Hyaluronan Supplementation Research Articles

Laminin and hyaluronan supplementation of collagen hydrogels enhances endothelial function and tight junction expression on three-dimensional cylindrical microvessel-on-a-chip

Vasculature-on-chip (VoC) models have become a prominent tool in the study of microvasculature functions because of their cost-effective and ethical production process. These models typically use a hydrogel in which the three-dimensional (3D) microvascular structure is embedded. Thus, VoCs are directly impacted by the physical and chemical cues of the supporting hydrogel. Endothelial cell (EC) response in VoCs is critical, especially in organ-specific vasculature models, in which ECs exhibit specific traits and behaviors that vary between organs. Many studies customize the stimuli ECs perceive in different ways; however, customizing the hydrogel composition accordingly to the target organ's extracellular matrix (ECM), which we believe has great potential, has been rarely investigated. We explored this approach to organ-specific VoCs by fabricating microvessels (MVs) with either human umbilical vein ECs or human brain microvascular ECs in a 3D cylindrical VoC using a collagen hydrogel alone or one supplemented with laminin and hyaluronan, components found in the brain ECM. We characterized the physical properties of these hydrogels and analyzed the barrier properties of the MVs. Barrier function and tight junction (ZO-1) expression improved with the addition of laminin and hyaluronan in the composite hydrogel.

Embryo Glue: The Use of Hyaluronan in Embryo Transfer Media.

EmbryoGlue is available to patients at many in vitro fertilization clinics, usually at an additional cost. The efficacy of hyaluronan-enriched transfer medium (HETM) is supported by moderate quality evidence that indicates a significant improvement in clinical outcomes such as live birth rates for patients, including poorer prognosis women (i.e., maternal age factor [>35 years] and recurrent implantation failure). An increased multiple pregnancy rate has been reported with the use of HETM; therefore, a single embryo transfer policy should be considered in conjunction with the use of EmbryoGlue. There is no evidence to suggest that HETM has any detrimental impact, and therefore the use of HETM in clinics may be justified for a specific demographic of patients. Further robust evidence, in the form of meta-analyses or large-scale randomized controlled trials, is needed to build a sufficient consensus regarding the benefit of hyaluronan supplementation in embryo transfer media.

Hyaluronan supplementation as a mechanical regulator of cartilage tissue development under joint-kinematic-mimicking loading.

Articular cartilage plays an essential role in joint lubrication and impact absorption. Through this, the mechanical signals are coupled to the tissue's physiological response. Healthy synovial fluid has been shown to reduce and homogenize the shear stress acting on the cartilage surfaces due to its unique shear-thinning viscosity. As cartilage tissues are sensitive to mechanical changes in articulation, it was hypothesized that replacing the traditional culture medium with a healthy non-Newtonian lubricant could enhance tissue development in a cartilage engineering model, where joint-kinematic-mimicking mechanical loading is applied. Different amounts of hyaluronic acid were added to the culture medium to replicate the viscosities of synovial fluid at different health states. Hyaluronic acid supplementation, especially at a physiologically healthy concentration (2.0 mg ml-1), promoted a better preservation of chondrocyte phenotype. The ratio of collagen II to collagen I mRNA was 4.5 times that of the control group, implying better tissue development (however, with no significant difference of measured collagen II content), with a good retention of collagen II and proteoglycan in the mechanically active region. Simulating synovial fluid properties by hyaluronic acid supplementation created a favourable mechanical environment for mechanically loaded constructs. These findings may help in understanding the influence of joint articulation on tissue homeostasis, and moreover, improve methods for functional cartilage tissue engineering.

