Microbiome single cell atlases generated with a commercial instrument.

Single cell sequencing is useful for resolving complex systems into their composite cell types and computationally mining them for unique features that are masked in pooled sequencing. However, while commercial instruments have made single cell analysis widespread for mammalian cells, analogous tools for microbes are limited. Here, we present EASi-seq (Easily Accessible Single microbe sequencing). By adapting the single cell workflow of the commercial Mission Bio Tapestri instrument, this method allows for efficient sequencing of individual microbes' genomes. EASi-seq allows thousands of microbes to be sequenced per run and, as we show, can generate detailed atlases of human and environmental microbiomes. The ability to capture large shotgun genome datasets from thousands of single microbes provides new opportunities in discovering and analyzing species subpopulations. To facilitate this, we develop a companion bioinformatic pipeline that clusters microbes by similarity, improving whole genome assembly, strain identification, taxonomic classification, and gene annotation. In addition, we demonstrate integration of metagenomic contigs with the EASi-seq datasets to reduce capture bias and increase coverage. Overall, EASi-seq enables high quality single cell genomic data for microbiome samples using an accessible workflow that can be run on a commercially available platform.

Viral load quantitation at the point-of-care with shaken digital droplet RT-LAMP.

Viral load quantitation is useful in clinical point-of-care settings to assess the status of patients with infectious disease, track response to treatment, and estimate infectiousness. However, existing methods for quantitating viral loads are complex and difficult to integrate into these settings. Here, we describe a simple, instrument-free approach for viral load quantitation suitable for point-of-care use. We develop a shaken digital droplet assay that can quantitate SARS-CoV2 with sensitivity comparable to gold standard qPCR.

Microfluidics-free single-cell genomics with templated emulsification.

Current single-cell RNA-sequencing approaches have limitations that stem from the microfluidic devices or fluid handling steps required for sample processing. We develop a method that does not require specialized microfluidic devices, expertise or hardware. Our approach is based on particle-templated emulsification, which allows single-cell encapsulation and barcoding of cDNA in uniform droplet emulsions with only a vortexer. Particle-templated instant partition sequencing (PIP-seq) accommodates a wide range of emulsification formats, including microwell plates and large-volume conical tubes, enabling thousands of samples or millions of cells to be processed in minutes. We demonstrate that PIP-seq produces high-purity transcriptomes in mouse-human mixing studies, is compatible with multiomics measurements and can accurately characterize cell types in human breast tissue compared to a commercial microfluidic platform. Single-cell transcriptional profiling of mixed phenotype acute leukemia using PIP-seq reveals the emergence of heterogeneity within chemotherapy-resistant cell subsets that were hidden by standard immunophenotyping. PIP-seq is a simple, flexible and scalable next-generation workflow that extends single-cell sequencing to new applications.

Polyhedral Particles with Controlled Concavity by Indentation Templating.

Current methods for fabricating microparticles offer limited control over size and shape. Here, we demonstrate a droplet microfluidic method to form polyhedral microparticles with controlled concavity. By manipulating Laplace pressure, buoyancy, and particle rheology, we generate microparticles with diverse shapes and curvatures. Additionally, we demonstrate the particles provide increased capture efficiency when used for particle-templated emulsification. Our approach enables microparticles with enhanced chemical and biological functionality.

Particle Templated Emulsification enables Microfluidic-Free Droplet Assays.

Reactions performed in monodispersed droplets afford enhanced accuracy and sensitivity compared to equivalent ones performed in bulk. However, the requirement of microfluidics to form controlled droplets imposes a barrier to non-experts, limiting their use. Here, we describe particle templated emulsification, an approach to generate monodisperse droplets without microfluidics. Using templating hydrogel spheres, we encapsulate samples in monodispersed droplets by simple vortexing. We demonstrate the approach by using it to perform microfluidic-free digital PCR.

Shape-fitting collagen-PLA composite promotes osteogenic differentiation of porcine adipose stem cells.

