CN115634314B - Unsupported bone repair gel microsphere and preparation method thereof - Google Patents
Unsupported bone repair gel microsphere and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a non-supporting bone repair gel microsphere and a preparation method thereof. The preparation method comprises the following steps: preparing SA-PLL and OSA respectively, preparing SA-PLL and OSA into aqueous solution with certain concentration, dispersing BP in the OSA solution, preparing microspheres with relatively uniform particle size by using SA-PLL solution and OSA solution containing BP as water phase and olive oil as oil phase and microfluidic chip, freeze-drying the microspheres, and adding the microspheres into CaCl 2 Soaking in the solution for 24h, and washing off excessive Ca with pure water 2+ The double cross-linked gel microsphere is obtained. The invention has the advantages of simple and easily obtained raw materials and low price; the gel microsphere prepared by double crosslinking of Schiff base and ion crosslinking has good mechanical properties, and BP can play a role in local heating and sterilization after near infrared light irradiation; with the degradation of BP and hydrogel, phosphate, which is the degradation product of BP, can react with Ca 2+ Calcium phosphate is formed, in-situ mineralization is realized, gel degradation is delayed, and bone repair is promoted.
Description
Technical Field
The invention relates to a bone repair material, in particular to an unsupported bone repair gel microsphere and a preparation method thereof.
Background
Bone defects caused by various reasons such as trauma, degenerative disease, congenital deformity, bone tumor, dental implant, periodontal disease, craniotomy and the like are quite common in clinic, and particularly are concentrated in patients in the fields of orthopaedics, stomatology, neurosurgery and the like. At present, most of orthopaedics clinic is to choose a 'split east wall and mend west wall' mode for taking autologous bones (common parts include ilium, ribs, fibula and the like) of patients for bone grafting treatment when treating bone defects; however, autogenous bone has a series of problems that cause secondary injury, donor area complications, prolonged surgery time, increased bleeding volume, limited bone mass, etc. in patients, and is not a perfect clinical solution. Therefore, the reduction of autologous bone in operation and the selection of artificial bone repair materials are the mainstream development trend of clinical bone grafting internationally.
According to the different components of the materials, the artificial bone repair materials can be mainly classified into metal materials, biological ceramics, calcium phosphate/calcium sulfate bone cement, biological glass, high polymer materials, composite materials and tissue engineering materials. The different materials have the characteristics of respective physicochemical properties and biological properties, and are widely applied to bone grafting operations in the fields of orthopaedics, oral and maxillofacial surgery, neurosurgery, plastic surgery and the like, and mainly play roles in filling bone defects, guiding bone regeneration and promoting bone healing.
To sum up, the prior art has the disadvantages that:
1) Autologous bone grafting has high cost and can cause secondary injury;
2) The mechanical properties of inorganic nonmetallic materials such as biological ceramics, calcium phosphate/calcium sulfate bone cement, bioglass and the like are weaker;
3) The metallic material graft is not degradable and the raw materials are expensive.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a preparation method of injectable bone repair gel microspheres, which can well fill bone defect parts and play the roles of resisting bacteria and promoting bone repair.
The aim and the technical problems of the invention are realized by adopting the following technical proposal.
The invention provides a non-supporting bone repair gel microsphere, which is prepared from SA-PLL solution and OSA solution containing BP and passes through CaCl 2 Soaking in the solution to obtain the double-crosslinked gel microsphere.
The invention provides a preparation method of a non-supporting bone repair gel microsphere, which comprises the following steps:
preparing SA-PLL and OSA respectively, preparing SA-PLL and OSA into aqueous solution with certain concentration, dispersing BP in the OSA solution, preparing microspheres with relatively uniform particle size by using SA-PLL solution and OSA solution containing BP as water phase and olive oil as oil phase and microfluidic chip, freeze-drying the microspheres, and adding the microspheres into CaCl 2 Soaking in the solution, and washing off excessive Ca with pure water 2+ The double cross-linked gel microsphere is obtained.
