CN110433336A - A kind of pre- mineralising natural polysaccharide based aquagel and the preparation method and application thereof - Google Patents

A kind of pre- mineralising natural polysaccharide based aquagel and the preparation method and application thereof Download PDF

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CN110433336A
CN110433336A CN201910778887.XA CN201910778887A CN110433336A CN 110433336 A CN110433336 A CN 110433336A CN 201910778887 A CN201910778887 A CN 201910778887A CN 110433336 A CN110433336 A CN 110433336A
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mineralising
natural polysaccharide
based aquagel
calcium
cap
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李立华
张舒昀
赵要武
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Jinan University
University of Jinan
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Jinan University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/025—Other specific inorganic materials not covered by A61L27/04 - A61L27/12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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    • A61L27/12—Phosphorus-containing materials, e.g. apatite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
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    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
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    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
    • A—HUMAN NECESSITIES
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    • A61L2430/12—Materials or treatment for tissue regeneration for dental implants or prostheses
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    • C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
    • C08J2305/04—Alginic acid; Derivatives thereof
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    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
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    • C08J2405/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2401/00 or C08J2403/00
    • C08J2405/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof

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Abstract

The invention discloses a kind of pre- mineralising natural polysaccharide based aquagels and the preparation method and application thereof.Calcium microcosmic salt is dissolved in acylated natural polysaccharide material aqueous solution, sulfhydrylation sodium alginate polysaccharide material aqueous solution is then added, after mixing, sodium β-glycerophosphate solution is added and adjusts pH to 7.0~8.0, obtains pre- mineralising natural polysaccharide based aquagel.After the freeze-drying of pre- mineralising natural polysaccharide based aquagel, the alkaline phosphatase enzyme solutions 12~48h of dialysis for being 1~10% in mass concentration, freeze-drying obtains the mineralising natural polysaccharide based aquagel timbering material of hydroxyl apatite.Pre- mineralising hydrogel obtained can mineralising in situ obtain the calcium microcosmic salt crystallization for being similar to native hydroxyl apatite in tooth, it more effectively can uniformly be deep into the inside of canaliculi dentales simultaneously, perplex to solve the odonthemodia due to caused by saprodontia to patient's bring, and repair deficiency dentine and enamel structure.

Description

A kind of pre- mineralising natural polysaccharide based aquagel and the preparation method and application thereof
Technical field
The invention belongs to field of tissue engineering technology, and in particular to a kind of pre- mineralising natural polysaccharide based aquagel and preparation method thereof With application.
Background technique
Dental caries (dental caries) are that one kind is extremely common, using bacterium as pathogen, while also along with many Occur caused by other causes of disease in the chronic of dental mineralization tissue, progressive, destructive disease.Clinical signs are tooth table The dentine that face enamel integrality is destroyed, and gradually corrodes even dental pulp forms cavity, and encountering caloric stimulation has allergy Shape can more seriously cause lesion tissue around dental pulp and the tip of a root.Dental caries usually occur in exposed dental surface, and existing bacterium is borrowed It helps and is attached to tooth surface in facing formation salivary pellicle and forms Dental plaque biofilm, the bacterium of the inside, which utilizes, enters plaque In sugar, by glycolysis process generate organic acid, the latter make plaque liquid environment change for tooth mineral it is unsaturated Chemical state forms defect so as to cause the dissolution of solid-state tooth mineral.Further, the organic matter in tooth is in bacterial secretory Related enzyme effect decline solution destroy, cavity is formd on facing after impaired dental tissue disintegration, cavity is deep into tooth sheet Dentinal tubule is exposed after matter, and then causes odonthemodia symptom of having a toothache.Nowadays it is most common to have had developed into oral cavity for dental caries One of disease makes sufferer be hurt to the fullest extent all the time.
Along with lower bound time stamp, traditional medical procedure is no longer satisfied the demand of people, and the mankind thirst for new material There is improving rejection phenomenon caused by implantation material at present or needs the surgical operation therapy feelings of second operation taking-up implant Condition.Hydrogel comes into being in the application of organizational project and regenerative medicine field, it is intended to utilize different specific function type hydrogels It repairs or replaces by a variety of unavoidable inside such as operation, wound, disease or outside cause, caused life body portion function It can be destroyed, the institutional framework that form is damaged.The infection brought due to surgical operation is effectively prevented, implant rejects, and two The problems such as implant is to sufferer bring pain, is taken out in secondary operation.Formed in situ hydrogel not only can by growth factor-loaded or The hydrogel material of drug brings the tissue defect site and formed in situ of higher depth into, can also reduce operation and create to sufferer bring Face, unavoidable infection risk during reduction can pass through the side of formed in situ at some defects position in irregular shape The molding of formula stickiness is to carry out repair.The injection aquagel preparation method for using more at present is mainly ultraviolet light Cause crosslinking or introduce chemical cross-linking agent, as Publication No. CN103524795A patent application in, the temperature sensitive type injectable chitosan Glycan hydrogel product need to introduce genipin cross-linked agent；In the patent application of Publication No. CN103937014A, the chitosan is double Network injection aquagel need to introduce double aldehyde compounds or bis-epoxy class compound is prepared for crosslinking agent.But no matter It is ultraviolet light cross-linking (photoinitiator) or introducing chemical cross-linking agent, can all has certain genotoxic potential to influence on cell.And physics The injection aquagel recovery of crosslinking is poor, and mechanical strength is poor, is unsuitable for surgical procedure.
Self-healing brings new thinking to bio-medical material, again fixed by way of introducing dynamic covalent bond The application field of adopted hydrogel, it will so that macromolecule hydrogel material is further strengthened.Either carrying medicine, three-dimensional cell training Support, or in terms of, even environment unfavorable for hydrogel in the past, as peracid crosses alkali (such as gastric mucosa Repair etc.), mechanical property requirements are excessively high (such as hard tissue repair), and it is wear-resistant to require excessively high (such as joint part tissue repair), The self-healing hydrogel for having different characteristics can overcome original adverse environment, reach the mesh of cross moulding again after destroying , with the continuous deepening of research, selection multiplicity, limitation is less, more preferably hydrogel will serve clinical medical inspection to performance It surveys and treats.Therefore, in order to reduce the damage of normal tissue, it is expensive and be unable to reach a group weaver that odonthemodia repair materials are solved The problems such as journey purpose remineralization develop the novel of a kind of abundant raw material sources, easy to operate, low immunogenicity or non-immunogenicity Tissue engineering material, then it is equipped with the effect of calcium microcosmic salt and bioactive molecule, obtaining one kind can be in repairing tooth defect closing tooth It can mineralising, simulation dentine regenerative process be in situ current for dental caries therapeutic while tubule alleviates odonthemodia symptom The important directions of department of stomatology medical material development.
