CN107137763B

CN107137763B - Vascularized tissue engineering bone and preparation method thereof

Info

Publication number: CN107137763B
Application number: CN201710308986.2A
Authority: CN
Inventors: 张华林; 周悦丽; 余娜
Original assignee: Ningxia Medical University
Current assignee: Ningxia Medical University
Priority date: 2017-05-04
Filing date: 2017-05-04
Publication date: 2020-05-22
Anticipated expiration: 2037-05-04
Also published as: CN107137763A

Abstract

The invention relates to a vascularized tissue engineering bone and a preparation method thereof, the vascularized tissue engineering bone is composed of a bracket which is composed of poly-lysine modified coral hydroxyapatite, and a vascularized endothelial cell membrane and an osteoblast membrane which are sequentially wrapped on the inner layer of the bracket. The invention uses Polylysine (PLL) to modify Coral Hydroxyapatite (CHA) to obtain an exogenous scaffold, which is beneficial to the creeping and growing of blood vessels and the final vascularization, provides a precondition for the long-term survival of cells in tissue engineering bone and simultaneously reduces the immune rejection and inflammatory reaction of the organism; the adipose-derived mesenchymal stem cells are selected to prepare the double-cell membrane, endothelial cells from different sources are not required to be additionally added, the cell source is rich, the separation and the acquisition are easy, the wound to a donor is small, the ethical principle is not violated, and the culture requirement is low. The preparation method of the vascularized tissue engineering bone is simple, the conditions are mild, the vascularized tissue engineering bone has good osteogenesis and vascularization performance, meets the application requirements in organisms, and has good application prospect as a novel vascularized tissue engineering bone.

Description

Vascularized tissue engineering bone and preparation method thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to a vascularized tissue engineering bone constructed by an osteoblast membrane and a hemangioblast endothelial cell membrane and a preparation method thereof.

Background

The treatment of massive bone defects caused by severe trauma, tumor resection, infection, congenital malformation, etc. is a difficult and enormous challenge for modern medicine, and has been an important subject of continuous and intensive research and exploration for human beings for centuries. At present, the commonly used repairing means in clinic comprise autologous bone transplantation, allogeneic bone transplantation, artificial bone use and the like, but the methods all have certain defects. Autologous bone grafting is a well-known gold standard for bone tissue repair, but patients suffer from trauma of autologous tissue grafting surgery and have limited supply areas, and therefore, autologous bone grafting cannot be regarded as an ideal method for repairing large-area bone defects; the allograft bone transplantation has the risks of immunological rejection, disease transmission and the like, and sometimes even endangers the life of a patient; the artificial bone implantation is easy to cause foreign body rejection reaction, infection and the like. Therefore, there is a need to find a new means for repairing a large bone defect.

Under the condition, the rise and the development of tissue engineering provide new possibility for repairing the bone defect and hope for compensating the defects of the current bone defect treatment method.

In recent years, research on bone tissue engineering has been greatly advanced, and methods for repairing small-sized bone defects by constructing bone tissue in small mammals using tissue engineering techniques have been developed, but for large-sized or poorly blood-supplied bone defects in a recipient area of large mammals, the osteogenic effect has not been stable due to the lack of independent blood supply in the early stage of the graft, insufficient nutrient permeation, slow callus formation, and the like. Research shows that the key to determining and restricting the curative effect of tissue engineering bone in repairing bone defect is the speed and degree of vascularization in vivo. sahota et al also believe that the main reason for failure of tissue engineering implants is due to the delay in vascularization. Therefore, how to establish effective blood supply and construct tissue engineering bone capable of vascularizing in vivo, shortening the bone healing time is an important direction of the current bone tissue engineering research and is also the key for restricting the large-scale clinical application of the tissue engineering bone.

The current vascularization strategies for tissue engineered bone mainly include: designing and developing a stent, applying growth factors, implanting in vivo, and culturing an arterial chamber and a whole culture system in vivo. Although these methods solve the vascularization problem to some extent during tissue construction, they all have disadvantages. For example, in the process of manufacturing the stent, the pore size and the interconnection inside the material are modified, which is beneficial to the ingrowth of the vascular network, but has inflammatory reaction, potential immunogenicity risk and the like, and the vascularization speed is not ideal; the application of the angiogenesis promoting factor has a regulation problem, the half-life period of the growth factor in vivo is short, the application of a large dose has teratogenesis possibility, and certain pathological processes such as hemangioma generation and the like can be accelerated; both the in vivo implantation and the in vivo arterial chamber need secondary operation, donor selection is difficult, potential disease transmission risk exists, and the clinical application is limited; the whole culture system is a perfect artificial organ, but the technical difficulty is high, and the realization is difficult.

