| 1. |
- Hjelmervik, Trond Ove R., et al.
(författare)
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The influence of the NOD Nss1/Idd5 loci on sialadenitis and gene expression in salivary glands of congenic mice
- 2007
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Ingår i: Arthritis Research and Therapy. - : Springer Science and Business Media LLC. - 1478-6362 .- 1478-6354. ; 9:5
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Tidskriftsartikel (refereegranskat)abstract
- The nonobese diabetic ( NOD) Nss1 and Idd5 loci have been associated with sialadenitis development in mice. In this study the NOD Nss1 and Idd5 loci were backcrossed onto the healthy control strain B10. Q by using the speed congenic breeding strategy, resulting in three congenic strains: B10. Q. Nss1, B10. Q. Nss1/Idd5 heterozygous and B10. Q. Nss1/Idd5 homozygous. We investigated the effects of the Nss1 and Idd5 loci on sialadenitis and gene expression in NOD congenic mice. One submandibular salivary gland from each mouse was used for histological analysis of sialadenitis, whereas the contralateral salivary gland was used for gene expression profiling with the Applied Biosystems Mouse Genome Survey chip v. 1.0. The results were validated using quantitative reverse transcriptase PCR. The NOD Nss1 and Idd5 loci had clear influence on the onset and progression of sialadenitis in congenic mice. Double congenic mice exhibited the most severe phenotype. We successfully identified several genes that are located in the NOD congenic regions to be differentially expressed between the congenic strains and the control strain. Several of these were found to be co-regulated, such as Stat1, complement component C1q genes and Tlr12. Also, a vast contingency of interferon-regulated genes ( such as Ltb, Irf7 and Irf8) and cytokine and chemokine genes ( such as Ccr7 and Ccl19) were differentially expressed between the congenic strains and the control strain. Over-representation of inflammatory signalling pathways was observed among the differentially expressed genes. We have found that the introgression of the NOD loci Nss1 and Idd5 on a healthy background caused sialadenitis in NOD congenic mouse strains, and we propose that genes within these loci are important factors in the pathogenesis. Furthermore, gene expression profiling has revealed several differentially expressed genes within and outside the NOD loci that are similar to genes found to be differentially expressed in patients with Sjogren's syndrome, and as such are interesting candidates for investigation to enhance our understanding of disease mechanisms and to develop future therapies.
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| 2. |
- Bartaula-Brevik, Sushma, et al.
(författare)
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Angiogenic and Immunomodulatory Properties of Endothelial and Mesenchymal Stem Cells
- 2016
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Ingår i: Tissue Engineering. Part A. - : Mary Ann Liebert. - 1937-3341 .- 1937-335X. ; 22:3-4, s. 244-252
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Bartaula-Brevik, Sushma, et al.
(författare)
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Leukocyte transmigration into tissue-engineered constructs is influenced by endothelial cells through Toll-like receptor signaling
- 2014
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Ingår i: Stem Cell Research & Therapy. - : Springer Science and Business Media LLC. - 1757-6512. ; 5, s. 143-
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Tidskriftsartikel (refereegranskat)abstract
- 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 difference in the number of neutrophils migrating into constructs, with the highest density found in the MSC/EC group. The number of macrophages positive for IL-6 and CD11b was significantly reduced in the MSC/EC group. Conclusions: The recruitment of leukocytes into tissue-engineered constructs with MSCs is strongly influenced by the addition of ECs via activation of leukocyte transmigration and Toll-like receptor pathways.
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| 4. |
- Bolstad, Anne Isine, et al.
