Enhanced articular cartilage regeneration using costal chondrocyte-derived scaffold-free tissue engineered constructs with ascorbic acid treatment.

Background: Cartilage tissue engineering faces challenges related to the use of scaffolds and limited seed cells. This study aims to propose a cost-effective and straightforward approach using costal chondrocytes (CCs) as an alternative cell source to overcome these challenges, eliminating the need for special culture equipment or scaffolds. Methods: CCs were cultured at a high cell density with and without ascorbic acid treatment, serving as the experimental and control groups, respectively. Viability and tissue-engineered constructs (TEC) formation were evaluated until day 14. Slices of TEC samples were used for histological staining to evaluate the secretion of glycosaminoglycans and different types of collagen proteins within the extracellular matrix. mRNA sequencing and qPCR were performed to examine gene expression related to cartilage matrix secretion in the chondrocytes. In vivo experiments were conducted by implanting TECs from different groups into the defect site, followed by sample collection after 12 weeks for histological staining and scoring to evaluate the extent of cartilage regeneration. Hematoxylin-eosin (HE), Safranin-O-Fast Green, and Masson's trichrome stainings were used to examine the content of cartilage-related matrix components in the in vivo repair tissue. Immunohistochemical staining for type I and type II collagen, as well as aggrecan, was performed to assess the presence and distribution of these specific markers. Additionally, immunohistochemical staining for type X collagen was used to observe any hypertrophic changes in the repaired tissue. Results: Viability of the chondrocytes remained high throughout the culture period, and the TECs displayed an enriched extracellular matrix suitable for surgical procedures. In vitro study revealed glycosaminoglycan and type II collagen production in both groups of TEC, while the TEC matrix treated with ascorbic acid displayed greater abundance. The results of mRNA sequencing and qPCR showed that genes related to cartilage matrix secretion such as Sox9, Col2, and Acan were upregulated by ascorbic acid in costal chondrocytes. Although the addition of Asc-2P led to an increase in COL10 expression according to qPCR and RNA-seq results, the immunofluorescence staining results of the two groups of TECs exhibited similar distribution and fluorescence intensity. In vivo experiments showed that both groups of TEC could adhere to the defect sites and kept hyaline cartilage morphology until 12 weeks. TEC treated with ascorbic acid showed superior cartilage regeneration as evidenced by significantly higher ICRS and O'Driscoll scores and stronger Safranin-O and collagen staining mimicking native cartilage when compared to other groups. In addition, the immunohistochemical staining results of Collgan X indicated that, after 12 weeks, the ascorbic acid-treated TEC did not exhibit further hypertrophy upon transplantation into the defect site, but maintained an expression profile similar to untreated TECs, while slightly higher than the sham-operated group. Conclusion: These results suggest that CC-derived scaffold-free TEC presents a promising method for articular cartilage regeneration. Ascorbic acid treatment enhances outcomes by promoting cartilage matrix production. This study provides valuable insights and potential advancements in the field of cartilage tissue engineering. The translational potential of this article: Cartilage tissue engineering is an area of research with immense clinical potential. The approach presented in this article offers a cost-effective and straightforward solution, which can minimize the complexity of cell culture and scaffold fabrication. This simplification could offer several translational advantages, such as ease of use, rapid scalability, lower costs, and the potential for patient-specific clinical translation. The use of costal chondrocytes, which are easily obtainable, and the scaffold-free approach, which does not require specialized equipment or membranes, could be particularly advantageous in clinical settings, allowing for in situ regeneration of cartilage.

Tumor-derived apoptotic extracellular vesicle-mediated intercellular communication promotes metastasis and stemness of lung adenocarcinoma.

