High formability Mg-Zn-Gd wire facilitates ACL reconstruction via its swift degradation to accelerate intra-tunnel endochondral ossification

After reconstructing the anterior cruciate ligament(ACL),unsatisfactory bone tendon interface healing may often induce tunnel enlargement at the early healing stage.With good biological features and high formability,Magnesium-Zinc-Gadolinium(ZG21)wires are developed to bunch the tendon graft for matching the bone tunnel during transplantation.Microstructure,tensile strength,degradation,and cytotoxicity of ZG21 wire are evaluated.The rabbit model is used for assessing the biological effects of ZG21 wire by Micro-CT,histology,and mechanical test.The SEM/EDS,immunochemistry,and in vitro assessments are performed to investigate the underlying mechanism.Material tests demonstrate the high formability of ZG21 wire as surgical suture.Micro-CT shows ZG21 wire degradation accelerates tunnel bone formation,and histologically with earlier and more fibrocartilage regeneration at the healing interface.The mechanical test shows higher ultimate load in the ZG21 group.The SEM/EDS presents ZG21 wire degradation triggered calcium phosphate(Ca-P)deposition.IHC results demonstrate upregulation of Wnt3a,BMP2,and VEGF at the early phase and TGFβ3 and Type II collagen at the late phase of healing.In vitro tests also confirmed the Ca-P in the metal extract could elevate the expression of Wnt3a,βcatenin,ocn and opn to stimulate osteogenesis.Ex vivo tests of clinical samples indicated suturing with ZG21 wire did not weaken the ultimate loading of human tendon tissue.In conclusion,the ZG21 wire is feasible for tendon graft bunching.Its degradation products accelerated intra-tunnel endochondral ossification at the early healing stage and therefore enhanced bone-tendon interface healing in ACL reconstruction.

Ca_v3.3-mediated endochondral ossification in a three-dimensional bioprinted Gel MA hydrogel

The growth plate(GP)is a crucial tissue involved in skeleton development via endochondral ossification(EO).The bone organoid is a potential research model capable of simulating the physiological function,spatial structure,and intercellular communication of native GPs.However,mimicking the EO process remains a key challenge for bone organoid research.To simulate this orderly mineralization process,we designed an in vitro sh Ca_(v)3.3 ATDC5-loaded gelatin methacryloyl(Gel MA)hydrogel model and evaluated its bioprintability for future organoid construction.In this paper,we report the first demonstration that the T-type voltage-dependent calcium channel(T-VDCC)subtype Ca_(v)3.3 is dominantly expressed in chondrocytes and is negatively correlated with the hypertrophic differentiation of chondrocytes during the EO process.Furthermore,Ca_(v)3.3 knockdown chondrocytes loaded with the Gel MA hydrogel successfully captured the EO process and provide a bioink capable of constructing layered and orderly mineralized GP organoids in the future.The results of this study could therefore provide a potential target for regulating the EO process and a novel strategy for simulating it in bone organoids.

Bio-integrated scaffold facilitates large bone regeneration dominated by endochondral ossification

Repair of large bone defects caused by severe trauma,non-union fractures,or tumor resection remains challenging because of limited regenerative ability.Typically,these defects heal through mixed routines,including intramembranous ossification(IMO)and endochondral ossification(ECO),with ECO considered more efficient.Current strategies to promote large bone healing via ECO are unstable and require high-dose growth factors or complex cell therapy that cause side effects and raise expense while providing only limited benefit.Herein,we report a bio-integrated scaffold capable of initiating an early hypoxia microenvironment with controllable release of low-dose recombinant bone morphogenetic protein-2(rhBMP-2),aiming to induce ECO-dominated repair.Specifically,we apply a mesoporous structure to accelerate iron chelation,this promoting early chondrogenesis via deferoxamine(DFO)-induced hypoxia-inducible factor-1α(HIF-1α).Through the delicate segmentation of click-crosslinked PEGylated Poly(glycerol sebacate)(PEGS)layers,we achieve programmed release of low-dose rhBMP-2,which can facilitate cartilage-to-bone transformation while reducing side effect risks.We demonstrate this system can strengthen the ECO healing and convert mixed or mixed or IMO-guided routes to ECO-dominated approach in large-size models with clinical relevance.Collectively,these findings demonstrate a biomaterial-based strategy for driving ECO-dominated healing,paving a promising pave towards its clinical use in addressing large bone defects.

