A comprehensive in vitro characterization of non-crosslinked, diverse tissue-derived collagen-based membranes intended for assisting bone regeneration

Collagen-based membranes are class III-medical devices widely used in dental surgical procedures to favour bone regeneration. Here, we aimed to provide biophysical and biochemical data on this type of devices to support their optimal use and design/manufacturing. To the purpose, four commercial, non-crosslinked collagen-based-membranes, obtained from various sources (equine tendon, pericardium or cortical bone tissues, and porcine skin), were characterized in vitro. The main chemical, biophysical and biochemical properties, that have significant clinical implications, were evaluated. Membranes showed similar chemical features. They greatly differed in morphology as well as in porosity and density and showed a diverse ranking in relation to these latter two parameters. Samples highly hydrated in physiological medium (swelling-ratio values in the 2.5–6.0 range) and, for some membranes, an anisotropic expansion during hydration was, for the first time, highlighted. Rheological analyses revealed great differences in deformability (150-1500kPa G’) also alerting about the marked variation in membrane mechanical behaviour upon hydration. Samples proved diverse sensitivity to collagenase, with the cortical-derived membrane showing the highest stability. Biological studies, using human-bone-derived cells, supported sample ability to allow cell proliferation and to prompt bone regeneration, while no relevant differences among membranes were recorded. Prediction of relative performance based on the findings was discussed. Overall, results represent a first wide panel of chemical/biophysical/biochemical data on collagen-based-membranes that 1) enhances our knowledge of these products, 2) aids their optimal use by providing clinicians with scientific basis for selecting products based on the specific clinical situation and 3) represents a valuable reference for optimizing their manufacturing

Biophysical and biological characterization of a new line of hyaluronan-based dermal fillers: A scientific rationale to specific clinical indications

Chemico-physical and biological characterization of hyaluronan-based dermal fillers is of key importance to differentiate between numerous available products and to optimize their use. These studies on fillers are nowadays perceived as a reliable approach to predict their performance in vivo. The object of this paper is a recent line of hyaluronic acid (HA)-based dermal fillers, Aliaxin®, available in different formulations that claim a complete facial restoration. The aim of the study is to provide biophysical and biological data that may support the clinical indications and allow to predict performance possibly with respect to similar available products. Aliaxin® formulations were tested for their content in soluble HA, water uptake capacity, rheological behavior, stability to enzymatic degradation, and for in vitro capacity to stimulate extracellular matrix components production. The formulations were found to contain a low amount of soluble HA and were equivalent to each other regarding insoluble hydrogel concentration. The different crosslinking degree declared by the producer was consistent with the trend in water uptake capacity, rigidity, viscosity. No significant differences in stability to enzymatic hydrolysis were found. In vitro experiments, using a full thickness skin model, showed an increase in collagen production in the dermoepidermal junction. Results support the claims of different clinical indications, the classification of products regarding hydro-, lift-action and the specifically suggested needle gauge for the delivery. The biological outcomes also support products effectiveness in skin structure restoration. These data predicted a better performance regarding hydro-action, tissue integration, clinical management during delivery, and a high durability of the aesthetic effect when compared to data on marketed similar products

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

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

The new normal of global mobility - flexibility, diversity & data mastery

<p>Blue: Nuclei (Hoescth), Green: ELS, red: Cytoskeleton (Phalloidin).</p

In vitro analysis of the effects on wound healing of high- and low-molecular weight chains of hyaluronan and their hybrid H-HA/L-HA complexes

