Electrical stimulation for cartilage tissue engineering - A critical review from an engineer's perspective.

Cartilage has a limited intrinsic healing capacity. Hence, cartilage degradation and lesions pose a huge clinical challenge, particularly in an ageing society. Osteoarthritis impacts a significant number of the population and requires the development of repair and tissue engineering methods for hyaline articular cartilage. In this context, electrical stimulation has been investigated for more than 50 years already. Yet, no well-established clinical therapy to treat osteoarthritis by means of electrical stimulation exists. We argue that one reason is the lack of replicability of electrical stimulation devices from a technical perspective together with lacking hypotheses of the biophysical mechanism. Hence, first, the electrical stimulation studies reported in the context of cartilage tissue engineering with a special focus on technical details are summarized. Then, an experimental and numerical approach is discussed to make the electrical stimulation experiments replicable. Finally, biophysical hypotheses have been reviewed on the interaction of electric fields and cells that are relevant for cartilage tissue engineering. With that, the aim is to inspire future research to enable clinical electrical stimulation therapies to fight osteoarthritis.

Chemofunctionalization of knitted silk to improve interface connection in a nano/micro scaffold based on polycaprolactone-chitosan-multi-walled carbon nanotube/silk.

Nano/micro hybrid scaffolds in long-term healing tissue engineering can simultaneously offer both mechanical and biological properties. In this study, a hybrid scaffold was fabricated through electrospinning of polycaprolactone (PCL)-chitosan (Cs)/ multi-walled carbon nanotubes (MWCNTs) based nanofibers onto a chemically functionalized knitted silk substrate (F-Silk) and the scaffold were evaluated with regard to morphology, chemical and crystalline structure, hydrophilicity, mechanical properties, bioactivity, biodegradability, and cellular behavior. Chemical functionalization of silk using N-hydroxysuccinimide (NHS) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) resulted in greater integrity in the formation of nanofibers onto the microfibers. The presence of MWCNTs significantly reduced the contact angle of the scaffolds from 79.72° ± 2.72 to 68.92° ± 5.63. Chemical functionalization of silk, the presence of nanofiber coating, and the presence of MWCNTs increased the ultimate tensile strength of the hybrid scaffolds by 18 %, 20 %, and 30 % compared to raw silk fabric, respectively. The presence of MWCNTs and chemical functionalization of knitted silk increased the bioactivity and reduced the degradation rate of hybrid scaffolds. The increase in the amount of carboxyl groups as a result of adding 0.5 wt% of MWCNTs significantly improved the adhesion, growth and proliferation of chondrocyte cells on the hybrid scaffolds as observed through cell morphology. According to the obtained results, hybrid scaffold based on PCL-Cs-MWCNTs/F-silk can be a suitable option for further research in cartilage tissue engineering.

Mechanical properties and biocompatibility characterization of 3D printed collagen type II/silk fibroin/hyaluronic acid scaffold.

Damage to articular cartilage is irreversible and its ability to heal is minimal. The development of articular cartilage in tissue engineering requires suitable biomaterials as scaffolds that provide a 3D natural microenvironment for the development and growth of articular cartilage. This study aims to investigate the applicability of a 3D printed CSH (collagen type II/silk fibroin/hyaluronic acid) scaffold for constructing cartilage tissue engineering. The results showed that the composite scaffold had a three-dimensional porous network structure with uniform pore sizes and good connectivity. The hydrophilicity of the composite scaffold was 1071.7 ± 131.6%, the porosity was 85.12 ± 1.6%, and the compressive elastic modulus was 36.54 ± 2.28 kPa. The creep and stress relaxation constitutive models were also established, which could well describe the visco-elastic mechanical behavior of the scaffold. The biocompatibility experiments showed that the CSH scaffold was very suitable for the adhesion and proliferation of chondrocytes. Under dynamic compressive loading conditions, it was able to promote cell adhesion and proliferation on the scaffold surface. The 3D printed CSH scaffold is expected to be ideal for promoting articular cartilage regeneration.

Mechanical and Biological Characterization of Ionic and Photo-Crosslinking Effects on Gelatin-Based Hydrogel for Cartilage Tissue Engineering Applications.

