System and method for assembling tissue grafts

Abstract

To provide a system and a device for assembling a plurality of tissue grafts.SOLUTION: This method includes the steps of collecting a plurality of minute tissue grafts from a donor site, arranging the plurality of minute tissue grafts in a desired orientation, forming a tissue construction object including the plurality of minute tissue grafts arranged in the desired orientation, and applying the tissue construction object to a recipient site.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims priority to U.S. Provisional Patent Application No. 62 / 414,405, filed Oct. 28, 2016, and U.S. Provisional Patent Application No. 62 / 478,207, filed Mar. 29, 2017, which are hereby incorporated herein by reference.

[0002] (Statement Regarding Federally Sponsored Research) This invention was made with government support under W81XWH - 13 - 2 - 0054 awarded by the Department of Defense. The government has certain rights in this invention.

Background Art

[0003] An autograft can refer to tissue transplanted from one part of an individual's body (e.g., a donor site) to another part (e.g., a recipient site). Autografts can be used, for example, to replace lost skin and other tissues and / or to facilitate healing from trauma, wounds, burns, surgery, and congenital defects. Generally, the transplantation procedure can be limited by the amount of tissue that can be removed from the donor site without causing excessive adverse effects. More specifically, the availability of tissue for autografting can be limited by the total area of tissue required, the healing behavior of the donor site, the similarity between the donor site and the recipient site, aesthetic considerations, and / or other characteristics of the candidate donor site and / or recipient site.

[0004] A sheet graft is a type of autograft that refers to a piece of tissue removed or harvested from an undamaged donor site. For example, a sheet graft can be obtained using an instrument designed to gently shave a piece of tissue from the skin at the donor site. The size of the donor skin piece used for the graft is approximately the same size as the damaged recipient site, slightly larger than the recipient site (to account for potential shrinkage of the graft tissue after harvesting), or smaller than the recipient site (by using a graft that can be expanded in a reticular manner, for example). Once harvested, the sheet graft can be applied to cover the wound at the recipient site, stapled or otherwise secured in place, and allowed to heal.

[0005] Sheet grafts can be full-thickness or segmented-thickness. For example, conventional segmented-thickness grafts can be formed by harvesting a sheet of epidermal and upper dermal tissue from a donor site, while full-thickness skin grafts can be formed using a sheet of tissue containing the entire epidermal layer and dermal components of variable thickness. The type of sheet graft used can affect healing at both the donor and recipient sites.

[0006] For example, in conventional split-thickness grafts, skin tissue may grow at the donor site through a process similar to that of healing a second-degree burn. Therefore, split-thickness grafts may be preferable to full-thickness grafts because the donor site can recover at least partially on its own, despite often being associated with scarring, pain, and other long-term side effects. However, skin tissue removed from a donor site for split-thickness skin autografts generally consists only of a thin epithelial layer, which may lack certain elements of the dermis that, upon healing, improve structural stability and normal appearance at the recipient site.

[0007] With conventional full-thickness grafts, more of the characteristics of normal skin, such as color, texture, and thickness, can be maintained at the recipient site after the transplantation procedure (i.e., the dermal components are (For example, these can be preserved in grafts.) Full-thickness grafts may contain more collagen, dermal vascular plexuses, and epithelial appendages compared to split-thickness grafts. Full-thickness grafts may also shrink less during healing. These properties can be important in more visible skin areas such as the face and hands. In addition, hair is more likely to grow from full-thickness grafts than from split-thickness grafts, and sweat glands and sebaceous glands are more likely to regenerate in full-thickness grafts than in split-thickness grafts, taking into account the sweating properties of the recipient site.

[0008] While full-thickness grafts can offer improved tissue quality at the recipient site, the donor site is completely sacrificed because there is no remaining dermis for skin regeneration. Therefore, the availability of potential donor sites is very limited, and donor sites for full-thickness grafts must be surgically closed. Furthermore, full-thickness grafts require more precise conditions for survival due to the large volume of tissue that necessitates vascular regeneration. Thus, conventional full-thickness skin grafts are generally limited to relatively small, uncontaminated wounds with good angiogenesis, and are not as suitable for as many types of grafting as segmented-thickness grafts. [Overview of the project] [Problems that the invention aims to solve]

[0009] In light of the above, it is desirable to provide a system and method for tissue harvesting and transplantation that provides efficient graft tissue with minimal donor site scarring while also appropriately replicating the microanatomical structure of normal tissue at the recipient site. Furthermore, it is desirable that such a system and method be scalable for use with recipient sites of various sizes and shapes. [Means for solving the problem]

[0010] The systems and methods of the present disclosure overcome the above and other drawbacks by providing partial tissue grafts in the form of full-thickness microtissue columns that maintain a desired orientation of individual tissue columns, such as a substantially vertical epidermal-dermal orientation, like substantial tissue columns. Multiple solid tissue constructs can be used as expandable building blocks arranged in a line to appropriately fit a wound of a desired size and geometric shape.

[0011] According to one aspect of the present disclosure, a method for assembling a plurality of microtissue grafts is provided. The method comprises the steps of: harvesting a plurality of microtissue grafts from a donor site; arranging the plurality of microtissue grafts in a desired orientation; forming a tissue construct comprising the plurality of microtissue grafts arranged in the desired orientation; and applying the tissue construct to a recipient site.

