CN110732042A - skin biological materials - Google Patents

Abstract

The invention relates to skin repair biomaterial, which is used for repairing and treating acute or chronic skin wound healing, and contains decellularized young animal tissues, such as dermis of newborn calf and SIS of weaned piglet.

Description

skin biological materials

The invention belongs to the field of biological repair materials, and particularly relates to skin repair biological materials.

Background art:

the research shows that the wound repair involves coordination and integration of series of complex physiological and biological processes, including cell migration, proliferation, extracellular matrix deposition and remodeling, and complex multicellular processes, including fibroblasts, keratinocytes, endothelial cells, inflammatory cells and the like, wherein the regeneration and activity of host wound cells are strong and weak, and have an important role in success or failure of wound repair, the main cells can release various cytokines and regulate the wound repair process.

Skin wounds include, for example, chronic, acute, traumatic, subacute, and dehiscent wounds, partial cortical burns, ulcers (such as diabetic ulcers, pressure ulcers, or venous insufficiency ulcers), there are several products/methods for the surgical clinical treatment of skin wounds, such as allogeneic or autologous skin grafts, which are classified as autologous skin grafts and xenogeneic skin grafts, although highly safe and less responsive to rejection, but often require creation of a second wound to provide a graft, which poses more pain to patients, and the latter risks immunity and disease transmission, the second product being a collagen sponge product prepared by enzymatic hydrolysis and purification of animal tissue as a raw material to produce highly pure collagen, which is then processed, although retaining the triple helical structure of collagen, but removing the natural spatial structure of membranous tissue, which is not conducive to regeneration and repair of skin tissue, and the sponge product is too fast in collagen degradation, cannot be synchronized with tissue regeneration, and although it has been used in clinical applications, but has great limitations, and the third product being a Acellular dermal patch, such as an Acellular dermal matrix (Acrill dermal matrix), which is the most widely used in the research of dermal matrix removal of dermal cell removal and dermal wound repair, and dermal cell replacement of dermal cell removal of dermal tissue, which was first reported by the first published studies of the biological dermal wound repair method for the removal of dermal tissue repair.

The patent documents of the existing skin patches retrieved are as follows:

a skin sticking patch with CN103272272A natural holes for treating skin diseases is prepared from pig skin or ox skin through removing the bristles from external surface, removing the skin bristles from skin to corium layer by 0.2-1.0mm, taking the skin tissue with 0.5-2.0mm thickness, immersing in surfactant solution, immersing in alkali solution, immersing in PBS buffer solution, immersing in radiation protecting reagent solution (pH5.8-7.2), freeze drying and radiation sterilizing.

The preparation method of CN107412867A heterogenous acellular dermal matrixes comprises the following steps of taking Bama miniature pig skin as a skin source, reversely taking a leather drum into an acellular dermal sheet, carrying out treatment such as peeling, acellular treatment and the like to obtain the acellular dermal, reacting the acellular dermal with carboxymethyl chitosan to prepare the acellular dermal matrix in step , and finally carrying out treatment by a softening agent and a humectant to make the prepared acellular dermal matrix closer to the natural state of human skin and improve the compliance of the use of the acellular dermal matrix.

The defects of the prior art are that in order to completely remove (exfoliate) cells, the used animal dermis is mostly from commercial livestock (pigs) which reach the market weight, the dermis is relatively compact, and the decellularization has difficulty and complexity, so that the decellularization is carried out for a long time or by using a mild reagent, the ECM structure in the patch is damaged, the contained active biological components are reduced, the tissue regeneration induction capability is seriously insufficient, and the generation of new vessels and granulation tissues is slow.

The ideal skin repair material has the following conditions of good skin tissue regeneration promoting function, relative easy absorption and degradation, safety, no toxicity, no infection, good histocompatibility, no immunological rejection, convenient use, simple operation, easy disinfection and sterilization, proper thickness, -pan material selection and low price.

The invention content is as follows:

the invention aims to solve the defects and shortcomings of the existing skin repair material, and active ingredients, cell growth factors and extracellular matrix (ECM) three-dimensional stereo structures in animal tissue source biological materials are seriously broken due to long-time, sometimes excessive and even severe cell removal, such as pancreatin digestion, or long-time treatment with chemical surfactants (detergents); further, the biological material (patch) HAs weak tissue regeneration promoting capability or less active components (such as hyaluronic acid HA) for promoting wound healing, and HAs poor tissue induction capability; weak ability to induce cell growth; the technical problems existing in the skin repair materials are solved; the inventors have focused on the selection of starting materials from patches, as well as the selection of decellularization reagents, optimization of the process, starting from these key technical features; simply and conveniently overcomes the defects of the prior art scheme and skillfully solves the problems in the prior art.

