The Effects of Fucoidan Derived from Sargassum filipendula and Fucus vesiculosus on the Survival and Mineralisation of Osteogenic Progenitors.

Osteosarcoma is a bone cancer primarily affecting teenagers. It has a poor prognosis and diminished quality of life after treatment due to chemotherapy side effects, surgical complications and post-surgical osteoporosis risks. The sulphated polysaccharide fucoidan, derived from brown algae, has been a subject of interest for its potential anti-cancer properties and its impact on bone regeneration. This study explores the influence of crude, low-molecular-weight (LMW, 10-50 kDa), medium-molecular-weight (MMW, 50-100 kDa) and high-molecular-weight (HMW, >100 kDa) fractions from Sargassum filipendula, harvested from the Colombian sea coast, as well as crude fucoidan from Fucus vesiculosus, on a specific human osteoprogenitor cell type, human embryonic-derived mesenchymal stem cells. Fourier transform infrared spectroscopy coupled with attenuated total reflection (FTIR-ATR) results showed the highest sulphation levels and lowest uronic acid content in crude extract from F. vesiculosus. There was a dose-dependent drop in focal adhesion formation, proliferation and osteogenic differentiation of cells for all fucoidan types, but the least toxicity was observed for LMW and MMW. Transmission electron microscopy (TEM), JC-1 (5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimi-dazolylcarbocyanine iodide) staining and cytochrome c analyses confirmed mitochondrial damage, swollen ER and upregulated autophagy due to fucoidans, with the highest severity in the case of F. vesiculosus fucoidan. Stress-induced apoptosis-like cell death by F. vesiculosus fucoidan and stress-induced necrosis-like cell death by S. filipendula fucoidans were also confirmed. LMW and MMW doses of <200 ng/mL were the least toxic and showed potential osteoinductivity. This research underscores the multifaceted impact of fucoidans on osteoprogenitor cells and highlights the delicate balance between potential therapeutic benefits and the challenges involved in using fucoidans for post-surgery treatments in patients with osteosarcoma.

A physiologically relevant, estradiol-17ß [E2]-responsive in vitro tissue-engineered model of the vaginal epithelium for vaginal tissue research.

AIMS: There are many situations where preclinical models of the human vagina would be valuable for in vitro studies into the pathophysiology of vaginally transmitted diseases, microbicide efficacy, irritability testing, and particularly, for assessing materials to be inserted in the vagina for support of the pelvic floor. The aim of this study is to develop a physiologically relevant, low cost, and ethically suitable model of the vagina using sheep vaginal tissue (SVT) to reduce the need for animal testing in gynecological research. METHODS: Tissue-engineered (TE) vaginal models were developed by culturing primary vaginal epithelial cells and vaginal fibroblasts, isolated from the native SVTs on decellularized sheep vaginal matrices at an air-liquid interface. Morphological analyses of the models were conducted by performing hematoxylin and eosin staining and further characterization was done by immunohistofluorescence (IHF) of structural proteins and cytokeratins. RESULTS: Histological analysis of the models revealed a gradual formation of a stratified epithelium on our decellularized matrices and cell metabolic activity remained high for 21 days as measured by the resazurin assay. Our models showed a dose-dependent response to estradiol-17ß [E2 ] with an increase in the vaginal epithelium thickness and cellular proliferation under higher E2 concentrations (100-400 pg/ml). The physiological relevance of these results was confirmed by the IHF analysis of Ki67, and cytokeratins 10 and 19 expression. CONCLUSION: In this study, we have developed an estradiol-responsive TE vaginal model that closely mimics the structural and physiological properties of the native SVT.

A Ruthenium(II) Polypyridyl Complex Disrupts Actin Cytoskeleton Assembly and Blocks Cytokinesis.

The dinuclear RuII complex [(Ru(phen)2 )2 (tpphz)]4+ (phen=1,10-phenanthroline, tpphz=tetrapyridophenazine) "RuRuPhen" blocks the transformation of G-actin monomers to F-actin filaments with no disassembly of pre-formed F-actin. Molecular docking studies indicate multiple RuRuPhen molecules bind to the surface of G-actin but not the binding pockets of established actin polymerisation inhibitors. In cells, addition of RuRuPhen causes rapid disruption to actin stress fibre organisation, compromising actomyosin contractility and cell motility; due to this effect RuRuPhen interferes with late-stage cytokinesis. Immunofluorescent microscopy reveals that RuRuPhen causes cytokinetic abscission failure by interfering with endosomal sorting complexes required for transport (ESCRT) complex recruitment.

