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Bone & Joint Research
Vol. 6, Issue 8 | Pages 489 - 498
1 Aug 2017
Mifuji K Ishikawa M Kamei N Tanaka R Arita K Mizuno H Asahara T Adachi N Ochi M

Objectives. The objective of this study was to investigate the therapeutic effect of peripheral blood mononuclear cells (PBMNCs) treated with quality and quantity control culture (QQ-culture) to expand and fortify angiogenic cells on the acceleration of fracture healing. Methods. Human PBMNCs were cultured for seven days with the QQ-culture method using a serum-free medium containing five specific cytokines and growth factors. The QQ-cultured PBMNCs (QQMNCs) obtained were counted and characterised by flow cytometry and real-time polymerase chain reaction (RT-PCR). Angiogenic and osteo-inductive potentials were evaluated using tube formation assays and co-culture with mesenchymal stem cells with osteo-inductive medium in vitro. In order to evaluate the therapeutic potential of QQMNCs, cells were transplanted into an immunodeficient rat femur nonunion model. The rats were randomised into three groups: control; PBMNCs; and QQMNCs. The fracture healing was evaluated radiographically and histologically. Results. The total number of PBMNCs was decreased after QQ-culture, however, the number of CD34+ and CD206+ cells were found to have increased as assessed by flow cytometry analysis. In addition, gene expression of angiogenic factors was upregulated in QQMNCs. In the animal model, the rate of bone union was higher in the QQMNC group than in the other groups. Radiographic scores and bone volume were significantly associated with the enhancement of angiogenesis in the QQMNC group. Conclusion. We have demonstrated that QQMNCs have superior potential to accelerate fracture healing compared with PBMNCs. The QQMNCs could be a promising option for fracture nonunion. Cite this article: K. Mifuji, M. Ishikawa, N. Kamei, R. Tanaka, K. Arita, H. Mizuno, T. Asahara, N. Adachi, M. Ochi. Angiogenic conditioning of peripheral blood mononuclear cells promotes fracture healing. Bone Joint Res 2017;6: 489–498. DOI: 10.1302/2046-3758.68.BJR-2016-0338.R1


Bone & Joint Research
Vol. 5, Issue 12 | Pages 594 - 601
1 Dec 2016
Li JJ Wang BQ Fei Q Yang Y Li D

Objectives. In order to screen the altered gene expression profile in peripheral blood mononuclear cells of patients with osteoporosis, we performed an integrated analysis of the online microarray studies of osteoporosis. Methods. We searched the Gene Expression Omnibus (GEO) database for microarray studies of peripheral blood mononuclear cells in patients with osteoporosis. Subsequently, we integrated gene expression data sets from multiple microarray studies to obtain differentially expressed genes (DEGs) between patients with osteoporosis and normal controls. Gene function analysis was performed to uncover the functions of identified DEGs. Results. A total of three microarray studies were selected for integrated analysis. In all, 1125 genes were found to be significantly differentially expressed between osteoporosis patients and normal controls, with 373 upregulated and 752 downregulated genes. Positive regulation of the cellular amino metabolic process (gene ontology (GO): 0033240, false discovery rate (FDR) = 1.00E + 00) was significantly enriched under the GO category for biological processes, while for molecular functions, flavin adenine dinucleotide binding (GO: 0050660, FDR = 3.66E-01) and androgen receptor binding (GO: 0050681, FDR = 6.35E-01) were significantly enriched. DEGs were enriched in many osteoporosis-related signalling pathways, including those of mitogen-activated protein kinase (MAPK) and calcium. Protein-protein interaction (PPI) network analysis showed that the significant hub proteins contained ubiquitin specific peptidase 9, X-linked (Degree = 99), ubiquitin specific peptidase 19 (Degree = 57) and ubiquitin conjugating enzyme E2 B (Degree = 57). Conclusion. Analysis of gene function of identified differentially expressed genes may expand our understanding of fundamental mechanisms leading to osteoporosis. Moreover, significantly enriched pathways, such as MAPK and calcium, may involve in osteoporosis through osteoblastic differentiation and bone formation. Cite this article: J. J. Li, B. Q. Wang, Q. Fei, Y. Yang, D. Li. Identification of candidate genes in osteoporosis by integrated microarray analysis. Bone Joint Res 2016;5:594–601. DOI: 10.1302/2046-3758.512.BJR-2016-0073.R1


The Journal of Bone & Joint Surgery British Volume
Vol. 89-B, Issue 1 | Pages 116 - 120
1 Jan 2007
Laing AJ Dillon JP Condon E Coffey JC Street JT Wang JH McGuinness AJ Redmond HP

