阅读说明：本技术 一种纳米凹凸棒石的骨修复支架制备方法及其应用 (A kind of bone repairing support preparation method and applications of nano-attapulgite stone ) 是由 李辰凯 秦文 赵红斌 倪新晔 周栋 陈文扬 于 2019-09-30 设计创作，主要内容包括：本发明公开了一种纳米凹凸棒石的骨修复支架制备方法及其应用,包括以下制备步骤：(1)、骨髓间充质干细胞的培养(2)、凹凸棒石纳米的制备(3)、电纺3D打印制备含纳米凹凸棒石骨修复支架(4)、骨髓间充质干细胞的种植本发明基于电纺3D打印技术,采用多材料、多维度方向发展的3D打印技术则为骨软骨修复,利用3D打印技术,可于时间和空间上精确、按需组合不同生理物理特性的材料(包括细胞、具有生物相容性且可降解的水凝胶、生长因子等),从而解决传统治疗方法存在的不足。(The invention discloses a kind of bone repairing support preparation method and applications of nano-attapulgite stone, including following preparation step: (1), the culture (2) of mesenchymal stem cell, the preparation (3) of palygorskite nano, electrospinning 3D printing prepares bone repairing support containing nano-attapulgite stone (4), the present invention is based on electrospinning 3D printing techniques for the plantation of mesenchymal stem cell, using more materials, the 3D printing technique that various dimensions direction is developed then is bone repair of cartilage, utilize 3D printing technique, it can be in the time and spatially accurate, material (including the cell of different physiology physical characteristics is combined on demand, with biocompatibility and degradable hydrogel, growth factor etc.), to solve deficiency existing for traditional therapy.)

1. a kind of bone repairing support preparation method and applications of nano-attapulgite stone, including following preparation step:

(1), the culture of mesenchymal stem cell

(2), the preparation of palygorskite nano

Using frequency conversion type planetary ball mill, calgon is grinding aid and dispersing agent, using pure water as abrasive media, 2000rpm Grinding concave convex rod powder 1~2 hour, by absorption, electrochemical action and chemical reaction, keeping concave-convex crystal fully dispersed is 200 ~500 nano crystals particles.

(3), electrospinning 3D printing prepares bone repairing support containing nano-attapulgite stone

The foundation of (3-1), model

Printer model is established using Auto CAD and Materialise Mimics combination 3D printer software kit, is multiple-layer stand Cube shape, each layer structural path gap are adjusted as needed, and print routine code is exported after preservation.

The preparation of (3-2), printed material

It takes appropriate bioabsorbable polymer material A under mild agitation, is slowly added in dispersant B to mixing.Continue stirring and it is slow The nano-attapulgite of 0.1%~30% (mass volume ratio) is added, dispersion liquid is made and 3D printer electrostatic spinning nozzle is added It is stand-by in barrel.

The parameter configuration of (3-3), printing device

The technological parameter for adjusting melting electrostatic spinning 3D printing equipment, accesses 3D after dispersion liquid made from step 2 is put into barrel Printer is heated to 65 DEG C~80 DEG C, passes through No. 20 syringe needle deliverings, spray head movement speed 25 with 0.8~5mL/hr of constant flow rate ~50mm/s.Needle point position applies 5~20kV of high pressure, and receiving surface applies the negative voltage of 2~15kV.Dispersion liquid is under voltage effect Go out to believe long fluid jet from spray head, when jet stream accelerates to receiving surface, solvent evaporation, electropolymer sweeps along nano-attapulgite Stone is deposited into 3D bracket in receiving surface by program setting.After the completion of printing, -40 DEG C of vacuum freeze-dryings 24~72.

(4), the plantation of mesenchymal stem cell: take appropriate mesenchymal stem cell plantation in each stent area.

