The total plasma alkaline phosphatase level has long been recognised as an indicator of osteoblastic activity, but lack of specificity makes it an insensitive index of the progress of disease and the response to treatment. Selective precipitation by wheatgerm lectin allows measurement of the plasma bone-specific alkaline phosphatase. We measured the plasma levels of this isoenzyme in 170 normal Chinese adolescents and adults, in 49 adults with fractures of a long bone, in 15 patients with osteosarcoma and in 38 patients with osteolytic metastases. The enzyme activity was also determined in 39 patients with liver disease. Of the patients with fractures, 94% had increased plasma activity during the healing process. The level was also increased in those with osteosarcoma but not in those with osteolytic bone metastases. There was no significant increase in activity in the patients with liver disease. We conclude that the plasma bone-specific alkaline phosphatase activity is a sensitive and reliable measure of osteoblastic activity

Experimentally produced fractures in long bones studied by light and electron microscopic histochemistry were found to heal by a process of enchondral calcification. There was intense proliferation in the cells of the cambium layer of the periosteum, with differentiation to chondroblasts and osteoblasts, suggesting that this layer was the primary tissue responsible for development of the callus. Cytoplasmic processes of the hypertrophic chondrocytes appeared to bud and produce matrix vesicles. Alkaline phosphatase activity was detected along the plasma membrane of the hypertrophic chondrocytes and around the matrix vesicles, before any signs of mineral deposition. Calcification took place by deposition of hydroxyapatite crystals in and around these matrix vesicles which frequently showed alkaline phosphatase activity. It is suggested that there is a close functional association between alkaline phosphatase activity and calcification in the process of fracture healing, which is another type of enchondral calcification mediated by matrix vesicles

Osteomalacia may be a contributory factor in some patients in the development of fractures of the femoral neck and complicate the subsequent management. The level of serum alkaline phosphatase is often valuable in the diagnosis of metabolic bone disease but rises after any uncomplicated fracture, and since such a rise may limit the diagnostic usefulness of this measurement in detecting osteomalacia its extent was assessed in 106 patients. In the majority serum levels were normal on admission, rising after seven to nine days to reach a maximum within a month after fracture. Elevated levels on admission were found in patients with osteomalacia, liver damage or where there had been a delay of several weeks between injury and admission. In a small number of patients normal levels on admission subsequently reached very high values, usually in association with comminution or instability of the fracture. Elevated levels persisted for six to twelve weeks after fracture, the major influence upon the level at this time being the maximum value achieved rather than the presence of osteomalacia. If patients are to be screened for osteomalacia, the alkaline phosphatase must be measured within the first week after a fracture to avoid the distorting influences of the fracture itself

Estimations of serum alkaline phosphatase were carried out prospectively on a series of patients having a total hip replacement. The levels of serum alkaline phosphatase before operation indicated a group of patients who subsequently developed heterotopic ossification. Levels of this enzyme after operation did not indicate those patients who were developing heterotopic ossification

The acid and alkaline phosphatase activity in fluid aspirated from solitary bone cysts in six patients was measured, and large increases in the concentration of acid phosphatase were found. In some cases this increase was reflected in venous blood concentrations. The significance of these findings for the pathogenesis and the management of solitary bone cyst is discussed

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. The involvement of long non-coding RNA (lncRNA) in bone marrow mesenchymal stem cell (MSC) osteogenic differentiation during osteoporosis (OP) development has attracted much attention. In this study, we aimed to disclose how LINC01089 functions in human mesenchymal stem cell (hMSC) osteogenic differentiation, and to study the mechanism by which LINC01089 regulates MSC osteogenesis. Methods. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting were performed to analyze LINC01089, miR-1287-5p, and heat shock protein family A (HSP70) member 4 (HSPA4) expression. The osteogenic differentiation of MSCs was assessed through alkaline phosphatase (ALP) activity, alizarin red S (ARS) staining, and by measuring the levels of osteogenic gene marker expressions using commercial kits and RT-qPCR analysis. Cell proliferative capacity was evaluated via the Cell Counting Kit-8 (CCK-8) assay. The binding of miR-1287-5p with LINC01089 and HSPA4 was verified by performing dual-luciferase reporter and RNA immunoprecipitation (RIP) experiments. Results. LINC01089 expression was reinforced in serum samples of OP patients, but it gradually diminished while hMSCs underwent osteogenic differentiation. LINC01089 knockdown facilitated hMSC osteogenic differentiation. This was substantiated by: the increase in ALP activity; ALP, runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN) messenger RNA (mRNA) levels; and level of ARS staining. Meanwhile, LINC01089 upregulation resulted in the opposite effects. LINC01089 targeted miR-1287-5p, and the LINC01089 knockdown-induced hMSC osteogenic differentiation was repressed by miR-1287-5p depletion. HSPA4 is a downstream function molecule of the LINC01089/miR-1287-5p pathway; miR-1287-5p negatively modulated HSPA4 levels and attenuated its functional effects. Conclusion. LINC01089 negatively regulated hMSC osteogenic differentiation, at least in part, via governing miR-1287-5p/HSPA4 signalling. These findings provide new insights into hMSC osteogenesis and bone metabolism. Cite this article: Bone Joint Res 2024;13(12):779–789

