CN109608667A - A kind of galapectite composite hydrogel and its preparation method and application with promotion bone defect healing effect - Google Patents
A kind of galapectite composite hydrogel and its preparation method and application with promotion bone defect healing effect Download PDFInfo
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Abstract
The invention discloses a kind of with the galapectite composite hydrogel and its preparation method and application for promoting bone defect healing effect.The present invention provides a kind of galapectite composite hydrogels that can be used as cell scaffold material, form photo-crosslinking under photoinitiator effect by the gelatin solution of incorporation galapectite and obtain；Wherein, the concentration of the galapectite is 0.1%~20%.Gelatin of the present invention with galapectite and through double bond modification is successfully synthesized with the composite hydrogel for promoting bone defect healing effect, the composite hydrogel can be used as excellent bone tissue engineering stent material, it is provided simultaneously with good mechanical performance, mechanical property and biology performance, it can support the growth and Osteoblast Differentiation of osteocyte, play the role of significantly promoting generation, the bone defect healing of area of new bone, the efficient treatment that can be used for the diseases such as periodontium or bone tissue defect has good potential applicability in clinical practice on bioengineering and field of biomedicine.
Description
Technical field
The invention belongs to biological engineering material technical fields.Having more particularly, to one kind promotes bone defect healing to make
Galapectite composite hydrogel and its preparation method and application.
Background technique
Since certain factor such as wound, infection, tumour etc. makes some sclerotin of bone loss, biggish gap is formed, referred to as
Bone defect.Clinically disease incidence is higher for bone defect, and treatment is the important technical problem of orthopaedics.The whole world is every year because serious
Wound, fracture concurrent infection, after fracture malpractice, bone tumour or with bone defect patient number caused by other complication with thousand
Ten thousand.In China, with the arrival of aging population society, the disease incidence of skeletal diseases is in rising trend, directly results in payment for medical care
With increase, production efficiency and quality of life decline.Although fractures early stage takes positive treatment means more in recent years, make
Bone nonunion and bone defect disease incidence are decreased obviously, but still are the problems that orthopedist needs to face.Currently, repairing bone defect
Method mainly have bone collection, tissue engineering technique, film inductive bone regeneration technology, gene therapy etc., but without thorough
The method for carrying out bone defect healing.Therefore, there is an urgent need to formulate effective alternative strategy, promote osteanagenesis in a secured manner,
To reach complete bone defect healing.
Study of various implant bone alternate material is the hot spot of Recent study, and self or allograph bone can be overcome intrinsic
The shortcomings that, and this exactly clinical problem in the urgent need to address.The characteristic that ideal bone renovating material should have first has:
(1) biocompatibility: it can be directly chemically combined with bone, not prevent osteocyte in the normal activity or interference on its surface
The Natural re generation process of its surrounding bone cells, absorbing to the decomposition of bone tissue has conductibility；
(2) there is plasticity and certain mechanical strength；
(3) biological degradability: being substituted by host bone within a certain period of time, does not influence the reparation of bone tissue, has no toxic side effect；
(4) regeneration induction: by itself or addition self-bone grafting factor, stimulation or induction bone growth.
Currently, clinically used artificial bone has various types of bone cements, bioceramic, tissue engineered bone etc..Its
In, bone cement class material mainly has calcium phosphate bone cement, BONE CEMENT BASED ON ACRYLIC RESIN etc..But bone cement due to mechanical strength not
Foot, portioned product heat of polymerization are excessively high and monomer release, water resistant dissolubility (blood dissolubility) poor, degradation speed and curing time are difficult to control
The disadvantages of, clinical application is subject to certain restrictions.Bioceramic mainly includes hydroxyapatite, bioactivity glass etc., hydroxyl
Apatite major defect is that the mechanical property of itself is poor, intensity is low, brittleness is big, this disadvantage affects it in clinical medicine
Extensive use；The brittleness and fatigability of bioactivity glass are also a problem to be solved, and its pass structure meeting
Oxygen radical, metabolin is caused to bleed around.Tissue engineered bone is then the novel people developed using tissue engineering technique
Work bone material.Artificial bone further include nano-crystal/collagen bone materials, calcium sulfate Osteo set particle bone transplantation substitute product,
Strontium-incorporated hydroxyapatite bone cement, nanometer calcium carbonate/Poly L-lactic acid composite material etc..These synthetic materials are different degrees of
On all there are problems that biocompatibility, sequelae caused by aseptic inflammation caused by physical stimulation and chronic rejection can be caused.
