Cartilage Tissue Engineering Research Articles

Double crosslinked hyaluronic acid and collagen as a potential bioink for cartilage tissue engineering

The avascular nature of hyaline cartilage results in limited spontaneous self-repair and regenerative capabilities when damaged. Recent advances in three-dimensional bioprinting have enabled the precise dispensing of cell-laden biomaterials, commonly referred to as ‘bioinks’, which are emerging as promising solutions for tissue regeneration. An effective bioink for cartilage tissue engineering needs to create a micro-environment that promotes cell differentiation and supports neocartilage tissue formation. In this study, we introduced an innovative bioink composed of photocurable acrylated type I collagen (COLMA), thiol-modified hyaluronic acid (THA), and poly(ethylene glycol) diacrylate (PEGDA) for 3D bioprinting cartilage grafts using human nasal chondrocytes. Both collagen and hyaluronic acid, being key components of the extracellular matrix (ECM) in the human body, provide essential biological cues for tissue regeneration. We evaluated three formulations – COLMA, COLMA+THA, and COLMA+THA+PEGDA – for their printability, cell viability, structural integrity, and capabilities in forming cartilage-like ECM. The addition of THA and PEGDA significantly enhanced these properties, showcasing the potential of this bioink in advancing applications in cartilage repair and reconstructive surgery.

Tuning the rheological properties of chitosan/alginate hydrogels for tissue engineering application

Biomaterials that mimic biological tissues are in great demand in tissue engineering. Hydrogels are rising as a conducive candidate in tissue engineering on account of their water-holding capacity, mechanical strength, and elasticity. Nevertheless, the development of hydrogels that mimic biological tissues with the desired stability and mechanical strength, remains a notable barrier. In this investigation, chitosan/alginate hydrogels were prepared by tuning the chitosan concentration from 0.5 % to 2.5 %w/v with a fixed 1 %w/v alginate concentration using 0.1 % glutaraldehyde as a gelling agent. The hydrogel system forms through covalent bonding between chitosan and alginate, mediated by aldehyde groups of the glutaraldehyde. The mechanical strength of the hydrogel was elucidated by probing its viscoelastic behavior through rheological analysis. The rheological investigations revealed that the prepared chitosan/alginate hydrogels are profoundly stable with shear-thinning characteristics. The increase in chitosan concentration enhanced the stability and viscoelastic attributes of the hydrogels. The yield stress values ranging from 0.93 kPa to 14.24 kPa indicate the possibility of using these hydrogels in bioadhesives and soft and bone tissue engineering. The complex modulus of the prepared chitosan/alginate hydrogels resembles the shear modulus of liver tissues, osteogenic cells, and articular cartilage, and hence, these hydrogels pose as suitable candidates in liver, bone, and cartilage tissue engineering. The chitosan/alginate hydrogels possess outstanding self-healing ability, along with biocompatibility, stability, and mechanical strength, rendering them highly advantageous for applications in biomedical tissue engineering and bioadhesives.

Biofabrication of Tissue-Engineered Cartilage Constructs Through Faraday Wave Bioassembly of Cell-Laden Gelatin Microcarriers.

Acoustic biofabrication is an emerging strategy in tissue engineering due to its mild and fast manufacturing process. Herein, tissue-engineered cartilage constructs with high cell viability are fabricated from cell-laden gelatin microcarriers (GMs) through Faraday wave bioassembly, a typical acoustic "bottom-up" manufacturing process. Assembly modules are first prepared by incorporating cartilage precursor cells, the chondrogenic cell line ATDC5, or bone marrow-derived mesenchymal stem cells (BMSCs), into GMs. Patterned structures are formed by Faraday wave bioassembly of the cell-laden GMs. Due to the gentle and efficient assembly process and the protective effects of microcarriers, cells in the patterned structures maintain high activity. Subsequently, tissue-engineered cartilage constructs are obtained by inducing cell differentiation of the patterned structures. Comprehensive evaluations are conducted to verify chondrocyte differentiation and the formation of cartilage tissue constructs in terms of cell viability, morphological analysis, gene expression, and matrix production. Finally, implantation studies with a rat cartilage defect model demonstrate that these tissue-engineered cartilage constructs are beneficial for the repair of articular cartilage damage in vivo. This study provides the first biofabrication of cartilage tissue constructs using Faraday wave bioassembly, extending its application to engineering tissues with a low cell density.

