Patent Application
20140242181
The skin is an organ consisting of three cell layers: epidermis, dermis and hypodermis, and by annexes: hair, nails, sebaceous and sweat glands (Castillo, 2001), as shown in
The dermis is located under the epidermis, and is composed of connective tissue ground substance, forming together with glycosaminoglycans and fibrillar protein extracellular matrix, cell, and primarily fibroblasts, which are responsible for producing collagen and elastin, which give support and elasticity to the skin (Green, 1991). It also has cells of the immune system that help in defense against pathogens. The dermis is divided into two areas:
Papillary dermis: contains vascular networks that serve two important functions: the first is nourishing the avascular epidermis, and the second is feathers. The vascular network is interfingered in areas called dermal papillae. In the papillary dermis, we also found sensory nerve endings, as for example, the Meissner corpuscle.
Reticular Dennis: consists of a dense, irregular connective tissue. It is important because it gives skin firmness and elasticity. In addition, it serves as support annexes skin, glands and hair follicles (Haake et al., 2001).
Also called adipose panniculus or subcutaneous tissue. It is made by fat cells, which are known with the name of adipocytes, which are arranged in lobes separated by connective tissue called Septum and interlobular Septum (Navarrete, 2003). In the hypodermis are: the vascular network deep, lower portions of some hair follicles, acinar glands, eccrine and apocrine, Vatter-Puccini sensory corpuscles, sensitive to pressure changes and heat-sensitive Ruffini corpuscles.
Among lesions that may affect this organ, burns are one of the diseases that most often involve the urgency consultations, being the variety of trauma, which leaves in the patient, in the more usual way, serious and permanent sequelae (Aguayo, 1999). Globally, burns are a serious public health problem. There are more than 300,000 deaths each year only by fire. Death by fire product Burns, is among 15 causes of death in children and young adults ages 5 to 29 years. In addition, millions of people are disabled or suffer disfigurement of life (WHO, 2008). In a schematic way, one can say that in Chile, 50,000 people a year suffer some burn, 5,000 of them are hospitalized, and 500 die. Of this total, two-thirds are children, the highest risk group, along with the older than sixty years of age (Castillo, 2003). The main feature of burns injuries, is the loss of cutaneous cover, either partial or total. In the case of severe burns, there is loss of electrolytes and fluids, and infection of the skin that can trigger a systemic infection (CIGNA Health Corporation, 2008).
Burns, according to the damage caused to the tissue were traditionally classified in:
However, a more recent scheme has been described. The injuries that affect only epidermis are called surface. Burns, also involving the dermis, are classified as partial thickness burns, and they may be superficial or deep (the first affect the papillary dermis and the latter both papillary dermis as a lattice). Burns that extend through all layers of the dermis and subcutaneous tissue, are called Burns full thickness (Ehrenreich and Ruszczak, 2006). Skin ulcers are another type of frequent injury and difficult to treat. Occur, usually because of a venous stasis, diabetes, vasculitis, or arterial occlusion. They are classified by stages 1-6; stage 1 that where the ulcer is not really formed intact skin is simply flushed, and stage 6 that where there is destruction of the outer layers of the skin (Bolivar-Flores and Kuri-Harcuch, 2000). Venous ulcers, which represent between 80-90% of the total vascular ulcers can be found within skin ulcers. They are more common in women, with a ratio of 1-3. Chronicity and recurrence are its most relevant clinical features, half remain open over the nine months, 20 one-third of the initially healed (CIGNA Health Corporation, 2008) are up to two years, and a 10 to five, reappearing it, within the twelve months following his cure.
Venous disease is a precursor of venous hypertension (hypertension, venous ulcers, constitute the highest percentage out of the total of the vascular, 75 to 90%), and a risk factor for the subsequent development of ulcers on the lower extremities.
