CN102293693B - Porous titanium alloy human cervical interbody fusion cage with bioactivity and preparation method thereof - Google Patents
Porous titanium alloy human cervical interbody fusion cage with bioactivity and preparation method thereof Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
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- A61F2/44—Joints for the spine, e.g. vertebrae, spinal discs
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/30—Joints
- A61F2002/30001—Additional features of subject-matter classified in A61F2/28, A61F2/30 and subgroups thereof
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Abstract
The invention provides a porous titanium alloy human cervical interbody fusion cage with bioactivity and a preparation method thereof. The method comprises the following steps: firstly, inputting model data into electron beam melting equipment according to a design requirement to prepare a porous titanium alloy human cervical interbody fusion cage; secondly, preparing gelatin microspheres; and immersing gelatin microsphere dry powder in an rhBMP-2 solution for gelatin coating modification, preparing a gelatin solution A in double distilled water, immersing the porous titanium alloy human cervical interbody fusion cage in the gelatin solution A, mixing the rhBMP-2 gelatin microspheres and absolute ethanol to obtain suspension B, and immersing the gelatin-coating-modified porous titanium alloy human cervical interbody fusion cage in the suspension B to prepare the porous titanium alloy human cervical interbody fusion cage internally containing an rhBMP-2 sustained-release system. The cervical interbody fusion cage prepared with the method has modulus of elasticity close to that of natural bone tissues, and the porous structure and the bioactivity factor sustained-release system inside the cervical interbody fusion cage can induce growth of new bone tissues, so that the binding problem of bone-material interfaces is solved. Therefore, the cervical interbody fusion cage has better mechanical compatibility and bone integration capability than those of a compact material.
Description
Technical field
The invention belongs to fixed network in spinal fusion, be specifically related to a kind of fusion device and preparation thereof between bioactive porous titanium alloy people cervical vertebra that have.
Background technology
The slippage of cervical vertebra degeneration, intervertebral segmental shakiness and discogenic disease etc. are common spinal surgery diseases, have a strong impact on patient's live and work, and the non-operative treatment nonresponder is generally needed to carry out fixedly fusion of cervical vertebra.The intervertebral fusion operation has been recognized for this class disease good therapeutic effect.The normal material adopted of intervertebral fusion mainly contains two classes: 1. bone-grafting material comprises autologous bone piece and the transplanting of allograph bone piece; 2. substitution material, i.e. Invasive lumbar fusion device (Interbody Fusion Cage).
Previously intervertebral fusion is normal adopts simple autologous bone piece to transplant, but autologous Qu Gu district complication comprises the residual pain in Qu Gu district, infection, sensory disturbance etc. up to 10%-20%.Although the allograph bone piece is transplanted and to be avoided Liao Qugu district complication, have that immunological rejection, aids infection are viral, expensive, allograph bone is substituted slowly, the little deficiency such as grade of area of new bone volume, clinical practice is restricted.(Lelze?EM，Hemmerle?J，Vo?egel?JC，etal.Characterization?and?histological?analyses?of?a?coral-co?llagen?composite?used?for?bone-replacement?graft?material：a?report?of?c?linical?cases.J?Mater?Sci?Mater?Med.1999：10(1)：47-51.)
For overcoming, adopt the simple bone graft material to carry out the deficiency of intervertebral fusion, between clinical main use cervical vertebra, fusion device carries out merging between cervical vertebra at present, mainly comprise: titanium alloy Invasive lumbar fusion device (Titanium Alloys-Cage), polyether-ether-ketone Invasive lumbar fusion device (Poily Ether Ether Ketone-Cage, PEEK-Cage), poly-D, Pfansteihl Invasive lumbar fusion device (Poly-(D, L-Lactide)-Cage, PDLLA-Cage).With the simple bone graft material, compare, Invasive lumbar fusion device can effectively reduce in art and post-operative complication, reduce patient's postoperative pain and accelerate operation in patients after functional rehabilitation.
