Abstract: Objective To observe the physical characteristics of decellularized porcine pulmonary valved conduits crosslinked by carbodiimide (EDC). Methods [WTBZ]Twenty porcine pulmonary valved arteries were mobilized on relative asepsis condition. They were cut longitudinally into three samples at the junction position of pulmonary valve (every sample was comprised of a part of the pulmonary conduit wall and the corresponding valve). The samples were randomly divided into three groups by lotdrawing method. Group A was the control group which was made up of the fresh porcine arterial valved conduit samples without any other treatments. Group B was comprised of porcine pulmonary samples decellularized by trypsindetergent digestion. Group Cincluded the decellularized porcine pulmonary samples crosslinked by EDC. We observed the water content, thickness, tensile strength, and shrinkage temperature of all the samples, based on which the physical characeteristics of these samples were analyzed. Results [WTBZ]Complete cellfree-pulmonary conduit matrix was achieved by trypsindetergent digestion. Compared with group A, in group B, the water content of pulmonary wall was significantly higher (P=0.000), while the water content of pulmonary valve was not significantly different; the thickness of pulmonary wall and valve (P=0.000，0.000) and tensile strength of pulmonary wall and valve (Plt;0.01） was significantly lower, while shrinkage temperature was not significantly different. Compared with group B, in group C, the water content of pulmonary wall was significantly lower (P=0.000), while the water content of pulmonary valve, and the thickness of pulmonary wall and valve were not significantly different; the tensile strength of pulmonary wall (Plt;0.01) and valve (P=0.000), and the shrinkage temperature of them (P=0.000, 0.000) were significantly higher. Compared with group A, in group C, the water content of pulmonary wall and valve, and the tensile strength of them were not statistically different, while the thickness of pulmonary wall and valve was significantly lower (P=0.000, 0.000), and the shrinkage temperature of them was significantly higher (P=0.000, 0.000). Conclusion [WTBZ]EDC crosslinking method is available for treating decellularized porcine pulmonary valved conduits in order to enhance its tensile strength, and decrease water content of pulmonary wall.
Objective To review the decellularized methods for obtaining extracellular matrix (ECM) and the applications of decellularized ECM scaffold in tissue engineering. Methods Recent and related literature was extensively and comprehensively reviewed. The decellularized methods were summarized and classified. The effects of different sterilization methods on decellularized scaffolds were analyzed; the evaluation criterion of extent of decellularization was put forward; and the application of decellularized ECM scaffold in different tissues and organs engineering field was summarized. Results The decellularized methods mainly include physical methods, chemical methods, and biological methods, and different decellularization methods have different effects on the extent of cell removal and ECM composition and structure. Therefore, the best decellularization method will be chosen according to the characteristics of the tissues and decellularization methods to achieve the ideal result. Conclusion It is very important to choose the appropriate decellularized method for preparing the biological materials desired by tissue engineering. The biological scaffolds prepared by decellularized methods will play an important role in tissue engineering and regenerative medicine.
Objective To investigate the feasibil ity of preparing the porous extracellular matrix (ECM) by use of some chemicals and enzymes to decellularize the porcine carotid artery. Methods The porcine carotid artery was procured, and warm ischemia time was less than 30 minunts. The porcine carotid artery was decellularized with 1% sodium dodecyl sulfate (SDS) for 60 hours to prepare common ECM; then common ECM was treated with 0.25% trypsin (for 6 hours) and 0.3 U/ mL collagenase (for 24 hours) to prepare porous ECM. The common ECM and porous ECM were stained with HE,Masson’s trichrome, and Orcein to evaluate the histological features. Then the mechanical property, cytotoxicity, and pore size of ECMs were determined. After 4 weeks of subcutaneous implantation in dogs, the histological examination was used for the study. Results Histological observation confirmed that 2 kinds of ECMs were decellularized completely and more porous structure was observed in porous ECM. Scanning electron microscope showed the pores in porous ECM were greater and the length of shorter axis in porous ECM ranged from 5 to 30 μm, the length of longer axis from 40 to 100 μm. The porosity of porous ECM (99.25%) was greater than that of common ECM (91.50%). The burst pressure of porous ECM decreased when compared with common ECM, showing significant difference [(0.154 3 ± 0.012 7) MPa vs [0.305 2 ± 0.015 7) MPa, P lt; 0.05]. There was no significant difference in suture retention strength between 2 kinds of ECMs (P gt; 0.05). The cytotoxicity test showed no obvious cytotoxicity in 2 kinds of ECMs. In vivo implantation test showed that the deeper host cells infiltration and more neo-microvessels in porous ECM were observed than in common ECM. Conclusion SDS and some enzymes can be used to prepare porous ECM as the scaffold for tissue engineered blood vessels.