Natural polysaccharide material such as sodium alginate, hyaluronic acid, chitosan polysaccharide are main in bioplex in nature The organic principle wanted, it is from a wealth of sources, it is cheap, and these materials are due to good biocompatibility and biodegradable Property etc. favor in Tissue Engineering Study by numerous researchers, therefore carry out organizational project by raw material of natural polysaccharide material Research is the effective ways for simulating natural tissues.
Therefore, develop that a kind of small toxicity, mechanical strength are high, gel time is short, controllably have self-healing, can simulate life Object mineralization process and good biocompatibility can in-situ injection shaped hydrogel repair field in saprodontia there is highly important meaning Justice.
Summary of the invention
To solve the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is that provide a kind of pre- mineralising natural The preparation method of polysaccharide based aquagel.
Another object of the present invention is to provide a kind of pre- mineralising natural polysaccharide based aquagels made from the above method.
A further object of the present invention is to provide a kind of above-mentioned pre- mineralising natural polysaccharide based aquagels to repair field in saprodontia In application.By external enzymatic mineralising method by its mineralising, it was demonstrated that the pre- mineralising natural polysaccharide based aquagel can be answered directly Filling sclerous tissues' defect time is being undertaken for reaching in saprodontia or the defect implantable clinical repair of Gu Deng sclerous tissues While multiple physiological function, has the purpose of tissue regeneration ability.The application can be filled alleviation tooth in tooth defect Condition susceptible, while mineralising in situ forms dentine hydroxyapatite structure, repair deficiency position reaches the mesh of regeneration 's.
Still a further object of the present invention is to provide a kind of hydrogel branch as made from above-mentioned pre- mineralising natural polysaccharide based aquagel Frame material and its preparation method and application.
The object of the invention is achieved through the following technical solutions:
A kind of preparation method of pre- mineralising natural polysaccharide based aquagel, comprising the following steps:
Calcium microcosmic salt is dissolved in acylated natural polysaccharide material aqueous solution, sulfhydrylation sodium alginate polysaccharide material is then added Aqueous solution is added sodium β-glycerophosphate solution and adjusts pH to 7.0~8.0, obtain pre- mineralising natural polysaccharide Ji Shui after mixing Gel；
Wherein, calcium microcosmic salt accounts for acylation natural polysaccharide material and sulfhydrylation alginic acid in acylated natural polysaccharide material aqueous solution 0.1~70% of sulfhydrylation sodium alginate polysaccharide material gross mass in sodium polysaccharide material aqueous solution, in the calcium microcosmic salt calcium with The molar ratio of phosphorus is 1~2.
Preferably, the calcium microcosmic salt accounts for acylation natural polysaccharide material and sulfhydrylation in acylated natural polysaccharide material aqueous solution 30~70% of sulfhydrylation sodium alginate polysaccharide material gross mass in sodium alginate polysaccharide material aqueous solution.
Preferably, the calcium microcosmic salt is the mixture of calcium salt and microcosmic salt, and the calcium salt is calcium chloride and/or phosphoglycerol Calcium, the microcosmic salt are at least one of calcium glycerophosphate, sodium glycero-phosphate and sodium dihydrogen phosphate.
Preferably, the double bond of the acylation natural polysaccharide material in the acylated natural polysaccharide material aqueous solution and sulfhydrylation sea The molar ratio of the sulfydryl of sulfhydrylation sodium alginate polysaccharide material in mosanom polysaccharide material aqueous solution is 4:1~1:4, preferably For 1:1.The concentration of the acylated natural polysaccharide material aqueous solution and sulfhydrylation sodium alginate polysaccharide material aqueous solution is 1~ 50mg/mL, preferably 35~40mg/mL.
Preferably, the acylation natural polysaccharide material in the acylated natural polysaccharide material aqueous solution is poly- for maleylation shell At least one of acylated Glucomannan of sugar, maleylation chitosan oligosaccharide, clothing health acylation chitosan and clothing health, more preferably Malaysia acyl Change at least one of chitosan, maleylation chitosan oligosaccharide and clothing health acylation chitosan.
The maleylation chitosan oligosaccharide is reacted to obtain by maleic anhydride with chitosan oligosaccharide；The clothing health acylation chitosan is by clothing health Acid anhydrides is obtained with chitosan reaction；The acylated Glucomannan itaconic anhydride of the clothing health reacts to obtain with Glucomannan.
The acylated natural polysaccharide material is by taking Nmaleoyl chitosan (CS-MA) as an example, preparation method are as follows: by chitosan (CS) it is dissolved in the acetic acid solution of 0.01~0.1mol/L, concentration is 0.1~40mg/mL, then according to CS repetitive unit Maleic anhydride is added with ratio that the molar ratio of maleic anhydride is 1:1, under nitrogen protection room temperature be protected from light 12~for 24 hours, then It is protected from light dialysis 3~7 days, -80 DEG C of 12~48h of freeze-drying obtain Nmaleoyl chitosan, and sealing is protected from light 4 DEG C of preservations.
Described to be protected from light in dialysis 3~7 days, the NaCl that dialyzate used in second day is pH=6.0, mass concentration is 1% is molten Liquid, dialyzate used in remaining dialysis time are the deionized water of pH=6.0, replace a dialyzate every 12h, purpose exists In removing impurity or unreacted small molecule.
The time being protected from light is preferably the time of the dialysis preferably 4 days for 24 hours；The bag filter of the dialysis Aperture be 5000~8000KDa of φ.
Preferably, the sulfhydrylation sodium alginate polysaccharide material in the sulfhydrylation sodium alginate polysaccharide material aqueous solution by with Lower section method is prepared: 2- (N- morpholine) is added into sodium alginate polysaccharide material (SA) aqueous solution that concentration is 5~20mg/mL Ethanesulfonic acid monohydrate (MES) adjusts pH to 6.0, and ethyl -3- (3- DimethylAminopropyl) carbodiimide hydrochloride is added (EDAC) it is protected from light with n-hydroxysuccinimide (NHS), adjusting pH to 5.0~6.0, is stirred to react 20min under room temperature, According to n-NH2Cysteine hydrochloride is added in the molar ratio of/n-COOH=1:1, adjusts pH to 5.0, be protected from light, room temperature condition Under be stirred to react 10h, be then protected from light dialysis 3~7 days, -80~-50 DEG C of 12~48h of freeze-drying obtain sulfhydrylation alginic acid Sodium polysaccharide material, sealing are protected from light 4 DEG C of preservations；Wherein concentration of the MES in SA aqueous solution be 0.1mol/L, EDAC, NHS and COO-Molar ratio be 1:1:1.
