CN115634314A - Non-supported bone repair gel microsphere and preparation method thereof - Google Patents
Non-supported bone repair gel microsphere and preparation method thereof Download PDFInfo
- Publication number
- CN115634314A CN115634314A CN202211331440.6A CN202211331440A CN115634314A CN 115634314 A CN115634314 A CN 115634314A CN 202211331440 A CN202211331440 A CN 202211331440A CN 115634314 A CN115634314 A CN 115634314A
- Authority
- CN
- China
- Prior art keywords
- solution
- osa
- gel
- pll
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an unsupported bone repair gel microsphere and a preparation method thereof. The preparation method comprises the following steps: preparation of SA-PLL, O separatelySA, preparing water solution of SA-PLL and OSA in certain concentration, dispersing BP in the OSA solution, preparing microsphere with relatively homogeneous particle size with SA-PLL solution and OSA solution containing BP as water phase and olive oil as oil phase in a micro flow control chip, freeze drying the microsphere, and adding the freeze dried microsphere into CaCl 2 Soaking in the solution for 24 hr, and washing off excessive Ca with pure water 2+ And obtaining the double-crosslinked gel microspheres. The raw materials are simple and easy to obtain, and the price is low; the gel microspheres prepared by double crosslinking of Schiff base and ionic crosslinking have good mechanical properties, and BP can play a role in local heating sterilization after near-infrared light irradiation; along with the degradation of BP and hydrogel, phosphate radical 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 can be promoted.
Description
Technical Field
The invention relates to a bone repair material, in particular to unsupported bone repair gel microspheres and a preparation method thereof.
Background
Bone defects caused by various reasons such as trauma, degenerative changes, congenital malformations, bone tumors, dental implants, periodontal diseases, craniotomy and the like are quite common in clinic, and patients in the fields of orthopedics, stomatology, neurosurgery and the like are concentrated. At present, most of orthopedics clinics still select a mode of removing the east wall and supplementing the west wall to perform bone grafting treatment by taking autologous bones of a patient (common parts comprise ilium, ribs, fibula and the like) when treating bone defects; however, the bone taken from the body has a series of problems of causing secondary damage to patients, complications in donor areas, prolonging operation time, increasing bleeding amount, limiting bone mass and the like, and is not a perfect clinical solution. Therefore, the main development trend of clinical bone grafting in the world is to reduce the taking of autologous bone in the operation and select more artificial bone repair materials.
According to different components of materials, the synthetic bone repair materials can be mainly divided into metal materials, biological ceramics, calcium phosphate/calcium sulfate bone cement, biological glass, high polymer materials, composite materials and tissue engineering materials. Different materials have respective physicochemical properties and biological performance characteristics, are widely applied to bone grafting operations in the fields of orthopedics, 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.
In summary, the prior art has the following disadvantages:
1) The autologous bone transplantation cost is high, and secondary damage can be caused;
2) Inorganic non-metallic materials such as biological ceramics, calcium phosphate/calcium sulfate bone cement, biological glass and the like have weaker mechanical properties;
3) Metallic implants are 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 better fill bone defect parts and play roles in resisting bacteria and promoting bone repair.
The purpose of the invention and the technical problem to be solved are realized by adopting the following technical scheme.
The invention provides non-support bone repair gel microspheres, which are prepared from SA-PLL solution and OSA solution containing BP, and are subjected to CaCl 2 Soaking in the solution to obtain the double-crosslinked gel microspheres.
The invention provides a preparation method of unsupported bone repair gel microspheres, which comprises the following steps:
respectively preparing SA-PLL and OSA, preparing aqueous solution of SA-PLL and OSA with certain concentration, dispersing BP in OSA solution, taking SA-PLL solution and OSA solution containing BP as water phase and olive oil as oil phase, preparing microspheres with relatively uniform particle size by microfluidic chip, freeze-drying, and adding into CaCl 2 Soaking in the solution, and washing off excessive Ca with pure water 2+ And obtaining the double-crosslinked gel microspheres.
