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ObjectiveStromal barriers, such as the abundant desmoplastic stroma that is characteristic of pancreatic ductal adenocarcinoma (PDAC), can block the delivery and decrease the tumour-penetrating ability of therapeutics such as tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), which can selectively induce cancer cell apoptosis. This study aimed to develop a TRAIL-based nanotherapy that not only eliminated the extracellular matrix barrier to increase TRAIL delivery into tumours but also blocked antiapoptotic mechanisms to overcome TRAIL resistance in PDAC.DesignNitric oxide (NO) plays a role in preventing tissue desmoplasia and could thus be delivered to disrupt the stromal barrier and improve TRAIL delivery in PDAC. We applied an in vitro–in vivo combinatorial phage display technique to identify novel peptide ligands to target the desmoplastic stroma in both murine and human orthotopic PDAC. We then constructed a stroma-targeted nanogel modified with phage display-identified tumour stroma-targeting peptides to co-deliver NO and TRAIL to PDAC and examined the anticancer effect in three-dimensional spheroid cultures in vitro and in orthotopic PDAC models in vivo.ResultsThe delivery of NO to the PDAC tumour stroma resulted in reprogramming of activated pancreatic stellate cells, alleviation of tumour desmoplasia and downregulation of antiapoptotic BCL-2 protein expression, thereby facilitating tumour penetration by TRAIL and substantially enhancing the antitumour efficacy of TRAIL therapy.ConclusionThe co-delivery of TRAIL and NO by a stroma-targeted nanogel that remodels the fibrotic tumour microenvironment and suppresses tumour growth has the potential to be translated into a safe and promising treatment for PDAC.

The short supply of donor corneas is exacerbated by the unsuitability of donors with insufficient endothelial cell density. Few studies have investigated promoting corneal endothelial cell proliferation to increase the endothelial cell density. We hypothesize that pre‐transplantation treatment of proliferative tissue‐cultivated corneas may increase corneal endothelial cell density. We observed that the airlift cultures were superior to immersion cultures with respect to both transparency and thickness. In this tissue culture system, we observed that lysophosphatidic acid increased the rabbit corneal endothelial cell density, number of BrdU‐positive cells and improve wound healing. We also observed an indirect effect of lysophosphatidic acid on corneal endothelial cell proliferation mediated by the stimulation of interleukin‐1β secretion from stromal cells. Human corneal tissues treated with lysophosphatidic acid or interleukin‐1β contained significantly more Ki‐67‐positive cells than untreated group. The lysophosphatidic acid‐ or interleukin‐1β‐treated cultured tissue remained hexagon‐shaped, with ZO‐1 expression and no evidence of the endothelial‐mesenchymal transition. Our novel protocol of tissue culture may be applicable for eye banks to optimize corneal grafting.

Nitric oxide (NO) is an essential endogenous signaling molecule regulating multifaceted physiological functions in the (cardio)vascular, neuronal, and immune systems. Due to the short half-life and location-/concentration-dependent physiological function of NO, translational application of NO as a novel therapeutic approach, however, awaits a strategy for spatiotemporal control on the delivery of NO. Inspired by the magnetic hyperthermia and magneto-triggered drug release featured by Fe

After the discovery of endogenous dinitrosyl iron complexes (DNICs) as a potential biological equivalent of nitric oxide (NO), bioinorganic engineering of [Fe(NO)2] unit has emerged to develop biomimetic DNICs [(NO)2Fe(L)2] as a chemical biology tool for controlled delivery of NO. For example, water-soluble DNIC [Fe2(μ-SCH2CH2OH)2(NO)4] (DNIC-1) was explored for oral delivery of NO to the brain and for the activation of hippocampal neurogenesis. However, the kinetics and mechanism for cellular uptake and intracellular release of NO, as well as the biocompatibility of synthetic DNICs, remain elusive. Prompted by the potential application of NO to dermato-physiological regulations, in this study, cellular uptake and intracellular delivery of DNIC [Fe2(μ-SCH2CH2COOH)2(NO)4] (DNIC-2) and its regulatory effect/biocompatibility toward epidermal cells were investigated. Upon the treatment of DNIC-2 to human fibroblast cells, cellular uptake of DNIC-2 followed by transformation into protein-bound DNICs occur to trigger the intracellular release of NO with a half-life of 1.8 ± 0.2 h. As opposed to the burst release of extracellular NO from diethylamine NONOate (DEANO), the cell-penetrating nature of DNIC-2 rationalizes its overwhelming efficacy for intracellular delivery of NO. Moreover, NO-delivery DNIC-2 can regulate cell proliferation, accelerate wound healing, and enhance the deposition of collagen in human fibroblast cells. Based on the in vitro and in vivo biocompatibility evaluation, biocompatible DNIC-2 holds the potential to be a novel active ingredient for skincare products.

