CN118557804A - Tissue repair material and preparation method thereof - Google Patents

Abstract

The invention belongs to the field of medical repairing materials, and relates to a decellularized medical material for tissue repair and a preparation method thereof. The innovation point of the invention is that the connective tissue or organ of the breeding stock is used, which has stronger toughness and better stability than the material from commercial meat stock; at the same time, the trouble of crosslinking and the residue of chemical reagent are avoided. The cell-removing material prepared by the method has better stability and mechanical property.

Description

Tissue repair material and preparation method thereof

The invention relates to a tissue repair material, in particular to a cell-free tissue repair material and a preparation method thereof.

Background

Medical biomaterials for tissue repair are now being clinically transformed from traditional non-absorbable materials to new biomaterials that are natural, degradable, and capable of actively inducing tissue regeneration; based on the principle of tissue engineering, animal tissue is taken as a raw material, cells in the tissue are removed, a relatively complete extracellular matrix (Extracellular Matrix, ECM) is reserved, and the decellularized medical material prepared by taking the animal tissue as the raw material is used for tissue repair and regeneration, so that the decellularized medical material is a main development direction of surgical medicine in the future.

The decellularized ECM generally has a three-dimensional structure, contains various macromolecular substances such as collagen, elastin, glycosaminoglycan (including hyaluronic acid and the like), growth factors and the like, can provide proper places and microenvironments for the survival and growth of cells, and can participate in regulating the growth, phenotypic expression, shape formation, metabolism, migration, proliferation and differentiation of various cells so as to regulate the functions of tissues and organs. The cell components and other immunogenic components in animal tissues are removed, the effective components in other ECMs are reserved, and an ideal tissue repair material, which is called a tissue source decellularized medical material in the industry, can be developed; at present, a plurality of acellular matrix materials are successfully applied to the fields of dural injury, abdominal wall repair, burn, pelvic floor repair, tendon repair, nerve repair and the like.

The ideal tissue repair material should have the following conditions: ① Has the advantages of safety, no toxicity, and no infection. ② Has good tissue compatibility and no immune rejection reaction. ③ Good compactness and no permeability. ④ Has toughness and is easy to be firmly sewed. ⑤ Can promote tissue regeneration, and has no adhesion. ⑥ Convenient use, simple operation and easy disinfection and sterilization. ⑦ Has wide material sources and low cost. ⑧ Does not cause acute and chronic inflammatory reactions.

At present, a plurality of tissue repair materials are used, mainly heterogeneous acellular matrixes, the medical materials are also called tissue acellular materials, and the initial raw materials are mainly derived from tissues such as small intestinal submucosa, dermis, urinary bladder submucosa, stomach submucosa, pericardium, meninges, amniotic membrane, visceral membranes, peritoneum and the like of mammals such as pigs, cattle, sheep, horses and the like; through a series of processing steps, including important process steps such as decellularization; specific reagents are also used to further remove DNA, and to remove immunogenic components such as the alpha-Gal antigen. The acellular matrix scaffold (or patch) obtained by this method is capable of retaining, on the one hand, a three-dimensional structure, while also containing some important active ingredients. The ideal biological cell removal material prepared from the cell removal matrix has good biocompatibility, degradability and absorbability; mechanical strength suitability, no toxicity and no immunity; can provide ideal space support and proper micro (nutrition) environment for chemotaxis, attachment, proliferation and differentiation of host cells, and is beneficial to structural repair and functional reconstruction of target tissues. In clinical application, the decellularized material prepared from the porcine Small Intestine Submucosa (SIS) is a better and ideal tissue repair material which is currently accepted by the industry and academia, and the application is earlier and more than the dermal tissue (dermal tissue) source medical material. For decellularized medical biomaterials, the decellularization process is the first technical difficulty in preparing quality biomedical materials. It is directly related to the quality of the decellularized medical material, i.e., the effectiveness and safety of the decellularized medical material. If the decellularization is incomplete, the decellularized medical material still has animal-derived cells, and has stronger immunogenicity, and can cause immune rejection and inflammation, such as serous swelling and other adverse reactions easily occurring after patch implantation, and certain potential safety hazard exists; on the other hand, if the cells in the tissue are thoroughly removed, a reagent with a relatively strong cell removal mode is often used, or the cell removal solution and the tissue are repeatedly acted (such as soaking, shaking, pouring and the like) for a long time, so that the natural three-dimensional structural stability of the ECM is affected to different degrees, the functional domains of various growth factors in the ECM (such as fibroblast growth factors, transforming growth factors, vascular endothelial growth factors and the like) are damaged, and the loss of active ingredients (such as hyaluronic acid HA and the like) are caused.

The good cell removal process is to remove various cells in animal tissues as much as possible, maintain a complete three-dimensional porous structure of ECM and keep the activities of various cell growth factors and active ingredients in the ECM which can promote tissue regeneration as much as possible; therefore, the method not only needs to consider the intrinsic factors of animal source tissues, such as the thickness of the tissues and the cell density, but also needs to fully know the advantages and disadvantages of various cell removing agents and methods in advance, and comprehensively compare and balance, scientifically and reasonably select and carefully optimize the cell removing scheme on the basis of being familiar with the main technical parameters of each cell removing agent.

The cell removing method in the prior art mainly comprises the following steps: physical, chemical and digestive enzymatic methods, several methods are combined or combined in practical application:

1) Physical method cell removal: including stirring and vibrating, repeated freezing and thawing, ultrasonic treatment, high pressure or ultrahigh pressure, pouring and the like;

2) Cell removal by chemical method: detergents (also known as detergents or surfactants), acidic or basic solvents are required;

3) Digestive enzyme method cell removal: such as using trypsin, neutral protease, etc.