Apoptosis has long been recognized as a significant mechanism for inhibiting tumor formation, and a plethora of stimuli can induce apoptosis during the progression and treatment of tumors. Moreover, tumor-derived apoptotic extracellular vesicles (apoEVs) are inevitably phagocytosed by live tumor cells, promoting tumor heterogeneity. Understanding the mechanism by which apoEVs regulate tumor cells is imperative for enhancing our knowledge of tumor metastasis and recurrence. Herein, we conducted a series of in vivo and in vitro experiments, and we report that tumor-derived apoEVs promoted lung adenocarcinoma (LUAD) metastasis, self-renewal and chemoresistance. Mechanistically, we demonstrated that apoEVs facilitated tumor metastasis and stemness by initiating the epithelial-mesenchymal transition program and upregulating the transcription of the stem cell factor SOX2. In addition, we found that ALDH1A1, which was transported by apoEVs, activated the NF-κB signaling pathway by increasing aldehyde dehydrogenase enzyme activity in recipient tumor cells. Furthermore, targeting apoEVs-ALDH1A1 significantly abrogated these effects. Collectively, our findings elucidate a novel mechanism of apoEV-dependent intercellular communication between apoptotic tumor cells and live tumor cells that promotes the formation of cancer stem cell-like populations, and these findings reveal that apoEVs-ALDH1A1 may be a potential therapeutic target and biomarker for LUAD metastasis and recurrence.

Osteocyte mitochondria regulate angiogenesis of transcortical vessels.

Transcortical vessels (TCVs) provide effective communication between bone marrow vascular system and external circulation. Although osteocytes are in close contact with them, it is not clear whether osteocytes regulate the homeostasis of TCVs. Here, we show that osteocytes maintain the normal network of TCVs by transferring mitochondria to the endothelial cells of TCV. Partial ablation of osteocytes causes TCV regression. Inhibition of mitochondrial transfer by conditional knockout of Rhot1 in osteocytes also leads to regression of the TCV network. By contrast, acquisition of osteocyte mitochondria by endothelial cells efficiently restores endothelial dysfunction. Administration of osteocyte mitochondria resultes in acceleration of the angiogenesis and healing of the cortical bone defect. Our results provide new insights into osteocyte-TCV interactions and inspire the potential application of mitochondrial therapy for bone-related diseases.

H3K36 methyltransferase NSD1 protects against osteoarthritis through regulating chondrocyte differentiation and cartilage homeostasis.

Osteoarthritis (OA) is one of the most common joint diseases, there are no effective disease-modifying drugs, and the pathological mechanisms of OA need further investigation. Here, we show that H3K36 methylations were decreased in senescent chondrocytes and age-related osteoarthritic cartilage. Prrx1-Cre inducible H3.3K36M transgenic mice showed articular cartilage destruction and osteophyte formation. Conditional knockout Nsd1Prrx1-Cre mice, but not Nsd2Prrx1-Cre or Setd2Prrx1-Cre mice, replicated the phenotype of K36M/+; Prrx1-Cre mice. Immunostaining results showed decreased anabolic and increased catabolic activities in Nsd1Prrx1-Cre mice, along with decreased chondrogenic differentiation. Transcriptome and ChIP-seq data revealed that Osr2 was a key factor affected by Nsd1. Intra-articular delivery of Osr2 adenovirus effectively improved the homeostasis of articular cartilage in Nsd1Prrx1-Cre mice. In human osteoarthritic cartilages, both mRNA and protein levels of NSD1 and OSR2 were decreased. Our results indicate that NSD1-induced H3K36 methylations and OSR2 expression play important roles in articular cartilage homeostasis and OA. Targeting H3K36 methylation and OSR2 would be a novel strategy for OA treatment.

Costal Cartilage Graft Repair Osteochondral Defect in a Mouse Model.

