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Silk fibroin (SF)–chondroitin sulfate (CS)-hybrid hydrogels (HHGs) containing kartogenin (KGN) were synthesized by the induction of di-tyrosine bonds, using enzymatic crosslinking in SF/CS blend solution. Then, physical crosslinking was performed to optimize the HHG properties. The results showed that the increasing of the β-sheet content as side effect of physical crosslinking from 10% to 40% affects KGN accumulative release percentage almost three times. Biological investigations using C28/I2 human chondrocyte cell line showed that the presence of KGN, CS, and optimized β-sheets content improved cell supporting behavior of HHG. Finally, KGN loaded HHG might be a promising candidate for cartilage repair. Schematic representation of fabrication of the dual crosslinked hybrid hydrogel containing silk fibroin and chondroitin sulfate by enzymatic crosslinking and physical crosslinking method for kartogenin release. Enzymatic crosslinking: shaping di-tyrosine formation bond between silk fibroin phenol groups and locking physically chondroitin sulfate between silk fibroin β-sheets. Physical crosslinking with water and/or ethanol treatments end up in increased β-sheets content. [ABSTRACT FROM AUTHOR]

Introduction: Kartogenin, a potent inducer of chondrogenic differentiation in mesenchymal stem cells and a key agent in cartilage regeneration, presents a viable therapeutic strategy for osteoarthritis management. Despite the abundance of literature on therapeutic potential of kartogenin, there is a paucity of studies characterizing the formulation specifics in microsphere fabrication. This exploration is pivotal to advances in regenerative medicine, particularly in the domain of cartilage regeneration, to assure clinical efficacy and safety. Methods: In this work, we fabricated kartogenin-loaded PLGA microspheres with diverse formulations and their particle size, size distribution, encapsulation efficiency, drug loading and release profiles were characterized. Ratio of polymer, drug, and solvent and the use of surfactant was used as variables, and in particular, the effect of surfactant on particles was investigated. Results: The average diameter of the spheres was 16.0–31.7 μm. Morphological variations from solid to porous surface structures depending on surfactant incorporation during the emulsification process was observed. Cumulative kartogenin release from microspheres ranged from 53.8% to 80.9% on day 28, and release profiles conform predominantly to the Korsmeyer-Peppas kinetics model. Discussion: This study provides a foundational framework for modulating kartogenin release dynamics, a critical consideration for optimizing therapeutic efficacy and minimizing adverse effects in cartilage tissue engineering applications. [ABSTRACT FROM AUTHOR]

Accumulating evidence supports the role of cartilage tissue engineering in cartilage defect repair, but the biological function has yet to be fully explained. In this work, kartogenin (KGN), an emerging chondroinductive nonprotein small molecule, was incorporated into a composite hydrogel of polyvinyl alcohol/nano‐hydroxyapatite (PVA/n‐HA) to fabricate an appropriate microenvironment for tendon‐bone healing after anterior cruciate ligament (ACL) reconstruction. KGN/PVA/n‐HA composite hydrogel scaffolds were prepared by in situ synthesis and physical adsorption, followed by characterization under a scanning electron microscope. The scaffolds were transplanted into healthy New Zealand White (NZW) rabbits. It was confirmed that KGN/PVA/n‐HA scaffolds were successfully prepared and exhibited good supporting properties and excellent biocompatibility. Unilateral ACL reconstruction was constructed with tendon autograft in NZW rabbits, and the morphology and diameter of collagen fiber were analyzed. The scaffolds were shown to promote ACL growth and collagen fiber formation. Furthermore, microcomputerized tomography analysis and bone formation histology were performed to detect new bone formation. KGN/PVA/n‐HA scaffolds effectively alleviated cartilage damage and prevented the occurrence of osteoarthritis. Meanwhile, ligament‐bone healing and bone formation were observed in the presence of KGN/PVA/n‐HA scaffolds. In conclusion, these results suggest that the KGN/PVA/n‐HA scaffolds can facilitate tendon‐bone healing after ACL reconstruction and might be considered novel hydrogel biomaterials in cartilage tissue engineering. [ABSTRACT FROM AUTHOR]

Loading drugs in drug delivery systems can increase their retention time and control their release within the knee cavity. Hence, we aimed to improve the therapeutic efficacy of celecoxib and kartogenin (KGN) through their loading in chitosan nanoparticles (CS NPs). Celecoxib-loaded nanoparticles (CNPs) and KGN-loaded nanoparticles (K-CS NPs) were prepared using the absorption method and covalent attachment, respectively, through an ionic gelation process. The morphology, particle size, zeta potential, polydispersity index (PDI), conjugation efficiency (CE), encapsulation efficiency (EE), the in vitro release of the drug from NPs, as well as MTT and hemolysis assays, were evaluated. Then, the IL-1β-stimulated chondrocytes were treated with CNPs and K-CS NPs, individually or in combination, to explore their potential chondroprotective and anti-inflammatory effects. CNPs and K-CS NPs showed sizes of 352.6 ± 22.5 and 232.7 ± 4.5 nm, respectively, suitable for intra-articular (IA) injection. Based on the hemolysis results, both NPs exhibited good hemocompatibility within the studied range. Results showed that treating IL-1β-pretreated chondrocytes with CNPs or K-CS NPs remarkably limited the negative effects of IL-1β, especially when both types of NPs were used together. Therefore, injecting these two NPs into the knee cavity may improve drug bioavailability, rapidly suppress inflammation and pain, and promote cartilage regeneration. Meanwhile, for the first time, the study investigated the effect of the simultaneous use of celecoxib and KGN to treat osteoarthritis (OA).

Highlights A multifactorial and oriented scaffolds was constructed to stimulate osteochondral regeneration. This is the first demonstration that both drug and gene nanoparticles were spatially deposited on a patterned film through metal-ligand interactions. For the first time, film-rolling approach was employed to construct osteochondral plug to mimick the Haversian canal structure of natural bone.

Kartogenin, a small and heterocyclic molecule, has emerged as a promising therapeutic agent for incorporation into biomaterials, owing to its unique physicochemical and biological properties. It holds potential for the regeneration of cartilage-related tissues in various common conditions and injuries. Achieving sustained release of kartogenin through appropriate formulation and efficient delivery systems is crucial for modulating cell behavior and tissue function. This review provides an overview of cutting-edge kartogenin-functionalized biomaterials, with a primarily focus on their design, structure, functions, and applications in regenerative medicine. Initially, we discuss the physicochemical properties and biological functions of kartogenin, summarizing the underlying molecular mechanisms. Subsequently, we delve into recent advancements in nanoscale and macroscopic materials for the carriage and delivery of kartogenin. Lastly, we address the opportunities and challenges presented by current biomaterial developments and explore the prospects for their application in tissue regeneration. We aim to enhance the generation of insightful ideas for the development of kartogenin delivery materials in the field of biomedical therapeutics and regenerative medicine by providing a comprehensive understanding of common preparation methods. [ABSTRACT FROM AUTHOR]

Highlights: A multifactorial and oriented scaffolds was constructed to stimulate osteochondral regeneration. This is the first demonstration that both drug and gene nanoparticles were spatially deposited on a patterned film through metal-ligand interactions. For the first time, film-rolling approach was employed to construct osteochondral plug to mimick the Haversian canal structure of natural bone. The integrated repair of bone and cartilage boasts advantages for osteochondral restoration such as a long-term repair effect and less deterioration compared to repairing cartilage alone. Constructing multifactorial, spatially oriented scaffolds to stimulate osteochondral regeneration, has immense significance. Herein, targeted drugs, namely kartogenin@polydopamine (KGN@PDA) nanoparticles for cartilage repair and miRNA@calcium phosphate (miRNA@CaP) NPs for bone regeneration, were in situ deposited on a patterned supramolecular-assembled 2-ureido-4 [lH]-pyrimidinone (UPy) modified gelation hydrogel film, facilitated by the dynamic and responsive coordination and complexation of metal ions and their ligands. This hydrogel film can be rolled into a cylindrical plug, mimicking the Haversian canal structure of natural bone. The resultant hydrogel demonstrates stable mechanical properties, a self-healing ability, a high capability for reactive oxygen species capture, and controlled release of KGN and miR-26a. In vitro, KGN@PDA and miRNA@CaP promote chondrogenic and osteogenic differentiation of mesenchymal stem cells via the JNK/RUNX1 and GSK-3β/β-catenin pathways, respectively. In vivo, the osteochondral plug exhibits optimal subchondral bone and cartilage regeneration, evidenced by a significant increase in glycosaminoglycan and collagen accumulation in specific zones, along with the successful integration of neocartilage with subchondral bone. This biomaterial delivery approach represents a significant toward improved osteochondral repair. [ABSTRACT FROM AUTHOR]

