Inventors: Atkins, Jane, Leek, Michael D, Kemp, Paul, Wolowacz, Richard, Teumer, Jeffrey
Assignee: Intercytex Limited
Publication date: 2007-11-01
Invention summary:
Use of a controlled delivery device for the direct delivery of inductive dermal sheath cells and/or inductive dermal papilla cells into the dermal layer to induce hair follicle formation in a dermal layer lying beneath an outer skin surface
Abstract:
Baldness or hair loss has been treated using pharmaceutical drugs and/or hair transplant surgery. The present invention relates to an improved method for treating baldness. In one aspect, there is provided a method for inducing hair follicle formation in a dermal layer lying beneath an outer skin surface, comprising delivering inductive dermal sheath cells and/or inductive dermal papilla cells directly into the dermal layer using a controlled delivery device. Delivery of the inductive cells need not be into pre-existing pores. Use of a controlled delivery device for the treatment of baldness is a further aspect of the invention
Description:
The present invention relates to delivery of inductive cells for generation of new organs, for example hair follicles.
Mammalian skin is composed of two layers, an outer layer called the epidermis and an inner layer called the dermis. The epidermis is several cell layers thick, is comprised of mainly keratinocyte cells, and has an external layer of dead cells that are constantly shed from the surface and replaced from below by a basal layer of cells, the stratum germinativum. The dermis comprises a network of collagenous extracellular material, elastic fibres, blood vessels, nerves and hair follicles with associated sebaceous glands.
During embryogenesis, the establishment of a dermal papilla is vital to the development of hair follicles and associated modified structures like sebaceous glands. The dermal papilla is a group of specialised dermal fibroblast cells, derived from the embryonic mesoderm. These dermal papilla cells begin to aggregate 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 fibre structures. Thus the development of a hair follicle requires a continuum through induction, initiation, elongation and differentiation stages.
A mature hair follicle comprises a bulb containing the dermal papilla cells, a hair shaft extending from the bulb through to the exterior of the epidermis, and a dermal sheath which provides an external covering of tissue around the bulb and along the length of the follicle. The hair follicle extends down through the dermis, a hypodermis (a loose layer of connective tissue below the dermis), and a fat or adipose layer. In adults, molecular signals between the dermal papilla and the epidermal component of a follicle cause the hair to enter an active (anagen) growth phase from an inactive (telogen) phase.
Baldness (known medically as alopecia) is defined as the absence of hair from an area of the body, especially where hair normally exists. Baldness can exist or arise for several reasons. Lack of hair can be caused by the non-presence of hair follicles, for example for genetic reasons. Hair loss can be caused by destruction (for example scarring), disease, infection and/or disruption of the natural hair growth cycle (for example, due to insensitivity to hormones).
For cosmetic and/or aesthetic reasons, several methods for treating baldness have been attempted. One approach has been to use pharmaceutical drugs (such as Minoxidil [RTM; Rogaine, Upjohn] and Finasteride [RTM; Propecia, Merck]). However, pharmaceuticals have achieved limited success in restoring natural hair growth.
Another approach, particularly for hair loss, has been hair transplantation, for example where tissue comprising hair follicles is transplanted from a site where the hair follicles are insensitive to dihydroxytestosterone (for example the back of the head) to a sensitive site where hair has been lost. This autograft approach is limited by the number of hair follicles which can be harvested for redistribution and the cosmetic results are not always consistent but may result in “doll-like hair”.
In other work, chimaeric hair has been generated by grafting tissue containing inductive dermal papilla or dermal sheath cells from a donor into the epidermis of a non-donor recipient (see for example WO01/32840). Such chimaeric hair tends to grow in variable directions and the method does not result in natural-looking hair.
Attempts have been made to inject donor cells into a recipient using a conventional hypodermic needle and syringe. The method does not allow cells to be delivered reproducibly or in controlled amounts into a subcutaneous compartment at an appropriate depth from the surface of the epidermis. Hair follicles induced with the bulb too close to the epidermal/dermal junction are susceptible to being pulled out when placed under a mechanical stress such as combing or brushing. Reproducibly obtaining the correct angle of hair shaft growth has also not been possible. In another approach (see WO02/060396), a “bleb” is formed between the epidermal and dermal layers of the skin by injecting a liquid to create a pocket into which cells can be injected.
