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

Introduction: The existence of circulating skeletal stem cells in the peripheral blood from different species including adult mouse and human has been found and documented. The circulating skeletal stem cells may provide a new source of stem cells that may be used for bone regeneration and tissue engineering applications. The aim of this study was to investigate the existence of circulating osteogenic stem cells in the rat peripheral blood, and to compare their osteogenic potentials with bone marrow mesenchymal stem cells (BMMSCs). Methods: Whole blood from twelve female 3-month old SD rats was harvested by cardiac puncture and bone marrows were also collected. Mononuclear cells from both bone marrow and peripheral blood (PBMNCs) were isolated by Lymphoprep density gradient centrifugation method, and plated at a density of 300000 to 400000/cm2 in flasks with á-MEM medium and 15% FCS. The colony forming efficiency (CFE) was calculated after 10–14 days culture. The osteogenic, adipogenic, and chondrogenic differentiation potential of both BMMSCs and peripheral blood mononuclear cell subset were examined and compared under different specific culture conditions. In addition, both BMMSCs and peripheral blood mononuclear cell subset were seeded into absorbable porous calcium phosphate substitute and implanted subcutaneously into SCID mice for 12 weeks, and the implants were examined histologically. Results: After 10–14 days in culture, the adherent fibroblast-like colonies were formed in the PBMNCs, with CFE ranging from 1.3 to 3.5 per 10000000 cells. Under osteogenic conditions, both BMMSCs and PBMNCs subset were positive for bone markers such as ALP, type I collagen and osteocalcin; bone nodules were formed in BMMSCs and PBMNCs subset long-term culture with positive Von Kossa and Alizarin Red S staining. Under adipogenic conditions, PBMNCs subset and BMMSCs were positive for Oil Red O and C/EBP á immunostaining. For chondrogenic differentiation studies, PBMNCs subset and BMMSCs were positive for type II collagen and they had Alcian blue positive nodules formation. After implantation with calcium phosphate substitutes in SCID mice for 12 weeks, osteoid and bony tissues were evident in the implants both loaded with PBMNCs subset and BMSCSs. Conclusions: A subset of mononuclear cells that have multi-differentiation potential similar to BMMSCs exists in the rat peripheral blood. Our present study has shown that these circulating stem cells possess osteogenic potential in vitro and in vivo. Further work is ongoing to investigate the roles of PBMNCs subset in fracture healing and their recruiting and homing mechanisms

Aims. To investigate the correlations among cytokines and regulatory T cells (T-regs) in ankylosing spondylitis (AS) patients, and their changes after anti-tumour necrosis factor-α (TNF-α) treatment. Methods. We included 72 AS patients with detailed medical records, disease activity score (Bath Ankylosing Spondylitis Disease Activity Index), functional index (Bath Ankylosing Spondylitis Functional Index), and laboratory data (interleukin (IL)-2, IL-4, IL-10, TNF-α, interferon (IFN)-γ, transforming growth factor (TGF)-β, ESR, and CRP). Their peripheral blood mononuclear cells (PBMCs) were marked with anti-CD4, anti-CD25, and anti-FoxP3 antibodies, and triple positive T cells were gated by flow cytometry as T-regs. Their correlations were calculated and the changes after anti-TNF-α therapy were compared. Results. The frequency of T-regs in PBMCs was positively correlated to ESR and CRP in AS (r = 0.35 and 0.43; p = 0.032 and 0.027, respectively), and there was also a significant correlation between serum level of TNF-α and CRP (p = 0.041). The frequency of T-regs in PBMCs positively correlated to serum levels of TNF-α, IL-10, and TGF-β, while IL-2, IL-4, and IFN-γ showed opposite results. After anti-TNF-α treatment, there were significantly lower serum levels of TNF-α, IL-10, TGF-β, and frequency of T-regs in PBMCs among these AS patients (p = 0.026, 0.032, 0.029, and 0.037, respectively). Conclusion. In AS patients, proinflammatory cytokine may give positive feedback to induce more T-reg production and anti-inflammatory cytokine secretion to suppress this inflammatory status, and they can be reversed by anti-TNF-α therapy. However, the detailed interactions among T-regs and complex cytokine networks in autoinflammatory diseases still need more studies and further functional assay. Cite this article: Bone Joint Res 2023;12(2):133–137

The authors wished to determine if macrophage activation and the release of osteolytic cytokines in response to orthopaedic wear debris could be suppressed pharmacologically with the use of anti-inflammatory and anti-oxidant agents.

