Cerebral small vessel disease burden and cognitive and functional outcomes after stroke: A multicenter prospective cohort study.

INTRODUCTION: It remains unknown whether the global small vessel disease (SVD) burden predicts post-stroke outcomes. METHODS: In a prospective multicenter study of 666 ischemic and hemorrhagic stroke patients, we quantified magnetic resonance imaging (MRI)-based SVD markers (lacunes, white matter hyperintensities, microbleeds, perivascular spaces) and explored associations with 6- and 12-month cognitive (battery of 15 neuropsychological tests) and functional (modified Rankin scale) outcomes. RESULTS: A global SVD score (range 0-4) was associated with cognitive impairment; worse performance in executive function, attention, language, and visuospatial ability; and worse functional outcome across a 12-month follow-up. Although the global SVD score did not improve prediction, individual SVD markers, assessed across their severity range, improved the calibration, discrimination, and reclassification of predictive models including demographic, clinical, and other imaging factors. DISCUSSION: SVD presence and severity are associated with worse cognitive and functional outcomes 12 months after stroke. Assessing SVD severity may aid prognostication for stroke patients. HIGHLIGHTS: In a multi-center cohort, we explored associations of small vessel disease (SVD) burden with stroke outcomes. SVD burden associates with post-stroke cognitive and functional outcomes. A currently used score of SVD burden does not improve the prediction of poor outcomes. Assessing the severity of SVD lesions adds predictive value beyond known predictors. To add predictive value in assessing SVD in stroke patients, SVD burden scores should integrate lesion severity.

Relationships of peripheral IGF-1, VEGF and BDNF levels to exercise-related changes in memory, hippocampal perfusion and volumes in older adults.

Animal models point towards a key role of brain-derived neurotrophic factor (BDNF), insulin-like growth factor-I (IGF-I) and vascular endothelial growth factor (VEGF) in mediating exercise-induced structural and functional changes in the hippocampus. Recently, also platelet derived growth factor-C (PDGF-C) has been shown to promote blood vessel growth and neuronal survival. Moreover, reductions of these neurotrophic and angiogenic factors in old age have been related to hippocampal atrophy, decreased vascularization and cognitive decline. In a 3-month aerobic exercise study, forty healthy older humans (60 to 77years) were pseudo-randomly assigned to either an aerobic exercise group (indoor treadmill, n=21) or to a control group (indoor progressive-muscle relaxation/stretching, n=19). As reported recently, we found evidence for fitness-related perfusion changes of the aged human hippocampus that were closely linked to changes in episodic memory function. Here, we test whether peripheral levels of BDNF, IGF-I, VEGF or PDGF-C are related to changes in hippocampal blood flow, volume and memory performance. Growth factor levels were not significantly affected by exercise, and their changes were not related to changes in fitness or perfusion. However, changes in IGF-I levels were positively correlated with hippocampal volume changes (derived by manual volumetry and voxel-based morphometry) and late verbal recall performance, a relationship that seemed to be independent of fitness, perfusion or their changes over time. These preliminary findings link IGF-I levels to hippocampal volume changes and putatively hippocampus-dependent memory changes that seem to occur over time independently of exercise. We discuss methodological shortcomings of our study and potential differences in the temporal dynamics of how IGF-1, VEGF and BDNF may be affected by exercise and to what extent these differences may have led to the negative findings reported here.

Inferior Frontal Sulcal Hyperintensities on Brain MRI Are Associated with Amyloid Positivity beyond Age-Results from the Multicentre Observational DELCODE Study.

