CN104667348A - Pharmaceutical composition containing sodium alginate and preparation method of pharmaceutical composition - Google Patents

Abstract

The invention provides a pharmaceutical composition containing sodium alginate for cartilage tissue repairing. As an active ingredient of the composition, bone morphogenetic protein-4 transfected fat is derived from stem cells, sodium alginate and calcium chloride; by virtue of the composition, gaps filled between cartilage rods as well as between the cartilage rods and peripheral cartilages and subchondral bones are completely filled by regenerated tissues and mechanical properties similar to that of normal cartilage are guaranteed; full fusion of mosaic bones and cartilages with peripheral cartilages and subchondral bones is achieved, and joint function improvement and level recovery in postoperative patients are significantly improved; in addition, the invention also provides a preparation method of the pharmaceutical composition.

Description

A kind of medicinal composition containing sodium alginate and its preparation method

technical field

The invention relates to a composition containing sodium alginate and a preparation method thereof, in particular to a medicinal composition containing sodium alginate which can promote bone and cartilage tissue repair.

Background technique

Intra-articular osteochondral compound injuries caused by diseases such as trauma and arthritis are very common in clinical practice. Once the cartilage tissue that occupies an important position in the joint is destroyed, it will greatly affect the normal function of the joint. The resulting vicious cycle leads to further disturbance of the internal environment of the joints, abnormal mechanical conduction leads to chain damage of normal articular cartilage, and finally leads to consequences such as restrictions on voluntary movement to varying degrees, which seriously affects the physical and mental health of the majority of patients, and also poses a threat to families and families. society is a heavy burden.

Due to the limited repair ability of cartilage after injury, it can only rely on surgical treatment, such as microfracture, drilling, and osteochondral autograft/allograft. However, there are more or less deficiencies in these treatment methods, and the ideal therapeutic effect cannot be achieved, especially for the treatment of large areas of cartilage damage, the deficiencies are more prominent.

At present, the Mosaicplasty technique (autologous osteochondral mosaic transplantation) is a widely used clinical method for repairing large-area osteochondral composite injuries. It uses autologous non-weight-bearing osteochondral column for mosaic transplantation to repair the osteochondral defect in the load-bearing area. The "integration" between the mosaic osteochondral columns and with the surrounding and basal bone and cartilage tissue is poor, which inevitably leads to the existence of many defect gaps, that is, "dead zones". Therefore, relying solely on Mosaicplasty technology cannot obtain a completely flat and continuous articular surface, and it is difficult to achieve better joint function improvement and recovery. In addition, the "donor area" (the bone harvesting area of the autologous osteochondral column) also has poor healing, resulting in pain in the "donor area" of the postoperative patient. Therefore, solving the "dead zone" between the osteochondral columns and the "donor area" of osteochondral growth and repair has become the key to the treatment of large-area osteochondral injuries.

Contents of the invention

Aiming at the shortcomings of osteochondral mosaic transplantation in the repair of large-area osteochondral injuries, the inventors have conducted a large number of scientific experimental studies and provided a medicinal product containing sodium alginate that can effectively promote the regeneration and repair of osteochondral tissue. Composition, the composition can make the gap between the filled bone columns, and the surrounding cartilage and subchondral bone be completely filled by new tissue, and has mechanical properties similar to normal cartilage, realizing the realization of mosaic osteochondral and The complete fusion of the surrounding cartilage and subchondral bone significantly improves the improvement and recovery of postoperative joint function.

The invention provides a pharmaceutical composition for osteochondral tissue repair, the active ingredient of which is: bone morphogenetic protein-4 (BMP-4) transgenic protein with a cell concentration not lower than 0.5×10 ⁷ /ml Adipose-derived stem cells (ADSCs), and sodium alginate with a content of 1-2% (w/v) and calcium chloride with a content of 0.3-0.5% (w/v).

The composition can also be called a composition containing sodium alginate for repairing osteochondral tissue or a pharmaceutical composition containing sodium alginate for repairing osteochondral tissue.

In order to solve the problems existing in the application of osteochondral mosaic grafting in the repair of large-area osteochondral injuries, the inventor once tried to apply calcium alginate gel in conjunction with this method, and the result was that the combination improved the "dead zone" phenomenon to a certain extent , but the new tissue in this part is not an osteochondral-like signal, but similar to fibrous granulation tissue, and its mechanical properties are much different from normal cartilage, which cannot meet the normal use of the repaired joint. In the process of continuous research and improvement, the inventors were pleasantly surprised to find that ADSCs transfected with BMP-4 in calcium alginate gel can significantly improve the integration effect between transplanted osteochondral and surrounding cartilage and subchondral bone. Produces hyaline cartilage-like tissue that allows the implant to integrate with bone or cartilage in the body. Thereby further improving the repair effect on cartilage damage and reducing the possibility of secondary osteoarthritis.

