3D printing of recombinant collagen/chitosan methacrylate/nanoclay hydrogels loaded with Kartogenin nanoparticles for cartilage regeneration.

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.

Biomimetic kartogenin containing κ-carrageenan hydrogel for nucleus pulposus regeneration.

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.

Kartogenin Induces Chondrogenesis in Cartilage Progenitor Cells and Attenuates Cell Hypertrophy in Marrow-Derived Stromal Cells.

INTRODUCTION: Kartogenin (KGN) is a synthetic small molecule that stimulates chondrogenic cellular differentiation by activating smad-4/5 pathways. KGN has been proposed as a feasible alternative to expensive biologic growth factors, such as transforming growth factor ß, which remain under strict regulatory scrutiny when it comes to use in patients. METHOD: This study reports the previously unexplored effects of KGN stimulation on cartilage- derived mesenchymal progenitor cells (CPCs), which have been shown to be effective in applications of cell-based musculoskeletal tissue regeneration. Our findings demonstrate that KGN treatment significantly increased markers of chondrogenesis, SOX9 and COL2 following 3-10 days of treatment in human CPCs. RESULT: KGN treatment also resulted in a significant dose-dependent increase in GAG production in CPCs. The same efficacy was not observed in human marrow-derived stromal cells (BM-MSCs); however, KGN significantly reduced mRNA expression of cell hypertrophy markers, COL10 and MMP13, in BM-MSCs. Parallel to these mRNA expression results, KGN led to a significant decrease in protein levels of MMP-13 both at 0-5 days and 5-10 days following KGN treatment. CONCLUSION: In conclusion, this study demonstrates that KGN can boost the chondrogenicity of CPCs and inhibit hypertrophic terminal differentiation of BM-MSCs.

A combination of chitosan nanoparticles loaded with celecoxib and kartogenin has anti-inflammatory and chondroprotective effects: Results from an in vitro model of osteoarthritis.

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-1ß-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-1ß-pretreated chondrocytes with CNPs or K-CS NPs remarkably limited the negative effects of IL-1ß, 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).

Self-Reinforced MOF-Based Nanogel Alleviates Osteoarthritis by Long-Acting Drug Release.

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. Zn2+ 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.

Nanomedicines Promote Cartilage Regeneration in Osteoarthritis by Synergistically Enhancing Chondrogenesis of Mesenchymal Stem Cells and Regulating Inflammatory Environment.

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive erosion of the articular cartilage and inflammation. Mesenchymal stem cells' (MSCs) transplantation in OA treatment is emerging, but its clinical application is still limited by the low efficiency in oriented differentiation. In our study, to improve the therapeutic efficiencies of MSCs in OA treatment by carbonic anhydrase IX (CA9) siRNA (siCA9)-based inflammation regulation and Kartogenin (KGN)-based chondrogenic differentiation, the combination strategy of MSCs and the nanomedicine codelivering KGN and siCA9 (AHK-CaP/siCA9 NPs) was used. In vitro results demonstrated that these NPs could improve the inflammatory microenvironment through repolarization of M1 macrophages to the M2 phenotype by downregulating the expression levels of CA9 mRNA. Meanwhile, these NPs could also enhance the chondrogenesis of bone marrow-derived mesenchymal stem cells (BMSCs) by upregulating the pro-chondrogenic TGF-ß1, ACAN, and Col2α1 mRNA levels. Moreover, in an advanced OA mouse model, compared with BMSCs alone group, the lower synovitis score and OARSI score were found in the group of BMSCs plus AHK-CaP/siCA9 NPs, suggesting that this combination approach could effectively inhibit synovitis and promote cartilage regeneration in OA progression. Therefore, the synchronization of regulating the inflammatory microenvironment through macrophage reprogramming (CA9 gene silencing) and promoting MSCs oriented differentiation through a chondrogenic agent (KGN) may be a potential strategy to maximize the therapeutic efficiency of MSCs for OA treatment.

Platelet-rich plasma in orthopedics: Bridging innovation and clinical applications for bone repair.

In the burgeoning domain of orthopedic therapeutic research, Platelet-Rich Plasma (PRP) has firmly established its position, transforming paradigms ranging from tissue regeneration to the management of chondral lesions. This review delves into PRP's recent integrations with cutting-edge interventions such as 3D-printed scaffolds, its role in bone and cartilage defect management, and its enhanced efficacy when combined with molecules like Kartogenin (KGN) for fibrocartilage zone repair. Significant attention is paid to tissue engineering for meniscal interventions, where a combination of KGN, PRP, and bone marrow-derived mesenchymal stem cells are under exploration. Within the sphere of osteochondral regenerative therapy, the synergy of PRP with Bone Marrow Aspirate Concentrate (BMAC) represents a noteworthy leap towards cartilage regeneration. The innovative incorporation of PRP with biomaterials like hydroxyapatite and graphene oxide further underscores its versatility in supporting structural integrity and ensuring sustained growth factor release. However, while PRP's autologous and nontoxic nature makes it an inherently safe option, concerns arising from its preparation methods, particularly with bovine thrombin, necessitate caution. As of 2023, despite the burgeoning promise of PRP in bone healing, the quest for its standardization, optimization, and substantiation through rigorous clinical trials continues. This comprehensive review elucidates the contemporary applications, challenges, and future trajectories of PRP in orthopedics, aiming to spotlight areas primed for further research and exploration.

Nanocrystal-chitosan particles for intra-articular delivery of disease-modifying osteoarthritis drugs.

Osteoarthritis is the most common chronic joint disease and a major health care concern due to the lack of efficient treatments. This is mainly related to the local and degenerative nature of this disease. Kartogenin was recently reported as a disease-modifying osteoarthritis drug that promotes cartilage repair, but its therapeutic effect is impeded by its very low solubility. Therefore, we designed a unique nanocrystal-chitosan particle intra-articular delivery system for osteoarthritis treatment that merges the following formulation techniques: nanosize reduction of a drug by wet milling and spray drying. The intermediate formulation (kartogenin nanocrystals) increased the solubility and dissolution rates of kartogenin. The final drug delivery system consisted of an easily resuspendable and ready-to-use microsphere powder for intra-articular injection. Positively charged chitosan microspheres with a median size of approximately 10 µm acted as a mothership drug delivery system for kartogenin nanocrystals in a simulated intra-articular injection. The microspheres showed suitable stability and a controlled release profile in synovial fluid and were nontoxic in human synoviocytes. The cartilage retention skills of the microspheres were also explored ex vivo using cartilage. This drug delivery system shows promise for advancement to preclinical stages in osteoarthritis therapy and scale-up production.

Reconstruction of tracheal window-shape defect by 3D printed polycaprolatone scaffold coated with Silk Fibroin Methacryloyl.

In this study, we aimed to utilize autologous tracheal epithelia and BMSCs as the seeding cells, utilize PCL coated with SilMA as the hybrid scaffold to carry the cells and KGN, which can selectively stimulate chondrogenic differentiation of BMSCs. This hybrid tracheal substitution was carried out to repair the tracheal partial window-shape defect. Firstly, SilMA with the concentration of 10%, 15% and 20% was prepared, and the experiment of swelling and degradation was performed. With the increase of the concentration, the swelling ratio of SilMA decreased, and the degradation progress slowed down. Upon the result of CCK-8 test and HE staining of 3D co-culture, the SilMA with concentration of 20% was selected. Next, SilMA and the cells attached to SilMA were characterized by SEM. Furthermore, in vitro cytotoxicity test shows that 20% SilMA has good cytocompatibility. The hybrid scaffold was then made by PCL coated with 20% SilMA. The mechanical test shows this hybrid scaffold has better biomechanical properties than native trachea. In vivo tracheal defect repair assays were conducted to evaluate the effect of the hybrid substitution. H&E staining, IHC staining and IF staining showed that this hybrid substitution ensured the viability, proliferation and migration of epithelium. However, it is sad that the results of chondrogenesis were not obvious. This study is expected to provide new strategies for the fields of tracheal replacement therapy needing mechanical properties and epithelization.

Biomimetic Injectable Hydrogel Based on Methacrylate-Modified Silk Fibroin Embedded with Kartogenin for Superficial Cartilage Regeneration.

The weak regeneration ability of chondrocytes is one of the main reasons that limit the therapeutic effect of clinical cartilage injury. Injectable hydrogels are potential scaffolds for cartilage tissue engineering with advantages such as minimally invasive surgery, porous structure, and drug sustained-release ability. At present, many biomaterials have been developed for the repair of deep cartilage defects. However, cartilage injury often begins on the surface, which requires us to propose a treatment strategy suitable for superficial cartilage injury repair. In this study, we fabricated a biomimetic injectable hydrogel based on methacrylate-modified silk fibroin (SilMA) embedded with kartogenin (KGN). The SilMA/KGN hydrogels have good biohistocompatibility and the ability to promote cartilage differentiation. In addition, SEM results show that it has a porous structure conducive to cell adhesion and proliferation. Most importantly, it has demonstrated remarkable superficial cartilage repair ability in vivo, showing potential in cartilage tissue engineering.

Kartogenin-loaded polyvinyl alcohol/nano-hydroxyapatite composite hydrogel promotes tendon-bone healing in rabbits after anterior cruciate ligament reconstruction.

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.

In Situ Deposition of Drug and Gene Nanoparticles on a Patterned Supramolecular Hydrogel to Construct a Directionally Osteochondral Plug.

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-3ß/ß-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.

Kartogenin potentially protects temporomandibular joints from collagenase-induced osteoarthritis via core binding factor ß and runt-related transcription factor 1 binding - A rat model study.

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 ß (CBFß) 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 CBFß-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-1ß (IL-1ß), 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 < .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-1ß (P < .01). Ro5-3335 did not aggravate the pathology of TMJOA but neutralized the therapeutic effects of kartogenin on TMJOA. Conclusion: Kartogenin has a potential therapeutic effect on TMJOA via promoting the CBFß-RUNX1 binding.

Kartogenin delivery systems for biomedical therapeutics and regenerative medicine.

