A site-selective hyaluronan-interferonα2a conjugate for the treatment of ovarian cancer.

While interferon alpha (IFNα) is used in several viral and cancer contexts, its efficacy against ovarian cancer (OC) is far from being incontrovertibly demonstrated and, more importantly, is hindered by heavy systemic side effects. To overcome these issues, here we propose a strategy that allows a targeted delivery of the cytokine, by conjugating IFNα2a with an aldehyde-modified form of hyaluronic acid (HA). The resulting HA-IFNα2a bioconjugate was biochemically and biologically characterized. The conjugation with HA did not substantially modified both the antiviral function and the anti-proliferative activity of the cytokine. Moreover, the induction of STAT1 phosphorylation and of a specific gene expression signature in different targets was retained. In vivo optical imaging biodistribution showed that the i.p.-injected HA-IFNα2a persisted into the peritoneal cavity longer than IFNα2a without being toxic for intraperitoneal organs, thus potentially enhancing the loco-regional therapeutic effect. Indeed, in OC xenograft mouse models bioconjugate significantly improved survival as compared to the free cytokine. Overall, HA-IFNα2a bioconjugate disclosed an improved anticancer efficacy, and can be envisaged as a promising loco-regional treatment for OC.

Drug conjugation to hyaluronan widens therapeutic indications for ovarian cancer.

Management of ovarian cancer still requires improvements in therapeutic options. A drug delivery strategy was tested that allows specific targeting of tumor cells in combination with a controlled release of a cytotoxic molecule. To this aim, the efficacy of a loco-regional intraperitoneal treatment with a bioconjugate (ONCOFID-S) derived by chemical linking of SN-38, the active metabolite of irinotecan (CPT-11), to hyaluronan was assessed in a mouse model of ovarian carcinomatosis. In vitro, the bioconjugate selectively interacted with ovarian cancer cells through the CD44 receptor, disclosed a dose-dependent tumor growth inhibition efficacy comparable to that of free SN-38 drug, and inhibited Topoisomerase I function leading to apoptosis by a mechanism involving caspase-3 and -7 activation and PARP cleavage. In vivo, the intraperitoneal administration of ONCOFID-S in tumor-bearing mice did not induce inflammation, and evidenced an improved therapeutic efficacy compared with CPT-11. In conclusion, SN-38 conjugation to hyaluronan significantly improved the profile of in vivo tolerability and widened the field of application of irinotecan. Therefore, this approach can be envisaged as a promising therapeutic strategy for loco-regional treatment of ovarian cancer.

Peritoneal tumor carcinomatosis: pharmacological targeting with hyaluronan-based bioconjugates overcomes therapeutic indications of current drugs.

Peritoneal carcinomatosis still lacks reliable therapeutic options. We aimed at testing a drug delivery strategy allowing a controlled release of cytotoxic molecules and selective targeting of tumor cells. We comparatively assessed the efficacy of a loco-regional intraperitoneal treatment in immunocompromised mice with bioconjugates formed by chemical linking of paclitaxel or SN-38 to hyaluronan, against three models of peritoneal carcinomatosis derived from human colorectal, gastric and esophageal tumor cell xenografts. In vitro, bioconjugates were selectively internalized through mechanisms largely dependent on interaction with the CD44 receptor and caveolin-mediated endocytosis, which led to accumulation of compounds into lysosomes of tumor cells. Moreover, they inhibited tumor growth comparably to free drugs. In vivo, efficacy of bioconjugates or free drugs against luciferase-transduced tumor cells was assessed by bioluminescence optical imaging, and by recording mice survival. The intraperitoneal administration of bioconjugates in tumor-bearing mice exerted overlapping or improved therapeutic efficacy compared with unconjugated drugs. Overall, drug conjugation to hyaluronan significantly improved the profiles of in vivo tolerability and widened the field of application of existing drugs, over their formal approval or current use. Therefore, this approach can be envisaged as a promising therapeutic strategy for loco-regional treatment of peritoneal carcinomatosis.