Described to be protected from light in dialysis 3~7 days, dialyzate used in second day is pH=6.0, contains 1wt%NaCl and concentration For three (2- carboxyethyl) phosphine (TCEP) solution of 0.1~1mmol/L；Dialyzate used in remaining dialysis time is pH=6.0, concentration For the TCEP solution of 0.1~1mmol/L.
The aperture of the bag filter of the dialysis is 5000~8000KDa of φ；The regulator for adjusting pH is 1mol/ The HCl solution or NaOH solution of L.
Preferably, the addition sulfhydrylation sodium alginate polysaccharide material aqueous solution preferably carries out under at the uniform velocity stirring condition.
Preferably, the concentration of the sodium β-glycerophosphate solution is 30~58g/100mL, preferably 58g/100mL.Institute It states sodium β-glycerophosphate solution to be preferably added dropwise under agitation, control is added complete in 30s.
Preferably, it after the addition sodium β-glycerophosphate solution adjusts pH to 7.0~8.0, can be obtained in 5~15min To pre- mineralising natural polysaccharide based aquagel.
A kind of pre- mineralising natural polysaccharide based aquagel made from the above method.
A kind of application of the above-mentioned pre- mineralising natural polysaccharide based aquagel in saprodontia repair materials field.
The pre- mineralising natural polysaccharide based aquagel is as saprodontia repair materials, application method are as follows: pre- mineralising is natural Polysaccharide based aquagel is coated in saprodontia or dentinal tubule surface, in the environment of 20~40 DEG C and human body contain alkaline phosphatase Block and mineralising in situ, the mineralising natural polysaccharide based aquagel timbering material of formation hydroxyl apatite, wherein mine in situ The time of change is 12~48h.
The original position mineralising preferably carries out in human oral cavity and vivo environment, and temperature is Human Physiology normal body temperature 36 ~37 DEG C.
Pre- mineralising natural polysaccharide based aquagel is coated in after saprodontia or dentinal tubule surface, can be in 5~15min Gel solids are frozen into, and will not be fallen off easily, mineralization process (12~48h) refers to that the calcium microcosmic salt of gel solids internal stent passes through The process of hydroxyapatite is formed with alkaline phosphatase enzyme effect.
A kind of preparation method of the mineralising natural polysaccharide based aquagel timbering material of hydroxyl apatite, including following step It is rapid:
After above-mentioned pre- mineralising natural polysaccharide based aquagel freeze-drying, the alkaline phosphatase for being 1~10% in mass concentration Enzyme solutions 12~48h of dialysis, freeze-drying obtain the mineralising natural polysaccharide based aquagel timbering material of hydroxyl apatite.
The temperature of the freeze-drying is -80 DEG C, and the time is 12~48h.
The aperture of the bag filter of the dialysis is 5000~8000KDa of φ, and the pH of the alkaline phosphatase enzyme solutions is 7.0~9.0, preferably pH=8.0；The temperature of the dialysis is 20 DEG C~120 DEG C, preferably 37~60 DEG C.
A kind of mineralising natural polysaccharide based aquagel timbering material of hydroxyl apatite made from the above method.
A kind of mineralising natural polysaccharide based aquagel timbering material of above-mentioned hydroxyl apatite is in saprodontia and bone defect healing Application in Material Field.
For preparation method of the present invention using modified natural polysaccharide as raw material, acylated natural polysaccharide material and sulfhydrylation sodium alginate are more Sugared material, by Michael addition reaction, using different functional groups mole when solution concentration, control gel swelling ratio, The controllable hydrogel of gelling performance is prepared in intensity, degradation rate etc..The unique structure of sodium alginate imparts solidifying in component The specific characteristics of colloid system calcium rush self-healing.Hydrogel abundance of the invention, easy to operate, gel time is short, reaction Mild condition, gelling performance are controllable, can carry out under the conditions of Human Physiology and gel can be realized without other chemical cross-linking agents Change.And the calcium microcosmic salt being rich in the hydrogel can make the hydrogel coated in both carious surfaces, be completed using the ALP of human body itself Mineralising in situ, achievees the purpose that reparation while filling up defect.The hydrogel that load calcium microcosmic salt is added can promote osteoblast Growth can effectively protect surgical wound surface, reduces surgical wound surface bleeding, has antiphlogistic antibacterial effect, promotes wound healing, can apply It as tissue engineering material, repairs especially suitable for saprodontia, surgical procedure is strong, automatic bonding in surgical procedure, without seam It closes and fixes, the surface of a wound of any shape, position can be all effectively protected.Meanwhile it being obtained by in-vitro simulated human body mineralization process A kind of mineralising natural polysaccharide based aquagel timbering material containing human body hard tissue main component hydroxyapatite, can be used as plant Enter the human body hard tissues defect such as the implantation of material direct surgical operation and tooth or bone, carry out function replacement, pattern reparation and Promote osteoblast enrichment, proliferation achievees the purpose that regeneration, and this embedded type natural polysaccharide hydrogel scaffold material is not necessarily to Second operation takes out, and avoids and brings physiology to sufferer and psychological injure again.
Compared with prior art, the present invention has the following advantages and beneficial effects:
(1) present invention preparation reaction system mild condition, easy to operate and control, raw material sources are abundant, at low cost, nothing Chemical cross-linking agent need to be added.
(2) present invention preparation is easy, plastic rapidly, convenient for surgical procedure, it is not necessary that any crosslinking agent is added, can be not The hydrogel of regular defect formed in situ not only has a good mechanical performance, porous network structure, and good biological is compatible The good characteristics such as property, simultaneously because in SA unique the eggshell state structure and high-valence cationic of guluronic acid section-" G sections " chelating Reaction can form secondary cross-linking site, so that this hydrogel is being squeezed shape by the external world but also with certain self-healing Shape can utilize human oral cavity or the free state Ca contained by itself when occurring damaged2+Self-regeneration is carried out, while in shape Has multistage plasticity.
(3) preparation method of the present invention is using modified natural polysaccharide as raw material, acylated natural polysaccharide material and sulfhydrylation alginic acid Sodium polysaccharide material using different functional groups mole when solution concentration, controls the swelling of gel by Michael addition reaction The controllable hydrogel of gelling performance is prepared in rate, intensity, degradation rate etc..
(4) the calcium microcosmic salt that the present invention is rich in can make the hydrogel coated in both carious surfaces, utilize the ALP of human body itself Mineralising in situ is completed, self-regeneration is achieved the purpose that while filling up defect, and the hydrogel that calcium microcosmic salt is added can promote into Bone cell growth can effectively protect surgical wound surface, reduces surgical wound surface bleeding, can especially fit using tissue engineering material is used as For in saprodontia reparation.
(5) present invention prepares that hydrogel surgical procedure is strong, automatic bonding in surgical procedure, fixes without suturing, to appointing What shape, position the surface of a wound can all be effectively protected.
(6) hydrogel prepared by the present invention has external mineralizing analogue ability, and one kind obtained contains human body hard tissue The mineralising natural polysaccharide based aquagel timbering material of main component hydroxyapatite can be directly as implantation material, filling implantation To human teeth or Gu Deng sclerous tissues defect, it is not necessarily to secondary taking-up, no cytotoxicity can substitute on pattern and composition Defect, and it is enriched with osteoblast, regeneration is carried out in defect.
(7) the pre- mineralising natural polysaccharide based aquagel of the present invention dissociates in human body under the action of alkaline phosphatase (ALP) The calcium microcosmic salt of state can simulate biomineralization process by template of the three-dimensional network-like structure of this hydrogel, fill up tooth defect Mineralising in situ is carried out while blocking canaliculi dentales obtains the calcium microcosmic salt crystallization for being similar to native hydroxyl apatite (HAP) in tooth. And the hydrogel of flowable state enough more effectively can uniformly be deep into the inside of canaliculi dentales excessively, to solve the tooth due to caused by saprodontia Sensitivity perplexs to patient's bring, and repair deficiency dentine and enamel structure.
Detailed description of the invention
Fig. 1 is pre- mineralising natural polysaccharide Ji Shui in the MS-CaP-0 and embodiment 1 of natural polysaccharide based aquagel in comparative example 1 The molding figure of gel MS-CaP-30, MS-CaP-50, MS-CaP-70.
Fig. 2 be comparative example 1 in natural polysaccharide based aquagel MS-CaP-0 and embodiment 1 in pre- mineralising natural polysaccharide Ji Shui Gel MS-CaP-30, MS-CaP-50, MS-CaP-70 micro-gel time diagram.
Fig. 3 is the pre- mineralising natural polysaccharide base in natural polysaccharide based aquagel MS-CaP-0 and embodiment 1 in comparative example 1 The scanning electron microscope (SEM) photograph (SEM) of hydrogel MS-CaP-30, MS-CaP-50, MS-CaP-70, wherein amplification factor is 300 times, figure 3a~d is followed successively by the SEM figure of MS-CaP-0, MS-CaP-30, MS-CaP-50 and MS-CaP-70.
Fig. 4 is MS-CaP-0, MS-CaP-30, MS-CaP-50 and MS-CaP-70 water absorption and swelling curve graph.
Fig. 5 is that pre- mineralising natural polysaccharide based aquagel MS-CaP-50 is coated in dental caries badly vertical section of exposed dentine in embodiment 1 Confocal microscope figure behind face.
Fig. 6 is that the saprodontia coated with mineralising natural polysaccharide based aquagel MS-CaP-50 pre- in embodiment 1 is sliced (transverse and longitudinal side Upward filling reparation) scanning electron microscope (SEM) photograph, amplification factor be 100 times.
Fig. 7 is in the MMS-0 in comparative example 2, MMS-30, MMS-50, MMS-70 in embodiment 2 and embodiment 3 The XRD diagram of MMS-12, MMS-24, MMS-48, MMS-72, wherein Fig. 7 a is the XRD of MMS-0, MMS-30, MMS-50, MMS-70 Figure, Fig. 7 b are the XRD diagram of MMS-12, MMS-24, MMS-48, MMS-72 and MMS-96.
MMS-30, MMS-50, MMS-70 in MMS-0, embodiment 2 in Fig. 8 comparative example 2, SEM figure, amplification factor It is 1000 times, length of the scale 200nm.
The SEM of MMS-12, MMS-24, MMS-48, MMS-72 in Fig. 9 embodiment 3 scheme, and amplification factor is 1000 times, mark Ruler length is 200nm.
Figure 10 is energy dispersive spectrum (EDS) figure of MMS-12, MMS-24, MMS-48, MMS-72 in embodiment 3.
Figure 11 is pre- mineralising natural polysaccharide based aquagel and hydroxyl apatite mineralising natural polysaccharide based aquagel bracket material Expect preparation process schematic diagram.
Figure 12 is cell viability point and line chart after MS-CaP-0, MS-CaP-30, MS-CaP-50 and MS-CaP-70 culture.
Figure 13 is cell viability difference analysis after MS-CaP-0, MS-CaP-30, MS-CaP-50 and MS-CaP-70 culture Histogram.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited In this.
Room temperature described in the embodiment of the present application is 25 DEG C unless otherwise specified.
Mouse bone-forming cell MC3T3-E1 used in embodiment is aseptically extracted from newborn mice cranium and is obtained, Mouse sheet comes from Jinan University Medical School animal experimental center.Cell line is placed in containing 10% (v/v) fetal calf serum (FBS) In the Dulbecco modified Eagle medium (DMEM) of 100U/mL penicillin/streptomycin, atmosphere (37 DEG C, 5% are moistened CO2) in secondary culture, obtain MC3T3-E1 cell.It is limited that chemical reagent used is purchased from Shanghai Aladdin biochemical technology share Company.Used medium presses DMEM low sugar culture solution: fetal calf serum: dual anti-reagent=44:5:1 volume ratio configuration gained.
The concentration of phosphate buffer described in the embodiment of the present application is pH=7.4.
Embodiment 1
(1) chitosan (CS) is dissolved in the acetic acid solution of 0.05mol/L, concentration of the chitosan in acetic acid solution For 10mg/mL, dissolved CS solution is placed in three-necked flask, is 1 according to the molar ratio of CS repetitive unit and maleic anhydride: Maleic anhydride is added in 1 ratio, and (maleic anhydride is dissolved in 15ml deionized water, is added in three times, and each additional amount is identical, often Secondary addition interval time be 5min), nitrogen is protected from light for 24 hours under room temperature, then with bag filter (φ 5000~ It 8000KDa) installs and is placed in dialyzate, dialysis 4 days is protected from light, wherein first and third, four day dialyzate is going for pH=6.0 Ionized water, the NaCl solution that second day dialyzate is pH=6.0, mass concentration is 1% replace a dialyzate every 12h, Impurity or unreacted small molecule are removed, dialysis is completed to be placed in culture dish, is freeze-dried for 24 hours at -80 DEG C, obtains Malaysia Acylation chitosan, sealing are protected from light 4 DEG C of preservations.
