CN121203949A - Application of thermosensitive hydroxypropyl chitosan hydrogel in the preparation of drugs for treating intervertebral disc degeneration - Google Patents
Application of thermosensitive hydroxypropyl chitosan hydrogel in the preparation of drugs for treating intervertebral disc degenerationInfo
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Abstract
本发明公开了温敏性羟丙基甲壳素水凝胶在制备治疗椎间盘退行性病变药物中的应用，属于生物医药技术领域。温敏性羟丙基甲壳素水凝胶具有良好的生物相容性和温敏性，在低温状态下为液体，进入体内迅速形成透明水凝胶，能够封闭破损的纤维环，防止药物泄露。成纤维细胞具有分泌细胞外基质、抑制炎症的功能，能够维持椎间盘的高度和稳定性，缓解临床症状。温敏性羟丙基甲壳素水凝胶与成纤维细胞联用可以促进成纤维细胞分泌细胞外基质，提供更强的生物力学功能，在纤维环发生破裂的患者中充分发挥疗效，填补泄露型椎间盘细胞治疗的空缺。
This invention discloses the application of thermosensitive hydroxypropyl chitosan hydrogel in the preparation of drugs for treating intervertebral disc degeneration, belonging to the field of biomedical technology. The thermosensitive hydroxypropyl chitosan hydrogel possesses good biocompatibility and thermosensitivity; it is liquid at low temperatures and rapidly forms a transparent hydrogel upon entering the body, effectively sealing the damaged annulus fibrosus and preventing drug leakage. Fibroblasts have the function of secreting extracellular matrix and inhibiting inflammation, maintaining the height and stability of the intervertebral disc and alleviating clinical symptoms. The combined use of thermosensitive hydroxypropyl chitosan hydrogel and fibroblasts can promote the secretion of extracellular matrix by fibroblasts, providing stronger biomechanical function and fully demonstrating therapeutic efficacy in patients with ruptured annulus fibrosus, filling the gap in cell therapy for leaking intervertebral discs.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of temperature-sensitive hydroxypropyl chitin hydrogel in preparation of a medicine for treating intervertebral disc degenerative disease.
Background
An intervertebral disc is a fibrocartilage tissue that is located between and connects two adjacent vertebral bodies, consisting of an outer Annulus Fibrosis (AF), an inner gelatinous nucleus pulposus, and cartilage endplates that connect the vertebral bodies. The outer annulus provides the mechanical and compressive strength of the disc to accommodate and maintain the osmotic pressure exerted by the nucleus pulposus. The annulus fibrosus surrounds a gelatinous nucleus pulposus, which is mainly composed of type II collagen and elastin fibers, which surround proteoglycan aggregates, the most abundant of which are the aggrecan. The high level of water molecules retained in the nucleus pulposus maintains a high hydrostatic expansion pressure, thereby resisting disc deformation and maintaining disc height. Cells within the intervertebral disc are present in a low density in the abundant extracellular matrix (ECM). Because the intervertebral disc is an anoxic and blood-supply-less environment, the number of cells and metabolism are low, and thus the intervertebral disc is difficult to regenerate and repair by itself after degeneration. Degeneration of the disc begins with degeneration of the nucleus pulposus tissue, and a variety of causative factors cause an inflammatory reaction in the disc at an early stage, so that the secretion of inflammatory factors is increased, and the inflammatory factors are expressed by a variety of matrix metalloproteinases, so that extracellular matrix in the nucleus pulposus is degraded. The degradation of extracellular matrix causes damage to the disc structure, stress concentration occurs, apoptosis is aggravated, and the disc is further caused to enter a malignant circulatory state of degeneration. The change of the microcosmic level can lead to pathological structural changes such as high loss, segment instability and the like of the intervertebral disc at the macroscopic level. At the same time, the fibrous ring is damaged and broken, and the nucleus pulposus tissue is extruded to bulge, protrude and even deviate from, so as to press spinal cord or nerve root, thereby causing lumbago and skelalgia symptoms. At present, the lumbago is a main cause of the loss of the work ability of individuals worldwide, and brings great social and economic burden to the world. Lumbar disc degeneration (DDD) is one of the main causes of lumbago, and has clinical manifestations of different degrees of lumbago and lumbar vertebra function limitation, and chronic lumbago patients can also be accompanied with mental symptoms such as anxiety or depression, and the like, so that the work and life quality are seriously affected.
At present, innovative therapeutic means for recovering, repairing and regenerating intervertebral discs are gradually emerging, such as growth factor treatment, cell treatment and gene treatment. The cell therapy method aims at transplanting living cells with extracellular matrix secretion and/or inflammation inhibition functions into the intervertebral disc, and can be used for refilling and repairing the degenerated intervertebral disc by singly or in combination with a carrier (such as hydrogel), so that the aims of repairing and treating are fulfilled, and the method mainly comprises mesenchymal stem cell treatment, fibroblasts and intervertebral disc cells. In the prior art, chinese patent publication No. CN110337490a, for example, discloses a method for increasing the efficacy of fibroblasts to regenerate cells and/or tissues, wherein regeneration includes regeneration of intervertebral discs, in particular contacting the fibroblasts with one or more bioactive substances, and/or culturing the fibroblasts under conditions that increase the efficacy of the fibroblasts for regenerating cells and/or tissues. In particular embodiments, the one or more bioactive substances include one or more cytokines, such as growth factors (e.g., FGF- α, FGF- β, and/or TGF- β family members), or include platelet rich plasma. Also, it discloses that the fibroblasts are contained in a pharmaceutically acceptable carrier selected from the group consisting of sterile solutions, hydrogels (such as PRP/HA/BTX hydrogels), implantable cell matrices, devices, and combinations thereof.
However, in this prior art, in order to enhance the therapeutic effect of the fibroblasts, it is necessary to contact the fibroblasts with a growth factor or plasma including platelet rich plasma. Such as inclusion of fibroblasts in PRP/HA/BTX hydrogels, where the activation of platelets in PRP (Platelet-RICH PLASMA, i.e., platelet rich plasma) releases large amounts of bioactive proteins such as Platelet-derived growth factors (PDGF), transforming growth factor-beta (TGF-beta), insulin-like growth factors (IGF), etc., which promote proliferation, differentiation and migration of cells. This clearly increases the cost of the treatment and some of the components in PRP (e.g. proteins, cytokines etc.) may still cause allergic reactions, presenting a potential safety risk.
Disclosure of Invention
1. Object of the invention
The invention aims to provide an application of thermosensitive hydroxypropyl chitin hydrogel in preparing a medicament for treating intervertebral disc degenerative disease, which is derived from unexpected findings of the applicant in research, and the applicant discovers that when the thermosensitive hydroxypropyl chitin hydrogel is used in combination with fibroblasts, the gene expression amounts of COL1A1 (type I collagen), COL3A1 (type III collagen) and FIBRINECTIN (fibronectin) of the fibroblasts are obviously increased, so that the secretion of extracellular matrixes by the fibroblasts can be obviously promoted, and the treatment effect of the fibroblasts is improved.
2. Technical proposal
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
In a first aspect, the invention provides the use of a temperature-sensitive hydroxypropyl chitin hydrogel to promote high expression of COL1A1, COL3A1 and FIBRINECTIN genes by fibroblasts.
Further, the above application is for non-diagnostic purposes.
In a second aspect, the invention provides the use of a temperature-sensitive hydroxypropyl chitin hydrogel in the preparation of a reagent that promotes fibroblast cells to highly express the COL1A1, COL3A1 and FIBRINECTIN genes.
In a third aspect, the invention provides the use of a temperature-sensitive hydroxypropyl chitin hydrogel to promote secretion of extracellular matrix by fibroblasts.
Further, the above application is for non-diagnostic purposes.
In a fourth aspect, the invention provides the use of a temperature-sensitive hydroxypropyl chitin hydrogel in the preparation of an agent that promotes secretion of extracellular matrix by fibroblasts.
In a fifth aspect, the invention provides an application of temperature-sensitive hydroxypropyl chitin hydrogel in preparing a medicament for treating intervertebral disc degenerative disease.
Further, the application comprises the step of mixing the temperature-sensitive hydroxypropyl chitin hydrogel with fibroblasts to form a fibroblast complex.
Further, the method further comprises culturing the fibroblast by using a culture medium containing temperature-sensitive hydroxypropyl chitin hydrogel before mixing the fibroblast.
Further, the medium is a complete medium.
Further, the mixing comprises mixing the temperature-sensitive hydroxypropyl chitin hydrogel with fibroblasts at a low temperature of 2-8 ℃.
Further, the mixing comprises the step of mixing the temperature-sensitive hydroxypropyl chitin hydrogel and the fibroblasts at a low temperature of 4-6 ℃.
Further, the mixing comprises mixing according to the proportion that the final concentration of the temperature-sensitive hydroxypropyl chitin hydrogel is 5-15 mg/mL and the final number of the fibroblasts is 5 multiplied by 10 6～2×107 cells/mL.
Further, the above mixing includes mixing in a ratio of a final concentration of the thermosensitive hydroxypropyl chitin hydrogel of 10mg/mL and a final number of fibroblasts of 1X 10 7 cells/mL.
In a sixth aspect, the present invention also provides a fibroblast complex comprising fibroblasts and a temperature-sensitive hydroxypropyl chitin hydrogel.
Further, the fibroblast is a fibroblast after being cultured in a culture medium containing temperature-sensitive hydroxypropyl chitin hydrogel. As a further explanation of the application, the temperature-sensitive hydroxypropyl chitin hydrogel can promote the fibroblast to highly express COL1A1, COL3A1 and FIBRINECTIN genes, and the cultured fibroblast has stronger activity of secreting extracellular matrix.
Further, the final concentration of the fibroblast compound and the temperature-sensitive hydroxypropyl chitin hydrogel is 5-15 mg/mL, and the final number of the fibroblasts is 5 multiplied by 10 6～2×107 cells/mL.
Further, the final concentration of the fibroblast compound and the temperature-sensitive hydroxypropyl chitin hydrogel is 10mg/mL, and the final number of the fibroblasts is 1×10 7 cells/mL.
In a seventh aspect, the invention also provides a pharmaceutical composition for treating degenerative disc disease, comprising fibroblasts and a temperature-sensitive hydroxypropyl chitin hydrogel, and a pharmaceutically acceptable carrier.