Further, the preparation method of the unsupported bone repair gel microsphere comprises the following steps:
1) Preparation of SA-PLL:
weighing sodium alginate, dissolving in water, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, adjusting pH, standing, adding N-hydroxysuccinimide, continuously stirring, weighing polylysine, adding into the solution, adjusting 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 dispersing and dissolving, and stirring uniformly; weighing sodium periodate in a dark place, dissolving the sodium periodate in deionized water to prepare a sodium periodate solution, slowly dripping the sodium periodate solution into the solution in the dark place, and reacting for 24 hours in the dark place;
after the reaction is finished, dropwise adding a small amount of glycol to terminate the reaction, weighing NaCl, adding the NaCl into the solution, then pouring the solution into absolute ethyl alcohol, separating out white solid, performing suction filtration, and washing the precipitate with water; dissolving the precipitate with a small amount of deionized water, dialyzing, and lyophilizing to obtain OSA;
3) Preparation of gel microspheres:
preparing an SA-PLL aqueous solution, and marking as a solution A; preparing an OSA aqueous solution, and dispersing BP in the OSA aqueous solution to be marked 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 the scaffolds on CaCl 2 Soaking in the solution for 24h, and washing with pure water to obtain Ca 2+ A cross-linked saturated gel.
Further, in the above preparation method of the non-supported bone repair gel microsphere, in the step 1), the sodium alginate is dissolved in water in an amount of 50mL per 1g of sodium alginate; the mass ratio of the sodium alginate to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 2:0.8 to 1.2; the mass ratio of the sodium alginate to the N-hydroxysuccinimide is as follows: 2:0.8 to 1.2; the mass ratio of the sodium alginate to the polylysine is as follows: 2:0.8 to 1.5.
In a further aspect, in the above preparation method of the gel microsphere for repairing an unsupported bone, in step 1), the mass ratio of the sodium alginate to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 2:1; the mass ratio of the sodium alginate to the N-hydroxysuccinimide is as follows: 2:1; the mass ratio of the sodium alginate to the polylysine is as follows: 2:1.
further, in the preparation method of the non-supported bone repair gel microsphere, in the step 1), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is added, the pH is adjusted to be 5.0-6.0, and the standing time is 15-30 min; adding N-hydroxysuccinimide, and stirring for 15-30 Min; adding polylysine, adjusting the pH to 7.4-10.0, and reacting for 12-24 h; the dialysis was performed for 1 day in sodium hydroxide solution with pH =10, and then for 3 days in pure water.
Further, in the above preparation method of the non-supported bone repair gel microsphere, 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; 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-supported bone repair gel microsphere comprises the following steps: in the step 2), the cleaning is three times; the dialysis was dialysis for three days.
Further, in the above preparation method of the unsupported bone repair gel microsphere, in the step 3), the preparation concentration of the SA-PLL aqueous solution is 0.1g/mL; the preparation concentration of the OSA aqueous solution is 0.02-0.06 g/mL; the solution A and the solution B are mixed according to a volume ratio of 1; the washing with pure water was repeated 5 times.
Further, in the above method for preparing the unsupported bone repair gel microsphere, in the step 3), the preparation concentration of the OSA aqueous solution is 0.04g/mL.
Further, in the above preparation method of the unsupported bone repair gel microsphere, in the step 3), the preparation concentration of BP in the solution B is 100 μ g/mL; the CaCl is 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 raw materials are simple and easy to obtain, and the price is low; the gel microspheres prepared by double crosslinking of Schiff base and ionic crosslinking have good mechanical properties, and BP can play a role in local heating sterilization after near-infrared light irradiation; along with the degradation of BP and hydrogel, phosphate radical which is the degradation product of BP can react with Ca 2+ Forming calcium phosphate to realize in-situ oreThe gel can delay the degradation of the gel and promote the repair of bones.
2) The gel has good mechanical property, biocompatibility and antibacterial activity, can be prepared into gel microspheres and filled in bone defect parts in an injection mode, and gel components can be degraded into components which can be absorbed by human bodies, namely Ca 2+ The existence of the cross-linked 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+ It also can promote proliferation of osteocyte. Meanwhile, the raw materials in the invention are simple and easy to obtain, the cost is lower, and the preparation process is relatively simple.
Drawings
FIG. 1 is a schematic view of a microfluidic chip;
FIG. 2 shows the results of the biocompatibility test of the hydrogels of the examples.
Detailed Description
The technical content scheme is as follows:
the gel microsphere takes Sodium Alginate (SA) as a main material, and utilizes carboxyl on a polysaccharide structure of the gel microsphere to graft with Polylysine (PLL) rich in an amino structure through amidation reaction to obtain amination modified SA-PLL; the SA is rich in hydroxyl and can be oxidized by sodium periodate to generate functional aldehyde group, so that Oxidized Sodium Alginate (OSA) is obtained. Both SA-PLL and OSA can generate imine structure through Schiff base reaction between amino and aldehyde groups, thereby generating 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 nanosheet (BP) with good photo-thermal property into a 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 properties of the SA-PLL/OSA/BP gel, the gel prepared by the method is soaked in CaCl 2 Soaking in the solution to make carboxyl and Ca in sodium alginate 2+ Ionic crosslinks are generated, forming a second, crosslinked network. Simultaneously, along with the gradual degradation of BP, the generated phosphate radical can be mixed with Ca 2+ Calcium phosphate is generated through reaction, and mineralization is carried out in situ in the gel, so that the bone repair is promoted.