(2) sodium alginate (SA) powder is dissolved in the water to obtain the SA aqueous solution that concentration is 10mg/mL, 2- (N- is added Morpholine) for ethanesulfonic acid monohydrate (MES) as surfactant, the concentration in SA aqueous solution is 0.1mol/L, use 1mol/ System is adjusted pH=6.0 by L hydrochloric acid solution.According to EDAC, NHS and the COO of finally feeding intake-Molar ratio be 1:1:1, successively plus Enter ethyl -3- (3- DimethylAminopropyl) carbodiimide hydrochloride (EDAC) and n-hydroxysuccinimide (NHS), adjusts body It is pH=6.0, after being protected from light, being stirred to react 20min under room temperature, according to n-NH2The molar ratio of/n-COOH=1:1, toward reaction Cysteine hydrochloride is added in system, and utilizes sodium hydroxide (NaOH) solution (1mol/L) regulation system pH=5.0.It is keeping away Light is stirred to react 10h under room temperature, places reaction liquid into bag filter (5000~8000KDa of φ), is protected from light dialysis 4 days, Three (2- carboxyethyl) phosphines (TCEP) that wherein first and third, four day dialyzate is pH=6.0, concentration is 0.1mmol/L are molten Liquid, second day dialyzate be pH=6.0, three (2- carboxyethyl) phosphine (TCEP) solution containing 1wt%NaCl (TCEP solution Concentration is 0.1mmol/L), a dialyzate is replaced every 12h, removes impurity or unreacted small molecule, postposition is completed in dialysis It in culture dish, is freeze-dried at -80 DEG C for 24 hours, obtains sulfhydrylation sodium alginate polysaccharide material, sealing is protected from light 4 DEG C of preservations.
(3) sulfhydrylation sodium alginate polysaccharide material in 0.4g step (2) is added in 10mL deionized water, it is sufficiently molten The A liquid that sulfhydrylation sodium alginate polysaccharide material mass concentration is 0.04g/mL is obtained after solution；By the Malaysia 0.35g acyl in step (1) Change chitosan to be added in 10mL deionized water, obtaining Nmaleoyl chitosan mass concentration after completely dissolution is 0.035g/mL B liquid.
(4) it will be dissolved in as calcium source-calcium chloride of phosphoric acid apatite presoma and phosphorus source-calcium glycerophosphate (Ca-GP) In the B liquid (10mL) of step (3), the A liquid (10mL) of step (3) is slowly added under agitation above-mentioned dissolved with phosphoric acid phosphorus In the B liquid of lime stone presoma, after 30s fullys shake, 500 μ L, mass concentration 58g/100mL are added dropwise under agitation Sodium β-glycerophosphate solution is added completely in 30s, pre- mineralising natural polysaccharide based aquagel is obtained, wherein added inanimate matter (phosphoric acid apatite presoma) gross mass accounts for 30%, the 50% of sulfhydrylation sodium alginate and Nmaleoyl chitosan gross mass respectively With 70%, the molar ratio of calcium and phosphorus is 1.67.According to the suitable of added inanimate matter gross mass content 30%, 50% and 70% The resulting pre- mineralising natural polysaccharide based aquagel of correspondence is successively labeled as MS-CaP-30, MS-CaP-50 and MS-CaP- by sequence 70。
Comparative example 1 does not add calcium microcosmic salt
Step (1)~(3) are the same as embodiment 1.
(4) the A liquid (10mL) of step (3) is slowly added under agitation in the B liquid of above-mentioned presoma, sufficiently After shaking 30s, 500 μ L are added dropwise under agitation, mass concentration is 58g/100mL sodium β-glycerophosphate solution, In It is added completely in 30s, obtains natural polysaccharide based aquagel, be labeled as MS-CaP-0.
By bottle anastrophe, MS-CaP-30, MS-CaP- in MS-CaP-0 and the embodiment 1 in comparative example 1 are observed 50, the pre- mineralising hydrogel material of MS-CaP-70, from flowable state to solidification state, (20 DEG C) time is no more than under normal temperature conditions 15min (Fig. 1 (a)).Further, by the extrusion molding from syringe by flowable state gel, coagulation forming on a glass, And deformation is not fallen off, the time, also within 15min (Fig. 1 (b)) the 1st, 2 figure was MS-CaP-0 in Fig. 1 b, and 3 be same hand As a result the pre- mineralising hydrogel of MS-CaP-30, MS-CaP-50, MS-CaP-70 of method injection molding proves that hydrogel can be with It is molded into different shapes and forms gel.And then the microscopic sdIBM-2+2q.p.approach of mechanics is carried out to the hydrogel of institute's injection molding, it takes respectively The pre- mineralising hydrogel material of MS-CaP-30, MS-CaP-50, MS-CaP-70 in MS-CaP-0 and embodiment 1 in comparative example 1 (the platform diameter on Kinexus Pro rotational rheometer platform is added dropwise in 1mL), subsequent gel sample is in 37 DEG C of items Strain sweep (f=1Hz) is carried out under part and determines linear viscoelastic region, scanning result is recorded and observe, with storage modulu and springform Amount intersection point is gel site, determines gel time and modulus change.The rheology by changing over time can be obtained according to interpretation of result (Fig. 2) shown in scanning figure, in 32s, the storage modulus (G') of MS-CaP-0 gradually increases and intersects with loss modulus (G "), card Hydrous gel gel site initially forms, and with the introducing of calcium microcosmic salt, Ca2+Rapid with the ionic reaction of SA, it reduce CS- Physical crosslinking effect and Michael addition reaction between MA and SA-SH, therefore gelation time slightly postpones, MS-CaP-30, The gel time of MS-CaP-50 and MS-CaP-70 is respectively 409s, 309s and 203s, wherein being continuously increased with calcium salt, Ca2+Chelation between SA-SH increases, therefore it can accelerate the gel-forming of MS-CaP gel, therefore gel site shape It is gradually shortened again at the time.And modulus change is little, and MS-CaP-50 mechanical property increases significantly, it was demonstrated that hydrogel tool Standby certain biomechanical property.
Fig. 3 is the pre- mineralising natural polysaccharide base in 1 gained natural polysaccharide based aquagel MS-CaP-0 of comparative example and embodiment 1 Hydrogel MS-CaP-30, MS-CaP-50, MS-CaP-70 observe the microstructure of gel by SEM electromicroscopic photograph, such as Fig. 3 (a- D), wherein Fig. 3 a is MS-CaP-0, and Fig. 3 b is MS-CaP-30, and Fig. 3 c is MS-CaP-50, and Fig. 3 d is MS-CaP-70, due to cold It is lyophilized dry, all samples show continuous porous structure, are consistent with pure water gel result.With phosphoric acid calcium concentration Increase, the diameter in hole reduces, this may be due to two main causes: 1. the calcium phosphate deposition in sample affects ice crystal It is formed, and also prevents heat transfer in freezing process, therefore compared with MS-CaP-0 sample, the shape of MS-CaP-70 hole At being restricted；2. in addition, Ca2+Chelation between SA increases the degree of cross linking during gel-forming, results in more Fine and close structure, this is consistent with swelling behavior test result.After absorbing moisture in Fig. 4, desiccant gel MS-CaP-0, MS- CaP-30, MS-CaP-50 and MS-CaP-70 can revert to hydrogel scaffold.Swelling ratio is tested using gravimetric method, point The balancing segment of line shows that all samples absorb water rapidly in 30 minutes, then progressivelyes reach balance.MS-CaP-0 gel has most Big swelling ratio, Qmax are 5.10 ± 0.15, and this is mainly due under not calcic microcosmic salt disturbed condition, crosslink density is minimum, Gel network is most loose in all samples.With the increase of calcium microcosmic salt proportion in hydrogel, aperture reduces, and water absorption is gradually Reduce, so the Qmax of MS-CaP-70 is reduced to 2.3 ± 0.23.