The current long-term results of total joint arthroplasty are limited by mechanical wear of the implants with an associated immune mediated bone lysis with subsequent loosening and eventual failure. It has been demonstrated that the osteolysis seen in cases of aseptic loosening is mediated by the immune system both directly and indirectly by activated macrophages. Macrophages indirectly cause osteolysis through release of the osteoclast activating cytokines TNFα, IL-1 and PGE2. They also directly resorb bone in small amounts when activated by wear particles.

We utilised established cell culture models of both peripherally derived monocyte/macrophages and lymphocyte enriched co-cultures and examined the effects of polymethylmethacrylate particles alone on the cells in culture. The effect of anti-inflammatory and anti-oxidant agents (dexamethasone, diclofenac and n-acetyl cysteine) in varying concentrations was then examined using ELISA of cytokine release and electron microscopy to examine ultra structural responses.

Cell viability was also measured in cultures over 24 hour periods (at 6, 12 and 24 hours) using Trypan blue exclusion and Coulter counter, while cell type and morphology were determined cytologically, including-naphthyl acetate esterase cytochemical identification and electron microscopy.

The use of N-acetyl cysteine was associated with very significant suppression of TNF, IL-1 and PGE2 in both macrophage and lymphocyte enriched co-culture with no effect on cell viability. While diclofenac was also associated with significant decreases in cytokine expression, it was associated with a decrease in cell viability that approached significance. Dexamethasone did not have a reliable effect on these cytokines. Ultra-structural electron microscopic examination of the cells also demonstrated signs of definite down-regulation of cytoplasmic and nuclear activation.

Novel anti-oxidant therapies and possibly other immune modulating drugs can eliminate the activation of macrophages in response to periprosthetic wear particles without any associated decrease in cell viability and thus may provide a means of reducing the incidence of loosening and failure of total joint arthroplasty.

Objectives. Traumatic brachial plexus injury causes severe functional impairment of the arm. Elbow flexion is often affected. Nerve surgery or tendon transfers provide the only means to obtain improved elbow flexion. Unfortunately, the functionality of the arm often remains insufficient. Stem cell therapy could potentially improve muscle strength and avoid muscle-tendon transfer. This pilot study assesses the safety and regenerative potential of autologous bone marrow-derived mononuclear cell injection in partially denervated biceps. Methods. Nine brachial plexus patients with insufficient elbow flexion (i.e., partial denervation) received intramuscular escalating doses of autologous bone marrow-derived mononuclear cells, combined with tendon transfers. Effect parameters included biceps biopsies, motor unit analysis on needle electromyography and computerised muscle tomography, before and after cell therapy. Results. No adverse effects in vital signs, bone marrow aspiration sites, injection sites, or surgical wound were seen. After cell therapy there was a 52% decrease in muscle fibrosis (p = 0.01), an 80% increase in myofibre diameter (p = 0.007), a 50% increase in satellite cells (p = 0.045) and an 83% increase in capillary-to-myofibre ratio (p < 0.001) was shown. CT analysis demonstrated a 48% decrease in mean muscle density (p = 0.009). Motor unit analysis showed a mean increase of 36% in motor unit amplitude (p = 0.045), 22% increase in duration (p = 0.005) and 29% increase in number of phases (p = 0.002). Conclusions. Mononuclear cell injection in partly denervated muscle of brachial plexus patients is safe. The results suggest enhanced muscle reinnervation and regeneration. Cite this article: Bone Joint Res 2014;3:38–47

