Preliminary Construction Of Bioactive Decellularizd Tendon Xenograft

Chapter one Preparation of decellularizd tendon xenograftObjective:Multiple decellularization cycles were carried out to remove bovine tenocytes. Then the decellularizd tendon xenograft was cross linked to enhance biomechanical property of the scaffold. The prepared scaffolds were finally sterilized by peracetic-acid ethanol combined with low dose y irradiation. Procedures were performed to determine the decellularization, deprivation of a-gal antigen, changes of biomechanical property, in vitro and in vivo cytocompatibility in order to provide experimental foundation for future application of decellularized tendon xenograft.Methods:Achilles tendons were harvested aseptically and stored at80℃for one month. Frozen tendons were thawed, and all adhering connective-tissue was removed. Then the tendons were decellularized as followed. Samples were washed with PBS, and then incubated in trypsin solution, which was terminated by DMEM with FBS. This was followed by incubation inTriton-X solution that contained Triton and NA-deoxycholate. This step was repeated. After washed with PBS, samples were incubated in EDTA for and washed by DMEM. The samples were then washed with PBS. After different cycles’washing, samples were treated by a-galactosidase. The samples were then washed with PBS and cross linked by incubation with Genipin solution. The solution was discarded and samples were washed with PBS. Samples were treated with PE solution which contained peracetic acid and ethanol. Samples were washed with PBS until residue of peracetic acid was accepted. After deep frozen, samples were freeze-dried and sterilized with15kGy y irradiation. Fresh-frozen and decellularised tendon scaffolds were processed for histological analysis. The sections were mounted on slides and stained with hematoxylin and eosin as well as4,6-diamidino-2-phenylindole (DAPI). The surface of each tendon was manually evaluated from the tendon sections mounted on glass slides and analyzed by optical microscopy, from which decellularization and3-D structure of tendons were evaluated. Quantity of a-gal antigen from fresh-frozen, a-galactosidase treated and decellularised tendon scaffolds was determined using a commercial available ILISA kit. Scanning electron microscopy was performed on the fresh-frozen tendons and the decellularised tendon scaffolds. Cross-sectional and longitudinal-sectional electron-micrographs were obtained to evaluate decellularization and3-D structure of tendons. Fresh-frozen and decellularised tendon scaffolds were freeze-dried and weighed. Then total DNA of specimens was isolated using E.Z.N.A.TM Tissue DNA Kit and determined using NANODROP2000spectrophometer. According to histological observation, SEM observation, concentration of a-gal antigen and quantity of DNA, the proper decellularization cycle was determined. Ninhydrin assay was performed to assess cross linking of genipin by determine the free amino group content. And the optical absorbance of the solution was recorded with a spectrophotometer at a wavelength of570nm. Using glycine at various known concentrations as standard, concentration of free amino was calculated, from which cross linking of genipin was determined. Isopropanol was used to dertermine porosity of fresh-frozen and decellularised tendon scaffolds. Uniaxial load-to-failure tests were performed with a digital universal testing machine. And tensile strength,%strain at ultimate failure load and elastic tensile modulus were compared to test bio mechanical property of fresh-frozen and decellularised tendon scaffolds. In order to evaluate in vitro cytocompatibility, CCK-8was used to determine the absorbance of the solution measured with an enzyme-linked immunoassay. For the cell viability assay, the neutral red solution was chosen to determine the absorbance measured at540nm using the96-well plate spectrophotometer noted above. The absorbance obtained was directly proportional to the viability of the cell populations and inversely proportional to the cytotoxicity of the material. A sterile surgical procedure was performed to implant the specimens subcutaneously in the dorsum of the rats. Decellularised tendon scaffold were experimental group, while fresh bovine tendon was positive control and collagen sponge was negative control. At3rd,7th,14th and21st day post-implantation, the animals were euthanised and the implants were harvested. In vivo host cell infiltration and inflammatory response were evaluated by hemalaun-eosin (H&E) staining with optical microscopy.Results:Cellular components were evident in fresh-frozen bovine tendon prior to decellularization after H&E staining. After processing, cellular components were not observed and more inter-fascicular and intra-fascicular space was present. DNA and RNA were evident in fresh-frozen bovine tendon prior to decellularization after DAPI staining, while after processing, DNA and RNA were not observed. Collagenous degeneration, rupture of collagen fibre and changes of natural structure of tendon were observed in over-decellularized samples. Scanning electron