Abstract Background: Recent studies have reported the roles of Hyaluronic acid (HA) chains of diverse length in wound repair, especially considering the simultaneous occurrence in vivo of both high- (H-HA) and low-molecular weight (L-HA) hyaluronan at an injury site. It has been shown that HA fragments (5 ≤ MW ≤ 20 kDa) usually trigger an inflammatory response that, on one hand, is the first signal in the activation of a repair mechanism but on the other, when it’s overexpressed, it may promote unwanted side effects. The present experimental research has aimed to investigate H-HA, L-HA and of a newly developed complex of the two (H-HA/L-HA) for stability (e.g. hyaluronidases digestion), for their ability to promote wound healing of human keratinocytes in vitro and for their effect on cellular biomarker expression trends. Results: Time-lapse video microscopy studies proved that the diverse HA was capable of restoring the monolayer integrity of HaCat. The H-HA/L-HA complex (0.1 and 1%w/v) proved faster in regeneration also in co-culture scratch test where wound closure was achieved in half the time of H-HA stimulated cells and 2.5-fold faster than the control. Gene expression was evaluated for transformation growth factor beta 1 (TGF-β1) proving that L-HA alone increased its expression at 4 h followed by restoration of similar trends for all the stimuli. Depending on the diverse stimulation (H-HA, L-HA or the complex), metalloproteinases (MMP-2, -9, -13) were also modulated differently. Furthermore, type I collagen expression and production were evaluated. Compared to the others, persistence of a significant higher expression level at 24 h for the H-HA/L-HA complex was found. Conclusions: The outcomes of this research showed that, both at high and low concentrations, hybrid complexes proved to perform better than HA alone thus suggesting their potential as medical devices in aesthetic and regenerative medicine. Keywords: Wound healing, Hyaluronan, MMPs, Hybrid complexe

Shape-​memory crosslinked polysaccharides for biomaterials

The invention relates to an innovative method for the prodn. of crosslinked polysaccharides which have the characteristic of maintaining the shape of the original solid matrix, and are therefore called shape-​memory polysaccharides.  The process according to the invention involves crosslinking at least one water-​sol. polysaccharide, in particular hyaluronic acid and hyaluronans, chondroitin, chondroitin sulfate and chitosan, with at least one polyfunctional epoxy crosslinking agent, operating in a biphasic system consisting of the polysaccharide matrix, which always remains in the solid state throughout the crosslinking process, and a non-​solvent system for the polysaccharide.  The materials obtained by the process according to the invention are insol. in aq. systems and maintain their original shape after hydration.  The process according to the invention is particularly important in the prepn. of crosslinked shape-​memory polysaccharides for clin. applications and in the field of aesthetic medicine.  For example, hyaluronic acid (HA) was crosslinked with 1,​4-​butanediol diglycidyl ether (BDDE) crosslinking agent in the presence of benzyl tri-​Me ammonium hydroxide (TMBAH) using the water-​acetone solvent system.  The reaction was stopped by adding concd. H3PO4 until neutralization.  The reaction yields of the crosslinked HA was dependent on the amt. of water in the reaction mixt., including the water present in the TMBAH: for the solvent mixts. comprising acetone-​water at 79.32:10.0 and 62.62:26.7 mL​/mL, resp., the yields of the crosslinked shape-​memory water-​insol. HA was 43.4​% and 79.4​%, resp.  The material obtained was cytocompatible

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. H-1-/C-13-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

Hyaluronan dermal fillers via crosslinking with 1,4-butandiol diglycidyl ether: Exploitation of heterogeneous reaction conditions.

Most of hyaluronan (HA)-based dermal fillers currently available on the market are produced through biopolymer crosslinking with 1,4-butandiol diglycidyl ether (BDDGE). Chemical modification is usually performed on the biopolymer dissolved in a highly alkaline aqueous medium (homogeneous conditions). Heterogeneous conditions for HA reaction with BDDGE were exploited here to obtain competitive HA fillers and to assess potential improvements in production process. Optimal parameters for effective reaction accomplishment were evaluated (e.g., medium composition, temperature and time of reaction). HA was modified with increasing BDDGE/HA equivalents (7–14%) achieving 66–74% (w/w) biopolymer insolubility. Hydrogels exhibited high swelling extent and outstanding resistance to enzymatic degradation decreasing and improving according to crosslinking degree, respectively. Once suspended in physiological solution (20 mg/mL), these products directly formed easy-to-extrude gels through 27–29 G needles. Gel particle dimensions were in the range 10–1000 mm. Rheological analyses revealed decreasing viscosity with the shear rate and G0 values in the range 1200– 1700 Pa. Overall, results of the in vitro characterization demonstrated the attainment of crosslinked HA particles suitable for application as dermal fillers. These new gels proved superior to similar commercialized products in terms of stability to enzymatic hydrolysis. Further, the protocol assessed allowed interesting improvements over conventional manufacturing procedures