Venous ulcers of the lower extremities are transformed into a clinical problem, affecting between 1 and 2 of the Western population (Castillo et al., 2004). The decrease in circulation and Neuropathies, are the leading cause of diabetic foot ulcers, and can lead to amputation of the extremity (CIGNA Health Corporation, 2008). In its pathogenesis, initial alterations occur in the venous macrocirculation, mainly for reflux and occasionally by obstruction. These alterations cause venous hypertension, which affects the microcirculation, secondarily causing pericapillary fibrin deposits and activating cytokines and proteases that promote inflammation. This determines the appearance of lipodermatosclerosis, consisting of a fibrous tissue, with poor cellularity and irrigation and the consequent ulceration (Herouy et al., 2000).
Wound healing, is a dynamic biological process that involves complex interactions of events cellular, molecular and biochemical (Lance, 2000). In this process, place interactions between the cells of the epidermis and the dermis, the extracellular matrix, controlled angiogenesis, and proteins derived from plasma, all coordinated by a number of cytokines and growth factors (Harding et al., 2002). This process has traditionally been divided into three distinct phases: inflammation, proliferation and remodeling (Schilling, 1976).
This stage occurs, immediately after the lesion or injury between days 4 and 6 produced. The first step is to stop the bleeding. There is contraction of the blood vessels damaged, and the endothelium and the area near platelets activate the intrinsic pathway of the coagulation cascade. The formed clot, is composed of collagen and platelet, Thrombin, fibronectin (Broughton et al., 2006). Trapped in the clot, mostly platelets, cells trigger the inflammatory response through the release of vasodilators and chemotactic and activation of the complement cascade (Clark, 1996). The fibrin clot, also serves as a prelude to invading cells like: neutral edges, monocytes, fibroblasts and endothelial cells (Kurkinen et al., 1980).
In the first stage of inflammation, predominantly neutrophils. They are attracted to the wound area by agents such as chemotactic: Interleukin 1 (IL-1), the tumor necrosis factor (TNF-α), factor (TGF-β) transforming growth, PF4 and bacterial products (Broughton et al., 2006;) Tamariz et al., 2002). Neutrophils removed bacteria and cellular debris from the wound, through the release of proteolytic enzymes that digest the bacteria and dead tissue (Broughton et al., 2006). The damaged extracellular matrix is also cleaned by Metalloproteinases matrix (MPP by its acronym in English), which are expressed by keratinocytes, fibroblasts, monocytes and macrophages in response to TNF-α (Abraham et al., 2000). Subsequently, the number of neutrophils decreases and these are replaced by macrophages. These activated macrophages, will act as mediators in angiogenesis (synthesizing endothelial growth factor vascular VEGF, FGF and TNF-α fibroblast growth factor), in the fibroplasia by synthesis of TGF-β, growth factor epidermal EGF growth factor derived from platelets PDGF, IL-1 and TNF-α and finally, synthesize nitric oxide (NO), through the activation of nitric oxide synthase inducible by IL-1 and TNF-α (Witte and snapper2002). Macrophages play a fundamental role, and mark the transition between the inflammatory phase, and the proliferative (Harding et al., 2002).
At this stage occur epithelization, angiogenesis and the formation of a temporary matrix, and this stage runs from the 4th to 14th. The predominant cells at this stage, are fibroblasts and endothelial cells (Broughton et al., 2006). Epithelization is stimulated, in principle, by inflammatory cytokines (IL-1 and TNF-α). Fibroblasts, on the other hand, synthesize and secrete (KGF-1 and KGF-2) keratinocyte growth factor and IL-6, which encourages neighbouring keratinocytes migrate to the damaged area, to proliferate and differentiate into the epidermis (Smola et al., 1993;) Broughton et al., 2006).
Fibroblasts migrate to the wound site from nearby tissues, and become active (PDGF and EGF are main signals to the fibroblasts), so they begin to synthesize collagen, and proliferate to finally transform into myofibroblasts, which cause the contraction of the wound (Sage, 2001). For the formation of the temporary array, the fibroblasts (stimulated by PDGF) begin to synthesize, in addition to collagen, fibronectin, and proteoglycans as for example, hyaluronic acid (Harding et al., 2002;) Lynch et al., 1989). TGF-β, leads also to the fibroblasts to synthesize collagen type I, also decreasing the production of Metalloproteinases (MMP) matrix and increasing the production of proteins in cell adhesion (Goldman, 2004).