The invention is characterized in that how to solve the technical problems that the skin patch has weak tissue regeneration induction capability, the ECM structure is seriously damaged essentially, the content of bioactive factors in the skin patch is low, chemical reagent residues possibly exist and degradation is influenced, and the inventor adopts a technical idea completely different from the prior art and directly takes the key factors of acellular tissue raw materials as the step of preparing the skin repair material to carry out deep research and analysis by taking the key factors as breakthrough points.

The main innovation point of the product is that the selection of animal tissue raw materials of the acellular patch is not conventional commercial grade meat livestock from slaughterhouses, but young animals such as newborn calves (the weight of the birth is 30-40 kg) and weaned piglets (6-8 kg, the day age is 20-30 days) are selected directly in a breeding farm, and the connective tissues of the young animals including or more of dermis, small intestine submucosa, bladder submucosa, stomach submucosa, bladder matrix, pericardium, meninges, peritoneum, fascia lata and parenchymal membrane are selected steps.

The secondary innovation point of the product is that the plant saponin which HAs mild action mode and no damage to ECM structural matrix is selected as a cell removal reagent, and the cell removal reagent can retain a more complete ECM three-dimensional structure and more effective active ingredients (such as HA) in the ECM for young tissues, particularly young animal dermis, young animal small intestine submucosa and young animal bladder; the tender tissues are very sensitive, the requirement on a cell removal reagent is high, and the type, working concentration, temperature during action and treatment time need to be considered; in short, the conditions are mild, and the method is proper, so that a more complete natural three-dimensional structure can be obtained and more active ingredients are reserved.

The third innovation point of the product of the invention is that the skin repair material is not limited to conventional sheet or film, but can be processed into kinds of powder, particles, colloid or paste or product combination by adopting physical, chemical and biological methods or the combination of , so that the dosage form of the skin repair material is richer, the using mode and method are more flexible and diversified, for example, the powder product does not need additional operation and has no technical difficulty, and even can be used to skin wounds or wound surfaces by a simple method directly without hospitals or doctors for guidance, so that the powder is contacted with the wound surfaces (including adsorption/adhesion) to promote early healing of the wound surfaces such as skin.

The invention is based on the insight that the inventors have given a particularly rapid tissue growth rate in young animals, which is characterised by or superior performance, that the growth rate of individual tissues is in the vigorous phase of growth in young animals, that recognizes the fact that we are directly reminded of the relative amounts (calculated as a percentage of dry matter) or effective activities of their cell growth factors and bioactive components (such as hyaluronic acid HA and fibronectin) in the tissues of young animals, which are significantly higher, times or several times higher than those of adult or preliminarily developed corresponding animal tissues, and that they have indeed been reported in foreign and related literature (Breen, m., Weinstein, h.g., bluck, l.j., Borcherding, m.s., and sitig, r.a.microanalysis and charectering of collagen from young animals, which have a high healing rate of cells and biological healing in young animals, which HAs a high healing rate of growth factors and biological activity in green tissue, green.

The young animal tissue, preferably dermis and small intestine submucosa are used as raw materials of the skin repair material, and the skin tissue repair material is prepared by decellularizing, and has the following advantages:

according to the principle of structural identity or similarity, the young animal dermis has high genetic homology with human skin, has homologous convertibility, and is very similar in various aspects such as microstructure and hierarchical distribution, ECM pore size, and composition and proportion of main structural components.

II, secondly: the dermis is rich in collagen, hyaluronic acid and elastin; the composition has obvious promotion effect on the formation of new blood vessels and basement membranes after skin injury; the repair of the damaged skin can be promoted even more effectively in the case of skin damage caused by any cause.

Compared with the skin of meat livestock which is already in market weight, the skin of young livestock is thinner, only parts of the skin of adult livestock is used, the degree of crosslinking is lower, the degradation speed is correspondingly improved to adapt to the growth speed of regenerated skin, but the regeneration capacity is strong, the biological active ingredients and cell growth factors with higher content, such as hyaluronic acid HA (calculated according to dry matter), the skin tissue regeneration and the wound healing are more facilitated, the skin tissue injury exposed outside is easier to cause infection and inflammation if the wound healing cannot be performed in time, and the healing and the comprehensive recovery of hearing are more difficult.