Simultaneous usage of sulforaphane nanoemulsion and tannic acid in ternary chitosan/gelatin/PEG hydrogel for knee cartilage tissue engineering: In vitro and in vivo study

The therapeutic potential of tissue engineering in addressing articular cartilage defects has been a focal point of research for numerous years. Despite its promising outlook, a persistent challenge within this domain is the lack of sufficient functional integration between engineered and natural tissues. This study introduces a novel approach that employs a combination of sulforaphane (SFN) nanoemulsion and tannic acid to enhance cartilage tissue engineering and promote tissue integration in a rat knee cartilage defect model. To substantiate our hypothesis, we conducted a series of in vitro and in vivo experiments. The SFN nanoemulsion was characterized using DLS, zeta potential, and TEM analyses. Subsequently, it was incorporated into a ternary polymer hydrogel composed of chitosan, gelatin, and polyethylene glycol. We evaluated the hydrogel with (H-SFN) and without (H) the SFN nanoemulsion through a comprehensive set of physicochemical, mechanical, and biological analyses. For the in vivo study, nine male Wistar rats were divided into three groups: no implant (Ctrl), H, and H-SFN. After inducing a cartilage defect, the affected area was treated with tannic acid and subsequently implanted with the hydrogels. Four weeks post-implantation, the harvested cartilage underwent histological examination employing H&E, safranin O/fast green, alcian blue, and immunohistochemistry staining techniques. Our results revealed that the SFN nanodroplets had an average diameter of 75 nm and a surface charge of −11.58 mV. Moreover, degradation, swelling rates, hydrophilicity, and elasticity features of the hydrogel incorporating SFN were improved. Histopathological analysis indicated a higher production of GAGs and collagen in the H-SFN group. Furthermore, the H-SFN group exhibited superior cartilage regeneration and tissue integration compared to the Ctrl and H groups. In conclusion, the findings of this study suggest the importance of considering cell protective properties in the fabrication of scaffolds for knee cartilage defects, emphasizing the potential significance of the proposed SFN nanoemulsion and tannic acid approach in advancing the field of cartilage tissue engineering.

Cartilage tissue engineering using decellularized biomatrix hydrogel containing TGF-β-loaded alginate microspheres in mechanically loaded bioreactor

Physiochemical tissue inducers and mechanical stimulation are both efficient variables in cartilage tissue fabrication and regeneration. In the presence of biomolecules, decellularized extracellular matrix (ECM) may trigger and enhance stem cell proliferation and differentiation. Here, we investigated the controlled release of transforming growth factor beta (TGF-β1) as an active mediator of mesenchymal stromal cells (MSCs) in a biocompatible scaffold and mechanical stimulation for cartilage tissue engineering. ECM-derived hydrogel with TGF-β1-loaded alginate-based microspheres (MSs) was created to promote human MSC chondrogenic development. Ex vivo explants and a complicated multiaxial loading bioreactor replicated the physiological conditions. Hydrogels with/without MSs and TGF-β1 were highly cytocompatible. MSCs in ECM-derived hydrogel containing TGF-β1/MSs showed comparable chondrogenic gene expression levels as those hydrogels with TGF-β1 added in culture media or those without TGF-β1. However, constructs with TGF-β1 directly added within the hydrogel had inferior properties under unloaded conditions. The ECM-derived hydrogel group including TGF-β1/MSs under loading circumstances formed better cartilage matrix in an ex vivo osteochondral defect than control settings. This study demonstrates that controlled local delivery of TGF-β1 using MSs and mechanical loading is essential for neocartilage formation by MSCs and that further optimization is needed to prevent MSC differentiation towards hypertrophy.

Adipose-derived mesenchymal stem cell-incorporated PLLA porous microspheres for cartilage regeneration.

