CN114652895A

CN114652895A - Preparation method of injectable tissue regeneration type chitosan composite gel scaffold

Info

Publication number: CN114652895A
Application number: CN202011537359.4A
Authority: CN
Inventors: 魏长征; 王意波; 谈浩琪; 蒋丽霞
Original assignee: Shanghai Qisheng Biological Preparation Co ltd
Current assignee: Shanghai Qisheng Biological Preparation Co ltd
Priority date: 2020-12-23
Filing date: 2020-12-23
Publication date: 2022-06-24

Abstract

The invention relates to a preparation method and application of an injectable tissue regeneration type chitosan composite gel scaffold. The main components of the gel are (1) chitosan or derivative microgel thereof and (2) electronegative mucopolysaccharide. The chitosan composite gel stent obtained by the invention can smoothly pass through a 27G needle, so that minimally invasive treatment is realized; the obtained chitosan composite gel scaffold can induce normal cells to grow in, and continuously provides more space to promote the generation of self normal tissues along with the absorption and degradation in vivo. The system material has the advantages of simple preparation process, strong preparation process operability, excellent biocompatibility and wide application prospect in the field of tissue regeneration.

Description

Preparation method of injectable tissue regeneration type chitosan composite gel scaffold

Technical Field

The invention belongs to the field of biomedical materials, and particularly relates to a preparation method and application of an injectable tissue regeneration type chitosan composite gel scaffold.

Background

The use of biological materials to promote or guide the repair and regeneration of tissues and organs has long received considerable attention. The physicochemical properties of the biomaterial, such as chemical groups, particle size, pore size, hydrophilicity and hydrophobicity, degradability, etc., play an important role in regulating and controlling tissue repair and regeneration. After implantation of a non-completely inert biological material, the body reacts more complexly to the material, which is reflected in the action of cells (such as macrophages, fibroblasts, endothelial cells, lymphocytes, etc.), the release of relevant factors, the activation of relevant pathways, which also determines the progress of tissue repair.

The chitosan is a natural alkaline polysaccharide, can be extracted from shrimp and crab shells and the like with abundant resources, and has degradability, good biocompatibility and antibacterial property. The chitosan and the derivatives thereof have important applications in hemostasis, adhesion prevention and tissue repair and regeneration of cartilage tissues, bone tissues, skin, nerves and the like. In order to achieve the effects of tissue repair and regeneration, researchers design various types of scaffold materials based on chitosan, such as an electrospinning fiber scaffold, a porous sponge scaffold, a hydrogel scaffold, and the like. Patent CN103893835B discloses chitosan biomembrane obtained by decalcification, deproteinization, acetyl and manual layering, which provides physical barrier and porous structure, and can be used as membrane for guiding tissue regeneration, promoting adhesion and growth of osteoblast and osteocyte. However, most stents are preformed stents, have no injectable properties and are generally only suitable for relatively regular wounds and tissue sites, and are poorly compliant for irregular areas to be repaired. Meanwhile, in order to relieve surgical wounds and the pain of people in treatment, accelerate the healing of wounds and improve the healing efficiency and quality of diseases, the injectable hydrogel stent becomes a new round of research hotspot in the field. To address this, researchers have explored injectable and in situ-forming implantable materials. Patent CN111068116A discloses a cartilage repair temperature-sensitive gel for injection and a preparation method thereof, and the method is based on a temperature-sensitive gel blended by chitosan and beta-glycerophosphate, and simultaneously adds collagen and cell growth factors, thereby being beneficial to the repair of cartilage defect. However, the complex synthetic process and the relatively complex components are involved, which is not favorable for the subsequent clinical application transformation. Patent WO20200237956a1 discloses a method for injecting a chitosan solution, wherein the chitosan is in a solution state at a pH of less than 6.2, and becomes insoluble and crystalline particles due to body fluid exchange when injected into the body, which can be used as a dermal filler and promote the reconstruction of dermal tissue, but the acidic solution is irritating to the human body, has strong solution fluidity, and cannot be well shaped in a short period of time. The invention innovatively controls the molecular structure of the chitosan raw material and the particle size of the microgel thereof, and mixes the polysaccharide with electronegativity and viscosity as an excipient with the chitosan microgel. The obtained chitosan composite gel scaffold has good support property, the electrostatic interaction between the chitosan microgel and the electronegative polysaccharide is destroyed through the shear force thinning effect, the scaffold material can smoothly pass through a 27G needle, the electrostatic interaction is recovered after injection to form the scaffold material again, and the minimally invasive delivery of the scaffold material is realized; in addition, through the control of the deacetylation degree of the chitosan raw material and the particle size of the microgel, the obtained chitosan composite gel scaffold can induce the growth of normal cells, and simultaneously provide proper inflammatory stimulation to promote the regeneration and formation of new tissues. The system material has the advantages of simple preparation process, strong preparation process operability, excellent biocompatibility and wide application prospect in the field of tissue regeneration.