Preparing dental caries, badly exposed dentine longitudinal section sample (sheet) is several, is polishing to 0.5mm thickness using polishing polishing machine Degree, until sample shows good translucency (such as Fig. 5).Obtained in 1g/L rhodamine B solution dye marker embodiment 1 MS-CaP-50 hydrogel after the label is coated on vertical profile tooth cross section, freezes at -80 DEG C by MS-CaP-50 hydrogel After 24 hours dry, it is cleaned by ultrasonic surface gel with deionized water, is coated with MS-CaP-50 water for above-mentioned with phthalic acid ester Slide is made in the tooth piece sample closing of gel, and slide is tipped upside down on laser confocal microscope sample stage, is in excitation wavelength The feelings that hydrogel closes infiltration on canaliculi dentales are observed and photographed to record to 546nm, launch wavelength under conditions of being 600nm Condition.Fig. 5 is picture of the dentinal tubule under laser confocal microscope, since rhodamine B shows under 546nm excitation wavelength Existing red fluorescence, and only have in MS-CaP-50 material on dentine and contain rhodamine B, it is displayed in red under Laser Scanning Confocal Microscope Part be exactly position existing for gel, observation feux rouges (Fig. 5) A place present with canaliculi dentales shape coincide elongated tubular product, and get over To the last dies down (B at) close to the following position red the few, it was demonstrated that hydrogel after permeating to a certain extent due to Surface tension and viscous effect do not permeate still further below, and occur lateral gel accumulation at C and be detained situation, we are initial guess C Place is that cross section coats plane.Gel under the effect of gravity along canaliculi dentales tube wall extend, penetration depth at 10~20 μm, this One result is consistent with expection, it was demonstrated that the hydrogel in the present invention can help it to load from flowable state to solidification this process of state Calcium microcosmic salt is uniformly deep into dentinal tubule depths, carries out more thorough and comprehensive canaliculi dentales closing, protects exposed tooth sheet Matter layer.
By gained MS-CaP-50 hydrogel sample in embodiment 1, it is coated in dental caries and badly exposes enamel and dentine transverse and longitudinal section On the tooth of face, by the tooth sample for being coated with hydrogel after mass concentration is to dialyse for 24 hours in 4% alkaline phosphatase enzyme solutions, use Deionized water carries out ultrasonic cleaning 30min in Ultrasound Instrument, and removal excess surface obtains hydrogel, is then fixed on together with conducting resinl On platform, metal spraying covering carries out morphology observation to sample using SEM (operating voltage 20.0kV).And selection region is targeted EDS scanning is carried out, selected areas chemical element and content are analyzed.MS-CaP-50 hydrogel is observed to canaliculi dentales (In by SEM Filling reparation on transverse and longitudinal direction) closing as a result, by finding out in Fig. 6, first row is enamel surface before and after coating hydrogel Mineralization, special fish scale-shaped structure is presented in enamel surface before applying, and is formed by hydroxyl phosphorus after applying hydrogel mineralising Lime stone forms new mineralized layer along certain orientation close-packed arrays, plays certain protective effect；Second row is that dentine is horizontal Cross-sectional sample, canaliculi dentales hole is clear before closing, the smooth of the edge, but as gel flows into hole in liquid, condenses rapidly At gel, and a large amount of inanimate matters are generated in subsequent mineralization process and replenish aperture position, finally show closing covering shape State, also performance is obvious in third row's picture longitudinal section sample for this phenomenon, arranges compact canaliculi dentales, edge becomes after closure It must obscure, groove part is filled by the covering of greatly degree.Prove that the natural based aquagel containing certain calcium microcosmic salt can promote Into the surface remineralization of dentine, it uniformly can in depth be filled into canaliculi dentales and play the role of closing and alleviate odonthemodia, from And it can be applied to the minimally-invasive treatment of saprodontia and hypersensitive dentin.
Embodiment 2
Embodiment 1 is obtained into pre- mineralising hydrogel MS-CaP-30, MS-CaP-50 and MS-CaP-70 and carries out extracorporal dialysis mine Change, specifically: by above-mentioned pre- mineralising hydrogel in -80 DEG C of pre-cooling 12h, -80 DEG C of freeze-dryings 24 obtain lyophilised gel h；With The alkaline phosphatase enzyme solutions (pH is adjusted to=8.0 through the NaOH solution of 1mol/L by the solution) that mass concentration is 4%, which are dialysed, to be frozen Xerogel is dialysed 24 hours, and temperature 60 C of dialysing, bag filter is 5000~8000KDa of φ.Later, with deionized water flushing water Gel is to neutrality and is freeze-dried, and is freeze-dried again for 24 hours with -80 DEG C, obtains containing human body hard tissue main component hydroxyl phosphorus The mineralising natural polysaccharide based aquagel timbering material of lime stone.According to added inanimate matter gross mass content 30%, 50% and 70% Sequence, by the resulting hydroxyl apatite mineralising natural polysaccharide based aquagel timbering material of correspondence be successively labeled as MMS-30, MMS-50 and MMS-70.
Comparative example 2
Comparative example 1 is obtained into natural polysaccharide based aquagel MS-CaP-0 and carries out extracorporal dialysis mineralising, specifically: by MS- CaP-0 obtains lyophilised gel in -80 DEG C of pre-cooling 12h, -80 DEG C of freeze-dryings 24；The alkaline phosphatase for being 4% with mass concentration Solution (pH is adjusted to 8.0 through the NaOH solution of 1mol/L by the solution) dialysis lyophilised gel, dialyses 24 hours, temperature 60 of dialysing DEG C, bag filter is 5000~8000KDa of φ.Later, it with deionized water flushing water gel to neutrality and is freeze-dried, with -80 DEG C It is freeze-dried again for 24 hours, obtains natural polysaccharide based aquagel timbering material, be labeled as MMS-0.
