Collagenase From Vibrio alginolyticus Cultures: Experimental Study and Clinical Perspectives.

Purpose The purpose of this study is to examine the activity of collagenase from cultures of Vibrio alginolyticus as in vitro as in biological samples and to evaluate clinical perspectives of this product about the treatment of fibroproliferative diseases like Dupuytren's contracture. Methods The experimental part of the study has been divided in 2 stages. In the first stage, the collagenase has been produced in laboratory, assessing its purity, verifying the in vitro degradation of collagen by the enzyme and measuring the size of the fragments; in the second part, an experimental injection into samples of fibrous cord typical of Dupuytren's disease has been performed in vitro. For the injection we used only collagenase, or collagenase after having subjected them to 2 types of mechanical stress or a collagenase combined with ethylenediamine tetra-acetic acid. Considering that the human samples have been treated in vitro, our institution does not require a specific informed consent. Results It appeared evident that the collagenase obtained from Vibrio alginolyticus (nonpathogenic bacterium) is highly pure (>98%) and does not contain nonspecific protease. The collagenase from Vibrio alginolyticus therefore has an excellent degradative capacity against the collagen and this activity takes on a dose- and time-dependent behavior. The collagenase from Vibrio alginolyticus does not act negatively on cell survival and collagen peptides obtained may provide a better proliferative stimulus compared to controls. Conclusions The collagenase from Vibrio alginolyticus, given its obvious ability in vitro and biological samples, could be an option in the nonsurgical treatment of Dupuytren's disease. Level of evidence Level III, therapeutic.

Preliminary report of in vitro reconstruction of a vascularized tendonlike structure: a novel application for adipose-derived stem cells.

INTRODUCTION: A greater supply of tendinous tissue can be obtained through tissue engineering technology with increasing application of adult stem cells. It is well known that adipose-derived stem cells (ADSCs), found in abundance in adipose tissue, have the same differentiating capacity as mesenchymal stem cells yet have the advantage of being easily isolated. In the present study, we combined the great facility of ADSCs to differentiate with the application of an external mechanical stimulus to successfully create an in vitro reconstructed tendonlike structure with a microcapillary network. MATERIALS AND METHODS: Hyalonect meshes (Fidia Advanced Biopolymers, Abano Terme, Padova, Italy) were used as scaffold. Human ADSCs were seeded onto the biomaterials, and the cell/scaffold constructs were cultured under mechanical stress for up to 15 days. Human tenocytes were used in the same conditions as control. Performance was assessed by histology, immunochemistry, ultrastructure, and biomolecular analysis. RESULTS: Adipose-derived stem cells seeded onto Hyalonect adhered and differentiated along the entire surface of the biomaterial and began to infiltrate within its structure. Subsequently, endothelial cells migrated, forming a capillary in the new extracellular matrix. CONCLUSIONS: This technique allowed for the creation of a vascularized tendon equivalent that could easily be detached from the bioreactor, thus facilitating its implant at the lesion site. These results highlight the biologic performance of biodegradable hyaluronic acid-based (HYAFF-11) scaffolds, which were shown to be suitable for deposition of the autologous extracellular matrix critical for ADSCs differentiation.

Hyaluronic acid three-dimensional scaffold for surgical revision of retracting scars: a human experimental study.

An observational study was carried out at the Plastic and Reconstructive Surgery Unit of the University of Pavia - Salvatore Maugeri Research and Care Institute, Pavia, Italy, to assess the clinical and histological long-term outcomes of autologous skin grafting of fresh surgical wounds following previous repair with a hyaluronic acid three-dimensional scaffold (Hyalomatrix®). Eleven fresh wounds from surgical release of retracted scars were enrolled in this study. A stable skin-like tissue cover was observed in all of the treated wounds in an average 1 month's time; at the end of this study, after an average of 12 months' time, all of the reconstructed areas were pliable and stable, although an average retraction rate of 51·62% was showed. Histological observation and immunohistochemical analysis displayed integration of the graft within the surrounding tissues. A regenerated dermis with an extracellular matrix rich in type I collagen and elastic fibres and with reduced type III collagen rate was observed. The epidermis and dermoepidermal junction featured a normal appearance with well-structured dermal papillae, too. Although the histological features would suggest regeneration of a skin-like tissue, with a good dermis and no signs of scarring, the clinical problem of secondary contracture is still unsolved.

