High- (H-HA; MW 1400 ± kDa, Mw/Mn = 1.5, Intrinsic viscosity =21.5 dl/g) and low-molecular weight hyaluronic acid (L-HA: Mw = 90 ± 5 kDa, Mw/Mn = 1.7 ± 0.07 Intrinsic viscosity =2.4 dl/g) were kindly provided by Altergon s.r.l., Italy. Altergon HA is a fermentative HA from Streptococcus equi ssp. equi, at pharmaceutical grade (e.g. purity >95 %, water content < 10 %, EU/mg < 0.05). This ensured that the products are controlled according to the pharmacopeia. Briefly in collaboration with Altergon, LHA 90 kDa was obtained through heterogeneous hydrolytic reaction in acidic conditions. The hydrodynamic characterization was performed using the SEC-TDA (Size Exclusion Chromatography-Triple Detector Array) equipment by Viscotek (Lab Service Analytica, Italy).
The H-HA/L-HA complex (1:1 weight ratio) was produced in our laboratory following the procedure described in patent application WO2011EP65633 [27 ], which was modified to obtain solutions (32 g/l) suitable for use in cell cultures (i.e., use of a physiological buffer solution, PBS). For all samples, pH and osmolality were measured in order to perform experiments in physiological conditions (i.e., pH 7.0–7.4; osmolality 300 mOsm). Endotoxin concentration (EU/mg) was evaluated with the Limulus test, and solutions were used only when the titer as <1 EU/mg. All solutions were sterilized in an autoclave at 1 bar for 12 min at 120 °C.
Bovine testicular hyaluronidase, BTH (EC 3.2.1.35), essentially salt free lyophilized powder with a specific activity of 1160U/mg was purchased from Sigma-Aldrich (Milan, Italy) (cat. N. H3884, lot. N. 057K7014).
HaCaT cells (Istituto Zooprofilattico, Brescia, Italy), a spontaneously transformed non-tumorigenic human keratinocyte cell line and human dermal fibroblast (HDF), a generous gift of Prof Caraglia, were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 % (v/v) heat inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/ml streptomycin. All materials were purchased from Flow Laboratories (Milan, Italy). The cells were grown on tissue culture plates (Corning Incorporated, New York, USA), using an incubator with a humidified atmosphere (95 % air/5%CO2 v/v) at 37 °C. Collagen was purchased from Sigma, Aldrich (Milan, Italy).
The H-HA/L-HA complex (1:1 weight ratio) was produced in our laboratory following the procedure described in patent application WO2011EP65633 [27 ], which was modified to obtain solutions (32 g/l) suitable for use in cell cultures (i.e., use of a physiological buffer solution, PBS). For all samples, pH and osmolality were measured in order to perform experiments in physiological conditions (i.e., pH 7.0–7.4; osmolality 300 mOsm). Endotoxin concentration (EU/mg) was evaluated with the Limulus test, and solutions were used only when the titer as <1 EU/mg. All solutions were sterilized in an autoclave at 1 bar for 12 min at 120 °C.
Bovine testicular hyaluronidase, BTH (EC 3.2.1.35), essentially salt free lyophilized powder with a specific activity of 1160U/mg was purchased from Sigma-Aldrich (Milan, Italy) (cat. N. H3884, lot. N. 057K7014).
HaCaT cells (Istituto Zooprofilattico, Brescia, Italy), a spontaneously transformed non-tumorigenic human keratinocyte cell line and human dermal fibroblast (HDF), a generous gift of Prof Caraglia, were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 % (v/v) heat inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/ml streptomycin. All materials were purchased from Flow Laboratories (Milan, Italy). The cells were grown on tissue culture plates (Corning Incorporated, New York, USA), using an incubator with a humidified atmosphere (95 % air/5%CO2 v/v) at 37 °C. Collagen was purchased from Sigma, Aldrich (Milan, Italy).
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