Shuhua Yang

Physicochemical characterization and biocompatibility in vitro of biphasic calcium phosphate/polyvinyl alcohol scaffolds prepared by freeze-drying method for bone tissue engineering applications

In this study, a well developed porous biphasic calcium phosphate (BCP)/polyvinyl alcohol (PVA) scaffold was prepared by emulsion foam freeze-drying method possessed moderate inter-connected pores and porosity. The SEM analysis showed that BCP nano-particles could disperse uniformly in the scaffolds, and the pore size, porosity, and compressive strength could be controlled by the weight ratio of BCP/PVA. The in vitro degradation and cytocompatibility of scaffolds were examined in this study. The degradation analysis showed the prepared scaffolds have a low variation of pH values (approximately 7.18-7.36) in SBF solution, and have the biodegradation rate of BCP/PVA scaffolds decreased with the increase of PVA concentration. Moreover, MTT assay indicated that the BCP/PVA porous scaffold has no negative effects on cells growth and proliferation, and the hBMSCs possessed a favorable spreading morphology on the BCP/PVA scaffold surface. The inter-connected pore structure, mechanical strength, biodegradation rate and cytocompatibility of the prepared BCP/PVA scaffold can meet essential requirements for blame bearing bone tissue engineering and regeneration.

ROCK1 as a potential therapeutic target in osteosarcoma

Osteosarcoma is the most common primary malignancy of bone. Patients with localized disease are routinely treated with surgery and chemotherapy. Unfortunately, many of these patients eventually relapse even after high‐dose pre‐ and postoperative chemotherapy. Upon recurrence of the tumor locally or distantly, they have limited treatment options that are usually unsuccessful. Our prior studies screening lentiviral shRNA libraries, searching for kinases involved in osteosarcoma cell growth and proliferation have identified the Rho‐associated coiled‐coil containing protein kinase 1 (ROCK1) as a possible hit. We show in this study that ROCK1 is highly expressed in various tumor cell lines and tumor tissues from osteosarcoma patients. ROCK1 knockdown by synthetic siRNA decreases cell proliferation, viability and induces apoptosis in osteosarcoma cell lines KHOS and U‐2OS. Finally, we established the relationship between expression levels of ROCK1 and clinical prognosis in osteosarcoma patients by using immunohistochemistry. There were significant differences in overall survival between cohorts of patients with ROCK1 levels categorized as high‐staining, moderate‐staining, and low‐staining. High levels of ROCK1 were associated with poor outcomes in clinical osteosarcoma. These findings suggest that knockdown of ROCK1 inhibits proliferation and induces apoptosis in osteosarcoma cell lines. ROCK1 may be a promising therapeutic target for the treatment of osteosarcoma patients.

Targeting CDK11 in osteosarcoma cells using the CRISPR-Cas9 system.

Osteosarcoma is the most common type primary malignant tumor of bone. Patients with regional osteosarcoma are routinely treated with surgery and chemotherapy. In addition, many patients with metastatic or recurrent osteosarcoma show poor prognosis with current chemotherapy agents. Therefore, it is important to improve the general condition and the overall survival rate of patients with osteosarcoma by identifying novel therapeutic strategies. Recent studies have revealed that CDK11 is essential in osteosarcoma cell growth and survival by inhibiting CDK11 mRNA expression with RNAi. Here, we apply the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)‐Cas9 system, a robust and highly efficient novel genome editing tool, to determine the effect of targeting endogenous CDK11 gene at the DNA level in osteosarcoma cell lines. We show that CDK11 can be efficiently silenced by CRISPR‐Cas9. Inhibition of CDK11 is associated with decreased cell proliferation and viability, and induces cell death in osteosarcoma cell lines KHOS and U‐2OS. Furthermore, the migration and invasion activities are also markedly reduced by CDK11 knockout. These results demonstrate that CRISPR‐Cas9 system is a useful tool for the modification of endogenous CDK11 gene expression, and CRISPR‐Cas9 targeted CDK11 knockout may be a promising therapeutic regimen for the treatment of osteosarcoma.

