Immunobiology of foreign body response to composite PLACL/gelatin electrospun nanofiber meshes with mesenchymal stem/stromal cells in a mouse model: Implications in pelvic floor tissue engineering and regeneration.

Pelvic Organ Prolapse (POP) is a common gynaecological disorder where pelvic organs protrude into the vagina. While transvaginal mesh surgery using non-degradable polymers was a commonly accepted treatment for POP, it has been associated with high rates of adverse events such as mesh erosion, exposure and inflammation due to serious foreign body response and therefore banned from clinical use after regulatory mandates. This study proposes a tissue engineering strategy using uterine endometrium-derived mesenchymal stem/stromal cells (eMSC) delivered with degradable poly L-lactic acid-co-poly ε-caprolactone (PLACL) and gelatin (G) in form of a composite electrospun nanofibrous mesh (P + G nanomesh) and evaluates the immunomodulatory mechanism at the material interfaces. The study highlights the critical acute and chronic inflammatory markers along with remodelling factors that determine the mesh surgery outcome. We hypothesise that such a bioengineered construct enhances mesh integration and mitigates the Foreign Body Response (FBR) at the host interface associated with mesh complications. Our results show that eMSC-based nanomesh significantly increased 7 genes associated with ECM synthesis and cell adhesion including, Itgb1, Itgb2, Vcam1, Cd44, Cdh2, Tgfb1, Tgfbr1, 6 genes related to angiogenesis including Ang1, Ang2, Vegfa, Pdgfa, Serpin1, Cxcl12, and 5 genes associated with collagen remodelling Col1a1, Col3a1, Col6a1, Col6a2, Col4a5 at six weeks post-implantation. Our findings suggest that cell-based tissue-engineered constructs potentially mitigate the FBR response elicited by biomaterial implants. From a clinical perspective, this construct provides an alternative to current inadequacies in surgical outcomes by modulating the immune response, inducing angiogenesis and ECM synthesis during the acute and chronic phases of the FBR.

Identification and characterisation of maternal perivascular SUSD2+ placental mesenchymal stem/stromal cells.

Mesenchymal stem cells (MSCs) that meet the International Society for Cellular Therapy (ISCT) criteria are obtained from placental tissue by plastic adherence. Historically, no known single marker was available for isolating placental MSCs (pMSCs) from the decidua basalis. As the decidua basalis is derived from the regenerative endometrium, we hypothesised that SUSD2, an endometrial perivascular MSC marker, would purify maternal perivascular pMSC. Perivascular pMSCs were isolated from the maternal placenta using SUSD2 magnetic bead sorting and assessed for the colony-forming unit-fibroblasts (CFU-F), surface markers, and in vitro differentiation into mesodermal lineages. Multi-colour immunofluorescence was used to colocalise SUSD2 and α-SMA, a perivascular marker in the decidua basalis. Placental stromal cell suspensions comprised 5.1%SUSD2+ cells. SUSD2 magnetic bead sorting of the placental stromal cells increased their purity approximately two-fold. SUSD2+ pMSCs displayed greater CFU-F activity than SUSD2- stromal fibroblasts (pSFs). However, both SUSD2+ pMSC and SUSD2- pSF underwent mesodermal differentiation in vitro, and both expressed the ISCT surface markers. Higher percentages of cultured SUSD2+ pMSCs expressed the perivascular markers CD146, CD140b, and SUSD2 than SUSD2- pSFs. These findings suggest that SUSD2 is a single marker that enriches maternal pMSCs, suggesting they may originate from eMSC. Placental decidua basalis can be used as an alternative source of MSC for clinical translation in situations where there is no access to endometrial tissue.

Immunobiology and Application of Aloe Vera-Based Scaffolds in Tissue Engineering.