In recent years, researchers at home and abroad construct vascularized tissue engineering bones by a technical method of combined culture of vascular endothelial cells and osteoblasts, so that the vascularized tissue engineering bones can be formed and vascularized simultaneously. However, the disadvantages of the conventional "cell-scaffold" strategy are: in the process of planting the seed cells on the scaffold material, 30-40% of the cells can not adhere to the scaffold and are lost, the cell adhesion rate is low, and the utilization rate is poor; the interaction among cells is little, the microenvironment due to the cells in the body is difficult to form, and after the cell scaffold compound is implanted into the body, bone formation always occurs at the periphery of the scaffold, so that the size and the quality of the bone formation are influenced. In order to solve the above problems, cell patch technology has been developed. It is characterized by that it utilizes the in vitro high-density culture of cell to make the cell grow into several layers and secrete lots of extracellular matrixes so as to form cell membrane formed from cell and extracellular matrixes. The cell patch technology is mainly applied to tissue engineering and has three advantages that firstly, the formation of the patch can reduce the loss and damage of cells in the process of tissue construction and improve the utilization rate of the cells; the cell membrane has certain mechanical strength, can be used for tissue construction under the condition of no support, avoids the problem of tissue compatibility caused by support materials, and is easy to operate and low in cost; and thirdly, the abundant extracellular matrix provides a proper growth environment for cells, stores and activates growth factors in the development process, and provides structural and functional help for the development of tissues. Therefore, if the osteoblast membrane can be combined with the endothelial cell membrane to construct the vascularized tissue engineering bone by a proper method, the vascularization and osteogenesis advantages are expected to be better than those of coculture of osteoblasts and endothelial cells.

Adipose-derived stem cells (ADSCs) are adult stem cells present in adipose tissue. Similar to bone marrow mesenchymal stem cells, the mesenchymal stem cells have the multi-differentiation potential of differentiating into adipogenic cells, chondrogenic cells, osteogenic cells, myogenic cells, neurogenic cells and the like, and are ideal seed cells. Compared with the mesenchymal stem cells, the mesenchymal stem cells have the advantages of wide sources, simple acquisition mode, low culture requirement, strong in vitro amplification capacity, short proliferation time, small wound to organisms and the like, and are expected to become more ideal cell sources for tissue engineering. At present, adipose-derived mesenchymal stem cells have been widely used for tissue regeneration and repair. Although the construction of the vascularized tissue engineering bone has been studied, few strategies have been involved in the preparation of a two-cell composite membrane having osteogenic and angiogenetic capabilities by using adipose-derived osteoblasts and endothelial cells.

In addition, in addition to cells, the biocompatibility and biodegradability of the stent material are also factors that influence vascularization. The scaffold material must have a good three-dimensional porous structure, so that blood vessels can creep into the scaffold material and finally become vascularized, and a precondition is provided for long-term survival of cells in the tissue engineering bone.

At present, the bone tissue engineering scaffold material comprises two main types of inorganic materials and organic materials.

Organic materials are first applied to bones in the field of hard tissue repair and replacement, and are widely used as bone repair materials, mainly comprising polylactic acid (PLA), polyacetic acid (PGA), polyglycolide-lactide copolymer (PLGA), poly-epsilon-caprolactone (PCL), polyanhydride, polyphosphazene, polyorthoester and the like. Among the organic materials, much research is done on polyhydroxy acids (mainly including PLA, PGA, and PLGA). This class of high molecular polymers has been approved by the FDA in the united states due to their good biocompatibility, and is widely used in the medical field. The PLGA is a high-molecular copolymer formed by poly PLA and PGA, and the ratio of PLA to PGA is changed, so that the mechanical strength of the PLGA and the degradation time of the PLGA in vivo can be adjusted. PLGA has excellent histocompatibility, has been approved for clinical use by the FDA in the united states, and is one of the most used bone repair materials to date. However, PLGA has poor mechanical strength, degradation products are slightly acidic, and inflammatory reaction in vivo is easily caused, and because PLGA has poor surface hydrophilicity, active functional groups are absent in molecular chains, and the bioactivity is slightly poor, interaction between PLGA and specific cells is difficult.

