Patent Publication Number: US-2013245716-A1

Title: Sleeve for stimulation of tissue regeneration

Description:
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH 
     This invention was made with government support under EB002520 awarded by the National Institutes of Health and W911NF-07-1-0572 awarded by the US Army. The government has certain rights in the invention. 
    
    
     BACKGROUND OF THE INVENTION 
     Tissue regeneration involves a cascade of biological events that combine to fully rebuild an excision or appendage that was lost during trauma or amputation. There is a distinct difference between a typical wound healing response and a regenerative response. These two processes, while similar in many aspects, result in completely different end products. During the course of normal wound healing, many complex biological structures, such as sweat glands, ducts and hair follicles, cannot be rebuilt since the biological machinery to do so is not available. In a typical adult mammalian skin wound, these structures are not regenerated since development of these tissues and organs require highly specific physiological processes to occur. In addition, normal wound closure and scar formation does not provide an adequate environment for these structures to regenerate. Epimorphic regeneration, on the other hand, is the process during which all original structures are replaced with replications of the originals. 
     While mammals and most higher vertebrates typically exhibit very limited and time-specific regenerative capacities, there are several model systems that exhibit epimorphic regeneration. These organisms and their ability to perform epimorphic regeneration are heavily studied; however, the exact pathways of regeneration remain obscure. Fortunately, several common motifs among regeneration schemes across a variety of species have been pieced together to generate a solid understanding of the general principles involved in limb regeneration. There are at least three requirements for any system to show epimorphic regeneration. The system must first contain mitotically active cells and, secondly, release signals to promote the proliferation of those cells. Thirdly, the system must be free of factors that can inhibit a regenerative response. These factors may include a dry external environment, bacterial infection, or an overwhelmingly efficient wound healing process that repairs the wound before a regeneration cascade can begin. 
     As the different stages of epimorphic regeneration progress, not only is there a biochemical response to the trauma, but a biophysical response also occurs that works in tandem with the biochemistry to synergistically repair the defect. The biophysical component of a regenerative response involves both physical mechanics and the various bioelectric events and phenomena such as mass depolarization of cellular membranes and the establishment of minute, long-range electric fields and biological wound currents. Alteration of the cellular transmembrane potential is known to trigger the cell to dedifferentiate and enter a highly mitotic state. Providing a longitudinal electric field is known to both drive an internal wound stump current and provide guidance cues for innervation and the migration of various other cell types near the wound site. While these biophysical phenomena do exist in a normal wound healing response, their presence is significantly more profound in the regeneration process. 
     SUMMARY 
     In some aspects, an apparatus is provided for stimulation of animal tissue regeneration at a wound site disposed on an end of an appendage. The apparatus includes a tubular sleeve, a cuff and an access port. The tubular sleeve includes an outer body that encloses the end of the appendage including the wound site and provides a sealed wound space between the wound site and the outer body. The cuff is disposed in an opening formed in the outer body, and the cuff is configured to conform to the size and shape of the appendage. The access port is disposed on the outer body and is configured to allow transfer of fluids to and from the wound space. 
     The apparatus may include one or more of the following features: The sleeve further includes an annular seal disposed between the cuff and the outer body, and the seal is configured to support the cuff with respect to the outer body and maintain a sealed closure of the opening. The outer body and cuff are each hollow cylinders, the cuff is disposed at a first end of the outer body, and spacing is provided between the cuff and a second end of the outer body, the second end being opposed to the first end. The access port includes a self-sealing body which closes the second end of the outer body. The outer body and the cuff are substantially coaxial. The apparatus further includes an electrical stimulation device having an anode and a cathode, the anode and cathode are configured to be electrically connected to corresponding terminals of a power source, and a portion of the cathode is disposed in the sleeve. The portion of the cathode is removable from the sleeve. 
     The apparatus may include one or more of the following additional features: The cuff is resilient. The outer body is resilient. The cuff, outer body and access port are integrally formed whereby the sleeve is a jointless and seamless structure. The access port includes a self-sealing septum which sealingly closes a second opening in the outer body and is configured to maintain a sealed closure of the second opening during and after needle puncture thereof. The access port includes an inlet portion and an outlet portion, the inlet portion configured to be connected to a fluid pump. The outer body is transparent. The outer body is configured to expand in a direction parallel to a longitudinal axis of the appendage. The outer body includes telescoping portions configured to expand the volume of the wound space. The apparatus further includes a rigid outer cover which encloses at least a portion of the sleeve. The apparatus further includes a treatment fluid disposed in the wound space and configured to stimulate tissue regeneration by controlling the ionic properties of cells of the wound site. The treatment fluid is disposed in the wound space and configured to stimulate tissue regeneration by inducing the cells of the wound cite to become mitotically active. 
     In other aspects, a method of stimulating animal tissue regeneration at a wound site is provided. The method includes the following method steps: Providing an apparatus for stimulation of animal tissue regeneration at a wound site, comprising a sleeve configured to enclose the wound site and provide a sealed wound space between the wound site and the sleeve, the sleeve including an access port configured to allow administration of a fluid to the wound space. Applying the sleeve to the wound site so as to enclose the wound site within the sleeve and form a sealed wound space between the wound site and the sleeve. Treating the wound by including in the wound space a predetermined fluid composition configured to stimulate tissue regeneration. 
     The method may include one or more of the following features: The predetermined fluid composition is configured to control the ionic properties of cells of the wound site. The predetermined fluid composition is configured to induce wound cells to become mitotically active. The predetermined fluid includes a composition including porcine urinary bladder matrix pepsin digest. The predetermined fluid includes at least two different fluids used sequentially. Moisture is constantly maintained in the wound space. The wound space is filled with the predetermined fluid composition. The predetermined fluid composition is a liquid. The wound site comprises an appendage stump resulting from an amputation of an end of the appendage, the sleeve includes a hollow cylindrical reservoir body, and the access port includes a septum which sealingly closes a first end of the body and is configured to maintain a sealed closure of the first end during and after needle puncture thereof. The sleeve further includes a hollow cylindrical cuff at least partially disposed within the body, and an annular seal disposed between the cuff and the body, and the seal is configured to support the cuff with respect to the body. The cuff is configured to receive the stump therein. 
     The method may include one or more of the following additional features: The access port includes an inlet portion and an outlet portion, the inlet portion is configured to be connected to a fluid pump, the outlet portion is configured to serve as a drain of the reservoir body, and the step of treating the wound includes providing a continuous flow of the predetermined fluid through the wound space via the inlet and outlet portions. The apparatus further includes an electrical stimulation device including an anode and a cathode configured to be electrically connected to a power source, the cathode being partially disposed within the sleeve. The method further includes applying electrical stimulation to the wound with the electrical stimulation device. The electrical stimulation is conducted to the wound through the predetermined fluid. The electrical stimulation device is configured to mimic electrical signals of biophysiological processes. The application of the electrical stimulation is performed periodically. 
     The regenerative sleeve provides a closed and controlled macro-environment around a wound or amputation site. By establishing a hydrated and controlled environment, it is possible to stimulate regeneration of tissues, reduce scarring and foster more rapid and direct tissue regeneration. 
     The regenerative sleeve couples a controlled macro-environment and pharmaceutical treatments with electrical stimulation. The electrical stimulation included with the regenerative sleeve allows for electrical stimulation to occur at the wound site and serves to mimic the biophysical processes of limb regeneration that has been observed in vertebrate systems that spontaneously undergo limb regeneration, such as urodeles, and juvenile frogs. The incorporation of an external power source, a cathode and an anode, provides an electric field aligned with a longitudinal axis of the limb, draws current out of the amputation site at the wound core and replicates the stump currents observed in amphibian models. 
     An additional form of electrical stimulation is provided by the regenerative sleeve which includes the utilization of a liquid pharmacological treatment composition designed to alter the cellular transmembrane potential of the cells at the wound site to help induce mitotic activity. 
     An example is provided in which the regenerative sleeve was studied in an animal model. In particular, the regenerative sleeve was used to enclose a surgically amputated murine digit. The regenerative sleeve provided an in utero-type environment conducive to promoting a regenerative response, including a controlled hydrated environment, electrical stimulation at the wound site, and the ability to administer a fluid treatment composition to the wound site to facilitate the recruitment and/or dedifferentiation of cells and blastema formation. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a perspective view of a regenerative sleeve disposed on a mouse digit. 
         FIG. 2  is an anatomical diagram of a mouse digit showing an amputation line. 
         FIG. 3  is an exploded view of the regenerative sleeve of  FIG. 1 . 
         FIG. 4  is a side sectional view of the regenerative sleeve of  FIG. 1 . 
         FIG. 5  is a diagram of the regenerative sleeve of  FIG. 1  including an electrical stimulation device. 
         FIG. 6  is a side sectional view of the regenerative sleeve of  FIG. 1  showing a cathode disposed in the reservoir. 
         FIG. 7  is a diagram of the regenerative sleeve of  FIG. 1  showing an alternative electrical stimulation device configuration. 
         FIG. 8  is a side sectional view of the regenerative sleeve of  FIG. 1  showing an alternative configuration of the cathode disposed in the reservoir. 
         FIG. 9  is a perspective view of the regenerative sleeve of  FIG. 1  showing a syringe and drain disposed in the access port. 
         FIG. 10  is a diagram of the regenerative sleeve of  FIG. 1  used with a fluid pump. 
         FIG. 11  is a perspective view of an alternative embodiment regenerative sleeve. 
         FIG. 12  is an exploded view of another alternative embodiment regenerative sleeve. 
         FIG. 13  is an exploded view of another alternative embodiment regenerative sleeve and a protective shroud. 
         FIG. 14  is a side sectional view of the regenerative sleeve of  FIG. 13  assembled with the shroud showing the connector in an unrolled, extended configuration. 
         FIG. 15  is a side sectional view of the regenerative sleeve of  FIG. 13  assembled with the shroud showing the connector in a retracted, rolled-back configuration. 
         FIG. 16  is a side view of an another alternative embodiment regenerative sleeve. 
         FIG. 17  is a diagram of the regenerative sleeve of  FIG. 16  including an electrical stimulation device. 
         FIGS. 18 and 19  illustrate an alternative configuration protective shroud configured for use with the regenerative sleeve of  FIG. 16 . 
         FIG. 20  is a histological image of a control wound tissue. 
         FIGS. 21 and 22  are histological images of regenerative tissues obtained by method of stimulating animal tissue regeneration at a wound site using the regenerative sleeve of  FIG. 1 . 
     