In facial plastic surgery, patients with nasal deformity are often treated by rib cartilage transplantation. In recent years, cartilage tissue engineering has developed as an alternative to complex surgery for patients with minor nasal defects via injection of nasal filler material. In this study, we prepared an injectable nasal filler material containing poly-L-lactic acid (PLLA) porous microspheres (PMs), hyaluronic acid (HA) and adipose-derived mesenchymal stem cells (ADMSCs). We seeded ADMSCs into as-prepared PLLA PMs using our newly invented centrifugation perfusion technique. Then, HA was mixed with ADMSC-incorporated PLLA PMs to form a hydrophilic and injectable cell delivery system (ADMSC-incorporated PMH). We evaluated the biocompatibility of PMH invitro and invivo. PMH has good injectability and provides a favorable environment for the proliferation and chondrogenic differentiation of ADMSCs. Invivo experiments, we observed that PMH has good biocompatibility and cartilage regeneration ability. In this study, a injectable cell delivery system was successfully constructed. We believe that PMH has potential application in cartilage tissue engineering, especially in nasal cartilage regeneration.

Electrical Stimulation of Mesenchymal Stem Cells as a Tool for Proliferation and Differentiation in Cartilage Tissue Engineering: A Scaffold-Based Approach.

Electrical stimulation (ES) is a widely discussed topic in the field of cartilage tissue engineering due to its ability to induce chondrogenic differentiation (CD) and proliferation. It shows promise as a potential therapy for osteoarthritis (OA). In this study, we stimulated mesenchymal stem cells (MSCs) incorporated into collagen hydrogel (CH) scaffolds, consisting of approximately 500,000 cells each, for 1 h per day using a 2.5 Vpp (119 mV/mm) 8 Hz sinusoidal signal. We compared the cell count, morphology, and CD on days 4, 7, and 10. The results indicate proliferation, with an increase ranging from 1.86 to 9.5-fold, particularly on day 7. Additionally, signs of CD were observed. The stimulated cells had a higher volume, while the stimulated scaffolds showed shrinkage. In the ES groups, up-regulation of collagen type 2 and aggrecan was found. In contrast, SOX9 was up-regulated in the control group, and MMP13 showed a strong up-regulation, indicating cell stress. In addition to lower stress levels, the control groups also showed a more spheroidic shape. Overall, scaffold-based ES has the potential to achieve multiple outcomes. However, finding the appropriate stimulation pattern is crucial for achieving successful chondrogenesis.

Enhancing 3D poly[ɛ]caprolactone scaffold properties through alkaline hydrolysis for improved chondrocyte culture: Morphological, physicochemical, and biocompatibility evaluations

This study investigates the impact of alkaline hydrolysis on the morphology, physicochemical properties, and mechanical strength of poly[ε]caprolactone (PCL) scaffolds fabricated using a melt-based electrohydrodynamic process. We systematically analyzed how a brief, 5-min alkaline hydrolysis treatment influenced scaffold characteristics compared to longer durations (15 and 30 min). Results demonstrated that the short-duration treatment significantly increased fiber diameter and reduced pore size while modifying the scaffold's surface chemistry. Importantly, the 5-min treatment optimized the water contact angle to 55–65°, enhancing hydrophilicity which facilitated superior cell attachment and proliferation of the C28/I2 immortalized human chondrocyte cell line. Mechanical testing revealed that while alkaline hydrolysis decreased the scaffold's Young's modulus, a 5-min treatment maintained sufficient mechanical integrity for potential tissue engineering applications, in contrast to the more substantial degradation observed with longer treatments.Biocompatibility evaluations showed that the scaffolds, particularly those treated for 5 min, supported cell viability and proliferation without inducing cytotoxic effects. The molecular analysis indicated a favorable upregulation of chondrogenic genes such as SOX9 and COL2A1, and a downregulation of COL1A1, suggesting potential benefits for cartilage tissue engineering. These findings highlight that precise control over the duration of alkaline hydrolysis can significantly enhance PCL scaffold properties, suggesting a scalable and cost-effective method for scaffold optimization in regenerative medicine applications. The results pave the way for further in vivo studies to validate these promising in vitro findings and explore the clinical applicability of the optimized scaffolds.