Mutual influence of selenium nanoparticles and FGF2-STAB® on biocompatible properties of collagen/chitosan 3D scaffolds: in vitro and ex ovo evaluation.

In a biological system, nanoparticles (NPs) may interact with biomolecules. Specifically, the adsorption of proteins on the nanoparticle surface may influence both the nanoparticles' and proteins' overall bio-reactivity. Nevertheless, our knowledge of the biocompatibility and risk of exposure to nanomaterials is limited. Here, in vitro and ex ovo biocompatibility of naturally based crosslinked freeze-dried 3D porous collagen/chitosan scaffolds, modified with thermostable fibroblast growth factor 2 (FGF2-STAB®), to enhance healing and selenium nanoparticles (SeNPs) to provide antibacterial activity, were evaluated. Biocompatibility and cytotoxicity were tested in vitro using normal human dermal fibroblasts (NHDF) with scaffolds and SeNPs and FGF2-STAB® solutions. Metabolic activity assays indicated an antagonistic effect of SeNPs and FGF2-STAB® at high concentrations of SeNPs. The half-maximal inhibitory concentration (IC50) of SeNPs for NHDF was 18.9 µg/ml and IC80 was 5.6 µg/ml. The angiogenic properties of the scaffolds were monitored ex ovo using a chick chorioallantoic membrane (CAM) assay and the cytotoxicity of SeNPs over IC80 value was confirmed. Furthermore, the positive effect of FGF2-STAB® at very low concentrations (0.01 µg/ml) on NHDF metabolic activity was observed. Based on detailed in vitro testing, the optimal concentrations of additives in the scaffolds were determined, specifically 1 µg/ml of FGF2-STAB® and 1 µg/ml of SeNPs. The scaffolds were further subjected to antimicrobial tests, where an increase in selenium concentration in the collagen/chitosan scaffolds increased the antibacterial activity. This work highlights the antimicrobial ability and biocompatibility of newly developed crosslinked collagen/chitosan scaffolds involving FGF2-STAB® and SeNPs. Moreover, we suggest that these sponges could be used as scaffolds for growing cells in systems with low mechanical loading in tissue engineering, especially in dermis replacement, where neovascularization is a crucial parameter for successful skin regeneration. Due to their antimicrobial properties, these scaffolds are also highly promising for tissue replacement requiring the prevention of infection.

Fucoidan Inhibition of Osteosarcoma Cells Is Species and Molecular Weight Dependent.

Fucoidan is a brown algae-derived polysaccharide having several biomedical applications. This study simultaneously compares the anti-cancer activities of crude fucoidans from Fucus vesiculosus and Sargassum filipendula, and effects of low (LMW, 10-50 kDa), medium (MMW, 50-100 kDa) and high (HMW, >100 kDa) molecular weight fractions of S. filipendula fucoidan against osteosarcoma cells. Glucose, fucose and acid levels were lower and sulphation was higher in F. vesiculosus crude fucoidan compared to S. filipendula crude fucoidan. MMW had the highest levels of sugars, acids and sulphation among molecular weight fractions. There was a dose-dependent drop in focal adhesion formation and proliferation of cells for all fucoidan-types, but F. vesiculosus fucoidan and HMW had the strongest effects. G1-phase arrest was induced by F. vesiculosus fucoidan, MMW and HMW, however F. vesiculosus fucoidan treatment also caused accumulation in the sub-G1-phase. Mitochondrial damage occurred for all fucoidan-types, however F. vesiculosus fucoidan led to mitochondrial fragmentation. Annexin V/PI, TUNEL and cytochrome c staining confirmed stress-induced apoptosis-like cell death for F. vesiculosus fucoidan and features of stress-induced necrosis-like cell death for S. filipendula fucoidans. There was also variation in penetrability of different fucoidans inside the cell. These differences in anti-cancer activity of fucoidans are applicable for osteosarcoma treatment.

Methyl-donor depletion of head and neck cancer cells in vitro establishes a less aggressive tumour cell phenotype.

