Decellularized Research Articles

The effects of decellularization and cross-linking techniques on the fatigue life and calcification of mitral valve chordae tendineae

In cases of severely diseased mitral valves (MV), the required treatment is often valve replacement. Bioprosthetic and stentless replacement valves are usually either fully or partially composed of animal derived tissue treated with a decellularization process, a cross-linking process, or both. In this study, we analysed the effects of these treatments on the fatigue properties of porcine MV chordae tendineae (CT), as well as on the calcification of the CT using an in vitro technique. CT were tested in 4 groups; (1) native, (2) decellularized (DC), (3) decellularized and cross-linked with glutaraldehyde (DC-GTH), and (4) decellularized and cross-linked with 1-ehtyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)(DC-EDC). CT were tested in both uniaxial tension, and in fatigue at 10MPa peak stress (1Hz). The cycles to failure (mean±SD) for the four groups are as follows; Native- 53,397±55,798, DC- 28,013±30,634, DC-GTH- 97,665±133,556, DC-EDC- 318,601±322,358. DC-EDC CT were found to have a slightly longer fatigue life than the native and DC groups. The DC-EDC group also had a marginally lower dynamic creep rate, meaning those CT elongate more slowly. After in vitro calcification, X-ray microtomography was used to determine relative levels of calcification. The DC-EDC and DC-GTH groups had the lowest volume of calcific deposits. Under uniaxial testing, the ultimate tensile strength (UTS) of the DC-GTH CT was statistically significantly reduced after calcification, while the UTS was relatively unchanged for the DC-EDC group. Overall, these results indicate that a treatment of decellularization plus cross-linking with EDC may improve the fatigue life of porcine CT, reduce the rate of elongation, and help the CT resist the negative effects of calcification. This may be a preferable treatment in the preparation of porcine MVs for the replacement of diseased MVs.

JoVE - Year in Review: 2015

In January 2015, JoVE Developmental Biology became the newest addition to the JoVE family, and featured a live-imaging protocol for visualizing the developing Drosophila eye. In February, JoVE Medicine showed state-of-the-art cranial ultrasound imaging in neonates. Advances in this technique have improved its diagnostic value, allowing timely therapeutic intervention. In March, JoVE Environment featured dendroecology, the science of using tree rings to study ecological effects of specific environmental factors. In April, JoVE Biology examined a mode of bacterial motility called swarming with a two-phase swarm assay. that can be used to study various aspects of bacterial growth. In May, JoVE Chemistry described the self-assembly of single-stranded DNA tiles into complex 2D shapes. This demonstrated the principles of DNA tiling as an approach for making programmable nanostructures. June 2015 marked the release of JoVE's 100th issue, and in JoVE Behavior, we featured protocols for assessing cognition, including a method of quantifying learning in young infants by tracking leg movements. In July, JoVE Medicine presented a method for measuring halitosis in dogs by taking breath samples, and performing gas chromatography. This assay could be used in trials of dog food formulated to fight bad breath in our canine companions. In August, JoVE Bioengineering contained a protocol for bioengineering kidney tissues. Kidneys are decellularized by perfusion, and repopulated with human renal cells, This promising technology can lead to kidney grafts made from a patient's own cells. In September, JoVE Environment examined herbicide resistance, which threatens commercial crop production. An excised leaf assay determines the rates of herbicide metabolism, which is useful for studying the metabolic basis of resistance. In October, JoVE Neuroscience featured a method for interfacing 3D neuronal cultures to micro-electrode arrays. The complex 3D assemblies closely approximate in vivo neural networks. November's edition of JoVE Behavior showcased the natural human ability to acquire new motor skills, which is central in a structured rehabilitation program for amputees as they learn how to use multifunctional prosthetics. In December, JoVE Immunology & Infection prepared us for the impending flu season with an easy protocol for sampling influenza in pigs. Because swine are important hosts for flu viruses, this method helps to monitor virus evolution and currently circulating strains. This Year in Review was just a sampling of more than 900 video-articles that JoVE published in 2015. Browse the JoVE archives for thousands of other videos, and come back each week to see brand-new material in JoVE: The Journal of Visualized Experiments.