Objective To investigate an optimal method for SD rat skeletal muscle decellularization. Methods Sixteen SD rats (male and female) weighing 180-200 g were used. Thirty-six skeletal muscle bundles obtained from 10 rats were randomly divided into 3 groups: normal group (group A, n=4) received non-decellularization; time group (group T, n=16) andconcentration group (group C, n=16) underwent decellularization using hypotonic-detergent method. Concentration of sodium dodecyl sulfate (SDS) was 1.0% for T group, which was subdivided into groups T1, T2, T3 and T4 (n=4 per subgroup) according to different processing durations (24, 48, 72 and 96 hours). Group C was treated for 48 hours and subdivided into groups C1, C2, C3 and C4 (n=4 per subgroup) according to different SDS concentrations (0.5%, 1.0%, 1.5% and 2.0%). The muscle bundles of each group underwent HE staining observation and hydroxyproline content detection in order to get the optimal decellularization condition. Seven of 14 complete skeletal muscle bundles obtained from 6 SD rats were treated with the optimal decellularization condition (experimental group), and the rest 7 muscle bundles served as normal control (control group). The muscle bundles of each group were evaluated with gross observation, Masson staining and biomechanical test. Results HE staining: there was no significant difference between groups T1, T2, C1, C2 and C3 and group A in terms of muscle fiber; portion of muscle fibers in group C4 were removed; muscle fibers in group T3 were fully removed with a complete basement membrane structure; muscle fibers of group T4 were fully removed, and the structure of basement membrane was partly damaged. Hydroxyprol ine content detection: there was no significant difference between group A and groups C1, C2, C3, T1 and T2 (P gt; 0.05); significant difference was evident between group A and groups C4, T3 and T4 (P lt; 0.05); the difference between group C4 and groups T3and T4 was significant (P lt; 0.05); no significant difference was evident between group T3 and group T4 (P gt; 0.05). The optimal decellularization condition was 4 , 1.0% SDS and 72 hours according to the results of HE staining and hydroxyproline content detection. Gross observation: the muscle bundles of the experimental group were pall id, half-transparent and fluffier comparing with the control group. Masson staining observation: the collagen fibers of the experimental group had a good continuity, and were fluffier comparing with control group. Biomechanics test: the maximum breaking load of the experimental group and the control group was (1.38 ± 0.35) N and (1.98 ± 0.77) N, respectively; the maximum extension displacement of the experimental group and the control group was (3.19 ± 3.23) mm and (3.56 ± 2.17) mm, respectively; there were no significant differences between two groups (P gt; 0.05). Conclusion Acellular matrix with intact ECM and complete removal of muscle fibers can be obtained by oscillatory treatment of rat skeletal muscle at 4℃ with 1% SDS for 72 hours.