High glucose induces adipogenic differentiation of muscle-derived stem cells.

Regeneration of mesenchymal tissues depends on a resident stem cell population, that in most cases remains elusive in terms of cellular identity and differentiation signals. We here show that primary cell cultures derived from adipose tissue or skeletal muscle differentiate into adipocytes when cultured in high glucose. High glucose induces ROS production and PKCbeta activation. These two events appear crucial steps in this differentiation process that can be directly induced by oxidizing agents and inhibited by PKCbeta siRNA silencing. The differentiated adipocytes, when implanted in vivo, form viable and vascularized adipose tissue. Overall, the data highlight a previously uncharacterized differentiation route triggered by high glucose that drives not only resident stem cells of the adipose tissue but also uncommitted precursors present in muscle cells to form adipose depots. This process may represent a feed-forward cycle between the regional increase in adiposity and insulin resistance that plays a key role in the pathogenesis of diabetes mellitus.

Hyaluronic acid biodegradable material for reconstruction of vascular wall: a preliminary study in rats.

The objective of this preliminary study was to develop a reabsorbable vascular patch that did not require in vitro cell or biochemical preconditioning for vascular wall repair. Patches were composed only of hyaluronic acid (HA). Twenty male Wistar rats weighing 250-350 g were used. The abdominal aorta was exposed and isolated. A rectangular breach (1 mm × 5 mm) was made on vessel wall and arterial defect was repaired with HA made patch. Performance was assessed at 1, 2, 4, 8, and 16 weeks after surgery by histology and immunohistochemistry. Extracellular matrix components were evaluated by molecular biological methods. After 16 weeks, the biomaterial was almost completely degraded and replaced by a neoartery wall composed of endothelial cells, smooth muscle cells, collagen, and elastin fibers organized in layers. In conclusion, HA patches provide a provisional three-dimensional support to interact with cells for the control of their function, guiding the spatially and temporally multicellular processes of artery regeneration.

In Vitro Effects of Low Doses of ß-Caryophyllene, Ascorbic Acid and d-Glucosamine on Human Chondrocyte Viability and Inflammation.

ß-caryophyllene (BCP), a plant-derived sesquiterpene, has been reported to have anti-inflammatory and antioxidant effects. The purpose of this study is to evaluate the effects of BCP in combination with ascorbic acid (AA) and d-glucosamine (GlcN) against macrophage-mediated inflammation on in vitro primary human chondrocytes. Changes in cell viability, intracellular ROS generation, gene expression of pro-inflammatory mediators, metalloproteinases (MMPs), collagen type II and aggrecan were analyzed in primary human chondrocytes exposed to the conditioned medium (CM) of activated U937 monocytes and subsequently treated with BCP alone or in combination with AA and GlcN. The CM-induced chondrocyte cytotoxicity was reduced by the presence of low doses of BCP alone or in combination with AA and GlcN. The exposure of cells to CM significantly increased IL-1ß, NF-κB1 and MMP-13 expression, but when BCP was added to the inflamed cells, alone or in combination with AA and GlcN, gene transcription for all these molecules was restored to near baseline values. Moreover, chondrocytes increased the expression of collagen type II and aggrecan when stimulated with AA and GlcN alone or in combination with BCP. This study showed the synergistic anti-inflammatory and antioxidative effects of BCP, AA and GlcN at low doses on human chondrocyte cultures treated with the CM of activated U937 cells. Moreover, the combination of the three molecules was able to promote the expression of collagen type II and aggrecan. All together, these data could suggest that BCP, AA and GlcN exert a chondro-protective action.

Neoarteries grown in vivo using a tissue-engineered hyaluronan-based scaffold.

