Hyaluronidase Solution Research Articles

Short- and long-term stability of synovial fluid calprotectin.

Information about analyte stability is of crucial importance. The aims of this study were to determine the short- and long-term stability of synovial fluid calprotectin at various temperature conditions (4-8 °C for 7 days, - 20 °C and - 80 °C for 6 weeks). Eleven samples from patients were included in this study. The samples were promptly transported at room temperature (RT) to the laboratory immediately after arthrocentesis. Upon arrival, the samples were transferred into plastic tubes without additives and pretreated with hyaluronidase solution. After centrifugation at 1500xg for 10 minutes at RT, the baseline calprotectin concentrations were determined. Seven aliquots were stored in LoBind tubes (Eppendorf) at 4-8 °C and the calprotectin was measured every day. Six additional aliquots were stored at temperatures - 20 °C and - 80 °C and the concentration of calprotectin was measured weekly. Analysis was done using Buhlmann fCAL turbo reagent on analyzer Siemens Atellica Solution (Siemens Healthcare, Erlangen, Germany). Data were analyzed by Microsoft Excel and MedCalc statistical software. The percentage difference (PD%) was calculated. The maximum permissible difference (MPD) was 9.1% for PD%. The PD% with the corresponding 95% confidence intervals were inside the predefined MPD. The instability equations and correlation coefficient for storage temperatures were PD% = 0.1644 x time (day), r = 0.06, P = 0.614 for 4-8°C, PD% = 0.5190 x time (week), r = - 0.22, P = 0.080 for - 20°C, and PD% = 0.1316 x time (week), r = 0.08, P = 0.545 for - 80°C. The calprotectin in the synovial fluid is stable when stored long-term for 6 weeks at - 20 °C or at - 80 °C or short-term (7 days) at 4-8 °C.

A photothermal surface modified with polyelectrolyte multilayers for gene transfection and cell harvest

Gene transfection, which involves introducing nucleic acids into cells, is a pivotal technology in the life sciences and medical fields, particularly in gene therapy. Surface-mediated transfection, primarily targeting cells adhering to surfaces, shows promise for enhancing cell transfection by localizing and presenting surface-bound nucleic acids directly to the cells. However, optimizing endocytosis for efficient delivery remains a persistent challenge. Additionally, ensuring efficient and non-traumatic cell harvest capability is crucial for applications such as ex vivo cell-based therapy. To address these challenges, we developed a photothermal platform with enzymatic degradation capability for efficient gene transfection and cell harvest. This platform is based on carbon nanotubes (CNTs) doped with poly(dimethylsiloxane) and modified with polyelectrolyte multilayers (PEMs) containing hyaluronic acid and quaternized chitosan, allowing for substantial loading of poly(ethyleneimine)/plasmid DNA (pDNA) complexes through electrostatic interactions. Upon irradiation of near-infrared laser, the photothermal properties of CNTs enable high transfection efficiency by delivering pDNA into attached cells via a membrane disruption mechanism. The engineered cells can be harvested by treating with a non-toxic hyaluronidase solution to degrade PEMs, thus maintaining good viability for further applications. This platform has demonstrated remarkable efficacy across various cell lines (including Hep-G2 cells, Ramos cells and primary T cells), achieving a transfection efficiency exceeding 95 %, cell viability exceeding 90 %, and release efficiency surpassing 95 %, highlighting its potential for engineering living cells.

Decellularized Umbilical Cord as a Scaffold to Support Healing of Full-Thickness Wounds.

