Medical/Surgical Implant

Abstract

The present invention relates to the field of implants. In particular, the present invention relates to an implant for tissue reconstruction which comprises a scaffold structure that includes a void system for the generation of prevascularized connective tissue with void spaces for cell and/or tissue transplantation. Moreover, the present invention relates to a method of manufacturing such an implant, to the internal architecture of such an implant, to a removal tool for mechanical removal of space-occupying structures from such an implant, to a kit comprising such an implant and such a removal tool, to a removal device for the removal of superparamagnetic or ferromagnetic space-occupying structures from such an implant, as well as to a guiding device for providing feedback to a surgeon during the procedure of introducing transplantation cells into the void spaces generated upon removal of space-occupying structures from such an implant.

Claims

1. A method for tissue reconstruction in the body of a patient, comprising the following steps in order: a) implanting an implant comprising a three-dimensional scaffold structure made of biodegradable material into said body of said patient at the site of intended tissue reconstruction; b) after a time period sufficient to allow connective tissue and/or host vasculature to penetrate into said three-dimensional scaffold structure introducing transplantation cells to the site of intended tissue reconstruction.

2. The method of claim 1, wherein said time period sufficient to allow connective tissue and/or host vasculature to penetrate into said three-dimensional scaffold structure is in the range of 4-12 weeks.

3. The method of claim 1, wherein said time period sufficient to allow connective tissue and/or host vasculature to penetrate into said three-dimensional scaffold structure is in the range of 6-8 weeks.

4. The method of claim 1, wherein said transplantation cells are a mixture of cells obtained by lipoaspiration.

5. The method of claim 4, further comprising a step of obtaining a mixture of cells by lipoaspiration, wherein the method comprises the additional steps of making an incision at the site where the fat is to be obtained, and aspirating fat using a blunt needle with multiple perforations and an aspirator.

6. The method of claim 4, further comprising a step of filtering the fat to separate one or more of blood, oil and local anesthetic from the fat cells.

7. The method of claim 1, wherein said transplantation cells are one of stem cells, progenitor cells and fully differentiated cells.

8. The method of claim 1, wherein said transplantation cells are individual cells which are not physically linked to each other.

9. The method of claim 1, wherein said presentation cells are formed by groups of cells that are physically linked to each other.

10. The method of claim 1, wherein said transplantation cells comprise cells selected from the group consisting of epidermic cells, pancreatic parenchymal cells, pancreatic duct cells, hepatic cells, blood cells, cardiac muscle cells, skeletal muscle cells, osteocytes, myocytes, neurons, vascular endothelial cells, pigment cells, smooth muscle cells, adipocytes, bone cells, chondrocytes, or combinations thereof.

11. The method of claim 1, wherein said transplantation cells are cardiomyocytes, cells derived from pancreas, or chondrocytes.

12. The method of claim 1, wherein said transplantation cells are selected from a group consisting of thymocytes, megakaryoblast, promegakaryocytes, lymphoblast, bone marrow precursor cells, normoblast, angioblasts, osteoblasts, skeletal myoblasts, myeloid progenitor cells, satellite cells found in muscles, and transit amplifying neural progenitors, or combinations thereof.

13. The method of claim 1, wherein said transplantation cells are selected from the group consisting of adipose tissue-derived precursor cells (APCs), bone marrow-derived precursor cells, periosteum-derived progenitor cells and Umbilical-cord-derived precursor cells, or combinations thereof.

14. The method of claim 1, wherein said transportation cells are of autologous origin with respect to the patient.

15. The method of claim 1, wherein said transplantation cells are syngeneic.

16. The method according to claim 1, wherein said biodegradable material is selected from the group consisting of polycaprolactone, poly(1,3-trimethylene carbonate), polylactide, polyglycolide, poly(ester amide), poly(ethylene glycol)/poly(butylene terephthalate), poly(glycerol sebacate), poly(1,8-octanediol-co-citric acid), poly(1,10-decanediol-co-D,L-lactic acid), poly(diol citrate), poly(glycolide-co-caprolactone), poly(1,3-trimethylene carbonate-co-lactide), poly(1,3-trimethylene carbonate-co-caprolactone) and a copolymer of at least two of these materials, wherein, more preferably said biodegradable material is either polycaprolactone or a copolymer of polycaprolactone and either poly-trimethylene carbonate or polylactide.

17. The method according to claim 1, wherein said implant is a soft tissue implant.

18. The method according to claim 1, wherein said implant is selected from the group consisting of a breast implant, an implant of the salivary gland, a pancreas implant, a bone implant, an implant to reconstruct an anterior cruciate ligament tear, a craniofacial reconstruction implant, a maxillofacial reconstruction implant, a complex jaw surgery implant, a post tumor-resection reconstruction implant, an implant for tissue reconstruction after removal of a melanoma, an implant for tissue reconstruction after removal of a head and neck cancer, an ear implant, a nose implant, a chest wall reconstruction implant, an orthopedic surgery implant, a cartilage reconstruction implant and a delayed burn reconstruction implant, wherein, more preferably, said implant is a breast implant.

19. The method according to claim 1, wherein said three-dimensional scaffold structure comprises a stack of multiple interconnected layers, each layer being composed of a plurality of, preferably parallel, bars, wherein a) said bars have a zigzag structure or a wiggled structure; or b) the bars of every n-th layer within said stack have a zigzag structure or a wiggled structure whereas the bars of all other layers are straight bars, wherein n is an integer in the range of from 2 to 5, preferably 2 or 3, more preferably 2; or c) each layer comprises bars that have a zigzag structure or a wiggled structure, wherein, preferably, at least 1/10, more preferably at least , more preferably at least , more preferably at least of the bars of each layer have a zigzag structure or a wiggled structure, whereas, preferably, all the other bars of said layer are straight bars; or d) each n-th layer within said stack comprises bars that have a zigzag structure or a wiggled structure, wherein, preferably, at least 1/10, more preferably at least , more preferably at least , more preferably at least of the bars of said each n-th layer have a zigzag structure or a wiggled structure, whereas, preferably, all the other bars of said each n-th layer within said stack and the bars of all other layers are straight bars, wherein n is an integer in the range of from 2 to 5, preferably 2 or 3, more preferably 2; or e) 1/10, preferably , more preferably , more preferably of the layers within said stack are layers that comprise bars having a zigzag structure or a wiggled structure, whereas, preferably, the other layers are layers that comprise only straight bars.

20. The method according to claim 1, wherein said three-dimensional scaffold structure comprises a stack of multiple interconnected layers, each layer being composed of a plurality of parallel bars, wherein the layers within said stack are arranged such that the parallel bars of any layer X within the stack and the parallel bars of the layer subsequent to said layer X (i.e. layer X+1) form an angle of (180/n), wherein n is an integer in the range of from 2 to 10, preferably 2, and wherein the bars of the n-th subsequent layer with respect to a certain layer Y within the stack (i.e. layer Y+n) are offset with respect to the bars of said layer Y by a distance of 1/m times the distance between the parallel bars of said layer Y, wherein m is an integer within the range of from 2 to 5, preferably 2.

21. The method of claim 20, wherein the bars of the layers within said stack are straight bars or the bars of the layers within said stack or the bars of every n-th layer within said stack have a zigzag structure or wiggled structure, whereas the bars of all other layers are straight bars.

22. The method of claim 1, wherein said three-dimensional scaffold structure is formed from a shape-memory polymer (SMP).

23. A method for tissue reconstruction in the body of a patient, comprising the following steps in order: a) implanting into said body of said patient at the site of intended tissue reconstruction an implant comprising a three-dimensional scaffold structure made of biodegradable material, wherein said three-dimensional scaffold structure comprises voids, and wherein said voids are filled with space-occupying structures that are removably attached to said three-dimensional scaffold structure and that are configured to prevent invasion of tissue and/or of individual cells into said voids; b) after an incubation time period sufficient to allow for connective tissue and/or host vasculature to one or both of penetrate into the scaffold structure and invade the space that was occupied by the biodegradable scaffold structure at the time of implantation, removing the space-occupying structures from said voids within the biodegradable scaffold structure or from the tissue that has replaced the biodegradable scaffold structure during the incubation time period, thus generating void spaces not filled with space-occupying structures; c) introducing transplantation cells into the void spaces not filled with space-occupying structures generated in step b).

24. The method according to claim 23, wherein said voids are interconnected with each other and are arranged in a convergent geometric orientation radiating from one origin, or wherein the voids are not interconnected and are arranged in a non-convergent geometric orientation.

25. The method according to claim 23, wherein said space-occupying structures are collapsible, wherein, preferably, said space-occupying structures comprise or consist of a liquid encased in a sheath that is impermeable to said liquid or a hydrogel encased in a sheath that is impermeable to said hydrogel.

26. The method according to claim 23, wherein said space-occupying structures comprise or consist of ferromagnetic or superparamagnetic material, preferably of a composite of a biocompatible polymeric material, more preferably polycaprolactone, and of a biocompatible ferromagnetic material, more preferably iron oxide.

27. The method according to claim 23, wherein said space-occupying structures are coated with a coating that prevents tissue attachment.

28. The method of claim 27, wherein said coating is a coating which comprises a cell proliferation inhibiting drug, preferably a coating that comprises one or more of the drugs tacrolimus, everolimus and mitomycin c.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0162] The invention is now described with reference to the attached figures, wherein:

[0163] FIG. 1 shows two exemplary embodiments of the present invention wherein the implant according to the invention is a breast implant. (A) Example of a breast implant according to the invention wherein the voids are interconnected with each other and arranged in a convergent geometric orientation radiating from one origin.

[0164] (B) Example of a breast implant according to the invention wherein the voids are not interconnected and arranged in a non-convergent (in this case parallel) geometric orientation.

[0165] FIG. 2 shows an embodiment of the invention in which the implant is a breast implant, as well as schematic depictions of steps during the use of such an implant. (A) Angular view (left) and side view (right) of a breast implant according to the invention wherein the voids are not interconnected and arranged in parallel (i.e. a non-convergent geometric orientation). (B) Removal of the space-occupying structures from the implant, in this case by punching out (left) and subsequently withdrawing (right) the space-occupying structures from the implant with a specialized removal tool. (C) Introduction of transplantation cells into the resulting void space not filled with a space-occupying structure, in this case by lipofilling (i.e. introduction of fat tissue) by injection with a syringe. Left: bringing the syringe into contact with the site of injection. Right: The void has been filled with fat tissue.

[0166] FIG. 3 Exemplary depiction of the preparation and use of an implant according to the invention, in this case a breast implant with interconnected voids in a convergent arrangement, filled with collapsible space-occupying structures. (A, B) The space-occupying structures are filled with a liquid or hydrogel (A: initial state; B: final state). (C) The implant has been implanted into the body of the patient at the site of desired breast reconstruction. (D, E) Removal of liquid or hydrogel from the collapsible space-occupying structures by means of aspiration with a syringe (D: initial state; E: final state), which causes the space-occupying structures to collapse and facilitates their subsequent removal. (F, G) Injection of transplantation cells (in this case fat isolated from a donor site within the patient) into the void spaces not filled by space-occupying structures (F: initial state; G: final state).

[0167] FIG. 4 shows the principle of an implant made from a shape-memory polymer by the example of a scaffold of a breast implant. (A) Fully-formed breast implant scaffold made from a shape-memory polymer (the fully-formed shape is the original, permanent shape which the implant will return to upon reaching the trigger temperature of 37 C.). (B) Side view of the deformed, disc-like shape of the breast implant scaffold made from an SMP material at room temperature.

[0168] FIG. 5 shows schematic depictions of different stages during implantation of a breast implant scaffold made from a shape-memory polymer. (A) Before (left) and right at the time of implantation (right) at the site of reconstruction, the scaffold exists in the deformed, disc-like shape that it can take at room temperature. (B) Upon reaching body temperature (37 C.), the scaffold returns to its original, permanent shape, i.e. the fully-formed shape of a breast implant.

[0169] FIG. 6 shows an embodiment of a removal device for the removal of ferromagnetic or superparamagnetic space-occupying structures from an implant according to the invention in an angular side view (A) and an angular bottom view (B).

[0170] FIG. 7 shows the construction principle of a conventional laydown pattern as commonly used in scaffolds of implants.

[0171] FIG. 8 shows examples for different types of bars that according to the invention may be employed for the formation of three-dimensional implant scaffolds. (A) Straight bar. (B, C) Examples of bars with regular zigzag structure. (D, E) Examples of bars with wiggled structure.

[0172] FIG. 9 shows different laydown patterns for three-dimensional scaffolds of implants according to the invention.

[0173] FIG. 10 shows schematic side views of three-dimensional scaffolds with a conventional laydown pattern without offset (A) and with an exemplary laydown pattern according to the invention in which every other layer is offset by a certain distance (B).

[0174] FIG. 11 shows stress versus strain curves of (A) a conventional three-dimensional scaffold with straight bars, (B) a three-dimensional scaffold according to the invention having bars with a wiggled structure, (C) a conventional three-dimensional scaffold without offset and (D) a three-dimensional scaffold according to the invention having an offset.

[0175] FIG. 12 shows the principle of the method for tissue reconstruction in the body of a patient according to the seventh aspect of the invention, illustrating the overall concept of prevascularisation and delayed fat injection. An empty scaffold is first implanted at the breast region without the addition of any cells or growth factors. Over the next e.g. 2-3 weeks, connective tissue and vasculature invades within the scaffold volume forming a bed of capillaries within the pores. Fat is then injected into the pores of the scaffold. Owing to the presence of the pre-formed vascular bed would allow the fat to remain stable at the implantation sites.

[0176] FIG. 13 (A) shows a scanning electron micrograph of the scaffold used in Example 1, showing the struts, pores and pore-interconnections. (B-F) Implantation process of the scaffolds as described in Example 1. (B) Liposuction procedure near the abdominal midline incision. (C, D) Process of injecting fat into the pores of the scaffold placed in the lipoaspirate only group. (C) shows an empty scaffold while (D) shows a completely filled scaffold. (E) shows the process of injecting fat into the prevascularisation+lipoaspirate group scaffolds. The scaffolds are placed empty into the implantation site and 2 weeks later, fat is injected into the scaffold pores while the scaffold remains implanted. (F) the final form of the scaffolds conforms highly to the natural breast shape. The physical and mechanical properties of the scaffolds used are shown in Table 1 below.

[0177] FIG. 14 shows explantation images taken from Example 1 described below, showing the integration of TECs (tissue engineered constructs) with the host tissue. The arrow in panel A points out a major blood vessel supplying blood to the TEC. (D, G) show empty scaffold-only group, (E, H) show lipoaspirate-only group, (F, I) show prevascularisation+lipoaspirate group. All scaffolds show good integration with the host tissues and large areas of fat (marked with +) and vascularisation (marked with ) were observed qualitatively on all scaffolds.

[0178] FIG. 15 (LEFT) shows representative images showing H&E staining of tissue explanted from the empty scaffold group (superficial layers) of Example 1. A majority of the tissue can be identified as being connective tissue and collagen with only very small patches of fat tissue. FIG. 15 (RIGHT) shows representative images showing H&E staining of tissue explanted from the empty scaffold group (deep layers) of Example 1. Adipose tissue is only seen at the edges of the construct and not in the central regions of the scaffold. Lymphatic structures (right panel, marked by arrows) were also observed in all groups mainly localised near scaffold strands.

[0179] FIG. 16 (LEFT) shows H&E stained sections of the lipoaspirate-only group (superficial layers) of Example 1. Overall, a higher percentage of fat tissue compared to overall tissue area, compared to empty scaffold group, was observed in this group. FIG. 16 (RIGHT) shows H&E stained sections of the lipoaspirate-only group (deep layers) of Example 1. Deeper layers of the scaffold showed lower relative adipose tissue areas and lower degrees of vascularisation.

[0180] FIG. 17 (LEFT) shows H&E stained sections of the prevascularisation+lipoaspirate group (superficial layers) of Example 1. This group showed the highest accumulation of adipose tissue interspersed between connective tissue. Tissue morphology also showed similarities with native tissue. FIG. 17 (RIGHT) shows H&E stained sections of the prevascularisation+lipoaspirate group (deep layers) of Example 1. Adipose tissue area was the highest among all other groups. Adipose tissue regions seemed to be better connected to each other and formed interconnected structures.

