CN111821518B - Preparation method of tissue engineering bone with regeneration capacity - Google Patents
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- CN111821518B CN111821518B CN201910297993.6A CN201910297993A CN111821518B CN 111821518 B CN111821518 B CN 111821518B CN 201910297993 A CN201910297993 A CN 201910297993A CN 111821518 B CN111821518 B CN 111821518B
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Abstract
The invention discloses a preparation method of a tissue engineering bone with regeneration capacity, which comprises the following steps: s1, preparing a collagen matrix; s2, preparing mesenchymal stem cells; s3, preparing bone marrow; s4, culturing and preparing the semi-finished tissue engineering bone; and S5, culturing and preparing the tissue engineering bone with regeneration capacity. The method has short osteogenesis induction time, high safety and low cost, is not only suitable for simple bone tissue preparation, but also can completely fuse the prepared tissue engineering bone with the in-vivo tissue after being embedded into the body so as to grow into a part of the tissue engineering bone, achieves the aim of tissue regeneration, completely avoids the problem that the growth and the degeneration of a fixed tissue material and an organism can not be synchronous, can also carry out shaping, splicing and in-vivo maturation on the tissue engineering bone, and is an ideal final product and material product of regenerative medicine.
Description
Technical Field
The invention relates to the technical field of medical materials, in particular to a preparation method of a tissue engineering bone with regeneration capacity.
Background
Bone loses some bone mass due to certain factors such as trauma, infection, tumor removal or congenital disease, creating a large gap called a bone defect. Bone defects are common in clinic, and the treatment of the bone defects is always a difficulty in clinical treatment. The bone grafting provides possibility for solving the problems, and the autologous bone is still the first choice of bone grafting source at present, but the supply of the autologous bone is limited, the dilemma of 'dismantling the east wall and supplementing the west wall' easily occurs during application, and the clinical requirement is difficult to meet. Therefore, finding a suitable bone graft substitute has been a primary problem in the study and repair of bone defects.
The tissue engineering bone technology is to inoculate seed cells on various support materials, and the secretion of extracellular matrix and the absorption of the materials are coordinated, so as to finally form a new tissue which is composed of the cells and the extracellular matrix and has the same shape as the material, thereby achieving the good repair of the structure and the function. With the development of the technology, absorbable and tissue-compatible materials are also used for treating bone defects, but at present, tissue engineering materials and tissue engineering bones are difficult to form blood vessels and form a balanced state of bone formation and absorption, regenerated tissues and normal tissues still have great difference in structure and function, the generation of blood vessels and the reconstruction of bone marrow are difficult to complete in the bone defect treatment, and the missing functions cannot be completely replaced.
Disclosure of Invention
In order to solve the above problems of the prior art, it is an object of the present invention to provide a method for preparing a tissue-engineered bone having a regenerating ability, which comprises preparing a regenerated tissue-engineered bone having angiogenesis and myelogenesis abilities, reconstructing the bone in the case that some bone materials are completely lost in vivo, and growing the bone into a functional bone material having bone marrow and blood vessels under a certain condition after being implanted in vivo.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a preparation method of a tissue engineering bone with regeneration capacity, which comprises the following steps:
s1, preparing a collagen matrix: adding aseptic, bioactive and undenatured type I collagen, type II collagen or compound collagen into a sterile tube, adding a low-sugar DMEM culture medium precooled at 4 ℃, fully mixing at 4 ℃, and standing for later use;
s2, preparing mesenchymal stem cells: selecting human mesenchymal stem cells with good state according to the ratio of 1 multiplied by 10 7 Individual cell/cm 2 Suspending the cell suspension in an induction culture medium to obtain a cell suspension;
s3, preparing bone marrow: suspending fresh bone marrow with 5 times of physiological saline, centrifuging, removing supernatant, and fully suspending the lower layer bone marrow precipitate with physiological saline to obtain bone marrow suspension;
s4, culturing and preparing the semi-finished tissue engineering bone: adding the collagen matrix obtained in the step S1 into the cell suspension obtained in the step S2, fully mixing, transferring the collagen matrix into a dialysis bag, fixing the dialysis bag, longitudinally penetrating through the dialysis bag by using an injection needle, and inserting a tubular semipermeable membrane into the perforated part for assembling; after the assembly is finished, putting the dialysis bag into a culture vessel, adding sufficient low-sugar DMEM induction culture medium, and culturing to obtain a semi-finished tissue engineering bone;
s5, culturing and preparing the tissue engineering bone with regeneration capacity: and (3) removing the dialysis bag in the culture vessel in the step (S4), extracting the tubular semipermeable membrane in the dialysis bag, adding the bone marrow suspension obtained in the step (S3) into the hole left after the tubular semipermeable membrane is extracted, then placing the dialysis bag in the culture vessel, adding a low-sugar DMEM induction culture medium containing 10% serum substitute to immerse the tissue, continuously culturing for 24 hours in an incubator at 37 ℃ and with the carbon dioxide content of 5%, and then removing the dialysis bag at the outer side to obtain the tissue engineering bone which is transplantable and has regeneration capacity.
