A novel calcium aluminate-melatonin scaffold enhances bone regeneration within a calvarial defect.

Over 500,000 bone graft or bio-implant procedures are performed annually in the United States. It has been reported that osseous autograft procurement may result in donor site complications and bio-implant allografts have been associated with disease transmission. Ceramic scaffolds are only osteoconductive, limiting their clinical use. The objective of this study was to create a bone filler substitute with regenerating properties similar to natural bone. Therefore, melatonin and platelet-rich plasma (PRP) were utilized for their known osteoinductive properties. It was hypothesized that melatonin and/or PRP would enhance the osteoinductive and osteoconductive properties of calcium aluminate (CA) scaffolds to promote bone regeneration in a model of calvarial defects. The biocompatibility of CA and CA-Mel scaffolds was tested in vitro and in vivo. Data show that CA-Mel scaffolds, in comparison with CA scaffolds, enhanced the adhesion, viability, and proliferation of normal human osteoblasts cells but not that of NIH3T3 fibroblasts. Data also showed that human adult mesenchymal stem cells grown on CA or CA-Mel scaffolds showed a time-dependent induction into osteoblasts over 14days revealed through scanning electron microscopy and by alkaline phosphatase analyses. Implantation of CA-Mel scaffolds into critical size calvarial defects in female, ovariectomized rats showed that the CA-Mel scaffolds were biocompatible, allowed for tissue infiltration, and showed evidence of scaffold biodegradation by 3 and 6months. Bone regeneration, assessed using fluorochrome labeling at 3 and 6months, was greatest in animals implanted with the CA-Mel scaffold. Overall, results from this study show that CA-Mel scaffolds were osteoconductive and osteoinductive.

Chemically-Induced Cross-Linking of Peptidic Fibrils for Scaffolding Polymeric Particles and Macrophages.

EAK16-II (EAK) is a self-assembling peptide (SAP) that forms ß-sheets and ß-fibrils through ionic-complementary interactions at physiological ionic strengths. The soft materials can be injected in vivo, creating depots of drugs and cells for rendering pharmacological and biological actions. The scope of the applications of EAK is sought to extend to tissues through which the flow of extracellular fluid tends to be limited. In such anatomical locales the rate and extent of the fibrilization are limited insofar as drug delivery and cellular scaffolding would be impeded. A method is generated utilizing a carbodiimide cross-linker by which EAK fibrils are pre-assembled yet remain injectable soft materials. It is hypothesized that the resulting de novo covalent linkages enhance the stacking of the ß-sheet bilayers, thereby increasing the lengths of the fibrils and the extent of their cross-linking, as evidenced in Diffuse Reflectance Infrared Fourier Transform (DRIFT) spectroscopy, scanning electron microscopy, and atomic force microscopy analyses. The cross-linked EAK (clEAK) retains polymeric microspheres with an average diameter of 1 µm. Macrophages admixed with clEAK remain viable and do not produce the inflammatory mediator interleukin-1ß. These results indicate that clEAK should be investigated further as a platform for delivering particles and cells in vivo.

Dynamic loading of human engineered heart tissue enhances contractile function and drives a desmosome-linked disease phenotype.

The role that mechanical forces play in shaping the structure and function of the heart is critical to understanding heart formation and the etiology of disease but is challenging to study in patients. Engineered heart tissues (EHTs) incorporating human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have the potential to provide insight into these adaptive and maladaptive changes. However, most EHT systems cannot model both preload (stretch during chamber filling) and afterload (pressure the heart must work against to eject blood). Here, we have developed a new dynamic EHT (dyn-EHT) model that enables us to tune preload and have unconstrained contractile shortening of >10%. To do this, three-dimensional (3D) EHTs were integrated with an elastic polydimethylsiloxane strip providing mechanical preload and afterload in addition to enabling contractile force measurements based on strip bending. Our results demonstrated that dynamic loading improves the function of wild-type EHTs on the basis of the magnitude of the applied force, leading to improved alignment, conduction velocity, and contractility. For disease modeling, we used hiPSC-derived cardiomyocytes from a patient with arrhythmogenic cardiomyopathy due to mutations in the desmoplakin gene. We demonstrated that manifestation of this desmosome-linked disease state required dyn-EHT conditioning and that it could not be induced using 2D or standard 3D EHT approaches. Thus, a dynamic loading strategy is necessary to provoke the disease phenotype of diastolic lengthening, reduction of desmosome counts, and reduced contractility, which are related to primary end points of clinical disease, such as chamber thinning and reduced cardiac output.

