A Mild Case of Autosomal Recessive Osteopetrosis Masquerading as the Dominant Form Involving Homozygous Deep Intronic Variations in the CLCN7 Gene.

Osteopetrosis is a heterogeneous group of rare hereditary diseases characterized by increased bone mass of poor quality. Autosomal-dominant osteopetrosis type II (ADOII) is most often caused by mutation of the CLCN7 gene leading to impaired bone resorption. Autosomal recessive osteopetrosis (ARO) is a more severe form and is frequently accompanied by additional morbidities. We report an adult male presenting with classical clinical and radiological features of ADOII. Genetic analyses showed no amino-acid-converting mutation in CLCN7 but an apparent haploinsufficiency and suppression of CLCN7 mRNA levels in peripheral blood mononuclear cells. Next generation sequencing revealed low-frequency intronic homozygous variations in CLCN7, suggesting recessive inheritance. In silico analysis of an intronic duplication c.595-120_595-86dup revealed additional binding sites for Serine- and Arginine-rich Splicing Factors (SRSF), which is predicted to impair CLCN7 expression. Quantitative backscattered electron imaging and histomorphometric analyses revealed bone tissue and material abnormalities. Giant osteoclasts were present and additionally to lamellar bone, and abundant woven bone and mineralized cartilage were observed, together with increased frequency and thickness of cement lines. Bone mineralization density distribution (BMDD) analysis revealed markedly increased average mineral content of the dense bone (CaMean T-score + 10.1) and frequency of bone with highest mineral content (CaHigh T-score + 19.6), suggesting continued mineral accumulation and lack of bone remodelling. Osteocyte lacunae sections (OLS) characteristics were unremarkable except for an unusually circular shape. Together, our findings suggest that the reduced expression of CLCN7 mRNA in osteoclasts, and possibly also osteocytes, causes poorly remodelled bone with abnormal bone matrix with high mineral content. This together with the lack of adequate bone repair mechanisms makes the material brittle and prone to fracture. While the skeletal phenotype and medical history were suggestive of ADOII, genetic analysis revealed that this is a possible mild case of ARO due to deep intronic mutation.

Equivalent values between anterior vertebral height, wedge ratio, and wedge angle in osteoporotic vertebral fractures.

INTRODUCTION: Vertebral mobility (V-mobility) has been used to diagnose fresh osteoporotic vertebral fractures (OVFs), and determine or predict bone union by setting cutoff values for these purposes. V-mobility is defined as the difference in shape of vertebral bodies between lateral radiographs taken in weight-bearing and non-weight-bearing positions. The parameters for V-mobility have varied in previous reports among anterior vertebral height (Ha, mm), wedge ratio (WR, %), and wedge angle (WA, degrees). The present study aimed to clarify WR and WA equivalent to Ha of 1.0 mm, and to compare the reported cutoff values for V-mobility presented as Ha, WR, or WA. MATERIALS AND METHODS: Lateral radiographs of 446 normal vertebrae (grade 0) and 146 deformed vertebrae (grade 1-3) from T11 to L2 were obtained from 183 female patients aged > 60 years. WR (%) values equivalent to Ha of 1.0 mm were calculated by Ha (1.0 mm)/Hp × 100 (Hp: posterior vertebral height). Corresponding WA values were calculated by trigonometric function using vertebral dimensions. RESULTS: The mean WR values equivalent to Ha of 1.0 mm in the vertebrae from T11 to L2 were 3.2%, 3.2%, 3.5%, and 3.7% for grades 0, 1, 2, and 3, respectively, and the corresponding WA values were 1.6°, 1.6°, 1.5°, and 1.4°. CONCLUSION: The equivalent values for V-mobility presented as Ha, WR, and WA were obtained. The mean WR and WA values equivalent to Ha of 1.0 mm in grade 1-3 vertebrae were 3.5% and 1.5°, respectively.

The paired-box homeodomain transcription factor Pax6 binds to the upstream region of the TRAP gene promoter and suppresses receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation.

