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.
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.
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 investigate the preparation of decellularized Achilles tendons and the effect of co-culture of human fibroblasts on the scaffold so as to provide a scaffold for the tissue engineered ligament reconstruction. Methods Achilles tendons of both hind limbs were harvested from 10 male New Zealand white rabbits (5-month-old; weighing, 4-5 kg). The Achilles tendons were decellularized using trypsin, Triton X-100, and sodium dodecyl sulfate (SDS), and then gross observation, histological examination, and scanning electron microscope (SEM) observation were performed; the human fibroblasts were seeded on the decellularized Achilles tendon, and then cytocompatibility was tested using the cell counting kit 8 method at 1, 3, 5, 7, and 9 days after co-culture. At 4 weeks after co-culture, SEM, HE staining, and biomechanical test were performed for observing cell-scaffold composite, and a comparison was made with before and after decellularization. ResultsAfter decellularization, the tendons had integrated aponeurosis and enlarged volume with soft texture and good toughness; there was no loose connective tissue and tendon cells between tendon bundles, the collagen fibers arranged loosely with three-dimensional network structure and more pores between tendon bundles; and it had good cytocompatibility. At 4 weeks after co-culture, cells migrated into the pores, and three-dimensional network structure disappeared. By biomechanical test, the tensile strength and Young’s elastic modulus of the decellularized Achilles tendon group decreased significantly when compared with normal Achilles tendons group and cell-scaffold composite group (P lt; 0.05), but no significant difference was found between normal Achilles tendons group and cell-scaffold composite group (P gt; 0.05). There was no significant difference in elongation at break among 3 groups (P gt; 0.05). ConclusionThe decellularized Achilles tendon is biocompatible to fibroblasts. It is suit for the scaffold for tissue engineered ligament reconstruction.
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.
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 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.
【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.
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.