Empowering Cartilage Restructuring with Biodegradable Magnesium Doped-Silicon Based-Nanoplatforms: Sustained Delivery and Enhanced Differentiation Potential.

Background: Cartilage-related diseases, such as hypoplastic chondrodysplasia a rare genetic disorder that affects newborns, causing abnormal cartilage development and restricted skeletal growth. However, the development of effective treatment strategies for chondrodysplasia still faces significant challenges due to limitations in the controlled drug delivery, biocompatibility, and biodegradability of nanomedicines. Methods: A biodegradable magnesium doped-silicon based-nanoplatforms based on silicon nanoparticles (MON) was constructed. Briefly, the MON was modified with sulfhydryl groups using MPTMS to form MOS. Further engineering of MOS was achieved by incorporating Mg2+ ions through the "dissolution-regrowth" method, resulting in MMOS. Ica was effectively loaded into the MMOS channels, and HA was anchored on the surface of MOS to obtain MMOS-Ica@HA nanoplatforms. Additionally, in vitro cell experiments and in vivo zebrafish embryo models were used to evaluate the effect of the nanoplatforms on cartilage differentiation or formation and the efficiency of treating chondrodysplasia. Results: A series of characterization tests including TEM, SEM, DLS, XPS, EDX, and BET analysis validate the successful preparation of MOS-Ica@HA nanoplatforms. The prepared nanoplatforms show excellent dispersion and controllable drug release behavior. The cytotoxicity evaluation reveals the good biocompatibility of MOS-Ica@HA due to the sustained and controllable release of Ica. Importantly, the presence of Ica and Mg component in MOS-Ica@HA significantly promote chondrogenic differentiation of BMSCs via the Smad5/HIF-1α signaling pathway. In vitro and in vivo experiments confirmed that the nanoplatforms improved chondrodysplasia by promoting cartilage differentiation and formation. Conclusion: The findings suggest the potential application of the developed biodegradable MMOS-Ica@HA nanoplatforms with acceptable drug loading capacity and controlled drug release in chondrodysplasia treatment, which indicates a promising approach for the treatment of chondrodysplasia.

Genome-wide DNA methylation analysis reveals layer-specific methylation patterns in deer antler tissue.

This paper explored using of deer antlers as a model for studying rapid growth and cartilage formation in mammals. The genes and regulatory mechanisms involved in antler chondrogenesis are poorly understood, however, previous research has suggested that DNA methylation played a key role in antler regeneration. By using fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP), this study measured DNA methylation levels in cartilage (CA) and reserve mesenchyme (RM) cells and tissues. Results showed that RM cells (RMCs) DNA methylation levels were significantly lower than those of CA, suggesting that DNA demethylation may be involved in antler fast cartilage differentiation. The study also identified 20 methylated fragments specific to RMCs or CA using the methylation-sensitive amplified polymorphism (MSAP) technique and confirmed these findings using southern blot analysis. The data provide the first experimental evidence of a link between epigenetic regulation and rapid cartilage differentiation in antlers.

Regulatory mechanism for the proliferation and differentiation of growth plates / 中华实用儿科临床杂志

The growth plate (cartilage tissue) is the key to bone development and linear growth.However, as the adolescence proceeds, the proliferation capacity of the growth plate will be continuously consumed, and finally the growth plate will be closed.A variety of regulatory factors control chondrocyte proliferation and differentiation through different mechanisms.Endocrine regulators (including growth hormone, insulin-like growth factor, thyroxine, sex hormone, glucocorticoid, etc.) and transcription factors play an important role in regulating the development of growth plates through systematic modulation.In addition, such local regulatory factors as Indian hedgehog protein, parathyroid hormone-related peptide, bone morphogenetic protein and fibroblast growth factor also regulate the development of the growth plate.In this paper, the regulatory mechanism for chondrocyte proliferation and differentiation was summarized.

Effects of Berberine on the Chondrogenic Differentiation of Embryonic Limb Skeletal Progenitors.