[0012] Another aspect of the present disclosure provides a method for assembling a plurality of microtissue grafts. The method comprises placing an apparatus having an array of needles, a matrix having holes corresponding to each of the needles, and a mesh on the matrix, over a donor site. The method also comprises applying a vacuum over the apparatus to draw the plurality of microtissue grafts through the array of needles into the holes of the matrix and causing the mesh to capture the plurality of microtissue grafts in the holes; removing the matrix from the mesh and the array of needles to form a tissue construct containing the plurality of microtissue grafts within the matrix; and applying the tissue construct to a recipient site.

[0013] Another aspect of this disclosure provides an apparatus for assembling multiple microtissue grafts into a tissue construct. The apparatus comprises an array of needles, a matrix, and a mesh. Each needle in the array is sized to harvest a respective microtissue graft from a donor site, and the matrix is ​​placed on top of the array of needles. The device is provided with holes configured to receive microtissue grafts. Furthermore, a mesh is placed on the matrix. The mesh is sized such that an applied vacuum allows air to pass through so that the microtissue grafts can be pulled into the matrix through the needle, and is also sized to accommodate the microtissue grafts within the matrix.

[0014] The above and other advantages of the present invention will become apparent from the following description. The description refers to the accompanying drawings which constitute part of this specification, and the drawings illustrate preferred embodiments of the present invention as examples. Such embodiments do not necessarily represent the entire scope of the present invention, and therefore refer to the claims and this specification to interpret the scope of the present invention. [Brief explanation of the drawing]

[0015] [Figure 1] This flowchart shows a method for organizing and assembling tissue grafts. [Figure 2] This figure shows a hydrophobic coating technique for orienting tissue grafts. [Figure 3] This figure shows hydrophobic coating and orbital motion techniques for orienting tissue grafts. [Figure 4A-F] Figures 4A-D show the tissue grafts in the solution at each step of the technique shown in Figure 3, and are top views of the tissue grafts. Figure 4E shows the tissue grafts in the solution at each step of the technique shown in Figure 3. Figure 4F shows the tissue grafts in the solution at each step of the technique shown in Figure 3, and is an isometric projection of the tissue grafts. [Figure 5] This figure shows a magnetic coating technique for orienting tissue grafts. [Figure 6A-C]FIG. 6A shows a rotation technique for orienting tissue grafts and is a side view of a support material wound with tissue grafts spaced apart. FIG. 6B shows a rotation technique for orienting tissue grafts and is a side view of a roll-shaped tissue construct including the support material and the oriented tissue grafts. FIG. 6C shows a rotation technique for orienting tissue grafts and is a top view of the roll-shaped tissue construct of FIG. 6B. [Figure 7] A chart showing the percentage of correctly aligned tissue grafts for each case assembled according to different orientation techniques. [Figure 8A] Top and bottom views of a tissue graft having a stained epidermal layer, showing the tissue graft in solution oriented according to the hydrophobic coating and orbital motion technique of FIG. 3. [Figure 8B] Top and bottom views of a tissue graft having a stained epidermal layer, showing an untreated tissue graft. [Figure 8C] Top and bottom views of a tissue graft having a stained epidermal layer, showing the tissue graft in solution oriented according to the orbital motion technique. [Figure 9A-C] Shows each step of a combination of tissue harvesting and orientation techniques using an apparatus comprising a needle array, a matrix, and a mesh cover. [Figure 10A-C] Shows each step of a combination of tissue harvesting and orientation techniques using an apparatus comprising a needle array, a two-layer matrix, and a mesh cover. [Figure 11A-B] FIG. 11A shows a top view of tissue grafts in a construct. FIG. 11B shows a side view of tissue grafts in a construct. [Figure 12A-B] FIG. 12A is a top view showing a structure including oriented tissue grafts and is a view showing an assembled construct. FIG. 12B is a top view showing a structure including oriented tissue grafts and is a view showing the construct applied to a wound. [Figure 13A-C]Figure 13A shows a top view of a wound healed by secondary intention, showing the wound at time zero. Figure 13B shows a top view of a wound healed by secondary intention, showing the wound 2 weeks after time zero. Figure 13C shows a top view of a wound healed by secondary intention, showing the wound 6 weeks after time zero. [Figure 14A-C] Figure 14A shows a plan view of a wound healed by randomly oriented microtissue grafts, showing the wound at time zero. Figure 14B shows a plan view of a wound healed by randomly oriented microtissue grafts, showing the wound 2 weeks after time zero. Figure 14C shows a plan view of a wound healed by randomly oriented microtissue grafts, showing the wound 6 weeks after time zero. [Figure 15A-F] Figure 15A shows a top view of a wound healed by microtissue grafts assembled in an epidermis-dermis orientation, showing the wound at time zero. Figure 15B shows a top view of a wound healed by microtissue grafts assembled in an epidermis-dermis orientation, showing the wound 1 week after time zero. Figure 15C shows a top view of a wound healed by microtissue grafts assembled in an epidermis-dermis orientation, showing the wound 3 weeks after time zero. Figure 15D shows a top view of a wound healed by microtissue grafts assembled in an epidermis-dermis orientation, showing the wound 4 weeks after time zero. Figure 15E shows a top view of a wound healed by microtissue grafts assembled in an epidermis-dermis orientation, showing the wound 6 weeks after time zero. Figure 15F shows a top view of a wound healed by microtissue grafts assembled in an epidermis-dermis orientation, showing the wound 8 weeks after time zero. **DETAILED DESCRIPTION OF THE INVENTION**

[0016] The present disclosure provides a system and method for organizing and assembling tissue grafts. More specifically, the system and method enable the assembly of a plurality of microtissue grafts in the form of biological microtissue columns into a larger tissue construct while maintaining the desired orientation of individual tissue columns.