The umbilical cord is a material that enhances regeneration and is devoid of age-related changes in the extracellular matrix (ECM). The aim of this work was to develop a biodegradable scaffold from a decellularized human umbilical cord (UC-scaffold) to heal full-thickness wounds. Decellularization was performed with 0.05% sodium dodecyl sulfate solution. The UC-scaffold was studied using morphological analysis methods. The composition of the UC-scaffold was studied using immunoblotting and Fourier transform infrared spectroscopy. The adhesion and proliferation of mesenchymal stromal cells were investigated using the LIVE/DEAD assay. The local reaction was determined by subcutaneous implantation in mice (n = 60). A model of a full-thickness skin wound in mice (n = 64) was used to assess the biological activity of the UC-scaffold. The proposed decellularization method showed its effectiveness in the umbilical cord, as it removed cells and retained a porous structure, type I and type IV collagen, TGF-β3, VEGF, and fibronectin in the ECM. The biodegradation of the UC-scaffold in the presence of collagenase, its stability during incubation in hyaluronidase solution, and its ability to swell by 1617 ± 120% were demonstrated. Subcutaneous scaffold implantation in mice showed gradual resorption of the product in vivo without the formation of a dense connective tissue capsule. Epithelialization of the wound occurred completely in contrast to the controls. All of these data suggest a potential for the use of the UC-scaffold.

Comparative evaluation of efficacy of 2% lidocaine containing adrenaline with and without hyaluronidase in inferior alveolar nerve blocks for patients requiring bilateral mandibular extractions

AbstractPurposeTo evaluate and compare the efficacy of 2% lidocaine containing adrenaline with and without hyaluronidase in inferior alveolar nerve blocks for patients requiring bilateral mandibular extractions.Patients and MethodsThis was the split‐mouth study conducted on 26 patients requiring bilateral mandibular teeth extraction. All the patients were assigned randomly to one of the two groups: (i) 2% lignocaine HCl + 1:2,00,000 adrenaline + hyaluronidase (75 IU) solution will be administered on one side which is the experimental side and (ii) 2% lignocaine HCl + 1:2,00,000 adrenaline solution on the other side is control side. In the first session, the patients randomly received the inferior alveolar nerve block with or without hyaluronidase in one side of the oral cavity. One week later, the same patient received the other technique which was not received in the first session. We also evaluated the onset, duration and need of any supplementary anaesthesia in both the groups.ResultsComparison of VAS score in the control group (2.73 ± 0.724) and experimental group (3 ± 0.693) showed statistically insignificant results (p = 0.177). Whereas the onset of local anaesthesia showed a statistically significant difference between the two groups (p < 0.001) mean of control group (2.73 ± 0.47) and experimental group (1.15 ± 0.23). Comparison of duration of anaesthesia showed a statistically significant difference (p < 0.001) with the mean duration of the experimental group being higher (106 ± 7.94) than the control group (86.27 ± 3.90). Comparison for the need for supplementary anaesthesia showed no statistically significant difference between the two groups.ConclusionWe conclude that if hyaluronidase is used, better blocks with smaller volumes are produced. LAs with hyaluronidase have been shown to improve the quality and effectiveness of anaesthetic blocks and shorten the time it takes for anaesthesia to begin, accelerate the onset of akinesia and lessen the need for additional anaesthesia. It also increases the duration of anaesthesia.

Antibacterial intraosseous implant surface coating that responds to changes in the bacterial microenvironment.

Bone implant-associated infection is one of the most challenging problems encountered by orthopedic surgeons. There is considerable interest in the development of drug-loaded antibacterial coatings for the surfaces of metal implants. However, it is difficult to achieve the stable local release of an effective drug dose for many antibacterial coatings. In the present study, analyses of the thickness and water contact angle of multiple layers confirmed the successful assembly of multilamellar membrane structures. Measurement of the zone of bacterial inhibition indicated gradual degradation of the (montmorillonite [MMT]/hyaluronic acid [HA])10 multilamellar film structure with concentration-dependent degradation during incubation with hyaluronidase solution and Staphylococcus aureus. In vivo results resembled the in vitro results. Overall, the findings confirm that the (MMT/HA-rifampicin)10 multilamellar film structure exhibits good antibacterial properties and excellent biocompatibility. Further studies of the clinical potential of the antibacterial coating prepared in this experiment are warranted.