Further, the preparation method of the unsupported bone repair gel microsphere comprises the following steps:
1) Preparation of SA-PLL:
dissolving sodium alginate in water, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, regulating pH, standing, adding N-hydroxysuccinimide, continuously stirring, weighing polylysine, adding into the solution, regulating pH, reacting, dialyzing the solution, and lyophilizing to obtain SA-PLL;
2) Preparation of OSA:
weighing sodium alginate, dissolving the sodium alginate in deionized water, adding ethanol for dispersion and dissolution, and uniformly stirring; weighing sodium periodate in a dark place, dissolving the sodium periodate in deionized water to prepare sodium periodate solution, slowly dripping the sodium periodate into the solution under the dark condition, and reacting for 24 hours in the dark;
after the reaction is finished, a small amount of glycol is dripped to terminate the reaction, naCl is weighed and added into the solution, then the solution is poured into absolute ethyl alcohol, white solid is separated out, suction filtration is carried out, and the precipitate is washed with water; dissolving the precipitate with a small amount of deionized water, dialyzing, and lyophilizing to obtain OSA;
3) Preparation of gel microspheres:
preparing SA-PLL aqueous solution, which is marked as solution A; preparing an OSA aqueous solution, dispersing BP in the OSA solution, and marking the solution as a solution B; uniformly mixing the solution A and the solution B to obtain initial gel, and freeze-drying to obtain a freeze-dried bracket; freeze-drying is supported on CaCl 2 Soaking in the solution for 24h, and cleaning with pure water to obtain Ca 2+ A crosslinked saturated gel.
Further, in the above method for preparing the unsupported bone repair gel microsphere, in the step 1), the sodium alginate is dissolved in water in an amount of 50mL per 1g sodium alginate; the mass ratio of the sodium alginate to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 2:0.8 to 1.2; mass ratio of the sodium alginate to the N-hydroxysuccinimide: 2:0.8 to 1.2; the mass ratio of the sodium alginate to the polylysine: 2:0.8 to 1.5.
In a further aspect, in the preparation method of the non-supporting bone repair gel microsphere described above, in the step 1), the mass ratio of the sodium alginate to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 2:1, a step of; mass ratio of the sodium alginate to the N-hydroxysuccinimide: 2:1, a step of; the mass ratio of the sodium alginate to the polylysine: 2:1.
in the step 1), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is added, the pH is regulated to 5.0-6.0, and the time is 15-30 min; the N-hydroxysuccinimide is added and the stirring time is 15-30 Min; the polylysine is weighed and added, the pH is regulated to 7.4-10.0, and the reaction time is 12-24 hours; the dialysis was performed in a sodium hydroxide solution having ph=10 for 1 day, followed by dialysis in pure water for 3 more days.
In the step 2), the sodium alginate is dissolved in deionized water, wherein each 1g of sodium alginate is dissolved in 40mL of water; the volume ratio of the ethanol to the deionized water is 1:4, a step of; the concentration of the sodium periodate solution is 0.5g/mL; the mass ratio of the sodium alginate to the NaCl is 1:1.
further, the preparation method of the non-supporting bone repair gel microsphere comprises the following steps: in the step 2), the cleaning is three times; the dialysis was three days of dialysis.
Further, in the above preparation method of the unsupported bone repair gel microsphere, in the step 3), the concentration of the SA-PLL aqueous solution is 0.1g/mL; the concentration of the OSA aqueous solution is 0.02-0.06 g/mL; the solution A and the solution B are mixed according to the volume ratio of 1:1; the washing with pure water is repeated for 5 times.
In the preparation method of the unsupported bone repair gel microsphere, in the step 3), the concentration of the aqueous OSA solution is 0.04g/mL.
