WO2021186181A1

WO2021186181A1 - Combinations, kits and methods for wound treatment

Info

Publication number: WO2021186181A1
Application number: PCT/GB2021/050675
Authority: WO
Inventors: Karin GRECO
Original assignee: Griffin Paste Research Limited
Priority date: 2020-03-19
Filing date: 2021-03-18
Publication date: 2021-09-23
Also published as: GB2593203A; GB202004003D0

Abstract

The invention provides a combination for use in wound treatment, the combination comprising: decellularized dermal tissue paste; and isolated skin cells and/or a patch comprising skin cells.

Description

COMBINATIONS, KITS AND METHODS FOR WOUND TREATMENT

Technical Field of the Invention

The present invention relates to combinations, kits and methods in relation to wound treatment.

In particular, the present invention relates to a therapeutic or pharmaceutical combination comprising decellularised dermal tissue and skin cells, for use in wound treatment; and to kits and methods relating thereto.

Background to the Invention

A wound is an injury to the body that typically involves laceration or breaking of a membrane and damage to the underlying tissue. Chronic wounds do not progress through the normal stages of healing, e.g. by remaining in an inflammatory stage for a long time relative to non-chronic wounds. Chronic wounds can be extremely painful, distressing and difficult to manage, and thus significantly reduce quality of life. Skin loss from thermal injuries alone is estimated to cause over 180,000 deaths annually worldwide.

Following injury, the skin is able to self-repair to an extent, but when the injury forms a large or deep wound, e.g. in third-degree burns, chronic diabetic wounds, trauma or in some genodermatoses, the skin may be unable to adequately self-repair without medical intervention.

Perhaps the most common procedure for treating extensive wounds is autologous full skin grafting, which involves transplanting unaffected skin from a donor site to an affected area about the wound. This procedure, however, has significant limitations. For example, it can be ineffective for treatment of large and deep wounds and only limited portions of the skin can be repaired in each procedure because additional injuries are created at the donor sites. Thus, multiples surgical procedures and thereby lengthened recovery can extend hospitalisation time, increase costs, result in comorbidities, and have a negative psychological impact on the patient.

In recent decades, the market for tissue (e.g. skin) replacements has rapidly developed to incorporate various matrices of biological origin and artificial design. Such matrices are used as provisional matrices to fill a wound void and act as a framework in which host cells can grow and regenerate tissue. An advantage of biological matrices is that they retain unique features similar to the body which are essential for the tissue regeneration.

Commercially available tissue replacements are typically provided in sheet- form. Whilst effective in some instances, sheet-form replacements can be difficult to apply to the complex contours of a wound. As a result, gaps can form between the dermal replacement and the wound, such that wound exudate and unwanted substances can enter the gaps and result in infection, and delay or avert healing.

In addition, sheet form tissue replacements may not enable adequate or optimal wound healing, due to inefficient or deficient wound filling by healing cells.

It is an object of the present invention to provide a tissue replacement or wound healing therapeutic or pharmaceutical composition or combination which overcomes or ameliorates one or more problems of the prior art.

Summary of the Invention

According to a first aspect of the present invention, there is provided a combination for use in wound treatment, the combination comprising: decellularised dermal tissue paste; and isolated skin cells and/or a patch comprising skin cells.

The combination may comprise a composition, kit or separate materials.

The combination may be a pharmaceutical or therapeutic combination.

In embodiments, application to a wound of the paste and isolated skin cells and/or a patch, is found to alleviate inflammation, induce neocollagen deposition, induce neovascularisation, alleviate tissue granulation and/or induce reepithelialisation. In addition, in embodiments, and without being bound by theory, it is believed that the patch can acts as a scab, and contributes to an excellent wound healing environment.

In embodiments, the present invention offers numerous advantages over existing sheet-form tissue replacements which can be difficult to apply to wounds having complex contours. For example, the paste can be closely integrated with the wound bed and ensure contact with the entire topography of the wound, increasing the likelihood of successful healing. In addition, the paste can be easily applied to a wound using various applicators, e.g. a syringe, and can conform to the shape of a wound without gaps. Furthermore, the paste can be added to large and/or deep wounds, whereas when using sheet-form tissue replacements, it can be necessary to mesh the sheet or use multiple sheets, which increases the risk of displacement and accumulation of fluid between the sheets. Moreover, the paste can accommodate wound exudate and does not allow for its accumulation in a wound dressing, and thereby reduces the risk of bacterial infection.

In embodiments, the components of the combination comprise naturally occurring materials that are present in the skin and can be rapidly produced (e.g. in less than 24 hours), which can result in an affordable and readily producible tissue replacement.

The combination may be for use in tissue replacement. The combination may be for use in human tissue replacement. The combination may be for use in animal tissue replacement. The combination may be for use in internal tissue replacement (e.g. to replace anal fissures). The combination may be for use in dermal replacement.

The particles and/or granules of the paste may have a viscosity of between 100 cP and 30><10⁶ cP, suitably 2500 cP and 30><10⁶ cP, suitably 2500 cP and 3xl0⁶ cP, suitably 10,000 cP and 30x l0⁶ cP, suitably 100,000 cP and 30x l0⁶ cP, suitably lxlO⁶ cP and 30xl0⁶ cP.

Herein, ‘decellularised dermal tissue paste’ can be defined as particles and/or granules of decellularised dermal tissue suspended in a fluid. Thus, the solid component of the paste is the particles and/or granules of decellularised dermal tissue, and any liquid component of the paste is generally extracellular fluid of the decellularised dermal tissue after decellularization. Accordingly, the paste can be formed from decellularised dermal tissue alone, without requiring addition of a further liquid component.

The particles and/or granules of the paste may have an average (median) diameter or length of at least 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 10 pm, 20 pm, 50 pm, 75 pm, or 100 pm. The particles or granules may have an average (median) diameter or length of between 10 pm and 500 pm, 25 pm and 500 pm, 50 pm and 500 pm, 100 pm and 500 pm, or 100 pm and 250 mih The average particle or granule size (diameter or length) may be measured using histological sections stained with Picro-Sirus Red (PSR) and a graticule, or any other suitable method known to persons skilled in the art.

The paste may comprise epidermis, dermis, hypodermis, basement membrane or any combination thereof. The paste may comprise intact skin. In a preferred embodiment, the paste comprises at least epidermis and dermis and may comprise substantially solely epidermis and dermis.

The paste may comprise at least 50%, 55%, 60%, 65%, 70%, 75% or 80% of the extracellular matrix proteins present in the dermal tissue prior to decellularisation.

The paste may comprise at least 75%, 80%, 85%, 90% or 95% of the collagens present in the dermal tissue prior to decellularisation.

The paste may comprise animal tissue, preferably porcine tissue. The paste may comprise human tissue.

The degree of cross-linking in the paste may be less than 5%, 4%, 3%, 2%, 1%, 0.1% or 0.01%. In embodiments, the paste may be substantially or completely free of cross-linking. The degree of cross-linking may be determined using a ninhydrin assay. Surprisingly, cross-linking is found to be detrimental to the physical and regenerative properties of the combination.

Herein, ‘decellularised’ may mean that at least 95%, 96%, 97%, 98%, 99%, 99.5%, to 99.6%, 99.7%, 99.8%, 99.9%, or substantially all cells have been removed from the dermal tissue.

The isolated skin cells and/or the patch may independently comprise animal- derived (e.g. porcine-derived) cells and/or human-derived cells. The isolated skin cells and/or the patch may independently comprise autologous cells. The isolated skin cells and/or the patch may independently comprise fibroblasts and/or keratinocytes and/or stem cells.

The patch may be a biological material. The patch may be a biocompatible material. The patch may comprise or be a skin graft. The skin graft may be a human skin graft. The skin graft may be a non-human animal skin graft (e.g. a porcine or bovine decellularised skin graft). Preferably, the skin graft is a split-thickness skin graft. The use of a split thickness skin graft, which includes the epidermis and part of the dermis, in combination with the particles or granules, can significantly expedite wound healing and thus provide an excellent tissue replacement.

In preferred embodiments, in use, the paste is deposited into a wound to partially fill (e.g. half-fill) the wound; fibroblasts are deposited onto the paste in the wound; more of the paste is deposited onto the fibroblasts in the wound; and keratinocytes and/or the skin graft, are applied onto the paste in the wound (proximal to the entrance of the wound), such that the wound is substantially filled and, where the skin graft is present, covered by the skin graft.

The isolated skin cells (e.g. fibroblasts) may be disposed between layers of the paste. The isolated skin cells (e.g. keratinocytes) may be disposed on a surface of the paste.

The isolated skin cells form one or more layers. For example, there may be one or more layers of isolated skin cells disposed within, or between layers of, the paste. For example, there may be one or more layers of isolated skin cells disposed on the paste (e.g. on an exposed outer surface thereof, distal the wound).

The patch may be disposed on the paste and, where present, on the isolated skin cells. Thus, in some embodiments the patch may be deposited directly on top of the paste (which may be in layers with one or more layers of isolated skin cells disposed between said layers); or the patch may be deposited on top of a layer of isolated skin cells, which in turn is on top of the paste.

In preferred embodiments, one or more layers of isolated skin cells (e.g. fibroblasts) are disposed within the paste, and one or more layers of isolated skin cells (e.g. keratinocytes) and/or the patch are disposed on the paste. Such embodiments facilitate/expedite wound healing and thus provide an excellent wound healing environment.

The combination may be a tissue replacement. The combination may be a dermal tissue replacement and/or an internal tissue replacement. As such, the present invention can be used to treat not only skin wounds, but also internal wounds such as anal fissures. Preferably, the tissue replacement is a dermal tissue replacement.

The combination may be sized (e.g. in terms of volume) to fill a full-thickness wound or to fill and cover a wound. The sizing may be determined using imaging means.

According to a second aspect of the present invention, there is provided a kit for use in wound treatment, the kit comprising: decellularised dermal tissue paste; and isolated skin cells and/or a patch comprising skin cells, optionally wherein the paste and the isolated cells and/or the patch, are contained prior to use.

The paste, the isolated skin cells and the patch may be as described for the first aspect of the invention.

The kit may be for use in tissue replacement. The kit may be for use in human tissue replacement. The kit may be for use in animal tissue replacement. The kit may be for use in internal tissue replacement (e.g. to replace anal fissures). The kit may be for use in dermal tissue replacement.

The paste and the isolated skin cells and/or the patch may be separately contained prior to use. For example, the paste may be contained in a first syringe and the isolated skin cells may be contained in a second syringe and/or the patch may be contained separately from the syringe or syringes, prior to use.

The isolated skin cells may be suspended in a liquid medium, suitably a biocompatible liquid medium. In this way, the isolated skin cells can be easily applied to a wound, e.g. using a syringe.

According to a third aspect of the present invention, there is provided a method of treating a wound, the method comprising: depositing decellularised dermal tissue paste and isolated skin cells and/or a patch comprising skin cells, on the wound.

The method may comprise depositing decellularised dermal tissue paste into the wound to partially fill (e.g. almost fill) the wound, and depositing isolated skin cells (e.g. keratinocytes; optionally as a layer) onto the paste in the wound, optionally such that the wound is substantially filled.

The method may comprise depositing decellularised dermal tissue paste into the wound to partially fill (e.g. half fill) the wound; depositing isolated skin cells (e.g. fibroblasts; optionally as a layer) onto the paste in the wound, and depositing decellularised dermal tissue paste onto the isolated skin cells in the wound, optionally such that the wound is substantially filled.

