Release of Applied Mechanical Loading Stimulates Intercellular Calcium Waves in Drosophila Wing Discs.

Mechanical forces are critical but poorly understood inputs for organogenesis and wound healing. Calcium ions (Ca2+) are critical second messengers in cells for integrating environmental and mechanical cues, but the regulation of Ca2+ signaling is poorly understood in developing epithelial tissues. Here we report a chip-based regulated environment for microorgans that enables systematic investigations of the crosstalk between an organ's mechanical stress environment and biochemical signaling under genetic and chemical perturbations. This method enabled us to define the essential conditions for generating organ-scale intercellular Ca2+ waves in Drosophila wing discs that are also observed in vivo during organ development. We discovered that mechanically induced intercellular Ca2+ waves require fly extract growth serum as a chemical stimulus. Using the chip-based regulated environment for microorgans, we demonstrate that not the initial application but instead the release of mechanical loading is sufficient, but not necessary, to initiate intercellular Ca2+ waves. The Ca2+ response depends on the prestress intercellular Ca2+ activity and not on the magnitude or duration of the mechanical stimulation applied. Mechanically induced intercellular Ca2+ waves rely on IP3R-mediated Ca2+-induced Ca2+ release and propagation through gap junctions. Thus, intercellular Ca2+ waves in developing epithelia may be a consequence of stress dissipation during organ growth.

Multiscale Porosity Directs Bone Regeneration in Biphasic Calcium Phosphate Scaffolds.

Large and load-bearing bone defects are challenging to treat and cause pain and disfigurement. The design of efficacious bone scaffolds for the repair of such defects involves a range of length scales from the centimeter down to the micrometer-scale. Here, we assess the influence on bone regeneration of scaffold rod spacing (>300 µm) and microporosity (<50 µm), as well as the combination of different structures and materials in the same scaffold, i.e., at the millimeter scale. We use four single-domain scaffolds, microporous (MP) or nonmicroporous (NMP) and with either a "small" or "large" rod spacing. Multidomain scaffolds combine four regions corresponding to the macro- and microarchitectures of the single-domain scaffolds. The scaffolds are implanted in pig mandibles for 3 weeks and bone regeneration is assessed by measuring the average bone volume fraction, BVF̅, the bone distribution and the trabecular thickness from micro-CT data. For the single-domain scaffolds, BVF̅ was 45 ± 3% for MP-small, 39 ± 2% for MP-large, 25 ± 2% for NMP-small, and 25 ± 2% for NMP-large. MP scaffolds have significantly higher BVF̅ and a more uniform bone distribution compared to NMP, regardless of rod spacing. The average trabecular thickness is significantly larger in MP compared to NMP, and in "large" compared to "small" scaffolds. Microporosity affects trabecular thickness throughout the scaffold, while rod spacing affects it only at the scaffold periphery. In multidomain scaffolds, MP-large and NMP-large domains have similar BVF̅ as compared to their respective single-domain counterparts. These results suggest that combining different architectures into one scaffold conserves the properties of each domain. Hence, bone growth and morphology can be tailored by controlling scaffold architecture from the millimeter down to the micrometer level. This will allow the customization of scaffold designs for the treatment of large and load-bearing bone defects.

Net shape fabrication of calcium phosphate scaffolds with multiple material domains.

Calcium phosphate (CaP) materials have been proven to be efficacious as bone scaffold materials, but are difficult to fabricate into complex architectures because of the high processing temperatures required. In contrast, polymeric materials are easily formed into scaffolds with near-net-shape forms of patient-specific defects and with domains of different materials; however, they have reduced load-bearing capacity compared to CaPs. To preserve the merits of CaP scaffolds and enable advanced scaffold manufacturing, this manuscript describes an additive manufacturing process that is coupled with a mold support for overhanging features; we demonstrate that this process enables the fabrication of CaP scaffolds that have both complex, near-net-shape contours and distinct domains with different microstructures. First, we use a set of canonical structures to study the manufacture of complex contours and distinct regions of different material domains within a mold. We then apply these capabilities to the fabrication of a scaffold that is designed for a 5 cm orbital socket defect. This scaffold has complex external contours, interconnected porosity on the order of 300 µm throughout, and two distinct domains of different material microstructures.

A microfluidic technique to probe cell deformability.

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~10(2) cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

Design and manufacture of combinatorial calcium phosphate bone scaffolds.

It is well known that pore design is an important determinant of both the quantity and distribution of regenerated bone in artificial bone tissue scaffolds. A requisite feature is that scaffolds must contain pore interconnections on the order of 100-1000 µm (termed macroporosity). Within this range, there is not a definitive optimal interconnection size. