Abstract:
Various inserts, called shapers and spacers, are provided for controlling tissue engineered heart valve (TEHV) leaflet geometry during culture. These inserts will prevent TEHV leaflet retraction during culture, be able to control the leaflet geometry during culture, enable culturing TEHV leaflets with a larger coaptation area, control the height of the coaptation area, maintain TEHV leaflet curvatures, and/or enable possibilities to culture TEHV leaflets in open configuration.

Description:
FIELD OF THE INVENTION 
       [0001]    This invention relates to devices for tissue engineering. In particular, the invention relates to devices for tissue engineering heart valves. 
       BACKGROUND OF THE INVENTION 
       [0002]    Tissue engineered heart valves (TEHV) are produced by seeding cells on a heart valve shaped scaffold material, followed by a culturing period in a bioreactor system. During culture, the cells will produce an extracellular matrix (ECM). 
         [0003]    So far no solutions are available to control heart valve geometry during culture. Contractile cells that are being used will compact the new-formed tissue in all possible directions of constrain. Any predefined scaffold geometry at the start of culture will therefore be lost during culture, resulting in an entirely different geometry after culture compared to the imposed starter geometry. 
         [0004]    There are two ways of culturing the TEHV. The first method is to culture the TEHVs in a so-called “open configuration”. This means that the individual heart valve leaflets are separated from each other during culture. The benefit of this approach is that the TEHV leaflets do not have to be separated after culture. The problem with this approach is that because cells will build up tension during culture, they will retract the leaflets, which results in shorted leaflets. In addition, because of the internal tension that builds up in the leaflets, the initially curved shape of the scaffold may be straightened thereby compromising the desired curvature of the leaflets and functionality of the valve. 
         [0005]    The second method is to culture the TEHV in a “closed configuration”. This means that the valve leaflets are attached to each other, which prevents shortening of the leaflets due to the internal tension that builds up in the leaflets during culture. However, it does not prevent ‘straightening’ or ‘flattening’ of the leaflets. In addition, it has been proven to be difficult to achieve a sufficiently large coaptation area between the leaflets in this way, which is crucial for in vivo functionality of the heart valve. 
         [0006]    The present invention addresses these problems and provides devices, which allow for the maintenance and control of heart valve geometry during culture. 
       SUMMARY OF THE INVENTION 
       [0007]    The present invention provides devices, methods of using these devices and systems for controlling tissue engineered heart valve leaflet geometry by using predefined inserts during tissue culture. The inserts are referred to herein as (leaflet) shapers and (leaflet) spacers, which can be used individually or in combination with each other mostly depending on the type of cells cultured with the tissue growth materials and level of geometry shaping/control. 
         [0008]    The first insert is a leaflet shaper and has been described herein with several different variations of embodiments. Since we observed that the cells build up tension in all constrained directions, we make use of this effect by inserting a rigid, concave construct that has the shape of the leaflet. The tension that develops in the leaflets will cause the leaflets to compact against the shaper, which acts as a constraint and is capable of controlling the curvature and coaptation of the leaflets. 
         [0009]    In some embodiments, the leaflet shaper is covered with small holes to achieve proper nutrient exchange between the medium and the tissue that compacts around the insert. The shaper does not cover the wall of the heart valve such that nutrients and oxygen can be supplied to the wall. Because the tissue compacts against the concave aspect of the shaper, there is no need for a second valve shaped insert/shaper on the other side of the valve leaflets. 
         [0010]    The second insert is a leaflet spacer and has been described herein as one embodiment that can be used in combination with the various shapers. When the leaflets are cultured in a closed configuration, the spacer will prevent retraction of the leaflets in the radial direction to constrain the height, and therefore control the size of the coaptation area. It will also enable maintenance of a predefined coaptation area. Hence, this leaflet spacer will constrain the height of the leaflets. A second advantage of the leaflet spacer is to prevent the leaflets from merging over the coaptation area during culture. Since the spacer will be positioned in between the individual leaflets, there is no chance for leaflet concrescence. 
         [0011]    An advantage of using the embodiments presented in this invention is that it can result in circumferential collagen orientation in the cultured heart valves, which is beneficial for heart valve functionality. 
         [0012]    Another advantage of using the embodiments presented in this invention is that it enables us to culture heart valves without the need of using a complex bioreactor system. In fact, the use of a simple jar would be sufficient. One of the functions of the bioreactor system was to impose the right geometry to the valves by dynamically loading them. But this inserts can achieve the same objective, which is to constrain the imposed geometry. 
     
    
     
       BRIEF DESCRIPTION OF THE D WINGS 
         [0013]      FIG. 1  shows according to an exemplary embodiment of the invention a three dimensional view of a shaper  100  for maintaining and controlling the shape of tissue growth material for three leaflets of a heart valve during tissue culture. 