The method may comprise depositing decellularised dermal tissue paste into the wound to partially fill (e.g. half fill) the wound; depositing isolated skin cells (e.g. fibroblasts; optionally as a layer) onto the paste in the wound; depositing decellularised dermal tissue paste onto the isolated skin cells in the wound; and depositing isolated skin cells (which may be the same as or different from the other isolated skin cells; and optionally as a layer) and/or the patch, onto the paste in the wound, optionally such that the wound is substantially filled and, where present, the patch optionally covers the wound.

The method may comprise, after the depositing step, monitoring and/or protecting the wound for at least 1 week, suitably at least 2 weeks, suitably at least 3 weeks, suitably at least 4 weeks. This may involve using equipment and/or dressings to protect the wound from damage whilst it is being treated.

The wound may be chronic.

The wound may be infected.

Any feature or features of any aspect of the present invention can be combined with any feature or features of any other aspect of the present invention.

For example, the skilled person will appreciate that the optional features in respect of the first aspect or other aspects of the present invention may apply in respect of the other aspects of the present invention.

The decellularised dermal paste may be prepared through a decellularizing step comprising subjecting the dermal tissue to osmotic shock. The decellularising step may comprise contacting the dermal tissue sequentially with hypotonic and hypertonic solutions (in any order) to promote cell lysis.

The decellularising step may comprise contacting or immersing the dermal tissue in the hypotonic and hypertonic solutions.

Contact or immersion of the dermal tissue with the hypotonic solutions may be repeated at least once, and preferably at least twice, three times or four times.

The hypertonic solution may comprise sodium chloride (NaCl). The hypotonic solution may further comprise ethylenediaminetetraacetate (EDTA) and/or Tris-HCl. The hypertonic solution may comprise between 0.5 M and 2M NaCl, such as around 1 M NaCl. The EDTA may be present at a concentration of between 10 mM and 100 mM, such as between 20 mM and 50 mM, or around 25 mM. The Tris-HCl may be present at a concentration of between 20 mM and 100 mM, such as between 25 mM and 75 mM, or around 50 mM. The hypertonic solution may comprise 1 M NaCl, and optionally 25 mM EDTA and 50 mM Tris-HCl.

The decellularising step may comprise contacting the dermal tissue with one or more nucleases. The nuclease may be contacted with a dermal tissue after the dermal tissue is contacted with the hypotonic and hypertonic solutions. The nuclease may be a DNase or an RNase or a combination of DNase and RNase. The decellularisation step may comprise washing the dermal tissue, preferably in saline solution, such as phosphate buffered saline (PBS).

After decellularisation, the dermal tissue may be immersed in a washing medium for at least 5 minutes, at least 10 minutes, or at least 15 minutes.

The hypotonic solution may comprise EDTA and/or Tris-HCl which may be present at concentrations as described hereinabove for the hypertonic solution.

After decellularization, the decellularised dermal tissue may be pasted by grinding (including freezing followed by grinding) or cryo-milling the dermal tissue into particles or granules.

The particles or granules may be prepared by milling, e.g. cryomilling. The particles or granules may be suspended in the carrier medium by mixing (e.g. spatulating) the particles or granules and the carrier medium.

Detailed Description of the Invention

In order that the invention may be more clearly understood one or more embodiments thereof will now be described, by way of example only, with reference to the accompanying drawings, of which:

Figure 1 A shows dermal paste implantation in full-thickness wounds at day 0;

Figure IB shows dermal paste implantation in full-thickness wounds after 4 weeks;

Figure 1C-N shows micrographs of the excised wounds of Fig. IB, wherein in C, F, I and L the wounds are stained with H&E; in D, G, J and M the wounds are stained with Picro-Sirius red and Miller elastin; and in E, H, K and N the wounds are stained with Picro-Sirius red and are under polarised light;

Figure 2A-P shows micrographs of histological sections of the excised wounds of Fig. IB, wherein in A, C, E, G, I, K, M and O the wounds are stained with H&E; in B, D, F, H, J, L, N and P the wounds are stained with Picro-Sirius and are under polarised light;

Figure 3 A shows a histometric score for different attributes of the wounds of Fig. IB, wherein results are expressed as the mean (n=4) of the scored values for each condition (± SD);

Figure 3B shows analysis of the progression of the epithelialisation process of the wounds of Fig. IB under different conditions of dermal paste implantation, wherein results are expressed as a percentage of re- epithelialisation observed in each condition (n=4); Figure 4 A-P shows immunofluorescence images of the wounds of Fig. IB, wherein A, C, E, G, I, K, M and O show staining for keratin 14 and B, D, F, H, J, K, N and P show staining for keratin 15; Figure 5A-P shows immunofluorescence images of the wounds of Fig. IB, wherein images A, C, E, G, I, K, M and O show staining for keratin 1-10 and images B, D, F, H, J, L, N and P show staining for laminin; and

Figure 6A-P shows immunohistochemistry images of the wounds of Fig. IB, wherein images A, C, E, G, I, K, M and O show staining for collagen type I and images B, D, F, H, J, L, N and P show staining for collagen type III.