Multidrug resistant osteosarcoma cell lines exhibit deficiency of GADD45α expression

To identify apoptosis genes involved in the multidrug resistance of osteosarcoma, a multidrug resistant human osteosarcoma cell line (U-2 OS MR) was established. Apoptosis gene microarray analysis demonstrated that GADD45α was significantly induced in U-2 OS cells after exposure to paclitaxel (Pxa0<xa00.0001). However, the induction of GADD45α did not occur in U-2 OS MR cells. Subsequent analysis by Western blot confirmed that the expression of GADD45α could be significantly induced by paclitaxel and doxorubicin in U-2 OS cells but not in U-2 OS MR cells. Furthermore, the paclitaxel or doxorubicin treated U-2 OS and KH OS cells have a higher percentage of apoptotic cells when compared with U-2 OS MR and KH OS R2 cells treated with the same drugs. When GADD45α was transfected into U-2 OS MR or KH OS R2 and expressed, the cells became more sensitive to chemotherapeutic drugs. These results suggest that GADD45α may play a role in drug-induced apoptosis, as well as multidrug resistance in osteosarcoma cells.

Establishment and characterization of a novel chordoma cell line: CH22

Chordoma is a rare primary malignant bone tumor and there exist only a few established human chordoma cell lines. The scarcity of robust chordoma cell lines has limited the ability to study this tumor. In this report, we describe the establishment of a novel chordoma cell line and characterize its in vitro and in vivo behaviors. The tumor tissue was isolated from a patient with recurrent chordoma of the sacrum. After 6 months in culture, the chordoma cell line, referred here as CH22, was established. Microscopic analysis of two‐dimensional culture confirmed that the CH22 cells exhibited a typical vacuolated cytoplasm similar to the well‐established chordoma cell line U‐CH1. Electron microscopy showed cohesive cells with numerous surface filopodia, pockets of glycogen and aggregates of intermediate tonofilaments in cytoplasm. Three‐dimensional culture revealed that the CH22 cells could grow and form clusters by day 8. The MTT assays demonstrated that, compared with sensitive osteosarcoma cell lines, CH22 cells were relatively resistant to conventional chemotherapeutic drugs. Western blotting and immunofluorescence analysis confirmed that the CH22 cells expressed brachyury, vimentin, and cytokeratin. Finally, histological analysis of CH22 xenograft tumor tissues demonstrated the appearance of physaliphorous cells and positive staining of brachyury, cytokeratin, and S100. By CT and MRI, imaging xenografts showed the typical appearances seen in human chordomas. These findings suggest that the established novel human chordoma cell line CH22 and its tumorigenecity in SCID nude mice may serve as an important model for studying chordoma cell biology and the development of new therapeutic modalities.

MiR-708 promotes steroid-induced osteonecrosis of femoral head, suppresses osteogenic differentiation by targeting SMAD3

Steroid-induced osteonecrosis of femoral head (ONFH) is a serious complication of glucocorticoid (GC) use. We investigated the differential expression of miRs in the mesenchymal stem cells (MSCs) of patients with ONFH, and aimed to explain the relationship between GC use and the development of MSC dysfunction in ONFH. Cells were collected from bone marrow of patients with ONFH. Samples were assigned to either GCs Group or Control Group at 1:1 matched with control. We then used miRNA microarray analysis and real-time PCR to identify the differentially expressed miRs. We also induced normal MSCs with GCs to verify the differential expression above. Subsequently, we selected some of the miRs for further studies, including miRNA target and pathway prediction, and functional analysis. We discovered that miR-708 was upregulated in ONFH patients and GC-treated MSCs. SMAD3 was identified as a direct target gene of miR-708, and functional analysis demonstrated that miR-708 could markedly suppress osteogenic differentiation and adipogenesis differentiation of MSCs. Inhibition of miR-708 rescued the suppressive effect of GC on osteonecrosis. Therefore, we determined that GC use resulted in overexpression of miR-708 in MSCs, and thus, targeting miR-708 may serve as a novel therapeutic biomarker for the prevention and treatment of ONFH.

