Correction to: Bone defect reconstruction with a novel biomaterial containing calcium phosphate and aluminum oxide reinforcement.

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Bone defect reconstruction with a novel biomaterial containing calcium phosphate and aluminum oxide reinforcement.

BACKGROUND: Reconstruction of metaphyseal fractures represents a clinical challenge for orthopedic surgeons. Especially in osteoporotic bone, these fractures are frequently accompanied by osseous substance defects. In order to ensure rapid mobilization of patients, high stability requirements must be met by osteosynthesis. Various bone graft materials have been introduced in the past, such as autologous bone or exogenous bone substitute materials. These are used as bone void fillers or as augmentation techniques to ensure safe fixation of osteosynthesis. New calcium phosphate-based bone void-filling materials could be a promising alternative to autologous bone or to the currently and widely used polymethylmethacrylate (PMMA)-based cement. The aim of this study was to evaluate a novel paste-like bone void filler in vivo and in vitro with regard to biocompatibility and osteoconductivity. METHODS: In addition to in vitro testing of cell compatibility using pre-osteoblasts (MC3T3-E1), 35 Wistar rats were treated in vivo with implantation of various material mixtures based on calcium phosphate and aluminum oxide reinforcement in a metaphyseal drill hole defect. After 4 weeks, an examination by micro-computed tomography (µCT) and histology was performed. RESULTS: The in vitro analysis showed good biocompatibility with a high cell survival of osteoblasts. In the in vivo experiments, a significantly higher bone ingrowth compared to the empty defect was shown by µCT and histological analysis. Here, the group receiving material reinforced with aluminum oxide (Al2O3) showed a bone volume/tissue volume (BV/TV) of 89.19% compared to a BV/TV of 83.14% for the empty defect (p = 0.0013). In the group treated with a polysaccharide matrix, no increase in BV/TV was observed given a mean ratio of 80.14%. Scoring of histological sections did not reveal a significant difference between CaP and CaP that was substituted with Al2O3. CONCLUSION: The results of this study show an encouraging first step towards the development of new pasty, bone void-filling materials. We demonstrated that a new paste-like bone-filling material, based on calcium phosphate granulates and aluminum oxide to provide strength, exhibits good biocompatibility and osteoconductivity. Further biomechanical test in an osteoporotic animal model will have to be performed, to prove feasibility in metaphyseal defects.

Bone regeneration of minipig mandibular defect by adipose derived mesenchymal stem cells seeded tri-calcium phosphate- poly(D,L-lactide-co-glycolide) scaffolds.

Reconstruction of bone defects represents a serious issue for orthopaedic and maxillofacial surgeons, especially in extensive bone loss. Adipose-derived mesenchymal stem cells (ADSCs) with tri-calcium phosphates (TCP) are widely used for bone regeneration facilitating the formation of bone extracellular matrix to promote reparative osteogenesis. The present study assessed the potential of cell-scaffold constructs for the regeneration of extensive mandibular bone defects in a minipig model. Sixteen skeletally mature miniature pigs were divided into two groups: Control group and scaffolds seeded with osteogenic differentiated pADSCs (n = 8/group). TCP-PLGA scaffolds with or without cells were integrated in the mandibular critical size defects and fixed by titanium osteosynthesis plates. After 12 weeks, ADSCs seeded scaffolds (n = 7) demonstrated significantly higher bone volume (34.8% ± 4.80%) than scaffolds implanted without cells (n = 6, 22.4% ± 9.85%) in the micro-CT (p < 0.05). Moreover, an increased amount of osteocalcin deposition was found in the test group in comparison to the control group (27.98 ± 2.81% vs 17.10 ± 3.57%, p < 0.001). In conclusion, ADSCs seeding on ceramic/polymer scaffolds improves bone regeneration in large mandibular defects. However, further improvement with regard to the osteogenic capacity is necessary to transfer this concept into clinical use.

Decreased Expression of the Human Urea Transporter SLC14A1 in Bone is Induced by Cytokines and Stimulates Adipogenesis of Mesenchymal Progenitor Cells.

