Promote Cartilage Formation Research Articles

Green synthesis, chemical characterization, anti-osteoarthritis properties of Cu nanoparticles

In our latest study, we assessed the effectiveness of a green copper nanoparticle formulation containing curcumin on the chick’s knee joint injury. The NPs were characterized using FE-SEM, TEM, XRD, and EDX techniques. The primary cultured chondrocytes were utilized to check the nanoparticles effectiveness on chondrogenesis in the orthopedic experiments. Subsequently, we assessed the functional restoration post copper nanoparticles transplantation using an osteoarthritis articular model in the knee. Histological, PCR, and western blot analyses were conducted to investigate the underlying mechanisms. The XRD results exhibited the CuO formula for CuNPs and a spherical shape with a size <60 nm was obtained for the NPs from the TEM and FE-SEM images. The presence signals in EDX diagram including signals at 0.22 keV for C Lα, 0.51 keV for O Lα, and 0.93 keV for Cu Lα approve the formation of Cu NPs. The use of nanoparticles effectively inhibited abnormal fibrosis and angiogenesis at the injury site. The nanoparticles presence in the histological test resulted in rised synthesis of cartilage matrix and proliferation of chondrocytes, while also reducing the formation of abnormal vasculature. The study unveiled that the use of nanoparticles accelerated the healing process following an injury after three weeks. Furthermore, we highlighted the beneficial effects of copper nanoparticles in promoting cartilage formation in a laboratory setting. In the immunological domain, the copper nanoparticles demonstrated a decrease in the quantities of IL-1β, TNF-α, p-p65, and MMPs expression. This phenomenon could potentially be attributed to the cellular redox homeostasis modulation associated with the Nrf2 pathway. In conclusion, the results of this research confirmed the positive efficacy of copper nanoparticles containing curcumin in accelerating the osteoarthritis articular cartilage repair.

A quick and innovative pipeline for producing chondrocyte-homing peptide-modified extracellular vesicles by three-dimensional dynamic culture of hADSCs spheroids to modulate the fate of remaining ear chondrocytes in the M1 macrophage-infiltrated microenvironment

BackgroundExtracellular vesicles (EVs) derived from human adipose-derived mesenchymal stem cells (hADSCs) have shown great therapeutic potential in plastic and reconstructive surgery. However, the limited production and functional molecule loading of EVs hinder their clinical translation. Traditional two-dimensional culture of hADSCs results in stemness loss and cellular senescence, which is unfavorable for the production and functional molecule loading of EVs. Recent advances in regenerative medicine advocate for the use of three-dimensional culture of hADSCs to produce EVs, as it more accurately simulates their physiological state. Moreover, the successful application of EVs in tissue engineering relies on the targeted delivery of EVs to cells within biomaterial scaffolds.Methods and ResultsThe hADSCs spheroids and hADSCs gelatin methacrylate (GelMA) microspheres are utilized to produce three-dimensional cultured EVs, corresponding to hADSCs spheroids-EVs and hADSCs microspheres-EVs respectively. hADSCs spheroids-EVs demonstrate excellent production and functional molecule loading compared with hADSCs microspheres-EVs. The upregulation of eight miRNAs (i.e. hsa-miR-486-5p, hsa-miR-423-5p, hsa-miR-92a-3p, hsa-miR-122-5p, hsa-miR-223-3p, hsa-miR-320a, hsa-miR-126-3p, and hsa-miR-25-3p) and the downregulation of hsa-miR-146b-5p within hADSCs spheroids-EVs show the potential of improving the fate of remaining ear chondrocytes and promoting cartilage formation probably through integrated regulatory mechanisms. Additionally, a quick and innovative pipeline is developed for isolating chondrocyte homing peptide-modified EVs (CHP-EVs) from three-dimensional dynamic cultures of hADSCs spheroids. CHP-EVs are produced by genetically fusing a CHP at the N-terminus of the exosomal surface protein LAMP2B. The CHP + LAMP2B-transfected hADSCs spheroids were cultured with wave motion to promote the secretion of CHP-EVs. A harvesting method is used to enable the time-dependent collection of CHP-EVs. The pipeline is easy to set up and quick to use for the isolation of CHP-EVs. Compared with nontagged EVs, CHP-EVs penetrate the biomaterial scaffolds and specifically deliver the therapeutic miRNAs to the remaining ear chondrocytes. Functionally, CHP-EVs show a major effect on promoting cell proliferation, reducing cell apoptosis and enhancing cartilage formation in remaining ear chondrocytes in the M1 macrophage-infiltrated microenvironment.ConclusionsIn summary, an innovative pipeline is developed to obtain CHP-EVs from three-dimensional dynamic culture of hADSCs spheroids. This pipeline can be customized to increase EVs production and functional molecule loading, which meets the requirements for regulating remaining ear chondrocyte fate in the M1 macrophage-infiltrated microenvironment.Graphical