Current more bone renovating materials primarily focus on " transhipment drug ", such as patent CN106730035A passes through dress
It carries correlation and facilitates bone drug, antibacterials isoreactivity substance, stimulation of host cell migration to bone defect position realizes bone in situ again
It is raw.And directly as cell carrier, the research at osteoblast or stem cell implantation bone defect position is less.Reason is to carry
Body itself can promote cell Proliferation to grow, and simultaneously participate in regulation stem cell differentiation, promote the high strength elastic bone of osteogenetic process
Repair materials are less.If the inventor Zhang Liqun seminar of patent CN106730035A is in " gelatin-based bioelastomer load Types of Medicine
The preparation of nanocomposite and performance characterization " in point out, in the gelatin base load Types of Medicine composite material of halloysite nanotubes, angstrom
Lip river stone nanotube has certain inhibiting effect to the growth of cell, and composite material inhibits the life of cell at this time
Long, cell is hardly grown on the surface of gelatin based composites, and many cells are all no longer the normal cellular morphologies such as shuttle-type
But dead either there is a situation where be broken.And different types of cell carrier bracket generates histiocytic function
Different influences.
Therefore, research and develop that a kind of cost performance is high, biocompatibility is good, Bioabsorbable is good and there is bon e formation to promote for itself
Bone renovating material into effect is used to have good innovation and application value on bone defect healing.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the defect of the above-mentioned prior art and deficiencies, provide a kind of cost performance
Height, biocompatibility is good, Bioabsorbable is good and the galapectite composite hydrogel with bone defect healing facilitation.
It is a further object of the present invention to provide the preparation methods of above-mentioned galapectite composite hydrogel.
Another object of the present invention is to provide the application of above-mentioned galapectite composite hydrogel.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
A kind of galapectite composite hydrogel can be used as cell scaffold material, by modified through double bond gelatin, photoinitiator and angstrom
Lip river stone carries out photo-crosslinking and obtains；Wherein, the concentration of the galapectite is 0.1%~20%.
Gelatin of the present invention with galapectite and through double bond modification is successfully synthesized with the compound of promotion bone defect healing effect
Hydrogel, the composite hydrogel can be used as excellent bone tissue engineering stent material, be provided simultaneously with good mechanical performance, mechanics
Performance and biology performance can support the growth and Osteoblast Differentiation of osteocyte, there is the apparent generation for promoting area of new bone, bone defect
The effect of reparation.
Further, in preferred embodiments of the present invention, concentration of the galapectite in composite hydrogel be 1%~
10%.For example, concentration of the galapectite in composite hydrogel can be 1%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%
Deng.
Further, in preferred embodiments of the present invention, concentration of the galapectite in composite hydrogel is 5%
~7%.
Further, in preferred embodiments of the present invention, the galapectite is in nano tubular structure.The present invention can not only
The hydrogel matrix for having suitable performance is targetedly constructed by chemical modification gelatin；Nanotube-shaped angstrom of Lip river is introduced simultaneously
Stone can prepare the periodontal for meeting different condition and bone defect tissue engineering bracket material.
Further, in preferred embodiments of the present invention, the length of the halloysite nanotubes is 200~1000
nm。
Further, in preferred embodiments of the present invention, the concentration of the gelatin through double bond modification is 5%~30%.
Further, in preferred embodiments of the present invention, it is described through double bond modification gelatin total concentration be 10%~
20%.For example, the concentration of the gelatin through double bond modification can for 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,
20% etc..
Further, in preferred embodiments of the present invention, the gelatin through double bond modification is methacrylated
Gelatin.
Further, in preferred embodiments of the present invention, methacrylated gelatin the preparation method comprises the following steps: 50 DEG C
Under water-bath, gelatin is dissolved in and is made into the solution that mass-volume concentration is 10% in phosphate buffer (PBS), presses quality volume afterwards
Methacrylic anhydride is reacted with gelatin solution than 5:1, methacrylic anhydride is added with the speed of 0.5 mL/min, reacts on 50
Continue 3~6 h in DEG C water-bath, the PBS that 4~6 times of amounts are added stop reaction；Gained liquid is dialysed after 4~7 d, is freeze-dried
To the spongy product of white, fluffy, obtaining the spongy product of white, fluffy after product dialysis is dry is methacrylate
Change gelatin (GelMa).