The keratinocytes, on the other hand, secrete VEGF (also secreted by macrophages, fibroblasts and platelets), which attracts endothelial cells located in the surroundings, thus beginning the formation of new capillaries. Endothelial cells also produce nitric oxide, which protects new tissue from the toxic effects of ischemia and damage by reperfusion, causing vasodilation of the endothelium (Broughton et al., 2006).
Clinically, the maturation and remodeling phase is perhaps the most important, and goes from the 8th until the year. The remodeling, or final stage is characterized by formation of the extracellular matrix, which is initially composed of fibrin and fibronectin. Then, fibroblasts begin to synthesize glycosaminoglycans, proteoglycans, and other proteins (Broughton et al., 2006).
This temporary array is then replaced by a more rigid and organized matrix composed mainly of collagen (in granulation tissue collagen type III reaches 30, while in the mature scar under 10). The proteinase present in matrix remodelling, are regulated by changes in the concentrations of TGF-β, PDGF, IL-1 and EGF.
MMP activity is also suppressed by MMP inhibitors produced by fibroblasts in tissue (Henry and Garner, 2003). The synthesis of collagen will continue for at least four to five weeks after that occurred the lesion (Diegelmann, 2003). The collagen in the scar, will never be as organized and structured (even after a year), as the collagen found in healthy skin. The strength and firmness of the skin will not be 100% normal (Broughton et al., 2006). After the closure of the wound, remodelling of the resulting scar can be months or years, and leave as a result, a reduction in the number of cells and a decrease in blood flow in the scar tissue (Harding et al., 2002). In conclusion, a full-thickness injury healing, requires resurfacing from the limits of the wound, through the proliferation and migration of keratinocytes, through a temporary extracellular matrix, which is then converted into tissue granulation, which will be finally remodeled neodermis (Clark, 1996;) Tamariz et al., 2002).
The need for rapid closure of wound in extensive burns, made to develop methods of expansion and reproduction cells autologous or heterologous of the dermis and epidermis in vitro (Green et al., 1979) so, as today, it is possible to make a culture on number of different cell formulas (pure keratinocyte or mixed with fibroblasts). From a small biopsy, human skin can be grown in large amounts of three to four weeks and used as autografts, and may cover the total body surface area of an adult (Cuenca and Alvarez, 2003). The use of autografts and allografts of cultured epidermis, has been established as part of the therapy of the burned patient. In fact, the guides clinics of the great burning, produced by Chilean experts to guide the treatment within the framework of the explicit health guarantees, including the use of cultured keratinocytes (MINSAL, 2007). Clinical studies demonstrate that the allograft of human epidermis, grown in vitro, act as a dressing biologically active, to reduce the time of epithelization in second-degree burns deep, donor areas of autografts, dermabrasion, and ulcers by venostasis or diabetes (Sosa et al., 1999). So, as today, a series of dermal substitutes, are available as shown in table 1:
Currently, a series of synthetic biomaterials that operate as extracellular microenvironments, imitating the regulatory features having natural extracellular matrix (ECMs) have been developed.
Biomaterials play a central role in modern strategies of regenerative medicine and tissue engineering, as a design of a biophysical and biochemical environment which leads cell behavior and functions. This can facilitate the restoration of structures or functions of the dysfunctional or damaged tissue.
Biomaterials, provide interim support interacting biomolecular with cells, guiding spatially and temporally complex multi-stage processes of formation and regeneration of tissues (Lütolf and Hubbell, 2005;) Bacakova et al., 2004). Therefore, the purpose of these biomaterials is mimic, to some extent, the processes that occur in viva. Dermal substitute or ideal synthetic biomaterial, must have or comply with the following characteristics: absence of antigenicity, compatibility with tissue, absence of local or systemic toxicity, impermeability against exogenous organisms, rapid and sustained adherence to the surface of the wound, elasticity, to allow the movement of the tissue, resistance, should be translucent to allow direct observation of healing have a low cost, causing minimal discomfort to the patient, which reduces the time of healing, among others. (Ehrenreich and Ruszczak, 2006; Smith et al., 1988).
