WO2015058318A1

WO2015058318A1 - Composition for accelerating or improving the healing of wounds, and method for accelerating or improving the healing of wounds, comprising the application of said composition

Info

Publication number: WO2015058318A1
Application number: PCT/CL2014/000055
Authority: WO
Inventors: Verónica Alejandra PALMA ALVARADO; Maria Rosa BONO MERINO
Original assignee: Universidad De Chile
Priority date: 2013-10-22
Filing date: 2014-10-20
Publication date: 2015-04-30
Also published as: CL2013003066A1

Abstract

The invention relates to a composition comprising a supporting matrix and mesenchymal stem cells from Wharton's jelly for treating wounds. The invention also relates to a method for treating wounds, comprising applying the composition to the wound to be treated.

Description

COMPOSITION TO ACCELERATE OR IMPROVE WOUND CICATRIZATION, AND A METHOD TO ACCELERATE OR IMPROVE WOUND CICATRIZATION THAT INCLUDES APPLYING SUCH COMPOSITION.

TECHNICAL FIELD

The present invention relates to a composition comprising a support matrix and Whaen's gelatin mesenchymal stem cells (WJ-MSC) for the treatment of wounds. And a method for the treatment of wounds that comprises applying the composition on the wound to be treated.

In a preferred embodiment, the wound to be treated is an ulcerative wound resulting from chronic ischemia, where the composition of the invention promotes neo-angiogenesis and tissue repair, so as to accelerate or improve healing, without incorporating the cells of the composition into the wound to treat.

BACKGROUND

Currently the treatment of chronic wounds is a problem that has not yet been satisfactorily resolved. Within these chronic wounds are vascular lesions, such as ulcerative wounds, and those associated with chronic ischemia, such as those that occur in diabetes, where the most important is the diabetic foot. Additionally, burns often require elements that accelerate wound healing.

Peripheral arterial disease (PAD) commonly affects the arteries that supply the legs and feet, a condition known as peripheral vascular disease in the extremities or lower limbs (Beard JD., 2000). In the EAP the decrease in blood flow, produced by arterial occlusion, can generate an imbalance between the contribution and the requirements of oxygen and nutrients at the level of the affected limb, a phenomenon known as ischemia. Ischemia of the lower limbs can manifest in acute or chronic form and its clinical consequences are varied. Acute ischemia is known as the sudden interruption of blood supply to a limb. The chronic form, on the other hand, is a consequence of the slow and progressive decrease in blood flow and is characterized by the appearance of muscle pain when walking, and in severe cases due to the appearance of ulcerations and gangrene, which can even lead to loss of the affected limb. Diabetes is one of the most important risk factors for chronic ischemia (Giménez et al., 2011). The consequences of diabetes as a disease are determined by metabolic abnormalities, characterized by hyperglycemia due to alterations in insulin secretion or defects in its action. In the vascular field, chronic hyperglycemia establishes a sequence of biochemical phenomena that finally manifest as micro and macrovascular diseases, such as nephropathy and retinopathy (microvascular diseases) and vascular diseases of various territories such as the heart, brain and lower extremities. (Julio R. et al., 2009). The poor circulation and the lower sensitivity in the feet of these patients result in a greater ease of chronic ulceration and infection of the ulcers. The diabetic foot is the complication that motivates the greatest number of hospitalizations in the diabetic population, being also recognized as the main cause of prolonged hospitalization in the medical and general surgery rooms (Brem & Tomic-canic, 2007).

In the United States and Europe, the annual incidence of chronic limb ischemia is in the range of 500 to 1,000 new cases per million people (Minar E., 2009), with diabetes being one of the most important risk factors of this pathology. It is estimated that in this population, its incidence is 20 times more frequent than in the population that does not have this disease (Spain Caparros G., 2000). The diabetic foot is the complication that motivates the greatest number of hospitalizations in the diabetic population, presents with a prevalence of between 5.3% and 10.5%, and corresponds to the first cause of major amputations of non-traumatic origin, presenting diabetics a risk of requiring an amputation 17 to 40 times higher compared to the general population (Lipsky, 2012; Vuorisalo S. et al., 2009; Fard AS et al., 2007).