Craniomaxillofacial bone defects can occur as a result of congenital, post-oncologic, and high-energy impact conditions. The scale and irregularity of such defects motivate new biomaterials to promote regeneration of the damaged bone. We have recently described a mineralized collagen scaffold capable of instructing stem cell osteogenic differentiation and new bone infill in the absence of traditional osteogenic supplements. Herein, we report the integration of a millimeter-scale reinforcing poly (lactic acid) frame fabricated via 3D-printing into the mineralized collagen scaffold with micron-scale porosity to form a multi-scale mineralized collagen-PLA composite. We describe modifications to the PLA frame design to increase the compressive strength (Young's Modulus, ultimate stress and strain) of the composite. A critical challenge beyond increasing the compressive strength of the collagen scaffold is addressing challenges inherent with the irregularity of clinical defects. As a result, we examined the potential for modifying the frame architecture to render the composite with increased compressive strength in one axis or radial compressibility and shape-fitting capacity in an orthogonal axis. A library of mineralized collagen-PLA composites was mechanically characterized via compression testing and push-out test to describe mechanical performance and shape-fitting capacity. We also report in vitro comparison of the bioactivity of porcine adipose derived stem cells in the mineralized collagen-PLA composite versus the mineralized collagen scaffold via metabolic activity, gene expression, and functional matrix synthesis. The results suggest that incorporation of the PLA reinforcing frame does not negatively influence the osteoinductive nature of the mineralized collagen scaffold. Together, these findings suggest a strategy to address often competing bioactivity, mechanical strength, and shape-fitting design requirements for biomaterials for craniomaxillofacial bone regeneration.

A Mineralized Collagen-Polycaprolactone Composite Promotes Healing of a Porcine Mandibular Defect.

A tissue engineering approach to address craniofacial defects requires a biomaterial that balances macro-scale mechanical stiffness and strength with the micron-scale features that promote cell expansion and tissue biosynthesis. Such criteria are often in opposition, leading to suboptimal mechanical competence or bioactivity. We report the use of a multiscale composite biomaterial that integrates a polycaprolactone (PCL) reinforcement structure with a mineralized collagen-glycosaminoglycan scaffold to circumvent conventional tradeoffs between mechanics and bioactivity. The composite promotes activation of the canonical bone morphogenetic protein 2 (BMP-2) pathway and subsequent mineralization of adipose-derived stem cells in the absence of supplemental BMP-2 or osteogenic media. We subsequently examined new bone infill in the acellular composite, scaffold alone, or PCL support in 10 mm dia. ramus mandibular defects in Yorkshire pigs. We report an analytical approach to quantify radial, angular, and depth bone infill from micro-computed tomography data. The collagen-PCL composite showed improved overall infill, and significantly increased radial and angular bone infill versus the PCL cage alone. Bone infill was further enhanced in the composite for defects that penetrated the medullary cavity, suggesting recruitment of marrow-derived cells. These results indicate a multiscale mineralized collagen-PCL composite offers strategic advantages for regenerative repair of craniofacial bone defects.

Nonmineralized and Mineralized Collagen Scaffolds Induce Differential Osteogenic Signaling Pathways in Human Mesenchymal Stem Cells.

The instructive capabilities of extracellular matrix components in progenitor cell differentiation have recently generated significant interest in the development of bioinspired materials for regenerative applications. Previously, a correlation was described between the osteogenic capabilities of nanoparticulate mineralized collagen glycosaminoglycan scaffolds (MC-GAG) and an autogenous activation of small mothers against decapentaplegic ( Smad1/5) in the canonical bone morphogenetic protein receptor (BMPR) pathway with a diminished extracellular signal regulated kinase 1/2 (ERK1/2) activation when compared to nonmineralized collagen glycosaminoglycan scaffolds (Col-GAG). This work utilizes a canonical BMPR inhibitor (dorsomorphin homologue 1, DMH1) and an inhibitor of the mitogen activated protein kinase/ERK kinase (MEK)/(ERK) cascade (PD98059) to characterize the necessity of each pathway for osteogenesis. While DMH1 inhibits runt-related transcription factor 2 (Runx2) and bone sialoprotein II (BSPII) gene expression of primary human mesenchymal stem cells (hMSCs) on MC-GAG, PD98059 inhibits BSPII expression on Col-GAG independent of Runx2 expression. DMH1 inhibits mineralization on both Col-GAG and MC-GAG, however, PD98059 only inhibits mineralization on Col-GAG. DMH1 inhibits both Smad1/5 phosphorylation and Runx2 protein expression, whereas PD98059 inhibits ERK1/2 and c-Jun amino-terminal kinase 1/2 (JNK1/2) phosphorylation without affecting Runx2. Thus, activation of the canonical BMPR signaling is necessary for osteogenic differentiation and mineralization of hMSCs on Col-GAG or MC-GAG. The MEK/ERK cascade, intimately tied to JNK activation, is necessary for Runx2-independent osteogenesis on Col-GAG, while completely dispensable in osteogenesis on MC-GAG.