In the process of preparing the decellularized medical material, the most important step is how to effectively decellularize, and the existing methods for decellularizing in the preparation of patches are as follows:

Zhao Zijian patent No. 201811545271.X, entitled a decellularized vascular matrix and method for preparing same; the method comprises the steps of sequentially subjecting vascular tissues to hypotonic treatment, trypsin treatment, detergent treatment and nuclease treatment to prepare vascular matrixes; zhao Bo, 201710126703.2, entitled biological tissue matrix material, method of preparation and use thereof, wherein the decellularized fluid used in the method comprises trypsin and PBS solution, and the decellularized fluid further comprises EDTA, EDTA2Na or EDTA 4Na; performing cell removal by treatment with a cell removal liquid in a multi-frequency ultrasonic environment;

Li Huaqiong, 201810140944.7, entitled a method for removing cells from bone material and a method for preparing the same; the method comprises the steps of incubating Tris HCl solution, incubating trypsin solution, incubating Tris HCl solution containing Sodium Dodecyl Sulfate (SDS), washing PBS buffer containing ethylenediamine tetraacetic acid and protease inhibitor, incubating Tris HCl solution containing sodium dodecyl sulfate, performing enzymolysis, washing PBS buffer and the like;

Wang Wenjia, 201710864637.9, entitled dermal deep filler, a method for its preparation and use, comprising the steps of: taking the amnion after delivery as a raw material, carrying out cell removal treatment by trypsin combined with Triton X100, grinding into particle fragments by a high-speed grinding instrument under the condition of liquid nitrogen freezing, and carrying out cobalt 60 irradiation sterilization to obtain amnion particles; inoculating umbilical cord mesenchymal stem cells on amniotic membrane particles, performing joint culture for 8-10 h to obtain a stem cell amniotic membrane particle mixture, and then taking an isolated blood sample to prepare platelet-rich plasma, namely PRP; before use, adding PRP into the mixture of the stem cell amniotic membrane particles to obtain a mixture of the three particles of the stem cell amniotic membrane particles of PRP, namely the deep dermis filler;

Yang Shuai patent with application number 201710247775.2, named as medical acellular dermal matrix, is prepared by removing flesh from animal skin, sequentially sterilizing, cleaning, treating with hypertonic salt, and soaking in mixed solution of alkali solution and hydrogen peroxide; then soaking in trypsin solution for enzyme digestion treatment, and then using a cross-linking agent for cross-linking treatment of collagen;

Sun Xinjun patent application No. 200310110871.0, named as a heterogeneous decellularized bone matrix material and a preparation method, wherein the bone matrix material is prepared by subjecting bones of pig ribs or limbs to physicochemical treatment, and removing heterogeneous proteins in cells and tissues after the bone is subjected to the action of one of preferred proteolytic enzymes combined with Triton-X100.

The non-patent documents are: xiaofeng Ye et al, "Impact ofdecellularization on porcine myocardium as scaffold for tissue engineeredheart tissue" published in 2016; in the report, trypsin reagent is adopted to remove cells in tissues, trypsin is serine proteinase extracted from pancreas of cattle, sheep and pigs, is endopeptidase, can cut off carboxyl side of lysine and arginine residues in polypeptide chains, namely mainly recognizes peptide chains adjacent to arginine and lysine, and hydrolyzes and cuts the peptide chains into independent small peptide chains at the site; the digestion and degradation effects of trypsin on proteins are not limited by the variety type and source of the proteins, the trypsin has no pertinence and selectivity on the proteins, and the trypsin has universality on corresponding enzyme cleavage sites on various proteins; therefore, when trypsin is used for cell removal, collagen, elastin, mucin and other protein components such as fibronectin and laminin in tissues are inevitably degraded and destroyed, and trypsin should be carefully used, rarely used or not used in the cell removal process because trypsin has certain degradation and destruction effects on structural proteins and functional proteins in extracellular matrix.

Trypsin also has a relatively slow cell removal effect compared to detergents, and is more damaging to collagen and elastin in the extracellular matrix, i.e. causes damage to the ultrastructure of the extracellular matrix, leading directly to a decrease in mechanical properties of the ECM.

The content of the effective active ingredients in the cell removal material is greatly different due to different cell removal modes (mechanical method, chemical method and enzymatic method) and different cell removal reagents; thomasW.Gilbert et al J Biomaterials 27 (2006) 3677 table 1 provides a detailed comparison of different decellularization methods and decellularization reagents. The detergent is synthesized chemically or semisynthetically, has strong detergency and good cell removing effect; but is prone to loss of active components such as GAGs, HA in the ECM, and to varying degrees of damage and disruption to the natural steric structure and integrity of the ECM. Reagents and methods used for tissue decellularization are compared to table 1 below:

Often the degree of decellularization and the degree of retention of the active factors are inversely related, with the loss of the active factors being accompanied to varying degrees at the same time as the decellularization; if the dosage of the reagent is too small, more cell components can remain, which is easy to cause the immune rejection reaction of the organism and possibly accompanied with adverse reactions such as adhesion of local tissues or calcification of tissue wrapping; if the cells are removed, the cells can be removed cleanly by using a large-dose reagent through long-time treatment, but the ultramicro three-dimensional structure can be damaged at the same time; therefore, how to thoroughly remove the cell and DNA components and simultaneously reduce the damage and injury to the aspects of the ultramicro three-dimensional structure, the bioactive components, the mechanical properties and the like as much as possible is the focus of the current research for preparing the acellular matrix.

In order to solve the above problems, there is an urgent need to adopt a decellularization method without side effects or with very low side effects in order to prepare a decellularized medical material more efficiently and safely.

Another technical difficulty that accompanies this is: because in order to ensure the high safety of medical materials, the production enterprises generally require thorough cell removal in actual operation, the cell removal process is very harsh and even excessive, which can cause more loss of active ingredients in the ECM and damage to the three-dimensional structure of the ECM to different degrees; thereby weakening the mechanical properties of the decellularized material.