In Chile, this pathology afflicts about 7% of the population (WHO, 2009). In 2000, the Central Metropolitan Health Service conducted a study to evaluate the "foot at risk" within the diabetic population, resulting in 63% of the diabetics studied had high-risk foot (20 or more points according to norm of evaluation year 1996), as well as 13.6% of amputations, being the rate of amputations within the general population of 40 / 100,000 adults aged 20 and over old. In 2002, it was estimated that around 6 million patients benefiting from the public system suffered from diabetes (MinSal, 2006). More recent studies indicate that in Chile the incidence of ischemia in the ulcers of the diabetic foot is close to 57%. Currently, there are no options available for reconstruction of tissue affected by ischemia resulting in ulcerations, and the treatments involved in the care of such wounds are extremely expensive (Hirsch et al., 2008). The latter is particularly relevant considering that more than 220 million people in the world suffer from diabetes, with more than 440 million cases projected by the year 2025.

The management of the diabetic foot requires in a first stage, antibiotic therapy, surgical drainage and resection of dead tissue, as the first priority is to control the infection locally. Once controlled, an evaluation of the patient is performed to detect the existence of ischemia at the level of the affected limb. If it is demonstrated that there is ischemia, the patient may undergo a percutaneous or surgical revascularization procedure, however, in a considerable proportion of patients (15%) revascularization is not an effective option. Of these patients, more than 40% will require a major amputation and 20% will die within 6 months. On the other hand, in those patients who undergo surgical intervention there is a high morbidity and mortality rate (Lipsky, 2012; Nikol S. et al., 2008; Fard AS et al., 2007; Faries PL. Et al. , 2004).

The cost of treating a simple ulcer varies between USD 5000 and USD 8000. Admission to a hospital because of an infected ulcer has an approximate cost of USD 15,000 and limb amputation costs between USD 50,000 to USD 150,000 (Fard AS et al., 2007). Currently, there are no options available for reconstruction of tissue affected by ischemia resulting in ulcerations or gangrene.

As already indicated, although the composition of the invention has its preferred application in the field of the treatment of ulcerative wounds such as the diabetic foot, it can be applied to any wound, regardless of its origin, to promote healing of the same. Among the wounds that can be treated with the composition of the invention are all ulcerative wounds, including varicose ulcers, burns, deep sharps wounds, bedsores, etc.

The possibility of using stem cells (SC) in medicine for regenerative purposes offers possibilities never previously imagined in this discipline and that is why the eyes have turned towards the use of SC for the development of eventual treatments (izuno H. et al. , 2010; Wu KH. Et al., 2007). In recent years the use of these cells has taken tremendous momentum in the clinical area, due to the great advances in the understanding of the mechanisms through which they exert their favorable effects, and the success achieved in clinical therapy studies. cell phone in which they have been used (Salem HK. And col., 2009).

The SC, are a specific type of undifferentiated cells, which have the ability to self-renew and the potential to differentiate into a wide range of cell lineages (Caplan I. et al., 2006; Flores-Figueroa E. et al., 2006). Depending on the development status of the individual from which they originate, they can be classified as embryonic (ESC), fetal (FSC) or adult (ASC) stem cells (Pappa Kl. Et al., 2009; Wacharaprechanont T., 2005). Within these last two groups it is possible to find a particular type of SC called mesenchymal stem cells (MSC) (Phinney & Prockop, 2007).

MSCs have been described as cells that have a number of characteristics that make them very attractive for therapeutic use:

- MSCs are multipotential cells, they have the ability to differentiate and give rise to different mesenchymal lineages, such as adipocytes, osteocytes, chondrocytes, myocytes, among others, and to transdifferentiate to other lineages (Uccelli A et al., 2008; Pountos I. et al., 2005; Minguell JJ et al., 2001).

- They have the ability to migrate to pathological areas, including wounds or ischemic areas (Roufosse CA. And col., 2004).

- They produce a series of bioactive factors (anti-fibrotic, anti-apoptotic, pro-angiogenic and mitotic) that go after the survival of damaged tissue (Caplan Al., 2009; Shabbir A. et al., 2009; Caplan Al And col. 2006), related to angiogenesis processes, migration to the wound site, cell regeneration damaged, proliferation and remodeling of the extracellular matrix (Burlacu et al., 2013; Ribeiro et al., 2012; Hematti, 2009; Valtieri & Sorrentino, 2008)

□ They are privileged cells in immunological terms, since among other characteristics they express low levels of the class I histocompatibility complex (MHC-I) and do not present levels of expression for MHC-ll or co-stimulatory molecules, which makes them hypo- immunogenic, then being tolerated by the recipient's immune system when used in allogeneic type transplants (Morandi et al., 2008; Uccelli A. et al., 2008; Ryan J. et al., 2005; Le Blanc K. and col., 2003).

