Articular cartilage (AC) possesses complex depth-dependent gradients in cell phenotype, matrix composition, metabolism, mechanics, and growth factors. These natural gradients are essential for cartilage function. Because of lack of blood vessels, the repair and treatment of AC defects have been a grand challenge for surgeons, the natural gradients inspired researchers to find solutioninthe gradient construction for AC repair in tissue engineering. This review emphasizes a critical shift from merely describing native gradients to actively implementing gradient-based strategies in scaffold design. Current biomimetic approaches focus on engineering controllable gradients-including cell density, metabolic factors (e.g., oxygen), matrix composition, mechanical properties, and growth factor concentrationswithinthe scaffold. These gradient-enabled constructs can be designed to guide zone-specific cellular responses and extracellular matrix formation, ultimately restoring the native structure and function of AC. We also highlight technologies such as 3D bioprinting, oxygen-releasing biomaterials, perfusion bioreactors, and graded microsphere systems that enable precise spatial control of scaffold properties. A successful gradient-based strategy integrates multiple interdependent gradients rather than relying on a single feature. This review provides a practical framework for developing next-generation biomimetic scaffolds capable of achieving functional AC regeneration for clinical translation.

The dedifferentiation of chondrocytes significantly restricts their functional performance and practical applications. In our previous research, an easily preparable microcavitary alginate hydrogel (MCG) was shown to effectively promote the redifferentiation of dedifferentiated chondrocytes. Building on this, the present study further investigates the transcriptomic changes during chondrocyte dedifferentiation, utilizing high-throughput RNA sequencing to explore how MCG regulates passage-four dedifferentiated porcine chondrocytes over a 28-day period. Integrated analysis of transcriptomic profiling data across multiple time points identified the p53 signaling pathway as a potentially central regulatory node. Key findings validated by quantitative real-time polymerase chain reaction, Western blot, and Cell Counting Kit-8 assays demonstrated the following: (1) MCG arrested the progression of dedifferentiation, downregulated fibrosis/degeneration markers (COL1A1, WNT5A/B), and partially restored chondrogenic gene expression relative to P4; (2) Time-series analysis revealed MCG’s influence on cell cycle regulation, extracellular matrix organization, DNA repair, and differentiation processes; (3) Crucially, MCG dynamically regulated the p53 pathway: early activation (TP53, p-p53 Ser15, MDM2) promoted DNA repair (RRM2B) and suppressed excessive inflammation/apoptosis (IL6/8, PMAIP1/CASP3), while subsequent attenuation of the pathway correlated with enhanced late-stage proliferation. In conclusion, the growth factor-free MCG microenvironment alleviates chondrocyte dedifferentiation and facilitates partial redifferentiation by orchestrating cellular behaviors through dynamic regulation of the p53 pathway—particularly via enhanced DNA repair—thereby offering a promising strategy for cell-based therapeutic approaches.

B-cell lymphoma 9 (BCL9) is a regulatory protein that plays a key role within the Wnt/β-catenin signaling pathway. In recent years, it has garnered significant attention across the domains of tissue repair and tumour biology. The Wnt/β-catenin signaling pathway is of great importance in regulating bone tissue formation and osteoblast differentiation. As a co-activator of β-catenin, BCL9 is capable of governing bone development and regeneration by augmenting the transcriptional activity of the Wnt signaling. In addition, BCL9 is also closely related to diverse biological processes, such as angiogenesis, muscle regeneration and tumour progression. Research findings have indicated potential correlations between BCL9 and the treatment of osteoporosis, fracture healing, as well as other bone-related ailments, thereby positioning it as a crucial target in the exploration of bone regeneration. In oncology research, BCL9 affects tumour cell proliferation and metastasis by regulating the Wnt/β-catenin pathway, thus emerging as a promising target for anticancer therapy. In this paper, we conducted a comprehensive review of the discovery, structure, and mechanism of action of BCL9 within the Wnt/β-catenin signaling pathway, along with its potential applications in bone development, tissue regeneration, and cancer therapy, with the intention of furnishing novel perspectives and a theoretical foundation for future investigations.