microscopy confirmed the dense micro-architecture observed in the histologic sections of the fresh-frozen tendons, as well as an increase in pore size and porosity following oxidative treatment. Quantity of a-gal antigen decreased gradually after decellularization. And a-gal antigen could not be quantified after the third cycle of decellularization. No a-gal antigen was detected in sample processed with a-galactosidase. DNA content of the decellularized/oxidized bovine tendon scaffolds was significantly decreased after decellularized and oxidative treatment when compared to the fresh-frozen tendons. DNA content decreased by2/3after the third cycle of decellularization and dropped3/4after the fourth cycle of decellularization. Combining histological observation, SEM observation and determination of a-gal antigen and DNA content,4-decellularization-cycle was chosen. After fixation, we noted that the color of the tissue fixed with genipin became dark blue. By determine the free amino group content, the rate tissue fixed by genipin was calculated as56.27%. Porosity increased after decellularization and significant difference between control and experimental group was noticed (t=-4.147, P=0.003). The tensile strength of fresh-frozen and decellularised tendon scaffolds was22.640±4.504MPa and29.733±4.623MPa respectively. No significant difference was found between two groups (t=-1.904, P=0.130).%strain at ultimate failure load of fresh-frozen and decellularised tendon scaffolds were15.200±4.071%and13.830±3.424%respectively. No significant difference was found between two groups (t=0.445, P=0.679). And elastic tensile modulus of fresh-frozen and decellularised tendon scaffolds were151.250±12.885MPa and218.680±23.833MPa respectively. Significant difference was noticed between two groups (t=-4.311, P=0.022). The results showed that tensile strength and tensile modulus increased, while%strain at ultimate failure load decreased after decellularization. In CCK-8tests, significant difference was noted between control and experimental group at different time (F=578.697, P<0.001). And the value of OD in experimental group is significantly higher than control group (F=4.011, P<0.05). No interaction effect was noticed betreen groups (F=1.650, P>0.05).The relative growth rate at2nd,5th, and7th day were105%,105%and110%respectively. The rating for cytotoxic reacting grade was0. And the rates of viability of the cell were99%and106%respectively for the3rd and7th day. Experiment for in vivo cytocompatibility, bovine xenograft without any decellularization showed a large volume of lymphocytes surrounding and infiltration. While the decellularized tendon scaffold and collagen sponge demonstrated similar reaction and response from the hosts. And compared to decellularized tendon scaffold, collagen sponge was absorbed more quickly. At the3rd day, grafts were surrounded with granulation tissue, where angiotelectasia, hyperaemia and leukocyte exudation was observed. The inflammatory cells were mainly neutrophil granulocyte and macrophagocyte. At the first week, grafts were still surrounded with granulation tissue, and the inflammatory cells decreased. Fibroblasts and collage fibres increased in the surrounding granulation tissue. In control, absorption of collagen sponge was observed. While in experimental group, infiltration of fibroblasts into grafts was noted. At the2nd week, the absorption of collagen sponge was obvious and only little collagen was found in control. The experimental grafts showed similar responses to the1st week. Slight absorption of graft was noticed in the experimental group. And more cell filtration was observed in the peripheral part of grafted tendon scaffolds. After three weeks, almost no collagen sponge was found. More cells infiltration into grafted scaffolds was observed in experimental group.Conclusion:The decellularized tendon scaffold was prepared by multiple decellularizations, genipin cross linking and sterilized by peracetic-acid ethanol combined y irradiation. This scaffold characterized by:(1) free of cells and a-gal antigen;(2) the natural structure of tendon was well maintained;(3) well preserved biomechanical property;(4) good cytocompatibility. Experimental foundation was provided for future application of decellularized tendon xenograft.Chapter two Preliminary construction of bioactive decellularizd tendon xenograftObjective:To explore the method of preparation for platelet rich plasma. Platelet rich plasma was activated and combined with decellularized tendon xenograft through agent characterized with potential of control release the growth factors in platelet rich plasma.Methods:Intraperitoneal injection of pentobarbital sodium was carried out to anesthetize SD rats. With the10ml syringe processed with anticoagulant in advance, arterial blood was procured from the heart anesthetized SD rats. Then platelet rich plasma was prepared through two centrifugation steps:The blood was centrifuged with a relative centrifugal force of200g for10minutes at a constant temperature of22℃. With a plastic transfer pipette, the whole supernatant, consisting of blood