Cross-linked Hyaluronan Research Articles

Cross-linked Hyaluronan Gel Reduces the Acute Rectal Toxicity of Radiotherapy for Prostate Cancer

Cross-linked Hyaluronan Gel Reduces the Acute Rectal Toxicity of Radiotherapy for Prostate Cancer

Preparation and characterization of cross-linked hyaluronan nanoparticles

The present investigation describes the synthesis and characterization of novel biodegradable nanoparticles based on hyaluronic acid (HA). The diamine, 2,2′(ethylenedioxy)bis(ethylamine) was used for cross-linking of the HA linear chains. The condensation reaction of amino groups and pendant carboxyl groups of HA was performed in aqueous media at room temperature using water-soluble carbodiimide. The prepared nanosystems, aqueous solutions, or dispersions of nanoparticles were stable, transparent, or mildly opalescent systems depending on the ratio of cross-linking, findings consistent with values of transmittance above 77%. The structure of products was determined by nuclear magnetic resonance spectroscopy, and the particle size was identified by laser light scattering (DLS) and transmission electron microscopy (TEM) measurements. Particle size measured by TEM varied less than 130 nm; in the swollen state, the average size of the particles measured by DLS was in the range of 30–140 nm depending on the ratio of cross-linking and the molecular weight of HA. Formation of cross-linked nanoparticles results in a viscosity drop compared to the viscosity of the corresponding solution of the HA, and this trend becomes decreasingly appreciable as the molecular weight of HA decreases.

Intratumoral Delivery of Paclitaxel in Solid Tumor from Biodegradable Hyaluronan Nanoparticle Formulations

In the current study, novel paclitaxel-loaded cross-linked hyaluronan nanoparticles were engineered for the local delivery of paclitaxel as a prototype drug for cancer therapy. The nanoparticles were prepared using a desolvation method with polymer cross-linking. In vitro cytotoxicity studies demonstrated that less than 75% of the MDA-MB-231 and ZR-75-1 breast cancer cells were viable after 2-day exposure to paclitaxel-loaded hyaluronan nanoparticles or free paclitaxel, regardless of the dose. These results suggest that hyaluronan nanoparticles maintain the pharmacological activity of paclitaxel and efficiently deliver it to the cells. Furthermore, in vivo administration of the drug-loaded nanoparticles via direct intratumoral injection to 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumor in female rats was studied. The paclitaxel-loaded nanoparticles treated group showed effective inhibition of tumor growth in all treated rats. Interestingly, there was one case of complete remission of tumor nodule and two cases of persistent reduction of tumor size that was observed on subsequent days. In the case of free paclitaxel-treated group, the mean tumor volume increased almost linearly (R(2) = 0.93) with time to a size that was 4.9-fold larger than the baseline volume at 57 days post-drug administration. Intratumoral administration of paclitaxel-loaded hyaluronan nanoparticles could be a promising treatment modality for solid mammary tumors.

Regeneration of intervertebral disc by the intradiscal application of cross-linked hyaluronate hydrogel and cross-linked chondroitin sulfate hydrogel in a rabbit model of intervertebral disc injury

The degeneration of intervertebral disc (IVD) is a major cause of low back pain. However, there is no satisfactory preventive treatment for degenerative disc disease (DDD). In this study, we examined the effects of a novel cross-linked hyaluronate hydrogel and cross-linked chondroitin sulfate (CS) hydrogel on a rabbit model of IVD injury. We injected 300 microl of phosphate buffer saline, 1% sodium hyaluronate, cross-linked hyaluronate hydrogel, or cross-linked CS hydrogel into the injured IVDs. One, three or six months after treatment, the whole spinal columns were dissected and magnetic resonance (MR) images of the IVDs were examined. It was noted that the IVD, which was injected with cross-linked hyaluronate hydrogel or cross-linked CS hydrogel mostly retained the normal signal intensity of the MR images. These IVDs exhibited a higher degree of staining with safranin-O than the control discs or 1% sodium hyaluronate-injected discs, suggesting that the intradiscal application of cross-linked hyaluronate hydrogel or cross-linked CS hydrogel probably inhibits the degenerative cascade of the DDD. The intradiscal administration of these drugs is safe, easy and costs less. In the near future, these intradiscal injections may become the standard therapy for the treatment of DDD instead of the spine surgeries.

