Comparative Study on Degradation Process of Acellular Porcine Small Intestinal Submucosa Matrix In Vivo and In Vitro

by Publications from Biosis Healing

Xiaolong Zheng1, Yi Chen1, Fumin Men1, Yang Zhang1, Yongsheng Li2, Hongquan Wang1,

2 College of Materials Science and Engineering, Hunan University, Changsha Hunan

Abstract

Degradation, In Vivo and In Vitro, Porcine Small Intestinal Submucosa, Biomaterial

1 Introduction
Acellular tissue matrix (ACTM) is a natural biodegradable material that removes the immunogenic substances in tissues from physicochemical and other methods, and retains the extracellular matrix (ECM). The primary role of ACTM materials is to provide a site for tissue cell growth while guiding tissue regeneration and providing tissue with some mechanical strength. Because the material is removed from the immunogenic substance, the tissue compatibility is good, and at the same time, it has a certain mechanical strength to support tissue reconstruction, so it is an ideal tissue repair material. The degradation rate of natural biodegradable materials has a great impact on the safety and effectiveness of material implantation. When the material degrades too fast, it can not provide biological and mechanical properties to the tissue, which leads to surgical failure and increased complications. Degradation is too slow, which will affect the regeneration and repair of the tissue [1], so the biological material should maintain its own characteristics before the tissue is completely repaired, so that the tissue can be reconstructed.
The acellular small intestinal submucosal matrix material (SIS) is a biological material obtained by removing the immune component from the small intestine of the pig source and retaining the ECM. The material is subject to its excellent physical and chemical properties, biocompatibility and degradable absorption capacity. More and more attention [2], SIS has been widely used in abdominal wall repair, tendon repair and dural repair. Zhang Xihai et al. studied the repair of abdominal wall defects in the small intestinal submucosa. The results showed that the animals had no adverse reactions and no sputum occurred. There was no rupture of the sputum patch at 12 weeks [3]. Song Zhicheng et al. performed tissue engineering scaffolding of small intestinal submucosa and tendon cells to repair the abdominal wall defect of rats. The experiment showed that the vascular growth and muscle tissue grew out at the junction of the stent and muscle tissue, and the mechanical properties showed that the mechanical strength of the stent was greater than that of SD rats. Abdominal wall strength [4]. Although the material has been proven to have good compatibility and high strength mechanical properties, the in vivo and in vitro degradation studies of materials have rarely been reported. In this paper, SIS was used to perform type I collagenase, proteinase K in vitro degradation experiments, subcutaneous implantation and abdominal wall repair experiments, to compare the degradation trend in vitro and in vivo, to explore the correlation between degradation and tissue repair, and to use SIS for tissue repair and degradation in vivo. Research provides theoretical basis

