Investigating the protective effect of tanshinone IIA against chondrocyte dedifferentiation: a combined molecular biology and network pharmacology approach.

BACKGROUND: Osteoarthritis (OA) is a common degenerative disease with multifactorial etiology. The dedifferentiation of chondrocytes can accelerate the progress of OA. Tanshinone IIA (TIIA) has been widely used to treat OA for many years and has proved to be effective in inhibiting chondrocyte dedifferentiation. Until now, the precise mechanism of TIIA's effect against dedifferentiation has not been well understood. METHODS: The targets of TIIA were explored from public databases using various methods. The related targets of OA were obtained from the GeneCards database and the Online Mendelian Inheritance in Man (OMIM) database. The potential targets and signaling pathways were determined using protein-protein interaction (PPI), Gene Ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Cell viability, proliferation, and metabolic activity were analyzed in vitro. The effects of TIIA on chondrocyte dedifferentiation were evaluated by assessing morphological changes, glycosaminoglycan (GAG) production, and messenger RNA (mRNA) levels of cartilage-related genes. After 48 hours of culture in medium with 100 µg/mL TIIA, chondrocytes/hydrogel spheres were implanted to repair cartilage defects in a rat model. The harvested specimens were examined with hematoxylin and eosin (H&E) staining and immunohistochemistry to evaluate cartilage regeneration. RESULTS: The results showed that there were 28 genes potentially interacting in the TIIA-chondrocyte dedifferentiation network, and nine hub genes were identified. In vitro experiments showed an inhibitory effect of TIIA on chondrocyte dedifferentiation. The proliferation and viability of chondrocytes were promoted by TIIA at a concentration of 100-200 µg/mL, but inhibited by TIIA at 400 µg/mL. Furthermore, the histology results showed that chondrocyte/hydrogel spheres pre-treated with TIIA had better cartilage repair. CONCLUSIONS: This study revealed a systematic network pharmacology approach and provided a basis for the future study of TIIA as an effective treatment for cartilage regeneration. Moreover, in vitro and in vivo results confirmed the protective effects of TIIA against chondrocyte dedifferentiation.

Prognostic value of tumoral and peritumoral magnetic resonance parameters in osteosarcoma patients for monitoring chemotherapy response.

OBJECTIVES: To evaluate parameters of diffusion-weighted imaging (DWI) and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) as early imaging indicators of tumor histologic response to pre-operative neoadjuvant chemotherapy and as probable prognostic factors for event-free survival (EFS) and overall survival in osteosarcoma (OS) in both tumoral and peritumoral areas. METHODS: Thirty-four OS patients who received three courses of neoadjuvant chemotherapy followed by surgery during 2014-2018 were enrolled in this study. All patients underwent baseline and post-chemotherapy DWI and DCE-MRI. Lesion region was defined as the tumoral area and peritumoral area. Parameters of apparent diffusion coefficient, capacity transfer constant (Ktrans), elimination rate constant, extravascular extracellular space volume ratio (Ve), and initial area under the curve as well as corresponding differences between pre- and post-chemotherapy in lesion regions were evaluated. Receiver operating characteristic analysis was used to evaluate the diagnostic performance of these parameters. The associations of all parameters with tumor histologic response, EFS, and overall survival were also calculated. RESULTS: In the tumor area, moderate evidence was found that post-Ktrans was lower in responders as compared with that in poor responders (p = 0.04, false discovery rate [FDR] corrected), and ΔKtrans exhibited significant between-groups differences (p = 0.04, Bonferroni corrected; or p = 0.006, FDR corrected). Weak evidence for the between-groups difference was found in the Ve in the peritumoral area (p = 0.025 before treatment and p = 0.021 after treatment, uncorrected). Furthermore, lower post-Ktrans in the tumoral area and lower pre-Ve in the peritumoral area were significant prognostic indicators for longer EFS (p = 0.002, p = 0.026) and overall survival (p = 0.003, p = 0.023). CONCLUSIONS: In OS, DWI and DCE-MRI parameters in both tumoral and peritumoral areas can reflect the chemotherapy response and prognosticate EFS and overall survival. KEY POINTS: • Peritumoral MRI parameters can reflect the chemotherapy response in OS patients. • Peritumoral MRI parameters can predict EFS and overall survival in OS patients. • MRI parameters may be predictive factors for evaluating chemotherapy efficacy and EFS.

