Critical size bone defects pose a serious clinical problem, as the intrinsic healing capacity of bone fails due to the size of the defect. Bone healing might be aided by addition of 1,25(OH)2 vitamin D3 (vitD3) to bone tissue engineering scaffolds. VitD3can promote osteogenic differentiation of human stem cells such as adipose stem cells (hASCs), which is a clinical-relevant source of mesenchymal stem cells. However, it is unknown which release kinetics of vitD3, i.e. short or sustained release from scaffolds, leads to the most optimal osteogenic differentiation of hASCs. We hypothesized that sustained release of vitD3 leads to more osteogenic differentiation of hASCs than shorter applications. hASCs (1×105, passage 3–4) were seeded on 20 ± 1 mg of calcium phosphate particles (day 0), cultured for 20 days, and treated with a total amount of 124 ng vitD3. This treatment was provided either during 30 min before seeding (pre-incubation, short stimulation: [200 nM]), after seeding, over the first 2 days (burst- release high: [100 nM]), or over the total culture period of 20 days (sustained-release: [10 nM]). In the extra condition: burst-release low the hASCs were treated for 2 days after seeding with 6.2 ng vitD3 ([10 nM]) per day. Live/dead staining followed by fluorescent microscopy showed that hASCs attached to the calcium phosphate particles and were mostly viable (±75 %) at day 2. VitD3 applied for any duration did not affect the proliferation of hASCs at day 7 and day 20, measured with an alamar blue assay. At day 7, sustained-release increased the release of active alkaline phosphatase on average by 3.5-fold, compared to all the other conditions. At day 20, this was increased 4.3-fold. At both day 7 and day 20 total protein levels were similar in all conditions. Our results suggest that sustained release of VitD3 from bone tissue engineered scaffolds may be beneficial for the osteogenic differentiation of human stem cells for the treatment of critical bone size defects

Aseptic loosening is a growing problem for orthopaedic surgeons and the importance of elevated hydrostatic pressure in its development in vivo is now well documented, but the mechanisms by which pressure could enhance loosening are unclear. We have demonstrated that hydrostatic pressures increased MP synthesis of cytokines, chemokines, PGE2 and M-CSF in vitro, all of which are implicated in bone resorption. 1,25-dihydroxy vitamin D3 (1,25D3) has a pivotal role in bone resorption. It stimulates osteoclastic bone resorption and formation, causes fusion of committed osteoclast precursor cells and activates mature osteoclasts in vitro. Under the correct conditions, macrophages (MP) have the ability to differentiate into osteoclasts. Research has shown that MP can synthesise 1,25D3 and changes in this synthesis occur during MP differentiation. We therefore examined how the application of hydrostatic pressure to MP in vitro influenced their synthesis of 1,25D3. In this study, normal human peripheral blood MP (5x105/ml) were cultured for 7 days then exposed to physiological pressure (34.5x10-3MPa) and/or UHMWPE particles (8mg/ml) and the effect on synthesis of 1,25D3 by endogenous 1a-hydroxylase (1aOHase) was studied. MP were incubated with H3-25, hydroxy vitamin D and 1,25D3 synthesis was analysed by HPLC. 1,25D3 synthesis was increased in cells under pressure by an average of 17% compared to static controls. In situ hybridisation (ISH) was used to demonstrate expression of 1aOHase. Image analysis showed a small increase in 1aOHase mRNA in response to pressure and to particles, and a larger increase to the two stimuli simultaneously. Expressed as % of maximum +Pressure + Particles 100%;+ Particles 59%; +Pressure 37%; No Stimulus < 0.1%. These results suggest that 1,25D3 may be one of the factors which stimulates osteoclastic bone resorption in aseptic loosening. As both these stimuli are likely to be present in vivo, such synthesis could further exacerbate loosening