An intriguing relation between periodontal and cardiovascular diseases

Periodontitis is a chronic inflammatory condition promoted by bacterial colonization of the gingiva that causes alveolar bone and tooth loss. Under these conditions, bacteria, as well as bacterial products and inflammatory mediators can move from the gingival pocket to the well vascularised periodontal tissues and into the circulation. In fact, recently a possible connection between oral infection and cardiovascular disease was suggested (1). One of the major pathogens involved in the progression of periodontal disease is the Porphyromonas gingivalis (LPS-G). The aim of the study was to induce endothelial differentiation in human periodontal ligaments stem cells (hPDLSCs) (2) on decellularized pig heart valve as scaffold and evaluate the role of LPS-G on cell cultures in terms of reactive oxygen species (ROS) production and NFKB pathway. Many studies have shown that ROS provoked oxidative stress plays a critical role in the development of cardiovascular disease. Excessive generation of ROS can cause cellular dysfunction and injury by directly oxidizing and damaging proteins, DNA and lipids, which ultimately result in cell death. To induce endothelial differentiation, human periodontal ligament stem cells (hPDLSCs) were cultured with endothelial growth medium (EGM-2MV) supplemented with vascular endothelial growth factor (VEGF) and seeded on decellularized pig heart valve. Valve leaflets were incubated for 30 min with 10 μM DCFH-DA at 37°C in humidified incubator. At the end of loading, the valve leaflets were observed using multiphoton microscope. Results of time lapse experiments revealed that after treatment, with 2,5 mg/ml LPS-G, a rapid sustained increase in ROS generation was observed mainly in differentiated hPDLSCs. Comparing the average response of undifferentiated and differentiated hPDLSCs is clearly evident that the latter showed an six fold increase in ROS production after LPS-G exposure, while on undifferentiated appeared to be negligible. Moreover, in endothelial differentiated cells the NFKB nuclear translocation in presence of LPS-G was evident. A reasonable conclusion could be that the treatment of periodontal disease not only improve dental but presumably also cardiovascular health.

Effect of three-dimensional acellular fibrotic lung matrices on fibroblast-to-myofibroblast differentiation

To explore the effect of three-dimensional (3D) and two-dimensional (2D) acellular fibrotic lung matrices on fibroblast-to-myofibroblast differentiation. Twenty adult SD rats were randomly divided into control group and idiopathic pulmonary fibrosis (IPF) group. The pulmonary fibrosis was induced by Bleomycin. Normal and fibrotic decellularized lungs were made, then sections with 500 µm (3D) and 10 µm (2D) thick were cut by a standard Vibratome. The lung fibroblasts were isolated from the lungs of SD rats at postnatal day 1, which were seeded dropwise into different slices (2D/3D normal and fibrotic scaffolds), then cultured for 72 hours in vitro. The protein expression of alpha smooth muscle actin (α-SMA) was measured by immunofluorescence staining and western blot. There was no spatial structure both in 2D normal scaffold and fibrotic scaffold. The spatial structure was different between 3D normal scaffold and fibrotic scaffold. The relative expression amount of α-SMA in 2D normal and fibrotic scaffold group and none scaffold group were (4.64 ± 0.36, 4.86 ± 0.37, 4.67 ± 0.35, respectively), there were no difference among them (P>0.05). In 3D fibrotic scaffold group, the relative expression amount of α-SMA was significantly increased compared with those in 3D normal scaffold group and 2D fibrotic scaffold group (6.29 ± 0.85 vs 4.85 ± 0.56, 4.86 ± 0.37, both P<0.05). 3D acellular fibrotic lung scaffold can promote fibroblast-to-myofibroblast differentiation.

Research progress of re-epithelialization in decellularized trachea matrix

Long-segment tracheal defects are difficult to be managed by conventional surgical means, which often requires tracheal transplantation.Studies have confirmed that the capacity of tissue engineering techniques in restoration of extensive tracheal defects in animals and human.Decellularized tracheal matrix has attracted widespread attention for its advantages, such as excellent biocompatibility.Re-epithelialization of the tissue engineered trachea, which remains the major obstacle in tracheal transplantation, plays an important role in protecting the grafts from infection and restenosis.To date, some research focuses on the re-epithelialization of the tissue engineered trachea, but achievements are still far from desired, and further studies are needed to ensure the visibility of the application of tissue engineered trachea. Key words: Tracheal transplantation; Tissue engineering; Decellularized trachea matrix; Re-epithelialization

Successful tissue engineering of competent allogeneic venous valves.

