Composite mesh design for delivery of autologous mesenchymal stem cells influences mesh integration, exposure and biocompatibility in an ovine model of pelvic organ prolapse.

The widespread use of synthetic transvaginal polypropylene mesh for treating Pelvic Organ Prolapse (POP) has been curtailed due to serious adverse effects highlighted in 2008 and 2011 FDA warnings and subsequent legal action. We are developing new synthetic mesh to deliver endometrial mesenchymal stem cells (eMSC) to improve mesh biocompatibility and restore strength to prolapsed vaginal tissue. Here we evaluated knitted polyamide (PA) mesh in an ovine multiparous model using transvaginal implantation and matched for the degree of POP. Polyamide mesh dip-coated in gelatin and stabilised with 0.5% glutaraldehyde (PA/G) were used either alone or seeded with autologous ovine eMSC (eMSC/PA/G), which resulted in substantial mesh folding, poor tissue integration and 42% mesh exposure in the ovine model. In contrast, a two-step insertion protocol, whereby the uncoated PA mesh was inserted transvaginally followed by application of autologous eMSC in a gelatin hydrogel onto the mesh and crosslinked with blue light (PA + eMSC/G), integrated well with little folding and no mesh exposure. The autologous ovine eMSC survived 30 days in vivo but had no effect on mesh integration. The stiff PA/G constructs provoked greater myofibroblast and inflammatory responses in the vaginal wall, disrupted the muscularis layer and reduced elastin fibres compared to PA + eMSC/G constructs. This study identified the superiority of a two-step protocol for implanting synthetic mesh in cellular compatible composite constructs and simpler surgical application, providing additional translational value.

Interaction of preservation methods and radiation sterilization in human skin processing, with particular insight on the impact of the final water content and collagen disruption. Part I: process validation, water activity and collagen changes in tissues cryopreserved or processed using 50, 85 or 98% glycerol solutions.

Current regulatory requirements demand an in-depth understanding and validation of protocols used in tissue banking. The aim of this work was to characterize the quality of split thickness skin allografts cryopreserved or manufactured using highly concentrated solutions of glycerol (50, 85 or 98%), where tissue water activity (aw), histology and birefringence changes were chosen as parameters. Consistent aw outcomes validated the proposed processing protocols. While no significant changes in tissue quality were observed under bright-field microscopy or in collagen birefringence, in-process findings can be harnessed to fine-tune and optimize manufacturing outcomes in particular when further radiation sterilization is considered. Furthermore, exposing the tissues to 85% glycerol seems to derive the most efficient outcomes as far as aw and control of microbiological growth.

Endometrial Mesenchymal Stem/Stromal Cells Modulate the Macrophage Response to Implanted Polyamide/Gelatin Composite Mesh in Immunocompromised and Immunocompetent Mice.

The immunomodulatory properties of human endometrial mesenchymal stem cells (eMSC) have not been well characterised. Initial studies showed that eMSC modulated the chronic inflammatory response to a non-degradable polyamide/gelatin mesh in a xenogeneic rat skin wound repair model, but the mechanism remains unclear. In this study, we investigated the immunomodulatory effect of eMSC on the macrophage response to polyamide/gelatin composite mesh in an abdominal subcutaneous wound repair model in C57BL6 immunocompetent and NSG (NOD-Scid-IL2Rgamma null ) immunocompromised mice to determine whether responses differed in the absence of an adaptive immune system and NK cells. mCherry lentivirus-labelled eMSC persisted longer in NSG mice, inducing longer term paracrine effects. Inclusion of eMSC in the mesh reduced inflammatory cytokine (Il-1ß, Tnfα) secretion, and in C57BL6 mice reduced CCR7+ M1 macrophages surrounding the mesh on day 3 and increased M2 macrophage marker mRNA (Arg1, Mrc1, Il10) expression at days 3 and 7. In NSG mice, these effects were delayed and only observed at days 7 and 30 in comparison with controls implanted with mesh alone. These results show that the differences in the immune status in the two animals directly affect the survival of xenogeneic eMSC which leads to differences in the short-term and long-term macrophage responses to implanted meshes.

Non-animal collagens as new options for cosmetic formulation.