A favorable microenvironment is of great significance for the repair of cartilage injury. In our previous study, a coculture system was devised, integrating genetically modified chondrocytes expressing transforming growth factor (TGF)-β3 with ATDC5 cells, which demonstrated an augmented chondrogenesis effect. In this study, a delivery platform for the controlled release of stromal cell-derived factor (SDF)-1α was constructed based on our microcavitary hydrogel system. Subsequently, it was combined with the coculture system to explore the release patterns of SDF-1α and TGF-β3 and investigate their synergistic impact on chondrogenesis. The findings indicated that both SDF-1α and TGF-β3 could be continuously and efficiently released from the delivery system throughout the culture period. Moreover, the combined application of SDF-1α and TGF-β3 was able to enhance cell proliferative activity, as demonstrated by the Cell Counting Kit-8 data. Synergistically, it led to superior chondrogenesis, as evidenced by real-time PCR, Western blot analysis, and immunohistochemistry staining assays. This study offers the insight that this delivery system, integrating the coculture system and microcavitary hydrogels, holds substantial potential for cell recruitment and cartilage repair, and may be applicable in regenerative medicine for diverse tissues.

Chondrocytes maintain the balance of the extracellular matrix by synthesizing glycoproteins, collagen, proteoglycans and hyaluronic acid. Chondrocyte dedifferentiation refers to a process in which chondrocytes lose their mature differentiated phenotype and transform into a fibroblast-like morphology with fewer differentiated stages and inferior function under external stimulation. The important mechanism of homeostasis loss in osteoarthritis (OA) is a change in the chondrocyte phenotype. The dedifferentiation markers of chondrocytes are upregulated in OA, and the pathogenic factors related to OA have also been shown to enhance chondrocyte dedifferentiation. In this review, we compile recent studies on chondrocyte dedifferentiation, with an emphasis on potential markers and the underlying mechanisms of dedifferentiation, as well as the current research progress in inhibiting dedifferentiation or achieving redifferentiation. A deep understanding of chondrocyte dedifferentiation would not only support the pathogenesis of OA theoretically but also provide insightful ideas for regenerative medicine to manipulate the functional phenotype of cells.

OBJECTIVES: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.RESULTS: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.CONCLUSIONS: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.

Bone/cartilage repair and regeneration have been popular and difficult issues in medical research. Tissue engineering is rapidly evolving to provide new solutions to this problem, and the key point is to design the appropriate scaffold biomaterial. In recent years, microsphere-based scaffolds have been considered suitable scaffold materials for bone/cartilage injury repair because microporous structures can form more internal space for better cell proliferation and other cellular activities, and these composite scaffolds can provide physical/chemical signals for neotissue formation with higher efficiency. This paper reviews the research progress of microsphere-based scaffolds in bone/chondral tissue engineering, briefly introduces types of microspheres made from polymer, inorganic and composite materials, discusses the preparation methods of microspheres and the exploration of suitable microsphere pore size in bone and cartilage tissue engineering, and finally details the application of microsphere-based scaffolds in biomimetic scaffolds, cell proliferation and drug delivery systems.

Osteoarthritis, the main cause of disability worldwide, involves not only cartilage injury but also subchondral bone injury, which brings challenges to clinical repair. Tissue engineering strategies provide a promising solution to this degenerative disease. Articular cartilage connects to subchondral bone through the osteochondral interfacial tissue, which has a complex anatomical architecture, distinct cell distribution and unique biomechanical properties. Forming a continuous and stable osteochondral interface between cartilage tissue and subchondral bone is challenging. Thus, successful osteochondral regeneration with engineering strategies requires intricately coordinated interplay between cells, materials, biological factors, and physical/chemical factors. This review provides an overview of the anatomical composition, microstructure, and biomechanical properties of the osteochondral interface. Additionally, the latest research on the progress related to osteochondral regeneration is reviewed, especially discussing the fabrication of biomimetic scaffolds and the regulation of biological factors for osteochondral defects.