The purpose of this study was to evaluate whether tissue-engineered human allogeneic vein valves have a normal closure time (competency) and tolerate reflux pressure in vitro. Fifteen human allogeneic femoral vein segments containing valves were harvested from cadavers. Valve closure time and resistance to reflux pressure (100 mm Hg) were assessed in an in vitro model to verify competency of the vein valves. The segments were tissue engineered using the technology of decellularization (DC) and recellularization (RC). The decellularized and recellularized vein segments were characterized biochemically, immunohistochemically, and biomechanically. Four of 15 veins with valves were found to be incompetent immediately after harvest. In total, 2 of 4 segments with incompetent valves and 10 of 11 segments with competent valves were further decellularized using detergents and DNAse. DC resulted in significant decrease in host DNA compared with controls. DC scaffolds, however, retained major extracellular matrix proteins and mechanical integrity. RC resulted in successful repopulation of the lumen and valves of the scaffold with endothelial and smooth muscle cells. Valve mechanical parameters were similar to the native tissue even after DC. Eight of 10 veins with competent valves remained competent even after DC and RC, whereas the two incompetent valves remained incompetent even after DC and RC. The valve closure time to reflux pressure of the tissue-engineered veins was <0.5 second. Tissue-engineered veins with valves provide a valid template for future preclinical studies and eventual clinical applications. This technique may enable replacement of diseased incompetent or damaged deep veins to treat axial reflux and thus reduce ambulatory venous hypertension.

Pilot study of a novel vacuum-assisted method for decellularization of tracheae for clinical tissue engineering applications.

Tissue engineered tracheae have been successfully implanted to treat a small number of patients on compassionate grounds. The treatment has not become mainstream due to the time taken to produce the scaffold and the resultant financial costs. We have developed a method for decellularization (DC) based on vacuum technology, which when combined with an enzyme/detergent protocol significantly reduces the time required to create clinically suitable scaffolds. We have applied this technology to prepare porcine tracheal scaffolds and compared the results to scaffolds produced under normal atmospheric pressures. The principal outcome measures were the reduction in time (9 days to prepare the scaffold) followed by a reduction in residual DNA levels (DC no-vac: 137.8±48.82 ng/mg vs. DC vac 36.83±18.45 ng/mg, p<0.05.). Our approach did not impact on the collagen or glycosaminoglycan content or on the biomechanical properties of the scaffolds. We applied the vacuum technology to human tracheae, which, when implanted in vivo showed no significant adverse immunological response. The addition of a vacuum to a conventional decellularization protocol significantly reduces production time, whilst providing a suitable scaffold. This increases clinical utility and lowers production costs. To our knowledge this is the first time that vacuum assisted decellularization has been explored. Copyright © 2015 John Wiley & Sons, Ltd.

Crosslinking of decellularized heart valve by procyanidins in the acidic buffer solution: Compliance, anti-calcification and biological compatibility

Crosslinking of decellularized heart valve by procyanidins in the acidic buffer solution: Compliance, anti-calcification and biological compatibility

Updates of application of decellularized liver bioscaffold

Replacement therapy is the most effective method for the treatment of end-stage liver disease, and decellularized liver bioscaffold broadens the research field of the replacement therapy. The present liver bioscaffold preparation is to perfuse chemical reagents (detergents, enzymes, et al) into the vascular structure of the liver under certain physical conditions, so as to remove cellular components and retain extracellular matrix and microvascular structure. Cells were reseeded into the decellularized liver scaffold to obtain the recellularized liver, which can be cultured and evaluated in vitro or in vivo by observing the adhesion of seeded cells, detecting the synthesis and secretion of the recellularized liver. Currently, the selection of seed cells, recellularization protocol and recellularized liver transplantation are still under exploration. In this review, the preparation, evaluation, detection and application of the decellularized liver bioscaffold are introduced for the further experimental study and clinical research. Key words: Decellularization; Liver; Bioscaffold; Manufacture; Application

Effects of combined decellularization and cryopreservation procedures on morpho-functional properties of porcine aortic valve allografts