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

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

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

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

In rheumatoid arthritis (RA) and other arthritic disorders e.g. gout, there is destruction of articular cartilage and juxta-articular bone. Osteoclasts are specialised multinucleated cells (MNCs) that carry out bone resorption. It has previously been shown that circulating monocytes and synovial macrophages in RA can be stimulated to differentiate into functional osteoclasts in the presence of RANKL and M-CSF. The aim of this study was to determine whether the mononuclear cells present in synovial fluid of RA patients are capable of differentiating into functional osteoclasts in the presence of osteogenic factors. Mononuclear cells were isolated from the synovial fluid obtained from patients with Ra, osteoarthritis (OA) gout and joint trauma. The cells were seeded onto dentine slices and coverslips and cultured for up to 21 days in the presence/absence of RANKL (30ng/ml) and M-CSF (25ng/ml). Cells cultured on coverslips for 24h, 14 and 21 days were assessed for the expression of the monocyte-macrophage antigen CD14 that is known to be expressed by osteoclasts, and the osteoclast associated markers; tartrate-resistant acid phosphatase (TRAP) and vitronectin receptor (VNR). After 21 days, dentine slices were assessed for evidence of osteoclastic lacunar resorption. After 24 h culture on coverslips mononuclear cells isolated from the synovial fluid of all the above joint conditions were largely CD14+, and entirely negative for TRAP and VNR. After 14 days culture, in the presence of RANKL and M-CSF these synovial fluid macrophages were stimulated to form multinucleated osteoclasts which were TRAP+ and VNR+ and capable of forming resorption pits on dentine slices. In the absence of either RANKL or M-CSF osteoclast formation did not occur. The osteogenic factors RANKL and M-CSF have been shown to be present in the synovial fluid of patients with RA, OA, gout and joint trauma. Results from this study demonstrate that CD14+ mononuclear cells (macrophages) in the synovial fluid of patients with the above conditions have the capacity to differentiate into functional multinucleated osteoclasts in the presence of RANKL and M-CSF. These findings show that one cellular mechanisms whereby bone erosions many occur in arthritic disorders is through increased osteoclast formation of synovial fluid macrophages; this process requires RANKL and m-CSF, both of which are produced by inflammatory cells e.g. T Cells found in the synovial fluid and the arthritic synovial membrane

The April 2012 Hip & Pelvis Roundup. 360. looks at osteoporotic hip fractures, retrotrochanteric pain, fibrin adhesive and reattachment of articular cartilage, autologous bone marrow mononuclear cells and avascular necrosis, bearing surfaces, stability after THR, digital templating, pelvic tilt after THR, custom-made sockets for DDH, and dogs and THR

Introduction Intervertebral disc degeneration may cause chronic low back pain. Disc degeneration is characterized by dysfunctional cells and a decrease in extra-cellular components. Bone marrow derived mononuclear cells are a heterogeneous cell population which contains mesenchymal stem cells. Transplantation of stem cells and progenitor cells may provide a new approach to treat disc degeneration, but it is unclear if transplanted cells can survive and differentiate in the non-vascularized disc. Methods Bone marrow was collected from syngeneic Sprague-Dawley rats and mononuclear cells were isolated. The cells were labelled with a fluorescence dye (Cell Tracker Orange) and suspended in PBS. 10–20μl of the cell suspension (1–2x10. 5. cells/disc) was transplanted into coccygeal discs in 12 syngeneic rats. For each rat two discs were cell transplanted and one disc served as control. The rats were sacrificed after 0, 7, 14 or 21 days. For each time point the discs from one animal were saved for routine histological staining. The cell transplanted discs of the other animals (n=4 discs per time point) were formalin-fixed, frozen and sectioned together with the control discs. Frozen disc sections were visualized with fluorescence microscopy and the number of transplanted cells assessed. Expression of collagen II, a marker of chondrocytes and chondrocyte-like cells in the disc, was assessed in the transplanted cells using immunofluorescence technique. Results All cell-suspension injected discs contained transplanted bone-marrow cells. The discs within each time-group demonstrated a large variation in number of detected cells. There was a decrease in detected cells at 7, 14 and 21 days compared to day 0. Transplanted cells expressed collagen II after 21 days but not after 7 and 14 days. Discussion The results suggest that transplanted bone marrow-derived mononuclear cells can survive and differentiate within the intervertebral disc. Further studies in models of disc degeneration are warranted to investigate the regenerative potential of the disc following cell transplantation

