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  • 1.
    Pettersson, Sofia
    Linköping University, Department of Clinical and Experimental Medicine, Surgery . Linköping University, Faculty of Health Sciences.
    Biodegradable gelatin microcarriers in tissue engineering: In vitro studies on cartilage and bone2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Tissue engineering is a multidisciplinary field that combines cells, biomaterial scaffolds and environmental factors to achieve functional tissue repair. This thesis focuses on the use of macroporous gelatin microcarriers as scaffolds in tissue engineering applications, with a special focus on cartilage and bone formation by human adult cells in vitro.

    In our first study, human articular chondrocytes were seeded on macroporous gelatin microcarriers. The microcarriers were subsequently encapsulated in coagulated blood-derived biological glues and cultured under free-swelling conditions for up to 17 weeks. Even in the absence of recombinant chondrogenic growth factors, the chondrocytes remained viable and metabolically active for the duration of the culture period, as indicated by an increased amount of cell nuclei and extracellular matrix (ECM). The ECM showed several cartilage characteristics, but lacked the cartilage specific collagen type II. Furthermore, ECM formation was seen primarily in a capsule surrounding the tissue-engineered constructs, leading to the conclusion that the used in vitro models were unable to support true cartilage formation.

    The capacity of human dermal fibroblasts to produce cartilage- and bone-like tissue in the previously mentioned model was also investigated. Under the influence of chondrogenic induction factors, including TGF-β1 and insulin, the fibroblasts produced cartilage specific molecules, as confirmed by indirect immunohistochemistry, however not collagen type II. Under osteogenic induction, by dexamethasone, ascorbate-2-phosphate and β–glycerophosphate, the fibroblasts formed a calcified matrix with bone specific markers, and an alkaline phosphatase assay corroborated a shift towards an osteoblast like phenotype. The osteogenic induction was enhanced by flow-induced shear stress in a spinner flask system.

    In addition, four different types of gelatin microcarriers, differing by their internal pore diameter and their degree of gelatin cross-linking, were evaluated for their ability to support chondrocyte expansion. Chondrocyte densities on the microcarriers were monitored every other day over a twoweek period, and chondrocyte growth was analyzed by piecewise linear regression and analysis of variance (ANOVA). No differences were seen between the different microcarriers during the first week. However, during the second week of culture both microcarrier pore diameter and gelatin crosslinking had significant impacts on chondrocyte density.

    Lastly, a dynamic centrifugation regime (f=12.5 mHz for 16 minutes every other day) was administered to chondrocyte-seeded microcarriers, with or without encapsulation in platelet rich plasma (PRP), to study the possible effect of dynamic stimuli on cartilage formation. Presence of PRP enhanced the structural stability of the tissue-engineered constructs, but we were not able to confirm any dose-response pattern between ECM formation and the applied forces. After 12 weeks, distinct gelatin degradation had occurred independent of both dynamic stimuli and presence of PRP.

    In summary, this thesis supports a plausible use for gelatin microcarriers in tissue engineering of cartilage and bone. Microcarrier characteristics, specifically gelatin cross-linking and pore diameter, have been shown to affect chondrocyte expansion. In addition, the use of human dermal fibroblasts as an alternative cell source for cartilage and bone formation in vitro was addressed.

    List of papers
    1. Human articular chondrocytes on macroporous gelatin microcarriers form structurally stable constructs with blood-derived biological glues in vitro
    Open this publication in new window or tab >>Human articular chondrocytes on macroporous gelatin microcarriers form structurally stable constructs with blood-derived biological glues in vitro
    2009 (English)In: JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, ISSN 1932-6254, Vol. 35, no 6, p. 450-460Article in journal (Refereed) Published
    Abstract [en]

    Biodegradable macroporous gelatin microcarriers fixed with blood-derived biodegradable glue are proposed as a delivery system for human autologous chondrocytes. Cell-seeded microcarriers were embedded in four biological glues - recalcified citrated whole blood, recalcified citrated plasma with or without platelets, and a commercially available fibrin glue - and cultured in an in vitro model under static conditions for 16 weeks. No differences could be verified between the commercial fibrin glue and the blood-derived alternatives. Five further experiments were conducted with recalcified citrated platelet-rich plasma alone as microcarrier sealant, using two different in vitro culture models and chondrocytes from three additional donors. The microcarriers supported chondrocyte adhesion and expansion as well as extracellular matrix (ECM) synthesis. Matrix formation occurred predominantly at sample surfaces under the static conditions. The presence of microcarriers proved essential for the glues to support the structural takeover of ECM proteins produced by the embedded chondrocytes, as exclusion of the microcarriers resulted in unstable structures that dissolved before matrix formation could occur. Immunohistochemical analysis revealed the presence of SOX-9- and S-100-positive chondrocytes as well as the production of aggrecan and collagen type I, but not of the cartilage-specific collagen type II. These results imply that blood-derived glues are indeed potentially applicable for encapsulation of chondrocyte-seeded microcarriers. However, the static in vitro models used in this study proved incapable of supporting cartilage formation throughout the engineered constructs.