Improved quality of porcine embryos cultured with hyaluronan due to the modification of the mitochondrial membrane potential and reactive oxygen species level

Although considerable progress has been made in pig embryo culture systems, the developmental competence and quality of the produced embryos are still lower than their in vivo-derived counterparts. Because hyaluronan (HA) regulates various cellular processes and possesses antioxidant properties, this glycosaminoglycan seems to be a promising supplement in culture media. However, until now, its beneficial influence on in vitro pig embryo development has been debatable. Hence, we aimed to investigate the effect of 0.25 mg/mL, 0.5 mg/mL and 1 mg/mL concentrations of HA on the developmental potential and quality of cultured porcine embryos. We found that 1 mg/mL HA supplementation significantly increased the obtained percentages of cleaved embryos to ∼95%, morulae to ∼87% and blastocysts to ∼77%. At 0.5 mg/mL and 1 mg/mL HA concentrations, we observed a significantly improved blastocyst quality, expressed as the total number of cells per blastocyst, number of cells in the inner cell mass, number of TUNEL-positive nuclei per blastocyst, the TUNEL index and the blastocyst diameter. Because the inner mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) level are important for proper embryo development, for the first time, we measured these two parameters in cultured embryos at various HA concentrations and during their development up to the expanded blastocyst stage. For blastocysts cultured with 1 mg/mL HA, the ΔΨm and ROS level were ∼1.6 and 2.7 times lower, respectively, than those of the control blastocysts. Both ΔΨm and the ROS level were increased in parallel during in vitro embryo development with and without HA, but this increase was less pronounced in the presence of HA. Hence, our quantitative data unequivocally show that supplementation of NCSU-23 culture medium with 1 mg/mL HA improves the developmental potential and quality of pig embryos. This effect results from a significant decrease in the ROS level induced by the HA-dependent ΔΨm reduction.

In vitro and ex vivo analysis of hyaluronan supplementation of Integra® dermal template on human dermal fibroblasts and keratinocytes.

Widespread application of collagen-glycosaminoglycan dermal templates in the treatment of cutaneous defects has identified the interval between initial engraftment and skin graft application as important for improvement. The aim of this study was to evaluate the effect of hyaluronan supplementation of Integra® dermal template on human dermal fibroblasts and keratinocytes in both in vitro and ex vivo models. This study utilized in vitro and ex vivo cell culture techniques to investigate supplementing Integra® Regeneration Template with hyaluronan (HA), as a strategy to decrease this interval. In vitro, Integra® was HA supplemented at 0.15, 1, 1.5 and 2 mg/mL-1. Primary human dermal fibroblast (PHDF) and keratinocyte proliferation, PHDF viability, migration and HA-induced signal transduction (phosphor-MAPK Array) were assessed. Ex vivo, wound models (wound diameter 4 mm) were created within 8 mm skin biopsies. Wounds were filled with Integra® or HA supplemented Integra®. Re-epithelialization was compared through hematoxylin and eosin-stained cross-sections at 7, 14 and 21 days in culture. Model viability was assessed through lactate dehydrogenase (LDH) assays. In vitro, PHDF and keratinocyte proliferation were enhanced significantly (p<0.001) when supplemented with HA. S-Phase and G2/M PHDFs in HA supplemented scaffolds increased. PHDF viability was enhanced to 72 hours culture with 1.5 mg/mL-1 HA (p = 0.016). PHDF migration was maximally enhanced at 1 mg/mL-1 and 1.5 mg/mL-1, whilst increased levels of phosphorylated Erk/MAPK proteins indicated increased metabolic activity. In ex vivo models, HA supplementation accelerated re-epithelialization at all concentrations. This ex vivo model provides a robust model for preclinical assessment of skin substitutes. HA supplementation to Integra® demonstrates increased in vitro growth, viability and migration. Whilst ex vivo data suggest HA supplementation of Integra® may increase rapidity of wound closure.