Enhanced bone regeneration via endochondral ossification using Exendin-4-modified mesenchymal stem cells

Nonunions and delayed unions pose significant challenges in orthopedic treatment,with current therapies often proving inadequate.Bone tissue engineering(BTE),particularly through endochondral ossification(ECO),emerges as a promising strategy for addressing critical bone defects.This study introduces mesenchymal stem cells overexpressing Exendin-4(MSC-E4),designed to modulate bone remodeling via their autocrine and paracrine functions.We established a type I collagen(Col-I)sponge-based in vitro model that effectively recapitulates the ECO pathway.MSC-E4 demonstrated superior chondrogenic and hypertrophic differentiation and enhanced the ECO cell fate in single-cell sequencing analysis.Furthermore,MSC-E4 encapsulated in microscaffold,effectively facilitated bone regeneration in a rat calvarial defect model,underscoring its potential as a therapeutic agent for bone regeneration.Our findings advocate for MSC-E4 within a BTE framework as a novel and potent approach for treating significant bone defects,leveraging the intrinsic ECO process.

The role of vascular endothelial growth factor in ossification

Osteogenesis and angiogenesis are two closely correlated processes during bone growth, development, remodelling and repair. Vascular endothelial growth factor （VEGF） is an essential mediator during the process of angiogenesis. Based on an extensive literature search, which was carried out using the PubMed database and the keywords of osteogenesis, VEGF, endochondral ossification and intramembranous ossification, this manuscript reviews the role of VEGF in ossification, with emphasis on its effect in endochondral and intramembranous ossification. Osteogenesis and angiogenesis are closely correlated processes. VEGF acts as an essential mediator durin~ these processes. It not only functions in bone an^io^enesis but also in various aspects of bone develooment.

Skeletal stem cells in bone development,homeostasis,and disease

Tissue-resident stem cells are essential for development and repair,and in the skeleton,this function is fulfilled by recently identified skeletal stem cells(SSCs).However,recent work has identified that SSCs are not monolithic,with long bones,craniofacial sites,and the spine being formed by distinct stem cells.Recent studies have utilized techniques such as fluorescence-activated cell sorting,lineage tracing,and single-cell sequencing to investigate the involvement of ssCs in bone development,homeostasis,and disease.These investigations have allowed researchers to map the lineage commitment trajectory of ssCs in different parts of the body and at different time points.Furthermore,recent studies have shed light on the characteristics of ssCs in both physiological and pathological conditions.This review focuses on discussing the spatiotemporal distribution of ssCs and enhancing our understanding of the diversity and plasticity of ssCs by summarizing recent discoveries.

HIF-1α and MIF enhance neutrophil-driven type 3 immunity and chondrogenesis in a murine spondyloarthritis model

The hallmarks of spondyloarthritis(SpA)are type 3 immunity-driven inflammation and new bone formation(NBF).Macrophage migration inhibitory factor(MIF)was found to be a key driver of the pathogenesis of SpA by amplifying type 3 immunity,yet MIF-interacting molecules and networks remain elusive.Herein,we identified hypoxia-inducible factor-1 alpha(HIF1A)as an interacting partner molecule of MIF that drives SpA pathologies,including inflammation and NBF.HIF1A expression was increased in the joint tissues and synovial fluid of SpA patients and curdlan-injected SKG(curdlan-SKG)mice compared to the respective controls.Under hypoxic conditions in which HIF1A was stabilized,human and mouse neutrophils exhibited substantially increased expression of MIF and IL-23,an upstream type 3 immunity-related cytokine.Similar to MIF,systemic overexpression of IL-23 induced SpA pathology in SKG mice,while the injection of a HIF1A-selective inhibitor(PX-478)into curdlan-SKG mice prevented or attenuated SpA pathology,as indicated by a marked reduction in the expression of MIF and IL-23.Furthermore,genetic deletion of MIF or HIF1A inhibition with PX-478 in IL-23-overexpressing SKG mice did not induce evident arthritis or NBF,despite the presence of psoriasis-like dermatitis and blepharitis.We also found that MIF-and IL-23-expressing neutrophils infiltrated areas of the NBF in curdlan-SKG mice.These neutrophils potentially increased chondrogenesis and cell proliferation via the upregulation of STAT3 in periosteal cells and ligamental cells during endochondral ossification.Together,these results provide supporting evidence for an MIF/HIF1A regulatory network,and inhibition of HIF1A may be a novel therapeutic approach for SpA by suppressing type 3 immunity-mediated inflammation and NBF.