Nanoparticulate Mineralized Collagen Scaffolds and BMP-9 Induce a Long-Term Bone Cartilage Construct in Human Mesenchymal Stem Cells.

Engineering the osteochondral junction requires fabrication of a microenvironment that supports both osteogenesis and chondrogenesis. Multiphasic scaffold strategies utilizing a combination of soluble factors and extracellular matrix components are ideally suited for such applications. In this work, the contribution of an osteogenic nanoparticulate mineralized glycosaminoglycan scaffold (MC-GAG) and a dually chondrogenic and osteogenic growth factor, BMP-9, in the differentiation of primary human mesenchymal stem cells (hMSCs) is evaluated. Although 2D cultures demonstrate alkaline phosphatase activity and mineralization of hMSCs induced by BMP-9, MC-GAG scaffolds do not demonstrate significant differences in the collagen I expression, osteopontin expression, or mineralization. Instead, BMP-9 increases expression of collagen II, Sox9, aggrecan (ACAN), and cartilage oligomeric protein. However, the hypertrophic chondrocyte marker, collagen X, is not elevated with BMP-9 treatment. In addition, histologic analyses demonstrate that while BMP-9 does not increase mineralization, BMP-9 treatment results in an increase of sulfated glycosaminoglycans. Thus, the combination of BMP-9 and MC-GAG stimulates chondrocytic and osteogenic differentiation of hMSCs.

Nanoparticulate mineralized collagen scaffolds induce in vivo bone regeneration independent of progenitor cell loading or exogenous growth factor stimulation.

Current strategies for skeletal regeneration often require co-delivery of scaffold technologies, growth factors, and cellular material. However, isolation and expansion of stem cells can be time consuming, costly, and requires an additional procedure for harvest. Further, the introduction of supraphysiologic doses of growth factors may result in untoward clinical side effects, warranting pursuit of alternative methods for stimulating osteogenesis. In this work, we describe a nanoparticulate mineralized collagen glycosaminoglycan scaffold that induces healing of critical-sized rabbit cranial defects without addition of expanded stem cells or exogenous growth factors. We demonstrate that the mechanism of osteogenic induction corresponds to an increase in canonical BMP receptor signalling secondary to autogenous production of BMP-2 and -9 early and BMP-4 later during differentiation. Thus, nanoparticulate mineralized collagen glycosaminoglycan scaffolds may provide a novel growth factor-free and ex vivo progenitor cell culture-free implantable method for bone regeneration.

CXCR4/CXCL12 signaling impacts enamel progenitor cell proliferation and motility in the dental stem cell niche.

Dental stem cells are located at the proximal ends of rodent incisors. These stem cells reside in the dental epithelial stem cell niche, termed the apical bud. We focused on identifying critical features of a chemotactic signal in the niche. Here, we report that CXCR4/CXCL12 signaling impacts enamel progenitor cell proliferation and motility in dental stem cell niche cells. We report cells in the apical bud express CXCR4 mRNA at high levels while expression is restricted in the basal epithelium (BE) and transit-amplifying (TA) cell regions. Furthermore, the CXCL12 ligand is present in mesenchymal cells adjacent to the apical bud. We then performed gain- and loss-of-function analyses to better elucidate the role of CXCR4 and CXCL12. CXCR4-deficient mice contain epithelial cell aggregates, while cell proliferation in mutant incisors was also significantly reduced. We demonstrate in vitro that dental epithelial cells migrate toward sources of CXCL12, whereas knocking down CXCR4 impaired motility and resulted in formation of dense cell colonies. These results suggest that CXCR4 expression may be critical for activation of enamel progenitor cell division and that CXCR4/CXCL12 signaling may control movement of epithelial progenitors from the dental stem cell niche.

Optimizing Collagen Scaffolds for Bone Engineering: Effects of Cross-linking and Mineral Content on Structural Contraction and Osteogenesis.