Dermal Paste Production

Fresh porcine dermis was obtained from large-white/Landrace cross breed pigs after euthanasia a clean environment. Dermal sheets were decellularised using a non- enzymatic protocol as described previously in Greco K V, Francis L, Greco G, et al. Characterization of porcine dermis scaffolds decellularised using a novel non- enzymatic method for biomedical applications. J Biomater Appl 2015; 30(2):239-253. The principle of osmotic shock was applied to the dermal sheets using hypertonic and hypotonic solutions followed by multiple washing steps. The decellularisation steps involved alternating between hypertonic (1 M NaCl, lOmM EDTA, 50mM Tris-HCl - 12h incubation) and hypotonic solutions (5mM EDTA, lOmM Tris-HCl - 12h incubation), with washing steps in between (PBS comprising 0.05% Tween-20, for 8 h; after each of the previous solutions). Decellularisation took less than two days and was carried out under agitation (Incubator shaker, SciQuip, Shropshire, UK), with 1% antibiotic/antimycotic solution (AA; Sigma-Aldrich, Dorset, UK) to minimise bio burden. After the final wash, under sterile conditions, the dermal sheets were placed into sterile flasks containing PBS+5% AA. The decellularised dermal sheets were removed from the PBS+5% antibiotic/antimycotic (AA; Sigma-Aldrich, UK) solution and washed 4 times with sterile PBS in order to remove any remaining AA. Samples were swabbed for microbiology assessment. If samples were shown to be sterile, they were cut into small pieces (0.5x0.5cm) and placed in sterile cryomil tubes (approximately 2g per tube). A cryomil machine (Spex SamplePrep Cryomill, Stanmore, UK) was set using one operating cycle at an impaction rate of 15 cps, with a run time of 2 minutes. The tubes were loaded and unloaded under a classified laminar flow hood in order to maintain sterility. The resulting milled dermal scaffolds (in powder form) were placed into petri dishes and spatulated several times (without addition of a further liquid component) until the paste was formed. Particle sizes were measured microscopically using a bright-field microscope and ranged from about 150 pm to about 250 pm (median average). The paste was packed into sterile 1 ml syringes, which were used for storage, measurement and delivery of the material into the wounds. The paste was cryocompatible.

Experimental Model

All experiments were approved by a local ethical review committee and carried out under UK Home Office approval and according to the Animals Scientific Procedures Act (1986). Female Large White pigs, with an initial weight of 30 kg to 45 kg were used. On arrival, the pigs were placed into individual pens and allowed to acclimatize for 7 days before initiation of any experiments. The pigs were pre-medicated with an intramuscular injection of 1 mg/kg of xylazine (Bayer, Newbury, UK) and 5 mg/kg of ketamine (Pharmacia Animal Health Ltd., Corby, UK), and anaesthetized with 2-5% isofluorane (Baxter Healthcare Ltd., Thetford, UK) in oxygen: nitrous oxide mixture (50:50). After clipping and shaving the designated area, each pig was washed with chi orhexi dine gluconate 0.5% w/v in 70% (v/v) DEB (Adams Healthcare, Leeds, UK), followed by Povidone-Iodine USP 10% (w/w) in water (Adams Healthcare, Leeds, UK). Under aseptic conditions, skin biopsies (12mm punch biopsy of approximately 2mm in depth) were collected from the neck (both the right and left-hand side) from each pig in order to isolate and expand sufficient number of skin cells (herein keratinocytes and fibroblasts). Postoperative analgesia was provided to each pig by a subcutaneous injection of 4 mg/kg of carprofen (Pfizer Ltd., Sandwich, UK) followed by further oral doses once daily for 2 days.

Fibroblasts and Keratinocytes Isolation and Expansion

Primary porcine keratinocytes and fibroblasts were isolated and cultured from the skin biopsies harvested from the neck of each pig approximately 14 days before the surgical procedure to create the full-thickness wounds. The skin was washed 4 times with PBS + 2% AA, and the dermis and epidermis were separated using dispase (5 U/ml; Stemcell Technologies, Cambridge, United Kingdom) by incubation for 60 minutes at 37°C. Keratinocytes were released from the epidermis by incubating the epidermal sheet in 0.5% trypsin 1:250 (ThermoFisher, Leicestershire, United Kingdom). The reaction was stopped by adding 10% foetal bovine serum (FBS) (Sigma- Aldrich, Dorset, United Kingdom).

Keratinocytes were separated from the remaining tissue using a 100 pm strainer (Greiner Bio-one, Gloucestershire, United Kingdom), then washing with Dulbecco's Modified Eagle's medium (DMEM; ThermoFisher Scientific, Leicestershire, United Kingdom) and centrifuging at 200 g for 5 minutes.

Subsequently, Fibroblasts were released from the dermis by 60 min incubation in a 0.3 % collagenase solution (w/v in PBS; Sigma-Aldrich, Dorset, United Kingdom). The reaction was stopped by adding 10 % foetal bovine serum (FBS) (Sigma-Aldrich, Dorset, United Kingdom). The solution was sieved with a 100 pm strainer (Greiner Bio-one, Gloucestershire, United Kingdom) then washed with DMEM and centrifuged at 200 G for 5 minutes.

Keratinocytes were seeded at a density of 3xl0⁴ cells/cm² in CnT 07 medium (Caltag Medsy stems, Buckingham, United Kingdom) and fibroblasts were seeded at 7xl0² cells/cm² in DMEM supplemented with 10% FBS. Flasks were reviewed daily and the CnT 07 medium was changed every 48 hours.

Keratinocytes reached 100% confluence after approximately 6-7 days and fibroblasts reached 100% confluence after 5-6 days. They were split at a ratio of 1:2 using 0.25% Trypsin-EDTA (Sigma Aldrich, UK) until reaching the approximate number of cells needed for the implants.

Both keratinocytes and fibroblasts were released from the flasks using a cell dissociation solution (Cell Dissociation Solution Nonenzymatic lx Sigma-Aldrich, Dorset, UK), counted and the correct number of cells for each wound were resuspended in 0.1 ml growth media (DMEM for fibroblasts; and CnT-07 for keratinocytes) specific for each cell (as described above) and stored in 1 ml syringes, ready for deployment into designated wounds. The correct number of cells was centrifuged and resuspended in phosphate buffered saline (an inert carrier to allow the cells to be stored in syringes and be delivered to the wounds)

Split-Thickness Skin Graft (STSG) Harvesting Prior to the surgical procedure for creating the wounds, the paravertebral area was shaved, scrubbed and cleaned with 0.5 % (v/v) chi orhexi dine solution and isotonic 0.9 % sodium chloride solution, washed twice with povidone-iodine and then covered with a povidone-iodine soaked sterile gauze for 5-10 mins to ensure adequate bacterial neutralization. The povidone-iodine was rinsed off with 70% alcohol and the area was left to air dry. Liquid paraffin (1ml) was applied for surface lubrication and a STSG approximately 10 cm long x 5 cm wide x 150 pm thick was harvested using a compressed medical air-driven Zimmer® Air Dermatome, at depth setting 3 (6/1000th of an inch). The harvested STSG were washed with sterile saline, trimmed to produce circles as the same diameter of the chambers to be inserted into the designated wounds, and kept in moist saline-soaked gauze until grafted.

