Preparation method of hydrogel microspheres based on photocuring silk fibroin

Abstract

A preparation method of hydrogel microspheres based on photocuring silk fibroin comprises the following steps: firstly, mixing a silk fibroin solution with a riboflavin aqueous solution to obtain a mixed solution, then adding a hydrogen peroxide solution into the mixed solution, and fully mixing to obtain a precursor solution; the preparation method comprises the following steps: preparing a precursor solution, putting the precursor solution into a push pump injector, spraying, enabling sprayed spherical-like liquid drops to fall into liquid nitrogen, continuously irradiating the spherical-like liquid drops by ultraviolet light in the whole process from spraying out of the injector to falling into the liquid nitrogen, and finally freezing and fixing the liquid drops falling into the liquid nitrogen by the liquid nitrogen, thereby obtaining the spherical-like liquid drops. And drying to obtain solid spheres, namely the silk fibroin hydrogel microspheres. According to the design, the manufacturing time is short, the manufacturing efficiency is high, the forming is high and good, and application and popularization in the biomedical field are facilitated.

Description

Technical Field

[0001] The invention relates to the preparation of silk fibroin hydrogel microspheres, belongs to the field of polymer gels, and particularly relates to a preparation method of silk fibroin hydrogel microspheres based on light-curing. Background Art

[0002] Hydrogels refer to materials that can absorb large amounts of water without dissolving. They are formed by chemical or physical crosslinking of hydrophilic natural or synthetic polymers. Due to their high water absorption and water retention properties, hydrogels have a wide range of applications, such as facial masks, fever-reducing patches, preservatives in the food industry, and drug carriers in the medical field. At present, there are many methods for preparing hydrogels reported at home and abroad, such as chemical crosslinking, physical crosslinking, biocatalysis, etc. Different preparation methods can prepare hydrogels with different properties to meet the application needs of different fields, such as hydrogel microspheres.

[0003] At present, the application of hydrogel microspheres in the medical field is increasing, such as controlling the delivery of cells and drugs, and tissue repair. The principle is to encapsulate bioactive substances or drugs in hydrogel microspheres to control their functions and realize their own unique characteristics. In addition, hydrogel microspheres are not only carriers for drug delivery, but also can prolong the drug release curve, maintain the biological activity of drugs, and improve the tolerance and therapeutic indicators of various drugs.

[0004] The Chinese invention patent application with application number 202211535863.X and application date 2022.12.02 discloses a hydrogel microsphere and its preparation method and application. The hydrogel microsphere is made of gelatin and polyethylene glycol acrylate derivatives as raw materials. The hydrogel microsphere has stability for long-term storage, and the chemical crosslinking and bonding between the microspheres can be initiated and controlled separately before use. The compression modulus can reach up to 100kPa, which improves the stability and mechanical properties of the microspheres. However, this patent application adopts a water-in-oil method to prepare hydrogel microspheres. Although the formed microspheres are relatively stable, it takes a long time and the formation speed is relatively slow. In addition, the parameters of the production process are difficult to control, which makes it difficult to meet the existing large-scale production needs.

[0005] The information disclosed in this background technology section is only intended to increase the understanding of the overall background of the application, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art already known to ordinary technicians in this field. Summary of the invention

[0006] The purpose of the present invention is to overcome the defects and problems of long production time and low efficiency in the prior art, and to provide a preparation method of light-cured silk fibroin hydrogel microspheres with short production time and high efficiency.

[0007] To achieve the above objectives, the technical solution of the present invention is: a preparation method based on light-cured silk fibroin hydrogel microspheres, the preparation method comprising the following steps: The first step: firstly, the silk fibroin solution and the riboflavin aqueous solution are mixed to obtain a mixed solution, and then the hydrogen peroxide solution is added to the mixed solution and mixed thoroughly to obtain a precursor solution; The second step: first place the precursor solution in a push pump syringe, then spray it, and let the sprayed spherical droplets fall into liquid nitrogen. The spherical droplets are continuously irradiated by ultraviolet light during the whole process from being sprayed to falling into liquid nitrogen, and then the droplets falling into it are frozen and fixed by liquid nitrogen to obtain solid balls, namely silk fibroin hydrogel microspheres.