A further method for reproduction of hair has been suggested in WO98/47471. The method involves removing hair in the anagen phase from a donor, culturing the hair follicle cells or keratinocytes in a culture medium and implanting the cultured hair follicle cells into pores of the receptor region. The method proposes to inject the cultured hair follicle cells into existing pores, optionally using a repeating-injection metering device. It remains unclear whether this approach will yield cosmetically useful hair restoration.
The prior art methods of treating baldness are therefore not optimal. The methods are restricted by the inability to control factors such as the density, orientation and positioning of induced or transplanted hair follicles. Furthermore, mechanical techniques (grafting, transplantation and injection) tend to be painful.
In the field of drug delivery, controlled delivery devices have provided safer, more reliable and more effective delivery of fluid drugs than the conventional hypodermic needle and syringe. Examples of such improvements are one or more high velocity driven needles (see WO00/09184), a high pressure fluid delivery system (see U.S. Pat. Nos. 5,540,657 and 6,224,567), a tracked injection needle (see U.S. Pat. No. 5,620,421), or needleless delivery means (see US20010027293 A1).
The present inventors have established that, unexpectedly, controlled delivery devices can be utilised to deliver appropriate cells directly into a recipient to provide effective treatments. In particular, controlled delivery devices are found to be useful for treating baldness.
According to the present invention there is provided a method for inducing hair follicle formation in a dermal layer lying beneath an outer skin surface, comprising delivering inductive dermal sheath cells and/or inductive dermal papilla cells directly into the dermal layer using a controlled delivery device.
The present method, particularly as a cosmetic method, is advantageous in that a physician or other practitioner will be able to repeatedly, accurately and precisely deliver cells in a cell suspension or bolus into a subcutaneous compartment. The method allows viable cells to be delivered in a reproducible volume of cell suspension while minimizing the amount of pain experienced by the patient. The method thus provides a significant improvement over prior art methods of treating baldness, in particular over using a traditional hypodermic needle and syringe and over other mechanical techniques such as grafting.
In one aspect, the invention provides a method for inducing hair follicle formation in a dermal layer beneath an outer skin surface, comprising the steps of removing a biopsy containing hair follicles, isolating inductive dermal sheath cells and/or inductive dermal papilla cells from the hair follicles by micro-dissection, expanding the inductive dermal sheath cells and/or inductive dermal papilla cells in culture under conditions which maintain their hair inductive phenotype, and delivering inductive dermal sheath cells and/or inductive dermal papilla cells directly into the dermal layer using a controlled delivery device.
The inductive dermal sheath cells and/or inductive dermal papilla cells do not need to be delivered into pre-existing pores. The invention described in WO98/47471 involves injecting mainly keratinocytes into pre-existing pores. Unlike the present invention, the method of WO98/47471 does not require induction and de novo formation of a new follicle. In the present invention, injected cultured demnal papilla or dermal sheath cells induce the development of new hair follicles, which requires cross-talk with the patient's host keratinocytes. Furthermore, in WO98/47471, when the words “hair follicle cells” are used the implication is clear that the reference is to keratinocytes and not to other cell types in the hair follicle. In paragraph 3 of the description, hair follicle cells are alternatively described as keratinocytes (“hair follicle cells or keratinocytes”) and they are described as cells that convert into the “tough and resilient material which is known as hair”, a reference to the hair shaft that is comprised completely of keratinocytes. In paragraph 4, reference is made that “hair follicle cells . . . form a differentiated epidermis or a fully developed epidermis . . . ” The epidermis does not contain any dermal papillae or sheath cells, so hair follicle cells that form a differentiated epidermis can only be keratinocytes. Another indication of the clear intent that “hair follicle cells” means “keratinocytes” is the use of serum-free keratinocyte culture medium as an example of media available for the growth in culture of “hair follicle cells”. Furthermore, the source of the cultured hair follicle cells is specified to be plucked hairs, which do not normally contain any of the dermal component of hair. Indeed, plucking of hairs by, for example, tape stripping is an experimental method of inducing the anagen phase of the hair cycle, which cannot take place without an intact dermal papilla.