The specific embodiment of the gel microsphere is as follows:
respectively preparing SA-PLL and OSA, preparing aqueous solution of SA-PLL and OSA with certain concentration, dispersing BP in OSA solution, taking SA-PLL solution and OSA solution containing BP as water phase and olive oil as oil phase, preparing microsphere with relatively uniform particle size by microfluidic chip shown in figure 1, lyophilizing microsphere, and adding into CaCl 2 Soaking in the solution for 24 hr, and washing off excessive Ca with pure water 2+ And obtaining the double-crosslinked gel microspheres.
Preparation of the above polylysine grafted sodium alginate (SA-PLL)
Weighing 2g of sodium alginate, dissolving in 100mL of water, adding 1g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) after the sodium alginate is dissolved, adjusting the pH to 5.5, activating carboxyl groups on hyaluronic acid, adding 1g of N-hydroxysuccinimide (NHS) after 15min, continuing stirring for 15min, weighing 1g of PLL, adding the PLL into the solution, adjusting the pH to 8.0, reacting overnight, filling the solution into a dialysis bag (molecular weight cut-off: 12-14 kDa), dialyzing in a sodium hydroxide solution with the pH =10 for 1 day, dialyzing in pure water for 3 days, and freeze-drying to obtain the polylysine grafted hyaluronic acid (SA-PLL).
Preparation of the above Oxidized Sodium Alginate (OSA)
Weighing 5g of sodium alginate, dissolving the sodium alginate in 200mL of deionized water, adding 50mL of ethanol for dispersing and dissolving, and uniformly stirring. 5g of sodium periodate is weighed away from light and dissolved in 10mL of deionized water to prepare a solution of 0.5g/mL, and the solution is slowly dripped into the solution away from light and reacted for 24 hours away from light.
After the reaction is finished, a small amount of glycol is dripped 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, the filtration is carried out, and the precipitate is washed with water for three times. Dissolving the precipitate with a small amount of deionized water, dialyzing the deionized water with a dialysis bag, dialyzing for three days, and freeze-drying to obtain the 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 concentrations: 0.05g/mL SA-PLL, 0.02g/mL OSA.
The hydrogel described in this example includes the following steps:
1. preparing an SA-PLL aqueous solution with the concentration of 0.1g/mL, and marking as a solution A;
2. preparing an OSA aqueous solution with the concentration of 0.04g/mL, and marking as a solution B;
3. uniformly mixing the solution A and the solution B according to a volume ratio of 1;
4. the freeze-dried scaffold was soaked in pure water for 24h to obtain a 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 concentrations: SA-PLL of 0.05g/mL, OSA of 0.02g/mL, ca 2+ 。
The hydrogel of the embodiment comprises the following steps:
1. preparing a SA-PLL aqueous solution with the concentration of 0.1g/mL, and marking as a solution A;
2. preparing an OSA aqueous solution with the concentration of 0.04g/mL, and marking as a solution B;
3. uniformly mixing the solution A and the solution B according to a volume ratio of 1;
4. freeze-drying the scaffolds in CaCl at a concentration of 0.1M 2 Soaking in the solution for 24 hr, and repeatedly washing with pure water for 5 times to obtain Ca 2 + A cross-linked saturated gel.
Example 3
As an example of the gel having a bone repair promoting function according to the present invention, the present example includes the following components in concentrations: 0.05g/mLSA-PLL, 0.01g/mLOSA, 50. Mu.g/mLBP, ca 2+ 。
The hydrogel described in this example includes the following steps:
1. preparing a SA-PLL aqueous solution with the concentration of 0.1g/mL, and marking as a solution A;
2. preparing an OSA aqueous solution with the concentration of 0.02g/mL, dispersing BP in the OSA aqueous solution to prepare a solution B with the concentration of 100 mug/mL;
3. uniformly mixing the solution A and the solution B according to a volume ratio of 1;
4. freeze-drying the scaffolds in CaCl at a concentration of 0.1M 2 Soaking in the solution for 24 hr, and repeatedly washing with pure water for 5 times to obtain Ca 2 + A cross-linked saturated gel.