PURPOSE: DNA methylation plays a fundamental role in the epigenetic control of carcinogenesis and is, in part, influenced by the availability of methyl donors obtained from the diet. In this study, we developed an in-vitro model to investigate whether methyl donor depletion affects the phenotype and gene expression in head and neck squamous cell carcinoma (HNSCC) cells. METHODS: HNSCC cell lines (UD-SCC2 and UPCI-SCC72) were cultured in medium deficient in methionine, folate, and choline or methyl donor complete medium. Cell doubling-time, proliferation, migration, and apoptosis were analysed. The effects of methyl donor depletion on enzymes controlling DNA methylation and the pro-apoptotic factors death-associated protein kinase-1 (DAPK1) and p53 upregulated modulator of apoptosis (PUMA) were examined by quantitative-PCR or immunoblotting. RESULTS: HNSCC cells cultured in methyl donor deplete conditions showed significantly increased cell doubling times, reduced cell proliferation, impaired cell migration, and a dose-dependent increase in apoptosis when compared to cells cultured in complete medium. Methyl donor depletion significantly increased the gene expression of DNMT3a and TET-1, an effect that was reversed upon methyl donor repletion in UD-SCC2 cells. In addition, expression of DAPK1 and PUMA was increased in UD-SCC2 cells cultured in methyl donor deplete compared to complete medium, possibly explaining the observed increase in apoptosis in these cells. CONCLUSION: Taken together, these data show that depleting HNSCC cells of methyl donors reduces the growth and mobility of HNSCC cells, while increasing rates of apoptosis, suggesting that a methyl donor depleted diet may significantly affect the growth of established HNSCC.

Polymersome-mediated delivery of combination anticancer therapy to head and neck cancer cells: 2D and 3D in vitro evaluation.

Polymersomes have the potential to encapsulate and deliver chemotherapeutic drugs into tumor cells, reducing off-target toxicity that often compromises anticancer treatment. Here, we assess the ability of the pH-sensitive poly 2-(methacryloyloxy)ethyl phosphorylcholine (PMPC)- poly 2-(diisopropylamino)ethyl methacrylate (PDPA) polymersomes to encapsulate chemotherapeutic agents for effective combinational anticancer therapy. Polymersome uptake and ability to deliver encapsulated drugs into healthy normal oral cells and oral head and neck squamous cell carcinoma (HNSCC) cells was measured in two and three-dimensional culture systems. PMPC-PDPA polymersomes were more rapidly internalized by HNSCC cells compared to normal oral cells. Polymersome cellular uptake was found to be mediated by class B scavenger receptors. We also observed that these receptors are more highly expressed by cancer cells compared to normal oral cells, enabling polymersome-mediated targeting. Doxorubicin and paclitaxel were encapsulated into pH-sensitive PMPC-PDPA polymersomes with high efficiencies either in isolation or as a dual-load for both singular and combinational delivery. In monolayer culture, only a short exposure to drug-loaded polymersomes was required to elicit a strong cytotoxic effect. When delivered to three-dimensional tumor models, PMPC-PDPA polymersomes were able to penetrate deep into the center of the spheroid resulting in extensive cell damage when loaded with both singular and dual-loaded chemotherapeutics. PMPC-PDPA polymersomes offer a novel system for the effective delivery of chemotherapeutics for the treatment of HNSCC. Moreover, the preferential internalization of PMPC polymersomes by exploiting elevated scavenger receptor expression on cancer cells opens up the opportunity to target polymersomes to tumors.

Combined mathematical modelling and experimentation to predict polymersome uptake by oral cancer cells.

This study is motivated by understanding and controlling the key physical properties underlying internalisation of nano drug delivery. We consider the internalisation of specific nanometre size delivery vehicles, comprised of self-assembling amphiphilic block copolymers, called polymersomes that have the potential to specifically deliver anticancer therapeutics to tumour cells. The possible benefits of targeted polymersome drug delivery include reduced off-target toxic effects in healthy tissue and increased drug uptake by diseased tissue. Through a combination of in vitro experimentation and mathematical modelling, we develop a validated model of nanoparticle uptake by cells via the clathrin-mediated endocytotic pathway, incorporating receptor binding, clustering and recycling. The model predicts how the characteristics of receptor targeting, and the size and concentration of polymersomes alter uptake by tumour cells. The number of receptors per cell was identified as being the dominant mechanism accounting for the difference between cell types in polymersome uptake rate. FROM THE CLINICAL EDITOR: This article reports on a validated model developed through a combination of in vitro experimentation and mathematical modeling of nanoparticle uptake by cells via the clathrin-mediated endocytotic pathway. The model incorporates receptor binding, clustering, and recycling and predicts how the characteristics of receptor targeting, the size and concentration alter polymersome uptake by cancer cells.