Recent advancements in cartilage tissue engineering innovation and translation.

Articular cartilage was expected to be one of the first successfully engineered tissues, but today, cartilage repair products are few and they exhibit considerable limitations. For example, of the cell-based products that are available globally, only one is marketed for non-knee indications, none are indicated for severe osteoarthritis or rheumatoid arthritis, and only one is approved for marketing in the USA. However, advances in cartilage tissue engineering might now finally lead to the development of new cartilage repair products. To understand the potential in this field, it helps to consider the current landscape of tissue-engineered products for articular cartilage repair and particularly cell-based therapies. Advances relating to cell sources, bioactive stimuli and scaffold or scaffold-free approaches should now contribute to progress in therapeutic development. Engineering for an inflammatory environment is required because of the need for implants to withstand immune challenge within joints affected by osteoarthritis or rheumatoid arthritis. Bringing additional cartilage repair products to the market willrequire an understanding of the translational vector for their commercialization. Advances thus far can facilitate the future translation of engineered cartilage products to benefit the millions of patients who suffer from cartilage injuries and arthritides.

Induced pluripotent stem cells in cartilage tissue engineering: a literature review.

A literature search was conducted using scientific search engines, PubMed, MEDLINE, and Google Scholar databases with the terms 'Cartilage tissue engineering' and 'stem cells' to retrieve published literature on chondrogenic differentiation and tissue engineering using MSCs, ESCs, and hiPSCs. hiPSCs may provide an effective and autologous treatment for focal chondral lesions, though further research is needed to explore the potential of such technologies. This review has provided a comprehensive overview of these technologies and the potential applications for hiPSCs in regenerative medicine.

3D Bioprinting of Biomimetic Alginate/Gelatin/Chondroitin Sulfate Hydrogel Nanocomposites for Intrinsically Chondrogenic Differentiation of Human Mesenchymal Stem Cells.

3D-printed hydrogel scaffolds biomimicking the extracellular matrix (ECM) are key in cartilage tissue engineering as they can enhance the chondrogenic differentiation of mesenchymal stem cells (MSCs) through the presence of active nanoparticles such as graphene oxide (GO). Here, biomimetic hydrogels were developed by cross-linking alginate, gelatin, and chondroitin sulfate biopolymers in the presence of GO as a bioactive filler, with excellent processability for developing bioactive 3D printed scaffolds and for the bioprinting process. A novel bioink based on our hydrogel with embedded human MSCs presented a cell survival rate near 100% after the 3D bioprinting process. The effects of processing and filler concentration on cell differentiation were further quantitatively evaluated. The nanocomposited hydrogels render high MSC proliferation and viability, exhibiting intrinsic chondroinductive capacity without any exogenous factor when used to print scaffolds or bioprint constructs. The bioactivity depended on the GO concentration, with the best performance at 0.1 mg mL-1. These results were explained by the rational combination of the three biopolymers, with GO nanoparticles having carboxylate and sulfate groups in their structures, therefore, biomimicking the highly negatively charged ECM of cartilage. The bioactivity of this biomaterial and its good processability for 3D printing scaffolds and 3D bioprinting techniques open up a new approach to developing novel biomimetic materials for cartilage repair.

A regenerative approach for temporomandibular joint repair: An invitro and exvivo study.