Bone tumors are currently a major clinical challenge. In recent decades, strategies using well-designed versatile biomaterials for the treatment of bone tumors have emerged and attracted extensive research interest. Suitable biomaterials not only facilitate repair for bone defects aroused by surgical intervention but also help deliver antineoplastic drugs to the target site or provide photothermal/magnetothermal therapy to kill bone tumor cells. Thus, the development of biomaterials exhibits a great perspective for future bone tumor treatment.We summarize the recent progress of versatile biomaterials for bone tumor therapy, with an emphasis on photothermal/magnetothermal therapy and drug delivery.With the further understanding and development of biomaterials, multifunctional biomaterials have been proposed for bone tumor treatment. Through the interdisciplinary cooperation from the fields of biomedicine, clinical medicine and engineering, multifunctional biomaterials will perfectly match individual bone defects in the clinic with low cost in the future.

Due to the lack of blood vessels, nerves and lymphatics, articular cartilage is difficult to repair once damaged. Tissue engineering is considered to be a potential strategy for cartilage regeneration. Successful tissue engineering strategies depend on the effective combination of biomaterials, seed cells and biological factors. In our previous study, a genetically modified coculture system with chondrocytes and ATDC5 cells in an alginate hydrogel has exhibited a superior ability to enhance chondrogenesis. In this study, we further evaluated the influence of chondrocytes at various passages on chondrogenesis in the coculture system. The results demonstrated that transfection efficiency was hardly influenced by the passage number of chondrocytes. The coculture system with passage 5 (P5) chondrocytes had a better effect on chondrogenesis of ATDC5 cells, while chondrocytes in this coculture system presented higher levels of dedifferentiation than other groups with P1 or P3 chondrocytes. Therefore, P5 chondrocytes were shown to be more suitable for the coculture system, as they accumulated in sufficient cell numbers with more passages and had a higher level of dedifferentiation, which was prone to form a favorable niche for chondrogenesis of ATDC5 cells. This study may provide fresh insights for future cartilage tissue engineering strategies with a combination of a coculture system and advanced biomaterials.

INTRODUCTION:Tissue engineering has brought hope for the repair of bone and cartilage injury. As potential therapeutic molecules for use in tissue engineering, chemokines promote the development of cell-free tissue engineering, avoiding dilemmas faced by cell-based tissue engineering. The main role of chemokines in tissue engineering is to recruit progenitor/stem cells to the site of damaged tissue in vivo and induce differentiation into the corresponding tissue, thus remodeling tissue function. In recent years, many studies have demonstrated the great potential of chemokines in the regeneration and repair of various tissues, such as heart, bone and cartilage tissue.AREAS COVERED:The classification, structure, and function of chemokines and the application of several common chemokines in diseases, especially in bone/cartilage tissue regeneration are discussed.EXPERT OPINION:Many studies have demonstrated that the combinatory use of cell chemotactic factors (CCFs) and growth factors can exert synergistic effects on chondrogenesis and osteogenesis. With further understanding of biomaterials and the development of powerful bio-fabrication techniques, intelligent biomaterials will be created to meet the requirements for controlled bioactive factor release and biomimetic architecture. Also, a better understanding of the biological cascade reactions and pathways of CCFs is beneficial to guide the design of innovative biomaterials.

Due to the lack of vascular distribution and the slow metabolism, cartilage tissue cannot repair itself, which remains a huge challenge in cartilage regeneration. Tissue engineering using stem cells appears to be a promising method for cartilage repair. Tissue engineers demonstrated that mechanical stimulation can enhance the quality of engineered cartilage, making it more similar to natural cartilage in structure and function. In this review, we summarize recent studies on the role of mechanical stimuli in chondrogenesis, focusing on the applications of extrinsic mechanical loading and the studies on mechanical properties of biomaterials in cartilage tissue engineering. This review will provide fresh insights into the potential use of mechanical stimuli for clinical use.