[0008] In the second step, the power of the ultraviolet lamp generating ultraviolet light is 100-900W, and each spherical droplet is continuously irradiated by the ultraviolet light for 30s-120s.

[0009] In the second step, the working parameters of the push pump syringe are: push injection speed is 1-10mL / h, and the voltage is 9V.

[0010] In the first step, the concentration of the silk fibroin solution is 1.5-7wt%, the concentration of the riboflavin aqueous solution is 0.05-10mmol / L, and the volume ratio of the silk fibroin solution to the riboflavin aqueous solution is 5-20:1.

[0011] In the first step, the concentration of the hydrogen peroxide solution is 20-30 vol%, and the volume ratio of the hydrogen peroxide solution to the mixed solution is 1:5-30.

[0012] In the first step, the method for preparing the silk fibroin solution is as follows: first, the raw silk is placed in a sodium carbonate aqueous solution and heated and immersed for degumming to obtain degummed silk fibroin fibers, and then the obtained degummed silk fibroin fibers are immersed in a LiBr aqueous solution and heated to dissolve to obtain a crude solution, and then the obtained crude solution is placed in a dialysis bag, and then the dialysis bag is placed in deionized water for dialyzation, and then the remaining solution in the dialysis bag after dialysis is filtered to obtain a filtrate, and then the filtrate is diluted to obtain a silk fibroin solution.

[0013] The concentration of the sodium carbonate aqueous solution is 0.1-0.5wt%, and the mass volume ratio of raw silk to the sodium carbonate aqueous solution is 1g / 10-100mL; The heating temperature of the degumming treatment is 45-100° C., and the time of the degumming treatment is 1-8 hours.

[0014] The concentration of the LiBr aqueous solution is 1-10 mol / L, and the mass volume ratio of the degummed silk fibroin fiber to the LiBr aqueous solution is 1 g / 1-20 mL; The temperature for heating and dissolving the degummed silk fibroin fibers in the LiBr aqueous solution is 45-85° C., and the heating and dissolving time is 0.5-3 hours.

[0015] The dialysis temperature is 0-4°C, and the dialysis time is 1-5 days.

[0016] In the first step, after the hydrogen peroxide solution is fully mixed in the mixed solution, the cell solution is added to obtain a precursor solution; In the second step, cells are contained both inside and on the outer wall of the silk fibroin hydrogel microspheres, and the number of cells contained inside the silk fibroin hydrogel microspheres is greater than the number of cells contained on the outer wall.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. In a preparation method of light-cured silk fibroin hydrogel microspheres of the present invention, a silk fibroin solution and a riboflavin aqueous solution are first mixed to obtain a mixed solution, and then a hydrogen peroxide solution is added to the mixed solution and mixed thoroughly to obtain a precursor solution, and then the precursor solution is placed in a push pump syringe, and then sprayed, and the sprayed spherical droplets are made to fall into liquid nitrogen, and the spherical droplets are continuously irradiated by ultraviolet light during the entire process from being sprayed out of the syringe to falling into the liquid nitrogen, and finally the droplets falling into the liquid nitrogen are frozen and fixed to obtain solid balls, i.e., silk fibroin hydrogel microspheres. The precursor solution used in this design is a blend of a silk fibroin solution, a riboflavin aqueous solution, and a hydrogen peroxide solution, wherein the silk fibroin Based on, riboflavin and hydrogen peroxide are a photocuring system, and the silk protein hydrogel is cured under ultraviolet light. During the reaction process, hydrogen peroxide will decompose and produce oxygen under ultraviolet light. Riboflavin is first excited to produce a triplet state under ultraviolet light, and then reacts with the oxygen produced by hydrogen peroxide to form oxygen free radicals, and then the oxygen free radicals promote the mutual entanglement between the silk protein macromolecules to form a gel (that is, riboflavin and H2O2 undergo a continuous photocrosslinking cycle reaction, promote the conversion of tyrosine to dityrosine, and play a catalytic gelation role). It can be seen that hydrogen peroxide, as an oxygen source, can provide more oxygen for the photocuring system, continuously promote the continuous progress of the reaction, greatly shorten the time of gel formation, and improve the production efficiency of hydrogel microspheres. Therefore, the present invention not only has a short production time, but also has a high production efficiency.

