Wound healing agent

Abstract

The present invention provides a D-deoxyribose sugar for use in promoting wound healing, wherein the sugar is provided in a carrier, the carrier being a biocompatible matrix material or hydrogel. [Selected Figure] Figure 1

Description

Technical Field

[0001] The present invention generally relates to the field of skin and hair restoration. More particularly, the present invention relates to the use of chemical compositions that promote wound healing, angiogenesis, vascularisation, and hair regrowth.

Background Art

[0002] There have been many studies examining methods to promote angiogenesis in wound beds. Among all angiogenic factors, the most potent factor commonly used in the development of biomaterials is vascular endothelial growth factor (VEGF) (Zhang et al., Biomaterials. 2015; Miyagi et al., Biomaterials. 2011; Khojasteh et al., Materials Science and Engineering: C. 2016; Johnson et al., Advances in Wound Care. 2014). This growth factor can be produced by recombinant technology and has been critically investigated in many studies (Neufeld et al., The FASEB journal. 1999; Long et al., Journal of theoretical biology. 2013; Xin et al., Cell. 2016). However, when growth factors are added alone, they are rapidly degraded or diluted and washed away, so the results have generally been disappointing. More recent strategies have involved delivering VEGF conjugated to biomaterials. Biomaterials can take various forms, including hydrogels, scaffolds, or particles (Chiu et al., Biomaterials. 2010; Cakir-Ozkan et al., Journal of Oral and Maxillofacial Surgery. 2017; Zhang et al., ACS Biomaterials Science & Engineering. 2016; Zhao et al., Advanced healthcare materials. 2016).

[0003] Several groups, including MacNeil's group, have attempted to deliver VEGF from heparin conjugated to a material. This is because heparin is a natural glycosaminoglycan that is present at high concentrations in wounds in the body and acts to bind VEGF and other pro-angiogenic factors (Wu et al., Biomacromolecules. 2016; Gigliobianco et al., Journal of biomaterials applications. 2015). Therefore, MacNeil's group has developed materials that can immobilize heparin electrostatically into a hydrogel (Gilmore et al., Biotechnology and bioengineering. 2013), or within the coating of each layer of an electrospun scaffold (Gigliobianco et al., Journal of biomaterials applications. 2015; Easton et al., Journal of Materials Chemistry B. 2014). In vivo heparin binds and releases VEGF and other pro-angiogenic mitogens (Ferrara N et al., Nature medicine. 2003). VEGF activates the proliferation and migration of endothelial cells in both normal and tumor tissues, leading to the rapid formation of new blood vessels (Harmey JH. Springer Science & Business Media; 2004; Hicklin et al., Journal of clinical oncology. 2005).Therefore, in the preparation of materials that require angiogenesis, such as the preparation of synthetic dermis for the treatment of full-thickness burns (Tan et al., Journal of tissue engineering and regenerative medicine. 2014; Xie et al., Acta biomaterialia. 2013; Guo R et al., Biomaterials. 2011), and to promote wound healing in chronic non-healing wounds such as diabetic ulcers where the microvascular structure is impaired, the potential of VEGF has been investigated.

[0004] VEGF stimulates angiogenesis, but is very expensive ($930 for 50 μg from Sigma-Aldrich) and unstable (Simon-Yarza et al., Theranostics. 2012; Thompson et al., Oxford Textbook of Vascular Surgery: Oxford University Press; 2016). Other angiogenesis-promoting growth factors involved in the formation of new blood vessels are known, but there is a complex cascade of factors that are produced and released in response to hypoxia.

[0005] Loss of the skin's barrier function can be life-threatening (Chua et al., Burns & trauma. 2016; Blais et al., Stem cells translational medicine. 2013). The first-choice treatment is usually the use of autologous split-thickness skin grafts, but in severely burned patients, the available grafts are insufficient to achieve rapid restoration of the barrier layer (Yi et al., Plastic and reconstructive surgery. 2015). Despite 30 years of research in the field, there is still a need for a functional and cost-effective permanent skin substitute to assist surgeons in managing patients with full-thickness wounds exceeding 30% (Chua et al., Burns & trauma. 2016).

[0006] When burns cover 30% or more than 40% of the total body surface area, burn surgeons will attempt to immediately cover the wound using natural and synthetic materials to assist in the ultimate replacement of both the dermis and epidermis (Sharma et al., Burns. 2014). Manufacturing tissue engineering materials equivalent to split-thickness skin grafts (containing all of the epidermis and a portion of the dermis) that will successfully "lie" on the wound bed remains technically very difficult (Bottcher-Haberzeth et al., Burns. 2010). The main challenge lies not in the production of materials in the laboratory (see Boyce et al), but in tissue engineering materials that can withstand engraftment on the wound bed, which requires rapid in-growth of new blood vessels from the underlying wound bed (Boyce et al., Annals of surgery. 2002; Supp et al., Clinics in dermatology. 2005). Practically speaking, graft survival is completely dependent on the growth of new blood vessels from the underlying wound bed into tissue engineering materials that lack any intrinsic vascular structure (Laschke et al., Tissue engineering. 2006; Hacker et al., Scientific reports. 2016).

[0007] In practice, full-thickness burns are usually treated in two stages (when autografting is insufficient). After a material for providing a vascularized dermal substitute is used, when the dermis is sufficiently vascularized, a thin split-thickness skin graft is placed on top of it (often by harvesting additional skin grafts from the initially healed donor site once, or by placing cultured cells over the vascularized dermis). The two most commonly used materials for providing vascularized dermis are the biomaterial Integra, developed for this purpose, and donor cadaver skin (Nguyen et al., Burns. 2010; Weigert et al., Journal of Hand Surgery (European Volume). 2011; Cleland et al., Burns. 2014). Integra consists of a bovine collagen matrix that has shark chondroitin sulfate, on which a silicon membrane is bonded (Chua et al., Burns & trauma. 2016). The burned tissue is clinically excised, Integra is placed in situ, and left in place until it becomes vascularized. This often takes more than three weeks. At this point, the surgeon can remove the silicon barrier membrane and place a thin split-thickness skin graft on top of it. The alternative material used is cadaver skin. This can be used to immediately cover the wound (after the burned tissue has been excised once) and restore the barrier function. Then, after it becomes vascularized within a few weeks, the donor's epidermal layer can be gently removed while leaving the donor's vascularized dermis in situ. And the epidermal barrier can be replaced with a patient-derived split-thickness skin graft or epidermal cells cultured from the patient (discussed in MacNeil, Nature. 2007). However, these materials are not always readily available to burn surgeons worldwide because there is no appropriately operated skin bank (for donor skin), or because the purchase of Integra is considered too expensive.

[0008] Hair loss is a common condition in humans, characterized by the loss or reduction of hair volume from the head or body. There are several different conditions that may be associated with hair loss. Several types of causes of hair loss are known, including infections, medications, trauma, and pregnancy, while the causes of other types of hair loss remain unknown. Some conventional treatments for hair loss may include drug therapies, such as those using medications like minoxidil or finasteride. Another treatment used is hair transplantation surgery, which involves harvesting hair follicles with hair fibers from a hairy part of the body and moving the follicles to a hairless part of the body.

[0009] In the management of severe burns, there is a need for new, effective, and affordable biomaterials that can provide a sufficiently vascularized dermal matrix. An important problem is that none of the current materials contain any intrinsic vascular structures, so in-growth of blood vessels is completely dependent on the blood vessels in the underlying wound bed (Supp et al., Clinics in dermatology. 2005; Sahota et al., Wound repair and regeneration. 2003). There is a need for a dermal matrix that can improve vascularization and increase the survival rate after transplantation. SUMMARY OF THE INVENTION

[0010] In one aspect, the present invention provides D - deoxyribose sugar for use in promoting wound healing, wherein the sugar is provided within a carrier, and the carrier is a biocompatible matrix material or a hydrogel. Preferably, the D - deoxyribose is 2 - deoxyribose.

[0011] In one embodiment, the carrier is a biodegradable carrier.

[0012] In one embodiment, the matrix material is an electrospun scaffold. Preferably, the electrospun scaffold comprises at least one of polylactic acid (PLA), polyglycolide (PGA), poly(lactic-co-glycolic acid) (PLGA), or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) PHBV.

[0013] In one embodiment, the hydrogel is a cross-linked hydrogel. Preferably, the hydrogel comprises at least one of chitosan, gelatin, alginate, agarose, methylcellulose, hyaluronan, or any combination thereof. In one embodiment, the hydrogel comprises chitosan and collagen. Additionally, the hydrogel may comprise polyvinyl alcohol, sodium polyacrylate, acrylate polymer, or any combination thereof. In one embodiment, the hydrogel comprises chitosan and polyvinyl alcohol.

[0014] In one embodiment, the carrier further comprises an antibacterial agent.

[0015] In one embodiment, the wound is a chronic wound.

[0016] In one embodiment, the wound is a full-thickness wound.

[0017] In one embodiment, the wound is a burn.

[0018] In one aspect, the present invention provides a biocompatible matrix material comprising D-deoxyribose sugar.

[0019] In one embodiment, the matrix material is an electrospun scaffold. Preferably, the electrospun scaffold comprises at least one of polylactic acid (PLA), polyglycolide (PGA), poly(lactic-co-glycolic acid) (PLGA), or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) PHBV.

[0020] In one aspect, the present invention provides a hydrogel containing D - deoxyribose sugar.

[0021] In one embodiment, the hydrogel is a cross - linked hydrogel. Preferably, the hydrogel contains at least one of chitosan, gelatin, alginate, agarose, methylcellulose, hyaluronan, or any combination thereof. In one embodiment, the hydrogel contains chitosan and collagen. Additionally, the hydrogel may contain polyvinyl alcohol, sodium polyacrylate, acrylate polymer, or any combination thereof. In one embodiment, the hydrogel contains chitosan and polyvinyl alcohol.

[0022] In one aspect, the present invention provides a biocompatible material of any one of the foregoing aspects or embodiments for use in promoting wound healing or treating alopecia.

[0023] In one aspect, the present invention provides a hydrogel of any one of the foregoing aspects or embodiments for use in promoting wound healing or treating alopecia.

[0024] In one aspect, the present invention provides a biocompatible material of any one of the foregoing aspects or embodiments for use in a method of increasing or inducing vascularization in a wound bed.

[0025] In one aspect, the present invention provides a hydrogel of any one of the foregoing aspects or embodiments for use in a method of increasing or inducing angiogenesis in a wound bed.

[0026] In one aspect, the present invention provides D - deoxyribose sugar for use in treating alopecia, wherein the sugar is provided in a carrier, and the carrier is a biocompatible matrix material or a hydrogel. Preferably, D - deoxyribose is 2 - deoxyribose.

[0027] In one embodiment, the carrier is a biodegradable carrier.

[0028] In one embodiment, the matrix material is an electrospun scaffold. Preferably, the electrospun scaffold comprises at least one of polylactic acid (PLA), polyglycolide (PGA), poly(lactic-co-glycolic acid) (PLGA), or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) PHBV.

[0029] In one embodiment, the hydrogel is a crosslinked hydrogel. Preferably, the hydrogel comprises at least one of chitosan, gelatin, alginate, agarose, methylcellulose, hyaluronan, or any combination thereof. In one embodiment, the hydrogel comprises chitosan and collagen. Additionally, the hydrogel may comprise polyvinyl alcohol, sodium polyacrylate, acrylate polymer, or any combination thereof. In one embodiment, the hydrogel comprises chitosan and polyvinyl alcohol.

[0030] In one embodiment, the carrier further comprises an antibacterial agent.

[0031] In one aspect, the present invention provides a non-therapeutic method for promoting hair regrowth, the method comprising the administration of D-deoxyribose sugar, the sugar being provided within a carrier, the carrier being a biocompatible matrix material or a hydrogel. Preferably, the D-deoxyribose is 2-deoxyribose.

[0032] In one embodiment, the carrier is a biodegradable carrier.

[0033] In one embodiment, the matrix material is an electrospun scaffold. Preferably, the electrospun scaffold comprises at least one of polylactic acid (PLA), polyglycolide (PGA), poly(lactic-co-glycolic acid) (PLGA), or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) PHBV.

[0034] In one embodiment, the hydrogel is a crosslinked hydrogel. Preferably, the hydrogel comprises at least one of chitosan, gelatin, alginate, agarose, methylcellulose, hyaluronic acid, or any combination thereof. In one embodiment, the hydrogel comprises chitosan and collagen. Additionally, the hydrogel may comprise polyvinyl alcohol, sodium polyacrylate, acrylate polymer, or any combination thereof. In one embodiment, the hydrogel comprises chitosan and polyvinyl alcohol.

[0035] In one embodiment, the carrier further comprises an antibacterial agent.

[0036] In one aspect, the present invention provides a wound dressing comprising a biocompatible matrix material according to any one of the foregoing aspects or embodiments.

[0037] In one aspect, the present invention provides a wound dressing comprising a hydrogel according to any one of the foregoing aspects or embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Embodiments of the present invention are further described below with reference to the accompanying drawings.

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Mode for Carrying Out the Invention

[0039] The inventors have surprisingly found that biomaterials carrying d - deoxyribose or L - deoxy sugars assist in the rapid formation of new blood vessels and aid in wound healing. Hydrogels carrying deoxyribose also promoted healing in an in vivo wound model. Wound healing was accompanied by new blood vessel formation. The ability of d - deoxyribose to promote wound healing is particularly surprising considering that other D - deoxy sugars such as 2 - deoxy - D - glucose are known to inhibit angiogenesis (Merchan J. et al., PLoS ONE 5(10): e13699).