Aloe vera (AV), a succulent plant belonging to the Liliaceae family, has been widely used for biomedical and pharmaceutical application. Its popularity stems from several of its bioactive components that have anti-oxidant, anti-microbial, anti-inflammatory and even immunomodulatory effects. Given such unique multi-modal biological impact, AV has been considered as a biomaterial for regenerative medicine and tissue engineering applications, where tissue repair and neo-angiogenesis are vital. This review outlines the growing scientific evidence that demonstrates the advantage of AV as tissue engineering scaffolds. We particularly highlight the recent advances in the application of AV-based scaffolds. From a tissue engineering perspective, it is pivotal that the implanted scaffolds strike an appropriate foreign body response to be well-accepted in the body without complications. Herein, we highlight the key cellular processes that regulate the foreign body response to implanted scaffolds and underline the immunomodulatory effects incurred by AV on the innate and adaptive system. Given that AV has several beneficial components, we discuss the importance of delving deeper into uncovering its action mechanism and thereby improving material design strategies for better tissue engineering constructs for biomedical applications.

Emerging Nano/Micro-Structured Degradable Polymeric Meshes for Pelvic Floor Reconstruction.

Pelvic organ prolapse (POP) is a hidden women's health disorder that impacts 1 in 4 women across all age groups. Surgical intervention has been the only treatment option, often involving non-degradable meshes, with variable results. However, recent reports have highlighted the adverse effects of meshes in the long term, which involve unacceptable rates of erosion, chronic infection and severe pain related to mesh shrinkage. Therefore, there is an urgent unmet need to fabricate of new class of biocompatible meshes for the treatment of POP. This review focuses on the causes for the downfall of commercial meshes, and discusses the use of emerging technologies such as electrospinning and 3D printing to design new meshes. Furthermore, we discuss the impact and advantage of nano-/microstructured alternative meshes over commercial meshes with respect to their tissue integration performance. Considering the key challenges of current meshes, we discuss the potential of cell-based tissue engineering strategies to augment the new class of meshes to improve biocompatibility and immunomodulation. Finally, this review highlights the future direction in designing the new class of mesh to overcome the hurdles of foreign body rejection faced by the traditional meshes, in order to have safe and effective treatment for women in the long term.

Electrospun Nanofiber Meshes With Endometrial MSCs Modulate Foreign Body Response by Increased Angiogenesis, Matrix Synthesis, and Anti-Inflammatory Gene Expression in Mice: Implication in Pelvic Floor.

PURPOSE: Transvaginal meshes for the treatment of Pelvic Organ Prolapse (POP) have been associated with severe adverse events and have been banned for clinical use in many countries. We recently reported the design of degradable poly L-lactic acid-co-poly ε-caprolactone nanofibrous mesh (P nanomesh) bioengineered with endometrial mesenchymal stem/stromal cells (eMSC) for POP repair. We showed that such bioengineered meshes had high tissue integration as well as immunomodulatory effects in vivo. This study aimed to determine the key molecular players enabling eMSC-based foreign body response modulation. METHODS: SUSD2+ eMSC were purified from single cell suspensions obtained from endometrial biopsies from cycling women by magnetic bead sorting. Electrospun P nanomeshes with and without eMSC were implanted in a NSG mouse skin wound repair model for 1 and 6 weeks. Quantitative PCR was used to assess the expression of extracellular matrix (ECM), cell adhesion, angiogenesis and inflammation genes as log2 fold changes compared to sham controls. Histology and immunostaining were used to visualize the ECM, blood vessels, and multinucleated foreign body giant cells around implants. RESULTS: Bioengineered P nanomesh/eMSC constructs explanted after 6 weeks showed significant increase in 35 genes associated with ECM, ECM regulation, cell adhesion angiogenesis, and immune response in comparison to P nanomesh alone. In the absence of eMSC, acute inflammatory genes were significantly elevated at 1 week. However, in the presence of eMSC, there was an increased expression of anti-inflammatory genes including Mrc1 and Arg1 by 6 weeks. There was formation of multinucleated foreign body giant cells around both implants at 6 weeks that expressed CD206, a M2 macrophage marker. CONCLUSION: This study reveals that eMSC modulate the foreign body response to degradable P nanomeshes in vivo by altering the expression profile of mouse genes. eMSC reduce acute inflammatory and increase ECM synthesis, angiogenesis and anti-inflammatory gene expression at 6 weeks while forming newly synthesized collagen within the nanomeshes and neo-vasculature in close proximity. From a tissue engineering perspective, this is a hallmark of a highly successful implant, suggesting significant potential as alternative surgical constructs for the treatment of POP.