Uncovering the relationship between macrophages and polypropylene surgical mesh.

Currently, in vitro testing examines the cytotoxicity of biomaterials but fails to consider how materials respond to mechanical forces and the immune response to them; both are crucial for successful long-term implantation. A notable example of this failure is polypropylene mid-urethral mesh used in the treatment of stress urinary incontinence (SUI). The mesh was largely successful in abdominal hernia repair but produced significant complications when repurposed to treat SUI. Developing more physiologically relevant in vitro test models would allow more physiologically relevant data to be collected about how biomaterials will interact with the body. This study investigates the effects of mechanochemical distress (a combination of oxidation and mechanical distention) on polypropylene mesh surfaces and the effect this has on macrophage gene expression. Surface topology of the mesh was characterised using SEM and AFM; ATR-FTIR, EDX and Raman spectroscopy was applied to detect surface oxidation and structural molecular alterations. Uniaxial mechanical testing was performed to reveal any bulk mechanical changes. RT-qPCR of selected pro-fibrotic and pro-inflammatory genes was carried out on macrophages cultured on control and mechanochemically distressed PP mesh. Following exposure to mechanochemical distress the mesh surface was observed to crack and craze and helical defects were detected in the polymer backbone. Surface oxidation of the mesh was seen after macrophage attachment for 7 days. These changes in mesh surface triggered modified gene expression in macrophages. Pro-fibrotic and pro-inflammatory genes were upregulated after macrophages were cultured on mechanochemically distressed mesh, whereas the same genes were down-regulated in macrophages exposed to control mesh. This study highlights the relationship between macrophages and polypropylene surgical mesh, thus offering more insight into the fate of an implanted material than existing in vitro testing.

Estradiol-17ß [E2] stimulates wound healing in a 3D in vitro tissue-engineered vaginal wound model.

Childbirth contributes to common pelvic floor problems requiring reconstructive surgery in postmenopausal women. Our aim was to develop a tissue-engineered vaginal wound model to investigate wound healing and the contribution of estradiol to pelvic tissue repair. Partial thickness scalpel wounds were made in tissue models based on decellularized sheep vaginal matrices cultured with primary sheep vaginal epithelial cells and fibroblasts. Models were cultured at an airliquid interface (ALI) for 3 weeks with and without estradiol-17ß [E2]. Results showed that E2 significantly increased wound healing and epithelial maturation. Also, E2 led to collagen reorganization after only 14 days with collagen fibers more regularly aligned and compactly arranged Additionally, E2 significantly downregulated α-SMA expression which is involved in fibrotic tissue formation. This model allows one to investigate multiple steps in vaginal wound healing and could be a useful tool in developing therapies for improved tissue healing after reconstructive pelvic floor surgery.

Surfactant-free gelatin-stabilised biodegradable polymerised high internal phase emulsions with macroporous structures.

High internal phase emulsion (HIPE) templating is a well-established method for the generation of polymeric materials with high porosity (>74%) and degree of interconnectivity. The porosity and pore size can be altered by adjusting parameters during emulsification, which affects the properties of the resulting porous structure. However, there remain challenges for the fabrication of polyHIPEs, including typically small pore sizes (∼20-50 µm) and the use of surfactants, which can limit their use in biological applications. Here, we present the use of gelatin, a natural polymer, during the formation of polyHIPE structures, through the use of two biodegradable polymers, polycaprolactone-methacrylate (PCL-M) and polyglycerol sebacate-methacrylate (PGS-M). When gelatin is used as the internal phase, it is capable of stabilising emulsions without the need for an additional surfactant. Furthermore, by changing the concentration of gelatin within the internal phase, the pore size of the resulting polyHIPE can be tuned. 5% gelatin solution resulted in the largest mean pore size, increasing from 53 µm to 80 µm and 28 µm to 94 µm for PCL-M and PGS-M respectively. In addition, the inclusion of gelatin further increased the mechanical properties of the polyHIPEs and increased the period an emulsion could be stored before polymerisation. Our results demonstrate the potential to use gelatin for the fabrication of surfactant-free polyHIPEs with macroporous structures, with potential applications in tissue engineering, environmental and agricultural industries.