In the preparation method of the unsupported bone repair gel microsphere, in the step 3), the preparation concentration of BP in the solution B is 100 mug/mL; the CaCl 2 The concentration of the solution was 0.1M.
By means of the technical scheme, the invention has the following advantages and beneficial technical effects:
1) The invention has the advantages of simple and easily obtained raw materials and low price; the gel microsphere prepared by double crosslinking of Schiff base and ion crosslinking has good mechanical properties, and BP can play a role in local heating and sterilization after near infrared light irradiation; with the degradation of BP and hydrogel, phosphate, which is the degradation product of BP, can react with Ca 2+ Calcium phosphate is formed, in-situ mineralization is realized, gel degradation is delayed, and bone repair is promoted.
2) The gel of the invention has better mechanical property, biocompatibility and antibacterial activity, can be filled in the bone defect part by injection through preparing gel microspheres, and the gel components can be degraded into components absorbable by human body, ca 2+ The existence of the crosslinked network prolongs the degradation time of the gel, the period of the composite bone repair, and the Ca released along with the degradation of the gel 2+ Can also promote proliferation of bone cells. Meanwhile, the raw materials in the invention are simple and easy to obtain, the cost is low, and the preparation process is relatively simple.
Drawings
FIG. 1 is a schematic diagram of a microfluidic chip;
FIG. 2 shows the results of the hydrogel biocompatibility test of each example.
Detailed Description
The technical proposal is as follows:
the gel microsphere takes Sodium Alginate (SA) as a main material, and uses carboxyl on a polysaccharide structure of the gel microsphere to be grafted with Polylysine (PLL) rich in an amino structure through amidation reaction to obtain an amination modified SA-PLL; SA is rich in hydroxyl groups and can be oxidized by sodium periodate to generate functional aldehyde groups, so that Oxidized Sodium Alginate (OSA) is obtained. Both SA-PLL and OSA can form imine structures by Schiff base reaction between amino groups and aldehyde groups, resulting in cross-linking to form SA-PLL/OSA gel.
On the premise of taking Schiff base hydrogel as a cross-linked network, the invention adds black phosphorus nanoplatelets (BP) with good photo-thermal property into the gel precursor solution, and the BP can be locally heated under the irradiation of near infrared light to play a role in killing bacteria. In order to enhance the mechanical properties and reduce the swelling performance of SA-PLL/OSA/BP gel, the invention immerses the prepared gel in CaCl 2 Soaking in solution to make carboxyl and Ca in sodium alginate 2+ Ionic crosslinking occurs, forming a second re-crosslinked network. Meanwhile, along with the gradual degradation of BP, the generated phosphate radical can be combined with Ca 2+ The reaction generates calcium phosphate, mineralizes in situ on the gel, and promotes bone repair.
The specific embodiment of the gel microsphere is as follows:
preparing SA-PLL and OSA respectively, preparing SA-PLL and OSA into aqueous solution with certain concentration, dispersing BP in OSA solution, preparing microsphere with relatively uniform particle diameter by micro-fluidic chip shown in figure 1 with SA-PLL solution and OSA solution containing BP as water phase and olive oil as oil phase, freeze-drying the microsphere, and dispersing in CaCl 2 Soaking in the solution for 24h, and washing off excessive Ca with pure water 2+ The double cross-linked gel microsphere is obtained.
Preparation of the polylysine grafted sodium alginate (SA-PLL) described above
2g of sodium alginate was weighed and dissolved in 100mL of water, after the dissolution, 1g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was added, the pH was adjusted to 5.5, the carboxyl group on the hyaluronic acid was activated, after 15min, 1g N-hydroxysuccinimide (NHS) was added, stirring was continued for 15min, 1g of PLL was weighed and added to the above solution, the pH was adjusted to 8.0, the reaction was carried out overnight, the solution was filled into a dialysis bag (molecular weight cut-off: 12-14 kDa), dialyzed in sodium hydroxide solution with pH=10 for 1 day, and then dialyzed in pure water for 3 days, and freeze-dried, to obtain polylysine grafted hyaluronic acid (SA-PLL).