Further, in step S4, the specific culture process is: after the assembly is finished, putting the dialysis bag into a culture vessel, adding sufficient low-sugar DMEM induction culture medium, immersing all tissues, injecting a tubular semipermeable membrane into the culture vessel, and sucking the induction culture medium to drive bubbles; and then, placing the culture vessel in an incubator with the temperature of 37 ℃ and the carbon dioxide content of 5% for culture, slowly sucking out culture supernatant after 48 hours, then adding a fresh low-sugar DMEM induction culture medium for continuous culture, replacing a fresh induction culture solution every 48 hours, and continuously culturing for 10 days to obtain the semi-finished tissue engineering bone.
Further, in the step S4, the length of the dialysis bag is matched with the length of the required tissue engineering bone, the diameter of the dialysis bag is 8mm, the cut-off molecular weight of the dialysis bag is 1000KD, and the diameter of the tubular semipermeable membrane is 5mm.
Further, in the step S2, the induction medium is a low-sugar DMEM medium containing glutamine, ascorbic acid, sodium pyruvate, dexamethasone, sodium beta-glycerophosphate, and a serum substitute.
Further, the human mesenchymal stem cells in the step S2 are selected from bone marrow, fat, skin, dental pulp, umbilical cord or amnion.
Further, the fresh bone marrow in step S3 is extracted from human, rat or mouse bone marrow by EDTA anticoagulation.
Compared with the prior art, the invention has the following beneficial effects:
the invention uses collagen with bioactivity as an engineering scaffold material, and the material can be absorbed and reconstructed by tissues after special treatment without inducing immune response; the tissue engineering cells select mesenchymal stem cells, the cells of the type have wide sources and no immunogenicity, and meanwhile, the cells of the type can be used, so that the immunity and other biological risks are further reduced; the bone marrow is directly transplanted into the semi-finished tissue engineering bone, so that the product has osteoblasts and osteoclasts, can reconstruct the hematopoietic capacity of the bone marrow and can ensure that the bone marrow has angiogenesis, and meanwhile, a permeable membrane system is used inside and outside in the process of preparing the tissue engineering bone, so that the nutrition supply in the process of bone formation is ensured, and the apoptosis and death of cells are avoided.
The method has the advantages of short osteogenesis induction time, high safety and low cost, is suitable for simple bone tissue production, and can produce an inner pipeline system with more branches and more complexity according to actual needs; the tissue engineering bone prepared by the method can be completely fused with the tissues in vivo after being embedded in the body, and further grows into a part of the bone, so that the aim of tissue regeneration is fulfilled, the problem that fixed tissue materials and the growth and degeneration of the body cannot be synchronous is completely avoided, the tissue engineering bone can be shaped, spliced and matured in the body, and the bone is an ideal final product and material product for regenerative medicine.
Detailed Description
The present invention will be described in detail with reference to the following embodiments.
The embodiment of the invention discloses a preparation method of a tissue engineering bone with regeneration capacity, which comprises the following steps:
s1, preparing a collagen matrix: adding 200 mg of sterile, bioactive and undenatured type I collagen, type II collagen or compound collagen into a 5mL sterile tube, adding 1mL of low-sugar DMEM culture medium precooled at 4 ℃, fully mixing at 4 ℃, and standing for later use;
s2, preparing mesenchymal stem cells: selecting good-state human mesenchymal stem cells (1 × 10) from patient fat 7 Individual cell/cm 2 Suspending the cell suspension in an induction culture medium to obtain a cell suspension;
s3, preparing bone marrow: extracting human bone marrow by EDTA anticoagulation to obtain fresh bone marrow, suspending fresh bone marrow with 5 times of physiological saline, centrifuging for 5 min at 300g, discarding supernatant, and suspending the lower layer bone marrow precipitate with physiological saline to obtain bone marrow suspension;
s4, culturing and preparing the semi-finished tissue engineering bone: adding the collagen matrix obtained in the step S1 into the cell suspension obtained in the step S2, fully mixing, moving the cell suspension into a dialysis bag with the diameter of 8mm and the cutoff molecular weight of 1000KD, fixing the dialysis bag, longitudinally penetrating through the dialysis bag by using an injection needle, and inserting a tubular semipermeable membrane with the diameter of 5mm into a perforated hole for assembling; after the assembly is finished, putting the dialysis bag into a culture vessel, adding sufficient low-sugar DMEM induction culture medium, immersing all tissues, injecting a tubular semipermeable membrane into the culture vessel, and sucking the induction culture medium to drive bubbles; and then placing the culture vessel in an incubator at 37 ℃ and with the carbon dioxide content of 5 percent for culture, slowly sucking out culture supernatant after 48 hours, then adding a fresh low-sugar DMEM induction culture medium for continuous culture, replacing fresh induction culture medium every 48 hours, and continuously culturing for 10 days to obtain the semi-finished tissue engineering bone.