[0040] The data presented indicate that a biomaterial carrying 2-deoxy-D-ribose supported the formation of new blood vessels within 7 days. This angiogenic property of deoxyribose was not shared to an appreciable extent by the other two deoxysugars investigated, namely deoxyfucose and deoxylamnose. However, the L-isomers of all three sugars were strongly angiogenic, while only the D-isomer of deoxyribose was strongly angiogenic. There are several advantages to using 2-deoxy-D-ribose or L-deoxysugars to stimulate VEGF production. For example, they are stable and inexpensive, and when introduced into a biomaterial, they can be sustained released over several days to stimulate the growth of new blood vessels. Also, most bacteria have been found to be unable to metabolize deoxyribose (Christensen et al., Journal of bacteriology. 2003).

[0041] The inventors have also found that common Gram-positive pathogens are unable to metabolize either the D- or L-isomers of the three deoxysugars tested (deoxyribose, deoxyfucose, and deoxylamnose), although some of these sugars can be metabolized by Gram-negative pathogenic species.

[0042] The inventors have demonstrated that a chitosan / collagen hydrogel carrying 2-deoxy-D-ribose stimulates the healing of an in vivo rat skin wound model. Wound healing was accompanied by new blood vessel formation.

[0043] The inventors have demonstrated that the gradual release of 2-deoxy-D-ribose from a biomaterial would promote the formation of new blood vessels and thus be of value in wound healing. The inventors sought to confirm whether the introduction of the D-sugar 2-deoxy-D-ribose could demonstrate pro-angiogenic properties and to evaluate whether this could be done using a wide range of clinically relevant biomaterials. Accordingly, this D-deoxysugar was incorporated into three biodegradable biomaterials, electrospun PCL nanofibers, a chitosan / PVA hydrogel, and a chitosan / collagen hydrogel.

[0044] The data show that this D-sugar, 2-deoxy-D-ribose, can be readily incorporated into three different biomaterials, and the inventors have confirmed that it is actually angiogenic using the CAM assay. Also, using this assay, the inventors investigated to what extent the angiogenic properties of the D-isomer of deoxyribose are shared with other deoxysugars. Here, the inventors found that all three L-isomers of deoxysugar (ribose, fucose, and rhamnose) are angiogenic, but only the D-isomer of deoxyribose is significantly angiogenic.

[0045] Noting the ability of these sugars to act as bacterial metabolic substrates, the inventors examined three strains of Staphylococcus aureus, but none of these strains were able to metabolize these sugars. This is encouraging news in attempting to develop biomaterials containing these sugars since these sugars are not bacterial nutrients.

[0046] The contribution of the D-isomer of deoxyribose to wound healing was evaluated in vivo using a chitosan / collagen hydrogel. For this, the inventors used a rat skin wound model. The inventors surprisingly found that the addition of D-deoxy-ribose to the chitosan / collagen hydrogel greatly promoted skin wound healing with an increase in vascularization detected by staining for CD34-positive cells. From this model, it was clear that the chitosan collagen gel itself stimulates wound healing, but this is significantly increased by the addition of the D-sugar. By day 17, wounds treated with the D-sugar were completely closed and the tissue structure showed the presence of very mature hair follicles. Based on this evidence, the inventors conclude that the release of the D-sugar from the chitosan / collagen gel stimulates angiogenesis and propose that the increased wound healing is a natural consequence.

[0047] Investigation of the macrophage response to deoxy sugars showed that by day 17, which indicates constructive remodeling, M2 macrophages were slightly more dominant than M1 macrophages. In the conduct of animal experiments, the first hydrogel was very robust to handle and could actually be sutured in place. Observation of the animal experiments on day 3 revealed that the hydrogel was very well adhered and in-growth of cells was also evident (Figure 8). The adhesion of the hydrogel to the surrounding tissue was better in the case of the sugar-bearing hydrogel. On day 9, the control hydrogel and the D-deoxyribose-bearing hydrogel were still intact. The hydrogel was gradually absorbed and by day 11, more than 50% of the wound area was healed with the D-deoxyribose hydrogel (see Figure 9), while the remaining hydrogel was still present on the wound (Figure 8). By day 14, there was no sign of D-deoxy sugar hydrogel remaining on the wound. By day 17, the D-deoxyribose transplanted wound was completely healed. This hydrogel seemed to be completely absorbed by the animal and could not be felt inside the healed skin upon palpation. The control hydrogel maintained its integrity until the end of the animal study.

[0048] These deoxy sugar-releasing hydrogels are a promising approach that stimulates angiogenesis in chronic non-healing wounds and also acts as a dermal substitute in the management of extensive full-thickness burns.

[0049] There are several advantages to stimulating VEGF production using this D-sugar; the D-sugar is stable and inexpensive and, when introduced into a biomaterial, can be sustained released over several days to stimulate the growth of new blood vessels.

[0050] The inventors have found that a chitosan-based hydrogel that releases D-deoxyribose can act as a substitute for the dermis as it stimulates in-growth of new blood vessels and provides a vascularized wound bed for subsequent transplantation with thin skin grafts or autologous cultured keratinocytes, resulting in the provision of a permanent skin barrier layer.

[0051] The positive effect of D - deoxyribose is surprising since in 2010 Merchan et al. reported the anti - angiogenic activity of 2 - deoxy - D - glucose (2 - DG). Merchan et al. reported that 2 - DG inhibits endothelial capillary formation and endothelial cell migration in vitro [.R. Merchan, K. Kovacs, J.W. Railsback, M. Kurtoglu, Y. Jing, Y. Pina, N. Gao, T.G. Murray, M.A. Lehrman, T.J. Lampidis, Antiangiogenic activity of 2 - deoxy - D - glucose, PLoS One. 5 (2010). doi:10.1371 / journal.pone.0013699].

[0052] The data presented herein indicate that for clinically useful products, rapid (within 5 days) post - transplantation neovascularization is important. For any TE construct over 200μm to survive in vivo, rapid ingrowth and infiltration of blood vessels are essential [C.K. Griffith, C. Miller, R.C.A. Sainson, J.W. Calvert, N.L. Jeon, C.C.W. Hughes, S.C.George, Diffusion Limits of an in Vitro Thick Prevascularized Tissue, Tissue Eng. 11 (2005) 257 - 266. doi:10.1089 / ten.2005.11.257]. The inventors have overcome this barrier by functionalizing tissue - engineering carriers with angiogenesis - promoting substances.

[0053] The inventors have generated a tissue - engineering carrier that overcomes the delay in neovascularization commonly seen after transplantation of tissue - engineering constructs.

[0054] The inventors firmly adhered the 2dDR - loaded CS / collagen hydrogel to the wound bed of full - thickness excisional wounds (20 mm) in rats by the third day. The wounds treated with 2dDR completely closed by the 17th day, and hair growth was distinct. On the other hand, the control wounds remained open, showing no evidence of epithelial tissue or hair growth. The tissue structure of the wound beds of all sacrificed animals on the 17th day showed complete healing in the case of 2dDR - treated wounds, with well - developed hair follicles. And the presence of new blood vessels in the healed wounds was confirmed by CD34 staining. [M. Yar, L. Shahzadi, M. Azra, M.I. Raheem, S. Roman, A.A. Chaudhry, I. ur Rehman, C.W.I. Douglas, S. MacNeil, Deoxy - sugar releasing biodegradable membranes and hydrogels promote angiogenesis and stimulate wound healing, Mater Today Commun.13 (2017) 295 - 305. doi:10.1016 / j.mtcomm.2017.10.015]. Carbohydrates can have D - and L - enantiomeric configurations determined by the configuration of the chiral carbon farthest from the anomeric carbon. When the linear form of a carbohydrate is depicted as a Fischer projection, the hydroxyl group attached to the chiral carbon can be arranged on either the left or right side of the carbon. If the hydroxyl is on the right side of the chiral carbon, the carbohydrate is annotated as a D - carbohydrate, and if the hydroxyl group is on the left side of the chiral carbon, the carbohydrate is annotated as an L - carbohydrate. The following example shows the constitution of the D - and L - enantiomers of glucose. [Chemical formula]

[0055] D-isomers of carbohydrates are the most common forms found to exist in nature, and many can be isolated from natural sources. In contrast, the non-naturally occurring L-isomers of carbohydrates can be costly to produce by chemical or enzymatic synthesis. However, some L-isomers of carbohydrates are also known to exist in nature.

[0056] A specific example of a naturally occurring D-sugar is D-ribose (chemical formula C5H 10 O5):

Chem.

[0057] The phosphorylated form of D-ribose is involved in amino acid synthesis, used in the pentose phosphate pathway, and is a component of ribonucleic acid (RNA).

[0058] Also, suitably, D-ribose can be dehydroxylated, for example, at the 2’, 3’, 4’, or 5’ positions. A notable example of dehydroxylated ribose is 2-D-deoxyribose where the hydroxyl group at the 2’ position is replaced by a hydrogen atom. This sugar is a component of deoxyribonucleic acid (DNA). Deoxy-D-ribose exists mainly in a cyclic form in solution rather than in a linear form.

Chem.

Chem.

[0059] Deoxy sugars are sugars in which a hydroxyl group is replaced by a hydrogen atom. Examples of deoxy sugars include L-2 deoxyribose, L-fucose (equivalent of 6-deoxy-L-galactose), and L-rhamnose (equivalent of 6-deoxy-L-mannose).

[0060] The L-enantiomers of deoxy sugars will become apparent for specific uses in the present invention.

[0061] The inventors have also surprisingly found that a biomaterial loaded with estradiol (E2) supports the rapid formation of new blood vessels and aids in wound healing. Estradiol (E2) plays an important role in neovascularization during the menstrual cycle [D.W. Losordo, J.M. Isner, Estrogen and Angiogenesis: A Review, Arterioscler 30 Thromb Vasc Biol. 21 (2001) 6-12. doi:10.1161 / 01.ATV.21.1.6, Y. Matsubara, K. Matsubara, Estrogen and progesterone play pivotal roles in endothelial progenitor cell proliferation, Reprod Biol Endocrinol. 10 (2012) 2. doi:10.1186 / 1477-7827-10-2]. Estradiol (E2) is clinically used in the treatment of osteoporosis and heart disease [M.L. Stefanick, Estrogens and progestins: Background and history, trends in use, and guidelines and regimens approved by the US Food and Drug Administration, in: Am J Med, 2005. doi:10.1016 / j.amjmed.2005.09.059].Furthermore, the blockade of the E2 receptor by adjuvants such as tamoxifen for estrogen receptor-positive tumors (where high estrogen aids in the growth and spread of cancer cells) is an effective method for reducing the tumor vasculature that has been used in clinics for many years, particularly for the treatment of breast cancer [B. Fisher, J. Costantino, C. Redmond, R. Poisson, D. Bowman, J. Couture, N. V Dimitrov, N. Wolmark, D.L. Wickerham, E.R. Fisher, A randomized clinical trial evaluating tamoxifen in the treatment of patients with node-negative breast cancer who have estrogen-receptor-positive tumors., N Engl J Med. 320 (1989) 479-84. doi:10.1056 / NEJM198902233200802, Early Breast Cancer Trialists Collaborative Group, Tamoxifen for early breast cancer: an overview of the randomised trials, Lancet. 351 (1998) 1451-1467. doi:10.1016 / S0140-6736(97)11423-4].E2 promotes endothelial cell migration and proliferation in vitro [K. Nikhil, S. Sharan, R. Wishard, S.R. Palla, R. Krishna Peddinti, P. Roy, Pterostilbene carboxaldehyde thiosemicarbazone, a resveratrol derivative inhibits 15 17β-Estradiol induced cell migration and proliferation in HUVECs, Steroids. 108 (2016) 17-30. doi:10.1016 / j.steroids.2016.01.020, G.M. Rubanyi, A. Johns, K. Kauser, Effect of estrogen on endothelial function and angiogenesis, Vascul Pharmacol. 38 (2002) 89-98. doi:10.1016 / S0306- 3623(02)00131-3], and has been shown to stimulate new blood vessel formation both in vitro and in vivo [D.E. Morales, K.A. McGowan, D.S. Grant, S. Maheshwari, D. Bhartiya, M.C. Cid, H.K. Kleinman, H. William Schnaper, Estrogen Promotes Angiogenic Activity in Human Umbilical Vein Endothelial Cells In Vitro and in a Murine Model, Circulation. 91 (1995) 755-63].The inventors confirmed that a poly-L-lactic acid (PLLA) scaffold carrying E2 is highly angiogenic using a CAM assay [N. Mangir, C.J. Hillary, C.R. Chapple, S. MacNeil, Oestradiol-releasing Biodegradable 25 Mesh Stimulates Collagen Production and Angiogenesis: An Approach to Improving Biomaterial Integration in Pelvic Floor Repair, Eur Urol Focus. (2017). doi:10.1016 / j.euf.2017.05.004].