Osteoclast formation is regulated by balancing between the receptor activator of nuclear factor-κB ligand (RANKL) expressed in osteoblasts and extracellular negative regulatory cytokines such as interferon-γ (IFN-γ) and interferon-ß (IFN-ß), which can suppress excessive bone destruction. However, relatively little is known about intrinsic negative regulatory factors in RANKL-mediated osteoclast differentiation. Here, we show the paired-box homeodomain transcription factor Pax6 acts as a negative regulator of RANKL-mediated osteoclast differentiation. Electrophoretic mobility shift and reporter assays found that Pax6 binds endogenously to the proximal region of the tartrate acid phosphatase (TRAP) gene promoter and suppresses nuclear factor of activated T cells c1 (NFATc1)-induced TRAP gene expression. Introduction of Pax6 retrovirally into bone marrow macrophages attenuates RANKL-induced osteoclast formation. Moreover, we found that the Groucho family member co-repressor Grg6 contributes to Pax6-mediated suppression of the TRAP gene expression induced by NFATc1. These results suggest that Pax6 interferes with RANKL-mediated osteoclast differentiation together with Grg6. Our results demonstrate that the Pax6 pathway constitutes a new aspect of the negative regulatory circuit of RANKL-RANK signaling in osteoclastogenesis and that the augmentation of Pax6 might therefore represent a novel target to block pathological bone resorption.

A secondary atypical diaphyseal femoral fracture after intramedullary nailing for an atypical subtrochanteric femoral fracture: A case report.

Intramedullary nailing is the gold standard of treatment for atypical femoral fractures, with a few reports of secondary atypical subtrochanteric femoral fractures following intramedullary nailing for atypical diaphyseal femoral fractures. However, there are no reports of secondary atypical diaphyseal femoral fractures following intramedullary nailing for atypical subtrochanteric femoral fractures. A 71-year-old woman with adult-onset Still's disease sustained a right atypical subtrochanteric femoral fracture and was treated with a mid-length intramedullary nail. One year after the surgery, the patient sustained a contralateral atypical diaphyseal femoral fracture and was treated with a long-length intramedullary nail. Moreover, 6 months after the second surgery, the patient complained of right-thigh pain, and a radiograph of the lateral view of the femur revealed a diaphyseal femoral fracture at the distal screw-insertion site. Revision surgery was performed using a long-length nail and screws directed toward the femoral head. Bony union of the bilateral diaphyseal femoral lesion was obtained, but the subtrochanteric lesion remained unhealed 1.5 years postoperatively. Mid-length intramedullary nailing for atypical subtrochanteric femoral fractures can cause secondary atypical diaphyseal fractures because of stress concentration at the distal screw-insertion site. For atypical subtrochanteric femoral fractures, the use of long-length nails and proximal screws directed toward the femoral head may be important to prevent secondary atypical diaphyseal femoral fractures.

Critical role of p38 MAPK for regeneration of the sciatic nerve following crush injury in vivo.

BACKGROUND: The physiological function of p38α, which is an isoform of p38 MAPK, has been investigated previously in several studies using pharmacological inhibitors. However, the results regarding whether p38α promotes or inhibits nerve regeneration in vivo have been controversial. METHODS: We generated novel p38α mutant mice (sem mice) with a point mutation in the region encoding the p38α substrate-docking-site, which serves as a limited loss-of-function model of p38α. In the present study, we utilized sem mice and wild-type littermates (wt mice) to investigate the physiological role of p38α in nerve regeneration following crush injuries. RESULTS: At four weeks after crush injury, the average axon diameter and the average axon area in sem mice were significantly smaller than those in wt mice. The average myelin sheath thickness in sem mice was reduced compared to wt mice, but no significant difference was observed in the G-ratio between the two groups. The sciatic functional index value demonstrated that functional nerve recovery in sem mice following crush injury was delayed, which is consistent with the histological findings. To investigate the underlying mechanisms of these findings, we examined inflammatory responses of the sciatic nerve by immunohistochemistry and western blotting. At an early phase following crush injury, sem mice showed remarkably lower expression of inflammatory cytokines, such as TNF-α and IL-1ß, than wt mice. The expression of Caspase-3 and Tenascin-C were also lower in sem mice. Conversely, at a late phase of the response, sem mice showed considerably higher expression of TNF-α and of IL-1ß with lower expression of S-100 than wt mice. CONCLUSIONS: This is the first study of the physiological role of p38 MAPK in nerve regeneration that does not rely on the use of pharmacological inhibitors. Our results indicate that p38α insufficiency may cause an inflammatory disorder, resulting in a delay of histological and functional nerve recovery following crush injury. We conclude that p38 MAPK has an important physiological role in nerve regeneration and may be important for controlling both initiation of inflammation and recovery from nerve injury.

