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Title: Assessment of human mesenchymal stem cells osteogenesis, depending on a scaffold type and perfusion


Bones constitute an important mechanical barrier protecting the soft tissues of the body but particularly sense different mechanical forces, regulating remodeling of the skeletal system. Bone remodeling (bone turnover) takes place under both physiological conditions and upon micro- and macro- bone injuries, and its imbalance, i.e. excessive or reduced bone resorption as well as formation affects the homeostasis of a whole organism. Treating of large bone defects or bone degenerative diseases (e.g. osteoporosis), manifesting with an impaired bone remodeling, still remains a challenging medical issue. Besides the classical protocols based on the use of bone auto- or allogenic grafts, bone tissue engineering offers several new, promising methods of bone defects treatment. This strategy applies appropriate osteoblast precursor cells cultured in vitro e.g. on three-dimensional biomaterials (scaffolds), in the presence of the selected osteoinductive factors, under mechanical forces applied in bioreactors. A major source of osteoblast progenitors with a great potential within bone tissue regeneration strategies are human bone marrow-derived mesenchymal stem cells (hBMSCs). The aim of this PhD dissertation was to investigate the mechanisms of osteogenesis in human bone marrow-derived mesenchymal stem cells (hBMSCs) in vitro cultures carried out on the selected scaffolds of a different ; chemical composition and bioactivity (experimental scaffolds based on a biodegradable poly-L-lactide-D-glycolide (PLGA) copolymer and two types of bioactive glasses: S2 or A2 or bioinert scaffolds made of polyurethane (PU) coated with gelatin) and the assessment of hBMSC mechanical stimulation in flow perfusion bioreactor in these 3D cultures. The research project presented was focused on the selection and optimisation of the in vitro culture conditions, most effectively promoting differentiation of human BMSCs towards a bone tissue. The experiments were carried out both in static conditions or under media flow perfusion stimulation in hBMSC cultures established on PLGA- or PU-based scaffolds. The cells were either cultured in a standard growth medium or stimulated with appropriate osteoinductive supplements: AA-2P, DEX, BGP and rhBMP-2. ; BGs-PLGA composites have exhibited high bioactivity, manifesting with the formation of calcium phosphate (apatite) precipitates inside the pores of the scaffolds. We also have characterised mechanical properties of both composites and shown they promoted superior hBMSC attachment to the scaffolds surface and stimulated the expression of several osteogenic markers in hBMSC compared to cells grown on unmodified PLGA. We concluded there were also marked differences in the response of hBMSCs to composite scaffolds, depending on chemical compositi ; ons of the scaffolds and culture treatments. Compared to silica-rich S2-PLGA composites, hBMSC grown on calcium-rich A2-PLGA scaffolds were overall less responsive to rhBMP-2 or DEX and the osteoinductive properties of these A2-PLGA scaffolds seemed to be partially dependent on activation of BMP signaling in untreated hBMSCs. Our composite scaffolds, specifically S2-PLGA also enhanced hBMSCs osteogenesis under a single, 2-hour session of unidirectional, steady, 2.5 ml/min flow perfusion stimulation and rhBMP-2 treatment. We also cultured hBMSCs on bioinert, porous, gelatine-coated, polyurethane scaffolds in the presence of the osteogenic supplements and stimulated them with a single 2-hour session of media flow perfusion at either early or late 3D culture stages. Some of the cells also were pre-treated in monolayers (2D cultures) with the osteogenic supplements to advance cell differentiation and followed by the osteogenic 3D cultures. These experiments were performed in order to characterize cell differentiation stage effects under flow perfusion stimulation. We noted that a single, short, unidirectional and steady media perfusion in hBMSC 3D cultures can significantly enhance expression of bone-related transcription and growth factors along with the enhancement of matrix components (collagen and mineral) production. However, it is the most effective when cells reach the pre-o ; steoblast or osteoblast differentiation stage. Based on the results obtained, we conclude it may become possible to modulate the osteogenic response of hBMSCs in 3D cultures, depending on the scaffolds chemistry (bioinert vs bioactive scaffolds of different CaO and SiO2 contents), cell treatment conditions (standard growth media without osteogenic supplements vs supplemented media; static vs dynamic cultures, stimulated with flow perfusion) and cell differentiation stage (undifferentiated precursors of osteoblasts vs differentiating osteoblasts). This knowledge may possibly contribute to the elaboration of new, clinically relevant protocols of bone defects treatment, based on bone tissue engineering strategy.

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Level of degree:

2 - studia doktoranckie

Degree discipline:

biologia ; biochemia

Degree grantor:

Wydział Nauk o Zdrowiu


Niedźwiedzki, Tadeusz ; Osyczka, Anna M.

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tylko w bibliotece

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Last modified:

Mar 16, 2023

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May 11, 2016

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ZB-124512 Mar 16, 2023


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