Much effort has been focused on novel nanomedicine for cancer therapy. However, tumor hypoxia limits the efficacy of various cancer therapeutics. Herein, we constructed a self-sufficient hybrid enzyme-based silk fibroin hydrogel system, consisting of Pt-decorated hollow Ag-Au trimetallic nanocages (HGN@Pt) and glucose oxidase (GOx), to supply O

Background The pumping function of corneal endothelial cells (CECs) plays a pivotal role in the maintenance of corneal water homeostasis. Corneal endothelial dysfunction (CED) leads to corneal edema and opacity, but with the exception of keratoplasty, no optimal therapeutic strategies have been established for CED. In this study, we aimed to investigate the ameliorative effect of ascorbic acid (AA) on CED and the underlying mechanism of action in the corneal endothelium. Methods Rabbit corneal endothelial damage was induced by anterior chamber injection of benzalkonium chloride (BAK). AA was topically administered to the corneal surface, and the transparency and thickness of the cornea were assessed by external eye photography, slit-lamp photography, and ultrasonic pachymetry. To further analyze the mechanism, rabbit CECs and immortalized human CECs (B4G12 cells) were cultured. A ferric reducing/antioxidant and AA (FRASC) assay was performed to measure the AA concentration. Cell proliferation was evaluated by cell counting and bromodeoxyuridine (BrdU) labeling assays, and protein expression was examined by liquid chromatography–mass spectrometry (LC/MS) and immunoblotting. The involvement of glucose transporter 1 (GLUT1) and phospho-ERK was evaluated via GLUT1-siRNA and phospho-ERK inhibitor (PD98059) treatment. Interpretation We observed that topical AA ameliorates BAK-induced rabbit corneal endothelial damage. Furthermore, we demonstrated that AA is transported into B4G12 cells via GLUT1, and afterward, AA increases ERK phosphorylation and promotes cell proliferation. Our findings indicate that CEC proliferation stimulated via the noncanonical AA-GLUT1-ERK axis contributes to AA-enhanced healing of CED.

Compromised pumping function of the corneal endothelium, due to loss of endothelial cells, results in corneal edema and subsequent visual problems. Clinically and experimentally, oxidative stress may cause corneal endothelial decompensation after phacoemulsification. Additionally, in vitro and animal studies have demonstrated the protective effects of intraoperative infusion of ascorbic acid (AA). Here, we established a paraquat-induced cell damage model, in which paraquat induced reactive oxygen species (ROS) production and apoptosis in the B4G12 and ARPE-19 cell lines. We demonstrate that oxidative stress triggered autophagic flux blockage in corneal endothelial cells and that addition of AA ameliorated such oxidative damage. We also demonstrate the downregulation of Akt phosphorylation in response to oxidative stress. Pretreatment with ascorbic acid reduced the downregulation of Akt phosphorylation, while inhibition of the PI3K/Akt pathway attenuated the protective effects of AA. Further, we establish an in vivo rabbit model of corneal endothelial damage, in which an intracameral infusion of paraquat caused corneal opacity. Administration of AA via topical application increased its concentration in the corneal stroma and reduced oxidative stress in the corneal endothelium, thereby promoting corneal clarity. Our findings indicate a perioperative strategy of topical AA administration to prevent oxidative stress-induced damage, particularly for those with vulnerable corneal endothelia.

Islet transplantation has been demonstrated to be a promising therapy for type 1 diabetes mellitus. Although it is a minimally invasive operating procedure and provides easy access for graft monitoring, subcutaneous transplantation of the islet only has limited therapeutic outcomes, owing to the poor capacity of skin tissue to foster revascularization in a short period. Herein, 3D cell spheroids of clinically accessible umbilical cord blood mesenchymal stem cells and human umbilical vein endothelial cells are formed and employed for codelivery with β cells subcutaneously. The 3D stem cell spheroids, which can secrete multiple proangiogenic and prosurvival growth factors, induce robust angiogenesis and prevent β cell graft death, as indicated by the results of in vivo bioluminescent tracking and histological analysis. These experimental data highlight the efficacy of the 3D stem cell spheroids that are fabricated using translationally applicable cell types in promoting the survival and function of subcutaneously transplanted β cells.

Ischemic stroke, and the consequent brain cell death, is a common cause of death and disability worldwide. Current treatments that primarily aim to relieve symptoms are relatively inefficient in achieving brain tissue regeneration and functional recovery, and thus novel therapeutic options are urgently needed. Although cell-based therapies have shown promise for treating the infarcted brain, a recurring challenge is the inadequate retention and engraftment of transplanted cells at the target tissue, thereby limiting the ultimate therapeutic efficacy. Here, we show that transplantation of preassembled three-dimensional (3D) spheroids of mesenchymal stem cells (MSCs) and vascular endothelial cells (ECs) results in significantly improved cell retention and survival compared with conventional mixed-cell suspensions. The transplanted 3D spheroids exhibit notable neuroprotective, proneurogenic, proangiogenic and anti-scarring potential as evidenced by clear extracellular matrix structure formation and paracrine factor expression and secretion; this ultimately results in increased structural and motor function recovery in the brain of an ischemic stroke mouse model. Therefore, transplantation of MSCs and ECs using the 3D cell spheroid configuration not only reduces cell loss during cell harvesting/administration but also enhances the resultant therapeutic benefit, thus providing important proof-of-concept for future clinical translation.

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.

Hydrogen gas can reduce cytotoxic reactive oxygen species (ROS) that are produced in inflamed tissues. Inspired by natural photosynthesis, this work proposes a multicomponent nanoreactor (NR) that comprises chlorophyll a, l-ascorbic acid, and gold nanoparticles that are encapsulated in a liposomal (Lip) system that can produce H

Patients with diabetes mellitus are prone to develop refractory wounds. They exhibit reduced synthesis and levels of circulating hydrogen sulfide (H

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.

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.

This article reviews recent progress in the development of carbon dioxide (CO

Focal infections that are caused by antibiotic-resistant bacteria are becoming an ever-growing challenge to human health. To address this challenge, a pH-responsive amphiphilic polymer of polyaniline-conjugated glycol chitosan (PANI-GCS) that can self-assemble into nanoparticles (NPs) in situ is developed. The PANI-GCS NPs undergo a unique surface charge conversion that is induced by their local pH, favoring bacterium-specific aggregation without direct contact with host cells. Following conjugation onto GCS, the optical-absorbance peak of PANI is red-shifted toward the near-infrared (NIR) region, enabling PANI-GCS NPs to generate a substantial amount of heat, which is emitted to their neighborhood. The local temperature of the NIR-irradiated PANI-GCS NPs is estimated to be approximately 5 °C higher than their ambient tissue temperature, ensuring specific and direct heating of their aggregated bacteria; hence, damage to tissue is reduced and wound healing is accelerated. The above results demonstrate that PANI-GCS NPs are practical for use in the photothermal ablation of focal infections.

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.

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.

Cell transplantation via local intramuscular injection is a promising therapy. However, the cells used are usually expanded in vitro and dissociated by trypsinization, which may be harmful to the cells. In the study, a novel method for the construction of fragmented sheets of mesenchymal stem cells (MSCs) with a uniform size, without treating with any enzymes, was reported. The obtained MSC sheets preserved the intercellular junctions and endogenous extracellular matrix and kept their cell phenotype. After injection through a needle, the fragmented MSC sheets maintained intact and retained their activity upon transferring to another growth surface, while the complete cell sheets were torn into pieces. Transplantation of fragmented MSC sheets in the skeletal muscle of a syngeneic rat model via local injection was evaluated. The transplanted MSC sheets were mainly localized at the site of injection, while the dissociated MSCs were scattered around. Additionally, there were significantly more MSCs retained in the local skeletal muscle for the group injected with fragmented MSC sheets than that injected with dissociated MSCs. These results indicated that the fragmented cell sheets might be used as a novel therapeutic cell-carrier for intramuscular administration.

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.

A recurring obstacle in cell-base strategies for treating ischemic diseases is the significant loss of viable cells that is caused by the elevated levels of regional reactive oxygen species (ROS), which ultimately limits therapeutic capacity. In this study, aggregates of human umbilical vein endothelial cells (HUVECs) and cord-blood mesenchymal stem cells (cbMSCs), which are capable of inducing therapeutic angiogenesis, are prepared. We hypothesize that the concurrent delivery of an antioxidant N-acetylcysteine (NAC) may significantly increase cell retention following the transplantation of HUVEC/cbMSC aggregates in a mouse model with hindlimb ischemia. Our in vitro results demonstrate that the antioxidant NAC can restore ROS-impaired cell adhesion and recover the reduced angiogenic potential of HUVEC/cbMSC aggregates under oxidative stress. In the animal study, we found that by scavenging the ROS generated in ischemic tissues, NAC is likely to be able to establish a receptive cell environment in the early stage of cell transplantation, promoting the adhesion, retention, and survival of cells of engrafted aggregates. Therapeutic angiogenesis is therefore enhanced and blood flow recovery and limb salvage are ultimately achieved. The combinatory strategy that uses an antioxidant and HUVEC/cbMSC aggregates may provide a new means of boosting the therapeutic efficacy of cell aggregates for the treatment of ischemic diseases.

Although the induction of neovascularization by cell-based approaches has demonstrated substantial potential in treating myocardial infarction (MI), the process of cell-mediated angiogenesis and its correlation with therapeutic mechanisms of cardiac repair remain elusive. In this work, three-dimensional (3D) aggregates of human umbilical vein endothelial cells (HUVECs) and cord-blood mesenchymal stem cells (cbMSCs) are constructed using a methylcellulose hydrogel system. By maximizing cell-cell and cell-ECM communications and establishing a hypoxic microenvironment in their inner cores, these cell aggregates are capable of forming widespread tubular networks together with the angiogenic marker αvβ3 integrin; they secret multiple pro-angiogenic, pro-survival, and mobilizing factors when grown on Matrigel. The aggregates of HUVECs/cbMSCs are exogenously engrafted into the peri-infarct zones of rats with MI via direct local injection. Multimodality noninvasive imaging techniques, including positron emission tomography, single photon emission computed tomography, and echocardiography, are employed to monitor serially the beneficial effects of cell therapy on angiogenesis, blood perfusion, and global/regional ventricular function, respectively. The myocardial perfusion is correlated with ventricular contractility, demonstrating that the recovery of blood perfusion helps to restore regional cardiac function, leading to the improvement in global ventricular performance. These experimental data reveal the efficacy of the exogenous transplantation of 3D cell aggregates after MI and elucidate the mechanism of cell-mediated therapeutic angiogenesis for cardiac repair.

Cell-based therapeutic neovascularization is a promising method for treating ischemic disorders. In this work, human umbilical vein endothelial cells (HUVECs) were thoroughly premixed with cord-blood mesenchymal stem cells (cbMSCs) and cultivated to form three-dimensional (3D) cell aggregates for cellular cardiomyoplasty. In the in vitro study, tubular networks were formed at day 1 after the co-culturing of dissociated HUVECs and cbMSCs on Matrigel; however, as time progressed, the grown tubular networks regressed severely. Conversely, when 3D cell aggregates were grown on Matrigel, mature and stable tubular networks were observed over time, under the influence of their intensive cell-extracellular matrix (ECM) interactions and cell-cell contacts. 3D cell aggregates were transplanted into the peri-infarct zones of rats with myocardial infarction (MI) via direct intramyocardial injection. Based on our pinhole single photon emission computed tomography (SPECT) myocardial-perfusion observations, echocardiographic heart-function examinations and histological analyses, the engrafted 3D cell aggregates considerably enhanced the vascular densities and the blood flow recovery in the ischemic myocardium over those of their dissociated counterparts, thereby reducing the size of perfusion defects and restoring cardiac function. These results demonstrate that the intramuscular delivery of 3D cell aggregates of HUVECs/cbMSCs can be a valuable cell-based regenerative therapeutic strategy against MI.

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.

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.

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.

Growing three-dimensional scaffolds that contain more than a few layers of seeded cells is crucial for the creation of thick and viable cardiac tissues. To achieve this goal, a bioengineered cardiac patch (the MSC patch) composed of a sliced porous biological scaffold inserted with multilayered mesenchymal stem cells (MSCs) was developed for myocardial repair in a syngeneic rat model. After culture, sliced layers of the scaffold were stuck together and seeded MSCs were redistributed throughout the scaffold. Immunofluorescence analyses indicated that cells were viable and tightly adhered to a robust fibronectin meshwork within the scaffold. Results of echocardiography and heart catheterization revealed that the MSC-patch group had a superior heart function to the infarct group. Cells together with neo-muscle fibers and neo-microvessels were clearly observed in the entire MSC patch to fill its original pores, indicating that the implanted patch became well integrated into the host. The thickness of the retrieved MSC patch increased significantly as compared to that before implantation. When compared with the infarct group, expressions of angiogenic cytokines (bFGF, vWF and PDGF-B) and cardioprotective factors (IGF-1 and HGF) were significantly increased in the MSC-patch group. The aforementioned results indicated that transplantation of the MSC patch could restore the dilated LV and preserve cardiac functions after infarction.