CN110585484A

CN110585484A - Composite bone powder for bone tissue and preparation method and application thereof

Info

Publication number: CN110585484A
Application number: CN201910968578.9A
Authority: CN
Inventors: 韩韦红; 葛翠兰; 钱锵; 张国强
Original assignee: SHANGHAI BAIYI BIOLOGICAL ENGINEERING Co Ltd
Current assignee: SHANGHAI BAIYI BIOLOGICAL ENGINEERING Co Ltd
Priority date: 2019-10-12
Filing date: 2019-10-12
Publication date: 2019-12-20

Abstract

The invention belongs to the field of bone tissue repair materials, relates to novel composite bone powder and a preparation method and application thereof, and overcomes the defects that the existing bone powder product does not contain bioactive components and growth factors, or has few and unstable growth factors and is not beneficial to chemotaxis, adhesion and growth of new bone cells. The invention additionally adds acellular matrix powder on the basis of the existing bone meal, and the acellular matrix powder not only contains a large amount of structural components such as collagen, has natural degradable three-dimensional structure characteristics, but also contains active component characteristics formed by various cell growth factors and various active components (such as glycosaminoglycan and the like); the two characteristics and the specific homologous similar structure of the bone powder form a microenvironment together, so that the microenvironment has strong inductivity on various cells, particularly osteocytes, is favorable for vascularization, and is favorable for chemotaxis, growth, differentiation and maturation of host osteocytes; has better osteogenesis performance, thereby shortening the bone healing time and improving the bone quality of regenerated bones.

Description

Composite bone powder for bone tissue and preparation method and application thereof

Technical Field

The invention belongs to the field of medical repair materials, and particularly relates to novel composite bone powder and a preparation method and application thereof.

Background

At present, more than 2 hundred million of patients with missing teeth exist in China, the number of missing teeth is increased every year, nearly half of patients receiving planting treatment have insufficient bone mass in a planting area, and bone repair materials are the most common and effective method for increasing the bone mass in the planting operation.

Cleft lip and palate is a common congenital developmental deformity of the oral and maxillofacial region, the disease rate in China is about 1.8 per thousand, and the cleft lip and palate is often accompanied by alveolar fissure. Usually, the oral cavity defect is manifested as alveolar process bone defect, dental arch integrity loss, dentition irregularity, anterior maxilla, slurred pronunciation caused by oral-nasal fistula, nasal wing collapse and other deformities due to lack of bone tissue support at the base of nasal wing, alveolar process defect, ectopic eruption and fissure teeth block eruption. With the improvement of the requirements of people on the quality of life and the appearance, the problems caused by the alveolar burst are gradually emphasized, and the restoration of the alveolar burst is one of the important steps in the sequence treatment of the cleft lip and palate. If most of the patients with cleft lip and palate are accompanied by alveolar fissure and need bone grafting, a bone repair material is needed.

Periodontitis is a common oral disease characterized by destruction of the periodontal tissue. Alveolar bone resorption and loss are among the most serious consequences of periodontitis and are generally difficult to repair themselves, as are conventional periodontal treatments. Regeneration and repair of periodontal bone defects have been successfully achieved clinically by implanting bone repair materials. Meanwhile, some false teeth with limited implant operation caused by insufficient bone mass and the like can be repaired.

The ideal oral bone repair material should have the following characteristics: good biocompatibility. No toxic or side effect on normal physiological functions of a human body, no immune rejection, no adverse effect and the like: ② has the function of inducing osteogenesis: good biomechanical properties, the material should be consistent with the elastic modulus, thermal expansion property and strength of bones: the self-biodegradable material can be replaced by new bone in a relatively short time: the utility model has a certain structure and can play the role of a bracket in the defect area: sixthly, the preparation or purchase is easy, and the cost is low: and the product is easy to sterilize and trim and form in operation.

Noun terms define: for a more accurate understanding of the present invention, the terms of scientific terminology used herein are defined and interpreted as follows to eliminate unnecessary divergence and misunderstandings and to prevent misunderstandings.

1. Extracellular Matrix (ECM, Extracellular Matrix): the complex organic three-dimensional integral structure is constructed by macromolecular components such as structural proteins, such as collagen, elastin and the like, functional proteins, such as fibronectin, laminin and the like, and components such as various cell growth factors, glycosaminoglycan, proteoglycan and the like, provides proper places and microenvironments for normal physiological activities of various cells, and regulates and controls the functions of tissues and organs; 50% of the cell's function is determined by the external microenvironment created by the extracellular matrix.

2. Acellular Matrix (ACTM, Acellular Tissue Matrix) or Acellular Tissue Matrix: the method is characterized in that specific reagents and treatment modes 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, preserve the integrity of the original natural three-dimensional structure to the maximum extent, and preserve cell growth factors and active functional components in the original matrix to the greatest extent; the acellular matrix is a novel tissue regeneration and repair material and has good biological scaffold performance.

3. Biological scaffolds (bioscaffolds) and biological patches, which differ only in terms of expression, are essentially acellular matrices for which the starting material belongs.

4. Glycosaminoglycan (also called mucopolysaccharide) is one of heteropolysaccharides, mainly exists in animal connective tissues, and is an important raw material participating in normal physiological activities such as bone and cartilage tissues; glycosaminoglycans can be classified into 5 major classes, Hyaluronic Acid (HA), Chondroitin Sulfate (CS), Dermatan Sulfate (DS), Keratan Sulfate (KS), heparan sulfate and Heparin (HP), according to the type of monosaccharide residues, the linkages between residues and the number and position of sulfate groups.

5. The composite material, bone graft material, bone substitute material are understood to be essentially the same material in the first place, unless otherwise specified and defined.

