The aim of this study was to examine whether the assessment BsALP as a biochemical parameter in the early posttraumatic phase may indicate the course of fracture healing. The methods used for monitoring the bone healing process have been based on the patient’s subjective evaluation and radiographic findings. The activity of bone-specific alkaline phosphatase was measured in the sera of 41 patients who had sustained fractures of long bones. All the patients had been treated surgically. The activity of BsALP was assessed every seven days over a four-week period. The same patients were subject to radiology follow-ups for several months. Our research showed that the increase of alkaline phosphatase correlated with an increase of BsALP levels. The volume of callus correlated with a decrease, no change or an increase in the level of ALP and BsALP in the same way. It can be concluded that the monitoring of changes in the biochemical parameters of alkaline phosphatase and bone-specific alkaline phosphatase allows the early detection of the fracture healing dynamics

Aims: The aim of the research is the functionalization of biosurfaces by anchoring on them biomolecules involved in the process of osteointegration (cellular adhesion, proliferation, differentiation, migration, matrix mineralization). Alkaline phosphatase (ALP) was used as model protein, because it is involved in the mineralization processes. The functionalized surfaces are biomimetic, because they show the biological signals triggering new tissue generation. A rapid osseointegration are the final goal and a good response and fast healing of bad quality bones is one of the main issues. The devices of interest for the research are dental or orthopaedic implants and substitutes of small bones. Methods: Bioactive glasses of various compositions were employed as substrates. Bioactive glasses, when in contact with biological fluids, stimulate the precipitation of a hydroxyapatite layer on their surfaces, which in turn promotes effective osteointegration of the implant. Since bioactive glasses are prone to hydroxylation, they could be successfully functionalized and grafted by biomolecules. So the biomimetic materials considered will be bioactive both from a physicochemical (osteoconduction and apatite precipitation) and from a biochemical (osteoinduction) point of view. The research was focused first of all on the methods for developing active sites on the substrates. In the case of bioactive glasses the surface must be cleaned of any contaminants and the reactive hydroxyls activated. Results: The immobilization of ALP was performed both with and without spacer molecules and a comparison among the different techniques will be presented. XPS was used for the analysis of the immobilized enzyme on titanium and bioglasses and specific signals for its identification were set. After the addition of the specific substrate, the ALP activity was evaluated by UV-VIS spectroscopy. Conclusions: ALP was successfully grafted on the surface of bioactive glasses with and without the use of an intermediate layer of spacer molecules. The presence of ALP was determined on all the samples, as well as its enzymatic activity. Further analyses are necessary to evaluate the opportunity of using a spacer molecule. Cell adhesion and proliferation tests are in progress

Mesenchymal stromal cells (MSCs) have been intensively researched in the orthopaedic field since they hold great promise for aiding the regeneration of musculoskeletal tissues. While there are a range of postulated surface markers to identify MSCs, currently there are no known cell markers that predict in vivo osteochondral potency. Runt-related transcription factor 2 (Runx2) is considered as an essential transcription factor in osteoblast differentiation [1] and has been shown to physically interact with retinoblastoma protein (pRb), which leads the loss of osteoblast proliferation and the activation of genes concerning terminal differentiation of osteoblasts [2]. The aim of this study was to use adenoviral-mediated gene overexpression/knockdown to investigate the interplay between Runx2 and pRb during in vitro osteogenic differentiation of human bone marrow (hBM)-MSCs. A first generation human adenovirus (hAd) serotype 5 dE/E3 carrying the gene of interest (Runx2 or shRNA-Runx2) were propagated and amplified in AD-293 cells, and purified over successive CsCl gradients. A second generation hAd serotype 5 carrying the gene of interest (Rb1) was generated. High efficiency single or double transduction of undifferentiated hBM-MSCs was achieved using lanthofection [3]. The transduced hBM-MSCs were then differentiated in osteogenic medium (OM) and osteogenic potency was assessed by quantification of alkaline phosphatase (ALP) activity (day 14) and Alizarin red staining (day 28). In addition, cell cultures were assessed for absorbance at OD 450nm, correlating to the refractive index of calcified areas, at days 0, 7, 14, 21 and 28 [4]. Quantitative RT-PCR was used to confirm expression of target genes following viral transduction. Basal medium was used as a control. Untransduced hBM-MSCs cultures grown in OM demonstrated peak calcium deposition at day 28, while the overexpression of either Runx2 or Rb1 accelerated peak calcium deposition to day 21. Consistent with this, Runx2 overexpression increased ALP activity of hBM-MSCs cultured in OM, while Rb1 overexpression enhanced ALP activity of hBM-MSCs cultured in both basal and osteogenic conditions. Co-expression of Runx2 and Rb1 did not further increase ALP activity compared to cells transduced with Runx2 or Rb1 alone. Alizarin red staining revealed that overexpression of either Runx2 or Rb1 increased mineral deposition in hBM-MSCs under basal conditions, although mineralisation was not enhanced above that of untransduced cells when cultured in OM. However, mineralisation was markedly enhanced above levels in untransduced cells when Runx2 and Rb1 were co-expressed in hBM-MSCs grown under both basal and osteogenic conditions. This study demonstrates an important stimulatory role of pRb in enhancing ALP activity of hBM-MSCs in the absence of osteogenic clues. However, pRb overexpression alone is insufficient to enhance mineralisation, requiring the co-expression of Runx2 in hBM-MSCs. The crucial nature of Runx2 for osteogenic differentiation of hBM-MSCs was demonstrated since knockdown of Runx2 prevented both mineral deposition and the increased ALP activity observed in untransduced cells grown in OM. Interestingly, overexpression of Rb1 could not compensate for the knockdown of Runx2 since Rb1 overexpression did not recover either mineral deposition or ALP activity in hBM-MSCs where Runx2 expression was inhibited