[0062] E2 has previously been reported by many groups and the inventors' research group to be angiogenic both in vitro and in vivo. Albrecht et al. suggested that E2 promotes angiogenesis through upregulation of VEGF. Albrecht et al. reported that administration of E2 to ovariectomized monkeys rapidly increases VEGF expression and cell permeability [E.D. Albrecht, J.S. Babischkin, Y. Lidor, L.D. Anderson, L.C. Udoff, G.J. Pepe, Effect 15 of estrogen on angiogenesis in co-cultures of human endometrial cells and microvascular endothelial cells., Hum Reprod. 18 (2003) 2039-2047. doi:10.1093 / humrep / deg415]. Similarly, an increase in VEGF mRNA expression levels was observed by Hyder et al. in ovariectomized rats after E2 treatment [S.M. Hyder, G.M. Stancel, C. Chiappetta, L. Murthy, H.L. Boettger-Tong, S. Makela, Uterine expression of vascular endothelial growth factor is increased by estradiol and 20 tamoxifen, Cancer Res. 56 (1996) 3954-3960].Similarly, Morales et al. reported that E2 promotes the migration of HUVECs and the formation of capillary-like networks on Matrigel [D.E. Morales, K.A. McGowan, D.S. Grant, S. Maheshwari, D. Bhartiya, M.C. Cid, H.K. Kleinman, H. William Schnaper, Estrogen Promotes Angiogenic Activity in Human Umbilical Vein Endothelial Cells In Vitro and in a Murine Model, Circulation. 91 (1995) 755-63]. Pence et al. showed that exogenous E2 promotes the endogenous production of VEGF by endometrial epithelial cells [J.C. Pence, K.B.H. Clancy, B.A.C. Harley, The induction of pro-angiogenic processes within a collagen scaffold via exogenous estradiol and endometrial epithelial cells, Biotechnol Bioeng. 112 (2015) 2185-2194. doi:10.1002 / bit.25622].More recently, the inventors' group demonstrated that E2 is released from both biodegradable (PLA [N. Mangir, C.J. Hillary, C.R. Chapple, S. MacNeil, Oestradiol-releasing Biodegradable 25 Mesh Stimulates Collagen Production and Angiogenesis: An Approach to Improving Biomaterial Integration in Pelvic Floor Repair, Eur Urol Focus. (2017). doi:10.1016 / j.euf.2017.05.004]) fibers and non-degradable (PU [S. Shafaat, N. Mangir, S.R. Regureos, C.R. Chapple, S. MacNeil, Demonstration of improved tissue integration and angiogenesis with an elastic, estradiol releasing polyurethane material designed for use in pelvic floor repair, Neurourol Urodyn. (n.d.) n / a-n / a. doi:10.1002 / nau.23510]) fibers, and that both electrospun scaffolds demonstrate good angiogenesis-promoting activity in the CAM assay.

[0063] As used herein, the term "promotion of wound healing" should be understood to mean restoring a disruption in the continuity of skin tissue (including any disorder, impairment, syndrome, abnormality, pathology, or abnormal condition characterized by any disease of the skin (dermis and epidermis) and / or underlying connective tissue). For example, a wound can be a small cut or abrasion; an intermediate wound; a composite / full-thickness wound; a traumatic wound such as a scratch or laceration; a surgical wound; a postoperative incision; a chronic / non-healing wound such as a pressure sore or a non-healing diabetic foot wound; an ulcer, particularly a venous ulcer, a decubitus ulcer, a skin ulcer due to infection, a decubitus ulcer, or a diabetic ulcer; an injury to connective tissue such as bone or cartilage; a chemical injury or a burn, particularly a third-degree burn; an accidental wound; a necrotic wound such as ischemic necrosis; an infected wound; the donor site of a full-thickness skin graft and a split-thickness skin graft; a bed sore; a diabetic-induced and age-induced failure of wound healing on the skin surface, as well as scars such as keloid scars, contracture scars, hypertrophic scars, and acne scars. A wound can be an open wound or a closed wound. As used herein, a "closed" wound means a wound that is open at one point (e.g., a surgical incision or an accidental cut) and is intentionally closed by sutures, staples, surgical adhesives, etc.

[0064] As used herein, the term "full-thickness wound" refers to the destruction of the dermis, tissue, and blood vessels in the dermis. In contrast, an "intermediate wound" refers only to the destruction of dermal tissue.

[0065] A specific use of D-deoxyribose or E2 is for use in extensive full-thickness wounds caused by burns.

[0066] According to the present invention, wound healing can be achieved by subjecting a subject to any suitable administration schedule, procedure, and / or route of administration of a composition containing D-deoxyribose sugar or E2 to the wound bed.

[0067] As used herein, the term "wound bed" refers to the exposed surface on the body where a disruption in the continuity of skin tissue has occurred.

[0068] Wound healing can mean complete or substantially complete healing of a wound, and the wound tissue is restored to substantially the same state as before the wound. Alternatively, wound healing can mean partial healing of a wound. In a further option, wound healing can include a reduction in the formation of fibrous scar tissue. Wound healing can be used for therapeutic purposes or for non-therapeutic [cosmetic] purposes.

[0069] As used herein, "induce" refers to the action of causing, promoting, forming, regulating, or activating a particular phenomenon. As used herein, "increase" refers to the action of promoting or enhancing a particular phenomenon.

[0070] D-deoxyribose or E2 may be used to increase or induce angiogenesis in a wound bed. Alternatively, D-deoxyribose or E2 may be used to increase or induce vascularization in a wound bed.

[0071] As used herein, "vascularization" includes the new formation, growth, development, or proliferation of blood vessels derived from undifferentiated or differentiating cells. Suitably, the blood vessels can be formed by the de novo generation of endothelial cells. As used herein, "angiogenesis" refers to the formation of new blood vessel structures (e.g., blood vessels; e.g., veins, arteries, venules, arterioles, capillaries) from existing blood vessel structures. For example, angiogenesis can occur by sprouting of new blood vessels from existing blood vessels (sprouting angiogenesis) and / or by branching of blood vessels (plexiform angiogenesis). Without wishing to be bound by a particular theory, D-deoxyribose may induce a chemical stimulus for angiogenesis via induction and / or stimulation and / or production of VEGF. VEGF is a known chemical stimulator of angiogenesis.

[0072] In addition to this, D-deoxyribose or E2 may also be used for the treatment of hair removal. Suitably, D-deoxyribose or E2 may be used to increase or induce hair regrowth. D-deoxyribose or E2 may also be used to delay, prevent or minimize hair removal. For this purpose, it is intended that D-deoxyribose may have both therapeutic and non-therapeutic [cosmetic] uses.

[0073] Suitably, D-deoxyribose or E2 may be used to treat alopecia. As used herein, "alopecia" is a general term encompassing all forms of medical hair removal. Alopecia can be caused by follicular absence, destruction (which may be scarring), disease, infection, shrinkage, dysregulation (changes in the hair cycle), or complete shutdown / dormancy. These changes can be temporary or permanent, partial, regional, and / or clearly global.

[0074] Examples of specific types of alopecia encompassed by the use of the term alopecia are selected from the group consisting of: androgenic alopecia; alopecia areata; alopecia totalis; alopecia universalis; telogen effluvium; anagen effluvium; traumatic alopecia; mechanical traction alopecia from hair styling routines; chemical-induced alopecia; heat-induced alopecia; radiation-induced alopecia; chemotherapy-induced alopecia; cicatricial alopecia; autoimmune disease-induced alopecia (e.g., from discoid lupus erythematosus or chronic cutaneous lupus erythematosus); disease-related alopecia (e.g., from hyperthyroidism or hypothyroidism, iron deficiency); drug-induced alopecia (e.g., antibiotics and antifungals; antidepressants, anticonvulsants; anticoagulants such as heparin and some LMWHs; NSAIDs such as aspirin; antihypertensives, alopecia induced by hormone replacement therapy), and syphilitic alopecia.

[0075] Examples of other conditions to be considered under this term are as follows: pituitary insufficiency such as Simmonds' disease and Sheehan's syndrome; other endocrine disorders such as hypothyroidism, diabetes, adrenogenital hypoplasia; chronic diseases such as lupus erythematosus, neoplasma; monilethrix; pseudomonilethrix; hereditary hypotrichosis simplex of the scalp; congenital triangular alopecia of the temporal region; craniosynostotic alopecia - Hallermann - Streiff syndrome; hereditary cicatricial alopecia such as Marie Unna type generalized alopecia, diffuse congenital cicatricial follicular keratosis; epidermal nevi - benign epithelial hyperplasia and / or malignant or benign tumors; physical trauma such as motor vehicle accidents, sports injuries; post - operative; anti - tumor agents such as cytostatic agents and / or alkylating agents; chemical trauma such as thioglycolate, antimetabolites; ionizing radiation; toxic drugs such as thallium, vitamin A; self - inflicted physical trauma such as trichotillomania; bacteria such as pyodermitis, impetigo of Bockhart; filamentous fungi / fungi such as candidiasis, dermatophytosis; accidental trauma / friction such as hats, beauty salons, etc. The related term is hypotrichosis which is defined as a decrease in hair production.

[0076] Suitably, a composition containing D - deoxyribose sugar or E2 may be applied to the isolated hair follicle. As used herein, the term "hair follicle" refers to a tubular structure derived from the epidermis in which a hair fiber can develop. A hair follicle typically consists of the following structures: papilla, matrix, root sheath, sebaceous gland, and optionally a hair fiber (consisting of a hair shaft and a hair root). The hair fiber may have a reduced diameter or may not be present at all depending on the degree of alopecia or the degree and stage of development of the formed hair follicle.

[0077] Optionally, a carrier may be provided for D-deoxyribose or E2. The carrier is preferably selected from gels, more preferably hydrogels, or biocompatible matrix materials, preferably electrospun scaffolds, particles, wound dressings, or cotton swabs, and is impregnated with a solution containing D-deoxyribose or E2. The carrier may consist of an amorphous material that does not have a defined surface and lacks a specific shape or solid matrix.

[0078] Optionally, a carrier that releases D-deoxyribose or E2 may stimulate in-growth of new blood vessels and provide a vascularized wound bed for subsequent transplantation with thin skin grafts or autologous cultured keratinocytes, thereby providing a permanent skin barrier layer and thus acting as a dermal substitute.

[0079] The carrier may further contain a substance that acts as an antibacterial agent, such as silver or cerium.

[0080] These carriers may be suitable for sustained release of D-deoxyribose or E2 into the wound. Preparations of D-deoxyribose or E2 with the carrier are particularly suitable for topical administration.

[0081] Optionally, D-deoxyribose or E2 may be formulated with or bound to a biocompatible matrix material. As used herein, the matrix material means a carrier or scaffold for cell mobilization, attachment, proliferation, and differentiation, as well as a potential delivery and storage device for D-deoxyribose or E2.

[0082] The preparation of biocompatible matrix materials is well known in the art. The biocompatible matrix preferably aids in wound healing and / or increases or induces angiogenesis or vascularization in the wound bed.

[0083] Suitably, the biocompatible matrix material may include a polymeric material. The polymeric material may include homopolymers or heteropolymers / copolymers of two or more different polymeric substances, or a combination of two. As used herein, the term "polymer" refers to a macromolecule formed by chemical bonding of five or more identical bonding units called monomers, such as sugars. Polymers can be of natural origin [e.g., polysaccharides such as starch, cellulose, pectin, seaweed rubber, plant rubber; polypeptides such as casein, albumin, globulin, keratin, insulin, DNA; and hydrocarbons], synthetic [e.g., thermoplastics (unvulcanized elastomers, nylon, polyvinyl chloride, poly(vinylidene fluoride trifluoroethylene) linear polyethylene, polystyrene, polypropylene, polyurethane, acrylate resins); thermosets (e.g., vulcanized elastomers, crosslinked polyethylene, phenol, alkyd, polyester), and semi-synthetic (e.g., cellulose-based such as rayon, methyl cellulose, cellulose acetate; and modified starch)]. The term "homopolymer" refers to a natural or synthetic polymer derived from a single monomer. The term "heteropolymer" refers to a natural or synthetic polymer (i.e., copolymer) derived from multiple monomer subunits. Unless otherwise specified, the term "polymer" is generally used to refer to both homopolymers and heteropolymers (i.e., copolymers) described herein.

[0084] As used herein, the term "biocompatible material" or "biocompatible matrix material" means that the matrix material does not stimulate a response, or stimulates only a mild and / or transient response, as opposed to a severe or stepwise response when the matrix material is placed or implanted in the desired wound bed.

[0085] Suitable biocompatible matrix materials include, for example, synthetic or naturally occurring matrices such as collagen, acellular matrices, crosslinked biomimetic molecules, tissue-based bioengineered structural frameworks, biomanufactured bioprostheses, and other implant structures such as vascular grafts suitable for cell infiltration and cell growth useful for promoting wound healing. Additional suitable biocompatible matrix materials can include collagen tissues chemically modified to reduce antigenicity and immunogenicity. Other suitable examples include collagen sheets for wound dressings, non-antigenic or antigen-reduced acellular matrices (Wilson G J et al. (1990) Trans Am Soc Artif Intern 36:340-343), or other biocompatible matrices designed to reduce the antigenic response to xenograft materials. Other matrices useful for promoting wound healing include, for example, processed bovine pericardial proteins containing insoluble collagen and elastin (Courtman DW et al. (1994) J Biomed Mater Res 28:655-666), and other acellular tissues that may be useful for providing a natural microenvironment for host cell migration to promote tissue regeneration (Malone J M et al. (1984) J Vasc Surg 1:181-91).

