CN100368026C - A kind of silk fibroin coated titanium mesh and its preparation method and application - Google Patents

Abstract

The invention belongs to the field of tissue engineering and medical materials, and in particular relates to a silk fibroin-coated titanium mesh, a preparation method thereof and an application in tracheal defect reconstruction. The invention adopts medical titanium mesh as supporting material and silk fibroin as coating to make a silk fibroin-coated titanium mesh complex. The invention utilizes the good biocompatibility of silk fibroin and the effect of promoting the growth of fibroblasts and mucosal cells, can accelerate healing, reduce complications, and can effectively support the lumen for a long time or permanently. The titanium mesh coated with silk fibroin of the present invention can be used as clinical tracheal defect repair, and can provide meaningful reference for exploring the role of silk fibroin in promoting tracheal mucosa repair and clinical reconstruction of tracheal defect, and reducing complications after tracheal reconstruction.

Description

A kind of silk fibroin coated titanium mesh and its preparation method and application

technical field

The invention belongs to the field of tissue engineering and medical materials, and in particular relates to a silk fibroin-coated titanium mesh, a preparation method thereof and an application in tracheal defect reconstruction.

Background technique

At present, when the body has extensive damage to the trachea due to inflammation, trauma, tumors or other lesions, and a long segment of the trachea is removed, the defective trachea needs to be repaired. For a long time, the problem of repairing tracheal cartilage defects has puzzled clinicians. One-stage end-to-end anastomosis is only suitable for the repair of local defects. When the length of the defect exceeds 6.0 cm, serious complications such as anastomotic leakage are likely to occur due to high anastomotic tension. The rapid development of tissue engineering technology research has brought new hope to tracheal reconstruction, but since the initial attempt in 1994, there has been no breakthrough. It is still in the stage of experimentation and exploration, and there are still problems to be solved. Many scholars have tried to apply methods such as allogeneic transplantation, autologous tissue transplantation and artificial material substitutes, but the results are not satisfactory. The key to the perfect application of artificial trachea is how to introduce materials that can regulate the growth and metabolism of tracheal mucosal cells on the surface of the artificial trachea lumen, so as to promote the crawling regeneration of mucosal epithelium. Among many biomaterials, collagen is considered to be an ideal choice. However, collagen is an allogeneic animal tissue extract, which has the potential to infect viruses and other diseases, and the production process is complicated, while silk fibroin is a natural protein with abundant sources and affordable prices. Foreign scholars have studied the growth of different human cell lines on silk fibroin membranes, and the results show that silk protein, like collagen, can promote cell growth.

Silk fibroin is a natural structural protein, mainly composed of three simple amino acids: glycine, alanine and serine, which account for about 85% of the total protein. The silk fibroin film has good biocompatibility in the skin, oral cavity and eyes, and its antigenicity is not obvious. The subcutaneous implantation test shows that there is no obvious inflammation or space occupying phenomenon. Studies have shown that because silk fibroin not only contains a considerable proportion of amino acid residues with polar groups such as OH, COOH, and NH ₂ , the regenerated silk fibroin membrane has strong hydrophilicity; It is rich in fibronectin, histone, etc., and has a strong ability to adhere to the surface of biological materials. The structural information carried on the surface of the regenerated silk fibroin membrane obviously plays an important role in the adhesion and proliferation of fibroblasts. On the other hand, it also contains a certain proportion of positively charged amino acid residues, which also creates good conditions for the adhesion and proliferation of fibroblasts on the surface of the regenerated silk fibroin membrane.

Silk fibroin has good biocompatibility, non-toxic, non-irritating, and has been widely used in biomedical fields, such as experimental research on medical silk fibroin skin regeneration membrane, contact lens, cell culture medium and artificial meninges etc. But so far, there is no report on the application of silk fibroin in the reconstruction of tracheal defects at home and abroad.

Contents of the invention

The object of the present invention is to provide a silk fibroin-coated titanium mesh. Another object of the present invention is to provide a method for preparing said titanium mesh. A further object of the present invention is to provide the application of the titanium mesh in clinical tracheal defect reconstruction.

The present invention adopts medical titanium mesh as supporting material, uses silk fibroin as coating to make silk fibroin-coated titanium mesh complex, and the concentration of said silk fibroin is 6-12% (weight/volume), and said The pores of the titanium mesh are not closed. The invention utilizes the good biocompatibility of silk fibroin and the effect of promoting the growth of fibroblasts and mucosal cells, can accelerate healing, reduce complications, and can effectively support the lumen for a long time or permanently.

