Adipose-derived stem cell-loaded L-chiral self-assembled hydrogel, preparation method thereof and application of hydrogel in periodontal tissue regeneration promoting medicine

Abstract

The invention relates to an adipose-derived stem cell-loaded L-chiral self-assembled hydrogel, a preparation method thereof and an application of the hydrogel in a medicine for promoting periodontal tissue regeneration. The hydrogel consists of adipose-derived stem cells and an L-chiral self-assembled hydrogel. According to the present invention, the L-chiral self-assembled hydrogel can promote the high expression of five osteogenesis-related genes such as ALP, OCN, DLX-5, RUNX-2 and BSP of the adipose-derived stem cells, the high expression of two angiogenesis-related genes such as VEGF and ANG-1, and the high expression of ALP, OCN, DLX-5 and RUNX-2 osteogenesis-related proteins and the high expression of VEGF angiogenesis proteins; the L-chiral self-assembled hydrogel provided by the invention can realize directional osteogenic differentiation of adipose-derived stem cells, and can accurately realize regeneration of periodontal bone tissues; the L-chiral self-assembly hydrogel loaded with the adipose-derived stem cells can improve vascularization of periodontium and promote regeneration of the periodontium in an inflammatory environment.

Description

Technical Field

[0001] The present invention relates to the technical field of periodontitis treatment, and in particular to a left-handed chiral self-assembly hydrogel carrying adipose stem cells, a preparation method thereof, and application of the hydrogel in drugs for promoting periodontal tissue regeneration. Background Art

[0002] Periodontitis is a microbial-related, host-mediated, multi-factorial chronic inflammatory disease that leads to periodontal attachment loss. According to the Fourth National Oral Health Epidemiology Survey Report, the detection rate of attachment loss in the 35-44 age group is 33.2%, and the detection rate of attachment loss in the 55-64 age group is 69.9%, while the detection rate of gingival bleeding is as high as 88.4%. Therefore, the periodontal health status of the middle-aged and elderly population in my country is very serious.

[0003] At present, the classic method of periodontal treatment includes periodontal basic treatment plus periodontal surgery. Although inflammation can be controlled after periodontal sequential treatment, the periodontal tissue defects are difficult to recover, which seriously affects the periodontal health and quality of life of patients. Due to the complex structure of periodontal tissue, the periodontal tissue is in an inflammatory microenvironment for a long time, and the oral pathogens of periodontitis patients have a disorder of flora homeostasis, etc., the effect of periodontal tissue regeneration for large-scale periodontal tissue defects is often limited. Summary of the invention

[0004] The purpose of the present invention is to provide a left-handed chiral self-assembled hydrogel loaded with adipose stem cells and a preparation method thereof, as well as its application in drugs for promoting periodontal tissue regeneration in order to overcome at least one of the defects of the above-mentioned prior art. The present invention realizes the regeneration of periodontal tissue under inflammatory conditions by preparing a left-handed chiral self-assembled hydrogel loaded with adipose stem cells, exerts an immunomodulatory effect on periodontal tissue, reduces the damage to periodontal tissue under inflammatory conditions, promotes tissue regeneration, and provides a new idea for the treatment of periodontal diseases.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] One of the purposes of the present invention is to provide a chiral self-assembly hydrogel carrying adipose-derived stem cells, wherein the hydrogel is composed of adipose-derived stem cells (ADSCs) and a left-handed chiral self-assembly hydrogel.

[0007] Furthermore, the adipose-derived stem cells are present in the left-handed chiral self-assembly hydrogel at a concentration of 10 7 / ml.

[0008] Furthermore, the preparation method of the left-handed chiral self-assembled hydrogel includes: the preparation method of the left-handed chiral self-assembled hydrogel includes: dissolving a left-handed gel factor in a dimethyl sulfoxide solution to obtain a left-handed chiral self-assembled hydrogel; wherein the left-handed gel factor is a C2-symmetric phenylalanine derivative hydrogel factor.

[0009] Furthermore, in the left-handed chiral self-assembled hydrogel, the concentration of the left-handed gel factor is 3 mg / ml.

[0010] Furthermore, in the left-handed chiral self-assembled hydrogel, the mass concentration of dimethyl sulfoxide is 3.3%.

[0011] Furthermore, the left-handed chiral self-assembled hydrogel has a porous structure of 10-80 microns, and the diameter of the nanofibers is 10-300 nanometers.

[0012] The second object of the present invention is to provide a method for preparing the above-mentioned left-handed chiral self-assembled hydrogel carrying adipose stem cells, comprising the following steps: mixing the adipose stem cell culture medium suspension with the chiral self-assembled hydrogel and then placing them in a 24-well plate, flattening them, placing them in a cell culture incubator and standing them to form a gel, thereby obtaining the left-handed chiral self-assembled hydrogel carrying adipose stem cells.

