Chitin-glucan improves important pathophysiological features of irritable bowel syndrome.

BACKGROUND: Irritable bowel syndrome (IBS) is one of the most frequent and debilitating conditions leading to gastroenterological referrals. However, recommended treatments remain limited, yielding only limited therapeutic gains. Chitin-glucan (CG) is a novel dietary prebiotic classically used in humans at a dosage of 1.5-3.0 g/d and is considered a safe food ingredient by the European Food Safety Authority. To provide an alternative approach to managing patients with IBS, we performed preclinical molecular, cellular, and animal studies to evaluate the role of chitin-glucan in the main pathophysiological mechanisms involved in IBS. AIM: To evaluate the roles of CG in visceral analgesia, intestinal inflammation, barrier function, and to develop computational molecular models. METHODS: Visceral pain was recorded through colorectal distension (CRD) in a model of long-lasting colon hypersensitivity induced by an intra-rectal administration of TNBS [15 milligrams (mg)/kilogram (kg)] in 33 Sprague-Dawley rats. Intracolonic pressure was regularly assessed during the 9 wk-experiment (weeks 0, 3, 5, and 7) in animals receiving CG (n = 14) at a human equivalent dose (HED) of 1.5 g/d or 3.0 g/d and compared to negative control (tap water, n = 11) and positive control (phloroglucinol at 1.5 g/d HED, n = 8) groups. The anti-inflammatory effect of CG was evaluated using clinical and histological scores in 30 C57bl6 male mice with colitis induced by dextran sodium sulfate (DSS) administered in their drinking water during 14 d. HT-29 cells under basal conditions and after stimulation with lipopolysaccharide (LPS) were treated with CG to evaluate changes in pathways related to analgesia (µ-opioid receptor (MOR), cannabinoid receptor 2 (CB2), peroxisome proliferator-activated receptor alpha, inflammation [interleukin (IL)-10, IL-1b, and IL-8] and barrier function [mucin 2-5AC, claudin-2, zonula occludens (ZO)-1, ZO-2] using the real-time PCR method. Molecular modelling of CG, LPS, lipoteichoic acid (LTA), and phospholipomannan (PLM) was developed, and the ability of CG to chelate microbial pathogenic lipids was evaluated by docking and molecular dynamics simulations. Data were expressed as the mean ± SEM. RESULTS: Daily CG orally-administered to rats or mice was well tolerated without including diarrhea, visceral hypersensitivity, or inflammation, as evaluated at histological and molecular levels. In a model of CRD, CG at a dosage of 3 g/d HED significantly decreased visceral pain perception by 14% after 2 wk of administration (P < 0.01) and reduced inflammation intensity by 50%, resulting in complete regeneration of the colonic mucosa in mice with DSS-induced colitis. To better reproduce the characteristics of visceral pain in patients with IBS, we then measured the therapeutic impact of CG in rats with TNBS-induced inflammation to long-lasting visceral hypersensitivity. CG at a dosage of 1.5 g/d HED decreased visceral pain perception by 20% five weeks after colitis induction (P < 0.01). When the CG dosage was increased to 3.0 g/d HED, this analgesic effect surpassed that of the spasmolytic agent phloroglucinol, manifesting more rapidly within 3 wk and leading to a 50% inhibition of pain perception (P < 0.0001). The underlying molecular mechanisms contributing to these analgesic and anti-inflammatory effects of CG involved, at least in part, a significant induction of MOR, CB2 receptor, and IL-10, as well as a significant decrease in pro-inflammatory cytokines IL-1b and IL-8. CG also significantly upregulated barrier-related genes including muc5AC, claudin-2, and ZO-2. Molecular modelling of CG revealed a new property of the molecule as a chelator of microbial pathogenic lipids, sequestering gram-negative LPS and gram-positive LTA bacterial toxins, as well as PLM in fungi at the lowesr energy conformations. CONCLUSION: CG decreased visceral perception and intestinal inflammation through master gene regulation and direct binding of microbial products, suggesting that CG may constitute a new therapeutic strategy for patients with IBS or IBS-like symptoms.