plasma, leucocytes, and thrombocytes, and also red blood cells above the buffy coat were drawn. The exceeding layer of erythrocytes was discarded. This solution was run at a relative centrifugal force of200g for10minutes. The supernatant was partly discarded and platelet rich plasma which concentration was five times of the whole blood. The concentration of platelet-derived growth factor (PDGF), vascular endothelial growth Factor (VEGF), Insulin growth factor-1(IGF-1), and transforming growth factor-β1(TGF-β1) was determined by ELISA. The standard curve was drawn by standard samples provides with the ELISA kit, and absorbance of the experimental samples were determined by enzyme-linked immunoassay. Then the concentration of samples could be determined from the standard curve. The actual concentration would be calculated by multiplying the dilution ratio. Collagen I was used to activate platelet rich plasma. And the solution of Collagen I and platelet rich plasma was injected into the decellularized tendon xenograft with syringe to prepare the bioactive decellularized tendon scaffold. The bioactive decellularized tendon scaffolds with potential system of control release were then immerge into distilled water with four times volume of the solution of Collagen I and platelet rich plasma. ELISA tests were performed at3rd,12th,24th and48th hour to determine the concentration of VEGF, PDGF, IGF-1and TGF-β1. The control release potential of the bioactive decellularized tendon scaffold was evaluated. The bioactive decellularized tendon scaffold was preceded with immunohistochemical staining to determine the existence of TGF-β and VEGF. The rat fibroblasts were co-cultured with the bioactive decellularized tendon scaffold. Then growth and proliferation of fibroblasts were observed with inverted phase contrast microscope. CCK-8was also used to determine the absorbance of the solution measured with an enzyme-linked immunoassay.Results:The results of ILISA showed that concentration of PDGF was3624.20±1453.62pg/ml in the prepared platelet rich plasma. And the concentration of IGF-1and TGF-β1was376.64±136.66pg/ml and1103.45±1149.31pg/ml respectively. However, the concentration of VEGF was not detected through ILES A test. The control release tests showed that the concentration of growth factors in the solution increased gradually. For PDGF, the concentration at48h was about three times of concentration at12h and24h. For IGF-1, the concentration at48h was around two times of concentration at12h and24h. For TGF-β1, the concentration at48h was around three times of concentration at12h and two times of concentration at24h. No obvious positive results were observed for immunohistochemical staining of TGF-β and VEGF. After co-cultured with the bioactive decellularized tendon scaffolds, fibroblast grew and proliferated well. CCK-8test showed that the absorbance of experimental group was3.280±0.235, while2.947±0.168for control. There was significant difference between two groups (t=-3.987, P=0.001). And the relative growth rate was111%.Conclusion:The preparation of platelet rich plasma was explored. The prepared bioactive decellularized tendon scaffolds possessed the potential of control releasing growth factors in platelet rich plasma. The prepared bioactive decellularized tendon scaffolds could improve growth and proliferation of rat fibroblasts.Chapter three Achilles tendon repair in a rabbit model with prepared bioactive decellularized tendon scaffoldsObjective:Animal tendon repair model was to be founded to evaluate the repairing potential of the bioactive decellularized tendon scaffolds.Methods:The whole blood was drawn from the rabbit ear central artery with pre-anti-coagulated syringe. Then platelet rich plasma was prepared followed the methods descripted in chapter two. The decellularized tendon scaffolds was prepared with the methods founded in chapter one. And the bioactive decellularized tendon scaffolds was constructed followed the procedure introduced in chapter two. Each rabbit was anesthetized with3%Pentobarbital,30mg/kg. The hind paws were shaved and prepared for sterile operative intervention. A2.5cm longitudinal incision of the skin was performed using a lateral paramedian approach followed by a longitudinal splitting of the crural fascia and the paratenon which surrounds the Achilles tendon complex. Subsequently, the Achilles tendon complex was exposed. The medial M. gastrocnemius tendon was separated from the lateral M. gastrocnemius tendon and the M. flexor digitalis superficialis tendon.2cm excision was created and repaired as study design, graft was sutured to host stumps with5-0 nylon in an "8" way strengthened by interrupted stitched. In group1, the medial M. gastrocnemius tendon was repaired with the bioactive decellularized tendon scaffolds. In group2, the medial M. gastrocnemius tendon was repaired with the opposite medial M. gastrocnemius tendon. In group3, the medial M. gastrocnemius tendon was repaired with the bovine tendon xenograft. For group4, the medial M. gastrocnemius tendon was not repaired. The tendon was reclined and the crural fascia/paratenon closed using1-0silk