[0017] For example, full-thickness skin tissue can be harvested from a donor site in the form of small columns (e.g., several hundred micrometers in diameter) without causing scarring at the donor site. These microtissue columns can be applied to the wound bed as "random" partial grafts to improve wound healing. However, since skin is structurally naturally polarized, transplanting microtissue columns to the wound bed as arrays with appropriate epidermal-dermal orientation can further improve healing by promoting re-epithelialization, recreating normal dermal structure, and reducing scarring. Thus, the methods and systems disclosed herein facilitate the orientation of microtissue columns and enable their assembly into three-dimensional full-thickness structures. The systems and methods also provide a practical and scalable solution for using a large number of microtissue columns of various sizes and shapes to improve wound healing.

[0018] Figure 1 shows a method 10 for assembling microtissue columns (MTCs) according to this disclosure. Generally, as shown in Figure 1, MTCs are collected from a donor site in step 12. In step 14, some or all of the MTCs S The MTCs are positioned in the desired orientation (for example, matching the epidermal-dermal polarity of normal skin). Then, in step 16, the oriented MTCs are applied to the recipient site. Note that the term microtissue columns, i.e., MTCs, used herein may be interchangeable with microtissue grafts or micrografts. Furthermore, when the tissue of interest is skin, MTCs may be called microcutaneous tissue columns (MSTCs).

[0019] Referring here to step 12, MTCs can be harvested from a donor site. More specifically, MTCs can be formed by removing an elongated, substantially cylindrical portion of tissue from a donor site, thereby leaving a pore in it. In some embodiments, the diameter or width of the MTC (microtissue column) is about 2 millimeters. The diameter or width may be less than 1 mm or about 1 mm. In some embodiments, the diameter or width may be less than about 0.5 mm, less than about 0.3 mm, or about 0.2 mm. In further embodiments, the diameter or width may be between about 0.8 mm and 0.3 mm. In other embodiments, the diameter or width may be between about 0.7 mm and 0.2 mm.

[0020] Each MTC can be a full-thickness graft containing both epidermal and dermal tissue from the donor site. Generally, it is best to avoid a significant amount of subcutaneous or muscular tissue while using epidermal and dermal tissue. It may be preferable to harvest MTCs from cutaneous tissue (however, in some applications, MTCs may include subcutaneous and / or muscle tissue). For example, each MTC can be approximately 3 mm in height, which can correspond to the entire depth of a typical skin layer (e.g., including both the epidermis and dermis, with the dermis containing hair follicles and sweat glands or sebaceous glands). Different heights can be used, such as between approximately 2 mm and 4 mm, based on the specific skin or tissue characteristics of the donor site. Furthermore, MTCs may contain stem cells throughout the dermal tissue (e.g., stem cells associated with hair follicles and sweat glands and / or stem cells in the lower part of the dermis, e.g., near the dermal / adipose tissue boundary).

[0021] Generally, MTCs can be harvested from donor sites in a way that minimizes or prevents scarring at the donor site. For example, the size of the pores in the donor site produced by each MTC can be selected so that the resulting minor damage heals quickly and / or without scarring. More specifically, the pores in each donor site can be small enough to heal quickly by regeneration; that is, the harvested tissue volume is replaced with new skin tissue that is normal in both structure and function, with little to no scarring. Furthermore, the size of the pores in the donor site formed by MTCs can be selected based on the formation of tissue portions that are small enough to promote viability when transplanted or placed in a growth medium, and large enough to form a sufficient amount of transplanted tissue and / or capture any tissue structures that may be present in the donor tissue.

[0022] In some embodiments, the portion of surface tissue removed from the donor site (which can correspond to the percentage of the donor site's surface area occupied by the pores) may be less than about 70%, less than or equal to about 50%, or more preferably about 10% to about 30%. The percentage of tissue removed is large enough to provide sufficient harvested MTCs to form a graft of appropriate size, but small enough to promote rapid healing at the donor site based on growth from the remaining undamaged tissue. Depending on factors such as the specific characteristics of the donor site, the required graft size, and the total amount of donor site tissue available, other percentages of tissue may be removed from the donor site.

[0023] According to some embodiments, MTCs can be collected using one or more harvesting needles, such as a 19-gauge coring needle. Furthermore, in some embodiments, MTCs can be collected using one or more double-pointed subcutaneous injection needles. However, different types or sizes of needles, individually or in arrays, may be contemplated within the scope of this disclosure. For example, MTCs can be collected using any of the tools and methods described in U.S. Patent No. 9,060,803, the entire contents of which are incorporated herein by reference.

[0024] As a result of step 12, a fractional skin graft containing multiple harvested MTCs is obtained. As described above, rather than a single large donor site wound, the fractional skin graft technique described above creates a small donor site wound that can heal with minimal scarring. Furthermore, in some embodiments, step 12 may include a step of pre-treating the donor site before harvesting the MTCs to assist in the orientation of the MTCs in step 14, as will be further described below.

[0025] Referring here to step 14, the harvested MTCs are assembled in the desired orientation, for example, to match the epidermal-dermal polarity of normal skin. More specifically, in step 14, the MTCs may be assembled into a three-dimensional full-thickness construct that maintains a substantially vertical orientation of the appropriate epidermal-dermal structure. In step 14, all or most of the columns are assembled spontaneously (e.g., by a hydrophobic coating floating on an aqueous medium) and / or using external factors (e.g., by an external magnet that orients the coating along magnetic field lines). This can be achieved by a self-assembly approach, which involves coating the surface of each tissue column with a material that induces organization into the desired orientation (by controlled stirring or fluid flow). Additionally or alternatively, supporting biomaterials can help maintain the overall structure and desired orientation of the assembled tissue columns and form a structure. These supporting materials can be applied in various ways, for example, by being initially introduced in liquid form and then induced to solidify around the assembled tissue columns, or by being used in solid form and combined with the tissue columns in layers or rolls.