80 REACTIVE OXYGEN SPECIES LEVEL IN PIG EMBRYOS CULTURED IN PRESENCE OF HYALURONAN

It has been reported that during in vitro embryo culture reactive oxygen species (ROS) are generated and are detrimental to embryo development. A recent study (Smorag et al., Proc. 9th ICPR, 2013, 110) demonstrated that an addition of 1 mg mL–1 of hyaluronan (HA) to porcine embryo culture medium improves the development of zygote to blastocyst stage and the quality of produced embryos. Moreover, the embryos cultured with HA showed lower inner mitochondrial membrane potential (Romek et al., 2015 Proc. Symp. Progress in Cell Biology: Mitochondria and Chloroplast, Krakow, 31). Based on the beneficial effect provided by supplementation of HA during embryo culture, we investigated the ROS level in porcine embryos cultured with HA. Porcine zygotes were obtained surgically after flushing the oviducts of superovulated and inseminated gilts. In the experimental group, zygotes were cultured up to the blastocyst stage in NCSU-23 medium supplemented with 1 mg mL–1 of HA (CROMA, Pharma GmbH, Leobendorf, Austria), in an atmosphere containing 5% CO2 in air, at 39°C. In the control group, HA supplementation was omitted. To measure ROS level, embryos at the stages 2–4 and 8–16 cell, morula, and blastocyst (experimental group) and zygote, 2–4 and 8–16 cell, morula, and blastocyst (control group) were labelled with 5 μM CM-H2DCFDA dye (Molecular Probes Inc., OR, USA) for 30 min at 39°C. Labelled embryos were then examined under a Nikon Eclipse microscope with a CCD camera. The total amount of fluorescence emitted from each individual embryos and proportional to the ROS level was measured in arbitrary units. The data were analysed using one-way ANOVA and post-hoc Tukey test. ROS level (mean ± standard error of the mean) in the experimental group was 8.21 ± 2.65 (n = 25), 10.31 ± 3.13 (n = 18), 9.08 ± 2.89 (n = 21), and 20.45 ± 2.38 (n = 31) for 2–4 cell, 8–16 cell, morula, and blastocyst, respectively, whereas in the control group was 9.15 ± 3.43 (n = 15), 7.11 ± 3.13 (n = 18), 8.67 ± 3.04 (n = 19), 11.47 ± 2.46 (n = 29), and 54.74 ± 2.89 (n = 21) for zygote, 2–4 cell, 8–16 cell, morula, and blastocyst, respectively. For experimental and control groups, ROS levels remained unchanged up to morula. On the contrary, at the blastocyst stage from the experimental group ROS level decreased significantly (P ≤ 0.05) in comparison with blastocysts from the control group. In conclusion, porcine blastocysts derived from zygotes cultured with supplementation of 1 mg of HA possess a significantly lower ROS level than blastocysts cultured without HA. This suggests that HA supplementation in culture medium can reduce the ROS level in porcine cultured blastocysts. The project was funded by the National Science Center based on decision number DEC-2012/07/B/NZ9/01326.

Ex vivo evaluation of acellular and cellular collagen-glycosaminoglycan flowable matrices

Collagen-glycosaminoglycan flowable matrices (CGFM) are increasingly finding utility in a diversifying number of cutaneous surgical procedures. Cellular in-growth and vascularisation of CGFM remain rate-limiting steps, increasing cost and decreasing efficacy. Through in vitro and ex vivo culture methods, this study investigated the improvement of injectable CGFM by the incorporation of hyaluronan (HA) and viable human cells (primary human dermal fibroblasts (PHDFs) and bone marrow-derived mesenchymal stem cells (BM-MSCs)). Ex vivo investigations included the development and evaluation of a human cutaneous wound healing model for the comparison of dermal substitutes. Cells mixed into the Integra Flowable Wound Matrix (IFWM), a commercially available CGFM, were confirmed to be viable and proliferative through MTT assays (p < 0.05). PHDFs proliferated with greater rapidity than BM-MSCs up to 1 week in culture (p < 0.05), with PHDF proliferation further enhanced by HA supplementation (p < 0.05). After scaffold mixing, gene expression was not significantly altered (qRT-PCR). PHDF and BM-MSC incorporation into ex vivo wound models significantly increased re-epithelialisation rate, with maximal effects observed for BM-MSC supplemented IFWM. HA supplementation to PHDF populated IFWM increased re-epithelialisation but had no significant effect on BM-MSC populated IFWM. In conclusion, when combined with PHDF, HA increased re-epithelialisation in IFWM. BM-MSC incorporation significantly improved re-epithelialisation in ex vivo models over acellular and PHDF populated scaffolds. Viable cell incorporation into IFWM has potential to significantly benefit wound healing in chronic and acute cutaneous injuries by allowing a point-of-care matrix to be formed from autologous or allogenic cells and bioactive molecules.

