CN114796619B

CN114796619B - Injectable hyaluronic acid microspheres and preparation method and application thereof

Info

Publication number: CN114796619B
Application number: CN202210394589.2A
Authority: CN
Inventors: 郭建俊; 宋春艳; 吕金艳; 余柳松; 司徒卫
Original assignee: Zhuhai Medfa Biotechnology Co ltd
Current assignee: Zhuhai Medfa Biotechnology Co ltd
Priority date: 2022-04-14
Filing date: 2022-04-14
Publication date: 2023-03-14
Anticipated expiration: 2042-04-14
Also published as: CN114796619A; WO2023197419A1

Abstract

The invention discloses an injectable hyaluronic acid microsphere and a preparation method and application thereof. The method of the present invention comprises preparing a first liquid phase by adding cross-linked hyaluronic acid to an aqueous solvent, the concentration of cross-linked hyaluronic acid in the first liquid phase being controlled to be 0.1-5% on a weight basis; adding the inner polyester into an organic solvent to prepare a second liquid phase, wherein the concentration of the inner polyester in the second liquid phase is controlled to be 0.1-500mg/mL; then, mixing the first liquid phase and the second liquid phase in a volume ratio of 3-15. Under the same condition, the microsphere is not easy to agglomerate and adhere, and has higher cell adhesion and histocompatibility.

Description

Injectable hyaluronic acid microspheres and preparation method and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to an injectable hyaluronic acid microsphere, a preparation method thereof and application thereof in the fields of tissue filling and the like.

Background

As people age or are affected by certain diseases, muscle and collagen tissues in human bodies can generate functional degeneration with different degrees, which causes problems of skin depression, gastric reflux and the like, so that people invent various fillers to fill the depressed skin or stimulate the regrowth of muscle and collagen through foreign matters, such as hyaluronic acid, bovine collagen and the like. However, these substances as fillers have a short retention time of the filling effect, and thus the filling effect thereof needs to be maintained by frequent injection.

In order to achieve the long-term filling effect, people try to use biodegradable materials to prepare microspheres as fillers, such as polyvinyl alcohol (PVA), polymethyl methacrylate (PMMA) and the like. Although the filling effect of these fillers is maintained for a significantly longer time, these materials remain in the body for too long, releasing harmful substances and thus causing a series of side reactions that are harmful to human health.

In recent years, biodegradable high polymer materials come into the visual field of people, and the material is nontoxic and has no rejection reaction to human bodies and can be gradually degraded along with the metabolism of the human bodies and then discharged out of the human bodies.

Polyhydroxyalkanoates, which is called polyhydroxyakanoates in English, called PHA for short, is a natural high-molecular biological material, and is an intracellular polyester synthesized by microorganisms. PHA is one of the most desirable biomedical materials due to its good biocompatibility and biodegradability.

In the prior art, when PHA material is prepared into injectable microspheres, the prepared microspheres are easy to agglomerate and adhere to form large massive substances, which brings inconvenience to subsequent injection, and the prepared microspheres have weak capability of adhering cells and can not be well compatible with tissues, thereby greatly hindering the application of biodegradable material microspheres as a filling agent. Therefore, a novel microsphere preparation scheme is urgently needed to solve the problems of low cell adhesion, easy agglomeration, adhesion, insufficient tissue compatibility and the like of the microsphere.

The information in this background is only for the purpose of illustrating the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art that is known to a person skilled in the art.

Disclosure of Invention

In order to solve at least part of technical problems in the prior art, the invention provides an injectable hyaluronic acid microsphere, which is not easy to agglomerate and adhere under the same conditions and has higher cell adhesion and histocompatibility. Specifically, the present invention includes the following.

In one aspect of the present invention, there is provided a method for preparing injectable hyaluronic acid microspheres, comprising the steps of:

(1) Adding cross-linked hyaluronic acid into an aqueous solvent to prepare a first liquid phase, wherein the concentration of the cross-linked hyaluronic acid in the first liquid phase is controlled to be 0.1-5% by weight;

(2) Adding the inner polyester into an organic solvent to prepare a second liquid phase, wherein the concentration of the inner polyester in the second liquid phase is controlled to be 0.1-500mg/mL; and

(3) Mixing the first liquid phase and the second liquid phase in a volume ratio of 3-15.