Heart valve disease is a major public health problem, with surgical valve replacement still representing the leading therapeutic option. In the last 20 years, implantation of cryopreserved human heart valves underwent so progressive increase that current availability of allogeneic valve substitutes is not sufficient to meet clinical demand. However, also valve allografts undergo long-term failure depending on host-versus-graft immune responses and degeneration of resident cells and extracellular matrix components with subsequent priming of mineralization processes. In a previous study, suitably decellularized porcine aortic valves implanted in the pulmonary position in Vietnamese pigs resulted to be permissive of in vivo spontaneous repopulation by recipient’s cells, showing tissue remodelling and satisfactory hemodynamic performance (1). Using the same animal model, decellularized valve allografts were compared with other ones which were additionally subjected to cryopreservation/thawing before implantation. Previous echocardiographic analysis showed near-normal hemodynamic behaviour for both allograft types. Here, microscopic examination revealed non-uniform outcomes for both treatments, because there was co-presence of native-like and altered regions. In the former, almost complete re-endothelialization and repopulation by cells with features of fibroblasts, myofibroblasts, and smooth muscle cells involved both cusps and aorta walls. Ultrastructural identification of typical canals of collagen fibrillogenesis and elastogenesis-related features revealed actual tissue remodelling to have been occurred. In aorta walls, incipient neo-vascularization and re-innervation of medial and adventitial tunicae were also detected. Conversely, altered regions showed fibrin deposits, inflammatory infiltrates, fragmented elastin fibers, and calcific loci in absence of cell repopulation, with these features mainly concerning cryopreserved allografts. In conclusion, cryopreservation was found to limit the favourable outcomes characterizing decellularized allografts, even if hemodynamic performance was not affected. However, both treatments were found to not compromise repopulation by host’s cells and tissue renewal. Thus, the obtained preclinical data suggest the feasibility to achieve cryobank-derived self-regenerating and hemodynamically functional allogeneic valve substitutes, after optimization of these two pre-implantation procedures or their mutual compatibility

Dextran sulphate crowding and sodium deoxycholate lysis of primary breast fibroblast cells achieve extracellular matrix deposition and decellularization for breast cancer stem cell culture

Abstrak Latar belakang: Lingkungan mikro yaitu sel stromal dam matriks ekstraseluler saat ini dinyatakansebagai kontributor dalam perkembangan tumor. Beberapa penelitian telah mengembangkan matriksekstraseluler yang mendukung perkembangan sel in vitro. Matriks ekstraseluler adalah suatu komplekssusunan supramolekuler dari berbagai macam glycoprotein dan proteoglycan. Matriks ekstraselulermenyediakan integritas jaringan, bertindak sebagai scaffold alami tempat sel melekat dan berinteraksiserta berperan sebagai reservoir pertumbuhan sel. Penelitian ini bertujuan untuk mendapatkan deposisidan deselularisasi yang optimal pada matriks ekstraseluler. Metode: Dalam penelitian ini, kami mengembangkan cells crowder untuk meningkatkan deposit matriksekstraseluler dari kultur sel primer fibroblast payudara yang diperoleh dari spesimen hasil operasimammoplasty. Dextran 500 kDa ditambahkan dalam media kultur DMEM lengkap yang telah ditambahkan0.5% FBS dan 100μM L-ascorbic acid 2-phosphate. Setelah tujuh hari, sel dilisis dengan menggunakanSodium Deoxycolate (DOC). Hasil: Deposisi matriks ekstraseluler dan proses deselulerisasi dari sel primer fibroblas payudara dapatterdeteksi dengan menggunakan antibodi Rabbit anti human fibronectin yang selanjutnya ditambahkandengan anti rabbit IgG yang telah dikonjugasi dengan Alexa Fluor 488. Kesimpulan: Penambahan dextran sulfat dan prosesing lysis dengan sodium deoxycolate dapatmeningkatkan deposisi dan menghasilkan deselularisasi matriks ekstraseluler. (Health Science Journalof Indonesia 2015;6:43-7) Kata kunci: matriks ekstra selular, kanker mammae, stem cell, sel fibroblast Abstract Background: The microenvironment including stromal cells and extracellular matrix (ECM) is now consideredan active contributor to tumor progression. Certain studies have developed ECM which supports a suitable cellulargrowth in vitro. The ECM is a complex supramolecular assembly of a variety of glycoproteins and proteoglycans.Extracellular matrix provides tissue integrity, acts as a native scaffold for cell attachment and interaction and alsoserves as reservoir for growth factors. The aim of this experiment w asto achieve the deposition and decellularizationof ECM. Methods: Cells crowder have been developed to increase ECM deposit in the primary breast fibroblast cells layerobtained from isolation of single cell from breast mammoplasty specimen. Five hundred kDa dextran was addedinto DMEM medium containing 0.5% fetal bovine serum (FBS) and 100 μm of L-ascorbic acid 2-phosphate. Afterseven days, cells were lysed by sodium deoxycholate (DOC). Results were observed in a fluorescence microscope. Results: Extracellular matrix deposition and decellularization of primary breast fibroblast cells were detected byusing extracellular matrix protein, fibronectin via rabbit anti human fibronectin and anti-rabbit IgG conjugated withAlexa Fluor 488. Conclusion: Dextran sulphate increased extracellular matrix deposit in primary breast fibroblast cell layer and thetreatment with sodium deoxycolate lysed cells resulted in extracellular matrix decellularization. (Health ScienceJournal of Indonesia 2015;6:43-7) Keywords: extracellular matrix, breast cancer stem cell, breast fibroblast cell