[0086] Suitably, the biocompatible matrix material can be formed by electrospinning to form a scaffold. The electrospinning process involves applying a high voltage to an injectable polymer solution to synthesize the polymer matrix material. The high voltage applied is sufficient to overcome the surface tension of the polymer solution, which results in fine fibers by stretching the polymer droplets, forming a non-woven matrix, mat, or mesh in a random orientation. Electrospinning typically produces a non-woven (i.e., mesh) matrix (also called a mat and / or scaffold) having a fiber diameter in the range of several hundred nanometers with interconnected pores. The electrospinning process typically results in the formation of a smooth surface substantially free of any beaded fibers. The matrix fibers may include a non-woven mesh of random and / or aligned nanofibers, or a combination thereof. Suitably, when D-deoxyribose sugar is carried, the nanofibers will typically have an increased diameter. For example, the carried fibers can have a diameter ranging from about 0.2 microns to about 5.0 microns.

[0087] The electrospun nanofibers may include polycaprolactone. Advantageously, polycaprolactone is biodegradable. Suitably, other biodegradable polymers may be used instead of polycaprolactone. Examples of other suitable biodegradable polymers that can be suitable alternatives include polylactic acid (PLA), polyglycolide (PGA), poly(lactic-co-glycolic acid) (PLGA), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) PHBV.

[0088] As used herein, the term "biodegradable" material is one that can be metabolized or excreted over a finite period under standard in vivo physiological conditions and can be broken down into components that have no harmful effects when decomposed in vivo.

[0089] Alternatively, the carrier may be a hydrogel. A hydrogel constitutes a network of hydrophilic functional groups attached to a natural or synthetic polymer backbone chain. The hydrogel may include a colloidal gel with an aqueous dispersion medium. The hydrophilic functional groups protruding from the polymer chain may make the hydrogel highly absorbent (some may contain more than 99.9% water). The high liquid content of the hydrogel may make the hydrogel flexible to a degree comparable to natural tissue. The absorbency of the hydrogel may also allow the hydrogel to act as a reservoir for local drug delivery. The hydrogel may be a crosslinked hydrogel. Crosslinking of the hydrogel may prevent dissolution. Crosslinking may be achieved by reacting with a suitable crosslinking agent. Suitable hydrogels may include, for example, a three-dimensional network of crosslinked hydrophilic polymers that are insoluble in water and interact with aqueous solutions by swelling. Exemplary hydrogels are highly compatible and permeable and, depending on their composition, may absorb various amounts of aqueous solutions. Suitably, the hydrogel may be non-adherent to the treatment site or may be treated to allow for easy removal.

[0090] Suitably, the hydrogel may preferably comprise at least one of chitosan, gelatin, alginate, agarose, methylcellulose, collagen, hyaluronan, or any combination thereof. Preferably, the hydrogel may comprise chitosan and collagen. In addition, the organochemical hydrogel may comprise polyvinyl alcohol, sodium polyacrylate, acrylate polymers, and copolymers with abundant hydrophilic groups.

[0091] Suitably, the carrier comprises ascorbic acid or a derivative thereof, such as L-ascorbic acid or ascorbic acid 2-phosphate (Mangir et al Acta Biomater. 2016 Jan 1; 29: 188-197. doi: 10.1016 / j.actbio.2015.10.019)

[0092] The term "wound dressing" refers to a dressing for topical application to a wound and excludes compositions suitable for systemic administration. For example, D - deoxyribose sugar or E2 may be dispersed in or on a solid sheet of a wound - contacting material such as a woven or non - woven fabric material, in a layer of a foam such as a polyurethane foam, or in a hydrogel, for example, a chitosan hydrogel, polyvinyl alcohol, polyacrylate hydrogel, gelatin, methylcellulose, agarose, alginate, hyaluronic acid hydrogel, or any combination thereof, for example, in a gel or an ointment. Suitably, D - deoxyribose sugar or E2 is dispersed in or on a biodegradable sheet material, for example, a sheet of a lyophilized chitosan / polyvinyl alcohol mixture, which achieves sustained release of the active ingredient into the wound.

[0093] Suitably, D - deoxyribose or E2 may be provided in the form of a liquid, semi - solid, or solid composition for direct application, or the composition may be applied to or incorporated into the surface of a solid contact layer such as a wound dressing, a hydrogel, a scaffold, or a matrix. The dressing composition may be provided, for example, in the form of a fluid or a gel.

[0094] A suitable amorphous hydrogel dressing may, for example, contain a formulation of water, polymer, and other formless components designed to provide moisture, maintain a moist healing environment, and / or re - hydrate the wound site. The hydrogel may be used in combination with a secondary dressing cover.

[0095] Examples of hydrogel dressings can include gauze and non - woven sponges, ropes, and strips saturated with amorphous hydrogel. Examples of suitable impregnated dressings can include gauze and non - woven sponges, ropes, and strips saturated with a solution, an emulsion, an oil, a gel, or some other pharmaceutically active compound or carrier agent (for example, physiological saline, oil, zinc salts, petrolatum, xeroform, and scarlet red, as well as the compounds described herein).

[0096] Suitable silicone gel sheets for bandages can include, for example, soft covers composed of cross-linked polymers reinforced with or bonded to a mesh or cloth.

[0097] Suitable liquid bandages can include, for example, a mixture of multi-protein materials and other elements found in the extracellular matrix. The solution may be applied to the treatment site after debridement and cleansing and then covered with an absorbent dressing or a non-adhesive pad.

[0098] Suitable transparent film bandages can include polymer films of various thicknesses coated with an adhesive on one side. The transparent film can be impermeable to liquids, water, and bacteria, but permeable to water vapor and atmospheric gases. The transparency of the film can allow visualization of the treatment site.

[0099] Suitable filler bandages can include, for example, beads, creams, foams, gels, ointments, pads, pastes, pillows, powders, strands, or other formulations. The filler may be non-sticky or may contain a sustained-release antimicrobial agent. Exemplary fillers can be useful for maintaining a moist environment, managing exudate, and treating, for example, partial-thickness and full-thickness wounds, infected wounds, draining wounds, and deep wounds that require packing.

[0100] D-deoxyribose or E2 may be provided in combination with conventional pharmaceutical excipients for topical application. Suitable pharmaceutical carriers include: Pluronic gels, poloxamer gels, hydrogels (containing cellulose derivatives such as hydroxyethyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, hydroxypropylmethyl cellulose, and mixtures thereof), and hydrogels containing polyacrylic acid (Carbopols).

[0101] Also, as other suitable carriers, creams / ointments used in topical pharmaceutical preparations can be mentioned, for example, creams based on cetomacrogol emulsifying ointment. The above carriers may contain alginate (as a thickener or stimulant), preservatives such as benzyl alcohol, buffers for controlling pH such as disodium hydrogen phosphate / sodium dihydrogen phosphate, agents for adjusting osmotic pressure such as sodium chloride, and stabilizers such as EDTA.

[0102] Preferred formulations include those for topical administration. Also included are compositions containing the incorporation of D-deoxyribose or E2 into liposomes, microemulsions, micelles, microparticles, or vesicles for long-term controlled delivery to the wound bed.

[0103] The pharmaceutical composition may, as described above, contain D-deoxyribose or E2 formulated with or bound to a biocompatible matrix, preferably containing collagen, gelatin, chitosan, and / or hyaluronan. The pharmaceutical composition may also contain a hydrogel having the above D-deoxyribose or E2.

[0104] The pharmaceutical composition may contain an antibacterial agent such as silver or cerium.

[0105] In particular, the present invention provides a pharmaceutical composition for use as a medicament. Specific uses are the above-mentioned wound healing and / or stimulation of angiogenesis. Another use is the treatment of the above-mentioned alopecia and other related disorders. A further option is the cosmetic treatment of hair removal.

[0106] Also provided is the use of an in vivo method, for example, to stimulate angiogenesis, for a subject having a wound or in need of such treatment.

[0107] As used herein, "topical administration" means administration to a defined surface, for example, directly to the wound surface, or if the wound is closed, to the area around or within the closure.

[0108] As used herein, the term "subject" in accordance with all aspects of the present invention refers to a vertebrate. The subject preferably refers to a mammalian animal including a human, a veterinary animal or a farm animal, a livestock or a pet, and an animal commonly used in clinical research (including non-human primates, dogs, rats, and mice). More specifically, the subject of the present invention may be a human.

[0109] Throughout the description and claims of this specification, the words "comprising" and "including" and their variations mean "including but not limited to", and are not intended (and do not exclude) other parts, additives, components, integers, or steps. Throughout the description and claims of this specification, the singular form includes the plural unless the context requires otherwise. In particular, when an indefinite article is used, the specification should be understood to contemplate plurality as well as singularity unless the context requires otherwise.

[0110] Features, integers, characteristics, compounds, chemical moieties, or groups described in connection with a particular aspect, embodiment, or example of the present invention are to be understood as applicable to any other aspect, embodiment, or example described herein, unless they are inconsistent therewith. All features disclosed in this specification (including the appended claims, abstract, and drawings), and / or all steps of any method or process disclosed therein, may be combined in any combination except combinations where at least some of such features and / or steps are mutually exclusive. The present invention is not limited to the details of any of the foregoing embodiments. The present invention extends to any novel one of the features disclosed in this specification (including the appended claims, abstract, and drawings), or any novel combination thereof, or any novel one of the steps of any method or process so disclosed, or any novel combination thereof.

[0111] The reader is to take note of all papers and documents that were filed simultaneously with or prior to this specification in connection with this application and that were made available to the public for inspection together with this specification, and the content of all such papers and documents is hereby incorporated by reference into this specification.

Example

[0112] [Experimental Section 1] Materials Polycaprolactone (Mw: 45,000), L-rhamnose, and NaOH were purchased from Sigma-Aldrich (USA). Chitosan (CS) was purchased from Mian Scientific Company, Lahore, Pakistan and further purified in our laboratory (Degree of deacetylation (DD) 84%; Mw: 87047.26 g / mol) (Farooq et al., Materials Science and Engineering: C. 2015). Collagen was purchased from the supplier Lahore of Mian Scientific. Poly(vinyl alcohol) (PVA) (Mw: 72,000, degree of hydrolysis 98%), hydrochloric acid (HCl), and sulfuric acid (H2SO4) were purchased from Merck (Germany). Triethyl orthoformate (98%) was purchased from Alfa Aesar (Germany). Glacial acetic acid (CH3COOH) was purchased from AnalaR BDH Laboratory Supplies (UK). Ethanol (99.8%) was purchased from Sigma-Aldrich (Germany). 2-Deoxy-D-ribose, D-fucose, and L-fucose were purchased from Sigma-Aldrich China, UK, and Slovakia, respectively. 2-Deoxy-L-ribose and D-rhamnose were products of Carbosynth (UK). Brain Heart Infusion broth and Tryptone Soya broth (1% w / v) were purchased from Oxoid Ltd. Phenol Red was purchased from Sigma-Aldrich UK.

[0113] Electrospinning PCL (10 wt%) was dissolved in 5 ml of DMF / DCM (4:1, 10% w / v), and 2-deoxy-D-ribose (1.8 wt%) was dissolved in DMF (0.5 mL). The two solutions were combined and left overnight on a rocking station to yield a homogeneous solution at room temperature. The next day, this solution was electrospun using a 4×5 mL syringe. It was placed horizontally on a programmable syringe pump (Kent Scientific, USA) and discharged at a rate of 40 μL / min. A high voltage power supply (Genvolt, UK) was used to supply 17 kV to the syringe. The fibrous scaffold was collected on a rotating drum with a diameter of 16 cm × 6 cm at a distance of 17 cm from the needle tip. The drum was rotated at 300 rpm to yield an 18×16 cm mat. The scaffold was fabricated at room temperature of approximately 20 °C.

[0114] Preparation of covalently crosslinked hydrogels: Chitosan / PVA hydrogels using variable amounts of crosslinker for D-deoxy sugar encapsulation CS (2.5% w / v) was dissolved in an acetic acid (1%) solution and magnetically stirred for 12 h. In a separate flask, PVA (10% w / v) was dissolved in distilled water at 80 °C under magnetic stirring. Next, the two solutions were mixed at a ratio of 80:20 of CS and PVA, respectively, and stirred for an additional 24 h. Next, the solution was poured into separate Petri dishes and the dishes were frozen at -80 °C for 24 h. The samples were lyophilized at -40 °C for 24 h in a lyophilizer. Next, the lyophilized hydrogels were rehydrated and immersed in various concentrations of TEOF (i.e., 0, 4, 8, and 16% w / v) with sulfuric acid (17% w / v) for 24 h. Next, the hydrogels were removed from the Petri dishes, treated with NaOH (12% w / v) for 1 h, washed three times with distilled water, and lyophilized for 24 h.

[0115] Preparation of crosslinked CS / collagen scaffolds by freeze gelation CS and collagen (0.5 g each) were dissolved in acetic acid (0.5 M, 20 mL) and stirred at room temperature until completely dissolved to obtain a clear solution. Triethyl orthoformate (4% v / v, 0.8 mL) was added as a crosslinking agent to this solution, and the solution was stirred further overnight to improve the homogeneity and crosslinkability of the material. Next, the mixture was poured into a Petri dish and frozen at -20°C overnight. Next, the frozen membrane was immersed in a pre-cooled ethanol solution of sodium hydroxide (3 M) and left at -20°C for another 24 hours. Thereafter, the frozen membrane was washed in a 50% (v / v) ethanol solution and then washed with distilled water to remove sodium hydroxide. The membrane was finally washed twice in absolute ethanol for 15 minutes each and dried at room temperature to obtain a porous scaffold.

[0116] FTIR analysis Infrared (FT-IR) spectra were recorded at a resolution of 8 cm -1 by 256 consecutive scans in the photoacoustic mode in the frequency range of 4000 - 400 cm -1 using a Thermo-Nicolet 6700 P FTIR Spectrometer (USA).

