Native Cartilage Tissue Research Articles

Report on a large animal study with Göttingen Minipigs where regenerates and controls for articular cartilage were created in a large number. Focus on the conditions of the operated stifle joints and suggestions for standardized procedures.

The characterization of regenerated articular cartilage (AC) can be based on various methods, as there is an unambiguous accepted criterion neither for the natural cartilage tissue nor for regenerates. Biomechanical aspects should be considered as well, leading to the need for more equivalent samples. The aim of the study was to describe a large animal model where 8 specimens of regenerated AC can be created in one animal plus the impact of two surgeries on the welfare of the animals. The usefulness of the inclusion of a group of untreated animals (NAT) was to analyzed. Based on the histological results the conditions of the regenerates were to be described and the impact on knee joints were to be explored in terms of degenerative changes of the cartilage. The usefulness of the statistical term “effect size” (ES) will be explained with histological results. We analyzed an animal model where 8 AC regenerates were obtained from one Göttingen Minipig, on both sides of the trochleae. 60 animals were divided into 6 groups of 10 each, where the partial thickness defects in the trochlea were filled with matrices made of Collagen I with or without autologous chondrocytes or left empty over the healing periods of 24 and 48 weeks. One additional control group consisting of 10 untreated animals was used to provide untouched “external” cartilage. We harvested 560 samples of regenerated tissue and “external” controls, besides that, twice the number of further samples from other parts of the joints referred to as “internal” controls were also harvested. The animals recovered faster after the 1st operation when the defects were set compared to the 2nd operation when the defects were treated. 9% of all animals were lost. Other complications were for example superficial infections, seroma, diarrhea, febrile state and an injury of a claw. The histological results of the treatments proved the robustness of the study design where we included an “external” control group (NAT) in which the animals were not operated. Comparable significant differences between treated groups and the NAT group were detected both after ½ year and after 1 year. Spontaneous regenerated AC as control revealed differences after an observation time of nearly 1 year. The impact of the treatment on cartilage adjacent to the defect as well as the remaining knee joint was low. The ES was helpful for planning the study as it is shown that the power of a statistical comparison seems to be more influenced by the ES than by the sample size. The ranking of the ES was done exemplarily, listing the results according to their magnitude, thus making the results comparable. We were able to follow the 3 R requirements also in terms of a numerical reduction of animals due to the introduction of a group of untreated animals. This makes the model cost effective. The presented study may contribute as an improvement of the standardization of large animal models for research and regulatory requirements for regenerative therapies of AC.

Condensation-Driven Chondrogenesis of Human Mesenchymal Stem Cells within Their Own Extracellular Matrix: Formation of Cartilage with Low Hypertrophy and Physiologically Relevant Mechanical Properties.

Mesenchymal stem cells (MSCs) represent a promising cell source to regenerate injured cartilage. In this study, MSCs are cultured under confluent conditions for 10 days to optimize the deposition of the extracellular matrix (mECM), which will serve as the scaffold to support MSC chondrogenesis. Subsequently, the MSC-impregnated mECM (MSC-mECM) composite is briefly treated with trypsin, allowing the MSCs to adopt a round morphology without being detached from their own mECM. The constructs are then cultured in a chondrogenic medium. Interestingly, after trypsin removal, the treated MSCs undergo an aggregation process, mimicking mesenchymal condensation during developmental chondrogenesis, specifically indicated by peanut agglutinin staining and immunodetectable N-cadherin expression, followed by robust chondrogenic differentiation. In comparison to conventional pellet culture, chondrogenically induced MSC-mECM displays a similar level of chondrogenesis, but with significantly reduced hypertrophy. The reparative capacity of the MSC-mECM derived construct is assessed using bovine cartilage explants. Mechanical testing and histology results show that engineered cartilage from MSC-mECM forms better integration with the surrounding native cartilage tissue and displays a much lower hypertrophic differentiation than that from pellet culture. Taken together, these findings demonstrate that MSC-mECM may be an efficacious stem cell-based product for the repair of hyaline cartilage injury without the use of exogenous scaffolds.

