Impact of Induced Pluripotent Stem Cells in Bone Repair and Regeneration.

PURPOSE OF REVIEW: The main objective of this article is to investigate the current trends in the use of induced pluripotent stem cells (iPSCs) for bone tissue repair and regeneration. RECENT FINDINGS: Pluripotent stem cell-based tissue engineering has extended innovative therapeutic approaches for regenerative medicine. iPSCs have shown osteogenic differentiation capabilities and would be an innovative resource of stem cells for bone tissue regenerative applications. This review recapitulates the current knowledge and recent progress regarding utilization of iPSCs for bone therapy. A review of current findings suggests that a combination of a three-dimensional scaffolding system with iPSC technology to mimic the physiological complexity of the native stem cell niche is highly favorable for bone tissue repair and regeneration.

Biomimetic Nanohydroxyapatite Synthesized With/Without Tris-Buffered Simulated Body Fluid: A Comparative Analysis.

Nano hydroxyapatite (nHAp) mimics the inorganic phase of hard tissue such as bone and teeth and, thus, has a wide range of clinical applications. The present study reports on the biomimetic synthesis of nHAp with and without Tris-buffered simulated body fluid (SBF) and investigated the role of buffering conditions on nHAp formation. The hypothesis of this study was that the nucleation and growth rate of nHAp may depend on buffering conditions during the precipitation process. The results of this study suggest that both of the above methods effectively synthesized carbonated "bone-like" nHAp. However, an increased incubation period of 8 hrs was necessary for nHAp synthesized using non Tris-buffered SBF as compared to Tris-buffered SBF which synthesized nHAp in just 3 hrs. Interestingly, there was no change in the chemical functionality for both samples. XRD and TGA analysis confirmed that Tris-buffered SBF facilitated more carbonate ion substitution than the non-Tris-buffered SBF approach. Therefore, this study concluded for the first time that the addition of Tris in SBF accelerates nHAp formation with more carbonate ion substitution. Nevertheless, carbonate ion substituted nHAp could also be synthesized using non Tris-buffered SBF, but would require longer incubation periods. This analysis highlights the importance of pH stability in the SBF for biomimetic nHAp synthesis which is useful for the synthesis of nHAp for a wide range of biomedical applications.

Development of Egg Shell Derived Carbonated Apatite Nanocarrier System for Drug Delivery.

Carbonated apatite has a chemical composition quite similar to biological apatite found in native bone. The incorporation of carbonate (CO2-3) ions groups into the apatitic crystal structure can tailor its crystallinity, solubility and biological activity that benefit the bone repair and regeneration. In this study, we report a simple and elegant method of synthesizing carbonated calcium deficient hydroxyapatite (ECCDHA) nanoparticles from egg shell wastes and its efficacy has been compared with synthetic calcium deficient hydroxyapatite (SCDHA) nanoparticles. Egg shell contains about 94% of calcium carbonate. Fourier transform infrared (FT-IR) spectroscopy results confirmed the carbonate substitution in the apatite as B-type and CHNS/O elemental analysis showed 6 wt.% of carbonate content in ECCDHA. Energy dispersive spectroscopy (EDS) analysis confirmed the presence of biologically relevant elements such as magnesium, strontium, fluoride, potassium etc., in ECCDHA inherited from the egg shell. In vitro cell culture studies confirmed that the ECCDHA is cellular compatible and it has enhanced cell adhesion and proliferation of L6 myoblast cells as compared to SCDHA. The potential of ECCDHA suitable for bone drug applications was tested with an antibiotic drug, doxycycline. The results showed higher drug loading and releasing for ECCDHA as compared to SCDHA during the period of study. Based on these results, the ECCDHA may be considered as a potential bone substitute and drug carrier system.

Development of Silver-Based Bactericidal Composite Nanofibers by Airbrushing.