[0117] Scanning electron microscope (SEM) The surface morphology of the composite matrix was investigated under a SEM, model JEOL JSM 6480. The samples were sputter-coated with gold and then tested at a wide range of magnifications.

[0118] Deoxy sugar loading on the scaffold For sugar loading, the hydrogel was immersed in an aqueous solution of 2-deoxy-D-ribose (1 mg / ml) at 37°C until all the liquid was absorbed by the hydrogel. After storing at -20°C overnight, the hydrogel was dried at -40°C in a freeze dryer (Christ, Alpha 1-2 LD plus, Germany) and then further characterized.

[0119] Evaluation of the angiogenesis properties of biomaterials using the CAM assay Fertilized eggs of Gallus domesticus were purchased from Big Bird (Raiwind road, Lahore, Pakistan) and incubated at 37 °C in a humidified incubator (HHD, 435, China) from the second day to the eighth day after fertilization. On the eighth day, a square window (1 cm 2 ) was cut out and removed from the shell, and a 2 cm 2 piece of electrospun scaffold or hydrogel (16% PCL electrospun scaffold / CS / PVA hydrogel / CS / collagen hydrogel) was placed on the CAM. Only one scaffold or hydrogel was transplanted into each egg.

[0120] The shell window was closed with parafilm (Bemis Flexible Packaging, USA) and sealed with adhesive tape. After transplantation, the eggs were placed again in a 40% humidified incubator at 37 °C until the 14th day. On the 14th day, the biomaterials were harvested and the eggs were sacrificed. Angiogenesis was quantified by taking optical micrographs immediately before scaffold harvest, and then blindly scored by four evaluators using histological images of the harvested materials. The number of eggs that were transplanted and survived is described in the caption of each CAM assay figure.

[0121] Full-thickness excisional rat wound model For in vivo experiments, a circle with a diameter of 20 mm and a thickness of 1.2 mm of the hydrogel was prepared. This sample was sterilized using an ethanol (70%) solution, air-dried, equilibrated in PBS for a short time, and then placed on the wound. For animal tests, male Sprague-Dawley (SD) rats weighing 140 - 170 g were used in the study. The animals were housed in the animal house facility of the Centre of Excellence in Molecular Biology (CEMB), Lahore, where they had free access to food and water. All animals were treated according to procedures approved by the Institutional Animal Ethics Committee of CEMB, Lahore, Pakistan. The animals were anesthetized with ketamine (100 mg / kg body weight) and xylazine (10 mg / kg body weight), and then the dorsal hair was removed using a hair trimmer (Dingling professional hair clipper, RF-608, China). The back of the rat was completely shaved for adhesive tape fixation and bandaging. After shaving, the back was cleaned with ethanol and then transferred to a clean sterile table. Three circular wounds were created by removing the skin in three marked areas using surgical scissors (Noorani Surgical Medical Supplies, Pakistan). Immediately after the excision process, one of these wounds was covered with a sterilized 2-deoxy-D-ribose-loaded (using 70% ethanol) membrane circle, and the other was covered with a membrane of the same material but containing no 2-deoxy-D-ribose. Both transplanted membranes were sutured in place using sterile braided silk thread (Mersilk, diameter 220 microns). The scaffold was covered with a dressing (Mepore, Sweden), further covered with cotton gauze, and finally fixed with white surgical adhesive tape (Nitto Denko Corporation, Japan) to prevent damage by self-grooming. One of these wounds was left open and covered only with a dressing (Mepore, Sweden) to function as a negative control.

[0122] Three days after membrane transplantation, the surgical bandage was removed. At 0, 3, 9, 11, 14, and 17 days post-wounding, the wounds were measured and photographed. The wound area was quantified using Image J software. By the end of the experiment, on day 17, the rats were euthanized with an overdose of anesthesia, and tissue sections were taken from the transplantation sites of the normal, sham, control, and 2-deoxy-D-ribose scaffold-treated groups. The samples were fixed in 3.7% paraformaldehyde (Sigma-Aldrich, USA) solution at room temperature for 24 hours. Dehydration was performed using various concentrations of ethanol from 70% to 100% and water to prepare paraffin blocks.

[0123] Histology Sections with a thickness of 6 μm were cut from the paraffin-embedded samples using a microtome (Leica TP 1020 Automatic Tissue Processor) and placed on Superfrost® plus slides (Menzel-Glaser, Denmark). After performing conventional hematoxylin and eosin (H&E) staining and Gouldner's trichrome staining (Yar et al., International Journal of Polymeric Materials and Polymeric Biomaterials. 2016), they were mounted with a coverslip in DPX mounting media (Fisher Scientific).

[0124] For immunohistochemistry, 6-μm sections were processed with a mouse / rabbit-specific HRP / DAB (ABC) detection IHC kit (Abcam). After the steps of rehydration, antigen retrieval, and protein blocking, the sections were incubated for 2 hours with three different monoclonal antibodies (rabbit anti-CD34 (Abcam) 1:1000, mouse anti-CD80 (Santa Cruz) 1:100, and mouse anti-CD163 (AbD Serotec) 1:300) (diluted in 1% BSA (Sigma-Aldrich)). Subsequently, they were incubated for 10 minutes with a secondary biotinylated goat anti-polyvalent antibody (Abcam Detection IHC Kit). After incubation with avidin and biotinylated horseradish peroxidase, the target proteins were visualized by incubation in a peroxidase substrate and DAB chromogen (Abcam Detection IHC Kit). Next, the samples were counterstained with hematoxylin, dehydrated, and mounted according to the H&E protocol. Controls consisted of samples incubated without primary and secondary antibodies or samples incubated with secondary antibody only. Semi-quantitative evaluation of the degree of immunostaining was performed on a blinded observer basis using a qualitative grading score; absent = 0, lightly present = 1, largely present = 2, abundant = 3, very abundant = 4. Five representative images from each sample at each time point were evaluated by two blinded investigators (n = 10). Exemplary photographs showing 0, 1, 2, 3, and 4 were provided for reference, and the median value from these scores was used. The M1 / M2 ratio was also calculated for each using the values from the blinded scoring of immunostaining.

[0125] Fermentation of sugars by bacteria. To evaluate the ability of bacteria to metabolize the L and D deoxy sugars studied in this investigation, the ability to ferment sugars to produce acids was determined.

[0126] Bacteria Five bacterial strains were employed in this study; four strains of Staphylococcus aureus, and one strain of Pseudomonas aeruginosa. The S. aureus strains were S235 - a recent clinical isolate used in the development of a bacterial infection skin model previously reported from this lab (Shepherd J et al., Tissue Engineering Part C: Methods. 2009). NCTC 6571 (Oxford) - a reference strain commonly used as a reference for antibiotic susceptibility testing. The Newman strain - clinically derived but defective in surface fibronectin binding protein. L-9879 - another clinical isolate but hydrophilic.

[0127] Growth, and sugar fermentation Bacteria were grown at 37°C for 16 h in Brain Heart Infusion broth. An aliquot (100 ml) of tryptone soya broth (1% w / v) supplemented with phenol red (0.02% w / v; Sigma-Aldrich UK) and the appropriate sugar (1% final concentration) was added to sterile 96-well plates. Next, 5 μl of an overnight broth culture of each bacterial strain was added to the wells and incubated at 37°C for 16 h. The ability of each strain to ferment each sugar was determined by acid production and the change in the phenol red pH indicator.

[0128] Statistics Differences between groups for immunostaining were tested by non-parametric Kruskal–Wallis test, and multiple comparisons between individual groups were tested using the Dunn test.

[0129] Results Incorporation of D-deoxy sugars into electrospun PLC membranes 2-Deoxy-D-ribose was encapsulated into electrospun nanofibers using DMF in DCM as the solvent for electrospinning. By electrospinning, a sheet of soft fibrous material carrying the sugar was produced (Figure 1). The average diameter of the sugar-free PCL fibers was 224 nm ± 82 nm, while the average diameter of the fibers carrying 2-deoxy-D-ribose increased significantly to 323 nm ± 25 nm (average diameter of 40 random fibers) (p ≤ 0.05). However, both types of fibers were randomly oriented with interconnected pores, and their surfaces were smooth and free of any beads. After sugar loading, the color of the membrane became bright blue.

[0130] Incorporation of D-sugars into chitosan / PVA hydrogels The method of crosslinking CS and PVA using triethyl orthoformate (TEOF) was previously reported by our group in 2015 (Yar et al., Materials Science and Engineering: C. 2015; Shahzadi et al., Journal of biomaterials applications. 2016). In this study, we loaded 2-deoxy-D-ribose onto CS / PVA hydrogels crosslinked with 16% TEOF by physical adsorption. In this study, non-crosslinked hydrogels loaded with 2-deoxy-D-ribose were used as controls. SEM micrographs showed that both crosslinking and sugar loading seemed to affect the pore size and morphology of this hydrogel (Figure 2). Before crosslinking, the hydrogel had a layered structure rather than interconnected pores. The average pore size was 103 μm ± 52 μm. After crosslinking, the pore size decreased significantly (p ≤ 0.05). Adding sugar in the absence of crosslinking changed the layered structure to distorted pores. The introduction of both crosslinking and sugar significantly reduced the average pore size to 68 μm ± 31 μm (p ≤ 0.05). These newly interconnected pores had a more uniform morphology, and the pores were well distributed throughout the hydrogel matrix.

[0131] Incorporation of D-deoxyribose into triethyl orthoformate-crosslinked chitosan / collagen hydrogels Chitosan and collagen were cross-linked using triethyl orthoformate and then immersed in a sugar aqueous solution (1 mg / ml) to load 2-deoxy-D-ribose. The average thickness of the hydrogel before submerging in physiological saline was 1.11 mm, and it increased to 1.37 mm after 24 hours of immersion. The values are the average of three independent experiments.

[0132] Finally, since the material will ultimately be handed over to the surgeon for application to the skin, it is important to evaluate the physical stability of the membrane. The surface of the membrane was smooth and had no cracks. Figure 3 below shows all the important parameters of the physical appearance of this membrane, as well as the strength, stretchability, and foldability that would be advantageous during surgery when applied to the wound site. Furthermore, the membrane can be easily cut into the desired shape with a scalpel or scissors.

[0133] The membrane was strong and exhibited excellent flexibility, stretchability, and handling characteristics.

[0134] To investigate how various sugars affect the structure and morphology of the hydrogel, which play important roles in cell infiltration and proliferation, the microscopic structure of these sugar-loaded hydrogels was investigated by SEM (Figure 4). A sheet-like structure with an average pore size of 75.07 ± 34.48 μm was observed in the case of the control hydrogel (Figure 4a). The average pore sizes of D- and L-deoxyribose were 100.04 ± 24.01 μm and 93.33 ± 23.49 μm, respectively (Figure 4b, Figure 4c). In the case of L-deoxylamnose (Figure 4e), the pore structure was intact, uniform, and isotropic, with an average pore size of 110.89 ± 20.56 μm, and there was no significant difference from the D-isomer (average pore size 102.45 ± 23.05 μm). D- and L-fucose showed average cross-sectional pore sizes of 72.18 ± 16.92 μm and 87.43 ± 14.11 μm, respectively. Therefore, the SEM micrographs showed that all the sugar-loaded hydrogels had similarly highly porous structures.

[0135] Characteristic evaluation FTIR The interaction between sugar and PCL microfibers was investigated using FTIR spectroscopy. Figure 5A shows that the main peak of D-ribose at approximately 3400 cm -1 was detected in the PCL fibers when sugar was added to the polymer before spinning. The main absorption band was observed at 1720 cm -1 corresponding to the carbonyl ester of PCL. The peaks of alkyl groups in both D-sugar and PCL showed absorption peaks at 2700 - 2900 cm -1 . The spectrum of D-sugar-loaded PCL fibers also indicates that the sugar was uniformly distributed in the electrospinning mixture.

[0136] The CS / PVA hydrogels without (Figure 5d) and with (Figure 5e) crosslinking agents contain characteristic peaks of chitosan and PVA (Figure 5B). The peak around 2900 cm -1 was assigned to C-H stretching vibration (Islam et al., Carbohydrate Polymers. 2012). The peak at 1530 cm -1 was assigned to the O-H bending vibration of PVA (Li et al., Polymer. 2000). The band appearing around 1400 cm -1 was due to C-H bending vibration. The peak at 1099 cm -1 was due to C-O-C stretching vibration. However, in the crosslinked hydrogel spectrum, O-H and N-H stretching also appeared as a broad peak at 3400 - 3200 cm -1 (Li et al., Polymer. 2000; Teli et al., International journal of biological macromolecules. 2012), but the intensity of this hump decreased significantly. This may be because the -NH2 of CS is utilized when forming the new -C=N bond. The new peak of the imine bond was at 1630 cm -1appeared in (Yar et al., Materials Science and Engineering: C. 2015; Li et al., Polymer. 2000; Rokhade et al., Carbohydrate Polymers. 2007).

[0137] Figure 5C shows the spectra of crosslinked CS / collagen carrying various sugars. The spectra show all the characteristic peaks of CS and collagen. The broad absorption band at 3200 - 3500 cm -1 was mainly due to O-H / N-H stretching vibrations. The broadness of the band was due to intermolecular hydrogen bonding. Peaks at 2600 - 2800 cm -1 appeared due to C-H stretching vibrations. The peak of amide I appeared at 1650 cm -1 The stretching vibrations of C-O-C and C-O of the ether bond also shifted to 1147 cm -1 and 1053 cm -1 Sugar loading probably did not affect the FTIR peaks of the hydrogel because the concentration of sugar in the polymer matrix was very low.