2. Experimental part
2.1. Main raw materials
Acellular porcine small intestinal submucosal matrix material (VIDASIS), product of Beijing Bohui Ruijin Biotechnology Co., Ltd.; proteinase K (Merck, Merck, Germany; type I collagenase (C0130, Sigma)).
Protease K: Accurately weigh 20 mg of enzyme, dissolve it to 100 mL with PBS, and take 2 mL of the volumetric solution to a volume of 100 mL with PBS solution. Type I collagenase: Accurately weigh 50 mg of type I collagenase, dissolve it in PBS, and dilute to a 100 mL volumetric flask.
2.2. Main instruments
The main instruments used in this study are shown in Table 1.
2.3. Type I collagenase degradation experiment
Take 1 × 2 cm2 SIS and weigh it into a 5 mL centrifuge tube. Add type I collagenase (sample: enzyme solution = 4 mg: 1 mL) in a certain ratio and react in a constant temperature shaker (37 ° C, 200). Rpm) at 3 h, 6 h, 9 h, 12 h, 20 h, 28 h, 36 h, 48 h,
At 60 h, 72 h, and 96 h, the samples were taken out and weighed to calculate the degradation rate.
2.4. Protease K Degradation Experiment
Take 2 × 0.7 cm2 SIS to dry and weigh, and add proteinase K according to a certain ratio (sample: enzyme solution = 5 mg: 1 mL), water bath 56 ° C, respectively at 15 min, 30 min, 45 min, 60 min, 75 min , 90 min, 105 min, 120 min, take the sample and weigh it, calculate the sample
Product degradation rate.
2.5. Subcutaneous degradation test in rats
Sixteen Wistar rats (conventional body weight 100 g~140 g, approved by the animal ethics of the National Laboratory for Blood Safety and Security of the Institute of Military Blood Transfusion, Chinese Academy of Military Medical Sciences) were randomly divided into 4 groups: the first week group, Week 4, Week 8 and Week 12 (4 in each group, half male and half female). Each rat was anesthetized by intraperitoneal injection of 3% sodium pentobarbital (30 mg/kg body weight). Under sterile conditions, the longitudinal incision was made in the middle of the abdomen, the subcutaneous tissue was bluntly separated on both sides, and 40 mm × 70 mm SIS was embedded, and the skin was sutured.
Dalbergine (20 mg / kg body weight), once a day, continuous administration for 3 days, normal feeding, eating and drinking water. At different times, rats were anesthetized and the absorption and degradation of SIS at the implant site were visually observed and recorded.
2.6. Rabbit abdominal wall implantation degradation experiment
New Zealand rabbits (conventional weight 2.5-3.0 kg, approved by the Animal Ethics Institute of Shandong Academy of Medical Sciences) were weighed and intravenously anesthetized with 3% pentobarbital sodium at a dose of 1.5 mL/kg. After the rabbit was anesthetized, it was fixed on the back, and the abdominal coat was removed, and the iodophor was disinfected. Spread a sterile hole towel,

Table 1. Mainly used instruments in this study

2. Experimental part
2.1. Main raw materials
Acellular porcine small intestinal submucosal matrix material (VIDASIS), product of Beijing Bohui Ruijin Biotechnology Co., Ltd.; proteinase K (Merck, Merck, Germany; type I collagenase (C0130, Sigma)).
Protease K: Accurately weigh 20 mg of enzyme, dissolve it to 100 mL with PBS, and take 2 mL of the volumetric solution to a volume of 100 mL with PBS solution. Type I collagenase: Accurately weigh 50 mg of type I collagenase, dissolve it in PBS, and dilute to a 100 mL volumetric flask.
2.2. Main instruments
The main instruments used in this study are shown in Table 1.
2.3. Type I collagenase degradation experiment
Take 1 × 2 cm2 SIS and weigh it into a 5 mL centrifuge tube. Add type I collagenase (sample: enzyme solution = 4 mg: 1 mL) in a certain ratio and react in a constant temperature shaker (37 ° C, 200). Rpm) at 3 h, 6 h, 9 h, 12 h, 20 h, 28 h, 36 h, 48 h,
At 60 h, 72 h, and 96 h, the samples were taken out and weighed to calculate the degradation rate.

2.4. Protease K Degradation Experiment
Take 2 × 0.7 cm2 SIS to dry and weigh, and add proteinase K according to a certain ratio (sample: enzyme solution = 5 mg: 1 mL), water bath 56 ° C, respectively at 15 min, 30 min, 45 min, 60 min, 75 min , 90 min, 105 min, 120 min, take the sample and weigh it, calculate the sample
Product degradation rate.
2.5. Subcutaneous degradation test in rats
Sixteen Wistar rats (conventional body weight 100 g~140 g, approved by the animal ethics of the National Laboratory for Blood Safety and Security of the Institute of Military Blood Transfusion, Chinese Academy of Military Medical Sciences) were randomly divided into 4 groups: the first week group, Week 4, Week 8 and Week 12 (4 in each group, half male and half female). Each rat was anesthetized by intraperitoneal injection of 3% sodium pentobarbital (30 mg/kg body weight). Under sterile conditions, the longitudinal incision was made in the middle of the abdomen, the subcutaneous tissue was bluntly separated on both sides, and 40 mm × 70 mm SIS was embedded, and the skin was sutured.
Dalbergine (20 mg / kg body weight), once a day, continuous administration for 3 days, normal feeding, eating and drinking water. At different times, rats were anesthetized and the absorption and degradation of SIS at the implant site were visually observed and recorded.
2.6. Rabbit abdominal wall implantation degradation experiment
New Zealand rabbits (conventional weight 2.5-3.0 kg, approved by the Animal Ethics Institute of Shandong Academy of Medical Sciences) were weighed and intravenously anesthetized with 3% pentobarbital sodium at a dose of 1.5 mL/kg. After the rabbit was anesthetized, it was fixed on the back, and the abdominal coat was removed, and the iodophor was disinfected. Spread a sterile hole towel,

