There is evidence that mesenchymal stem cells (MSCs) can differentiate towards an intervertebral disc (IVD)-like phenotype. We compared the standard chondrogenic protocol using transforming growth factor beta-1 (TGFß) to the effects of hypoxia, growth and differentiation factor-5 (GDF5), and coculture with bovine nucleus pulposus cells (bNPC). The effi cacy of molecules recently discovered as possible nucleus pulposus (NP) markers to differentiate between chondrogenic and IVD-like differentiation was evaluated. MSCs were isolated from human bone marrow and encapsulated in alginate beads. Beads were cultured in DMEM (control) supplemented with TGFß or GDF5 or under indirect coculture with bNPC. All groups were incubated at low (2 %) or normal (20 %) oxygen tension for 28 days. Hypoxia increased aggrecan and collagen II gene expression in all groups. The hypoxic GDF5 and TGFß groups demonstrated most increased aggrecan and collagen II mRNA levels and glycosaminoglycan accumulation. Collagen I and X were most up-regulated in the TGFß groups. From the NP markers, cytokeratin-19 was expressed to highest extent in the hypoxic GDF5 groups; lowest expression was observed in the TGFß group. Levels of forkhead box F1 were down-regulated by TGFß and up-regulated by coculture with bNPC. Carbonic anhydrase 12 was also down-regulated in the TGFß group and showed highest expression in the GDF5 group cocultured with bNPC under hypoxia. Trends in gene expression regulation were confi rmed on the protein level using immunohistochemistry. We conclude that hypoxia and GDF5 may be suitable for directing MSCs towards the IVD-like phenotype.

The results suggest that immediately after cement injection, there was a reflex fall in heart rate and arterial pressure. The second fall in arterial pressure was a consequence of fat emboli passing through the heart and getting trapped in the lungs.

No abstract

Human bone marrow-derived mesenchymal stem cells (MSCs) have limited growth potential in vitro and cease to divide due to replicative senescence, which from a tissue-engineering perspective has practical implications, such as defining the correct starting points for differentiation and transplantation. Time spent in culture before the loss of required differentiation potential is different and reflects patient variability, which is a problem for cell expansion. This study aimed to develop a score set which can be used to quantify the senescent state of MSCs and predict whether cells preserve their ability to differentiate to osteogenic, adipogenic and chondrogenic phenotypes, based on colony-forming unit (CFU) assay, population doubling time (PDT), senescence-associated β-galactosidase (SA-β-Gal) activity, cell size, telomere length and gene expression of MSCs cultured in vitro over 11 passages. This set of morphological, physiological and genetic senescence markers was correlated to the ability of MSCs to differentiate. Differentiation efficiency was assessed by marker genes and protein expression. CFUs decreased with increasing passage number, whereas SA-β-Gal activity and PDT increased; however, the correlation with MSCs' differentiation potential was sometimes unexpected. The expression of genes related to senescence was higher in late-passage cells than in early-passage cells. Early-passage cells underwent efficient osteogenic differentiation, with mid-passage cells performing best in chondrogenic differentiation. Late-passage cells preserve only adipogenic differentiation potential. Based on this marker set, we propose a senescence score in which combined markers give a reliable quality control of MSCs, not depending only on mechanistic passage number.

Research efforts aim at enhancing early osseointegration of cementless implants to improve early fixation and, thus, reduce the risk of loosening. The aim of the present study was to investigate whether bone morphogenetic protein (BMP) 2 had a positive effect on the osseointegration of hydroxyapatite-coated implants. Hydroxyapatite (HA) implants (perforated hollow cylinders and solid rods) were coated with BMP-2 and hyaluronic acid (HY) as the carrier or with HY alone. Uncoated HA implants served as controls. The osseointegration of the implants was evaluated either by light microscopy or by pullout tests after 1, 2, and 4 weeks of unloaded implantation in the cancellous bone of 24 sheep. The BMP-2 coating significantly increased bone growth into the implant perforations compared with HA-coated implants at 2 and 4 weeks. Bone-implant contact and interface shear strength of BMP-2 implants were lower than HA implants at 2 weeks. At 4 weeks, there was no significant difference in bone-implant contact and shear strength between BMP-2 and HA-coated implants. The BMP-2 coating enhanced gap healing but had no positive or even an inhibitory effect (at 2 weeks) on bone-implant contact and interface shear strength. In the clinical situation, a perfect press-fit implantation cannot be achieved, and BMP-2 may be beneficial for enhancing bone growth into gaps around cementless implants.

No abstract

Introduction Cell-based therapies for regeneration of the degenerated intervertebral disc (IVD) are an alternative to current surgical intervention. Mesenchymal stem cells (MSCs), in combination with a scaffold, might be ideal candidates for regenerating nucleus pulposus (NP), the pressure-distributing part of the IVD. While the use of growth factors for MSCs differentiation currently receives major attention, in this study we compare the performance of sponge-like matrixes in supporting cell differentiation into NP-like cells. Materials and methods Four types matrixes approved as medical devices for other applications were tested as scaffolds for MSCs: two made of equine or porcine collagen, one of gelatin and one of chitosan. Bone marrowderived human MSCs were seeded in these scaffolds or embedded in alginate, as a three-dimensional control. After five weeks in culture, NP-like differentiation of the cellscaffold constructs was analyzed by qRT-PCR, histology, total DNA quantification, proteoglycan accumulation and immunohistochemistry. Results MSCs in collagen matrixes and gelatin produced more mRNA and proteins of the chondrogenic markers collagen type I, collagen type II (COL2) and aggrecan (ACAN), when compared with cells embedded in alginate or chitosan. Proteoglycan accumulation and cell survival were also higher in collagen and gelatin matrixes. Gene expression results were also confirmed by histological and immunohistochemical staining. In contrast to alginate control, the gene expression of the undesired bone marker osteopontin was lower in all tested groups. In porcine collagen supports, MSC expression ratio between COL2/ ACAN closely resembled the expression of nucleus pulposus cells, but gene expression of recently described NP markers keratin19, PAX1 and FOXF1 was lower. Conclusions Collagen supports provide a readily available, medically approved and effective scaffold for chondrogenic differentiation in vitro, but the phenotype of differentiated MSCs is not yet completely equivalent to that of NP cells.

Concerns have been raised that the use of calcium phosphate (CaP) cements for the augmentation of fractured, osteoporotic bones may aggravate cardiovascular deterioration in the event of pulmonary cement embolism by stimulating coagulation. The aim of the present study was therefore to investigate the cardiovascular changes after pulmonary embolism of CaP cement using an animal model. In 14 sheep, 2.0 mL CaP or polymethylmethacrylate cement were injected intravenously. Cardiovascular parameters and antithrombin levels were monitored until 60 min postinjection. Postmortem, lungs were subjected to CT scanning, and 3D reconstruction of the cement was performed. Intravenous injection of CaP cement resulted in a more severe increase in pulmonary arterial pressure and decrease in arterial blood pressure. Disintegration of the CaP cement seemed to be the reason for the more severe reaction. There was no evidence of thromboembolism. Disintegration of CaP cement in circulating blood does not only compromise the mechanical properties, but also represents a risk of cardiovascular complications. Reliable cohesion of CaP cements in an aqueous environment is essential for clinical applications such as osteoporotic bone augmentation.