Ectopic bone formation in cell-seeded poly(ethylene oxide)/poly(butylene terephthalate) copolymer scaffolds of varying porosity

Scaffolds from poly(ethylene oxide) and poly(butylene terephthalate), PEOT/PBT, with a PEO molecular weight of 1,000 and a PEOT content of 70 weight% (1000PEOT70PBT30) were prepared by leaching salt particles (425–500 μm). Scaffolds of 73.5, 80.6 and 85.0% porosity were treated with a CO2 gas plasma and seeded with rat bone marrow stromal cells (BMSCs). After in vitro culture for 7 days (d) in an osteogenic medium the scaffolds were subcutaneously implanted for 4 weeks in nude mice. Poly(d, l-lactide) (PDLLA) and biphasic calcium phosphate (BCP) scaffolds were included as references. After 4 weeks (wks) all scaffolds showed ectopic formation of bone and bone marrow. For the scaffolds of different porosities, no significant differences were observed in the relative amounts of bone (7–9%) and bone marrow (6–11%) formed, even though micro computed tomography (μ-CT) data showed considerable differences in accessible pore volume and surface area. 1000PEOT70PBT30 scaffolds with a porosity of 85% could not maintain their original shape in vivo. Surprisingly, 1000PEOT70PBT30 scaffolds with a porosity of 73.5% showed cartilage formation. This cartilage formation is most likely due to poorly accessible pores in the scaffolds, as was observed in histological sections. μ-CT data showed a considerably smaller accessible pore volume (as a fraction of the total volume) than in 1000PEOT70PBT30 scaffolds of 80.6 and 85.0% porosity. BMSC seeded PDLLA (83.5% porosity) and BCP scaffolds (29% porosity) always showed considerably more bone and bone marrow formation (bone marrow formation is approximately 40%) and less fibrous tissue ingrowth than the 1000PEOT70PBT30 scaffolds. The scaffold material itself can be of great influence. In more hydrophobic and rigid scaffolds like the PDLLA or BCP scaffolds, the accessibility of the pore structure is more likely to be preserved under the prevailing physiological conditions than in the case of hydrophilic 1000PEOT70PBT30 scaffolds. Scaffolds prepared from other PEOT/PBT polymer compositions, might prove to be more suited

Controlled release of proteins from degradable poly(ether-ester) multiblock copolymers

A new series of multiblock poly(ether-ester)s based on poly(ethylene glycol) (PEG), butylene terephthalate (BT) and butylene succinate (BS) segments were introduced as matrices for controlled release applications. The release of two model proteins, lysozyme and bovine serum albumin (BSA), from poly(ether-ester) films were evaluated and correlated to the swelling and degradation characteristics of the polymer matrices. First- and zero-order profiles were found for the release of lysozyme, depending on the composition of the polymer matrix. The initial diffusion coefficient was correlated to the swelling of the matrix, which increased with longer PEG segments and lower BT/BS ratios of the polymer. High swelling matrices released the lysozyme according to diffusion-controlled first-order release profiles. Zero-order release profiles were obtained from less swollen matrices due to a combination of diffusion and degradation of the matrix. In contrast to the release of lysozyme, BSA was released from the poly(ether-ester) matrices via delayed release profiles. Both the delay time and the release rate could be tailored by varying the matrix composition. The BSA release rate was mainly determined by the degradation, whereas the delay time was determined by a combination of the swelling and the degradation rate of the polymer matrix

Expansion of mesenchymal stem cells using a microcarrier-based cultivation system: growth and metabolism

For the continuous and fast expansion of mesenchymal stem cells (MSCs), microcarriers have gained increasing interest. The aim of this study was to evaluate the growth and metabolism profiles of MSCs, expanded in a microcarrier-based cultivation system. We investigated various cultivation conditions to expand goat mesenchymal stem cells on Cytodex 1 microcarriers. These conditions differed in feeding regime, i.e. the addition of fresh proliferation medium, with or without new microcarriers. For all conditions, cell attachment, cell proliferation, energy source consumption, metabolite production, and cell distribution on the microcarriers were studied. Attachment efficiencies of 40% were obtained followed by successful expansion up to 15 cultivation days. Depending on the feeding regime, an exponential growth, stationary growth, and decline growth phase could be distinguished. Addition of 30% fresh medium containing microcarriers every three days showed the longest continuous proliferation of goat MSCs on microcarriers. This feeding regime has the advantage that metabolites, such as ammonia, are diluted and that new energy sources, such as glucose and glutamine, and additional surface area are provided to the cells. In addition, by adding extra microcarriers a more homogenous cell distribution on the microcarriers is obtained as a result of bead-to-bead transfer. A correlation between nutrient consumption, metabolite production and cell growth was observed. The decreasing yield of lactate from glucose over time indicated a possible shift in cellular metabolism

Human tissue-engineered bone produced in clinically relevant amounts using a semi-automated perfusion bioreactor system: a preliminary study