Hyaluronan derivatives: Alkyl chain length boosts viscoelastic behavior to depolymerization.

Five amide derivatives of Hyaluronic Acid (HA) were synthesized with C8, C12, C15, C16 and C18 linear alkyl-amines. These polymers (Hyadd) were tested against thermal, oxidative and hyaluronidase degradation by means of rheological experiments and SEC analysis and compared to non-modified HA. First of all, no free hexadecylamine was detected in the treated samples, meaning that under these stressing conditions only cleavage of glycosidic bonds occurs. Then, viscoelastic properties were assessed during thermal degradation and their variation as a function of time was expressed by means of a decay constant k(G'): while no significant difference in the decrease rate was observed between Hyadd-C8 and Hyadd-C12, a marked stabilization of viscoelastic properties during thermal treatment was detected for Hyadd-C15, Hyadd-C16 and Hyadd-C18. On the other hand, no difference was observed between the MW decrease rate (kMW decay constant) of HA and Hyadd-C12 to-C18; the depolymerization takes place on the backbone of the polymers independently whether they are derivatized or not, but longer alkyl chains lead to higher viscoelasticity in the depolymerized products. Finally, both oxidative and enzymatic degradation were carried out analyzing the changes in elastic modulus and in dynamic viscosity: once again, the amide side chain came out with similar behavior to chemical cross-linked HA (HBC) and with improved performances respect to linear HA in terms of preservation of viscoelasticity after chain depolymerization.

Conjugation of hyaluronan to proteins.

Polymer conjugation has been widely exploited to prolong half-life and reduce immunogenicity of therapeutic proteins. Here, the potentials of hyaluronic acid (HA) have been investigated by studying the conjugates with two model enzymes, trypsin and RNase A, and with insulin. As the direct coupling of proteins to the HA's carboxylic groups can cause cross-linking problems, a hyaluronan-aldehyde derivative has been synthesized for N-terminal site-selective conjugation. HA conjugation, termed HAylation, preserved the activities of enzymes and their thermal stabilities. Insulin HAylation was studied by preparing two conjugates with different peptide loadings (32% and 17%, w/w). Noticeably, the conjugate with the lower loading showed the greater effect on blood glucose level. The 17% HA-insulin conjugate showed a lowering effect on blood glucose level for up to 6h, while free insulin exhausted its action after 1h. This study highlights the potentials of hyaluronan-aldehyde for protein delivery.

Paclitaxel-hyaluronan hydrosoluble bioconjugate: mechanism of action in human bladder cancer cell lines.

OBJECTIVES: A previously described hydrosoluble paclitaxel-hyaluronan bioconjugate appears particularly well suited for treatment of superficial bladder cancer because of its in vitro cytotoxic profile against urothelial carcinoma (UC) cell lines and in vivo biocompatibility. The aim of this work was to assess the mechanism of action of the bioconjugate in UC cells. MATERIALS AND METHODS: Expression of CD44 and RHAMM hyaluronan-binding receptors in RT-4 and RT-112/84 UC cell lines, interaction of fluorochrome-labeled bioconjugate with tumor cells, CD44 modulation upon incubation with the compound or free hyaluronan, and caspase activation were assessed by flow cytometry. Cytotoxicity was studied by the MTT assay. Analysis of bioconjugate intracellular localization and effects on ß-tubulin organization was carried out by confocal microscopy. RESULTS: The paclitaxel-hyaluronan bioconjugate bound to UC tumor cells entered intracellular compartments through a saturable and energy-dependent mechanism that involved CD44, as assessed by blocking with specific antibody. Upon internalization, the bioconjugate accumulated into lysosomes where the esteric bond between paclitaxel and the hyaluronan moiety was cleaved, leading to cytoplasmic diffusion of the free drug, caspase activation, and disruption of the ß-tubulin microtubular mesh with subsequent cell death. CONCLUSIONS: Conjugation of paclitaxel to hyaluronan results in a new chemical entity, characterized by selective targeting to polymer receptors on plasma membrane and cell entry through receptor-mediated endocytosis, followed by lysosomal accumulation. Ultimately, the active molecule is released, fully preserving the cytotoxic potential and profile of clinically used free paclitaxel.

