Dedifferentiation Of Chondrocytes Research Articles

Cell‐preserving scheme for mechanobiological research on dedifferentiation of chondrocytes

AbstractIn the present work, we introduce a scheme for efficient mechanobiological research on the dedifferentiation of chondrocytes (CHs) using a deep neural network (DNN) model that does not require sacrificing the cells and thus, saving resources. Cells are a part of active biological systems subjected to physical stimuli such as mechanical loading. Such loading affects cellular processes including proliferation, differentiation, and the interplay with the surrounding environment. CHs are mechanosensitive cells that produce and maintain the cartilaginous matrix that allows cartilage to bear and distribute mechanical loads in joints; their role in cartilage regeneration and treatment of osteoarthritis (OA) has been the focus of research projects. Nevertheless, it remains an open challenge to fully understand the effect of mechanical stimuli on CHs. One of the unresolved mechanobiological behaviors of CHs is dedifferentiation. Dedifferentiation of CHs is the phenomenon where isolated CHs alter their phenotype when cultured in a 2D in vitro environment over passages. While intact, cobblestone‐shaped CHs mainly produce collagen II, dedifferentiated CHs with elongated shapes produce fibroblastic collagen I. This limits the scalability of a promising treatment for OA, called ‘autologous chondrocyte implantation (ACI)’. To overcome this limit, research is being carried out to understand the dedifferentiation mechanism in detail. This proposed scheme is composed of three parts: (i) the cell‐seeded specimens are loaded in a bioreactor system, (ii) an optical microscope is used to obtain the phase‐contrast images of living cells, and (iii) the images are analyzed using a DNN. This scheme provides an efficient tool to analyze the ratio of intact to dedifferentiated CHs while preserving cells on our way to elucidate the effects of mechanical loading on the dedifferentiation of CHs.

Targeting Fascin1 maintains chondrocytes phenotype and attenuates osteoarthritis development

Osteoarthritis (OA) is the most common form of arthritic disease, and phenotypic modification of chondrocytes is an important mechanism that contributes to the loss of cartilage homeostasis. This study identified that Fascin actin-bundling protein 1 (FSCN1) plays a pivotal role in regulating chondrocytes phenotype and maintaining cartilage homeostasis. Proteome-wide screening revealed markedly upregulated FSCN1 protein expression in human OA cartilage. FSCN1 accumulation was confirmed in the superficial layer of OA cartilage from humans and mice, primarily in dedifferentiated-like chondrocytes, associated with enhanced actin stress fiber formation and upregulated type I and III collagens. FSCN1-inducible knockout mice exhibited delayed cartilage degeneration following experimental OA surgery. Mechanistically, FSCN1 promoted actin polymerization and disrupted the inhibition of Decorin on TGF-β1, leading to excessive TGF-β1 production and ALK1/Smad1/5 signaling activation, thus, accelerated chondrocyte dedifferentiation. Intra-articular injection of FSCN1-overexpressing adeno-associated virus exacerbated OA progression in mice, which was mitigated by an ALK1 inhibitor. Moreover, FSCN1 inhibitor NP-G2-044 effectively reduced extracellular matrix degradation in OA mice, cultured human OA chondrocytes, and cartilage explants by suppressing ALK1/Smad1/5 signaling. These findings suggest that targeting FSCN1 represents a promising therapeutic approach for OA.

Selenium deficiency and T-2 toxin trigger ferroptosis in cartilage from Kashin-Beck diseases