[0181] FIG. 18 depicts representative H&E-stained micrographs of regions around the scaffold strands showing non-specific minor granulomatose reactions. (A) shows the empty scaffold-only group, (B) shows the lipoaspirate-only group (C) shows the prevascularisation+lipoaspirate group. Arrow heads point to macrophages.

[0182] FIG. 19 shows representative images of Masson's Trichrome stained tissue sections obtained from Example 1. In Masson's Trichrome staining, green colour indicates collagen fibres, red colour indicates muscle fibres and dark brown shows cell nuclei. (A, D) show the empty scaffold group (B, E) show the prevascularisation+lipoaspirate group (C, F) show the lipoaspirate-only group. Besides the adipose tissue, a majority of the tissue filling the pores of the implant consisted of connective tissue. Smooth muscle tissue was also detected lining the strands of the scaffold. These smooth muscle layers had the highest thickness in case of the prevascularisation+lipoaspirate group. (G) Column plot showing the adipose tissue area relative to total tissue area over 24 weeks. Negative control scaffold-only group had the lowest relative area of adipose tissue (8.31%8.94) which was significantly lower than both the lipoaspirate-only (39.67%2.04) and the prevascularisation+lipoaspirate group (47.32%4.12) and also compared to native breast tissue (44.97%14.12) (p<0.05, p<0.01 and p<0.01 respectively). No statistically significant difference in relative adipose tissue area was observed between the native breast tissue, lipoaspirate-only and prevascularisation+lipoaspirate groups. (H) Graph showing blood vessel density in the tissue sections from different groups. Highest blood vessel density was observed in the prevascularisation+lipoaspirate group (38.01/mm.sup.22.02), however the density was not statistically significantly higher than the scaffold-only (33.13/mm.sup.212.03), lipoaspirate-only (26.67/mm.sup.21.6) or control breast tissue (35.45/mm.sup.21.93). (I) Histogram showing the distribution of adipose cells according to the cell surface area. In all groups, the histograms were skewed to the right suggesting that a majority of adipose cell surface areas lay in the range of 100-700 m.sup.2. The distribution of the cell sizes in control breast tissue was considerably different compared to the other groupswith the highest percentage of cells in the 100-200, 300-400 and 500-600 m.sup.2 range. The empty scaffold and lipoaspirate-only groups had a low number of adipose cells whose surface areas were larger than 800 m.sup.2; however, the prevascularisation+lipoaspirate group showed a more equalised distribution with a significantly large number of cells having a surface area larger than 1000 m.sup.2. (J) Clustered column graph showing tissue composition at week 24 in various groups. TECs from the empty scaffold group contained an estimated 4.99 cm.sup.3 (2.71) of adipose tissue, TECs from the lipoaspirate-only group contained an estimated 23.85 cm.sup.3 (1.22) of adipose tissue, whereas TECs from the prevascularisation+lipoaspirate group contained an estimated 28.391 cm.sup.3 (2.48) of adipose tissue. (K) Column graph showing estimated fold increase in adipose tissue volume compared to initial injected lipoaspirate volume (4 cm.sup.3) in the lipoaspirate-only and prevascularisation+lipoaspirate groups. The prevascularisation+lipoaspirate group had a higher fold increase in adipose tissue volume (6.10.62) compared to lipoaspirate-only group (4.950.31); however, the difference was not statistically significant (p=0.143).

[0183] FIG. 20 shows H & E stained sections of untreated control breast tissue (healthy breast tissue, no scaffold implantation or application of lipoaspirate).

[0184] FIG. 21 shows a fabricated breast shaped scaffold containing voids and space-occupying structures and the removal of the space occupying structures. (A) Fabricated breast shaped scaffold made out of biodegradable poly-lactic acid (white material in FIG. 21) containing solid regions of low porosity made of poly-lactic acid with a black dye (i.e. space-occupying structures, seen as black dots in FIG. 21 A). The space-occupying structures had basically 0% porosity and were loosely attached with the main body of the scaffold (0.4 mm gap between the main body and the space occupying structures). (B) A cutting tool is used to punch out the regions of low porosity and mechanical integrity (i.e. to remove the space-occupying structures from the scaffold). While for illustrative purposes FIG. 21 B shows the removal procedure with a scaffold outside of the body of a patient, the same procedure of punching out the space-occupying structures is also used for a scaffold that has been implanted into the body. (C) The void space left behind by removal of the low porosity regions (highlighted with circle) can be used for lipofilling.

[0185] FIG. 22 shows (TOP) photographic images of scaffolds containing void spaces (no implantation) (left) and with adipose tissue injected into the void spaces (right). (BOTTOM) Hematoxylin and Eosin stained section of a scaffold explanted after 6 months implantation into minipigs. Inset on top left shows a cut out of the area surrounding a randomly selected void filled with adipose tissue (adipose tissue encircled). The corresponding area in the histological section, also encircled, shows healthy well vascularised adipose tissue at the injection site with no signs of necrosis.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0186] For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is intended thereby, such alterations and further modifications in the device and methods and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur now or in the future to one skilled in the art to which the invention relates.

[0187] Moreover, it is to be understood that features and advantages described with regard to one aspect of the invention may also be implied by other aspects of the invention.

[0188] FIG. 1 shows two exemplary embodiments of implants according to the present invention. Both embodiments represented are breast implants the overall shape of which is formed from a three-dimensional scaffold structure made from biodegradable or non-biodegradable material. As seen in FIG. 1, in both embodiments the three-dimensional scaffold structure comprises voids that are filled with space-occupying structures.

[0189] In the embodiment of FIG. 1A, the voids are interconnected with each other and arranged in a convergent geometric orientation radiating from one origin. Entry through the origin of the void structure (from the top of the implant as depicted) allows access to all voids of the void structure, for example to introduce transplantation cells after removal of the space-occupying structures. Moreover, this arrangement may also allow to fill a collapsible space-occupying structure with liquid or hydrogel during preparation of an implant with collapsible space-occupying structures through a single access point, to aspirate the liquid or hydrogel from collapsible space-occupying structures through a single access point or to remove the space-occupying structures through a single incision wound at the point of origin. Thus, in several aspects this arrangement allows to simplify the steps carried out for use of the implant and to minimize the injuries inflicted to the patient during surgical procedures.

[0190] The specific embodiment of FIG. 1A further includes a handle (depicted at the top of the implant) that the surgeon can use to remove the entire space-occupying structure assembly after he/she has aspirated the liquid or hydrogel and the space-occupying structures have collapsed. The handle helps the surgeon to lift the collapsed assembly out of the scaffold and the patient's body. Other shapes of the handle than the conceptualised shape shown in FIG. 1A are contemplated as well.

[0191] In the embodiment of FIG. 1B, the voids within the implant are not interconnected, but arranged in parallel along one axis of the scaffold (i.e. arranged in a non-convergent geometric orientation). While such an arrangement requires multiple access tunnels for removal of the space-occupying structures or the introduction of transplantation cells, it makes removal of the space-occupying structures technically easier, because each space-occupying structure can be accessed directly and lies directly beneath the skin surface.

[0192] It is to be understood that the arrangements of the voids as shown in the embodiments depicted in FIG. 1 are merely of exemplary nature, and diverse other arrangements of the voids (convergent, non-convergent or combinations thereof) also lie within the scope of the present invention. Moreover, as the skilled person will appreciate, the breast implants shown in FIG. 1 (and some of the other Figures) are only examples, and the present invention also relates to implants for reconstruction of other parts or tissues of the body, such as reconstruction after anterior cruciate ligament tear, craniofacial reconstruction, maxillofacial reconstruction, complex jaw surgery, tissue reconstruction after removal of melanoma or head and neck cancer, chest wall reconstruction, delayed burn reconstruction etc. Naturally, such implants will differ in their structure, shape and characteristics from the breast implants depicted and will, while still constructed and used according to the principles of the present invention, be specifically adapted to the required purposes.