Pharmacokinetic profile of Oncofid-S after intraperitoneal and intravenous administration in the rat.

OBJECTIVES: Oncofid-S is a bio-conjugate molecule obtained from the binding of campthotecin, SN-38, to hyaluronic acid. In view of a possible clinical development for loco-regional treatment of peritoneal carcinomatosis, this study aimed to establish the pharmacokinetic profile of Oncofid-S after single intraperitoneal or intravenous administration in the rat. METHODS: Single-dose intraperitoneal or intravenous administrations of Oncofid-S were performed. Groups of six rats were sacrificed at various times (up to 24 and 72 h in i.p. and i.v. study, respectively) after drug injection. Trunk blood, livers and spleens were collected for subsequent analysis. Total SN-38 was assayed by HPLC. KEY FINDINGS: We found that Oncofid-S was poorly absorbed after intraperitoneal injection, the estimated AUC0­72 being less than 2%. The drug was distributed in liver,but not spleen, and was eliminated with a terminal half-life of 16 h. After intravenous dosing, Oncofid-S was found in liver as well as in spleen. CONCLUSIONS: Here we have demonstrated that Oncofid-S administered intraperitoneally in the rat was poorly absorbed into the systemic circulation, even after the administration of an extremely high dose. This finding reinforces the rationale for developing Oncofid-S in the loco-regional intraperitoneal treatment of peritoneal carcinomatosis in man.

CD44-targeting for antitumor drug delivery: a new SN-38-hyaluronan bioconjugate for locoregional treatment of peritoneal carcinomatosis.

An innovative approach for cancer therapy implies the use of drugs covalently conjugated to macromolecular carriers that specifically target molecules over-expressed on tumor cells. This drug delivery strategy may allow a controlled release of the drug and a high targeting selectivity on tumor cells, increasing drug cytotoxicity and decreasing its undesirable side effects. We provide in vitro and in vivo preclinical data on the antitumor efficacy of ONCOFID™-S, a new bioconjugate of hyaluronic acid (HA) with SN-38 (the CPT11 active metabolite), that support the validity of the drug delivery strategy implying the use of HA as macromolecular carrier of antineoplastic drugs, an approach based on the over-expression of its target CD44 (the receptor for HA-mediated motility) in a wide variety of cancers. We show that ONCOFID™-S exerts a strong in vitro anti-proliferative activity on CD44 over-expressing rat DHD/K12/trb colon adenocarcinoma cells, as well as on gastric, breast, oesophageal, ovarian and lung human cancer cells, higher than that exerted by unconjugated SN-38. We also demonstrated the in vivo anti-tumor efficacy of locoregional treatment with ONCOFID™-S on two pre-clinical models of colorectal cancer (CRC) in BDIX rats: a) syngeneic model of subcutaneous tumor; b) syngeneic model of metastatic tumor induced by injection of cells into the peritoneal cavity, mimicking the clinical situation of peritoneal carcinomatosis. Specifically, in the latter model ONCOFID™-S is able to dramatically reduce all parameters indicative of a poor prognosis in peritoneal metastatization of CRC without any myelotoxicity or mesothelial inflammation. We propose this CD44-targeted therapeutic strategy for locoregional treatment of peritoneal carcinomatosis from CRC, against which systemic chemotherapy results almost inefficient.

Hyaluronic acid-paclitaxel: effects of intraperitoneal administration against CD44(+) human ovarian cancer xenografts.