Kashin-Beck disease (KBD) is a prevalent, endemic, and degenerative cartilage injury disorder, characterized by high rates of teratogenicity and disability. The etiology and pathogenesis of KBD are not fully understood, although research suggests that selenium deficiency and exposure to T-2 toxin are significant environmental risk factors. The initial pathological changes of KBD manifest as necrosis of deep chondrocytes, dedifferentiation of chondrocytes, excessive apoptosis of chondrocytes, and subsequent disruption of extracellular matrix metabolism. However, the precise pathogenic mechanisms of chondrocyte damage in KBD remain incompletely understood. Ferroptosis is auniqueform of programmed cell death triggered byiron-dependent lipid peroxide accumulation. It has been shown to contribute to cartilage damage and chondrocyte death in various osteoarticular conditions, particularly osteoarthritis (OA). Notably, KBD not only exhibits clinical and pathologicalsimilarities with OA, but also indicates a potential association with ferroptosis in morphological and molecular similarities. Additionally, the environmental risk factors T-2 toxin exposure and selenium deficiency are also significant contributors toferroptosis. Consequently, it is plausible to postulate that environmental risk factors may trigger ferroptosis, leading to the initiation of cartilage damage in KBD. Our hypothesis can be verified through both in vitro and in vivo experiments. Chondrocyte injury induced by ferroptosis may be a novel finding in KBD, which is important for clarifying its etiology and developing effective therapeutic strategies.

Arthroscopic Shaver-based Harvest of Minced Cartilage Results in Reduced Chondrocyte Viability and Reduced Quality of Cartilaginous Repair Tissue Compared With Open Harvest and Conventional Fragmentation

To characterize and compare the quality of regenerative cartilage tissue (ReCT) after conventional minced cartilage (CMC) and arthroscopic minced cartilage (AMC), in terms of cell viability, gene expression, and matrix synthesis and to investigate the influence of different shaver types. Chondral tissue was harvested from the knees of 8 porcine donors. Porcine specimens were euthanized one day before harvest. AMC was created with 2 shaver blades in 2 operating modes (oscillating vs forward) and compared with a scalpel-fragmented CMC control. Before histologic analysis, 50% of the tissue was digested to prevent dedifferentiation of chondrocytes to fibroblasts. Cells were cultured and analyzed for cell viability, gene expression of cartilage-specific markers (aggrecan [ACAN], collagen type II, alpha1 [COL2A1], collagen type I, alpha1 [COL1A1], fibronectin-1 [FN1]), and matrix synthesis (Alcian-blue). AMC tissue contained fewer viable chondrocytes (41%-54% vs 91%; P= .001-.048) compared with CMC. After culture, CMC showed greater expressions of ACAN (27 virtual copy numbers [VCN]/housekeeping gene) and COL2A1 (30 VCN) compared with AMC (ACAN 2-9 VCN, COL2A1 2-7 VCN, P= .001-.039). AMC presented greater expressions of COL1A1 (9-21 VCN) and FN1 (12-17 VCN) than CMC (1 and 6 VCN, P= .001-.050). The signal intensity of the cartilage matrix formed by CMC (86/mm2) was greater than by AMC (7-10 mm2, P= .001-.032). CMC contained high numbers of viable chondrocytes, resulting in high-quality, hyaline-like ReCT. In contrast, AMC showed impaired chondrocyte quantity and viability, showing greater expressions of fibroblast markers and a decreased formation of mature cartilage matrix in porcine samples. The high chondrogenic potential of CMC to form hyaline-like ReCT was not confirmed for AMC. On the basis of our findings, arthroscopic harvest of minced cartilage leads to reduced chondrocyte viability and ReCT quality. Accordingly, CMC and AMC cannot be regarded as synonymous techniques, as arthroscopic techniques seem to be less efficacious.

Latest Advances in Chondrocyte-Based Cartilage Repair.

Chondrocyte-based cell therapy has been used for more than 30 years and is still considered to be a promising method of cartilage repair despite some limitations. This review introduces the latest developments of four generations of autologous chondrocyte implantation and current autologous chondrocyte products. The regeneration of cartilage from adult chondrocytes is limited by culture-induced dedifferentiation and patient age. Cartibeads is an innovative three-step method to produce high-quality hyaline cartilage microtissues, and it is developed from adult dedifferentiated chondrocytes with a high number of cell passages. In addition, allogeneic chondrocyte therapies using the Quantum hollow-fiber bioreactor and several signaling pathways involved in chondrocyte-based cartilage repair are mentioned, such as WNT signaling, the BMP-2/WISP1 pathway, and the FGF19 pathway.