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.

Kartogenin-loaded liposomes coated with alkylated chondroitin sulfate for cartilage repair.

Cartilage loss is a common clinical problem, which leads to significant pain, dysfunction, and even disability. As a result, there is growing interest in using small, non-protein molecules to protect or repair cartilage. Kartogenin (KGN), a small hydrophobic molecule, shows chondroprotective and chondrogenic properties. In this study, we embedded KGN in liposomes, and the whole system was stabilized by covering it with n-octadecylated (at two different substitution degrees) chondroitin sulfate (CS) derivatives. We investigated the interactions of empty liposomes and KGN-loaded liposomes with both CS derivatives using various physicochemical techniques, which revealed that hydrophobically modified CSs can interact with both neutral lipid membrane and negatively charged loaded-KGN lipid membrane. The cytotoxicity and chondrogenic properties of the polysaccharides and liposome-CS formulations of KGN were analyzed towards mesenchymal stem cells (MSCs). The results showed that the alkylated CS exhibited cytotoxic properties. The higher substituted CS self-assembles into stable nanoaggregates that can form a corona on the surface of liposomes, eliminating the overall cytotoxicity of this polymer. However, all tested chondrogenic markers' expression levels are enhanced for KGN-loaded liposomes and coated by lower substituted CS. Furthermore, the undesirable hypertrophy effect for this formulation significantly decreased compared to pure polymeric derivative.

Incorporation of kartogenin and silk fibroin scaffolds promotes rat articular cartilage regeneration through enhancement of antioxidant functions.

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.

Kartogenin-enhanced dynamic hydrogel ameliorates intervertebral disc degeneration via restoration of local redox homeostasis.

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)-1ß, 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 â€‹µM) in the presence of IL-1ß. 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-1ß-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.

Cytomodulin-10 modified GelMA hydrogel with kartogenin for in-situ osteochondral regeneration.

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-ß. However, the high cost, instability, and immunogenicity of TGF-ß would limit its combined effect with KGN in clinical application. In this study, a composite hydrogel CM-KGN@GelMA, which contained TGF-ß1 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-ß1, 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-ß1 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. STATEMENT OF SIGNIFICANCE: Kartogenin and TGF-ß 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-ß1 mimicking peptide CM-10 and KGN. CM-10 in hydrogel retained an activity like TGF-ß1 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.

[Effect of Kartogenin combined with adipose-derived stem cells on tendon-bone healing after anterior cruciate ligament reconstruction].

Objective: To investigate the effect of Kartogenin (KGN) combined with adipose-derived stem cells (ADSCs) on tendon-bone healing after anterior cruciate ligament (ACL) reconstruction in rabbits. Methods: After the primary ADSCs were cultured by passaging, the 3rd generation cells were cultured with 10 µmol/L KGN solution for 72 hours. The supernatant of KGN-ADSCs was harvested and mixed with fibrin glue at a ratio of 1∶1; the 3rd generation ADSCs were mixed with fibrin glue as a control. Eighty adult New Zealand white rabbits were taken and randomly divided into 4 groups: saline group (group A), ADSCs group (group B), KGN-ADSCs group (group C), and sham-operated group (group D). After the ACL reconstruction model was prepared in groups A-C, the saline, the mixture of ADSCs and fibrin glue, and the mixture of supernatant of KGN-ADSCs and fibrin glue were injected into the tendon-bone interface and tendon gap, respectively. ACL was only exposed without other treatment in group D. The general conditions of the animals were observed after operation. At 6 and 12 weeks, the tendon-bone interface tissues and ACL specimens were taken and the tendon-bone healing was observed by HE staining, c-Jun N-terminal kinase (JNK) immunohistochemical staining, and TUNEL apoptosis assay. The fibroblasts were counted, and the positive expression rate of JNK protein and apoptosis index (AI) were measured. At the same time point, the tensile strength test was performed to measure the maximum load and the maximum tensile distance to observe the biomechanical properties. Results: Twenty-eight rabbits were excluded from the study due to incision infection or death, and finally 12, 12, 12, and 16 rabbits in groups A-D were included in the study, respectively. After operation, the tendon-bone interface of groups A and B healed poorly, while group C healed well. At 6 and 12 weeks, the number of fibroblasts and positive expression rate of JNK protein in group C were significantly higher than those of groups A, B, and D (P<0.05). Compared with 6 weeks, the number of fibroblasts gradually decreased and the positive expression rate of JNK protein and AI decreased in group C at 12 weeks after operation, with significant differences (P<0.05). Biomechanical tests showed that the maximum loads at 6 and 12 weeks after operation in group C were higher than in groups A and B, but lower than those in group D, while the maximum tensile distance results were opposite, but the differences between groups were significant (P<0.05). Conclusion: After ACL reconstruction, local injection of a mixture of KGN-ADSCs and fibrin glue can promote the tendon-bone healing and enhance the mechanical strength and tensile resistance of the tendon-bone interface.

Fabrication of Injectable Kartogenin-Conjugated Composite Hydrogel with a Sustained Drug Release for Cartilage Repair.

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.