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. The aim of this study was to report the long-term prognosis of patients with multiple Langerhans cell histiocytosis (LCH) involving the spine, and to analyze the risk factors for progression-free survival (PFS). Methods. We included 28 patients with multiple LCH involving the spine treated between January 2009 and August 2021. Kaplan-Meier methods were applied to estimate overall survival (OS) and PFS. Univariate Cox regression analysis was used to identify variables associated with PFS. Results. Patients with multiple LCH involving the spine accounted for 15.4% (28/182 cases) of all cases of spinal LCH: their lesions primarily involved the thoracic and lumbar spines. The most common symptom was pain, followed by neurological dysfunction. All patients presented with osteolytic bone destruction, and 23 cases were accompanied by a paravertebral soft-tissue mass. The incidence of vertebra plana was low, whereas the oversleeve-like sign was a more common finding. The alkaline phosphatase was significantly higher in patients with single-system multifocal bone LCH than in patients with multisystem LCH. At final follow-up, one patient had been lost to follow-up, two patients had died, three patients had local recurrence, six patients had distant involvement, and 17 patients were alive with disease. The median PFS and OS were 50.5 months (interquartile range (IQR) 23.5 to 63.1) and 60.5 months (IQR 38.0 to 73.3), respectively. Stage (hazard ratio (HR) 4.324; p < 0.001) and chemotherapy (HR 0.203; p < 0.001) were prognostic factors for PFS. Conclusion. Pain is primarily due to segmental instability of the spine from its destruction by LCH. Chemotherapy can significantly improve PFS, and radiotherapy has achieved good results in local control. The LCH lesions in some patients will continue to progress. It may initially appear as an isolated or single-system LCH, but will gradually involve multiple sites or systems. Therefore, long-term follow-up and timely intervention are important for patients with spinal LCH. Cite this article: Bone Joint J 2023;105-B(6):679–687

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. 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

Objectives

Effects of insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 2 (BMP2) on the expression of genes involved in the proliferation and differentiation of osteoblasts in culture were analysed. The best sequence of growth factor addition that induces expansion of cells before their differentiation was sought.

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

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

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. This meta-analysis and systematic review aimed to comprehensively investigate the effects of vitamin K supplementation on bone mineral density (BMD) at various sites and bone metabolism in middle-aged and older adults. Methods. The databases of PubMed, Web of Science, and Cochrane Library were thoroughly searched from inception to July 2023. Results. The results revealed that vitamin K supplementation increased BMD at the lumbar spine (p = 0.035). Moreover, the pooled effects demonstrated a notable increase in carboxylated osteocalcin (cOC) (p = 0.004), a decrease in uncarboxylated osteocalcin (ucOC) (p < 0.001), and no significant effect on total osteocalcin (tOC) (p = 0.076). Accordingly, the ratio of cOC to ucOC (p = 0.002) significantly increased, while the ratio of ucOC to tOC decreased (p = 0.043). However, there was no significant effect of vitamin K supplementation on other bone metabolism markers, such as cross-linked telopeptide of type 1 collagen (NTx), bone alkaline phosphatase (BAP), and procollagen I N-terminal propeptide (PINP). Subgroup analysis revealed that vitamin K notably enhanced bone health in females by increasing lumbar spine BMD (p = 0.028) and decreasing ucOC (p < 0.001). Vitamin K, especially vitamin K2, exhibited effects on maintaining or increasing lumbar spine BMD, and influencing the balance of cOC and ucOC. Conclusion. This review suggests that the beneficial effects of vitamin K supplementation on bone health primarily involve enhancing the carboxylation of OC rather than altering the total amount of OC. Cite this article: Bone Joint Res 2024;13(12):750–763

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

We examined cultured osteoblasts derived from paired samples from the greater tuberosity and acromion from eight patients with large chronic tears of the rotator cuff. We found that osteoblasts from the tuberosity had no apparent response to mechanical stimulation, whereas those derived from the acromion showed an increase in alkaline phosphatase activity and nitric oxide release which is normally a response of bone cells to mechanical strain. By contrast, we found that cells from both regions were able to respond to dexamethasone, a well-established promoter of osteoblastic differentiation, with the expected increase in alkaline phosphatase activity. Our findings indicate that the failure of repair of the rotator cuff may be due, at least in part, to a compromised capacity for mechanoadaptation within the greater tuberosity. It remains to be seen whether this apparent decrease in the sensitivity of bone cells to mechanical stimulation is the specific consequence of the reduced load-bearing history of the greater tuberosity in these patients

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. 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. 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

Aims. Here we introduce a wide and complex study comparing effects of growth factors used alone and in combinations on human mesenchymal stem cell (hMSC) proliferation and osteogenic differentiation. Certain ways of cell behaviour can be triggered by specific peptides – growth factors, influencing cell fate through surface cellular receptors. Methods. In our study transforming growth factor β (TGF-β), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor (VEGF) were used in order to induce osteogenesis and proliferation of hMSCs from bone marrow. These cells are naturally able to differentiate into various mesodermal cell lines. Effect of each factor itself is pretty well known. We designed experimental groups where two and more growth factors were combined. We supposed cumulative effect would appear when more growth factors with the same effect were combined. The cellular metabolism was evaluated using MTS assay and double-stranded DNA (dsDNA) amount using PicoGreen assay. Alkaline phosphatase (ALP) activity, as early osteogenesis marker, was observed. Phase contrast microscopy was used for cell morphology evaluation. Results. TGF-β and bFGF were shown to significantly enhance cell proliferation. VEGF and IGF-1 supported ALP activity. Light microscopy showed initial extracellular matrix mineralization after VEGF/IGF-1 supply. Conclusion. A combination of more than two growth factors did not support the cellular metabolism level and ALP activity even though the growth factor itself had a positive effect. This is probably caused by interplay of various messengers shared by more growth factor signalling cascades. Cite this article: Bone Joint Res 2020;9(7):412–420

