Using a new, non-invasive method, we measured the patellofemoral force (PFF) in cadaver knees mounted in a rig to simulate weight-bearing. The PFF was measured from 20° to 120° of flexion before and after implanting three designs of knee prosthesis.

Medial unicompartmental arthroplasty with a meniscal-bearing prosthesis and with retention of both cruciate ligaments caused no significant change in the PFF. After arthroplasty with a posterior-cruciate-retaining prosthesis and division of the anterior cruciate ligament, the PFF decreased in extension and increased by 20% in flexion. Implantation of a posterior stabilised prosthesis and division of both cruciate ligaments produced a decrease in the PFF in extension but maintained normal load in flexion.

There was a direct relationship between the PFF and the angle made with the patellar tendon and the long axis of the tibia. The abnormalities of the patellar tendon angle which resulted from implantation of the two total prostheses explain the observed changes in the PFF and show how the mechanics of the patellofemoral joint depend upon the kinematics of the tibiofemoral articulation.

This is part of a larger study designed to investigate the action of particulate metals of orthopaedic interest on tissues. Damaging effects were determined by cytological examination and the assay of two enzymes. Lactic dehydrogenase (LDH) if released into the supernatant indicates a damaged cell membrane; decreased intracellular levels of glucose-6-phosphate dehydrogenase (G6PD) indicates a lowered phagocytic capacity of the cells. Soluble and wear products around implanted prostheses could facilitate late infections by impairing local reactions to bacteria. Particulate cobalt, nickel and cobalt-chromium alloy were found to damage the cells and to cause LDH release. G6PD was found to have a lower activity in the cells exposed to these materials. In contrast, titanium, chromium and molybdenum were well tolerated by macrophages and had no effect on the distribution and activity of either enzyme. The solubility of these metals in the culture medium was also measured.

Sterilisation by gamma irradiation in the presence of air causes free radicals generated in polyethylene (PE) to react with oxygen, which could lead to loss of physical properties and reduction in fatigue strength. Tissue retrieved from failed total hip replacements often has large quantities of particulate PE and most particles associated with peri-implant osteolysis are oxidised. Consequently, an understanding of the cellular responses of oxidised PE particles may lead to clarification of the pathogenesis of osteolysis and aseptic loosening.

We have used the agarose system to demonstrate the differential effects of oxidised and non-oxidised PE particles on the release of proinflammatory products such as interleukin-1β (IL-1β), IL-6, and tumour necrosis factor-α (TNF-α) from monocytes/ macrophages (M/M). Oxidised PE particles were shown to stimulate human M/M to phagocytose and to release cytokines. Oxidation may alter the surface chemistry of the particles and enhance the response to specific membrane receptors on macrophages, such as scavenger-type receptors.

We exposed human macrophages isolated from the peripheral blood of healthy donors to metal and bone-cement particles from 0.2 to 10 μm in size.

Zymography showed that macrophages exposed to titanium alloy and polymethylmethacrylate (PMMA) particles released a 92- and 72-kDa gelatinase in a dose- and time-dependent manner. Western immunoblotting confirmed that the 92- and 72-kDa gelatinolytic activities corresponded to matrix metalloproteinase-9 and matrix metalloproteinase-2 (MMP-9, MMP-2), respectively. Western immunoblotting also indicated that titanium alloy and PMMA particles increased the release of MMP-1. Northern blotting showed elevated mRNA signal levels for MMP-1, MMP-2, and MMP-9 after exposure to both types of particle. Collagenolytic activity also increased in the macrophage culture medium in response to both types of particle.

Our findings support the hypothesis that macrophages release MMPs in proportion to the amount of particulate debris within periprosthetic tissues.

1. Methods for culturing cells isolated from slices of arthritic human or normal mammalian cancellous bone are described.

2. The capacity of the cultured cells to take up and hydroxylate labelled proline has been investigated.

3. Sections of the partially decalcified bone and of the isolated cells have been examined by transmission electron microscopy.

