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.

JMJD3 promotes chondrocyte proliferation and hypertrophy during endochondral bone formation in mice

JMJD3(KDM6B)is an H3K27me3 demethylase and counteracts polycomb-mediated transcription repression.However,the function of JMJD3 in vivo is not well understood.Here we show that JMJD3 is highly expressed in cells of the chondrocyte lineage,especially in prehypertrophic and hypertrophic chondrocytes,during endochondral ossification.Homozygous deletion of Jmjd3 results in severely decreased proliferation and delayed hypertrophy of chondrocytes,and thereby marked retardation of endochondral ossification in mice.Genetically,JMJD3 associates with RUNX2 to promote proliferation and hypertrophy of chondrocytes.Biochemically,JMJD3 associates with and enhances RUNX2 activity by derepression of Runx2 and Ihh transcription throughits H3K27me3 demethylase activity.These results demonstrate that JMJD3 is a key epigenetic regulator in the process of cartilage maturation during endochondral bone formation.

Postnatal ex vivo rat model for longitudinal bone growth investigations

Background: Chondrocytes in the growth plate(GP) undergo increases in volume during different cascades of cell differentiation during longitudinal bone growth. The volume increase is reported to be the most significant variable in understanding the mechanism of long bone growth.Methods: Forty-five postnatal Sprague-Dawley rat pups, 7-15 days old were divided into nine age groups(P7-P15). Five pups were allocated to each group. The rats were sacrificed and tibia and metatarsal bones were harvested. Bone lengths were measured after 0, 24, 48, and 72 hours of ex vivo incubation. Histology of bones was carried out, and GP lengths and chondrocyte densities were determined.Results: There were significant differences in bone length among the age groups after 0 and 72 hours of incubation. Histological sectioning was possible in metatarsal bone from all age groups, and in tibia from 7-to 13-day-old rats. No significant differences in tibia and metatarsal GP lengths were seen among different age groups at 0 and 72 hours of incubation. Significant differences in chondrocyte densities along the epiphyseal GP of the bones between 0 and 72 hours of incubation were observed in most of the age groups.Conclusion: Ex vivo growth of tibia and metatarsal bones of rats aged 7-15 days old is possible, with percentage growth rates of 23.87 ± 0.80% and 40.38 ± 0.95%measured in tibia and metatarsal bone, respectively. Histological sectioning of bones was carried out without the need for decalcification in P7-P13 tibia and P7-P15 metatarsal bone. Increases in chondrocyte density along the GP influence overall bone elongation.

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.