Patent Description:
This invention was made with government support under W81XWH-<NUM>-<NUM>-<NUM> awarded by the Department of Defense. The government has certain rights in the invention.

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 missing skin and other tissue and/or to accelerate healing resulting from trauma, wounds, burns, surgery, and birth defects. Generally, grafting procedures can be limited by the amount of tissue that can be removed from the donor site without causing excessive adverse effects. More specifically, availability of tissue for autografting can be limited by a total area of tissue needed, healing behavior of the donor site, similarity of the donor and recipient sites, aesthetic considerations, and/or other characteristics of candidate donor and/or recipient sites.

A sheet graft is one type of autograft and refers to a piece of tissue that is removed, or harvested, from an undamaged donor site. For example, a sheet graft may be obtained using an instrument structured 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 may be about the same size as the damaged recipient site, slightly larger than the recipient site (e.g., to account for potential shrinkage of the graft tissue after harvesting), or smaller than the recipient site (e.g., with grafts that can be meshed and expanded). Once harvested, the sheet graft can be applied over the recipient site wound, stapled or otherwise fastened in place, and allowed to heal.

Sheet grafts can be full-thickness or split-thickness. For example, a conventional split-thickness graft can be formed by harvesting a sheet of epidermis and upper dermal tissue from a donor site, whereas full-thickness skin grafts can be formed using sheets of tissue that include the entire epidermis layer and a dermal component of variable thickness. The type of sheet graft used can affect healing at both the donor site and the recipient site.

For example, in conventional split-thickness grafts, the skin tissue may grow back at the donor site in a process similar to that of healing a second-degree burn. Split-thickness grafts may thus be preferable to full-thickness grafts because the donor site can at least partially recover on its own, albeit often with scarring, pain, and other long-term side effects. However, skin tissue removed from the donor site for a split-thickness skin autograft generally includes only a thin epithelial layer, which can lack certain elements of the dermis that would improve structural stability and normal appearance at the recipient site once healed.

In conventional full-thickness grafts, more characteristics of normal skin, such as color, texture, and thickness, can be maintained at the recipient site following the grafting procedure (i.e., because the dermal component can be preserved in such grafts). For example, full-thickness grafts can contain a greater collagen content, dermal vascular plexus, and epithelial appendages as compared to split-thickness grafts. Full-thickness grafts may also undergo less contraction while healing. These properties can be important on more visible skin areas, such as the face and hands. Additionally, hair can be more likely to grow from full-thickness grafts than from split-thickness grafts, and sweat glands and sebaceous glands can be more likely to regenerate in full-thickness grafts than in split-thickness grafts, taking on the sweating characteristics of the recipient site.

While full-thickness grafts can provide improved tissue quality at the recipient site, the donor site is completely sacrificed because there is no dermis left for skin to regenerate from. Thus, there is a very limited availability of potential donor sites, and donor sites for full-thickness grafts must be surgically closed. Additionally, full-thickness grafts require more precise conditions for survival because of the greater amount of tissue requiring revascularization. As such, conventional full-thickness skin grafts are generally limited to relatively small, uncontaminated, well-vascularized wounds, and may not be appropriate for as many types of graft procedures as split-thickness grafts.

In light of the above, it may be desirable to provide systems and methods for tissue harvesting and grafting that provide efficient graft tissue with minimal donor site scarring while also properly replicating normal tissue microanatomy at the recipient site. Additionally, it is desirable for such systems and methods to be scalable for use at recipient sites of various sizes and shapes. methods for tissue harvesting and grafting that provide efficient graft tissue with minimal donor site scarring while also properly replicating normal tissue microanatomy at the recipient site. Additionally, it is desirable for such systems and methods to be scalable for use at recipient sites of various sizes and shapes.

<CIT> describes a method for obtaining one or more portions of biological tissue ("micrografts") to form grafts. For example, a hollow tube can be inserted into tissue at a donor site, and a pin provided within the tube can facilitate controlled removal of the micrograft from the tube. Micrografts can be harvested and directly implanted into an overlying biocompatible matrix through coordinated motion of the tube and pin. A needle can be provided around the tube to facilitate a direct implantation of a micrograft into a remote recipient site or matrix.

<CIT> relates to methods for applying a skin graft, which involve harvesting an epidermal skin graft, and applying the epidermal skin graft to a recipient site such that the basal layer of the skin graft makes direct contact with the recipient site.

<CIT> relates to apparatus for affecting an appearance of skin by harvesting small portions of tissue from a donor (first) site and applying them at a recipient (second) site. A plurality of micrografts can be formed from a piece of graft tissue and attached to a dressing material. The dressing material can then be expanded to increase a separation distance between the micrografts, and the dressing material having spaced-apart micrografts attached thereto can be applied to a prepared recipient site.