The present invention also provides the preparation methods of the galapectite composite hydrogel, comprising the following steps:
S1. after the gelatin modified through double bond being sterilized and being dissolved with solvent, the gelatin solution after being modified；To after modification
Photoinitiator and galapectite is added in gelatin solution, mixes, obtains pre-polymerization liquid；
S2. it after pre-polymerization liquid being mixed, removing bubble, is placed in mold, photo-crosslinking is carried out under ultraviolet light
Obtain the galapectite composite hydrogel.
Further, in preferred embodiments of the present invention, in step S2, the time of the ultraviolet light is 5~200
s。
Further, in preferred embodiments of the present invention, in step S2, time of the ultraviolet light is 15~
30 s。
Further, in preferred embodiments of the present invention, the photoinitiator is I2959, benzophenone, styrax fourth
One of ether, isopropyl thioxanthone, diphenylethan or methyl benzoylformate are a variety of.
Further, in preferred embodiments of the present invention, the photoinitiator is I2959.
Further, in preferred embodiments of the present invention, solvent described in step S1 is pure water.
Further, in preferred embodiments of the present invention, in step S1, after the photoinitiator is first dissolved in DMSO,
In gelatin solution after adding modification.
The present invention can pass through the items such as the concentration of change ultraviolet light time, the dosage of photoinitiator, type or pre-polymerization liquid
Part regulates and controls the crosslinking rate and plastic speed of hydrogel.
Galapectite composite hydrogel of the present invention has fine and close porous three-dimensional network structure.
Further, in preferred embodiments of the present invention, the elasticity modulus of the galapectite composite hydrogel is 0.05
~1 MPa.
Further, in preferred embodiments of the present invention, the average pore diameter of the galapectite composite hydrogel is big
Small is 100 μm.
The galapectite composite hydrogel as or prepare application in cell carrier, and its repaired as cell carrier
Application in multiple periodontal or bone tissue defect, also within protection scope of the present invention.For example, the galapectite composite hydrogel
It can be prepared into cartilage tissue substitute, be covered on the cartilaginous lesion site of human or animal, interim protective effect is played, micro-
Fracture Technique is treated in the operation of cartilage damage, and operation puncturing position can be covered in, and absorbs bone marrow cell, while as cell
These cells are fixed on damage position by bracket, promote the regeneration of cartilaginous tissue；It can also be prepared into cell-scaffold compound,
The cartilage cell of human or animal or stem cell are seeded on timbering material, cell-scaffold compound is formed, cultivates in vitro
A period of time is grafted directly to cartilaginous lesion site, promotes the regeneration and reparation of cartilaginous tissue or defect bone tissue.
Compared with prior art, the invention has the following advantages:
The present invention is prepared for having unique property galapectite and hydrogel complex using light polymerization process, can be used as biological branch
Frame is used for the efficient treatment of the diseases such as periodontium or bone tissue defect, and application prospect is very wide.
Compound hydrogel material obtained by the present invention overcomes the inhibiting effect that halloysite nanotubes itself grow cell,
Not only have the characteristics that conventional hydrogels (porosity, degradability etc.), but also with itself many good characteristics: (1) having
The porous microstructure for having aperture suitable, provides enough spaces for the adherency of osteocyte, migration, growth and proliferation, maximum
Degree rebuilds surrounding tissue environment；(2) there is suitable swelling ratio, supply advantageous stability and water imbibition for cytotrophy,
Regenerated Bone can participate in dissolution-reconstruction circulation with the activity that normal bone maintains like, to have when facing foreign impacts
Centainly " elasticity "；(3) mechanical property of hydrogel is improved, enough intensity can be provided for bone tissue regeneration；(4) acellular poison
Property, good biocompatibility and osteoinductive are had both, the Proliferation, Differentiation of osteocyte can be accelerated, promotes bone tissue regeneration；(5) originally
Crosslinking rate and the gelation time for inventing galapectite composite hydrogel obtained are controllable (can realize gel in-situ as needed),
The friendship of the conditional regulatories hydrogels such as the concentration by change ultraviolet light time, the dosage of photoinitiator, type or pre-polymerization liquid
Connection rate and plastic speed.
