FeS2-incorporated 3D PCL scaffold improves new bone formation and neovascularization in a rat calvarial defect model.

199Three-dimensional (3D) scaffolds composed of various biomaterials, including metals, ceramics, and synthetic polymers, have been widely used to regenerate bone defects. However, these materials possess clear downsides, which prevent bone regeneration. Therefore, composite scaffolds have been developed to compensate these disadvantages and achieve synergetic effects. In this study, a naturally occurring biomineral, FeS2, was incorporated in PCL scaffolds to enhance the mechanical properties, which would in turn influence the biological characteristics. The composite scaffolds consisting of different weight fractions of FeS2 were 3D printed and compared to pure PCL scaffold. The surface roughness (5.77-fold) and the compressive strength (3.38-fold) of the PCL scaffold was remarkably enhanced in a dose-dependent manner. The in vivo results showed that the group with PCL/ FeS2 scaffold implanted had increased neovascularization and bone formation (2.9-fold). These results demonstrated that the FeS2 incorporated PCL scaffold might be an effective bioimplant for bone tissue regeneration.

Advances in the development of tubular structures using extrusion-based 3D cell-printing technology for vascular tissue regenerative applications.

Until recent, there are no ideal small diameter vascular grafts available on the market. Most of the commercialized vascular grafts are used for medium to large-sized blood vessels. As a solution, vascular tissue engineering has been introduced and shown promising outcomes. Despite these optimistic results, there are limitations to commercialization. This review will cover the need for extrusion-based 3D cell-printing technique capable of mimicking the natural structure of the blood vessel. First, we will highlight the physiological structure of the blood vessel as well as the requirements for an ideal vascular graft. Then, the essential factors of 3D cell-printing including bioink, and cell-printing system will be discussed. Afterwards, we will mention their applications in the fabrication of tissue engineered vascular grafts. Finally, conclusions and future perspectives will be discussed.

Overcome the barriers of the skin: exosome therapy.

Exosomes are nano-sized cargos with a lipid bilayer structure carrying diverse biomolecules including lipids, proteins, and nucleic acids. These small vesicles are secreted by most types of cells to communicate with each other. Since exosomes circulate through bodily fluids, they can transfer information not only to local cells but also to remote cells. Therefore, exosomes are considered potential biomarkers for various treatments. Recently, studies have shown the efficacy of exosomes in skin defects such as aging, atopic dermatitis, and wounds. Also, exosomes are being studied to be used as ingredients in commercialized skin treatment products. In this review, we discussed the need for exosomes in skin therapy together with the current challenges. Moreover, the functional roles of exosomes in terms of skin treatment and regeneration are overviewed. Finally, we highlighted the major limitations and the future perspective in exosome engineering.

3D-printed gelatin methacrylate (GelMA)/silanated silica scaffold assisted by two-stage cooling system for hard tissue regeneration.

Among many biomaterials, gelatin methacrylate (GelMA), a photocurable protein, has been widely used in 3D bioprinting process owing to its excellent cellular responses, biocompatibility and biodegradability. However, GelMA still shows a low processability due to the severe temperature dependence of viscosity. To overcome this obstacle, we propose a two-stage temperature control system to effectively control the viscosity of GelMA. To optimize the process conditions, we evaluated the temperature of the cooling system (jacket and stage). Using the established system, three GelMA scaffolds were fabricated in which different concentrations (0, 3 and 10 wt%) of silanated silica particles were embedded. To evaluate the performances of the prepared scaffolds suitable for hard tissue regeneration, we analyzed the physical (viscoelasticity, surface roughness, compressive modulus and wettability) and biological (human mesenchymal stem cells growth, western blotting and osteogenic differentiation) properties. Consequently, the composite scaffold with greater silica contents (10 wt%) showed enhanced physical and biological performances including mechanical strength, cell initial attachment, cell proliferation and osteogenic differentiation compared with those of the controls. Our results indicate that the GelMA/silanated silica composite scaffold can be potentially used for hard tissue regeneration.

