DS_10.1177_0363546518756087 – Supplemental material for Anterior Cruciate Ligament Transection–Induced Cellular and Extracellular Events in Menisci: Implications for Osteoarthritis

<p>Supplemental material, DS_10.1177_0363546518756087 for Anterior Cruciate Ligament Transection–Induced Cellular and Extracellular Events in Menisci: Implications for Osteoarthritis by Jing Xie, Demao Zhang, Yunfeng Lin, Quan Yuan and Xuedong Zhou in The American Journal of Sports Medicine</p

Softening Substrates Promote Chondrocytes Phenotype via RhoA/ROCK Pathway

Due to its evascular, aneural, and alymphatic conditions, articular cartilage shows extremely poor regenerative ability. Thus, directing chondrocyte toward a desired location and function by utilizing the mechanical cues of biomaterials is a promising approach for effective tissue regeneration. However, chondrocytes cultured on Petri dish will lose their typical phenotype which may lead to compromised results. Therefore, we fabricated polydimethylsiloxane (PDMS) materials with various stiffness as culture substrates. Cell morphology and focal adhesion of chondrocytes displayed significant changes. The cytoskeletal tension of the adherent cells observed by average myosin IIA fluorescent intensity increased as stiffness of the underlying substrates decreased, consistent with the alteration of chondrocyte phenotype in our study. Immunofluorescent images and q-PCR results revealed that chondrocyte cultured on soft substrates showed better chondrocyte functionalization by more type II collagen and aggrecan expression, related to the lowest mRNA level of Rac-1, RhoA, ROCK-1, and ROCK-2. Taken together, this work not only points out that matrix elasticity can regulate chondrocyte functionalization via RhoA/ROCK pathway, but also provides new prospect for biomechanical control of cell behavior in cell-based cartilage regeneration

BACE1 Aptamer-Modified Tetrahedral Framework Nucleic Acid to Treat Alzheimer’s Disease in an APP-PS1 Animal Model

Alzheimer’s disease is a neurodegenerative disease caused by excessive amyloid β protein-induced neurotoxicity. However, drugs targeting amyloid β protein production face many problems, such as the low utilization rate of drugs by cells and the difficulty of drugs in penetrating the blood–brain barrier. A tetrahedral framework nucleic acid is a new type of nanonucleic acid structure that functions as a therapy and drug carrier. Here, we synthesized a BACE1 aptamer-modified tetrahedral framework nucleic acid and tested its therapeutic effect on Alzheimer’s disease in vitro and in vivo. Our results demonstrated that the tetrahedral framework nucleic acid could be used as a carrier to deliver the BACE1 aptamer to the brain to reduce the production of amyloid β proteins. It also played an antiapoptotic role by reducing the production of reactive oxygen species. Thus, this nanomaterial is a potential drug for Alzheimer’s disease

Wnt5a promotes the adhesion but inhibits the migration of hDPCs.

<p>A: A total of 25,000 cells were seeded for the indicated times and nonadherent cells were rinsed off. Adherent cells were stained with crystal violet and were analyzed spectrophotometrically. The number of adherent cells is shown as mean±SD from three independent experiments, with the number of DMEM-treated cells set as 100%. B: Confluent monolayers of hDPCs were scratched with a pipette tip and cultured in different medium containing 5% FBS for 12 hr. The relative migration distance of the wound edge was shown as mean±SD of three independent experiments, with the migration distance of DMEM-treated cells set as 100%. Bars, 100 µm. C: HDPCs were plated on glass coverslips coated with type I collagen and cultured with different medium for the indicated times. For FACs immunostaining, anti-vinculin antibody was used, and for F-actin staining, rhodamine-phalloidin was used, arrowheads mark FACs. Bars,10 µm. D: Confluent hDPCs were incubated with Wnt5a for the indicated times and Western analyses were used to detect the expression of vinculin, phospho-paxillin, phospho-MLC, with GAPDH, total-paxillin and total-MLC as loading control. The relative protein expression at 0 min is designated 1.0. *<i>p</i> < 0.05, n=3.</p

Wnt5a activated JNK signaling is dependent and independent of RhoA signaling.

<p>A: Lysates from hDPCs were obtained following transfection with adenoviral vectors encoding GFP, RhoA WT, RhoA T19N and RhoA Q63L for 48 hr and the levels of phospho-JNK and total-JNK were measured by Western blot analysis. The normalized amount of phospho-JNK in GFP adenovirus infected hDPCs is designated 1.0. B: HDPCs infected with adenoviruses encoding the RhoA mutant for 48 hr were cultured with Wnt5a CM and the cell lysates were obtained at the indicated times, with the level of phospho-JNK and total-JNK measured by Western analyses. The relative amount of phospho-JNK is normalized to the total amount, at 0min, is designated 1.0. *<i>p</i> < 0.05, n=3.</p

The role of the JNK pathway in Wnt5a-dependent adhesion, migration and formation of FACs.