[0193] FIG. 2 shows individual steps to be carried out during the use of an implant according to the invention, illustrated for the example of a breast implant comprising a three-dimensional scaffold structure made of biodegradable material. In the exemplary embodiment of the implant used, the voids are not interconnected and are arranged in parallel, i.e. in a non-convergent geometric orientation (FIG. 2A). While in the example shown in FIG. 2 solid space-occupying structures are used, other types of space-occupying structures are equally possible, as defined in the embodiments of the invention above.

[0194] For breast reconstruction or augmentation, the implant is surgically implanted at the desired site. After several weeks of incubation (such as 6-8 weeks), the biodegradable scaffold material will be partially degraded and connective tissue and host vasculature will have penetrated into the scaffold structure and the space emerging due to scaffold structure degradation. At this point, the solid space-occupying structures are surgically removed. As shown in FIG. 2B, this may be achieved by a specialized removal tool according to the invention.

[0195] According to the embodiment shown in FIG. 2B, the removal tool has a grip to which a blade is attached at its distal end. The blade is designed as a biopsy punch blade with a circular shape. To reduce scar formation through the biopsy punch blade, a small linear incision is inflicted to the skin and tissue overlying the space-occupying structure through which the blade of the removal tool is inserted. The shape and size of the circular blade mirrors the round shape and the diameter of the cross-section of the space-occupying structures. Thus, by applying the removal tool with correct positioning, it allows to accurately excise the space-occupying structure (FIG. 2B, left; Note that the depictions of FIG. 2 are schematic illustrations that show the depicted steps at the isolated implant. In practice, the implant will of course be located in the body of a patient during the removal step of FIG. 2B and the step of introducing the transplantation cells of FIG. 2C). The removal tool further includes an appliance that allows grasping the excised space-occupying structure (not visible in the depiction of FIG. 2B). Upon grasping the excised space-occupying structure, the removal tool is withdrawn and thus the excised space-occupying structure is removed, leaving behind a void space that was previously filled with the space-occupying structure (FIG. 2B, right) and that, due to the previous occupation by the space-occupying structure, is free of invaded connective tissue and vasculature. Subsequently, the space-occupying structures will also be removed from the other voids of the implant.

[0196] As a following step, transplantation cells, i.e. cells of the desired cell type (differentiated cells or precursor cells) that are to be introduced for tissue reconstruction, are introduced into this void space. In the case of a breast implant as shown in FIG. 2, the transplantation cells may be fat tissue obtained from a donor site of the same patient that is injected with a syringe into the void space. Since in the example of FIG. 2 the voids are arranged in parallel arrangement and are not interconnected, an individual injection has to be carried out for each void space separately.

[0197] Implants according to the invention and their use as exemplified above result in the creation of a pre-formed bed of connective tissue and vasculature into which the transplantation cells are introduced. Thus, a stable association of the introduced cells with the implantation site, optimal supply of the transplanted cells with oxygen and metabolites, and minimal necrosis and resorption are achieved. At the same time, the inclusion of voids and space-occupying structures in the implant makes sure that there is sufficient space for introduction of an adequate amount of transplantation cells into the pre-formed bed of vascularized connective tissue upon removal of the space-occupying structures.

[0198] FIG. 3 provides another example of the preparation and use of an implant according to the invention. In contrast to the embodiment described in FIG. 2, FIG. 3 shows the use of a breast implant with interconnected voids in a convergent arrangement (FIG. 3A, B). Moreover, while FIG. 2 shows an implant with solid space-occupying structures, the space-occupying structures of the implant of FIG. 3 consist of a liquid- or hydrogel-filled sheath and thus are collapsible. The sheath consists of a biocompatible polymeric material that is impermeable to the liquid or hydrogel.

[0199] The implant is produced with voids containing a sheath in the shape of a tubing, and the sheath is subsequently filled with a liquid or hydrogel (FIG. 3A, B). Since in the embodiment of FIG. 3 the voids (and also the space-occupying structures) are interconnected, all the space-occupying structures can be filled with the liquid or hydrogel through a single access point, which is subsequently sealed.

[0200] The implant is then implanted at the site of intended tissue reconstruction, in the embodiment of FIG. 3 the area of the breast of a patient where breast reconstruction/augmentation is desired (FIG. 3C). Upon implantation, the implantation site is left to heal for several weeks. During this time, connective tissue and blood vessels will invade the implant, which, if the implant comprises a three-dimensional scaffold structure made of biodegradable material, is concurrently gradually degraded.

[0201] After 6-8 weeks, the fluid within the space-occupying structures is removed, in the exemplary procedure of FIG. 3 by piercing of the space-occupying structures and aspiration of the fluid with a syringe (FIG. 3D, E). Since the voids and space-occupying structures are interconnected, the complete volume of liquid or hydrogel can be removed from the space-occupying structures through the single access point at the origin of the convergent void system. Upon removal of the liquid or hydrogel, the space-occupying structure collapses to an empty sheath of polymeric material.

[0202] Whereas the specialized removal tool shown in FIG. 2B is particularly useful for the removal of solid space-occupying structures, the collapsible space-occupying structures of the embodiment depicted in FIG. 3 can be removed after aspiration of the liquid/hydrogel simply by grasping the interconnected space-occupying structures at the origin of the convergent void system with surgical forceps and withdrawing them from the implantation site. Removal of the space-occupying structures is further simplified and collateral tissue damage during removal is reduced, if the space-occupying structures used have a surface coating that rejects tissue and cells from invasion, such as a coating with the drug tacrolimus (not shown in the embodiment of FIG. 3).

[0203] Upon removal of the space-occupying structures, void spaces are left behind into which transplantation cells (in case of the breast reconstruction shown in FIG. 3 fat tissue isolated from a different site of the patient's body) are injected (FIG. 3F, G).

[0204] Due to the convergent arrangement of the voids and space-occupying structures, in the embodiment of the implant shown in FIG. 3 aspiration of the liquid/hydrogel from the space-occupying structures, removal of the collapsed space-occupying structures and injection of the fat tissue can all be carried out through a single, small incision at the origin of the convergent arrangement by a minimally invasive procedure.

[0205] The use of (biodegradable) shape-memory polymer (SMP) materials for construction of three-dimensional scaffolds of implants according to the invention is highly advantageous, in particular with regard to minimizing the tissue and skin damage that is necessary for surgical insertion of the implant. FIG. 4 shows an example of a scaffold structure of a breast implant made from a shape-memory polymer to illustrate the principle of an SMP implant. The original, permanent shape of the scaffold corresponds to the extended shape of the fully-formed breast implant, as shown in FIG. 4A. Below a certain trigger temperature, the scaffold can be deformed into other shapes, such as the more compact, disc-like shape depicted in FIG. 4B. However, once the temperature of the scaffold is increased and reaches or surpasses the trigger temperature, the scaffold will return to its original, permanent shape, which is the extended shape of the breast implant of FIG. 4A. As the skilled person will appreciate, the trigger temperature in the context of the present invention should equal are be just below the body temperature of the patient undergoing transplantation.

[0206] The practical application of an implant according to the invention comprising an SMP scaffold is exemplified in FIG. 5 (to simplify the depiction, only the scaffold component of the breast implant is shown). A breast implant scaffold with the characteristics as described in FIG. 4 (i.e. the original, permanent shape of the scaffold corresponds to the extended shape of the fully-formed breast implant; and the trigger temperature of the SMP material is identical to body temperature) is deformed to a compact, disc-like shape at room temperature (FIG. 5A, left). Owing to its compact structure, the deformed scaffold can be implanted by the surgeon more easily and through a smaller incision than an implant with an extended scaffold structure and placed at the desired site of implantation (FIG. 5A, right). As soon as the implant is inside the body of the patient, the SMP implant adapts from room temperature to the patient's body temperature. Once it reaches body temperature, the SMP material returns to its original, permanent shape which is the extended shape of a fully-shaped breast implant (FIG. 5B). The surgeon can then close the small incision safely.

[0207] While removal of the space-occupying structures may occur with a specialized removal tool as described in the procedure shown in FIG. 2B above or by withdrawal of collapsible space-occupying structures as described in connection with FIG. 3 above, the present invention also provides for a specialized removal device for the removal of ferromagnetic or superparamagnetic space-occupying structures from an implant according to the invention.