Injectable PNIPAM/Hyaluronic acid hydrogels containing multipurpose modified particles for cartilage tissue engineering: Synthesis, characterization, drug release and cell culture study

Novel injectable thermosensitive PNIPAM/hyaluronic acid hydrogels containing various amounts of chitosan-g-acrylic acid coated PLGA (ACH-PLGA) micro/nanoparticles were synthesized and designed to facilitate the regeneration of cartilage tissue. The ACH-PLGA particles were used in the hydrogels to play a triple role: first, the allyl groups on the chitosan-g-acrylic acid shell act as crosslinkers for PNIPAM and improved the mechanical properties of the hydrogel to mimic the natural cartilage tissue. Second, PLGA core acts as a carrier for the controlled release of chondrogenic small molecule melatonin. Third, they could reduce the syneresis of the thermosensitive hydrogel during gelation. The optimum hydrogel with the minimum syneresis and the maximum compression modulus was chosen for further evaluations. This hydrogel showed a great integration with the natural cartilage during the adhesion test, and also, presented an interconnected porous structure in scanning electron microscopy images. Eventually, to evaluate the cytotoxicity, mesenchymal stem cells were encapsulated inside the hydrogel. MTT and Live/Dead assay showed that the hydrogel improved the cells growth and proliferation as compared to the tissue culture polystyrene. Histological study of glycosaminoglycan (GAG) showed that melatonin treatment has the ability to increase the GAG synthesis. Overall, due to the improved mechanical properties, low syneresis, the ability of sustained drug release and also high bioactivity, this injectable hydrogel is a promising material system for cartilage tissue engineering.

Bi-layered micro-fibre reinforced hydrogels for articular cartilage regeneration

Articular cartilage has limited capacity for regeneration and when damaged cannot be repaired with currently available metallic or synthetic implants. We aim to bioengineer a microfibre-reinforced hydrogel that can capture the zonal depth-dependent mechanical properties of native cartilage, and simultaneously support neo-cartilage formation. With this goal, a sophisticated bi-layered microfibre architecture, combining a densely distributed crossed fibre mat (superficial tangential zone, STZ) and a uniform box structure (middle and deep zone, MDZ), was successfully manufactured via melt electrospinning and combined with a gelatin–methacrylamide hydrogel. The inclusion of a thin STZ layer greatly increased the composite construct’s peak modulus under both incongruent (3.2-fold) and congruent (2.1-fold) loading, as compared to hydrogels reinforced with only a uniform MDZ structure. Notably, the stress relaxation response of the bi-layered composite construct was comparable to the tested native cartilage tissue. Furthermore, similar production of sulphated glycosaminoglycans and collagen II was observed for the novel composite constructs cultured under mechanical conditioning w/o TGF-ß1 supplementation and in static conditions w/TGF-ß1 supplementation, which confirmed the capability of the novel composite construct to support neo-cartilage formation upon mechanical stimulation. To conclude, these results are an important step towards the design and manufacture of biomechanically competent implants for cartilage regeneration. Statement of SignificanceDamage to articular cartilage results in severe pain and joint disfunction that cannot be treated with currently available implants. This study presents a sophisticated bioengineered bi-layered fibre reinforced cell-laden hydrogel that can approximate the functional mechanical properties of native cartilage. For the first time, the importance of incorporating a viable superficial tangential zone (STZ) – like structure to improve the load-bearing properties of bioengineered constructs, particularly when in-congruent surfaces are compressed, is demonstrated. The present work also provides new insights for the development of implants that are able to promote and guide new cartilaginous tissue formation upon physiologically relevant mechanical stimulation.

Microporous acellular extracellular matrix combined with adipose-derived stem cell sheets as a promising tissue patch promoting articular cartilage regeneration and interface integration

BackgroundAcute or chronic injury of articular cartilage leads to localized destruction. Difficulties with interface integration between the implant and native cartilage tissue can lead to an undesirable outcome. To improve cartilage repair and interface integration, we explored the therapeutic efficacy of microporous acellular extracellular matrix (ECM) combined with adipose-derived stem cell (ASC) sheets. MethodsMethods for fabricating ASC sheets and microporous acellular ECM were explored before transplanting the constructed ASC sheet/matrix in vivo and in vitro, respectively. After the operation, distal femur samples were collected at 6 and 12 weeks for further analysis. ResultsThe decellularization process removed 90% of the DNA but retained 82.4% of glycosaminoglycans (GAGs) and 82.8% of collagen, which are the primary components of cartilage matrix. The acellular matrix/ASC sheet construct treatment in vivo showed better interface integration, cartilage regeneration, and collagenous fiber arrangement, which resembles the native structure. There was a significant increase in GAG and collagen accumulation at the zone of regeneration and integration compared to other groups. Gene expression analysis showed that the mRNA level associated with cartilage formation significantly increased in the acellular matrix/ASC sheet group (p<0.05), which is consistent with the histological analysis. DiscussionASC sheets promote interface integration between the implant and native tissue. This effect, together with the acellular matrix as a graft, is beneficial for cartilage defect repair, which suggests that acellular matrix/ASC sheet bioengineered cartilage implants may be a better approach for cartilage repair due to their enhanced integration.

