Chieh-Cheng Huang

The use of injectable spherically symmetric cell aggregates self-assembled in a thermo-responsive hydrogel for enhanced cell transplantation.

Typical cell transplantation techniques involve the administration of dissociated cells directly injected into muscular tissues; however, retention of the transplanted cells at the sites of the cell graft is frequently limited. An approach, using spherically symmetric aggregates of cells with a relatively uniform size self-assembled in a thermo-responsive methylcellulose hydrogel system, is reported in the study. The obtained cell aggregates preserved their endogenous extracellular matrices (ECM) and intercellular junctions because no proteolytic enzyme was used when harvesting the cell aggregates. Most of the cells within aggregates (with a radius of approximately 100 microm) were viable as indicated by the live/dead staining assay. After injection through a needle, the cell aggregates remained intact and the cells retained their activity upon transferring to another growth surface. The cell aggregates obtained under sterile conditions were transplanted into the skeletal muscle of rats via local injection. The dissociated cells were used as a control. It was found that the cell aggregates can provide an adequate physical size to entrap into the muscular interstices and offer a favorable ECM environment to enhance retention of the transplanted cells at the sites of the cell graft. These results indicated that the spherically symmetric cell aggregates developed in the study may serve as a cell delivery vehicle for therapeutic applications.

An Implantable Depot That Can Generate Oxygen in Situ for Overcoming Hypoxia-Induced Resistance to Anticancer Drugs in Chemotherapy

In the absence of adequate oxygen, cancer cells that are grown in hypoxic solid tumors resist treatment using antitumor drugs (such as doxorubicin, DOX), owing to their attenuated intracellular production of reactive oxygen species (ROS). Hyperbaric oxygen (HBO) therapy favorably improves oxygen transport to the hypoxic tumor tissues, thereby increasing the sensitivity of tumor cells to DOX. However, the use of HBO with DOX potentiates the ROS-mediated cytotoxicity of the drug toward normal tissues. In this work, we hypothesize that regional oxygen treatment by an implanted oxygen-generating depot may enhance the cytotoxicity of DOX against malignant tissues in a highly site-specific manner, without raising systemic oxygen levels. Upon implantation close to the tumor, the oxygen-generating depot reacts with the interstitial medium to produce oxygen in situ, effectively shrinking the hypoxic regions in the tumor tissues. Increasing the local availability of oxygen causes the cytotoxicity of DOX that is accumulated in the tumors to be significantly enhanced by the elevated production of ROS, ultimately allaying the hypoxia-induced DOX resistance in solid malignancies. Importantly, this enhancement of cytotoxicity is limited to the site of the tumors, and this feature of the system that is proposed herein is unique.

Injectable PLGA porous beads cellularized by hAFSCs for cellular cardiomyoplasty

Cellular cardiomyoplasty has been limited by poor graft retention after cell transplantation. To ensure good retention of the engrafted cells, a microfluidic device was used to fabricate spherical porous beads of poly(D,L-lactic-co-glycolic acid) as a platform for cell delivery. The beads thus obtained had a relatively uniform size, a highly porous structure, and a favorably interconnected interior architecture, to facilitate the transportation of oxygen and nutrients. These porous beads were loaded with human amniotic fluid stem cells (hAFSCs) to generate cellularized microscaffolds. Live/dead assay demonstrated that most of the cells in the porous constructs were viable. The hAFSCs that were grown in beads formed a complex three-dimensional organization with well-preserved extracellular matrices (ECM) according to their porous structure. Retention of the administered beads was clearly identified at the site of engraftment following an experimentally induced myocardial infarction in a rat model. The results of echocardiography, magnetic resonance imaging, and histological analyses suggest that the transplantation of hAFSC beads into an infarcted heart could effectively maintain its gross morphology, prevent successive ventricular expansion, and thereby improve the post-infarcted cardiac function. Immunofluorescent staining revealed that the microenvironment that was provided by the infarcted myocardium might offer cues for the induction of the engrafted hAFSCs into angiogenic and cardiomyogenic lineages. Our results demonstrate that the cellularized beads with endogenously secreted ECM were of sufficient physical size to be entrapped in the interstitial tissues following transplantation, thereby benefiting the infarcted heart.

Three-dimensional cell aggregates composed of HUVECs and cbMSCs for therapeutic neovascularization in a mouse model of hindlimb ischemia

The proximity of cells in three-dimensional (3D) organization maximizes the cell-cell communication and signaling that are critical for cell function. In this study, 3D cell aggregates composed of human umbilical vein endothelial cells (HUVECs) and cord-blood mesenchymal stem cells (cbMSCs) were used for therapeutic neovascularization to rescue tissues from critical limb ischemia. Within the cell aggregates, homogeneously mixed HUVECs and cbMSCs had direct cell-cell contact with expressions of endogenous extracellular matrices and adhesion molecules. Although dissociated HUVECs/cbMSCs initially formed tubular structures on Matrigel, the grown tubular network substantially regressed over time. Conversely, 3D HUVEC/cbMSC aggregates seeded on Matrigel exhibited an extensive tubular network that continued to expand without regression. Immunostaining experiments show that, by differentiating into smooth muscle cell (SMC) lineages, the cbMSCs stabilize the HUVEC-derived tubular network. The real-time PCR analysis results suggest that, through myocardin, TGF-β signaling regulates the differentiation of cbMSCs into SMCs. Transplantation of 3D HUVEC/cbMSC aggregates recovered blood perfusion in a mouse model of hindlimb ischemia more effectively compared to their dissociated counterparts. The experimental results confirm that the transplanted 3D HUVEC/cbMSC aggregates enhanced functional vessel formation within the ischemic limb and protected it from degeneration. The 3D HUVEC/cbMSC aggregates can therefore facilitate the cell-based therapeutic strategies for modulating postnatal neovascularization.

Vascularization and restoration of heart function in rat myocardial infarction using transplantation of human cbMSC/HUVEC core-shell bodies.

Cell transplantation is a promising strategy for therapeutic treatment of ischemic heart diseases. In this study, cord blood mesenchymal stem cells (cbMSCs) and human umbilical vein endothelial cells (HUVECs) in the form of core-shell bodies (cbMSC/HUVEC bodies) were prepared to promote vascularization and restore heart functions in an experimentally-created myocardial infarction (MI) rat model. Saline, cbMSC bodies and HUVEC bodies were used as controls. In vitro results indicated that cbMSC/HUVEC bodies possessed the capability of heterotypic assembly of cbMSCs and HUVECs into robust and durable tubular networks on Matrigel. The up-regulated gene expressions of VEGF and IGF-1 reflected the robust expansion of tubular networks; in addition, the augmented levels of SMA and SM22 suggested smooth muscle differentiation of cbMSCs, possibly helping to improve the durability of networks. Moreover, according to the in vivo echocardiographic, magnetic resonance and computed-tomographic results, transplantation of cbMSC/HUVEC bodies benefited post-MI dysfunction. Furthermore, the vascularization analyses demonstrated the robust vasculogenic potential of cbMSC/HUVEC bodies in vivo, thus contributing to the greater viable myocardium and the less scar region, and ultimately restoring the cardiac function. The concept of core-shell bodies composed of perivascular cells and endothelial cells may serve as an attractive cell delivery vehicle for vasculogenesis, thus improving the cardiac function significantly.

A strategy for fabrication of a three-dimensional tissue construct containing uniformly distributed embryoid body-derived cells as a cardiac patch.

Growing three-dimensional (3D) scaffolds that contain more than a few layers of seeded cells in vitro is crucial for the creation of thick and viable cardiac tissues in vivo. Embryonic stem cells (ESCs) have been used as an alternative cell source for cardiac repair; however, dissociated ESCs show poor viability in the scaffold and do not form the embryoid body (EB)-like structures. In this study, a strategy intended for cultivating EB-derived cells (EBDCs) uniformly in a porous 3D tissue scaffold was developed. This strategy employed techniques of formation of spherically symmetric EBs in a thermo-responsive hydrogel system, production of cell sheets of EBDCs in a similar hydrogel system coated with collagen and fabrication of sliced porous tissue scaffolds. The prepared EBs were collected and plated evenly in the cell-sheet culture system. After 8 days in culture, a continuous sheet of EBDCs with cell beating was obtained; our qPCR and flow cytometric analyses showed that the collagen-coated on the cell-sheet culture system can significantly enhance the population of cardiac-lineage cells. The produced EBDC sheets were then sandwiched into the sliced porous tissue scaffold. After reculture, the seeded EBDCs were redistributed uniformly throughout the scaffold, with a significant increase in mechanical strength. Cardiac-specific myosin heavy chain and alpha-actinin were expressed for some cells grown in the scaffold, while connexin 43 was clearly expressed at the cell borders. Additional studies such as employing purification techniques to enrich the population of cardiomyocytes are needed to further improve the developed tissue constructs as a bioengineered cardiac patch.

Hypoxia-induced therapeutic neovascularization in a mouse model of an ischemic limb using cell aggregates composed of HUVECs and cbMSCs

Cell transplantation for therapeutic neovascularization holds great promise for treating ischemic diseases. This work prepared three-dimensional aggregates of human umbilical vein endothelial cells (HUVECs) and cord-blood mesenchymal stem cells (cbMSCs) with different levels of internal hypoxia by a methylcellulose hydrogel system. We found that few apoptosis occurred in these cell aggregates, despite developing a hypoxic microenvironment in their inner cores. Via effectively switching on the hypoxia-inducible factor-1α-dependent angiogenic mechanisms, culturing the internally hypoxic HUVEC/cbMSC aggregates on Matrigel resulted in formation of extensive and persistent tubular networks and significant upregulation of pro-angiogenic genes. As the level of internal hypoxia created in cell aggregates increased, the robustness of the tubular structures developed on Matrigel increased, and expression levels of the pro-angiogenic genes also elevated. Transplantation of hypoxic HUVEC/cbMSC aggregates into a mouse model of an ischemic limb significantly promoted formation of functional vessels, improved regional blood perfusion, and attenuated muscle atrophy and bone losses, thereby rescuing tissue degeneration. Notably, their therapeutic efficacy was clearly dependent upon the level of internal hypoxia established in cell aggregates. These analytical results demonstrate that by establishing a hypoxic environment in HUVEC/cbMSC aggregates, their potential for therapeutic neovascularization can be markedly enhanced.

A translational approach in using cell sheet fragments of autologous bone marrow-derived mesenchymal stem cells for cellular cardiomyoplasty in a porcine model

Based on a porcine model with surgically created myocardial infarction (MI) as a pre-clinical scheme, this study investigates the clinical translation of cell sheet fragments of autologous mesenchymal stem cells (MSCs) for cellular cardiomyoplasty. MSC sheet fragments retaining endogenous extracellular matrices are fabricated using a thermo-responsive methylcellulose hydrogel system. Echocardiographic observations indicate that transplantation of MSC sheet fragments in infarcted hearts can markedly attenuate the adverse ventricular dilation and preserve the cardiac function post MI, which is in contrast to the controlled groups receiving saline or dissociated MSCs. Additionally, histological analyses suggest that administering MSC sheet fragments significantly prevents the scar expansion and left ventricle remodeling after MI. Immunohistochemistry results demonstrate that the engrafted MSCs can differentiate into endothelial cells and smooth muscle cells, implying that angiogenesis and the subsequent regional perfusion improvement is a promising mechanism for ameliorating post-infarcted cardiac function. However, according to the data recorded by an implantable loop recorder, the transplanted MSCs may provoke arrhythmia. Nevertheless, the proposed approach may potentially lead to the eventual translation of MSC-based therapy into practical and effective clinical treatments.

Synergistic antibacterial effects of localized heat and oxidative stress caused by hydroxyl radicals mediated by graphene/iron oxide-based nanocomposites.

UNLABELLED This work develops a composite system of reduced graphene oxide (rGO)-iron oxide nanoparticles (rGO-IONP) that can synergistically induce physical and chemical damage to methicillin-resistant Staphylococcus aureus (MRSA) that are present in subcutaneous abscesses. rGO-IONP was synthesized by the chemical deposition of Fe(2+)/Fe(3+) ions on nanosheets of rGO in aqueous ammonia. The antibacterial efficacy of the as-prepared rGO-IONP was evaluated in a mouse model with MRSA-infected subcutaneous abscesses. Upon exposure to a near-infrared laser in vitro, rGO-IONP synergistically generated localized heat and large amounts of hydroxyl radicals, which inactivated MRSA. The in vivo results reveal that combined treatment with localized heat and oxidative stress that is caused by hydroxyl radicals accelerated the healing of wounds associated with MRSA-infected abscesses. The above results demonstrate that an rGO-IONP nanocomposite system that can effectively inactivate multiple-drug-resistant bacteria in subcutaneous infections was successfully developed. FROM THE CLINICAL EDITOR The emergence of methicillin-resistant S. aureus (MRSA) has posed a significant problem in the clinical setting. Thus, it is imperative to develop new treatment strategies against this. In this study, the authors described the use of reduced graphene oxide (rGO)-iron oxide nanoparticles (rGO-IONP) to induce heat and chemical damage to MRSA. This approach may provide a platform the design of other treatment modalities against multiple-drug-resistant bacteria.

Injectable Cell Constructs Fabricated via Culture on a Thermoresponsive Methylcellulose Hydrogel System for the Treatment of Ischemic Diseases

Cell transplantation via direct intramuscular injection is a promising therapy for patients with ischemic diseases. However, following injections, retention of transplanted cells in engrafted areas remains problematic, and can be deleterious to cell-transplantation therapy. In this Progress Report, a thermoresponsive hydrogel system composed of aqueous methylcellulose (MC) blended with phosphate-buffered saline is constructed to grow cell sheet fragments and cell bodies for the treatment of ischemic diseases. The as-prepared MC hydrogel system undergoes a sol-gel reversible transition upon heating or cooling at ≈32 °C. Via this unique property, the grown cell sheet fragments (cell bodies) can be harvested without using proteolytic enzymes; consequently, their inherent extracellular matrices (ECMs) and integrative adhesive agents remain well preserved. In animal studies using rats and pigs with experimentally created myocardial infarction, the injected cell sheet fragments (cell bodies) become entrapped in the interstices of muscular tissues and adhere to engraftment sites, while a minimal number of cells exist in the group receiving dissociated cells. Moreover, transplantation of cell sheet fragments (cell bodies) significantly increases vascular density, thereby improving the function of an infarcted heart. These experimental results demonstrate that cell sheet fragments (cell bodies) function as a cell-delivery construct by providing a favorable ECM environment to retain transplanted cells locally and consequently, improving the efficacy of therapeutic cell transplantation.