阅读说明：本技术 一种交联复合胶原支架及其制备方法 (Cross-linked composite collagen scaffold and preparation method thereof ) 是由 白新鹏 王国锭 于 2021-01-05 设计创作，主要内容包括：本发明提供了一种交联复合胶原支架及其制备方法,通过将猪脱细胞真皮外基质与牛I型胶原交联来制备复合胶原支架。与传统的胶原蛋白支架相比,此支架具有均匀的多孔结构,高孔隙率,强吸水能力和强持水能力,同时,它具有良好的抗拉伸强度和热稳定性,可以更好地满足伤口敷料的需求。生物相容性结果表明,该复合胶原支架没有细胞毒性,适合细胞粘附和增殖。它是一种更好的胶原蛋白复合支架,能够代替传统的胶原蛋白支架。(The invention provides a cross-linked composite collagen scaffold and a preparation method thereof. Compared with the traditional collagen scaffold, the scaffold has the advantages of uniform porous structure, high porosity, strong water absorption capacity and strong water holding capacity, and meanwhile, the scaffold has good tensile strength and thermal stability and can better meet the requirements of wound dressings. The biocompatibility result shows that the composite collagen scaffold has no cytotoxicity and is suitable for cell adhesion and proliferation. The collagen composite scaffold is a better collagen composite scaffold and can replace the traditional collagen scaffold.)

1. A cross-linked composite collagen scaffold is prepared by cross-linking pig skin with extracellular matrix and bovine type I collagen.

2. The crosslinked composite collagen scaffold according to claim 1, wherein the weight ratio of porcine skin extracellular matrix to bovine type I collagen is 1: 6-1: 10.

3. The cross-linked composite collagen scaffold according to claim 1, wherein said cross-linking agent is glutaraldehyde with a working concentration of 0.2-0.3%.

4. The cross-linked composite collagen scaffold according to any one of claims 1 to 3, wherein the cross-linking temperature is 37 ℃ and the cross-linking time is 0.5 to 1 hour.

5. A method for preparing a cross-linked composite collagen scaffold comprises the step of cross-linking pig skin extracellular matrix and bovine type I collagen to prepare the composite collagen scaffold.

6. The method for preparing a cross-linked composite collagen scaffold according to claim 5, comprising: dissolving bovine type I collagen in an acetic acid solution, adding pigskin to remove extracellular matrix, crosslinking with glutaraldehyde for 0.5-1 hour at 37 ℃, and washing with deionized water.

7. The method for preparing a crosslinked composite collagen scaffold according to claim 6, wherein the weight to volume ratio of bovine type I collagen to acetic acid solution is 3: 100, the weight ratio of the pig skin extracellular matrix to the bovine type I collagen is 1: 6-1: 10.

8. The method for preparing a cross-linked composite collagen scaffold according to claim 6, wherein the working concentration of glutaraldehyde is 0.2% -0.3%.

9. The method for preparing a cross-linked composite collagen scaffold according to any one of claims 5 to 8, wherein the pig skin is prepared by removing extracellular matrix by the following method:

1) removing epidermis and subcutaneous fat of pig skin to obtain dermis;

2) preparing a cell removing solution by using TritonX-100 and sodium hydroxide, putting pig dermis into a reactor, stirring for 1 hour, and changing the solution once in 1 hour for 6 times in total;

3) the obtained sample was dried and sterilized.

10. The method for preparing a cross-linked composite collagen scaffold according to claim 9, wherein the ratio of the porcine dermal material to the decellularized fluid in step 2) is 10: 1; more preferably, the weight percentage of sodium hydroxide in the cell-removing solution is 2%, and the weight percentage of TritonX-100 is 1%.

Technical Field

The invention belongs to the technical field of biomedical materials, and relates to a cross-linked composite collagen scaffold and a preparation method thereof.

Background

Collagen is a structural protein that provides mechanical strength to bone, cartilage, skin and tissues, while supporting organs and playing an important role in cell differentiation, cell migration and connective tissue repair. Collagen is widely used in biomedical materials and clinical medicine due to its natural low toxicity, low antigenicity and low immunity, inhibits epithelial cell migration, directs cell regeneration, and has good histocompatibility. Collagen is also widely used in medicine, cosmetics, foods and other fields because of its many excellent properties. To date, 28 different types of collagen have been genetically reported, with type I collagen accounting for more than 90% of the total protein content of the human body. According to the current literature, the commercially available collagen used in the above formulations is bovine type I collagen, while other types of collagen have been prepared only in experimental studies, and are not suitable for large-scale production due to their high price.

