Development of a 3D polymer reinforced calcium phosphate cement scaffold for cranial bone tissue engineering

The repair of critical-sized cranial bone defects represents an important clinical challenge. The limitations of autografts and alloplastic materials make a bone tissue engineering strategy desirable, but success depends on the development of an appropriate scaffold. Key scaffold properties include biocompatibility, osteoconductivity, sufficient strength to maintain its structure, and resorbability. Furthermore, amenability to rapid prototyping fabrication methods is desirable, as these approaches offer precise control over scaffold architecture and have the potential for customization. While calcium phosphate cements meet many of these criteria due to their composition and their injectability, which can be leveraged for scaffold fabrication via indirect casting, their mechanical properties are a major limitation. Thus, the overall goal of this work was to develop a 3D polymer reinforced calcium phosphate cement scaffold for use in cranial bone tissue engineering. Dicalcium phosphate dihydrate (DCPD) setting cements are of particular interest because of their excellent resorbability. We demonstrated for the first time that DCPD cement can be prepared from monocalcium phosphate monohydrate (MCPM)/hydroxyapatite (HA) mixtures. However, subsequent characterization revealed that MCPM/HA cements rapidly convert to HA during degradation, which is undesirable and led us to choose a more conventional formulation for scaffold fabrication. In addition, we developed a novel method for calcium phosphate cement reinforcement that is based on infiltrating a pre-set cement structure with a polymer, and then crosslinking the polymer in situ. Unlike prior methods of cement reinforcement, this method can be applied to the reinforcement of 3D scaffolds fabricated by indirect casting. Using our novel method, composites of poly(propylene fumarate) (PPF) reinforced DCPD were prepared and demonstrated as excellent candidate scaffold materials, as they had increased strength and ductility and were biocompatible in vitro. Furthermore, 3D PPF reinforced DCPD scaffolds had strengths comparable to trabecular bone. Based on these results, 3D PPF reinforced DCPD scaffolds were evaluated in vivo using a rabbit calvarial defect model. Although bone formation was not enhanced by the addition of mesenchymal stem cells, significant bone ingrowth from the surrounding tissue was observed. The results of this work provide a foundation for future research on 3D polymer reinforced calcium phosphate cement scaffolds

Shooting for the moon: Using tissue-mimetic hydrogels to gain new insight on cancer biology and screen therapeutics

Tissue engineering holds great promise for advancing cancer research and achieving the goals of the Cancer Moonshot by providing better models for basic research and testing novel therapeutics. This paper focuses on the use of hydrogel biomaterials due to their unique ability to entrap cells in three-dimensional (3D) matrix that mimics tissues and can be programmed with physical and chemical cues to recreate key aspects of tumor microenvironments. The chemistry of some commonly used hydrogel platforms is discussed, and important examples of their use in tissue engineering 3D cancer models are highlighted. Challenges and opportunities for future research are also discussed.</p

Coumarin-Based Photodegradable Hydrogel: Design, Synthesis, Gelation, and Degradation Kinetics

The design, synthesis, and characterization of a new class of coumarin-based photodegradable hydrogels are reported. Hydrogel formation was achieved rapidly and efficiently under aqueous conditions using copper-catalyzed click chemistry, which afforded excellent control over the rate of network formation. Rapid photodegradation, to the point of reverse gelation, was observed using both 365 and 405 nm light, and micrometer-scale features were eroded using two-photon irradiation at wavelengths as long as 860 nm

Synthetically Tractable Click Hydrogels for Three-Dimensional Cell Culture Formed Using Tetrazine–Norbornene Chemistry

The implementation of bio-orthogonal click chemistries is a topic of growing importance in the field of biomaterials, as it is enabling the development of increasingly complex hydrogel materials capable of providing dynamic, cell-instructive microenvironments. Here, we introduce the tetrazine–norbornene inverse electron demand Diels–Alder reaction as a new cross-linking chemistry for the formation of cell laden hydrogels. The fast reaction rate and irreversible nature of this click reaction allowed for hydrogel formation within minutes when a multifunctional PEG-tetrazine macromer was reacted with a dinorbornene peptide. In addition, the cytocompatibility of the polymerization led to high postencapsulation viability of human mesenchymal stem cells, and the specificity of the tetrazine–norbornene reaction was exploited for sequential modification of the network via thiol–ene photochemistry. These advantages, combined with the synthetic accessibility of the tetrazine molecule compared to other bio-orthogonal click reagents, make this cross-linking chemistry an interesting and powerful new tool for the development of cell-instructive hydrogels for tissue engineering applications

Photocontrolled Nanoparticles for On-Demand Release of Proteins

We describe here light-regulated swelling and degradation features of polymeric nanoparticles that are produced using an inverse microemulsion polymerization method. We demonstrate the phototriggered release characteristics of the nanoparticles by sequestering protein molecules and releasing them using light as a trigger. Furthermore, the intracellular translocation of the nanoparticles, along with its fluorescent protein payload, was achieved using a cell-penetrating peptide-based surface modification. We expect that the noncovalent encapsulation of proteins using nanoparticles and their photo triggered release using an external light would provide opportunities for achieving intracellular release of molecular therapeutics for on-demand requirements

Sequential Thiol–Ene and Tetrazine Click Reactions for the Polymerization and Functionalization of Hydrogel Microparticles

Click chemistry is a versatile tool for the synthesis and functionalization of polymeric biomaterials. Here, we describe a versatile new strategy for producing bioactive, protein-functionalized poly­(ethylene glycol) (PEG) hydrogel microparticles that is based on sequential thiol–ene and tetrazine click reactions. Briefly, tetra-functional PEG-norbornene macromer and dithiothreitol (SH) cross-linker were combined at a 0.75:1 [SH]:[norbornene] ratio, emulsified in a continuous Dextran phase, and then photopolymerized to form PEG hydrogel microparticles that varied from 8 to 30 μm in diameter, depending on the PEG concentration used. Subsequently, tetrazine-functionalized protein was conjugated to unreacted norbornene groups in the PEG microparticles. Tetrazine-mediated protein tethering to the microparticles was first demonstrated using fluorescein-labeled ovalbumin as a model protein. Subsequently, bioactive protein tethering was demonstrated using alkaline phosphatase (ALP) and glucose oxidase (GOx). Enzyme activity assays demonstrated that both ALP and GOx maintained their bioactivity and imparted tunable bioactivity to the microparticles that depended on the amount of enzyme added. ALP-functionalized microparticles were also observed to initiate calcium phosphate mineralization <i>in vitro</i> when incubated with calcium glycerophosphate. Collectively, these results show that protein-functionalized hydrogel microparticles with tunable bioactive properties can be easily synthesized using sequential click chemistry reactions. This approach has potential for future applications in tissue engineering, drug delivery, and biosensing