Structure-function relationships of fetal ovine articular cartilage

It is crucial that the properties of engineered neocartilage match healthy native cartilage to promote the functional restoration of damaged cartilage. To accurately assess the quality of neocartilage and the degree of biomimicry achieved, its properties must be evaluated against native cartilage and tissue from which the cells for neocartilage formation were sourced. Fetal ovine cartilage is a promising and translationally relevant cell source with which to engineer neocartilage, yet, it is largely non-characterized. The influence of biomechanics during cartilage development, as well as their potential impact on structure-function relationships in utero motivates additional study of fetal cartilage. Toward providing tissue engineering design criteria and elucidating structure-function relationships, 11 locations across four regions of the fetal ovine stifle were characterized. Locational and regional differences were found to exist. Although differences in GAG content were observed, compressive stiffness did not vary or correlate with any biochemical component. Patellar cartilage tensile stiffness and strength were significantly greater than those of the medial condyle. Tensile modulus and UTS significantly correlated with pyridinoline content. More advanced zonal organization, more intense collagen II staining, and greater collagen and pyridinoline contents in the trochlear groove and patella suggest these regions exhibit a more advanced maturational state than others. Regional differences in functional properties and their correlations suggest that structure-function relationships emerge in utero. These data address the dearth of information of the fetal ovine stifle, may serve as a repository of information for cartilage engineering strategies, and may help elucidate functional adaptation in fetal articular cartilage. Statement of SignificanceEngineered neocartilage must be evaluated against healthy native cartilage and cell source tissue to determine its quality and degree of biomimicry. While fetal ovine cartilage has emerged as a promising and translationally relevant cell source with which to engineer neocartilage, it is largely non-characterized. Therefore, 11 locations across four regions (medial condyle, lateral condyle, trochlear groove, and patella) of the fetal ovine stifle were characterized. Importantly, locational and regional differences in functional properties were observed, and significant correlations of tensile properties to collagen and crosslink contents were detected, suggesting that functional adaptation begins in utero. This study provides a repository of quantitative information, clarifies the developmental order of cartilage functional properties, and informs future cartilage engineering efforts.

Hydrazone covalent adaptable networks modulate extracellular matrix deposition for cartilage tissue engineering

Cartilage tissue engineering strategies often rely on hydrogels with fixed covalent crosslinks for chondrocyte encapsulation, yet the resulting material properties are largely elastic and can impede matrix deposition. To address this limitation, hydrazone crosslinked poly(ethylene glycol) hydrogels were formulated to achieve tunable viscoelastic properties and to study how chondrocyte proliferation and matrix deposition vary with the time-dependent material properties of covalent adaptable networks. Hydrazone equilibrium differences were leveraged to produce average stress relaxation times from hours (4.01 × 103 s) to months (2.78 × 106 s) by varying the percentage of alkyl-hydrazone (aHz) and benzyl-hydrazone (bHz) crosslinks. Swelling behavior and degradation associated with adaptability were characterized to quantify temporal network changes that can influence the behavior of encapsulated chondrocytes. After four weeks, mass swelling ratios varied from 36 ± 3 to 17 ± 0.4 and polymer retention ranged from 46 ± 4% to 92 ± 5%, with higher aHz content leading to loss of network connectivity with time. Hydrogels were formulated near the Flory-Stockmayer bHz percolation threshold (17% bHz) to investigate chondrocyte response to distinct levels of covalent architecture adaptability. Four weeks post-encapsulation, formulations with average relaxation times of 3 days (2.6 × 105s) revealed increased cellularity and an interconnected articular cartilage-specific matrix. Chondrocytes embedded in this adaptable formulation (22% bHz) deposited 190 ± 30% more collagen and 140 ± 20% more sulfated glycosaminoglycans compared to the 100% bHz control, which constrained matrix deposition to pericellular space. Collectively, these findings indicate that incorporating highly adaptable aHz crosslinks enhanced regenerative outcomes. However, connected networks containing more stable bHz bonds were required to achieve the highest quality neocartilaginous tissue. Statement of SignificanceCovalently crosslinked hydrogels provide robust mechanical support for cartilage tissue engineering applications in articulating joints. However, these materials traditionally demonstrate purely elastic responses to deformation despite the dynamic viscoelastic properties of native cartilage tissue. Here, we present hydrazone poly(ethylene glycol) hydrogels with tunable viscoelastic properties and study covalent adaptable networks for cartilage tissue engineering. Using hydrazone equilibrium and Flory-Stockmayer theory we identified average relaxation times leading to enhanced regenerative outcomes and showed that extracellular matrix deposition was biphasic as a function of the hydrazone covalent adaptability. We also showed that the incorporation of highly adaptable covalent crosslinks could improve cellularity of neotissue, but that a percolating network of more stable bonds was required to maintain scaffold integrity and form the highest quality neocartilaginous tissue.