According to the method for preparing injectable hyaluronic acid microspheres of the present invention, preferably, the cross-linking degree of the cross-linked hyaluronic acid is 5% to 50%.

According to the method for preparing injectable hyaluronic acid microspheres of the present invention, preferably, the average molecular weight of the crosslinked hyaluronic acid is 9-2 million daltons.

According to the method for preparing injectable hyaluronic acid microspheres of the present invention, preferably, the inner polyester has a weight average molecular weight of 2-100 ten thousand daltons, and is selected from one or more of poly (3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate), poly (3-hydroxybutyrate-4-hydroxybutyrate) and poly (3-hydroxybutyrate-co-3-hydroxyvalerate).

According to the method for preparing injectable hyaluronic acid microspheres, the particle size of the microspheres is preferably 20-60 μm.

According to the preparation method of injectable hyaluronic acid microspheres of the invention, preferably, the aqueous solvent in step (1) is water, and the organic solvent in step (2) is one or more selected from N-methylpyrrolidone, dichloromethane, chloroform and acetonitrile.

In another aspect of the present invention, there is provided an injectable hyaluronic acid microsphere prepared by the method according to the first aspect.

Preferably, the injectable hyaluronic acid microspheres of the present invention have a young's modulus of 1000Pa or more.

The injectable hyaluronic acid microspheres according to the invention preferably have a prolonged degradation time.

In yet another aspect of the invention, there is provided the use of injectable hyaluronic acid microspheres for the preparation of a tissue filler.

The injectable hyaluronic acid microsphere has higher Young modulus reaching more than 1000Pa under the same condition, has prolonged degradation time, is not easy to agglomerate and adhere, and has higher cell adhesion and histocompatibility.

Drawings

FIG. 1 is a flow chart of an exemplary method of making the present invention.

FIG. 2 is an electron micrograph of microspheres prepared in example 1 of the present invention.

FIG. 3 shows the results of testing the microspheres obtained in example 1 of the present invention for particle size and span using a laser particle sizer.

FIG. 4 is a standard curve of bovine serum albumin adsorption experiment.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but rather as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.

[ injectable hyaluronic acid microspheres ]

In one aspect of the present invention, there is provided an injectable hyaluronic acid microsphere, which may sometimes be referred to herein simply as a "microsphere of the present invention". The microsphere is not easy to agglomerate and adhere, but has higher cell adhesion and histocompatibility.

The microspheres of the present invention comprise an outer hydrophilic layer and an inner hydrophobic layer. Wherein the hydrophilic layer is a crosslinked hyaluronic acid layer, the hydrophobic layer is an inner polyester compound, and the hydrophobic coating is prepared by coating an oil phase containing the inner polyester compound with a water phase containing crosslinked hyaluronic acid.

Crosslinked hyaluronic acid is used in the present invention, not uncrosslinked hyaluronic acid. In the invention, hyaluronic acid molecules are more agglomerated through crosslinking, so that the supporting force of hyaluronic acid is enhanced, and the hyaluronic acid becomes a good filling material. Cross-linked hyaluronic acid also greatly improves cell adhesion and histocompatibility compared to uncrosslinked hyaluronic acid.

The crosslinking degree of the crosslinked hyaluronic acid in the present invention is not particularly limited, but is generally controlled to 5 to 55%, preferably 10 to 50%, more preferably 20 to 40%. If the degree of crosslinking is outside the above range, it is disadvantageous for the effect of the present invention. On the one hand, when the crosslinking degree becomes low, for example, less than 5%, cell adhesion becomes weak, and the Young's modulus becomes low. On the other hand, when the degree of crosslinking becomes high, for example, more than 55%, the produced microspheres tend to be unstable and even to suffer from breakage. The degree of crosslinking can be controlled by, for example, the amount of the crosslinking agent used and the reaction time.