Background/purpose: Temporomandibular joint (TMJ) osteoarthritis (TMJOA) is a chronic disease with progressive destruction of articular cartilage. This study aimed to explore the therapeutic effects of kartogenin on TMJOA via promoting the binding of core binding factor β (CBFβ) and runt-related transcription factor 1 (RUNX1). Materials and methods: Type II collagenase was injected into 35 8-week-old male Sprague Dawley rat TMJs to establish the TMJOA model. Kartogenin, or the CBFβ-RUNX1 complex inhibitor (Ro5-3335), was also delivered via intra-articular injection. Subchondral bone was analyzed by MicroCT. The hematoxylin-eosin, Safranin O, and toluidine blue O staining were used to observe histopathology. Immunohistochemical staining of proliferating cell nuclear antigen (PCNA), caspase-3 (CASP3), interleukin-1β (IL-1β), and collagen II (COL2) was performed. Results: TMJOA was established in rats by intra-articular injection of type II collagenase. The condylar cartilage and subchondral bone were damaged, with decreased PCNA and COL2 and increased CASP3 and IL-1 (P

Introduction: Over-activation of oxidative stress due to impaired antioxidant functions in nucleus pulpous (NP) has been identified as a key factor contributing to intervertebral disc degeneration (IVDD). While Kartogenin (KGN) has previously demonstrated antioxidant properties on articular cartilage against osteoarthritis, its effects on NP degeneration have yet to be fully understood. Objectives: This study aimed to investigate the protective effects of KGN on nucleus pulpous cells (NPCs) against an inflammatory environment induced by interleukin (IL)-1β, as well as to explore the therapeutic potential of KGN-enhanced dynamic hydrogel in preventing IVDD. Methods: NPCs were isolated from rat caudal IVDs and subjected to treatment with KGN at varying concentrations (ranging from 0.01 to 1 ​μM) in the presence of IL-1β. The expression of extracellular matrix (ECM) anabolism markers was quantitatively assessed at both the mRNA and protein levels. Additionally, intracellular reactive oxygen species and antioxidant enzyme expression were evaluated, along with the role of nuclear factor erythroid 2-related factor 2 (NRF2). Based on these findings, a dynamic self-healing hydrogel loaded with KGN was developed through interconnecting networks. Subsequently, KGN-enhanced dynamic hydrogel was administered into rat caudal IVDs that had undergone puncture injury, followed by radiographic analysis and immunohistochemical staining to evaluate the therapeutic efficacy. Results: In vitro treatments utilizing KGN were observed to maintain ECM synthesis and inhibit catabolic activities in IL-1β-stimulated NPCs. The mechanism behind this protective effect of KGN on NPCs was found to involve the asctivation of NRF2 and downstream antioxidant enzymes, including glutathione peroxidase 1 and heme oxygenase 1. This was further supported by the loss of both antioxidant and anabolic effects upon pharmacological inhibition of NRF2. Furthermore, a self-healing hydrogel was developed and loaded with KGN to achieve localized and sustained release of the compound. The injection of KGN-enhanced hydrogel effectively ameliorated the degradation of NP ECM and mitigated inflammation in a rat model of puncture-induced IVDD. Conclusions: Our results indicate that KGN exhibits potential as a therapeutic agent for NP degeneration, and that KGN-enhanced dynamic hydrogel represents a novel approach for treating IVDD by restoring redox homeostasis in NP.The translational potential of this article: The dysregulation of oxidant and antioxidant balance has been shown to impede the repair and regeneration of NP, thereby hastening the progression of IVDD following injury. The present investigation has demonstrated that the sustained release of KGN promotes the synthesis of ECM in vitro and mitigates the progression of IVDD in vivo by restoring redox equilibrium, thereby presenting a novel therapeutic candidate based on the antioxidant properties of KGN for the treatment of IVDD.

AbstractKartogenin, a small and heterocyclic molecule, has emerged as a promising therapeutic agent for incorporation into biomaterials, owing to its unique physicochemical and biological properties. It holds potential for the regeneration of cartilage-related tissues in various common conditions and injuries. Achieving sustained release of kartogenin through appropriate formulation and efficient delivery systems is crucial for modulating cell behavior and tissue function. This review provides an overview of cutting-edge kartogenin-functionalized biomaterials, with a primarily focus on their design, structure, functions, and applications in regenerative medicine. Initially, we discuss the physicochemical properties and biological functions of kartogenin, summarizing the underlying molecular mechanisms. Subsequently, we delve into recent advancements in nanoscale and macroscopic materials for the carriage and delivery of kartogenin. Lastly, we address the opportunities and challenges presented by current biomaterial developments and explore the prospects for their application in tissue regeneration. We aim to enhance the generation of insightful ideas for the development of kartogenin delivery materials in the field of biomedical therapeutics and regenerative medicine by providing a comprehensive understanding of common preparation methods.

The incidence of osteochondral defect is increasing year by year, but there is still no widely accepted method for repairing the defect. Hydrogels loaded with bioactive molecules have provided promising alternatives for in-situ osteochondral regeneration. Kartogenin (KGN) is an effective and steady small molecule with the function of cartilage regeneration and protection which can be further boosted by TGF-β. However, the high cost, instability, and immunogenicity of TGF-β would limit its combined effect with KGN in clinical application. In this study, a composite hydrogel CM-KGN@GelMA, which contained TGF-β1 analog short peptide cytomodulin-10 (CM-10) and KGN, was fabricated. The results indicated that CM-10 modified on GelMA hydrogels exerted an equivalent role in enhancing chondrogenesis as TGF-β1, and this effect was also boosted when combined with KGN. Moreover, it was revealed that CM-10 and KGN had a synergistic effect on promoting the chondrogenesis of BMSCs by up-regulating the expression of RUNX1 and SOX9 at both mRNA and protein levels in vitro. Finally, the composite hydrogel exhibited a satisfactory osteochondral defect repair effect in vivo , showing similar structures close to the native tissue. Taken together, this study has revealed that CM-10 may serve as an alternative for TGF-β1 and can collaborate with KGN to accelerate chondrogenesis, which suggests that the fabricated CM-KGN@GelMA composite hydrogel can be acted as a potential scaffold for osteochondral defect regeneration. Kartogenin and TGF-β have shown great value in promoting osteochondral defect regeneration, and their combined application can enhance the effect and show great potential for clinical application. Herein, a functional CM-KGN@GelMA hydrogel was fabricated, which was composed of TGF-β1 mimicking peptide CM-10 and KGN. CM-10 in hydrogel retained an activity like TGF-β1 to facilitate BMSC chondrogenesis and exhibited boosting chondrogenesis by up-regulating RUNX1 and SOX9 when being co-applied with KGN. In vivo , the hydrogel promoted cartilage regeneration and subchondral bone reconstruction, showing similar structures as the native tissue, which might be vital in recovering the bio-function of cartilage. Thus, this study developed an effective scaffold and provided a promising way for osteochondral defect repair. [Display omitted] [ABSTRACT FROM AUTHOR]

Temporomandibular joint (TMJ) osteoarthritis (TMJOA) is a chronic disease with progressive destruction of articular cartilage. This study aimed to explore the therapeutic effects of kartogenin on TMJOA via promoting the binding of core binding factor β (CBFβ) and runt-related transcription factor 1 (RUNX1). Type II collagenase was injected into 35 8-week-old male Sprague Dawley rat TMJs to establish the TMJOA model. Kartogenin, or the CBFβ-RUNX1 complex inhibitor (Ro5-3335), was also delivered via intra-articular injection. Subchondral bone was analyzed by MicroCT. The hematoxylin-eosin, Safranin O, and toluidine blue O staining were used to observe histopathology. Immunohistochemical staining of proliferating cell nuclear antigen (PCNA), caspase-3 (CASP3), interleukin-1β (IL-1β), and collagen II (COL2) was performed. TMJOA was established in rats by intra-articular injection of type II collagenase. The condylar cartilage and subchondral bone were damaged, with decreased PCNA and COL2 and increased CASP3 and IL-1 (P <.001). Compared with the OA group, kartogenin alleviated the destruction of cartilage and subchondral bone, rescued the expression of PCNA and COL2, and decreased the expression of CASP3 and IL-1β (P <.01). Ro5-3335 did not aggravate the pathology of TMJOA but neutralized the therapeutic effects of kartogenin on TMJOA. Kartogenin has a potential therapeutic effect on TMJOA via promoting the CBFβ-RUNX1 binding. [ABSTRACT FROM AUTHOR]

Intervertebral disc (IVD) degeneration (IDD) and the consequent low back pain present a major medical challenge. Stem cell-based tissue engineering is promising for the treatment of IDD. However, stem cell-based treatment is severely impaired by the increased generation of reactive oxygen species (ROS) in degenerative disc, which can lead to a high level of cell dysfunction and even death. In this study, a kartogenin (KGN)@PLGA-GelMA/PRP composite hydrogel was designed and used as a carrier of ADSCs-based therapies in disc repair. Injectable composite hydrogel act as a carrier for controlled release of KGN and deliver ADSCs to the degenerative disc. The released KGN can stimulate the differentiation of ADSCs into a nucleus pulposus (NP) -like phenotype and boost antioxidant capacity of ADSCs via activating Nrf2/TXNIP/NLRP3 axis. Furthermore, the composite hydrogel combined with ADSCs attenuated the in vivo degeneration of rat IVDs, maintained IVD tissue integrity and accelerated the synthesis of NP-like extracellular matrix. Therefore, the KGN@PLGA-GelMA/PRP composite hydrogel is a promising strategy for stem cell-based therapies of IDD. [Display omitted] • KGN@PLGA-GelMA/PRP composite hydrogel combined with ADSCs attenuated the in vivo degeneration of rat IVDs. • KGN can stimulate the differentiation of ADSCs and boost antioxidant capacity of ADSCs via activating Nrf2/TXNIP/NLRP3 axis. • We chose to perform cell therapy one weeks after puncture, which is more realistic in terms of clinical practice. [ABSTRACT FROM AUTHOR]