In practical applications, such decellularized medical materials often do not achieve the mechanical strength required for tissue repair at certain specific sites (e.g., hernias, tendons, etc.) in terms of mechanical properties; therefore, in the process of preparing the decellularized medical material, the following two methods are commonly adopted at present to enhance the mechanical properties of the decellularized medical biological material so as to meet the technical requirements of good toughness and high tensile strength. Firstly, it is: in the chemical (biochemical) method, various cross-linking agents such as epoxide, glutaraldehyde, genipin, glutamine transaminase (TGase) and the like are used in the preparation process of the cell removal material so as to improve the biomechanical property of the cell removal patch. For example, the biological patch product of Guangdong Guanhao uses bovine pericardium as a raw material, and is used for tissue repair products in dura mater operation and thoracoabdominal operation repair, and a cross-linking process is adopted in preparation to ensure that the product has certain mechanical properties. For example, the hernia patch product of Beijing qingyuan Wei industry is made of dermis and also is prepared by adopting a cell removal process; in order to ensure adequate mechanical properties of the finished product, a crosslinking process is also used. For another example, applicant is the responsible company of the leifu biotechnology limited in beijing , the patent of publication No. CN108261565 of inventor No. Sun Jihuang, and the patent of publication No. CN105999411 of inventor No. Liu Bowen, both of which are optimized in the cell removal (decellularization) process, and both of which use a crosslinking agent in order to meet the mechanical requirements of the decellularized material. The cross-linking degree is difficult to control, the residual cross-linking agent has potential cytotoxicity, and the cross-linking type decellularized material has the advantages of good mechanical property, strong resistance, difficult degradation or slow degradation, degradation and tissue regeneration can not be synchronously matched, and is easy to cause adverse reactions such as fibrosis, chronic inflammation and the like; the tissue repair material of Guangdong company is processed by pericardium or pleura through epoxy crosslinking treatment and the like, and the decellularized material has enough mechanical strength but slower degradation, can cause fibrosis and chronic inflammatory reaction, and has risks of adhesion with skull or brain tissue and the like; and because of its hardness, it cannot perfectly adhere to the brain tissue surface.

Secondly, it is: the physical method, such as transverse or longitudinal single-layer sheets and overlapping multi-layer crossed parts, is that the inventor is Zhou Ninghui, the patent with publication number of CN106039404A, and the whole decellularized material prepared by the method has improved tensile strength, but more decellularized raw materials are used, on one hand, the raw materials are wasted, and on the other hand, the decellularized material is too thick, so that the whole time for completely degrading the decellularized material is prolonged, and the repair and functional rehabilitation of target tissues are affected. In addition, the patent CN109248339A disclosed by the inventor has the innovation points that the cell-removing raw material is prepared into a thin strip shape and then twisted into a wire, and then the cell-removing material is prepared in a braiding mode; the stretching strength of the cell-free material prepared by braiding is obviously improved, but the cell-free material is required to be prepared into thin strips firstly and then prepared by braiding, so that the cell-free material is difficult in technology and complicated in step.

Disclosure of Invention

The invention aims at solving the problems that the partial decellularized medical material has poor stability, is easy to degrade or is fast to degrade when being used in medical clinic, cannot effectively meet the actual requirements in medical clinic, for example, a biological patch is fast to degrade, and is not beneficial to being used as a reliable and stable cell scaffold to assist the generation of a new tissue structure at a damaged part and the recovery of functions.

The second purpose of the invention is to solve the problems that the decellularized medical material has poor and weak mechanical properties in the process of repairing the tissue injury of the part, is easy to break, tear or other accidents in actual clinical use, cannot effectively meet the actual requirements of medical clinic, for example, a hernia patch, a tendon patch, a rotator cuff patch, a dura mater patch and the like with poor mechanical properties can generate swelling, pulling or cracking, and serious effusion leakage can occur.

The third object of the present invention is to avoid the possible residual cross-linking agent and the possible toxicity to host cells in the preparation of medical materials using cross-linking agents; meanwhile, the fine weaving reprocessing which is required to be performed with high effort to enhance the mechanical strength of the decellularized medical material is avoided.

The fourth object of the present invention is to increase the content of tissue repair promoting components such as active components (e.g., HA) in decellularized medical materials, which is more advantageous for chemotaxis, adhesion, proliferation, maturation and differentiation of host cells.

The fifth object of the present invention is to improve the imitation and integrity of ECM three-dimensional structures in decellularized medical materials, so as to facilitate early and better induction of repair regeneration and functional comprehensive rehabilitation of damaged tissue structures.

The method aims at avoiding the problems that in the cell removal process, more active ingredients in the ECM are lost, and the three-dimensional structure of the ECM is damaged and destroyed, so that the stability of the product is poor, the mechanical property is weak, the regeneration capacity of the induced host tissue is insufficient after the use, and meanwhile, the method also comprises the steps of avoiding possible residues of the detergents and potential damage to the host cells.

In order to effectively solve the technical problems listed above, the above-mentioned objects are achieved; the inventor tightly surrounds the key technical characteristics of biomechanical strength, stability, effective active ingredient content, integrity of an ECM three-dimensional structure and the like of the acellular medical biomaterial, and solves the technical problems of insufficient stability, weak mechanical property, more loss of effective active ingredient, less storage quantity and more damage to the ECM structure of the acellular medical biomaterial skillfully and conveniently by continuously and massively reading professional documents at home and abroad, combining deep research and careful analysis of academic theory by teams and adding the rich working experience of team members overseas for many years.

In order to achieve the object of the present invention, in one aspect, the present invention provides a medical decellularized biological material, characterized in that the decellularized biological material contains a tissue, an organ or a combination thereof of a seed animal;

further, the decellularized reagent mainly comprises saponin;

Further, the breeding stock is a sow, a cow, a ewe, a mare, a camel, a donkey and a mule which are produced by a warp;

Further, the breeding stock is a healthy eliminated sow and cow;

Further, the tissue of the breeding stock is one or more of small intestine submucosa, bladder submucosa, stomach submucosa, dermis matrix, pericardium, meninges, amniotic membrane, visceral membrane, peritoneum and bladder membrane;

Further, the organs of the breeding stock are one or a combination of a plurality of liver, pancreas, heart, kidney, lung and trachea; on the other hand, the invention provides a preparation method of the medical acellular biological material, which is characterized in that the acellular reagent in the acellular process mainly comprises a plant source nonionic surfactant;

Further, the decellularizing agent is one of plant-derived pentacyclic triterpene saponin and steroid saponin or a combination thereof;

Further, the decellularization reagent is one of Quil-A and tea saponin or a combination thereof;

further, the effective working concentration weight ratio of the decellularizing reagent is 0.05-1%, the action time with the patch raw material is 10-60 minutes each time, and the action temperature is 4-15 ℃;

furthermore, the raw materials of the preparation method of the medical acellular biological material mainly comprise tissues, organs or a composition of the tissues, organs or the composition of the tissues and the organs of the breeding stock.