[0018] 2. In the preparation method of the light-cured silk fibroin hydrogel microspheres of the present invention, whether it is the mixing of the silk fibroin solution, the riboflavin aqueous solution, and the hydrogen peroxide solution, or the spraying, ultraviolet irradiation, and liquid nitrogen freezing after obtaining the precursor solution, there are no parameters that are difficult to control, such as the rotation speed and concentration, nor are there high-precision equipment requirements and temperature requirements that are difficult to reach or control. It is very easy to control, which not only makes the design easy to implement and facilitates popularization and application, but also helps to improve the molding effect of the hydrogel microspheres and improve the uniformity of the size of the microspheres. Therefore, the present invention is not only easy to operate, but also has good product formability.

[0019] 3. In a preparation method based on light-cured silk fibroin hydrogel microspheres of the present invention, light curing combined with a push pump injection method is used to prepare silk fibroin hydrogel microspheres, and cross-linking is formed by mutual entanglement between silk fibroin macromolecules, and riboflavin and H2O2 are used to catalyze this process, which can not only ensure that the droplets are transformed into stable microspheres with good formability, but also the internal crystalline structure of the microspheres is relatively low, mainly a silk I structure, so that the hydrogel microspheres have good light transmittance and excellent biocompatibility, and are particularly suitable for the field of biomedical materials. Therefore, the present invention not only has good formability, but also has excellent light transmittance.

[0020] 4. In the preparation method of light-cured silk fibroin hydrogel microspheres of the present invention, the raw materials used are mainly silk fibroin solution, riboflavin aqueous solution, and hydrogen peroxide solution, wherein the silk fibroin solution and the riboflavin aqueous solution have good biocompatibility, and hydrogen peroxide is basically consumed in the gel process, and its content is negligible, non-toxic to cells, so that there is no organic reagent residue in the hydrogel microspheres finally prepared, no biological toxicity, and good market application prospects in the fields of cell control, drug delivery, tissue repair, etc. Therefore, the present invention has no biological toxicity and is convenient for use in the field of biomedicine.

[0021] 5. In a preparation method based on photocurable silk fibroin hydrogel microspheres of the present invention, after the hydrogen peroxide solution is fully mixed in the mixed solution, a cell solution can be added to obtain a precursor solution. At this time, the silk fibroin hydrogel microspheres finally obtained contain cells on the inside and the outer wall, and the number of cells contained in the silk fibroin hydrogel microspheres is greater than the number of cells contained in the outer wall. The design makes full use of the short preparation time of the hydrogel microspheres in this design, so that the mixed solution can be mixed with the cell solution and then photocured, so that most of the cells are evenly coated inside the hydrogel microspheres, and the microspheres are used as cell carriers to achieve the purpose of controlling cells, drug delivery and tissue repair, and expand the application of silk fibroin-based hydrogel microspheres in the biomedical field. Therefore, the present invention can carry cells in large quantities and evenly inside the microspheres, which is conducive to the promotion and application in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a physical picture of the hydrogel microspheres prepared in Example 1 of the present invention.

[0023] Figure 2 yes Figure 1 Actual image after rhodamine staining.

[0024] Figure 3 yes Figure 2 Schematic diagram of hydrogel microspheres after staining under fluorescence microscope.

[0025] Figure 4 This is a SEM image of the hydrogel microspheres prepared in Example 1 of the present invention after freeze-drying.

[0026] Figure 5 yes Figure 4 An enlarged schematic diagram of .

[0027] Figure 6 This is the FTIR graph of the hydrogel microspheres prepared in Example 1 of the present invention.

[0028] Figure 7 This is the XRD diagram of the hydrogel microspheres prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0030] See also Figure 1 — Figure 7 A method for preparing light-cured silk fibroin hydrogel microspheres comprises the following steps: The first step: firstly, the silk fibroin solution and the riboflavin aqueous solution are mixed to obtain a mixed solution, and then the hydrogen peroxide solution is added to the mixed solution and mixed thoroughly to obtain a precursor solution; The second step: first place the precursor solution in a push pump syringe, then spray it, and let the sprayed spherical droplets fall into liquid nitrogen. The spherical droplets are continuously irradiated by ultraviolet light during the whole process from being sprayed to falling into liquid nitrogen, and then the droplets falling into it are frozen and fixed by liquid nitrogen to obtain solid balls, namely silk fibroin hydrogel microspheres.