The titanium mesh coated with silk fibroin of the present invention can be used as clinical tracheal defect repair, and can provide meaningful reference for exploring the role of silk fibroin in promoting tracheal mucosa repair and clinical reconstruction of tracheal defect, and reducing complications after tracheal reconstruction.

The silk fibroin-coated titanium mesh of the present invention is prepared by the following methods and steps:

1. Preparation of silk fibroin

Degumming the raw silk of waste mulberry silkworms in NaHCO ₃ (0.5wt%) aqueous solution, dissolving the degummed silk in 9.3mol/L LiBr aqueous solution, and stirring until the silk is completely dissolved. Filtrate to remove undissolved silk and impurities to obtain silk fibroin protein salt solution, dialyze with deionized water to remove LiBr in the solution, obtain silk protein with a concentration of 4%, then concentrate, dialyze with PEG, and remove the obtained silk fibroin protein solution by centrifugation Impurities, keep the supernatant. The obtained silk fibroin has a concentration of 20%, and is stored in a refrigerator at 4° C. for future use. The concentration of the silk fibroin solution is determined by a weighing method, and can be diluted to 6-12% solutions with different concentrations according to needs, while avoiding conformational changes.

2. Preparation of silk fibroin-coated titanium mesh

Use medical titanium mesh (commercially available) to make the shape of the trachea-like ring-like structure with the same curvature and thickness as the experimental animal trachea. After ethylene oxide fumigation and sterilization, the titanium mesh is repeatedly dipped in the above-mentioned low temperature. (4 degrees centigrade) the silk fibroin solution with a diluted concentration of 6-12% is stored, taken out, put into an oven to dry at 60 degrees, and repeat this for 6-10 times, so that the surface is evenly coated with silk fibroin material, and the titanium mesh The pores are not closed, and the above processes are performed aseptically.

3. Animal experiments on tracheal defect reconstruction

25 clean-grade New Zealand male white rabbits (provided by the Animal Experiment Center of Fudan University), weighing 2.3-2.5KG, with an average of 2.4KG, were used for 3 months, and were randomly divided into: titanium mesh coated with silk fibroin group (n=12), pure titanium mesh group (n=12). Normal rabbits (n=1) were used as controls.

result:

1) Titanium mesh reconstruction set with silk fibroin coating:

All the rabbits had no dyspnea after operation, and samples were taken at 4 weeks, 8 weeks, and 12 weeks after operation, and it was found that the structure of the tracheal reconstruction was ideal, the surface of the silk titanium mesh was covered with translucent fibrous tissue, and microvessels grew in the tissue. , and no adhesion to surrounding tissues.

Tracheal endoscopy showed smooth mucous membrane in the tracheal cavity, no obvious narrowing of the lumen, no scar and growth of granulation tissue.

No obvious tracheal stenosis was found in the CT examination of the trachea or the CT examination of the trachea excised and reconstructed, and there was only slight localized thickening of the tracheal mucosa in CT.

Histopathological section examination showed that the mucosal cell composition of the defect reconstruction site was not significantly different from that of the normal tracheal mucosa, mainly composed of ciliated columnar cells and goblet cells.

Scanning electron microscopy showed that the growth was good at 8 weeks, and the cilia were longer and denser, but the directionality was still slightly poorer and shorter and sparser than normal tracheal mucosal cilia, and returned to normal at 12 weeks, with no significant difference from normal tracheal mucosal cilia.

2) Pure titanium mesh reconstruction group

All rabbits were complicated with cervical subcutaneous emphysema to varying degrees after operation, among which 2 cases were complicated with systemic subcutaneous emphysema, 1 rabbit had diarrhea 5 days after implantation and 14 days after reconstruction, and the other rabbit had diarrhea after 14 days after implantation. Seventeen days after reconstruction, he developed diarrhea, did not eat, gradually lost weight, and died. Another rabbit had a subcutaneous mass in the neck after surgery, and scar tissue on the surface of the titanium mesh at the site of tracheal reconstruction was seen when the material was collected.

Tracheal endoscopy in dead rabbits showed the formation of sticky pus plugs in the tracheal cavity and the growth of red inflammatory granulation tissue at the reconstruction site. The lumen was obviously narrow, with scars, granulation tissue growth, and obvious secretions.

CT examination of the trachea showed severe stenosis of the tracheal cavity, or even no cavity at all.

