Proangiogenic Hypoxia-Mimicking Agents Attenuate Osteogenic Potential of Adipose Stem/Stromal Cells

BACKGROUND: Insufficient vascularization hampers bone tissue engineering strategies for reconstructing large bone defects. Delivery of prolyl-hydroxylase inhibitors (PHIs) is an interesting approach to upregulate vascular endothelial growth factor (VEGF) by mimicking hypoxic stabilization of hypoxia-inducible factor-1alpha (HIF-1α). This study assessed two PHIs: dimethyloxalylglycine (DMOG) and baicalein for their effects on human adipose tissue-derived mesenchymal stem/stromal cells (AT-MSCs). METHODS: Isolated AT-MSCs were characterized and treated with PHIs to assess the cellular proliferation response. Immunostaining and western-blots served to verify the HIF-1α stabilization response. The optimized concentrations for long-term treatment were tested for their effects on the cell cycle, apoptosis, cytokine secretion, and osteogenic differentiation of AT-MSCs. Gene expression levels were evaluated for alkaline phosphatase (ALPL), bone morphogenetic protein 2 (BMP2), runt-related transcription factor 2 (RUNX2), vascular endothelial growth factor A (VEGFA), secreted phosphoprotein 1 (SPP1), and collagen type I alpha 1 (COL1A1). In addition, stemness-related genes Kruppel-like factor 4 (KLF4), Nanog homeobox (NANOG), and octamer-binding transcription factor 4 (OCT4) were assessed. RESULTS: PHIs stabilized HIF-1α in a dose-dependent manner and showed evident dose- and time dependent antiproliferative effects. With doses maintaining proliferation, DMOG and baicalein diminished the effect of osteogenic induction on the expression of RUNX2, ALPL, and COL1A1, and suppressed the formation of mineralized matrix. Suppressed osteogenic response of AT-MSCs was accompanied by an upregulation of stemness-related genes. CONCLUSION: PHIs significantly reduced the osteogenic differentiation of AT-MSCs and rather upregulated stemness-related genes. PHIs proangiogenic potential should be weighed against their longterm direct inhibitory effects on the osteogenic differentiation of AT-MSCs.Peer reviewe

Monocyte-derived extracellular vesicles stimulate cytokinesecretion and gene expression of matrixmetalloproteinases by mesenchymal stem/stromal cells

Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. DatabaseGene expression data are available in the GEO database under the accession number .Peer reviewe

Growth Response and Differentiation of Bone Marrow-Derived Mesenchymal Stem/Stromal Cells in the Presence of Novel Multiple Myeloma Drug Melflufen

Mesenchymal stem/stromal cells (MSCs) are self-renewing and multipotent progenitors, which constitute the main cellular compartment of the bone marrow stroma. Because MSCs have an important role in the pathogenesis of multiple myeloma, it is essential to know if novel drugs target MSCs. Melflufen is a novel anticancer peptide–drug conjugate compound for patients with relapsed refractory multiple myeloma. Here, we studied the cytotoxicity of melflufen, melphalan and doxorubicin in healthy human bone marrow-derived MSCs (BMSCs) and how these drugs affect BMSC proliferation. We established co-cultures of BMSCs with MM.1S myeloma cells to see if BMSCs increase or decrease the cytotoxicity of melflufen, melphalan, bortezomib and doxorubicin. We evaluated how the drugs affect BMSC differentiation into adipocytes and osteoblasts and the BMSC-supported formation of vascular networks. Our results showed that BMSCs were more sensitive to melflufen than to melphalan. The cytotoxicity of melflufen in myeloma cells was not affected by the co-culture with BMSCs, as was the case for melphalan, bortezomib and doxorubicin. Adipogenesis, osteogenesis and BMSC-mediated angiogenesis were all affected by melflufen. Melphalan and doxorubicin affected BMSC differentiation in similar ways. The effects on adipogenesis and osteogenesis were not solely because of effects on proliferation, seen from the differential expression of differentiation markers normalized by cell number. Overall, our results indicate that melflufen has a significant impact on BMSCs, which could possibly affect therapy outcome

Growth Response and Differentiation of Bone Marrow-Derived Mesenchymal Stem/Stromal Cells in the Presence of Novel Multiple Myeloma Drug Melflufen

