Blended Nanostructured Degradable Mesh with Endometrial

Subscriber access provided by University of Winnipeg Library

Article

Blended Nanostructured Degradable Mesh with Endometrial Mesenchymal Stem Cells Promotes Tissue Integration and AntiInflammatory Response in Vivo for Pelvic Floor Application Shayanti Mukherjee, Saeedeh Darzi, Anna Rosamilia, Vinod Kadam, Yen Truong, Jerome A. Werkmeister, and Caroline E. Gargett Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01661 • Publication Date (Web): 04 Dec 2018 Downloaded from http://pubs.acs.org on December 10, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Blended Nanostructured Degradable Mesh with Endometrial Mesenchymal Stem Cells Promotes Tissue Integration and Anti-Inflammatory Response in Vivo for Pelvic Floor Application

Shayanti Mukherjee†,‡,§,*, Saeedeh Darzi†, Anna Rosamilia‡, ⊥, Vinod Kadam§, Yen Truong§, Jerome A. Werkmeister†,‡,§ and Caroline E. Gargett†,‡

†The

Ritchie Centre, Hudson Institute of Medical Research, Clayton, Australia 3168

‡Department

§CSIRO

⊥Pelvic

of Obstetrics and Gynaecology, Monash University, Clayton Australia 3168

Manufacturing, Clayton, Australia 3168

Floor Disorders Unit, Monash Health Australia

KEYWORDS: Electrospinning, Mesenchymal Stem Cells, Tissue Engineering, Foreign Body Response, Pelvic Organ Prolapse, Biomaterials

1 ACS Paragon Plus Environment

Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 49

ABSTRACT

The current urogynaecological clinical meshes trigger unfavourable foreign body response which leads to graft failure in the long term. To overcome the present challenge we applied a tissue engineering strategy using endometrial SUSD2+ Mesenchymal stem cells (eMSCs) with high regenerative properties. This study delves deeper into foreign body response to SUSD2+ eMSC based degradable PLACL/Gelatin nanofiber meshes using a mice model targeted at understanding immunomodulation and mesh integration in the long term. Delivery of cells with nanofiber mesh provides a unique topography that enables entrapment of therapeutic cells for upto 6 weeks that promotes substantial cellular infiltration of host anti-inflammatory macrophages. As a result, degradation rate and tissue integration are highly impacted by eMSCs, revealing an unexpected level of implant integration over 6 weeks in vivo. From a clinical perspective, such immunomodulation may aid in overcoming the current challenges and provide an alternative to an unmet women’s urogynaecological health need.


Page 3 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

INTRODUCTION Pelvic Organ Prolapse (POP) is a common childbirth induced urogynaecological disorder that affects 1 in 4 women across all age groups, and over 50% of parous women over the age of 50 years.1 Until recently, synthetic polypropylene meshes were often used to augment pelvic floor reconstruction surgeries as transvaginal meshes to treat POP. However, vaginal meshes have been associated with serious adverse events such as inflammation, dyspareunia, infection, retraction, exposure and erosion. Erosion and exposure of vaginally implanted mesh are serious adverse events associated with mesh-augmented POP surgery. Mesh erosion is defined as the unwanted protrusion of the mesh into another organ eg bladder or bowel. Mesh exposure occurs when the mesh protrudes through the vaginal epithelium into the vaginal cavity. Such mesh complications require morbid re-operations for removal of the exposed/eroded mesh. FDA warnings on these adverse events led to market withdrawals and ban on several synthetic nondegradable vaginal mesh products in several countries. A growing body of evidence suggests that the risk of complications outweighs the long-term benefits of non-degradable meshes and there is a need for superior biocompatible surgical therapies that foster tissue integration and overcome the hurdle of mesh exposure/erosion.

2, 3

An improved understanding of mesh

behaviour in vivo and its impact on the vagina and surrounding organs is imperative to obtain improved outcomes for women with POP.4 5All implanted biomaterials trigger a foreign body response that ultimately determines rejection or integration of the implanted biomaterial.6 The inability of polypropylene meshes to fully integrate with host tissue due to the chronic inflammatory response they evoke may be attributed to their lack of biomimetic character that ultimately leads to mesh erosion.7 Hence, it is pivotal that the new generation of pelvic floor biomaterial constructs offering optimal healing, integration and even immunomodulation rather than piquing an undesirable chronic foreign body response. Our observations from 3 ACS Paragon Plus Environment


Page 4 of 49

collagen coated polypropylene meshes in sheep vagina for 6 months have clearly highlighted that material design directly affects the innate immune response.8 If the foreign body response is controlled, neo-vascularization promoted and new collagen deposition boosted, better integration of the mesh is achieved. This emphasises the need to further influence tissue remodelling by tissue engineering methods to the design of new vaginal meshes as highlighted by clinicians and experts in the medical practice.9 In the last decade, several clinical trial outcomes have highlighted that multipotent and clonogenic mesenchymal stem cells (MSCs) from various tissue sources can mitigate inflammation and influence the microenvironment in reparative processes.10-15. We discovered a rare population of perivascular MSCs in the endometrial layer of the uterus,16,

17

easily

obtainable from endometrial biopsies procured in an office based procedure even from postmenopausal elderly women. We have previously shown in a phase IV clinical trial that postmenopausal women can regrow endometrial tissue and hence eMSCs make an attractive choice for cell therapy for the treatment of POP.18 We also identified a unique marker, SUSD2 to purify these rare perivascular cells19 using the W5C5 antibody. Furthermore, we showed that a small molecule TGF receptor inhibitor maintains clonogenic SUSD2+ eMSCs and prevents spontaneous fibroblast differentiation during culture expansion,20 a major advance for therapeutic applications such as pelvic tissue regeneration. Here we report, that SUSD2+ eMSCs co-localize with perivascular smooth muscle cells within human endometrium, confirming studies with ovine eMSCs.21 Previously, we have shown that eMSCs reduce the foreign body reaction to a novel non-degradable mesh by modulating inflammatory cytokine secretion and promoting macrophage polarization from M1 to M2 phenotype.22 These immunomodulatory effects are primarily mediated by cross-talk between eMSCs and macrophages, exerted via paracrine mechanisms in response to the microenvironment. Given that vaginal meshes have been associated with inadequate tissue integration and prolonged 4 ACS Paragon Plus Environment