The therapeutic advances that have been made to the products developed by engineering in tissues, are based on three strategies:
the use of cells without matrix (such as autologous cell transplantation or stem cell therapy);
the use of synthetic polymers with or without growth factors and cytokines; and the use of a three-dimensional matrix with cells in it (Jimenez and Jiménez, 2004).
There are a number of these experimental systems available. These range from matrix derived from cells or tissues (e.g.: Matrigel), hydrogels of synthetic polymer, to matrix composed of recombinant proteins. Bioinert surfaces are also been developed, using molecules such as protein albumin anti adhesive, hydrogels based on hyaluronic acid or poly(hydroxyl ethyl methacrylate), Poly, polyacrylamide, dextran and alcohol particularly polyethylene glycol (PEG) (Bacakova et al., 2004).
Thus, biomaterials derived from natural ECM, can be used as supports for transplanted cells, which will be subsequently implemented on injured tissue, and also to induce regeneration and remodeling in vivo. For example, collagen and fibrin as arrays, are clinically established and approved by the FDA (Food and Drug Administration), in the treatment of burns, for the healing of wounds and tissue repair, respectively (Lütolf and Hubbell, 2005;) Van Dorp et al., 1998). Fibrin products, contain two components isolated from human plasma: Fibrinogen and Thrombin. The combination of these two components gives as a result, the formation of a fibrin clot, which is used to achieve hemostasis, and the closure of the wound in surgical processes (Geer et al., 2002).
In a study conducted by Cox et al., 2004, analyzed the fibrin clot as a means of transport of human dermal fibroblasts, and also assessed, proliferation and migration of these in different formulations of clots, varying the concentrations of Fibrinogen and Thrombin, coming to the conclusion that the variation in the concentrations of both components, affected the behavior of the fibroblasts in three-dimensional fibrin clots.
The first commercially available dermal substitute, used similar collagen based on bovine collagen and Chondroitin-6-sulfate with an outer covering of Silastic® (Burke J et al., 1981).
Dermagraft® is a modification of the previously composed dermal substitute, where instead of bovine collagen, fibroblasts obtained from the foreskin of newborn infants, are cultivated on a nylon mesh and covered with an outer silicone layer (Dermagraft Transitional Covering). It is designed to cover the dermal layer of the skin and to stimulate an improvement in the wound healing process (Mansbridge et al., 1998).
There are also other types of dermal substitutes, which incorporate both epidermal components as dermal (mixed or composite substitutes). The first composite substitute was developed by Ortec International Inc., and was a member of fibroblasts and keratinocytes of newborns, grown on a porous matrix criss cross with bovine collagen type 1. However, the use of this substitute is limited, since the FDA has approved it only for two indications: the treatment of reconstruction of hand in patients who suffer from Epidermolysis Bullosa recessive, and as aid in the healing of donor areas of autografts in burned patients (Pham et al., 2002;) Eisenbud et al., 2004). Grañskin (Apligraj®) it is also a joint replacement, but with multiple applications. It was developed for the coverage of full thickness wounds. It is a graft mixed allogeneic consisting of human epidermal cells, human fibroblasts and bovine collagen type 1, which delivers 4 components: epidermal keratinocytes, a stratum corneum well differentiated, matrix extracellular and viable allogeneic dermal fibroblasts (Pham et al., 2002; Vêves et al., 2001). On the other hand, Mylan Laboratories, Inc., developed Biobrane, which is a biosynthetic product, consisting of an ultra-thin semi-permeable silicone film, with nylon fibers partially embedded in the film, linked chemically to collagen from porcine (UDL Labs, 2008). It is used, mainly in patients with partial thickness burns and wounds relatively fresh (24-48 hours) (Gerding et al., 1990). Another product of Bioengineering, which was approved by the FDA in 1997, but has a high cost, is TransCyte®, composed of newly born human fibroblasts, grown under aseptic conditions on the component of Biobrane nylon mesh. Fibroblasts secrete into the mesh, components of the extracellular matrix like fibronectin, collagen type 1, decorin and growth factors linked to the matrix (Ehrenreich and Ruszczak, 2006;) Noordenbos et al., 1999). However, in most of the cases, it is difficult to argue the use of these expensive products (for example in donor areas of skin) when there is more economic and effective alternatives that are already available, like Biobrane and conventional implants for wound coverage. In extensively burned patients, where the donor areas of autografts are limited or are not appropriate (to use them requires two or more weeks), could justify the use of biomaterials that will provide a temporary wound coverage, thus, promoting a faster and better quality healing (Sosa et al., 1999; Bar-Meir et al., 2006).