<CIT> describes devices and methods for obtaining a plurality of skin tissue particles for use in skin grafting.

<CIT> relates devices for generating and transferring micrografts and methods of use thereof. In certain embodiments, devices of this document include a housing having an open configuration and a closed configuration, a micrograft generating station, and a micrograft transferring station.

No methods for treatment of the human or animal body by surgery or therapy are claimed. Embodiments and examples not covered by the claims are meant to illustrate, and facilitate the understanding of, the claimed invention. The methods of the present disclosure overcome the above and other drawbacks by providing fractional tissue grafts, in the form of full-thickness micro tissue columns, in a tissue construct that maintains a desired orientation of the individual tissue columns, such as a substantially vertical, epidermal-dermal orientation. Multiple solid tissue constructs can be used as scalable building blocks arranged in a side-by-side manner to properly fit a desired size and geometry of a wound.

In accordance with one aspect of the present invention, a method for assembling a plurality of harvested micro tissue grafts is provided. The method includes a) arranging the plurality of micro tissue grafts in a desired orientation; and b) forming a tissue construct containing the plurality of micro tissue grafts arranged in the desired orientation, the tissue construct being a solid, three-dimensional construct that maintains the plurality of micro tissue grafts therein in the desired orientation for application of the tissue construct to a recipient site. The tissue construct includes the plurality of micro tissue grafts arranged in the desired orientation within a supportive material, and step b) includes rolling a strip of the supportive material and attaching the plurality of micro tissue grafts attached to the strip at spaced-apart intervals during rolling.

In accordance with an embodiment of the present invention, the supportive material is a biocompatible matrix.

In accordance with an embodiment of the preset invention, the desired orientation is a substantially vertical epidermal-dermal orientation.

The foregoing and other advantages of the invention will appear from the following description. In the description, reference is made to the accompanying drawings that form a part hereof, and in which there is shown by way of illustration a preferred embodiment of the invention. Such embodiment does not necessarily represent the full scope of the invention, however, and reference is made therefore to the claims and herein for interpreting the scope of the invention.

The disclosure provides exemplary systems and methods for organizing and assembling tissue grafts. More specifically, the present exemplary systems and methods enable assembling multiple micro tissue grafts, in the form of biological micro tissue columns, into a larger tissue construct in a way that maintains a desired orientation of the individual tissue columns.

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 micro tissue columns can be applied to wound beds as "random" fractional grafts to improve wound healing. However, because skin is naturally polarized in architecture, engrafting micro tissue columns as an array having a proper epidermal-dermal orientation into the wound bed can further improve healing by accelerating re-epithelialization processing, recapitulating normal dermal architecture, and reducing scarring. As such, the methods and exemplary systems disclosed herein facilitate the orientation of micro tissue columns, and enable their assembly into three-dimensional, full-thickness constructs. The present exemplary systems and methods also provide a practical, scalable solution for using large numbers of micro tissue columns to improve healing wounds of various sizes and shapes.

<FIG> illustrates a method <NUM> for assembling micro tissue columns (MTCs) in accordance with the present disclosure. Generally, as shown in <FIG>, MTCs are harvested from a donor site at step <NUM>. At step <NUM>, some or all MTCs are arranged in a desired orientation (e.g., matching an epidermal-dermal polarity of normal skin). And at step <NUM>, the oriented MTCs are applied to a recipient site. While the term micro tissue columns, or MTCs, is used herein, it should be noted that this term may be interchangeable with micro tissue grafts or micrografts. Furthermore, when the subject tissue is skin, MTCs may be referred to as micro skin tissue columns (MSTCs).

Referring now to step <NUM>, the MTCs can be harvested from a donor site. More specifically, MTCs can be formed by removing elongated, substantially cylindrical portions of tissue from the donor site, thus leaving holes therein. In some embodiments, a diameter or width of an MTC can be less than about <NUM> millimeters (mm) or less than about <NUM>. In some embodiments, the diameter or width can be less than about <NUM>, less than about <NUM>, or about <NUM>. In further embodiments, the diameter or width can be between about <NUM> and <NUM>. In other embodiments, the diameter or width can be between about <NUM> and <NUM>.