The present study shows that the phenomenon is helpful for the healing of wound surface, see the article named Fetal Microchimaer in skin surrounding health published in the year of Danny Nassar, Kiarash Khostroman and Selim Arctingi 2012, and the article named Microchim clinical cells a in bone in skin surrounding health published in other person 2014, kinds of micro vesicles secreted by cells (immune cells, nerve cells, stem cells) and so on, the fact that the newborn baby animal passes the Exosome to the body fluid such as blood, breast milk, etc., the extracellular vesicles pass through the body fluid such as the blood, breast milk, etc., the extracellular vesicles are secreted by the cells (immune cells, nerve cells, stem cells) and even the extracellular vesicles are secreted by the immune cells, the RNA, RNA and RNA contained in the cells, and the RNA of the immune cell receptor can be directly involved in the cell reaction, cell apoptosis, and even the RNA and RNA of the immune cell receptor can be directly involved in the cell-mediated apoptosis reaction.

Fifthly: the Small Intestinal Submucosa (SIS) mainly comprises type I fibrous collagen, and also comprises type III, IV and VI collagen, particularly important type IV collagen, and has obvious promotion effect on the formation of new blood vessels and basement membranes; meanwhile, according to various articles of United states STEPHEN F.BADYLAK, the degradation product of the SIS patch has stronger antibacterial activity and has the function similar to that of swine defensin (pBD-1); and article "excellular Matrix bioscafolds for building Tissue" in georges s s.hussey 2018 reported that SIS patch degradation products also have a cell chemotactic and mitotic inducing effect.

The invention principle is as follows:

it is characterized by that the growth speed of young animal tissue is very high, and the growth speed of every tissue is in the growth stage of , and this suggests that the relative contents (calculated by dry matter percentage) of cell growth factor and bioactive component (such as HA and FN) of young animal tissue are much higher than those of commercial meat animal which HAs reached marketing weight or meat animal which HAs been developed initially, and the young animal tissue HAs strong regeneration capacity, short injury healing time and high wound healing rate, so that it also can fully confirm that the cell growth factor and bioactive component contents in young animal tissue are high or their activities are strong.

II, secondly: because of inherent structural characteristics (such as low crosslinking degree and loose structure) and development characteristics of young animal tissues and the biological materials prepared from the young animal tissues, the young animal tissues are relatively loose in structure and tender compared with the biological materials of meat animal tissue sources which reach the market weight, so that the young animal tissues are more favorable for the immersion, growth and propagation of host fibroblasts; is more easily degraded and absorbed, and is more beneficial to the formation of new blood vessels and the generation of granulation tissues in skin wound tissues.

The method is characterized in that the plant-derived saponin is selected as a cell removing reagent, the cell removing mode is strong in pertinence mainly because the plant-derived saponin is a natural nonionic surfactant, the cell removing mode is mainly through destroying lipid cell membranes and organelle membranes, the action mode is strong in pertinence, other components are not damaged, the activity performance of functional proteins and the stability of structural proteins are not influenced, and the ECM structure is not damaged, so that the method is very critical to the good repair of the skin structure.

Fourthly, the method comprises the following steps: in the present invention, selected tissues of the young animal include dermis, small intestine submucosa, urinary bladder submucosa, stomach submucosa, urinary bladder matrix, pericardium, meninges, peritoneum, fascia lata. Dermal tissue, Small Intestinal Submucosa (SIS), and bladder matrix (UBM) are preferred as raw materials for decellularization.

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

1) the invention adopts young animal tissues as raw materials of skin repair materials, contains hyaluronic acid HA and cell growth factors with higher content, can obviously promote the regeneration of wound tissues and cells, and can accelerate the growth and function recovery of postoperative skin tissues.

2) The skin repair material of the invention takes the tissues of young stock as raw materials, and the finished product of biological material has loose texture, is easy to grow cells in and degrade relatively, which means easier absorption and effective utilization.

3) According to the technical scheme, no protein digestive enzyme and chemical detergent are used, mild plant saponin is used for removing cells, the collagen structure in the ECM is not damaged and destroyed, and a relatively complete extracellular matrix three-dimensional structure can be reserved; the biological material has better functions of inducing cells and blood vessels to grow in, and is relatively easy to degrade when new tissues grow in;

4) according to the technical scheme, the plant saponin is used as a cell removing reagent, a synthetic reagent is not used for removing cells, no chemical residue is generated, and the plant saponin is safe and has no side effect.