There are, on the contrary, lower cost biomaterials, and which have been used widely in tissue engineering, as in the case of the Chitosan and hyaluronic acid. The first is a complex, non-toxic, and biodegradable carbohydrate derived from chitin, which has shown mucoadhesive activity, this makes it an excellent Hemostatic agent (Wedmore et al., 2006). Also has other biological properties (antibacterial and antifungal) and affects the macrophages function, which helps fast healing. Also, has ability to stimulate cell proliferation and tissue histoarchitectonics organization (Paul and Sharma, 2004;) Fukasawa et al., 1992). On the other hand, the hyaluronic acid (HA), is a glycosaminoglycan which is normal skin in the intercellular spaces of the skin, except in the granular layer and stratum corneum. Besides being a matrix in which the cells are embedded, it was discovered that it has numerous functions on the skin: can retain water in the tissues, due to this change the volume and compressibility dermal; It can also improve proliferation and cell differentiation and the repair of damaged tissue (Juhlin, 1997;) Laurent and Fraser, 1992).
The developed system is called integrated, because the cells are not located on the surface of the support but embedded in it (Young et al., 2006). This system has proven an excellent adhesion to the area of the lesion and, on the other hand, no observed toxicity reactions to the system components.
In addition, studies published by the own inventors of this application are encapsulated into fibrin, skin cells to exhibit a growth pattern different from cells cultured in conventional culture bottles (
The technical issue raised in this application consists of providing a product and system preferably autologous allowing immobilize, proliferate and conveyed cells within a matrix for the purpose of tissue regeneration.
The search of biomaterials that cell growth is a fertile ground. Required materials that mimic the extracellular cell with its three-dimensional features. Within these biomaterials collagen, gelatin, alginate and other materials have been used. Among these, the use of fibrin has shown extraordinary properties to promote the growth of skin cells. However, in the clinical application of these cultures for the treatment of skin lesions is with the disadvantage that only clinical grade materials should be used. The latter exist and are called Tissucol® and Beriplast® in the clinic is used as fibrin sealants in different surgical procedures. Found these products maintain the properties of cell growth promoting detected with the combination of Thrombin and Fibrinogen level in vitro research. However the above the cost of these products, given the complex microbiological analysis that require, makes them incompatible with the possibility of creating a commercial product containing fibrin, and cover large areas of damaged skin. Materials that are safe for human beings, free of animal components and risk of transmission of known and emerging diseases are also required. A product of autologous origin will always be more secure from the microbiological standpoint than one heterologous, since although this polluted, will return to the same host. On the other hand, from the immunological point of view, antibodies and other proteins present in it will be those already existing in the individual, eliminating the possibility of immune rejection. The present invention is directed to generate a fibrin gel for proliferation and cell conduction from the patient's own blood or blood compatible. The blood rate of 200 μl per cm of implant to prepare is taken using sodium citrate as an anticoagulant in a reason of 0.09 g per mL of blood. Plasma citrated is separated by centrifugation. The cells are then suspended again in this plasma. To form the gel either as isolated clot inside a porous matrix is to add a CaCl2 solution. The product thus obtained is of much lower cost than commercial “fibrin glue” or “fibrin sealant” and for being an autologous product, the risk of infection and immune rejection is avoided, and although it may be difficult to obtain large volumes of blood from a patient, this can be overcome using compatible blood. This system has been used to treat skin lesions with positive results. The application will be for purposes of cell growth in skin lesions implants, cartilage, osseous and gingival lesions among others. It can be anticipated that any application in the field of tissue engineering can use this technology.