But traditional Invasive lumbar fusion device still has some limitations: 1. the titanium alloy Invasive lumbar fusion device mainly adopts Ti6Al4V to make, although intensity is high, but elastic modelling quantity (110GPa) is far away higher than natural bone (the about 3-12GPa of cortical bone), there is the interface problem of metal-bone, can produce stress shielding, cause bone resorption and that implant occurs is loosening.2. PEEK-Cage elastic modelling quantity and vertebral body cortical bone approach, avoided stress shielding, but it and titanium alloy all belong to bio-inert material, osseous tissue can only be attached to material surface and can't enter its inside and reach good bone and integrate, cause bone-material interface bond strength deficiency, easily occur loosening.The surface modification of PEEK-Cage is (as at surface spraying titanium silk, hydroxyapatite (HA), utilize the micropore spline structure of coating to induce osseous tissue to grow into) although solved to a certain extent the problem of bone-material interface bond strength deficiency, but face coat adds under the load effect at physiological environment, or may be absorbed by body or due to stress concentration, generation of interfaces crackle along porous vesicular surface and ground, cause and come off.3. PDLLA-Cage has the elastic modelling quantity close with sclerotin, the product of degradable for being absorbed by the body, thus be absorbed gradually and, by the surrounding bone tissue substitute, there is good biological activity, there do not is bone-material interface problem.But its mechanical strength is lower, it is cracked that the inherent fragility of material itself causes it to be easy in operation technique.And its early stage macromolecules degradation product can cause material proliferation of fibrous tissue on every side, has to a certain degree delayed early stage skeletonization.
In sum, a kind of desirable Invasive lumbar fusion device should possess following characteristics: 1) have good biocompatibility; 2) there is enough mechanical strengths and anti-fatigue ability; 3) there is suitable elastic modelling quantity, be beneficial to bone-material interface mechanotransduction; 4) there is certain bone growth inducing etc.In order to reach above requirement, porous titanium alloy material and bio-modification thereof become the focus of research.Porous titanium alloy had both had enough mechanical strengths, had again vesicular texture, and the many kinds of parameters such as shape that can be by controlling porosity, pore size, hole and distribution effectively reduce the elastic modelling quantity of titanium alloy, realize being complementary with the elastic modelling quantity of natural bone.Add bioactie agent in hole, as bone morphogenetic protein (BMP) etc. can be induced the osseous tissue hole of growing into, strengthen the machinery locking of porous titanium alloy implant, thereby obtain better long-term fixed effect, so porous titanium alloy is the desirable Invasive lumbar fusion device material for preparing.
Summary of the invention
The object of the present invention is to provide and a kind ofly there is enough support strengths to maintain disc height and vertebral body stability, can stimulate new bone growth by the induced osteogenesis effect of its inner bioactie agent slow-released system again, integrate thereby reach good bone, promote the fusion device and preparation method thereof between bioactive porous titanium alloy people cervical vertebra that has of bone fusion between vertebral body.