One-step soaking strategy toward mechanobiological double-network hydrogel with improving chondrogenesis capacity

Cartilage tissue engineering (CTE) is a promising strategy for addressing the persistent challenges in healing articular cartilage defects in the clinic, aiming to manufacture scaffolds with ideal mechanical characteristics that can provide a conducive microenvironment for the reconstruction or replacement of damaged cartilage defects. Easy operation, excellent mechanical strength, remarkable biocompatibility, and inherent capacity to promote cartilage formation are essential in CTE. This study designed a mechanobiological double-network (DN) hydrogel (KCS-PAA-Mn2+) through a one-step soaking strategy of immersing the composite hydrogel (KCS-PAA) of kartogenin (KGN)-conjugated short-chain chitosan (CS) covalent with polyacrylic acid (PAA) in manganese chloride (MnCl2) solution. Due to the strong physical interactions of CS chain-entanglement network and carboxylate-Mn2+ complex inducing polymer aggregation state transitions, the KCS-PAA-Mn2+ DN hydrogel exhibited exceptional mechanical characteristics, such as high tensile property (strength: 0.24 MPa at a strain of 552 %; toughness: 0.66 MPa), compressibility (strength: 51.98 MPa at a strain of 95 %; compressive modulus: 0.09 MPa), controllable swelling behavior (~327 %), and sustained drug release (>2 weeks). Moreover, in vitro biological studies demonstrated that the KCS-PAA-Mn2+ DN hydrogel not only promoted cell growth and proliferation but also enhanced the expression of genes specific to cartilage and the release of cartilage extracellular matrix (ECM) by bone mesenchymal stem cells (BMSCs), thus enabling a continuous concentration for a long period and persistently enhancing its chondrogenic efficiency. Overall, this soaking methodology reported here is universal to construct mechanically bioactive scaffolds that could expand their bio-applicability with simultaneous advantages of mechanical supports, tailorable swelling, sustainable drug release and long-lasting biological function, thus facilitating the advancement of cartilage-engineered hydrogel biomaterials to a higher level.

Engineered hsa-miR-455-3p-Abundant Extracellular Vesicles Derived from 3D-Cultured Adipose Mesenchymal Stem Cells for Tissue-Engineering Hyaline Cartilage Regeneration.

Efforts are made to enhance the inherent potential of extracellular vesicles (EVs) by utilizing 3D culture platforms and engineered strategies for functional cargo-loading. Three distinct types of adipose mesenchymal stem cells-derived EVs (ADSCs-EVs) are successfully isolated utilizing 3D culture platforms consisting of porous gelatin methacryloyl (PG), PG combined with sericin methacryloyl (PG/SerMA), or PG combined with chondroitin sulfate methacryloyl (PG/ChSMA). These correspond to PG-EVs, PG/SerMA-EVs, and PG/ChSMA-EVs, respectively. Unique microRNA (miRNA) profiles are observed in each type of ADSCs-EVs. Notably, PG-EVs encapsulate higher levels of hsa-miR-455-3p and deliver more hsa-miR-455-3p to chondrocytes, which results in the activation of the hsa-miR-455-3p/PAK2/Smad2/3 axis and the subsequent hyaline cartilage regeneration. Furthermore, the functionality of PG-EVs is optimized through engineered strategies, including agomir/lentivirus transfection, electroporation, and Exo-Fect transfection. These strategies, referred to as Agomir-EVs, Lentivirus-EVs, Electroporation-EVs, and Exo-Fect-EVs, respectively, are ranked based on their efficacy in encapsulating hsa-miR-455-3p, delivering hsa-miR-455-3p to chondrocytes, and promoting cartilage formation via the hsa-miR-455-3p/PAK2/Smad2/3 axis. Notably, Exo-Fect-EVs exhibit the highest efficiency. Collectively, the 3D culture conditions and engineered strategies have an impact on the miRNA profiles and cartilage regeneration capabilities of ADSCs-EVs. The findings provide valuable insights into the mechanisms underlying the promotion of cartilage regeneration by ADSCs-EVs.