Exploring a tissue engineering strategy as a novel approach for haemophilic arthropathy treatment

Among the most disabling complications of Haemophilia, repeated and sponta- neous intra-articular haemorrhages may cause irreversible damage to the joint. This leads to haemophilic arthropathy, a polyarticular disease characterized by joint stiff- ness, chronic pain and a severely limited range of motion. Occurrence of haemophilic arthropathy can be avoided by the prophylactic administration of clotting factors to prevent articular haemorrhages, but it can also be addressed using anti-inflammatory drugs and surgery to alleviate the effects of articular damage, up to arthroplasty as resolute option [1]. However, innovative strategies for the prevention and treatment of this common and serious complication are still required, due to some important limits of current therapies, first of all inhibitor development. In this work, we inves- tigated a tissue engineering approach to regenerate articular focal lesions in Haemo- philic patients by in vitro development of an autologous bio-hybrid prosthesis. For this purpose, we isolated articular chondrocytes from Haemophilic patients (HaeCs) and characterized them for the first time in literature, to verify whether they were altered by blood exposure. Using healthy chondrocytes as control, optical microscope morphological analysis, flow cytometry immunophenotype evaluation and gene expression study by qRT-PCR were performed. After that, an innovative compos- ite scaffold was obtained by combining decellularized Wharton’s Jelly (W’s J) from human umbilical cord with a novel biodegradable polyvinyl alcohol (PVA) hydrogel [2]. Finally, we assessed HaeCs capacity to re-populate biosynthetic scaffolds by Scan- ning Electron Microscopy and MTT assay on cells seeded on supports. Taken togeth- er, our results contributed to define HaeCs phenotype, highlighting the possibility to use these cells for autologous implant. What is more, HaeCs capacity to growth and proliferate on composite scaffolds set the stage for planning the development of autologous tissue substitutes for haemophilic cartilage regeneration.

C214 肝移植に向けた肝臓の脱細胞化及び再細胞化の検討(C2-3 生物工学、バイオデバイス2)

C214 肝移植に向けた肝臓の脱細胞化及び再細胞化の検討(C2-3 生物工学、バイオデバイス2)

Biomechanical and angiogenic properties of tissue-engineered rabbit trachea using genipin cross-linked decellularized tissue