Introduction: Osteoclast-like multinucleated giant cells (MNGCs) are found in a number of soft tissue sarcomas including malignant fibrous histiocytoma and leiomyosarcoma. The nature of these MNGCs is poorly understood and the cellular mechanisms underlying their accumulation in sarcomas is not known. Methods: We analysed by immunohistochemistry the expression of osteoclast, macrophage and smooth muscle markers by mononuclear and multinucleated cells in two cases of giant cell-rich leiomyosarcoma. We also characterised the role of mononuclear stromal cells and tumour-associated macrophages in the formation of MNGCs by RT-PCR, cell culture studies and immunohistochemistry/histochemistry for macrophage, osteoclast and smooth muscle markers. Results: MNCGs in giant cell-rich leiomyosarcoma expressed an osteoclast-like phenotype, being negative for smooth muscle actin and CD14 but positive for tartrate-resistant acid phophatase (TRAP), CD45, CD68 and vitronectin receptor (VNR). Scattered mononuclear cells expressing an osteoclast-like antigenic phenotype were also noted. An analysis of 25 conventional (non-giant cell-containing) leiomyosarcomas found isolated CD68+ MNGCs in three cases (approximately 12%); all of these cases were Grade-II/III leiomyosarcomas in which there was a prominent tumour-associated macrophage (TAM) infiltrate. Leiomyosarcoma TAMs isolated from two cases of conventional leiomyosarcoma and cultured in the presence of the osteoclastogenic factors RANKL and M-CSF differentiated into TRAP+/VNR+ MNGCs that were capable of lacunar resorption. RT-PCR studies showed that cultured leiomyosarcoma mononuclear stromal cells expressed RANKL, OPG and TRAIL. Discussion: These findings show that the MNGCs which are found in leiomyosarcomas are osteoclast-like in nature and that these MNGCs are formed from TAMs by a RANKL dependent mechanism which involves an interaction with RANKL-expressing mononuclear stromal cells. A similar mechanism is likely to account for MNGC accumulation in other soft tissue sarcomas

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

Giant cell tumour of bone (GCTB) is a primary tumour of bone characterised by a proliferation of mononuclear stromal cells and infiltrating macrophages and osteoclast-like giant cells. GCTB has a variable and unpredictable course and can produce metastatic lesions, mostly in the lungs, in up to 3% of cases. Whether these represent tumour implants rather than true neoplastic secondaries is uncertain. In this study, we analysed morphological and immunophenotypic features of primary GCTBs which metastasised to the lung as well as the metastatic lesions themselves in order to determine if these would provide a clue as to the mechanism of lung metastasis in GCTB. 17 cases of primary GCTB which metastasised to the lung and the lung metastases in these cases were obtained from IOR, Bologna. Morphologically, primary tumours showed variable features, often containing both giant cell-rich and mononuclear stromal cell-rich areas. Mononuclear cells showed frequent mitotic activity and a degree of nuclear pleomorphism; none of the tumours showed cytological features of malignancy. The tumours were highly vascular and frequently contained dilated thin-walled blood vessels and large areas of haemorrhage. GCTB lung metastases were generally small and contained osteoclast-like giant cells and mononuclear stromal cells which showed typical mitotic activity; cytologically, the metastatic tumours were relatively bland and showed little nuclear pleomorphism. Expression of HLA-DR (an allele of which has been associated with a more aggressive GCTB phenotype) and smooth muscle actin (SMA) was noted in stromal cells in primary and secondary GCTBs; frequently, the same pattern of SMA expression was seen in both primary and secondary lesions. Osteoclasts were vitronectin receptor+, CD14-HLA-DR- in both primary and secondary GCTBs. Our findings indicate that mononuclear stromal cells in lung metastases of GCTB often recapitulate the immunophenotype of the primary tumours from which they derive. Taken with the morphological finding that many primary GCTBs are highly vascular and contain areas of haemorrhage, it is possible that the lung “secondaries” of GCTB more likely represent tumour implants than true neoplastic metastases

