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Perivascular cells are multilineage cells located around the vessel wall and important for wall stabilization. In this study, we evaluated a stem cell media and a perivascular cell-specific media for the culture of primary perivascular cells regarding their cell morphology, doubling time, stem cell properties, and expression of cell type-specific markers. When the two cell culture media were compared to each other, perivascular cells cultured in the stem cell medium had a more elongated morphology and a faster doubling rate and cells cultured in the pericyte medium had a more typical morphology, with several filopodia, and a slower doubling rate. To evaluate stem cell properties, perivascular cells, CD146 cells, and mesenchymal stem cells (MSCs) were differentiated into the adipogenic, osteogenic, and chondrogenic lineages. It was seen that perivascular cells, as well as CD146 cells and MSCs, cultured in stem cell medium showed greater differentiation than cells cultured in pericyte-specific medium. The expression of pericyte-specific markers CD146, neural/glial antigen 2 (NG2), platelet-derived growth factor receptor-β (PDGFR-β), myosin, and α-smooth muscle actin (α-SMA) could be found in both pericyte cultures, as well as to varying amounts in CD146 cells, MSCs, and endothelial cells. The here presented work shows that perivascular cells can adapt to their in vitro environment and cell culture conditions influence cell functionality, such as doubling rate or differentiation behavior. Pericyte-specific markers were shown to be expressed also from cells other than perivascular cells. We can further conclude that CD146⁺ perivascular cells are inhomogeneous cell population probably containing stem cell subpopulations, which are located perivascular around capillaries.
Critical size bone defects and non-union fractions are still challenging to treat. Cell-loaded bone substitutes have shown improved bone ingrowth and bone formation. However, a lack of methods for homogenously colonizing scaffolds limits the maximum volume of bone grafts. Additionally, therapy robustness is impaired by heterogeneous cell populations after graft generation. Our aim was to establish a technology for generating grafts with a size of 10.5 mm in diameter and 25 mm of height, and thus for grafts suited for treatment of critical size bone defects. Therefore, a novel tailor-made bioreactor system was developed, allowing standardized flow conditions in a porous poly(L-lactide co-caprolactone) material. Scaffolds were seeded with primary human mesenchymal stem cells derived from four different donors. In contrast to static experimental conditions, homogenous cell distributions were accomplished under dynamic culture. Additionally, culture in the bioreactor system allowed the induction of osteogenic lineage commitment after one week of culture without addition of soluble factors. This was demonstrated by quantitative analysis of calcification and gene expression markers related to osteogenic lineage. In conclusion, the novel bioreactor technology allows efficient and standardized conditions for generating bone substitutes that are suitable for the treatment of critical size defects in humans.
Bone homeostasis is maintained by osteoblasts (bone formation) and osteoclasts (bone resorption). While there have been numerous studies investigating mesenchymal stem cells and their potential to differentiate into osteoblasts as well as their interaction with different bone substitute materials, there is only limited knowledge concerning in vitro generated osteoclasts. Due to the increasing development of degradable bone-grafting materials and the need of sophisticated in vitro test methods, it is essential to gain deeper insight into the process of osteoclastogenesis and the resorption functionality of human osteoclasts. Therefore, we focused on the comparison of osteoclastogenesis and resorption activity on tissue culture polystyrene (TCPS) and bovine extracellular bone matrices (BMs). Cortical bone slices were used as two-dimensional (2D) substrates, whereas a thermally treated cancellous bone matrix was used for three-dimensional (3D) experiments. We isolated primary human monocytes and induced osteoclastogenesis by medium supplementation. Subsequently, the expression of the vitronectin receptor (αVβ3) and cathepsin K as well as the characteristic actin formation on TCPS and the two BMs were examined. The cell area of human osteoclasts was analyzed on TCPS and on BMs, whereas significantly larger osteoclasts could be detected on BMs. Additionally, we compared the diameter of the sealing zones with the measured diameter of the resorption pits on the BMs and revealed similar diameters of the sealing zones and the resorption pits. We conclude that using TCPS as culture substrate does not affect the expression of osteoclast-specific markers. The analysis of resorption activity can successfully be conducted on cortical as well as on cancellous bone matrices. For new in vitro test systems concerning bone resorption, we suggest the establishment of a 2D assay for high throughput screening of new degradable bone substitute materials with osteoclasts.
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.
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.
