Enhanced Osteogenic Potential of Noggin Knockout C2C12 Cells on BMP-2 Releasing Silk Scaffolds.

The CRISPR/Cas9 mechanism offers promising therapeutic approaches for bone regeneration by stimulating or suppressing critical signaling pathways. In this study, we aimed to increase the activity of BMP-2 signaling through knockout of Noggin, thereby establishing a synergistic effect on the osteogenic activity of cells in the presence of BMP-2. Since Noggin is an antagonist expressed in skeletal tissues and binds to subunits of bone morphogenetic proteins (BMPs) to inhibit osteogenic differentiation, here Noggin expression was knocked out using the CRISPR/Cas9 system. In accordance with this purpose, C2C12 (mouse myoblast) cells were transfected with CRISPR/Cas9 plasmids. Transfection was achieved with Lipofectamine and confirmed with intense fluorescent signals in microscopic images and deletion in target sequence in Sanger sequencing analysis. Thus, Noggin knockout cells were identified as a new cell source for tissue engineering studies. Then, the transfected cells were seeded on highly porous silk scaffolds bearing BMP-2-loaded silk nanoparticles (30 ng BMP-2/mg silk nanoparticle) in the size of 288 ± 62 nm. BMP-2 is released from the scaffolds in a controlled manner for up to 60 days. The knockout of Noggin by CRISPR/Cas9 was found to synergistically promote osteogenic differentiation in the presence of BMP-2 through increased Coll1A1 and Ocn expression and mineralization. Gene editing of Noggin and BMP-2 increased almost 2-fold Col1A1 expression and almost 3-fold Ocn expression compared to the control group. Moreover, transfected cells produced extracellular matrix (ECM) containing collagen fibers on the scaffolds and mineral-like structures were formed on the fibers. In addition, mineralization characterized by intense Alizarin red staining was detected in transfected cells cultured in the presence of BMP-2, while the other groups did not exhibit any mineralized areas. As has been demonstrated in this study, the CRISPR/Cas9 mechanism has great potential for obtaining new cell sources to be used in tissue engineering studies.

Random/aligned electrospun PCL fibrous matrices with modified surface textures: Characterization and interactions with dermal fibroblasts and keratinocytes.

Micro- or nano-surface topography of a biomaterial can improve various cellular activities for obtaining functional tissues. Electrospun fibers can gain further functionality when introduced topographic details to their surfaces. In this regard, we produced random and aligned polycaprolactone (PCL) micron/submicron fibers by the electrospinning method. Simultaneously, the surface structure of the fibers was altered by applying phase separation processes including non-solvent-induced phase separation (NIPS) and vapor-induced phase separation (VIPS) mechanisms. As a result, PCL fibers with porous, wrinkled, grooved, and crater-like morphology were obtained. Human dermal fibroblasts (BJ cells) and human keratinocytes (HS2) were cultured onto the fiber surfaces and the data were evaluated in terms of cell-material interactions. Results showed that not only the orientation of fibers but also fiber topography affected both cell-fiber and cell-cell interactions in different manners. It was observed that the wrinkled topography is the most suitable for both dermal fibroblasts and keratinocytes in terms of cell attachment and proliferation. We also concluded that cellular behavior was varied according to the morphology of the cells used. Morphological observations showed that HS2 cells proliferated more intensively on all surfaces compared to BJ cells. All these findings can be evaluated in terms of the design of tissue scaffolds, especially in skin tissue engineering.

Investigation of the synergistic effect of platelet-rich plasma and polychromatic light on human dermal fibroblasts seeded chitosan/gelatin scaffolds for wound healing.

Conventional wound healing treatments are insufficient for chronic wounds caused by factors such as senescence of fibroblasts, reduced growth factor synthesis, and poor angiogenesis. Recently, tissue engineering approaches have been investigated to develop effective therapies. In this study, a biochemical/biophysical stimulant-based 3D system was developed for the healing of chronic wounds. In this direction, genipin crosslinked chitosan (CHT)/gelatin (GEL) scaffolds were fabricated by freeze-drying and loaded with platelet-rich plasma (PRP). The scaffolds were seeded with human dermal fibroblasts and then, polychromatic light in near infrared region (NIR) was applied to the scaffolds for activating the platelets and stimulating the fibroblasts (photoactivation, PAC). Thus, fibroblasts were stimulated both chemically and physically by PRP and light, respectively. Cell migration, proliferation, morphology, gene expressions and reactive oxygen species (ROS) activity were evaluated in-vitro. Laminin and collagen 4 expressions that are important for extracellular matrix (ECM) formation, and PDGF (Platelet-derived growth factor) and VEGF (Vascular endothelial growth factor) expressions that are important for vascularization significantly increased in the presence of both PRP and light. Besides, PRP and light improved cell migration in 3D core-and shell model synergistically. Hydrogen peroxide content decreased in both PRP and light, indicating inhibition of ROS production. It was concluded that the stimulation of platelets with light in the NIR has a great potential to use for both platelets activation and stimulation of fibroblasts. As a result, an effective therapy can be developed for chronic wounds by using scaffold-based 3D systems together with PRP and photostimulation.

Enhanced osteogenic effect in reduced BMP-2 doses with siNoggin transfected pre-osteoblasts in 3D silk scaffolds.

Bone morphogenetic proteins (BMPs), especially BMP-2, are being increasingly used in bone tissue engineering due to its osteo-inductive effects. Although recombinant human BMP-2 (rhBMP-2) was approved by Food and Drug Administration (FDA) to use for bone repair, its high doses cause undesired side effects. In order to reduce the BMP-2 dose for enhanced osteogenic differentiation, in this study we decided to suppress the synthesis of Noggin protein, the primary antagonist of BMP-2, on the MC3T3-E1 cells using Noggin targeted small interfering RNA (siRNA). Unlike other studies, Noggin siRNA (siNoggin) transfected cells were seeded on silk scaffolds, and osteogenic differentiation was investigated for a long-term period (21 days) with MTT, qPCR, SEM/EDS, and histological analysis. Besides, siNoggin transfected MC3T3-E1 cells were evaluated as a new cell source for tissue engineering studies. It was determined that Nog gene expression was suppressed in the siNoggin group and Ocn gene expression increased 5-fold compared to the control group (*p < 0.05). The osteogenic effect of BMP-2 was clearly observed in siNoggin transfected cells. According to the SEM/EDS analysis, the siNoggin group has mineral structures clustered on cells, which contain intense Ca and P elements. Histological staining showed that the siNoggin group has a more intense mineralized area than that of the control group. In conclusion, this study indicated that Noggin silencing by siRNA induces osteogenic differentiation in reduced BMP-2 doses for scaffold-based bone regeneration. This non-gene integration strategy has as a safe therapeutic potential to enhance tissue regeneration.

Photostimulation of osteogenic differentiation on silk scaffolds by plasma arc light source.

Low-level laser therapy (LLLT) has been used for more than 30 years to heal wounds. In recent years, LLLT or photostimulation has been indicated as an effective tool for regenerative and dental medicine by using monochromatic light. The aim of this study is to indicate the usability of plasma arc light source for bone regeneration. This is why we used polychromatic light source providing effective wavelengths in the range of 590-1500 nm for cellular response and investigated photostimulation effects on osteogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on 3D silk scaffolds. Cellular responses were examined by using cell culture methods in terms of proliferation, differentiation, and morphological analyses. The results showed that photostimulation with a polychromatic light source (applied for 5 min from the 3rd day after seeding up to the 28th day in 2-day intervals with 92-mW/cm2 power from 10-cm distance to the cells) enhanced osteogenic differentiation of hMSCs according to higher alkaline phosphatase (ALP) activity, collagen and calcium content, osteogenic gene expressions, and matrix mineralization. In conclusion, we suggest that the plasma arc light source that was used here has a great potential for bone regeneration.

Sequential IGF-1 and BMP-6 releasing chitosan/alginate/PLGA hybrid scaffolds for periodontal regeneration.

The goal of periodontal tissue engineering is to repair or regenerate the destructed or lost periodontium by improving functions of cells in the remaining tissue. For continuty of cell growth process, two group of growth factors, i.e. competence factors and progression factors, are needed to act together. However, the short biological half-life of these factors limits their effects on cells and their clinical efficacy. The purpose of this study is to develop different microparticles-loaded chitosan carriers/scaffolds for controlled and sequential delivery of a competence factor, insulin-like growth factor (IGF-1), and progression factor, bone morphogenetic factor-6 (BMP-6). Alginate and poly (lactic-co-glycolic acid) (PLGA) microparticles provided release of IGF-1 and BMP-6 for early short period and for long period, respectively. The cell culture studies showed that, chitosan/alginate/PLGA hybrid scaffolds induced proliferation and osteoblastic differentiation of cementoblasts when compared with IGF-1 and BMP-6 free chitosan scaffold.

RGD-bearing peptide-amphiphile-hydroxyapatite nanocomposite bone scaffold: an in vitro study.

In this study, a fibrous nanocomposite scaffold was developed by combining hydroxyapatite (HA) fibers produced by electrospinning method and arginine-glycine-aspartic acid (RGD)-bearing peptide-amphiphile (PA) gel (PA-RGD) produced by self-assembly and gelation induced by calcium ions. Scanning electron microscope, transmission electron microscope and atomic force microscopy imaging confirmed the successful production of inorganic and organic components of this nanocomposite material. Within the HA, the presence of a CaCO3 phase, improving biodegradation, was shown by x-ray diffraction analysis. The in vitro effectiveness of the PA-RGD/HA scaffold was determined on MC3T3-E1 preosteoblast cultures in comparison with HA matrix and PA-RGD gel. The highest cellular proliferation was obtained on PA-RGD gel, however, alkaline phosphatase activity results denoted that osteogenic differentiation of the cells is more favorable on HA containing matrices with respect to PA-RGD itself. Microscopic observations revealed that all three matrices support cell attachment and proliferation. Moreover, cells form bridges between the HA and PA-RGD components of the nanocomposite scaffold, indicating the integrity of the biphasic components. According to the real time-polymerase chain reaction (RT-PCR) analyses, MC3T3-E1 cells expressed significantly higher osteocalcin on all matrices. Bone sialoprotein (BSP) expression level is ten-fold higher on PA-RGD/HA nanocomposite scaffolds than that of HA and PA-RGD scaffolds and the elevated expression of BSP on PA-RGD/HA nanocomposite scaffolds suggested higher mineralized matrix on this novel scaffold. Based on the results obtained in this study, the combination of HA nanofibers and PA-RGD gel takes advantage of good structural integrity during the cell culture, besides the osteoinductive and osteoconductive properties of the nanofibrous scaffold.