In vitro Effect of Geranylgeraniol (GGOH) on Bisphosphonate-Induced Cytotoxicity of Oral Mucosa Cells.

Medication-related osteonecrosis of the jaw (MRONJ) is an often-severe complication found in patients receiving bisphosphonates in the management of Paget's, osteoporosis and metastatic bone cancer. Mucosal breakdown with bone exposure is a primary clinical presentation of MRONJ linked to the inhibitory effect of nitrogen-containing bisphosphonates (N-BP) on the mevalonate pathway. Geranylgeraniol (GGOH) has demonstrated a rescue effect on N-BP-treated osteoclasts but the biological effects on oral soft tissues and cells remain unclear. This study aimed to determine whether GGOH could prevent bisphosphonate induced toxicity to oral mucosa cells in vitro. Primary oral fibroblasts and keratinocytes were exposed to different GGOH concentrations or GGOH in combination with two nitrogen-containing bisphosphonates, zoledronic acid (ZA) or pamidronic acid (PA), for 72 h. The metabolic activity of each cell type was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. GGOH without bisphosphonates significantly reduced the metabolic activity of oral mucosa cells. Fibroblasts treated with GGOH and ZA in combination showed a slight increase in metabolic status compared to fibroblasts treated with ZA alone, however this positive effect was not observed in keratinocytes. In the presence of PA, GGOH was unable to increase the metabolic activity of either cell type. These findings demonstrate that GGOH is toxic to oral mucosa cells and that GGOH was not able to prevent bisphosphonate induced toxicity. These data show that GGOH does not have therapeutic potential for bisphosphonate-induced soft tissue toxicity in MRONJ and the use of GGOH as an MRONJ treatment should be strongly reconsidered.

A Ruthenium(II) Polypyridyl Complex Disrupts Actin Cytoskeleton Assembly and Blocks Cytokinesis.

The dinuclear RuII complex [(Ru(phen)2)2(tpphz)]4+ (phen=1,10-phenanthroline, tpphz=tetrapyridophenazine) "RuRuPhen" blocks the transformation of G-actin monomers to F-actin filaments with no disassembly of pre-formed F-actin. Molecular docking studies indicate multiple RuRuPhen molecules bind to the surface of G-actin but not the binding pockets of established actin polymerisation inhibitors. In cells, addition of RuRuPhen causes rapid disruption to actin stress fibre organisation, compromising actomyosin contractility and cell motility; due to this effect RuRuPhen interferes with late-stage cytokinesis. Immunofluorescent microscopy reveals that RuRuPhen causes cytokinetic abscission failure by interfering with endosomal sorting complexes required for transport (ESCRT) complex recruitment.

A Review Into the Effects of Pamidronic Acid and Zoledronic Acid on the Oral Mucosa in Medication-Related Osteonecrosis of the Jaw.

Medication-related osteonecrosis of the jaw (MRONJ) is a growing problem without an effective treatment, presenting as necrotic bone sections exposed via lesions in the overlying soft tissue. There is currently a lack of clarity on how the factors involved in MRONJ development and progression contribute to disease prognosis and outcomes. Bisphosphonates (BPs), the most common cause of MRONJ, affect bone remodeling, angiogenesis, infection, inflammation and soft tissue toxicity, all of which contribute to MRONJ development. This article reviews the cellular mechanisms through which BPs contribute to MRONJ pathology, with a focus on the effects on cells of the oral mucosa. BPs have been shown to reduce cell viability, reduce proliferation, and increase apoptosis in oral keratinocytes and fibroblasts. BPs have also been demonstrated to reduce epithelial thickness and prevent epithelial formation in three-dimensional tissue engineered models of the oral mucosa. This combination of factors demonstrates how BPs lead to the reduced wound healing seen in MRONJ and begins to uncover the mechanisms through which these effects occur. The evidence presented here supports identification of targets which can be used to develop novel treatment strategies to promote soft tissue wound healing and restore mucosal coverage of exposed bone in MRONJ.

A novel characterisation approach to reveal the mechano-chemical effects of oxidation and dynamic distension on polypropylene surgical mesh.