These properties have generated intense research in the use of these cells for the regeneration and neovascularization of damaged tissues, either by their mere tissue integration or by the localized secretion of growth factors or survival factors (Sukpat et al., 2013; Danner et al., 2012; Salem & Thiemermann 2010; Azari et al., 2011; Volarevic et al., 2011; Bartosh et al., 2010).

Considering the pathophysiological abnormalities present in chronic wounds, as is the case of the diabetic foot, MSCs arise as an attractive alternative for the treatment of this type of lesions, since they could help the reconstitution of the dermis, vascularization and other required components for an optimal cure (Volk S., 2009; Dash NR. and col., 2009).

According to the report developed by the SC Industry, Select Bíosciences, in 2007-2008, the types of CS used most frequently in research and commercialization activities are the MSCs obtained from adult individuals and the ESC, both of human origin (Select Biosciences , 2008). However, the use of these cells has some limitations.

In the case of the ESC, its obtaining is strongly limited by ethical issues and regulate us, when obtained from the early stages of embryonic development (Insausti CL. Et al., 2010; Flores-Figueroa E. et al., 2006) .

On the other hand, obtaining a therapeutic amount of ASC, extracted mainly from the bone marrow (BM) and adipose tissue, is associated with the performance of a surgical procedure that requires anesthesia and hospitalization (Insausti CL. Et al., 2010 ; Wu KH. Et al., 2007). Additionally, in the case of ASCs It has been shown that there is a decrease in their proliferation capacity and their potential for differentiation in direct relation to the age of the patient from which they are extracted (Wagner W. et al., 2009; Fehrer C. et al., 2005). In the same way, cells obtained from individuals suffering from chronic diseases do not have the same potential as those obtained from healthy individuals. For example, it has been documented that MSCs obtained from the BM of patients with chronic Hepatitis B or cirrhosis have a low proliferation capacity (Zhong YS. Et al., 2010). On the other hand, the MSCs obtained from diabetic patients have low proliferation capacity, decrease in the potential for differentiation, and a large percentage of them are in a senescent state (Stolzing A. et al., 2010; Kume S. et al., 2005). Finally, the MSCs obtained from BM or adipose tissue of patients with diabetes, anemia or renal failure, have a decreased angiogenic potential (Li TS. Y col., 2010; El-Ftesi S. et al., 2009). The decrease of the capacities and potentialities of ASCs with respect to the patient's age and suffering from chronic diseases limits their use in autologous transplants. In view of the existence of these limitations in the use of ESCs and ASCs, an alternative source of MSC is necessary. For this, the identification of new sources of MSCs is crucial and the definition of their potential use is the focus of our proposal.

The umbilical cord is a rich source of specific tissue SC. It has been used as a source of HSC for 20 years to restore hematopoiesis, both in malignant and non-malignant disorders (Pappa Kl. Et al, 2009).

The umbilical cord has been considered an attractive source of MSC cells (Pappa Kl. Et al., 2009; Erices A. et al., 2000). The clinical use of the MSC derived from the umbilical cord is attractive because they are easy to obtain and do not have the limitations presented by those obtained from other sources. MSCs have been isolated from several compartments of the umbilical cord, specifically from the blood, the sub-endothelium of the vein that runs through it and recently from Wharton's jelly (WJ) (Troyer DL. Et al., 2008). WJ is called the connective-gelatinous tissue that surrounds the arteries and vein inside the umbilical cord. Obtaining MSC from umbilical cord blood has a low efficiency in comparison to its obtaining from BM, since the number of MSCs in cord blood mononuclear cells is very low (Musina RA. and col., 2007). However, obtaining MSC from the WJ is very efficient, since these cells are much more abundant in this tissue than in cord blood or BM. On the other hand, WJ MSCs (WJ-MSC) have a greater capacity to be expanded in vitro (Pappa Kl. Et al., 2009; Troyer DL. Et al., 2008; Secco M. et al., 2008) . Additionally, it has been reported that a percentage close to 20% of the WJ-MSC being expanded does not express the genes of either the MHC-I or the MHC-II, which could make them even better tolerated in an allogeneic transplant than those obtained from adult tissues (Pappa Kl., 2009; Gucciardo L. et al., 2009).