In this article, we report a simple, cost-effective and eco-friendly method of airbrushing for the fabrication of antibacterial composite nanofibers using Nylon-6 and silver chloride (AgCl). The Nylon-6 is a widely used polymer for various biomedical applications because of its excellent biocompatibility and mechanical properties. Similarly, silver has also been known for their antibacterial, antifungal, antiviral, and anti-inflammatory properties. In order to enhance the antibacterial functionality of the Nylon-6, composite nanofibers in combination with AgCl have been fabricated using airbrush method. The chemical functional groups and morphological studies of the airbrushed Nylon-6/AgCl composite nanofibers were carried out by FTIR and SEM, respectively. The antibacterial activity of airbrushed Nylon-6/AgCl composite nanofibers was evaluated using Gram +ve (Staphylococcus aureus) and Gram -ve (Escherichia coli) bacterial strains. The results showed that the airbrushed Nylon-6/AgCl composite nanofibers have better antibacterial activity against the tested bacterial strains than the airbrushed Nylon-6 nanofibers. Therefore, the airbrushed Nylon-6/AgCl composite nanofibers could be used as a potential antibacterial scaffolding system for tissue engineering and regenerative medicine.

Spatial control of self-organizing vascular networks with programmable aptamer-tethered growth factor photopatterning.

Given the dynamic nature of engineered vascular networks within biofabricated tissue analogues, it is instrumental to have control over the constantly evolving biochemical cues within synthetic matrices throughout tissue remodeling. Incorporation of pro-angiogenic vascular endothelial growth factor (VEGF165) specific aptamers into cell-instructive polymer networks is shown to be pivotal for spatiotemporally controlling the local bioactivity of VEGF that selectively elicit specific cell responses. To harness this effect and quantitatively unravel its spatial resolution, herein, bicomponent micropatterns consisting of VEGF165 specific aptamer-functionalized gelatin methacryloyl (GelMA) (aptamer regions) overlaid with pristine GelMA regions using visible-light photoinitiators (Ru/SPS) were fabricated via two-step photopatterning approach. For the 3D co-culture study, human umbilical vein-derived endothelial cells and mesenchymal stromal cells were used as model cell types. Bicomponent micropatterns with spatially defined spacings (300/500/800 â€‹µm) displayed high aptamer retention, aptamer-fluorescent complementary sequence (CSF) molecular recognition and VEGF sequestration localized within patterned aptamer regions. Stiffness gradient at the interface of aptamer and GelMA regions was observed with high modulus in aptamer region followed by low stiffness GelMA regions. Leveraging aptamer-tethered VEGF's dynamic affinity interactions with CS that upon hybridization facilitates triggered VEGF release, co-culture studies revealed unique characteristics of aptamer-tethered VEGF to form spatially defined luminal vascular networks covered with filopodia-like structures in vitro (spatial control) and highlights their ability to control network properties including orientation over time using CS as an external trigger (temporal control). Moreover, the comparison of single and double exposed regions within micropatterns revealed differences in cell behavior among both regions. Specifically, the localized aptamer-tethered VEGF within single exposed aptamer regions exhibited higher cellular alignment within the micropatterns till d5 of culture. Taken together, this study highlights the potential of photopatterned aptamer-tethered VEGF to spatiotemporally regulate vascular morphogenesis as a tool for controlling vascular remodeling in situ.

Spatiotemporally controlled, aptamers-mediated growth factor release locally manipulates microvasculature formation within engineered tissues.

Spatiotemporally controlled growth factor (GF) delivery is crucial for achieving functional vasculature within engineered tissues. However, conventional GF delivery systems show inability to recapitulate the dynamic and heterogeneous nature of developing tissue's biochemical microenvironment. Herein, an aptamer-based programmable GF delivery platform is described that harnesses dynamic affinity interactions for facilitating spatiotemporal control over vascular endothelial GF (VEGF165) bioavailability within gelatin methacryloyl matrices. The platform showcases localized VEGF165 sequestration from the culture medium (offering spatial-control) and leverages aptamer-complementary sequence (CS) hybridization for triggering VEGF165 release (offering temporal-control), without non-specific leakage. Furthermore, extensive 3D co-culture studies (human umbilical vein-derived endothelial cells & mesenchymal stromal cells), in bi-phasic hydrogel systems revealed its fundamentally novel capability to selectively guide cell responses and manipulate lumen-like microvascular networks via spatiotemporally controlling VEGF165 bioavailability within 3D microenvironment. This platform utilizes CS as an external biochemical trigger for guiding vascular morphogenesis which is suitable for creating dynamically controlled engineered tissues.

Designing vascular supportive albumen-rich composite bioink for organ 3D printing.

Bioink plays a major role in 3D printing of tissues and organs. Alginate is a widely used component for bioinks but its cellular responses are limited, which limits its clinical translation. In this study, we demonstrate the printability and cellular compatibility of composite bioink consists of sodium alginate (NaAlg) and egg white, also called albumen. The experimental conditions necessary for 3D printing composite bioink were optimized by changing different concentration ratios of Albumen/NaAlg and their various physicochemical properties were studied. The structural characteristics of the 3D printed scaffold was also investigated. In vitro experiments showed that human umbilical vein endothelial cells can successfully attach to the printed scaffold and maintain high viability during the course of study. Interestingly, vascular sprouting and neovascular network formation was observed inbetween fibers within the printed scaffold. In conclusion, the results of this study demonstrate that 3D printed Albumen/NaAlg composite bioinks with favorable biological functionality hold a great potential in tissue and organ engineering.

Enhanced proliferation of human bone marrow derived mesenchymal stem cells on tough hydrogel substrates.

Stem cell plays a significant role in tissue engineering and regenerative medicine. However, one of the major limitations in translation of stem cell technologies for clinical applications is limited cell survival and growth upon implantation. To address this limitation, authors have made an attempt to design polyacrylamide/alginate (PAAm/Algi) based tough hydrogel substrates and studied their impact on the survival and proliferation of human bone marrow-derived mesenchymal stem cells (hBMSCs). The PAAm/Algi hydrogel substrates have been prepared by initiator-induced free radical polymerization with mechanical properties quite similar to human soft tissues. To evaluate the efficacy of hydrogel substrates in support of cellular functions, hBMSCs were cultured on the PAAm/Algi hydrogel substrate (Gel system) and conventional tissue culture plate (TcP system) under defined conditions. The results of this study demonstrated that the cells cultured on the Gel and TcP systems showed 80-90% of cell viability throughout the period of study. The cells cultured on the Gel system showed 25% increase in proliferation after 7days of culture, whereas the TcP system showed only an increase of 10%. These results confirm the cellular compatibility and enhanced cell proliferative nature of the hydrogel substrates, due the fact that the hydrogel substrates provided necessary microenvironmental cues to the cells as compared the conventional TcP system. The overall results suggest that the PAAm/Algi based hydrogels could be used as a potential substrate for hBMSCs culture and expansion.

Development of decellularized scaffolds for stem cell-driven tissue engineering.

Organ transplantation is an effective treatment for chronic organ dysfunctioning conditions. However, a dearth of available donor organs for transplantation leads to the death of numerous patients waiting for a suitable organ donor. The potential of decellularized scaffolds, derived from native tissues or organs in the form of scaffolds has been evolved as a promising approach in tissue-regenerative medicine for translating functional organ replacements. In recent years, donor organs, such as heart, liver, lung and kidneys, have been reported to provide acellular extracellular matrix (ECM)-based scaffolds through the process called 'decellularization' and proved to show the potential of recellularization with selected cell populations, particularly with stem cells. In fact, decellularized stem cell matrix (DSCM) has also emerged as a potent biological scaffold for controlling stem cell fate and function during tissue organization. Despite the proven potential of decellularized scaffolds in tissue engineering, the molecular mechanism responsible for stem cell interactions with decellularized scaffolds is still unclear. Stem cells interact with, and respond to, various signals/cues emanating from their ECM. The ability to harness the regenerative potential of stem cells via decellularized ECM-based scaffolds has promising implications for tissue-regenerative medicine. Keeping these points in view, this article reviews the current status of decellularized scaffolds for stem cells, with particular focus on: (a) concept and various methods of decellularization; (b) interaction of stem cells with decellularized scaffolds; (c) current recellularization strategies, with associated challenges; and (iv) applications of the decellularized scaffolds in stem cell-driven tissue engineering and regenerative medicine. Copyright © 2015 John Wiley & Sons, Ltd.

Covalently immobilized VEGF-mimicking peptide with gelatin methacrylate enhances microvascularization of endothelial cells.

Clinically usable tissue-engineered constructs are currently limited due to their inability of forming microvascular networks necessary for adequate cellular oxygen and nutrient supply upon implantation. The aim of this study is to investigate the conditions necessary for microvascularization in a tissue-engineered construct using vascular endothelial growth factor (VEGF). The construct was made of gelatin methacrylate (GelMA) based cell-laden hydrogel system, which was then covalently linked with VEGF-mimicking peptide (AcQK), using human umbilical vein endothelial cells (HUVECs) as the model cell. The results of the mechanics and gene expression analysis indicated significant changes in mechanical properties and upregulation of vascular-specific genes. The major finding of this study is that the increased expression of vascular-specific genes could be achieved by employing AcQK in the GelMA based hydrogel system, leading to accelerated microvascularization. We conclude that GelMA with covalently-linked angiogenic peptide is a useful tissue engineered construct suitable for microvascularization. STATEMENT OF SIGNIFICANCE: (1) This study reports the conditions necessary for microvascularization in a tissue-engineered construct using vascular endothelial growth factor (VEGF). (2) The construct was made of gelatin methacrylate based cell-laden hydrogel system. (3) There is a significant change observed in mechanical properties and upregulation of vascular-specific genes, in particular CD34, when AcQK is used. (4) The major finding of this study is that the increased expression of vascular-specific genes, i.e., CD34 could be achieved by employing AcQK in the GelMA based hydrogel system, leading to accelerated microvascularization.

Design and fabrication of auxetic PCL nanofiber membranes for biomedical applications.

The main objective of this study was to fabricate poly (ε-caprolactone) (PCL)-based auxetic nanofiber membranes and characterize them for their mechanical and physicochemical properties. As a first step, the PCL nanofibers were fabricated by electrospinning with two different thicknesses of 40µm (called PCL thin membrane) and 180µm (called PCL thick membrane). In the second step, they were tailored into auxetic patterns using femtosecond laser cut technique. The physicochemical and mechanical properties of the auxetic nanofiber membranes were studied and compared with the conventional electrospun PCL nanofibers (non-auxetic nanofiber membranes) as a control. The results showed that there were no significant changes observed among them in terms of their chemical functionality and thermal property. However, there was a notable difference observed in the mechanical properties. For instance, the thin auxetic nanofiber membrane showed the magnitude of elongation almost ten times higher than the control, which clearly demonstrates the high flexibility of auxetic nanofiber membranes. This is because that the auxetic nanofiber membranes have lesser rigidity than the control nanofibers under the same load which could be due to the rotational motion of the auxetic structures. The major finding of this study is that the auxetic PCL nanofiber membranes are highly flexible (10-fold higher elongation capacity than the conventional PCL nanofibers) and have tunable mechanical properties. Therefore, the auxetic PCL nanofiber membranes may serve as a potent material in various biomedical applications, in particular, tissue engineering where scaffolds with mechanical cues play a major role.

Surface functionalization of nanobiomaterials for application in stem cell culture, tissue engineering, and regenerative medicine.

Stem cell-based approaches offer great application potential in tissue engineering and regenerative medicine owing to their ability of sensing the microenvironment and respond accordingly (dynamic behavior). Recently, the combination of nanobiomaterials with stem cells has paved a great way for further exploration. Nanobiomaterials with engineered surfaces could mimic the native microenvironment to which the seeded stem cells could adhere and migrate. Surface functionalized nanobiomaterial-based scaffolds could then be used to regulate or control the cellular functions to culture stem cells and regenerate damaged tissues or organs. Therefore, controlling the interactions between nanobiomaterials and stem cells is a critical factor. However, surface functionalization or modification techniques has provided an alternative approach for tailoring the nanobiomaterials surface in accordance to the physiological surrounding of a living cells; thereby, enhancing the structural and functional properties of the engineered tissues and organs. Currently, there are a variety of methods and technologies available to modify the surface of biomaterials according to the specific cell or tissue properties to be regenerated. This review highlights the trends in surface modification techniques for nanobiomaterials and the biological relevance in stem cell-based tissue engineering and regenerative medicine. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:554-567, 2016.