Tribological Characterization of Polyacrylamide-Alginate Hybrid Hydrogels as a Potential Candidate for Cartilage Replacement

Various hydrogels such as polyvinyl alcohol (PVA) and poly (2-hydroxyethyl methacrylate) were assessed as articular cartilage replacement in the past. In the current study, the biphasic, biocompatible, and mechanically tunable Polyacrylamide-Alginate (PAAm-Alg) hybrid hydrogel was evaluated with different parameters such as load, speed and lubrication to study friction and wear performance of the material. Dried mass loss of hydrogel with lubrication was in the range of 2-9 wt %. The average friction coefficient of hydrogel under bovine serum lubrication was measured as 0.03, which is fairly close to native cartilage tissue. SEM studies revealed adhesion as the dominant wear mechanism due to excessive plastic deformation, independent of load and speed condition while fatigue wear mechanism was more noticeable under 1.3MPa applied pressure. However, the lubrication has dramatically decreased the wear rate and as a result, it was difficult to distinguish the worn surface of hydrogel samples from the unworn surface and just a light depression was observed on the samples, which is promising for load-bearing applications such as cartilage replacement.

Collagen Scaffolds in Cartilage Tissue Engineering and Relevant Approaches for Future Development.

Cartilage tissue engineering (CTE) aims to obtain a structure mimicking native cartilage tissue through the combination of relevant cells, three-dimensional scaffolds, and extraneous signals. Implantation of 'matured' constructs is thus expected to provide solution for treating large injury of articular cartilage. Type I collagen is widely used as scaffolds for CTE products undergoing clinical trial, owing to its ubiquitous biocompatibility and vast clinical approval. However, the long-term performance of pure type I collagen scaffolds would suffer from its limited chondrogenic capacity and inferior mechanical properties. This paper aims to provide insights necessary for advancing type I collagen scaffolds in the CTE applications. Initially, the interactions of type I/II collagen with CTE-relevant cells [i.e., articular chondrocytes (ACs) and mesenchymal stem cells (MSCs)] are discussed. Next, the physical features and chemical composition of the scaffolds crucial to support chondrogenic activities of AC and MSC are highlighted. Attempts to optimize the collagen scaffolds by blending with natural/synthetic polymers are described. Hybrid strategy in which collagen and structural polymers are combined in non-blending manner is detailed. Type I collagen is sufficient to support cellular activities of ACs and MSCs; however it shows limited chondrogenic performance than type II collagen. Nonetheless, type I collagen is the clinically feasible option since type II collagen shows arthritogenic potency. Physical features of scaffolds such as internal structure, pore size, stiffness, etc. are shown to be crucial in influencing the differentiation fate and secreting extracellular matrixes from ACs and MSCs. Collagen can be blended with native or synthetic polymer to improve the mechanical and bioactivities of final composites. However, the versatility of blending strategy is limited due to denaturation of type I collagen at harsh processing condition. Hybrid strategy is successful in maximizing bioactivity of collagen scaffolds and mechanical robustness of structural polymer. Considering the previous improvements of physical and compositional properties of collagen scaffolds and recent manufacturing developments of structural polymer, it is concluded that hybrid strategy is a promising approach to advance further collagen-based scaffolds in CTE.

