Synthesis and <i>in vitro</i> evaluation of thermosensitive hydrogel scaffolds based on (PNIPAAm-PCL-PEG-PCL-PNIPAAm)/Gelatin and (PCL-PEG-PCL)/Gelatin for use in cartilage tissue engineering

<p><b>Background</b>: Biodegradable thermosensitive hydrogel scaffolds based on novel three-block PCL-PEG-PCL and penta block PNIPAAm-PCL-PEG-PCL-PNIPAAm copolymers blended with gelatin were prepared and examined on functional behavior of chondrocytes. <b>Methods</b>: In this work, we compared two different thermosensitive hydrogel scaffolds (PNIPAAm-PCL-PEG-PCL-PNIPAAm)/Gelatin and (PCL-PEG-PCL)/Gelatin prepared by TIPS (thermally induced phase separation) method. The feature of copolymers was characterized by FT-IR, ¹H NMR. The lower critical solution temperatures (LCSTs) of aqueous solutions of copolymers were measured by cloud point (turbidity) measurements. We also examined water absorption capacity and swelling ratio. Mechanical features of the prepared hydrogels were evaluated by stress-strain measurements. Thereafter, isolated chondrocytes were cultured on each scaffold for a period of 10 days and cell arrangement and morphology studied pre-and post-plating. Cell survival assay was done by using MTT assay. The transcription level of genes Sox-9, Collagen-II, COMP, MMP-13 and oligomeric matrix protein was monitored by real-time PCR assay. The samples were also stained by Toluidine blue method to monitor the synthesis of proteoglycan. <b>Results</b>: Data demonstrated an increased survival rate in cells coated seeded on scaffolds, especially (PNIPAAm-PCL-PEG-PCL-PNIPAAm)/Gelatin as compared to control cells on the plastic surface. (PNIPAAm-PCL-PEG-PCL-PNIPAAm)/Gelatin had potential to increase the expression of genes Sox-6, Collagen-II, COMP and after 10 days <i>in vitro</i>. <b>Conclusion</b>: Thermosensitive PCEC/Gel and (PNIPAAm-PCEC-PNIPAAm)/Gel hydrogel scaffolds that fabricated by TIPS method possesses useful hydrophilic properties for growth and cell embedding and secretion of extracellular matrix. It can serve as an ideal strategy to promote the formation of cartilage tissue.</p

A–B: Effects of Src-, PI-3K-, AKT-inhibitors and IGF-1 or/and PDGF-bb on IL-1β-stimulated phosphorylation of NF-κB in chondrocytes.

<p>Primary chondrocytes were either stimulated with 10 ng/ml IL-1β, pre-stimulated with PP1 (10 µM), wortmannin (20 nM) and SH-5 (10 µM) for 1 h (A), or with 10 ng/ml IGF-1, 10 ng/ml PDGF-bb or a combination of both growth factors (5 ng/ml each) for 12 h (B) and then incubated with IL-1β for 30 min. Nuclear extracts were subjected to 10% SDS-PAGE (500 ng protein per lane), transferred to nitrocellulose membranes and then probed using an antiserum reactive with an anti-phospho-p65 or anti-PARP polyclonal antibody (housekeeping control). Similar results were obtained in three independent experiments.</p

A–C: Effects of IGF-1 or/and PDGF-bb on the IL-1β-induced p65 acetylation in chondrocytes in monolayer cultures.

<p>Primary chondrocytes were either stimulated with 10 ng/ml IL-1β, pre-stimulated with 10 ng/ml IGF-1, 10 ng/ml PDGF-bb or a combination of both growth factors (5 ng/ml each) for 12 h and treated with 10 ng/ml IL-1β for the indicated times. Whole-cell extracts were prepared and immunoprecipitated with an anti-p65 antibody. Western blot analysis was then performed with an anti-acetyl-lysine antibody or with an anti-p65 antibody. The results shown are representative of three independent experiments.</p

Effects of IGF-1 or/and PDGF-bb on IL-1β-induced NF-κB-dependent pro-inflammatory, pro-apoptotic and matrix degrading gene products in chondrocytes.

<p>To determine whether <i>IGF-1</i> or/and <i>PDGF-bb</i>exert effects on IL-1β-induced NF-κB-dependent expression of pro-inflammatory, pro-apoptotic and matrix degrading gene products, primary chondrocytes were either stimulated with 10 ng/ml IL-1β, 10 ng/ml PDGF-bb, 10 ng/ml IGF-1 or combination of both growth factors (5 ng/ml each) or pre-stimulated for 12 h with 10 ng/ml PDGF-bb, 10 ng/ml IGF-1 or combination of both growth factors (5 ng/ml each) followed by 10 ng/ml IL-1β for 24. Equal amounts of total proteins were separated by SDS-PAGE and analyzed by immunoblotting using antibodies raised against COX-2, MMP-9 and MMP-13 and active caspase-3. Stimulation with IL-1β resulted in production of COX-2, MMP-9, MMP-13 and caspase-3 cleavage. Pre-treatment with a combination of both IGF-1 or/and PDGF-bb downregulated COX-2, MMP-9, MMP-13 and cleaved caspase-3.</p

A–D: Effects of IGF-1 or/and PDGF-bb or IKK-inhibitor (BMS) on the IL-1β-induced IKK activation in chondrocytes in monolayer cultures.

<p>Primary chondrocytes were either stimulated with 10 ng/ml IL-1β, pre-stimulated with 10 ng/ml IGF-1, 10 ng/ml PDGF-bb, a combination of both growth factors (5 ng/ml each) or 5 µM BMS-345541 (BMS) for 12 h and treated with 10 ng/ml IL-1β for the indicated times. Whole-cell extracts were immunoprecipitated with an antibody against IκB kinase (IKK)and then analyzed by an immune complex kinase assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028663#s2" target="_blank">Materials and Methods</a>. To examine the effect of <i>IGF-1 or/and PDGF-bb or</i> BMS on the level of activation of IKK proteins, whole-cell extracts were fractionated (500 ng protein per lane) on SDS–PAGE and examined by western blot analysis using anti-IKK-α and anti-IKK-β antibodies. The results shown are representative of three independent experiments.</p

Effects of IGF-1 or/and PDGF-bb on IL-1β-induced inhibition of signaling proteins expression in chondrocytes.

<p>To evaluate the effects of IGF-1 and/or PDGF-bb on IL-1β-induced inhibition of MAPK signaling proteins in chondrocytes, whole cell lysates were probed with antibodies to Shc, Erk1/2 and SOX-9. Primary chondrocytes were either stimulated with 10 ng/ml IL-1β, 10 ng/ml PDGF-bb, 10 ng/ml IGF-1 or combination of both growth factor (5 ng/ml each) or pre-treated for 12 h with 10 ng/ml PDGF-bb, 10 ng/ml IGF-1 or combination of both (5 ng/ml each) followed by 10 ng/ml IL-1β for 24. Untreated cultures showed high expression of Shc, Erk1/2, and SOX-9, while IL-1β alone resulted in inhibition of Erk1/2, Shc as well as SOX-9 production. However, pre-treatment of cultures with IGF-1 or/and PDGF-bb inhibited the adverse effects of IL-1β and chondrocytes produced large amounts of Shc, Erk1/2 and SOX-9 at levels similar to control cultures. The results were confirmed by quantitative densitometry.</p

A–C: Effects of IGF-1 or/and PDGF-bb on IL-1β-induced p65 phosphorylation and nuclear translocation in chondrocytes in monolayer cultures.

<p>Primary chondrocytes were either stimulated with 10 ng/ml IL-1β, pre-stimulated with 10 ng/ml PDGF-bb, 10 ng/ml IGF-1 or combination of both growth factors (5 ng/ml each) for 12 h and treated with 10 ng/ml IL-1β for the indicated times. Nuclear extracts were prepared, fractionated (500 ng protein per lane) on 10% SDS–PAGE and electrotransferred onto nitrocellulose membranes. Western blot analysis was performed with anti-phospho-specific-p65, anti-p65 antibodies and anti-PARP (control). The results shown are representative of three independent experiments.</p

A: a–j: Effects of IGF-1 or/and PDGF-bb on IL-1β-induced apoptosis and cellular degeneration in chondrocytes.

