Engineered bone tissue to recreate the continuity of damaged skeletal segments is one of the field of interest of tissue engineering. Trabecular titanium has very good mechanical properties and high in vitro and in vivo biocompatibility: it can be used in biomedical applications to promote osteointegration demonstrating that it can be successfully used for regenerative medicine in orthopaedic surgery (1). Purpose of this investigation was to evaluate the behavior of adipose tissue derived stem cells (hASCs) cultured on scaffolds of Trabecular TitaniumTM (Lima-Lto) (TT). hASCs are considered to be multipotent mesenchymal stem cells that are easily induced to differentiate into functional osteoblasts both in vitro and in vivo (2). The hASCs were obtained from the subcutaneous adipose tissue of healthy donors during total hip replacement procedures after digestion with collagenase. They were seeded on monolayer and on the TT scaffolds, and incubated at 37 degrees C in 5% CO2 with osteogenic medium or control medium. The expression of bone-related genes using RT-PCR, time course of alkaline phosphatase activity and morphological investigation with Scanning Electron Microscopy (SEM) were performed to evaluate the osteogenic differentiation of hASCs. Alkaline phosphatase activity, marker of the differentiation toward the osteogenic pattern, was significantly higher in hASCs grown with osteogenic medium than in cells grown with control medium, both in monolayer and TT scaffolds; moreover, also alkaline phosphatase of hASCs grown on TT scaffolds in the presence of control medium increased with time, differently from that of cells grown on monolayer. The osteogenic differentiated hASCs expressed the bone-related genes type I collagen, osteocalcin, Runx-2 and alkaline phosphatase. SEM observations showed that hASCs differentiated toward osteoblast-like cells: they produced a big amount of extracellular matrix that covered the surface of the porous scaffolds with bridges between the pore walls. These data suggest that hASCs are able to adhere to TT scaffolds, to acquire an osteoblastic phenotype and to produce abundant extracellular matrix, with but also without osteogenic medium. We can therefore conclude that this material carries osteinductive properties being responsible of ostegenic differentiation; consequently, this scaffold/cells construct is effective to regenerate damaged tissue and to restore the function of bone tissue

Early mortality in patients with hip fractures due to bony metastases is unknown. The aim was to quantify 30 and 90-day mortality in patients with metastatic hip fractures and identify markers associated with early death. Consecutive patients referred to orthopaedics with a metastatic proximal femoral fracture/impending fracture over a six-year period were compared to a matched control group of non-malignant hip fractures. Minimum follow-up was 1 year and data was analysed using the student´s t-test (significance p<0.05). From Jan 2010-Dec 2015, 163 patients were referred with metastatic proximal femoral lesions. 90-day mortality was three times higher than controls (44% 71/163 vs. 12% 4/33, p<0.01). Mean time from referral to surgery was longer in impending versus completed fractures (11 and 4 days respectively, p<0.05). Multiple biochemical markers were associated with early mortality in the metastatic group. Patients who died early were more likely to demonstrate low haemoglobin and albumin, and high c-reactive protein, platelets, urea, alkaline phosphatase and calcium (p<0.05). Several biochemical markers associated with early mortality reached clinical and statistical significance. These markers were combined into a score out of 7 and indicated a higher early mortality in metastatic patients compared to controls. Patients with a score of 5–6/7 were 31 times more likely to die within 90 days versus controls. This scoring system could be utilised to predict early mortality and guide management. The average delay to surgery of 4 days (completed) and 11 days (impending fractures) identifies a window to intervene and correct these abnormalities to improve survival

Introduction. Infection of endoprostheses is a serious complication in orthopedic surgery. As silver is known for its antibactierial effects, silver-coated endoprostheses have gained increased attention to decrease infection rates. However, cytotoxic effects of silver on bone cells have not been investigated in detail. We aimed to investigate whether silver nano-/microparticles and ionic silver exert cytotoxic effects on osteoblasts and osteoclasts in vitro and to correlate potential effects with the antibacterial effect on Staph. epidermidis. Methods. Murine osteoclasts (OC) and murine osteoblasts (OB) were treated with silver particles (avg. sizes: 50nm, 3μm, 30μm, 8μg/ml–500μg/ml) and Ag+NO3- (0.5μg/ml–500μg/ml). Silver treatment started on day 3 to prevent interference with cell adhesion. XTT assays were performed to assess cell viability. Tartrate resistant acidic phosphatase (TRAP) activity and alkaline phosphatase (ALP) activity served as measures for OC and OB differentiation, respectively. The release of silver ions from silver particles was quantified with atomic emission spectometry (AES). Titanium particles (avg. sizes: 50nm and 30μm) were used as controls to investigate whether potential silver effects were particle- or ion-mediated. The antimicrobial activity of silver ions and particles was tested with Staph. epidermidis agar inhibition assays. Results. Ionic silver had the strongest impact on cell differentiation and viability of OC and OB (OC differentiation: mean IC50 = 5 μg/ml, OC viability: mean IC50 = 14 μg/ml, OB differentiation: mean IC50 = 1 μg/ml, OB viability: mean IC50 = 1 μg/ml). Silver nanoparticles decreased cell differentiation and viability in a dose dependent manner (OC differentiation: mean IC50 = 5μg/ml, OC viability: mean IC50 = 14μg/ml, OB differentiation: mean IC50 = 1μg/ml, OB viability: mean IC50 = 1μg/ml). Silver microparticles as well as titanium nano- and microparticles had no effect on cell differentiation and viability. AES showed a size and dose dependent release of silver ions from silver nano- and microparticles. Agar inhibition assays showed a dose correlation of the antibacterial effect of silver with the cytotoxic effects on OB and OC. Conclusion. Silver nanoparticles and silver ions exert dose-dependent cytotoxic effects on OB and OC in vitro resulting in a severe alteration of cell differentiation and viability. The effect of silver on OB and OC seems to be mediated primarily by silver ions and correlates with the substance's antibacterial effects. The cytotoxicity of silver nanoparticles is mediated primarily by the size-dependent liberation of silver ions. Disturbance of OB and OC survival may have deleterious effects on the osseointegration of orthopedic implants. Further in vivo studies are needed to investigate the osseointegration of silver coated implants prior to their widespread clinical application

Introduction. iPSCs represent a promising cell source for bone regeneration. To generate osteoprogenitor cells, most protocols use the generation of embryoid bodies (EBs). However, these protocols give rise to heterogeneous population of different cell lineage. Hypothesis. We hypothesized that a direct plating method without EB formation step could be an efficient protocol for generating a homogeneous population of osteoprogenitor cells from iPSCs. Materials & Methods. Murine iPSC colonies were dissociated with trypsin-EDTA, and obtained single cells were cultured on gelatin-coated plates in MSC medium and FGF-2. Adherent cells obtained by this direct-plating technique were termed as direct-plated cells (DPCs). DPCs were evaluated for cell-surface protein expression using flow cytometry. Expression levels of Oct-3/4 mRNA in iPSCs and DPCs were analyzed by real-time PCR. DPCs were cultured for 14 days in osteogenic medium. Osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity, real-time PCR, and alizarin red S staining. Results. Expression level of Oct-3/4 in DPCs was robustly down-regulated compared to that in iPSCs. Flow cytometry analysis revealed DPCs had similar characteristics to MSC, suggesting DPCs lost pluripotency. Moreover, the DPCs exhibited high osteogenic potential. Discussion & Conclusion. Our novel direct plating method in the absence of EB formation step could be amenable to large-scale production of osteoprogenitor cells for bone regeneration

Introduction. Parathyroid hormone 1–34 (PTH) has been reported to accelerate fracture healing. Previously, we demonstrated human fracture hematoma contained osteo-/chondro-progenitor cells. To date, there has been no study investigating the effect of PTH on fracture hematoma-derived cells (HCs) in vitro. Hypothesis. We hypothesized PTH treatment affected osteogenesis and chondrogenesis of HCs. Materials & Methods. HCs were divided into 3 groups: control (growth medium), PTH (−) (osteogenic or chondrogenic medium (OM or CM)), and PTH (+) group (OM or CM with PTH). Cell proliferation was assessed by MTS assay. Osteogenesis was assessed by alkaline phosphatase (ALP) activity, real-time PCR, and Alizarin red S staining. Chondrogenesis was assessed by real-time PCR and Safranin-O staining. Results. There was no significant difference in proliferation among 3 groups. ALP activity and expression levels of ALP and Runx2 in PTH (+) group were comparable with PTH (−) group. HCs in PTH (−) and PTH (+) group were strongly stained with Alizarin red S staining. The expression levels of collagen-II and -X in PTH (+) group were significantly lower than PTH (−) group. Pellets in PTH (+) group were slightly stained with Safranin-O staining. Discussion & Conclusion. Our results revealed that PTH treatment did not affect osteogenesis and inhibited chondrogenesis of HCs. PTH treatment after fracture may positively affect other cells such as periosteum-derived cells and circulating stem cells