The main innovation point of the invention is that the tissue or organ of the breeding stock is selected as the raw material for cell removal, rather than the commercial meat stock. Since the existing raw materials for cell removal of animal sources, such as small intestine, dermis, pericardium, peritoneum, pleura, tendon, etc., the freshly slaughtered animal tissues used are all commercial pork pigs, beef cattle, mutton sheep just coming out of the fence; for example, commercial pork pigs to be slaughtered in a large-scale slaughter house, most of which are just slaughtered, are mainly breeds of external ternary hybridization such as Duchesness and the like, and also have some internal ternary pork breeds; the weight of the slaughtered pork pig is 90-120 kg; the feeding day age is 5-6 months, and the weight and the number of days of the pigs going out from the fence are changed according to seasons and pig price quotations.

In order to achieve the aim of the product of the invention, namely to solve the technical problems of poor stability and insufficient mechanical properties of the tissue repair material, the inventor adopts a completely different technical idea from the prior art, and neither does the technical idea of stopping at the physical method during processing the cell removal material, such as the mode of overlapping (longitudinal/transverse/oblique), the overlapping ratio, the number of layers of overlapping, the thickness of raw wires used during braiding, the modes of plain weave, twill weave, twisting weave and the like, nor the optimization of the technical parameters of variety selection, proportioning, concentration improvement, crosslinking time and the like of stopping at the crosslinking agent.

In order to achieve the aim of the invention, the inventor adopts a new technical scheme, namely, the raw materials are grasped from the first step of preparing the cell-removing biological cell-removing material, and the raw materials are taken as technical breakthrough points or points of force for intensive research from the original animal sources, and the main innovation point of the product is that the raw materials of animal tissues of the cell-removing material are selected, so that the commercial-grade meat animals used at present are not selected, but breeding animals, preferably, the breeding animals are selected, and most preferably, sows are eliminated healthily due to the reduction of reproductive performance; the inventor in the first line of deep pig raising, after fully communicating with the responsible person in a large pig farm, according to the described raising mode (usually self-breeding and autotrophic) and raising period (pigs come out from the fence for 5 to 6 months and weigh about 90 to 120 kg), sows are gradually eliminated after eight to ten fetuses due to reduced reproductive performance, at this time, the inventor detects the actual situation that the mechanical performance difference of two cell-removing materials prepared from the eliminated sow SIS and the pig SIS through analysis and comparison, and discovers that the tensile strength (namely toughness) of the cell-removing materials of the sow SIS is significantly higher than that of the commercial pig SIS, and meanwhile, the toughness (tensile strength) of the cell-removing materials of the eliminated sow SIS from the eliminated sow SIS is better than that of the cell-removing materials of the post-production sow SIS with less fetuses, and comprehensively compares, and the low-cost eliminated sows are preferred as cell-removing raw materials.

The preferred tissue site of the product is Small Intestine Submucosa (SIS), and the secondary choice is connective tissue such as dermis, bladder, pericardium, peritoneum, other visceral membranes and the like, and cell-removing materials prepared by removing cells from the connective tissue generally contain similar natural three-dimensional structures, but compared with other tissue sites, the composition of the Small Intestine Submucosa (SIS) mainly contains I-type fiber collagen and also contains III, IV, VI-type collagen, particularly contains important IV-type collagen, and has obvious promotion effect on the formation of new blood vessels and basal membranes.

In addition, according to the papers published by STEPHENF. BADYLAK doctor, the degradation products of SIS decellularized material have strong antibacterial activity and function similar to defensins (porcinedefensin, pBD-1); the article Extracellular Matrix Bioscaffolds for BuildingGastrointestinal Tissue by georges.humisey 2018 reports that SIS decellularized material degradation products also have the effect of inducing cell chemotaxis and mitosis.

In order to achieve the purposes of avoiding more loss of active ingredients (such as hyaluronic acid HA) in the ECM of the cell removing material, reducing damage to the three-dimensional structure of the ECM, and enhancing the stability and mechanical strength of the cell removing material. The preparation method of the invention has two innovation points, the first is: in this critical step of decellularization, natural agents of plant origin, such as nonionic surfactants, are used instead of chemical or semisynthetic detergents; no protease is used for achieving the purpose of cell removal; the second is: the raw materials for cell removal are connective tissues or organs of breeding stock, not tissues or organs of commercial meat stock.

The first innovation point of the method is that in the process of preparing the cell removing material, a plant source non-enzyme cell removing agent is used, and the plant source non-enzyme cell removing agent comprises natural non-ionic surfactant, such as plant source natural saponin; can effectively remove cells in tissues; the surfactant HAs strong cell removal mode, namely, the cell removal effect is thorough mainly by destroying lipid cell membranes and membranes of organelles, and the action mode is mild, so that the structure of the ECM is not obviously damaged, and the obvious loss of effective active ingredients in the ECM is not caused, more effective active ingredients in the ECM can be reserved, including various cell growth factors (such as FGF-2, VEGF and the like) and effective active ingredients (such as hyaluronic acid HA, heparin and the like), and further the cell chemotaxis, growth and tissue repair and regeneration are more favorably induced; the cell is destroyed by cell removal under the low-temperature condition, so that the degradation and destruction of ECM possibly caused by endogenous enzymes released after cell disruption can be greatly reduced and slowed down.

The saponin is also called as saponin or saponin in the invention; is a natural high-quality nonionic surfactant, has double functional groups, contains water-soluble groups and fat-soluble groups in the molecular structure, and is a natural cleaning agent or surfactant. The saponin can combine with cholesterol and lipid on cell membrane to destroy cell surface structure; in addition, the saponin is helpful for solubility, and can promote the dissolution of other components in water. The saponin is widely distributed in plants, particularly in Chinese medicinal materials (such as famous ginseng, pseudo-ginseng and other main active ingredients are all saponins), in recent years, the research on the saponin is continuously and widely carried out in the field of Chinese medicaments, for example, eighteen ginsenosides such as Rb1, rb2, rd, rc, re, rg1, rg2 and Rh1 are separated and purified from ginseng at present, and Rg1 and Rb1 are found to have the most medicinal activity; saponin compounds such as the vaccine adjuvant QS-21 known from the company ANTIGENIS in the United states of America have a monomer molecular weight of 1990. The saponin has been confirmed to have various pharmacological activities and biological activities, such as antibiosis, antiphlogosis, bidirectional immunoregulation, plasma cholesterol reduction, blood pressure reduction and other pharmacological actions; meanwhile, saponin is a very good natural surfactant, which can reduce the surface tension of liquid, so that people can also use the saponin as a foaming agent, an emulsifying agent, a flavoring agent and an antioxidant.