3. Results
3.1. Type I collagenase degradation
As shown in Fig. 2, the in vitro degradation process of type I collagenase showed that the sample quality decreased gradually with the prolongation of degradation time. The sample in the degradation solution was gradually reduced with the prolongation of degradation time, and the solid debris of the experimental sample was hydrolyzed after 96 h. The quality is almost zero. It can be seen from the degradation rate and degradation curve that the degradation rate of the sample at 6 h is about 29%, the degradation rate at 40 h is 40%, the degradation rate at 20 h is about 68%, and the degradation rate of the sample at 96 h is over 90%.
                    

                 

                       Figure 2. In vitro degradation curve of SIS dissolved in protease solution of type I collagenase

3.2. Protease K degradation
Further in vitro degradation experiments using proteinase K are shown in Figure 3. Compared to type I collagenase, proteinase K has a stronger enzymatic hydrolysis capacity for SIS materials and a faster degradation process in vitro. Through the degradation curve, it can be found that the degradation rate is 29.67% at 15 min and more than half at 60 min.
The product has been degraded and the degradation rate is 56.33%. At 90 min, most of the samples have been degraded, the degradation rate is 71.62%, and the degradation rate is over 90% at 120 min.
3.3. Subcutaneous degradation in rats
The subcutaneous implantation of rats was used to observe the degradation of SIS in vivo. After 1, 4, 8 and 12 weeks of SIS implantation in the abdominal of rats, SIS was gradually absorbed with the extension of the implantation cycle. After 1 week of implantation, the complete SIS was visible, surrounded by a small amount of connective tissue.
Easy to peel off; after 4 weeks of implantation, the volume of SIS began to decrease (approximately one-fifth of the reduction by visual observation), and the connective tissue around the sample increased, but it was easy to peel off; after 8 weeks of implantation, the volume of SIS was significantly reduced (about five 2), the sample is surrounded by a large amount of connective tissue, still peelable;
After 12 weeks, the SIS basically completed tissue repair and reconstruction, and the sample (retained slightly more than one-half) was tightly wrapped around the connective tissue and was not easily peeled off. In this way, the material degradation rates of SIS implanted at 1, 4, 8, and 12 weeks were 0%, 20%, 40%, and 45%, respectively.
As shown in Figure 4.

Figure 3. Degradation curve of SIS dissolved in protease K solution

3.4. Rabbit abdominal wall implantation experiment
The SIS was implanted into the rabbit peritoneum to observe the degradation process of the sample in the peritoneum. It was found that there was no adhesion between the SIS and the surrounding tissues, no prolapse, deformation and displacement. No blood clots were seen around the repaired piece, and no fibrous envelope was formed around it. The degradation curve is shown in Figure 5. After 2 weeks, 4 weeks, and 8 weeks of implantation of the intraperitoneal wound in the animal, the SIS was soft and compliant, and no shrinkage was found. At 16 and 24 weeks, the material was fused with the tissue. Unable to separate. After 2 weeks of implantation, the intact SIS was visible, with no degradation, and the material was infiltrated with a small amount of fibrous tissue, which was easily separated from the surrounding area. After 4 weeks of implantation, the SIS part was replaced by tissue (visual observation was reduced by about a quarter) ), the fibrous tissue around the material is increased, but it is easy to peel off; after 8 weeks of implantation, the SIS is increased by the tissue replacement part (about one-half), and the material is infiltrated by a large amount of fibrous tissue, and can still be peeled off;
After 16 weeks of implantation, the SIS basically completed tissue repair and reconstruction. The material (about four-fifths) was completely fused with the neonatal peritoneum and could not be separated. After 24 weeks of implantation, the peritoneal tissue was almost completely regenerated and no material could be observed. ,As shown in Figure 6.