Histopathological examination showed a large number of granulation tissue hyperplasia and a small amount of inflammatory cell infiltration. The submucosal fibrous tissue layer was thicker than that of the silk fibroin-coated titanium mesh reconstruction group, and the cilia were sparser.

Scanning electron microscopy showed that compared with the silk fibroin-coated titanium mesh reconstruction group, the cilia were sparser, shorter, less directional, and less self-cleaning, with a little secretion attached to the surface

3) Using SAS6.12 statistical software, using cmh chi-square, the scoring method is the modified ridit score, and using the time factor to test the difference in the cross-sectional area of the hyperplastic tissue at the narrowest part of the reconstructed trachea between the two groups and the corresponding trachea The difference in the internal cross-sectional area of the two groups; the difference in other postoperative complications (death by asphyxia; subcutaneous emphysema; neck infection) between the two groups was tested by the exact probability method. Table 1 is the postoperative complication "asphyxiation death" comparison.

Table 2 shows the postoperative complications of neck infection. Table 3 is the situation of subcutaneous emphysema.

Table 1

choking death No choking Total Silk fibroin-coated titanium mesh reconstruction group Pure titanium mesh reconstruction group 05 116 1111 Total 5 17 twenty two

Exact probability method test: the statistical difference is significant (p<0.05).

Table 2

neck infection No neck infection Total Silk fibroin-coated titanium mesh reconstruction group Pure titanium mesh reconstruction group 02 119 1111 Total 2 20 twenty two

Exact probability method test: the statistical difference was not significant (p>0.05).

table 3

subcutaneous emphysema No subcutaneous emphysema Total Silk fibroin-coated <u>titanium mesh</u> reconstruction group Titanium mesh alone reconstruction group 311 80 1111 Total 14 8 twenty two

Exact probability method test: the statistical difference is significant (p<0.05=.

4) Use 16-slice spiral CT (manufactured by GE, USA) AW4.0 work station for image analysis, and calculate the cross-sectional area of the hyperplastic tissue at the narrowest part of the rabbit reconstructed trachea and the internal cross-sectional area of the corresponding trachea. Table 4 shows the cross-sectional area (mm2) of the hyperplastic tissue at the most narrow part of the reconstructed trachea in rabbits. Table 5 is the inner cross-sectional area of the corresponding trachea.

Table 4

Asphyxiated rabbits at and within 4 weeks of reconstitution At 8 weeks of reconstruction At 12 weeks of reconstruction Silk Fibroin Coated Titanium Mesh Reconstruction Group 4.02, 1.35, 1.41 4.05, 5.31, 3.35, 5.78 3.56, 2.29, 2.25, 1.24 Pure titanium mesh reconstruction group 28.56, 21.75, 26.31, 25.76, 12.78, 16.65 14.80, 24.89, 9.21 14.56, 4.35

The results showed that the cross-sectional area of the hyperplastic tissue at the most narrow part of the trachea in rabbits reconstructed with silk fibroin-coated titanium mesh: the minimum value was 1.24mm ² , the maximum value was 5.78mm ² , the average value was 3.15±1.58mm ² , the middle The number of digits is 3.35.

The cross-sectional area of the hyperplastic tissue at the most narrow part of the reconstructed trachea in the pure titanium mesh reconstruction group: the minimum value was 4.35mm ² , the maximum value was 28.56mm ² , the average value was 18.15±7.85mm ² , and the median value was 16.65.

Two groups of data: using cmh chi square, the scoring method is the modified ridit score, and the difference in the cross-sectional area of the hyperplastic tissue at the narrowest part of the reconstructed trachea between the two groups was tested stratified by the time factor, and the statistical difference was significant (p< 0.05).

table 5:

Asphyxiated rabbits at and within 4 weeks of reconstitution At 8 weeks of reconstruction At 12 weeks of reconstruction Silk Fibroin Coated Titanium Mesh Reconstruction Group 4.02, 1.35, 1.41 4.05, 5.31, 3.35, 5.78 3.56, 2.29, 2.25, 1.24 Pure titanium mesh reconstruction group 28.56, 21.75, 26.31, 25.76, 12.78, 16.65 14.80, 24.89, 9.21 14.56, 4.35

The results showed that the internal cross-sectional area of the tracheal reconstruction site in the rabbit trachea reconstruction group coated with silk fibroin: the minimum value was 26.79mm ² , the maximum value was 29.69mm ² , and the average value was 28.37±0.86mm ² .