Detailed description of the invention
Fig. 1 is that the scanning electron microscope of galapectite composite hydrogel of the present invention amplifies 100 times of figures.
Fig. 2 be galapectite composite hydrogel of the present invention in PBS solution at any time and swelling broken line graph.
Fig. 3 is that galapectite composite hydrogel tensile property of the present invention detects broken line graph.
Fig. 4 is the comparative survival rate of cells figure (mtt assay detection) of galapectite composite hydrogel of the present invention.
Fig. 5 is to detect cell biological compatibility figure using fluorescence microscope dead cell stain living.
Fig. 6 is that Western blotting detects skeletonization correlative protein expression result figure after cell is grown on gel.
For repairing situation map after Rat calvarial defect 2 months after Fig. 7 galapectite composite hydrogel of the present invention.
Specific embodiment
Further illustrate the present invention below in conjunction with specific embodiment, but embodiment the present invention is not done it is any type of
It limits.Without departing from the spirit and substance of the case in the present invention, to made by the method for the present invention, step or condition simply modification or
Replacement, all belongs to the scope of the present invention；Unless otherwise specified, technological means used in embodiment is those skilled in the art institute
Well known conventional means.
Unless stated otherwise, following embodiment agents useful for same and material are commercially available.
The preparation of 1 galapectite composite hydrogel of embodiment
1, a kind of galapectite composite hydrogel that can be used as cell scaffold material, preparation method includes the following steps:
(1) it prepares pre-polymerization liquid: by gelatin after methacrylated is modified, being configured to the first that concentration is 15% with pure water dissolution
Base acroleic acid esterification gelatin solution, be added 0.1%(w/v) the photoinitiator I2959 for being dissolved in DMSO, add concentration be 5% angstrom
Lip river stone nanotube, three is mixed, and obtains pre-polymerization liquid；
Wherein, by the method for gelatin methacrylated are as follows: the superfine gelatin in pigskin source is dissolved in PBS under 50 DEG C of water-baths
In buffer, it is prepared into 10%(w/v) solution of concentration, methacrylic anhydride is slowly added dropwise with the speed of 0.5 mL/min, with
The ratio of gelatin is 5:1(w/v), after 50 DEG C of stirred in water bath react 6 h plus the PBS of 4 times of amounts stops reaction；Gained is milky white
Color liquid is fitted into bag filter (8~14 KD), and 7 d that dialyse in distilled water dialyse after solution to transparence, is freeze-dried
To the spongy product of white, fluffy, as methacrylated gelatin (GelMa) material；Wherein, methacrylated
The grafting rate of gelatin is 50%~80%；
(2) preparation of composite hydrogel: pre-polymerization liquid obtained above removing bubble being placed in mold, 30 s of ultraviolet lighting,
It can be obtained with fine and close porous three-dimensional network structure, can be used as cell scaffold material can accelerate osteocyte and periodontal cell
Proliferation, Differentiation promotes the galapectite composite hydrogel of bone tissue and paradenlal tissue regeneration.
The preparation of 2 galapectite composite hydrogel of embodiment
The reaction being similar in embodiment 1 is carried out, in addition to the concentration of halloysite nanotubes in embodiment 1 is become 7%.It is logical
The reaction is crossed, obtaining has fine and close porous three-dimensional network structure, and can be used as cell scaffold material can accelerate osteocyte and periodontal
The Proliferation, Differentiation of cell promotes the galapectite composite hydrogel of bone tissue and paradenlal tissue regeneration.
The preparation of 3 galapectite composite hydrogel of embodiment
The reaction being similar in embodiment 1 is carried out, in addition to the concentration of halloysite nanotubes in embodiment 1 is become 3%.It is logical
The reaction is crossed, obtaining has fine and close porous three-dimensional network structure, and can be used as cell scaffold material can accelerate osteocyte and periodontal
The Proliferation, Differentiation of cell promotes the galapectite composite hydrogel of bone tissue and paradenlal tissue regeneration.
The preparation of 4 galapectite composite hydrogel of embodiment
The reaction being similar in embodiment 1 is carried out, in addition to the concentration of halloysite nanotubes in embodiment 1 is become 10%.It is logical
The reaction is crossed, obtaining has fine and close porous three-dimensional network structure, and can be used as cell scaffold material can accelerate osteocyte and periodontal
The Proliferation, Differentiation of cell promotes the galapectite composite hydrogel of bone tissue and paradenlal tissue regeneration.