[0026] Therefore, in some embodiments, a coating is used to orient the MTCs. For example, the surface of the donor site is covered with a coating before graft harvesting in step 12. The coating can be hydrophobic, hydrophilic, or any type of coating that exhibits phase separation in solution. The coating can be non-toxic and / or biologically inert, and in some applications, silicone-based. Once coated, MTCs can be extracted and immersed in a solution that aligns them in the epidermal-dermal direction. More specifically, due to the properties of the coating, the coated epidermis of some or all MTCs spontaneously aligns in the solution, orienting itself toward the top of the solution.

[0027] Figure 2 shows an exemplary coating technique 20. As shown in Figure 2, in step 22, a donor site 24 can be selected. In step 26, a hydrophobic coating 28, such as petrolatum or another suitable coating, can be applied to the surface 30 of the donor site 24 (for example, to cover the epidermal layer 32 of the donor site). In step 33, MTCs 34 are harvested from the donor site 24 according to step 12 above. For example, the MTCs 34 may be full-thickness grafts including the epidermal layer 32 as well as the dermal layer 36, and optionally a portion of the dermal / fatty layer boundary 38. In step 40, the MTCs 34 are placed in a solution 42 (e.g., a well plate). Due to the hydrophobicity of the coating 28, the coated epidermis 32 of some or all of the MTCs 34 generally align vertically in the solution 42 in an epidermal-dermal orientation. In step 44, the solution 42 (or a different solution) is induced to solidify around the assembled MTCs 34 to create a construct 48 of the oriented MTCs 34.

[0028] In some embodiments, solution 42 may be saline solution or another suitable solution such as a biocompatible and / or biodegradable polymer that can solidify after a certain period of time (e.g., polymers may solidify for a certain period after mixing) or in response to induction (e.g., by the application of a crosslinking agent). Furthermore, in some embodiments, a different solution may be used in step 44. For example, this other solution may be a supporting biomaterial such as a biocompatible matrix or collagen solution that can solidify after incubation. The coating 28 may be washed off after alignment in some applications, but may not be necessary in others. (For example, the coating 28 may remain on the donor site 24 after wound application and be able to peel off during the natural metabolic turnover of the epidermis).

[0029] In some embodiments, the above coating technique may be combined with a stirring step. For example, agitation may stir MTCs that may have settled in the solution. This helps to stir the MTCs, increasing their chances of floating on the fluid surface. Once they reach the fluid surface, the hydrophobic coating keeps the MTCs in the desired orientation. Furthermore, the oscillating motion increases the chances that floating MTCs are close enough to cluster together (i.e., due to the effect of surface tension around small floating particles, also known as the "Cheerios effect").

[0030] For example, Figure 3 shows the coating and orbital motion technology 50. As shown in Figure 3, in steps 52 and 54, donor sites 24 are selected and hydrophobic coatings 28 are applied. The coated MTCs 34 are collected in step 56. Steps 52-56 in Figure 3 are substantially equivalent to steps 22, 26, and 33 in Figure 2. However, following step 56, in step 58, the collected MTCs 34 can be immersed in solution 42 and oscillated to enhance the clustering of the MTCs 34 in each other's directions (e.g., towards the center of the well plate). Such oscillation can be achieved, for example, by adding orbital motion using an orbital shaker (not shown). In one particular application, oscillation can be achieved using an orbital shaker at 150 revolutions per minute (RPM) for about 30 seconds. However, other applications use different orbital shaker parameters. Furthermore, in step 60, the epidermal-dermal oriented MTCs 34 can be positioned on the supporting biocompatible material 46. For example, the epidermal-dermal oriented MTCs can be transferred to a new culture plate containing the supporting biocompatible material 46, such as a liquid collagen solution or other biocompatible matrix, and then orbited again (e.g., using an orbital shaker at 200 RPM for about 30 seconds, or with other RPM and timing parameters). In step 62, the collagen solution 46 containing the appropriately oriented MTCs 34 can be induced to form a solid structure 48. For example, in one application, the collagen solution 46 can be incubated at 37°C for about 45 minutes to form a solid structure 48.

[0031] Figures 4A to 4F show MTCs34 using the above technique in Figure 3, where donor The epidermis 32 of the Ito is stained with ink before MTC harvesting to indicate orientation. Figure 4A shows MTCs 34 suspended in solution 42 in a well plate 64, coated with a hydrophobic coating, with several epidermal layers 32 facing upward (corresponding, e.g., to step 40 above). Figure 4B shows the MTCs 34 (indicated by their stained epidermal layers 32) gathering toward the center of the well plate 64 after orbital motion has been applied (corresponding, e.g., to step 58 above). Figures 4C and 4D show the MTCs 34 before and after orbital motion, transferred to a new culture plate 66 in collagen solution 46 (corresponding, e.g., to step 60 above), respectively. Thus, Figure 4D shows, This shows that the MTCs34 are clustered toward the center of the well plate 66 after orbital motion is applied. Figures 4E and 4F show a top view and an isometric view, respectively, of the MTCs34 correctly oriented within the coagulated partial skin graft construct 48 (corresponding, for example, to step 62 above, in which the collagen solution 46 is induced to coagulate).