OBJECTIVE: Osteochondral defects develop into osteoarthritis without intervention. Costal cartilage can be utilized as an alternative source for repairing osteochondral defect. Our previous clinical study has shown the successful osteochondral repair by costal cartilage graft with integration into host bone bed. In this study, we investigate how cartilaginous graft adapt to osteochondral environment and the mechanism of bone-cartilage interface formation. DESIGN: Costal cartilage grafting was performed in C57BL/6J mice and full-thickness osteochondral defect was made as control. 3D optical profiles and micro-CT were applied to evaluate the reconstruction of articular cartilage surface and subchondral bone as well as gait analysis to evaluate articular function. Histological staining was performed at 2, 4, and 8 weeks after surgery. Moreover, costal cartilage from transgenic mice with fluorescent markers were transplanted into wild-type mice to observe the in vivo changes of costal chondrocytes. RESULTS: At 8 weeks after surgery, 3D optical profiles and micro-CT showed that in the graft group, the articular surface and subchondral bone were well preserved. Gait analysis and International Cartilage Repair Society (ICRS) score evaluation showed a good recovery of joint function and histological repair in the graft group. Safranin O staining showed the gradual integration of graft and host tissue. Costal cartilage from transgenic mice with fluorescent markers showed that donor-derived costal chondrocytes turned into osteocytes in the subchondral area of host femur. CONCLUSION: Costal cartilage grafting shows both functional and histological repair of osteochondral defect in mice. Graft-derived costal chondrocytes differentiate into osteocytes and contribute to endochondral ossification.

SERPINB5 is a prognostic biomarker and promotes proliferation, metastasis and epithelial-mesenchymal transition (EMT) in lung adenocarcinoma.

BACKGROUND: Serine protease inhibitors clade B serpins (SERPINBs) are the largest subclass of protease inhibitors, once thought of as a tumor suppressor gene family. However, some SERPINBs exhibit functions unrelated to the inhibition of catalytic activity. METHODS: The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Gene Set Cancer Analysis (GSCA), and cBioPortal databases were utilized to investigate SERPINBs expression, prognostic correlation, and genomic variation in 33 cancer types. We also conducted a comprehensive transcriptome analysis in multiple lung adenocarcinoma (LUAD) cohorts to reveal the molecular mechanism of SERPINB5 in LUAD. Then, qPCR and immunohistochemistry were used to verify the expression and prognostic value of SERPINB5 in LUAD patients. Furthermore, knockdown and overexpression of SERPINB5 in LUAD cell lines were performed to evaluate cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). RESULTS: The expression of SERPINB5 was upregulated and demethylated in LUAD, and its abnormally high expression was significantly correlated with poor overall survival (OS). In addition, the expression of SERPINB5 was analyzed to determine its prognostic value in LUAD and confirmed that SERPINB5 was an independent predictor of LUAD in TCGA and GEO cohorts and qPCR validation with 106 clinical samples. At last, A knockdown of SERPINB5 in LUAD cells reduced proliferation, migration, and EMT. Proliferation, migration, and invasion are promoted by the overexpression of SERPINB5. CONCLUSION: Therefore, SERPINB5 has shown potential as a prognostic biomarker for LUAD, and it may become a potential therapeutic target for lung adenocarcinoma.

Intrinsic Immunogenic Tumor Cell Death Subtypes Delineate Prognosis and Responsiveness to Immunotherapy in Lung Adenocarcinoma.

Recent studies have highlighted the combination of activation of host immunogenic cell death (ICD) and tumor-directed cytotoxic strategies. However, overall multiomic analysis of the intrinsic ICD property in lung adenocarcinoma (LUAD) has not been performed. Therefore, the aim of this study was to develop an ICD-based risk scoring system to predict overall survival (OS) and immunotherapeutic efficacy in patients. In our study, both weighted gene co-expression network analysis (WGCNA) and LASSO-Cox analysis were utilized to identify ICDrisk subtypes (ICDrisk). Moreover, we identify genomic alterations and differences in biological processes, analyze the immune microenvironment, and predict the response to immunotherapy in patients with pan-cancer. Importantly, immunogenicity subgroup typing was performed based on the immune score (IS) and microenvironmental tumor neoantigens (meTNAs). Our results demonstrate that ICDrisk subtypes were identified based on 16 genes. Furthermore, high ICDrisk was proved to be a poor prognostic factor in LUAD patients and indicated poor efficacy of immune checkpoint inhibitor (ICI) treatment in patients with pan-cancer. The two ICDrisk subtypes displayed distinct clinicopathologic features, tumor-infiltrating immune cell patterns, and biological processes. The ISlowmeTNAhigh subtype showed low intratumoral heterogeneity (ITH) and immune-activated phenotypes and correlated with better survival than the other subtypes within the high ICDrisk group. This study suggests effective biomarkers for the prediction of OS in LUAD patients and immunotherapeutic response across Pan-cancer and contributes to enhancing our understanding of intrinsic immunogenic tumor cell death.