Further, the fibroblast is a fibroblast after being cultured in a culture medium containing temperature-sensitive hydroxypropyl chitin hydrogel.
In an eighth aspect, the invention provides a method of promoting secretion of extracellular matrix by fibroblasts, the method comprising contacting the fibroblasts with a temperature-sensitive hydroxypropyl chitin hydrogel.
Further, the fibroblasts are derived from foreskin tissue.
Further, the foreskin tissue is a healthy tissue.
Further, the foreskin tissue is derived from a surgical sample of a patient aged no more than 18 years.
Further, the above patients were examined medically for no infectious viral infection, and no steroid hormone was used within 6 months prior to surgery.
Furthermore, the foreskin-derived fibroblasts have the characteristics of high purity (the positive rate of fibrosis related proteins CD90, FSP1, COL1A1 and Vimentin is more than 90%, the positive rate of blood cell related protein CD31 is less than 2%), low immunogenicity (the positive rate of HLA-DR/DP/DQ related to immunogenic MHC class II molecules is less than 2%), good safety (no in vivo and in vitro nodulation), and play roles in structural support and inflammation inhibition in the process of treating intervertebral disc degenerative diseases.
3. Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
(1) The application of the thermosensitive hydroxypropyl chitin hydrogel in preparing the medicine for treating intervertebral disc degenerative disease provided by the invention comprises the steps of firstly, preparing the thermosensitive hydroxypropyl chitin hydrogel into COL1A1 (type I collagen), COL3A1 (type III collagen) and FIBRINECTIN (fibronectin) genes for promoting fibroblasts to secrete extracellular matrixes, and secondly, preparing the thermosensitive hydroxypropyl chitin hydrogel into liquid at a low temperature state, and rapidly forming transparent hydrogel in a body, so that broken fibrous rings can be sealed, and medicine leakage is prevented.
(2) The application of the thermosensitive hydroxypropyl chitin hydrogel in preparing the medicine for treating the intervertebral disc degenerative disease provided by the invention combines the thermosensitive hydroxypropyl chitin hydrogel with the fibroblasts, can promote the fibroblasts to secrete extracellular matrixes, can reduce the need of the fibroblasts to be contacted with bioactive substances or various cytokines in advance, can provide stronger biomechanical functions, fully plays the curative effect in patients with the rupture of the fibrous ring (full-thickness ring tearing of the target intervertebral disc level determined by intervertebral disc radiography), and fills the gap of the treatment of the leaked intervertebral disc cells.
(3) The application of the thermosensitive hydroxypropyl chitin hydrogel in preparing the medicine for treating the intervertebral disc degenerative disease provided by the invention has the advantages that the fibroblast and the thermosensitive hydroxypropyl chitin hydrogel are combined to show good treatment advantages, the water content of the rat degenerative intervertebral disc is obviously improved, the height and the stability of the intervertebral disc are maintained, and the degeneration of the intervertebral disc is delayed. The humanized preparation is injected into a rat body, the rat does not have rejection reaction, and the mixed preparation of the fibroblast and the temperature-sensitive hydroxypropyl chitin hydrogel has no immunogenicity and good safety. Therefore, the combined use of the fibroblast and the temperature-sensitive hydroxypropyl chitin hydrogel is expected to bring new hope for the treatment of the intervertebral disc degenerative disease, and can fill the gap of the treatment of the leakage type intervertebral disc cell.
Drawings
FIG. 1 is a photograph of a P8 generation fibroblast (10X).
FIG. 2 is a flow chart of a fibroblast detection profile.
Fig. 3 is a temperature-sensitive picture of different concentrations of temperature-sensitive hydroxypropyl chitin.
FIG. 4 is a photograph of fibroblasts co-cultured with different concentrations of temperature sensitive hydroxypropyl chitin.
FIG. 5 is a graph of LDH activity in fibroblasts co-cultured with varying concentrations of temperature sensitive hydroxypropyl chitin.
FIG. 6 is a diagram showing the expression of extracellular matrix-related genes in fibroblasts.
Fig. 7 is an discography X-ray view.
Fig. 8 is a flow chart for the treatment and detection of degenerative disc disease in rats.
Fig. 9 is an X-ray diagram of a rat intervertebral disc.
Fig. 10 is a statistical plot of rat disc height.
Fig. 11 is a rat spinal disc MRI diagram.
Fig. 12 is a graph of rat disc relative signal intensity statistics.
FIG. 13 is an immunofluorescence assay of rat disc extracellular matrix.
Detailed Description
The invention is further described below in connection with specific embodiments.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, and the term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
As used herein, the term "about" is used to provide the flexibility and inaccuracy associated with a given term, metric or value. The degree of flexibility of a particular variable can be readily determined by one skilled in the art.
As used herein, the term "is intended to be synonymous with" one or more of ". For example, "at least one of A, B and C" explicitly includes a only, B only, C only, and their respective combinations.
Concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a numerical range of about 1 to about 4.5 should be interpreted to include not only the explicitly recited limits of 1 to about 4.5, but also include individual numbers (such as 2,3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges reciting only one numerical value, such as "less than about 4.5," which should be construed to include all such values and ranges. Moreover, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.