    Keywords
    cartilage, chondrocyte, microcarrier, tissue engineering, fibrin glue, platelet-rich plasma
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-20597 (URN)10.1002/term.179 (DOI)
    Available from: 2009-09-15 Created: 2009-09-15 Last updated: 2015-06-29
    2. Engineering three-dimensional cartilage- and bone-like tissues using human dermal fibroblasts and macroporous gelatine microcarriers
    Open this publication in new window or tab >>Engineering three-dimensional cartilage- and bone-like tissues using human dermal fibroblasts and macroporous gelatine microcarriers
    Show others...
    2010 (English)In: Journal of plastic, reconstructive & aesthetic surgery : JPRAS, ISSN 1878-0539, Vol. 63, no 6, p. 1036-1046Article in journal (Refereed) Published
    Abstract [en]

    The creation of tissue-engineered cartilage and bone, using cells from an easily available source seeded on a suitable biomaterial, may have a vast impact on regenerative medicine. While various types of adult stem cells have shown promising results, their use is accompanied by difficulties associated with harvest and culture. The proposed inherent plasticity of dermally derived human fibroblasts may render them useful in tissue-engineering applications. In the present study, human dermal fibroblasts cultured on macroporous gelatine microcarriers encapsulated in platelet-rich plasma into three-dimensional constructs were differentiated towards chondrogenic and osteogenic phenotypes using specific induction media. The effect of flow-induced shear stress on osteogenic differentiation of fibroblasts was also evaluated. The generated tissue constructs were analysed after 4, 8 and 12 weeks using routine and immunohistochemical stainings as well as an enzyme activity assay. The chondrogenic-induced tissue constructs were composed of glycosaminoglycan-rich extracellular matrix, which stained positive for aggrecan. The osteogenic-induced tissue constructs were composed of mineralised extracellular matrix containing osteocalcin and osteonectin, with cells showing an increased alkaline phosphatase activity. Increased osteogenic differentiation was seen when applying flow-induced shear stress to the culture. Un-induced fibroblast controls did not form cartilage- or bone-like tissues. Our findings suggest that primary human dermal fibroblasts can be used to form cartilage- and bone-like tissues in vitro when cultured in specific induction media.

    Keywords
    Dermal fibroblast; Chondrogenesis; Osteogenesis; Microcarrier; Tissue engineering; Regenerative medicine
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-54113 (URN)10.1016/j.bjps.2009.02.072 (DOI)000277356900025 ()19329368 (PubMedID)
    Available from: 2010-02-23 Created: 2010-02-23 Last updated: 2010-10-29
    3. Cell expansion of human articular chondrocytes on macroporous gelatine scaffolds: — impact of micro carrier selection on cell proliferation
    Open this publication in new window or tab >>Cell expansion of human articular chondrocytes on macroporous gelatine scaffolds: — impact of micro carrier selection on cell proliferation
    2011 (English)In: Biomedical Materials, ISSN 1748-6041, E-ISSN 1748-605X, Vol. 6, no 6, p. 065001-Article in journal (Refereed) Published
    Abstract [en]

    This study investigates human chondrocyte expansion on four macroporous gelatine microcarriers (CultiSpher) differing with respect to two manufacturing processes—the amount of emulsifier used during initial preparation and the gelatine cross-linking medium. Monolayer-expanded articular chondrocytes from three donors were seeded onto the microcarriers and cultured in spinner flask systems for a total of 15 days. Samples were extracted every other day to monitor cell viability and establish cell counts, which were analysed using analysis of variance and piecewise linear regression. Chondrocyte densities increased according to a linear pattern for all microcarriers, indicating an ongoing, though limited, cell proliferation. A strong chondrocyte donor effect was seen during the initial expansion phase. The final cell yield differed significantly between the microcarriers and our results indicate that manufacturing differences affected chondrocyte densities at this point. Remaining cells stained positive for chondrogenic markers SOX-9 and S-100 but extracellular matrix formation was modest to undetectable. In conclusion, the four gelatine microcarriers supported chondrocyte adhesion and proliferation over a two week period. The best yield was observed for microcarriers produced with low emulsifier content and cross-linked in water and acetone. These results add to the identification of optimal biomaterial parameters for specific cellular processes and populations.