The composition of the present invention also contains a pH regulator, which is a commonly used pH regulator in the field, such as 1N hydrochloric acid, 1N sodium hydroxide and the like.

As we all know, calcium alginate is usually made of water-soluble alginate and calcium ions. Calcium alginate gel is viscous and has poor fluidity. The gelation speed and viscous state are related to the type, ratio and pH value of raw materials. And the speed of gelation and its strength are crucial for the field of application of the present invention. On the one hand, the viscous state of the cross-linked calcium alginate gel makes it unable to meet the filling requirements of the small, complex and irregular spaces between the mosaic cartilage and its own bone/cartilage. On the other hand, if its strength does not meet the requirements, the gel will be broken, and it will not be able to provide a growth scaffold for the seed cells carrying the target gene, and it will be difficult to well fix and support the implanted bone column, which will directly affect the operation. Effect.

In order to take into account factors such as the crosslinking (gel) speed, the strength of the gel, and the negative impact of excessive calcium ions on cells, the inventor obtained the combination of sodium alginate and calcium chloride described in the composition of the present invention through repeated tests. Ratio, and suitable pH range, that is, 1-2% sodium alginate and 0.3-0.5% calcium chloride by weight, and a pH regulator for adjusting the pH value of the composition to 7.35-7.45. The gelation time and gel strength are moderate after the two parts of this ratio are mixed, and the crosslinking can be completed in about 20-30 seconds. Moreover, the composition of the present invention is applied by mixing the sodium alginate part and the calcium chloride part before use and injecting it into the body immediately. It takes about 10-15 seconds from the mixing to the completion of the injection. The mixture at this time It is still in a relatively thin state, and can achieve the purpose of completely filling any small and far-reaching gaps in the surgical wound, and there is no need to wait deliberately after the filling is completed. The composition has become a gel with a certain strength, and can be sutured. Even if all the gaps are filled completely without prolonging the wound opening time, and the gel has appropriate strength, it is enough to protect the position and shape of the implanted cartilage column, and a small external force will not cause its deformation, providing a scaffold and suitable for the early growth of cells. growth environment.

ADSCs transfected with BMP-4 can continue to secrete cytokines in vivo for a long time (about 4-6 weeks), stimulate their division and proliferation, start and accelerate the repair process, improve the pathological environment in the joint, and enhance the quality of repair; while ADSCs The bidirectional differentiation potential enables it to differentiate into corresponding chondrocytes or osteocytes in different microenvironments of the joint cavity and the subchondral bone bed.

The calcium alginate gel of ADSCs transfected with BMP-4 was injected into the "dead zone". While fixing and protecting the implanted cartilage column, the gel provided a three-dimensional growth environment similar to the physiological state for the implanted stem cells. Evenly distributed in the gel, it can exchange nutrients and metabolites. Stimulated by the strain force of the gel, it is conducive to proliferation and differentiation and the formation of extracellular matrix. Finally, the new cartilage tissue will completely fill up the "dead area", so that the implanted part can be compared with the autologous body. The integration greatly improves the operability and success rate of one-stage complete repair of osteochondral compound tissue damage, and more importantly, significantly improves the prognosis of patients with large-scale cartilage damage and improves the quality of life of such patients.

The composition of the present invention is used in conjunction with osteochondral mosaic transplantation for 2-3 months, and new cartilage-like tissue will form on the edge of the cartilage column. There are a large number of chondrocytes in the gel part, and this part is combined with the implanted cartilage column and autologous cartilage. Firm, good joint repair. In addition, the inventors also directly apply the composition of the present invention to the filling of small or tiny areas of cartilage damage, which also achieves a good repair effect.

It should be noted that the adipose-derived stem cells transfected with bone morphogenetic protein-4 used in the present invention are cells prepared within 48 hours, that is, the transfection step is completed within 48 hours before the composition is used, preferably prepared within 24 hours Finish.

In order to make the composition of the present invention have an osmotic pressure compatible with the human body and a constant pH value, the composition also contains the following auxiliary materials: 0.5-0.7% sodium chloride, and 0.3-0.5% 4 - Hydroxyethylpiperazineethanesulfonic acid (HEPES).