           [0014]      FIG. 2  shows according to an exemplary embodiment of the invention a top view of the shaper as shown in  FIG. 1 . The dimensions are in mm. 
           [0015]      FIG. 3  shows according to an exemplary embodiment of the invention a bottom view of the shaper as shown in  FIG. 1 . The dimensions are in mm. 
           [0016]      FIGS. 4-5  show according to an exemplary embodiment of the invention side views of the shaper as shown in  FIG. 1 . The dimensions are in mm. 
           [0017]      FIG. 6  shows according to an exemplary embodiment of the invention a three dimensional view of a shaper  600  for maintaining and controlling the shape of tissue growth material for three leaflets of a heart valve during tissue culture. 
           [0018]      FIG. 7  shows according to an exemplary embodiment of the invention a top view of the shaper as shown in  FIG. 6 . The dimensions are in mm. 
           [0019]      FIG. 8  shows according to an exemplary embodiment of the invention a bottom view of the shaper as shown in  FIG. 6 . The dimensions are in mm. 
           [0020]      FIGS. 9-10  show according to an exemplary embodiment of the invention side views of the shaper as shown in  FIG. 6 . The dimensions are in mm. 
           [0021]      FIG. 11  shows according to an exemplary embodiment of the invention a detailed aspect of a meshed surface area with holes from  FIG. 7 . The dimensions are in mm. 
           [0022]      FIG. 12  shows according to an exemplary embodiment of the invention a three dimensional view of a shaper  1200  for maintaining and controlling the shape of tissue growth material for three leaflets of a heart valve during tissue culture. 
           [0023]      FIG. 13  shows according to an exemplary embodiment of the invention a top view of the shaper as shown in  FIG. 12 . The dimensions are in mm. 
           [0024]      FIG. 14  shows according to an exemplary embodiment of the invention a bottom view of the shaper as shown in  FIG. 12 . The dimensions are in mm. 
           [0025]      FIGS. 15-16  show according to an exemplary embodiment of the invention side views of the shaper as shown in  FIG. 12 . The dimensions are in mm. 
           [0026]      FIG. 17  shows according to an exemplary embodiment of the invention a three dimensional view of a spacer  1700  for further maintaining and controlling the shape of tissue growth material for three leaflets of a heart valve during tissue culture. 
           [0027]      FIGS. 18-19  show according to an exemplary embodiment of the invention top views of the spacer as shown in  FIG. 17 . The dimensions are in mm. 
           [0028]      FIGS. 20-21  show according to an exemplary embodiment of the invention side views of the spacer as shown in  FIG. 17 . The dimensions are in mm. 
           [0029]      FIG. 22  shows according to an exemplary embodiment of the invention the various shapers  100 ,  600  and  1200  with spacer  1700 , and tissue growth material  2200  and how they fit and can be used together. 
           [0030]      FIG. 23  shows according to an exemplary embodiment of the invention a change from random collagen orientation towards circumferential aligned collagen orientation, due to the leaflet shaper insert during culture. 
           [0031]      FIGS. 24-25  show according to exemplary embodiments of the invention in vitro results of TEHVs cultured with the use of, for example, but not limited to shaper  1200 . 
           [0032]      FIG. 26  shows according to an exemplary embodiment of the invention long term in vivo results of TEHVs cultured with the use of, for example, but not limited to shaper  1200 . 
       
    
    
     DETAILED DESCRIPTION 
       [0033]      FIGS. 1-5  show a first embodiment of a shaper  100  for maintaining and controlling heart valve geometry during culture. Shaper  100  is intended for a heart valve with three leaflets and distinguishes a support base  110  and three inner arms  112  each capable of supporting a tissue growth material (not shown) to faun one of the leaflets of the heart valve. In this embodiment, it is the mid-axis of the heart valve leaflets that will be constrained and controlled during culture. 
         [0034]    Each of the inner arms  112  has a first portion  112 ′ and a second portion  112 ″, which is only indicated for one of the inner arms for clarity purposes. First portion  112 ′ is disposed normal to support base  110  and disposed proximal to a center of support base  110 . Second portion  112 ″ is nonlinear and disposed distal to support base  110  and bends away from the center of support base  110 . 
         [0035]    The inner arms  112  are distributed in a triangular pattern at support base  110  and are spaced from each other, as is evident in  FIG. 1 , to define enough space to fit at least the respective tissue growth materials. In other words, the tissue growth materials are placed over and against their respective inner arms  112  at the medial aspects  114  of inner arms  112  ( 114  is only indicated for one of the inner arms  112  for clarity purposes). In this embodiment, the respective tissue growth material is extended (not shown) to area  116 ′ and  116 ″ forming a wedge-shape growth material and a canopy (e.g. concave) draped over second portion  112 ″. 
