CN112933296A

CN112933296A - Injection type fibrin gel regeneration material capable of loading cells

Info

Publication number: CN112933296A
Application number: CN202110112137.6A
Authority: CN
Inventors: 江飞; 周芷萱; 谭俊灵; 沈铭; 杨益
Original assignee: Affiliated Stomatological Hospital of Nanjing Medical University
Current assignee: Affiliated Stomatological Hospital of Nanjing Medical University
Priority date: 2021-01-27
Filing date: 2021-01-27
Publication date: 2021-06-11

Abstract

The invention provides a preparation method and application of injection type cell-loaded fibrin gel for inducing tissue regeneration. The invention provides an injection type regeneration repair material prepared by wrapping seed cells capable of promoting regeneration by using a fibrin solution capable of being catalyzed into gel by thrombin. The injectable gel material has good biocompatibility, expansibility, simple operation, and rapid gelling process, and can effectively fill irregular defects. Is beneficial to the local survival and colonization of the carried stem cells and promotes the regeneration of tissues.

Description

Injection type fibrin gel regeneration material capable of loading cells

Technical Field

The invention belongs to the field of tissue engineering and regenerative medicine, and particularly relates to a preparation method and application of an injection type fibrin gel regenerative material capable of loading cells.

Background

The current clinical study of bone defect repair tends to use tissue engineering strategies to replace autologous bone grafts. Calcium-phosphorus bulk scaffold materials or granular scaffold materials are commonly used, but the materials have certain osteo-guidance property and lack osteo-inductivity; the finished product block material has large brittleness and is not easy to shape, and the granular material is loose and is not easy to shape; meanwhile, the defects of complex operation of loading factors and seed cells, low loading rate and the like are not beneficial to the effective osteogenesis of the bone defect area with complex structure defect (lacunar property). Therefore, injectable or hydrogel materials have been the focus of research because they can compensate for the above-mentioned drawbacks.

Fibrin is marketed as a finished product (Sigma company) and can be used for research, and a patent (publication number: CN2824875Y) discloses an injection type tissue engineering bone graft prepared by applying fibrin gel to load autologous Platelet Rich Plasma (PRP) and Bone Marrow Mesenchymal Stem Cells (BMMSCs). But the acquisition mode of PRP and BMMSCs is invasive, and the operation procedure is more complicated. The overall number of BMMSCs is extremely limited in bone marrow, and the isolation process is susceptible to contamination by peripheral blood cells, and BMMSCs lose some of their stem cell characteristics as donors age. Therefore, the search for a better way to obtain growth factors by selecting other suitable seed cells is a problem to be solved urgently.

The placenta is usually abandoned as medical waste after the delivery of the pregnant women, has sufficient sources and easy acquisition, and the acquisition does not violate ethical principles. Because the placenta tissue plays a key role in maternal/fetal immune tolerance, the amniotic mesenchymal stem cells (hAMSCs) derived from the placenta tissue have no obvious immune rejection and have possibility of allograft transplantation. hAMSCs have the capacity of multipotential differentiation as stem cells and can secrete various growth factors such as: angiogenesis promoting factors and chemokines such as platelet growth factor (PDGF), angiopoietin 1(Ang-1), stromal cell derived factor (SDF-1), interleukin 8 (IL-8). After the hAMSCs are transplanted at the bone defect part, autologous osteogenesis related cells can be recruited through the paracrine effect to carry out effective vascularized bone repair, so that the hAMSCs can be used for simultaneously delivering seed cells and growth factors, and the technical difficulty and high cost caused by additional loading of the growth factors are avoided. However, when hAMSCs are simply applied to the local bone defect, cells are easy to lose. The hAMSCs are loaded on fibrin gel and injected into the bone defect to solve the problem of cell loss. Currently, no report is found on the application of the fibrin gel loaded hAMSCs in vascularized bone regeneration.

Disclosure of Invention

The first purpose of the invention is to provide a concentration optimization scheme of the injection type fibrin gel regeneration material capable of loading cells.

The second purpose of the invention is to provide an optimization scheme of the cell loading density of the injection type cell-loadable fibrin gel regeneration material.

The third purpose of the invention is to provide the application of the injection type fibrin gel loaded hAMSCs in bone regeneration.

