63 research outputs found

    Brief communication: Effects of conditioned media from human platelet lysate cultured MSC on osteogenic cell differentiation in vitro.

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    Culturing mesenchymal stromal cells (MSC) in human platelet lysate (HPL) supplemented media can enhance their osteogenic differentiation potential. The objective of this study was to test the hypothesis that conditioned media (CM) derived from HPL-cultured MSC also have pro-osteogenic effects. Pooled CM was prepared from HPL-cultured human bone marrow MSC (BMSC) of multiple donors and applied on BMSC of different donors (than those used for CM preparation), with or without additional supplementation [HPL, fetal bovine serum (FBS)] and osteogenic stimulation. At various time-points, cell proliferation, alkaline phosphatase (ALP) activity, osteogenic gene expression and in vitro mineralization were assessed. BMSC in standard unstimulated growth media served as controls. After 3-7 days, CM alone did not promote BMSC proliferation or ALP activity; supplementation of CM with HPL slightly improved these effects. After 2 and 7 days, CM alone, but not CM supplemented with HPL, promoted osteogenic gene expression. After 14 days, only CM supplemented with FBS and osteogenic stimulants supported in vitro BMSC mineralization; CM alone and CM supplemented with HPL did not support mineralization, regardless of osteogenic stimulation. In summary, CM from HPL-cultured BMSC promoted osteogenic gene expression but not in vitro mineralization in allogeneic BMSC even when supplemented with HPL and/or osteogenic stimulants. Future studies should investigate the role and relevance of supplementation and osteogenic induction in in vitro assays using CM from MSC

    Engineering 3D degradable, pliable scaffolds toward adipose tissue regeneration; optimized printability, simulations and surface modification

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    We present a solution to regenerate adipose tissue using degradable, soft, pliable 3D-printed scaffolds made of a medical-grade copolymer coated with polydopamine. The problem today is that while printing, the medical grade copolyesters degrade and the scaffolds become very stiff and brittle, being not optimal for adipose tissue defects. Herein, we have used high molar mass poly(L-lactide-co-trimethylene carbonate) (PLATMC) to engineer scaffolds using a direct extrusion-based 3D printer, the 3D Bioplotter¼. Our approach was first focused on how the printing influences the polymer and scaffold’s mechanical properties, then on exploring different printing designs and, in the end, on assessing surface functionalization. Finite element analysis revealed that scaffold’s mechanical properties vary according to the gradual degradation of the polymer as a consequence of the molar mass decrease during printing. Considering this, we defined optimal printing parameters to minimize material’s degradation and printed scaffolds with different designs. We subsequently functionalized one scaffold design with polydopamine coating and conducted in vitro cell studies. Results showed that polydopamine augmented stem cell proliferation and adipogenic differentiation owing to increased surface hydrophilicity. Thus, the present research show that the medical grade PLATMC based scaffolds are a potential candidate towards the development of implantable, resorbable, medical devices for adipose tissue regeneration.publishedVersio

    Proteomic Analysis of Mesenchymal Stromal Cells Secretome in Comparison to Leukocyte- and Platelet-Rich Fibrin.

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    Secretomes of mesenchymal stromal cells (MSCs) are emerging as a novel growth factor (GF)-based strategy for periodontal and bone regeneration. The objective of this study was to compare the secretome of human bone marrow MSC (BMSC) to that of leukocyte- and platelet-rich fibrin (L-PRF), an established GF-based therapy, in the context of wound healing and regeneration. Conditioned media from human BMSCs (BMSC-CM) and L-PRF (LPRF-CM) were subjected to quantitative proteomic analysis using liquid chromatography with tandem mass spectrometry. Global profiles, gene ontology (GO) categories, differentially expressed proteins (DEPs), and gene set enrichment (GSEA) were identified using bioinformatic methods. Concentrations of selected proteins were determined using a multiplex immunoassay. Among the proteins identified in BMSC-CM (2157 proteins) and LPRF-CM (1420 proteins), 1283 proteins were common. GO analysis revealed similarities between the groups in terms of biological processes (cellular organization, protein metabolism) and molecular functions (cellular/protein-binding). Notably, more DEPs were identified in BMSC-CM (n = 550) compared to LPRF-CM (n = 118); these included several key GF, cytokines, and extracellular matrix (ECM) proteins involved in wound healing. GSEA revealed enrichment of ECM (especially bone ECM)-related processes in BMSC-CM and immune-related processes in LPRF-CM. Similar trends for intergroup differences in protein detection were observed in the multiplex analysis. Thus, the secretome of BMSC is enriched for proteins/processes relevant for periodontal and bone regeneration. The in vivo efficacy of this therapy should be evaluated in future studies