Mesenchymal stem cells (MSC) are suitable candidates for the cell-based cartilage reconstruction and have been isolated from different sources such as bone marrow (BMSC), adipose tissue (ATSC) and synovium (SMSC). The aim of this study was to analyse the tendency of BMSC, ATSC and SMSC to undergo hypertrophy during chondrogenic induction in vitro and to evaluate their in vivo development after ectopic transplantation into SCID mice in order to determine which cell source is most suitable for cartilage regeneration. Human BMSC, ATSC and SMSC were cultured under chondrogenic conditions for five weeks. Differentiation was evaluated based on histology, gene expression, and analysis of alkaline phosphatase activity (ALP). Pellets were transplanted subcutaneously into SCID mice after chondrogenic induction for 5 weeks and analysed 4 weeks later by histology. Similar COL2A1:COL10A1 mRNA ratios were found in BMSC, ATSC and SMSC. BMSC displayed the highest ALP activities, SMSC had lower and heterogenic ALP activities in vitro which correlated with calcification of spheroids in vivo. Most SMSC transplants specifically lost their collagen type II in vivo or were fully degraded. BMSC and ATSC pellets always underwent vascular invasion and calcification in vivo. Single BMSC samples had the capacity to develop into woven bone or fully developed ossicles with hematopoietic tissue surrounded by a bone capsule. Neither BMSC nor ATSC or SMSC were able to form stable ectopic cartilage. While BMSC and ATSC underwent developmental processes related to endochondral ossification instead of stable ectopic cartilage formation, SMSC tended to undergo fibrous dedifferentiation or degradation. Besides appropriate induction of chondrogenesis, locking of cells in the desired differentiation state is, thus, a further challenge for adult stem cell-based cartilage repair

Objective: The clinical significance of biochemical bone markers in the diagnosis and severity of Osteoarthritis remains still unknown. The relationship between biochemical bone turnover markers and commonly recognizable radiographic features of knee and hip osteoarthritis remains unclear. Purpose: We evaluated the serum levels of Receptor Activator of Nuclear Factor-κB Ligand (RANKL), Bone-specific Alkaline Phosphatase (b-ALP), Osteocalcin and Osteoprotegerin in two groups of patients suffering from osteoarthritis of the Knee or Hip respectively, aiming to correlate these results with the radiographically assessed severity of the disease and the patients’ age. The results between the two groups were also compared. Patients-Methods: Between March 2007 and February 2009, a total of 175 patients suffering from Knee or Hip Osteoarthritis were enrolled in the study. Following proper radiographic evaluation, the osteoarthritic changes of patients were graded by 3 orthopaedic surgeons according to the system of Kellgren and Lawrence; at the same time the serum levels of biochemical markers were determined. Results: Osteoprotegerin was found to be positively correlated with age in both the Knee (r=0.376, p=0.000) and Hip (r=0.425, p=0.001) group, whether Osteocalcin was significantly correlated with the age in the group of Knee Osteoarthritis(r=0.218, p=0.02). No other significant correlation was noted between the serum level of markers and age of patients in both groups. There was not significant difference in the mean serum level of biochemical markers among patients belonging to each of the four different levels of severity of hip and knee OA. There was no significant impact of the type of Osteoarthritis, to the serum level of all biochemical markers. Conclusions: Based on our results, it seems that none of the serum biochemical markers studied can be used (either independently or in combination with the others) as surrogates for radiographic imaging in Hip and Knee osteoarthritis