Articular cartilage degeneration poses a significant public health challenge; techniques such as 3D bioprinting are being explored for its regeneration in vitro. Gelatin-based hydrogels represent one of the most promising biopolymers used in cartilage tissue engineering, especially for its collagen composition and tunable mechanical properties. However, there are no standard protocols that define process parameters such as the crosslinking method to apply. To this aim, a reproducible study was conducted for exploring the influence of different crosslinking methods on 3D bioprinted gelatin structures. This study assessed mechanical properties and cell viability in relation to various crosslinking techniques, revealing promising results particularly for dual (photo + ionic) crosslinking methods, which achieved high cell viability and tunable stiffness. These findings offer new insights into the effects of crosslinking methods on 3D bioprinted gelatin for cartilage applications. For example, ionic and photo-crosslinking methods provide softer materials, with photo-crosslinking supporting cell stretching and diffusion, while ionic crosslinking preserves a spherical stem cell morphology. On the other hand, dual crosslinking provides a stiffer, optimized solution for creating stable cartilage-like constructs. The results of this study offer a new perspective on the standardization of gelatin for cartilage bioprinting, bridging the gap between research and clinical applications.

Cell Mediated Reactions Create TGF-ß Delivery Limitations in Engineered Cartilage.

During native cartilage development, endogenous TGF-ß activity is tightly regulated by cell-mediated chemical reactions in the extracellular milieu (e.g., matrix and receptor binding), providing spatiotemporal control in a manner that is localized and short acting. These regulatory paradigms appear to be at odds with TGF-ß delivery needs in tissue engineering (TE) where administered TGF-ß is required to transport long distances or reside in tissues for extended durations. In this study, we perform a novel examination of the influence of cell-mediated reactions on the spatiotemporal distribution of administered TGF-ß in cartilage TE applications. Reaction rates of TGF-ß binding to cell-deposited ECM and TGF-ß internalization by cell receptors are experimentally characterized in bovine chondrocyte-seeded tissue constructs. TGF-ß binding to the construct ECM exhibits non-linear Brunauer-Emmett-Teller (BET) adsorption behavior, indicating that as many as seven TGF-ß molecules can aggregate at a binding site. Cell-mediated TGF-ß internalization rates exhibit a biphasic trend, following a Michaelis-Menten relation (Vmax=2.4 molecules cell-1 s-1, Km=1.7 ng mL-1) at low ligand doses (≤130ng/mL), but exhibit an unanticipated non-saturating power trend at higher doses (≥130ng/mL). Computational models are developed to illustrate the influence of these reactions on TGF-ß spatiotemporal delivery profiles for conventional TGF-ß administration platforms. For TGF-ß delivery via supplementation in culture medium, these reactions give rise to pronounced steady state TGF-ß spatial gradients; TGF-ß concentration decays by ∼90% at a depth of only 500 µm from the media-exposed surface. For TGF-ß delivery via heparin-conjugated affinity scaffolds, cell mediated internalization reactions significantly reduce the TGF-ß scaffold retention time (160 to 360-fold reduction) relative to acellular heparin scaffolds. This work establishes the significant limitations that cell-mediated chemical reactions engender for TGF-ß delivery and highlights the need for novel delivery platforms that account for these reactions to achieve optimal TGF-ß exposure profiles. STATEMENT OF SIGNIFICANCE: During native cartilage development, endogenous TGF-ß activity is tightly regulated by cell-mediated chemical reactions in the extracellular milieu (e.g., matrix and receptor binding), providing spatiotemporal control in a manner that is localized and short acting. However, the effect of these reactions on the delivery of exogenous TGF-ß to engineered cartilage tissues remains not well understood. In this study, we demonstrate that cell-mediated reactions significantly restrict the delivery of TGF-ß to cells in engineered cartilage tissue constructs. For delivery via media supplementation, reactions significantly limit TGF-ß penetration into constructs. For delivery via scaffold loading, reactions significantly limit TGF-ß residence time in constructs. Overall, these results illustrate the impact of cell-mediated chemical reaction on TGF-ß delivery profiles and support the importance of accounting for these reactions when designing TGF-ß delivery platforms for promoting cartilage regeneration.

ALB-PRF facilitates chondrogenesis by promoting chondrocytes migration, proliferation and differentiation.

