 
     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. 
       FIG. 9C  illustrates a cross sectional view of the implant assembly  1  having cells  130  attached to the inner chamber walls. These cells  130  are preferably selected, such as genetically modified, for producing and releasing at least one agent of medical interest as is described further herein. 
     The cross sectional views of  FIGS. 9A to 9C  also illustrate that the outer end of the frame structure  10  is preferably smooth or rounded. This further reduces the risk or irritation and connective tissue growth following implantation. As is seen in the figures, the implant assembly  1  does not present any sharp edges to the outside and instead has a smooth finish. 
     The teachings of the different embodiment described above and illustrated in the drawings may be combined. For example, the implant assemblies illustrated in  FIGS. 4 ,  6 ,  7  and  9  can be equipped with a port as illustrated in  FIG. 3 . Furthermore, any of the disclosed assemblies could use an internal cell matrix, such as titanium granulates or net provided in the enclosed implant chamber. Also a laminate structure as illustrated in  FIG. 4-6  or  8  can be used in any of the implant assemblies. The tissue anchoring structure of  FIG. 6  can be applied to other embodiments of the implant assembly. 
     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&#39;s body in response to the sensor measurements. 
       FIG. 10  is a flow diagram illustrating a method of producing a bioartificial implant assembly according to the present invention. The method starts in step S 1 , where the frame structure of the invention having the inwardly protruding support flange and the raised frame portions is provided. This frame structure is manufactured from any of the previously described materials, preferably by injection molding the frame around the port or injection molding the frame and the port in one piece, if a port is used. 
     A next step S 2  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 S 4  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 S 2  to S 4  are performed can differ from what is illustrated in  FIG. 10 . For instance, the sealing in connection with the first semipermeable membrane can be performed before positioning the second membrane onto the support flange. The sealing of the membranes could alternatively be performed in parallel or in series. 
     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. 
       FIG. 11  is a flow diagram illustrating an embodiment of the sealing step of  FIG. 10 . The method continues from step S 3  of  FIG. 10 . In this embodiment the sealing is effected by heat-pressing (welding) the extending frame-backbone portions down towards the flange to form the implant seal. The material of the frame portions will partly melt through the semipermeable membranes or the membrane-containing laminate structures to connect with and attach to the flange material. 
       FIG. 12  is a flow diagram illustrating another embodiment of the sealing step of  FIG. 10 . The method continues from step S 3  of  FIG. 10 . A first foil ring having an outer circumference (diameter) matching (being equal to or close to) the circumference (diameter) of the first frame portion and an inner circumference/diameter being smaller than the outer circumference/diameter of the first membrane is positioned on the first membrane or the first laminate structure. In a next step S 21 , a corresponding foil ring is positioned on the second membrane or laminate structure. A first seal is effected by welding the first foil ring to the first frame portion and to the first membrane or laminate structure in step S 22 . A second seal is likewise performed by welding the second foil ring to the second frame portion and to the second membrane or laminate structure in step S 23 . 
     The order of the operation steps S 20  to S 23  can be different from what is illustrated in the figure. For instance, the welding of step S 22  can be performed prior to the positioning step S 21 . Alternatively, steps S 20  and S 21  are performed in parallel and steps S 22  and S 23  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. 
     Experiment 
     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. 
       FIG. 13  is a graph showing hemoglobin values in mice that have received the implant assembly containing EPO-producing cells (n=9) or control implant assembly (n=6) without any EPO-producing cells. There is an apparent increase in hemoglobin concentration for those mice having received EPO-producing cells as compared to control mice, thereby illustrating that the enclosed EPO-producing cells are viable in the implant assembly following implantation. The lowered hemoglobin value in the control group between day  11  and day  14  is due to sample collection. Mice in the EPO group were protected from anemia by the released EPO. 
       FIG. 14  illustrates the results of the comparative study of implant assembly having or not having a titanium net attached on the outside of the Biopore membrane. There is an apparent higher measured hemoglobin concentration for those mice (n=3) having received an implant assembly with titanium net than for those mice (n=3) with an assembly without any net. This indicates that the titanium net is able to improve the cell survival in the implant assembly during implantation. 
     Extensive vessel observation could be seen, see  FIG. 15 , in the skin tissue of the mice that during the implantation period was in direct contact with the chamber. The left panel of  FIG. 15  illustrates the removal of the skin tissue. The vessel formation in the skin tissue facing the implant assembly is clearly seen in the middle panel of  FIG. 15 . When studied in microscope it was also found that blood vessels had been formed between the titanium net and the Biopore membrane of the implant assembly as is seen in the right panel of  FIG. 15 . 
       FIG. 16  provides photographs showing cross-sections of the implant assembly in 100× magnification stained with hematoxylin and eosin. The two upper panels show two different implant assemblies without any titanium net while the lower to panels show two different implants with titanium nets. The black and white arrows in the figure indicate presence of blood vessels. It is seen in the figure that the inclusion of a titanium net causes increased vessel formation around the implant assembly. The arrows are also concentrated to the areas around the threads of the net, further indicating that it is the titanium that promotes the formation of vessels. 
     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&lt;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. 
       FIGS. 17 to 19  illustrates, in left panel, the implant assembly wall facing the skin and, in the right panel, the wall facing the abdomen for the implant assembly with 0.12 mm thread size ( FIG. 17 ), 0.10 mm thread size ( FIG. 18 ) and 0.10 mm thread size+titanium powder coating ( FIG. 19 ).  FIG. 20  is the cross-sectional view of the implant wall facing the abdomen at 100× magnification for the 0.12 mm thread size implant. Titanium granulates can be seen above and sectioned threads below the membrane (grey).  FIG. 21  illustrate cross-sections of titanium powder coating on the implant with titanium net with 0.10 mm threads and titanium powder at 200× magnification (left panel) and 400× magnification (right panel). 
     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.