[0208] An exemplary embodiment of a removal device for removing ferromagnetic or superparamagnetic space-occupying structures from a breast implant is shown in FIG. 6. The removal device has the shape of a flattened can which is traversed by twelve rod-shaped electromagnets. The bottom surface of the container has a bell-shaped indentation, thus providing for a good fit to the breast.

[0209] To remove ferromagnetic or superparamagnetic space-occupying structures from an implant at a transplantation site, small incisions are made through the overlying tissue at the positions where the space-occupying structures reside, thus creating a path through the tissue along which the space-occupying structures may be removed. Then the indented surface of the removal device is brought into contact with the implantation site. The space-occupying structures to be removed have the same spatial distribution as the electromagnets on the removal device, such that the electromagnets of the removal device are in perfect orientation for interacting with the space-occupying structures to be removed. Moreover, since the surface of the removal device that contacts the breast has a breast-shaped indentation, a tight fit of the surface of the removal device to the breast is ensured, thus bringing the electromagnets into direct contact with the space-occupying structures to be removed.

[0210] At this point, the electromagnets of the removal device are turned on. The strong magnetic forces exerted by the powerful electromagnets attract the ferromagnetic or superparamagnetic space-occupying structures, such that they get attached to the electromagnets, and the removal device is withdrawn from the body of the patient. The space-occupying structures move along with the removal device and are thus removed from the patient's body.

[0211] The present invention also provides special laydown patterns for implant scaffolds that allow to adapt the three-dimensional scaffold of the implant to the specific needs of an implant with voids and space-occupying structures.

[0212] FIG. 7 shows the principle of a conventional laydown pattern for implant scaffolds. Pictured are two layers (a bottom layer of equidistantly arranged, parallel bars oriented along the y axis and a top layer of equidistantly arranged, parallel bars oriented along the x axis) that illustrate the construction principle of such a conventional laydown pattern. The individual bars are physically connected at the points of contact with other bars. The pattern of the two layers is repeated in the z direction (i.e. out of the plane of the paper), resulting in a three-dimensional scaffold structure as shown in FIG. 9A (see below).

[0213] FIG. 8 shows examples for different types of bars that may be used for the formation of three-dimensional scaffold structures of implants according to the invention. This includes straight bars as they are also used in conventional laydown patterns (FIG. 8A), bars with regular zigzag structure (FIG. 8B), bars with regular zigzag structure, wherein the zigzag structure has a staircase-shape (FIG. 8C), bars with regular wiggled structure (FIG. 8D) and bars with irregular wiggled structure (FIG. 8E). As the skilled person will appreciate, the bars shown in FIG. 8 are only of exemplary nature, and bars with various other regular or irregular zigzag structures, wiggled structures or combinations thereof are also contemplated by the present invention.

[0214] The central axis of the bars depicted in FIGS. 8 B-E is indicated as a dashed line. In a parallel arrangement of bars with zigzag or wiggled structure, the bars will be oriented such that the central axis of the bars will be parallel.

[0215] FIG. 9 shows different laydown patterns for three-dimensional scaffold structures of implants formed from stacks of multiple interconnected layers, each layer being composed of a plurality of parallel bars: FIG. 9A is an illustration of a three-dimensional scaffold structure with a conventional laydown pattern, as it is obtained if the construction principle of FIG. 7 is followed. FIG. 9B, in contrast, shows an embodiment of the three-dimensional scaffold structure according to the invention, wherein the parallel bars of every other layer have a regular, staircase-shaped zigzag structure and wherein the bars of subsequent layers with straight bars are offset with respect to each other. FIG. 9C is a depiction of an alternative embodiment of the three-dimensional scaffold structure according to the invention, wherein the parallel bars of every other layer have a wiggled structure. In the three-dimensional scaffold structure of FIG. 9D, a scaffold according to the invention is represented, wherein all bars have a wiggled structure.

[0216] The scaffold structures shown in FIG. 9 B-D represent only examples, and various other scaffold structures also fall within the scope of the present invention. Thus, for example bars of various other shapes and combinations of bars with zigzag structure and wiggled structure are contemplated as well. Moreover, the layers within the stacks of FIGS. 9 B-D are all arranged such that the bars of any layer have a perpendicular arrangement with respect to the bars of the subsequent layer. According to other (not depicted) embodiments of the invention, however, subsequent layers may also be rotated by other angles, for example by an angle of 60, such that the third layer after any layer X (i.e. layer X+3) has again an orientation of its bars that is parallel to the bars of said layer X.

[0217] In FIG. 10 schematic depictions of different three-dimensional scaffold structures are provided in side view representation. FIG. 10A is a scaffold with conventional laydown pattern without offset. This scaffold structure is identical to the one shown in FIG. 9A. In contrast, FIG. 10B shows an exemplary laydown pattern according to the invention in which, of those layers that have bars oriented along the y axis (i.e. pointing out of the paper plane), every layer is, with respect to the previous layer, offset by a distance of times the distance between the bars of said layer (i.e. the bars of the layer are parallel-shifted within the plane of the layer, which in this depiction means shifted along the x axis). This means that every other layer with bars oriented along they axis is again vertically in line, i.e. the bars of such layers can geometrically be brought to congruency by a simple translation along the z-axis.

[0218] While the embodiment shown in FIG. 10B has a scaffold structure with a repetition after every second layer of the same bar orientation (i.e. every fourth layer in FIG. 10B, if all layers are counted, independent of the orientation of their bars), the present invention also embraces embodiments with other repetition patterns. For example, if the layers with the same orientation of their bars are shifted by a distance of times the distance between the bars, repetition is achieved after every third layer, and if the layers with the same orientation of their bars are shifted by a distance of 1/m times the distance between the bars, repetition is achieved after every m-th layer of the same orientation.

[0219] FIG. 11 shows experimental data obtained from compression testing performed on scaffolds with conventional laydown structure as shown in FIG. 10A (data in FIG. 11A) and with a laydown pattern according to the invention having an offset as shown in FIG. 10B (data in FIG. 11D). In these experiments, 3 sets of 20 equal-sized square sheets of porous scaffolds were fabricated from polycaprolactone. The porosity, strut/bar size and strut spacing were kept constant across all the groups; however, one group (Group A) was formed with a conventional laydown structure as shown in FIG. 10A, another group (Group B) was formed with a laydown pattern according to the invention having an offset as shown in FIG. 10B and the third group (Group C) was formed with a wiggled laydown pattern according to the invention as shown in FIG. 9C. Compression testing was performed on the explanted scaffolds using an Instron 5848 microtester fitted with a 500N load cell. All scaffolds from Group B were compressed in the Z direction (axial compression), all scaffolds from Group C in the X direction (transverse compression) whereas 50% of the Group A scaffolds were compressed in Z-direction and the remaining were compressed in the X direction. The testing protocol consisted of a 2 mm compression of the scaffolds at a rate of 0.6 mm/min.

[0220] The data obtained from the microtester was used to plot a stress-strain curve which, as a skilled person will appreciate, corresponds to the stiffness of the construct. FIGS. 11A and 11B show a stress vs strain plot of Group A and Group C scaffolds, respectively, whereas FIGS. 11C and 11D show a stress vs strain plot of Group A and Group B scaffolds, respectively.

[0221] From these data, it can be concluded that the scaffold structure according to the invention having a zigzag laydown pattern is more flexible in the XY direction, can take the same stress as the control scaffold and displays a higher range of elastic deformations as compared to control scaffolds with a conventional laydown pattern fabricated with the same parameters.

[0222] Similarly, scaffold structures with an offset in the Z direction according to the invention are more flexible in their axial Z direction.

[0223] FIG. 12 is an exemplary depiction of the different steps of the method for tissue reconstruction in the body of a patient according to the seventh aspect of the invention, illustrated for the example of a breast reconstruction. A breast implant comprising a three-dimensional scaffold structure made from a biodegradable material is implanted at the desired site of breast reconstruction (a). The scaffold structure is allowed to remain at the implantation site for a period of 6-8 weeks during which connective tissue and especially host vasculature penetrates into the scaffold structure (b). After this period, fat is isolated from a donor site within the patient's body and injected into the scaffold structure (c). The presence of a pre-formed bed of connective tissue and vasculature allows the fat to remain stably within the implantation site with minimal tissue necrosis and resorption (d). Moreover, such a structure would also better mimic the internal architecture of the breast.