    Contact osteogenesis by biodegradable 3D-printed poly(lactide-co-trimethylene carbonate)

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    Background To support bone regeneration, 3D-printed templates function as temporary guides. The preferred materials are synthetic polymers, due to their ease of processing and biological inertness. Poly(lactide-co-trimethylene carbonate) (PLATMC) has good biological compatibility and currently used in soft tissue regeneration. The aim of this study was to evaluate the osteoconductivity of 3D-printed PLATMC templates for bone tissue engineering, in comparison with the widely used 3D-printed polycaprolactone (PCL) templates. Methods The printability and physical properties of 3D-printed templates were assessed, including wettability, tensile properties and the degradation profile. Human bone marrow-derived mesenchymal stem cells (hBMSCs) were used to evaluate osteoconductivity and extracellular matrix secretion in vitro. In addition, 3D-printed templates were implanted in subcutaneous and calvarial bone defect models in rabbits. Results Compared to PCL, PLATMC exhibited greater wettability, strength, degradation, and promoted osteogenic differentiation of hBMSCs, with superior osteoconductivity. However, the higher ALP activity disclosed by PCL group at 7 and 21 days did not dictate better osteoconductivity. This was confirmed in vivo in the calvarial defect model, where PCL disclosed distant osteogenesis, while PLATMC disclosed greater areas of new bone and obvious contact osteogenesis on surface. Conclusions This study shows for the first time the contact osteogenesis formed on a degradable synthetic co-polymer. 3D-printed PLATMC templates disclosed unique contact osteogenesis and significant higher amount of new bone regeneration, thus could be used to advantage in bone tissue engineering.publishedVersio

    Comparison of bone regenerative capacity of donor-matched human adipose–derived and bone marrow mesenchymal stem cells

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    Adipose-derived stem cells (ASC) have been used as an alternative to bone marrow mesenchymal stem cells (BMSC) for bone tissue engineering. However, the efficacy of ASC in bone regeneration in comparison with BMSC remains debatable, since inconsistent results have been reported. Comparing ASC with BMSC obtained from different individuals might contribute to this inconsistency in results. Therefore, this study aimed to compare the bone regenerative capacity of donor-matched human ASC and BMSC seeded onto poly(L-lactide-co-Δ-caprolactone) scaffolds using calvarial bone defects in nude rats. First, donor-matched ASC and BMSC were seeded onto the co-polymer scaffolds to evaluate their in vitro osteogenic differentiation. Seeded scaffolds and scaffolds without cells (control) were then implanted in calvarial defects in nude rats. The expression of osteogenesis-related genes was examined after 4 weeks. Cellular activity was investigated after 4 and 12 weeks. Bone formation was evaluated radiographically and histologically after 4, 12, and 24 weeks. In vitro, ASC and BMSC demonstrated mineralization. However, BMSC showed higher alkaline phosphatase activity than ASC. In vivo, human osteogenesis–related genes Runx2 and collagen type I were expressed in defects with scaffold/cells. Defects with scaffold/BMSC had higher cellular activity than defects with scaffold/ASC. Moreover, bone formation in defects with scaffold/BMSC was greater than in defects with scaffold/ASC, especially at the early time-point. These results suggest that although ASC have the potential to regenerate bone, the rate of bone regeneration with ASC may be slower than with BMSC. Accordingly, BMSC are more suitable for bone regenerative applications.publishedVersio

    Understanding of how the properties of medical grade lactide based copolymer scaffolds influence adipose tissue regeneration: Sterilization and a systematic in vitro assessment