Hyaluronan-Tethered Opioid Depots: Synthetic Strategies and Release Kinetics In Vitro and In Vivo

We proposed the use of opioid drug bound covalently to hyaluronan (HA) via ester linkages as a method to prolong drug delivery and to possibly increase the quality of perioperative pain management. The in vitro release profile of morphine conjugated to HA (1.3 million MW) was studied. The influence of parameters such as conjugation site and steric protection of the labile ester bonds was investigated in phosphate buffered saline (PBS) medium. HA--codeine and HA--naloxone conjugates were used as structural controls. Codeine and morphine conjugated via the allylic hydroxyl group had a release half-life of 14.0 days in PBS. Naloxone conjugated via the phenolic hydroxyl group showed a half-life of 0.3 days, and all drugs admixed in HA showed half-lives of 0.1 days. Methyl, ethyl, or n-propyl introduced in vicinal position to the ester bond prolonged release of naloxone with half-lives of 0.5, 4.0, and 4.0 days in PBS, respectively. Incorporation of a methyl group prolonged codeine release with a half-life of 55.0 days in PBS. Drugs were released chemically unaltered from the conjugates as confirmed by LC-MS/MS. Further, morphine was conjugated to divinylsulfone cross-linked HA (Hylan B) particles and the release profiles in rat plasma were studied in vitro and in vivo. Release in rat plasma was faster than in PBS with a half-life of 2.5 days, but the release was similar (ca. 12 days) when a cocktail of protease inhibitors was added to the plasma. Sustained release of morphine was observed in a rat surgical model over 30 h. Morphine was released chemically unaltered from the conjugate and morphine intermediates were not detected in significant amounts as confirmed by LC-MS/MS. These results suggest that the morphine release profile from the HA conjugates depends on the alkyl groups vicinal to the ester and the nature of the leaving group. In rat plasma, hydrolysis seems to be controlled by esterase activity.

Adipose tissue engineering in vivo with adipose‐derived stem cells on naturally derived scaffolds

Placental decellular matrix (PDM) and PDM combined with cross-linked hyaluronan (XLHA) scaffolds, seeded with primary human adipose-derived stem cells (ASC), were investigated in a subcutaneous athymic mouse model. The in vivo response at 3 and 8 weeks was characterized using histological and immunohistochemical staining. Fibrous capsule formation was assessed and the relative number of adipocytes in each scaffold was quantified. Undifferentiated ASC were localized using immunostaining for human vimentin. Unilocular and multilocular adipocytes were identified by intracellular lipid accumulation. Staining for murine CD31 assessed implant vascularization. Both scaffolds macroscopically maintained their three-dimensional volume and supported mature adipocyte populations in vivo. There was evidence of implant integration and a host contribution to the adipogenic response. The results suggested that incorporating the XLHA had a positive effect in terms of angiogenesis and adipogenesis. Overall, the PDM and PDM with XLHA scaffolds showed great promise for adipose tissue regeneration.

Proliferation and differentiation of adipose-derived stem cells on naturally derived scaffolds

A tissue-engineered substitute that facilitates large-volume regeneration of the subcutaneous adipose tissue layer is needed for reconstructive plastic surgery. Towards this goal, we describe the in vitro culture of primary human adipose-derived stem cells (ASC) seeded into placental decellular matrix (PDM) and cross-linked hyaluronan (XLHA) scaffolds. Specifically, we evaluated cellular proliferation and adipogenic differentiation in the PDM, XLHA, and PDM combined with XLHA scaffolds. Cellular proliferation, viability, and glucose consumption were determined prior to the induction of differentiation. Adipogenesis within each of the scaffolds was investigated through gene expression analysis using end point and real time reverse transcriptase polymerase chain reaction (RT-PCR). The results indicate that the cell-adhesive PDM scaffolds facilitated proliferation and viability, while differentiation was augmented when the cells were encapsulated in the non-adhesive XLHA gels.