The experimental results show that the content of sodium alginate in the composition directly affects the growth effect of cells (fat-derived stem cells and chondrocytes), and this effect is not a simple proportional or inverse relationship, but within a specific range, The content of sodium alginate in the composition is preferably 1.4-1.8%.

The concentration of the BMP-4-transfected adipose-derived stem cells (ie, BMP-4-transfected ADSCs) is preferably (1˜2)×10 ⁷ /ml.

The composition of the present invention is adipose-derived stem cells transfected with bone morphogenetic protein-4 at a cell concentration of (1-1.5)×10 ⁷ /ml, and the content is 1.6-1.7% sodium alginate, 0.35-0.45 % calcium chloride, 0.5-0.7% sodium chloride, 0.3-0.5% 4-hydroxyethylpiperazineethanesulfonic acid and an appropriate amount of pH regulator. The application state of the composition is a gel agent, that is, the sodium alginate solution part and the calcium chloride solution part are mixed to undergo a crosslinking reaction to form a calcium alginate gel.

Histological and biomechanical test results prove that the composition of the present invention cooperates with Mosaicplasty technology to repair cartilage defects, and the regenerated tissue is no different from normal articular cartilage in terms of tissue structure and functionality (biomechanics), achieving an unprecedented repair effect. Further perfected the large area cartilage defect repair technology.

Another object of the present invention is to provide a preparation method of the composition, the method comprising the following steps:

(1) Extracting and preparing bone morphogenetic protein-4 transfected adipose-derived stem cells;

(2) Preparation of sodium alginate solution;

(3) prepare calcium chloride solution;

(4) suspending the cells obtained in step (1) in the sodium alginate solution obtained in step (2) before use;

(5) Mix the products of step (4) and step (3) at a volume ratio of 2:1 to obtain the composition of the present invention.

Step (1) described in the above-mentioned preparation method comprises:

a. Extraction, isolation and culture of autologous adipose tissue stem cells, and cells of 3-10 generations are taken;

b. bone morphogenetic protein-4 recombinant adenovirus;

c. BMP-4 recombinant adenovirus transfected adipose-derived stem cells in vitro;

d. To detect the transfection effect, take the cells with a transfection rate greater than 70% and store them at -65--75°C for later use.

The methods for extracting, isolating and culturing the adipose tissue stem cells in the above step a are well-known techniques in the art, and various commonly used methods can be used. If the following method is used:

Autologous or allogeneic adipose tissue from the groin. Rinse with a large amount of PBS solution to remove surface blood, cut into pieces with ophthalmic scissors in a 1:1 configuration of PBS and 0.1% type Ⅰ collagenase, digest at 37°C for 30 minutes, neutralize the digestion solution with DMEM containing 10% fetal bovine serum, take Centrifuge at 300g for 5min, discard the suspension containing mature adipocytes, add 160mM NH4Cl erythrocyte lysate to the sediment containing adipose tissue-derived stem cells, let it stand at room temperature for 10min, pass it through a 200-lm nylon sieve, collect the filtrate, Use DMEM medium containing 10% FBS, 1% blue chain double antibody in 37 ℃/5% CO2 incubator overnight. The primary cells were cultured for 4-5 days, and after they grew confluently, they were subcultured at a ratio of 1:3. The primary cells were marked as passage 0, and the cells used in the experiment were passages 3-10. The isolated cells have differentiation into fat and bone cells.

The recombination and transfection methods in the above steps b and c are also known in the art, and various common methods can be used. The transfection method can adopt the following method: the third-generation adipose tissue stem cells are planted in 6-well plates in equal amounts, and after reaching 90% confluence, the growth medium is discarded, and cells are digested in 3 wells and counted. Calculate the amount of virus (bone morphogenetic protein-4 recombinant adenovirus) required for each well according to MOI=100, 250, and 400, and let the stored virus spontaneously thaw on ice, then add different amounts of virus to 400 μl serum-free in DMEM solution. Discard the complete culture medium in the 6-well plate, wash it twice with PBS, then add the virus solution, shake the culture plate in a cross shape to allow the virus solution to fully contact the cells, incubate at 37°C for 3 hours, and shake the culture plate every 20 minutes during the period. After 3 hours, aspirate the virus solution and add 1ml of complete culture solution to each well to continue culturing.

In order to improve the chondrocyte differentiation ability and cartilage repair ability of the composition of the present invention, BMP-4 transfected ADSCs with a compound transfection rate greater than 85% in calcium alginate gel are preferred.

The step (2) of the preparation method is specifically: dissolving sodium chloride and 4-hydroxyethylpiperazineethanesulfonic acid in deionized water with a total volume of 70-95%, heating to 50-80°C, adding sodium alginate , stirred to obtain a homogeneous solution, placed at room temperature, added a pH regulator to adjust the pH value to 7.35-7.45, made to volume with deionized water, and set aside.

The step (3) of the preparation method is specifically: take calcium chloride and 4-hydroxyethylpiperazineethanesulfonic acid, add 50-95% deionized water to make a solution, adjust the pH value to 7.35-7.45, and add deionized water Constant volume, filter with 0.22μm filter membrane, set aside.

Wherein, the concentration of 4-hydroxyethylpiperazineethanesulfonic acid in the sodium alginate solution obtained in step (2) and the calcium chloride solution obtained in step (3) is about 2:1.

The cell suspension method in step (4) of the preparation method is a conventional method to make the cell concentration reach above 0.75×10 ⁷ /ml.

According to the method described in the step (5), the composition of the present invention is obtained after mixing evenly. After mixing, the alginate starts to cross-link immediately, and the mixture should be injected into the bone suture immediately, and it can be sutured after filling.

The present invention also provides the application of the composition in the preparation of osteochondral tissue repair medicine and/or medical material.

The inventor further verified the functional effect of the product of the present invention through specific experiments.

Reiterate again: the following experiment is just an exemplary experiment in many experiments in the development process of the present invention, and does not cover and exhaust all experiments done by the inventor for the present invention. The purpose is only to illustrate the beneficial effect of the product of the present invention with those data.

Experimental method: Taking miniature pigs as the research object, an osteochondral defect with a diameter of 8 mm and a depth of 5 mm was made in the load-bearing area of the medial femoral condyle; three osteochondral columns with a diameter of 3.5 mm and a length of 5 mm were respectively taken from the non-functional area of the femoral trochlear edge and filled with Mosaicplasty technology Most of the defect area. The experiment was randomly divided into 3 groups for restoration. Scanning electron microscopy, histological scoring and biomechanical observation were performed at 12 and 24 weeks after operation. Quantitative results were expressed as mean ± standard deviation, and SPSS software was used for statistical analysis using Mann–Whitney test, and P<0.05 was considered statistically significant

Group a: pure Mosaicplasty group;

Group b: Mosaicplasty+calcium alginate gel group;

Group c: Mosaicplasty+composite BMP-4 transfected ADSCs calcium alginate gel (composition of the present invention described in Example 1) group.

1. Scanning electron microscopy

Inspection method: After the animals were killed, the repaired cartilage defects were quickly removed and rinsed with normal saline. Put into pre-cooled 2% glutaraldehyde solution for fixation for 8 hours. Wash thoroughly with distilled water, and fix with 1% osmium tetroxide for 1 hour. Aspirate the fixative, add distilled water to wash thoroughly for 1 hour, and change the distilled water every 15 minutes. Aspirate the distilled water, dehydrate with ethanol step by step, aspirate the ethanol, add isoamyl acetate ethanol mixture (the ratio of isoamyl acetate to ethanol is 1:1) after 20 minutes, inhale the liquid, and add pure isoamyl acetate for 30 minutes. Blot the surface of the sample with isoamyl acetate on the filter paper, transfer the sample to a critical point dryer, and dry it at a critical state of 31°C and 72.8 atmospheres. The surface is gold-plated by ion sputtering. Scanning electron microscope observation.

Inspection results: (see attached picture 1)

At 12 weeks, the boundary between the transplanted osteochondral column and its surroundings was clear in group a, and the deformation of the osteochondral column was obvious (Fig. The arrangement of tissue fibers was disordered, and there were still gaps between the osteochondral column and its surroundings (Fig. 1-b1); in group c, the deformation of the osteochondral column was small, and there was new cartilage-like tissue at the edge of the cartilage column, and the surface of the new tissue was similar to the surface of the cartilage column. The cartilage columns were not fully fused, but the gap was significantly smaller than the former two groups (Fig. 1-c1);

At 24 weeks, the gaps between the transplanted osteochondral columns and the surrounding normal cartilage tissue in group a were clear, and the deformation of the osteochondral column was obvious (Fig. 1-a2); in group b, the gap between the transplanted osteochondral columns and the surrounding normal cartilage tissue Some fissures were basically filled up by the regenerated tissue, but the surface was uneven, the fibers were not arranged neatly, and there were deep grooves (Fig. 1-b2). In group c, the deformation of the osteochondral columns was small, the gaps between the cartilage columns disappeared, the osteochondral tissues were fully fused, and the surface was continuous and complete. The surface of the regenerated tissue was uniform and fibrous, relatively flat, and the fibers were arranged neatly, similar to normal cartilage. (Fig. 1-c2).

