CN109381742A - Three-dimensional artificial interverbebral disc and its preparation method and application based on patterning bacteria cellulose - Google Patents
Three-dimensional artificial interverbebral disc and its preparation method and application based on patterning bacteria cellulose Download PDFInfo
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Abstract
The present invention provides it is a kind of based on patterning bacteria cellulose three-dimensional artificial interverbebral disc, and its preparation method and application.Interverbebral disc provided by the invention can reverse-engineer fibrous ring picture on surface, diameter and the number of plies with the method according to the size of the interverbebral disc of different patients, thus realize can in analogue body annulus fibrosus disci intervertebralis anisotropic structure.Of the invention constructs three-dimensional artificial total spinal disc bracket based on patterning bacteria cellulose and collagen gel, since material has good biocompatibility, bracket is after implanting, it can be good at same in-vivo tissue to combine, and mechanics support is provided, having development is the potential quality of implantable artificial intervertebral disk.
Description
Technical field
The invention belongs to biomedical engineering fields, and in particular to a kind of three-dimensional artificial based on patterning bacteria cellulose
Interverbebral disc, and its preparation method and application.
Background technique
Chronic neck and flank pain are the high illnesss of disease incidence, and annual a huge sum of money all spends in intervertebral disc disorder and draws
In the treatment for sending out disease.Different degrees of neck and back pain are suffering from according to 50 years old or more the individual that statistics is more than 97%
Bitterly.The most common waist and cervical pain are mainly caused by vertebral disc portion lesion.Degenerative disc disease is controlled at present
Treating includes conservative therapy (drug and physical therapy) to operative treatment (diskectomy, spinal fusion and intervertebral disc replacement), however
These treatments can relieve pain or restore disc material function, can not fundamentally cure.
Human body shares 23 interverbebral discs (intervertebral disc): interverbebral disc is by upper and lower ends cartilage endplate, periphery
Collagen-rich fibrous ring and center are formed rich in three parts of nucleus pulposus of proteoglycan.
Fibrous ring has complicated multilayer concentric cyclic structure, and each layer of internal fiber is in 30-45 ° along same cross section
It is arranged in parallel, and the arrangement of adjacent ring is opposite.Fibrous ring is very firm, is adhering closely on cartilage endplate, keeps the steady of backbone
It is qualitative.Nucleus pulposus is the translucent colloid of milky, between two cartilage plates and fibrous ring.By cartilage cell and proteoglycan base
Texture at elastic gel substance.
With the development of organizational project regenerative medicine, have by the theoretical method using biology and engineering science to prepare
This target of the artificial intervertebral disk of the mechanism and function of normal tissue and organ is intended to possibility.There are large quantities of science both at home and abroad
Researcher is engaged in the research of engineered artificial intervertebral disk, and engineered artificial intervertebral disk is concentrated mainly on organizational project
Change Single Fiber ring or single nucleus pulposus.Timbering material currently used for constructing tissue-engineering fiber ring mainly includes natural biological
Material, synthetic material and composite material three categories.Natural biologic material has decellularization fibrous ring, collagen, silk
Fibroin gel etc.；Synthetic material has and has polyamide, polycaprolactone etc. including synthetic material, and composite material such as polylactic acid/
Hyaluronic acid, elastin laminin/glycosaminoglycan/collagen etc..Artificial nucleus is mainly with natural material such as seaweed gel, collagen
II type gel, aggrecanase and hyaluronic acid isogel material.The artificial intervertebral disk reported at present still cannot good mould
Intend the three-dimensional structure of internal interverbebral disc, and forms functional implantation substitute.It is main during developing bionical disc tissue
Want facing challenges as follows: (1) the anisotropic multilevel structure of fibrous ring in analogue body, the i.e. each ring of the fibrous ring of concentric annular
All in the same direction+30 ° or -30 ° oriented parallel arrangements of interior collagenous fibres, cut the nanofiber arrangement side in adjacent two layers ring
To opposite；(2) the functional total spinal disc being made of annulus fibrosus portion and nucleus pulposus portion is constructed.
Bacteria cellulose (Bacterial Cellulose, BC) have ultra-fine reticular structure, have high moisture holding capacity,
The advantages that high-mechanical property, high osmosis, degradability and hypotoxicity, leads in artificial skin, cartilage tissue engineered material, nerve
Pipe, blood vessel and Dental implantion material etc. have good application.But natural BC is that acetobacter xylinum ferments in its natural state
It generates, in the perforated grill structure of disorderly and unsystematic arrangement, the high-sequential three-dimensional structure of in-vivo tissue cannot be simulated well, because
How Induction of bacterial makes the BC of secretion that ordered arrangement be presented for this, and further inducing cell arranges Challenge along BC.