Page 5 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

inflammatory responses years after mesh implantation,23 the use of eMSCs to improve tissue repair, reduce the foreign body response and promote early mesh integration is an important advance in improving outcomes for treating POP.15 Moreover, eMSCs can be safely harvested from the post-menopausal uterus and they have similar properties to premenopausal eMSCs.18 They are an ideal source of autologous therapeutic cells for the treatment of POP using novel tissue engineering approaches due to their ease of acquisition.12 While tissue engineering using polymeric materials and MSCs has been studied for various biomedical applications, the crosstalk between MSCs and polymeric materials and its overall impact on foreign body response have not been fully elucidated. This study aims to develop a tissue engineered POP treatment that exploits the immunomodulatory properties of eMSC to overcome mesh related foreign body responses. In particular, this study explores the properties and interactions at the interface of materials and cell biology critical for successful clinical application in urogynecology such as tissue integration and immune response to implanted mesh constructs that remain unknown, despite many years of research in the field of tissue engineering. Clinical trials of cell therapies using MSCs and other cells over the last decade have shown that mere injection of therapeutic cells is not sufficient to cure disease.24-26 In nature, cell behaviour and tissue structural development are supported by the nanoscale arrangement of the extracellular matrix (ECM) architecture that provides a larger surface area to adsorb proteins and present binding sites for cell membrane receptors.27 In order to overcome the damaged tissue microenvironment resulting from POP, surgical constructs that mimic the properties of normal ECM at the nanoscale are likely to integrate better and lead to a healing type of foreign body response. Ultrafine and biomimetic meshes with ECM-like topography can be achieved by electrospinning biomaterials. Such nanofibrous meshes made of biocompatible materials with nanoscale architecture provide larger surface areas to adsorb proteins and provide more binding sites to cell membrane receptors, unlike microscale and flat surfaces.28 Following the 5 ACS Paragon Plus Environment


Page 6 of 49

controversies associated with the use of polypropylene mesh for pelvic reconstruction, electrospun nanofiber meshes of degradable and non-degradable formats have been explored by researchers as an alternative solution.29-34 These studies have evaluated the potential of noncellular polymer based nanofiber meshes in urogynecology. Our study combines use of nanotechnology and cell based therapy to design a tissue engineered treatment for POP. We have further enhanced the biological properties of the nanofiber meshes using a biomacromolecule, gelatin. The impact of eMSCs on degradable nanofiber mesh integration and foreign body response has never been explored, neither for vaginal nor other tissue applications. Moreover, how and why therapeutic cells together with nanomaterials enhance outcomes remains unknown. Although cell based tissue engineering has been studied for several years, it remains unknown how mesh composition influences stem/progenitor cell penetration, which ultimately impacts long term tissue integration. From a clinical perspective, the mechanism of these cell-material interactions is imperative for the translation of tissue engineered constructs for treatment of POP. This study investigates the potential of degradable blended polymeric and natural biocomposite nanofiber meshes as a vaginal mesh and the impact of purified SUSD2+ eMSCs on the foreign body response when implanted using a subcutaneous NOD SCID gamma (NSG) mouse model. Vaginal surgeries are extremely challenging in rodent models owing to the small size of the vagina. Although a recent study reports nanofiber implantation vaginal surgery in a rat model, there is a lack of evidence of in vivo histology of mesh implantation or the surgery. 32 To this end, we have translated our ovine surgical methods in a simple subcutaneous mouse model to study and predict mesh performance in the vagina. Here we report, for the first time, the development, analysis and foreign body response of a nano-topographically controlled degradable mesh made of poly( L -lactic acid)- co -poly( ε -caprolactone) (PLACL) and gelatin boosted with MSCs of reproductive origin aimed at vaginal wall repair for POP treatment. 6 ACS Paragon Plus Environment

Page 7 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Recently, PLACL based meshes have shown promising results for pelvic floor application in clinical trial particularly symptom improvement and no erosion.35 Given our interest in future clinical translation, we used FDA approved polymers PLA and PCL in combination with the biomacromolecule gelatin to fabricate nanofiber meshes using electrospinning. To our knowledge, this is the first in vitro and in vivo study of the foreign body response and tissue integration of a SUSD2+ eMSC-based degradable PLACL and PLACL+G nanofiber construct designed for potential use in a urogynecological application. This study shows how hydrophilic nanofibers containing biomacromolecule, PLACL/Gelatin, improves stem cell retention in vivo addressing a major shortfall of clinical cell therapy. Furthermore, we report for the first time that eMSC (a) can control the fate of nanofibers in vivo using quantitative assessments at the nanoscale and (b) modulate the M1 and M2 macrophage response to TE constructs depending on polymer composition.