    Place, publisher, year, edition, pages
    Bristol, UK: Institute of Physics Publishing Ltd., 2011
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-54114 (URN)10.1088/1748-6041/6/6/065001 (DOI)000298241800001 ()
    Note
    Funding agencies|Swedish foundation for strategic research| A302:319 |Linkoping University and Landstinget i Ostergotland||Available from: 2010-02-23 Created: 2010-02-23 Last updated: 2017-12-12
    4. The Role of Platelet Rich Plasma and Dynamic Centrifugation on Extracellular Matrix Formation of Human Articular Chondrocytes on Macroporous Gelatin Microcarriers in Pellet Culture
    Open this publication in new window or tab >>The Role of Platelet Rich Plasma and Dynamic Centrifugation on Extracellular Matrix Formation of Human Articular Chondrocytes on Macroporous Gelatin Microcarriers in Pellet Culture
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Platelet rich plasma (PRP) has been investigated for its beneficial use in cartilage tissue engineering previously. Here, we address the effect of using PRP as encapsulating agent for gelatin-supported chondrocyte pellet culture in vitro. Furthermore, the concept of using dynamic centrifugation to stimulate extracellular matrix (ECM) formation of the chondrocytes is explored. Human articular chondrocytes were expanded on macroporous gelatin microcarriers in a spinner flask system. The cell-seeded microcarriers were allowed to form pellets with or without re-calcified citrated PRP, and subjected to dynamic centrifugation (f = 0.0125 Hz) for a total of 16 min every other day using a standard tabletop centrifuge. Three acceleration curves with differing top speeds (corresponding to 500 g, 1500 g and 3000 g respectively) were used for the experimental groups and unstimulated controls were set for comparison. Pellets were kept in culture for up to 12 weeks, paraffin embedded and sectioned for histological and immunohistochemical analysis. Results showed increasing numbers of cells and ECM with time, as well as a gradual degradation of the gelatin microcarriers, indicating ongoing cell proliferation and metabolism throughout the culture period. Cell densities and ECM formation were more pronounced in the PRP-containing groups after four weeks, although this difference diminished with time. At the last time point several cartilage markers were found in the produced ECM, however including the fibrocartilaginous marker collagen type I. Dynamic centrifugation did not visibly increase the ECM accumulation over the 12-week duration of this experiment, although non-conclusive indications of collagen fiber organization were seen in the two groups with the highest acceleration limits at the last time point.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-54115 (URN)
    Available from: 2010-02-23 Created: 2010-02-23 Last updated: 2015-06-29
  • 2.
    Pettersson, Sofia
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Surgery . Linköping University, Faculty of Health Sciences.
    Wetterö, Jonas
    Linköping University, Department of Clinical and Experimental Medicine, Rheumatology . Linköping University, Faculty of Health Sciences.
    Kratz, Gunnar
    Linköping University, Department of Clinical and Experimental Medicine, Plastic Surgery, Hand Surgery and Burns . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of Plastic Surgery, Hand surgery UHL.
    The Role of Platelet Rich Plasma and Dynamic Centrifugation on Extracellular Matrix Formation of Human Articular Chondrocytes on Macroporous Gelatin Microcarriers in Pellet CultureManuscript (preprint) (Other academic)
    Abstract [en]

    Platelet rich plasma (PRP) has been investigated for its beneficial use in cartilage tissue engineering previously. Here, we address the effect of using PRP as encapsulating agent for gelatin-supported chondrocyte pellet culture in vitro. Furthermore, the concept of using dynamic centrifugation to stimulate extracellular matrix (ECM) formation of the chondrocytes is explored. Human articular chondrocytes were expanded on macroporous gelatin microcarriers in a spinner flask system. The cell-seeded microcarriers were allowed to form pellets with or without re-calcified citrated PRP, and subjected to dynamic centrifugation (f = 0.0125 Hz) for a total of 16 min every other day using a standard tabletop centrifuge. Three acceleration curves with differing top speeds (corresponding to 500 g, 1500 g and 3000 g respectively) were used for the experimental groups and unstimulated controls were set for comparison. Pellets were kept in culture for up to 12 weeks, paraffin embedded and sectioned for histological and immunohistochemical analysis. Results showed increasing numbers of cells and ECM with time, as well as a gradual degradation of the gelatin microcarriers, indicating ongoing cell proliferation and metabolism throughout the culture period. Cell densities and ECM formation were more pronounced in the PRP-containing groups after four weeks, although this difference diminished with time. At the last time point several cartilage markers were found in the produced ECM, however including the fibrocartilaginous marker collagen type I. Dynamic centrifugation did not visibly increase the ECM accumulation over the 12-week duration of this experiment, although non-conclusive indications of collagen fiber organization were seen in the two groups with the highest acceleration limits at the last time point.