Combination of Mesenchymal Stem Cells, Cartilage Pellet and Bioscaffold Supported Cartilage Regeneration of a Full Thickness Articular Surface Defect in Rabbits.

Mesenchymal stem cells (MSCs) and/or biological scaffolds have been used to regenerate articular cartilage with variable success. In the present study we evaluated cartilage regeneration using a combination of bone marrow (BM)-MSCs, HyalofastTM and/or native cartilage tissue following full thickness surgical cartilage defect in rabbits. Full-thickness surgical ablation of the medial-tibial cartilage was performed in New Zealand white (NZW) rabbits. Control rabbits (Group-I) received no treatment; Animals in other groups were treated as follows. Group-II: BM-MSCs (1×106 cells) + HyalofastTM; Group-III: BMMSCs (1×106 cells) + cartilage pellet (CP); and Group-IV: BM-MSCs (1×106 cells) + HyalofastTM + CP. Animals were sacrificed at 12weeks and cartilage regeneration analyzed using histopathology, International Cartilage Repair Society (ICRS-II) score, magnetic resonance observation of cartilage repair tissue (MOCART) score and biomechanical studies. Gross images showed good tissue repair (Groups IV > III > Group II) and histology demonstrated intact superficial layer, normal chondrocyte arrangement, tidemark and cartilage matrix staining (Groups III and IV) compared to the untreated control (Group I) respectively. ICRS-II score was 52.5, 65.0, 66 and 75% (Groups I-IV) and the MOCART score was 50.0, 73.75 and 76.25 (Groups II-IV) respectively. Biomechanical properties of the regenerated cartilage tissue in Group IV closed resembled that of a normal cartilage. HyalofastTM together with BM-MSCs and CP led to efficient cartilage regeneration following full thickness surgical ablation of tibial articular cartilage in vivo in rabbits. Presence of hyaluronic acid in the scaffold and native microenvironment cues probably facilitated differentiation and integration of BM-MSCs.

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.

Biomimetic design and fabrication of multilayered osteochondral scaffolds by low-temperature deposition manufacturing and thermal-induced phase-separation techniques

Integrative osteochondral repair is a useful strategy for cartilage-defect repair. To mimic the microenvironment, it is necessary that scaffolds effectively mimic the extracellular matrix of natural cartilage and subchondral bone. In this study, biomimetic osteochondral scaffolds containing an oriented cartilage layer, a compact layer, and a three-dimensional (3D)-printed core-sheath structured-bone layer were developed. The oriented cartilage layer was designed to mimic the structural and material characteristics of native cartilage tissue and was fabricated with cartilage matrix-chitosan materials, using thermal-induced phase-separation technology. The 3D-printed core-sheath structured-bone layer was fabricated with poly(L-lactide-co-glycolide)/β-tricalcium phosphate-collagen materials by low-temperature deposition technology, using a specially designed core-sheath nozzle, and was designed to mimic the mechanical characteristics of subchondral bone and improve scaffold hydrophilicity. The compact layer was designed to mimic the calcified-layer structure of natural cartilage to ensure the presence of different suitable microenvironments for the regeneration of bone and cartilage. A dissolving-bonding process was developed to effectively combine the three parts together, after which the bone and cartilage scaffolds exhibited good mechanical properties and hydrophilicity. Additionally, goat autologous bone mesenchymal stem cells (BMSCs) were isolated and then seeded into the bone and cartilage layers, respectively, and following a 1 week culture in vitro, the BMSC-scaffold constructs were implanted into a goat articular-defect model. Our results indicated that the scaffolds exhibited good biocompatibility, and 24 weeks after implantation, the femoral condyle surface was relatively flat and consisted of a large quantity of hyaloid cartilage. Furthermore, histological staining revealed regenerated trabecular bone formed in the subchondral bone-defect area. These results provided a new method to fabricate biomimetic osteochondral scaffolds and demonstrated their effectiveness for future clinical applications in cartilage-defect repair.