For achieving the above object, the technical solution used in the present invention is:
1) porous titanium alloy Invasive lumbar fusion device preparation
At first by the designing requirement of fusion device between the porous titanium alloy people cervical vertebra of required preparation, model data is inputted in electron beam melting equipment, and add the titanium alloy ti6al4v powder in electron beam melting equipment, at the 600-800 ℃ of electronic beam current with 20-40mA, scanning speed preheating titanium alloy powder with 10000-18000mm/s, then with the electronic beam current of 5-7mA, the scanning speed of 300-500mm/s is the melting titanium alloy powder successively, prepare with required porous titanium alloy people cervical vertebra between the consistent porous titanium alloy people Invasive lumbar fusion device of fusion device shape, it is characterized in that: between this bioactivity, porous titanium alloy people cervical vertebra, the peripheral part (1) of fusion device has regular hole, average pore size 300-400 μ m, porosity 40-50%, central part (2) has the irregular hole of bone trabecula sample, average pore size 400-1000 μ m, and porosity 60-80%,
2) the bioactie agent slow-released system builds
2.1 be loaded with the preparation of rhBMP-2's (rhBMP-2) gelatine microsphere
2.1.1 the preparation of gelatine microsphere
Pharmagel is dissolved in to the gelatin solution that in the distilled water of 40-60 ℃, ultrasonic discrete mass concentration is 5%-15%, at 40-60 ℃, gelatin solution is mixed with the volume ratio of liquid paraffin by 1: 5, add the emulsifying agent Span-80 of gelatin solution and paraffin mixed liquor cumulative volume 2% to stir simultaneously and make suspension, suspension is cooled to rapidly to 0 ℃ also to be continued to stir 20 minutes, under agitation in suspension, by every 1g gelatin, add the genipin precuring that the 3-6ml mass concentration is 1% to stop after 1 hour stirring, repeatedly with acetone, dewater, thoroughly washing paraffin final vacuum pump off with absolute ether drains, and then add by every 1g gelatin standing the solidifying 24 hours of genipin room temperature lucifuge that the 3-6ml mass concentration is 1%, with vacuum drying under room temperature after the residual genipin of the thorough cleaning of dehydrated alcohol, sieve,
60obtain gelatine microsphere after the Co radiation sterilization,
2.1.2 gelatine microsphere loads rhBMP-2
At first utilize the rhBMP-2 solution of PBS (PH=6.0) preparation 100-300ng/ml, ratio according to the every 1.0mg gelatine microsphere of 100 μ l rhBMP-2 dry powder, gelatine microsphere dry powder is soaked in rhBMP-2 solution, and 37 ℃ of shaking tables are hatched, and spend the night, PBS rinses, centrifugal 10 minutes of 2500rmp, clean gelatine microsphere 2 times, collects the rhBMP-2 gelatine microsphere, the negative pressure lyophilizing
60seal lucifuge-80 ℃ preservation after the Co radiation sterilization;
2.2.rhBMP-2/ the combination of gelatine microsphere and porous titanium alloy internal void
2.2.1 the gelatin coating of porous titanium alloy is modified
The gelatin solution A that the preparation mass concentration is 5-10% in the distilled water of 50 ℃, to after the first ultrasonic cleaning of porous titanium alloy people Invasive lumbar fusion device, immerse in the NaOH solution of 0.5mol/L, PBS cleans 3 times, then immerse in gelatin solution A and process 10 minutes under negative pressure, soak after 30 minutes, take out in the baking box that material is placed on 50 ℃ and dry;
2.2.2rhBMP-2/ the porous titanium alloy internal void of gelatine microsphere after gelatin is modified is combined
RhBMP-2 gelatine microsphere and dehydrated alcohol are prepared to dehydrated alcohol suspension B according to 30mg/ml, ultrasonic lower discrete 2 hours, porous titanium alloy people Invasive lumbar fusion device after gelatin coating is modified immerses in suspension B 24 hours, under-93.3kPa negative pressure, and the evacuation lyophilizing.
60the Co radiation sterilization obtains the porous titanium alloy people Invasive lumbar fusion device that contains the rhBMP-2 slow-released system in inside.
The peripheral part of the Invasive lumbar fusion device of making by preparation method of the present invention has regular hole, average pore size 300-400 μ m, porosity 40-50%; Central part has the irregular hole of bone trabecula sample, average pore size 400-1000 μ m, and porosity 60-80%, and be attached with the rhBMP-2/ gelatine microsphere in the hole of peripheral part and central part.
The Invasive lumbar fusion device that preparation method of the present invention is made has with natural bone organizes close elastic modelling quantity, loose structure allows transmission and the freshman bone tissue of body fluid to grow into, solved bone-material interface in conjunction with problem, had than the better mechanical compatibility of dense material and bone integration ability.The small-bore design of its peripheral part makes it have higher intensity, and regular porous structure stress is even, can reduce material fragility, is convenient to operation technique.Central part has the irregular pore structure close to the bone trabecula tissue, and has larger aperture and porosity, and the more bone space of growing into is provided, and is beneficial to that nutrient substance enters and metabolic waste is got rid of.RhBMP-2 gelatine microsphere slow-released system is realized controlled, stable release by progressively decomposing or dissolving of diffusion, carrier, thereby can bring into play lasting bone inductive effect, promotes bone fusion between early stage bone integration and vertebral body, reaches interpyramidal long-term stability.In addition, because the medicine carrying gelatine microsphere will mainly be distributed in the central part that hole is larger, make bone inductive effect mainly occur in Invasive lumbar fusion device inside, can prevent fusion device surrounding bone excessive tissue growth compressing vertebral body important structure on every side.