Osteoporosis following ovariectomy has been suggested to modulate bone response to polyethylene wear debris. In this work, we evaluate the influence of estrogen deficiency on experimental particle-induced osteolysis. Polyethylene (PE) particles were implanted onto the calvaria of wild-type (WT), sham-ovariectomized (OVX), OVX mice and OVX mice supplemented with estrogen (OVX+E2) (12 mice per group). Sham-implanted mice served as internal controls. After 14 days, seven skulls per group were analyzed with a high-resolution micro-computed tomography (CT) and by histomorphometry, and for tartrate-specific alkaline phosphatase. Five calvariae per group were cultured for the assay of IL-1, IL-6, TNF- and RANKL secretion using quantitative ELISA. The expression of RANKL and OPG mRNA were evaluated using real-time PCR. As assessed by CT and by histomorphometry, PE particles induced an extensive bone resorption and an intense inflammatory reaction in WT, sham-OVX and OVX+E2 mice. In OVX mice group, these features appeared considerably attenuated. In WT, sham-OVX and OVX+E2 mice, PE particles induced an increase in serum IL-6, in TNF-and RANKL local concentrations, and resulted in a two-fold increase in RANKL/OPG mRNA ratio. Conversely, these parameters remained unchanged in OVX mice after PE implantation. The combination of two well-known bone resorptive mechanisms ultimately attenuated osteolytic response, suggesting a protective effect of estrogen deficiency on particle-induced osteolysis. This paradoxical phenomenon was associated with a downregulation of pro-resorptive cytokines. It is hypothesized that excessive inflammatory response was controlled, illustrated by the absence of increase of serum IL-6 in OVX mice after PE implantation

Aims

Currently, periprosthetic fractures are excluded from the American Society for Bone and Mineral Research (ASBMR) definition of atypical femoral fracture (AFFs). This study aims to report on a series of periprosthetic femoral fractures (PFFs) that otherwise meet the criteria for AFFs. Secondary aims were to identify predictors of periprosthetic atypical femoral fractures (PAFFs) and quantify the complications of treatment.

Aims

The aims of this study were to characterize the frequency of missing data in the National Surgical Quality Improvement Program (NSQIP) database and to determine how missing data can influence the results of studies dealing with elderly patients with a fracture of the hip.

Aims

Little is known about the effect of haemorrhagic shock and resuscitation on fracture healing. This study used a rabbit model with a femoral osteotomy and fixation to examine this relationship.

Neurogenic heterotopic ossification (NHO) is a disorder of aberrant bone formation affecting one in five patients sustaining a spinal cord injury or traumatic brain injury. Ectopic bone forms around joints in characteristic patterns, causing pain and limiting movement especially around the hip and elbow. Clinical sequelae of neurogenic heterotopic ossification include urinary tract infection, pressure injuries, pneumonia and poor hygiene, making early diagnosis and treatment clinically compelling. However, diagnosis remains difficult with more investigation needed. Our pathophysiological understanding stems from mechanisms of basic bone formation enhanced by evidence of systemic influences from circulating humor factors and perhaps neurological ones. This increasing understanding guides our implementation of current prophylaxis and treatment including the use of non-steroidal anti-inflammatory drugs, bisphosphonates, radiation therapy and surgery and, importantly, should direct future, more effective ones.

Objectives

The biomembrane (induced membrane) formed around polymethylmethacrylate (PMMA) spacers has value in clinical applications for bone defect reconstruction. Few studies have evaluated its cellular, molecular or stem cell features. Our objective was to characterise induced membrane morphology, molecular features and osteogenic stem cell characteristics.