4. The possible significance of the results and observations are discussed. We are deeply grateful to Dame Janet Vaughan, who very kindly read this manuscript and made several valuable suggestions and criticisms. We are much obliged to Dr Sylvia Fitton-Jackson for her advice on the techniques of tissue culture and for giving us the composition of her chemically defined medium. Dr Palfrey kindly allowed one of us, M. J. Dickens, to learn transmission electron microscopy in his department at St Thomas's Hospital Medical School under the expert tuition of Mr G. Maxwell. Mr R. Hockhan and Mr M. Hepburn of the University of Surrey Structural Studies Unit helpfully instructed in the operation of the transmission electron microscope. Our special thanks are due to Mr E. P. Morris for his competent and enthusiastic technical assistance.

It is well recognised that meniscal tears situated within the inner, avascular region do not heal. We investigated the potential effect of insulin-like growth factor-I (IGF-I) in promoting regeneration of meniscal tissue in the inner, middle and outer zones of the meniscus. Sheep menisci were harvested and monolayer cell cultures prepared. Various concentrations of IGF-I were used in the presence or absence of 10% fetal calf serum (FCS). We measured the uptake of radioactive thymidine, sulphur, and proline to assess cell proliferation and formation of extracellular matrix (ECM). IGF-I, in the presence or absence of FCS, increased the formation of DNA and ECM in all meniscal zones. However, the response of the cells from the avascular zone was greater than that from the vascular zone. Our findings indicate that fibrochondrocytes cultured from avascular meniscal tissue have the ability to regenerate when exposed to anabolic cytokines such as IGF-I.

Aims. In this investigation, we administered oxidative stress to nucleus pulposus cells (NPCs), recognized DNA-damage-inducible transcript 4 (DDIT4) as a component in intervertebral disc degeneration (IVDD), and devised a hydrogel capable of conveying small interfering RNA (siRNA) to IVDD. Methods. An in vitro model for oxidative stress-induced injury in NPCs was developed to elucidate the mechanisms underlying the upregulation of DDIT4 expression, activation of the reactive oxygen species (ROS)-thioredoxin-interacting protein (TXNIP)-NLRP3 signalling pathway, and nucleus pulposus pyroptosis. Furthermore, the mechanism of action of small interfering DDIT4 (siDDIT4) on NPCs in vitro was validated. A triplex hydrogel named siDDIT4@G5-P-HA was created by adsorbing siDDIT4 onto fifth-generation polyamidoamine (PAMAM) dendrimer using van der Waals interactions, and then coating it with hyaluronic acid (HA). In addition, we established a rat puncture IVDD model to decipher the hydrogel’s mechanism in IVDD. Results. A correlation between DDIT4 expression levels and disc degeneration was shown with human nucleus pulposus and needle-punctured rat disc specimens. We confirmed that DDIT4 was responsible for activating the ROS-TXNIP-NLRP3 axis during oxidative stress-induced pyroptosis in rat nucleus pulposus in vitro. Mitochondria were damaged during oxidative stress, and DDIT4 contributed to mitochondrial damage and ROS production. In addition, siDDIT4@G5-P-HA hydrogels showed good delivery activity of siDDIT4 to NPCs. In vitro studies illustrated the potential of the siDDIT4@G5-P-HA hydrogel for alleviating IVDD in rats. Conclusion. DDIT4 is a key player in mediating pyroptosis and IVDD in NPCs through the ROS-TXNIP-NLRP3 axis. Additionally, siDDIT4@G5-P-HA hydrogel has been found to relieve IVDD in rats. Our research offers an innovative treatment option for IVDD. Cite this article: Bone Joint Res 2024;13(5):247–260