P-145 Hyaluronidase concentration and its effect on embryo quality

Abstract Study question Does hyaluronidase concentration used during denudation affect any success rate parameter? Summary answer There is an increase in the good quality blastocyst rates as well as an increment of maximum quality blastocysts when using less than 40 IU/ml. What is known already Denudation is a key process in IVF treatments. Combining chemical with mechanical forces allows the classification of mature and immature oocytes. Hyaluronidase dissolves the hyaluronic acid found between the cumulus cells aiding the denudation process. Time under exposure to this acid must be minimized in order to avoid potential harm to the oocytes. Available commercial media report a concentration of 80 IU/ml. However, some laboratories have described benefits when diluting hyaluronidase even further. This statement makes sense when considering that damage to oocytes has been reported when incubating oocytes with hyaluronidase for too long. Study design, size, duration Prospective study designed to analyze the effect of different concentrations of hyaluronidase into egg donation treatments between August 2020 and November 2021. Time under the hyaluronidase solution was limited to a maximum of 30 seconds, after that denudation tips of decreasing diameter were used to complete the denudation process. We analyzed the percentage of mature oocytes, fecundation rates, good quality blastocyst rates, blastocyst quality and clinical success rates. Participants/materials, setting, methods This study includes 743 egg donation treatments cultured in a time-lapse incubator. In the control group (CG = 277 tratments) we used a solution of 80 IU/ml of hyaluronidase, group 1 (G1=196 treatments) consisted of those denudations carried out using a solution 40 IU/ml, group 2 (G2=123 treatments) using 28.6 IU/ml of hyaluronidase, and group 3 (G3=147 tratments) using 20 IU/ml of hyaluronidase. Main results and the role of chance Both metafase II rates (CG = 79.99%; G1=78.95%; G2=76.72%; G3=76.74%; p = 0.648) and fertilization rates (GC = 74.50%; G1=73.84%; G2=74.03%; G3=72.24%; p = 0.667) were not affected by the different dilutions. Still, good quality blastocyst rates increased with lower concentrations of hyaluronidase (CG = 59.96%; G1=64.34%; G2=63.61%; G3=63.28%; p < 0.01). When considering clinical results, we did not see any differences between the implantation rates of the embryos resulting from these different denudation solutions (CG = 50.56%; G1=50.00%; G2=44.74%; G3=47.95%; p = 0.92). A deeper analysis into embryo quality allowed us to observe an incremented number of embryos with a top quality Inner Cell Mass (Gardner Score A) (CG = 59.56%; G1=62.70%; G2=69.50%; G3=66.46%; p < 0.01), as well as an incremented number of embryos with a maximum quality trophectoderm (CG = 45.99%; G1=42.4%; G2=54.1%; G3=56.6; p < 0.01). Limitations, reasons for caution Analyze the cumulative pregnancy rates for the different groups would be ideal to assess if the different hyaluronidase solutions affect the efficiency of in vitro fertilization treatments. Wider implications of the findings This results show an improvement in embryo quality when lower concentrations of hyaluronidase are used. This allows both to increase the treatment efficiency as well as to reduce the economic impact of these. Trial registration number Not applicable

Influence of anticholinergic drugs on the development of an experimental periodontitis model.

The endogenous microbiome of the oral cavity plays an essential role in the development of periodontal disease. It also has a significant pathogenic effect on the inner-vation of the oral cavity organs. The experimental determination of the effectiveness of various drugs is required for the effective treatment of periodontal disease, and this involves the creation of a model of experimental periodontitis. The objective of this series of studies was to determine the possibility of reproduction of the experimental model of periodontitis and the study of the effects of anticholinergic drugs on the development of an experimental periodontitis model. The reproduction of the experimental model of periodontitis was performed by injecting the gums of rats with solutions of pathogenic factors: lipopolysaccharide, hyaluronidase and trypsin. We aimed to study the effect of anticholinergic drugs (pilocarpine and atropine) on the development of an experimental model of periodontitis after the injection of a hyaluronidase solution (2 mg/ml) into the rats' gums. The study was performed on white Wistar rats. Elastase activity, malonic dialdehyde content, urease activity (bacterial contamination index), lysozyme activity (an indicator of nonspecific immunity), and catalase activity (an antioxidant enzyme) were determined in the homogenate of the studied tissues. The results of a comparative study of the effect of three pathogenic factors (lipopolysaccharide, hyaluronidase, and trypsin) on the activity of elastase in different tissues of experimental animals (gums, tooth pulp, serum, and gastric mucosa) showed that hyaluronidase has the greatest proinflammatory effect. The action of pilocarpine and atropine was determined with an underline experimental periodontitis model. It was shown that both anticholinergic drugs stimulate the inflammatory process in the periodontium and that anticholinergic drugs enhance the proinflammatory effect of hyaluronidase.

Cross‐linked hyaluronic acid filler hydrolysis with hyaluronidase: Different settings to reproduce different clinical scenarios

Skin necrosis is the most severe complication arising from hyaluronic acid (HA) injection. To avoid skin necrosis, hyaluronidase should be injected along the course of the involved artery, to allow blood flow restoration. We evaluated the ability of hyaluronidase to degrade a HA filler in two simulated clinical situations-a compression case and an embolization case-to identify differences in the hyaluronidase injection. In the compression case, a bolus of HA filler was directly soaked in hyaluronidase solution; in the embolization case, a vein harvested from a living patient was filled with the same HA filler and then soaked in hyaluronidase. We then evaluated the quantity of HA remaining after 2 hr. While we found hydrolysis of HA in both cases, in the compression case, we detected almost complete hydrolysis, whereas in the embolization case we observed a reduction of the 60%. Our results support the hypothesis that vessel compression can be resolved with only one injection of hyaluronidase, while in the case of vascular embolization, repeated perivascular injections should be performed owing to the reduction of hyaluronidase activity.

An In Vitro Model Assessing the Penetration of Hyaluronidase through Optic Nerve Dura for Management of Hyaluronic Acid Facial Filler Embolism.

Blindness from ophthalmic or central retinal artery embolism is one of the most devastating complications of cosmetic filler facial injections. A proposed therapy to mitigate visual loss is prompt retrobulbar injection of hyaluronidase into the retrobulbar space. Despite Zhu et al. showing a lack of evidence and very limited published literature for reversing visual loss with this intervention, it is still widely accepted as a treatment for filler-related emboli. The purpose of this study was to evaluate the penetration of hyaluronidase through optic nerve dura using an in vitro model. At study conclusion, five 1-cm-long segments of fresh optic nerve were obtained and injected with highly crosslinked hyaluronic acid filler, then ligated on both ends in a watertight fashion. The sections were immersed in three concentrations of hyaluronidase solution for 24 hours. Histopathologic examination of the specimen was performed to assess the presence of filler. The optic nerve sections were 1.1 cm (range, 0.8 to 1.2 cm). Three were immersed in 20 ml of 1500 IU/ml hyaluronidase solution and two were immersed in saline as control. After 24 hours, there was a persistence of hyaluronic acid within the optic nerves. There is a lack of evidence for penetration of optic nerve sheath by hyaluronidase. This raises question about the effectiveness of retrobulbar injection of hyaluronidase in reversing filler-related blindness. Further studies are needed before this can be adopted as the treatment of choice. Therapeutic, V.

Construction of High Drug Loading and Enzymatic Degradable Multilayer Films for Self-Defense Drug Release and Long-Term Biofilm Inhibition.

Bacterial infections and biofilm formation on the surface of implants are important issues that greatly affect biomedical applications and even cause device failure. Construction of high drug loading systems on the surface and control of drug release on-demand is an efficient way to lower the development of resistant bacteria and biofilm formation. In the present study, (montmorillonite/hyaluronic acid-gentamicin)10 ((MMT/HA-GS)10) organic/inorganic hybrid multilayer films were alternately self-assembled on substrates. The loading dosage of GS was as high as 0.85 mg/cm2, which could be due the high specific surface area of MMT. The obtained multilayer film with high roughness gradually degraded in hyaluronidase (HAS) solutions or a bacterial infection microenvironment, which caused the responsive release of GS. The release of GS showed dual enzyme and bacterial infection responsiveness, which also indicated good drug retention and on-demand self-defense release properties of the multilayer films. Moreover, the GS release responsiveness to E. coli showed higher sensitivity than that to S. aureus. There was only ∼5 wt % GS release from the film in PBS after 48 h of immersion, and the amount quickly increased to 30 wt % in 105 CFU/mL of E. coli. Importantly, the high drug dosage, smart drug release, and film peeling from the surface contributed to the efficient antibacterial properties and long-term biofilm inhibition functions. Both in vitro and in vivo antibacterial tests indicated efficient sterilization function and good mammalian cell and tissue compatibility.

Enzymatic hydrolysis of chitosan films in water and physiological solution

It was shown that the processes of enzymatic hydrolysis of chitosan in aqueous acetic acid and on the surface of chitosan films in a solution of hyaluronidase in acetic acid are described by uniform kinetic constants. Kinetic parameters of enzymatic hydrolysis of the chitosan film samples in water and in physiological solution (Ringer–Locke’s solution) were determined. It was found that the introduction of medicinal agents and low-molecular-weight electrolytes to a chitosan-based film material reduces the rate of enzymatic hydrolysis of the films, which may indicate a possible increase in their service life when used on the wound surface.

Influência das fases de anestro e diestro na competência meiótica oocitária de cadelas

<p>A cadela apresenta particularidades reprodutivas que a diferencia de outras espécies. Diversos experimentos têm sido realizados visando estabelecer protocolos eficientes para a maturação, entretanto os resultados mostram-se insatisfatórios. Nesse aspecto, a fase reprodutiva da fêmea doadora deve ser considerada, já que pode ser um fator de variabilidade dos achados ate então presenciados nessa espécie. O objetivo deste estudo foi avaliar a influencia das fases do ciclo estral (anestro e diestro) na maturação <em>in vitro </em>(MIV) de cadelas. Os ovários foram transportados em solução de cloreto de sódio 0,9% e seccionados em solução salina fosfato tamponado (PBS) e os complexos <em>cumulus</em>-oócito (COCs) selecionados em meio de cultura de tecidos (TCM) 199 suplementado com HEPES. Foram obtidos 469 oócitos grau 1 de cadelas em anestro e diestro. Esses oócitos selecionados foram transferidos para o meio de maturação por um período de 72 horas, sendo posteriormente submetidos a solução de hialuronidase e corados com HOESCHT 33342 para avaliação da configuração nuclear. A comparação das fases de anestro e diestro não revelou diferença (p > 0,05) entre os estágios de maturação nuclear. Dessa maneira, a fase do ciclo estral não influenciou na maturação in vitro de oócitos caninos, incrementando os índices de M-II nesta espécie.</p>