[0224] One disadvantage of the method depicted in FIG. 12 is that, since the invading connective tissue may take over the majority of the volume initially occupied by the biodegradable scaffold structure, no further volume may remain for the secondary injection of fat tissue injected during step (c) (or other transplantation cells to be injected). As the skilled person will appreciate, the use of an implant according to the present invention (comprising a three-dimensional scaffold structure with voids and removable space-occupying structures as described above) and, accordingly, a method for tissue reconstruction using such an implant (as defined in the eighth aspect of the invention above) overcomes such problems, because the space-occupying structures will protect void spaces that cannot be invaded by connective tissue or vasculature and that, upon removal of the space-occupying structures, become available for introduction of fat tissue or other transplantation cells.

EXAMPLES

Example 1

[0225] This example combines delayed fat injection with an acellular biodegradable scaffold. In this method of implantation, the scaffold is first implanted with no fat tissue into the implantation site. A fibrin clot is formed immediately after implantation of the scaffold from the hematoma caused by the surgical procedure (Henkel et al., 2013; Salgado et al., 2004). The clot consists of platelets embedded in a mesh of cross-linked fibres, together with a growth-factor rich cocktail of fibronectin, vitronectin and thrombospondin. The fibrin clot and the associated growth-factor cocktail may stimulate a strong angiogenic response and induce highly organised connective tissue to penetrate into the scaffold. After a fixed period of time, fat is isolated from a donor site within the patient's body and injected into the scaffold (see FIG. 12 for a visualisation of this concept). The amount of fat that can be harvested from the patient without encountering donor site morbidity depends on the body composition of the patientwhereby a larger volume of fat can be extracted from patients with higher body fat percentage. In this study, based on the expertise of our surgical team and a literature search, 4 cm of adipose tissue was considered to be the maximum amount that can be harvested from a patient with a very low body fat percentage without encountering donor-site complications. Therefore, the scaffolds were seeded with 4 cm.sup.3 of fat isolated from the donorrepresenting 5.23% of the total volume of the scaffolds.

[0226] The study of Example 1 characterised adipose tissue retention in large 75 cm.sup.3 acellular polycaprolactone-based scaffolds subjected to a delayed fat injection implanted in a large animal model (pigs) for a period of 24 weeks.

Study Design and Sample Size Rationale

[0227] A randomised and blinded animal study was carried out, evaluating the adipose tissue regenerative potential of large 75 cm.sup.3 biodegradable scaffolds for 24-weeks using a subglandular swine animal model.

[0228] Three experimental groups were included in this study: [0229] 1) Empty scaffold (negative control). [0230] 2) Scaffold containing 4 cm.sup.3 lipoaspirate. [0231] 3) Empty scaffold+2 week prevascularisation period. After 2 weeks of prevascularisation, 4 cm.sup.3 of lipoaspirate was injected into scaffolds.

[0232] The primary endpoint evaluated was the percentage of adipose tissue area compared to overall tissue area (AA/TA). In an optimal case, no statistically significant difference in mean AA/TA between the experimental groups (prevascularisation+lipoaspirate and lipoaspirate-only to groups) and the healthy breast tissue group (<10% difference in means) would be detected, while, at the same time, a statistically significant difference between the AA/TA of negative control (empty scaffold) group and healthy breast tissue group would be detected. For an expected standard deviation of 5 (5 point scale), a sample size of 12 used in this study gives a statistical power of 85.7%. Statistical Power calculations were performed using Researcher's Toolkit Statistical Power Calculator (DSS Research, Fort Worth, USA).

Rules for Stopping Data Collection

[0233] Data collection was stopped and the scaffolds were excluded from further analysis if one of the two following conditions were met (all signs verified by experienced plastic and veterinary surgeons): [0234] 1) Detection of infection. [0235] 2) Long-standing signs of haematoma or seroma.

Selection of Endpoint

[0236] Since adipose tissue undergoes remodelling multiple times during the wound healing process, in this study a primary endpoint of 24 weeks was chosen to be adequate in terms of addressing tissue permanence mechanisms.

Randomisation and Blinding

[0237] Two study parameters were randomised: [0238] 1) Allocation of a scaffold to an experimental group. [0239] 2) Allocation of a scaffold to a subglandular pocket.

[0240] For both parameters, randomisation sequence was created using Excel 2010 (Microsoft, Redmond, USA) with a 1:1 allocation using random block sizes of 2 and 4 by an independent researcher. Except for the plastic surgeon operating on the animals, all researchers were kept blind to the allocation of scaffold and subglandular pockets to the experimental groups. Geographical separation ensured minimal contact between the operating surgeon and the researcher performing histological and qualitative analyses. Upon explantation, the operating surgeon coded each scaffold with an ID (JT-n; where n=1 to 12) and kept the key hidden from the researchers performing downstream analyses. The key was revealed to the researchers only upon completion of the data analysis. In summary, all study outcomes were assessed in a blinded manner.

Design & Fabrication of Scaffolds

[0241] Rapid prototyped hemisphere-shaped polycaprolactone-based scaffolds were designed and manufactured by Osteopore International Pte Ltd (Singapore). All scaffolds were produced using medical-grade polycaprolactone adhering to ISO 11137 (Sterilisation), 13485 (Quality Systems), 11607 (Packaging), and 14644-1 (Clean Room) standards.

In Vivo Implantation into Minipigs

[0242] The animal experiments were performed under GMP conditions at PWG Laboratories, Singapore with ethical approval from PWG Laboratories which, in turn, is maintained in accordance with NIH Guide for the Care and Use of Laboratory Animals. Two female adult immunocompetent minipigs were used in this study. The operation was performed under general anaesthesia, following the standard protocol of sterility requirements for breast augmentation procedures. Careful homeostasis was also maintained throughout the surgical procedure. 3 separate subglandular pockets were created on each side of the mammary region via a longitudinal incision. 6 implants were randomly placed in each animal. Prior to implantation, all scaffolds were trimmed by 1 mm from the outer boundary at the operating table by the surgeon to ease the implantation process and gain access to the inner pores by removing the outer shells of the scaffolds.

[0243] In groups 2 and 3, a midline incision was made and adipose tissue was obtained via the Tulip system (Tulip Medical Products, San Diego, USA). The lipoaspirate was injected directly into the interconnected pore architecture of the scaffoldsusing a 10-cm.sup.3 Tulip cell-friendly injector.

[0244] After the placement of the implants each pocket was closed with absorbable vicryl sutures, such that the implants were fixed stably and had no contact to each other. Finally, the skin was sutured with interrupted 2.0 Ethilon sutures.

Histological and Histomorphometrical Analyses

Hematoxylin & Eosin (H & E)

[0245] Implants were harvested from the minipigs after 24 weeks and were fixed with 4% PFA (paraformaldehyde), cut into 10 mm10 mm cube sections, dehydrated and embedded in paraffin using a tissue processor (Excelsior ES, Thermo Scientific, Waltham, USA). Constructs were horizontally sliced to 5 deparaffinised with xylene, rehydrated with a decreasing series of ethanol and stained with H & E (Hematoxylin and eosin stain). Stained slides were scanned with a BIOREVO BZ-9000 microscope (Keyence, Itasca, USA) at 5 magnification.

Massons Trichrome Staining

[0246] The slides were deparaffinised with xylene, rehydrated with a decreasing series of ethanol and re-fixed in Bouin's solution at room temperature overnight. After rinsing in tap water for 10 minutes, the slides were stained in Weigert's iron hematoxylin for 10 minutes, rinsed in running warm tap water, stained in Biebrich scarlet-acid fuchsin solution for 10 minutes and transferred directly into aniline blue solution and stained for 10 minutes. The slides were rinsed briefly in distilled water and differentiated into 1% acetic acid solution for 5 minutes.