[0138] Evaluation of the angiogenic promoting response of deoxysugar-carrying biomaterials using the chorioallantoic membrane (CAM) assay Electrospun fibers and hydrogels carrying D-deoxyribose The CAM assay was used to investigate the angiogenic potential of the synthesized materials. In the case of 2-deoxy-D-ribose carrying fibers, extensive new blood vessel growth was observed under the material (see Figure 6A(b)). This confirmed the chemotactic and angiogenic potential of the 2-deoxy-D-ribose carrying materials.

[0139] 2-Deoxy-D-ribose was loaded into a chitosan / PVA triethyl orthoformate crosslinked hydrogel by immersing it in an aqueous solution of D-deoxy sugar. After drying in a freeze dryer, the hydrogel was transplanted into fertilized eggs on the 8th day, and the eggs were sacrificed on the 14th day. The 2-deoxy-D-ribose support was observed to promote angiogenesis by judging from observing more new blood vessels compared to the control material (Figure 6A).

[0140] Chitosan / collagen crosslinked hydrogels loaded with D and L sugars (3 sugars, 6 isomers) Generally, biomaterials that aid in the rapid growth and invasion of blood vessels are considered to exhibit better biocompatibility than biomaterials that show delayed angiogenesis and persistent inflammation (Wolf et al., Biomaterials. 2014). As a result, this is an absolutely essential property for tissue engineering materials to ensure tissue survival.

[0141] The D and L isomers of three deoxy sugars (ribose, fucose, and rhamnose) were loaded onto a porous freeze-gelled triethyl orthoformate (4%) covalently crosslinked chitosan / collagen (1:1) hydrogel. The hydrogel was immersed in an aqueous solution of the sugar (1 mg / ml) and further freeze-dried to produce a porous membrane. Regarding the electrospun scaffold, the hydrogel was transplanted into fertilized eggs on the 8th day, and the eggs were sacrificed after the 14th day to investigate the effect of the sugar on angiogenesis.

[0142] As can be seen from Figure 7, all three of the L isomers stimulated angiogenesis, but for the D isomers, only the D isomer of deoxyribose had strong angiogenesis, the response to the D isomer of deoxyfucose was not significant, and the response to the D isomer of deoxyrhamnose was only slight.

[0143] Evaluation of the wound healing response to D-deoxyribose-loaded hydrogels using a rat skin wound model Wound healing in rats was promoted by applying 2-deoxy-D-ribose-loaded hydrogels. After marking a 20-mm area, full-thickness wounds were created by performing an excision under anesthesia using surgical scissors. Each material was applied to the wound and sutured in place, first covered with Mepore (Sweden) surgical tape and finally with a cotton bandage. All rats were dressed with the same bandage that was removed 3 days after the wound. Digital photographs of each wound were taken at specific time points and further quantified using Image J software. Representative macroscopic photographs of the wounds are shown in Figure 8. Shown are control hydrogels (crosslinked CS / collagen scaffolds) and 2-deoxy-D-ribose-loaded hydrogels at 3, 9, 11, 14, and 17 days post-wounding.

[0144] On day 3, the 2-deoxy-D-ribose-loaded scaffolds were very firmly attached to the surrounding tissue compared to the other test materials (Figure 8), and when stretched by hand, good mechanical strength of the wound was shown. By day 9, the wounds treated with D-deoxyribose were approximately 50% of the wound area (Figure 9). On days 11 and 14, the wound area further decreased, and by day 17, for the wounds treated with D-deoxyribose, the wounds were completely closed in contrast to the control wounds and wounds treated with hydrogel only.

[0145] Histological and immunohistochemical analysis of excised wound tissue Samples from all groups were harvested on day 17 and fixed overnight in paraformaldehyde. After dehydration, the samples were embedded in paraffin to make blocks, which were sectioned at 6 μm. Next, the slides were stained with H&E, Gouldner's trichrome, and DAB peroxidase kit (to examine the detection of three antigens, CD34 for progenitor endothelial cells, CD80 for M1 macrophages, and CD163 for M2 macrophages). Finally, the slides were covered with DPX mounting medium and glass coverslips and then imaged with an optical microscope. The sham (wound without any treatment) group, control (wound treated with chitosan and collagen scaffolds) group, and D - deoxyribose (wound treated with scaffolds loaded with 2 - deoxy - D - ribose) group were compared with normal skin from untreated rats.

[0146] On day 17, wound healing in the group treated with 2 - deoxy - D - ribose was essentially complete, and the structure of the explant tissue was similar to normal skin as shown by hematoxylin and eosin (H&E) staining (Figure 10). Goldren's trichrome stains collagen blue / green, while muscle, epithelium, hair follicles, and blood vessels are stained red. Except for the stained muscle layer under the skin as shown for the normal group, the rest appears very similar to the D - deoxyribose - treated group where epithelium and hair follicles were already formed. In contrast, the sham group and the control group still showed unorganized granulation wound tissue by day 17 (Figure 10).

[0147] Macrophages expressing M1 response markers are associated with graft rejection and activation of the chronic inflammatory response, while M2 response markers are expressed by macrophages with an inflammatory response associated with constructive remodeling (Badylak et al., Tissue Engineering Part A. 2008).

[0148] As expected, for the normal non-wounded group, neither M1 macrophages nor M2 macrophages were stained (Figure 11). As expected after wound or transplantation of materials into animals, some M1 macrophages and M2 macrophages were seen in the other groups. The table shown in Figure 12 shows the evaluation of the degree of macrophage presence obtained by the blinded laboratory staff. The M2 to M1 ratio of presence was approximately 1 for wounded animals and animals treated with non-supported chitosan. For the group treated with chitosan carrying 2-deoxy-D-ribose, the ratio of presence was slightly higher (not statistically significant) (Figure 11).

[0149] The ability of bacteria to metabolize L- and D-deoxy sugars Six sugars under investigation were compared to glucose with respect to their action as substrates for bacterial metabolism. Four strains of Staphylococcus aureus (S. aureus) and one strain of Pseudomonas aeruginosa were investigated as representatives of common pathogens in wound infections. The results for all four strains of S. aureus were the same in that all could use glucose as a substrate for fermentation, but none of these strains could use the L or D isomers of the three deoxy sugars under investigation. However, P. aeruginosa was able to ferment all sugars except the L isomers of deoxyfucose and deoxylamnose.

[0150] The reason for investigating the ability of these sugars to act as substrates for bacteria is that if preparing biomaterials to be used for burns or chronic wounds, it is preferable to use materials that cannot be easily metabolized by bacteria.

[0151]

Table 1

[0152] Conclusion This study demonstrated the angiogenic ability of 2-deoxy-D-ribose when delivered in either electrospun PCL fibers or chitosan-based hydrogels. This was in contrast to the D-isomers of deoxyfucose or deoxylaminose, although all three L-isomers of the 2-deoxy sugars tested were angiogenic. 2-Deoxy-D-ribose could be metabolized by Pseudomonas aeruginosa, but not by four strains of Staphylococcus aureus. The addition of D-deoxy-ribose to chitosan / collagen hydrogels promoted wound healing and follicle recovery in full-thickness wounds in rats.

[0153] [Experimental Section 2] Ex-ovo CAM assay Incubation of eggs All CAM experiments were performed in accordance with the guidelines of the UK Home Office. A schematic of the ex-ovo CAM assay is shown in Figure 13. Fertilized eggs of Gallus domesticus were purchased from Henry Stewart & Co. (MedEggs, Norwich, UK). The eggs were carefully wiped with a hand paper towel and 20% industrial methylated spirit (IMS) solution to remove dirt and feathers from the shell. The eggs were then placed horizontally in a humidified incubator (RCOM King SURO, P&T Poultry, Powys, Wales) and incubated at 37.5 °C until embryonic development day (EDD) 3.

[0154] Transfer of embryo into Petri dish On EDD3, the upper surface of the egg was marked with a felt pen. The egg was held horizontally (with the marked surface up) and cracked at the edge of a 1000 ml glass beaker and held near the bottom of a Petri dish. The embryo was then gently transferred into a sterile Petri dish and stored in a humidified incubator (Binder, Tuttlingen, Germany) at 38 °C.

[0155] Application of substances onto CAM A plastic ring (diameter approximately 6.5 mm) was used as a reservoir for the substance and a marker for the implantation area, placed on the CAM, and a 20 μl volume of the substance was applied to the CAM twice a day, giving a total volume of 40 μl between EDD7 and EDD11. An image of the ring was obtained using a digital microscope at EDD11, and then 20% lens culinaris agglutinin (LCA) (Vector Laboratories, Peterborough, UK) was injected into the circulatory system using a 30G needle under a dissecting microscope (Wild Heerbrugg, Heerbrugg, Switzerland) to label the intravascular endothelial cells. The CAM was then removed and fixed in a 3.7% formaldehyde solution. The embryos were sacrificed at the end of EDD11. Next, the fixed CAM samples were imaged under a confocal microscope (Zeiss LSM 510 Meta, Jena, Germany) to investigate the effect of the substance on the microvascular structure of the CAM.

[0156] Quantification of angiogenesis After the image was segmented to include all identifiable blood vessels [C. Roma-Rodrigues, A. Heuer-Jungemann, A.R. Fernandes, A.G. Kanaras, P. V. Baptista, Peptide-coated gold nanoparticles for modulation of angiogenesis in vivo, IntJ Nanomedicine. 11 (2016) 2633-2639. doi:10.2147 / IJN.S108661], the blood vessels were quantified by counting the bifurcation points and calculating the average vessel length through multiple image processing steps [D. Ribatti, B. Nico, A. Vacca, M. Presta, The gelatin sponge-chorioallantoic membrane assay, Nat Protoc. 1 (2006) 85-91. doi:10.1038 / nprot.2006.13, P. Brooks, A.P. Montgomery, D. Cheresh, Use of the 10-Day-Old Chick Embryo Model for Studying Angiogenesis, Integrin Protoc. 129 (1999) 257-269. doi:10.1385 / 1-59259-249-X:257]. First, the inner region of the ring was cut out, and the red, green, and blue channels were segmented using Adobe Photoshop CS6 (ADOBE Systems Inc., San Jose, California, USA). Next, the green channel was imported into ImageJ (Wayne Rasband, National Institutes of Health, USA) for further analysis including unsharp mask filtering, local contrast enhancement, noise removal, and segmentation. Finally, the quantification software (AngioTool, National Cancer Institute) was used to quantify the number of bifurcation points (Figure 14A), and the average vessel length was calculated using the known pixel / mm ratio within ImageJ (Wayne Rasband, National Institutes of Health, USA) with a binary image histogram.

[0157] As shown in Fig. 14B, the percentage of vascular area (VA%) of the microvascular system of the CAM was quantified using confocal images of rhodamine-labeled lectin-injected CAM samples. To do this, the images were imported into ImageJ (Wayne Rasband, National Institutes of Health, USA), converted to binary images after filtering and smoothing processes, and then quantified. The VA% was calculated using the histogram list of the black and white areas within the images.

[0158] Determination of the optimal concentrations of E2 and 2dDR on the CAM before loading onto the scaffolds Unless otherwise specified, all chemicals were purchased from Sigma Aldrich. The recombinant VEGF165 stock solution was diluted to a concentration of 2 ng / μl (VEGF - 80 ng). E2 was dissolved in methanol and then the working solution was prepared with phosphate-buffered saline (PBS) at concentrations of (a) 100 ng / day (E2 - 100 ng), (b) 200 ng / day (E2 - 200 ng), and (b) 600 ng / day (E2 - 600 ng). The 2dDR solution was prepared by dissolving it in PBS to final concentrations of (a) 20 μg / day (2dDR - 20), (b) 200 μg / day (2dDR - 200), and (c) 1000 μg / day (2dDR - 1000). Sorafenib malate (sorafenib) was dissolved in DMSO and diluted with PBS to a final concentration of 50 ng / μl. The working solutions of all substances were prepared immediately before EDD7. The angiogenesis efficiency of various substance concentrations was evaluated by directly applying the substances to the CAM (2 doses / day / embryo). The angiogenesis activity was determined by quantifying the number of branch points and calculating the average vessel length.

[0159] Comparison of the angiogenesis potential of the E2-releasing scaffolds and 2dDR-releasing scaffolds on the CAM Electrospinning of PHBV scaffolds loaded with E2 and 2dDR Preparation of solutions

[0160] After preparing a 10% (w / w) PHBV loading solution, electrospinning was carried out. 1 g of PHBV granules (Goodfellow, London, UK) was dissolved in 1 g of methanol (Fisher Scientific, Massachusetts, USA) and 8 g of DCM (Fisher Scientific, Massachusetts, USA) in a fume hood. After preparing four 10% PHBV solutions, substances were added. Finally, 25 mg of E2, 50 mg of E2, 250 mg of 2dDR, and 500 mg of 2dDR per 1 g of PHBV were added to each solution. The mixture was stirred magnetically overnight.

[0161] Electrospinning The solution (about 10 ml) was loaded into a 10 ml syringe fitted with a syringe tip of 0.6 mm inner diameter. Next, the syringe was placed inside a syringe pump (GenieTM Plus, Kent Scientific, Connecticut, USA). An aluminum foil was used as a collector and placed 17 cm away from the needle tip. The pump was set at 40 μl / min and a voltage of 17 kV was applied to both the collector and the tip. Electrospinning was carried out at room temperature until all the polymer solutions were used up.