In Northern China, Ca intake and serum vitamin-D level of adolescents are low due to non-dairy-based diets and insufficient sunshine exposure. Maximisation of bone mineral accretion in childhood and adolescence requires adequate dietary calcium (Ca) intake and body vitamin-D status. This study focused on nutritional adaptation in Chinese adolescents under these adversed conditions by determining Ca absorption (CaAbn) and urinary calcium excretion (CaEx). 16 healthy individuals (12 girls, 4 boys) aged 9–17-y were recruited from Beijing during December. CaAbn was determined by a dual stable-isotope technique (44Ca and 42Ca) coupled with a Thermal-Ionization -Mass-Spectrometer. Mean ± . sd. Ca intake, 24-h CaEx, and serum 25-(OH) vitamin D3 were 603 ±158 mg/d, 87.5 ± 59.2 mg/24-h and 13.7 ± 4.8 ng/mL respectively. Mean serum 25-(OH) vitamin D3 reached the lower normal-limit of 11 ng/mL. 24-h-CaEx (< 100 mg/d) reflected a higher efficiency of Ca retention. CaAbn was found 57.4 ± 15.4% which was significantly higher than the U.S. counterparts (25–34%; Ca intake: 925 mg/d), P< 0.05. However, CaAbn in the current study was comparable to a group of healthy Hong Kong children aged 7-y (CaAbn: 54.8%, Ca intake: 862 mg/d, serum 25-(OH) vitamin D3:33.3 ng/mL). The study showed that growing individuals with suboptimal vitamin D status are still capable of enhancing calcium absorption and reducing urinary calcium excretion to allow adequate bone Ca accretion

Introduction. Vitamin D deficiency is common in patients undergoing total hip (THA) or total knee arthroplasty (TKA) which may affect prosthesis survival and 90-day readmission rates. The purpose of this study was to assess whether preoperative Vitamin D deficiency or insufficiency have an influence on revision, readmission, and complication rates following THA and TKA. We hypothesized that low Vitamin D levels in patients undergoing THA and TKA have a negative effect on revision rates. Methods. Patients who underwent primary THA or TKA in a 2-year period university hospital were identified and stratified into 3 groups based on preoperative 25-hydroxyvitamin D serum levels: normal levels of 30 ng/ml or greater, (2) deficient levels of 20–29.9 ng/ml, and (3) insufficient levels of less than 20 ng/ml. Patient demographics and postoperative course were collected from the electronic medical record. Results. This study found that 45% out of 197 THA had Vitamin D levels less than 30ng/ml and significantly higher odds (14.1, p=0.018) of requiring revision surgery at a mean follow-up of 34 ± 11.2 months. Out of 167 TKA, 46% were Vitamin deficient/insufficient without an influence on revision rate. Vitamin D levels did not influence 90-day readmissions, wound complications, or reaching discharge goals. Low Vitamin D levels correlated with high BMI and young patient age for THA. Conclusion. Based on the findings of this study, the authors recommend preoperative Vitamin D3 supplementation (2,000–4,000 IU daily) for patients with a BMI greater than 30kg/m. 2. undergoing THA. Patient with Vitamin D deficiency may require referral for endocrinologic work-up. Based on the findings of this study, the authors have adopted postoperative Vitamin D3 supplementation with 2,000–4,000 IU daily for 3 months as part of the rehabilitation protocol for all patients undergoing THA

To determine whether elderly patients presenting with a fracture of the proximal femur have an underlying vitamin D deficiency. We identified 59 consecutive cases of a fracture of the proximal femur over a 10-week period. 16 patients were excluded as they had a secondary underlying cause of bone loss which included chronic renal disease, rheumatoid arthritis, thyroid/parathyroid disorders, long term steroid usage and malignancy. Of the 43 that were eligible for the study, 7 were men and the average age was 81 years. 9 had sustained previous osteoporotic fractures. The majority mobilised independently or with 1 stick prior to the fall and the mechanism in all cases was a low velocity injury from standing height or less. The mean vitamin D3 level in these cases was 28. 3 nmol/ l. 28 of the 43 had a pathologically low level of vitamin D3 as defined as < 30nmol/l. The mean PTH level was 53. 7 nmol/ l. 15 of the 43 had an elevated PTH and all 15 were also deficient in vitamin D. The mean Albumin, an indicator of nutritional status, was 29 g/l. This study highlights that 65% of the patients who present with a fracture of the proximal femur are depleted in vitamin D. The ageing process is associated with a reduction in the intake of vitamin D, gut absorption and its sunlight activation. Repletion of vitamin D and suppression of parathyroid hormone, both prophylactically or at the time of injury, may reduce future fracture risk and assist in fracture repair