    
    
     DETAILED DESCRIPTION 
     Referring to  FIGS. 1 and 2 , a regenerative sleeve  100  for stimulating tissue regeneration is shown. The sleeve  100  is used to enclose an end of an appendage, and more particularly to sealingly enclose a wound site  9  corresponding to an amputation of the end of the appendage. In the illustrated embodiment, the sleeve  100  is described with respect to enclosure of mouse digit  3  in which the tip  2  has been amputated along a line  5  through at least a portion of the distal phalange  4 , whereby regenerated tissue may include among other tissues, bone tissue, muscle tissue and skin tissue. 
     Referring to  FIGS. 3 and 4 , the sleeve  100  includes a reservoir body  110  and a cuff  170  disposed in an opening in the reservoir body  110 . The reservoir body  110  is a hollow cylindrical member that encloses the end of the appendage  3  including the wound site  9  and provides a sealed wound space  10  between the wound site  9  and the reservoir body  110 . The reservoir body  110  includes an open first end  114 , and an open second end  116  opposed to the first end  114 . The reservoir body  110  is transparent to permit observation of the wound site while the sleeve  100  is in use. In addition, the reservoir body  110  is sufficiently rigid to prevent any deflection or indentation of the body walls during use so as ensure that a desired wound space volume is maintained, and to protect the wound site  9 . In the illustrated embodiment, the reservoir body  110  is formed of transparent nylon tubing having a length of about 0.25 inches, and an inner diameter of about 0.085 inches. 
     The reservoir body  110  includes an access port  130  disposed on the reservoir body  110  and configured to allow administration of fluids to, and drainage of fluids from, the wound space  10 . The access port  130  is a thin, flat circular disk which is disposed in the open second end  116  of the reservoir body  110 , and is sized and shaped to sealingly close the open second end  116 . The access port  130  is fitted in the open second end  116  such that a peripheral edge  138  of the access port  130  abuts an inner surface  118  of the reservoir body  110 , and an outward facing side  134  of the access port  130  is generally aligned with the end  116  of the reservoir body  110 . The access port  130  is formed of a material which is impermeable to gases and liquids, and which is self-sealing. The term self-sealing is used here to describe a material that can be punctured by a needle, which forms a seal about the needle when punctured, and which sealingly closes the puncture hole after the needle is withdrawn. In the illustrated embodiment, the access port  130  is a silicone disk having a thickness of about 0.038 inches. 
     The sleeve  100  also includes a cuff  170  disposed in the open first end  114  of the reservoir body  110 . The cuff  170  is supported within the reservoir body by an annular seal  150 . The annular seal  150  is disposed at the first end  114  of the reservoir body  110 , and extends between an outer surface  180  of the cuff  170  and the inner surface  118  of the reservoir body  110 . The annular seal  150  maintains a sealed closure of the space between cuff  170  and the reservoir body  110  at the first end  114  thereof. The annular seal  150  is resilient, whereby cuffs  170  of varying sizes may be easily accommodated. In some embodiments, the annular seal  150  is formed of silicone. 
     The cuff  170  is an elongate, thin-walled hollow cylinder which is used to provide a connection between the reservoir body  110  and the appendage  3 . The cuff  170  includes a first end  172 , a second end  174  that is opposed to the first end  172 , and a mid portion  176  disposed between the first and second ends  172 ,  174 . The annular seal  150  supports the cuff  170  with respect to the reservoir body  110  so that the outer surface  180  of the cuff  170  is spaced apart from the inner surface  118  of the reservoir body  110 , the mid portion  176  of the cuff  170  resides within a plane defined by the open first end  114  of the reservoir body  110 , and the second end  174  of the cuff  170  is spaced apart from the second end  116  of the reservoir body  110 . More particularly, the second end  174  of the cuff  170  is spaced apart from an inward facing side  136  of the access port  130 . In addition, the cuff  170  is arranged in the reservoir body  110  so that a longitudinal axis of the cuff  170  is coaxial with a longitudinal axis  122  of the reservoir body. 
     The cuff  170  includes an inner diameter dimensioned to engage and surround a periphery of the appendage  3 . The length of the cuff  170  is dependent on the specific application. For example, in use, an adhesive is provided on the inner surface  178  of the cuff  170  to maintain the cuff  170  on the appendage  3 , and thus the cuff  170  must be sufficiently long to provide an adhesive bond area sufficient to prevent inadvertent dislocation of the sleeve  100  from the appendage  3 . In the illustrated embodiment, the length of the cuff  170  is about 0.10 inches long. In the illustrated embodiment, the cuff  170  is formed of a polyamide tube. 
     Referring to  FIG. 5 , in some embodiments, the sleeve  100  also includes an electrical stimulation device  32  to establish a longitudinal electrical field through the wound site  9 , which is considered to provide an internal wound stump current and to provide electrical guidance cues for innervation and migration of cell types near the wound site. The electrical stimulation device  32  includes an anode  38  and a cathode  36  that are electrically connected to corresponding terminals of a power source  34  through leads  40 ,  42 . 
     Referring also to  FIG. 6 , the cathode  36  is in the form of a stranded stainless steel wire which is disposed in the wound space  10 . The cathode  36  includes a distal end  50 , a proximal end  52 , and a mid portion  54  between the proximal and distal ends  50 ,  52 . In some embodiments, a mid portion of the cathode may be coated, for example with polytetrafluoroethylene such as that sold under the trademark Teflon® manufactured by E.I. du Pont de Nemours and Company, Wilmington, Del., leaving the proximal  52  and distal  50  ends exposed. The proximal end  52  resides outside the sleeve  100  and is connectable to the lead  40 . The mid portion  54  extends through the end  114  of the reservoir body  110  between the cuff  170  and the annular seal  150 . In some embodiments, the mid portion  54  is fixed at this location using an adhesive (e.g., epoxy). The distal end  50  is disposed within the body  112  of the reservoir body  110  at a location adjacent to the second end  174  of the cuff  170 , so that in use, the distal end is closely adjacent to the wound site  9 . In some embodiments, wire strands forming the distal end  50  of the cathode  36  are unwound and arranged in a fan shape in order to maximize contact area in the vicinity of the wound site  9 . 