The above results show that: only using the method of group a, the research object cannot complete the fusion growth between the implanted part and itself, the injected part is easy to shift and deform, and the surgical effect is very unsatisfactory; although the effect of group b is improved compared with group a, However, the fusion is poor and the stability is poor, which cannot meet the stimulation brought by normal activities; while the group c using the composition of the present invention regenerates cartilage-like tissue and realizes complete fusion between osteochondral columns.

2. Histological examination

Inspection method: After the animals were anesthetized, the specimens were taken out from the implanted sites, and the redundant soft tissues were carefully removed, rinsed with PBS, and fixed in 4% paraformaldehyde for 48 hours. Take out the specimen from the fixative, rinse with tap water for 20 minutes, and wash with distilled water twice. The ascending gradient alcohol was dehydrated sequentially, soaked in wax after being transparent in xylene, embedded to make a wax block and sectioned, and observed by H.E., toluidine blue staining and type II collagen staining.

Inspection results: (see attached picture 2)

At 12 weeks: in group a, there was no new cartilage at the junction of the transplanted osteochondral columns, a very small amount of fibrous tissue filled the subchondral bone, and there were gaps between the transplanted cartilage columns, which failed to fuse (Fig. 2-a1); in group b, the transplanted cartilage could be seen The subchondral bone between the cartilage columns healed well, and fibrous tissue filled the fissures between the cartilage tissues still existed (Fig. 2-b1). Both toluidine blue and type II collagen staining were negative. In group C, it can be seen that the junction of grafted osteochondral column is repaired by cartilage-like tissue, the surface is relatively smooth, and there are a large number of chondrocytes in the cartilage lacuna, arranged in columns or clusters; endochondral ossification can be seen in deep cartilage, subchondral bone Partial reconstruction; there is no cell loss zone at the junction with the surrounding normal cartilage, but the boundary is obvious (Fig. 2-c1). Both toluidine blue and type II collagen staining were positive.

At 24 weeks: in group a, the gap at the junction of the transplanted osteochondral column was narrowed and filled with a small amount of fibrous tissue, and the gap still existed (Fig. 2-a2). Toluidine blue staining and type II collagen staining were negative. In group b, it can be seen that the fissure at the junction of the transplanted osteochondral column was repaired by a mixture of fibrocartilage-like tissue, the surface was uneven, and there were chondrocytes and fibroblasts scattered in the regenerated tissue (Fig. 2-b2). Toluidine blue, Masson staining and type II collagen staining were weakly positive. In group c, it can be seen that the junction of the transplanted osteochondral column is repaired by cartilage tissue, the surface is smooth, and there are a large number of chondrocytes inside. The superficial cells are parallel to the surface, and the deep cells are arranged in columns, tending to be normal. The lower tide line of cartilage can be seen; it is different from the surrounding normal cartilage There is no cell defect zone at the junction, and the boundary is unclear (Fig. 2-c2). Toluidine blue staining, Masson staining and type II collagen staining were all positive.

The above results indicated that only the new repair tissue in group C had the histological characteristics of normal cartilage.

The histological score adopts the O’Driscol scoring standard, and the higher the score, the better the repair effect, and the full score is 24 points. Among them, at 12 weeks, the score of group a was 5.2±1.60, the score of group b was 8.8±3.12, and the score of group c had reached 16.2±2.63; at 24 weeks, the score of group a was 7.2±1.32, and the score of group b was 12±3.12 4.56, while the score of group c reaches 21.4±1.35, which is close to the full score.

The above results indicated that the new repair tissue in group c was histologically close to normal tissue.

3. Biomechanical testing

Inspection method: nanoindentation biomechanical assay

Three indicators are mainly used to evaluate the biomechanical properties of the repaired tissue, including contact stiffness (contact stiffness), reduced modulus (elastic modulus), and hardness (hardness). Contact stiffness is mainly used to evaluate the ability of repair tissue to resist deformation caused by external force. The elastic modulus is mainly used to evaluate the elasticity of the repaired tissue. The hardness is an index for evaluating the hardness of the repaired tissue.

The experimental results showed (see Figure 3-6), the mechanical properties of the repaired tissue in group c were significantly better than those of the other two groups in these three indexes, and were close to normal cartilage. Although the mechanical properties of the repaired tissue in b are stronger than those in group a, they are far from those in group c. The biomechanical properties of repaired tissue in groups a and b were far from normal cartilage and filling areas, and could not meet the needs of cartilage repair. It shows that the composition of the present invention can continuously induce and promote the differentiation of regenerated tissue to osteochondral, and restore the histological and functional characteristics of the osteochondral defect area in a short period of time.

