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Identification of articular cartilage progenitor cells (ACPCs) has opened up new opportunities for cartilage repair. These cells may be used as alternatives for or in combination with mesenchymal stromal cells (MSCs) in cartilage engineering. However, their potential needs to be further investigated, since only a few studies have compared ACPCs and MSCs when cultured in hydrogels. Therefore, in this study, we compared chondrogenic differentiation of equine ACPCs and MSCs in agarose constructs as monocultures and as zonally layered co-cultures under both normoxic and hypoxic conditions. ACPCs and MSCs exhibited distinctly differential production of the cartilaginous extracellular matrix (ECM). For ACPC constructs, markedly higher glycosaminoglycan (GAG) contents were determined by histological and quantitative biochemical evaluation, both in normoxia and hypoxia. Differential GAG production was also reflected in layered co-culture constructs. For both cell types, similar staining for type II collagen was detected. However, distinctly weaker staining for undesired type I collagen was observed in the ACPC constructs. For ACPCs, only very low alkaline phosphatase (ALP) activity, a marker of terminal differentiation, was determined, in stark contrast to what was found for MSCs. This study underscores the potential of ACPCs as a promising cell source for cartilage engineering.
In this study, we evaluate hydrogels based on oxidized hyaluronic acid, cross-linked with adipic acid dihydrazide, for their suitability as bioinks for 3D bioprinting. Aldehyde containing hyaluronic acid (AHA) is synthesized and cross-linked via Schiff Base chemistry with bifunctional adipic acid dihydrazide (ADH) to form a mechanically stable hydrogel with good printability. Mechanical and rheological properties of the printed and casted hydrogels are tunable depending on the concentrations of AHA and ADH cross-linkers.
Many different biofabrication approaches as well as a variety of bioinks have been developed by researchers working in the field of tissue engineering. A main challenge for bioinks often remains the difficulty to achieve shape fidelity after printing. In order to overcome this issue, a homogeneous pre-crosslinking technique, which is universally applicable to all alginate-based materials, was developed. In this study, the Young’s Modulus after post-crosslinking of selected hydrogels, as well as the chemical characterization of alginate in terms of M/G ratio and molecular weight, were determined. With our technique it was possible to markedly enhance the printability of a 2% (w/v) alginate solution, without using a higher polymer content, fillers or support structures. 3D porous scaffolds with a height of around 5 mm were printed. Furthermore, the rheological behavior of different pre-crosslinking degrees was studied. Shear forces on cells as well as the flow profile of the bioink inside the printing nozzle during the process were estimated. A high cell viability of printed NIH/3T3 cells embedded in the novel bioink of more than 85% over a time period of two weeks could be observed.
Biointerface engineering is a wide-spread strategy to improve the healing process and subsequent tissue integration of biomaterials. Especially the integration of specific peptides is one promising strategy to promote the regenerative capacity of implants and 3D scaffolds. In vivo, these tailored interfaces are, however, first confronted with the innate immune response. Neutrophils are cells with pronounced proteolytic potential and the first recruited immune cells at the implant site; nonetheless, they have so far been underappreciated in the design of biomaterial interfaces. Herein, an in vitro approach is introduced to model and analyze the neutrophil interaction with bioactivated materials at the example of nano-bioinspired electrospun surfaces that reveals the vulnerability of a given biointerface design to the contact with neutrophils. A sacrificial, transient hydrogel coating that demonstrates optimal protection for peptide-modified surfaces and thus alleviates the immediate cleavage by neutrophil elastase is further introduced.
The Multiweek Thermal Stability of Medical-Grade Poly(ε-caprolactone) During Melt Electrowriting
(2022)
Melt electrowriting (MEW) is a high-resolution additive manufacturing technology that places unique constraints on the processing of thermally degradable polymers. With a single nozzle, MEW operates at low throughput and in this study, medical-grade poly(ε-caprolactone) (PCL) is heated for 25 d at three different temperatures (75, 85, and 95 °C), collecting daily samples. There is an initial increase in the fiber diameter and decrease in the jet speed over the first 5 d, then the MEW process remains stable for the 75 and 85 °C groups. When the collector speed is fixed to a value at least 10% above the jet speed, the diameter remains constant for 25 d at 75 °C and only increases with time for 85 and 95 °C. Fiber fusion at increased layer height is observed for 85 and 95 °C, while the surface morphology of single fibers remain similar for all temperatures. The properties of the prints are assessed with no observable changes in the degree of crystallinity or the Young's modulus, while the yield strength decreases in later phases only for 95 °C. After the initial 5-d period, the MEW processing of PCL at 75 °C is extraordinarily stable with overall fiber diameters averaging 13.5 ± 1.0 µm over the entire 25-d period.
3D bioprinting often involves application of highly concentrated polymeric bioinks to enable fabrication of stable cell-hydrogel constructs, although poor cell survival, compromised stem cell differentiation, and an inhomogeneous distribution of newly produced extracellular matrix (ECM) are frequently observed. Therefore, this study presents a bioink platform using a new versatile dual-stage crosslinking approach based on thiolated hyaluronic acid (HA-SH), which not only provides stand-alone 3D printability but also facilitates effective chondrogenic differentiation of mesenchymal stromal cells. A range of HA-SH with different molecular weights is synthesized and crosslinked with acrylated (PEG-diacryl) and allylated (PEG-diallyl) polyethylene glycol in a two-step reaction scheme. The initial Michael addition is used to achieve ink printability, followed by UV-mediated thiol–ene reaction to stabilize the printed bioink for long-term cell culture. Bioinks with high molecular weight HA-SH (>200 kDa) require comparably low polymer content to facilitate bioprinting. This leads to superior quality of cartilaginous constructs which possess a coherent ECM and a strongly increased stiffness of long-term cultured constructs. The dual-stage system may serve as an example to design platforms using two independent crosslinking reactions at one functional group, which allows adjusting printability as well as material and biological properties of bioinks.
