Introduction. The therapeutic potential of hematopoietic stem cells for fracture healing has been demonstrated with mechanistic insight of vasculogenesis and osteogenesis enhancement. Lnk has recently been proved an essential inhibitory signaling molecule in SCF-c-Kit signaling pathway for stem cell self-renewal demonstrating enhanced hematopoietic and osteogenic reconstitution in Lnk-deficient mice. We investigated the hypothesis that down regulation of Lnk enhances regenerative response via vasculogenesis and osteogenesis in fracture healing. Methods. A reproducible model of femoral fracture was created in mice. Immediately after fracture creation, mice received local administration of the following materials with AteloGene, 10μM (1)Lnk siRNA, (2)control siRNA. Results. Lnk group demonstrated more prompt fracture repair than control group. The functional bone healing was also significantly greater in Lnk group. Immunohistochemical staining and the mRNA expressions in fracture sites indicated the superior ability for angiogenesis and osteogenesis in Lnk group. Moreover, Lnk siRNA transfected cells showed high capacity of colony formation in vitro. Conclusion. We clarified that negatively controlled Lnk system contributed to a favorable environment for fracture healing by enhancing vasculogenesis and osteogenesis. These findings suggest that down regulation of Lnk may have a clinical potential for faster fracture healing, which contributes to reduce delayed union or nonunion

Introduction. Locking plates have been especially useful in the treatment of osteoporotic fractures. This study aimed to investigate the role of locking plate in femoral fractures in patients with osteogenesis imperfecta. Material & Methods. 6 male patients with osteogenesis imperfecta were operated for femoral fractures using locking compression plate. Their age ranged between 4 and 14 years. They were kept non weight bearing till the fracture united. Results. 5 patients out of 6 patients had refracture, as soon as they started walking. All 5 patients were reoperated using intramedullary titanium elastic nails. All of them united later on and were walking full weight bearing at the end of one year. Conclusion. Our study indicates that locking plates should not be used in the treatment of fractures in patients with osteogenesis imperfecta

The burden of osteoporosis (OP), and its accompanied low energy fractures, is ever increasing. Targeted therapies are under development to stem the tide of the disease, with microRNAs identified as biomarkers and potential targets. Assessing the functional capacity of bone marrow mesenchymal stromal cells (BMSC) from patients with low energy neck of femur fractures (NOF) will identify the expected outcomes to be achieved from new, targeted osteogenic therapies. Two patient groups were assessed; low energy NOF and osteoarthritic. Bone marrow aspirates were taken at time of arthroplasty surgery. The adherent fraction was cultured and assessed by flow cytometry, microRNA expression and differentiation functionality. Both patient groups demonstrated characteristic extracellular markers of BMSCs. 3 key markers were significantly reduced in their expression in the NOF group (CD 90, 13, 166 P=0.0286). Reduced differentiation capacity was observed in the NOF group when cultured in osteogenic and adipogenic culture medium. 105 microRNAs were seen to be significantly dysregulated, with microRNAs known to be crucial to osteogenesis and disease process such as osteoporosis abnormally expressed. This data demonstrates the impaired functional capacity of BMSCs and their abnormal microRNA expression in patients who suffer a low energy NOF. Future targeted therapies for OP must address this to maximise their restorative effect on diseased bone. The important role microRNAs can play as biomarkers and target sites has been further reinforced

In biomaterial engineering the surface of an implant can influence cell differentiation, adhesion and affinity towards the implant. Increased bone marrow derived mesenchymal stromal cell (BMSC) differentiation towards bone forming osteoblasts, on contact with an implant, can improve osteointegration. The process of micropatterning has been shown to improve osteointegration in polymers, but there are few reports surrounding ceramics. The purpose of this study was to establish a co-culture of BMSCs with osteoclast progenitor cells and to observe the response to micropatterned zirconia toughened alumina (ZTA) ceramics with 30 µm diameter pits. The aim was to establish if the pits were specifically bioactive towards osteogenesis or were generally bioactive and would also stimulate osteoclastogenesis that could potentially lead to osteolysis. We demonstrate specific bioactivity of micropits towards osteogenesis with more nodule formation and less osteoclastogenesis. This may have a role when designing ceramic orthopaedic implants