Page 8 of 49

EXPERIMENTAL SECTION Fabrication of Nanofiber Meshes Nanofiber meshes of PLACL and PLACL+G were fabricated by electrospinning. PLACL polymer of 70:30 ratio (Resomer, Evonik) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). PLACL and gelatin from porcine skin (Sigma Catalogue No. G2500) were dissolved and mixed in HFP at a ratio of 1:1 (w/w) to make final concentrations of 10% (w/v). Electrospinning was performed using solutions in syringes were attached to 23 G blunted stainless steel needle (Terumo Corporation, Japan) controlled using a syringe pump (NE1000, New Era Pump Systems, Inc. USA) at 1 ml/hour and voltage of 18 kV (Spellman SL150, USA) over a distance of 12.5 cm. Nanofibers were collected on grounded aluminum foil with or without 15 mm cover slips. All samples were dried and stored overnight in a vacuum oven for experimental use. Mechanical Characterization of Nanofibers All mechanical characterization studies were performed on dry nanofiber samples. To determine the molecular composition of a nanofibrous membrane and to investigate chemical functional groups, Fourier Transform Infrared (FTIR) spectroscopic analysis was performed on Avatar 380 (Thermo Nicolet, Waltham, MA, USA) over a range of 500–3800 cm-1 at a resolution of 2 cm-1. The powder form of polymer and gelatin and nanofibers served as controls. Gelatin nanofibers used as control were fabricated using 18% (w/v) at 1 ml/hour and voltage of 22 kV over a distance of 20 cm. Static Sessile drop water contact angle measurements (pocket goniometer PG-3, Fibro system) were done to investigate the hydrophilic nature of the electrospun nanofibrous membranes. Samples received a drop of water and the contact angle was recorded for 1 min using the equipment software. Pore size distribution of the nanofibrous scaffolds was measured by Capillary Flow Porometer (Porous 8 ACS Paragon Plus Environment

Page 9 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Materials Inc, USA) on the basis of wet-up/dry-up method. The membranes were wetted through with Galwick (Porous Materials Inc, USA) before measuring and the data was evaluated by using Automated Capillary Flow Porometer system software. Tensile properties of nanofibers were measured using uniaxial tester (Instron 3345, Canton, MA, USA) with a load of 10 N. Samples were loaded in dry condition and clipped at both ends. As the load was applied, the sample was uniformly subjected to a crosshead speed of 10 mm/min until breakpoint. Tensile properties were generated by Bluehill software in built in the system. Human Tissue Collection All SUSD2+ eMSC were isolated from endometrial biopsies obtained women undergoing laparoscopic surgery for non-endometrial gynaecological conditions under informed consent as per approval from the Monash Health and Monash University Human Research Ethics committees (09270B). All methods were performed in accordance with National Health and Medical Research Council guidelines. Each patient biopsy was used to generate a single eMSC cell line and served as n=1. Isolation and purification of SUSD2+ eMSCs Endometrial tissues were minced and digested using 5% collagenase type II and 40μg/ml deoxyribonuclease type I (DNAse I) (Worthington-Biochemical Corporation) at 37°C in Dulbecco’s modified Eagle’s Medium/F12 medium (DMEM/F12) containing 15 μM Hepes buffer (Invitrogen) in a humidified incubator at 37°C on a rotating MACSmix (Miltenyi Biotech) for 60- 90 minutes as per our previous protocols.22 Single cell suspensions were incubated with PE- conjugated SUSD2 (formerly W5C5) antibody (2μg/ml) (Biolegend) followed by anti- magnetic beads separation (Miltenyi Biotec) using a column and magnet (Miltenyi Biotec) to collect SUSD2+ eMSC. 9 ACS Paragon Plus Environment


Page 10 of 49

In vitro eMSC culture on nanofibers Each nanofiber scaffold of PLACL or PLACL+G collected on 15 mm coverslip was placed in a 24-well plate and secured with a stainless steel ring to ensure complete contact of the scaffolds with wells. Confluent cultures of SUSD2+ eMSCs grown in 75 cm2 cell culture flasks were detached using TryPLE (Gibco) containing 0.1% EDTA and 7500 cells were seeded on the scaffolds, based on our optimization results. Plain coverslips without nanofibers served as tissue culture plate (TCP) control. In vitro cell proliferation Assay Growth and proliferation of eMSCs on nanofiber meshes were evaluated by CellTiter 96 Aqueous One Solution Cell Proliferation-MTS Assay (Promega, Madison, WI, USA) as per manufacturer’s protocol over 7 and 14 days. Plain tissue culture plastic (TCP) coverslip without nanofibers was used as a positive control. On the day of analysis, samples were thoroughly washed with 1X PBS and 10% MTS solution in serum-free medium were added to incubate for 2.5 hours at 370C in a humidified dark incubator. Thereafter, volumes of 100 μl from each sample were pipetted into 96 well plates and absorbance read in a spectrophotometric plate reader (FLUOstar OPTIMA, BMG Lab Technologies, Germany) at 492 nm. m-Cherry Lentivirus Transduction and Sorting of eMSC To detect eMSC in vivo, the cells were transduced with a mCherry lentivirus as per our previous protocols.22 Following 48–72 hours transduction, eMSC were washed with 2% FBS/PBS, trypsinized with TrypLETM (Life Technologies) and then incubated with APC-conjugated mouse anti-human SUSD2 antibody (2 µg/ml) for 30 minutes in the dark on ice. Cells coexpressing mCherry and SUSD2 were sorted using a Beckman XDP cell sorter (Beckman


Page 11 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Coulter, Life Science, Australia) and the data were analysed using Summit Cytomation software version 5.2. Animal Mesh implantation Surgeries All animal husbandry, housing and experimental procedure were performed as approved by Monash Medical Centre Animal Ethics Committee A (2017/05). NSG mice were housed in the animal house at Monash Medical Centre according to the National Health and Medical Research Council of Australia guidelines for the care and use of laboratory animals. 40 NSG mice aged 8-16 weeks were randomly divided into 5 experimental groups namely (i) PLACL (ii) PLACL+G (iii) PLACL+eMSCs (iv) PLACL+G+eMSCs and (v) sham. The mice were anaesthetized with 3% w/v Isoflurane® and carprofen (5 mg/kg body weight) was used as analgesia. A longitudinal skin incision was performed in the lower abdomen and nanofiber mesh of 1.5 x 1cm size each was sutured into each of the two pockets of each animal for group i-iv. For animal groups receiving cells, 1x106 eMSCs labelled with m-Cherry lentivirus (1 eMSC patient cell line/replicate mouse for all groups and time points) in 20μl sterile PBS was carefully distributed onto the sutured mesh by pipette, based on our previously optimized protocols.22 Animals were euthanized in a CO2 chamber and mesh-tissue areas were harvested at 1 or 6 weeks (4mice/group/time-point) for histological analysis. The harvested tissues were fixed using 4% para-formaldehyde (PFA) embedded in OCT medium (Tissue-Tek, USA) and cut into 8μm sections.