Inorganic materials for bone tissue engineering scaffolds mainly include Hydroxyapatite (HA), tricalcium phosphate (TCP), and other kinds of ceramic materials. The biological ceramic material is widely used as a bone grafting substitute due to good biological activity and biocompatibility. Although it has the advantages of good biocompatibility, certain degradability, higher chemical stability, stronger bone conduction and bone induction, etc. However, the material has the defects of difficult shaping, insufficient strength, large brittleness, low degradation rate and the like.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the vascularized tissue engineering bone which is constructed by utilizing stem cells and a cell membrane technology, has good osteogenesis and angiogenisis capabilities, abundant cell sources, easy separation and acquisition, small wound to a donor and low culture requirement and effectively reduces immune reactions such as inflammation, rejection and the like generated to a host.

The invention also aims to provide a preparation method of the vascularized tissue engineering bone.

The technical scheme adopted for realizing the aim of the invention is as follows:

the vascularized tissue engineering bone is characterized by being composed of a stent formed by polylysine modified coral hydroxyapatite and a blood vessel forming endothelial cell membrane (inner layer) and an osteoblast membrane (outer layer) which are sequentially wrapped on the surface of the stent.

The endothelial cells for preparing the hemangioblast endothelial cell membrane and the osteoblast for preparing the osteoblast membrane are both derived from adipose mesenchymal stem cells.

The preparation method of the vascularized tissue engineering bone is characterized by comprising the following steps:

1) preparation of poly-lysine modified coral hydroxyapatite

Soaking coral hydroxyapatite in 0.25-1.25 wt% polylysine solution at 0-4 deg.c for 15-24 hr, vacuum filtering, freezing in refrigerator at-20 deg.c overnight, and freeze drying;

2) preparation of osteoblast membrane

Digesting the third generation or fourth generation adipose-derived mesenchymal stem cells with trypsin until most cells become round, falling off from the bottom of the flask, terminating the digestion with DMEM/F12 medium at 1 × 10⁶～3×10⁶Per cm²Inoculating the mixture into a culture dish, adding DMEM/F12 culture medium containing 10-12% fetal calf serum and 1-1.5% double antibody (penicillin and streptomycin), and placing the mixture at 37 ℃ and 5% CO₂Culturing for 24-48 h in a constant-temperature incubator, and then culturing for 10-14 days by using an osteoblast induction culture solution;

3) preparation of vascular endothelial cell membrane

Taking the adipose-derived mesenchymal stem cells of the third generation or the fourth generation according to the ratio of 2 multiplied by 10⁶～4×10⁶Per cm²The density of the culture medium is inoculated in a DMEM/F12 culture medium containing 10-12% fetal calf serum and 1-1.5% double antibody for culturing for 24-48 h, and then the culture medium is continuously cultured for 10-14 days by using a vascular endothelial cell induction solution;

4) construction of vascularized tissue-engineered bone

Winding the angioblasts obtained in the step 3) on the surface of the polylysine modified coral hydroxyapatite scaffold obtained in the step 1) for a circle, then winding the angioblasts obtained in the step 2) outside the angioblasts for a circle to form a coil-like complex, and winding and fixing the coil-like complex by silk threads.

In the above process 1), shaking the soaking solution evenly every 3 hours.

In the process 1), the negative pressure suction filtration is carried out until no bubbles overflow from the surface pores of the coral hydroxyapatite.

In the above process 1), the freeze-drying means: placing the coral hydroxyapatite which is frozen overnight and soaked in the poly-lysine solution into a freeze dryer, pre-cooling for 30min at the temperature of-42 ℃ to-47 ℃ and under the negative pressure of 20-28Pa, then starting to vacuumize, controlling the vacuum degree to be 25-28 Pa, and freeze-drying for 48-72 h.

In the process 2), the osteoblast induction culture solution comprises 170-180 ml of DMEM/F12 culture medium, 17-22 ml of fetal bovine serum, 1.7-2.7 ml of diabase, 2-3 ml of glutamine, 550-580 ul of ascorbic acid, 2-3 ml of β -sodium glycerophosphate and 15-25 ul of dexamethasone.

