Low oxygen tension is a more potent promoter of chondrogenic differentiation than dynamic compression

During fracture healing and microfracture treatment of cartilage defects mesenchymal stem cells (MSCs) infiltrate the wound site, proliferate extensively and differentiate along a cartilaginous or an osteogenic lineage in response to local environmental cues. MSCs may be able to directly sense their mechanical environment or alternatively, the mechanical environment could act indirectly to regulate MSC differentiation by inhibiting angiogenesis and diminishing the supply of oxygen and other regulatory factors. Dynamic compression has been shown to regulate chondrogenesis of MSCs. In addition, previous studies have shown that a low oxygen environment promotes in vitro chondrogenesis of MSCs. The hypothesis of this study is that a low oxygen environment is a more potent promoter of chondrogenic differentiation of MSCs embedded in agarose hydrogels compared to dynamic compression. In MSC-seeded constructs supplemented with TGF-β3, GAG and collagen accumulation was higher in low oxygen conditions compared to normoxia. For normoxic and low oxygen culture GAG accumulation within the agarose hydrogel was inhomogeneous, with low levels of GAG measured in the annulus of constructs maintained in normoxic conditions. Dynamic compression did not significantly increase GAG or collagen accumulation in normoxia. However under low oxygen conditions, dynamic compression reduced GAG accumulation compared to free-swelling controls, but remained higher than comparable constructs maintained in normoxic conditions. This study demonstrates that continuous exposure to low oxygen tension is a more potent pro-chondrogenic stimulus than 1 h/day of dynamic compression for porcine MSCs embedded in agarose hydrogels.     

Sox9-Regulated miRNA-574-3p Inhibits Chondrogenic Differentiation of Mesenchymal Stem Cells

The aim of this study was to identify new microRNAs (miRNAs) that are modulated during the differentiation of mesenchymal stem cells (MSCs) toward chondrocytes. Using large scale miRNA arrays, we compared the expression of miRNAs in MSCs (day 0) and at early time points (day 0.5 and 3) after chondrogenesis induction. Transfection of premiRNA or antagomiRNA was performed on MSCs before chondrogenesis induction and expression of miRNAs and chondrocyte markers was evaluated at different time points during differentiation by RT-qPCR. Among miRNAs that were modulated during chondrogenesis, we identified miR-574-3p as an early up-regulated miRNA. We found that miR-574-3p up-regulation is mediated via direct binding of Sox9 to its promoter region and demonstrated by reporter assay that retinoid X receptor (RXR)α is one gene specifically targeted by the miRNA. In vitro transfection of MSCs with premiR-574-3p resulted in the inhibition of chondrogenesis demonstrating its role during the commitment of MSCs towards chondrocytes. In vivo, however, both up- and down-regulation of miR-574-3p expression inhibited differentiation toward cartilage and bone in a model of heterotopic ossification. In conclusion, we demonstrated that Sox9-dependent up-regulation of miR-574-3p results in RXRα down-regulation. Manipulating miR-574-3p levels both in vitro and in vivo inhibited chondrogenesis suggesting that miR-574-3p might be required for chondrocyte lineage maintenance but also that of MSC multipotency.     

Spontaneous Differentiation of Human Mesenchymal Stem Cells on Poly-Lactic-Co-Glycolic Acid Nano-Fiber Scaffold

IntroductionMesenchymal stem cells (MSCs) have immunosuppressive activity and can differentiate into bone and cartilage; and thus seem ideal for treatment of rheumatoid arthritis (RA). Here, we investigated the osteogenesis and chondrogenesis potentials of MSCs seeded onto nano-fiber scaffolds (NFs) in vitro and possible use for the repair of RA-affected joints.MethodsMSCs derived from healthy donors and patients with RA or osteoarthritis (OA) were seeded on poly-lactic-glycolic acid (PLGA) electrospun NFs and cultured in vitro.ResultsHealthy donor-derived MSCs seeded onto NFs stained positive with von Kossa at Day 14 post-stimulation for osteoblast differentiation. Similarly, MSCs stained positive with Safranin O at Day 14 post-stimulation for chondrocyte differentiation. Surprisingly, even cultured without any stimulation, MSCs expressed RUNX2 and SOX9 (master regulators of bone and cartilage differentiation) at Day 7. Moreover, MSCs stained positive for osteocalcin, a bone marker, and simultaneously also with Safranin O at Day 14. On Day 28, the cell morphology changed from a spindle-like to an osteocyte-like appearance with processes, along with the expression of dentin matrix protein-1 (DMP-1) and matrix extracellular phosphoglycoprotein (MEPE), suggesting possible differentiation of MSCs into osteocytes. Calcification was observed on Day 56. Expression of osteoblast and chondrocyte differentiation markers was also noted in MSCs derived from RA or OA patients seeded on NFs. Lactic acid present in NFs potentially induced MSC differentiation into osteoblasts.ConclusionsOur PLGA scaffold NFs induced MSC differentiation into bone and cartilage. NFs induction process resembled the procedure of endochondral ossification. This finding indicates that the combination of MSCs and NFs is a promising therapeutic technique for the repair of RA or OA joints affected by bone and cartilage destruction.     