Equivalent values between anterior vertebral height, wedge ratio, and wedge angle for evaluating vertebral mobility and deformity in osteoporotic vertebral fractures: a conventional observational study.

BACKGROUND: Vertebral mobility (V-mobility) has been used to diagnose fresh osteoporotic vertebral fractures (OVFs) and determine bone union by setting cutoff values for these purposes. V-mobility is the difference in vertebral height on dynamic radiographs taken in the sitting and lateral decubitus or supine positions. The dimensions for V-mobility were presented as anterior vertebral height (Ha; mm), wedge ratio (WR; %), and wedge angle (WA; °) in previous reports. This study was performed to obtain WR and WA values equivalent to V-mobility of 1.0 mm in Ha. METHODS: Lateral radiographs of 284 OVFs (grade 1-3 deformed vertebrae) from T11 to L2 were obtained from 77 patients with OVF. V-mobility presented as Ha, posterior vertebral height, and WA was obtained by the difference in these dimensions on dynamic radiographs. The WR and WA values equivalent to 1.0 mm in Ha were obtained by dividing the V-mobility values for WR and WA by that for Ha. RESULTS: The mean WR values corresponding to 1.0 mm in Ha for grade 1, 2, and 3 vertebrae were 3.2% ± 1.4%, 3.2% ± 0.9%, and 3.4% ± 1.0%, respectively, and the corresponding value for grade 1-3 vertebrae was 3.3% ± 1.0%. The mean WA values corresponding to 1.0 mm in Ha for grade 1, 2, and 3 vertebrae were 1.5° ± 0.8°, 1.5° ± 0.6°, and 1.5° ± 0.8°, respectively, and the corresponding value for grade 1-3 vertebrae was 1.5° ± 0.7°. CONCLUSIONS: The WR and WA values equivalent to V-mobility of 1.0 mm in Ha were 3.3% and 1.5°, respectively, in grade 1-3 vertebrae. These findings may be useful to secure a reliable value of V-mobility of OVFs using simultaneous measurements in three dimensions (Ha, WR, and WA) in clinical practice and to establish cutoff values for V-mobility to determine bone union.

Effect of gait training using Hybrid Assistive Limb on gait ability and the risk for overwork weakness in the lower limb muscles in patients with neuromuscular disease: a proof-of-concept study.

BACKGROUND: Few previous studies have reported the efficacy of robot rehabilitation for improving gait ability or its adverse events in patients with neuromuscular diseases. AIM: The aim of the present study was to elucidate the effects of gait training with a hybrid assistive limb (HAL) on gait ability and to investigate serum enzyme levels associated with skeletal muscle damage. DESIGN: Proof-of-concept study. SETTING: Department of rehabilitation medicine in university hospital. POPULATION: Twenty-one patients with neuromuscular disease (NMD, 13 males and 8 females, mean age of 60.6 years). METHODS: All patients underwent 1 to 5 series of gait rehabilitation which consisted of 9 sessions of HAL training. Gait ability was assessed with the 10-meter walk test and the 2-min walk test before and after HAL training, while serum creatine phosphokinase, aspartate aminotransferase, and lactic acid dehydrogenase values were measured before, midway through, and after HAL training. RESULTS: Gait velocity and step length for 10-meter walk test, and 2-min walk distance were significantly improved after HAL gait training. There was no significant change in serum level of all 3 measured enzymes between the three time points. CONCLUSIONS: HAL gait training with the practical setting as this study improved gait ability in patients with progressive NMD and did not damage skeletal muscle, as indicated by no significant change in serum level of muscle enzymes. CLINICAL REHABILITATION IMPACT: Robot assisted gait training could be safely applied to the patients with NMD, as one of the effective rehabilitation programs to improve gait ability.