Summary of the invention
Therefore, the purpose of the present invention is to overcome the defects in the prior art, provides a kind of based on patterning bacterial fibers
The three-dimensional artificial interverbebral disc of element, and its preparation method and application.
Before illustrating the content of present invention, it is as follows to define term used herein:
Term " bacteria cellulose (Bacterial Cellulose, BC) " refers to: being produced during fermented and cultured by bacterium
Raw secondary metabolite native cellulose.
Term " PDMS " refers to: dimethyl silicone polymer (Polydimethylsiloxane).
Term " BC " refers to: bacteria cellulose (Bacterial Cellulose).
Term " AFCs " refers to: annulus fibrosis cells (annulus fibrosus cells).
Term " IVD " refers to: interverbebral disc (intervertebral disc).
Term " NP " refers to: nucleus pulposus (nucleus pulposus).
To achieve the above object, the first aspect of the present invention provides a kind of three-dimensional artificial interverbebral disc, the interverbebral disc packet
It includes:
Artificial fiber ring based on patterning bacteria cellulose；With
Artificial nucleus, the nucleus pulposus are made of one or more gel rubber materials selected from the following: seaweed gel, collagen
Gel, aggrecanase and hyaluronic acid.
Interverbebral disc according to a first aspect of the present invention, wherein the gel rubber material is collagen gel, the collagen egg
White preferably typeⅡ Collagen.
Interverbebral disc according to a first aspect of the present invention, wherein the fibrous ring is the patterning bacterial fibers along strip
The annulus that element rolls, each layer of intra-striate all along 0~90 ° of angular range, be most preferably+30 ° and -30 ° align, phase
Direction in two layers adjacent is arranged along different directions.
Interverbebral disc according to a first aspect of the present invention, wherein the bacterium is selected from one or more of: acetic acid Pseudomonas,
Agrobacterium, rhizobium and Sarcina；Preferably acetic acid Pseudomonas (Acetobacter)；Most preferably, described thin
Bacterium is acetobacter xylinum (ATCC 53582).
The second aspect of the present invention provides the preparation method of interverbebral disc described in first aspect, which can wrap
Include following steps:
(1) bacterial fermentation culture prepares patterned bacteria cellulose, purification process and sterilizes；
(2) it being rolled along patterning bacteria cellulose one end of strip, each layer of intra-striate all aligns, and adjacent two
Micro- pattern is arranged along different directions in layer；
(3) gel solution is led into ring, is put 37 DEG C of incubator cultures into and is formed gel.
Preparation method according to a second aspect of the present invention, wherein described to prepare patterned bacterium in the step (1)
The method of cellulose the following steps are included:
(a) culture medium and Zymolysis Equipment are subjected to sterilization treatment；
(b) it takes the fluid nutrient medium of logarithmic phase to be centrifuged, removes supernatant, fresh culture is then added and prepares bacterium
Suspension, in which:
The centrifugal rotational speed is preferably 5000~30000rpm, more preferably 8000~15000rpm, is most preferably
10000rpm, and/or
The centrifugation time is preferably 5~30 minutes, more preferably 8~15 minutes, is most preferably 10 minutes；
(c) bacterial suspension is poured into the culture tank of stainless steel, covers figuratum PDMS template, figuratum one side
It is adjacent to liquid level and ensures that bubble-free generates；
(d) equipment after will be inoculated puts constant incubator into, cultivates 4~6 days under the conditions of 30 DEG C；
(e) after cultivating 4~6 days, it will form the film of white gels shape bacteria cellulose at PDMS- culture medium interface, to it
Cleaning purifies, and the film bubble after purification process is put into sample bottle in ultrapure water, places 4 after 121 DEG C of high pressure steam sterilizations
DEG C refrigerator cryo-conservation.
Preferably, in the step (c), the preparation method of the figuratum PDMS template the following steps are included:
(I) pattern of photo etched mask is designed with AutoCAD, and through high-resolution laser printer on film film
It carries out printing and prepares photo etched mask；
(II) lithography process is carried out on silicon wafer using the photo etched mask prepared in step (I) prepare silicon template；
(III) PDMS deaeration being stirred evenly to be poured in the silicon template prepared in step (II), vacuum outgas, be heating and curing molding,
It takes the PDMS being cured off and obtains the figuratum PDMS template.