Influence of fetuin and hyaluronan on the post-thaw quality and fertilizing ability of Holstein bull semen

It was determined that fetuin and hyaluronan supplementation did not provide any significant effect on the post-thaw subjective and CASA motility percentages and sperm motion characteristics, in comparison to the controls (P>0.05).Sperm acrosome and total abnormalities were similar in all groups (P>0.05). Groups M (hyaluronan+fetuin) and H (hyaluronan) displayed a higher rate of sperm membrane integrity, compared to that of Group C (control) (P<0.01). According to the results of the comet assay, the lowest percentage of sperm with damaged DNA was achieved in Group H, when compared to all of the experimental groups (P<0.01). Furthermore, all of the additives resulted in a lower rate of sperm with damaged DNA than that of the controls, and thus, reduced DNA damage (P<0.01). For pregnancy rates, there were no significant differences between the extender groups (P>0.05).MDA formation was found to be lower in Groups M and F (P<0.01). In Group M, SOD activity was determined to have significantly increased (23.61±5.62U/ml) compared to the other groups (P<0.01). All experimental groups had a GSH-Px activity higher than that of the control group (P<0.01).

131 EFFECTS OF HIGH MOLECULAR WEIGHT HYALURONAN ON SHEEP EMBRYO DEVELOPMENT AND SURVIVAL AFTER CRYOPRESERVATION

Hyaluronan (HA), a component of extracellular matrix in mammalian tissues including that of the reproductive system, has been shown to support embryo development. HA is produced in various sizes with distinct physiological functions. Cleaved sheep embryos produced after in vitro maturation and in vitro fertilization were cultured in serum free synthetic oviduct fluid medium supplemented with increasing concentrations (0, 0.25, 0.5, and 1 mg mL–1) of large size HA (Healon; 6 × 106 Da). Development to blastocyst stage was recorded at Day 7 when a group of the embryos were fixed and stained by differential staining combined with TUNEL labelling to analyse embryo quality. The remainder of the blastocysts from each treatment/repeat were vitrified in open pulled straws and then cultured for an extra period of 48 h to analyse their survival rate and quality after cryopreservation. SPSS version 20 software (SPSS Inc., Chicago, IL, USA) was used for analyzing the data with generalized linear model. Healon did not change blastocyst (33 ± 5.7, 32 ± 6.0, 35 ± 5.5; P ≥ 0.05) or survival rates (63 ± 17.1, 83 ± 15.2, 58 ± 14.2; P ≥ 0.05) as compared to the respective controls (25 ± 5.2, 38 ± 17.1). It increased the total cell (TC) number (83.6 ± 4.6, 100.7 ± 3.8, 97.2 ± 3.7, 105.0 ± 3.9; P ≤ 0.05) and trophectoderm cells (TE) (58.4 ± 3.8, 74.2 ± 3.2, 75.6 ± 3.3, 80.1 ± 3.4; P ≤ 0.05) but had no effect on the number of inner cell mass (ICM) and apoptotic cells. The ICM : TE ratio was not affected (0.45 ± 0.04, 0.36 ± 0.03, 0.33 ± 0.03, 0.30 ± 0.03; P ≥ 0.05). Surviving embryos had higher TC (63.2 ± 3.7, 130.8 ± 3.6, 113.9 ± 5.2, 149.8 ± 5.4; P ≤ 0.05), TE (42.9 ± 3.0, 96.7 ± 3.1, 85.2 ± 4.5, 111.9 ± 4.7; P ≤ 0.05) and ICM (20.3 ± 2.2, 32.9 ± 1.8, 27.7 ± 2.6, 36.5 ± 2.7; P ≤ 0.05). The apoptotic cell numbers and ICM : TE ratio of the survived embryos after cryopreservation were not affected by HA supplementation. The results indicate that large size HA improves the embryo viability and quality, which may have implication for improving embryo transfer.