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

Introduction: Cell-based strategies for regeneration and reconstitution of musculoskeletal tissues are gaining interest. The difficulty in obtaining the required amount of mesenchymal stem cells (MSC) stems from their scarcity and the time needed to grow them in culture. We developed a rapid and efficient method to isolate MSC from bone marrow aspirate based on their surface markers, as a platform for future cell based therapy. Methods: Bone marrow was aspirated from the iliac crest of fifteen adult subjects undergoing surgeries involving this bone. 15 ml samples were obtained, fractionated for mononuclear cells and then subjected to immunomagnetic isolation using microbeads of directly conjugated mouse anti–human CD105 antibodies. Recovered cell fraction was analyzed for phenotype and functional parameters. Results: The samples yielded an average of 14.6±2.5x10. 6. mononuclear cells per ml. Of these, fraction of CD105 positive cells consisted of 2.3±0.45%, which accounts for 0.25±0.06x10. 6. cells per ml. Post isolation analysis shows that 79±3.2% were positively stained for CD105 and 36±5.8% stained positive for CD45. These cells generated 6.3±1.4 Colony Forming Units (CFU) per 10. 5. cells. MSC concentration is higher in males and lower in smokers. Processing time is approximately 3 hours. Discussion and Conclusion: Regeneration of mesenchymal tissues using progenitor cells with appropriate matrix and signals was shown feasible, however large numbers of these rare cells are needed. An effective and safe method for purification of autologous MSC enables us to avoid the risks and the time span associated with culture expansion. We conclude that this method is both effective and rapid

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

Giant cell tumors of bone (GCTs) are locally aggressive tumors with recurrence potential that represent up to 10% of primary tumors of the bone. GCTs pathogenesis is driven by neoplastic mononuclear stromal cells that overexpress receptor activator of nuclear factor kappa-B/ligand (RANKL). Treatment with specific anti-RANKL antibody (denosumab) was recently introduced, used either as a neo-adjuvant in resectable tumors or as a stand-alone treatment in unresectable tumors. While denosumab has been increasingly used, a percentage of patients do not improve after treatment. Here, we aim to determine molecular and histological patterns that would help predicting GCTs response to denosumab to improve personalized treatment. Nine pre-treatment biopsies of patients with spinal GCT were collected at 2 centres. In 4 patients denosumab was used as a neo-adjuvant, 3 as a stand-alone and 2 received denosumab as adjuvant treatment. Clinical data was extracted retrospectively. Total mRNA was extracted by using a formalin-fixed paraffin-embedded extraction kit and we determined the transcript profile of 730 immune-oncology related genes by using the Pan Cancer Immune Profiling panel (Nanostring). The gene expression was compared between patients with good and poor response to Denosumab treatment by using the nSolver Analysis Software (Nanostring). Immunohistochemistry was performed in the tissue slides to characterize cell populations and immune response in CGTs. Two out of 9 patients showed poor clinical response with tumor progression and metastasis. Our analysis using unsupervised hierarchical clustering determined differences in gene expression between poor responders and good responders before denosumab treatment. Poor responding lesions are characterized by increased expression of inflammatory cytokines as IL8, IL1, interferon a and g, among a myriad of cytokines and chemokines (CCL25, IL5, IL26, IL25, IL13, CCL20, IL24, IL22, etc.), while good responders are characterized by elevated expression of platelets (CD31 and PECAM), coagulation (CD74, F13A1), and complement classic pathway (C1QB, C1R, C1QBP, C1S, C2) markers, together with extracellular matrix proteins (COL3A1, FN1,. Interestingly the T-cell response is also different between groups. Poor responding lesions have increased Th1 and Th2 component, but good responders have an increased Th17 component. Interestingly, the checkpoint inhibitor of the immune response PD1 (PDCD1) is increased ~10 fold in poor responders. This preliminary study using a novel experimental approach revealed differences in the immune response in GCTs associated with clinical response to denosumab. The increased activity of checkpoint inhibitor PD1 in poor responders to denosumab treatment may have implications for therapy, raising the potential to investigate immunotherapy as is currently used in other neoplasms. Further validation using a larger independent cohort will be required but these results could potentially identify the patients who would most benefit from denosumab therapy

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