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,
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.
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
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