Approximately 5 – 10% of all bone fractures are associated with impaired healing. It is thought that regenerative medicine has the potential to improve on existing treatments for non-union fractures, and the European market for such treatments is projected to reach £2.2 billion in 2010. The use of scaffolds for the delivery of both growth factors and human Marrow Stromal Cells (hMSCs) is thought to be a promising approach. It may be desirable to promote proliferation and chemotaxis of hMSCs at the defect site shortly after implantation, and differentiation in the longer term. This is likely to require a dual delivery system, capable of releasing multiple drugs with different release profiles. Our aim has been to develop a polymer scaffold capable of releasing bioactive molecules that are able to direct the differentiation of primary hMSCs down the osteoblastic lineage. We have examined two mutually compatible drug delivery systems: collagen coating for short term release, and polymer encapsulation for longer term release. Polymer scaffolds were manufactured and coated with Type I Collagen containing BMP-7. hMSCs from three different patient sources were exposed to the scaffolds for 14 days. The cells were then histochemically stained for Alkaline Phosphatase (ALP) and photographed. The areas of ALP staining were then normalised against the total cell count. Normalised ALP expression was increased compared to the controls for three different patients (‘110 ± 39% SE, n=6, p=0.005’, ‘540 ± 270% SE, n=6, p=0.001’, and ‘32 ± 17% SE, n=6’). Scaffolds were also manufactured either with 1,25 Vitamin D3 (another active compound) in a coating of Collagen, or encapsulated using proprietary methodologies. It was found that both treatments significantly increased normalised Alkaline Phosphatase expression within the 14d experimental period demonstrating release of the active 1,25 Vitamin D3 (’88 ± 37% SE, n=6, p=0.012’ and ‘100 ± 32% SE, n=6, p=0.012’ respectively). Our findings suggest that, subject to future testing and development, such bioactive scaffolds could form the basis for a dual drug delivery system, suitable for applications in bone regenerative medicine

The physiological effects of 1,25 vitamin D3 (1,25D) are well known and the previously held dogma was that this was the only active vitamin D metabolite. A number of methods have been employed to demonstrate the effects of 24,25-dihydroxyvitamin D3 (24,25D) on osteoblast maturation responses, in the presence of FHBP, ((3S) 1-Fluoro-3-hydroxy-4-(oleoyloxy)butyl-1-phosphonate), an agonist of lysophosphatidic acid (LPA). These include alkaline phosphatase (ALP) expression and investigation of the role of CYP27B1, which is the enzyme responsible for converting 24,25D to 1,24,25D. Ketoconazole, which inhibits the actions of CYP27B1, as well as an enzyme-linked immunosorbant assay (ELISA) for CYP27B1 were used. The results clearly demonstrate that 24,25D stimulates maturation of MG63 cells when combined with FHBP. It has also been shown that the metabolite is not converted to another active form (for example, 1,24,25D) within osteoblasts, due to the absence of CYP27B1. 24,25D is an active vitamin D metabolite and exerts its effects in a bone fide manner, rather than following conversion to another active metabolite in osteoblasts. Given it is non-calcaemic, this metabolite has the exciting potential of being used in a bone regenerative setting in orthopaedic applications