         [0036]      FIGS. 2-5  show an exemplary embodiment of some dimensions of shaper  100 , which are not limited to the invention as a person skilled in the art would readily appreciate that heart valves/leaflets would vary in dimensions and shape. A paper by the same group as the current inventors provides guidelines for some of the dimensions. The paper is entitled “Effects of valve geometry and tissue anisotropy on the radial stretch and coaptation area of tissue-engineered heart valves” by Loerakker et al. and published in Journal of Biomechanics 46 (2013) 1792-1800. 
         [0037]    Depending on the type of cells used with the tissue growth material for shaper  100 , there might be a desire to further control the shape and/or spacing between the tissue growth materials draped against the inner arms  112 . For this purpose, spacer  1700  is designed with three surfaces  1710  distributed/oriented with respect to each other in the same triangular pattern as how inner arms  112  are distributed. Side  1720  of spacer  1700  can be placed towards the top of support base  110  and will then sit at the top of the support base  100  (see also  FIG. 22 ). Surfaces  1710  fit in the space left to fit at least the tissue growth material to separate the tissue growth materials supported by each of the linear portions  112 ′ of the inner arms  112 . In other words, surfaces  1710  will separate the tissue growth materials. 
         [0038]      FIGS. 6-11  show a second embodiment of a shaper  600  for maintaining and controlling heart valve geometry during culture, where shaper  600  could be viewed as an extension of shaper  100  with similar structural components. Shaper  600  is intended for a heart valve with three leaflets and distinguishes a support base  610  and three canopy growth surfaces  620  expanded from the second portions of their respective inner arms  112 . It is noted that only one inner arm  112  is indicated in  FIG. 6  for clarity purposes. 
         [0039]    Each canopy growth surface  620  is capable of supporting a tissue growth material (not shown) to form one of the leaflets of the heart valve. The canopy growth surfaces  620  define a concave surface when moving away from the center of support base  610  in outer direction. 
         [0040]    The canopy growth surfaces  620  are supported by the respective first portions of the inner arms  112  and a pair of outer arms  612 ′,  612 ″ defined for each of the inner arms. Each of the outer arms  612 ′,  612 ″ have a first portion disposed normal to support base  610  and disposed distal to the center of support base  610 . 
         [0041]    In other words, each of the canopy growth surfaces  620  further span to the base of support surface  610  along the radial separation of the respective outer arms  612 ′,  612 ″ and inner arm  112  such that each span is capable of supporting the respective growth material. Differently stated, the combinations of each of the first portions of the inner arms  112  with their respective pair of outer arms  612 ′,  612 ″ define wedge-shape growth surfaces each capable of supporting the respective growth material. As a result the tissue growth material for the heart valve leaflets will be constraint and controlled during culture. Open area  630  (indicated for only one of the leaflet canopy growth surfaces for clarity purposes) is left open as it could enhance tissue formation. Holes  640  are intended to allow for improved exchange of nutrients. 
         [0042]    The three canopy growth surfaces  620  are distributed in a triangular pattern at support base  610  and are spaced  650  from each other forming a star design, as is evident from e.g.  FIGS. 6-8  especially looking from the top down. The space is defined to fit at least the respective tissue growth materials. In other words, the tissue growth materials are placed over and against their canopy growth surfaces  620  at the medial aspects of canopy growth surfaces  620 . In this embodiment, the respective tissue growth material is extended forming a wedge-shape growth material and a canopy (e.g. concave) draped over the canopy growth surfaces  620 . 
         [0043]      FIGS. 7-11  show an exemplary embodiment of some dimensions of shaper  600 , which are not limited to the invention as a person skilled in the art would readily appreciate that heart valves/leaflets would vary in dimensions and shape. The same paper mentioned supra provides guidelines for some of the dimensions. 
         [0044]    Depending on the type of cells used with the tissue growth material for shaper  600 , there might be a desire to further control the shape and/or spacing between the tissue growth materials draped against the canopy growth surfaces  620 . For this purpose, spacer  1700  is designed with three surfaces  1710  distributed/oriented with respect to each other in the same triangular pattern as how canopy growth surfaces  620  are distributed. Side  1720  of spacer  1700  can be placed towards the top of support base  610  and will then sit at the top of the support base  610  (see also  FIG. 22 ). Surfaces  1710  fit in the space left to fit at least the tissue growth material to separate the tissue growth materials supported by each of the canopy growth surfaces  620 . In other words, surfaces  1710  will separate the tissue growth materials. 
         [0045]      FIGS. 12-16  show a third embodiment of a shaper  1200  for maintaining and controlling heart valve geometry during culture, where shaper  1200  could be viewed as an extension of shapers  100  and  600  by having some structural components in common. Shaper  1200  is intended for a heart valve with three leaflets and distinguishes a support base  610  and three canopy growth surfaces  620  expanded from their respective inner arms  112 . It is noted that only one inner arm  112  is indicated in  FIG. 12  for clarity purposes. 