OBJECTIVE: To examine the potential of non-animal collagens as a new option for cosmetic applications. METHODS: Non-animal collagens from three species, Streptococcus pyogenes, Solibacter usitatus and Methylobacterium sp 4-46, have been expressed as recombinant proteins in Escherichia coli using a cold-shock, pCold, expression system. The proteins were purified using either metal affinity chromatography or a simple process based on precipitation and proteolytic digestion of impurities, which is suitable for large-scale production. Samples were examined using a range of analytical procedures. RESULTS: Analyses by gel electrophoresis and mass spectrometry were used to examine the purity and integrity of the products. Circular dichroism spectroscopy showed stabilities around 38°C, and calculated pI values were from 5.4 to 8.6. UV-visible light spectroscopy showed the clarity of collagen solutions. The collagens were soluble at low ionic strength between pH 5 and pH 8, but were less soluble under more acidic conditions. At lower pH, the insoluble material was well dispersed and did not form the fibrous associations and aggregates found with animal collagens. The materials were shown to be non-cytotoxic to cells in culture. CONCLUSIONS: These novel, non-animal collagens may be potential alternatives to animal collagens for inclusion in cosmetic formulations.

Temporal changes in the biomechanical properties of endometrial mesenchymal stem cell seeded scaffolds in a rat model.

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.

Mechanical evaluation and cell response of woven polyetheretherketone scaffolds.

Polyetheretherketone (PEEK) is a high performance polymer, with high melting temperature and high resistance to wear. PEEK biomedical devices are typically manufactured to produce nonflexible structures. In this study, we fabricated flexible PEEK scaffolds from multifilament and monofilament yarns, using weaving technologies. Scaffolds were compared for structural and mechanical properties, and assessed for in vitro biological response to L929 mouse fibroblast cells. PEEK scaffolds were found to support fibroblast cell attachment and proliferation, with similar cell numbers to a polyethylene terephthalate scaffold. The large pores (261-280 µm) of the monofilament scaffold prevented pore coverage by cells, confining cells to filaments, whereas the smaller pores (81-100 µm) of the multifilament scaffold permitted partial pore coverage. Poor cell adhesion, due to large filament curvature angles, created a checkered pattern on the woven surface, a previously undocumented phenomenon. The multifilament scaffold was found to be lighter, thinner, and less porous, with better mechanical properties (load at break: 657 N, elastic recovery: 66%, burst strength: 492 N) than the monofilament scaffold (load at break: 534 N, elastic recovery: 30%, burst strength: 401 N). Results indicate that flexible PEEK woven structures may find application as tissue engineering scaffolds, particularly for engineering soft tissues.

Impact of heparan sulfate chains and sulfur-mediated bonds on the mechanical properties of bovine lens capsule.

We assessed the importance of glycosaminoglycans and sulfur-mediated bonds for the mechanical properties of lens capsules by comparing the stress-strain responses from control and treated pairs of bovine source. No significant change in mechanical properties was observed upon reduction of disulfide bonds. However, removal of glycosaminoglycan chains resulted in a significantly stiffer lens capsule, whereas high concentrations of reducing agent, which is expected to reduce the recently reported sulfilimine bond of collagen IV, resulted in a significantly less stiff lens capsule. A comparison of the diffraction patterns of the control and strongly reduced lens capsules indicated structural rearrangements on a nanometer scale.

Biodegradable and injectable cure-on-demand polyurethane scaffolds for regeneration of articular cartilage.

This paper describes the synthesis and characterization of an injectable methacrylate functionalized urethane-based photopolymerizable prepolymer to form biodegradable hydrogels. The tetramethacrylate prepolymer was based on the reaction between two synthesized compounds, diisocyanato poly(ethylene glycol) and monohydroxy dimethacrylate poly(epsilon-caprolactone) triol. The final prepolymer was hydrated with phosphate-buffered saline (pH 7.4) to yield a biocompatible hydrogel containing up to 86% water. The methacrylate functionalized prepolymer was polymerized using blue light (450 nm) with an initiator, camphorquinone and a photosensitizer, N,N-dimethylaminoethyl methacrylate. The polymer was stable in vitro in culture media over the 28 days tested (1.9% mass loss); in the presence of lipase, around 56% mass loss occurred over the 28 days in vitro. Very little degradation occurred in vivo in rats over the same time period. The polymer was well tolerated with very little capsule formation and a moderate host tissue response. Human chondrocytes, seeded onto Cultispher-S beads, were viable in the tetramethacrylate prepolymer and remained viable during and after polymerization. Chondrocyte-bead-polymer constructs were maintained in static and spinner culture for 8 weeks. During this time, cells remained viable, proliferated and migrated from the beads through the polymer towards the edge of the polymer. New extracellular matrix (ECM) was visualized with Masson's trichrome (collagen) and Alcian blue (glycosaminoglycan) staining. Further, the composition of the ECM was typical for articular cartilage with prominent collagen type II and type VI and moderate keratin sulphate, particularly for tissue constructs cultured under dynamic conditions.