Objective To investigate the preparation of the decellularized tracheal scaffold in rabbit,and investigate the impact of biomechanical and angiogenic properties of tissue-engineered rabbit trachea using genipin cross-linked decellularized tissue.Methods New Zealand rabbits tracheae were decellularized using a modified detergent-enzymatic treatment.Glycosaminoglycan content,histoarchitecture,and mechanical properties were evaluated.Then evaluate mechanical properties and in vivo pro-angiogenic properties of genipin-treated tracheal matrices.Results After 7 detergent-enzymatic cycles,almost complete decellularized tracheae,retaining the hierarchical and mechanical properties of the native tissues,could be obtained.DNA concentration in the tissue was substantially decreased 92.78％ from (586.78 ± 53.56) ng/mg in native trachea to (42.39-±6.66) ng/mg after 7 cycles of decellularization (P ＜0.01).A significant (P ＜ 0.05) increase of the secant modulus for the cross-linked tracheae,compared to native and decellularized samples,was computed,especially in the case of samples treated with 1％ geninin.In particular,the increase induced by tracheal matrices treated with genipin 1％ resulted significantly different from the one induced by all the other samples.Conclusion In conclusion,this work suggests that 7 cycles of the modified detergent-enzymatic method (DEM) generates a bioengineered rabbit tracheal matrix that is structurally and mechanically similar to native trachea.The treatment with genipin increased mechanical properties,in term of secant modulus,without neither altering the pro-angiogenic properties of decellularized airway matrices or eliciting an in vivo inflammatory response. Key words: Tissue engineering; Decellularization; Angiogenesis ; Mechanical properties; Genipin

Management of fungal corneal ulcer by corneal collagen cross-linking technique

Background Fungal corneal ulcer is a visual-threatening eye disease,and drug therapy has a limiting efficacy.Corneal transplantation or eye enucleation sometimes is necessary to the severe patients.Corneal collagen cross-linking (CXL) is an effective method for some corneal diseases,but the study on CXL for fungal corneal ulcer is lack.Objective This study was to evaluate the clinical effectiveness and safety CXL for fungal corneal ulcer.Methods Fifteen 8-week-old healthy New Zealand white rabbits were used in this study and other 5 rabbits served as normal controls.Fungal corneal ulcer models were established in the right eyes of other 10 rabbits by infecting sickle bacteria liquid after corneal scratching and removing corneal epithelium,then decellularized ostrich corneal patch covered the defected cornea.The models were randomly divided into the non-treatment group and the CXL treatment group.Corneal lesions were examined under the slit lamp microscope every day,and cornea was pictured by laser scanning confocal microscope on the 3rd,7th,14th,21st and 28th day individually after CXL.All rabbits were sacrificed and corneal tissues were obtained 4 weeks after treatment,and the collagen fiber diameter and fibrocytes were observed under the scanning electron microscope.Results Fungal corneal ulcer models were successfully established by corneal scratching and decellularized ostrich cornea covering.The gray ulcer lesions and hypbae like bean pod were seen by slit lamp microscope and laser scanning confocal microscope 3 days after modeling.Corneal ulcer deepened and expanded 1 week later,and there were a large number of spore and hyphae criss-crossing as short rod in shallow stroma.Inflammatory cells were observed in corneal endothelial cells and ocular anterior chamber.In the CXL treatment group,the range of corneal epithelial deficiency was less than that in the nontreatment group on the 3rd,7th,14th,and 21st (all at P＜ 0.05).The diameters of collagen fibers were (24.6± 1.8) nm,(24.9 ± 1.9) nm and (43.0 ± 7.4) nm in the normal control group,non-treatment group and CXL treatment group,showing a significant difference among the 3 groups (F =27.05,P =0.00),and the collagen diameters were thicker in the CXL treatment group than those in the normal control group and non-treatment group (t =5.40,-5.30,both at P＜0.05),and fibrocytes were seen among the collagen fibers.No significant difference was found in the collagen diameters between the non-treatment group and normal control group,and the fibrocytes were less in the non-treatment group.Conclusions CXL therapy can treat fungal corneal ulcer by enhancing collagen,promoting fibrocytes proliferation,suppressing fungus and inflammatory response and accelerating tissue repair. Key words: Corneal collagen cross-linking; Fungus; Corneal ulcer/therapy; Laser scanning confocal microscope; Collagen fiber; Disease model/animal; Rabbit

Property analysis of decellularized tracheal scaffold with preserved cartilage in rabbits