         [0046]    Shapers  600  and  1200  are similar with the difference that for shaper  1200  each of the canopy growth surfaces  620  further span to the base of support surface  610  with meshes surfaces  1210  between the respective outer arms  612 ′,  612 ″ and inner arm  112 . Only one of the meshed surfaces is indicated for clarity purposes. It is also noted that a wedge shaped surface forms the basis for each of the concave parts of the canopy growth surfaces. 
         [0047]    Another difference is that the meshes surface  1210  have holes, like holes  640 , to allow exchange of nutrients. Each of these canopy growth surfaces  620  is capable of supporting the respective growth material. Similar to shaper  600 , spacer  1700  can be used for shaper  1200  to fit in the space  650  left to fit at least the tissue growth material to separate the tissue growth materials supported by the meshed surfaces. 
         [0048]      FIGS. 13-16  show an exemplary embodiment of some dimensions of shaper  1200 , which are not limited to the invention as a person skilled in the art would readily appreciate that heart valves/leaflets would vary in dimensions and shape. The same paper mentioned supra provides guidelines for some of the dimensions. 
         [0049]    In summary,  FIG. 22  shows the various shapers  100 ,  600  and  1200  with spacer  1700 , and tissue growth material  2200  and how they fit and can be used together. There are various variations one could imagine, such as that these embodiments can be constructed for a single-leaflet heart valve, bi-leaflet (two leaflet) heart valve or multiple-leaflet heart valve. The design principles for these different heart valves would be similar to the tri-leaflet heart valve with the difference of the number of inner arms for shaper  100 , the number of canopy growth surfaces for shaper  600  and  1200 , the shape of the space with the growth surfaces and various others as a person skilled in the art would readily appreciate. In addition, dimensions (including the radius/angles of the canopy growth surfaces) shown in the exemplary embodiments could be varied to fit the desired objective for the tissue engineered heart valves. 
         [0050]    The manufacturing of the inserts could be via conventional computer numerical control (CNC) milling technology with biocompatible materials such as polyether ether ketone (PEEK) or via rapid prototyping techniques like three-dimensional printing with materials such as acrylonitrile butadiene styrene (ABS) or more biocompatible materials such as PLA. However, other conventional manufacturing techniques would still suffice. In addition, the shapers and spacers could be made as modular components that could be assembled to for example come up for a single-leaflet, bi-leaflet or tri-leaflet design. 
       Circumferential Collagen Alignment 
       [0051]    Circumferential collagen alignment in TEHVs will result in radial leaflet stretch while being hemodynamically loaded, which is beneficial for the opening and closing behavior of the valve. As shown in  FIG. 23 , the starter matrix of the TEHV contains mainly randomly organized scaffold fibers. When the cells are seeded onto the construct, they will start producing randomly organized collagen matrix along these scaffold fibers. During culture, the scaffold material will hydrolyze and lose mechanical functionality. From this point on cells will start pulling in the direction of constrained. Since the leaflet shaper insert is a rigid body, cells will compact around this insert and realign the collagen in the direction of constrain. This will result in circumferentially aligned collagen orientation ( FIG. 23 ). 
       Static Valve Culture 
       [0052]    Currently TEHVs are being cultured in a sophisticated bioreactor system. This system is regulating pulsatile pressures onto the leaflets in combination with regulated medium flow to enhance tissue formation. We found out that by using the insert as presented herein during culture, the bioreactor system can be replaced by a simple jar. Since the insert is required to maintain the initial heart valve geometry, it is hampering the pulsatile pressures exerted on the leaflets, which makes the main function of the bioreactor system redundant or obsolete. It seems that when the fluid flow is maintained, it would still be possible to culture functional TEHVs. This finding can have a big impact in the way TEHVs can be produced in a future commercial way. Without the use of a complicated bioreactor system, valve production can be up scaled easily and will lower the production costs. 
       Results 
       [0053]      FIGS. 24-25  show examples of in vitro results of TEHVs cultured with the use of for example shaper  1200 . After removal of shaper  1200  the TEHV maintained the imposed geometry.  FIG. 24  shows results for a closed configuration with no leaflet retraction  2410 , maintenance of leaflet curvature  2420  and a controlled coaptation area  2430 .  FIG. 25  shows results for an open configuration with leaflets shaped around the shaper insert  2510 , maintenance of leaflet curvature  2520  and a controlled coaptation area  2530 . 
         [0054]      FIG. 26  shows an example of long term in vivo results of TEHVs cultured with the use of for example shaper  1200 . Up to 24 weeks, the heart valve maintained its initial geometry and showed no signs of leaflet retraction. These results confirm that the initial geometry of the heart valve after culture is decisive for the final long-term outcome, which can only be obtained by using the leaflet shaper insert during culture.