In the present application, the temperature-sensitive hydroxypropyl chitin hydrogel means a temperature-sensitive hydroxypropyl chitin solution (low temperature) or a temperature-sensitive hydroxypropyl chitin gel (body temperature), which can be judged as a solution or gel by a person skilled in the art according to temperature.
In the present application, the source of the fibroblast is not limited, and one skilled in the art can isolate it from skin tissue such as foreskin tissue and culture it in vitro. As an example, the present application provides a method of isolated culture of fibroblasts:
(1) Initial organizational nature and source legitimacy declarations
The waste prepuce tissue after the prepuce circumcision of the clinical healthy teenagers is used as an initial source.
The selected foreskin tissue is healthy tissue, and is derived from a surgical sample of a patient aged less than 18 years, the patient is not infected by infectious viruses through medical examination, and the patient does not use steroid hormone medicine within 6 months before surgery. The patient was fully informed of the purpose of the acquisition of the surgical sample prior to surgery and signed an informed consent form.
(2) Isolated culture of fibroblasts
Using sterile PBS buffer solution to wash the foreskin tissue twice, sterilizing the foreskin tissue by povidone iodine and ethanol (placing the foreskin tissue in a sterilizing solution with the active ingredient of 5% povidone iodine, washing in a centrifuge tube for 2-3 minutes, adjusting the time according to the size of tissue blocks, replacing sterile forceps, taking skin tissue in a new cell culture dish, washing by PBS, and transferring the skin tissue into a centrifuge tube containing 75% ethanol, soaking for 1-2 minutes (the cell viability is lost due to excessive sterilization));
the tissue is washed for 2 times after sterilization, soaked in 2mg/mL type XI collagenase (sigma, C7657-500 mg) and gently shaken and digested for 2-3 hours (digestion time is judged according to the turbidity degree of the cell suspension) by a sterile PBS buffer, the cell suspension is subjected to screen filtration by a 70 mu m cell filter with the aid of a high-sugar DMEM medium (Shanghai Yuanzhi Biotechnology Co., ltd., L110 KJ), the filtrate is collected and mucus and undigested tissue are removed, then 500g is centrifuged for 5 minutes, the supernatant is discarded, cell sediment is collected, and the cell sediment is resuspended by a complete medium (complete medium formula: 7.5% hPL+92.5% high-sugar DMEM medium+20 ng/mL TGF-. Beta.1; hPL: brand Sexton, product number PL-NH-100) to obtain Pr fibroblasts;
Cell counting, inoculating the cells into a 6-well plate according to the density of 2X 10 4cells/cm2, adding 2mL of complete culture medium into each well to obtain P0 generation, carrying out cell passage after the cell confluency reaches 80% -90%, inoculating the cells into a new culture container according to the density of 1.2X 10 4cells/cm2, and sequentially increasing the cell generation times, wherein the cell generation times are sequentially marked as P1 generation, P2 generation and P3 generation.
As shown in fig. 1, microscopic pictures of the P8 generation fibroblasts were taken, from which it can be seen that the fibroblasts were uniform in morphology, and had a fusiform or star-like shape, and grown on the wall.
(3) Fibroblast identification
Surface markers and intracellular markers of P8 generation fibroblasts were tested to identify fibroblasts.
The surface marker logistics detection step comprises the steps of sucking and discarding a culture medium, using 5mL of sterile PBS buffer solution for rinsing, then using 2mL of pancreatin digestion solution to be dripped into a culture dish for digestion treatment to obtain a cell mixture, placing the cell mixture into a 15mL centrifuge tube, placing the centrifuge tube into a centrifuge, centrifuging at the rotating speed of 300g for 5 minutes, and discarding supernatant to obtain cell sediment. To the cell pellet, 400. Mu.L of staining buffer (BD, 554656) was added to resuspend the cell pellet, and the resuspended cell pellet was transferred to 3 1.5mL centrifuge tubes, 100. Mu.L/tube. 10 mu L of the antibody to be tested is added into the 3 1.5mL centrifuge tubes respectively, and the mixture is blown and evenly mixed. And placing the centrifuge tubes in a refrigerator at a temperature of 2-8 ℃ for 30 minutes, adding sterile PBS buffer solution into each centrifuge tube according to the dosage of 800 mu L/tube, and then placing the centrifuge tubes into the centrifuge, and performing centrifugation treatment at a rotating speed of 300g for 5 minutes. After centrifugation, the supernatant was discarded, 400. Mu.L of staining buffer was added to each centrifuge tube to resuspend the cell pellet, and then transferred to a flow tube, and the resuspended cell mixture was subjected to flow detection of surface markers. Antibody names used for surface marker flow assay are CD90 (BD, 555596), CD31 (BD, 560984) and HLA-DR/DP/DQ (BD, 555811).
