Effect of Integrin Binding Peptide on Vascularization of Scaffold-Free Microtissue Spheroids.

BACKGROUND: Three-dimensional (3D) biomimetic models via various approaches can be used by therapeutic applications of tissue engineering. Creating an optimal vascular microenvironment in 3D model that mimics the extracellular matrix (ECM) and providing an adequate blood supply for the survival of cell transplants are major challenge that need to be overcome in tissue regeneration. However, currently available scaffolds-depended approaches fail to mimic essential functions of natural ECM. Scaffold-free microtissues (SFMs) can successfully overcome some of the major challenges caused by scaffold biomaterials such as low cell viability and high cost. METHODS: Herein, we investigated the effect of soluble integrin binding peptide of arginine-glycine-aspartic acid (RGD) on vascularization of SFM spheroids of human umbilical vein endothelial cells. In vitro-fabricated microtissue spheroids were constructed and cultivated in 0 mM, 1 mM, 2 mM, and 4 mM of RGD peptide. The dimensions and viability of SFMs were measured. RESULTS: Maximum dimension and cell viability observed in 2 mM RGD containing SFM. Vascular gene expression of 2 mM RGD containing SFM were higher than other groups, while 4 mM RGD containing SFM expressed minimum vascularization related genes. Immunofluorescent staining results indicating that platelet/endothelial cell adhesion molecule and vascular endothelial growth factor protein expression of 2 mM RGD containing SFM was higher compared to other groups. CONCLUSION: Collectively, these findings demonstrate that SFM spheroids can be successfully vascularized in determined concentration of RGD peptide containing media. Also, soluble RGD incorporated SFMs can be used as an optimal environment for successful prevascularization strategies.

Enhanced osteogenesis of human mesenchymal stem cells by self-assembled peptide hydrogel functionalized with glutamic acid templated peptides.

Self-assembling peptide (SAP) hydrogel has been shown to be an excellent biological material for three-dimensional cell culture and stimulatie cell migration and differentiation into the scaffold, as well as for repairing bone tissue defects. Herein, we designed one of the SAP scaffolds KLD (KLDLKLDLKLDL) through direct coupling to short bioactive motif O1 (EEGGC) and O2 (EEEEE) of which bioactivity on osteogenic differentiation was previously demonstrated and self-assembled in different concentrations (0.5%, 1%, and 2%). Our aim was to enhance osteogenesis and biomineralization of injectable SAP hydrogels with controlled mechanical properties so that the peptide hydrogel also becomes capable of being injected to bone defects. The molecular integration of the nanofibrous peptide scaffolds was observed using atomic force microscopy (AFM) and scanning electron microscopy (SEM). The rheological properties and degradation profile of SAP hydrogels were evaluated to ensure stability of SAPs. Compared with pure KLD scaffold, we found that these designed bioactive peptide scaffolds significantly promoted hMSCs proliferation depicted by biochemical analysis of alkaline phosphatase (ALP) activity, total calcium deposition. Moreover, key osteogenic markers of ALP activity, collagen type I (COL-1), osteopontin (OP), and osteocalcin (OCN) expression levels determined by real-time polymerase chain reaction (PCR) and immunofluorescence analysis were also significantly increased with the addition of glutamic acid residues to KLD. We demonstrated that the designed SAP scaffolds promoted the proliferation and osteogenic differentiation of hMSCs. Our results suggest that these designed bioactive peptide scaffolds may be useful for promoting bone tissue regeneration.

Evaluation of efficacy of non-thermal atmospheric pressure plasma in treatment of periodontitis: a randomized controlled clinical trial.

OBJECTIVES: In this clinical study, we aim to evaluate the effectiveness of non-thermal atmospheric pressure plasma (NAPP), which is a novel procedure used in periodontal pocket decontamination adjunctive to non-surgical periodontal treatment (NSPT). METHODS: The study included 25 systemically healthy periodontitis patients. In the split-mouth design, NAPP application into the pockets, in addition to NSPT, was performed. Clinical periodontal data, gingival crevicular fluid, and subgingival plaque samples of patients were taken before and during the first and third months of treatment. Biochemical assays were conducted using enzyme-linked immunosorbent assay. Analysis of bacteria was performed with polymerase chain reaction method. RESULTS: There was more clinical attachment level (CAL) gain in the 3rd month in the test group (deep pockets: 3.90 mm, pockets ≥ 5 mm: 2.72 mm) compared to the control group (deep pockets: 3.40 mm, pockets ≥ 5 mm: 2.58 mm) (p < 0.05), but no significant difference between groups in CAL. Clinical periodontal parameters improved in both study groups (p < 0.05). However, the gingival index (GI) and the bleeding on probing (BOP) rate decreased more in the test group (GI: 0.55, BOP: 9.48%, and GI: 0.38, BOP: 8.46% in the 1st and 3rd months, respectively) compared to the control group (GI: 0.68, BOP: 13.43%, and GI: 0.52, BOP: 14.58%) (p < 0.05). In addition, there was no significant difference in probing depth and biochemical markers between groups (p > 0.05). It was observed that NAPP reduced the number of bacteria more than the control group in the 1st and 3rd months. CONCLUSIONS: It was seen that the single-time NAPP application concurrent with NSPT provided additional CAL gain, elimination of putative periodontopathogens and reduced their recolonization. Longitudinal studies with larger population and longer time are required. CLINICAL RELEVANCE: NSPT is an effective method for the treatment of periodontitis but bacteria recolonization that causes recurrence of the periodontal disease occurs within a short period. NAPP can reduce the recurrence of periodontal disease by providing better bacterial elimination and should, therefore, be used in maintenance of periodontitis.

Guiding neural extensions of PC12 cells on carbon nanotube tracks dielectrophoretically formed in poly(ethylene glycol) dimethacrylate.

The PC12 cell line has been widely used as an in vitro model for studying neuronal differentiation and identifying the factors affecting the process. It has the ability to differentiate in the presence of nerve growth factor (NGF), resulting in neural extensions called dendrites and axons. In this study, first the impact of randomly distributed multi-walled carbon nanotubes (MWCNTs) in poly(ethylene glycol) dimethacrylate (PEGDMA) on PC12 cell differentiation was investigated in terms of neurite length, number of neurite per cell and differentiation marker gene expression profile. Then, dielectrophoretically aligned MWCNTs in PEGDMA was used to guide and support the neuronal differentiation of PC12 cells in the presence of NGF. The method is expected to be useful in revealing the nanotopographical role in fundamental studies and understanding of nanotopographical effects for biomedical applications on nerve regeneration.

Aspartic and Glutamic Acid Templated Peptides Conjugation on Plasma Modified Nanofibers for Osteogenic Differentiation of Human Mesenchymal Stem Cells: A Comparative Study.

Optimization of nanofiber (NF) surface properties is critical to achieve an adequate cellular response. Here, the impact of conjugation of biomimetic aspartic acid (ASP) and glutamic acid (GLU) templated peptides with poly(lactic-co-glycolic acid) (PLGA) electrospun NF on osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) was evaluated. Cold atmospheric plasma (CAP) was used to functionalize the NF surface and thus to mediate the conjugation. The influence of the CAP treatment following with peptide conjugation to the NF surface was assessed using water contact angle measurements, Fourier-Transform Infrared Spectroscopy (FTIR) and X-ray Photoelectron Spectroscopy (XPS). The effect of CAP treatment on morphology of NF was also checked using Scanning Electron Microscopy (SEM). Both the hydrophilicity of NF and the number of the carboxyl (-COOH) groups on the surface increased with respect to CAP treatment. Results demonstrated that CAP treatment significantly enhanced peptide conjugation on the surface of NF. Osteogenic differentiation results indicated that conjugating of biomimetic ASP templated peptides sharply increased alkaline phosphatase (ALP) activity, calcium content, and expression of key osteogenic markers of collagen type I (Col-I), osteocalcin (OC), and osteopontin (OP) compared to GLU conjugated (GLU-pNF) and CAP treated NF (pNF). It was further depicted that ASP sequences are the major fragments that influence the mineralization and osteogenic differentiation in non-collagenous proteins of bone extracellular matrix.