Disclosure of Invention

In order to overcome the defects, the invention provides a preparation method of an injectable tissue regeneration type chitosan composite gel scaffold, the size of the chitosan microgel is 20-60 mu m, and the scaffold has minimally invasive injectability after an excipient is added, and has good support property and degradability after injection; the scaffold is of a porous structure and can induce cells to grow in, the pH value of the gel scaffold is 6.8-7.4, is close to the pH value of body fluid of normal tissues of a human body, and the gel scaffold has wide application prospects in the field of tissue regeneration of bone tissues, cartilage tissues, skin and the like.

In order to achieve the above purpose, the technical scheme adopted in the experiment is as follows:

the preparation method of the injectable tissue regeneration type chitosan composite gel scaffold comprises the following steps:

(1) the chitosan or chitosan derivative is used as a raw material, and a clear solution is obtained by acid dissolution.

(2) Adding alkali liquor for precipitation to obtain chitosan physical gel

(3) Granulating by a granulator to obtain the chitosan microgel.

(4) And (3) cleaning the microgel to be neutral, adding an electronegative polysaccharide (excipient) solution with certain viscosity, mixing and homogenizing to obtain the injectable chitosan composite gel scaffold.

In order to control the inflammation level and tissue regeneration of the injectable tissue regeneration type chitosan composite gel scaffold, the selection of chitosan or chitosan derivatives and excipients and the size of chitosan microgel need to be strictly controlled.

The deacetylation degree of the chitosan or the chitosan derivative is 85-99%.

The granulation process comprises crushing, passing through a squeezing plate, sieving and the like.

The particle size of the chitosan microgel is 20-60 mu m.

The excipient comprises one or more of sodium hyaluronate, carboxymethyl chitosan, sodium alginate and sodium carboxymethyl cellulose, and the viscosity is 14000mPa & s.

The chitosan microgel concentration in the injectable tissue regeneration type chitosan composite gel scaffold system is 4-10%, the excipient concentration is 1-5%, and the pH value is 6.8-7.4.

The injectable tissue regeneration type chitosan composite gel scaffold system can pass through a 25G and thinner needle head, and the pushing force is less than 25N.

Description of the terms

And (3) chitosan: the chitosan is a product of natural polysaccharide chitin with partial acetyl removed, and has multiple physiological functions of biodegradability, biocompatibility, nontoxicity, immunity enhancement and the like.

Degree of deacetylation: in order to deacetylate the natural polysaccharide chitin, the higher the deacetylation degree is, the higher the molecular chain amino (NH)₂) The more the content is, the more the amine groups are protonated, the more the charged groups of the chitosan in the diluted acid solution are, the more the polyelectrolyte charge density is increased, and the result is that the structure, the property and the performance of the chitosan are necessarily changed.

Excipients: the excipient is a viscous polysaccharide solution, the relatively high viscosity of the solution can provide a lubricating effect for the chitosan microgel during injection, and stable mechanical properties of the material are provided through intermolecular forces (hydrogen bonds, electrostatic interactions and the like) between the mucopolysaccharide and the chitosan microgel after injection.

Tissue engineering scaffolds: the tissue engineering scaffold material is a material which can be combined with tissue living cells, can be implanted into different tissues of a living body and can be used for replacing functions of the tissues. In order to proliferate and differentiate seed cells, it is necessary to provide a cell scaffold composed of a biomaterial, which corresponds to an artificial extracellular matrix.