In the above step 2), the culture is carried out by changing the culture medium every other day when the induction culture medium is used for the culture.

In the above process 3), the vascular endothelial cell-inducing solution consists of: 90-110 ml of endothelial cell growth medium (EGM-2), 4.5-5.5 ml of fetal bovine serum, 0.09-0.11 ml of vitamin C, 0.09-0.11 ml of gentamicin-amphotericin B (GA-1000), 0.036-0.044 ml of hydrocortisone, 0.09-0.11 ml of human epidermal growth factor (hEGF), 0.45-0.55 ml of human fibroblast growth factor (hFGF-B), 0.09-0.11 ml of insulin-like growth factor (IGF-I) and 0.09-0.11 ml of Vascular Endothelial Growth Factor (VEGF).

In the above process 3), in the process of culturing with the vascular endothelial cell inducing solution, the solution culture is changed every 2-3 days.

In the process 4), the polylysine modified coral hydroxyapatite scaffold is soaked in a DMEM/F12 culture medium containing 10-12% fetal calf serum and 1-1.5% double antibody (penicillin and streptomycin) for 12-24 hours before use.

Coral Hydroxyapatite (CHA) is a product obtained by coral through a hydrothermal exchange reaction, retains the characteristics of coral porosity and high porosity, remarkably increases the mechanical strength of the original coral material, and can adjust the in vivo degradation rate of the CHA by controlling the conditions of the hydrothermal exchange reaction. CHA not only overcomes the defects of rapid degradation, fragile texture and the like of natural coral, but also has simple preparation, good osteoinductivity and biocompatibility, can be compounded with various growth factors or molecules such as surface modified protein and the like, and has good application prospect in the aspect of repairing bone defect.

Polylysine (PLL) is a polyvalent cationic polymer formed by lysine monomers, is polymerized from a plurality of amino acid fragments, can be decomposed into lysine in vivo to participate in human metabolism, and has no toxic or side effect, the polylysine is often used for surface modification of bone tissue engineering scaffold materials, the main reasons are that ① lysine residues have positive charges on the surface of the materials and can enhance the adhesion capability of fibroblasts with negative charges, amino groups, hydroxyl groups and the like contained in ② can simulate extracellular matrix, so that the surface activity of the materials is improved, the cell adhesion rate is improved, the cell growth and reproduction are promoted, ③ can promote cartilage formation, ④ is safe and good in tissue compatibility, the decomposers are essential amino acids for human bodies, ⑤ has strong hydrophilicity and can improve the adhesion between cells and the materials, and the like.

According to the idea of material optimization design, Polylysine (PLL) is used for modifying Coral Hydroxyapatite (CHA), so that an exogenous scaffold which is good in biocompatibility and has a three-dimensional porous structure is obtained; the characteristic that the adipose-derived mesenchymal stem cells have the multidirectional differentiation potential is utilized, and the adipose-derived mesenchymal stem cells are induced to differentiate to prepare a double-cell membrane with osteogenic capacity and angiogenisis capacity; fully using stem cell and cell membrane technology and bone tissue development process for reference, discussing the idea and method for compositely constructing the innovative vascularized tissue engineering bone, and finally constructing the vascularized tissue engineering bone with good osteogenic and vascularizing effects by using the double-cell membrane composite PLL/CHA. The technical advantages of the vascularized tissue engineering bone of the present invention are embodied in the following aspects:

1. the exogenous scaffold obtained by modifying Coral Hydroxyapatite (CHA) with Polylysine (PLL) is beneficial to the creeping and growing of blood vessels and the final vascularization, provides a precondition for the long-term survival of cells in tissue engineering bone, and simultaneously reduces the immune rejection and inflammatory reaction of an organism;

2. the adipose-derived mesenchymal stem cells are selected to prepare the double-cell membrane, endothelial cells from different sources are not required to be additionally added, the cell source is rich, the separation and the acquisition are easy, the wound to a donor is small, the ethical principle is not violated, and the culture requirement is low;

3. the preparation method of the vascularized tissue engineering bone is simple, the conditions are mild, the vascularized tissue engineering bone has good osteogenesis and vascularization performance, meets the application requirements in organisms, and has good application prospect as a novel vascularized tissue engineering bone.