Histomorphometry

[0247] Histomorphometrical analyses were carried out with the Osteomeasure histomorphometry analysis system (Osteometrics Inc., Decatur, Ga., USA). All measurements were performed blinded on 8 randomly chosen sections from each scaffold from each group (4 from the superficial regions and 4 from the deep regions). To determine the average adipose tissue area, the total area of the adipose tissue was first calculated (A). Secondly, the total area occupied by the scaffold struts was measured (S). Finally, the combined area of the tissue section was measured (C). The ratio of adipose tissue area to total tissue area (R) was calculated using the following formula (Chhaya et al., 2015):

[00001]$R=\frac{A}{\left(C-S\right)}*1.Math.0.Math.0.Math.\%$

[0248] ImageJ (National Institutes of Health, MA, USA), in conjunction with Adipocyte Tools plugin developed by Montpellier RIO Imaging (Montpellier, France), was used for all automated calculations involving cell size distribution. The field of view (FOV) from each histological section was kept uniform. Background was first removed from each histological section by the pre-processing macro within the Adipocyte Tools plugin using the thresholding method. Minimum size of each cell was chosen to be 80 m, maximum size as 800 m and the number of dilates were set to be 10. These threshold values were kept constant across all samples and groups. The same threshold was also chosen to automatically set regions of interest (ROI) around the adipose cells. The automated method generated a small number of ROI artefacts. Artefacts that could be detected visually were manually removed. In order to remove the remaining artefacts, 10% of the smallest and 10% of the largest ROIs were excluded from any further analysis.

[0249] In order to calculate the blood vessel density, all blood vessels that showed red erythrocytes within the lumen were counted. The number of blood vessels was divided by the total tissue area to get the density. Values based on 4 stitched microphotographs from each scaffold per experimental condition.

Estimation of Adipose Volume in TEC (Tissue Engineered Constructs)

[0250] Since the entire volume of the scaffold was filled with host tissue, it is reasonable to assume that each scaffold held 60 cm.sup.3 of total tissue volume at the end of the implantation period (75 cm.sup.3 total volume80% porosity=60 cm.sup.3 volume available for tissue growth; scaffold degradation has not been taken into account in order to simplify calculations).

[0251] The relative adipose tissue fraction values shown in FIG. 19G have been calculated from 8 randomly chosen tissue sections, each 40 mm25 mm in dimensions. The estimated volume fraction of adipose tissue in each group was extrapolated from these adipose tissue area fraction values.

Statistical Analysis

[0252] All data are represented as meanSD and are subjected to one-way analyses of variance (one-way ANOVA) and Tukey's post-hoc test (Prism 6, GraphPad, San Diego, USA). Significance levels were set at p<0.05. All error bars represent standard deviation.

Clinical Observations

[0253] The surgery and implant placement were tolerated well by all animals and no apparent clinical signs of infection were observed throughout the implantation period. 12 weeks after the initiation of the study, one scaffold was observed to have seroma accumulation in the surgically-created pocket and was therefore excluded from further analysis.

Scaffold Characterisation

[0254] The overall geometry of the scaffold was similar to that of a silicone implant used for breast augmentation (FIG. 13). The high porosity value of the scaffolds (obtained from the manufacturer) implies that more volume is available for tissue ingrowth.

Scaffold Explantation and Degradation

[0255] As pointed out above, three study groups were evaluated in this study. After 6 months of implantation, the Tissue Engineered Constructs (TECs) were retrieved for histological analysis. The scaffolds were well integrated with the surrounding tissue and there was a widespread invasion of host vasculature into the constructs (FIG. 14C). Visual examination revealed that the overall shape of the scaffolds did not change drastically over the implantation period. All scaffolds showed good integration with the host tissues and large areas of fat and vascularisation were observed qualitatively on all scaffolds. Qualitatively, it was also clear that the prevascularisation+lipoaspirate group (FIG. 14 F, I) had the highest degree of vascularisation and fat tissue deposits, followed by the lipoaspirate-only group (FIG. 14 E, H). Although the empty scaffold-only group also showed deposits of adipose tissue (FIG. 14 D, G), they were not as widespread as in the other groups.

Formation of Vascularised Adipose Tissue

[0256] FIGS. 15-17 show representative H&E stained images of all scaffold groups after 24 weeks in vivo. All sections showed the typical ring-like morphology of fat tissue. Overall, multiple areas of well-vascularised adipose tissue were found in all groups.

[0257] H&E staining of tissue explanted from the empty scaffold group showed that although the newly infiltrated tissue was highly vascular, a majority of the tissue was connective tissue and collagen with only very small patches of fat tissue (FIG. 15) identified in the micrographs by their typical ring-like morphology and the empty vacuole in the middle of the cell. The deeper layers of the empty scaffolds also showed similar results.

[0258] FIG. 16 shows the H&E stained sections of the lipoaspirate-only group. Overall, a higher percentage of fat tissue compared to overall tissue area (referred to herein as relative tissue area) was observed in this group. The superficial layers of the scaffold especially showed widespread distribution of adipose tissue whose relative tissue area matched closely to that of native breast tissue. However, the deeper layers of the scaffold showed lower relative adipose tissue areas and lower degrees of vascularisation.

[0259] FIG. 17 shows the H&E stained sections of the prevascularisation+lipoaspirate group. This group showed the highest amount of fat tissue compared to all other groups. There were large highly vascularised regions of fat tissue interspersed between connective tissue. This tissue morphology was highly similar to that of native breast tissue (see FIG. 20). Furthermore, the relative adipose tissue area was also considerably higher in the deeper layers of this group compared to all other groups. These adipose tissue regions seemed to be better connected to each other and formed interconnected structures.

[0260] While no major signs of chronic inflammation were observed in the tissue sections or in the gross morphology of the constructs, non-specific localised low-grade granulomatose reactions were observed in the vicinity of the localised scaffold strands (FIG. 18). Lymphatic structures (FIG. 15, right panel) and leucocytes were also observed in all groups localised mainly near scaffold strands.

[0261] To identify the nature and composition of the connective tissue, Masson's trichrome staining was performed (FIG. 19A-F). In this staining, green colour indicates collagen fibres, red colour indicates muscle fibres and dark brown shows cell nuclei. As can be seen from the micrographs, besides the adipose tissue, a majority of the tissue filling the pores of the implant consisted of collagen fibres.

[0262] Thin layers of smooth muscle tissue were also observed, however it was only lining the boundaries of the scaffold strands. These smooth muscle layers had the highest thickness in case of the prevascularisation+lipoaspirate group (FIG. 19C).

[0263] In order to quantify adipose tissue regeneration, the total area of the adipose tissue relative to the total tissue area was counted on all slides (FIG. 19G). The negative control empty scaffold group had the lowest relative area of adipose tissue (8.31%8.94) which was significantly lower than in both the lipoaspirate-only (39.67%2.04) and the prevascularisation+lipoaspirate group (47.32%4.12) and also compared to native breast tissue (44.97%14.12) (p<0.05, p<0.01 and p<0.01 respectively). However, there was no statistically significant difference in relative adipose tissue area between the native breast tissue, lipoaspirate-only and prevascularisation+lipoaspirate group.

[0264] To quantify neovascularisation, blood vessels were counted on all slides (FIG. 19H). These blood vessels were identified by a ring/tubular structure, with only those lined with red blood cells included in the count as functional blood vessels. In general, all constructs, including the empty scaffold-only group, showed a substantial ingression of neovascularisation. The highest blood vessel density was observed in the prevascularisation+lipoaspirate group (38.01/mm.sup.22.02), however the density was not statistically significantly higher than the scaffold-only (33.13/mm.sup.212.03), lipoaspirate-only (26.67/mm.sup.21.6) or control breast tissue (35.45/mm.sup.21.93). H&E sections of constructs also showed blood vessels on and parallel to the surface of the constructs, suggesting that new capillaries are likely to have sprouted from these larger vessels that penetrated into the scaffolds.

[0265] Quantification of adipose cell area allowed the visualisation of the distribution of different-sized cells as a histogram (FIG. 19I). In all groups, the histograms were skewed to the right suggesting that a majority of adipose cell surface areas lay in the range of 100-700 m.sup.2. The distribution of the cell sizes in control breast tissue was considerably different compared to the other groupswith the highest percentage of cells in the 100-200, 300-400 and 500-600 m.sup.2 range. The empty scaffold and lipoaspirate-only groups had a low number of adipose cells having a surface area larger than 800 m.sup.2; whereas, the prevascularisation+lipoaspirate group showed a considerably higher number of cells having a surface area larger than 800 m.sup.2.