     The anode  38  is a fine conductive wire that may be inserted in the animal  1  at a location distant from the wound site  9 . In the illustrated embodiment in which the sleeve  100  is disposed on a mouse digit  3 , the anode  38  is disposed in the upper portion of the limb (rear leg) from which the digit  3  extends. In the illustrated embodiment, the anode  38  is a 0.003 inch diameter, uncoated Platinum/Iridium alloy wire which is connected to the power source  32  via the lead  42 . The anode can be permanently implanted, or temporarily inserted as needed. 
     The power source  34  includes a battery pack  31  and circuitry  33 , both of which are enclosed in a housing  35  and configured to provide a constant, low level current to the electrodes  36 ,  38  when connected thereto. In the illustrated embodiment, the power source  34  resides externally of the animal  1 , and the electrodes  36 ,  38  are configured to be detachably connectable to the power source  34 . In this arrangement, when electrical stimulation is required, the cathode  36  and anode  38  are electrically connected to the power source  34  for the duration of the electrical stimulation treatment, and then disconnected between electrical stimulation treatments. Since the power source  32  and leads  40 ,  42  may be detached from the respective electrodes  36 ,  38 , this arrangement conveniently reduces the overall bulk of the combined sleeve  100  and electrical stimulation device  32  during treatment paradigms in which electrical stimulation is used only intermittently. 
     Referring to  FIG. 7 , in other embodiments, the cathode  36  and anode  38  may be configured to be permanently connected to the power source  34 . This arrangement is suitable for providing a continuous electrical stimulation of the wound site. In such an arrangement, the power source  34  can be configured to be implantable into the animal body, and the electrical leads  40 ,  42  that connect the cathode  36  and anode  38  to the power source  34  can also be configured to be implanted. By doing so, the overall bulk of the combined sleeve  100  and electrical stimulation device  32  can be reduced. 
     Referring to  FIG. 8 , an alternative cathode configuration is described. In this embodiment, the cathode  36 ′ is an uncoated, stranded stainless steel wire that is disposed in the wound space  10  at a location spaced apart from the second end  174  of the cuff  170 . In particular, the cathode  36 ′ is arranged so that the mid portion  54 ′ extends across the wound space in a direction transverse to the longitudinal axis  122  of the sleeve  100 . In this embodiment, the distal end  50 ′ of the cathode  36 ′ is fixed to the body  112 , and the proximal end  52 ′ passes through the body  112  at a location that is generally diametrically-opposed to the distal end  50 ′. By arranging the cathode  36 ′ at a location spaced apart from the second end  174  of the cuff  170 , electrical stimulation applied through the cathode  36 ′ is conducted indirectly, through the fluids within the wound space  10 . 
     Referring to  FIG. 9 , the wound space  10  is filled with a fluid to at least maintain a moist wound site  9 . More particularly, the wound space  10  is continuously filled with a fluid treatment composition. Applying fluids to the wound space  10  is accomplished by transferring the treatment composition into the wound space  10  through the access port  130  using a syringe  28 . A drain  30  may be simultaneously inserted through the access port  130  to permit drainage of wound exudate or treatment fluids from the reservoir body  110 , and to permit the wound space to be flushed and/or filled with new treatment fluids. 
     The fluid treatment composition is formulated to stimulate tissue regeneration by controlling the ionic properties of cells of the wound site and inducing the cells of the wound cite to become mitotically active, as discussed further below. In some embodiments, the treatment composition is applied to wound site immediately upon application of the sleeve  100  to the amputated digit  3 . The treatment composition is maintained within the wound space throughout the duration of use of the sleeve  100  due to the sealed configuration of the reservoir body  110 . The same treatment fluid may be kept within the reservoir body  110  for the duration of use of the sleeve  100 , or the treatment composition may be periodically replaced during the duration of use of the sleeve  100 . The replacement fluid may be the original treatment composition, or may consist of a different treatment composition. For example, at least two different treatment compositions may used sequentially. In this case, the selection and ordering of treatment compositions may be determined based at least in part upon the stage of regeneration. 
     The specific function of the treatment composition is dependent on its particular components. However, the general function of each drug mixture is to chemically stimulate the cells at the wound site  9  to enter a regenerative state. This can be accomplished via several different pathways. For example, the transmembrane potential of the wound cite cells can be targeted to induce the cells to enter a differentiated and highly mitotic state by using depolarization compositions, hyperpolarization combinations, MRL matrix combination, or combinations thereof. Methods and compositions for promoting tissue regeneration by administration of a composition effective to modulate cell membrane potential, for example by increasing intracellular sodium concentration in cellular tissue, are disclosed in U.S. provisional application Nos. 61/273,193, filed Jul. 31, 2009, and 61/227,708, filed Jul. 22, 2009, which are incorporated herein by reference. 
     An example of a depolarization treatment composition includes sodium (Na + ) and potassium (K + ) concentrations of 150 mM and 170 mM, respectively. This is an increase in the normal extracellular ion concentrations for Na +  and K +  of 150 mM and 5 mM, respectively. The composition is prepared using NaCl and KCl added to basic physiological buffered saline (PBS) solution containing both CA 2+  and Mg 2+  to promote normal cell signaling function. 
     An example of a hyperpolarization treatment composition includes andenosine triphosphate (ATP)-sensitive K+ channel openers such as Pinacidil and Diazoxide. In the presence of ATP, these molecules cause K +  channels in the cellular membranes to open up, allowing free K +  to flow down a concentration gradient and out of the cell. This outward flux of positive ions causes the transmembrane potential to become more negative, thereby hyperpolarizing the cell. 