Example 4
As an example of the gel having a bone repair promoting function according to the present invention, the present example includes the following components in concentrations: SA-PLL of 0.05g/mL, OSA of 0.015g/mL, BP of 50. Mu.g/mL, ca 2+ 。
The hydrogel described in this example includes the following steps:
1. preparing a SA-PLL aqueous solution with the concentration of 0.1g/mL, and marking as a solution A;
2. preparing an OSA aqueous solution with the concentration of 0.03g/mL, dispersing BP in the OSA aqueous solution to prepare a solution B with the concentration of 100 mug/mL;
3. uniformly mixing the solution A and the solution B according to a volume ratio of 1;
4. freeze-dried scaffolds were incubated with 0.1M CaCl 2 Soaking in the solution for 24 hr, and repeatedly washing with pure water for 5 times to obtain Ca 2 + A cross-linked saturated gel.
Example 5
As an example of the gel having a bone repair promoting function according to the present invention, the present example includes the following components in concentrations: SA-PLL of 0.05g/mL, OSA of 0.02g/mL, BP of 50. Mu.g/mL, ca 2+ 。
The hydrogel of the embodiment comprises the following steps:
1. preparing an SA-PLL aqueous solution with the concentration of 0.1g/mL, and marking as a solution A;
2. preparing an OSA aqueous solution with the concentration of 0.04g/mL, dispersing BP in the OSA aqueous solution to prepare a solution B with the concentration of 100 mu g/mL;
3. uniformly mixing the solution A and the solution B according to a volume ratio of 1;
4. freeze-dried scaffolds were incubated with 0.1M CaCl 2 Soaking in the solution for 24 hr, and repeatedly washing with pure water for 5 times to obtain Ca 2 + A cross-linked saturated gel.
Example 6
As an example of the gel having a bone repair promoting function according to the present invention, the present example includes the following components in concentrations: SA-PLL of 0.05g/mL, OSA of 0.025g/mL, BP of 50. Mu.g/mL, ca 2+ 。
The hydrogel described in this example includes the following steps:
1. preparing a SA-PLL aqueous solution with the concentration of 0.1g/mL, and marking as a solution A;
2. preparing an OSA aqueous solution with the concentration of 0.05g/mL, dispersing BP in the OSA aqueous solution to prepare a solution B with the concentration of 100 mug/mL;
3. uniformly mixing the solution A and the solution B according to a volume ratio of 1;
4. freeze-drying the scaffolds in CaCl at a concentration of 0.1M 2 Soaking in the solution for 24 hr, and repeatedly washing with pure water for 5 times to obtain Ca 2 + A cross-linked saturated gel.
Example 7
As an example of the gel having a bone repair promoting function according to the present invention, the present example includes the following components in concentrations: SA-PLL of 0.05g/mL, OSA of 0.03g/mL, BP of 50. Mu.g/mL, ca 2+ 。
The hydrogel described in this example includes the following steps:
1. preparing an SA-PLL aqueous solution with the concentration of 0.1g/mL, and marking as a solution A;
2. preparing an OSA aqueous solution with the concentration of 0.06g/mL, dispersing BP in the OSA aqueous solution to prepare a solution B with the concentration of 100 mug/mL;
uniformly mixing the solution A and the solution B according to a volume ratio of 1;
4. freeze-drying the scaffolds in CaCl at a concentration of 0.1M 2 Soaking in the solution for 24 hr, and repeatedly washing with pure water for 5 times to obtain Ca 2 + A cross-linked saturated gel.
Examples of Effect test
1. Swelling Performance test
Taking the prepared regular cylindrical hydrogel, measuring its initial diameter d 0 And an initial height h 0 Freeze-drying, soaking in solution until water is saturated, and measuring the diameter d of the solution after swelling saturation 1 And height h 1 The swelling volume Δ V and swelling degree SR of the gel were calculated according to the following formulas:
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 results of the swelling tests of the gels prepared in each example 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 in example 1 did not use Ca 2+ The crosslinking forms a second, heavily crosslinked network, the degree of swelling of which is much greater than in the other examples, the degree of swelling being unfavourable for the mechanical strength of the gel, and the degree of swelling of the gel decreasing from example 3 to example 7, on the one hand, as the concentration of OSA increases and the degree of crosslinking with SA-PLL increases, and on the other hand, as the concentration of OSA increases and the content of carboxyl groups in the gel increases, and thus with Ca 2+ The degree of crosslinking is also increased as a result of the interaction of the schiff base crosslinking with the ionic crosslinking.
Meanwhile, from the results of examples 2 and 5, it is seen that the degree of gel swelling is reduced after BP is added, which may be Ca 2+ With part BPThe degradation products of the gel are interacted to generate calcium phosphate in situ, thereby strengthening the crosslinking 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 sample, placing the sample under a special gel strength probe, extruding the hydrogel by the gel probe until the hydrogel is broken, recording the force F and the height change delta h 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 of the hydrogel at the critical point of rupture was recorded, as well as the Strain, strain = Δ h/h × 100%.
The results of the compression performance test of each example are shown in table 2 below.
Table 2 compression performance test results of each example
As can be seen from Table 2, the gel of example 1 did not form a second heavy ion crosslinked network, which broke at a strain of about 13.23%, while the other examples added Ca 2+ The mechanical strength of the gel was significantly increased by the second crosslinking, indicating that the mechanical strength of the gel was primarily dependent on the ionic crosslinked network, and that the mechanical strength of the gel increased with increasing concentration of OSA.
3. Test for degradation Properties
The isovolumetric saturated gel was freeze-dried and weighed as W 0 Simultaneously, the initial hydrogel was soaked in a PBS solution containing 1000U/mL lysozyme, placed in a constant temperature shaker (37 ℃,70 rpm), and the time to complete degradation of the hydrogel was recorded.
The results of the degradation performance test for each example are shown in table 3 below.
TABLE 3 in vitro degradation test results for each example
As seen from Table 3, only example 1, which is a Schiff base crosslinked network, completely degraded in about 12.67 days, and after the gel was degraded, the bone defect part lacked filler and cells could not be attached to grow, which is not favorable for bone repair. The degradation time of each embodiment added with the second heavy ion crosslinking network is obviously prolonged, and the degradation time is increased along with the increase of the crosslinking degree of the ion crosslinking network; from the results of examples 2 and 5, the increase in BP also delayed the gel degradation time, since as BP was degraded, its degradation products were associated with Ca 2+ The reaction produces calcium phosphate, which produces mineralization in situ, thus prolonging the degradation time of the gel. The ionic crosslinked network is adjusted by adjusting the concentration of OSA, thereby adjusting the approximate degradation time of OSA so that it coincides with the period of bone repair, which is an ideal treatment for bone repair.
4. Test of antibacterial Property
The antibacterial properties of the hydrogels were evaluated with gram-positive staphylococcus aureus and gram-negative escherichia coli. The OD value of the bacteria in the antibacterial test was adjusted to 0.1. Co-culturing the hydrogel sample and bacterial suspension in a biochemical incubator at 37 deg.C, irradiating the hydrogel with 808nm near infrared light for 5min, and setting the power of the near infrared light at 1.5W/cm 2 And after 12h, measuring the OD value of the mixed bacteria liquid. 100 μ L of the bacterial suspension was diluted and plated on LB agar plates. The number of culturable colonies was counted after 24h incubation at 37 ℃ using the following formula:
AR(％)＝(Nc-Ns)/Nc×100；
the Antibacterial Ratio (AR) was calculated, where Nc is the average number of colonies of the control sample and Ns is the average number of bacterial colonies of the hydrogel sample.
The results of the in vitro antibacterial test for each example are shown in table 4 below.
TABLE 4 in vitro antibacterial test results for each example
As seen from table 4, the gel with BP added shows good antibacterial performance after irradiation with near infrared light, while the gel without BP added also shows a certain antibacterial performance, mainly because PLL exists in the gel component, although the free amino group in PLL participates in the schiff base reaction, the structure of PLL also contains secondary amine and imine structure, and has a certain inhibitory effect on bacteria.
5. Biocompatibility testing
The cultured mesenchymal stem cells of rabbit bone marrow were digested with 0.25% pancreatin and suspended at a density of 2X 10 per well 4 one/mL of the cell suspension was seeded in 48-well plates. Culturing for 12 hr, taking out stock culture solution, transferring hydrogel sample to 48-well plate, inoculating 10-containing gel on the hydrogel 5 100 mu L of each cell suspension per mL, at least 5 holes are arranged in each group, the liquid is changed every 24h, three time points of 1d, 4d and 7d are set in the experiment, and the group without hydrogel is used as a blank control. The specific operation method comprises the following steps:
cell survival rate:
cell viability was quantified using CCK 8. The corresponding well plate was removed at the indicated time intervals, 100. Mu.L of CCK8 working solution was added to each well, and carbon dioxide incubator (containing 5% CO) was maintained at a constant temperature of 37 ℃ 2 ) After incubation for 1-2 h, measuring the absorbance (OD) at the wavelength of 450nm by using an enzyme-labeling instrument, and calculating the cell survival rate according to the formula:
cell viability (%) = OD Experimental group /OD Control group ×100％；
FIG. 2 shows the results of biocompatibility tests of hydrogels of the respective examples, and it can be seen from FIG. 2 that the viability of cells in examples 3 and 4 is relatively low, and the cells show a certain cytotoxicity, which may be caused by the presence of free amino groups in the gel and a certain toxicity to the cells, and the viability of the cells is relatively increased as the concentration of OSA is increased and the free amino groups are bonded.