S5, culturing and preparing the tissue engineering bone with regeneration capacity: removing the dialysis bag in the culture vessel in the step S4, extracting the tubular semipermeable membrane in the dialysis bag, adding the bone marrow suspension obtained in the step S3 to the hole left after the tubular semipermeable membrane is extracted until the hole is filled, then placing the dialysis bag in the culture vessel, adding a low-sugar DMEM induction culture medium containing 10% of serum substitute to immerse the tissue, continuously culturing for 24 hours in an incubator at 37 ℃ and with the carbon dioxide content of 5%, and then removing the dialysis bag at the outer side to obtain the tissue engineering bone which is used in a transplantable way and has the regeneration capacity;
s6, using the tissue engineering bone: the obtained tissue engineering bone can be directly transplanted to a bone defect part in vivo after the size and the shape of the bone are trimmed according to actual requirements; or matching with other tissue engineering bones, etc. to be made into a target shape and then transplanted. Clinical experiments prove that the tissue engineering bone can be transplanted to a defective bone of a bone defect animal model or to a muscle, kidney and other parts with rich blood supply, and a functional bone tissue can be formed about 30 days after the tissue engineering bone is transplanted into a body.
S7, storing the tissue engineering bone: the tissue engineering bone tissue is suitable for immediate use after being prepared, is not suitable for being stored for too long time, and can be stored for 12 hours at 4 ℃.
Wherein the induction culture medium is a low-sugar DMEM culture medium containing glutamine, ascorbic acid, sodium pyruvate, dexamethasone, beta-sodium glycerophosphate and a serum substitute.
The invention uses collagen with bioactivity as an engineering scaffold material, and the material can be absorbed and reconstructed by tissues after special treatment without inducing immune response; the tissue engineering cells select mesenchymal stem cells, the cells of the type have wide sources and no immunogenicity, and self cells can be used, so that the immunity and other biological risks are further reduced; bone marrow is directly transplanted into the semi-finished tissue engineering bone, so that the product has osteoblast and osteoclast, can reconstruct the hematopoietic capability of the bone marrow and can generate blood vessels, and meanwhile, a permeable membrane system is used inside and outside in the process of preparing the tissue engineering bone, so that the nutrition supply in the process of generating the bone is ensured, and the apoptosis and the death of the cells are avoided.
The method has short osteogenesis induction time, high safety and low cost, is suitable for simple bone tissue manufacture, and can manufacture an inner pipeline system with more branches and more complexity according to actual needs; the tissue engineering bone prepared by the method can be completely fused with the tissues in vivo after being embedded in the body, and further grows into a part of the bone, so that the aim of tissue regeneration is fulfilled, the problem that fixed tissue materials and the growth and degeneration of the body cannot be synchronous is completely avoided, the tissue engineering bone can be shaped, spliced and matured in the body, and the bone is an ideal final product and material product for regenerative medicine.
The present invention has been described in terms of specific examples, which are provided to aid in understanding the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.
Claims (5)
1. A preparation method of a tissue engineering bone with regeneration capability is characterized by comprising the following steps:
s1, preparing a collagen matrix: adding aseptic, bioactive and undenatured type I collagen, type II collagen or compound collagen into a sterile tube, adding a low-sugar DMEM culture medium precooled at 4 ℃, fully mixing at 4 ℃, and standing for later use;
s2, preparing mesenchymal stem cells: selecting the human mesenchymal stem cells with good state according to the ratio of 1 multiplied by 10 7 Individual cell/cm 2 Suspending the cell suspension in an induction culture medium to obtain a cell suspension;
s3, preparing bone marrow: suspending fresh bone marrow with 5 times of physiological saline, centrifuging, removing supernatant, and fully suspending the lower layer bone marrow precipitate with physiological saline to obtain bone marrow suspension;
s4, culturing and preparing the semi-finished tissue engineering bone: adding the collagen matrix obtained in the step S1 into the cell suspension obtained in the step S2, fully mixing, transferring into a dialysis bag, fixing the dialysis bag, longitudinally penetrating through the dialysis bag by using an injection needle, and inserting a tubular semipermeable membrane into a perforation for assembling; after the assembly is finished, putting the dialysis bag into a culture vessel, adding sufficient low-sugar DMEM induction culture medium, immersing all tissues, injecting a tubular semipermeable membrane into the culture vessel, and sucking the induction culture medium to drive bubbles; then placing the culture vessel in an incubator with the temperature of 37 ℃ and the carbon dioxide content of 5% for culture, slowly sucking out culture supernatant after 48 hours, then adding a fresh low-sugar DMEM induction culture medium for continuous culture, replacing a fresh induction culture solution every 48 hours, and continuously culturing for 10 days to obtain a semi-finished tissue engineering bone;
s5, culturing and preparing the tissue engineering bone with regeneration capacity: and (5) removing the dialysis bag in the culture vessel in the step (S4), extracting the tubular semipermeable membrane in the dialysis bag, adding the bone marrow suspension obtained in the step (S3) into the hole left after the tubular semipermeable membrane is extracted, then placing the dialysis bag in the culture vessel, adding a low-sugar DMEM induction culture medium containing 10% serum substitute to immerse the tissue, continuously culturing for 24 hours in an incubator at 37 ℃ and with the carbon dioxide content of 5%, and then removing the outer dialysis bag to obtain the tissue engineering bone with regeneration capacity and capable of being transplanted.