Inferior frontal sulcal hyperintensities (IFSHs) on fluid-attenuated inversion recovery (FLAIR) sequences have been proposed to be indicative of glymphatic dysfunction. Replication studies in large and diverse samples are nonetheless needed to confirm them as an imaging biomarker. We investigated whether IFSHs were tied to Alzheimer's disease (AD) pathology and cognitive performance. We used data from 361 participants along the AD continuum, who were enrolled in the multicentre DELCODE study. The IFSHs were rated visually based on FLAIR magnetic resonance imaging. We performed ordinal regression to examine the relationship between the IFSHs and cerebrospinal fluid-derived amyloid positivity and tau positivity (Aß42/40 ratio ≤ 0.08; pTau181 ≥ 73.65 pg/mL) and linear regression to examine the relationship between cognitive performance (i.e., Mini-Mental State Examination and global cognitive and domain-specific performance) and the IFSHs. We controlled the models for age, sex, years of education, and history of hypertension. The IFSH scores were higher in those participants with amyloid positivity (OR: 1.95, 95% CI: 1.05-3.59) but not tau positivity (OR: 1.12, 95% CI: 0.57-2.18). The IFSH scores were higher in older participants (OR: 1.05, 95% CI: 1.00-1.10) and lower in males compared to females (OR: 0.44, 95% CI: 0.26-0.76). We did not find sufficient evidence linking the IFSH scores with cognitive performance after correcting for demographics and AD biomarker positivity. IFSHs may reflect the aberrant accumulation of amyloid ß beyond age.

Bioactive factors in platelet-rich plasma obtained by apheresis.

INTRODUCTION: The use of platelet-rich plasma (PRP) in regenerative approaches in cartilage repair is becoming more common. Information about PRP composition and its content of putative bioactive chondrogenic growth factors (GF) that may support cartilage regeneration is scarce. METHODS: GF composition of a pool of 6 PRP preparations was determined using Protein Antibody Membrane Arrays covering 507 GF, signaling molecules, and receptors. To verify the chondrogenic GF variability in PRP, Growth Factor Antibody Membrane Arrays covering 26 GF were applied to 6 individual PRP preparations. Selected GF involved in chondrogenic differentiation were quantified by Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: 417 out of 507 possible detectable proteins were present in the PRP pool, including 76 GF. Quantification of selected chondrogenic GF by ELISA showed an average of 0.31 ng/ml bone morphogenetic protein-2, 0.50 ng/ml connective tissue growth factor, 0.76 ng/ml fibroblast growth factor-2, and 0.59 ng/ml transforming growth factor-ß3. CONCLUSION: PRP as a therapeutic option in regenerative cartilage repair strategies is a powerful tool for the local application of chondrogenic GF to the site of injury. Chondrogenic GF are present in PRP and may support cartilage repair by inducing cell differentiation and cartilage matrix formation.

Microvascular Impairment in Patients With Cerebral Small Vessel Disease Assessed With Arterial Spin Labeling Magnetic Resonance Imaging: A Pilot Study.

In this pilot study, we investigated microvascular impairment in patients with cerebral small vessel disease (CSVD) using non-invasive arterial spin labeling (ASL) magnetic resonance imaging (MRI). This method enabled us to measure the perfusion parameters, cerebral blood flow (CBF), and arterial transit time (ATT), and the effective T1-relaxation time (T1eff) to research a novel approach of assessing perivascular clearance. CSVD severity was characterized using the Standards for Reporting Vascular Changes on Neuroimaging (STRIVE) and included a rating of white matter hyperintensities (WMHs), lacunes, enlarged perivascular spaces (EPVSs), and cerebral microbleeds (CMBs). Here, we found that CBF decreases and ATT increases with increasing CSVD severity in patients, most prominent for a white matter (WM) region-of-interest, whereas this relation was almost equally driven by WMHs, lacunes, EPVSs, and CMBs. Additionally, we observed a longer mean T1eff of gray matter and WM in patients with CSVD compared to elderly controls, providing an indication of impaired clearance in patients. Mainly T1eff of WM was associated with CSVD burden, whereas lobar lacunes and CMBs contributed primary to this relation compared to EPVSs of the centrum semiovale. Our results complement previous findings of CSVD-related hypoperfusion by the observation of retarded arterial blood arrival times in brain tissue and by an increased T1eff as potential indication of impaired clearance rates using ASL.

Engraftment Effects after Intra-Bone Marrow versus Intravenous Allogeneic Stem Cell Transplantation in a Reduced-Intensity Conditioning Dog Leukocyte Antigen-Identical Canine Model.