The association and underlying mechanism of the digit ratio(2D:4D)in hypospadias

The second-to-fourth digit(2D:4D)ratio is thought to be associated with prenatal androgen exposure.However,the relationship between the 2D:4D ratio and hypospadias is poorly understood,and its molecular mechanism is not clear.In this study,by analyzing the hand digit length of 142 boys with hypospadias(23 distal,68 middle,and 51 proximal)and 196 controls enrolled in Shanghai Children’s Hospital(Shanghai,China)from December 2020 to December 2021,we found that the 2D:4D ratio was significantly increased in boys with hypospadias(P<0.001)and it was positively correlated with the severity of the hypospadias.This was further verified by the comparison of control mice and prenatal low testosterone mice model obtained by knocking out the risk gene(dynein axonemal heavy chain 8[DNAH8])associated with hypospadias.Furthermore,the discrepancy was mainly caused by a shift in 4D.Proteomic characterization of a mouse model validated that low testosterone levels during pregnancy can impair the growth and development of 4D.Comprehensive mechanistic explorations revealed that during the androgen-sensitive window,the downregulation of the androgen receptor(AR)caused by low testosterone levels,as well as the suppressed expression of chondrocyte proliferation-related genes such as Wnt family member 5a(Wnt5a),Wnt5b,Smad family member 2(Smad2),and Smad3;mitochondrial function-related genes in cartilage such as AMP-activated protein kinase(AMPK)and nuclear respiratory factor 1(Nrf-1);and vascular development-related genes such as myosin light chain(MLC),notch receptor 3(Notch3),and sphingosine kinase 1(Sphk1),are responsible for the limitation of 4D growth,which results in a higher 2D:4D ratio in boys with hypospadias via decreased endochondral ossification.This study indicates that the ratio of 2D:4D is a risk marker of hypospadias and provides a potential molecular mechanism.

Sox9 augments BMP2-induced chondrogenic differentiation by downregulating Smad7 in mesenchymal stem cells(MSCs)

Cartilage injuries caused by arthritis or trauma pose formidable challenges for effective clinical management due to the limited intrinsic proliferative capability of chondrocytes.Autologous stem cell-based therapies and transgene-enhanced cartilage tissue engineering may open new avenues for the treatment of cartilage injuries.Bone morphogenetic protein 2(BMP2)induces effective chondrogenesis of mesenchymal stem cells(MSCs)and can thus be explored as a potential therapeutic agent for cartilage defect repair.However,BMP2 also induces robust endochondral ossification.Although the precise mechanisms through which BMP2 governs the divergence of chondrogenesis and osteogenesis remain to be fully understood,blocking endochondral ossification during BMP2-induced cartilage formation may have practical significance for cartilage tissue engineering.Here,we investigate the role of Sox9-donwregulated Smad7 in BMP2-induced chondrogenic differentiation of MSCs.We find that overexpression of Sox9 leads to a decrease in BMP2-induced Smad7 expression in MSCs.Sox9 inhibits BMP2-induced expression of osteopontin while enhancing the expression of chondrogenic marker Col2a1 in MSCs.Forced expression of Sox9 in MSCs promotes BMP2-induced chondrogenesis and suppresses BMP2-induced endochondral ossification.Constitutive Smad7 expression inhibits BMP2-induced chondrogenesis in stem cell implantation assay.Mouse limb explant assay reveals that Sox9 expands BMP2-stimulated chondrocyte proliferating zone while Smad7 promotes BMP2-intitated hypertrophic zone of the growth plate.Cell cycle analysis indicates that Smad7 induces significant early apoptosis in BMP2-stimulated MSCs.Taken together,our results strongly suggest that Sox9 may facilitate BMP2-induced chondrogenesis by downregulating Smad7,which can be exploited for effective cartilage tissue engineering.

New perspective of skeletal stem cells

Tissue-resident stem cells are a group of stem cells distinguished by their capacity for self-renewal and multilineage differentiation capability with tissue specificity.Among these tissue-resident stem cells,skeletal stem cells(SSCs)were discovered in the growth plate region through a combination of cell surface markers and lineage tracing series.With the process of unravelling the anatomical variation of SSCs,researchers were also keen to investigate the developmental diversity outside the long bones,including in the sutures,craniofacial sites,and spinal regions.Recently,fluorescence-activated cell sorting,lineage tracing,and single-cell sequencing have been used to map lineage trajectories by studying SSCs with different spatiotemporal distributions.The SSC niche also plays a pivotal role in regulating SSC fate,such as cell-cell interactions mediated by multiple signalling pathways.This review focuses on discussing the spatial and temporal distribution of SSCs,and broadening our understanding of the diversity and plasticity of SSCs by summarizing the progress of research into SSCs in recent years.