Influence of Polymeric Microparticle Size and Loading Concentration on 3D Printing Accuracy and Degradation Behavior of Composite Scaffolds.

Successful employment of 3D printing for delivery of therapeutic biomolecules requires protection of their bioactivity on exposure to potentially inactivating conditions. Although intermediary encapsulation of the biomolecules in polymeric particulate delivery vehicles is a promising strategy for this objective, the inclusion of such particles in 3D printing formulations may critically impact the accuracy or precision of 3D printed scaffolds relative to their intended designed architectures, as well as the degradation behavior of both the scaffolds and the included particles. The present work aimed to elucidate the effect of poly(d,l-lactic-co-glycolic acid) particle size and loading concentration on material accuracy, machine precision, and degradation of 3D printed poly(ɛ-caprolactone)-based scaffolds. Using a main effects analysis, the sizes and loading concentrations of particle delivery vehicles investigated were found to have neither a beneficial nor disadvantageous influence on the metrics of printing quality such as material accuracy and machine precision. Meanwhile, particle loading concentration was determined to influence degradation rate, whereas printing temperature affected the trends in composite weight-average molecular weight. Neither of the two particle-related parameters (concentration nor diameter) was found to exhibit a significant effect on intra-fiber nor inter-fiber porosity. These findings evidence the capacity for controlled loading of particulate delivery vehicles in 3D printed scaffolds while preserving construct accuracy and precision, and with predictable dictation of composite degradation behavior for potential controlled release of encapsulated biomolecules.

Development of photoreactive demineralized bone matrix 3D printing colloidal inks for bone tissue engineering.

Demineralized bone matrix (DBM) has been widely used clinically for dental, craniofacial and skeletal bone repair, as an osteoinductive and osteoconductive material. 3D printing (3DP) enables the creation of bone tissue engineering scaffolds with complex geometries and porosity. Photoreactive methacryloylated gelatin nanoparticles (GNP-MAs) 3DP inks have been developed, which display gel-like behavior for high print fidelity and are capable of post-printing photocrosslinking for control of scaffold swelling and degradation. Here, novel DBM nanoparticles (DBM-NPs, ∼400 nm) were fabricated and characterized prior to incorporation in 3DP inks. The objectives of this study were to determine how these DBM-NPs would influence the printability of composite colloidal 3DP inks, assess the impact of ultraviolet (UV) crosslinking on 3DP scaffold swelling and degradation and evaluate the osteogenic potential of DBM-NP-containing composite colloidal scaffolds. The addition of methacryloylated DBM-NPs (DBM-NP-MAs) to composite colloidal inks (100:0, 95:5 and 75:25 GNP-MA:DBM-NP-MA) did not significantly impact the rheological properties associated with printability, such as viscosity and shear recovery or photocrosslinking. UV crosslinking with a UV dosage of 3 J/cm2 directly impacted the rate of 3DP scaffold swelling for all GNP-MA:DBM-NP-MA ratios with an ∼40% greater increase in scaffold area and pore area in uncrosslinked versus photocrosslinked scaffolds over 21 days in phosphate-buffered saline (PBS). Likewise, degradation (hydrolytic and enzymatic) over 21 days for all DBM-NP-MA content groups was significantly decreased, ∼45% less in PBS and collagenase-containing PBS, in UV-crosslinked versus uncrosslinked groups. The incorporation of DBM-NP-MAs into scaffolds decreased mass loss compared to GNP-MA-only scaffolds during collagenase degradation. An in vitro osteogenic study with bone marrow-derived mesenchymal stem cells demonstrated osteoconductive properties of 3DP scaffolds for the DBM-NP-MA contents examined. The creation of photoreactive DBM-NP-MAs and their application in 3DP provide a platform for the development of ECM-derived colloidal materials and tailored control of biochemical cue presentation with broad tissue engineering applications.

Hydrogel viscoelasticity modulates migration and fusion of mesenchymal stem cell spheroids.

Multicellular spheroids made of stem cells can act as building blocks that fuse to capture complex aspects of native in vivo environments, but the effect of hydrogel viscoelasticity on cell migration from spheroids and their fusion remains largely unknown. Here, we investigated the effect of viscoelasticity on migration and fusion behavior of mesenchymal stem cell (MSC) spheroids using hydrogels with a similar elasticity but different stress relaxation profiles. Fast relaxing (FR) matrices were found to be significantly more permissive to cell migration and consequent fusion of MSC spheroids. Mechanistically, inhibition of ROCK and Rac1 pathways prevented cell migration. Moreover, the combination of biophysical and biochemical cues provided by fast relaxing hydrogels and platelet-derived growth factor (PDGF) supplementation, respectively, resulted in a synergistic enhancement of migration and fusion. Overall, these findings emphasize the important role of matrix viscoelasticity in tissue engineering and regenerative medicine strategies based on spheroids.