Characterizing viscoelasticity of unhydrolyzed chicken sternal cartilage extract suspensions: Towards development of injectable therapeutics formulations

Exogenous delivery of cartilage extract is being explored as a promising candidate for knee arthritis treatment as it biomimics native cartilage tissue characteristics. In this study, we report on the rheological characterization of aqueous suspensions constituted from a powdered form of unhydrolyzed chicken sternum extract. The effect of particle size (as-received vs. milled), suspension fluid (water vs. PBS), and temperature (37°C vs. 4°C), on the viscoelastic properties of the sternum extract based particulate suspensions were evaluated. Results showed that these suspensions exhibit shear-thinning characteristics as shear rate (γ̇) increases, while viscosity (η), storage (G′), and loss (G″) moduli of the suspensions increased with increasing particulate loading (ϕ: 2.5–10wt%). Reducing the as-received particle size by milling decreased Gʹ, G, and η of the suspensions and increased the influence of ϕ on these properties, possibly due to improved particle packing. Replacing water with PBS had no significant effect on the rheological properties, but temperature reduction from 37°C to 4°C increased Gʹ, Gʺ, and η of the suspensions and lowered the impact of powder loading on viscoelastic properties. The suspension's time-dependent response was typical of viscoelastic materials, characterized by an asymptotical approach to a final stress (stress relaxation) or strain (creep). Results were fit to a power-law model for creep, a general relaxation model for exponential decay in stress, Carreau-Yasuda models for flow curves, and a two-parameter Liu model to identify the maximum powder loading (ϕm). Among the various forces involved in particle-particle interactions within these suspensions, electrostatic forces appeared to dominate the most. Such characterization of the viscoelastic nature of these suspensions would help in formulating stable injectable cartilage extract based therapeutics for in vivo applications.

Investigating cellulose derived glycosaminoglycan mimetic scaffolds for cartilage tissue engineering applications.

Articular cartilage has a limited capacity to heal and, currently, no treatment exists that can restore normal hyaline cartilage. Creating tissue engineering scaffolds that more closely mimic the native extracellular matrix may be an attractive approach. Glycosaminoglycans, which are present in native cartilage tissue, provide signalling and structural cues to cells. This study evaluated the use of a glycosaminoglycan mimetic, derived from cellulose, as a potential scaffold for cartilage repair applications. Fully sulfated sodium cellulose sulfate (NaCS) was initially evaluated in soluble form as an additive to cell culture media. Human mesenchymal stem cell (MSC) chondrogenesis in pellet culture was enhanced with 0.01% NaCS added to induction media as demonstrated by significantly higher gene expression for type II collagen and aggrecan. NaCS was combined with gelatine to form fibrous scaffolds using the electrospinning technique. Scaffolds were characterized for fibre morphology, overall hydrolytic stability, protein/growth factor interaction and for supporting MSC chondrogenesis in vitro. Scaffolds immersed in phosphate buffered saline for up to 56days had no changes in swelling and no dissolution of NaCS as compared to day 0. Increasing concentrations of the model protein lysozyme and transforming growth factor-β3 were detected on scaffolds with increasing concentrations of NaCS (p<0.05). MSC chondrogenesis was enhanced on the scaffold with the lowest NaCS concentration as seen with the highest collagen type II production, collagen type II immunostaining, and expression of cartilage-specific genes. These studies demonstrate the feasibility of cellulose sulfate as a scaffolding material for cartilage tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd.

Generating Mechanically Stable, Pediatric, and Scaffold-Free Nasal Cartilage Constructs In Vitro.

Traditional methods of cartilage tissue engineering rely on the use of scaffolds. Although successful chondrogenesis has been reported in scaffold-based constructs, the use of exogenous materials has limited their application due to eliciting host immunogenic responses and potentially resulting in construct failure. As a result, tissue engineering approaches, which aim to generate scaffold-free cartilaginous constructs, have become of particular interest. Here, we generated stable three-dimensional scaffold-free cartilaginous constructs by cultivating expanded pediatric nasal chondrocyte multilayers in a slow turning lateral vessel bioreactor system under chemically defined media. Bioreactor cultivation resulted in increased construct cellularity, fourfold tissue thickness, and 200% sulfated glycosaminoglycan deposition with respect to static culture equivalent cultures. These improvements led to significantly enhanced mechanical and biochemical properties of bioreactor-cultivated constructs, allowing them to support their own weight, while static culture constructs remained fragile. Consequently, bioreactor-cultivated constructs closely resembled native nasal cartilage tissue histologically, mechanically, and biochemically. We propose that this method of cartilage construct formation could be used to obtain readily available human scaffold-free cartilaginous constructs.

Fundamental biomaterial properties of tough glycosaminoglycan-containing double network hydrogels newly developed using the molecular stent method.