CDKN1A regulation on chondrogenic differentiation of human chondrocytes in osteoarthritis through single-cell and bulk sequencing analysis

ObjectiveChondrocyte death is the hallmark of cartilage degeneration during osteoarthritis (OA). However, the specific pathogenesis of cell death in OA chondrocytes has not been elucidated. This study aims to validate the role of CDKN1A, a key programmed cell death (PCD)-related gene, in chondrogenic differentiation using a combination of single-cell and bulk sequencing approaches. DesignOA-related RNA-seq data (GSE114007, GSE55235, GSE152805) were downloaded from Gene Expression Omnibus database. PCD-related genes were obtained from GeneCards database. RNA-seq was performed to annotate the cell types in OA and control samples. Differentially expressed genes (DEGs) among those cell types (scRNA-DEGs) were screened. A nomogram of OA was constructed based on the featured genes, and potential drugs targeting the featured genes were predicted. The presence of key genes was confirmed using Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR), Western blot (WB), and immunohistochemistry (IHC). Micromass culture and Alcian blue staining were used to determine the effect of CDKN1A on chondrogenesis. ResultsSix cell types, namely HomC, HTC, RepC, preFC, FC, and RegC, were annotated in scRNA-seq data. Five featured genes (JUN, CDKN1A, HMGB2, DDIT3, and DDIT4) were screened by multiple biological information analysis methods. TAXOTERE had the highest ability to dock with DDIT3. Functional analysis indicated that CDKN1A was enriched in processes related to collagen catabolism and acts as a positive regulator of autophagy. Additionally, CDKN1A was found to be associated with several KEGG pathways, including those involved in acute myeloid leukemia and autoimmune thyroid disease. CDKN1A was confirmed down-regulated in the joint tissues of OA mouse model and OA model cell. Inhibiting the expression of CDKN1A can significantly suppress the differentiation of OA chondrocytes. ConclusionOur findings highlight the critical role of CDKN1A in promoting cartilage formation in both in vivo and in vitro and suggest its potential as a therapeutic target for OA treatment.

Skeletal growth is enhanced by a shared role for SOX8 and SOX9 in promoting reserve chondrocyte commitment to columnar proliferation

SOX8 was linked in a genome-wide association study to human height heritability, but roles in chondrocytes for this close relative of the master chondrogenic transcription factor SOX9 remain unknown. We undertook here to fill this knowledge gap. High-throughput assays demonstrate expression of human SOX8 and mouse Sox8 in growth plate cartilage. In situ assays show that Sox8 is expressed at a similar level as Sox9 in reserve and early columnar chondrocytes and turned off when Sox9 expression peaks in late columnar and prehypertrophic chondrocytes. Sox8-/- mice and Sox8fl/flPrx1Cre and Sox9fl/+Prx1Cre mice (inactivation in limb skeletal cells) have a normal or near normal skeletal size. In contrast, juvenile and adult Sox8fl/flSox9fl/+Prx1Cre compound mutants exhibit a 15 to 20% shortening of long bones. Their growth plate reserve chondrocytes progress slowly toward the columnar stage, as witnessed by a delay in down-regulating Pthlh expression, in packing in columns and in elevating their proliferation rate. SOX8 or SOX9 overexpression in chondrocytes reveals not only that SOX8 can promote growth plate cell proliferation and differentiation, even upon inactivation of endogenous Sox9, but also that it is more efficient than SOX9, possibly due to greater protein stability. Altogether, these findings uncover a major role for SOX8 and SOX9 in promoting skeletal growth by stimulating commitment of growth plate reserve chondrocytes to actively proliferating columnar cells. Further, by showing that SOX8 is more chondrogenic than SOX9, they suggest that SOX8 could be preferred over SOX9 in therapies to promote cartilage formation or regeneration in developmental and degenerative cartilage diseases.

Effect of glycosaminoglycans with different degrees of sulfation on chondrogenesis.