Type I collagen has good biological properties as well as good material properties, including adhesion and fiber impermeability, water absorption, and relatively high porosity, compared to other conventional biomaterials. High porosity can provide space for new tissue formation, and collagen fibers can be integrated into the new tissue matrix. Currently, various forms of collagen are used in biomaterials. The commercialized collagen solution can be used for preparing various gels, membranes and scaffolds, for tissue engineering, as a drug controlled release carrier, and for wound repair and healing.

Commercial collagen dressings currently on the market are typically prepared using bovine tissue. The collagen sponge scaffold may be prepared by freeze-drying a solution containing 0.5-5 w/v% dry matter. Different structures can be controlled by adjusting the freeze-drying conditions. The collagen dressing stimulates cell migration and promotes the development of new tissue, and may promote the deposition and organization of newly formed collagen, creating an environment that promotes wound healing by activating the chemotactic properties of wound fibroblasts. However, conventional bovine type I collagen dressings still have some disadvantages, such as poor thermal stability and poor mechanical properties.

Disclosure of Invention

In view of the above problems, an object of the present invention is to provide a cross-linked composite collagen scaffold prepared by cross-linking porcine acellular dermal extracellular matrix with bovine type I collagen, and a method for preparing the same. Compared with the traditional collagen scaffold, the collagen scaffold has good tensile strength and thermal stability, and can better meet the requirements of a wound dressing scaffold.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a cross-linked composite collagen scaffold which is prepared by cross-linking pig skin extracellular matrix and bovine type I collagen.

Preferably, the weight ratio of the pig skin extracellular matrix to the bovine type I collagen is 1: 6-1: 10.

Preferably, the cross-linking agent is glutaraldehyde with a working concentration of 0.2% -0.3%.

Preferably, the crosslinking temperature is 37 ℃ and the crosslinking time is 0.5 h-1 h.

The invention also provides a preparation method of the cross-linked composite collagen scaffold, which comprises the step of cross-linking the pig skin extracellular matrix and the cattle type I collagen to prepare the composite collagen scaffold.

Further, comprising: dissolving bovine type I collagen in an acetic acid solution, adding pigskin to remove extracellular matrix, crosslinking with glutaraldehyde for 0.5-1 hour at 37 ℃, and washing with deionized water.

Preferably, the weight to volume ratio (g/mL) of bovine type I collagen to acetic acid solution is 3: 100, the weight ratio of the pig skin extracellular matrix to the bovine type I collagen is 1: 6-1: 10.

Preferably, the working concentration of glutaraldehyde is 0.2% to 0.3%.

Preferably, the pig skin extracellular matrix is prepared by the following method:

1) removing epidermis and subcutaneous fat of pig skin to obtain dermis;

2) preparing a cell removing solution by using TritonX-100 and sodium hydroxide, putting pig dermis into a reactor, stirring for 1 hour, and changing the solution once in 1 hour for 6 times in total;

3) the obtained sample was put into a freeze dryer to be dried and sterilized.

Preferably, in the step 2), the feed-liquid ratio (g/mL) of the pig dermal raw material to the acellular liquid is 10: 1.

more preferably, in the step 2) cell removing solution, the weight percentage of sodium hydroxide is 2 percent, and the weight percentage of TritonX-100 is 1 percent.

The invention has the following beneficial effects:

the invention designs and researches a novel cross-linked composite collagen scaffold, which is beneficial to solving the current situation that a large amount of pigskin is wasted and improving the performance of the existing collagen dressing scaffold. The results show that the scaffold has a uniform porous structure, high porosity, strong water absorption capacity and strong water holding capacity. Compared with the traditional collagen dressing, the collagen dressing has good tensile strength and thermal stability, and can better meet the requirements of wound dressings. The biocompatibility result shows that the composite collagen scaffold has no cytotoxicity and is suitable for cell adhesion and proliferation. The collagen composite scaffold is a better collagen composite scaffold and can replace the traditional collagen dressing scaffold.

Drawings

Fig. 1 is a scanning microscope view of a cross-linked composite collagen scaffold prepared according to an embodiment of the present invention, and a, b, and c are different microscope observation results of a conventional collagen dressing, respectively. d, e and f are respectively different microscope observation results of the cross-linked composite collagen scaffold.