Several bisphosphonates are now available for the treatment of osteoporosis. Porous hydroxyapatite/collagen (HA/Col) composite is an osteoconductive bone substitute which is resorbed by osteoclasts. The effects of the bisphosphonate alendronate on the formation of bone in porous HA/Col and its resorption by osteoclasts were evaluated using a rabbit model. Porous HA/Col cylinders measuring 6 mm in diameter and 8 mm in length, with a pore size of 100 μm to 500 μm and 95% porosity, were inserted into a defect produced in the lateral femoral condyles of 72 rabbits. The rabbits were divided into four groups based on the protocol of alendronate administration: the control group did not receive any alendronate, the pre group had alendronate treatment for three weeks prior to the implantation of the HA/Col, the post group had alendronate treatment following implantation until euthanasia, and the pre+post group had continuous alendronate treatment from three weeks prior to surgery until euthanasia. All rabbits were injected intravenously with either saline or alendronate (7.5 μg/kg) once a week. Each group had 18 rabbits, six in each group being killed at three, six and 12 weeks post-operatively. Alendronate administration suppressed the resorption of the implants. Additionally, the mineral densities of newly formed bone in the alendronate-treated groups were lower than those in the control group at 12 weeks post-operatively. Interestingly, the number of osteoclasts attached to the implant correlated with the extent of bone formation at three weeks.

In conclusion, the systemic administration of alendronate in our rabbit model at a dose-for-weight equivalent to the clinical dose used in the treatment of osteoporosis in Japan affected the mineral density and remodelling of bone tissue in implanted porous HA/Col composites.

The scarcity of mesenchymal stem cells (MSCs) in iliac crest bone marrow aspirate (ICBMA), and the expense and time in culturing cells, has led to the search for alternative harvest sites. The reamer-irrigation-aspirator (RIA) provides continuous irrigation and suction during reaming of long bones. The aspirated contents pass via a filter, trapping bony fragments, before moving into a ‘waste’ bag from which MSCs have been previously isolated. We examined the liquid and solid phases, performed a novel digestion of the solid phase, and made a comparative assessment in terms of number, phenotype and differentiation capacity with matched ICBMA.

The solid fraction from the filtrate was digested for 60 minutes at 37°C with collagenase. Enumeration was performed via the colony-forming unit fibroblast (CFU-F) assay. Passage (P2) cells were differentiated towards osteogenic, adipogenic and chondrogenic lineages, and their phenotypes assessed using flow cytometry (CD33, CD34, CD45, CD73, CD90, and CD105).

MSCs from the RIA phases were able to differentiate at least as well as those from ICBMA, and all fractions had phenotypes consistent with other established sources. The median number of colonies for the three groups was: ICBMA = 8.5 (2 to 86), RIA-liquid = 19.5 (4 to 90), RIA-solid = 109 (67 to 200) per 200 μl. The mean total yield of cells for the three groups was: ICBMA = 920 (0 to 4275), RIA-liquid = 114 983 (16 500 to 477 750), RIA-solid = 12 785 (7210 to 28 475).

The RIA filtrate contains large numbers of MSCs that could potentially be extracted without enzymatic digestion and used for bone repair without prior cell expansion.

There have been recent reports linking alendronate and a specific pattern of subtrochanteric insufficiency fracture. We performed a retrospective review of all subtrochanteric fractures admitted to our institution between 2001 and 2007. There were 20 patients who met the inclusion criteria, 12 of whom were on long-term alendronate. Alendronate-associated fractures tend to be bilateral (Fisher’s exact test, p = 0.018), have unique radiological features (p < 0.0005), be associated radiologically with a pre-existing ellipsoid thickening of the lateral femoral cortex and are likely to be preceded by prodromal pain. Biomechanical investigations did not suggest overt metabolic bone disease. Only one patient on alendronate had osteoporosis prior to the start of therapy. We used these findings to develop a management protocol to optimise fracture healing. We also advocate careful surveillance in individuals at-risk, and present our experience with screening and prophylactic fixation in selected patients.