Hyaluronan hydrogel: An appropriate three-dimensional model for evaluation of anticancer drug sensitivity

The extracellular polysaccharide hyaluronan (HA) controls cell migration, differentiation and proliferation, and contributes to the invasiveness of human cancers. In order to investigate the sensitivity of cancer cells to antimitotic agents, we developed a cross-linked HA hydrogel, a three-dimensional matrix in which cells can invade and grow. We have studied three cell lines (SA87, NCI-H460 and H460M), from primary tumors and metastases, that migrated into the HA hydrogel and proliferated giving rise to clusters and colonies. Concurrently, we studied the growth of these cell lines in a usual monolayer culture system. In these two models, increasing concentrations of doxorubicin and 5-fluorouracil were evaluated for their ability to inhibit tumor cell growth and colony formation. Taken together, our data suggest that the cancer cells were more resistant in the three-dimensional model than in monolayer cell systems. The antimitotic drugs were efficient after 24 h of treatment in the monolayer cultures, whereas they were significantly efficient only after one week of incubation in the HA hydrogels. Herein, we show that this cross-linked matrix provides a three-dimensional model particularly appropriate for investigating mechanisms involved in cancer cell line sensitivity to antimitotic drugs.

Injectable Form of Cross-Linked Hyaluronan is Effective for Middle Ear Wound Healing

Two studies were designed to investigate a hyaluronan (HA) gel for middle ear (ME) wound healing. We used a guinea pig model of ME wound healing. In a long-term study, we performed a comparison of hearing and ME inflammation in 3 groups. Group 1 (n = 8) underwent bilateral wounding of ME mucosa and unilateral packing of the ME with HA gel (Sepragel). Group 2 (n = 6) was the same as group 1 except that the packing was absorbable bovine collagen sponges (Gelfoam). Group 3, the control group (n = 14), had operated, unpacked ears. In a short-term study, we investigated ME retention of HA gel at 1 and 2 weeks (n = 16). At 1 week, all ears showed decreased distortion product otoacoustic emissions (DPOAEs) and auditory brain stem responses (ABRs) secondary to ME packing and postsurgical inflammation. The controls recovered preoperative DPOAEs and ABRs by week 2. Group 1 had decreased low-frequency DPOAEs at weeks 2 and 6, but their high-frequency DPOAEs and ABRs recovered to preoperative values by week 6. Group 2 had hearing losses that persisted throughout the study. Group 1 showed normal ME and inner ear histologic characteristics. Group 2 showed inflammatory cells within the ME and cochleas. Group 1 showed less packing retention than did group 2 at week 6 (p = .016). Eighty-five percent of the HA packing remained at 1 week, and 73% at 2 weeks. Hyaluronan gel was a relatively safe and effective ME packing material in our animal model.

Peritoneal adhesion prevention with an in situ cross-linkable hyaluronan gel containing tissue-type plasminogen activator in a rabbit repeated-injury model

Postoperative peritoneal adhesions can have serious, potentially lethal consequences. Pharmacotherapy and barrier devices can reduce adhesion formation to varying degrees, but their efficacy is limited by rapid clearance from the peritoneum and lack of biological activity, respectively. To overcome these limitations, we have delivered tissue-type plasminogen activator (tPA), which is deficient in the first 2–3 postoperative days, using a highly cross-linked in situ forming hyaluronan gel (HAX hx). We demonstrated this formulation's anti-adhesion activity in a rigorous animal model that involved recurrent adhesions. While non-treated or saline-treated animals developed widespread adhesions (frequency, both 100%; median adhesion area, 12.7 and 15.4 cm 2, respectively), tPA delivered by HAX hx (tPA-HAX hx) was highly effective in preventing recurrent adhesions (frequency, 44%; median adhesion area, 0.1 cm 2). HAX hx itself, tPA solution, and inactivated tPA in HAX hx did not provide comparable anti-adhesion activity. tPA-HAX hx is a system that is easy to use and potentially promising for adhesion prevention.