6. Extracellular Vesicles (EVs) are present in the Extracellular matrix and can be classified according to the vesicle biosynthesis or release pathway; such as Microvesicles (MVs) and Exosomes (Exosomes, Exs), apoptotic bodies (apoptotic bodies); these vesicular bodies, which are shed from cell membranes or secreted from cells, have a double-layer membrane structure, and have diameters varying from 40nm to 1000 nm. Recent research shows that extracellular vesicles serving as goods of specific components are wrapped and carry important active molecules such as micro RNA, protein and lipid, and can efficiently transmit physiological signals among cells and regulate and control the activities of the cells.

The prior art is as follows:

firstly, the method comprises the following steps: the applicant, xuelian, Chinese patent application No. CN02145493.0, discloses a preparation method, a coating and a cell compounding method of an absorbable biphasic calcined bone. The biphasic calcined bone takes cancellous bone as a raw material, and Hydroxyapatite (HAP) in the calcined bone is partially or completely converted into beta-tricalcium phosphate (beta-TCP) by impregnation, one-time calcination and reduction of calcination temperature; then, coating the surface of the calcined bone and the porous material with collagen gel; the compounding method of the cells, the calcined bone and the porous material comprises the steps of collecting mesenchymal stem cells of the bone marrow, completely absorbing cell suspension into the material, incubating in an incubator, finally adding culture solution containing serum, continuing to incubate for 3-8 days, and then implanting into the body.

II, secondly: the applicant's chinese patent CN200910022842.6, king love army, discloses a method for preparing a humanized active calcined bone, which is characterized in that a coating formed by synthesizing and secreting extracellular matrix by human body cells is compounded on the surface of the calcined bone, the main component of which is hydroxyapatite; the preparation method comprises four steps, wherein in the first step, the calcined bone is granulated, then the calcined bone granules are placed in any solution of bovine serum, or collagen, or polylysine, or RGD polypeptide to be soaked for not less than 1 hour, and then the calcined bone granules are dried and sterilized; secondly, preparing a human body cell culture solution; thirdly, cell inoculation is carried out, and the dried calcined bone particles are added into a human cell culture solution for culture; the fourth step: washing the cultured calcined bone particles with a phosphate buffer solution, and then carrying out vacuum drying and sterilization; through the four steps, the humanized calcined bone particles which are compounded with extracellular matrix synthesized and secreted by human cells and cell growth factors are obtained.

Thirdly, the method comprises the following steps: the applicant's Chinese patent with application number 200810116023.3 of von qingling discloses a bone repair material containing nano hydroxyapatite/collagen particles and a preparation method thereof; the bone repair material contains 1-2 parts by weight of nano hydroxyapatite/collagen particles, 1 part by weight of chitosan and 2-3 parts by weight of BETA-sodium glycerophosphate, and when the bone repair material is used, the bone repair material is placed at 37 ℃ or injected into a body, so that gel can be formed in situ.

Fourthly, the method comprises the following steps: collagen-supplemented bone meal products are currently marketed, such as Bio-Os Collagen, a product of Gauss, Switzerland, which contains 90% hydroxyapatite and 10% Collagen.

The defects of the prior art scheme are as follows:

although the technical proposal is improved on the basis of the original bone meal product, the technical proposal still has some more or less defects; in the products according to the first and second embodiments, the product is intended to contain the cytokine, but the content thereof is small and unstable; the third scheme and the products of Swiss and Gauss company are added with collagen, but do not contain cell growth factors and active functional components, and the specific technical defects and defect analysis are as follows:

scheme one, there are four significant disadvantages: firstly, a collagen coating is used in the scheme, but the collagen coating is in contact with bone materials, namely an organic phase and an inorganic phase, only on the surface, so that effective firm combination is difficult to form; secondly, after the bone meal is compounded with cells, live cells or residual cell fragments have certain immunogenicity and may have certain rejection reaction and other side effects; thirdly, the culture of living cells is difficult, the technical difficulty is high, professional culture media and skilled technical operators are needed, and when the method is clinically used, the pretreatment of products is very complicated, the consumed time is long, the cost is high, and the popularization and the promotion are difficult. Fourthly, the method comprises the following steps: in practical applications, it is difficult to perform irregular defects into desired shapes, and these disadvantages limit the practical clinical application of this material.

Scheme two, there are at least two significant disadvantages: firstly, living cells need to be cultured in the production process, the living cells are cultured in vitro on an unconventional culture medium, the growth conditions of the cells are harsh, the growth is slow, and factors secreted outwards are few; the technology has higher difficulty, needs professional culture medium and skilled technical operators, has higher failure rate, very complicated production process of products, long time consumption and high cost, and is difficult to popularize and promote. Secondly, the following steps: the calcined bone particles, which are difficult to cell culture, are washed with phosphate buffered saline, and the growth factors secreted from the cells, which are otherwise very small, are washed away, resulting in further loss of active growth factors.

The third scheme has at least five disadvantages, namely, from the physical aspect, the nano-hydroxyapatite/collagen particles destroy the natural porous structure of the nano-hydroxyapatite/collagen particles, and are not beneficial to the attachment and growth of cells; secondly, from the chemical composition perspective, the extract contains components which may not be biological sources, such as BETA-sodium glycerophosphate which may be chemically synthesized, and has safety risk. Thirdly, the proportion of the hydroxyapatite is obviously lower than 25% -40% from the aspect of the proportion of the product components, while the weight of the hydroxyapatite in normal bones is 70% -90%. And fourthly, the composition does not contain any accepted bioactive factor andor active ingredient capable of promoting cell growth. Fifthly, the product contains high-content sodium BETA-glycerophosphate; sodium BETA-glycerophosphate generally has five crystal waters, and is also easily soluble in water, and therefore, there is an unstable situation, thereby affecting the quality of the product.

Fourthly, the method comprises the following steps: Bio-Os Collagen, a product of Gauss Switzerland, in which Collagen has been processed to a liquid and then mixed with bone meal. On one hand, after the collagen in the product is liquefied, the natural three-dimensional grid structure of the collagen is destroyed, and the induction and attachment capacity to bone cells is greatly reduced; on the other hand, the product does not contain various cell factors, functional proteins and other effective components for promoting cell growth; cannot effectively induce and promote the growth, differentiation and maturation of bone cells.