Mesenchymal stem/stromal cells (MSCs) are self-renewing and multipotent progenitors, which constitute the main cellular compartment of the bone marrow stroma. Because MSCs have an important role in the pathogenesis of multiple myeloma, it is essential to know if novel drugs target MSCs. Melflufen is a novel anticancer peptide-drug conjugate compound for patients with relapsed refractory multiple myeloma. Here, we studied the cytotoxicity of melflufen, melphalan and doxorubicin in healthy human bone marrow-derived MSCs (BMSCs) and how these drugs affect BMSC proliferation. We established co-cultures of BMSCs with MM.1S myeloma cells to see if BMSCs increase or decrease the cytotoxicity of melflufen, melphalan, bortezomib and doxorubicin. We evaluated how the drugs affect BMSC differentiation into adipocytes and osteoblasts and the BMSC-supported formation of vascular networks. Our results showed that BMSCs were more sensitive to melflufen than to melphalan. The cytotoxicity of melflufen in myeloma cells was not affected by the co-culture with BMSCs, as was the case for melphalan, bortezomib and doxorubicin. Adipogenesis, osteogenesis and BMSC-mediated angiogenesis were all affected by melflufen. Melphalan and doxorubicin affected BMSC differentiation in similar ways. The effects on adipogenesis and osteogenesis were not solely because of effects on proliferation, seen from the differential expression of differentiation markers normalized by cell number. Overall, our results indicate that melflufen has a significant impact on BMSCs, which could possibly affect therapy outcome.Peer reviewe

Chitosan/collagen/Mg, Se, Sr, Zn-substituted calcium phosphate scaffolds for bone tissue engineering applications : A growth factor free approach

According to the biomimetic bone scaffold design paradigm, a scaffold resembling natural bone tissue with molecular, structural and biological compatibility is needed to allow effective regeneration of bone tissue. Continuing our previous studies regarding scaffolds with chitosan matrix containing Mg, Se, Sr, Zn-substituted calcium phosphates (CaPs), the focus of this work was to further improve the properties of these growth factor-free scaffolds. By addition of collagen into the chitosan matrix at weight ratios of 100:0, 75:25, 50:50, 25:75 and 0:100, we aimed to better resemble natural bone tissue. Highly porous composite scaffolds based on chitosan and collagen, with 30 wt% of Mg, Se, Sr, Zn-substituted CaPs, were prepared by the freeze-gelation method. The scaffolds show a highly porous structure, with interconnected pores in the range of 20–350 μm and homogeneously dispersed CaPs. The added collagen further enhanced the stability measured during 28 days in simulated biological conditions. Live/dead and CyQUANT assays confirmed good viability and proliferation of human bone marrow-derived mesenchymal stem/stromal cells, while successful osteogenic differentiation was confirmed by alkaline phosphatase quantification and type I collagen immunocytochemical staining. Results indicated that the addition of collagen into the chitosan matrix containing Mg, Se, Sr, Zn-substituted CaPs improved the physicochemical and biological properties of the scaffolds.publishedVersionPeer reviewe

Growth Response and Differentiation of Bone Marrow-Derived Mesenchymal Stem/Stromal Cells in the Presence of Novel Multiple Myeloma Drug Melflufen

Mesenchymal stem/stromal cells (MSCs) are self-renewing and multipotent progenitors, which constitute the main cellular compartment of the bone marrow stroma. Because MSCs have an important role in the pathogenesis of multiple myeloma, it is essential to know if novel drugs target MSCs. Melflufen is a novel anticancer peptide–drug conjugate compound for patients with relapsed refractory multiple myeloma. Here, we studied the cytotoxicity of melflufen, melphalan and doxorubicin in healthy human bone marrow-derived MSCs (BMSCs) and how these drugs affect BMSC proliferation. We established co-cultures of BMSCs with MM.1S myeloma cells to see if BMSCs increase or decrease the cytotoxicity of melflufen, melphalan, bortezomib and doxorubicin. We evaluated how the drugs affect BMSC differentiation into adipocytes and osteoblasts and the BMSC-supported formation of vascular networks. Our results showed that BMSCs were more sensitive to melflufen than to melphalan. The cytotoxicity of melflufen in myeloma cells was not affected by the co-culture with BMSCs, as was the case for melphalan, bortezomib and doxorubicin. Adipogenesis, osteogenesis and BMSC-mediated angiogenesis were all affected by melflufen. Melphalan and doxorubicin affected BMSC differentiation in similar ways. The effects on adipogenesis and osteogenesis were not solely because of effects on proliferation, seen from the differential expression of differentiation markers normalized by cell number. Overall, our results indicate that melflufen has a significant impact on BMSCs, which could possibly affect therapy outcome

