UNLABELLED:Pelvic Organ Prolapse (POP) is the herniation of pelvic organs into the vagina. Despite broad acceptance of mesh use in POP surgical repair, the complication rate is unacceptable. We hypothesized that collagen-containing polypropylene (PP) mesh types could modulate mesh-tissue integration and reduce long-term inflammation, thereby reducing mesh-associated complications. This study compared the long-term tissue response to an unmodified PP mesh and two collagen containing meshes in an ovine model which has similar pelvic anatomy and vaginal size to human. Three commercially available macroporous PP meshes, uncoated PP mesh (Avaulta Solo) (PP), the same textile PP mesh layered with a sheet of cross-linked porcine acellular matrix (Avaulta Plus) (PP-ACM) and a different yet also macroporous PP (Sofradim) mesh coated with solubilized atelocollagen (Ugytex) (PP-sCOL) were implanted in the ovine vagina and tissue explanted after 60 and 180days. The macrophage phenotype and response to implanted meshes, and vascularity were quantified by immunostaining and morphometry. We quantified changes in extracellular matrix composition biochemically and collagen organisation and percentage area around the interface of the mesh implants by Sirius Red birefringence and morphometry. PP-ACM induced a more sustained inflammatory response, indicated by similar CD45(+) leukocytes but reduced CD163(+) M2 macrophages at 60days (P<0.05). PP-sCOL increased Von Willebrand Factor (vWF)-immunoreactive vessel profiles after 60days. At the micro-molecular level, collagen birefringence quantification revealed significantly fewer mature collagen fibrils (red, thick fibrils) at the mesh-tissue interface than control tissue for all mesh types (P<0.001) but still significantly greater than the proportion of immature (green thin fibrils) at 60days (P<0.05). The proportion of mature collagen fibrils increased with time around the mesh filaments, particularly those containing collagen. The total collagen percent area at the mesh interface was greatest around the PP-ACM mesh at 60days (P<0.05). By 180days the total mature and immature collagen fibres at the interface of the mesh filaments resembled that of native tissue. In particular, these results suggest that both meshes containing collagen evoke different types of tissue responses at different times during the healing response yet both ultimately lead to physiological tissue formation approaching that of normal tissue. STATEMENT OF SIGNIFICANCE:Pelvic organ prolapse (POP) is the descent of the pelvic organs to the vagina. POP affects more than 25% of all women and the lifetime risk of undergoing POP surgery is 19%. Although synthetic polypropylene (PP) meshes have improved the outcome of the surgical treatment for POP, there was an unacceptable rate of adverse events including mesh exposure and contracture. It is hypothesized that coating the PP meshes with collagen would provide a protective effect by preventing severe mesh adhesions to the wound, resulting in a better controlled initial inflammatory response, and diminished risk of exposure. In this study we assessed the effect of two collagen-containing PP meshes on the long-term vaginal tissue response using new techniques to quantify these tissue responses.

The fabrication of three-dimensional (3D) constructs relies heavily on the use of biomaterial-based scaffolds. These are required as mechanical supports as well as to translate two-dimensional cultures to 3D cultures for clinical applications. Regardless of the choice of scaffold, timely degradation of scaffolds is difficult to achieve and undegraded scaffold material can lead to interference in further tissue development or morphogenesis. In cartilage tissue engineering, hydrogel is the highly preferred scaffold material as it shares many similar characteristics with native cartilaginous matrix. Hence, we employed gelatin microspheres as porogens to create a microcavitary alginate hydrogel as an interim scaffold to facilitate initial chondrocyte 3D culture and to establish a final scaffold-free living hyaline cartilaginous graft (LhCG) for cartilage tissue engineering.

The biomacromolecule, gelatin, has increasingly been used in biomedicine—beyond its traditional use in food and cosmetics. The appealing advantages of gelatin, such as its cell-adhesive structure, low cost, off-the-shelf availability, high biocompatibility, biodegradability and low immunogenicity, among others, have made it a desirable candidate for the development of biomaterials for tissue engineering and drug delivery. Gelatin can be formulated in the form of nanoparticles, employed as size-controllable porogen, adopted as surface coating agent and mixed with synthetic or natural biopolymers forming composite scaffolds. In this article, we review recent advances in the versatile applications of gelatin within biomedical context and attempt to draw upon its advantages and potential challenges.