The purpose of the invention is as follows:

because the traditional bone meal product does not contain any bioactive factor, the bone meal product belongs to a pure mineralized inorganic product and does not contain any organic components such as protein, amino acid and the like; is more free of any growth factor, and does not facilitate the adhesion and proliferation of cells per se; the existing improved bone powder or surface modified bone powder is only coated or soaked with cell factors on the surface of the bone powder, and due to the fact that technical steps are multiple, or the bone powder is complex, or operation difficulty is high, an actual product is unstable and reliable, and the defects of difficulty in storage, short shelf life and the like exist, the micro-improved bone powder is actually poor or very limited in capability in inducing bone cell chemotaxis, promoting bone cell growth and the like, and the defects of slow growth of new bone tissues, difficulty in bone healing and long recovery period of patients are still difficult to overcome effectively; it is difficult to meet the higher technical requirements of modern medicine on bone regeneration and repair materials.

Aiming at overcoming the defects of weak capability in inducing chemotaxis and differentiation of osteocytes and poor expression in promoting growth and maturation of the osteocytes of the existing improved bone powder products; the method solves the technical problems that the growth of new bone tissues is slow and the bone healing takes a long time, so that the inventor makes a great deal of intensive research, exploration and development, and continuously searches and tries for years all the time, so that the method breaks through to some extent and achieves certain success.

Disclosure of Invention

Aiming at the defects of the existing products and the technical scheme, the inventor takes acellular matrix, bone tissue repair and regeneration and the like as subject words again on the basis of the original technology accumulated by the company for many years, combines the technical characteristics of the existing bone powder and the technical requirements of bone repair, makes extensive and deep reading on related scientific and technical literatures at home and abroad, and researches and develops the novel bone repair material on the basis of careful analysis and full comparison.

The invention relates to a novel composite bone powder and a preparation method thereof, and the composite bone powder (bone repair material) is prepared by additionally adding a certain proportion of biological supplement powder or acellular matrix powder on the basis of various conventional bone powders or composite bone powders. The matrix powder not only contains a large amount of extracellular network components such as collagen, elastin, fibrillarin and the like, but also particularly, important active functional components are carried in the porous network structure of the matrix powder, and comprise various cell growth factors such as fibroblast growth factor (FGF-2), transforming growth factor (TGF-beta) and Vascular Endothelial Growth Factor (VEGF), functional protein molecules such as fibronectin, laminin and the like, and also comprise some glycosaminoglycan components such as heparin, heparan sulfate, chondroitin sulfate, hyaluronic acid and the like. The acellular matrix powder has natural grid structure characteristics and active ingredient characteristics consisting of a plurality of cell growth factors and a plurality of active ingredients contained and distributed in the acellular matrix powder, and the two characteristics, namely a microenvironment formed by the grid structure characteristics and the active ingredient characteristics, are very favorable for chemotaxis, growth, differentiation and maturation of host bone cells. The novel bone powder material effectively overcomes the defects of weak inductivity of the existing product to osteocytes, slow growth of the osteocytes and poor osteogenesis, thereby shortening the bone healing time and improving the bone quality of new bone tissues.

The invention has specific technical requirements on acellular matrix powder, and in the acellular process, the specific natural grid three-dimensional structure of the extracellular matrix is kept as far as possible, and various cell growth factors and active functional components in the matrix are also kept as far as possible; it is worth noting that: when the acellular matrix is processed into powder, the powder needs to be processed at low temperature by adopting special processing and crushing equipment; so as to prevent the large amount of heat generated by friction in the crushing process from damaging the grid structure and various active ingredients in the acellular matrix.

The invention principle is as follows:

the inventor considers that the prior bone meal product lacks the cell growth factors and active ingredients with quality and quantity preservation, and the bone meal product has weak or limited capability of inducing the chemotaxis of the bone cells and promoting the growth of the bone cells; meanwhile, because the bone tissue has certain particularity (compact structure), the blood vessel distribution is less, the blood flow is slower, the total blood flow is less, and the growth of bone cells is slow. After the bone is damaged, if osteoblasts are not induced by cell growth factors, other active ingredients and the like and the growth and differentiation of the osteoblasts are promoted, the growth of new osteoblasts and bone tissues at the damaged part is slower, and the bone healing period is longer.

After the inventor consults a large amount of literature data and latest research results at home and abroad, the acellular matrix is considered to have a natural degradable biological net-shaped three-dimensional structure and can provide good scaffold performance for the attachment and growth of cells; more importantly, the acellular matrix reticular structure also contains a plurality of cell growth factors and various effective active ingredients for promoting tissue healing, which has important key effects on inducing chemotaxis, attachment, positioning and growth of osteocytes.

The acellular matrix has the unique advantages in the two aspects, and can completely overcome and make up the defects of the existing bone meal and bone meal collagen composite product; the acellular matrix powder and the bone powder are used in a combined manner, so that the bone repairing powder has a synergistic repairing effect and can exert better effects of promoting bone growth and bone healing; the compound bone powder has good application prospect.

The acellular matrix is the main technical innovation of the present invention, and further description and explanation are necessary here to facilitate understanding of the characteristics of the present invention; the acellular matrix is a potential multipurpose tissue repair and regeneration material, and when the acellular matrix is used as a tissue repair material, the acellular matrix generally contains four types of effective active ingredients, and the specific structure and the function of the acellular matrix are introduced as follows:

firstly, the method comprises the following steps: acellular matrices contain large amounts of structural proteins, such as collagen types I, III, IV, VI and elastin; the structural proteins form a main body of a net structure in a matrix, and a net framework taking collagen as a main body has good biocompatibility and safety; meanwhile, the reticular structure is completely degradable, can be modified and reused, has extremely high bioavailability, and plays a critical determining role in the rapid repair and regeneration of tissue cells.