Single-cell RNA sequencing reveals different chondrocyte states in femoral cartilage between osteoarthritis and healthy individuals.

Cartilage injury is the main pathological manifestation of osteoarthritis (OA). Healthy chondrocyte is a prerequisite for cartilage regeneration and repair. Differences between healthy and OA chondrocyte types and the role these types play in cartilage regeneration and OA progression are unclear. This study conducted single-cell RNA sequencing (scRNA-seq) on the cartilage from normal distal femur of the knee (NC group) and OA femur (OA group) cartilage, the chondrocyte atlas was constructed, and the differences of cell subtypes between the two groups were compared. Pseudo-time and RNA velocity analysis were both performed to verify the possible differentiation sequence of cell subtypes. GO and KEGG pathway enrichment analysis were used to explore the potential functional characteristics of each cell subtype, and to predict the functional changes during cell differentiation. Differences in transcriptional regulation in subtypes were explored by single-cell regulatory network inference and clustering (SCENIC). The distribution of each cell subtype in cartilage tissue was identified by immunohistochemical staining (IHC). A total of 75,104 cells were included, they were divided into 19 clusters and annotated as 11 chondrocyte subtypes, including two new chondrocyte subtypes: METRNL+ and PRG4+ subtype. METRNL+ is in an early stage during chondrocyte differentiation, and RegC-B is in an intermediate state before chondrocyte dedifferentiation. With cell differentiation, cell subtypes shift from genetic expression to extracellular matrix adhesion and collagen remodeling, and signal pathways shift from HIF-1 to Hippo. The 11 subtypes were finally classified as intrinsic chondrocytes, effector chondrocytes, abnormally differentiated chondrocytes and dedifferentiated chondrocytes. IHC was used to verify the presence and distribution of each chondrocyte subtype. This study screened two new chondrocyte subtypes, and a novel classification of each subtype was proposed. METRNL+ subtype is in an early stage during chondrocyte differentiation, and its transcriptomic characteristics and specific pathways provide a foundation for cartilage regeneration. EC-B, PRG4+ RegC-B, and FC are typical subtypes in the OA group, and the HippO-Taz pathway enriched by these cell subtypes may play a role in cartilage repair and OA progression. RegC-B is in the intermediate state before chondrocyte dedifferentiation, and its transcriptomic characteristics may provide a theoretical basis for intervening chondrocyte dedifferentiation.

Targeting F-actin stress fibers to suppress the dedifferentiated phenotype in chondrocytes

Actin is a central mediator of the chondrocyte phenotype. Monolayer expansion of articular chondrocytes on tissue culture polystyrene, for cell-based repair therapies, leads to chondrocyte dedifferentiation. During dedifferentiation, chondrocytes spread and filamentous (F-)actin reorganizes from a cortical to a stress fiber arrangement causing a reduction in cartilage matrix expression and an increase in fibroblastic matrix and contractile molecule expression. While the downstream mechanisms regulating chondrocyte molecular expression by alterations in F-actin organization have become elucidated, the critical upstream regulators of F-actin networks in chondrocytes are not completely known. Tropomyosin (TPM) and the RhoGTPases are known regulators of F-actin networks. The main purpose of this study is to elucidate the regulation of passaged chondrocyte F-actin stress fiber networks and cell phenotype by the specific TPM, TPM3.1, and the RhoGTPase, CDC42. Our results demonstrated that TPM3.1 associates with cortical F-actin and stress fiber F-actin in primary and passaged chondrocytes, respectively. In passaged cells, we found that pharmacological TPM3.1 inhibition or siRNA knockdown causes F-actin reorganization from stress fibers back to cortical F-actin and causes an increase in G/F-actin. CDC42 inhibition also causes formation of cortical F-actin. However, pharmacological CDC42 inhibition, but not TPM3.1 inhibition, leads to the re-association of TPM3.1 with cortical F-actin. Both TPM3.1 and CDC42 inhibition, as well as TPM3.1 knockdown, reduces nuclear localization of myocardin related transcription factor, which suppresses dedifferentiated molecule expression. We confirmed that TPM3.1 or CDC42 inhibition partially redifferentiates passaged cells by reducing fibroblast matrix and contractile expression, and increasing chondrogenic SOX9 expression. A further understanding on the regulation of F-actin in passaged cells may lead into new insights to stimulate cartilage matrix expression in cells for regenerative therapies.