Cartilage injury is common in orthopedics and cartilage tissue engineering provides a therapeutic direction for cartilage regeneration. Albumin (ALB)-platelet-rich fibrin (PRF) is speculated to be an ideal natural scaffold material for cartilage tissue engineering theoretically as a product derived from human venous blood. Through in vitro and in vivo experiments, it was demonstrated that ALB-PRF displayed porous structure and slowly released growth factors (TGF-ß1, PDGF-AA, PDGF-AB, PDGF-BB, EGF, IGF-1 and VEGF), ALB-PRF conditioned media promoted proliferation, migration, adhesion, phenotype maintenance and extracellular matrix secretion of rabbit chondrocytes. Moreover, ALB-PRF facilitated chondrogenesis in vivo, the regenerative cartilage formed by ALB-PRF/chondrocytes was histologically similar to that of natural knee joint cartilage, the regenerative cartilage expressed cartilage differentiation marker (SOX9, ACAN and COL II), and proliferation marker PCNA and secreted abundant glycosaminoglycans (GAGs) in extracellular matrix. In conclusion, ALB-PRF promoted the migration, proliferation and phenotype maintenance of chondrocytes in vitro. Its loose, porous structure and rich growth factors contained enhanced cell adhesion and growing into the materials. ALB-PRF facilitated chondrogenesis of chondrocytes in vivo.

What is the context? Cartilage injury is a common problem in orthopedics and the self-healing ability of cartilage is limited.At present, the treatment methods for cartilage injury are limited, and surgical treatments also has defects. Cartilage tissue engineering brings hope for cartilage regeneration. At present, there are also certain disadvantages in the scaffold materials for cartilage tissue engineering and it is urgent to develop new scaffold materials.Platelet-rich fibrin (PRF) is a second-generation platelet concentrate produced from autologous blood which can form a fibrin network containing high concentrations of growth factors.The improved PRF, also known as ALB­PRF, has superior properties compared to PRF. Further research is needed to determine whether it can become an excellent scaffold material for cartilage tissue engineering.What is new? ALB­PRF displayed porous structure and slow-released growth factors.ALB­PRF promoted proliferation, migration, adhesion, phenotype maintenance and extracellular matrix secretion of rabbit chondrocytes.ALB­PRF facilitated chondrogenesis in vivo, the regenerative cartilage formed by ALB­PRF/chondrocytes was similar to that of natural knee joint cartilage.What is the impact? ALB­PRF can be utilized as an excellent scaffold material for cartilage tissue engineering, but further research is needed to improve ALB­PRF and "seed cells.".

Cartilage Tissue Engineering in Multilayer Tissue Regeneration.

The functional and structural integrity of the tissue/organ can be compromised in multilayer reconstructive applications involving cartilage tissue. Therefore, multilayer structures are needed for cartilage applications. In this review, we have examined multilayer scaffolds for use in the treatment of damage to organs such as the trachea, joint, nose, and ear, including the multilayer cartilage structure, but we have generally seen that they have potential applications in trachea and joint regeneration. In conclusion, when the existing studies are examined, the results are promising for the trachea and joint connections, but are still limited for the nasal and ear. It may have promising implications in the future in terms of reducing the invasiveness of existing grafting techniques used in the reconstruction of tissues with multilayered layers.

Rapid preparation of injectable dual-network hydrogels for biomedical applications using UV-triggered sulfhydryl click reactions.

The use of hydrogels to mimic natural cartilage implantation can effectively solve the current problems of insufficient cartilage donors and low rate of injury healing. In particular, injectable hydrogels are less invasive in clinical applications and better able to fill uneven injury surfaces. Here, we prepared NorCS and CS-SH by modifying chitosan with 5-norbornene-2-carboxylic acid and N-Acetyl-L-cysteine, respectively. Dual-network hydrogels were prepared by using UV-triggered thiol-ene click reaction between NorCS and CS-SH and the metal coordination between SA and Ca2+. The prepared hydrogels can be cross-linked quickly and exhibit excellent degradability, self-healing and injectable properties. At the same time, the hydrogel also showed good cytocompatibility and could significantly restore the motor function of mice. This study provides an effective strategy for preparing injectable hydrogels capable of rapid cross-linking.

Delivery Strategies of Growth Factors in Cartilage Tissue Engineering.