Following conventional i.v. hematopoietic stem cell transplantation (IV-HSCT), most of the hematopoietic stem cells get trapped in peripheral organs and do not reach the bone marrow niche. A promising approach to overcome this cell loss during the homing process seems to be the infusion of hematopoietic stem cells directly into the bone marrow cavity (intra-bone marrow [IBM]-HSCT). This study aimed to investigate the engraftment efficiency of IBM-HSCT compared with IV-HSCT following reduced-intensity conditioning in a canine HSCT model. Furthermore, the impact of 2 different graft infusion rates during IBM-HSCT on the engraftment was evaluated. Dogs received 4.5 Gy total body irradiation for conditioning at day -1 and 15 mg/kg cyclosporin A twice daily at days -1 to +35 as immunosuppression. The IV-HSCT group (n = 7) received unmodified bone marrow. The IBM-HSCT cohorts received buffy coat-enriched bone marrow that was applied into the humerus and femur simultaneously with an infusion time of either 10 minutes (IBM10; n = 8) or 60 minutes (IBM60; n = 7). Statistical analyses were performed using the Kruskal-Wallis test followed by the Mann-Whitney U test with Bonferroni correction for multiple comparisons. Statistical significance was declared at Bonferroni-adjusted P < .017. All dogs initially engrafted. One dog of the IBM10 cohort died at day +15 from infection. All 21 evaluable dogs developed a durable mixed donor chimerism over the course of 112 days. Engraftment kinetics did not differ significantly across the 3 groups. Leukocyte and platelet nadirs, as well as the durations of leukopenia and thrombocytopenia, were comparable in the 3 groups. Signs of toxicity for ingestion, body temperature, activity, and defecation did not show statistically significant differences among the 3 groups; only weight loss was greater in the IBM60 group compared with the IV group. IBM-HSCT following reduced-intensity conditioning resulted in an engraftment efficiency and hematopoietic recovery comparable to that seen with conventional IV-HSCT. In addition, modification of the graft infusion rate had no impact on engraftment and hematopoietic recovery in the canine IBM-HSCT model.

Reliability and Reproducibility of Hadamard Encoded Pseudo-Continuous Arterial Spin Labeling in Healthy Elderly.

The perfusion parameters cerebral blood flow (CBF) and arterial transit time (ATT) measured with arterial spin labeling (ASL) magnetic resonance imaging (MRI) provide valuable essentials to assess the integrity of cerebral tissue. Brain perfusion changes, due to aging, an intervention, or neurodegenerative diseases for example, could be investigated in longitudinal ASL studies with reliable ASL sequences. Generally, pseudo-continuous ASL (pCASL) is preferred because of its larger signal-to-noise ratio (SNR) compared to pulsed ASL (PASL) techniques. Available pCASL versions differ regarding their feature details. To date only little is known about the reliability and reproducibility of CBF and ATT measures obtained with the innovative Hadamard encoded pCASL variant, especially if applied on participants in old age. Therefore, we investigated an in-house developed Hadamard encoded pCASL sequence on a group of healthy elderly at two different 3 Tesla Siemens MRI systems (Skyra and mMR Biograph) and evaluated CBF and ATT reliability and reproducibility for several regions-of-interests (ROI). Calculated within-subject coefficients of variation (wsCV) demonstrated an excellent reliability of perfusion measures, whereas ATT appeared to be even more reliable than CBF [e.g., wsCV(CBF) = 2.9% vs. wsCV(ATT) = 2.3% for a gray matter (GM) ROI on Skyra system]. Additionally, a substantial agreement of perfusion values acquired on both MRI systems with an inter-session interval of 78 ± 17.6 days was shown by high corresponding intra-class correlation (ICC) coefficients [e.g., ICC(CBF) = 0.704 and ICC(ATT) = 0.754 for a GM ROI]. The usability of this novel Hadamard encoded pCASL sequence might improve future follow-up perfusion studies of the aging and/or diseased brain.

Chemokine profile of human serum from whole blood: migratory effects of CXCL-10 and CXCL-11 on human mesenchymal stem cells.