Vascular tissue engineering has emerged as a promising technology for the design of an ideal, responsive, living conduit with properties similar to that of native tissue. The missing link in tissue-engineered blood vessels is elastin biosynthesis. Several biomaterials are currently used but few support elastin biosynthesis in a 3-D array. In previous studies, we demonstrated that a hyaluronan-based scaffold (HYAFF-11) grafted in the infrarenal rat aorta successfully guided the complete regeneration of a well-functioning small-diameter (2 mm) neoartery. The aim of the present study was to test the ability of HYAFF-11 biodegradable grafts to develop into neovessels of larger size (4 mm) in a porcine model, focusing on extracellular matrix (ECM) remodeling and elastin biosynthesis. HYAFF-11 tubes (diameter 4 mm, length 5 cm) were implanted in an end-to-end fashion in the common carotid artery. Grafts were analyzed for patency with a Duplex scan every 15 days. ECM components were evaluated by histological and molecular biological methods. All the animals survived the observation period without complications. Intimal hyperplasia (initiating at the anastomotic site) and graft thrombosis led to 3 cases of partial or complete occlusion, as demonstrated by histological examination. There were no signs of stenoses or aneurysms in the remaining grafts. After 5 months, the biomaterial was almost completely degraded and replaced by a neoartery segment composed of mature smooth muscle cells, collagen, and elastin fibers organized in layers and was completely covered on the luminal surface by endothelial cells (vWF(+)). Whereas in previous small animal studies, patency rates were not optimal, those obtained in the present study using hyaluronan-based grafts of larger size confirmed the ability of these constructs to guide the development of a well-functioning neoartery, with the remarkable additional attribute of facilitating the formation of organized layers of elastin fibers.

Hyaluronan benzyl ester as a scaffold for tissue engineering.

Tissue engineering is a multidisciplinary field focused on in vitro reconstruction of mammalian tissues. In order to allow a similar three-dimensional organization of in vitro cultured cells, biocompatible scaffolds are needed. This need has provided immense momentum for research on "smart scaffolds" for use in cell culture. One of the most promising materials for tissue engineering and regenerative medicine is a hyaluronan derivative: a benzyl ester of hyaluronan (HYAFF). HYAFF can be processed to obtain several types of devices such as tubes, membranes, non-woven fabrics, gauzes, and sponges. All these scaffolds are highly biocompatible. In the human body they do not elicit any adverse reactions and are resorbed by the host tissues. Human hepatocytes, dermal fibroblasts and keratinocytes, chondrocytes, Schwann cells, bone marrow derived mesenchymal stem cells and adipose tissue derived mesenchymal stem cells have been successfully cultured in these meshes. The same scaffolds, in tube meshes, has been applied for vascular tissue engineering that has emerged as a promising technology for the design of an ideal, responsive, living conduit with properties similar to that of native tissue.

Effects of intra-articular hyaluronic acid associated to Chitlac (arty-duo®) in a rat knee osteoarthritis model.

Among conventional osteoarthritis (OA) treatments, intra-articular (i.a) viscosupplementation with hyaluronic acid (HA) is used to restore joint viscoelasticity. However, the rapid clearance and elimination of HA may limit its application. The aim of this study was to verify the improved efficacy of HA within the joint, using a lactose-modified chitosan (chitlac) as a potentially chondroprotective additive. Four weeks after induction of experimental OA by destabilization of the medial meniscus (DMM), 12-week-old Sprague Dawley male rats (n = 30), received once a week, for three weeks, i.a injections of: (i) HA associated to chitlac (ARTY-DUO®), (ii) HA; and (iii) sodium chloride (NaCl). Five animals for each group were euthanized 4 weeks after the first i.a injection, while the remaining five were euthanized 8 weeks after the first i.a injection. The restoration of physiological joint microenvironment was tested by histology, histomorphometry, immunohistochemistry, and microtomography (micro-CT). At 4 and even more at 8 weeks, histological analysis showed a significant decrease in OARSI and Mankin scores, with weaker matrix metalloproteinase (MMP)-3, MMP-13, and Galectin-3 in ARTY-DUO® group versus NaCl and HA groups. A reduction in Galectin-1 and a stronger Collagen II staining was seen in both ARTY-DUO® and HA versus NaCl. A reduction in Kreen-modified score, for synovium inflammation, was observed in the ARTY-DUO® group. Micro-CT measurements did not shown significant differences between the groups. The present results show that i.a ARTY-DUO® injections produce a significant improvement in knee articular cartilage degeneration and synovium inflammation in a rat model of DMM-induced OA. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

New 3D hyaluronan-based scaffold for in vitro reconstruction of the rat sciatic nerve.

