Aspects of a Suspended Bioprinting System Affect Cell Viability and Support Bath Properties.

Suspended hydrogel printing is a growing method for fabricating bioprinted hydrogel constructs, largely due to how it enables nonviscous hydrogel inks to be used in extrusion printing. In this work, a previously developed poly(N-isopropylacrylamide)-based thermogelling suspended bioprinting system was examined in the context of chondrocyte-laden printing. Material factors such as ink concentration and cell concentration were found to have a significant effect on printed chondrocyte viability. In addition, the heated poloxamer support bath was able to maintain chondrocyte viability for up to 6 h of residence within the bath. The relationship between the ink and support bath was also assessed by measuring the rheological properties of the bath before and after printing. Bath storage modulus and yield stress decreased during printing as nozzle size was reduced, indicating the likelihood that dilution occurs over time through osmotic exchange with the ink. Altogether this work demonstrates the promise for printing high-resolution cell-encapsulating tissue engineering constructs, while also elucidating complex relationships between the ink and bath, which must be taken into consideration when designing suspended printing systems.

Thermogelling hydrogel charge and lower critical solution temperature influence cellular infiltration and tissue integration in an ex vivo cartilage explant model.

Thermogelling hydrogels based on poly(N-isopropyl acrylamide) (p[NiPAAm]) and crosslinked with a peptide-bearing macromer poly(glycolic acid)-poly(ethylene glycol)-poly(glycolic acid)-di(but-2-yne-1,4-dithiol) (PdBT) were fabricated to assess the role of hydrogel charge and lower critical solution temperature (LCST) over time in influencing cellular infiltration and tissue integration in an ex vivo cartilage explant model over 21 days. The p(NiPAAm)-based thermogelling polymer was synthesized to possess 0, 5, and 10 mol% dimethyl-γ-butyrolactone acrylate (DBA) to raise the LCST over time as the lactone rings hydrolyzed. Further, three peptides were designed to impart charge into the hydrogels via conjugation to the PdBT crosslinker. The positively, neutrally, and negatively charged peptides K4 (+), zwitterionic K2E2 (0), and E4 (-), respectively, were conjugated to the modular PdBT crosslinker and the hydrogels were evaluated for their thermogelation behavior in vitro before injection into the cartilage explant models. Samples were collected at days 0 and 21, and tissue integration and cellular infiltration were assessed via mechanical pushout testing and histology. Negatively charged hydrogels whose LCST changed over time (10 mol% DBA) were demonstrated to promote the greatest tissue integration when compared to the positive and neutral gels of the same thermogelling polymer formulation due to increased transport and diffusion across the hydrogel-tissue interface. Indeed, the negatively charged thermogelling polymer groups containing 5 and 10 mol% DBA demonstrated cellular infiltration and cartilage-like matrix deposition via histology. This study demonstrates the important role that material physicochemical properties play in dictating cell and tissue behavior and can inform future cartilage tissue engineering strategies.

A dual-gelling poly(N-isopropylacrylamide)-based ink and thermoreversible poloxamer support bath for high-resolution bioprinting.

Extrusion bioprinting is a popular method for fabricating tissue engineering scaffolds because of its potential to rapidly produce complex, bioactive or cell-laden scaffolds. However, due to the relatively high viscosity required to maintain shape fidelity during printing, many extrusion-based inks lack the ability to achieve precise structures at scales lower than hundreds of micrometers. In this work, we present a novel poly(N-isopropylacrylamide) (PNIPAAm)-based ink and poloxamer support bath system that produces precise, multi-layered structures on the tens of micrometers scale. The support bath maintains the structure of the ink in a hydrated, heated environment ideal for cell culture, while the ink undergoes rapid thermogelation followed by a spontaneous covalent crosslinking reaction. Through the combination of the PNIPAAm-based ink and poloxamer bath, this system was able to produce hydrogel scaffolds with uniform fibers possessing diameters tunable from 80 to 200 µm. A framework of relationships between several important printing factors involved in maintaining support and thermogelation was also elucidated. As a whole, this work demonstrates the ability to produce precise, acellular and cell-laden PNIPAAm-based scaffolds at high-resolution and contributes to the growing body of research surrounding the printability of extrusion-based bioinks with support baths.

Evaluating the physicochemical effects of conjugating peptides into thermogelling hydrogels for regenerative biomaterials applications.

Thermogelling hydrogels, such as poly(N-isopropylacrylamide) [P(NiPAAm)], provide tunable constructs leveraged in many regenerative biomaterial applications. Recently, our lab developed the crosslinker poly(glycolic acid)-poly(ethylene glycol)-poly(glycolic acid)-di(but-2-yne-1,4-dithiol), which crosslinks P(NiPAAm-co-glycidyl methacrylate) via thiol-epoxy reaction and can be functionalized with azide-terminated peptides via alkyne-azide click chemistry. This study's aim was to evaluate the impact of peptides on the physicochemical properties of the hydrogels. The physicochemical properties of the hydrogels including the lower critical solution temperature, crosslinking times, swelling, degradation, peptide release and cytocompatibility were evaluated. The gels bearing peptides increased equilibrium swelling indicating hydrophilicity of the hydrogel components. Comparable sol fractions were found for all groups, indicating that inclusion of peptides does not impact crosslinking. Moreover, the inclusion of a matrix metalloproteinase-sensitive peptide allowed elucidation of whether release of peptides from the network was driven by hydrolysis or enzymatic cleavage. The hydrophilicity of the network determined by the swelling behavior was demonstrated to be the most important factor in dictating hydrogel behavior over time. This study demonstrates the importance of characterizing the impact of additives on the physicochemical properties of hydrogels. These characteristics are key in determining design considerations for future in vitro and in vivo studies for tissue regeneration.