And (3) performing intracellular marker logistics detection, namely sucking and discarding a culture medium, using 5mL of sterile PBS buffer solution for rinsing, then dripping 2mL of pancreatin digestion solution into a culture dish for digestion treatment to obtain a cell mixture, placing the cell mixture into a 15mL centrifuge tube, placing the centrifuge tube into a centrifuge, centrifuging at a rotating speed of 300g for 5 minutes, and discarding supernatant of the cell mixture after centrifugation to obtain a cell precipitate. 1mL of a fixing/membrane penetrating fluid (BD, 554714) is added to the cell sediment, the centrifuge tube is placed in a refrigerator at a temperature of 2-8 ℃ for standing for 50 minutes, 2mL of a staining buffer (BD, 554656) is added to the centrifuge tube, the centrifuge tube is subjected to centrifugation at a rotational speed of 300g for 5 minutes, and after centrifugation, 400 mu L of the staining buffer is added to the centrifuge tube for resuspension. The resuspended cell pellet was transferred to 3 1.5mL centrifuge tubes, 100. Mu.L/tube format. 10 mu L of the antibody to be tested is added into the 3 1.5mL centrifuge tubes respectively, the mixture is blown and evenly mixed, and the centrifuge tubes are placed into a 37 ℃ incubator for standing for 30 minutes. After incubation, a staining buffer was added to each centrifuge tube at a dose of 800. Mu.L/tube, and the tubes were placed in a centrifuge and centrifuged at 300g for 5 minutes. After centrifugation, the supernatant was discarded, 400. Mu.L of staining buffer was added to each centrifuge tube to resuspend the cell pellet, and then transferred to a flow tube, and the resuspended cell mixture was subjected to flow detection of intracellular markers. The antibodies used for the flow assay of intracellular markers were named FSP1 (Proteintech, CL 488-16105), COL1A1 (LSBio, LS-C721218-100) and Vimentin (BD, 562337).
As shown in FIG. 2, the positive rates of CD90, FSP1 (fibroblast-specific protein 1), COL1A1 (type I collagen) and Vimentin (Vimentin) are above 90%, and the positive rates of HLA-DR/DP/DQ related to the blood cell related marker CD31 and the immunogenic MHC class II molecule are below 2%, so that the isolated and cultured fibroblast of the system has the characteristics of high purity and low immunogenicity.
In the present application, the temperature-sensitive hydroxypropyl chitin hydrogel is not limited, and those skilled in the art can refer to the prior art. As an example. The application provides a temperature-sensitive hydroxypropyl chitin hydrogel and a preparation method thereof, wherein the temperature-sensitive hydroxypropyl chitin hydrogel comprises the following components:
(1) Preparation method
2G of chitin powder (MCE, HY-126389) is added into 20mL of precooled 50% (w/w) NaOH solution, and the mixture is continuously stirred (100-200 rpm/min) at the temperature of-20 ℃ for reaction for 12h. After the reaction is finished, filtering the solution to obtain the blocky or flocculent alkaline chitin. The alkaline chitin is transferred to a beaker, 100mL of isopropanol is added to start stirring (200-300 rpm/min), 30mL of propylene oxide is slowly added dropwise during stirring, and then the reaction is carried out for 6 hours in a constant temperature box at 65 ℃.
The hydroxylation reaction equation is:
chitin-OH+NaOH- & gt chitin-ONa+H 2 O,
Chitin-ona+h 2O+H2C-CH-CH3 →chitin-O-CH 2-CH(OH)-CH3 O.
After the reaction is finished, cooling the system to room temperature, neutralizing the pH value of the reaction system to 7-7.5 by glacial acetic acid, slowly pouring the reaction mixture into 800mL of glacial ethanol with intense stirring (> 500 rpm/min), and separating out the temperature-sensitive hydroxypropyl chitin in a fibrous or flocculent form. Filtering with a Buchner funnel, collecting precipitate, repeatedly washing with 80% (V/V) ethanol solution for 5-6 times until the filtrate is detected to be free of white precipitate (indicating no chloride ions) with silver nitrate solution, and the filtrate is neutral. And redissolving the Wen Minqiang propyl chitin crude product in 200mL of deionized water, stirring overnight to fully dissolve the product, filling the solution into a dialysis bag (with the molecular weight cutoff of 8-14 kDa), and dialyzing for 3-5 days to thoroughly remove all small molecular impurities and salts. Freezing the dialyzed solution, and then placing the frozen solution in a freeze dryer for sublimation drying for 48 hours to obtain the temperature-sensitive hydroxypropyl chitin solid.
20Mg of temperature-sensitive hydroxypropyl chitin is taken and dissolved in 2mL and 4mL of aqueous solution respectively to prepare 10mg/mL and 5mg/mL of sol.
(2) Temperature sensitive performance detection
The fluidity of the sol at low temperature (2-8 ℃) and high temperature (37+/-2 ℃) is detected, and the result is shown in a figure 3, wherein the sol of 5mg/mL and the sol of 10mg/mL are both in a liquid state at low temperature, have strong fluidity, are in a solidification state at high temperature, have no fluidity and have good temperature sensitivity, and realize the transformation behavior of sol-gel.
(3) Cytotoxicity detection
Cytotoxicity of the temperature sensitive hydroxypropyl chitin hydrogels was detected using a Lactate Dehydrogenase (LDH) detection kit (Shanghai bi yunshan biotechnology limited, C0016).
Adding 20 mu L of temperature-sensitive hydroxypropyl chitin hydrogel with the concentration of 10mg/mL, 5mg/mL and 2mg/mL into a 96-well plate, paving the bottom of the well, placing the mixture in a 37 ℃ incubator for half an hour, inoculating fibroblasts into the 96-well plate according to the ratio of 1X 10 4, sucking the supernatant when the cell density is 80-90%, washing the mixture once by PBS, adding 150 mu L of LDH release reagent provided by a kit diluted 10 times by PBS, properly shaking the culture plate, mixing the mixture, and then continuously incubating the mixture in the cell incubator for 1 hour. The cell culture plates were then centrifuged for 5min with 400g in a multi-well plate centrifuge. The supernatant from each well, 120 μl, was added to a new 96-well plate corresponding well, and then the sample was assayed. 60 mu L of LDH detection working solution is added to each hole, the mixture is uniformly mixed, and the mixture is incubated at room temperature (25 ℃) for 30min in a dark place. The absorbance was then measured at 490 nm.
As shown in FIG. 4, the fibroblasts were co-cultured with different concentrations of temperature sensitive hydroxypropyl chitin hydrogel, each group of cells had good adherence, supernatant showed no floating dead cells, and cell proliferation confluency showed no significant difference.
The lactate dehydrogenase activity in each constituent fiber cell was detected using a lactate dehydrogenase assay kit, with higher OD values indicating a greater cell number. As shown in FIG. 5, the fibroblasts were co-cultured with temperature-sensitive hydroxypropyl chitin hydrogels at different concentrations, and the activity of lactate dehydrogenase in each group of cells was not significantly different from that in the control group, indicating that the temperature-sensitive hydroxypropyl chitin hydrogels did not affect the proliferation of fibroblasts and did not produce cytotoxicity.
Example 1
This example provides a study of thermosensitive hydroxypropyl chitin hydrogel to promote secretion of extracellular matrix by fibroblasts.
The confluence of the fibroblasts reaches 90%, the supernatant is discarded, PBS is used for cleaning once, pancreatin digestive juice is added, digestion is carried out for 3min at 37 ℃, all cell suspensions are collected, 500g is centrifuged for 5min, and the centrifugal supernatant is discarded;
Adding 250 mu L of mixed solution of temperature-sensitive hydroxypropyl chitin hydrogel and fibroblast complete culture medium (10 mg/mL of mixed solution of temperature-sensitive hydroxypropyl chitin hydrogel and fibroblast complete culture medium according to the volume ratio of 1:1, and pre-cooling at 4 ℃), and re-suspending cell sediment with the cell density of 1 multiplied by 10 7 cells/mL;
Inoculating into 6-well plate (6-well plate is preheated at 37 ℃ in advance), adding 100 mu L of cell mixture into each well, placing into a 37 ℃ incubator for 20min, adding 2mL of complete culture medium preheated at 37 ℃, and placing into a 37 ℃ and 5% CO 2 incubator for culturing for 48h.
Setting a hyaluronic acid control, comprising:
The confluence of the fibroblasts reaches 90%, the supernatant is discarded, PBS is used for cleaning once, pancreatin digestive juice is added, digestion is carried out for 3min at 37 ℃, all cell suspensions are collected, 500g is centrifuged for 5min, and the centrifugal supernatant is discarded;
Adding 250 μl of mixed solution of hyaluronic acid and fibroblast complete medium (10 mg/mL hyaluronic acid (manufactured and bioengineered (Shanghai) Co., ltd., A003907-0100) and fibroblast complete medium mixed according to a volume ratio of 1:1, and pre-cooling at 4deg.C), re-suspending cell pellet with cell density of 1×10 7 cells/mL;
Inoculating into 6-well plate (6-well plate is preheated at 37 ℃ in advance), adding 100 mu L of cell mixture into each well, placing into a 37 ℃ incubator for 20min, adding 2mL of complete culture medium preheated at 37 ℃, and placing into a 37 ℃ and 5% CO 2 incubator for culturing for 48h.
A blank control was set:
The confluence of the fibroblasts reaches 90%, the supernatant is discarded, PBS is used for cleaning once, pancreatin digestive juice is added, digestion is carried out for 3min at 37 ℃, all cell suspensions are collected, 500g is centrifuged for 5min, and the centrifugal supernatant is discarded;
Adding complete culture medium to re-suspend the cell sediment, wherein the cell density is 1×10 7 cells/mL;
Inoculated in 6-well plates with 2mL each, and incubated in a 37℃5% CO 2 incubator for 48h.
After the culture, the fibroblasts cultured in each system are collected, RNA lysate is added, RNA is extracted and inverted into cDNA, and the gene expression of extracellular matrix related genes COL1A1 (type I collagen), COL3A1 (type III collagen) and FIBRINECTIN (fibronectin) are detected by RT-qPCR method.
The total RNA extraction kit is Promega, LS1040;
reverse transcription kit, promega, LS2052;
qPCR reaction kit, shanghai Biyun biotechnology Co., ltd., D7265;
qPCR reaction system:
| Component (A) | Volume of |
| qPCRmix | 10μL |
| CDNA template | 2μL |
| Primer F (10. Mu.M) | 0.5μL |
| Primer R (10. Mu.M) | 0.5μL |
| Sterilizing water | 7μL |
QPCR reaction procedure, pre-denaturation at 95℃for 2min, denaturation at 95℃for 15sec, annealing/extension at 60℃for 30sec, repeated denaturation and annealing/extension for a total of 40 cycles.