4. The successful development of the invention can provide experimental basis and theoretical basis for the development of ideal vascularized tissue engineering bone and the regeneration treatment of massive bone defect, especially the proposal and test of fat stem cell induced double-cell membrane, because of relatively simple operation technology, lower cost, small wound and better osteogenesis and vascularization effect, the application of the vascularized tissue engineering bone has wider prospect to meet the requirement of repairing various clinical hard tissue defects.

Transplanting the vascularized tissue engineering bone prepared by the invention to the back subcutaneous of an immunodeficient nude mouse. After operation, gross observation, SEM and histomorphology analysis are respectively carried out to detect the ectopic osteogenesis capacity and the influence on the blood vessel regeneration. The results show that: at week 8, the stent pores in the experimental group were filled with collagen-like tissue and had vascularity. Areas of varying degrees of calcification appeared at week 12, with extensive vascular cavities visible in the tissue. Masson staining is shown in: the scaffold at the 8 th week is deep in color and high in density, and mainly comprises nascent collagen fibers. At week 12, the fibers were mineralized to different degrees, and a large number of vascular lumens were observed, with sparse collagen fibers. SEM showed that the CHA pores were filled to varying degrees with fibrous connective tissue (. times.100). At 5000-fold, the results show: at week 12, a large amount of hydroxyapatite-like structural substances are formed on the surface of the fiber, the coverage area is wide, and the mineralization is mature. The vascularization tissue engineering bone constructed by the coral hydroxyapatite scaffold and the double-cell membrane has good osteogenic and vascularization performance.

Drawings

FIG. 1 is an SEM image of CHA (left) and PLL/CHA (right);

FIG. 2 is a pictorial representation of an osteoblast membrane (left) and an endothelial membrane (right);

FIG. 3 is a diagram of a vascularized tissue engineering bone object constructed by combining a PLL/CHA scaffold and a bi-cellular membrane;

FIG. 4 shows the implantation of PLL/CHA/double cell patch complex subcutaneously in nude mice;

FIG. 5 is HE staining (x 200) at 8 weeks (left) and 12 weeks (right) of group A transplantation;

FIG. 6 is a scanning electron micrograph of group A at 8 weeks (left) and 12 weeks (right) (. times.100) of transplantation.

Detailed description of the invention

The invention is illustrated below by way of examples, which are to be understood as being illustrative and not limiting. The scope and core content of the invention are to be determined by the claims.

Preparation and characterization of poly-lysine modified coral hydroxyapatite

(1) Primary reagent

Coral Hydroxyapatite (CHA) (china, beijing jeffea-hayata), Polylysine (PLL) (Sigma, usa).

(2) Apparatus and device

Ultrasonic cleaning machine (Kunshan instruments and Equipment works, China), vacuum freeze dryer (Shanghai Bilang instruments, China), s-3400N scanning electron microscope (HITACHI, Japan).

(3) Experimental methods

① PLL soaked CHA

PLL was prepared to a concentration of 0.25% to 1.25% w/v with double distilled water, and sterilized by filtration through a 0.22 μm filter. Soaking the CHA in the prepared PLL solution for 15-24 h at 0-4 ℃, shaking the soaking solution uniformly every 3h to ensure that the PLL and the CHA are in full contact.

② negative pressure suction filtration

Placing the container soaked with CHA in a sterile drying dish connected with a negative pressure suction filtration bottle and a negative pressure suction filtration machine, sealing the joint and the cover of the drying dish, continuously performing negative pressure suction filtration for 5h at 20 ℃ until no bubbles overflow from the pores on the surface of the CHA, taking out the soaking container, and freezing in a refrigerator at-20 ℃ overnight.

③ Freeze drying

And (3) placing the soaking container in a freeze dryer, presetting the temperature of minus 42-minus 47 ℃ and the negative pressure of 20-28Pa, pre-cooling for 30min, and then starting vacuumizing to ensure that the vacuum degree is 25-28 Pa. Freeze drying for 48 hr, weighing and packaging the material, labeling, and storing in a low temperature refrigerator at-20 deg.C.

