Vertebral body versus iliac crest bone marrow as a source of multipotential stromal cells: Comparison of processing techniques, tri-lineage differentiation and application on a scaffold for spine fusion.

The potential use of bone progenitors, multipotential stromal cells (MSCs) helping spine fusion is increasing, but convenient MSC sources and effective processing methods are critical factors yet to be optimised. The aim of this study was to test the effect of bone marrow processing on the MSC abundance and to compare the differentiation capabilities of vertebral body-bone marrow (VB-BM) MSCs versus iliac crest-bone marrow (IC-BM) MSCs. We assessed the effect of the red blood cell lysis (ammonium chloride, AC) and density-gradient centrifugation (Lymphoprep™, LMP), on the extracted VB-BM and IC-BM MSC numbers. The MSC abundance (indicated by colony counts and CD45lowCD271high cell numbers), phenotype, proliferation and tri-lineage differentiation of VB-BM MSCs were compared with donor-matched IC-BM MSCs. Importantly, the MSC attachment and osteogenesis were examined when VB-BM and IC-BM samples were loaded on a beta-tricalcium phosphate scaffold. In contrast to LMP, using AC yielded more colonies from IC-BM and VB-BM aspirates (p = 0.0019 & p = 0.0201 respectively). For IC-BM and VB-BM, the colony counts and CD45lowCD271high cell numbers were comparable (p = 0.5186, p = 0.2640 respectively). Furthermore, cultured VB-BM MSCs exhibited the same phenotype, proliferative and adipogenic potential, but a higher osteogenic and chondrogenic capabilities than IC-BM MSCs (p = 0.0010 and p = 0.0005 for calcium and glycosaminoglycan (GAG) levels, respectively). The gene expression data confirmed higher chondrogenesis for VB-BM MSCs than IC-BM MSCs, but osteogenic gene expression levels were comparable. When loaded on Vitoss™, both MSCs showed a similar degree of attachment and survival, but a better osteogenic ability was detected for VB-BM MSCs as measured by alkaline phosphatase activity (p = 0.0386). Collectively, the BM processing using AC had more MSC yield than using LMP. VB-BM MSCs have a comparable phenotype and proliferative capacity, but higher chondrogenesis and osteogenesis with or without using scaffold than donor-matched IC-BM MSCs. Given better accessibility, VB-BM could be an ideal MSC source for spinal bone fusion.

Release of growth factors and the effect of age, sex, and severity of injury after long bone fracture. A preliminary report.

BACKGROUND AND PURPOSE: The systemic response after fracture is regulated by a complex mechanism involving numerous growth factors. In this study, we analyzed the kinetics of key growth factors following lower-limb long bone fracture. MATERIALS AND METHODS: Human serum was isolated from 15 patients suffering from lower-limb long bone fracture (tibia/femur) requiring surgical fixation. The levels of platelet-derived growth factor (PDGF-BB), vascular edothelial growth factor (VEGF), insulin growth factor-I (IGF-I), and transforming growth factor ß1 (TGF-ß1) were assayed by colorimetric ELISA at different time points during the first week after fracture. 10 healthy volunteers made up the control group of the study. Serum levels of the growth factors measured were compared to age, sex, and injury severity score. RESULTS: We found that there was a decline in the levels of PDGF-BB, IGF-I and TGF-ß1 during the first 3 days after fracture. However, VEGF levels remained unchanged. The levels of all the growth factors studied then increased, with the highest concentrations noted at day 7 after surgery. No correlation was found between circulating levels of growth factors and age, injury severity score (ISS), blood loss, or fluid administration. INTERPRETATION: There are systemic mitogenic and osteogenic signals after fracture. Important growth factors are released into the peripheral circulation, but early after surgery it appears that serum levels of key growth factors fall. By 7 days postoperatively, the levels had increased considerably. Our findings should be considered in cases where autologous serum is used for ex vivo expansion of mesenchymal stem cells. There should be further evaluation of the use of these molecules as biomarkers of bone union.

B cell biomarkers of rituximab responses in systemic lupus erythematosus.