Objective To investigate the optimum concentration of NaClO4 treatment in order to prepare tracheal scaffold.Methods Thirty healthy New Zealand rabbits were randomly divided into 5 groups (n =6),the tracheas of group A was treated with nothing as control group,the tracheas of group B,C,D and E were treated respectively with different concentrations of NaClO4(3％,5％,6％,7％).We obtained the histologic appearance via optical microscope,the ultrastructure was observed through scanning electron microscope (SEM) and survey the structural integrity and biomechanical property of tracheal matrix.Results Histology staining showed vividly lots of epithelial cells in group A,while removed clearly in group C.SEM emerged distinctly the cilium of epithelial cells in group A.The internal surface of group B were still rough,and became smooth in group C.However the matrix in group D and E fractured more or less.Compared to the native group,the number of cell nucleus in group C were obviously decreased,the decline of DNA was statistical significance (P ＜ 0.05).There were not significantly elimination of the collagen,elastic fibers and reticular fibers in group C,as well as the contents of glycosaminoglycan (GAG) (P ＞ 0.05).We found the chondrocyte viability reserved well,but exhibited a significantly lower value of maximum tensile strength,tensile strain at break and elastic modulus with the increased concentration of NaClO4 (P ＜ 0.05).Conclusion Decellularized rabbit tracheal scaffold acquired via 5 ％ NaClO4 concentration treatment showed its structural integrity,antigen cell removal and a fine biomechanical property,which would be suitable for the application in tissue engineering tracheal allograft. Key words: Tissue engineering; Decellularization ; Tracheal scaffold ; Chondrocyte

Repairing peripheral nerve defect with prevascularized artificial nerve

Objective To evaluate the effectiveness of prevascularized acellular nerve graft combined with autologous differentiated adipose-derived stem cells on repairing nerve defect in rats.Methods Adiposederived stem cells (ADSCs) were isolated from rat inguinal fat pad.Vascular endothelial growth factor (VEGF) encapsulated nanoparticles were injected into the chemically decellularized nerve graft which in turn was buried into the incision for fat harvesting.The rat was kept for 2 weeks to allow prevascularization of the acellular nerve graft.The ADSCs were cultured under neurogeneic conditions for 2 weeks to differentiate into Schwann cells.A 15 mm sciatic nerve gap in rats was repaired by VEGF nanoparticle prevascularized acellular nerve graft injected with differentiated ADSCs (Group A),by non-VEGF nanoparticle prevascularized acellular nerve injected with differentiated ADSCs (Group B),by acellular nerve injected with differentiated ADSCs (Group C),by autologous nerve graft (Group D),and by VEGF nanoparticle prevascularized acellular nerve graft (Group E).On postoperative day 7 grafts from Groups A,B and C were harvested.Microvessel density (MVD) and graft cell apoptosis were examined.After 12 weeks,nerve conduction study,triceps surae muscle wet weight,and neuromorphometry analysis were carried out to evaluate the extent of nerve regeneration.Results Seven days postoperatively,graft cell apoptosis was most prominent in group C.Apoptosis in group A was less severe than Group B.MVD was highest in group A,followed by group B and then group C.Evaluations at 12 weeks postoperatively showed significantly higher nerve conduction velocity,larger action potential amplitude,higher triceps surae muscle weight recovery rate and larger myelinated nerve fiber count in Group A than in Group B,Group C and Group E (P ＜ 0.05).Conclusion There is a synergistic effect of acellular nerve and differentiated ADSCs in repairing nerve gap in rats.The prevascularized acellular nerve improves survival of transplanted cells,which is benefit for nerve regeneration. Key words: Nerve regeneration; Stem cells, mesenchymal; Prevascularization; Acellular nerve

Decellularized kidney scaffold-mediated renal regeneration

Renal regeneration approaches offer great potential for the treatment of chronic kidney disease, but their availability remains limited by the clinical challenges they pose. In the present study, we used continuous detergent perfusion to generate decellularized (DC) rat kidney scaffolds. The scaffolds retained intact vascular trees and overall architecture, along with significant concentrations of various cytokines, but lost all cellular components. To evaluate its potential in renal function recovery, DC scaffold tissue was grafted onto partially nephrectomized rat kidneys. An increase of renal size was found, and regenerated renal parenchyma cells were observed in the repair area containing the grafted scaffold. In addition, the number of nestin-positive renal progenitor cells was markedly higher in scaffold-grafted kidneys compared to controls. Moreover, radionuclide scan analysis showed significant recovery of renal functions at 6 weeks post-implantation. Our results provide further evidence to show that DC kidney scaffolds could be used to promote renal recovery in the treatment of chronic kidney disease.

