Biomechanical Properties Of Porcine Bile Duct And Its Feasibility As Tissue Engineered Bile Duct

Background:Biliary tract surgery is one of the difficult points in surgical operation, and the intraoperative repair of bile duct, post-repair restricture and treatment of malignant stricture are always the major concerns in abdominal surgery. Efforts have been made to design a type of artificial bile duct to replace the diseased bile duct as a solution to bile duct repair and reconstruction. Allograft transplantation has been widely used in clinical practice. However, there are still many problems in the prevention and treatment of rejection after operation. The postoperative stricture and obstruction together with functional restoration of Oddi sphincter could not be completely addressed by bile duct transplantation. Xenotransplantation has become a new field of organ transplantation due to the shortage of human organs in recent years. It is necessary to discover new donor materials to effectively overcome the shortage of organs. Pig is considered as the animal most suitable for human xenotransplantation. Pig has been used in experimental research and clinical practice of xenotransplantation by domestic and overseas scholars. However, there have been few reports on comparative research of the mechanical characteristics of porcine bile duct in xenotransplantation. Genetically modified animals are the candidate donors of human organs. The genetically modified pig which can express human complement regulatory protein has been cultured successfully, and the problem of hyperacute rejection has been solved. This makes pig very attractive and challenging to serve as donors for human xenotransplantation. It is necessary for xenotransplantation donor to possess the physiological functions and overcome the immunogenicity, in addition to the anatomical and biomechanical compatibility. Therefore, the study on the feasibility of porcine common bile duct to be used as human xenotransplantation donor or in tissue engineering should be carried out from the biomechanical perspective.Part1Biomechanical evaluation of porcine bile duct as human bile duct graft substitute materialsObjective:Through the comparative study of biomechanics of common bile duct in pig and human, to explore the relationship of biomechanics of the common bile duct between the human and pig, and evaluation from the aspects of biomechanics porcine bile duct as human bile duct transplant feasibility of alternative materials.Methods:Totally50healthy Chinese Hubei white pigs aged2months (30males and20females) were randomly divided into10groups.50pigs were housed under standard husbandry conditions,5pigs (3males,2females) were killed via an intramuscular injection of ketamine (5mg/kg) at3,4,5,6,7,8,9,10,11and12months respectively. The common bile duct was cut after anatomical isolation and in situ measurement of respective in vivo length. The bile ducts of humans were obtained from5deceased donors (3human men,2human women) without hepatic diseases, who were between20and40years old. Each sample sliced into5μm frozen sections was stained with hematoxylin-eosin (H&E). Geometric morphological indices (wall thickness and diameter) of common bile duct were measured with a computer image analysis system. The opening angles of common bile duct were measured using computer software of e-ruler. The pressure-diameter experiment of common bile duct was tested with the biomechanical test equipment. The corresponding parameters were calculated, including incremental elastic modulus (Einc), pressure-strain elastic modulus (Ep), volume elastic modulus (Ev), and compliance. The common bile duct was stained with immunohistochemical methods, the content of microstructural components (collagen, smooth muscle and elastin) of common bile duct were detected and calculated with laser confocal microscope.Results:1. Changes in the body weight of pig, weight and volume of porcine livers at different month ages. The body weight of pig, weight and volume of porcine livers gradually increase with the increased age. There was no significant difference in body weight of pig, weight and volume of porcine livers between pigs aged6-7months and adult human (F=109.15, P=0.08).2. Comparison of geometric morphological indices of common bile duct between pigs and humans. There were significant differences in wall thickness(F=6.46, P=0.00), diameters (F=9.56, P=0.00), and length (F=13.29, P=0.00) of the common bile ducts between humans and pigs in different ages. The wall thickness and diameters of common bile duct of pigs aged3-6were smaller than that of adult human (P<0.01). There was no significant difference in the wall thickness and diameters of common bile duct between pigs aged7-10months and adult humans (P>0.05). The length of the porcine common bile ducts in different ages was shorter than that of adult human (P<0.01).3. Comparison of opening angles of common bile duct between pigs and humans. The opening angles of porcine common bile duct increased gradually from3to12months (P<0.05), and tended to be stabilize when the pigs aged7months. There was no significant difference in the opening angles of common bile duct between pigs aged7-10months and adult humans (P>0.05). 4. Relation between elastic modulus of the common bile duct of humans and pigs and pressure. The Einc, Ep and Ev of common bile duct of pigs and humans increased with increasing pressure. When the pressure reached4kPa and higher, the diameters of common bile duct did not change any more, and the elastic modulus did not change or slightly changed, and the slopes of the curves become gentle.5. Comparison of elastic modulus of the common bile duct between humans and pigs. The one-factor analysis of variance showed that there were significant differences in Einc (F=502.08, P=0.00),Ep (F=137.42,P=0.00),andEv(F=134.59, P=0.00) of the common bile ducts between humans and pigs in different ages. The Einc, Ep, and Ev of common bile duct of pigs aged3-6,11-12months were higher than that of adult humans (P<0.01). There was no significant difference in the elastic modulus of the common bile duct between pigs aged7-10months and adult humans (P>0.05).6. Changes in compliance of porcine common bile duct at different ages. The compliance of porcine common bile duct gradually increase with increased age, however, after the pigs aged10months, the compliance gradually decrease with increased age. The changes in compliance of porcine common bile duct with age were opposed to the changes in elastic modulus.7. Comparison of compliance of common bile duct between humans and pigs. The one-factor analysis of variance