Evidences for correlation between the reduced VCAM-1 expression and hyaluronan synthesis during cellular senescence of human mesenchymal stem cells

Mesenchymal stem cells (MSCs) undergo cellular senescence during in vitro expansion culture, which accompanies the loss of migration and homing abilities. In this study, we analyzed expression levels of several surface markers of human MSCs at different passages of expansion culture. It has been shown that expression of vascular cell adhesion molecule-1 (VCAM-1) was most markedly decreased among the tested markers in the senescent MSCs. Interestingly the reduced VCAM-1 expression could be restored by applying hyaluronan, a major glycosaminoglycan ligand of CD44, to the culture. It was found that the hyaluronan level in extracellular and pericellular matrices was greatly reduced in the senescent MSCs, mainly due to the decreased expression of hyaluronan synthases, suggesting a correlation between the reduced VCAM-1 expression and hyaluronan synthesis. In fact, when hyaluronan synthases were knock-downed by siRNA transfection, the VCAM-1 expression was also reduced. Our results indicate that VCAM-1 expression in the senescent MSCs was down-regulated because of the reduced synthesis of hyaluronan. Thus, we suggest that hyaluronan supplementation in expansion culture of MSCs would compensate adverse effects induced by its decreased synthesis and subsequently enhance cell adhesion and migration abilities.

Efficacy of intra-articular polynucleotides in the treatment of knee osteoarthritis: a randomized, double-blind clinical trial

This randomized, double-blind clinical trial was conducted over 16 weeks to assess the efficacy and safety profile of intra-articular polynucleotides gel injections in the treatment of knee osteoarthritis associated with persistent knee pain. 60 patients were enrolled and randomized to receive intra-articular polynucleotides (n = 30) or hyaluronan (n = 30); patients received five weekly intra-articular knee injections and the follow-up period was 3 months after the end of treatment. Primary endpoint was to determine polynucleotides (PN) efficacy in reducing knee pain at the end of the study, over baseline value and over standard hyaluronan viscosupplementation (HA). Pain levels were measured using a 0-10 cm Visual Analogue Scale (VAS). Secondary endpoints included Knee Osteoarthritis Outcome Score (KOOS), NSAIDs consumption, crackling during movement and articular mobility limitation. The mean global VAS pain decreased from 5.7 + or - 1.9 cm (T0) to 1.9 + or - 1.5 cm (T16) in polynucleotide group and from 4.9 + or - 2.0 cm (T0) to 2.1 + or - 1.4 cm (T16) in hyaluronan group. The reduction in pain was statistically significant for both groups. KOOS increases from baseline values were statistically significant in both groups. No significant adverse events were reported. These findings suggest that intra-articular polynucleotides can be a valid alternative to traditional hyaluronan supplementation for the treatment of knee osteoarthritis.

