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Bone tissue, by definition, is an organic–inorganic nanocomposite, where metabolically active cells are embedded within a matrix that is heavily calcified on the nanoscale. Currently, there are no strategies that replicate these definitive characteristics of bone tissue. Here we describe a biomimetic approach where a supersaturated calcium and phosphate medium is used in combination with a non-collagenous protein analog to direct the deposition of nanoscale apatite, both in the intra- and extrafibrillar spaces of collagen embedded with osteoprogenitor, vascular, and neural cells. This process enables engineering of bone models replicating the key hallmarks of the bone cellular and extracellular microenvironment, including its protein-guided biomineralization, nanostructure, vasculature, innervation, inherent osteoinductive properties (without exogenous supplements), and cell-homing effects on bone-targeting diseases, such as prostate cancer. Ultimately, this approach enables fabrication of bone-like tissue models with high levels of biomimicry that may have broad implications for disease modeling, drug discovery, and regenerative engineering. Bone tissue is a complex organic-inorganic nanocomposite and strategies that replicate the characteristics of bone tissue are scarce. Here the authors demonstrate the deposition of nanoscale apatite in collagen embedded with mesenchymal, vascular and nerve cells, using a protein-guided biomineralization approach.

Engineered tissue constructs require the fabrication of highly perfusable and mature vascular networks for effective repair and regeneration. In tissue engineering, stem cells are widely employed to create mature vascularized tissues in vitro. Pericytes are key to the maturity of these vascular networks, and therefore the ability of stem cells to differentiate into pericyte-like lineages should be understood. To date, there is limited information regarding the ability of stem cells from the different tissue sources to differentiate into pericytes and form microvascular capillaries in vitro. Therefore, here we tested the ability of the stem cells derived from bone marrow (BMSC), dental pulp (DPSC) and dental apical papilla (SCAP) to engineer pericyte-supported vascular capillaries when encapsulated along with human umbilical vein endothelial cells (HUVECs) in gelatin methacrylate (GelMA) hydrogel. Our results show that the pericyte differentiation capacity of BMSC was greater with high expression of α-SMA and NG2 positive cells. DPSC had α-SMA positive cells but showed very few NG2 positive cells. Further, SCAP cells were positive for α-SMA while they completely lacked NG2 positive cells. We found the pericyte differentiation ability of these stem cells to be different, and this significantly affected the vasculogenic ability and quality of the vessel networks. In summary, we conclude that, among stem cells from different craniofacial regions, BMSCs appear more suitable for engineering of mature vascularized networks than DPSCs or SCAPs.

The requirement for immediate vascularization of engineered dental pulp poses a major hurdle towards successful implementation of pulp regeneration as an effective therapeutic strategy for root canal therapy, especially in adult teeth. Here, we demonstrate a novel strategy to engineer pre-vascularized, cell-laden hydrogel pulp-like tissue constructs in full-length root canals for dental pulp regeneration. We utilized gelatin methacryloyl (GelMA) hydrogels with tunable physical and mechanical properties to determine the microenvironmental conditions (microstructure, degradation, swelling and elastic modulus) that enhanced viability, spreading and proliferation of encapsulated odontoblast-like cells (OD21), and the formation of endothelial monolayers by endothelial colony forming cells (ECFCs). GelMA hydrogels with higher polymer concentration (15% w/v) and stiffness enhanced OD21 cell viability, spreading and proliferation, as well as endothelial cell spreading and monolayer formation. We then fabricated pre-vascularized, full-length, dental pulp-like tissue constructs by dispensing OD21 cell-laden GelMA hydrogel prepolymer in root canals of extracted teeth and fabricating 500 µm channels throughout the root canals. ECFCs seeded into the microchannels successfully formed monolayers and underwent angiogenic sprouting within 7 days in culture. In summary, the proposed approach is a simple and effective strategy for engineering of pre-vascularized dental pulp constructs offering potentially beneficial translational outcomes.

Bone homeostasis depends on spatially orchestrated interactions among osteoclasts, osteoblasts, and osteocytes that are embedded within a unique extracellular matrix that is mineralized on the nanoscale to define the structure and function of bone. Reconstructing these interactions to enable autonomous cell differentiation and tissue remodeling has remained a significant challenge towards mimicking adequate bone physiology in-vitro. Here, we present an engineered model that spatially defines the paracrine communication of heterogeneous cell populations within bone tissue that support the rapid maturation of primary osteoblasts into osteocytes, the differentiation of macrophages into osteoclasts, and calcified tissue resorption within a mineralized cell-laden bone-like tissue. We demonstrate that nanoscale mineralization of cell-laden collagen hydrogels on-a-chip enhances osteoblast to osteocyte differentiation, whereas osteocytes in the matrix accelerate osteoclastogenesis and remodeling in a spatially defined manner without the need for exogenous growth factors. Osteocyte-dependent osteoclastogenesis on-a-chip outperformed conventional stimulation with RANKL and M-CSF, reproduced the clinical response of anti-resorptive drugs, and mimicked established tumor-bone interactions observed in invasive oral cancer. By replicating essential aspects of bone composition and function, this system provides a robust, self-regulated microphysiologic model to investigate bone remodeling, cancer-bone crosstalk, and therapeutic interventions.