1. A quantitative study of phosphatase distribution in the limb bones of growing rabbits is reported. 2. Alkaline phosphatase is present in high concentrations in areas of deposition of new bone. Both local concentrations and the total alkaline phosphatase content of a bone are found to decrease with age. There is good correlation between total alkaline phosphatase activity and monthly increment of weight. 3. Acid phosphatase is present in these bones in greatly less concentrations than alkaline phosphatase. 4. The acid phosphatase of bone shows nearly full activity in the presence of 0·5 per cent formaldehyde. It can be subdivided into two enzymes with characteristically different distributions by the effect of M/100 tartrate on activity. 5. The formaldehyde-stable and tartrate-stable acid phosphatase of rabbit bone (FTS) has a distribution very similar to that of alkaline phosphatase, though very much less in amount, and, like the latter, declines in activity as the bone matures. 6. Tartrate-inhibited, formaldehyde-stable acid phosphatase (FSTI) is found mainly in red marrow and cancellous bone, and full activity persists in mature bone. This enzyme may be associated with resorption and remodelling of bone, or it may represent residual activity under these conditions of the acid phosphatase of developing erythrocytes in the marrow

1. Alkaline and acid phosphatase, non-specific esterase and beta-glucuronidase have been estimated and demonstrated histochemically in a series of bone tumours and allied lesions, of which ten were osteogenic sarcomata, ten were giant-cell lesions, eleven were fibroblastic lesions and seven were tumours of cartilage. 2. Osteogenic sarcoma was found to be characterised by high levels of alkaline phosphatase, with rich staining for this enzyme in the tumour cells. Similar high levels of alkaline phosphatase were found in other bone-forming lesions, such as fibrous dysplasia, a giant-cell sarcoma with osteogenic matrix, and fracture callus. 3. Giant-cell lesions were characterised by high levels of acid phosphatase, and intense staining for this enzyme in the osteoclasts. These cells were also found to be rich in non-specific esterase (as shown by the alpha-naphthyl acetate method) and in beta-glucuronidase, but almost or entirely lacking in alkaline phosphatase. High levels of alkaline phosphatase were not found in giant-cell lesions except in relation to osteogenic matrix. 4. Fibroblastic tumours were characterised by moderate levels of all four enzymes, with little or no staining for phosphatases in the tumour cells; non-specific esterase was generally present in a proportion of the cells. 5. In certain lesions intermediate stages in the differentiation of fibroblasts to osteoblasts were found, notably in fibrous dysplasia, in which the biochemical change preceded the histological. In such lesions high total levels of alkaline phosphatase were found. 6. Cartilaginous tumours were characterised by low levels of all four enzymes, and little histochemical staining except in hypertrophied cells in areas of ossification. 7. It was found in general that the enzyme distributions in these neoplasms and other lesions reflected the findings in comparable reactive and growing normal tissues

Aims. The purpose of this study was to identify prognostic indicators of outcome at presentation to the orthopaedic surgeon, in patients with metastatic prostate cancer. Our aim was to use this information in a pragmatic, clinic-based approach so that surgical decision making could be optimized to benefit the patient in their remaining lifetime. Patients and Methods. A cohort analysis was undertaken of all patients with metastatic disease of the prostate who presented to a regional orthopaedic centre in the United Kingdom between 2003 and 2016. Biochemical data were collected in addition to disease and demographic data. These included: prostate-specific antigen (PSA) at orthopaedic presentation; haemoglobin (Hb); platelets (plt); alkaline phosphatase (ALP); albumin (Alb); and corrected calcium (CaC). Statistical analysis included Kaplan–Meier survival analysis, and a Cox proportional hazards model was fitted to the data. Results. From the departmental database, 137 episodes were identified in 136 patients with a median age at presentation of 72 years (interquartile range (IQR) 66 to 78). Most patients had stage IV disease (n = 98, 72%), and most did not undergo surgical intervention. At one-year follow-up, 50% of patients had died. Biomarkers found to be independently associated with poor survival were: low Hb, low Alb, relatively low PSA (< 30 mmol/l), and a raised ALP. Patients who needed surgical intervention had a poorer survival rate than patients who were managed nonoperatively. Conclusion. The study findings are important for orthopaedic clinical practice in the management of patients with metastatic prostate cancer. The interpretation of routine blood tests can help to predict survival in patients who present with orthopaedic manifestations of prostate cancer. A lower PSA is not necessarily a good prognostic sign. We believe that simple blood testing should be carried out routinely when assessing a patient, guiding potential surgical management and palliative care in the future