The preparation of 5 galapectite composite hydrogel of embodiment
The reaction being similar in embodiment 1 is carried out, in addition to by methacrylated gelatin solution and Ai Luo in embodiment 1
The concentration of stone nanotube becomes 10% and 0.1% respectively.By the reaction, obtaining has fine and close porous three-dimensional network structure, can
It can accelerate the Proliferation, Differentiation of osteocyte and periodontal cell as cell scaffold material, promote angstrom of bone tissue and paradenlal tissue regeneration
Lip river stone composite hydrogel.
The preparation of 6 galapectite composite hydrogel of embodiment
The reaction being similar in embodiment 1 is carried out, in addition to by methacrylated gelatin solution and Ai Luo in embodiment 1
The concentration of stone nanotube becomes 20% and 20% respectively.By the reaction, obtaining has fine and close porous three-dimensional network structure, can make
It can accelerate the Proliferation, Differentiation of osteocyte and periodontal cell for cell scaffold material, promote the Ai Luo of bone tissue and paradenlal tissue regeneration
Stone composite hydrogel.
The preparation of 7 galapectite composite hydrogel of embodiment
(1) it prepares pre-polymerization liquid: by gelatin after methacrylated is modified, being configured to the methyl that concentration is 5% with pure water dissolution
Acroleic acid esterification gelatin solution, be added 0.3%(w/v) the photoinitiator benzophenone for being dissolved in DMSO, add concentration be 5% angstrom
Lip river stone nanotube, three is mixed, and obtains pre-polymerization liquid；
(2) preparation of composite hydrogel: pre-polymerization liquid obtained above removing bubble being placed in mold, 15 s of ultraviolet lighting,
It can be obtained with fine and close porous three-dimensional network structure, can be used as cell scaffold material can accelerate osteocyte and periodontal cell
Proliferation, Differentiation promotes the galapectite composite hydrogel of bone tissue and paradenlal tissue regeneration.
The preparation of 8 galapectite composite hydrogel of embodiment
(1) it prepares pre-polymerization liquid: by gelatin after methacrylated is modified, being configured to the first that concentration is 30% with pure water dissolution
Base acroleic acid esterification gelatin solution, be added 0.5%(w/v) the photoinitiator benzophenone for being dissolved in DMSO, add concentration be 5%
Halloysite nanotubes mix three, obtain pre-polymerization liquid；
(2) preparation of composite hydrogel: pre-polymerization liquid obtained above removing bubble is placed in mold, 5 s of ultraviolet lighting, i.e.,
It can obtain with fine and close porous three-dimensional network structure, can be used as cell scaffold material can accelerate the increasing of osteocyte and periodontal cell
Differentiation is grown, the galapectite composite hydrogel of bone tissue and paradenlal tissue regeneration is promoted.
Comparative example 1
The reaction being similar in embodiment 1 is carried out, in addition to the concentration of halloysite nanotubes in embodiment 1 is become 0%(i.e.
It is added without halloysite nanotubes).By the reaction, compared with galapectite composite hydrogel, this comparative example obtain hydrogel its
Swelling ratio (Fig. 2) and mechanical property are poor (shown in Fig. 3).
The Electronic Speculum observation and property test of 9 hydrogel of embodiment
1, hydrogel Electronic Speculum detects
By embodiment 1(5% halloysite nanotubes), embodiment 2(7% halloysite nanotubes), embodiment 3(3% galapectite nanometer
Pipe), embodiment 4(10% halloysite nanotubes) galapectite composite hydrogel and the hydrogel of comparative example 1 (0% galapectite is received
Mitron) scanning electron microscope diagram amplify 100 times, as shown in Figure 1.As can be seen from the figure galapectite Compound Water of the invention
Gel has fine and close porous three-dimensional network structure.Through counting, the average pore diameter size of the galapectite composite hydrogel
It is 100 μm.
2, the equilibrium swelling experiments of hydrogel
(1) method
Equilibrium swelling experiments show the ability of the absorption moisture of hydrogel.The swelling ratio of hydrogel is measured by weight method.It will do
Dry samples weighing is placed in PBS solution, is subsequently placed in 37 DEG C of water bath with thermostatic control, under the time of setting, is taken out sample, is used
Filter paper wipes the excessive moisture for doing its surface, weighing, and the swelling ratio of hydrogel: the matter before and after swelling ratio (%)=swelling is calculated as follows
Quality × 100% before measuring difference/swelling.