     An example of a Murphy Roths Large (MRL) mouse cell matrix treatment composition is obtained by isolating and decellularizing the extracellular matrix from the blastema-like cells of MRL mice that have received a 2 mm ear punch wound and exposing it to a pepsin digest. MRL mice possess a stunning regenerative capacity compared to other strains of mice, and the MRL regenerative process shows striking resemblances to amphibian processes such as blastema formation. 
     An example of a urinary bladder matrix treatment composition is obtained by harvesting the extracellular matrix from a porcine urinary bladder and exposing it to a pepsin digest. 
     Referring to  FIG. 10 , fluid treatment delivery to the reservoir body  110  is not limited to manual injection. For example, in some embodiments, a continuous flow of a fluid treatment composition into the wound space  10  through the access port  130  is achieved using a fluid pump  22  connected to the access port  130  via tubing  24 . In small animal applications where animal mobility and tampering-prevention are of concern, the fluid pump  22  may supported on the animal  1  using a mounting jacket  26 . In addition, for the same reasons, the tubing  24  may be at least partially implanted within the animal  1 . 
     Although the illustrated embodiment employs a cuff  170  arranged so that a first end  172  of the cuff is disposed outside the reservoir body  110 , the mid portion  176  of the cuff  170  is disposed in the open first end  114  of the reservoir body  110 , and the second end  174  is disposed within the interior space of the reservoir body  110 , the sleeve  10  is not limited to this arrangement. For example, in some embodiments, the cuff  170  is disposed in the reservoir body  110  so that the first cuff end  172  lies substantially flush with the first end  114  of the reservoir body  110 . In other embodiments, the second cuff end  174  lies substantially flush with the first end of the reservoir body. 
     In the illustrated embodiment, the cuff  170  is formed of a generally inelastic polyamide tube, and the inner diameter of the cuff  170  is dimensioned as required to accommodate appendages  3  of different diameters. In some embodiments, sleeve  10  may be provided in multiple, pre-sized versions. For example, some sleeves  10  may having a cuff  170  of a small inner diameter, while other sleeves  10  may have a cuff  170  of a medium or large inner diameter. In other embodiments, the cuff  170  is formed of an elastic material, whereby one size cuff  170  can accommodate appendages  3  of various sizes. 
     In the illustrated embodiment, due to the relatively small size of the sleeve  10  used to enclose a wound site on a mouse digit, the electrical stimulation device  30  including the battery pack  32  is housed separately from the sleeve  10 , and only the cathode  34  is incorporated into the sleeve assembly. However, for applications in which a sleeve is increased in size to accommodate larger appendages, it is well within the scope of the invention to incorporate the battery pack  32  into the reservoir body  110 . 
     Referring to  FIG. 11 , an alternative embodiment regenerative sleeve  200  is shown. Similar in form and function to the regenerative sleeve  100  described above, the alterative embodiment regenerative sleeve  200  includes a reservoir body  210 , an access port  230 , an annular seal  250  and a cuff  270 . However, the exterior surface  258  of the annular seal  250  is tapered inward toward the cuff  270 , and the outer surface of the cuff  270  includes reinforcing ribs  272 . The ribs  272  protrude radially outward and extend along an axial direction of the cuff  270 . In addition, the ribs  272  are circumferentially spaced apart. For example, in the illustrated embodiment, ribs  272  are provided on opposed sides of the cuff  270 . The outer surface of the cuff  270  is also provided with a pair of opposed, elongated guides  274  which extend along an axial direction of the cuff and protrude axially beyond the cuff end  214 . The guides  274  are circumferentially disposed between the ribs  272 , and aid in applying the sleeve  200  to the appendage  3 . For example, the guides  274  may direct the appendage  3  to the open end of the cuff  270 , or may be used to manually grasp and pull the sleeve over the appendage  3 . 
     Unlike the regenerative sleeve  100  described above, in the sleeve  200 , the reservoir body  210 , access port  230 , seal  250  and cuff  270  are all formed integrally of a single piece of material. In addition, the material used to form the sleeve  200  is flexible, permitting the cuff  270  to be applied to appendages  3  of varied sizes, and also permitting elastic expansion of the reservoir body  210 . In addition, the material is transparent, self-sealing, easily castable and biocompatible. For example, the material may be a silicon elastomer such as that sold under the registered trademark Dragon Skin® 
     By forming the regenerative sleeve  200  integrally of a single piece of material, assembly of individual sleeve components is avoided. This is particularly advantageous here due to the very small size of the individual sleeve components. In addition, due to the nature of an amputation wound and the need to apply the treatment fluid to the wound site very quickly after amputation, the difficult and time consuming assembly of the small sleeve components during a surgical procedure is also avoided. 
     Referring to  FIG. 12 , an alternative embodiment regenerative sleeve  300  is formed of a reservoir body  110  and access port  130  (not shown in this figure) as described above for the regenerative sleeve  100 . In addition, the regenerative sleeve  300  also includes a modified cuff  310  in which a cuff  370  and annular seal  350  are integrally formed of a single piece of material. In the modified cuff  310 , the annular seal  350  is disposed at the first end  114  of the reservoir body  110 , and extends between an outer surface  380  of the cuff  370  and the inner surface  118  of the reservoir body  110 . The annular seal  350  maintains a sealed closure of the space between the modified cuff  310  and the first end  114  of the reservoir body  110 . The annular seal  350  includes circumferentially-spaced buttresses  358  that extend between a periphery  360  of the annular seal  350  and an outer surface of the cuff  370 . The buttresses  358  protrude radially outward from the outer surface of the cuff  370 , extend along an axial direction of the cuff  370 , and taper inward from the periphery  360  of the annular seal to the outer surface of the cuff  370 . In addition, the outer surface of the cuff  370  includes reinforcing ribs  372 . The ribs  372  protrude radially outward and extend along an axial direction of the cuff  370 . In addition, the ribs  372  are circumferentially spaced apart. For example, in the illustrated embodiment, two pairs of ribs  372 a,  372 b are provided, the ribs  372  of a given pair being disposed on opposed sides of the cuff  370 . The buttresses  358  and ribs  372  provide axial and radial structural reinforcement to the modified cuff  310 . 
