Development of a nasal mucosa-removal model for evaluating cell therapy.

INTRODUCTION: Endoscopic sinus surgery is an effective surgical procedure for treating chronic sinusitis; however, extensive exposure of the bone in the nasal cavity can result in permanent disability postoperatively. Particularly, closure of the sinus drainage pathway due to bone hyperplasia associated with bone exposure can trigger the recurrence of sinusitis. It is essential to regenerate the nasal mucosa after surgery to avoid bone hyperplasia. Regenerative medicine, including cell therapy, could be one of the leading options for nasal mucosa regeneration. To date, there is a lack of effective models for evaluating treatments for prevention of bone hyperplasia that occurs after sinus surgery. The purpose of this study was to develop a model of nasal mucosal removal to evaluate cellular therapies. METHODS: The model was created in rabbits, a species with a wide nasal structure, and was generated by approaching the maxillary sinus from the nasal bone side and solely removing the maxillary sinus mucosa without destroying the structures in the nasal cavity. Adipose-derived mesenchymal stromal cell sheets prepared in temperature-responsive cell culture dishes were examined for the effect of transplantation in the animal model. Intranasal evaluation was assessed by micro-computed tomography and tissue staining. RESULTS: Significant bone hyperplasia in the maxillary sinus occurred on the side of mucosal removal, and no bone hyperplasia occurred in the control sham side in the same rabbits on postoperative day 28. Bone hyperplasia was observed over a short time period, with the presence of bone hyperplasia in the maxillary sinus on day 14 and calcification of the bone on day 28. The adipose-derived mesenchymal stromal cell (ADSC) sheet was transplantable in a nasal mucosa-removal model. No significant differences in bone hyperplasia were found between the transplantation side and the sham side in terms of the effect of transplantation of the ADSC sheet; however, bone hyperplasia tended to be suppressed on the transplantation side. CONCLUSIONS: This animal model is simple, highly reproducible, and does not require special equipment or drugs. In addition, this model can be used for various therapeutic interventions, including cell therapy. The presence or absence of the nasal mucosa affects bone remodeling, which highlights the importance of regeneration of the nasal mucosa. In the nasal mucosal regeneration therapy, the ADSC sheet had an inhibitory effect on bone hyperplasia. The nasal mucosa-removal model allows observation of conditions associated with nasal mucosa removal and evaluation of the effectiveness of cell therapy.

A novel rat model of inflammatory bowel disease developed using a device created with a 3D printer.

OBJECTIVE: Inflammatory bowel disease (IBD) is an intractable condition. Existing models of experimental IBD are limited by their inability to create consistent ulcers between animals. The aim of this study was to develop a novel model of experimental colitis with ulcers of reproducible size. DESIGN: We used a 3D printer to fabricate a novel device containing a small window (10 × 10 mm) that could be inserted rectally to facilitate the creation of a localized ulcer in the rat intestinal mucosa. The mucosa within the window of the device was exposed to 2,4,6-trinitrobenzene sulfonic acid (TNBS) to generate ulceration. We evaluated the effects of conventional drug therapies (mesalazine and prednisolone) and local transplantation of allogeneic adipose-derived mesenchymal stem cells (ASCs) on ulcer size (measured from photographic images using image analysis software) and degree of inflammation (assessed histologically). RESULTS: The novel method produced localized, circular or elliptical ulcers that were highly reproducible in terms of size and depth. The pathological characteristics of the lesions were similar to those reported previously for conventional models of TNBS-induced colitis that show greater variation in ulcer size. Ulcer area was significantly reduced by the administration of mesalazine or prednisolone as an enema or localized injection of ASCs. CONCLUSION: The new model of TNBS-induced colitis, made with the aid of a device fabricated by 3D printing, generated ulcers that were reproducible in size. We anticipate that our new model of colitis will provide more reliable measures of treatment effects and prove useful in future studies of IBD therapies.

Enhanced migration of murine fibroblast-like 3T3-L1 preadipocytes on type I collagen-coated dish is reversed by silibinin treatment.

Migration of fibroblast-like preadipocytes is important for the development of adipose tissue, whereas excessive migration is often responsible for impaired adipose tissue related with obesity and fibrotic diseases. Type I collagen (collagen I) is the most abundant component of extracellular matrix and has been shown to regulate fibroblast migration in vitro, but its role in adipose tissue is not known. Silibinin is a bioactive natural flavonoid with antioxidant and antimetastasis activities. In this study, we found that type I collagen coating promoted the proliferation and migration of murine 3T3-L1 preadipocytes in a dose-dependent manner, implying that collagen I could be an extracellular signal. Regarding the mechanisms of collagen I-stimulated 3T3-L1 migration, we found that NF-κB p65 is activated, including the increased nuclear translocation of NF-κB p65 as well as the upregulation of NF-κB p65 phosphorylation and acetylation, accompanied by the increased expressions of proinflammatory factors and the generation of reactive oxygen species (ROS). Reduction of collagen I-enhanced migration of cells by treatment with silibinin was associated with suppression of NF-κB p65 activity and ROS generation, and negatively correlated with the increasing sirt1 expression. Taken together, the enhanced migration of 3T3-L1 cells induced on collagen I-coated dish is mediated by the activation of NF-κB p65 function and ROS generation that can be alleviated with silibinin by upregulation of sirt1, leading to the repression of NF-κB p65 function and ROS generation.

The role of Tsukushi (TSK), a small leucine-rich repeat proteoglycan, in bone growth.

INTRODUCTION: Endochondral ossification is one of a key process for bone maturation. Tsukushi (TSK) is a novel member of the secreted small leucine-rich repeat proteoglycan (SLRP) family. SLRPs localize to skeletal regions and play significant roles during whole phases of bone development. Although prior evidence suggests that TSK may be involved in the regulation of bone formation, its role in skeletal development has not yet been elucidated. METHODS: In the present study, we examined TSK's function during bone growth by comparing skeletal growth of TSK deficient (TSK-/-) mice and wild type (WT) mice. And an in vitro experiment using siRNA transfection of a chondrogenic cell line was performed. RESULTS: TSK-/- mice exhibited decreased weight and short stature at 3 weeks of age due to decreased longitudinal bone growth coupled with low bone mass. Furthermore, an in vitro experiment using siRNA transfection into a chondrogenic cell line revealed that decreased TSK expression induced down-regulation of key chondrogenic marker gene expression and up-regulation of mid-to-late chondrogenic markers gene expression. CONCLUSIONS: Our results reveal that TSK regulates bone elongation and bone mass by modulating growth plate chondrocyte function and consequently, overall body size.

Membrane-Permeable Calpain Inhibitors Promote Rat Oral Mucosal Epithelial Cell Proliferation by Inhibiting IL-1α Signaling.

To standardise regenerative medicine using cultured cells, the use of serum-free, chemically defined media will be necessary. We have reported that IL-1α inhibits the growth of epithelial cells in culture and that recombinant IL-1 receptor antagonist (IL-1RA) significantly promotes epithelial cell growth in no feeder layer condition. In this study, we examined inhibitors of calpain, a cysteine proteinase that plays crucial roles in various cellular functions, including IL-1α maturation and secretion. The culturing of epithelial cells in serum-free media supplemented with a membrane-permeable calpain inhibitor significantly promoted growth while suppressing IL-1α maturation and secretion. By contrast, non-membrane-permeable calpain inhibitor treatment did not have these effects. Interestingly, immunoblotting analysis revealed that immature, untruncated, IL-1α expression was also downregulated by cell-permeable calpain inhibitor treatment, and the difference in IL-1α gene expression increased from day 2 to day 6. Although IL-1RA has been reported to promote epithelial cell growth, we detected no synergistic promotion of epithelial cell growth using a calpain inhibitor and IL-1RA. These findings indicate that calpain inhibitors promote epithelial cell proliferation by inhibiting IL-1α maturation at an early phase of epithelial cell culture and by suppressing the positive feedback-mediated amplification of IL-1α signalling.

Improved enzymatic treatment for accurate cell counting from extracellular matrix-rich periodontal ligament cell sheets.

PURPOSE: The objective of this study was to establish a method for accurate cell counting from matrix-rich cell sheets in the clinical setting. MATERIALS AND METHODS: Human periodontal ligament (HPDL) cells were obtained from healthy donors to prepare PDL cell sheets. To obtain single cell suspensions, the cell sheets were treated with three different enzymatic formulations: collagenase alone, trypsin-ethylenediaminetetraacetic acid (EDTA) alone, and a combination of collagenase and trypsin-EDTA. After cell dispersion, cell numbers and cell survival rates were measured. To evaluate damage to the cell surfaces from the enzymes, the dispersed cells were analyzed by a flow cytometer with an anti-alkaline phosphatase antibody. RESULTS: Treatment with collagenase alone or trypsin-EDTA alone dispersed few cells from HPDL cell sheets. In contrast, combined treatment with collagenase and trypsin-EDTA successfully produced a large amount of single cells from cell sheets. Flow cytometry analysis showed that single cells obtained by combined use of collagenase and trypsin-EDTA preserved alkaline phosphatase epitopes on the cell surfaces. CONCLUSIONS: Cell sheets rich with extracellular matrix were dispersed via combined treatment with collagenase and trypsin-EDTA without destroying the expression of cell surface markers. The results suggest that this method would be useful for determining the accurate cell number of cell sheets for cell therapies and should also be applicable for other kinds of matrix-rich cell sheets.

An autoclave treatment reduces the solubility and antigenicity of an allergenic protein found in buckwheat flour.

The effects of an autoclave treatment of buckwheat flour on a 24-kDa allergenic protein were investigated by measuring reduction in solubility and antibody binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the intensity of the major bands, including that of the 24-kDa allergen, was reduced by the autoclave treatment. The protein solubility in buckwheat flour was variably decreased by the autoclave treatment. Enzyme-linked immunosorbent assay analysis using a monoclonal antibody specific for buckwheat 24-kDa protein showed that the reactivity of protein extracts (10 µg/ml) from buckwheat flour was lowered by the autoclave treatment. The autoclave treatment may reduce the major allergen content of buckwheat. Future studies will determine if autoclaving treatments affect the allergenicity of the 24-kDa buckwheat protein.

Induction of putative stratified epithelial progenitor cells in vitro from mouse-induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells generally exhibit a normal karyotype, are transcriptionally and epigenetically similar to embryonic stem (ES) cells, and maintain the potential to differentiate into derivatives of all germ layers. Recently, the use of different types of cell or tissue derived from iPS cells for transplantation has become a possibility. However, the differentiation of epithelial lineages from iPS cells has not yet been demonstrated. We attempted to establish a culture technique for the induction of epithelial progenitors from mouse iPS cells. Mouse iPS cells were cultured on dishes coated with type IV collagen in keratinocyte culture medium (KCM) supplemented with or without bone morphogenic protein-4 (BMP-4) or combined with pretreatment of retinoic acid (RA) and BMP-4 in the undifferentiated state. Markers for undifferentiated cells (Oct3/4, Nanog) and for differentiation (p63, cytokeratin14) were analyzed by immunofluorescence staining and real-time RT-PCR. Putative epithelial progenitors were successfully induced in vitro from iPS cells. These progenitors expressed p63, a transcription factor necessary for maintenance of regenerative epithelia and cytokeratin 14 constitutively present in the basal layer of stratified epithelia. Enhancement of putative epithelial progenitor commitment was observed when cultured in KCM with BMP-4 following pretreatment of RA and BMP-4. The differentiation efficiency of putative epithelial progenitors from iPS cell cultures was similar to that of ES cell cultures. This report is the first to demonstrate in vitro differentiation of iPS cells into putative epithelial progenitors. These iPS-derived putative epithelial progenitors provide a powerful tool for understanding the mechanisms of epithelial lineage differentiation.

Cell delivery in regenerative medicine: the cell sheet engineering approach.

Recently, cell-based therapies have developed as a foundation for regenerative medicine. General approaches for cell delivery have thus far involved the use of direct injection of single cell suspensions into the target tissues. Additionally, tissue engineering with the general paradigm of seeding cells into biodegradable scaffolds has also evolved as a method for the reconstruction of various tissues and organs. With success in clinical trials, regenerative therapies using these approaches have therefore garnered significant interest and attention. As a novel alternative, we have developed cell sheet engineering using temperature-responsive culture dishes, which allows for the non-invasive harvest of cultured cells as intact sheets along with their deposited extracellular matrix. Using this approach, cell sheets can be directly transplanted to host tissues without the use of scaffolding or carrier materials, or used to create in vitro tissue constructs via the layering of individual cell sheets. In addition to simple transplantation, cell sheet engineered constructs have also been applied for alternative therapies such as endoscopic transplantation, combinatorial tissue reconstruction, and polysurgery to overcome limitations of regenerative therapies and cell delivery using conventional approaches.

Influence of insulin immobilization to thermoresponsive culture surfaces on cell proliferation and thermally induced cell detachment.

Temperature-responsive culture dishes immobilized with insulin have been fabricated and studied to shorten cell culture periods by facilitating more rapid cell proliferation. Cells are recovered as contiguous cell sheets simply by temperature changes. Functionalized culture dishes were prepared by previously reported electron beam grafting copolymerization of N-isopropylacrylamide (IPAAm) with its carboxylate-derivatized analog, 2-carboxyisopropylacrylamide (CIPAAm), having similar molecular structure to IPAAm but with carboxylate side chains to tissue culture polystyrene dishes. Insulin was then immobilized onto culture dishes through standard amide bond formation with CIPAAm carboxylate groups. Adhesion and proliferation of bovine carotid artery endothelial cells (ECs) were examined on these insulin-immobilized dishes. Insulin immobilization was shown to promote cell proliferation in serum-supplemented medium. Increasing the grafted CIPAAm content on the tissue culture surfaces reduces cell adhesion and proliferation, even though these surfaces contained increased amounts of immobilized insulin. This result implies that a discrete balance exists between the amount of CIPAAm-free carboxylate groups and immobilized insulin for optimum cell proliferative stimulation. Cells grown on the insulin-immobilized surfaces can be recovered as contiguous cell monolayers simply by lowering culture temperature, without need for exogenous enzyme or calcium chelator additions. In conclusion, insulin-modified thermoresponsive culture dishes may prove useful for advanced cell culture and tissue engineering applications since they facilitate cell proliferation, and cultured cells can be recovered as viable contiguous monolayers by merely reducing culture temperature.

Micropatterned surfaces prepared using a liquid crystal projector-modified photopolymerization device and microfluidics.

A commercial liquid crystal device projector was modified for photopolymerization using its on-board intense light source and a precision optical control circuit. This device projects reduced images generated by a typical personal computer onto the stage where photopolymerization on a surface occurs. This all-in-one device does not require expensive photomasks and external light sources. However, light scattering and diffraction through glass substrates resulted in undesired reactions in areas corresponding to masked (black) domains in mask patterns, limiting pattern resolution. To overcome this shortcoming, two-step surface patterning was developed. First, three-dimensional microstructures of crosslinked silicone elastomer were fabricated with this device and adhered onto silanized glass substrate surfaces, forming microchannels in patterns on the glass support. Then, acrylamide monomer solution containing photoreactive initiator was flowed into these micromold channels and reacted in situ. The resultant polyacrylamide layer was highly hydrophilic and repelled protein adsorption. Cell seeding on these patterns in serum-supplemented culture medium produced cells selectively adhered to different patterns: cells attached and spread only on unpolymerized silanized glass surfaces, not on the photopolymerized acrylamide surfaces. This technique should prove useful for inexpensive, rapid prototyping of surface micropatterns from polymer materials.

Fabrication of pulsatile cardiac tissue grafts using a novel 3-dimensional cell sheet manipulation technique and temperature-responsive cell culture surfaces.

Recent progress in cell transplantation therapy to repair impaired hearts has encouraged further attempts to bioengineer 3-dimensional (3-D) heart tissue from cultured cardiomyocytes. Cardiac tissue engineering is currently pursued utilizing conventional technology to fabricate 3-D biodegradable scaffolds as a temporary extracellular matrix. By contrast, new methods are now described to fabricate pulsatile cardiac grafts using new technology that layers cell sheets 3-dimensionally. We apply novel cell culture surfaces grafted with temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), from which confluent cells detach as a cell sheet simply by reducing temperature without any enzymatic treatments. Neonatal rat cardiomyocyte sheets detached from PIPAAm-grafted surfaces were overlaid to construct cardiac grafts. Layered cell sheets began to pulse simultaneously and morphological communication via connexin43 was established between the sheets. When 4 sheets were layered, engineered constructs were macroscopically observed to pulse spontaneously. In vivo, layered cardiomyocyte sheets were transplanted into subcutaneous tissues of nude rats. Three weeks after transplantation, surface electrograms originating from transplanted grafts were detected and spontaneous beating was macroscopically observed. Histological studies showed characteristic structures of heart tissue and multiple neovascularization within contractile tissues. Constructs transplanted into 3-week-old rats exhibited more cardiomyocyte hypertrophy and less connective tissue than those placed into 8-week-old rats. Long-term survival of pulsatile cardiac grafts was confirmed up to 12 weeks. These results demonstrate that electrically communicative pulsatile 3-D cardiac constructs were achieved both in vitro and in vivo by layering cardiomyocyte sheets. Cardiac tissue engineering based on this technology may prove useful for heart model fabrication and cardiovascular tissue repair. The full text of this article is available at http://www.circresaha.org.