5) Compared with chemical detergents, the skin repair material of the invention contains more cell growth factors and effective active ingredients, and has good function of inducing tissue regeneration.

6) The skin repair material can be made into preset sizes and thicknesses with different specifications, for example, 1 layer or multiple layers of original patches are overlapped, the patches can be completely degraded and absorbed, and are favorable for regeneration of various skin wound tissues, meanwhile, the skin repair material is not limited to conventional sheet or film shapes, but can also be processed into types or product combinations in powder, granules, jelly and paste by adopting a physicochemical method, so that the preparation form of the skin repair material is richer, the using mode and method are more flexible and diversified, for example, a powdery product does not need operations and training, has no technical difficulty, can be directly used by family or friends without hospitals or even under the condition of no guidance of doctors, can directly use the skin repair material powder on skin wounds or wound surfaces by a simple method, and can ensure that the powder is fully contacted with the wound surfaces (including adsorption/adhesion) to promote early healing of the wound parts such as skin and the like.

The terms/nouns are preferably understood in the following written description unless otherwise specified; other terms are understood to have their meaning at the level of ordinary skill in the art.

() tissue repair materials, tissue regeneration materials, biological patches (membranes), biological patches, biological scaffolds, degradable patches, absorbable patches Bio-Mesh, Bio-Patch, bioscafold, wherein the terms or terms are different from each other in surface but are substantially -like in purpose and use, and the connotations of the terms are substantially equivalent unless otherwise specified.

(II) skin: is composed of epidermis, dermis and hypodermis; the epidermis is mainly composed of epithelial cells derived from the ectoderm, which are also called keratinocytes since they form keratin-containing keratinocytes in the final stage of their generation and differentiation, and dendritic cells mainly including langerhans cells and melanocytes. The keratinocytes can be divided into five layers from the surface to the bottom, namely, the stratum corneum, the stratum lucidum, the stratum granulosum, the stratum spinosum and the stratum basale, because of different differentiation stages and characteristics. The dermis is the most important tissue component in the skin, is positioned below the epidermis and is used for supporting the skin structure and mainly comprises fibroblasts, collagen fibers, elastic fibers, glycosaminoglycan (including hyaluronic acid HA and the like), fibronectin and the like; extracellular matrix components are mainly composed of fibroblast secretion products.

Extracellular matrix ECM (extracellular matrix) is a non-cellular component present in all tissues and organs, and not only provides necessary physical support for cellular tissues, provides suitable sites and microenvironment for normal physiological activities of various cells, but also plays an important role in leveraging morphogenesis of tissues, chemotaxis and differentiation of cells, and important aspects of physiological biochemistry and biomechanics, so as to influence or regulate functions of tissues and organs, 50% of the functions of cells are determined by the external microenvironment created by extracellular matrix, and the extracellular matrix is composed of structural proteins such as Collagen (Collagen), elastin (elastins), fibrillarin, and other macromolecular components as main framework structures, and functional proteins such as fibronectin FN (fibrinectins), laminin (lamins), and the like, and various cell growth factors (such as fibroblast growth factor FGF, transforming growth factor TGF, VEGF, and possibly very small amount of endothelial growth factor, but also contains very small amount of epidermal growth factor, and glycosaminoglycan-like (IGF-1-like) and also contains other active components such as IGF 1-type cell growth factors.

And (IV) exosomes (exosomes) which are microvesicles secreted from cells, have a diameter of about 30-200nm, have a cup-shaped form and a double-layer membrane structure, and naturally exist in biological fluids such as blood, urine, saliva, breast milk, cell culture media and the like. Including almost all types of cells (immune cells, neural cells, stem cells), can produce and release exosomes; the exosomes contain protein rRNA and microRNA related to cell sources, can directly activate receptor cells through cell membrane receptors, and can also transport protein, mRNA, miRNA, IncRNA, circRNA, even organelles into receptor cells to participate in intercellular communication. Exosomes play a key role in physiological processes such as immune response, inflammatory response, angiogenesis, apoptosis, coagulation and the like, and RNA and protein components contained in exosomes from different cell sources are different.