Effects of gait training with a voluntary-driven wearable cyborg, Hybrid Assistive Limb (HAL), on quality of life in patients with neuromuscular disease, able to walk independently with aids.

Robot-assisted gait training using a voluntary-driven wearable cyborg, Hybrid Assistive Limb (HAL), has been shown to improve the mobility of patients with neurological disorders; however, its effect on the quality of life (QOL) of patients is not clear. The aim of this study was to assess the effects of HAL-assisted gait training on QOL and mobility in patients with neuromuscular diseases (NMDs). Ten patients with NMDs (seven men and three women, mean age: 57 ± 11 years), with impairment in mobility but could walk alone with aids underwent two courses of gait training with HAL over 6 months, and the single course consisted of nine sessions of training for 4 weeks. We compared the findings of the 2 min walk test, 10 m walk test, the Short Form-36 (SF-36) questionnaire, and the Hospital Anxiety and Depression Scale at baseline, after the 1st training, before the 2nd training, and after the 2nd training using the Friedman test. A significant improvement was observed in the 2 min walking distance from baseline (93 ± 50 m) to after the 2nd training (115 ± 48 m, P = 0.034), as well as in the domains of vitality (P = 0.019) and mental component summary score (P = 0.019) of SF-36. The improvement in 10 m walking speed was significantly correlated with that in the physical functioning (R = 0.831, P = 0.003) and role physical (R = 0.697, P = 0.025) domains in the SF-36. Our findings suggest that HAL-assisted gait training is effective in improving QOL associated with mental health as well as gait ability in selected patients with NMDs.

Vertebral mobility is a valuable indicator for predicting and determining bone union in osteoporotic vertebral fractures: a conventional observation study.

BACKGROUND: Conservative treatments for osteoporotic vertebral fractures (OVFs) have not been standardized, and criteria for determining bone union have not been established. To determine bone union, we have adopted a cutoff value of 1.0 mm for vertebral mobility (V-mobility), defined as the difference in anterior vertebral height (Ha) between lateral radiographs taken in weight-bearing and non-weight-bearing positions. The present study aimed to investigate the usefulness of V-mobility for determining bone union and predicting bone union at 6 months after OVF onset. METHODS: The study included 54 acute OVFs from T11 to L3 in 53 patients (12 males, 41 females; mean age 82 years; age range 55-97 years) who were hospitalized at ≤ 3 weeks after OVF onset. Vertebral deformity (V-deformity) and V-mobility were evaluated in accordance with Ha on lateral radiographs taken in the sitting position (SIT), lateral decubitus position (DEC), and supine position (SUP). OVFs showing V-mobility of ≤ 1.0 mm between SIT and DEC radiographs and no intravertebral cleft on DEC radiograph were defined as semi-union, while those showing V-mobility of ≤ 1.0 mm between SIT and SUP radiographs and no intravertebral cleft on SUP radiograph were defined as bone union. We calculated the bone union rates including semi-unions associated with V-mobility cutoff values of 1.0 mm, 1.5 mm, and 2.0 mm and estimated cutoff values for V-mobility at 5 weeks after OVF onset to predict bone union at 6 months after OVF onset. RESULTS: The cumulative number of bone unions including semi-unions was more influenced by the different V-mobility cutoff values in Ha for determining bone union in the earlier period compared with the later period in the time course of OVF. Receiver-operating characteristic curve analyses revealed that V-mobility cutoff value of 2.1 mm in Ha between SIT and DEC radiographs at 5 weeks after OVF had moderate accuracy for predicting bone union including semi-union at 6 months after OVF. The mean V-deformity value on SIT radiographs did not progress significantly. CONCLUSION: V-mobility in the early stage after OVF can predict bone union at 6 months after OVF and is a useful quantitative indicator for determining bone union.

The effects of ball impact position on shoulder muscle activation during spiking in male volleyball players.

BACKGROUND: The ball impact position during spiking in volleyball may influence the pattern of activation of shoulder girdle muscles and, therefore, could be a significant risk factor for shoulder injury. METHODS: Activation of 10 muscles in the dominant shoulder was evaluated using surface electromyography (EMG) in 11 male volleyball players, during spiking in a static standing position, with the goal being to precisely control the specified ball impact positions, without a run-up or ball setting. The following 4 ball impact positions were evaluated: standard, posterior, medial, and lateral. The EMG amplitude, normalized to the maximal voluntary isometric contraction of the respective muscles, was compared for each phase of the spiking movement between the standard position and the other 3 different impact positions, using the Dunnett test. RESULTS: The following between-position differences were noted for the deltoid muscle: increased activation of the anterior deltoid during the acceleration phase for the posterior position (P = .041), increase in the posterior deltoid during the acceleration phase for the lateral position (P = .04), and increase in the middle deltoid during the deceleration phase for the lateral position (P = .005). CONCLUSION: A posterior or lateral shift in the position of ball impact may cause an increase in the activity of the deltoid muscle that would cause a decrease in the centripetal force of the humeral head through the acceleration and deceleration phases. As such, neuromuscular exercises, combined with strengthening of the rotator cuff muscle, might reduce the risk of shoulder injury during performance of the volleyball spiking movement.

Evidence for altered osteoclastogenesis in splenocyte cultures from VDR knockout mice.

The indirect action of 1α,25(OH)2-vitamin-D3 (1,25D) on the osteoclast through stromal signalling is well established. The role of vitamin D in osteoclasts through direct 1,25D-VDR signalling is less well known. We showed previously that local 1,25D synthesis in osteoclasts modified osteoclastogenesis and osteoclastic resorptive activity. In this study, we hypothesised that osteoclasts lacking VDR expression would display an enhanced resorptive capacity due to the loss of 1,25D signalling. Splenocytes were cultured under osteoclast-differentiating conditions from mice with global deletion of the Vdr gene (VDRKO) and this was compared with age-matched wild-type littermate controls (WT). In VDRKO cultures, osteoclastogenesis was reduced, as indicated by fewer TRAP-positive multinucleated cells at all time points measured (p<0.05) compared to WT levels. However, VDRKO osteoclasts demonstrated greater resorption on a per cell basis than their WT counterparts. VDRKO cultures expressed greatly increased c-Fos mRNA compared to WT. In addition, the ratio of expression of the pro-apoptotic gene Bax to the pro-survival gene Bcl-2 was decreased in VDRKO cultures, implying that these osteoclasts may survive longer than WT osteoclasts. Our data indicate abnormal osteoclastogenesis due to the absence of Vdr expression, consistent with direct effects of vitamin D signalling being important for regulating the maturation and resorptive activities of osteoclasts.

Time dependent loss of trabecular bone in human tibial plateau fractures.