Wound Chamber Model

Full-thickness, circular wounds, 2 cm in diameter (six per animal), were created between scapula and iliac fossa on the dorsal area of four of the pigs. Using sharp dissection with a scalpel (blade 11), full-thickness skin was removed, the skin comprising epidermis, dermis, subcutaneous fat and superficial fascia. Bleeding was kept to a minimum using pressure and dry gauze. The wound edges were approximately 2.5 cm to allow polytetrafluoroethylene (PTFE) chambers (custom designed in house, with 2cm diameter) to be inserted under the skin and stitched in place with 2.0 prolene sutures (Ethicon Ltd, Livingston, UK). These chambers were inserted to restrict wound contraction and to prevent healing by epithelialization from the wound margins. The fascia over the skeletal muscle acted as the wound bed, and 0.7 ml of the dermal paste (from the syringes) was placed over the wound bed and smoothed to create a uniform surface.

A patch, in the form of a split-thickness skin graft (STSG) and/or autologous cells (herein keratinocytes and/or fibroblasts) were applied to the paste. Where fibroblasts were applied, half of the paste (0.35 ml) was loaded on the wound bed first, then fibroblasts (2.5x10^s per cm² resuspended in 100 mΐ of PBS) were applied to the surface of the paste, which were then covered with the second half of the paste. Where keratinocytes were applied, the paste was loaded onto the wound bed first, such that the wound was substantially filled with the paste (with or without fibroblasts therein), then keratinocytes (2.5x10^s per cm² resuspended in 100 mΐ of PBS) were applied on the top surface of the dermal paste. Where a STSG was applied, the paste was loaded onto the wound bed first, such that the wound was substantially filled with the paste (with or without fibroblasts therein and with or without keratinocytes thereon), then the STSG was applied to the top surface of the paste (to act as a ‘cover’).

Each wound was covered with pre-cut sheets of Teflaclear® followed by Jelonet. Over the top of the Jelonet, chambers were packed with saline-moist sterile swabs, and the chamber structure above the skin was wrapped with povidone-iodine soaked swabs.

The final dressing applied to all chambers was made with orthopaedic wool (Velband™) Mefix® and Elastplast® adhesive tape. Finally, custom-made plastic rigid jackets were applied and secured in place with Velcro straps to protect the experimental wounds from abrasion and accidental displacement. Once per week the pigs were anaesthetised as described above and the dressings were changed for identical fresh dressings.

Clinical and Histomorphometric Analysis

Once per week, every experimental wound was macroscopically assessed for inflammatory reaction, infection, and graft take (where applied). Photographs of the wounds were taken with a Canon EOS 300D digital camera to document wound appearance and for further wound assessment. After four weeks, excision of the entire wounds was performed, and routine histological analyses were carried out.

Five-micrometre sections were cut and stained with both Haematoxylin and Eosin (H&E) and with Picro-Sirius red (PSR) combined with Miller’s elastin for histomorphometric evaluation. Structural integrity of collagen was assessed in the sections stained with PSR using polarised light microscopy.

Histomorphometric evaluation of the in vivo biointegration of dermal paste was carried out and attributes such as biomaterial engraftment, degradation, host cellular invasion, inflammatory reaction, neocollagen deposition, neovasculature formation, granulation tissue formation, split-thickness skin graft survival, and wound re-epithelialisation were assessed. A histometric scoring system was applied to individual tissue sections (stained with either haematoxylin and eosin (H&E) or Picro-Sirius Red/Miller elastin (PSR)) using a range of grades 0-4 (where 0=absent, l=minimal, 2=mild, 3=moderate, 4=extensive/marked presence of the assessed attribute) was adopted. Results were expressed as the mean of the scored values for each treatment group (n=4).

Immuno- Analysis

All samples used for staining were fixed in neutral buffered formalin, embedded in paraffin wax and sectioned at 5 pm thickness before mounting on positively charged glass slides and baked at 50°C for 24-48 hours. On the day of immunostaining, tissue sections were dewaxed through xylene and decreasing alcohol concentrations before rinsing in tap water.

Immunofluorescence staining (IFS): antigen retrieval was performed in Proteinase K (ab64220, Abeam, Cambridge, UK) at 37°C for 20 mins followed by permeabilisation in Tris-EDTA with Triton-XlOO (0.5%; Sigma-Aldrich, UK) before blocking in 2.5% Normal Horse Serum (NHS) (Vector Laboratories Inc., Burlingame, USA). Sections were washed in PBS between steps. Primary antibodies were diluted in the blocking solution and applied to the sections, which were then incubated overnight at 4°C. Secondary antibodies were diluted in the blocking solution and sections were incubated for 2 hours at room temperature. Sections were mounted with either Vectashield Vibrance (Vector Laboratories Inc., Burlingame, USA) or ProLong Gold mounting media (Invitrogen, Carlesbad, CA) before imaging on a fluorescence microscope (Olympus BX43, Tokyo, Japan).