Polypropylene (PP) surgical mesh, used successfully for the surgical repair of abdominal hernias, is associated with serious clinical complications when used in the pelvic floor for repair of stress urinary incontinence or support of pelvic organ prolapse. While manufacturers claim that the material is inert and non-degradable, there is a growing body of evidence that asserts PP fibres are subject to oxidative damage and indeed explanted material from patients suffering with clinical complications has shown some evidence of fibre cracking and oxidation. It has been proposed that a pathological cellular response to the surgical mesh contributes to the medical complications; however, the mechanisms that trigger the specific host response against the material are not well understood. Specifically, this study was constructed to investigate the mechano-chemical effects of oxidation and dynamic distension on polypropylene surgical mesh. To do this we used a novel advanced spectroscopical characterisation technique, secondary electron hyperspectral imaging (SEHI), which is based on the collection of secondary electron emission spectra in a scanning electron microscope (SEM) to reveal mechanical-chemical reactions within PP meshes.

Healing and Angiogenic Properties of Collagen/Chitosan Scaffolds Enriched with Hyperstable FGF2-STAB® Protein: In Vitro, Ex Ovo and In Vivo Comprehensive Evaluation.

Wound healing is a process regulated by a complex interaction of multiple growth factors including fibroblast growth factor 2 (FGF2). Although FGF2 appears in several tissue engineered studies, its applications are limited due to its low stability both in vitro and in vivo. Here, this shortcoming is overcome by a unique nine-point mutant of the low molecular weight isoform FGF2 retaining full biological activity even after twenty days at 37 °C. Crosslinked freeze-dried 3D porous collagen/chitosan scaffolds enriched with this hyper stable recombinant human protein named FGF2-STAB® were tested for in vitro biocompatibility and cytotoxicity using murine 3T3-A31 fibroblasts, for angiogenic potential using an ex ovo chick chorioallantoic membrane assay and for wound healing in vivo with 3-month old white New Zealand rabbits. Metabolic activity assays indicated the positive effect of FGF2-STAB® already at very low concentrations (0.01 µg/mL). The angiogenic properties examined ex ovo showed enhanced vascularization of the tested scaffolds. Histological evaluation and gene expression analysis by RT-qPCR proved newly formed granulation tissue at the place of a previous skin defect without significant inflammation infiltration in vivo. This work highlights the safety and biocompatibility of newly developed crosslinked collagen/chitosan scaffolds involving FGF2-STAB® protein. Moreover, these sponges could be used as scaffolds for growing cells for dermis replacement, where neovascularization is a crucial parameter for successful skin regeneration.

Synthetic Hydroxyapatite Inhibits Bisphosphonate Toxicity to the Oral Mucosa In Vitro.

Medication-related osteonecrosis of the jaw (MRONJ) is a side effect of bisphosphonate therapy, characterised by exposed necrotic bone. The soft tissues of the oral mucosa no longer provide a protective barrier and MRONJ patients experience pain, infections and difficulties eating. We hypothesised that hydroxyapatite (Ca5(PO4)3(OH)) could reduce bisphosphonate concentrations and protect the oral mucosa by exploiting bisphosphonate's calcium binding affinity. The effect of zoledronic acid (ZA) and pamidronic acid (PA) on the metabolism of oral fibroblasts, oral keratinocytes and three-dimensional oral mucosa models was investigated and then repeated in the presence of hydroxyapatite granules. Without hydroxyapatite, ZA and PA significantly reduced the metabolic activity of oral cells in a dose-dependent manner. Both drugs reduced epithelial thickness and 30 µM ZA resulted in loss of the epithelium. Hydroxyapatite granules had a protective effect on oral cells, with metabolic activity retained. Oral mucosa models retained their multi-layered epithelium when treated with ZA in the presence of hydroxyapatite granules and metabolic activity was comparable to controls. These results demonstrate hydroxyapatite granules protected oral soft tissues from damage caused by bisphosphonate exposure. Porous hydroxyapatite granules are currently used for socket preservation and this data suggests their potential to prevent MRONJ in at-risk patients.

The effect of porous structure on the cell proliferation, tissue ingrowth and angiogenic properties of poly(glycerol sebacate urethane) scaffolds.

