Manufacturing process of biological membrane based on fibrin, denatured albumin and growth factors

Abstract

This invention relates to a process for manufacturing a biological membrane based on fibrin, denatured albumin, and growth factors. The present invention falls within the field of biotechnology and bioengineering, relating to a method for preparing biomaterials useful in clinical and healthcare applications. More specifically, the present biological membrane is a material based on fibrin, a fibrous protein involved in the blood coagulation cascade, obtained through the processing of blood serum by centrifugation, in association with albumin, a soluble protein of blood plasma, which is thermally and / or chemically denatured, and may be characterized as a bioactive, moldable, malleable, stable, sustained-release material and / or possessing the capacity to carry cells, biomolecules, and / or drugs.The biomaterial can thus be defined as a moldable biological membrane based on fibrin, obtained by fractionating blood serum, associated with denatured albumin, with regenerative potential and great versatility of application in sites that are candidates for tissue therapy.

Description

Descriptive Report of Invention Patent Manufacturing process for a biological membrane based on fibrin, denatured albumin, and growth factors. Field of Invention

[0001] The present invention is situated in the field of Biotechnology and Bioengineering, relating to a method for preparing biomaterials useful in clinical and healthcare applications. More specifically, the present biological membrane is a fibrin-based material, a fibrous protein involved in the blood coagulation cascade, obtained through the processing of blood serum in association with albumin, a soluble protein of blood plasma, which is denatured, and can be characterized as a bioactive, moldable, malleable, stable, sustained-release material and / or possessing the capacity to carry cells, biomolecules and / or drugs. The biomaterial is thus defined as a moldable biological membrane based on fibrin obtained by fractionating blood serum associated with denatured albumin, with regenerative potential for application in sites suitable for guided tissue therapy. Background of the Invention

[0002] Bioengineering has been extensively investigated as a tool for therapeutic strategies involving soft and bone tissues, including maxillofacial and orthopedic procedures. For this purpose, cells are associated with growth factors and scaffolds composed of biocompatible biomaterials capable of promoting tissue regeneration (Bottino MC, Thomas V, Schmidt G, Vohra YK, Chu TM, Kowolik MJ, et al. Recent advances in the development of GTR / GBR membranes for periodontal regeneration - A materials perspective. Dent Mater 2012;28:703-21). In this context, platelet-rich fibrin (PRF) membranes are configured as scaffolds. Petition 870190065649, dated 12 / 07 / 2019, page 16 / 35: autologous fibrin-based compounds serve as carriers of immune cells and platelets for therapeutic purposes due to the production and release of growth factors (Dohan DM, Choukroun J, Diss A, Dohan SL, Dohan AJ, Mouhyi J, et al. Platelet-rich fibrin (PRF): A second-generation platelet concentrate. Part III: Leukocyte activation: A new feature for platelet concentrates? Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101:e51-5.). However, their effectiveness as a barrier and scaffold for tissue regeneration, especially bone and cartilage tissues, is limited by their short residence time in the tissues.

[0003] Blood concentrates are a promising and clinically relevant method, offering an alternative source of minimally invasive autologous regeneration. Platelet aggregates can be generated from the patient's own peripheral blood and concentrated by centrifugation, capable of releasing different growth factors. Platelet-rich fibrin (PRF) constitutes the second generation of platelet concentrates (Dohan DM, Choukroun J, Diss A, Dohan SL, Dohan AJ, Mouhyi J, et al. Platelet-rich fibrin (PRF): A second-generation platelet concentrate. Part II: Platelet-related biologic features. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101:e45-50.), characterized as a simple and low-cost technique obtained from a single centrifugation without the addition of anticoagulants (Dohan Ehrenfest DM, de Peppo GM, Doglioli P, Sammartino G).Slow release of growth factors and thrombospondin-1 in Choukroun's platelet-rich fibrin (PRF): A gold standard to achieve for all surgical platelet concentrate technologies. Growth Factors 2009;27:63-9). During centrifugation, a solid fibrin matrix is ​​generated by platelet activation through interaction with the tube surface, culminating in the release of different growth factors. The resulting PRF matrix consists of a fibrin scaffold that includes platelets, leukocytes, and plasma proteins, with good documentation in the literature regarding its use as a membrane for tissue protection. Petição 870190065649, de 12 / 07 / 2019, pág. 17 / 35 moles e em regeneração óssea guiada (Bolukbasi N, Ersanli S, Keklikoglu N, Basegmez C, Ozdemir T. Sinus augmentation with platelet-rich fibrin in combination with bovine bone graft versus bovine bone graft in combination with collagen membrane. J Oral Implantol 2015;41:586-95.; Soydan SS, Uckan S. Management of bisphosphonate-related osteonecrosis of the jaw with a platelet-rich fibrin membrane: Technical report. J Oral Maxillofac Surg 2014;72:322-6.: Tajima N, Ohba S, Sawase T, Asahina I. Evaluation of sinus floor augmentation with simultaneous implant placement using platelet-rich fibrin as sole grafting material. Int J Oral Maxillofac Implants 2013;28:77-83).

