Hyaluronic Acid Biomaterials Research Articles

Evaluation of Platelet-Derived Extracellular Vesicles in Gingival Fibroblasts and Keratinocytes for Periodontal Applications.

Gingival regeneration aims at restoring the architecture and functionality of oral damaged tissue. Different biomaterials or biological materials have been tested for tissue repair, such as platelet concentrates such as PL. In this article, the use of extracellular vesicles (EVs) derived from platelet lysate (PL) and their combination with hyaluronic acid biomaterials (HA) in an in vitro wound healing assay is investigated. EVs were isolated by size exclusion chromatography from PL. In addition, HA gels were formulated with PL or EVs. EVs or HA combined with EVs (HA-EVs) were tested in vitro in gingival fibroblasts and keratinocytes for biocompatibility (LDH activity and metabolic activity) and by an in vitro wound-healing assay and gene expression analysis. EVs and EVs-HA treatments were biocompatible in gingival fibroblasts and keratinocytes and showed an increase in wound healing in vitro compared to control. Moreover, changes in gene expression related to extracellular matrix remodeling were observed after the treatment with EVs. EVs can be combined with HA biomaterials, showing good biocompatibility and preserving their activity and functionality. Therefore, platelet-derived EVs could emerge as a new application for periodontal regeneration in combination with biomaterials in order to enhance their clinical use.

16822 Effect of commonly antiseptics used in clinic in an artificial autologous skin model based on hyaluronic acid biomaterial

Artificial bioengineered skin substitutes (ABSS) are the main treatment used in addition to autografts when skin injuries involve a large body surface area. An antiseptic/antibiotic treatment is necessary to prevent infections in the ABSS implant area. This study aimed to evaluate the effect of clinical antiseptics on cell viability, structural integrity and epidermal barrier function in ABSS based on hyaluronic acid during 28 days of follow-up. Keratinocytes (KT) and dermal fibroblasts (DF) were enzymatically isolated from 9-cm2 skin biopsies (n = 3).

Hyaluronic acid biomaterial for human tissue-engineered skin substitutes: Preclinical comparative in vivo study of wound healing.

There is not an ideal biomaterial for tissue-engineered skin substitutes (TESSs), and most of the studies or existing therapies use xenogeneic origin natural biomaterials or biosynthetic scaffolds. To analyse clinical, histological integration and homeostasis parameters of a human TESS manufactured with fibrin-hyaluronic acid biomaterial (HA-Skin), grafted in immunodeficient mice for 8weeks, and compared with the gold standard treatment (Autograft), a human TESS manufactured with fibrin-agarose biomaterial (AG-Skin) and secondary wound healing dressings. Human TESSs and autografts were implanted into BALB/c mice after surgical excision. Secondary wound healing approach was achieved with biosynthetic collagen wound dressing (Biobrane® ) and fibrin-hyaluronic acid or fibrin-agarose biomaterial without cells (Total N=44). Clinical integration and homeostasis parameters were evaluated every two weeks for two months. Histological and immunohistochemical analyses were performed four and eight weeks after grafting. HA-Skin, AG-Skin and Autograft groups showed a proper clinical integration and epithelization eight weeks later. Scar evaluation revealed better results for Autograft and HA-Skin. Homeostasis analysis indicated similar values of transepidermal water loss and elasticity between HA-Skin (6.42±0.75g/h/m2 , 0.42±0.08AU), Autograft (6.91±1.28g/h/m2 , 0.40±0.08AU) and healthy mouse skin (6.40±0.43g/h/m2 , 0.35±0.03AU). Histological results showed that human TESSs and autografts presented better skin structuration and higher expression of cytokeratins. This study suggests that human TESS based on fibrin-hyaluronic acid biomaterial could be suitable for clinical application in the treatment of several dermatological pathologies (wound healing).