Cell dedifferentiation is the process by which cells grow reversely from a partially or terminally differentiated stage to a less differentiated stage within their own lineage. This extraordinary phenomenon, observed in many physiological processes, inspires the possibility of developing new therapeutic approaches to regenerate damaged tissue and organs. Meanwhile, studies also indicate that dedifferentiation can cause pathological changes. In this review, we compile the literature describing recent advances in research on dedifferentiation, with an emphasis on tissue-specific findings, cellular mechanisms, and potential therapeutic applications from an engineering perspective. A critical understanding of such knowledge may provide fresh insights for designing new therapeutic strategies for regenerative medicine based on the principle of cell dedifferentiation.

Cell dedifferentiation is the process by which cells grow reversely from a partially or terminally differentiated stage to a less differentiated stage within their own lineage. This extraordinary phenomenon, observed in many physiological processes, inspires the possibility of developing new therapeutic approaches to regenerate damaged tissue and organs. Meanwhile, studies also indicate that dedifferentiation can cause pathological changes. In this review, we compile the literature describing recent advances in research on dedifferentiation, with an emphasis on tissue-specific findings, cellular mechanisms, and potential therapeutic applications from an engineering perspective. A critical understanding of such knowledge may provide fresh insights for designing new therapeutic strategies for regenerative medicine based on the principle of cell dedifferentiation.

Critical size bone defects are one of the most serious complications in orthopedics due to the lack of effective osteogenesis treatment. We fabricated carboxymethyl cellulose with phenol moieties (CMC-ph) microcapsules loaded with gene-modified rat bone mesenchymal stem cells (rBMSCs) that secrete hBMP2 following doxycycline (DOX) induction. The results showed that the morphology of microcapsules was spherical, and their diameters have equally distributed in the range of 100-150 μm; the viability of rBMSCs was unchanged over time. Through real-time PCR and Western blot analyses, the rBMSCs in microcapsules were found to secrete hBMP2 and to have upregulated mRNA and protein expression of osteogenesis-related genes in vitro and in vivo. Furthermore, the in vivo results suggested that the group with the middle concentration of cells expressed the highest amount of osteogenic protein over time. In this study, we showed that gene-modified rBMSCs in CMC-ph microcapsules had good morphology and viability. The BMP2-BMSCs/CMC-Ph microcapsule system could upregulate osteogenic mRNA and protein in vitro and in vivo. Further analysis demonstrated that the medium concentration of cells had a suitable density for transplantation in nude mice. Therefore, BMP2-BMSCs/CMC-Ph microcapsule constructs have potential for bone regeneration in vivo.

Articular cartilage repair after injury is a great challenge worldwide due to its nerveless and avascular features. Tissue engineering is proposed as a promising alternative for cartilage regeneration. In this study, an adenoviral vector carrying the transforming growth factor-β3 (TGF-β3) gene was constructed and introduced into dedifferentiated chondrocytes, which were then cocultured with ATDC5 cells in an alginate hydrogel system. The results showed that the experimental groups exhibited better cell viability and higher levels of cartilage-related genes than the control groups. In this coculture system, the chondrogenic differentiation of ATDC5 cells was effectively induced by TGF-β3 and other latent cytokines that were produced by the transfected chondrocytes. Thus, this method can avoid the degradation of exogenous TGF-β3, and it can protect ATDC5 cells during virus transfection to maintain cell viability and chondrogenic differentiation capability. Taken together, this study provides fresh insights for applying this genetically manipulated coculture system to cartilage repair in the future.