(2) result
The swelling ratio result of above-mentioned hydrogel is shown in Fig. 2.In 700 min swelling test, it can be observed that Examples 1 to 4 each group
Galapectite composite hydrogel swelling ratio no significant difference.Compared with galapectite composite hydrogel, 1 hydrogel (0% of comparative example
Halloysite nanotubes) swelling ratio it is poor.
3, the compression performance detection of hydrogel
(1) method
By the slitting shape of above-mentioned hydrogel (40 × 10 mm), extension test is carried out on H5K-S type universal testing machine,
Rate is 50 mm/min, until sample fracture stops.Each sample only detects 3 times, and results are averaged.The stretching of hydrogel
Intensity is the ratio of gel maximum tensile stress and sample in cross section area.
(2) result
The compression performance result of hydrogel is shown in Fig. 3.The result shows that with the increase of galapectite content, galapectite composite hydrogel
Tensile strength with enhancing.Compared with galapectite composite hydrogel, the stretching of 1 hydrogel of comparative example (0% halloysite nanotubes)
Intensity is poor.
The above results illustrate that galapectite compound hydrogel material obtained by the present invention has suitable swelling ratio, are cell
The advantageous stability of nutrition supply and water imbibition, Regenerated Bone can participate in dissolution-reconstruction with the activity that normal bone maintains like
Circulation, to have certain " elasticity " when facing foreign impacts, while improving the mechanical property of hydrogel, can be bone tissue
Regeneration provides enough intensity.Galapectite composite hydrogel obtained by the present invention is provided simultaneously with good mechanical performance, mechanical property
Energy and biology performance.
The biocompatibility of 10 galapectite composite hydrogel of embodiment detects
In order to investigate the biocompatibility of hydrogel, determines cell using mtt assay and dead cell stain living and trained altogether with hydrogel
After supporting, the survival rate of cell changes.
1, method
Material: embodiment 1(5% halloysite nanotubes), embodiment 2(7% halloysite nanotubes), embodiment 3(3% galapectite nanometer
Pipe), embodiment 4(10% halloysite nanotubes) galapectite composite hydrogel and the hydrogel of comparative example 1 (0% galapectite is received
Mitron).
(1) the above-mentioned hydrogel after sterilizing is laid in orifice plate bottom first, then by cell seeding in 24 orifice plates, density
104Cells/well, every hole contain the DMEM(10% FBS of 200 μ L, containing penicillin and each 100 U/mL of streptomysin)；
(2) by above-mentioned culture plate be placed in carbon dioxide incubator (37 DEG C, 5% CO2) in, the time of culture to setting, removal training
It is cleaned three times after supporting base with PBS, the DMEM containing 5 mg/mL MTT reagents is added, continued after cultivating 4 h, remove culture medium, often
The DMSO solution of 200 μ L of Kong Zhongjia measures absorbance value after the dissolution of purple substance in microplate reader；
(3) group directly in tissue culture plate culture is defined as control group；It is added without the of the cell that hydrogel is cultivated
Survival rate is defined as 100% within one day, remaining each group numerical value in contrast, observe 1,4,7 day comparative survival rate of cells respectively.
2, result
(1) as shown in figure 4, comparative survival rate of cells is high, illustrate that cell can be preferably on galapectite composite hydrogel of the present invention
Growth, cellular morphology is good, and the galapectite composite hydrogel of preparation has good cell compatibility without overt toxicity.
(2) simultaneously, after at 1,4,7 day using dead cell stain reagent dyeing cell living, inverted fluorescence microscope observation is obtained
Similar result out: galapectite compound hydrogel material obtained by the present invention overcomes halloysite nanotubes itself and grows to cell
Inhibiting effect, have good cell biological compatibility (see figure 5).
11 hydrogel of embodiment facilitates bone performance detection
Material: embodiment 1(5% halloysite nanotubes), embodiment 2(7% halloysite nanotubes), embodiment 3(3% galapectite nanometer
Pipe), embodiment 4(10% halloysite nanotubes) galapectite composite hydrogel and the hydrogel of comparative example 1 (0% galapectite is received
Mitron).