It is highly preferred that the pattern of the photo etched mask is designed to the alternate pattern of equidistant convex-concave in the step (I)；
Preferably, the protrusion of the pattern and the spacing of groove are 1~200 μm, preferably 1~100 μm, are more preferably 5~45 μm, most
Preferably 10 μm.
The third aspect of the present invention provides three-dimensional artificial interverbebral disc described in first aspect or according to described in second aspect
Method preparation three-dimensional artificial interverbebral disc preparing the application in tissue engineering material.
Preferably, the tissue engineering material is the material for implantable artificial interverbebral disc.
It is template that the present invention provides one kind based on patterning PDMS microfluidic channel, utilizes aerobic bacterium acetobacter xylinum
Aerotaxis feature, the gas-liquid interface microchannel that induction acetobacter xylinum is attached to PDMS- culture medium, which grows and secretes, generates BC, thus
The pattern of PDMS is reprinted and prepares patterned BC (such as Fig. 1) on the surface of BC.It is thin that we further pass through culture fibrous ring
Born of the same parents (Annulus fibrosus cell, AFCs) are on the patterned surface BC, and inducing cell is along patterning BC surface topology knot
Structure oriented growth successfully simulates the cell directional growth of the fibrous annulus tissue of interverbebral disc.We tie according to the 3D of interverbebral disc complexity
Structure reverse-engineers the pattern of 2D BC, so that the concentric ring BC rolled has anisotropic structure identical with internal fibrous ring
(such as Fig. 2): being made of multilayer ring-type, and the surface fiber of each layer of ring aligns+30 ° or -30 °, and two neighboring ring
Orientation it is opposite.Our further fibrous ring of the building based on BC and nucleus pulposus compositions based on typeⅡ Collagen gel
The artificial intervertebral disk (such as Fig. 3) of 3D, and its function and histocompatbility for making to implant is evaluated, the people of surface this method preparation
It is the potential quality and application of implantable artificial interverbebral disc that work interverbebral disc, which has latency development,.
Three-dimensional artificial interverbebral disc based on patterning bacteria cellulose of the invention can have but be not limited to following beneficial
Effect:
1, the present invention provides a kind of reverse method for constructing complicated annulus fibrosus disci intervertebralis from 2 d-to-3 d, Ke Yigen
According to the size of the interverbebral disc of different patients, fibrous ring picture on surface, diameter and the number of plies are reverse-engineered with the method, to realize energy
The anisotropic structure of annulus fibrosus disci intervertebralis in enough analogue bodies.
2, the present invention provides one kind constructs three-dimensional artificial total spinal disc based on patterning bacteria cellulose and collagen gel
Bracket, since material has good biocompatibility, bracket can be good at same in-vivo tissue and be incorporated into after implanting
Together, and mechanics support is provided, having development is the potential quality of implantable artificial intervertebral disk.
Detailed description of the invention
Hereinafter, carrying out the embodiment that the present invention will be described in detail in conjunction with attached drawing, in which:
Fig. 1 shows the preparation patterning bacteria cellulose preparation principle figure of the method based on biometric print.
Fig. 2 shows the schematic diagrames for the silicon wafer die plate pattern for preparing 2D fibrous ring.
Fig. 3 shows building and the implantation flow chart of prosthetic total disc.
It is respectively 10 μm that Fig. 4, which shows (a-d),30 μm, andThe difference for patterning bacteria cellulose is aobvious
Micro mirror observes result.(e) the patterning bacteria cellulose pattern width of different groups measures knot
It is 10 × 10 μm of square lattice shapes of silicon wafer template and 10 × 10 μm respectively that Fig. 5, which shows figure (a) and figure (b),
Parallel groove just sets micro- sem observation figure.Scheming (c) with figure (d) is that 10 × 10 μm of square lattice shapes and 10 μm are parallel respectively
Observation figure of the channel patterns BC under inverted phase contrast microscope.
Fig. 6 shows the surface topography characterization result of patterning BC and non-patterned BC.Figure (a) and (b) be respectively 10 ×
10 μm of patterning BC SEM under 500 × and 50000 × amplification factor is observed as a result, figure (c) and (d) are respectively non-patterned BC
500 × and 50000 × amplification factor under SEM observe result.
Fig. 7 shows (a) degree of order of AFCs cell and (b) cell shape index on different size patterns and is analyzed.