Immunohistochemistry (IHC): antigen retrieval was performed at 95°C for 20 mins in citrate buffer and left to stand for a further 10 mins at room temperature before proceeding with staining. Sections were then washed three times in PBS for 3 mins each time. Endogenous peroxidase was blocked with 3% H2O2 (in methanol) over 30 mins followed by a second PBS wash. The sections were then blocked in 2.5% normal horse serum (NHS; Vector Laboratories, Peterborough, UK) for 30 mins and excess blocker tapped off the slides before addition of primary antibody. Collagen I primary antibody was incubated for 2hrs at room temperature and collagen III was incubated overnight. At the end of the primary incubation period, the slides were washed in PBS as above and the secondary antibodies were added; lhr for collagen type I and 30min for collagen III, both at room temperature. A PBS wash was done by adding DAB substrate for 1 minute followed by a final PBS wash before counter-staining in haematoxylin and dehydrating and mounting in DPX Mountant. Primary antibody dilutions were made in PBS whilst the NHS and secondary antibodies came as ready to use stocks.

Results

In vivo integration of the porcine dermal paste after implantation in full-thickness wounds

Animals were clinically well throughout the study. Macroscopical analysis indicated that wound closure took place within the 4 weeks. Neither bleeding, discharge nor seroma was visualized over the course of the study (see Fig. 1 A, B).

Histological analysis showed that the dermal paste allowed host cell infiltration (see Fig. IF, inset) and supported wound healing (see Fig. IF). In some of the experimental wounds, the implanted dermal paste could be detected after 4 weeks implantation (characterized by coarser collagen bundles - see Fig. 1J, M), whereas in others it seemed completely integrated within host tissue (Fig. 1G), especially in the wounds where neo-epithelium was seen (see Fig. IF). Vascularization of the paste was observed in most of the samples (see Fig. II, L, insets). Cytomorphological observations showed controlled granulation tissue formation in this network (see Fig. II, L). The wounds where the split-thickness skin grafts (STSG) were placed on top of the paste advantageously displayed a similar histological structure to the native skin after 4 weeks, and also showed an undulating and highly proliferative basal layer of keratinocytes and stratum corneum formation (even when the STSG was not retained; see Fig. 2C, E). Lack of epithelialization where STSG was added with the paste was observed in one experimental wound only (see Fig. 20). The addition of autologous keratinocytes and/or fibroblasts advantageously induced the formation of a multi layered proliferative neo-epithelium (see Fig. 2G, I, K), but not in all the wounds and not as prominently as shown with STSG implants. Implantation of paste alone displayed more abundant granulation tissue (see Fig. II). PSR staining was used for collagen and elastic fibres visualization. PSR enhances the natural birefringence of normal collagen fibres and demonstrates their thickness and quality when the stained tissue is viewed under polarized light. Newly formed collagen fibres often display a yellow-green (thinner fibres) colour and when collagen is denatured it loses its natural birefringence and appears darker than undenatured collagen. Some samples showed retention of part of implanted paste, surrounded by the native dermis and no excessive contraction was seen (see Fig. 1G, J, M). The arrangement of collagen fibres in the wounds were generally well preserved but some wounds showed an area with less organised fibres in the middle of the wound where the newly formed collagen fibres were laid, mainly in the samples where the epithelialisation process was not completed (see Fig. 2L, N, P vs Fig 2B, control). However, no collagen denaturation was observed in the experimental wounds. Neo-deposition of collagen took place in most of the experimental wounds, which was evident by PSR stained sections showing yellow-green fibres under polarized light (see Fig. 2D, F, H, J, L, N, P).

Histomorphometric Analysis

Histological assessment of the dermal paste was carried out individually by 3 people and attributes such as engraftment, degradation, host cellular invasion, inflammatory reaction, neo-collagen deposition, neovasculature formation, granulation tissue formation, STSG survival, and wound re-epithelialisation were noted. The range of grades were 0-4, where 0 - absent, 1- minimal, 2- mild, 3- moderate and 4- extensive/marked presence of the assessed attribute (Fig. 3). Results (n=4 for each condition; see Figs. 3A and 3B) showed engraftment of the paste and host cellular invasion were assessed close to the maximum score (AV=3.94±0.15 and 3.96±0.1, respectively) in all conditions. Neo-collagen deposition and neovascularization was moderate (AV= 3.3± 0.7 and 3.5±0.7, respectively). Paste degradation showed to be at minimum to mild levels, and the presence of granulation tissue was inversely proportional to re-epithelialisation.

Iininu n o-A n alysis

With reference to Fig. 4A-P, keratins (K) staining revealed that K14-positive cells were present in the basal layer in the experimental wounds, showing highly proliferative epidermis, where either paste + fibroblasts + STSG (see Fig. 4C), paste + STSG (see Fig. 4E), or autologous cells (mainly in the presence of keratinocytes - see Fig. 4G, I) were applied.

In contrast, we observed that either paste + fibroblasts (see Fig. 4K), paste alone (see Fig. 4M) or STSG where re-epithelialisation seemed to be more premature or absent (see Fig. 4P), a higher expression of KR15-positive cells - not confined to the basal layer of keratinocytes - was observed.

Overall, in our experimental settings KR15-positve cells were shown to be more abundant in all conditions when compared with control tissue where the positive cells were present mainly in the hair follicle (see Fig. 2B, inset).

With reference to Figs 4A-P and Figs. 5A-P, where the epithelialisation process was shown to be more mature after implantation of paste associated with either fibroblasts + STSG, paste + STSG, combination of fibroblasts + keratinocytes or paste + keratinocytes, the intermediate filaments KRl-10 were more abundant (see Fig. 5C, E, G, I, respectively) when compared with paste + fibroblasts (see Fig. 4K) or paste alone (see Fig. 4M). Laminin was clearly expressed and assembled along with the basement membrane (comparatively with control - see Fig. 5B) only when paste + fibroblasts + STSG (see Fig. 5C) or paste + STSG (see Fig. 5E) was applied. When we used either paste + fibroblasts + keratinocytes (see Fig.5G) or combination of paste + keratinocytes (see Fig. 5J) we could see clear positive staining but not as a defined layer.