Post-natal vasculogenesis, the process by which vascular committed bone marrow stem cells or endothelial precursor cells migrate, differentiate and incorporate into the nacent endothelium and thereby contribute to physiological and pathological neurovascularisation, has stimulated much interest. Its contribution to neovascularisation of tumours, wound healing and revascularisation associated with ischaemia of skeletal and cardiac muscles is well established. We evaluated the responses of endothelial precursor cells in bone marrow to musculoskeletal trauma in mice. Bone marrow from six C57 Black 6 mice subjected to a standardised, closed fracture of the femur, was analysed for the combined expression of cell-surface markers stem cell antigen 1 (sca-1. +. ) and stem cell factor receptor, CD117 (c-kit. +. ) in order to identify the endothelial precursor cell population. Immunomagnetically-enriched sca-1. +. mononuclear cell (MNC. sca-1+. ) populations were then cultured and examined for functional vascular endothelial differentiation. Bone marrow MNC. sca-1+,c-kit+. counts increased almost twofold within 48 hours of the event, compared with baseline levels, before decreasing by 72 hours. Sca-1. +. mononuclear cell populations in culture from samples of bone marrow at 48 hours bound together Ulex Europus-1, and incorporated fluorescent 1,1′-dioctadecyl- 3,3,3,’3′-tetramethylindocarbocyanine perchlorate-labelled acetylated low-density lipoprotein intracellularily, both characteristics of mature endothelium. Our findings suggest that a systemic provascular response of bone marrow is initiated by musculoskeletal trauma. Its therapeutic manipulation may have implications for the potential enhancement of neovascularisation and the healing of fractures


Orthopaedic Proceedings
Vol. 106-B, Issue SUPP_1 | Pages 134 - 134
2 Jan 2024
Häusner S Horas K Blunk T Herrmann M
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Autografts containing bone marrow (BM) are current gold standard in the treatment of critical size bone defects, delayed union and bone nonunion defects. Although reaching unprecedented healing rates in bone reconstruction, the mode of action and cell-cell interactions of bone marrow mononuclear cell (BM-MNC) populations have not yet been described. BM-MNCs consist of a heterogeneous mixture of hematopoetic and non-hematopoetic lineage fractions. Cell culture in a 3D environment is necessary to reflect on the complex mix of these adherend and non-adherend cells in a physiologically relevant context. Therefore, the main aim of this approach was to establish conditions for a stable 3D BM-MNC culture to assess cellular responses on fracture healing strategies. BM samples were obtained from residual material after surgery with positive ethical vote and informed consent of the patients. BM-MNCs were isolated by density gradient centrifugation, and cellular composition was determined by flow cytometry to obtain unbiased data sets on contained cell populations. Collagen from rat tail and human fibrin was used to facilitate a 3D culture environment for the BM-MNCs over a period of three days. Effects on cellular composition that could improve the regenerative potential of BM-MNCs within the BM autograft were assessed using flow cytometry. Cell-cell-interactions were visualized using confocal microscopy over a period of 24 hours. Cell localization and interaction partners were characterized using immunofluorescence labeled paraffin sectioning. Main BM-MNC populations like Monocytes, Macrophages, T cells and endothelial progenitor cells were determined and could be conserved in 3D culture over a period of three days. The 3D cultures will be further treated with already clinically available reagents that lead to effects even within a short-term exposure to stimulate angiogenic, osteogenic or immunomodulatory properties. These measures will help to ease the translation from “bench to bedside” into an intraoperative protocol in the end


Orthopaedic Proceedings
Vol. 106-B, Issue SUPP_18 | Pages 72 - 72
14 Nov 2024
Uvebrant K Andersen C Lim HC Vonk L Åkerlund EL
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Introduction. Homogenous and consistent preparations of mesenchymal stem cells (MSCs) can be acquired by selecting them for integrin α10β1 (integrin a10-MSCs). Safety and efficacy of intra-articular injection of allogeneic integrin a10-MSCs were shown in two post-traumatic osteoarthritis horse studies. The current study investigated immunomodulatory capacities of human integrin a10-MSCs in vitro and their cell fait after intra-articular injection in rabbits. Method. The concentration of produced immunomodulatory factors was measured after licensing integrin a10-MSCs with pro-inflammatory cytokines. Suppression of T-cell proliferation was determined in co-cultures with carboxyfluorescein N-succinimidyl ester (CFSE) labelled human peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3/CD28 and measuring the CFSE intensity of CD4+ cells. Macrophage polarization was assessed in co-cultures with differentiated THP-1 cells stimulated with lipopolysaccharide and analysing the M2 macrophage cell surface markers CD163 and CD206. In vivo homing and regeneration were investigated by injecting superparamagnetic iron oxide nanoparticles conjugated with Rhodamine B-labeled human integrin a10-MSCs in rabbits with experimental osteochondral defects. MSC distribution in the joint was followed by MRI and fluorescence microscopy. Result. The production of the immunomodulatory factors indoleamine 2,3-dioxygenase and prostaglandin E2 was increased after inflammatory licensing integrin a10-MSCs. Co-cultures with integrin a10-MSCs suppressed T-cell proliferation and increased the frequency of M2 macrophages. In vivo injected integrin a10-MSCs homed to osteochondral defects and were detected in the repair tissue of the defects up to 10 days after injection, colocalized with aggrecan and type II collagen. Conclusion. This study showed that human integrin a10-MSCs have immunomodulatory capacities and in vivo can home to the site of osteochondral damage and directly participate in cartilage regeneration. This suggests that human integrin α10β1-selected MSCs may be a promising therapy for osteoarthritis with dual mechanisms of action consisting of immunomodulation and homing to damage followed by early engraftment and differentiation into chondrocyte-like cells that deposit hyaline cartilage matrix molecules