Preparation of the Oxidized Sodium Alginate (OSA)
5g of sodium alginate is weighed and dissolved in 200mL of deionized water, and 50mL of ethanol is added for dispersion and dissolution, and the mixture is stirred uniformly. 5g of sodium periodate is weighed out in a dark place, dissolved in 10mL of deionized water to prepare a solution with the concentration of 0.5g/mL, slowly dripped into the solution under the dark condition, and reacted for 24 hours in the dark.
After the reaction, a small amount of ethylene glycol is added dropwise to terminate the reaction, 5g of NaCl is weighed and added into the solution, then the solution is poured into absolute ethyl alcohol, white solid is separated out, suction filtration is carried out, and the precipitate is washed three times with water. Dissolving the precipitate with a small amount of deionized water, dialyzing with a dialysis bag for three days, and lyophilizing to obtain OSA.
Example 1
As an example of the gel having a function of promoting bone repair according to the present invention, the present example includes the following components in the following concentrations: 0.05g/mL SA-PLL, 0.02g/mL OSA.
The hydrogel of this example comprises the following steps:
1. preparing SA-PLL aqueous solution with the concentration of 0.1g/mL, and recording as solution A;
2. preparing an aqueous OSA solution with the concentration of 0.04g/mL, and marking the aqueous OSA solution as a solution B;
3. uniformly mixing the solution A and the solution B according to the volume ratio of 1:1 to obtain initial gel, and freeze-drying to obtain a freeze-dried bracket;
4. soaking the freeze-dried bracket in pure water for 24 hours to obtain saturated gel.
Example 2
As an example of the gel having a function of promoting bone repair according to the present invention, the present example includes the following components in the following concentrations: 0.05g/mL SA-PLL, 0.02g/mL OSA, ca 2+ 。
The hydrogel of this example comprises the following steps:
1. preparing SA-PLL aqueous solution with the concentration of 0.1g/mL, and recording as solution A;
2. preparing an aqueous OSA solution with the concentration of 0.04g/mL, and marking the aqueous OSA solution as a solution B;
3. uniformly mixing the solution A and the solution B according to the volume ratio of 1:1 to obtain initial gel, and freeze-drying to obtain a freeze-dried bracket;
4. freeze-dried scaffolds were incubated at a concentration of 0.1M CaCl 2 Soaking in the solution for 24h, and repeatedly cleaning with pure water for 5 times to obtain Ca 2 + A crosslinked saturated gel.
Example 3
As an example of the gel having a function of promoting bone repair according to the present invention, the present example includes the following components in the following concentrations: 0.05g/mLSA-PLL, 0.01g/mLOSA, 50. Mu.g/mLBP, ca 2+ 。
The hydrogel of this example comprises the following steps:
1. preparing SA-PLL aqueous solution with the concentration of 0.1g/mL, and recording as solution A;
2. preparing an aqueous OSA solution with the concentration of 0.02g/mL, dispersing BP in the OSA solution, and preparing a solution B with the concentration of 100 mu g/mL;
3. uniformly mixing the solution A and the solution B according to the volume ratio of 1:1 to obtain initial gel, and freeze-drying to obtain a freeze-dried bracket;
4. freeze-dried scaffolds were incubated at a concentration of 0.1M CaCl 2 Soaking in the solution for 24h, and repeatedly cleaning with pure water for 5 times to obtain Ca 2 + A crosslinked saturated gel.
Example 4
As an embodiment of the gel with the function of promoting bone repair according to the invention, the embodiment comprisesThe following components in concentration: SA-PLL 0.05g/mL, OSA 0.015g/mL, BP 50. Mu.g/mL, ca 2+ 。
The hydrogel of this example comprises the following steps:
1. preparing SA-PLL aqueous solution with the concentration of 0.1g/mL, and recording as solution A;
2. preparing an aqueous OSA solution with the concentration of 0.03g/mL, dispersing BP in the OSA solution, and preparing a solution B with the concentration of 100 mug/mL;
3. uniformly mixing the solution A and the solution B according to the volume ratio of 1:1 to obtain initial gel, and freeze-drying to obtain a freeze-dried bracket;
4. freeze-dried scaffolds were incubated at a concentration of 0.1M CaCl 2 Soaking in the solution for 24h, and repeatedly cleaning with pure water for 5 times to obtain Ca 2 + A crosslinked saturated gel.
Example 5
As an example of the gel having a function of promoting bone repair according to the present invention, the present example includes the following components in the following concentrations: SA-PLL 0.05g/mL, OSA 0.02g/mL, BP 50 μg/mL, ca 2+ 。
The hydrogel of this example comprises the following steps:
1. preparing SA-PLL aqueous solution with the concentration of 0.1g/mL, and recording as solution A;
2. preparing an aqueous OSA solution with the concentration of 0.04g/mL, dispersing BP in the OSA solution, and preparing a solution B with the concentration of 100 mug/mL;
3. uniformly mixing the solution A and the solution B according to the volume ratio of 1:1 to obtain initial gel, and freeze-drying to obtain a freeze-dried bracket;
4. freeze-dried scaffolds were incubated at a concentration of 0.1M CaCl 2 Soaking in the solution for 24h, and repeatedly cleaning with pure water for 5 times to obtain Ca 2 + A crosslinked saturated gel.
Example 6
As an example of the gel having a function of promoting bone repair according to the present invention, the present example includes the following components in the following concentrations: SA-PLL 0.05g/mL, OSA 0.025g/mL, BP 50 μg/mL, ca 2+ 。
The hydrogel of this example comprises the following steps:
1. preparing SA-PLL aqueous solution with the concentration of 0.1g/mL, and recording as solution A;
2. preparing an aqueous OSA solution with the concentration of 0.05g/mL, dispersing BP in the OSA solution, and preparing a solution B with the concentration of 100 mug/mL;
3. uniformly mixing the solution A and the solution B according to the volume ratio of 1:1 to obtain initial gel, and freeze-drying to obtain a freeze-dried bracket;
4. freeze-dried scaffolds were incubated at a concentration of 0.1M CaCl 2 Soaking in the solution for 24h, and repeatedly cleaning with pure water for 5 times to obtain Ca 2 + A crosslinked saturated gel.
Example 7
As an example of the gel having a function of promoting bone repair according to the present invention, the present example includes the following components in the following concentrations: SA-PLL 0.05g/mL, OSA 0.03g/mL, BP 50 μg/mL, ca 2+ 。
The hydrogel of this example comprises the following steps:
1. preparing SA-PLL aqueous solution with the concentration of 0.1g/mL, and recording as solution A;
2. preparing an aqueous OSA solution with the concentration of 0.06g/mL, dispersing BP in the OSA solution, and preparing a solution B with the concentration of 100 mu g/mL;
uniformly mixing the solution A and the solution B according to the volume ratio of 1:1 to obtain initial gel, and freeze-drying to obtain a freeze-dried bracket;
4. freeze-dried scaffolds were incubated at a concentration of 0.1M CaCl 2 Soaking in the solution for 24h, and repeatedly cleaning with pure water for 5 times to obtain Ca 2 + A crosslinked saturated gel.
Effect test example
1. Swelling Performance test
Taking prepared regular cylindrical hydrogel, and measuring initial diameter d 0 And an initial height h 0 Freeze-drying, soaking in the solution until water absorption is saturated, and measuring diameter d after swelling saturation 1 And height h 1 According to the following formulaThe swelling volume Δv and the swelling degree SR of the gel were calculated:
ΔV＝3.14×(d 1 /2) 2 ×h 1 -3.14×(d 0 /2) 2 ×h 0 ；
SR＝(ΔV/3.14×(d 0 /2) 2 ×h 0 )×100％；
the swelling test results of the gels prepared in the examples are shown in table 1 below.