<p>A: HDPCs were pretreated with 10 µM or 30 µM SP600125 for 30 min and the levels of phospho-JNK and total JNK were measured by Western blot analysis. The relative protein expression without SP600125 treatment is designated 1.0. B ,C: Cell adhesion and wound healing assays were performed as in Figure 1, but the cells were hDPCs which were pretreated with 30 µm SP600125 for 30 min, and the observed time in the wound healing assay was 16 hr. Bars, 100 µm. D: Vinculin immunostaining and phalloidin staining were performed at 15min as in Figure 1C, but the hDPCs were pretreated with 30µM SP600125 for 30 min before being seeded onto glass slides. Arrowheads mark FACs. The number of FACs and the relative fluorescence were analyzed as in Figure 1C. Bars,10 µm. E: Pretreatment with 30 µM SP600125 for 30 min, hDPCs were incubated with Wnt5a CM for the indicated times, the cell lysates were collected and immunoblotted with antibodies to phospho-MLC, phospho-paxillin, total-paxillin, total-MLC and vinculin. The promotion of phospho-paxillin by Wnt5a CM was delayed until after 60 min, but no changes were seen in the expression of phospho-MLC. *<i>p</i> < 0.05, n = 3.</p

RhoA signaling contributed to the Wnt5a-dependent adhesion, migration changes and formation of FACs.

<p>A: After infection with RhoA mutant adenoviruses, hDPCs were cultured with GFP CM or Wnt5a CM and cell adhesion assays were performed, as in Figure 1A. B: Confluent hDPCs infected with RhoA mutant adenoviruses for 48 hr were scratched and cultured with GFP CM or Wnt5a CM for 20 hr to observe the effect of Wnt5a CM on the cell. Bars,100 µm. C: Vinculin immunostaining and phalloidin staining were performed as in Figure 1C, but the hDPCs were infected with different RhoA mutant adenoviruses and the secondary antibody was Alexa 546-labeled. Arrowheads mark FACs. The number of FACs and the relative fluorescence were analyzed as if Figure 1C. Bars, 10 µm. D: After infection with RhoA T19N or RhoA Q63L adenoviruses for 48 hr, hDPCs were incubated with Wnt5a CM for the indicated times and collected for protein extraction. Western analyses of phospho-MLC, phospho-paxillin, total-paxillin, total-MLC and vinculin in hDPCs. a <i>p</i> < 0.05, n = 3, compared with RhoA WT at the same time point. *<i>p</i> < 0.05, n = 3.</p

Wnt5a has no effect on β-catenin expression or translocation in hDPCs.

<p>A: Western analyses of β-catenin in cytosolic or nuclear fractions of cells cultured in Wnt5a CM for indicated times. Cytosolic and nuclear signals were normalized to GAPDH and H3, respectively. The relative expression of β-catenin at 0min is designated 1.0. B: Immunofluorescence microscopy of hDPCs following culture with rhWnt5a or Wnt5a CM for 1 hr. β-catenin signal is in green. Bars,30 µm. C: Wnt5a up-regulates the expression of GTP-RhoA and phospho-JNK. RhoA activity stimulated by Wnt5a was detected by GST-Pull down assay at the indicated times, the expression of total RhoA, phospho-JNK, and total-JNK was detected by Western blot analysis. The relative protein expression at 0min is designated 1.0. *<i>p</i> < 0.05, n=3.</p

Fabrication of Calcium Phosphate Microflowers and Their Extended Application in Bone Regeneration

The structure of materials is known to play an important role in material function. Nowadays, flowerlike structures have gained attention for studies not only in analytical chemistry, but also in biomaterial design. In this study, flowerlike structures were applied in bone regeneration in the form of calcium phosphate microflowers. The material was synthesized by a simple and environmentally friendly method. We characterized the structure and properties of the microflower using various methods. Cytotoxicity and osteogenesis-related gene regulations of the microflower were investigated <i>in vitro</i>. Cell uptake was observed by immunofluorescence. Rat calvarial critical-size defect models were successfully established to further confirm the enhanced bone regeneration ability of this material. We expect that this novel study will be of practical importance for the extended application of flowerlike materials and will provide new insights into the optimization of the morphology of calcium phosphate materials

The role of the RhoA and JNK pathways in Wnt5a-dependent hDPC motility.

<p>The role of the RhoA and JNK pathways in Wnt5a-dependent hDPC motility.</p