LGR5 Modulates Differentiated Phenotypes of Chondrocytes Through PI3K/AKT Signaling Pathway.

Tissue engineering is increasingly viewed as a promising avenue for functional cartilage reconstruction. However, chondrocyte dedifferentiation during in vitro culture remains an obstacle for clinical translation of tissue engineered cartilage. Re-differentiated induction have been employed to induce dedifferentiated chondrocytes back to their original phenotype. Regrettably, these strategies have been proven to be only moderately effective. To explore underlying mechanism, RNA transcriptome sequencing was conducted on primary chondrocytes (P0), dedifferentiated chondrocytes (P5), and redifferentiated chondrocytes (redifferentiation-induction of P5, P5.R). Based on multiple bioinformatics analysis, LGR5 was identified as a target gene. Subsequently, stable cell lines with LGR5 knocking-down and overexpression were established using P0 chondrocytes. The phenotypic changes in P1 and P5 chondrocytes with either LGR5 knockdown or overexpression were assessed to ascertain the potential influence of LGR5 dysregulation on chondrocyte phenotypes. Regulatory mechanism was then investigated using bioinformatic analysis, protein-protein docking, immunofluorescence co-localization and immunoprecipitation. The current study found that dysregulation of LGR5 can significantly impact the dedifferentiated phenotypes of chondrocytes (P5). Upregulation of LGR5 appears to activate the PI3K/AKT signal via increasing the phosphorylation levels of AKT (p-AKT1). Moreover, the increase of p-AKT1 may stabilize β-catenin and enhance the intensity of Wnt/β-catenin signal, and help to restore the dedifferentated phenotype of chondrocytes. LGR5 can modulate the phenotypes of chondrocytes in P5 passage through PI3K/AKT signaling pathway.

Collagen and Alginate Hydrogels Support Chondrocytes Redifferentiation In Vitro without Supplementation of Exogenous Growth Factors.

Focal cartilage defects are a prevalent knee problem affecting people of all ages. Articular cartilage (AC) possesses limited healing potential, and osteochondral defects can lead to pain and long-term complications such as osteoarthritis. Autologous chondrocyte implantation (ACI) has been a successful surgical approach for repairing osteochondral defects over the past two decades. However, a major drawback of ACI is the dedifferentiation of chondrocytes during their in vitro expansion. In this study, we isolated ovine chondrocytes and cultured them in a two-dimensional environment for ACI procedures. We hypothesized that 3D scaffolds would support the cells' redifferentiation without the need for growth factors so we encapsulated them into soft collagen and alginate (col/alg) hydrogels. Chondrocytes embedded into the hydrogels were viable and proliferated. After 7 days, they regained their original rounded morphology (aspect ratio 1.08) and started to aggregate. Gene expression studies showed an upregulation of COL2A1, FOXO3A, FOXO1, ACAN, and COL6A1 (37, 1.13, 22, 1123, and 1.08-fold change expression, respectively) as early as day one. At 21 days, chondrocytes had extensively colonized the hydrogel, forming large cell clusters. They started to replace the degrading scaffold by depositing collagen II and aggrecan, but with limited collagen type I deposition. This approach allows us to overcome the limitations of current approaches such as the dedifferentiation occurring in 2D in vitro expansion and the necrotic formation in spheroids. Further studies are warranted to assess long-term ECM deposition and integration with native cartilage. Though limitations exist, this study suggests a promising avenue for cartilage repair with col/alg hydrogel scaffolds.