(V) glycosaminoglycan GAGs (glycosaminoglycans), also called mucopolysaccharides, are kinds of heteropolysaccharides, mainly present in animal connective tissues, and are important raw materials involved in normal physiological activities of tissues and tissue damage repair and regeneration, and are important components in ECM, and according to the types of monosaccharide residues and the bonds between residues and the number and positions of sulfate groups, glycosaminoglycan can be divided into 5 main categories, namely Hyaluronic Acid (HA), Chondroitin Sulfate (CS), Dermatan Sulfate (DS), Keratan Sulfate (KS), heparan sulfate and Heparin (HP).

(VI) Hyaluronic Acid (HA), also known as Hyaluronic acid or hyaluronate, is glycosaminoglycans, is , a major component of extracellular matrix (ECM), and is involved in many cellular physiology and tissue repair processes such as tissue remodeling, cell space expansion, inflammatory response and the like, studies have shown that Hyaluronic acid HAs significant cellular effects, significantly contributing to cell chemotaxis and migration, and cell proliferation, by a mechanism that occurs upon binding of cell surface HA to HA via a cell surface HA binding protein, and the adhesion molecule CD44 is a cell surface receptor for HA, is a class of cell surface glycoproteins of pan distribution, which are involved in specific adhesion between cells and between cell matrices, Ru-MingLiu, et al, ExperimentalResearch Volume 345, Issue 2,15 July 2016, Pages 218-229, which was published on Hyaluronic acid 229, and HAs been shown that Hyaluronic acid can promote proliferation by activating Wnt/β -desmoplasmon signaling pathways, and can be derived from large biological tissues, including large biological molecular weight animal tissues, such as large molecular weight, and large molecular weight, including large molecular weight, and large molecular weight, including large molecular weight, and large molecular weight.

Fibronectin (Fn) is a macromolecular extracellular membrane protein on the surface of animal cells, and is a major non-collagenous glycoprotein in extracellular matrix and basement membrane, plays a central role in cell adhesion, can regulate cell polarity, differentiate and grow high molecular weight glycoproteins in extracellular matrices, is major cell adhesion molecules, and plays structural and adhesive roles in a cell fiber matrix, can promote cell growth, increase cell adhesion rate, enhance cell metabolism, shorten cell growth time, increase cell fusion rate, and make cell morphology and structure good, is mainly in three forms, namely plasma Fibronectin generated by liver cells or endothelial cells, cellular Fibronectin secreted and synthesized by fibroblasts and early mesenchymal cells, and fetal Fibronectin in placenta and amniotic tissues, Fibronectin participates in the processes of skin dermal cell migration, adhesion, proliferation, hemostasis, aging, damaged skin tissue repair, etc., regulates the mononuclear cell system in blood to remove damaged tissue, has a harmful substance, has a growth factor, Fibronectin growth factor, increases the synthesis rate of Fibronectin and collagen, increases the fibroblast adhesion rate, collagen adhesion rate increase, collagen adhesion rate increase, collagen adhesion rate.

(eighth) acellular matrix ACTM (Acellular Tissue matrix) or acellular Tissue matrix, which means that specific reagents and treatment methods are adopted to fully remove or inactivate components which can generate immunological rejection reaction, such as cells, viruses, DNA and the like in animal organs or tissues, so that the integrity of the original natural three-dimensional structure is kept to the maximum extent, and cell growth factors and active functional components in the original matrix are kept to the maximum extent, the acellular matrix has the characteristics of degradation by a host, easiness in inducing the migration (easiness) of receptor stem cells and the like due to the natural three-dimensional (3D) structure, and is applied to clinic for the repair and regeneration (congenital defect and acquired wound) of tissues, and the acellular matrix is a novel Tissue regeneration repair material and has good biological scaffold performance.

The skin biomaterial includes but is not limited to biogenic materials for various injuries (whether internal or external, acute or chronic) of superficial or deep skin, mainly refers to ECM stereo structure obtained from animal tissue after decellularization process and the like, contains various cytokines and bioactive components, animal tissue raw materials can be derived from dermis, small intestine submucosa, bladder matrix, pericardium or other tissues and the like, and the appearance form can be kinds of powder, granules, jelly, paste, membrane or the combination thereof.

(ten) young animals: means that the livestock are usually born before the initial stage, are in a developmental stage, grow and develop rapidly, and are greatly different from adult livestock in terms of physiological functions to body conditions, and young livestock comprise weaned livestock and non-developed livestock, and comprise livestock in a lactation stage and at the beginning of birth; livestock that have matured or ended soon after development are excluded.