Self-Healing Injectable Hydrogels for Tissue Regeneration.

Biomaterials with the ability to self-heal and recover their structural integrity offer many advantages for applications in biomedicine. The past decade has witnessed the rapid emergence of a new class of self-healing biomaterials commonly termed injectable, or printable in the context of 3D printing. These self-healing injectable biomaterials, mostly hydrogels and other soft condensed matter based on reversible chemistry, are able to temporarily fluidize under shear stress and subsequently recover their original mechanical properties. Self-healing injectable hydrogels offer distinct advantages compared to traditional biomaterials. Most notably, they can be administered in a locally targeted and minimally invasive manner through a narrow syringe without the need for invasive surgery. Their moldability allows for a patient-specific intervention and shows great prospects for personalized medicine. Injected hydrogels can facilitate tissue regeneration in multiple ways owing to their viscoelastic and diffusive nature, ranging from simple mechanical support, spatiotemporally controlled delivery of cells or therapeutics, to local recruitment and modulation of host cells to promote tissue regeneration. Consequently, self-healing injectable hydrogels have been at the forefront of many cutting-edge tissue regeneration strategies. This study provides a critical review of the current state of self-healing injectable hydrogels for tissue regeneration. As key challenges toward further maturation of this exciting research field, we identify (i) the trade-off between the self-healing and injectability of hydrogels vs their physical stability, (ii) the lack of consensus on rheological characterization and quantitative benchmarks for self-healing injectable hydrogels, particularly regarding the capillary flow in syringes, and (iii) practical limitations regarding translation toward therapeutically effective formulations for regeneration of specific tissues. Hence, here we (i) review chemical and physical design strategies for self-healing injectable hydrogels, (ii) provide a practical guide for their rheological analysis, and (iii) showcase their applicability for regeneration of various tissues and 3D printing of complex tissues and organoids.

Viscoelastic Biomaterials for Tissue Regeneration.

The extracellular matrix (ECM) mechanical properties regulate key cellular processes in tissue development and regeneration. The majority of scientific investigation has focused on ECM elasticity as the primary mechanical regulator of cell and tissue behavior. However, all living tissues are viscoelastic, exhibiting both solid- and liquid-like mechanical behavior. Despite increasing evidence regarding the role of ECM viscoelasticity in directing cellular behavior, this aspect is still largely overlooked in the design of biomaterials for tissue regeneration. Recently, with the emergence of various bottom-up material design strategies, new approaches can deliver unprecedented control over biomaterial properties at multiple length scales, thus enabling the design of viscoelastic biomaterials that mimic various aspects of the native tissue ECM microenvironment. This review describes key considerations for the design of viscoelastic biomaterials for tissue regeneration. We provide an overview of the role of matrix viscoelasticity in directing cell behavior toward regenerative outcomes, highlight recent strategies utilizing viscoelastic hydrogels for regenerative therapies, and outline remaining challenges, potential solutions, and emerging applications for viscoelastic biomaterials in tissue engineering and regenerative medicine. Impact statement All living tissues are viscoelastic. As we design viscoelastic biomaterials for tissue engineering and regenerative medicine, we must understand the effect of matrix viscoelasticity on in vitro cell behavior and in vivo regenerative outcomes. Engineering the next generation of biomaterials with tunable viscoelasticity to direct cell and tissue behavior will contribute to the development of in vitro tissue models and in vivo regenerative therapies to address unmet clinical needs.

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.

Engineering the Dynamics of Cell Adhesion Cues in Supramolecular Hydrogels for Facile Control over Cell Encapsulation and Behavior.

The extracellular matrix (ECM) forms through hierarchical assembly of small and larger polymeric molecules into a transient, hydrogel-like fibrous network that provides mechanical support and biochemical cues to cells. Synthetic, fibrous supramolecular networks formed via non-covalent assembly of various molecules are therefore potential candidates as synthetic mimics of the natural ECM, provided that functionalization with biochemical cues is effective. Here, combinations of slow and fast exchanging molecules that self-assemble into supramolecular fibers are employed to form transient hydrogel networks with tunable dynamic behavior. Obtained results prove that modulating the ratio between these molecules dictates the extent of dynamic behavior of the hydrogels at both the molecular and the network level, which is proposed to enable effective incorporation of cell-adhesive functionalities in these materials. Excitingly, the dynamic nature of the supramolecular components in this system can be conveniently employed to formulate multicomponent supramolecular hydrogels for easy culturing and encapsulation of single cells, spheroids, and organoids. Importantly, these findings highlight the significance of molecular design and exchange dynamics for the application of supramolecular hydrogels as synthetic ECM mimics.