Aims. Acquired heterotopic ossification (HO) is a debilitating disease characterized by abnormal extraskeletal bone formation within soft-tissues after injury. The exact pathogenesis of HO remains unknown. It was reported that BRD4 may contribute to osteoblastic differentiation. The current study aims to determine the role of BRD4 in the pathogenesis of HO and whether it could be a potential target for HO therapy. Methods. Achilles tendon puncture (ATP) mouse model was performed on ten-week-old male C57BL/6J mice. One week after ATP procedure, the mice were given different treatments (e.g. JQ1, shMancr). Achilles tendon samples were collected five weeks after treatment for RNA-seq and real-time quantitative polymerase chain reaction (RT-qPCR) analysis; the legs were removed for micro-CT imaging and subsequent histology. Human bone marrow mesenchymal stem cells (hBMSCs) were isolated and purified bone marrow collected during surgeries by using density gradient centrifugation. After a series of interventions such as knockdown or overexpressing BRD4, Alizarin red staining, RT-qPCR, and Western Blot (Runx2, alkaline phosphatase (ALP), Osx) were performed on hBMSCs. Results. Overexpression of BRD4 enhanced while inhibition of Brd4 suppressed the osteogenic differentiation of hBMSCs in vitro. Overexpression of Brd4 increased the expression of mitotically associated long non-coding RNA (Mancr). Downregulation of Mancr suppressed the osteoinductive effect of BRD4. In vivo, inhibition of BRD4 by JQ1 significantly attenuated pathological bone formation in the ATP model (p = 0.001). Conclusion. BRD4 was found to be upregulated in HO and Brd4-Mancr-Runx2 signalling was involved in the modulation of new bone formation in HO. Cite this article: Bone Joint Res 2021;10(10):668–676

Objectives. Up to 10% of fractures result in undesirable outcomes, for which female sex is a risk factor. Cellular sex differences have been implicated in these different healing processes. Better understanding of the mechanisms underlying bone healing and sex differences in this process is key to improved clinical outcomes. This study utilized a macrophage–mesenchymal stem cell (MSC) coculture system to determine: 1) the precise timing of proinflammatory (M1) to anti-inflammatory (M2) macrophage transition for optimal bone formation; and 2) how such immunomodulation was affected by male versus female cocultures. Methods. A primary murine macrophage-MSC coculture system was used to demonstrate the optimal transition time from M1 to M2 (polarized from M1 with interleukin (IL)-4) macrophages to maximize matrix mineralization in male and female MSCs. Outcome variables included Alizarin Red staining, alkaline phosphatase (ALP) activity, and osteocalcin protein secretion. Results. We found that 96 hours of M1 phenotype in male cocultures allowed for maximum matrix mineralization versus 72 hours in female cocultures. ALP activity and osteocalcin secretion were also enhanced with the addition of IL-4 later in male versus female groups. The sex of the cells had a statistically significant effect on the optimal IL-4 addition time to maximize osteogenesis. Conclusion. These results suggest that: 1) a 72- to 96-hour proinflammatory environment is critical for optimal matrix mineralization; and 2) there are immunological differences in this coculture environment due to sex. Optimizing immunomodulation during fracture healing may enhance and expedite the bone regeneration response. These findings provide insight into precise immunomodulation for enhanced bone healing that is sex-specific. Cite this article: K. Nathan, L. Y. Lu, T. Lin, J. Pajarinen, E. Jämsen, J-F. Huang, M. Romero-Lopez, M. Maruyama, Y. Kohno, Z. Yao, S. B. Goodman. Precise immunomodulation of the M1 to M2 macrophage transition enhances mesenchymal stem cell osteogenesis and differs by sex. Bone Joint Res 2019;8:481–488. DOI: 10.1302/2046-3758.810.BJR-2018-0231.R2

Objectives. There is increasing application of bone morphogenetic proteins (BMPs) owing to their role in promoting fracture healing and bone fusion. However, an optimal delivery system has yet to be identified. The aims of this study were to synthesise bioactive BMP-2, combine it with a novel α-tricalcium phosphate/poly(D,L-lactide-co-glycolide) (α-TCP/PLGA) nanocomposite and study its release from the composite. Methods. BMP-2 was synthesised using an Escherichia coli expression system and purified. In vitro bioactivity was confirmed using C2C12 cells and an alkaline phosphatase assay. The modified solution-evaporation method . was used to fabricate α-TCP/PLGA nanocomposite and this was characterised using X-ray diffraction and scanning electron microscopy. Functionalisation of α-TCP/PLGA nanocomposite by adsorption of BMP-2 was performed and release of BMP-2 was characterised using an enzyme-linked immunosorbent assay (ELISA). Results. Alkaline phosphatase activity of C2C12 cells was increased by the presence of all BMP-2/nanocomposite discs compared with the presence of a blank disc (p = 0.0022), and increased with increasing incubation concentrations of BMP-2, showing successful adsorption and bioactivity of BMP-2. A burst release profile was observed for BMP-2 from the nanocomposite. . Conclusions. Functionalisation of α-TCP/PLGA with BMP-2 produced osteoinduction and was dose-dependent. This material therefore has potential application as an osteoinductive agent in regenerative medicine

1. Histochemical studies have been made of the distribution of alkaline phosphatase, glycogen and acid mucopolysaccharides in normal growing bones (mice, rats and men) and also in forty cases of pathological bone processes (neoplastic and dystrophic). 2. The study of normal material confirmed that alkaline phosphatase is plentiful in calcification of cartilage and even more plentiful in bone formation (whether enchondral or direct). 3. It was observed that glycogen increased in the cartilage areas about to be calcified, and that it disappeared in those calcified. It seemed that osteoblasts did not always contain glycogen. 4. In the pathological material (tumours and dystrophic processes) there was great phosphatase activity in the osteogenic areas and also in the cartilage about to be calcified. Whereas glycogen was plentiful in some cases of neoplastic or reactive osteogenesis, it was absent from others. 5. In every area of normal or pathological ossification, the presence of phosphatase seems to be a rule; glycogen is often but not always present. 6. It appears that alkaline phosphatase plays an important role in the formation of the protein matrix of bone, but is not associated with the elaboration of the mucoprotein cartilage matrix. We believe it is premature to draw any definite conclusion on the behaviour and role of the metachromatic substances in the processes of calcification and ossification. The histochemical study of alkaline phosphatase has shown that this is a valuable method in the detection of reactionary or pathological osteogenic processes which in some cases are difficult to demonstrate with the usual histological methods