[0013] Furthermore, after being flattened, the thickness of the mixed solution of the adipose stem cell culture medium suspension and the chiral self-assembled hydrogel is 2-3 mm.

[0014] The third purpose of the present invention is to use a left-handed chiral self-assembled hydrogel loaded with adipose stem cells in a drug for promoting periodontal tissue regeneration. The left-handed chiral self-assembled hydrogel loaded with adipose stem cells is the left-handed chiral self-assembled hydrogel loaded with adipose stem cells as described in any one of claims 1-6.

[0015] Furthermore, the left-handed chiral self-assembled hydrogel promotes the high expression of five osteogenic-related genes, ALP, OCN, DLX-5, RUNX-2, and BSP, and the high expression of two angiogenic-related genes, VEGF and ANG-1, in adipose stem cells, and promotes the high expression of osteogenic-related proteins, ALP, OCN, DLX-5, and RUNX-2, and the high expression of VEGF angiogenic protein.

[0016] The present invention has the following positive effects:

[0017] (1) Left-handed chiral self-assembled hydrogels can achieve directional osteogenic differentiation of adipose-derived stem cells and accurately achieve the regeneration of periodontal bone tissue.

[0018] (2) L-chiral self-assembled hydrogel loaded with adipose-derived stem cells can enhance the vascularization of periodontal tissue and promote periodontal tissue regeneration under inflammatory conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The effect of the chiral self-assembled hydrogel on the proliferation of rat adipose stem cells at 1, 3, and 7 days in the example;

[0020] Figure 2 The chiral self-assembled hydrogel in the example shows the alkaline phosphatase staining result of rat ADSC;

[0021] Figure 3 Download one of the expression of osteogenesis and angiogenesis-related genes of adipose stem cells left-handed chiral self-assembled hydrogel in the inflammatory environment of the embodiment;

[0022] Figure 4 Download the expression of osteogenesis and angiogenesis-related proteins of left-handed self-assembled hydrogels of adipose stem cells in the inflammatory environment of the embodiment;

[0023] Figure 5 Download the expression of osteogenesis and angiogenesis-related proteins of left-handed self-assembled hydrogels of adipose stem cells in the inflammatory environment of the embodiment;

[0024] Figure 6 This is a Micro-CT image of the left-handed chiral self-assembled hydrogel loaded with adipose stem cells applied to periodontal bone defects in rats in the example;

[0025] Figure 7 This is the Micro-CT imaging data analysis of the left-handed chiral self-assembled hydrogel of adipose stem cells applied to periodontal bone defects in rats in the example. DETAILED DESCRIPTION

[0026] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0027] The endpoints and any values ​​of the ranges disclosed in the present invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in the present invention.

[0028] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. If the specific conditions are not specified, they shall be carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially. The left-handed chiral self-assembled hydrogel, right-handed chiral self-assembled hydrogel, and racemic chiral self-assembled hydrogel groups in the following embodiments and analysis examples are all preparation methods that can be obtained by technicians in this field from published literature or purchased related products.

[0029] Example

[0030] Step 1: Prepare adipose-derived stem cells

[0031] After routine disinfection of the operating table, bilateral inguinal adipose tissue of 3-4 week-old rats was taken, and repeatedly rinsed with sterile PBS to remove residual fascia and blood clots in the fat. After controlling the water, it was placed in a sterile culture dish, and the fat in the centrifuge tube was cut into a paste using ophthalmic scissors. The adipose tissue cut into a paste was poured into a 15mL centrifuge tube, and then a type I collagenase solution with a mass concentration of 0.25% was added. After the two were fully mixed, they were placed in a 37°C constant temperature shaker for digestion for 60min. Subsequently, the centrifuge tube was taken out and an equal volume of cell culture medium was added to terminate the digestion, and then it was filtered with a sterile 70um cell filter. The filtrate was centrifuged at 1000r / min for 5min, the supernatant was discarded, the cells were resuspended with 1mL cell culture medium, added to a cell culture dish, cell culture medium was added, and placed in a cell culture incubator (37°C, 5% CO2 concentration) for culture to obtain a suspension of adipose stem cell culture medium.

[0032] Step 2: Preparation of left-handed chiral self-assembled hydrogel

[0033] The left-handed chiral self-assembled hydrogel (phenylalanine derivative hydrogel with C2 symmetry as the left-handed gel factor) powder was dissolved in dimethyl sulfoxide (DMSO) solution to make the final concentration of the material 3 mg / ml and the final concentration of DMSO 3.3%, thereby obtaining the left-handed chiral self-assembled hydrogel.