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.

Effect of 3D Printing Temperature on Bioactivity of Bone Morphogenetic Protein-2 Released from Polymeric Constructs.

Growth factors such as bone morphogenetic protein-2 (BMP-2) are potent tools for tissue engineering. Three-dimensional (3D) printing offers a potential strategy for delivery of BMP-2 from polymeric constructs; however, these biomolecules are sensitive to inactivation by the elevated temperatures commonly employed during extrusion-based 3D printing. Therefore, we aimed to correlate printing temperature to the bioactivity of BMP-2 released from 3D printed constructs composed of a model polymer, poly(propylene fumarate). Following encapsulation of BMP-2 in poly(DL-lactic-co-glycolic acid) particles, growth factor-loaded fibers were fabricated at three different printing temperatures. Resulting constructs underwent 28 days of aqueous degradation for collection of released BMP-2. Supernatants were then assayed for the presence of bioactive BMP-2 using a cellular assay for alkaline phosphatase activity. Cumulative release profiles indicated that BMP-2 released from constructs that were 3D printed at physiologic and intermediate temperatures exhibited comparable total amounts of bioactive BMP-2 release as those encapsulated in non-printed particulate delivery vehicles. Meanwhile, the elevated printing temperature of 90 °C resulted in a decreased amount of total bioactive BMP-2 release from the fibers. These findings elucidate the effects of elevated printing temperatures on BMP-2 bioactivity during extrusion-based 3D printing, and enlighten polymeric material selection for 3D printing with growth factors.

Machine Learning-Guided Three-Dimensional Printing of Tissue Engineering Scaffolds.

Various material compositions have been successfully used in 3D printing with promising applications as scaffolds in tissue engineering. However, identifying suitable printing conditions for new materials requires extensive experimentation in a time and resource-demanding process. This study investigates the use of Machine Learning (ML) for distinguishing between printing configurations that are likely to result in low-quality prints and printing configurations that are more promising as a first step toward the development of a recommendation system for identifying suitable printing conditions. The ML-based framework takes as input the printing conditions regarding the material composition and the printing parameters and predicts the quality of the resulting print as either "low" or "high." We investigate two ML-based approaches: a direct classification-based approach that trains a classifier to distinguish between low- and high-quality prints and an indirect approach that uses a regression ML model that approximates the values of a printing quality metric. Both modes are built upon Random Forests. We trained and evaluated the models on a dataset that was generated in a previous study, which investigated fabrication of porous polymer scaffolds by means of extrusion-based 3D printing with a full-factorial design. Our results show that both models were able to correctly label the majority of the tested configurations while a simpler linear ML model was not effective. Additionally, our analysis showed that a full factorial design for data collection can lead to redundancies in the data, in the context of ML, and we propose a more efficient data collection strategy.

Fiber engraving for bioink bioprinting within 3D printed tissue engineering scaffolds.

In this work, we describe a new 3D printing methodology for the fabrication of multimaterial scaffolds involving the combination of thermoplastic extrusion and low temperature extrusion of bioinks. A fiber engraving technique was used to create a groove on the surface of a thermoplastic printed fiber using a commercial 3D printer and a low viscosity bioink was deposited into this groove. In contrast to traditional extrusion bioinks that rely on increased viscosity to prevent lateral spreading, this groove creates a defined space for bioink deposition. By physically constraining bioink spreading, a broader range of viscosities can be used. As proof-of-concept, we fabricated and characterized a multimaterial scaffold containing poly(ε-caprolactone) (PCL) as the thermoplastic polymer and a gelatin-based bioink. A 7.5 w/v% gelatin methacryloyl (GelMA) bioink loaded with either 5 w/v% poly(lactic-co-glycolic acid) (PLGA) microparticles containing fluorescent albumin or mouse fibroblasts (1 × 106 cell/mL) was printed at 24 °C. The structure of the composite scaffolds had no significant decrease in porosity or mechanical properties as compared to the PCL control scaffolds, demonstrating the engraving technique did not significantly compromise the mechanical or structural integrity of the scaffold. The encapsulated PLGA microparticles were homogeneously distributed in the GelMA and remained in the scaffolds after incubation in PBS for 24 h at 37 °C. In addition, the viability of the fibroblasts encapsulated in the GelMA bioink and printed in the grooves of the PCL scaffolds was confirmed after 24 h of incubation. Overall, this work provides a new methodology for the preparation of 3D printed scaffolds containing a robust thermoplastic structure in combination with low viscosity bioinks.