In England and Wales in 2012 over 160,000 primary total hip and knee replacements were performed with 57% of hip replacements utilising uncemented prostheses. The main cause of failure, affecting approximately 10% of patients, is aseptic loosening. Previous research has found that functionalising titanium with lysophosphatidic acid (LPA) induces an increase in human osteoblast maturation on the implant surface through co-operation with active metabolites of vitamin D3. This feature, the small size of the LPS molecule and its affinity to readily bind to titanium and hydroxylapatite makes it an especially desirable molecule for bone biomaterials. Nevertheless biomaterials that also demonstrate anti-microbial properties are highly desirable. To test the antimicrobial efficacy of the LPA-functionalised titanium, a clinical isolate of Staphylococcus aureus, obtained from an infected revision surgery, was cultured on the surface of titanium discs functionalised with 0, 0.1. 0.5, 1, 2 and 5μM LPA. Bacterial adhesion was quantified at 1, 2, 6, 12 and 24 hours by live/dead counts and biofilm mass quantified by crystal violet staining after 24, 48, 72 and 96 hours culture. To elucidate the mechanisms of action of LPA, proteomic analysis of adhered bacteria was performed using SDS-PAGE and Western blots. 500nM to 1μM LPA were the optimum concentrations to significantly inhibit bacterial adhesion (ANOVA, p<0.001). These concentrations also reduced biofilm mass on the surface of the titanium. Proteomic analysis highlighted an increase in low molecular weight proteins as a result of optimal LPA surface concentrations. Fatty acid chains as found in LPA have previously been associated with causing leakage of low molecular weight proteins through increased cell membrane permeability. LPA coatings have the potential to enhance implant osseointegration whilst simultaneously reducing bacterial attachment. This technology may reduce both septic and aseptic failure of cementless joint prostheses, ultimately prolonging implant longevity and patient quality of life

Successful osseointegration requires the production of a mechanically competent collagenous matrix, by osteoblasts, at the implant site. Lysophosphatidic acid (LPA) is a bioactive lipid which we discovered interacts with vitamin D3 (D3) to secure human osteoblast (hOB) maturation on both titanium (Ti) and hydroxyapatite. We therefore covalently attached LPA and a related compound, (3S) 1-fluoro-3-hydroxy-4-butyl-1-phosphate (FHBP), to both solid and porous Ti discs and seeded them with hOBs to assess their ability to support D3-induced cell maturation. Solid functionalised discs were washed and reused a further two times, whilst other discs were stored for 6 months. Increased alkaline phosphatase (ALP) activity indicated that both LPA and FHBP-modified Ti serve as superior substrates for securing D3-induced hOB maturation compared to unmodified metal (p < 0.001). Although total ALP activity was less for cells on recycled discs and after storage, enzyme levels were still significantly greater compared to hOBs grown on control Ti. LPA and D3 co-treatment also resulted in an increase in osteocalcin (∼17ng/ml versus 6ng/ml for D3 alone, P < 0.001) and collagen synthesis (∼310pg/ml versus <10pg/ml for D3 alone, P < 0.001). Research is ongoing to evaluate the efficacy of our modified Ti surfaces to secure hOB formation from their stem cell progenitors

Introduction. Assessment for and treatment of osteoporosis is recommended following hip fracture. All forms of osteoporosis treatment require an adequate calcium intake and normal vitamin D levels. This study assesses vitamin D levels in patients with hip fractures and describes guidelines on how to manage low vitamin D levels with high dose oral vitamin D3 (cholecalciferol). Materials and methods. Circulating 25-hydroxyvitamin D levels were measured in consecutive patients with a hip fracture over an 18 month period. Substitution therapy with high dose oral cholecalciferol was started in 2 selected cohorts; one group received substitution therapy for 3 days, the second group for 7 days. Results. 381 patients with 387 hip fractures were included. Only 27 patients had sufficient (>75 nmol/L) vitamin D levels (mean 91.2 nmol/L) and of these 22 were taking supplements. The remainder, 354 patients, had low vitamin D levels (mean 26.4 nmol/L). Substitution with 50,000 IU cholecalciferol daily for 3 days in 14 patients resulted in an increase in vitamin D levels from 29.6 nmol/L to 81.4 nmol/L (p < 0.0001), at a mean of 14 days. 71% of patients achieved levels above the desired threshold of 75 nmol/L. Substitution with 50,000 IU cholecalciferol for 7 days in 54 patients resulted in an increase in vitamin D levels from 31.4 nmol/L to 131.1 nmol/L (p < 0.0001), at a mean of 16 days. 100% of patients achieved levels above the desired threshold. No clinical or biochemical side effects were reported. Discussion. Virtually all patients who are not taking vitamin D supplements and sustain a hip fracture have abnormally low circulating vitamin D levels and all require substitution. The routine measurement of vitamin D levelsmay be unnecessary. Substitution with 50,000 IU oral cholecalciferol daily for 7 days increases vitamin D levels rapidly, safely and consistently