Embodiment 3
By the pre- mineralising natural polysaccharide based aquagel MS-CaP-50 in embodiment 1 in -80 DEG C of pre-cooling 12h, then -80 DEG C freeze-drying for 24 hours, obtain lyophilised gel, with mass concentration be 4% alkaline phosphatase enzyme solutions (solution is through 1mol/L's PH is adjusted to=8.0 by NaOH solution) dialysis lyophilised gel, temperature 60 C of dialysing, bag filter is 5000~8000KDa of φ, thoroughly Analysis the time be respectively 12h, for 24 hours, 48h, 72h and 96h.Later, it with deionized water flushing water gel to neutrality and is freeze-dried, It is freeze-dried again with -80 DEG C and finally obtains the mineralising natural polysaccharide containing human body hard tissue main component hydroxyapatite for 24 hours Based aquagel timbering material.According to dialysis time 12h, for 24 hours, the sequence of 48h, 72h and 96h, successively be labeled as MMS-12, MMS- 24, MMS-48, MMS-72 and MMS-96.
By the MMS-0 in the comparative example 2 of equal volume, MMS-30, MMS-50, MMS-70 in embodiment 2, and implement MMS-12, MMS-24, MMS-48, MMS-72, MMS-96 brittle failure in -196 DEG C of liquid nitrogen in example 3, using X-ray diffraction Instrument, with voltage 36kV, Guan Liu 20mA, CuK α target, 2 θ=4 °~60 ° of scanning range are that condition carries out X-ray diffraction point to sample Sample XRD spectrum is observed in analysis.Such as Fig. 7 (a) (sample MMS-0, MMS-30, MMS-50, MMS-70), display gained mineralization product With the XRD diffracting spectrum comparative analysis of business pure ha (HAP) powder, all samples in addition to MMS-0 are not all With showing HAP characteristic diffraction peak in degree, and with the increase of calcium microcosmic salt concentration, its characteristic diffraction peak becomes more sharp, becomes Be bordering on pure HAP commodity diffraction maximum, with shown in arrow for annotation about 32 ° three peaks correspond to plane (211), (112), (002), close to 46 ° peak correspond to plane (222), close to 26 ° of peaks correspond to plane (002), and grow (211), (222) and (002).Dialyse in ALP solution for MS-CaP-50 sample and the 12h that dialyses respectively, for 24 hours, 48h, 72h and 96h Afterwards, check that it corresponds to mineralization product in resulting MMS-12, MMS-24, MMS-48, MMS-72 and MMS-96 sample with XRD Crystallinity, as shown in Fig. 7 (b) based on same concentration calcium microcosmic salt, with the extension of mineralising time, product crystallization Degree and purity are also higher.This result directly indicates in the solution using ALP as medium that Ca-GP and calcium chloride can succeed Transformation of the self assembly to HAP.This process of Fig. 7 successfully simulates vivo biodistribution mineralising, it was demonstrated that prepared is hard containing human body Organize main component hydroxyapatite mineralising natural polysaccharide based aquagel timbering material after implanting, can in body fluid Generally existing ALP combined launch Ca-GP and CaCl2Conversion to HAP forms the production of new anthropoid HAP structure and composition Object, to achieve the purpose that hard tissue repair.
By the MMS-0 in comparative example 2, the MMS- in MMS-30, MMS-50, MMS-70 and embodiment 3 in embodiment 2 12, MMS-24, MMS-48, MMS-72 hydrogel sample are fixed on the copper platform for posting conducting resinl, and metal spraying covering utilizes SEM (work Make voltage 20.0kV) morphology observation is carried out to sample.And selection region targetedly carries out EDS scanning, analyzes selected areas Chemical element and content.The form for observing MMS-0, MMS-30, MMS-50, MMS-70 hydrogel respectively with SEM, such as Fig. 8 institute Show, wherein Fig. 8 a is MMS-0, and Fig. 8 b is MMS-30, and Fig. 8 c is MMS-50, and Fig. 8 d is MMS-70.Ca-GP and CaCl2As can Soluble is embedded in MS-CaP hydrogel and is uniformly distributed in inside, after with ALP dialysis for 24 hours, calcium ion and phosphate radical from Son from Ca-GP decompose, react and be then first nucleated in MMS hydrogel, after extended by substrate template of hydrogel network Growth, is finally translated into synthos.The surface of hydrogel otherwise smooth is after mineralising by the inorganic of graininess or flower clusters The covering of matter group polymers, and with the increase of calcium microcosmic salt concentration, the shape of these aggregations becomes more regular, and edge becomes It is thinner, this result highly significant in MMS-70 hydrogel sample.Using SEM, for MS-CaP-50 sample in different time sections Product (as MMS-12, MMS-24, MMS-48, MMS-72) under the conditions of same temperature after mineralising carries out morphology characterization, as a result See that Fig. 9 is shown, wherein Fig. 9 a is MMS-12, and Fig. 9 b is MMS-24, and Fig. 9 c is MMS-48, and Fig. 9 d is MMS-72, when with mineralising Between extend, mineralising deposit gradually becomes rule first by significantly improving, in form in quantity, and edge gradually becomes thin, Petal-shaped, sheet, needle-shaped, tufted this result is presented being conducive to gained mineralization product to be neatly closely spaced, and then, make (Figure 10) is scanned to these mineralising deposits with energy dispersive spectrometry (EDS) and is corresponding in turn to MMS-12, MMS-24, MMS- 48, MMS-72) correspond to the different mineralising times deposited inorganic matter calcium and phosphorus ratio: be respectively 0.956,1.199, 1.276 and 1.463, these results further demonstrate gained from side all in 1.0~1.67 range of human body HAP calcium-phosphorus ratio The ingredient of mineralization product is really hydroxyapatite, it was confirmed that the present invention can simulate biomineralization process using the intracorporal ALP of people, Its application in terms of Dental Erosion is widened.
1 gained MS-CaP-0 hydrogel of comparative selection example and embodiment 1 gained MS-CaP-30, MS-CaP-50 and MS- CaP-70 hydrogel explores the bio-compatible of pre- mineralising natural polysaccharide based aquagel prepared in the present invention for cell culture Property.It is to test positive group with the orifice plate all containing material hydrogel, only having culture medium with not aqueous gel, (culture medium is by DMEM Low sugar culture solution: fetal calf serum: dual anti-reagent=44:5:1 volume ratio configuration gained) orifice plate as compareing negative group.It is all Hydrogel sample uses 75% (v/v) ethyl alcohol to impregnate 2h, and then sterilize 6h under ultraviolet light.After ethyl alcohol volatilizees completely, water-setting Glue sample is rinsed 3 times with phosphate buffered saline (PBS) (PBS, pH=7.4).MS-CaP-0, MS-CaP-30, MS-CaP- after sterilizing 50 and MS-CaP-70 hydrogel is respectively placed in 24 orifice plates, and every group of 3 Duplicate Samples are spare.