Current clinical approaches to regenerate temporomandibular joint (TMJ) articulating cartilage defects only treat the symptoms (i.e. pain and dysfunction) and do not seek to restore joint integrity for long-term relief. Therefore, we investigated a novel self-assembling tissue-engineered cartilage to overcome this significant clinical issue for TMJ regenerative purposes. Examine the maturation of dynamic self-regenerating cartilage (dSRC) using auricular chondrocytes and evaluate a novel combinatorial approach with fractional laser treatment and dSRC implantation for TMJ cartilage repair. A suspension of 107 freshly harvested rabbit ear chondrocytes was cultured under a continuous reciprocating motion to form the dSRC. After 2, 4 and 8 weeks of culture, dSRC samples were stained with H&E, Safranin-O and Toluidine Blue. Immunohistochemistry (IHC) was performed for collagens type I and II. Channels (300-500 μm diameter and 1.2-1.5 mm depth) were created in six freshly harvested condyles using a fractional Erbium laser. Two groups were tested: dSRC in a laser-ablated lesion (experimental) and an empty laser-ablated channel (control). TMJ condyles were cultured for up to 8 weeks and analysed as described above. H&E staining showed a high cell density in dSRC compared to native cartilage. All dSRC groups demonstrated intense Safranin-O staining, indicating high glycosaminoglycan (GAG) production and intense Toluidine Blue staining showed high proteoglycan content. IHC confirmed that dSRC consisted predominantly of collagen type II. The experimental group showed improved cartilage repair at both time points compared to the empty channels. dSRC viability and successful matrix formation were demonstrated invitro. The combination of fractional laser ablation and dSRC implantation enhanced cartilage repair.

One-step soaking strategy toward mechanobiological double-network hydrogel with improving chondrogenesis capacity

Cartilage tissue engineering (CTE) is a promising strategy for addressing the persistent challenges in healing articular cartilage defects in the clinic, aiming to manufacture scaffolds with ideal mechanical characteristics that can provide a conducive microenvironment for the reconstruction or replacement of damaged cartilage defects. Easy operation, excellent mechanical strength, remarkable biocompatibility, and inherent capacity to promote cartilage formation are essential in CTE. This study designed a mechanobiological double-network (DN) hydrogel (KCS-PAA-Mn2+) through a one-step soaking strategy of immersing the composite hydrogel (KCS-PAA) of kartogenin (KGN)-conjugated short-chain chitosan (CS) covalent with polyacrylic acid (PAA) in manganese chloride (MnCl2) solution. Due to the strong physical interactions of CS chain-entanglement network and carboxylate-Mn2+ complex inducing polymer aggregation state transitions, the KCS-PAA-Mn2+ DN hydrogel exhibited exceptional mechanical characteristics, such as high tensile property (strength: 0.24 MPa at a strain of 552 %; toughness: 0.66 MPa), compressibility (strength: 51.98 MPa at a strain of 95 %; compressive modulus: 0.09 MPa), controllable swelling behavior (~327 %), and sustained drug release (>2 weeks). Moreover, in vitro biological studies demonstrated that the KCS-PAA-Mn2+ DN hydrogel not only promoted cell growth and proliferation but also enhanced the expression of genes specific to cartilage and the release of cartilage extracellular matrix (ECM) by bone mesenchymal stem cells (BMSCs), thus enabling a continuous concentration for a long period and persistently enhancing its chondrogenic efficiency. Overall, this soaking methodology reported here is universal to construct mechanically bioactive scaffolds that could expand their bio-applicability with simultaneous advantages of mechanical supports, tailorable swelling, sustainable drug release and long-lasting biological function, thus facilitating the advancement of cartilage-engineered hydrogel biomaterials to a higher level.

Chitosan-Chitosan derivative for cartilage associated disorders: Protein interaction and biodegradability

Chitosan is a well-known biomaterial in the field of cartilage tissue engineering because of its flexibility. The construction of chitosan-based scaffolds is reviewed in several production methods, including freeze-drying, gelation, salt leaching, and electrospinning. In this review, many benefits of chitosan are discussed, including the interaction of chitosan with other materials and their effects, its adaptability, high reproducibility, biocompatibility, role in cellular differentiation, interactions with TGF-β (transforming growth factor-β), protein interactions, biodegradability, influencing osteoblast expression, and potential for treating bone diseases. The study also provides information on how mathematical models are used to analyze how MSC (mesenchymal stem cells) and chondrocyte distribution in multilayer hydrogels change in response to TGF-β diffusion. Results showed that collagen has weak mechanical properties and easily disintegrates during bone tissue formation, it is difficult to use alone as a biomaterial, and new scaffolds made of whey protein isolate (WPI) and chitosan may help repair osteochondral tissue. However, the thorough analysis reveals chitosan's crucial contribution to the development of cartilage tissue engineering as well as its potential to address issues in the field.