The novelty lies in the incorporation of the cells into a fibrin clot using plasma instead of purified fibrin and thrombin. The fibrin glues contain both components and some others from blood, but they are no equivalent to plasma since they are purified. Also they incorporate additives, such as protease inhibitors that are not present in plasma.
All shown references are ways for generating the fibrin clot, but not ways for integrating cells in a fibrin clot with purposes of tissue engineering.
There are many publications that support the potential of growing cells in fibrin clots. For this, commercial fibrinogen and thrombin are acquired or commercial preparations known as fibrin glues are used, the most popular being Tissucol (Baxter) or Beriplast (CSL Behring). These products allow the cells to be suspended in thrombin and then fibrinogen is added for making a cell containing clot. The problem is that for doing this with clinical and commercial purposes a lot of money should be expended because the products of clinical grade are very expensive. It is also difficult to be 100% sure that the clot will not transmit any virus or unknown microorganism for which there are no screening tests with appropriate sensitivity.
Thus, the idea of generating fibrin for cell growth from the patient's own blood to form the gel, either as an isolated clot or within a porous matrix, arises.
According to the above, the product generated in the present invention has as main advantages that it has much lower cost than the commercial “fibrin glues” and for being and autologous product, it eliminates the risk of infection and immune rejection. On the other hand, the disadvantage that it can be difficult to obtain large volumes of blood from a compromised patient, is solved using compatible blood.
For the support of the invention, a number of clinical trials have been developed in which the cells are incorporate into a porous matrix by the use of autologous blood. This system has been used to treat skin lesions with positive results.
An object of the present invention is the application for the purpose of cell growth in implants for skin, cartilage, gingival and bone lesions, among others.
The patent literature shows homologous products that differ of the invention in fundamental aspects. A description of the most relevant known in patents follows:
Beretta & Grippi in the patent applications numbers US 2009258056 and WO 2007021344 and Beretta & Lodi in patent number U.S. Pat. No. 6,368,298, developed methods for regenerating tissue in a living organism, which include contacting the affected area with a net of solid fibrin containing platelets that release growth factors. The gel is a-cellular and contains platelets derived from autologous blood.
Similarly, Baugh & Lim in patent applications numbers US 2005152886 and US 2002159985, and in the patent number U.S. Pat. No. 6,444,228, and Hirsch & Johnston in patent number EP0820314, describe methods for obtaining autologous fibrin sealant, which does not contain incorporated cells but only platelets. The patent number U.S. Pat. No. 5,185,001 by Galanakis D., discloses a kit which contains all the elements to produce a fibrin clot from autologous plasma, in order to use it to induce hemostasis (clotting in a wound), but no reports the inclusion of cells in it.
Moreover, in the Chilean patent application CL 1439-2002, obtaining of a dermal substitute from plasma, using calcium chloride during gelation is described, but just as same as previously disclosed inventions, the cell component from a culture is not incorporated. It is mentioned that it allows the invasion by cells and blood vessels, but it refers to those of the wound bed and not exogenously provided.
Additionally, there is a significant amount of scientific articles, as well as some patents already mentioned, pursuing the research of the same technical problem. However, they are all ways to generate the fibrin clot and not ways for integrating the cells into a fibrin clot with tissue engineering purposes.
The examples below indicated, are incorporated by way of illustration only, to facilitate understanding of the invention, and not meant to limit in any way the scope of the requested claims.