Bilayered, peptide-biofunctionalized hydrogels for in vivo osteochondral tissue repair.

Osteochondral defects present a unique clinical challenge due to their combination of phenotypically distinct cartilage and bone, which require specific, stratified biochemical cues for tissue regeneration. Furthermore, the articular cartilage exhibits significantly worse regeneration than bone due to its largely acellular and avascular nature, prompting significant demand for regenerative therapies. To address these clinical challenges, we have developed a bilayered, modular hydrogel system that enables the click functionalization of cartilage- and bone-specific biochemical cues to each layer. In this system, the crosslinker poly(glycolic acid)-poly(ethylene glycol)-poly(glycolic acid)-di(but-2-yne-1,4-dithiol) (PdBT) was click conjugated with either a cartilage- or bone-specific peptide sequence of interest, and then mixed with a suspension of thermoresponsive polymer and mesenchymal stem cells (MSCs) to generate tissue-specific, cell-encapsulated hydrogel layers targeting the cartilage or bone. We implanted bilayered hydrogels in rabbit femoral condyle defects and investigated the effects of tissue-specific peptide presentation and cell encapsulation on osteochondral tissue repair. After 12 weeks implantation, hydrogels with a chondrogenic peptide sequence produced higher histological measures of overall defect filling, cartilage surface regularity, glycosaminoglycan (GAG)/cell content of neocartilage and adjacent cartilage, and bone filling and bonding compared to non-chondrogenic hydrogels. Furthermore, MSC encapsulation promoted greater histological measures of overall defect filling, cartilage thickness, GAG/cell content of neocartilage, and bone filling. Our results establish the utility of this click functionalized hydrogel system for in vivo repair of the osteochondral unit. STATEMENT OF SIGNIFICANCE: Osteochondral repair requires mimicry of both cartilage- and bone-specific biochemical cues, which are highly distinct. While traditional constructs for osteochondral repair have mimicked gross compositional differences between the cartilage and bone in mineral content, mechanical properties, proteins, or cell types, few constructs have recapitulated the specific biochemical cues responsible for the differential development of cartilage and bone. In this study, click biofunctionalized, bilayered hydrogels produced stratified presentation of developmentally inspired peptide sequences for chondrogenesis and osteogenesis. This work represents, to the authors' knowledge, the first application of bioconjugation chemistry for the simultaneous repair of bone and cartilage tissue. The conjugation of tissue-specific peptide sequences successfully promoted development of both cartilage and bone tissues in vivo.

Evaluation of tissue integration of injectable, cell-laden hydrogels of cocultures of mesenchymal stem cells and articular chondrocytes with an ex vivo cartilage explant model.

This study investigated the chondrogenic activity of encapsulated mesenchymal stem cells (MSCs) and articular chondrocytes (ACs) and its impact on the mechanical properties of injectable poly(N-isopropylacrylamide)-based dual-network hydrogels loaded with poly( l -lysine) (PLL). To this effect, an ex vivo study model was employed to assess the behavior of the injected hydrogels-specifically, their surface stiffness and integration strength with the surrounding cartilage. The highest chondrogenic activity was observed from AC-encapsulated hydrogels, while the effect of PLL on MSC chondrogenesis was not apparent from biochemical analyses. Mechanical testing showed that there were no significant differences in either surface stiffness or integration strength among the different study groups. Altogether, the results suggest that the ex vivo model can allow further understanding of the relationship between biochemical changes within the hydrogel and their impact on the hydrogel's mechanical properties.

Chondrogenesis of cocultures of mesenchymal stem cells and articular chondrocytes in poly(l-lysine)-loaded hydrogels.

This work investigated the effect of poly(l-lysine) (PLL) molecular weight and concentration on chondrogenesis of cocultures of mesenchymal stem cells (MSCs) and articular chondrocytes (ACs) in PLL-loaded hydrogels. An injectable dual-network hydrogel composed of a poly(N-isopropylacrylamide)-based synthetic thermogelling macromer and a chondroitin sulfate-based biological network was leveraged as a model to deliver PLL and encapsulate the two cell populations. Incorporation of PLL into the hydrogel did not affect the hydrogel's swelling properties and degradation characteristics, nor the viability of encapsulated cells. Coculture groups demonstrated higher type II collagen expression compared to the MSC monoculture group. Expression of hypertrophic phenotype was also limited in the coculture groups. Histological analysis indicated that the ratio of MSCs to ACs was an accurate predictor of the degree of long-term chondrogenesis, while the presence of PLL was shown to have a more substantial short-term effect. Altogether, this study demonstrates that coculturing MSCs with ACs can greatly enhance the chondrogenicity of the overall cell population and offers a platform to further elucidate the short- and long-term effect of polycationic factors on the chondrogenesis of MSC and AC cocultures.

Polymeric Systems for Bioprinting.

Bioprinting is rapidly being adopted as a major method for fabricating tissue engineering constructs. Through the precise deposition of cell- and bioactive molecule-laden materials, bioprinting offers researchers a means to create biological constructs with enhanced spatial complexity that more closely mimics native tissue. The vast majority of materials used in bioprinting have been polymers due to their suitability toward resembling the cellular environment and the variety of methods available to process polymeric systems in ambient or relatively mild chemical and environmental conditions. In this review, we will discuss in detail the wide variety of natural and synthetic polymers that have been employed as inks in bioprinting. We will review recent bioprinting innovations, such as increasing architectural complexity and cell viability in heterogeneous tissue constructs, which allow for the investigation of biological questions that could not be addressed before. We will also survey nascent fields of study that promise to further advance the development of novel biofabrication technologies in the field, such as 4D bioprinting and the inclusion of nanomaterials. To conclude, we will examine some of the necessary steps that must take place to bring this technology to commercial markets and facilitate its use in clinical therapies.