This study aims to investigate the effects and mechanisms of chondroitin sulfate (CS), dermatan sulfate (DS), and heparin (HEP) on chondrogenesis of murine chondrogenic cell line (ATDC5) cells and the maintenance of murine articular cartilage in vitro. ATDC5 and articular cartilage tissue explant were cultured in the medium containing different sulfated glycosaminoglycans. Cell proliferation, differentiation, cartilage formation, and mechanism were observed using cell proliferation assay, Alcian blue staining, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blot, respectively. Results showed that HEP and DS primarily activated the bone morphogenetic protein (BMP) signal pathway, while CS primarily activated the protein kinase B (AKT) signal pathway, further promoted ATDC5 cell proliferation and matrix production, and increased Sox9, Col2a1, and Aggrecan expression. This study investigated the differences and mechanisms of different sulfated glycosaminoglycans in chondrogenesis and cartilage homeostasis maintenance. HEP promotes cartilage formation and maintains the normal state of cartilage tissue in vitro, while CS plays a more effective role in the regeneration of damaged cartilage tissue.

Exosomes Derived From Kartogenin-Preconditioned Mesenchymal Stem Cells Promote Cartilage Formation and Collagen Maturation for Enthesis Regeneration in a Rat Model of Chronic Rotator Cuff Tear

Background: Poor tendon-to-bone healing in chronic rotator cuff tears (RCTs) is related to unsatisfactory outcomes. Exosomes derived from mesenchymal stem cells reportedly enhance rotator cuff healing. However, the difficulty in producing exosomes with a stronger effect on enthesis regeneration must be resolved. Purpose: To study the effect of exosomes derived from kartogenin (KGN)-preconditioned human bone marrow mesenchymal stem cells (KGN-Exos) on tendon-to-bone healing in a rat model of chronic RCT. Study Design: Controlled laboratory study. Methods: Exosome-loaded sodium alginate hydrogel (SAH) was prepared. Moreover, exosomes were labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) or 1,1′-dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate (Dil) for in vivo tracking. Bilateral rotator cuff repair (RCR) was conducted in an established chronic RCT rat model. A total of 66 rats were randomized to control, untreated exosome (un-Exos), and KGN-Exos groups to receive local injections of pure SAH, un-Exos, or KGN-Exos SAH at the repaired site. The presence of DiR/Dil-labeled exosomes was assessed at 1 day and 1 week, and tendon-to-bone healing was evaluated histologically, immunohistochemically, and biomechanically at 4 and 8 weeks. Results: Both un-Exos and KGN-Exos exhibited sustained release from SAH for up to 96 hours. In vivo study revealed that un-Exos and KGN-Exos were localized to the repaired site at 1 week. Moreover, the KGN-Exos group showed a higher histological score and increased glycosaminoglycan and collagen II expression at 4 and 8 weeks. In addition, more mature and better-organized collagen fibers with higher ratios of collagen I to collagen III were observed at 8 weeks in the tendon-to-bone interface compared with those in the control and un-Exos groups. Biomechanically, the KGN-Exos group had the highest failure load (28.12 ± 2.40 N) and stiffness (28.57 ± 2.49 N/mm) among the 3 groups at 8 weeks. Conclusion: Local injection of SAH with sustained KGN-Exos release could effectively promote cartilage formation as well as collagen maturation and organization for enthesis regeneration, contributing to enhanced biomechanical properties after RCR. Clinical Relevance: KGN-Exos injection may be used as a cell-free therapeutic option to accelerate tendon-to-bone healing in chronic RCT.

Progress on knee cartilage repair by knee joint distraction

Knee joint distraction is a new technology for the treatment of knee osteoarthritis in recent years. It could reduce knee pain and improve knee function, which is inseparable from the role of cartilage repair. The mechanism and influencing factors of knee joint distraction in repairing cartilage are the focus of current research. In this paper, the author reviewed literature and found that knee joint distraction could reduce knee joint load and provide a appropriate mechanical environment for cartilage repair, and it is resulting hydrostatic pressure fluctuation in the knee joint not only helps cartilage to absorb nutrients, but also promotes cartilage formation genes and inhibits cartilage matrix degrading enzyme gene expression. In addition, knee joint distraction creates conditions for synovial mesenchymal stem cells to be collected to cartilage injury, and improves ability of synovial mesenchymal stem cells to proliferate and differentiate into a chondrogenic lineage. Knee joint distraction could reduce inflammatory reaction and cartilage injury of knee joint by reducing content of inflammatory factors and inhibiting expression of inflammatory genes. At present, it is known that the factors affect repair of cartilage by knee joint distraction include, increasing weight-bearing activity and height and time of distraction is helpful for cartilage repair, male patients and patients with higher severity of knee osteoarthritis have better cartilage repair effect after knee joint distraction.The better efficacy of cartilage repair on the first year after knee joint distraction predicts a higher long-term survival rate of knee joint distraction with knee preservation. However, the research on the above hot spots is only at the initial stage and further exploration is still needed, in order to better guide clinical application of knee joint distraction.