2. a kind of bone repairing support preparation method and applications of nano-attapulgite stone according to claim 1, feature Be: the bioabsorbable polymer material A is by polylactic acid, polycaprolactone, poly lactide-glycolide acid and gathers to dioxocyclohex One or more of ketone is constituted.

3. a kind of bone repairing support preparation method and applications of nano-attapulgite stone according to claim 1, feature Be: the dispersant B is one in polyvinyl alcohol, polyethylene glycol, methylene chloride, hexafluoroisopropanol and polyvinylpyrrolidone Kind or a variety of mixtures.

4. a kind of bone repairing support preparation method and applications of nano-attapulgite stone according to claim 1, feature Be: the implantation methods of the mesenchymal stem cell are as follows: removing and train around the mesenchymal stem cell of adherent growth Base is supported, is washed twice using 0.01M PBS, is added and is digested 3 minutes for 0.25% (mass percent) pancreatin in right amount, observation cell is de- Situation is fallen, stops pancreatin effect using serum-containing media in due course.Cell suspension is collected, supernatant is abandoned in centrifugation, after culture medium is resuspended, It is inoculated in each stent area, fresh culture is replaced after 24 hours, hereafter liquid was changed every 2 days and continues to cultivate, to subsequent experimental.

Technical field

The present invention relates to bone cartilage technical field of biological materials, in particular to a kind of bone repairing supports of nano-attapulgite stone Preparation method and applications.

Background technique

Important component of the bone cartilage as joint can avoid intercartilaginous impact and friction.In the special pressure such as joint Cartilage under force environment, influence the problems such as vulnerable to wound, disease and damage and lesion occurs.Its mainly with cartilage cell's nutrition The factors such as metabolism, extracellular matrix are degraded extremely, biomechanical balance is broken are related.Due in cartilaginous tissue lack blood vessel with Nerve, the self-repairing capability after causing cartilage damage is fairly limited, often causes the diseases such as osteoarthritis, causes function of joint Obstacle seriously affects minimal invasive treatment.The inflammation that concomitant injury causes causes Apoptosis, can also be by inhibiting II Collagen Type VI to close At promoting the secreting, expressing of IL-1 β, TNF-α and MMP-13, to indirectly lead to subtracting for cartilage supporting role and protection mechanism It is weak, accelerate the regression of articular cartilage, convert normal cartilage cell to osteoarthritis like cell, or aggravates arthritic pathology Change.Research to the osteocartilaginous and subchondralo bone injury origin cause of formation and reparation a, it has also become research emphasis of regenerative medicine.

Existing osteocartilaginous and subchondralo bone injury restorative procedure mainly has conventional method and 3D printing two major classes.Traditional cartilaginous tissue treatment Method includes conservative therapy and surgical intervention.Wherein, surgical intervention include self cartilaginous tissue cell transplantation, it is microfrature, soft Drilling decompression, joint fusion etc. under bone, but above method there are donors limited, immunological rejection, power of regeneration are weak etc. Problem.

Existing to be applied to clinical metal, ceramics, inorganic composite materials bracket, the physics for being often more conceived to material is special Property, better supportive and durability are pursued under the conditions of meeting biocompatibility, though space is provided for bone repair of cartilage Lack the facilitation to chondrocyte proliferation reparation.In order to solve the above problem, scientific research personnel passes through more materials and use, change material The modes such as structure, decorative material molecular radical and the different crosslinking agents of application of material, have carried out various trials, these stock supports exist Physical characteristic, printing shaping feasibility, osteocyte and the cartilage cell of each material are illustrated in varying degrees in different support material Survival, proliferation on material, differentiation capability.But still have following problems at present: (1) biocompatibility of different materials is different, Some material components under various concentration even larger cytotoxicity；(2) material physical property, degradation property are different, influence soft Osteanagenesis；(3) microenvironment locating for cartilage cell cannot be simulated completely.

Summary of the invention

The technical problem to be solved by the present invention is to provide a kind of bone repairing support of nano-attapulgite stone in view of the above problems Preparation method and applications.

