WO2007042818A1

WO2007042818A1 - Delivery device

Info

Publication number: WO2007042818A1
Application number: PCT/GB2006/003797
Authority: WO
Inventors: Gunasekar Vuppalapati
Original assignee: Gunasekar Vuppalapati
Priority date: 2005-10-12
Filing date: 2006-10-12
Publication date: 2007-04-19
Also published as: GB0520757D0

Abstract

The present invention relates in one aspect to a delivery device comprising a plurality of needles mounted on a substrate, the device being adapted to deliver cells through the needles into a biological tissue. The invention also relates to an implant apparatus adapted to co-operate with the delivery device, and associated methods and uses.

Description

DELIVERY DEVICE

The present invention relates to the field of devices, apparatus and methods for injecting substances into living biological tissues, in particular for the in vivo delivery of cells, ground substance and/or growth factors into tissues or organs such as the skin of a subject, for example in order to restore their form and/or function by regenerating tissue de novo.

A unit of living being is a cell. Various types of cells together in a scaffold of interstitial ground substance in an organised manner forms the tissue. The type and function of the tissue varies according the type of the cells it contains. An organ is formed of more than one tissue laid in a typical architecture. Optimal wellbeing of a living being depends on the normal anatomy, physiology of each organ and thus biochemistry.

Tissue engineering technology is able to develop some of these tissues in the lab in order to replace the loss or failure. There have been limitations in being able develop whole tissues/organs in vitro. The limitations include difficulties developing an ideal scafold, integration of cells into the scafold, vascularisation of such tissue, transfer of such developed tissue into the body, integration of such tissue into the body and maintenace of the viability of this tissue through all the stages until it is fully integrated to begin its function in the body. This concept of "In Vivo- De Novo Tissue Engineering" can be applied to regenration of any tissue in the body. One of the demands of this technology is in hair follicle/hair regeneration. Hair follicle/hair regeneration as described in the present application may be considered to be an example of a "In Vivo — De Novo Tissue Engineering" process. Thus various other tissues can also be produced by related tissue engineering processes.

Hair provides no vital function for humans, but its psychological effect is nearly immeasurable. Luxurious scalp hair expresses femininity for women and masculinity for men. Male pattern baldness, although accepted in our society, is still distressing to most men as it remains source of attention. Men often go to great lengths to preserve, restore, or regrow hair on their scalps, needless to mention the impact on women with hair loss.

The total number of hair follicles for an adult human is estimated at 5 million with 1 million on the head of which 100,000 alone cover the scalp. The incidence of male pattern baldness (Androgenic Alopecia in medical terms) is estimated to be 25% of population at 25 years age group, reaching 50% by fifties¹.

Hair is one of the skin appendages. The skin has two layers, (1) Epidermis and (2) Dermis. The epidermis is a stratified squamous epithelium containing multiple layers of cells on a basement membrane that mainly serves as a protective barrier. The epidermis is about 0.1 mm thick. The dermis is a tough supportive connective tissue matrix containing numerous specialized structures. The dermal thickness varies between 0.04 mm to 4 mm. The papillary dermis - the thin upper layer - of the dermis - lies directly below and interdigitates with the epidermal rete ridges. The papillary dermis is composed of loosely interwoven collagen. The second deeper and thicker reticular dermis has coarser and horizontally running bundles of collagen.

Embryogenesis of hair in the fetus involves aggregation and differentiation of mesenchymal stem cells into a dermal papilla (DP) in the dermis just below the epidermis. Above the dermal papilla an epidermal plug, or peg of cells develops and proliferates growing into the dermis towards the dermal papilla. The mesoderm-derived dermal papilla and the ectoderm-derived epidermal plug communicate via molecular signals with the result of further proliferation of epidermal matrix cells and differentiation into the various sheath and hair fiber structures. Thus the development of a hair follicle requires a continuum through induction, proliferation, communication, elongation and differentiation stages.

DP cells can also interact with adult epidermis to induce the development of new hair follicles. In the established hair follicle the DP cells act in conjunction with epidermal cells via mechanisms similar to those in embryogenesis to permit hair follicle cycling through hair production and resting phases. DP cells are almost unique in maintaining their embryogenic regenerative properties in adults making them potentially attractive for investigation with a view to gaining an insight on organ/limb regeneration and similar studies.

Methods of isolation and culturing these inductive dermal papilla/dermal sheath cells (Oliver et al-1990 US Patent No: 4,919,664; Jahoda et al-2004 US Patent app No: 20040057937) and inductive epidermal matrix cells (Luo et al-1996, US Patent No: Hl, 610) are however well established. It is the next three important requirements that required to be achieved i.e. effective and controlled delivery of cells for induction, maintaining the in vivo micro-environment during regeneration of hair follicle and controlling the direction of hair growth.

To achieve such embryogenesis of hair in the adult requires that multiple conditions are fulfilled, in order to recreate the conditions which promote embryogenic hair growth. Some of these conditions have been met by various inventors during the past 10 years, yet the objective of growing head full of hairs has not been achieved so far due to various limitations.

These limitations include failure of attempts to grow the hair in the lab, difficulties of keeping viability throughout the process in vitro hair follicle regeneration, problems with the transfer of partly generated follicle to a desired location, and disappointment over the unpredictable and suboptimal quality of regenerated hair in the undesirable direction.

Prior art approaches have included methods of introducing cells into the scalp, for instance through the use of a needle mounted on a special syringe as a manual cell delivery device. However technical difficulties with such an approach include the extensive expertise and time required to perform such implantation of inductive cells for each hair one by one. This would involve manual repeated implantation for as many as 25,000 times in extreme baldness (class VII, Norwood).

For instance some approaches focus on the use of a manual syringe injecting through a hypodermic needle or their variants. US 2005/0147652 Al discloses a controlled delivery system with reproducibility of precise volume, cell count and depth of the delivery deposit. A Hamilton syringe is loaded with multiple doses and the delivery is controlled and assisted by a micro-pump device. Although it is claimed to be faster than the previous methods of follicular unit hair transplantation, this technique still needs considerable expertise and is time consuming, because the device can only transfer cells to a single site in the skin at one time. To transfer cells to multiple sites, the needle needs to be removed and reinserted repeatedly.

Moreover, following transfer of cells to the skin according to the prior art methods, the conditions for cell growth and hair formation are not necessarily as optimal as embryo genie hair growth. A further problem with the prior art methods is that injected cells do not necessarily induce hair growth in a direction which results in natural-looking hair. Whilst the direction of natural hair growth varies over the scalp, the prior art methods typically do not take this into account. If the direction of hair growth is not controlled by the implantation process, the induced hair growth typically does not have a natural appearance.

Embodiments of the present invention aim to overcome one or more of the problems of the prior art methods. In particular, the present invention aims to provide a delivery device, apparatus and methods which can be used to provide an improved or more convenient delivery of cells, and/or improved cell growth and differentiation, in living biological tissues such as skin.

Accordingly, the present invention provides a delivery device comprising a plurality of needles mounted on a substrate, the device being adapted to deliver cells through the needles into a biological tissue.

hi a further aspect, the present invention provides an implant apparatus comprising a plurality of elongate members extending away from a base element, each elongate member enclosing a first lumen, the implant apparatus being adapted to co-operate with a delivery device as defined above, such that the needles of the delivery device can be accommodated at least partially within the first lumina of the elongate members.

In a further aspect, the present invention provides an assembly comprising one or more delivery devices as defined above and an implant apparatus as defined above.

In a further aspect, the present invention provides a method for delivering cells into a biological tissue, comprising delivering the cells through an injection device comprising a plurality of needles attached to a substrate.

In a further aspect, the present invention provides use of a delivery device comprising a plurality of needles attached to a substrate, to deliver cells into a biological tissue.