[0032] In some embodiments, the orbital motion described above can be used to orient the MTCs, while in other embodiments, other types of agitation or fluid flow can be used. For example, in one embodiment, harvested MTCs are delivered from the harvesting needle through a microfluidic channel or a flow channel having a tapered shape (not shown) to maintain their epidermal-dermal orientation from the extraction. The channel may also be oriented in a manner that facilitates closer grouping of the MTCs. That is, the channel may be oriented to reduce the spacing between MTCs compared to their original spacing when extracted from the donor site. From these channels, the epidermal-dermal-oriented MTCs can be transferred to a culture plate containing a biocompatible matrix (such as a collagen solution) and incubated to form a solid construct. In some embodiments, these additional agitation and fluid flow examples can also be combined with any of the coating techniques described herein.

[0033] Furthermore, in other embodiments, magnetic or ferromagnetic coatings are used to orient the MTCs. In this example, the surface of the donor site can be coated with magnetic paint or iron oxide particles, etc., before graft harvesting. The MTCs are then extracted and immersed in a solution (such as saline solution, biocompatible matrix, collagen solution, or other supporting biomaterial). The MTCs can then be oriented in the solution using an external magnet. Due to the magnetic properties of the coating, the coated epidermis of some or all of the MTCs is aligned. The magnets align themselves according to the magnetic field lines generated by the magnets, and therefore orient themselves towards the top of the solution. Thus, the patterning of MTCs can be controlled with great precision using external magnets. Furthermore, in some applications, for example, an array of magnets (i.e., rather than a single magnet) can be used to create areas of MTCs with different patterns or densities within the same tissue structure.

[0034] Figure 5 shows an exemplary partial covering technique 68. As shown in Figure 5, in step 70, a donor site 24 can be selected. In step 72, an adhesive coating 74, such as ostomy adhesive or other suitable adhesive, can be applied to the surface 30 of the donor site 24. In step 76, iron oxide particles 78 are applied to the adhesive. In step 80, an additional coating 82, such as a spray bandage, is applied on top of the iron oxide particles 78. Although not shown in Figure 5, following step 80, MTCs can be taken from the donor site (e.g., as described above according to step 12) and placed in a solution. An external magnet can then be placed on the solution so that some or all of the MTCs are vertically aligned in the solution in an epidermal-dermal orientation. That is, due to the magnetic properties of the coating, the coated epidermis of some or all of the MTCs align according to the magnetic field lines generated by the magnet and orient themselves toward the top of the solution. The solution is then allowed to solidify around the assembled MTCs to create a construct of oriented MTCs.

[0035] As described above, supporting biomaterials (such as collagen solutions or biocompatible matrices) are used to orient MTCs and / or maintain MTC orientation within the construct. More specifically, the aforementioned supporting materials can be used to create constructs that maintain the overall structure and orientation of assembled tissue columns. As a result, these constructs create grafts that are easier to handle and, in some applications, may allow physicians to add drugs, other components, or other cell types as needed.

[0036] Therefore, consistent with the above techniques, MTCs can be introduced into a support material in liquid form and then induced to solidify around the tissue column (e.g., by incubation or other suitable techniques).

[0037] However, in other embodiments, the supporting biocompatible material may be used in solid form and combined with MTCs in layers or rolls. For example, the supporting material may be used with a rolling technique that preserves the orientation of the MTCs. More specifically, as shown in Figure 6A, the supporting material 84 (such as a matrix or other type of biomaterial strip) can be rolled up with oriented MTCs 86 spaced apart on the material 84. This rolling technique can result in a construct 88 having a jelly roll arrangement, as shown in Figures 6B and 6C. The size of the construct 88 can be smaller or larger (e.g., by less or more rolling) according to the diameter and / or shape of the desired wound. In some embodiments, a rolling device (not shown) is used to support the supporting material 84 substantially vertically, while the rolling device rolls up the supporting material 84, allowing an operator to place the MTCs 86 on the supporting material 84 at a predetermined distance from each other. Alternatively, a pick-and-place gantry machine (not shown) can be used to automatically position the MTC86 against vertically positioned strips of matrix material 84 as the MTC86 rolls onto it.

[0038] The above example includes the step of fabricating a structure having MTCs in a support material, but in some embodiments, the structure includes MTCs formed together (in a desired orientation) in a different way. Thus, these structures have MTCs that are properly oriented but dispersed amongst the MTCs. This may include MTCs that are not supported by a causative material. Therefore, in some embodiments, the solid structure may be formed by a material or tool that positions and orientations the MTCs by contacting or communicating with the upper surface of the MTCs. For example, after orienting the MTCs, an adhesive bandage can be applied to the epidermal surface to "pick up" all the oriented MTCs as a solid structure. In another example, the MTCs can be coated with a magnetic layer as described above, and then all the oriented MTCs can be picked up as a solid structure using a magnet. In these applications, once the oriented MTCs have been picked up and the structure has been formed, the structure can be applied directly to the recipient site (as further described below with respect to step 16).