     Like the preceding embodiment, the modified cuff  310  is formed of a flexible material, permitting the cuff  270  to be applied to appendages  3  of varied sizes. In addition, the modified cuff  310  is formed of a material that is easily castable and biocompatible. For example, the material may formed of Dragon Skin® silicon elastomer. 
     Referring to  FIG. 13 , an alternative embodiment regenerative sleeve  400  is formed of the reservoir body  110 , a supplemental reservoir  490 , an access port  430 , and a cuff member  470 . The supplemental reservoir  490  is a transparent, rigid cylindrical body that includes an open first end  494 , and an open second end  496  opposed to the first end  494 . The supplemental reservoir  490  surrounds at least the second end  116  of the reservoir body  110 , and is dimensioned to be press fit on the outer surface  120  of the reservoir body  110  so that the reservoir body  110  and supplemental reservoir  490  are coaxially arranged. In addition, the supplemental reservoir  490  is axially longer than the reservoir body  110 . In this embodiment, the second end  116  of the reservoir body  110  remains open, and the access port  430  is disposed in the second end  496  of the supplemental reservoir  490 . The access port  430  is substantially the same in function and structure as the access port  130  described above, but has been increased in size to provide a sealed closure to the second end  496  of the supplemental reservoir  490 . 
     In the regenerative sleeve  400 , the cuff member  470  is substantially modified relative to earlier embodiments. In particular, the cuff member  470  is formed of an annular-shaped medical grade foam material. The outer diameter of the cuff member  470  is dimensioned to correspond to the inner diameter of the reservoir body  110 . Due to the resilient compliance of the cuff member material, the inner diameter of the cuff member  470  can be made less than the outer diameter of the appendage  3  to ensure a good seal and fit about the appendage  3  while still providing wearer comfort and avoiding pressure-related tissue damage. Moreover, the soft foam accommodates any increases or decreases in swelling of the appendage while maintaining a fluid-sealed closure of the first end  114  of the reservoir body  110 . 
     By providing the reservoir body  110  with the superstructure  490 , the volume of the wound space is increased, permitting larger volumes of the treatment composition to be applied to the wound site  9 . In addition, due to the press-fit, coaxial arrangement of the reservoirs  110 ,  490 , the overall length of the regenerative sleeve  400  can be increased by telescopically sliding the supplemental reservoir  490  axially relative to the reservoir  100 . In particular, this adjustment can be made while the sleeve  400  is in use, for example to accommodate regenerative tissue growth and/or to further adjust the volume of the wound space. 
     With reference to  FIGS. 14 and 15 , in some applications in which the regenerative sleeve  400  is used in small mammals, it can be advantageous to provide a transparent protective shroud  500  over the regenerative sleeve  400  to protect it from animal tampering. The shroud  500  includes a transparent cylindrical jacket  510  dimensioned to surround the superstructure  490  of the regenerative sleeve  400 , and further includes a flexible connector  520  used to secure the jacket  510  to the appendage  3 . The connector  520  is formed of a flexible tubing which is disposed on a first end  514  of the jacket  510 , and extends longitudinally from the jacket first end  514  toward appendage  3 . For example, the connector  520  may be formed of a silicone tube. 
     The connector  520  can conveniently be rolled back on itself ( FIG. 15 ) to simplify assembly, and then can be unrolled ( FIG. 14 ) to cover at least a portion of the appendage  3 . Although illustrated here as connecting the jacket  510  to the mouse digit  3 , this arrangement is not limiting. For example, by providing the connector  520  with sufficient length, the connector  520  can be used provide a connection to the animal limb proximal to the digit  3 , for example by surrounding the limb at, or proximal to, the paw. 
     Referring again to  FIG. 13 , in some embodiments the shroud  500  further includes a rigid cylindrical shell  530  which can surround the connector  520 , leaving the transparent jacket  510  uncovered to permit viewing of the wound site. The shell  500  is used to protect the soft connector  520  from animal tampering. 
     In the illustrated embodiment, the regenerative sleeve  100 ,  200 ,  300 ,  400  has been described with respect to enclosure of an amputated digit  3  of a mouse  1 . However, the sleeve is not limited to this application and may be adapted, for example through appropriate scaling of components, for use on appendages other than digits, as discussed further below. Moreover, the sleeve may be adapted for use on appendages of larger animals, and for use on non-appendage wound sites. In addition, the sleeve may be useful in managing growth at wound sites that do not originate from a traumatic injury such as limb amputation. For example, the sleeve may be useful for treatment of organs or appendages that are insufficient due to birth defect, disease or infection. 
     Referring to  FIG. 16 , another embodiment regenerative sleeve  600  is shown which is particularly adapted for use on a murine tail  12 . The regenerative sleeve  600 , like the regenerative sleeve  200  is formed integrally of a single cast piece of material, and includes an elongated cuff  670  sized and shaped to receive a length of the tail  12 , and a reservoir portion  610  connected to the cuff  670 . The reservoir  610  is a hollow cylindrical member that encloses the amputated end of the tail  12  including the wound site  9  and provides a sealed wound space  10  between the wound site  9  and the reservoir  610 . The reservoir  610  may be transparent to permit observation of the wound site while the sleeve  600  is in use. 
     Referring to  FIG. 17 , the regenerative sleeve  600  may include an electrical stimulation device  32  to establish a longitudinal electrical field through the wound site  9 . As described above in more detail, the electrical stimulation device  32  includes an anode  38  and a cathode  36  that are electrically connected to corresponding terminals of a power source  34  through corresponding leads  40 ,  42 . 