Embodiments of the present invention, in particular the use of a delivery device comprising a plurality of needles, can provide an advantage over the prior art methods by enhancing the rate and/or convenience of cell delivery to biological tissues, for instance in the delivery of cells in a hair regeneration method. In one aspect, the use of a delivery device comprising multiple needles enables parallel delivery of cells, growth factors and/or ground substances to multiple (for instance several thousand) sites at a single time point, compared to serial delivery to individual sites in the prior art.

Moreover, aspects of the present invention enable the growth and/or differentiation of injected cells to be facilitated or regulated in vivo by providing an implant apparatus which is co-operates mechanically with the injection device and which is biocompatible. This allows the micro-environment of cell regeneration to be controlled by delivering the necessary cells, growth factors and/or ground substance which are required at various time points/stages. Thus different agents can be delivered at various stages of the regeneration and differentiation process, by intermittent delivery and/or continuous irrigation through the implant apparatus.

This concept of induction and regulation of tissue regeneration within the body by external supply of necessary cells, growth factors/cytokines and/or ground substance is termed "in vivo-de novo tissue engineering" in the present application.

Embodiments of the present invention also enable the direction of cell growth (e.g. the direction of hair growth in a hair regeneration method) to be controlled by regulating the angle at which the needles of the injection device are implanted into the skin, and maintaining hair growth in this direction by the use of the implant apparatus.

By "delivery" or "delivering" in the present invention it is intended to refer to the introduction of cells or other materials into a biological tissue, for instance by injection or infusion/infiltration. Thus the delivery device may for example be an injection device. References to "needles" are intended to include any pointed element capable of penetrating into a biological tissue, and through which a material may be delivered into the tissue.

The needles can be constructed from a variety of materials, including metals, ceramics or composites. Preferably the needles are made of a rigid material. The needles can have straight or tapered shafts having any suitable shape in cross-section (for instance circular, square, triangular) and may have one or more bores.

By "micro needle" it is typically meant a needle having an internal diameter of less than 100 μm and a length of less than 20 mm. Suitable micro needles, substrates, fluidic systems and their methods of manufacture which may be used in the present invention are disclosed in general terms for example in WO 99/64580 and WO 01/49346.

Both the delivery device and implant assembly can be made of any biocompatible material, preferably one that is used in micro fabrication processes. Micro needles are preferably made of material strong enough to penetrate the skin. Micro sheaths are preferably made of polypyrrole-hyaluronic acid composite biomaterials, or alternatively any biocompatible polymer, more preferably one which preferably has some electrical conduction properties. However according to the present invention, the needles are arranged for delivery of cells into a biological tissue. Typically the micro needles used in the present invention are longer than those used in prior art in order to enable them to penetrate deeper into the skin to where cell deposit is required, for instance in a hair restoration method. Thus the micro needles of the present invention are preferably of length 0.5 to 20 mm, more preferably 2 to 5 mm.

Moreover, the needles used in the present invention are preferably oriented at predetermined angles with respect to the substrate. In some embodiments the needles may be oriented perpendicular to the substrate. More preferably, one or more of the needles, or more preferably all of the needles in the device, are oriented at an angle other than perpendicular to the substrate. For instance, the needles may be oriented at an angle of 15 to 85°, more preferably 20 to 80°, most preferably 45 to 75° with respect to the substrate. The angles mentioned above refer to the smaller angle between a plane defined by the substrate and an axis running along the length of a shaft of the needle.

By "substrate" is meant any element on which the needles may be mounted and which supports or holds the needles in position. For instance in certain embodiments the substrate may be adapted to hold the needles in a desired orientation, e.g. at a particular angle with respect the substrate. The substrate may be made of any suitable material and may be planar or non-planar in form. In preferred embodiments the substrate is substantially planar in form, with needles extending away at predetermined angles from one surface. The needles may be mounted on the substrate in any desired manner, for instance they may be attached or fixed to the substrate by adhesive or they may mechanically co-operate with the substrate so as to lock into position. By "mounted" it is also intended to encompass embodiments where the needles are unitary or integral with the substrate, i.e. the needles and substrate are formed or moulded from a single, continuous material.

"Implant" is intended to refer to any device, at least part of which is introduced into a biological tissue, and which remains in the tissue for a period of time. The implant typically remains in the tissue only temporarily, for instance for a period of 1 to 30 days, more preferably 3 to 10 days, but may in certain circumstances remain in the tissue for shorter or longer periods.

By "elongate member" it is intended to include any longitudinally extended, filamentous, tubular, cylindrical, sheath-like or rod-shaped structure, provided that the elongate member comprises an internal lumen (referred to hereinafter as a first lumen). Typically the first lumen comprises a cavity, channel, cannula, sac or recess running along the length of the elongate member. The first lumen may be of any suitable shape or dimensions, provided that it is capable of at least partially accommodating a needle of a delivery device.

The implant apparatus is designed to co-operate with a delivery device as described above. By "co-operate" it is meant that the implant apparatus is adapted to be connected to, juxtaposed or interlocked with a delivery device such that they can together perform their functions. In particular the implant apparatus is shaped or configured such that it is complementary to a delivery device, so that the needles of the delivery device can be accommodated in the elongate members. By "accommodating" it is meant that the first lumen of the elongate member surrounds at least part of the shaft of the needle, for instance by forming a sheath surrounding the needle. However the elongate member need not encompass the entire shaft of the needle. For instance, in some embodiments a tip of the needle emerges through an aperture at an end of the lumen distal to the base element.

In some embodiments the distal end of the lumen may optionally comprise a micro valve, such that the lumen can be closed if required following disconnection of the delivery device from the implant apparatus, i.e. following removal of the needles from the elongate members. The micro valve may be closed while the implant apparatus remains implanted in the biological tissue. The elongate member is preferably a micro sheath. "Micro sheath" means a sheath-like structure which is a configured such that is capable of accommodating a micro needle. Thus the dimensions and shape of the micro sheath are preferably compatible with those described above for micro needles. The elongate members or micro sheaths are preferably made of a flexible material such that they are capable of adapting to the orientation of the needles or micro needles of the delivery device.

By "base element" it is intended to refer to any element which forms a base to which the elongate members are connected. Thus the elongate members may be unitary or integral with the material from which the base element is formed, or may be detachably mounted on the base element.

In some embodiments, both the delivery device and implant assembly of the present invention is designed to incorporate a micro fluidic system within the substrate and/or base element. The micro fluidic system may comprise fluid reservoirs, channels, micro pumps and/or further components for delivery of fluids, including fluids comprising cells, through the micro needles and/or elongate members.

In certain embodiments of the present invention, delivery device and/or implant apparatus may comprise one or more monitoring means or sensors for detecting conditions at the injection/implant site, and regulating fluid and/or cell delivery accordingly. US2005/0137536 discloses a micro needle device which may comprise a sensor. The sensor may generate a signal capable of operating a dose control system or flow meter that controls or allows the flow of a drug to the patient. This sensor detects the effect of growth factors including, without limitation, fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), nerve derived growth factor (NGF), insulin like growth factor (IGF) and cytokines modulate cellular behavior via interaction with cell surface receptors. The interaction with the cell surface receptor results in the activation of signal transduction pathways which result in changes in cellular behavior. In the case of growth factors, these changes in cellular behavior include changes in cell survival, changes in cell proliferation, and changes in cell migration. The interaction between the growth factor and its receptor results in a change in conformation, and often a change in phosphorylation, of the receptor and/or the growth factor itself. Similar sensors may be used in the methods of the present invention, for instance in the needles or elongate members to control flow. Optionally, the sensor may control an alarm or indicator that may be visual, or auditory.

The role of growth factors in signaling, induction, proliferation, migration and differentiation of various cells is well known to the scientific community. These growth factors include Epidermal growth factor (EGF), Fibroblast growth factor (FGF), various angiogenic growth factors and other cytokines. Such growth factors may used in the present invention, for instance they may be infused through the implant apparatus into the tissue or may be combined with the cells during the cell delivery through the micro needles.