FRESH 3D Bioprinting a Full-Size Model of the Human Heart.

Recent advances in embedded three-dimensional (3D) bioprinting have expanded the design space for fabricating geometrically complex tissue scaffolds using hydrogels with mechanical properties comparable to native tissues and organs in the human body. The advantage of approaches such as Freeform Reversible Embedding of Suspended Hydrogels (FRESH) printing is the ability to embed soft biomaterials in a thermoreversible support bath at sizes ranging from a few millimeters to centimeters. In this study, we were able to expand this printable size range by FRESH bioprinting a full-size model of an adult human heart from patient-derived magnetic resonance imaging (MRI) data sets. We used alginate as the printing biomaterial to mimic the elastic modulus of cardiac tissue. In addition to achieving high print fidelity on a low-cost printer platform, FRESH-printed alginate proved to create mechanically tunable and suturable models. This demonstrates that large-scale 3D bioprinting of soft hydrogels is possible using FRESH and that cardiac tissue constructs can be produced with potential future applications in surgical training and planning.

De novo lung biofabrication: clinical need, construction methods, and design strategy.

Chronic lung disease is the 4th leading cause of death in the United States. Due to a shortage of donor lungs, alternative approaches to support failing, native lungs have been attempted, including mechanical ventilation and various forms of artificial lungs. However, each of these support methods causes significant complications when used for longer than a few days and are thus not capable of long-term support. For artificial lungs, complications arise due to interactions between the artificial materials of the device and the blood of the recipient. A potential new approach is the fabrication of lungs from biological materials, such that the gas exchange membranes provide a more biomimetic blood-contacting interface. Recent advancements with three-dimensional, soft-tissue biofabrication methods and the engineering of thin, basement membranes demonstrate the potential of fabricating a lung scaffold from extracellular matrix materials. This scaffold could then be seeded with endothelial and epithelial cells, matured within a bioreactor, and transplanted. In theory, this fully biological lung could provide improved, long-term biocompatibility relative to artificial lungs, but significant work is needed to perfect the organ design and construction methods. Like artificial lungs, biofabricated lungs do not need to follow the shape and structure of a native lung, allowing for simpler manufacture. However, various functional requirements must still be met, including stable, efficient gas exchange for a period of years. Design decisions depend on the disease state, how the organ is implanted, and the latest biofabrication methods available in a rapidly evolving field.

Natural Biomaterials for Corneal Tissue Engineering, Repair, and Regeneration.

Corneal blindness is a major cause of vision loss, estimated to affect over 10 million people worldwide. Once impaired through clouding or shape change, the best treatment option for restoring vision is corneal transplantation using full or partial thickness cadaveric grafts. However, donor corneas are globally limited and face rejection and graft failure, similar to other transplanted organs. Thus, there is a need for viable alternatives to donor corneas in order to increase supply, reduce rejection, and to minimize variability in tissue quality. To address this, researchers have developed new materials and strategies to tissue engineer full or partial thickness cornea grafts in order to repair, regenerate, or replace the diseased cornea. This progress report first reviews the anatomy and physiology of the cornea to frame the biological requirements and discuss the injuries and diseases that necessitate the need fortransplantation, as well as the requirements for a suitable donor tissue alternative. This is followed by recent progress using naturally derived biomaterials including silk, collagen, amniotic membranes, and decellularized corneas. Finally, remaining challenges in the field as they relate to the biomaterials discussed are identified, and the future research directions that should result in further advances in restoring corneal vision are highlighted.