Hybrid particles derived from alendronate and bioactive glass for treatment of osteoporotic bone defects.

Osteoporosis is the most widespread metabolic bone disease which represents a major public health burden. Consequently, novel biomaterials with a strong capacity to regenerate osteoporotic bone defects are urgently required. In view of the anti-osteoporotic and osteopromotive efficacy of alendronate and 45S5 bioactive glass, respectively, we investigated the feasibility to synthesize novel hybrid particles by exploiting the strong interactions between these two compounds. Herein, we demonstrate the facile preparation of a novel class of hybrid particles of tunable morphology, chemical composition and structure. These hybrid particles (i) release alendronate and various inorganic elements (Ca, Na, Si, and P) in a controlled manner, (ii) exhibit a strong anti-osteoclastic effect in vitro, and (iii) stimulate regeneration of osteoporotic bone in vivo. Consequently, this novel class of hybrid biomaterials opens up new avenues of research on the design of bone substitutes with specific activity to facilitate regeneration of bone defects in osteoporotic patients.

Fiber-reinforced colloidal gels as injectable and moldable biomaterials for regenerative medicine.

Hydrogels are the preferred material choice for various strategies in regenerative medicine. Nevertheless, due to their high water content and soft nature, these materials are often mechanically weak, which limits their applicability. This study demonstrates mechanical reinforcement of colloidal gels at microscale using discrete polyester fibers, as confirmed by rheological, compression and nanoindentation tests. This reinforcement strategy results in injectable and moldable colloidal gels with improved mechanical performance. The fully organic gels presented here are cytocompatible and can maintain their mechanical integrity under physiological conditions. Consequently, these gels exhibit a strong potential for applications in tissue engineering and regenerative medicine.

Development of a PCL-silica nanoparticles composite membrane for Guided Bone Regeneration.

The pivotal step in Guided Bone Regeneration (GBR) therapy is the insertion of a membrane for support and barrier functions. Here, we studied the effect of the addition of silica nanoparticles (Si-NPs) in electrospun poly(ε-caprolactone) (PCL) membranes to improve the mechanical and osteoconductive properties of the membranes. To this end, Si-NPs were firstly synthesized and then suspended in PCL solutions containing a polar solvent (2,2,2-trifluroethanol) and water with the addition of an anionic surfactant. Nanocomposite membranes were fabricated from the solutions through an electrospinning technique. Morphology, structure and chemical composition, and tensile properties of the membranes were analyzed. Membrane stability was determined by visual examination of the membranes after immersion in phosphate buffered saline. The effect of the materials on osteoblastic differentiation was evaluated by in vitro culture of the membranes with MC3T3-E1 osteoblastic cells. The results indicated that Si-NPs were successfully incorporated in the interior of the PCL electrospun fibers during the electrospinning process. Tensile modulus was significantly increased for composition S50 and tensile strength significantly increased for compositions S25 and S50. Membranes containing Si-NPs have shown to be cytocompatible. The results obtained demonstrate that the Si-NPs were homogeneously incorporated in the electrospun fibers, resulting in an improvement of the tensile properties of the prepared materials.

Nanostructured raspberry-like gelatin microspheres for local delivery of multiple biomolecules.

Multicompartment particles, which are particles composed of smaller building units, have gained considerable interest during the past decade to facilitate simultaneous and differential delivery of several biomolecules in various applications. Supercritical carbon dioxide (CO2) processing is an industrial technology widely used for large-scale synthesis and processing of materials. However, the application of this technology for production of multicompartment particles from colloidal particles has not yet been explored. Here, we report the formation of raspberry-like gelatin (RLG) microparticles composed of gelatin nanoparticles as colloidal building blocks through supercritical CO2 processing. We show that these RLG microparticles exhibit a high stability upon dispersion in aqueous media without requiring chemical cross-linking. We further demonstrate that these microparticles are cytocompatible and facilitate differential release of two different model compounds. The strategy presented here can be utilized as a cost-effective route for production of various types of multicompartment particles using colloidal particles with suitable interparticle interactions. STATEMENT OF SIGNIFICANCE: Multicompartment particles have gained considerable interest during the past decade to facilitate simultaneous and differential delivery of multiple biomolecules in various biomedical applications. Nevertheless, common methods employed for the production of such particles are often complex and only offer small-scale production. Here, we report the formation of raspberry-like gelatin (RLG) microparticles composed of gelatin nanoparticles as colloidal building blocks through supercritical CO2 processing. We show that these microparticles are cytocompatible and facilitate differential release of two model compounds with different molecular sizes, promising successful applications in various biomedical areas. Summarizing, this paper presents a novel strategy that can be utilized as a cost-effective route for production of various types of multicompartment particles using a wide range of colloidal building blocks.