[0031] In the second step, the power of the ultraviolet lamp generating ultraviolet light is 100-900W, and each spherical droplet is continuously irradiated by the ultraviolet light for 30s-120s.

[0032] In the second step, the working parameters of the push pump syringe are: push injection speed is 1-10mL / h, and the voltage is 9V.

[0033] In the first step, the concentration of the silk fibroin solution is 1.5-7wt%, the concentration of the riboflavin aqueous solution is 0.05-10mmol / L, and the volume ratio of the silk fibroin solution to the riboflavin aqueous solution is 5-20:1.

[0034] In the first step, the concentration of the hydrogen peroxide solution is 20-30 vol%, and the volume ratio of the hydrogen peroxide solution to the mixed solution is 1:5-30.

[0035] In the first step, the method for preparing the silk fibroin solution is as follows: first, the raw silk is placed in a sodium carbonate aqueous solution and heated and immersed for degumming to obtain degummed silk fibroin fibers, and then the obtained degummed silk fibroin fibers are immersed in a LiBr aqueous solution and heated to dissolve to obtain a crude solution, and then the obtained crude solution is placed in a dialysis bag, and then the dialysis bag is placed in deionized water for dialyzation, and then the remaining solution in the dialysis bag after dialysis is filtered to obtain a filtrate, and then the filtrate is diluted to obtain a silk fibroin solution.

[0036] The concentration of the sodium carbonate aqueous solution is 0.1-0.5wt%, and the mass volume ratio of raw silk to the sodium carbonate aqueous solution is 1g / 10-100mL; The heating temperature of the degumming treatment is 45-100° C., and the time of the degumming treatment is 1-8 hours.

[0037] The concentration of the LiBr aqueous solution is 1-10 mol / L, and the mass volume ratio of the degummed silk fibroin fiber to the LiBr aqueous solution is 1 g / 1-20 mL; The temperature for heating and dissolving the degummed silk fibroin fibers in the LiBr aqueous solution is 45-85° C., and the heating and dissolving time is 0.5-3 hours.

[0038] The dialysis temperature is 0-4°C, and the dialysis time is 1-5 days.

[0039] In the first step, after the hydrogen peroxide solution is fully mixed in the mixed solution, the cell solution is added to obtain a precursor solution; In the second step, cells are contained both inside and on the outer wall of the silk fibroin hydrogel microspheres, and the number of cells contained inside the silk fibroin hydrogel microspheres is greater than the number of cells contained on the outer wall.

[0040] The supplementary technical features of the present invention are as follows: The LiBr aqueous solution in the present invention refers to a lithium bromide aqueous solution.

[0041] The reason why the concentration of the silk fibroin solution is limited to 1.5-7wt% in the present invention is that if the concentration is too low, the final microspheres will be soft and difficult to shape, and if the concentration is too high, it is not suitable to use the electrospray method to eject spherical droplets.

[0042] The reason why the volume ratio of the silk fibroin solution to the riboflavin aqueous solution is limited to 5-20:1 in the present invention is that if it is too low, the gel effect is poor and it is difficult to form, and if it is too high, materials are wasted and costs are increased.

[0043] The reason why the concentration of the hydrogen peroxide solution is limited to 20-30 vol% in the present invention is that if the concentration is too low, the gelation promoting effect cannot be achieved, and if the concentration is too high, materials are wasted and costs are increased.

[0044] In the process of preparing microspheres by electrospraying, liquid nitrogen mainly plays the role of freezing and rapid solidification. Specifically, after the solution forms microdroplets through electrospraying technology, these microdroplets will quickly enter the liquid nitrogen. Due to the extremely low temperature of liquid nitrogen (-196°C), the microdroplets will be frozen and fixed at the moment of contact with liquid nitrogen, thereby maintaining the spherical shape of the microdroplets and quickly forming solid microspheres. This rapid freezing process is crucial to the formation of microspheres because it prevents the deformation of the microdroplets during drying or curing. In addition, the low temperature environment of liquid nitrogen also helps to maintain the stability and biological activity of the internal structure of the microspheres, which is particularly important for the preparation of microspheres containing sensitive biological molecules (such as proteins, enzymes or cells). In the present invention, the silk fibroin hydrogel microspheres are generally taken out from the liquid nitrogen one or two minutes after the microsphere preparation is completed.

