Double printing of hyaluronic acid/poly(glycidol) hybrid hydrogels with poly(ε-caprolactone) for MSC chondrogenesis

This study investigates the use of allyl-functionalized poly(glycidol)s (P(AGE-co-G)) as a cytocompatible cross-linker for thiol-functionalized hyaluronic acid (HA-SH) and the optimization of this hybrid hydrogel as bioink for 3D bioprinting. The chemical cross-linking of gels with 10 wt.% overall polymer concentration was achieved by a UV-induced radical thiol-ene coupling between the thiol and allyl groups. The addition of unmodified high molecular weight HA (1.36 MDa) enabled the rheology to be tuned for extrusion-based bioprinting. The incorporation of additional HA resulted in hydrogels with a lower Young's modulus and a higher swelling ratio, especially in the first 24 h, but a comparable equilibrium swelling for all gels after 24 h. Embedding of human and equine mesenchymal stem cells (MSCs) in the gels and subsequent in vitro culture showed promising chondrogenic differentiation after 21 d for cells from both origins. Moreover, cells could be printed with these gels, and embedded hMSCs showed good cell survival for at least 21 d in culture. To achieve mechanically stable and robust constructs for the envisioned application in articular cartilage, the formulations were adjusted for double printing with thermoplastic poly(ε-caprolactone) (PCL)

Double printing of hyaluronic acid / poly(glycidol) hybrid hydrogels with poly(ε-caprolactone) for MSC chondrogenesis

This study investigates the use of allyl-functionalized poly(glycidol)s (P(AGE-co-G)) as cytocompatible cross-linker for thiol-functionalized hyaluronic acid (HA-SH) and the optimization of this hybrid hydrogel as bioink for 3D bioprinting. Chemical cross-linking of gels with 10 wt.% overall polymer concentration was achieved by UV-induced radical thiol-ene coupling between the thiol and allyl groups. Addition of unmodified high molecular weight HA (1.36 MDa) allowed tuning of the rheology for extrusion based bioprinting. Incorporation of additional HA resulted in hydrogels with lower Young's modulus and higher swelling ratio especially in the first 24 h, but a comparable equilibrium swelling for all gels after 24 h. Embedding of human and equine mesenchymal stem cells (MSCs) in the gels and subsequent in vitro culture showed promising chondrogenic differentiation after 21 d for cells from both origins. Moreover, cells could be printed with these gels, and embedded hMSCs showed good cell survival for at least 21 d in culture. To achieve mechanical stable and robust constructs for the envisioned application in articular cartilage, the formulations were adjusted for double printing with the thermoplastic poly--caprolactone (PCL)

Regulation of neuronal differentiation by proteins associated with nuclear bodies.

Nuclear bodies are large sub-nuclear structures composed of RNA and protein molecules. The Survival of Motor Neuron (SMN) protein localizes to Cajal bodies (CBs) and nuclear gems. Diminished cellular concentration of SMN is associated with the neurodegenerative disease Spinal Muscular Atrophy (SMA). How nuclear body architecture and its structural components influence neuronal differentiation remains elusive. In this study, we analyzed the effects of SMN and two of its interaction partners in cellular models of neuronal differentiation. The nuclear 23 kDa isoform of Fibroblast Growth Factor - 2 (FGF-2(23)) is one of these interacting proteins - and was previously observed to influence nuclear bodies by destabilizing nuclear gems and mobilizing SMN from Cajal bodies (CBs). Here we demonstrate that FGF-2(23) blocks SMN-promoted neurite outgrowth, and also show that SMN disrupts FGF-2(23)-dependent transcription. Our results indicate that FGF-2(23) and SMN form an inactive complex that interferes with neuronal differentiation by mutually antagonizing nuclear functions. Coilin is another nuclear SMN binding partner and a marker protein for Cajal bodies (CBs). In addition, coilin is essential for CB function in maturation of small nuclear ribonucleoprotein particles (snRNPs). The role of coilin outside of Cajal bodies and its putative impacts in tissue differentiation are poorly defined. The present study shows that protein levels of nucleoplasmic coilin outside of CBs decrease during neuronal differentiation. Overexpression of coilin has an inhibitory effect on neurite outgrowth. Furthermore, we find that nucleoplasmic coilin inhibits neurite outgrowth independent of SMN binding revealing a new function for coilin in neuronal differentiation

SMN inhibits FGF-2²³ activation of Nurr1-dependent transcription.

