Jianghong Huang

Evaluation of the novel three-dimensional porous poly (L-lactic acid)/nano-hydroxyapatite composite scaffold.

To determine the optimal ratio of nano-hydroxyapatite (n-HA) to polylactic acid (PLLA) in the novel three-dimensional porous PLLA/n-HA composite scaffolds, low-temperature rapid prototyping technology was employed to fabricate the composite materials with different n-HA contents. Mechanical properties and degradation behaviors of the composites were examined, and the scaffold microstructure and n-HA dispersion were observed by scanning electron microscope (SEM). Mechanical tests demonstrated that the tensile strength of the composite material gradually decreased with an increase in n-HA content. When the n-HA content reached 20 wt%, the bending strength of the composite material peaked at 138.5 MPa. SEM images demonstrated that the optimal content of n-HA was 20 wt% as the largest interconnected pore size that can be seen, with a porosity as high as 80%. In vitro degradation experiments demonstrated that the pH value of the material containing solution gradually decreased in a time-dependent manner, with a simultaneous weakening of the mechanical properties. In vitro study using rat osteoblast cells showed that the composite scaffolds were biocompatible; the 20 wt% n-HA scaffold offered particular improvement to rat osteoblast cell adhesion and proliferation compared to other compositions. It was therefore concluded that 20 wt% n-HA is the optimal nano-hydroxyapatite (n-HA) to polylactic acid (PLLA) ratio, with promise for bone tissue engineering.

Investigation of the in vitro degradation of a novel polylactide/nanohydroxyapatite composite for artificial bone

We prepared the poly-L-lactic acid (PLLA)/nanohydroxyapatite (n-HA) composite and investigated the in vitro degradation of pure PLLA material and PLLA/n-HA composites in order to identify a suitable and ideal artificial bone tissue repair material. The water uptake, weight loss, and changes in the PBS pH value and in the mechanical properties of material were measured during the processes that PLLA and PLLA/n-HA biological composites were degraded in PBS. We also performed electron microscopic scanning of the material fracture surface and observed the microscopic morphologies of materials during the degradation process. We found that the degradation rate of the PLLA/n-HA material was slower than the PLLA material, and there was a little degradation of the PLLA/n-HA material at early stages. The PLLA/n-HA material also maintained the initial mechanical strength better than the pure PLLA material. The PLLA/n-HA material is thus a better material for artificial bone than the pure PLLA material.

Effect of inorganic/organic ratio and chemical coupling on the performance of porous silica/chitosan hybrid scaffolds

Inorganic/organic hybrid scaffolds have great potential for tissue engineering applications due to controllable mechanical properties and tailorable biodegradation. Here, silica/chitosan hybrid scaffolds were fabricated through the sol-gel method with a freeze drying process. 3-Glycidoxypropyl trimethoxysilane (GPTMS) and tetraethylorthosilicate (TEOS) were used as the covalent inorganic/organic coupling agent and the separate inorganic source, respectively. Hybrid scaffolds with various inorganic/organic weight ratios (I/Os) and molar ratios of chitosan and GPTMS (GCs) were examined and compared in this study. FTIR showed that higher GPTMS content resulted in the increased covalent cross-linking of the chitosan and the silica network in hybrids. Compression testing indicated that increasing the GPTMS content greatly improved the compressive strength of scaffold. LIVE/DEAD assay showed that enhanced cytocompatibility was obtained as the silica content increased. Therefore, the results confirmed that the two parameters I/O and GC can largely influence the scaffold performance, which can be used to tailor the hybrid properties.

Low-temperature deposition manufacturing: A novel and promising rapid prototyping technology for the fabrication of tissue-engineered scaffold

Developed in recent years, low-temperature deposition manufacturing (LDM) represents one of the most promising rapid prototyping technologies. It is not only based on rapid deposition manufacturing process but also combined with phase separation process. Besides the controlled macropore size, tissue-engineered scaffold fabricated by LDM has inter-connected micropores in the deposited lines. More importantly, it is a green manufacturing process that involves non-heating liquefying of materials. It has been employed to fabricate tissue-engineered scaffolds for bone, cartilage, blood vessel and nerve tissue regenerations. It is a promising technology in the fabrication of tissue-engineered scaffold similar to ideal scaffold and the design of complex organs. In the current paper, this novel LDM technology is introduced, and its control parameters, biomedical applications and challenges are included and discussed as well.