Orthopaedic Proceedings
Vol. 104-B, Issue SUPP_14 | Pages 8 - 8
1 Dec 2022
Caravaggio F Antonelli M Depalmi F
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Chronic Achilles tendinopathy is characterised by sub-acute inflammation with pro-inflammatory type 1 macrophages (M1), tissue degeneration and consequent partial or total tendon injury. Control of the inflammatory response and M1-to-M2 macrophage polarisation can favour tendon healing both directly and indirectly, by allowing for the regenerative process driven by local mesenchymal stem cells. Ten patients (3 females and 7 males aged between 32 and 71 years old) with partial Achilles tendon injury were treated with injections of autologous peripheral blood mononuclear cells (PB-MNCs). The cell concentrate was obtained from 100-120 cc of each patient's blood with a selective point-of-care filtration system. PB-MNCs remained trapped in the filter and were injected immediately after sampling. Around 60% of the PB-MNC concentrate was injected directly into the injured area, while the remaining 40% was injected in smaller amounts into the surrounding parts of the Achilles tendon affected by tendinosis. All patients were evaluated both clinically with the help of the American Orthopaedic Foot & Ankle Society (AOFAS) scale, and radiologically (MRI examination) at baseline and 2 months after the PB-MNC injection. A clinical reassessment with the AOFAS scale was also performed 6 months after the intervention. The rehabilitation protocol implied full weight-bearing walking immediately after the procedure, light physical activity 3-4 days after the injection, and physiotherapist-assisted stretching exercises and eccentric training. In all patients, functional and radiological signs of tendon healing processes were detected as early as 2 months after a single treatment and the AOFAS scale rose from the initial mean value of 37.5 (baseline) to 85.4 (6 months). Our preliminary results indicate that regenerative therapies with PB-MNCs can prove useful for partial Achilles tendon injuries as a valid alternative to surgical options, especially when other conservative approaches have failed. Advantages of this therapy include rapid execution, no need for an operating theatre, easy reproducibility, quick recovery and good tolerability regardless of the patient's age (the procedure is not to be performed in subjects who are below 18 years old). Further studies on the topic are recommended to confirm these observations


Bone & Joint Research
Vol. 5, Issue 7 | Pages 314 - 319
1 Jul 2016
Xiao X Hao J Wen Y Wang W Guo X Zhang F

Objectives. The molecular mechanism of rheumatoid arthritis (RA) remains elusive. We conducted a protein-protein interaction network-based integrative analysis of genome-wide association studies (GWAS) and gene expression profiles of RA. Methods. We first performed a dense search of RA-associated gene modules by integrating a large GWAS meta-analysis dataset (containing 5539 RA patients and 20 169 healthy controls), protein interaction network and gene expression profiles of RA synovium and peripheral blood mononuclear cells (PBMCs). Gene ontology (GO) enrichment analysis was conducted by DAVID. The protein association networks of gene modules were generated by STRING. Results. For RA synovium, the top-ranked gene module is HLA-A, containing TAP2, HLA-A, HLA-C, TAPBP and LILRB1 genes. For RA PBMCs, the top-ranked gene module is GRB7, consisting of HLA-DRB5, HLA-DRA, GRB7, CD63 and KIT genes. Functional enrichment analysis identified three significant GO terms for RA synovium, including antigen processing and presentation of peptide antigen via major histocompatibility complex class I (false discovery rate (FDR) = 4.86 × 10 – 4), antigen processing and presentation of peptide antigen (FDR = 2.33 × 10 – 3) and eukaryotic translation initiation factor 4F complex (FDR = 2.52 × 10 – 2). Conclusion. This study reported several RA-associated gene modules and their functional association networks. Cite this article: X. Xiao, J. Hao, Y. Wen, W. Wang, X. Guo, F. Zhang. Genome-wide association studies and gene expression profiles of rheumatoid arthritis: an analysis. Bone Joint Res 2016;5:314–319. DOI: 10.1302/2046-3758.57.2000502


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_VIII | Pages 22 - 22
1 Mar 2012
Yamasaki T Yasunaga Y Hamaki T Yoshida T Oshima S Hori J Yamasaki K Ochi M
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Introduction. Since 2005, we have performed implantation of bone marrow-derived mononuclear cells for osteonecrosis of the femoral head in order to improve vascularization and bone repair. This study focused on early bone repair of osteonecrosis of the femoral head after transplantation of bone marrow-derived mononuclear cells (BMMNC). Patients and Methods. Twenty-two patients (30 joints) who had bilateral osteonecrosis followed for more than 2 years after BMMNC implantation were evaluated. Eight women and 14 men were included. Their mean age at surgery was 41 years (range, 18 to 64 years) and the mean follow-up period was 31 months. Pre-operative stage according to the ARCO classification was Stage 2 in 25 joints and Stage 3 in 5 joints. The mean volume ratio of osteonecrosis was 21%. For preparing BMMNC, about 700ml of bone marrow was aspirated from the ilium and centrifuged using a Spectra cell separator (Gambro). The BMMNC were seeded to interconnected porous calcium hydroxyapatite (IP-CHA) and implanted to the osteonecrotic lesion. As a control, cell-free IP-CHA was implanted for 8 patients (9 joints). A woman and 7 men were included. The mean age at surgery was 49 years (range, 28 to 73 years) and the mean follow-up period was 37 months. Preoperative stage was stage 2 in all patients. The mean volume ratio of osteonecrosis was 22%. At post-operative evaluations; progression of collapse, consolidation at reactive zone, post-operative course of volume rate of osteonecrosis, and bone absorption at osteonecrosis was assessed. Results. Shrinkage of osteonecrosis has been observed subsequent to bone consolidation at the transitional zone which progressed from 3 to 6 months post-operatively in the BMMNC-seeded group. Progression of collapse more than 2 mm was detected in 4 joints (13%), and hip arthroplasty was performed in 1 joint (3%). Consolidation at the reactive zone was detected in 28 joints (93%) and the volume rate of osteonecrosis significantly decreased by 12 months after surgery. Bone resorption at the osteonecrotic lesion was observed in 14 joints (47%). Meanwhile, subtle bone consolidation was detected after 12 months post-operatively in the control group. Progression of collapse was observed in 6 joints (67%) and further surgical treatments were needed in 3 joints (33%). Conclusion. This study found that BMMNC was beneficial to osteonecrosis of the femoral head from the viewpoint of prevention of collapse