Morphological characteristics and biological mechanics analysis of rabbit decellularized allograft trachea matrix-derived material

Objective To study the different concentrations of Triton X-100 combined with nuclease to take off the cells from rabbit allograft trachea matrix and analyze the morphological and biomechanical properties.Methods Thirty healthy New Zealand rabbits were randomly divided into 5 groups (n =6each).The tracheas in group A were treated with nothing as control group,and those in groups B,C,D and E were treated with different concentrations of Triton X-100 (1％,2％,3％,and 4％) respectively.The histologic findings were observed with HE stain,and the tracheal inner and outer surface was observed by scanning electron microscope.Length,thickness,transverse diameter and longitudinal diameter of transversal surface,maximum drawing force and elastic modulus were observed.The native trachea and decellularized trachea obtained by 2％ concentration of Triton X-100 were embedded in the back of SD rats,and at 1st,4th and 8th week the tracheas were observed by HE staining.Results There were many epithelial cells and cartilage cells in the tracheas of group A.Cartilage cells in the tracheas of groups C,D and E were less than in groups A and B under the histological observation.There were many epithelium cilia in the tracheas of group A,and in the groups B and C,epithelium cilia disappeared,and basement membrane was intact.Basement membrane was broken in the tracheas of groups D and E.Maximum drawing force and elastic modulus were declined throughout the increase of concentration,but there was significant difference only between group E and group A (P ＜ 0.05).Implanted decellularized trachea showed good biocompatibility.Conclusion Decellularized rabbit trachea matrix obtained by 2％ Triton X-100 has obvious advantage in morphology and mechanical aspects. Key words: Decellularized matrix; Trachea; Morphology ; Biomechanics; Biocompatibility

Preparation and biomechanical property of genipin-crosslinked rat acellular spinal cord scaffolds

Objective To construct genipin-crosslinked rat acellular spinal cord scaffolds and evaluate their enzymatic degradation rate,biomechanical properties and cytotoxicity.Methods Rat spinal cord scaffolds were decellularized by chemical extraction and chemically crosslinked with 5 g/L genipin solution.Micro-structure of the uncrosslinked and genipin-crosslinked acellular spinal cord scaffolds were observed by HE staining and scanning electron microscopy and properties of pore size,porosity,water ratio,and degradation rate in 2.5 g/L trypsin enzyme solution were examined.Ultimate tensile strength and elastic modulus of normal rat thoracic spinal cord,uncrosslinked and genipin-crosslinked acellular spinal cord scaffolds were determined on Instron mechanical testing instrument.Rat bone marrow mesenchymal stem cells were cultured in lixivium of uncrosslinked and genipin-crosslinked acellular spinal cord scaffolds and MTT assay for relative cell growth rate was test to evaluate the cytotoxicity of scaffolds.Results The uncrosslinked and the genipin-crosslinked acellular spinal cord scaffolds possessed a similar three-dimensional mesh-porous structure with a mean pore diameter about 30 μm and a porosity over 80％,but there was a statistical difference between the two groups(P ＞ 0.05).Water ratio of genipincrosslinked scaffolds was (229.7 ± 12.5) ％,far lower than (283.4 ± 11.2) ％ of uncrosslinked scaffolds (P ＜ O.05) ; genipin-crosslinked acellular spinal cord scaffolds had lower weight loss at each time point than the uncrosslinked acellular spinal cord scaffolds (P ＜ 0.05),but the stability in trypsin,ultimate tensile strength and elastic modulus of acellular spinal cord scaffolds were significantly enhanced by genipin-crosslinking (P ＜ 0.05).Furthermore,no obvious cytotoxicity was observed in the uncroslinked and genipin-crosslinked scaffolds.Conclusions Rat acellular spinal cord scaffolds present no obvious change in structure after genipin-crosslinking,but there is significant improvement in the biomechanical properties and ability against enzymatic degradation and no marked cytotoxicity.Hence,the genipincrosslinked scaffolds are promising in tissue engineering for spinal injury. Key words: Tissue scaffold; Biomechanics; Acellular spinal cord