Use of synthetic clinical meshes in pelvic organ prolapse (POP) repair can lead to poor mechanical compliance in vivo, as a result of a foreign body reaction leading to excessive scar tissue formation. Seeding mesh with mesenchymal stem cells (MSCs) prior to implantation may reduce the foreign body reaction and lead to improved biomechanical properties of the mesh-tissue complex. This study investigates the influence of seeding human endometrial mesenchymal stem cells (eMSCs) on novel gelatin-coated polyamide scaffolds, to identify differences in scaffold/tissue biomechanical properties and new tissue growth following up to 90 days' implantation, in a subcutaneous rat model of wound repair. Scaffolds were subcutaneously implanted, either with or without eMSCs, in immunocompromised rats and following 7, 30, 60 and 90 days were removed and assessed for their biomechanical properties using uniaxial tensile testing. Following 7, 30 and 90 days' implantation scaffolds were assessed for tissue ingrowth and organization using histological staining and scanning electron microscopy. The eMSCs were associated with altered collagen growth and organization around the mesh filaments of the scaffold, affecting the physiologically relevant tensile properties of the scaffold-tissue complex, in the toe region of the load-elongation curve. Scaffolds seeded with eMSCs were significantly less stiff on initial stretching than scaffolds implanted without eMSCs. Collagen growth and organization were enhanced in the long-term in eMSC-seeded scaffolds, with improved fascicle formation and crimp configuration. Results suggest that neo-tissue formation and remodelling may be enhanced through seeding scaffolds with eMSCs.

A series of high refractive index (RI) ZnS/PVP/PDMAA hydrogel nanocomposites containing ZnS nanoparticles (NPs) were successfully synthesized via a simple ultraviolet-light-initiated free radical co-polymerization method. The average diameter of the ZnS NPs is ∼3 nm and the NPs are well dispersed and stabilized in the PVP/PDMAA hydrogel matrix up to a high content of 60 wt.% in the hydrogel nanocomposites. The equilibrium water content of ZnS/PVP/PDMAA hydrogel nanocomposites varied from 82.0 to 66.8 wt.%, while the content of mercaptoethanol-capped ZnS NPs correspondingly varied from 30 to 60 wt.%. The resulting nanocomposites are clear and transparent and their RIs were measured to be as high as 1.58–1.70 and 1.38–1.46 in the dry and hydrated states, respectively, which can be tuned by varying the ZnS NPs content. In vitro cytotoxicity assays suggested that the introduction of ZnS NPs added little cytotoxicity to the PVP/PDMAA hydrogel and all the hydrogel nanocomposites exhibited minimal cytotoxicity towards common cells. The hydrogel nanocomposites implanted in rabbit eyes can be well tolerated over 3 weeks. Hence, the high RI ZnS/PVP/PDMAA hydrogel nanocomposites with adjustable RIs developed in this work might potentially be a candidate material for artificial corneal implants.

Pelvic organ prolapse is a major hidden burden affecting almost one in four women. It is treated by reconstructive surgery, often augmented with synthetic mesh. To overcome the growing concerns of using current synthetic meshes coupled with the high risk of reoperation, a tissue engineering strategy has been developed, adopting a novel source of mesenchymal stem cells. These cells are derived from the highly regenerative endometrial lining of the uterus (eMSCs) and will be delivered in vivo using a new gelatin-coated polyamide scaffold. In this study, gelatin properties were optimized by altering the gelatin concentration and extent of crosslinking to produce the desired gelation and degradation rate in culture. Following cell seeding of uncoated polyamide (PA) and gelatin-coated meshes (PA + G), the growth rate of eMSCs on the PA + G scaffolds was more than that on the PA alone, without compromising cell shape. eMSCs cultured on the PA + G scaffold retained their phenotype, as demonstrated by W5C5/SUSD2 (eMSC-specific marker) immunocytochemistry. Additionally, eMSCs were induced to differentiate into smooth muscle cells (SMC), as shown by immunofluorescence for smooth muscle protein 22 and smooth muscle myosin heavy chain. eMSCs also differentiated into fibroblast-like cells when treated with connective tissue growth factor with enhanced detection of Tenascin-C and collagen type I as well as new tissue formation, as seen by Masson’s trichrome. In summary, it was demonstrated that the PA + G scaffold is an appropriate platform for eMSC delivery, proliferation and differentiation into SMC and fibroblasts, with good biocompatibility and the capacity to regenerate neo-tissue.

