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Engineering three-dimensional cartilage- and bone-like tissues using human dermal fibroblasts and macroporous gelatine microcarriers
Linköping University, Department of Clinical and Experimental Medicine, Plastic Surgery, Hand Surgery and Burns . Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Surgery . Linköping University, Faculty of Health Sciences.
Department of Surgery, Section of Plastic Surgery and Burn Centre, Haukeland University Hospital, Bergen, Norway.
Department of Surgery, Section of Plastic Surgery and Burn Centre, Haukeland University Hospital, Bergen, Norway.
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2010 (English)In: Journal of plastic, reconstructive & aesthetic surgery : JPRAS, ISSN 1878-0539, Vol. 63, no 6, 1036-1046 p.Article 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.

Place, publisher, year, edition, pages
2010. Vol. 63, no 6, 1036-1046 p.
Keyword [en]
Dermal fibroblast; Chondrogenesis; Osteogenesis; Microcarrier; Tissue engineering; Regenerative medicine
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-54113DOI: 10.1016/j.bjps.2009.02.072ISI: 000277356900025PubMedID: 19329368OAI: oai:DiVA.org:liu-54113DiVA: diva2:299690
Available from: 2010-02-23 Created: 2010-02-23 Last updated: 2010-10-29
In thesis
1. Biodegradable gelatin microcarriers in tissue engineering: In vitro studies on cartilage and bone
Open this publication in new window or tab >>Biodegradable gelatin microcarriers in tissue engineering: In vitro studies on cartilage and bone
2009 (English)Doctoral 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.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2009. 68 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1147
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-54116 (URN)978-91-7393-557-9 (ISBN)
Public defence
2009-09-25, Berzeliussalen, Hälsouniversitet, Campus US, Linköpings universitet, Linköping, 13:00 (Swedish)
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Available from: 2010-02-23 Created: 2010-02-23 Last updated: 2015-06-29Bibliographically approved

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Pettersson, SofiaKratz, GunnarJunker, Johan P E

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