Saponins can be divided into two classes depending on the nature of the sapogenin bound to the sugar (hexose, pentose or uronic acid): triterpene saponins (triterpenoidal saponins, C27, triterpene linked to sugar chains via carbon-oxygen bonds) and steroid saponins (steroidal saponins, C30, steroid linked to sugar chains via carbon-oxygen bonds). The domestic cheap and good triterpene saponin is tea saponin (TeaSaponin) which is prepared by refining and extracting tea seed cake which is a byproduct of tea oil processing; more than 90% of tea saponin (HPLC grade purity) is easy to prepare and obtain, such as the invention patent of gold Germany, the application number of which is CN201610990197, the name of which is: a method for producing high-purity tea saponin; in addition, sapindaceae plant-derived saponin can be selected. Another preferred saponin is QuillajaSaponariaMolina plant extract, also known as Quil-A, CAS number 8047-15-2, available from a variety of commercial sources.

The decellularization reagent used in the invention, saponin, not only can select triterpene saponin or steroid saponin, but also can select the mixture of two kinds of saponin as the decellularization reagent; the amount of the cell removing agent is calculated by taking pure saponin (saponine) as an active ingredient, and the working concentration of the cell removing agent is 0.05% -1%, preferably 0.25% -0.5%.

In addition, since the mode of action of saponin is mild, there may be partial reversibility of the binding with cell membrane lipids; if the subsequent flushing liquid or soaking liquid has low saponin content or no saponin, the cell removal and debris removal effect can be reduced; therefore, the next washing liquid or soaking liquid after cell removal also needs to use a solution containing saponin; the saponin solution of original concentration can be selected to be washable or soaked so as to remove cells and cell fragments thereof more thoroughly.

The principle of the invention:

The acellular medical material has the advantages that the acellular raw material sources are tissues or organs of breeding stock, such as sows with several fetuses, and the feeding time is usually longer than 24 months; about 2.2 fetuses are produced by a normal sow every year, the average gestation period of the sow is 114 days, the nonpregnant period is 10-15 days, and the lactation period of the sow is 21-28 days; the elimination of sows is mainly due to the reduction of reproductive performance, such as less number of farrowing, more weaning, difficult hybridization or less milk, light weight of weaning litter and other factors, and the sows are usually eliminated, and the feeding month age of the sows reaches more than 40 months; compared with commercial pork pigs, the tissue and organ development of the produced sow in all aspects is very complete, and the real sense, full and complete maturity is realized in the indexes of sub-organ level and structure, mesoscopic and microscopic properties, histological strength and the like; further, from the molecular level and molecular structure, the degree of hydroxylation of the collagen is possibly high, the stability of the triple helix structure is better, the natural crosslinking degree between collagens is high, and the thermal stability is better; the macroscopic appearance of the material is that the mechanical property of connective tissue is better, and the prepared cell-removing material has high tensile strength.

The sources of the cell-free raw materials are commercial meat animals such as pork pigs, the common feeding period is short, the time is only 5-6 months, the weight of the slaughter is 90-120 kg, and compared with the weight and the size of the produced sow (including 180-200 kg of the obsolete sow), the weight of the commercial meat pig is obviously lighter, and the volume of the commercial meat pig is smaller; the raising time of commercial pork pigs is only 5-6 months when slaughtering and slaughtering, and the raising time of multiparous sows is at least more than 12 months (calculated by only producing a litter) and 40 months when the sows are eliminated; the feeding time is 2 to 8 times of that of commercial pork pigs; meanwhile, compared with the sow produced by the commercial pig, the tissue organs of the commercial pig are only formed on the surface of a primary scale, but the indexes such as internal structure, microscopic performance, histological strength and the like of the commercial pig are not really, thoroughly and fully mature, and further, the hydroxylation degree of collagen is possibly low, the stability of a triple helix structure is poor, the natural crosslinking degree among collagens is low and the thermal stability is poor from the aspects of molecular level and molecular structure; the macroscopic appearance is that the mechanical property of connective tissue is poor, the prepared decellularized material has low tensile strength.

In the method, a plant-derived nonionic surfactant such as plant-derived saponin, preferably triterpene saponin such as Quil-A and tea saponin is used as the cell removing reagent, and the cell removing mode of the reagent has strong pertinence, mainly by destroying lipid cell membranes and organelle membranes, has strong pertinence, does not damage ECM structures, and does not cause loss of active ingredients (such as hyaluronic acid) in ECM; compared with other chemical or semisynthetic detergents, the plant saponin has thorough cell removal effect, but the action mode of the plant saponin is unique and mild, and more effective components in ECM can be reserved by using the plant saponin for cell removal; compared with the cell removing material prepared by the chemical industry detergent cell removing, the cell removing material prepared by the method HAs obviously high content of Hyaluronic Acid (HA).

Compared with the prior art, the invention has the following remarkable advantages and beneficial effects:

1. The tissue repair material product takes the animal tissue as the raw material, and is not derived from commercial meat animals, so that the tissue repair material product has better natural crosslinking degree, and the decellularized material has good toughness, high tensile strength and difficult rupture, and can prevent cerebrospinal fluid from leaking;

2. the tissue repair material disclosed by the invention takes the small intestine submucosa as a main raw material, reserves the three-dimensional structure, various functional proteins, growth factors, hyaluronic acid and other components in an extracellular matrix, has a good tissue regeneration induction function, and can accelerate the growth of postoperative tissues and the reconstruction of functions;

3. The product of the invention has no residue of cross-linking agent and synthetic detergent, no potential cytotoxicity and no fibrosis and chronic inflammation;

4. The tissue repair material has the functions of inducing cells and blood vessels to grow in the three-dimensional structure of the ECM, and can gradually degrade the ECM when new tissues grow in the ECM, so that polypeptide components of degradation products have antibacterial performance, and inflammation and infection after implantation can be reduced; 5. in the method, the plant-derived nonionic surfactant is used as a cell removal reagent to replace chemical detergents, so that more loss of active ingredients in the ECM is avoided, the three-dimensional structure of the ECM is not damaged, and the mechanical property is not reduced. 6. Economically, the raw materials can be multiparous sows which are eliminated due to the reduced reproductive performance, and the price of the raw materials is obviously lower than that of commercial pork pigs; for the mass purchase of raw materials in the future, the use of the obsolete sow as the raw material can save great expense for enterprises, which is one of innovation points.