A Visible Light Driven Nickel Carbonylation Catalyst: The Synthesis of Acid Chlorides from Alkyl Halides.

We describe here the development of a visible light driven nickel carbonylation catalyst. The combination of the large bite-angle Xantphos ligand with nickel(0) generates a catalyst capable of activating alkyl halides toward carbonylation at ambient temperature in the presence of blue light irradiation, and the reductive elimination of high energy acid chloride products. Unlike classical carbonylations, where the coordination of carbon monoxide inhibits the reactivity of earth abundant nickel catalysts, a CO-associated nickel is found to be the active catalyst in the reaction. Coupling the build-up of acid chlorides with nucleophile addition can be used to access various amides, esters and thioesters, including those of sterically encumbered substrates or with metal-reactive functionalities.

Effects of Ambient Illuminance on Explicit and Implicit Altruism: The Mediation Roles of Perceived Anonymity and Satisfaction with Light.

Ambient light plays a key role in social interactions, and the effects of ambient light on explicit altruism have been widely documented. However, whether ambient light affects implicit altruism and the potential mechanisms underlying the effect remain largely unknown. The current study aimed to explore the effects of ambient illuminance on explicit and implicit altruism simultaneously, and to determine the potential mediation role of subjective mood, state self-control perceived anonymity and satisfaction with light. A one-factor (Illuminance: dim (100 lx) vs. bright (1000 lx) at eye level), between-subjects design was employed in the current study, during which seventy-eight undergraduates (52 females, 18-25 years old) were assigned to two groups, with participants in each group undergoing both the dictator game assessing explicit altruism and the implicit association test (IAT) assessing implicit altruism under one of two illuminance conditions. Meanwhile, subjective mood, state self-control, perceived anonymity and satisfaction with light were also assessed with questionnaires at the beginning or/and at the end of the experiment. Results revealed that participants tended to allocate more money in the dictator game and showed a higher state self-control, satisfaction with light and lower perceived anonymity under bright versus dim illuminance condition, whereas the performance in IAT and subjective mood revealed no statistically significant effects of illuminance. The promoting effect of bright illuminance on explicit altruism was partially mediated by perceived anonymity and satisfaction with light, but not by state self-control. These findings suggest that ambient light holds the potential to regulate psychological well-being and thus facilitate prosocial behavior, but such benefits are dependent on the type of task.

Case Report: Diagnosis of Mucopolysaccharidosis Type IVA With Compound Heterozygous Galactosamine-6 Sulfatase Variants and Biopsy of Replaced Femoral Heads.