Aims. Astragalus polysaccharide (APS) participates in various processes, such as the enhancement of immunity and inhibition of tumours. APS can affect osteoporosis (OP) by regulating the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs). This study was designed to elucidate the mechanism of APS in hBMSC proliferation and osteoblast differentiation. Methods. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were performed to determine the expression of microRNA (miR)-760 and ankyrin repeat and FYVE domain containing 1 (ANKFY1) in OP tissues and hBMSCs. Cell viability was measured using the Cell Counting Kit-8 assay. The expression of cyclin D1 and osteogenic marker genes (osteocalcin (OCN), alkaline phosphatase (ALP), and runt-related transcription factor 2 (RUNX2)) was evaluated using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mineral deposits were detected through Alizarin Red S staining. In addition, Western blotting was performed to detect the ANKFY1 protein levels following the regulation of miR-760. The relationship between miR-760 and ANKFY1 was determined using a luciferase reporter assay. Results. The expression of miR-760 was upregulated in OP tissues, whereas ANKFY1 expression was downregulated. APS stimulated the differentiation and proliferation of hBMSCs by: increasing their viability; upregulating the expression levels of cyclin D1, ALP, OCN, and RUNX2; and inducing osteoblast mineralization. Moreover, APS downregulated the expression of miR-760. Overexpression of miR-760 was found to inhibit the promotive effect of APS on hBMSC differentiation and proliferation, while knockdown of miR-760 had the opposite effect. ANKFY1 was found to be the direct target of miR-760. Additionally, ANKFY1 participated in the APS-mediated regulation of miR-760 function in hBMSCs. Conclusion. APS promotes the osteogenic differentiation and proliferation of hBMSCs. Moreover, APS alleviates the effects of OP by downregulating miR-760 and upregulating ANKFY1 expression. Cite this article: Bone Joint Res 2023;12(8):476–485

Background: The clinical significance of bone turnover markers is well recognized, at least in several diseases affecting the bone metabolism. However, their clinical significance (if any) remains still unknown in patients undergoing Total Joint Arthroplasty (TJA). Changes in the levels of some markers have been reported in the early postoperative period after Total Hip Arthroplasty; however their exact postoperative course has not been clearly documented yet. In order to assess the clinical value of biochemical markers when trying to determine the fixation of orthopaedic implants, it is necessary to clarify their normal postoperative course.

The aim of this study was to extend the evaluation of the course of bone turnover markers over a longer period (12 postoperative months) following a TJA, and to assess the postoperative course for two of them (RANKL and Osteoprotegerin) for the first time.

Methods: The serum levels of RANKL, Osteocalcin, Osteoprotegerin and bALP were determined one day preoperatively and several times during the first postoperative year in patients suffering from idiopathic osteoarthritis that underwent total knee (n=23) and hip arthroplasties (n=24).

Results: There were statistically significant changes in the serum levels of all markers over time (p< 0,001). RANKL values initially increased and then gradually decreased. Following an initial decrease, Osteocalcin values continuously increased until the 2nd postoperative month and then continuously decreased. Osteoprotegerin initially increased, then decreased until the 4th postoperative month and then increased again reaching a peak 8 months postoperatively. Bone-specific ALP decreased until the 7th postoperative day. After that time it continuously increased, reaching a peak at the 8th month, and then it gradually decreased. There were no major differences in the postoperative course of all markers between the hip and knee arthroplasties.

Conclusions: The levels of all bone markers did not uniformly ‘return’ to their preoperative values one year postoperatively. A one-year period is not enough, when assessing an orthopaedic implant’s fixation with the use of bone turnover markers.

Aims. The aim of this study is to determine the predictors of overall survival (OS) and predictive factors of poor prognosis of conventional high-grade osteosarcoma of the limbs in a single-centre in South Africa. Methods. We performed a retrospective cross-sectional analysis to identify the prognostic factors that predict the OS of patients with histologically confirmed high-grade conventional osteosarcoma of the limbs over ten years. We employed the Cox proportional regression model and the Kaplan-Meier method for statistical analysis. Results. This study comprised 77 patients at a three-year minimum follow-up. The predictors of poor OS were: the median age of ≤ 19 years (hazard ratio (HR) 0.96; 95% confidence interval (CI) 0.92 to 0.99; p = 0.021); median duration of symptoms ≥ five months (HR 0.91; 95% CI 0.83 to 0.99; p < 0.037); metastasis at diagnosis (i.e. Enneking stage III) (HR 3.33; 95% CI 1.81 to 6.00; p < 0.001); increased alkaline phosphatase (HR 3.28; 95% CI 1.33 to 8.11; p < 0.010); palliative treatment (HR 7.27; 95% CI 2.69 to 19.70); p < 0.001); and amputation (HR 3.71; 95% CI 1.12 to 12.25; p < 0.032). In contrast, definitive surgery (HR 0.11; 95% CI 0.03 to 0.38; p < 0.001) and curative treatment (HR 0.18; 95% CI 0.10 to 0.33; p < 0.001) were a protective factor. The Kaplan-Meier median survival time was 24 months, with OS of 57.1% at the three years. The projected five-year event-free survival was 10.3% and OS of 29.8% (HR 0.76; 95% CI 0.52 to 1.12; p = 0.128). Conclusion. In this series of high-grade conventional osteosarcoma of the appendicular skeleton from South Africa, 58.4% (n = 45) had detectable metastases at presentation; hence, an impoverished OS of five years was 29.8%. Large-scale future research is needed to validate our results. Cite this article: Bone Jt Open 2024;5(3):210–217

Aims. Accumulated evidence indicates that local cell origins may ingrain differences in the phenotypic activity of human osteoblasts. We hypothesized that these differences may also exist in osteoblasts harvested from the same bone type at periarticular sites, including those adjacent to the fixation sites for total joint implant components. Methods. Human osteoblasts were obtained from the acetabulum and femoral neck of seven patients undergoing total hip arthroplasty (THA) and from the femoral and tibial cuts of six patients undergoing total knee arthroplasty (TKA). Osteoblasts were extracted from the usually discarded bone via enzyme digestion, characterized by flow cytometry, and cultured to passage three before measurement of metabolic activity, collagen production, alkaline phosphatase (ALP) expression, and mineralization. Results. Osteoblasts from the acetabulum showed lower proliferation (p = 0.034), cumulative collagen release (p < 0.001), and ALP expression (p = 0.009), and produced less mineral (p = 0.006) than those from the femoral neck. Osteoblasts from the tibia produced significantly less collagen (p = 0.021) and showed lower ALP expression than those from the distal femur. Conclusion. We have demonstrated for the first time an anatomical regional variation in the biological behaviours of osteoblasts on either side of the hip and knee joint. The lower osteoblast proliferation, matrix production, and mineralization from the acetabulum compared to those from the proximal femur may be reflected in differences in bone formation and implant fixation at these sites. Cite this article: Bone Joint Res 2021;10(9):611–618

Aims

Long non-coding RNAs (lncRNAs) act as crucial regulators in osteoporosis (OP). Nonetheless, the effects and potential molecular mechanism of lncRNA PCBP1 Antisense RNA 1 (PCBP1-AS1) on OP remain largely unclear. The aim of this study was to explore the role of lncRNA PCBP1-AS1 in the pathogenesis of OP.

Aims. The association of auraptene (AUR), a 7-geranyloxycoumarin, on osteoporosis and its potential pathway was predicted by network pharmacology and confirmed in experimental osteoporotic mice. Methods. The network of AUR was constructed and a potential pathway predicted by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) terms enrichment. Female ovariectomized (OVX) Institute of Cancer Research mice were intraperitoneally injected with 0.01, 0.1, and 1 mM AUR for four weeks. The bone mineral density (BMD) level was measured by dual-energy X-ray absorptiometry. The bone microstructure was determined by histomorphological changes in the femora. In addition, biochemical analysis of the serum and assessment of the messenger RNA (mRNA) levels of osteoclastic markers were performed. Results. In total, 65.93% of the genes of the AUR network matched with osteoporosis-related genes. Osteoclast differentiation was predicted to be a potential pathway of AUR in osteoporosis. Based on the network pharmacology, the BMD and bone mineral content levels were significantly (p < 0.05) increased in the whole body, femur, tibia, and lumbar spine by AUR. AUR normalized the bone microstructure and the serum alkaline phosphatase (ALP), bone-specific alkaline phosphatase (bALP), osteocalcin, and calcium in comparison with the OVX group. In addition, AUR treatment reduced TRAP-positive osteoclasts and receptor activator of nuclear factor kappa-B ligand (RANKL). +. nuclear factor of activated T cells 1 (NFATc1). +. expression in the femoral body. Moreover, the expressions of initiators for osteoclastic resorption and bone matrix degradation were significantly (p < 0.05) regulated by AUR in the lumbar spine of the osteoporotic mice. Conclusion. AUR ameliorated bone loss by downregulating the RANKL/NFATc1 pathway, resulting in improvement of osteoporosis. In conclusion, AUR might be an ameliorative cure that alleviates bone loss in osteoporosis via inhibition of osteoclastic activity. Cite this article: Bone Joint Res 2022;11(5):304–316