     Referring to  FIGS. 18 and 19 , a protective shroud  700  may be provided that is adapted to prevent animal tampering with the regenerative sleeve  600 . The protective shroud  700  is formed of a tough, lightweight material such as plastic, and includes a base portion  710  which encloses the cuff  670  of the sleeve  600 , and further includes a conical shaped collar  720  which protrudes from an end of the base portion  710 , and which surrounds the reservoir  610  of the sleeve  600 . The collar  720  extends from the base portion  710  to a location that is distal to the reservoir  610 , making difficult for the animal to gain access to the sleeve  600 . The conical collar  720  is arranged so that the widest portion is located at the distal end of the sleeve  600 . This arrangement advantageously permits easy access to the access port  630  so that treatment fluids and electrical stimulation can be administered to the wound site  9 . The shroud  700  is provided as two halves  732 ,  734  hinged along one axial side, permitting the shroud  700  to be opened as shown in  FIG. 19  to receive the regenerative sleeve  600  while use on the murine tail  12 . Once the sleeve  600  is received in the shroud  600 , the halves  732 ,  734  are assembled as shown in  FIG. 18  and fixed, for example by using adhesive. 
     A method of stimulating animal tissue regeneration at a wound site  9  using the regenerative sleeve  100 ,  200 ,  300 ,  400 ,  600  will now be described. In the method described herein, the renerative sleeve is used to enclose a wound site  9  formed by amputation of the end  2  of a murine digit  3 . In particular, the amputation is provided along an amputation line  5  through the distal phalange  4  of a murine digit  3  as shown in  FIG. 2 . 
     The method of stimulating animal tissue regeneration at a wound site  9  includes the following method steps: 
     The method includes providing a regenerative sleeve  100 ,  200 ,  300 ,  400 ,  600  as described above. The regenerative sleeve is applied to the end of the digit  3  immediately following amputation of the end  2  of the digit  3 , so as to enclose the wound site  9  within the sleeve and form a sealed wound space between the wound site  9  and the sleeve. 
     The method includes treating the wound site  9  by filling the wound space  10  with a predetermined fluid composition configured to maintain a moist wound site  9 , and to stimulate tissue regeneration at the wound site  9 . In some embodiments, the fluid composition is administered by puncturing the access port  130  with a pair of hypodermic syringes, where one syringe is used to deliver the fluid composition to the wound space  10  and the other syringe is open-ended and serves as a vent ( FIG. 9 ). During this step, care is taken to prevent formation of air bubbles within the wound space  10 . 
     The predetermined fluid composition is configured to control the ionic properties of cells of the wound site to induce wound cells to become mitotically active. In some embodiments, the predetermined fluid composition includes porcine urinary bladder matrix pepsin digest, but this is not limiting. Treating the wound may include continuous treatment of the wound site  9  with a single fluid composition throughout the duration of use of the sleeve. Alternatively, the treatment composition may be periodically replaced during the duration of use of the sleeve. The replacement fluid may be the original treatment composition, or may consist of a different treatment composition. For example, at least two different treatment compositions may used sequentially. In this case, the selection and ordering of treatment compositions may be determined based at least in part upon the stage of regeneration. In some embodiments, a continuous flow of the predetermined fluid through the wound space may be provided using fluid pump. 
     The method further includes treating the wound site  9  by applying electrical stimulation to the wound site  9  using the electrical stimulation device  32 . Electrical stimulation can be achieved by directly contacting the wound site with a cathode  36 . Alternatively, electrical stimulation can be achieved disposing the cathode  36  in the wound space  10  whereby the electrical stimulation is conducted to the wound through the treatment fluid. The electrical stimulation is applied in such a way as to mimic electrical signals of biophysiological processes, in terms of current intensity (up to 10 uA maximum), flow direction, and temporal pattern. In some embodiments, application of the electrical stimulation is performed periodically. For example, stimulation can be provided on alternating days for a duration of 15-30 minutes on each of those days. In other embodiments, application of the electrical stimulation is performed continuously. 
     Although in the method described herein, the regenerative sleeve is used to enclose a wound site  9  formed by amputation of the end of a murine digit  3 , this is not limiting. For example, it is well within the scope of the invention to apply the disclosed method to amputation wounds of other appendages, to such wounds in the appendages of other animals, and to wounds not resulting from amputation (for example, crush injuries). 
     EXAMPLE 
     Animal Study 
     The effectiveness of the regenerative sleeve  10  described above to biochemically and biophysically stimulate tissue regeneration in a murine toe amputations was studied. 
     In particular, twelve 6-8 week old male mice, weighing approximately 20-25 g. were used in this study. Even though female mice are known to demonstrate slightly more developed regenerative capacities, males were selected since the majority of the intended long-term beneficiaries of this study are injured male soldiers than have suffered limb loss in conflict. 
     The mice were anesthetized via intraperitoneal injection with Ketamine (90-120 mg/kg) and Xylazine (10 mg/kg) prior to surgery. Lubricating eye drops are administered to the eyes of sedated mice to prevent dehydration. After anesthetization, the mice were prepared for surgery by repeatedly cleaning the right hind foot with 70% ethanol and then with a 10% povidone iodine solution. Fur near the anode insertion site is removed with an electric trimmer and razor. The surgical sites are also cleaned with ethanol and povidone iodine after fur removal. The digit amputation was performed at the midline of the 2nd phalange of the right, hind middle digit (digit 3, Standard US Nomenclature) with extra-fine bone scissors. Amputations took place under a Leica EZ 4D microscope. Scissors and other surgical instruments were sterilized between mice using alcohol and a hot glass bead sterilizer. Digit tips were properly discarded after amputation. The surgical time for each mouse averaged between 10 and 15 minutes from the onset of sedation, excluding time required for electrical stimulation. 
     This investigation involved two treatment groups containing six mice each (see Table 1). Group 1 received a Regenerative sleeve  10  containing the urinary bladder matrix (UBM) digest control treatment. Group 2 received a Regenerative sleeve  10  containing the UBM digest treatment. Electrical stimulation was provided to all subjects on days 0, 1 and 3. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Treatment Matrix 
               