Each MTC can be a full-thickness graft, including both epidermal tissue and dermal tissue from the donor site. In general, it can be preferable to harvest MTCs with epidermal tissue and dermal tissue, while avoiding a significant amount of subcutaneous tissue or muscle tissue (though, in some applications, MTCs can include subcutaneous tissue and/or muscle tissue). For example, each MTC can be about <NUM> in height, which can correspond to a total depth of a typical skin layer (e.g., including both epidermal and dermal layers, where the dermal layer includes hair follicles and sweat or sebaceous glands). A different height may be used, such as between about <NUM> and about <NUM>, based on the particular skin or tissue characteristics of the donor site. Additionally, MTCs can include stem cells throughout the dermal tissue (e.g., stem cells associated with hair follicles and sweat glands and/or stem cells in a lower portion of the dermal layer, for example, near a dermal/fatty layer boundary).

Generally, the MTCs can be harvested from the donor site in a way that minimizes or prevents scarring at the donor site. For example, a size of a donor site hole created by a respective MTC can be selected so that the minor damage created heals rapidly and/or without scarring. More specifically, each donor site hole can be small enough to heal quickly by regeneration, that is, by replacement of the harvested tissue volume with new skin tissue that is normal in both structure and function, without or with minimal scarring. Additionally, the size of the donor site holes created by the MTCs can be selected based on creating portions of tissue that can be small enough to promote viability when transplanted or placed in a growth medium, and large enough to form a sufficient amount of graft tissue and/or to capture tissue structures that may be present in the donor tissue.

In some embodiments, a fraction of surface tissue removed from the donor site (which can correspond to a fractional surface area of the donor site occupied by the holes) can be less than about <NUM>%, less than or equal to about <NUM>%, or more preferably between about <NUM>% and about <NUM>%. The fraction of tissue removed can be sufficiently large to provide enough harvested MTCs to form an appropriately sized graft, but small enough to facilitate rapid healing at the donor site based on growth from the remaining undamaged tissue. Other fractions of tissue can be removed from a donor site depending on factors such as, for example, the particular characteristics of the donor site, the size of the graft needed, and the overall amount of donor site tissue available.

According to some embodiments, the MTCs can be harvested using one or more harvesting needles, such as, for example, <NUM>-gauge coring needles. Furthermore, in some embodiments, the MTCs may be harvested using one or more double-pointed hypodermic needles. However, needles of different types or sizes, individually or grouped in arrays, may be contemplated within the scope of this disclosure. For example, MTCs may be harvested using any of the tools and methods described in <CIT>.

The result of step <NUM> is a fractional skin graft that includes a plurality of harvested MTCs. As described above, rather than a single, large donor site wound, the fractional skin grafting techniques described above create minor donor site wounds that can heal with minimal to no scarring. Additionally, in some embodiments, step <NUM> can include pre-treating the donor site prior to harvesting the MTCs to assist MTC orientation at step <NUM>, as further described below.

Referring now to step <NUM>, the harvested MTCs are assembled in a desired orientation, for example, matching an epidermal-dermal polarity of normal skin. More specifically, at step <NUM>, the MTCs can be assembled into a three-dimensional, full-thickness construct maintaining proper epidermal-dermal, substantially vertical orientation. Step <NUM> can be accomplished via a self-assembly approach by coating the surface of each tissue column with a substance that induces all, or most, columns to organize in the desired orientation either spontaneously (e.g., by a hydrophobic coating that would float to the top of an aqueous medium) and/or using external factors (e.g., by an external magnet that causes a coating to orient along magnetic field lines, or by controlled agitation or fluid flow). In addition or alternatively, supportive biomaterials can help maintain the overall structure and desired orientation of the assembled tissue columns, forming a construct. These supportive materials can be applied in different ways, such as, for example, first introduced in liquid form then induced to solidify around the assembled tissue columns, or used in solid form and combined with tissue columns in layers or rolls.

Accordingly, in some embodiments, a coating is used to orient MTCs. For example, a surface of the donor site is coated with a coating prior to graft harvesting at step <NUM>. The coating may be a hydrophobic coating, a hydrophilic coating, or any type of coating that exhibits a phase separation in a solution. The coating can be non-toxic and/or biologically inert and, in some applications, silicone-based. Once coated, the MTCs can be extracted and submerged in a solution that causes the MTCs to align in an epidermal-dermal orientation. More specifically, due to the properties of the coating, the coated epidermis of some or all MTCs will spontaneously align in the solution, orienting itself toward the top of the solution.