Autologous human serum is used in cartilage repair and may exert its effect by the recruitment of mesenchymal stem and progenitor cells (MSC). Aim of our study was to analyze the chemokine profile of human serum and to verify chemotactic activity of selected chemokines on MSC. Human MSC were isolated from iliac crest bone marrow aspirates. Chemotactic activity of human serum made from whole blood and pharma grade serum was tested in 96-well chemotaxis assays and chemokine levels were analyzed using human chemokine antibody membrane arrays. The chemotactic potential of selected chemokines on MSC was tested dose dependently using chemotaxis assays. Human serum derived from whole blood significantly attracted human MSC, while pharma grade serum did not recruit MSC. Human chemokine antibody array analysis showed that the level of chemokines CXCL-3, 5, 7-8, 10-12, 16; CCL- 2, 5, 11, 13, 16-20, 24-25, 27; as well as XCL-1 was elevated (fold change >1.5) in serum derived from whole blood compared to nonrecruiting pharma grade serum. Chemotaxis assays showed that the chemokines IP-10/CXCL-10 and I-TAC/CXCL-11 significantly recruit human MSC. PARC/CCL-18, HCC-4/CCL-16, CTACK/CCL-27, and Lymphotactin/XCL-1 showed no chemotactic effect on MSC. Therefore, human serum derived from whole blood contains chemokines that may contribute to serum-mediated recruitment of human mesenchymal progenitors from bone marrow.

Human periosteum-derived progenitor cells express distinct chemokine receptors and migrate upon stimulation with CCL2, CCL25, CXCL8, CXCL12, and CXCL13.

For bone repair, transplantation of periosteal progenitor cells (PCs), which had been amplified within supportive scaffolds, is applied clinically. More innovative bone tissue engineering approaches focus on the in situ recruitment of stem and progenitor cells to defective sites and their subsequent use for guided tissue repair. Chemokines are known to induce the directed migration of bone marrow CD34(-) mesenchymal stem cells (MSCs). The aim of our study was to determine the chemokine receptor expression profile of human CD34(-) PCs and to demonstrate that these cells migrate upon stimulation with selected chemokines. PCs were isolated from periosteum of the mastoid bone and displayed a homogenous cell population presenting an MSC-related cell-surface antigen profile (ALCAM(+), SH2(+), SH3(+), CD14(-), CD34(-), CD44(+), CD45(-), CD90(+)). The expression profile of chemokine receptors was determined by real-time PCR and immunohistochemistry. Both methods consistently demonstrated that PCs express receptors of all four chemokine subfamilies CC, CXC, CX(3)C, and C. Migration of PCs and a dose-dependent migratory effect of the chemokines CCL2 (MCP1), CCL25 (TECK), CXCL8 (IL8), CXCL12 (SDF1alpha), and CXCL13 (BCA1), but not CCL22 (MDC) were demonstrated using a 96-multiwell chemotaxis assay. In conclusion, for the first time, here we report that human PCs express chemokine receptors, present their profile, and demonstrate a dose-dependent migratory effect of distinct chemokines on these cells. These results are promising towards in situ bone repair therapies based on guiding PCs to bone defects, and encourage further in vivo studies.

Age-related functional changes in domain-specific medial temporal lobe pathways.