INTRODUCTION: Berberine (BBR) is an isoquinoline plant alkaloid with demonstrated anti-inflammatory, anti-tumor and immunosuppressive pharmacological properties that functions via multiple signaling pathways and epigenetic modulators. Numerous studies have proposed BBR as a promising therapeutic agent for joint cartilage degeneration, and other connective tissue diseases. PURPOSE AND METHODS: This work aimed to evaluate the effects of BBR on the growth and differentiation of embryonic skeletal progenitors using the limb mesoderm micromass culture assay. RESULTS: Our findings show that at difference of its apoptotic influence on a variety of tumor tissues, cell death was not induced in skeletal progenitors by the addition of 12 or 25 µM BBR concentration to the culture medium. Morphological and transcriptional analysis revealed dual and opposite effects of BBR treatments on chondrogenesis depending on the stage of differentiation of the cultured progenitors. At early stage of culture, BBR was a potent chondrogenic inhibitor, while chondrogenesis was intensified in treatments at advanced stages of culture. The chondrogenic promoting effect was accompanied by a moderate upregulation of gene markers of prehypertrophic cartilage, including ColXa1, alkaline phosphatase Alpl, Runx2, and Indian Hedgehog Ihh. We further observed a positive transcriptional influence of BBR in the expression of DNA methyltransferase genes, Dnmt1, Dnmt3a and Dnmt3b, suggesting a potential involvement of epigenetic factors in its effects. CONCLUSION: Our study uncovers a new pharmacological influence of BBR in cartilage differentiation that must be taken into account in designing clinical protocols for its employment in the treatment of cartilage degenerative diseases.

Regulating effect of Circ_ATRNL1 on the promotion of SOX9 expression to promote chondrogenic differentiation of hAMSCs mediated by MiR-145-5p.

Circ_ATRNL1 is significantly highly expressed in cartilage tissues of patients with osteoarthritis. This study explored the role and mechanism of circ_ATRNL1 in cartilage differentiation of human adipose-derived mesenchymal stem cells (hAMSCs). hAMSCs were isolated and identified by flow cytometry. The degree of chondrocyte and adipogenic differentiation was assessed using Alcian blue staining and Oil Red O staining, respectively. The expressions of cartilage- and adipogenic-related genes, circ_ATRNL1, and SOX9 were detected by reverse transcription quantitative polymerase chain reaction. The correlation between SOX9 and circ_ATRNL1 was analyzed using Pearson test. Bioinformatics and luciferase analysis were used to detect the overlapped target miRNAs of circ_ATRNL1 and SOX9. The role of circ_ATRNL1/miRNA/SOX9 was examined using functional rescue assays. hAMSCs were identified as CD90-, CD105-, and CD44-positive. The degree of cartilage differentiation of hAMSCs was significantly enhanced after 2 weeks. Cartilage-related genes, circ_ATRNL1 and SOX9, were significantly upregulated, and positively correlated with each other. Circ_ATRNL1 overexpression enhanced hAMSC proliferation and differentiation into chondrogenesis, and promoted the expressions of COL2, Aggrecan, and SOX9. Overexpression of circ_ATRNL1 inhibited the adipogenic differentiation of hAMSCs and the expressions of adipogenic-related genes. miR-145-5p was a target miRNA for circ_ATRNL1 and SOX9. miR-145-5p mimic inhibited hAMSC differentiation toward cartilage, and inhibited the expression of cartilage-related factors. miR-145-5p mimic effectively reversed the regulating effect of circ_ATRNL1 on hAMSCs. Circ_ATRNL1 regulates the promotion of SOX9 expression to promote chondrogenic differentiation of hAMSCs mediated by miR-145-5p.

Hyaluronic Acid Hydrogel with Adjustable Stiffness for Mesenchymal Stem Cell 3D Culture via Related Molecular Mechanisms to Maintain Stemness and Induce Cartilage Differentiation.

The stemness and differentiation characteristics of bone marrow mesenchymal stem cells (BMSCs) in three-dimensional (3D) culture are of great significance for stem cell therapy and cartilage tissue engineering repair. Moreover, due to their mechanical sensitivity, scaffold materials play important roles in various cell behaviors in 3D culture. In this study, the mechanical strength of hydrogel scaffolds was adjusted by changing the molecular weight of hyaluronic acid (HA). It was proven that BMSCs in a low-strength hydrogel could maintain stemness properties by activating the Wnt/ß-catenin pathway for 1 week, while the high-molecular-weight hydrogel with a higher mechanical strength had the potential to promote the direction of cartilage differentiation of BMSCs by opening transient receptor potential vanilloid 4 (TRPV4)/Ca2+ molecular channels, also increasing the expression of type II collagen and SOX9 in BMSCs. This research has a certain reference value for the design of biomaterials for BMSCs' delivery in vivo, as well as the formulation of cartilage repair drug delivery programs based on molecular mechanisms.

Sequence of chondrocranial development in the oriental fire bellied toad Bombina orientalis.