The main advantages of the invention are:

(1) the injectable tissue regeneration type chitosan composite gel scaffold provided by the invention has good injectability, and can be injected from a 25G needle and a smaller needle, so that the minimally invasive property is realized.

(2) The injectable tissue regeneration type chitosan composite gel scaffold provided by the invention has good plasticity and degradability, is loose and porous in the scaffold, can support cell growth, and does not generate toxic and harmful substances.

(3) The chitosan microgel has the particle size of 20-60 mu m, and can effectively control the inflammation level and promote the tissue regeneration.

(4) The preparation process of the injectable tissue regeneration type chitosan composite gel scaffold provided by the invention is simple and feasible, is suitable for large-scale industrial production, and has the potential of clinical application.

Drawings

FIG. 1 particle size of chitosan microgel;

FIG. 2 extrusion force of injectable chitosan composite gel scaffolds;

FIG. 3 is a diagram of an injectable tissue regeneration type chitosan composite gel scaffold implanted subcutaneously for 20 days;

FIG. 4H & E tissue staining after subcutaneous implantation of injectable tissue regeneration type chitosan composite gel scaffolds.

Detailed Description

The features of the present invention and other related features are described in further detail below by way of examples to facilitate understanding by persons skilled in the art, but are not to be construed as limiting the invention in any way. Those skilled in the art should also realize that such changes, modifications, additions and substitutions can be made without departing from the spirit and scope of the invention.

Example 1

And (3) dissolving chitosan with deacetylation degree of 90% in 1% glacial acetic acid solution to obtain 0.7% chitosan solution, and uniformly dissolving. Then, 1M sodium hydroxide solution was added dropwise to precipitate a physical gel of chitosan. After the precipitation was completed, the precipitate was dispersed with pure water, pulverized in a pulverizer for 20min, and the particle size distribution was measured using a laser particle sizer, the median of the particle size was 33.71 μm, as shown in FIG. 1.

Example 2

The chitosan gel in example 1 was repeatedly washed with pure water, and after pH was neutralized, further granulation and homogenization were performed using a 200 μm squeeze plate to obtain a chitosan physical gel. And (3) uniformly mixing 7% of chitosan microgel and 1% of sodium carboxymethylcellulose (with the viscosity of 6000mPa & s), and homogenizing through 100-mesh spun silk to obtain the injectable chitosan composite gel scaffold. The pushing force is tested by a universal tensile machine, the pushing speed is 30mm/min, the test is carried out for 3 times, and the needle head is a 27G needle head (BD Hypoint TM, 0.4 x 13 mm). The extrusion force is shown in figure 2, and the average extrusion force in the test range is 9.27N.

Example 3

The chitosan gel of example 1 was repeatedly washed with pure water, and after the pH was neutralized, further granulation and homogenization were performed using 500 μm and 200 μm squeeze plates to obtain a chitosan physical gel. And (3) uniformly mixing 8% of chitosan microgel and 1.7% of hydroxypropyl methylcellulose solution to obtain the injectable chitosan composite gel scaffold.

Example 4

The chitosan gel in example 1 was repeatedly washed with pure water until the pH became neutral, to obtain a chitosan physical gel. Uniformly mixing 5% of chitosan microgel and 1.5% of sodium carboxymethylcellulose solution (the viscosity is 1000mPa & s), and homogenizing by using a 200-micron extrusion plate to obtain the injectable chitosan composite gel scaffold.

Example 5

The injectable tissue regeneration type chitosan composite gel scaffold of example 4 was implanted under the skin of rabbits, and was dissected and observed 20 days after implantation. As can be seen from the real figure (FIG. 3), the color of the gel scaffold is slightly yellow compared with that before the gel scaffold is implanted, and blood vessels and tissues are formed on the surface of the gel scaffold. H & E it can be seen (FIG. 4) that vessel walls and red blood cells, etc. appear on and within the material, indicating the formation of blood vessels; fibroblasts, macrophages and a small amount of cellular infiltration into the interior of the gel are present at the interface of the material and the tissue. The new blood vessel is a ligament of nutrition supply and is the basis of tissue regeneration, so the injectable chitosan composite gel scaffold is beneficial to promoting tissue regeneration.