Epidermal barrier function evaluation in an artificial autologous skin model based on hyaluronic acid biomaterial

Background & Aim The aim of this study was evaluated the epidermal barrier function in an artificial bioengineered skin substitutes (ABSS) based on hyaluronic acid biomaterial. The study was performed evaluating specific skin ointments at different test period. Methods, Results & Conclusion Dermal fibroblasts (DF) and Keratinocytes (KT) were enzymatically isolated from 9 cm2 skin biopsies. After 4-5 weeks in culture, DF were part of dermal fibrin-hyaluronic acid stroma. KT (obtained using feeder-free method) were seeded on the surface to establish ABSS. Flow cytometry assay was carried out to phenotypically evaluate of keratinocytes: CD49f (integrin α6) and CD90 (fibroblast cell surface marker). Cell viability (LIVE/DEAD® assay), structural integrity (histological evaluation) and epidermal barrier function (transepidermal water loss, TEWL: Tewameter®) were measured after 5, 15 and 30 minutes to apply beclomethasone, mupirocin and vaseline. Acellular ABSS were used as controls. Specific cell culture parameters were evaluated after final recovery of keratinocytes. The cell expansion rate was significantly higher (p≤0.001%) with the CNT-57S media were supplemented with human platelet lysate. CD49f percentage was higher in keratinocytes with CNT-57S (95.2%). The CD90 percentage was lower in keratinocytes expanded with CNT-57S human lysate supplemented (1.60%). A viability assay was carried out in ABSS at first (85.1%) and second week (87.8%). Finally, barrier function evaluation of ABSS was carried out by measuring TEWL in the first and in the second week of maintenance. TEWL values in acellular ABSS were higher than in cellular ABSS, although the difference was not significant. Cells improved ABSS barrier function, thus favouring the reduction of water loss. Regarding the evaluation period, significant differences of TEWL were observed, decreasing the values in the second week. This may be due to the growth of the epithelial layer in cellular ABSS, which contributes to improvement of the barrier function. TEWL measures also decreased over the measurement time, although the differences were not significant. No difference was observed with respect to the type of ointment. In conclusion, keratinocytes culture was optimized using animal component-free and feeder-free method. A novel way to evaluate keratinocytes contribution to epidermal barrier function has been development in vitro.

Generation of novel hyaluronic acid biomaterials for study of pain in third molar intervention: a review.

Hyaluronic acid (HA) has long been studied in diverse applications. It is a naturally occurring linear polysaccharide in a family of unbranched glycosaminoglycans, which consists of repeating di-saccharide units of N-acetyl-D-glucosamine and D-glucuronic acid. It is almost ubiquitous in humans and other vertebrates, where it participates in many key processes, including cell signaling, tissue regeneration, wound healing, morphogenesis, matrix organization, and pathobiology. HA is biocompatible, biodegradable, muco-adhesive, hygroscopic, and viscoelastic. These unique physico-chemical properties have been exploited for several medicinal purposes, including recent uses in the adjuvant treatment for chronic inflammatory disease and to reduce pain and accelerate healing after third molar intervention. This review focuses on the post-operative effect of HA after third molar intervention along with its various physio-chemical, biochemical, and pharmaco-therapeutic uses.

Biohybrids of scaffolding hyaluronic acid biomaterials plus adipose stem cells home local neural stem and endothelial cells: Implications for reconstruction of brain lesions after stroke.

Endogenous neurogenesis in stroke is insufficient to replace the lost brain tissue, largely due to the lack of a proper biological structure to let new cells dwell in the damaged area. We hypothesized that scaffolds made of hyaluronic acid (HA) biomaterials (BM) could provide a suitable environment to home not only new neurons, but also vessels, glia and neurofilaments. Further, the addition of exogenous cells, such as adipose stem cells (ASC) could increase this effect. Athymic mice were randomly assigned to a one of four group: stroke alone, stroke and implantation of BM, stroke and implantation of BM with ASC, and sham operated animals. Stroke model consisted of middle cerebral artery thrombosis with FeCl3 . After 30 days, animals underwent magnetic resonance imaging (MRI) and were sacrificed. Proliferation and neurogenesis increased at the subventricular zone ipsilateral to the ventricle and neuroblasts, glial, and endothelial cells forming capillaries were seen inside the BM. Those effects increased when ASC were added, while there was less inflammatory reaction. Three-dimensional scaffolds made of HA are able to home newly formed neurons, glia, and endothelial cells permitting the growth neurofilaments inside them. The addition of ASC increase these effects and decrease the inflammatory reaction to the implant. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1598-1606, 2019.

Light-activatable prodrugs based on hyaluronic acid biomaterials

Photosensitive in situ cross-linked hyaluronan (HA) hydrogels are prepared by modular chemoselective assembly from the biopolymer precursors and novel heterobifunctional linkers with middle photo-labile ortho-nitrobenzyl group and orthogonally reactive terminals. Starting from the thiol-modified HA and a linker with activated disulfide and hydrazide terminals, a photo-degradable HA hydrogel was prepared by the hydrazone cross-linking reaction. Moreover, a light-triggered drug-releasing hydrogel prodrug was constructed by an orthogonal conjugation of dopamine (DA) via a photo-labile linker to HA dually modified with thiol and hydrazide groups (hy-HA-SH) and a subsequent cross-linking with aldehyde-derivatized HA (HA-al). On-demand release of DA from the hydrogel was achieved upon exposure of the hydrogel to UV light whereas 11-fold less release of the drug was observed in the absence of light. The mechanical properties of the hydrogels, photodegradation kinetics, photorelease of the model drugs were studied by rheology, spectrophotometry, chromatography, and mass spectrometry. For the first time, integration of photolabile components into an actual polysaccharide of extracellular matrix was implemented for the light-controlled release of drug molecules.

Uptake of silica covered Quantum Dots into living cells: Long term vitality and morphology study on hyaluronic acid biomaterials

Quantum Dots (QDs) are promising very bright and stable fluorescent probes for optical studies in the biological field but water solubility and possible metal bio-contamination need to be addressed. In this work, a simple silica-QD hybrid system is prepared and the uptake in bovine chondrocytes living cells without any functionalization of the external protective silica shield is demonstrated. Moreover, long term treated cells vitality (up to 14days) and the transfer of silica-QDs to the next cell generations are here reported. Confocal fluorescence microscopy was also used to determine the morphology of the so labelled cells and the relative silica-QDs distribution. Finally, we employ silica-QD stained chondrocytes to characterize, as proof of concept, hydrogels obtained from an amphiphilic derivative of hyaluronic acid (HA-EDA-C18) functionalized with different amounts of the RGD peptide.

Proteomic Profiling of Neuroblastoma Cells Adhesion on Hyaluronic Acid-Based Surface for Neural Tissue Engineering.

The microenvironment of neuron cells plays a crucial role in regulating neural development and regeneration. Hyaluronic acid (HA) biomaterial has been applied in a wide range of medical and biological fields and plays important roles in neural regeneration. PC12 cells have been reported to be capable of endogenous NGF synthesis and secretion. The purpose of this research was to assess the effect of HA biomaterial combining with PC12 cells conditioned media (PC12 CM) in neural regeneration. Using SH-SY5Y cells as an experimental model, we found that supporting with PC12 CM enhanced HA function in SH-SY5Y cell proliferation and adhesion. Through RP-nano-UPLC-ESI-MS/MS analyses, we identified increased expression of HSP60 and RanBP2 in SH-SY5Y cells grown on HA-modified surface with cotreatment of PC12 CM. Moreover, we also identified factors that were secreted from PC12 cells and may promote SH-SY5Y cell proliferation and adhesion. Here, we proposed a biomaterial surface enriched with neurotrophic factors for nerve regeneration application.

Effect of hyaluronic acid incorporation method on the stability and biological properties of polyurethane–hyaluronic acid biomaterials

The high failure rate of small diameter vascular grafts continues to drive the development of new materials and modification strategies that address this clinical problem, with biomolecule incorporation typically achieved via surface-based modification of various biomaterials. In this work, we examined whether the method of biomolecule incorporation (i.e., bulk versus surface modification) into a polyurethane (PU) polymer impacted biomaterial performance in the context of vascular applications. Specifically, hyaluronic acid (HA) was incorporated into a poly(ether urethane) via bulk copolymerization or covalent surface tethering, and the resulting PU-HA materials characterized with respect to both physical and biological properties. Modification of PU with HA by either surface or bulk methods yielded materials that, when tested under static conditions, possessed no significant differences in their ability to resist protein adsorption, platelet adhesion, and bacterial adhesion, while supporting endothelial cell culture. However, only bulk-modified PU-HA materials were able to fully retain these characteristics following material exposure to flow, demonstrating a superior ability to retain the incorporated HA and minimize enzymatic degradation, protein adsorption, platelet adhesion, and bacterial adhesion. Thus, despite bulk methods rarely being implemented in the context of biomolecule attachment, these results demonstrate improved performance of PU-HA upon bulk, rather than surface, incorporation of HA. Although explored only in the context of PU-HA, the findings revealed by these experiments have broader implications for the design and evaluation of vascular graft modification strategies.

