Gelatin Hydrogel Microspheres Research Articles

Comparing embolic particles for prostatic artery embolization to treat lower urinary tract symptoms in patients with benign prostatic hyperplasia.

Compare the safety and efficacy of polyvinyl alcohol particles (PVA) versus trisacryl gelatin microspheres (Embospheres) versus hydrogel microspheres coated with polyzene-F (Embozenes) for prostatic artery embolization (PAE) to treat patients with benign prostatic hyperplasia (BPH). A single-center prospective cohort study from 2019 to 2023, including patients with international prostate symptom score (IPSS) ≥ 15 and/or quality of life score (QoL) ≥ 4. Allocation to embolic agents was performed chronologically: 100-300 µm PVA (n = 53), followed by 300-500 µm Embospheres (n = 50), and finally, 400 µm Embozenes (n = 50). All patients were evaluated at baseline and at 1 and 6 months after PAE with IPSS/QoL; peak urinary flow rate, post-void residual volume, and prostate volume with ultrasound and prostate-specific antigen. Adverse events and the need for prostatic re-interventions were assessed. There were no significant baseline differences between the three groups except for patient age (62.5 years PVA; 66.1 years Embospheres and 66.6 years Embozenes; p = 0.019). There were no major adverse events and no differences between groups regarding minor adverse events. All outcome measures improved significantly from baseline, with no significant differences between groups. Mean ± standard deviation IPSS/QoL improvement at 6 months: -10.7 ± 7.9/-2.2 ± 1.7 PVA; -10.4 ± 7.3/-2.0 ± 1.5 Embospheres; -10.4 ± 7.0/-2.2 ± 1.6 Embozenes (p = 0.987). Re-intervention rates after 6 months: 9% (n = 5/53) PVA; 14% (n = 7/50) Embospheres; 8% (n = 4/50) Embozenes (p = 0.591). PAE with PVA particles, Embospheres, and Embozenes is equally safe and effective in treating BPH-related lower urinary tract symptoms. This is the first prospective study showing equivalence between the most frequently used embolic agents for prostatic artery embolization. Different particles can be used interchangeably for prostatic artery embolization. The improvements in measured metrics were the same between groups, with no differences in adverse events. The need for prostatic medication and re-intervention rates were the same at 1 and 6 months after embolization.

The hyaluronic acid-gelatin hierarchical hydrogel for osteoporotic bone defect repairment

Osteoporotic bone defects are serious medical problems due to their sparse bone structure, difficulty in restoration and reconstruction, and high recurrence rates, which also result in heavy economic and social burdens. Herein, we developed a hierarchical hydrogel composed of alendronate sodium (AS)/Mg2+-loaded inverse opal methylpropenylated gelatin (GelMA) hydrogel microspheres (IOHM-AS-Mgs) within methylpropenylated poly(hyaluronic acid) (HAMA) for osteoporotic bone defect treatment. The IOHM-AS-Mgs displayed good cytocompatibility and cell adhesion and strongly stimulated osteogenesis at the transcriptomic and protein levels. When this treatment was applied to the osteoporotic bone defect area, HAMA was used to fix the microspheres. The results of the microcomputed tomography (micro-CT) and histological analyses indicated that the hierarchical hydrogel had the best therapeutic effect. Therefore, this hydrogel is a new candidate for osteoporotic bone defect treatment.

Innovative Bio-based Hydrogel Microspheres Micro-Cage for Neutrophil Extracellular Traps Scavenging in Diabetic Wound Healing.

Neutrophil extracellular traps (NETs) seriously impede diabetic wound healing. The disruption or scavenging of NETs using deoxyribonuclease (DNase) or cationic nanoparticles has been limited by liberating trapped bacteria, short half-life, or potential cytotoxicity. In this study, a positive correlation between the NETs level in diabetic wound exudation and the severity of wound inflammation in diabetic patients is established. Novel NETs scavenging bio-based hydrogel microspheres 'micro-cage', termed mPDA-PEI@GelMA, is engineered by integrating methylacrylyl gelatin (GelMA) hydrogel microspheres with cationic polyethyleneimine (PEI)-functionalized mesoporous polydopamine (mPDA). This unique 'micro-cage' construct is designed to non-contact scavenge of NETs between nanoparticles and the diabetic wound surface, minimizing biological toxicity and ensuring high biosafety. NETs are introduced into 'micro-cage' along with wound exudation, and cationic mPDA-PEI immobilizes them inside the 'micro-cage' through a strong binding affinity to the cfDNA web structure. The findings demonstrate that mPDA-PEI@GelMA effectively mitigates pro-inflammatory responses associated with diabetic wounds by scavenging NETs both in vivo and in vitro. This work introduces a novel nanoparticle non-contact NETs scavenging strategy to enhance diabetic wound healing processes, with potential benefits in clinical applications.