The establishment of adipose tissue test systems is still a major challenge in the investigation of cellular and molecular interactions responsible for the pathogenesis of inflammatory diseases involving adipose tissue. Mature adipocytes are mainly involved in these pathologies, but rarely used in vitro, due to the lack of an appropriate culture medium which inhibits dedifferentiation and maintains adipocyte functionality. In our study, we showed that Dulbecco's Modified Eagle's Medium/Ham's F-12 with 10% fetal calf serum (FCS) reported for the culture of mature adipocytes favors dedifferentiation, which was accompanied by a high glycerol release, a decreasing release of leptin, and a low expression of the adipocyte marker perilipin A, but high expression of CD73 after 21 days. Optimized media containing FCS, biotin, pantothenate, insulin, and dexamethasone decelerated the dedifferentiation process. These cells showed a lower lipolysis rate, a high level of leptin release, as well as a high expression of perilipin A. CD73-positive dedifferentiated fat cells were only found in low quantity. In this work, we showed that mature adipocytes when cultured under optimized conditions could be highly valuable for adipose tissue engineering in vitro.
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.
Sunlight has various effects on human health. Several important metabolic processes are only enabled by sunlight. But longtime sun bathing and extended outdoor activities can cause skin irritation, inflammation or even skin cancer due to high radiation dose. We developed in vitro skin models of different complexity to investigate UV-light associated skin damage. Substances and their phototoxic, sun protective or photo-sensitizing potential can be analyzed to prevent white skin cancer.
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.
In bioprinting approaches, the choice of bioink plays an important role since it must be processable with the selected printing method, but also cytocompatible and biofunctional. Therefore, a crosslinkable gelatin-based ink was modified with hydroxyapatite (HAp) particles, representing the composite buildup of natural bone. The inks’ viscosity was significantly increased by the addition of HAp, making the material processable with extrusion-based methods. The storage moduli of the formed hydrogels rose significantly, depicting improved mechanical properties. A cytocompatibility assay revealed suitable ranges for photoinitiator and HAp concentrations. As a proof of concept, the modified ink was printed together with cells, yielding stable three-dimensional constructs containing a homogeneously distributed mineralization and viable cells.
The development of in vitro adipose tissue constructs is highly desired to cope with the increased demand for substitutes to replace damaged soft tissue after high graded burns, deformities or tumor removal. To achieve clinically relevant dimensions, vascularization of soft tissue constructs becomes inevitable but still poses a challenge. Adipose-derived stem cells (ASCs) represent a promising cell source for the setup of vascularized fatty tissue constructs as they can be differentiated into adipocytes and endothelial cells in vitro and are thereby available in sufficiently high cell numbers.
This review summarizes the currently known characteristics of ASCs and achievements in adipogenic and endothelial differentiation in vitro. Further, the interdependency of adipogenesis and angiogenesis based on the crosstalk of endothelial cells, stem cells and adipocytes is addressed at the molecular level. Finally, achievements and limitations of current co-culture conditions for the construction of vascularized adipose tissue are evaluated.
Blood vessel reconstruction is still an elusive goal for the development of in vitro models as well as artificial vascular grafts. In this study, we used a novel photo curable cytocompatible polyacrylate material (PA) for freeform generation of synthetic vessels. We applied stereolithography for the fabrication of arbitrary 3D tubular structures with total dimensions in the centimeter range, 300 µm wall thickness, inner diameters of 1 to 2 mm and defined pores with a constant diameter of approximately 100 µm or 200 µm. We established a rinsing protocol to remove remaining cytotoxic substances from the photo-cured PA and applied thio-modified heparin and RGDC-peptides to functionalize the PA surface for enhanced endothelial cell adhesion. A rotating seeding procedure was introduced to ensure homogenous endothelial monolayer formation at the inner luminal tube wall. We showed that endothelial cells stayed viable and adherent and aligned along the medium flow under fluid-flow conditions comparable to native capillaries. The combined technology approach comprising of freeform additive manufacturing (AM), biomimetic design, cytocompatible materials which are applicable to AM, and biofunctionalization of AM constructs has been introduced as BioRap® technology by the authors.
Large, deep full-thickness skin wounds from high-graded burns or trauma are not able to reepithelialize sufficiently, resulting in scar formation, mobility limitations, and cosmetic deformities. In this study, in vitro-constructed tissue replacements are needed. Furthermore, such full-skin equivalents would be helpful as in vivo-like test systems for toxicity, cosmetic, and pharmaceutical testing. Up to date, no skin equivalent is available containing the underlying subcutaneous fatty tissue. In this study, we composed a full-skin equivalent and evaluated three different media for the coculture of mature adipocytes, fibroblasts, and keratinocytes. Therefore, adipocyte medium was supplemented with ascorbyl-2-phosphate and calcium chloride, which are important for successful epidermal stratification (Air medium). This medium was further supplemented with two commercially available factor combinations often used for the in vitro culture of keratinocytes (Air-HKGS and Air- KGM medium). We showed that in all media, keratinocytes differentiated successfully to build a stratified epidermal layer and expressed cytokeratin 10 and 14. Perilipin A-positive adipocytes could be found in all tissue models for up to 14 days, whereas adipocytes in the Air-HKGS and Air-KGM medium seemed to be smaller. Adipocytes in all tissue models were able to release adipocyte-specific factors, whereas the supplementation of keratinocyte-specific factors had a slightly negative effect on adipocyte functionality. The permeability of the epidermis of all models was comparable since they were able to withstand a deep penetration of cytotoxic Triton X in the same manner. Taken together, we were able to compose functional three-layered fullskin equivalents by using the Air medium.
The composition of vascularized adipose tissue is still an ongoing challenge as no culture medium is available to supply adipocytes and endothelial cells appropriately. Endothelial cell medium is typically supplemented with epidermal growth factor (EGF) as well as hydrocortisone (HC). The effect of EGF on adipocytes is discussed controversially. Some studies say it inhibits adipocyte differentiation while others reported of improved adipocyte lipogenesis. HC is known to have lipolytic activities, which might result in mature adipocyte dedifferentiation. In this study, we evaluated the influence of EGF and HC on the co-culture of endothelial cells and mature adipocytes regarding their cell morphology and functionality. We showed in mono-culture that high levels of HC promoted dedifferentiation and proliferation of mature adipocytes, whereas EGF seemed to have no negative influence. Endothelial cells kept their typical cobblestone morphology and showed a proliferation rate comparable to the control independent of EGF and HC concentration. In co-culture, HC promoted dedifferentiation of mature adipocytes, which was shown by a higher glycerol release. EGF had no negative impact on adipocyte morphology. No negative impact on endothelial cell morphology and functionality could be seen with reduced EGF and HC supplementation in co-culture with mature adipocytes. Taken together, our results demonstrate that reduced levels of HC are needed for co-culturing mature adipocytes and endothelial cells. In co-culture, EGF had no influence on mature adipocytes. Therefore, for the composition of vascularized adipose tissue constructs, the media with low levels of HC and high or low levels of EGF can be used.
In vitro cultured cells produce a complex extracellular matrix (ECM) that remains intact after decellularization. The biological complexity derived from the variety of distinct ECM molecules makes these matrices ideal candidates for biomaterials. Biomaterials with the ability to guide cell function are a topic of high interest in biomaterial development. However, these matrices lack specific addressable functional groups, which are often required for their use as a biomaterial. Due to the biological complexity of the cell-derived ECM, it is a challenge to incorporate such functional groups without affecting the integrity of the biomolecules within the ECM. The azide-alkyne cycloaddition (click reaction, Huisgen-reaction) is an efficient and specific ligation reaction that is known to be biocompatible when strained alkynes are used to avoid the use of copper (I) as a catalyst. In our work, the ubiquitous modification of a fibroblast cell-derived ECM with azides was achieved through metabolic oligosaccharide engineering by adding the azide-modified monosaccharide Ac4GalNAz (1,3,4,6 tetra-O-acetyl-N-azidoacetylgalactosamine) to the cell culture medium. The resulting azide-modified network remained intact after removing the cells by lysis and the molecular structure of the ECM proteins was unimpaired after a gentle homogenization process. The biological composition was characterized in order to show that the functionalization does not impair the complexity and integrity of the ECM. The azides within this ‘‘clickECM” could be accessed by small molecules (such as an alkyne modified fluorophore) or by surface-bound cyclooctynes to achieve a covalent coating with clickECM.
In vitro composed vascularized adipose tissue is and will continue to be in great demand e.g. for the treatment of extensive high-graded burns or the replacement of tissue after tumor removal. Up to date, the lack of adequate culture conditions, mainly a culture medium, decelerates further achievements. In our study, we evaluated the influence of epidermal growth factor (EGF) and hydrocortisone (HC), often supplemented in endothelial cell (EC) specific media, on the co-culture of adipogenic differentiated adipose derived stem cells (ASCs) and microvascular endothelial cells (mvECs). In ASCs, EGF and HC are thought to inhibit adipogenic differentiation and have lipolytic activities. Our results showed that in indirect co-culture for 14 days, adipogenic differentiated ASCs further incorporated lipids and partly gained an univacuolar morphology when kept in media with low levels of EGF and HC. In media with high EGF and HC levels, cells did not incorporate further lipids, on the contrary, cells without lipid droplets appeared. Glycerol release, to measure lipolysis, also increased with elevated amounts of EGF and HC in the culture medium. Adipogenic differentiated ASCs were able to release leptin in all setups. MvECs were functional and expressed the cell specific markers, CD31 and von Willebrand factor (vWF), independent of the EGF and HC content as long as further EC specific factors were present. Taken together, our study demonstrates that adipogenic differentiated ASCs can be successfully co-cultured with mvECs in a culture medium containing low or no amounts of EGF and HC, as long as further endothelial cell and adipocyte specific factors are available.