We investigated if time between injury and surgery affects cancellous bone properties in patients suffering tibial plateau fractures (TPF), in terms of structural integrity and gene expression controlling bone loss. A cohort of 29 TPF, operated 1-17 days post-injury, had biopsies from the fracture and an equivalent contralateral limb site, at surgery. Samples were assessed using micro-computed tomography and real-time RT-PCR analysis for the expression of genes known to be involved in bone remodeling and fracture healing. Significant decreases in the injured vs control side were observed for bone volume fraction (BV/TV, -13.5 ± 6.0%, p = 0.011), trabecular number (Tb.N, -10.5 ± 5.9%, p = 0.041) and trabecular thickness (Tb.Th, -4.6 ± 2.5%, p = 0.033). Changes in these parameters were more evident in patients operated 5-17 days post-injury, compared to those operated in the first 4 days post-injury. A significant negative association was found between Tb.Th (r = -0.54, p < 0.01) and BV/TV (r = -0.39, p < 0.05) in relation to time post-injury in the injured limb. Both BV/TV and Tb.Th were negatively associated with expression of key molecular markers of bone resorption, CTSK, ACP5, and the ratio of RANKL:OPG mRNA. These structure/gene expression relationships did not exist in the contralateral tibial plateau of these patients. This study demonstrated that there is a significant early time-dependent bone loss in the proximal tibia after TPF. This bone loss was significantly associated with altered expression of genes typically involved in the process of osteoclastic bone resorption but possibly also bone resorption by osteocytes. The mechanism of early bone loss in such fractures should be a subject of further investigation. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2865-2875, 2018.

Anodized 3D-printed titanium implants with dual micro- and nano-scale topography promote interaction with human osteoblasts and osteocyte-like cells.

The success of implantation of materials into bone is governed by effective osseointegration, requiring biocompatibility of the material and the attachment and differentiation of osteoblastic cells. To enhance cellular function in response to the implant surface, micro- and nano-scale topography have been suggested as essential. In this study, we present bone implants based on 3D-printed titanium alloy (Ti6Al4V), with a unique dual topography composed of micron-sized spherical particles and vertically aligned titania nanotubes. The implants were prepared by combination of 3D-printing and anodization processes, which are scalable, simple and cost-effective. The osseointegration properties of fabricated implants, examined using human osteoblasts, showed enhanced adhesion of osteoblasts compared with titanium materials commonly used as orthopaedic implants. Gene expression studies at early (day 7) and late (day 21) stages of culture were consistent with the Ti substrates inducing an osteoblast phenotype conducive to effective osseointegration. These implants with the unique combination of micro- and nano-scale topography are proposed as the new generation of multi-functional bone implants, suitable for addressing many orthopaedic challenges, including implant rejection, poor osseointegration, inflammation, drug delivery and bone healing. Copyright © 2016 John Wiley & Sons, Ltd.

Drug-releasing nano-engineered titanium implants: therapeutic efficacy in 3D cell culture model, controlled release and stability.

There is an ongoing demand for new approaches for treating localized bone pathologies. Here we propose a new strategy for treatment of such conditions, via local delivery of hormones/drugs to the trauma site using drug releasing nano-engineered implants. The proposed implants were prepared in the form of small Ti wires/needles with a nano-engineered oxide layer composed of array of titania nanotubes (TNTs). TNTs implants were inserted into a 3D collagen gel matrix containing human osteoblast-like, and the results confirmed cell migration onto the implants and their attachment and spread. To investigate therapeutic efficacy, TNTs/Ti wires loaded with parathyroid hormone (PTH), an approved anabolic therapeutic for the treatment of severe bone fractures, were inserted into 3D gels containing osteoblast-like cells. Gene expression studies revealed a suppression of SOST (sclerostin) and an increase in RANKL (receptor activator of nuclear factor kappa-B ligand) mRNA expression, confirming the release of PTH from TNTs at concentrations sufficient to alter cell function. The performance of the TNTs wire implants using an example of a drug needed at relatively higher concentrations, the anti-inflammatory drug indomethacin, is also demonstrated. Finally, the mechanical stability of the prepared implants was tested by their insertion into bovine trabecular bone cores ex vivo followed by retrieval, which confirmed the robustness of the TNT structures. This study provides proof of principle for the suitability of the TNT/Ti wire implants for localized bone therapy, which can be customized to cater for specific therapeutic requirements.

Evidence for altered osteoclastogenesis in splenocyte cultures from Cyp27b1 knockout mice.