(författare)
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Association between genetic variants in the tumour necrosis factor/lymphotoxin α/lymphotoxin β locus and primary Sjogren's syndrome in Scandinavian samples
- 2012
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Ingår i: Annals of the Rheumatic Diseases. - : BMJ. - 0003-4967 .- 1468-2060. ; 71:6, s. 981-988
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVES: Lymphotoxin β (LTB) has been found to be upregulated in salivary glands of patients with primary Sjögren's syndrome (pSS). An animal model of pSS also showed ablation of the lymphoid organisation and a marked improvement in salivary gland function on blocking the LTB receptor pathway. This study aimed to investigate whether single-nucleotide polymorphisms (SNP) in the lymphotoxin α (LTA)/LTB/tumour necrosis factor (TNF) gene clusters are associated with pSS.METHODS:527 pSS patients and 532 controls participated in the study, all of Caucasian origin from Sweden and Norway. 14 SNP markers were genotyped and after quality control filtering, 12 SNP were analysed for their association with pSS using single marker and haplotype tests, and corrected by permutation testing.RESULTS:Nine markers showed significant association with pSS at the p=0.05 level. Markers rs1800629 and rs909253 showed the strongest genotype association (p=1.64E-11 and p=4.42E-08, respectively, after correcting for sex and country of origin). When the analysis was conditioned for the effect of rs1800629, only the association with rs909253 remained nominally significant (p=0.027). In haplotype analyses the strongest effect was observed for the haplotype rs909253G_rs1800629A (p=9.14E-17). The associations were mainly due to anti-Ro/SSA and anti-La/SSB antibody-positive pSS.CONCLUSIONS:A strong association was found between several SNP in the LTA/LTB/TNFα locus and pSS, some of which led to amino acid changes. These data suggest a role for this locus in the development of pSS. Further studies are needed to examine if the genetic effect described here is independent of the known genetic association between HLA and pSS.
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| 5. |
- Entesarian, Miriam, et al.
(författare)
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Mutations in the gene encoding fibroblast growth factor 10 are associated with
- 2005
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Ingår i: Nat Genet. - 1061-4036. ; 37:2, s. 125-7
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Tidskriftsartikel (refereegranskat)abstract
- Autosomal dominant aplasia of lacrimal and salivary glands (ALSG; OMIM 180920 and OMIM 103420) is a rare condition characterized by irritable eyes and dryness of the mouth. We mapped ALSG to 5p13.2-5q13.1, which coincides with the gene fibroblast growth factor 10 (FGF10). In two extended pedigrees, we identified heterozygous mutations in FGF10 in all individuals with ALSG. Fgf10(+/-) mice have a phenotype similar to ALSG, providing a model for this disorder. We suggest that haploinsufficiency for FGF10 during a crucial stage of development results in ALSG.
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| 6. |
- Entesarian, Miriam, et al.
(författare)
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Mutations in the gene encoding fibroblast growth factor 10 are associated with aplasia of lacrimal and salivary glands.
- 2005
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Ingår i: Nat Genet. ; 37:2, s. 125-7
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Tidskriftsartikel (refereegranskat)abstract
- Autosomal dominant aplasia of lacrimal and salivary glands (ALSG; OMIM 180920 and OMIM 103420) is a rare condition characterized by irritable eyes and dryness of the mouth. We mapped ALSG to 5p13.2-5q13.1, which coincides with the gene fibroblast growth factor 10 (FGF10). In two extended pedigrees, we identified heterozygous mutations in FGF10 in all individuals with ALSG. Fgf10(+/-) mice have a phenotype similar to ALSG, providing a model for this disorder. We suggest that haploinsufficiency for FGF10 during a crucial stage of development results in ALSG.
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| 7. |
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| 8. |
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| 9. |
- Idris, Shaza B., et al.
(författare)
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Biocompatibility of Polyester Scaffolds with Fibroblasts and Osteoblast-like Cells for Bone Tissue Engineering
- 2010
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Ingår i: Journal of bioactive and compatible polymers (Print). - : SAGE Publications. - 0883-9115 .- 1530-8030. ; 25:6, s. 567-583
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to evaluate the in vitro cytotoxicity and cytocompatibility of the developed aliphatic polyester co-polymer scaffolds: poly(L-lactide-co-epsilon-caprolactone) and poly(L-lactide-co-1,5-dioxepan-2-one). The scaffolds were produced by solvent casting and particulate leaching, and tested by direct and indirect contact cytotoxicity assays on human osteoblast-like cells and mouse fibroblasts. Cell morphology was documented by light and scanning electron microscopy. Viability was assessed by the MTT, neutral red uptake, lactic dehydrogenase and apoptosis assays. Extraction tests confirmed that the scaffolds did not have a cytotoxic effect on the cells. The cells grew and spread well on the test scaffolds with good cellular attachment and viability. The scaffolds are noncytotoxic and biocompatible with the two cell types and warrant continued investigation as potential constructs for bone tissue engineering.