The accompanying drawing explanation
The top view that Fig. 1 is the embodiment of the present invention;
The side sectional view that Fig. 2 is Fig. 1;
The front view that Fig. 3 is Fig. 1;
Fig. 4 is schematic diagram after compound rhBMP-2 gelatine microsphere.
The specific embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail.
1) porous titanium alloy Invasive lumbar fusion device preparation
At first by the designing requirement of fusion device between the porous titanium alloy people cervical vertebra of required preparation, model data is inputted in electron beam melting equipment, and add the titanium alloy ti6al4v powder in electron beam melting equipment, at the 600-800 ℃ of electronic beam current with 20-40mA, scanning speed preheating titanium alloy powder with 10000-18000mm/s, then with the electronic beam current of 5-7mA, the scanning speed of 300-500mm/s is the melting titanium alloy powder successively, prepare with required porous titanium alloy people cervical vertebra between the consistent porous titanium alloy people Invasive lumbar fusion device of fusion device shape;
The present embodiment is as Fig. 1, Fig. 2, and the wedge shape Invasive lumbar fusion device shown in Fig. 3, specification is: long (L): 16mm, wide (W): 13mm, high (H): 5/6/7/8mm, wedge angle (angle shown in a): 17 °.Its cross section is similar to the vertebral body cross section, and wedge-shaped design is convenient in art, it be implanted in intervertebral space.The inclined-plane that upper surface is 3-4 ° (angle shown in b), be beneficial to maintain the cervical vertebrae physiological bending.It is 3mm that there are 2 diameters front portion, the aperture 3 that the degree of depth is 5mm, and for clamping Invasive lumbar fusion device, pitch of holes (d) is 8mm.Peripheral part 1 thickness (D) is 2mm, has regular hole, average pore size 300-400 μ m, porosity 40-50%; Central part 2 has the irregular hole of bone trabecula sample, average pore size 400-1000 μ m, porosity 60-80%.
2) the bioactie agent slow-released system builds
2.1 be loaded with the preparation of rhBMP-2's (rhBMP-2) gelatine microsphere
2.1.1 the preparation of gelatine microsphere
Pharmagel is dissolved in to the gelatin solution that in the distilled water of 40-60 ℃, ultrasonic discrete mass concentration is 5%-15%, at 40-60 ℃, gelatin solution is mixed with the volume ratio of liquid paraffin by 1: 5, add the emulsifying agent Span-80 of gelatin solution and paraffin mixed liquor cumulative volume 2% to stir simultaneously and make suspension, suspension is cooled to rapidly to 0 ℃ also to be continued to stir 20 minutes, under agitation in suspension, by every 1g gelatin, add the genipin precuring that the 3-6ml mass concentration is 1% to stop after 1 hour stirring, repeatedly with acetone, dewater, thoroughly washing paraffin final vacuum pump off with absolute ether drains, and then add by every 1g gelatin standing the solidifying 24 hours of genipin room temperature lucifuge that the 3-6ml mass concentration is 1%, with vacuum drying under room temperature after the residual genipin of the thorough cleaning of dehydrated alcohol, sieve,
60obtain gelatine microsphere after the Co radiation sterilization,
2.1.2 gelatine microsphere loads rhBMP-2