There is now converging evidence from studies in animals and humans that the medial temporal lobes (MTLs) harbor anatomically distinct processing pathways for object and scene information. Recent functional magnetic resonance imaging studies in humans suggest that this domain-specific organization may be associated with a functional preference of the anterior-lateral part of the entorhinal cortex (alErC) for objects and the posterior-medial entorhinal cortex (pmErC) for scenes. As MTL subregions are differentially affected by aging and neurodegenerative diseases, the question was raised whether aging may affect the 2 pathways differentially. To address this possibility, we developed a paradigm that allows the investigation of object memory and scene memory in a mnemonic discrimination task. A group of young (n = 43) and healthy older subjects (n = 44) underwent functional magnetic resonance imaging recordings during this novel task, while they were asked to discriminate exact repetitions of object and scene stimuli from novel stimuli that were similar but modified versions of the original stimuli ("lures"). We used structural magnetic resonance images to manually segment anatomical components of the MTL including alErC and pmErC and used these segmented regions to analyze domain specificity of functional activity. Across the entire sample, object processing was associated with activation of the perirhinal cortex (PrC) and alErC, whereas for scene processing, activation was more predominant in the parahippocampal cortex and pmErC. Functional activity related to mnemonic discrimination of object and scene lures from exact repetitions was found to overlap between processing pathways and suggests that while the PrC-alErC pathway was more involved in object discrimination, both pathways were involved in the discrimination of similar scenes. Older adults were behaviorally less accurate than young adults in discriminating similar lures from exact repetitions, but this reduction was equivalent in both domains. However, this was accompanied by significantly reduced domain-specific activity in PrC in older adults compared to what was observed in the young. Furthermore, this reduced domain-specific activity was associated to worse performance in object mnemonic discrimination in older adults. Taken together, we show the fine-grained functional organization of the MTL into domain-specific pathways for objects and scenes and their mnemonic discrimination and further provide evidence that aging might affect these pathways in a differential fashion. Future experiments will elucidate whether the 2 pathways are differentially affected in early stages of Alzheimer's disease in relation to amyloid or tau pathology.

Regeneration of ovine articular cartilage defects by cell-free polymer-based implants.

The aim of our study was the evaluation of a cell-free cartilage implant that allows the recruitment of mesenchymal stem and progenitor cells by chemo-attractants and subsequent guidance of the progenitors to form cartilage repair tissue after microfracture. Chemotactic activity of human serum on human mesenchymal progenitors was tested in 96-well chemotaxis assays and chondrogenic differentiation was assessed by gene expression profiling after stimulating progenitors with hyaluronan in high-density cultures. Autologous serum and hyaluronan were combined with polyglycolic acid (PGA) scaffolds and were implanted into full-thickness articular cartilage defects of the sheep pre-treated with microfracture. Defects treated with microfracture served as controls. Human serum was a potent chemo-attractant and efficiently recruited mesenchymal progenitors. Chondrogenic differentiation of progenitors upon stimulation with hyaluronan was shown by the induction of typical chondrogenic marker genes like type II collagen and aggrecan. Three months after implantation of the cell-free implant, histological analysis documented the formation of a cartilaginous repair tissue. Controls treated with microfracture showed no formation of repair tissue. The cell-free cartilage implant consisting of autologous serum, hyaluronan and PGA utilizes the migration and differentiation potential of mesenchymal progenitors for cartilage regeneration and is well suited for the treatment of cartilage defects after microfracture.

Synovial fluid recruits human mesenchymal progenitors from subchondral spongious bone marrow.

Microfracture is a frequently used reparative technique that induces a healing response in articular cartilage defects. Penetration of the subchondral bone leads to blood clot formation, allows multipotent mesenchymal cells to access the defect and, subsequently, leads to cartilaginous repair tissue. The aim of our study was to analyze the chemotactic recruitment of human subchondral spongious bone marrow-derived cells by synovial fluid (SF) from normal donors (ND), patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Subchondral spongious bone marrow-derived mesenchymal progenitors were isolated from bone cylinders after high tibial osteotomy and analyzed for the presence of stem cell-related cell surface antigens by flow cytometry. Recruitment of subchondral progenitors by normal SF and SF from donors with degenerated joint diseases was documented by using a modified Boyden chamber chemotaxis assay. The chemotaxis assay demonstrated that synovial fluid has the potential to recruit mesenchymal progenitors in vitro. SF from normal donors and patients with OA showed no difference in the potential to stimulate cell migration. SF obtained from RA donors showed significantly reduced cell recruitment compared to SF derived from OA patients (p = 0.0054) and normal donors (p < 0.0001). The chemotactic activity of SF obtained from normal donors and from patients with degenerative joint diseases suggests that SF may be actively involved in the migration of progenitors in cartilage defects after microfracture.

How adults with an intellectual disability experience bereavement and grief: a qualitative exploration.