<p>Transmission electron microscopy was performed to study the effects of IGF-1 or/and PDGF-bb on IL-1β-stimulated primary chondrocytes. Untreated control cultures consist of vital, active cells containing a well-developed rough endoplasmic reticulum, mitochondria and a well-organized cytoplasm after 24 or 48 h in culture (a and b). In contrast, stimulation with IL-1β for 24 h led to degenerative changes including condensation of heterochromatin, swelling of the rough endoplasmic reticulum and mitochondria (c). Longer exposure (48 h) to IL-1β resulted in more severe degenerative features including formation of apoptotic bodies and cell lysis (d). Pre-treatment of IL-1β-stimulated chondrocytes with IGF-1 or/and PDGF-bb for 24 or 48 h inhibited the degenerative effects of IL-1β (e–j). After 48 h (f, h and j), chondrocytes exhibited as large, viable and flattened cells with numerous tiny cytoplasmic processes, mitochondria, rough endoplasmic reticulum and other cytoplasmic organelles compared to control chondrocytes. <b>B: a–e: Redifferentiation of IL-1β-treated chondrocytes in high-density culture by IGF-1 or/and PDGF-bb.</b> Primary chondrocytes were either left untreated (a) or were treated with 10 ng/ml IL-1β (b), pre-treated with 10 ng/ml IGF-1 (c), 10 ng/ml PDGF-bb (d) or 5 ng/ml PDGF-bb and 5 ng/ml IGF-1 (e) for 12 h and then stimulated with IL-1β for another 24 h. The cells were transferred to high-density culture for seven days. Ultrastructural morphology was evaluated by electron microscopy. Control cultures showed characteristic features, including chondrocytes (c) embedded in a well-developed ECM (m) (<b>a</b>). Treatment with IL-1β for 24 h led to matrix breakdown and cell lysis (<b>b</b>). Pre-treatment with IGF-1 (<b>c</b>), PDGF-bb (<b>d</b>) or both growth factors in combination (<b>e</b>) resulted in a marked improvement of chondrocyte phenotype and the formation of cartilage nodules. The formation of a dense ECM (m) surrounding well-developed chondrocytes (c) was observed. ×5000; Bars: 1 µm.</p

A–D: Effects of PP1 and IGF-1 or/and PDGF-bb on IL-1β-induced c-Src phosphorylation in chondrocytes in monolayer cultures.

<p>Primary chondrocytes were either stimulated with 10 ng/ml IL-1β, pre-stimulated with 10 ng/ml IGF-1, 10 ng/ml PDGF-bb or a combination of both growth factors (5 ng/ml each) for 12 h, or with PP1 (10 µM) for 1 h and treated with 10 ng/ml IL-1β for the indicated times. The cell lysates were subjected to 10% SDS-PAGE (500 ng protein per lane), transferred to nitrocellulose membranes and then probed using anti-phospho-Src and anti-β-actin (as an indicator of protein loading in each lane) antibodies. Identical results were obtained in three independent experiments.</p

A–C: Effects of IGF-1 or/and PDGF-bb on the IL-1β-induced Akt activation in chondrocytes in monolayer cultures.

<p>Primary chondrocytes were either stimulated with 10 ng/ml IL-1β, pre-stimulated with 10 ng/ml IGF-1, 10 ng/ml PDGF-bb or a combination of both growth factors (5 ng/ml each) for 12 h and treated with 10 ng/ml IL-1β for the indicated times. Whole cell extracts were immunoprecipitated with anti-IKK-α antibody followed by western blot analysis using anti-Akt, anti-phospho-specific Akt and anti-IKK-α antibodies. The results shown are representative of three independent experiments.</p