Photoactivated Composite Biomaterial for Soft Tissue Restoration in Rodents and in Humans

Soft tissue reconstruction often requires multiple surgical procedures that can result in scars and disfiguration. Facial soft tissue reconstruction represents a clinical challenge because even subtle deformities can severely affect an individual's social and psychological function. We therefore developed a biosynthetic soft tissue replacement composed of poly(ethylene glycol) (PEG) and hyaluronic acid (HA) that can be injected and photocrosslinked in situ with transdermal light exposure. Modulating the ratio of synthetic to biological polymer allowed us to tune implant elasticity and volume persistence. In a small-animal model, implanted photocrosslinked PEG-HA showed a dose-dependent relationship between increasing PEG concentration and enhanced implant volume persistence. In direct comparison with commercial HA injections, the PEG-HA implants maintained significantly greater average volumes and heights. Reversibility of the implant volume was achieved with hyaluronidase injection. Pilot clinical testing in human patients confirmed the feasibility of the transdermal photocrosslinking approach for implantation in abdomen soft tissue, although an inflammatory response was observed surrounding some of the materials.

Optimization of Molecularly Imprinted Polymers of Serotonin for Biomaterial Applications

We prepared molecularly imprinted polymers (MIPs) of serotonin (5-hydroxytryptamine or 5-HT), a neurotransmitter and mood modulator, using a combination of neutral (methacrylamide or acrylamide) and positively charged (methacrylic acid) functional monomers. Water, PBS, acidified methanol and sodium dodecyl sulfate were compared as rinsing solvents for the removal of serotonin from the MIPs. Methacrylamide MIPs rinsed in acidified methanol (92% serotonin removal) produced the highest imprinting factor (3.1) in equilibrium batch rebinding experiments using a combination of 2% water and 98% MeCN as the rebinding solvent. For the first time, these MIPs were assessed for cytocompatibility using mouse mesencephalon neural progenitor cells (NPC) and NIH 3T3 fibroblasts. Although MIP particles decreased NPC viability to <70%, MIP particles did not significantly reduce fibroblast viability when incorporated into a hyaluronic acid biomaterial. MIP microparticles were incorporated into a cross-linked hyaluronic acid biomaterial to present one way in which molecularly imprinted polymers may be used in vivo in future biomaterials or biosensors applications.

Cellular and molecular events during chondrogenesis of human mesenchymal stromal cells grown in a three-dimensional hyaluronan based scaffold

Mesenchymal stromal cells (MSCs) seem to be a good alternative to chondrocytes for cartilage regeneration. To obtain new information on the sequence of cellular and molecular events during in vitro chondrogenic differentiation we analysed MSCs on a widely used hyaluronic acid biomaterial (Hyaff ®-11). Cellular differentiation was induced using two different concentrations of TGF β 1 (10 and 20 ng/ml) and the process was analysed at different time points (24 h, and 7, 14, 21 and 28 days) using techniques of light and electron microscopy, real-time PCR and immunohistochemistry. We found that without TGF β MSCs did not survive while in the presence of TGF β the cells significantly proliferated from day 7 until day 28. Light and electron microscopy showed that TGF β at 20 ng/ml better induced the formation of cartilage-like tissue. Real-time PCR showed an increased expression of collagen type II, IX and aggrecan associated to a down-regulation of collagen type I. Immunohistochemical analysis confirmed that collagen type I was down-modulated while collagen type II increased from day 14 to day 28. These data clearly showed that higher concentrations of TGF β (20 ng/ml) induce chondrogenesis of MSCs on Hyaff ®-11 scaffold better than 10 ng/ml of TGF β . This process is characterized by a sequence of cellular and molecular events that deal with the in vitro formation of a cartilage-like tissue.