The association between increased serum 25-hydroxyvitamin D (25D) and reduced osteoclastic bone resorption is well known. Previously, we have demonstrated that mechanism by which this occurs, may include the conversion of 25D to 1,25-dihydroxyvitamin D (1,25D) by osteoclasts, catalysed by the CYP27B1 enzyme. Local 1,25D synthesis in osteoclasts was shown to regulate osteoclastogenesis and moderating resorptive activity. Thus, we hypothesised that osteoclasts differentiated from mice with global deletion of the Cyp27b1 gene (Cyp27b1 KO) would display enhanced resorptive capacity due to the lack of an ameliorating effect of 1,25D. Splenocytes isolated from Cyp27b1 KO mice or their wild-type (WT) littermates between 6 and 8 weeks of age were cultured under osteoclast-forming conditions for up to 14 days. Osteoclast formation was measured by staining for the osteoclast marker tartrate resistant acid phosphatase (TRAP). Bone resorption activity was measured by plating the cells on a bone-like substrate. In Cyp27b1 KO cultures, osteoclastogenesis was reduced, as indicated by fewer TRAP-positive multinucleated cells at all time points measured (p<0.05) when compared to wild-type (WT) levels. However, Cyp27b1 KO osteoclasts demonstrated greater resorption on a per cell basis than their WT counterparts (p<0.03). In addition, the ratio of expression of the pro-apoptotic gene Bax to the pro-survival gene Bcl-2 was decreased in Cyp27b1 KO cultures, implying that these smaller osteoclasts survive longer than WT osteoclasts. Our data indicate abnormal osteoclastogenesis due to the absence of CYP27B1 expression, consistent with the notion that endogenous metabolism of 25D optimises osteoclastogenesis and ameliorates the resulting activity of mature osteoclasts.

Drug diffusion, integration, and stability of nanoengineered drug-releasing implants in bone ex-vivo.

To treat skeletal conditions such as bone infections, osteoporotic fractures, and osteosarcoma, it would be ideal to introduce drugs directly to the affected site. Localized drug delivery from the bone implants is a promising alternative to systemic drug administration. In this study we investigated electrochemically nanoengineered Ti wire implants with titania nanotubes (TNTs), as minimally invasive drug-releasing implants for the delivery of drugs directly into the bone tissue. Since trabecular bone in vivo contains a highly interconnected bone marrow, we sought to determine the influence of marrow on drug release and diffusion. Electrochemical anodization of Ti wires (length 10 mm) was performed to create an oxide layer with TNTs on the surface, followed by loading with a fluorescent model drug, Rhodamine B (RhB). Cores of bovine trabecular bone were generated from the sternum of a young steer, and were processed to have an intact bone marrow, or the marrow was removed. RhB-loaded TNTs/Ti wires were inserted into the bone cores, which were then cultured ex vivo using the ZetOS™ bioreactor system to maintain bone viability. Release and diffusion of RhB inside the bone was monitored using fluorescence imaging and different patterns of drug transport in the presence or absence of marrow were observed. Scanning electron microscopy of the implants after retrieval from bone cores confirmed survival of the TNTs structures. Histological investigation showed the presence of bone cells adherent on the implants. This study shows a potential of Ti drug-releasing implants based on TNTs technology towards localized bone therapy. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 714-725, 2016.

Evidence that osteocyte perilacunar remodelling contributes to polyethylene wear particle induced osteolysis.