suture and continuous suture. The subcutaneous tissue was closed using1-0silk suture then the skin using1-0silk suture. Antibiosis was performed with intramuscularly injected gentamicin (20mg/kg) continuously three days post-operative. Immobilization of the animals was conducted for three weeks. The animals were housed individually in a standard rabbit cage and kept at the same conditions of temperature, humidity and light, and subjected to comprehensive veterinary care. Clinical parameters including appetite, diarrhea, infection, wound healing and adhesion were monitored and assessed. On3rd day and at2nd,4th,8th and12th week post-implantation, animals were euthanized and the implants were harvested. Gross and histological evaluation was performed for the repaired Achilles tendon.Results:All rabbits showed normal appetite, and except for1animal in group2caught infections, there was no evidence of clinical complications such as local infection or diarrhea. In all groups there existed muscle atrophy of immobilized limb, but recovered after immobilization was removed. Group1and group2shared similar macroscopic alterations:no tendon rupture existed and no elongation at the interfere zone of grafts and host tendons were found. At the first few weeks, color of grafts was translucent and dull white, while at12w the color was close to host tendons and similar appearance was observed between grafted and host tendon. Grafts integrated tightly into stumps of host Achilles tendon and slight adhesion with the surrounding tissue was found. In group3, the xenograft was objected by host and no healing of the excised tendon in group4. Therefore the histological observation was compared between group1and group2. On the third day post-operation, granulation tissue was found grew perpendicular to the long axis of tendons. Main cellular components were neutrophils and macrophages and cells infiltration into graft was observed. Fibril vascular capsule formed along grafted and host tendons; meanwhile micro angiogenesis was confirmed at the periphery of the graft. Group1and group2exhibited similar alterations. No much leukocytes infiltration and cellular necrosis existed around grafted tendons. Interface between graft and host Achilles tendon:At2nd week post operation, maturation of granulation tissue, irregularly arranged cells were observed, which included few inflammatory cells, micro vessels and lots of fibroblasts. New disorganized collagen fibrils were perpendicular to the long axis of tendons. Remodeling was active near interface, lots of micro vessels, fibroblasts and macrophages found. At4th week, inflammatory cells inside granulation tissue decreased, while micro vessels and fibroblasts increased. In group1inflammatory cell was a little more and micro vessels were slightly less compared to group2. At8th week, few inflammatory cells existed at interface zone, on contrary lots of fibroblasts scattered in long axis of tendons. New collagen fibrils were dense and paralleled in the direction of long axis of tendons. At12th week, collagen fibrils were denser and more organized paralleled, but still less mature than normal tendon. In the whole observed period, group1and group2presented similar alteration. Grafted tendons:At2nd week, fibril vascular capsule formed along grafted tendons for both group. Angiogenesis and collagen synthesis occurred at periphery of the grafted tendons. For both groups tendons, at remote part from interface or central part of grafted tendons, dense regularly arranged collagen fibrils were observed in sections, and little or no cell infiltration was found. Cell infiltration and angiogenesis increased at4th week. Less fibroblasts and micro vessels were observed in group1. At8th week, remodeling was evident that lots of new collagens were replacing grafted tendons not only at periphery but also in the central part of grafted tendons. New collagen fibrils were small but dense. At12weeks, remodeling was more evident and new collagen fibrils were close to normal tendons. Within observed period, similar alteration occurred for experimental and control group. After Masson staining new collagen fibrils exhibited green, while original ones presented red. It is observed that new collagen fibrils gradually increased and replacing original ones. At2nd week, most of collagen fibrils were stained red, while fibrils at periphery or near interface exhibited green. At4th week, red stained fibrils still were majority, but green stained fibrils increased and were found at inner part of grafted tendons. At8th week, only few fibrils were stained red, most of which were replaced by green stained fibrils.Conclusion:In the rabbit model, the responses of host to tendon autograft and the bioactive decellularized tendon scaffolds were similar when the two grafts were used to repair the medial M. gastrocnemius tendon of the Achilles tendon complex. And the two grafts also experienced the similar histological alteration. Therefore, and the bioactive decellularized tendon scaffold was believed to repair the excised tendon effectively, which provided experimental support for its future application.