The purpose of this study was to clarify fundamental mechanical properties and biological responses of the sodium hyaluronate-containing double network (HA-DN) gel and chondroitin sulfate-containing double network (CS-DN) gel, which were newly developed using the molecular stent method. This study discovered the following facts. First, these hydrogels had high mechanical performance comparable to the native cartilage tissue, and the mechanical properties were not affected by immersion in the saline solution for 12weeks. Secondly, the mechanical properties of the CS-DN gel were not significantly reduced at 12weeks in vivo, while the mechanical properties of the HA-DN gel were significantly deteriorated at 6weeks. Thirdly, the degree of inflammation around the HA-DN gel was the same as that around the negative control. The CS-DN gel showed a mild but significant foreign body reaction, which was significantly greater than the negative control and less than the positive control at 1week, while the inflammation was reduced to the same level as the negative control at 4 and 6weeks. Fourthly, these gels induced differentiation of the ATDC5 cells into chondrocytes in the culture with the insulin-free maintenance medium. These findings suggest that these tough hydrogels are potential biomaterials for future application to therapeutic implants such as artificial cartilage. The present study reported fundamental biomaterial properties of the sodium hyaluronate-containing double network (HA-DN) gel and chondroitin sulfate-containing double network (CS-DN) gel, which were newly developed using the molecular stent method. Both the HA- and CS-DN gels had high mechanical properties comparable to the cartilage tissue and showed the ability to induce chondrogenic differentiation of ATDC5 cells in vitro. They are potential biomaterials that may meet the requirements of artificial cartilage concerning the material properties. Further, these DN gels can be also applied to the implantable inducer for cell-free cartilage regeneration therapy.

Engineering biomechanically functional neocartilage derived from expanded articular chondrocytes through the manipulation of cell-seeding density and dexamethasone concentration.

Recent work has established methods to engineer self-assembled, scaffold-free neocartilage from an expanded articular chondrocyte (AC) cell source. In continuing such work, the objective of the present study was to investigate the effects of cell-seeding density and dexamethasone concentration on these neocartilage constructs. Neocartilage discs (5mm diameter) were formed by self-assembling passaged leporine articular chondrocytes into non-adherent agarose moulds. The cell-seeding densities (2, 3, 4, 5 and 6 million cells/construct) and dexamethasone concentrations (10 and 100nm) in the culture medium were varied in a full-factorial study. After 4weeks, the neocartilage constructs were assessed for morphological, biochemical and biomechanical properties. The cell-seeding density profoundly affected neocartilage properties. The two dexamethasone concentrations explored did not induce overall significant differences. Constructs formed using lower cell-seeding densities possessed much higher biochemical and biomechanical properties than constructs seeded with higher cell densities. Notably, the 2 million cells/construct group formed hyaline-like neocartilage with a collagen wet weight (WW) content of ~7% and a Young's modulus of ~4MPa, representing the high end of values achieved in self-assembled neocartilage. Excitingly, the mechanical properties of these constructs were on a par with that of native cartilage tissues tested under similar conditions. Through optimization of cell-seeding density, this study shows for the first time the use of expanded ACs to form homogeneous self-assembled neocartilage with exceptionally high tensile strength. With such functional properties, these engineered neocartilage constructs provide a promising alternative for treating articular lesions. Copyright © 2016 John Wiley & Sons, Ltd.

Approaching the compressive modulus of articular cartilage with a decellularized cartilage-based hydrogel