In order to verify the functional effect of the composition of the present invention, the inventor is carrying out repeated experiments. Unlike the above experiments, the dosage of each component and the concentration of cells are different (including Examples 2-6), and the parameters in the preparation method are fine-tuned . Wherein, the floating range of the dosage (w/v) of each component is: 1-2% sodium alginate, 0.3-0.5% calcium chloride, 0.5-0.7% sodium chloride, and 0.3-0.5% 4-hydroxyethylpiperazineethanesulfonic acid; the concentration range of ADSCs transfected with BMP-4 is (0.5-2)×10 ⁷ /ml.

The results of completed examples 1-3 show that after adjusting the dosage of each component and the cell concentration within a certain range, it still has a good effect of promoting osteocartilage tissue repair. The data of statistical analysis on this basis shows that the compositions within the above floating range have similar effects on promoting the repair of osteochondral tissue.

Due to limited space, the methods, steps and related data of the above experiments will not be repeated here.

Description of drawings

Figure 1: Atlas of scanning electron microscopy, where a, b, and c represent groups respectively, the first row is 12-week-old animal specimens, and the second row is 24-week-old animal specimens;

Figure 2: HE staining pattern of histological examination, where a, b, and c represent groups respectively, the first row is 12-week-old animal specimens, and the second row is 24-week-old animal specimens; N is normal cartilage, R is new cartilage;

Figure 3: Biomechanical examination of group a, Normal is the normal cartilage area, Mosaic is the transplanted osteochondral area, and blank is the defect area during surgery;

Figure 4: Biomechanical examination of group b, Normal is the normal cartilage area, Mosaic is the transplanted osteochondral area, and Alginate is the area filled with calcium alginate gel during surgery;

Figure 5: Biomechanical examination of group c, Normal is the normal cartilage area, Mosaic is the transplanted osteochondral area, Alginate+ADSC is the area filled with the composition of the present invention during operation.

Figure 6: Biomechanical comparison between the three groups.

Detailed ways

Example 1:

150ml composition formula:

Preparation:

(1) Extracting and preparing bone morphogenetic protein-4 transfected adipose-derived stem cells;

Obtained from fat tissue in the groin of the body. Rinse with a large amount of PBS solution to remove surface blood, cut into pieces with ophthalmic scissors in a 1:1 configuration of PBS and 0.1% type Ⅰ collagenase, digest at 37°C for 30 minutes, neutralize the digestion solution with DMEM containing 10% fetal bovine serum, take Centrifuge at 300g for 5min, discard the suspension containing mature adipocytes, add 160mM NH4Cl erythrocyte lysate to the sediment containing adipose tissue-derived stem cells, let it stand at room temperature for 10min, pass it through a 200-lm nylon sieve, collect the filtrate, Use DMEM medium containing 10% FBS, 1% blue chain double antibody in 37 ℃/5% CO ₂ incubator overnight. The primary cells were cultured for 4-5 days, and after they grew confluently, they were subcultured at a ratio of 1:3. The primary cells were marked as passage 0, and the cells used were passage 3.

Transfection method (operate within 24 hours before the composition is used): the third generation adipose tissue stem cells were planted in 6-well plates in equal amounts, and after reaching 90% confluence, the growth medium was discarded, and the cells were digested and counted in 3 wells. Calculate the amount of virus (bone morphogenetic protein-4 recombinant adenovirus) required for each well according to MOI=100, 250, and 400, and let the stored virus spontaneously thaw on ice, then add different amounts of virus to 400 μl serum-free in DMEM solution. Discard the complete culture medium in the 6-well plate, wash it twice with PBS, then add the virus solution, shake the culture plate in a cross shape to allow the virus solution to fully contact the cells, incubate at 37°C for 3 hours, and shake the culture plate every 20 minutes during the period. After 3 hours, the virus liquid was discarded and 1ml of complete culture medium was added to each well to continue culturing.

The ADSCs transfected with BMP-4 with a transfection rate greater than 85% were stored at about -70°C for future use.

⑵Preparation of sodium alginate solution: (100ml)

Weigh 0.48g of HEPES and 0.88g of sodium chloride, dissolve them in 80ml of deionized water, heat to 60°C, keep warm, add 2.5g of sodium alginate, and keep stirring until the solution is evenly mixed. Adjust the pH to 7.4 with 1N hydrochloric acid, add deionized water to 100ml, and set aside.