[0004] In almost 20 years since its proposal, different protocols for producing PRF have been presented in the scientific literature with the aim of improving the biological properties of these autologous biomaterials (Dohan Ehrenfest DM, Andia I, Zumstein MA, Zhang CQ, Pinto NR, Bielecki T, et al. Classification of platelet concentrates (Platelet-rich plasma-PRP, platelet-rich fibrin-PRF) for topical and infiltrative use in orthopedic and sports medicine: Current consensus, clinical implications and perspectives. Muscles Ligaments Tendons J 2014;4:3-9). The protocols for producing platelet aggregates seek to increase cellularity within the fibrin mesh, attempting to improve the distribution and production of cytokines, mainly growth factors, at the receptor site.However, while these are often suitable for specific clinical applications, limitations in the stability of the fibrin mesh may compromise their applicability to surgical procedures that require stability for extended periods.

[0005] The use of serum albumin, an abundant and easily isolated protein in humans, in the field of tissue engineering is widely reported. It is obtained by precipitating blood plasma with high purity and homogeneity (Horváthy DB, Simon M, Schwarz CM, Masteling M, Vácz G, Hornyák I, et al. Serum albumin as a local therapeutic agent in cell therapy and tissue engineering. Biofactors 2017;43:315-30). In addition to providing a compatible structure for cell proliferation, biomaterials enriched with albumin Petition 870190065649, dated 12 / 07 / 2019, page 18 / 35 shows little reduction in their dimensions over time (dimensional stability), suggesting less degradation in vitro, since albumin can modulate the ultrastructure and permeability of the fibrin network, inducing thicker fibers and a dense nodular appearance (van Gelder JM, Nair CH, Dhall DP. Colloid determination of fibrin network permeability. Blood Coagul Fibrinolysis 1996;7:747-60.).

[0006] The present invention establishes a new autologous biomaterial manufacturing process consisting of applying centrifugation following specific parameters, capable of producing platelet-rich fibrin from blood aliquots processed in both horizontal and variable-angle rotor centrifuges and fixed-angle rotor centrifuges, in association with denatured serum albumin. The incorporation of albumin into the structure imparts new properties to the fibrin-based scaffold, configuring a potentially autologous, biocompatible material with greater stability, durability, and duration of action.

[0007] Searches of the patent literature have revealed documents particularly relevant to the present invention, which, however, present substantial differences in the processes of obtaining and treating the material, as well as in the essence of their claim. In this context, the present invention is a new alternative to the objects of the patent applications cited in this document, to the methods and equipment already used commercially. The background cited in the aforementioned applications, as well as the applications themselves, when applicable, constitute the state of the art closest to the present invention.

[0008] Application WO 2014114995A1 is entitled “Albumin tissue scaffold”. This document presents a tissue structure in which albumin-based material is obtained from animal tissue. The albumin scaffold is defined as a three-dimensional porous material, useful in tissue engineering to provide a structure for cell attachment, proliferation, and the formation of new tissues. According to the document, two approaches to synthesizing albumin polymers are demonstrated, Petition 870190065649, dated 12 / 07 / 2019, page 19 / 35 through the use of a chemical agent and crosslinking enzyme. However, this material, due to the absence of platelets and leukocytes, does not present biological inducers such as growth factors and chemokines, which promote biological events such as migration, adhesion, proliferation and cell differentiation.

[0009] US application RE43134E1 is entitled “Biocompatible denatured albumin lamina”. This document discloses a denatured albumin lamina, useful for repairing lesions in solid visceral organs. The lamina comprises human serum albumin, formed into a thin, flexible, and denatured sheet. The denatured lamina can be sterilized and stored until use. In addition, it can be impregnated with a variety of bioagents. Its mechanical properties make it especially suitable for use in tissue cauterization in solid visceral organs.In another aspect of this document, a method for manufacturing denatured albumin slides is provided. The method comprises placing a quantity of viscous albumin solution between two non-porous sheets, then spreading the albumin solution between the sheets to a selected and substantially uniform thickness. The albumin solution is thus formed and placed in a container, which is then evacuated. The solution is then heated by autoclaving or immersion in a water bath of at least 86°C. Denaturation of the retained albumin solution changes its state from a viscous liquid to a flexible solid. However, this material, due to the absence of platelets and leukocytes, does not contain biological inducers, growth factors, and / or chemokines.