【Abstract】 Objective To develop a novel cartilage acellular matrix (CACM) scaffold and to investigate its performance for cartilage tissue engineering. Methods Human cartilage microfilaments about 100 nm-5 μm were prepared after pulverization and gradient centrifugation and made into 3% suspension after acellularization treatment. After placing the suspension into moulds, 3-D porous CACM scaffolds were fabricated using a simple freeze-drying method. The scaffolds were cross-l inked by exposure to ultraviolet radiation and immersion in a carbodiimide solution 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysucinimide. The scaffolds were investigated by histological staining, SEM observation and porosity measurement, water absorption rate analysis. MTT test was also done to assess cytotoxicity of the scaffolds. After induced by conditioned medium including TGF-β1, canine BMSCs were seeded into the scaffold. Cell prol iferation and differentiation were analyzed using inverted microscope and SEM. Results The histological staining showed that there are no chondrocytefragments in the scaffolds and that toluidine blue, safranin O and anti-collagen II immunohistochemistry staining werepositive. The novel 3-D porous CACM scaffold had good pore interconnectivity with pore diameter (155 ± 34) μm, 91.3% ± 2.0% porosity and 2 451% ± 155% water absorption rate. The intrinsic cytotoxicity assessment of novel scaffolds using MTT test showed that the scaffolds had no cytotoxic effect on BMSCs. Inverted microscope showed that most of the cells attached to the scaffold. SEM micrographs indicated that cells covered the scaffolds uniformly and majority of the cells showed the round or ell iptic morphology with much matrix secretion. Conclusion The 3-D porous CACM scaffold reserved most of extracellular matrix after thoroughly decellularization, has good pore diameter and porosity, non-toxicity and good biocompatibil ity, which make it a suitable candidate as an alternative cell-carrier for cartilage tissue engineering.
【Abstract】 Objective To investigate the feasibil ity of applying enzymatic method to prepare decellularizedporcine aorta and to evaluate its biomechanical properties, immunogenicity and cell compatibil ity. Methods 0.1% trypsin- 0.01% EDTA was appl ied to extract cells from porcine aorta under 37 continuously vibrating condition and its histology and microstructure were observed. The thickness, stress-strain curve, ultimate tension stress (UTS) and strain of failure (SOF) were compared before and after decellularization for 48, 96 and 120 hours under uniaxial tensile tests, respectively. The histological change was observed at 1, 3 and 6 weeks after the decellularized tissue was implanted subcutaneously in 3 dogs. According to the HE stains and a semi-quantitative Wakitani grading method, gross changes, category and amounts of infiltrated cells and neo-capillaries were compared between pre- and post-decellularization of porcine aortae. Endothel ial cells from canine external jugular vein were seeded onto the decellularized patches to observe the cell compatibil ity. Results Microscopy and electron microscopies examination identified that cell components was completely removed from the fresh porcine aorta and Masson’ strichrome showed that the structure of matrix (fibrins) was maintained intact at 96 hours using the decellularization method. There were no significant differences in the thickness, UTS and SOF between before and after decellularization (P gt; 0.05). However, The UTS values showed a decrease tendency and SOF showed an increase tendency. The stress-strain curve also verified a decrease tendency in mechanical intensity and an increase one in ductil ity after decellularization. After implanting the acellularized matrix subcutaneously in canine, moderately lymphocyte infiltration was seen at the 1st week and the infiltration was replaced by fibroblasts accompanied by neocapillary formation at the 6th week. A semi-quantity histological evaluation showed that there were differences in gross observation, category and the numbers of the infiltrated cells between decellularized and non-decellularized tissues（P lt; 0.05). A cell monolayer was identified by HE staining and scanning electron microscopywhen the endothel ial cells were seeded onto the inner luminal surface of the scaffold, al igned at the same direction on the whole. Conclusion The decellularized porcine aortic scaffold, prepared by trypsin-EDTA extraction under continuously vibrating condition, could meet the requirements of tissue-engineering graft in biomechanical properties, immunogenicity and cell compatibil ity.