The human urea transporter SLC14A1 (HUT11/UT-B) has been suggested as a marker for the adipogenic differentiation of bone cells with a relevance for bone diseases. We investigated the function of SLC14A1 in different cells models from bone environment. SLC14A1 expression and cytokine production was investigated in bone cells obtained from patients with osteoporosis. Gene and protein expression of SLC14A1 was studied during adipogenic or osteogenic differentiation of human mesenchymal progenitor cells (hMSCs) and of the single-cell-derived hMSC line (SCP-1), as well as in osteoclasts and chondrocytes. Localization was determined by histochemical methods and functionality by urea transport experiments. Expression of SLC14A1 mRNA was lower in cells from patients with osteoporosis that produced high levels of cytokines. Accordingly, when adding a combination of cytokines to SCP-1 SLC14A1 mRNA expression decreased. SLC14A1 mRNA expression decreased after both osteogenic and more pronounced adipogenic stimulation of hMSCs and SCP-1 cells. The highest SLC14A1 expression was determined in undifferentiated cells, lowest in chondrocytes and osteoclasts. Downregulation of SLC14A1 by siRNA resulted in an increased expression of interleukin-6 and interleukin-1 beta as well as adipogenic markers. Urea influx through SLC14A1 increased expression of osteogenic markers, adipogenic markers were suppressed. SLC14A1 protein was localized in the cell membrane and the cytoplasm. Summarizing, the SLC14A1 urea transporter affects early differentiation of hMSCs by diminishing osteogenesis or by favoring adipogenesis, depending on its expression level. Therefore, SLC14A1 is not unequivocally an adipogenic marker in bone. Our findings suggest an involvement of SLC14A1 in bone metabolism and inflammatory processes and disease-dependent influences on its expression.

Growth factor-mediated augmentation of long bones: evaluation of a BMP-7 loaded thermoresponsive hydrogel in a murine femoral intramedullary injection model.

BACKGROUND: Due to our aging population, an increase in proximal femur fractures can be expected, which is associated with impaired activities of daily living and a high risk of mortality. These patients are also at a high risk to suffer a secondary osteoporosis-related fracture on the contralateral hip. In this context, growth factors could open the field for regenerative approaches, as it is known that, i.e., the growth factor BMP-7 (bone morphogenetic protein 7) is a potent stimulator of osteogenesis. Local prophylactic augmentation of the proximal femur with a BMP-7 loaded thermoresponsive hydrogel during index surgery of an osteoporotic fracture could be suitable to reduce the risk of further osteoporosis-associated secondary fractures. The present study therefore aims to test the hypothesis if a BMP-7 augmented hydrogel is an applicable carrier for the augmentation of non-fractured proximal femurs. Furthermore, it needs to be shown that the minimally invasive injection of a hydrogel into the mouse femur is technically feasible. METHODS: In this study, male C57BL/6 mice (n = 36) received a unilateral femoral intramedullary injection of either 100 µl saline, 100 µl 1,4 Butan-Diisocyanat (BDI)-hydrogel, or 100 µl hydrogel loaded with 1 µg of bone morphogenetic protein 7. Mice were sacrificed 4 and 12 weeks later. The femora were submitted to high-resolution X-ray tomography and subsequent histological examination. RESULTS: Analysis of normalized CtBMD (Cortical bone mineral density) as obtained by X-ray micro-computed tomography analysis revealed significant differences depending on the duration of treatment (4 vs 12 weeks; p < 0.05). Furthermore, within different anatomically defined regions of interest, significant associations between normalized TbN (trabecular number) and BV/TV (percent bone volume) were noted. Histology indicated no signs of inflammation and no signs of necrosis and there were no cartilage damages, no new bone formations, or new cartilage tissues, while BMP-7 was readily detectable in all of the samples. CONCLUSIONS: In conclusion, the murine femoral intramedullary injection model appears to be feasible and worth to be used in subsequent studies that are directed to examine the therapeutic potential of BMP-7 loaded BDI-hydrogel. Although we were unable to detect any significant osseous effects arising from the mode or duration of treatment in the present trial, the effect of different concentrations and duration of treatment in an osteoporotic model appears of interest for further experiments to reach translation into clinic and open new strategies of growth factor-mediated augmentation.