At first utilize the rhBMP-2 solution of PBS (PH=6.0) preparation 100-300ng/ml, ratio according to the every 1.0mg gelatine microsphere of 100 μ l rhBMP-2 dry powder, gelatine microsphere dry powder is soaked in rhBMP-2 solution, and 37 ℃ of shaking tables are hatched, and spend the night, PBS rinses, centrifugal 10 minutes of 2500rmp, clean gelatine microsphere 2 times, collects the rhBMP-2 gelatine microsphere, the negative pressure lyophilizing
60seal lucifuge-80 ℃ preservation after the Co radiation sterilization;
2.2.rhBMP-2/ the combination of gelatine microsphere and porous titanium alloy internal void
2.2.1 the gelatin coating of porous titanium alloy is modified
The gelatin solution A that the preparation mass concentration is 5-10% in the distilled water of 50 ℃, to after the first ultrasonic cleaning of porous titanium alloy people Invasive lumbar fusion device, immerse in the NaOH solution of 0.5mol/L, PBS cleans 3 times, then immerse in gelatin solution A and process 10 minutes under negative pressure, soak after 30 minutes, take out in the baking box that material is placed on 50 ℃ and dry;
2.2.2rhBMP-2/ the porous titanium alloy internal void of gelatine microsphere after gelatin is modified is combined, rhBMP-2 gelatine microsphere and dehydrated alcohol are prepared to dehydrated alcohol suspension B according to 30mg/ml, ultrasonic lower discrete 2 hours, porous titanium alloy people Invasive lumbar fusion device after gelatin coating is modified immerses in suspension B 24 hours, under-93.3kPa negative pressure, the evacuation lyophilizing.
60the Co radiation sterilization obtains the porous titanium alloy people Invasive lumbar fusion device that contains the rhBMP-2 slow-released system in inside.After the compound rhBMP-2 gelatine microsphere of loose structure, Fig. 4 is shown in by schematic diagram.
RhBMP-2 (rhBMP-2) slow-released system that the bioactie agent slow-released system of porous titanium alloy fusion device of the present invention adopts the gelatine microsphere slow release method to set up.First by improved emulsifying condensation method, make gelatin form the small chondritic of diameter 10-30 μ m left and right, and make rhBMP-2 be dispersed in gelatine microsphere inside, build the medicine carrying gelatine microsphere.Adopt again the disperse method will be loaded with gelatine microsphere and the embedded material of rhBMP-2 compound, gelatine microsphere is attached among the material hole, build the rhBMP-2 slow-released system.The distribution of medicine carrying gelatine microsphere in porous material is relevant with material aperture and porosity, and the larger recombination rate of material aperture and porosity is higher, so gelatine microsphere will mainly be distributed in the central part of Invasive lumbar fusion device.Gelatin is the main organic principle of osseous tissue---the hydrolyzate of collagen, and it can refine obtain through hydrolysis from animal collagen, and cheap and easy to get, no antigen, coefficient of friction are low, biodegradable, use safe and effective.RhBMP-2 has stronger induced osteogenesis effect, and in clinical acquisition application, it can significantly promote immature osteoblast and bone precursor (MSC) to change to ripe osteocyte, has very strong dystopy, original position osteogenic ability.Metal composite rhBMP-2, no matter in vitro for osteoblastic growth, still, in vivo for the formation of freshman bone tissue, all have obvious bone inductive effect.(Herford?AS.rhBMP-2?as?an?option?for?reconstructing?mandibular?continuity?defects.J?Oral?Maxillofac?Surg.2009?Dec；67(12)：2679-84.)