Human fibroblasts are obtained from foreskins of surgeries due to phimosis in children under 6 years. The biopsy is washed in a sterile Petri dish with 5 ml of phosphate buffered saline solution, 0.1 M pH 7.4 (PBS) (GIBCO®), containing a mixture of penicillin/streptomycin antibiotics 100 U/ml/100 Invitrogen®) and amphotericin B 250 μg/ml (Fungizone®, Invitrogen). To obtain separate fibroblasts and keratinocytes, the sample is incubated in trypsin 0.5%, 5.3 mM EDTA (GIBCO®) or 30 minutes at 37° C. in incubator Thermo Forma®. Subsequently, using sterile forceps, the dermis and the epidermis are mechanically separated. The dermis is treated for 20 min with collagenase type I (Invitrogen®) 2 mg/ml at 37° C. Then, the obtained cell suspension is centrifuged and the precipitated obtained is washed with PBS to remove the enzyme. Its cell viability is determined by stain exclusion with trypan blue. This is accomplished by microscopic examination of an aliquot of cell suspension mixed with trypan blue stain in a 1:1 ratio. Then this suspension is placed in a Neubauer chamber and viable cells are counted by observation in inverted microscope Lieder®. Human fibroblasts obtained after enzymatic digestion, are re-suspended in Eagle culture medium and modified by Dulbecco (DMEM, GIBCO®) supplemented with 10% of fetal bovine serum ((INVITROGEN®), 50% Ham F12 medium (GIBCO®) and 10 ml/ml Biomyc 1 (Biological Industries®) in culture flasks T-25 (Falcon®), that are incubated in an incubator thermoformed, in a humidified atmosphere with 5% CO2 and a temperature of 37° C. 50% medium F12 of Ham (GIBCO®) and 10 μl\ml of Biomyc 1 (Biological Industries®) in culture flasks T-25 (Falcone), that are incubated in an incubator ThermoForma, in a humidified atmosphere with 5% of CO, and a temperature of 37° C.
Obtaining the plasma. The plasma is separated from whole blood collected into tubes containing sodium citrate at 3.2%, centrifuged at 3000 rpm for 5 minutes. The separated plasma in sterile conditions under bio-safety hood, stored at −20° C. until use.
Clotting test by visual observation: Initial tests of clotting, are performed in microcentrifuge tubes by testing various concentrations of calcium, named formulations (F). For this, a constant volume of citrated plasma 100 μl is used with different volumes of dilutions of CaCl2, to obtain different final concentrations of the same, generating 30 formulations as illustrated in Table 2:
After preparing the 30 formulations, these are incubated with the constant volume of plasma at 37° C. for 20 minutes. They are visually evaluated, whether is coagulation in each formulation. Furthermore, each clot is removed from the microcentrifuge tube with forceps, for evaluation of its mechanical strength, and thus the best 6 formulations are selected, based on the existence of clot and the greatest mechanical strength. For the determination of the clotting time a clot timer (BBL Fibrosystem) is used, which allows to determine, more precisely, the time in which the clot forms. This test is performed only in the 6 preselected formulations in the preliminary stage. The stability of the clot is determined in 6 clots of the formulations selected with culture medium (DMEM, GIBCO®), supplemented with 10% of fetal bovine serum (INVITROGEN®), 50% medium F12 of Ham (GIBCO®). Subsequently, it is incubated at 37° C. The degree of disintegration is evaluated visually on day 3 (
Preparation of base polymer: For the base polymer, the following solutions are prepared: gelatin 1% w/v, chitosan 2% w/v in acetic acid 1% v/v, and hyaluronic acid 0.01% w/v according to the following protocol:
The gelatin solution with chitosan and hyaluronic acid is mixed, and homogenized for 30 minutes with magnetic stirring at 50° C.
Subsequently, the mixture is poured into Petri dishes that are refrigerated at 4° C. until the formation of gel. Then, the gel is frozen slowly at −20° C. for 8 hours. Then, the polymer is carried to −80° C. for 8 hours.
The polymer is slowly immersed in liquid nitrogen for 3 minutes. Finally, it is lyophilized for 48 hours in Liobras LT01 lyophilizer.
Reticulation: For the base polymer reticulation, the following procedure is performed:
The lyophilisate is immersed in MES 50 mM (2-morpholinoethane sulphonic acid), for 30 minutes. Then, this solution is discarded and reticulant solution composed by MES 50 mM, EDC 30 mM (1-ethyl-[3,3-dimethylaminopropyl]carbodiimide) and NHS 8 mM (N-hydroxysuccinimide) are added. It is left to react for 2 hours.
Then it is washed with ethanol and frozen.
Once frozen, it is immersed in liquid nitrogen for 3 minutes.
Finally, it is lyophilized for 24 hours.