Cartilage tissue engineering has attracted great attention in defect repair and regeneration. The utilization of bioactive scaffolds to effectively regulate the phenotype and proliferation of chondrocytes has become an elemental means for cartilage tissue regeneration. On account of the simultaneous requirement of mechanical and biological performances for tissue-engineered scaffolds, in this work we prepared a naturally derived hydrogel composed of a bioactive kartogenin (KGN)-linked chitosan (CS-KGN) and an aldehyde-modified oxidized alginate (OSA) via the highly efficient Schiff base reaction and multifarious physical interactions in mild conditions. On the basis of the rigid backbones and excellent biocompatibility of these two natural polysaccharides, the composite hydrogel demonstrated favorable morphology, easy injectability, good mechanical strength and tissue adhesiveness, low swelling ratio, long-term sustainable KGN release, and facilitated bone marrow mesenchymal stem cell activity, which could simultaneously provide the mechanical and biological supports to promote chondrogenic differentiation and repair the articular cartilage defects. Therefore, we believe this work can offer a designable consideration and potential alternative candidate for cartilage and other soft tissue implants. [ABSTRACT FROM AUTHOR]

Osteoarthritis is a prevalent musculoskeletal disorder in the elderly, which leads to high rates of morbidity. Mesenchymal stem cells (MSCs) are a promising approach to promote tissue regeneration in the absence of effective long-term treatments. Small molecules are relatively inexpensive and can selectively alter stem cell behavior during their differentiation, making them an attractive option for clinical applications. In this study, we developed an extracellular matrix (ECM)-based biphasic scaffold (BPS) loaded with two small-molecule drugs, kartogenin (KGN) and metformin (MET). This cell-free biomimetic biphasic scaffold consists of a bone (gelatin/hydroxyapatite scaffold embedded with metformin [GHSM]) and cartilage (nano-gelatin fiber embedded with kartogenin [NGFK]) layer designed to stimulate osteochondral regeneration. Extracellular matrix (ECM)-based biomimetic scaffolds can promote native cell recruitment, infiltration, and differentiation even in the absence of additional growth factors. The biphasic scaffold (BPS) showed excellent biocompatibility in vitro, with mesenchymal stem cells (MSCs) adhering, proliferating, and differentiated on the biomimetic biphasic scaffolds (GHSM and NGFK layers). The biphasic scaffolds upregulated both osteogenic and chondrogenic gene expression, sulfated glycosaminoglycan (sGAG), osteo- and chondrogenic biomarker, and relative mRNA gene expression. In an in vivo rat model, histo-morphological staining showed effective regeneration of osteochondral defects. This novel BPS has the potential to enhance both subchondral bone repair and cartilage regeneration, demonstrating excellent effects on cell homing and the recruitment of endogenous stem cells. [ABSTRACT FROM AUTHOR]

Introduction: Kartogenin (KGN) is a small-molecule compound that has been reported to improve the chondrogenic differentiation of mesenchymal stem cells in vitro and to alleviate knee joint osteoarthritis in animal models. However, whether KGN has any effect on temporomandibular joint osteoarthritis (TMJOA) remains unclear. Methods: We first performed partial temporomandibular joint (TMJ) discectomy to induce TMJOA in rats. Histological analysis, tartrate-resistant acid phosphatase staining, and immunohistochemistry were used to assess the therapeutic effect of KGN on TMJOA in vivo. CCK8 and pellet cultures were used to determine whether KGN treatment could promote the proliferation and differentiation of FCSCs in vitro. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to determine the expression of aggrecan, Col2a1, and Sox9 in FCSCs. Furthermore, we performed western blot to analysis the effect of KGN treatment on the expression of Sox9 and Runx2 in FCSCs. Results and discussion: Histological analysis, tartrate-resistant acid phosphatase staining, and immunohistochemistry showed that intra-articular injection of KGN attenuated cartilage degeneration and subchondral bone resorption in vivo. Further analyses of the underlying mechanisms revealed that KGN enhanced chondrocyte proliferation, increased the number of cells in both superficial and proliferative zones of TMJ condylar cartilage in vivo, enhanced the proliferation and chondrogenic differentiation of fibrocartilage stem cells (FCSCs), and upregulated the expression of chondrogenesis-related factors in vitro. Collectively, in our study, KGN was shown to promote FCSC chondrogenesis and restore TMJ cartilage, suggesting that KGN injections might be a potential treatment for TMJOA. [ABSTRACT FROM AUTHOR]

Zengxin Jiang,1,&ast; Zeng Zhang,1,&ast; Shuo Li,2,&ast; Sen Lin,3 Hengfeng Yuan1 1Department of Orthopaedics, Shanghai Jiaotong University Affiliated Sixth People’s Hospital, Shanghai, People’s Republic of China; 2Department of Joint Bone Disease Surgery, Changhai Hospital, Second Military Medical University, Shanghai, People’s Republic of China; 3National Engineering Research Center, East China University of Science and Technology, Shanghai, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Sen Lin; Hengfeng Yuan, Email linsen@ecust.edu.cn; yuanhf@shsmu.edu.cnBackground: Degenerative joint disease or osteoarthritis (OA) is a leading cause of disability worldwide. Intra-articular injection is the mainstay nonsurgical treatment for OA. However, dense cartilage and a lack of vasculature often limit the ability of drugs to reach cell or tissue targets at the concentrations necessary to elicit the desired biological response. Kartogenin (KGN), a small molecular compound, possesses a strong capacity to promote chondrogenic differentiation of mesenchymal stem cells (MSCs). However, the rapid clearance of KGN from the intra-articular cavity limits its feasibility.Materials and Methods: We constructed a magnetically guided biodegradable nanocarrier system (MNP) which enabled intracartilaginous delivery of KGN to promote chondrogenic differentiation by MSCs embedded within the articular matrix. Moreover, in preclinical models of OA, KGN-loaded MNPs exhibited increased tissue penetration and retention within the joint matrix under external magnetic guidance.Results: Histological examination showed that compared with KGN alone, KGN-loaded MNPs enhanced chondrogenic differentiation and improved the structural integrity of both articular cartilage and subchondral bone.Conclusion: This study demonstrates a practical method for intracartilaginous delivery using engineered nanocarriers, thus providing a new strategy to improve the efficacy of molecular therapeutic agents in the treatment of OA.Graphical Abstract: Keywords: osteoarthritis, kartogenin, magnetic nanoparticles, intracartilaginous

Background: Poor tendon-to-bone healing in chronic rotator cuff tears (RCTs) is related to unsatisfactory outcomes. Exosomes derived from mesenchymal stem cells reportedly enhance rotator cuff healing. However, the difficulty in producing exosomes with a stronger effect on enthesis regeneration must be resolved. Purpose: To study the effect of exosomes derived from kartogenin (KGN)-preconditioned human bone marrow mesenchymal stem cells (KGN-Exos) on tendon-to-bone healing in a rat model of chronic RCT. Study Design: Controlled laboratory study. Methods: Exosome-loaded sodium alginate hydrogel (SAH) was prepared. Moreover, exosomes were labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) or 1,1′-dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate (Dil) for in vivo tracking. Bilateral rotator cuff repair (RCR) was conducted in an established chronic RCT rat model. A total of 66 rats were randomized to control, untreated exosome (un-Exos), and KGN-Exos groups to receive local injections of pure SAH, un-Exos, or KGN-Exos SAH at the repaired site. The presence of DiR/Dil-labeled exosomes was assessed at 1 day and 1 week, and tendon-to-bone healing was evaluated histologically, immunohistochemically, and biomechanically at 4 and 8 weeks. Results: Both un-Exos and KGN-Exos exhibited sustained release from SAH for up to 96 hours. In vivo study revealed that un-Exos and KGN-Exos were localized to the repaired site at 1 week. Moreover, the KGN-Exos group showed a higher histological score and increased glycosaminoglycan and collagen II expression at 4 and 8 weeks. In addition, more mature and better-organized collagen fibers with higher ratios of collagen I to collagen III were observed at 8 weeks in the tendon-to-bone interface compared with those in the control and un-Exos groups. Biomechanically, the KGN-Exos group had the highest failure load (28.12 ± 2.40 N) and stiffness (28.57 ± 2.49 N/mm) among the 3 groups at 8 weeks. Conclusion: Local injection of SAH with sustained KGN-Exos release could effectively promote cartilage formation as well as collagen maturation and organization for enthesis regeneration, contributing to enhanced biomechanical properties after RCR. Clinical Relevance: KGN-Exos injection may be used as a cell-free therapeutic option to accelerate tendon-to-bone healing in chronic RCT. [ABSTRACT FROM AUTHOR]

