Hypothesis / aims of study
Tissue engineering is the field of medicine in which cells, (bio)materials and biochemical signals are used to repair or replace biological functions. Our research focus is tissue engineering for urethral reconstruction in patients with complicated urethral stricture disease or hypospadias.
In more severe urethral stricture, besides fibrotic changes of the urethral wall and obstruction of the lumen, also tissue surrounding the urethra, i.e. corpus spongiosum (CS), is affected by fibrosis. In hypospadias , the distal CS is absent to a greater or lesser extent. In urethral reconstruction using oral mucosal or preputial grafts, only the urethral epithelium and subepithelium is replaced. Currently, no substitution for the surrounding CS is provided. We hypothesize that replacement of CS-like tissue in urethral reconstruction might improve outcomes of urethral reconstruction.
To recreate CS that mimics healthy native CS the best possible, the exact structure and composition of healthy native CS should be known. The CS is composed of several layers, which could be mimicked using 3D-bioprinting. For this, a hydrogel is required as printing ink. Existing hydrogels like Gelatin Methacryloyl (GelMA) have good mechanical properties for printing, but lack the required biochemical cues for cells. By enriching the biomaterials with natural components of the extracellular matrix (ECM), regeneration is enhanced . The ECM is the essential non-cellular component of the tissue microenvironment of cells, comprised of a network of macromolecules including polysaccharide glycosaminoglycans (GAGs) and proteins such as collagens, laminins, and fibronectin. Ideally, the graft used in urethral reconstruction promotes healthy healing. By studying the difference of ECM proteins in spongiofibrosis and in healthy CS, a hydrogel can be developed with relevant ECM proteins steering towards healthy healing.
Our hypothesis is that understanding the composition of the ECM in healthy and diseased CS can lead to a tissue specific hydrogel. Here we focus on the molecular composition of the ECM using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to analyze tissue on protein level. We compare ECM components both qualitatively and quantitatively in healthy and fibrotic CS tissue.
Study design, materials and methods
Surgical waste was collected under local biobank protocol. Both fibrotic lesions obtained from excision and primary anastomosis urethral surgery and resected penile urethra after gender conformation surgery were used. Decellularization was performed using Sodium Dodecyl Sulphate (SDS). Decellularized tissue was stored in phosphate buffered saline before processing to LC-MS/MS analysis. The differences in amounts of ECM protein were investigated for three groups: healthy CS, fibrotic CS and normal-looking CS that was located next to the fibrotic part.
Samples were processed as described before . MS/MS spectra were extracted out of raw data files and were analyzed by using MaxQuant software. Results were analyzed using Perseus (version 220.127.116.11). By cross referencing with the Human Matrisome Project the results were analyzed using gene ontology analysis (geneontoly.org) and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) network analysis (string-db.org).
In parallel, surgical waste was fixated and embedded in paraffin. Tissue sections of 3 μm were stained with 4′,6-diamidino-2-phenylindole (DAPI), Hematoxylin-Eosin and used in immunohistochemistry for ECM components.
Elastin (ELN) was found in all three comparisons, it was highest expressed in fibrotic tissue, lower in normal-looking tissue and the lowest in transgender tissue. The proteins fibrilin 2 (FBN2) and emilin 2 (EMILIN2) were expressed higher in healthy (transgender) tissue. TNC, COMP, CILP, FGB, COL8A1, THBS4, AEBP1, ELN, FGF7, WNT9A and CHRDL1 were expressed higher in fibrotic tissue. No clear interaction between this group of proteins was found in the GO analysis nor the STRING. A few proteins were identified up in the two other comparisons (transgender - normal looking and fibrotic - normal looking). Expression of FBN2 was low in fibrotic tissue, ITIH1 was low in normal-looking tissue, and TNC, COMP, CILP and FGB were expressed low in transgender tissue. Findings from the MS/MS could be confirmed by immunohistochemistry. Most striking was the aberrant distribution of ELN in fibrotic samples, in contrast to healthy tissue, no bundles could be identified.
Interpretation of results
Some differences were found between the protein distribution, however, despite the different origin of the samples, the overall protein intensities were comparable. FBN2 and EMILIN2 are candidates for a scaffold to stimulate generation of healthy CS, whereas TNC, COMP, CILP, FGB, COL8A1, THBS4, AEBP1, ELN, FGF7, WNT9A and CHRDL1 are higher in fibrotic tissue, indicating these should be downregulated for regeneration of healthy CS. ELN was highest expressed in fibrotic tissue, this was unexpected as elastin is a protein that establishes strength and flexibility in tissues. ELN and FNB2 are interacting proteins in the formation of the elastic ECM network, low expression of FNB2 could contribute to the aberrant distribution of ELN in fibrotic tissue. Limitation of our research is the use of waste material from gender conformation surgery. This tissue is hormone treated and may not represent healthy tissue. On the other hand, only few significant differences were found when this tissue was compared to healthy tissue from urethroplasty. In addition, there are few other ways to obtain healthy human penile tissue for research purposes.
In this study we compared ECM components of healthy and fibrotic CS in order to be able to compose the ideal hydrogel for 3D-bioprinting for tissue engineering of the urethra and CS.
We suggest that FBN2 and EMILIN2 may stimulate regeneration, whereas TNC, COMP, CILP, FGB, COL8A1, THBS4, AEBP1, ELN, FGF7, WNT9A and CHRDL1 should be downregulated for regeneration of healthy CS.