Cartilage plays an important role in supporting soft tissues, reducing joint friction, and distributing pressure. However, its self-repair capacity is limited due to the lack of blood vessels, nerves, and lymphatic systems. Tissue engineering offers a potential solution to promote cartilage regeneration by combining scaffolds, seed cells, and growth factors. Among these, growth factors play a critical role in regulating cell proliferation, differentiation, and extracellular matrix remodeling. However, their instability, susceptibility to degradation and potential side effects limit their effectiveness. This article reviews the main growth factors used in cartilage tissue engineering and their delivery strategies, including affinity-based delivery, carrier-assisted delivery, stimuli-responsive delivery, spatial structure-based delivery, and cell system-based delivery. Each method shows unique advantages in enhancing the delivery efficiency and specificity of growth factors but also faces challenges such as cost, biocompatibility, and safety. Future research needs to further optimize these strategies to achieve more efficient, safe, and economical delivery of growth factors, thereby advancing the clinical application of cartilage tissue engineering.

Autoinduction-Based Quantification of In Situ TGF-ß Activity in Native and Engineered Cartilage.

Transforming growth factor beta (TGF-ß) is a potent growth factor that regulates the homeostasis of native cartilage and is administered as an anabolic supplement for engineered cartilage growth. The quantification of TGF-ß activity in live tissues in situ remains a significant challenge, as conventional activity assessments (e.g., Western blotting of intracellular signaling molecules or reporter cell assays) are unable to measure absolute levels of TGF-ß activity in three-dimensional tissues. In this study, we develop a quantification platform established on TGF-ß's autoinduction response, whereby active TGF-ß (aTGF-ß) signaling in cells induces their biosynthesis and secretion of new TGF-ß in its latent form (LTGF-ß). As such, cell-secreted LTGF-ß can serve as a robust, non-destructive, label-free biomarker for quantifying in situ activity of TGF-ß in live cartilage tissues. Here, we detect LTGF-ß1 secretion levels for bovine native tissue explants and engineered tissue constructs treated with varying doses of media-supplemented aTGF-ß3 using an isoform-specific ELISA. We demonstrate that: 1) LTGF-ß secretion levels increase proportionally to aTGF-ß exposure, reaching 7.4- and 6.6-fold increases in native and engineered cartilage, respectively; 2) synthesized LTGF-ß exhibits low retention in both native and engineered cartilage tissue; and 3) secreted LTGF-ß is stable in conditioned media for 2 weeks, thus enabling a reliable biological standard curve between LTGF-ß secretion and exposed TGF-ß activity. Accordingly, we perform quantifications of TGF-ß activity in bovine native cartilage, demonstrating up to 0.59 ng/mL in response to physiological dynamic loading. We further quantify the in situ TGF-ß activity in aTGF-ß-conjugated scaffolds for engineered tissue, which exhibits 1.81 ng/mL of TGF-ß activity as a result of a nominal 3 µg/mL loading dose. Overall, cell-secreted LTGF-ß can serve as a robust biomarker to quantify in situ activity of TGF-ß in live cartilage tissue and can be potentially applied for a wide range of applications, including multiple tissue types and tissue engineering platforms with different cell populations and scaffolds.

Collagen binding and mimetic peptide-functionalized self-assembled peptide hydrogel enhance chondrogenic differentiation of human mesenchymal stem cells.

The avascular structure and low cell migration to the damaged area due to the low number of cells do not allow spontaneous repair of the articular cartilage tissue. Therefore, functional scaffolds obtained from biomaterials are used for the regeneration of cartilage tissue. Here, we functionalized one of the self-assembling peptide (SAP) scaffolds KLD (KLDLKLDLKLDL) with short bioactive motifs, which are the α1 chain of type II collagen binding peptide WYRGRL (C1) and the triple helical collagen mimetic peptide GFOGER (C2) by direct coupling. Our goal was to develop injectable functional SAP hydrogels with proper mechanical characteristics that would improve chondrogenesis. Scanning electron microscopy (SEM) was used to observe the integration of peptide scaffold structure at the molecular level. To assure the stability of SAPs, the rheological characteristics and degradation profile of SAP hydrogels were assessed. The biochemical study of the DNA, glycosaminoglycan (GAG), and collagen content revealed that the developed bioactive SAP hydrogels greatly increased hMSCs proliferation compared with KLD scaffolds. Moreover, the addition of bioactive peptides to KLD dramatically increased the expression levels of important chondrogenic markers such as aggrecan, SOX-9, and collagen Type II as evaluated by real-time polymerase chain reaction (PCR). We showed that hMSC proliferation and chondrogenic differentiation were encouraged by the developed SAP scaffolds. Although the chondrogenic potentials of WYRGRL and GFOGER were previously investigated, no study compares the effect of the two peptides integrated into 3-D SAP hydrogels in chondrogenic differentiation. Our findings imply that these specifically created bioactive peptide scaffolds might help enhance cartilage tissue regeneration.

