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 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.
【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.
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.
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.
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.
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.
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 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 design a novel small-cal iber vascular graft using a decellularized allogeneic vascularscaffold pre-loaded with bFGF. Methods The decellularized canine common carotid were obtained by a detergent-enzymatic procedure, then the scaffolds were covalently l inked with heparin and pre-loaded with bFGF, the amount of binding bFGF and releasing curve were assayed by ELISA. Canine BMSCs expanded in vitro were seed on the scaffolds to observe the effects of binding bFGF on prol iferation. Both bFGF pre-loaded and non-pre-loaded decellularized grafts were implanted in canines as carotid artery interposition for 8 weeks, the patency was examined by digital subtraction angiography and histological method. Results Histology and electron microscopic examination of the decellularized scaffolds showed that cellular components were removed completely and that the extracellular matrix structure remained intact. The amount of binding bFGF positively related to the concentration of bFGF. There was a significant difference in the amount of binding bFGF between two different scaffoldsthroughout all bFGF concentrations(P lt; 0.05), and up to 100 ng/mL, the local and sustained release of bFGF from the heparin treated scaffolds were assayed up to 20 days. Additionally, MTT test showed the bFGF-preloaded scaffolds significantly enhanced the prol iferation of seeded BMSCs in vitro compared with non-bFGF-preloaded scaffolds at 3 days after seeding and thereafter(P lt; 0.01). Furthermore, in vivo canine experiments revealed that all 8 bFGF-pre-loaded scaffolds remained patent after 8 weeks of implantation, and host cell l ined the lumen and populated the wall. Only 1 non-bFGF-pre-loaded scaffold was patent, and the other 7 grafts were occluded because of thrombsus formation. Conclusion This study provides a new strategy to develop a small diameter vascular graft with excellent biocompatibil ity and high patency rate.