There is a lot of scientific evidence of using electrical stimulation in inducing and accelerating the cellular proliferation, migration and other wound repair events. The relationship between direct current electricity and cellular mitosis and cellular growth has become better understood during the latter half of the twentieth century. Weiss, in Weiss, Daryl S., et. al., Electrical Stimulation and Wound Healing, Arch Dermatology, 126:222 (February 1990), points out that living tissues naturally possess direct current electro potentials that regulate, at least in part, the wound healing process. Following tissue damage, a current of injury is generated that is thought to trigger biological repair. This current of injury has been extensively documented in scientific studies. It is believed that this current of injury is instrumental in ensuring that the necessary cells are drawn to the wound location at the appropriate times during the various stages of wound healing.

Localized exposure to low levels of electrical current that mimic this naturally occurring current of injury has been shown to enhance the healing of soft tissue wounds in both human subjects and animals. It is thought that these externally applied fields enhance, augment, or take the place of the naturally occurring biological field in the wound environment, thus fostering the wound healing process. This electrical stimulation has been so far utilised in the prior art for modulating the wound healing in a macro- environment (US Patent No: 5,433,735; 4,982,742; 4,911,688 and others). However this has not been used in the prior art for modulating the micro-environment of in vivo tissue engineering or wound healing.

Another useful beneficial addition to electrical stimulation are silver ions as observed by R O Becker, New York in his abstract titled "Effects of electrically generated silver ions on human cells and wound healing". A method of producing local antibiotic effects by means of an ionophoretic technique using free silver ions has been evaluated in vitro and in vivo for more than two decades. The antibiotic properties of the technique have proved useful in both animal and human studies. In the course of determining the optimal clinical methodology for infected open wounds, a significant growth stimulation property resembling local tissue regeneration was noted. This has been traced to either the apparent production of dedifferentiation of normal mature cells or the stimulation of preexisting stem cells in the wound, resulting in the production of large numbers of progenitor cells.

Hence in certain embodiments of the present invention, the elongate members e.g. micro sheaths may incorporate a substrate to release silver ions and to deliver controlled electrical currents along the canal.

The present invention is able to deliver to the necessary inductive cells at a precise depth in the dermis instantly at up to 100,000 sites, for instance in the case of multiple delivery devices into a subject's scalp. Preferably a hair regeneration method may use around 25,000 micro needles with a density of around 50 sites/sq centimeter. In addition, the optional implant apparatus part of the assembly is designed to stay in situ for up to 2 weeks to facilitate formation of a canal for each regenerating hair in the desirable/optimal direction. This canal remains open for a while even after removing this devise because of the epidermal in-growth on a tubular connective tissue. This epidermal in-growth in addition contributes to the formation and growth of the hair in the desirable direction. The in-growth/down-growth of the epidermal epithelium is a natural phenomenon seen in surgical practice as suture marks/hatch marks as a result of sutures left unremoved longer than a week on the face or longer elsewhere. This epithelisation of suture track was also demonstrated in animal studies⁴. The in situ (implant apparatus) component of this device, while allowing the natural in-growth of epidermal lining, is also designed to provide further supply of exogenous or cultured fibroblasts/ epidermal matrix cells /keratinocytesin sequence, continuous or intermittent irrigation of each canal with growth factors such as epidermal growth factor (EGF), anesthetic/antibiotic agents. The hair follicle development process in this micro-environment can be monitored by means of micro-bioassay (lab on chip), Scanning near field optical microscopy (SNOM), nonlinear optical microscopy (super resolution), fiber optics, laser trapping, fiber-optical biosensors, biochips. lasing in micro-cavities; near-field Mie scattering; computer-aided data acquisition; multi-dimensional image visualisation.

The invention will now be described by way of example only with reference to the following specific embodiments, including those shown in the Figures in which:

Figure 1 shows an individual's bald scalp with various components of the present invention, before assembly and application to the bald scalp;

Figure 2 shows the individual's scalp with the assembly applied;

Figure 3 shows the individual's scalp with an implant assembly only in place after removal of delivery devices;

Figure 4 shows the individual's scalp after removal of the implant assembly following completion of a period of in situ location and irrigation;

Figure 5 shows an overview of the assembly when not applied to a scalp;

Figure 6 shows a view of a vertical section through the delivery device, viewed from the side;

Figure 7 shows the under-surface of a delivery device;

Figure 8 shows a side view of one unit of the device assembly;

Figure 9 shows a view of a transverse section through one unit of the device assembly;

Figure 10 shows a front view of an assembly; Figure 11 shows a side view of an assembly;

Figure 12 shows a vertical section through a delivery device;

Figure 13 shows a magnified vertical section through a micro fluidic base part of a delivery device;

Figure 14 shows a view of the underside of a micro fluidic base part of a delivery device;

Figure 15 shows a side view of a strip of micro needles;

Figure 16 shows a side view of two micro needles;

Figure 17 shows a segment of a part of a delivery device after completed assembly of micro needle strips into the micro fluidic base part.

Figure 18 shows a vertical section shows a vertical section through a part of the implant apparatus component B;

Figure 19 shows a vertical section through an assembly according to the present invention;

Figure 20 shows an oblique view from above of a segment of an implant assembly;

Figure 21 shows the flow of materials through one unit of the assembly;

Figure 22 shows various stages of in vivo hair regeneration in the skin by means of one unit of the device.

One embodiment of this micro system is an assembly of many microstructures into 2 detachable sub assemblies A and B. The sub assembly/Primary component 'A' is meant for supporting the whole assembly for the lodgment and instant delivery of first set of cells as a bolus (one time delivery). This subassembly is removed after bolus delivery, while leaving the second, softer biocompatible polymeric subassembly/secondary component 'B' in situ for in-growth of epidermal lining and maintaining the microenvironment by infusion/infiltration of necessary factors at various stages of the process.

In this embodiment the whole assembly is an array of hollow, round, strong micro needles of 'A' (Bolus delivery system) made of for example, carbon, metal, fiber or crystal. The micro needles are designed to be inserted into the central lumen of two luminae micro sheaths (elongate members) of 'B' (In-situ Irrigation system) with porous outer layer made of bio compatible polymer or silicon. Refer to the drawings to follow the description. Each micro needle with its micro sheath is an unit of this Microsystems. The number of the units in the system is determined by the number of hairs to be grown and within safety limits that is without compromising blood circulation to the recipient skin.

The length of these micro needle and micro sheath units are purposefully made unequal within a preferred range between 2 to 5 mm. This length of the each unit determines depth of bolus deposit and future hair follicle. The variable depth for this instant delivery is better accommodated than depositing the whole volume at the same depth. The diameter of each unit is that of an average hair, e.g. preferably between 30 to 100 microns.

These units are supported on the substrate base which has incorporated micro fluidic system. Each unit is mounted on this base at an angle pre determined by the desired angle the future hair.

Hence the total number of the units, their density and obliquity are custom designed for each individual for optimal aesthetic outcome. This is achieved by computer aided optical reading of the skin(scalp) to record existing hair density and pattern(direction and angle) and automated generation of reciprocal copy. The computer needs a denominator for the number hairs in order to generate an automated reciprocal copy. This is determined by the density of hair the individual patient wish to have and maximum density that can be achieved by this approach, preferably 50 hairs/ sq cm. So with this denomination of 50, if he/she has 12 hairs in a given sq cm (fixed point in the grid), the computer generated reciprocal copy will have 38 units marked. Similarly the direction and angle are opposite on the reciprocal copy. Thus generated reciprocal copy is the prototype for micro fabrication of the whole array of microneedles+Amicrosheaths.

Alternatively, a prefabricated devise having some common/popular patterns and density can be offered for those individuals with no residual hairs or those who wish save on the overall cost.

The micro fluidic systems are separate for 'A' and 'B' sub assemblies as they are detachable. It also allows isolated use of 'A' alone if the instant cell delivery alone is required.

The micro fluidic system in the irrigation system (B) consists of two to five main micro trunks along the borders and midline of the base with alternating interdigitating microchannels. The fluid through this system is driven by the incorporated micro pumps, while the volume, cell counts, concentrates are titrated by the microprocessor.

Bolus delivery system (A) unlike 'B' has a hard base, hence made as more than one block to accommodate the contour of the scalp. Depending on size of the area, the whole assembly may need up to 5 blocks. The micro fluidic system for each block is independent, simple, and preloaded.

The micro needle devices can further include a flow meter or other dose control system to monitor flow and optionally control flow through the micro needles and to coordinate use of the pumps and valves.

The device is incorporated with multiple reservoirs with ports to fill. Each reservoir is dedicated to contain specific cell group or growth factor, for delivery through the micro sheath via micro fluidics. The reservoir may be a hollow vessel, a porous matrix, or a solid form including content which is transported there from. The reservoir can be formed from a variety of materials that are compatible with the content contained therein. Preferred materials include natural and synthetic polymers, metals, ceramics, semiconductors, organics, and composites.

Monitoring devises such as lab-on-chip, SNOM/ lasing in micro-cavities are incorporated in some selected sample units. These sensors generate biochemical and optical data feedback to microprocessor for auto regulation of the irrigation system. This data can be recorded for research and further development of this system.

Useful sensors may include sensors of pressure, temperature, chemicals, and/or electromagnetic fields. Biosensors can be employed, and in one arrangement, are located on the micro needle surface, inside a hollow or porous micro needle, or inside a device in communication with the body tissue via the micro needle (solid, hollow, or porous). These micro needle biosensors may include any suitable transducers, including but not limited to potentiometer, amperometric, optical, magnetic and physiochemical. An amperometric sensor monitors currents generated when electrons are exchanged between a biological system and an electrode.

Materials and methods of manufacture which may be utilized in manufacture of a delivery device of the present invention are disclosed in general terms, for example, in WO 99/64580, WO 00/74763, WO 01/49346, US Application No. 60/323,417, US Application No. 60/323,852, US Application No. 60/325,522, each of which is hereby incorporated by reference.

A number of versatile techniques of micro fabrication of these microstructure/MEMS assemblies exists in the prior art and it is rapidly advancing. Traditional and most widely used technique by most of the manufactures currently is photolithography with many variations. Some of these which have been applied to developing a range of micro fluidic systems are US Patent No: 6,824,697; 5,389,196; 6,821,475; 6,877,964; 6,890,493; 6,896,821 and many others.

Currently various innovative methods being developed to make this micro fabrication ever versatile, rapid and less expensive. The following references highlight these developments.

1. Hui Yu, of Boston university in his abstract titles "Flexible Fabrication of Three- dimensional Multi-layered Microstructures Using a Scanning Laser System" described a most versatile micro fabrication using a scanning laser system. This system rapid processing of freeform multi-layered microstructures. More importantly it enables rapid prototyping of three-dimensional (3D) micro-devices at low cost. The capabilities of three-dimensional manufacturing, inclined patterning, and multi-layered manufacturing are demonstrated. Specifically, both in-plane and out-of-plane processing is feasible using spot-by-spot controllable laser pulsing. The laser processing perpendicular to the specimen surface is realized by fine tuning the focus level and laser intensity. 3D microstructures requiring gaps between layers, such as cantilever beams and embedded channels, are demonstrated in a single layer. Furthermore, a variety of 3D microstructures and microchips including micro concave and convex lens, micro needle arrays, micro valve and micro fluidic capture chip, are manufactured with a bio-benign material, SU-8 and characterized. Those individually developed microstructures and microchips are readily to be integrated as disposable components in a variety of "lab-on-chip" applications. Compared to the existing manufacturing techniques, our direct laser writing method greatly simplifies fabrication processes, potentially reducing the design-to- fabrication cycle to a few hours.

2. A.P. Sudarsan et al of Texas A&M University in their abstract titled "Micro fabrication of 3D Structures Using Novel Thermoplastic Elastomers" described another verstile option. The said method involves the use of novel thermoplastic elastomers synthesized by dissolving readily available polystyrene-(polyethylene/ polybutylene)- polystyrene (SBS/SEBS) triblock copolymers (e.g., Kraton™ G series) in hydrocarbon oils for which the ethylene/butylene midblocks are selectively miscible. The insoluble styrene endblocks phase separate into localized domains, resulting in the formation of a 3-D gel network. The resulting gels are elastic solids at room temperature and share all of the desirable features of PDMS (e.g., biocompatibility, electric neutrality, optical transparency). They also have the advantage of being melt-processable in the vicinity of 100 ⁰C. Fabrication of micro fluidic devices is accomplished taking an impression from a pre-heated master onto a slab of the elastomer. We demonstrate the suitability of these elastomeric materials as substrates for microfluidic applications by constructing devices for DNA electrophoresis and diffusive transport studies. We are also able to easily assemble a variety of complex multilayered structures in only a few minutes. A further advantage involves making multi-height structures in a single micro-device.

3. S. Hardt of Darmstadt University of Technology, "Design Paradigms and Methodologies for Microfluidics" This paper reviews and compares different strategies for designing and building up microfluidic systems, with a special emphasis on the system aspect as opposed to single components capable of performing only special operations. An overwhelming number of microfluidic components have been reported in the literature in the past few years, but convincing concepts to integrate those components to a unit performing complex microfluidic operations seem to be quite rare. Opposed to such bottom-up approaches there are some concepts of top-down character, in which a rather monolithic system is able to perform a number of complex operations. Another competition occurs between continuous-flow and droplet-based systems. Droplet-based microfluidics is a quite attractive concept when small sample amounts need to be processed in a well-defined manner. However the physics of moving droplets is much more involved than that continuous flow. Correspondingly, the design and simulation of droplet-based microfluidic systems poses considerable challenges, and effects such as contact-angle hysteresis can make a well-controlled transport of such droplets quite difficult. The last major competition considered here is that between pressure-driven flow and electrokinetic flow, possibly the two most popular concepts for fluid transport on the microscale. In this work the current status of the competition between different paradigms in microfluidics is reviewed, and the advantages and disadvantages of the approaches are compared. It is hoped that by virtue of such an analysis of the state-of-the- art in microfluidics, future directions might become clearer.

A hair regeneration process by using this in vivo tissue engineering model will now be described in general terms. A whole assembly of microneedles with microsheath segment of this devise is inserted into premarked and topically anesthetised scalp. Instant delivery of the inductive cells from 'A' into the dermis at a depth ranging from 2 to 4 mm predetermined by the length of the microneedle units is done at all the sites at once. This is the beginning of the whole process. Now the microneedle of the component 'A' with its base is removed soon after delivery of the inductive cells. This leaves porous microsheaths with its microfluidic base of component 'B' in situ. Due to the flexible nature of the base of 'B', it drapes well on the convex contour of the scalp as a thin layer.

The microneedle-microsheath unit is held in the skin at a predetermined angle, delivering an aggregate of inductive cells and angiogenic growth factors at the tip of the microneedle. At this stage, inductive cells begin to proliferate at this de novo site and the canal created by insertion of the microneedle is maintained by the presence of microsheath.

Regenerative process also begins at epidermis epithelium on the surface 24-48 hrs later, as this regenerated epithelium can not bridge the whole due to presence of microsheath in situ. This microsheath redirects the direction of the advancing end of the proliferating epithelium in to the canal around the microsheath resulting in epithelial ingrowth over weeks. While proliferation of cells is taking place at the two ends of this canal, an inflammation process followed by repair process sets in around canal in an effort to obliterate this canal. Again due to the presence of microsheath, this repair process lays collagen connective tissue around this canal instead of obliteration. This regeneration and repair processes takes place over a 3 weeks period in unmodulated normal circumstances. So, at 1 week these events at their early stages.