The molecular weight of the crosslinked hyaluronic acid in the present invention is not particularly limited, and may be controlled generally between 0.9 million and 2 million daltons, preferably between 1 million and 1000 million daltons, and more preferably between 5 million and 500 million daltons. The larger the molecular weight, the less susceptible to degradation, but if the molecular weight is too large, the cell adhesion becomes poor.

The internal polyester compound refers to a natural polymer biological material existing in microorganisms, and has good cell compatibility with cells in vivo. The type of the internal polyester compound is not particularly limited, and examples thereof include, but are not limited to, poly (3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate), poly (3-hydroxybutyrate-4-hydroxybutyrate), and poly (3-hydroxybutyrate-co-3-hydroxyvalerate). The present invention may employ one or more of the above ingredients. When a plurality of components are used, the amount ratio of the components is not particularly limited, and can be freely blended by a person skilled in the art as needed.

The molecular weight of the inner polyester-based compound in the present invention is not particularly limited, but is generally controlled to be in the range of 2 to 100 ten thousand daltons, preferably 5 to 90 ten thousand daltons, more preferably 10 to 80 ten thousand daltons, for example, 20 ten thousand, 30 ten thousand, 40 ten thousand, 50 ten thousand, 60 ten thousand, 70 ten thousand, etc.

The particle size of the microspheres in the invention is controlled to be 20-60 μm, preferably 30-50 μm. A particle size of 20-60 μm is essential for the purposes of the present invention. If the diameter of the microspheres is less than 20 microns, the microspheres may be phagocytosed by human cells. On the other hand, if the microspheres are too large, injection is not facilitated, and in severe cases, the needle may be blocked, even causing skin breakdown. Although the particle size of the microspheres may vary within the above ranges, the microspheres of the present invention have a more uniform particle size distribution.

The microspheres of the invention have higher Young's modulus under the same conditions. The microspheres of the present invention preferably have a young's modulus of 1000Pa or more, for example 1100Pa or more, 1200Pa or more, 1300Pa or more, and the like.

[ production method ]

In another aspect of the present invention, there is provided a method for preparing injectable hyaluronic acid microspheres, which includes, but is not limited to, the following steps:

In the present invention, the step (1) is a step of preparing a first liquid phase, which is an aqueous phase. The step (1) comprises dissolving or dispersing the crosslinked hyaluronic acid in an aqueous solvent. Wherein the aqueous solvent includes water and mixtures thereof with other aqueous solvents. Water, such as purified or distilled water, is preferably used in the present invention. The dissolution or dispersion can be accelerated by, for example, stirring. The concentration of the cross-linked hyaluronic acid in the first liquid phase of the present invention is generally controlled to be 0.1 to 5%, preferably 0.5 to 4%, more preferably 1 to 3%, such as 1%, 1.5%, 2%, 2.5%, 3%, etc. on a weight basis.

In the present invention, the step (2) is a step of preparing a second liquid phase, which is an oil phase. And (2) dissolving the required amount of the inner polyester in an organic solvent to prepare a solution with a certain concentration. Among them, examples of the organic solvent include N-methylpyrrolidone, dichloromethane, chloroform and acetonitrile. The present invention may use one or more of the above solvents. When a plurality of solvents are used, the amount ratio of each solvent is not particularly limited, and can be freely set by a skilled person as needed. In an exemplary step (2) which involves dissolving 0.8g of P34HB in 20mL of methylene chloride, a 40mg/mL PHA/organic solvent solution is prepared.

In the present invention, the step (3) is a step of preparing microspheres by mixing two phases. The volume of the first liquid phase is greater than the volume of the second liquid phase, and the volume ratio of the two phases is generally in the range of (3-15): 1, preferably in the range of 4-10, and more preferably in the range of 5-8:1. The first liquid phase is either too small or too large in volume to facilitate microsphere formation. For example, if the volumes of the first liquid phase and the second liquid phase are equal, or smaller than the second liquid phase, the hyaluronic acid microspheres of the present invention may not be formed.

In the present invention, the mixing of the first liquid phase and the second liquid phase may be performed by a membrane emulsifier. The emulsifying pressure is controlled to be between 0.01 and 0.1MPA, preferably between 0.02 and 0.05MPA, when the mixing is carried out using a membrane emulsifier. The mixing and emulsifying time is generally between 60 and 240 minutes, preferably between 90 and 200 minutes, more preferably between 100 and 150 minutes. The stirring speed during mixing and emulsifying can be controlled at 100-500r/min, preferably 150-400r/min, and more preferably 200-350r/min. After the emulsification is completed, further curing can be performed.

In an exemplary mixed emulsification scheme, the volume ratio of the second liquid phase to the first liquid phase is controlled to be 1/5, the emulsification pressure is 0.04MPA, the length of the membrane tube is 15 μm, and the emulsification time is 120min; the length of the magneton is 5cm, and the emulsifying and stirring speed is 350r/min. And after emulsification is finished, starting curing, wherein an overhead stirrer and a 10cm stirring paddle are used for curing, 280mL of water is additionally added, the stirring speed is 120r/min, and the curing time is 24h, so that the injectable PHA microspheres are obtained.

It will be understood by those skilled in the art that the order of steps (1) and (2) is not particularly limited as long as the object of the present invention can be achieved. Further, both steps may be performed simultaneously. In addition, it will be understood by those skilled in the art that other steps or operations may be included before, after, or between any of the above steps (1) - (3), such as to further optimize and/or improve the methods of the present invention.

[ use ]

The invention also provides the use of injectable hyaluronic acid microspheres, preferably for the preparation of a tissue filler. The microsphere has good biocompatibility and biodegradability, has good cell compatibility with cells in vivo, can well grow on the microsphere, and can be degraded into CO ₂ And H ₂ O, no harmful component is generated.

Example 1

The preparation method of this example is as follows:

s1, preparing an oil phase: dissolving 0.8g of P34HB in 20mL of dichloromethane to prepare 40mg/mL of PHA/organic solvent solution, wherein the weight average molecular weight of P34HB is 15 ten thousand;

s2, preparing a water phase: preparing a cross-linked hyaluronic acid (the cross-linking degree is 30%) water solution with the mass fraction of 3%;

s3, emulsifying the oil phase into a rotating water phase through a membrane emulsifier, controlling the volume ratio of the oil phase to the water phase to be 1/5, the emulsifying pressure to be 0.04MPA, the length of a membrane tube to be 15 mu m, and the emulsifying time to be 120min; the length of the magneton is 5cm, and the emulsifying and stirring speed is 350r/min. And after emulsification is finished, starting curing, wherein an overhead stirrer and a 10cm stirring paddle are used for curing, 280mL of water is additionally added, the stirring speed is 120r/min, and the curing time is 24h, so that the injectable PHA microspheres are obtained.

Example 2

The preparation method of this example is as follows:

s1, preparing an oil phase: dissolving 100mg of P34HB in 20mL of trichloromethane to prepare a 500mg/mL PHA/organic solvent solution, wherein the weight average molecular weight of P34HB is 10 ten thousand;

s2, preparing a water phase: preparing a cross-linked hyaluronic acid (the cross-linking degree is 40%) water solution with the mass fraction of 4%;

s3, controlling the volume ratio of the oil phase to the water phase to be 1/3, mixing the oil phase and the water phase, and preparing the microspheres by a conventional emulsification and volatilization method, wherein the method specifically comprises the following steps: the method comprises the steps of firstly stirring oil-water mixed liquor by magnetic force at the rotating speed of 300r/min for 5min, then reducing the rotating speed to 150r/min, stirring for 24h to volatilize the organic solvent trichloromethane, finally centrifuging the mixed liquor, collecting and cleaning microspheres, and freeze-drying to obtain the injectable P34HB microspheres.