[0010] Application WO 2018212758A1 is entitled “Platelet-rich fibrin as a carrier system for biomaterials and biomolecules”. This document discloses a method for preparing an isolated serum fraction of liquid PRF comprising centrifuging a whole blood sample to separate the erythrocytes; separating a liquid PRF sample from the whole blood sample without the addition of any additives; collecting the liquid PRF sample instantaneously by centrifugation performed at 20 to 950 G for 2 to 8 minutes; and collecting an isolated serum fraction containing the liquid PRF and ensuring its immediate effectiveness. Petition 870190065649, dated 12 / 07 / 2019, p. 20 / 35 using an application device. This process configures a liquid, injectable material of low stability without the presence of an albumin scaffold.

[0011] Application KR20100105356 is entitled “Bio filler producing method using plasma gel”. This document discloses a closed system for preparing filler material by filtering and heating human plasma at a temperature of 70-90°C for 9 minutes, followed by cooling in water at 4-12°C. This system produces a resorbable bio-gel applicable in aesthetic fillers, but not for therapeutic purposes, since the process results in the denaturation of all components present, implying cell death and loss of all growth factors.

[0012] The present invention differs from the aforementioned documents for several technical reasons. In one aspect, the present invention differs from the prior art due to the source of albumin, and regarding the methods of protein denaturation and platelet aggregate production, as well as the combination of these methods generating a new material with their combined properties. Other characteristics and technical effects of the invention also differ substantially from the prior art, as will be shown in the detailed description below.

[0013] Based on patent and non-patent literature, there is a clear need to search for new alternative solutions regarding tissue therapy to those already existing, in order to overcome the limitations of current methods and establish a new process for obtaining a scaffold based on fibrin rich in growth factors and cells, having its structure modified through the inclusion of denatured serum albumin, configuring a totally autologous, bioactive, moldable and stable material for prolonged periods. The present patent application reveals a solution to these problems. From what can be deduced from the literature researched, only the scientific publication related to this document previously presented by the inventors themselves was found (Mourão, Carlos Fernando de Almeida Barros; Gheno, Ezio; Lourenço, Emanuelle Stellet; Barbosa, Renata de Lima; Kurtzman, Gregorim; Petition 870190065649, dated 12 / 07 / 2019, p. 21 / 35 Javid, Kayvon; Mavropoulos, Elena; Benedicenti, Stefano; Calasans-Maia, Monica Diuana; Machado, Rafael Coutinho de Mello; Alves, Gutemberg Gomes. Characterization of a new membrane from concentrated growth factors associated with denatured albumin (Alb-Cgf) for clinical applications: A preliminary study. International Journal of Growth Factors and Stem Cells in Dentistry, 2018.), anticipating or suggesting the implementation of the protocol based on the teachings of the present invention, which, in the eyes of the inventors, possesses novelty and inventive activity compared to the state of the art. Summary of the Invention

[0014] The invention process provides for obtaining a dense fibrin membrane, exhibiting the trapping of platelets, leukocytes and plasma proteins in its structure, allowing the potential gradual release of growth factors.

[0015] The association with denatured serum albumin confers new properties to the platelet-rich fibrin (PRF) based scaffold, configuring a fully autologous, biocompatible, malleable, moldable material with greater durability and duration of action.

[0016] The invention provides several technical advantages: it provides an accessible means of reducing costs, since albumin is an abundant protein in the human body and is easily isolated from precipitation of blood plasma with high purity and homogeneity, and from the use of blood collection tubes without the addition of any substance, accessible in any medical-hospital supply distribution establishment.

[0017] In one embodiment, processing 9 mL blood aliquots at a force of 200 to 700 RCF (400G) for 8 to 12 minutes in a horizontal rotor centrifuge with variable angle (Figure 1), in association with an equivalent volume of denatured serum albumin in a glass container (Figure 2), can generate a material with characteristics suitable as a scaffold for tissue engineering, serving as support and allowing Petition 870190065649, dated 12 / 07 / 2019, page 22 / 35: migration, proliferation and cell differentiation, supporting angiogenesis and tissue neoformation, being subsequently reabsorbed, giving way to newly formed tissue.