Characterization of human telomerase reverse transcriptase immortalized anterior cruciate ligament cell lines.

BACKGROUND: The anterior-cruciate-ligament (ACL) contains mesenchymal stem cells (ACL-MSCs), suggesting the feasibility of regenerative treatments of this tissue. The immortalization of isolated cells results in cell-lines applicable to develop cell-based therapies. Immortal cell lines eliminate the need for frequent cell isolation from donor tissues. The objective of this study was to characterize cell lines that were generated from isolated ACL-MSCs using TERT gene transfer. METHODS: We isolated ACL-MSCs from human ACLs derived at the time of ACL reconstruction surgery or total knee arthroplasty. We generated cell lines and compared them to non-immortalized ACL-MSCs. We assessed the cellular morphology and we detected surface antigen expression. The resistance to senescence was inferred using the beta galactosidase activity. Histology, immunohistochemistry, and reverse transcriptase polymerase chain reaction (RT-PCR) were used to evaluate the multilineage differentiation capacity. RESULTS: The morphology of hTERT-ACL-MSCs was similar to ACL up to the last assessment at passage eight. We detected a strong surface expression of CD44, CD90, CD105, and STRO-1 in hTERT-ACL-MSCs. No substantial reduction in the ATP activity was observed in hTERT-ACL-MSCs. CONCLUSION: Cell lines generated from ACL-MSCs maintain their morphology, surface antigen expression profile, and proliferative capacity; while markers of senescence appear to be reduced. These cell-lines maintained their multilineage differentiation capacity. The demonstrated model systems can be used for further development of new cell-based regenerative approaches in anterior cruciate ligament research, which may lead to new therapeutic strategies in the future.

In Vitro Comparison of 2D-Cell Culture and 3D-Cell Sheets of Scleraxis-Programmed Bone Marrow Derived Mesenchymal Stem Cells to Primary Tendon Stem/Progenitor Cells for Tendon Repair.

The poor and slow healing capacity of tendons requires novel strategies to speed up the tendon repair process. Hence, new and promising developments in tendon tissue engineering have become increasingly relevant. Previously, we have established a tendon progenitor cell line via ectopic expression of the tendon-related basic helix-loop-helix (bHLH) transcription factor Scleraxis (Scx) in human bone marrow mesenchymal stem cells (hMSC-Scx). The aim of this study was to directly compare the characteristics of hMSC-Scx cells to that of primary human tendon stem/progenitors cells (hTSPCs) via assessment of self-renewal and multipotency, gene marker expression profiling, in vitro wound healing assay and three-dimensional cell sheet formation. As expected, hTSPCs were more naive than hMSC-Scx cells because of higher clonogenicity, trilineage differentiation potential, and expression of stem cell markers, as well as higher mRNA levels of several gene factors associated with early tendon development. Interestingly, with regards to wound healing, both cell types demonstrate a comparable speed of scratch closure, as well as migratory velocity and distance in various migration experiments. In the three-dimensional cell sheet model, hMSC-Scx cells and hTSPCs form compact tendinous sheets as histological staining, and transmission electron microscopy shows spindle-shaped cells and collagen type I fibrils with similar average diameter size and distribution. Taken together, hTSPCs exceed hMSC-Scx cells in several characteristics, namely clonogenicity, multipotentiality, gene expression profile and rates of tendon-like sheet formation, whilst in three-dimensional cell sheets, both cell types have comparable in vitro healing potential and collagenous composition of their three-dimensional cell sheets, making both cell types a suitable cell source for tendon tissue engineering and healing.

IL-6, IL-1ß, and TNF-α only in combination influence the osteoporotic phenotype in Crohn's patients via bone formation and bone resorption.