The restoration of cartilage damage is a slow and not always successful process. Kartogenin (KGN) has significant potential in this space—it is able to induce the chondrogenic differentiation of stem cells and protect articular chondrocytes. In this work, a series of poly(lactic-co-glycolic acid) (PLGA)-based particles loaded with KGN were successfully electrosprayed. In this family of materials, PLGA was blended with a hydrophilic polymer (either polyethyleneglycol (PEG) or polyvinylpyrrolidone (PVP)) to control the release rate. Spherical particles with sizes in the range of 2.4–4.1 µm were fabricated. They were found to comprise amorphous solid dispersions, with high entrapment efficiencies of >93%. The various blends of polymers had a range of release profiles. The PLGA-KGN particles displayed the slowest release rate, and blending with PVP or PEG led to faster release profiles, with most systems giving a high burst release in the first 24 h. The range of release profiles observed offers the potential to provide a precisely tailored profile via preparing physical mixtures of the materials. The formulations are highly cytocompatible with primary human osteoblasts. [ABSTRACT FROM AUTHOR]

Despite extensive studies, hydrogels are unable to meet the mechanical and biological requirements for successful outcomes in cartilage tissue engineering. In the present study, beta cyclodextrin (β-CD)-modified alginate/cartilage extracellular matrix (ECM)-based interpenetrating polymer network (IPN) hydrogel was developed for sustained release of Kartogenin (KGN). Furthermore, the hydrogel was incorporated within a 3D-printed poly (ε-caprolactone) (PCL)/starch microfiber network in order to reinforce the construct for cartilage tissue engineering. All the synthesized compounds were characterized by H1-NMR spectroscopy. The hydrogel/microfiber composite with a microfiber strand size and strand spacing of 300 μm and 2 mm, respectively showed a compressive modulus of 17.2 MPa, resembling the properties of the native cartilage tissue. Considering water uptake capacity, degradation rate, mechanical property, cell cytotoxicity and glycosaminoglycan secretions, β-CD-modified hydrogel reinforced with printed PCL/starch microfibers with controlled release of KGN may be considered as a promising candidate for using in articular cartilage defects. [ABSTRACT FROM AUTHOR]

Improving chondrogenic differentiation while inhibiting hypertrophic differentiation of mesenchymal stem cells (MSCs) is of vital importance to effectively repairing cartilage injury. Accordingly, kartogenin (KGN) and antioxidants, which promote chondrogenic differentiation and inhibit hypertrophic differentiation, respectively, have shown great potential in promoting cartilage repair. However, KGN is poorly soluble in water, hindering efficient intracellular delivery. Near-infrared light (NIR)-responsive mesoporous polydopamine nanoparticles (MPDA) reportedly exert antioxidative effects by eliminating reactive oxygen species (ROS). In this study, we assessed whether KGN loaded in MPDA can accelerate cartilage injury repair in rats. Specifically, a thermosensitive phase-change material (PCM, melting point 39 °C) combined with KGN was loaded into MPDA as a gatekeeper (KGN@MPDA-PCM). The results showed that KGN@MPDA-PCM exhibited excellent photothermal properties under NIR irradiation, which induced PCM melting and consequent KGN release. In combination with antioxidative therapy, NIR-triggered KGN release enabled the nanocomposite to accelerate MSC chondrogenic differentiation and inhibit hypertrophic differentiation. Importantly, negligible damage to primary organs and satisfactory biocompatibility were observed in the rat model. RNA sequencing found that this MPDA-based platform promotes chondrogenic differentiation by promoting fibronectin-1 (Fn1) expression and activating the PI3K/Akt pathway. Collectively, these findings highlight the therapeutic potential of this MPDA-based platform for accelerating cartilage injury repair.

Due to the unique physical characteristics of intervertebral disc degeneration (IVDD) and the pathological microenvironment that it creates, including inflammation and oxidative stress, effective self-repair is impossible. During the process of intervertebral disc degeneration, there is an increase in the infiltration of M1 macrophages and the secretion of proinflammatory cytokines. Here, we designed a novel injectable composite hydrogel scaffold: an oligo [poly (ethylene glycol) fumarate]/sodium methacrylate (OPF/SMA) hydrogel scaffold loaded with dual-drug/sustained-release PLGA microspheres containing IL-4 (IL-4-PLGA) and kartogenin (KGN-PLGA). This scaffold exhibited good mechanical properties and low immunogenicity while also promoting the sustained release of drugs. By virtue of the PLGA microspheres loaded with IL-4 (IL-4-PLGA), the composite hydrogel scaffold induced macrophages to transition from the M1 phenotype into the M2 phenotype during the early induced phase and simultaneously exhibited a continuous anti-inflammatory effect through the PLGA microspheres loaded with kartogenin (KGN-PLGA). Furthermore, we investigated the mechanisms underlying the immunomodulatory and anti-inflammatory effects of the composite hydrogel scaffold. We found that the scaffold promoted cell proliferation and improved cell viability in vitro. While ensuring mechanical strength, this composite hydrogel scaffold regulated the local inflammatory microenvironment and continuously repaired tissue in the nucleus pulposus via the sequential release of drugs in vivo. When degenerative intervertebral discs in a rat model were injected with the scaffold, there was an increase in the proportion of M2 macrophages in the inflammatory environment and higher expression levels of type II collagen and aggrecan; this was accompanied by reduced levels of MMP13 expression, thus exhibiting long-term anti-inflammatory effects. Our research provides a new strategy for promoting intervertebral disc tissue regeneration and a range of other inflammatory diseases. [ABSTRACT FROM AUTHOR]

Cartilage regeneration after injury is still a great challenge in clinics, which suffers from its avascularity and poor proliferative ability. Herein we designed a novel biocompatible cellulose nanocrystal/GelMA (gelatin-methacrylate anhydride)/HAMA (hyaluronic acid-methacrylate anhydride)-blended hydrogel scaffold system, loaded with synthetic melanin nanoparticles (SMNP) and a bioactive drug kartogenin (KGN) for theranostic purpose. We found that the SMNP-KGN/Gel showed favorable mechanical property, thermal stability, and distinct magnetic resonance imaging (MRI) contrast enhancement. Meanwhile, the sustained release of KGN could recruit bonederived mesenchymal stem cells to proliferate and differentiate into chondrocytes, which promoted cartilage regeneration in vitro and in vivo. The hydrogel degradation and cartilage restoration were simultaneously monitored by multiparametric MRI for 12 weeks, and further confirmed by histological analysis. Together, these results validated the multifunctional hydrogel as a promising tissue engineering platform for noninvasive imaging-guided precision therapy in cartilage regenerative medicine. [ABSTRACT FROM AUTHOR]

Treating articular cartilage defects in patients remains a challenging task due to the absence of blood vessels within the cartilage tissue. The regenerative potential is further compromised by an imbalance between anabolism and catabolism, induced by elevated levels of reactive oxygen species. However, the advent of tissue engineering introduces a promising strategy for cartilage regeneration, offering viable solutions such as mechanical support and controlled release of chondrogenic molecules or cytokines. In this study, we developed an antioxidant scaffold by incorporating natural silk fibroin (SF) and kartogenin (KGN)-loaded liposomes (SF-Lipo@KGN). The scaffold demonstrated appropriate pore size, connectivity, and water absorption and the sustained release of KGN was achieved through the encapsulation of liposomes. In vitro experiments revealed that the SF-Lipo@KGN scaffolds exhibited excellent biocompatibility, as evidenced by enhanced cell adhesion, migration, and proliferation of chondrocytes. The SF-Lipo@KGN scaffolds were found to stimulate cartilage matrix synthesis through the activation of the nuclear factor erythroid-2-related factor 2/heme oxygenase-1 antioxidant signaling pathway. In vivo experiments demonstrated the effective promotion of articular cartilage regeneration by the SF-Lipo@KGN scaffolds, which enhanced extracellular matrix anabolism and restored the intrinsic redox homeostasis. Overall, this study successfully developed biomimetic KGN-loaded scaffolds that restore cartilage redox homeostasis, indicating promising prospects for cartilage tissue engineering. [ABSTRACT FROM AUTHOR]