Comparative example 1

s2, preparing a water phase: preparing a hyaluronic acid (molecular weight is 10 w) water solution with the mass fraction of 3%;

Comparative example 2

s2, preparing a water phase: preparing a hyaluronic acid (molecular weight 7 w) water solution with the mass fraction of 3%;

s3, emulsifying the oil phase into a rotating water phase through a membrane emulsifier, controlling the volume ratio of the oil phase to the water phase to be 1/5, the emulsifying pressure to be 0.04MPA, the length of a membrane tube to be 15 mu m, and the emulsifying time to be 120min; the length of the magneton is 5cm, and the emulsifying and stirring speed is 350r/min. And after the emulsification is finished, starting curing, wherein an overhead stirrer and a 10cm stirring paddle are used for curing, 280mL of water is additionally added, the stirring speed is 120r/min, and the curing time is 24h, so that the injectable PHA microspheres are obtained.

Test example 1

As can be seen from the electron micrograph of the injectable PHA microspheres prepared in example 1, the obtained microspheres have good dispersibility, no adhesion among themselves, uniform morphology and spherical shape.

Next, the particle size and span values were measured using a laser particle sizer, and FIG. 3 is a particle size distribution graph of the injectable PHA microspheres obtained in example 1. As can be seen from fig. 3, the microspheres obtained in example 1 had an average particle size of 33.7 μm, and table 1 shows the span value (span value is the degree of dispersion of particle size distribution) of example 1 measured by a laser particle sizer, and the measurement result shows that the span value is 0.801, which indicates that the injectable PHA microspheres of example 1 have uniform particle sizes.

TABLE 1

Test example 2

To further examine the protein adsorption of injectable PHA microspheres, the following experiments were performed:

(1) Drawing a protein growth curve: BSA was used to prepare a 3mg/ml solution, which was scanned at full wavelength using a spectrophotometer to detect the maximum absorption wavelength of BSA at 278.58nm. Then, a series of concentration gradients of 0.5, 1, 1.5, 3, 4, 5mg/ml solutions were prepared, and the absorbance of each concentration of solution was measured at the maximum absorption wavelength to prepare a standard curve, which is shown in FIG. 4, where the abscissa of FIG. 4 is represented by x, the ordinate is represented by y, x is the BSA concentration, and y is the absorbance.

(2) Preparing 5mg/ml BSA solution, incubating injectable PHA microspheres, measuring the light absorption value of the BSA solution to be 2.941 before incubation, centrifuging and taking supernate after incubation for 5 hours, measuring the light absorption value of the BSA solution (supernate solution), and calculating the specific adsorption concentration, wherein the result is as follows: after 5 hours of incubation, the concentration of BSA in the supernatant was 4.79mg/mL, the absorbance was 2.892, and the difference in concentration between before and after incubation indicated that BSA protein was adsorbed by injectable PHA microspheres.

(3) The value of protein adsorbed per gram of microspheres was calculated (higher values indicate better protein adsorption).

The value of the adsorption protein of the injectable PHA microspheres obtained in example 1 was 3600ug/g, which is compared with the adsorption value of 600ug/g of CT1 charge spheres (CT 1 charge spheres are commonly used cell culture microcarriers, and the main component of the charge spheres is GE Healthcare, DEAE-sephadex, which is known as Cytodex 1, ct1), thus it can be seen that the injectable PHA microspheres provided in example 1 can be well adsorbed on the cell surface, and the adsorption effect is better than that of the traditional CT1 spheres, which is about 10 times of the adsorption capacity of CT1 spheres.

TABLE 2

Group of	BSA adsorption (μ g/g)
		Example 1	3600
Example 2	4000
		Comparative example 1	1200
Comparative example 2	900

When the biomaterial is in contact with the physiological environment, the biomaterial is not rejected in the body, has no serious filler-related side effects, and needs good cell adhesion, and a very important point in the cell adhesion is that a large amount of protein is adhered to the cell surface, and the protein adsorption is an important cell adhesion driving force. The CT1 charge ball is a commonly used cell culture microcarrier, and the higher the value, the better the protein adsorption. The value of the CT1 charge ball adsorbed bovine serum albumin is 600 mug/g. As shown in Table 2, the adsorption concentration of the PHA microspheres prepared in example 1 to bovine serum albumin was 3600. Mu.g/g; the adsorption concentration of PHA microspheres prepared in example 2 to bovine serum albumin was 4000. Mu.g/g. Through comparative analysis, the PHA microsphere prepared by the invention has excellent protein adsorbability, and the adsorption concentration is 6-7 times of that of a CT1 charge ball, so that the PHA microsphere has excellent cell adhesion and can be used for medical treatment, cosmetic filling, implantation of medical instruments and the like.