Regeneration of Subcutaneous Cartilage in a Swine Model Using Autologous Auricular Chondrocytes and Electrospun Nanofiber Membranes Under Conditions of Varying Gelatin/PCL Ratios.

The scarcity of ideal biocompatible scaffolds makes the regeneration of cartilage in the subcutaneous environment of large animals difficult. We have previously reported the successful regeneration of good-quality cartilage in a nude mouse model using the electrospun gelatin/polycaprolactone (GT/PCL) nanofiber membranes. The GT/PCL ratios were varied to generate different sets of membranes to conduct the experiments. However, it is unknown whether these GT/PCL membranes can support the process of cartilage regeneration in an immunocompetent large animal model. We seeded swine auricular chondrocytes onto different GT/PCL nanofiber membranes (GT:PCL = 30:70, 50:50, and 70:30) under the sandwich cell-seeding mode. Prior to subcutaneously implanting the samples into an autologous host, they were cultured in vitro over a period of 2 weeks. The results revealed that the nanofiber membranes with different GT/PCL ratios could support the process of subcutaneous cartilage regeneration in an autologous swine model. The maximum extent of homogeneity in the cartilage tissues was achieved when the G5P5 (GT: PC = 50: 50) group was used for the regeneration of cartilage. The formed homogeneous cartilage tissues were characterized by the maximum cartilage formation ratio. The extents of the ingrowth of the fibrous tissues realized and the extents of infiltration of inflammatory cells achieved were found to be the minimum in this case. Quantitative analyses were conducted to determine the wet weight, cartilage-specific extracellular matrix content, and Young’s modulus. The results indicated that the optimal extent of cartilage formation was observed in the G5P5 group. These results indicated that the GT/PCL nanofiber membranes could serve as a potential scaffold for supporting subcutaneous cartilage regeneration under clinical settings. An optimum GT/PCL ratio can promote cartilage formation.

Senolytic Peptide FOXO4-DRI Selectively Removes Senescent Cells From in vitro Expanded Human Chondrocytes.

Autologous chondrocyte implantation (ACI) is a procedure used to treat articular cartilage injuries and prevent the onset of post-traumatic osteoarthritis. In vitro expansion of chondrocytes, a necessary step in ACI, results in the generation of senescent cells that adversely affect the quality and quantity of newly formed cartilage. Recently, a senolytic peptide, fork head box O transcription factor 4-D-Retro-Inverso (FOXO4-DRI), was reported to selectively kill the senescent fibroblasts. In this study, we hypothesized that FOXO4-DRI treatment could remove the senescent cells in the expanded chondrocytes, thus enhancing their potential in generating high-quality cartilage. To simulate the in vitro expansion for ACI, chondrocytes isolated from healthy donors were expanded to population doubling level (PDL) 9, representing chondrocytes ready for implantation. Cells at PDL3 were also used to serve as the minimally expanded control. Results showed that the treatment of FOXO4-DRI removed more than half of the cells in PDL9 but did not significantly affect the cell number of PDL3 chondrocytes. Compared to the untreated control, the senescence level in FOXO4-DRI treated PDL9 chondrocytes was significantly reduced. Based on the result from standard pellet culture, FOXO4-DRI pre-treatment did not enhance the chondrogenic potential of PDL9 chondrocytes. However, the cartilage tissue generated from FOXO4-DRI pretreated PDL9 cells displayed lower expression of senescence-relevant secretory factors than that from the untreated control group. Taken together, FOXO4-DRI is able to remove the senescent cells in PDL9 chondrocytes, but its utility in promoting cartilage formation from the in vitro expanded chondrocytes needs further investigation.