Redifferentiation of genetically modified dedifferentiated chondrocytes in a microcavitary hydrogel.

We genetically modified dedifferentiated chondrocytes (DCs) using lentiviral vectors and adenoviral vectors encoding TGF-β3 (referred to as transgenic groups below) and encapsulated these DCs in the microcavitary hydrogel and investigated the combinational effect on redifferentiation of the genetically manipulated DCs. The Cell Counting Kit-8 data indicated that both transgenic groups exhibited significantly higher cell viability in the first week but inferior cell viability in the subsequent timepoints compared with those of the control group. Real-time polymerase chain reaction and western blot analysis results demonstrated that both transgenic groups had a better effect on redifferentiation to some extent, as evidenced by higher expression levels of chondrogenic genes, suggesting the validity of combination with transgenic DCs and the microcavitary hydrogel on redifferentiation. Although transgenic DCs with adenoviral vectors presented a superior extent of redifferentiation, they also expressed greater levels of the hypertrophic gene type X collagen. It is still worth further exploring how to deliver TGF-β3 more efficiently and optimizing the appropriate parameters, including concentration and duration. The results demonstrated the better redifferentiation effect of DCs with the combinational use of transgenic TGF-β3 and a microcavitary alginate hydrogel and implied that DCs would be alternative seed cells for cartilage tissue engineering due to their easily achieved sufficient cell amounts through multiple passages and great potential to redifferentiate to produce cartilaginous extracellular matrix.

Effect of Passaging on Bovine Chondrocyte Gene Expression and Engineered Cartilage Production.

Tissue engineering strategies show great potential for repairing osteochondral defects in osteoarthritic joints; however, these approaches often rely on passaging cells multiple times to obtain enough cells to produce functional tissue. Unfortunately, monolayer expansion culture causes chondrocyte dedifferentiation, which is accompanied by a phenotypical and morphological shift in chondrocyte properties that leads to a reduction in the quality of de novo cartilage produced. Thus, the objective of this study was to evaluate transcriptional variations during in vitro expansion culture and determine how differences in cell phenotype from monolayer expansion alter development of functional engineered cartilage. We used an unbiased approach to explore genome-wide transcriptional differences in chondrocyte phenotype at passage 1 (P1), P3, and P5, and then seeded cells into hydrogel scaffolds at P3 and P5 to assess cells' abilities to produce cartilaginous extracellular matrix in three dimensional (3D). We identified distinct phenotypic differences, specifically for genes related to extracellular organization and cartilage development. Both P3 and P5 chondrocytes were able to produce chondrogenic tissue in 3D, with P3 cells producing matrix with greater compressive properties and P5 cells secreting matrix with higher glycosaminoglycan/DNA and collagen/DNA ratios. Furthermore, we identified 24 genes that were differentially expressed with passaging and enriched in human osteoarthritis (OA) genome-wide association studies, thereby prioritizing them as functionally relevant targets to improve protocols that recapitulate functional healthy cartilage with cells from adult donors. Specifically, we identified novel genes, such as TMEM190 and RAB11FIP4, which were enriched with human hip OA and may play a role in chondrocyte dedifferentiation. This work lays the foundation for several pathways and genes that could be modulated to enhance the efficacy for chondrocyte culture for tissue regeneration, which could have transformative impacts for cell-based cartilage repair strategies.

Novel insight on IRE1 in the regulation of chondrocyte dedifferentiation through ER stress independent pathway.