II, secondly: the acellular matrix contains a certain amount of active functional protein, such as Fibronectin (FN), Laminin (LN) and the like; for example, several domains of fibronectin can be combined with fibrin, DNA and integrin on the cell surface, and further influence the activities of cell adhesion, migration and the like through the bidirectional signal transduction of the integrin; further, the mitosis, differentiation and growth of cells can be influenced.

Thirdly, the method comprises the following steps: the acellular matrix also contains glycosaminoglycan, which is a functional component with unique activity, such as heparin, heparan sulfate, chondroitin sulfate, hyaluronic acid and the like; these active ingredients, all containing the disaccharide aminocaproic-uronic acid unit, have physiological functions including the retention of water in connective tissue; regulating the distribution of cations such as Ca ions in the tissue to maintain normal physiological functions; the glycosaminoglycan has certain viscosity, and has good lubrication and protection effects on cells/tissues after being used alone or combined with protein; heparin among glycosaminoglycans has an anti-coagulant effect and has an effect of promoting wound healing.

Fourthly, the method comprises the following steps: some important growth factors secreted by cells to the outside have been detected in acellular matrices; for example, in the porcine Small Intestinal Submucosa (SIS) acellular matrix, fibroblast growth factor (FGF-2), transforming growth factor (TGF-beta) and Vascular Endothelial Growth Factor (VEGF) have been detected, and the content reaches high, so that the porcine Small Intestinal Submucosa (SIS) acellular matrix has practical clinical application value; the cell growth factors have obvious promotion effect on the cell growth and the functional recovery of damaged tissues.

Meanwhile, new scientific research shows that acellular matrix derived from porcine Small Intestinal Submucosa (SIS) can release crypt defense peptide (matricryptic peptide) with unique bioactivity after degradation; some of the crypt defense peptides and the defense peptides and lysozyme secreted by small intestinal Pangolin cells (Paneth cells) to intestinal tracts have certain homology and have similarity in function; for example, all have certain anti-tumor activity and antibacterial activity and can participate in immune regulation; defensins can also selectively aggregate monocytes, T lymphocytes, dendritic cells and mast cells chemotactic to sites of inflammation to promote adaptive immunity; defensins may also induce proliferation of fibroblasts and epithelial cells to promote wound tissue repair

In addition, more and more research reports prove that collagen in acellular matrixes (such as porcine small intestine submucosa acellular matrixes) can be degraded under physiological conditions or under the action of other biological factors, and secondary active ingredients such as Arrestin (Arrestin), tumstatin, endostatin, kanpitkin factor (cantatin, a novel angiogenesis inhibitor) and the like can be generated; these degradation products have obvious inducing and chemotactic effects on cells (injured tissues), and are beneficial to reconstruction and repair of cells and tissues.

Research data show that when the porcine Small Intestinal Submucosa (SIS) acellular Matrix is used as a tissue repair material in animal experiments, the content of Matrix-Metal-proteases (MMPs) can be obviously reduced, and the endogenous Matrix proteolytic enzymes, especially MMP-1, 2 and 9, can block the generation of new collagen, thereby influencing the regeneration and repair of tissues. The principle of action may be that some macromolecules in the acellular matrix, such as certain specific structural domains in collagen, can be specifically bound with active sites in MMPs, thereby causing MMPs to lose activity; thus, MMPs can not prevent the formation of new collagen; the practical application has the specific effect that the wound tissue can heal faster and better after the acellular matrix is used as a tissue repair material.

In addition, the latest research shows that the acellular matrix contains important Extracellular Vesicles (EVs) which are used as goods packages of specific components and carry important active molecules such as micro RNA, protein, lipid and the like, so that physiological signals can be efficiently transmitted among cells, and the activity of the cells can be regulated; such as promoting macrophage polarization (activation) and stem cell differentiation; the extracellular vesicles have important positive significance for tissue repair.

Therefore, high-quality acellular matrix powder with a certain proportion is additionally, scientifically and reasonably added in addition to the bone meal and the collagen; the combined use and compound proportion of the three raw materials can complement each other structurally and functionally, so that the regeneration and repair effect mainly based on bone powder is more carefully and fully exerted in bone tissue repair, which is more favorable for the rapid growth of bone cells, the formation of new bone tissue structure and the recovery of various physiological functions.

Has the advantages that:

compared with the existing product, the novel composite bone powder prepared by the invention has the following advantages: not only can induce chemotaxis, growth, differentiation and calcification of bone cells; can also induce the regeneration of non-bone tissues such as ossein and the like, namely has the functions of comprehensively inducing the regeneration and the repair of bone target tissues; on one hand, the collagen can provide a microstructure similar to human bone, a better calcium-phosphorus ratio and an ideal easily-absorbed calcium-phosphorus source, and simultaneously provides high-quality animal-derived collagen; more importantly, the bone marrow cell also contains a plurality of cell growth factors and active functional components, can induce chemotaxis, attachment and positioning of osteoblasts at the first time, and the induced chemotaxis on the osteoblasts is very critical to the subsequent growth and repair of bone tissues. The acellular matrix powder can also provide an absorbable biological scaffold for the attachment positioning and rapid growth of osteocytes; the composite bone powder containing the acellular matrix has better effect than the existing products, and is mainly attributed to the acellular matrix; the main technical features and unique effects of acellular matrices are now introduced as follows:

the first is that: the acellular matrix powder has good biological scaffold performance due to the specific natural latticed structure. The three-dimensional net structure mainly composed of collagen has good biocompatibility, and is familiar and adaptable to living host cells; on one hand, the cell is easy to adhere and grow, on the other hand, the degradation and absorption of the cell are facilitated, namely, the cell also has good plasticity, tissue compliance and high-efficiency (re) availability; the characteristics of the three-dimensional biological scaffold are very beneficial to the growth of new cells and the formation of new tissues; the degradation and the regression of the original grid structure, the generation and the reconstruction of new extracellular matrix and the transition between the new and the old are naturally and smoothly realized, and the new and the old can be efficiently and organically unified with each other to thoroughly organize the fusion and the functional reconstruction.