Drosophila PEBP1 inhibits intestinal stem cell aging via suppression of ERK pathway.

The intestine is a high cellular turnover tissue largely dependent on the regenerative function of stem cell throughout life, and a signaling center for the health and viability of organisms. Therefore, better understanding of the mechanisms underlying the regulation of intestinal stem cell (ISC) regenerative potential is essential for the possible intervention of aging process and age-related diseases. Drosophila midgut is a well-established model system for studying the mechanisms underlying ISC regenerative potential during aging. Here, we report the requirement of Drosophila phosphatidylethanolamine binding protein 1 (PEBP1) in ISC regenerative potential. We showed that PEBP1 was strongly expressed in enterocytes (ECs) of guts and its decrease with age and oxidative stress. Furthermore, the downregulation of PEBP1 in ECs accelerates ISC aging, as evidenced by ISC hyper-proliferation, γH2AX accumulation, and centrosome amplification, and intestinal hyperplasia. The decrease in PEBP1 expression was associated with increased extracellular signal-regulated kinase (ERK) activity in ECs. All these phenotypes by EC-specific depletion of PEBP1 were rescued by the concomitant inhibition of ERK signaling. Our findings evidence that the age-related downregulation of PEBP1 in ECs is a novel cause accelerating ISC aging and that PEBP1 is an EC-intrinsic suppressor of epidermal growth factor receptor (EGFR)/ERK signaling. Our study provides molecular insights into the tight regulation of EGFR/ERK signaling in niches for stem cell regenerative potential.

Effect of heterochromatin stability on intestinal stem cell aging in Drosophila.

Chromatin change is one of the crucial causes of aging. Specifically, maintenance of heterochromatin stability is critical for cellular integrity, and its loss induces genomic instability and cellular aging. However, the causes and effects of heterochromatin instability in multicellular tissue aging still remain unclear. Here, in the adult Drosophila midgut, we report age-related loss of heterochromatin stability in enterocytes (ECs) due to the loss and dispersion of tri-methylated histone H3 Lys9 (H3K9me3) and heterochromatin protein 1 (HP1). Our study further shows that EC-specific knockdown of Su(var)3-9, histone lysine methyltransferase for H3K9me3 formation, or HP1a leads to intestinal stem cell (ISC) aging through genomic stress, JNK signaling, and apoptotic death in ECs. Our findings revealed the plausible causes of age-related loss of heterochromatin stability in ECs, including oxidative stress and nutrient-sensing AKT/TOR signaling. Taken together, the loss of heterochromatin stability may be the crucial niche aging mechanism for ISC aging which is the prime determinant of intestinal tissue aging. Furthermore, our study provides new clues on the link between heterochromatin and aging.

Efficacy of rhBMP-2 Loaded PCL/ß-TCP/bdECM Scaffold Fabricated by 3D Printing Technology on Bone Regeneration.

This study was undertaken to evaluate the effect of 3D printed polycaprolactone (PCL)/ß-tricalcium phosphate (ß-TCP) scaffold containing bone demineralized and decellularized extracellular matrix (bdECM) and human recombinant bone morphogenetic protein-2 (rhBMP-2) on bone regeneration. Scaffolds were divided into PCL/ß-TCP, PCL/ß-TCP/bdECM, and PCL/ß-TCP/bdECM/BMP groups. In vitro release kinetics of rhBMP-2 were determined with respect to cell proliferation and osteogenic differentiation. These three reconstructive materials were implanted into 8 mm diameter calvarial bone defect in male Sprague-Dawley rats. Animals were sacrificed four weeks after implantation for micro-CT, histologic, and histomorphometric analyses. The findings obtained were used to calculate new bone volumes (mm3) and new bone areas (%). Excellent cell bioactivity was observed in the PCL/ß-TCP/bdECM and PCL/ß-TCP/bdECM/BMP groups, and new bone volume and area were significantly higher in the PCL/ß-TCP/bdECM/BMP group than in the other groups (p < .05). Within the limitations of this study, bdECM printed PCL/ß-TCP scaffolds can reproduce microenvironment for cells and promote adhering and proliferating the cells onto scaffolds. Furthermore, in the rat calvarial defect model, the scaffold which printed rhBMP-2 loaded bdECM stably carries rhBMP-2 and enhances bone regeneration confirming the possibility of bdECM as rhBMP-2 carrier.