Engineered Co-culture Strategies Using Stem Cells for Facilitated Chondrogenic Differentiation and Cartilage Repair

Osteoarthritis (OA) is a chronic disease in elders and athletes due to limited regenerative capacities of cartilage tissues and subsequently insufficient recovery of damaged sites. Recent clinical treatments for OA have utilized progenitor cell-based therapies for cartilage tissue regeneration. Administration of a single type of cell population such as stem cells or chondrocytes does not guarantee a full recovery of cartilage defects. Therefore, current tissue engineering approaches using co-culture techniques have been developed to mimic complex and dynamic cellular interactions in native cartilage tissues and facilitate changes in cellular phenotypes into chondrogenesis. Therefore, this paper introduces recently developed co-culture systems using two major cell populations, mesenchymal stem cells (MSCs) and chondrocytes. Specifically, a series of examples to describe (1) synergistic in vitro activations of MSCs by paracrine signaling molecules from adult chondrocytes in co-culture systems and (2) functional in vivo tissue regeneration via co-administration of both cell types were reviewed. Based on these findings, it could be speculated that engineered co-culture systems using MSC/ chondrocyte is a promising and feasible cell-based OA therapy in clinical aspects.     

Fabrication of Biomimetic Bone Tissue Using Mesenchymal Stem Cell-Derived Three-Dimensional Constructs Incorporating Endothelial Cells

The development of technologies to promote vascularization of engineered tissue would drive major developments in tissue engineering and regenerative medicine. Recently, we succeeded in fabricating three-dimensional (3D) cell constructs composed of mesenchymal stem cells (MSCs). However, the majority of cells within the constructs underwent necrosis due to a lack of nutrients and oxygen. We hypothesized that incorporation of vascular endothelial cells would improve the cell survival rate and aid in the fabrication of biomimetic bone tissues in vitro. The purpose of this study was to assess the impact of endothelial cells combined with the MSC constructs (MSC/HUVEC constructs) during short- and long-term culture. When human umbilical vein endothelial cells (HUVECs) were incorporated into the cell constructs, cell viability and growth factor production were increased after 7 days. Furthermore, HUVECs were observed to proliferate and self-organize into reticulate porous structures by interacting with the MSCs. After long-term culture, MSC/HUVEC constructs formed abundant mineralized matrices compared with those composed of MSCs alone. Transmission electron microscopy and qualitative analysis revealed that the mineralized matrices comprised porous cancellous bone-like tissues. These results demonstrate that highly biomimetic bone tissue can be fabricated in vitro by 3D MSC constructs incorporated with HUVECs.     

Functional properties of cartilaginous tissues engineered from infrapatellar fat pad-derived mesenchymal stem cells

Articular cartilage has a poor intrinsic capacity for self-repair. The advent of autologous chondrocyte implantation has provided a feasible method to treat cartilage defects. However, the associated drawbacks with the isolation and expansion of chondrocytes from autologous tissue has prompted research into alternative cell sources such as mesenchymal stem cells (MSCs) which have been found to exist in the bone marrow as well as other joint tissues such as the infrapatellar fat pad (IFP), synovium and within the synovial fluid itself. In this work we assessed the chondrogenic potential of IFP-derived porcine cells over a 6 week period in agarose hydrogel culture in terms of mechanical properties, biochemical content and histology. It was found that IFP cells underwent robust chondrogenesis as assessed by glycosaminoglycan (1.47±0.22% w/w) and collagen (1.44±0.22% w/w) accumulation after 42 days of culture. The 1 Hz dynamic modulus of the engineered tissue at this time point was 272.8 kPa (±46.8). The removal of TGF-β3 from culture after 21 days was shown to have a significant effect on both the mechanical properties and biochemical content of IFP constructs after 42 days, with minimal increases occurring from day 21 to day 42 without continued supplementation of TGF-β3. These findings further strengthen the case that the IFP may be a promising cell source for putative cartilage repair strategies.     