This multicentre prospective clinical trial aimed to determine whether early administration of alendronate (ALN) delays fracture healing after surgical treatment of fractures of the distal radius. The study population comprised 80 patients (four men and 76 women) with a mean age of 70 years (52 to 86) with acute fragility fractures of the distal radius requiring open reduction and internal fixation with a volar locking plate and screws. Two groups of 40 patients each were randomly allocated either to receive once weekly oral ALN administration (35 mg) within a few days after surgery and continued for six months, or oral ALN administration delayed until four months after surgery. Postero-anterior and lateral radiographs of the affected wrist were taken monthly for six months after surgery. No differences between groups was observed with regard to gender (p = 1.0), age (p = 0.916), fracture classification (p = 0.274) or bone mineral density measured at the spine (p = 0.714). The radiographs were assessed by three independent assessors. There were no significant differences in the mean time to complete cortical bridging observed between the ALN group (3.5 months (se 0.16)) and the no-ALN group (3.1 months (se 0.15)) (p = 0.068). All the fractures healed in the both groups by the last follow-up. Improvement of the Quick-Disabilities of the Arm, Shoulder and Hand (QuickDASH) score, grip strength, wrist range of movement, and tenderness over the fracture site did not differ between the groups over the six-month period. Based on our results, early administration of ALN after surgery for distal radius fracture did not appear to delay fracture healing times either radiologically or clinically.

Cite this article: Bone Joint J 2013;95-B:1544–50.

Heterotopic ossification (HO) is perhaps the single most significant obstacle to independence, functional mobility, and return to duty for combat-injured veterans of Operation Enduring Freedom and Operation Iraqi Freedom. Recent research into the cause(s) of HO has been driven by a markedly higher prevalence seen in these wounded warriors than encountered in previous wars or following civilian trauma. To that end, research in both civilian and military laboratories continues to shed light onto the complex mechanisms behind HO formation, including systemic and wound specific factors, cell lineage, and neurogenic inflammation. Of particular interest, non-invasive in vivo testing using Raman spectroscopy may become a feasible modality for early detection, and a wound-specific model designed to detect the early gene transcript signatures associated with HO is being tested. Through a combined effort, the goals of early detection, risk stratification, and development of novel systemic and local prophylaxis may soon be attainable.

Heterotopic ossification (HO) of the hip after injury to the central nervous system can lead to joint ankylosis. Surgery is usually delayed to avoid recurrence, even if the functional status is affected. We report a consecutive series of patients with HO of the hip after injury to the central nervous system who required surgery in a single, specialised tertiary referral unit. As was usual practice, they all underwent CT to determine the location of the HO and to evaluate the density of the femoral head and articular surface. The outcome of surgery was correlated with the pre-, peri- and post-operative findings.

In all, 183 hips (143 patients) were included of which 70 were ankylosed. A total of 25 peri-operative fractures of the femoral neck occurred, all of which arose in patients with ankylosed hips and were associated with intra-articular lesions in 18 and severe osteopenia of the femoral head in seven. All the intra-articular lesions were predicted by CT and strongly associated with post-operative complications.

The loss of the range of movement before ankylosis is a more important factor than the maturity of the HO in deciding the timing of surgery. Early surgical intervention minimises the development of intra-articular pathology, osteoporosis and the resultant complications without increasing the risk of recurrence of HO.

Between 2000 and 2006 we performed salvage tibiotalar arthrodesis in 17 diabetic patients (17 ankles) with grossly unstable ankles caused by bimalleolar fractures complicated by Charcot neuro-arthropathy. There were ten women and seven men with a mean age of 61.6 years (57 to 69). A crossed-screw technique was used. Two screws were used in eight patients and three screws in nine. Additional graft from the malleoli was used in all patients. The mean follow-up was 26 months (12 to 48) and the mean time to union was 5.8 months (4 to 8). A stable ankle was achieved in 14 patients (82.4%), nine of whom had bony fusion and five had a stiff fibrous union. The results were significantly better in underweight patients, in those in whom surgery had been performed three to six months after the onset of acute Charcot arthropathy, in those who had received anti-resorptive medication during the acute stage, in those without extensive peripheral neuropathy, and in those with adequate peripheral oxygen saturation (> 95%). The arthrodesis failed because of avascular necrosis of the talus in only three patients (17.6%), who developed grossly unstable, ulcerated hindfeet, and required below-knee amputation.