Fig. 8 shows AFCs and sees in the cytoskeleton and nuclear targeting of the patterning surface BC (a) and non-patterned BC (b)
It examines.Work dead fluorescent staining picture of the AFCs in the patterning surface BC (c) and non-patterned BC (d) surface.
Fig. 9 shows the stretching experiment of (a) micro-patterning and non-patterned BC.(b) based on patterning and it is non-patterned
The incompressible experiment of tubular artificial fibrous ring.
Figure 10 shows the phase contrast microscope observation result that 2D artificial fiber ring shows pattern
Figure 11 shows the sectional view of (a, b) based on BC fibrous ring.(b) be (a) enlarged drawing in situ.(c) nucleus pulposus cell
The dead fluorescent staining microscope photograph of work in II collagen type gel.
Figure 12 shows the building of 3D artificial fiber ring tissue.AFCs is in the surface BC of different pattern edge and -30 ° respectively
(a) and+30 ° (b) oriented growths and arrangement.(c) the interface confocal microscopy figure of fibrous annulus tissue, blue and green
Layer inner cell is respectively along same horizontal axis in -30 ° and+30 ° of oriented growths and arrangement.AFCs uses two kinds of cell dye Calcein respectively
AM green (green) and Calcein AM violet (blue) dyeing.
Figure 13 shows rat tails interverbebral disc 0,1,3 month after surgery of artificial intervertebral disk implantation group and discectomy group
NMR imaging result
It is respectively that normal disc tissue and artificial intervertebral disk are implanted into the slice HE after January that Figure 14, which shows (a) and (b, c),
Colored graph.It (c) is the observation result under the amplification factor of (b).(d) and (e, f) is respectively normal disc tissue and artificial vertebra
Disk is implanted into the slice HE colored graph after March.It (f) is the observation result under the amplification factor of (e).(g), (h) distinguish with (i)
Enrolled fibrous ring, cartilage endplate and the boundary of centrum are planted for normal disc tissue, disc removal group and artificial intervertebral disk
The HE colored graph of face slice.
Specific embodiment
Present invention will be further explained by specific examples below, it should be understood, however, that, these embodiments are only
It is used, is but should not be understood as present invention is limited in any form for specifically describing in more detail.
This part carries out general description to the material and test method that arrive used in present invention test.Although being
It realizes many materials used in the object of the invention and operating method is it is known in the art that still the present invention still uses up herein
It may detailed description.It will be apparent to those skilled in the art that within a context, if not specified, material therefor of the present invention and behaviour
It is well known in the art as method.
Reagent and instrument used in the following embodiment are as follows:
Reagent name
Instrument
Acetobacter xylinum (ATCC 53582) is bought in American Type Culture collection warehousing.
SD rat is purchased from Beijing HFK Bio-Technology Co., Ltd..
Embodiment 1
The present embodiment is for illustrating template design method of the present invention.
Simulating natural intervertebral disc institutional framework is to pass for preparing engineered artificial organ with the same function
Important.In order to simulate the multi-level structure of fibrous ring in natural intervertebral disc, we construct the concentric of three-dimensional using micro- pattern BC
Ring structure, wherein very low power adjacent area distribution in+- 30 ° be alternately arranged.The protrusion and groove spacing of very low power are all 5
μm.Ensure that each rectangle with identical pattern parallel corresponds to a ring of interverbebral disc annulus by calculating, in rings different in this way
The degree of order of pattern is also in the arrangement of opposite direction.We first devise and pattern BC in two-dimentional level, wherein in adjacent area
In the microflute that is aligned on 30 ° of directions alternately, a ring (Fig. 5 a) of each Region Matching AF thin slice, then we surround
Mandrel rolls the BC (diameter 2mm) of micro-patterning and forms concentric ring structure, and wherein microflute is in contiguous slices to take on the contrary
It is arranged to (+/- 30 °).
N and RnIt is related to the number of plies and the n-th wheel radius of concentric ring counted in AF tissue from innermost circle respectively.3D thin slice
The perimeter of n-th wheel (Wn) is equal to the length of the n-th part in 2D mother matrix, needs using accuracy computation below:
Rn=R1+(n-1)D (1)
Wn=2 π Rn (2)
Photo etched mask is prepared according to the fibrous ring 2D pattern of design later.
Embodiment 2
The present embodiment is used to illustrate the processing preparation of Lithographic template of the present invention.
The preparation process of lithography process micro-fluidic chip mainly has following steps, and process is as follows.
1. with the pattern of AutoCAD design photo etched mask, and through high-resolution laser printer on film film
The printing system of progress prepares photo etched mask；Pattern is designed to 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm
The alternate pattern of equidistant convex-concave.