The vertebrate head as a major novelty is directly linked to the evolutionary success of the vertebrates. Sequential information on the embryonic pattern of cartilaginous head development are scarce, but important for the understanding of its evolution. In this study, we use the oriental fire bellied toad, Bombina orientalis, a basal anuran to investigate the sequence and timing of larval cartilaginous development of the head skeleton from the appearance of mesenchymal Anlagen in post-neurulation stages until the premetamorphic larvae. We use different methodological approaches like classic histology, clearing and staining, and antibody staining to examine the larval skeletal morphology. Our results show that in contrast to other vertebrates, the ceratohyals are the first centers of chondrification. They are followed by the palatoquadrate and the basihyal. The latter later fuses to the ceratohyal and the branchial basket. Anterior elements like Meckel's cartilage and the rostralia are delayed in development and alter the ancestral anterior posterior pattern observed in other vertebrates. The ceratobranchials I-IV, components of the branchial basket, follow this strict anterior-posterior pattern of chondrification as reported in other amphibians. Chondrification of different skeletal elements follows a distinct pattern and the larval skeleton is nearly fully developed at Gosner Stage 28. We provide baseline data on the pattern and timing of early cartilage development in a basal anuran species, which may serve as guidance for further experimental studies in this species as well as an important basis for the understanding of the evolutionary changes in head development among amphibians and vertebrates.

The Murine Limb Bud in Culture as an In Vitro Teratogenicity Test System.

There is widespread interest today in the use of in vitro methods to study normal and abnormal development. The limb is attractive in this context since much is known about pattern formation during limb development. The murine limb bud culture technique described in this chapter was developed and refined in the 1970s. In this culture system, limb development mimics the in vivo process, although at a slower rate, where growth and cartilage differentiation lead to the formation of proximal and distal structures with an "in vivo-like" 3D shape. Uniform developmental stages are selected for assessment, exposures are controlled precisely, and the confounding influences of maternal metabolism and transport are avoided. The existence of transgenic mice with fluorescent markers for the different stages of endochondral ossification adds a further dimension to the technique by allowing striking time course observations of the developing limb. Today, limb bud cultures are used to study the roles of genes during embryogenesis and the mechanisms by which chemicals interfere with critical signalling pathways.

Skeletal development of the chondrocranium in the tongue sole Cynoglossus semilaevis (Pleuronectiformes: Cynoglossidae).

This study provides a comprehensive description of chondrocranial development before, during and after larval metamorphosis in the tongue sole Cynoglossus semilaevis, a commercially valuable flatfish in China. Samples were collected at regular intervals ranging from 1 to 23 days post hatching (dph). Based on observations of cleared and double-stained specimens and images from sections stained with safranin O-fast green, major morphological events during early development were described. No cartilaginous structure was visible at hatching. From 2 dph onwards, cartilaginous structures such as the trabecular bar and some elements of the mandibular, hyoid and branchial arches appeared. At this time also, cartilaginous structures of the neurocranium started to form. Hypertrophic chondrocytes could be observed in many splanchnocranium elements at 5 dph. The start of ossification was indicated by alizarin red stain visible at 14 dph. At 17 dph, most of the cartilaginous skeleton was ossified. Soon after, the right eye started to migrate and pass through a slit beneath the dorsal-fin base and above the skull. Metamorphosis was complete at 20 dph, at which time the dorsal-fin base cartilage extended onto the anterior region of the head. Meanwhile, extremities of the hyoid and branchial arch elements remained cartilaginous. At 23 dph, endochondral ossification of the splanchnocranium was nearly complete. Unlike previous observations of other Pleuronectiformes, our study indicates that endochondral ossification of C. semilaevis skull cartilage occurs before metamorphosis.

Sequence and timing of early cranial skeletal development in Xenopus laevis.

Xenopus laevis is widely used as a model organism in biological research. Morphological descriptions of the larval cartilaginous skeleton are more than half a century old and comprehensive studies of early cartilage differentiation and development are missing. A proper understanding of early cranial skeletal development in X. laevis requires a detailed description that can function as a baseline for experimental studies. This basis makes it possible to evaluate skeletal defects produced by experiments on gene interactions, such as gain- or loss-of function experiments. In this study, we provide a detailed description of the pattern and timing of early cartilage differentiation and development in the larval head of X. laevis. Methods used include antibody staining, confocal laser scanning microscopy and 3D-reconstruction. Results were than compared to earlier studies based on classical histological approaches and clearing-and-staining. The first cartilage to chondrify is, in contrast to other vertebrates investigated so far, the ceratohyal. The components of the branchial basket chondrify in anterior-to-posterior direction as reported for other amphibians. Chondrification of different cartilages begins at different stages and the majority of cartilages are fully developed at Ziermann and Olsson stage 17. Our baseline data on the pattern and timing of early cartilaginous development in X. laevis is useful for evaluation of experiments which alter head skeletal development as well as for identifying heterochronic shifts in head development in other amphibians.