Exploring New Horizons: Advancements in Cartilage Tissue Engineering Under Space Microgravity.

Novel investigations of how microgravity affects cellular and tissue development have recently been made possible by the multidisciplinary fusion of tissue engineering and space science. This review examines the intersection of cartilage tissue engineering (CTE) and space science, focusing on how microgravity affects cartilage development. Space microgravity induces distinct physiological changes in chondrocytes, including a 20-30% increase in cell diameter, a 1.5- to 2-fold increase in proliferation rates, and up to 3-fold increases in chondrogenic markers such as SOX9 and collagen type II. These cellular alterations impact extracellular matrix composition and tissue structure. Space-optimized bioreactors using dynamic culture methods replicate physiological conditions and enhance tissue growth, but the absence of gravity raises concerns about the mechanical properties of engineered cartilage. Key research areas include the role of growth factors in cartilage development under microgravity, biocompatibility and degradation of scaffold materials in space, and in situ experiments on space stations. This review highlights the opportunities and challenges in leveraging microgravity for CTE advancements, emphasizing the need for continued research to harness space environments for therapeutic applications in cartilage regeneration. The multidisciplinary fusion of tissue engineering and space science opens novel avenues for understanding and improving cartilage tissue engineering, with significant implications for the future of biomedical applications in space and on Earth.

Macroporous PEG-Alginate Hybrid Double-Network Cryogels with Tunable Degradation Rates Prepared via Radical-Free Cross-Linking for Cartilage Tissue Engineering.

Trauma or repeated damage to joints can result in focal cartilage defects, significantly elevating the risk of osteoarthritis. Damaged cartilage has an inherently limited self-healing capacity and remains an urgent unmet clinical need. Consequently, there is growing interest in biodegradable hydrogels as potential scaffolds for the repair or reconstruction of cartilage defects. Here, we developed a biodegradable and macroporous hybrid double-network (DN) cryogel by combining two independently cross-linked networks of multiarm polyethylene glycol (PEG) acrylate and alginate.Hybrid DN cryogels are formed using highly biocompatible click reactions for the PEG network and ionic bonding for the alginate network. By judicious selection of various structurally similar cross-linkers to form the PEG network, we can generate hybrid DN cryogels with customizable degradation kinetics. The resulting PEG-alginate hybrid DN cryogels have an interconnected macroporous structure, high mechanical strength, and rapid swelling kinetics. The interconnected macropores in the cryogels support efficient mesenchymal stem cell infiltration at a high density. Finally, we demonstrate that PEG-alginate hybrid DN cryogels allow sustained release of chondrogenic growth factors and support chondrogenic differentiation of mouse mesenchymal stem cells. This study provides a novel method to generate macroporous hybrid DN cryogels with customizable degradation rates and a potential scaffold for cartilage tissue engineering.

Comparative Study of Autogenic and Allogenic Chondrocyte Transplants on Polyethersulfone Scaffolds for Cartilage Regeneration.

The aim of this study was to evaluate the chondrogenic potential of chondrocyte transplants cultured in vitro on polyethersulfone (PES) membranes. Forty-eight rabbits (96 knee joints) were used in the project. The synthetic, macro-porous PES membranes were used as scaffolds. Fragments of articular cartilage were harvested from non-weight-bearing areas of the joints of the animals. Chondrocytes were isolated and then cultivated on PES scaffolds for 3 weeks. The animals were divided into four groups. All the lesions in the articular cartilage were full thickness defects. In Group I, autogenic chondrocytes on PES membranes were transplanted into the defect area; in Group II, allogenic chondrocytes on PES membranes were transplanted into the defect area; in Group III, pure PES membranes were transplanted into the defect area; and in Group IV, lesions were left untreated. Half of the animals from each group were terminated after 8 weeks, and the remaining half were terminated 12 weeks postoperatively. The samples underwent macroscopic evaluation using the Brittberg scale and microscopic evaluation using the O'Driscoll scale. The best regeneration was observed in Groups II and I. In Group I, the results were achieved with two surgeries, while in Group II, only one operation was needed. This indicates that allogenic chondrocytes do not require two surgeries, highlighting the importance of further in vivo studies to better understand this advantage. The success of the study and the desired properties of PES scaffolds are attributed mainly to the presence of sulfonic groups in the structure of the material. These groups, similar to chondroitin sulfate, which naturally occurs in hyaline cartilage, likely enable mutual affinity between the scaffold and cells and promote scaffold colonization by the cells.