The internal cross-sectional area of the rabbit tracheal reconstruction in the simple titanium mesh reconstruction group: the minimum value was 26.31mm ² , the maximum value was 29.46mm ² , and the average value was 28.13±1.01mm ² .

Two groups of data: the difference in the internal cross-sectional area of the trachea at the reconstructed trachea between the two groups was tested by time factor stratification, and the statistical difference was not significant (p=0.19>0.05), and the time stratified factor test showed no statistical difference Significance (p=0.35>0.05).

The results of animal experiments of the present invention show that silk fibroin can significantly promote the growth of fibroblasts, and fibroblasts are closely attached to the surface of silk fibroin with the same direction and growth with polarity. The experimental results of the present invention also show that capillaries and trachea Mucosal cells grew well on the surface of fibroblasts induced by silk fibroblasts without hyperplasia of granulation tissue. The present invention finds that fibroblasts growing on the surface of silk fibroin do not proliferate unlimitedly, but have a certain polarity, layer and thickness, but the surface thickness of silk fibroin in different parts and areas is completely different.

The invention adopts medical titanium mesh as the supporting material, which can not only utilize the good biocompatibility of silk fibroin and the effect of promoting the growth of fibroblasts and mucosal cells, accelerate healing, reduce complications, but also effectively support the lumen for a long time or permanently. Compared with pure titanium mesh, titanium mesh with silk fibroin coating has more advantages for tracheal reconstruction in rabbits. Since silk can promote the growth of fibroblasts and capillaries, the trachea can be implanted two weeks before The translucent fibrous tissue on the surface of the final titanium mesh completely closes all the mesh holes. At this time, it is used for tracheal reconstruction and can completely seal the tracheal wall so that the inside and outside cannot communicate, avoiding the occurrence of subcutaneous emphysema and local infection. The mucosa of the normal trachea at the edge of the tracheal defect grows along the surface of the thin layer of fibroblasts on the surface of the silk fibroin-coated titanium mesh, covering the luminal surface of the stent completely in time and quickly, effectively preventing the growth of granulation, tracheal stenosis, and suffocation death due to stenosis and other complications. Histological examination showed that the mucosa growing on the surface of the material grew well. Electron microscopy showed that the cilia had good directionality, and the self-cleaning function was the same as that of normal tracheal mucosa. Moreover, the titanium mesh as a scaffold could maintain a certain degree of elasticity, length and sufficient hardness to resist Under negative pressure, the silk fibroin-coated titanium mesh was used for tracheal reconstruction, and no displacement of the titanium mesh was found. The growth of fibroblasts on the surface of the material and the residual tracheal edge tissue of the body were well fused.

The present invention uses silk fibroin-coated titanium mesh for tracheal reconstruction, which solves the problem of introducing materials that can regulate the growth and metabolism of tracheal mucosal cells on the surface of the artificial tracheal cavity, can promote the crawling of mucosal epithelium, and avoid postoperative tracheal stenosis , achieved satisfactory results, with five criteria for an ideal tracheal substitute: it is a synthetic material with high biocompatibility; the blood vessels are suitable for growing in the graft; it has epithelial tissue or is suitable for the growth of epithelial tissue; it has sufficient length To meet the different degrees of defect repair; with enough hardness to resist negative pressure. It can be further extended to clinical application in the reconstruction of large circular tracheal tissue defects.

Description of drawings

Figure 1-3 Endoscopic inspection results,

Among them, a: silk fibroin-coated titanium mesh reconstruction at 12 weeks, b: granulation hyperplasia after pure titanium mesh reconstruction, c: stenosis at 12 weeks after pure titanium mesh reconstruction.

Figure 2 shows the mucocilia at the site of silk fibroin-coated titanium mesh reconstruction,

Where a: ×2000 reconstruction at 4 weeks after operation, b: ×2000 reconstruction at 8 weeks after operation, c: reconstruction at ×2000 at 12 weeks after operation, it has returned to normal.

Figure 3 shows the mucosal cilia at the site of simple titanium mesh reconstruction.

Where a: ×2000 reconstruction at 4 weeks after operation, b: ×2000 reconstruction at 8 weeks after operation, c: reconstruction at ×2000 at 12 weeks after operation.