INTRODUCTION: Osseous defects of the craniofacial skeleton occur frequently in congenital, posttraumatic, and postoncologic deformities. The field of scaffold-based bone engineering emerged to address the limitations of using autologous bone for reconstruction of such circumstances. In this work, the authors evaluate 2 modifications of three-dimensional collagen-glycosaminoglycan scaffolds in an effort to optimize structural integrity and osteogenic induction. METHODS: Human mesenchymal stem cells (hMSCs) were cultured in osteogenic media on nonmineralized collagen-glycosaminoglycan (C-GAG) and nanoparticulate mineralized collagen-glycosaminoglycan (MC-GAG) type I scaffolds, in the absence and presence of cross-linking. At 1, 7, and 14 days, mRNA expression was analyzed using quantitative real-time -reverse-transcriptase polymerase chain reaction for osteocalcin (OCN) and bone sialoprotein (BSP). Structural contraction was measured by the ability of the scaffolds to maintain their original dimensions. Mineralization was detected by microcomputed tomographic (micro-CT) imaging at 8 weeks. Statistical analyses were performed with Student t-test. RESULTS: Nanoparticulate mineralization of collagen-glycosaminoglycan scaffolds increased expression of both OCN and BSP. Cross-linking of both C-GAG and MC-GAG resulted in decreased osteogenic gene expression; however, structural contraction was significantly decreased after cross-linking. Human mesenchymal stem cells-directed mineralization, detected by micro-CT, was increased in nanoparticulate mineralized scaffolds, although the density of mineralization was decreased in the presence of cross-linking. CONCLUSIONS: Optimization of scaffold material is an essential component of moving toward clinically translatable engineered bone. Our current study demonstrates that the combination of nanoparticulate mineralization and chemical cross-linking of C-GAG scaffolds generates a highly osteogenic and structurally stable scaffold.

Mineralized collagen scaffolds induce hMSC osteogenesis and matrix remodeling.

Biomaterials for bone tissue engineering must be able to instruct cell behavior in the presence of the complex biophysical and biomolecular environments encountered in vivo. While soluble supplementation strategies have been identified to enhance osteogenesis, they are subject to significant diffusive loss in vivo or the need for frequent re-addition in vitro. This investigation therefore explored whether biophysical and biochemical properties of a mineralized collagen-GAG scaffold were sufficient to enhance human mesenchymal stem cell (hMSC) osteogenic differentiation and matrix remodeling in the absence of supplementation. We examined hMSC metabolic health, osteogenic and matrix gene expression profiles, as well as matrix remodeling and mineral formation as a function of scaffold mineral content. We found that scaffold mineral content enhanced long term hMSC metabolic activity relative to non-mineralized scaffolds. While osteogenic supplementation or exogenous BMP-2 could enhance some markers of hMSC osteogenesis in the mineralized scaffold, we found the mineralized scaffold was itself sufficient to induce osteogenic gene expression, matrix remodeling, and mineral formation. Given significant potential for unintended consequences with the use of mixed media formulations and potential for diffusive loss in vivo, these findings will inform the design of instructive biomaterials for regenerative repair of critical-sized bone defects, as well as for applications where non-uniform responses are required, such as in biomaterials to address spatially-graded interfaces between orthopedic tissues.

Osteogenesis on nanoparticulate mineralized collagen scaffolds via autogenous activation of the canonical BMP receptor signaling pathway.

Skeletal regenerative medicine frequently incorporates deliverable growth factors to stimulate osteogenesis. However, the cost and side effects secondary to supraphysiologic dosages of growth factors warrant investigation of alternative methods of stimulating osteogenesis for clinical utilization. In this work, we describe growth factor independent osteogenic induction of human mesenchymal stem cells (hMSCs) on a novel nanoparticulate mineralized collagen glycosaminoglycan scaffold (MC-GAG). hMSCs demonstrated elevated osteogenic gene expression and mineralization on MC-GAG with minimal to no effect upon addition of BMP-2 when compared to non-mineralized scaffolds (Col-GAG). To investigate the intracellular pathways responsible for the increase in osteogenesis, we examined the canonical and non-canonical pathways downstream from BMP receptor activation. Constitutive Smad1/5 phosphorylation with nuclear translocation occurred on MC-GAG independent of BMP-2, whereas Smad1/5 phosphorylation depended on BMP-2 stimulation on Col-GAG. When non-canonical BMPR signaling molecules were examined, ERK1/2 phosphorylation was found to be decreased in MC-GAG but elevated in Col-GAG. No differences in Smad2/3 or p38 activation were detected. Collectively, these results demonstrated that MC-GAG scaffolds induce osteogenesis without exogenous BMP-2 addition via endogenous activation of the canonical BMP receptor signaling pathway.

