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.
ObjectiveTo explore the possibility of constructing tissue engineered adipose by adipose tissue derived extracellular vesicles (hAT-EV) combined with decellularized adipose tissue (DAT) scaffolds, and to provide a new therapy for soft tissue defects.MethodsThe adipose tissue voluntarily donated by the liposuction patient was divided into two parts, one of them was decellularized and observed by HE and Masson staining and scanning electron microscope (SEM). Immunohistochemical staining and Western blot detection for collagen type Ⅰ and Ⅳ and laminin were also employed. Another one was incubated with exosome-removed complete medium for 48 hours, then centrifuged to collect the medium and to obtain hAT-EV via ultracentrifugation. The morphology of hAT-EV was observed by transmission electron microscopy; the nanoparticle tracking analyzer (NanoSight) was used to analyze the size distribution; Western blot was used to analyse membrane surface protein of hAT-EV. Adipose derived stem cells (ADSCs) were co-cultured with PKH26 fluorescently labeled hAT-EV, confocal fluorescence microscopy was used to observe the uptake of hAT-EV by ADSCs. Oil red O staining was used to evaluate adipogenic differentiation after hAT-EV and ADSCs co-cultured for 15 days. The DAT was scissored and then injected into the bilateral backs of 8 C57 mice (6-week-old). In experimental group, 0.2 mL hAT-EV was injected weekly, and 0.2 mL PBS was injected weekly in control group. After 12 weeks, the mice were sacrificed, and the new fat organisms on both sides were weighed. The amount of new fat was evaluated by HE and peri-lipoprotein immunofluorescence staining to evaluate the ability of hAT-EV to induce adipogenesis in vivo.ResultsAfter acellularization of adipose tissue, HE and Masson staining showed that DAT was mainly composed of loosely arranged collagen with no nucleus; SEM showed that no cells and cell fragments were found in DAT, and thick fibrous collagen bundles could be seen; immunohistochemical staining and Western blot detection showed that collagen type Ⅰ and Ⅳ and laminin were retained in DAT. It was found that hAT-EV exhibited a spherical shape of double-layer envelope, with high expressions of CD63, apoptosis-inducible factor 6 interacting protein antibody, tumor susceptibility gene 101, and the particle size of 97.9% hAT-EV ranged from 32.67 nmto 220.20 nm with a peak at 91.28 nm. Confocal fluorescence microscopy and oil red O staining showed that hAT-EV was absorbed by ADSCs and induced adipogenic differentiation. In vivo experiments showed that the wet weight of fat new organisms in the experimental group was significantly higher than that in the control group (t=2.278, P=0.048). HE staining showed that the structure of lipid droplets in the experimental group was more than that in the control group, and the collagen content in the control group was higher than that in the experimental group. The proportion of new fat in the experimental group was significantly higher than that in the control group ( t=4.648, P=0.017).ConclusionDAT carrying hAT-EV can be used as a new method to induce adipose tissue regeneration and has a potential application prospect in the repair of soft tissue defects.
ObjectiveTo explore an optimized protocol of decellularization to fabricate an ideal scaffold derived from porcine skeletal muscle acellular matrix.
MethodsSerial-step protocol of homogenating-milling-detergent method was used to fabricate decellularized porcine muscle tissue (DPMT) derived from native porcine skeletal muscle tissue from adult pig waist. Histological method was used to assess the effects of decellularization and degreasing. Sirius red staining was used to analyze collagen components. Scanning electron microscopy, BCA assay, and PicoGreen assay were used to evaluate the ultrastructure, total protein content, and DNA content in DPMT. The adipose derived stem cells (ADSCs), NIH3T3 cells, and human umbilical vein endothelial cells (HUVECs) were cultured in extraction liquor of DPMT in different concentrations for 1, 3, and 5 days, then the relative growth rate was calculated with cell counting kit 8 to assess the toxicity in vitro. Live/dead cell staining was used to evaluate the cytocompatibility by seeding HUVECs on the surface of DPMT and co-cultured in vitro for 3 days. For in vivo test, DPMT was subcutaneously implanted at dorsal site of male specific-pathogen free Sprague Dawley rats and harvested after 3, 7, 14, and 28 days. Gross obersvation was done and transverse diameter of remained DPMT in vivo was determined. HE staining and immunohistochemical staining of CD31 were used to assess inflammatory response and new capillary rings formation.
ResultsDecellularization of the porcine skeletal muscle tissue by homogenating-milling-detergent serial steps protocol was effective, time-saving, and simple, which could be finished within only 1 day. The decellularizarion and degreasing effect of DPMT was complete. The main component of DPMT was collagen type I and type IV. The DNA content in DPMT was (15.902±1.392) ng/mg dry weight, the total protein content was 68.94% of DPMT dry weight, which was significantly less than those of fresh skeletal muscle tissue[(140.727±10.422) ng/mg and 93.14%] (P<0.05). The microstructure of DPMT was homogeneous and porous. The result of cytocompatibility revealed that the cytotoxicity of DPMT was 0-1 grade, and HUVECs could stably grow on DPMT. In vivo study revealed DPMT could almost maintain its structural integrity at 14 days and it degraded completely at 28 days after implantation. The inflammatory response peaked at 3 days after implantation, and reduced obviously at 7 days. Difference was significant in the number of inflammatory cells between 2 time points (P<0.05). Neovascularization was observed at 7 days after implantation and the number of new vessels increased at 14 days, showing significant difference between at 7 and 14 days (P<0.05).
ConclusionThe homogenating-milling-detergent serial-steps protocol is effective, time-saving, and reproducible. The DPMT reveals to be cell and lipid free, with highly preserved protein component. DPMT has good biocompatibility both in vitro and in vivo and may also have potential in promoting neovascularization.