Meanwhile, it is seen from the figure that Ca is added in example 2 compared with example 1 2+ Thereafter, a significantly high cell survival rate was exhibited at day 7, indicating that Ca 2+ The existence of the gel is beneficial to the proliferation of the bone marrow mesenchymal stem cells, and the proper proportion of the gel component is feasible for bone repair.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, so that any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.
Claims (10)
1. An unsupported bone repair gel microsphere, which is characterized in that: the gel microspheres are prepared from SA-PLL solution and OSA solution containing BP, and are subjected to CaCl 2 Soaking in the solution to obtain the double-crosslinked gel microspheres.
2. The preparation method of the unsupported bone repair gel microsphere is characterized by comprising the following steps:
respectively preparing SA-PLL and OSA, preparing aqueous solution of SA-PLL and OSA with certain concentration, dispersing BP in OSA solution, taking SA-PLL solution and OSA solution containing BP as water phase and olive oil as oil phase, preparing microspheres with relatively uniform particle size by microfluidic chip, freeze-drying, and adding into CaCl 2 Soaking in the solution, and washing off excessive Ca with pure water 2+ And obtaining the double-crosslinked gel microspheres.
3. The method of claim 2, comprising the steps of:
1) Preparation of SA-PLL:
weighing sodium alginate, dissolving in water, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, adjusting pH, standing, adding N-hydroxysuccinimide, continuously stirring, weighing polylysine, adding into the solution, adjusting 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 dispersing and dissolving, and stirring uniformly; weighing sodium periodate in a dark place, dissolving the sodium periodate in deionized water to prepare a sodium periodate solution, slowly dripping the sodium periodate solution into the solution in the dark place, and reacting for 24 hours in the dark place;
after the reaction is finished, dropwise adding a small amount of glycol to terminate the reaction, weighing NaCl and adding the NaCl into the solution, then pouring the solution into absolute ethyl alcohol, separating out white solid, performing suction filtration, and washing the precipitate with water; dissolving the precipitate with a small amount of deionized water, dialyzing, and lyophilizing to obtain OSA;
3) Preparation of gel microspheres:
preparing an SA-PLL aqueous solution, and marking as a solution A;
preparing an OSA aqueous solution, and dispersing BP in the OSA aqueous solution to be marked 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 the scaffolds on CaCl 2 Soaking in the solution for 24 hr, and washing with pure water to obtain Ca 2+ A cross-linked saturated gel.
4. The production method according to claim 3, characterized in that: in the step 1), the sodium alginate is dissolved in water, wherein 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;
the mass ratio of the sodium alginate to the N-hydroxysuccinimide is as follows: 2:0.8 to 1.2;
the mass ratio of the sodium alginate to the polylysine is as follows: 2:0.8 to 1.5.
5. The production method according to claim 3, characterized in that: in the step 1), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is added, the pH is adjusted to be 5.0-6.0, and the standing time is 15-30 min;
adding N-hydroxysuccinimide, and stirring for 15-30 min;
adding polylysine, adjusting the pH to 7.4-10.0, and reacting for 12-24 h;
the dialysis was performed for 1 day in sodium hydroxide solution with pH =10, and then for 3 days in pure water.
6. The production method according to claim 3, characterized in that: 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;
the concentration of the sodium periodate solution is 0.5g/mL;
the mass ratio of the sodium alginate to the NaCl is 1:1.
7. the production method according to claim 3, characterized in that: in the step 2), the cleaning is three times;
the dialysis was dialysis for three days.
8. The production method according to claim 3, characterized in that: in the step 3), the preparation concentration of the SA-PLL aqueous solution is 0.1g/mL;
the preparation concentration of the OSA aqueous solution is 0.02-0.06 g/mL;
the solution A and the solution B are mixed according to a volume ratio of 1;
the washing with pure water was repeated 5 times.
9. The production method according to claim 3, characterized in that: in the step 3), the preparation concentration of the OSA aqueous solution is 0.04g/mL.
