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Human adipose-derived stem cells (hASCs) have become an important cell source for the use in tissue engineering and other medical applications. Not every biomaterial is suitable for human cell culture and requires surface modifications to enable cell adhesion and proliferation. Our hypothesis is that chemical surface modifications introduced by low-discharge plasma enhance the adhesion and proliferation of hASCs. Polystyrene (PS) surfaces were modified either by ammonia (NH3), carbon dioxide (CO2) or acrylic acid (AAc) plasma. The results show that the initial cell adhesion is significantly higher on all modified surfaces than on unmodified material as evaluated by bright field microscopy, live/dead staining, total DNA amount and scanning electron microscopy. The formation of focal adhesions was well pronounced on the Tissue Culture PS, NH3-, and CO2 plasma modified samples. The number of matured fibrillar adhesions was significantly higher on NH3 plasmamodified surfaces than on all other surfaces. Our study validates the suitability of chemical plasma activation and represents a method to enhance hASCs adhesion and improved cell expansion. All chemical modification promoted hASCs adhesion and can therefore be used for the modification of different scaffold materials whereby NH3-plasma modified surfaces resulted in the best outcome concerning hASCs adhesion and proliferation.
Methacrylated gelatin and mature adipocytes are promising components for adipose tissue engineering
(2016)
In vitro engineering of autologous fatty tissue constructs is still a major challenge for the treatment of congenital deformities, tumor resections or high-graded burns. In this study, we evaluated the suitability of photo-crosslinkable methacrylated gelatin (GM) and mature adipocytes as components for the composition of three-dimensional fatty tissue constructs. Cytocompatibility evaluations of the GM and the photoinitiator Lithium phenyl-2,4,6 trimethylbenzoylphosphinate (LAP) showed no cytotoxicity in the relevant range of concentrations. Matrix stiffness of cell-laden hydrogels was adjusted to native fatty tissue by tuning the degree of crosslinking and was shown to be comparable to that of native fatty tissue. Mature adipocytes were then cultured for 14 days within the GM resulting in a fatty tissue construct loaded with viable cells expressing cell markers perilipin A and laminin. This work demonstrates that mature adipocytes are a highly valuable cell source for the composition of fatty tissue equivalents in vitro. Photo-crosslinkable methacrylated gelatin is an excellent tissue scaffold and a promising bioink for new printing techniques due to its biocompatibility and tunable properties.
Thin radio-frequency magnetron sputter deposited nano-hydroxyapatite (HA) films were prepared on the surface of a Fe-tricalcium phosphate (Fe-TCP) bioceramic composite, which was obtained using a conventional powder injection moulding technique. The obtained nano-hydroxyapatite coated Fe-TCP biocomposites (nano HA-Fe-TCP) were studied with respect to their chemical and phase composition, surface morphology, water contact angle, surface free energy and hysteresis. The deposition process resulted in a homogeneous, single-phase HA coating. The ability of the surface to support adhesion and the proliferation of human mesenchymal stem cells (hMSCs) was studied using biological short-term tests in vitro. The surface of the uncoated Fe-TCP bioceramic composite showed an initial cell attachment after 24 h of seeding, but adhesion, proliferation and growth did not persist during 14 days of culture.However, the HA-Fe-TCP surfaces allowed cell adhesion, and proliferation during 14 days. The deposition of the nano-HA films on the Fe-TCP surface resulted in higher surface energy, improved hydrophilicity and biocompatibility compared with the surface of the uncoated Fe-TCP. Furthermore, it is suggested that an increase in the polar component of the surface energy was responsible for the enhanced cell adhesion and proliferation in the case of the nano-HA Fe-TCP biocomposites.