Ghrelin accelerates the cartilagic differentiation of rabbit mesenchymal stem cells through the ERK1/2 pathway.

Mesenchymal stem cells (MSCs) can differentiate into chondroblasts, adipocytes, or cartilage under appropriate stimulation. Identifying a mechanism triggering the differentiation of MSCs into cartilage may help develop novel therapeutic approaches for treating heterotopic ossification, the pathological formation of lamellar bone in soft tissue outside the skeleton that can lead to debilitating immobility. Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, stimulates growth hormone secretion, and has both orexigenic and adipogenic effects. This study sought to understand the potential involvement of the ERK1/2 signaling pathway in the ghrelin-induced growth of rat MSCs (rMSCs). We applied various concentrations of ghrelin to cultured rMSCs by observing the changes in the phosphorylation state of ERK1/2, p38, JNK as well as the type II collagen expression levels by western blot. The highest expression level for both type II collagen was obtained with 600 ng/mL ghrelin at 24 h. We found that the ghrelin-induced differentiation of rMSCs into cartilage was promoted primarily by the ERK1/2 pathway. Our study suggests that ghrelin induced differentiation of rMSCs into cartilage primarily through the ERK1/2 pathway.

miR-127-5p promotes chondrogenic differentiation in rat bone marrow mesenchymal stem cells.

The effect and related mechanisms of miR-127-5p on the cartilage differentiation of rat bone marrow mesenchymal stem cells (BMSCs) was investigated. Rat BMSCs were generated and transfected with miR-127-5p, RT-PCR and Safranin O staining were used to detect the effect of miR-127-5p on the cartilage differentiation of rat BMSCs. Western blot analysis was used to detect the related mechanisms of miR-127-5p on the cartilage differentiation of rat BMSCs. Genes related to cartilage differentiation such as Sox9, collagen II and aggrecan were significantly increased in the group which were transfected with miR-127-5p, while collagen X, which was related to cartilage hypertrophy, was decreased in the miR-127-5p transfected group. Safranin O staining revealed that the expression of chondroitin sulfate was significantly increased in the group of miR-127-5p, than the miRNA control group. Western blot analysis showed that miR-127-5p transfection promoted the expression of Sox9, while decreased the expression of Runx2 of rat BMSCs. In conclusion, via increasing the expression of Sox9 and decreasing the expression of Runx2, miR-127-5p could promote cartilage differentiation and decrease cartilage hypertrophy of rat BMSCs.

Cx43- and Smad-Mediated TGF-ß/ BMP Signaling Pathway Promotes Cartilage Differentiation of Bone Marrow Mesenchymal Stem Cells and Inhibits Osteoblast Differentiation.

BACKGROUND/AIMS: The aim of this study was to investigate the influence of Cx43- and Smad-mediated TGF-ß/BMP signaling pathway on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into cartilage and inhibition of ossification. METHODS: BMSCs of Wistar rats were cultured and assigned into 5 groups for transfection with adenoviruses. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were employed to detect mRNA and protein expressions of target genes. The condition of cartilage and ossification were measured by a series of staining methods. Subcutaneous injection of mesenchymal stem cells (MSCs) into nude rats was performed. RESULTS: After transfection, compared to the AdGFP group, the corresponding target mRNAs were overexpressed in the AdBMP2, AdSmad1, AdCx43 + AdSmad1 and AdCx43 + AdSmad1 + AdBMP2 groups, and overexpression of BMP2 at the mRNA and protein expression was observed in the AdSmad1 and AdCx43 + AdSmad1 groups. The mRNA expressions of aggrecan (ACAN) and collagen type II alpha 1 (Col2a1), the glycosaminoglycan content of the extracellular matrix and the expression of type II collagen, Col2a1, osteopontin (OPN) and osteocalcin (OC) were higher in the AdBMP2, AdSmad1, AdCx43 + AdSmad1 and AdCx43 + AdSmad1 + AdBMP2 groups than in the AdGFP group; alkaline phosphatase (ALP) activity and mRNA and protein expressions of Runx2 were also higher in these groups than in the AdGFP group. Heterotopic osteogenesis tests demonstrated evident cartilage differentiation ability in the AdCx43 + AdSmad1 + AdBMP2 groups. In comparison, the AdCx43 + AdSmad1 and AdSmad1 groups exhibited weaker cartilage differentiation abilities. CONCLUSION: Cx43 and Smad1 promote BMP-induced cartilage differentiation of BMSCs and inhibit osteoblast differentiation, which provide a new strategy for cartilage tissue engineering using exogenous Cx43 and Smad1.