Both these events are accelerated by the irrigation and stimulation system, by providing fibroblasts to lay collagen around the canal followed by the supply of epidermal matrix cells/cultured keratinocytes for lining of this canal and electrical stimulation. In addition to irrigation with Fibroblast growth factor (FGF), Epidermal growth factors(EGF) and other angiogenetic factors at appropriate stages. The process of hair follicle differentiation, formation of connective tissue sheath and outer root sheath with epidermal lining is accelerated. At this stage, remaining 'B' component of the devise is also withdrawn.

The canal remains open even after removal of the microsheath as it is fully lined by outer root sheath on connective tissue sheath. This provides the path for emerging hair. Although in the above the invention has been particularly described with reference to hair regeneration methods, the invention is not limited to such applications. The delivery devices, implant apparatus and assembly of the present invention may also be used in further applications where is desired to regenerate tissue in vivo, in particular where a controlled microenvironment can be used to stimulate cell growth and differentiation.

Applications of this in vivo tissue engineering in the light of current knowledge and evidence are 1. Hair regeneration, 2. Islets of Langerhans for treating Diabetes Mellitus, 3. Skin regeneration, 4. Cartilage regeneration, 5. Myocardium regeneration, 6. Bone regeneraion, 7. Wound healing etc.

Another embodiment of this invention is to have similar assembly at a smaller scale than scalp at a convenient location in the subcutaneous fat plane. This requires modification of the dimensions of the array of microneedle/microsheath, supplying appropriate stem cells with alpha and beta cell lineage, ground substance and angiogenic factors. In this embodiment, the canal is irrigated with epidermal inhibiting factors to avoid unnecessary epithelialisation of track.

Similarly, in other embodiments of the present invention, assemblies and cell lineages are appropriately modified or chosen according to the tissue that is required to be regenerated based on the same concept.

One object of the present invention is to create a bioreactor micro-environment within the body by instant and/or controlled delivery of cells, ground substance and mediators. The present invention enables a novel concept of "In vivo-de novo tissue engineering" such as regeneration of hair, skin, islets of langerhans, bone, cartilage, Myocardium and others.

This embodiment is concerned with creating a controlled bioreactor micro-environment with in the body to achieve the objective of regeneration of tissues. This embodiment involves delivery of adult or embryonic stem cells/gene vector, ground substance, cytokines including appropriate growth factors and angiogentic factors to a place where the tissue would otherwise exist or to an alternative location. The delivery can be controlled in terms on timing (instant/continous/intermittent/any combination), cell counts, cell types and volume/concentation/types of the cytokines. The process of tissue regeneration can be controlled by monitoring with bioassay and direct visualisation which in turn control the delivery.

By this develop ement, it is possible to overcome the key limitations such as need for a scaffold, vascularisation of regenerating tissue, transfer of tissue, integretion, maintainance of regenerated tissue through the whole process. Because, the body responds to controlled supply of the cells and mediators by laying the scaffold, provides nutrition, immunity and blood supply by ingrowth of new blood vessels and gradually incorporates into the body to benefit from its function or presence.

However, the concept of In vivo-De novo regeneration of certain tissues such as bone, cartilage, myocardium and any other deeper tissue may be preferably achieved by using biodegradable implant device which dissolves as the tissue is being formed or biocompatible implant device which may remain as scafold. These delivery devices for deeply located tissues may require minimal access or open surgery to place the implant device. Thus placed device may or may not be required to be removed after the process depending on the material such as biodegradable, biocompatible scafold or not.

The design/devise to achieve the objective of this invention is a custom made assembly of microfluidics and a single or an array of hollow/solid microneedles with porous microsheath. The distribution and delivery of cells/mediators through this micro- assembly is facilitated and controlled by micropumps, lab-on-chip, microoptics, microprocessor, microcontainers/sinks and any other microstuctures as required.

This micro-assembly can be custom made as required by the architecture of the said tissue to be regenerated, as demanded by the researcher, physician and the individual patient. This objective can be achieved by micro fabrication using various methods of nano technology which includes scanning laser, photolithography, moulding and others. A preferred embodiment of this invention is its application in vivo hair regeneration for restoring the lost hair due to baldness, burns, trauma and other causes.

Understanding the embryogenesis of hair in fetus was vital in developing this invention. This requires mesenchymal stem cells with hair follicle inductive phenotype, undifferentiated/dedifferentiated epidermal matrix cells capable of differentiation into the various sheaths, hair fiber structures and a hair canal for emergence of regenerated hair and factors (cytokines) signaling between these two groups of cells. This also requires suitable method of delivering the said cells and factors at appropriate stages of hair regeneration as well as creating a micro-environment close to fetus skin.

There are no successful prior art methods to ensure another important requirement of creating a micro-environment to simulate the bioreactor with in the skin for optimising and controlling the environment for healthy hair follicle regeneration and guiding each budding hair in the right direction.

References:

1. Rushton DH, Ramsay ID, Norris MJ, Gilkes JJ. Natural progression of male pattern baldness in young men. CHn Exp Dermatol 1991; 16: 188-192.

2. Li L, Hoffman RM. The feasibility of targeted selective gene therapy of the hair follicle. Nature Med 1995; 1: 705-706.

3.McElwee KJ, Hoffmann R. Growth factors in early hair follicle morphogenesis. Eur J Dermatol 2000 Jul-Aug;10(5):341-50

4. Ordman LJ, Gillman T: Studies in the healing of cutaneous wounds: II. The healing of epidermal, appendageal, and dermal injuries inflicted by suture needles and by suture material in the skin of pigs. Arch Surg 93:883, 1966

The invention will now be described more particularly with respect to the embodiments shown in the Figures. In one embodiment, an assembly according to the present invention is designed to comprise 2 major components. A first or primary component (Bolus delivery system or component A) comprises a solid or flexible microfludic base or substrate from which an array of needles emerges at defined angles. Secondly, a secondary element (implant apparatus or In-situ irrigation system or component B) comprises a flexible/mouldable microfludic base with an array of elongated cylindrical members or sacs.

The primary component is designed to achieve penetration of needles in the desired/predetermined angles and depth to start delivery of cells, growth factors and/or ground substance.

The secondary component is made of bioinert/biocompatible material to be mounted on the primary element for penetration and to remain in the needle tracks much longer after withdrawal of primary component, for intermittant delivery or continuous irrigation of needle tracks with varoius growth factors, cells and/or ground substance.

The primary component can be used in isolation or in combination with the secondary component.

As shown in Figure 1, an assembly according to the present invention comprises several delivery devices A and an implant assembly or scaffold B. Each delivery device A comprises a plurality of microneedles 1 mounted on a substrate. The substrate may comprise a solid or flexible base part, containing a microfluidic system as described below.

The microneedles 1 are mounted on the substrate at predetermined angles. The orientation of the needles may vary between needles comprised in the same delivery device A and also between delivery devices A. Each delivery device may be arranged to be contacted with the scalp of an individual at a predetermined region, such that an angle or direction in which each microneedle penetrates the scalp of the individual is dependent on the position on the scalp. The angle of penetration of each microneedle is chosen in order to correspond to the direction of natural hair growth at each position on the individual's scalp. Thus in the example shown a first delivery device A (temple) is arranged to be contacted with the individual's temple region. The microneedles of the first delivery device are oriented towards the midline of the individual's scalp. A second delivery device A (vertex) is arranged to be contacted with a vertex region of the scalp and contains needles oriented towards the crown. A third delivery device A (crown) is arranged to be contacted with the crown region of the scalp and contains needles oriented in a swirl pattern. Further delivery devices are adapted to be contacted with the frontal and occipital regions and contain needles oriented towards the crown region.