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| 10. |
- Idris, Shaza B., et al.
(författare)
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Global Gene Expression Profile of Osteoblast-Like Cells Grown on Polyester Copolymer Scaffolds
- 2011
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Ingår i: Tissue Engineering. Part A. - NEW ROCHELLE, NY : Mary Ann Liebert. - 1937-3341 .- 1937-335X. ; 17:21-22, s. 2817-2831
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Tidskriftsartikel (refereegranskat)abstract
- One of the principal goals in tissue engineering is to produce scaffold materials that will guide cells to differentiate and regenerate functional replacement tissue at the site of injury. Poly(l-lactide-co-1,5-dioxepan-2-one) [Poly(LLA-co-DXO)], a potential scaffolding material for bone tissue engineering, has high hydrophilicity. Previous in vitro studies using human osteoblast-like cells (HOBs) demonstrated greater cytocompatibility and enhanced osteogenic differentiation when HOBs were seeded onto Poly(LLA-co-DXO) compared to Poly(llactide) [P(LLA)] scaffolds. The aim of the study was to identify the gene expression profiles of HOBs obtained from alveolar bone and grown on Poly(LLA-co-DXO) biodegradable polymer scaffolds compared to P(LLA) one. Illumina HumanWG-6 v3.0 Expression BeadChips were used for the gene expression analysis. Several genes were found as differentially expressed at 24 h and at 21 days. Expression of genes related to cell adhesion, cytoskeleton, antiapoptosis, proliferation, and bone mineralization was influenced by adding the monomer 1,5-dioxepan-2-one to the l-lactide. Genes related to three biological pathways involving Integrin, Notch, and Ras were found to be upregulated. For selected genes, results were confirmed by quantitative reverse transcriptase– polymerase chain reaction. Further, calcium content analysis revealed a significant enhancement of calcium deposition on both tested scaffolds. This observation was confirmed by Von Kossa and Alizarin Red S staining. Findings of this study are relevant to a better understanding of the molecular mechanisms underlying the behavior of HOBs in bone regenerative procedure.
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| 11. |
- Idris, Shaza Bushra, et al.
(författare)
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Polyester copolymer scaffolds enhance expression of bone markers in osteoblast-like cells
- 2010
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Ingår i: J BIOMED MATER RES PART A. - : Wiley. - 1549-3296. ; 94A:2, s. 631-639
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Tidskriftsartikel (refereegranskat)abstract
- 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
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| 12. |
- Isine Bolstad, Anne, et al.
