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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.
Artificial adipose tissue (AT) constructs are urgently needed to treat severe wounds, to replace removed tissue, or for the use as in vitro model to screen for potential drugs or study metabolic pathways. The clinical translation of products is mostly prevented by the absence of a vascular component that would allow a sustainable maintenance and an extension of the construct to a relevant size. With this study, we aimed to evaluate the suitability of a novel material based on bacterial cellulose (CBM) on the defined adipogenic differentiation of human adipose-derived stem cells (ASCs) and the maintenance of the received adipocytes (diffASCs) and human microvascular endothelial cells (mvECs) in mono- and coculture. A slight acceleration of adipogenic differentiation over regular tissue culture polystyrene (TCPS) was seen on CBM under defined conditions, whereas on the maintenance of the generated adipocytes, comparable effects were detected for both materials. CBM facilitated the formation of vascular like structures in monoculture of mvECs, which was not observed on TCPS. By contrast, vascular-like structures were detected in CBM and TCPS in coculture by the presence of diffASCs. Concluding, CBM represents a promising material in vascularized AT engineering with the potential to speed up and simplify the in vitro setup of engineered products.
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.
Size and function of bioartificial tissue models are still limited due to the lack of blood vessels and dynamic perfusion for nutrient supply. In this study, we evaluated the use of cytocompatible methacryl-modified gelatin for the fabrication of a hydrogel-based tube by dip-coating and subsequent photo-initiated cross-linking. The wall thickness of the tubes and the diameter were tuned by the degree of gelatin methacryl-modification and the number of dipping cycles. The dipping temperature of the gelatin solution was adjusted to achieve low viscous fluids of approximately 0.1 Pa s and was different for gelatin derivatives with different modification degrees. A versatile perfusion bioreactor for the supply of surrounding tissue models was developed, which can be adaped to several geometries and sizes of blood-vessel mimicking tubes. The manufactured bendable gelatin tubes were permeable for water and dissolved substances, like Nile Blue and serum albumin. As a proof of concept, human fibroblasts in a three-dimensional collagen tissue model were sucessfully supplied with nutrients via the central gelatin tube under dynamic conditions for 2 days. Moreover, the tubes could be used as scaffolds to build-up a functional and viable endothelial layer. Hence, the presented tools can contribute to solving current challenges in tissue engineering.
Tissue constructs of physiologically relevant scale require a vascular system to maintain cell viability. However, in vitro vascularization of engineered tissues is still a major challenge. Successful approaches are based on a feeder layer (FL) to support vascularization. Here, we investigated whether the supporting effect on the self‐assembled formation of prevascular‐like structures by microvascular endothelial cells (mvECs) originates from the FL itself or from its extracellular matrix (ECM). Therefore, we compared the influence of ECM, either derived from adipose‐derived stem cells (ASCs) or adipogenically differentiated ASCs, with the classical cell‐based FL. All cell‐derived ECM (cdECM) substrates enabled mvEC growth with high viability. Prevascular‐like structures were visualized by immunofluorescence staining of endothelial surface protein CD31 and could be observed on all cdECM and FL substrates but not on control substrate collagen I. On adipogenically differentiated ECM, longer and higher branched structures could be found compared with stem cell cdECM. An increased concentration of proangiogenic factors was found in cdECM substrates and FL approaches compared with controls. Finally, the expression of proteins associated with tube formation (E‐selectin and thrombomodulin) was confirmed. These results highlight cdECM as promising biomaterial for adipose tissue engineering by inducing the spontaneous formation of prevascular‐like structures by mvECs.
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.
Bone tissue is highly vascularized. The crosstalk of vascular and osteogenic cells is not only responsible for the formation of the strongly divergent tissue types but also for their physiological maintenance and repair. Extrusion-based bioprinting presents a promising fabrication method for bone replacement. It allows for the production of large-volume constructs, which can be tailored to individual tissue defect geometries. In this study, we used the all-gelatin-based toolbox of methacryl-modified gelatin (GM), non-modified gelatin (G) and acetylated GM (GMA) to tailor both the properties of the bioink towards improved printability, and the properties of the crosslinked hydrogel towards enhanced support of vascular network formation by simple blending. The vasculogenic behavior of human dermal microvascular endothelial cells (HDMECs) and human adipose-derived stem cells (ASCs) was evaluated in the different hydrogel formulations for 14 days. Co-culture constructs including a vascular component and an osteogenic component (i.e. a bone bioink based on GM, hydroxyapatite and ASCs) were fabricated via extrusion-based bioprinting. Bioprinted co-culture constructs exhibited functional tissue-specific cells whose interplay positively affected the formation and maintenance of vascular-like structures. The setup further enabled the deposition of bone matrix associated proteins like collagen type I, fibronectin and alkaline phosphatase within the 30-day culture.