Orthopaedic Proceedings
Vol. 102-B, Issue SUPP_11 | Pages 16 - 16
1 Dec 2020
Kontakis MG Schou J Hailer N
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Bone tissue engineering attempts at substituting critical size bone defects with scaffolds that can be primed with osteogenic cells, usually mesenchymal stem cells (MSC) from the bone marrow. Although overlooked, peripheral blood is a valuable source of MSC and circulating osteoprogenitors (COP), bearing a significant regenerative potential, and peripheral blood is easier to access than bone marrow. We thus studied osteodifferentiation of peripheral blood mononuclear cells (pbMNC) under different culture conditions, and how they compared to primary human osteoblasts. pbMNC were isolated from healthy adult volunteers by Ficoll density gradient centrifugation, and they were then cultured using media supplemented with 100nM Dexamethasone, 10mM sodium β-glycero phosphate and ascorbic acid (either 40mM or 0.05mM). For comparison, primary osteoblasts were isolated from the femoral heads of patients undergoing hip arthroplasty. After 4 weeks of culture, osteogenic activation was quantified with spectrometric measurement of alkalic phosphatase (ALP) and lactate dehydrogenase (LDH) levels. The extent of osteoid mineralization was measured with Alizarin red staining. We studied the effects of 1) varying cell concentration at seeding, 2) surface coating of culture wells with collagen and 3) high compared to low ascorbic acid (40mM and 0.05mM) media. Higher numbers of pbMNC (0.5–5.9 versus 0.062–0.25 million cells per well) at seeding resulted in a lower ALP/LDH-ratio (mean ± standard deviation), 0.39 ± 0.33 arbitrary units (AU) versus 1.36 ± 1.06 AU, but led to higher amount of osteoid production, 0.10 ± 0.06 versus 0.065 ± 0.02 AU, p < 0.05. Culture of pbMNC on collagen did not confer any difference in ALP/LDH-ratios, with 0.43 ± 0.3 AU for collagen-coated and 0.43 ± 0.41 AU for uncoated wells (p = 0.95), and we also observed no relevant difference in osteoid production (0.07 ± 0.01 AU for collagen-coated versus 0.1 ± 0.08 AU for uncoated wells, p = 0.28). Cultures of pbMNC on collagen in media supplemented with a higher concentration of ascorbic acid showed a 130% higher ALP/LDH-ratio when compared to cultures exposed to a lower ascorbic acid concentration (p < 0.05). Cultures with a low initial concentration of pbMNC (0.5 − 1 million cells) had no significantly different ALP/LDH-ratio when compared to primary human osteoblasts, but the cultures of pbMNC resulted in a 90% increase in osteoid mineralization when compared to primary human osteoblasts (p < 0.05). These findings indicate that progenitor cells derived from peripheral blood have a significant osteogenic potential, rendering them interesting candidates for seeding of scaffolds intended to fill critical sized bone defects. pbMNC produced almost double the amount of osteoid as primary osteoblasts. The isolation of pbMSC and COP is non-invasive and easy, and they might be seeded directly onto scaffolds without prior ex-vivo expansion, a question that we intend to pursue further


Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_14 | Pages 26 - 26
1 Nov 2018
Bastos R do Amaral RJFC Mathias M Andrade R Bastos R Balduino A Schott V Rodeo S Mendes JE
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Intra-articular injections of human mesenchymal stromal cells (MSCs) and platelet-rich plasma (PRP) have been intensively investigated as therapies for knee osteoarthritis (OA) with positive outcomes. In this work we evaluated weather a combination of the treatments (MSCs + PRP) would be beneficial compared to MSCs alone (MSCs) and standard corticosteroid injection (Control group). Forty seven patients (24 males and 23 females; 53.3 ± 10.7 years old) with radiographic symptomatic knee OA (Dejour grades II–IV) were randomized to receive intra-articular injections of MSCs (n = 16), MSCs + PRP (n = 14) or corticosteroid (n=17). MSCs were obtained after mononuclear cells separation from bone marrow aspiration collected from both posterior iliac crests using Sepax automated closed system and expanded in culture until reaching the number of 4 × 10. 7. PRP was obtained by double-centrifugation of whole blood according to a protocol developed in house. After 12 months follow-up, the MSCs and MSCs+PRP groups achieved higher percentages of expected improvement when comparing to the corticosteroid group for the KOOS-symptoms, pain, function and daily living, domains and global score. For the population older or equal to 60 years old the MSCs+PRP group showed significant superiority for the KOOS-ADL domain at 12 months. Cytokines quantification evidenced anti-inflammatory aspects of the treatments. This work evidences the safety and efficacy of intra-articular injection of MSCs for the treatment of early knee OA, with greater improvement with PRP addition particularly to the older population


Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_14 | Pages 115 - 115
1 Nov 2018
Müller S Nicholson L Jone E Dickinson A Dalgarno K Wang X
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Mesenchymal stromal cells (MSCs) are widely used in clinical trials for the treatment of many bone defects. Apatite-wollastonite glass ceramic (A-W) is an osteoconductive biomaterial shown to be compatible with MSCs. This is the first study comparing the osteogenic potential of two MSC populations, heterogeneous plastic adherence MSCs (PA-MSCs) and CD271-enriched MSCs (CD271-MSCs), when cultured on A-W 3D scaffold. The paired MSC populations were assessed for their attachment, growth kinetics and ALP activity using confocal or scanning electron microscopy and the quantifications of DNA contents and p-nitrophenyl (pNP) production. While the PA-MSCs and CD271-MSCs had similar expansion and tri-lineage differentiation capacity during standard 2D culture, they showed different proliferation kinetics when seeded on the A-W scaffolds. PA-MSCs displayed a well-spread attachment with more elongated morphology compared to CD271-MSCs, signifying a different level of interaction between the cell populations and the scaffold surface. PA-MSCs also fully integrated into the scaffold surface and showed a stronger propensity for osteogenic differentiation on the A-W scaffold as indicated by higher ALP activity than CD271-MSCs. Furthermore, A-W scaffold seeded uncultured bone marrow mononuclear cells (BM-MNCs) demonstrated a higher proliferation rate and greater ALP activity compared to freshly isolated CD271-enriched BM-MNCs. Our findings suggest that enrichment of CD271-positive population is not beneficial for osteogenesis when the cells are seeded on A-W scaffold. Furthermore, unselected heterogeneous MSCs or BM-MNCs are more promising for A-W scaffold-based bone regeneration, providing novel insight with potential clinical implications in regenerative medicine for bone defects using an innovative tissue engineering approach


Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_16 | Pages 7 - 7
1 Nov 2018
Kim S
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ONFH with large or lateral-located lesion is challenging due to difficulty of regeneration. We introduce novel tissue engineering technique using ex vivo expanded bone marrow stromal cell seeded on calcium metaphosphate (CMP) scaffold to regenerate dead bone for these challenging cases. Ten millilitres of bone marrow was aspirated from iliac crest and mononuclear cells were collected. These cells were expanded and differentiated to osteoblast-lineage cells using osteogenic media and autologous serum for 2–4 weeks ex vivo. Porous bead-form scaffolds were made of CMP and cells were seeded in a density of million/ml³ into 20 to 30 beads for 1 hour. The necrotic area was curetted and the beads were implanted through core tract in 9 hips (Steinberg IIc in 5 hips and IVc in 4 hips which involved greater than 30% of whole head; JIC classification C1 in 4 hips, and C2 in 5 hips which involved weight bearing area). The tract was blocked with a CMP rod. The age of patients ranged from 16 to 37. Associated factors were; steroid in 4, idiopathic in 3, alcoholic in 1 and traumatic in 1 hip, respectively. Kerboul combined necrotic angle was more than 200° in all hips. We compared preoperative and annual radiographs and MRI images to check dome depression of femoral head and signal change of osteonecrotic area. Follow-up period ranged from 8 to 14 years. Two IIc lesions progressed and were converted to THA at two and six years postoperatively. We could get clinical and radiographic success in 7 hips (78%). Follow-up radiographs and MRI showed partial or nearly complete regeneration of necrotic bone, prevention of collapse, and reduction in necrotic lesion. This can be a good strategy for bone regeneration of unmet need as in a human model


Orthopaedic Proceedings
Vol. 101-B, Issue SUPP_2 | Pages 42 - 42
1 Jan 2019
Lal S Hall R Tipper JL
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Since 2010, there has been a sharp decline in the use of metal-on-metal joint replacement devices due to adverse responses associated with the release of metal wear particles and ions in patients. Surface engineered coatings offer an innovative solution to this problem by covering metal implant surfaces with biocompatible and wear resistant materials. The present study tests the hypothesis whether surface engineered coatings can reduce the overall biological impact of a device by investigating recently introduced silicon nitride coatings for joint replacements. Biological responses of peripheral blood mononuclear cells (PBMNCs) to Si3N4 model particles, SiNx coating wear particles and CoCr wear particles were evaluated by testing cytotoxicity, inflammatory cytokine release, oxidative stress and genotoxicity. Clinically relevant wear particles were generated from SiNx-on-SiNx and CoCr-on-CoCr bearing combinations using a multidirectional pin-on-plate tribometer. All particles were heat treated at 180°C for 4 h to destroy endotoxin contamination. Whole peripheral blood was collected from healthy donors (ethics approval BIOSCI 10–108, University of Leeds). The PBMNCs were isolated using Lymphoprep (Stemcell) and incubated with particles at various volumetric concentrations (0.5 to 100 µm3 particles/cell) for 24 h in 5% (v/v) CO2 at 37°C. After incubation, cell viability was measured using the ATPlite assay (Perkin Elmer); TNF-alpha release was measured by ELISA (Invitrogen); oxidative stress was measured using H2DCFDA (Abcam); and DNA damage was measured by comet assay (Tevigen). The results were expressed as mean ± 95% confidence limits and the data was analysed using one-way ANOVA and Tukey-Kramer post-hoc analysis. No evidence of cytotoxicity, oxidative stress, TNF-alpha release, or DNA damage was observed for the silicon nitride particles at any of the doses. However, CoCr wear particles caused cytotoxicity, oxidative stress, TNF-alpha release and DNA damage in PBMNCs at high doses (50 µm3 particles per cell). This study has demonstrated the in-vitro biocompatibility of SiNx coatings with primary human monocytic cells. Therefore, surface engineered coatings have potential to significantly reduce the biological impact of metal components in future orthopaedic devices