Cartilage shear dynamics during tibio-femoral articulation: effect of acute joint injury and tribosupplementation on synovial fluid lubrication

To determine the effects of acute injury and tribosupplementation by hyaluronan (HA) on synovial fluid (SF) modulation of cartilage shear during tibio-femoral articulation. Human osteochondral blocks from the lateral femoral condyle (LFC) and tibial plateau (LTP) were apposed, compressed 13%, and subjected to sliding under video microscopy. Tests were conducted with equine SF from normal joints (NL-SF), SF from acutely injured joints (AI-SF), and AI-SF to which HA was added (AI-SF+HA). Local and overall shear strain (E(xz)) and the lateral displacement (Deltax) at which E(xz) reached 50% of peak values (Deltax(1/2)) were determined. During articulation, LFC and LTP cartilage E(xz) increased with Deltax and peaked when surfaces slid, with peak E(xz) being maintained during sliding. With AI-SF as lubricant, surface and overall Deltax(1/2) were approximately 40% and approximately 20% higher, respectively, than values with NL-SF and AI-SF+HA as lubricant. Also, peak E(xz) was markedly higher with AI-SF as lubricant than with NL-SF as lubricant, both near the surface (approximately 80%) and overall (50-200%). Following HA supplementation to AI-SF, E(xz) was reduced from values with AI-SF alone by 30-50% near the surface and 20-30% overall. Magnitudes of surface and overall E(xz) were markedly (approximately 50 to 80%) higher in LTP cartilage than LFC cartilage for all lubricants. Acute injury impairs SF function, elevating cartilage E(xz) markedly during tibio-femoral articulation; such elevated E(xz) may contribute to post-injury associated cartilage degeneration. Since HA partially restores the function of AI-SF, as indicated by E(xz), tribosupplements may be beneficial in modulating normal cartilage homeostasis.

Effects of Hyaluronan Supplementation on Cryopreserved Equine Spermatozoa Hyaluronan and Cryopreserved Equine Spermatozoa

Addition of hyaluronan, a nonsulfated glycosaminoglycan, to fresh and frozen thawed human semen results in substantial retention of motility over time. Hyaluronan also has been reported to preserve postthaw viability and maintain membrane stability of boar spermatozoa. Therefore, experiments were designed to investigate the use of a commercially available hyaluronan (Map-5, Bioniche Animal Health, Inc., Athens, GA) in freezing extender for cryopreservation of equine spermatozoa. In experiment 1, aliquots from ejaculates were supplemented before freezing with one of four levels of hyaluronan: 100 μg/mL, 200 μg/mL, 400 μg/mL, and 1000 μg/mL along with an untreated control. No differences in sperm motility, assessed by computer-assisted sperm motility analysis (CASA), were found for any treatment at times 0, 30, or 60 minutes postthaw. Decreases in motility were noted in the highest hyaluronan group (1,000 μg/mL) after 90 and 120 minutes of incubation. Sperm viability, as assessed using SYBR-14/propidium iodide staining, was decreased ( P < .05) when treated with 1,000 μg/mL compared with the control (37.1% and 46.1%, respectively). Motility parameters tended to remain elevated in those ejaculates treated with 200 μg/mL at various time points. Experiment 2, therefore, further investigated the effects of hyaluronan at 200 μg/mL on motility parameters and acrosome integrity and zona pellucida binding. Total (TM) and progressive (PM) motility of treated sperm immediately after thawing and at 60 minutes post-thaw were higher compared with control ( P < .05). A tendency ( P < .1) to maintain TM at 90 and 120 minutes post-thaw also was noted. No differences were noted for the mean number of spermatozoa bound to bovine oocytes for control or treated sperm (22 ± 14 vs 25 ± 17, respectively). Acrosome integrity also was unchanged between the two groups based on fluorescein isothiocyanate (FITC)−peanut agglutinin (PNA)/propidium iodide staining. All samples contained <1% live acrosome-damaged spermatozoa. In the final experiment, the effects of hyaluronan supplementation post-thaw was investigated using hyaluronan concentrations of 100, 200, and 400 μg/mL. Motility parameters studied over an 8-hour period at 37°C yielded no consistent differences. In conclusion, addition of hyaluronan at a concentration of 200 μg/mL before freezing increased spermatozoal post-thaw motility. High concentration of hyaluronan (1,000 μg/mL) appeared to be detrimental to post-thaw motility. Effects of hyaluronan on fertility are beyond the scope of this study and have yet to be determined.

