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.
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 compare the effect of fabricating decellularized scaffold of homograft bioprosthetic tube valved (HBTV) with two kinds of cell detergents and to provide a homograft bioprosthetic scaffold for fabrication of tissueengineering heart valve (TEHV). Methods The active cells in the HBTV, which conserved by liquid nitrogen, were decellularized by low osmotic pressure of Tris buffer, in which containing sodium dodecylsulphate (SDS) and deoxycholic acid (DOA) respectively. The leaflets or aortic wall was fixed with fixative and stained with hematoxylin and eosin, collagen fibers or elastic fibers for observation and photographs by light microscope or by scanning electron microscope (SEM) after decellularized. Results When the leaflets of HBTV were incubated togetherwith 0.03% SDS or 0.5% DOA of Tris buffer respectively for 48 hours, the activeendothelial cells (ECs) in the leaflets were not only decellularized completely, but also reserved the collagen fibers or elastic fibers integrally, which is two of the main components of extracellular matrix (ECM). A part of fibroblast inthe center leaflets was reserved. The morphologic structure of leaflets after decellularized was not significantly different from that before decellularized. The concentration of SDS was increased to 0.1% when decellularized the cells of aortic wall, but DOA was still kept 0.5%. Conclusion The better decellularizedscaffold of HBTV obtained was disposed by 0.03%-0.1% SDS or 0.5% DOA, which wasadvantageous to adhesiveness and amplification of implantation cells on the decellularized scaffold of HBTV in order that HBV reendothelialized or for the TEHVfabricated in vitro.
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.
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 investigate a method for preparing decellularized rat heart scaffold, and to detect and evaluate the decellularized scaffold. Methods The decellularized rat heart scaffold was prepared by retrograde perfusion with a combination of enzymatic and Triton X-100 detergent methods to remove the populations of resident cells, and then the decellularized scaffold was observed by gross, toluidine blue staining, HE staining, scanning electron microcope (SEM), Alcian blue staining, and immunohistochemisty staining to evaluate the structure and essential component of extracellular maxtix (ECM) in the scaffold. Results Tissue engineered scaffold based on decellularized whole heart ECM was successfully prepared, which maintained not only the gross morphology of the heart, but also the intact vascular structure and ultrastructural conformation that certified by toluidine blue staining, HE staining, and SEM analyses. Alcian blue staining and immunohistochemisty staining showed that the essential components of ECM, such as collagen type I, glycosaminoglycan, fibronectin, and Laminin were remained in decellularized whole heart matrix. Conclusion The decellularized whole heart ECM prepared by method mentioned can maintain the intact structure of rat heart and basic compositions of extracellular matrices, so it could be suitable for further studies of tissue engineered scaffolds for whole heart reconstruction.
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 develop a tissue engineering scaffold by using 4arm branched polyethylene glycol-VS (PEG-VS) crosslinked with decellularized valved conduits (DVC), and to research on its mechanical and biological functions. Methods The valved aortic conduits of rabbits were taken and decellularized by trypsin method and then were crosslinked with 4arm branched PEG-VS to construct the composite scaffolds (CS). The functions of decellularized valved conduits and the composite scaffolds were tested by mechanics test system. Thirty New Zealand white rabbits were equally and randomly assigned to one of the three groups: the control group, the DVC group, and the CS group. Valved aortic conduits, decellularized valved conduits and composite scaffoldswere transplanted into the common carotid artery of the abovementioned three groups of rabbits respectively. Twentyeight days after the operation, patency of the transplants was tested by Color Doppler ultrasound; micromorphology and inflammatory infiltration were observed by hematoxylin eosin（HE） staining andscanning electron microscope (SEM)，and endothelialization of composite scaffolds was detected by immunofluorescent staining. Results A series of biomechanical analyses revealed that the composite scaffolds had highly similar mechanical properties as fresh tissue, and had superior elastic modulus (P=3.1×10-9) and tensile strength (P=1.1×10-6) compared with decellularized valved conduits. Color Doppler ultrasound revealed that the graft patency for the CS group was better than the control group (P=0.054) and the DVC group (P=0.019), and the intraaortic thrombosis rate and distortion rate decreased significantly. HE staining and SEM showed that the endothelialization of composite scaffolds in the CS group was significantly higher than the other two groups with the endothelial cells evenly distributed on the scaffolds. The [CM(159mm]immunofluorescent staining indicated that the positive rate of the endothelial cell marker CD34 was higher than the other two groups. Conclusion The composite scaffolds using 4arm branched PEGVS crosslinked with DVC have great mechanical and biological properties.
Objective To produce a decellularized cartilage matrix (DCM) and investigate its possibil ity to be used as a scaffold for tissue engineering. Methods Fresh bovine articular cartilage from knee joints was sl iced, freeze-dried and freeze-ground into fine powder, and then was treated sequentially with Trypsin, Triton X-100 and hypotonic solution for decellularization. The decellularized matrix was freeze-dried for shaping and cross-l inked by UV radiation. Histological, immunohistological, SEM, porosity assays and biomechanical assays were used to characterize the DCM. BMSCs were isolated from rabbit bone marrow aspirate and cultured in DCM extraction medium of different concentration (100%, 10% and 1%) for 0, 24, 48 and 72 hours, respectively, to detect the release rate of the lactate dehydrogenase (LDH). The DMEM medium (5% FBS) served as the control. Biocompatibil ity was evaluated using BMSCs (1 × 107/mL) cultured with DCM. Results The DCM showed white spongy appearances, and histological analysis showed that the material was constructed by cartilage particles without any cells or cell fragments left in the matrix. Immunohistology staining and alcian blue staining showed that DCM retained the collagen and glycosaminoglycan components of cartilaginous matrix. SEM scanning showed that DCM had a porous spongy-l ike structure with the aperture ranging 30-150 μm .The porosity assay showed that the average porosity was 89.37% and the average aperture was 90.8 μm. The mechanical assay showed that there was no difference for the compress module before and after the decellularization process, which was (17.91 ± 0.98) MPa and (15.12 ± 0.77) MPa, respectively (P gt; 0.05), but were both statistical different from normal articular cartilage [(26.30 ± 1.98) MPa, P lt; 0.05]. The LDH release rate in the DCM extraction medium of different concentration was not significantly different from that in the normal DMEM medium (P gt; 0.05). The cell adhesion test showed BMSCs grew well on DCM without any signs of growth inhibition. Conclusion Articular cartilage can be decellularized and fabricated into a scaffold which retains the major components of cartilaginous matrix. DCM has goodbiochemical, biophysical characteristics and good biocompatibil ity with cultured BMSCs and may be used as a novel scaffold for tissue engineering studies.