This qualitative study explored the lived experiences of bereavement of 13 adults with an intellectual disability and found that their experiences could be situated within the concept of disenfranchised grief. The latter mediated participants' meaning making of the grieving process illustrated in the themes of intra- and interpersonal bereavement experiences, core beliefs about life and death, level of inclusion, and maintaining a continuing relationship with the deceased. The results suggest that participants experience bereavement and grief in a manner similar to that of the general population and suggest the need for open communication, facilitation of informed choice, and a culture of inclusion.

Chondrogenic differentiation of human subchondral progenitor cells is affected by synovial fluid from donors with osteoarthritis or rheumatoid arthritis.

BACKGROUND: Microfracture is a first-line treatment option for cartilage repair. In microfracture, subchondral mesenchymal cortico-spongious progenitor cells (CSP) enter the defect and form cartilage repair tissue. The aim of our study was to investigate the effects of joint disease conditions on the in vitro chondrogenesis of human CSP. METHODS: CSP were harvested from the subchondral bone marrow. CSP characterization was performed by analysis of cell surface antigen pattern and by assessing the chondrogenic, osteogenic and adipogenic differentiation potential, histologically. To assess the effect of synovial fluid (SF) on chondrogenesis of CSP, micro-masses were stimulated with SF from healthy (ND), osteoarthritis (OA) and rheumatoid arthritis donors (RA) without transforming growth factor beta 3. RESULTS: CSP showed the typical cell surface antigen pattern known from mesenchymal stem cells and were capable of osteogenic, adipogenic and chondrogenic differentiation. In micro-masses stimulated with SF, histological staining as well as gene expression analysis of typical chondrogenic marker genes showed that SF from ND and OA induced the chondrogenic marker genes aggrecan, types II and IX collagen, cartilage oligomeric matrix protein (COMP) and link protein, compared to controls not treated with SF. In contrast, the supplementation with SF from RA donors decreased the expression of aggrecan, type II collagen, COMP and link protein, compared to CSP treated with SF from ND or OA. CONCLUSION: These results suggest that in RA, SF may impair cartilage repair by subchondral mesenchymal progenitor cells in microfracture, while in OA, SF may has no negative, but a delaying effect on the cartilage matrix formation.

An ovine in vitro model for chondrocyte-based scaffold-assisted cartilage grafts.

BACKGROUND: Scaffold-assisted autologous chondrocyte implantation is an effective clinical procedure for cartilage repair. From the regulatory point of view, the ovine model is one of the suggested large animal models for pre-clinical studies. The aim of our study was to evaluate the in vitro re-differentiation capacity of expanded ovine chondrocytes in biomechanically characterized polyglycolic acid (PGA)/fibrin biomaterials for scaffold-assisted cartilage repair. METHODS: Ovine chondrocytes harvested from adult articular cartilage were expanded in monolayer and re-assembled three-dimensionally in PGA-fibrin scaffolds. De- and re-differentiation of ovine chondrocytes in PGA-fibrin scaffolds was assessed by histological and immuno-histochemical staining as well as by real-time gene expression analysis of typical cartilage marker molecules and the matrix-remodelling enzymes matrix metalloproteinases (MMP) -1, -2 and -13 as well as their inhibitors. PGA scaffolds characteristics including degradation and stiffness were analysed by electron microscopy and biomechanical testing. RESULTS: Histological, immuno-histochemical and gene expression analysis showed that dedifferentiated chondrocytes re-differentiate in PGA-fibrin scaffolds and form a cartilaginous matrix. Re-differentiation was accompanied by the induction of type II collagen and aggrecan, while MMP expression decreased in prolonged tissue culture. Electron microscopy and biomechanical tests revealed that the non-woven PGA scaffold shows a textile structure with high tensile strength of 3.6 N/mm2 and a stiffness of up to 0.44 N/mm2, when combined with gel-like fibrin. CONCLUSION: These data suggest that PGA-fibrin is suited as a mechanically stable support structure for scaffold-assisted chondrocyte grafts, initiating chondrogenic re-differentiation of expanded chondrocytes.