A reappraisal of the biological effects of hyaluronan on human dermal fibroblast

Hyaluronan (HA) is a fundamental component of many biomaterials. Reports on the biological effects of HA are, however, both inconsistent and contradictory. This particularly refers to fibroblast contraction in the presence of high concentrations of HA. In this study we used a broad range of molecular weight (MW) and concentrations of HA to assay its influence on human dermal fibroblasts in both two and three-dimensional culture models. High-MW HA in high concentrations demonstrated inhibitory effects on both cell attachment and proliferation. HA-coated surfaces appeared less adhesive to fibroblasts than collagen-coated surfaces. In the fibroblast-populated collagen lattice, HA supplementation dose-dependently retarded gel contraction, which was associated with reduced cell division and spreading. Although gene transcripts of collagen type I and III were not significantly upregulated in the monolayer culture as determined by real-time PCR analysis, more type III collagen was present in HA-incorporated collagen gels by Sirius red staining. Type III collagen was homogeneously distributed and well organized in the presence of HA. Our data indicates that HA has both a qualitative and quantitative effect on cell-matrix interactions. Optimum parameters in terms of MW and concentration need to be determined when HA-based biomaterials are being prepared for tissue-repair purposes.

Fragment size- and dose-specific effects of hyaluronan on matrix synthesis by vascular smooth muscle cells

Tissue engineering of vascular elastin matrices disrupted by mechanical injury, disease, or congenitally absent, is among other factors, limited by the lack of suitable cell scaffolds to up-regulate and guide innately poor elastin synthesis by adult vascular smooth muscle cells (SMCs). Evidence suggests that scaffolds based on hyaluronan (HA), a glycosaminoglycan, may be useful to elicit elastogenic cell responses, although these effects appear to be dictated by HA fragment size and/or dose. This study investigates the efficacy of a simple, frequently adopted exogenous HA supplementation model to test this hypothesis. Rat aortic SMCs were cultured with HA (2×10 6 Da (HMW)⩾MW⩽2.2×10 4 Da) supplemented at doses between 0.2 and 200 μg/ml. Cell layers were biochemically assayed for DNA, elastin and collagen content. Fragmented, but not high molecular weight (HMW) HA, stimulated cell proliferation in inverse correlation fragment size while the opposite effect was observed for synthesis of soluble and matrix elastin; almost no dose effects were observed within any group. SDS-Page/Western Blot and a desmosine assay semi-quantitatively confirmed the observed biochemical trends for tropoelastin and matrix elastin, respectively. Quantitative differences in elastin deposition were mirrored in TEM micrographs. Elastin was mostly deposited in the form of amorphous clumps but fibers were increasingly present in cell layers cultured with HMW HA. HA and its fragments did not disrupt normal fibrillin-mediated mechanisms of elastin matrix deposition. While the current outcomes confirm that the effects of HA on elastin synthesis are fragment size-specific, this study shows that an exogenous supplementation model does not necessarily simulate cellular matrix synthesis responses to HA-based biomaterial scaffolds.

Intra-articular Hyaluronan Following Autogenous Osteochondral Grafting of the Knee

The aim of this study was to assess whether intra-articular hyaluronan viscosupplementation after osteochondral grafting (mosaic arthroplasty) of the knee allowed better graft integration and function in a sheep model. Experimental study. Twelve adult sheep underwent a mosaic arthroplasty procedure to a standardized osteochondral defect on the medial femoral condyle. One week after surgery the animals were randomized to receive a course of weekly intra-articular injections for 5 weeks of either sodium-hyaluronate solution 25 mg/2.5 mL (HA group) or of a 2.5 mL buffer solution (control group). Synovial fluid samples were taken preoperatively, preceding each injection, and at 8 and 12 weeks. The animals were euthanized at 12 weeks, the knees dissected out, and biomechanical and histologic assessments were made. There was no difference in the synovial fluid leukocyte or total protein concentration between the groups, but the hyaluronan concentration was statistically higher in the HA group. Aggregate moduli of the articular surface were statistically higher in the graft articular cartilage in the HA group than in the control group, as were the sulphated glycosaminoglycan levels. Histologic assessment found more articular cartilage flow in the HA group, whereas there was more interstitial tissue present in the interstices between the grafts in the control group. Results from this study suggest that hyaluronan viscosupplementation following osteochondral grafting does convey some beneficial effects on graft cartilage in the early postoperative period in an ovine model. This study suggests that, in the early postoperative period, hyaluronan supplementation improves articular cartilage survival after osteochondral grafting.