3.5. In vitro and in vivo fitting
The results of in vivo and in vitro degradation experiments indicate that SIS is a degradable material. At 12 weeks, the material can basically complete tissue repair and reconstruction. The corresponding type I collagenase degradation time is about 12 h, and the degradation in vitro corresponds to in vitro degradation. h, while the body degrades for 24 weeks
The in vitro degradation time should be 96 h. From the analysis of the amount of degradation of materials, the key time point of in vitro degradation time is 12 hours (corresponding to 8~12 weeks in vivo). At this time, most of the degradation of the material appears, which means that most of the materials in the body are degraded and absorbed, and the mechanical properties are poor. Complete material reinforcement and repair
For the purpose of complex and reconstructed defect tissue, the in vitro simulated degradation process of the material should be controlled at 12 h with less than 50% degradation (type I collagenase), or 60 min degradation less than 56.33% (proteinase K). Week corresponds to 6 h (type I collagenase) and 30 min (proteinase K) in the in vitro degradation experiment, and 16 weeks after the implantation is equivalent to 48 h (type I collagenase) and 105 min (proteinase K) in vitro degradation experiments. The complete degradation time in vivo corresponds to 96 h (type I collagenase) and 120 min (proteinase K) in vitro degradation experiments. The degradation data are shown in Table 2. The degradation time in vitro for 12 weeks compared with the in vitro degradation is shown in the dotted line. The time for complete degradation of the body corresponding to in vitro degradation is shown in the solid-line box in Figure 7.