For this experiment, the technique is MTT [(3-4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] was used, which allows quantification of viable cells. This technique is based on the reduction of MTT, only by those metabolically active cells, in a compound called “formazan” a soluble purple blue crystal, which can be quantified by spectrophotometry at 540 nm.
MTT assay: Obtaining fibroblasts to this test is performed as follows:
Culture flasks T-75 (Falcon®) with fibroblasts are washed with PBS, and then treated with trypsin. The cell suspension is placed in a 50 ml disposable centrifuge tube, and centrifuged at 3000 rpm for 5 minutes. Then, the precipitate obtained is re-suspended in order to verify the number of viable cells.
Once realized the cell counting, the suspension is centrifuged again, and a portion of the cells are re-suspended in culture medium (DMEM, GIBCO®), supplemented with 10% of fetal bovine serum (INVITROGEN®), 50% of medium F12 of Ham (GIBCO®) and a mixture of penicillin/streptomycin antibiotics (100 U/ml/100 μg/ml), INVITROGEN®), and the other in plasma. This assay is performed in 96 flat bottom wells (Falcon®), where the proliferation of fibroblasts is evaluated, using different surfaces for its cultivation. The assay is performed from day 0 to day 3, i.e. at 0, 24, 48 and 72 hours, and each condition is evaluated in triplicate with its level control for each day. Also, only the two best previously selected formulations are considered for testing.
Detailed below is the MTT assay to examine the proliferation of fibroblasts on different surfaces:
Monolayer: human fibroblasts re-suspended in medium (5×106 cells per well), are cultured directly in the well with 150 μl of culture medium.
Polymer Matrix: the polymer matrix is placed in each well and the re-suspended human fibroblast are delivered in medium (5×106 cells per well). Culture medium is added until complete 150 μl.
Clot F17: 30 μl of plasma with humans fibroblasts are deposited (5×106 cells per well), and to this 45 μl of CaCl2 30 mM is added. Is incubated at 37° C. until the clot formation and culture medium is then added until complete 150 μl.
Clot F27: 30 μl of plasma with humans fibroblasts are deposited (5×106 cells per well), and to this 22.5 μl of CaCl2 50 mM is added. Is incubated at 37° C. until the clot formation, and the culture medium is then added until complete 150 μl.
SU with F17: on the polymer matrix, 30 μl of plasma with human fibroblasts (5×106 cells per well) and 45 μl of CaCl2 30 mM are deposited. Is incubated at 37° C. until clot formation, and then the culture medium is added until complete 150 μl.
SU with F27: on the polymeric matrix, 30 μl of plasma with human fibroblasts (5×106 cells per well), and 22.5 μl of 50 CaCl2 mM are deposited. Is incubated at 37° C. until clot formation, and then the culture medium is added until complete 150 μl.
MTT procedure: Once assembled the experiment with the different cell culture conditions, MTT assay for day 0 (0 hours) is performed to establish values of cell viability at the time of the beginning of the experiment. This procedure is subsequently repeated after 24, 48 and 72 hours. The procedure consists of:
Add 50 μl of MTT solution to each well, corresponding to the evaluation day and incubate at 37° C. for 4 hours.
Subsequently, each solution in the wells is removed and transferred into the corresponding microcentrifuge tubes for each sample.
150 μl of trypsin 10% is added to each well. The plate is again lead to the incubator at 37° C. for 1 hour.
The tubes are maintained at −20° C. until photometric quantification. To do this, microfuge tubes with samples of each day are thawed.
After thawing the tubes, these are agitated vigorously by vortexing.
Then, ultrasound is applied for 30 minutes.
The tubes are homogenized again by vortexing.
Ultrasound is reapplied for 30 minutes.
Subsequently, centrifuged at 14,000 rpm for 15 minutes.
Carefully, particle-free supernatants from each tube are extracted and 200 μl of each sample are deposited into a 96 wells plate.
Finally, the absorbance is read on plate reader ELISA SENSISCAN (MERK®) at 540 nm.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1870-2011 | Aug 2011 | CL | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CL2012/000039 | 8/3/2012 | WO | 00 | 4/10/2014 |