In order to achieve the above object of the present invention, the present invention describes the following technical solutions:

1. an injectable fibrin gel regenerative material capable of loading cells is formed by enzyme catalysis of cell suspension containing stem cells and fibrin gel.

2. The injectable cell-loadable fibrin gel regeneration material according to 1, wherein the cell suspension is a cell suspension containing hAMSCs; optionally, the enzyme is thrombin.

3. The injectable cell-loadable fibrin gel regeneration material according to any one of claims 1-2, wherein the concentration of the fibrin gel is 1mg/ml to 10 mg/ml.

4. The injectable cell-loadable fibrin gel regeneration material according to 3, wherein the concentration of the fibrin gel is 5 mg/ml.

5. The injectable cell-loadable fibrin gel regeneration material according to any one of claims 1-2, wherein the concentration of the cells is 2 x 10⁶One/ml to 10X 10⁶One per ml.

6. Injectable loadable according to 5Fibrin gel regeneration material of cells with concentration of 2 × 10⁶One per ml.

7. A pharmaceutical composition comprising an injectable cell-loadable fibrin gel regeneration material according to any one of claims 1-6.

8. A preparation method of an injection type fibrin gel regeneration material capable of loading cells is characterized by comprising the following steps:

culturing the cell;

digesting and centrifuging the cells, and carrying out heavy suspension by using a culture solution to obtain a cell suspension;

and carrying out enzyme catalysis on the cell suspension and the fibrin gel to obtain the regenerated material.

In a specific embodiment, the preparation method of the injection type cell-loadable fibrin gel regeneration material comprises the following steps:

obtaining and culturing mesenchymal stem cells;

digesting and centrifuging the mesenchymal stem cells obtained in the step, then re-suspending the mesenchymal stem cells by using a culture solution, mixing the obtained cell suspension with 5mg/ml fibrin gel solution 1:1, dripping 50U/ml thrombin, using a syringe for injection, and carrying out catalytic gelling by standing 5 mul thrombin at 37 ℃ for 5min for every 100 mul fibrin gel solution.

9. The method according to claim 8, wherein the cell suspension is a cell suspension containing hAMSCs; optionally, the enzyme is thrombin.

10. Use of an injectable cell-loadable fibrin gel regeneration material according to any one of claims 1-6 or a composition according to 7 for the manufacture of a medicament for promoting bone regeneration or treating a bone defect.

The hAMSCs-loaded fibrin gel can repair bone defects through injection alone or in combination with a traditional bracket, and meanwhile, the injection mode can reduce surgical trauma, simplify the implantation mode of bone grafting materials, and avoid complications such as wound infection and the like.

In one particular embodiment, the present invention first optimizes the fibrin gelThe gel forming time under the catalysis of thrombin is determined to be preferably 1-3min according to the convenience of clinical operation. After the proper concentration of the fibrin gel is determined, the density of the loaded cells is optimized, and the cell density is finally determined to be 2 x 10 because the cell density is too high and can influence the gelatinization of the fibrin gel⁶One per ml. And finally, verifying the bone regeneration effect of the injection type fibrin gel regeneration material capable of loading cells on a rabbit skull defect model.

The invention has the advantages that: the invention provides a concept and a preparation method of an injection type fibrin gel regeneration material capable of loading cells for promoting tissue regeneration, on one hand, the injection mode can reduce surgical trauma, simplify the implantation mode of a bone graft material and avoid complications such as wound infection and the like; on the other hand, the gel is used for wrapping the local planting of the defect of the seed cell and protecting the local survival of the seed cell, thereby being beneficial to the seed cell to play a role in the defect area and promoting the regeneration of local tissues; finally, the injection type fibrin gel regeneration material capable of loading cells is applied to a rabbit skull defect model, so that newly-grown bones in a defect area are obviously increased, and the repair effect is obviously improved. The invention does not need special, complex and expensive equipment, has simple operation flow and is beneficial to popularization and use.

Drawings

FIG. 1: thrombin catalytic doses are preferred. A50U/ml thrombin solution was chosen to catalyze the fibrin gel according to the thrombin recommendation from Sigma. The gelation occurs within 3 minutes when catalyzed by 2 mul and 5 mul thrombin solution, which is in line with the expected operation time. The catalytic gelation is too fast by 10 mul thrombin solution, partial gelation appears within 1min after adding the gel hemozyme, which is not beneficial to practical application.