[0039] In some embodiments, one or more of the above examples can be combined or replaced entirely or partially to orient MTCs. In some applications, combining techniques can increase the amount of properly oriented MTCs. For example, Figure 7 provides a chart 90 showing the percentage of properly aligned MTCs: when assembled using hydrophobic coating and orbital motion 92, about 80% to 90% were properly aligned; with hydrophobic coating alone 94, about 55% to 65% were properly aligned; with iron oxide and external magnet 96, about 35% were properly aligned; with magnetic paint and external magnet 98, about 30% to 40% were properly aligned; and with mineral oil interface 100, about 10% to 15% were properly aligned. The mineral oil interface contained a mixture of an aqueous fluid (e.g., ordinary saline solution) and an organic fluid (e.g., mineral oil). As the mixture separates into layers (or phases), this interface can orient MTCs accordingly. This is because MTCs typically consist of a mostly hydrophilic portion (i.e., the dermis) sandwiched between two hydrophobic portions (i.e., the epidermis at one end and the subcutaneous fat at the other).

[0040] As shown in Figure 7, the use of hydrophobic coating and orbital motion (according to the technique in Figure 3) significantly increases the proportion of properly aligned MTCs compared to the other methods illustrated. To further illustrate these results, Figure 8A shows top view 102 and bottom view 104 of a culture plate containing MTCs 106 with stained epidermis, treated with hydrophobic coating and orbital motion. Figure 8B shows a culture plate containing untreated MTCs 106 (i.e., hydrophobic). Figure 8C shows top and bottom views 108 and 110 of a culture plate (without hydrophobic coating or orbital motion), and Figure 8C shows top and bottom views 112 and 114 of a culture plate containing MTCs 106 treated with orbital motion only. As shown in Figures 8A–8C, a considerable number of MTCs 106 treated with hydrophobic coating and orbital motion are oriented with their epidermis facing upward (as shown by multiple stained epidermis in the top view of Figure 8A) compared to the MTCs 106 shown in Figures 8B and 8C. However, as stated above, the techniques disclosed herein are not mutually exclusive, and one or more techniques can be combined or replaced entirely or partially to further increase the total percentage of properly aligned MTCs and / or achieve desired properties. For example, in one application, magnetic particles can be applied to a hydrophobic coating to provide the ability to create a large number of properly aligned MTCs (i.e., by hydrophobic coating techniques) as well as a precise pattern of MTCs (i.e., by using magnetic techniques).

[0041] The above technique orients the MTCs spontaneously and / or using external factors after they have been collected in step 12. However, in some embodiments, steps 12 and 14 may be combined so that the collection and orientation of the MTCs are completed in a single step. For example, as shown in Figure 9A, the collection and assembly apparatus 116 may comprise a coring needle 118 array, a pre-formed matrix 120, and a mesh material 122. The coring needle 118 array can be sized and positioned to collect MTCs from a donor site, and the matrix 120 may be positioned on the coring needle 118 array and may include a plurality of holes 121, each hole corresponding to a respective coring needle 118 It is aligned and has a diameter approximately equal to the inner diameter of the coring needle 118. The mesh material 122 can be positioned to cover the matrix 120, for example, to act as a cover that covers the matrix holes 121 while allowing suction through the matrix 120.

[0042] During operation, as shown in Figure 9A, the coring needle 118 is positioned within the donor site tissue and pulls the MTCs 126 through the coring needle 118 into the matrix 120. A vacuum 124 is applied over the mesh material 122. As shown in Figure 9B, the mesh material 122 can trap the MTCs 126 within the matrix 120 while still allowing the vacuum to pass through the mesh material 122. As a result, the MTCs 126 remain within the matrix 120 and are correctly aligned in an epidermal-dermal orientation. Then, as shown in Figure 9C, the matrix 120 can be removed from the coring needle array 118 and the mesh material 122, resulting in a tissue construct 128. In other words The matrix material 120 acts as a supporting biomaterial that maintains the overall structure and desired orientation of the assembled MTCs 126.

[0043] Therefore, the matrix 120 may be biocompatible, and as a result, the entire matrix structure 128 may be placed directly within the wound (according to step 16, as further described below). Examples of biocompatible matrices include decellularized tissues (e.g., skin, intestines, amniotic membrane, or other tissues processed to remove all living cells, so the remainder of the original tissue is extracellular components), various forms of natural biomolecules (collagen, fibrin, hyaluronan, etc., used alone or in combination) (spun into gels or fibers), and synthetic materials that are biodegradable and have specific biomimetic properties (e.g., functionalized at the cell adhesion site). Examples include, but are not limited to, biodegradable polymers and matrices containing collagen, hydrogels, fibrin gels, or carbon scaffolds. Furthermore, any of the above examples may contain growth factors and / or oxygen concentration enhancing materials (e.g., CaO2) and / or other substances.

[0044] Furthermore, in some embodiments, as shown in Figures 10A to 10C, the sampling and assembly apparatus 130 may include a two-layer matrix 132 comprising an upper layer 134 and a lower layer 136. In such embodiments, as shown in Figure 10A, a suction force can be applied to draw the MTCs 126 from the donor site into the two-layer matrix 132. As a result, the MTCs 126 are trapped within the two-layer matrix 132, as shown in Figure 10B. The needle array 118, the mesh material 122, and one of the matrix layers, such as the lower layer 136, can then be removed. Consequently, the MTCs 126 remain within the upper layer 134 with their proper epidermal-dermal orientation, with the lower end of each dermis exposed, forming a structure 138 as shown in Figure 10C. When applied to a wound (as further described below), this type of structure allows the exposed dermal layer to come into direct contact with the wound bed, which increases the likelihood of good re-establishment of blood flow from the wound bed to the MTCs (which can be important for long-term tissue survival).