Each scalp area (temple, vertex, etc.) may require a number of individual delivery devices to provide full coverage, depending on the size of each delivery device. In embodiments where the substrate of each delivery device is substantially flat or planar, it is preferable to use a number of small delivery devices to cover each area, in order to accommodate the contours of the skull and to achieve the desired direction of hair growth in each region. An alternative option is to use one or more delivery devices whose substrates are configured to be complementary to a portion of, or the whole of the scalp.

The microneedles are formed from a rigid material such that they maintain their orientation following insertion into the implant apparatus. Each microneedle comprises a central bore and has a sharp tip to facilitate penetration into a biological tissue.

Implant apparatus B comprises a plurality of elongated/cylindrical members or sacs in the form of microsheaths 2 which are attached to a base element. The base element is a mouldable microfluidic system. Hence, the implant apparatus adapts to the shape to fit onto the individual's scalp. The microsheaths 2 extend away from a lower surface of the implant apparatus. The microsheaths in the element 'B' are spaced similar to that of microneedles in the delivery devices 'A'. Each microsheath comprises a central cannula which can accommodate (i.e. at least partially surround) the shaft of a microneedle. The central cannula/lumen of each microsheath comprises an opening 3 at an end proximal to the base element, in order to permit insertion of the microneedle through the base member into its internal cavity. The microsheaths are formed from a flexible material such that they can adapt to an orientation determined by the microneedle following its insertion. The microsheaths may have blunt (i.e. not sharp) tips, and may be porous along their length.

Figure 2 shows how it looks when whole devise assembly is applied to the bald scalp. This is achieved by inserting the microneedles 1 of primary element 'A' into a central lumen or cannula of microsheaths 2 of secondary element 'B'. At this stage flexible B is mounted on rigid A i e, the devise is now completely assembled. Such assembled devise is still flexible due to the joints between various parts/blocks (temple, vertex, occiput etc) of the base of primary element 'A'. The flexibility allows careful insertion of microneedle-microsheath complex of each block separately. The figure 2 is the picture after completing the insertion of all the blocks/parts of the devise.

Following delivery of cells through the microneedles, the delivery devices A are removed leaving the implant/irrigation apparatus B in situ in the subject's scalp as shown in Figure 3. The implant apparatus B comprises a fluid transport means, for example a microfluidic system, integrated into the base element for transferring fluids through the microsheaths and into the scalp. Components of the microfluidic system visible in Figure 3 include a primary channels 4, secondary channels 5 and an input means 6. Fluid may be introduced into the implant apparatus via the input means 6, and distributed throughout the apparatus by means of the primary channels which subsequently connect to the secondary channels. The secondary channels supply fluid to individual microsheaths 2 and thus into the scalp of the subject.

Figure 4 shows the individual's scalp after removal of the implant apparatus B following a period of in situ irrigation. Epithelial tracks 7 induced by the microneedles and microsheaths are visible on the patient's scalp.

Figure 5 shows a perspective view of an assembly comprising a number of delivery devices A connected to an implant apparatus B, but without being applied to a scalp. The underside of the apparatus (which would be contacted with the scalp) is visible, including the microneedles and microsheaths that protrude through the lower surface of the implant apparatus.

As illustrated in Figure 6, the delivery device A and implant apparatus B interconnect such that the microneedles 1 are accommodated within the microsheaths 2. As the microsheaths are flexible, they adapt to the orientation of the microneedles and extend away from the lower surface of implant apparatus at predefined angles. Figure 7 shows a view from below of the same delivery device, whose base element is in the form of a square of dimensions lcm by lcm.

A close-up view of a single unit of the assembly can be seen in Figure 8, a transverse section of which is shown in Figure 9. A "unit" here refers to a single microneedle 1 accommodated in a single microsheath 2. The microneedle 1 has a bore 12, which is continuous with a fluid transport means (e.g. a microfluidic system) integrated into the delivery device. The shaft of the microneedle 1 is accommodated within (i.e. at least partially surrounded by) a central lumen or cannula 13 of the microsheath, the tip of the microneedle protruding through an aperture 11 at an end of the microsheath distal to the base element.

The microsheath 2 comprises an inner wall 9 and an outer wall 8, which together enclose an internal cavity or peripheral cannula 14. The walls of the microsheath comprise pores 10 which permit fluid and/or cell transfer from the internal cavity of the microsheath to the exterior. The microsheath 2 is also connected to a microfluidic system separate from that of the delivery device, the microfluidic system supplying fluid to the internal cavity of each microsheath.

Figures 10 and 11 show front and side views respectively of an assembly comprising delivery devices A and implant apparatus B. Figure 12 shows a cross-section through a 1 cm by 1 cm delivery device A as shown above, but apart from an implant apparatus. The figure illustrates how strips of needles 1 may be connected to a microfluidic system in the substrate of the device. The substrate comprising the microfluidic system is further illustrated in Figures 13 and 14 (Figure 13 is an inverted vertical section through the substrate shown in Figure 14), whereas a strip of microneedles to be mounted into the substrate is shown in Figure 15. Figures 13 to 15 are intended to be viewed together in the relative positions shown to better illustrate the alignment of the components shown therein.

A lower surface of the substrate comprises a plurality of parallel ridges 15 running transversely across the surface. The lower surface is facing upwards in Figure 14 but is facing downwards in Figure 13. The ridges 15 appear V-shaped (forming a triangular profile) when viewed in a vertical section as shown in Figure 13. A rectangular groove 16 runs along one side of each ridge, in which are set apertures 17 at predetermined positions/intervals. Parts of the groove 16 between apertures 17 are marked 18 in the figures. Each aperture 17 opens into a bore 19 in the ridge 15, which is continuous with a microfluidic channel 20 running across the substrate in a direction perpendicular to the ridges. The substrate may comprise a plurality of parallel microfluidic channels 20 running orthogonally to the ridges, and optionally further microfluidic channels running parallel to the ridges as shown.

As shown in Figure 15, a series of microneedles are connected in a strip by a narrow connecting member 21. A nozzle 22 of each needle is located proximal to the connecting member 21. The dimensions (e.g. length and width) of the connecting member 21 are selected to be complementary to those of the grooves 16 in the ridges of the substrate.

The strip of needles is mounted into the substrate such that the connecting member 21 fits into the groove 16. The nozzle 22 connects the bore of each microneedle 1 to an aperture 17, permitting fluid transfer from the microfluidic system into the microneedle bore. Figure 17 further illustrates how the strip of microneedles and substrate interconnect. Thus the slope (or angle) of the ridges containing the grooves 16 can be used to set the orientation of a strip of microneedles 1 with respect to the substrate. Figure 17 shows how the microneedles are oriented at an angle a with respect to a plane X defined by the substrate. As several strips of microneedles can be mounted in the substrate shown, the orientation of each strip of microneedles can be individually set by the slope of the groove/ridge into which it fits. As shown in Figure 16, the microneedle shape and length can also be variable.

Figure 18 shows a corresponding vertical section through an implant apparatus B illustrating the microfluidic system comprised therein. A microfluidic channel 25 supplies fluid to the peripheral cannula 14 of the microsheaths, and a microvalve 24 keeps the opening to the central cannula 13 closed when it does not contain a microneedle.

Figures 19 shows a sectional view of a 1 cm by 1 cm delivery device connected to an implant apparatus, including the separate microfluidic systems of each component. Figure 20 shows a view of a 1 cm by 1 cm part of an implant apparatus B.

Figure 21 illustrates the function of the assembly by reference to a single unit thereof. The arrows with a solid head 26 indicate how, following implantation of the assembly into the scalp of a subject, a fluid containing cells (such as dermal papilla cells) is delivered via the microfluidic channel 20 of the delivery device A and the microneedle 1 into the tissue. The cells are delivered to a location at the bottom of the microneedle track.