Taking into account the above, the inventors have developed a new composition for the treatment of wounds comprising a support matrix and Wharton's jelly mesenchymal stem cells (WJ-MSC), where the composition when applied to the wound promotes neo- angiogenesis and tissue repair, in order to accelerate or improve healing, without incorporating the cells of the composition into the patient's circulation. This is achieved by the factors secreted by the WJ-MSC, retained in the patch, which are transferred from the composition of the invention to the damaged tissue promoting healing. PRIOR ART

The present invention relates to a support structure (matrix) containing mesenchymal stem cells obtained from Wharton's jelly. This structure is applied to wounds to promote neo-angiogenesis, in order to accelerate or improve healing. A differentiating feature of the present invention is that the integration of foreign cells into the patient is avoided or minimized, so that the technical effect is exerted through the diffusion of angiogenic factors produced by the cells towards the wound, without Consider the integration of the cells themselves to the patient.

Prior to the development of the present invention, healing patches had been developed using mesenchymal cells of the same patient to be treated, in order to generate an autologous transplant, which incorporated the cells of the composition as part of the repaired tissue. For example, the document AU2009320446 (A1) describes a matrix where progenitor mesenchymal cells obtained from muscle processes of the same patient are incorporated, to improve wound healing. This approach has serious limitations, as previously discussed, since in patients with chronic diseases that require this type of treatment, bone marrow or adipose tissue mesenchymal cells have low proliferation capacity.

The inventors have solved this technical problem by not trying to incorporate mesenchymal cells as a transplant, but instead using them as a natural source of factors that promote healing and angiogenesis. In this way exogenous cells can be used, with high production capacity of these factors, such as the WJ-MSC.

In the state of the art, no document has been found that contains each and every one of the elements that make up the present invention.

First of all, it is known in the state of the art to obtain mesenchymal stem cells from Wharton's jelly, this is described in WO2012131618 (A1), where the WJ-MSC are formulated in a pharmaceutical composition which is useful for handling disorders of immunological character, by the immunomodulatory properties of these cells.

On the other hand, compositions for healing that include mesenchymal stem cells, but not WJ-MSC, were known. For example, application US2011256089 (A1) describes a hydrogel composition for applying cells to a damaged area to promote healing. It is indicated that mesenchymal stem cells can be used, among many other types of cells, and the strategy is that the gel cells integrate into the tissue to be repaired. This same strategy of integrating cells into the tissue to be repaired is used in the application US2012134965, which discloses compositions for the treatment of vascular diseases in general, where the composition contains stem cells as an active component, the WJ-MSC is not mentioned and as We indicate the objective is that the cells are incorporated into the tissue to be treated.

Another strategy in the state of the art is that used in the application AU2012205269 (A1), where the application of mesenchymal cells is described to promote angiogenesis and improve healing among other applications, through their systemic administration. The claims describe cell doses per kilogram, and it is further indicated that administration may be intravenous.

As can be seen in any of the documents analyzed neither by themselves nor by the combination of 2 or more documents, the composition of the invention is anticipated, nor is the method for treating wounds comprising applying the composition on the wound to be treated without incorporating The composition cells in the wound to be treated.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. (A) The stains of 2 matrices (IM and collagen), after incubation of the MTT assay in the absence and presence of WJ-MSC (1 x 10 ⁵ cells), at different in vitro incubation times are shown. (B) Quantification of WJ-MSC metabolic activity seeded in IM and collagen, at different incubation times. The values correspond to the average ± SEM (n = 6). * P <0.05 IM vs. collagen

Figure 2. Abundance of VEGF protein in cell extracts obtained from WJ-MSC grown for 24 hours in collagen and IM matrices in a serum-free culture medium. The symbols represent (-) absence or (+) presence of WJ-MSC. The values correspond to the average ± S.E.M. (n = 6). * P <0.05 IM vs. collagen

Figure 3. {A) Representative photo of the secretoma (conditioned medium) from WJ-MSC grown in the absence of serum (24 hours) obtained from the cells sown in the collagen (left) and IM (right). (B) The graph shows the levels of relative secretion that were grouped according to their cellular function. The different pixel intensities were normalized by the positive control (internal points of known location in each membrane). The values correspond to the average ± S.E.M. (n = 3). * P <0.05, IM vs. collagen

Figure 4. (A) Representative photo of CAM assay for IM Matrix + DMEM control culture medium (left), IM + VEGF Matrix (center) and the composition of the invention IM Matrix + WJ-MSC cells (right). (B) The graph shows the quantification of the blood vessels in the three conditions at 24, 48, 72 and 96 hours after introduced the composition. The difference in the average number of blood vessels generated by the matrices seeded with WJ-MSC and DMEM determined by ANOVA * P> 0.05

Figure 5. (A) Representative photo of human cells adhered to CAM after 96 hours with IM Matrix + DMEM culture medium by immunohistochemistry, using human anti-Presinilin 1 antibody (1 mm scale bar at A, and 100 um A ' ) (β) Representative photo of human cells adhered to CAM after 96 hours with IM Matrix + WJ-MSC cells by immunohistochemistry, using human anti-Presinilin 1 antibody (1 mm scale bar in B, and 100 um B ').