2. the pattern designed by pair carries out lithography process in clean working room.
Dehumidifying: surface dirt is removed with nitrogen gun first, then silicon wafer is placed on 150 DEG C of flat plate heats of setting and is heated 1 hour
To remove residual moisture in silicon wafer.
Gluing: after silicon wafer is slowly dropped to room temperature, by the negative photoresist of SU-8 2010 down on four cun of silicon wafer centers,
Then by silicon wafer as spin coating on desk-top sol evenning machine.Using the spin coating on four cun of silicon wafers of SU-8 type negtive photoresist 2010,500rpm is pre- even
10s, 3500rpm spin coating 30s.
The purpose of front baking front baking is in order to which the gamma-butyrolacton solvent that will be mixed in photoresist evaporates, to enhance light
Adhesiveness between photoresist and silicon wafer.Even good silicon wafer is placed in baking 3 min of piece in 95 DEG C of thermal station；
Exposure by the silicon wafer after front baking in thermal station cooled to room temperature, later using two-sided orderly ultraviolet photolithographic machine into
Row photoetching, operation put the mask in silicon chip surface, select vacuum contact mode, expose 30s (290j/cm2).According to institute
The relational graph of the light exposure and depth shown, the basic parameter for setting litho machine is: light exposure 10mJcm–2·s–1, when exposure
Between be 30s.
It dries for the silicon wafer of end exposure to be placed in 95 DEG C of thermal station afterwards and further dries piece 3min；
The rear silicon wafer terminated that dries is placed on cooled to room temperature in thermal station by development, is developed in SU-8 developer
1~2min, developing process are fixed judge whether developing process is complete using isopropanol, and the region not being completely dissolved can go out
Existing white precipitate.For not developing, adequately part will develop repeatedly in this way, be fixed, until not having white precipitate substance, present
Clearly pattern, N2Silicon wafer is placed in holding 2h post bake on 150 DEG C of flat plate heats by drying post bake, and silicon wafer is placed in 150 DEG C of flat plate heats
Upper holding 2h.
Silicon wafer is placed on 150 DEG C of flat plate heats and keeps 2h by post bake.
The silicon wafer of post bake thickness after plasma is handled, is put into vacuum, is then dripped by the purpose of silanization silanization
Upper 10ul 1H, 1H, 2H, 2H ,-perfluoro decyl triethoxysilane vacuumize placement 24 hours, make silicon wafer in trimethylchloro-silicane
The processing that surface silanization is carried out in the atmosphere of alkane, it is stand-by to handle silicon template well.
Embodiment 3
The present embodiment is used to illustrate the preparation of PDMS chip of the present invention.
What above-mentioned lithography step obtained is the template of overmolded.The step of overmolded, is as follows:
1. by the precursor of dimethyl silicone polymer (PDMS, Polydimethylsiloxane) and curing agent according to 10:1's
Mass ratio configuration, carries out stirring evenly deaeration being later poured in template, thickness is in 3mm or so again on deaeration machine；
2. degassing: PDMS being put into vacuum desiccator and continues extraction vacuum 2min；
3. curing molding: PDMS being placed in 100 DEG C of thermal station and heats 15min；
4. taking off film: taking the PDMS being cured off, pattern is transferred on PDMS.With blade according to suitably sized cutting.
Embodiment 4
The present embodiment is used to illustrate the preparation of present invention patterning BC.
Prepare Hestrin&Schramm (HS) fluid nutrient medium: glucose 20g, yeast powder 5g, albumen need to be added in every 1L water
Peptone 5g, citric acid 1.5g, Na2HPO4·12H2O 6.8g, the preparation of agarose solid medium be then by HS fluid nutrient medium by
Agarose is added according to 2% ratio, i.e., every 100ml fluid nutrient medium is added 2g agarose and is prepared.Then 121 DEG C of sterilizings
20min is spare.
We prepare patterning BC by fermented and cultured acetobacter xylinum, provide growth with stainless steel groove for culture bacterium
Space, the PDMS that will be patterned into covers above culture medium (such as Fig. 1) as template, since PDMS is chemical inertness, and has
There are gas permeability, hydrophobicity.So during the fermentation, bacterium is attached to the surface PDMS to obtain the required O of growth2, pass through one
The fermented and cultured of section time, will form a pattern layers BC film at PDMS/ culture medium interface.