Collagens type I and III were assessed and results showed that collagen I is highly expressed when the wounds were re-epithelialised (see Fig. 6C, E, G, I) whereas collagen III was more evident when no epithelialisation has taken place (see Fig. 6N), regardless of addition of STSG (see Fig.6P). Initial stages of re-epithelialisation as seen in Fig.4K, L (paste + fibroblasts) was not enough to show this switch in collagen types.

Discussion

Underlying conditions such as metabolic disease (e.g. diabetes mellitus), peripheral vascular disease, compromised immune function or previous local injury (e.g. radiation therapy) can compromise wound healing resulting in chronic or recalcitrant wounds. Acute trauma, cancer resections, infection or thermal injuries can also result in significant loss of the dermal layer causing vulnerability to infections, thermal deregulation, and fluid loss.

The dermis provides structural support to the skin, and it is an essential component for keratinocytes adhesion. Therefore, in order to have successful long-term wound healing in various conditions, replacement of the dermis is paramount. Understanding wound healing, especially from an extracellular matrix (ECM) perspective, can empower the design of a pro-regenerative and bioactive scaffold that can restore the structural and mechanical support lost in these cutaneous wounds. Although biologic scaffolds composed of naturally occurring ECM have received significant attention in the past decades, their entire potential in promoting a constructive remodelling in chronic conditions/secondary healing is still poorly understood.

Designing a pro-regenerative ECM substitute that allows complete healing of full thickness wounds, as well as understanding the factors that affect skin regeneration can be of great help in Tissue Engineering. Factors that appear important for the constructive remodelling of ECM biologic scaffolds are an ability to degrade with generation of downstream bioactive molecules in a timely manner; bio-inductive properties of its functional molecules supporting host cell attachment, proliferation and differentiation; and an ability to produce mechanical support at the implantation site through preservation of its collagen fibres.

We have developed a dermal paste derived from porcine dermal scaffolds that underwent a decellularisation process using osmotic shock with no enzymatic digestion. Our previous results (SDS-PAGE, SEM, TEM, and MS) revealed that these scaffolds retained structural properties comparable to a native dermal ECM.

The majority of decellularisation methods using enzymes can result in disruption of tissue architecture and potential loss of surface structure and composition, compromising the ability of the scaffold to provide mechanical support during the remodelling process.

We reported the protein profile of our porcine dermal scaffolds, which showed retention of approximately 60% of the extracellular proteins that promote cell-matrix interaction, and over 90% of the main collagen types (I, II and III) in the matrix after decellularisation. These molecules are of major importance as the orientation of collagen fibres can profoundly influence the directed migration of cells, possibly by potentiating growth factor receptor signalling or by mechanically reinforcing cell migration.

The model used herein resembles a healing by secondary intention where greater tissue proliferation is required and therefore it may take longer to heal. Our dermal paste, however, showed to facilitate the wound closure, inducing a pronounced host cells infiltration and differentiation within 4 weeks. We observed that in the wounds where STSG was not applied, the granulation phase was more pronounced, lasted longer and less epithelialisation was seen. Nevertheless, a well-developed microvasculature network and neocollagen deposition was clearly observed in all experimental conditions. Neo-collagen deposition was observed by PSR staining, which revealed increasing birefringence of newly-formed collagen fibres deposited within the biomateriaTs meshwork when tissue sections were examined using polarised light microscopy. Brighter-red coloured collagen bundles were also observed within the new remodelled dermis, which are most likely related to the implanted paste that had not been degraded. This may still be active and signalling to promote further healing, especially when restoration of epithelial continuity was shown to be incomplete. Epithelialisation is an essential component of wound healing used as a defining parameter of a successful wound closure. In our samples using paste and STSG implantation it seems that keratinocytes populated the wound bed below the STSG either (or both) by migration from the latter to the dermal paste or from precursors cells recruited to the wound bed. Finding an ideal niche, the cells proliferated and differentiated forming a multi-layered neo-epithelium, which was highly proliferative, including the formation of stratum corneum. Where paste and autologous cells were implanted, an initial stage of neo-epithelium was also observed in some of the experimental wounds, which makes the hypothesis of precursor cells recruitment more plausible in this experimental condition. As the STSG survival was minimal in all the wounds, we suggest that it might have worked as a ‘viable scab’, prolonging presence of proteinases and growth factors-rich fluid locally. This would create an ideal environment for cell growth and differentiation. The expression of different keratins (KR), which are the main components of cytoskeleton intermediate filaments, has been used to define keratinocyte maturation and different phenotypic subtypes participating in the wound healing process. Our results with native skin controls are in accordance with other studies showing that, in normal stratified epithelium, expression of KR15 is restricted to the basal layer and is present in small patches, whereas cells overlying dermal papillae show little or no expression. Our experimental conditions however allowed for evidencing a marked number of KR15-positive cells in the wound bed. Especially when the epithelialisation process was not complete, we observed a large number of KRl 5-positive cells scattered throughout the entire wound. Advanced molecular biology techniques have identified the importance of stem cell differentiation and initiation of epithelial stratification, showing derivation of keratinocytes from induced pluripotent cells and vice-versa. Several authors have described KRl 5 as a putative epidermal stem cell marker, with few contradictions. Since our model used isolating PTFE chambers, it does not allow migration of keratinocytes from the wound edges to cover the wound. Our results support the idea that migration of K 15-positive cells to the wound bed seems to be crucial to neo-epithelialisation, due to the marked presence of those cells preceding this phenomenon. Nevertheless, more detailed long-term studies using autologous cells in combination with dermal paste should provide further information regarding the molecular cues that drive this entire process.