[0045] Referring again to the method in Figure 2, once the MTCs have been harvested and oriented in steps 12 and 14 according to any of the techniques described above, they are applied to the recipient site (e.g., a wound) in step 16. More specifically, following steps 12 and 14, one or more three-dimensional full-thickness constructs 48 (or 88, 128, 138) are available for wound healing, and as shown in Figures 11A and 11B, these constructs contain substantially vertical epidermal-dermal oriented MTCs 34 (or 86, 106, 126). These constructs are three-dimensional because they have usable width, length, and height, and are full-thickness because they contain epidermal and dermal layers 32, 36 (as shown in Figures 11A and 11B). In some embodiments, the construct 48 may be circular, as shown in Figure 12A. However, in other embodiments, the structure may be rectangular, square, or other suitable shape.

[0046] According to step 16, the MTCs 34 can be placed in or on the wound to completely or at least partially cover the wound. In some embodiments, as shown in Figure 12B, a single construct 48 may completely cover the wound 140 at the recipient site 142. In other embodiments, multiple constructs, each containing multiple MTCs, can be arranged side by side to conform to the geometric shape of the wound. For example, a single MTC roll construct 88 (formed, e.g., by the rolling technique described above) can conform to the geometric shape of the wound. Alternatively, multiple MTC roll constructs 88 can be arranged side by side to conform to the shape of the wound. Thus, the method may be scalable to accommodate use in large and / or asymmetrical wounds by providing one or more solid constructs, each formed of multiple MTCs and arranged side by side at the recipient site.

[0047] In light of the above, the method of the present invention enables the assembly of multiple MTCs into a solid three-dimensional tissue construct in a desired orientation. Furthermore, one or more systems can be provided to carry out the above method completely or partially. When such a construct is applied to a recipient site, full-thickness MTCs grow to complete the sweat glands and other complex features of the harvested tissue. Thus, these MTCs can be used to aid and improve tissue healing at a recipient site (such as a wound). More specifically, properly oriented MTCs can improve treatment by promoting re-epithelialization, recreating normal skin structure, and / or reducing scarring compared to untreated wounds and treated wounds treated with randomly oriented MTCs.

[0048] In particular, while harvested MTCs can be applied randomly to the wound bed, i.e., without maintaining the normal epidermal-dermal polarity of the skin, MTCs organized with a clear epidermal-dermal orientation may be advantageous in promoting wound healing by providing more efficient cell and tissue proliferation as well as more faithful replication of the fine anatomical structure of normal tissue (for example, complex structures in full-thickness tissue grafts, such as hair follicles, have a fixed polarity and are generally less tolerant to being transplanted in a mis-oriented manner). Thus, while randomly oriented MTCs have been shown to improve healing compared to untreated wounds (e.g., by healing more quickly with less contraction), MTCs assembled and oriented according to the above systems and methods further improve healing time, contraction response, skin appearance, and / or structural properties. This configuration can be improved.

[0049] For example, Figures 13A to 13C show the results at 0, 2 weeks, and 6 weeks, respectively. Figures 14A to 14C show the typical secondary intentional healing process of an untreated skin wound 144. Figures 14A to 14C show the healing process of a skin wound 146 treated with randomly oriented MTCs 147 at 0, 2 weeks, and 6 weeks, respectively. As shown in Figure 13C, after 6 weeks, the untreated wound 144 healed slowly, mainly by contraction, with a portion of the wound 144 148 still open. On the other hand, as shown in Figure 14C, after 6 weeks, the wound 146 treated with randomly oriented MTCs closed and healed completely, healing faster and with less contraction than the untreated wound 144.

[0050] Compared to randomly oriented MTCs, MTC constructs positioned in an epidermal-dermal orientation can provide faster healing times with less contraction response and result in healing wounds that better match normal tissue coloration and structure (for example, it better matches the appearance and structure of the tissue surrounding the recipient site). For example, Figures 15A to 15F illustrate the above. According to the method (for example, using a properly oriented solid structure of MTCs152) The healing process of skin wounds 150 treated with appropriately oriented MTCs 152 at time points 0, 1 week, 3 weeks, 4 weeks, 6 weeks, and 8 weeks, respectively, is shown. In general, wounds reconstructed with appropriately oriented MTCs tend to appear more oval or round (as shown, for example, in Figures 15D–15F). The rounder appearance of appropriately oriented MTC wounds may indicate a less severe contraction response.

[0051] As another example, a study comparing collagen staining of untreated, randomly MTC-treated, and oriented MTC-treated skin wounds demonstrates that, according to the systems and methods of this disclosure, oriented MTC-treated wounds heal to better match normal tissue. For example, a comparison of collagen staining of untreated (i.e., secondary intentionally healed wounds) and randomly MTC-treated wounds shows that the healed areas of both types of wounds are distinctly different in color from the surrounding normal tissue. Furthermore, randomly MTC-treated wounds had a more undulating epidermis-dermal (DE) junction and were more similar to normal skin compared to secondary intentionally closed wounds, which showed a disappearance of the DE junction (scar-matching). Such comparisons indicated that secondary intentional healing destroys the collagen structure of the wound, and the collagen fibers are thinned and haphazardly organized. In randomly oriented MTCs, some collagen structure was observed, but it was abnormal compared to the surrounding tissue.

[0052] However, in wounds treated with properly oriented MTCs, the DE junction closely resembles that of normal skin, and the dermal staining (e.g., as given by Herovici's stain) is much closer to that of normal skin compared to wounds treated with random MTCs or secondary intentional treatment. Furthermore, in wounds treated with properly oriented MTCs, the collagen fibers are thicker, closely match the staining of normal collagen fibers, and are organized in a way that is much closer to that of normal skin compared to wounds treated with random MTCs or secondary intentional treatment.