Therapeutic potential of human induced pluripotent stem cell–derived cardiac tissue in an ischemic model with unloaded condition mimicking left ventricular assist device

ObjectiveThis study aimed to explore the therapeutic potential of human induced pluripotent stem cell (hiPSC)-derived cardiac tissues (HiCTs) in the emerging approach of bridge to recovery for severe heart failure with ventricular assist devices. We used a rat model of heterotopic heart transplantation (HTx) to mimic ventricular assist device support and heart unloading. MethodsHiCTs were created by inserting gelatin hydrogel microspheres between cell sheets made from hiPSC-derived cardiovascular cells. Male athymic nude rats underwent myocardial infarction (MI) and were divided into the following groups: MI (loaded, untreated control), MI + HTx (unloaded, untreated control), MI + HTx + HiCT (unloaded, treated), and MI + HiCT (loaded, treated). HiCTs were placed on the epicardium of the heart in treated groups. We evaluated HiCT engraftment, fibrosis, and neovascularization using histologic analysis. ResultsAfter 4 weeks, HiCTs successfully engrafted in 5 of 6 rats in the MI + HTx + HiCT group (83.3%). The engrafted HiCT area was greater under unloaded conditions (MI + HTx + HiCT) than loaded conditions (MI + HiCT) (P < .05). MI + HTx + HiCT had a significantly smaller infarct area compared with MI and MI + HTx. The MI + HTx + MiCT group exhibited greater vascular density in the border zone than MI and MI + HTx. HiCT treatment suppressed cardiomyocyte atrophy due to left ventricular unloading (P = .001). The protein level of muscle-specific RING finger 1, an atrophy-related ubiquitin ligase, was lower in the MI + HTx + HiCT group than in MI + HTx (P = .036). ConclusionsTransplanting HiCTs into ischemic hearts under unloaded conditions promoted engraftment, neovascularization, attenuated infarct remodeling, and suppressed myocyte atrophy. These results suggest that HiCT treatment could contribute to future advancements in bridge to recovery.

A Novel Bioimplant Comprising Ad-BMP9-Transfected BMSCs and GelMA Microspheres Produced from Microfluidic Devices for Bone Tissue Engineering

Oral and maxillofacial bone defect repair in patients remains challenging in clinical treatment due to the different morphologies of bone defects. An injectable hydrogel of microspheres with sustained bone morphogenetic protein 9 (BMP9) expression for oral and maxillofacial bone defect repair has been developed. This study is bioinspired by the substantial osteogenesis property of recombinant adenoviruses expressing bone morphogenetic protein 9 (Ad-BMP9) and minimally invasive treatment by injection. A novel scaffold encompassing bone mesenchymal stem cells (BMSCs) transfected with Ad-BMP9 was produced and cocultured on a superficial surface of monodisperse photocrosslinked methacrylate gelatin hydrogel microspheres (GelMA/MS, produced with microfluidic technology). The biological tests including live/dead cell staining, phalloidin staining, cell counting kit-8 (CCK-8) assay, alkaline phosphatase (ALP) activity and staining, alizarin red S staining, and quantitative real-time polymerase chain reaction (RT-qPCR), revealed that the hydrogel microspheres exhibited good biocompatibility and remarkably promoted the osteogenic differentiation of BMSCs in vitro. In addition, a small needle was injected the innovative scaffold beneath the nude mice’s skin. The micro-CT and histological staining assay results demonstrated that the new implant, with high blood vessel formation markers (CD31-positive cells) expression over four and eight weeks, achieved significant vascularized bone-like tissue formation. Consequently, the injectable hydrogel microspheres, cocultured with BMSC transfected with Ad-BMP9, enhanced vascularized bone regeneration, therefore representing a facile and promising technique for the minimally invasive treatment of oral and maxillofacial bone defects.