Deficiency of Atg6 impairs beneficial effect of metformin on intestinal stem cell aging in Drosophila.

Age-related changes of adult stem cell are crucial for tissue aging and age-related diseases. Thus, clarifying mechanisms to prevent adult stem cell aging is indispensable for healthy aging. Metformin, a drug for type 2 diabetes, has been highlighted for its anti-aging and anti-cancer effect. In Drosophila intestinal stem cell (ISC), we previously reported the inhibitory effect of metformin on age-related phenotypes of ISC. Here, we showed that knockdown of Atg6, a crucial autophagy-related factor, in ISC induces age-related phenotypes of ISC such as hyperproliferation, centrosome amplification, and DNA damage accumulation. Then, we revealed that metformin inhibits ISC aging phenotypes in Atg6-dependent manner. Taken together, our study suggests that Atg6 is required for the inhibitory effect of metformin on ISC aging, providing an intervention mechanism of metformin on adult stem cell aging.

Deficiency in DNA damage response of enterocytes accelerates intestinal stem cell aging in Drosophila.

Stem cell dysfunction is closely linked to tissue and organismal aging and age-related diseases, and heavily influenced by the niche cells' environment. The DNA damage response (DDR) is a key pathway for tissue degeneration and organismal aging; however, the precise protective role of DDR in stem cell/niche aging is unclear. The Drosophila midgut is an excellent model to study the biology of stem cell/niche aging because of its easy genetic manipulation and its short lifespan. Here, we showed that deficiency of DDR in Drosophila enterocytes (ECs) accelerates intestinal stem cell (ISC) aging. We generated flies with knockdown of Mre11, Rad50, Nbs1, ATM, ATR, Chk1, and Chk2, which decrease the DDR system in ECs. EC-specific DDR depletion induced EC death, accelerated the aging of ISCs, as evidenced by ISC hyperproliferation, DNA damage accumulation, and increased centrosome amplification, and affected the adult fly's survival. Our data indicated a distinct effect of DDR depletion in stem or niche cells on tissue-resident stem cell proliferation. Our findings provide evidence of the essential role of DDR in protecting EC against ISC aging, thus providing a better understanding of the molecular mechanisms of stem cell/niche aging.

Porosity effect of 3D-printed polycaprolactone membranes on calvarial defect model for guided bone regeneration.

The appropriate porosity and pore size of barrier membranes were associated with the transportation of biomolecules required for new bone formation and angiogenesis. In this study, we fabricated three-dimensional (3D)-printed resorbable polycaprolactone (PCL) membranes with different porosities (30%, 50%, and 70%) to evaluate the effective pore size for guided bone regeneration (GBR) membranes. To analyze mechanical properties and cytocompatibility, PCL membranes prepared using extrusion-based 3D printing technology were compared in dry and wet conditions and tested in vitro. The proliferation rates and pattern of fibroblasts and preosteoblasts on PCL membranes with different porosities were determined using a cell counting kit-8 assay and scanning electron microscopy. PCL membrane porosity did not affect cell proliferation, but osteogenic differentiation and mechanical properties were increased with lower porosity (30%) on day 14 (p < 0.001). Similar results were found in an in vivo calvarial defect model; new bone formation was significantly higher in PCL membranes with lower porosity (p < 0.001). These results indicate that 3D-printed PCL with 30% porosity (130 µm pore size) is an excellent pore size for GBR membranes.

Alternately plasma-roughened nanosurface of a hybrid scaffold for aligning myoblasts.

For successful skeletal muscle tissue regeneration, inducing alignment and fusion of myoblasts into multinucleated myotubes is critical. Many studies are ongoing to induce myoblast alignment using various micro/nanopatternings on scaffold surfaces, mechanically stretching scaffolds, or aligned micro/nanofibers. In this study, we have developed a simple method to induce myoblast alignment using a modified plasma treatment on a hybrid PCL scaffold consisting of melt-printed perpendicular PCL struts and an electrospun PCL fibrous mat. For the hybrid scaffold, the surface of the electrospun mat was selectively roughened with a plasma process supplemented with a template. The cell alignment of myoblasts using this system was enhanced significantly when compared to results from the use of a hybrid scaffold with a non-roughened electrospun fiber surface or a hybrid scaffold where the whole surface of the electrospun fibers was roughened. This new type of plasma-treated hybrid scaffold has strong potential as a biomaterial for use in muscle tissue regeneration.

Requirement of ATR for maintenance of intestinal stem cells in aging Drosophila.

The stem cell genomic stability forms the basis for robust tissue homeostasis, particularly in high-turnover tissues. For the genomic stability, DNA damage response (DDR) is essential. This study was focused on the role of two major DDR-related factors, ataxia telangiectasia-mutated (ATM) and ATM- and RAD3-related (ATR) kinases, in the maintenance of intestinal stem cells (ISCs) in the adultDrosophila midgut. We explored the role of ATM and ATR, utilizing immunostaining with an anti-pS/TQ antibody as an indicator of ATM/ATR activation, γ-irradiation as a DNA damage inducer, and the UAS/GAL4 system for cell type-specific knockdown of ATM, ATR, or both during adulthood. The results showed that the pS/TQ signals got stronger with age and after oxidative stress. The pS/TQ signals were found to be more dependent on ATR rather than on ATM in ISCs/enteroblasts (EBs). Furthermore, an ISC/EB-specific knockdown of ATR, ATM, or both decreased the number of ISCs and oxidative stress-induced ISC proliferation. The phenotypic changes that were caused by the ATR knockdown were more pronounced than those caused by the ATM knockdown; however, our data indicate that ATR and ATM are both needed for ISC maintenance and proliferation; ATR seems to play a bigger role than does ATM.

Metformin inhibits age-related centrosome amplification in Drosophila midgut stem cells through AKT/TOR pathway.

We delineated the mechanism regulating the inhibition of centrosome amplification by metformin in Drosophila intestinal stem cells (ISCs). Age-related changes in tissue-resident stem cells may be closely associated with tissue aging and age-related diseases, such as cancer. Centrosome amplification is a hallmark of cancers. Our recent work showed that Drosophila ISCs are an excellent model for stem cell studies evaluating age-related increase in centrosome amplification. Here, we showed that metformin, a recognized anti-cancer drug, inhibits age- and oxidative stress-induced centrosome amplification in ISCs. Furthermore, we revealed that this effect is mediated via down-regulation of AKT/target of rapamycin (TOR) activity, suggesting that metformin prevents centrosome amplification by inhibiting the TOR signaling pathway. Additionally, AKT/TOR signaling hyperactivation and metformin treatment indicated a strong correlation between DNA damage accumulation and centrosome amplification in ISCs, suggesting that DNA damage might mediate centrosome amplification. Our study reveals the beneficial and protective effects of metformin on centrosome amplification via AKT/TOR signaling modulation. We identified a new target for the inhibition of age- and oxidative stress-induced centrosome amplification. We propose that the Drosophila ISCs may be an excellent model system for in vivo studies evaluating the effects of anti-cancer drugs on tissue-resident stem cell aging.

Age-related change in γH2AX of Drosophila muscle: its significance as a marker for muscle damage and longevity.

Muscle aging is closely related to unhealthy late-life and organismal aging. Recently, the state of differentiated cells was shown to be critical to tissue homeostasis. Thus, understanding how fully differentiated muscle cells age is required for ensuring healthy aging. Adult Drosophila muscle is a useful model for exploring the aging process of fully differentiated cells. In this study, we investigated age-related changes of γH2AX, an indicator of DNA strand breaks, in adult Drosophila muscle to document whether its changes are correlated with muscle degeneration and lifespan. The results demonstrate that γH2AX accumulation increases in adult Drosophila thoracic and leg muscles with age. Analyses of short-, normal-, and long-lived strains indicate that the age-related increase of γH2AX is closely associated with the extent of muscle degeneration, cleaved caspase-3 and poly-ubiquitin aggregates, and longevity. Further analysis of muscle-specific knockdown of heterochromatin protein 1a revealed that the excessive γH2AX accumulation in thoracic and leg muscles induces accelerated degeneration and decreases longevity. These data suggest a strong correlation between age-related muscle damage and lifespan in Drosophila. Our findings indicate that γH2AX may be a reliable biomarker for assessing muscle aging in Drosophila.

Increased centrosome amplification in aged stem cells of the Drosophila midgut.

Age-related changes in long-lived tissue-resident stem cells may be tightly linked to aging and age-related diseases such as cancer. Centrosomes play key roles in cell proliferation, differentiation and migration. Supernumerary centrosomes are known to be an early event in tumorigenesis and senescence. However, the age-related changes of centrosome duplication in tissue-resident stem cells in vivo remain unknown. Here, using anti-γ-tubulin and anti-PH3, we analyzed mitotic intestinal stem cells with supernumerary centrosomes in the adult Drosophila midgut, which may be a versatile model system for stem cell biology. The results showed increased centrosome amplification in intestinal stem cells of aged and oxidatively stressed Drosophila midguts. Increased centrosome amplification was detected by overexpression of PVR, EGFR, and AKT in intestinal stem cells/enteroblasts, known to mimic age-related changes including hyperproliferation of intestinal stem cells and hyperplasia in the midgut. Our data show the first direct evidence for the age-related increase of centrosome amplification in intestinal stem cells and suggest that the Drosophila midgut is an excellent model for studying molecular mechanisms underlying centrosome amplification in aging adult stem cells in vivo.

Functional modification of Drosophila intestinal stem cells by ionizing radiation.

Although the diverse effects of ionizing radiation on biological and pathological processes at various levels ranging from molecular to whole body are well studied, the effects on adult stem cells by ionizing radiation remain largely unknown. In this study, we characterized the functional modifications of adult Drosophila midgut intestinal stem cells after ionizing radiation treatment. A dose of 10 Gy of radiation decreased the proliferative capacity of intestinal stem cells. Interestingly, after irradiation at 2 Gy, the intestinal stem cells exhibited increased proliferative activity, misdifferentiation and γH2AvD and 8-oxo-dG levels. In addition, the guts irradiated with 2 Gy showed increased JNK and AKT activities. Furthermore, we showed that 2 Gy of ionizing radiation induced centrosome amplification in intestinal stem cells of adult midguts. Our data gives molecular insights into the effects of ionizing radiation on functional modifications of stem cells. The adult Drosophila midgut intestinal stem cells offer a potentially rich new system for the exploration of the biological effects of ionizing radiation.

A surface-modified poly(ɛ-caprolactone) scaffold comprising variable nanosized surface-roughness using a plasma treatment.

Melt-plotted poly (ɛ-caprolactone) (PCL) has been widely applied in various tissue regenerations. However, its hydrophobic nature has hindered its usage in wider tissue engineering applications. In this study, we present the development of a porous and multilayered PCL scaffold, which shows outstanding hydrophilic properties and has a roughened surface consisting of homogeneously distributed nanosized pits. The scaffold was obtained using an innovative oxygen plasma treatment. This technology can induce variable nanoscale surface roughness, which is difficult from traditional plasma treatment. Osteoblast-like cells were cultured on the scaffolds and several cellular responses (cell viability, fluorescence images [live/dead cells, nucleus, and actin cytoskeleton], ALP activity, and calcium mineralization) were assessed for untreated PCL and conventionally plasma-treated PCL scaffolds. The data indicated that an appropriate roughness (654 ± 91 nm) of the PCL scaffold processed with the new plasma treatment induced more advantageous responses for the cells, compared with untreated scaffolds and traditional plasma-treated scaffolds.

A mini-review: Cell response to microscale, nanoscale, and hierarchical patterning of surface structure.

Cellular behavior can be influenced by the chemical and physical surface characteristics of biomedical substrates. To understand the relationships between various topographical surface patterns and cellular activities, various types of pattern models have been developed and examined in a range of sizes (microscale, nanoscale, and hierarchical structures consisting of both) and shapes (pillar, hole, groove, grate, grid, and island). Here, we review fabrication methods for obtaining physically patterned microscale and nanoscale surfaces, and discuss the relationships between cellular responses and physically patterned surfaces, which could be applied to various biomedical scaffolds used in tissue engineering applications.

Preparation and characterization of an electrospun polycaprolactone (PCL) fibrous mat and multi-layered PCL scaffolds having a nanosized pattern-surface for tissue regeneration.

A method for fabrication of nanoscale surface patterns on electrospun polycaprolactone (PCL) fibers and melt-plotted PCL struts was developed. Physical patterns were achieved using selective plasma treatment in the presence of an anodic aluminum oxide (AAO) template (800 nm). The nanoscale-patterned surfaces were evaluated using X-ray photoelectron spectroscopy (XPS) and topological analyses. The roughness (Ra) of the fabricated patterns on the electrospun PCL fiber surfaces was 716 ± 43 nm, while normally plasma-treated surfaces exhibited relatively low roughness (Ra = 126 ± 13 nm). To evaluate the feasibility of using a microfibrous PCL mat with a nanoscale-roughened surface as a biomedical scaffold, osteoblast-like cells (MG63) were cultured and analyzed using fluorescence analysis (live/dead and 4',6-diamidino-2-phenylindole (DAPI)/phalloidin analyses), alkaline phosphatase (ALP) activity determination, and calcium deposition. The selectively plasma-treated PCL mats exhibited outstanding biological activities, such as cell proliferation and differentiation, compared with untreated PCL fibrous mats (control) and normally plasma-treated fibrous mats.

Mechanism of metformin: inhibition of DNA damage and proliferative activity in Drosophila midgut stem cell.

Age-related changes in stem cells could have a profound impact on tissue aging and the development of age-related diseases such as cancer. However, the effects of metformin, a recently recognized anti-cancer drug, on stem cell aging remain largely unknown. In the present study, an experiment was set up to investigate the underlying mechanism of metformin's beneficial effects on age-related changes in intestinal stem cells (ISCs) derived from Drosophila midgut. Results showed that metformin reduced age- and oxidative stress-related accumulation of DNA damage marked by Drosophila γH2AX foci and 8-oxo-dG in ISCs and progenitor cells. Metformin also inhibited age and- oxidative stress-related ISC hyperproliferation as well as intestinal hyperplasia. Our study further revealed that the inhibitory effects of metformin on DNA damage accumulation may be due to the down-regulation of age-related and oxidative stress-induced AKT activity. These data indicate that metformin has beneficial effects on age-related changes in ISCs derived from Drosophila midgut. Further, our results suggest a possible impact of DNA damage on stem cell genomic instability, which leads to the development of age-related diseases. Additionally, our study suggests that Drosophila midgut stem cells can be a suitable model system for studying stem cell biology and stem cell aging.