ObjectiveTo prepare the aortic extracellular matrix (ECM) scaffold by using different methods to decellularize porcine ascending aorta and to comprehensively compare the efficiency of decellularization and the damage of ECM, evaluation of biomechanical property and biocompatibil ity. MethodsThirty specimens of fresh porcine ascending aorta were randomly divided into 6 groups (n=5). The porcine ascending aorta was decellularized by 5 different protocols in groups A-E: 0.1% trypsin/0.02% ethylenediamine tetraacetic acid (EDTA)/PBS was used in group A, 1%Triton X-100/0.02% EDTA/ distilled water in group B, 1% sodium deoxycholic acid/distilled water in group C, 0.5% sodium deoxycholic acid/0.5% sodium dodecyl sulfate/distilled water in group D, and 1% deoxycholic acid/distilled water in group E; and the porcine ascending aorta was not decellularized as control in group F. The ascending aorta scaffolds were investigated by gross examination, HE staining, DNA quantitative analysis, immunohistochemistry, and scanning electron microscopy were used to observe the efficiency of decellularization, microstructure of the ECM, the damage of collagen type Ⅰ and elastin, the structure of intimal surface, and biomechanical property. The 90 Sprague Dawley rats were randomly divided into 6 groups (n=15). Each scaffold was implanted in the abdominal muscles of rats respectively to evaluate the immunogenicity and biocompatibil ity. ResultsHE staining and quantitative analysis of DNA showed that the cells were completely removed only in groups A and D. The expression of collagen type Ⅰ in group A was significantly lower than that in the other 5 groups (P < 0.05), and serious damage of the basement membrane and decreased beomechanical property were observed. The maximum stress and tensile strength in group A was significantly lower than those in the other groups (P < 0.05), and elongation at break was significantly higher than that in the other groups (P < 0.05). The destruction of collagen type Ⅰ was significant (P < 0.05) in group D, but the basement membrane was integrity, the biomechanical properties were close to the natural blood vessels (group F) (P > 0.05). Implantation results showed that the scaffold of group D had superior immunogenicity and histocompatibility to the scaffold of the other groups. The inflammatory reaction was gentle and the number of the inflammatory cell infiltration was lower in group D than in other groups (P < 0.05). ConclusionIt is concludes that 0.5% sodium deoxycholic acid/0.5% sodium dodecyl sulfate/distilled water is more suitable for the decellularization of porcine aorta, by which the acquired ECM scaffold has the potential for constructing tissue engineered vessel.
ObjectiveTo analyze the effectiveness of a new type of decellularized allogeneic bone in the application of anterior cervical discectomy and fusion (ACDF). MethodsA retrospective analysis was made on the clinical data of 73 patients with single segmental cervical spondylosis treated with ACDF between January 2009 and December 2013. Of 73 cases, autologous iliac bone was used in 22 cases (group A), new decellularized allogeneic bone transplantation (Bio-Gene) in 22 cases (group B), and normal allogeneic bone (Xin Kang Chen) in 24 cases (group C). There was no significant difference in gender, age, type of cervical spondylosis, course of disease, and involved segment among 3 groups (P>0.05). The operation time, intraoperative blood loss, and complications were compared between groups; X-ray films and CT images were taken to observe the bone fusion, and Japanese Orthopaedic Association (JOA) score was used to assess the clinical efficacy. ResultsThe operation time and intraoperative blood loss of group A were significantly more than those of groups B and C (P<0.05), but no significant difference was found between groups B and C (P>0.05). Pain and numbness at donor site occurred in 12 cases, and poor healing in 1 case of group A; red swelling and exudate were observed in 1 case of group B and in 6 cases of group C; and there was significant difference in complications among 3 groups (χ2=18.82, P=0.00). All patients were followed up 6-54 months (mean, 30 months). The graft fusion rate was 100% in groups A and B, and was 95.8% in group C, showing no significant difference (χ2=2.04, P=0.36). The JOA score at 6 months after operation were significantly improved when compared with preoperative score in 3 groups (P<0.05), but no significant difference was found among the 3 groups at preoperation and 6 months after operation (P>0.05). The excellent and good rates of groups A, B, and C were 90.9%, 88.9%, and 87.5% respectively, showing no significant difference (χ2=0.14, P=0.93). ConclusionNew type of decellularized allogeneic bone in ACDF has the advantages of shorter operation time, less blood loss, and better early effectiveness. But whether there is a chronic rejection or delayed rejection needs further studies.