In order to achieve the object of the present invention, the present invention provides a medical decellularized biological material, which is characterized in that the decellularized biological material contains a tissue, an organ or a combination thereof of a seed animal;

further, the decellularized reagent mainly comprises saponin;

Further, the breeding stock is a sow, a cow, a ewe, a mare, a camel, a donkey and a mule which are produced by a warp;

Further, the breeding stock is a healthy eliminated sow and cow;

Further, the tissue of the breeding stock is one or more of small intestine submucosa, bladder submucosa, stomach submucosa, dermis matrix, pericardium, meninges, amniotic membrane, visceral membrane, peritoneum and bladder membrane;

Further, the organs of the breeding stock are one or a combination of a plurality of liver, pancreas, heart, kidney, lung and trachea; on the other hand, the invention provides a preparation method of the medical acellular biological material, which is characterized in that the acellular reagent in the acellular process mainly comprises a plant source nonionic surfactant;

Further, the decellularizing agent is one of plant-derived pentacyclic triterpene saponin and steroid saponin or a combination thereof;

Further, the decellularization reagent is one of Quil-A and tea saponin or a combination thereof;

further, the effective working concentration weight ratio of the decellularizing reagent is 0.05-1%, the action time with the patch raw material is 10-60 minutes each time, and the action temperature is 4-15 ℃;

furthermore, the raw materials of the preparation method of the medical acellular biological material mainly comprise tissues, organs or a composition of the tissues, organs or the composition of the tissues and the organs of the breeding stock.

The invention also provides a preparation method of the tissue repair material, which comprises the following steps:

1) Drawing materials and cleaning: collecting connective tissue of livestock, and cleaning thoroughly;

2) Pretreatment: mechanically scraping off non-connective tissue and discarding it; washing the target tissue, soaking in weak acid solution to obtain the cell-free material to be pretreated;

3) Pre-sterilization: soaking cell-free raw materials in a mixed solution containing peroxyacetic acid and ethanol under the conditions of ultrasound and room temperature, and sterilizing; ultrasonically cleaning with purified water;

4) Degreasing: soaking cell-free raw materials in ethanol solution under ultrasonic and normal temperature conditions, and then ultrasonically cleaning with water for injection;

5) Cell removal: soaking the cell-free raw material in a solution containing a plant-derived surfactant at a low temperature and under ultrasound; then soaking the cell-free raw material with a new plant source surfactant solution with the same concentration; the ratio of the decellularized raw material to the solution is 1:10 (W/V); washing the cell material with PBS-EDTA ultrasonic wave, wherein the ratio of the cell material to the solution is 1:10 (W/V); the cell removal can be repeated for 1 to 3 times according to the actual situation;

6) DNA and alpha-Gal antigen removal: soaking the cell-free raw material in the solution containing DNase at 36 ℃ for 15-40 minutes; soaking the cell-free raw material in the solution containing alpha-galactosidase for 15-40 min after cleaning;

7) Soaking the cell-free raw material in 10mMNaOH solution at normal temperature under ultrasonic conditions; ultrasonically cleaning to neutrality by using PBS;

8) The semi-finished product cell-removing material is made into a sheet shape, and is overlapped and fixed on a mould, and then the cell-removing material can be obtained through freeze drying, packaging and irradiation sterilization.

The further concrete operation method is as follows:

1. drawing materials and cleaning: taking small intestines of breeding animals, and fully cleaning;

2. Pretreatment: mechanically scraping off mucous membrane layer, muscular layer, serosa layer and lymph node of small intestine, and separating submucosa; washing the small intestine submucosa, soaking in acetic acid solution for 30-120 min, wherein the ratio of the small intestine submucosa to the acetic acid solution is 1:5-1:10; obtaining a raw material to be used for pretreatment;

3. Pre-sterilization: soaking cell-free raw materials in a mixed solution containing peroxyacetic acid and ethanol under the conditions of ultrasound and room temperature, and sterilizing; the concentration of the peracetic acid is 0.5-1.5%, the concentration of the ethanol is 15-25%, and the ratio of the cell removing raw material to the mixed aqueous solution is 1:5-1: 10, soaking time is 30-120 minutes; ultrasonically cleaning with purified water;

4. degreasing: soaking the cell-removing raw material with alcohol solution under ultrasonic and normal temperature conditions, wherein the concentration of the alcohol is 90-100%, the ratio of the cell-removing raw material to the alcohol is 1:5-1:10, and the normal temperature soaking time is 0.5-6 h; then using water for injection to carry out ultrasonic cleaning;

5. Cell removal: soaking cell-free raw material in solution containing plant saponin at 4-15deg.C under ultrasound for 10-60 min; the ratio of the decellularized raw material to the solution is 1:10 (W/V); then soaking the cell-free raw material for 5-60 minutes by using fresh saponin solution with the same concentration; soaking with PBS-EDTA for 10-60 min; repeating the cell removal for 1-3 times;

6. DNA and alpha-Gal antigen removal: soaking the cell-free raw material in the aqueous solution containing DNase at 36 ℃ for 15-40 minutes; cleaning, and soaking in alpha-galactosidase solution for 15-40 min;

7. soaking in 10mMNaOH aqueous solution under ultrasonic condition at room temperature; ultrasonically cleaning with PBS until the solution is neutral;

8. the semi-finished product cell-removing material is made into a sheet shape, and is fixed on a mould in a cross overlapping way, and the cell-removing material can be obtained through freeze drying, packaging and irradiation sterilization.

Further, in the above described decellularization process (step 5), the following technical parameters are preferable:

The effective saponin content in the saponin solution is 0.05-1% (W/W), the ratio of the cell removing raw material to the plant saponin solution is 1:5-1:10, and the plant saponin solution is soaked for 20-45 minutes at the low temperature of 4-10 ℃ under the ultrasonic condition; then soaking the cell-free raw material for 5-30 minutes by using fresh saponin solution with the same concentration; soaking with PBS-EDTA for 10-30 min; decellularization was repeated 1 time. Further, in the decellularization process (step 4), more preferably, the following technical parameters are:

The content of effective saponin in saponin solution is 0.25-0.5% (W/W), the ratio of cell removing raw material to plant saponin solution is 1:10, and soaking for 20-45 min at low temperature of 4deg.C under ultrasonic condition;

Further, the plant-derived surfactant is one or a combination of plant-derived steroid saponin and triterpene saponin;

Further, the plant-derived triterpene saponin is Quil-A source, tea saponin or their combination;

Further, the working concentration of the saponin is 0.25-0.5% (W/W), note that the effective working concentration is not calculated as commercial product; further, the soaking time required for cell removal of the saponin solution is 20-30 minutes; the operating temperature was 4 ℃.