BACKGROUND: Crohn´s disease (CD) is associated with a higher prevalence of osteoporosis. The pathogenesis of bone affliction remains controversial, especially if inflammatory cytokines or glucocorticoid therapy are the main contributors. In postmenopausal osteoporosis, bone resorption is induced by IL-6, IL-1ß and TNF-α. In contrast, in children with CD, IL-6 exclusively decreased bone formation without affecting bone resorption. OBJECTIVES: The objective of this study was to further clarify the pathophysiology of bone affliction in adult patients with CD with the use of an osteoblast and osteoclast cell model. MATERIAL AND METHODS: Inflammatory cytokines IL-6, IL-1ß, and TNF-α were measured in adult CD patients' serum. Mean values of these cytokines were applied with or without dexamethasone to the human cell line SCP-1 (osteoblastic cell model). Also, the effect of cytokines on primary human osteoclast differentiation and activity was determined. RESULTS: The combined cytokine application increased the receptor activator of NF-κB ligand/osteoprotegerin (RANKL/OPG) ratio 2-fold after 2 and 14 days. Additional application of dexamethasone to SCP-1 cells further increased the RANKL/OPG ratio 3-fold, but decreased IL-6 and IL-1ß expression to 10% and 50%, respectively. TNF-α expression was maximally suppressed to 16% by dexamethasone in the presence of cytokines. In osteoclasts, the combined cytokine treatment decreased expression of characteristic genes to approx. 30%, while increasing osteoclast resorption activity to 148%. In addition, a cytokine stimulated osteoblast cell culture-generated supernatant stimulated osteoclast resorption activity by 170%. CONCLUSIONS: Our results suggest that IL-6, IL-1ß, and TNF-α only in combination induced osteoclaststimulating activity represented by the RANKL/OPG ratio in osteoblasts. Dexamethasone further increased this effect in osteoblasts, while decreasing cytokine expression. The results in osteoclasts support a direct and osteoblast-mediated effect on bone resorption. Our in vitro results differentiate for the first time the effect of cytokines on bone turnover as measured in adult CD patients from the additional dexamethasone effect on osteoblasts as part of the pathophysiology of osteoporosis.

Functionalized thermosensitive hydrogel combined with tendon stem/progenitor cells as injectable cell delivery carrier for tendon tissue engineering.

Thermosensitive hydrogels have been studied for potential application as promising alternative cell carriers in cell-based regenerative therapies. In this study, a thermosensitive butane diisocyanate (BDI)-collagen hydrogel (BC hydrogel) was designed as an injectable cell delivery carrier of tendon stem/progenitor cells (TSPCs) for tendon tissue engineering. We functionalized the BDI hydrogel with the addition of 20% (v/v) collagen I gel to obtain the thermosensitive BC hydrogel, which was then seeded with TSPCs derived from human Achilles tendons. The BC hydrogel compatibility and TSPC behavior and molecular response to the 3D hydrogel were investigated. Collagen (COL) I gel served as a control group. Our findings demonstrated that the BC hydrogel was thermosensitive, and hardened above 25 °C. It supported TSPC survival, proliferation, and metabolic activity with satisfactory dimension stability and biocompatibility, as revealed by gel contraction assay, live/dead staining, DNA quantification, and resazurin metabolic assay. Phalloidin-based visualization of F-actin demonstrated that the TSPCs were stretched within COL I gel with classical spindle cell shapes; similar cell morphologies were also found in the BC hydrogel. The gene expression profile of TSPCs in the BC hydrogel was comparable with that in COL I gel. Moreover, the BC hydrogel supported capillary-like structure formation by human umbilical vein endothelial cells (HUVECs) in the hydrogel matrix. Taken together, these results suggest that the thermosensitive BC hydrogel holds great potential as an injectable cell delivery carrier of TSPCs for tendon tissue engineering.

Tenomodulin is essential for prevention of adipocyte accumulation and fibrovascular scar formation during early tendon healing.