4. Discussion
With the increase of clinical demand, biological materials have a series of complications, such as intestinal infarction, intestinal fistula, abdominal wall adhesion, etc. The study found that the complications have a great relationship with the degradation rate of the material, so the material degradation law is clarified. It is of great significance for follow-up work [8]. Material degradation includes in vivo degradation and in vitro simulated degradation. In vivo degradation usually involves implanting the material into the subcutaneous or repair site, observing and recording the degradation of the material and tissue reconstruction. This method is closer to clinical application, but it has high cost and long cycle. And the disadvantages of individual differences between different animals, and in vitro degradation experiments generally immerse the material in simulated body fluids (such as PBS, SBF, etc.) or enzyme solutions (such as collagenase, protease, etc.) to observe the degradation of the material. The method adopts a simplified model, no cells and other participation, and has the advantages of good controllability and short cycle. As a rapid evaluation method for animal experiments and pre-clinical methods, it reduces the cost of animal use and animal experiment research and shortens the material development process. The material development cycle has an important role [5] [6] [7]. Therefore, establishing the correlation between the degradation of SIS materials in vitro and in vivo is of great significance for understanding the degradation behavior, tissue compatibility and material development of SIS materials. In this experiment, type I collagenase and proteinase K solution were used to simulate the in vivo degradation experiment, and the in vivo degradation behavior of the material was studied by subcutaneous implantation experiment and rabbit abdominal repair experiment. The in vitro and in vivo degradation curves of the material were fitted to establish the correlation between degradation in vitro and in vivo. SIS materials contain type I collagen, a small amount of type III and type IV collagen, and also contain a variety of cytokines, such as fibroblast growth factor, transforming growth factor, vascular endothelial growth factor, etc., in addition to elastic fibers, mucin, Ingredients such as GAGs [9]. Type I collagenase is a specific hydrolase of type I collagen. Collagen content in SIS materials is over 93%, and type I collagen accounts for about 40% of total collagen. From the in vitro degradation process data of type I collagenase, it can be found that the material becomes smaller and the structure becomes more and more loose with the prolongation of degradation time, and the rate is faster in the initial stage of degradation, and the degradation rate of 40 h after enzymatic hydrolysis reaches 40%, degradation at 20 h. The rate is about 68%, and the late speed begins to slow down. This is mainly because the initial type I collagen content is more. With the degradation, the type I collagen content is gradually reduced, and the degradation rate of the enzymatic hydrolysis 96 h is over 90%. Complete degradation, this may be because type I collagen as a main component of SIS has a certain maintenance effect on the material structure, when it is destroyed, the material loses support and the three-dimensional structure is destroyed. Further, proteinase K was used for in vitro degradation experiments. Compared with type I collagenase, proteinase K is a broad-cut serine protease that can be used to digest various proteins and is a broad-spectrum protein digestive enzyme. It can be seen from the experimental data that the digestion rate of proteinase K is significantly higher than that of type I collagenase. This is because SIS materials contain other proteins in addition to type I collagen, including type III collagen, type IV collagen and some elastin. Etc., these can be used as the digestion object of proteinase K. In proteinase K, the degradation rate of the material was 56.33% at 60 min and the degradation rate was over 90% at 120 min.
After the material was implanted into the subcutaneous or abdominal wall of the rat, the degradation process of the material in the two tissues was similar. After the SIS was implanted, the SIS was gradually absorbed and bound to the surrounding tissue as the implantation time prolonged. Complete the repair and reconstruction of the organization. By observing the changes of the material in the body, it was found that the material did not change after 1~2 weeks of implantation. By 4 weeks, the material began to degrade. By 12 weeks, the material degraded by about 50%, and the peritoneal tissue was almost completely completed by 24 weeks. Regeneration, no material can be observed, the material can be considered to have been completely degraded.
A comprehensive comparison of the results of in vitro and in vivo degradation shows that the in vitro and in vivo degradation curves of SIS materials are similar. From the analysis of degradation amount, the initial 4 weeks of in vivo implantation corresponds to 6 h of in vitro degradation experiments (type I collagenase), 30 Min (proteinase K), equivalent to 12 weeks after implantation
Degradation of 12 h (collagenase), or 60 min (proteinase K), and the 16 weeks after implantation corresponds to 48 h (collagenase) and 105 min (proteinase K) in vitro degradation experiments. The complete degradation time in vivo is equivalent to in vitro. Degradation experiments of 96 h (collagenase), 120 min (proteinase K). The in vitro degradation rate is faster than the in vivo degradation rate. The possible reason is that the in vitro enzyme concentration is higher than the in vivo concentration, and the enzyme is easily accessible to the entire body of the material in a liquid environment in vitro. Sun et al. studied the biodegradability of polyglycolide lactide in vitro and in vivo and found that the in vitro degradation rate is lower than the in vivo degradation rate.
Rate [10], the reason that the results of this study differ from other studies may be due to the different mechanisms of degradation of synthetic materials and extracellular matrix materials in vivo. The degradation of synthetic materials is mainly carried out by hydrolysis, while extracellular matrix materials The degradation is mainly enzymatic degradation [11].
In summary, by studying the degradation behavior of SIS materials in vitro and in vivo, it is found that the in vitro degradation behavior has a certain correlation with the degradation behavior in vivo, and the degradation rate in vitro is faster than the degradation rate in vivo. This study preliminarily reveals the correlation between in vitro and in vivo degradation, and provides a reference for in vitro simulation of in vivo degradation, which helps to quickly evaluate the degradation behavior of materials in vitro and shorten the development cycle of biodegradable bio-patches.

Thanks
This study was supported by the National 863 Program New Materials Area (2015AA033602) and the Science and Technology SME Innovation Fund (Z14010101281).
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https://doi.org/10.1161/01.RES.0000070112.80711.3D