[0053] In light of the above, small columns of full-thickness skin tissue can be harvested, and each donor wound is small enough to heal rapidly by regeneration with minimal scarring. While such columns can be applied randomly to the wound bed to promote wound healing, using tissue columns organized with a clear epidermal-dermal orientation may be advantageous by providing more efficient cell and tissue growth and a more faithful replication of normal tissue microanatomy. Furthermore, the above methods and systems for transplanting and assembling MTCs are simple and non-toxic, forming solid constructs that can be used as expandable building blocks that can be appropriately fitted to the desired size and geometric shape of the recipient site using biocompatible support materials.

[0054] The methods and systems described above can be used for a variety of wound healing applications, including but not limited to burns, abrasions, and surgical wounds, or for other grafting applications, including but not limited to vitiligo. Furthermore, although the methods and systems described above are based on skin grafts, the principles described herein can be similarly applied to other tissue types. For example, the methods and systems described above can be used with other types of tissue, including but not limited to liver, kidney, or heart tissue, to provide microtissue columns positioned in a desired orientation.

[0055] While the present invention has been described in relation to one or more preferred embodiments, many equivalents, substitutes, variations, and modifications are possible within the scope of the invention, beyond those explicitly stated. Furthermore, the term “about” as used herein means a range of ±20%, more preferably ±10%, even more preferably ±5%, and most preferably ±2% from a given value. Alternatively, as is known in the art, the term “about” means equal to half of the smallest increment of the measurement available during the measurement process of such a value using a given measuring tool. This shows the deviation from the specified value.

Claims

1. A system for assembling multiple microtissue grafts, The apparatus includes an array of needles, a matrix having holes corresponding to each of the needles, and a mesh arranged on the matrix to cover the holes, Vacuum is applied through the mesh to draw the plurality of microtissue grafts into the holes of the matrix through the array of needles. The mesh is configured such that the plurality of microtissue grafts are trapped within the pores of the matrix and maintain an epidermal-dermal orientation that matches the epidermal-dermal polarity of normal skin and corresponds to the orientation of the plurality of microtissue grafts already harvested by the needle array. The system is characterized in that the matrix is ​​removable from the mesh and the array of needles so as to form a tissue construct, and the system includes the matrix and a plurality of microtissue grafts within the matrix, the tissue construct being used as a supporting biomaterial.

2. The aforementioned matrix is ​​a two-layer matrix, The system according to claim 1, characterized in that one layer of the two-layer matrix is ​​removable in order to form the tissue construct containing the plurality of microtissue grafts within the remaining layer of the two-layer matrix.

3. The system according to claim 1, wherein the mesh is formed to dimensions that allow air to pass through so that the applied vacuum draws the plurality of microtissue grafts into the matrix through each of the needles.

4. The system according to claim 1, characterized in that the matrix is ​​biocompatible and is used in a solid form.

5. The system according to claim 4, characterized in that the matrix comprises decellularized tissue, collagen, hyaluronan, biodegradable polymer, fibrin gel, or carbon scaffold.

6. The system according to claim 5, wherein the matrix further comprises growth factors and / or oxygen concentration enhancing materials.

7. The system according to claim 2, characterized in that the remaining layer of the two-layer matrix is ​​formed to such dimensions that the lower end of each of the plurality of microtissue grafts is exposed to the outside of the remaining layer.

8. The system according to claim 1, characterized in that the needle array is an array of coring needles.

9. The system according to claim 1, characterized in that each needle in the array of needles has an inner diameter substantially equal to the diameter of the hole in the matrix.

10. A device for assembling multiple microtissue grafts into a tissue construct, An array of needles, each small enough to collect a micro-tissue graft, A matrix having holes arranged on an array of needles and configured to receive the microtissue grafts from the needles, wherein each of the holes is aligned with the corresponding needle in the array of needles and has a diameter substantially equal to the inner diameter of the needle, The matrix comprises a mesh disposed on the matrix, which allows the applied vacuum to allow air to pass through and to pull the microtissue graft into the matrix through the needle, but prevents the microtissue graft from passing through in order to capture the microtissue graft within the matrix, The apparatus is characterized by assembling the tissue construct such that the matrix used as a molded supporting biomaterial contains a plurality of microtissue grafts that are trapped within the matrix and maintain an epidermal-dermal orientation that matches the epidermal-dermal polarity of normal skin and corresponds to the orientation when already harvested by the needle array.

11. The apparatus according to claim 10, characterized in that the matrix includes a biocompatible material.

12. The apparatus according to claim 10, characterized in that the matrix is ​​biocompatible and used in a solid form.

13. The apparatus according to claim 12, characterized in that the matrix comprises decellularized tissue, collagen, hyaluronan, biodegradable polymer, fibrin gel, or carbon scaffold.

14. The apparatus according to claim 13, wherein the matrix further comprises growth factors and / or oxygen concentration enhancing materials.

15. The aforementioned matrix is ​​a two-layer matrix, One layer of the two-layer matrix is ​​removable from the tissue structure. The apparatus according to claim 10, characterized in that the tissue structure comprises the plurality of microtissue grafts within the remaining layers of the two-layer matrix.

16. The apparatus according to claim 15, characterized in that the remaining layer of the two-layer matrix is ​​formed to such dimensions that the lower end of each of the plurality of microtissue grafts is exposed to the outside of the remaining layer.

17. The apparatus according to claim 10, characterized in that the needle array is an array of coring needles.

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