BACKGROUND Pelvic organ prolapse (POP) is defined as the descent of one or more of the pelvic structures into the vagina and includes uterine, vaginal vault, and anterior or posterior vaginal wall prolapse. The treatment of POP may include implantation of a synthetic mesh. However, the long-term benefit of mesh surgery is controversial due to complications such as mesh exposure or pain. The aim of this study was to use a tissue engineering (TE) approach to assess the in vivo biological and biomechanical behavior of a new gelatin/polyamide mesh, seeded with a novel source of mesenchymal stem cells in a subcutaneous rat model of wound repair. METHODS W5C5-enriched human endometrial mesenchymal stem cells (eMSC) were seeded onto meshes (gelatin-coated polyamide knit) at 100,000 cells/cm². Meshes, with or without cells were subcutaneously implanted dorsally in immunocompromised rats for 7, 30, 60, and 90 days. Flow cytometry was used to detect DiO labeled cells after explantation. Immunohistochemical assessment of foreign body reaction and tissue integration were conducted. Total collagen and the levels of collagens type III and type I were determined. Uniaxial tensiometry was performed on explanted meshes, originally seeded with and without cells, at days 7 and 90. RESULTS Implanted meshes were well tolerated, with labeled cells detected on the mesh up to 14 days postimplantation. Meshes with cells promoted significantly more neovascularization at 7 days (p<0.05) and attracted fewer macrophages at 90 days (p<0.05). Similarly, leukocyte infiltration was significantly lower in the cell-seeded meshes at 90 days (p<0.05). Meshes with cells were generally less stiff than those without cells, after 7 and 90 days implantation. CONCLUSION The TE approach used in this study significantly reduced the number of inflammatory cells around the implanted mesh and promoted neovascularization. Seeding with eMSC exerts an anti-inflammatory effect and promotes wound repair with new tissue growth and minimal fibrosis, and produces mesh with greater extensibility. Cell seeding onto polyamide/gelatin mesh improves mesh biocompatibility and may be an alternative option for future treatment of POP.

ObjectiveTo undertake a comprehensive analysis of the biochemical tissue composition and passive biomechanical properties of ovine vagina and relate this to the histo-architecture at different reproductive stages as part of the establishment of a large preclinical animal model for evaluating regenerative medicine approaches for surgical treatment of pelvic organ prolapse.MethodsVaginal tissue was collected from virgin (n = 3), parous (n = 6) and pregnant sheep (n = 6; mean gestation; 132 d; term = 145 d). Tissue histology was analyzed using H+E and Masson's Trichrome staining. Biochemical analysis of the extracellular matrix proteins used a hydroxyproline assay to quantify total collagen, SDS PAGE to measure collagen III/I+III ratios, dimethylmethylene blue to quantify glycosaminoglycans and amino acid analysis to quantify elastin. Uniaxial tensiometry was used to determine the Young's modulus, maximum stress and strain, and permanent strain following cyclic loading.ResultsVaginal tissue of virgin sheep had the lowest total collagen content and permanent strain. Parous tissue had the highest total collagen and lowest elastin content with concomitant high maximum stress. In contrast, pregnant sheep had the highest elastin and lowest collagen contents, and thickest smooth muscle layer, which was associated with low maximum stress and poor dimensional recovery following repetitive loading.ConclusionPregnant ovine vagina was the most extensible, but the weakest tissue, whereas parous and virgin tissues were strong and elastic. Pregnancy had the greatest impact on tissue composition and biomechanical properties, compatible with significant tissue remodeling as demonstrated in other species. Biochemical changes in tissue protein composition coincide with these altered biomechanical properties.