Scaffoldless tissue-engineered cartilage for studying transforming growth factor beta-mediated cartilage formation.

Reduced transforming growth factor beta (TGF-β) signaling is associated with osteoarthritis (OA). TGF-β is thought to act as a chondroprotective agent and provide anabolic cues to cartilage, thus acting as an OA suppressor in young, healthy cartilage. A potential approach for treating OA is to identify the factors that act downstream of TGF-β's anabolic pathway and target those factors to promote cartilage regeneration or repair. The aims of the present study were to (a) develop a scaffoldless tissue-engineered cartilage model with reduced TGF-β signaling and disrupted cartilage formation and (b) validate the system for identifying the downstream effectors of TGF-β that promote cartilage formation. Sox9 was used to validate the model because Sox9 is known to promote cartilage formation and TGF-β regulates Sox9 activity. Primary bovine articular chondrocytes were grown in Transwell supports to form cartilage tissues. An Alk5/TGF-β type I receptor inhibitor, SB431542, was used to attenuate TGF-β signaling, and an adenovirus encoding FLAG-Sox9 was used to drive the expression of Sox9 in the in vitro-generated cartilage. SB431542-treated tissues exhibited reduced cartilage formation including reduced thicknesses and reduced proteoglycan staining compared with control tissue. Expression of FLAG-Sox9 in SB431542-treated cartilage allowed the formation of cartilage despite antagonism of the TGF-β receptor. In summary, we developed a three-dimensional in vitro cartilage model with attenuated TGF-β signaling. Sox9 was used to validate the model for identification of anabolic agents that counteract loss of TGF-β signaling. This model has the potential to identify additional anabolic factors that could be used to repair or regenerate damaged cartilage.

Regulation of articular chondrocyte catabolic genes by growth factor interaction.

Osteoarthritis is characterized by a loss of articular cartilage homeostasis in which degradation exceeds formation. Several growth factors have been shown to promote cartilage formation by augmenting articular chondrocyte anabolic activity. This study tests the hypothesis that such growth factors also play an anticatabolic role. We transferred individual or combinations of the genes encoding insulin-like growth factor-I, bone morphogenetic protein-2, bone morphogenetic protein-7, transforming growth factor-β1, and fibroblast growth factor-2, into adult bovine articular chondrocytes and measured the expression of catabolic marker genes encoding A disintegrin and metalloproteinase with thrombospondin motifs-4 and -5, matrix metalloproteinases-3 and -13, and interleukin-6. When delivered individually, or in combination, these growth factor transgenes differentially regulated the direction, magnitude, and time course of expression of the catabolic marker genes. In concert, the growth factor transgenes regulated the marker genes in an interactive fashion that ranged from synergistic inhibition to synergistic stimulation. Synergistic stimulation prevailed over synergistic inhibition, reaching maxima of 15.2- and 2.7-fold, respectively. Neither the magnitude nor the time course of the effect of the transgene combinations could be predicted on the basis of the individual transgene effects. With few exceptions, the data contradict our hypothesis. The results demonstrate that growth factors that are traditionally viewed as chondrogenic tend also to promote catabolic gene expression. The competing actions of these potential therapeutic agents add an additional level of complexity to the selection of regulatory factors for restoring articular cartilage homeostasis or promoting repair.

ITGBL1 modulates integrin activity to promote cartilage formation and protect against arthritis.

Developing and mature chondrocytes constantly interact with and remodel the surrounding extracellular matrix (ECM). Recent research indicates that integrin-ECM interaction is differentially regulated during cartilage formation (chondrogenesis). Integrin signaling is also a key source of the catabolic reactions responsible for joint destruction in both rheumatoid arthritis and osteoarthritis. However, we do not understand how chondrocytes dynamically regulate integrin signaling in such an ECM-rich environment. Here, we found that developing chondrocytes express integrin-β-like 1 (Itgbl1) at specific stages, inhibiting integrin signaling and promoting chondrogenesis. Unlike cytosolic integrin inhibitors, ITGBL1 is secreted and physically interacts with integrins to down-regulate activity. We observed that Itgbl1 expression was strongly reduced in the damaged articular cartilage of patients with osteoarthritis (OA). Ectopic expression of Itgbl1 protected joint cartilage against OA development in the destabilization of the medial meniscus-induced OA mouse model. Our results reveal ITGBL1 signaling as an underlying mechanism of protection against destructive cartilage disorders and suggest the potential therapeutic utility of targeting ITGBL1 to modulate integrin signaling in human disease.

Effect of polycaprolactone-ascobic acid scaffold in repairing articular cartilage defects in rabbits

To investigate the effect of polycaprolactone-ascobic acid (PCL-AA) scaffolds in promoting repair of articular cartilage defects in a rabbit model. The cartilage defects (3.5 mm in diameter and 3.0 mm in depth) were created in the trochlear groove of the bilateral knees of eight 6-month-old male New Zealand white rabbits. The rabbit models were then randomized into 3 groups to receive implantation of PCL-AA scaffolds (group A, n=8), implantation of PCL scaffolds without AA (group B, n=5), or no treatment (group C, n=3). In groups A and B, the mixture of fibrin gel (10 µg) and thrombinogen (10 µg) was injected into the defects to fix the scaffolds during the surgery. Histological analyses and quantitative assessments of defect repair were conducted at 6 and 12 weeks after implantation of the scaffold. At 6 weeks after scaffold implantation, macroscopic observation showed better filling of the cartilage defects in group A than in group B, while no obvious defect repair was observed in group C. The rabbits in group A showed a significant improvement of the Wakitani score than those in group B (4.05∓1.11 vs 7.05∓0.98, P<0.05). HE staining revealed the presence of newly generated cells in and around the PCL-AA scaffolds without inflammatory cells. Safranin O staining showed a significantly greater ECM of the newly regenerated tissue in groups A and B than in group C (P<0.05), and the volume of the regenerated cartilage and cells was significantly greater in group A than in group B (P<0.05). Samples harvested at 12 weeks showed more hyalione-like cartilage formation than that at 6 weeks in group A. PCL-AA scaffolds have a good biocompatibility and promotes the healing of articular cartilage defects. Adding ascorbic acid into PCL scaffolds better promotes cartilage formation in terms of both quantity and quality of the regenerated tissues. PCL-AA scaffolds can serve as a promising biomaterial to promote the regeneration of articular cartilage using tissue engineering techniques.

Fracture healing physiology and the quest for therapies for delayed healing and nonunion.

ABSTRACTDelayed healing and nonunion of fractures represent enormous burdens to patients and healthcare systems. There are currently no approved pharmacological agents for the treatment of established nonunions, or for the acceleration of fracture healing, and no pharmacological agents are approved for promoting the healing of closed fractures. Yet several pharmacologic agents have the potential to enhance some aspects of fracture healing. In preclinical studies, various agents working across a broad spectrum of molecular pathways can produce larger, denser and stronger fracture calluses. However, untreated control animals in most of these studies also demonstrate robust structural and biomechanical healing, leaving unclear how these interventions might alter the healing of recalcitrant fractures in humans. This review describes the physiology of fracture healing, with a focus on aspects of natural repair that may be pharmacologically augmented to prevent or treat delayed or nonunion fractures (collectively referred to as DNFs). The agents covered in this review include recombinant BMPs, PTH/PTHrP receptor agonists, activators of Wnt/β‐catenin signaling, and recombinant FGF‐2. Agents from these therapeutic classes have undergone extensive preclinical testing and progressed to clinical fracture healing trials. Each can promote bone formation, which is important for the stability of bridged calluses, and some but not all can also promote cartilage formation, which may be critical for the initial bridging and subsequent stabilization of fractures. Appropriately timed stimulation of chondrogenesis and osteogenesis in the fracture callus may be a more effective approach for preventing or treating DNFs compared with stimulation of osteogenesis alone. © 2016 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 35:213–223, 2017.

Statins give bone growth a boost

The development of stem-cell-based models of two diseases that cause dwarfism reveals that statins — drugs that are used to treat high levels of blood cholesterol — may also promote cartilage formation and bone growth. See Article p.507 Some of the most common causes of dwarfism or skeletal dysplasia in humans are the result of gain-of-function mutations in the fibroblast growth factor receptor 3 gene (FGFR3). Noriyuki Tsumaki and colleagues have reprogrammed fibroblasts from patients with two such conditions — thanatophoric dysplasia type 1 (TD1) and achondroplasia (ACH) — to produce induced pluripotent stem cells (iPSCs). Chondrogenic differentiation of TD1 iPSCs resulted in the formation of degraded cartilage. A screen for molecules with the ability to rescue chondrogenically differentiated TD1 iPSCs from the degraded cartilage phenotype identified statins — drugs normally used to reduce blood cholesterol levels — as the most effective. Statins were included as candidate molecules because they have been reported to have anabolic effects on chondrocytes. In addition, statin treatment led to significant recovery of bone growth in a mouse model of ACH. These findings suggest that statins may have potential as a medical treatment for infants and children with TD1 and ACH.