            
           
           
               
               
               
               
            
               
                 Group 
                 Liquid Treatment 
                 n 
                 Electrical Stimulation 
               
               
                   
               
               
                 1 
                 UBM digest control 
                 6 
                 6.4 uA for 15 minutes on 
               
               
                   
                 (neutralized pepsin 
                   
                 days 0, 1 and 3 
               
               
                   
                 buffer) 
               
               
                 2 
                 UBM digest 
                 6 
                 6.4 uA for 15 minutes on 
               
               
                   
                   
                   
                 days 0, 1 and 3 
               
               
                   
               
            
           
         
       
     
     The UBM pepsin digest treatment and control treatment, a neutralized pepsin buffer, were the only treatments administered. The UBM treatments have been shown to perform well as scaffolds and promotion of regenerative healing. The preparation of the UBM treatments was accomplished by harvesting the ECM from a porcine urinary bladder and, then exposing the ECM to a pepsin mediated enzymatic deigestion. While a fully characterized composition the UBM digest remains unknown, various molecules such as collagen, glycosoaminoglycans (GAGs), matrix metalloproteinases (MMPs) and a variety of growth factors are present and serve as a physical scaffold for cell growth. 
     Treatment Delivery: 
     The ECM digest treatments were administered by puncturing the distal silicone septum of the Regenerative sleeve  10  with a pair of 30.5 gauge hypodermic syringes. One syringe was used to deliver the liquid cocktail treatment while the second syringe was open-ended and serves as a vent, allowing the air within the Regenerative sleeve  10  reservoir to escape as treatment is added. At this time, the system is checked for leaks. Care was taken to avoid the formation of air bubbles within the Regenerative sleeve  10  reservoir. Electrical stimulation was administered while the animals were sedated. Immediately prior to electrical stimulation, the 0.18 mm diameter electrical stimulation acupuncture needle was inserted into the haunch ipsilateral with the Regenerative sleeve  10 . This anode was inserted at an angle to ensure it did not pierce the muscle layer. The stainless steel cathode was built in to the Regenerative sleeve  10  reservoir as shown in  FIG. 1 . By definition, electrical current flows from the anode towards the cathode. 
     The electrical connection was made to the anode using a mini alligator clip, and the anode was promptly removed and discarded after stimulation. Electrical stimulation began promptly after the anode was inserted. After the power supply was adjusted to deliver the proper current (6.4 uA), the mouse was electrically connected to the power supply and stimulated for 15 minutes. To check for proper function of the electrical stimulation system, the initial current flow was verified at the start of the electrical stimulation session using an in-line Ammeter. After electrical stimulation, the mouse was relocated from the surgical area to the heated stimulation area for recovery. Per protocol, mice received buprenorphine as an analgesic to reduce pain following the surgical procedures. The buprenorphine was administered subcutaneously at 0.05 mg/kg immediately after all surgical procedures have taken place. 
     Recovery and Euthanasia: 
     Recovery from surgery and electrical stimulation occurred on a heating pad. Mice were intermittently monitored by visual analysis and toe-pinch reflex to determine level of consciousness. After the mice began to move on their own, they were placed in individual housing containers for the duration of the study with free access to food and water in a temperature controlled room. During the initial bedding period, soft Kimwipe® bedding was provided. Euthanasia was performed by CO2 inhalation prior to collections of samples for histology. 
     Histology: 
     On day fourteen, the amputated digits along with an adjacent digit for a control were isolated for histology. Due to the presence of the bone in the tissue samples, all samples are decalcified using the Decalcifier I® treatment (Surgipath Inc.). After decalcification, the samples were placed in 10% formalin until they were paraffin embedded. The digits were then sectioned along the proximal-distal axis, stained with trichrome, and imaged at low and high magnifications. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Result Summary 
               