Tissue Engineering Approaches for the Treatment of Degenerated Intervertebral Discs

This dissertation describes novel tissue engineering approaches for the treatment of intervertebral disc (IVD) degeneration. Defects in the outer part of the IVD, the so-called annulus fibrosus (AF), cannot be restored using the current surgical methods. AF tissue cannot be regenerated using autologous AF cells, because of their limited availability and expansion capacity. We compared the potency of transforming growth factor-beta (TGF-beta) type 1 and type 3 to stimulate differentiation of adipose tissue derived-mesenchymal stem cells (AT-MSCs) towards an AF-like phenotype. We engineered AF tissue in vitro by seeding and stimulating AT-MSCs inside AF-mimetic polymer scaffolds. Several techniques to seed cells in the scaffolds were compared. Cells release membrane vesicles called extracellular vesicles (EVs). EVs have gained interest as a biomimetic tool to induce lineage-specific differentiation of stem cells. We evaluated gene expression in AT-MSCs after exposure to EVs from activated monocytes or osteoclasts. Neither TGF- beta 1 nor TGF- beta 3 increased matrix synthesis by AT-MSCs. Nevertheless, TGF- beta 3 supported cell proliferation and thereby matrix accumulation. Seeding of AT-MSCs into the scaffolds using fibrin gel resulted in superior cell distribution, proliferation and AF-like matrix production compared to other seeding strategies. The formed collagen was aligned into bundles within the pore channels of the scaffolds. Activated monocytes promoted the chemotaxis of immune cells by AT-MSCs via EV-mediated signalling, showing the importance of EVs in controlling the function of the immune system. Monocyte-derived EVs upregulated the expression of matrix metalloproteinases in AT-MSCs. The results show the potential of AT-MSCs as a multipotent cell source in tissue engineering applications for the treatment of degenerated IVDs. The extracellular matrix deposited in the cell-seeded scaffolds had a composition and structure similar to that in native AF tissue, demonstrating the high potential of this strategy in AF repair. Furthermore, the study sheds light on the mechanism by which EVs regulate the immune system and bone remodelling.Väitöskirjan aiheena on kudosteknologiset sovellukset nikamavälilevyn rappeuman hoitoon. Nykyisillä kirurgisilla menetelmillä ei voida korjata nikamavälilevyn ulomman sitkeän syykehän eli niin sanotun annulus fibrosuksen (AF) vaurioita. Tässä tutkimuksessa vertasimme TGF-beta-kasvutekijän tyyppien 1 ja 3 kykyä ja tehokkuutta rasvakudoksen mesenkymaalisten kantasolujen erilaistamisessa AF-soluiksi. Tuotimme uutta AF-kudosta laboratoriossa istuttamalla kantosoluja AF-kudosta jäljitteleviin polymeerimateriaalista valmistettuihin kehikkoihin (engl. scaffold). Vertailimme myös useita erilaisia solujen istutusmenetelmiä. Solut erittävät kalvorakkuloita, niin sanottuja solunulkoisia vesikkeleitä. Kiinnostus solunulkoisiin vesikkeleihin on lisääntynyt kudosteknologian alalla, koska niitä voidaan käyttää luontaisina työkaluina kantasolujen erilaistamisessa. Arvioimme geenien ilmentymistä kantasoluissa, jotka oli altistettu monosyyttien (valkosolujen) ja osteoklastien (luun syöjäsolujen) solunulkoisille vesikkeleille. TGF-beta 1 ja TGF-beta 3 eivät kumpikaan lisänneet kantasolujen soluväliaineen synteesiä. TGF-beta 3 kuitenkin tuki solujen lisääntymistä ja siten soluväliaineen muodostumista. Kantasolujen istuttaminen kehikkoihin fibriinigeelin avulla osoittautui parhaaksi testatuista istutusmenetelmistä solujen levinneisyyden, lisääntymisen ja soluväliaineen muodostumisen suhteen. Muodostetut kollageenikimput seurasivat kehikkojen pieniä kanavia. Aktivoidut monosyytit tukivat solunulkoisten vesikkelien avulla prosesseja, joissa kantasolut vetävät valkosoluja puoleensa. Monosyyttien solunulkoiset vesikkelit lisäsivät myös kollageenia hajottavien entsyymien, matriksin metalloproteinaasien, ilmentymistä. Tämä väitöskirja esittelee rasvakudoksen mesenkymaalisten kantasolujen mahdollisuuksia monipotentiaalisena solulähteenä kudosteknologisissa sovelluksissa nikamavälilevyn rappeuman hoidossa. Soveltuvissa olosuhteissa kantasolut tuottivat soluväliainetta, jolla oli samanlainen koostumus ja rakenne kuin alkuperäisessä AF-kudoksessa. Lisäksi tutkimus valottaa sitä, miten solunulkoiset vesikkelit ohjaavat immuunipuolustusjärjestelmän toimintaa sekä luukudoksen muodostumista ja hajoamista