[0045] Embodiment 1: A preparation method based on light-cured silk fibroin hydrogel microspheres, the preparation method comprising the following steps: Step 1: First, a silk fibroin solution (1.5 wt%) and a riboflavin aqueous solution (0.5 mmol / L) are mixed at a volume ratio of 20:1 to obtain a mixed solution, and then a hydrogen peroxide solution (30 vol%) is added to the mixed solution at a volume ratio of 1:30 to the mixed solution, and the mixture is fully mixed to obtain a precursor solution; The second step: first place the precursor solution in the syringe of the push pump, and then spray it (the injection speed of the push pump is 5mL / h, the voltage is 9V), and let the sprayed spherical droplets fall into liquid nitrogen. The spherical droplets are continuously irradiated by ultraviolet light during the whole process from being sprayed to falling into liquid nitrogen, and then the droplets falling into it are frozen and fixed by liquid nitrogen to obtain solid balls, namely silk fibroin hydrogel microspheres.

[0046] Figure 1 This is a physical picture of the hydrogel microspheres prepared in Example 1. Figure 2 This is a real picture of the hydrogel microspheres after being stained with rhodamine. Figure 3 The schematic diagram of the dyed hydrogel microspheres under a fluorescence microscope shows that the hydrogel microspheres prepared by the electrospray method not only have good molding effect, but also have uniform size, especially Figure 3It can be seen that the hydrogel microspheres have good morphology and a diameter of about 300-400 μm.

[0047] To further observe the morphological characteristics of the hydrogel microspheres, the technicians also freeze-dried them and took SEM photos. The results are as follows: Figure 4 , Figure 5 As shown in the figure, it can be seen that the dried hydrogel microspheres have a porous structure. These pores can become channels for transporting nutrients during cell culture. At the same time, the diameter of the pores is less than 10μm, which can firmly lock the cells inside the microspheres.

[0048] The hydrogel microspheres prepared in Example 1 were characterized by infrared spectroscopy. Figure 6 As shown in the figure, it can be seen that the absorption bands mainly appear at 1656cm-1 (amide I presents a random structure), 1539cm-1 (amide II presents a random structure) and 1240cm-1 (amide III presents a random structure), indicating that the secondary structure of the hydrogel microspheres is mainly a random structure, and the secondary structure of SF is not changed during the gelation process.

[0049] The hydrogel microspheres prepared in Example 1 were further characterized by XRD. Figure 7 As shown in the figure, it can be seen that the prepared hydrogel microspheres have a diffraction peak at 19.7°, which mainly shows a silk I structure, indicating that the silk fibroin mainly reacts in the non-crystalline area during the formation of hydrogel.

[0050] Embodiment 2: The basic content is the same as that of Example 1, except that: The preparation method of the silk fibroin solution is as follows: first, 30 g of raw silk is placed in 900 mL of a 0.1 wt% sodium carbonate aqueous solution, boiled and soaked for 1.5 hours to perform degumming treatment to obtain degummed silk fibroin fibers, and then the obtained degummed silk fibroin fibers are immersed in 300 mL of a 10 mol / L LiBr aqueous solution, heated to 80° C. and dissolved for 3 hours to obtain a crude solution, and then the obtained crude solution is placed in a dialysis bag, and then the dialysis bag is placed in deionized water for dialysis, and the dialysis is performed in 0° C. deionized water for 5 days, and then the remaining solution in the dialysis bag after dialysis is filtered to obtain a filtrate, and then the filtrate is diluted with deionized water to obtain a 1.5 wt% silk fibroin solution.

[0051] Embodiment 3: The basic content is the same as that of Example 1, except that: In the first step, after the hydrogen peroxide solution is fully mixed in the mixed solution, the cell solution is added to obtain a precursor solution; In the second step, cells are contained both inside and on the outer wall of the silk fibroin hydrogel microspheres, and the number of cells contained inside the silk fibroin hydrogel microspheres is greater than the number of cells contained on the outer wall.