(3) Preparation of calcium chloride solution: 50ml

Weigh 0.57g of calcium chloride and 0.12g of HEPES, dissolve them in 40ml of deionized water, add 1N hydrochloric acid dropwise to make the pH value 7.4, add deionized water to 50ml, filter through a 0.22μm filter membrane, and set aside.

(4) Suspend the cells obtained in step (1) in the sodium alginate solution obtained in step (2) before use. Specifically: digest the cells from the culture dish with trypsin, neutralize the trypsin with serum, centrifuge the supernatant, and re-spin the centrifuged cells with the medium. For specific steps, refer to cell culture; make the cell concentration reach 2×10 ⁷ /mL.

(5) Mix the product of step (4) and step (3) according to the volume ratio of 2:1, and obtain the composition of the present invention after mixing. In practical application, the composition of the present invention should be injected into the filling area of the body immediately after mixing, and the cross-linking is basically completed after the injection, and it is in a gel state.

Example 2:

150ml composition formula:

Preparation:

The difference from Example 1 is:

(1) Extract and prepare adipose-derived stem cells transfected with bone morphogenetic protein-4; the primary cells are marked as passage 0, and the cells used are passage 5; ADSCs transfected with BMP-4 with a transfection rate greater than 90% were obtained at about -65 Store under the environment of ℃ (the transfection step is carried out within 48 hours before the application of the composition), and it is ready for use.

⑵Preparation of sodium alginate solution: (100ml)

Weigh 0.6g of HEPES, 0.75g of sodium chloride, dissolve in 70ml of deionized water, heat to 70°C, keep warm, add 2.1g of sodium alginate, keep stirring until the solution is evenly mixed, after cooling to room temperature, add 1N Hydrochloric acid, adjust the pH to 7.35, add deionized water to 100ml, set aside.

(3) Preparation of calcium chloride solution: 50ml

Weigh 0.45g of calcium chloride and 0.15g of HEPES, dissolve them in 47.5ml of deionized water, add 1N hydrochloric acid dropwise to make the pH value 7.35, add deionized water to 50ml, filter through a 0.22μm filter membrane, and set aside.

(4) Suspend the cells obtained in step (1) in the sodium alginate solution obtained in step (2) before use; make the cell concentration reach 1.5×10 ⁷ /mL.

Example 3:

150ml composition formula:

Preparation:

The difference from Example 1 is:

(1) Extract and prepare adipose-derived stem cells transfected with bone morphogenetic protein-4; the primary cells are marked as passage 0, and the cells used are passage 7; ADSCs transfected with BMP-4 with a transfection rate of more than 70% are taken at about -75 Store in ℃ environment, spare

⑵Preparation of sodium alginate solution: (100ml)

Weigh 0.36g of HEPES, 1.05g of sodium chloride, dissolve in 95ml of deionized water, heat to 50°C, keep warm, add 2.7g of sodium alginate, keep stirring until the solution is evenly mixed, after cooling to room temperature, add 1N Adjust the pH to 7.45 with hydrochloric acid, add deionized water to 100ml, and set aside.

(3) Preparation of calcium chloride solution: 50ml

Weigh 0.75g of calcium chloride and 0.09g of HEPES, dissolve them in 25ml of deionized water, add dropwise 1N hydrochloric acid to make the pH value 7.45, add deionized water to 50ml, filter through a 0.22μm filter membrane, and set aside.

(4) Suspend the cells obtained in step (1) in the sodium alginate solution obtained in step (2) before use; make the cell concentration reach 0.75×10 ⁷ /mL.

Example 4:

150ml composition formula:

The difference between the preparation method and Example 1 is:

(1) Extract and prepare adipose-derived stem cells transfected with bone morphogenetic protein-4; the primary cells are marked as the 0th generation, and the cells used are the 10th generation; the ADSCs transfected with BMP-4 with a transfection rate greater than 80% were obtained at about -70 Store in ℃ environment, spare

⑵Preparation of sodium alginate solution: (100ml)

Weigh 0.46g of HEPES and 0.9g of sodium chloride, dissolve them in 90ml of deionized water, heat to 80°C, keep warm, add 2.4g of sodium alginate, keep stirring until the solution is evenly mixed, after cooling to room temperature, add hydrochloric acid dropwise to adjust pH value to 7.4, add deionized water to 100ml, set aside.

