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Cell coculture strategies can promote angiogenesis within tissue engineering constructs. This study aimed to test the angiogenic potential of human umbilical vein endothelial cells (HUVEC) cocultured with gingiva-derived progenitor cells (GPC) as spheroids in a xeno-free environment. Human platelet lysate (HPL) was used as a cell culture supplement and as a hydrogel matrix (HPLG) for spheroid encapsulation. HUVEC and HUVEC + GPC (1:1 or 5:1) spheroids were encapsulated in various HPLG formulations. Angiogenesis was assessed via in vitro sprouting and in vivo chick chorioallantoic membrane (CAM) assays. HUVEC revealed characteristic in vitro sprouting in HPL/HPLG and this was significantly enhanced in cocultures with GPC (p < 0.05). A trend for greater sprouting was observed in 5:1 vs 1:1 HUVEC + GPC spheroids and in certain HPLG formulations (p > 0.05). Both HUVEC and HUVEC + GPC spheroids in HPLG revealed abundant and comparable neoangiogenesis in the CAM assay (p > 0.05). Spheroid coculture of HUVEC + GPC in HPLG represents a promising strategy to promote angiogenesis.

Gingiva has been identified as a minimally invasive source of multipotent progenitor cells (GPCs) for use in bone tissue engineering (BTE). To facilitate clinical translation, it is important to characterize GPCs in xeno-free cultures. Recent evidence indicates several advantages of three-dimensional (3D) spheroid cultures of mesenchymal stromal cells (MSCs) over conventional 2D monolayers. The present study aimed to characterize human GPCs in xeno-free 2D cultures, and to test their osteogenic potential in 3D cultures, in comparison to bone marrow MSCs (BMSCs). Primary GPCs and BMSCs were expanded in human platelet lysate (HPL) or fetal bovine serum (FBS) and characterized based onin vitroproliferation, immunophenotype and multi-lineage differentiation. Next, 3D spheroids of GPCs and BMSCs were formed via self-assembly and cultured in HPL. Expression of stemness- (SOX2, OCT4, NANOG) and osteogenesis-related markers (BMP2, RUNX2, OPN, OCN) was assessed at gene and protein levels in 3D and 2D cultures. The cytokine profile of 3D and 2D GPCs and BMSCs was assessed via a multiplex immunoassay. Monolayer GPCs in both HPL and FBS demonstrated a characteristic MSC-like immunophenotype and multi-lineage differentiation; osteogenic differentiation of GPCs was enhanced in HPL vs. FBS. CD271(+)GPCs in HPL spontaneously acquired a neuronal phenotype and strongly expressed neuronal/glial markers. 3D spheroids of GPCs and BMSCs with high cell viability were formed in HPL media. Expression of stemness- and osteogenesis-related genes was significantly upregulated in 3D vs. 2D GPCs/BMSCs; the latter was independent of osteogenic induction. Synthesis of SOX2, BMP2 and OCN was confirmed via immunostaining, andin vitromineralization via Alizarin red staining. Finally, secretion of several growth factors and chemokines was enhanced in GPC/BMSC spheroids, while that of pro-inflammatory cytokines was reduced, compared to monolayers. In summary, monolayer GPCs expanded in HPL demonstrate

It has been suggested that the effect of implanted cells on the local environment is important when selecting the appropriate cell type for tissue regeneration. Our aim was to compare the local tissue response to implanted human mesenchymal stem cells (MSC) and human umbilical vein endothelial cells (EC). MSC and EC were cultured in poly(l-lactide-co-1,5-dioxepan-2-one) scaffolds for 1 week in a bioreactor system, after which they were implanted subcutaneously in NOD/SCID mice. After 3 weeks, scaffolds were retrieved, and the mRNA expression of selected genes involved in hypoxia and inflammation was examined by real-time reverse transcription polymerase chain reaction and correlated with immunofluorescent staining for corresponding proteins. The Toll-like receptor signaling pathway was examined by superarray hybridization. The expression of 53 angiogenesis-related proteins was investigated by a proteome profiler angiogenesis antibody array kit. Vascularization was quantified using immunohistochemistry for CD31. The expression of hypoxia-inducible factors and biomarkers for angiogenesis was more strongly upregulated in response to implanted EC than to MSC, suggesting a higher sensitivity to low oxygen tension among EC. Hypoxic signaling was increased after implantation of EC compared with MSC, leading to a prolonged acute inflammatory phase that promoted ingrowth of vascular cells and establishment of the circulation. Inflammatory cytokines were also differently expressed at the gene and protein levels in the two experimental groups, resulting in altered recruitment of acute and chronic inflammatory cells. The end result of these differences was increased vessel formation within the constructs in the EC group., QC 20160308. QC 20160319