④ characterization

And taking a composite bracket sample, spraying gold coating on the surface of the composite bracket sample by using an ion sputtering instrument, and observing the surface appearance of the bracket by using SEM.

⑤ results

SEM results show that both CHA and PLL/CHA are porous structures with interconnected pores having a diameter of 200-500 μm. Under a high power lens, spherical hydroxyapatite crystals (with the diameter of 500-4000nm) consisting of leaf crystals are arranged on the CHA surface, and besides the hydroxyapatite crystals, a layer of irregular crystals of PLL is also arranged on the PLL/CHA surface.

Secondly, inducing differentiation by using rabbit ADSCs (adipose-derived mesenchymal stem cells) to respectively construct osteoblast membranes and hemangioblast membranes

(1) Primary reagent

Thiazole Blue (Alarmar Blue) (Shanghai, assist in san Francisco), ascorbic acid (Ascrobic acid) (Beijing Soilebao Biotech Co., Ltd.), Direct Red 80(Direct Red 80) (Sigma in USA), DMEM/F12 medium, Phosphate Buffered Saline (PBS), Fetal Bovine Serum (FBS) (Hyclone in USA).

(4) Apparatus and device

Infinite M200Pro microplate reader (Switzerland, Nano Quan), H-7650 Transmission Electron microscope (Hitachi), s-3400N scanning electron microscope (Hitachi), polarizing microscope DM2500P (Germany, Leica), upright fluorescence microscope BX-511250CCD (Japan, Olympus), CO2 incubator (Heraeus, Germany).

(3) Experimental methods

① preparation of osteoblast membrane

Fourth generation ADSCs (adipose-derived mesenchymal stem cells) with good proliferation state are conventionally digested with 1% trypsin until most of the cells become round and fall off from the bottom of the bottle, and then digestion is stopped with DMEM/F12 medium. Adjusting cell density to 1X 10⁶～3×10⁶Per cm²Inoculating into a culture dish coated with 1% gelatin in advance, adding DMEM/F12 culture medium containing 10-12% fetal calf serum and 1-1.5% double antibody (penicillin, streptomycin), placing at 37 deg.C and 5% CO₂Culturing in a constant temperature incubator, and replacing with osteoblast induction culture solution (DMEM/F12 culture medium 170-180 ml, fetal bovine serum 17-22 ml, double antibiotics (penicillin, streptomycin) 1.7-2.7 ml, glutamine 2-3 ml, ascorbic acid 550-580 ul, β -sodium glycerophosphate 2-3 ml after 24-48 hml, 15-25 ul of dexamethasone), changing liquid every other day, observing the formation process and formation condition of the membrane, continuously culturing for 10-14 days, allowing a semitransparent milky white film sample to be visible at the bottom of the dish, slightly scraping the membrane by using a cell scraper when a plurality of white knots with different sizes exist in the membrane, and separating the membrane from the bottom of the dish to obtain the osteoblast membrane.

② preparation of vascular endothelial cell membrane

Taking the adipose-derived mesenchymal stem cells of the third generation or the fourth generation according to the ratio of 2 multiplied by 10⁶～4×10⁶Per cm²The cell density is inoculated in a DMEM/F12 culture medium containing 10-12% fetal bovine serum and 1-1.5% double antibody (penicillin and streptomycin) for culturing for 24-48 h, and then the culture solution is replaced by vascular endothelial cell induction solution (endothelial cell growth medium (EGM-2) 90-110 ml, fetal bovine serum 4.5-5.5 ml, vitamin C0.09-0.11 ml, gentamicin-amphotericin B (GA-1000) 0.09-0.11 ml, hydrocortisone (hydrocortisone) 0.036-0.044 ml, human epidermal growth factor (hEGF) 0.09-0.11 ml, human fibroblast growth factor (hFGF-B) 0.45-0.55 ml, insulin-like growth factor (IGF-I) 0.09-0.11 ml, Vascular Endothelial Growth Factor (VEGF) 0.09-0.11 ml), cell morphology change is observed and recorded, and the cell morphology change is performed every 2-3 days. The culture is continued for 14 days, and endothelial cell membranes can be obtained.

Third, PLL/CHA/double-cell membrane sheet construction vascularization tissue engineering bone and animal in-vivo ectopic transplantation experiment

(1) Primary reagent

Fast dormancy new II injection (Jilin, Santa), Su 3 injection (Jilin, Santa), Power DryHeto LL3000 vacuum freeze dryer (U.S., Thermo Fisher), the rest of the reagents are analytical.