Secondly, the following steps: the grid structure of the acellular matrix can reserve various cell growth factors and active functional components to the maximum extent. Functionally, the active ingredients have good inducing and chemotactic effects on the stressed cells at the damaged part and assist in creating a good extracellular microenvironment; help the cells restore normal metabolism and promote the growth, differentiation and maturation of the cells; such as fibronectin, has cell adhesion effect; the fibroblast growth factor has the functions of regulating cell polarity, differentiation and the like.

Thirdly, the method comprises the following steps: the grid structure of the acellular matrix has a natural slow release effect. The grid-shaped structure can temporarily 'net or bind' various growth factors and active ingredients secreted by protocells; on one hand, low loss and low loss of the active factors are ensured, and high fidelity and high effectiveness of the active factors are ensured; on the other hand, the reticular structure can lead the cell factors to be released in batches, slowly and continuously, thus achieving the effect of natural long-acting; rather than a high concentration for a short period of time, which may cause undesirable irritation; the natural slow release mode can lead the growth and differentiation of new cells to continuously obtain the positive support of exogenous growth factors, and promote the growth of the new cells and the reconstruction of new tissues.

Fourthly, the method comprises the following steps: the ordered grid structure of the acellular matrix is beneficial to endogenous new cells, particularly small stem cells or red blood cells and the like to freely migrate along a certain way, such as a line or a track, so that unnecessary 'collision' and disordered competition among host cells are reduced; the inhibition of the cells for competing for space resources and the inhibition of competing for nutrient resources are avoided, and the rapid growth, differentiation and maturation and tissue reconstruction and regeneration of the cells can be more effectively promoted.

Compared with the existing bone meal products, the novel composite bone meal has the specific advantages mainly expressed in the aspect of efficacy and performance.

The novel composite bone powder of the invention not only retains the basic advantages of the first generation (traditional) bone powder product such as good biocompatibility, promotion of calcium and phosphorus absorption, increase of bone mass and the like; meanwhile, the bone adhesion and growth of bone cells can be induced, which is beneficial to the delivery of nutrient substances and can promote the regeneration of bone tissues and the repair of injuries; the bone powder and the acellular matrix form a microenvironment beneficial to bone cell regeneration together under the optimized combination and synergistic effect of the two components; the good microenvironment is very beneficial to the rapid repair of new tissue structures and the overall rehabilitation of functions.

The novel composite bone powder can better promote chemotaxis, attachment, growth, differentiation and maturation of osteocytes and help osteocyte bone tissues to grow better; thereby promoting the healing and rehabilitation of bones, shortening the medical treatment time, reducing the pain of patients and saving the medical treatment expense.

In addition, in terms of safety: the selected pharmaceutical-grade bone powder and acellular matrix powder are approved and mature tissue regeneration repair materials, and the safety of the materials is generally approved. The medical pure bone powder is a natural porous structure, belongs to an inorganic phase, and mainly comprises hydroxyapatite or tricalcium phosphate; acellular matrix powder also belongs to a porous latticed structure and belongs to an organic phase; the bone meal and the acellular matrix powder can not generate any chemical reaction, the bone meal can be uniformly mixed with the acellular matrix powder, and a part of the matrix powder can be adsorbed by the porous bone meal; even if some adsorption occurs, the surface contact is also the weak connection which is easy to separate, the bone meal and the acellular matrix powder can not generate any chemical reaction, and the compatibility is good; meanwhile, the two components can not cause immune reaction, and both have no pyrogen, so that the safety of the product is sufficiently guaranteed.

In terms of convenience of use: because the final product is of powder type, the appearance of the product is not greatly different from that of the conventional bone meal, but the color is slightly different. The composite bone powder is still very convenient to use, and the composite bone powder can be molded into any preset shape by using a curable material according to the requirement, so that the diversified requirements of bone defect repair at different parts and different degrees in clinic can be met, and the composite bone powder can also be used as a carrier of bone tissue engineering.

In terms of product stability: the medical pure bone powder is inorganic phase, and has extremely low water content, which is usually less than 0.4%; acellular matrix powders, although organic, have a low water content, typically less than 4%; the water content of the composite bone meal is about 0.5 percent, the composite bone meal and the composite bone meal do not have any chemical reaction, and the composite bone meal have excellent compatibility, so that the stability of the composite bone meal product is sufficiently ensured.

In addition, the compound bone powder is processed by pure biodegradable materials, is completely degradable, absorbable and utilizable, and has no residue; no foreign body is left in the body, and the body does not need to be taken out after a secondary operation.

The novel composite bone powder and the preparation method thereof have the following specific operation steps:

the production method of the compound bone powder comprises the steps of uniformly mixing the bone powder (raw material I), the acellular matrix powder (raw material II) and the adhesive (raw material III) in a mechanical stirring manner, and then carrying out low-temperature freeze-drying, subpackaging, sterilizing, packaging and the like.

Furthermore, in the preferred composite bone powder, 70-90% (W/W) of the bone powder raw material and 5-15% (W/W) of acellular matrix powder are selected;

furthermore, the bone powder raw material in the composite bone powder is prepared by calcining, processing and mineralizing mammal cancellous bone at high temperature, mainly using hydroxyapatite or tricalcium phosphate, mechanically crushing, processing and sieving.

Further, the particle size of the processed bone meal is 0.1-8 mm, and the preferred particle size is 0.2-2.5 mm.

Furthermore, the acellular matrix is prepared by taking mammal tissues or organs as raw materials and carrying out the steps of acellular, virus killing, DNA removal and the like.