Periprosthetic osteolysis (PO) leading to aseptic loosening, is the most common cause of failure of total hip replacement (THR) in the mid- to long-term. Polyethylene (PE) particulates from the wear of prosthesis liners are bioactive and are implicated in the initiation and or progression of osteolysis. Evidence exists that cells of the osteoblast/osteocyte lineage are affected by PE particles and contribute to the catabolic response by promoting osteoclastic bone resorption. In this study, we hypothesised that osteocytes contribute directly to PO by removing bone from their perilacunar matrix. Osteocyte responses to ultra-high molecular weight PE (UHMWPE) particles were examined in vitro in human primary osteocyte-like cultures, in vivo in the mouse calvarial osteolysis model, and in the acetabulum of patients undergoing revision total hip replacement (THR) surgery for PO. Osteocytes exposed to UHMWPE particles showed upregulated expression of catabolic markers, MMP-13, carbonic anhydrase 2 (CA2), cathepsin K (CTSK) and tartrate resistant acid phosphatase (TRAP), with no effect on cell viability, as assessed by Caspase 3 activity. Consistent with this catabolic activity causing perilacunar bone loss, histological analysis of calvarial sections from mice exposed to UHMWPE revealed a significant (p<0.001) increase in osteocyte lacunar area (Lac.Ar) compared to sham-operated animals. Furthermore, acetabular biopsies from patients with PO also showed significantly (p<0.001) increased osteocyte lacunar size in trabecular bone adjacent to PE particles, compared with osteocyte lacunar size in bone from primary THR patients. Together, these findings suggest a previously unrecognised action of UHMWPE wear particles on osteocytes, which directly results in a loss of osteocyte perilacunar bone. This action may exacerbate the indirect pro-osteoclastic action of UHMWPE-affected osteocytes, previously shown to contribute to aseptic loosening of orthopaedic implants. STATEMENT OF SIGNIFICANCE: This study addresses the clinical problem of periprosthetic osteolysis, bone loss in response to polyethylene wear particles derived from materials used in orthopaedic implants. Periprosthetic osteolysis has been thought to be due largely to wear particles stimulating the activity of bone resorbing osteoclasts. However, in this study we demonstrate for the first time that polyethylene particles stimulate another type of bone loss, mediated by the direct activity of bone mineral embedded osteocytes, termed osteocytic osteolysis or osteocyte perilacunar remodelling. This study provides new mechanistic insight into wear-particle mediated bone loss and represents a new paradigm for the way in which bone cells, namely osteocytes, the key controlling cell type in bone, react to biomaterials.

[Osteoporosis and alcohol intake].

Current understanding suggests that moderate quantities of alcohol use may have beneficial effects on bone mineral density, irrespective of age and sex, and would not be a risk factor of osteoporotic fracture. Heavy alcohol intake or alcoholism, however, frequently disrupts calcium and bone homeostasis, which leads to reduce bone mineral density and increase the incidence of fragility fracture. Since the habits of alcohol intake is closely associated with other diet and lifestyle factors, the exact mechanism underlying alcohol-related bone loss still remained to be clarified. The effects of alcohol use on osteoporosis should be studied more in detail.

Regulation of FGF23 expression in IDG-SW3 osteocytes and human bone by pro-inflammatory stimuli.

Fibroblast growth factor-23 (FGF23), produced by osteocytes, is the key physiological regulator of phosphate homeostasis. Sepsis patients often experience transient hypophosphataemia, suggesting the regulation of FGF23 levels by pro-inflammatory factors. Here, we used the osteocyte-like cell line IDG-SW3 to investigate the effect of pro-inflammatory stimuli on FGF23 production. In differentiated IDG-SW3 cultures, basal Fgf23 mRNA was dose-dependently up-regulated by pro-inflammatory cytokines TNF, IL-1ß and TWEAK, and bacterial LPS. Similar effects were observed in human bone samples. TNF- and IL-1ß-induced Fgf23 expression was NF-κB-dependent. Conversely, mRNA encoding negative regulators of FGF23, Phex, Dmp1 and Enpp1, were suppressed by TNF, IL-1ß, TWEAK and LPS, independent of NF-κß signalling. Galnt3, the protein product of which protects intact FGF23 protein from furin/furin-like proprotein convertase cleavage, increased in response to these treatments. C-terminal FGF23 and intact FGF23 protein levels also increased, the latter only in the presence of Furin inhibitors, suggesting that enzymatic cleavage exerts critical control of active FGF23 secretion by osteocytes. Our results demonstrate in principle that pro-inflammatory stimuli are capable of increasing osteocyte secretion of FGF23, which may contribute to hypophosphataemia during sepsis and possibly other inflammatory conditions.