Analysis of collagen expression during chondrogenic induction of human bone marrow mesenchymal stem cells

Adult mesenchymal stem cells (MSCs) are currently being investigated as an alternative to chondrocytes for repairing cartilage defects. As several collagen types participate in the formation of cartilage-specific extracellular matrix, we have investigated their gene expression levels during MSC chondrogenic induction. Bone marrow MSCs were cultured in pellet in the presence of BMP-2 and TGF-β3 for 24 days. After addition of FGF-2, at the fourth passage during MSC expansion, there was an enhancing effect on specific cartilage gene expression when compared to that without FGF-2 at day 12 in pellet culture. A switch in expression from the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of the collagen type II gene was observed at day 24. A short-term addition of FGF-2 followed by a treatment with BMP-2/TGF-β3 appears sufficient to accelerate chondrogenesis with a particular effect on the main cartilage collagens.     

Morphogenetic signals from chondrocytes promote chondrogenic and osteogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are potentially useful cells for musculoskeletal tissue engineering. However, controlling MSC differentiation and tissue formation in vivo remains a challenge. There is a significant need for well-defined and efficient protocols for directing MSC behaviors in vivo. We hypothesize that morphogenetic signals from chondrocytes may regulate MSC differentiation. In micromass culture of MSCs, incubation with chondrocyte-conditioned medium (CCM) significantly enhanced the production of cartilage specific matrix including type II collagen. In addition, incubation of MSCs with conditioned medium supplemented with osteogenic factors induced more osteogenesis and accumulation of calcium and increased ALP activity. These findings reveal that chondrocyte-secreted factors promote chondrogenesis as well as osteogenesis of MSCs during in vitro micromass culture. Moreover, when MSCs expanded with chondrocyte-conditioned medium were encapsulated in hydrogels and subsequently implanted into athymic mice, basophilic extracellular matrix deposition characteristic of neocartilage was evident. These results indicate that articular chondrocytes produce suitable morphogenetic factors that induce the differentiation program of MSCs in vitro and in vivo.     

Rescue of proinflammatory cytokine-inhibited chondrogenesis by the antiarthritic effect of melatonin in synovium mesenchymal stem cells via suppression of reactive oxygen species and matrix metalloproteinases

Cartilage repair by mesenchymal stem cells (MSCs) often occurs in diseased joints in which the inflamed microenvironment impairs chondrogenic maturation and causes neocartilage degradation. In this environment, melatonin exerts an antioxidant effect by scavenging free radicals. This study aimed to investigate the anti-inflammatory and chondroprotective effects of melatonin on human MSCs in a proinflammatory cytokine-induced arthritic environment. MSCs were induced toward chondrogenesis in the presence of interleukin-1 β (IL-1β) or tumor necrosis factor α (TNF-α) with or without melatonin. Levels of intracellular reactive oxygen species (ROS), hydrogen peroxide, antioxidant enzymes, and cell viability were then assessed. Deposition of glycosaminoglycans and collagens was also determined by histological analysis. Gene expression of chondrogenic markers and matrix metalloproteinases (MMPs) was assessed by real-time polymerase chain reaction. In addition, the involvement of the melatonin receptor and superoxide dismutase (SOD) in chondrogenesis was investigated using pharmacologic inhibitors. The results showed that melatonin significantly reduced ROS accumulation and increased SOD expression. Both IL-1β and TNF-α had an inhibitory effect on the chondrogenesis of MSCs, but melatonin successfully restored the low expression of cartilage matrix and chondrogenic genes. Melatonin prevented cartilage degradation by downregulating MMPs. The addition of luzindole and SOD inhibitors abrogated the protective effect of melatonin associated with increased levels of ROS and MMPs. These results demonstrated that proinflammatory cytokines impair the chondrogenesis of MSCs, which was rescued by melatonin treatment. This chondroprotective effect was potentially correlated to decreased ROS, preserved SOD, and suppressed levels of MMPs. Thus, melatonin provides a new strategy for promoting cell-based cartilage regeneration in diseased or injured joints.     