<FIG> illustrates an example coating technique <NUM>. As shown in <FIG>, at step <NUM>, a donor site <NUM> can be selected. At step <NUM>, a hydrophobic coating <NUM>, such as petroleum jelly or another suitable coating, can be applied to a surface <NUM> of the donor site <NUM> (for example, over an epidermal layer <NUM> of the donor site). At step <NUM>, MTCs <NUM> are harvested from the donor site <NUM> in accordance with step <NUM> described above. For example, the MTCs <NUM> can be full-thickness grafts, including the epidermal layer <NUM> as well as a dermal layer <NUM> and, optionally, a portion of a dermal/fatty layer boundary <NUM>. At step <NUM>, the MTCs <NUM> are placed in a solution <NUM> (for example, in a well plate). Due to the hydrophobic properties of the coating <NUM>, the coated epidermis <NUM> of some or all MTCs <NUM> will generally align vertically within the solution <NUM> in an epidermal-dermal orientation. At step <NUM>, the solution <NUM> (or a different solution) is induced to solidify around the assembled MTCs <NUM> to create a construct <NUM> of oriented MTCs <NUM>.

In some embodiments, the solution <NUM> can be saline or another suitable solution, such as a biocompatible and/or biodegradable polymer capable of solidifying after a time period (e.g., the polymer can solidify a time period after being mixed), or in response to induction (e.g., through application of a crosslinking agent). Additionally, in some embodiments, a different solution may be used at step <NUM>. For example, this other solution may be a supportive biomaterial, such as a biocompatible matrix or collagen solution capable of solidifying after incubation. While the coating <NUM> may be washed off after alignment in some applications, it may not be necessary in other applications (e.g., the coating <NUM> may remain on the donor site <NUM> after wound application and be allowed to slough off during the natural turnover of the epidermis).

In some embodiments, the above coating technique may be combined with an agitation step. For example, agitation can help stir MTCs that may have sunk down into the solution, increasing their chances of floating up to the fluid surface. Once at the fluid surface, the hydrophobic coating would cause the MTCs to stay in the desired orientation. Additionally, agitation can increase the likelihood that MTCs floating at the surface will get close enough to each other to cluster together (i.e., due to the effects of surface tension around small floating objects, also known as the "Cheerios effect").

For example, <FIG> illustrates a coating and orbital motion technique <NUM>. As shown in <FIG>, a donor site <NUM> is selected and coated with a hydrophobic coating <NUM> at steps <NUM> and <NUM>, respectively, and coated MTCs <NUM> are harvested at step <NUM>. Steps <NUM>-<NUM> of <FIG> can be generally equivalent to above-described steps <NUM>, <NUM>, and <NUM> of <FIG>. Following step <NUM>, however, the harvested MTCs <NUM> can be submerged in a solution <NUM> and agitated to enhance clustering of the MTCs <NUM> toward each other (e.g., toward the center of the well plate) at step <NUM>. Such agitation can be accomplished, for example, by applying orbital motion using an orbital shaker (not shown). In one specific application, agitation can be accomplished using an orbital shaker at <NUM> rotations per minute (RPM) for about thirty seconds; however, other orbital shaker parameters may be used in other applications. Additionally, at step <NUM>, the epidermal-dermal oriented MTCs <NUM> can be placed in a supportive biocompatible material <NUM>. For example, the epidermal-dermal oriented MTCs can be transferred to a new culture plate containing the supportive biomaterial <NUM>, such as a liquid collagen solution or other biocompatible matrix, and again subjected to orbital motion (for example, using an orbital shaker at <NUM> RPM for about thirty seconds or at other RPM and timing parameters). At step <NUM>, the collagen solution <NUM>, including properly oriented MTCs <NUM>, can be induced to form a solid construct <NUM>. For example, in one application, the collagen solution <NUM> can be incubated at <NUM> degrees Celsius for about forty-five minutes to form the solid construct <NUM>.

<FIG> illustrate MTCs <NUM> in accordance with the above technique of <FIG>, where the epidermis <NUM> of the donor site was stained with ink before MTC harvesting to illustrate orientation. <FIG> illustrates the MTCs <NUM> coated with a hydrophobic coating and floating in a solution <NUM> in a well plate <NUM> with some epidermal layers <NUM> oriented upward (e.g., corresponding to step <NUM> above).

<FIG> illustrates the MTCs <NUM> (shown by their stained epidermal layers <NUM>) clustered toward the center of the well plate <NUM> after orbital motion was applied (e.g., corresponding to step <NUM> above). <FIG> illustrate the MTCs <NUM> transferred to a new culture plate <NUM> in a collagen solution <NUM> before and after orbital motion, respectively (e.g., corresponding to step <NUM> above). Accordingly,.

<FIG> illustrates the MTCs <NUM> clustered toward the center of the well plate <NUM> after orbital motion was applied. <FIG> illustrate top and isometric views, respectively, of the MTCs <NUM> correctly oriented in a solidified fractional skin graft construct <NUM> (e.g., corresponding to step <NUM> above, where the collagen solution <NUM> was induced to solidify).