Claims (2)
1. the preparation method with fusion device between bioactivity, porous titanium alloy people cervical vertebra is characterized in that:
1) porous titanium alloy Invasive lumbar fusion device preparation
At first by the designing requirement of fusion device between the porous titanium alloy people cervical vertebra of required preparation, model data is inputted in electron beam melting equipment, and add the titanium alloy ti6al4v powder in electron beam melting equipment, at the 600-800 ℃ of electronic beam current with 20-40mA, scanning speed preheating titanium alloy powder with 10000-18000mm/s, then with the electronic beam current of 5-7mA, the scanning speed of 300-500mm/s is the melting titanium alloy powder successively, prepare with required porous titanium alloy people cervical vertebra between the consistent porous titanium alloy people Invasive lumbar fusion device of fusion device shape;
2) the bioactie agent slow-released system builds
2.1 be loaded with the preparation of rhBMP-2's gelatine microsphere
2.1.1 the preparation of gelatine microsphere
Pharmagel is dissolved in to the gelatin solution that in the distilled water of 40-60 ℃, ultrasonic discrete mass concentration is 5%-15%, at 40-60 ℃, gelatin solution is mixed with the volume ratio of liquid paraffin by 1:5, add the emulsifying agent Span-80 of gelatin solution and paraffin mixed liquor cumulative volume 2% to stir simultaneously and make suspension, suspension is cooled to rapidly to 0 ℃ also to be continued to stir 20 minutes, under agitation in suspension, by every 1g gelatin, add the genipin precuring that the 3-6ml mass concentration is 1% to stop after 1 hour stirring, repeatedly with acetone, dewater, thoroughly washing paraffin final vacuum pump off with absolute ether drains, and then add by every 1g gelatin standing the solidifying 24 hours of genipin room temperature lucifuge that the 3-6ml mass concentration is 1%, with vacuum drying under room temperature after the residual genipin of the thorough cleaning of dehydrated alcohol, sieve,
60obtain gelatine microsphere after the Co radiation sterilization,
2.1.2 gelatine microsphere loads the rhBMP-2
At first utilize rhBMP-2's solution of the PBS preparation 100-300ng/ml of pH=6.0, ratio according to the every 1.0mg gelatine microsphere of the rhBMP-2 of 100 μ l dry powder, gelatine microsphere dry powder is soaked in rhBMP-2's solution, and 37 ℃ of shaking tables are hatched, and spend the night, PBS rinses, centrifugal 10 minutes of 2500rmp, clean gelatine microsphere 2 times, collects the gelatine microsphere that is loaded with the rhBMP-2, the negative pressure lyophilizing
60seal lucifuge-80 ℃ preservation after the Co radiation sterilization;
2.2. be loaded with the combination of rhBMP-2's gelatine microsphere and porous titanium alloy internal void
2.2.1 the gelatin coating of porous titanium alloy is modified
The gelatin solution A that the preparation mass concentration is 5-10% in the distilled water of 50 ℃, to after the first ultrasonic cleaning of porous titanium alloy people Invasive lumbar fusion device, immerse in the NaOH solution of 0.5mol/L, PBS cleans 3 times, then immerse in gelatin solution A and process 10 minutes under negative pressure, soak after 30 minutes, take out in the baking box that material is placed on 50 ℃ and dry;
2.2.2 being loaded with rhBMP-2's the porous titanium alloy internal void of gelatine microsphere after gelatin is modified is combined
The gelatine microsphere and the dehydrated alcohol that are loaded with the rhBMP-2 are prepared to dehydrated alcohol suspension B according to 30mg/ml, ultrasonic lower discrete 2 hours, porous titanium alloy people Invasive lumbar fusion device after gelatin coating is modified immerses in suspension B 24 hours, under-93.3kPa negative pressure, the evacuation lyophilizing
60the Co radiation sterilization obtains the porous titanium alloy people Invasive lumbar fusion device that contains the gelatine microsphere that is loaded with the rhBMP-2 in inside.
2. fusion device between the bioactivity, porous titanium alloy people cervical vertebra that the preparation method with fusion device between bioactivity, porous titanium alloy people cervical vertebra as claimed in claim 1 is made, it is characterized in that: between this bioactivity, porous titanium alloy people cervical vertebra, the peripheral part (1) of fusion device has regular hole, average pore size 300-400 μ m, porosity 40-50%; Central part (2) has the irregular hole of bone trabecula sample, average pore size 400-1000 μ m, and porosity 60-80%, and be attached with the gelatine microsphere that is loaded with the rhBMP-2 in the hole of peripheral part (1) and central part (2).
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