[0052] The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiment. Any equivalent modifications or changes made by ordinary technicians in this field based on the contents disclosed by the present invention should be included in the protection scope recorded in the claims.

Claims

1. A method for preparing light-cured silk fibroin hydrogel microspheres, characterized in that: The preparation method comprises the following steps: The first step: firstly, the silk fibroin solution and the riboflavin aqueous solution are mixed to obtain a mixed solution, and then the hydrogen peroxide solution is added to the mixed solution and mixed thoroughly to obtain a precursor solution; The second step: first place the precursor solution in a push pump syringe, then spray it, and let the sprayed spherical droplets fall into liquid nitrogen. The spherical droplets are continuously irradiated by ultraviolet light during the whole process from being sprayed to falling into liquid nitrogen, and then the droplets falling into it are frozen and fixed by liquid nitrogen to obtain solid balls, namely silk fibroin hydrogel microspheres.

2. The method for preparing light-cured silk fibroin hydrogel microspheres according to claim 1, characterized in that: In the second step, the power of the ultraviolet lamp generating ultraviolet light is 100-900W, and each spherical droplet is continuously irradiated by the ultraviolet light for 30s-120s.

3. A method for preparing light-cured silk fibroin hydrogel microspheres according to claim 1 or 2, characterized in that: In the second step, the working parameters of the push pump syringe are: push injection speed is 1-10mL / h, and the voltage is 9V.

4. A method for preparing light-cured silk fibroin hydrogel microspheres according to claim 1 or 2, characterized in that: In the first step, the concentration of the silk fibroin solution is 1.5-7wt%, the concentration of the riboflavin aqueous solution is 0.05-10mmol / L, and the volume ratio of the silk fibroin solution to the riboflavin aqueous solution is 5-20:

1.

5. The method for preparing light-cured silk fibroin hydrogel microspheres according to claim 4, characterized in that: In the first step, the concentration of the hydrogen peroxide solution is 20-30 vol%, and the volume ratio of the hydrogen peroxide solution to the mixed solution is 1:5-30.

6. A method for preparing light-cured silk fibroin hydrogel microspheres according to claim 1 or 2, characterized in that: In the first step, the silk fibroin solution is prepared by: First, the raw silk is placed in a sodium carbonate aqueous solution and heated and immersed for degumming to obtain degummed silk fibroin fibers, and then the degummed silk fibroin fibers are immersed in a LiBr aqueous solution and heated to dissolve to obtain a crude solution, and then the obtained crude solution is placed in a dialysis bag, and then the dialysis bag is placed in deionized water for dialyzation, and then the remaining solution in the dialysis bag after dialysis is filtered to obtain a filtrate, and then the filtrate is diluted to obtain a silk fibroin solution.

7. The method for preparing light-cured silk fibroin hydrogel microspheres according to claim 6, characterized in that: The concentration of the sodium carbonate aqueous solution is 0.1-0.5wt%, and the mass volume ratio of raw silk to the sodium carbonate aqueous solution is 1g / 10-100mL; The heating temperature of the degumming treatment is 45-100° C., and the time of the degumming treatment is 1-8 hours.

8. The method for preparing light-cured silk fibroin hydrogel microspheres according to claim 6, characterized in that: The concentration of the LiBr aqueous solution is 1-10 mol / L, and the mass volume ratio of the degummed silk fibroin fiber to the LiBr aqueous solution is 1 g / 1-20 mL; The temperature for heating and dissolving the degummed silk fibroin fibers in the LiBr aqueous solution is 45-85° C., and the heating and dissolving time is 0.5-3 hours.

9. The method for preparing light-cured silk fibroin hydrogel microspheres according to claim 6, characterized in that: The dialysis temperature is 0-4°C, and the dialysis time is 1-5 days.

10. A method for preparing light-cured silk fibroin hydrogel microspheres according to claim 1 or 2, characterized in that: In the first step, after the hydrogen peroxide solution is fully mixed in the mixed solution, the cell solution is added to obtain a precursor solution; In the second step, cells are contained both inside and on the outer wall of the silk fibroin hydrogel microspheres, and the number of cells contained inside the silk fibroin hydrogel microspheres is greater than the number of cells contained on the outer wall.

Images