Osteoblast growth and differentiation are central to the formation and maintenance of healthy bone tissue. The search for novel mechanisms resulting in osteoblast maturation are highly desirable on several fronts. Firstly they provide potentially important information on the normal development of bone, in addition they may offer alternative therapies for bone diseases like osteoporosis and finally they may facilitate ex-vivo manipulation of cells for the subsequent improvement of oseointegration in transplantation/tissue engineering regimens. Recently we have been addressing how calcitriol, an active metabolite of vitamin D3, integrates with the signalling of epidermal growth factor (EGF) following reports that calcitriol can influence EGF receptor trafficking, expression and ligand binding. We have also extended our studies to investigating how other growth factors known to signal via receptor tyrosine kinases (RTKs) interact with calcitriol in controlling osteoblast growth and differentiation. The co-treatment of human pre-osteoblasts (MG63) with EGF and calcitriol resulted in the synergistic induction of their differentiation as supported by demonstrable increases in alkaline phosphatase activity and osteocalcin. The intracellular components responsible for eliciting the maturation response included protein kinase C and MEK 1/2 since the addition of calphostin C or UO126, respectively, blocked the differentiation response. Other ligands known to signal via RTKs, namely IGF1, VEGF and FGF1 could not induce differentiation in the presence of calcitriol. These findings support the specific integration of calcitriol/EGF signalling in osteoblast maturation. Collectively we have identified a novel, integrated, signalling pathway that drives terminal differentiation of osteoblasts. Our findings support earlier predictions (Yoneda 1996) in identifying novel actions of EGF in bone that will lead to advances in the field. Yoneda, T. 1996. Local regulators of bone: Epidermal growth factor – transforming growth factor-α. In Principles of bone biology (ed. J.P. Bilezikian, L.G. Raisz and G.A. Rodan.), pp. 729–738. Academic press Ltd

Fracture prevention has so far been studied in patients included on the basis of low bone density, and not after a fracture. In this study the inclusion criteria was a new hip fracture irrespective of bone density. An international, multicenter, randomized, double-blind, placebo-controlled, parallel-group trial (HORIZON-RFT) studied whether the bisphosphonate, zoledronic acid (ZOL) 5 mg, reduced subsequent clinical fractures in men and women ≥50 yrs after a hip fracture. Methods: Patients with hip fracture were included. They received daily vitamin D3 and calcium supplements. Of 2127 randomized, 2111 were treated with once-yearly IV infusions of ZOL 5 mg (n=1054) or placebo (PBO; n=1057) and followed until 211 experienced new clinical fractures (the primary efficacy endpoint). Results: Baseline characteristics were similar. Median age was 76 yrs (range, 50–98); 76% were women. Clinical fractures occurred in 92 ZOL and 139 PBO patients. 2-year cumulative event rates were 8.59% and 13.88%, respectively (Kaplan-Meier); relative risk reduction was 35% (HR=0.65; 95% CI: 0.50–0.84; P=.0012). ZOL reduced risk for clinical vertebral and nonvertebral fractures vs. PBO by 46% (HR=0.54; 95% CI: 0.32–0.92; P=.0210) and 27% (HR=0.73; 95% CI: 0.55–0.98; P=.0338), respectively. ZOL reduced risk of hip fractures by 30% vs. PBO (HR=0.70; 95% CI: 0.41–1.19; P=NS). AEs and SAEs were comparable between groups. There were no significant differences in cardiovascular parameters or long-term renal function. No cases of ONJ were reported. Death occurred in 9.58% of ZOL patients vs 13.34% PBO, a 28% lower mortality risk (HR=0.72; 95% CI: 0.56–0.93, P=.0117). Conclusions: Subjects with a new hip fracture treated with annual IV ZOL infusions experienced significantly fewer clinical fractures vs. placebo. ZOL was well tolerated with a favorable safety profile. This is the first trial demonstrating a mortality benefit for an antiresorptive agent