Engineered Basement Membranes for Regenerating the Corneal Endothelium.

Basement membranes are protein-rich extracellular matrices (ECM) that are essential for epithelial and endothelial tissue structure and function. Aging and disease cause changes in the physical properties and ECM composition of basement membranes, which has spurred research to develop methods to repair and/or regenerate these tissues. An area of critical clinical need is the cornea, where failure of the endothelium leads to stromal edema and vision loss. Here, an engineered basement membrane (EBM) is developed that consists of a dense layer of collagen IV and/or laminin ≈5-10 nm thick, created using surface-initiated assembly, conformally attached to a collagen I film. These EBMs are used to engineer a corneal endothelium (CE) that mimics the structure of Descemet's membrane with a thin stromal layer, toward use as a graft for lamellar keratoplasty. Results show that bovine and human CE cells form confluent monolayers on the EBM, express ZO-1 at the cell-cell borders, and achieve a density of ≈1600 cells mm-2 for 28 and 14 d, respectively. These results demonstrate that the technique is capable of fabricating EBMs with structural and compositional properties that mimic native basement membranes and that EBM may be a suitable carrier for engineering transplant quality CE grafts.

In vitro expansion of corneal endothelial cells on biomimetic substrates.

Corneal endothelial (CE) cells do not divide in vivo, leading to edema, corneal clouding and vision loss when the density drops below a critical level. The endothelium can be replaced by transplanting allogeneic tissue; however, access to donated tissue is limited worldwide resulting in critical need for new sources of corneal grafts. In vitro expansion of CE cells is a potential solution, but is challenging due to limited proliferation and loss of phenotype in vitro via endothelial to mesenchymal transformation (EMT) and senescence. We hypothesized that a bioengineered substrate recapitulating chemo-mechanical properties of Descemet's membrane would improve the in vitro expansion of CE cells while maintaining phenotype. Results show that bovine CE cells cultured on a polydimethylsiloxane surface with elastic modulus of 50 kPa and collagen IV coating achieved >3000-fold expansion. Cells grew in higher-density monolayers with polygonal morphology and ZO-1 localization at cell-cell junctions in contrast to control cells on polystyrene that lost these phenotypic markers coupled with increased α-smooth muscle actin expression and fibronectin fibril assembly. In total, these results demonstrate that a biomimetic substrate presenting native basement membrane ECM proteins and mechanical environment may be a key element in bioengineering functional CE layers for potential therapeutic applications.

Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels.

We demonstrate the additive manufacturing of complex three-dimensional (3D) biological structures using soft protein and polysaccharide hydrogels that are challenging or impossible to create using traditional fabrication approaches. These structures are built by embedding the printed hydrogel within a secondary hydrogel that serves as a temporary, thermoreversible, and biocompatible support. This process, termed freeform reversible embedding of suspended hydrogels, enables 3D printing of hydrated materials with an elastic modulus <500 kPa including alginate, collagen, and fibrin. Computer-aided design models of 3D optical, computed tomography, and magnetic resonance imaging data were 3D printed at a resolution of ~200 µm and at low cost by leveraging open-source hardware and software tools. Proof-of-concept structures based on femurs, branched coronary arteries, trabeculated embryonic hearts, and human brains were mechanically robust and recreated complex 3D internal and external anatomical architectures.

Shrink Wrapping Cells in a Defined Extracellular Matrix to Modulate the Chemo-Mechanical Microenvironment.