Aims. Transcription factor nuclear factor kappa B (NF-κB) plays a major role in the pathogenesis of chronic inflammatory diseases in all organ systems. Despite its importance, NF-κB targeted drug therapy to mitigate chronic inflammation has had limited success in preclinical studies. We hypothesized that sex differences affect the response to NF-κB treatment during chronic inflammation in bone. This study investigated the therapeutic effects of NF-κB decoy oligodeoxynucleotides (ODN) during chronic inflammation in male and female mice. Methods. We used a murine model of chronic inflammation induced by continuous intramedullary delivery of lipopolysaccharide-contaminated polyethylene particles (cPE) using an osmotic pump. Specimens were evaluated using micro-CT and histomorphometric analyses. Sex-specific osteogenic and osteoclastic differentiation potentials were also investigated in vitro, including alkaline phosphatase, Alizarin Red, tartrate-resistant acid phosphatase staining, and gene expression using reverse transcription polymerase chain reaction (RT-PCR). Results. Local delivery of NF-κB decoy ODN in vivo increased osteogenesis in males, but not females, in the presence of chronic inflammation induced by cPE. Bone resorption activity was decreased in both sexes. In vitro osteogenic and osteoclastic differentiation assays during inflammatory conditions did not reveal differences among the groups. Receptor activator of nuclear factor kappa Β ligand (Rankl) gene expression by osteoblasts was significantly decreased only in males when treated with ODN. Conclusion. We demonstrated that NF-κB decoy ODN increased osteogenesis in male mice and decreased bone resorption activity in both sexes in preclinical models of chronic inflammation. NF-κB signalling could be a therapeutic target for chronic inflammatory diseases involving bone, especially in males. Cite this article: Bone Joint Res 2024;13(1):28–39

Aims. The antidiabetic agent metformin inhibits fibrosis in various organs. This study aims to elucidate the effects of hyperglycaemia and metformin on knee joint capsule fibrosis in mice. Methods. Eight-week-old wild-type (WT) and type 2 diabetic (db/db) mice were divided into four groups without or with metformin treatment (WT met(-/+), Db met(-/+)). Mice received daily intraperitoneal administration of metformin and were killed at 12 and 14 weeks of age. Fibrosis morphology and its related genes and proteins were evaluated. Fibroblasts were extracted from the capsules of 14-week-old mice, and the expression of fibrosis-related genes in response to glucose and metformin was evaluated in vitro. Results. The expression of all fibrosis-related genes was higher in Db met(-) than in WT met(-) and was suppressed by metformin. Increased levels of fibrosis-related genes, posterior capsule thickness, and collagen density were observed in the capsules of db/db mice compared with those in WT mice; these effects were suppressed by metformin. Glucose addition increased fibrosis-related gene expression in both groups of mice in vitro. When glucose was added, metformin inhibited the expression of fibrosis-related genes other than cellular communication network factor 2 (Ccn2) in WT mouse cells. Conclusion. Hyperglycaemia promotes fibrosis in the mouse knee joint capsule, which is inhibited by metformin. These findings can help inform the development of novel strategies for treating knee joint capsule fibrosis. Cite this article: Bone Joint Res 2024;13(7):321–331

Aims. After a few passages of in vitro culture, primary human articular chondrocytes undergo senescence and loss of their phenotype. Most of the available chondrocyte cell lines have been obtained from cartilage tissues different from diarthrodial joints, and their utility for osteoarthritis (OA) research is reduced. Thus, the goal of this research was the development of immortalized chondrocyte cell lines proceeded from the articular cartilage of patients with and without OA. Methods. Using telomerase reverse transcriptase (hTERT) and SV40 large T antigen (SV40LT), we transduced primary OA articular chondrocytes. Proliferative capacity, degree of senescence, and chondrocyte surface antigen expression in transduced chondrocytes were evaluated. In addition, the capacity of transduced chondrocytes to synthesize a tissue similar to cartilage and to respond to interleukin (IL)-1β was assessed. Results. Coexpression of both transgenes (SV40 and hTERT) were observed in the nuclei of transduced chondrocytes. Generated chondrocyte cell lines showed a high proliferation capacity and less than 2% of senescent cells. These cell lines were able to form 3D aggregates analogous to those generated by primary articular chondrocytes, but were unsuccessful in synthesizing cartilage-like tissue when seeded on type I collagen sponges. However, generated chondrocyte cell lines maintained the potential to respond to IL-1β stimulation. Conclusion. Through SV40LT and hTERT transduction, we successfully immortalized chondrocytes. These immortalized chondrocytes were able to overcome senescence in vitro, but were incapable of synthesizing cartilage-like tissue under the experimental conditions. Nonetheless, these chondrocyte cell lines could be advantageous for OA investigation since, similarly to primary articular chondrocytes, they showed capacity to upregulate inflammatory mediators in response to the IL-1β cytokine. Cite this article: Bone Joint Res 2023;12(1):46–57

