A randomized clinical trial for the assessment of the efficacy and safety of guluronic acid (G2013) in patients with rheumatoid arthritis.

Objective: To evaluate the safety, efficacy and tolerability of guluronic acid (G2013) in order to treat the rheumatoid arthritis patients who had inadequate response to conventional drugs. Methods: A randomized, 12-week clinical trial with two treatment arms: guluronic acid (G2013) and conventional treatment was performed. The diagnosed RA patients according to the ACR/European League against Rheumatism 2010 classification criteria, with an active disease at baseline that had inadequate response to conventional therapy were considered for the study. G2013 was administered orally twice a day with capsules of 500 mg during a period of 12 weeks and the patients were followed up for the safety and efficacy. Results: Our data showed that, the mean changes in the G2013 and control groups were -7.54 and -2.5 for tender joint count; -7.59 and -3.59 for swollen joint count; -30 and -0.9 for physician global assessment; -23.18 and -1.81 for patient global assessment; -14.45 and -1.45 for erythrocyte sedimentation rate, respectively. Improvements seen with G2013 were significantly greater than those with conventional drugs. In total, in 15.3% of G2013-treated patients and 69.2% of conventional-treated patients adverse events (AEs) occurred in this study. Conclusion: These data from routine rheumatology clinical practice highlight the effectivenessof G2013 in combination with conventional therapy with more desirable safety profile compared to the conventional-treated patients. Therefore, G2013 therapy could be an appropriate choice in order to manage the RA disease. (Clinical trial identifier: IRCT2016092813739N5).

Randomized controlled trial of steroid-soaked absorbable calcium alginate nasal packing following endoscopic sinus surgery.

OBJECTIVES/HYPOTHESIS: To evaluate the potential efficacy of steroid-soaked, absorbable calcium alginate nasal packing following endoscopic sinus surgery. STUDY DESIGN: Prospective, randomized, single-blinded, placebo-controlled trial. METHODS: Twenty-two patients (44 nostrils) who had chronic rhinosinusitis with polyps underwent bilateral endoscopic sinus surgery. Only those with an intersinus difference in Lund-Mackay severity score of 1 or less were included. In each patient, one randomly selected nostril was packed with calcium alginate soaked with 2 mL of triamcinolone (40 mg/mL) (triamcinolone group), whereas the contralateral nostril received an identical packing soaked in 2 mL of normal saline (saline group). Two independent investigators blinded to the packing allocation scored the surgical field using the validated Perioperative Sinus Endoscopy (POSE) scores 1, 4, and 8 weeks after surgery. RESULTS: All 44 nostrils were analyzed; the Lund-Mackay scores did not differ significantly between the groups before surgery. Eight weeks after surgery, the total POSE scores were significantly lower in the triamcinolone group (P = .014). The POSE scoring parameters were then compared between groups, and the following variables were significantly different: middle turbinate synechiae with the lateral wall (P = .037), polypoid degeneration of the ethmoid cavity (P = .006), and sphenoid sinus severity (P = .036). CONCLUSIONS: This study demonstrated that steroid-soaked, absorbable nasal packing can be used to enhance wound healing after endoscopic sinus surgery and to prevent polypoid changes in the nasal mucosa. LEVEL OF EVIDENCE: 1b. Laryngoscope, 128:311-316, 2018.

Reactogenicity and safety assessment of an attenuated nanovaccine against scorpion envenomation: Preclinical study.

An attenuated nanovaccine (Nps - V∗) has been developed to protect humans from fatal scorpion envenomation in at-risk regions. This study was conducted to evaluate the toxicity and the local reactogenicity of the Nps - V∗ nanovaccine developed against Androctonus australis hector (Aah) venom. Assessment of the systemic inflammatory response and serum cytokine levels were evaluated in vaccinated mice with 100µg of irradiated Aah venom (V∗) encapsulated or not into polymeric calcium-alginate nanoparticles (Nps) and injected by subcutaneous (s.c) route. The local reactogenicity was evaluated by dermal Draize observations and skin tissue analysis at the injection site of vaccinated rabbits with 250 or 500µg of V∗-loaded into Nps. All animals gained weight and had normal food consumption during the study. Additionally, results showed that the nanoformulation Nps - V∗ did not cause clinical evidence of systemic toxicity in mice or rabbits, a transient edema/erythema at the injection site was only recorded as treatment-related reactogenicity. These results indicated a favorable safety profile for Nps - V∗ and supported its use in superior animal tests, then in a Phase 1 clinical trial.

Drug-Loadable Calcium Alginate Hydrogel System for Use in Oral Bone Tissue Repair.

This study developed a drug-loadable hydrogel system with high plasticity and favorable biological properties to enhance oral bone tissue regeneration. Hydrogels of different calcium alginate concentrations were prepared. Their swelling ratio, degradation time, and bovine serum albumin (BSA) release rate were measured. Human periodontal ligament cells (hPDLCs) and bone marrow stromal cells (BMSCs) were cultured with both calcium alginate hydrogels and polylactic acid (PLA), and then we examined the proliferation of cells. Inflammatory-related factor gene expressions of hPDLCs and osteogenesis-related gene expressions of BMSCs were observed. Materials were implanted into the subcutaneous tissue of rabbits to determine the biosecurity properties of the materials. The materials were also implanted in mandibular bone defects and then scanned using micro-CT. The calcium alginate hydrogels caused less inflammation than the PLA. The number of mineralized nodules and the expression of osteoblast-related genes were significantly higher in the hydrogel group compared with the control group. When the materials were implanted in subcutaneous tissue, materials showed favorable biocompatibility. The calcium alginate hydrogels had superior osteoinductive bone ability to the PLA. The drug-loadable calcium alginate hydrogel system is a potential bone defect reparation material for clinical dental application.

Preclinical and pharmacotoxicology evaluation of α-l-guluronic acid (G2013) as a non-steroidal anti-inflammatory drug with immunomodulatory property.

CONTEXT: Therapeutic effects of α-l-guluronic acid with the greatest tolerability and efficacy (G2013) have been shown in experimental model of multiple sclerosis and other in vitro and in vivo examinations regarding α-l-guluronic acid; there are no toxicological researches on its safety although the pharmacological impacts have been recorded. OBJECTIVE: This study was designed to determine the acute and sub chronic toxicity of α-l-guluronic acid in healthy male and female BALB/c mice. MATERIALS AND METHODS: For the acute toxicity study, the animals orally received five different single doses of α-l-guluronic acid and were kept under observation for 14 d. In the sub-chronic study, 24 male and female BALB/c mice were divided into four groups and treated daily with test substance preparation at dose levels of 0, 50, 250, and 1250 mg/kg body weight for at least 90 consecutive days. The mortality, body weight changes, clinical signs, hematological and biochemical parameters, gross findings, histopathological, and organs weight determinants were monitored during this study. RESULTS: The results of acute toxicity indicated that the LD50 of α-l-guluronic acid is 4.8 g/kg. We found no mortality or abnormality in clinical signs, body weight, relative organs weight, or necropsy in any of the animals in the subchronic study. Additionally, the results showed no significant difference in hematological, biochemical, and histopathological parameters in rats. CONCLUSIONS: Our results suggest that α-l-guluronic acid has high safety when administered orally in animals.

Immunological Challenges Facing Translation of Alginate Encapsulated Porcine Islet Xenotransplantation to Human Clinical Trials.

Transplantation of alginate-encapsulated islets has the potential to treat patients suffering from type I diabetes, a condition characterized by an autoimmune attack against insulin-secreting beta cells. However, there are multiple immunological challenges associated with this procedure, all of which must be adequately addressed prior to translation from trials in small animal and nonhuman primate models to human clinical trials. Principal threats to graft viability include immune-mediated destruction triggered by immunogenic alginate impurities, unfavorable polymer composition and surface characteristics, and release of membrane-permeable antigens, as well as damage associated molecular patterns (DAMPs) by the encapsulated islets themselves. The lack of standardization of significant parameters of bioencapsulation device design and manufacture (i.e., purification protocols, surface-modification grafting techniques, alginate composition modifications) between labs is yet another obstacle that must be overcome before a clinically effective and applicable protocol for encapsulating islets can be implemented. Nonetheless, substantial progress is being made, as is evident from prolonged graft survival times and improved protection from immune-mediated graft destruction reported by various research groups, but also with regard to discoveries of specific pathways involved in explaining observed outcomes. Progress in the latter is essential for a comprehensive understanding of the mechanisms responsible for the varying levels of immunogenicity of certain alginate devices. Successful translation of encapsulated islet transplantation from in vitro and animal model testing to human clinical trials hinges on application of this knowledge of the pathways and interactions which comprise immune-mediated rejection. Thus, this review not only focuses on the different factors contributing to provocation of the immune reaction by encapsulated islets, but also on the defining characteristics of the response itself.

In Vivo Assessment of Bone Regeneration in Alginate/Bone ECM Hydrogels with Incorporated Skeletal Stem Cells and Single Growth Factors.

The current study has investigated the use of decellularised, demineralised bone extracellular matrix (ECM) hydrogel constructs for in vivo tissue mineralisation and bone formation. Stro-1-enriched human bone marrow stromal cells were incorporated together with select growth factors including VEGF, TGF-ß3, BMP-2, PTHrP and VitD3, to augment bone formation, and mixed with alginate for structural support. Growth factors were delivered through fast (non-osteogenic factors) and slow (osteogenic factors) release PLGA microparticles. Constructs of 5 mm length were implanted in vivo for 28 days within mice. Dense tissue assessed by micro-CT correlated with histologically assessed mineralised bone formation in all constructs. Exogenous growth factor addition did not enhance bone formation further compared to alginate/bone ECM (ALG/ECM) hydrogels alone. UV irradiation reduced bone formation through degradation of intrinsic growth factors within the bone ECM component and possibly also ECM cross-linking. BMP-2 and VitD3 rescued osteogenic induction. ALG/ECM hydrogels appeared highly osteoinductive and delivery of angiogenic or chondrogenic growth factors led to altered bone formation. All constructs demonstrated extensive host tissue invasion and vascularisation aiding integration and implant longevity. The proposed hydrogel system functioned without the need for growth factor incorporation or an exogenous inducible cell source. Optimal growth factor concentrations and spatiotemporal release profiles require further assessment, as the bone ECM component may suffer batch variability between donor materials. In summary, ALG/ECM hydrogels provide a versatile biomaterial scaffold for utilisation within regenerative medicine which may be tailored, ultimately, to form the tissue of choice through incorporation of select growth factors.

Nanoparticles based on oleate alginate ester as curcumin delivery system.

Hydrophobic alginate derivative was prepared by the modification of alginate with methyl oleate. The synthesized oleate alginate ester (OAE) conjugate was characterized by FTIR and (1)HNMR analysis. Results of critical aggregation concentration (CAC) revealed that OAE conjugate had low CAC and was prone to form self-assembled nanoparticles in aqueous medium. Curcumin loaded OAE nanoparticles (Cur-OAE Nps) were developed by a simple sonication method and the physicochemical parameters of the nanoparticles such as zeta potential, size distribution and drug encapsulation were characterized. The results showed that zeta potential of the prepared nanoparticles was -55.4±0.91 mV and the average size was about 200 nm. A significant enhancement in aqueous solubility and stability of curcumin were observed after encapsulation into OAE nanoparticles. With the increase of curcumin concentration, loading efficiency increased but encapsulation efficiency decreased. The in vitro release profile exhibited significant sustained release pattern with initial burst release followed by a slower release over a period of one week. Cytotoxicity assay against MCF-7cells showed that Cur-OAE Nps had slow and continuous cytotoxic effect. Furthermore, in vitro cell uptake study revealed that cell uptake of curcumin from OAE nanoparticles was sustained and both were time and concentration dependent. Therefore, the developed Cur-OAE Nps might be promising candidates for curcumin delivery to cancer cells.

Development and characterization of alginate coated low molecular weight chitosan nanoparticles as new carriers for oral vaccine delivery in mice.

In the present study, nanoparticles of low MW chitosan (CS) were formulated in which measles antigen was entrapped and subsequently coated with sodium alginate. The size and surface properties of the nanoparticle can be tuned with different MW of CS. In vitro release studies showed initial burst release followed by extended release, best fitted in the Makoid-Banakar model (R(2)>0.98). SDS-PAGE assay revealed that alginate coating could effectively protect antigen in acidic condition for at least 2h. Cell viability was assessed using MTT assay into HT 29 cell line. Formulations were orally administered to mice and immunological responses were evaluated using ELISA method. Obtained results showed that measles antigen-loaded CS nanoparticles induced strong immune response and significant correlation was observed between the immune response with CS MW. Protecting ability of antigen in gastric environment, sustained release kinetics, systemic and mucosal immune responses and low cytotoxicity observed for the alginate coated nanoparticles demonstrated that LMW CS could be promising platform for oral vaccine delivery.

Dual responsive dysprosium-doped hydroxyapatite particles and toxicity reduction after functionalization with folic and glucuronic acids.

The development of probes for biomedical applications demands materials with low toxicity levels besides fluorescence or magnetic properties to be detected by confocal microscopes or MRI resonators. Several drug delivery systems or other biomedical materials prepared with hydroxyapatite have been proposed, however, toxicity effects might arise when the size of particles is nanometric. In this study, hydroxyapatite functionalized with glucuronic or folic acids presented lower oxidative stress, measured from lipoperoxides and nitric oxide indicators in rats than pure hydroxyapatite. In separated experiments, hydroxyapatite was doped with dysprosium cations by coprecipitation producing a single crystal phase with fluorescent properties easily visualized by confocal microscopy when excited at 488nm. These particles also presented the ability to modify the proton relaxation time in T1 maps collected by magnetic resonance imaging. These modified hydroxyapatite nanoparticles could be candidates to design bimodal probes with low toxicity.

Adverse effects of fillers and their histopathology.

Injectable fillers nowadays represent a pillar in facial rejuvenation and make a significant contribution to the success of the treatment. Despite their obvious benefits, a wide range of possible complications such as immediate, late, delayed, temporary, or irreversible adverse effects have to be respected. Differentiating the various filler materials, these effects are assigned to histopathology findings and currently available treatment options.

Intracoronary delivery of injectable bioabsorbable scaffold (IK-5001) to treat left ventricular remodeling after ST-elevation myocardial infarction: a first-in-man study.

BACKGROUND: We aimed to test, for the first time, the feasibility of intracoronary delivery of an innovative, injectable bioabsorbable scaffold (IK-5001), to prevent or reverse adverse left ventricular remodeling and dysfunction in patients after ST-segment-elevation myocardial infarction. METHODS AND RESULTS: Patients (n=27) with moderate-to-large ST-segment-elevation myocardial infarctions, after successful revascularization, were enrolled. Two milliliters of IK-5001, a solution of 1% sodium alginate plus 0.3% calcium gluconate, was administered by selective injection through the infarct-related coronary artery within 7 days after myocardial infarction. IK-5001 is assumed to permeate the infarcted tissue, cross-linking into a hydrogel and forming a bioabsorbable cardiac scaffold. Coronary angiography, 3 minutes after injection, confirmed that the injection did not impair coronary flow and myocardial perfusion. Furthermore, IK-5001 deployment was not associated with additional myocardial injury or re-elevation of cardiac biomarkers. Clinical assessments, echocardiographic studies, 12-lead electrocardiograms, 24-hour Holter monitoring, blood tests, and completion of Minnesota Living with Heart Failure Questionnaires were repeated during follow-up visits at 30, 90, and 180 days after treatment. During a 6-month follow-up, these tests confirmed favorable tolerability of the procedure, without device-related adverse events, serious arrhythmias, blood test abnormalities, or death. Serial echocardiographic studies showed preservation of left ventricular indices and left ventricular ejection fraction. CONCLUSIONS: This first-in-man pilot study shows that intracoronary deployment of an IK-5001 scaffold is feasible and well tolerated. Our results have promoted the initiation of a multicenter, randomized controlled trial to confirm the safety and efficacy of this new approach in high-risk patients after ST-segment-elevation myocardial infarction. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01226563.

Alginate gelation-induced cell death during laser-assisted cell printing.

Modified laser-induced forward transfer has emerged as a promising bioprinting technique. Depending on the operating conditions and cell properties, laser cell printing may cause cell injury and even death, which should be carefully elucidated for it to be a viable technology. This study has investigated the effects of alginate gelation, gelation time, alginate concentration, and laser fluence on the post-transfer cell viability of NIH 3T3 fibroblasts. Sodium alginate and calcium chloride are used as the gel precursor and gel reactant solution to form cell-laden alginate microspheres. It is found that the effects of gelation depend on the duration of gelation. Two-minute gelation is observed to increase the cell viability after 24 h incubation, mainly due to the protective cushion effect of the forming gel membrane during droplet landing. Despite the cushion effect from 10 min gelation, it is observed that the cell viability decreases after 24 h incubation because of the forming thick gel membrane that reduces nutrient and oxygen diffusion from the culture medium. In addition, the cell viability after 24 h incubation decreases as the laser fluence or alginate concentration increases.

Microencapsulation of lectin anti-cancer agent and controlled release by alginate beads, biosafety approach.

Hepatocellular carcinoma (HCC) is considered as one of the most aggressive cancer worldwide. In Egypt, the prevalence of HCC is increasing during last years. Recently, drug-loaded microparticles were used to improve the efficiency of various medical treatments. This study is designed to evaluate the anticancer potentialities of lectins against HCC while hinting to its safety usage. The aim is also extended to encapsulate lectins in alginate microbeads for oral drug delivery purposes. The extracted lectins showed anti-proliferative effect against HCC with a percentage of 60.76% by using its nontoxic dose with an up-regulation of P53 gene expression. Concerning the handling of lectin alginate microbeads for oral drug delivery, the prepared lectin alginate beads were ∼100µm in diameter. The efficiency of the microcapsules was checked by scanning electron microscopy, the SEM showed the change on the alginate beads surface revealing the successful lectin encapsulation. The release of lectins from the microbeads depended on a variety of factors as the microbeads forming carriers and the amount-encapsulated lectins. The Pisum sativum extracted lectins may be considered as a promising agent in controlling HCC and this solid dosage form could be suitable for oral administration complemented with/or without the standard HCC drugs.

Control of multi-drug-resistant pathogens with non-thermal-plasma-treated alginate wound dressing.

BACKGROUND: Non-thermal dielectric-barrier discharge plasma (non-thermal plasma) is being investigated for use in wound healing. Alginate gel, already in clinical use, is non-toxic but has no meaningful antimicrobial property. This study reports that a non-thermal-plasma-treated alginate wound dressing has strong antimicrobial properties. METHODS: Alginate gel was treated with non-thermal plasma in room air and inoculated with bacterial pathogens. At 15 min after this, bacterial cell viability was determined by colony assay or 2,3-bis-(2-methoxy-4- nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. The anti-biofilm efficacy of the non-thermal-plasma-treated alginate gel was investigated and the treated gel was tested against vascular endothelial cells for a cytotoxic effect. The proliferation and migration of bacterial cells before and after exposure to the treated gel were investigated with an in vitro wound testing assay. Scanning electron microscopy was used to observe changes in the gel surface associated with exposure to bacterial pathogens. The treated gel was tested against Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, S. epidermidis, Candida albicans, and C. glabrata as representative pathogens (at 10(6)-10(9) colony-forming units [CFU]/mL), and the thickness of a plasma-treated gel dressing and distance between a glass dielectric-barrier discharge plasma probe and the gel surface were kept constant. RESULTS: Non-thermal-plasma-treated alginate gel exhibited a strong biocidal property and inactivated all of the pathogens included in the study at counts of 10(8) CFU/mL and within 15 sec of treatment. The treated gel inactivated 10(9) CFU/mL of the organisms within 1 min, and 3 min of exposure to the treated gel inactivated pathogens embedded in biofilms. The plasma-treated gel showed no significant cytotoxicity, and endothelial cells exposed to the treated gel proliferated and migrated well across a wound area over a period of time. Dressings made with the treated gel retained their biocidal effects for about a month. Scanning electron microscopy showed no damage to the surfaces of treated gels, but damage to the bacterial pathogens on plasma exposure. CONCLUSION: A non-thermal-plasma-treated alginate gel dressing has the clinical potential to decontaminate wounds, prevent surgical site infection, and promote wound healing.

[Comparative study of osteoplastic materials based on chitosan, alginate or fibrin with tricalcium phosphate].

The study presents comparative analysis of porous composites made of chitosan, alginate, fibrin with beta-tricalcium phosphate. Histological findings on Wistar rat condyles showed that fibrin-beta-TCP-based composite had the most effective positive biological response.

Vacuum-assisted wound closure versus alginate for the treatment of deep perivascular wound infections in the groin after vascular surgery.

BACKGROUND: Vacuum-assisted wound closure (VAC) therapy may heal wounds faster than conventional dressings after surgical debridement of perivascular groin infections after vascular surgery. METHODS: Patients with deep infected wounds (Szilagyi grade III) were surgically revised and left open for secondary healing, then randomized to either VAC or alginate (Sorbalgon) therapy, between February 2007 and November 2011. To test the hypothesis, it was calculated that 42 patients needed to be included (90% power, 5% level of significance). It was decided to perform an interim analysis after inclusion of 20 patients. RESULTS: Among 66 patients undergoing groin revision, 20 patients were included in this study. Patients were randomized to VAC (n = 10) or alginate (n = 10). The two groups were comparable in patient and wound characteristics. Time to full skin epithelialization was significantly shorter in the VAC group (median, 57 days) compared with the alginate group (median, 104 days; P = .026). The number of positive wound cultures of bacteria and C-reactive protein values decreased equally in both groups between surgical revision and day 21. One femur amputation was performed in each group as a consequence of the groin infection, one patient died during the in-hospital stay in the alginate group, and none died in the VAC group. CONCLUSIONS: VAC achieves faster healing than alginate therapy after wound debridement for deep perivascular wound infections in the groin after vascular surgery. This finding does not allow further inclusion of patients from an ethical point of view, and this study was, therefore, stopped prematurely.

Gastroesophageal reflux in pregnancy: a systematic review on the benefit of raft forming agents.

The prevalence of gastroesophageal reflux disease (GERD) symptoms in pregnancy is very high, up to 80%, with a maximum peak during the third trimester. Together with lifestyle modifications, antacids and antisecretive agents, such as proton pump inhibitors (PPIs) and histamine H2 receptor antagonists (H2RAs), are commonly prescribed in non-pregnant, adult population. In certain Countries these drugs are not allowed in or are allowed only during the late stages of pregnancy. Alginate-based formulations have been used for the symptomatic treatment of heartburn for decades, as they usually contain sodium or potassium bicarbonate. In the presence of gastric acid, a foamy raft is created above the gastric contents. The alginate raft moves into the esophagus in place or ahead of acidic gastric contents during reflux episodes physically preventing reflux of gastric contents into the esophagus. Alginate-based formulations are allowed with no restrictions also in pregnancy: their safety profile make them a very valid option taking into account the risk/benefit ratio for both parturient and unborn baby. This systematic review paper aims to explore the use of medications for treating GERD in pregnancy, including alginate raft-forming-agents, highlighting the benefits for both the mother and the fetus. Electronic search in databases was conducted on databases such as Medline, PubMed, Ovid retrieving data concerning the reflux treatments in pregnancy, with a special focus on alginate raft forming antireflux agents. From the literature on alginate use in pregnancy, no particular risks have been shown to date for both parturient and unborn baby when alginate had been administered during all the pregnancy trimesters. The physical mode of action ensures the maximum esophageal protection by the neutral foam floating in the stomach, maintaining physiological pH values at stomach level, without interfering with the digestive processes. The symptoms' healing has been markedly improved during the weeks of observation; the symptoms monitored in all studies were: heartburn, regurgitation, pain (chest). After four weeks of treatment little or no change was observed in maternal mean sodium or potassium concentrations. No sodium restriction diet has been adopted. No edema of lower limbs or weight gain occurred. No adverse reactions related to the testing drug had been reported and all the authors concluded that alginate was safe for the unborn baby. Nowadays pharmacological treatments for GER are available as OTC drugs, including antacids, antisecretive agents, PPIs and H2RAs, and as medical devices, such as alginate raft forming antireflux agents (i.e.: Reflubloc™, Novartis NCH Italy). On this last product, considering the specific indication in pregnancy and the safety profile, without restrictions of administration during the whole pregnancy period, furthermore the physical mode of action, it gives the gynecologists a very important option in treating GER in pregnancy, taking care of both pregnant and fetus. Raft-forming-antireflux agents are safe and effective in GER treatment during pregnancy.

The role of pathogen-associated molecular patterns in inflammatory responses against alginate based microcapsules.

Alginate-based microcapsules are used for immunoisolation of cells to release therapeutics on a minute-to-minute basis. Unfortunately, alginate-based microcapsules are suffering from varying degrees of success, which is usually attributed to differences in tissue responses. This results in failure of the therapeutic cells. In the present study we show that commercial, crude alginates may contain pathogen-associated molecular patterns (PAMPs), which are recognized by the sensors of the innate immune system. Known sensors are Toll-like receptors (TLRs), NOD receptors, and C-type lectins. By using cell-lines with a non-functional adaptor molecule essential in Toll-like receptor signaling, i.e. MyD88, we were able to show that alginates signal mainly via MyD88. This was found for low-G, intermediate-G, and high-G alginates applied in calcium-beads, barium-beads as well as in alginate-PLL-alginate capsules. These alginates did stimulate TLRs 2, 5, 8, and 9 but not TLR4 (LPS receptor). Upon implantation in rats these alginates provoked a strong inflammatory response resulting in fibrosis of the capsules. Analysis demonstrated that commercial alginates contain the PAMPs peptidoglycan, lipoteichoic acid, and flagellin. By applying purification procedures, these PAMPs were largely removed. This was associated with deletion of the inflammatory tissue responses as confirmed by an implantation experiment in rats. Our data also show that alginate itself does not provoke TLR mediated responses. We were able to unravel the sensor mechanism by which contaminants in alginates may provoke inflammatory responses.

Design and characterization of core-shell mPEG-PLGA composite microparticles for development of cell-scaffold constructs.

Appropriate scaffolds capable of providing suitable biological and structural guidance are of great importance to generate cell-scaffold constructs for cell-based tissue engineering. The aim of the present study was to develop composite microparticles with a structure to provide functionality as a combined drug delivery/scaffold system. Composite microparticles were produced by incorporating either alginate/dermatan sulfate (Alg/DS) or alginate/chitosan/dermatan sulfate (Alg/CS/DS) particles in mPEG-PLGA microparticles using coaxial ultrasonic atomization. The encapsulation and distribution of Alg/DS or Alg/CS/DS particles in the mPEG-PLGA microparticles were significantly dependent on the operating conditions, including the flow rate ratio (Qout/Qin) and the viscosity of the polymer solutions (Vout, Vin) between the outer and the inner feeding channels. The core-shell composite microparticles containing the Alg/DS particles or the Alg/CS/DS particles displayed 40% and 65% DS release in 10 days, respectively, as compared to the DS directly loaded microparticles showing 90% DS release during the same time interval. The release profiles of DS correlate with the cell proliferation of fibroblasts, i.e. more sustainable cell growth was induced by the DS released from the core-shell composite microparticles comprising Alg/CS/DS particles. After seeding fibroblasts onto the composite microparticles, excellent cell adhesion was observed, and a successful assembly of the cell-scaffold constructs was induced within 7 days. Therefore, the present study demonstrates a novel strategy for fabrication of core-shell composite microparticles comprising additional particulate drug carriers in the core, which provides controlled delivery of DS and favorable cell biocompatibility; an approach to potentially achieve cell-based tissue regeneration.