[0266] From data showing the percentage of adipose tissue area relative to total tissue area, the fold increase in adipose tissue volume was calculated (FIG. 19J, K). The prevascularisation+lipoaspirate group showed a higher fold increase in adipose tissue volume (6.10.62) compared to the lipoaspirate-only group (4.950.31); however, the difference was not statistically significant (p=0.143). Data for empty scaffold group has not been included because lipoaspirate was not injected into these scaffolds.

[0267] While cell-seeded anatomically shaped scaffolds are promising for the regeneration of complex, living tissue, they also lead to several disadvantages with problems ranging from scaling up of tissue culture to requiring complex GMP-approved laboratories for tissue culturing. The approach described in Example 1 circumvents such problems while scaling up the volumes of adipose tissue being regenerated by implanting an acellular scaffold and using the patient's body as a bioreactor. However, in the absence of a strong adipogenic stimulus, the scaffold gets filled with mostly non-specific fibrovascular tissue.

[0268] Here we have overcome the lack of adipogenic stimulus by injecting a small volume of lipoaspirate with no additional growth factors, cell transplantation or ligated vascular pedicles and introducing a completely novel prevascularisation technique that uses the patient's own body as a bioreactor and a source of blood vessels. Based on surgical expertise and the literature (Venkataram, 2008; Hanke et al., 1995; Gilliland and Coates, 1997; Housman et al., 2002), it was determined that 4 cm.sup.3 of adipose tissue is close to the maximum amount of fat that can be safely harvested from patients with low body fat. In terms of percentage, it represents 5.3% of total volume of the scaffold at the time of implantation.

[0269] The delayed lipo-injection technique allowed the formation of a bed of vascular and connective tissue within the scaffold volume. Such a vascular and connective tissue supports early adipogenesis, provided sufficient mesenchymal stem cells or adipose progenitor cells have been recruited to the implantation site. Consequently, in the study of Example 1 the adipose tissue, when injected into the already prevascularised scaffold, remained stably within the implantation sites with no tissue necrosis and resorption. Over a period of 24 weeks, the fold increase in adipose tissue volume was found to be 4.950.31 in case of lipoaspirate-only and 6.10.62 in case of prevascularisation+lipoaspirate group.

[0270] For aesthetic breast augmentation, it may be advantageous if the regenerated tissue consists mainly of adipose tissue with smaller amounts of organised connective tissue in order to maintain the natural tactile sensation of the breast. In case of post-mastectomy breast reconstruction, it may be advantageous if the regenerated tissue is mostly composed of highly organised connective tissue, if adipose progenitor cells infiltrating into the scaffold are suspected to stimulate breast cancer recurrence via HGF/c-Met signalling. The results of this study indicate that the morphology of the regenerated tissue can be reproducibly controlled depending on the initial scaffold treatment strategy (empty scaffold vs. prevascularisation+lipoaspirate)whereby empty scaffolds yield highly organised connective tissue whereas scaffolds containing lipoaspirate yield tissue rich in adipose tissue. In this way, scaffolds can truly be tailored for either an aesthetic augmentation procedure or a total reconstruction procedure.

[0271] Contrary to musculoskeletal systems, where tissue such as bone and muscle grow in response to mechanical forces, adipogenesis seems to be inhibited by mechanical forces. The scaffolds used in this study had a stiffness value that was 3 orders of magnitude higher than native breast tissue. By using mechanically robust scaffolds, a shielding effect can be exerted on the newly formed adipose tissue and the effects of the compressive, tensile and shear forces acting on the fat tissue can be reduced. This decreased mechanical stimuli can allow the cells to maintain a round morphology which, in turn, further promotes adipogenesis of the adipose progenitor cells (Nava et al., 2012).

[0272] As the skilled person is aware, the stiffness of the scaffolds may also be chosen dependent upon their placement. In case of most cosmetic augmentations whereby the implants are placed in a subglandular pocket, it is advantageous if the scaffold remains elastomeric and flexible so as to not cause patient discomfort; whereas in case of most post-mastectomy breast reconstruction procedures whereby the implants are placed in a submuscular pocket and no other supporting tissue remains, it is advantageous if relatively stiff implants are used in order to properly support the regeneration of the entire breast region (Vazquez et al., 1987).

[0273] Non-specific localised low-grade granulomatose reactions were observed in the vicinity of the localised scaffold strands. A granuloma is an organised collection of macrophages (Mukhopadhyay et al., 2012). While the roles of macrophages in angiogenesis are not yet completely understood, various research groups have shown that macrophages have the potential to contribute in angiogenesis. More specifically, M1 macrophages secrete VEGF which initiates the process of angiogenesis, M2a macrophages secrete PDGF-BB known to be involved in later stages of angiogenesis, while M2c macrophages secrete high levels of MMP-9 known to have a role in remodelling of vasculature. It has also been reported in the literature that macrophages can secrete alpha smooth muscle actin and can transdifferentiate into smooth muscle cells. All treatment groups examined showed accumulation of smooth muscle tissue around the scaffold strands (FIG. 19A-C) which indicates that macrophages may have played a role in angiogenesis and consequently higher adipogenesis in this group. Since the constructs were placed in PFA for an extended period of time post explantation, the proteins within the samples were denatured and immunohistology could therefore not be undertaken to provide direct evidence of this effect.

[0274] While no major outward signs of chronic inflammation were observed clinically or in the gross morphology of the constructs, lymphatic structures and leucocytes were detected in the histology of all treatment groupswhich is to be expected because the study used an immunocompetent animal model. Polycaprolactone has met FDA approval and been proven in multiple independent studies to be cytocompatible. The increased leucocyte count may be explained by the fact that during the lipoaspiration process, adipose cells may have formed non-viable aggregates in the syringe which, when injected into the scaffold, triggered an auto-immune reaction from the host aiming to break them down, ultimately leading to the ingression of lymphatic vessels.

[0275] Amongst others, Example 1 shows that the prevascularisation and delayed fat injection technique can be used for efficient regeneration of large volumes of adipose tissue for long periods of time. Thus, the approach combining delayed fat injection with a biodegradable scaffold can be used for long-standing regeneration of clinically relevant volumes of adipose tissue.

Example 2

[0276] Breast shaped scaffolds made of poly(D,L)-lactide polymer and containing voids and space-occupying structures essentially as shown in FIG. 21, scaled up to a scaffold volume of 125 cm.sup.3, were prepared. The scaffolds were fabricated with a 3D printer fitted with two extruders (one for printing poly(D,L)-lactide polymer for the scaffold structure, one for printing polylactic acid including a black dye for the space-occupying structures). Such a dual 3D print strategy also allows to prepare complex channel designs, if desired (e.g. a radially convergent design). Since the space-occupying structures are made of solid material, they are not degraded as quickly as the scaffold and thus are capable of preventing tissue/cell invasion within the rather short period of prevascularization in this example. With space-occupying structures made of an undegradable material, prevention of tissue/cell invasion is even better.

[0277] With such scaffolds, a pilot study was undertaken whereby n=6 scaffolds (volume=125 cm.sup.3 each) were implanted in immunocompetent minipigs.

[0278] After 2 weeks of prevascularisation, the surgeon used a commonly used biopsy punch (FIG. 21 B) to remove the space-occupying structures. Adipose tissue was injected into the generated void spaces.

[0279] Upon explantation (24 weeks after implantation), it was observed that the scaffolds had been well integrated with the surrounding tissue and that there was a widespread invasion of host vasculature into the constructs. Visual examination revealed that the overall shape of the scaffolds did not change drastically over the implantation period. Histological evaluation showed large areas of fat and vascularisation at and around the sites where adipose tissue had been injected into the void spaces on all scaffolds (see FIG. 22).

Tables

[0280]

TABLE-US-00001 TABLE 1 Physical and mechanical properties of the scaffolds used in Example 1. Elastic Scaffold Modulus Porosity Volume Pore size [MPa] [%] [mm.sup.3] [mm] 21.5 2.2 79.9 1.56 75 10.sup.3 0.46

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