Aims. The goal was to evaluate tibiofemoral knee joint kinematics during stair descent, by simulating the full stair descent motion in vitro. The knee joint kinematics were evaluated for two types of knee implants: bi-cruciate retaining and bi-cruciate stabilized. It was hypothesized that the bi-cruciate retaining implant better approximates native kinematics. Methods. The in vitro study included 20 specimens which were tested during a full stair descent with physiological muscle forces in a dynamic knee rig. Laxity envelopes were measured by applying external loading conditions in varus/valgus and internal/external direction. Results. The laxity results show that both implants are capable of mimicking the native internal/external-laxity during the controlled lowering phase. The kinematic results show that the bi-cruciate retaining implant tends to approximate the native condition better compared to bi-cruciate stabilized implant. This is valid for the internal/external rotation and the anteroposterior translation during all phases of the stair descent, and for the compression-distraction of the knee joint during swing and controlled lowering phase. Conclusion. The results show a better approximation of the native kinematics by the bi-cruciate retaining knee implant compared to the bi-cruciate stabilized knee implant for internal/external rotation and anteroposterior translation. Whether this will result in better patient outcomes remains to be investigated. Cite this article: Bone Joint Res 2023;12(4):285–293

Aims. There is a lack of biomaterial-based carriers for the local delivery of rifampicin (RIF), one of the cornerstone second defence antibiotics for bone infections. RIF is also known for causing rapid development of antibiotic resistance when given as monotherapy. This in vitro study evaluated a clinically used biphasic calcium sulphate/hydroxyapatite (CaS/HA) biomaterial as a carrier for dual delivery of RIF with vancomycin (VAN) or gentamicin (GEN). Methods. The CaS/HA composites containing RIF/GEN/VAN, either alone or in combination, were first prepared and their injectability, setting time, and antibiotic elution profiles were assessed. Using a continuous disk diffusion assay, the antibacterial behaviour of the material was tested on both planktonic and biofilm-embedded forms of standard and clinical strains of Staphylococcus aureus for 28 days. Development of bacterial resistance to RIF was determined by exposing the biofilm-embedded bacteria continuously to released fractions of antibiotics from CaS/HA-antibiotic composites. Results. Following the addition of RIF to CaS/HA-VAN/GEN, adequate injectability and setting of the CaS/HA composites were noted. Sustained release of RIF above the minimum inhibitory concentrations of S. aureus was observed until study endpoint (day 35). Only combinations of CaS/HA-VAN/GEN + RIF exhibited antibacterial and antibiofilm effects yielding no viable bacteria at study endpoint. The S. aureus strains developed resistance to RIF when biofilms were subjected to CaS/HA-RIF alone but not with CaS/HA-VAN/GEN + RIF. Conclusion. Our in vitro results indicate that biphasic CaS/HA loaded with VAN or GEN could be used as a carrier for RIF for local delivery in clinically demanding bone infections. Cite this article: Bone Joint Res 2022;11(11):787–802

Aims. This study aimed to demonstrate the promoting effect of elastic fixation on fracture, and further explore its mechanism at the gene and protein expression levels. Methods. A closed tibial fracture model was established using 12 male Japanese white rabbits, and divided into elastic and stiff fixation groups based on different fixation methods. Two weeks after the operation, a radiograph and pathological examination of callus tissue were used to evaluate fracture healing. Then, the differentially expressed proteins (DEPs) were examined in the callus using proteomics. Finally, in vitro cell experiments were conducted to investigate hub proteins involved in this process. Results. Mean callus volume was larger in the elastic fixation group (1,755 mm. 3. (standard error of the mean (SEM) 297)) than in the stiff fixation group (258 mm. 3. (SEM 65)). Pathological observation found that the expression levels of osterix (OSX), collagen, type I, alpha 1 (COL1α1), and alkaline phosphatase (ALP) in the callus of the elastic fixation group were higher than those of the stiff fixation group. The protein sequence of the callus revealed 199 DEPs, 124 of which were highly expressed in the elastic fixation group. In the in vitro study, it was observed that a stress of 200 g led to upregulation of thrombospondin 1 (THBS1) and osteoglycin (OGN) expression in bone marrow mesenchymal stem cells (BMSCs). Additionally, these genes were found to be upregulated during the osteogenic differentiation process of the BMSCs. Conclusion. Elastic fixation can promote fracture healing and osteoblast differentiation in callus, and the ability of elastic fixation to promote osteogenic differentiation of BMSCs may be achieved by upregulating genes such as THBS1 and OGN. Cite this article: Bone Joint Res 2024;13(10):559–572

Aims. There is a considerable challenge in treating bone infections and orthopaedic device-associated infection (ODAI), partly due to impaired penetration of systemically administrated antibiotics at the site of infection. This may be circumvented by local drug administration. Knowledge of the release kinetics from any carrier material is essential for proper application. Ceftriaxone shows a particular constant release from calcium sulphate (CaSO. 4. ) in vitro, and is particularly effective against streptococci and a large portion of Gram-negative bacteria. We present the clinical release kinetics of ceftriaxone-loaded CaSO. 4. applied locally to treat ODAI. Methods. A total of 30 operations with ceftriaxone-loaded CaSO. 4. had been performed in 28 patients. Ceftriaxone was applied as a single local antibiotic in 21 operations and combined with vancomycin in eight operations, and in an additional operation with vancomycin and amphotericin B. Sampling of wound fluid was performed from drains or aspirations. Ceftriaxone concentrations were measured by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Results. A total of 37 wound fluid concentrations from 16 operations performed in 14 patients were collected. The ceftriaxone concentrations remained approximately within a range of 100 to 200 mg/l up to three weeks. The median concentration was 108.9 mg/l (interquartile range 98.8 to 142.5) within the first ten days. No systemic adverse reactions were observed. Conclusion. Our study highlights new clinical data of locally administered ceftriaxone with CaSO. 4. as carrier material. The near-constant release of ceftriaxone from CaSO. 4. observed in vitro could be confirmed in vivo. The concentrations remained below known local toxicity thresholds. Cite this article: Bone Joint Res 2022;11(11):835–842