Cartilage Derived from Bone Marrow Mesenchymal Stem Cells Expresses Lubricin In Vitro and In Vivo

Objective

Lubricin expression in the superficial cartilage will be a crucial factor in the success of cartilage regeneration. Mesenchymal stem cells (MSCs) are an attractive cell source and the use of aggregates of MSCs has some advantages in terms of chondrogenic potential and efficiency of cell adhesion. Lubricin expression in transplanted MSCs has not been fully elucidated so far. Our goals were to determine (1) whether cartilage pellets of human MSCs expressed lubricin in vitro chondrogenesis, (2) whether aggregates of human MSCs promoted lubricin expression, and (3) whether aggregates of MSCs expressed lubricin in the superficial cartilage after transplantation into osteochondral defects in rats.

Methods

For in vitro analysis, human bone marrow (BM) MSCs were differentiated into cartilage by pellet culture, and also aggregated using the hanging drop technique. For an animal study, aggregates of BM MSCs derived from GFP transgenic rats were transplanted to the osteochondral defect in the trochlear groove of wild type rat knee joints. Lubricin expression was mainly evaluated in differentiated and regenerated cartilages.

Results

In in vitro analysis, lubricin was detected in the superficial zone of the pellets and conditioned medium. mRNA expression of Proteoglycan4 (Prg4), which encodes lubricin, in pellets was significantly higher than that of undifferentiated MSCs. Aggregates showed different morphological features between the superficial and deep zone, and the Prg4 mRNA expression increased after aggregate formation. Lubricin was also found in the aggregate. In a rat study, articular cartilage regeneration was significantly better in the MSC group than in the control group as shown by macroscopical and histological analysis. The transmission electron microscope showed that morphology of the superficial cartilage in the MSC group was closer to that of the intact cartilage than in the control group. GFP positive cells remained in the repaired tissue and expressed lubricin in the superficial cartilage.

Conclusion

Cartilage derived from MSCs expressed lubricin protein both in vitro and in vivo. Aggregation promoted lubricin expression of MSCs in vitro and transplantation of aggregates of MSCs regenerated cartilage including the superficial zone in a rat osteochondral defect model. Our results indicate that aggregated MSCs could be clinically relevant for therapeutic approaches to articular cartilage regeneration with an appropriate superficial zone in the future.     

The role of mechanical signals in regulating chondrogenesis and osteogenesis of mesenchymal stem cells

It is becoming increasingly clear that mesenchymal stem cell (MSC) differentiation is regulated by mechanical signals. Mechanical forces generated intrinsically within the cell in response to its extracellular environment, and extrinsic mechanical signals imposed upon the cell by the extracellular environment, play a central role in determining MSC fate. This article reviews chondrogenesis and osteogenesis during skeletogenesis, and then considers the role of mechanics in regulating limb development and regenerative events such as fracture repair. However, observing skeletal changes under altered loading conditions can only partially explain the role of mechanics in controlling MSC differentiation. Increasingly, understanding how epigenetic factors, such as the mechanical environment, regulate stem cell fate is undertaken using tightly controlled in vitro models. Factors such as bioengineered surfaces, substrates, and bioreactor systems are used to control the mechanical forces imposed upon, and generated within, MSCs. From these studies, a clearer picture of how osteogenesis and chondrogenesis of MSCs is regulated by mechanical signals is beginning to emerge. Understanding the response of MSCs to such regulatory factors is a key step towards understanding their role in development, disease and regeneration. Birth Defects Research (Part C) 90:75–85, 2010. © 2010 Wiley‐Liss, Inc.     

Mesenchymal stem cells in the treatment of articular cartilage degeneration: New biological insights for an old-timer cell

Osteoarthritis (OA) is a debilitating, degenerative joint disease characterized by progressive destruction of articular cartilage. Given the poor repair capacity of articular cartilage and the associated local destructive immune/inflammatory responses involving all joint structures, OA frequently ends up as a “whole joint failure” requiring prosthetic replacement. Current pharmacological efforts, belatedly started, mainly aim at symptomatic pain relief, underscoring the need for novel therapeutic schemes designed to modify the course of the disease. Mesenchymal stem cell (MSC)–based therapy has gained significant interest, sparking the design of multiple trials proving safety while providing promising preliminary efficacy results. MSCs possess ‘medicinal signaling cell’ properties related to their immunomodulatory and anti-inflammatory effects, which induce the establishment of a pro-regenerative microenvironment at the injured tissue. Those trophic effects are paralleled by the long-established chondroprogenitor capacity that can be harnessed to ex vivo fabricate engineered constructs to repair damaged articular cartilage. The present review focuses on these two aspects of the use of MSCs for articular cartilage damage, namely, cell therapy and tissue engineering, providing information on their use criteria, advancements, challenges and strategies to overcome them.     