OBJECTIVE: Rituximab appears to be effective in many studies of systemic lupus erythematosus (SLE), with variable initial clinical response and time to relapse. However, results of a randomized controlled trial of rituximab were negative. This study was undertaken to evaluate the effectiveness of rituximab in SLE, using highly sensitive flow cytometry (HSFC), which can define B cell numbers 50-100 times lower than conventional techniques and predicts responses in rheumatoid arthritis. METHODS: Thirty-nine patients with active SLE were started on a standard regimen of rituximab with intravenous and oral steroids. Clinical response and relapse were defined using the British Isles Lupus Assessment Group (BILAG) index with criteria for major clinical response, partial clinical response, and nonresponse. HSFC, including analysis of B cell subsets, was performed. RESULTS: There was a significant reduction from baseline in global BILAG score at all time points analyzed (P<0.0001), and major clinical response and partial clinical response rates were 51% and 31%, respectively. Time to relapse was highly variable. Fifty percent of the patients relapsed after 6-18 months (earlier relapse); the remainder relapsed at a slower rate (later relapse). B cell depletion and repopulation were variable and were predictive of these clinical outcomes. There was a persistent B cell presence in 21 patients after 2 infusions of rituximab, which included all 7 patients with no response (P=0.012 versus patients with complete depletion of B cells). Memory B cell (P=0.02) and plasmablast (P<0.001) repopulation after 26 weeks was markedly faster in patients with earlier relapse versus patients with later relapse. CONCLUSION: Our findings indicate that rituximab is effective in SLE, and clinical responses are supported by close correlation with B cell numbers. HSFC is a valuable tool in the assessment and prediction of response in SLE.

Reduced-dose rituximab in rheumatoid arthritis: efficacy depends on degree of B cell depletion.

OBJECTIVE: Studies comparing 500 mg rituximab and 1,000 mg rituximab doses in rheumatoid arthritis have yielded conflicting data on clinical outcomes, but in all of these studies a subgroup of patients has had excellent responses at the lower dose. Historically, it was considered that rituximab uniformly depleted B cells at both doses. Using highly sensitive assays, we have shown that B cell depletion is variable and predictive of clinical response. Using the same techniques, we undertook the present study to test the hypothesis that the level of B cell depletion, rather than the rituximab dose, determines clinical response. METHODS: Nineteen patients were treated with two 500-mg infusions of rituximab, and 61 patients were treated with two 1,000-mg infusions of rituximab. Highly sensitive flow cytometry was performed at 0, 2, 6, 14, and 26 weeks. European League Against Rheumatism (EULAR) response rates at 6 months were compared between patients with and those without complete depletion at each dose. RESULTS: The median B cell count was numerically higher at all time points following therapy in the 500 mg rituximab group. Twenty-five percent of patients in the 500 mg rituximab group had complete depletion at 2 weeks, compared with 49% of those in the 1,000 mg rituximab group. Complete depletion at 2 weeks after treatment with 500 mg rituximab was associated with lower baseline preplasma cell counts (P = 0.047). Most patients responded after either dose, but response was related to B cell depletion. Notably, in the 500 mg rituximab group all patients with complete depletion had a EULAR good response (P = 0.011). CONCLUSION: This pilot study suggests that the degree of B cell depletion, rather than the dose of rituximab, determines clinical response. It may be possible to predict which patients will respond to lower-dose rituximab, and this may allow more cost-effective treatment.

Effects of antithrombotic drugs fondaparinux and tinzaparin on in vitro proliferation and osteogenic and chondrogenic differentiation of bone-derived mesenchymal stem cells.

An unexpected side effect of some classes of anticoagulants has been osteoporosis which may be, at least in part, related to deranged mesenchymal stem cell (MSC) function. The aim of the present study was to compare the effect of fondaparinux (FDP), a novel antithrombotic with a traditional widely used low molecular weight heparin, tinzaparin (TZP) on MSC proliferation and differentiation. MSCs were isolated from trabecular bone of 14 trauma patients by a collagenase-based digestion procedure and expanded in standard conditions until passage 3. Proliferation and differentiation of MSCs to chondrocytes and osteoblasts was assessed with or without the addition of FDP and TZP using standard in vitro assays and a broad range of drug concentrations. Flow cytometry was used for MSC phenotyping. In the age studied group (17-74 years old) the MSC frequency in collagenase-released fractions was 641/10(6) cells (range 110-2,158) and their growth characteristics were ∼4 days/population doubling. Cultures had a standard MSC phenotype (CD73+, CD105+, CD146+, CD106+, and CD166+). Cell proliferation was assessed by both colony-forming unit-fibroblast (CFU-F) and colorimetric tetrazolium salt XTT assays. In both assays, MSC proliferation was inhibited by the addition of TZP, particularly at high concentrations. In contrast, FDP had no effect on MSC proliferation. Osteogenic differentiation and chondrogenic differentiation were not affected by the addition of either TZP or FDP. Whilst MSC proliferation, but not differentiation, is negatively affected by TZP, there was no evidence for adverse effects of FDP in this in vitro model system which argues well for its use in the orthopedic setting.

The effect of bone morphogenetic protein-2, bone morphogenetic protein-7, parathyroid hormone, and platelet-derived growth factor on the proliferation and osteogenic differentiation of mesenchymal stem cells derived from osteoporotic bone.

INTRODUCTION: It has been previously shown that in patients with osteoporosis, mesenchymal stem cell (MSC) growth rate and osteogenic potential is decreased contributing to inferior fracture consolidation. The aim of this study was to investigate the effect of bone morphogenetic protein-2 (BMP-2), BMP-7, parathyroid hormone (PTH), and platelet-derived growth factor (PDGF) on proliferation and osteogenic differentiation of MSCs derived from patients with osteoporosis. MATERIALS AND METHODS: Trabecular bone was obtained from 10 patients (four males, mean age 76 years) with lower extremity osteoporotic fractures. MSCs were isolated by enzymatic digestion. Functional assays of proliferation and osteogenic differentiation were performed under the influence of a wide range of concentrations of BMP-2, BMP-7, PTH, and PDGF-BB. Proliferation was assessed using CFU-F and XTT assays. Osteogenic differentiation was assessed by alkaline phosphatase activity and total calcium production. RESULTS: MSC proliferation was found to be stimulated by supplementation with BMP-7 and PDGF-BB, whereas BMP-2 and PTH had little effect. The largest increase in proliferation rate was observed after administration 100 ng/mL of BMP-7. All four molecules induced alkaline phosphatase activity and calcium production in growing osteoblasts with a dose-dependent effect noted. BMP-2 and BMP-7 at their highest studied concentration (100 ng/mL) produced a threefold increase in the osteogenic potential of MSCs. CONCLUSION: BMP-7, BMP-2, PTH, and PDGF-BB were observed to have a positive effect on osteogenic differentiation of MSCs. BMP-7 and PDGF-BB (in high doses) could be considered most potentially advantageous because they enhance both proliferation and osteogenic differentiation of MSCs derived from elderly osteoporotic bone.

Highly sensitive B cell analysis predicts response to rituximab therapy in rheumatoid arthritis.

OBJECTIVE: In rheumatoid arthritis (RA), B cell depletion occurs in all patients treated with rituximab, but the clinical responses to rituximab are variable. A highly sensitive assay was used to test the hypothesis that B cell depletion is variable, and that incomplete depletion leads to a poorer outcome. METHODS: Sixty patients with active RA unresponsive to anti-tumor necrosis factor agents received two 1-gram infusions of rituximab. B cell numbers were measured by highly sensitive flow cytometry before and after each infusion and at 3-month intervals thereafter. A reduction in B cell levels below 0.0001x10(9)/liter was defined as complete depletion (compared with 0.05x10(9)/liter by conventional cytometry). Clinical responses were measured using the European League Against Rheumatism (EULAR) criteria. RESULTS: At 6 months, 92% of patients had a moderate-to-good clinical response according to the EULAR criteria. B cells were detected in 63% of patients after the first infusion of rituximab (median level 0.0009x10(9)/liter [range<0.0001-0.0015x10(9)/liter), and these patients had poorer clinical outcomes than patients with complete depletion. At 9 months, 82% of patients with complete depletion had a moderate-to- good EULAR response, compared with 43% of those with partial depletion (P=0.01). At 12 months, 59% of complete responders had a moderate-to-good EULAR response, compared with 21% of those with partial depletion (P=0.01). Patients in whom B cells were depleted only after the second infusion did no better than those in whom depletion was never complete and had poorer clinical outcomes than those in whom depletion was initially complete. CONCLUSION: This study is the first to show, using a highly sensitive analysis, that rituximab therapy is associated with variable diminution in B cell numbers. A lack of complete depletion of B cells after 1 infusion was associated with a poorer outcome.