The arrows without a solid head 27 indicate how fluid flow through the implant apparatus B proceeds via a microfluidic channel 25 into the microsheath 2 and out into the tissue via the pores 10. Note that the site of irrigation in the needle track is determined by the locations of the pores 10 in the microsheath. Thus the pores can be arranged such the whole, or only a part of (for instance only a lower part) the needle track is irrigated. The fluid irrigated through the implant apparatus B may comprise growth factors (e.g. EGF, FGF, PDGF) which are known to skilled person to enhance or control hair follicle growth or cell differentiation. Different growth factors or cells can be irrigated into the tissue at different times following implantation, as required by the particular stage of regeneration while the implant apparatus is implanted in the individual's scalp.

The various stages of in vivo hair regeneration in the skin using the present assembly are shown diagrammatically in Figure 22. Figure 22 represents a timeline showing the development from left to right of a hair in a single needle track, except that the normal anatomy of a hair follicle 28 and hair in an adult is shown on the left for reference. Thus each track shown in Figure 22 represents a particular stage of regeneration of a single hair at the specified number of days after starting the procedure.

At the start of the procedure on day 1, a unit of the present assembly, comprising a microneedle surrounded by a microsheath, penetrates the skin creating a needle entry wound 31 and a needle track 30. The orientation of the microneedle in the Delivery device may determine the angle, at which the microneedle penetrates the skin, and thus the angle and direction of the needle track 30.

A standardized number of dermal papilla cells and/or sheath cells 29 are delivered through the microneedle and deposited at the base of the needle track. The delivery device including the microneedle is then removed leaving the implant apparatus including the microsheath in place in the needle track.

After the first day, the deposited dermal papilla/sheath cells 29 start to proliferate to produce proliferated mass of cells 32, as shown in Figure 22 at "Day 3". The needle track 30 is irrigated with growth factors and/or cytokines (e.g. EGF, FGF). In response to a breach in the skin epithelium at wound 31, multiplication of skin epithelial cells is triggered at the edge thereof. Epithelial cells 33 proliferate and migrate down the needle track wall 30. This process continues due to lack of contact inhibition and is accelerated by the growth factors/cytokines supplied via the microsheath.

Cells may also be supplied through the microsheath into the needle track. For instance an exogenous supply of cultured keratinocytes and/or dermal sheath cells 34 may be delivered through the microsheath to enhance the epithelial lining of the needle track.

Typically the supply of exogenous cultured cells and growth factors dramatically reduces the amount of time required for complete epithelialisation of the needle track, for instance from around 14 days to 7 days or less. As shown in Figure 22, by day 5 the wall of the needle track 30 is almost completely lined by epithelial cells 33, derived either from exogenous cells introduced via the microsheath or ingrowth of endogenous epithelial cells. Further proliferation and/or differentiation of the deposited dermal papilla cells has by this stage led to a mass of cells 35 with the beginning of neovascularisation. The microsheath is kept in place at this stage, mainly to support the needle track and to keep it open and oriented at the correct angle, until epithelialisation is complete or it is capable remaining open and correctly oriented in the absence of support from the microsheath.

By day 7, epithelialisation of the needle track is virtually complete. Collagen has also been deposited around the track by this stage, which supports the track and makes it more rigid. For these reasons it is now possible to consider removing the implant apparatus comprising the microsheath. The epithelialisation and collagen deposition have together strengthened the track such that it is likely to remain open after removal of the microsheath, without collapsing or distorting such that its orientation is substantially altered. Even if epithelialisation is incomplete, the collagen deposition may be sufficient to keep the track open by this stage after removal of the microsheath.

Also by day 7 the deposited dermal papilla cells have become a well differentiated cell mass 36, and keratin 37 has started to form on the summit of the dermal papillae, signaling the start of hair shaft growth.

At day 14, the microsheath has been removed for about a week. A well-formed hair follicle 38 is in the process of further definition and maturation. The deposited dermal papilla cells have developed into a well-demarcated group of cells 39 within the follicle. The needle track has now developed into fully lined epithelial track 41 simulating a natural hair canal, within which a hair 40 is beginning to grow. The needle wound 31 has developed into pore 42 in the skin, allowing the growing hair to emerge in the desired angle dictated by the orientation of the microneedle in the delivery device.

Claims

1. A delivery device comprising a plurality of needles mounted on a substrate, the device being adapted to deliver cells through the needles into a biological tissue.

2. A delivery device according to claim 1, wherein each needle is oriented at a predetermined angle with respect to the substrate.

3. A delivery device according to claim 1 or claim 2, wherein one or more of the needles is oriented at an angle other than perpendicular to the substrate.

4. A delivery device according to any preceding claim, wherein the needles are oriented in a plurality of different directions with respect to the substrate.

5. A delivery device according to claim 4, wherein the needles are oriented along a plurality of axes which form different angles with a plane defined by the substrate.

6. A delivery device according to any preceding claim, wherein each needle extends away from a common surface of the substrate.

7. A delivery device according to any preceding claim, wherein a length of each needle is 0.5 to 20 mm.

8. A delivery device according to claim 8, wherein the length of each needle is 2 to 5 mm.

9. A delivery device according to any preceding claim, wherein each needle is oriented at an angle of 20 to 90° with respect to a plane defined by the substrate.

10. A delivery device according to any preceding claim, comprising a plurality of needles of different lengths.

11. A delivery device according to any preceding claim, wherein an internal diameter of each needle is 10 to 100 μm.

12. A delivery device according to any preceding claim, wherein the plurality of needles comprises an array of microneedles.

13. A delivery device according to claim 12, wherein the microneedles are hollow.

14. A delivery device according to any preceding claim, comprising 10 to 150 needles per square centimeter of substrate.

15. A delivery device according to claim 14, comprising 20 to 80 needles per square centimeter of substrate.

16. A delivery device according to any preceding claim, further comprising one or more reservoirs for holding a liquid for further delivery through the needles.

17. A delivery device according to any preceding claim, further comprising a control means for controlling flow of a liquid through the needles.

18. A delivery device according to any preceding claim, further comprising a monitoring means for detecting one or more conditions, and regulating the control means in response to the detected conditions.

19. A delivery device according to claim 18, wherein the monitoring means comprises a sensor located on a surface of or inside a needle.

20. A delivery device according to claim 19, wherein the sensor detects pressure, temperature, or biochemical, optical, or electromagnetic conditions at a delivery site.

21. An implant apparatus comprising a plurality of elongate members extending away from a base element, each elongate member enclosing a first lumen, the implant apparatus being adapted to co-operate with a delivery device comprising a plurality of needles mounted on a substrate, such that the needles of the delivery device can be accommodated at least partially within the first lumina of the elongate members.

22. An implant apparatus according to claim 21, wherein each elongate member comprises a second lumen, the second lumen being adapted to permit delivery of a liquid and/or cells into a biological tissue in which the apparatus is implanted.

23. An implant apparatus according to claim 22, wherein an outer wall of the second lumen of each elongate member is porous, thereby permitting migration of cells and/or fluids from the second lumen into the tissue.

24. An implant apparatus according to any of claims 21 to 23, wherein the elongate members extend away from a lower surface of the base element, and an upper surface of the base element is adapted to contact the substrate of the delivery device.

25. An implant apparatus according to any of claims 21 to 24, wherein the first lumen of each elongate member comprises a first aperture at an end proximal to the base element, such that a needle of the delivery device can pass through the aperture into the first lumen of the elongate member.

26. An implant apparatus according to any of claims 21 to 25, wherein the first lumen of each elongate member comprises a second aperture at a distal end, such that when the implant apparatus is fully engaged with the delivery device, a tip of the needle protrudes out of the distal end of the elongate member through the second aperture.