Fibrin gel concentration is preferred. The fibrin gel with the concentration of 1mg/ml has poor gelling quality, and obvious hydration phenomenon of the fibrin gel can be seen at the 9 th min after 5 mul of gel hemozyme is added. The fibrin gel with the concentration of 5mg/ml has better gelling quality when the gel hemozyme is added for 5 min. The fibrin gel with the concentration of 10mg/ml is gelatinized too fast, and partial gelatinization can be realized within 1min after adding gel hemozyme. Each set of experiments was repeated three times.

FIG. 2: 5mg/ml fiberProtein gel-loaded stem cell densities are preferred. At 1 × 10⁶After the stem cell concentration of each ml is mixed with the fibrin gel, the formed gel has better quality. At 2X 10⁶The mixing of the stem cell concentration per ml and the fibrin gel has the advantage of good gel quality although the gel forming speed is reduced. At 5X 10⁶The mixture of individual/ml of stem cell concentration with fibrin gel was not stable to gelling. Each set of experiments was repeated three times.

FIG. 3: staining of the dried cells with 5mg/ml fibrin gel for stem cell toxicity test (A) live and dead cells, the results show: the number of viable cells of hAMSCs cultured on 5mg/ml fibrin gel was approximately equal to that of viable cells cultured under normal culture conditions. (B) Statistical analysis of the proportion of viable cells in the two culture conditions using two independent sample t-tests using GraphPad 8.0.1 software showed no statistical difference.

FIG. 4: and 2w after the loaded hAMSCs fibrin gel is transplanted to the skull of the rabbit, and the result of MHC class I staining of a human specific index of the sample is obtained. The results show that: hAMSCs were confirmed to survive by positive staining for human MHC Class I in 2w rabbit cranial specimens (see FIG. 4F).

FIG. 5: the loaded hAMSCs fibrin gel can repair the experimental result of rabbit skull defect, (A) the reconstruction result of 2w, 4w and 12w CT data after operation; (B) the GraphPad 8.0.1 software statistically analyzed each set of BV/TV results from CT scans with one-way analysis of variance. (x, p < 0.05).

Detailed Description

Definition of

The terms "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification can mean "one," but can also mean "one or more," at least one, "and" one or more than one.

As used in the claims and specification, the terms "comprising," "having," "including," or "containing" are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Throughout this specification, the term "about" means: a value includes the standard deviation of error for the device or method used to determine the value.

Although the disclosure supports the definition of the term "or" as merely an alternative as well as "and/or," the term "or" in the claims means "and/or" unless expressly indicated to be merely an alternative or a mutual exclusion between alternatives.

When used in the claims or specification, the term "range of values" is selected/preferred to include both the end points of the range and all natural numbers subsumed within the middle of the end points of the range with respect to the aforementioned end points of values.

EXAMPLE one preference for fibrin gel concentration

Preparing fibrin gel solutions of 1mg/ml, 5mg/ml and 10mg/ml, catalyzing 400 mul with thrombin of 2 mul, 5 mul and 10 mul respectively, observing the gelling condition of the solutions at different time points at the bottom of a glass bottle and taking pictures. The gel is preferably fixed and coagulated on the bottom of the bottle. The thrombin catalysis gelling speed effect of 2 mul and 5 mul 50U/ml is better. And the gelling speed and quality of the 5mg/ml fibrin gel solution were screened to be optimal (see fig. 1).