While the above-described orbital motion may be used to orient MTCs in some embodiments, other types of agitation or fluid flow may be used in other embodiments. For example, in one embodiment, harvested MTCs be routed from the harvesting needles through microfluidic channels or flow channels having a tapered geometry (not shown) in order to maintain their epidermal-dermal orientation from extraction. The channels may also be oriented in a way to facilitate a closer grouping between MTCs. That is, the channels may be oriented to decrease a spacing between MTCs compared to their original spacing when extracted from the donor site. From these channels, the epidermal-dermal oriented MTCs may 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 may also be combined with any of the coating techniques described herein.

Additionally, in other embodiments, a magnetic or ferromagnetic coating is used to orient MTCs. In this example, a surface of a donor site can be coated with the coating prior to graft harvesting, such as with a magnetic paint or iron oxide particles. The MTCs are then extracted and submerged in a solution (such as saline, a biocompatible matrix, a collagen solution, or another supportive biomaterial), and an external magnet can be used to orient the MTCs within the solution. Due to the magnetic properties of the coating, the coated epidermis of some or all MTCs will align according to magnetic field lines created by the magnet, thus orienting itself toward the top of the solution. Accordingly, the external magnet can be used to control patterning of the MTCs very precisely. Additionally, in some applications, an array of magnets (that is, rather than a single magnet) can be used, for example, to create regions of different patterns or different densities of MTCs within the same tissue construct.

<FIG> illustrates an example partial coating technique <NUM>. As shown in <FIG>, at step <NUM>, a donor site <NUM> can be selected. At step <NUM>, an adhesive coating <NUM>, such as ostomy glue or another suitable adhesive, can be applied to a surface <NUM> of the donor site <NUM>. At step <NUM>, iron oxide particles <NUM> are applied to the adhesive. At step <NUM>, an additional coating <NUM>, such as a spray-on bandage, is applied over the iron oxide particles <NUM>. While not shown in <FIG>, following step <NUM>, MTCs can be harvested from the donor site (e.g., as described above in accordance with step <NUM>) and placed in a solution. An external magnet can then be positioned over the solution so that some or all MTCs generally align vertically within the solution in an epidermal-dermal orientation. That is, due to the magnetic properties of the coating, the coated epidermis of some or all MTCs will align according to magnetic field lines created by the magnet, orienting itself toward the top of the solution. The solution is then induced to solidify around the assembled MTCs to create a construct of oriented MTCs.

As described above, supportive biomaterials (such as a collagen solution or biocompatible matrix) are used to orient the MTCs and/or maintain MTC orientation in a construct. More specifically, the above-described supportive materials can be used to create a construct that maintains the overall structure and orientation of the assembled tissue columns. As a result, these constructs create a more easily handled graft and, in some applications, can allow for physicians to add drugs, other components, or other cell types as needed.

Accordingly, in line with the above-described techniques, MTCs can be introduced into a supportive material in liquid form, and then the material can be induced to solidify around the tissue columns (for example, by incubation or other suitable techniques).

In other embodiments, however, supportive biocompatible materials can be used in solid form and combined with MTCs in layers or rolls. In the invention, a supportive material is used with a rolling technique that preserves the orientation of the MTCs. More specifically, as shown in <FIG>, a supportive material <NUM> (such as a matrix or other type of biomaterial strip) is rolled up while oriented MTCs <NUM> are placed onto the material <NUM> at spaced-apart intervals. This rolling technique can result in a construct <NUM> having a jelly roll arrangement, as shown in <FIG>. The size of the construct <NUM> can be made smaller or larger (e.g., by less or more rolling) according to a desired wound diameter and/or shape. In some embodiments, a rolling device (not shown) can be used to support the supportive material <NUM> in a substantially vertical orientation while allowing an operator to place the MTCs <NUM> at predetermined distances from each other onto the supportive material <NUM> as the rolling device rolls up the supportive material <NUM>. Alternatively, a pick and place gantry machine (not shown) can be used to automatically place MTCs <NUM> against a vertically positioned strip of matrix material <NUM> that would roll along as the MTCs <NUM> were placed on it.

While the above examples include creating a construct having MTCs in supportive materials, in some embodiments, constructs include MTCs formed together (in the desired orientation) in another manner. As such, these constructs can include MTCs that are oriented properly, but not supported by exogenous materials dispersed between MTCs. Accordingly, in some embodiments, a solid construct may be formed by a material or tool that maintains MTCs arranged and oriented by contacting or communicating with an upper surface of the MTCs. For example, after orienting MTCs, an adhesive dressing can be applied to the epidermal surface to "pick up" all of the oriented MTCs as a solid construct. In another example, MTCs can be coated with a magnetic layer, as described above, and then a magnet can be used to pick up all of the oriented MTCs as a solid construct. In these applications, once the oriented MTCs are picked up, thus forming the construct, the construct may be directly applied to a recipient site (as further described below with respect to step <NUM>).