Figure 6. (A) Representative photographs of tissue transillumination and digital segmentation of tissues treated with compositions comprising IM Matrix (Control), IM Matrix + WJ-MSC cells (invention), IM Matrix + AD-MSC cells (comparative example) ( n = 12 for control, 13 for WJ-MSC and 3 for AD-MSC Scale bars: 5 mm. (B) Quantification of digital segmentation showed significantly higher levels of vascularization for compositions containing WJ-MSC. with respect to the control of IM matrix only and for compositions containing AD-MSC Values were normalized with normal skin and expressed as mean ± SEM.

SUMMARY OF THE INVENTION

The present invention relates to a composition comprising a support matrix and Whaen's gelatin mesenchymal stem cells for wound treatment. And a method for the treatment of wounds that comprises applying the composition on the wound to be treated.

The wound to be treated is chosen among chronic or acute wounds, such as all ulcerative wounds, including varicose ulcers and ulcers associated with ischemia such as the diabetic foot; burns, deep sharps wounds, bedsores, etc. In a preferred embodiment, the wound to be treated is an ulcerative wound resulting from chronic ischemia, where the composition of the invention promotes neo-angiogenesis and tissue repair, so as to accelerate or improve healing, without incorporating the cells of the composition into the wound to treat.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a composition useful for the treatment of chronic or acute wounds, such as all ulcerative wounds, including varicose ulcers and ulcers associated with ischemia such as the diabetic foot; burns, deep sharps wounds, bedsores, etc. In particular, the composition of the invention is useful in the treatment of chronic wounds or difficult healing, as is the case of wounds caused by prolonged ischemia, such as the diabetic foot. The composition comprises two essential elements, a support matrix and Whaen's gelatin mesenchymal stem cells.

The support matrix must be flexible and allow the integration of live WJ-MSCs. The matrix can be composed of any biocompatible polymer that is not biadsorbable, since the WJ-MSC must not be integrated into the wound. Examples of biocompatible polymers suitable for the support matrix of the present invention are: substantially pure collagen or in combination with other compounds such as glycosaminoglycans and semi-permeable polysiloxane (silicone), pectin, carboxycellulose, chitosan, combinations thereof or any other biocompatible polymer that It exists in art. In one embodiment, the commercial support matrix, IntegraTM atrix (IM) is used.

The second component of the composition of the invention are Wharton's jelly mesenchymal stem cells or WJ-MSC, which are isolated from the umbilical cord.

The WJ-MSC are responsible for the therapeutic effect of the composition since they secrete different factors that favor wound healing, such as: pro-angiogenesis factors such as angiogenin, VEGF, uPA, IGFBP-3; of migration factors such as TIMP-1, Serpina E1, of inflammatory response factors such as IL-8, MCP-1, Pentraxin-3, and tissue repair factors such as HGF and Thrombospondin-1.

Optionally, the matrix may comprise other components that allow to increase the survival of the WJ-MSC or components with pharmacological action for the treatment of wounds. Among the components that can increase the survival of the WJ-MSC are the culture medium for human cells, such as the DMEM medium, or a buffer composition of physiological pH (pH 7.2) and nutrients like glucose, essential amino acids, vitamins and minerals. However, it has been observed that WJ-MSC can grow in hypoxic conditions and survive without supply of nutrients for up to 10 days.

Among the components of healing pharmacological action that the composition of the invention can optionally comprise are: argumentative sulfadiazine, poly (n-acetylgalactosamine-d-glucuronic) sodium polysulfate, chloramphenicol, retinol palmitate, benzalkonium chloride, benzoate benzyl, amino acids: alanine , cysteine, vitamins C, E, F. Other healing components that can be added to the composition are components that are also secreted by the WJ-MSC in order to increase its concentration, such as: angiogenin, VEGF, uPA, IGFBP-3, TIMP-1, Serpina E1, IL-8, MCP-1, Pentraxin-3, HGF and / or Thrombospondin-1.

