Collagen is the primary component of the extracellular matrix in the human body. It has proved challenging to fabricate collagen scaffolds capable of replicating the structure and function of tissues and organs. We present a method to 3D-bioprint collagen using freeform reversible embedding of suspended hydrogels (FRESH) to engineer components of the human heart at various scales, from capillaries to the full organ. Control of pH-driven gelation provides 20-micrometer filament resolution, a porous microstructure that enables rapid cellular infiltration and microvascularization, and mechanical strength for fabrication and perfusion of multiscale vasculature and tri-leaflet valves. We found that FRESH 3D-bioprinted hearts accurately reproduce patient-specific anatomical structure as determined by micro–computed tomography. Cardiac ventricles printed with human cardiomyocytes showed synchronized contractions, directional action potential propagation, and wall thickening up to 14% during peak systole.

Automated visual-tracking of cell populations in vitro using time-lapse phase contrast microscopy enables quantitative, systematic, and high-throughput measurements of cell behaviors. These measurements include the spatiotemporal quantification of cell migration, mitosis, apoptosis, and the reconstruction of cell lineages. The combination of low signal-to-noise ratio of phase contrast microscopy images, high and varying densities of the cell cultures, topological complexities of cell shapes, and wide range of cell behaviors poses many challenges to existing tracking techniques. This paper presents a fully automated multi-target tracking system that can efficiently cope with these challenges while simultaneously tracking and analyzing thousands of cells observed using time-lapse phase contrast microscopy. The system combines bottom-up and top-down image analysis by integrating multiple collaborative modules, which exploit a fast geometric active contour tracker in conjunction with adaptive interacting multiple models (IMM) motion filtering and spatiotemporal trajectory optimization. The system, which was tested using a variety of cell populations, achieved tracking accuracy in the range of 86.9-92.5%.

Genetic studies place the transcription factor Osterix (Osx) downstream of Runx2, but limited information is available about Osx regulation during osteoblastic differentiation. An important role for bone morphogenetic protein-2 (BMP-2) and insulin-like growth factor-I (IGF-I) on Osx expression and the requirement for p38 for the BMP-2-mediated effect was reported previously by our group. In this study, we continued to investigate the molecular mechanisms by which BMP-2 and IGF-1 regulate Osx expression during osteoblast lineage progression. IGF-I-mediated Osx expression required all three MAPK components (Erk, p38, and JNK), whereas BMP-2 required p38 and JNK signaling. As a common mediator of growth factor signaling, we also investigated the involvement of protein kinase C/D (PKC/D) signaling. BMP-2-and IGF-I-mediated Osx expression was blocked in response to a PKD inhibitor. A selective inhibitor of conventional PKCs had no effect on the BMP-2-mediated Osx expression. BMP-2 and IGF-I induced a selective phosphorylation of PKD, and PKD was required for mineralization. PKC/D and MAPK signaling also mediate Runx2 activity. Therefore, to document the implication for Runx2 in Osx regulation, we blocked Runx2 activity using a dominant negative Runx2 construct and an ubiquitination mediator for Runx2 degradation. We showed that blocking Runx2 activity inhibited the BMP-2-mediated induction of Osx. These studies implicated that multiple signaling pathways mediate Osx, a critical gene for osteoblast differentiation and bone formation. In addition to Runx2, other signaling components may be necessary to regulate Osx during osteoblast lineage progression.

In vivo, growth factors exist both as soluble and as solidphase molecules, immobilized to cell surfaces and within the extracellular matrix. We used this rationale to develop more biologically relevant approaches to study stem cell behaviors. We engineered stem cell microenvironments using inkjet bioprinting technology to create spatially defined patterns of immobilized growth factors. Using this approach, we engineered cell fate toward the osteogenic lineage in register to printed patterns of bone morphogenetic protein (BMP) 2 contained within a population of primary musclederived stem cells (MDSCs) isolated from adult mice. This patterning approach was conducive to patterning the MDSCs into subpopulations of osteogenic or myogenic cells simultaneously on the same chip. When cells were cultured under myogenic conditions on BMP-2 patterns, cells on pattern differentiated toward the osteogenic lineage, whereas cells off pattern differentiated toward the myogenic lineage. Time-lapse microscopy was used to visualize the formation of multinucleated myotubes, and immunocytochemistry was used to demonstrate expression of myosin heavy chain (fast) in cells off BMP-2 pattern. This work provides proof-of-concept for engineering spatially controlled multilineage differentiation of stem cells using patterns of immobilized growth factors. This approach may be useful for understanding cell behaviors to immobilized biological patterns and could have potential applications for regenerative medicine.

Background-There is a clinical need for bone replacement strategies because of the shortfalls endemic to autologous bone grafting, especially in the pediatric patient population. For the past 25 years, the animal model that has been used to test bone replacement strategies has been the calvarial critical-sized defect (CSD), based on the initial size of the bone defect. This study was undertaken to test the concept of the critical-size in several different models. A review of the theoretical and scientific bases for the CSD was also undertaken.