Microbial Poly(hydroxybutyrate-co-hydroxyvalerate) Scaffold for Periodontal Tissue Engineering.

In this study, we fabricated three dimensional (3D) porous scaffolds of poly(hydroxybutyrate-co-hydroxyvalerate) with 50% HV content. P(HB-50HV) was biosynthesized from bacteria Cupriavidus necator H16 and the in vitro proliferation of dental cells for tissue engineering application was evaluated. Comparisons were made with scaffolds prepared by poly(hydroxybutyrate) (PHB), poly(hydroxybutyrate-co-12%hydroxyvalerate) (P(HB-12HV)), and polycaprolactone (PCL). The water contact angle results indicated a hydrophobic character for all polymeric films. All fabricated scaffolds exhibited a high porosity of 90% with a sponge-like appearance. The P(HB-50HV) scaffolds were distinctively different in compressive modulus and was the material with the lowest stiffness among all scaffolds tested between the dry and wet conditions. The human gingival fibroblasts (HGFs) and periodontal ligament stem cells (PDLSCs) cultured onto the P(HB-50HV) scaffold adhered to the scaffold and exhibited the highest proliferation with a healthy morphology, demonstrating excellent cell compatibility with P(HB-50HV) scaffolds. These results indicate that the P(HB-50HV) scaffold could be applied as a biomaterial for periodontal tissue engineering and stem cell applications.

Exogenous LIN28 Is Required for the Maintenance of Self-Renewal and Pluripotency in Presumptive Porcine-Induced Pluripotent Stem Cells.

Porcine species have been used in preclinical transplantation models for assessing the efficiency and safety of transplants before their application in human trials. Porcine-induced pluripotent stem cells (piPSCs) are traditionally established using four transcription factors (4TF): OCT4, SOX2, KLF4, and C-MYC. However, the inefficiencies in the reprogramming of piPSCs and the maintenance of their self-renewal and pluripotency remain challenges to be resolved. LIN28 was demonstrated to play a vital role in the induction of pluripotency in humans. To investigate whether this factor is similarly required by piPSCs, the effects of adding LIN28 to the 4TF induction method (5F approach) on the efficiency of piPSC reprogramming and maintenance of self-renewal and pluripotency were examined. Using a retroviral vector, porcine fetal fibroblasts were transfected with human OCT4, SOX2, KLF4, and C-MYC with or without LIN28. The colony morphology and chromosomal stability of these piPSC lines were examined and their pluripotency properties were characterized by investigating both their expression of pluripotency-associated genes and proteins and in vitro and in vivo differentiation capabilities. Alkaline phosphatase assay revealed the reprogramming efficiencies to be 0.33 and 0.17% for the 4TF and 5TF approaches, respectively, but the maintenance of self-renewal and pluripotency until passage 40 was 6.67 and 100%, respectively. Most of the 4TF-piPSC colonies were flat in shape, showed weak positivity for alkaline phosphatase, and expressed a significantly high level of SSEA-4 protein, except for one cell line (VSMUi001-A) whose properties were similar to those of the 5TF-piPSCs; that is, tightly packed and dome-like in shape, markedly positive for alkaline phosphatase, and expressing endogenous pluripotency genes (pOCT4, pSOX2, pNANOG, and pLIN28), significantly high levels of pluripotent proteins (OCT4, SOX2, NANOG, LIN28, and SSEA-1), and a significantly low level of SSEA-4 protein. VSMUi001-A and all 5F-piPSC lines formed embryoid bodies, underwent spontaneous cardiogenic differentiation with cardiac beating, expressed cardiomyocyte markers, and developed teratomas. In conclusion, in addition to the 4TF, LIN28 is required for the effective induction of piPSCs and the maintenance of their long-term self-renewal and pluripotency toward the development of all germ layers. These piPSCs have the potential applicability for veterinary science.

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model.

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

Human Mesenchymal Stromal Cell Sheets Induce Macrophages Predominantly to an Anti-Inflammatory Phenotype.

Tissue healing is a highly complex process involving a cascade of biochemical and cellular events. Excessive inflammation can impair the healing response. Previous in vitro studies have shown that mesenchymal stromal cells can modulate macrophage-induced inflammation and, therefore, are promising candidates for cell-based therapies aimed at promoting tissue repair. Recently, cell sheets were introduced as a new method of delivering stromal cells to the repair site. The goal of the current study was to compare the effect of different types of stromal cell sheets on the inflammatory state of macrophages in vitro. We compared the effects of adipose tissue-derived stromal cell (ASC) sheets, bone marrow derived stromal cell (BMSC) sheets, and fibroblast sheets on macrophage functional phenotype using flow cytometric analysis, gene expression, as well as cell sheet protein secretion. This was evaluated with and without inflammatory stimulation. Viability and senescence for the different types of sheet were also evaluated. Macrophages cultured in ASC sheet conditioned medium (CM) displayed a higher fluorescence intensity of the anti-inflammatory CD206 surface marker than when cultured in BMSC sheet CM and expressed more CCL18 and IL1RA than when cultured in fibroblast sheet CM. Moreover, ASC sheets had higher cell viability and less senescent cells than BMSC sheets and fibroblast sheets. Taken together, ASC and BMSC can stimulate the anti-inflammatory macrophage (M2) phenotype to a better extent than fibroblasts. It is suggested that ASC sheets might outperform BMSC sheets in an inflammatory situation since ASC sheet CM induced-macrophages have more M2 characteristics, and ASC in the sheet was more viable.

Adipose Tissue-Derived Stem Cell Sheet Application for Tissue Healing In Vivo: A Systematic Review.

Adipose tissue-derived stem cells (ASCs) are known to be tissue-healing promoters due to their cellular plasticity and secretion of paracrine factors. Cultured ASC sheets provide a novel method of ASC application and can retain ASCs at the targeted tissue. The purpose of this systematic review is to evaluate preclinical studies using ASC sheet transplantation therapy for promoting tissue healing. First, we searched databases to identify studies of ASC sheet therapy in different experimental animal models, and then determined the quality score of studies using SYRCLE's risk bias tool. A total of 18 included studies examined the role of ASC sheets on tissue healing and function in models for myocardial infarction, dilated cardiomyopathy, full-thickness skin wounds, hind limb ischemia, esophageal strictures, and oral ulcers. ASC sheet application after myocardial infarction improved survival rate, cardiac function, and capillary density and reduced the extent of fibrosis. Application of ASC sheets to a full-thickness skin wound decreased the wound size and stimulated wound maturation. In the hind limb ischemia model, ASC sheet application improved limb perfusion and capillary density, and decreased the amount of ischemic tissue and inflammation. ASC sheet application to mucosal wounds of the digestive tract accelerated wound healing and decreased the degree of stricture and fibrosis. Taken together, transplanted ASC sheets had a positive effect on tissue healing and reconstruction in these preclinical studies. The reported favorable effects of ASC sheet therapy in various tissue healing applications may be implemented in future translational studies. It is suggested that future preclinical animal model studies of ASC sheet therapy should concern standardization of culture techniques and investigate the mechanisms of action. In addition, clearly indicated experimental setups according to the SYRCLE's guidelines should improve study quality and validity.

Effects of adipose stem cell sheets on colon anastomotic leakage in an experimental model: Proof of principle.

The most dreaded complication of colorectal surgery is anastomotic leakage. Adipose tissue-derived stem cell sheets (ASC sheets) prepared from temperature-responsive culture surfaces can be easily transplanted onto tissues. These sheets are proposed to improve cell transplant efficiency and enhance wound healing. The aim of this study was to investigate whether application of ASC sheets could prevent leakage of sutured colorectal anastomoses. Insufficient suturing of colorectal anastomoses was performed in Wistar rats to create a colorectal anastomotic leakage model. Rats were randomized to ASC sheet application or control group. Leakage, abscess formation, adhesion formation, anastomotic bursting pressure (ABP), and histology were evaluated on postoperative day 3 or 7. ASC sheet application significantly reduced anastomotic leakage compared to controls, without increased adhesion formation. ASC sheet transplantation resulted in more CD3+ T-cells and CD163+ anti-inflammatory macrophages at the anastomotic site than the control group. ABP, vessel density and collagen deposition were not different between groups. Using cell sheet technology, we generated ASC sheets that prevented disruption of sutured colorectal anastomoses as shown by reduced leakage. Increased numbers of anti-inflammatory macrophages and T-cells might have contributed to this positive effect.

Effect of Cell Seeding Density and Inflammatory Cytokines on Adipose Tissue-Derived Stem Cells: an in Vitro Study.

Adipose tissue-derived stem cells (ASCs) are known to be able to promote repair of injured tissue via paracrine factors. However, the effect of cell density and inflammatory cytokines on the paracrine ability of ASCs remains largely unknown. To investigate these effects, ASCs were cultured in 8000 cells/cm2, 20,000 cells/cm2, 50,000 cells/cm2, and 400,000 cells/cm2 with and without 10 or 20 ng/ml tumor necrosis factor alpha (TNFα) and 25 or 50 ng/ml interferon gamma (IFNγ). ASC-sheets formed at 400,000 cells/cm2 after 48 h of culture. With increasing concentrations of TNFα and IFNγ, ASC-sheets with 400,000 cells/cm2 had increased production of angiogenic factors Vascular Endothelial Growth Factor and Fibroblast Growth Factor and decreased expression of pro-inflammatory genes TNFA and Prostaglandin Synthase 2 (PTGS2) compared to lower density ASCs. Moreover, the conditioned medium of ASC-sheets with 400,000 cells/cm2 stimulated with the low concentration of TNFα and IFNγ enhanced endothelial cell proliferation and fibroblast migration. These results suggest that a high cell density enhances ASC paracrine function might beneficial for wound repair, especially in pro-inflammatory conditions.