Collagen Scaffolds Incorporating Coincident Gradations of Instructive Structural and Biochemical Cues for Osteotendinous Junction Engineering.

A fully 3D biomaterial containing overlapping gradations of structural, compositional, and biomolecular cues as seen in native orthopedic interfaces is described for the first time. A multi-compartment collagen scaffold is created for engineering tendon-bone junctions connected by a continuous interface that can induce spatially specific MSC differentiation down tenogenic and osteogenic lineages without the use of differentiation media.

Collagen scaffold arrays for combinatorial screening of biophysical and biochemical regulators of cell behavior.

Arrays of 3D macroporous collagen scaffolds with orthogonal gradations of structural and biomolecular cues are described. Gradient maker technology is applied to create linear biomolecular gradients within microstructurally distinct sections of a single CG scaffold array. The array set up is used to explore cell behaviors including proliferation and regulation of stem cell fate.

Photopatterning of vascular endothelial growth factor within collagen-glycosaminoglycan scaffolds can induce a spatially confined response in human umbilical vein endothelial cells.

Biomolecular signals within the native extracellular matrix are complex, with bioactive factors found in both soluble and sequestered states. In the design of biomaterials for tissue engineering applications it is increasingly clear that new approaches are required to locally tailor the biomolecular environment surrounding cells within the matrix. One area of particular focus is strategies to improve the speed or quality of vascular ingrowth and remodeling. While the addition of soluble vascular endothelial growth factor (VEGF) has been shown to improve vascular response, strategies to immobilize such signals within a biomaterial offer the opportunity to optimize efficiency and to explore spatially defined patterning of such signals. Here we describe the use of benzophenone (BP) photolithography to decorate three-dimensional collagen-glycosaminoglycan (CG) scaffolds with VEGF in a spatially defined manner. In this effort we demonstrate functional patterning of a known agonist of vascular remodeling and directly observe phenotypic effects induced by this immobilized cue. VEGF was successfully patterned in both stripes and square motifs across the scaffold with high specificity (on:off pattern signal). The depth of patterning was determined to extend up to 500 µm into the scaffold microstructure. Notably, photopatterned VEGF retained native functionality as it was shown to induce morphological changes in human umbilical vein cells indicative of early vasculogenesis. Immobilized VEGF led to greater cell infiltration into the scaffold and the formation of immature vascular network structures. Ultimately, these results suggest that BP-mediated photolithography is a facile method to spatially control the presentation of instructive biological cues to cells within CG scaffolds.

The influence of collagen-glycosaminoglycan scaffold relative density and microstructural anisotropy on tenocyte bioactivity and transcriptomic stability.

Biomaterials for orthopedic tissue engineering must balance mechanical and bioactivity concerns. This work describes the fabrication of a homologous series of anisotropic collagen-GAG (CG) scaffolds with aligned tracks of ellipsoidal pores but increasing relative densities (ρ(∗)/ρ(s)), and we report the role scaffold relative density plays in directing tenocyte bioactivity. Scaffold permeability and mechanical properties, both in tension and compression, were significantly influenced by relative density in a manner predicted by cellular solids models. Equine tenocytes showed greater levels of attachment, metabolic activity, soluble collagen synthesis, and alignment as well as less cell-mediated scaffold contraction in anisotropic CG scaffolds of increasing relative density. Notably, the lowest density scaffolds experienced significant cell-mediated contraction with associated decreases in tenocyte number as well as loss of microstructural integrity, aligned contact guidance cues, and preferential tenocyte orientation over a 14 day culture period. Gene expression analyses suggested tenocyte de-differentiation in the lowest density scaffold while indicating that the highest density scaffold supported significant increases in COMP (4-fold), tenascin-C (3-fold), and scleraxis (15-fold) expression as well as significant decreases in MMP-1 (9-fold) and MMP-13 (13-fold) expression on day 14. These results suggest that anisotropic scaffold relative density can help to modulate the maintenance of a more tendon-like microenvironment and aid long-term tenocyte transcriptomic stability. Overall, this work demonstrates that relative density is a critical scaffold parameter, not only for insuring mechanical competence, but also for directing cell transcriptomic stability and behavior.