There are currently two primary tissue engineering strategies for bladder reconstruction: one is the use of basic scaffolds, whereas the other is the use of scaffolds seeded along with cells or cytokines. Various studies have shown that the last mentioned strategy is far better for restoration of the anatomical construction and function of the bladder [ 20 – 22 ].
However , several issues can not be solved by the bladder reconstruction method developed in the current research. First, although the bladder tissue regeneration was found making use of morphology detection, the restoration of bladder function remains difficult. Second, although cell seeding techniques have been used in many bladder repair experiments, the presence of seeded cells within vivo are scarce after 4 weeks of implantation [ 3 , 23 ]. Two reasons may account for this trend: either the number of seeded cells is not sufficient, or the majority of the seeded cells are lost or die due to immediate contact with the abdominal cavity or the urine in the urinary lumen.
On this study, the cell-seeding technique was combined with a subcutaneous prefabrication strategy to investigate whether AM-SF scaffolds could market ASC survival in vivo and restore bladder functionality. After subcutaneous prefabrication for 1 week, the BAMG-SF was surrounded by transversely arranged myofibroblasts. A bag-shaped AM-SF scaffold structure was acquired by removing the particular BAMG. This bag-shaped AM-SF scaffold provides sufficient area for ASC implantation, and the dense AM acts as a water-resistant barrier that prevents the ASCs from flowing to the abdominal cavity or bladder lumen. Furthermore, the remaining porous SF increases the contact area between the inner wall as well as the ASCs, which contributes to cells migration and proliferation. All of us implemented these novel technologies to improve the survival associated with ASCs and to ensure that ASCs were retained in the restoration area. We believe that this novel scaffold may be an appealing biomaterial for bladder reconstruction. In addition , the results demonstrated the particular utility of this technique.
Of the initial 42 rats in this study, 4 died within the first week after implantation. The success rate of the two groups was the same, and no rodents died in the cystotomy group. Autopsy revealed evidence of dehiscence at the suture line between the scaffold and the native urinary wall and the leakage of urine in the abdominal tooth cavity was obvious. Gross observation of the bladders revealed minor adhesion of the suture area to adjacent fat. Minimal scar formation and graft shrinkage were observed in the particular regeneration site of the AM-SF-ASCs 12 weeks postoperation. In comparison, marked shrinkage by approximately 50% was observed in the particular AM-SF group. The contracting of myofibroblasts may are the reason for the shrinkage of the repair area. However , stem tissue may prevent myofibroblasts from contracting and prevent tissue fibrosis using a paracrine function [ 24 – 26 ]. In addition , varying degrees of urinary stone formation were observed in the two groups during the 2nd week after implantation. Infrared spectrometer analysis revealed the calculus composition was ammonium magnesium phosphate, which may be brought on by the inflammatory response [ 27 , 28 ]. No stones had been found in the cystotomy group 12 weeks after implantation. Although some studies have suggested that stem cells can attenuate tissue inflammatory responses [ 29 ], ASCs were not noticed to reduce the rate of stone formation in this study. These types of findings demonstrated that the inflammation of scaffold materials performs a more important role in stone formation. In addition , degradation from the SF layer was similar to that reported in our prior study [ 17 ].
At week 2, there was no significant difference involving the two groups regarding regeneration of the urothelium, smooth muscle mass, or nerve bundles. However , at weeks 4 plus 12, the CK/SM22α /NeuN-positive area in the AM-SF-ASC team was superior to that of the control group. Regeneration from the urothelium may be explained as follows: during re-epithelialization, when basal cells undergo initial proliferation and migration across the problem site [ 5 ], a small amount of urothelium was found on the luminal surface of the repair area in the two groups from week 2 . However , due to the excessive proliferation of basal/intermediate cells [ 5 , 30 ], a multilayered urothelial lining had been detected across the luminal surface in the AM-SF-ASC group in weeks 4 and 12, which may be due to endocrine plus paracrine functions of the ASCs. The regeneration of steady muscle was remarkable in the experimental group, reaching beliefs similar to those of the cystotomy group at week twelve. Unfortunately, even after 12 weeks, apparent innervation in the AM-SF-ASC group was still significantly lower than that in the cystotomy group.
Because of the limited transdifferentiation of ASCs, the paracrine and endocrine roles of ASCs at the site of regeneration tend to be more important [ 31 ]. Bladder augmentation and construction utilizing the AM-SF-ASCs achieved excellent regeneration of smooth muscle plus vessels. However , ASCs possess redundant function on the urothelium and insufficient function on nerves. Nevertheless, the features of ASCs on neuranagenesis were positive compared to the AM-SF scaffolds. Therefore , ASCs may promote angiogenesis and neural axon growth in vivo via secreting VEGF, NGF, BDNF, and multiple other cytokines, as previously proven [ 13 ].
Urodynamic tracing analysis performed at 12 weeks right after implantation demonstrated differences among the three groups. The cystotomy and AM-SF-ASC groups demonstrated more normal waveforms compared to control group. Bladder capacity was augmented by minimum 30% with the AM-SF scaffolds and AM-SF-ASCs. Although almost half of the bladder was resected, and shrinkage from the regeneration zone was observed in the control group, the particular bladder itself maintains a certain volume by means of compensation: along with decreased strength of the bladder wall, hypertrophy and hyperplasia of the bladder smooth muscle in the normal region might play a major role [ 32 ]. Similar to the process of myocardial remodeling, this inevitably results in decreased compliance and eventually leads to myocardial fibrosis [ 33 ]. Hypertrophy and hyperplasia of bladder smooth muscle in the normal region might account for the lower bladder compliance in the AM-SF group, even though additional experiments are needed to verify this mechanism. In comparison, in the experimental group, quantification of urodynamic parameters highlighted that bladder capacity and compliance were higher than the particular cystotomy group, which may be explained by a neurogenic bladder because of limited innervation. The Aδ fibres, which respond to urinary wall distention and trigger micturition [ 34 ], had been insufficient because of the few innervations in the regeneration site. Therefore, the areflexia of de novo regenerated SMCs happens and eventually leads to a large capacity and low pressure urinary. Therefore , urodynamic normalization of the bladder supported by AM-SF-ASC scaffolds would likely require more than 12 weeks to achieve adequate innervation.
The particular highlight of our study is to create a novel AM-SF scaffold consisting of autologous myofibroblasts and porous silk fibroin construction. The scaffolds have the advantage of being water-proof and of an appropriate mechanical strength. The bag-shaped structure of the AM-SF scaffold was shown to improve the survival of ASCs in vivo for at least 12 weeks. We confirmed that this story scaffold combined with ASCs can be successfully used for bladder tissues regeneration and promote the recovery of bladder functionality. Importantly, ASCs promoted bladder regeneration rapidly and enhanced urinary function, and possessed the ability to differentiate into adult SMCs in vivo . Therefore , we believe that this method has great prospects meant for future clinical applications.
A limitation of our study is the lack of the comparable result with the simple AM or SF scaffolds. In addition , the technique reported here possesses some disadvantages, such as the complicated procedure for repairing and damage caused by several surgeries, the incidence of stone formation, and the restricted innervations for bladder regeneration. Future studies will concentrate on evaluating the long-term efficacy of our procedure in big animal models to translate this scaffold technology in order to clinical applications.