Prevention of peritoneal adhesions with an in situ cross-linkable hyaluronan hydrogel delivering budesonide

Peritoneal adhesions are tissue connections that form within the abdominopelvic cavity following surgery or other injuries. They can cause major medical complications. Barrier devices and pharmacological agents have been used to prevent adhesion formation, with mixed success. We hypothesize that an adhesion barrier which also delivers anti-adhesion drugs can address both physical and physiological causes for adhesion formation. Here, we describe an in situ cross-linking hyaluronan hydrogel (barrier device) containing the glucocorticoid receptor agonist budesonide. Budesonide was chosen because of the known role of inflammation in adhesion formation, hyaluronan because of its known biocompatibility in the peritoneum. The system, consisting of two cross-linkable precursor liquids, was applied using a double-barreled syringe, forming a flexible and durable hydrogel in less than 5 s. We applied this formulation or controls to the injured sites after the second injury in a severe repeat sidewall defect–cecum abrasion model of peritoneal adhesion formation in the rabbit. Large adhesions (median area 15.4 cm 2) developed in all saline-treated animals. Adhesion formation and area were slightly mitigated in animals treated with budesonide in saline (median area 5.0 cm 2) or the hydrogel without budesonide (median area 4.9 cm 2). The incidence and area of adhesions were dramatically reduced in animals treated with budesonide in the hydrogel (median area 0.0 cm 2). In subcutaneous injections in rats, budesonide in hydrogel reduced inflammation compared to hydrogel alone. In summary, budesonide in a hyaluronan hydrogel is easy to use and highly effective in preventing adhesions in our severe repeated injury model. It is a potentially promising system for post-surgical adhesion prevention, and suggests that the effectiveness of barrier devices can be greatly enhanced by concurrent drug delivery.

Determining the Molecular Basis for the pH-dependent Interaction between the Link Module of Human TSG-6 and Hyaluronan

TSG-6 is an inflammation-associated hyaluronan (HA)-binding protein that has anti-inflammatory and protective functions in arthritis and asthma as well as a critical role in mammalian ovulation. The interaction between TSG-6 and HA is pH-dependent, with a marked reduction in affinity on increasing the pH from 6.0 to 8.0. Here we have investigated the mechanism underlying this pH dependence using a combined approach of site-directed mutagenesis, NMR, isothermal titration calorimetry and microtiter plate assays. Analysis of single-site mutants of the TSG-6 Link module indicated that the loss in affinity above pH 6.0 is mediated by the change in ionization state of a histidine residue (His(4)) that is not within the HA-binding site. To understand this in molecular terms, the pH-dependent folding profile and the pK(a) values of charged residues within the Link module were determined using NMR. These data indicated that His(4) makes a salt bridge to one side-chain oxygen atom of a buried aspartate residue (Asp(89)), whereas the other oxygen is simultaneously hydrogen-bonded to a key HA-binding residue (Tyr(12)). This molecular network transmits the change in ionization state of His(4) to the HA-binding site, which explains the loss of affinity at high pH. In contrast, simulations of the pH affinity curves indicate that another histidine residue, His(45), is largely responsible for the gain in affinity for HA between pH 3.5 and 6.0. The pH-dependent interaction of TSG-6 with HA (and other ligands) provides a means of differentially regulating the functional activity of this protein in different tissue microenvironments.

Total knee replacement delayed with Hylan G-F 20 use in patients with grade IV osteoarthritis.