Tenomodulin (Tnmd) is the best-known mature marker for tendon and ligament lineage cells. It is important for tendon maturation, running performance and has key implications for the resident tendon stem/progenitor cells (TSPCs). However, its exact functions during the tendon repair process still remain elusive. Here, we established an Achilles tendon injury model in a Tnmd knockout (Tnmd-/-) mouse line. Detailed analyses showed not only a very different scar organization with a clearly reduced cell proliferation and expression of certain tendon-related genes, but also increased cell apoptosis, adipocyte and blood vessel accumulation in the early phase of tendon healing compared with their wild-type (WT) littermates. In addition, Tnmd-/- tendon scar tissue contained augmented matrix deposition of biglycan, cartilage oligomeric matrix protein (Comp) and fibronectin, altered macrophage profile and reduced numbers of CD146-positive cells. In vitro analysis revealed that Tnmd-/- TSPCs exhibited significantly reduced migration and proliferation potential compared with that of WT TSPCs. Furthermore, Tnmd-/- TSPCs had accelerated adipogenic differentiation accompanied with significantly increased peroxisome proliferator-activated receptor gamma (Pparγ) and lipoprotein lipase (Lpl) mRNA levels. Thus, our results demonstrate that Tnmd is required for prevention of adipocyte accumulation and fibrovascular scar formation during early tendon healing.

Effect of hypoxia on the proliferation of porcine bone marrow-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells in 2- and 3-dimensional culture.

OBJECTIVE: Bone marrow-derived mesenchymal stem cells (MSCs) and adipose-derived mesenchymal stem cells (ASCs) currently represent a promising tool for the regeneration of large bony defects. Therefore, it is pivotal to find the best cell source within the body and the best conditions for in vitro cellular expansion. This study compared cellular response of MSCs and ASCs from a porcine animal in normoxic (21% O2) and hypoxic (2% O2) cell culture conditions via 2D and 3D experimental settings. MATERIALS AND METHODS: The effect of constant exposure to hypoxia on primary pig stem cells was evaluated by two methods. First, a cumulative population doublings (cumPD) over a period of 40 days, a metabolic activity assay in both 2D and 3D beta-TCP-PHB scaffolds, followed by analysis of osteogenic differentiation potential in cell monolayers. RESULTS: Our results displayed enhanced cell culture proliferation in 2% O2 for both MSCs and ASCs, with impaired osteogenic differentiation of MSCs. The impact of constant hypoxia on porcine MSCs and ASCs exhibited a statistically significant decrease in osteogenic differentiation under hypoxic conditions with the MSCs. CONCLUSIONS: Our data suggest that MSCs and ASCs expanded in hypoxic culture conditions, might be more suitable for use in the clinical setting where large cell numbers are required. When differentiated in normoxic conditions, MSCs showed the highest osteogenic differentiation potential and might be the best choice of cells with consideration to bone repair.

Oxygen mapping: Probing a novel seeding strategy for bone tissue engineering.

Bone tissue engineering (BTE) utilizing biomaterial scaffolds and human mesenchymal stem cells (hMSCs) is a promising approach for the treatment of bone defects. The quality of engineered tissue is crucially affected by numerous parameters including cell density and the oxygen supply. In this study, a novel oxygen-imaging sensor was introduced to monitor the oxygen distribution in three dimensional (3D) scaffolds in order to analyze a new cell-seeding strategy. Immortalized hMSCs, pre-cultured in a monolayer for 30-40% or 70-80% confluence, were used to seed demineralized bone matrix (DBM) scaffolds. Real-time measurements of oxygen consumption in vitro were simultaneously performed by the novel planar sensor and a conventional needle-type sensor over 24 h. Recorded oxygen maps of the novel planar sensor revealed that scaffolds, seeded with hMSCs harvested at lower densities (30-40% confluence), exhibited rapid exponential oxygen consumption profile. In contrast, harvesting cells at higher densities (70-80% confluence) resulted in a very slow, almost linear, oxygen decrease due to gradual achieving the stationary growth phase. In conclusion, it could be shown that not only the seeding density on a scaffold, but also the cell density at the time point of harvest is of major importance for BTE. The new cell seeding strategy of harvested MSCs at low density during its log phase could be a useful strategy for an early in vivo implantation of cell-seeded scaffolds after a shorter in vitro culture period. Furthermore, the novel oxygen imaging sensor enables a continuous, two-dimensional, quick and convenient to handle oxygen mapping for the development and optimization of tissue engineered scaffolds. Biotechnol. Bioeng. 2017;114: 894-902. © 2016 Wiley Periodicals, Inc.