Claims

1. A preparation method of an injectable tissue regeneration type chitosan composite gel scaffold is characterized by comprising the following steps:

(1) microgel of chitosan group or derivative thereof

(2) Electronegative mucopolysaccharide (excipient)

(3) And (3) a gelling buffer solvent.

2. The material as set forth in claim 1, wherein the chitosan or chitosan derivative material has deacetylation degree of 85-99%, and weight average molecular weight of 1 x 10⁵Da-1×10⁶Da. Can be dissolved in an acidic reagent, and is insoluble in an aqueous solution with neutral pH or a salt solution, and comprises chitosan, alkylated chitosan, water-insoluble carboxymethyl chitosan, N-succinylated chitosan, hydroxypropyl chitosan and the like.

3. The method for preparing an injectable tissue regeneration type chitosan composite gel scaffold, which is the material according to claim 1, wherein the chitosan or chitosan derivative microgel has a dispersion particle size of (20-60) μm in water.

4. The preparation method of the injectable tissue regeneration type chitosan composite gel scaffold, which is the material according to claim 1, wherein the viscous polysaccharide has electronegativity, such as one or more of sodium hyaluronate, carboxymethyl chitosan, sodium alginate and sodium carboxymethyl cellulose, but not limited thereto, and the solution dynamic viscosity of the polysaccharide is 14000mPa & s.

5. The preparation method of an injectable tissue regeneration type chitosan composite gel scaffold, which is prepared from the material according to claim 1, wherein the gelling buffer solution is a phosphate buffer solution, and the main components are sodium chloride, disodium hydrogen phosphate and sodium dihydrogen phosphate.

6. The preparation method of the injectable tissue regeneration type chitosan composite gel scaffold is characterized in that the chitosan microgel concentration in the injectable chitosan composite gel scaffold system is 5-10% (wt/v), the excipient concentration is 1-5% (wt/v), the pH is 6.8-7.4, and the osmotic pressure is 270-350 mOsmol/kg.

7. The preparation method of the injectable tissue regeneration type chitosan composite gel scaffold is characterized in that the injectable chitosan composite gel scaffold system can pass through 27G and thicker needles, and the pushing force is less than 20N.

8. A method for preparing an injectable tissue regeneration type chitosan composite gel scaffold, the method for preparing the hydrogel material according to claim 1, comprising the steps of:

(1) dissolving chitosan or chitosan derivative as a raw material in an acid solution to obtain a clear and transparent chitosan solution;

(2) adding an alkaline solution into the solution to separate out chitosan to obtain chitosan block gel;

(3) granulating the chitosan block gel to obtain chitosan microgel;

(4) cleaning the waste water to be neutral, and squeezing out excessive free water;

(5) adding excipient solution with certain viscosity, mixing and homogenizing to obtain the injectable chitosan composite gel scaffold.

9. The method as claimed in claim 8, wherein the acid is dissolved in one or more of glacial acetic acid, hydrochloric acid, lactic acid and glycolic acid, the concentration of the dissolved chitosan solution is 0.5-2% (wt/v), and the pH value is 4.0-6.0.

10. The method for preparing the injectable tissue regeneration type chitosan composite gel scaffold according to claim 8, wherein the alkaline precipitation method uses one or more of sodium hydroxide, potassium hydroxide and sodium carbonate solution, and the molar ratio of the alkaline agent to the acidic agent added is about 1.2: 1.0.

11. The method as claimed in claim 8, wherein the granulation process comprises crushing, cutting, grinding, etc., and the size (median) of the chitosan microgel is 20-60 μm.

12. The method of claim 8, wherein the homogenization process comprises stirring, extruding through a plate, etc., but is not limited thereto.

13. The method for preparing the injectable tissue regeneration type chitosan composite gel scaffold is characterized in that the chitosan composite gel scaffold can be subjected to minimally invasive injection delivery, has certain support property after delivery, can induce normal cell growth, has lighter inflammatory reaction and can be absorbed and degraded in a body, and continuously provides more space to promote the generation of normal tissues per se. The material can be applied to the field of tissue regeneration of bone tissues, muscles, skin and other parts.