(författare)
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Association between genetic variants in the tumour necrosis factor/lymphotoxin alpha/lymphotoxin beta locus and primary Sjogrens syndrome in Scandinavian samples
- 2012
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Ingår i: Annals of the Rheumatic Diseases. - : BMJ Publishing Group. - 0003-4967 .- 1468-2060. ; 71:6, s. 981-988
-
Tidskriftsartikel (refereegranskat)abstract
- Objectives Lymphotoxin beta (LTB) has been found to be upregulated in salivary glands of patients with primary Sjogrens syndrome (pSS). An animal model of pSS also showed ablation of the lymphoid organisation and a marked improvement in salivary gland function on blocking the LTB receptor pathway. This study aimed to investigate whether single-nucleotide polymorphisms (SNP) in the lymphotoxin alpha (LTA)/LTB/tumour necrosis factor (TNF) gene clusters are associated with pSS. less thanbrgreater than less thanbrgreater thanMethods 527 pSS patients and 532 controls participated in the study, all of Caucasian origin from Sweden and Norway. 14 SNP markers were genotyped and after quality control filtering, 12 SNP were analysed for their association with pSS using single marker and haplotype tests, and corrected by permutation testing. less thanbrgreater than less thanbrgreater thanResults Nine markers showed significant association with pSS at the p=0.05 level. Markers rs1800629 and rs909253 showed the strongest genotype association (p=1.64E-11 and p=4.42E-08, respectively, after correcting for sex and country of origin). When the analysis was conditioned for the effect of rs1800629, only the association with rs909253 remained nominally significant (p=0.027). In haplotype analyses the strongest effect was observed for the haplotype rs909253G_rs1800629A (p=9.14E-17). The associations were mainly due to anti-Ro/SSA and anti-La/SSB antibody-positive pSS. less thanbrgreater than less thanbrgreater thanConclusions A strong association was found between several SNP in the LTA/LTB/TNF alpha locus and pSS, some of which led to amino acid changes. These data suggest a role for this locus in the development of pSS. Further studies are needed to examine if the genetic effect described here is independent of the known genetic association between HLA and pSS.
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| 13. |
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| 14. |
- Johansson, Åsa, et al.
(författare)
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Genetic control of collagen-induced arthritis in a cross with NOD and C57Bl/10 mice is dependent on gene regions coding for complement factor 5 and FcγRIIb and is not associated with loci controlling diabetes.
- 2001
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Ingår i: European Journal of Immunology. - 1521-4141. ; 31:6, s. 1847-1856
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Tidskriftsartikel (refereegranskat)abstract
- The nonobese diabetic (NOD) mouse spontaneously develops autoimmune-mediated diseases such as diabetes and Sjögren′s syndrome. To investigate whether NOD genes also promote autoimmune-mediatedarthritis we established a NOD strain with an MHC class II fragment containing the Aq class II gene predisposing for collagen induced arthritis (NOD.Q). However, this mouse was resistant to arthritis in contrast to other Aq expressing strains such as B10.Q and DBA/1. To determine the major resistance factor/s, a genetic analysis was performed. (NOD.Q×B10.Q)F1 mice were resistant, whereas 27% of the (NOD.Q×B10.Q)F2 mice developed severe arthritis. Genetic mapping of 353 F2 mice revealed two loci associated with arthritis. One locus was found on chromosome 2 (LOD score 9.8), at the location of the complement factor 5 (C5) gene. The susceptibility allele was from B10.Q, which contains a productive C5 encoding gene in contrast to NOD.Q. The other significant locus was found on chromosome 1 (LOD score 5.6) close to the Fc-gamma receptor IIb gene, where NOD carried the susceptible allele. An interaction between the two loci was observed, indicating that they operate on the same or on interacting pathways. The genetic control of arthritis is unique in comparison to diabetes, since none of these loci have been identified in analysis of diabetes susceptibility.
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| 15. |
- Johansson, Åsa, et al.
(författare)
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The genetic control of sialadenitis versus arthritis in a NOD.QxB10.Q F2 cross.
- 2002
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Ingår i: European Journal of Immunology. - 1521-4141 .- 0014-2980. ; 32:1, s. 243-250
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Tidskriftsartikel (refereegranskat)abstract
- The non-obese diabetic (NOD) mouse spontaneously develops diabetes and sialadenitis. The sialadenitis is characterized by histopathological changes in salivary glands and functional deficit similar to Sjögren's syndrome. In humans, Sjögren's syndrome could be associated with other connective tissue disorders, such as rheumatoid arthritis. In the present study the genetic control of sialadenitis in mice was compared to that of arthritis. We have previously reported a NOD locus, identified in an F2 cross with the H2(q) congenic NOD (NOD.Q) and C57BL/10.Q (B10.Q) strains, that promoted susceptibility to collagen-induced arthritis. The sialadenitis in NOD.Q showed a similar histological phenotype as in NOD, whereas no submandibular gland infiltration was found in B10.Q. The development of sialadenitis was independent of immunization with type II collagen and established arthritis. To identify the genetic control of sialadenitis, a gene segregation experiment was performed on an (NOD.QxB10.Q)F2 cross and genetic mapping of 353 F2 mice revealed one significant locus associated with sialadenitis on chromosome 4, LOD score 4.7. The NOD.Q allele-mediated susceptibility under a recessive inheritance pattern. The genetic control of sialadenitis seemed to be unique in comparison to diabetes and arthritis, as no loci associated with these diseases have been identified at the same location.