Growth differentiation factor 5 modulation of chondrogenesis of self‐assembled constructs involves gap junction‐mediated intercellular communication

A novel scaffold‐free self‐assembled cartilage construct has been generated and used to repair particular chondral defects effectively. However, the mechanisms related to the construction of these self‐assembled cartilages have not yet been fully elucidated. We hypothesize that gap junction intercellular communication (GJIC) plays a critical role in the development of self‐assembled constructs upon GDF‐5 induction. In this study, we investigated the effect of connexin 43 (C×43) mediated GJIC on GDF‐5 modulation of chondrogenesis from two aspects, cell monolayer culture and 3‐D self‐assembly culture. We induced cells or self‐assembled constructs with chondrogenic media (CM), growth differentiation factor 5 (GDF‐5) or 1‐heptanol for 3 weeks. At the end of that time, the results of quantitative fluorescence redistribution after photobleaching (FRAP) assay and immunofluorescence demonstrated that GDF‐5 improved both GJIC and chondrogenic differentiation to a significant degree while 1‐heptanol nearly offset the expected improvements in chondrogenesis. Biochemical assay and histology showed that GDF‐5 can obviously enhance GAG, C×43 and type II collagen expressions. Conversely, we also showed that while 1‐heptanol weakened GAG and type II collagen expression in self‐assembled constructs, it had no effect on C×43 expression. Furthermore, real‐time polymerase chain reaction showed that GDF‐5 enhanced GAG and type II collagen transcription while 1‐heptanol reduced them, but was affectless on C×43 transcription. This suggests that the generation of scaffold‐free self‐assembled cartilage from human mesenchymal stem cells upon GDF‐5 induction may be mediated, at least in part, via the modulation of GJIC.

The Involvement of Protease Nexin-1 (PN1) in the Pathogenesis of Intervertebral Disc (IVD) Degeneration

Protease nexin-1 (PN-1) is a serine protease inhibitor belonging to the serpin superfamily. This study was undertaken to investigate the regulatory role of PN-1 in the pathogenesis of intervertebral disk (IVD) degeneration. Expression of PN-1 was detected in human IVD tissue of varying grades. Expression of both PN-1 mRNA and protein was significantly decreased in degenerated IVD, and the expression levels of PN-1 were correlated with the grade of disc degeneration. Moreover, a decrease in PN-1 expression in primary NP cells was confirmed. On induction by IL-1β, the expression of PN-1 in NP cells was decreased at day 7, 14, and 21, as shown by western blot analysis and immunofluorescence staining. PN-1 administration decreased IL-1β-induced MMPs and ADAMTS production and the loss of Agg and Col II in NP cell cultures through the ERK1/2/NF-kB signaling pathway. The changes in PN-1 expression are involved in the pathogenesis of IVD degeneration. Our findings indicate that PN-1 administration could antagonize IL-1β-induced MMPs and ADAMTS, potentially preventing degeneration of IVD tissue. This study also revealed new insights into the regulation of PN-1 expression via the ERK1/2/NF-kB signaling pathway and the role of PN-1 in the pathogenesis of IVD degeneration.

Clinical result of structural augmentation with cannulated bone screws for the treatment of osteonecrosis of the femoral head.

Objective:u2002 To evaluate the results of treatment of osteonecrosis of the femoral head by structural augmentation through a routine core decompression procedure combined with insertion of cannulated bone screws incorporating autogenous bone graft and biomaterial containing decalcified bone matrix.

Total hip arthroplasty for treatment of elderly patients with comminuted intertrochanteric fracture accompanied by femoral head necrosis.

OBJECTIVEnTo assess the curative effect and investigate the indications of total hip arthroplasty for treatment of comminuted intertrochanteric fractures.nnnMETHODSnTotal hip arthroplasty was carried out in 9 cases of severe intertrochanteric fracture. The patients included two men and seven women. The average age of the patients was 68 years (48-75 years). The period from fracture to operation was 5 days (2-10 days). The mean follow-up period lasted for 11 months (3 months-2 years). There was one patient with comminuted intertrochanteric fracture accompanied by femoral head necrosis and 2 patients with intertrochanteric fracture and stroke. Other 6 patients had severe osteoporosis. The Harris score before operation was 63 points (45-71 points).nnnRESULTSnAt the last follow-up, the patients gained 86 points (70-100 points) according to the Harris score. The effects of the 8 cases were good. The Harris score of all patients improved after treatment. Only two hemiplegia patients needed sticks to walk. The others could walk without hip pain. No radiographic evidence of acetabular wear and prosthesis dislocation or other major complications happened during the follow-up.nnnCONCLUSIONSnProsthetic replacements can well treat unstable intertrochanteric fracture if operative indication is correctly selected. It is suitable for elderly patients and the operation should be performed by experienced surgeons.