Cell-matrix interactions are important for the physical integration of cells into tissues and the function of insoluble, mechanosensitive signaling networks. Studying these interactions in vitro can be difficult because the extracellular matrix (ECM) proteins that adsorb to in vitro cell culture surfaces do not fully recapitulate the ECM-dense basement membranes to which cells such as cardiomyocytes and endothelial cells adhere to in vivo. Towards addressing this limitation, we have developed a surface-initiated assembly process to engineer ECM proteins into nanostructured, microscale sheets that can be shrink wrapped around single cells and small cell ensembles to provide a functional and instructive matrix niche. Unlike current cell encapsulation technology using alginate, fibrin or other hydrogels, our engineered ECM is similar in density and thickness to native basal lamina and can be tailored in structure and composition using the proteins fibronectin, laminin, fibrinogen, and/or collagen type IV. A range of cells including C2C12 myoblasts, bovine corneal endothelial cells and cardiomyocytes survive the shrink wrapping process with high viability. Further, we demonstrate that, compared to non-encapsulated controls, the engineered ECM modulates cytoskeletal structure, stability of cell-matrix adhesions and cell behavior in 2D and 3D microenvironments.

Co-immobilization of active antibiotics and cell adhesion peptides on calcium based biomaterials.

Two bioactive molecules with unrelated functions, vancomycin and a cell adhesion peptide, were immobilized on the surface of a potential bone scaffold material, calcium aluminum oxide. In order to accomplish immobilization and retain bioactivity three sequential surface functionalization strategies were compared: 1.) vancomycin was chemically immobilized before a cell adhesion peptide (KRSR), 2.) vancomycin was chemically immobilized after KRSR and 3.) vancomycin was adsorbed after binding the cell adhesion peptide. Both molecules remained on the surface and active using all three reaction sequences and after autoclave sterilization based on osteoblast attachment, bacterial turbidity and bacterial zone inhibition test results. However, the second strategy was superior at enhancing osteoblast attachment and significantly decreasing bacterial growth when compared to the other sequences.

Development of polydimethylsiloxane substrates with tunable elastic modulus to study cell mechanobiology in muscle and nerve.

Mechanics is an important component in the regulation of cell shape, proliferation, migration and differentiation during normal homeostasis and disease states. Biomaterials that match the elastic modulus of soft tissues have been effective for studying this cell mechanobiology, but improvements are needed in order to investigate a wider range of physicochemical properties in a controlled manner. We hypothesized that polydimethylsiloxane (PDMS) blends could be used as the basis of a tunable system where the elastic modulus could be adjusted to match most types of soft tissue. To test this we formulated blends of two commercially available PDMS types, Sylgard 527 and Sylgard 184, which enabled us to fabricate substrates with an elastic modulus anywhere from 5 kPa up to 1.72 MPa. This is a three order-of-magnitude range of tunability, exceeding what is possible with other hydrogel and PDMS systems. Uniquely, the elastic modulus can be controlled independently of other materials properties including surface roughness, surface energy and the ability to functionalize the surface by protein adsorption and microcontact printing. For biological validation, PC12 (neuronal inducible-pheochromocytoma cell line) and C2C12 (muscle cell line) were used to demonstrate that these PDMS formulations support cell attachment and growth and that these substrates can be used to probe the mechanosensitivity of various cellular processes including neurite extension and muscle differentiation.

Increased osteoblast adhesion on physically optimized KRSR modified calcium aluminate.

Calcium aluminate (CA) is a porous biocompatible material easily cast at room temperature. Through this casting process, the average surface pore size of CA was varied from an average of 100 to 290 microns. The optimal surface pore size of the hydrated CA for cell viability was determined to be 100 microns. Further, a three step-solution deposition technique was developed to covalently immobilize cell adhesion peptides, RGD, and KRSR to the CA surface. Cell adhesion for 1-, 4-, and 7-day time periods was tested with primary osteoblasts and NIH 3T3 fibroblasts. Both peptides were found to increase fibroblast adhesion to the CA surface. However, only KRSR increased osteoblast adhesion to the surface of the CA, which may aid in bone formation after implantation.