Page 12 of 49

Scanning Electron Microscopy All biological samples from in vitro and in vivo experiments; 4% PFA fixed, on coverslips and tissue sections respectively, were washed with deioninsed water and then dehydrated with ascending concentrations of ethanol (30%, 50%, 75%, 95%, 100%) 2 times for 15 minutes each. All samples were finally treated with hexamethyldisilazane (HMDS) and air-dried overnight. All samples were coated with thin gold layer (Bal-Tec SCD 005, Leica USA) or platinum layer (Cressington 208 HR, UK) prior to examination under the scanning electron microscope (Nova NanoSEM, FEI, USA). Immunofluorescence The samples were permeabilized with 0.1% Triton X-100 (for Vinculin and Col I ), blocked for nonspecific staining using protein block (Dako) for 1 hour at room temperature. Primary antibodies were incubated with samples for 1 hour at room temperature followed by 3 washes with PBS. In case of unconjugated antibodies, species specific Alexa-Fluor-488 and AlexaFluor-647-conjugated secondary antibodies were incubated for 30 minutes at room temperature followed by 3 washes with PBS. Nuclei were stained with Hoechst 33258 (Molecular Probes) for 3 minutes and the slides were mounted with fluorescent mounting medium (Dako) and observed under a confocal microscope (Olympus Fluoview FV1200). For quantification of cellular infiltration in meshes, images were analysed using Image J. Morphometric Analysis of Nanofibers and Cellular Characteristics Cellular size was evaluated by outlining the perimeter of individual cells using pencil tool on Image J. Appropriate scale size was fed to the software and area measurement of the selected area was calculated using the area measurement tool. For cell size measurement of fluorescence images, F-actin and bright-field channels were used to draw the perimeter around the cells. F12 ACS Paragon Plus Environment

Page 13 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

actin fibrils and in vivo fibre diameter measurements were measured using the line measurement tool. At least 10 measurements/image were made to obtain an average. In vivo cellular infiltration values were obtained by image J analysis of confocal images. Mesh area was identified using the bright-field channel and area was selected in that zone. Image J macro plug in was used to identify and calculate fluorescent cells per unit area. Statistical Analysis All data presented are expressed as mean ± standard errors and analyzed using Graph Pad Prism v7. Data were analyzed using non-parametric Mann- Whitney U test; One-way ANOVA, Two-Way ANOVA or and Three-way ANOVA with Tukey’s multiple comparison tests, based on the comparisons made. A value of P ≤ 0.05 was considered to be statistically significant.



Page 14 of 49

RESULTS AND DISCUSSION Fabrication and Characterization of Nanofiber Meshes Electrospinning enables the design of scalable nanoscale meshes that mimic the structural and mechanical cues of tissue extracellular matrix (ECM). Moreover, the inclusion of natural biomaterials such as gelatin provides sites for integrin-mediated cell attachment thereby imparting excellent cell binding properties to meshes.28 Therefore, we aimed to fabricate degradable nanostructured meshes comprising PLACL and G from porcine origin (PLACL+G) using electrospinning to mimic the precise features of native tissue dimensions. Macroscopically, the electrospun nanofiber meshes appeared similar to a sheet of tissue paper (Fig S1). Scanning electron microscopy (SEM) micrographs

36

confirmed that both PLACL

and PLACL+G fabricated nanofiber meshes were porous and beadless, with uniform random nanofibers (Fig 1A and B) without any phase separation. The most critical factor of the fabrication process over other electrospinning parameters (i.e. voltage or flow rate) that eliminated formation of beads and non-uniform fibers was the distance between the Taylor’s cone and collection plate (Fig S2). The blending of G with PLACL was confirmed by chemical characteristics and functional group analysis using Fourier-transform infrared spectroscopy (FTIR). High-spectral-resolution data of wide spectral range revealed gelatin-specific N-H and C-H stretches between 3000cm-1 and 3500cm-1 (Fig 1C). Blended PLACL+G also showed the characteristic peak of amide I between 1600 and 1700 cm-1 and amide II between 1510 and 1580 cm-1 as seen in controls of gelatin powder and gelatin nanofiber. The amide region corresponds to the triple helix structure of collagen which is one of the key structural proteins found in the ECM of many connective tissues including skin and vagina, making up about 25% to 35% of the whole-body protein content.37 Despite being a denatured form of collagen, G has almost identical biological properties following electrospinning, at a much lower cost.38 Our 14 ACS Paragon Plus Environment

Page 15 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

results confirm that electrospinning enables complete blending of G with synthetic polymer PLACL to form uniform meshes that can be handled with ease for surgical applications. Nanofiber composition impacts Biophysical and Mechanical properties of mesh Following mesh fabrication, we further characterized and compared PLACL and PLACL+G meshes to evaluate the effect of blending. The characterization of the nanofibers highlighted that blending G with PLACL had significant impact on several biophysical and mechanical nanofiber properties. Analysis of SEM images and mechanical characterization revealed that while both PLACL and PLACL+G formed uniform fibers with interconnected pores, fiber diameter dimensions (Fig 2 A) and pore size (Fig 2 B) were significantly increased in PLACL+G. PLACL nanofibers had an average fiber diameter of 576 nm and pore size of 1.47μm where as PLACL+G meshes resulted in nanofibers with a higher average fiber diameter of 726nm and pore size of 2.1 μm. Furthermore, we analysed the hydrophilicity of the nanofiber meshes using a sessile drop water contact angle measurement. The water contact angle of PLACL+G almost immediately dropped below 900, while PLACL stayed over 900 for several minutes (Fig 2C). Our results suggest that G blending significantly increased the hydrophilicity of the meshes. Ideal meshes are expected to provide a compliant and hydrated environment similar to soft tissues having a high water content to facilitate cellular diffusion of nutrients and cellular waste. Thus, tailoring the surface hydrophilicity of the biomaterial meshes is likely to facilitate stronger host-mesh interaction and thereby reducing risk of erosion and providing better mesh performance. Tensile properties of vaginal meshes are a critical factor in determining long term performance.39 The human vagina is primarily comprised of dense connective tissue of elastin and collagen fibrils with random alignment in the lamina propria layer just beneath the surface epithelium and a deeper smooth muscle layer that provides mechanical support.40, 41 Both old 15 ACS Paragon Plus Environment