The Study on Biocompatibility of Porous nHA/PLGA Composite Scaffolds for Tissue Engineering with Rabbit Chondrocytes In Vitro

Objective. To examine the biocompatibility of a novel nanohydroxyapatite/poly[lactic-co-glycolic acid] (nHA/PLGA) composite and evaluate its feasibility as a scaffold for cartilage tissue engineering. Methods. Chondrocytes of fetal rabbit were cultured with nHA/PLGA scaffold in vitro and the cell viability was assessed by MTT assay first. Cells adhering to nHA/PLGA scaffold were then observed by inverted microscope and scanning electron microscope (SEM). The cell cycle profile was analyzed by flow cytometry. Results. The viability of the chondrocytes on the scaffold was not affected by nHA/PLGA comparing with the control group as it was shown by MTT assay. Cells on the surface and in the pores of the scaffold increased in a time-dependent manner. Results obtained from flow cytometry showed that there was no significant difference in cell cycle profiles between the coculture group and control (P > 0.05). Conclusion. The porous nHA/PLGA composite scaffold is a biocompatible and good kind of scaffold for cartilage tissue engineering.

Osteogenic differentiation of bone marrow mesenchymal stem cells by magnetic nanoparticle composite scaffolds under a pulsed electromagnetic field

This study was conducted to investigate the effect of magnetic nanoparticle composite scaffold under a pulsed electromagnetic field on bone marrow mesenchymal stem cells (BMSCs), which was achieved by examining the biological behaviors of cell adhesion, proliferation and differentiation on the surface of the scaffolds. This may provide some experimental evidence for the use of magnetic nanoparticles in medical application. The magnetic nanoparticle composite scaffolds were evaluated and characterized by the following indexes: the cell proliferation was detected by the CCK-8 method, the alkaline phosphatase (ALP) activity was examined by a detection kit, and the expression of type I collagen and osteocalcin gene were evaluated by RT-PCR. The CCK-8 test showed that there was no significant difference in Group A (BMSCs-seeded magnetic scaffolds under the electromagnetic field), B (BMSCs-seeded magnetic scaffolds) and C (BMSCs cultured alone) (P > 0.05). The value for the ALP activity in Group A was higher than the other two groups. In addition, the RT-PCR results showed that the expression of type I collagen gene in Group A was enhanced (P < 0.05), suggesting that the magnetic nanoparticles combined with the pulsed electromagnetic field had a positive effect on the osteogenic differentiation of BMSCs. However, the expression of osteocalcin was not significantly different in three groups (P > 0.05). To conclude, magnetic nanoparticles may induce the osteogenic differentiation with the action of the pulsed electromagnetic field.

MicroRNA-33a-5p suppresses growth of osteosarcoma cells and is downregulated in human osteosarcoma

A body of evidence has indicated that microRNAs (miRNAs) may have significant roles in cancer. Aberrant expression of miRNAs has frequently been observed in various human malignancies, including osteosarcoma (OS). However, the roles of miRNAs in OS remain poorly understood. In the present study, high-throughput deep sequencing was performed to screen for deregulated miRNAs in OS. Screening identified 310 miRNAs which were significantly overexpressed and 41 miRNAs which were significantly downregulated (>2-fold) in OS samples, compared with adjacent non-tumor bone tissues. Among these miRNAs, miR-33a-5p was notably downregulated. TaqMan reverse transcription-polymerase chain reaction analysis further verified that miR-33a-5p expression was significantly reduced in a large cohort of human OS samples. Enhancing miR-33a-5p expression via transfection with miR-33a-5p precursor significantly inhibited OS cell growth, suggesting potential antitumor properties of miR-33a-5p. The results of the present study provide novel insights into the miRNAs involved in OS, and suggest that miR-33a-5p may function as a tumor suppressor in OS. Therefore, miR-33a-5p may be able to serve as a diagnostic and therapeutic target for OS treatment.

MicroRNA‑124 acts as a tumor‑suppressive miRNA by inhibiting the expression of Snail2 in osteosarcoma

The aim of the present study was to investigate the clinical significance of hsa-microRNA-124-3p (miR-124) in osteosarcoma (OS), and examine its role in cell growth and invasion. Using a microRNA chip array, the expression of miR-124 was detected in samples of surgically resected OS and matched against the levels of expression in tumor-adjacent normal tissues. The levels of miR-124 were upregulated in the OS cells through the transfection of miR-124 mimics. Cell proliferation and Transwell assays were performed to determine cell proliferation and invasion; Reverse transcription-quantitative polymerase chain reaction, western blot and luciferase assays were then used to detect the expression of the target gene snail family zinc finger 2 (Snail2). The expression of miR-124 was significantly lower in the OS tissues, compared with that in the tumor-adjacent normal tissues; and the expression of miR-124 in the tumor tissues was significantly associated with tumor size. miR-124 directly repressed the expression of Snail2, and resulted in a significant inhibition of cell proliferation and invasion. In a mouse model, the overexpression of miR-124 significantly inhibited U2OS cell proliferation and invasion. Taken together, miR-124 was associated with the adverse clinical and pathological features observed in OS. It acted as a tumor suppressor to regulate the proliferation and invasion of OS cells by targeting Snail2, suggesting that miR-124 may be key in the progression of OS.

TGFβ1 induces hypertrophic change and expression of angiogenic factors in human chondrocytes

The transforming growth factor β1 (TGFβ1) plays an important role in cartilage development. However, whether TGFβ1 stimulates chondrocyte proliferation and cartilage regeneration in osteoarthritis (OA) remains elusive, especially in the context of different treatment and tissue environments. In the present study, we investigated the role of TGFβ1 in human chondrocyte culture in vitro, focusing on the morphological change of chondrocytes and the expression of angiogenic factors upon TGFβ1 stimulation. We found increased expression of biomarkers indicating chondrocyte hypertrophy and the chondrocytes aggregated to form networks when they were treated with TGFβ1. DNA microarray analysis revealed significantly increased expression of genes related to blood vessel formation in TGFβ1 treatment group compared to control group. Matrigel assay further demonstrated that chondrocytes had the potential to form network-like structure. These results suggested that TGFβ1 induces the hypertrophic change of chondrocytes culture in vitro and induce expression of angiogenic biomarkers. Therefore, application of TGFβ1 for chondrocyte culture in practice should be considered prudentially and targeting TGFβ1 or relevant receptors to block the signaling pathway might be a strategy to prevent or alleviate progression of osteoarthritis.

DNA Methylation Profiling in Chondrocyte Dedifferentiation In Vitro

DNA methylation has emerged as a crucial regulator of chondrocyte dedifferentiation, which severely compromises the outcome of autologous chondrocyte implantation (ACI) treatment for cartilage defects. However, the full‐scale DNA methylation profiling in chondrocyte dedifferentiation remains to be determined. Here, we performed a genome‐wide DNA methylation profiling of dedifferentiated chondrocytes in monolayer culture and chondrocytes treated with DNA methylation inhibitor 5‐azacytidine (5‐AzaC). This research revealed that the general methylation level of CpG was increased while the COL‐1A1 promoter methylation level was decreased during the chondrocyte dedifferentiation. 5‐AzaC could reduce general methylation levels and reverse the chondrocyte dedifferentiation. Surprisingly, the DNA methylation level of COL‐1A1 promoter was increased after 5‐AzaC treatment. The COL‐1A1 expression level was increased while that of SOX‐9 was decreased during the chondrocyte dedifferentiation. 5‐AzaC treatment up‐regulated the SOX‐9 expression while down‐regulated the COL‐1A1 promoter activity and gene expression. Taken together, these results suggested that differential regulation of the DNA methylation level of cartilage‐specific genes might contribute to the chondrocyte dedifferentiation. Thus, the epigenetic manipulation of these genes could be a potential strategy to counteract the chondrocyte dedifferentiation accompanying in vitro propagation. J. Cell. Physiol. 232: 1708–1716, 2017.