[0034] Step 3: Mix the adipose stem cell culture medium suspension obtained in step 1 and the left-handed chiral self-assembled hydrogel obtained in step 2 to adjust the adipose stem cell concentration to 10 7 / ml, mix well and place in a 24-well plate, spread flat to make the mixture 2-3mm thick, place in a cell culture incubator and let stand to gel, add cell culture medium after gelation for use in subsequent research, change the medium every 2-3 days to obtain left-handed chiral self-assembled hydrogel loaded with adipose stem cells.

[0035] Analytical Example 1 In vitro study

[0036] This analysis example studies the effect of the left-handed chiral self-assembled hydrogel obtained in Example 1 on the proliferation and osteogenic differentiation of adipose stem cells, as well as the osteogenesis and angiogenesis in a periodontitis environment simulated by gingipain.

[0037] 1. Effect of chiral self-assembled hydrogel on proliferation of rat adipose-derived stem cells

[0038] To investigate the effect of chiral self-assembled hydrogel on the proliferation of rat adipose-derived stem cells, the adipose-derived stem cells obtained in step 1 of Example 1 were seeded in a well plate pre-coated with chiral self-assembled hydrogel (1×10 4 Cell proliferation was detected by MTT on days 1, 3, and 7.

[0039] Figure 1 The effect of chiral self-assembled hydrogel on the proliferation of rat adipose stem cells at 1, 3, and 7 days, A: 1 day; B: 3 day; C: 7 day; Con: blank control group; D: right-handed chiral self-assembled hydrogel group (DPG group); L: left-handed chiral self-assembled hydrogel group (LPG group); R: racemic chiral self-assembled hydrogel group (RPG group: DPG+LPG). (*P<0.05).

[0040] The results showed that the left-handed chiral self-assembled hydrogel had no significant effect on the proliferation of rat adipose stem cells on the first day compared with the blank control group ( Figure 1 .A), and significantly promoted the proliferation of rat adipose stem cells on the 3rd and 7th days (P<0.05) ( Figure 1 .B,1.C). The right-handed chiral self-assembled hydrogel group and the racemic chiral self-assembled hydrogel group inhibited the proliferation of adipose stem cells.

[0041] 2. Effect of chiral self-assembled hydrogel on osteogenesis of rat adipose-derived stem cells

[0042] To detect the effect of chiral self-assembled hydrogel on the osteogenesis of rat adipose-derived stem cells, the adipose-derived stem cells obtained in step 1 of Example 1 were seeded in a well plate pre-coated with chiral self-assembled hydrogel (1×10 5 After 7 days of culture, the cells were detected by alkaline phosphatase staining. ALP is an important indicator for early detection of cell osteogenic differentiation. The observation of alkaline phosphatase staining reflects the osteogenic effect of cells.

[0043] Figure 2 The results of alkaline phosphatase staining of rat ADSCs by chiral self-assembled hydrogels: A. blank control group; B. right-handed chiral self-assembled hydrogel group (DPG group); C. left-handed chiral self-assembled hydrogel group (LPG group); D. racemic chiral self-assembled hydrogel group (RPG group: DPG+LPG).

[0044] Figure 2The experimental results showed that at 7 days, the left-handed chiral self-assembled hydrogel group (LPG group) could significantly upregulate the alkaline phosphatase activity of rat adipose stem cells and promote the osteogenic differentiation of adipose stem cells.

[0045] 3. Osteogenesis and angiogenesis of left-handed self-assembled hydrogels derived from adipose-derived stem cells in an inflammatory microenvironment simulated by gingipain

[0046] Adipose-derived stem cells were seeded in a well plate pre-coated with chiral self-assembled hydrogel (1×10 5 cells), and used 3 μg / ml gingipain to pretreat the cells to simulate an inflammatory microenvironment. After culturing with osteogenic induction solution for 3 days and 7 days, PCR and Western blot techniques were used to detect the expression of osteogenic and angiogenic related genes and proteins in the left-handed chiral self-assembled hydrogel of adipose stem cells in the inflammatory microenvironment.

[0047] Figure 3 and Figure 4 Download the expression of osteogenesis and angiogenesis-related genes in left-handed self-assembled hydrogels of adipose stem cells for inflammatory environment. Figure 5 The inflammatory environment downloads the expression of osteogenesis and angiogenesis related proteins of adipose stem cells in left-handed self-assembled hydrogel.

[0048] Figure 3-5 The experimental results show that at 3 and 7 days, the left-handed chiral self-assembled hydrogel group loaded with adipose stem cells can significantly upregulate the expression of osteogenic and angiogenic related genes and proteins, and promote new bone formation and vascularization of periodontal tissues. Specifically, the left-handed chiral self-assembled hydrogel can promote the high expression of five osteogenic related genes ALP, OCN, DLX-5, RUNX-2, and BSP in adipose stem cells, as well as the high expression of two angiogenic related genes VEGF and ANG-1. It can also promote the high expression of ALP, OCN, DLX-5, RUNX-2 osteogenic related proteins and the high expression of VEGF angiogenic protein.