1, skeletonization GAP-associated protein GAP (COL-1, RUNX2, BMP4, BSP) expression
(1) after dental pulp stem cell being incubated at above-mentioned hydrogel, 14 days osteogenic inductions are carried out, collect total protein of cell；It carries out
Western blotting detects skeletonization GAP-associated protein GAP (COL-1, RUNX2, BMP4, BSP) expression.
(2) the result shows that: to overcome halloysite nanotubes itself right for galapectite compound hydrogel material obtained by the present invention
The inhibiting effect of bone cell growth, can be obviously promoted skeletonization GAP-associated protein GAP in dental pulp stem cell (COL-1, RUNX2, BMP4,
BSP expression) can support the growth and Osteoblast Differentiation of osteocyte, there is the apparent generation for promoting area of new bone.Wherein 7% galapectite
The skeletonization GAP-associated protein GAP content of dental pulp stem cell expression in composite hydrogel is higher than other group of (see figure 6).
2, bone tissue defect repair situation
(1) SPF grades SD rat 30 are injected intraperitoneally phenobarbital (3.5 mg/100 g), make notch in skull surface skin, cruelly
Reveal skull, and is bored with low speed gear division and make the defect that a diameter is 5 mm in skull；Defect at 30 is randomly divided into 6 groups (n=5), yin
Property control group with no treatment, hydrogel group be implanted into cell hydrogel scaffold complex, after 2 months take out skull carry out
Micro-CT scanning；Micro-CT scanning: carrying out Micro-CT scanning for skull, to avoid sample shift in scanning process,
Foam pad is placed around sample to fix.Design parameter is provided that image resolution ratio 1024 × 1024,20 μm of interlamellar spacing.CT value
It is set to bone tissue higher than 225, carries out CT three-dimensional reconstruction, observes skull defeci newborn bone amount.
(2) as a result, it has been found that, as shown in fig. 7, galapectite composite hydrogel of the present invention can participate in regulation stem cell differentiation,
It is obviously promoted adherency, migration, growth and the proliferation of osteocyte, utmostly rebuilds surrounding tissue environment, there is apparent promote newly
Raw ostosis, the effect of bone defect healing, can be used for the efficient treatment of the diseases such as periodontium or bone tissue defect.Wherein, tooth
The more group of marrow stem cell area of new bone is 5% galapectite composite hydrogel group and 7% galapectite composite hydrogel group.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of galapectite composite hydrogel that can be used as cell scaffold material, which is characterized in that by modified through double bond gelatin,
Photoinitiator and galapectite carry out photo-crosslinking in a solvent and obtain；Wherein, the concentration of the galapectite is 0.1%~20%.
2. galapectite composite hydrogel according to claim 1, which is characterized in that the concentration of the galapectite be 1%~
10%。
3. galapectite composite hydrogel according to claim 1, which is characterized in that the galapectite is in nanotube-shaped knot
Structure.
4. galapectite composite hydrogel according to claim 1, which is characterized in that it is described through double bond modification gelatin it is dense
Degree is 5%~30%.
5. galapectite composite hydrogel according to claim 1, which is characterized in that the gelatin through double bond modification is first
Base acroleic acid esterification gelatin.
6. galapectite composite hydrogel according to claim 1, which is characterized in that the galapectite composite hydrogel has
Fine and close porous three-dimensional network structure；Its elasticity modulus is 0.05~1 MPa.
7. galapectite composite hydrogel according to claim 1, which is characterized in that prepared by method comprising the following steps
It obtains:
S1. after the gelatin modified through double bond being sterilized and being dissolved with solvent, the gelatin solution after being modified；To after modification
Photoinitiator and galapectite is added in gelatin solution, mixes, obtains pre-polymerization liquid；
S2. it after pre-polymerization liquid being mixed, removing bubble, is placed in mold, photo-crosslinking is carried out under ultraviolet light
Obtain the galapectite composite hydrogel.
8. galapectite composite hydrogel according to claim 7, which is characterized in that in step S2, the ultraviolet light
Time be 5~200 s.
9. any galapectite composite hydrogel of claim 1~8 as or prepare application in cell carrier material.
10. any galapectite composite hydrogel of claim 1~8 is repairing the application in periodontal or bone tissue defect.
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