Aims. Continuous local antibiotic perfusion (CLAP) has recently attracted attention as a new drug delivery system for orthopaedic infections. CLAP is a direct continuous infusion of high-concentration gentamicin (1,200 μg/ml) into the bone marrow. As it is a new system, its influence on the bone marrow is unknown. This study aimed to examine the effects of high-concentration antibiotics on human bone tissue-derived cells. Methods. Cells were isolated from the bone tissue grafts collected from six patients using the Reamer-Irrigator-Aspirator system, and exposed to different gentamicin concentrations. Live cells rate, apoptosis rate, alkaline phosphatase (ALP) activity, expression of osteoblast-related genes, mineralization potential, and restoration of cell viability and ALP activity were examined by in vitro studies. Results. The live cells rate (the ratio of total number of cells in the well plate to the absorbance-measured number of live cells) was significantly decreased at ≥ 500 μg/ml of gentamicin on day 14; apoptosis rate was significantly increased at ≥ 750 μg/ml, and ALP activity was significantly decreased at ≥ 750 μg/ml. Real-time reverse transcription-polymerase chain reaction results showed no significant decrease in the ALP and activating transcription factor 4 transcript levels at ≥ 1,000 μg/ml on day 7. Mineralization potential was significantly decreased at all concentrations. Restoration of cell viability was significantly decreased at 750 and 1,000 μg/ml on day 21 and at 500 μg/ml on day 28, and ALP activity was significantly decreased at 500 μg/ml on day 28. Conclusion. Our findings suggest that the exposure concentration and duration of antibiotic administration during CLAP could affect cell functions. However, further in vivo studies are needed to determine the optimal dose in a clinical setting. Cite this article: Bone Joint Res 2024;13(3):91–100

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. 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

Introduction and Objective. Global prevalence of obesity has risen almost three-fold between 1975 and 2016. Alongside the more well-known health implications of obesity such as cardiovascular disease, cancer and type II diabetes, is the effect of male obesity on testosterone depletion and hypogonadism. Hypogonadism is a well-known contributor to the acceleration of bone loss during aging, and obesity is the single biggest risk factor for testosterone deficiency in men. Understanding the micro and macro structural changes to bone in response to testosterone depletion in combination with a high fat ‘Western’ diet, will advance our understanding of the relationship between obesity and bone metabolism. This study investigated the impact of surgically induced testosterone depletion and subsequent testosterone treatment upon bone remodelling in mice fed a high fat diet. Materials and Methods. Male ApoE. −/−. mice were split into 3 groups at 7 weeks of age and fed a high fat diet: Sham surgery with placebo treatment, orchiectomy surgery with placebo treatment, and orchiectomy surgery with testosterone treatment. Surgeries were performed at 8 weeks of age, followed by fortnightly testosterone treatment via injection. Mice were sacrificed at 25 weeks of age. Tibiae were collected and scanned ex-vivo at 4.3μm on a SkyScan 1272 Micro-CT scanner (Bruker). Left tibiae were used for assessment of trabecular and cortical Volumes of Interest (VOIs) 0.2mm and 1.0mm respectively from the growth-plate bridge break. Tibiae were subsequently paraffin embedded and sectioned at 4μm prior to immunohistochemical evaluation of alkaline phosphatase. Results. Trabecular bone volume and mineral density were significantly reduced in orchiectomised mice compared to sham-operated controls; and these parameters were normalised to control levels in orchiectomised mice treated with testosterone. In contrast, Trabecular thickness was significantly higher in testosterone depleted animals. Cortical bone parameters and body weights did not significantly differ between groups. Levels of alkaline phosphatase did not differ significantly in cortical or trabecular osteoblasts between groups. Conclusions. Findings suggest that testosterone deficiency significantly reduces trabecular bone parameters, and testosterone therapy may be a useful intervention for the loss of bone mass in testosterone deficient males. These results indicate that testosterone therapy may be useful for the treatment of trabecular bone frailty in testosterone deficient males. Observed changes in trabecular bone do not appear to be due to decreased mineralisation caused by osteoblast alkaline phosphatase. Ongoing work includes histology analysis to elucidate the mechanisms underpinning the changes seen in the bones of testosterone deficient animals