1. Histochemical staining and correlated biochemical estimations of five hydrolytic enzymes were done on eighteen benign and twenty malignant fibroblastic lesions of bone and soft tissue. 2. Alkaline phosphatase activity was moderate in a fibroma and very high in fibrous dysplasia. In a typical fibrosarcoma the fibroblasts showed no enzyme activity and estimations were low. Exceptions indicated an osteogenic potential in the tumour. 3. ß-glucuronidase, leucine aminopeptidase, and to a less extent non-specific esterase, were more active in malignant than in benign lesions, and the highest activities were found in sarcomata arising in Paget's disease of bone. 4. Acid phosphatase showed no correlation with malignancy and was generally unremarkable except for high activity in osteoclasts, but was raised in two sarcomata that occurred after irradiation of giant-cell tumours. 5. A non-osteogenic fibroma and a fibrous cortical defect, though poorly represented in this series, are not uncommon; they sometimes lead to pathological fracture, but sarcoma is very rare in such lesions. They tend to show more alkaline phosphatase than fibrosarcoma but not the very high activity of fibrous dysplasia, which is related to its osteogenic potential. 6. Fibrous dysplasia most often presents in the five to fifteen age group but seldom leads to malignancy, though this may occur, usually as osteosarcoma, which has a similar high content of alkaline phosphatase. Fibrosarcoma is typically negative or very weak in this enzyme: the exceptional cases with high activity were tumours which were in part osteosarcoma. Generally the demonstration of high alkaline phosphatase activity in a fibroblastic lesion of bone, in the absence of trauma or inflammation, suggests the diagnosis of fibrous dysplasia

In an attempt to increase the life of cementless prostheses, an hydroxyapatite-coated implant which releases a bisphosphonate has been suggested as a drug-delivery system. Our in vitro study was designed to determine the maximum dose to which osteoblasts could be safely exposed. Our findings demonstrated that zoledronate did not impair the proliferation of human osteoblasts when used at concentrations below 1 μ. m. Murine cells can be exposed to concentrations as high as 10 μ. m. . A concentration of 0.01% of titanium particles did not impair the proliferation of either cell line. Zoledronate affected the alkaline phosphatase activity of murine osteoblasts through a chelation phenomenon. The presence of titanium particles strongly decreased the alkaline phosphatase activity of murine osteoblasts. We did not detect any synergic effect of zoledronate and titanium particles on the behaviour of both human and murine osteoblasts

Objectives. Osteoporosis is a systemic bone metabolic disease, which often occurs among the elderly. Angelica polysaccharide (AP) is the main component of angelica sinensis, and is widely used for treating various diseases. However, the effects of AP on osteoporosis have not been investigated. This study aimed to uncover the functions of AP in mesenchymal stem cell (MSC) proliferation and osteoblast differentiation. Methods. MSCs were treated with different concentrations of AP, and then cell viability, Cyclin D1 protein level, and the osteogenic markers of runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), alkaline phosphatase (ALP), bone morphogenetic protein 2 (BMP-2) were examined by Cell Counting Kit-8 (CCK-8) and western blot assays, respectively. The effect of AP on the main signalling pathways of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Wnt/β-catenin was determined by western blot. Following this, si-H19#1 and si-H19#2 were transfected into MSCs, and the effects of H19 on cell proliferation and osteoblast differentiation in MSCs were studied. Finally, in vivo experimentation explored bone mineral density, bone mineral content, and the ash weight and dry weight of femoral bone. Results. The results revealed that AP significantly promoted cell viability, upregulated cyclin D1 and increased RUNX2, OCN, ALP, and BMP-2 protein levels in MSCs. Moreover, we found that AP notably activated PI3K/AKT and Wnt/β-catenin signalling pathways in MSCs. Additionally, the relative expression level of H19 was upregulated by AP in a dose-dependent manner. The promoting effects of AP on cell proliferation and osteoblast differentiation were reversed by H19 knockdown. Moreover, in vivo experimentation further confirmed the promoting effect of AP on bone formation. Conclusion. These data indicate that AP could promote MSC proliferation and osteoblast differentiation by regulating H19. Cite this article: X. Xie, M. Liu, Q. Meng. Angelica polysaccharide promotes proliferation and osteoblast differentiation of mesenchymal stem cells by regulation of long non-coding RNA H19: An animal study. Bone Joint Res 2019;8:323–332. DOI: 10.1302/2046-3758.87.BJR-2018-0223.R2

The alkaline phosphatase activity of pre-osseous tibial cartilage of rachitic bone stored in the deep freeze for two weeks at -25 degrees centigrade was only slightly less than that of fresh controls from the same animals. The deep frozen pre-osseous tissue did not calcify in in vitro calcifying media containing either inorganic phosphorus or organic phosphate ester. The fresh controls calcified equally well in both media. In addition, after deep-freeze storage the tissue hydrolysed the organic phosphate to the same degree as did the fresh tissue. Bones heated at 65 degrees centigrade will calcify in vitro after calcium chloride treatment despite the destruction of phosphatase activity. It appears unlikely that a relationship exists between alkaline phosphatase and the minimal system required for calcification of pre-osseous cartilage in vitro. These findings do not exclude the possibility that alkaline phosphatase plays some critical role in vivo