The chemokines CXCL10 and XCL1 recruit human annulus fibrosus cells.

STUDY DESIGN: Human annulus fibrosus tissue and cells were analyzed for the presence of chemokine receptors and the migratory effect of selected chemokines. OBJECTIVE: To investigate spontaneous repair mechanisms and underlying cell recruitment in response to annular tears and degenerative defects. SUMMARY OF BACKGROUND DATA: Resorption of herniated disc tissue and the attempt to close annulus tears with repair tissue occur spontaneously. Although chemokines are suggested to play a role in resorption of herniated disc tissue, the role of chemokines in annulus fibrosus homeostasis and repair remains unclear. METHODS: Cells were isolated from annulus fibrosus tissue and expanded in the presence of human serum. Multiwell chemotaxis assays were used to analyze the migratory effect of human serum and 0 to 1000 nM concentrations of the chemokines CXCL7, CXCL10, CXCL12, CCL25, and XCL1 on annulus fibrosus cells (AFCs) (n = 9 per chemokine and dose). Presence of corresponding chemokine receptors in AFCs was determined by real-time polymerase chain reaction analysis and immunohistochemistry. RESULTS: Serum (0.1%-10%) significantly (P < 0.01) stimulates the migration of AFCs. Compared with untreated cells, the migration of cells was significantly (P < 0.01) enhanced upon stimulation with 100 to 1000 nM CXCL10 and 1000 nM XCL1. Chemokine receptors showed low expression levels in expanded AFCs as assessed by polymerase chain reaction. Immunohistochemical staining of the CXCL10 receptor CXCR3 and the XCL1 receptor XCR1 showed that the presence of the particular receptors in AFCs expanded under conventional cell culture conditions. In native annulus fibrosus tissue, CXCR3 was evident, whereas XCR1 could not be detected. CONCLUSION: The findings suggest that chemokines, in particular CXCL10, effectively recruit isolated AFCs. This suggests that chemokines are involved in annulus fibrosus homeostasis and potentially in spontaneous annulus repair attempts. This might have important implications for biological annulus-sealing strategies.

Chondrogenic differentiation of human mesenchymal stem cells in micro-masses is impaired by high doses of the chemokine CXCL7.

Chemokines have been shown to recruit human mesenchymal stem cells (MSCs) and are suggested to be promising candidates for in situ tissue engineering. The aim of our study was to analyse the effect of CXCL7, a chemokine that has the capacity to recruit MSCs, on the chondrogenic differentiation of MSCs. Bone marrow-derived MSCs were cultured in high-density micro-masses under serum-free conditions and were co-stimulated with 0-100 nM CXCL7 in the presence of 10 ng/ml transforming growth factor-ß3 (TGFß3). Micro-masses stimulated without growth factors and chemokines served as controls. Histological staining of proteoglycan, immunostaining of type II collagen, staining of mineralized matrix according to von Kossa as well as real-time gene expression analysis of typical chondrogenic and osteogenic marker genes showed that the TGFß3-mediated chondrogenic development of MSCs was not impaired by 0-50 nM CXCL7. Micro-masses stimulated with TGFß3 and CXCL7 developed chondrogenic cells and formed a cartilaginous matrix rich in proteoglycans, accompanied by the induction of typical chondrogenic marker genes, such as cartilage oligomeric matrix protein, aggrecan, type IIα1 collagen and by regulation of matrix metalloproteinases and their inhibitors. As assessed by histological staining, MSCs showed a significantly reduced deposition of proteoglycan and a mildly mineralized matrix when stimulated with TGFß3 in the presence of 100 nM CXCL7. Induction of osteogenic marker genes such as osteocalcin was not evident. These results suggest that low doses of CXCL7 do not impair the chondrogenic differentiation of bone marrow-derived stem cells and may suited for in situ cartilage tissue engineering.

Chondrogenic differentiation of human subchondral progenitor cells is impaired by rheumatoid arthritis synovial fluid.