(2) Apparatus and device

SHZ-D (III) circulating water type vacuum pump (consolidation, sublimation).

(3) Experimental methods

① PLL/CHA/double-cell membrane complex for constructing vascularized tissue engineering bone

Cutting PLL/CHA into cuboid of 0.5 × 2cm, sterilizing with ethylene oxide, and aseptically packaging. Before use, the mixture is soaked in a DMEM/F12 culture medium containing 10-12% of fetal calf serum and 1-1.5% of double antibiotics (penicillin and streptomycin) for 12-24 hours.

The double-cell membranes (osteoblast membrane and hemangioblast membrane) prepared by the above method were carefully scraped from the periphery of the bottom wall of the culture dish by a cell scraper to separate the membrane from the dish.

Winding a blood vessel endothelial cell membrane on the inner surface of a stent (PLL modified coral hydroxyapatite) for wrapping a circle, then winding an osteogenic cell membrane outside the blood vessel endothelial cell membrane for wrapping a circle to form a coil-like complex, winding and fixing the complex by a silk thread, and marking the complex as a complex A, which is an experimental group; the complex formed by wrapping the composite scaffold with the single osteoblast membrane and the single endothelial cell membrane for one circle is respectively marked as a complex B and a complex C, and is used as a control group. Culturing for 3 days for later use.

② transplant experiment for vascularized tissue engineering bone without exogenous scaffold

36 nude mice were randomly divided into a group a, a group B, and a group C, and examined 8 weeks and 12 weeks after subcutaneous implantation of PLL/CHA/double cell patch complex nude mice (n ═ 6/group/time point). Subcutaneous implantation of nude mice: the diluted fast-sleep new liquid is anesthetized by intramuscular injection according to 0.1ml-0.2ml/20g, a transverse incision is made on the back, and the construct is implanted subcutaneously. The experimental group is complex a, and the control group is complex B and patch complex C.

③ characterization

General observation: observing the living state of a nude mouse, the healing condition of a cut and the change of a graft after the tissue engineering bone is implanted; before drawing materials, the relationship between the dermis, the surrounding tissues and the graft is observed.

And (3) histological detection: the removed graft was divided into two portions, one portion was fixed with 2.5% glutaraldehyde for 24 hours, SEM samples were prepared, and the specimen cross-sectional structure was observed. Embedding a part of conventional paraffin, making 5-micrometer continuous sections, selecting 4 sections at the central part of a tissue block, performing HE staining, counting microvessels according to a method reported by Weidner and the like, and performing statistical analysis on the result by using SPSS 20.0; and selecting 4 sections for Masson dyeing, and observing the mineralization condition of the fibers.

④ results

The results show that the two-cell patch complex is tightly attached to the scaffold after being compounded. After the implant is implanted into the subcutaneous part of a nude mouse, no inflammatory reaction is seen in an incision. At week 8 post-surgery, adhesion of tissue surrounding the graft, with capillaries, was seen. At week 12, there was an increase in tissue surrounding the graft and an increase in surface attachment of small blood vessels; the CHA pores were completely filled with yellowish tissue, into which capillaries were visible. HE staining results were as follows: at week 8, the pores of the scaffold were filled with collagen-like tissue, and the tissue was scattered in blood vessels of different distribution numbers, which showed the morphology of erythrocytes and osteoblasts. The tissue was seen to have a large increase in cell numbers at week 12, with A, B groups showing areas of varying degrees of calcification, with group a mineralizing most clearly. No mineralized area was found in group C, and a large number of effectively perfused blood vessels were visible in the tissues. Masson staining results showed: in week 8, the pores of the stent are filled with unequal amounts of collagen fibers, the fibers are sparsely distributed, and blood vessels are formed. No significant differences were seen between the three groups. At week 12, the tissue fibers were seen to be mineralized to varying degrees, with group A being the most evident. In group C, a large number of vascular cavities were visible, with sparse collagen fibers. SEM results showed that at week 8, three groups of CHA pores were filled to varying degrees with fibrous connective tissue. At week 12, the three groups of CHA pores were completely filled with fibrous tissue and the tissue was tightly bound to the scaffold. The microvascular count results show: at 8 and 12 weeks, the differences among A, B, C groups of microvascular numbers have statistical significance, and the differences between the C group and A, B group at two time points have statistical significance (P <0.05), which indicates that the blood vessel forming ability of the C group is obviously higher than that of the other two groups. At week 8, the difference between group A and group B was also statistically significant (P <0.05), indicating that group A had a higher number of microvasculature than group B at week 8. The result shows that the vascularized tissue engineering bone constructed by the PLL/CHA/double-cell membrane complex has good osteogenic and angiogenizing performances.