Inositol-requiring enzyme-1 (IRE1) is the master regulator of the unfolded protein response pathway, associated with the endoplasmic reticulum (ER) in sensing and regulating cell stress. The activity of IRE1 is highly explored and well-characterized in cancer and other cells. However, the IRE1 molecular mechanism in chondrocytes is poorly understood. The present study explored the effect of IRE1 on chondrocytes regarding its chondrogenic gene expression and its correlation with different cellular pathways and cell behavior. Chondrocytes transfected with the cDNA of IRE1 reduced the expression of type II collagen, disrupting chondrocyte differentiation as confirmed by western blotting and immunofluorescence. Upon siRNA treatment, the influence of IRE1 on chondrocyte differentiation is restored by reviving the normal expression of type II collagen. Different molecular pathways were explored to investigate the role of IRE1 in causing chondrocyte dedifferentiation. However, we found no significant correlation, as IRE1 induces dedifferentiation through independent pathways. In response to various endoplasmic reticulum (ER) agonists (2-deoxy-D-glucose), and ER stress antagonists (tauroursodeoxycholic acid and salubrinal), IRE1 overexpression did not affect GRP78/94, as implicated in the pathogenesis of ER stress. Moreover, when IRE1 overexpression was correlated with the inflammation pathway, nuclear factor-kappa B (NFκB), IRE1 substantially increased the expression of p50 while decreasing the expression of nuclear factor kappa light polypeptide alpha (IκBα). These results suggest that IRE1 induces dedifferentiation in chondrocytes by modulating inflammatory pathways that cause dedifferentiation by disrupting type II collagen expression.

Epigenetic Priming Enhances Chondrogenic Potential of Expanded Chondrocytes.

Expansion of chondrocytes presents a major obstacle in the cartilage regeneration procedure, such as matrix-induced autologous chondrocyte implantation. Dedifferentiation of chondrocytes during the expansion process leads to the emergence of a fibrotic (chondrofibrotic) phenotype that decreases the chondrogenic potential of the implanted cells. We aim to (1) determine the extent that chromatin architecture of H3K27me3 and H3K9me3 remodels during dedifferentiation and persists after the transfer to a three-dimensional (3D) culture; and (2) to prevent this persistent remodeling to enhance the chondrogenic potential of expanded bovine chondrocytes, used as a model system. Chromatin architecture remodeling of H3K27me3 and H3K9me3 was observed at 0 population doublings, 8 population doublings, and 16 population doublings (PD16) in a two-dimensional (2D) culture and after encapsulation of the expanded chondrocytes in a 3D hydrogel culture. Chondrocytes were treated with inhibitors of epigenetic modifiers (epigenetic priming) for PD16 and then encapsulated in 3D hydrogels. Chromatin architecture of chondrocytes and gene expression were evaluated before and after encapsulation. We observed a change in chromatin architecture of epigenetic modifications H3K27me3 and H3K9me3 during chondrocyte dedifferentiation. Although inhibiting enzymes that modify H3K27me3 and H3K9me3 did not alter the dedifferentiation process in 2D culture, applying these treatments during the 2D expansion did increase the expression of select chondrogenic genes and protein deposition of type II collagen when transferred to a 3D environment. Overall, we found that epigenetic priming of expanded bovine chondrocytes alters the cell fate when chondrocytes are later encapsulated into a 3D environment, providing a potential method to enhance the success of cartilage regeneration procedures.

Assessing the advantages of 3D bioprinting and 3D spheroids in deciphering the osteoarthritis healing mechanism using human chondrocytes and polarized macrophages

The molecular niche of an osteoarthritic microenvironment comprises the native chondrocytes, the circulatory immune cells, and their respective inflammatory mediators. Although M2 macrophages infiltrate the joint tissue during osteoarthritis (OA) to initiate cartilage repair, the mechanistic crosstalk that dwells underneath is still unknown. Our study established a co-culture system of human OA chondrocytes and M2 macrophages in 3D spheroids and 3D bioprinted silk-gelatin constructs. It is already well established that Silk fibroin-gelatin bioink supports chondrogenic differentiation due to upregulation of the Wnt/β-catenin pathway. Additionally, the presence of anti-inflammatory M2 macrophages significantly upregulated the expression of chondrogenic biomarkers (COL-II, ACAN) with an attenuated expression of the chondrocyte hypertrophy (COL-X), chondrocyte dedifferentiation (COL-I) and matrix catabolism (MMP-1 and MMP-13) genes even in the absence of the interleukins. Furthermore, the 3D bioprinted co-culture model displayed an upper hand in stimulating cartilage regeneration and OA inhibition than the spheroid model, underlining the role of silk fibroin-gelatin in encouraging chondrogenesis. Additionally, the 3D bioprinted silk-gelatin constructs further supported the maintenance of stable anti-inflammatory phenotype of M2 macrophage. Thus, the direct interaction between the primary OAC and M2 macrophages in the 3D context, along with the release of the soluble anti-inflammatory factors by the M2 cells, significantly contributed to a better understanding of the molecular mechanisms responsible for immune cell-mediated OA healing.

