The effect of multi-material architecture on the ex vivo osteochondral integration of bioprinted constructs.

Extrusion bioprinted constructs for osteochondral tissue engineering were fabricated to study the effect of multi-material architecture on encapsulated human mesenchymal stem cells' tissue-specific matrix deposition and integration into an ex vivo porcine osteochondral explant model. Two extrusion fiber architecture groups with differing transition regions and degrees of bone- and cartilage-like bioink mixing were employed. The gradient fiber (G-Fib) architecture group showed an increase in chondral integration over time, 18.5 ± 0.7 kPa on Day 21 compared to 9.6 ± 1.6 kPa on Day 1 for the required peak push-out force, and the segmented fiber (S-Fib) architecture group did not, which corresponded to the increase in sulfated glycosaminoglycan deposition noted only in the G-Fib group and the staining for cellularity and tissue-specific matrix deposition at the fiber-defect boundary. Conversely, the S-Fib architecture was associated with significant mineralization over time, but the G-Fib architecture was not. Notably, both fiber groups also had similar chondral integration as a re-inserted osteochondral tissue control. While architecture did dictate differences in the cells' responses to their environment, architecture was not shown to distinguish a statistically significant difference in tissue integration via fiber push-out testing within a given time point or explant region. Use of this three-week osteochondral model demonstrates that these bioink formulations support the fabrication of cell-laden constructs that integrate into explanted tissue as capably as natural tissue and encapsulate osteochondral matrix-producing cells, and it also highlights the important role that spatial architecture plays in the engineering of multi-phasic tissue environments. STATEMENT OF SIGNIFICANCE: Here, an ex vivo model was used to interrogate fundamental questions about the effect of multi-material scaffold architectural choices on osteochondral tissue integration. Cell-encapsulating constructs resembling stratified osteochondral tissue were 3D printed with architecture consisting of either gradient transitions or segmented transitions between the bone-like and cartilage-like bioink regions. The printed constructs were assessed alongside re-inserted natural tissue plugs via mechanical tissue integration push-out testing, biochemical assays, and histology. Differences in osteochondral matrix deposition were observed based on architecture, and both printed groups demonstrated cartilage integration similar to the native tissue plug group. As 3D printing becomes commonplace within biomaterials and tissue engineering, this work illustrates critical 3D co-culture interactions and demonstrates the importance of considering architecture when interpreting the results of studies utilizing spatially complex, multi-material scaffolds.

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.

Dissecting the recruitment and self-organization of αSMA-positive fibroblasts in the foreign body response.

The foreign body response (FBR) is a clinically relevant issue that can cause malfunction of implanted medical devices by fibrotic encapsulation. Whereas inflammatory aspects of the FBR have been established, underlying fibroblast-dependent mechanisms remain unclear. We here combine multiphoton microscopy with ad hoc reporter mice expressing α-smooth muscle actin (αSMA) protein to determine the locoregional fibroblast dynamics, activation, and fibrotic encapsulation of polymeric materials. Fibroblasts invaded as individual cells and established a multicellular network, which transited to a two-compartment fibrotic response displaying an αSMA cold external capsule and a long-lasting, inner αSMA hot environment. The recruitment of fibroblasts and extent of fibrosis were only incompletely inhibited after depletion of macrophages, implicating coexistence of macrophage-dependent and macrophage-independent mediators. Furthermore, neither altering material type or porosity modulated αSMA+ cell recruitment and distribution. This identifies fibroblast activation and network formation toward a two-compartment FBR as a conserved, self-organizing process partially independent of macrophages.

Human gelatin-based composite hydrogels for osteochondral tissue engineering and their adaptation into bioinks for extrusion, inkjet, and digital light processing bioprinting.