Breath volatile metabolome reveals the impact of dietary fibres on the gut microbiota: Proof of concept in healthy volunteers.

BACKGROUND: Current data suggest that dietary fibre (DF) interaction with the gut microbiota largely contributes to their physiological effects. The bacterial fermentation of DF leads to the production of metabolites, most of them are volatile. This study analyzed the breath volatile metabolites (BVM) profile in healthy individuals (n=15) prior and after a 3-week intervention with chitin-glucan (CG, 4.5 g/day), an insoluble fermentable DF. METHODS: The present exploratory study presents the original data related to the secondary outcomes, notably the analysis of BVM. BVM were analyzed throughout the test days -in fasting state and after standardized meals - using selected ion flow tube mass spectrometry (SIFT-MS). BVM production was correlated to the gut microbiota composition (Illumina sequencing, primary outcome), analyzed before and after the intervention. FINDINGS: The data reveal that the post-prandial state versus fasting state is a key determinant of BVM fingerprint. Correlation analyses with fecal microbiota spotlighted butyrate-producing bacteria, notably Faecalibacterium, as dominant bacteria involved in butyrate and other BVM expiration. CG intervention promotes interindividual variations of fasting BVM, and decreases or delays the expiration of most exhaled BVM in favor of H2 expiration, without any consequence on gastrointestinal tolerance. INTERPRETATION: Assessing BVM is a non-invasive methodology allowing to analyze the influence of DF intervention on the gut microbiota. FUNDING: FiberTAG project was initiated from a European Joint Programming Initiative "A Healthy Diet for a Healthy Life" (JPI HDHL) and was supported by the Service Public de Wallonie (SPW-EER, convention 1610365, Belgium).

Chitin-glucan supplementation improved postprandial metabolism and altered gut microbiota in subjects at cardiometabolic risk in a randomized trial.

Chitin-glucan (CG), an insoluble dietary fiber, has been shown to improve cardiometabolic disorders associated with obesity in mice. Its effects in healthy subjects has recently been studied, revealing its interaction with the gut microbiota. In this double-blind, randomized, cross-over, twice 3-week exploratory study, we investigated the impacts of CG on the cardiometabolic profile and gut microbiota composition and functions in 15 subjects at cardiometabolic risk. They consumed as a supplement 4.5 g of CG daily or maltodextrin as control. Before and after interventions, fasting and postprandial metabolic parameters and exhaled gases (hydrogen [H2] and methane [CH4]) were evaluated. Gut microbiota composition (16S rRNA gene sequencing analysis), fecal concentrations of bile acids, long- and short-chain fatty acids (LCFA, SCFA), zonulin, calprotectin and lipopolysaccharide binding protein (LBP) were analyzed. Compared to control, CG supplementation increased exhaled H2 following an enriched-fiber breakfast ingestion and decreased postprandial glycemia and triglyceridemia response to a standardized test meal challenge served at lunch. Of note, the decrease in postprandial glycemia was only observed in subjects with higher exhaled H2, assessed upon lactulose breath test performed at inclusion. CG decreased a family belonging to Actinobacteria phylum and increased 3 bacterial taxa: Erysipelotrichaceae UCG.003, Ruminococcaceae UCG.005 and Eubacterium ventriosum group. Fecal metabolites, inflammatory and intestinal permeability markers did not differ between groups. In conclusion, we showed that CG supplementation modified the gut microbiota composition and improved postprandial glycemic response, an early determinant of cardiometabolic risk. Our results also suggest breath H2 production as a non-invasive parameter of interest for predicting the effectiveness of dietary fiber intervention.

Chitin Glucan Shifts Luminal and Mucosal Microbial Communities, Improve Epithelial Barrier and Modulates Cytokine Production In Vitro.

The human gut microbiota has been linked to the health status of the host. Modulation of human gut microbiota through pro- and prebiotic interventions has yielded promising results; however, the effect of novel prebiotics, such as chitin-glucan, on gut microbiota-host interplay is still not fully characterized. We assessed the effect of chitin-glucan (CG) and chitin-glucan plus Bifidobacterium breve (CGB) on human gut microbiota from the luminal and mucosal environments in vitro. Further, we tested the effect of filter-sterilized fecal supernatants from CG and CGB fermentation for protective effects on inflammation-induced barrier disruption and cytokine production using a co-culture of enterocytes and macrophage-like cells. Overall, CG and CGB promote health-beneficial short-chain fatty acid production and shift human gut microbiota composition, with a consistent effect increasing Roseburia spp. and butyrate producing-bacteria. In two of three donors, CG and CGB also stimulated Faecalibacterium prausniitzi. Specific colonization of B. breve was observed in the lumen and mucosal compartment; however, no synergy was detected for different endpoints when comparing CGB and CG. Both treatments included a significant improvement of inflammation-disrupted epithelial barrier and shifts on cytokine production, especially by consistent increase in the immunomodulatory cytokines IL10 and IL6.

Noninvasive monitoring of fibre fermentation in healthy volunteers by analyzing breath volatile metabolites: lessons from the FiberTAG intervention study.

The fermentation of dietary fibre (DF) leads to the production of bioactive metabolites, the most volatile ones being excreted in the breath. The aim of this study was to analyze the profile of exhaled breath volatile metabolites (BVM) and gastrointestinal symptoms in healthy volunteers after a single ingestion of maltodextrin (placebo) versus chitin-glucan (CG), an insoluble DF previously shown to be fermented into short-chain fatty acids (SCFA) by the human microbiota in vitro. Maltodextrin (4.5 g at day 0) or CG (4.5 g at day 2) were added to a standardized breakfast in fasting healthy volunteers (n = 15). BVM were measured using selected ion flow tube mass spectrometry (SIFT-MS) throughout the day. A single ingestion of 4.5 g CG did not induce significant gastrointestinal discomfort. Untargeted metabolomics analysis of breath highlighted that 13 MS-fragments (among 408 obtained from ionizations of breath) discriminated CG versus maltodextrin acute intake in the posprandial state. The targeted analysis revealed that CG increased exhaled butyrate and 5 other BVM - including the microbial metabolites 2,3-butanedione and 3-hydroxybutanone - with a peak observed 6 h after CG intake. Correlation analyses with fecal microbiota (Illumina 16S rRNA sequencing) spotlighted Mitsuokella as a potential genus responsible for the presence of butyric acid, triethylamine and 3-hydroxybutanone in the breath. In conclusion, measuring BMV in the breath reveals the microbial signature of the fermentation of DF after a single ingestion. This protocol allows to analyze the time-course of released bioactive metabolites that could be proposed as new biomarkers of DF fermentation, potentially linked to their biological properties. Trial registration: Clinical Trials NCT03494491. Registered 11 April 2018 - Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT03494491.

Development of a Repertoire and a Food Frequency Questionnaire for Estimating Dietary Fiber Intake Considering Prebiotics: Input from the FiberTAG Project.

Most official food composition tables and food questionnaires do not provide enough data to assess fermentable dietary fibers (DF) that can exert a health effect through their interaction with the gut microbiota. The aim of this study was to develop a database and a food frequency questionnaire (FFQ) allowing detailed DF intake estimation including prebiotic (oligo)saccharides. A repertoire of DF detailing total, soluble DF, insoluble DF and prebiotic (oligo)saccharides (inulin-type fructans, fructo-oligosaccharides and galacto-oligosaccharides) in food products consumed in Europe has been established. A 12 month FFQ was developed and submitted to 15 healthy volunteers from the FiberTAG study. Our data report a total DF intake of 38 g/day in the tested population. Fructan and fructo-oligosaccharides intake, linked notably to condiments (garlic and onions) ingestion, reached 5 and 2 g/day, respectively, galacto-oligosaccharides intake level being lower (1 g/day). We conclude that the FiberTAG repertoire and FFQ are major tools for the evaluation of the total amount of DF including prebiotics. Their use can be helpful in intervention or observational studies devoted to analyze microbiota-nutrient interactions in different pathological contexts, as well as to revisit DF intake recommendations as part of healthy lifestyles considering specific DF.

Metabolite profiling reveals the interaction of chitin-glucan with the gut microbiota.

Dietary fibers are considered beneficial nutrients for health. Current data suggest that their interaction with the gut microbiota largely contributes to their physiological effects. In this context, chitin-glucan (CG) improves metabolic disorders associated with obesity in mice, but its effect on gut microbiota has never been evaluated in humans. This study explores the effect of a 3-week intervention with CG supplementation in healthy individuals on gut microbiota composition and bacterial metabolites. CG was given to healthy volunteers (n = 15) for three weeks as a supplement (4.5 g/day). Food diary, visual analog and Bristol stool form scales and a "quality of life" survey were analyzed. Among gut microbiota-derived metabolites, bile acids (BA), long- and short-chain fatty acids (LCFA, SCFA) profiling were assessed in stool samples. The gut microbiota (primary outcome) was analyzed by Illumina sequencing. A 3-week supplementation with CG is well tolerated in healthy humans. CG induces specific changes in the gut microbiota composition, with Eubacterium, Dorea and Roseburia genera showing the strongest regulation. In addition, CG increased bacterial metabolites in feces including butyric, iso-valeric, caproic and vaccenic acids. No major changes were observed for the fecal BA profile following CG intervention. In summary, our work reveals new potential bacterial genera and gut microbiota-derived metabolites characterizing the interaction between an insoluble dietary fiber -CG- and the gut microbiota.

Chitosan enriched three-dimensional matrix reduces inflammatory and catabolic mediators production by human chondrocytes.

This in vitro study investigated the metabolism of human osteoarthritic (OA) chondrocytes encapsulated in a spherical matrix enriched of chitosan. Human OA chondrocytes were encapsulated and cultured for 28 days either in chitosan-alginate beads or in alginate beads. The beads were formed by slowly passing dropwise either the chitosan 0.6%-alginate 1.2% or the alginate 1.2% solution through a syringe into a 102 mM CaCl2 solution. Beads were analyzed histologically after 28 days. Interleukin (IL)-6 and -8, prostaglandin (PG) E2, matrix metalloproteinases (MMPs), hyaluronan and aggrecan were quantified directly in the culture supernatant by specific ELISA and nitric oxide (NO) by using a colorimetric method based on the Griess reaction. Hematoxylin and eosin staining showed that chitosan was homogeneously distributed through the matrix and was in direct contact with chondrocytes. The production of IL-6, IL-8 and MMP-3 by chondrocytes significantly decreased in chitosan-alginate beads compared to alginate beads. PGE2 and NO decreased also significantly but only during the first three days of culture. Hyaluronan and aggrecan production tended to increase in chitosan-alginate beads after 28 days of culture. Chitosan-alginate beads reduced the production of inflammatory and catabolic mediators by OA chondrocytes and tended to stimulate the synthesis of cartilage matrix components. These particular effects indicate that chitosan-alginate beads are an interesting scaffold for chondrocytes encapsulation before transplantation to repair cartilage defects.

Development of a procedure to simultaneously isolate RNA, DNA, and proteins for [corrected] characterizing cells invading or cultured on chitosan scaffolds.

For many years, chitosan and its derivatives have been considered to be promising biomaterials for tissue engineering and repair. However, information regarding their biological effect on cell phenotype is usually limited to evaluation of cell proliferation and survival, overlooking proteomic and transcriptomic analysis. This is largely related to the lack of efficient and quantitative procedures for protein and nucleic acid purification from cells cultured on, or inside, chitosan scaffold. Here we describe an ultracentrifugation procedure enabling the simultaneous and quantitative recovery of high quality RNA, DNA and proteins from cells growing in close contact of biomaterial matrices containing chitosan.

Evaluation of human bone marrow stromal cell growth on biodegradable polymer/bioglass composites.

Bone tissue engineering using human bone marrow mesenchymal stem cells (HBMCs) and biocompatible materials provides an attractive approach to regenerate bone tissue to meet the major clinical need. The aim of this study was to examine the effects of novel porous biodegradable composite materials consisting of a bioactive phase (45S5 Bioglass, 0, 5, and 40 wt%) incorporated within a biodegradable poly(dl-lactic acid) matrix, on HBMCs growth. Cell adhesion, spreading, and viability was examined using Cell Tracker Green/Ethidium Homodimer-1. Bone formation was assessed using scaffolds seeded with stro-1 positive HBMCs in nude mice. In vitro biochemistry indicated that with minimal scaffold pre-treatment osteoblast activity falls with increasing Bioglass content. However, 24h scaffold pre-treatment with serum resulted in a significant increase in alkaline phosphatase specific activity in 5 wt% Bioglass composites relative to the 0 and 40 wt% Bioglass groups. In vivo studies indicate significant new bone formation throughout all the scaffolds, as evidenced by immunohistochemistry.

Limitations in transplantation of astroglia-biomatrix bridges to stimulate corticospinal axon regrowth across large spinal lesion gaps.

Regrowth of injured axons across rather small spinal cord lesion gaps and subsequent functional recovery has been obtained after many interventions. Long-distance regeneration of injured axons across clinically relevant large spinal lesion gaps is relatively unexplored. Here, we aimed at stimulating long-distance regrowth of the injured corticospinal (CS) tract. During development, an oriented framework of immature astrocytes is important for correct CS axon outgrowth. Furthermore, a continuous growth promoting substrate may be needed to maintain a CS axon regrowth response across relatively large spinal lesion gaps. Hence, we acutely transplanted poly(D,L)-lactide matrices, which after seeded with immature astrocytes render aligned astrocyte-biomatrix complexes (R. Deumens, et al. Alignment of glial cells stimulates directional neurite growth of CNS neurons in vitro. Neuroscience 125 (3) (2004) 591-604), into 2-mm long dorsal hemisection lesion gaps. In order to create a growth promoting continuum, astrocyte suspensions were also injected rostral and caudal to the lesion gap. During 2 months, locomotion was continuously monitored. Histological analysis showed that astrocytes injected into host spinal tissue survived, but did not migrate. None of the astrocytes on the biomatrices survived within the lesion gap. BDA-labeled CS axons did not penetrate the graft. However, directly rostral to the lesion gap, 120.9+/-38.5% of the BDA-labeled CS axons were present in contrast to 12.8+/-3.9% in untreated control animals. The observed anatomical changes were not accompanied by locomotor improvements as analyzed with the BBB and CatWalk. We conclude that although multifactorial strategies may be needed to stimulate long-distance CS axon regrowth, future studies should focus on enhancing the viability of cell/biomatrix complexes within large spinal lesion gaps.

Surface modification of metallic cardiovascular stents by strongly adhering aliphatic polyester coatings.

This article reports on a novel two-step strategy for the coating of cardiovascular stents by strongly adhering biocompatible and biodegradable aliphatic polyesters. First, a precoating of poly(ethylacrylate) (PEA) was electrografted onto the metallic substrate by cathodic reduction of the parent monomer in dimethylformamide (DMF). The electrodeposition of PEA, in a good solvent of it, was confirmed by both Infra-red and Raman spectroscopies. The pendant ester groups of PEA were then chemically reduced into aluminum alkoxides, able to initiate the ring-opening polymerization (ROP) of either D,L-lactide (LA) or epsilon-caprolactone (CL). Growth of biodegradable PLA or PCL coatings from the adhering precoating was confirmed by both Infra-red and Raman spectroscopies, and directly observed by scanning electron microscopy (SEM). This type of coating can act as an anchoring layer for the subsequent casting of drug-loaded polyester films allowing the controlled release of antiproliferative agents for the treatment of in-stent restenosis.

Poly (D,L-lactic acid) macroporous guidance scaffolds seeded with Schwann cells genetically modified to secrete a bi-functional neurotrophin implanted in the completely transected adult rat thoracic spinal cord.

Freeze-dried poly(D,L-lactic acid) macroporous scaffold filled with a fibrin solution containing Schwann cells (SCs) lentivirally transduced to produce and secrete D15A, a bi-functional neurotrophin with brain-derived neurotrophic factor and neurotrophin-3 activity, and to express green fluorescent protein (GFP) were implanted in the completely transected adult rat thoracic spinal cord. Control rats were similarly injured and then implanted with scaffolds containing the fibrin solution with SCs lentivirally transduced to produce express GFP only or with the fibrin solution only. Transgene production and biological activity in vitro, SC survival within the scaffold in vitro and in vivo, scaffold integration, axonal regeneration and myelination, and hind limb motor function were analyzed at 1, 2, and 6 weeks after implantation. In vitro, lentivirally transduced SCs produced 87.5 ng/24 h/10(6) cells of D15A as measured by neurotrophin-3 activity in ELISA. The secreted D15A was biologically active as evidenced by its promotion of neurite outgrowth of dorsal root ganglion neurons in culture. In vitro, SCs expressing GFP were present in the scaffolds for up to 6 h, the end of a typical surgery session. Implantation of SC-seeded scaffolds caused modest loss of spinal nervous tissue. Reactive astrocytes and chondroitin sulfate glycosaminoglycans were present in spinal tissue adjacent to the scaffold. Vascularization of the scaffold was ongoing at 1 week post-implantation. There were no apparent differences in scaffold integration and blood vessel formation between groups. A decreasing number of implanted (GFP-positive) SCs were found within the scaffold during the first 3 days after implantation. Apoptosis was identified as one of the mechanisms of cell death. At 1 week and later time points after implantation, few of the implanted SCs were present in the scaffold. Neurofilament-positive axons were found in the scaffold. At 6 weeks post-grafting, myelinated axons were observed within and at the external surface of the scaffold. Axons did not grow from the scaffold into the caudal cord. All groups demonstrated a similar improvement of hind limb motor function. Our findings demonstrated that few seeded SCs survived in vivo, which could account for the modest axonal regeneration response into and across the scaffold. For the development of SC-seeded macroporous scaffolds that effectively promote axonal regeneration in the injured spinal cord, the survival and/or total number of SCs in the scaffold needs to be improved.

In vitro and in vivo analysis of macroporous biodegradable poly(D,L-lactide-co-glycolide) scaffolds containing bioactive glass.

Recent studies have demonstrated the angiogenic potential of 45S5 Bioglass. However, it is not known whether the angiogenic properties of Bioglass remain when the bioactive glass particles are incorporated into polymer composites. The objectives of the current study were to investigate the angiogenic properties of 45S5 Bioglass particles incorporated into biodegradable polymer composites. In vitro studies demonstrated that fibroblasts cultured on discs consisting of specific quantities of Bioglass particles mixed into poly(D,L-lactide-co-glycolide) secreted significantly increased quantities of vascular endothelial growth factor. The optimal quantity of Bioglass particles determined from the in vitro experiments was incorporated into three-dimensional macroporous poly(D,L-lactide-co-glycolide) foam scaffolds. The foam scaffolds were fabricated using either compression molding or thermally induced phase separation processes. The foams were implanted subcutaneously into mice for periods of up to 6 weeks. Histological assessment was used to determine the area of granulation tissue around the foams, and the number of blood vessels within the granulation tissue was counted. The presence of Bioglass particles in the foams produced a sustained increase in the area of granulation tissue surrounding the foams. The number of blood vessels surrounding the neat foams was reduced after 2 weeks of implantation; however, compression-molded foams containing Bioglass after 4 and 6 weeks of implantation had significantly more blood vessels surrounding the foams compared with foams containing no Bioglass at the same time points. These results indicate that composite polymer foam scaffolds containing Bioglass particles retain granulation tissue and blood vessels surrounding the implanted foams. The use of this polymer composite for tissue engineering scaffolds might provide a novel approach for ensuring adequate vascular supply to the implanted device.

In vivo characterisation of a novel bioresorbable poly(lactide-co-glycolide) tubular foam scaffold for tissue engineering applications.

Polylactide-co-glycolide (PLGA) foams of tubular shape were assessed for their use as soft-tissue engineering scaffolds in vitro and in vivo. Porous membranes were fabricated by a thermally induced phase separation process of PLGA solutions in dimethylcarbonate. The parameters investigated were the PLGA concentration and the casting volume of solution. Membranes produced from 5 wt/v % polymer solutions and a 6 ml casting volume of polymer solution were selected for fabricating tubes of 3 mm diameter, 20 mm length and a nominal wall thickness of 1.5 mm. Scanning electron microscopy revealed that the structure of the tubular foams consisted of radially oriented and highly interconnected pores with a large size distribution (50-300 microm). Selected tubes were implanted subcutaneously into adult male Lewis rats. Although the lumen of the tubes collapsed within one week of implantation, histological examination of the implanted scaffolds revealed that the foam tubes were well tolerated. Cellular infiltration into the foams, consisting mainly of fibrovascular tissue, was evident after two weeks and complete within eight weeks of implantation. The polymer was still evident in the scaffolds after eight weeks of implantation. The results from this study demonstrate that the PLGA tubular foams may be useful as soft-tissue engineering scaffolds with modification holding promise for the regeneration of tissues requiring a tubular shape scaffold such as intestine.

Characterisation of bioactive and resorbable polylactide/Bioglass composites by FTIR spectroscopic imaging.

Formation, size and distribution of hydroxyapatite domains in resorbable composites made of poly(DL-lactide) foams and Bioglass particles after exposure to a solution of phosphate-buffer saline (PBS) for different periods of time have been analysed with FTIR imaging using the micro-ATR-IR approach. The spectral information of 4096 spectra measured simultaneously with the IR microscope equipped with a focal plane infrared array detector allowed us to obtain chemical images showing the distribution of Bioglass particles in the composites. FTIR imaging in micro-ATR mode allowed to obtain images with enhanced spatial resolution. A random distribution of hydroxyapatite domains with average size of ca. 10 microm on the surface of the composites was found after exposure to PBS for 14 and 28 days. The further growth of the hydroxyapatite domains after exposure to PBS for 63 days was detected. The spectroscopic imaging method introduced here promises to become a powerful method for characterisation of resorbable polymer composites containing bioactive inorganic phases developed for bone tissue engineering scaffolds. The accurate detection of hydroxyapatite domains and the imaging of their location in the scaffold structure is required to provide an assessment of the composites bioactivity.

Tissue engineering of biphasic cartilage constructs using various biodegradable scaffolds: an in vitro study.

Biological restoration of osteochondral defects requires suitable subchondral support material that also allows the induction of hyaline cartilage tissue. Biphasic implants consisting of pre-fabricated neocartilage and an underlying biodegradable osteoconductive base may meet these requirements. Here we explore various candidate biodegradable support materials onto which neo-cartilage was produced in vitro. Porcine chondrocytes were seeded in a closed and static bioreactor with a base of biomaterial consisting of either poly-L-lactide [P(L)LA], poly-d,l-lactide [P(D,L)LA] or Collagen-hydroxyapatite [Col-HA] and were cultured for 15 weeks. Viable neo-cartilage was produced on each biomaterial with differing amounts of cellular colonisation. P(D,L)LA breakdown was more rapid and uneven among the three biomaterials, leading to constructs of irregular shape. Little or no breakdown or chondrocyte colonisation was evident in P(L)LA. Col-HA constructs were superior in terms of viability, implant morphology and integration between neo-cartilage and biomaterial. These results indicate that our reported system has potential for producing biphasic implants that may be adequate for the repair of osteochondral defects.

PDLLA/Bioglass composites for soft-tissue and hard-tissue engineering: an in vitro cell biology assessment.

The aim of this study was to examine the effect of increased content of 45S5 Bioglass (0-40 wt%) in poly(dl-lactic acid) (PDLLA) porous foams on the behaviour of MG-63 (human osteosarcoma cell line) and A549 cells (human lung carcinoma cell line). The ability of these cell lines to grow on bioactive composites was quantitatively investigated in order to assess the potentiality for their use in hard and soft-tissue engineering. Two hours after cell seeding, an increase of cell adhesion according to the increased content of Bioglass((R)) present in the foams for both cell types was observed. Cell proliferation studies performed over a period of 4 weeks showed a better aptitude of the A549 cells to proliferate on PDLLA foams containing 5 wt% Bioglass when compared to the proliferation on foams with 40 wt% Bioglass. A lower proliferation rate was obtained for cells on pure PDLLA. Scanning electron microscopy analysis showed for both cell types the presence of cells inside the porous structure of the foams. These results confirmed the biocompatibility of PDLLA/Bioglass composite foams and the positive effect of Bioglass on MG-63 cell behaviour and also showed for the first time the possibility for human lung epithelial type II cells to adhere and proliferate on these porous scaffolds. In addition, we describe a positive effect of 45S5 Bioglass on A549 cell behaviour in a dose-dependent manner, indicating the potential of using PDLLA/Bioglass composites with an optimal concentration of 45S5 Bioglass not only in bone tissue engineering but also in lung tissue engineering.

Freeze-dried poly(D,L-lactic acid) macroporous guidance scaffolds impregnated with brain-derived neurotrophic factor in the transected adult rat thoracic spinal cord.

The effects of poly(D,L-lactic acid) macroporous guidance scaffolds (foams) with or without brain-derived neurotrophic factor (BDNF) on tissue sparing, neuronal survival, axonal regeneration, and behavioral improvements of the hindlimbs following implantation in the transected adult rat thoracic spinal cord were studied. The foams were embedded in fibrin glue containing acidic-fibroblast growth factor. One group of animals received fibrin glue with acidic-fibroblast growth factor only. The foams were prepared by a thermally induced polymer-solvent phase separation process and contained longitudinally oriented macropores connected to each other by a network of micropores. Both foams and fibrin only resulted in a similar gliotic and inflammatory response in the cord-implant interfaces. With BDNF foam, up to 20% more NeuN-positive cells in the spinal nervous tissue close to the rostral but not caudal spinal cord-implant interface survived than with control foam or fibrin only at 4 and 8 weeks after implantation. Semithin plastic sections and electron microcopy revealed that cells and axons more rapidly invaded BDNF foam than control foam. Also, BDNF foam contained almost twice as many blood vessels than control foam at 8 weeks after implantation. Tissue sparing was similar in all three implantation paradigms; approximately 42% of tissue was spared in the rostral cord and approximately 37% in the caudal cord at 8 weeks post grafting. The number of myelinated and unmyelinated axons was low and not different between the two types of foams. Many more axons were found in the fibrin only graft. Serotonergic axons were not found in any of the implants and none of the axons regenerated into the caudal spinal cord. The behavioral improvements in the hindlimbs were similar in all groups. These findings indicated that foam is well tolerated within the injured spinal cord and that the addition of BDNF promotes cell survival and angiogenesis. However, the overall axonal regeneration response is low. Future research should explore the use of poly(D,L-lactic acid) foams, with or without axonal growth-promoting factors, seeded with Schwann cells to enhance the axonal regeneration and myelination response.