In order to solve the above technical problems, technical solution provided by the invention are as follows: a kind of Bone Defect Repari branch of nano-attapulgite stone Frame preparation method and applications, including following preparation step:

(1), the culture of mesenchymal stem cell

(2), the preparation of palygorskite nano

Using frequency conversion type planetary ball mill, calgon is grinding aid and dispersing agent, using pure water as abrasive media, 2000rpm is ground concave convex rod powder 1-2 hours, by absorption, electrochemical action and chemical reaction, keeps concave-convex crystal fully dispersed For 200-500 nano crystals particle.

(3), electrospinning 3D printing prepares bone repairing support containing nano-attapulgite stone

The foundation of (3-1), model

Printer model is established using Auto CAD and Materialise Mimics combination 3D printer software kit, is more Layer cubic, each layer structural path gap are adjusted as needed, and print routine code is exported after preservation.

The preparation of (3-2), printed material

It takes appropriate bioabsorbable polymer material A under mild agitation, is slowly added in dispersant B to mixing.Continue stirring simultaneously It is slowly added to the nano-attapulgite of 0.1%-30% (mass volume ratio), dispersion liquid is made and the spray of 3D printer electrostatic spinning is added It is stand-by in head barrel.

The parameter configuration of (3-3), printing device

The technological parameter for adjusting melting electrostatic spinning 3D printing equipment, is put into barrel for dispersion liquid made from step 2 and is followed by Enter 3D printer, be heated to 65 DEG C -80 DEG C, No. 20 syringe needle deliverings, spray head movement speed are passed through with constant flow rate 0.8-5mL/hr 25-50mm/s.Needle point position applies high pressure 5-20kV, and receiving surface applies the negative voltage of 2-15kV.Dispersion liquid is under voltage effect Go out to believe long fluid jet from spray head, when jet stream accelerates to receiving surface, solvent evaporation, electropolymer sweeps along nano-attapulgite Stone is deposited into 3D bracket in receiving surface by program setting.After the completion of printing, -40 DEG C of vacuum freeze-drying 24-72.

(4), the plantation of mesenchymal stem cell: take appropriate mesenchymal stem cell plantation in each stent area.

The advantages of the present invention over the prior art are that: the present invention is based on electrospinning 3D printing technique, using more materials, more The 3D printing technique that dimension direction is developed then is bone repair of cartilage, using 3D printing technique, can it is in the time and spatially accurate, press The material of different physiology physical characteristics need to be combined (including cell, with biocompatibility and degradable hydrogel, growth factor Deng), to solve deficiency existing for traditional therapy.

As an improvement, the bioabsorbable polymer material A by polylactic acid, polycaprolactone, poly lactide-glycolide acid and One or more of polydioxanone is constituted.

As an improvement, the dispersant B is polyvinyl alcohol, polyethylene glycol, methylene chloride, hexafluoroisopropanol and polyethylene pyrrole One of pyrrolidone or a variety of mixtures.

As an improvement, the implantation methods of the mesenchymal stem cell are as follows: removing the medulla mesenchyma of adherent growth Stem cell surrounding media is washed twice using 0.01M PBS, is added and is digested 3 points in right amount for 0.25% (mass percent) pancreatin Clock observes cell detachment situation, stops pancreatin effect using serum-containing media in due course.Cell suspension is collected, supernatant is abandoned in centrifugation, After culture medium is resuspended, it is inoculated in each stent area, fresh culture is replaced after 24 hours, hereafter liquid was changed every 2 days and continues to cultivate, To subsequent experimental.

Detailed description of the invention

Fig. 1 is a kind of bone repairing support of the bone repairing support preparation method and applications of nano-attapulgite stone.

Fig. 2 is a kind of microcosmic knot of bone repairing support of the bone repairing support preparation method and applications of nano-attapulgite stone Structure.