Synovial fluid of osteoarthritis promotes the differentiation of synovial mesenchymal stem cells / 第二军医大学学报

Objective To explore the effect of synovial fluid of osteoarthritis (OA) on the differentiation of synovium-derived stem cells (SDSCs). Methods The SDSCs from the synovium of OA patients were identified and were induced to cartilage differentiation. The SDSCs were cultured with OA joint fluid (experimental group) or without OA joint fluid (control group). The diameter of the cartilage was measured. The mRNA and protein expressions of COL II, Aggrecan and SOX9 were determined by RT-PCR and Western blotting analysis. Results The diameter of the cartilage in the experimental group was greater than that in the control 16 days after cultured with OA joint fluid (P<0.05). And the levels of COLII, Aggrecan and SOX9 mRNA and protein in the experimental group was significantly higher than those in the control group (P<0.05). Conclusion The OA joint fluid can promote differentiation of SDSCs.

Effects of BIO on proliferation and chondrogenic differentiation of mouse marrow-derived mesenchymal stem cells.

In vitro expansion of mesenchymal stem cell (MSCs) into large number is necessary for their application in cell-based treatment of articular cartilage defects. On the other hand, some studies have indicated that BIO (6-Bromoindirubin-3-Oxime) possesses mitogenic effects on cell culture. The objective of the present study was to examine the effect of BIO on in vitro expansion and chondrogenic differentiation of mouse marrow-derived MSCs. The culture was established using bone marrow tissue obtained from 10 NMRI mice. MSC nature of the isolated cells was verified according to the minimal criteria proposed for MSC. Passaged-3 cells were seeded in 24-well culture plates and treated by 0.05, 0.01, 0.1, 1.0 and 1.5 µM BIO for seven days. The culture without BIO was taken as the control. At the end of cultivation period, the cultures were examined for viable cell number which was then used to calculate population doubling time (PDT). The BIO with higher proliferation-promoting effect was investigated for its chondrogenic effect on MSC culture. There was significantly more viable cells at the cultures treated by 0.1 µM BIO. At this culture the cells tended to double their population in rapid rate (each 43.07 hr) than the cells treated with the other BIO concentrations (p < 0.05). Interestingly treatment of MSC chondrogenic culture with 0.1 µM BIO led to the up-regulation of cartilage specific genes including aggrecan, collagen II and Sox9. In conclusion BIO at 0.1 µM could enhance mouse MSC in vitro proliferation as well as their chondrogenic differentiation. These findings would be of great importance for the field of regenerative medicine.

Expression of Chicken Cartilage Derived Matrix Protein 10 (CCMP 10) in Chondrogenesis / 체질인류학회지

Over the past few years, considerable progress has been achieved about the extracellular elements and intracellular regulatory molecules that are involved in the regulation of chondrogenesis. However, little is known about the molecular mechanism of how these molecules influence the gene activities during cartilage differentiation. Recently we isolated a Chicken Cartilage derived Matrix Protein (CCMP 10), a novel protein, from chicken prechondrogenic mesenchyme. To further understand the function of CCMP-10 in cartilage development, we investigated the expression of CCMP-10 during the prechondrocyte differentiation in chick embryos and micromass cultured prechondrogenic cells, using a variety of methods such as transient transfection of CCMP 10, immunohistochemical localization, northern analysis, and western analysis. When transiently transfected, CCMP 10 was expressed in both nucleus and cytoplasm, with stronger intensity in the nucleus. In an immunohistochemical study, CCMP 10 was expressed in prechondrogeinc mesenchymal cell, perichondrium, and resting and proliferative zone of the growth plate of long bone, while no expression of CCMP 10 was observed in upper mature chondrocytes and hypertrophic chondrocytes. Northern analysis of micromass cultured prechondrogenic cells showed the expression of CCMP-10 mRNA for first 2 days, while Col 2a1, aggrecan, and CMP mRNAs, known genes to express in mature chondrocyte, initiated the expression at day 2 and continued to express by day 5. In western analysis, CCMP-10 was detected at initial stage and continued to express by day 3, while Col 2al protein began to express only one day after, and continued to express. Taken together, our data suggest that CCMP-10 may play a significant role in the early cartilage development.