An advanced ‘clickECM’ that can be modified by the inverse-electron demand Diels-Alder reaction
(2021)
The extracellular matrix (ECM) represents the natural environment of cells in tissue and therefore is a promising biomaterial in a variety of applications. Depending on the purpose, it is necessary to equip the ECM with specific addressable functional groups for further modification with bioactive molecules, for controllable cross-linking and/or covalent binding to surfaces. Metabolic glycoengineering (MGE) enables the specific modification of the ECM with such functional groups without affecting the native structure of the ECM. In a previous approach (S. M. Ruff, S. Keller, D. E. Wieland, V. Wittmann, G. E. M. Tovar, M. Bach, P. J. Kluger, Acta Biomater. 2017, 52, 159–170), we demonstrated the modification of an ECM with azido groups, which can be addressed by bioorthogonal copper-catalyzed azide-alkyne cycloaddition (CuAAC). Here, we demonstrate the modification of an ECM with dienophiles (terminal alkenes, cyclopropene), which can be addressed by an inverse-electron-demand Diels-Alder (IEDDA) reaction. This reaction is cell friendly as there are no cytotoxic catalysts needed. We show the equipment of the ECM with a bioactive molecule (enzyme) and prove that the functional groups do not influence cellular behavior. Thus, this new material has great potential for use as a biomaterial, which can be individually modified in a wide range of applications.
In recent years, the development and application of decellularized extracellular matrices (ECMs) for use as biomaterials have grown rapidly. These cell-derived matrices (CDMs) represent highly bioactive and biocompatible materials consisting of a complex assembly of biomolecules. Even though CDMs mimic the natural microenvironment of cells in vivo very closely, they still lack specifically addressable functional groups, which are often required to tailor a biomaterial functionality by bioconjugation. To overcome this limitation, metabolic glycoengineering has emerged as a powerful tool to equip CDMs with chemical groups such as azides. These small chemical handles are known for their ability to undergo bioorthogonal click reactions, which represent a desirable reaction type for bioconjugation. However, ECM insolubility makes its processing very challenging. In this contribution, we isolated both the unmodified ECM and azide-modified clickECM by osmotic lysis. In a first step, these matrices were concentrated to remove excessive water from the decellularization step. Next, the hydrogel-like ECM and clickECM films were mechanically fragmentized, resulting in easy to pipette suspensions with fragment sizes ranging from 7.62 to 31.29 μm (as indicated by the mean d90 and d10 values). The biomolecular composition was not impaired as proven by immunohistochemistry. The suspensions were used for the reproducible generation of surface coatings, which proved to be homogeneous in terms of ECM fragment sizes and coating thicknesses (the mean coating thickness was found to be 33.2 ± 7.3 μm). Furthermore, they were stable against fluid-mechanical abrasion in a laminar flow cell. When primary human fibroblasts were cultured on the coated substrates, an increased bioactivity was observed. By conjugating the azides within the clickECM coatings with alkyne-coupled biotin molecules, a bioconjugation platform was obtained, where the biotin–streptavidin interaction could be used. Its applicability was demonstrated by equipping the bioactive clickECM coatings with horseradish peroxidase as a model enzyme.
Gelatin is one of the most prominent biopolymers in biomedical material research and development. It is frequently used in hybrid hydrogels, which combine the advantageous properties of bio‐based and synthetic polymers. To prevent the biological component from leaching out of the hydrogel, the biomolecules can be equipped with azides. Those groups can be used to immobilize gelatin covalently in hydrogels by the highly selective and specific azide–alkyne cycloaddition. In this contribution, we functionalized gelatin with azides at its lysine residues by diazo transfer, which offers the great advantage of only minimal side‐chain extension. Approximately 84–90% of the amino groups are modified as shown by 1H‐NMR spectroscopy, 2,4,6‐trinitrobenzenesulfonic acid assay as well as Fourier‐transform infrared spectroscopy, rheology, and the determination of the isoelectric point. Furthermore, the azido‐functional gelatin is incorporated into hydrogels based on poly(ethylene glycol) diacrylate (PEG‐DA) at different concentrations (0.6, 3.0, and 5.5%). All hydrogels were classified as noncyctotoxic with significantly enhanced cell adhesion of human fibroblasts on their surfaces compared to pure PEG‐DA hydrogels. Thus, the new gelatin derivative is found to be a very promising building block for tailoring the bioactivity of materials.