Three-Dimensional Scaffold-Free Fusion Culture: the Way to Enhanced Chondrogenesis of in vitro Propagated Human Articular Chondrocytes

Cartilage regeneration based on isolated and culture-expanded chondrocytes has been studied in various in vitro models, but the quality varies with respect to the morphology and the physiology of the synthesized tissues. The aim of our study was to promote in vitro chondrogenesis of human articular chondrocytes using a novel three-dimensional (3-D) cultivation system in combination with the chondrogenic differentiation factors transforming growth factor beta 2 (TGF-β2) and L-ascorbic acid. Articular chondrocytes isolated from six elderly patients were expanded in monolayer culture. A single-cell suspension of the dedifferentiated chondrocytes was then added to agar-coated dishes without using any scaffold material, in the presence, or absence of TGF-β2 and/or L-ascorbic acid. Three-dimensional cartilage-like constructs, called single spheroids, and microtissues consisting of several spheroids fused together, named as fusions, were formed. Generated tissues were mainly characterized using histological and immunohistochemical techniques. The morphology of the in vitro tissues shared some similarities to native hyaline cartilage in regard to differentiated S100-positive chondrocytes within a cartilaginous matrix, with strong collagen type II expression and increased synthesis of proteoglycans. Finally, our innovative scaffold-free fusion culture technique supported enhanced chondrogenesis of human articular chondrocytes in vitro. These 3-D hyaline cartilage-like microtissues will be useful for in vitro studies of cartilage differentiation and regeneration, enabling optimization of functional tissue engineering and possibly contributing to the development of new approaches to treat traumatic cartilage defects or osteoarthritis.Key words: in vitro cartilage, 3-D cell culture, fusion culture technique, tissue engineering, cell differentiation, extracellular matrix, immunohistochemistry     

Mesenchymal stem cell therapy to rebuild cartilage

Disorders affecting cartilage touch almost the whole population and are one of the leading causes of invalidity in adults. To repair cartilage, therapeutic approaches initially focused on the implantation of autologous chondrocytes, but this technique proved unsatisfactory because of the limited number of chondrocytes obtained at harvest. The discovery that several adult human tissues contain mesenchymal stem cells (MSCs) capable of differentiating into chondrocytes raised the possibility of injecting MSCs to repair cartilages. The important data published recently on the factors controlling chondrocyte commitment must be thoroughly considered to make further progress towards this therapeutic approach. The potential application of MSC therapy provides new hope for the development of innovative treatments for the repair of cartilage disorders.     

Dose-Response of Superparamagnetic Iron Oxide Labeling on Mesenchymal Stem Cells Chondrogenic Differentiation: A Multi-Scale In Vitro Study

Aim

The aim of this work was the development of successful cell therapy techniques for cartilage engineering. This will depend on the ability to monitor non-invasively transplanted cells, especially mesenchymal stem cells (MSCs) that are promising candidates to regenerate damaged tissues.

Methods

MSCs were labeled with superparamagnetic iron oxide particles (SPIO). We examined the effects of long-term labeling, possible toxicological consequences and the possible influence of progressive concentrations of SPIO on chondrogenic differentiation capacity.

Results

No influence of various SPIO concentrations was noted on human bone marow MSC viability or proliferation. We demonstrated long-term (4 weeks) in vitro retention of SPIO by human bone marrow MSCs seeded in collagenic sponges under TGF-β1 chondrogenic conditions, detectable by Magnetic Resonance Imaging (MRI) and histology. Chondrogenic differentiation was demonstrated by molecular and histological analysis of labeled and unlabeled cells. Chondrogenic gene expression (COL2A2, ACAN, SOX9, COL10, COMP) was significantly altered in a dose-dependent manner in labeled cells, as were GAG and type II collagen staining. As expected, SPIO induced a dramatic decrease of MRI T2 values of sponges at 7T and 3T, even at low concentrations.

Conclusions

This study clearly demonstrates (1) long-term in vitro MSC traceability using SPIO and MRI and (2) a deleterious dose-dependence of SPIO on TGF-β1 driven chondrogenesis in collagen sponges. Low concentrations (12.5–25 µg Fe/mL) seem the best compromise to optimize both chondrogenesis and MRI labeling.     