FIG. 2 shows the results of the water absorption test of the present invention, wherein COL is an abbreviation of conventional collagen dressing scaffold, and CCS is an abbreviation of novel cross-linked composite collagen scaffold. The designations a, b are significant.

Detailed Description

The technical solutions of the present invention are described below clearly and completely with reference to specific embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In addition, unless otherwise specified, the materials and methods employed below are all conventional in the art, and are within the ordinary skill of the art.

Example 1

Process for preparing pig skin without extracellular matrix

1. Separating epidermis from dermis, namely removing epidermis and subcutaneous fat of the pig skin to obtain the dermis.

2. TritonX-100 and sodium hydroxide are used for preparing a cell removal solution, and pig dermis is placed into a reaction kettle to be stirred for 1 hour at the temperature of 37 ℃, and the solution is changed once every 1 hour for 6 times.

In the step, the weight percentage (wt/wt) of sodium hydroxide in the acellular solution is 2%, the weight percentage (wt/wt) of TritonX-100 in the acellular solution is 1%, and the feed-liquid ratio of the pig dermis raw material to the acellular liquid is 10: 1.

3. drying in a freeze dryer for 24h, and sterilizing for storage.

The pig skin prepared by this method was also used to remove extracellular matrix in examples 2 and 3.

(II) Process for preparing Cross-Linked composite scaffolds

1.3 g of bovine type I collagen was dissolved in 100ml of acetic acid (0.5mol/L), and 0.5g of pigskin was added after being subjected to extracellular matrix removal.

2. Crosslinking with glutaraldehyde (working concentration 0.2%) at 37 ℃ for 0.5 hour.

Glutaraldehyde (GTA) is a homologous bifunctional cross-linking agent that forms schiff bases with two-CHO groups and two primary amino groups of two identical or different molecules and connects the two molecules through a five-carbon bridge. The high concentration of GTA reacts with the amino groups of lysine or hydroxylysine residues E2 of the collagen molecule to form intermolecular crosslinks. The low concentration of GTA and collagen molecules form intramolecular crosslinks. GTA crosslinked collagen membranes have a higher strain modulus and a significantly improved resistance to degradation by collagenase compared to uncrosslinked collagen membranes.

3. After crosslinking was complete, rinse with deionized water.

4. Drying in a freeze dryer, sterilizing and storing.

The scanning microscope view of the finally obtained cross-linked composite collagen scaffold is shown in fig. 1. The novel cross-linked composite collagen scaffold has a three-dimensional porous structure, relatively uniform pore size and good connectivity. The good porosity and connectivity are beneficial to the transmission of water vapor and drug delivery, so that the stent material can be used as a drug delivery carrier to promote the recovery of wounds.

Example 2

1. 4g of bovine type I collagen was dissolved in 100ml of acetic acid (0.5mol/L), and 0.5g of pigskin was added after being subjected to extracellular matrix removal.

2. Crosslinking with glutaraldehyde (working concentration 0.25%) at 37 ℃ for 1 hour.

3. After crosslinking was complete, rinse with deionized water.

4. Drying in a freeze dryer, sterilizing and storing.

The structural characteristics of the finally obtained cross-linked composite collagen scaffold are consistent with those of example 1.

Example 3

1.3 g of bovine type I collagen was dissolved in 100ml of acetic acid (0.5mol/L), and 0.3g of pigskin was added after being subjected to extracellular matrix removal.

2. Crosslinking with glutaraldehyde (working concentration 0.3%) at 37 ℃ for 45 minutes.

3. After crosslinking was complete, rinse with deionized water.

4. Drying in a freeze dryer, sterilizing and storing.

The structural characteristics of the finally obtained cross-linked composite collagen scaffold are consistent with those of example 1.

The mechanical properties, Water Vapor Transmission Rate (WVTR), and water absorption rate of the crosslinked composite collagen scaffold prepared in example 1 were compared with bovine type I collagen by test examples, using example 1 as an example. In addition, biological evaluation was performed by cytotoxicity and proliferation experiments.

Test example (I) mechanical Property measurement

Mechanical properties are one of the important characteristics of stent materials. Tensile strength is the ability to resist breaking under tension.

