Zhenwei Zou

Repair of rat cranial bone defects with nHAC/PLLA and BMP‐2‐related peptide or rhBMP‐2

An ideal artificial substitute has good biocompatibility properties and is able to provide for rapid bone formation. Bone morphogenetic protein‐2 (BMP‐2) is considered as one of the most important growth factors for bone regeneration. In this study, a synthetic BMP‐2‐related peptide (designated P24) corresponding to residues of the knuckle epitope of BMP‐2 was introduced into a bioactive scaffold based on nano‐hydroxyapatite/collagen/poly(L‐lactic acid) (nHAC/PLLA); its in vitro release kinetics was then measured. A 5 mm diameter cranial bone defect was created in the calvariae of 30 rats and randomly implanted with three groups of biomaterials: Group A (nHAC/PLLA alone); Group B (P24/nHAC/PLLA composite); and Group C (recombinant human BMP‐2 (rhBMP‐2)/nHAC/PLLA composite). The P24/nHAC/PLLA implants significantly stimulated bone growth similarly to the rhBMP‐2/nHAC/PLLA implants based on the radiographic and three‐dimensional CT evaluation and histological examination, thereby confirming the enhanced bone healing rate of these compounds compared with the stand‐alone nHAC/PLLA scaffold material. The osteoinductive ability of 3 mg P24 was similar to that of 1 µg rhBMP‐2. P24/nHAC/PLLA is a promising scaffold biomaterial for bone tissue regeneration. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1745–1752, 2011

Biocompatibility and bioactivity of designer self‐assembling nanofiber scaffold containing FGL motif for rat dorsal root ganglion neurons

We report here a designer self-assembling peptide nanofiber scaffold developed specifically for nerve tissue engineering. We synthesized a peptide FGL-RADA containing FGL (EVYVVAENQQGKSKA), the motif of neural cell adhesion molecule (NCAM), and then attended to make a FGL nanofiber scaffold (FGL-NS) by assembling FGL-RADA with the peptide RADA-16 (AcN-RADARADARADARADA-CONH2). The microstructures of the scaffolds were tested using atomic force microscopy (AFM), and rheological properties of materials were accessed. Then we demonstrated the biocompatibility and bioactivity of FGL-NS for rat dorsal root ganglion neurons (DRGn). We found that the designer self-assembling peptide scaffold was noncytotoxic to neurons and able to promote adhesion and neurite sprouting of neurons. Our results indicate that the designer peptide scaffold containing FGL had excellent biocompatibility and bioactivity with adult sensory neurons and could be used for neuronal regeneration.

Biocompatibility of functionalized designer self‐assembling nanofiber scaffolds containing FRM motif for neural stem cells

Peptide self-assembling scaffolds have been widely used in tissue engineering. Much work has been focused on modifying the self-assembling scaffolds with functional motifs for desired biological activities. We report here the development of a biological material designed specifically for neural tissue engineering (NTE). Using RADA-16 (AcN-RADARADARADARADA-CONH2) as a base scaffold, we synthesized a 31 amino acid peptide RADA-FRM (AcN-RADARADARADARADAGGSIDRVEPYSSTAQ-CONH2) containing the neural cell adhesion molecule (NCAM)-derived mimetic peptide FRM (SIDRVEPYSSTAQ), which could undergo self-assembly into a nanofiber scaffold. We tested the characterization of the nanofiber scaffold using atomic force microscopy (AFM) and accessed the rheological properties of FRM-containing nanofiber scaffold (FRM-NS). Then we examined its biocompatibility on neural stem cells (NSCs) from neonatal rats. Regrettably, we found that FRM-NS had no effect on differentiation of NSCs. However, we tested that FRM-NS was noncytotoxic. Furthermore, compared to pure RADA-16 scaffold, we found that the designer self-assembling peptide scaffold containing FRM motif could significantly promote NSCs proliferation and stimulate NSCs migration into the three-dimensional scaffold. Our results indicate that the novel designer peptide scaffold containing FRM had excellent biocompatibility with NSCs and may be useful for central nervous tissue repair.

SHP1-mediated cell cycle redistribution inhibits radiosensitivity of non-small cell lung cancer

BackgroundRadioresistance is the common cause for radiotherapy failure in non-small cell lung cancer (NSCLC), and the degree of radiosensitivity of tumor cells is different during different cell cycle phases. The objective of the present study was to investigate the effects of cell cycle redistribution in the establishment of radioresistance in NSCLC, as well as the signaling pathway of SH2 containing Tyrosine Phosphatase (SHP1).MethodsA NSCLC subtype cell line, radioresistant A549 (A549S1), was induced by high-dose hypofractionated ionizing radiations. Radiosensitivity-related parameters, cell cycle distribution and expression of cell cycle-related proteins and SHP1 were investigated. siRNA was designed to down-regulate SHP1expression.ResultsCompared with native A549 cells, the proportion of cells in the S phase was increased, and cells in the G0/G1 phase were consequently decreased, however, the proportion of cells in the G2/M phase did not change in A549S1 cells. Moreover, the expression of SHP1, CDK4 and CylinD1 were significantly increased, while p16 was significantly down-regulated in A549S1 cells compared with native A549 cells. Furthermore, inhibition of SHP1 by siRNA increased the radiosensitivity of A549S1 cells, induced a G0/G1 phase arrest, down-regulated CDK4 and CylinD1expressions, and up-regulated p16 expression.ConclusionsSHP1 decreases the radiosensitivity of NSCLC cells through affecting cell cycle distribution. This finding could unravel the molecular mechanism involved in NSCLC radioresistance.

A novel peptide‐modified and gene‐activated biomimetic bone matrix accelerating bone regeneration

The osteogenic differentiation of bone marrow stromal cells (BMSCs) can be regulated by systemic or local growth factor, especially by transforming growth factor beta 1 (TGF-β1). However, how to maintain the bioactivity of exogenous TGF-β1 is a great challenge due to its short half-life time. The most promising solution is to transfer TGF-β1 gene into seed cells through transgenic technology and then transgenic cells to continuously secret endogenous TGF-β1 protein via gene expression. In this study, a novel non-viral vector (K)16GRGDSPC was chemically linked to bioactive bone matrices PLGA-[ASP-PEG]n using cross-linker to construct a novel non-viral gene transfer system. TGF-β1 gene was incubated with this system and subsequently rabbit-derived BMSCs were co-cultured with this gene-activated PLGA-[ASP-PEG]n, while co-cultured with PLGA-[ASP-PEG]n modified with (K)16GRGDSPC only and original PLGA-[ASP-PEG]n as control. Thus we fabricated three kinds of composites: Group A (BMSCs-TGF-β1DNA-(K)16GRGDSPC-PLGA-[ASP-PEG]n composite); Group B (BMSCs-(K)16GRGDSPC-PLGA-[ASP-PEG]n composite); and Group C (BMSCs-PLGA-[ASP-PEG]n composite). TGF-β1 and other osteogenic phenotype markers of alkaline phosphatase, osteocalcin, osteopontin and type I collagen in Group A were all significantly higher than the other two groups ex vivo. In vivo, 15-mm long segmental rabbit bone defects were created and randomly implanted the aforementioned composites separately, and then fixed with plate-screws. The results demonstrated that the implants in Group A significantly accelerated bone regeneration compared with the other implants based on X-rays, histological and biomechanical examinations. Therefore, we conclude this novel peptide-modified and gene-activated biomimetic bone matrix of TGF-β1DNA-(K)16GRGDSPC-PLGA-[ASP-PEG]n is a very promising scaffold biomaterial for accelerating bone regeneration.

Effect of IKVAV Peptide Nanofiber on Proliferation, Adhesion and Differentiation into Neurocytes of Bone Marrow Stromal Cells *

SummaryThis study examined the effect of IKVAV peptide nanofiber on proliferation, adhesion and differentiation into neurocytes of bone marrow stromal cells (BMSCs). IKVAV Peptide-amphiphile was synthesized and purified. Then, hydrogen chloride was added to the diluted aqueous solutions of PA to induce spontaneous formation of nanofiber in vitro. The resultant samples was observed under transmission electron microscope. BMSCs were cultured with IKVAV peptide nanofiber. The effect of IKVAV nanofiber on the proliferation, adhesion and induction differentiation of BMSCs was observed by inverted microscopy, calcein-AM/PI staining, cell counting and immunofluorescence staining. The results demonstrated that IKVAV peptide-amphiphile could self-assemble to form nanofiber gel. BMSCs cultured in combination with IKVAV peptide nanofiber gel grew well and the percentage of live cells was over 90%. IKVAV peptide nanofiber gel exerted no influence on the proliferation of BMSCs and could promote the adhesion of BMSCs and raise the ratio of neurons when BMSCs were induced to differentiate into neurocytes. It is concluded that BMSCs could proliferate and adhere well and yield more neurons during when induced to differente into neurocytes on IKVAV peptide nanofiber gel.This study examined the effect of IKVAV peptide nanofiber on proliferation, adhesion and differentiation into neurocytes of bone marrow stromal cells (BMSCs). IKVAV Peptide-amphiphile was synthesized and purified. Then, hydrogen chloride was added to the diluted aqueous solutions of PA to induce spontaneous formation of nanofiber in vitro. The resultant samples was observed under transmission electron microscope. BMSCs were cultured with IKVAV peptide nanofiber. The effect of IKVAV nanofiber on the proliferation, adhesion and induction differentiation of BMSCs was observed by inverted microscopy, calcein-AM/PI staining, cell counting and immunofluorescence staining. The results demonstrated that IKVAV peptide-amphiphile could self-assemble to form nanofiber gel. BMSCs cultured in combination with IKVAV peptide nanofiber gel grew well and the percentage of live cells was over 90%. IKVAV peptide nanofiber gel exerted no influence on the proliferation of BMSCs and could promote the adhesion of BMSCs and raise the ratio of neurons when BMSCs were induced to differentiate into neurocytes. It is concluded that BMSCs could proliferate and adhere well and yield more neurons during when induced to differente into neurocytes on IKVAV peptide nanofiber gel.

Bone induction by surface-double-modified true bone ceramics in vitro and in vivo

True bone ceramic (TBC), obtained by twice sintering fresh bovine cancellous bone at high temperatures, is an osteoconductive and bioactive bone substitute material that exhibits excellent biocompatibility with hard tissue. The authors have previously synthesized a novel BMP-2-related peptide, P24, and found that it could enhance the osteoblastic differentiation of cells. The objective of the present study was to construct a double-modified TBC via mineralization into simulated body fluid and P24 incorporation for enhanced bone formation. In vitro experiments revealed that surface mineralization-modified (SMM) TBC scaffolds demonstrated efficiency for sustained release of P24. The P24/SMM-TBC composite exhibited increased osteogenic activity by cell adhesion rate determination, MTT assay, alkaline phosphatase staining, and calcium nodule staining with alizarin red compared with SMM-TBC and TBC. In vivo studies showed that the P24/SMM-TBC composite scaffold promoted significant bone defect repair, in marked contrast to stand-alone SMM-TBC and TBC, based on the results of radiographic evaluation and histological examination. These findings indicate that SMM-TBC is a good scaffold for the controlled release of P24 and that the P24/SMM-TBC composite could improve the adhesion, proliferation and differentiation of cells and repair bone defects. The double-modified P24/SMM-TBC composite biomaterial shows potential for clinical application in bone tissue engineering.

Tumor Ablation and Therapeutic Immunity Induction by an Injectable Peptide Hydrogel

Immunosuppressive tumor microenvironments (TMEs) create tremendous obstacles for an effective cancer therapy. Herein, we developed a melittin-RADA32 hybrid peptide hydrogel loaded with doxorubicin (DOX) for a potent chemoimmunotherapy against melanoma through the active regulation of TMEs. The formed melittin-RADA32-DOX (MRD) hydrogel has an interweaving nanofiber structure and exhibits excellent biocompatibility, controlled drug release properties both in vitro and in vivo, and an enhanced killing effect to melanoma cells. A single-dose injection of MRD hydrogel retarded the growth of primary melanoma tumors by more than 95% due to loaded melittin and DOX, with concomitant recruitment of activated natural killer cells in the tumors. Furthermore, MRD hydrogel can activate dendritic cells of draining lymph nodes, specifically deplete M2-like tumor-associated macrophages (TAMs), and produce active, cytotoxic T cells to further defend the cells against remaining tumors, providing potent anticancer efficacy against subcutaneous and metastatic tumors in vivo. Multidose injection of MRD hydrogel eliminated 50% of the primary tumors and provided a strong immunological memory effect against tumor rechallenge after eradication of the initial tumors. Owing to its abilities to perform controlled drug release, regulate innate immune cells, deplete M2-like TAMs, direct anticancer and immune-stimulating capabilities, and reshape immunosuppressive TMEs, MRD hydrogel may serve as a powerful tool for anticancer applications.

Increased expression of SHP-1 is associated with local recurrence after radiotherapy in patients with nasopharyngeal carcinoma

Abstract Background. Nasopharyngeal carcinoma (NPC) is a major cancer in southern China. Src homology phosphatase-1 (SHP-1) is a tyrosine phosphatase that regulates growth, differentiation, cell cycle progression, and oncogenesis. We determined the clinical significance of SHP-1 expression in the tumours of NPC patients from southern China who were treated with radiotherapy. Patients and methods. SHP-1 expression was determined by real-time polymerase chain reaction (PCR) and western blotting of NPC tissue samples of 50 patients and nasopharyngeal tissues of 50 non-NPC patients who had chronic nasopharyngeal inflammation. SHP-1 expression was measured in NPC tissue samples of 206 patients by immunohistochemistry and survival analysis was performed. Results. The tumours of NPC patients had significantly increased expression of SHP-1 at mRNA and protein levels relative to patients with chronic nasopharyngeal inflammation. Survival analysis of NPC patients indicated that SHP-1 expression was significantly associated with poor local recurrence-free survival (p = 0.008), but not with nodal recurrence- free survival, distant metastasis-free survival, or overall survival. Conclusions. SHP-1 appears to be associated with radiation resistance of NPC cells and can be considered as a candidate marker for prognosis and/or therapeutic target in patients with this type of cancer.

Melatonin suppresses thyroid cancer growth and overcomes radioresistance via inhibition of p65 phosphorylation and induction of ROS.

Thyroid cancer is the most common endocrine carcinoma with increasing incidence worldwide and anaplastic subtypes are frequently associated with cancer related death. Radioresistance of thyroid cancer often leads to therapy failure and cancer-related death. In this study, we found that melatonin showed potent suppressive roles on NF-κB signaling via inhibition of p65 phosphorylation and generated redox stress in thyroid cancer including the anaplastic subtypes. Our data showed that melatonin significantly decreased cell viability, suppressed cell migration and induced apoptosis in thyroid cancer cell lines in vitro and impaired tumor growth in the subcutaneous mouse model in vivo. By contrast, irradiation of thyroid cancer cells resulted in elevated level of phosphorylated p65, which could be reversed by cotreatment with melatonin. Consequently, melatonin synergized with irradiation to induce cytotoxicity to thyroid cancer, especially in the undifferentiated subgroups. Taken together, our results suggest that melatonin may exert anti-tumor activities against thyroid carcinoma by inhibition of p65 phosphorylation and induction of reactive oxygen species. Radio-sensitization by melatonin may have clinical benefits in thyroid cancer.