Aims. Bacterial infection activates neutrophils to release neutrophil extracellular traps (NETs) in bacterial biofilms of periprosthetic joint infections (PJIs). The aim of this study was to evaluate the increase in NET activation and release (NETosis) and haemostasis markers in the plasma of patients with PJI, to evaluate whether such plasma induces the activation of neutrophils, to ascertain whether increased NETosis is also mediated by reduced DNaseI activity, to explore novel therapeutic interventions for NETosis in PJI in vitro, and to evaluate the potential diagnostic use of these markers. Methods. We prospectively recruited 107 patients in the preoperative period of prosthetic surgery, 71 with a suspicion of PJI and 36 who underwent arthroplasty for non-septic indications as controls, and obtained citrated plasma. PJI was confirmed in 50 patients. We measured NET markers, inflammation markers, DNaseI activity, haemostatic markers, and the thrombin generation test (TGT). We analyzed the ability of plasma from confirmed PJI and controls to induce NETosis and to degrade in vitro-generated NETs, and explored the therapeutic restoration of the impairment to degrade NETs of PJI plasma with recombinant human DNaseI. Finally, we assessed the contribution of these markers to the diagnosis of PJI. Results. Patients with confirmed PJI had significantly increased levels of NET markers (cfDNA (p < 0.001), calprotectin (p < 0.001), and neutrophil elastase (p = 0.022)) and inflammation markers (IL-6; p < 0.001) in plasma. Moreover, the plasma of patients with PJI induced significantly more neutrophil activation than the plasma of the controls (p < 0.001) independently of tumour necrosis factor alpha. Patients with PJI also had a reduced DNaseI activity in plasma (p < 0.001), leading to a significantly impaired degradation of NETs (p < 0.001). This could be therapeutically restored with recombinant human DNaseI to the level in the controls. We developed a model to improve the diagnosis of PJI with cfDNA, calprotectin, and the start tail of TGT as predictors, though cfDNA alone achieved a good prediction and is simpler to measure. Conclusion. We confirmed that patients with PJI have an increased level of NETosis in plasma. Their plasma both induced NET release and had an impaired ability to degrade NETs mediated by a reduced DNaseI activity. This can be therapeutically restored in vitro with the approved Dornase alfa, Pulmozyme, which may allow novel methods of treatment. A combination of NETs and haemostatic biomarkers could improve the diagnosis of PJI, especially those patients in whom this diagnosis is uncertain. Cite this article: Bone Joint J 2024;106-B(9):1021–1030

Aims. It has been established that mechanical stimulation benefits tendon-bone (T-B) healing, and macrophage phenotype can be regulated by mechanical cues; moreover, the interaction between macrophages and mesenchymal stem cells (MSCs) plays a fundamental role in tissue repair. This study aimed to investigate the role of macrophage-mediated MSC chondrogenesis in load-induced T-B healing in depth. Methods. C57BL/6 mice rotator cuff (RC) repair model was established to explore the effects of mechanical stimulation on macrophage polarization, transforming growth factor (TGF)-β1 generation, and MSC chondrogenesis within T-B enthesis by immunofluorescence and enzyme-linked immunosorbent assay (ELISA). Macrophage depletion was performed by clodronate liposomes, and T-B healing quality was evaluated by histology and biomechanics. In vitro, bone marrow-derived macrophages (BMDMs) were stretched with CELLOAD-300 load system and macrophage polarization was identified by flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR). MSC chondrogenic differentiation was measured by histochemical analysis and qRT-PCR. ELISA and qRT-PCR were performed to screen the candidate molecules that mediated the pro-chondrogenic function of mechanical stimulated BMDMs. Results. Mechanical stimulation promoted macrophage M2 polarization in vivo and in vitro. The conditioned media from mechanically stimulated BMDMs (MS-CM) enhanced MSC chondrogenic differentiation, and mechanically stimulated BMDMs generated more TGF-β1. Further, neutralizing TGF-β1 in MS-CM can attenuate its pro-chondrogenic effect. In vivo, mechanical stimulation promoted TGF-β1 generation, MSC chondrogenesis, and T-B healing, which were abolished following macrophage depletion. Conclusion. Macrophages subjected to appropriate mechanical stimulation could polarize toward the M2 phenotype and secrete TGF-β1 to promote MSC chondrogenesis, which subsequently augments T-B healing. Cite this article: Bone Joint Res 2023;12(3):219–230