[0018] In one embodiment, the material obtained from a similar centrifugation protocol, associated with denatured serum albumin from a 15-minute treatment in a rotary bath at 75°C for 10 minutes, can produce a biodegradable material for guided tissue regeneration procedures, especially guided bone regeneration, where a barrier is needed to prevent the penetration of soft tissue (connective) cells, this barrier being configured by the high density offered by the denatured albumin (Figure 3).

[0019] In another embodiment, when produced from the proposed protocol, and with the addition of different drugs, such as antibiotics, anti-inflammatories, and tissue regeneration inducers, incorporated during membrane processing, it can generate a bioabsorbable material with therapeutic properties in addition to those already expected for tissue repair.

[0020] In one embodiment, the proposed process can be carried out using any blood processing device characterized as a horizontal rotor centrifuge with variable angle, widely used in hospital settings such as hematology, and generally accessible in clinical and surgical procedures. Thus, a membrane with biological properties similar to those produced with commercial centrifuges dedicated exclusively to PRF production will be produced at low cost, characterized by a high presence of leukocytes and continuous release of growth factors (Figures 4 and 5). Brief Description of the Figures

[0021] Figure 1 shows details of an embodiment of the invention. A whole blood sample is processed in a fixed-angle horizontal rotor centrifuge. The platelet-poor plasma [PPP] portion is used for Petition 870190065649, dated 12 / 07 / 2019, page 23 / 35, to produce albumin gel through plasma activation in the APAG machine. This gel is mixed with the liquid phase of the growth factor concentrate and buffy coat (containing CD34+ cells) in a glass container. Contact with the glass contributes to producing a denser and more stable fibrin mesh in the form of a dense, yet malleable membrane (albumin concentrate and growth factors).

[0022] Figure 2 shows images of the final stage of production of the growth factor-rich albumin membrane, being manipulated in a glass container (a), forming a solid, yet malleable membrane (b).

[0023] Figure 3 shows scanning electron micrographs of the growth factor-rich albumin membrane, obtained at 15 kV, at different magnifications. (aeb) Very dense surface, with very evident deposition of a layer of denatured protein (ced), which is clearly coated with fibrin fibers (cee); f) The fibers that surround the trapped cells and platelets.

[0024] Figure 4 shows an estimate of cellularity in the growth factor-rich albumin membrane 7 days after production. Cell nuclei were evidenced by fluorescence microscopy after staining with 4',6-diamidino-2-phenylindole at the left edge (a), center (b) or right edge of the membrane (c) (images obtained with a χ²0 objective). (d) The average number of nucleated cells (n = 3) for each portion of the membrane. No statistical difference was found (P <0.05).

[0025] Figure 5 shows the evaluation of growth factor release by the growth factor-rich albumin membrane in cell culture medium, indicating that growth factors continue to be produced and released even one week after membrane production. Bars represent mean ± standard deviation of three biological replicates and three techniques. Detailed Description of the Invention Petition 870190065649, dated 12 / 07 / 2019, page 24 / 35

[0026] An object of the invention is a process comprising: Collecting samples of peripheral blood using tubes specifically designed for blood collection without the addition of any substance. After collection, at least one tube is subjected to a centrifugation process to obtain growth factors in liquid phase. Upon completion of this process, it is possible to observe the plasma and the remaining decanted blood material, containing red blood cells.

[0027] Albumin is obtained by collecting a small aliquot of the initial portion of plasma, which is called the platelet-poor portion [PPP]. The PPP is subjected to a process that results in the protein denaturation of the albumin present, which can be obtained through physical methods using a device that promotes heating and acts at a constant temperature to denature the albumin present, or chemical processes, such as heating at different temperatures and pH variations.

[0028] Subsequently, plasma is collected from the region near the fog zone so as not to homogenize the material. The albumin is placed in a container with the desired shape, in order to obtain the desired form for use. After waiting for the fibrin polymerization process, the membrane is obtained.

[0029] The invention process further comprises: processing blood aliquots in centrifuge-type equipment with a variable angle horizontal rotor.

[0030] The protocol presented in this invention has as one of its essential elements a specific method for denaturing serum albumin, using equipment that operates at a constant temperature, between 60°C and 200°C throughout the entire time employed, ensuring the efficient and uniform processing of the serological content.

[0031] Additionally, the invention protocol further comprises: processing for denaturation of serum albumin by both physical methods Petition 870190065649, dated 12 / 07 / 2019, page 25 / 35 and / or chemicals, examples of which are pH variation and heating methods.

[0032] In one embodiment, the protocol proposed by the invention results in a malleable, colloidal material that is resistant to cellular penetration and can be used as a biological barrier in guided tissue regeneration procedures, especially guided bone regeneration, which is widely explored in the field of Periodontics.