27. An implant apparatus according to claim 26, wherein each elongate member comprises a valve at the distal end of the first lumen, the valve being capable of regulating opening and closing the second aperture.

28. An implant apparatus according to any of claims 21 to 27, wherein the elongate members and/or the base element are formed from a biocompatible material.

29. An implant apparatus according to any of claims 21 to 28, wherein the elongate members are formed from an electrically conductive material.

30. An implant apparatus according to any of claims 21 to 29, wherein the elongate members are formed from a polypyrrole-hyaluronic acid composite material.

31. An implant apparatus according to any of claims 21 to 30, wherein the elongate members extend away from a lower surface of the base element, the lower surface of the base element being adapted to contact an outer surface of a biological tissue into which the elongate members extend when the apparatus is implanted into the tissue.

32. An implant apparatus according to claim 31, wherein the biological tissue is human skin.

33. An implant apparatus according to claim 31 or claim 32, wherein the base element is formed from a flexible material.

34. An implant apparatus according to any of claims 21 to 33, wherein the base element and/or elongate members are formed from a flexible material, such that when the delivery device is engaged with the implant apparatus, the elongate members are capable of individually adapting to orientations compatible with predefined orientations of the needles which are accommodated in the first lumina of the elongate members.

35. An implant apparatus according to any of claims 21 to 34, wherein the plurality of elongate members comprises an array of microsheaths.

36. An implant apparatus according to any of claims 21 to 35, wherein a length of each elongate member is 0.5 to 10 mm.

37. An implant apparatus according to claim 36, wherein the length of each elongate member is 2 to 5 mm.

38. An implant apparatus according to any of claims 21 to 37, comprising a plurality of elongate members of different lengths.

39. An implant apparatus according to any of claims 21 to 38, wherein an internal diameter of each elongate member is 10 to 100 μm.

40. An implant apparatus according to any of claims 21 to 39, comprising 10 to 150 elongate members per square centimeter of base element.

41. An implant apparatus according to any of claims 21 to 40, comprising 20 to 80 elongate members per square centimeter of base element.

42. An implant apparatus according to any of claims 22 to 41, further comprising one or more reservoirs for holding cells and/or fluids, the reservoirs being connected to the second lumina of the elongate members such that the cells and/or fluids can be passed through the elongate members for irrigation of a biological tissue into which the apparatus is implanted.

43. An implant apparatus according to claim 42, further comprising a control means for controlling flow of the fluid through the elongate members.

44. An assembly comprising one or more delivery devices according to any of claims 1 to 20 connected to an implant apparatus according to any of claims 21 to 43.

45. A method for delivering cells into a biological tissue, comprising delivering the cells through a delivery device comprising a plurality of needles mounted on a substrate.

46. A method according to claim 45, wherein the delivery device is as defined in any of claims 1 to 20.

47. A method according to claim 45 or claim 46, wherein the method is a cosmetic treatment method.

48. A method according to claim 47, wherein the method is for inducing hair regeneration and/or treating hair loss.

49. A method according to claim 48, wherein the cells comprise stem cells.

50. A method according to claim 48 or claim 49, wherein the cells comprise inductive dermal papilla cells, epidermal cells, fibroblasts and/or epidermal matrix cells.

51. A method according to any of claims 48 to 50, wherein the length and orientation of the needles of the delivery device are selected such that the cells are injected into the dermal layer of skin.

52. A method according to any of claims 48 to 51, wherein the cells are injected into skin on the scalp of a subject.

53. A method according to any of claims 45 to 52, comprising:

(a) connecting one or more delivery devices to an implant apparatus as defined in any of claims 21 to 43 to form an assembly in which the needles of the delivery device are accommodated in the elongate members of the implant apparatus;

(b) implanting the assembly into a biological tissue, such that the needles of the delivery device and the elongate members of the implant apparatus penetrate into the tissue;

(c) delivering the cells into the tissue through the needles of the delivery device.

54. A method according to claim 53, further comprising detaching the delivery device from the implant apparatus whilst the implant apparatus remains implanted in the biological tissue.

55. A method according to claim 54, further comprising introducing further cells and/or biologically active agents into the tissue through the elongate members of the implant apparatus after the delivery device has been removed.

56. A method according to claim 54, wherein the biologically active agents comprise cytokines, growth factors or angiogenic factors.

57. A method according to claim 54, wherein the method is for regeneration of scalp hair, and the implant apparatus is retained in the scalp for a period sufficient to permit ingrowth of an epidermal epithelium into a cavity in the scalp surrounding the elongate member.

58. A method according to claim 57, wherein the implant apparatus is retained in the scalp for 1 to 30 days, preferably 4 to 10 days.

59. A method according to claim 56, wherein the biologically active agents comprise agents which promote hair growth.

60. A method according to claim 57, further comprising inducing an electric current in the elongate members after the implant apparatus has been implanted into the scalp.

61. A method according to claim 60, wherein the electric current is generated by means of release of silver ions by the elongate members.

62. A method according to any of claims 48 to 61 , further comprising scanning a patient's scalp in order to determine one or more properties of existing hair growth, and configuring the needles of the delivery device according to the determined properties.

63. A method according to claim 62, wherein the property to be determined comprises a density of hairs per unit area at particular locations on the patients scalp, and the configuring comprises setting a number of needles per unit area of the substrate of the delivery device in order to regenerate a sufficient number of hairs at each location such that the density of hairs on the patient's scalp is restored to a normal level.

64. A method according to claim 62, wherein the property to be determined comprises a direction of hair growth at particular locations on the patients scalp, and the configuring comprises setting orientations of the needles in the delivery device such that hair growth is induced in predefined directions at the particular locations, the predefined directions corresponding to the determined directions of natural hair growth at the particular locations.

65. A method according to any of claims 48 to 64, comprising configuring the number of needles per unit area and/or orientation of needles in the delivery device in order to achieve a desired density of hairs and/or direction of hair growth at particular locations on a patient's scalp.

66. A method according to claim 65, wherein the direction of hair growth is determined by an angle or direction at which the needle penetrates the skin at each location.

67. A method according to claim 65 or claim 66, wherein the direction of hair growth at each location is determined by the orientation of an elongate member of an implant apparatus implanted in the scalp at each location.

68. A method according to any of claims 45 to 67, further comprising anaesthetizing the biological tissue before introducing the delivery device.

69. A method according to claim 44 and 45, wherein the method is for skin regeneration.

70. A method according to claim 44 and 45, wherein the method is for cartilage and/ or bone regeneration.

71. A method according to claim 44 and 45, wherein the method is for the treatment of diabetes mellitus.

72. A method according to claim 71, wherein the method is for the regeneration of islets of Langerhans cells in the tissue.

73. A method according to claim 72, wherein alpha and/or beta islet cells are introduced into the tissue.

74. A method according to claim 45, wherein the method is for regeneration of myocardium.

75. A method according to claim 45, wherein the cells are derived from stem cells.

76. A method according to claim 53, wherein the implant apparatus is biodegradable or dissolvable.

77. A method according to claim 53, further comprising removing the implant apparatus from the biological tissue.

78. A method according to claim 45, for the treatment of stuck and/or non-healing wounds.

79. Use of a delivery device comprising a plurality of needles attached to a substrate, to deliver cells into a biological tissue.

80. Use of a delivery device according to claim 79, further comprising using the device to deliver growth factors, cytokines and/or extracellular matrix into the tissue.

81. Use of a delivery device according to claim 79 or claim 80, further comprising using the device to deliver genes and/or gene vectors into the tissue.

82. A delivery device comprising a plurality of needles mounted on a substrate, the device being adapted to deliver growth factors, cytokines, extracellular matrix, genes or gene vectors through the needles into a biological tissue.

83. A delivery device according to claim 1 or claim 82, wherein the biological tissue is a living mammalian tissue.

84. A delivery device according to claim 83, wherein the biological tissue is a living human tissue.