Biofabrication, including printing technologies, has emerged as a powerful approach to the design of disease models, such as in cancer research. In breast cancer, adipose tissue has been acknowledged as an important part of the tumor microenvironment favoring tumor progression. Therefore, in this study, a 3D-printed breast cancer model for facilitating investigations into cancer cell-adipocyte interaction was developed. First, we focused on the printability of human adipose-derived stromal cell (ASC) spheroids in an extrusion-based bioprinting setup and the adipogenic differentiation within printed spheroids into adipose microtissues. The printing process was optimized in terms of spheroid viability and homogeneous spheroid distribution in a hyaluronic acid-based bioink. Adipogenic differentiation after printing was demonstrated by lipid accumulation, expression of adipogenic marker genes, and an adipogenic ECM profile. Subsequently, a breast cancer cell (MDA-MB-231) compartment was printed onto the adipose tissue constructs. After nine days of co-culture, we observed a cancer cell-induced reduction of the lipid content and a remodeling of the ECM within the adipose tissues, with increased fibronectin, collagen I and collagen VI expression. Together, our data demonstrate that 3D-printed breast cancer-adipose tissue models can recapitulate important aspects of the complex cell–cell and cell–matrix interplay within the tumor-stroma microenvironment
Hyaluronic acid (HA)-based hydrogels are very commonly applied as cell carriers for different approaches in regenerative medicine. HA itself is a well-studied biomolecule that originates from the physiological extracellular matrix (ECM) of mammalians and, due to its acidic polysaccharide structure, offers many different possibilities for suitable chemical modifications which are necessary to control, for example, network formation. Most of these chemical modifications are performed using the free acid function of the polymer and, additionally, lead to an undesirable breakdown of the biopolymer’s backbone. An alternative modification of the vicinal diol of the glucuronic acid is oxidation with sodium periodate to generate dialdehydes via a ring opening mechanism that can subsequently be further modified or crosslinked via Schiff base chemistry. Since this oxidation causes a structural destruction of the polysaccharide backbone, it was our intention to study a novel synthesis protocol frequently applied to selectively oxidize the C6 hydroxyl group of saccharides. On the basis of this TEMPO/TCC oxidation, we studied an alternative hydrogel platform based on oxidized HA crosslinked using adipic acid dihydrazide as the crosslinker.
Dual setting cements composed of an in situ forming hydrogel and a reactive mineral phase combine high compressive strength of the cement with sufficient ductility and bending strength of the polymeric network. Previous studies were focused on the modification with non-degradable hydrogels based on 2-hydroxyethyl methacrylate (HEMA). Here, we describe the synthesis of suitable triblock degradable poly(ethylene glycol)-poly(lactide) (PEG-PLLA) cross-linker to improve the resorption capacity of such composites. A study with four different formulations was established. As reference, pure hydroxyapatite (HA) cements and composites with 40 wt% HEMA in the liquid cement phase were produced. Furthermore, HEMA was modified with 10 wt% of PEG-PLLA cross-linker or a test series containing only 25% cross-linker was chosen for composites with a fully degradable polymeric phase. Hence, we developed suitable systems with increased elasticity and 5-6 times higher toughn ess values in comparison to pure inorganic cement matrix. Furthermore, conversion rate from alpha-tricalcium phosphate (alpha-TCP) to HA was still about 90% for all composite formulations, whereas crystal size decreased. Based on this material development and advancement for a dual setting system, we managed to overcome the drawback of brittleness for pure calcium phosphate cements.
In 3D bioprinting for cartilage regeneration, bioinks that support chondrogenic development are of key importance. Growth factors covalently bound in non-printable hydrogels have been shown to effectively promote chondrogenesis. However, studies that investigate the functionality of tethered growth factors within 3D printable bioinks are still lacking. Therefore, in this study, we established a dual-stage crosslinked hyaluronic acid-based bioink that enabled covalent tethering of transforming growth factor-beta 1 (TGF-β1). Bone marrow-derived mesenchymal stromal cells (MSCs) were cultured over three weeks in vitro, and chondrogenic differentiation of MSCs within bioink constructs with tethered TGF-β1 was markedly enhanced, as compared to constructs with non-covalently incorporated TGF-β1. This was substantiated with regard to early TGF-β1 signaling, chondrogenic gene expression, qualitative and quantitative ECM deposition and distribution, and resulting construct stiffness. Furthermore, it was successfully demonstrated, in a comparative analysis of cast and printed bioinks, that covalently tethered TGF-β1 maintained its functionality after 3D printing. Taken together, the presented ink composition enabled the generation of high-quality cartilaginous tissues without the need for continuous exogenous growth factor supply and, thus, bears great potential for future investigation towards cartilage regeneration. Furthermore, growth factor tethering within bioinks, potentially leading to superior tissue development, may also be explored for other biofabrication applications.