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The Role of Platelet Rich Plasma and Dynamic Centrifugation on Extracellular Matrix Formation of Human Articular Chondrocytes on Macroporous Gelatin Microcarriers in Pellet Culture
Linköpings universitet, Institutionen för klinisk och experimentell medicin, Kirurgi. Linköpings universitet, Hälsouniversitetet.
Linköpings universitet, Institutionen för klinisk och experimentell medicin, Reumatologi. Linköpings universitet, Hälsouniversitetet.ORCID-id: 0000-0002-6916-5490
Linköpings universitet, Institutionen för klinisk och experimentell medicin, Hand och plastikkirurgi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Rekonstruktionscentrum, Hand- och plastikkirurgiska kliniken US.
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
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.

Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
URN: urn:nbn:se:liu:diva-54115OAI: oai:DiVA.org:liu-54115DiVA, id: diva2:299698
Tillgänglig från: 2010-02-23 Skapad: 2010-02-23 Senast uppdaterad: 2015-06-29
Ingår i avhandling
1. Biodegradable gelatin microcarriers in tissue engineering: In vitro studies on cartilage and bone
Öppna denna publikation i ny flik eller fönster >>Biodegradable gelatin microcarriers in tissue engineering: In vitro studies on cartilage and bone
2009 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Linköping: Linköping University Electronic Press, 2009. s. 68
Serie
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1147
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
urn:nbn:se:liu:diva-54116 (URN)978-91-7393-557-9 (ISBN)
Disputation
2009-09-25, Berzeliussalen, Hälsouniversitet, Campus US, Linköpings universitet, Linköping, 13:00 (Svenska)
Opponent
Handledare
Tillgänglig från: 2010-02-23 Skapad: 2010-02-23 Senast uppdaterad: 2015-06-29Bibliografiskt granskad

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Pettersson, SofiaWetterö, JonasKratz, Gunnar

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KirurgiHälsouniversitetetReumatologiHand och plastikkirurgiHand- och plastikkirurgiska kliniken US
Medicin och hälsovetenskap

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