OBJECTIVE: For peripheral nerve regeneration, three-dimensional distribution and growth of cells within the porous scaffold are of clinical significance. The purpose of this study was to test in vitro a novel hyaluronic acid-based tubular conduit (HYAFF-11 biomaterials: 1 x 10 mm) as a nerve guide. METHODS: Human fibroblasts, RN22 Schwann cell lines, human umbilical vein endothelial cells and primary nerve cells, obtained from neonatal rat sciatic nerve, were harvested and seeded on HYAFF-11 devices. Histologic (hematoxylin-eosin), immunohistochemical (antibodies to S100, CD31 and Von Willebrand factor) and PCR analyses were performed after 7 and 14 days from cell seeding onto biomaterials. MTT-based (thiazolyl blue) and DELFIA cell proliferation kit tests were performed to observe the biocompatibility of the cells cultured within the biomaterial devices. RESULTS: We concluded that the conduits were not cytotoxic and demonstrated that cultured RN22 Schwann cells and rat Schwann cells grow in vitro on new artificial nerve conduits. We thus inferred that the HYAFF-11 conduit was a suitable biomaterial able to support nerve cell growth in vitro and after 14 days of cultivation, remained circular with a round lumen, maintaining the size and shape of its original architecture. Finally, attachment and proliferation of endothelial cells attested to the feasibility of developing a coculture system to promote in vivo integration of a microvascularized nerve substitute. DISCUSSION: HYAFF-11 pre-seeded with Schwann and endothelial cells has the potential to be an alternative to autografting for the repair of long peripheral nerve defects.

Tissue engineering approaches for the construction of a completely autologous tendon substitute.

Tissue engineering is a multidisciplinary field that involves the application of the principles and methods of engineering and life sciences towards i) the fundamental understanding of structure-function relationships in normal and pathological mammalian tissues and ii) the development of biological substitutes that restore, maintain or improve tissue function. The goal of tissue engineering is to surpass the limitations of conventional treatments based on organ transplantation and biomaterial implantation. The field of tendon tissue engineering is relatively unexplored due to the difficulty in in vitro preservation of tenocyte phenotype. Only recently has mechanobiology allowed us to gain a better understanding of the fundamental role of in vitro mechanical stimuli in maintaining the phenotype of tendinous tissue. This review analyzes the techniques used so far for in vitro regeneration of tendinous tissue.

In vivo regeneration of small-diameter (2 mm) arteries using a polymer scaffold.

The difficulty of obtaining significant long-term patency and good wall mechanical strength in vivo has been a significant obstacle in achieving small-diameter vascular prostheses. The aim of the present study was to develop a prosthetic graft that could perform as a small-diameter vascular conduit. Tubular structures of hyaluronan (HYAFF-11 tubules, 2 mm diameter, 1 cm length) were grafted in the abdominal aorta of 30 rats as temporary absorbable guides to promote regeneration of vascular structures. Performance was assessed by histology, immunohistochemistry, and ultra-structural analysis. These experiments resulted in three novel findings: 1) complete endothelialization of the tube's luminal surface occurred; 2) sequential regeneration of vascular components led to complete vascular wall regeneration 15 days after surgery; and 3) the biomaterial used created the ideal environment for the delicate regeneration process during the critical initial phases, yet its biodegradability allowed for complete degradation of the construct four months after implantation, at which time, a new artery remained to connect the artery stumps. This study assesses the feasibility to create a completely biodegradable vascular regeneration guide in vivo, able to sequentially orchestrate vascular regeneration events needed for very small artery reconstruction.

Maturation of tissue engineered cartilage implanted in injured and osteoarthritic human knees.