Hammasimplanttihoito Suomessa vuosina 2005-2015 : aineisto implanttirekisteristä

Suomessa suoritetuista hammasimplanttien asetusleikkauksista sekä poistoleikkauksista on kerätty tietoja lakisääteiseen hammasimplanttirekisteriin vuodesta 1994 lähtien. Terveyden ja hyvinvoinnin laitos (THL) aloitti implanttirekisterin ylläpidon 01.11.2009. Tätä ennen rekisterinpitäjänä toimi Lääkelaitos. Kustannussyistä THL lopetti implanttirekisterin ylläpidon vuonna 2016. Tutkimuksemme tarkoituksena oli selvittää kyseisellä aikavälillä asetettujen sekä poistettujen implanttien valmistajat sekä lukumäärät. Erityisenä kiinnostuksen kohteena oli tarkastella biomateriaalien käyttöä implanttihoidossa ja luusiirteiden sekä kalvomenetelmien vaikutuksia implanttien ennusteeseen. Myös leikkausmenetelmien sekä muiden taustatekijöiden, kuten yleissairauksien tai säännöllisen päivittäisen tupakoinnin vaikutukset implanttien ennusteeseen haluttiin selvittää. Aineisto perustuu THL:n implanttirekisteriin kerättyihin tietoihin hammasimplanteista vuodesta 2005 lähtien aina rekisterin ylläpidon lopetukseen kesällä 2016. Lomakkeita saatiin yhteensä 107 257. Vuoden 2016 aineisto jätettiin sen vähäisyyden vuoksi analyysin ulkopuolelle. Tiedoista analysoitiin implantoinnin yleisiä ja paikallisia taustatekijöitä sekä implanttien poistosyitä. Tarkasteluajanjaksolla rekisterin tietojen mukaan Suomessa asetettiin 166 842 implanttia. Biomateriaaleja (luusiirteitä ja kalvomateriaaleja) käytettiin 25 304 kpl. Poistoilmoituksia saatiin 1938 kpl ja poistettuja implantteja oli 2351 kpl. Yli puolet implanteista asetettiin useimmiten 40-69 -vuotiaille, naisille useammin kuin miehille. Biomateriaalien käyttö lisäsi selvästi komplikaatioiden määriä. Ilmoituksien perusteella luukorvikkeita tai muita luun määrää lisääviä menetelmiä käytettiin lähes neljäsosassa leikkauksista. Biomateriaalien käyttö lisäsi primaarikomplikaatioiden riskiä. Naissukupuoli, ikä ja tupakointi ovat riskitekijöitä, jotka tulee huomioida implanttihoidon suunnittelussa. Jatkossa Kanta-arkiston toivotaan parantavan implanttihoitojen tilastointia.The objective of this study was find out the use of dental implants, bone grafts and membrane materials used in implant dentistry between 2005-2015 in Finland. The statutory implant registry was maintained by the National Institute for Health and Welfare. Variables from the implant registry included the patient's gender, age, medical history, indications for implantation, augmentation area, installed fixtures, bone grafts and membranes, complications, removed fixtures and removal indications. Variables were analyzed by using statistical analysis software IBM® SPSS® Statistics (v24). During the time period 166 842 implants were installed and 2351 were removed. The most frequently used bone graft materials were bovine bone graft and autograft, whereas collagen membrane was the most used membrane material. Biomaterials were often used with two-step procedure and mostly in the maxilla. Patients age, gender, biomaterial use, irradiation and smoking were considered risk factors for implant survial. Information data about dental implants and biomaterial use were collected based on voluntary reports from the clinics, therefore the registry is not complete and does not represent the overall statistics

Differentiation of adipose stem cells seeded towards annulus fibrosus cells on a designed poly(trimethylene carbonate) scaffold prepared by stereolithography

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