285 REGULATION OF OOCYTE MEIOTIC RESUMPTION USING cAMP MODULATORS IN BOVINE IN VITRO MATURATION

In vitro maturation (IVM) is a reproductive technique critical to in vitro embryo production (IVP). Currently, IVP has low efficiency due to an inadequate IVM system where premature meiotic resumption results in low oocyte viability. Meiotic arrest is regulated primarily by 3′,5′-cyclic adenosine monophosphate (cAMP). Some successful methods of improving IVM have utilised cAMP modulators to maintain high intraoocyte cAMP, delaying the onset of nuclear maturation and allowing cytoplasmic maturation to occur. The current experiment is a follow-up to previous work in which an extended 2-step maturation system was examined. In the previous experiments, we found that meiotic resumption was significantly delayed, but the overall maturation rates of extended IVM were about half those of standard IVM, suggesting that the effects of the modulators on the oocytes were not completely reversible. The current experiment compares cAMP concentrations of oocytes in this extended IVM to standard IVM to determine whether high cAMP is the cause of the low maturation rate. Bovine oocytes (n = 686) were obtained from mixed-breed cattle by transvaginal ultrasound-guided aspiration. Oocytes from each cow were divided into 2 groups: standard IVM and extended IVM. Standard IVM consists of a 23-h maturation composed of TCM-199 supplemented with 10% fetal bovine serum, sodium pyruvate, pen/strep, glutamine, and FSH, and cultured at 39°C in 5% CO2. Extended IVM consists of 2 steps: a pre-IVM phase composed of HEPES-TALP supplemented with 100 µM forskolin (FSK) and 500 µM 3-isobutyl-1-methylxanthine (IBMX) for 2 h at 39°C, followed by an extended IVM phase composed of standard IVM media supplemented with 20 µM cilostamide for 31 h (39°C, 5% CO2). Additionally, oocytes in the extended IVM treatment group where held in HEPES-TALP media with FSK and IBMX during the 2-h oocyte collection period in order to prevent any decrease in cAMP before the oocytes could be placed in the extended IVM media. Oocytes in standard IVM were sampled at 0, 8, and 23 h of maturation, while oocytes in extended IVM were sampled at 0, 8, 18, and 33 h of maturation. Cumulus cells were removed from all oocytes by vortexing in hyaluronidase solution. Oocytes were stored in groups of 10 at –80°C. A cAMP enzyme immunoassay (GE Healthcare) was performed to determine cAMP concentrations throughout standard and extended IVM. Assay results were analysed using an ANOVA followed by a Tukey's pairwise test (Sigma Stat 3.5) to detect significant differences (P < 0.05). Results indicated significantly higher cAMP levels in extended IVM oocytes during the first 3 h after collection using FSK and IBMX in the holding media (0.505 v. 1.006 fmol/oocyte, P = 0.035) but cAMP levels were not maintained in the cilostamide-only extended IVM medium. This suggests that high cAMP levels were not the cause of low maturation rates in extended IVM, since cAMP concentrations did decrease after 3 h. Possible negative effect of cilostamide on these oocytes may be suggested and need to be analysed.

Gamma ray-induced synthesis of hyaluronic acid/chondroitin sulfate-based hydrogels for biomedical applications

Hyaluronic acid (HA)/chondroitin sulfate (CS)/poly(acrylic acid) (PAAc) hydrogel systems were synthesized by gamma-ray irradiation without the use of additional initiators or crosslinking agents to achieve a biocompatible hydrogel system for skin tissue engineering. HA and CS derivatives with polymerizable residues were synthesized. Then, the hydrogels composed of glycosaminoglycans, HA, CS, and a synthetic ionic polymer, PAAc, were prepared using gamma-ray irradiation through simultaneous free radical copolymerization and crosslinking. The physicochemical properties of the HA/CS/PAAc hydrogels having various compositions were investigated to evaluate their feasibility as artificial skin substitutes. The gel fractions of the HA/CS/PAAc hydrogels increased in absorbed doses up to 15kGy, and they exhibited 91–93% gel fractions under 15kGy radiation. All of the HA/CS/PAAc hydrogels exhibited relatively high water contents of over 90% and reached an equilibrium swelling state within 24h. The enzymatic degradation kinetics of the HA/CS/PAAc hydrogels depended on both the concentration of the hyaluronidase solution and the ratio of HA/CS/PAAc. The in vitro drug release profiles of the HA/CS/PAAc hydrogels were significantly influenced by the interaction between the ionic groups in the hydrogels and the ionic drug molecules as well as the swelling of the hydrogels. From the cytotoxicity results of human keratinocyte (HaCaT) cells cultured with extracts of the HA/CS/PAAc hydrogels, all of the HA/CS/PAAc hydrogel samples tested showed relatively high cell viabilities of more than 82%, and did not induce any significant adverse effects on cell viability.

Transitioning between Denudation Media: Is There a Difference in the Fractured Zona Damage Rates?

Removal of cumulus cells surrounding oocytes, denudation, is a necessary step prior to some Assisted Reproductive Technology procedures. An important aspect during the chemical/physical removal of cumulus cells is to minimize the risk of fractured zona pellucida (FZ) damage. In our labratory, two denudation medias have been used to facilitate this action: Irvine Scientific's Hyaluronidase Solution (HYAL) and Origio's ICSI Cumulase® (CUMU). Over a 12 month time period we initially used HYAL, then transitioned to CUMU and finally returned back to HYAL.

Hyaluronic acid/chondroitin sulfate-based hydrogel prepared by gamma irradiation technique

Gamma-ray irradiation of novel hydrogels was used to develop a biocompatible hydrogel system for skin tissue engineering. These novel hydrogels are composed of natural polymers including hyaluronic acid (HA) and chondroitin sulfate (CS), and the synthetic polymer, poly(vinyl alcohol) (PVA). The γ-ray irradiation method has advantages, such as relatively simple manipulation without need of any extra reagents for polymerization and cross-linking. We synthesized HA and CS derivatives with polymerizable residues. The HA/CS/PVA hydrogels with various compositions were prepared by using γ-ray irradiation technique and their physicochemical properties were investigated to evaluate the feasibility of their use as artificial skin substitutes. HA/CS/PVA hydrogels showed an 85–88% degree of gelation under 15kGy radiation. All HA/CS/PVA hydrogels exhibited more than 90% water content and reached an equilibrium swelling state within 24h. Hydrogels with higher concentrations of hyaluronidase solution and HA/CS content had proportionally higher enzymatic degradation rates. The drug release behaviors from HA/CS/PVA hydrogels were influenced by the composition of the hydrogel and drug properties. Exposure of human keratinocyte (HaCaT) culture to the extracts of HA/CS/PVA hydrogels did not significantly affect the cell viability. All HaCaT cell cultures exposed to the extracts of HA/CS/PVA hydrogels exhibited greater than 92% cell viability. The HaCaT growth in HA/CS/PVA hydrogels gradually increased as a function of culture time. After 7 days, the HaCaT cells in all HA/CA/PVA hydrogels exhibited more than 80% viability compared to the control group HaCaT culture on a culture plate.

A new method for determination of hyaluronidase activity in biological samples using capillary zone electrophoresis

The aim of the study was to develop a new capillary zone electrophoresis (CZE) method for determination of enzymatic activity of hyaluronidase. The method permits monitoring of the process of hyaluronic acid digestion by hyaluronidase. Studies were performed using CZE instrument equipped with capillary of 64.5 cm total length, 56 cm effective length and internal diameter 75 µm. Separation was performed in the phosphate buffer (pH 8.10) in the electric field of 20 kV, λ = 220 nm. The procedure was based on mixing a known quantity of hyaluronic acid and an aliquot of hyaluronidase solution, followed by obtaining CZE profiles after a known period of incubation (0.5 h). The activity of hyaluronidase was calculated using multiple regression analysis in which sizes of the peaks of the main degradation products were used. The newly developed method was fully validated and it is appropriate to evaluate the activity of hyaluronidase originating from different sources with high precision and accuracy. t-Tests showed that there were no significant differences between results obtained using turbidimetric, viscosimetric and the new CZE method. The developed method is characterized by a short duration of analysis, low volume of analyzed sample, small amount of buffers used and low cost of analysis.