The effectiveness of existing tissue‐engineering cartilage (TEC) is known to be hampered by weak integration of biocompatibility, biodegradation, mechanical strength, and microenvironment supplies. The strategy of hydrogel‐based TEC holds considerable promise in circumventing these problems. Herein, a non‐toxic, biodegradable, and mechanically optimized double‐network (DN) hydrogel consisting of polyethylene glycol (PEG) and kartogenin (KGN)‐conjugated chitosan (CHI) is constructed using a simple soaking strategy. This PEG‐CHI‐KGN DN hydrogel possesses favorable architectures, suitable mechanics, remarkable cellular affinity, and sustained KGN release, which can facilitate the cartilage‐specific genes expression and extracellular matrix secretion of peripheral blood‐derived mesenchymal stem cells (PB‐MSCs). Notably, after tracing the transplanted cells by detecting the rabbit sex‐determining region Y‐linked gene sequence, the allogeneic PB‐MSCs are found to survive for even 3 months in the regenerated cartilage. Here, the long‐term release of KGN is able to efficiently and persistently activate multiple genes and signaling pathways to promote the chondrogenesis, chondrocyte differentiation, and survival of PB‐MSCs. Thus, the regenerated tissues exhibit well‐matched histomorphology and biomechanical performance such as native cartilage. Consequently, it is believed this innovative work can expand the choice for developing the next generation of orthopedic implants in the loadbearing region of a living body. [ABSTRACT FROM AUTHOR]

• Developed a core–shell structured KGN/Gel@Cur/PLCL nanomembrane using coaxial electrospinning to enhance the chondrogenic stability of Bone Marrow Stem Cell (BMSC)-Regenerated Cartilage (BRC). • The shell layer of the nanomembrane contains curcumin (Cur) and poly(L-lactide-co-caprolactone) (PLCL), known for their potent immunomodulatory and anti-angiogenic effects; the core layer contains Kartogenin (KGN) and gelatin (Gel), recognized chondrogenic inducers. • The nanomembrane encapsulated BRCs induced from goat BMSCs in vitro and was subcutaneously implanted into autologous goats. The physical barrier of the nanomembrane and the release of Cur from the shell layer prevented vascular invasion and inflammatory infiltration. • KGN release from the core layer maintained the BRC chondrogenic phenotype and promoted cartilage maturation, significantly inhibiting BRC ossification and preserving its chondrogenic stability. • This study highlights the crucial role of the core-shell structured KGN/Gel@Cur/PLCL nanomembrane in preventing BRC ossification and preserving the structural and functional stability of BRC, which is essential for the clinical translation of stem cell therapy into cartilage repair. Bone marrow stem cell (BMSC)-regenerated cartilage (BRC) shows promising prospects for clinically repairing cartilage defects. However, when subcutaneously implanted into immunocompetent large animals, BRC is prone to ossification due to vascular invasion and inflammatory infiltration, compromising its chondrogenic stability. To address this challenge, we developed a core–shell structured KGN/Gel@Cur/PLCL nanomembrane using coaxial electrospinning technology to enhance the chondrogenic stability of BRC in subcutaneous niche. The shell layer comprises curcumin (Cur), known for its potent immunomodulatory and anti-angiogenic effects, and poly(L-lactide-co-caprolactone) (PLCL). The core layer comprises Kartogenin (KGN), a recognized chondrogenic inducer, and gelatin (Gel). This nanomembrane encapsulated BRCs induced from goat BMSCs in vitro and was subcutaneously implanted into autologous goats. The nanomembrane's physical barrier and Cur release from the shell layer prevented vascular invasion and inflammatory infiltration. Additionally, KGN release from the core layer maintained the BRC's chondrogenic phenotype and promoted cartilage maturation. These combined mechanisms significantly inhibited BRC ossification and preserved its chondrogenic stability. chondrogenic phenotype and promoted cartilage maturation. These combined mechanisms significantly inhibited BRC ossification and maintained its chondrogenic stability. Overall, this study highlights the effectiveness of the core–shell structured KGN/Gel@Cur/PLCL nanomembrane in providing sequential immune and vascular isolation, followed with chondrogenic induction, to prevent BRC ossification and preserve the chondrogenic stability of subcutaneously implanted BRC in large animal. This is crucial for the clinical translation of stem cell therapy into subcutaneous cartilage repair. [ABSTRACT FROM AUTHOR]

• The adverse immune microenvironment ensuing from cartilage injury, coupled with the intrinsic property of the tissue characterized by limited cellularity, has posed a significant challenge to the reconstruction of cartilage defect. • Enzymatic treatment converts small intestinal submucosa (SIS) into a hydrogel form, not only providing a scaffold for cells engaged and achieving prolonged drug release, but also exhibiting excellent immunomodulatory effects. • Kartogenin (KGN) recruits the endogenous BMSCs of the host and directly enhances their chondrogenic differentiation, thereby achieving cartilage repair and regeneration without cell transplantation. • Leveraging the combined effects of the SIS hydrogel and KGN, this innovative composite material provides a simple yet effective method for expediting the repair and regeneration of cartilage defects. Articular cartilage is crucial for the functional integrity of joints yet vulnerable to a spectrum of injuries. The adverse immune microenvironment ensuing from injury, coupled with the intrinsic property of the tissue characterized by limited cellularity, poses a significant challenge to the reconstruction of cartilage defect. In this study, an extracellular matrix hydrogel derived from porcine small intestinal submucosa (SIS) with encapsulation of Kartogenin (KGN) has been designed for the cartilage repair and regeneration. The SIS hydrogel, which possessed a three-dimensional porous structure akin to the natural cartilage, has provided a scaffold essential for the progenitor cells engaged in the repair process and sustained release of KGN. As shown by in vitro experiments, the KGN could recruit host endogenous bone marrow-derived mesenchymal stem cells (BMSCs) and induce them to differentiate into chondrocytes, thus enabling in situ cartilage regeneration without cell transplantation. Notably, the bioactive component of SIS was further revealed to possess excellent immunomodulatory capacity to induce the phenotypic shift from M1 to M2 macrophages, which could enhance the chondrogenic differentiation of BMSCs indirectly. Additionally, in vivo experiments showed that the regenerated tissues of the composite SIS hydrogel group were close to the natural hyaline cartilage. Leveraging the combined effects of KGN and SIS hydrogel, this innovative composite material shows great potential as a biomaterial for effective cartilage repair and regeneration. [ABSTRACT FROM AUTHOR]

Intervertebral disc degeneration is a clinical disease that reduces the quality of patient's life. The degeneration usually initiates in the nucleus pulposus (NP), hence the use of hydrogels represents a promising therapeutic approach. However, the viscoelastic nature of hydrogel and its ability to provide biomimetic architecture and biochemical cues influence the regeneration capability. This study focused on tuning the physical nature of a glycosaminoglycan hydrogel (κ-carrageenan) as well as the release kinetics of a chondrogenic factor (kartogenin - KGN) through physical cross-linking. For this, κ-carrageenan was cross linked with 2.5 % and 5 % potassium chloride (KCl) for 15 and 30 min and loaded with KGN molecule at 50 μM and 100 μM. The tight network structure with low water retention and degradation property was seen in hydrogel cross-linked with increased KCl concentration and time. However, optimal degradation along with NP mimicking viscoelastic nature was exhibited by 5 wt% KCl treated hydrogel (H3 hydrogel). All hydrogel groups exhibited burst KGN release at 24 h followed by a sustained release for 5 days. However, hydrogel cross-linked with 5 wt% KCl enhanced chondrogenic differentiation, mainly at lower KGN dose. In summary, this study shows the potential application of biomimetic KGN laden carrageenan hydrogel in NP regeneration. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract The effectiveness of existing tissue‐engineering cartilage (TEC) is known to be hampered by weak integration of biocompatibility, biodegradation, mechanical strength, and microenvironment supplies. The strategy of hydrogel‐based TEC holds considerable promise in circumventing these problems. Herein, a non‐toxic, biodegradable, and mechanically optimized double‐network (DN) hydrogel consisting of polyethylene glycol (PEG) and kartogenin (KGN)‐conjugated chitosan (CHI) is constructed using a simple soaking strategy. This PEG‐CHI‐KGN DN hydrogel possesses favorable architectures, suitable mechanics, remarkable cellular affinity, and sustained KGN release, which can facilitate the cartilage‐specific genes expression and extracellular matrix secretion of peripheral blood‐derived mesenchymal stem cells (PB‐MSCs). Notably, after tracing the transplanted cells by detecting the rabbit sex‐determining region Y‐linked gene sequence, the allogeneic PB‐MSCs are found to survive for even 3 months in the regenerated cartilage. Here, the long‐term release of KGN is able to efficiently and persistently activate multiple genes and signaling pathways to promote the chondrogenesis, chondrocyte differentiation, and survival of PB‐MSCs. Thus, the regenerated tissues exhibit well‐matched histomorphology and biomechanical performance such as native cartilage. Consequently, it is believed this innovative work can expand the choice for developing the next generation of orthopedic implants in the loadbearing region of a living body.