Unless the context clearly dictates otherwise, as used in this specification and the appended claims, the singular forms " and" the "include plural referents and thus, for example," the method "includes or methods, and/or steps, which are of the type described herein and/or which will be apparent to those of skill in the art upon reading this disclosure the term" about "or" approximately "refers to a range of values in a statistical sense and can range, within orders of magnitude, generally within 50% of the specified value or range, further refers to within 20%, still more generally within 10%, even more generally within 5%, the permissible variations encompassed by the terms" about "or" approximately "are readily apparent to those of skill in the art, the permissible variations are due to the study of the particular systems.

The present invention provides skin repair materials, and more particularly, skin repair materials, which comprise a decellularized young animal tissue, that is, a young animal tissue which is processed by a process such as a decellularization treatment, etc., and , the young animal is a livestock which is usually born before the first episode, is in a developmental stage, grows rapidly, and is greatly different from an adult livestock in terms of physiological functions and physical conditions.

At step , the young animals include young animals that have been weaned but not yet developed, animals that are in the lactation stage and are born, and animals that have matured or have completed development are excluded.

, the young livestock is young pig, calf, lamb, camel, donkey, or rabbit.

, the tissue is selected from the group consisting of dermis, small intestine submucosa, bladder submucosa, stomach submucosa, meninges, peritoneum, fascia lata, and pericardium .

, the young animal tissue refers to dermis of newborn calf and weaned piglet.

Further , the process comprises three main steps of pre-treatment, decellularization and post-processing in sequence of operations.

, the pretreatment in the processing includes washing, sterilizing and defatting.

, the decellularization treatment in the process refers to the use of a plant-derived surfactant as a decellularization agent.

, the subsequent processing is to adopt physicochemical method to remove cell tissue, and process into kinds of powder, granule, sponge, gel, paste, and membrane or their combination.

, the process includes the step , which is the pre-treatment, including washing the connective tissue of young animal, washing thoroughly, disinfection with acid liquid and/or alkali liquid, defatting with organic solvent, the step two, which is the cell eliminating treatment, of soaking the young animal tissue material with plant source surfactant solution under ultrasonic condition and washing with PBS, and the step three, which is the subsequent processing, of the semi-finished product after cell eliminating treatment, and the physical process to produce the product in required shape.

Further , optionally, after the decellularization process, the tissue material is soaked with an aqueous solution containing DNase to remove DNA, washed with PBS, and then soaked with an aqueous solution containing α -galactosidase to remove α -Gal antigen.

, pre-treating during processing, which comprises cleaning young animal dermis, small intestine, and bladder tissue, mechanically scraping or shearing to remove non-connective tissue, washing to obtain target tissue, soaking in acetic acid solution at a ratio of 1:10(W/W) for 15-90 min, sterilizing by soaking in solution containing peroxyacetic acid and alcohol for 10-45 min, ultrasonic cleaning with water, defatting by soaking in alcohol solution at normal temperature for 0.5-2 hr at a ratio of 1:5-1:10, and ultrasonic cleaning with water;

the skin repairing material is prepared through the cell eliminating treatment including soaking tissue material in plant source surfactant solution at low temperature and ultrasonic wave for 20-60 min, soaking tissue material in new plant source surfactant solution in the ratio of 1 to 10(W/V), and cell eliminating for 1 time.

The optimized cell-removing treatment operation method comprises soaking biological material in solution containing plant saponin at 4-15 deg.C under ultrasound for 20-50 min; the ratio of biological raw material to solution is 1:10 (W/V); then soaking the biological raw material for 5-30 minutes by using a fresh saponin solution with the same concentration.

The following technical parameters are preferred: the content of effective saponin in the saponin solution is 0.1-1% (W/W), the ratio of the biological raw material to the plant source saponin solution is 1:5-1:10, and the biological raw material and the plant source saponin solution are soaked for 20-30 minutes at the low temperature of 4-10 ℃ under the ultrasonic condition; then soaking the biological raw material for 5-30 minutes by using fresh saponin solution with the same concentration; then soaking for 15-30 minutes by PBS-EDTA. Preferred plant-derived surfactants are plant-derived triterpene saponins, more preferably Quil-A; the working concentration of saponin is 0.1-0.5% (W/W).

the principles and aspects of the present invention will be further described with reference to specific examples, which are intended to be illustrative only and not to be limiting, and which are not intended to limit the scope of the invention in any way, and which are not to be construed as limiting the scope of the invention, wherein the specific procedures and methods described in detail are those well known in the art and may include omitting routine procedures and methods used in the medical and biological materials arts.