Effects of Mechanical Stretch on Cell Proliferation and Matrix Formation of Mesenchymal Stem Cell and Anterior Cruciate Ligament Fibroblast.

Mesenchymal stem cells (MSCs) and fibroblasts are two major seed cells for ligament tissue engineering. To understand the effects of mechanical stimulation on these cells and to develop effective approaches for cell therapy, it is necessary to investigate the biological effects of various mechanical loading conditions on cells. In this study, fibroblasts and MSCs were tested and compared under a novel Uniflex/Bioflex culture system that might mimic mechanical strain in ligament tissue. The cells were uniaxially or radially stretched with different strains (5%, 10%, and 15%) at 0.1, 0.5, and 1.0 Hz. The cell proliferation and collagen production were compared to find the optimal parameters. The results indicated that uniaxial stretch (15% at 0.5 Hz; 10% at 1.0 Hz) showed positive effects on fibroblast. The uniaxial strains (5%, 10%, and 15%) at 0.5 Hz and 10% strain at 1.0 Hz were favorable for MSCs. Radial strain did not have significant effect on fibroblast. On the contrary, the radial strains (5%, 10%, and 15%) at 0.1 Hz had positive effects on MSCs. This study suggested that fibroblasts and MSCs had their own appropriate mechanical stimulatory parameters. These specific parameters potentially provide fundamental knowledge for future cell-based ligament regeneration.

Effect of thickness of HA-coating on microporous silk scaffolds using alternate soaking technology.

Hydroxyapatite (HA) can be coated on various materials surface and has the function of osteogenicity. Microporous silk scaffold has excellent biocompatibility. In this study, alternate soaking technology was used to coat HA on microporous silk scaffolds. However, the cell proliferation was found to decrease with the increasing thickness (cycles of soaking) of HA-coating. This study aims to determine the best thickness (cycles of soaking) of HA-coating on microporous silk scaffolds. The SEM observation showed that group with one cycle of alternate soaking (1C-HA) has the most optimal porosity like non-HA-modified microporous silk scaffolds. The proliferation of osteoblasts has no significant difference between noncoated HA (N-HA) and 1C-HA groups, which are both significantly higher than those in two cycles of soaking (2C-HA) and three cycles of soaking (3C-HA) groups. The transcription levels of specific genes (runx2 and osteonectin) in osteoblasts of 1C-HA group were significantly higher than those of N-HA group. Moreover, the levels showed no significant difference among 1C-HA, 2C-HA, and 3C-HA groups. In conclusion, microporous silk scaffold with 1 cycle of HA-coating can combine the biocompatibility of silk and osteogenicity of HA.

Immobilized lentivirus vector on chondroitin sulfate-hyaluronate acid-silk fibroin hybrid scaffold for tissue-engineered ligament-bone junction.

The lack of a fibrocartilage layer between graft and bone remains the leading cause of graft failure after anterior cruciate ligament (ACL) reconstruction. The objective of this study was to develop a gene-modified silk cable-reinforced chondroitin sulfate-hyaluronate acid-silk fibroin (CHS) hybrid scaffold for reconstructing the fibrocartilage layer. The scaffold was fabricated by lyophilizing the CHS mixture with braided silk cables. The scanning electronic microscopy (SEM) showed that microporous CHS sponges were formed around silk cables. Each end of scaffold was modified with lentiviral-mediated transforming growth factor- ß 3 (TGF- ß 3) gene. The cells on scaffold were transfected by bonded lentivirus. In vitro culture demonstrated that mesenchymal stem cells (MSCs) on scaffolds proliferated vigorously and produced abundant collagen. The transcription levels of cartilage-specific genes also increased with culture time. After 2 weeks, the MSCs were distributed uniformly throughout scaffold. Deposited collagen was also found to increase. The chondral differentiation of MSCs was verified by expressions of collagen II and TGF- ß 3 genes in mRNA and protein level. Histology also confirmed the production of cartilage extracellular matrix (ECM) components. The results demonstrated that gene-modified silk cable-reinforced CHS scaffold was capable of supporting cell proliferation and differentiation to reconstruct the cartilage layer of interface.