The tensile strength of the scaffold was tested using a texture analyzer using a 50mm x 10mm sample. Both ends are clamped and suspended at both ends of a test head of a universal testing machine. The tensile strength of the stent was measured at a tensile rate of 1mm/min and the tensile curve on the recorder was observed until the material broke. Three samples were tested in parallel for each set of materials and the results are expressed as mean ± SD.

The tensile strength test of the collagen scaffold shows that the tensile strength of the traditional collagen scaffold is 1.56 plus or minus 0.08MPa, while the tensile strength of the novel cross-linked composite collagen scaffold is 2.76 plus or minus 0.06MPa, which is statistically significantly higher than that of the traditional collagen scaffold (P < 0.05). Good tensile strength facilitates further processing of the stent material.

(II) detection of water vapor transmission rate and Water absorption

Water Vapor Transmission Rate (WVTR): the dried collagen sponge holder was placed in the nozzle of a cylindrical test tube having a diameter of 18mm, which was filled with 10mL of distilled water, and the test tube was placed in an incubator at 37 ℃ and a relative humidity of 35%. The calculation formula is as follows:

wherein A is the area of the sponge (m)²) S is the slope of weight loss with respect to time (g/h)

As a result: the WVTR of the traditional collagen scaffold is 859.53g/m²Day, while the WVTR of the novel cross-linked composite collagen scaffold was 942.75g/m²·day。

Water absorption (U): both scaffolds were soaked in phosphate buffer at room temperature for at least 30 minutes to achieve complete absorption, retrieved, wiped with filter paper to gently absorb surface moisture, and then weighed. The calculation formula is as follows:

wherein m is₁Is the weight of the dry sponge, m₂Is the weight of the swollen sponge.

As a result: according to the graph of fig. 2, the water absorption capacity of the traditional collagen scaffold is 28.6 plus or minus 0.57, and the water absorption capacity of the novel cross-linked composite collagen scaffold is 31.3 plus or minus 1.0, i.e. the water absorption performance of the novel cross-linked composite scaffold is obviously higher than that of the traditional collagen dressing scaffold.

(III) porosity measurement

Appropriate specified volumes of ethanol (V1) were placed in two measuring cylinders, and 2 × 2cm collagen sponges were immersed. The subsequent volume of ethanol was measured (V2). After 5 minutes, the sample was withdrawn and the volume of remaining ethanol (V3) was measured. Then, the porosity of the two groups of collagen sponges was obtained using the following formula.

P＝(V1-V3)/(V2-V3)×100％

Through tests, the porosity of the traditional collagen scaffold is 60.3 +/-2.0%, the porosity of the novel cross-linked composite collagen scaffold is 65.6 +/-1.2%, and the difference has statistical significance (P is less than 0.05). The higher porosity allows the scaffold to be used as a carrier to promote nutrient exchange and cell growth and metabolism.

(IV) evaluation of cytotoxicity

Mouse fibroblast L929 cells were used for cytotoxicity assessment. L929 cells were maintained in Dulbecco's modified Eagle's Medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin/streptomycin antibiotic. The collagen sponge scaffold was cut into pieces of 1X 1 cm. The ratio of the sample to the culture medium was 1 mg/mL. Cells in logarithmic growth phase were enzymolyzed and formulated to a concentration of 10⁵Individual cells/mL of cell suspension. For each set of materials, an aliquot of 100 μ Ι _ of cell suspension was added to each well of a 96-well plate of three parallel wells. Cells were cultured in DMEM for 24 hours, and then the culture solution was aspirated and replaced with sterile solution. After 24 hours incubation in the incubator, 10 μ LCCk-8 reagent was added to each well and incubated in the incubator for another 3 hours. The absorbance at 450nm of each well was measured using a microplate reader. Cell viability was calculated using the following formula:

wherein As is the absorbance of the test well (cell containing cells, CCK-8, toxic substance), A_cIs the absorbance of a control well (cells containing cells, CCK-8, non-toxic substance), A_bBlank wells (cell-free medium and toxic medium, containing CCK-8).

Culturing L929 cells in the support leaching liquor to carry out enzyme-labeling instrument detection, and finally detecting that the cell survival rate is more than 80 percent, namely the support material has no toxicity.

The test effect of the composite collagen scaffolds prepared in examples 2 and 3 is substantially the same as that of the test example in example 1, and no obvious difference exists, and the detailed description is omitted here.