Objective To develop a new small-caliber vascular xenograft and evaluate the feasibility of xenogenic artery for coronary artery bypass grafting. Methods Canine carotid arteries were decellularized by detergent and enzymatic extraction. All decellularized xenografts were randomly divided into two groups. Heparin-linked group （n=24）： grafts were then covalently linked with heparin. Non-heparin-linked group （n=24）： as control. Xenografts in two groups were implanted in rabbits＇ left and right carotid artery respectively as bypass grafts. Graft patency was checked by ultrasonography after 3 weeks, 3 and 6 months. Grafts were harvested after 3 and 6 months. Microscopic observation and immunohistochemical staining were performed. Results All the cells were removed while the extracellular matrix were well preserved observed. Heparin was successfully linked to the grafts through their whole thickness. There was no obstruction at both sides after implantation of the grafts, while less thrombus was found in the decellularized heparin-linked grafts than in the other side. Smooth muscle cells densely populated the graft wall and endothelial cells covered the lumen at 3 months after implantation. Conclusion Canine common carotid artery treated by detergent and enzymatic extraction and heparin linkage may be a new small-caliber vascular xenograft for coronary artery bypass grafting.
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.
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.
【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.
Objective To observe whether Cyclo-RGDfK (Arg-Gly-Asp-D-Phe-Lys) could enhance the adhesion of myofibroblast to decellularized scaffolds and upregulate the expression of Integrin αVβ3 gene. Methods Myofibroblast from the rat thoracic aorta was acquired by primary cell culture. The expression of Vimentin and α-smooth muscle actin(α-SMA) has been detected by immunoflurescent labeling. Decellularized valves have been randomly divided into three groups (each n=7). Group A (blank control): valves do not receive any pretreatment; Group B: valves reacted with linking agent NEthylN(3dimethylaminopropyl)carbodiimide hydrochloride (EDC) for 36 hours before being seeded; Experimental group: Cyclo-RGD peptide has been covalently immobilized onto the surface of scaffolds by linking agent EDC. The fifth generation of myofibroblast has been planted on the scaffolds of each group. The adhesion of myofibroblast to the scaffolds was evaluated by HE staining and electron scanning microscope. The expression of Integrin αVβ3 was quantified by halfquantitative reverse transcriptionpolymerase china reaction (RT-PCR). Results We can see that myofibroblast has exhibited b positive staining for Vimentin and α-SMA. Besides, it has been shown that the expression of Integrin αVβ3 was much higher in the experimental group than that of the group A and group B(Plt;0.05). There was no statistically difference in group A and group B (P=0.900). Conclusion RGD pretreatment does enhance the adhesive efficiency of seeding cells to the scaffolds and this effect may be related to the upregulation of Integrin αVβ3.
ObjectiveExtracting the endothelial cells or all endothelial cells and interstitial cells from the cryopreserved homograft valves (HV), to evaluate the immunogenicity of this two kinds of decellular HV.
MethodsFor extracting the endothelial cells, the leaflet and wall of the HV were decellularized by a 4-step detergent-enzymatic extraction method involving the 1% triton in combination with RNase (1μg/ml) and DNase (10μg/ml). For extracting the endothelial cells and interstitial cells, the leaflet and wall of the HV were decellularized by a 3-step detergent-enzymatic extraction method involving the 1% deoxycholic acid (DOA) in combination with RNase (20μg/ml) and DNase (200μg/ml). HLA-DR antigen expression was detected by using immunohistochemical techniques. The valve and wall of the HV were transplanted subcutaneously in the mice for 8 weeks, and the histology, calcium assay and calcium content were examined.
ResultsFor the staining of the HLA-DR antigens, the immunogenic potential of the HV with extracting all endothelial cells and interstitial cells or only the endothelial cells was lower than cryopreserved HV, but it more obviously decreased for the HV with extracting all endothelial cells and interstitial cells. After 8 weeks embedded in the mice, the histological signs of the inflammatory reactions and the calcification extent to the cryopreserved HV and the HV with only extracting endothelial cells were stronger than the HV with extracting all endothelial cells and interstitial cells predominantly. And calcification extent or the inflammatory reactions to the wall of the HV were more severe than those of the leaflet.
ConclusionsThe immunogenicity of the HV with extracting all endothelial cells and interstitial cells is much less than HV with only extracting endothelial cells. The histological signs of the inflammatory reactions and the calcification extent in vivo experiments is obviously decreased. For the HV with only extracting endothelial cells, though the histological signs of the inflammatory reactions slightly decrease, the calcification extent in vivo experiments is more severe, especially for the wall. The interstitial cells may be the important factor for the donor-reactive immune responses that is related to the graft calcification or destruction after implantation.
ObjectiveTo review the properties of bio-derived hydrogels and their application and research progress in tissue engineering.
MethodsThe literature concerning the biol-derived hydrogels was extensively reviewed and analyzed.
ResultsBio-derived hydrogels can be divided into single-component hydrogels (collagen,hyaluronic acid,chitosan,alginate,silk fibroin,etc.) and multi-component hydrogels[Matrigel,the extract of extracellular matrix (ECM),and decellularized ECM].They have favorable biocompatibility and bioactivity because they are mostly extracted from the ECM of biological tissue.Among them,hydrogels derived from decellularized ECM,whose composition and structure are more in line with the requirements of bionics,have incomparable advantages and prospects.This kind of scaffold is the closest to the natural environment of the cell growth.
ConclusionBio-derived hydrogels have been widely used in tissue engineering research.Although there still exist many problems,such as the poor mechanical properties,rapid degradation,the immunogenicity or safety,vascularization,sterilization methods,and so on,with the deep-going study of optimization mechanism,desirable bio-derived hydrogels could be obtained,and thus be applied to clinical application.