Evaluation of photo-crosslinked fibrinogen as a rapid and strong tissue adhesive.

Tissue adhesives and sealants are commonly used in surgery either as an adjunct to, or replacement for, sutures. Previously, we have shown that fibrinogen can be crosslinked rapidly to give a high-strength bond in the presence of a ruthenium(II) complex, a persulfate and irradiation with visible light, and that the crosslinked fibrinogen is nontoxic to cells in vitro. This approach addresses limitations to current fibrin sealants that typically have relatively slow curing times and low bond strengths. In the present study, we have evaluated the efficacy and safety of this new biological scaffold sealant in various animal models. When placed as solid implants into rats, the crosslinked fibrinogen persisted for at least 8 weeks but was fully resorbed by 18 weeks with minimal inflammatory responses. When used as a tissue adhesive for repair of skin incisions in rats or as an arterial haemostat in pig, the photo-crosslinked fibrinogen sealed tissue or arrested bleeding within 20 s of application. For the skin incisions, the fibrinogen sealant promoted rapid tissue vascularization and cellular infiltration with no adverse foreign body cell generation. New collagen deposition occurred and with time the matrix had remodelled to acquire large mature collagen fiber bundles which were accompanied by maximum regenerated tensile strength. This biomaterial system may find useful applications in surgical procedures where rapid curing and/or high strength tissue sealing is required.

Use of biodegradable urethane-based adhesives to appose meniscal defect edges in an ovine model: a preliminary study.

OBJECTIVE: To evaluate the biological response to two urethane-based adhesives used to repair full thickness meniscal wounds created in the partially vascularised (red-white) zone. DESIGN: An ovine bilateral meniscal defect model was used to evaluate the initial biological response of the meniscal cartilage and synovium over a 1-month period. A 10-mm full-thickness defect was created in the medial meniscus of each femorotibial joint. The defects were either left untreated or repaired using the urethane-based adhesives. Synovial fluid, synovial membrane and the meniscal cartilages were retrieved at necropsy for cytological and histological assessment. RESULTS: The ovine model proved to be a suitable system for examining meniscal repair. Untreated defects showed no tissue apposition or cellular healing response, whereas all eight defects repaired with the two urethane-based adhesive formulations showed signs of repair and tissue regeneration with indications of cell infiltration and new collagen deposition in and around the polymer. No adverse cellular response to the adhesives was observed in the meniscal defect or in the synovial membrane and fluid. CONCLUSION: Trauma to the knee commonly results in tears to the meniscal cartilage, with the majority of these occurring in the partially vascularised (red-white) or non-vascularised (white) zones of the meniscus. Repair, and subsequent healing, of these tears is poor because of the reduced vascularity and limited surgical access. The present data indicate that an ovine model is a suitable system for examining meniscal repair, and that development of urethane-based adhesives offers a strategy that may be clinically effective for the treatment of these injuries.

Synthetic biodegradable microparticles for articular cartilage tissue engineering.

Articular cartilage tissue engineering procedures require the transplantation of chondrocytes that have been expanded in vitro. The expansion is carried out for a considerable time and can lead to a modulation of cell phenotype. However, microcarrier cultures have been shown to allow cell expansion while maintaining the phenotype. Here, we have used the biodegradable polyester poly(lactide-co-glycolide) (PLGA) in the form of microspheres and irregular shaped microparticles with a diameter between 47 and 210 microm. Surface modification of particles was carried out by ammonia plasma treatment and subsequent adsorption of collagen. Alternatively, particles were modified by partial hydrolysis and subsequent immobilization of an amine-terminated dendrimer. Each surface modification step was characterized by X-ray photoelectron spectroscopy. The effectiveness of the surface modification procedures was demonstrated by in vitro cell culture experiments using sheep articular cartilage chondrocytes. A significant influence of both the particle shape and the surface chemistry on the proliferation rate was observed while the phenotype was maintained independent of the surface chemistry or particle shape. Chondrocytes cultured on PLGA microspheres were further assessed for cartilage tissue formation in collagen type I gels in nude mice. The tissue that were formed showed the appearance of a hyaline-like cartilage and the presence of the microspheres substantially reduced the degree of collagen gel contraction over 1-2 months.

Sequence requirements for the activity of membrane-active peptides.