Interleukin-4-Loaded Gelatin Methacryloyl Hydrogel Promotes Subcutaneous Chondrogenesis of Engineered Auricular Cartilage in a Rabbit Model.

Tissue engineering technology offers a promising solution for ear reconstruction; however, it faces the challenge of foreign body reaction and neocartilage malformation. This study investigates the impact of interleukin-4 (IL-4), an anti-inflammatory factor, on cartilage regeneration of hydrogel encapsulating autologous auricular chondrocytes in a rabbit subcutaneous environment. Initially, we assessed the influence of IL-4 on chondrocyte proliferation and determined the appropriate concentration using the CCK-8 test in vitro. Subsequently, we loaded IL-4 into gelatin methacryloyl (GelMA) hydrogel containing chondrocytes and measured its release profile through ELISA. The constructs were then implanted autologously into rabbits' subcutis, and after 3, 7, 14, and 28 days, cartilage matrix formation was evaluated by histological examinations, and gene expression levels were detected by qRT-PCR. Results demonstrated that IL-4 promotes chondrocyte proliferation in vitro, and maximum release from constructs occurred during the first week. In the rabbit subcutaneous implantation model, IL-4-loaded constructs (20 ng/mL) maintained a superior chondrocytic phenotype compared to controls with increased expression of anti-inflammatory factors. These findings highlight IL-4 as a potential strategy for promoting chondrogenesis in a subcutaneous environment and improving ear reconstruction.

Fabrication and Characterization of Porous PEGDA Hydrogels for Articular Cartilage Regeneration.

Functional articular cartilage regeneration remains an unmet medical challenge, increasing the interest for innovative biomaterial-based tissue engineering (TE) strategies. Hydrogels, 3D macromolecular networks with hydrophilic groups, present articular cartilage-like features such as high water content and load-bearing capacity. In this study, 3D porous polyethylene glycol diacrylate (PEGDA) hydrogels were fabricated combining the gas foaming technique and a UV-based crosslinking strategy. The 3D porous PEGDA hydrogels were characterized in terms of their physical, structural and mechanical properties. Our results showed that the size of the hydrogel pores can be modulated by varying the initiator concentration. In vitro cytotoxicity tests showed that 3D porous PEGDA hydrogels presented high biocompatibility both with human chondrocytes and osteoblast-like cells. Importantly, the 3D porous PEGDA hydrogels supported the viability and chondrogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cell (hBM-MSC)-based spheroids as demonstrated by the positive staining of typical cartilage extracellular matrix (ECM) (glycosaminoglycans (GAGs)) and upregulation of chondrogenesis marker genes. Overall, the produced 3D porous PEGDA hydrogels presented cartilage-like mechanical properties and supported MSC spheroid chondrogenesis, highlighting their potential as suitable scaffolds for cartilage TE or disease modelling strategies.

Acceleration of chondrogenic differentiation utilizing biphasic core-shell alginate sulfate electrospun nanofibrous scaffold.

Engineering new biomedical materials with tailored physicochemical, mechanical and biological virtues in order to differentiate stem cells into chondrocytes for cartilage regeneration has garnered much scientific interest. In this study, core/shell nanofibrous scaffold based on poly(ɛ-caprolactone) (PCL) as a core material and alginate sulfate (AlgS)-poly(vinyl alcohol) (PVA) blend as shell materials (AlgS-PVA/PCL) was fabricated by emulsion electrospinning. In this vein, the influence of AlgS to PVA ratio (30:70, 50:50), organic to aqueous phase ratio (1:2, 1:3 and 1:5) and acid concentration (0, 10, 20, 30, 40 and 50 %) on nanofibers morphology were investigated. SEM images depicted that AlgS to PVA ratio of 30:70 and 50:50, organic to aqueous phase ratio of 1:3 and 1:5 and acid concentration of 30 % led to uniform, bead-free fibrous mats. AlgS-PVA/PCL scaffolds with AlgS to PVA ratio of 30:70 and organic to aqueous phase ratio of 1:3, showed admirable mechanical features, high porosity (>90 %) with desirable swelling ratio in wet condition. In vitro assays indicated that the AlgS-PVA/PCL scaffold surface had desirable interaction with stem cells and promotes cells attachment, proliferation and differentiation. Thus, we envision that this salient structure could be an intriguing construction as a cartilage tissue-engineered scaffold.