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| 16. |
- Prokunina, Ludmila, et al.
(författare)
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A regulatory polymorphism in PDCD1 is associated with susceptibility to systemic lupus erythematosus in humans
- 2002
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Ingår i: Nature Genetics. - : Springer Science and Business Media LLC. - 1061-4036 .- 1546-1718. ; 32:4, s. 666-669
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Recension (övrigt vetenskapligt/konstnärligt)abstract
- Systemic lupus erythematosus (SLE, OMIM 152700) is a complex autoimmune disease that affects 0.05% of the Western population, predominantly women. A number of susceptibility loci for SLE have been suggested in different populations, but the nature of the susceptibility genes and mutations is yet to be identified. We previously reported a susceptibility locus (SLEB2) for Nordic multi-case families. Within this locus, the programmed cell death 1 gene (PDCD1, also called PD-1) was considered the strongest candidate for association with the disease. Here, we analyzed 2,510 individuals, including members of five independent sets of families as well as unrelated individuals affected with SLE, for single-nucleotide polymorphisms (SNPs) that we identified in PDCD1. We show that one intronic SNP in PDCD1 is associated with development of SLE in Europeans (found in 12% of affected individuals versus 5% of controls; P = 0.00001, r.r. (relative risk) = 2.6) and Mexicans (found in 7% of affected individuals versus 2% of controls; P = 0.0009, r.r. = 3.5). The associated allele of this SNP alters a binding site for the runt-related transcription factor 1 (RUNX1, also called AML1) located in an intronic enhancer, suggesting a mechanism through which it can contribute to the development of SLE in humans.
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| 17. |
- Schander, Kerstin, et al.
(författare)
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Response of Bone and Periodontal Ligament Cells to Biodegradable Polymer Scaffolds In Vitro
- 2010
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Ingår i: Journal of bioactive and compatible polymers (Print). - : SAGE Publications. - 0883-9115 .- 1530-8030. ; 25:6, s. 584-602
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Tidskriftsartikel (refereegranskat)abstract
- In this in vitro study, the initial response of human periodontal ligament (PDL) cells and alveolar osteoblast-like cells (HOB) to three biodegradable polymers with varying pore size and different mechanical properties were evaluated. Scaffolds were synthesized from poly(L-lactide), [poly(LLA)], poly(L-lactide-co-1,5-dioxepan-2-one), [poly(LLA-co-DXO)], poly(L-lactide-co-epsilon-caprolactone), and [poly(LLA-co-CL)] with pore sizes greater or less than 90 mm by salt leaching. Cells were obtained from patients undergoing routine oral surgery. After 2-4 passages, the cells were grown on scaffolds and in culture plates (control) for 3 h (PDL cells), 3 days (PDL cells and HOB), 10 and 14 days (HOB), respectively. The cellular morphology and spreading were determined by scanning electron microscopy (SEM) and the attachment and proliferation were evaluated by MTT assays. The SEM images revealed heterogeneous cellular morphology and good spreading. Cellular attachment and proliferation were significantly higher on poly(LLA-co-DXO) and poly(LLA-co-CL) than on poly(LLA) scaffolds (p = 0.003) and highest for poly(LLA-co-DXO). Expression of bone formation markers, collagen-I (COL-I), transforming growth factor-beta(1) (TGF-beta(1)), and osteocalcin (OCN), was determined by ELISA. The expression of COL-1 was similar for HOB and PDL cells, but significantly higher for pore size >90 mm while the HOB expression of TGF-beta(1) and OCN was greater on poly(LLA-co-CL) and poly(LLA-co-DXO) than on poly(LLA) scaffolds.
