Improved Cell Adhesion and Osteogenesis of op-HA/PLGA Composite by Poly(dopamine)-Assisted Immobilization of Collagen Mimetic Peptide and Osteogenic Growth Peptide

A nanocomposite of poly­(lactide-<i>co</i>-glycolide) (PLGA) and hydroxyapatite (HA) with a different grafting ratio of l-lactic acid oligomer (op-HA) showed better interface compatibility, mineralization, and osteogenetic abilities. However, surface modification of the composite is crucial to improve the osteointegration for bone regeneration. In this study, a biomimetic process via poly­(dopamine) coating was utilized to prepare functional substrate surfaces with immobilized bioactive peptides that efficiently regulate the osteogenic differentiation of preosteoblasts (MC3T3-E1). Our study demonstrated that incorporation of collagen mimetic peptide significantly enhanced cell adhesion and proliferation. The immobilization of osteogenic growth peptide induced the osteodifferentiation of cells, as indicated by the alkaline phosphate activity test, quantitative real-time polymerase chain reaction analysis, and immunofluorescence staining. The mineralization on the peptide-modified substrates was also enhanced greatly. Findings from this study revealed that this biofunctionalized layer on op-HA/PLGA substrate improved mineralization and osteogenic differentiation. In conclusion, the surface modification strategy with bioactive peptides shows potential to enhance the osteointegration of bone implants

Enhanced in Vitro Mineralization and in Vivo Osteogenesis of Composite Scaffolds through Controlled Surface Grafting of l‑Lactic Acid Oligomer on Nanohydroxyapatite

Nanocomposite of hydroxyapatite (HA) surface grafted with l-lactic acid oligomer (LAc oligomer) (op-HA) showed improved interface compatibility, mechanical property, and biocompatibility in our previous study. In this paper, composite scaffolds of op-HA with controlled grafting different amounts of LAc oligomer (1.1, 5.2, and 9.1 wt %) were fabricated and implanted to repair rabbit radius defects. The dispersion of op-HA nanoparticles was more uniform than n-HA in chloroform and nanocomposites scaffold. Calcium and phosphorus exposure, in vitro biomineralization ability, and cell proliferation were much higher in the op-HA_1.1 wt %/PLGA scaffolds than the other groups. The osteodifferentiation and bone fusion in animal tests were significantly enhanced for op-HA_5.2 wt %/PLGA scaffolds. The results indicated that the grafted LAc oligomer of 5.2 or 9.1 wt %, which formed a barrier layer on the HA surface, prevented the exposure of nucleation sites. The shielded nucleation sites of op-HA particles (5.2 wt %) might be easily exposed as the grafted LAc oligomer was decomposed easily by enzyme systems in vivo. Findings from this study have revealed that grafting 1.1 wt % amount of LAc oligomer on hydroxyapatite could improve in vitro mineralization, and 5.2 wt % could promote in vivo osteogenesis capacity of composite scaffolds

Modulation of Osteogenesis in MC3T3-E1 Cells by Different Frequency Electrical Stimulation

<div><p>Electrical stimulation (ES) is therapeutic to many bone diseases, from promoting fracture regeneration to orthopedic intervention. The application of ES offers substantial therapeutic potential, while optimal ES parameters and the underlying mechanisms responsible for the positive clinical impact are poorly understood. In this study, we assembled an ES cell culture and monitoring device. Mc-3T3-E1 cells were subjected to different frequency to investigate the effect of osteogenesis. Cell proliferation, DNA synthesis, the mRNA levels of osteosis-related genes, the activity of alkaline phosphatase (ALP), and intracellular concentration of Ca²⁺ were thoroughly evaluated. We found that 100 Hz could up-regulate the mRNA levels of collagen I, collagen II and Runx2. On the contrary, ES could down-regulate the mRNA levels of osteopontin (OPN). ALP activity assay and Fast Blue RR salt stain showed that 100 Hz could accelerate cells differentiation. Compared to the control group, 100 Hz could promote cell proliferation. Furthermore, 1 Hz to 10 Hz could improve calcium deposition in the intracellular matrix. Overall, these results indicate that 100Hz ES exhibits superior potentialities in osteogenesis, which should be beneficial for the clinical applications of ES for the treatment of bone diseases.</p></div

Quantitative DNA assay.

<p>Mc-3T3-E1 cells were subjected to different frequency ES for 3 days. Pico-green DNA assay was used to quantified DNA concentration of cells. 100 Hz group has the higher DNA concentration than other ES groups and control.</p

Effects of ES frequency on regulating Intracellular [Ca²⁺].

<p>MC-3T3-E1 cells subjected to different ES frequency for 1 day and 3 days. Fluo-4 NW was used to assay relative Ca²⁺ concentration. 1 Hz and 10 Hz groups shown an increasing regulation on intracellular Ca²⁺ concentration compared to control group. * P < 0.05, indicated statistically significant difference compared to 1Hz and 10Hz.</p

Effects of ES on ALP activity of MC3T3-E1 cells.

<p>Cells were exposed to different frequency ES for 30min a day, continued 3 days. Values are the mean ± SD of three independent cultures. *P < 0.05 vs. 1Hz, 10Hz, 100 kHz group.</p

Cells were stained for ALP.

<p>ALP activity increased in a ES groups reached the highest level at 100 Hz ES for 3 days.(A)1 Hz,(B)10 Hz,(C)100 Hz,(D)1 KHz,(E)10 KHz,(F)100 KHz, bar = 100um.</p

List of Genes and Primer Nucleotide Sequences.

<p>List of Genes and Primer Nucleotide Sequences.</p

Assessment of Proliferation in MC-3T3-E1 cells after ES.

<p>MTT assay to assess proliferation of the MC-3T3-E1 cells after ES. 100 Hz group has the highest proliferation rate at days 1, 3 compared to the other frequency and control group. 1K Hz group showed similar proliferation compared to control group at days 1, 3. 1 Hz, 10 Hz, 10 KHz, and 100 KHz showed lower proliferation than control group at day 1. (P < 0.05, indicated statistically significant difference)</p

Preparation, Pharmacokinetics, Biodistribution, Antitumor Efficacy and Safety of Lx2-32c-Containing Liposome

<div><p>Lx2-32c is a novel taxane that has been demonstrated to have robust antitumor activity against different types of tumors including several paclitaxel-resistant neoplasms. Since the delivery vehicles for taxane, which include cremophor EL, are all associated with severe toxic effects, liposome-based Lx2-32c has been developed. In the present study, the pharmacokinetics, biodistribution, antitumor efficacy and safety characteristics of liposome-based Lx2-32c were explored and compared with those of cremophor-based Lx2-32c. The results showed that liposome-based Lx2-32c displayed similar antitumor effects to cremophor-based Lx2-32c, but with significantly lower bone marrow toxicity and cardiotoxicity, especially with regard to the low ratio of hypersensitivity reaction. In comparing these two delivery modalities, targeting was superior using the Lx2-32c liposome formulation; it achieved significantly higher uptake in tumor than in bone marrow and heart. Our data thus suggested that the Lx2-32c liposome was a novel alternative formulation with comparable antitumor efficacy and a superior safety profiles to cremophor-based Lx2-32c, which might be related to the improved pharmacokinetic and biodistribution characteristics. In conclusion, the Lx2-32c liposome could be a promising alternative formulation for further development.</p></div