Adipose tissue is related to the development and manifestation of multiple diseases, demonstrating the importance of suitable in vitro models for research purposes. In this study, adipose tissue lobuli were explanted, cultured, and used as an adipose tissue control to evaluate in vitro generated adipose tissue models. During culture, lobule exhibited a stable weight, lactate dehydrogenase, and glycerol release over 15 days. For building up in vitro adipose tissue models, we adapted the biomaterial gelatin methacryloyl (GelMA) composition and handling to homogeneously mix and bioprint human primary mature adipocytes (MA) and adipose-derived stem cells (ASCs), respectively. Accelerated cooling of the bioink turned out to be essential for the homogeneous distribution of lipid-filled MAs in the hydrogel. Last, we compared manual and bioprinted GelMA hydrogels with MA or ASCs and the explanted lobules to evaluate the impact of the printing process and rate the models concerning the physiological reference. The viability analyses demonstrated no significant difference between the groups due to additive manufacturing. The staining of intracellular lipids and perilipin A suggest that GelMA is well suited for ASCs and MA. Therefore, we successfully constructed physiological in vitro models by bioprinting MA-containing GelMA bioinks.
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.
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.
Due to its availability and minimal invasive harvesting human adipose tissue-derived extracellular matrix (dECM) is often used as a biomaterial in various tissue engineering and healthcare applications. Next to dECM, cell-derived ECM (cdECM) can be generated by and isolated from in vitro cultured cells. So far both types of ECM were investigated extensively toward their application as (bio)material in tissue engineering and healthcare. However, a systematic characterization and comparison of soft tissue dECM and cdECM is still missing. In this study, we characterized dECM from human adipose tissue, as well as cdECM from human adipose-derived stem cells, toward their molecular composition, structural characteristics, and biological purity. The dECM was found to exhibit higher levels of collagens and lower levels of sulfated glycosaminoglycans compared with cdECMs. Structural characteristics revealed an immature state of the fibrous part of cdECM samples. By the identified differences, we aim to support researchers in the selection of a suitable ECM-based biomaterial for their specific application and the interpretation of obtained results.
The extracellular matrix (ECM) naturally surrounds cells in humans, and therefore represents the ideal biomaterial for tissue engineering. ECM from different tissues exhibit different composition and physical characteristics. Thus, ECM provides not only physical support but also contains crucial biochemical signals that influence cell adhesion, morphology, proliferation and differentiation. Next to native ECM from mature tissue, ECM can also be obtained from the in vitro culture of cells. In this study, we aimed to highlight the supporting effect of cell-derived- ECM (cdECM) on adipogenic differentiation. ASCs were seeded on top of cdECM from ASCs (scdECM) or pre-adipocytes (acdECM). The impact of ECM on cellular activity was determined by LDH assay, WST I assay and BrdU assay. A supporting effect of cdECM substrates on adipogenic differentiation was determined by oil red O staining and subsequent quantification. Results revealed no effect of cdECM substrates on cellular activity. Regarding adipogenic differentiation a supporting effect of cdECM substrates was obtained compared to control. With these results, we confirm cdECM as a promising biomaterial for adipose tissue engineering.
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 models of human adipose tissue may serve as beneficial alternatives to animal models to study basic biological processes, identify new drug targets, and as soft tissue implants. With this approach, we aimed to evaluate adipose-derived stem cells (ASC) and mature adipocytes (MA) comparatively for the application in the in vitro setup of adipose tissue constructs to imitate native adipose tissue physiology. We used human primary MAs and human ASCs, differentiated for 14 days, and encapsulated them in collagen type I hydrogels to build up a three-dimensional (3D) adipose tissue model. The maintenance of the models was analyzed after seven days based on a viability staining. Further, the expression of the adipocyte specific protein perilipin A and the release of leptin and glycerol were evaluated. Gene transcription profiles of models based on dASCs and MAs were analyzed with regard to native adipose tissue. Compared to MAs, dASCs showed an immature differentiation state. Further, gene transcription of MAs suggests a behavior closer to native tissue in terms of angiogenesis, which supports MAs as preferred cell type. In contrast to native adipose tissue, genes of de novo lipogenesis and tissue remodeling were upregulated in the in vitro attempts.
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.
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.
Background aims: In vitro engineered adipose tissue is in great demand to treat lost or damaged soft tissue or to screen for new drugs, among other applications.However, today most attempts depend on the use of animal-derived sera. To pave the way for the application of adipose tissue-engineered
products in clinical trials or as reliable and robust in vitro test systems, sera should be completely excluded from the production process. In this study, we aimed to develop an in vitro adipose tissue model in the absence of sera and maintain its function long-term.
Methods: Human adipose tissue-derived stem cells were expanded and characterized in a xeno- and serum-free environment. Adipogenic differentiation was induced using a completely defined medium. Developed adipocytes were maintained in a completely defined maturation medium for additional 28 days. In addition to cell-viability and adherence, adipocyte-specific markers such as perilipin A expression of leptin release were evaluated.
Results: The defined differentiation medium enhanced cell adherence and lipid
accumulation at a significant level compared with the corresponding negative control. The defined maturation medium also significantly supported cell adherence and functional adipocyte maturation during the long-term culture period.
Conclusions: The process described here enables functional adipocyte generation and maintenance without the addition fo unknown or unimal-derived constituents, achieving an important milestone in the introduction of adipose tissue engineered products into clinical trials or in vitro screening.