10. The production method according to claim 3, characterized in that: in the step 3), the preparation concentration of BP in the solution B is 100 mug/mL;
the CaCl is 2 The concentration of the solution was 0.1M.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211331440.6A CN115634314B (en) | 2022-10-28 | 2022-10-28 | Unsupported bone repair gel microsphere and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211331440.6A CN115634314B (en) | 2022-10-28 | 2022-10-28 | Unsupported bone repair gel microsphere and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115634314A true CN115634314A (en) | 2023-01-24 |
| CN115634314B CN115634314B (en) | 2023-08-04 |
Family
ID=84947053
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211331440.6A Active CN115634314B (en) | 2022-10-28 | 2022-10-28 | Unsupported bone repair gel microsphere and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115634314B (en) |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020114795A1 (en) * | 2000-12-22 | 2002-08-22 | Thorne Kevin J. | Composition and process for bone growth and repair |
| US20170313827A1 (en) * | 2014-11-27 | 2017-11-02 | Yu Sun | Preparation method, product and application of non-free radical photo-crosslinked hydrogel material |
| US20190008775A1 (en) * | 2016-03-29 | 2019-01-10 | Jiangnan University | Method for Preparing Modified Sodium Alginate Embolization Microsphere |
| CN109498838A (en) * | 2018-12-29 | 2019-03-22 | 广州贝奥吉因生物科技有限公司 | A kind of injectable cartilage repair hydrogel and preparation method thereof |
| CN110075348A (en) * | 2019-04-11 | 2019-08-02 | 温州医科大学 | It is used to prepare the sol system, hydrogel and application of pH sensitivity double-network hydrogel |
| CN110917391A (en) * | 2019-12-26 | 2020-03-27 | 广东泰宝医疗科技股份有限公司 | Polypeptide modified sodium alginate/PVA hydrogel dressing and preparation method thereof |
| CN110917395A (en) * | 2019-12-24 | 2020-03-27 | 福建师范大学 | Preparation method of two-dimensional nano black phosphorus functionalized modified polylactic acid nano fiber scaffold |
| CN111150880A (en) * | 2020-01-08 | 2020-05-15 | 广州贝奥吉因生物科技股份有限公司 | Antibacterial composite hydrogel and preparation method thereof |
| CN111265714A (en) * | 2020-03-11 | 2020-06-12 | 四川大学 | Black phosphorus functionalized injectable hydrogel and preparation method and application thereof |
| US20210309810A1 (en) * | 2020-04-03 | 2021-10-07 | Iucf-Hyu (Industry-University Cooperation Foundation Hanyang University) | Double-crosslinked self-healing hydrogel |
| CN114805854A (en) * | 2022-05-06 | 2022-07-29 | 南京工业大学 | Polylysine conductive hydrogel with ROS removing and multiple antibacterial functions |
| CN115028903A (en) * | 2022-07-07 | 2022-09-09 | 广州创赛生物医用材料有限公司 | Hydrogel and preparation method and application thereof |
-
2022
- 2022-10-28 CN CN202211331440.6A patent/CN115634314B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020114795A1 (en) * | 2000-12-22 | 2002-08-22 | Thorne Kevin J. | Composition and process for bone growth and repair |
| US20170313827A1 (en) * | 2014-11-27 | 2017-11-02 | Yu Sun | Preparation method, product and application of non-free radical photo-crosslinked hydrogel material |
| US20190008775A1 (en) * | 2016-03-29 | 2019-01-10 | Jiangnan University | Method for Preparing Modified Sodium Alginate Embolization Microsphere |
| CN109498838A (en) * | 2018-12-29 | 2019-03-22 | 广州贝奥吉因生物科技有限公司 | A kind of injectable cartilage repair hydrogel and preparation method thereof |
| CN110075348A (en) * | 2019-04-11 | 2019-08-02 | 温州医科大学 | It is used to prepare the sol system, hydrogel and application of pH sensitivity double-network hydrogel |
| CN110917395A (en) * | 2019-12-24 | 2020-03-27 | 福建师范大学 | Preparation method of two-dimensional nano black phosphorus functionalized modified polylactic acid nano fiber scaffold |
| CN110917391A (en) * | 2019-12-26 | 2020-03-27 | 广东泰宝医疗科技股份有限公司 | Polypeptide modified sodium alginate/PVA hydrogel dressing and preparation method thereof |
| CN111150880A (en) * | 2020-01-08 | 2020-05-15 | 广州贝奥吉因生物科技股份有限公司 | Antibacterial composite hydrogel and preparation method thereof |
| CN111265714A (en) * | 2020-03-11 | 2020-06-12 | 四川大学 | Black phosphorus functionalized injectable hydrogel and preparation method and application thereof |
| US20210309810A1 (en) * | 2020-04-03 | 2021-10-07 | Iucf-Hyu (Industry-University Cooperation Foundation Hanyang University) | Double-crosslinked self-healing hydrogel |