PURPOSE: Hyaluronan (HA)-receptors (mainly CD44 and RHAMM) are overexpressed in a wide variety of cancers including ovarian tumors, and HA-bioconjugates have been developed to enhance selective entry of cytotoxic drugs into HA receptor-expressing cancerous cells. Here, we evaluated the potential application of a new HA-paclitaxel bioconjugate, ONCOFID-P, for intraperitoneal (IP) treatment of ovarian cancer. METHODS: In vitro cytotoxic effect of ONCOFID-P was first assessed on CD44(+) OVCAR-3 and SKOV-3 human ovarian cancer cell lines. Studies were performed in female Balb/c athymic mice IP implanted with OVCAR-3 or SKOV-3 and treated with IP ONCOFID-P, and IP and intravenous (IV) free paclitaxel, at their maximum tolerated dose (MTD 168, 80 and 80 mg/kg, total dose, respectively). The potential detrimental effect of the IP ONCOFID-P and IP free paclitaxel on hematopoiesis was also assessed on peripheral blood, bone marrow and spleen. RESULTS: Results show that ONCOFID-P cytotoxicity against both OVCAR-3 and SKOV-3 cell lines was somewhat less effective than free paclitaxel. Conversely, in in vivo experiments, IP treatment with ONCOFID-P was overall more effective than IV and IP free paclitaxel in inhibiting intra-abdominal tumor dissemination, abrogating ascites, prolonging survival and curing mice. ONCOFID-P and IP free paclitaxel were equivalent in terms of myelotoxicity, although the former was administered at a two-fold higher dose. CONCLUSIONS: Present data strongly support the development of ONCOFID-P for locoregional treatment of ovarian cancer.

A new viscosupplement based on partially hydrophobic hyaluronic acid: a comparative study.

A novel partially hydrophobized derivative of hyaluronic acid (HYADD® 4), containing a low number of C16 side-chains per polysaccharide backbone, provides injectable hydrogels stabilized by side-chain hydrophobic interactions. The rheological properties of Hymovis®, a physical hydrogel based on the hyaluronic acid derivative HYADD® 4, were evaluated using as reference a solution of the parent natural polysaccharide, hyaluronic acid. The rheological measurements were performed both in flow and oscillation regimes at the physiological frequency values of the knee, typically spanning the range from 0.5 Hz (walking frequency) to 3 Hz (running frequency). Moreover, the viscoelastic features of Hymovis® were compared with the market-available viscosupplementation products in view of its use in joint diseases.The different behavior of the investigated materials in crossover frequency measurements and in structure recovery experiments can be explained on the basis of the structural and dynamic properties of the polymeric systems.

Biodistribution imaging of a paclitaxel-hyaluronan bioconjugate.

INTRODUCTION: Gamma-ray detectors represent sensitive and noninvasive instruments to evaluate in vivo the metabolic trapping of radiopharmaceuticals. This study aimed to assess the imaging biodistribution of a [(99m)Tc]-radiolabelled new prototype bioconjugate composed of paclitaxel linked to hyaluronan (ONCOFID-P). METHODS: A small gamma camera providing high-resolution images was employed. Imaging of biodistribution following intravenous, intraperitoneal, intravesical and oral administration was carried out for a 2-h period in anesthetized mice receiving [(99m)Tc]ONCOFID-P. At the end of the observation time, radioactivity in organs was directly measured. As a control, groups of mice were treated with free [(3)H]paclitaxel given according to the same administration routes, and organ biodistribution of the drug was assessed after 2 h. RESULTS: Intravenous inoculation of [(99m)Tc]ONCOFID-P was followed by a rapid and strong liver uptake. In fact, almost 80% of the imaging signal was detected in this organ 10 min after injection and such value remained constant thereafter, thus indicating that the bioconjugate given through the intravenous route could be well suited to targeting primary or metastatic liver neoplasias. Imaging of the bladder, abdomen and gastrointestinal tract after local administration disclosed that the radiolabelled compound remained confined to the cavities, suggesting a potential regional application for transitional bladder cell carcinomas, ovarian cancers and gastric tumors, respectively. Free [(3)H]paclitaxel biodistribution profoundly differed from that of [(99m)Tc]ONCOFID-P. CONCLUSIONS: Conjugation of drugs with polymers results in new chemical entities characterized by a modified biodistribution pattern. Therefore, preclinical studies based on imaging analysis of such new compounds can suggest novel therapeutic applications.