Total knee replacement (TKR), a last resort for treating knee pain due to osteoarthritis (OA), is not always medically indicated or preferred by many patients. Hylan G-F 20 is a cross-linked hyaluronan derivative approved for the treatment of pain due to OA of the knee after other conservative approaches have failed. The objectives of this study were to (1) determine the effect of hylan G-F 20 on patient need for TKR as measured by time from hylan G-F 20 injection to TKR, and (2) assess patient factors that might influence time from hylan G-F 20 therapy to TKR. This is a retrospective case series review of the medical records of patients seen in 1 orthopedic specialty practice. The incidence and time to TKR in patients who were TKR candidates (100% grade IV OA [severe]) treated with 1 or more courses of intra-articular hylan G-F 20 injections (3 weekly injections per course) were determined from October 1997 to November 2003. Survival analysis was used to evaluate time to TKR and the effects of age, gender, ethnicity, body mass index (BMI), and presence of effusion on this outcome. Logistic regression was also used to assess these covariates. The incidence of TKR in hylan G-F 20-treated knees (1,187 knees; 863 patients) was 19% (n=225 knees). The median time to TKR in these patients was 638 days (1.8 years; minimum of 14 days, maximum of 2,147 days). For patients in whom a TKR had not yet occurred during the observation time, the median time of hylan G-F 20 treatment and patient follow-up was 810 days (2.2 years; minimum of 7 days, maximum of 2,222 days). A total of 1,978 courses of hylan G-F 20 given to 1,187 knees (average 1.67 courses per knee) resulted in an average cost of $1,419.76 per knee to delay TKR by a median of 2.1 years (772 days, minimum 7, maximum 2,222), the median time of all knees to either TKR or time of last observation. Survival analysis showed that 75% of knees had not had a TKR by 1,370 days (3.8 years). Survival analysis and logistic regression indicated that of age, gender, ethnicity, BMI, and presence of effusion, only age significantly affected time to TKR. In patients who are candidates for TKR, the need for TKR can be delayed with hylan G-F 20 when used for the treatment of OA knee pain.

Comparison of the sensitivity of 11 crosslinked hyaluronic acid gels to bovine testis hyaluronidase

Crosslinked hyaluronan gels are used in various applications where their stability is a prerequisite. The sensitivity of such gels to hyaluronidase can be determined as an index of stability by several approaches: chromatography, electrophoresis, and viscometry. We describe here a test based on the colorimetric determination of the N-acetyl- d-glucosamine released by hyaluronidase in standardized conditions. The sensitivities to bovine testicular hyaluronidase of 11 different gels used to fill skin wrinkles (Restylane; Perlane; Juvéderm 18, 24, 24HV, 30, and 30HV; Surgiderm 18, 24XP, 30, and 30XP) were compared. The method was reproducible, easy to perform, not time-consuming and allowed us to demonstrate that the sensitivity to testicular hyaluronidase was dependent on the degree of crosslinking of the gels and also on their monophasic/biphasic nature. Under our conditions, Surgiderm 30, 24XP and 30XP were the most resistant gels. We propose to retain the hyaluronidase test to predict the in situ stability of a crosslinked gel used to fill skin wrinkles.

Molecular Stenting with a Crosslinked Hyaluronan Derivative Inhibits Collagen Gel Contraction

Adult burn wounds, which lack hyaluronan (HA), often undergo excessive tissue remodeling and contraction. In contrast fetal wounds, which contain large amounts of HA, undergo remodeling that culminates in a scarless repair or regeneration. Therefore, adding a HA derivative to burn wounds would better mimic the fetal extracellular matrix and could reduce contraction. To test this hypothesis, we determined the effects of HA and its two derivatives on fibroblast-mediated, collagen gel contraction, an assay widely used to mimic in vivo wound contraction. Interestingly, high molecular weight HA (HMW HA) facilitated collagen gel contraction, whereas a thiol-functionalized derivative HA-DTPH weakly inhibited contraction. In contrast, polyethylene glycol diacrylate (PEGDA)-crosslinked HA-DTPH (HA-DTPH-PEGDA) strongly inhibited contraction in a concentration-dependent manner. Immunofluorescence staining of cellular actin showed that this inhibition was not owing to reduced cell attachment or spreading. Furthermore, the supernatant of contracted collagen-HMW HA gels contained greater amounts of HA than those found in the supernatant of collagen-HA-DTPH-PEGDA gels, suggesting that HMW HA facilitates contraction by effectively diffusing out of the collagen gels. Therefore, the results suggest that the crosslinking of HA-DTPH enhances the structural mechanics of collagen/HA-DTPH composites, which resists the fibroblast contractile forces and may, therefore, be able to reduce excessive wound contraction observed in pathological conditions.