In a second aspect the invention relates to a method for treating wounds which comprises applying the composition directly on the wound to be treated. The composition of the invention can remain active and stable on the wound for a period of up to 10 days or more, after that time the composition begins to degrade, and fall from the wound naturally, without any WJ transfers. -MSC to the treated wound tissue. At 10 days or even before, depending on the evolution of the wound, the composition can be applied again on the wound, without removing the remains of the first applied composition. This treatment can be repeated as many times as necessary until wound healing is achieved, as in the case of wounds that are difficult to heal, such as the diabetic foot.

In a preferred embodiment, the wound to be treated is an ulcerative wound resulting from chronic ischemia, where the composition of the invention promotes neo-angiogenesis and dermal repair, so as to accelerate or improve healing, without incorporating the cells of the composition into the wound to treat.

The fact that WJ-MSC is not incorporated into the tissue to be treated is one of the great advantages of the composition, since it eliminates the possible rejections of the patient to exogenous cells and only gives the tissue that needs to heal the factors that promote healing and angiogenesis The support matrix must comprise the biocompatible polymer in a conformation that ensures its flexibility.

The WJ-MSC are incorporated into the support matrix at a concentration of between 1 x 10 ² to 1 x 10 ⁸ cells per 100pL and 1 mL of matrix. Especially preferred are compositions with a concentration of WJ-MSC of between 1 x 10 ⁴ to 1 x 10 ⁶ cells per 00pL and 1 mL of matrix.

The WJ-MSCs are incorporated into the support matrix at a concentration of between 1 x 10 ² to 1 x 10 ⁸ cells per 1 to 100 pL of matrix. Especially preferred are compositions with a concentration of WJ-MSC of between 1 x 10 ⁴ to 1 x 10 ⁶ cells per 1 and 100 pL of matrix.

Subsequently the composition is spread on a non-stick surface to form sheets of the composition. The nature of the support matrix allows the composition to adhere to the wound to be treated without adding adhesion compounds thereto. Optionally, biocompatible adhesive components can be added.

As already indicated, the composition of the invention is presented as sheets of the support matrix with the WJ-MSC inserted therein, and if necessary with the additional components, of a thickness of between 0.1-1 mm. The shape and measurements of the sheets are variable, and do not affect the effectiveness of the invention. Conveniently, the composition of the invention is presented in sheets of an area between 4 mm ² to 100 cm ² .

Once a sheet of the composition of the invention has been obtained, it is applied to the wound to be treated, by gently pressing it so that it is adhered to the clean wound, no additional procedure for the treatment of the wound is necessary.

The examples show that the composition of the invention effectively promotes angiogenesis and healing, solving this important technical problem of the Pharmaceutical Industry.

APPLICATION EXAMPLES

Example 1. Obtaining the wound healing composition, a) Using collagen support matrix. The composition of the invention was generated using collagen I support matrix, where collagen I was obtained in the laboratory from rat tail. The composition of the invention was generated in a 7: 2: 1 ratio, where 70% corresponds to the preparation of collagen, 20% to DMEM medium (4X, pH 7.2) and the remaining 10% to WJ- MSC (1 x 10 ⁴ ) resuspended in complete medium.

Quickly, 80 μΙ of this composition was deposited in 96-well plates and then incubated at 37 ° C for a period of 5-10 minutes to allow the collagen to polymerize.

b) Using IntegraTM Matrix (IM) support matrix.

The composition of the invention was generated using a commercial support matrix, IntegraTM Matrix (IM) matrices (Integra®, LifeScience Corporation). IM is an FDA-approved device for advanced wound care, consisting of a porous matrix of cross-linked bovine tendon collagen and glycosaminoglycans and semi-permeable polysiloxane (silicone). For this, 6 mm diameter IM pieces were used and dried with sterile gauze, then deposited in a 24-well plate where 1 x 10 ⁴ WJ-MSC cells resuspended in complete culture medium were seeded and incubated for 1 time at 37 ° C to allow WJ-MSC cells to integrate into the IM support matrix.

Example 2. Evaluation of the viability of the WJ-MSC in the wound healing composition.

The in vitro metabolic activity of the WJ-MSCs in the compositions of the invention generated in Example 1 was first evaluated as an indicator of their viability. The metabolic activity of the WJ-MSC in both matrices was quantified by an MTT assay, based on the metabolic reduction of the reagent bromide of 3- (4,5-dimethylthiazol-2-yl) -2.5 diphenyltetrazolium (MTT). After each incubation time (12, 24 and 48 hours, as well as 5 and 10 days) and in the absence of fetal bovine serum (FBS), metabolic activity was quantified by absorbance at 550 nm (OD 500nm) . The results obtained show that the metabolic activity of the WJ-MSCs was maintained in both compositions of the invention until 10 days after they were generated (Figure 1). The results show that the metabolic activity of the WJ-MSC remained significantly higher in the compositions of the invention using IM support matrix.