Page 16 of 49

and young vaginal tissues have viscoelastic properties that exhibit nonlinear stress–strain relations and Mullin’s effects.41 Characterization of the tensile properties of nanofiber meshes revealed that there was no difference between PLACL and PLACL+G meshes and that the latter matched the Young’s modulus of vaginal tissues (Fig 2D). There was also no difference between the PLACL and PLACL+G for other mesh tensile properties (Fig 2E, F). Our results show that while blending of G alters some PLACL mesh properties, the tensile properties are not significantly different. Human vaginal Young’s modulus ranges from 6 to12 MPa depending on age, reproductive phase and extent of prolapse. With age and weight gain, the stiffness of prolapsed vaginal tissue also reduces.42-44 Our results show that PLACL+G had mechanical properties similar to human vagina at all reproductive stages and hence may be a suitable choice for vaginal application. Mesh properties influence endometrial mesenchymal stem cell growth and protein expression in vitro Perivascular eMSCs largely contribute to the regeneration of endometrium following menstruation every month. Analysis of human endometrial biopsies revealed that SUSD2+ eMSCs co-localized with alpha smooth muscle actin (α-SMA) around the blood vessels in the endometrium (Fig 3A). Using dissociated endometrial biopsy tissue, we specifically purified eMSCs using SUSD2 magnetic bead sorting for this study. As expected, the isolated and purified eMSCs showed typical spindle shaped morphology in tissue culture (Fig 3B) and reached confluency within 2 weeks. Moreover, these eMSCs exhibited typical MSC surface phenotype; positive for CD73, CD90 and CD105 and negative for CD34 and CD45 by flow cytometry (Fig 3B) as per International Society of Stem Cell Research (ISCT) criteria. eMSCs are also multi-potent, giving rise to cells of osteogenic, adipogenic, chondrogenic and myogenic lineages like bone marrow MSCs.17, 19, 45 While our previous research has shown 16 ACS Paragon Plus Environment

Page 17 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

their beneficial immunomodulatory effects of eMSC on non-degradable polyamide based meshes11, 22, 46-48, their effect on nanofiber mesh has never been evaluated. To this end, we evaluated the cell-biomaterial interactions between eMSCs and nanofiber meshes of PLACL and PLACL+G in vitro. Cell interaction with the substratum forms the basis of tissue organisation in vivo. Vaginal mesh erosion results from sub-optimal mesh integration in the body. If cells adhere and penetrate meshes, tissue integration is likely to be superior and more effective. We found that eMSC-nanofiber interaction was influenced by nanofiber mesh composition (Fig 4). At day 7, eMSCs attach and start extending filapodias that partially penetrated the PLACL mesh surface (Fig 4A). By day 14, eMSCs were still on the surface and initiating cell-cell communication using the nanofiber architecture of the PLACL mesh (Fig 4B). In contrast, PLACL+G enabled complete eMSC penetration by day 7, resembling a “behind bars” feature. The growth and penetration of single eMSCs through a mesh with pores almost 10 times smaller than the cells themselves (Fig 4C) emphasizes that PLACL+G enables cellular penetration soon after seeding when the average cell size is less than 50μm (Fig S3). The gelatin component providing integrin binding sites that interact with cell integrin receptors invariably improves cell attachment and spreading (Fig 4). The larger pore size of PLACL+G mesh (Fig 2B) also contributed to the greater penetration and growth of eMSCs inside the PLACL+G nanofiber mesh compared with PLACL (Fig S4). After 14 days, there was complete integration of the cellular monolayer within PLACL+G nanofiber mesh (Fig 4D, S4). Image analysis of the SEM micrographs revealed a significant increase in cellular spreading on PLACL+G compared with PLACL alone indicating the importance of the gelatin component (Fig 4E). The cell-material biomaterial interactions were further characterized for focal adhesion interactions by the expression of cytoskeletal F-actin and Vinculin proteins. Focal adhesions 17 ACS Paragon Plus Environment


Page 18 of 49

are responsible for anchoring cells on substrates and are regulated by cellular transmembrane proteins and ECM ligands.49 The structure of F-actin and vinculin produced by eMSCs on PLACL+G closely resembled that on positive control of tissue culture plastic (TCP) (Fig 5A). In contrast, eMSCs on PLACL lacked continuous spreading of filamentous F-actin and vinculin over the mesh surface as the cells stayed rounded due to the paucity of cell-cell and cellmaterial interactions (Fig 4B). The eMSC cellular size, calculated as area covered by the spreading cell (Fig 5B) and F-actin filament length, calculated as an average filamentous length expressed on cells (Fig 5C) on PLACL+G was significantly higher than PLACL and closely matched that of TCP positive control. While focal adhesions signify cellular anchoring on substrates in stationary conditions, they also serve as support points during spreading and migration that suppress membrane contraction (rounding of cells) and promote protrusion at the leading edge. Cellular therapy aims to use therapeutic cells which retain their intrinsic properties. The focal adhesion patterns of eMSC on PLACL+G closely resembles positive control TCP, signifying that these meshes do not compromise their intricate migration properties and are likely to enhance mesh integration following implantation. This hypothesis was also supported by the growth and proliferation of eMSCs on nanofiber meshes in comparison to TCP over 7 and 14 days (Fig 5D). Our results indicate that eMSC growth is similar on both nanofiber meshes at 7 days. However, by 14 days, eMSC growth on PLACL+G was significantly greater than that on PLACL alone. The growth of eMSCs significantly increased from 7 day to 14 day for both PLACL and PLACL+G. Together, this suggests that eMSC proliferate more slowly on PLACL than PLACL+G meshes, but also that neither meshes are toxic or have a detrimental effect on eMSCs. To prepare a successful cell based mesh construct, it is of utmost importance that the mesh scaffolding supports cell proliferation and maintains normal protein expression. Our results confirm that PLACL+G nanofiber meshes provide a suitable scaffolding where cells can interact with the mesh, each other as well as host 18 ACS Paragon Plus Environment