Introduction: Between the elderly affected by femoral neck or diaphyseal fractures are emerging few serious complications as delate union, instability of osteosynthesis, re-fracture or periprosthetic fracture. In addition the co-morbidity elevate ulteriorly the risks of the new operation which is often impossible or refused. Recently it has been recommended to orthopaedic surgeons the managment and treatment of osteoporosis. The aim of this study was to consider at 2 years follow-up the functional outcome of femoral fracture in osteoporotic elderly treated with a surgical procedure followed by daily assumption of teriparatide, an anabolic agent increasing bone mineral content, density and strength. Materials and Methods: 21 compliant female between 63 and 94 years-old presenting a femoral fracture were recruited. Before operation they undergone to a routinary instrumental examins completed by bone metabolism screening. This was constituted by biochemical bone turnover markers, standard radiograms of dorsal and lumbar spine. Lumbar and contralateral femoral BMD was measured by DXA during hospitalization before the assumption of anabolic agent. They received daily subcutaneous teriparatide (20 microg) per day for 18 months, 1g of calcium and 800 UI of vitamin D3 daily as oral supplementation from day 15 by operation. All the patients repeated: xrays of affected segment at 2, 4, 6 months; biochemical bone markers 1, 3, 6, 12, 18 and 24 months; DEXA at first and second year. The evaluation of the quality of life was evaluated in terms of recovery of walking, need of re.operation, occurance of new fracture and with a questionnaire. Results: eleven lateral femoral neck fracture treated with endomedullary nail, six medial femoral neck fracture treated with cemented endoprosthesis, four periprosthetic fracture of the femur treated with plaque and screws. The healing was detected with radiograms before 4th month. The vitamin D was at lower levels at admission but the supplementation was sufficient to normalize. The other biochemical variables of bone formation and resorption peaked within the consolidation process then remained normalized for two years. Lumbar and contralateral femoral BMD were increased after 12 months and maintained at 24 months. At 2 years follow-up all patients live, walk in autonomy without or with stick and none of them have needed a re-operation or was afflicted by new vertebral or non vertebral fracture. Conclusions: The clinical relevance of the present study is the significant improvement of functional outcome and quality of life after femoral fracture in osteoporotic elderly with post-operative assumption of anbolic agent as Teriparatide

Aims

This study investigated the effects of β-caryophyllene (BCP) on protecting bone from vitamin D deficiency in mice fed on a diet either lacking (D-) or containing (D+) vitamin D.

Aims

This study was designed to develop a model for predicting bone mineral density (BMD) loss of the femur after total hip arthroplasty (THA) using artificial intelligence (AI), and to identify factors that influence the prediction. Additionally, we virtually examined the efficacy of administration of bisphosphonate for cases with severe BMD loss based on the predictive model.

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.

Aims

Low-energy distal radius fractures (DRFs) are the most common upper arm fractures correlated with bone fragility. Vitamin D deficiency is an important risk factor associated with DRFs. However, the relationship between DRF severity and vitamin D deficiency is not elucidated. Therefore, this study aimed to identify the correlation between DRF severity and serum 25-hydroxyvitamin-D level, which is an indicator of vitamin D deficiency.

Aims

Bone turnover markers (BTMs) follow distinct trends after fractures and limited evidence suggests differential levels in BTMs in patients with delayed healing. The effect of vitamin D, and other factors that influence BTMs and fracture healing, is important to elucidate the use of BTMs as surrogates of fracture healing. We sought to determine whether BTMs can be used as early markers of delayed fracture healing, and the effect of vitamin D on BTM response after fracture.

Aims

Despite the interest in the association of gut microbiota with bone health, limited population-based studies of gut microbiota and bone mineral density (BMD) have been made. Our aim is to explore the possible association between gut microbiota and BMD.

Aims

Orthopaedic infection is a potentially serious complication of elective and emergency trauma and orthopaedic procedures, with a high associated burden of morbidity and cost. Optimization of vitamin D levels has been postulated to be beneficial in the prevention of orthopaedic infection. This study explores the role of vitamin D in orthopaedic infection through a systematic review of available evidence.