Scaffold-free Scleraxis-programmed tendon progenitors aid in significantly enhanced repair of full-size Achilles tendon rupture.

AIM: Currently there is no effective approach to enhance tendon repair, hence we aimed to identify a suitable cell source for tendon engineering utilizing an established clinically relevant animal model for tendon injury. MATERIALS & METHODS: We compared, by in-depth histomorphometric evaluation, the regenerative potential of uncommitted human mesenchymal stem cells (hMSC) and Scleraxis (Scx)-programmed tendon progenitors (hMSC-Scx) in the healing of a full-size of rat Achilles tendon defect. RESULTS: Our analyses clearly demonstrated that implantation of hMSC-Scx, in contrast to hMSC and empty defect, results in smaller diameters, negligible ectopic calcification and advanced cellular organization and matrix maturation in the injured tendons. CONCLUSION: Scaffold-free delivery of hMSC-Scx aids in enhanced repair in a clinically translatable Achilles tendon injury model.

Influence of osteogenic stimulation and VEGF treatment on in vivo bone formation in hMSC-seeded cancellous bone scaffolds.

BACKGROUND: Tissue engineering approaches for reconstruction of large bone defects are still technically immature, especially in regard to sufficient blood supply. Therefore, the aim of the present study was to investigate the influence of osteogenic stimulation and treatment with VEGF on new bone formation and neovascularization in hMSC-loaded cancellous bone scaffolds in vivo. METHODS: Cubic scaffolds were seeded with hMSC and either cultured in stem cell medium or osteogenic stimulation medium. One osteogenically stimulated group was additionally treated with 0.8 µg VEGF prior to subcutaneous implantation in athymic mice. After 2 and 12 weeks in vivo, constructs and selected organs were harvested for histological and molecular analysis. RESULTS: Histological analysis revealed similar vascularization of the constructs with and without VEGF treatment and absence of new bone formation in any group. Human DNA was detected in all inoculated scaffolds, but a significant decrease in cells was observed after 2 weeks with no further decrease after 12 weeks in vivo. CONCLUSION: Under the chosen conditions, osteogenic stimulation and treatment with VEGF does not have any influence on the new bone formation and neovascularization in hMSC-seeded cancellous bone scaffolds.

Mesenchymal stem cells from osteoporotic patients reveal reduced migration and invasion upon stimulation with BMP-2 or BMP-7.

Fractures to the osteoporotic bone feature a delay in callus formation and reduced enchondral ossification. Human mesenchymal stem cells (hMSC), the cellular source of fracture healing, are recruited to the fracture site by cytokines, such as BMP-2 and BMP-7. Aim of the study was to scrutinize hMSC for osteoporosis associated alterations in BMP mediated migration and invasion as well as in extracellular matrix (ECM) binding integrin expression. HMSC were isolated from 18 healthy or osteoporotic donors. Migration was assessed using a collagen IV coated micro-slide linear gradient chamber and time-lapse microscopy. Invasion was analyzed utilizing an ECM coated transmembrane invasion assay. Quantitative real-time RT PCR was performed for the ECM binding integrins α1, α2, α3, α4, α5, α11, αv and ß1. HMSC from osteoporotic patients showed a significant increase of migration upon BMP-2 or FCS stimulation, as well as a significant increase of invasion upon BMP-2, BMP-7 or FCS stimulation. Nevertheless, the migration and invasion capacity was significantly decreased compared to healthy controls. Out of all integrins analyzed, collagen binding integrin α2 was significantly downregulated in hMSC from osteoporotic patients. In conclusion, we here demonstrate for the first time osteoporosis associated alterations in BMP mediated hMSC recruitment. These findings may underlie the reduced healing of osteoporotic fractures. Nevertheless, the maintained migration and invasion response upon BMP stimulation illustrates the therapeutic potential of these clinically approved substances in the treatment of osteoporotic fractures. Another therapeutic target may be the downregulation of the collagen binding integrin α2 in hMSC from osteoporotic patients.