Primer information:
| primer name | Primer sequences | SEQ ID NO |
| COL1A1-F | CGGTGAACCTGGTGCTCCTG | 1 |
| COL1A1-R | GCTCCTCGCTTTCCTTCCTCTC | 2 |
| COL3A1-F | ATCTTGGTCAGTCCTATGCGG | 3 |
| COL3A1-R | TTTCTGGGTTGGGGCAGTCTA | 4 |
| FIBRONECTIN-F | AGAGGCATAAGGTTCGGGAAGAGG | 5 |
| FIBRONECTIN-R | CGAGTCATCCGTAGGTTGGTTCAAG | 6 |
| GAPDH-F | TCGGAGTCAACGGATTTGGT | 7 |
| GAPDH-R | TTCCCGTTCTCAGCCTTGAC | 8 |
As shown in FIG. 6, after the temperature-sensitive hydroxypropyl chitin was co-cultured with fibroblasts, the gene expression levels of COL1A1 (type I collagen), COL3A1 (type III collagen) and FIBRINECTIN (fibronectin) were significantly increased, and the potential of the fibroblasts for secreting extracellular matrix was not changed after the co-culture of hyaluronic acid with fibroblasts. The temperature-sensitive hydroxypropyl chitin can obviously promote the fibroblast to secrete extracellular matrix.
Example 2
This example provides a study of temperature sensitive hydroxypropyl chitin hydrogels in broken annulus seals.
The method specifically comprises the following steps:
s1, pre-cooling a contrast agent at 2-8 ℃ in advance, uniformly mixing 10mg/mL of temperature-sensitive hydroxypropyl chitin hydrogel and hyaluronic acid with the contrast agent according to a ratio of 1:1, and temporarily storing at 2-8 ℃;
S2, animal modeling, namely after the rat is anesthetized, positioning the CO7/8 intervertebral disc, puncturing the intervertebral disc by a 20G needle head for 2mm, rotating for 360 degrees, and then withdrawing, so as to damage the integrity of the intervertebral disc and break the annulus fibrosus;
S3, disc radiography, namely, re-penetrating (non-original channel) the molded disc by using a micro injector, respectively injecting 12 mu L (6 mu L of hydrogel+6 mu L of contrast agent) of the contrast agent containing the temperature-sensitive hydroxypropyl chitin hydrogel and the hyaluronic acid in the S1 into the molded disc, and checking the position of the contrast agent by using X-ray.
As shown in fig. 7, the intervertebral disc injected with hyaluronic acid and contrast agent leaks, the intervertebral disc injected with temperature-sensitive hydroxypropyl chitin hydrogel and contrast agent does not leak, and the injection solution is gathered in the center of the intervertebral disc, which indicates that the temperature-sensitive hydroxypropyl chitin hydrogel can quickly turn into a coagulated state and seal broken fibrous rings in vivo.
Example 3
The present example provides a study of the therapeutic effect of a cell complex of temperature-sensitive hydroxypropyl chitin hydrogel and fibroblasts on SD rat disc degeneration.
SD rats are taken as objects, an intervertebral disc degenerative disease model is constructed, and a cell complex of temperature-sensitive hydroxypropyl chitin hydrogel and fibroblasts (the temperature-sensitive hydroxypropyl chitin is taken as a carrier) is injected into the degenerated intervertebral disc to verify the effect of the intervertebral disc degenerative disease. The flow is shown in fig. 8. The method specifically comprises the following steps:
(1) Experimental methods and procedures
A) Grouping animals
| Group of | Grouping | Dosage and formulation |
| 1 | Control group (Normal mouse) | / |
| 2 | Degeneration group | 12 Mu L physiological saline |
| 3 | Fibroblast group | 12 Mu L of fibroblast solution |
| 4 | Thermosensitive hydroxypropyl chitin hydrogel group | 12 Mu L temperature-sensitive hydroxypropyl chitin hydrogel |
| 5 | Fibroblast + temperature sensitive hydroxypropyl chitin hydrogel group | 12 Mu L cell complex containing temperature-sensitive hydroxypropyl chitin hydrogel |
| 6 | Fibroblast + hyaluronan group | 12 Μl of cell complex containing hyaluronic acid |
The volume of the intervertebral disc injectable preparation injected into 1 SD rats was 12. Mu.L, and thus a total of 12. Mu.L of each preparation was injected.
And (2) injection:
The fibroblast solution is prepared by resuspending the fibroblasts in normal saline, wherein the number of the fibroblasts is 1 multiplied by 10 7 cells/mL, and the number of the fibroblasts in 12 mu L of the fibroblast solution is 1.2X10 5 cells/L, and the density is 1 multiplied by 10 7 cells/mL;
The cell compound containing the thermosensitive hydroxypropyl chitin hydrogel comprises the steps of re-suspending fibroblast sediment by 1mL of precooled thermosensitive hydroxypropyl chitin hydrogel with the concentration of 10mg/mL, wherein the number of the fibroblasts is 1 multiplied by 10 7 cells/mL, the number of the fibroblasts in 12 mu L of cell compound containing the thermosensitive hydroxypropyl chitin hydrogel is 1.2X10 5 cells/mL, the density is 1 multiplied by 10 7 cells/mL, the using amount of the thermosensitive hydroxypropyl chitin hydrogel is 0.12 mg/mL, and the final concentration is 10mg/mL;
The cell complex containing hyaluronic acid was prepared by resuspending a fibroblast pellet with 1mL of pre-chilled hyaluronic acid having a concentration of 10mg/mL, the number of fibroblasts was 1X 10 7 cells/mL, the number of fibroblasts in 12. Mu.L of the cell complex containing hyaluronic acid was 1.2X10 5 cells/mL, the density was 1X 10 7 cells/mL, the amount of hyaluronic acid used was 0.12 mg/mL, and the final concentration was 10mg/mL.