用成体干细胞修复软骨损伤是目前的研究热点,其中骨髓间充质干细胞( bone marrow mesenchymal stem cells, BM-MSCs)可以诱导成软骨细胞,转化生长因子 β( transforming growth factorβ,TGF-β)可以通过活化成软骨标志物诱导BM-MSCs成软骨,但分化软骨细胞容易肥大且生长因子可能引发免疫反应. 有研究者致力于寻找适合的药物来诱导BM-MSCs成软骨分化,有报道视黄醇酸受体是一种可能的药理作用靶点[1]. LE135是一种视黄醇酸受体拮抗剂,化学式为C29 H30 N2 O2 ,分子质量438. 56,不溶于水,溶于二甲亚砜. 本研究就LE135在大鼠BM-MSCs成软骨分化诱导中的可能作用进行了初步的探索.

Objective To investigate the effect of alginate hydrogel 3D co-culture on redifferentiation of dedifferentiated chondrocytes. Methods The first generation (P1) chondrocytes and the fourth generation (P4) chondrocytes were co-cultured with ATDC5 at the ratio of 3: 1, and wee named as AP1 and AP4 respectively. Another 3D culture system consisting of single P1, P4 and ATDC5 was established, and was named as P1, P4 and ATDC, respectively. All the groups were cultured in the chondrocyte-inducing medium with the supply of TGF-β3. After 28 days, the expression of collagen typeⅠ (ColⅠ), collagen typeⅡ (ColⅡ) and aggrecan (Acan) genes was assayed by Q-PCR. Histological staining was performed. The cartilage-related genes (Col Ⅱ and Acan) of each group were compared. The results were analyzed by Bonferroni test. Results The expression of ColⅡ and Acan in AP4 group was significantly up-regulated compared to P1 and P4 groups (ColⅡ: F=38.41, P<0.01, Acan: F=5, P<0.01 ), and the deposition of relative protein products could also be clearly observed on histological staining. Conclusion The 3D co-culture system can make the dedifferentiated chondrocytes regain their unique phenotype, the mechanism of which involves cellular factors and the cell-cell connection. Key words: Transforming growth factor beta3; Hydrogel; Collagen type Ⅰ; Collagen type Ⅱ; Biocompatible materials; Chondrocyte; Dedifferentiation

INTRODUCTION:Cartilage tissue engineering has rapidly developed in recent decades, exhibiting promising potential to regenerate and repair cartilage. However, the origin of a large amount of a suitable seed cell source is the major bottleneck for the further clinical application of cartilage tissue engineering. The use of a monoculture of passaged chondrocytes or mesenchymal stem cells results in undesired outcomes, such as fibrocartilage formation and hypertrophy. In the last two decades, co-cultures of chondrocytes and a variety of mesenchymal stem cells have been intensively investigated in vitro and in vivo, shedding light on the perspective of co-culture in cartilage tissue engineering.AREAS COVERED:We summarize the recent literature on the application of heterologous cell co-culture systems in cartilage tissue engineering and compare the differences between direct and indirect co-culture systems as well as discuss the underlying mechanisms.EXPERT OPINION:Co-culture system is proven to address many issues encountered by monocultures in cartilage tissue engineering, including reducing the number of chondrocytes needed and alleviating the dedifferentiation of chondrocytes. With the further development and knowledge of biomaterials, cartilage tissue engineering that combines the co-culture system and advanced biomaterials is expected to solve the difficult problem regarding the regeneration of functional cartilage.

Cartilage tissue engineering appears as an important strategy for repairing articular cartilage.However, it faces the problems of insufficient number of chondrocytes, dedifferentiation in vitro, and difficulty in controlling the direction of differentiation.The co-culture system provides a new solution to these problems.A series of studies have reported that the co-culture system can prevent the dedifferentiation of chondrocytes cultured in vitro, promote the re-differentiation of dedifferentiated chondrocytes, and facilitate the chondrogenic differentiation of stem cells in the system as well as inhibit the process of hypertrophy.This review summarized the literatures about the application of co-culture system in cartilage tissue engineering, discussed the role of co-culture system in cartilage tissue engineering, and explored the underlying mechanism.