Gel-like structure of a hexadecyl derivative of hyaluronic acid for the treatment of osteoarthritis.

Hyaluronic acid is a polysaccharide with viscoelastic and mechanical properties that are crucial for the normal functioning of osteoarticular junctions. It is demonstrated that introduction of a hexadecyl side chain into HA yields an injectable polysaccharide capable of forming physical hydrogels, which are stable at very low polymer concentrations, whereas native hyaluronic acid forms viscous solutions at concentrations that are ten times higher. Characterization of this system showed that the driving force for its gel-like behavior is the occurrence of hydrophobic interactions involving aliphatic side chains, despite the low degree of substitution, as confirmed by molecular dynamics simulations of HYADD4 and HA hydrogels.

The in vitro rabbit whole bladder as a model to investigate the urothelial transport of anticancer agents The ONCOFID-P paradigm.

Intravesical instillation of BCG or anticancer agents after transurethral resection is currently considered a standard of therapy. However, this approach is basically empirical; none of the anticancer agents used in this setting was specifically formulated for intravesical therapy. Moreover, concern is raised by the kinetic features of water soluble drugs, because of poor transport across the mucosal barrier, or of liphophylic compounds, for the increased risks of systemic toxicity. A need exists to improve the pre-clinical and clinical approaches used at present to test anticancer agents undergoing specific development for intravesical use. We used in vitro rabbit whole bladders as a new pre-clinical model to investigate the kinetics of locally administered anticancer agents. In this study, we investigated the rate of urothelial transport of a novel paclitaxel derivative, Oncofid-P. Male New Zealand albino rabbits were used. Bladders were rapidly explanted, filled with vehicle alone or vehicle containing graded concentrations of Oncofid-P, and kept for various times under standardized incubation conditions. At the end of experiments, drug concentrations were assessed by high-pressure-liquid-chromatography technique in the intravesical and external bath solutions, as well as in bladder wall homogenates. We found that less than 1% of the drug additioned to the intravesical solution is recovered within the bladder wall in the form of paclitaxel; experiments carried out collecting different areas from the same bladders showed that Oncofid-P is uniformly distributed over the internal surface of bladder mucosa. Isolated rabbit bladders may be a useful pre-clinical model to investigate the rate of transport of chemotherapeutic agents administered by intravesical route. In this paradigm, Oncofid-P displays a kinetic profile that is predictive of local activity over the whole urothelial surface, and low or absent systemic absorption.

A paclitaxel-hyaluronan bioconjugate targeting ovarian cancer affords a potent in vivo therapeutic activity.

PURPOSE: This study was designed to evaluate the pharmacologic and biological properties of a paclitaxel-hyaluronan bioconjugate (ONCOFID-P) against IGROV-1 and OVCAR-3 human ovarian cancer xenografts following i.p. administration. EXPERIMENTAL DESIGN: In vitro tumor sensitivity to ONCOFID-P was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, whereas bioconjugate interaction with cells was studied cytofluorimetrically and by confocal microscopy. In vivo toxicity was assessed by a single-dose maximum-tolerated dose, peripheral blood cell count determination and by histologic analysis. Biodistribution of the compound was evaluated with a small animal-dedicated scintigraphy gamma camera following injection of 99mTc-labeled ONCOFID-P. Pharmacokinetic analysis was also carried out. Female severe combined immunodeficiency mice implanted with ovarian cancer cells underwent treatment with ONCOFID-P or free paclitaxel starting from day 7 or 14 after tumor injection, and survivals were compared. RESULTS: ONCOFID-P interacted with CD44, entered cells through a receptor-mediated mechanism, and exerted a concentration-dependent inhibitory effect against tumor cell growth. After i.p. administration, the bioconjugate distributed quite uniformly within the peritoneal cavity, was well-tolerated, and was not associated with local histologic toxicity. Pharmacokinetic studies revealed that blood levels of bioconjugate-derived paclitaxel were much higher and persisted longer than those obtained with the unconjugated free drug. Intraperitoneal treatment of tumor-bearing mice with the bioconjugate revealed that ONCOFID-P exerted a relevant increase in therapeutic activity compared with free drug. CONCLUSIONS: ONCOFID-P significantly improved results obtained with conventional paclitaxel, in terms of in vivo tolerability and therapeutic efficacy; these data strongly support its development for locoregional treatment of ovarian cancer.