showed that there was significant differences in compliance (F=62.93, P=0.00) of the bile ducts between humans and pigs in different ages. There was no significant difference in compliance of the common bile duct between pigs aged7-10months and adult humans (P>0.05), but the compliance of common bile duct of pigs aged3-6,11and12months was lower than that of adult humans (P<0.01).8. Content of microstructural components of common bile duct of humans and pigs. The organization structure of porcine common bile duct was similar to that of human. Changes in content of smooth muscle (F=89.85, P=0.001), collagen (F=273.69, P=0.003) and elastin (F=182.60, P=0.006) of porcine common bile duct with ages. The content of smooth muscles and elastins increased with the increased age, whereas the content of collagens and collagen/elastin (C/E) ratio decreased gradually with the increased age. There were no significant differences in the content of smooth muscles, collagens and elastins of the common bile duct between pigs aged7-10months and adult humans (P>0.05).Conclusions:Changes in biomechanical properties of porcine common bile duct with age. The biomechanical properties of geometric morphological indices, opening angles, elastic modulus, compliance, and microstructural components of the common bile duct of pigs aged7-10months match that of adult human. The correlation between age and biomechanical properties of common bile duct in pigs could imply that it was possible for the common bile duct of pigs aged7-10months to be used as the donor for bile duct xenotransplantation.Part2Preparation and histological and biomechanical evaluation of decellularized porcine bile ductObjective:The common bile ducts were treated with different acellular matrix. The histological and biomechanical characteristics of porcine common bile ducts before and after acellular matrix treatment were evaluated, and an appropriate acellular matrix was explored to provide theoretical and experimental basis for the application of bile duct scaffold materials for tissue engineering.Methods:1. Grouping:Thirty porcine common bile ducts were made into samples of30mm x12mm along the vertical axis and were divided into5groups (6in each) randomly:fresh control group A, acellular matrix group B, acellular matrix group C, acellular matrix group D and acellular matrix group E.2. Acellular matrix treatment:Four different acellular matrix treatments were used (group B:0.05%trypsin+nuclease, group C:0.1%SDS+nuclease, group D:1.0%triton X-100+nuclease, group E:1.0%triton X-100+0.1%SDS+nuclease). All the steps were performed under constant temperature of37℃with continuous agitation, and the samples were washed repeatedly using PBS solution.3. Observation of acellular matrix treatment effect:After HE dyeing, the tissue structure of acellular matrix and the residual cells were observed under light microscope.4. Determination of nuclear acid (DNA) content of acellular matrix:The DNA content of acellular matrix was detected using UV spectrophotometry, and the acellular rate was calculated.5. Biomechanical experiment:The samples were cut into stripes of20mm×10mm along the vertical axis. The loading-unloading experiment and ultimate tensile strength experiment were performed using TestResources biomechanical tester. Indexes such as biomechanical material constant (α1,β1,α2,β2), elastic modulus, ultimate tensile strength (UTS) and breaking elongation rate were calculated.Results:1. The cells of porcine common bile ducts were removed to different degrees after the four treatments as indicated by light microscope observation. Few residual cells and fiber damage were observed in group B. All the cells were removed and no obvious damages were done in group C, group D and group E.2. The DNA content of group A was71.24±2.56μg/100mg. The DNA contents of four acellular matrix treatment groups B, C, D, E were statistically different from that of group A (F=15.29, P=0.00). There was obvious acellular matrix treatment effect in the four groups (P<0.01). The acellular matrix treatment effect of group B was slightly worse that of group C, group D, group E (p<0.05); the acellular rate of group E was up to99.03%.3. There were no statistical differences in the biomechanical material constants (α1, β1,α2, β2) of the common bile ducts between group A and two acellular matrix treatment groups D and E (F=12.21, P=0.06). The biomechanical material constants of group B and group D were lower than that of group A, group C and group E (P<0.01). There was no obvious difference between the two acellular matrix treatment groups D and E (P>0.05), with group E closer to the level of group A.4. The elastic module of common bile ducts of the two acellular matrix treatment groups D and E were a little larger than that of group A, but without significant difference (P>0.05). The levels of group B and group C were lower than that of group A (P<0.05), and the difference between group D and group E was not significant (P>0.05).5. There were no significant differences in UTS value and SOF value between the acellular matrix treatment groups D, E, and group A (P>0.05). The UTS value of acellular matrix treatment groups B and C was significantly lower than that of group A (P<0.05), and the SOF value was significantly higher than that of group A (P<0.05). There were no obvious differences in UTS value and SOF value between acellular matrix treatment groups D and E (P>0.05).Conclusions:1. The acellular matrix treatment effect of0.05%trypsin+nuclease was unsatisfactory with high residue amount of DNA; the EMC structural integrity of common bile duct was damaged, and the changes of biomechanical characteristics were manifested as reduced material constants, lower stiffness, obviously decreased strength and the increased extensibility.2. The acellular matrix treatment effect of0.1%SDS+nuclease was better, but a similar changing trend occurred to the biomechanical characteristics as in group B.3. The acellular matrix treatment effect of1.0%Triton X-100+nuclease and1.0%Triton X-100+0.1%SDS+nuclease was quite satisfactory, and the biomechanical characteristics of porcine common bile duct were not affected. The acellular rate of the former was up to95.42%, the acellular rate of the later was up to99.03%, and the immunogenicity of common bile duct can be better reduced. Therefore, it is an ideal acellular method for porcine common bile ducts.4. From the views of immunogenicity and biomechanics, the porcine common bile ducts prepared by the acellular method of1.0%Triton X-100+0.1%SDS+nuclease provides a good scaffold material for the construction of bile duct by tissue engineering.