            
           
           
               
               
               
            
               
                 Treatment 
                 Day 14 Result Summary 
                 Images 
               
               
                   
               
               
                 True Control 
                 Lymphocytes: significant presence 
                 FIG. 20 
               
               
                 (−) Regenerative sleeve 10 
                 of cells Wound epithelium 
               
               
                 (−) electrical stimulation 
                 and new glands: relatively thin 
               
               
                 (−) pharmacological 
                 wound epithelium with minimal 
               
               
                 treatment 
                 new gland formation within 
               
               
                   
                 the re-growth region. 
               
               
                   
                 Large mononuclear eosinophilic 
               
               
                   
                 cells: significant presence of 
               
               
                   
                 LMECs without any indication 
               
               
                   
                 of advanced organization 
               
               
                 UBM pepsin digest control 
                 Lymphocytes: low presence of 
                 FIG. 21 
               
               
                 (+) Regenerative sleeve 10 
                 immune cells Wound epithelium 
               
               
                 (+) UBM pepsin digest 
                 and new glands: slightly thicker 
               
               
                 control cocktail 
                 wound epithelium with increased 
               
               
                 (+) electrical stimulation 
                 new gland formation within 
               
               
                   
                 the re-growth regions 
               
               
                   
                 Large mononuclear eosinophilic 
               
               
                   
                 cells: strong presence of LMECs 
               
               
                   
                 with some samples showing 
               
               
                   
                 increased organization and 
               
               
                   
                 formation of possible lacuna-type 
               
               
                   
                 regions 
               
               
                 UBM pepsin digest 
                 Lymphocytes: low presence of 
                 FIG. 22 
               
               
                 (+) Regenerative sleeve 10 
                 immune cells Wound epithelium 
               
               
                 (+) UBM pepsin digest 
                 and new glands: thickest wound 
               
               
                 cocktail 
                 epithelium with strongest 
               
               
                 (+) electrical stimulation 
                 evidence of new gland formation 
               
               
                   
                 and vascularization within 
               
               
                   
                 the re-growth region. 
               
               
                   
                 Large mononuclear eosinophilic 
               
               
                   
                 cells: Strongest presence of 
               
               
                   
                 LMEC&#39;s with a high degree of 
               
               
                   
                 organization and lacuna-type 
               
               
                   
                 regions adjacent to original bone. 
               
               
                   
               
            
           
         
       
     
     The regenerative sleeve  100  was designed to address the following issues in a murine model system: wound site hydration, drug delivery, electrical stimulation, subject ambulation and stress management, tamper prevention, and simplicity of installation. The materials and configuration for the device were chosen to minimize potential damage caused by gnawing, scratching, normal movement and exposure to the rodent housing environment. The regenerative sleeve  100  was streamlined to minimize complexity of the device for fabrication and handling reasons and to reduce the number and severity of possible complications that could arise during any part of the surgical procedure. 
     Study results indicate that the regenerative sleeve  100  provides a protected and hydrated environment by encompassing the wound site. Qualitative analysis of the histological data indicates that the presence of the regenerative sleeve&#39;s  100  well-hydrated environment plays a crucial role in enhancing digit regeneration. Also, administration of electrical stimulation to the wound site enhances this response to the extent where highly organized structures indicative of bone remodeling were observed as early as day 14 in most subjects. Subject receiving a regenerative sleeve  100  with UBM pepsin digest control solution with electrical stimulation showed evidence of enhanced regeneration ( FIG. 21 ) over the control digit ( FIG. 20 ).  FIG. 20  shows a histological image take 14 post amputation in a Subjects receiving a regenerative sleeve  100  with UBM pepsin digest and electrical stimulation exhibited an even greater evidence of regeneration over the UBM control treatment as indicated by a more pronounced network of collagen deposition and large eosinophilic mononuclear cells ( FIG. 22 ).  FIG. 20  shows a histological image taken 14 post amputation on a C57bl/6 mouse. This mouse was a true control and received no treatment (pharmacological or physical) or wound dressing post amputation. The distal region of the digit is indicated near point (D), and the adjacent digit is indicated at (G). The amputation line shows the approximate line of amputation on Day 0, and the regrowth region is defined as the region of tissue distal to the amputation line. (A) Original, mature bone; (B) Proliferative large mononuclear eosinophilic cells; (C) New hair follicle and sebaceous glands; (D) Wound epithelium; (E) Lympyhocytes; (F) New collagen disposition, possibly scar tissue formation.  FIGS. 21 and 22  show (A, C) Low and (B, D) high magnification images of two different digit tips in the UBM pepsin digest control with electrical stimulation group. Scale bars: 100 um (A,C), 50 um (B, D). 
     A selected illustrative embodiment of the invention is described above in some detail. It should be understood that only structures considered necessary for clarifying the present invention have been described herein. Other conventional structures, and those of ancillary and auxiliary components of the system, are assumed to be known and understood by those skilled in the art. Moreover, while a working example of the present invention has been described above, the present invention is not limited to the working example described above, but various design alterations may be carried out without departing from the present invention as set forth in the claims.