Objective: There are increasing numbers of reports describing human vaginal tissue composition in women with and without pelvic organ prolapse with conflicting results. The aim of this study was to compare ovine and human posterior vaginal tissue in terms of histological and biochemical tissue composition and to assess passive biomechanical properties of ovine vagina to further characterise this animal model for pelvic organ prolapse research.Study Design: Vaginal tissue was collected from ovariectomised sheep (n = 6) and from postmenopausal women (n = 7) from the proximal, middle and distal thirds. Tissue histology was analyzed using Masson's Trichrome staining; total collagen was quantified by hydroxyproline assays, collagen III/I+III ratios by delayed reduction SDS PAGE, glycosaminoglycans by dimethylmethylene blue assay, and elastic tissue associated proteins (ETAP) by amino acid analysis. Young's modulus, maximum stress/strain, and permanent strain following cyclic loading were determined in ovine vagina.Results: Both sheep and human vaginal tissue showed comparable tissue composition. Ovine vaginal tissue showed significantly higher total collagen and glycosaminoglycan values (p<0.05) nearest the cervix. No significant differences were found along the length of the human vagina for collagen, GAG or ETAP content. The proximal region was the stiffest (Young's modulus, p<0.05), strongest (maximum stress, p<0.05) compared to distal region, and most elastic (permanent strain).Conclusion: Sheep tissue composition and mechanical properties showed regional differences along the postmenopausal vaginal wall not apparent in human vagina, although the absolute content of proteins were similar. Knowledge of this baseline variation in the composition and mechanical properties of the vaginal wall will assist future studies using sheep as a model for vaginal surgery.

Remedying patellofemoral osteochondral defects using clinical therapy remains challenging. Construct-based and cell-based regenerative medicine with in vitro physical stimuli has been progressively implemented. However, the effect of physical stimuli in situ in knee joints with degradable constructs is still not well-documented. Therefore, we studied whether it was practical to achieve articular cartilage repair using a poly(lactic-co-glycolic acid) (PLGA) construct in addition to early short-term continuous passive motion (CPM) for treatment of full-thickness osteochondral defects in the lower-weigh bearing (LWB) zone of the femoral trocheal groove. Twenty-six rabbits were randomly allocated into either intermittent active motion (IAM) or CPM treatment groups with or without PLGA constructs, termed PLGA construct-implanted (PCI) and empty defect knee models, respectively. Gross observation, histology, inflammatory cells, which were identified using H&E staining, total collagen and alignment, studied qualitatively using Masson’s trichrome staining, glycosaminoglycan (GAG), identified using Alcian blue staining, and newly formed bone, observed using micro-CT, were evaluated at 4 and 12 weeks after surgery. Repair of osteochondral defects in the PCI-CPM group was more promising than all other groups. The better osteochondral defect repair in the PCI-CPM group corresponded to smooth cartilage surfaces, no inflammatory reaction, hyaline cartilaginous tissues composition, sound collagen alignment with positive collagen type II expression, higher GAG content, mature bone regeneration with osteocyte, clear tidemark formation, and better degradation of PLGA. In summary, the use of a simple PLGA construct coupled with passive motion promotes positive healing and may be a promising clinical intervention for osteochondral regeneration in LWB defects.

Pelvic organ prolapse (POP) is the herniation of bladder, bowel and/or uterus into the vagina causing urinary incontinence and sexual dysfunction. The outcome of POP reconstructive surgery treatment is poor and the use of synthetic polypropylene meshes has caused further complications. Our aim is to produce a tissue engineering construct using Endometrial Mesenchymal Stromal Cells (eMSCs) from the highly regenerative uterine lining to be delivered in new scaffold designs for effective treatment of POP.