Aims

Heterotopic ossification (HO) is a common complication after elbow trauma and can cause severe upper limb disability. Although multiple prognostic factors have been reported to be associated with the development of post-traumatic HO, no model has yet been able to combine these predictors more succinctly to convey prognostic information and medical measures to patients. Therefore, this study aimed to identify prognostic factors leading to the formation of HO after surgery for elbow trauma, and to establish and validate a nomogram to predict the probability of HO formation in such particular injuries.

Introduction. Failures in fracture healing are mainly caused by a lack of neovascularization. We have previously demonstrated that G-CSF-mobilized peripheral blood (GM-PB) CD34+ cells, an endothelial progenitor enriched cell population, contributed to fracture healing via vasculogenesis and osteogenesis. We postulated the hypothesis that local transplantation of culture expanded bone marrow (cEx-BM) CD34+ cells could exhibit therapeutic potential for fracture healing. Materials. BM CD34+ cells were cultured in specific medium with 5 growth factors for 1week. A reproducible model of femoral fracture was created in nude rats with periosteum cauterization, which leads to nonunion at 8 weeks post-fracture. Rats received local administration of the following cells or PBS alone(1)cEx-BM, (2)BM, (3)GM-PB CD34+ cells or (4)PBS. Results. Our 7-day culture expansion technique allowed us to obtain 23 times of BM CD34+ cells maintaining 60% purity of CD34 positivity. cEx-BM CD34+ cells exhibited striking therapeutic efficacy for unhealing fracture promoting neovascularization and osteogenesis in sites of fracture. Moreover, cEx-BM CD34+ cells showed high capacity of colony formation and osteogenic differentiation. Conclusion. BM CD34+ cells can be obtained from the fracture site at the time of primary operation and stored for further use, autologous culture expanded BM CD34+ cell transplantation therapy would be not only a simple but also powerful therapeutic strategy for unhealing fracture

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

Purpose. Surgical correction of upper limb deformities in severe osteogenesis imperfecta (OI) is technically difficult and less absolving, hence we aimed to analyse the surgical complications of rodding the humeri in severe OI. Methods and results. Retrospective analysis was carried out for consecutive humeral roddings for severe OI in last 3 years. Surgical technique for all humeri included retrograde telescopic nailing (female or both of FD or TST rods) with entry from olecranon fossa, exploration of radial nerve followed by osteotomies. Deformities were quantified and sub-classified as per level of deformity). Variables such as number of osteotomies, radiological union, intraoperative and postoperative complications, improvement in ROM and subjective patient satisfaction were recorded. Total 18 humeri in 12 patients with type III OI (except 1) with mean age of 8.9 years underwent nailing. All patients achieved radiological union at 6–10 weeks. Total 8 complications (44.4 %) were reported within mean 8.4 months follow up. Four segments (22.2 %) had intraoperative fractures at distal third of the humerus while negotiating the nail. Significantly higher intraoperative complications were encountered in humeri fixed with both components combined and upper third level deformities, deformities > 900 and more than 2 osteotomies. Other complications were prominent implant at upper end (2) with growth and one each of contralateral fracture and distal humeral varus. All patients reported improvement in ROM and functional status. Conclusion. Significant complications are associated with humeral nailing for severe OI. Quantification of the deformity with meticulous surgical planning and execution is advised. Despite these complications outcomes show benefits of the surgical treatment. Level of evidence. Therapeutic III

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. Methods. Following Institutional Review Board approval, biomembrane specimens were obtained from 12 patient surgeries for management of segmental bony defects (mean patient age 40.7 years, standard deviation 14.4). Biomembranes from nine tibias and three femurs were processed for morphologic, molecular or stem cell analyses. Gene expression was determined using the Affymetrix GeneChip Operating Software (GCOS). Molecular analyses compared biomembrane gene expression patterns with a mineralising osteoblast culture, and gene expression in specimens with longer spacer duration (> 12 weeks) with specimens with shorter durations. Statistical analyses used the unpaired student t-test (two tailed; p < 0.05 was considered significant). Results. Average PMMA spacer in vivo time was 11.9 weeks (six to 18). Trabecular bone was present in 33.3% of the biomembrane specimens; bone presence did not correlate with spacer duration. Biomembrane morphology showed high vascularity and collagen content and positive staining for the key bone forming regulators, bone morphogenetic protein 2 (BMP2) and runt-related transcription factor 2 (RUNX2). Positive differentiation of cultured biomembrane cells for osteogenesis was found in cells from patients with PMMA present for six to 17 weeks. Stem cell differentiation showed greater variability in pluripotency for osteogenic potential (70.0%) compared with chondrogenic or adipogenic potentials (100% and 90.0%, respectively). Significant upregulation of BMP2 and 6, numerous collagens, and bone gla protein was present in biomembrane compared with the cultured cell line. Biomembranes with longer resident PMMA spacer duration (vs those with shorter residence) showed significant upregulation of bone-related, stem cell, and vascular-related genes. Conclusion. The biomembrane technique is gaining favour in the management of complicated bone defects. Novel data on biological mechanisms provide improved understanding of the biomembrane’s osteogenic potential and molecular properties. Cite this article: Dr H. E. Gruber. Osteogenic, stem cell and molecular characterisation of the human induced membrane from extremity bone defects. Bone Joint Res 2016;5:106–115. DOI: 10.1302/2046-3758.54.2000483

The Fassier-Duval (FD) rod, which offers a single-entry design and allows elongation for growth, has been widely adopted in paediatric deformity correction over the past decade, although evidence is limited in literature regarding the associated complications from its use. All FD roddings carried out in a Scottish tertiary referral centre were identified. The electronic records and radiographs of each procedure were reviewed. The follow-up duration, indications for surgery, complications arisen and further operations were recorded. 21 procedures in 11 patients were identified between 2009–2016. The mean age at operation was 6 years and 2 months. The median follow-up period was 3 years and 9 months. The main underlying pathology was osteogenesis imperfecta (71.4%, n=15). The main indication of surgery was deformity correction (61.9%, n=13). 11 (52.4%) FD roddings were for femur and the remainder were for tibia. The commonest complication was proximal migration (n=6, 28.6%). In our cohort we did not have negative telescoping or non-union. Two procedures (9.5%) were complicated by deep infections which were successfully treated. There were 3 further operations (14.3%), including one revision to a locked intramedullary nail for fracture and one below knee amputation for recurrent pseudarthrosis. We compared our results with those from Birke and co (J Paediatr Orthop 2011) from Australia. Our results are comparable and with a longer follow-up period. Although FD rodding allows children to maintain their mobility and prevent fractures, there are significant complications associated with its use. We hope in the future other centres can publish their results to allow improvements in surgical practice and implant design

We describe a series of 20 patients with ununited fractures of the femoral neck following neglected trauma or failed primary internal fixation who were seen at a mean of 7.5 months (2 to 18) following injury. Open reduction and internal fixation of the fracture was performed in all patients, together with a myoperiosteal flap on the quadratus femoris muscle pedicle. Union occurred at a mean of 4.9 months (2 to 10) in all patients. The mean follow-up was for 70 months (14 to 144). There was no further progression in six of seven patients with pre-operative radiological evidence of osteonecrosis of the femoral head. One patient had delayed collapse and flattening of the femoral head ten years after union of the fracture, but remained asymptomatic. This study demonstrates the orthopaedic application of myoperiosteal grafting for inducing osteogenesis in a difficult clinical situation

Aims

The primary aim of this study was to identify independent predictors associated with nonunion and delayed union of tibial diaphyseal fractures treated with intramedullary nailing. The secondary aim was to assess the Radiological Union Scale for Tibial fractures (RUST) score as an early predictor of tibial fracture nonunion.