Novel, porous, biodegradable biomaterials which support tissue integration and angiogenesis and which have elastomeric properties are needed to repair and replace soft tissues in dynamic environments. In this study poly(glycerol sebacate urethane) (PGSU) scaffolds with different porous structures were fabricated using freeze-drying by varying the polymer concentration of the freeze-drying solution, during which the polymer was further crosslinked. The effect of the porous structure on the physical properties, cell proliferation, tissue ingrowth and angiogenic properties was investigated. By increasing the polymer concentration in the freeze-drying solution from 5 w/v% to 10 w/v% and 15 w/v%, the porosity and pore size of the scaffold decreased, resulting in porosities ranging between 88 - 96% and pore sizes 6.4-28.2 µm. The mechanical properties increased with the polymer concentration, with ultimate tensile strength and Young's modulus between 0.05 - 0.86 MPa and 0.05-0.65 MPa respectively and negligible loss of tensile strength after 100 cycles of loading. Enzymatic degradation over 28 days demonstrated linear degradation kinetics with mass loss between 19.1 - 52.3%. All PGSU scaffolds provided a viable environment for cell attachment, in which cell metabolic activity increased over time indicating cell proliferation. The cells adhered to PGSU scaffolds produced and deposited high quantities of collagen, reaching 7.5% of the sample's dry mass after 14 days culture for the scaffold with the highest porosity. Additionally, the scaffolds with the polymer concentration of 5 w/v% implanted onto the chick chorioallantoic membrane supported rapid tissue ingrowth and new blood vessel formation within the porous scaffold. These results demonstrate that PGSU scaffolds have potential for use in many areas of soft tissue engineering.

Diffusion studies of nanometer polymersomes across tissue engineered human oral mucosa.

PURPOSE: To measure the diffusion of nanometer polymersomes through tissue engineered human oral mucosa. METHODS: In vitro models of full thickness tissue engineered oral mucosa (TEOM) were used to assess the penetration properties of two chemically different polymersomes comprising two of block copolymers, PMPC-PDPA and PEO-PDPA. These copolymers self-assemble into membrane-enclosed vesicular structures. Polymersomes were conjugated with fluorescent rhodamine in order to track polymersome diffusion. Imaging and quantification of the diffusion properties were assessed by confocal laser scanning microscopy (CLSM). RESULTS: TEOM is morphologically similar to natural oral mucosa. Using CLSM, both formulations were detectable in the TEOM within 6 h and after 48 h both penetrated up to 80 microm into the TEOM. Diffusion of PMPC-PDPA polymersomes was widespread across the epithelium with intra-epithelial uptake, while PEO-PDPA polymersomes also diffused into the epithelium. CONCLUSIONS: CLSM was found to be an effective and versatile method for analysing the level of diffusion of polymersomes into TEOM. The penetration and retention of PMPC-PDPA and PEO-PDPA polymersomes means they may have potential for intra-epithelial drug delivery and/or trans-epithelial delivery of therapeutic agents.

Synergistic effect of bovine platelet lysate and various polysaccharides on the biological properties of collagen-based scaffolds for tissue engineering: Scaffold preparation, chemo-physical characterization, in vitro and ex ovo evaluation.

Crosslinked 3D porous collagen-polysaccharide scaffolds, prepared by freeze-drying, were modified with bovine platelet lysate (BPL) and evaluated in terms of chemical, physical and biological properties. Natural antibacterial polysaccharides like chitosan, chitin/chitosan-glucan complex and calcium salt of oxidized cellulose (CaOC) incorporated in collagen scaffolds affected not only chemo-physical properties of the composite scaffolds but also improved their biological properties, especially when BPL was presented. Lipophilic BPL formed microspheres in porous scaffolds while reduced by half their swelling ratio. The resistance of collagen sponges to hydrolytic degradation in water depended strongly on chemical crosslinking varying from 60 min to more than one year. According to in-vitro tests, chemically crosslinked scaffolds exhibited a good cellular response, cell-matrix interactions, and biocompatibility of the material. The combination of collagen with natural polysaccharides confirmed a significant positive synergistic effect on cultivation of cells as determined by MTS assay and PicoGreen method, as well as on angiogenesis evaluated by ex ovo Chick Chorioallantoic Membrane (CAM) assay. Contrary, modification only by BLP of pure collagen scaffolds exhibited decreased biocompatibility in comparison to unmodified pure collagen scaffold. We propose that the newly developed crosslinked collagen sponges involving bioactive additives could be used as scaffold for growing cells in systems with low mechanical loading in tissue engineering, especially in dermis replacement, where neovascularization is a crucial parameter for successful skin regeneration.