It is 2 × 10 by density4The cell suspension of a MC3T3-E1 cell/mL is added dropwise in each hole, and every hole adds 200 μ L incubates two hours (CO at 37 DEG C of incubation conditions in incubate box2Atmosphere) after every hole 1ml culture medium is added, change 1 times/day of liquid, temperature It educates in case and cultivates 1,3,5 and 7 day.It is raw that control group is set as the cell that monitoring anhydrous gel sample contains only under culture conditions It is long.After reaching the predetermined time, every culture hole is rinsed 3 times with PBS (pH=7.4), and 1mL culture medium and 100 μ L CCK-8 examination is added Agent then incubates 3 hours (CO at 37 DEG C of incubation conditions2Atmosphere).The supernatant in each hole is transferred in 96 well culture plates later. Absorbance of the hydrogel sample at 450nm is measured with enzyme standard items liquid, and 1 after cell adherence, the control of calculating in 3,5,7 days Optical density (OD) absorbance of group and hydrogel sample group.Percent viability is calculated using following formula:
Cell activity %=(absorbance in daily experimental group)/(absorbance in daily control group) × 100%
Cell proliferation rate %=(cell activity in daily experimental group)/(cell activity in daily control group) × 100%
Supernatant ultraviolet absorptivity OD value after being incubated for by CCK-8 calculates cell viability and with histogram and dotted line chart It shows.From in Figure 12 it is clear that it is all feminine gender blank control and positive test group in cell all show increase with time Proliferation trend, and with the increase of calcium microcosmic salt concentration in hydrogel, cell viability also increases.Especially for MS-CaP-50 and The cell proliferative conditions of MS-CaP-70, numerical value is very close to control group result.In addition, the survival rate of cell is according to GB/T 16886.5-2003 (ISO 10993-5:1999) is calculated, as shown in every group of cell of Figure 13 and same day control group comparing result, institute Have that experimental group is all lower in first day survival rate, but with the extension of incubation time, numerical value is sharply increased.Meanwhile with dampening The increase of calcium microcosmic salt concentration, survival rate also increase in gel.7th day MS-CaP-50 and MS-CaP-70 respectively reach 97.1 (± 5.50) % and 97.2 (± 2.49) %, this shows that synthos can promote the proliferation of osteoblast, and the experiment for meeting us is pre- Phase.Difference analysis, analytical standard: * * * *=P < 0.0001, * * *=P < are carried out to daily cell survival rate 0.001, * *=P < 0.01, *=P < 0.5, ns=P > 0.5, the proliferative conditions daily to cell carry out difference analysis, knot Fruit (Figure 13) shows that MA-CS/SA-SH, MS-CaP-0, MS-CaP-30, MS-CaP-50 and MS-CaP-70 hydrogel can make Cell steady-state growth but low concentration inanimate matter hydrogel is bigger with respect to the otherness of reference group, but after inanimate matter is added especially For hydrogel in the case of inanimate matter concentration 70% almost with reference group no significant difference, this shows the introducing of inanimate matter not only The proliferation promotion property for not bringing cytotoxicity to enhance it instead to MC3T3-E1 cell to hydrogel material, is conducive to cell Growth, also further demonstrates applicability of this material under rich calcium environment.
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 preparation method of pre- mineralising natural polysaccharide based aquagel, which comprises the following steps:
Calcium microcosmic salt is dissolved in acylated natural polysaccharide material aqueous solution, it is water-soluble that sulfhydrylation sodium alginate polysaccharide material is then added Liquid is added sodium β-glycerophosphate solution and adjusts pH to 7.0~8.0, obtain pre- mineralising natural polysaccharide base water-setting after mixing Glue；
Wherein, calcium microcosmic salt accounts for acylation natural polysaccharide material in acylated natural polysaccharide material aqueous solution and sulfhydrylation sodium alginate is more 0.1~70% of sulfhydrylation sodium alginate polysaccharide material gross mass in sugared material aqueous solution, calcium and phosphorus in the calcium microcosmic salt Molar ratio is 1~2.
2. a kind of preparation method of pre- mineralising natural polysaccharide based aquagel according to claim 1, which is characterized in that the calcium Microcosmic salt accounts for acylation natural polysaccharide material in acylated natural polysaccharide material aqueous solution and sulfhydrylation sodium alginate polysaccharide material is water-soluble 30~70% of sulfhydrylation sodium alginate polysaccharide material gross mass in liquid.
3. a kind of preparation method of pre- mineralising natural polysaccharide based aquagel according to claim 2, which is characterized in that the calcium Microcosmic salt is the mixture of calcium salt and microcosmic salt, and the calcium salt is calcium chloride and/or calcium glycerophosphate, and the microcosmic salt is phosphoglycerol At least one of calcium, sodium glycero-phosphate and sodium dihydrogen phosphate.
4. a kind of preparation method of pre- mineralising natural polysaccharide based aquagel according to Claims 2 or 3, which is characterized in that institute State the double bond and sulfhydrylation sodium alginate polysaccharide material water of the acylation natural polysaccharide material in acylated natural polysaccharide material aqueous solution The molar ratio of the sulfydryl of sulfhydrylation sodium alginate polysaccharide material in solution is 4:1~1:4；The acylated natural polysaccharide material water The concentration of solution and sulfhydrylation sodium alginate polysaccharide material aqueous solution is 1~50mg/mL；
The concentration of the sodium β-glycerophosphate solution is 30~58g/100mL；The sodium β-glycerophosphate solution is in stirring condition Under be added dropwise, control is added complete in 30s.
5. a kind of preparation method of pre- mineralising natural polysaccharide based aquagel according to Claims 2 or 3, which is characterized in that institute State acylation natural polysaccharide material in acylated natural polysaccharide material aqueous solution be Nmaleoyl chitosan, maleylation chitosan oligosaccharide, At least one of clothing health acylation chitosan and the acylated Glucomannan of clothing health.
6. a kind of pre- mineralising natural polysaccharide based aquagel made from any one of Claims 1 to 5 the method.
7. a kind of application of the pre- mineralising natural polysaccharide based aquagel described in claim 6 in saprodontia repair materials field.
8. a kind of preparation method of the mineralising natural polysaccharide based aquagel timbering material of hydroxyl apatite, which is characterized in that packet Include following steps:
After the freeze-drying of pre- mineralising natural polysaccharide based aquagel described in claim 6, the alkalinity for being 1~10% in mass concentration Phosphoric acid enzyme solutions 12~48h of dialysis, freeze-drying obtain the mineralising natural polysaccharide based aquagel bracket material of hydroxyl apatite Material.
9. a kind of mineralising natural polysaccharide based aquagel timbering material of hydroxyl apatite made from claim 8 the method.
10. a kind of mineralising natural polysaccharide based aquagel timbering material of hydroxyl apatite described in claim 9 is in saprodontia and bone Application in impairment renovation material field.
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Application publication date: 20191112