Objectives. Laser-engineered net shaping (LENS) of coated surfaces can overcome the limitations of conventional coating technologies. We compared the in vitro biological response with a titanium plasma spray (TPS)-coated titanium alloy (Ti6Al4V) surface with that of a Ti6Al4V surface coated with titanium using direct metal fabrication (DMF) with 3D printing technologies. Methods. The in vitro ability of human osteoblasts to adhere to TPS-coated Ti6Al4V was compared with DMF-coating. Scanning electron microscopy (SEM) was used to assess the structure and morphology of the surfaces. Biological and morphological responses to human osteoblast cell lines were then examined by measuring cell proliferation, alkaline phosphatase activity, actin filaments, and RUNX2 gene expression. Results. Morphological assessment of the cells after six hours of incubation using SEM showed that the TPS- and DMF-coated surfaces were largely covered with lamellipodia from the osteoblasts. Cell adhesion appeared similar in both groups. The differences in the rates of cell proliferation and alkaline phosphatase activities were not statistically significant. Conclusions. The DMF coating applied using metal 3D printing is similar to the TPS coating, which is the most common coating process used for bone ingrowth. The DMF method provided an acceptable surface structure and a viable biological surface. Moreover, this method is automatable and less complex than plasma spraying. Cite this article: T. Shin, D. Lim, Y. S. Kim, S. C. Kim, W. L. Jo, Y. W. Lim. The biological response to laser-aided direct metal-coated Titanium alloy (Ti6Al4V). Bone Joint Res 2018;7:357–361. DOI: 10.1302/2046-3758.75.BJR-2017-0222.R1

Aims. Exosomes derived from bone marrow mesenchymal stem cells (BMSCs) have been reported to be a promising cellular therapeutic approach for various human diseases. The current study aimed to investigate the mechanism of BMSC-derived exosomes carrying microRNA (miR)-136-5p in fracture healing. Methods. A mouse fracture model was initially established by surgical means. Exosomes were isolated from BMSCs from mice. The endocytosis of the mouse osteoblast MC3T3-E1 cell line was analyzed. CCK-8 and disodium phenyl phosphate microplate methods were employed to detect cell proliferation and alkaline phosphatase (ALP) activity, respectively. The binding of miR-136-5p to low-density lipoprotein receptor related protein 4 (LRP4) was analyzed by dual luciferase reporter gene assay. HE staining, tartrate-resistant acid phosphatase (TRAP) staining, and immunohistochemistry were performed to evaluate the healing of the bone tissue ends, the positive number of osteoclasts, and the positive expression of β-catenin protein, respectively. Results. miR-136-5p promoted fracture healing and osteoblast proliferation and differentiation. BMSC-derived exosomes exhibited an enriched miR-136-5p level, and were internalized by MC3T3-E1 cells. LRP4 was identified as a downstream target gene of miR-136-5p. Moreover, miR-136-5p or exosomes isolated from BMSCs (BMSC-Exos) containing miR-136-5p activated the Wnt/β-catenin pathway through the inhibition of LRP4 expression. Furthermore, BMSC-derived exosomes carrying miR-136-5p promoted osteoblast proliferation and differentiation, thereby promoting fracture healing. Conclusion. BMSC-derived exosomes carrying miR-136-5p inhibited LRP4 and activated the Wnt/β-catenin pathway, thus facilitating fracture healing. Cite this article: Bone Joint Res 2021;10(12):744–758