Bone & Joint Research
Vol. 3, Issue 3 | Pages 76 - 81
1 Mar 2014
Okabe YT Kondo T Mishima K Hayase Y Kato K Mizuno M Ishiguro N Kitoh H

Objectives. In order to ensure safety of the cell-based therapy for bone regeneration, we examined in vivo biodistribution of locally or systemically transplanted osteoblast-like cells generated from bone marrow (BM) derived mononuclear cells. Methods. BM cells obtained from a total of 13 Sprague-Dawley (SD) green fluorescent protein transgenic (GFP-Tg) rats were culture-expanded in an osteogenic differentiation medium for three weeks. Osteoblast-like cells were then locally transplanted with collagen scaffolds to the rat model of segmental bone defect. Donor cells were also intravenously infused to the normal Sprague-Dawley (SD) rats for systemic biodistribution. The flow cytometric and histological analyses were performed for cellular tracking after transplantation. Results. Locally transplanted donor cells remained within the vicinity of the transplantation site without migrating to other organs. Systemically administered large amounts of osteoblast-like cells were cleared from various organ tissues within three days of transplantation and did not show any adverse effects in the transplanted rats. Conclusions. We demonstrated a precise assessment of donor cell biodistribution that further augments prospective utility of regenerative cell therapy. Cite this article: Bone Joint Res 2014;3:76–81


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_2 | Pages 107 - 107
1 Jan 2017
Girolami M Brodano GB Babbi L Cenacchi A Gasbarrini A Bandiera S Terzi S Ghermandi R Boriani S
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The nature of the Aneurysmal Bone Cyst (ABC) is still controversial among benign tumor, often identifiable in the “aggressive” form (Enneking stage 3) or pseudotumoral lesion. It is well known instead the very high risk of intraoperative bleeding, indicating a strongly unfavorable relationship between the surgical morbidity and the nature of the disease. Recently, excellent results have been obtained in the treatment of ABC by repeated arterial embolizations (SAE), without any surgery, while initial experiences with administration of denosumab and doxycycline are still under study. This study presents the results of our initial experience in the treatment of vertebral ABC through the use of concentrated autologous mesenchymal stem cells (MSCs). Two teenagers aged 15 years, male, and 14 years, female, came to our attention both with diagnosis of ABC in C2 vertebra which was histologically confirmed. They were both neurologically intact, the girl complained of neck pain. The arteriography showed in both cases close relationships between the pathological ABC vascularization and the vertebral and cervical ascending arteries, making treatment by selective arterial embolization unsuitable. After discussion with the parents of patients, we jointly decided to undertake the treatment by direct injection of MSCs preceded, in the same operative session, by harvesting from the iliac crest of 60 cc of bone marrow (by needle aspiration) and its separation with the use of concentration system Res-Q ™ 60 BMC. In the second case the treatment was repeated two times at distance of 4 months. The clinical and radiological follow-up is to of 30 months from the first treatment in both cases. In the first case the presence of newly formed bone within the ABC appeared as a clear sign of recovery just a month after the first treatment and increased gradually, until the cyst appeared completely ossified one year after the treatment, with associated disappearance of the pain. In the second case an initial sclerotic peripheral margin appeared after the second treatment and later ossification progressed, concurrently with the disappearance of the pain. Treatment with selective serial arterial embolization is considered effective in the treatment of ABC even if not without risks, mainly related to the frequent and repeated exposure to ionizing radiation. Furthermore, in a certain percentage of cases the procedure is not technically executable, especially for the presence of arteries afferent to the medullar vascularization. Inconsistent results were obtained with other procedures: the injection of calcitonin, steroid, alcoholic solutions, or the use of sclerosing substances. Radiation therapy, though very effective, it is not considered the first choice. Recently, promising results have been achieved by the injection of mononuclear cells derived from bone marrow in the treatment of Aneurysmal Bone Cyst. Based on the early results obtained in the two cases described, the injection of MSCs can be considered a valid alternative in the treatment of vertebral ABCs untreatable by embolization