Basic fibroblast growth factor-loaded methacrylate gelatin hydrogel microspheres for spinal nerve regeneration.

Spinal cord injury is a severe central nervous system injury, and developing appropriate drug delivery platforms for spinal nerve regeneration is highly anticipated. Here, we propose a basic fibroblast growth factor (bFGF)-loaded methacrylate gelatin (GelMA) hydrogel microsphere with ideal performances for spinal cord injury repair. Benefitting from the precise droplet manipulation capability of the microfluidic technology, the GelMA microspheres possess uniform and satisfactory size and good stability. More importantly, by taking advantage of the porous structures and facile chemical modification of the GelMA microspheres, bFGF could be easily loaded and gradually released. By co-culturing with neural stem cells, it is validated that the bFGF-loaded GelMA microspheres could effectively promote the proliferation and differentiation of neural stem cells. We also confirm the effective role of the bFGF-loaded GelMA microspheres in nerve repair of spinal cord injury in rats. Our results demonstrate the potential value of the microspheres for applications in repairing central nervous system injuries.

Living and Injectable Porous Hydrogel Microsphere with Paracrine Activity for Cartilage Regeneration.

Paracrine is an important mechanism in mesenchymal stem cells (MSCs) that promotes tissue regeneration. However, anoikis is attributed to unsuitable adhesion microenvironment hindered this paracrine effect. In this study, a living and injectable porous hydrogel microsphere with long-term paracrine activity is constructed via the freeze-drying microfluidic technology and the incorporation of platelet-derived growth factor-BB (PDGF-BB) and exogenous MSCs. Benefiting from the porous structure and superior mechanical property of methacrylate gelatin (GelMA) hydrogel microspheres (GMs), exogenous stem cells are able to adhere and proliferate on GMs, thereby facilitating cell-to-extracellular matrix (ECM) and cell-to-cell interactions and enhancing paracrine effect. Furthermore, the sustained release of PDGF-BB can recruit endogenous MSCs to prolong the paracrine activity of the living GMs. In vitro and in vivo experiments validated that the living GMs exhibit superior secretion properties and anti-inflammatory efficacy and can attenuate osteoarthritis (OA) progression by favoring the adherent microenvironment and utilizing the synergistic effect of exogenous and endogenous MSCs. Overall, a living injectable porous hydrogel microsphere that can enhance the paracrine activity of stem cells is fabricated and anticipated to hold the potential of future clinical translation in OA and other diseases.

Immunosuppressive mesenchymal stem cells aggregates incorporating hydrogel microspheres promote an invitro invasion of cancer cells.

IntroductionThe objective of this study is to design a co-culture system of cancer cells and three-dimensional (3D) mesenchymal stem cells (MSC) aggregates for the in vitro evaluation of cancer invasion.MethodsFirst, the MSC of an immunosuppressive phenotype (MSC2) were prepared by the MSC stimulation of polyriboinosinic polyribocytidylic acid. By simple mixing MSC2 and gelatin hydrogel microspheres (GM) in a U-bottomed well of 96 well plates which had been pre-coated with poly (vinyl alcohol), 3D MSC2 aggregates incorporating GM were obtained. The amount of chemokine (C–C motif) ligand 5 (CCL5) secreted from the MSC2 aggregates incorporating GM. Finally, an invasion assay was performed to evaluate the cancer invasion rate by co-cultured cancer cells and the 3D MSC2 incorporating GM.ResultsThe amount of CCL5 secreted for the 3D MSC2 aggregates incorporating GM was significantly higher than that of two-dimensional (2D) MSC, 2D MSC2, and 3D MSC aggregates incorporating GM. When MDA-MB-231 human breast cancer cells were co-cultured with the 3D MSC2 aggregates incorporating GM, the invasion rate of cancer cells was significantly high compared with that of 2D MSC or 2D MSC2 and 3D MSC aggregates incorporating GM. In addition, high secretion of matrix metalloproteinase-2 was observed for the 3D MSC2 aggregates/cancer cells system.ConclusionsIt is concluded that the co-culture system of 3D MSC2 aggregates incorporating GM and cancer cells is promising to evaluate the invasion of cancer cells in vitro.

Therapeutic potential of clinical-grade human induced pluripotent stem cell-derived cardiac tissues

ObjectivesTo establish a protocol to prepare and transplant clinical-grade human induced pluripotent stem cell (hiPSC)-derived cardiac tissues (HiCTs) and to evaluate the therapeutic potential in an animal myocardial infarction (MI) model. MethodsWe simultaneously differentiated clinical-grade hiPSCs into cardiovascular cell lineages with or without the administration of canonical Wnt inhibitors, generated 5- layer cell sheets with insertion of gelatin hydrogel microspheres (GHMs) (HiCTs), and transplanted them onto an athymic rat MI model. Cardiac function was evaluated by echocardiography and cardiac magnetic resonance imaging and compared with that in animals with sham and transplantation of 5-layer cell sheets without GHMs. Graft survival, ventricular remodeling, and neovascularization were evaluated histopathologically. ResultsThe administration of Wnt inhibitors significantly promoted cardiomyocyte (CM) (P < .0001) and vascular endothelial cell (EC) (P = .006) induction, which resulted in cellular components of 52.0 ± 6.1% CMs and 9.9 ± 3.0% ECs. Functional analyses revealed the significantly lowest left ventricular end-diastolic volume and highest ejection fraction in the HiCT group. Histopathologic evaluation revealed that the HiCT group had a significantly larger median engrafted area (4 weeks, GHM(-) vs HiCT: 0.4 [range, 0.2-0.7] mm2 vs 2.2 [range, 1.8-3.1] mm2; P = .005; 12 weeks, 0 [range, 0-0.2] mm2 vs 1.9 [range, 0.1-3.2] mm2; P = .026), accompanied by the smallest scar area and highest vascular density at the MI border zone. ConclusionsTransplantation of HiCTs generated from clinical-grade hiPSCs exhibited a prominent therapeutic potential in a rat MI model and may provide a promising therapeutic strategy in cardiac regenerative medicine.

Efficient cell transplantation combining injectable hydrogels with control release of growth factors.

IntroductionThe objective of this study is to investigate the effect of gelatin microspheres incorporating growth factors on the therapeutic efficacy in cell transplantation. The strength of this study is to combine gelatin hydrogel microspheres incorporating basic fibroblast growth factor and platelet growth factor mixture (GM/GF) with bioabsorbable injectable hydrogels (iGel) for transplantation of adipose-derived stem cells (ASCs).MethodsThe rats ASCs suspended in various solutions were transplanted in masseter muscle. Rats were euthanized 2, 7, 14 days after injection for measurement of the number of ASCs retention in the muscle and morphological evaluation of muscle fibers and the inflammation of the injected tissue by histologic and immunofluorescent stain.ResultsFollowing the injection into the skeletal muscle, the GM/GF allowed the growth factors to release at the injection site over one week. When ASCs were transplanted into skeletal muscle using iGel incorporating GM/GF (iGel+GM/GF), the number of cells grafted was significantly high compared with other control groups. Moreover, for the groups to which GM/GF was added, the cells transplanted survived, and the Myo-D expression of a myoblast marker was observed at the region of cells transplanted.ConclusionsThe growth factors released for a long time likely enhance the proliferative and differentiative capacity of cells. The simple combination with iGel and GM/GF allowed ASCs to enhance their survival at the injected site and consequently achieve improved therapeutic efficacy in cell transplantation.

Biomimetic injectable hydrogel microspheres with enhanced lubrication and controllable drug release for the treatment of osteoarthritis

The occurrence of osteoarthritis (OA) is highly associated with the reduced lubrication property of the joint, where a progressive and irreversible damage of the articular cartilage and consecutive inflammatory response dominate the mechanism. In this study, bioinspired by the super-lubrication property of cartilage and catecholamine chemistry of mussel, we successfully developed injectable hydrogel microspheres with enhanced lubrication and controllable drug release for OA treatment. Particularly, the lubricating microspheres (GelMA@DMA-MPC) were fabricated by dip coating a self-adhesive polymer (DMA-MPC, synthesized by free radical copolymerization) on superficial surface of photo-crosslinked methacrylate gelatin hydrogel microspheres (GelMA, prepared via microfluidic technology), and encapsulated with an anti-inflammatory drug of diclofenac sodium (DS) to achieve the dual-functional performance. The tribological test and drug release test showed the enhanced lubrication and sustained drug release of the GelMA@DMA-MPC microspheres. In addition, the functionalized microspheres were intra-articularly injected into the rat knee joint with an OA model, and the biological tests including qRT-PCR, immunofluorescence staining assay, X-ray radiography and histological staining assay all revealed that the biocompatible microspheres provided significant therapeutic effect against the development of OA. In summary, the injectable hydrogel microspheres developed herein greatly improved lubrication and achieved sustained local drug release, therefore representing a facile and promising technique for the treatment of OA.

Abstract 14377: Therapeutic Potential of Clinical Grade Human Induced Pluripotent Stem Cell-derived Cardiac Tissues for a Rat Myocardial Infarction Model: A Pre-clinical Study for a Cell Transplantation Therapy

Introduction: Transplantation of three-dimensional bioengineered cardiac tissues composed of pluripotent stem cell-derived cardiovascular cell lineages is reported to hold potential for functional recovery on pre-clinical studies. We aimed to evaluate therapeutic and myocardial regenerative potential of clinical grade human induced pluripotent stem cell (hiPSC)-derived cardiac tissues (HiCTs) on a rat myocardial infarction (MI) model. Methods: Clinical grade hiPSC lines established from a healthy volunteer were simultaneously differentiated into cardiovascular cell lineages. We seeded the cells on temperature responsive culture dishes to form cell sheets. HiCTs were generated by stacking 5 cell sheets with insertion of gelatin hydrogel microspheres (GHMs) to promote oxygen and nutrition supply and transplanted onto an athymic rat MI model (n=6). Echocardiography and histological analysis at 12 weeks after surgery were conducted and compared to those in animals with sham surgery (n=9) and with cell sheet stacks without GHMs [GHM(-), n=6]. Some of HiCT-transplanted rats were subjected to tissue clearing and two-photon excitation microscopy (TPEM) to assess graft vascularization. Results: Flow cytometry revealed cellular components after differentiation as follows: 52.0±1.4% of cardiomyocytes (cardiac isoform of troponin-T + :cTnT), 9.9±0.7% of vascular endothelial cells (VE-cadherin + ) and 14.1±1.8% of vascular mural cells (PDGFRβ + ). Echocardiography revealed significantly lower left ventricular end diastolic volume (LVEDV) and higher left ventricular ejection fraction (LVEF) in HiCT group [sham vs GHM(-) vs HiCT: LVEDV; 1.5±0.1 vs 1.3±0.05 vs 0.9±0.03 mL, p&lt;0.0001 / LVEF; 59.7±2.2 vs 67.4±0.6 vs 82.7±0.9 %, p&lt;0.0001]. HiCT group showed significantly larger engraftment [GHM(-) vs HiCT; 12 week; 0.1 ± 0.1 vs 1.8 ± 0.6 mm 2 ; p&lt;0.05]. Engrafted graft tissues were composed of cTnT / α-Actinin-positive cardiomyocytes which exhibited obvious striated structure. TPEM revealed host to graft vascular connection at 2 weeks after HiCT transplantation. Conclusions: HiCTs derived from clinical grade hiPSC potentially serve as a stem cell-derived cellular product in cardiac regenerative therapy for foreseeable clinical applications.

Transplantation of clinical-grade human induced pluripotent stem cell derived cardiac tissues contributes to functional recovery in a rat myocardial infarction model

Abstract Background Clinical-grade human induced pluripotent stem cells (iPSCs) established from a healthy volunteer are currently being considered as a quality controlled cell source for regenerative therapy. Transplantation of three-dimensional bioengineered cardiac tissues composed of human iPSC-derived cardiovascular cell lineages is reported to hold potential for cardiac functional recovery. Purpose The aims of this study were to evaluate tissue conformation and cellular viability of human iPSC-derived cardiac tissues (HiCTs) generated from clinical-grade cells and to validate functional efficacy of HiCT transplantation. Methods Clinical-grade human iPSC lines were simultaneously differentiated into cardiovascular cell lineages by a high-density monolayer culture. The differentiation efficacy was analyzed by flow cytometry. We seeded the cells on temperature responsive culture dishes to form cell sheets. HiCTs are generated by stacking 5 cell sheets with insertion of gelatin hydrogel microspheres (GHMs) between each sheet to promote oxygen and nutrition supply. Characteristics of the HiCTs are histologically and immunohistochemically evaluated. The HiCTs were transplanted onto an athymic nude rat myocardial infarction (MI) model. Cardiac function was evaluated by echocardiography and cardiac magnetic resonance imaging (MRI) until 4 weeks after surgery, and compared to those in animals with sham operation and with cell sheet stacks without GHMs [GHM(−)]. Results Flow cytometry at differentiation day15 revealed cellular components as follows: 52.5±1.4% of cardiomyocytes (cardiac isoform of troponin-T+), 9.8±0.7% of vascular endothelial cells (VE-cadherin+), 14.8±1.8% of vascular mural cells (PDGFRβ+) and 0.2±0.1% of undifferentiated cells (TRA-1-60+). HiCTs were significantly thicker [GHM(−) vs HiCT: 357.3±81.5 vs 723.0±84.0μm, p&amp;lt;0.05], composed of higher area of cardiomyocytes (27.7±7.9 vs 71.9±15.5mm2, p&amp;lt;0.05) and endothelial cells (CD31+) (1.6±0.7 vs 9.2±1.5mm2, p&amp;lt;0.05), free from hypoxia (HIF-1α+) (3.1±0.1 vs 0.8±0.2%, p&amp;lt;0.05) and cell death (TUNEL+) (3.2±0.1 vs 1.4±0.3%, p&amp;lt;0.05) after 7 days of in vitro culture. Echocardiography revealed significantly lower left ventricular end diastolic volume (LVEDV) and higher left ventricular ejection fraction (LVEF) in HiCT group [sham (n=27) vs GHM(−) (n=12) vs HiCT (n=12): LVEDV; 1.4±0.1 vs 1.3±0.1 vs 0.9±0.1mL, p&amp;lt;0.0001/LVEF; 55.3±1.1 vs 58.2±2.3 vs 78.2±1.5%, p&amp;lt;0.0001]. Cardiac MRI showed lower LVEDV and higher LVEF as well [sham (n=8) vs GHM(−) (n=6) vs HiCT (n=6): LVEDV; 0.7±0.03 vs 0.7±0.03 vs 0.6±0.02mL, p&amp;lt;0.01 / LVEF; 39.2±2.1 vs 43.8±1.4 vs 54.0±2.8%, p&amp;lt;0.001]. Conclusions We conclude that HiCTs generated from clinical-grade cells hold sufficient viability and tissue conformation suitable for functional recovery validated by a rat MI model. Clinical-grade human iPSCs potentially serve as a reasonable cell source for stem cell-derived product transplantation therapy with foreseeable clinical applications. Funding Acknowledgement Type of funding source: Other. Main funding source(s): Japan Agency for Medical Research and Development (AMED), Invited Research Project of Institute for Advancement of Clinical Transnational Science, Kyoto University Hospital

A novel topical treatment for bone metastases using a gelatin hydrogel incorporating cisplatin as a sustained release system.

Management of bone metastasis is becoming increasingly important. Thus, local and systemic treatment options have been developed for control. Although systemic administration of anticancer agents is effective for bone metastasis, it is often stopped because of poor general conditions or side effects. Therefore, it is highly desirable to develop a more effective and safer local treatment for bone metastasis. The purpose of the current study was to investigate the antitumor effects and safety of gelatin hydrogel microspheres incorporating cisplatin (GM-CDDP), which we developed as a sustained release system without harmful substances. First, we assessed GM-CDDP for its in vitro degradability and potential for sustained release. Second, in vivo antitumor and side effects were evaluated using a murine bone metastasis model of MDA-MB-231 human breast cancer cells incorporating GFP. In vitro, initial bursts were observed within 2 h and CDDP was released gradually with gelatin hydrogel degradation, which reached 100% at 48 h. In vivo, local administration of GM-CDDP (2 mg/kg) significantly suppressed tumor growth and bone osteolysis compared with the control, and local and systemic administration of free CDDP (2 mg/kg; p < 0.05). Local administration of GM-CDDP significantly reduced loss of body weight and elevation of blood urea nitrogen compared with the systemic administration of free CDDP (p < .05). The current study suggests that local administration of GM-CDDP achieves higher antitumor effects with a potential for lesser side effects compared with local or systemic administration of free CDDP.

A Co-Culture System of Three-Dimensional Tumor-Associated Macrophages and Three-Dimensional Cancer-Associated Fibroblasts Combined with Biomolecule Release for Cancer Cell Migration.

The objective of this study is to design a cancer invasion model by making use of cancer-associated fibroblasts (CAF) or tumor-associated macrophages (TAM) and gelatin hydrogel microspheres (GM) for the sustained release of drugs. The GM containing adenosine (A) (GM-A) were prepared and cultured with TAM to obtain three-dimensional (3D) TAM aggregates incorporating GM-A (3D TAM-GM-A). The GM-A incorporation enabled TAM to enhance the secretion level of vascular endothelial growth factor. When co-cultured with HepG2 liver cancer cells in an invasion assay, the 3D TAM-GM-A promoted the invasion rate of cancer cells. In addition, the E-cadherin expression level decreased to a significantly greater extent compared with that co-cultured with TAM aggregates incorporating GM, whereas the significantly higher expression of N-cadherin and Vimentin was observed. This indicates that the epithelial-mesenchymal transition event was induced. The GM containing transforming growth factor-β1 (TGF-β1) were prepared to incorporate into 3D CAF (3D CAF-GM-TGF-β1). Following a co-culture of mixed 3D CAF-GM-TGF-β1 and 3D TAM-GM-A and every HepG2, MCF-7 breast cancer cell, or WA-hT lung cancer cell, the invasion rate of every cancer cell enhanced depending on the mixing ratio of 3D TAM-GM-A and 3D CAF-GM-TGF-β1. The amount of matrix metalloproteinase-2 (MMP-2) secreted also enhanced, and the enhancement was well corresponded with that of cancer cell invasion rate. The higher MMP secretion assists the breakdown of basement membrane, leading to the higher rate of cancer cell invasion. This model is a promising 3D culture system to evaluate the invasion ability of various cancer cells in vitro. Impact statement This study proposes a cell culture system to enhance the tumor-associated macrophage function based on the combination of three-dimensional (3D) cell aggregates and gelatin hydrogel microspheres (GM) for adenosine delivery. An additional combination of 3D cancer-associated fibroblasts incorporating GM containing transforming growth factor-β1 allowed cancer cells to enhance their invasion rate. This co-culture system is promising to evaluate the ability of cancer cell invasion for anticancer drug screening.

Preparation of cell aggregates incorporating gelatin hydrogel microspheres of sugar-responsive water solubilization.

The objective of this study is to design hydrogel microspheres of a cell scaffold, which not only function as a scaffold to form cell aggregates of three-dimensional culture but also can disappear to release growth factors in the well-controlled manner by noncytotoxic stimulation in any timing. The hydrogel microspheres were prepared by a water-in-oil emulsion method from m-aminophenylboronic acid (APBA)-introduced gelatin (APBA-gelatin) with or without poly(vinyl alcohol) (PVA) mixing. Irrespective of the PVA concentration, the microspheres with the same diameter were prepared. The microspheres were water solubilized only by adding sorbitol of a sugar although the solubilization extent depended on the PVA concentration. When cocultured with the microspheres, mesenchymal stem cells formed cell aggregates homogeneously incorporating the microspheres. Upon adding sorbitol in the culture medium, mixed APBA-gelatin-PVA hydrogel microspheres disappeared with time in the cell aggregates. The microspheres containing basic fibroblast growth factor or bone morphogenetic protein-2 released the respective growth factor accompanied with the microspheres disappearance. It is concluded that the present microspheres of sugar-responsive water solubilization are promising scaffold of cell aggregates and have an ability to allow growth factors to be released in the cell aggregates when it is required.

A cancer invasion model of cancer-associated fibroblasts aggregates combined with TGF-β1 release system.

IntroductionThe objective of this study is to design a cancer invasion model where the cancer invasion rate can be regulated in vitro.MethodsCancer-associated fibroblasts (CAF) aggregates incorporating gelatin hydrogel microspheres (GM) containing various concentrations of transforming growth factor-β1 (TGF-β1) (CAF-GM-TGF-β1) were prepared. Alpha-smooth muscle actin (α-SMA) for the CAF aggregates was measured to investigate the CAF activation level by changing the concentration of TGF-β1. An invasion assay was performed to evaluate the cancer invasion rate by co-cultured of cancer cells with various CAF-GM-TGF-β1.ResultsThe expression level of α-SMA for CAF increased with an increased in the TGF-β1 concentration. When co-cultured with various types of CAF-GM-TGF-β1, the cancer invasion rate was well correlated with the α-SMA level. It is conceivable that the TGF-β1 concentration could modify the level of CAF activation, leading to the invasion rate of cancer cells. In addition, at the high concentrations of TGF-β1, the effect of a matrix metalloproteinase (MMP) inhibitor on the cancer invasion rate was observed. The higher invasion rate would be achieved through the higher MMP production.ConclusionsThe present model is promising to realize the cancer invasion whose rate can be modified by changing the TGF-β1 concentration.

A Cancer Invasion Model Combined with Cancer-Associated Fibroblasts Aggregates Incorporating Gelatin Hydrogel Microspheres Containing a p53 Inhibitor.

The objective of this study is to design a cancer invasion model based on an interaction between cancer cells and cancer-associated fibroblasts (CAF) aggregates. The strength of this study is to incorporate gelatin hydrogel microspheres (GM) containing pifithrin-α (PFT) of a p53 inhibitor (GM-PFT) with the CAF aggregates. Incorporation of GM-PFT allowed CAF aggregates to enhance the alpha-smooth muscle actin expression level at a high concentration of PFT. When the cancer cells were cocultured with the CAF aggregates incorporating GM-PFT, the invasion rate of cancer cells was significantly high compared with CAF aggregates or CAF aggregates incorporating GM with or without the same dose of free PFT as well as two-dimension cultured CAF with or without the same dose of PFT. In addition, an inhibitor of matrix metalloproteinase decreased the cancer invasion rate for the CAF aggregates incorporating GM-PFT. It is concluded that the interaction between cancer cells and CAF aggregates incorporating GM-PFT of biological activation needs to realize the invasion of cancer cells even in vitro. Impact Statement The strength of this study is to combine with a three-dimensional cell culture system and a drug delivery system technology for a cancer invasion model. The combination enabled cancer-associated fibroblasts to enhance the biological functions. This cancer invasion model is a promising tool to mimic the tumor microenvironment for anticancer drug screening.

Regenerative potential of basic fibroblast growth factor contained in biodegradable gelatin hydrogel microspheres applied following vocal fold injury: Early effect on tissue repair in a rabbit model

IntroductionPostoperative dysphonia is mostly caused by vocal fold scarring, and careful management of vocal fold surgery has been reported to reduce the risk of scar formation. However, depending on the vocal fold injury, treatment of postoperative dysphonia can be challenging. ObjectiveThe goal of the current study was to develop a novel prophylactic regenerative approach for the treatment of injured vocal folds after surgery, using biodegradable gelatin hydrogel microspheres as a drug delivery system for basic fibroblast growth factor. MethodsVideoendoscopic laryngeal surgery was performed to create vocal fold injury in 14 rabbits. Immediately following this procedure, biodegradable gelatin hydrogel microspheres with basic fibroblast growth factor were injected in the vocal fold. Two weeks after injection, larynges were excised for evaluation of vocal fold histology and mucosal movement. ResultsThe presence of poor vibratory function was confirmed in the injured vocal folds. Histology and digital image analysis demonstrated that the injured vocal folds injected with gelatin hydrogel microspheres with basic fibroblast growth factor showed less scar formation, compared to the injured vocal folds injected with gelatin hydrogel microspheres only, or those without any injection. ConclusionA prophylactic injection of basic fibroblast growth factor -containing biodegradable gelatin hydrogel microspheres demonstrates a regenerative potential for injured vocal folds in a rabbit model.

Influence of shaking culture on the biological functions of cell aggregates incorporating gelatin hydrogel microspheres

The objective of this study is to investigate the influence of shaking culture on the biological functions of cell aggregates incorporating gelatin hydrogel microspheres in terms of the microspheres/cells ratio. The mixture of MC3T3-E1 cells and the microspheres was cultured in the U-bottomed wells of 96-well plate pre-coated with poly (vinyl alcohol) (PVA) to form cell aggregates incorporating microspheres. When incubated in the static or shaking culture, the size of cell aggregates increased with amounts of gelatin hydrogel microspheres but was similar between the two cultures. At the smaller ratio of microspheres to cells, the viability of cell aggregates under the shaking culture was significantly higher than that of static culture. On the other hand, there was no significant difference in the viability between them at the higher ratio. Gelatin hydrogel microspheres enabled to enhance ATP and mitochondrial activities of cell aggregates under the shaking culture. The effect was high at the smaller microspheres/cells ratio. It is concluded that the shaking culture was promising to allow cells to enhance their activities.