<p>In this reporter-gene assay, the effects of FGF-2²³, Nurr1 and SMN on transcription driven from the Nurr1 monomer binding responsive element (NBRE) were assessed by transfection of neuroblastoma cells NB. For this purpose, expression vectors for each protein and the NBRE-Luc reporter were used. Empty vector pcDNA3.1 was employed as a FGF-2 expression negative control and pβ-gal as a negative control for SMN constructs. Full-length-SMN (SMN1-294) inhibits transcriptional activation mediated by FGF-2²³, whereas coexpression of the SMN mutant protein SMN235-294 (without the N-terminal FGF-2²³-binding sequence and comprising amino acid residues 235-294) did not exhibit an inhibitory effect. Data represent the mean ± SEM of the ratio of firefly to <i>Renilla</i> luciferase activity (Fluc/Rluc) for n = 3 experiments, each performed in quadruplicate. Results were analyzed using one-way ANOVA followed by Tukeýs posthoc test (means ± SEM; ***, p<0.001; *, p<0.05; compared with pcDNA3.1/pNurr1.</p

Regulation of the SMN-interacting protein coilin.

<p>Neuroblastoma SK-N-BE(2) cells were differentiated for different periods with retinoic acid (5 µM), lysed by sonification in modified RIPA-buffer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082871#pone.0082871-Forthmann1" target="_blank">[7]</a> and analyzed by Western blot for coilin, SMN and α-Tubulin (A). (B) Endogenous coilin protein levels were decreased significantly to 52% after 72h of differentiation, (C) while SMN protein levels were increased non-significantly after the same time of differentiation. (D) Cells were treated with RA and stained with anti-SMN and anti-coilin antibodies. Coilin-positive (SMN-negative Cajal bodies), SMN-positive (nuclear gems) as well as coilin- and SMN-positive dots (SMN-positive CBs) in the nucleus of differentiated NB cells were counted and compared with a non-differentiated control. No significant changes of absolute numbers of these nuclear bodies were detected. (E) Intensity correlation analyses of coilin and SMN in the nucleus of differentiated cells were performed and compared to a non-differentiated control. No change in colocalization of coilin and SMN was detected (B, C: n = 7, means ± SEM; *, p<0.05; **, p<0.01; unpaired, two tailed t-test; D: control, n = 18; 24h RA, n = 13; 72h RA, n = 20; E: control, n = 20; 24h RA, n = 15; 72h RA, n = 22).</p

Expression of coilin decreases neurite outgrowth.

<p>(A) PC12 cells were transfected with pEGFP or pCoilin-EGFP. After incubation in differentiation medium with nerve growth factor (NGF) for three days, neurite lengths were measured and their relative changes analyzed. (B) N-terminal EGFP-tagged wild-type and mutant coilin-constructs decreased the length of neurites significantly in the same experiment. For statistical evaluation, averaged relative neurite lengths of n>100 cells were compared, pooled from three individual experiments (means ± SEM; n.s.; A: **, p<0.01; Mann-Whitney test; B: n.s., non-significant; *, p<0.05; **, p<0.01; ***, p<0.001; Dunn’s test for comparison of multiple groups).</p

Interaction of SMN with FGF-2²³ negatively regulates neurite outgrowth.

<p>PC12 cells were transfected with pSMN-EGFP or pEGFP control vector, respectively. Cells were additionally transfected with pFGF-2¹⁸-DsRed2 (SMN non-interacting control), pFGF-2²³-DsRed2 (SMN-interacting) or pDsRed2 control (grey, transfections with control plasmids; blue, FGF-2¹⁸ and, red, FGF-2²³ transfections). After incubation with nerve growth factor (NGF) in differentiation medium for three days, neurite lengths were measured and the relative changes of lengths analyzed. For statistical evaluations, average relative neurite lengths of n>100 cells were compared. Levels of significance on top of each bar represent comparison to pEGFP/pDsRed2 controls; means ± SEM; n.s., non-significant; *, p<0.05; **, p<0.01; Dunn’s test for comparison of multiple groups.</p