In some embodiments, one or more of the above examples may be combined or fully or partially interchanged in order to orient MTCs. In some applications, combining techniques can increase an amount of properly oriented MTCs. For example, <FIG> provides a chart <NUM> illustrating a percentage of correctly aligned MTCs when assembled using: hydrophobic coating plus orbital motion <NUM> (resulting in about <NUM>%-<NUM>% correctly aligned); hydrophobic coating alone <NUM> (resulting in about <NUM>%-<NUM>% correctly aligned); iron oxide with an external magnet <NUM> (resulting in about <NUM>% correctly aligned); magnetic paint with an external magnet <NUM> (resulting in about <NUM>%-<NUM>% correctly aligned); and a mineral oil interphase <NUM> (resulting in about <NUM>%-<NUM>% correctly aligned). The mineral oil interphase included a mixture of aqueous fluid (e.g., normal saline) and organic fluid (e.g., mineral oil). This interphase can cause MTCs to orient accordingly as the mixture separates into layers (or phases) because MTCs naturally consist of a mostly hydrophilic portion (i.e., the dermis), sandwiched between two hydrophobic portions (i.e., the epidermis on one end, and the subcutaneous fat on the other end).

As shown in <FIG>, using a hydrophobic coating plus orbital motion (in accordance with the technique of <FIG>) significantly increases the percentage of correctly aligned MTCs compared to the other methods shown. To further illustrate these results, <FIG> illustrates top and bottom views <NUM>, <NUM> of culture plates including MTCs <NUM>, with a stained epidermis, treated with hydrophobic coating and orbital motion. <FIG> illustrates top and bottom views <NUM>, <NUM> of culture plates including untreated MTCs <NUM> (that is, no hydrophobic coating and no orbital motion), and <FIG> illustrates top and bottom views <NUM>, <NUM> of culture plates including MTCs <NUM> treated only with orbital motion. As shown in <FIG>, significantly more of the MTCs <NUM> treated with hydrophobic coating and orbital motion are oriented with their epidermis upward (as shown by the plurality of stained epidermises in the top view of <FIG>) compared to the MTCs <NUM> shown in <FIG>. However, as noted above, the techniques disclosed herein are not mutually exclusive and one or more techniques may be combined or fully or partially interchanged to further increase the total percentage of correctly aligned MTCs and/or achieve desired characteristics. For example, in one application, magnetic particles may be applied to a hydrophobic coating to provide a high amount of properly oriented MTCs (that is, caused by the hydrophobic coating technique) as well as the capability to create precise patterns of MTCs (that is, using the magnetic techniques).

The above techniques orient MTCs, spontaneously and/or using external factors, after they have been harvested at step <NUM>. However, in some embodiments, steps <NUM> and <NUM> may be combined so that MTC harvesting and orienting are completed in a single step. For example, as shown in <FIG>, a harvesting and assembling apparatus <NUM> can include an array of coring needles <NUM>, a pre-molded matrix <NUM>, and a mesh material <NUM>. The array of coring needles <NUM> may be sized and arranged to harvest MTCs from a donor site, and the matrix <NUM> may be arranged over the array of coring needles <NUM> and include a plurality of holes <NUM>, where each hole is aligned with a respective coring needle <NUM> and includes a diameter substantially equal to an inner diameter of the coring needle <NUM>. The mesh material <NUM> can be arranged over the matrix <NUM>, for example, to act as a covering over the matrix holes <NUM> while still permitting suction therethrough.

In operation, as shown in <FIG>, the coring needles <NUM> are placed into the donor site tissue and a vacuum <NUM> is applied from above the mesh <NUM> to pull MTCs <NUM> through the coring needles <NUM> and into the matrix <NUM>. As shown in <FIG>, the mesh material <NUM> can trap the MTCs <NUM> within the matrix <NUM> while still allowing a vacuum to pass through the mesh material <NUM>. As a result, the MTCs <NUM> remain in the matrix <NUM> and are correctly aligned in the proper epidermal-dermal orientation. The matrix material <NUM> can then be removed from the needle array <NUM> and the mesh material <NUM>, as shown in <FIG>, resulting in a tissue construct <NUM>. In other words, the matrix material <NUM> acts as a supportive biomaterial that maintains the overall structure and desired orientation of the assembled MTCs <NUM>.