Changes in the expression of different keratins during wound healing are particularly relevant to show the development of the neo-epidermis. In the basal layer, interfilaments KR5 and KRl 4 are expressed in the mitotically active basal keratinocytes. As these cells enter terminal differentiation, becoming post-mitotic and suprabasal, KR5 and KR14 are substituted by KRl and KR10.37. Our results demonstrated that as the epithelialisation process was complete (especially when paste + STSG and paste + STSG combination with autologous cells were applied), a clear layer of KRl/10 positive cells were present in the outermost layer of the new epithelium. Similarly, the presence of positive staining for laminin, a protein present in the basement membrane, was shown to be correlated with more mature epithelialisation. Another characteristic of wound remodelling is the change of extracellular matrix composition. In the remodelling phase, collagen fibres become more organized, fibronectin decreases markedly, while type III collagen is replaced by type I collagen. These events allow collagen fibres to become closer, promoting natural collagen cross-linking and thereafter decreasing scar thickness. This process is very important as it results in increased wound strength. We therefore assessed the expression of the collagen I and III in response to dermal paste implantation and the results showed that the transition from collagen type III to I was correlated with the re- epithelialisation process.

There are several advantages in using an acellular dermal paste:

(i) Paste can be added to extensive and deep wounds, whereas when using sheet-form matrices (allografts, xenografts or autografts), in order to cover the entire wound bed, it is necessary to either mesh it or add multiple sheets, with risk of displacement and accumulation of fluid between them.

(ii) For deep wounds with irregular contours and multiple interdigitations, a flowable dermal paste can be applied with a syringe into the wound bed of any shape with assurance of contact with the entire topography.

(iii) Wounds must have an adequate inflow of oxygen, enzymes, nutrients and cells in order to heal. A formulation that allows interchangeable communication with the host tissue and environment (allowing for better oxygenation) is ideal.

(iv) Oedema or excess of exudates decreases cell migration and prevents bactericidal activity of leukocytes. Hence, a formulation that can accommodate excess fluid and does not allow for its accumulation in the wound with consequent increase of the bioburden and healing impairment is paramount.

(v) Using any skin/dermal implants that can cause any complications described above can lead to multiple surgical procedures, adding other co-morbidities and increasing the overall costs and patient distress.

Improving wound healing in adverse conditions, minimizing the impact of multiple surgical procedures and patient distress, as well as rehabilitating tissue function remain a central interest in clinical wound management. After implantation (in a model that resembles second intention healing), our bioactive porcine dermal paste integrated closely with the wound topography, provided the framework for cell infiltration and differentiation, facilitated neo-collagen deposition and encouraged timely switching of keratins and collagen types.

Overall, the results suggest that the optimum biological properties of the non- crosslinked dermal paste formulation described herein can be an attractive and cost- effective alternative to current dermal substitutes for full-thickness wounds, allowing also for prompt grafting and regenerative cell-based therapy throughput.

The one or more embodiments are described above by way of example only. Many variations are possible without departing from the scope of protection afforded by the appended claims.

Claims

1 A combination for use in wound treatment, the combination comprising: decellularised dermal tissue paste; and isolated skin cells and/or a patch comprising skin cells.

2 The combination as claimed in claim 1 wherein the paste comprises animal dermal tissue.

3. The combination as claimed in claim 1 or 2 wherein the paste comprises porcine or human dermal tissue.

4. The combination as claimed in any preceding claim wherein the degree of cross- linking in the paste is less than 5%.

5. The combination as claimed in any preceding claim wherein the paste is substantially or completely free of cross-linking.

6 The combination as claimed in any preceding claim comprising the isolated skin cells, wherein the isolated skin cells comprise animal derived skin cells.

7 The combination as claimed in any preceding claim comprising the patch, wherein the patch comprises animal derived skin tissue.

8 The combination as claimed in any preceding claim comprising the isolated skin cells, wherein one or more layers of isolated skin cells are disposed within the paste.

9 The combination as claimed in any preceding claim comprising the isolated skin cells, wherein one or more layers of isolated skin cells are disposed on the paste.

10 The combination as claimed in any preceding claim comprising the isolated skin cells, wherein isolated skin cells are disposed within the paste and isolated skin cells are disposed on the paste.

11 The combination as claimed in any preceding claim comprising the patch, wherein the patch is disposed on the paste.

12 The combination as claimed in any preceding claim comprising the isolated skin cells, wherein the isolated skin cells comprise autologous cells.

13. The combination as claimed in any preceding claim comprising the patch, wherein the patch comprises autologous cells.

14. The combination as claimed in any preceding claim comprising the isolated skin cells, wherein the isolated skin cells comprise fibroblasts.

15. The combination as claimed in any preceding claim comprising the isolated skin cells, wherein the isolated skin cells comprise keratinocytes.

16. The combination as claimed in any preceding claim comprising the isolated skin cells, wherein the isolated skin cells comprise stem cells.

17. The combination as claimed in any preceding claim comprising the isolated skin cells, wherein the isolated skin cells comprise fibroblasts and keratinocytes.

18. The combination as claimed in any preceding claim comprising the patch, wherein the patch comprises fibroblasts and keratinocytes.

19. The combination as claimed in any preceding claim comprising the patch, wherein the patch comprises a skin graft.

20. The combination as claimed in any preceding claim comprising the isolated skin cells and the patch, wherein the isolated skin cells comprise fibroblasts and the patch comprises a skin graft.

21 The combination as claimed in claim 19 or 20 wherein the skin graft is a split thickness skin graft.

22 The combination as claimed in claim 20 or claim 21 when dependent on claim 20, wherein one or more layers of fibroblasts are disposed within the paste, and the skin graft is disposed on the paste.

23. The combination as claimed in any preceding claim wherein the combination is a tissue replacement.

24. A kit for use in wound treatment, the kit comprising: decellularised dermal tissue paste; and isolated skin cells and/or a patch comprising skin cells, optionally wherein the paste and the isolated cells and/or the patch, are contained prior to use.

25. A kit as claimed in claim 24 wherein the paste and the isolated skin cells and/or the patch, are separately contained prior to use.

26. A kit as claimed in claim 24 or 25 wherein the isolated skin cells are suspended in a biocompatible liquid medium.