Fermented and cultured specific steps are as follows: (1) by the sterilization treatment of culture medium and Zymolysis Equipment；(2) logarithm is taken
The fluid nutrient medium of phase carries out 10000rpm and is centrifuged 10min, removes supernatant, and fresh culture is then added and prepares bacterial suspension
Liquid；(3) bacterial suspension is poured into the culture tank of stainless steel, then covers PDMS template, it is figuratum to be adjacent to liquid level on one side
And ensure that bubble-free generates.(4) equipment after will be inoculated puts constant incubator into, cultivates 4~6 days under the conditions of 30 DEG C.(5)
After culture 4~6 days, the BC film of white gels shape will form at PDMS- culture medium interface.It first repeatedly impregnated, cleaned with ultrapure water
It is adsorbed on residual liquid culture medium therein to remove, film is placed in the NaOH solution with 1%wt boils 0.5 hour later
(twice) purification process is later repeatedly impregnated film with ultrapure water with removing thallus and culture medium residual remaining among BC film
Until measuring PH as neutrality.(6) sample saves: the film bubble after purification process is put into sample bottle in ultrapure water, by 121
4 DEG C of refrigerator cryo-conservations are placed after DEG C high pressure steam sterilization.
Embodiment 5
The present embodiment is used to illustrate the building of artificial intervertebral disk of the present invention.
Fibrous ring (annulus fibrosus, AF) major function is subject to dmm, and fiber is in centrum cross section
Diagonal arrangement is in concentric circles (20~25 layers), and collagenous fibres are arranged in diagonal between centrum, are respectively formed with cross section +/-
30 ° of direction arrangements, and adjacent two layers are arranged in opposite direction, are assigned its powerful tensile resistance, nucleus pulposus can be prevented to evagination
Out, deformation occurs for nucleus pulposus when while allowing spinal motion in it, to control the various movements of backbone.
Further preparation has with the fibrous ring based on patterning BC and is based on typeⅡ Collagen gel nucleus pulposus structure for we
At total spinal disc implant, this method preparation process is following (such as Fig. 3):
(1) patterned BC is prepared first with acetobacter xylinum fermented and cultured, purification process and sterilize spare.
It (2) is that 2mm circular shaft rolls around diameter along one end patterning BC of strip, it is every due to designing 2D pattern in advance
The width at one position, each layer of intra-striate of the annulus rolled are all aligned along+30 ° or -30 °, and micro- in adjacent two layers
Pattern is arranged along different directions.
(3) collagen solution is led into ring after, puts 37 DEG C of incubators, 1 hour formation gel into.It is formed and is based on glue
The artificial NP that former protein gel the is constituted and full IVD formed based on anisotropic artificial AF.
Test example 1
This test example carries out in-vitro evaluation based on artificial intervertebral disk prepared by Examples 1 to 5.
Material characterization phase contrast microscope observes micro- pattern on bacteria cellulose surface.
Cell evaluation
We extract nucleus pulposus and annulus fibrosis cells (annulus fibrosus cells, AFCs) from rat disc,
Then the cytotoxicity of fibrous ring and collagen gel based on bacteria cellulose is evaluated.We are the figure on research material surface
Influence of the case to annulus fibrosis cells taxis, by AFCs with 105-106ml-1The table of density being inoculated into respectively in patterning BC
On face, again 37 DEG C later, cultivated under 5% carbon dioxide culture 3 days, then with 488 phalloidine of acti-TM to the F- of cell
Actin filament dyeing.With confocal laser scanning microscope cellular morphology.
The orientation angles of the nucleus dyed by hoechst under 40 amplification factors are analyzed by using Image J software,
To analyze the degree of order of cell on different substrates.Cell orientation angle indicates Nanowire of the long axis of cell body relative to orientation
The angle in the direction of dimension, and the lower degree of order for indicating cell of cell angle is higher.In order to analyze, we are by the angle of AFCs
Be divided into 10~90 equal 9 groups with 10 ° for gradient, when nucleus angle within 10 ° it is considered that cell is ordered into arrangement.
And we are got over the shape index of Image J software analysis cell, the ductility of the smaller superficial cell of the shape index of cell
It is good.
Test example 2
According to Examples 1 to 5, the fibrous ring that micro- pattern spacing is 10 μm and the artificial vertebra constituted based on collagen gel are prepared
Disk.Cell evaluation such as test example 1.