The investigation of novel hydrogel systems allows for the study of relationships between biomaterials, cells, and other factors within osteochondral tissue engineering. Three-dimensional (3D) printing is a popular research method that can allow for further interrogation of these questions via the fabrication of 3D hydrogel environments that mimic tissue-specific, complex architectures. However, the adaptation of promising hydrogel biomaterial systems into 3D-printable bioinks remains a challenge. Here, we delineated an approach to that process. First, we characterized a novel methacryloylated gelatin composite hydrogel system and assessed how calcium phosphate and glycosaminoglycan additives upregulated bone- and cartilage-like matrix deposition and certain genetic markers of differentiation within human mesenchymal stem cells (hMSCs), such as RUNX2 and SOX9. Then, new assays were developed and utilized to study the effects of xanthan gum and nanofibrillated cellulose, which allowed for cohesive fiber deposition, reliable droplet formation, and non-fracturing digital light processing (DLP)-printed constructs within extrusion, inkjet, and DLP techniques, respectively. Finally, these bioinks were used to 3D print constructs containing viable encapsulated hMSCs over a 7 d period, where DLP printed constructs facilitated the highest observed increase in cell number over 7 d (∼2.4×). The results presented here describe the promotion of osteochondral phenotypes via these novel composite hydrogel formulations, establish their ability to bioprint viable, cell-encapsulating constructs using three different 3D printing methods on multiple bioprinters, and document how a library of modular bioink additives affected those physicochemical properties important to printability.

Bioinspired electrospun decellularized extracellular matrix scaffolds promote muscle regeneration in a rat skeletal muscle defect model.

Volumetric muscle loss is a debilitating injury that can leave patients with long-lasting or permanent structural and functional deficits. With clinical treatments failing to address these shortcomings, there is a great need for tissue-engineered therapies to promote skeletal muscle regeneration. In this study, we aim to assess the potential for electrospun decellularized skeletal muscle extracellular matrix (dECM) to promote skeletal muscle regeneration in a rat partial thickness tibialis anterior defect model. Aligned electrospun scaffolds with varying degrees of crosslinking density were implanted into the defect site and compared to an empty defect control. After 8 weeks, muscles were harvested, weighed, and cellular and morphological analyses were performed via histology and immunohistochemistry. Cell infiltration, angiogenesis, and myogenesis were observed in the defect site in both dECM groups. However, favorable mechanical properties and slower degradation kinetics resulted in greater support of tissue remodeling in the more crosslinked scaffolds and preservation of existing myofiber area in both dECM groups compared to the empty defect control. More sustained release of pro-regenerative degradation products also promoted greater myofiber formation in the defect site. This study allowed for a greater understanding of how electrospun skeletal muscle scaffolds interact with existing skeletal muscle and can inform their potential as a therapy in a wide variety of soft tissue applications.

3D printed colloidal biomaterials based on photo-reactive gelatin nanoparticles.

Biomaterials-based strategies have shown great promise for tissue regeneration. 3D printing technologies can deliver unprecedented control over architecture and properties of biomaterial constructs when combined with innovative material design strategies. Colloidal gels made of polymeric nanoparticles are attractive injectable and self-healing systems, but their use as bio-inks for extrusion-based printing is largely unexplored. Here, we report 3D printing of novel biomaterial constructs with shape memory behavior using photo-reactive gelatin nanoparticles as colloidal building blocks. These nanoparticles are stabilized with intraparticle covalent crosslinks, and also contain pendant methacryloyl groups as photo-reactive moieties. While non-covalent interactions between nanoparticles enable formation of colloidal gel inks that are printable at room temperature, UV-induced covalent interparticle crosslinks based on methacryloyl moieties significantly enhance mechanical properties of printed constructs. Additionally, the UV crosslinking modality enables remarkable control over swelling, degradation, and biomolecule release behavior of 3D constructs. Finally, by exploiting the mechanical properties of colloidal biomaterials after UV crosslinking, 3D constructs can be designed with shape memory properties, returning to their original programmed geometry upon re-hydration. Accordingly, these novel colloidal inks exhibit great potential to serve as bio-inks for 3D printing of biomaterials with shape-morphing features for a wide range of tissue engineering and regenerative medicine applications.

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.