[0033] In one embodiment, the invention protocol further allows: the application of the fibrin and denatured albumin framework in procedures for the regeneration of hard tissues, such as filling dental alveoli and bone defects surrounded or not by bone walls, surgeries for lifting the floor of maxillary sinuses and in surgical procedures for correcting oroantral communications.

[0034] In one embodiment, the invention protocol further allows: the application of the fibrin and denatured albumin scaffold in procedures for soft tissue regeneration, such as root coverage, regeneration of intraoral surgical sites and periodontal plastic surgical procedures.

[0035] In one embodiment, the invention protocol further allows: the application of the fibrin and denatured albumin scaffold in auxiliary local hemostasis procedures.

[0036] In one embodiment, the invention protocol further comprises: the possibility of applying the resulting membrane in facial harmonization procedures.

[0037] In one embodiment, the invention protocol further comprises: the possibility of applying the resulting membrane in procedures within the field of orthopedics, as a regenerative device for cartilage and tendons.

[0038] The applicant, by filing this patent application with the competent / guarantor body, seeks and intends to: (i) name the inventors with respect to Petition 870190065649, dated 12 / 07 / 2019, pp. 26 / 35, their respective moral rights; (ii) unequivocally indicate that they possess the trade secret and hold any form of intellectual property derived therefrom and desired by the applicant; (iii) describe in detail the content of the secret, proving its existence in physical and legal terms; (iv) establish the relationship between the examples / concretizations and the inventive concept according to the applicant's understanding and context, to clearly demonstrate the scope of their protected and / or protectable intangible asset; (v) request and obtain the additional rights provided for in patents, if the applicant chooses to proceed with the administrative procedure to the end.

[0039] The eventual future publication of the patent application does not, in itself, constitute authorization for commercial use by third parties. Even if the content becomes part of the physical world accessible to third parties, the publication of the patent application under the terms of the law does not eliminate the legal status of secrecy, serving only and solely the spirit of the Law to: (i) unequivocally indicate its owner / holder and inventor(s); (ii) inform third parties of the existence of said industrial secret, the content for which patent protection is sought and the date of its filing, from which the term of patent exclusivity will begin; and (iii) assist in the technological and economic development of the country, based on the authorization of the use of the secret solely and exceptionally for the purposes of studies and / or development of new improvements, thereby avoiding parallel reinvestment by third parties in the development of the same asset.

[0040] It is hereby warned that any commercial use requires authorization from the owner / holder and that unauthorized use will result in penalties as provided by law. In this context, given the extensive detail with which the concept and examples have been revealed by the applicant, those skilled in the art may, without much effort, consider other ways of implementing the present invention in ways not identical to those merely exemplified above. However, such ways are or may be considered as within the scope of one or more of the appended claims. Petition 870190065649, dated 12 / 07 / 2019, page 27 / 35

Claims

1 / 2 Claims 1. Biological membrane manufacturing process CHARACTERIZED by the production of a platelet-rich fibrin network by centrifugation with the addition of denatured serum albumin, comprising the following steps: I. collection of peripheral blood samples; II. applying a centrifugation process to obtain growth factors in liquid phase; III. collecting a small aliquot of the initial portion of plasma; IV. subjecting the PPP to a protein denaturation process of the albumin present; V. collecting plasma from the region near the cloud zone so as not to homogenize the material; VI. placing the albumin in a container with the desired shape; VII. waiting for the fibrin polymerization process.

2. Manufacturing Process according to claim 1 CHARACTERIZED by employing a centrifuge with a variable angle horizontal rotor in the processing of blood aliquots that give rise to the biological membrane in fibrin and denatured albumin.

3. Manufacturing Process according to claim 1 CHARACTERIZED by comprising: the use of one or more processes that provide protein denaturation of the albumin to be incorporated into the fibrin structure, allowing uniform processing of the serological content.

4. Manufacturing process according to claim 1 CHARACTERIZED by generating a membrane with: the ability to carry drugs within the membrane structure and their subsequent local distribution.

5. Manufacturing Process according to claim 1 CHARACTERIZED by the denaturation of serum albumin occurring through equipment that operates at a constant temperature, between 60°C and 200°C throughout the entire time employed. Petition 870260072532, dated 07 / 21 / 2026, page 10 / 11 2 / 2 6. Manufacturing Process according to claim 1 CHARACTERIZED by the processing for denaturation of serum albumin occurring through physical and / or chemical methods, such as pH variation and heating methods. Petition 870260072532, dated 07 / 21 / 2026, p. 11 / 11

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