3D bioprinted endometrial stem cells on melt electrospun poly ε-caprolactone mesh for pelvic floor application promote anti-inflammatory responses in mice.

Endometrial mesenchymal stem/stromal cells (eMSCs) exhibit excellent regenerative capacity in the endometrial lining of the uterus following menstruation and high proliferative capacity in vitro. Bioprinting eMSCs onto a mesh could be a potential therapy for Pelvic Organ Prolapse (POP). This study reports an alternative treatment strategy targeting vaginal wall repair using bioprinting of eMSCs encapsulated in a hydrogel and 3D melt electrospun mesh to generate a tissue engineering construct. Following a CAD, 3D printed poly ε-caprolactone (PCL) meshes were fabricated using melt electrospinning (MES) at different temperatures using a GMP clinical grade GESIM Bioscaffolder. Electron and atomic force microscopies revealed that MES meshes fabricated at 100 °C and with a speed 20 mm/s had the largest open pore diameter (47.2 ±â€¯11.4 µm) and the lowest strand thickness (121.4 ±â€¯46 µm) that promoted optimal eMSC attachment. An Aloe Vera-Sodium Alginate (AV-ALG) composite based hydrogel was optimised to a 1:1 mixture (1%AV-1%ALG) and eMSCs, purified from human endometrial biopsies, were then bioprinted in this hydrogel onto the MES printed meshes. Acute in vivo foreign body response assessment in NSG mice revealed that eMSC printed on MES constructs promoted tissue integration, eMSC retention and an anti-inflammatory M2 macrophage phenotype characterised by F4/80+CD206+ colocalization. Our results address an unmet medical need highlighting the potential of 3D bioprinted eMSC-MES meshes as an alternative approach to overcome the current challenges with non-degradable knitted meshes in POP treatment. STATEMENT OF SIGNIFICANCE: This study presents the first report of bioprinting mesenchymal stem cells derived from woman endometrium (eMSCs) to boost Pelvic Organ Prolapse (POP) treatment. It impacts over 50% of elderly women with no optimal treatment at present. The overall study is conducted in three stages as fabricating a melt electrospun (MES) mesh, bioprinting eMSCs into a Ca2+ free Aloe Vera-Alginate (AV-Alg) based hydrogel and in vivo study. Our data showed that AV-ALG hydrogel potentially suppresses the foreign body response and further addition of eMSCs triggered a high influx of anti-inflammatory CD206+ M2 macrophages. Our final construct demonstrates a favourable foreign body response to predict expected tissue integration, therefore, provides a potential for developing an alternative treatment for POP.

Mesenchymal stem cell-based bioengineered constructs: foreign body response, cross-talk with macrophages and impact of biomaterial design strategies for pelvic floor disorders.

An excessive foreign body response (FBR) has contributed to the adverse events associated with polypropylene mesh usage for augmenting pelvic organ prolapse surgery. Consequently, current biomaterial research considers the critical role of the FBR and now focuses on developing better biocompatible biomaterials rather than using inert implants to improve the clinical outcomes of their use. Tissue engineering approaches using mesenchymal stem cells (MSCs) have improved outcomes over traditional implants in other biological systems through their interaction with macrophages, the main cellular player in the FBR. The unique angiogenic, immunomodulatory and regenerative properties of MSCs have a direct impact on the FBR following biomaterial implantation. In this review, we focus on key aspects of the FBR to tissue-engineered MSC-based implants for supporting pelvic organs and beyond. We also discuss the immunomodulatory effects of the recently discovered endometrial MSCs on the macrophage response to new biomaterials designed for use in pelvic floor reconstructive surgery. We conclude with a focus on considerations in biomaterial design that take into account the FBR and will likely influence the development of the next generation of biomaterials for gynaecological applications.

In vivo evaluation of injectable calcium phosphate cement composed of Zn- and Si-incorporated ß-tricalcium phosphate and monocalcium phosphate monohydrate for a critical sized defect of the rabbit femoral condyle.