Detailed Description

Example (newborn calf dermis + Saponin)

Selecting healthy newborn calves, taking dermis of the calves, and preparing a skin biological patch, wherein the skin biological patch comprises the following specific steps:

1) pretreatment: selecting healthy newborn calf from a cattle farm, taking the skin in the middle of the abdomen of the newborn calf after blood is taken to prepare serum and the newborn calf is killed, fully cleaning, mechanically removing fat and the outer surface layer of the inner surface layer, washing the skin by deionized water, and taking dermal tissue (comprising a papilla layer, a basement membrane connected with the papilla layer and a reticular layer) with the thickness of 0.3mm by a skin taking machine; soaking in 0.5% acetic acid solution for 20 min at a weight ratio of 1:8, and washing with purified water for 3 times; then, the dermis material was soaked in a mixed solution containing 1.0% peracetic acid and 15% ethanol at room temperature for 45 minutes under ultrasonic conditions for sterilization, and the ratio of the dermis material to the mixed solution was 1: 10. Then purified water is used for ultrasonic cleaning for 3 times; degreasing, and soaking for 45 minutes at normal temperature by using 90% ethanol under the ultrasonic condition; the weight ratio of the dermal material to the ethanol is 1:10, and then the mixture is ultrasonically cleaned for 3 times by water;

2) decellularization by soaking the biological material in a solution containing 0.2% saponin (commercial Quil-A, calculated as the concentration indicated by the commercial product and calculated as the working concentration based on the pure saponin content) at 4 ℃ under ultrasonic conditions for 20 minutes, rinsing the biological material with a solution containing 0.2% saponin at the same concentration for 10 minutes, then soaking the patch in a PBS-EDTA solution for 10 minutes, then after decellularization, soaking the tissue material in an aqueous solution containing DNase to remove DNA, after PBS rinsing, then soaking in an aqueous solution containing α -galactosidase to remove α -Gal antigen.

3) And (3) subsequent processing: overlapping 2, 3, 4 and 5 layers of the decellularized dermis flaky raw material according to the thickness requirement, fixing on a mold, freeze-drying at low temperature, cutting according to different preset sizes and dimensions, such as 10cm by 10cm, 8cm by 8cm and 5cm by 5cm, packaging, sterilizing, and labeling to obtain the finished product.

Example two: (weaned pig SIS + saponin)

Selecting healthy weaned piglets, taking small intestinal submucosa thereof, and preparing a skin biological material, wherein the method comprises the following specific steps:

1) pretreatment: selecting and cleaning materials, selecting healthy ternary hybrid piglets which reach the weaning day age and the weight, taking small intestines of the ternary hybrid piglets, and cleaning the small intestines to be used as young animal tissue raw materials; through deep knowledge, the days of weaning piglets are usually 21-30 days, and the weaning days are different due to different seasons, slightly shorter in summer and slightly longer in winter; the weight is about six to eight kilograms, the piglets mainly eat breast milk before weaning, and the complete feed (which is called creep feed or piglet milk powder) is little. Removing mucosa layer, muscular layer, serous layer, lymph node, etc. of small intestine jejunum by mechanical scraping method, cutting into segments to obtain Small Intestine Submucosa (SIS) raw material, soaking in 0.8% acetic acid solution for 45 min, wherein the weight ratio of pig small intestine submucosa to acetic acid solution is 1:8, and washing with purified water for 3 times; then, disinfection is carried out by soaking the SIS material in a mixed solution containing 1.0 percent of peroxyacetic acid and 15 percent of ethanol for 90 minutes at room temperature under the ultrasonic condition, and the ratio of the SIS material to the mixed solution is 1: 10. Then purified water is used for ultrasonic cleaning for 3 times; degreasing, and soaking for 90 minutes at normal temperature by using 90% ethanol under the ultrasonic condition; the weight ratio of the SIS material to the ethanol is 1:10, and then the mixture is ultrasonically cleaned for 3 times by water;

2) and (3) cell removal: soaking the biological material in 0.4% saponin solution (commercial Quil-A, converted according to the concentration marked by the commercial product, and working concentration calculated by pure saponin content) at 4 deg.C under ultrasonic condition for 30 min; then flushing the biological raw material for 10 minutes by using a saponin solution with the same concentration of 0.4 percent; the patch was then soaked for another 20 minutes in PBS-EDTA solution.