TABLE 1 swelling volume and swelling ratio test results for each example
As can be seen from Table 1, the gel of example 1 did not use Ca 2+ Crosslinking to form a second re-crosslinked network, the swelling degree being much greater than in the other examples, too high a swelling degree being detrimental to the mechanical strength of the gel, the swelling degree of the gel gradually decreasing from example 3 to example 7, on the one hand because the concentration of OSA increases, the degree of crosslinking with SA-PLL increases, and on the other hand, the concentration of OSA increases, the carboxyl content of the gel correspondingly increases, thereby reacting with Ca 2+ As well as the degree of crosslinking of the polymer, as a result of the combination of schiff base crosslinking and ionic crosslinking.
Meanwhile, from the results of example 2 and example 5, the gel swelling degree was reduced after BP was added, which may be Ca 2+ Interaction with partial BP degradation products occurs to generate calcium phosphate in situ, thereby strengthening the crosslinked network of the gel and reducing the water absorption performance of the gel.
2. Compression performance test
Taking a prepared cylindrical saturated hydrogel sample, measuring the initial height h and the bottom radius r of the cylindrical saturated hydrogel sample, placing the cylindrical saturated hydrogel sample under a gel strength special probe, extruding the hydrogel by the gel probe until the hydrogel is broken, recording the force F and the height change deltah of the hydrogel in the compression process, and calculating the compression modulus P (KPa) of the hydrogel in the compression process according to the following formula:
P＝F×h/(3.14×r 2 ×Δh×1000)；
the compressive modulus and Strain of the hydrogel at the break critical point were recorded, strain = Δh/h x 100%.
The compression performance test results of each example are shown in table 2 below.
Table 2 compression performance test results for various embodiments
As can be seen from Table 2, the gel of example 1 did not form a second heavy ion crosslinked network, which was broken when strain was about 13.23%, whereas other examples were carried out with Ca added 2+ The mechanical strength of the gel is significantly enhanced by the creation of the second secondary cross-links, which suggests that the mechanical strength of the gel is primarily dependent on the ionomer network, as the concentration of OSA increases.
3. Degradation Performance test
The mass of the saturated gel after freeze-drying of an equal volume is designated as W 0 At the same time, the initial hydrogel was immersed in 1000U/mL lysozyme in PBS, placed in a constant temperature shaker (37 ℃,70 rpm), and the time for complete degradation of the hydrogel was recorded.
The degradation performance test results of each example are shown in table 3 below.
Table 3 results of in vitro degradation test of various examples
As can be seen from Table 3, example 1 of the Schiff base crosslinked network was completely degraded in about 12.67 days, and after gel degradation, the bone defect site was lack of filler, and cells were not able to grow in an adhering manner, which was disadvantageous for bone repair. WhileThe degradation time of each embodiment of the second heavy ionic crosslinked network is obviously prolonged, and the degradation time is increased along with the increase of the crosslinking degree of the ionic crosslinked network; from the results of example 2 and example 5, the increase in BP also delays the degradation time of the gel, because the degradation products and Ca are reduced as BP degrades 2+ The reaction generates calcium phosphate, and mineralization is generated in situ, so that the degradation time of gel is prolonged. The concentration of OSA is regulated to regulate the ion crosslinking network, so that the approximate degradation time is regulated to be consistent with the period of bone repair, which is an ideal treatment scheme for bone repair.