Bone remodeling can be mimicked in vitro by co-culture models. Based on bone cells, such co-cultures help to study synergistic morphological changes and the impact of materials and applied substances. Hence, we examined the formation of osteoclasts on bovine bone materials to prove the bone resorption functionality of the osteoclasts in three different co-culture set-ups using human monocytes (hMCs) and (I) human mesenchymal stem cells (hMSCs), (II) osteogenic differentiated hMSCs (hOBs), and (III) hOBs in addition of soluble monocyte-colony stimulating factor (M CSF) and cytokine receptor activator of NFkB ligand (RANKL).We detected osteoclast-specific actin morphology, as well as the expression of cathepsin K and CD51/61 in single cells in set-up II and in numerous cells in set-up III. Resorption pits on bone material as characteristic proof of functional osteoclasts were not found in set-up I and II, but we detected such resorption pits in set–up III. We conclude in co culture models without M-CSF and RANKL that monocytes can differentiate into osteoclasts that show the characteristic actin structures and protein expression. However, to receive functional bone resorbing osteoclasts in vitro, the addition of M-CSF and RANKL is needed. Moreover, we suggest the use of bone or bone-like materials for future studies evaluating osteoclastogenesis.
The world population is growing and alternative ways of satisfying the increasing demand for meat are being explored, such as using animal cells for the fabrication of cultured meat. Edible biomaterials are required as supporting structures. Hence, we chose agarose, gellan and a xanthan-locust bean gum blend (XLB) as support materials with pea and soy protein additives and analyzed them regarding material properties and biocompatibility. We successfully built stable hydrogels containing up to 1% pea or soy protein. Higher amounts of protein resulted in poor handling properties and unstable gels. The gelation temperature range for agarose and gellan blends is between 23–30 °C, but for XLB blends it is above 55 °C. A change in viscosity and a decrease in the swelling behavior was observed in the polysaccharide-protein gels compared to the pure polysaccharide gels. None of the leachates of the investigated materials had cytotoxic effects on the myoblast cell line C2C12. All polysaccharide-protein blends evaluated turned out as potential candidates for cultured meat. For cell-laden gels, the gellan blends were the most suitable in terms of processing and uniform distribution of cells, followed by agarose blends, whereas no stable cell-laden gels could be formed with XLB blends.
In the current study the in vitro outcome of a degradable magnesium alloy (AZ91D) and standard titanium modified by nanostructured-hydroxyapatite (n-HA) coatings concerning cell adhesion and osteogenic differentiation was investigated by direct cell culture. The n-HA modification was prepared via radio-frequency magnetron sputtering deposition and proven by field emission scanning electron microscopy and X-ray powder diffraction patterns revealing a homogenous surface coating. Human mesenchymal stem cell (hMSCs) adhesion was examined after one and 14 days displaying an enhanced initial cell adhesion on the n-HA modified samples. The osteogenic lineage commitment of the cells was determined by alkaline phosphatase (ALP) quantification. On day one n-HA coated AZ91D exhibited a comparable ALP expression to standard tissue culture polystyrene samples. However, after 14 days solely little DNA and ALP amounts were measurable on n-HA coated AZ91D due to the lack of adherent cells. Titanium displayed excellent cell adhesion properties and ALP was detectable after 14 days. An increased pH of the culture was measured for AZ91D as well as for n-HA coated AZ91D. We conclude that n-HA modification improves initial cell attachment on AZ91D within the first 24 h. However, the effect does not ersist for 14 days in in vitro conditions.
Engineering of large vascularized adipose tissue constructs is still a challenge for the treatment of extensive high-graded burns or the replacement of tissue after tumor removal. Communication between mature adipocytes and endothelial cells is important for homeostasis and the maintenance of adipose tissue mass but, to date, is mainly neglected in tissue engineering strategies. Thus, new coculture strategies are needed to integrate adipocytes and endothelial cells successfully into a functional construct. This review focuses on the cross-talk of mature adipocytes and endothelial cells and considers their influence on fatty acid metabolism and vascular tone. In addition, the properties and challenges with regard to these two cell types for vascularized tissue engineering are highlighted.