| CN114805854A (en) * | 2022-05-06 | 2022-07-29 | 南京工业大学 | Polylysine conductive hydrogel with ROS removing and multiple antibacterial functions |
| CN115028903A (en) * | 2022-07-07 | 2022-09-09 | 广州创赛生物医用材料有限公司 | Hydrogel and preparation method and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| GE L, ET AL: "Antibacterial dialdehyde sodium alginate/ε-polylysine microspheres for fruit preservation", FOOD CHEMISTRY, vol. 387, pages 7 * |
| HU X, ET AL: "Preparation and characterization of composite hydrogel beads based on sodium alginate", POLYMER BULLETIN, vol. 72, no. 11, pages 3 - 4 * |
| 朱享波;武欣;杨国强;汪建新;: "γ聚谷氨酸/海藻酸钠双网络水凝胶的制备及性能研究", 化学推进剂与高分子材料, no. 04 * |
| 李云洁;滕彬宏;赵艳红;杨强;王连永;黄颖;: "软骨组织工程用羧甲基壳聚糖/氧化海藻酸钠复合水凝胶的制备及体外评估", 华西口腔医学杂志, no. 03, pages 1 - 13 * |
| 王见伟;宋利锋;赵瑾;原续波;: "基于多肽结构的聚合物水凝胶", 化学进展, no. 04 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115634314B (en) | 2023-08-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Florczyk et al. | Evaluation of three‐dimensional porous chitosan–alginate scaffolds in rat calvarial defects for bone regeneration applications | |
| CN111097068B (en) | Bionic hydroxyapatite powder/gelatin/sodium alginate composite 3D printing support and preparation method thereof | |
| CN107349470B (en) | Preparation method of inorganic nanoparticle reinforced hydrogel and application of inorganic nanoparticle reinforced hydrogel in artificial periosteum | |
| CN101474424A (en) | High-artificial tissue engineering nerve repair material NGCS and preparation method thereof | |
| CN112023120B (en) | Injectable pre-filled bone repair particle and preparation method and application thereof | |
| CN110548171B (en) | Gelatin-based bone tissue adhesive, and preparation method and application thereof | |
| CN110152055A (en) | The functional drug that alginic acid amination derivative/bacteria cellulose nanocomposite gel is constructed is sustained medical dressing | |
| CN102671237B (en) | High-simulation tissue engineering nerve-repair material and preparation method | |
| CN109331223B (en) | Medicine-carrying bioactive glass composite calcium phosphate bone cement and application thereof | |
| Wang et al. | Injectable antibacterial Ag-HA/GelMA hydrogel for bone tissue engineering | |
| CN112138216B (en) | Hybrid cross-linked skull repairing porous membrane with highly bionic bone matrix and preparation method thereof | |
| CN105797211A (en) | Preparation method of hydrogel, osteoblast containing hydrogel and preparation method of osteoblast containing hydrogel | |
| CN115634314B (en) | Unsupported bone repair gel microsphere and preparation method thereof | |
| CN108478874B (en) | Preparation method of hydroxyethyl chitosan nano composite bone scaffold material | |
| WO2023065474A1 (en) | Calcium phosphate-based organic-inorganic composite bioactive material and preparation method therefor | |
| CN115475283B (en) | Tissue engineering bone constructed based on hydrogel material and preparation method and application thereof | |
| CN113061255B (en) | Polyoxyethylene polyoxypropylene-chitosan segmented copolymer, bone hemostatic material and preparation method thereof | |
| JP2014124416A (en) | Bone regeneration material kit, pasty bone regeneration material, bone regeneration material and bone junction material | |
| CN102671238B (en) | High-emulation tissue-engineered nerve repair material | |
| CN114432492B (en) | Tissue engineering scaffold suitable for cartilage and preparation method thereof | |
| KR20220019775A (en) | Means for use in the production of hydrogels based on hydroxyphenyl derivatives of hyaluronan, methods for producing hydrogels and uses thereof | |
| CN113559331A (en) | High-activity injectable material and preparation method and application thereof | |
| de Sousa et al. | Recent advances in methods of synthesis and applications of bacterial cellulose/calcium phosphates composites in bone tissue engineering: Biomaterials; Biocomposites; Tissue Engineering; Bacterial cellulose Calcium Phosphates | |
| CN114028615B (en) | Drug-loaded ceramic artificial bone with extremely-small curved surface structure and preparation method thereof | |
| CN117679555B (en) | Larch cellulose hydrogel, preparation method thereof and application of larch cellulose hydrogel in promoting bone repair |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |