Highly Concentrated Stabilized Hybrid Complexes of Hyaluronic Acid: Rheological and Biological Assessment of Compatibility with Adipose Tissue and Derived Stromal Cells towards Regenerative Medicine.

Cells and extracts derived from adipose tissue are gaining increasing attention not only in plastic surgery and for aesthetic purposes but also in regenerative medicine. The ability of hyaluronan (HA) to support human adipose stromal cell (hASC) viability and differentiation has been investigated. However, the compatibility of adipose tissue with HA-based formulation in terms of biophysical and rheological properties has not been fully addressed, although it is a key feature for tissue integration and in vivo performance. In this study, the biophysical and biochemical properties of highly concentrated (45 mg/mL) high/low-molecular-weight HA hybrid cooperative complex were assessed with a further focus on the potential application in adipose tissue augmentation/regeneration. Specifically, HA hybrid complex rheological behavior was observed in combination with different adipose tissue ratios, and hyaluronidase-catalyzed degradation was compared to that of a high-molecular-weight HA (HHA). Moreover, the HA hybrid complex's ability to induce in vitro hASCs differentiation towards adipose phenotype was evaluated in comparison to HHA, performing Oil Red O staining and analyzing gene/protein expression of PPAR-γ, adiponectin, and leptin. Both treatments supported hASCs differentiation, with the HA hybrid complex showing better results. These outcomes may open new frontiers in regenerative medicine, supporting the injection of highly concentrated hybrid formulations in fat compartments, eventually enhancing residing staminal cell differentiation and improving cell/growth factor persistence towards tissue regeneration districts.

Multi-step Strategies Toward Regioselectively Sulfated M-Rich Alginates.

Sulfated alginates (ASs), as well as several artificially sulfated polysaccharides, show interesting bioactivities. The key factors for structure-activity relationships studies are the degree of sulfation and the distribution of the sulfate groups along the polysaccharide backbone (sulfation pattern). The former parameter can often be controlled through stoichiometry, while the latter requires the development of suitable chemical or enzymatic, regioselective methods and is still missing for ASs. In this work, a study on the regioselective installation of several different protecting groups on a d-mannuronic acid enriched (M-rich) alginate is reported in order to develop a semi-synthetic access to regioselectively sulfated AS derivatives. A detailed structural characterization of the obtained ASs revealed that the regioselective sulfation could be achieved complementarily at the O-2 or O-3 positions of M units through multi-step sequences relying upon a silylating or benzoylating reagent for the regioselective protection of M-rich alginic acid, followed by sulfation and deprotection.

Self-esterified hyaluronan hydrogels: Advancements in the production with positive implications in tissue healing.

Hyaluronan-(HA) short half-life in vivo limits its benefits in tissue repair. Self-esterified-HA is of great interest because it progressively releases HA, promoting tissue-regeneration longer than the unmodified-polymer. Here, the 1-ethyl-3-(3-diethylaminopropyl)carbodiimide(EDC)-hydroxybenzotriazole(HOBt) carboxyl-activating-system was evaluated for self-esterifying HA in the solid state. The aim was to propose an alternative to the time-consuming, conventional reaction of quaternary-ammonium-salts of HA with hydrophobic activating-systems in organic media, and to the EDC-mediated reaction, limited by by-product formation. Additionally, we aimed to obtain derivatives releasing defined molecular-weight(MW)-HA that would be valuable for tissue renewal. A 250 kDa-HA(powder/sponge) was reacted with increasing EDC/HOBt amounts. HA-modification was investigated through Size-Exclusion-Chromatography-Triple-Detector-Array-analyses, FT-IR/1H NMR and the products(XHAs) extensively characterized. Compared to conventional protocols, the set procedure is more efficient, avoids side-reactions, allows for an easier processing to diverse clinically-usable 3D-forms, leads to products gradually releasing HA under physiological conditions with the possibility to tune the MW of the biopolymer-released. Finally, the XHAs exhibit sound stability to Bovine-Testicular-Hyaluronidase, hydration/mechanical properties suitable for wound-dressings, with improvements over available matrices, and prompt in vitro wound-regeneration, comparably to linear-HA. To the best of our knowledge, the procedure is the first valid alternative to conventional protocols for HA self-esterification with advances in the process itself and in product performance.

Hyaluronan Hydrogels: Rheology and Stability in Relation to the Type/Level of Biopolymer Chemical Modification.

BDDE (1,4-butanediol-diglycidylether)-crosslinked hyaluronan (HA) hydrogels are widely used for dermo-aesthetic purposes. The rheology and stability of the gels under physiological conditions greatly affect their clinical indications and outcomes. To date, no studies investigating how these features are related to the chemistry of the polymeric network have been reported. Here, four available HA-BDDE hydrogels were studied to determine how and to what extent their rheology and stability with respect to enzymatic hydrolysis relate to the type and degree of HA structural modification. 1H-/13C-NMR analyses were associated for the quantification of the "true" HA chemical derivatization level, discriminating between HA that was effectively crosslinked by BDDE, and branched HA with BDDE that was anchored on one side. The rheology was measured conventionally and during hydration in a physiological medium. Sensitivity to bovine testicular hyaluronidase was quantified. The correlation between NMR data and gel rheology/stability was evaluated. The study indicated that (1) the gels greatly differed in the amounts of branched, crosslinked, and overall modified HA, with most of the HA being branched; (2) unexpectedly, the conventionally measured rheological properties did not correlate with the chemical data; (3) the gels' ranking in terms of rheology was greatly affected by hydration; (4) the rheology of the hydrated gels was quantitatively correlated with the amount of crosslinked HA, whereas the correlations with the total HA modification level and with the degree of branched HA were less significant; (5) increasing HA derivatization/crosslinking over 9/3 mol% did not enhance the stability with respect to hyaluronidases. These results broaden our knowledge of these gels and provide valuable information for improving their design and characterization.

Potential of Biofermentative Unsulfated Chondroitin and Hyaluronic Acid in Dermal Repair.

Chondroitin obtained through biotechnological processes (BC) shares similarities with both chondroitin sulfate (CS), due to the dimeric repetitive unit, and hyaluronic acid (HA), as it is unsulfated. In the framework of this experimental research, formulations containing BC with an average molecular size of about 35 KDa and high molecular weight HA (HHA) were characterized with respect to their rheological behavior, stability to enzymatic hydrolysis and they were evaluated in different skin damage models. The rheological characterization of the HHA/BC formulation revealed a G' of 92 ± 3 Pa and a G″ of 116 ± 5 Pa and supported an easy injectability even at a concentration of 40 mg/mL. HA/BC preserved the HHA fraction better than HHA alone. BTH was active on BC alone only at high concentration. Assays on scratched keratinocytes (HaCaT) monolayers showed that all the glycosaminoglycan formulations accelerated cell migration, with HA/BC fastening healing 2-fold compared to the control. In addition, in 2D HaCaT cultures, as well as in a 3D skin tissue model HHA/BC efficiently modulated mRNA and protein levels of different types of collagens and elastin remarking a functional tissue physiology. Finally, immortalized human fibroblasts were challenged with TNF-α to obtain an in vitro model of inflammation. Upon HHA/BC addition, secreted IL-6 level was lower and efficient ECM biosynthesis was re-established. Finally, co-cultures of HaCaT and melanocytes were established, showing the ability of HHA/BC to modulate melanin release, suggesting a possible effect of this specific formulation on the reduction of stretch marks. Overall, besides demonstrating the safety of BC, the present study highlights the potential beneficial effect of HHA/BC formulation in different damage dermal models.

Hyaluronan and Derivatives: An In Vitro Multilevel Assessment of Their Potential in Viscosupplementation.

In this research work, viscosupplements based on linear, derivatized, crosslinked and complexed HA forms were extensively examined, providing data on the hydrodynamic parameters for the water-soluble-HA-fraction, rheology, sensitivity to enzymatic hydrolysis and capacity to modulate specific biomarkers' expression in human pathological chondrocytes and synoviocytes. Soluble HA ranged from 0 to 32 mg/mL and from 150 to 1330 kDa MW. The rheological behavior spanned from purely elastic to viscoelastic, suggesting the diversity of the categories that are suitable for restoring specific/different features of the healthy synovial fluid. The rheological parameters were reduced in a diverse manner upon dilution and hyaluronidases action, indicating different durations of the viscosupplementation effect. Bioactivity was found for all the samples, increasing the expression of different matrix markers (e.g., hyaluronan-synthase); however, the hybrid cooperative complexes performed better in most of the experiments. Hybrid cooperative complexes improved COLII mRNA expression (~12-fold increase vs. CTR), proved the most effective at preserving cell phenotype. In addition, in these models, the HA samples reduced inflammation. IL-6 was down-regulated vs. CTR by linear and chemically modified HA, and especially by hybrid complexes. The results represent the first comprehensive panel of data directly comparing the diverse HA forms for intra-articular injections and provide valuable information for tailoring products' clinical use as well as for designing new, highly performing HA-formulations that can address specific needs.

Gelatin-biofermentative unsulfated glycosaminoglycans semi-interpenetrating hydrogels via microbial-transglutaminase crosslinking enhance osteogenic potential of dental pulp stem cells.

Gelatin hydrogels by microbial-transglutaminase crosslinking are being increasingly exploited for tissue engineering, and proved high potential in bone regeneration. This study aimed to evaluate, for the first time, the combination of enzymatically crosslinked gelatin with hyaluronan and the newly developed biotechnological chondroitin in enhancing osteogenic potential. Gelatin enzymatic crosslinking was carried out in the presence of hyaluronan or of a hyaluronan-chondroitin mixture, obtaining semi-interpenetrating gels. The latter proved lower swelling extent and improved stiffness compared to the gelatin matrix alone, whilst maintaining high stability. The heteropolysaccharides were retained for 30 days in the hydrogels, thus influencing cell response over this period. To evaluate the effect of hydrogel composition on bone regeneration, materials were seeded with human dental pulp stem cells and osteogenic differentiation was assessed. The expression of osteocalcin (OC) and osteopontin (OPN), both at gene and protein level, was evaluated at 7, 15 and 30 days of culture. Scanning electron microscopy (SEM) and two-photon microscope observations were performed to assess bone-like extracellular matrix (ECM) deposition and to observe the cell penetration depth. In the presence of the heteropolysaccharides, OC and OPN expression was upregulated and a higher degree of calcified matrix formation was observed. Combination with hyaluronan and chondroitin improved both the biophysical properties and the biological response of enzymatically crosslinked gelatin, fastening bone deposition.

Hyaluronan Hydrogels for Injection in Superficial Dermal Layers: An In Vitro Characterization to Compare Performance and Unravel the Scientific Basis of Their Indication.

BACKGROUND: Skinboosters represent the latest category of hyaluronan (HA) hydrogels released for aesthetic purposes. Different from originally developed gels, they are intended for more superficial injections, claiming a skin rejuvenation effect through hydration and possibly prompting biochemical effects in place of the conventional volumetric action. Here, three commercial skinboosters were characterized to unravel the scientific basis for such indication and to compare their performances. METHODS: Gels were evaluated for water-soluble/insoluble-HA composition, rheology, hydration, cohesivity, stability and effect, in vitro, on human dermal fibroblasts towards the production of extracellular matrix components. RESULTS: Marked differences in the insoluble-hydrogel amount and in the hydrodynamic parameters for water-soluble-HA chains were evidenced among the gels. Hydration, rigidity and cohesivity also varied over a wide range. Sensitivity to hyaluronidases and Reactive Oxygen Species was demonstrated allowing a stability ranking. Slight differences were found in gels' ability to prompt elastin expression and in ColIV/ColI ratio. CONCLUSIONS: A wide panel of biophysical and biochemical parameters for skinboosters was provided, supporting clinicians in the conscious tuning of their use. Data revealed great variability in gels' behavior notwithstanding the same clinical indication and unexpected similarities to the volumetric formulations. Data may be useful to improve customization of gel design toward specific uses.

Evaluation of the Volumizing Performance of a New Volumizer Filler in Volunteers with Age-Related Midfacial Volume Defects.

PURPOSE: The primary aim of this study was to evaluate the performance of the study product, in terms of volumizing activity as well as the duration of the effect, in women with age-related midfacial volume defects. In addition, the study allowed the evaluation of the tolerability of the product by both volunteers and investigators. PATIENTS AND METHODS: Twenty-two female volunteers, aged 42-60 years, participated in this study, which was performed under dermatological control in a single center. After an initial visit at baseline to verify adherence to the protocol criteria, volunteers received an injection of Aliaxin® SV (IBSA Farmaceutici Italia Srl), followed 3-4 weeks later by a second touch-up treatment to treat eventual asymmetries. Four subsequent visits, the last performed 9 months from the first injection, were performed to evaluate clinically and instrumentally the efficacy of the treatment. RESULTS: Clinical and statistically significant improvement in cheek volume was recorded after the first postinjection visit, and the effect was maintained until the end of the study period. A clinically measurable amelioration of wrinkle severity was also observed. By 3D picture recording and subsequent quantitative analysis, it was possible to determine the efficacy in terms of increased facial volume, which was already appreciable at the first visit, was further increased at the second and third visits and was maintained at the fourth and last visits. The injections were very well tolerated by the volunteers, as determined by their self-evaluation questionnaires. CONCLUSION: The results of the study confirm the esthetic performance of the study product on age-related midfacial volume defects. The very strong high-volumizing activity of the study product was not only properly determined by the investigators but also confirmed by self-evaluation by the volunteers. These effects were obtained with no appreciable undesired effects.

Corrigendum to "Novel Hybrid Gels Made of High and Low Molecular Weight Hyaluronic Acid Induce Proliferation and Reduce Inflammation in an Osteoarthritis In Vitro Model Based on Human Synoviocytes and Chondrocytes".

[This corrects the article DOI: 10.1155/2019/4328219.].

Hyaluronan Dermal Fillers: Efforts Towards a Wider Biophysical Characterization and the Correlation of the Biophysical Parameters to the Clinical Outcome.

INTRODUCTION: Hyaluronic Acid (HA) fillers are among the most used products in cosmetic medicine. Companies offer different formulations to allow full facial treatment and/or remodeling. Gels are being studied to establish the biophysical properties behind the specific clinical use and a correlation between the gel biophysical properties and their clinical performance. Clinicians' awareness is growing about the potential benefit deriving from such biophysical characterization. AIM: The Aliaxin® line of HA dermal fillers is the object of this study. The study aimed to widen the biophysical characterization of these gels by investigating a variety of properties to better support their optimal use. Further, we aimed to provide some clinical findings to gain a deeper insight into the correlation between filler features and clinical outcome. METHODS: The four gels of the line were investigated, for the first time, for their cohesivity and stability to Reactive Oxygen Species (ROS). Additional secondary rheological parameters; evidence of relative water-uptake ability; and some clinical findings on product safety, palpability and duration of the aesthetic effect are provided. RESULTS AND CONCLUSION: The gels proved highly cohesive and sensitive to ROS action with stability declining with the decrease in the overall gel elasticity. The G* and complex viscosity values at clinically relevant frequencies and gel water-uptake ability are consistent with the relative clinical indication related to gel projection and hydration capacity. Clinical outcomes showed the safety of the products and a perception of palpability well correlating with the cohesive/viscosity properties of the gels. A similar duration of the aesthetic effect (up to 1 year) was observed despite the diverse in vitro gel stability. The results broaden our knowledge of these gels and may contribute to optimize their clinical use towards the improvement of patient safety and satisfaction. Initial clinical observation indicated that gel biophysical properties allow for a reliable prediction of gel palpability, while in vitro data on gel stability cannot be related to the duration of the observed skin improvement. The latter finding further corroborates the idea of a skin restoration process activated by the gels besides the physical volumetric action.

Hyaluronan-based hydrogels via ether-crosslinking: Is HA molecular weight an effective means to tune gel performance?

Hyaluronan (HA)-based hydrogels obtained by crosslinking the biopolymer via ether bonds are widely used in clinical practice. There is interest in improving the design of these gels to match specific properties. Here, the possibility to tune HA-hydrogel behavior by adjusting the molecular weight distribution of the biopolymer undergoing crosslinking was investigated. Three HA samples (500, 1100 and 1600 kDa) underwent reaction with 1,4-butandioldiglycidyl-ether(BDDE) under reported conditions and the crosslinked products were characterized for chemical modification extent, swelling, rheological behavior, cohesivity, sensitivity to enzymatic degradation and effect on Human Dermal Fibroblasts (HDF). HA hydrolysis, under the highly alkaline crosslinking conditions, was also studied for the first time. The main achievements are that 1) varying HA chain length affects hydrogel behavior less than expected, due to the de-polymerization occurring alongside crosslinking, that reduces the differences in sample size 2) when differences in chain length persist notwithstanding hydrolysis, lowering HA size is a means to prepare more concentrated formulations, expected to exhibit longer duration and better cohesivity in vivo, while retaining a certain rigidity, preserving biocompatibility and slightly influencing HDF behavior in relation to CollagenI production. The study shed light on aspects concerning BDDE-HA gel manufacturing and contributed to the improvement of their design.

Levan from a new isolated Bacillus subtilis AF17: Purification, structural analysis and antioxidant activities.

A strain of Bacillus subtilis AF 17 with high exopolysaccharide (EPS) production ability was isolated and identified based on morphological and physiological characteristics and phylogenetic analysis of 16S rDNA sequences. EPS was isolated from the strain fermentation broth by alcohol precipitation and gel-filtration chromatography. Its structural characteristics were investigated and elucidated by methylation analysis, gas chromatography mass spectrometry and nuclear magnetic resonance spectroscopy. Based on the obtained data, the EPS was found to be a levan containing a backbone of 6-substituted ß-fructoses, with a low grade of branching at position 1 (linear/branched ratio 20:1). Levan showed a molecular weight of about 20 MDa. The antioxidant activity of this biopolymer was studied and revealed that levan showed an interesting 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging capacity (IC50 levan = 1.42 mg/mL), reducing power, and also a strong total antioxidant activity. Overall, the results suggest that levan is a promising source of natural antioxidants and can be used as additive in food and pharmaceutical preparations.

In Vitro Evaluation of Novel Hybrid Cooperative Complexes in a Wound Healing Model: A Step Toward Improved Bioreparation.

The effectiveness of hyaluronic acid (HA), also called as hyaluronan, and its formulations on tissue regeneration and epidermal disease is well-documented. High-molecular-weight hyaluronan (HHA) is an efficient space filler that maintains hydration, serves as a substrate for proteoglycan assembly, and is involved in wound healing. Recently, an innovative hybrid cooperative complex (HCC) of high- and low-molecular-weight hyaluronan was developed that is effective in wound healing and bioremodeling. The HCC proposed here consisted of a new formulation and contained 1.6 ± 0.1 kDa HHA and 250 ± 7 kDa LHA (low molecular weight hyaluronic acid). We investigated the performance of this HCC in a novel in vitro HaCaT (immortalized human keratinocytes)/HDF (human dermal fibroblast) co-culture model to assess its ability to repair skin tissue lesions. Compared to linear HA samples, HCC reduced the biomarkers of inflammation (Transforming Growth Factor-ß (TGF-ß), Tumor Necrosis Factor receptor-α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8)), and accelerated the healing process. These data were confirmed by the modulation of metalloproteases (MMPs) and elastin, and were compatible with a prospectively reduced risk of scar formation. We also examined the expression of defensin-2, an antimicrobial peptide, in the presence of hyaluronan, showing a higher expression in the HCC-treated samples and suggesting a potential increase in antibacterial and immunomodulatory functions. Based on these in vitro data, the presence of HCC in creams or dressings would be expected to enhance the resolution of inflammation and accelerate the skin wound healing process.

Novel Hybrid Gels Made of High and Low Molecular Weight Hyaluronic Acid Induce Proliferation and Reduce Inflammation in an Osteoarthritis In Vitro Model Based on Human Synoviocytes and Chondrocytes.

High molecular weight hyaluronan (H-HA) has a pivotal role in the maintenance of normal functions of synovial fluid and structure of the articular joint, but it has been shown that its concentration is reduced in patients affected by degenerative cartilage diseases, such as osteoarthritis (OA). The aim of this study was to investigate the anti-inflammatory effects and properties of hybrid cooperative complexes based on high and low molecular weight hyaluronan (HCC) compared to H-HA on human primary cells derived by pathological joints. In addition, the rheological behavior of HCC was evaluated in order to define their potential as viscosupplement gel in degenerated joints. The experiments were performed using an in vitro model of OA based on human chondrocytes and synoviocytes isolated from degenerated joints of patients hospitalized for surgical replacement. In order to assess the anti-inflammatory effects of HCC, we evaluated NF-kB, COMP-2, IL-6, and IL-8 as specific markers at the transcriptional and/or protein level. Moreover, the proliferative properties of HCC were assessed using time lapse video microscopy. We showed that chondrocytes and synoviocytes clearly presented an altered cytokine profile compatible with a severe ongoing inflammation status. H-HA and, above all, HCC significantly reduced levels of the specific biomarkers evaluated and improved cartilage healing. The rheological profile indicated HCC suitability for intra-articular injection in joint diseases. HCC viscoelastic properties and the protective/anti-inflammatory effect on human chondrocytes and synoviocytes suggest the novel HCC-based gels as a valid support for OA management.

Hyaluronan-based hydrogels as dermal fillers: The biophysical properties that translate into a "volumetric" effect.

Biophysical and biochemical data on hyaluronan (HA)-based dermal fillers strongly support their optimal use and design to meet specific requisites. Here, four commercially available (in Europe) HA "volumetric" fillers, among the most used in the clinical practice, have been characterized in vitro. Analyses revealed the highest amounts of water-soluble HA reported so far and provided hydrodynamic data for these soluble polymeric fractions. Volumetric gels exhibit a wide range of rigidity with most of them showing G' values around 200-300Pa. They greatly differ in cohesivity. 1mL of gel hydrates up to 2.4-3.2mL. The products completely solubilize due to Bovine Testicular Hyaluronidase (BTH)'s action, thus predicting in vivo complete resorption. For the first time, filler degradation due to reactive oxygen species (ROS) was studied by rheological measurements and a rank in stability was established. Studies using Human Dermal Fibroblasts (HDF) indicated a positive biological response to the HA networks. Further, gel capacity to prompt collagen I, elastin and aquaporin3 synthesis was demonstrated, thus suggesting a positive effect on skin elasticity and hydration, besides the physical volumetric action. The findings are the first wide assessment of features for the volumetric class of HA-fillers and include first data on their resistance to degradation by ROS and biological effects on HDF. The study represents a valuable contribution to the understanding of HA-fillers, useful to optimize their use and manufacture.

A biophysically-defined hyaluronic acid-based compound accelerates migration and stimulates the production of keratinocyte-derived neuromodulators.

Hyaluronic acid (HA) preparations are widely used in clinical practice and recent data suggest that commercially available HA-based compounds promote ulcer re-epithelialization and induce pain relief. However, the pathophysiological basis of these effects remains poorly understood. In the present study, we investigated the biophysical, biomolecular and functional properties of a HA preparation combined with a pool of collagen precursor synthetic aminoacids, namely l-proline, l-leucine, l-lysine and glycine (Aminogam®). Hydrodynamic characterization of Aminogam® by size exclusion chromatography-triple detector array (SEC-TDA) revealed an average molecular weight in the range of 700-1700 kDa. Rheological measurements of the 1700kDa Mw lot showed a pseoudoplastic behaviour with a zero-shear viscosity (η0) equal to 90 ± 9 Pa∙s at 25°C and 55 ± 6 Pa∙s at 37°C. Automated time-lapse videomicroscopy studies in a fibroblast-free system demonstrated that 1% (v/v) Aminogam® significantly reduced the healing time of wounded keratinocyte monolayers. In AKGOS assays, Aminogam® stimulated cellular locomotion (chemokinesis) and directional migration (chemotaxis) of keratinocytes. Analysis of microarray data suggested that keratinocytes had a functional neuroendocrine machinery, and this was confirmed by testing the secretion of six neuroactive molecules by ELISA, namely α-MSH, ß-endorphins, melatonin, substance P, cortisol, and neurotensin. Interestingly, Aminogam® regulated the production of several neuropeptides, including ß-endorphins. In conclusion, our data shed light on the epithelial-dependent mechanisms that underlie the efficacy of Aminogam®, particularly in reference to wound healing and nociception.

Macroporous alginate foams crosslinked with strontium for bone tissue engineering.

Nowadays, the need of novel strategies to repair and regenerate bone defects in the field of biomedical applications has increased. Novel approaches include the design of natural bioactive scaffolds mimicking bone tissue. These bioactive scaffolds have to possess biophysical properties suitable to address biological response towards newly bone tissue formation. In particular, scaffold porosity and pore size play a pivotal role in cell migration, adhesion and proliferation, thus increasing cell-material surface interaction and osteogenic signals transmission. Here we propose the development of macroporous alginate foams (MAFs) with porous and well interconnected structure, useful to enhance growth and osteogenic differentiation of human Mesenchymal Stem Cells (hMSCs). Moreover, in this study we report a new method for MAFs fabrication based on the combination of internal gelation technique with gas foaming. Strontium was employed in combination with calcium as cross-linking agent for the alginate chains and as enhancer of the osteogenic differentiation. The influence of strontium ions on the gelation kinetics, physical properties and degradation in physiological medium of MAFs was investigated. Our results suggest that the combination of internal gelation technique with gas foaming followed by freeze-drying is an easy and straightforward procedure to prepare alginate foams with high porosity and interconnectivity, able to support cell infiltration. Finally, biological assays showed how scaffolds with high strontium content are able to support cell growth and differentiation in long times by promoting osteogenic marker expression.

In Vitro Evaluation of Hybrid Cooperative Complexes of Hyaluronic Acid as a Potential New Ophthalmic Treatment.

PURPOSE: The purpose of this in vitro study was to assess the potential benefits of eye drops based on hybrid cooperative complexes (HCCs) obtained from high and low molecular weight hyaluronic acid (HA). METHODS: Rheological measurements were performed to adjust the HCC concentration toward optimal resistance to drainage from the ocular surface. The viscosity and mucoadhesion profiles of the optimized preparation were derived. Primary porcine corneal epithelial cells were used for biological studies. Cells were exposed to dehydration after being pretreated with the HCC solution, and protection from desiccation was evaluated using cell viability assays. Time-lapse experiments were carried out to evaluate the ability of the HCC preparation to promote corneal wound healing. The characterization studies were performed in comparison with a control HA solution representative of commercial HA-based products. RESULTS: The HCC formulation is able to deliver twice the amount of biopolymer compared with conventional products while avoiding discomfort due to excessive viscosity. The viscosity and mucoadhesion profiles allowed the authors to predict the longer in vivo retention and, therefore, an improved HCC formulation bioavailability. The new preparation also proved superior in protecting porcine corneal epithelial cells from desiccation and in hastening corneal cell wound repair in vitro. CONCLUSIONS: The results suggest that the developed formulation may be a promising topical ophthalmic medical treatment.