Sox2 is a potent inhibitor of osteogenic and adipogenic differentiation in human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are a promising target for cell-based bone regeneration. However, their application for clinical use is limited because hMSCs lose their ability for cell division and differentiation during longer in vitro cultivation. The osteogenic differentiation is regulated through a complex network of molecular signal transduction pathways where the canonical Wnt pathway plays an important role. Sox2, a known key factor for maintenance of cellular pluripotency in stem cells, is supposed to influence the Wnt pathway in osteoblasts. In this study, we overexpressed Sox2 in immortalized hMSCs by lentiviral gene transfer. Sox2 overexpression significantly reduced the osteogenic and adipogenic differentiation potentials. This effect was abolished by knockdown of Sox2 overexpression. In addition, Oct4 and Nanog, other key transcription factors for pluripotency, are strongly upregulated when Sox2 is overexpressed. Furthermore, Dkk1, a target gene of the Sox2-Oct4 heterodimer and a Wnt antagonist, is downregulated. Sox2 overexpression causes higher expression levels of ß-catenin, the central transcription factor of the canonical Wnt pathway. These results suggest that Sox2 keeps hMSCs in an undifferentiated state by influencing the canonical Wnt pathway. Regulated expression of Sox2 may be a promising tool to cultivate hMSCs in sufficient quantities for cell and gene therapy applications.

In vivo mesenchymal stem cell tracking with PET using the dopamine type 2 receptor and 18F-fallypride.

UNLABELLED: Human mesenchymal stem cells (hMSCs) represent a promising treatment approach for tissue repair and regeneration. However, little is known about the underlying mechanisms and the fate of the transplanted cells. The objective of the presented work was to determine the feasibility of PET imaging and in vivo monitoring after transplantation of dopamine type 2 receptor-expressing cells. METHODS: An hMSC line constitutively expressing a mutant of the dopamine type 2 receptor (D2R80A) was generated by lentiviral gene transfer. D2R80A messenger RNA expression was confirmed by reverse transcriptase-polymerase chain reaction. Localization of the transmembrane protein was analyzed by confocal fluorescence microscopy. The stem cell character of transduced hMSCs was investigated by adipogenic and osteogenic differentiation. Migration capacity was assessed by scratch assays in time-lapse imaging. In vitro specific binding of ligands was tested by fluorescence-activated cell sorting analysis and by radioligand assay using (18)F-fallypride. Imaging of D2R80A overexpressing hMSC transplanted into athymic rats was performed by PET using (18)F-fallypride. RESULTS: hMSCs showed long-term overexpression of D2R80A. As expected, the fluorescence signal suggested the primary localization of the protein in the membrane of the transduced cells. hMSC and D2R80A retained their stem cell character demonstrated by their osteogenic and adipogenic differentiation capacity and their proliferation and migration behavior. For in vitro hMSCs, at least 90% expressed the D2R80A transgene and hMSC-D2R80A showed specific binding of (18)F-fallypride. In vivo, a specific signal was detected at the transplantation site up to 7 d by PET. CONCLUSION: The mutant of the dopamine type 2 receptor (D2R80A) is a potent reporter to detect hMSCs by PET in vivo.

MicroRNA-23a mediates post-transcriptional regulation of CXCL12 in bone marrow stromal cells.