Example II isolation and culture of hAMSCs

After fresh placenta tissue without infectious diseases is obtained, the placenta tissue is quickly placed in an ice box and sealed for storage. Return to the laboratory as soon as possible. Placing the placenta tissue which is refrigerated and preserved in a biological safety cabinet which is disinfected by ultraviolet, carefully distinguishing and separating the amniotic membrane layer on the surface of the placenta, and temporarily storing the separated amniotic membrane in an alpha-MEM complete culture medium. Amniotic membrane surface blood clots, decidua were removed in PBS containing a solution of double penicillin-streptomycin. The cleaned amniotic tissue was transferred to fresh PBS solution and minced to small tissue pieces approximately 2mm by 2mm in size. The minced amniotic membrane tissue was transferred to a neutral protease/PBS solution at a concentration of 2.4U/mL and digested at 37 ℃ for 10 min. The digested amniotic membrane tissue is allowed to stand in alpha-MEM complete medium for 10 min. After standing, the amniotic membrane tissue was again transferred to the alpha-MEM complete culture solution with collagenase D concentration of 1mg/mL and LDNA enzyme concentration of 20. mu.g/mL, and digested at 37 ℃ for 2.5-3 h. After completion of digestion, containing amniotic tissueFiltering the alpha-MEM complete culture solution by a cell sieve of 100 mu m, and removing the amnion tissues which are not completely degraded. After filtration, the liquid fraction containing hAMSCs was transferred to a 15mL centrifuge tube and centrifuged at 200 Xg for 10 min. The centrifuged cell pellet was suspended in α -MEM complete medium. Inoculating into a Petri dish and placing in 5% CO₂And cultured in a cell culture chamber at 37 ℃. And after 24-48h, changing the solution according to the adherent condition of the cells. Complete culture solution prepared from alpha-MEM culture solution, 5% (v/v) Human Platelet Lysate (HPL), 1% (v/v) penicillin-streptomycin solution and 2U/mL heparin solution, and 5% CO₂And cultured at 37 ℃. The solution was changed 1 time every 3 days. When the cell recovery degree reaches 90%, passage is carried out at a ratio of 1: 4. Cells were discarded after passage 5.

EXAMPLE III 5mg/ml fibrin gel solution load Stem cell Density optimization

10mg/ml fibrin gel solution was prepared. The adherent culture of hAMSCs were digested, centrifuged to collect cells at 2X 10⁶4X 10 pieces/ml⁶Each/ml and 10X 10⁶The dry cells were resuspended in culture medium at a concentration of one/ml. Mixing the cell suspensions with different concentrations with 10mg/ml fibrin gel solution 1:1, respectively taking 400 μ l, catalyzing with 5 μ l 50U/ml thrombin, observing the gelation condition of the solution at different time points at the bottom of the glass bottle, and taking pictures. The gel is preferably fixed and coagulated on the bottom of the bottle. And screening out 2X 10⁶The rate and quality of gelation was optimal for individual/ml stem cell concentrations (see FIG. 2).

EXAMPLE four examination of the toxic Effect of 5mg/ml fibrin gel on hAMSCs

And (3) paving the 5mg/ml fibrin gel at the bottom of a cell culture dish before gelling, and after gelling, normally passaging hAMSCs on the surface of the fibrin gel, wherein the hAMSCs cultured by normal adherence are used as a control. After collecting the cells at 14d, the samples were washed 1-2 times according to the product Calcein-AM/PI live/dead cell double stain kit (san Jose biosciences, Shanghai) instructions, 1 × Assay Buffer, and incubated for 30min at 37 ℃ in the dark using a staining solution containing 2 μ M Calcein-AM (Calcein-acetylmethoxymethyl ester) and 4.5 μ M PI (propidium iodide). Live cells were detected under a fluorescence microscope using a 490. + -.10 nm excitation filter, dead cells were observed using a 545nm emission filter, and images were taken. No obvious difference is observed between the surface of the gel and the normally cultured hAMSCs, and the injection type fibrin gel regeneration material capable of loading cells is proved to have good biocompatibility (as shown in figure 3).

Example five Effect of loading hAMSCs fibrin gel to promote repair of rabbit skull defect

Step one, preparation of hAMSCs-loaded fibrin gel

Based on the results of examples one, two, and three, we loaded hAMSCs with good activity on fibrin gel to prepare a gel solution with a final concentration of fibrin of 5mg/ml and a cell density of hAMSCs of 2X 10⁶Each cell/ml is catalyzed by thrombin with the concentration of 50U/ml, and the cell is injected into a skull defect of a rabbit before gelling, and is marked as an hAMSCs group. The fibrin gel group was used as a control group.

Step two, preparation of rabbit skull defect and implantation of loaded hAMSCs fibrin gel

New Zealand white rabbits of 4-6 months age were weighed and anesthetized with 3% sodium pentobarbital solution at 30 mg/kg. After corneal reflection disappears, shaving hair, disinfecting a skull top operation area by 75% alcohol, making a sagittal incision along a skull median suture to a periosteum, separating the periosteum by a periosteum stripper, preparing circular bone defects with the diameter of about 10mm along two sides of the skull median suture, and respectively injecting hAMSCs-fibrin gel and acellular fibrin gel into the defects, wherein the defects are respectively marked as an hAMSCs group and a control group. The gel volume was about 150 μ l, with each group giving a sample size n of 3 per time point. Stopping bleeding with gauze, implanting two groups of fibrin gels into defect area, covering collagen membrane on the surface, drawing periosteum together, and suturing periosteum skin. The operation area is disinfected again, the breathing and the expression of the rabbits are observed, the stable state of the rabbits is confirmed, and the rabbits are placed back in the rabbit cage after being marked.

Step three, survival condition of the loaded hAMSCs fibrin gel implanted into skull defect area of rabbit

After operation 2w, the rabbit was euthanized, the skull specimen was fixed in formalin fixing solution, decalcified, embedded in paraffin, sectioned, and immunohistochemically stained for pedestrian specific protein (major histocompatibility complex class I, i.e., MHC class I) in the specimen. The results show that: there were a large number of positive cells in the hAMSCs group, as seen in fig. 4 (F). The hAMSCs have good biocompatibility after transplantation, can survive for at least two weeks, and can play a biological role in the bone defect.

Step four, evaluating the repair effect of the skull defect of the rabbit

After operation, 2w, 4w and 12w, the rabbits are euthanized, and skull specimens are taken and fixed in formalin fixing liquid; fixing and then performing imaging detection (the result is shown in figure 5); and then, detecting the formation condition of new bones by using a micro CT (micro computed tomography), and analyzing the bone formation effect of each group.

The experimental results are shown in FIG. 5(A), and it can be seen from FIG. 5(A) that at 2w, the bone repair effect of the group of hAMSCs and the control group is not significantly different; at 4w, the hAMSCs group bone defect region has the blocking projection, and the bone defect central region shows certain new bone formation; at 12w, the bone defect area in the hAMSCs group is basically covered by new bone tissue, and a larger area of the center of the bone defect area in the control group is not covered by the bone tissue. As shown in fig. 5(B), BV/TV (%), 2w, 5.366 ± 1.062 for hAMSCs and 5.348 ± 0.990 for control; at 4w, the group of hAMSCs is 11.017 + -3.011, and the control group is 6.801 + -1.613; at 12w, the group of hAMSCs is 42.539 + -1.382, and the control group is 25.068 + -2.159; in conclusion, the new bone mass of the hAMSCs fibrin gel group is obviously higher than that of the control group.

All technical features disclosed in the present specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

Furthermore, from the foregoing description, one skilled in the art can readily appreciate the key features of the disclosure from the present disclosure, that numerous modifications can be made to adapt the invention to various usages and conditions without departing from the spirit and scope of the disclosure, and therefore, such modifications are intended to fall within the scope of the appended claims.

Claims

1. An injectable fibrin gel regeneration material capable of loading cells, which is characterized in that the regeneration material is formed by enzyme catalysis of cell suspension containing stem cells and fibrin gel.

2. The injectable cell-loadable fibrin gel regeneration material according to claim 1, wherein the cell suspension is a cell suspension comprising hAMSCs; optionally, the enzyme is thrombin.

3. An injectable cell-loadable fibrin gel regeneration material according to any of claims 1-2, wherein the concentration of the fibrin gel is between 1mg/ml and 10 mg/ml.

4. The injectable cell-loadable fibrin gel regeneration material according to claim 3, wherein the concentration of the fibrin gel is 5 mg/ml.

6. The injectable cell-loadable fibrin gel regeneration material according to claim 5, wherein the concentration of said cells is 2 x 10⁶One per ml.

7. A pharmaceutical composition comprising the injectable cell-loadable fibrin gel regeneration material according to any one of claims 1 to 6.

culturing the cell;

9. The method of claim 8, wherein the cell suspension is a cell suspension comprising hAMSCs; optionally, the enzyme is thrombin.

10. Use of an injectable cell-loadable fibrin gel regeneration material according to any one of claims 1-6 or a composition according to claim 7 in the preparation of a medicament for promoting bone regeneration or treating a bone defect.