Material characterization
It is observed by micro- pattern of the phase contrast microscope to bacteria cellulose surface.With cold field emission scanning electron microscopy
Mirror (10KV, 7.0A) observes material surface.Micro- figure under hygrometric state is measured with general-purpose tensilometer (Instron5567, USA)
The tensile strength of case and non-patterned BC.BC film is cut into dumbbell specimens by us, and the distance between two fixtures are set as
50mm.Under 25 DEG C, 50% relative humidity, with 20mmmin-1Tensile speed test all samples.Based on 10 μm of patterns
The compression modulus of the BC fibrous ring of change is with dynamic mechanical analysis machine (DMA, Q800, TA instrument, US) with 0.2mm/min's (24 DEG C)
Compression speed measurement.Every layer of microflute is alternately ± 30 ° relative to horizontal axis, using non-patterned multilayer BC as control.Test
Outer diameter is 4mm, is highly the tubulose BC ring of 2mm.
We simulate human fiber's ring and construct mutually isostructural engineered artificial fiber ring, and thin for each layer
Born of the same parents' arrangement and cyclic structure characterize, and specific preparation process is as follows；
(1) it prepares patterned BC first, purification process and sterilizes spare.
(2) the AFCs suspension of preparation different dyes label.When density reaches 80% in culture dish, two ware AFCs are taken
Dye marker is carried out with the calcein-AM violet (blue) of 1mg/ml and calcein-AM (green) respectively.37 DEG C of dyeing
After 15min, extra dye solution is removed, cell is digested with pancreatin later, fresh culture preparation is added after centrifugation
105Cell suspending liquid A (calcein-AM violet, blue markings) and cell suspending liquid B (calcein-AM, the green of/ml
Label).
(3) it designs according to PDMS with PDMS template of same size, PDMS template is covered on patterning BC, so that different
Frame edge can be completely bonded with the edge of patterned cellulosic.
(4) upper different cell is planted respectively into frame, the cell of different colours in adjacent area kind.
(5) nucleus and PDMS are placed in culture dish together and are cultivated, every other day change a subculture.Culture 3
After it, PDMS is removed, starts to start scrolling along circular shaft along the shortest one end strip pattern BC, forms multi-layer cylinder shape structure.
Test example 3
Zoopery
After preparing micro- pattern spacing and being 10 μm of artificial intervertebral disk IVD, we are in rat (SD, female, 250g) big rat-tail
The original interverbebral disc of artificial disc replacement that the BC/ collagen of micro-patterning is manually constituted is implanted into the interverbebral disc of 3/4 position of vertebra,
To carry out including TE-IVD implantation group (rat number n=12) up to 3 months implantation effect assessments, while being provided with interverbebral disc
Excision group is as a control group (rat number n=12).Use 3% (wt/vol) yellow Jackets 10ml 100g-1To rat into
Row anesthesia.For TE-IVD implantation group, original interverbebral disc is first removed, then implantation based on BC fibrous ring and is based on collagen nucleus pulposus
Intervertebral disk prosthesis implantation material substitution；Blank control group needs to dissect taking-up interverbebral disc, is then no longer implanted into interverbebral disc, as
Blank control group.
To ensure that artificial intervertebral disk is still able to maintain in the position of interverbebral disc after performing the operation, each group of tail bone customization
Extending apparatus is fixed to be fixed, to ensure that implantation material is able to maintain in the space of interverbebral disc between convalescence, and at 1
After month, when local recovery, wound portion, fixed device is removed.We carry out the observation and analysis up to 3 months after the procedure.
MRI imagings are carried out to two experimental groups at 0,1,3 month, come the interverbebral disc at observation experiment position recovery we use
BioSpec70/20USR magnetic resonance imaging system analyzes the form of IVD and adjacent vertebrae.It obtains within 0,1 and 2 month after the implantation
High-resolution T2 imaging results (resolution ratio: 137m × 47m × 1mm).
We carried out histologic analysis to TE-IVD and control when 1,3 month, put to death 6 each experimental groups respectively
Animal is to carry out histopathological analysis.Steps are as follows:
The fixed muscle and tendon fiber for removing tail portion is immersed in 2% paraformaldehyde solution 2 days with abundant fixing organization
Decalcification by sample ultrapure water it is clean after, be placed in tris hydrochloric acid progress decalcification processing, it is known that tissue softens
It can be sliced
Slice by the sample paraffin embedding after decalcification, be then cut into the thin slice of 5 μ m thicks, we using hematoxylin-she
Red colouring observes TE-IVD and self spinal tissues contact interface, is taken pictures later with just setting microscope and be scanned.