Test example 3

Young's modulus of the microspheres was measured using an Antopa MCR302 rheometer. The instrument was first set to a constant temperature amplitude scanning mode, the flat rotor of appropriate diameter (25 mm) and sample application stage height h (1 mm) were selected, frequency (1 Hz) was fixed, and then the shear strain γ was set to vary logarithmically from 0.01% to 10%. The specific detection steps are as follows:

and placing the microsphere sample in the center of the sample adding platform, lowering the rotor to a measuring position, starting to measure after sample scraping, and covering a sealing device around to reduce the water loss in the measuring process to obtain a curve graph of the storage modulus G 'and the loss modulus G' along with the change of the shear strain. Calculating the Young modulus E according to the following formula; e =2G (1+v), v =0.5

TABLE 3

Group of	Young's modulus (Pa)
		Example 1	1108.4
Example 2	1198.3
		Comparative example 1	100.3
Comparative example 2	90.4

Test example 4

In order to simulate the influence of body fluid circulation on the degradation degree in a physiological state, a dynamic degradation system is designed to carry out an in-vitro dissolution experiment according to the peristaltic pump principle. 5g of microspheres are put into 200ml of normal saline, subjected to constant-temperature water bath at 37 ℃, two transfusion tubes are respectively used as a dropping tube and a dropping tube, and the dropping and flowing speeds are adjusted to reach 1000ml/h simultaneously. Observing the degradation condition of the microspheres in 50, 100, 150, 200, 250, 300, 350 and 400 days respectively, taking the mixed solution for centrifugation, if the microspheres with precipitates are present, the microspheres are not completely degraded, if the microspheres without precipitates are completely degraded, the microspheres are completely degraded, and recording the complete degradation time. The degradation time of PHA microspheres is greatly prolonged by using the cross-linked hyaluronic acid on the experimental surface.

TABLE 4

Group of	Degradation time (Tian)
		Example 1	300
Example 2	350
		Comparative example 1	200
Comparative example 2	150

While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Many modifications and variations may be made to the exemplary embodiments of the present description without departing from the scope or spirit of the present invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.

Claims

1. A method of improving cell adhesion of injectable hyaluronic acid microspheres, comprising the steps of:

(1) Adding cross-linked hyaluronic acid to an aqueous solvent to prepare a first liquid phase, wherein the concentration of the cross-linked hyaluronic acid in the first liquid phase is controlled to be 0.1-5% by weight;

(3) Mixing the first liquid phase and the second liquid phase in a volume ratio of 3-15; the crosslinking degree of the crosslinked hyaluronic acid is 5-50%.

2. The method of claim 1, wherein the cross-linked hyaluronic acid has a mean weight molecular weight of 9 kilo-daltons to 2 kilo-daltons.

3. The method of claim 1, wherein the inner polyester has a weight average molecular weight of 2 to 100 kilodaltons and is selected from one or more of poly (3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate), poly (3-hydroxybutyrate-4-hydroxybutyrate), and poly (3-hydroxybutyrate-co-3-hydroxyvalerate).

4. The method of claim 1, wherein the microspheres have a particle size of 20-60 μm.

5. The method according to claim 1, wherein the aqueous solvent in step (1) is water, and the organic solvent in step (2) is one or more selected from the group consisting of N-methylpyrrolidone, dichloromethane, chloroform and acetonitrile.

6. Injectable hyaluronic acid microspheres with improved cell adhesion obtained by the method according to any of claims 1-5.

7. The injectable hyaluronic acid microsphere of claim 6, wherein the Young's modulus of the injectable hyaluronic acid microsphere is above 1000 Pa.

8. The injectable hyaluronic acid microsphere of claim 6, characterized in that it has a prolonged degradation time.

9. Use of injectable hyaluronic acid microspheres according to claim 6 for the preparation of a tissue filler.