Characterization of Segment Defect Regeneration in the Axolotl Fibula

We determined the critical size defect (CSD) for regeneration of the axolotl fibula by surgically removing bone segments comprising 10, 20, 40, and 50% of the length of the fibula in 15–20 cm axolotls. The limbs were first subjected to X‐ray and micro‐CT imaging. Half the specimens were stained with methylene blue/alizarin red for cartilage/bone and the other half sectioned and stained with H&amp;E. H&amp;E staining revealed initiation of cartilage formation in 10% and 20% defects by one month; cartilage and bone were regenerated by three months. None of the 40% defects regenerated cartilage by two months post‐operation, and 7/8 (87.5%) failed to regenerate cartilage at three months. None of the 50% defects regenerated cartilage after three months. H&amp;E staining showed that the 40% and 50% defects filled in with fibrous soft tissue. We concluded that the CSD is slightly less than 40% of the length of the fibula. A pig small intestine submucosa (SIS) scaffold did not promote regeneration across a 50% defect. The scaffold degraded by two months after implantation while the defect was filled by connective tissue and disorganized muscle fibers. Our aim is to use the axolotl fibula as a new, convenient model to screen for the optimum combination of growth factors/scaffold that promotes cartilage formation in a CSD, thus mimicking the first step of normal endochondral bone development and fracture repair.

Intermittent Administration of Human Parathyroid Hormone before Osteosynthesis Stimulates Cancellous Bone Union in Ovariectomized Rats

It has been reported that intermittent administration of human parathyroid hormone (h-PTH) promotes bone healing after surgery for osteoporotic fractures. If bone healing is promoted by the administration of h-PTH during pre-operative waiting period, we can prevent prolonged bed rest. Therefore, we evaluated the effects of pre-operative h-PTH treatment on cancellous bone union and its mechanism for fracture healing in ovariectomized rats as a model for osteoporosis. Ovariectomized 7-month-old female Sprague-Dawley rats underwent an osteotomy of the proximal tibia as a fracture model, and h-PTH (30 μg/kg body weight) or vehicle was administered as a pre-operative treatment for one week. After the one-week treatment, tibiae were fixed with wire for osteosynthesis, and h-PTH or vehicle was administered for 1 or 3 weeks following wire fixation. In addition to bone histomorphometry, we used alcian blue/hematoxylin stained sections for evaluating cartilage volume and immunostained sections for analyzing the expression of proliferating cell nuclear antigen (PCNA) for cell proliferation and that of Sox9 and Runx2, differentiation markers for cartilage cells and osteoblasts, respectively. Pre-operative treatment with PTH significantly increased bone volume. Pre-operative and pre- to post-operative treatment with PTH for 2 weeks significantly promoted bone union. Pre-operative treatment with PTH significantly increased cartilage volume, and pre- to post-operative treatment with PTH for 2 weeks significantly increased the percentage of cells positive for Runx2 (p < 0.01), but not PCNA or Sox9. Pre-operative administration of h-PTH enhances bone union by promoting cartilage formation and cell differentiation to osteoblasts, but not by promoting cell proliferation.

Advances in the research of gene therapy for chondral lesions

Cartilage injury has a very limited capacity of recovery by itself due to the lack of blood vessel in cartilage tissues. Current studies indicate that many kinds of cytokines have the function of promoting cartilage formation or repairing the injured cartilage with cartilage-like tissues. Cell-mediated transfer of the respective genes may ideally combine the supply of a chondrogenic cell population with the production of certain factors se-creted from the lesion and to promote the repairing of the lesion, which is considered the best treatment. Gene therapy based on this technology has developed rapidly in recent years. This review aims to summarize some of the development of the research in the field. Key words: Gene therapy; Cartilage repair; Cytokine