B) Animal model
SD rats were anesthetized, coccyx Co7/8 intervertebral discs were positioned, groups 2-6 were subjected to injection of the intervertebral discs by a 20G injection needle 2mm, then rotated 360 degrees and withdrawn for molding, and group 1 was not treated (i.e., normal mice).
C) Animal treatment
After the molding, groups 2 to 6 were re-injected (non-original) with 12. Mu.L of each formulation using a microinjector. All the preparations are stored at 2-8 ℃ before use, and are uniformly mixed by blowing and beating during use. After the injection is completed, the injection site is sterilized with iodophor.
D) Detection index
1 Month (30 days) after animal treatment, all animals were scanned by X-ray and MRI to observe the water content and height of the intervertebral disc, and the degeneration of the intervertebral disc was analyzed.
(2) Analysis of results
A) X-ray detection results:
As shown in FIG. 9, the intervertebral disc limit of the degeneration group was blurred and the intervertebral disc height was seriously lost 1 month after the operation of the animals, the intervertebral disc limit of the fibroblast + temperature sensitive hydroxypropyl chitin hydrogel group was clear and the intervertebral disc height was maintained.
As shown in figure 10, the statistical result of the height of the rat intervertebral disc is shown in figure 10, the height of the intervertebral disc of the degeneration group is obviously reduced compared with the control group, the height of the intervertebral disc of the fibroblast+temperature-sensitive hydroxypropyl chitin hydrogel group is slightly reduced, the reduction degree of the height of the intervertebral disc of the temperature-sensitive hydroxypropyl chitin hydrogel group, the fibroblast group and the fibroblast+hyaluronic acid group is more obvious compared with the reduction degree of the height of the intervertebral disc of the fibroblast+temperature-sensitive hydroxypropyl chitin hydrogel group, and the treatment of the fibroblast+temperature-sensitive hydroxypropyl chitin hydrogel proves that the height of the intervertebral disc can be well maintained and the loss of the height of the intervertebral disc is prevented.
B) MRI detection results:
The results are shown in fig. 11, and the signal intensity of the intervertebral disc of the degeneration group is obviously weakened 1 month after the operation of the animal, which indicates that the intervertebral disc is severely degenerated, and the signal of the intervertebral disc of the control group is good.
As shown in fig. 12, the disc relative signal intensity statistics result shows that the disc relative signal intensity Value (RELATIVE GRAY Value) is analyzed, the signal intensity of the disc in the degeneration group is obviously reduced compared with that of the control group, the disc signal intensity of the fibroblast+temperature-sensitive hydroxypropyl chitin hydrogel group is obviously reduced compared with that of the degeneration group, the disc signal reduction degree of the temperature-sensitive hydroxypropyl chitin hydrogel group, the fibroblast group and the fibroblast+hyaluronic acid group is more obvious compared with that of the fibroblast+temperature-sensitive hydroxypropyl chitin group, and the signal intensity of the disc is maintained by the fibroblast+temperature-sensitive hydroxypropyl chitin hydrogel through secreting extracellular matrix, so that disc degeneration is delayed.
C) Histological examination:
after 1 month of animal operation, the extracellular matrix immunofluorescence detection is carried out by taking each group of intervertebral disc tissues, and the detection is carried out by the Wohai Weibull biotechnology Co.
As shown in figure 13, the intervertebral disc morphology of the degeneration group and the independent temperature-sensitive hydroxypropyl chitin hydrogel group is lost, the boundaries of the nucleus pulposus and the annulus fibrosus are fuzzy, the intervertebral disc morphology treated by the fibroblast and the hydrogel carrier is good, and the boundaries are clear, wherein the intervertebral disc treated by the fibroblast and the temperature-sensitive hydroxypropyl chitin hydrogel detects a large amount of secretion of extracellular matrixes such as aggrecan, type I collagen and the like, and fully shows that the temperature-sensitive hydroxypropyl chitin hydrogel and the fibroblast can be combined to promote the fibroblast to secrete the extracellular matrixes, so that the intervertebral disc degeneration disease treatment method has better curative effect.
In conclusion, the thermosensitive hydroxypropyl chitin can promote fibroblasts to secrete extracellular matrixes, can seal broken fibrous rings and prevent medicine leakage. The compound of the thermosensitive hydroxypropyl chitin and the fibroblasts is injected into the degenerated intervertebral disc of rats, so that the damaged intervertebral disc can be repaired, the height and the signal intensity of the intervertebral disc can be maintained, and the degeneration of the intervertebral disc can be delayed.
Claims (10)
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