  • 3.
    Pettersson, Sofia
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Plastic Surgery, Hand Surgery and Burns. Linköping University, Faculty of Health Sciences.
    Wetterö, Jonas
    Linköping University, Department of Clinical and Experimental Medicine, Rheumatology. Linköping University, Faculty of Health Sciences.
    Tengvall, Pentti
    Institute of Clinical Sciences, Department of Biomaterials, The Sahlgrenska Academy at University of Gothenburg, Gothenburg.
    Kratz, Gunnar
    Linköping University, Department of Clinical and Experimental Medicine, Plastic Surgery, Hand Surgery and Burns. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of Plastic Surgery, Hand surgery UHL.
    Cell expansion of human articular chondrocytes on macroporous gelatine scaffolds: — impact of micro carrier selection on cell proliferation2011In: Biomedical Materials, ISSN 1748-6041, E-ISSN 1748-605X, Vol. 6, no 6, p. 065001-Article in journal (Refereed)
    Abstract [en]

    This study investigates human chondrocyte expansion on four macroporous gelatine microcarriers (CultiSpher) differing with respect to two manufacturing processes—the amount of emulsifier used during initial preparation and the gelatine cross-linking medium. Monolayer-expanded articular chondrocytes from three donors were seeded onto the microcarriers and cultured in spinner flask systems for a total of 15 days. Samples were extracted every other day to monitor cell viability and establish cell counts, which were analysed using analysis of variance and piecewise linear regression. Chondrocyte densities increased according to a linear pattern for all microcarriers, indicating an ongoing, though limited, cell proliferation. A strong chondrocyte donor effect was seen during the initial expansion phase. The final cell yield differed significantly between the microcarriers and our results indicate that manufacturing differences affected chondrocyte densities at this point. Remaining cells stained positive for chondrogenic markers SOX-9 and S-100 but extracellular matrix formation was modest to undetectable. In conclusion, the four gelatine microcarriers supported chondrocyte adhesion and proliferation over a two week period. The best yield was observed for microcarriers produced with low emulsifier content and cross-linked in water and acetone. These results add to the identification of optimal biomaterial parameters for specific cellular processes and populations.

  • 4.
    Pettersson, Sofia
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Surgery. Linköping University, Faculty of Health Sciences.
    Wetterö, Jonas
    Linköping University, Department of Clinical and Experimental Medicine, Rheumatology. Linköping University, Faculty of Health Sciences.
    Tengvall, Pentti
    University of Gothenburg.
    Kratz, Gunnar
    Linköping University, Department of Clinical and Experimental Medicine, Plastic Surgery, Hand Surgery and Burns. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of Plastic Surgery, Hand surgery UHL.
    Human articular chondrocytes on macroporous gelatin microcarriers form structurally stable constructs with blood-derived biological glues in vitro2009In: JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, ISSN 1932-6254, Vol. 35, no 6, p. 450-460Article in journal (Refereed)
    Abstract [en]

    Biodegradable macroporous gelatin microcarriers fixed with blood-derived biodegradable glue are proposed as a delivery system for human autologous chondrocytes. Cell-seeded microcarriers were embedded in four biological glues - recalcified citrated whole blood, recalcified citrated plasma with or without platelets, and a commercially available fibrin glue - and cultured in an in vitro model under static conditions for 16 weeks. No differences could be verified between the commercial fibrin glue and the blood-derived alternatives. Five further experiments were conducted with recalcified citrated platelet-rich plasma alone as microcarrier sealant, using two different in vitro culture models and chondrocytes from three additional donors. The microcarriers supported chondrocyte adhesion and expansion as well as extracellular matrix (ECM) synthesis. Matrix formation occurred predominantly at sample surfaces under the static conditions. The presence of microcarriers proved essential for the glues to support the structural takeover of ECM proteins produced by the embedded chondrocytes, as exclusion of the microcarriers resulted in unstable structures that dissolved before matrix formation could occur. Immunohistochemical analysis revealed the presence of SOX-9- and S-100-positive chondrocytes as well as the production of aggrecan and collagen type I, but not of the cartilage-specific collagen type II. These results imply that blood-derived glues are indeed potentially applicable for encapsulation of chondrocyte-seeded microcarriers. However, the static in vitro models used in this study proved incapable of supporting cartilage formation throughout the engineered constructs.