2. The method for preparing tissue engineering bone with regeneration capability of claim 1, wherein in step S4, the length of dialysis bag is matched with the length of required tissue engineering bone, the diameter of dialysis bag is 8mm, the cut-off molecular weight of dialysis bag is 1000KD, and the diameter of tubular semipermeable membrane is 5mm.
3. The method for preparing tissue engineered bone with regeneration ability according to claim 1, wherein in step S2, the induction medium is low sugar DMEM medium containing glutamine, ascorbic acid, sodium pyruvate, dexamethasone, sodium β -glycerophosphate, and serum replacement.
4. The method for preparing a tissue-engineered bone with regenerative capacity according to claim 1, wherein the human mesenchymal stem cells in step S2 are selected from bone marrow, fat, skin, dental pulp, umbilical cord and amniotic membrane.
5. The method for preparing tissue-engineered bone with regeneration capability according to claim 1, wherein the fresh bone marrow in step S3 is bone marrow extracted from human, rat or mouse by EDTA anticoagulation.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005334456A (en) * | 2004-05-28 | 2005-12-08 | Olympus Corp | Implant material for regeneration of biological tissue and method of manufacturing the same |
| CN104096266A (en) * | 2014-07-25 | 2014-10-15 | 中国人民解放军第三军医大学 | Tissue-engineered bone based on entochondrostosis system and construction method thereof |
| CN104225681A (en) * | 2014-09-19 | 2014-12-24 | 浙江大学 | Tissue-engineered bone and preparation method thereof |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006123579A1 (en) * | 2005-05-17 | 2006-11-23 | National University Corporation Nagoya University | Method of preparing cell for bone tissue formation and application of cell for bone tissue formation |
| US9192695B2 (en) * | 2008-11-20 | 2015-11-24 | Allosource | Allografts combined with tissue derived stem cells for bone healing |
| US20160067377A1 (en) * | 2009-12-10 | 2016-03-10 | Allosource | Stem Cell Seeded Natural Substrates and Methods Relating Thereto |
| US8574614B2 (en) * | 2010-02-04 | 2013-11-05 | Hwa-Chang Liu | Surgical grafts for repairing chondral defects |
| CN103768656A (en) * | 2014-01-10 | 2014-05-07 | 中国医学科学院整形外科医院 | Tissue engineered bone constructed from allogeneic bone marrow mesenchymal stem cells and application thereof |
| CN104740686A (en) * | 2015-04-01 | 2015-07-01 | 上海交通大学医学院附属第九人民医院 | Step-by-step tissue engineering bone building method |
| CN105561395B (en) * | 2016-01-05 | 2018-08-21 | 广州医科大学附属第三医院 | A kind of structure of close coupled type study of vascularized tissue engineering bone and its application |
| CN107129969B (en) * | 2017-05-17 | 2021-04-23 | 四川大学 | Method for inducing bone marrow mesenchymal stem cells to directionally differentiate into osteoblasts |
| CN107446885B (en) * | 2017-09-06 | 2021-02-26 | 大连医科大学 | Mesenchymal stem cell in-vitro osteogenic induced differentiation scaffold material and application thereof |
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2019
- 2019-04-15 CN CN201910297993.6A patent/CN111821518B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005334456A (en) * | 2004-05-28 | 2005-12-08 | Olympus Corp | Implant material for regeneration of biological tissue and method of manufacturing the same |
| CN104096266A (en) * | 2014-07-25 | 2014-10-15 | 中国人民解放军第三军医大学 | Tissue-engineered bone based on entochondrostosis system and construction method thereof |
| CN104225681A (en) * | 2014-09-19 | 2014-12-24 | 浙江大学 | Tissue-engineered bone and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 大鼠BMSCs成骨诱导及复合支架材料构建组织工程骨组织的研究;年争好 等;《生物骨科材料与临床研究》;20150228;第12卷(第1期);第1-6页 * |
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