Introduction: Mucopolysaccharidosis Type IVA (MPS IVA) or Morquio A Syndrome, is a rare metabolic disorder caused by compromised galactosamine-6 sulfatase (GALNS) encoded by GALNS gene (NM_000512.5), leading to keratin sulfate (KS), and chondroitin-6-sulfate accumulation in various organs. We present a 17-year-old woman with progressive bilateral hip pain and radiographic evidence of spondyloepiphyseal dysplasia. Methods: Diagnosis of MPS IVA was made based on whole-exome sequencing (WES) of blood samples collected from the patient and family members, high urinary glycosaminoglycan excretion, supportive clinical manifestations, radiographic examinations, including whole-body X-rays, cervical MRI, and pelvic CT. The patient underwent bilateral total hip arthroplasties sequentially, at a 1-month interval. Femoral heads were preserved for the micro-CT (µCT) analysis and the osteochondral histology examination. Results: The patient presented with multiple skeletal deformities, including vertebras and long bone deformities. WES disclosed compound heterozygous variants at exon 11 (c.1156C>T) and exon 12 (c.1288C>G) of the GALNS (NM_000512.5). The µCT analysis revealed significant bone quantity loss and microarchitectural change in both weight-bearing area (WBA) and non-weight-bearing area (NWBA) of the femoral heads, while histological analysis showed structural abnormity of articular cartilage in the WBA of the femoral heads. Conclusion: We have found compound heterozygous variants of GALNS. This is also the first study to report the microarchitectural and histological changes of both subchondral bone and articular cartilage of the femoral head in a patient with MPS IVA.

Co-culture pellet of human Wharton's jelly mesenchymal stem cells and rat costal chondrocytes as a candidate for articular cartilage regeneration: in vitro and in vivo study.

BACKGROUND: Seeding cells are key factors in cell-based cartilage tissue regeneration. Monoculture of either chondrocyte or mesenchymal stem cells has several limitations. In recent years, co-culture strategies have provided potential solutions. In this study, directly co-cultured rat costal chondrocytes (CCs) and human Wharton's jelly mesenchymal stem (hWJMSCs) cells were evaluated as a candidate to regenerate articular cartilage. METHODS: Rat CCs are directly co-cultured with hWJMSCs in a pellet model at different ratios (3:1, 1:1, 1:3) for 21 days. The monoculture pellets were used as controls. RT-qPCR, biochemical assays, histological staining and evaluations were performed to analyze the chondrogenic differentiation of each group. The 1:1 ratio co-culture pellet group together with monoculture controls were implanted into the osteochondral defects made on the femoral grooves of the rats for 4, 8, 12 weeks. Then, macroscopic and histological evaluations were performed. RESULTS: Compared to rat CCs pellet group, 3:1 and 1:1 ratio group demonstrated similar extracellular matrix production but less hypertrophy intendency. Immunochemistry staining found the consistent results. RT-PCR analysis indicated that chondrogenesis was promoted in co-cultured rat CCs, while expressions of hypertrophic genes were inhibited. However, hWJMSCs showed only slightly improved in chondrogenesis but not significantly different in hypertrophic expressions. In vivo experiments showed that all the pellets filled the defects but co-culture pellets demonstrated reduced hypertrophy, better surrounding cartilage integration and appropriate subchondral bone remodeling. CONCLUSION: Co-culture of rat CCs and hWJMSCs demonstrated stable chondrogenic phenotype and decreased hypertrophic intendency in both vitro and vivo. These results suggest this co-culture combination as a promising candidate in articular cartilage regeneration.

Anti-hypertrophic effect of synovium-derived stromal cells on costal chondrocytes promotes cartilage repairs.