Figure 4 is the CT examination result of silk fibroin-coated titanium mesh reconstruction,

Where a: reconstruction at 4 weeks, b: reconstruction at 8 weeks, c: reconstruction at 12 weeks, d: sagittal reconstruction, e: AIR STRUCTURE shows tracheal stenosis at the reconstruction site (arrow points).

Figure 5 is the CT examination result of simple titanium mesh reconstruction.

Among them, a: reconstruction of asphyxia at 8 weeks, b: reconstruction of stenosis at 12 weeks, c: coronal reconstruction, d: sagittal reconstruction, e: AIR STRUCTURE shows tracheal stenosis at reconstruction (arrow points).

Detailed ways

Embodiment 1 prepares silk fibroin

Use waste mulberry silkworm raw silk to degumming in 100°C NaHCO ₃ (0.5wt%) aqueous solution for 45 minutes, and dissolve the degummed silk in 9.3mol/L LiBr aqueous solution, and keep stirring for about 2 hours at 45°C until the silk is completely dissolve. Filter with eight layers of medical gauze to remove undissolved silk and impurities. The obtained silk fibroin protein salt solution is dialyzed with deionized water for four days to remove LiBr in the solution. The cut-off molecular weight of the dialysis bag is 12,000-14,000Da. The prepared silk fibroin has a concentration of about 4%, further concentrating it, continuing to dialyze with 10% PEG for one day, centrifuging the dialyzed silk fibroin solution at 6000 rpm for 15 minutes to remove impurities, and leaving the supernatant. The obtained silk fibroin has a concentration of 20%, and it is stored in a refrigerator at 4° C. as a storage solution for future use. The concentration of the silk fibroin solution is determined by a weighing method, and can be diluted into solutions with different concentrations of 6-12% as required. The dialyzed sample should try to avoid strong stirring and shaking, as well as contact with organic solvents, etc., to prevent conformational changes, and try to avoid excessive stirring during the dilution process.

Example 2 Preparation of silk fibroin coated titanium mesh

Use medical titanium mesh (commercially available) to make the shape of a trachea-like ring-like structure with the same curvature and thickness as the experimental animal rabbit trachea. After ethylene oxide fumigation and sterilization, the titanium mesh is repeatedly dipped in The above-mentioned silk fibroin solution with a dilution concentration of 6% stored at low temperature is taken out and placed in an oven to dry at 60 degrees. Repeat this 6-10 times to make the surface evenly coated with silk fibroin material and keep the pores of the titanium mesh unsealed. All of the above processes were performed aseptically.

Example 3

Use medical titanium mesh (commercially available) to make the shape of a trachea-like ring-like structure with the same curvature and thickness as the experimental animal rabbit trachea. After ethylene oxide fumigation and sterilization, the titanium mesh is repeatedly dipped in The above-mentioned silk fibroin solution with a dilution concentration of 12% stored at low temperature is taken out and placed in an oven to dry at 60 degrees. Repeat this 6-8 times to make the surface evenly coated with silk fibroin material and keep the pores of the titanium mesh unsealed. All of the above processes were performed aseptically.

Example 4

Use medical titanium mesh (commercially available) to make the shape of a trachea-like ring-like structure with the same curvature and thickness as the experimental animal rabbit trachea. After ethylene oxide fumigation and sterilization, the titanium mesh is repeatedly dipped in The above-mentioned silk fibroin solution with a dilution concentration of 10% stored at low temperature is taken out and put into an oven to dry at 60 degrees. Repeat this 8 times so that the surface is evenly coated with silk fibroin material and the pores of the titanium mesh are not closed. The above process All aseptically operated.

Embodiment 5 animal experiments

25 clean-grade New Zealand male white rabbits (provided by the Animal Experiment Center of Fudan University), weighing 2.3-2.5KG, with an average of 2.4KG, were all 3 months old, and were randomly divided into: silk fibroin-coated titanium mesh group ( n=12), pure titanium mesh group (n=12). Normal rabbits (n=1) were used as controls.