Fig. 3 is a kind of bone repairing support electron microscope of the bone repairing support preparation method and applications of nano-attapulgite stone (×100)。

Fig. 4 is a kind of bone repairing support electron microscope of the bone repairing support preparation method and applications of nano-attapulgite stone (×1000)。

Fig. 5 is electron microscope of the cell in rack surface adherency growth.

Fig. 6 is the result figure that CCK8 method detection support cell proliferation influences.

Fig. 7 is that qRT-PCR detects Bone formation-related gene result figure.

Fig. 8 is that Westernblot method detects skeletonization correlative protein expression result figure.

Specific embodiment

Present invention will be described in further detail below with reference to the accompanying drawings.

The present invention in the specific implementation, a kind of bone repairing support preparation method and applications of nano-attapulgite stone, including Following preparation step:

(1), the culture of mesenchymal stem cell

(2), the preparation of palygorskite nano

Using frequency conversion type planetary ball mill, calgon is grinding aid and dispersing agent, using pure water as abrasive media, 2000rpm is ground concave convex rod powder 1-2 hours, by absorption, electrochemical action and chemical reaction, keeps concave-convex crystal fully dispersed For 200-500 nano crystals particle.

(3), electrospinning 3D printing prepares bone repairing support containing nano-attapulgite stone

The foundation of (3-1), model

Printer model is established using Auto CAD and Materialise Mimics combination 3D printer software kit, is more Layer cubic, each layer structural path gap are adjusted as needed, and print routine code is exported after preservation.

The preparation of (3-2), printed material

It takes appropriate bioabsorbable polymer material A under mild agitation, is slowly added in dispersant B to mixing.Continue stirring simultaneously It is slowly added to the nano-attapulgite of 0.1%-30% (mass volume ratio), dispersion liquid is made and the spray of 3D printer electrostatic spinning is added It is stand-by in head barrel.

The parameter configuration of (3-3), printing device

The technological parameter for adjusting melting electrostatic spinning 3D printing equipment, is put into barrel for dispersion liquid made from step 2 and is followed by Enter 3D printer, be heated to 65 DEG C -80 DEG C, No. 20 syringe needle deliverings, spray head movement speed are passed through with constant flow rate 0.8-5mL/hr 25-50mm/s.Needle point position applies high pressure 5-20kV, and receiving surface applies the negative voltage of 2-15kV.Dispersion liquid is under voltage effect Go out to believe long fluid jet from spray head, when jet stream accelerates to receiving surface, solvent evaporation, electropolymer sweeps along nano-attapulgite Stone is deposited into 3D bracket in receiving surface by program setting.After the completion of printing, -40 DEG C of vacuum freeze-drying 24-72.

(4), the plantation of mesenchymal stem cell: take appropriate mesenchymal stem cell plantation in each stent area.

The implantation methods of the mesenchymal stem cell are as follows: removing around the mesenchymal stem cell of adherent growth Culture medium is washed twice using 0.01M PBS, is added and is digested 3 minutes for 0.25% (mass percent) pancreatin in right amount, and cell is observed Fall off situation, stops pancreatin effect using serum-containing media in due course.Cell suspension is collected, supernatant is abandoned in centrifugation, and culture medium is resuspended Afterwards, it is inoculated in each stent area, fresh culture is replaced after 24 hours, hereafter liquid was changed every 2 days and continues to cultivate, to subsequent reality It tests.

The bioabsorbable polymer material A is by polylactic acid, polycaprolactone, poly lactide-glycolide acid and gathers to dioxy One or more of cyclohexanone is constituted.

The dispersant B is in polyvinyl alcohol, polyethylene glycol, methylene chloride, hexafluoroisopropanol and polyvinylpyrrolidone One or more mixtures.

The working principle of the invention: it the present invention is based on electrospinning 3D printing technique is based on, is sent out using more materials, various dimensions direction The 3D printing technique of exhibition is then bone repair of cartilage.