Highly Elastic and Self-Healing Composite Colloidal Gels.

Composite colloidal gels are formed by the pH-induced electrostatic assembly of silica and gelatin nanoparticles. These injectable and moldable colloidal gels are able to withstand substantial compressive and tensile loads, and exhibit a remarkable self-healing efficiency. This study provides new, critical insight into the structural and mechanical properties of composite colloidal gels and opens up new avenues for practical application of colloidal gels.

Exploiting Bisphosphonate-Bioactive-Glass Interactions for the Development of Self-Healing and Bioactive Composite Hydrogels.

Hydrogels are widely recognized as promising candidates for various biomedical applications, such as tissue engineering. Recently, extensive research efforts have been devoted to the improvement of the biological and mechanical performance of hydrogel systems by incorporation of functional groups and/or inorganic particles in their composition. Bisphosphonates are a class of drugs, commonly used for treatment of osteoporosis, which exhibit a strong binding affinity for hydroxyapatite. In this study, the binding affinity of a bisphosphonate-functionalized polymer, hyaluronan, toward a bioactive glass (i.e., 45S5 Bioglass) is evaluated using force-distance measurements with atomic force microscopy. The strong interaction between bisphosphonate and bioactive glass is then exploited to develop organic-inorganic composite hydrogels and the viscoelastic and self-healing ability of these materials are investigated. Finally, the stability and mineralization behavior of these hydrogels are evaluated in simulated body fluid. Following this approach, injectable, bioactive and self-healing organic-inorganic composite hydrogels are produced, which mineralize abundantly and rapidly in simulated body fluid. These properties render these composite gels suitable for applications in bone-tissue engineering.

Electrophoretic Deposition of Chitosan Coatings Modified with Gelatin Nanospheres To Tune the Release of Antibiotics.

Orthopedic and dental implants are increasingly used in the medical field in view of their high success rates. Implant-associated infections, however, still occur and are difficult to treat. To combat these infections, the application of an active coating to the implant surface is advocated as an effective strategy to facilitate sustained release of antibacterial drugs from implant surfaces. Control over this release is, however, still a major challenge. To overcome this problem, we deposited composite coatings composed of a chitosan matrix containing gelatin nanospheres loaded with antibiotics onto stainless steel plates by means of the electrophoretic deposition technique. The gelatin nanospheres were distributed homogeneously throughout the coatings. The surface roughness and wettability of the coatings could be tuned by a simple adjustment of the weight ratio between the gelatin nanospheres and chitosan. Vancomycin and moxifloxacin were released in sustained and burst-type manners, respectively, while the coatings were highly cytocompatible. The antibacterial efficacy of the coatings containing different amounts of antibiotics was tested using a zone of inhibition test against Staphylococcus aureus, which showed that the coatings containing moxifloxacin exhibited an obvious inhibition zone. The coatings containing a high amount of vancomycin were able to kill bacteria in direct contact with the implant surface. These results suggest that the antibacterial capacity of metallic implants can be tuned by orthogonal control over the release of (multiple) antibiotics from electrophoretically deposited composite coatings, which offers a new strategy to prevent orthopedic implant-associated infections.

Gelatin Nanoparticles with Enhanced Affinity for Calcium Phosphate.

Gelatin nanoparticles can be tuned with respect to their drug loading efficiency, degradation rate, and release kinetics, which renders these drug carriers highly suitable for a wide variety of biomedical applications. The ease of functionalization has rendered gelatin an interesting candidate material to introduce specific motifs for selective targeting to specific organs, but gelatin nanoparticles have not yet been modified to increase their affinity to mineralized tissue. By means of conjugating bone-targeting alendronate to biocompatible gelatin nanoparticles, a simple method is developed for the preparation of gelatin nanoparticles which exhibit strong affinity to mineralized surfaces. It has been shown that the degree of alendronate functionalization can be tuned by controlling the glutaraldehyde crosslinking density, the molar ratio between alendronate and glutaraldehyde, as well as the pH of the conjugation reaction. Moreover, it has been shown that the affinity of gelatin nanoparticles to calcium phosphate increases considerably upon functionalization with alendronate. In summary, gelatin nanoparticles have been developed, which exhibit great potential for use in bone-specific drug delivery and regenerative medicine.