1) Silk fibroin-coated titanium mesh reconstruction set:

7 days after implantation and 12 days after reconstruction, intramuscular injection of penicillin 100,000 u/time twice a day. Eat very little food within three days after the operation, and the food intake is basically normal on the fifth day. The postoperative body temperature is normal, basically around 39.3-39.5°C. 3 cases of postoperative slight neck subcutaneous emphysema, all rabbits in this group have no postoperative infection Complications occurred. Diarrhea occurred in a rabbit 14 days after reconstruction, and 10,000 units of gentamicin was given intramuscularly per day. After 3 days, the diarrhea stopped, but he did not eat, and died after gradually losing weight. All rabbits had no symptoms after surgery. Difficulty breathing, mild wheezing within 1 week after operation, which gradually disappeared after 1 week, samples were taken at 4 weeks, 8 weeks, and 12 weeks after operation, it can be seen that the structure of the tracheal reconstruction is ideal, and the surface of the silk titanium mesh covers half of the growth. Transparent fibrous tissue, and microvascular growth is seen in this thin layer of tissue, and there is no adhesion with the surrounding tissue.

Tracheal endoscopy:

Examination at 4 weeks, 8 weeks, and 12 weeks after operation showed that the mucosa in the tracheal cavity was smooth without obvious stenosis, no scar and growth of granulation tissue, and there was a little secretion on the surface of the mucosa at 4 weeks.

CT examination of the trachea after anesthesia and fixation in rabbits or CT examination of the trachea at the site of resection and reconstruction:

No obvious tracheal stenosis was found in the tracheal CT examination of all rabbits at 4, 8, and 12 weeks after operation, and there was only slight localized thickening of some tracheal mucosa on CT. AIR STRUCTURE imaging reconstruction also showed no stenosis in the reconstructed tracheal lumen .

Histopathological examination showed that the mucosal cell composition of the defect reconstruction site was not significantly different from that of the normal tracheal mucosa, mainly composed of ciliated columnar cells and goblet cells. Sparse, well covered at 8 weeks, mostly normal at 12 weeks. Fibroblasts in the submucosal thin layer grow orderly, it can be seen that the incompletely absorbed silk fibroin material has good biocompatibility, fibroblast tissue grows around the silk fibroin, a few monocytes are occasionally seen, and the antigenic reaction is not obvious or there is no obvious foreign body rejection reaction . Compared with the normal tracheal pathological section (HE×400), the former had a thin layer of fibrous tissue under the mucosa and abundant capillaries.

Scanning electron microscope examination: at 4 weeks after operation, the cilia were sparse and short, with poor directionality, poor self-cleaning function, more secretions attached to the surface, and some areas were exposed without cilia; at 8 weeks, the cilia grew well, and the cilia were longer and denser, but The directionality is still slightly worse and shorter and sparser than normal tracheal mucous membrane cilia, the self-cleaning function is better, and there is no obvious secretion on the surface; it returns to normal at 12 weeks, and there is no significant difference with normal tracheal mucous membrane cilia.

2) Pure titanium mesh reconstruction group

7 days after implantation and 12 days after reconstruction, intramuscular injection of penicillin 100,000 u/time twice a day, began to eat a little on the third day after the operation, and the food intake was basically normal on the seventh day. The body temperature behind the ears of the rabbits was around 40.5°C on the 2nd to 4th day after the operation. On the 4th day after the operation, 1 case died of suffocation due to the formation of sticky pus embolism in the trachea and the granulation at the reconstruction site, and the other case died one week after the operation. Subcutaneous infection in the front of the neck formed a fistula, and the body temperature of other rabbits was normal after operation, basically around 39.3-39.5°C. Another 4 rabbits died of asphyxiation on the 5th day, 7th day, 17th day and 60th day after operation respectively. All the rabbits in this group were complicated with neck subcutaneous emphysema to varying degrees after operation, and 2 cases were complicated with systemic subcutaneous emphysema, with the maximum pumping out about 300ml of gas from the subcutaneous back of their back every day. Diarrhea reappeared 14 days and 14 days after reconstruction, and gentamicin was injected intramuscularly at 10,000 units/day. After 3 days, the diarrhea stopped, and gradually began to eat and recovered. Another rabbit had diarrhea 17 days after reconstruction. , given intramuscular injection of gentamicin 10,000 units/day 2 days later, the diarrhea stopped, but he did not eat, gradually lost weight, and died after 3 days. Another case of postoperative subcutaneous mass in the neck of a rabbit, about 2cm in diameter, spherical with a smooth capsule. Scar tissue on the surface of the titanium mesh at the site of tracheal reconstruction can be seen during sampling.