Hyaline articular cartilage trauma and degeneration are major causes of suffering and pose great threat to the life quality of human beings worldwide. In cell-based therapy, TGF-beta 3 could be dosed to synovium-derived mesenchymal stem cells to induce differentiation into chondrocytes. However, type I collagen is intrinsically expressed and upregulated during monolayer culture by the introduction of TGF-beta 3, which would result in the formation of fibrous cartilage. Therefore, RNA interference strategy was adopted to suppress the expression of type I collagen by degrading its mRNA in a posttranscriptional pathway. In our study, we constructed a dual-functioning lentiviral vector that can express TGF-beta 3 and inhibit type I collagen expression. Based on this lentiviral vector, protocols for lentiviral titration, transduction and TGF-beta 3 quantification in the active form with modified ELISA would be specifically formulated and provided. Optimal medium would also be selected for the culture of transduced cells. Particularly, as the lentiviral nanoparticles are encapsulated in negative-charged envelope, there would be electrical repulsion between the lentiviral nanoparticles and the cells during viral transduction. Therefore, positive-charged polybrene has been added to mitigate the repulsion and facilitate lentiviral transduction. In our study, we tried several polybrene concentrations and determined the optimal one for lentiviral transduction.

A novel living hyaline cartilage graft (LhCG) with controllable dimensions and free of non-cartilaginous constituents for articular regeneration is developed. As a living graft for regenerative medicine, LhCG is purely living tissue based and truly scaffold-free. The process of neotissue formation in LhCG is mediated by an interim biomaterial-based novel scaffolding system. This design highlights a philosophy of using biomaterials in engineered regenerative medicine as a transient guiding facility rather than a permanent part of substitute. The fabrication is designed and practiced in a continuous and integrated process, which attributes to its simplicity in operation. Because of the intrinsic non-cell-adhesive property of hydrogel scaffolds, articular chondrocytes phenotype is always preserved throughout the whole procedure, which has been tested and approved both in vitro and in vivo. In situ grafting trials in a rabbit model showcase high success rates in both cartilage repair and graft-host integration. Beyond cartilage repair, this LhCG model may provide a living-tissue-based open platform or niche for multi-tissue regenerations.

To regenerate articular cartilage tissue from degeneration and trauma, synovial mesenchymal stem cells (SMSCs) were used in this study as therapeutic progenitor cells to induce therapeutic chondrogenesis. To accomplish this, chondrocytes pre-transduced with adenoviral vectors carrying the transforming growth factor (TGF) β3 gene were selected as transgenic companion cells and co-cultured side-by-side with SMSCs in a 3D environment to provide chondrogenic growth factors in situ. We adopted a mixed co-culture strategy for this purpose. Transgenic delivery of TGF-β3 in chondrocytes was performed via recombinant adenoviral vectors. The mixed co-culture of SMSCs and transgenic chondrocytes was produced in alginate gel constructs. Gene expression in both SMSCs and chondrocytes were characterized. Biochemical assays in vitro and in vivo showed that release of TGF-ß3 from transgenic chondrocytes not only induced SMSC differentiation into chondrocytic cells but also preserved the chondrocytic phenotype of chondrocytes from suspected dedifferentiation. As a result, this mixed co-culture strategy in conjunction with TGF-ß3 gene delivery could be a promising approach in cartilage tissue engineering.