Viscoelastic measurements after vocal fold scarring in rabbits—short-term results after hyaluronan injection

Conclusions. The scarring model resulted in significant damage and elevated viscoelasticity of the lamina propria. Hyaluronan preparations may alter viscoelasticity in scarred rabbit vocal folds. Objectives. Vocal fold scarring results in stiffness of the lamina propria and severe voice problems. The aims of this study were to examine the degree of scarring achieved in the experiment and to measure the viscoelastic properties after injection of hyaluronan in rabbit vocal folds. Materials and methods. Twenty-two vocal folds from 15 New Zealand rabbits were scarred, 8 vocal folds were controls. After 8 weeks 12 of the scarred vocal folds received injections with 2 types of cross-linked hyaluronan products and 10 scarred folds were injected with saline. After 11 more weeks the animals were sacrificed. After dissection, 15 vocal folds were frozen for viscoelastic measurements, whereas 14 vocal folds were prepared and stained. Measurements were made of the lamina propria thickness. Viscoelasticity was measured on intact vocal folds with a linear skin rheometer (LSR) adapted to laryngeal measurements. Results. Measurements on the digitized slides showed a thickened lamina propria in the scarred samples as compared with the normal vocal folds (p <0.05). The viscoelastic analysis showed a tendency to stiffening of the scarred vocal folds as compared with the normal controls (p =0.05). There was large variation in stiffness between the two injected hyaluronan products.

Composition of Hyaluronan Affects Wound Healing in the Rabbit Maxillary Sinus

Hyaluronan (HA) is a ubiquitous component of the extracellular matrix. HA and its derivatives have been used in the sinuses to reduce scarring and possibly promote wound healing. However, in recent animal studies, HA esters exhibited inflammatory effects. Mitomycin C (MMC) is another potential antiscarring treatment. This study prospectively evaluated the effects of three different HA constructs on wound healing in the rabbit maxillary sinus: (i) a novel cross-linked HA hydrogel, (ii) the cross-linked HA gel containing covalently bound MMC, and (iii) a commercially available woven HA ester (Merogel). Ostia were created with a 4-mm otologic drill in the maxillary sinuses of 15 New Zealand white rabbits with one side randomly chosen for treatment. After 14 or 21 days the size of the maxillary ostia were recorded and the tissue was examined under light microscopy. Sinuses treated with the novel HA and HA-MMC hydrogels showed an increased ostial diameter compared with untreated controls. Woven HA ester-treated sinuses showed no improvement, with a trend toward a smaller ostium than controls. Histological examination showed that woven HA ester tended to cause increased fibrosis and granulomatous inflammation, and heterophilia was slightly increased in the HA hydrogel-treated sinuses. Blinded observation noted foamy macrophages surrounding the residual woven HA ester in each specimen while no similar reaction was noted near the residual HA or HA-MMC hydrogels. This study suggests that the degree of ostial narrowing, inflammation, and fibrosis depends on the formulation of the HA used. Minimal, if any, additional benefit is seen with addition of MMC to the HA hydrogel in this pilot study.