Intra-articular injection of drugs is an effective strategy for osteoarthritis (OA) treatment. However, the complex microenvironment and limited joint space result in rapid clearance of drugs. Herein, a nanogel-based strategy is proposed for prolonged drug delivery and microenvironment remodeling. Nanogel is constructed through the functionalization of hyaluronic acid (HA) by amide reaction on the surface of Kartogenin (KGN)-loaded zeolitic imidazolate framework-8 (denoted as KZIF@HA). Leveraging the inherent hydrophilicity of HA, KZIF@HA spontaneously forms nanogels, ensuring extended drug release in the OA microenvironment. KZIF@HA exhibits sustained drug release over one month, with low leakage risk from the joint cavity compared to KZIF, enhanced cartilage penetration, and reparative effects on chondrocytes. Notably, KGN released from KZIF@HA serves to promote extracellular matrix (ECM) secretion for hyaline cartilage regeneration. Zn 2+ release reverses OA progression by promoting M2 macrophage polarization to establish an anti-inflammatory microenvironment. Ultimately, KZIF@HA facilitates cartilage regeneration and OA alleviation within three months. Transcriptome sequencing validates that KZIF@HA stimulates the polarization of M2 macrophages and secretes IL-10 to inhibit the JNK and ERK pathways, promoting chondrocytes recovery and enhancing ECM remodeling. This pioneering nanogel system offers new therapeutic opportunities for sustained drug release, presenting a significant stride in OA treatment strategies., (© 2024 Wiley‐VCH GmbH.)

Cartilage defects are frequently caused by trauma, illness and degradation of the cartilage. If these defects are not sufficiently treated, the joints will degrade irreversibly, possibly resulting in disability. Articular cartilage lacks blood vessels and nerves and is unable to regenerate itself, so the repair of cartilage defects is extremely challenging in clinical treatment. Tissue engineering technology is an emerging technology in cartilage repair and cartilage regeneration. 3D-printed hydrogels show great potential in cartilage tissue engineering for the fabrication of 3D cell culture scaffolds to mimic extracellular matrix. In this study, we construct a 3D-printed hydrogel loaded with nanoparticles by electrostatic interaction and photo cross-linking for the regeneration of cartilage, which has adaptable and drug-continuous release behavior. A photopolymerizable bioink was prepared using recombinant collagen, chitosan, nanoclay Laponite-XLG and nanoparticles loaded with Kartogenin (KGN). This bioink was added with KGN, a small molecule drug that promotes cartilage differentiation, and as a result, the 3D-printed CF/CM/3%LAP/KGN scaffolds obtained by extrusion printing is expected to be used for cartilage repair. It was shown that the 3D-printed scaffolds had good cytocompatibility for human bone marrow mesenchymal stem cells (hBMSCs) and exhibited excellent antimicrobial properties, the continuous release of KGN in the scaffold induced the hBMSCs differentiation into chondrocytes, which significantly enhanced the expression of collagen II and glycosaminoglycan. In vivo studies have shown that implantation of KGN-loaded scaffolds into cartilage-injured tissues promoted cartilage tissue regeneration. This study demonstrated that 3D-printed CF/CM/3%LAP/KGN scaffolds can be used for cartilage repair, which is expected to lead to new healing opportunities for cartilage injury-based diseases., (© The Author(s) 2024. Published by Oxford University Press.)

Cartilage tissue engineering has attracted great attention in defect repair and regeneration. The utilization of bioactive scaffolds to effectively regulate the phenotype and proliferation of chondrocytes has become an elemental means for cartilage tissue regeneration. On account of the simultaneous requirement of mechanical and biological performances for tissue-engineered scaffolds, in this work we prepared a naturally derived hydrogel composed of a bioactive kartogenin (KGN)-linked chitosan (CS-KGN) and an aldehyde-modified oxidized alginate (OSA) via the highly efficient Schiff base reaction and multifarious physical interactions in mild conditions. On the basis of the rigid backbones and excellent biocompatibility of these two natural polysaccharides, the composite hydrogel demonstrated favorable morphology, easy injectability, good mechanical strength and tissue adhesiveness, low swelling ratio, long-term sustainable KGN release, and facilitated bone marrow mesenchymal stem cell activity, which could simultaneously provide the mechanical and biological supports to promote chondrogenic differentiation and repair the articular cartilage defects. Therefore, we believe this work can offer a designable consideration and potential alternative candidate for cartilage and other soft tissue implants.

Osteoarthritis is a prevalent musculoskeletal disorder in the elderly, which leads to high rates of morbidity. Mesenchymal stem cells (MSCs) are a promising approach to promote tissue regeneration in the absence of effective long-term treatments. Small molecules are relatively inexpensive and can selectively alter stem cell behavior during their differentiation, making them an attractive option for clinical applications. In this study, we developed an extracellular matrix (ECM)-based biphasic scaffold (BPS) loaded with two small-molecule drugs, kartogenin (KGN) and metformin (MET). This cell-free biomimetic biphasic scaffold consists of a bone (gelatin/hydroxyapatite scaffold embedded with metformin [GHSM]) and cartilage (nano-gelatin fiber embedded with kartogenin [NGFK]) layer designed to stimulate osteochondral regeneration. Extracellular matrix (ECM)-based biomimetic scaffolds can promote native cell recruitment, infiltration, and differentiation even in the absence of additional growth factors. The biphasic scaffold (BPS) showed excellent biocompatibility in vitro, with mesenchymal stem cells (MSCs) adhering, proliferating, and differentiated on the biomimetic biphasic scaffolds (GHSM and NGFK layers). The biphasic scaffolds upregulated both osteogenic and chondrogenic gene expression, sulfated glycosaminoglycan (sGAG), osteo- and chondrogenic biomarker, and relative mRNA gene expression. In an in vivo rat model, histo-morphological staining showed effective regeneration of osteochondral defects. This novel BPS has the potential to enhance both subchondral bone repair and cartilage regeneration, demonstrating excellent effects on cell homing and the recruitment of endogenous stem cells.

In this study, kartogenin was incorporated into an electrospun blend of polycaprolactone and poly(lactic-co-glycolic acid) (1:1) to determine the feasibility of this system for sustained drug delivery. Kartogenin is a small-molecule drug that could enhance the outcome of microfracture, a cartilage restoration procedure, by selectively stimulating chondrogenic differentiation of endogenous bone marrow mesenchymal stem cells. Experimental results showed that kartogenin did not affect the electrospinnability of the polymer blend, and it had negligible effects on fiber morphology and scaffold mechanical properties. The loading efficiency of kartogenin into electrospun membranes was nearly 100%, and no evidence of chemical reaction between kartogenin and the polymers was detected by Fourier transform infrared spectroscopy. Analysis of the released drug using high-performance liquid chromatography–photodiode array detection indicated an abundance of kartogenin and only a small amount of its major hydrolysis product. Kartogenin displayed a typical biphasic release profile, with approximately 30% being released within 24 h followed by a much slower, constant rate of release up to 28 days. Although additional development is needed to tune the release kinetics and address issues common to electrospun scaffolds (e.g., high fiber density), the results of this study demonstrated that a scaffold electrospun from biodegradable synthetic polymers is a suitable kartogenin delivery vehicle. [ABSTRACT FROM AUTHOR]

An emulsification method was developed for fabricating core-shell microspheres with a thick shell layer. Kartogenin (KGN) and platelet-derived growth factor BB (PDGF-BB) were respectively loaded into the core portion and the shell layer of the microspheres with high loading efficiency. The optimally built microspheres were combined with chitosan (CH) and silk fibroin (SF) to construct a new type of composite hydrogel with enhanced strength and elasticity, using genipin or/and tyrosinase as crosslinkers for the intended use in cartilage tissue engineering. The composite hydrogels were found to be thermo-responsive at physiological temperature and pH with well-defined injectability. Rheological measurements revealed that they had an elastic modulus higher than 6 kPa with a high ratio of elastic modulus to viscous modulus, indicative of their mechanically strong features. Compressive measurements demonstrated that they possessed well-defined elasticity. In addition, some gels had the ability to administer the temporal separation release of PDGF-BB and KGN in an approximately linear manner for several weeks. The released PDGF-BB was found to be bioactive based on its effects on Balb/c 3T3 cells. The composite gels supported the growth of seeded chondrocytes while preserving their phenotype. The results suggest that these composite gels have the potential for endogenous cartilage repair. [ABSTRACT FROM AUTHOR]

Background and Objectives Cartilage tissue engineering, by using stem cells, scaffolding and appropriate growth factors, seek to produce natural cartilage tissue to replace damaged tissue and solve the problems that exist in the treatment of cartilage damage. This study aims to compare chondrogenic differentiation of adipose-derived stem cells under the influence of the transforming growth factor beta 3 (TGF-β3), Kartogenin, and avocado/soybean unsaponifiables (ASU) on the fibrin scaffold in the laboratory and animal models. Subjects and Methods In this experimental laboratory study, after extraction of stem cells from human adipose tissue, induction of chondrogenic differentiation was done for 14 days on the fibrin scaffold in laboratory. The cells differentiated in the fibrin scaffold were transplanted subcutaneously under the skin of male rats for two weeks. Then, comparison of histological and immunohistochemical studies was performed in both laboratory and animal models. Results A significant increase in the density of Toluidine blue dye accumulation was observed in TGF-β3, Kartogenin and ASU groups in the animal model compared to the laboratory model. Immunohistochemical results for the collagen type X accumulation in the TGF-β3 group showed a significant increase in the animal model compared to the laboratory model. In the Kartogenin and ASU groups, the accumulation of collagen type Χ showed a significant decrease in the animal model compared to the laboratory model. Conclusion The implantation of differentiated cartilage cells in laboratory before subcutaneous transfer to the skin can help mature and complete the characteristics of the constructed cartilage. [ABSTRACT FROM AUTHOR]