Zinc (Zn) enhances bone formation with mineralization and is an essential element of osteoblastic proliferation. Silicon (Si) is important in apatite formation coupled with the promotion of osteogenesis. The primary focus of this work was the assessment of the bone healing capacity of calcium phosphate cements (CPC) composed of Zn- and Si-incorporated ß-tri calcium phosphate (TCP) and mono calcium phosphate mono hydrate (MCPM). Zn- and Si-incorporated ß-TCP was synthesized through a sol gel process with varying amounts of Zn: (3, 6, or 9% w/w) and 15% w/w Si. Fabricated CPC samples were characterized by scanning electron microscopy, setting time, injectability, compressive strength and initial pH change with time. Compositional analysis and the effects of Zn and Si on cellular interaction were evaluated by energy dispersive X-ray spectroscopy mapping, viability determination and F-actin assay. The data were used to optimize the CPC formulation. The efficacy of bone healing was investigated via implantation into critical sized rabbit femoral condyle defects for 4 and 8 weeks. CPC cement with 6% (w/w) Zn content was the best candidate for faster bone healing (bone to tibial volume ratio in 8 weeks: 22.78% ± 0.02). Significantly faster degradation was also revealed. Bone healing was significantly delayed when CPC cement with 9% (w/w) Zn was used. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 260-271, 2017.

A Study of BMP-2-Loaded Bipotential Electrolytic Complex around a Biphasic Calcium Phosphate-Derived (BCP) Scaffold for Repair of Large Segmental Bone Defect.

A bipotential polyelectrolyte complex with biphasic calcium phosphate (BCP) powder dispersion provides an excellent option for protein adsorption and cell attachment and can facilitate enhanced bone regeneration. Application of the bipotential polyelectrolyte complex embedded in a spongy scaffold for faster healing of large segmental bone defects (LSBD) can be a promising endeavor in tissue engineering application. In the present study, a hollow scaffold suitable for segmental long bone replacement was fabricated by the sponge replica method applying the microwave sintering process. The fabricated scaffold was coated with calcium alginate at the shell surface, and genipin-crosslinked chitosan with biphasic calcium phosphate (BCP) dispersion was loaded at the central hollow core. The chitosan core was subsequently loaded with BMP-2. The electrolytic complex was characterized using SEM, porosity measurement, FTIR spectroscopy and BMP-2 release for 30 days. In vitro studies such as MTT, live/dead, cell proliferation and cell differentiation were performed. The scaffold was implanted into a 12 mm critical size defect of a rabbit radius. The efficacy of this complex is evaluated through an in vivo study, one and two month post implantation. BV/TV ratio for BMP-2 loaded sample was (42±1.76) higher compared with hollow BCP scaffold (32±0.225).

Augmenting in vitro osteogenesis of a glycine-arginine-glycine-aspartic-conjugated oxidized alginate-gelatin-biphasic calcium phosphate hydrogel composite and in vivo bone biogenesis through stem cell delivery.

A functionally modified peptide-conjugated hydrogel system was fabricated with oxidized alginate/gelatin loaded with biphasic calcium phosphate to improve its biocompatibility and functionality. Sodium alginate was treated by controlled oxidation to transform the cis-diol group into an aldehyde group in a controlled manner, which was then conjugated to the amine terminus of glycine-arginine-glycine-aspartic. Oxidized alginate glycine-arginine-glycine-aspartic was then combined with gelatin-loaded biphasic calcium phosphate to form a hydrogel of composite oxidized alginate/gelatin/biphasic calcium phosphate that displayed enhanced human adipose stem cell adhesion, spreading and differentiation. 1H nuclear magnetic resonance and electron spectroscopy for chemical analysis confirmed that the glycine-arginine-glycine-aspartic was successfully grafted to the oxidized alginate. Co-delivery of glycine-arginine-glycine-aspartic and human adipose stem cell in a hydrogel matrix was studied with the results indicating that hydrogel incorporated modified with glycine-arginine-glycine-aspartic and seeded with human adipose stem cell enhanced osteogenesis in vitro and bone formation in vivo.