Mesenchymal stem cells (MSCs) reside around blood vessels in all organs. This reservoir of progenitors can be ‘recruited’ in response to injury. The ability to manipulate stem cells therapeutically within injured tissue provides an attractive alternative to transplantation. Stem cells are regulated by neighbouring cells. We hypothesized that endothelial cells (ECs) influence MSC differentiation into bone and fat. MSCs were sorted from fat using fluorescent activated sorting. Their capacity to differentiate into bone, fat and cartilage was used to confirm MSC phenotype. MSCs and ECs were cultured in two-dimensions (standard culture dishes) and three-dimensions (vascular networks suspended in gel). Cocultures were exposed to osteogenic and adipogenic media. The role of EC-released factors on MSC differentiation was determined using a system in which cells share media but do not contact. Wnt pathway modulators were used to investigate the role of Wnt signalling. MSCs differentiated into bone, fat and cartilage. MSCs and ECs integrated in two- and three-dimensions. MSCs and ECs formed vessel-like structures in three-dimensions. When cultured with ECs, MSC differentiation to bone was accelerated while differentiation to fat was inhibited. This effect on osteogenesis was maintained when cells shared media but did not contact. Coculture with Wnt modulators confirmed that this effect is in part, mediated through Wnt signalling. Our data suggest that ECs influence MSC differentiation. Therapeutic targeting of EC-MSCs signalling may enable manipulation of MSCs in vivo avoiding the need for cell transplantation. This could enable trauma and orthopaedic patients who have healthy resident stem cells to self-repair

Introduction. The delay looks radiographically as a fracture callus not very evident or absent 6 months after osteosynthesis. Patients undergo a long period of immobilization and this fact causes the increase the social cost of the disease. The technique we suggest aims to the reduce the period of immobilization and as a consequence the management costs of the disease. Materials and methods. Our technique includes the infiltration of the delay focus with platelet rich fibrin, bone marrow concentrated and demineralized bone matrix. Outpatients and radiographic checks were carried out 3, 6 and 12 months after surgery. The treatment was considered fail in case of absence of bone callus at 3 out of 4 corticals at the rx after 6 months from surgery. Results. From November 2008 we treated 14 patients (average age 35 years, range 18 to 53). The treatment was performed after an average period of 14 months (range 5 to 38) from the fracture. The average follow up was 12 months (range 3–30). After 3 months from surgery, seven patients bear full load, did not feel pain and X-ray showed an increase of osteogenesis. Two cases failed; in one of them the patient underwent another infiltration and in the other one we replaced the fixation. Conclusion. Our tecnique lets a reduction of healing times in patients with delay and can be performed through the One Day Surgery programme

Introduction. Low-intensity pulsed ultrasound (LIPUS) has been reported to enhance healing of fracture and nonunion. Bone morphogenetic protein-7 (BMP-7) has also been reported to promote bone formation. Recently, we demonstrated progenitor cells with osteogenic/chondrogenic differentiation potential existed in human fracture hematoma and nonunion tissue. Hypothesis. We hypothesised the combined application of LIPUS and BMP-7 would cause major effect on osteogenesis of hematoma-derived cells (HCs) and nonunion tissue-derived cells (NCs). Materials & Methods. HCs and NCs were isolated, and cultured. The cells were divided into two groups: (1) BMP-7 group: cells cultured in osteogenic medium (OM), and (2) BMP-7 + LIPUS group: cells cultured in OM with LIPUS treatment. LIPUS (30 mW/cm2, intensity at 1.5 MHz) was given for 20 minutes daily. Osteogenic differentiation potential and proliferation were analysed. Results. ALP activity, the gene expression of osteogenic genes, and mineralisation of HCs and NCs were shown to be higher in BMP-7 + LIPUS group than in BMP-7 group. There was no significant difference in cell proliferation between the two groups. Discussion. Our findings demonstrated the significant effect of LIPUS on the osteogenic differentiation of HCs and NCs induced by BMP-7. This study may provide significant evidence for the clinical combined application of BMP-7 and LIPUS for the treatment of severe bone fracture and nonunion