Hyaluronan as carrier of carboranes for tumor targeting in boron neutron capture therapy.

Boron neutron capture therapy (BNCT) represents a promising approach for tumor therapy. A critical requirement for BNCT is tumor targeting, a goal that is currently addressed with the development of low and high molecular weight agents capable of interacting with receptors expressed by cancer cells. Here, we describe a new bioconjugate (HApCB) composed by n-propyl carborane linked to hyaluronan (HA) via an ester linkage for a degree of substitution of approximately 30%, leading to a water-soluble derivative. The structure and main physicochemical characteristics of the new HA derivative were determined by means of Fourier transform infrared, fluorescence, and 1H, 13C, and 10B NMR analysis and are herein reported in detail. As HA is recognized by the CD44 antigen, densely populating the surface of many tumor cells, HApCB is expected to deliver boron atoms from the locally released carborane cages directly to target cells for antitumor application in BNCT. In vitro biological experiments showed that HApCB was not toxic for a variety of human tumor cells of different histotypes, specifically interacted with CD44 as the native unconjugated HA, and underwent uptake by tumor cells, leading to accumulation of amounts of boron atoms largely exceeding those required for a successful BNCT approach. Thus, HApCB may be regarded as a promising new BNCT agent for specific targeting of cancer cells overexpressing the CD44 receptor.

HYTAD1-p20: a new paclitaxel-hyaluronic acid hydrosoluble bioconjugate for treatment of superficial bladder cancer.

OBJECTIVE: To report the development of a new water-soluble paclitaxel-hyaluronic acid bioconjugate, HYTAD1-p20, for intravesical treatment of superficial bladder cancer. MATERIALS AND METHODS: HYTAD1-p20 was synthesized by carboxyl esterification of hyaluronic acid with paclitaxel, and its physicochemical and biologic properties were characterized. RESULTS: Paclitaxel loading was optimized at 20% w/w; this procedure increased by 500-fold the paclitaxel concentration in the resulting water-soluble biomaterial. In vitro, HYTAD1-p20 exerted a much higher dose-dependent inhibitory effect against RT-4 and RT-112/84 bladder carcinoma cell growth than that of free drug, and directly interacted with CD44 expressed by bladder tumor cells. In vivo, results of pharmacokinetic studies performed in mice after bladder catheterization and intravesical instillation of HYTAD1-p20 disclosed that drug leakage was negligible during a 2-hour analysis. Histologic examination of drug-instilled bladders revealed that HYTAD1-p20 was extremely well tolerated, while paclitaxel alone produced mucosal disruption and submucosal infiltration of inflammatory cells. Treatment of severe combined immunodeficient mice bearing subcutaneous RT-112/84 tumors with maximum tolerated doses of bioconjugate or paclitaxel showed that HYTAD1-p20 exerted a therapeutic activity comparable to that of free drug. CONCLUSIONS: These data suggest that HYTAD1-p20 significantly improved results obtained with conventional paclitaxel in terms of hydrosolubility, in vitro activity against human bladder cancer cells, and in vivo biocompatibility. This bioconjugate is a potentially useful treatment for superficial urothelial malignancy.

Pharmacokinetic behaviour of ACP gel, an autocrosslinked hyaluronan derivative, after intraperitoneal administration.

Autocrosslinked polysaccharide (ACP) gel is a fully biocompatible cross-linked derivative of hyaluronic acid, which has prolonged in vivo residence time and improved mechanical properties with respect to native hyaluronan for use in various surgical applications. The objective of this study was to assess the pharmacokinetic behaviour of ACP gel in dogs after intraperitoneal administration. Seven beagle dogs received intraperitoneal injections of tritium-labelled ACP gel. Blood samples were taken, and urine and faeces were collected until sacrifice, scheduled at various time points from 3 to 192 h after administration. Organs were removed from the animals at autopsy. Bodily fluid and organ samples were analysed for total and non-volatile radioactivity. Non-volatile radioactivity slowly appeared in plasma, with a median T(max) of 12 h, and then declined with a mean half-life of 69 h. Total radioactivity in plasma peaked later and declined more slowly, consistent with the formation of tritiated water. Little non-volatile radioactivity was found in any organs except the liver, where about 16% of the dose was present 72 h after administration, and the intestines, where the presence of radioactivity was probably due to a retention effect. A minor amount of non-volatile radioactivity was also found in the bone marrow. In summary, ACP gel administered into the peritoneal cavity is removed slowly by active initial catabolism at the injection site, and is then catabolised by well described physiological pathway of hyaluronan degradation with final release of simple molecules such as CO(2) and H(2)O. Given its in vivo residence time, ACP gel may be considered an ideal implantable surgical device.

Tissue-specific gene expression in chondrocytes grown on three-dimensional hyaluronic acid scaffolds.

The re-differentiation capacities of human articular and chick embryo sternal chondrocytes were evaluated by culture on HYAFF-11 and its sulphate derivative, HYAFF-11-S, polymers derived from the benzyl esterification of hyaluronate. Initial results showed that the HYAFF-11-S material promoted the highest rate of chondrocyte proliferation. RNA isolated from human and chick embryo chondrocytes cultured in Petri dishes, HYAFF-11 or HYAFF-11-S were subjected to semi-quantitative RT-PCR analyses. Human collagen types I, II, X, human Sox9 and aggrecan, chick collagen types I, II, IX and X were analysed. Results showed that human collagen type II mRNA expression was upregulated on HYAFF-11 biomaterials. In particular, a high level of collagen type IIB expression was associated with three-dimensional culture conditions, and the HYAFF-11 material was the most supportive for human collagen type X mRNA expression. Human Sox9 mRNA levels were constantly maintained in monolayer cell culture conditions over a period of 21 days, while these were upregulated when chondrocytes were cultured on HYAFF-11 and HYAFF-11S. Furthermore, chick collagen type IIA and IIB mRNA expression was detected after only 7 days of HYAFF-11 culture. Chick collagen type IX mRNA expression decreased in scaffold cultures over time. Histochemical staining performed in engineered cartilage revealed the presence of a de novo synthesized glycosaminoglycan-rich extracellular matrix; immunohistochemistry confirmed the deposition of collagen type II. This study showed that the three-dimensional HYAFF-11 culture system is both an effective chondrocyte delivery system for the treatment of articular cartilage defects, and an excellent in vitro model for studying cartilage differentiation.

Atomic force microscopy evaluation of aqueous interfaces of immobilized hyaluronan.

Hyaluronan (HA) was immobilized on aminated glass surfaces in three different ways: by simple ionic interaction and by covalent linking at low density and at full density. In agreement with previous reports, in vitro experiments show that the outcome of fibroblast adhesion tests is markedly affected by the details of the coupling procedure, suggesting that different interfacial forces are operating at the aqueous/HA interface in the three cases investigated. The interfacial properties of the HA-coated surfaces were probed by force-distance curves obtained with the atomic force microscope (AFM). This approach readily shows significant differences among the tested samples, which are directly related to the coupling strategy and to results of cell adhesion tests. In particular, the range of interaction between the tip and the surface is much lower when HA is covalently linked than when it is ionically coupled, suggesting a more compact surface structure in the former case. Increasing HA surface density minimizes the interaction force between the surface and the AFM tip, likely reflecting more complete shielding by the HA chains of the underlying substrate. In summary, these measurements clearly show the different nature of the aqueous interfaces tested, and underline the role of this analytical approach in the development and control of finely tuned biomaterial surfaces.