The aim of this study was to evaluate a semi-automated perfusion bioreactor system for the production of clinically relevant amounts of human tissue-engineered bone. Human bone marrow stromal cells (hBMSCs) of eight donors were dynamically seeded and proliferated in a perfusion bioreactor system in clinically relevant volumes (10 cm3) of macroporous biphasic calcium phosphate scaffolds (BCP particles, 2–6 mm). Cell load and distribution were shown using methylene blue staining. MTT staining was used to demonstrate viability of the present cells. After 20 days of cultivation, the particles were covered with a homogeneous layer of viable cells. Online oxygen measurements confirmed the proliferation of hBMSCs in the bioreactor. After 20 days of cultivation, the hybrid constructs became interconnected and a dense layer of extracellular matrix was present, as visualized by scanning electron microscopy (SEM). Furthermore, the hBMSCs showed differentiation towards the osteogenic lineage as was indicated by collagen type I production and alkaline phosphatase (ALP) expression. We observed no significant differences in osteogenic gene expression profiles between static and dynamic conditions like ALP, BMP2, Id1, Id2, Smad6, collagen type I, osteocalcin, osteonectin and S100A4. For the donors that showed bone formation, dynamically cultured hybrid constructs showed the same amount of bone as the statically cultured hybrid constructs. Based on these results, we conclude that a semi-automated perfusion bioreactor system is capable of producing clinically relevant and viable amounts of human tissue-engineered bone that exhibit bone-forming potential after implantation in nude mice

Quantifying in vitro growth and metabolism kinetics of human mesenchymal stem cells using a mathematical model

Better quantitative understanding of human mesenchymal stem cells (hMSCs) metabolism is needed to identify, understand, and subsequently optimize the processes in expansion of hMSCs in vitro. For this purpose, we analyzed growth of hMSCs in vitro with a mathematical model based on the mass balances for viable cell numbers, glucose, lactate, glutamine, and glutamate. The mathematical modeling had two aims: (1) to estimate kinetic parameters of important metabolites for hMSC monolayer cultures, and (2) to quantitatively assess assumptions on growth of hMSCs. Two cell seeding densities were used to investigate growth and metabolism kinetics of MSCs from three human donors. We analyzed growth up to confluency and used metabolic assumptions described in literature. Results showed a longer initial phase, a slower growth rate, and a higher glucose, lactate, glutamine, and glutamate metabolic rates at the lower cell seeding density. Higher metabolic rates could be induced by a lower contact inhibition effect when seeding at 100cells/cm2 than when seeding at 1000cells/cm2. In addition, parameter estimation describing kinetics of hMSCs in culture, depending on the seeding density, showed doubling times in the order of 17–32h, specific glucose consumption in the order of 1.25×10-1 to 3.77×10-1pmol/cell/h, specific lactate production in the order of 2.48×10-1 to 7.67×10-1pmol/cell/h, specific glutamine production in the order of 7.04×10-3 to 2.27pmol/cell/h, and specific glutamate production in the order of 4.87×10-1 to 23.4pmol/cell/h. Lactate-to-glucose yield ratios confirmed that hMSCs use glucose via anaerobic glycolysis. In addition, glutamine and glutamate metabolic shifts were identified that could be important for understanding growth of hMSCs in vitro. This study showed that the mathematical modeling approach supports quantitative analysis of important mechanisms in proliferation of hMSCs in vitr

Quantifying in vitro growth and metabolism kinetics of human mesenchymal stem cells using a mathematical model

Better quantitative understanding of human mesenchymal stem cells (hMSCs) metabolism is needed to identify, understand, and subsequently optimize the processes in expansion of hMSCs in vitro. For this purpose, we analyzed growth of hMSCs in vitro with a mathematical model based on the mass balances for viable cell numbers, glucose, lactate, glutamine, and glutamate. The mathematical modeling had two aims: (1) to estimate kinetic parameters of important metabolites for hMSC monolayer cultures, and (2) to quantitatively assess assumptions on growth of hMSCs. Two cell seeding densities were used to investigate growth and metabolism kinetics of MSCs from three human donors. We analyzed growth up to confluency and used metabolic assumptions described in literature. Results showed a longer initial phase, a slower growth rate, and a higher glucose, lactate, glutamine, and glutamate metabolic rates at the lower cell seeding density. Higher metabolic rates could be induced by a lower contact inhibition effect when seeding at 100cells/cm2 than when seeding at 1000cells/cm2. In addition, parameter estimation describing kinetics of hMSCs in culture, depending on the seeding density, showed doubling times in the order of 17–32h, specific glucose consumption in the order of 1.25×10-1 to 3.77×10-1pmol/cell/h, specific lactate production in the order of 2.48×10-1 to 7.67×10-1pmol/cell/h, specific glutamine production in the order of 7.04×10-3 to 2.27pmol/cell/h, and specific glutamate production in the order of 4.87×10-1 to 23.4pmol/cell/h. Lactate-to-glucose yield ratios confirmed that hMSCs use glucose via anaerobic glycolysis. In addition, glutamine and glutamate metabolic shifts were identified that could be important for understanding growth of hMSCs in vitro. This study showed that the mathematical modeling approach supports quantitative analysis of important mechanisms in proliferation of hMSCs in vitr