Introduction: Inflammation plays a crucial role in tissue regeneration, wound healing, and the success of tissue-engineered constructs. The aim of this study was to investigate the influence of human umbilical vein endothelial cells (ECs) on leukocyte transmigration when co-cultured with primary human bone marrow-derived multipotent stromal cells (MSCs). Methods: MSCs with and without ECs were cultured in poly (L-lactide-co-1, 5-dioxepan-2-one) (poly (LLA-co-DXO)) scaffolds for 1 week in vitro in a bioreactor system, after which they were implanted subcutaneously in non-obese diabetic/severe combined immunodeficient mice. After 1 and 3 weeks, scaffolds were retrieved, and the mRNA expression of interleukin 1-beta (IL-1 beta), IL-6, IL-10, hypoxia-inducible factor 1-alpha (HIF-1 alpha), HIF-1 beta, and mammalian target of rapamycin was examined by real-time reverse transcription-polymerase chain reaction. Furthermore, immunofluorescent staining was performed for IL-1 beta, IL-6, neutrophils, and CD11b. In addition, Western blotting was done for IL-1 beta and IL-6. Leukocyte transmigration genes and genes in Toll-like receptor pathways, expressed by MSCs cultured in vitro with or without ECs, were further investigated with a microarray dataset. Results: In vitro, genes involved in leukocyte transmigration and Toll-like receptor pathways were clearly influenced by the addition of ECs. Platelet/endothelial cell adhesion molecule-1 (PECAM-1) and cadherin-5 (CDH5), both genes involved in leukocyte transmigration, were expressed significantly higher in the MSC/EC group. In vivo, the MSC/EC group showed higher mRNA expression of hypoxia-inducible factors HIF-1 alpha and HIF-1 beta. The mRNA expression of anti-inflammatory cytokine IL-10 showed no significant difference, whereas the mRNA and protein expression of pro-inflammatory cytokines IL-1 beta and IL-6 were lower in the MSC/EC group. The quantitative analysis of immunofluorescent staining revealed a significant differe, QC 20150303

In tissue engineering, the resorbable aliphatic polyester poly(L-lactide) (PLLA) is used as scaffolds in bone regeneration. Copolymers of poly(L-lactide)-co-(epsilon-caprolactone) [poly(LLA-co-CL)] and poly(L-lactide)-co-(1,5-dioxepan-2-one) [poly(LLA-co-DXO)], with superior mechanical properties to PLLA, have been developed to be used as scaffolds, but the influence on the osteogenic potential is unclear. This in vitro study of test scaffolds of poly(LLA-co-CL) and poly(LLA-co-DXO) using PLLA scaffolds as a control demonstrates the attachment and proliferation of human osteoblast-like cells (HOB) as measured by SEM and a methylthiazol tetrazolium (MTT) colorimetric assay, and the progression of HOB osteogenesis for up to 3 weeks; expressed as synthesis of the osteoblast differentiation markers: collagen type 1 (Col 1), alkaline phosphatase, bone sialoprotein, osteocalcin (OC), osteopontin and runt related gene 2 (Runx2). Surface analysis disclosed excellent surface attachment, spread and penetration of the cells into the pores of the test scaffolds compared to the PLLA. MTT results indicated that test scaffolds enhanced the proliferation of HOBs. Cells grown on the test scaffolds demonstrated higher synthesis of Col 1 and OC and also increased bone markers mRNA expression. Compared to scaffolds of PLLA, the poly(LLA-co-CL) and poly(LLA-co-DXO) scaffolds enhanced attachment, proliferation, and expression of osteogenic markers by HOBs in vitro. Therefore, these scaffolds might be appropriate carriers for bone engineering. (C) 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 94A: 631-639, 2010, QC 20101229