Do human osteoblasts grow into open-porous titanium?

A titanium foam for spine fusion and other applications was tested by cell culture. Its high porosity and surface roughness should enable bone cells to grow through it, resulting in a better fixation of the vertebral body.The foam was tested by in vitro experiments with human osteoblasts under static culture conditions and in a perfused system. By means of cell number, viability, scanning electron microscopy and histological staining, cell proliferation could be observed. The expression of osteogenic genes like collagen-I, alkaline phosphatase and osteocalcin was proven by reverse transcription polymerase chain reaction (RT-PCR) as well as in the case of alkaline phosphatase with biochemical methods.The conducted experiments showed that human osteoblasts could grow through the interconnected porosity of the metal foam and that they expressed an osteoblast like phenotype. The results suggest that in vivo osteoblasts are likely to form a trabecular bone bridge through this titanium foam. Consequently, with this osteoconductive material, there may be a reduced need for autologous bone in spinal fusion procedures

Cell-seeded polyurethane-fibrin structures--a possible system for intervertebral disc regeneration

Nowadays, intervertebral disc (IVD) degeneration is one of the principal causes of low back pain involving high expense within the health care system. The long-term goal is the development of a medical treatment modality focused on a more biological regeneration of the inner nucleus pulposus (NP). Hence, interest in the endoscopic implantation of an injectable material took center stage in the recent past. We report on the development of a novel polyurethane (PU) scaffold as a mechanically stable carrier system for the reimplantation of expanded autologous IVD-derived cells (disc cells) to stimulate regenerative processes and restore the chondrocyte-like tissue within the NP. Primary human disc cells were seeded into newly developed PU spheroids which were subsequently encapsulated in fibrin hydrogel. The study aims to analyze adhesion properties, proliferation capacity and phenotypic characterization of these cells. Polymerase chain reaction was carried out to detect the expression of genes specifically expressed by native IVD cells. Biochemical analyses showed an increased DNA content, and a progressive enhancement of total collagen and glycosaminoglycans (GAG) was observed during cell culture. The results suggest the synthesis of an appropriate extracellular matrix as well as a stable mRNA expression of chondrogenic and/or NP specific markers. In conclusion, the data presented indicate an alternative medical approach to current treatment options of degenerated IVD tissue

In vitro evaluation of a thermoreversible hyaluronan-based hydrogel loaded with cell-seeded polyurethane particles for nucleus pulposus repair

Introduction Thermoreversible hyaluronan (HA) based hydrogels represent a promising carrier for IVD repair because of the mild gel formation allowing for encapsulation of viable cells and biological stimulation of cells by HA.1,2 Additionally, polyurethane (PU) foams can provide elasticity, which is of key importance for the restoration of IVD mechanical function, as the latter experiences repeated loading during the daily human activities. Finally, the addition of relevant cells may promote a faster restoration of the IVD function. Therefore, the aim of this study was to compare the single and combined effect of PU particles and HA hydrogel on the proliferation and differentiation of human IVD and mesenchymal stem cells. Materials and Methods Human bone marrow stromal cells (hMSCs) or human disk cells (hIVD cells) (P2-P4) were seeded on PU particles; after 12 hours, the seeded particles were suspended in either a 12% w/v thermoreversible hyaluronan-poly(N-isopropylacrylamide) hydrogel (HA-pNIPAM)2 or in a fibrinogen/thrombin solution.3 Each sample contained 50 µL hydrogel, 2.5 mg PU particles, and 2.5 × 105 cells. Controls included hMSC (or hIVD cells) seeded HA-pNIPAM, hMSC (or hIVD cells) seeded fibrin, and hMSC (or hIVD cells) seeded PU. After gelling for 1 hour at 37°C, samples were cultured in chondrogenic medium (DMEM high glucose, 1% ITS, 130 µM ascorbic acid, 100 nM dexamethasone and 10 ng/mL TGF-β1) for one week and analyzed for DNA (by Picogreen), sulfated glycosaminoglycan (GAG) (by 1,9 dimethylmethylene blue), mRNA expression, and histology (toluidine blue) at day 3 and 7. Three independent experiments were performed in triplicates for each group and cell type. After confirming the normality of data distribution and exclusion of the outliers, data were analyzed with a univariate general linear model and post hoc Bonferroni test ( p &lt; 0.05). Results In the MSC cultures, a slight DNA increase was observed between day 3 and 7 for both PU/HA-pNIPAM and PU/fibrin samples. At day 7, the highest amounts of DNA were found for the PU/fibrin group, followed by fibrin and PU groups ( Fig. 1a ). The amount of DNA was higher in the PU group than PU/HA-pNIPAM and HA-pNIPAM, suggesting a lower cell proliferation in the presence of the thermoreversible hydrogel. A similar DNA profile was obtained when hIVD cells were used instead of hMSCs. A significantly increased amount of GAG was observed in hMSC-seeded PU/HA-pNIPAM samples (Fig.1B) and there was an increase of GAG from day 3 to day 7. All other groups had a much lower amount of glycosaminoglycans and there was only a slight increase of GAG from day 3 to day 7. The GAG retention (GAG in sample divided by the sum of GAG in the sample and GAG released in the medium) at day 7 was comprised between 50% and 80% for all groups. Disk cells produced significantly less GAG in the PU/HA-pNIPAM system compared to hMSC, indicating that MSCs were more responsive to the combination of these two biomaterials ( Fig. 1B ). Moreover, the GAG retention at day 7 was lower with human IVD cells (20–60%) compared to hMSCs in all groups. This suggests that an early and higher GAG accumulation is associated to a better GAG retention in the hydrogel. This finding may be related to the fact that while MSCs were obtained from patients undergoing various types of surgeries, IVD cells were obtained from (degenerate) disks of patients undergoing total IVD replacement. The gene expression profile confirmed MSC chondrogenic differentiation with a strong upregulation of collagen type II and, to a lower extent, of aggrecan and SOX9 (compared to cells collected on the day of seeding) without significant differences between the two gels. Histological sections revealed cell migration from PU particles to the hydrogels. [Figure: see text] Conclusion Human disk cells from degenerating disks synthesized less GAG than hMSCs, indicating that MSCs may represent a suitable cell type for the biological repair of IVDs. A synergic effect for MSC differentiation was observed when a thermoreversible hyaluronan-based hydrogel was combined with polyurethane particles. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Mortisen D, et al. Biomacromolecules 2010;11:1261–1272 Peroglio M, et al. European Spine Journal Epub ahead of print Mauth C, et al. European Cells and Material 2009;18:27–38 </jats:sec