  • 5.
    Sommar, Pehr
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Plastic Surgery, Hand Surgery and Burns . Linköping University, Faculty of Health Sciences.
    Pettersson, Sofia
    Linköping University, Department of Clinical and Experimental Medicine, Surgery . Linköping University, Faculty of Health Sciences.
    Ness, Charlotte
    Department of Surgery, Section of Plastic Surgery and Burn Centre, Haukeland University Hospital, Bergen, Norway.
    Johnson, Hans
    Department of Surgery, Section of Plastic Surgery and Burn Centre, Haukeland University Hospital, Bergen, Norway.
    Kratz, Gunnar
    Linköping University, Department of Clinical and Experimental Medicine, Plastic Surgery, Hand Surgery and Burns . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of Plastic Surgery, Hand surgery UHL.
    Junker, Johan P E
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Engineering three-dimensional cartilage- and bone-like tissues using human dermal fibroblasts and macroporous gelatine microcarriers2010In: Journal of plastic, reconstructive & aesthetic surgery : JPRAS, ISSN 1878-0539, Vol. 63, no 6, p. 1036-1046Article in journal (Refereed)
    Abstract [en]

    The creation of tissue-engineered cartilage and bone, using cells from an easily available source seeded on a suitable biomaterial, may have a vast impact on regenerative medicine. While various types of adult stem cells have shown promising results, their use is accompanied by difficulties associated with harvest and culture. The proposed inherent plasticity of dermally derived human fibroblasts may render them useful in tissue-engineering applications. In the present study, human dermal fibroblasts cultured on macroporous gelatine microcarriers encapsulated in platelet-rich plasma into three-dimensional constructs were differentiated towards chondrogenic and osteogenic phenotypes using specific induction media. The effect of flow-induced shear stress on osteogenic differentiation of fibroblasts was also evaluated. The generated tissue constructs were analysed after 4, 8 and 12 weeks using routine and immunohistochemical stainings as well as an enzyme activity assay. The chondrogenic-induced tissue constructs were composed of glycosaminoglycan-rich extracellular matrix, which stained positive for aggrecan. The osteogenic-induced tissue constructs were composed of mineralised extracellular matrix containing osteocalcin and osteonectin, with cells showing an increased alkaline phosphatase activity. Increased osteogenic differentiation was seen when applying flow-induced shear stress to the culture. Un-induced fibroblast controls did not form cartilage- or bone-like tissues. Our findings suggest that primary human dermal fibroblasts can be used to form cartilage- and bone-like tissues in vitro when cultured in specific induction media.

  • 6.
    Wetterö, Jonas
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Rheumatology . Linköping University, Faculty of Health Sciences.
    Pettersson, Sofia
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Surgery .
    Holmgren Peterson, Kajsa
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    A cellular imaging CDIO project for 2nd semester students in engineering biology2006In: World Transactions on Engineering and Technology Education, ISSN 1446-2257, Vol. 5, no 2, p. 279-282Article in journal (Refereed)
    Abstract [en]

    The demand for exact engineering within the life sciences is growing and the Engineering Biology programme at Linköping University, Linköping, Sweden, prepares students for a career at this interface. Conceive – Design – Implement – Operate (CDIO) was recently pioneered in an introductory project course. Groups of six to seven students apply a LIPS scalable project model from traditional engineering educational environments on, for example, a cellular imaging task in a hospital setting, prior to taking courses in cell biology/optics. Besides facilitating the implementation of CDIO in higher courses, students gain early career insight and enhance their communication skills. A customer (senior teacher) needs to visualise structures in cells, and the student group is contracted to deliver an applied and optimised method to meet specified requirements. The customer reviews deliverables before the tollgates and communicates with the student project leader. Other students are responsible for documentation and subsystems. The project is allocated laboratory facilities and hardware, and two fictitious subcontractors supply samples and consumables. Extra teachers perform supervision and methodological consultation. In summary, CDIO is indeed applicable and rewarding in cellular imaging, yet is also challenging.

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