The chemokine CXCL12 regulates the interaction between hematopoietic stem and progenitor cells and bone marrow stromal cells. Although its relevance in the bone marrow niche is well recognized, the regulation of CXCL12 by microRNA is not completely understood. We transfected a library of 486 microRNA in the bone marrow stromal cell line SCP-1 and studied the expression of CXCL12. Twenty-seven microRNA were shown to downregulate expression of CXCL12. Eight microRNA (miR-23a, 130b, 135, 200b, 200c, 216, 222, and 602) interacted directly with the 3'UTR of CXCL12. Next, we determined that only miR-23a is predicted to bind to the 3'UTR and is strongly expressed in primary bone marrow stromal cells. Modulation of miR-23a changes the migratory potential of hematopoietic progenitor cells in co-culture experiments. We discovered that TGFB1 mediates its inhibitory effect on CXCL12 levels by upregulation of miR-23a. This process was partly reversed by miR-23a molecules. Finally, we determined an inverse expression of CXCL12 and miR-23a in stromal cells from patients with myelodys-plastic syndrome indicating that the interaction has a pathophysiological role. Here, we show for the first time that CXCL12-targeting miR23a regulates the functional properties of the hematopoietic niche.

Comparison of different strategies for in vivo seeding of prevascularized scaffolds.

Scaffolds seeded with multipotent precursor cells were hypothesized to heal critically sized bone defects. However, the success of this concept was limited by low cell survival after transplantation due to a lack of nutrients and oxygen. In vivo prevascularization of scaffolds before cell seeding may improve cell survival, yet the best seeding technique and time point of cell application remain elusive. Thus, the aim of this study was to compare different strategies. Demineralized bone matrix scaffolds were implanted around the saphenous arteriovenous (AV) bundle in nude mice. In vivo seeding was performed 0, 5, or 21 days after implantation using enhanced green fluorescent protein (eGFP)-expressing mesenchymal stem cells (MSCs). Cells were applied either by injection or the repetitive dripping technique. In vitro seeded and subcutaneously implanted scaffolds served as controls. Fourteen days after cell application, the fluorescence intensity of transplanted cells and the extent of newly formed vessels were quantified. We found that the AV flow through model as well as cell application increased vessel formation. In vitro seeding resulted in significantly higher cell numbers than in vivo seeding. With increasing time of prevascularization, the number of cells declined dramatically. In vivo seeding by cell injection was superior to the repetitive dripping protocol. On subcutaneously implanted scaffolds, significantly, more cells were found than on axially perfused scaffolds. We conclude that in vitro seeding is more efficient compared to the two novel in vivo seeding techniques of prevascularized scaffolds. With increasing time of prevascularization, the seeding efficiency for the in vivo methods further decreases, presumably due to the ingrowth of connective tissue. Even though, the presence of MSCs and the longer period of prevascularization enhances vessel formation, this conceivable advantage is limited supposedly by the inferior seeding efficiency.

MiR-134-mediated ß1 integrin expression and function in mesenchymal stem cells.

The composition of the hematopoietic stem cell (HSC) niche within the bone marrow is highly dynamic, tightly regulated, and of importance for various HSC properties. Integrins are important molecules within this niche that influence those properties through the interactions of HSCs and mesenchymal stem cells (MSCs). Here we investigated the function of miR-134 in integrin regulation in MSCs. In MSCs, miR-134 post-transcriptionally regulated ß1 integrin expression. This negative regulation of ß1 integrin was mediated by the binding of miR-134 to its 3' untranslated region, which contains two conserved binding sites for miR-134. The miR-134-mediated silencing of ß1 integrin in MSCs was shown by atomic force microscopy to decrease the adhesion of 32D cells to MSCs transfected with miR-134. Furthermore, the adhesion of MSCs to fibronectin was reduced after transfection with miR-134. MSCs from patients with myelodysplastic syndrome (MDS) revealed highly significant miR-134 overexpression compared with MSCs from healthy bone marrow donors. MSCs from MDS patients showed lower ß1 integrin protein, but not lower mRNA, expression, suggesting post-transcriptional regulation. The present study demonstrates miR-134-mediated negative regulation of ß1 integrin that influences cell adhesion to and of MSCs. These results further contribute to our understanding of the complexity of MDS.