Impact of Different Culture Media on Proliferation and Dedifferentiation of Fatal Chondrocytes

Impact of Different Culture Media on Proliferation and Dedifferentiation of Fatal Chondrocytes

Priming chondrocytes during expansion alters cell behavior and improves matrix production in 3D culture

ObjectiveCartilage tissue engineering strategies that use autologous chondrocytes require in vitro expansion of cells to obtain enough cells to produce functional engineered tissue. However, chondrocytes dedifferentiate during expansion culture, limiting their ability to produce chondrogenic tissue and their utility for cell-based cartilage repair strategies. The current study identified conditions that favor cartilage production and the mechanobiological mechanisms responsible for these benefits. DesignChondrocytes were isolated from juvenile bovine knee joints and cultured with (primed) or without (unprimed) a growth factor cocktail. Gene expression, cell morphology, cell adhesion, cytoskeletal protein distribution, and cell mechanics were assessed. Following passage 5, cells were embedded into agarose hydrogels to evaluate functional properties of engineered cartilage. ResultsPriming cells during expansion culture altered cell phenotype and chondrogenic tissue production. Unbiased RNA-sequencing analysis suggested, and experimental studies confirmed, that growth factor priming delays dedifferentiation associated changes in cell adhesion and cytoskeletal organization. Priming also overrode mechanobiological pathways to prevent chondrocytes from remodeling their cytoskeleton to accommodate the stiff, monolayer microenvironment. Passage 1 primed cells deformed less and had lower YAP1 activity than unprimed cells. Differences in cell adhesion, morphology, and cell mechanics between primed and unprimed cells were mitigated by passage 5. ConclusionsPriming suppresses mechanobiologic cytoskeletal remodeling to prevent chondrocyte dedifferentiation, resulting in more cartilage-like tissue-engineered constructs

Evaluation of chondrocyte dedifferentiation mechanisms using confocal Raman microscopy

Progression to metastases remains the overriding cause of cancer-associated mortality. Metastatic cancer is not amenable to surgery and its treatment is further complicated by the development of therapy resistance often observed at advanced cancer stages. Early detection of metastases is therefore critical but has been limited by the lack of probes that can effectively localize them. Similar challenges persist with therapeutics specifically targeting metastasized cancer cells. Thus, agents that specifically target disseminating tumor cells at an early stage could produce new theranostic applications and be transformative for the survival of patients with advanced cancers. Recent studies have described new approaches for early detection and targeted eradication of metastatic cancer. Here we summarize the results from preclinical validation of the experimental probes reported to date.

Tanshinone IIA Facilitates Efficient Cartilage Regeneration under Inflammatory Factors Caused Stress via Upregulating LncRNA NEAT1_2.