Further, preferred tissues or organs are small intestine submucosa, bladder submucosa, stomach submucosa, dermal matrix, pericardium, meninges, amnion, organ membrane, peritoneal pericardium, and the like.

Further, preferred animal tissues are small intestine submucosa tissues of adult swine.

Furthermore, the acellular matrix powder is prepared by grinding and crushing the acellular matrix in an ultralow temperature environment.

Further, the grinding is carried out by an ultra-low temperature freezing grinder, the frequency is 10-30 revolutions per second, and the time is 15-20 minutes each time.

Further, the size of the acellular matrix powder is 0.1-5 mm, and the preferable size is 0.1-2 mm.

Furthermore, the acellular matrix powder is of a three-dimensional grid structure, and the diameter or the aperture of the grid is 20-50 um.

Furthermore, the acellular matrix contains one or more of fibroblast growth factor, vascular endothelial growth factor and transforming growth factor.

Furthermore, the content of at least one of the acellular matrix, the fibroblast growth factor, the vascular endothelial growth factor and the transforming growth factor reaches 40 ng/g.

Further, the acellular matrix powder contains at least 5mg/g of glycosaminoglycan.

Further, the adhesive is common medical adhesive or collagen gel, is prepared from animal tissue by extracting, purifying, salting out, dialyzing, or can be prepared from commercial collagen sponge by adding water, and the concentration of collagen is 0.3-5%.

Raw material I: the processing and preparation method of the bone meal is briefly introduced as follows:

firstly, placing the animal bone powder raw material which is subjected to pretreatment such as degreasing, deproteinization and the like, such as cattle, pig or sheep bones and the like, into a high-temperature furnace to calcine for 2-3 times at the temperature of 600-; and (4) sorting the bone meal particles with different sizes and grades by using a screen, drying again, and sterilizing for later use. The calcined bone powder is mainly composed of hydroxyapatite or tricalcium phosphate.

And (2) raw material II: the preparation method of the acellular matrix powder is briefly introduced as follows:

(1) pretreatment: taking tissues or organs of corresponding animals (such as organs containing tissues of small intestine submucosa, bladder submucosa, stomach submucosa, dermal matrix, pericardium, meninges, amnion, visceral membrane, peritoneum and pericardium) and repeatedly washing with water until the tissues are completely cleaned, soaking with acetic acid solution, and removing other tissues such as mucosa, muscle layer, serous layer, lymph node and other unwanted tissues of small intestine jejunum by using a physical scraping method; separating to obtain relatively pure connective tissue, and washing with purified water for at least 3 times to obtain acellular matrix material.

(2) Virus inactivation: soaking the raw material of the acellular matrix in a mixed aqueous solution containing peroxyacetic acid and ethanol under the ultrasonic condition and the room temperature condition to inactivate viruses; and then ultrasonically cleaning for at least 3 times by using purified water.

(3) Degreasing: soaking the raw materials obtained in the last step by using solvents such as ethanol and the like at room temperature under the ultrasonic condition; and then ultrasonically cleaning for at least 3 times by using water.

(4) Decellularized, DNA-removed and alpha-Gal antigens

Soaking the raw material obtained in the previous step at 37 ℃ for 20 minutes by using a solution containing 0.02% of trypsin and 0.02% of EDTA under the ultrasonic condition; and ultrasonically washed 3 times by using PBS.

Soaking the raw material obtained in the previous step in a solution containing DNA enzyme at 37 ℃ under the ultrasonic condition; followed by 3 ultrasonic washes using PBS rinsing.

Soaking the raw material obtained in the previous step in a solution containing alpha-galactosidase at 30 ℃ under ultrasonic conditions, and ultrasonically cleaning for 3 times by using PBS.

Soaking the raw materials obtained in the previous step in 20mM NaOH solution at normal temperature under ultrasonic condition, and then ultrasonically cleaning with PBS until the raw materials are neutral.

(5) Freeze drying, processing, shaping, pulverizing, and sterilizing

Fixing the processed sheet acellular matrix on a mold, freeze-drying, grinding by using an ultralow temperature freeze grinder to reach the particle size of less than 1000um, and then sub-packaging and sterilizing.

Raw material III: the adhesive is collagen gel, and the raw material is used for uniformly adhering bone powder and acellular matrix powder, and the collagen has no side effect and can be completely absorbed to help the formation of new collagen. The density difference between the bone meal and the acellular matrix powder is large, if the bone meal and the acellular matrix powder are simply and physically mixed, the bone meal and the acellular matrix powder are not easily mixed uniformly, and the two components are easily layered, so that the collagen gel is adopted for adhesion; the collagen gel is prepared from animal tissues such as bovine achilles tendon, pig dermis and other tissue raw materials by the steps of extraction, purification, salting out, dialysis and the like, and can also be prepared from commercialized collagen sponge by adding water; when in use, the concentration of the collagen in the collagen gel is 0.3-5%.

Drawings

FIG. 1 is a sectional view of the tissues of the experimental group of the eighth example.

FIG. 2 is a sectional view of the control group in the eighth embodiment.

FIG. 3 is a three-dimensional scanning image of Micro-CT of the experimental group of the eighth embodiment.

FIG. 4 is a three-dimensional scan of Micro-CT of the control group of example eight.

Detailed Description

In order to more accurately represent the technical solution of the present invention, the technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

The first embodiment is as follows: processing and preparation method of bovine-derived bone meal

Firstly, putting the ox cancellous bone which is subjected to pretreatment such as degreasing, deproteinization and the like into a muffle furnace, calcining for 3 times at about 950 ℃ for 3 hours each time to obtain a powder block of the calcined bone, and mechanically crushing the powder block into fine particles with the size of about 0.35 mm; and (5) drying again, and sterilizing for later use.