Claims

1. A vascularized tissue engineering bone is characterized by comprising a bracket formed by polylysine modified coral hydroxyapatite, and a hemangioblast endothelial cell membrane and an osteoblast membrane which are sequentially wrapped on the surface of the bracket, wherein endothelial cells used for preparing the hemangioblast endothelial cell membrane and osteoblast cells used for preparing the osteoblast membrane are both derived from adipose mesenchymal stem cells, and the specific technological process comprises the following steps:

1) preparation of poly-lysine modified coral hydroxyapatite

2) preparation of osteoblast membrane

Digesting the third generation or fourth generation adipose-derived mesenchymal stem cells by trypsin and then stopping digestion by DMEM/F12 culture medium according to the proportion of 1 × 10⁶～3×10⁶Per cm²Inoculating the mixture into a culture dish, adding the mixture into a DMEM/F12 culture medium containing 10-12% fetal calf serum and 1-1.5% double antibody, placing the mixture at 37 ℃ and 5% CO₂Culturing for 24-48 h in a constant-temperature incubator, and then culturing for 10-14 days by using an osteoblast induction culture solution;

3) preparation of vascular endothelial cell membrane

4) construction of vascularized tissue-engineered bone

2. The vascularized tissue-engineered bone according to claim 1, wherein the soaking solution is shaken every 3 hours in the process 1).

3. The vascularized tissue engineered bone of claim 1, wherein in process 1), said negative pressure is suction filtered until no bubbles are visible to escape from the surface pores of the coral hydroxyapatite.

4. The vascularized tissue engineered bone according to claim 1, wherein said freeze-drying in process 1) is: placing the coral hydroxyapatite which is frozen overnight and soaked in the poly-lysine solution into a freeze dryer, pre-cooling for 30min at the temperature of-42 ℃ to-47 ℃ and under the negative pressure of 20-28Pa, then starting to vacuumize, controlling the vacuum degree to be 25-28 Pa, and freeze-drying for 48-72 h.

5. The vascularized tissue-engineered bone according to claim 1, wherein in the process 2), the osteoblast induction culture solution comprises 170-180 ml of DMEM/F12 culture medium, 17-22 ml of fetal bovine serum, 1.7-2.7 ml of diabesin, 2-3 ml of glutamine, 550-580 ul of ascorbic acid, 2-3 ml of β -sodium glycerophosphate, and 15-25 ul of dexamethasone.

6. The vascularized tissue-engineered bone according to claim 1, wherein in the step 2), the culture is carried out in the inducing culture medium by changing the culture medium every other day.

7. The vascularized tissue-engineered bone according to claim 1, wherein in the process 3), the vascular endothelial cell inducing solution consists of: 90-110 ml of endothelial cell growth culture medium, 4.5-5.5 ml of fetal bovine serum, 0.09-0.11 ml of vitamin C, 0.09-0.11 ml of gentamicin-amphotericin B, 0.036-0.044 ml of hydrocortisone, 0.09-0.11 ml of human epidermal growth factor, 0.45-0.55 ml of human fibroblast growth factor, 0.09-0.11 ml of insulin-like I growth factor and 0.09-0.11 ml of vascular endothelial cell growth factor.

8. The vascularized tissue-engineered bone according to claim 1, wherein in the step 3), the culture is performed by changing the culture medium every 2 to 3 days in the course of the culture with the vascular endothelial cell inducing medium.

9. The vascularized tissue-engineered bone according to claim 1, wherein in the step 4), the polylysine-modified coral hydroxyapatite scaffold is immersed in a DMEM/F12 medium containing 10 to 12% fetal bovine serum and 1 to 1.5% diabase for 12 to 24 hours before use.