Engineering Blood Vessels from Lumenized Vascular Tissue Spheroids

We have applied the principle of VEGF-mediated vascular fusion (Drake and Little, Proc Natl Acad Sci, 1995) to engineer blood vessels. Using the murine allantois as a source of endothelial precursor cells, spheroids were generated in hanging drop culture (HDC) that consist of an inner network of capillary-sized vessels (CSV) and outer layers of alpha smooth muscle actin-positive cells (SMAC). To transform spheroids into blood vessel-like structures (BVLS), VEGF was used to induce the fusion of the CSV. This resulted in the formation of BVLS having a single endothelial-lined lumen (dia. >300μm) surrounded by SMAC. Further, when two spheroids were exposed to VEGF in HDC, a single BVLS with an increased luminal diameter was formed. Essential to the use of spheroids in vascular engineering are 3D-matrices that are permissive for both spheroid formation and vascular fusion. In studies evaluating a collagen hydrogel (CH) versus a cross-linkable hyaluronan hydrogel (HH), we observed that HH but not CH was permissive for the formation of BVLS from spheroids. Finally, to demonstrate the utility of lumenized spheroids as modules to engineer blood vessels, we show that when multiple spheroids were exposed to VEGF in HH, they formed a single lumenized vascular tube-like structure surrounded by SMAC. These findings validate our mathematical model of lumenized vascular spheroid fusion and demonstrate the feasibility of a tissue self-assembly approach as a means of engineering blood vessels.

Temperature-Sensitive Micrometer-Thick Layers of Hyaluronan Grafted on Microspheres

The giant polyelectrolyte glycosaminoglycan hyaluronan (1-10 MDa) is a major component of the pericellular coat on a variety of cells, where it is an important modulator and mediator of early cell adhesion events. This pericellular layer can reach 5 mum thickness on cells that produce cartilage (chondrocytes), and up to 2 mum on Xenopus laevis kidney epithelial cells (A6). We are interested in generating model systems for the pericellular coat in order to learn more about the structure and function of hyaluronan on biological or artificial surfaces. We report here the synthesis of model systems where a coat of coordinatively cross-linked hyaluronan of up to 2 mum thickness was covalently photografted onto polystyrene microspheres. The hydrated coat was imaged directly by environmental scanning electron microscopy (ESEM) at close to 100% relative humidity. The key feature of the procedure is the reversible reverse-temperature phase transition of hyaluronan induced by trivalent lanthanide cations, which is exploited to achieve sufficient density for grafting of thick layers. The microsphere-grafted coat shows a temperature-dependent swelling when labeled with lanthanide ions (Gd(3+) or Tb(3+)). We directly observed a volume contraction of 20% with increasing temperature between 1 and 11 degrees C by wet-mode ESEM.

Dual growth factor-induced angiogenesis in vivo using hyaluronan hydrogel implants

Crosslinked hyaluronan (HA) hydrogels preloaded with two cytokine growth factors, vascular endothelial growth factor (VEGF) and keratinocyte growth factor (KGF), were employed to elicit new microvessel growth in vivo. As a major glycosaminoglycan (GAG) component of extracellular matrix (ECM), HA is an excellent biopolymeric building block for new biomimetic, biocompatible therapeutic materials. HA hydrogel film samples were surgically implanted in the ear pinnae of mice, and the ears were harvested at 7 or 14 days post-implantation. Histologic analysis showed that each of the groups receiving an implant demonstrated significantly more microvessel density than control ears undergoing surgery but receiving no implant ( p < 0.001 ). Treatment groups receiving either co-delivery of both KGF and VEGF, an HA hydrogel lacking a growth factor or HA hydrogels containing a single cytokine were statistically unchanged with time, whereas treatment with KGF alone produced continuing increases in vascularization from day 7 to day 14. Strikingly, presentation of both VEGF and KGF in crosslinked HA generated intact microvessel beds with well-defined borders. In addition, an additive response to co-delivery of both cytokines in the HA hydrogel was observed. The HA hydrogels containing KGF+VEGF produced the greatest angiogenic response of any treatment group tested ( NI = 5.4 at day 14, where NI is a neovascularization index). This was 33% greater vessel density than in the next largest treatment group, that received HA+KGF ( NI = 4.0 , p < 0.002 ). New therapeutic approaches for numerous pathologies could be notably enhanced by the localized, sustained angiogenic response produced by release of both VEGF and KGF from crosslinked HA films.