[0162] Scanning electron microscope (SEM) The surface morphology of the E2 release scaffold and the 2dDR release scaffold was observed under SEM (Philips / FEI XL-20 SEM; Cambridge, UK). The samples were imaged after coating with gold using a gold sputter (Edwards sputter coater S150B, Crawley, England). The fiber diameter and pore size were measured using ImageJ.

[0163] Release of E2 and 2dDR from the scaffold The scaffolds were cut into pieces to fit into a 6-well plate, weighed, and submerged in 4 ml of PBS. The concentrations of E2 and 2dDR released from each group (25 mg E2, 50 mg E2, 250 mg 2dDR, 500 mg 2dDR) were measured fluorimetrically using a UV-VIS spectrophotometer (Thermo Fischer Evolution 220, Massachusetts, USA) at 238 nm for 2dDR and at 220 nm for E2. The absorbance values were converted to concentrations using standard curves of known concentrations of E2 and 2dDR.

[0164] Implantation of E2-releasing scaffolds and 2dDR-releasing scaffolds onto the CAM The scaffolds were cut into circles with a diameter of 5.5 mm using a laser cutter (Epilog Laser Cutter, Clevedon, UK), sterilized under UV light for 1 hour, and then implanted. Two circular scaffolds were placed onto the CAM at EDD8. Images of the scaffolds were obtained at EDD12 and EDD14 using a digital microscope. Microinjection of rhodamine-labeled lectin into the circulatory system was performed, the CAM was removed, and fixed in a 3.7% formaldehyde solution. Next, the embryos were sacrificed at the end of EDD14. Angiogenesis was quantified by counting all the blood vessels converging towards the implants.

[0165] Histological evaluation of E2-releasing scaffolds and 2dDR-releasing scaffolds on the CAM Hematoxylin and eosin (H&E) staining was performed on the cell-impregnated scaffolds by modifying a standard protocol

[47] . Briefly, the fixed samples were embedded in optimal cutting temperature (OCT) and frozen in liquid nitrogen for 3 minutes. Sections were cut to a thickness of 8 - 10 μm using a cryostat (Leica Biosystems Nussloch, Germany) at -20°C. Next, the sections were stained with hematoxylin for 90 seconds and eosin for 5 minutes. Finally, H&E images were obtained under an optical microscope (Motic DM-B1, Xiamen, China). The total number of blood vessels adjacent to the scaffolds was quantified by counting the blood vessels in the H&E sections [A. Minajeva, M. Kase, M. Saretok, A. Adamson-Raieste, S. Kase, K. Niinepuu, M Vardja, T. Asser, J. Jaal, Impact of Blood Vessel Quantity and Vascular Expression of CD133 and ICAM-1 on Survival of Glioblastoma Patients, Neurosci J. 2017 (2017) 8 pages]. Briefly, all distinguishable blood vessels adjacent to the scaffolds were counted by two independent investigators using two independent microscopes at 10x magnification from a total of six different slides for each group and six different regions of interest from each slide.

[0166] Comparison of the mechanical properties of the E2 release scaffolds and the 2dDR release scaffolds Biomechanical test samples were prepared by cutting 20 mm × 10 mm fragments from the scaffolds. The clamps of the device were placed 10 mm apart from each other, and the width and thickness of each scaffold were measured. The test samples were clamped with two grips inside a tensile meter (BOSE Electroforce Test Instruments, Minnesota, USA). A tensile test was performed on each sample at a speed of 0.1 mm / second until the sample broke (n = 4). Stress-strain graphs and load-displacement graphs were drawn using the raw data of these tests. The ultimate tensile strength (UTS) was calculated from the stress (σ) and strain (ε) curves of each sample, while the stiffness was calculated from the load (F) and displacement (ΔL) curves.

[0167] Also, to confirm the effects of E2 and 2dDR on the wettability of the scaffolds, a wettability test of the drug-releasing electrospun scaffolds was carried out using a droplet shape analyzer (Kruss DSA100, Germany) under ambient laboratory conditions. Briefly, 5 μl droplets were dropped onto the scaffold surface, and the retention time of the droplets on the scaffold until they were completely absorbed was calculated from the recorded test movie. For each type of sample, the retention times for at least 3 droplets on 3 different substrates were measured.

[0168] Statistics Statistical analysis was performed using an unpaired Student's t-test. A P-value < 0.05 was considered statistically significant, and the degree of significance was indicated by the number of stars ( **** P ≤ 0.0001, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, ns P ≥ 0.05).

[0169] Results Evaluation of the angiogenic activity of E2 and 2dDR against CAM Quantification of the macro images of the CAM showed that for the E2-100 ng, E2-200 ng, and E2-600 ng groups, the number of branch points increased 1.3-fold, 1.5-fold, and 1.4-fold, respectively, and for all concentrations, the average vessel length increased 1.2-fold over 4 days compared to the control. Similarly, for 2dDR-20 μg and 2dDR-200 μg, the number of branch points increased 1.3-fold and 1.4-fold, respectively. For both concentrations, the average vessel length increased 1.2-fold compared to the control scaffold, while there was no significant difference for the 2dDR-1000 μg group. Quantification of the branch points and average vessel length is shown in FIGS. 15A and 15B, respectively. Macro images of the CAM at the most effective concentrations of E2 and 2dDR are shown in FIG. 16.

[0170] In the microvascular evaluation of the CAM samples, it was shown that over 4 days compared to the control, for the groups treated with E2 and 2dDR, the VA% increased from 55.3% ± 3% to 79.5% ± 5% and 71.7% ± 3%, respectively (FIG. 16). VEGF and sunitinib were used as positive and negative controls, respectively (FIG. 16).

[0171] Effect of including E2 and 2dDR on the microstructure of the PHBV scaffold SEM images of the E2- and 2dDR-releasing PHBV scaffolds can be seen in FIG. 17. As shown in the graph in the lower right corner of FIG. 17, the fiber diameter increased significantly when substances were added to all groups compared to the PHBV control group (0.66 ± 0.16 μm) (25 mg E2 (0.83 ± 0.17 μm), 50 mg E2 (0.98 ± 0.35 μm), 250 mg 2dDR (0.89 ± 0.19 μm), 500 mg 2dDR (1.22 ± 0.28 μm) were added to the PHBV scaffold).

[0172] Release of E2 and 2dDR from the PHBV scaffold over 30 days As shown in Fig. 18, the release rates of E2 and 2dDR from the scaffolds were evaluated over 30 days. By the 7th day, the 2dDR release from the scaffolds was 81.3% and 86.5% of the 2dDR present in the polymer solution for the 250 mg and 500 mg 2dDR scaffolds, respectively (Fig. 18A). In contrast, the total E2 release from the scaffolds within 7 days represented 1.3% and 1.6% of the initial E2 present in the polymer solutions for the 25 mg and 50 mg E2 scaffolds, respectively (Fig. 18B).

[0173] Comparison of the effects of E2 and 2dDR on the mechanical properties of the scaffolds As can be seen from Fig. 19A, when all substances were added, the UTS of the PHBV scaffolds increased significantly compared to the plain PHBV scaffolds. The most significant increase in UTS was observed for the 2dDR 250 mg group. Similarly, the stiffness of the scaffolds carrying 2dDR and E2 was significantly higher compared to the non - carrying PHBV scaffolds.

[0174] As shown in Fig. 19B, the loading of 250 mg 2dDR maximally increased the stiffness of the PHBV scaffolds. The wettability of the drug - releasing scaffolds was investigated by the droplet retention time calculated using a droplet shape analyzer. The retention time of water droplets on the drug - releasing scaffolds is shown in Fig. 19C. This indicated that adding 2dDR increased the wettability of the scaffolds, while adding E2 decreased the wettability of the scaffolds.

[0175] Evaluation of the angiogenic activity of the E2 - releasing scaffolds and 2dDR - releasing scaffolds on the CAM The evaluation of the E2 - releasing scaffolds and 2dDR - releasing scaffolds on the CAM showed that, as can be seen in Fig. 20, for all groups, the number of distinguishable blood vessels growing towards the scaffolds was at least doubled compared to the plain PHBV scaffolds. The average number of blood vessels for the 25 mg E2 - carrying scaffolds and 50 mg E2 - carrying scaffolds, when compared to the control group (average number of blood vessels: 23.1 (±1.24)), was 49.5 (±0.92)( **** P≦0.0001) and 37.9 (±1.05)( ****On the other hand, for the 2dDR-supported scaffolds of 250 mg and 500 mg, they were 48.6 (±1.02) ( **** P ≤ 0.0001) and 37.1 (±1.37) ( **** P ≤ 0.0001), respectively. None of the supported substances affected the embryo survival rate, and it exceeded 75% for each group.

[0176] Histological analysis of E2-releasing scaffolds and 2dDR-releasing scaffolds on the CAM The average number of blood vessels adjacent to the scaffolds increased significantly according to all concentrations of both the E2-releasing scaffolds and the 2dDR-releasing scaffolds when compared with the control group and the group with only the CAM (see Figures 21 and 22).

[0177] Regardless of whether they carried an angiogenesis promoter or not, all scaffolds showed good adhesion to the CAM, and all membranes showed similar cell infiltration. Figure 21 shows an image of a representative tissue structure.

[0178] Conclusion From the data, it can be concluded that both the direct administration of 2dDR and E2 and the gradual release of these factors from PHBV fibers stimulated angiogenesis in the ex-ovo CAM assay. These two small stabilizing factors were easily incorporated into electrospun fibers, such as those with high potential for use in tissue engineering constructs and for the functionalization of TE scaffolds that promote angiogenesis in vivo.

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[0180] Certain aspects and embodiments of the present invention are described in the following paragraphs.

[0181] Paragraph 1. D-Deoxyribose sugar and / or estradiol used for promoting wound healing.

[0182] Paragraph 2. D-Deoxyribose sugar and / or estradiol for use according to Paragraph 1, wherein the D-deoxyribose is 2-deoxyribose.

[0183] Paragraph 3. L-Deoxysugar used for promoting wound healing. Paragraph 4. L-Deoxysugar for use according to Paragraph 3, wherein the L-deoxysugar is selected from L-deoxyribose, L-deoxyfucose, and L-deoxylamnose. Paragraph 5. The sugar is for topical administration, the D-deoxyribose sugar and / or estradiol for use according to any one of Paragraphs 1 to 2, or the L-deoxysugar for use according to Paragraph 3 or 4.

[0184] Paragraph 6. The sugar is prepared in a carrier, the D-deoxyribose sugar, estradiol, or L-deoxysugar for use according to any of the above paragraphs.

[0185] Paragraph 7. The carrier is a biocompatible matrix material, the D-deoxyribose sugar, estradiol, or L-deoxysugar for use according to Paragraph 6.

[0186] Paragraph 8. The matrix material is an electrospun scaffold, the D-deoxyribose sugar, estradiol, or L-deoxysugar for use according to Paragraph 7.

[0187] Paragraph 9. The electrospun scaffold is a polycaprolactone scaffold, the D-deoxyribose sugar, estradiol, or L-deoxysugar for use according to Paragraph 8.

[0188] Paragraph 10. The carrier is a hydrogel, for use according to claim 6, D-deoxyribose sugar, estradiol, or L-deoxy sugar.

[0189] Paragraph 11. The hydrogel is a crosslinked hydrogel, for use according to paragraph 10, D-deoxyribose sugar, estradiol, or L-deoxy sugar.

[0190] Paragraph 12. The hydrogel contains at least one of chitosan, gelatin, alginate, agarose, methylcellulose, hyaluronic acid, or any combination thereof, for use according to paragraph 10 or paragraph 11, D-deoxyribose sugar, estradiol, or L-deoxy sugar.

[0191] Paragraph 13. The hydrogel contains chitosan and collagen, for use according to any one of paragraphs 10 to 12, D-deoxyribose sugar, estradiol, or L-deoxy sugar.

[0192] Paragraph 14. The hydrogel contains polyvinyl alcohol, sodium polyacrylate, acrylate polymer, or any combination thereof, for use according to any one of paragraphs 10 to 13, D-deoxyribose sugar, estradiol, or L-deoxy sugar.

[0193] Paragraph 15. The hydrogel contains chitosan and polyvinyl alcohol, for use according to any one of paragraphs 10 to 14, D-deoxyribose sugar, estradiol, or L-deoxy sugar.

[0194] Paragraph 16. The carrier further contains an antibacterial agent, for use according to any one of paragraphs 6 to 15, D-deoxyribose sugar, estradiol, or L-deoxy sugar.

[0195] Paragraph 17. The wound is a chronic wound, for use according to any of the preceding paragraphs, D-deoxyribose sugar, estradiol, or L-deoxy sugar.

[0196] Paragraph 18. The wound is a full-thickness wound, and the D-deoxyribose sugar, estradiol, or L-deoxy sugar for use according to any of the preceding paragraphs.

[0197] Paragraph 19. The wound is a burn, and the D-deoxyribose sugar, estradiol, or L-deoxy sugar for use according to any of the preceding paragraphs.

[0198] Paragraph 20. A biocompatible matrix material containing D-deoxyribose sugar and / or estradiol.

[0199] Paragraph 21. A biocompatible matrix material containing L-deoxy sugar.

[0200] Paragraph 22. The matrix material is an electrospun scaffold, and the biocompatible matrix material according to Paragraph 20 or Paragraph 21.

[0201] Paragraph 23. The electrospun scaffold is a polycaprolactone scaffold, and the biocompatible matrix material according to Paragraph 22.