The cartilage has poor ability to mount a sufficient healing response. Herein, kartogenin (KGN), an emerging stable non-protein compound with the ability to recruit bone marrow mesenchyme stem cells (BMSCs) to promote chondrogenic differentiation, was grafted onto dopamine-Fe(III) chelating nanoparticles, followed by involving a gelatin- and dextran-based injectable hydrogel to mimic the extracellular matrix to promote cartilage repair. The in vitro results demonstrated that KGN underwent long-term sustained release behavior and availably promoted the deep migration of BMSC cells in yielding hydrogels. Furthermore, in vivo New Zealand white rabbits’ cartilage defect model repairing results showed that cartilage defect obtained significant regeneration post operation in the 12th week, and the defect edge almost disappeared compared to adjacent normal cartilage tissue. Meanwhile, the T2-weighted magnetic resonance imaging (MRI) property resulting from dissociative Fe (III) can significantly monitor the degradation degree of the implanted hydrogels in the defect site. This integrated diagnosis and treatment system gives insight into cartilage regeneration.

Fibrocartilaginous regeneration at the tendon-to-bone interface is a major challenge for rotator cuff tear repair. We fabricated a decellularized umbilical cord Wharton’s jelly (DUCWJ) scaffold by decellularization method and performed histological and proteomic analyses. We then synthesized a kartogenin-conjugated DUCWJ (DUCWJ-KGN) scaffold using an EDC/NHS catalyst. 1H NMR, Fluorescence staining, and FTIR confirmed that the carboxyl group of KGN was covalently coupled with the free amine groups in the Wharton’s jelly (WJ) matrix, and the in vitro release profiles of KGN from the DUCWJ-KGN scaffold showed sustained release for 6 weeks. Both the DUCWJ and DUCWJ-KGN scaffolds had excellent biocompatibility and biofunctionality, supporting cell attachment, migration, and proliferation, and DUCWJ-KGN scaffolds the KGN-loaded scaffold could induce higher chondrogenic markers expression. The DUCWJ-KGN scaffold could also promote fibrocartilaginous regeneration at the rotator cuff tendon-to-bone interface, which indicates that the DUCWJ-KGN scaffold is a promising enthesis to enhance fibrocartilaginous interface regeneration.

[Display omitted] • Gelatin methacrylate (GelMA)/poly(2-ethyl-2-oxazoline) (PEtOx) hydrogel was prepared. • The swelling and porosity of GelMA/PEtOx is very close to the ECM of cartilage tissue. • The elastic modulus of GelMA (20 wt%)/PEtOx (45 wt%) is similar to that of cartilage. • The release rate of KGN drug from the hydrogel is stable and gradual within 2 weeks. • The hydrogel showed high cell compatibility and no cytotoxicity. Gelatin methacrylate (GelMA) is a biocompatible, and biodegradable macromolecule developed from a natural polymer, gelatin, via covalent bonding with methacrylic groups. In this research, GelMA/poly(2-ethyl-2-oxazoline) (PEtOx) hydrogel loaded with Kartogenin (KGN) drug was developed with the aim to regenerate the extracellular matrix of cartilage. The morphology, swelling ratio, degree of hydrophilicity, biodegradability, elastic modulus, blood compatibility, drug release and cytotoxicity were assessed. The hydrogel was found to be hydrophilic, highly porous and biodegradable, with 92 % porosity and ∼ 43 % weight loss within 14 days. Mechanical testing revealed that the elastic modulus of GelMA (20 wt%)/PEtOx (45 wt%) was close to that of cartilage. Good blood compatibility and sustained drug release in the damaged cartilage tissue environment were also detected. According to flow cytometry and MTT tests, due to the presence of KGN, up to 75 % of the cells can survive and easily adhere to the developed scaffold. The results confirm the ability of GelMA/PEtOx hydrogel containing KGN to increase cell adhesion by preserving cell population combined with minimal cytotoxicity, which may be advantageous for use in cartilage regeneration following in-vivo implantation. [ABSTRACT FROM AUTHOR]

Synthesized process of UCNP/KGN nanocomposites and UCNP/VitroGel for "integrated diagnosis and treatment" of OA. [Display omitted] • Multifunctional design: combination of upconversion nanoparticles and hydrogels. • ROS response of detection and KGN control release for diagnosis and treatment. • "Integrated diagnosis and treatment" of OA. Osteoarthritis (OA) is a common degenerative joint disease for which there is no definitive treatment. Mesenchymal stem cells (MSCs) can be differentiated into many types of cells such as chondrocytes, which have been used for OA treatment. How to keep the cell viability and promote the chondrogenic differentiation after MSCs transplantation into the knee joint cavity is crucial. In this study, upconversion nanocomposite hydrogel was developed for integrated diagnosis and treatment of OA. The Er doped core–shell upconversion nanoparticles (UCNPs) were synthesized first and coated on mesoporous silica (UCNP@mSiO 2). Then, an H 2 O 2 -sensitive thioketal (TK) linker (MAL-PEG-TK-NHS) was attached to the UCNP@mSiO 2 surface to link capsular β-cyclodextrin (NH 2 -β-CD) and black hole quencher (NH 2 -BHQ 3) to form functionalized UCNPs. After loading the drug kartogenin (KGN) that can induce chondrogenic differentiation of MSCs, the UCNP/KGN nanocomposites were mixed with VitroGel to formed the upconversion nanocomposite hydrogel (UCNP/VitroGel). The UCNP/VitroGel could allow MSCs to be implanted well and maintain good survival in vitro. In the presence of hydrogen peroxide (H 2 O 2), the TK bond breakage and triggered release BHQ to recover the fluorescence of UCNPs for ROS detection, and the β-CD also left from the surface of UCNPs to release KGN for inducing the chondrogenic differentiation of MSCs. Thus, the UCNP/VitroGel could detect the ROS after inject into the knee joint cavity for OA diagnosis first via the fluorescence recovery of UCNPs under the 980 nm near-infrared light. And then the KGN release from UCNP/KGN nanocomposites in the environment of OA to induce the chondrogenic differentiation of MSCs for the treatment of OA. This UCNP/VitroGel will be a powerful tool for "integrated diagnosis and treatment" of OA. [ABSTRACT FROM AUTHOR]

The restoration of cartilage damage is a slow and not always successful process. Kartogenin (KGN) has significant potential in this space—it is able to induce the chondrogenic differentiation of stem cells and protect articular chondrocytes. In this work, a series of poly(lactic-co-glycolic acid) (PLGA)-based particles loaded with KGN were successfully electrosprayed. In this family of materials, PLGA was blended with a hydrophilic polymer (either polyethyleneglycol (PEG) or polyvinylpyrrolidone (PVP)) to control the release rate. Spherical particles with sizes in the range of 2.4–4.1 µm were fabricated. They were found to comprise amorphous solid dispersions, with high entrapment efficiencies of >93%. The various blends of polymers had a range of release profiles. The PLGA-KGN particles displayed the slowest release rate, and blending with PVP or PEG led to faster release profiles, with most systems giving a high burst release in the first 24 h. The range of release profiles observed offers the potential to provide a precisely tailored profile via preparing physical mixtures of the materials. The formulations are highly cytocompatible with primary human osteoblasts.

We report a chitosan-based nanocomposite thermogel with superior shear modulus resembling that of cartilage and dual pro-chondrogenic and anti-inflammatory functions. Two therapeutic agents, kartogenin (KGN) and diclofenac sodium (DS), are employed to promote chondrogenesis of stem cells and suppress inflammation, respectively. To extend the release time in a controlled manner, KGN is encapsulated in the uniform-sized starch microspheres and DS is loaded into the halloysite nanotubes. Both drug carriers are doped into the maleimide-modified chitosan hydrogel to produce a shear modulus of 167 ± 5 kPa that is comparable to that of articular cartilage (50–250 kPa). Owing to the hydrogel injectability and relatively suitable gelation time (5 ± 0.5 min) at 37 °C, this system potentially constitutes a manageable platform for clinical practice. Moreover, sustained linear drug release for over a month boosts chondro-differentiation of stem cells to eliminate the necessity for multiple administrations. Considering virtues such as thermogel strength and ability to co-deliver anti-inflammatory and chondro-inductive biomolecules continuously, the materials and strategy have promising potential in functional cartilage tissue engineering. [Display omitted] • Novel injectable chitosan thermogel with high shear modulus comparable to cartilage • Development of kartogenin encapsulated starch microspheres by droplet microfluidics. • Extended linear kartogenin release from the thermogel for cartilage regeneration • Sustained diclofenac sodium release from the thermogel for pain reduction [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a chronic disease caused by the damage of articular cartilage. Kartogenin (KGN) is a well-recognized small molecule which could induce MSCs chondrogenesis and promote cartilage repair treatments. Nano-level micells could be a suitable drug carrier technology for the treatments. In this study, the acid-responsive methoxy poly(ethylene oxide)-hydrazone-poly(ε-caprolactone) copolymers, mPEG-Hz-b-PCL, were synthesized. The structure was characterized by 1H NMR. The evaluation of a designed kartogenin drug delivery system (DDS) of hydrazone-linkage-based pH responsive mPEG-Hz-b-PCL nanomicelles for treatment of osteoarthritis could be carried out.

