Stimulation of intervertebral disc cells in alginate bead culture with bone morphogenetic protein 2 and/or L51P

INTRODUCTION: In clinics, Bone Morphogenetic Protein 2 (BMP2) was applied for the support during spinal fusion. Further BMP2 was tested in IVD models and showed potential for IVD regeneration. The aim of this study is the investigation of BMP2 and the BMP2 analogue, L51P, on different cell types of the human IVD in 3D alginate beads, particularly their plasticity to undergo bone formation. METHODS: Human nucleus pulposus (NPC), annulus fibrosus (AFC) and cartilaginous endplate cells (CEPC) were each encapsulated in 1.2% alginate at a density of 4 Mio/mL. NPC, AFC, and CEPC beads were then cultured in α-MEM or osteogenic medium (OM) supplemented with 10% FBS and 100 ng/mL BMP2 and/or L51P for 21 days. Medium supplemented with cytokines was refreshed twice per week. Beads were snap frozen with liquid nitrogen after 7 days for mRNA analysis of Aggrecan (ACAN), Collagen type1 (COL1), Collagen type 2 (COL2) and runt-related transcription factor 2 (RUNX2) qPCR. Further, beads were stained with Alcian Blue after 21 days. RESULTS & DISCUSSION: ACAN expression was the highest up-regulated in IVD cells stimulated with OM and 100 ng/mL BMP2 and L51P compared to the negative control (basal medium only) in NPC, AFC and CEPC (mean ± SEM NP: 18.95 ± 15.65). The same was true for COL2 expression (NP: 72.47 ± 62.95). COL1 remained unaffected (N=2). CONCLUSIONS: In this study, we showed the trend of an increase in ACAN and COL2 gene expression in stimulated cells. Interestingly the co-treatment of BMP2 and L51P showed a cumulative effect towards an increased ECM production

A photopolymerized composite hydrogel and surgical implanting tool for a nucleus pulposus replacement.

Nucleus pulposus replacements have been subjected to highly controversial discussions over the last 40 years. Their use has not yet resulted in a positive outcome to treat herniated disc or degenerated disc disease. The main reason is that not a single implant or tissue replacement was able to withstand the loads within an intervertebral disc. Here, we report on the development of a photo-polymerizable poly(ethylene glycol)dimethacrylate nano-fibrillated cellulose composite hydrogel which was tuned according to native tissue properties. Using a customized minimally-invasive medical device to inject and photopolymerize the hydrogel insitu, samples were implanted through an incision of 1 mm into an intervertebral disc of a bovine organ model to evaluate their long-term performance. When implanted into the bovine disc model, the composite hydrogel implant was able to significantly re-establish disc height after surgery (p < 0.0025). The height was maintained after 0.5 million loading cycles (p < 0.025). The mechanical resistance of the novel composite hydrogel material combined with the minimally invasive implantation procedure into a bovine disc resulted in a promising functional orthopedic implant for the replacement of the nucleus pulposus

FACS reveals more Pluripotent Intervertebral Disc Progenitor Cells compared to MACS and pluriSelect

INTRODUCTION: Nucleus Pulposus Progenitor Cells (NPPCs), positive for the angiopoietin-1 receptor (Tie2), were demonstrated in human, mouse, canine and bovine NP tissue [1,2,3]. Tie2+ NPPCs possess a multi-lineage differentiation potential, and regeneration potential is attributed to them. However, the isolation of Tie2+ NPPCs can be cumbersome. Hence, three isolation methods were compared. METHODS: Bovine NP cells were isolated from 10-14-month-old animals. Cell sorting was performed with an antibody against Tie2 (bs-1300R, Bioss) using FACS, magnetic-activated cell sorting (MACS) and pluriSelect, a size-based sorting method. Outcomes were evaluated by cell yield of Tie2+ cells, the ability of sorted cells to form colonies and tri-lineage differentiation assays. RESULTS & DISCUSSION: FACS resulted in the highest Tie2+ cell yield (5.0 ± 4.0%) followed by MACS (1.6 ± 2.9%) and pluriSelect (1.1 ± 1.4%). Colony forming ability did not differ between Tie2+ and Tie2- cells for any isolation method. However, Tie2+ cells obtained by MACS tended to have more colonies than FACS and pluriSelect. Osteogenic and adipogenic differentiation of Tie2+ and Tie2- cells did not result in a clear distinction for MACS and pluriSelect; Tie2+ FACS-sorted cells demonstrated superior osteogenic and adipogenic differentiation over Tie2- cells. Also for chondrogenesis, the Tie2+ FACS-sorted Tie2+ NPPCs tended to produce more proteoglycan versus Tie2- NPPCs, whereas for MACS and pluriSelect no difference was found. CONCLUSIONS: Based on the parameters tested, isolation of NPPC is possible with all three methods. However, cell yields differed widely. FACS although most invasive, appears to be the most specific sorting method for these Tie2 + cells among the tested methods as Tie2 + cells do not demonstrate osteogenic and adipogenic differentiation. As for cell yield MACS seems to reveal the most, possibly this is due to inclusion of cells expressing Tie2 less strongly

Effect of Heat Treatment on the Electrochemical and Mechanical Behavior of the Ti6Al4V Alloy

The effects of heat treatment on the hardness and electrochemical behavior of the Ti6Al4V alloy were studied. Two heat treatments were performed: one below (800 °C) and the other above (1050 °C) its beta transformation temperature (T= 980 ± 20 °C) and cooled using three conditions: water quenching, normalizing, and furnace. A microstructure observed using Optical Microscopy showed dependence on the heat treatment temperature applied; mainly three microstructures were obtained: martensitic, globular, and lamellar. Besides, alpha and beta phases were characterized by X-ray diffraction (XRD) technique. The Berkovich tests were performed to measure the hardness and reduced modulus (E). The Ti6Al4V alloy treated at 1050 °C and air-cooled exhibited hardness values closer to those of the Ti6Al4V as-received alloy. Electrochemical tests were carried out to analyze the electrochemical behavior after 7-day immersion in Hank’s solution at 37 °C and pH 7.40. Open Circuit Potentials (E) showed less negative values for Ti6Al4V and Ti6Al4V alloys, suggesting ennoblement of these materials. Furthermore, these alloys exhibited an outstanding electrochemical behavior compared to the Ti6Al4V as-received alloy by Electrochemical Impedance Spectroscopy (EIS) technique.The authors thank David Pérez-Risco for sharing his experience and useful advice on Berkovich Nano-indenter at the National Metallurgical Research Center (CENIM-CSIC Madrid, Spain), allowing this work to be carried out. Mercedes Paulina Chávez-Díaz thanks the National Science and Technology Council (CONACyT) for a doctoral fellowship to hold a research stay at CENIM-CSIC. This work was supported by the Government of Spain, through the Ministry of Economy and Competitiveness [MAT2015-67750-C3-1]. Elsa Miriam Arce-Estrada and Román Cabrera-Sierra wish to thank the National Research System (SNI) for the distinction of its members and the stipend received