Page 19 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

cells. Moreover, bioengineered vaginal meshes would likely need pre-seeded eMSCs prior to surgical implantation. Hence, from a clinical perspective, it is paramount that vaginal meshes facilitate cell-material interaction for optimal tissue integration. eMSCs enhance cellular infiltration and tissue integration of meshes in vivo Regardless of their origin, the therapeutic value of MSCs are defined by their trophic, paracrine and immunomodulatory properties in regulating tissue repair. Many reports support the consensus that implanted MSCs are hardly retained 50 but yet they benefit organ and tissue repair, mostly through paracrine effects51. Most clinical trials have also demonstrated that retention of MSCs is a major clinical translation hurdle 52 and the quest for the ideal strategy to attain this goal is still on-going. Strongly suggested by our in vitro data, we hypothesized that nanotopography and mesh composition would enhance cellular retention of eMSCs in vivo, which will invariably improve mesh integration in the host tissue. Here we used NSG mice as they are immunodeficient, lacking components of the adaptive immune system such as T cells, B cells and Natural Killer cells. NSG mice are an attractive model to study innate macrophage responses and are commonly used as models in cell transplantation studies. Recently, we compared the transplantation and immunomodulatory properties of eMSCs using immunocompromised and immunocompetent mice that support the use of this model for tissue engineering studies aimed at understanding the foreign body response.22 In the present study, nanofiber meshes with or without eMSCs were transplanted subcutaneously in our NSG mouse model. SUSD2 purified eMSCs cultures (Fig 6) were genetically labelled with m-Cherry lentivirus for transplantation to enable in vivo detection. Double positive SUSD2+ and mCherry+ cells were sorted by flow cytometry (Fig 6A) and expanded in culture until confluency prior to in vivo implantation (Fig 6B). Nanofiber meshes of PLACL and PLACL+G were transplanted with or without m-Cherry-labelled eMSCs for 1 and 6 weeks (Fig 6C). Analysis 19 ACS Paragon Plus Environment


Page 20 of 49

of eMSC retention and host cell infiltration was performed by staining tissue sections with Hoechst 22358 for the nucleus (blue) and observing under florescence microscope (Fig 7). We detected many m-Cherry+ eMSCs (red) in multiple locations along the mesh- host tissue borders at 1 week for both PLACL and PLACL+G implanted mice (Fig 7A-B). eMSCs were also found distant to implanted PLACL+G (Fig S5) suggesting some have migrated from the mesh and incorporated into the host tissue. However, at 6 weeks, m-Cherry eMSCs were detected in very low quantity, in PLACL+G implanted mice only (Fig 7C-D), mostly inside the mesh. This may be attributed to rapid penetration of eMSCs into the PLACL+G nanofiber meshes soon after seeding, through its larger pores and integrin mediated interactions with the cells, as observed in vitro (Fig 4C). This feature is likely to have trapped the cells and kept them in the implanted zone for 6 week as seen in Fig 7D. It also indicates that while xenografted human eMSCs are mostly removed by the innate immune system (macrophages), PLACL+G enables longer retention. Meshes implanted in the subcutaneous pocket between the skin and abdominal wall serve as an ideal model to evaluate nanostructured mesh driven tissue integration and the influence of added MSC on this process. Without external stimuli, the abdominal wall and skin following explantation are completely detached as seen in sham controls (Fig S6). Therefore, any level of tissue integration is solely driven by the properties on the implant itself, including the eMSC present in the construct. Evaluation of vaginal meshes in a small animal pre-clinical model is extremely challenging due to the small size of rodent vagina and associated animal welfare issues. Hence, the subcutaneous site in mice is an ideal surrogate small animal preclinical model to investigate mesh properties prior to advancing to a suitable large animal model such as sheep.8 Using fluorescence microscopy of explanted mesh-tissue sections and image quantification we assessed the extent of host cellular infiltration into the implanted nanofiber meshes (Fig 7 E-L) at 1 and 6 weeks. Cellular infiltration in implants from all groups 20 ACS Paragon Plus Environment

Page 21 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

only changed marginally with time, except for PLACL+G+eMSC (Fig 7 M), highlighting that eMSCs positively influence host responses to PLACL+G meshes. At 1 week, PLACL mesh without eMSCs had the least cellular infiltration and lowest tissue integration (Fig 7 E). More specifically, Fig 7M shows the cellular infiltration per unit area was significantly lower compared to all other groups. There was no difference in the cellular infiltration between PLACL+G, PLACL+eMSC and PLACL+G+eMSC at 1 week. At 1 week the presence of eMSC significantly improved the poor cellular infiltration of the PLACL mesh (Fig 7G). In contrast at 6 weeks, the extent of cellular infiltration differed between all groups (Fig7M), particularly in presence of eMSCs. Both PLACL and PLACL+G nanofiber meshes with eMSCs had a significantly higher cellular infiltration compared to the nanofiber meshes alone. We also observed that material composition impacted the extent of cellular infiltration with significantly more cells/mm2 for PLACL+G compared to PLACL and for PLACL+G+eMSC versus PLACL+eMSC. Strikingly, PLACL+G+eMSC nanofiber meshes were completely infiltrated with cells to its core (Fig 7L). It is plausible that this effect is mediated by the retention of eMSC by the mesh even at 6 weeks. These results emphasize that nanofiber mesh implants with eMSCs significantly improve tissue integration by aiding cellular infiltration that invariably leads to formation of robust fully integrated mesh-tissue complexes. The initial foreign body response to implanted biomaterials is characterized by infiltration of neutrophils, followed by monocytes that later differentiate and promote recruitment of macrophages and fibroblasts in the later stages. Monocytes have the capacity to transmigrate across tight blood vessel walls into tissue through a series highly regulated interactions between the immune and the endothelial cells.53 Therefore, host monocyte infiltration can be attributed to more than just mesh pore size, although function of the G component in the blended PLACL, which provides integrin binding sites is likely to be in influence. Moreover, the immunomodulatory effects of the eMSCs also influence the foreign body response.22, 46 Together, these three components 21 ACS Paragon Plus Environment


Page 22 of 49

result in a superior host-compatible and cell-inducing composite meshes that promotes superior tissue integration, particularly those seeded with eMSC. From the vaginal tissue engineering and clinical translation perspective, PLACL+G shows significant promise as a new degradable vaginal mesh that may overcome the current challenge of mesh erosion and/or exposure owing to its enhanced cell retention and infiltration properties. Material composition and eMSCs control ECM formation and degradation of nanofiber meshes in vivo While the use of natural biomaterials in combination with polymers imparts many desirable properties that boost host cellular responses, it also hastens degradation. An ideal vaginal mesh needs to degrade slowly to ensure sufficient tissue re-organization required for vaginal wall strengthening occurs prior to complete mesh degradation. To investigate this, we analysed mesh morphology of explanted tissue sections using SEM to reveal effects of in vivo degradation by quantitative changes in fiber diameter after tissue explantation. Several studies have predicted the degradation rate of nanofibers, mostly, measured as weight loss over time in vitro.54-56 However, the precise change of individual fiber dimensions following in vivo implantation has not been previously studied. Herein, we depict the fate of implanted nanofiber meshes following in vivo implantation in NSG mice over 6 weeks. SEM micrographs revealed significant changes in nanofiber morphology, which was accentuated by the presence of eMSCs (Fig 8 A-H). Quantification of SEM micrographs (Fig 8 I) showed that both PLACL and PLACL+G meshes underwent significant but opposite morphological changes following in vivo implantation.

At 1 week, PLACL fibers (Fig 8A) appear more separated than

PLACL+G (Fig 8C), which had a more integrated appearance. At 6 weeks, however, PLACL (Fig 8E) was more integrated with the host tissue. To our surprise, PLACL+G (Fig 8G) appeared much thinner compared to pre-transplanation meshes. In the absence of eMSCs, 22 ACS Paragon Plus Environment

Page 23 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

PLACL mesh fibres exhibited significant swelling, PLACL+G had reduced to almost 50% of its implanted size by 6 weeks. Interestingly, eMSCs mitigated both these effects on the nanofiber meshes (Fig 8I). PLACL +eMSCs (Fig 8B, F) had the highest swelling at 1week, but this significantly reduced from 1week to 6 weeks (Fig 8I). On the other hand, eMSCs prevented the thinning of PLACL+G at 1 and 6 weeks (Fig 8D,H, I). PLACL+G was characterized by a heavy host cellular response at 6 weeks that is likely from the enzymatic action of infiltrated macrophages on the nanofibers (Fig 8G). eMSCs appeared to control PLACL+G thinning suggesting that eMSCs may have an impact on the host cellular response that may regulate the degradation of natural biomaterials such as gelatin. While most natural biomaterials support cell growth and spreading in vitro, their in vivo degradation rate is relatively rapid, occurring before the healing process has completed. This may be a critical limitation of using degradable mesh for a chronic disorder such as POP where biomaterial support is key. Although there are no optimal standards for POP repair or treatment, it would be desirable for degradable biomaterials to last 6-12 months until new tissue of desired stiffness has been regenerated. Our results show that eMSCs limit the rapid thinning of PLACL+G , an effect likely due to the paracrine effect of MSCs that regulate many host responses.51 Although, in vivo tissue microenvironment are known to maintain the tissue homeostasis through enzymatic action of ECM proteins such as MMPs and TIMPs, further studies are required to fully understand the mechanism and enzymes that are responsible. Such studies are likely to shed light on the effect of eMSCs on ECM biochemical cues and other microenvironment effects. These significant observations from our present study suggest that nanofiber meshes provide a suitable scaffolding for eMSCs which in turn aid in the precise control over fiber thinning and swelling that critically affect in vivo performance. Collagen deposition and neovascularization are also important aspects of healthy tissue regeneration. We confirmed collagen deposition inside the meshes at 6 weeks as revealed by immunofluorescence and SEM imaging (Fig S7). 23 ACS Paragon Plus Environment


Page 24 of 49

Neovascularization was mainly seen around individual mesh nanofibers (Fig S8) for all groups except PLACL+G+eMSC where we found CD31+ endothelial cells inside the mesh at 6 weeks. These features may be of critical importance for practical considerations for a new generation of vaginal repair surgical meshes. eMSCs influence the macrophage-based foreign body response to nanofiber meshes in vivo The physical, biological and chemical properties of biomaterials; pore-size, topography, surface chemistry, degradation rate and use of immunomodulatory cells such as MSCs greatly influences the nature of foreign body response and fate of the implant. To this end, our unique research strategy in vaginal mesh design has focussed on early and proactive interaction with the immune system to drive a desirable host response. Recently we showed that the immunomodulatory properties of eMSCs predominantly impact macrophage polarization and cytokine profiles using immunodeficient and immunocompetent mice models.22 Our studies indicated that the NSG mice model is optimal for understanding innate foreign body response to cell tissue engineered constructs due to longer eMSC retention times and lack of an adaptive immune system in these mice. As the foreign body response progresses, macrophages assemble at the site of implantation, further amplifying chemo-attractive signals that play a critical role in wound healing and tissue regeneration.