Pericytes stabilize blood vessels and promote vascular barrier function. However, vessels subjected to pro-inflammatory conditions have impaired barrier function, which has been suggested to potentially expose perivascular cells to SARS-CoV-2. To test this hypothesis, we engineered pericyte-supported vascular capillaries on-a-chip, and determined that the extravasation and binding of spike protein (S1) on perivascular cells of inflamed vessels to be significantly higher that in healthy controls, indicating a potential target to understand COVID-19 vascular complications. Competing Interest Statement The authors have declared no competing interest.

Bone defects may occur in different sizes and shapes due to trauma, infections, and cancer resection. Autografts are still considered the primary treatment choice for bone regeneration. However, they are hard to source and often create donor-site morbidity. Injectable microgels have attracted much attention in tissue engineering and regenerative medicine due to their ability to replace inert implants with a minimally invasive delivery. Here, we developed novel cell-laden bioprinted gelatin methacrylate (GelMA) injectable microgels, with controllable shapes and sizes that can be controllably mineralized on the nanoscale, while stimulating the response of cells embedded within the matrix. The injectable microgels were mineralized using a calcium and phosphate-rich medium that resulted in nanoscale crystalline hydroxyapatite deposition and increased stiffness within the crosslinked matrix of bioprinted GelMA microparticles. Next, we studied the effect of mineralization in osteocytes, a key bone homeostasis regulator. Viability stains showed that osteocytes were maintained at 98% viability after mineralization with elevated expression of sclerostin in mineralized compared to non-mineralized microgels, indicating that mineralization effectively enhances osteocyte maturation. Based on our findings, bioprinted mineralized GelMA microgels appear to be an efficient material to approximate the bone microarchitecture and composition with desirable control of sample injectability and polymerization. These bone-like bioprinted mineralized biomaterials are exciting platforms for potential minimally invasive translational methods in bone regenerative therapies.Bone defects may occur in different sizes and shapes due to trauma, infections, and cancer resection. Autografts are still considered the primary treatment choice for bone regeneration. However, they are hard to source and often create donor-site morbidity. Injectable microgels have attracted much attention in tissue engineering and regenerative medicine due to their ability to replace inert implants with a minimally invasive delivery. Here, we developed novel cell-laden bioprinted gelatin methacrylate (GelMA) injectable microgels, with controllable shapes and sizes that can be controllably mineralized on the nanoscale, while stimulating the response of cells embedded within the matrix. The injectable microgels were mineralized using a calcium and phosphate-rich medium that resulted in nanoscale crystalline hydroxyapatite deposition and increased stiffness within the crosslinked matrix of bioprinted GelMA microparticles. Next, we studied the effect of mineralization in osteocytes, a key bone homeostasis regulator. Viability stains showed that osteocytes were maintained at 98% viability after mineralization with elevated expression of sclerostin in mineralized compared to non-mineralized microgels, indicating that mineralization effectively enhances osteocyte maturation. Based on our findings, bioprinted mineralized GelMA microgels appear to be an efficient material to approximate the bone microarchitecture and composition with desirable control of sample injectability and polymerization. These bone-like bioprinted mineralized biomaterials are exciting platforms for potential minimally invasive translational methods in bone regenerative therapies.

It has long been proposed that recapitulating the extracellular matrix (ECM) of native human tissues in the laboratory may enhance the regenerative capacity of engineered scaffolds in-vivo. Organ- and tissue-derived decellularized ECM biomaterials have been widely used for tissue repair, especially due to their intrinsic biochemical cues that can facilitate repair and regeneration. The main purpose of this study was to synthesize a new photocrosslinkable human bone-derived ECM hydrogel for bioprinting of vascularized scaffolds. To that end, we demineralized and decellularized human bone fragments to obtain a bone matrix, which was further processed and functionalized with methacrylate groups to form a photocrosslinkable methacrylate bone ECM hydrogel – BoneMA. The mechanical properties of BoneMA were tunable, with the elastic modulus increasing as a function of photocrosslinking time, while still retaining the nanoscale features of the polymer networks. The intrinsic cell-compatibility of the bone matrix ensured the synthesis of a highly cytocompatible hydrogel. The bioprinted BoneMA scaffolds supported vascularization of endothelial cells and within a day led to the formation of interconnected vascular networks. We propose that such a quick vascular network formation was due to the host of pro-angiogenic biomolecules present in the bone ECM matrix. Further, we also demonstrate the bioprintability of BoneMA in microdimensions as injectable ECM-based building blocks for microscale tissue engineering in a minimally invasive manner. We conclude that BoneMA may be a useful hydrogel system for tissue engineering and regenerative medicine.