Purpose: The objective of this study is to develop and apply an innovative, tissue engineered, living hyaline cartilaginous graft (LhCG) that is free of synthetic material components and composed of phenotypically bona fide hyaline cartilaginous neo-tissues. During the process of neo-tissue formation in LhCG, an interim biomaterial based novel scaffolding system plays an intermediate role before a complete removal prior to transplantation. The social-economic importance of this work lies in contribution of a man-made living and qualified cartilaginous graft to make up for the limited regenerations in this musculoskeletal tissue so that it may substantially serve as a possible solution to the ailments faced by current ageing population - particularly for regenerative treatment of osteoarthritis and other joint related disorders or lesions. Methods: Technically, two steps of processes are performed for the preparation of LhCG (as reported before). 1) Phase transfer cell culture (PTCC) in hydrogel scaffold: microspherical pores are created in chondrocytes-encapsulating alginate gel bulk via a controlled clearance of co-encapsulated gelatin microspheres. By proliferative expansion, the encapsulated chondrocytic isogenic groups adjacent to the gel/pore interface grow out of gel phase and fill up the pores, where neo-chondral nodules are formed. And, 2) LhCG formation by hydrogel removal: expansion and fusion of PTCC induced neo-tissue nodules within hydrogel bulk constructs macroscopic 3D network of a LhCG that interpenetrates the alginate scaffolds (gel) and maintains structural integrity during the removal of alginate components by a dissolving treatment with sodium citrate buffer. Results: Successful fabrication of LhCG provides a scaffold-free porous construct made of pure living cartilaginous tissues to act as a graft for implantation. The results indicate that, by mediation within non-cell-adhesive hydrogel scaffolds, the delivered chondrocytic phenotype is well preserved from the very beginning to the end. This performance is approved both in vitro and in vivo. In situ implantation in animal (rabbit knee joint) models has demonstrated high success rates in both cartilage repair and graft-host integration. Conclusion: The results suggest that LhCG is a promising cartilaginous graft for cartilage repair. Additionally, with a living tissue based nature, LhCG may also play a role as a niche for multi-tissue regenerations as well as an implantable transgenic platform for controlled release in situ.

Stromal cell-derived factor-1 (SDF-1), also known as a homing factor, is a potent chemokine that activates and directs mobilization, migration, and retention of certain cell species via systemic circulation. The responding homing cells largely consist of activated stem cells, so that, in case of tissue lesions, such SDF-1-induced cell migration may execute recruitment of endogenous stem cells to perform autoreparation and compensatory regeneration in situ. In this study, a recombinant adenoviral vector carrying SDF-1 transgene was constructed and applied to transduce a novel scaffold-free living hyaline cartilage graft (SDF-t-LhCG). As an engineered transgenic living tissue, SDF-t-LhCG is capable of continuously producing and releasing SDF-1 in vitro and in vivo. The in vitro trials were examined with ELISA, while the in vivo trials were subsequently performed via a subcutaneous implantation of SDF-t-LhCG in a nude mouse model, followed by series of biochemical and biological analyses. The results indicate that transgenic SDF-1 enhanced the presence of this chemokine in mouse's circulation system; in consequence, SDF-1-induced activation and recruitment of endogenous stem cells were also augmented in both peripheral blood and SDF-t-LhCG implant per se. These results were obtained via flow cytometry analyses on mouse blood samples and implanted SDF-t-LhCG samples, indicating an upregulation of the CXCR4(+)(SDF-1 receptor) cell population, accompanied by upregulation of the CD34(+), CD44(+), and Sca-1(+) cell populations as well as a downregulation of the CD11b(+) cell population. With the supply of SDF-1-recruited endogenous stem cells, enhanced chondrogenesis was observed in SDF-t-LhCG implants in situ.

Large and complex bone defects or voids cannot rely on natural bone healing process for recovery. They require natural or engineered grafts to facilitate osteo-progenitor cell recruitment and development. In this study, we have employed an in vitro macro-sized 3D cell-based platform for investigation and application of osteogenesis. The model is based on a porous construct made of engineered living cartilaginous tissue named living hyaline cartilaginous graft (LhCG). It is scaffold-free and is solely made up of living chondrocytes and their extra cellular matrix (ECM). To evaluate the efficiency of LhCG as a viable platform for bone formation, osteoblast and human mesenchymal stem cell (hMSC) were seeded respectively into LhCG constructs, establishing a co-culture system consisting of osteo-progenitors and chondrocytes. The results showed that LhCG could support both osteoblast and hMSC maturation and differentiation to the osteogenic lineage respectively. Successful osteogenesis is also observed after subcutaneous implantation in nude mice model suggesting that bone formation could be achieved both in vitro and in vivo. Additionally, with exposure to osteogenic medium, LhCG construct without any further cell seeding expressed similar levels of osteogenic phenotype markers as the ones with hMSC seeded on. It suggests the existence of an osteoprogenitor sub-population residing within LhCG chondrocytes. Hence, it is demonstrated that LhCG, as a cartilage template, could serve as a dynamic platform to support osteogenesis and its intrinsic phenotypic flexibility may also permit a wide range of applications for stem cell research and processing.