57

Hence, we aimed to

determine the effect of eMSCs on macrophage phenotype and foreign body response to nanofiber mesh implants. We analysed macrophage phenotypes within and around the implanted nanofiber mesh constructs at 1 and 6 weeks (Fig 9). The inflammatory M1 phenotype was detected by colocalisation of F4/80 (pan macrophage marker) and CCR7 using immunofluorescence and confocal microscopy, whereas wound healing M2 phenotype was identified by F4/80 and CD206 colocalisation. In the absence of eMSCs, a small number of 24 ACS Paragon Plus Environment

Page 25 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

M1 macrophages were observed inside PLACL meshes at 1 week (Fig 9A), which was reduced by 6 weeks (Fig 9B). In contrast, an increased M1 macrophage response was observed in and around PLACL+G mesh from 1 to 6 weeks (Fig 9C, D). This may be due to the gelatin component and the likely reason for increased nanofiber degradation. Cells with macrophage morphology (F4/80+) were also seen in SEM micrographs of PLACL+eMSCs and PLACL+G +eMSCs at 6 weeks (Fig 9F, H). Both PLACL and PLACL+G had few M2 macrophages around the nanofiber meshes in the absence of eMSCs (Fig 9I, J, K, L). In contrast, both PLACL and PLACL+G with eMSCs appeared to have more M2 macrophages than M1 in and around the implants (Fig 9, E-H, M-P). In particular, PLACL+G+eMSCs had more M2 macrophages around and inside the mesh at 1 week (Fig 9G, O). Mesh implantation triggers the immune system and circulating white blood cells, mainly neutrophils and monocytes, rapidly infiltrate the implantation site 57. The initial M1 macrophage response is necessary for the first few days after biomaterial implantation to remove dead cells and tissue debris resulting from the surgical incision. Eventually, the M1/M2 paradigm plays a crucial and dynamic role in the outcome of biomaterial implantation 58. We observed a very high M2 macrophage and minimal M1 response around the PLACL+G+eMSC meshes at 6 weeks (Fig 9H, 9P) in comparison to all other groups. Strikingly, the cells that infiltrated the mesh did not stain for any of the macrophage markers including F4/80 (Fig 9H,P). The lack of host macrophage markers and implanted m-Cherry+ cells suggests that these infiltrated cells are likely host fibroblasts. Macrophages mediate the migration and proliferation of fibroblasts into wound sites and constitute the initial steps toward effective tissue regeneration.14 Apart from phagocytosis of wound debris, these processes entail release of cytokines and growth factors important for tissue reorganization. A recent landmark study of samples from women with controversial mesh complication, highlighted that the presence of macrophages, elevated cytokines, chemokines, and MMPs in tissue-mesh complexes results in ongoing chronic host 25 ACS Paragon Plus Environment


Page 26 of 49

inflammatory response for several years following polypropylene implantation.23 In order to improve optimal vaginal repair and mesh performace, our results from the present study suggest that eMSCs promote a healing and regenerative host response to PLACL+G nanofiber meshes within 6 weeks. Moreover, our data confirms that degradable PLACL+G nanofiber mesh provides a suitable microenvironment which acts like a platform to retain therapeutic cells for a longer time as well as initiate host fibroblasts influx to eventually enable mesh integration. Such a microenvironment also controls the macrophage-tissue response that impacts on mesh degradation more favourably. Furthermore, the nanoscale cues from the ECM component of meshes such as PLACL+G provide a robust scaffolding for therapeutic SUSD2+ eMSCs that improves in vivo mesh performance and have potential use in vaginal meshes for the treatment of POP, with more longer term studies.


Page 27 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

CONCLUSION The most serious adverse effects of recent and current vaginal meshes are mesh erosion and exposure, and chronic pelvic pain that may necessitate surgical removal of implanted meshes. To avoid such an undesirable prolonged response, it is pivotal that implanted meshes promote the release of cytokines and growth factors that guide the tissue repair and reorganization. This study demonstrated that a nanofibrous biocomposite mesh that supports and retains endometrium-derived SUSD2+ eMSC at the site of implantation ultimately boosts tissue integration via host cellular infiltration in a subcutaneous mouse model. The hydrophilic, biocompatible blended PLACL+G nanofibrous meshes stimulate superior attachment, growth and infiltration of therapeutic eMSCs that significantly improves cell–material interactions without attenuating their functional activity. These properties enable longer retention of eMSCs and promote host cell infiltration that in turn retards mesh degradation, promotes mesh integration and a superior macrophage-mediated foreign body response in vivo. Our study suggests that bioengineering PLACL+G using a women’s own (autologous) eMSCs may aid in overcoming the current challenges facing clinicians and holds immense promise for POP treatment and possibly other graft augmentation surgeries. For successful clinical translation, it is imperative that appropriate studies using larger animal models are conducted to assess the impact of mechanical loading, and dynamics of daily human life activities on the mesh efficacy in the long term.



Page 28 of 49

FIGURES

Figure 1: Fabrication of PLACL and PLACL+G electrospun nanofiber meshes. Scanning Electron Micrograph show uniform and beadless electrospun nanofiber morphology of (A) PLACL and (B) PLACL+G. (C) FTIR overlay confirms blending of gelatin with PLACL revealing characteristic peak of amide I between 1600 and 1700 cm-1 and amide II between 1510 and 1580 cm-1 corresponding to gelatin in PLACL+G, similar to gelatin powder and gelatin nanofiber controls.


Page 29 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 2: Biophysical and Mechanical characterization of nanofiber meshes. PLACL (pink bars) and PLACL+G (blue bars) nanofibers show significant difference in (A) fiber diameter, (B) pore size diameter and (C) hydrophilicity. Addition of gelatin to PLACL did not alter tensile properties; (D) Young’s Modulus, (E) maximum elongation and (F) percentage



Page 30 of 49

elongation at break point. Data are mean ± SEM of n=3-5 samples/group analysed by MannWhitney U test; *p

Blended Nanostructured Degradable Mesh with Endometrial

Recommend Documents