We have investigated whether cells derived from haemarthrosis caused by injury to the anterior cruciate ligament could differentiate into the osteoblast lineage in vitro. Haemarthroses associated with anterior cruciate ligament injuries were aspirated and cultured. After treatment with β-glycerophosphate, ascorbic acid and dexamethasone or 1,25 (OH). 2. D. 3. , a significant increase in the activity of alkaline phosphatase was observed. Matrix mineralisation was demonstrated after 28 days and mRNA levels in osteoblast-related genes were enhanced. Our results suggest that the haemarthrosis induced by injury to the anterior cruciate ligament contains osteoprogenitor cells and is a potential alternative source for cell-based treatment in such injury

Objectives. Enhanced micromotions between the implant and surrounding bone can impair osseointegration, resulting in fibrous encapsulation and aseptic loosening of the implant. Since the effect of micromotions on human bone cells is sparsely investigated, an in vitro system, which allows application of micromotions on bone cells and subsequent investigation of bone cell activity, was developed. Methods. Micromotions ranging from 25 µm to 100 µm were applied as sine or triangle signal with 1 Hz frequency to human osteoblasts seeded on collagen scaffolds. Micromotions were applied for six hours per day over three days. During the micromotions, a static pressure of 527 Pa was exerted on the cells by Ti6Al4V cylinders. Osteoblasts loaded with Ti6Al4V cylinders and unloaded osteoblasts without micromotions served as controls. Subsequently, cell viability, expression of the osteogenic markers collagen type I, alkaline phosphatase, and osteocalcin, as well as gene expression of osteoprotegerin, receptor activator of NF-κB ligand, matrix metalloproteinase-1, and tissue inhibitor of metalloproteinase-1, were investigated. Results. Live and dead cell numbers were higher after 25 µm sine and 50 µm triangle micromotions compared with loaded controls. Collagen type I synthesis was downregulated in respective samples. The metabolic activity and osteocalcin expression level were higher in samples treated with 25 µm micromotions compared with the loaded controls. Furthermore, static loading and micromotions decreased the osteoprotegerin/receptor activator of NF-κB ligand ratio. Conclusion. Our system enables investigation of the behaviour of bone cells at the bone-implant interface under shear stress induced by micromotions. We could demonstrate that micromotions applied under static pressure conditions have a significant impact on the activity of osteoblasts seeded on collagen scaffolds. In future studies, higher mechanical stress will be applied and different implant surface structures will be considered. Cite this article: J. Ziebart, S. Fan, C. Schulze, P. W. Kämmerer, R. Bader, A. Jonitz-Heincke. Effects of interfacial micromotions on vitality and differentiation of human osteoblasts. Bone Joint Res 2018;7:187–195. DOI: 10.1302/2046-3758.72.BJR-2017-0228.R1

The aim of our study was to investigate the effect of platelet-rich plasma on the proliferation and differentiation of rat bone-marrow cells and to determine an optimal platelet concentration in plasma for osseous tissue engineering. Rat bone-marrow cells embedded in different concentrations of platelet-rich plasma gel were cultured for six days. Their potential for proliferation and osteogenic differentiation was analysed. Using a rat limb-lengthening model, the cultured rat bone-marrow cells with platelet-rich plasma of variable concentrations were transplanted into the distraction gap and the quality of the regenerate bone was evaluated radiologically. Cellular proliferation was enhanced in all the platelet-rich plasma groups in a dose-dependent manner. Although no significant differences in the production and mRNA expression of alkaline phosphatase were detected among these groups, mature bone regenerates were more prevalent in the group with the highest concentration of platelets. Our results indicate that a high platelet concentration in the platelet-rich plasma in combination with osteoblastic cells could accelerate the formation of new bone during limb-lengthening procedures

Systemic factors are believed to be pivotal for the development of heterotopic ossification in severely-injured patients. In this study, cell cultures of putative target cells (human fibroblastic cells, osteoblastic cells (MG-63), and bone-marrow stromal cells (hBM)) were incubated with serum from ten consecutive polytraumatised patients taken from post-traumatic day 1 to day 21 and with serum from 12 healthy control subjects. The serum from the polytraumatised patients significantly stimulated the proliferation of fibroblasts, MG-63 and of hBM cells. The activity of alkaline phosphatase in MG-63 and hBM cells was significantly decreased when exposed to the serum of the severely-injured patient. After three weeks in 3D cell cultures, matrix production and osteogenic gene expression of hBM cells were equal in the patient and control groups. However, the serum from the polytraumatised patients significantly decreased apoptosis of hBM cells compared with the control serum (4.3% vs 19.1%, p = 0.031). Increased proliferation of osteoblastic cells and reduced apoptosis of osteoprogenitors may be responsible for increased osteogenesis in severely-injured patients