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_1 | Pages 35 - 35
1 Jan 2017
Lopa S Bongio M Gilardi M Bersini S Mondadori C Moretti M
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We developed a 3D vascularized bone remodeling model embedding human osteoblast and osteoclast precursors and endothelial cells in a mineralized matrix. All the cells included in the model exerted their function, resulting in a vascularized system undergoing mineralized matrix remodeling. Bone remodeling is a dynamic process relying on the balance between the activity of osteoblasts and osteoclasts which are responsible for bone formation and resorption, respectively. This process is also characterized by a tight coupling between osteogenesis and angiogenesis, indicating the existence of a complex cross-talk between endothelial cells and bone cells. We have recently developed microscale in vitro hydrogel-based models, namely the 3D MiniTissue models, to obtain bone-mimicking microenvironments including a 3D microvascular network formed by endothelial cell self-assembly [1–2]. Here, we generated a vascularized 3D MiniTissue bone remodeling model through the coculture of primary human cells in a 3D collagen/fibrin (Col/Fib) matrix enriched with CaP nanoparticles (CaPn) to mimic bone mineralized matrix. Human umbilical vein endothelial cells (HUVECs), bone marrow mesenchymal stem cells (BMSCs), osteoblast (OBs) and osteoclast (OCs) precursors were cocultured in plain and CaPn-enriched Col/Fib according to the following experimental conditions: a) HUVECs-BMSCs; b) OBs-OCs; c) HUVECs-BMSCs-OBs-OCs. Undifferentiated BMSCs were used to support HUVECs in microvascular network formation. BMSCs and peripheral blood mononuclear cells were respectively pre-differentiated into OB and OC precursors through 7 days of culture in osteogenic or osteoclastogenic medium. Needle-shaped CaPn (Ø ∼20 nm, length ∼80 nm) were added to a collagen/fibrinogen solution. Cells were resuspended in a thrombin solution and then mixed with plain or CaPn-enriched collagen/fibrinogen. The cell-laden mix was injected in U-shaped PMMA masks and let to polymerize to generate constructs of 2×2×5 mm. 3. Samples were cultured for 10 days. Microvascular network formation was evaluated by confocal microscopy. OB differentiation was analyzed by quantification of Alkaline Phosphatase (ALP) and cell-mediated mineralization. OC differentiation was assessed by Tartrate-Resistant Acid Phosphatase (TRAP) and cell-mediated phosphate release quantification. HUVECs developed a robust 3D microvascular network and BMSCs differentiated into mural cells supporting vasculogenesis. The presence of CaPn enhanced OB and OC differentiation, as demonstrated by the significantly higher ALP and TRAP levels and by the superior cell-mediated mineralization and phosphate release measured in CaPn-enriched than in plain Col/Fib. The coculture of OBs and OCs with HUVECs and BMSCs further enhanced ALP and TRAP levels, indicating that the presence of HUVECs and BMSCs positively contributed to OB and OC differentiation. Remarkably, higher values of ALP and TRAP activity were measured in the tetraculture in CaPn-enriched Col/Fib compared to plain Col/Fib, indicating that also in the tetraculture the mineralized matrix stimulated OB and OC differentiation. The 3D MiniTissue bone remodeling model developed in this study is a promising platform to investigate bone cell and endothelial cell cross-talk. This system allows to minimize the use of cells and reagents and is characterized by a superior ease of use compared to other microscale systems, such as microfluidic models. Finally, it represents a suitable platform to test drugs for bone diseases and can be easily personalized with patient-derived cells further increasing its relevance as drug screening platform


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 329 - 329
1 Jul 2014
Beckmann R Hartz C Tohidnezhad M Neuss-Stein S Ventura Ferreira M Rath B Tingart M Pries F Varoga D Pufe T
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Aim of the study was to evaluate if abrasion-arthroplasty (AAP) and abrasion-chondroplasty (ACP) leads to a release of mesenchymal stem cell (MSC) like cells from the bone marrow to the joint cavity where they probably differentiate into a chondrogenic phenotype. Introduction. Cartilage demage is a sever problem in our aging society. About 5 million people only in Germany are affected. Osteoathritis is a degeneration of cartilage caused by aging or traumata 50 % of the people over 40 have signs of osteoarthritis. But the ability of self-regeneration of cartilage is strongly limited. There are different approaches to therapy osteoathritic lesions. Arthroscopic treatment of OA includes bone marrow stimulation technique such as abrasion arthroplasty (AAP) and microfracturing (MF). Beside the support of chondrocyte progenitor cells the environment is also important for the commitment to chondrocytes. Therefore insulin-like growth factor-1 (IGF-1) and transforming growth factor beta-1 (TGF-β1) are important factors during the regeneration process. In the present study we characterised the heamarthrosis and the released cells after AAP and its ability to differentiate into the chondrocyte lineage. Material and Methods. Postoperative haemarthrosis was taken 5, 22 or 44 hours after surgery. 7.5 mg Dexamethasone (Corticosteroid) was administered into the knee joint to prevent postoperative inflammation. Mononuclear cells were isolated from haemarthrosis from the drainage bottle by ficoll density gradient centrifugation. The isolated cells were characterised using fluorescence-activated cell-sorting (FACS) analysis for characteristic markers of MSC such as CD 44, 73, 90, 105. After expanding cells were cultured in a pellet culture. After 3 weeks, histochemistry and immunohistochemistry against Sox9, collagen II and proteoglycan were performed. The release of IGF1, BMP4 and BMP7 was analysed in haemarthrosis serum by ELISA and Luminex technology. Results. The isolated cells after AAP are positive for the mesenchymal stem cell marker CD105, CD90, CD73, CD 44 and negative for the marker of hematopoetic stem cells CD 34. Isolated cells after ACP couldn't be expanded for further characterizations. The staining of the 3D-culture revealed a positive signal for the chondrogen transcription factor Sox9 and the expression of extracellular markerproteins like collagen type II and proteoglycan. Both surgery techniques, AAP and ACP provides a chondrogenic environment. We were able to detect IGF-1, TGFß, BMP4 and BMP7 in the haemarthrosis. Discussion. The benefit of abrasion arthroplasty surgery and microfracturing is controversial discussed because they do not consistently result in hyaline cartilage. But the opening of the bone marrow allows the release of monocytic cells which have the potential to differentiate into a chondrogenic phenotype. In 3D-culture these cells express Sox9 and a collagen proteoglycan rich matrix. The haemarthrosis provides also a cartilage-stimulating environment. We could detect IGF1, TGFβ, BMP4 and 7 which could enhance the commitment concerning differentiation of MSCs to a chondrogenic lineage concerning the production of cartilage specific extracellular matrix. Taken together our study provides the evidence for a therapeutic benefit of opening bone marrow in order to generate neocartilage after AAP