4. Antibacterial property test
Gram-positive staphylococcus aureus and gram-negative escherichia coli were used to evaluate the antimicrobial properties of hydrogels. The OD value of the bacteria in the antibacterial test was adjusted to 0.1. Co-culturing the hydrogel sample and bacterial suspension in biochemical incubator at 37deg.C, irradiating hydrogel with 808nm near infrared light for 5min, setting near infrared light power to 1.5W/cm 2 After 12 hours, OD value measurement is carried out on the mixed bacterial liquid. 100. Mu.L of the bacterial suspension was diluted and inoculated onto LB agar plates. After incubation at 37℃for 24 hours, the number of culturable colonies was counted using the following formula:
AR(％)＝(Nc-Ns)/Nc×100；
the Antibacterial Ratio (AR) was calculated, where Nc is the average colony count of the control sample and Ns is the average bacterial colony count of the hydrogel sample.
The results of the in vitro antimicrobial test of each example are shown in table 4 below.
Table 4 results of in vitro antimicrobial testing of various examples
As can be seen from table 4, the gel with BP added shows good antibacterial performance after near infrared light irradiation, while the gel group without BP added also shows a certain antibacterial performance mainly because PLL exists in the gel component, and although free amino group in PLL participates in schiff base reaction, the structure also contains secondary amine and imine structure, and also has a certain inhibition effect on bacteria.
5. Biocompatibility testing
After the cultured mesenchymal stem cells of rabbit were suspended by digestion with 0.25% pancreatin, the density per well was 2×10 4 The cell suspension was seeded on a 48-well plate at a volume of one/mL. After 12h of incubation, the stock broth was removed, the hydrogel samples were transferred to a 48-well plate and 10-fold was inoculated on the hydrogel 5 100 μl of each cell suspension was prepared, each group was provided with at least 5 wells, the liquid was changed once every 24 hours, and three time points of 1d, 4d and 7d were set for the experiment, and the group without hydrogel was used as a blank. The specific operation method is as follows:
cell viability:
the viability of the cells was quantified using CCK 8. The corresponding well plates were removed at designated time intervals, 100. Mu.L of CCK8 working fluid was added to each well, and the temperature was maintained in a carbon dioxide incubator (containing 5% CO at 37 DEG C 2 ) After 1-2 h incubation, the absorbance (OD) was measured at 450nm wavelength with a microplate reader, and the cell viability was calculated according to the formula:
cell viability (%) =od Experimental group /OD Control group ×100％；
Fig. 2 shows the results of the biocompatibility test of the hydrogels of the examples, and it can be seen from fig. 2 that the cell viability in examples 3 and 4 is relatively low, showing a certain cytotoxicity, which is probably that free amino groups are also present in the gel, and a certain toxicity is generated for the cells, and as the concentration of OSA increases, the free amino groups are bonded, and the cell viability is also relatively increased.
Meanwhile, as can be seen from the figure, example 2 was added with Ca as compared with example 1 2+ After that, a significantly high cell viability was exhibited on day 7, indicating Ca 2+ The presence of (3) is beneficial for proliferation of bone marrow mesenchymal stem cells, and a suitable proportion of gel component is feasible for bone repair.
The present invention is not limited to the preferred embodiments, but can be modified, equivalent, and modified in any way without departing from the technical scope of the present invention.
Claims (9)
1. An unsupported bone repair gel microsphere, characterized in that: the gel microsphere is prepared from a polylysine grafted sodium alginate SA-PLL solution and an oxidized sodium alginate OSA solution containing black phosphorus nanoplatelets BP, and is prepared by CaCl 2 Soaking in the solution to obtain double-crosslinked gel microspheres;
the unsupported bone repair gel microsphere is prepared by the following process steps:
preparing SA-PLL and OSA respectively, preparing SA-PLL and OSA into aqueous solution with certain concentration, dispersing BP in the OSA solution, preparing microspheres with relatively uniform particle size by using SA-PLL solution and OSA solution containing BP as water phase and olive oil as oil phase and microfluidic chip, freeze-drying the microspheres, and adding the microspheres into CaCl 2 Soaking in the solution, and washing off excessive Ca with pure water 2+ The double cross-linked gel microsphere is obtained.