Aims. To investigate whether idiopathic osteonecrosis of the femoral head (ONFH) is related to impaired osteoblast activities. Methods. We cultured osteoblasts isolated from trabecular bone explants taken from the femoral head and the intertrochanteric region of patients with idiopathic ONFH, or from the intertrochanteric region of patients with osteoarthritis (OA), and compared their viability, mineralization capacity, and secretion of paracrine factors. Results. Osteoblasts from the intertrochanteric region of patients with ONFH showed lower alkaline phosphatase (ALP) activity and mineralization capacity than osteoblasts from the same skeletal site in age-matched patients with OA, as well as lower messenger RNA (mRNA) levels of genes encoding osteocalcin and bone sialoprotein and higher osteopontin expression. In addition, osteoblasts from patients with ONFH secreted lower osteoprotegerin (OPG) levels than those from patients with OA, resulting in a higher receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) ligand (RANKL)-to-OPG ratio. In patients with ONFH, osteoblasts from the femoral head showed reduced viability and mineralized nodule formation compared with osteoblasts from the intertrochanteric region. Notably, the secretion of the pro-resorptive factors interleukin-6 and prostaglandin E. 2. as well as the RANKL-to-OPG ratio were markedly higher in osteoblast cultures from the femoral head than in those from the intertrochanteric region. Conclusion. Idiopathic ONFH is associated with a reduced mineralization capacity of osteoblasts and increased secretion of pro-resorptive factors. Cite this article: Bone Joint Res 2021;10(9):619–628

Aims. Circular RNAs (circRNAs) are a novel type of non-coding RNA that plays major roles in the development of diverse diseases including osteonecrosis of the femoral head (ONFH). Here, we explored the impact of hsa_circ_0066523 derived from forkhead box P1 (FOXP1) (also called circFOXP1) on bone mesenchymal stem cells (BMSCs), which is important for ONFH development. Methods. RNA or protein expression in BMSCs was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot, respectively. Cell Counting Kit 8 (CCK8) and 5-ethynyl-2’-deoxyuridine (EdU) were used to analyze cell proliferation. Alkaline phosphatase (ALP) activity, ALP staining, and Alizarin Red S staining were employed to evaluate the osteoblastic differentiation. Chromatin immunoprecipitation (ChIP), luciferase reporter, RNA pull down, and RNA immunoprecipitation (RIP) assays were combined for exploring molecular associations. Results. Circ_0066523 was upregulated in osteogenic induction process of BMSCs. Silencing circ_0066523 restrained the proliferation and osteogenic differentiation of BMSCs. Mechanistically, circ_0066523 activated phosphatidylinositol-4,5-bisphosphate 3-kinase / AKT serine/threonine kinase 1 (PI3K/AKT) pathway via recruiting lysine demethylase 5B (KDM5B) to epigenetically repress the transcription of phosphatase and tensin homolog (PTEN). Functionally, AKT signalling pathway agonist or PTEN knockdown counteracted the effects of silenced circ_0066523 on BMSC proliferation and differentiation. Conclusion. Circ_0066523 promotes the proliferation and differentiation of BMSCs by epigenetically repressing PTEN and therefore activating AKT pathway. This finding might open new avenues for the identification of therapeutic targets for osteoblast differentiation related diseases such as ONFH. Cite this article: Bone Joint Res 2021;10(8):526–535

Aims. Platelet concentrates, like platelet-rich plasma (PRP) and platelet lysate (PL), are widely used in regenerative medicine, especially in bone regeneration. However, the lack of standard procedures and controls leads to high variability in the obtained results, limiting their regular clinical use. Here, we propose the use of platelet-derived extracellular vesicles (EVs) as an off-the-shelf alternative for PRP and PL for bone regeneration. In this article, we evaluate the effect of PL-derived EVs on the biocompatibility and differentiation of mesenchymal stromal cells (MSCs). Methods. EVs were obtained first by ultracentrifugation (UC) and then by size exclusion chromatography (SEC) from non-activated PL. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and the expression of CD9 and CD63 markers by western blot. The effect of the obtained EVs on osteoinduction was evaluated in vitro on human umbilical cord MSCs by messenger RNA (mRNA) expression analysis of bone markers, alkaline phosphatase activity (ALP), and calcium (Ca. 2+. ) content. Results. Osteogenic differentiation of MSCs was confirmed when treated with UC-isolated EVs. In order to disprove that the effect was due to co-isolated proteins, EVs were isolated by SEC. Purer EVs were obtained and proved to maintain the differentiation effect on MSCs and showed a dose-dependent response. Conclusion. PL-derived EVs present an osteogenic capability comparable to PL treatments, emerging as an alternative able to overcome PL and PRP limitations. Cite this article: Bone Joint Res 2020;9(10):667–674