Synthetic peptides were constructed with the sequence of the first 20 residues of melittin and terminating with a range of different amino acid amides. These were found to have haemolytic and cytolytic activity similar to that of melittin, provided that certain charge constraints were observed. The nature of the 21st residue was not critical except when the residue introduced a negative charge. The presence of at least two positive charges in the molecule was found to be essential for activity. One of these charges could be the amino-terminal amine. Peptides could be inactivated by the addition of a non-acidic presequence which was acetylated at the N-terminus. Introducing a protease cleavable sequence into an N-terminal extension of the peptides produced analogues with low haemolytic activity that could be activated by proteolytic action. A peptide with extra positive charges introduced on the hydrophilic face of the helix possessed a haemolytic activity that was greater than that of melittin.

Effects of mesh modification on the structure of a mandrel-grown biosynthetic vascular prosthesis.

Mandrel-grown, mesh-reinforced vascular prostheses require adequate tissue coverage of the mesh for effective clinical function, particularly in low blood flow situations. Development of the ovine collagen-based Omniflowtrade mark vascular prosthesis has shown that the extent of this tissue cover is dependent on the interactions of the mandrel and the mesh with the sheep host. In the present study, the effects of chemical changes to the mesh have been examined. These data indicate that certain treatments of the mesh, particularly collagen or heparin, lead to increased tissue coverage while the number of sheep cells present and the ultrastructure of the resulting vessel remain unchanged.

In vivo evaluation of modified mandrel-grown vascular prostheses.

The Omniflowtrade mark Vascular Prosthesis (OVP) has been manufactured and extensively tested in animal and human trials. It has mechanical and biological qualities superior to synthetic and biological conduits, particularly in low flow conditions. For further development into the smaller diameter coronary prostheses, the inner luminal surface is of paramount importance. In a previous study this inner surface was modified to produce a more uniformly thicker nonundulating surface. In this study the mandrels of these modified OVPs were treated with either collagen or heparin; the OVPs were evaluated for patency, tissue integration and wound healing, and endothelialization using a dog model comparable to that used to evaluate the unmodified OVP. In all instances, each of the modified prostheses were fully patent and had no signs of any deleterious effects caused by these modifications; no thrombus or aneurysms were visible. The tissue response was rapid with excellent new host collagen deposition within the vessel wall and minimal inflammatory and foreign body giant cells. Endothelialization was noted at the earliest explant time point in central regions of the prostheses, albeit that the histological picture at this time point appeared to reflect a complex atypical intimal layer.

Production of recombinant hydroxylated human type III collagen fragment in Saccharomyces cerevisiae.

A recombinant hydroxylated fragment of human type III collagen has been produced in Saccharomyces cerevisiae by coordinated coexpression of a collagen gene fragment together with both the alpha- and beta-subunit genes for prolyl-4-hydroxylase (EC 1.14.11.2). The collagen fragment consisted of 255 residues of the helical domain and the complete C-telopeptide and C-propeptide domains. It was inserted under the control of the ethanol-inducible ADH2 promoter in a multicopy, TRP1-selectable, yeast expression vector, YEpFlag1. The prolyihydroxylase subunit genes were cloned on either side of a bidirectional galactose-inducible promoter in a low-copy minichromosome yeast expression vector, pYEUra3, which is URA3 selectable. Coordinated expression of the three different gene products after cotransformation into S. cerevisiae was detected by immunoblotting. Amino acid analysis of an immunoreactive collagen fraction demonstrated the presence of hydroxyproline, while the presence of a triple-helical domain in the collagen fragment was demonstrated by its resistance to pepsin proteolysis.

Modulation of the tissue reaction to biomaterials. II. The function of T cells in the inflammatory reaction to crosslinked collagen implanted in T-cell-deficient rats.

Unwanted tissue reactions are often observed resulting in events such as early resorption of the biomaterial, loosening of the implant, or a chronic (immunologic) response. From immunologic studies it is known that inflammatory reactions can be modulated by use of (anti)-growth factors or anti-inflammatory drugs. Before this can be employed with respect to biomaterials, the role of individual factors (humoral and cellular) has to be studied. In this part of the investigation, the role of T cells was studied by use of T-cell-deficient (nude) rats and control (AO) rats. Hexamethylenediisocyanate-crosslinked dermal sheep collagen (HDSC) was selected as the test material. The results showed that T cells or T cell-related factors played a prominent role in the attraction of macrophages and the formation of giant cells, their antigen presentation, and their phagocytotic capacity. As a consequence, degradation of HDSC was strongly delayed. This study also showed that infiltration of fibroblasts and creation of stromal areas in HDSC was restricted to areas subjected to degradation. However, in time, absence of T cells resulted in increased formation and maturation of autologous rat collagen. Results obtained suggest that the inflammatory reaction to biomaterials might be modulated by controlling T-cell activation.