Chondroitin sulfate based niches for chondrogenic differentiation of mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (MSCs) have strong potential in regeneration of musculoskeletal tissues including cartilage and bone. The microenvironment, comprising of scaffold and soluble factors, plays a pivotal role in determining the efficacy of cartilage tissue regeneration from MSCs. In this study, we investigated the effect of a three-dimensional synthetic-biological composite hydrogel scaffold comprised of poly (ethylene glycol) (PEG) and chondroitin sulfate (CS) on chondrogenesis of MSCs. The cells in CS-based bioactive hydrogels aggregated in a fashion which mimicked the mesenchymal condensation and produced cartilaginous tissues with characteristic morphology and basophilic extracellular matrix production. The aggregation of cells resulted in an enhancement of both chondrogenic gene expressions and cartilage specific matrix production compared to control PEG hydrogels containing no CS-moieties. Moreover, a significant down-regulation of type X collagen expression was observed in PEG/CS hydrogels, indicating that CS inhibits the further differentiation of MSCs into hypertrophic chondrocytes. Overall, this study demonstrates the morphogenetic role of bioactive scaffold-mediated microenvironment on temporal pattern of cartilage specific gene expressions and subsequent matrix production during MSC chondrogenesis.     

Phenotypic analysis of bovine chondrocytes cultured in 3D collagen sponges: effect of serum substitutes

Repair of damaged cartilage usually requires replacement tissue or substitute material. Tissue engineering is a promising means to produce replacement cartilage from autologous or allogeneic cell sources. Scaffolds provide a three-dimensional (3D) structure that is essential for chondrocyte function and synthesis of cartilage-specific matrix proteins (collagen type II, aggrecan) and sulfated proteoglycans. In this study, we assessed porous, 3D collagen sponges for in vitro engineering of cartilage in both standard and serum-free culture conditions. Bovine articular chondrocytes (bACs) cultured in 3D sponges accumulated and maintained cartilage matrix over 4 weeks, as assessed by quantitative measures of matrix content, synthesis, and gene expression. Chondrogenesis by bACs cultured with Nutridoma as a serum replacement was equivalent or better than control cultures in serum. In contrast, chondrogenesis in insulin-transferrin-selenium (ITS⁺³) serum replacement cultures was poor, apparently due to decreased cell survival. These data indicate that porous 3D collagen sponges maintain chondrocyte viability, shape, and synthetic activity by providing an environment favorable for high-density chondrogenesis. With quantitative assays for cartilage-specific gene expression and biochemical measures of chondrogenesis in these studies, we conclude that the collagen sponges have potential as a scaffold for cartilage tissue engineering.     

Hyaluronic Acid Enhances the Mechanical Properties of Tissue-Engineered Cartilage Constructs

There is a need for materials that are well suited for cartilage tissue engineering. Hydrogels have emerged as promising biomaterials for cartilage repair, since, like cartilage, they have high water content, and they allow cells to be encapsulated within the material in a genuinely three-dimensional microenvironment. In this study, we investigated the mechanical properties of tissue-engineered cartilage constructs using in vitro culture models incorporating human chondrocytes from osteoarthritis patients. We evaluated hydrogels formed from mixtures of photocrosslinkable gelatin-methacrylamide (Gel-MA) and varying concentrations (0–2%) of hyaluronic acid methacrylate (HA-MA). Initially, only small differences in the stiffness of each hydrogel existed. After 4 weeks of culture, and to a greater extent 8 weeks of culture, HA-MA had striking and concentration dependent impact on the changes in mechanical properties. For example, the initial compressive moduli of cell-laden constructs with 0 and 1% HA-MA were 29 and 41 kPa, respectively. After 8 weeks of culture, the moduli of these constructs had increased to 66 and 147 kPa respectively, representing a net improvement of 69 kPa for gels with 1% HA-MA. Similarly the equilibrium modulus, dynamic modulus, failure strength and failure strain were all improved in constructs containing HA-MA. Differences in mechanical properties did not correlate with glycosaminoglycan content, which did not vary greatly between groups, yet there were clear differences in aggrecan intensity and distribution as assessed using immunostaining. Based on the functional development with time in culture using human chondrocytes, mixtures of Gel-MA and HA-MA are promising candidates for cartilage tissue-engineering applications.