The controlled delivery device may deliver inductive dermal sheath and/or inductive dermal papilla cells in small volumes repeatably, rapidly and consistently. The inductive dermal sheath and/or inductive dermal papilla cells may be delivered in a volume between 0.5 ul and 10 ul, preferably between 1 ul and 2 ul. An issue with existing hair transplantation procedures is the length of time it takes a hair surgeon to place a follicle into the patient's scalp. Typically, the surgeon also requires the assistance of technicians who will harvest the follicles by dissection from the donor biopsy. Once the follicles are dissected, the bald area is sterilised, an incision is made with a scalpel blade, and the donor hair follicle is implanted. In the prior art, a surgeon can typically implant approximately 500 follicles per hour. It is envisaged that the controlled delivery device of the present method in one aspect delivers significant advantages in terms of time for the hair surgeon, due to the reproducible and consistent delivery of small volumes (preferably 1-2 ul of viable inductive DP or DS cells).
The cells preferably remain viable after delivery. For example, loading the cells into the cell delivery device may be performed carefully so as to maintain cell viability. Cells may be sensitive to shear forces when they are forced through the needle of any device, so this step may be monitored also.
Furthermore, the cells used for implanting need to retain their hair inductive capacity. A number of approaches are known including culture with keratinocyte conditioned medium.
The controlled delivery device may be sterilisable if it is to be used on more than one patient to prevent cross contamination of infectious agents from dermal papilla or dermal sheath cells from previous surgical procedures. Typically sterilisation can be achieved by autoclaving. In specific embodiments described below, the PB-600 repeating dispenser of a modified Hamilton syringe is sterilisable, as is the glass syringe barrel. The device described in Example 7 is fully steam sterilisable. The controlled delivery device is preferably constructed from non-corrodable materials. For example, surgical grade stainless steel is preferable to aluminium. Alternatively, components which will corrode need to be sufficiently cheap to be single patient use or disposable.
The inductive dermal sheath cells and/or inductive dermal papilla cells may be derived from a variety of sources. One source is mesenchymal stem cells derived from bone marrow (available from Osiris, for example). Another source is bone marrow mesodermal progenitor cells (see WO01/11011—Catharine Verfaillie's multipotent adult progenitor cells). Yet a further source is hematopoietic stem cells derived from human bone marrow. Another source of cells are pluripotent cells derived from the skin (Toma, J. G. et al., 2001, Nature Cell Biol. 3: 778-784; Aegera Therapeutices Inc. & Curis Inc [both US]). Alternatively, the inductive dermal sheath cells and/or inductive dermal papilla cells may be derived from embryonic stem cells. Another source is embryonic carcinoma cells which have been suitably differentiated towards a DP phenotype for hair using known methods. (Teratomas from which embryonic carcinoma cell lines can be derived have hair and teeth-like structures: embryonic carcinoma cells are commercially available from Layton Biosciences [US], for example.) A further source is reprogrammed cells, for example, autologous cells such fibroblasts which have been “reprogrammed” by dermal papilla cells or embryonic carcinoma cells to induce hair formation (for reprogramming of cells, see WO00/49138).
Cells with a desired functionality of hair inducibility may be stably maintained in culture using known methods (see for example: U.S. Pat. No. 5,851,831, for long term subculture of dermal papilla cells; and the methods disclosed in WO01/74164).
The inductive dermal sheath cells and/or inductive dermal papilla cells may be delivered to a depth from an outer surface where normal hair follicles form in vitro (e.g. in cultured skin) or in vivo (i.e. in a person or other mammal). For example, the depth may be 0.5-4.0 mm into human tissue. The controlled delivery device allows the depth of delivery to be precisely determined and consistently reproduced, but is also adjustable for a particular delivery situation. Delivery of the inductive cells to the correct depth allows induced hair follicles to be imbedded in the dermis so that developed hairs will be better anchored and less susceptible to mechanical stresses such as pulling, combing or brushing.
The inductive dermal sheath cells and/or inductive dermal papilla cells may be delivered at a given angle within the dermal layer.
A growing hair follicle will not necessarily automatically orientate itself properly. According to the invention the inductive dermal sheath cells and/or inductive dermal papilla cells may be delivered in a track (or channel) formed by the controlled delivery device and oriented towards an outer surface. The track may be contiguous with the host epidermis. A track provides a pathway which allows a nascent hair follicle to grow in the correct direction towards the surface of the skin and connect with the surface epidermis surrounding the track. In addition, the angle of the track can be varied, allowing the nascent hair follicle to grow at an appropriate angle relative to the outer surface. This achieves a good cosmetic result because hair follicles grow at different angles in different regions of the scalp and a more robust hair follicle. In one embodiment, the controlled delivery device used to generate the track has a needle with blunt end and an orifice on the lateral side near the tip (for example, an orifice approximately 0.5 mm from the tip), allowing cells to be implanted along the needle track.