2. The unsupported bone repair gel microsphere of claim 1, wherein the preparation method specifically comprises the following steps:
1) Preparation of SA-PLL:
dissolving sodium alginate in water, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, regulating pH, standing, adding N-hydroxysuccinimide, continuously stirring, weighing polylysine, adding into the solution, regulating pH, reacting, dialyzing the solution, and lyophilizing to obtain SA-PLL;
2) Preparation of OSA:
weighing sodium alginate, dissolving the sodium alginate in deionized water, adding ethanol for dispersion and dissolution, and uniformly stirring; weighing sodium periodate in a dark place, dissolving the sodium periodate in deionized water to prepare sodium periodate solution, slowly dripping the sodium periodate into the solution under the dark condition, and reacting for 24 hours in the dark;
after the reaction is finished, a small amount of glycol is dripped to terminate the reaction, naCl is weighed and added into the solution, then the solution is poured into absolute ethyl alcohol, white solid is separated out, suction filtration is carried out, and the precipitate is washed with water; dissolving the precipitate with a small amount of deionized water, dialyzing, and lyophilizing to obtain OSA;
3) Preparation of gel microspheres:
preparing SA-PLL aqueous solution, which is marked as solution A;
preparing an OSA aqueous solution, dispersing BP in the OSA solution, and marking the solution as a solution B;
uniformly mixing the solution A and the solution B to obtain initial gel, and freeze-drying to obtain a freeze-dried bracket;
freeze-drying is supported on CaCl 2 Soaking in the solution for 24h, and cleaning with pure water to obtain Ca 2+ A crosslinked saturated gel.
3. The unsupported bone repair gel microsphere of claim 2 wherein: in the step 1), the sodium alginate is dissolved in water, namely, each 1g of sodium alginate is dissolved in 50mL of water;
the mass ratio of the sodium alginate to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 2:0.8 to 1.2;
mass ratio of the sodium alginate to the N-hydroxysuccinimide: 2:0.8 to 1.2;
the mass ratio of the sodium alginate to the polylysine: 2:0.8 to 1.5.
4. The unsupported bone repair gel microsphere of claim 3 wherein: in the step 1), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is added, the pH is regulated to be 5.0-6.0, and the standing time is 15-30 min;
the N-hydroxysuccinimide is added and stirred for 15 to 30 minutes;
the polylysine is weighed and added, the pH is regulated to 7.4-10.0, and the reaction time is 12-24 hours;
the dialysis was performed in a sodium hydroxide solution having ph=10 for 1 day, followed by dialysis in pure water for 3 more days.
5. The unsupported bone repair gel microsphere of claim 3 wherein: in the step 2), the sodium alginate is dissolved in deionized water, wherein every 1g of sodium alginate is dissolved in 40mL of water;
the volume ratio of the ethanol to the deionized water is 1:4, a step of;
the concentration of the sodium periodate solution is 0.5g/mL;
the mass ratio of the sodium alginate to the NaCl is 1:1.
6. the unsupported bone repair gel microsphere of claim 3 wherein: in the step 2), the cleaning is three times; the dialysis was three days of dialysis.
7. The unsupported bone repair gel microsphere of claim 3 wherein: in the step 3), the preparation concentration of the SA-PLL aqueous solution is 0.1g/mL;
the concentration of the OSA aqueous solution is 0.02-0.06 g/mL;
the solution A and the solution B are mixed according to the volume ratio of 1:1;
the washing with pure water is repeated for 5 times.
8. The unsupported bone repair gel microsphere of claim 3 wherein: in step 3), the concentration of the OSA aqueous solution is 0.04g/mL.
9. The unsupported bone repair gel microsphere of claim 3 wherein: in the step 3), the preparation concentration of BP in the solution B is 100 mug/mL; the CaCl 2 The concentration of the solution was 0.1M.
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