Although present invention has been a degree of descriptions, it will be apparent that, do not departing from the spirit and scope of the present invention
Under the conditions of, the appropriate variation of each condition can be carried out.It is appreciated that the present invention is not limited to the embodiments, and it is attributed to right
It is required that range comprising the equivalent replacement of each factor.
Claims (10)
1. a kind of three-dimensional artificial interverbebral disc, which is characterized in that the interverbebral disc includes:
Artificial fiber ring based on patterning bacteria cellulose；With
Artificial nucleus, the nucleus pulposus are made of one or more gel rubber materials selected from the following: seaweed gel, collagen are solidifying
Glue, aggrecanase and hyaluronic acid.
2. interverbebral disc according to claim 1, which is characterized in that the gel rubber material is collagen gel, the glue
Former albumen is preferably typeⅡ Collagen.
3. interverbebral disc according to claim 1 or 2, which is characterized in that the fibrous ring is thin along the patterning of strip
The annulus that fungin rolls, each layer of intra-striate all along 0~90 ° of angular range, be most preferably that+30 ° and -30 ° orientations are arranged
It arranges, micro- pattern is arranged along different directions in adjacent two layers.
4. interverbebral disc described in any one of claim 1 to 3, which is characterized in that the bacterium be selected from it is following a kind of or
It is a variety of: acetic acid Pseudomonas, Agrobacterium, rhizobium and Sarcina；Preferably, the bacterium is acetic acid Pseudomonas；It is optimal
Selection of land, the bacterium are acetobacter xylinum.
5. according to the preparation method of interverbebral disc according to any one of claims 1 to 4, which is characterized in that the method
The following steps are included:
(1) bacterial fermentation culture prepares patterned bacteria cellulose, purification process and sterilizes；
(2) it is rolled along patterning bacteria cellulose one end of strip, each layer of intra-striate all aligns, in adjacent two layers
Micro- pattern is arranged along different directions；
(3) gel solution is led into ring, is put 37 DEG C of incubator cultures into and is formed gel.
6. according to the method described in claim 5, it is characterized in that, in the step (1), described to prepare patterned bacterium fine
Tie up element method the following steps are included:
(a) culture medium and Zymolysis Equipment are subjected to sterilization treatment；
(b) it takes the fluid nutrient medium of logarithmic phase to be centrifuged, removes supernatant, fresh culture is then added and prepares bacterial suspension
Liquid, in which:
The centrifugal rotational speed is preferably 5000~30000rpm, more preferably 8000~15000rpm, is most preferably 10000rpm,
And/or
The centrifugation time is preferably 5~30 minutes, more preferably 8~15 minutes, is most preferably 10 minutes；
(c) bacterial suspension is poured into the culture tank of stainless steel, covers figuratum PDMS template, it is figuratum to be adjacent on one side
Liquid level simultaneously ensures that bubble-free generates；
(d) equipment after will be inoculated puts constant incubator into, cultivates 4~6 days under the conditions of 30 DEG C；
(e) after cultivating 4~6 days, it will form the film of white gels shape bacteria cellulose at PDMS- culture medium interface, it cleaned
It purifies, the film bubble after purification process is put into sample bottle in ultrapure water, and 4 DEG C of ice are placed after 121 DEG C of high pressure steam sterilizations
Case cryo-conservation.
7. according to according to the method described in claim 6, it is characterized in that, in the step (c), the figuratum PDMS mould
The preparation method of plate the following steps are included:
(I) pattern of photo etched mask is designed with AutoCAD, and is carried out on film film by high-resolution laser printer
Printing prepares photo etched mask；
(II) lithography process is carried out on silicon wafer using the photo etched mask prepared in step (I) prepare silicon template；
(III) PDMS is stirred evenly deaeration to be poured in the silicon template prepared in step (II), vacuum outgas, be heating and curing molding, takes off
The PDMS being cured obtains the figuratum PDMS template.
8. the method according to the description of claim 7 is characterized in that in the step (I), the design of the photo etched mask
At the alternate pattern of equidistant convex-concave；Preferably, the pattern protrusion and groove spacing be 1~200 μm, preferably 1~
100 μm, more preferably 5~45 μm, most preferably 10 μm.
9. according to any one of claims 1 to 4 or according to made from any one of claim 5~8 the method
Three-dimensional artificial interverbebral disc is preparing the application in tissue engineering material.
10. application according to claim 9, the tissue engineering material is the material for implantable artificial interverbebral disc.
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| CN113017936A (en) * | 2021-03-08 | 2021-06-25 | 吉林大学 | Variable-rigidity bionic intervertebral disc |
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