ECM-based materials are appealing for tissue engineering strategies because they may promote stem cell recruitment, cell infiltration, and cell differentiation without the need to supplement with additional biological factors. Cartilage ECM has recently shown potential to be chondroinductive, particularly in a hydrogel-based system, which may be revolutionary in orthopedic medicine. However, hydrogels composed of natural materials are often mechanically inferior to synthetic materials, which is a major limitation for load-bearing tissue applications. The objective was therefore to create an unprecedented hydrogel derived entirely from native cartilage ECM that was both mechanically more similar to native cartilage tissue and capable of inducing chondrogenesis. Porcine cartilage was decellularized, solubilized, and then methacrylated and UV photocrosslinked to create methacrylated solubilized decellularized cartilage (MeSDCC) gels. Methacrylated gelatin (GelMA) was employed as a control for both biomechanics and bioactivity. Rat bone marrow-derived mesenchymal stem cells were encapsulated in these networks, which were cultured in vitro for 6weeks, where chondrogenic gene expression, the compressive modulus, swelling, and histology were analyzed. One day after crosslinking, the elastic compressive modulus of the 20% MeSDCC gels was 1070±150kPa. Most notably, the stress strain profile of the 20% MeSDCC gels fell within the 95% confidence interval range of native porcine cartilage. Additionally, MeSDCC gels significantly upregulated chondrogenic genes compared to GelMA as early as day 1 and supported extensive matrix synthesis as observed histologically. Given that these gels approached the mechanics of native cartilage tissue, supported matrix synthesis, and induced chondrogenic gene expression, MeSDCC hydrogels may be promising materials for cartilage tissue engineering applications. Future efforts will focus on improving fracture mechanics as well to benefit overall biomechanical performance. Statement of SignificanceExtracellular matrix (ECM)-based materials are appealing for tissue engineering strategies because they may promote stem cell recruitment, cell infiltration, and cell differentiation without the need to supplement with additional biological factors. One such ECM-based material, cartilage ECM, has recently shown potential to be chondroinductive; however, hydrogels composed of natural materials are often mechanically inferior to synthetic materials, which is a major limitation for load-bearing tissue applications. Therefore, this work is significant because we were the first to create hydrogels derived entirely from cartilage ECM that had mechanical properties similar to that of native cartilage until hydrogel failure. Furthermore, these hydrogels had a compressive modulus of 1070±150kPa, they were chondroinductive, and they supported extensive matrix synthesis. In the current study, we have shown that these new hydrogels may prove to be a promising biomaterial for cartilage tissue engineering applications

The Synergistic Effects of Matrix Stiffness and Composition on the Response of Chondroprogenitor Cells in a 3D Precondensation Microenvironment.

Improve functional quality of cartilage tissue engineered from stem cells requires a better understanding of the functional evolution of native cartilage tissue. Therefore, a biosynthetic hydrogel was developed containing RGD, hyaluronic acid and/or type-I collagen conjugated to poly(ethylene glycol) acrylate to recapitulate the precondensation microenvironment of the developing limb. Conjugation of any combination of the three ligands did not alter the shear moduli or diffusion properties of the PEG hydrogels; thus, the influence of ligand composition on chondrogenesis could be investigated in the context of varying matrix stiffness. Gene expression of ligand receptors (CD44 and the b1-integrin) as well as markers of condensation (cell clustering and N-cadherin gene expression) and chondrogenesis (Col2a1 gene expression and sGAG production) by chondroprogenitor cells in this system were modulated by both matrix stiffness and ligand composition, with the highest gene expression occurring in softer hydrogels containing all three ligands. Cell proliferation in these 3D matrices for 7 d prior to chondrogenic induction increased the rate of sGAG production in a stiffness-dependent manner. This biosynthetic hydrogel supports the features of early limb-bud condensation and chondrogenesis and is a novel platform in which the influence of the matrix physicochemical properties on these processes can be elucidated.

Multiscale Biofabrication of Articular Cartilage: Bioinspired and Biomimetic Approaches.

Articular cartilage is the load-bearing tissue found inside all articulating joints of the body. It vastly reduces friction and allows for smooth gliding between contacting surfaces. The structure of articular cartilage matrix and cellular composition is zonal and is important for its mechanical properties. When cartilage becomes injured through trauma or disease, it has poor intrinsic healing capabilities. The spectrum of cartilage injury ranges from isolated areas of the joint to diffuse breakdown and the clinical appearance of osteoarthritis. Current clinical treatment options remain limited in their ability to restore cartilage to its normal functional state. This review focuses on the evolution of biomaterial scaffolds that have been used for functional cartilage tissue engineering. In particular, we highlight recent developments in multiscale biofabrication approaches attempting to recapitulate the complex 3D matrix of native articular cartilage tissue. Additionally, we focus on the application of these methods to engineering each zone of cartilage and engineering full-thickness osteochondral tissues for improved clinical implantation. These methods have shown the potential to control individual cell-to-scaffold interactions and drive progenitor cell differentiation into a chondrocyte lineage. The use of these bioinspired nanoengineered scaffolds hold promise for recreation of structure and function on the whole tissue level and may represent exciting new developments for future clinical applications for cartilage injury and restoration.