Objective
To investigate the effect of repeated freezing and thawing combining nuclease treatment on the decellularization of bovine tendons, and the morphology, structure, biochemical compositions, and mechanical properties of the decellularized tendons.
Methods
A total of 48 fresh 1-day-old bovine Achilles tendons were randomly divided into 3 groups (n=16): fresh normal tendons (group A), repeated freezing and thawing for 5 times (liquid nitrogen refrigeration/37℃ thawing, group B), and repeated freezing and thawing combining nuclease processing for 24 hours (group C). In each group, 2 tendons were used for scanning electron microscope (SEM), 3 tendons for histological and immunohistochemical observations, 3 tendons for DNA content detection, and 8 tendons for biomechanical testing.
Results
SEM observation indicated the intact, aligned, and densely packed collagen fibers with no disruption in groups A and B, and the slightly loose collagen fibers with little disruption in group C. The alcian blue staining, sirius red staining, and immunohistochemical staining showed that the most of glycosaminoglycan, collagen type I, collagen type III, and fibronectin in group C were retained after decellularization treatment. HE and DAPI staining showed that the cell nuclei between the collagen fibers were clearly visible in groups A and B; however, the cell nuclei between collagen fibers almost were invisible with a few residual nuclei on the endotendineum in group C. DNA quantitative detection confirmed that DNA content in group C [(0.05 ± 0.02) μg/mg] was significantly lower than those in group A [(0.24 ± 0.12) μg/mg] and group B [(0.16 ± 0.07) μg/mg] (P lt; 0.05). Biomechanical testing showed that the values of tensile strength, failure strain, stiffness, and elastic modulus were different among 3 groups, but no significant difference was found (P gt; 0.05).
Conclusion
Repeated freezing and thawing combining nuclease processing can effectively remove the component of cells, and simultaneously retain the original collagen fibrous structure, morphology, most of the extracellular matrix compositions, and mechanical properties of the bovine tendons.
Objective To study and test novel hybrid valves in vitro and in vivo, and provide basis for clinical use in future. Methods The hybrid valves were fabricated from decellularized porcine aortic valves coated with poly (3-hydroxybutyrate-co-3hydroxyhexanoate, PHBHHx).（1）In the mechanical test in vitro, the uniaxial tensile biomechanics test of the fresh (n=12), uncoated (n=12) and hybrid valve leaflets (n=12) were investigated. (2）In study in vivo, hybrid valves(n=5) implanted in pulmonary position in sheep without cardiopulmonary bypass. Uncoated grafts (n=5) used as control. The specimens of the hybrid or uncoated valve in sheep were explanted and examined by scanning electron microscopy, histology, calcium content and immunofluorescence staining 18 weeks after surgery. Results The mechanical test in vitro revealed that coating with PHBHHx increased maximal tensile strength of hybrid valves compared with the fresh and uncoated state (P＜0.05). The results in vivo indicated the hybrid valves maintained original shape and softness. Immunofluorescence staining for CD31 confirmed that the surface of hybrid valve was covered by confluent CD31+ cells.The interstitium of hybrid valve indicated that smooth muscle actin (SMA)+ cells population were similar to native valvular tissue. The calcium content of hybrid valve was significantly lower than that of uncoated valve leaflets (P＜0.05). Conclusion Decellularized porcine aortic valves coated with PHBHHx have good biological and biomechanical characteristics. The hybrid valve may provide superior valve replacement with current techniques.
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.
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.
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.
【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 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 investigate the effect of canine decellularized tendon slices (DTSs) on tendon-bone healing in repairing rotator cuff injury of rabbit.
Methods
Canine DTSs were prepared by repetitive freeze/thaw 5 times combined with nuclease processing for 12 hours from the adult Beagles Achilles tendons. Histological observation and cytocompatibility evaluation for the canine DTSs were performed in vitro. Twenty-four mature male New Zealand white rabbits, weighing 2.5-3.0 kg, were randomly selected. U-shaped defect of more than 50% of normal tendon in width and 8 mm in length was made in infraspinatus tendons of unilateral limb as the experimental group; the canine DTSs were used to repair defect, and the insertion of infraspinatus tendon on greater tuberosity of humerus was reconstructed in the experimental group. No treatment was done on the contralateral limb as the control group. At 4, 8, and 12 weeks after operation, the specimens were harvested for histological observation and biomechanical test.
Results
Histological examination showed that collagen fibers of canine DTSs were well preserved, without residual cells. The cytocompatibility examination showed that fibroblasts attached well to canine DTSs. Biomechanical test showed that the maximum load and stiffness increased significantly with time, and the maximum load and stiffness at 12 weeks were significantly higher than those at 4 and 8 weeks (P lt; 0.05). The maximum load and stiffness of the experimental group at 4 and 8 weeks were significantly lower than those of the control group (P lt; 0.05). The stiffness of the experimental group at 12 weeks was significantly lower than that of the control group (t=
—
5.679, P=0.000), but no significant difference was found in the maximum load at 12 weeks between 2 groups (t=0.969, P=0.361). Histological observation showed that the control group displayed a 4-layer structure of the tendon-bone insertion. In the experimental group at 4 weeks, the tendon-bone interface was filled with granulation tissue, and a small amount of Sharpey’s fibers-like connected the tendon to bone; granulation tissue disappeared, and fibroblasts, Sharpey’s fiber, new cartilage, and chondrocytes significantly increased with time; tendon-bone interface became mature, but the tide line was not observed between the unmineralized fibrocartilage and mineralized fibrocartilage.
Conclusion
Canine DTSs prepared by repetitive freeze/thaw 5 times combined with nuclease processing for 12 hours, can enhance the healing of host tendon-bone and improve the biomechanical characteristics of the rabbit infraspinatus tendon.
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 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.