Evaluation of a collagen-based biosynthetic material for the repair of abdominal wall defects.

A collagen tissue polymer composite manufactured in sheep and prepared in two different forms (wet and dry) was compared to polypropylene mesh and to a control group for effectiveness in the repair of an abdominal wall defect in a rabbit model. The wet and dry patches were shown to differ significantly in their pore size. The wet material was shown to retain its natural porosity and promoted neovascularization, tissue integration, cellular infiltration, and neomatrix formation compared to the dry collagen-polymer patch. This material was superior to the polypropylene mesh implant, which was associated with significant adhesions. The appearance of type VI collagen was the earliest sign of new cell infiltration and neomatrix formation within the implant. New deposition of type VI collagen was apparent throughout the thickness of the implant within 4 weeks, followed by type III collagen accumulation. Decreased porosity of the collagen component in the dry patches resulted in a totally nonintegrated implant. This induced a foreign-body capsule with minimal cellular tissue infiltration and no deposition of collagen types VI and III within the implant.

Identification of the epitope for a monoclonal antibody that blocks platelet aggregation induced by type III collagen.

A library of eight conformation-dependent monoclonal antibodies that react with distinct epitopes on native human type III collagen has been examined for the ability of these antibodies to inhibit platelet aggregation induced by this collagen. Six of these antibodies had no effects; one, 1E7-D7/Col3, delayed the onset and slowed the rate of platelet aggregation, while another, 2G8-B1/Col3, completely inhibited aggregation. In order to identify the epitope recognized by this inhibitory antibody, a series of peptides that could fold to form triple-helical fragments was examined. Each peptide included six Gly-Xaa-Yaa triplets from the human type III collagen sequence, where Xaa and Yaa represent the particular amino acids in the sequence, and a C-terminal (Gly-Pro-Hyp)4 sequence to enhance triple-helical stability. Using these peptides we have identified the epitope as a nine-amino-acid sequence, GLAGAOGLR (where O is the one-letter code for 4-hydroxyproline), starting at position 520 in the human type III collagen helical domain. This sequence is proximal to the site proposed for the interaction of type III collagen with alpha2beta1-integrin of platelets.

In vivo evaluation of a collagenous membrane as an absorbable adhesion barrier.

An absorbable membrane made from purified, pepsin-soluble collagen was compared to Interceed, an absorbable cellulose-based product, and to a control group for effectiveness in inhibiting the formation of adhesions between peritoneal surface injuries in adult rats. An adhesion scoring system was used to evaluate and compare the performance of the test materials with the control group in regard to the extent, tenacity, and type of any adhesions evident at 28 days following surgery. The collagen group performed significantly better (p < 0.05) than either the Interceed or control groups, showing fewer, less extensive adhesions. The collagen membranes resulted in either no or weak adhesions between the body wall and caecum. Adhesions in the Interceed group were quite variable and characterized by a marked peritoneal reaction in the caecal and body walls adjacent to adhesions. Control samples were characterized by close, dense fibrotic adhesions between the caecum and body wall. Both of the test materials showed some deficiencies in respect to their physical and handling properties that could be further improved for this indication.

Organization of fibrillar collagen in the human and bovine cornea: collagen types V and III.

The localization and fibrillar organization of collagen types V and III in the human and bovine corneal stromas were studied. In the chicken cornea, type V co-assembles with type I collagen as heterotypic fibrils and this interaction is involved in the regulation of fibril diameter necessary for corneal transparency. To determine whether this is a regulatory mechanism common to the corneas of different species the human and bovine corneal stroma were studied. Collagen type V was found in the epithelium and Bowman's membrane in the untreated adult human and bovine cornea using immunofluorescence microscopy. In the absence of any treatment, there was no type V reactivity within the stroma. However, type V collagen was detected homogeneously throughout the corneal stroma after treatments that partially disrupt fibril structure. The reactivity was strongest in the cornea, weaker in the limbus and weakest in the sclera. Fetal corneas showed similar reactivity for type V collagen, but unlike the adult, the stroma was slightly reactive. Immunoelectron microscopy demonstrated that type V collagen was associated with disrupted, but not with intact, fibrils in both human and bovine corneal stroma. Type III collagen reactivity was not detected in the cornea, but was present subepithelially in the limbus and in the scleral stroma. These data indicate that type V collagen is a component of striated collagen fibrils throughout the human and bovine corneal stromas. The interaction of type I and V collagen as heterotypic fibrils masks the helical epitope recognized by the monoclonal antibody against type V collagen. The heterotypic interactions of collagen type V indicate a role in the regulation of fibril diameter analogous to that described in the avian cornea.