A wide range of conditions, including chronic inflammatory diseases and cancer, are characterized by the fibrotic microarchitecture and increased stiffness of collagen type I extracellular matrix. These conditions are typically accompanied by altered vascular function, including vessel leakiness, abnormal capillary morphology and stability. The dynamic cell-matrix interactions that regulate vascular function in healthy tissues have been well documented. However, our understanding of how the gradual mechanical and structural alterations in collagen type I affect vascular homeostasis remains elusive, especially as a function of the interactions between endothelial and perivascular cell with the altered matrix. Here we hypothesized that perivascular cells might function as mechano-structural sensors of the microvasculature by mediating the interaction between endothelial cells and altered collagen type I. To test that, we utilized an organotypic model of perivascular cell-supported vascular capillaries in collagen scaffolds of controlled microarchitecture and mechanics. Our results demonstrate that capillaries cultured in soft reticular collagen exhibited consistent pericyte differentiation, endothelial cell-cell junctions, and barrier function. In contrast, capillaries embedded in stiff and bundled collagen fibrils to mimic a more fibrotic matrix induced abluminal migration of perivascular cells, increased leakage, and marked expression of vascular remodeling and inflammatory markers. These patterns, however, were only observed when endothelial capillaries were engineered with perivascular cells. Silencing of NOTCH3, a mediator of endothelial-perivascular cell communication, largely re-established normal vascular morphology and function. In summary, our findings point to a novel mechanism of perivascular regulation of vascular dysfunction in fibrotic tissues which may have important implications for anti-angiogenic and anti-fibrotic therapies in cancer, chronic inflammatory diseases and regenerative medicine.Competing Interest StatementThe authors have declared no competing interest.

Biomaterial scaffolds have served as the foundation of tissue engineering and regenerative medicine. However, scaffold systems are often difficult to scale in size or shape in order to fit defect-specific dimensions, and thus provide only limited spatiotemporal control of therapeutic delivery and host tissue responses. Here, a lithography-based three-dimensional (3D) printing strategy is used to fabricate a novel miniaturized modular LEGO-like cage scaffold system, which can be assembled and scaled manually with ease. Scalability is based on an intuitive concept of stacking modules, like conventional LEGO blocks, allowing for literally thousands of potential geometric configurations, and without the need for specialized equipment. Moreover, the modular hollow-cage design allows each unit to be loaded with biologic cargo of different compositions, thus enabling controllable and easy patterning of therapeutics within the material in 3D. In summary, the concept of miniaturized cage designs with such straight-forward assembly and scalability, as well as controllable loading properties, is a flexible platform that can be extended to a wide range of materials for improved biological performance. 3D printed LEGO-like hollow microcages can be easily assembled, adjoined, and stacked-up to suit the complexity of defect tissues; aid spatial loading of cells and biomolecules; instruct cells migration three-dimensionally; and facilitate cell invasion and neovascularization in-vivo, thus accelerating the process of tissue healing and new tissue formation. biorxiv;2020.03.02.974204v1/UFIG1F1ufig1

Genomic variation across cancers scales with tissue stiffness: meta-analyses show tumors in stiff tissues such as lung and bone exhibit up to 100-fold more variation than tumors in soft tissues such as marrow and brain. Here, nuclear lamina damage and DNA double-strand breaks (DSBs) result from invasive migration of cancer cells through stiff constrictions. DSBs increase with lamin-A knockdown and require micro-pores sufficiently small for lamins to impede migration. Blebs in the vast majority of post-migration nuclei are enriched in lamin-A but deficient in lamin-B and an age-associated form of lamin-A. Validation of DSBs by an electrophoretic comet assay calibrates against a cancer line having nuclease sites engineered in chromosome-1, and DSB-bound repair factors in nuclei pulled into constrictions show folded chromatin orients, extends, and concentrates without fragmentation. Mobile repair proteins simultaneously segregate away from pore-condensed chromatin. Global squeeze-out of repair factors and loss with lamin-A-dependent rupture explains why overexpression of repair factors cannot rescue DSBs in migration through stiff constrictions, ultimately favoring genomic variation.