Aims. To explore the novel molecular mechanisms of histone deacetylase 4 (HDAC4) in chondrocytes via RNA sequencing (RNA-seq) analysis. Methods. Empty adenovirus (EP) and a HDAC4 overexpression adenovirus were transfected into cultured human chondrocytes. The cell survival rate was examined by real-time cell analysis (RTCA) and EdU and flow cytometry assays. Cell biofunction was detected by Western blotting. The expression profiles of messenger RNAs (mRNAs) in the EP and HDAC4 transfection groups were assessed using whole-transcriptome sequencing (RNA-seq). Volcano plot, Gene Ontology, and pathway analyses were performed to identify differentially expressed genes (DEGs). For verification of the results, the A289E/S246/467/632 A sites of HDAC4 were mutated to enhance the function of HDAC4 by increasing HDAC4 expression in the nucleus. RNA-seq was performed to identify the molecular mechanism of HDAC4 in chondrocytes. Finally, the top ten DEGs associated with ribosomes were verified by quantitative polymerase chain reaction (QPCR) in chondrocytes, and the top gene was verified both in vitro and in vivo. Results. HDAC4 markedly improved the survival rate and biofunction of chondrocytes. RNA-seq analysis of the EP and HDAC4 groups showed that HDAC4 induced 2,668 significant gene expression changes in chondrocytes (1,483 genes upregulated and 1,185 genes downregulated, p < 0.05), and ribosomes exhibited especially large increases. The results were confirmed by RNA-seq of the EP versus mutated HDAC4 groups and the validations in vitro and in vivo. Conclusion. The enhanced ribosome pathway plays a key role in the mechanism by which HDAC4 improves the survival rate and biofunction of chondrocytes. Cite this article: Bone Joint Res 2023;12(7):433–446

Aims. To verify whether secretory leucocyte protease inhibitor (SLPI) can promote early tendon-to-bone healing after anterior cruciate ligament (ACL) reconstruction. Methods. In vitro: the mobility of the rat bone mesenchymal stem cells (BMSCs) treated with SLPI was evaluated by scratch assay. Then the expression levels of osteogenic differentiation-related genes were analyzed by real-time quantitative PCR (qPCR) to determine the osteogenic effect of SLPI on BMSCs. In vivo: a rat model of ACL reconstruction was used to verify the effect of SLPI on tendon-to-bone healing. All the animals of the SLPI group and the negative control (NC) group were euthanized for histological evaluation, micro-CT scanning, and biomechanical testing. Results. SLPI improved the migration ability of BMSCs and upregulated the expression of genes related to osteogenic differentiation of BMSCs in vitro. In vivo, the SLPI group had higher histological scores at the tendon-bone interface by histological evaluation. Micro-CT showed more new bone formation and bone ingrowth around the grafted tendon in the SLPI group. Evaluation of the healing strength of the tendon-bone connection showed that the SLPI group had a higher maximum failure force and stiffness. Conclusion. SLPI can effectively promote early tendon-to-bone healing after ACL reconstruction via enhancing the migration and osteogenic differentiation of BMSCs. Cite this article: Bone Joint Res 2022;11(7):503–512

Aims. Alcoholism is a well-known detrimental factor in fracture healing. However, the underlying mechanism of alcohol-inhibited fracture healing remains poorly understood. Methods. MicroRNA (miR) sequencing was performed on bone mesenchymal stem cells (BMSCs). The effects of alcohol and miR-19a-3p on vascularization and osteogenic differentiation were analyzed in vitro using BMSCs and human umbilical vein endothelial cells (HUVECs). An in vivo alcohol-fed mouse model of femur fracture healing was also established, and radiological and histomorphometric analyses were used to evaluate the role of miR-19a-3p. The binding of miR-19a-3p to forkhead box F2 (FOXF2) was analyzed using a luciferase reporter assay. Results. miR-19a-3p was identified as one of the key regulators in the osteogenic differentiation of BMSCs, and was found to be downregulated in the alcohol-fed mouse model of fracture healing. In vitro, miR-19a-3p expression was downregulated after ethanol administration in both BMSCs and HUVECs. Vascularization and osteogenic differentiation were independently suppressed by ethanol and reversed by miR-19a-3p. In addition, the luciferase reporter assay showed that FOXF2 is the direct binding target of miR-19a-3p. In vivo, miR-19a-3p agomir stimulated callus transformation and improved the alcohol-impaired fracture healing. Conclusion. This study is the first to demonstrate that the miR-19a-3p/FOXF2 axis has a pivotal role in alcohol-impaired fracture healing, and may be a potential therapeutic target. Cite this article: Bone Joint Res 2022;11(6):386–397