BACKGROUND: Costal chondrocytes (CCs), as a promising donor cell source for cell-based therapy for cartilage repair, have strong tendency of hypertrophy and calcification, which limited CCs from further application in cartilage regenerative medicine. Synovium-derived stromal cells (SDSCs), have shown their beneficial effect for chondrocytes to maintain phenotype. This study aims to investigate whether SDSCs could help CCs to maintain chondrogenic phenotype and suppress hypertrophic differentiation in cartilage repairs. METHODS: CCs were directly cocultured with SDSCs in pellet or indirectly cocultured using a conditioned medium in vitro for 3 weeks. Cartilage matrix formation and hypertrophic differentiation of CCs were analyzed by RT-PCR, biochemical assays, and histological staining. Cocultured pellets were implanted into the osteochondral defects made on the femoral groove of the rats. Then, macroscopic and histological evaluations were performed. RESULTS: Pellets formed by CCs alone and CCs cocultured with SDSCs reveal equal cartilage matrix deposition. However, the gene expression of type X collagen was significantly downregulated in cocultured pellets. Immunohistochemistry analysis revealed suppressed expression of type X collagen in cocultured pellets, indicating SDSCs may suppress hypertrophic differentiation of chondrocytes. Further in indirect coculture experiment, SDSCs suppressed type X collagen expression as well and promoted the proliferation of CCs, indicating SDSCs may influence CCs by paracrine mechanism. The pellets implanted in the osteochondral defects showed good restoration effects, whereas the grafts constructed with CCs and SDSCs showed lower type X expression levels. CONCLUSION: These results suggest that SDSCs may maintain the phenotype of CCs and prevent the hypertrophic differentiation of CCs in cartilage repair.The Translational Potential of this Article: CCs is a promising donor cell source for cell-based therapy for cartilage repair. Based on our study, cocultured with SDSCs weakened the tendency of hypertrophy and calcification of CCs, which provide a potential usage of SDSCs in CCs-based cartilage repair therapy to suppress newly formed cartilage calcification and improve clinical outcomes.

Serum Amyloid A Correlates With the Osteonecrosis of Femoral Head by Affecting Bone Metabolism.

Osteonecrosis of femoral head (ONFH) is a progressive hip joint disease without disease-modifying treatment. Lacking understanding of the pathophysiological process of ONFH has become the humper to develop therapeutic approach. Serum amyloid A (SAA) is an acute phase lipophilic protein during inflammation and we found that SAA is increased for the first time in the serum of ONFH patients through proteomic studies and quantitatively verified by ELISA. Treating rBMSCs with SAA inhibited the osteogenic differentiation via Wnt/ß-catenin signaling pathway deactivation and enhanced the adipogenic differentiation via MAPK/PPARγ signaling pathway activation. Finally, bilateral critical-sized calvarial-defect rat model which received SAA treated rBMSCs demonstrated reduction of bone formation when compared to untreated rBMSCs implantation control. Hence, SAA is a vital protein in the physiological process of ONFH and can act as a potential therapeutic target to treat ONFH.

Synthesis of oxazoles by silver catalysed oxidative decarboxylation-cyclization of α-oxocarboxylates and isocyanides.

A silver catalysed synthesis of oxazoles by the oxidative decarboxylation-cyclization of α-oxocarboxylates and isocyanides was developed. This method provided a novel strategy to construct oxazole rings compared to traditional methods. Mechanistic investigations such as operando IR, EPR and radical inhibition experiments were carefully done and confirmed the acyl cation and Ag(II) as the intermediates in this transformation, and the involvement of a radical decarboxylative process.

Iron-Catalyzed Oxidative C-H/C-H Cross-Coupling between Electron-Rich Arenes and Alkenes.

A novel oxidative C-H/C-H cross-coupling reaction between electron-rich arenes and alkenes is established utilizing FeCl3 as the catalyst and DDQ as the oxidant. Interestingly, direct arylation products are obtained with diaryl-ethylenes and double arylation products are obtained with styrene derivatives, which show high chemoselectivity and good substrate scope. A radical trapping experiment and EPR (electron paramagnetic resonance) experiments indicate that this reaction proceeds through a radical pathway in which DDQ plays a key role in the aryl radical formation. XAFS (X-ray absorption fine structure) experiments reveal that the oxidation state of the iron catalyst does not change during the reaction, suggesting that FeCl3 might be used as a Lewis acid. Finally, a detailed mechanism is proposed for this transformation.

Relay cooperation of K2S2O8 and O2 in oxytrifluoromethylation of alkenes using CF3SO2Na.

A new radical oxytrifluoromethylation of alkenes via an aerobic Cvinyl-heteroatom bond oxygenation process is reported, in which O2 and a catalytic amount of K2S2O8 work in concert to activate CF3SO2Na. Mechanistic investigation disclosed that CF3SO2˙ could react with O2 to reinitiate radical chain process.