The results indicate that the metabolic activity of the WJ-MSC in the initial condition (12 hours) is similar in both compositions of the invention, and that as expected this decays over time, however over a period as long as the Ten days after the compositions were generated, an important metabolic activity is still observed in the WJ-MSC cells of both compositions of the invention. Where in the compositions of the invention comprising collagen the activity is 25% of the initial activity, while in the compositions of the invention comprising IM the activity is 70% of the initial activity. (Figure 1).

Example 3. Evaluation of the production of VEGF in the wound healing composition.

The compositions of the invention are useful for accelerating or improving wound healing if WJ-MSC cells secrete angiogenic factors. For this reason, the production of VEGF, a very important angiogenic factor in both compositions of the invention generated in Example 1, was evaluated after 24 hours of culture.

The expression of VEGF in the compositions of the invention, with collagen and IM was determined and the results were normalized with the expression of β-actin, as matrices without WJ-MSC cells. The results are shown in Figure 2, in both compositions of the invention a high expression of VEGF is observed in Cellular Used. This result demonstrates that the compositions of the invention can promote angiogenesis since they supply a large amount of VEGF on the wound in which the composition is applied.

Example 4. Evaluation of the production of other trophic factors in the wound healing composition.

The production of other trophic factors that promote angiogenesis in the 2 compositions of the invention generated in Example 1 was analyzed. To assess this, conditioned media of the compositions of the invention were collected 24 hours after they were generated for Proteome Profiler assays. Array according to the indications of Manufacturer (Human Angiogenesis Array Kit, Protome ProfilerTM Antibody Arrays, R&D Systems ARY007). This kit allowed to detect simultaneously, quickly and sensitively, the expression levels of 55 proteins related to angiogenic processes. The kit provides nitrocellulose membranes that contain specific antibodies attached at different points in duplicate, so to detect the signal of said proteins the sample must be prepared with a cocktail of biotin-coupled antibodies. To perform this test, the membrane was incubated with the conditioned medium overnight. Subsequently, washings were performed (3 x 10 minutes) and incubated with the secondary antibody coupled to horseradish peroxidase (HRP) that recognizes biotin. Finally, by means of a chemiluminescence reaction, the amount of each of the analytes present in the conditioned medium of the compositions of the invention was detected. Our results indicated that the compositions of the invention secrete a wide range of trophic factors, in addition to VEGF, which favor healing, see Figure 3.

Figure 3 B shows the relative secretion levels of the analyzed proteins, all related to the angiogenic processes, which were grouped according to their cellular function, in both compositions of the invention. The different pixel intensities were normalized by the positive control (internal points of known location in each membrane). The values correspond to the average ± S.E.M. (n = 3). * P <0.05, IM vs. collagen Although it is observed that the compositions of the invention that employ IM support matrix show a greater secretion of the analyzed proteins, both compositions of the invention secrete significant amounts of pro-angiogenesis factors such as angiogenin, VEGF, uPA, IGFBP-3; of migration factors such as TIMP-1, Serpina E1, of inflammatory response factors such as IL-8, MCP-1, Pentraxin-3, and tissue repair factors such as HGF and Thrombospondin-1.

Example 5. Evaluation of angiogenesis in vivo with the wound healing composition.

To evaluate the in vivo effect of the compositions of the invention, a chicken embryo chorioallantoic membrane assay, known as the CAM test, was performed. This method is a classic model to study the neovascularization The test consisted of introducing an IM matrix membrane with different compositions to be analyzed, control: IM Matrix + DMEM Culture Medium, IM + VEGF Matrix (10 ng / μΙ) and the composition of the invention IM Matrix + WJ-MSC cells (5 x 10 ⁵ ), on the chorioallantoic membrane of chicken embryo through a hole in the eggshell from day 8 to day 12 of embryonic development (E8-E12). Subsequently, the angiogenesis in the membrane was quantified by quantifying the vessels that cross in a perimeter of 4 mm around the membrane. The results show that the composition of the invention overcomes the angiogenesis generated by the support matrix in combination with the pure VEGF angiogenic factor, used as a positive control (Figure 4).