Osteochondral lesions represent a major clinical challenge, especially in the elderly. Traditional treatment strategies, such as arthroplasty or tissue engineering, have limitations and drawbacks. In this study, we presented a new treatment concept for the application of an innovative porous bioactive prosthesis with regenerative activity for the treatment of osteoarticular lesions. For regenerative activity, we fabricated chitosan/mesoporous silica nanoparticles (CS/MSNs) composite microspheres via the microfluidic method as a dual-factor carrier for the sequential release of platelet-derived growth factor BB (PDGF-BB) and kartogenin (KGN). We then integrated the factor carrier and a nondegradable polyetheretherketone (PEEK) scaffold through a surface modification technique to construct the porous sulfonated PEEK (SPK) @polydopamine (polydopamine)-CS/MSNs scaffold. We systematically evaluated the biocompatibility and biofunctionality of the SPK@PDA-CS/MSNs scaffold and implanted the scaffold in an in vivo cartilage defect model in rabbits. These results suggest that the SPK@PDA-CS/MSNs scaffold is biocompatible, promotes cell migration, enhances chondrogenic differentiation of BMSCs in vitro, and promotes cartilage regeneration in vivo. The porous bioactive prosthesis with regenerative activity presented first in this study may comprise a new therapeutic concept for osteoarticular lesions.

Osteoarthritis (OA) is a major degenerative joint disease characterized by progressive loss of articular cartilage, synovitis, subchondral bone changes, and osteophyte formation. Currently there is no treatment for OA except temporary pain relief and end-stage joint replacement surgery. We performed a pilot study to determine the effect of kartogenin (KGN, a small molecule) on both cartilage and subchondral bone in a rat model of OA using multimodal imaging techniques. OA was induced in rats (OA and KGN treatment group) by anterior cruciate ligament transection (ACLT) surgery in the right knee joint. Sham surgery was performed on the right knee joint of control group rats. KGN group rats received weekly intra-articular injection of 125 μM KGN 1 week after surgery until week 12. All rats underwent in vivo magnetic resonance imaging (MRI) at 3, 6, and 12 weeks after surgery. Quantitative MR relaxation measures (T1ρ and T2 ) were determined to evaluate changes in articular cartilage. Cartilage and bone turnover markers (COMP and CTX-I) were determined at baseline, 3, 6, and 12 weeks. Animals were sacrificed at week 12 and the knee joints were removed for micro-computed tomography (micro-CT) and histology. KGN treatment significantly lowered the T1ρ and T2 relaxation times indicating decreased cartilage degradation. KGN treatment significantly decreased COMP and CTX-I levels indicating decreased cartilage and bone turnover rate. KGN treatment also prevented subchondral bone changes in the ACLT rat model of OA. Thus, kartogenin is a potential drug to prevent joint deterioration in post-traumatic OA. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1780-1789, 2016.

BACKGROUND: The small molecule drug Kartogenin is an activator of the Smad4/Smad5 pathway. It has a strong ability to promote bone differentiation and can promote the directional differentiation of bone marrow mesenchymal stem cells into osteoblasts, but its drug effect is relatively limited. OBJECTIVE: We designed and developed Kartogenin/Pluronic F127 micelles to observe its effect on the directional osteogenic differentiation of bone marrow mesenchymal stem cells. METHODS: Kartogenin was dissolved in dimethyl sulfoxide and mixed with Pluronic F127 solution. The organic solvent was removed by vacuum rotary evaporator to form a dry drug lipid membrane. PBS was added to prepare Kartogenin/Pluronic F127 micelles. The bone marrow mesenchymal stem cells of rats in the logarithmic growth phase were divided into four groups, which were treated with conventional cell culture medium (blank group), cell culture medium containing Pluronic F127 micelle solution (Pluronic F127 group), cell culture medium containing Kartogenin (drug group), and cell culture medium containing Kartogenin/Pluronic F127 micelle solution (experimental group). Among them, the drug concentration in the drug group and the experimental group was 1 μmol/L, and the micelle concentration in Pluronic F127 group was the same as that in the experimental group. Cell proliferation, apoptosis and expression of osteogenic related proteins were detected. RESULTS AND CONCLUSION: (1) Transmission electron microscope revealed that Kartogenin/Pluronic F127 micelles had irregular spherical shape and uniform particle size distribution. The particle size ranged from 32.6 nm to 118.9 nm, most of which were concentrated at 77.3 nm. (2) MTT assay showed that with the extension of culture time, the cell survival rate of the four groups decreased, but remained above 90%, and there was no significant difference in the cell survival rate among the four groups (P > 0.05). (3) Flow cytometry after 24 hours of treatment showed that there was no significant difference in the cell apoptosis rate among the four groups (P > 0.05). (4) After 24 hours of treatment, western blot assay showed that the expression levels of osteopontin, alkaline phosphatase, bone morphogenetic protein-2 and matrix metalloproteinase-2 in drug group and experimental group were higher than those in Pluronic F127 micelle group and blank group (P < 0.05). The expression levels of alkaline phosphatase, bone morphogenetic protein-2 and matrix metalloproteinase-2 in experimental group were higher than those in drug group (P < 0.05). (5) The results showed that Kartogenin/Pluronic F127 micelles could promote the osteogenic differentiation of bone marrow mesenchymal stem cells. [ABSTRACT FROM AUTHOR]

Abstract Background Regeneration of articular cartilage poses a tremendous challenge due to its limited self-repair capability and inflammation at the damaged site. To generate the desired structures that mimic the structure of native tissue, microtissues with repeated functional units such as cell aggregates have been developed. Multicellular aggregates of mesenchymal stem cells (MSCs) can be used as microscale building blocks of cartilage due to their potential for cell-cell contact, cell proliferation, and differentiation. Methods Chondrogenic microtissues were developed through incorporation of kartogenin-releasing poly (lactic-co-glycolic acid) (PLGA) microparticles (KGN-MP) within the MSC aggregates. The chondrogenic potential of KGN-MP treated MSC aggregates was proven in vitro by studying the chondrogenic markers at the RNA level and histological analysis. In order to address the inflammatory responses at the defect site, the microtissues were delivered in vivo via an injectable, anti-inflammatory hydrogel that contained gelatin methacryloyl (GelMA) loaded with curcumin (Cur). Results The KGN-MPs were fabricated to support MSCs during cartilage differentiation. According to real-time RT-PCR analysis, the presence of KGN in the aggregates led to the expression of cartilage markers by the MSCs. Both toluidine blue (TB) and safranin O (SO) staining demonstrated homogeneous glycosaminoglycan production throughout the KGN-MP incorporated MSC aggregates. The curcumin treatment efficiently reduced the expressions of hypertrophy markers by MSCs in vitro. The in vivo results showed that implantation of chondrogenic microtissues (KGN-MP incorporated MSC aggregates) using the curcumin loaded GelMA hydrogel resulted in cartilage tissue regeneration that had characteristic features close to the natural hyaline cartilage according to observational and histological results. Conclusions The use of this novel construct that contained chondrogenic cell blocks and curcumin is highly desired for cartilage regeneration. Graphical abstract

Objective To explore the effect of kartogenin (KGN) on proliferation and chondrogenic differentiation of human umbilical cord mesenchymal stem cells (hUCMSC) in vitro, and the synergistic effects of KGN and transforming growth factor (TGF)‐β3 on hUCMSC. Methods Human umbilical cord mesenchymal stem cells were isolated and cultured. Then the differentiation properties were identified by flow cytometry analysis. HUCMSC were divided into four groups: control group, KGN group, TGF‐β3 group, and TK group (with TGF‐β3 and KGN added into the medium simultaneously). Cells in all groups were induced for 21 days using the suspension ball culture method. Hematoxylin and eosin, immunofluorescence, and Alcian blue staining were used to analyze chondrogenic differentiation. Real‐time reverse transcriptase polymerase chain reaction was performed to investigate genes associated with chondrogenic differentiation. Result Hematoxylin and eosin staining showed that cells in the TGF‐β3 group and the TK group had formed cartilage‐like tissue after 21 days of culture. The results of immunofluorescence and Alcian blue staining showed that compared with the control group, cells in the KGN and TGF‐β3 groups demonstrated increased secretion of aggrecan after 21 days of culture. In addition, cells in the group combining KGN with TGF‐β3 (5.587 ± 0.27, P