Accordingly, the matrix material <NUM> may be biocompatible so that the entire matrix construct <NUM> may be placed directly into a wound (in accordance with step <NUM>, as further described below). Example biocompatible matrices include, but are not limited to, decellularized tissue (e.g., skin, gut, amnion, or other tissue that has been processed to remove all living cells, so all that's left of the original tissue are the extracellular components), matrices made from natural biomolecules (collagen, fibrin, hyaluronan, etc., used alone or in combination) in various forms (e.g., in a gel or spun into fibers), synthetic materials that are biodegradable and have certain bio-mimicking properties (e.g., biodegradable polymers functionalized with cell adhesion moieties), and matrices including collagen, hydrogels, fibrin gels, or carbon scaffolds. Additionally, any of the above examples can include growth factor and/or oxygen concentration enhancing material (e.g., CaO2) and/or other substances.

Furthermore, in some embodiments, as shown in <FIG>, a harvesting and assembling apparatus <NUM> can include bilayer matrix <NUM>, including an upper layer <NUM> and a lower layer <NUM>. In such embodiments, suction can be applied to pull the MTCs <NUM> from a donor site into the bilayer matrix <NUM>, as shown in <FIG>, resulting in the MTCs <NUM> being trapped in the bilayer matrix <NUM>, as shown in <FIG>. The needle array <NUM>, the mesh material <NUM>, and one of the matrix layers, such as the lower layer <NUM>, can then be removed. As a result, the MTCs <NUM> remain in the upper layer <NUM>, in their proper epidermal-dermal orientation with the lower end of each dermis exposed, to form a construct <NUM>, as shown in <FIG>. This type of construct, when applied to a wound (as further described below), permits the exposed dermal layer to come into direct contact with the wound bed, which may increase the likelihood of successful reestablishment of blood flow from the wound bed to the MTCs (which can be important for long-term tissue survival).

Referring back to the method of <FIG>, once MTCs are harvested and oriented at steps <NUM> and <NUM> in accordance with any of the above-described techniques, they are applied to a recipient site (such as a wound) at step <NUM>. More specifically, following steps <NUM> and <NUM>, one or more three-dimensional, full-thickness constructs <NUM> (or <NUM>, <NUM>, <NUM>) are available for wound healing, and these constructs include MTCs <NUM> (or <NUM>, <NUM>, <NUM>) in substantially vertical, epidermal-dermal orientation as shown in <FIG>. These constructs are three-dimensional because they have a usable width, length, and height and are full-thickness because they include epidermal and dermal layers <NUM>, <NUM> (as shown in <FIG>). In some embodiments, as shown in <FIG>, a construct <NUM> may be round. However, in other embodiments, constructs may be rectangular, square, or another suitable shape.

According to step <NUM>, the MTCs <NUM> can be placed in or on a wound in order to entirely, or at least partially, cover the wound. In some embodiments, a single construct <NUM> may entirely cover a wound <NUM> at a recipient site <NUM>, as shown in <FIG>. In other embodiments, multiple constructs, each containing a plurality of MTCs, can be arranged side-by-side in order to fit the geometry of a wound. For example, a single MTC roll construct <NUM> (e.g., formed by the rolling technique described above) can fit the geometry of a wound. Alternatively, multiple MTC roll constructs <NUM> can be arranged side-by-side to fit the geometry of a wound. Accordingly, the present methods can be scalable for use in large and/or asymmetrical wounds by providing one or more solid constructs, each formed with a plurality of MTCs, to be arranged side-by-side at a recipient site.

In light of the above, the present methods allow for assembling multiple MTCs, in a desired orientation, into solid, three-dimensional tissue constructs. Furthermore, one or more systems may be provided to fully or partially execute the above-described methods. When such constructs are applied to a recipient site, the full-thickness MTCs can grow, complete with sweat glands and other complex features of the harvested tissue. Accordingly, these MTCs can be used to assist and improve tissue healing at the recipient site (such as a wound). More specifically, properly oriented MTCs can improve healing by accelerating re-epithelialization processing, recapitulating normal dermal architecture, and/or reducing scarring, as compared to healed untreated wounds and healed wounds treated with randomly oriented MTCs.

In particular, while harvested MTCs can be applied to wound beds randomly, that is, without maintaining the normal epidermal-dermal polarity of skin, MTCs organized in a defined epidermal-dermal orientation can be advantageous to accelerate wound healing by providing for more efficient cell and tissue growth and more faithful replication of normal tissue microanatomy (for example, complex structures in full-thickness tissue grafts, like hair follicles, have defined polarities and are generally less tolerant of being implanted in the wrong orientation). Thus, while randomly oriented MTCs have been shown to improve healing compared to untreated wounds (e.g., by healing faster with less contraction), MTCs assembled and oriented in accordance with the exemplary systems and methods described above can further improve healing time, contractile response, skin appearance, and/or structural organization.