Additionally, the transfer of cells from the composition to the chorioallantoic membrane of chicken embryo was studied, by immunohistochemistry, using the human anti-Presinylin 1 antibody. Figure 5 shows the distribution of human cells in the Matrix IM + DMEM culture medium assay and with the composition of the invention Matrix IM + WJ-MSC cells after 96 h in contact with it, evidencing that most of the cells are identified in the IM and not in the CAM so it can be said that the migration of WJ-MSC is not detectable. Example 6. Comparison of the composition of the invention with the empty IM matrix and with I + AD-ÍUISC cells.

To compare the effects of the composition of the invention with the regenerative effects associated with the IM matrix, and the use of mesenchymal cells from adult adipose tissue, which corresponds to the usual practice of autologous transplants, a dermal regeneration experiment was performed. in murine model evaluating the 3 conditions: IM matrix (Control), IM matrix + WJ-MSC cells (invention), IM matrix + AD-MSC cells (comparative example). The percentage of vascularization was quantified at 5 and 10 days after the application of the composition. The results are shown in Figure 6, where it can be seen that the composition of the invention shows an angiogenesis significantly greater than that obtained in the other 2 control conditions. Photographs of the blood vessels are shown in Figure 6A and their quantification is shown in Figure 6B. INDUSTRIAL APPLICATION

As previously described, the present invention, referring to a composition comprising a support matrix and Wharton's gelatin mesenchymal stem cells, is useful in the pharmaceutical industry for wound treatment, especially for inducing neo-angiogenesis, of way to accelerate or improve healing, without incorporating the cells of the composition into the wound to be treated.

REFERENCES

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Claims

REVINDICAC! ONES

1. Composition for the treatment of wounds comprising a support matrix and mesenchymal stem cells of Wharton's jelly (WJ-MSC).

2. Composition for the treatment of wounds, according to claim 1 wherein the support matrix is a biodegradable and pharmacologically acceptable material.

3. Wound treatment composition according to claim 2 wherein the support matrix is chosen from collagen, substantially pure or in combination with other compounds such as glycosaminoglycans and semi-permeable polysiloxane (silicone), pectin, carboxycellulose, chitosan, combinations of them or any other biocompatible polymer that exists in the art.

4. Composition for wound treatment according to claim 3 wherein the support matrix is collagen combined with glycosaminoglycans and semi-permeable polysiloxane (silicone).

5. Composition for wound treatment according to claim 4 wherein the support matrix is IntegraTM Matrix (IM).

6. Composition for the treatment of wounds according to claim 1 wherein the WJ-MSC are in a concentration of between 1 x 10 ² to 1 x 10 ⁸ cells per 1 ml_ of matrix.

7. Composition for wound treatment according to claim 6 wherein the WJ-MSC are in a concentration of between 1 x 10 ⁴ to 1 x 10 ⁶ cells per

1 mL matrix

8. A wound treatment composition according to claim 1, further comprising other components such as culture medium, physiological pH buffer, or nutrients such as glucose, essential amino acids, vitamins and minerals.

9. Composition for the treatment of wounds according to claim 1 which optionally comprises other components of healing pharmacological action such as argumentative sulfadiazine, poly (n-acetylgalactosamine-d-glucuronic) sodium polysulfate, chloramphenicol, retinol palmitate, benzalkonium chloride, Benzyl benzoate, amino acids: alanine, cysteine, vitamins C, E, F, angiogenin, VEGF, uPA, IGFBP-3, TIMP-1, Serpina E1, IL-8, MCP-1, Pentraxin-3, HGF and / or Thrombospondin -one.

10. Composition for the treatment of wounds, according to claim 1 which is presented in sheets of a thickness between 0.1-1 mm.

11. A method for treating wounds which comprises applying the composition of claim 1 onto the wound, which comprises a support matrix and Wharton's gelatin mesenchymal stem cells.

12. Method for treating wounds according to claim 11 wherein the wound to be treated is a vascular lesion.

13. Method for treating wounds according to claim 11 wherein the wound to be treated is an ulcerative wound.

14. Method for treating wounds according to claim 11 wherein the wound to be treated is a burn.

15. Method for treating wounds, according to claim 1, wherein the wound to be treated is a diabetic foot.

16. Method for treating wounds according to claim 1, wherein the wound to be treated is a chronic wound.

17. Method for treating wounds according to claim 11 wherein the wound to be treated is an acute wound.

18. Method for treating wounds according to claim 1 wherein the composition is applied to the wound and acts for a period of up to 10 days or more.