In microfracture, subchondral progenitors enter the cartilage defect and form cartilage repair tissue. We hypothesize that synovial fluid (SF) from rheumatoid arthritis (RA) donors affects chondrogenesis of human subchondral progenitors stimulated with transforming growth factor-beta3 (TGFB3), whereas SF from normal and osteoarthritis (OA) donors do not. Human progenitors from subchondral cortico-spongious bone (pool of n = 4) were cultured in micromasses under serum-free conditions and were stimulated with 10 ng/mL TGFB3 and with 5% SF from normal, OA, and RA donors (pool of n = 7, each). Histological staining of proteoglycan and immunostaining of type II collagen showed that progenitors stimulated with SF from RA donors show significantly reduced cartilage matrix formation compared to progenitors treated with TGFB3 or with SF from normal and OA donors (n = 3, each). Gene expression analysis of typical chondrocyte marker genes and genes encoding matrix modifying enzymes showed that SF from OA and RA donors influence the onset of TGFB3-mediated chondrogenesis (pool of 20 micromasses), but had no effect on the gene expression profile after prolonged culture in micromasses. These results suggest that SF from RA patients may impair the chondrogenic development of mesenchymal progenitors in microfracture, whereas osteoarthritic SF may has no negative effect on the cartilage matrix formation.

Gene expression profile of adult human bone marrow-derived mesenchymal stem cells stimulated by the chemokine CXCL7.

A variety of chemokines has been shown to recruit human bone marrow-derived mesenchymal stem cells (MSC) and may be potential candidates for chemokine-based tissue regeneration approaches. The aim of our study was to determine whether the chemokine CXCL7 stimulates migration of human bone marrow-derived MSC and to analyze the effect of CXCL7 on the recruitment of MSC on the broad molecular level. Chemotaxis assays documented that high doses of CXCL7 significantly recruited MSC. Gene expression profiling using oligonucleotide microarrays showed that MSC treated with CXCL7 differentially expressed genes related to cell migration, cell adhesion and extracellular matrix remodeling. Pathway analysis showed that CXCL7 induced the expression of all chemokines binding the interleukin (IL) receptors A and B, CXCR1 and CXCR2, as well as the IL6 signal transducer (gp130) and its ligands IL6 and leukemia inhibitory factor (LIF). Induction of differentially expressed chemokines CXCL1-3, CXCL5, and CXCL6 as well as LIF and gp130 in MSC by CXCL7 was verified by real-time polymerase chain reaction. Immunoassay of cell culture supernatants confirmed elevated levels of the interleukins 6 and 8 in MSC upon treatment with CXCL7. Chemotaxis assays showed that interleukin 6 did not recruit MSC. In conclusion, CXCL7 significantly stimulates the migration of human MSC in vitro. Pathway analysis suggests that recruitment of human MSC by CXCL7 is supported by the induction of ligands of the interleukin 8 receptors, synergistically activating the respective signaling pathways.

Formation of cartilage repair tissue in articular cartilage defects pretreated with microfracture and covered with cell-free polymer-based implants.

The aim of our study was to evaluate the mid-term outcome of a cell-free polymer-based cartilage repair approach in a sheep cartilage defect model in comparison to microfracture treatment. Cell-free, freeze-dried implants (chondrotissue) made of a poly-glycolic acid (PGA) scaffold and hyaluronan were immersed in autologous serum and used for covering microfractured full-thickness articular cartilage defects of the sheep (n = 4). Defects treated with microfracture only served as controls (n = 4). Six months after implantation, cartilage implants and controls were analyzed by immunohistochemical staining of type II collagen, histological staining of proteoglycans, and histological scoring. Histological analysis showed the formation of a cartilaginous repair tissue rich in proteoglycans. Histological scoring documented significant improvement of repair tissue formation when the defects were covered with the cell-free implant, compared to controls treated with microfracture. Immunohistochemistry showed that the cell-free implant induced cartilaginous repair tissue and type II collagen. Controls treated with microfracture showed marginal formation of a mixed-type repair tissue consisting of cartilaginous tissue and fibro-cartilage. Covering of microfractured defects with the cell-free polymer-based cartilage implant is suggested to be a promising treatment option for cartilage defects and improves the regeneration of articular cartilage.