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| 18. |
- Shanbhag, Siddharth, et al.
(författare)
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Characterization of Human Gingiva- and PDL-derived Progenitor Cells in Xeno-free Conditions
- 2018
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Objectives: Periodontal tissues represent a clinically relevant and less invasive source of progenitor cells compared to bone marrow, for periodontal- and/or alveolar bone-tissue engineering (P-/BTE). The safety and efficacy of clinical-grade stem cells can be enhanced via substitution of xenogeneic supplements and three-dimensional (3-D) cell culture, respectively, to simulate the in vivo microenvironment more closely. The objective of this study was to comprehensively characterize progenitor cells derived from human gingiva (GPCs) and periodontal-ligament (PDL; PPCs) in xeno-free conditions for use in P-/BTE. Methods: In preliminary experiments, pooled human platelet lysate (PL) was identified as the optimal xeno-substitute for fetal bovine serum (FBS) in stem cell cultures. Primary GPCs and PPCs were isolated and expanded as monolayers in PL- and FBS-supplemented media; passage 3-5 cells were used in experiments. Bone marrow mesenchymal stem cells (BMSCs) were used as a reference. Growth kinetics were compared via cell proliferation and colony forming-unit (CFU) assays. GPCs and PPCs were characterized via cytometric expression of stromal markers, multi-lineage (osteogenic, adipogenic and chondrogenic) differentiation potential, and secretory cytokine profiles. 3-D sphere cultures of GPCs and PPCs were established, and the expression of stemness- and osteogenesis-related markers in 3-D and monolayer cultures was evaluated via gene expression and immunocytochemistry. Results: GPCs and PPCs in both FBS and PL showed characteristic fibroblastic morphology and stromal phenotype (highly positive for CD105/CD90/CD73 and negative for CD34/CD45/HLA-DR). Cell proliferation and CFU efficiency were superior in PL compared to FBS. GPCs and PPCs expanded in both PL and FBS showed multi-lineage differentiation comparable to BMSCs; notably, osteogenic differentiation was enhanced in GPCs expanded in PL. 3-D spheres of GPCs and PPCs were formed and maintained for up to 7 days in xeno-free suspension culture. Notably, expression of stemness- (Sox2, Oct4, Nanog) and osteogenesis-related markers (Runx2, Osx, BMP2) was significantly upregulated in GPC- and PPC-derived spheres vs. monolayers. Conclusions: PPCs and particularly GPCs, due to easy access, cultured in 3-D xeno-free conditions represents a promising strategy for P-/BTE. Student Presenter This abstract is based on research that was funded entirely or partially by an outside source: International Team for Implantology (117/2015), Helse Vest Norway (912048) Disclosure Statement: The submitter must disclose the names of the organizations with which any author have a relationship, the nature of the relationship, and the clinical or research area involved. The following is submitted: NONE
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| 19. |
- Shanbhag, Siddharth, et al.
(författare)
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Spheroid Coculture of Human Gingiva-Derived Progenitor Cells With Endothelial Cells in Modified Platelet Lysate Hydrogels
- 2021
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Ingår i: Frontiers in Bioengineering and Biotechnology. - : Frontiers Media S.A.. - 2296-4185. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 20. |
- Shanbhag, Siddharth, et al.
(författare)
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Xeno-Free Spheroids of Human Gingiva-Derived Progenitor Cells for Bone Tissue Engineering
- 2020
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Ingår i: Frontiers in Bioengineering and Biotechnology. - : Frontiers Media S.A.. - 2296-4185. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- 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 enhanced osteogenic differentiation potential, comparable to that of BMSCs. Xeno-free spheroid culture further enhances stemness- and osteogenesis-related gene expression, and cytokine secretion in GPCs, comparable to that of BMSCs.
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