A novel tropoelastin-based resorbable surgical mesh for pelvic organ prolapse repair.

Pelvic organ prolapse is a common condition that affects 1 in 4 women across all age groups. It is mainly caused by vaginal birth injury and can be exacerbated by obesity and increased age. Until recently, treatment strategies often used non-degradable synthetic meshes for reconstructive surgery. However, owing to their frequent, unacceptable rate of adverse events such as mesh erosion, transvaginal meshes have been banned in many countries. Recent reports have highlighted the urgent need for biocompatible design of meshes for a safe and effective treatment in the long term. This study reports the design and evaluation of a novel, elastin based degradable mesh using an ovine model of POP as a potential surgical treatment. Elastin is a protein component of the ECM and provides elasticity to tissues throughout the body. Tropoelastin, the monomer subunit of elastin, has been used with success in electrospun constructs as it is a naturally cell interactive polymer. Biomaterials that incorporate tropoelastin support cell attachment and proliferation, and have been proven to encourage elastogenesis and angiogenesis in vitro and in vivo. The biological properties of tropoelastin were combined with the physical properties of PCL, a degradable synthetic polymer, with the aim of producing, characterizing and assessing the performance of continuous tropoelastin:PCL electrospun yarns. Using a modified spinneret electrospinning system and adjusting settings based on relative humidity, four blends of tropoelastin:PCL yarns were fabricated with concentration ratios of 75:25, 50:50, 25:75 and 0:100. Yarns were assessed for ease of manufacture, fibrous architecture, protein/polymer content, yarn stability - including initial tropoelastin release, mechanical strength, and ability to support cell growth. Based on overall favorable properties, a mesh woven from the 50:50 tropoelastin:PCL yarn was implanted into the vagina of a parous ewe with vaginal wall weakness as a model of pelvic organ prolapse. This mesh showed excellent integration with new collagen deposition by SEM and a predominant M2 macrophage response with few pro-inflammatory M1 macrophages after 30 days. The woven tropoelastin:PCL electrospun mesh shows potential as an alternative to non-degradable, synthetic pelvic organ prolapse mesh products.

Building Elastin. Incorporation of recombinant human tropoelastin into extracellular matrices using nonelastogenic rat-1 fibroblasts as a source for lysyl oxidase.

The purpose of this study was to assess the feasibility of crosslinking exogenously produced tropoelastin, the precursor of insoluble elastin, into existing elastin. Tritiated recombinant human tropoelastin (rhTE) was added to neonatal rat aorta smooth-muscle cell (NNRSMC) cultures. As much as 12% of the added rhTE was incorporated into the NNRSMC-derived insoluble elastin with the formation of the elastin crosslinks desmosine (DES) and isodesmosine (IDES) in a time-dependent fashion. The ratio of radioactivity found in DES and IDES crosslinks to that found in lysyl residues increased from 0.18 immediately after incubation with rhTE to 0.76 after 14 d. Crosslinking of rhTE into elastin and the accompanying formation of tritiated water was inhibited by beta-aminoproprionitrile, a potent inhibitor of lysyl oxidase, an enzyme critical for the post-translational processing of elastin and collagen. Acellular NNRSMC matrices were produced and replated with Rat-1 fibroblasts, cells that were found to express lysyl oxidase but not tropoelastin. At 14 d after incubation with rhTE, the ratio of DES and IDES radioactivity to that of lysine in the insoluble elastin was 0.38. We show for the first time that cells expressing lysyl oxidase, but not elastin, as well as elastogenic cells can incorporate rhTE into insoluble elastin with the formation of elastin crosslinks. This novel approach might be used to augment elastin repair in certain pathologic states.

Tandem integration of multiple ILV5 copies and elevated transcription in polyploid yeast.

An industrial yeast strain was modified by introducing DNA into brewing yeast such that the derived cells contain only yeast DNA. Thus selectable markers and bacterial sequences are not present in the final strain, making this procedure attractive for the development of generally acceptable brewing yeast. Linear DNA containing the cloned ILV5 gene was introduced into lager yeast along with an unlinked circular bifunctional plasmid containing a dominant resistance marker. Resistant colonies were screened for site-directed integration of the ILV5 DNA. Candidates were examined by several methods including Southern transfer and polymerase chain reaction. In this way, a strain WM56 was identified containing three tandem copies of ILV5. The amplified ILV5 region is stable during repeated subculturing in the absence of selective pressure. Correspondingly elevated levels of ILV5 transcript in strain WM56 compared to the control (i.e. non-tandem) parental strain led to increased amounts of encoded acetohydroxyacid reductoisomerase as evidenced by significantly lower diacetyl production. WM56 appears to be identical to the parental strain judged by CHEF, total restriction digestion patterns, and probing, but differs in the ILV5 region of the chromosome. The method is generally applicable to other yeast strains, and if desired, is amenable to iterated cycles of integration to increase the number of copies.