Objectives. This study aimed to assess the effect of age and osteoporosis on the proliferative and differentiating capacity of bone-marrow-derived mesenchymal stem cells (MSCs) in female rats. We also discuss the role of these factors on expression and migration of cells along the C-X-C chemokine receptor type 4 (CXCR-4) / stromal derived factor 1 (SDF-1) axis. Methods. Mesenchymal stem cells were harvested from the femora of young, adult, and osteopenic Wistar rats. Cluster of differentiation (CD) marker and CXCR-4 expression was measured using flow cytometry. Cellular proliferation was measured using Alamar Blue, osteogenic differentiation was measured using alkaline phosphatase expression and alizarin red production, and adipogenic differentiation was measured using Oil red O. Cells were incubated in Boyden chambers to quantify their migration towards SDF-1. Data was analyzed using a Student’s t-test, where p-values < 0.05 were considered significant. Results. CD marker expression and proliferation of the MSCs from the three groups was not significantly different. The young MSCs demonstrated significantly increased differentiation into bone and fat and superior migration towards SDF-1. The migration of SDF-1 doubled with young rats compared with the adult rats (p = 0.023) and it was four times higher when compared with cells isolated from ovariectomized (OVX) osteopenic rats (p = 0.013). Conclusion. Young rat MSCs are significantly more responsive to osteogenic differentiation, and, contrary to other studies, also demonstrated increased adipogenic differentiation compared with cells from adult and ostopenic rats. Young-rat-derived cells also showed superior migration towards SDF-1 compared with MSCs from OVX and adult control rats. Cite this article: A. Sanghani-Kerai, L. Osagie-Clouard, G. Blunn, M. Coathup. The influence of age and osteoporosis on bone marrow stem cells from rats. Bone Joint Res 2018;7:289–297. DOI: 10.1302/2046-3758.74.BJR-2017-0302.R1

Objectives. Oxidative stress plays a major role in the onset and progression of involutional osteoporosis. However, classical antioxidants fail to restore osteoblast function. Interestingly, the bone anabolism of parathyroid hormone (PTH) has been shown to be associated with its ability to counteract oxidative stress in osteoblasts. The PTH counterpart in bone, which is the PTH-related protein (PTHrP), displays osteogenic actions through both its N-terminal PTH-like region and the C-terminal domain. Methods. We examined and compared the antioxidant capacity of PTHrP (1-37) with the C-terminal PTHrP domain comprising the 107-111 epitope (osteostatin) in both murine osteoblastic MC3T3-E1 cells and primary human osteoblastic cells. Results. We showed that both N- and C-terminal PTHrP peptides at 100 nM decreased reactive oxygen species production and forkhead box protein O activation following hydrogen peroxide (H. 2. O. 2. )-induced oxidation, which was related to decreased lipid oxidative damage and caspase-3 activation in these cells. This was associated with their ability to restore the deleterious effects of H. 2. O. 2. on cell growth and alkaline phosphatase activity, as well as on the expression of various osteoblast differentiation genes. The addition of Rp-cyclic 3′,5′-hydrogen phosphorothioate adenosine triethylammonium salt (a cyclic 3',5'-adenosine monophosphate antagonist) and calphostin C (a protein kinase C inhibitor), or a PTH type 1 receptor antagonist, abrogated the effects of N-terminal PTHrP, whereas protein phosphatase 1 (an Src kinase activity inhibitor), SU1498 (a vascular endothelial growth factor receptor 2 inhibitor), or an anti osteostatin antiserum, inhibited the effects of C-terminal PTHrP. Conclusion. These findings indicate that the antioxidant properties of PTHrP act through its N- and C-terminal domains and provide novel insights into the osteogenic action of PTHrP. Cite this article: S. Portal-Núñez, J. A. Ardura, D. Lozano, I. Martínez de Toda, M. De la Fuente, G. Herrero-Beaumont, R. Largo, P. Esbrit. Parathyroid hormone-related protein exhibits antioxidant features in osteoblastic cells through its N-terminal and osteostatin domains. Bone Joint Res 2018;7:58–68. DOI: 10.1302/2046-3758.71.BJR-2016-0242.R2

In this study 201 elderly patients with femoral neck fractures were compared with 30 osteomalacic patients with the same injury. Hypocalcaemia and a raised alkaline phosphatase level are common biochemical abnormalities in elderly patients with femoral neck fractures. In only a minority of patients, however, were they associated with histologically proven osteomalacia. By using the combination of hypocalcaemia and a raised alkaline phosphatase it is possible to identify a subgroup (approximately 10% of all admissions) in whom osteomalacia is relatively likely

1. A case of hypophosphatasia in a boy who sustained a fractured left femur is described. 2. The literature is reviewed, and the reported cases are found to fall into severe, moderately severe and mild forms. 3. The diagnostic features of the disease are the radiological picture, which resembles that of rickets, very low serum alkaline phosphatase, and excessive phosphoethanolamine excretion in the urine. 4. Other clinical features may be a failure to thrive in early infancy, premature loss of deciduous teeth, hypercalcaemia and renal damage. 5. The function of alkaline phosphatase in bone metabolism in relation to this disease has been discussed

A study of two siblings with a severe infantile form of familial idiopathic hyperphosphatasia is reported. A girl aged one year was followed for two years while receiving intermittent treatment with porcine calcitonin. This induced a clinical remission, a reduction of both the high serum level of alkaline phosphatase and the raised urinary excretion of hydroxyproline, and a remarkable improvement in bone structure as seen radiologically. Her sister aged two months received porcine calcitonin for three weeks, during which clinical improvement, no change in the serum level of alkaline phosphatase and a marked decrease of the excretion of hydroxyproline were recorded