Synovial fluid mesenchymal stem cells in health and early osteoarthritis: detection and functional evaluation at the single-cell level.

OBJECTIVE: Arthritic synovial fluid (SF) contains mesenchymal stem cells (MSCs), which could simply reflect their shedding from diseased joint structures. This study used the bovine model to explore SF MSCs in health and enumerated them at the earliest stages of human osteoarthritis (OA) in radiographically normal joints. METHODS: Clonogenicity and multipotentiality of normal bovine SF MSCs were compared with donor-matched bone marrow (BM) MSCs at the single-cell level. The colony-forming unit-fibroblastic assay was used for MSC enumeration. The XTT assay was employed to assess cell proliferation, and flow cytometry was used to investigate the marker phenotype of bovine and human SF MSCs. RESULTS: Single MSCs were present in normal bovine SF, and 96% of them were able to expand at least 1 million-fold. These cells were CD271-, multipotential, considerably more clonogenic, and less adipogenic than matched BM MSCs. In both pellet assays and on polyglycolic acid scaffolds, SF clones displayed consistent chondrogenic differentiation, while BM clones were variable. MSCs were present in arthroscopically normal human joints and were increased 7-fold in early OA (P = 0.034). Their numbers correlated with numbers of free microscopic synovial tissue fragments (r = 0.826, P < 0.0001). OA SF had a growth-promoting effect on synovial MSCs. CONCLUSION: This study confirms the presence of MSCs in normal SF and shows their numerical increase in early human OA. SF MSCs are likely to originate from synovium. These findings provide a platform for the exploration of the potential role of SF MSCs in joint homeostasis and for investigation of their utility in novel joint regeneration strategies.

Enumeration and phenotypic characterization of synovial fluid multipotential mesenchymal progenitor cells in inflammatory and degenerative arthritis.

OBJECTIVE: To evaluate synovial fluid (SF) for the presence of mesenchymal progenitor cells (MPCs), to compare SF MPCs with bone marrow (BM) MPCs, and to enumerate these cells in both inflammatory arthritis and osteoarthritis (OA). METHODS: SF from 100 patients with arthritis (53 rheumatoid arthritis [RA], 20 OA, and 27 other arthropathies) was evaluated. To establish multipotentiality, polyclonal and single cell-derived cultures of SF fibroblasts were examined by standard and quantitative differentiation assays. Their phenotype before and after expansion was determined by multiparameter flow cytometry. A colony-forming unit-fibroblast assay was used for SF MPC enumeration. RESULTS: Regardless of the nature of the arthritis, both polyclonal and single cell-derived cultures of SF fibroblasts possessed trilineage mesenchymal differentiation potentials. The number of MPCs in a milliliter of SF was higher in OA (median 37) than in RA (median 2) (P < 0.00001). No significant differences in MPC numbers were found between early and established RA (median 3 and 2 cells/ml, respectively). Culture-expanded SF and BM MPCs had the same phenotype (negative for CD45 and positive for D7-FIB, CD13, CD105, CD55, and CD10). Rare, uncultured SF fibroblasts were CD45(low) and expressed low-affinity nerve growth factor receptor, similar to in vivo BM MPCs. CONCLUSION: Our findings prove the presence of rare tripotential MPCs, at the single-cell level, in the SF of patients with arthritis. SF MPCs are clonogenic and multipotential fibroblasts that, despite the pathologic environment within a diseased joint, have a phenotype similar to that of uncultured BM MPCs. The higher prevalence of MPCs in OA SF suggests their likely origin from disrupted joint structures. These findings could determine the role of MPCs in the pathogenesis of inflammatory arthritis, together with their role in attempted joint regeneration in degenerative arthritis, which has yet to be established.