The regeneration of damaged organs requires that engineered tissues mature when implanted at sites of injury or disease. We have used new analytic techniques to determine the extent of tissue regeneration after treatment of knee injury patients with a novel cartilage tissue engineering therapy and the effect of pre-existing osteoarthritis on the regeneration process. We treated 23 patients, with a mean age of 35.6 years, presenting with knee articular cartilage defects 1.5 cm2 to 11.25 cm2 (mean, 5.0 cm2) in area. Nine of the patients had X-ray evidence of osteoarthritis. Chondrocytes were isolated from healthy cartilage removed at arthroscopy. The cells were cultured for 14 days, seeded onto esterified hyaluronic acid scaffolds (Hyalograft C), and grown for a further 14 days before implantation. A second-look biopsy was taken from each patient after 6 to 30 months (mean, 16 months). After standard histological analysis, uncut tissue was further analyzed using a newly developed biochemical protocol involving digestion with trypsin and specific, quantitative assays for type II collagen, type I collagen, and proteoglycan, as well as mature and immature collagen crosslinks. Cartilage regeneration was observed as early as 11 months after implantation and in 10 out of 23 patients. Tissue regeneration was found even when implants were placed in joints that had already progressed to osteoarthrosis. Cartilage injuries can be effectively repaired using tissue engineering, and osteoarthritis does not inhibit the regeneration process.

In vitro reconstruction of an endothelialized skin substitute provided with a microcapillary network using biopolymer scaffolds.

Successful in vitro reconstruction of skin requires the inclusion of several cell types that give rise in coculture to the specific elements present in native skin, and the appropriate scaffolding structure to house and support these cells. In addition to the two main structural components, epidermis and dermis, one critical apparatus of the skin is a capillary network that guarantees adequate perfusion of nutrients and oxygen. The aim of the present study was to develop an in vitro coculture system that assumed the human dermal-epidermal architecture and included a microcapillary network in a three-dimensional biomaterial that guaranteed ease of handling in a clinical setting. Endothelialized skin (ES) was prepared by coculturing three human cell types: keratinocytes, fibroblasts, and endothelial cells, obtained from human full-thickness skin samples, in scaffolds produced from modified hyaluronic acid. Results were evaluated by histological and immunohistochemical analyses at different time points. In vitro, engineered skin obtained with this composite culture developed into a well-differentiated upper layer of stratified keratinocytes lining a dermal-like structure, in which fibroblasts, extracellular matrix and a microvascular network were present. Furthermore, the biodegradable fabric produced from hyaluronic acid and used as the scaffolding support for this in vitro constructed skin graft greatly facilitated handling in the perioperative period.

Urokinase plasminogen activator, uPa receptor, and its inhibitor in vernal keratoconjunctivitis.

PURPOSE: Plasminogen activators play a role, not only in fibrinolysis but also in events such as chemotaxis, collagen degradation, and cell spreading. The serine protease urokinase (uPA) is a potent chemoattractant for leukocytes that may be involved in the pathogenesis of severe forms of allergic conjunctivitis such as vernal keratoconjunctivitis (VKC). METHODS: Tear and peripheral blood samples were obtained from 20 patients with active VKC and from 19 normal subjects who formed the control group. Levels of plasminogen activity, uPA, tissue plasminogen activator (tPA), and their inhibitor, plasminogen activator inhibitor type-1 (PAI-1) were measured in tears and plasma of patients with VKC. The presence of tPA, uPA, and urokinase receptor (uPAR) in conjunctival tissues were evaluated by immunohistochemistry. uPA, uPAR, and PAI-1 expression and production were measured in conjunctival epithelial cell and fibroblast cultures treated with cytokines. RESULTS: Tear levels of uPA and tPA and tear plasminogen activity levels were significantly greater in patients with VKC than in control subjects. Increased staining for uPA and uPAR was found in VKC tissues compared with normal conjunctiva. Both conjunctival epithelial cells and fibroblasts demonstrated an increased expression of uPAR after exposure to IL-4 or -13, whereas uPA was highly expressed by epithelial cells exposed to IL-4. PAI-1 levels in culture medium were increased in IL-4-exposed epithelial cells compared to nonstimulated cells and were decreased in fibroblast culture. CONCLUSIONS: Increased expression of fibrinolytic system components and imbalance between plasminogen activators and PAI may be involved in the pathogenesis of severe allergic conjunctivitis, thus contributing to inflammatory cell migration and tissue remodeling.