(1) Background: Osteoarthritis (OA) is a crippling condition characterized by chondrocyte dedifferentiation, cartilage degradation, and subsequent cartilage defects. Unfortunately, there is a lack of effective medicines to facilitate the repair of cartilage defects in OA patients. In this study, we investigated the role of lncRNA NEAT1_2 in maintaining the chondrocyte phenotype and identified tanshinone IIA(TAN) as a natural medicine that enhances NEAT1_2 levels, resulting in efficient cartilage regeneration under inflammatory cytokines. (2) Methods: The transcriptional levels of NEAT1_2 and cartilage phenotype-related genes were identified by RT-qPCR. The siRNA interference approach was utilized to silence NEAT1_2; the Alamar Blue assay was performed to determine chondrocyte viability under inflammatory conditions. To evaluate the concentrations of collagen type II and glycosaminoglycans distributed by chondrocytes in vitro and in vivo, immunohistochemical staining and Safranin O staining were used. (3) Results: IL-1β suppresses NEAT1_2 and genes related to the chondrocytic phenotype, whereas TAN effectively upregulates them in a NEAT1_2-dependent manner. Consistently, TAN alleviated chondrocyte oxidative stress inhibited cartilage degradation by modulating the relevant genes and promoted efficient cartilage regeneration in vitro and in vivo when chondrocytes are exposed to inflammatory cytokines. (4) Conclusions: TAN enhances the expression of NEAT1_2 inhibited by IL-1β and affects the transcription of chondrocytic phenotype-related genes, which promotes cartilage regeneration in an inflammatory environment.

Kruppel-Like Factor 5 Acetylation Upregulates Runx2 Expression and Accelerates the Dedifferentiation Process of Chondrocyte in Monolayer Culture

This study aimed to investigate the role of KLF5 (Kruppel-like factor 5) in the abnormal dedifferentiation of chondrocytes (CHs) observed in osteoarthritis (OA) and monolayer culture conditions. Human primary CHs were cultured in a monolayer for 14 days, and various analyses were conducted. The area and aspect ratio of the cells were measured, and protein expression levels of KLF5, acetylated KLF5 (Ac-KLF5), and Runx2 were assessed using western blotting. Additionally, mRNA levels of chondrogenic genes (type II collagen, Col-II), hypertrophic genes (type X collagen, Col-X), and MMP-13 were determined through RT-PCR. The effects of TGF-β and Am80 supplementation on KLF5 acetylation and Runx2 expression were examined, and siRNA was used to silence Runx2 gene expression. Results showed that CHs exhibited dedifferentiation after 7 days of culture, characterized by increased cell size, larger aspect ratio, elevated Ac-KLF5 levels, decreased Col-II expression, and increased Col-X and MMP-13 expression. TGF-β treatment enhanced Ac-KLF5 and Runx2 expression, thereby accelerating dedifferentiation within 3 days. In contrast, Am80 suppressed Ac-KLF5 and Runx2 expression, leading to delayed dedifferentiation over 14 days. Silencing Runx2 mitigated KLF5 acetylation-induced CH dedifferentiation without affecting Ac-KLF5 levels. In conclusion, KLF5 acetylation promotes Runx2 expression and subsequently contributes to CH dedifferentiation during monolayer culture.

Cartilage Lacuna-Inspired Microcarriers Drive Hyaline Neocartilage Regeneration.

Cartilage equivalents from hydrogels containing chondrocytes exhibit excellent potential in hyaline cartilage regeneration, yet current approaches have limited success at reconstituting the architecture to culture nondifferentiated chondrocytes in vitro. In this study, specially designed lacunar hyaluronic acid microcarriers (LHAMCs) with mechanotransductive conditions that rapidly form stable hyaluronic acid (HA) N-hydroxy succinimide ester (NHS-ester) are reported. Specifically, carboxyl-functionalized HA is linked to collagen type I via amide-crosslinking, and gas foaming produced by ammonium bicarbonate forms concave surface of the microcarriers. The temporal 3D culture of chondrocytes on LHAMCs uniquely remodels the extracellular matrix to induce hyaline cartilaginous microtissue regeneration and prevents an anaerobic-to-aerobic metabolism transition in response to the geometric constraints. Furthermore, by inhibiting the canonical Wnt pathway, LHAMCs prevent β-catenin translocation to the nucleus, repressing chondrocyte dedifferentiation. Additionally, the subcutaneous implantation model indicates that LHAMCs display favorable cytocompatibility and drive robust hyaline chondrocyte-derived neocartilage formation. These findings reveal a novel strategy for regulating chondrocyte dedifferentiation. The current study paves the way for a better understanding of geometrical insight clues into mechanotransduction interaction in regulating cell fate, opening new avenues for advancing tissue engineering.