Tracheal endoscopy examination of rabbits who died of asphyxiation 4 days after operation showed the formation of sticky pus plugs in the trachea cavity and the growth of red inflammatory granulation tissue at the reconstruction site, and the lumen was obviously narrowed. The rabbits died of suffocation on the 1st, 17th, and 60th days respectively. Tracheal endoscopy showed obvious granulation tissue formation in different degrees in the tracheal lumen, and the lumen was very narrow; after 4 weeks, 8 weeks, and 12 weeks after the operation, It can be seen that the mucosa in the tracheal cavity is not smooth, the lumen is narrowed to varying degrees, there are scars and granulation tissue growth, and there are obvious secretions on the surface of the tracheal mucosa at 4 weeks.

Postoperative CT examinations of rabbits including asphyxia death showed severe stenosis of the trachea, or even no cavity at all. Tracheal CT examinations at 4 weeks, 8 weeks, and 12 weeks after surgery showed different degrees of tracheal stenosis, and AIR STRUCTURE reconstruction also showed that the reconstruction site Tracheal lumen narrowing.

Histopathological examination of tracheal hyperplasia in 5 rabbits who died of asphyxia after operation, a large number of granulation tissue hyperplasia and a small amount of inflammatory cell infiltration can be seen; after reconstruction, there is fibrous scar tissue hyperplasia at the defect reconstruction site, and other cell composition is similar to that of normal trachea There was no significant difference in the mucosa, which mainly consisted of ciliated columnar cells and goblet cells. The growth of cilia on the mucosal surface: at 4 weeks after surgery, the cilia on the surface of the scar tissue grew poorly and the density was sparse; at 8 weeks, the cilia on the surface of the scar tissue grew slightly better, but It was worse than the silk fibroin-coated titanium mesh reconstruction group; at 12 weeks, the submucosal fibrous tissue layer was thicker than the silk fibroin-coated titanium mesh reconstruction group, and the cilia were also sparse.

The growth of cilia in the tracheal mucosa of rabbits was observed by scanning electron microscopy: compared with the silk fibroin-coated titanium mesh reconstruction group, the cilia were sparser, shorter, poorer in direction, poor in self-cleaning function at 4 weeks after operation, and there was a little secretion on the surface Attachment; the growth was slightly better at 8 weeks, the cilia were slightly longer and denser, but still worse than the silk fibroin-coated titanium mesh reconstruction group, some areas were not completely covered by cilia, and the directionality was not good; it was not fully recovered at 12 weeks Normal, compared with the normal tracheal mucosa and silk fibroin-coated titanium mesh reconstruction group, the directionality is not as good as that of the latter two groups, and the cilia are shorter than the latter two groups.

Claims (5)

1. A silk fibroin-coated titanium mesh is characterized in that a medical titanium mesh is used as a supporting material, and silk fibroin is used as a coating to make a silk fibroin-coated titanium mesh composite, and the weight of the silk fibroin is The volume concentration is 6-12%, and the pores of the titanium mesh are not closed. 2. The silk fibroin-coated titanium mesh according to claim 1, characterized in that the weight/volume concentration of the silk fibroin is 10%. 3. The silk fibroin-coated titanium mesh according to claim 1, characterized in that said titanium mesh is in the shape of a tracheal ring-like structure. 4. the preparation method of silk fibroin coating titanium mesh as claimed in claim 1 is characterized in that by following methods and steps: 1) Preparation of silk fibroin Degumming the waste mulberry silkworm silk in 0.5wt% NaHCO ₃ aqueous solution, dissolving the degummed silk in 9.3mol/L LiBr aqueous solution, and stirring until the silk dissolves. Filtrate to remove undissolved silk and impurities to obtain silk fibroin salt solution, dialyze with deionized water to remove LiBr in the solution, concentrate again, PEG dialyze, centrifuge, leave the supernatant to obtain a concentration of 20% silk fibroin, place Store in a refrigerator at 4°C for later use, measure the concentration of silk fibroin solution by weighing method, and dilute to 6-12% solutions with different concentrations according to needs, so as to avoid conformational changes; 2) Preparation of silk fibroin-coated titanium mesh The medical titanium mesh is used to make the shape of a trachea-like ring-like structure with the same curvature and thickness as the trachea. After ethylene oxide fumigation and sterilization, the titanium mesh is repeatedly dipped in the above-mentioned low-temperature storage at a dilution concentration of 6. -12% silk fibroin solution, take it out and put it in an oven to dry at 60 degrees, dip and dry it repeatedly for 6-10 times, so that the surface is evenly coated with silk fibroin material, and the pores of the titanium mesh are not closed. 5. The application of the silk fibroin-coated titanium mesh according to claim 1 in preparing a trachea for defect reconstruction.

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