Effect of hyaluronan supplementation on boar sperm motility and membrane lipid architecture status after cryopreservation

We investigated the effect of supplementing extended boar semen with different amounts of hyaluronan (HA) prior to freezing on post-thaw sperm characteristics. Using a split sample design, the effect of HA at a final concentration of 500 or 1000 μg/ml semen on post-thaw motility parameters, and membrane lipid architecture status assessed by merocyanine-540/YOPRO-1 and flow cytometry were evaluated. HA-supplementation improved motility parameters ( P<0.05 to P<0.001) and decreased the percentage of hyperactivated spermatozoa ( P<0.05). HA-supplemented samples had more spermatozoa showing high lipid membrane stability as assessed with merocyanine-540. In conclusion, HA appeared to preserve post-thaw spermatozoa viability in vitro and maintained membrane stability after cryopreservation.

Intraperitoneal Hyaluronan Administration in Conscious Rats: Absorption, Metabolism, and Effects on Peritoneal Fluid Dynamics

Hyaluronan (HA) is a major component of interstitial tissue that participates in fluid homeostasis, response to inflammation, and wound healing. Previous studies have shown that intraperitoneal administration of HA can affect peritoneal fluid transport during short peritoneal dialysis exchanges in anesthetized rats. We sought to investigate the effect of high molecular weight HA on peritoneal permeability in conscious rats during dialysis exchanges up to 8 hours in duration. In addition, we sought to investigate the absorption of HA from the peritoneal cavity, its accumulation in peritoneal tissues, and its metabolism in normal and uremic rats. Experiments were performed on male Wistar rats infused with 30 mL peritoneal dialysis solution (Dianeal, Baxter Healthcare; Castelbar, Ireland) containing 10 mg/dL HA or with Dianeal alone (control). Peritoneal fluid removal (net ultrafiltration), permeability to glucose, creatinine, and total proteins, and tissue and blood levels of HA were determined in separate groups of rats at 1,2, 4, 6, and 8 hours after intraperitoneal infusion. Hyaluronan appearance and disappearance from plasma were also studied for 24 hours in separate groups of normal and uremic rats. Net ultrafiltration was significantly greater (27%) in rats infused with HA at 4, 6, and 8 hours (p < 0.01) compared to controls. Transperitoneal equilibration of protein was reduced by 27% (p < 0.001) at 4 hours and by 30% (p < 0.01) at 8 hours. During the 8-hour exchange, peritoneal clearance of creatinine increased by 27% (p < 0.01), whereas the clearance of total protein decreased by 27% (p < 0.005). After 8 hours, 25.7% +/- 3.1% of the administered HA was absorbed from the peritoneal cavity, peritoneal tissue HA concentration was increased by 117% (p < 0.001), and plasma HA levels increased by 435% (p < 0.001). Plasma HA levels returned to normal within 24 hours after intraperitoneal administration in both healthy and uremic rats. Hyaluronan added to dialysis fluid is absorbed from the peritoneal cavity and accumulates in peritoneal tissues. Hyaluronan supplementation produces changes in peritoneal permeability, leading to higher net ultrafiltration and peritoneal creatinine clearance, whereas total protein clearance decreases. The HA that is absorbed from the peritoneal cavity appears to be rapidly metabolized in both healthy and uremic rats.