Improved stability and tumor targeting of 5‐fluorouracil by conjugation with hyaluronan

To circumvent the rapid clearance in vivo and consequent low tumor targeting of 5‐fluorouracil (5‐Fu), hyaluronan‐5‐fluorouracil conjugate (HA‐5‐Fu) was firstly synthesized and characterized. The stability of HA‐5‐Fu in vitro was evaluated by incubation with phosphate buffered saline, hyaluronidase solution, and mice plasma, respectively. The tumor targeting was tested by in vitro cytotoxicity evaluation and in vivo pharmacokinetics study in plasma and tumor. HA‐5‐Fu with drug loading of 87.674 mg/g was successfully obtained and confirmed. HA‐5‐Fu showed high stability in acidic environment and moderate stability under enzymatic cleavage. The enhanced cytotoxicity of HA‐5‐Fu over 5‐Fu depended on drug concentration, incubation time, and cell lines type. The t1/2 of HA‐5‐Fu in plasma after injection of prodrug was extended up to 10 times compared with that of 5‐Fu. Notably, AUC0–t in tumors of HA‐5‐Fu was 3.6 times higher than 5‐Fu, demonstrating its excellent tumor targeting and quite promising prospect in anti‐cancer therapy. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 130: 927‐932, 2013

A feasibility study for evaluation of mechanical properties of articular cartilage with a two-electrode electrical impedance method

BackgroundSince articular cartilage has important mechanical properties such as load-bearing, shock absorption and lubrication for activities in daily life, it is important to evaluate the mechanical properties of repaired cartilage in terms of whether those properties are the same as those of natural cartilage. The purpose of this study was to investigate the effectiveness of an electrical impedance method for quantitatively measuring the mechanical properties of cartilage. MethodsCartilage specimens were harvested from porcine knee joint, and two kinds of enzyme-treated cartilages were prepared to investigate the correlation between mechanical and electrical properties resulting from changes in the structure of the extracellular matrix. Collagenase solution and hyaluronidase solution were used to digest the collagen fibril and proteoglycan, respectively. Electrical impedance measurement, indentation test and biochemical analysis were carried out for the enzyme-treated cartilages. ResultsThe water content increased with enzyme treatment time, and the permeability of the treated cartilages increased with decreasing glycosaminoglycan content for both types of enzyme-treated cartilages. The aggregate modulus and the electrical resistivity for both types of enzyme-treated cartilages decreased with increasing permeability after 12-h treatment. The aggregate modulus and the electrical resistivity for both types of treated cartilages decreased with increasing water content and permeability after 24-h treatment. The electrical resistivity and the aggregate modulus of articular cartilage depended not only on the water content, but also on the permeability, and the electrical resistivity for both types of enzyme-treated cartilages was found to be significantly linearly correlated with the aggregate modulus. ConclusionsThese results showed that the aggregate modulus of articular cartilage can be estimated by measuring its electrical impedance.

Can the Use of Hyaluronidase Reduce Capsule Formation?

The purpose of this study was to find whether the use of hyaluronidase reduces capsule formation. Ten New Zealand White rabbits were used. Eight pockets were created using an aseptic technique, four on the right side and four on the left side, along the vertebral column of every animal. One piece of silicone from a silicone block was inserted inside every pocket. The dimensions of each piece were 3.5×2×1.5cm. In every pocket on the right side of each animal we placed 0.5ml of hyaluronidase solution. The animals were sacrificed 5months postoperatively. Capsule formation in the each side of the animals was compared. Two rabbits presented infection in two pockets and were excluded from the study. There was a statistically significant difference between groups concerning the capsule thickness variable using parametric (P=0.003) and nonparametric (P=0.001) analysis [capsule thickness on the right side: 256.46±114.88 (mean±SD) and on the left side: 369.10±147.81 (mean±SD); capsule thickness on the right side: 235.69 (104.72) [median (IQR)] and on the left side: 332.12 (188.68) median (IQR)]. The use of hyaluronidase may reduce capsule formation around implants.