Photo-crosslinked chitosan-gelatin xerogel-like coating onto “cold” plasma functionalized poly(lactic acid) film as cell culture support

This paper reports on biofunctionalisation of a poly(lactic acid) (PLA) film by surface activation through cold plasma treatment followed by coating with a chitosan-gelatin xerogel. The UV cross-linking of the xerogel precursor was simultaneously performed with the fixation onto the PLA support. This has a strong effect on surface properties, in terms of wettability, surface free energy, morphology and micromechanical features. The hydrophilic – hydrophobic character of the surface, determined by contact angle measurements, was tuned along the process, passing from moderate hydrophobic PLA to enhanced hydrophilic plasma activated surface, which favors coating adhesion, then to moderate hydrophobic chitosan-gelatin coating. The coating has a Lewis amphoteric surface, with a porous xerogel-like morphology, as revealed by scanning electron microscopy images. By riboflavin mediated UV cross-linking the chitosan-gelatin coating becomes high adhesive and with a more pronounced plasticity, as shown by AFM force-distance spectroscopy. Thus prepared surface-coated PLA supports were successfully tested for growth of dermal fibroblasts, which are known for their induction potential of chondrogenic cells, which is very important in cartilage tissue engineering.

Silk-Based 3D Porous Scaffolds for Tissue Engineering.

Silk fibroin, extracted from the silk of the Bombyx mori silkworm, stands out as a biomaterial due to its nontoxic nature, excellent biocompatibility, and adjustable biodegradability. Porous scaffolds, a type of biomaterial, are crucial for creating an optimal microenvironment that supports cell adhesion and proliferation, thereby playing an essential role in tissue remodeling and repair. Therefore, this review focuses on 3D porous silk fibroin-based scaffolds, first summarizing their preparation methods and then detailing their regenerative effects on bone, cartilage, tendon, vascular, neural, skin, hepatic, and tracheal epithelial tissue engineering in recent years.

Glycosaminoglycan-Mediated Interactions in Articular, Auricular, Meniscal, and Nasal Cartilage.

Glycosaminoglycans (GAGs) are ubiquitous components in the cartilage extracellular matrix (ECM). Ultrastructural arrangement of ECM and GAG-mediated interactions with collagen are known to govern the mechanics in articular cartilage, but these interactions are less clear in other cartilage types. Therefore, this article reviews the current literature on ultrastructure of articular, auricular, meniscal, and nasal septal cartilage, seeking insight into GAG-mediated interactions influencing mechanics. Ultrastructural features of these cartilages are discussed to highlight differences between them. GAG-mediated interactions are reviewed under two categories: interactions with chondrocytes and interactions with other fibrillar macromolecules of the ECM. Moreover, efforts to replicate GAG-mediated interactions to improve mechanical integrity of tissue-engineered cartilage constructs are discussed. In conclusion, studies exploring cartilage specific GAGs are poorly represented in the literature, and the ultrastructure of nasal septal and auricular cartilage is less studied compared with articular and meniscal cartilages. Understanding the contribution of GAGs in cartilage mechanics at the ultrastructural level and translating that knowledge to engineered cartilage will facilitate improvement of cartilage tissue engineering approaches.

Challenges in Nasal Cartilage Tissue Engineering to Restore the Shape and Function of the Nose.

The repair of nasal septal cartilage is a key challenge in cosmetic and functional surgery of the nose, as it determines its shape and its respiratory function. Supporting the dorsum of the nose is essential for both the prevention of nasal obstruction and the restoration of the nose structure. Most surgical procedures to repair or modify the nasal septum focus on restoring the external aspect of the nose by placing a graft under the skin, without considering respiratory concerns. Tissue engineering offers a more satisfactory approach, in which both the structural and biological roles of the nose are restored. To achieve this goal, nasal cartilage engineering research has led to the development of scaffolds capable of accommodating cartilaginous extracellular matrix-producing cells, possessing mechanical properties close to those of the nasal septum, and retaining their structure after implantation in vivo. The combination of a non-resorbable core structure with suitable mechanical properties and a biocompatible hydrogel loaded with autologous chondrocytes or mesenchymal stem cells is a promising strategy. However, the stability and immunotolerance of these implants are crucial parameters to be monitored over the long term after in vivo implantation, to definitively assess the success of nasal cartilage tissue engineering. Here, we review the tissue engineering methods to repair nasal cartilage, focusing on the type and mechanical characteristics of the biomaterials; cell and implantation strategy; and the outcome with regard to cartilage repair.