(3) Preparation of calcium chloride solution: 50ml

Weigh 0.6g of calcium chloride and 0.12g of HEPES, dissolve them in 35ml of deionized water, add hydrochloric acid dropwise to make the pH value 7.4, add deionized water to 50ml, filter through a 0.22μm filter membrane, and set aside.

(4) Suspend the cells obtained in step (1) in the sodium alginate solution obtained in step (2) before use; make the cell concentration reach 3×10 ⁷ /mL.

Example 5:

150ml composition formula:

Preparation:

The difference from Example 1 is:

(1) Extract and prepare adipose-derived stem cells transfected with bone morphogenetic protein-4; the primary cells are marked as generation 0, and the cells used are the 4th generation; BMP-4 transfected ADSCs with a transfection rate greater than 90% were obtained at about -70 Store in ℃ environment, spare

(4) Suspend the cells obtained in step (1) in the sodium alginate solution obtained in step (2) before use; make the cell concentration reach 2.25×10 ⁷ /mL.

Embodiment 6:

150ml composition formula:

The preparation method is the same as in Example 1.

The present invention is not limited to the above-mentioned embodiments, and any other products identical or similar to the present invention obtained by anyone under the inspiration of the present invention are not excluded from the protection scope of the present invention.

Claims (10)

1. contain a Pharmaceutical composition for sodium alginate for osteochondral tissue reparation, it is characterized in that, the active component of said composition is: cell concentration is not less than 0.5 × 10 ⁷the adipose-derived stem cells of the BMP-4 transfection of/ml, and content is the calcium chloride of the sodium alginate and 0.3 ~ 0.5% (w/v) of 1 ~ 2% (w/v).

2. compositions as claimed in claim 1, it is characterized in that, this compound also comprises the adjuvant of following content: the sodium chloride of 0.5 ~ 0.7% (w/v), and the 4-hydroxyethyl piperazine ethanesulfonic acid of 0.3 ~ 0.5% (w/v).

3. compositions as claimed in claim 1 or 2, it is characterized in that, the content of described sodium alginate is 1.4 ~ 1.8%.

4. compositions as claimed in claim 1 or 2, it is characterized in that, the concentration of the adipose-derived stem cells of described BMP-4 transfection is (1 ~ 2) × 10 ⁷/ ml.

5. the compositions as described in claim 1-4 arbitrary, is characterized in that, said composition is (1 ~ 1.5) × 10 by cell concentration ⁷the adipose-derived stem cells of the BMP-4 transfection of/ml, content is the sodium alginate of 1.6 ~ 1.7%, calcium chloride, the sodium chloride of 0.5 ~ 0.7%, the 4-hydroxyethyl piperazine ethanesulfonic acid of 0.3 ~ 0.5% and the qs pH adjuster of 0.35 ~ 0.45% is made.

6. the preparation method of compositions according to claim 1, is characterized in that, the method comprises the following steps:

(1) extract and prepare the adipose-derived stem cells of BMP-4 transfection;

(2) prepare sodium alginate soln;

(3) prepare calcium chloride solution;

(4) in the sodium alginate soln that (2) cell suspension step (1) obtained before use obtains in step;

By step (4) with the ratio mix homogeneously of step (3) gains 2:1 by volume, obtain the present composition.

7. preparation method as claimed in claim 6, it is characterized in that, (1) described step comprises:

A. the extracting and developing of Adipose Tissue and cultivation, get 3 ~ 10 generation cell;

B. BMP-4 recombinant adenovirus;

C. BMP-4 recombinant adenovirus in-vitro transfection adipose-derived stem cells;

D. detect transfection, get the cell that transfection efficiency is greater than 70% and store under-65 ~-75 DEG C of environment, for subsequent use.

8. preparation method as claimed in claim 6, it is characterized in that, (2) described step is specially: be dissolved in the deionized water of 70 ~ 95% by sodium chloride, 4-hydroxyethyl piperazine ethanesulfonic acid, be heated to 50 ~ 80 DEG C, insulation, add sodium alginate, stir, obtain uniform solution, place room temperature, add pH adjusting agent adjust ph to 7.35 ~ 7.45, use deionized water standardize solution, for subsequent use.

9. preparation method as claimed in claim 6, it is characterized in that, (3) described step is specially: get calcium chloride and 4-hydroxyethyl piperazine ethanesulfonic acid, add 50 ~ 95% deionized waters and make solution, adjust ph to 7.35 ~ 7.45, deionized water standardize solution, 0.22 μm of membrane filtration, for subsequent use.

10. compositions according to claim 1 is preparing the application in osteochondral tissue repair medicine and/or medical material.