The Chinese and English terms/nouns are understood by the following text descriptions unless otherwise specified; other terms are to be construed as having a meaning that is well within the level of ordinary skill in the art.

Chinese and English terms noun interpretation:

1) And (3) breeding animals: is aimed at commercial meat animals; the main purpose of the breeding is to breed young animals, not to breed as commercial meat animals for a short period (such as the pig breeding time is about 5-6 months); the main purpose of raising commercial meat animals is to grow meat for supply; the breeding stock comprises pig, cattle, sheep, horse, donkey, camel, and dog; the sires referred to in this patent are adult, capable of reproduction and fertility, and are usually kept for at least one year or more; for example, breeding pigs include sows and boars, where sows refer to adult sows that can reproduce, excluding young replacement gilts;

2) A female livestock of warp production: part of the breeding stock is at least one female stock; the backup female animals are mainly young female animals which have not been pregnant because the age or month of age has not been reached;

3) Female animals were eliminated: the feed belongs to a part of breeding stock; in a farm, a manager considers that when the reproductive performance of female animals is reduced to a certain degree, such as small number of farrowing, low milk yield, light weight of weaning litter, and comprehensively considers various factors such as the utilization rate of the female animal, the price of the obsolete sow and the like; from the economic point of view, the value obtained by continuously breeding the female animals is considered to be obviously reduced, and the elimination of the female animals is more cost-effective; each farm has a certain difference in the evaluation standard of the eliminated female animals; for example, sows are usually gradually eliminated after delivery of 8 fetuses; slight differences exist among pigs of different varieties;

4) Tissue repair material, tissue regeneration material, biological patch, decellularized medical material, biological patch, biological scaffold, degradable patch, absorbable implant, and Bio-Mesh, bio-Patch, patch, bioscaffold, which are terms or terms of Chinese and English, are different on the surface, but the purpose and use are basically the same; unless specifically stated otherwise, the terms or meanings mentioned above are essentially identical or equivalent, except for differences in the habit of each person;

5) Small intestinal submucosa SIS (SMALL INTESTINAL Submucosa): the small intestine tissue comprises jejunum and ileum, and the rest part after removing small intestine mucosa layer, muscle layer and serosa layer contains collagen as main component, accounting for more than 80%;

6) Dermis (Dermis): a portion belonging to the skin, interposed between the epidermis and the subcutaneous layer; from mesoderm differentiation, there is no obvious boundary between the papillary layer and the reticular layer. The dermis is generally 1-2 mm thick; mainly comprises fibroblast and collagen fiber and matrix produced by the fibroblast, and has blood vessel, lymphatic vessel and nerve adhesion. The matrix is composed of a compound composed of mucopolysaccharide such as hyaluronic acid, chondroitin sulfate and the like and protein, is filled in gaps among collagen fibers and fiber bundles and among cells, and has hydrophilicity;

7) Decellularized biological material: removing cells from animal tissues or organs in a physicochemical mode to make the animal tissues or organs in a cell-free state without immune rejection reaction; can be used for repairing and regenerating medical tissues or organs; can also be used with stem cells for the direct or indirect treatment of certain diseases; the physical state can be solid single-layer or multi-layer sheet, powder, gel or liquid;

8) Extracellular matrix ECM (Extracellular Matrix): is a non-cellular component existing in all tissues and organs, which not only provides necessary physical support for the cellular tissues, but also provides proper places and microenvironments for normal physiological activities of various cells; but also plays an important lever regulation role in aspects of tissue morphogenesis, cell chemotaxis and differentiation, important physiological biochemistry, biomechanics and the like, thereby affecting or regulating the functions of tissues and organs. The function of 50% of the cells is determined by the external microenvironment created by the extracellular matrix.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and/or steps, of the type described herein and/or as would be apparent to one of skill in the art upon reading this disclosure. The term "about" or "approximately" refers to a range of values in a statistical sense, which may be within an order of magnitude, typically within 50% of the specified value or range, further within 20%, still more typically within 10%, even more typically within 5%. The terms "about" or "approximately" encompass permissible variations depending on the particular system under investigation, as would be readily recognized by one of skill in the art.

The principles and aspects of the present invention are described further below in connection with specific embodiments; it is to be understood that these examples are for illustrative purposes only and are not limiting to the inventive concept; the examples, in which the various processes and methods not described in detail are methods of common general knowledge used in the art, are not intended to limit the scope of the present invention in any way; it is also possible to omit through manipulations and methods used in the field of medical biomaterials; for example, the conception and the principle of the invention and the corresponding technical scheme are applied to the middle-large-scale farmed animals such as the ewes, the cows and the like of other mammals, and the farmed animals also comprise various sires, and obviously, the invention is also included in the protection scope of the invention.

Detailed Description

Example 1 (elimination of sow SIS+0.25% Saponin)

The preparation of the porcine small intestine submucosa tissue repair material comprises the following specific steps:

1) Drawing materials: selecting fresh small intestines of the obsolete sow as a cell removal raw material in a slaughter house; through deep knowledge, the eliminated binary hybrid sow has the common gestation age of more than 8, and is nonpregnant with the weight of about 180 kg; raising for more than 40 months;

2) Pretreatment: removing mucous membrane layer, muscle layer, serosa layer and lymph node of small intestine of pig by physical scraping method, separating out mucous membrane layer, soaking in 0.5% acetic acid solution for 30 min at a ratio of small intestine to acetic acid solution of 1:5, soaking in purified water for 3 times to obtain biological cell removing material, namely small intestine mucous membrane layer, hereinafter abbreviated as SIS material;

3) And (3) disinfection: the mixed solution containing 1.0% of peracetic acid and 15% of ethanol is used, the ratio of SIS material to the mixed aqueous solution is 1:10, and the SIS material is soaked for 100 minutes at room temperature under ultrasonic conditions for sterilization. Then using purified water to ultrasonically clean for 3 times;