Example two: processing and preparation of pig source bone meal

Firstly, placing the pig cancellous bone which is subjected to pretreatment such as degreasing, deproteinization and the like into a muffle furnace to calcine for 2 times at about 850 ℃ for 4 hours each time to obtain a powder block of the calcined bone, and mechanically crushing the powder block into fine particles with the size of about 0.25 mm; and (5) drying again, and sterilizing for later use.

Example three: preparation of porcine small intestine acellular matrix powder

(1) Pretreatment: taking fresh small intestine of pig, washing with water repeatedly until it is completely clean, soaking in acetic acid solution, and mechanically scraping off unnecessary tissues such as mucosa layer, muscle layer, serous layer, lymph node, etc. of small intestine; separating to obtain pure small intestine submucosa tissue, and washing with purified water at least 3 times to obtain acellular matrix material.

(3) Degreasing: soaking the material obtained in the last step by using ethanol at room temperature under an ultrasonic condition; and then ultrasonically cleaning for at least 3 times by using water.

(4) Decellularized, DNA-removed and alpha-Gal antigens

Soaking the raw material obtained in the previous step in a solution containing alpha-galactosidase at 30 ℃ under ultrasonic conditions, and then ultrasonically cleaning the soaked raw material for 3 times by using PBS (phosphate buffer solution).

(5) Freeze drying, processing, shaping, and sterilizing

Fixing the acellular matrix processed by the four steps on a mould, freeze-drying, grinding by using an ultralow-temperature freeze grinder at the grinding frequency of 20 revolutions per second for 15 minutes each time to obtain fine particles of about 600um, subpackaging, packaging and sterilizing.

Example four: preparation of bovine pericardium acellular matrix powder

(1) Pretreatment: taking fresh bovine pericardium, repeatedly washing with water until the bovine pericardium is completely clean, adding acetic acid solution for soaking, and removing other unnecessary tissues by using a mechanical scraping method; separating to obtain pure bovine pericardium, and washing with purified water at least 3 times to obtain acellular matrix raw material.

(4) Decellularized, DNA-removed and alpha-Gal antigens

Soaking the raw material obtained in the previous step in a solution containing alpha-galactosidase at 30 ℃ under ultrasonic conditions, and ultrasonically cleaning the soaked raw material for 3 times by using PBS (phosphate buffer solution).

(5) Freeze drying, processing, shaping, and sterilizing

Fixing the acellular matrix processed by the four steps on a mould, freeze-drying, grinding by using an ultralow temperature freeze grinder at the grinding frequency of 15 revolutions per second for 20 minutes each time to obtain fine particles of about 800 microns, and then sub-packaging and sterilizing.

Example five: preparation of compound bovine bone powder

The bovine bone powder prepared in the first embodiment and the porcine small intestine acellular matrix powder prepared in the third embodiment are prepared by taking 85 g (85%) of the bovine bone powder, 10 g (10%) of the porcine small intestine acellular matrix powder and 250 g (2% of collagen, 5 g of effective collagen and 5% w/w) of porcine dermal collagen gel according to weight percentage, uniformly mixing by adopting a mechanical stirring manner, and then carrying out freeze-drying, subpackaging, sterilizing, packaging and the like to prepare blocks, or processing the blocks into granules with different sizes.

Example six: preparation of pig compound bone meal

The pig bone powder prepared in the second embodiment and the bovine pericardium acellular matrix powder prepared in the fourth embodiment are mixed uniformly by adopting a mechanical stirring mode according to the weight percentage that the pig bone powder accounts for 75 g (80% w/w), the porcine small intestine acellular matrix powder accounts for 10 g (10%) and the bovine achilles tendon collagen gel accounts for 500 g (the collagen concentration is 3%, the effective collagen is 15 g and accounts for 15% w/w), and then the mixture is subjected to freeze-drying, subpackaging, sterilizing, packaging and the like to form blocks, and can also be processed into granules with different sizes.

Example seven: detection of active ingredients in composite bone meal

And detecting the indexes of the active ingredients of the compound bone meal products prepared in the fifth and sixth embodiments.

The contents of fibroblast growth factor and glycosaminoglycan (GAGs) were measured by ELISA.

The detection results of the compound bone meal sample of the fifth embodiment are as follows: the average value of Fibroblast Growth Factor (FGF) is 4.2 ng/g; glycosaminoglycans (GAGs) have an average value of: 0.65 mg/g.

The detection results of the compound bone meal sample of example six are as follows: the average value of Fibroblast Growth Factor (FGF) is 3.9 ng/g; glycosaminoglycans (GAGs) have an average value of: 0.71 mg/g.

Example eight: animal test of composite bone meal

The composite bone powder prepared in example five was used as a test product, and commercial dental bone powder containing collagen, which was commercially available, was used as a control product. The expected composite bone powder product is used on a human body, and the anatomical size and physiological characteristics of the periodontal tissues of the dog are higher in similarity with those of the human body, so that the using effect of the bone repair material can be better evaluated, and therefore, an animal test model can be set manually to simulate clinical periodontal defects so as to preliminarily evaluate the clinical using effect of the composite bone powder product.

Test animals: adult Beagle dogs, three, around three years old, were tested for three months.

Preparation of the test: firstly, manufacturing alveolar bone defects of a dog, taking third molar teeth on two sides of the lower jaw of the dog as target observation teeth, turning a flap, fully exposing the bone wall on the buccal side, and then removing part of alveolar bone by using a split drill and an osteotome; alveolar bone defects range from: the alveolar bone U-shaped defect with the vertical height of about 5mm and the horizontal height of about 3.5mm exposes the buccal side root surface, and incisures are prepared on the near-far root surface corresponding to the bottom of the bone defect and used as mark points for tissue observation, periodontal ligament and cementum exposed on the root surface above the incisures are scraped, and the root surface is flattened and washed. The experimental and control products were applied to the left and right sides of the mandible of the same dog, respectively.