[0202] Paragraph 24. A hydrogel containing D-deoxyribose sugar and / or estradiol.

[0203] Paragraph 25. A hydrogel containing L-deoxy sugar.

[0204] Paragraph 26. The hydrogel contains at least one of chitosan, gelatin, alginate, agarose, methylcellulose, hyaluronan, or any combination thereof, and the hydrogel according to Paragraph 24 or Paragraph 25.

[0205] Paragraph 27. The hydrogel contains chitosan and collagen, and the hydrogel according to any one of Paragraphs 24 to 26.

[0206] Paragraph 28. The hydrogel is a hydrogel according to any one of paragraphs 24 to 27, including polyvinyl alcohol, sodium polyacrylate, an acrylate polymer, or any combination thereof.

[0207] Paragraph 29. The hydrogel is a hydrogel according to any one of paragraphs 24 to 28, including chitosan and polyvinyl alcohol.

[0208] Paragraph 30. A biocompatible material according to paragraphs 20 to 23, which is used for promoting wound healing or treating alopecia.

[0209] Paragraph 31. A hydrogel according to paragraphs 24 to 29, which is used for promoting wound healing or treating alopecia.

[0210] Paragraph 32. A biocompatible material according to paragraphs 20 to 23, which is used in a method for increasing or inducing vascularization in a wound bed.

[0211] Paragraph 33. A hydrogel according to paragraphs 24 to 29, which is used in a method for increasing or inducing angiogenesis in a wound bed.

[0212] Paragraph 34. A method for increasing or inducing angiogenesis in a wound bed, including administering D-deoxyribose sugar and / or estradiol to the wound.

[0213] Paragraph 35. A method for increasing or inducing angiogenesis in a wound bed, including administering L-deoxy sugar to the wound.

[0214] Paragraph 36. The method according to paragraph 34 or paragraph 35, wherein the sugar and / or estradiol is locally administered to the wound bed.

[0215] Paragraph 37. D-deoxyribose sugar and / or estradiol used for treating alopecia.

[0216] Paragraph 38. The D-deoxyribose sugar for use according to Paragraph 37 is 2-deoxyribose.

[0217] Paragraph 39. The L-deoxy sugar used for the treatment of alopecia.

[0218] Paragraph 40. The L-deoxy sugar for use according to Paragraph 39 is selected from L-deoxyribose, L-deoxyfucose, and L-deoxylamnose.

[0219] Paragraph 41. The sugar is for topical administration, the D-deoxyribose sugar and / or estradiol for use according to any one of Paragraphs 37 to 38, or the L-deoxy sugar for use according to Paragraphs 39 to 40.

[0220] Paragraph 42. The sugar is prepared in a carrier, the D-deoxyribose sugar and / or estradiol for use according to any one of Paragraphs 37 to 38 or 41, or the L-deoxy sugar for use according to Paragraphs 39 to 41.

[0221] Paragraph 43. The carrier is a biocompatible matrix material, the D-deoxyribose sugar, and estradiol or L-deoxy sugar for use according to Paragraph 42.

[0222] Paragraph 44. The matrix material is an electrospun scaffold, the D-deoxyribose sugar, and estradiol or L-deoxy sugar for use according to Paragraph 43.

[0223] Paragraph 45. The electrospun scaffold is a polycaprolactone scaffold, the D-deoxyribose sugar, and estradiol or L-deoxy sugar for use according to Paragraph 44.

[0224] Paragraph 46. The carrier is a hydrogel, D-deoxyribose sugar for use according to Paragraph 43, and estradiol or L-deoxy sugar.

[0225] Paragraph 47. The hydrogel is a crosslinked hydrogel, D-deoxyribose sugar for use according to Paragraph 46, and estradiol or L-deoxy sugar.

[0226] Paragraph 48. The hydrogel contains at least one of chitosan, gelatin, alginate, agarose, methylcellulose, hyaluronic acid, or any combination thereof, D-deoxyribose sugar for use according to Paragraph 46 or Paragraph 47, and estradiol or L-deoxy sugar.

[0227] Paragraph 49. The hydrogel contains chitosan and collagen, D-deoxyribose sugar for use according to any one of Paragraphs 46 to 48, and estradiol or L-deoxy sugar.

[0228] Paragraph 50. The hydrogel contains polyvinyl alcohol, sodium polyacrylate, acrylate polymer, or any combination thereof, D-deoxyribose sugar for use according to any one of Paragraphs 46 to 49, and estradiol or L-deoxy sugar.

[0229] Paragraph 51. The hydrogel contains chitosan and polyvinyl alcohol, D-deoxyribose sugar for use according to any one of Paragraphs 42 to 36, and estradiol or L-deoxy sugar.

[0230] Paragraph 52. The carrier further contains an antibacterial agent, D-deoxyribose sugar for use according to any one of Paragraphs 42 to 51, and estradiol or L-deoxy sugar.

[0231] Paragraph 53. A non-therapeutic method for promoting hair regeneration, the method comprising administering a composition comprising D-deoxyribose sugar and / or estradiol.

[0232] Paragraph 54. The method according to paragraph 53 for promoting hair regeneration, the method comprising administering a composition comprising D-deoxyribose sugar and / or estradiol to an isolated hair follicle.

[0233] Paragraph 55. The non-therapeutic method for promoting hair regeneration according to paragraph 53 or paragraph 54, wherein D-deoxyribose is 2-deoxyribose.

[0234] Paragraph 56. A non-therapeutic method for promoting hair regeneration, the method comprising administering a composition comprising an L-deoxysugar.

[0235] Paragraph 57. The method according to paragraph 56 for promoting hair regeneration, the method comprising administering a composition comprising an L-deoxysugar to an isolated hair follicle.

[0236] Paragraph 58. The non-therapeutic method for promoting hair regeneration according to paragraph 46 or paragraph 57, wherein the L-deoxysugar is selected from L-deoxyribose, L-deoxyfucose, and L-deoxylaminose.

[0237] Paragraph 59. The non-therapeutic method for promoting hair regeneration according to any one of paragraphs 53 to 58, wherein the sugar and / or estradiol is for topical administration.

[0238] Paragraph 60. The non-therapeutic method for promoting hair regeneration according to any one of paragraphs 53 to 59, wherein the sugar or estradiol is prepared in a carrier.

[0239] Paragraph 61. The non-therapeutic method for promoting hair regeneration according to paragraph 60, wherein the carrier is a biocompatible matrix material.

[0240] Paragraph 62. A non-therapeutic method for promoting hair regeneration according to Paragraph 61, wherein the matrix material is an electrospun scaffold.

[0241] Paragraph 63. A non-therapeutic method for promoting hair regeneration according to Paragraph 62, wherein the electrospun scaffold is a polycaprolactone scaffold.

[0242] Paragraph 64. A non-therapeutic method for promoting hair regeneration according to Paragraph 63, wherein the carrier is a hydrogel.

[0243] Paragraph 65. A non-therapeutic method for promoting hair regeneration according to Paragraph 64, wherein the hydrogel is a crosslinked hydrogel.

[0244] Paragraph 66. A non-therapeutic method for promoting hair regeneration according to Paragraph 64 or Paragraph 65, wherein the hydrogel contains at least one of chitosan, gelatin, alginate, agarose, methylcellulose, hyaluronic acid, or any combination thereof.

[0245] Paragraph 67. A non-therapeutic method for promoting hair regeneration according to any one of Paragraphs 64 to 65, wherein the hydrogel contains chitosan and collagen.

[0246] Paragraph 68. A non-therapeutic method for promoting hair regeneration according to any one of Paragraphs 64 to 67, wherein the hydrogel contains polyvinyl alcohol, sodium polyacrylate, acrylate polymer, or any combination thereof.

[0247] Paragraph 69. A non-therapeutic method for promoting hair regeneration according to any one of Paragraphs 64 to 68, wherein the hydrogel contains chitosan and polyvinyl alcohol.

[0248] Paragraph 70. A non-therapeutic method for promoting hair regeneration according to any one of Paragraphs 60 to 69, wherein the carrier further contains an antibacterial agent.

[0249] A pharmaceutical composition containing D - deoxyribose sugar or estradiol, or a pharmaceutical salt or derivative thereof, which is used for promoting wound healing or treating alopecia.

[0250] Paragraph 72. A pharmaceutical composition containing L - deoxy sugar, or a pharmaceutical salt or derivative thereof, which is used for promoting wound healing or treating alopecia.

[0251] Paragraph 73. A pharmaceutical composition according to Paragraph 71 or Paragraph 72, wherein the composition further contains an antibacterial agent.

[0252] Paragraph 74. A pharmaceutical composition according to any one of Paragraphs 71 to 73, wherein the composition contains isolated hair follicles.

[0253] Paragraph 75. A wound dressing containing a biocompatible matrix material according to any one of Paragraphs 20 to 23.

[0254] Paragraph 76. A wound dressing containing a hydrogel according to any one of Paragraphs 24 to 29.

[0255] Paragraph 77. A wound dressing containing a pharmaceutical composition according to any one of Paragraphs 71 to 74.

[0256] Paragraph 78. D - deoxyribose sugar for use substantially as described herein with reference to the accompanying drawings.

[0257] Paragraph 79. L - deoxy sugar for use substantially as described herein with reference to the accompanying drawings.

[0258] Paragraph 80. A hydrogel substantially as described herein with reference to the accompanying drawings.

[0259] Paragraph 81. A hydrogel substantially as described herein with reference to the accompanying drawings.

[0260] The pharmaceutical composition substantially described in this specification with reference to the accompanying drawings in paragraph 82.

Claims

1. Use of D - deoxyribose sugar for the manufacture of a medicament for promoting wound healing, wherein said sugar is provided in a carrier, and said carrier is a biocompatible matrix material or a hydrogel, The D-deoxyribose sugar is released from the biocompatible matrix material or hydrogel at a rate of about 20 to about 200 μg / 0.33 cm ２ / day, and Use of D - deoxyribose sugar, wherein said medicament is for topical administration.

2. The use according to claim 1, wherein said D - deoxyribose is 2 - deoxyribose.

3. The use according to claim 1 or 2, wherein said carrier is a biodegradable carrier.

4. The use according to any one of claims 1 to 3, wherein said matrix material is an electrospun scaffold.

5. The use according to claim 4, wherein said electrospun scaffold comprises at least one of polylactic acid (PLA), polyglycolide (PGA), poly(lactic - co - glycolic acid) (PLGA), or poly(3 - hydroxybutyrate - co - 3 - hydroxyvalerate) PHBV.

6. The use according to claim 1 or 2, wherein said hydrogel is a cross - linked hydrogel.

7. The use according to claim 1 or 6, wherein said hydrogel comprises at least one of chitosan, gelatin, alginate, agarose, methylcellulose, hyaluronan, or any combination thereof.

8. The use according to claim 7, wherein said hydrogel comprises chitosan and collagen.

9. The use according to claim 7 or 8, wherein said hydrogel comprises polyvinyl alcohol, sodium polyacrylate, acrylate polymer, or any combination thereof.

10. The use according to any one of claims 6 to 9, wherein said hydrogel comprises chitosan and polyvinyl alcohol.

11. The use according to any one of claims 1 to 10, wherein said carrier further comprises an antibacterial agent.

12. The use according to any one of claims 1 to 11, wherein said wound is a chronic wound.

13. The use according to any one of claims 1 to 12, wherein said wound is a full - thickness wound.

14. The use according to any one of claims 1 to 13, wherein said wound is a burn.

15. A biocompatible matrix material for topical administration used for promoting wound healing, comprising a sugar, and said sugar consisting of D - deoxyribose sugar, A biocompatible matrix material in which the D - deoxyribose sugar is released from the biocompatible matrix material at a rate of about 20 to about 200 μg / 0.33 cm² / day.

16. The biocompatible matrix material according to claim 15, wherein the matrix material is an electrospun scaffold.

17. The biocompatible matrix material according to claim 16, wherein the electrospun scaffold comprises at least one of polylactic acid (PLA), polyglycolide (PGA), poly(lactic - co - glycolic acid) (PLGA), or poly(3 - hydroxybutyrate - co - 3 - hydroxyvalerate) PHBV.

18. A hydrogel for topical administration used for promoting wound healing, comprising a sugar, wherein the sugar consists of D - deoxyribose sugar, The hydrogel in which the D - deoxyribose sugar is released from the hydrogel at a rate of about 20 to about 200 μg / 0.33 cm² / day.

19. The hydrogel according to claim 18, wherein the hydrogel comprises at least one of chitosan, gelatin, alginate, agarose, methylcellulose, hyaluronan, or any combination thereof.

20. The hydrogel according to claim 18 or 19, wherein the hydrogel comprises chitosan and collagen.

21. The hydrogel according to any one of claims 18 - 20, wherein the hydrogel comprises polyvinyl alcohol, sodium polyacrylate, acrylate polymer, or any combination thereof.

22. The hydrogel according to any one of claims 18 - 21, wherein the hydrogel comprises chitosan and polyvinyl alcohol.

23. The biocompatible material according to any one of claims 15 - 17, used in a method for increasing or inducing vascularization in a wound bed by topical administration.

24. The hydrogel according to any one of claims 18 - 22, used in a method for increasing or inducing angiogenesis in a wound bed by topical administration.

25. A wound dressing comprising the biocompatible matrix material according to any one of claims 15 - 17.

26. A wound dressing comprising the hydrogel according to any one of claims 18 - 22.

Images