[0049] Analytical Example 2 In vivo study

[0050] This analysis example studied the regenerative effect of the adipose stem cell-loaded left-handed chiral self-assembled hydrogel obtained in Example 1 on periodontal bone defects in rats.

[0051] A rat alveolar bone defect model was established using 8-week-old SD rats. Surgery was performed after intraperitoneal injection of isoflurane at a volume concentration of 3%. An intrasulcular incision and a horizontal incision extending distally were made along the left maxillary incisor of the rat. A periosteal stripper was used to open the full-thickness flap to expose the bone surface. A ball drill was used to create a 1mm*1mm*0.5mm bone defect 1mm lateral to the top root of the alveolar ridge of the maxillary incisor. The study was divided into three groups, including the Con group (bone defect only), the L group (bone defect + left-handed chiral self-assembled hydrogel), and the L+adipose stem cell group (bone defect + left-handed chiral self-assembled hydrogel + adipose stem cells). Micro-CT scanning was performed 4 weeks after surgery, and the results were as follows: Figure 6 , Figure 7 shown. Figure 6 Micro-CT imaging of adipose-derived stem cell-loaded left-handed self-assembled hydrogel applied to periodontal bone defects in rats; Figure 7 Micro-CT imaging data analysis of left-handed chiral self-assembled hydrogel of adipose stem cells applied to periodontal bone defects in rats.

[0052] The Micro-CT results of animal experiments showed that the osteogenesis effect of the adipose stem cell-loaded left-handed chiral self-assembled hydrogel group was significantly better than that of the control group and the group with only left-handed chiral self-assembled hydrogel.

[0053] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A chiral self-assembled hydrogel loaded with adipose-derived stem cells, characterized in that: The hydrogel consists of adipose stem cells and left-handed chiral self-assembled hydrogel.

2. The left-handed chiral self-assembled hydrogel loaded with adipose-derived stem cells according to claim 1, characterized in that: The adipose-derived stem cells are present in the left-handed chiral self-assembled hydrogel at a concentration of 10 7 / ml.

3. The adipose-derived stem cell-loaded left-handed self-assembled hydrogel according to claim 1, characterized in that: The preparation method of the left-handed chiral self-assembled hydrogel comprises: dissolving a left-handed gel factor in a dimethyl sulfoxide solution to obtain a left-handed chiral self-assembled hydrogel; Wherein, the left-handed gel factor is a C2-symmetrical phenylalanine derivative hydrogel factor.

4. The adipose-derived stem cell-loaded left-handed self-assembled hydrogel according to claim 3, characterized in that: In the left-handed chiral self-assembled hydrogel, the concentration of the left-handed gel factor is 3 mg / ml.

5. The adipose-derived stem cell-loaded left-handed self-assembled hydrogel according to claim 3, characterized in that: In the left-handed chiral self-assembled hydrogel, the mass concentration of dimethyl sulfoxide is 3.3%.

6. The adipose-derived stem cell-loaded left-handed self-assembled hydrogel according to claim 1, characterized in that: The left-handed chiral self-assembled hydrogel has a porous structure of 10-80 microns, and the diameter of the nanofiber is 10-300 nanometers.

7. A method for preparing the left-handed chiral self-assembled hydrogel loaded with adipose stem cells according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: mixing adipose stem cell culture medium suspension and chiral self-assembled hydrogel, placing the mixture in a 24-well plate, flattening the mixture, placing the mixture in a cell culture box and standing to form a gel, thereby obtaining adipose stem cell-loaded left-handed chiral self-assembled hydrogel.

8. The adipose-derived stem cell-loaded left-handed self-assembled hydrogel according to claim 7, characterized in that: After paving, the thickness of the mixed solution of the adipose stem cell culture medium suspension and the chiral self-assembled hydrogel is 2-3 mm.

9. Application of a left-handed chiral self-assembled hydrogel loaded with adipose stem cells in promoting periodontal tissue regeneration, characterized in that: The adipose stem cell-loaded left-handed chiral self-assembled hydrogel is the adipose stem cell-loaded left-handed chiral self-assembled hydrogel as claimed in any one of claims 1 to 6.

10. The use of a left-handed chiral self-assembled hydrogel loaded with adipose stem cells in a drug for promoting periodontal tissue regeneration according to claim 9, characterized in that: The left-handed chiral self-assembly hydrogel promotes the high expression of five osteogenic-related genes, ALP, OCN, DLX-5, RUNX-2, and BSP, and the high expression of two angiogenic-related genes, VEGF and ANG-1, in adipose stem cells, and promotes the high expression of osteogenic-related proteins, ALP, OCN, DLX-5, and RUNX-2, and the high expression of VEGF angiogenic protein.

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