Though bioprinting is a forward-looking approach in bone tissue engineering, the development of bioinks which are on the one hand processable with the chosen printing technique, and on the other hand possess the relevant mechanical as well as osteoconductive features remains a challenge. In the present study, polymer solutions based on methacrylated gelatin and methacrylated hyaluronic acid modified with hydroxyapatite (HAp) particles (5 wt%) were prepared. Encapsulation of primary human adipose derived stem cells in the HAp-containing gels and culture for 28 d resulted in a storage moduli significantly increased to 126% ± 9.6% compared to the value on day 1 by the sole influence of the HAp. Additional use of osteogenic media components resulted in an increase of storage module up to 199% ± 27.8%. Similarly, the loss moduli was increased to 370% ± 122.1% under the influence of osteogenic media components and HAp. Those changes in rheological material characteristics indicate a distinct change in elastic and viscous hydrogel properties, and are attributed to extensive matrix production in the hydrogels by the encapsulated cells, what could also be proven by staining of bone matrix components like collagen I, fibronectin, alkaline phosphatase and osteopontin. When using the cell-laden polymer solutions as bioinks to build up relevant geometries, the ink showed excellent printability and the printed grid structure's integrity remained intact over a culture time of 28 d. Again, an intense matrix formation as well as upregulation of osteogenic markers by the encapsulated cells could be shown. In conclusion, we demonstrated that our HAp-containing bioinks and hydrogels on basis of methacrylated gelatin and hyaluronic acid are on the one hand highly suitable for the build up of relevant three-dimensional geometries with microextrusion bioprinting, and on the other hand exhibit a significant positive effect on bone matrix development and remodeling in the hydrogels, as indicated by rheological measurements and staining of bone components. This makes the developed composite hydrogels an excellent material for bone bioprinting approaches.
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.
To date, special interest has been paid to composite scaffolds based on polymers enriched with hydroxyapatite (HA). However, the role of HA containing different trace elements such as silicate in the structure of a polymer scaffold has not yet been fully explored. Here, we report the potential use of silicate-containing hydroxyapatite (SiHA) microparticles and microparticle aggregates in the predominant range from 2.23 to 12.40 μm in combination with polycaprolactone (PCL) as a hybrid scaffold with randomly oriented and well-aligned microfibers for regeneration of bone tissue. Chemical and mechanical properties of the developed 3D scaffolds were investigated with XRD, FTIR, EDX and tensile testing. Furthermore, the internal structure and surface morphology of the scaffolds were analyzed using synchrotron X-ray μCT and SEM. Upon culturing human mesenchymal stem cells (hMSC) on PCL-SiHA scaffolds, we found that both SiHA inclusion and microfiber orientation affected cell adhesion. The best hMSCs viability was revealed at 10 day for the PCL-SiHA scaffolds with well-aligned structure (~82%). It is expected that novel hybrid scaffolds of PCL will improve tissue ingrowth in vivo due to hydrophilic SiHA microparticles in combination with randomly oriented and well-aligned PCL microfibers, which mimic the structure of extracellular matrix of bone tissue.
Completely defined co-culture of adipogenic differentiated ASCs and microvascular endothelial cells
(2018)
Vascularized adipose tissue models are in high demand as alternatives to animal models to elucidate the mechanisms of widespread diseases, screen for new drugs or assess drug safety levels. Animal-derived sera such as fetal bovine serum (FBS), which are commonly used in these models, are associated with ethical concerns, risk of contaminations and inconsistencies of their composition and impact on cells. In this study, we developed a serum-free, defined co culture medium and implemented it in an adipocyte/endothelial cell (EC) co culture model.
Human adipose-derived stem cells were differentiated under defined conditions (diffASCs) and, like human microvascular ECs (mvECs), cultured in a defined co culture medium in mono-, indirect or direct co-culture for 14 days. The defined co-culture medium was superior when compared to mono-culture media and facilitated the functional maintenance and maturation of diffASCs including perilipin A expression, lipid accumulation, and also glycerol and leptin release. The medium also allowed mvEC maintenance, confirmed by the expression of CD31 and von Willebrand factor (vWF), and by acetylated low density lipoprotein (acLDL) uptake. Thereby, mvECs showed strong dependence on EC-specific factors. Additionally, mvECs formed vascular structures in direct co-culture with diffASCs.
The completely defined co-culture system allows for the serum-free culture of adipocyte/EC co-cultures and thereby represents a valuable and ethically acceptable tool for the culture and study of vascularized adipose tissue models.