Test grouping and defect site treatment:

experimental groups: the third molar defect on the left side of the lower jaw of the dog is an experimental part and is filled with the composite bone powder prepared in the fifth embodiment; then covering the trimmed oral cavity biological patch on the surface of the composite bone powder material, and restoring the full-thickness valve crown in a manner that the edge exceeds the peripheral bone wall by 3mm according to the GTR operation requirement, and adopting intermittent suture gum tissue.

Control group: filling a bone powder collagen composite product at the third molar defect part on the right side of the lower jaw of the dog as a control part, covering the trimmed oral biological patch on the surface of a bone repair material, enabling the edge to exceed the peripheral bone wall by 3mm according to the GTR operation requirement, finally restoring the full-thickness valve in a coronal direction, and suturing gum tissues by adopting intermission.

The indexes of test observation recording are as follows:

at the end of the test, hard tissue slice examination and three-dimensional scanning examination are performed on soft and hard tissues including the defect repair part.

Tissue section analysis: staining with methylene blue-acid fuchsin.

The results show that: generally speaking, the defect boundary line of the alveolar bone of the test group is very fuzzy and difficult to be distinguished; some sheet-shaped new bones can be seen at the defect position, fibrous tissues are attached and filled on the surfaces and gaps of the new bones, and the tooth bones are slightly worn, which is shown in figure 1.

The alveolar bone defect boundary of the control group is fuzzy, less flaky new bones can be seen at the defect position, and fibrous tissues are attached and filled on the bone surface and in the gaps, which is shown in figure 2.

The test results can preliminarily show that the alveolar bone growth effect of the test group product is obviously better than that of the control group product.

Meanwhile, the new alveolar bone is measured by Micro-CT three-dimensional scanning, and the result shows that:

the alveolar bone of the experimental group can be filled with a small amount of defects or without defects, and the defect is shown in figure 3; significant defect filling of the alveolar bone was seen in the control group and the binding was tight, see fig. 4.

In addition, the height of the new alveolar bone (distance from the incisal trace at the bottom of the defect to the crest of the new alveolar bone) is shown in Table 1.

Table 1: scanning detection values of observation site and new alveolar bone

The test summary is as follows: preliminarily, the repair condition of the bone-related tissues in the test group is better than that of the alveolar bone in the control group; this can help to show that the compound bone powder of the invention has better inducing bone cell chemotaxis, differentiation, growth and calcification and better promoting effect on the growth and healing of bone tissue compared with the control group product because of adding the acellular matrix powder.

The contents of the present invention are not limited to those listed in the above embodiments, which are only typical examples. The composite bone powder material containing the acellular matrix prepared by the invention not only can keep good performance of bone powder and provide collagen, but also can be beneficial to inducing adhesion, differentiation and growth of osteocytes, and better promote bone tissue repair.

The same inventive idea and direct inspiration are obtained by those skilled in the art through reading the description of the present invention, and without any creative thinking, only simple equivalent replacement is adopted for the technical scheme of the present invention, such as only adding additional cell growth factors and/or active functional components, or repeatedly adding various exogenous cell growth promoting factors, and such simple replacement or change is all within the protection scope of the claims of the present invention. Meanwhile, the idea of the invention can be simply extended to the application field of other orthopedic repair materials; namely, the bone powder can be applied to other orthopedic fields only by simply adjusting the proportion of the bone powder and the matrix removing powder. Meanwhile, the two types of raw materials of the invention have wide sources and low cost, the preparation method of the semi-finished product is not complicated, and the production process of the composite bone powder is simple; is suitable for large-scale industrialized production, and can reduce the economic burden of patients.

Claims

1. The composite bone powder for bone tissue repair is characterized by comprising acellular matrix powder, bone powder and an adhesive.

2. The composite bone meal according to claim 1, wherein the weight of the acellular matrix powder is 5-20%, and the weight of the bone meal is 70-90%.

3. The composite bone meal according to claim 1, wherein the composite bone meal contains at least 0.5mg/g glycosaminoglycan.

4. The acellular matrix powder according to claim 1, wherein the acellular matrix is a powder having a particle size of 0.2-2 mm.

5. The acellular matrix powder according to claim 1, wherein the acellular matrix powder is obtained by physical pulverization at ultra-low temperature.

6. The acellular matrix powder according to claim 1, wherein the acellular matrix powder is prepared by mixing one or more of mammalian dermis, pericardium, blood vessel, bladder and small intestine submucosa.

7. The acellular matrix powder according to claim 1, wherein the acellular matrix powder is prepared by carrying out acellular processing on porcine small intestine submucosa by a pancreatin and EDTA mixed solution, and then carrying out low-temperature crushing.

8. The acellular matrix powder according to claim 1, wherein the acellular matrix powder is prepared by acellular processing of porcine small intestine submucosa; the acellular matrix powder at least contains one or more of fibroblast growth factor, vascular endothelial growth factor and transforming growth factor.

9. The bone meal according to claim 1, wherein the bone meal is prepared by calcining and pulverizing a raw material of mammal cancellous bone, and the main component of the bone meal is hydroxyapatite and/or tricalcium phosphate.

10. A production method of composite bone powder for bone tissue repair is characterized by comprising the following steps,

1): preparing bone powder, namely taking cancellous bone of mammals as a raw material, and calcining and crushing the cancellous bone to obtain the bone powder;

2): preparing acellular matrix powder, namely taking small intestine submucosa, bladder submucosa, stomach submucosa, dermal matrix, pericardium, meninges, amnion, visceral membrane, peritoneal pericardium and the like of mammals, processing the materials by using the processes of acellular treatment and the like, drying the materials, and grinding and crushing the materials in an ultralow temperature environment; the grinding frequency of the ultra-low temperature freezing grinder is 10-30 revolutions per second, and the grinding time is 15-20 minutes each time;

3): mixing bone powder, acellular matrix powder, and binder, stirring, lyophilizing, packaging, sterilizing, and packaging.