For example, <FIG> illustrate a typical secondary intention healing process of an untreated skin wound <NUM> at time zero, two weeks, and six weeks, respectively. <FIG> illustrate a healing process of a skin wound <NUM> treated with randomly oriented MTCs <NUM> at time zero, two weeks, and six weeks, respectively. As shown in <FIG>, after six weeks, the untreated wound <NUM> has slowly healed, mostly by contraction, with a portion <NUM> of the wound <NUM> still open. On the other hand, as shown in <FIG>, at six weeks, the wound <NUM> treated with randomly oriented MTCs is completed closed, healing faster than the untreated wound <NUM> and with less contraction.

In comparison to randomly oriented MTCs, MTC constructs arranged in an epidermal-dermal orientation can provide faster healing time with less contractile response, and result in a healed wound that better matches normal tissue coloring and structure (e.g., that better matches an appearance and structure of the tissue that surrounds the recipient site). For example, <FIG> illustrate a healing process of a skin wound <NUM> at time zero, one week, three weeks, four weeks, six weeks, and eight weeks, respectively, treated using properly oriented MTCs <NUM> in accordance with the methods described above (e.g., using a solid construct of properly oriented MTCs <NUM>). Generally, wounds reconstructed with properly oriented MTCs tend to appear more oval or round (for example, as shown in <FIG>), compared to wounds reconstructed with randomly oriented MTCs, which tend to be shaped with more pointed ends. The rounder appearance of properly oriented MTC wounds may indicate a less severe contractile response.

As another example, studies comparing collagen staining of untreated, random-MTC treated, and oriented-MTC treated skin wounds illustrate that oriented-MTC treated wounds, in accordance with exemplary systems and methods of the present disclosure, heal in a way that better matches normal tissue. For example, comparisons of collagen staining of untreated (that is secondary intention-healed wounds) and random-MTC treated wounds illustrate that the healed areas of both types of wounds were a distinctly different color than the surrounding normal tissue. Additionally, the random-MTC treated wounds had a more undulating dermoepidermal (DE) junction, more similar to normal skin, compared to wounds closed by secondary intention, which showed effacement of the DE junction (consistent with scarring). Such comparisons showed that, with secondary intention healing, the collagen structure of the wound was disrupted and the collagen fibers were thin and haphazardly organized. With randomly oriented MTCs, some collagen structure was seen, but was abnormal compared to the surrounding tissue.

However, in wounds treated with properly oriented MTCs, the DE junction appears much more like that of normal skin and dermal staining color (e.g., given by Herovici's stain) is much closer to normal skin, compared to wounds treated by random MTCs or secondary intention. Additionally, in wounds treated with properly oriented MTCs, collagen fibers are thicker, better match staining of normal collagen fibers, and are organized in a manner that is much closer to normal skin compared to random-MTC or secondary intention wounds.

In light of the above, small columns of full-thickness skin tissue can be harvested, with each donor wound being small enough to heal quickly by regeneration with minimal to no scarring. While such columns can be applied to wound beds randomly to accelerate wound healing, using tissue columns organized in a defined epidermal-dermal orientation can be advantageous by providing for more efficient cell and tissue growth and more faithful replication of normal tissue microanatomy. Furthermore, the above methods and exemplary systems for grafting and assembling MTCs are simple and nontoxic, using biocompatible supportive materials to form solid constructs that can be used as scalable building blocks capable of properly fitting a desired size and geometry of a recipient site.

The above methods and exemplary systems may be used in different wound healing applications, such as, but not limited to, burns, abrasions, and surgical wounds, or other grafting applications, such as, but not limited to, vitiligo. Additionally, while the above methods and exemplary systems have been described with respect to skin grafts, the principles described herein may applied to other tissue types as well. For example, the above methods and exemplary systems may be used with other types of tissue, such as, but not limited to, tissue of the liver, kidney, or heart, to provide micro tissue columns arranged in a desired orientation.

Claim 1:
A method for assembling a plurality of harvested micro tissue grafts (<NUM>), the method comprising:
a) arranging the plurality of micro tissue grafts in a desired orientation; and
b) forming a tissue construct (<NUM>) containing the plurality of micro tissue grafts arranged in the desired orientation, the tissue construct being a solid, three-dimensional construct that maintains the plurality of micro tissue grafts therein in the desired orientation for application of the tissue construct to a recipient site,
wherein the tissue construct includes the plurality of micro tissue grafts arranged in the desired orientation within a supportive material (<NUM>), and
characterised in that
step b) includes rolling a strip of the supportive material and attaching the plurality of micro tissue grafts attached to the strip at spaced-apart intervals during rolling.