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    Aliphatic polyesters are the synthetic polymers most commonly used in the development of resorbable medical implants/devices. Various three-dimensional (3D) scaffolds have been fabricated from these polymers and used in adipose tissue engineering. However, their systematic evaluation altogether lacks, which makes it difficult to select a suitable degradable polymer to design 3D resorbable implants and/or devices able to effectively mimic the properties of adipose tissue. Additionally, the impact of sterilization methods on the medical devices, if any, must be taken into account. We evaluate and compare five different medical-grade resorbable polyesters with l-lactide content ranging from 50 to 100 mol% and exhibiting different physiochemical properties depending on the comonomer (d-lactide, Δ-caprolactone, glycolide, and trimethylene carbonate). The salt-leaching technique was used to prepare 3D microporous scaffolds. A comprehensive assessment of physical, chemical, and mechanical properties of the scaffolds was carried out in PBS at 37 °C. The cell-material interactions and the ability of the scaffolds to promote adipogenesis of human adipose tissue-derived stem cells were assessed in vitro. The diverse physical and mechanical properties of the scaffolds, due to the different composition of the copolymers, influenced human adipose tissue-derived stem cells proliferation and differentiation. Scaffolds made from polymers which were above their glass transition temperature and with low degree of crystallinity showed better proliferation and adipogenic differentiation of stem cells. The effect of sterilization techniques (electron beam and ethylene oxide) on the polymer properties was also evaluated. Results showed that scaffolds sterilized with the ethylene oxide method better retained their physical and chemical properties. Overall, the presented research provides (i) a detailed understanding to select a degradable polymer that has relevant properties to augment adipose tissue regeneration and can be further used to fabricate medical devices/implants; (ii) directions to prefer a sterilization method that does not change polymer properties.publishedVersio

    Conditioned Medium from Bone Marrow Mesenchymal Stem Cells Restored Oxidative Stress-Related Impaired Osteogenic Differentiation

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    Oxidative stress from high levels of intracellular reactive oxygen species (ROS) has been linked to various bone diseases. Previous studies indicate that mesenchymal stem cells (MSC) secrete bioactive factors (conditioned medium (MSC-CM)) that have antioxidant effects. However, the antioxidant role of MSC-CM on osteogenesis has not been fully studied. We aimed to identify antioxidant proteins in MSC-CM using mass spectrometry-based proteomics and to explore their effects on osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSC) exposed to oxidative stress induced by hydrogen peroxide (H2O2). Our analysis revealed that MSC-CM is comprised of antioxidant proteins that are involved in several biological processes, including negative regulation of apoptosis and positive regulation of cell proliferation. Then, hBMSC exposed to H2O2 were treated with MSC-CM, and the effects on their osteogenic differentiation were evaluated. MSC-CM restored H2O2-induced damage to hBMSC by increasing the antioxidant enzyme-SOD production and the mRNA expression level of the anti-apoptotic BCL-2. A decrease in ROS production and cellular apoptosis was also shown. MSC-CM also modulated mRNA expression levels of osteogenesis-related genes, runt-related transcription factor 2, collagen type I, bone morphogenic protein 2, and osteopontin. Furthermore, collagen type I protein secretion, alkaline phosphatase activity, and in vitro mineralization were increased. These results indicate that MSC-CM contains several proteins with antioxidant and anti-apoptotic properties that restored the impaired hBMSC osteogenic differentiation associated with oxidative stress.publishedVersio

    The use of mesenchymal stromal cell secretome to enhance guided bone regeneration in comparison with leukocyte and platelet-rich fibrin.

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    OBJECTIVES Secretomes of mesenchymal stromal cells (MSC) represent a novel strategy for growth-factor delivery for tissue regeneration. The objective of this study was to compare the efficacy of adjunctive use of conditioned media of bone-marrow MSC (MSC-CM) with collagen barrier membranes vs. adjunctive use of conditioned media of leukocyte- and platelet-rich fibrin (PRF-CM), a current growth-factor therapy, for guided bone regeneration (GBR). METHODS MSC-CM and PRF-CM prepared from healthy human donors were subjected to proteomic analysis using mass spectrometry and multiplex immunoassay. Collagen membranes functionalized with MSC-CM or PRF-CM were applied on critical-size rat calvaria defects and new bone formation was assessed via three-dimensional (3D) micro-CT analysis of total defect volume (2 and 4 weeks) and 2D histomorphometric analysis of central defect regions (4 weeks). RESULTS While both MSC-CM and PRF-CM revealed several bone-related proteins, differentially expressed proteins, especially extracellular matrix components, were increased in MSC-CM. In rat calvaria defects, micro-CT revealed greater total bone coverage in the MSC-CM group after 2 and 4 weeks. Histologically, both groups showed a combination of regular new bone and 'hybrid' new bone, which was formed within the membrane compartment and characterized by incorporation of mineralized collagen fibers. Histomorphometry in central defect sections revealed greater hybrid bone area in the MSC-CM group, while the total new bone area was similar between groups. CONCLUSION Based on the in vitro and in vivo investigations herein, functionalization of membranes with MSC-CM represents a promising strategy to enhance GBR
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