3) And (3) performing subsequent processing, namely layers, or two layers, or three layers, or even multiple layers of the acellular SIS sheet raw material are overlapped according to the thickness requirement, then fixing the raw material on a mould, performing low-temperature freeze drying, cutting according to different preset sizes (such as 6cm by 4cm and the like), packaging, finally sterilizing, and labeling the product name to obtain the finished product.

EXAMPLE III (control , commercial pork pig)

The specific operation in the preparation method is basically the same as that in the second embodiment, and the main difference is as follows: the pigs are different in age in days and weight, commercial pork pigs from slaughterhouses are selected in the embodiment, the weight is nearly 100 kg, and the feeding days are about 150 days and 160 days; other processing methods, pretreatment (including pretreatment, disinfection, degreasing), decellularization, subsequent processing and other steps, methods and using reagents, concentrations and action time are completely the same as the second embodiment.

Example four: (control group two, trypsin)

The preparation method is basically the same as the second embodiment, and the main difference is that the second step is selected, the cell removing reagent is selected, the plant saponin is not used, the conventional test is used, for example, the working concentration of the trypsin is 0.05%, the acting time is the same and is 30 minutes, and the soaking is not carried out again by using the trypsin with the same concentration, namely, only the trypsin solution is used for acting times (30 minutes).

Example five: detection of bioactive factors in skin biomaterials

The skin biomaterial samples prepared in examples 1 to 4 were subjected to detection of bioactive factors; detecting the content of an active ingredient Hyaluronic Acid (HA) by adopting a commercial kit; the sample pretreatment method is to adopt a low-temperature grinding method for treatment, and the detection results are as follows:

group of	Tissue source and decellularization reagent	Hyaluronic acid HA (ug/mg)
			Example 1	Newborn calf dermis + saponin	3.28
Example 2	Weaned piglet SIS + saponin	3.57
			Example 3 (control )	Commercial pork pig SIS + saponin	2.25
Example 4 (control group two)	Weaned piglet SIS + trypsin	2.11

Example five: making into different dosage forms

And (3) cutting the dried sheet products prepared in the examples and II manually or mechanically at low temperature (5-20 ℃ below zero) for 5 minutes at the rotating speed of 5 revolutions per second, processing the dried sheet products into biological chips, biological particles or biological powder with different specifications and sizes, wherein the diameters and the widths are 0.3mm,0.4mm,0.5mm,0.6mm,0,7mm,0.8mm,0.9mm,1mm, 1.5mm and 2mm, sieving and grading the biological chips, and then packaging, sterilizing and labeling the biological chips to obtain the biological powder.

It should be emphasized that the present invention, while certain novel features of the invention are set forth and described below in the appended claims, and thus, no attempt is made to limit the invention to the specific details, since it will be understood by those skilled in the art that various omissions, modifications, substitutions and changes in the form and details of the invention illustrated, and in its implementation, may be made without departing in any way from the spirit of the invention.

Claims (9)

1, skin biomaterials, which is characterized by comprising decellularized young animal tissues, wherein the young animals refer to young livestock from birth to the initial stage.

2. The dermal biomaterial of claim 1, wherein the young animal tissue is treated with a decellularizing agent.

3. The dermal biomaterial of claim 1, wherein the young animals are selected from the group consisting of, but not limited to, pigs, cattle, sheep, horses, camels, donkeys, rabbits, cats, dogs, right after birth, pre-weaning, right after weaning.

4. The skin biomaterial of claim 1, wherein the tissue is or more selected from dermis, small intestine submucosa, bladder submucosa, stomach submucosa, meninges, peritoneum, and fascia lata.

5. The dermal biomaterial of claim 1, wherein the pup tissue refers to species or combinations in the dermis of newborn calves, the small intestinal submucosa of weaned piglets.

6. The dermal biomaterial of claim 2, wherein the decellularizing agent is based on a plant-derived nonionic surfactant.

7. The dermal biomaterial of claim 2, wherein the decellularization reagent consists essentially of triterpene saponin, steroid saponin or a complex thereof.

8. The skin biomaterial of claim 2, wherein the decellularizing reagent consists essentially of Quil-a; the cell removing reagent has effective working concentration of 0.1-1 wt%, and has an action time of 5-60 min with biological material and a cell removing action temperature of 2-20 deg.C.

9, skin biomaterials, characterized in that the dosage form can be or combination of powder, granule, colloid, paste and film, the application method is flexible, and the application method can be operated or directly applied without operation.