Tear levels and activity of matrix metalloproteinase (MMP)-1 and MMP-9 in vernal keratoconjunctivitis.

PURPOSE: To study levels and activity of matrix metalloproteinase (MMP)-1 and -9 and their tissue inhibitor (TIMP-1) in tears of patients with vernal keratoconjunctivitis (VKC), with and without severe corneal damage. METHODS: Tear samples were obtained from 16 patients with active VKC and 10 normal control subjects, after clinical evaluation and tear cytology. Tear levels of pro-MMP-1, pro-MMP-9, and TIMP-1 were measured by enzyme-linked immunosorbent assay (ELISA). Collagenase and gelatinase activity were measured in tears by MMP activity assays. Immunohistochemistry was performed on a fragment of superficial keratectomy from two vernal corneal ulcers. RESULTS: Tear levels of pro-MMP-1 and pro-MMP-9 were significantly increased in patients with VKC compared with control subjects (P < 0.001). MMP-1/TIMP-1 and MMP-9/TIMP-1 molar ratios were significantly increased (P < 0.001) in VKC. MMP-1 and MMP-9 activities were significantly increased in VKC tears compared with control samples (P < 0.005). MMP-9 activity correlated significantly with corneal involvement and giant papillae formation. Immunohistochemistry showed positive staining for MMP-9, fibronectin, and eosinophil cationic protein (ECP) on the superficial corneal stroma of the ulcer bed, but no inflammatory cells. CONCLUSIONS: Increased levels and activity of MMP-1 and -9 and an imbalance between MMPs and TIMP may be involved in the pathogenesis of VKC.

Effects of Th2 cytokines on expression of collagen, MMP-1, and TIMP-1 in conjunctival fibroblasts.

PURPOSE: To determine whether cytokines involved in chronic allergic conjunctival disorders may affect formation of giant papillae and tissue remodeling. METHODS: Conjunctival fibroblast cultures were challenged with different concentrations of human recombinant interleukin (IL)-4, IL-13, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha. Procollagens I (PIP) and III (PIIIP), matrix metalloproteinase (MMP)-1 and -9, and tissue inhibitor of metalloproteinase (TIMP)-1 were measured in supernatants, and their respective mRNAs were evaluated by RT-PCR. RESULTS: IL-4 and -13 (10 ng/mL) significantly increased production and expression of PIP compared with nonstimulated cells, whereas IFN-gamma elicited the opposite effect, at both the protein and mRNA levels. Both IL-4 and -13 significantly decreased production of MMP-1 and increased that of TIMP-1, whereas TNF-alpha increased production of MMP-1 and -9. Expression of MMP-1 was reduced by IL-4 and increased by the other tested cytokines, whereas expression of TIMP-1 was increased by all tested cytokines. CONCLUSIONS: IL-4 and -13 increased production of collagen and modified the equilibrium between MMP-1 and its inhibitor, TIMP-1. These effects were partially opposed by IFN-gamma and TNF-alpha.

Hyaluronan-based scaffolds (Hyalograft C) in the treatment of knee cartilage defects: preliminary clinical findings.

Hyalograft C is an innovative tissue-engineering approach for the treatment of knee cartilage defects involving the implantation of laboratory expanded autologous chondrocytes grown on a three-dimensional hyaluronan-based scaffold. This technique has recently been introduced into clinical practice, with more than 600 patients treated so far. Because no periosteal coverage is required to keep the graft in place, surgical time and morbidity are reduced, and handling of the graft is much simpler than currently available autologous chondrocyte implantation techniques. The safety profile of the treatment appears positive, with a limited number of adverse events reported. Here we discuss the clinical, arthroscopic and histological results from a cohort of 67 patients treated with Hyalograft C (mean follow-up time from implantation of 17.5 months). Results are reported based on four endpoints: patients' subjective evaluation of knee conditions (97% of patients improved) and quality of life (94% improved), surgeons' knee functional test (87% of patients with the best scores), arthroscopic evaluation of cartilage repair (96.7% biologically acceptable) and histological assessment of the grafted site (majority of specimens hyaline-like). The positive clinical results obtained indicate that Hyalograft C may be a viable therapeutic option for the treatment of acute cartilage lesions.

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.