The haematoma occurring at the site of a fracture is known to play an important role in bone healing. We have recently shown the presence of progenitor cells in human fracture haematoma and demonstrated that they have the capacity for multilineage mesenchymal differentiation. There have been many studies which have shown that low-intensity pulsed ultrasound (LIPUS) stimulates the differentiation of a variety of cells, but none has investigated the effects of LIPUS on cells derived from human fracture tissue including human fracture haematoma-derived progenitor cells (HCs). In this in vitro study, we investigated the effects of LIPUS on the osteogenic activity of HCs. Alkaline phosphatase activity, osteocalcin secretion, the expression of osteoblast-related genes and the mineralisation of HCs were shown to be significantly higher when LIPUS had been applied but without a change in the proliferation of the HCs. These findings provide evidence in favour of the use of LIPUS in the treatment of fractures

Bone loss around replacement prostheses may be related to the activation of mononuclear phagocytes (MNP) by prosthetic wear particles. We investigated how osteoblast-like cells were regulated by human MNP stimulated by particles of prosthetic material. Particles of titanium-6-aluminium-4-vanadium (TiAlV) stimulated MNP to release interleukin (IL)-1β, tumour necrosis factor (TNF)α, IL-6 and prostaglandin E. 2. (PGE. 2. ). All these mediators are implicated in regulating bone metabolism. Particle-activated MNP inhibited bone cell proliferation and stimulated release of IL-6 and PGE. 2. The number of cells expressing alkaline phosphatase, a marker associated with mature osteo-blastic cells, was reduced. Experiments with blocking antibodies showed that TNFα was responsible for the reduction in proliferation and the numbers of cells expressing alkaline phosphatase. By contrast, IL-1β stimulated cell proliferation and differentiation. Both IL-1β and TNFα stimulated IL-6 and PGE. 2. release from the osteoblast-like cells. Our results suggest that particle-activated mono-nuclear phagocytes can induce a change in the balance between bone formation and resorption by a number of mechanisms

Particulate wear debris can induce the release of bone-resorbing cytokines from cultured macrophages and fibroblasts in vitro, and these mediators are believed to be the cause of the periprosthetic bone resorption which leads to aseptic loosening in vivo. Much less is known about the effects of particulate debris on the growth and metabolism of osteoblastic cells. We exposed two human osteoblast-like cell lines (SaOS-2 and MG-63) to particulate cobalt, chromium and cobalt-chromium alloy at concentrations of 0, 0.01, 0.1 and 1.0 mg/ml. Cobalt was toxic to both cell lines and inhibited the production of type-I collagen, osteocalcin and alkaline phosphatase. Chromium and cobalt-chromium were well tolerated by both cell lines, producing no cytotoxicity and no inhibition of type-I collagen synthesis. At the highest concentration tested (1.0 mg/ml), however, chromium inhibited alkaline phosphatase activity, and both chromium and cobalt-chromium alloy inhibited osteocalcin expression. Our results clearly show that particulate metal debris can modulate the growth and metabolism of osteoblastic cells in vitro. Reduced osteoblastic activity at the bone-implant interface may be an important mechanism by which particulate wear debris influences the pathogenesis of aseptic loosening in vivo

We investigated the effect of calcitonin in the prevention of acute bone loss after a pertrochanteric fracture and its ability to reduce the incidence of further fractures in the same patient. Fifty women aged between 70 and 80 years who had a pertrochanteric fracture of the hip were randomly allocated to group A (200 IU of nasal salmon calcitonin daily for three months) or group B (placebo). Patients in group A showed a significantly higher level of total alkaline phosphatase and osteocalcin on the 15th day after injury and a significantly higher level of bone alkaline phosphatase on the 90th day after surgery. These patients also had significantly lower levels of urinary C-telopeptide (CrossLaps) on the 15th, 45th and 90th days after injury and lower levels of urinary hydroxyproline on the 15th and 45th days after injury. Patients in group A had significantly higher bone mineral density at all recorded sites except the greater trochanter at three months and one year after operation. After a four-year period of clinical observation, five patients (24%) in group B sustained a new fracture, in four of whom (20%) it was of the contralateral hip. Our findings show that calcitonin reduces acute bone loss in patients with pertrochanteric fractures and may prevent the occurrence of new fractures of the contralateral hip in the elderly

Peri-prosthetic osteolysis and subsequent aseptic loosening is the most common reason for revising total hip replacements. Wear particles originating from the prosthetic components interact with multiple cell types in the peri-prosthetic region resulting in an inflammatory process that ultimately leads to peri-prosthetic bone loss. These cells include macrophages, osteoclasts, osteoblasts and fibroblasts. The majority of research in peri-prosthetic osteolysis has concentrated on the role played by osteoclasts and macrophages. The purpose of this review is to assess the role of the osteoblast in peri-prosthetic osteolysis. In peri-prosthetic osteolysis, wear particles may affect osteoblasts and contribute to the osteolytic process by two mechanisms. First, particles and metallic ions have been shown to inhibit the osteoblast in terms of its ability to secrete mineralised bone matrix, by reducing calcium deposition, alkaline phosphatase activity and its ability to proliferate. Secondly, particles and metallic ions have been shown to stimulate osteoblasts to produce pro inflammatory mediators in vitro. In vivo, these mediators have the potential to attract pro-inflammatory cells to the peri-prosthetic area and stimulate osteoclasts to absorb bone. Further research is needed to fully define the role of the osteoblast in peri-prosthetic osteolysis and to explore its potential role as a therapeutic target in this condition. Cite this article: Bone Joint J 2013;95-B:1021–5