Polysaccharide base electrospun nanofibrous scaffolds for cartilage tissue engineering: Challenges and opportunities.

Polysaccharides, known as naturally abundant macromolecular materials which can be easily modified chemically, have always attracted scientists' interest due to their outstanding properties in tissue engineering. Moreover, their intrinsic similarity to cartilage ECM components, biocompatibility, and non-harsh processing conditions make polysaccharides an excellent option for cartilage tissue engineering. Imitating the natural ECM structure to form a fibrous scaffold at the nanometer scale in order to recreate the optimal environment for cartilage regeneration has always been attractive for researchers in the past few years. However, there are some challenges for polysaccharides electrospun nanofibers preparation, such as poor solubility (Alginate, cellulose, chitin), high viscosity (alginate, chitosan, and Hyaluronic acid), high surface tension, etc. Several methods are reported in the literature for facing polysaccharide electrospinning issues, such as using carrier polymers, modification of polysaccharides, and using different solvent systems. In this review, considering the importance of polysaccharide-based electrospun nanofibers in cartilage tissue engineering applications, the main achievements in the past few years, and challenges for their electrospinning process are discussed. After careful investigation of reported studies in the last few years, alginate, chitosan, hyaluronic acid, chondroitin sulfate, and cellulose were chosen as the main polysaccharide base electrospun nanofibers used for cartilage regeneration.

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.

Physiologic Doses of Transforming Growth Factor-ß Improve the Composition of Engineered Articular Cartilage.

Conventionally, for cartilage tissue engineering applications, transforming growth factor beta (TGF-ß) is administered at doses that are several orders of magnitude higher than those present during native cartilage development. While these doses accelerate extracellular matrix (ECM) biosynthesis, they may also contribute to features detrimental to hyaline cartilage function, including tissue swelling, type I collagen (COL-I) deposition, cellular hypertrophy, and cellular hyperplasia. In contrast, during native cartilage development, chondrocytes are exposed to moderate TGF-ß levels, which serve to promote strong biosynthetic enhancements while mitigating risks of pathology associated with TGF-ß excesses. Here, we examine the hypothesis that physiologic doses of TGF-ß can yield neocartilage with a more hyaline cartilage-like composition and structure relative to conventionally administered supraphysiologic doses. This hypothesis was examined on a model system of reduced-size constructs (∅2 × 2 mm or ∅3 × 2 mm) comprised of bovine chondrocytes encapsulated in agarose, which exhibit mitigated TGF-ß spatial gradients allowing for an evaluation of the intrinsic effect of TGF-ß doses on tissue development. Reduced-size (∅2 × 2 mm or ∅3 × 2 mm) and conventional-size constructs (∅4-∅6 mm × 2 mm) were subjected to a range of physiologic (0.1, 0.3, 1 ng/mL) and supraphysiologic (3, 10 ng/mL) TGF-ß doses. At day 56, the physiologic 0.3 ng/mL dose yielded reduced-size constructs with native cartilage-matched Young's modulus (EY) (630 ± 58 kPa) and sulfated glycosaminoglycan (sGAG) content (5.9 ± 0.6%) while significantly increasing the sGAG-to-collagen ratio, leading to significantly reduced tissue swelling relative to constructs exposed to the supraphysiologic 10 ng/mL TGF-ß dose. Furthermore, reduced-size constructs exposed to the 0.3 ng/mL dose exhibited a significant reduction in fibrocartilage-associated COL-I and a 77% reduction in the fraction of chondrocytes present in a clustered morphology, relative to the supraphysiologic 10 ng/mL dose (p < 0.001). EY was significantly lower for conventional-size constructs exposed to physiologic doses due to TGF-ß transport limitations in these larger tissues (p < 0.001). Overall, physiologic TGF-ß appears to achieve an important balance of promoting requisite ECM biosynthesis, while mitigating features detrimental to hyaline cartilage function. While reduced-size constructs are not suitable for the repair of clinical-size cartilage lesions, insights from this work can inform TGF-ß dosing requirements for emerging scaffold release or nutrient channel delivery platforms capable of achieving uniform delivery of physiologic TGF-ß doses to larger constructs required for clinical cartilage repair.