Introduction. Nonunion is a common and costly fracture outcome. Intricate reciprocity between angiogenesis and osteogenesis means vascular cell-based therapy offers a novel approach to stimulating bone regeneration. Hypothesis. The current study compared early and late outgrowth endothelial progenitor cell subtypes (EPCs vs OECs) for fracture healing potential in vitro and in vivo. Methods. Primary cell cultures were isolated and characterized by endothelial assays, immunosorbent assays, and multi-color flow cytometry. Co-cultures of EPC subtypes with/without primary osteoblasts (pObs) were analyzed for tube length and connectivity. In vivo, EPCs or OECs (1×10. 6. ) seeded on a gelfoam scaffold were implanted in a rat model of nonunion. Radiography was used to monitor callus formation. Results. OECs expressed more BMP-2 and less VEGF than EPCs (p<0.05). Analysis of surface markers showed decreased CD34+/CD133+/Flk-1+, CD133+ and CD45+ populations in OECs while CD34+/CD31+/Flk-1+ cells increased. pObs significantly inhibited the strong tubulogenesis of OECs while enhancing connectivity and sprout length of EPCs. In vivo, 0/6 scaffold-control and 1/5 OEC rats achieved union at 10 weeks. In comparison, all EPC rats achieved full or partial union. Discussion and Conclusion. Despite favorable tubulogenic and osteoconductive profiles of OECs, EPCs display enhanced fracture healing in vivo. Differences in CXCR4 expression and cell-mediated effects may contribute to this result

Aims

To clarify the effectiveness of the induced membrane technique (IMT) using beta-tricalcium phosphate (β-TCP) for reconstruction of segmental bone defects by evaluating clinical and radiological outcomes, and the effect of defect size and operated site on surgical outcomes.

A multicenter retrospective analysis of patients treated for tibial fracture was conducted to develop a score that correlates with fracture healing time and, ultimately, to identify the risk gradient of delayed healing. The clinical records of 93 patients treated for tibial fracture at three orthopaedic centers were evaluated. Patients were considered healed when full weight bearing was allowed and no further controls were scheduled. For the purpose of our analysis, we separated patients healed within or after 180 days. Patient's risk factors known to be associated to delay healing, as well as fracture morphology and orthopaedic treatment were recorded in an electronic Case Report Form (e-CRF). Information available in the literature was used to weight the relative risk (RR) associated to each risk factor; values were combined to calculate a score to be correlated to the fracture healing time: L-ARRCO (Literature-Algoritmo Rischio Ritardo Consolidazione Ossea). Among all information collected in e-CRFs, we identified other risk factors, associated to delayed healing, that were used to calculate a new score: ARRCO. Univariate logistic analysis was used to determine a correlation between the score and healing time. Analysis by receiver operating characteristic (ROC) and calculation of the area under the curve (AUC) were used for sensitivity and specificity. Complete information was available for 53 patients. The mean value of the L-ARRCO score among patients healed within 180 days was 5.78 ± 1.59 and 7.05 ± 2.46 among those healed afterwards, p=0.044. The mean value of the ARRCO score of patients healed within 180 days was 5.92 ± 1.78 and 9.03 ± 2.79 among those healed afterwards, p<0.0001. The ROC curve shows an AUC of 0.62±0.09 for L-ARRCO and an AUC of 0.82±0.07 for ARRCO, (p<0.0001). We have shown that the ARRCO score value is significantly correlated to fracture healing time. The score may be used to identify fractures at risk of delayed healing, thus allowing surgeon's early intervention to stimulate osteogenesis

Aims

The aim of this double-blind prospective randomised controlled trial was to assess whether low intensity pulsed ultrasound (LIPUS) accelerated or enhanced the rate of bone healing in adult patients undergoing distraction osteogenesis.

Aims

The management of a significant bony defect following excision of a diaphyseal atrophic femoral nonunion remains a challenge. We present the outcomes using a combined technique of acute femoral shortening, stabilized with a long retrograde intramedullary nail, accompanied by bifocal osteotomy compression and distraction osteogenesis with a temporary monolateral fixator.