4) Degreasing: using 90% ethanol, wherein the ratio of SIS raw material to ethanol is 1:10, and soaking for 2 hours at normal temperature under ultrasonic condition; then ultrasonic cleaning is carried out for 3 times by using water for injection;

5) Cell removal: using a solution containing 0.25% saponin (from Quil-a, working concentration calculated as pure saponin content), the decellularized material was soaked for 30 min at 4 ℃ and under ultrasound; then washing the cell-removing raw material for 10 minutes by using a saponin solution with the same concentration of 0.5%; then soaking the cell-removing material in PBS-EDTA solution for 20 minutes; repeating the step of removing cells once for about 120 minutes;

6) DNA and alpha-Gal antigen removal: using an aqueous solution containing 5U/ml DNase, wherein the ratio of SIS material to DNase solution is 1:5, and soaking for 20 minutes at 37 ℃ under ultrasonic conditions; then washed 3 times with PBS; using an aqueous solution containing 5U/ml of alpha-galactosidase, wherein the ratio of SIS material to alpha-galactosidase solution is 1:5, and soaking for 20 minutes at 30 ℃ under ultrasonic conditions; then rinsing with PBS solution;

7) Using 10mM NaOH aqueous solution, wherein the ratio of SIS material to NaOH solution is 1:20, and soaking for 50 minutes at normal temperature under ultrasonic condition; then using PBS to ultrasonically clean until the solution is neutral;

8) Shaping, freeze-drying and sterilizing: and (3) longitudinally and transversely crossing the acellular lamellar raw materials, overlapping and fixing the acellular lamellar raw materials on a die, freeze-drying, packaging and finally sterilizing.

Example 2 (commercial pig SIS from the fence +0.25% Saponin)

The cell-free raw material in the embodiment is small intestine tissue of commercial pork pig, and the preparation method comprises the steps of pretreatment of small intestine tissue of pig, disinfection, degreasing, cell removal, DNA removal, alpha-Gal antigen removal, freeze drying, sterilization and the like, which are completely the same as the embodiment I; the difference is only on the animal month age, in this example, fresh small intestine from slaughter house commercial pork pig was selected as decellularized raw material; as known, most commercial pork pigs are of external ternary varieties, such as Du Changda and the like, and the weight of the commercial pork pigs is 90-110 kg; the day of raising is about 150-180 days.

Example 3 (commercial pig SIS in the out-fence+0.25% SDS)

The cell-free raw material in the embodiment is small intestine tissue of commercial pork pig, and the preparation method comprises the steps of pretreatment of small intestine tissue of pig, disinfection, degreasing, DNA removal, alpha-Gal antigen removal, freeze drying, sterilization and the like, which are completely the same as those in the second embodiment; the difference is that in the fourth step of the selection of the decellularizing reagent, 0.25% SDS was used instead of the 0.25% saponin solution in the first embodiment.

Example 4 (obsolete sow dermis+0.5% tea saponin)

The steps of pretreatment of raw materials for cell removal, disinfection, degreasing, cell removal, DNA removal, alpha-Gal antigen removal, freeze drying, sterilization and the like are basically the same as those of the first embodiment;

But differs in the first, fifth and eighth steps.

The difference points of the first step are that the types of raw materials are different, the skin of healthy eliminated sows is selected as the raw material in the embodiment, and the material-taking part is the middle part area of the back of the pork pig; the skin is treated by an electric skin removing machine, the cuticle and the epidermis are removed, and the thickness of the dermis is made to be 0.5mm by adopting a mechanical method, so that the subsequent treatments are facilitated.

The second difference is that in the fifth step, 0.5% tea saponin is selected as the decellularizing agent, and the saponin source is not Quil-A.

The third difference is that in the eighth step, two pieces of cell-removed sheet raw material are taken out instead of four layers like SIS, which are overlapped and fixed on a mold, freeze-dried, packaged and finally sterilized.

Example 5 (commercial pork pig dermis +0.5% tea saponin)

The steps of pretreatment, disinfection, degreasing, cell removal, DNA removal, alpha-Gal antigen removal, freeze-drying, sterilization and the like of the cell-free raw material in this embodiment are basically the same as those of the fourth embodiment. The only difference is that in the first step, the type of material is different, the present example selects dermis from slaughterhouse healthy commercial pigs instead of dermis from obsolete sows as the initial material.

Example 6: and (5) detecting the mechanical properties of the tissue repair material.

The samples of tissue repair materials prepared in examples 1-5 were tested for tensile strength.

The method comprises the following steps: cutting the sample into 10mm width along two directions; after cutting, the test is carried out after the mixture is placed for 2 hours in an environment with the relative humidity of 40 to 60 percent and the temperature of 22 plus or minus 2 ℃. The clamp spacing was 25mm, the ends of the test specimen were fixed to the jaws of a tensile tester, and the tensile was performed at a speed of 100 mm/min, and the maximum force value at break was recorded.

The results are shown in Table 2 below:

group of	Cell-free material source and treatment mode	Toughness (tensile strength) unit: n
			Example 1	Eliminated sow SIS+0.25% saponin (from Quil-A)	53.0
Example 2	Commercial pork SIS+0.25% saponin (from Quil-A)	34.1
			Example 3	Commercial pork pig SIS+0.25% SDS	29.7
Example 4	Eliminating sow dermis and 0.5% tea saponin	41.5
			Example 5	Commercial pork pig dermis+0.5% tea saponin	32.6

Numerous simple variations or modifications and combinations of the present invention will be apparent to those of ordinary skill in the art in light of the above teachings; accordingly, certain details of the embodiments are set forth without departing from the spirit of the invention, which is defined by the following claims.

Claims (5)

1. A medical decellularized biological material comprising a tissue, organ or combination thereof of a seed animal.

2. The material of claim 1, wherein the sire is a multiparous sow, cow, ewe, mare, camel, donkey, mule.

3. The material of claim 1, wherein the sires are healthy replacement sows, cows.

4. The material of claim 1, wherein the animal's organ is one or more of liver, pancreas, heart, kidney, lung, trachea; the tissue of the breeding stock is one or more of small intestine submucosa, stomach submucosa, dermis, pericardium, visceral membrane, peritoneum and bladder membrane.

5. The method according to any one of claims 1 to 4, wherein the raw materials of the method mainly consist of the tissue, organ or combination thereof of the breeding stock.