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 103 - 103
1 Jul 2014
Avnet S Salerno M Zini N Gibellini D Baldini N
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Summary. We demonstrate that osteoclast-like cells of GCT result from the spontaneous fusion and differentiation of CD14+ cells of the monoblastic lineage by an autocrine mechanism mediated by RANKL, rather than induced by stromal cells. This process is further enhanced by the simultaneous impairment of the negative feed-back regulation of osteoclastogenesis by interferon β. Introduction. Giant cell tumor of bone (GCT) is a benign osteolytic lesion with a complex histology, comprising prominent multinucleated osteoclast-like cells (OC), mononuclear stromal cells (SC), and monocyte-like elements. So far, most studies have focused on SC as the truly transformed elements that sustain osteoclast differentiation, while less attention has been paid on the monocyte-like cell fraction. On the contrary, we have previously shown that SC are non-transformed element that can induce osteoclastogenesis of monocytes at levels that do not exceed that of normal mesenchymal stromal cells. We therefore focused on CD14+ monocyte-like cells as an alternative key candidate for the pathogenesis of GCT. Methods. We isolated CD14+ enriched cell fraction from tumor samples by immunomagnetic separation. We analyzed CD14+ cells for ultrastructural morphology, mRNA levels of haematopoietic, monocytic, and dendritic markers, and for RANKL, and M-CSF. Due to the very high number of OC in GCT, we hypothesised that the IFN-b pathway might be impaired. In fact, IFN-b functions as a negative-feedback regulator that inhibits osteoclast differentiation. We assayed IFN-b mRNA and protein expression in both cultures and tumor samples. Finally, we verified the ability of CD14+ cells to spontaneously form osteoclasts. Results. In the CD14+ enriched fraction we identified two different cell populations, both positive for TRACP activity and negative for Ki-67 nuclear localization, one with an undefined histotype and the other showing characteristics of the monoblastic lineage, mainly monoblasts and promonocytes. Isolated cells were positive for CD45, MSE-1, RANK, CD14, and CD80, and negative for CD144, and were able to spontaneously form collagen-resorbing multinucleated cells, a process that was strongly impaired by the addition of osteoprotegerin. The expression of RANKL and M-CSF mRNA in cultured cells demonstrated the presence of an autocrine circuit inducing osteoclast formation. Finally, we found very low expression of IFN-b both in the in vitro formed OC and in tissue samples. Conclusions. These data show that CD14+ cells in GCT are monocyte-like cells that can spontaneously form bone-resorbing multinucleated cells through impaired IFN-b expression. Taken together, these data raise questions regarding the role of the CD14+ cell component and of their regulating mechanisms that may be relevant for the development of effective therapeutic strategies


The Journal of Bone & Joint Surgery British Volume
Vol. 92-B, Issue 10 | Pages 1475 - 1479
1 Oct 2010
Gortzak Y Kandel R Deheshi B Werier J Turcotte RE Ferguson PC Wunder JS

Various chemicals are commonly used as adjuvant treatment to surgery for giant-cell tumour (GCT) of bone. The comparative effect of these solutions on the cells of GCT is not known. In this study we evaluated the cytotoxic effect of sterile water, 95% ethanol, 5% phenol, 3% hydrogen peroxide (H2O2) and 50% zinc chloride (ZnCI2) on GCT monolayer tumour cultures which were established from six patients. The DNA content, the metabolic activity and the viability of the cultured samples of tumour cells were assessed at various times up to 120 hours after their exposure to these solutions.

Equal cytotoxicity to the GCT monolayer culture was observed for 95% ethanol, 5% phenol, 3% H2O2 and 50% ZnCI2. The treated samples showed significant reductions in DNA content and metabolic activity 24 hours after treatment and this was sustained for up to 120 hours. The samples treated with sterile water showed an initial decline in DNA content and viability 24 hours after treatment, but the surviving cells were viable and had proliferated. No multinucleated cell formation was seen in these cultures.

These results suggest that the use of chemical adjuvants other than water could help improve local control in the treatment of GCT of bone.


Bone & Joint Research
Vol. 6, Issue 2 | Pages 98 - 107
1 Feb 2017
Kazemi D Shams Asenjan K Dehdilani N Parsa H

Objectives

Mesenchymal stem cells have the ability to differentiate into various cell types, and thus have emerged as promising alternatives to chondrocytes in cell-based cartilage repair methods. The aim of this experimental study was to investigate the effect of bone marrow derived mesenchymal stem cells combined with platelet rich fibrin on osteochondral defect repair and articular cartilage regeneration in a canine model.

Methods

Osteochondral defects were created on the medial femoral condyles of 12 adult male mixed breed dogs. They were either treated with stem cells seeded on platelet rich fibrin or left empty. Macroscopic and histological evaluation of the repair tissue was conducted after four, 16 and 24 weeks using the International Cartilage Repair Society macroscopic and the O’Driscoll histological grading systems. Results were reported as mean and standard deviation (sd) and compared at different time points between the two groups using the Mann-Whitney U test, with a value < 0.05 considered statistically significant.