Co-culture of RhoA-overexpressed microtia chondrocytes and adipose-derived stem cells in the construction of tissue-engineered ear-shaped cartilage.

Microtia is a congenital auricle dysplasia with a high incidence and tissue engineering technology provides a promising strategy to reconstruct auricles. We previously described that the engineered cartilage constructed from microtia chondrocytes exhibited inferior levels of biochemical and biomechanical properties, which was proposed to be resulted of the decreased migration ability of microtia chondrocytes. In the current study, we found that Rho GTPase members were deficient in microtia chondrocytes. By overexpressing RhoA, Rac1, and CDC42, respectively, we further demonstrated that RhoA took great responsibility for the decreased migration ability of microtia chondrocytes. Moreover, we constructed PGA/PLA scaffold-based cartilages to verify the chondrogenic ability of RhoA overexpressed microtia chondrocytes, and the results showed that overexpressing RhoA was of limited help in improving the quality of microtia chondrocyte engineered cartilage. However, coculture of adipose-derived stem cells (ADSCs) significantly improved the biochemical and biomechanical properties of engineered cartilage. Especially, coculture of RhoA overexpressed microtia chondrocytes and ADSCs produced an excellent effect on the wet weight, cartilage-specific extracellular matrix, and biomechanical property of engineered cartilage. Furthermore, we presented that coculture of RhoA overexpressed microtia chondrocytes and ADSCs combined with human ear-shaped PGA/PLA scaffold and titanium alloy stent fabricated by CAD/CAM and 3D printing technology effectively constructed and maintained auricle structure in vivo. Collectively, our results provide evidence for the essential role of RhoA in microtia chondrocytes and a developed strategy for the construction of patient-specific tissue-engineered auricular cartilage.

Elevated nutrient availability enhances chondrocyte metabolism and biosynthesis in tissue-engineered cartilage

ObjectiveChondrocytes, which typically rely on anaerobic metabolism, exhibit upregulated biosynthetic activity when subjected to conditions that elicit mixed aerobic-anaerobic metabolism. Previously, we observed that increasing media volume resulted in the transition from anaerobic to mixed aerobic-anaerobic metabolism. Maximal extracellular matrix (ECM) accumulation occurred at this transition as a result of changes in HIF-1α signaling and associated hypoxic gene expression. This study aimed to explore the effect of further increases in media availability on ECM synthesis and chondrocyte metabolism. MethodsPrimary bovine chondrocytes were grown in 3D high-density tissue culture under varying levels of media availability (4 – 16 mL/106 cells). Changes in ECM accumulation and metabolism were determined through biochemical assays and 13C-metabolic flux analysis (13C-MFA). ResultsIncreasing media volumes resulted in higher accumulation of cartilaginous extracellular matrix (collagen and proteoglycans) and cellularity. Extracellular metabolite measurements revealed that elevated media availability led to increased glucose and glutamine metabolism, along with increased anaerobic activity. 13C-MFA utilizing [U-13C] glucose demonstrated that increased media availability significantly impacted central carbon metabolism, upregulating all glucose-related metabolic pathways (glycolysis, lactate fermentation, the TCA cycle, hexosamine biosynthetic pathway, and the malate-aspartate shuttle). Furthermore, 13C-MFA indicated that glutamine was donating carbons to the TCA cycle, and additional studies involving [U-13C] glutamine tracing supported this notion. ConclusionsElevated media availability upregulates extracellular matrix synthesis and leads to significant changes in metabolic phenotype. Glutamine plays an important role in chondrocyte metabolism and increases in glutamine metabolism correlate with increases in extracellular matrix accumulation.