The controlled delivery device may dispense in suspension between 1000 and 40000 inductive dermal sheath cells and/or inductive dermal papilla cells per delivery. The controlled delivery device may dispense inductive dermal sheath cells and/or inductive dermal papilla cells in suspension at a cell density of between 5×105 and 4×107 cells/ml per delivery. In the specific embodiments described below, experiments were performed to determine the effect of these various parameters.
The controlled delivery device may dispense pre-formed aggregates of inductive dermal sheath cells and/or inductive dermal papilla cells. For example, one to three pre-formed aggregates may be dispensed per delivery. Each pre-formed aggregates may contain between 1.5×103 and 1×104 cells. In these embodiment, cells are allowed to aggregate, or inducing cells to aggregate, into pre-formed aggregates, i.e. clumps of cells of the appropriate volume and cell number, before being injected subcutaneously. Cells or aggregates of cells may be placed in a formulation, such as hyaluronic acid or glycosaminoglycans, that includes a substance (or substances) which increases the viscosity of the injected material in order to protect the cells during handling and injection. Cells or aggregates of cells may be placed in a formulation, for example one including fibrin, fibronectin and/or collagen or other extra-cellular matrix molecules known to those skilled in the art, that enhances the microenvironment of cells after implantation, in order to facilitate cell migration or cell-cell interaction.
The inductive dermal sheath cells and/or inductive dermal papilla cells may be from a source autologous or allogeneic to the dermal layer.
Preferably, the controlled delivery device comprises one or more high velocity driven needles (for example as claimed in WO00/09184), a high pressure fluid delivery system (for example as claimed in U.S. Pat. No. 5,540,657 or U.S. Pat. No. 6,224,567), a tracked injection needle (for example as claimed in U.S. Pat. No. 5,620,421) or is needleless (for example as claimed in US20010027293 A1).
Alternatively, the controlled delivery device may be a Hamilton syringe with a controlled volume delivery modification (for example, a Hamilton PB-600 repeating dispenser).
In a further embodiment, the controlled delivery device comprises a micropump dispensing mechanism. Certain prior art devices, such as medication delivery pens, have been developed to improve upon the traditional syringe injection delivery system. These devices are repeatable and typically use a piston mechanism similar to a syringe. However, unlike a typical syringe, an actuator mechanism is used to exert a defined axial force on the piston to inject only the set dosage of medication. However, these devices may be unable to repeatedly inject quantities smaller than 50 μL and to control the depth of injection. However, a controlled delivery device with a micropump dispensing mechanism, as used previously in the microfluidics field to create, for example, microsized bioassay systems (“lab-on-a-chip”), cell microarrays and miniaturized chemical analysis systems, may be used in the present invention for delivering a viable cell suspension or cluster of cells. Preferably the controlled delivery device with a micropump dispensing mechanism provides at least one of the following: (i) the ability to deliver minute (<10 μL) amounts of cell suspension, (ii) the ability to deliver such amounts in a repeatable fashion so as to facilitate multiple injections and to avoid the need to refill the device numerous times, and (iii) the ability to determine and control the depth of the injection so as to ensure the proper delivery of cells to a desired injection site. A preferred specific embodiment is shown in FIGS. 14-16 and described further below.
Further provided according to the present invention is a method as described herein additionally including one or more steps resulting in the development of a mature hair follicle.
Also provided according to the present invention is the use of a controlled delivery device for the delivery of inductive dermal sheath cells and/or inductive dermal papilla cells into a dermal layer to induce hair follicle formation. The features pertaining to the method elaborated herein are also applicable for this use.
It will also be appreciated that the invention described herein may be more generally applicable so as to provide a method for inducing organ or tissue formation. The method may comprise delivering inductive cells into an organ or tissue regenerative cellular environment using a controlled delivery device. Furthermore, the invention covers the use of a controlled delivery device for the delivery of inductive cells into a regenerative cellular environment to induce organ or tissue formation. Using a controlled delivery device also allows for cells to be delivered to a specific location for therapeutic purposes. |
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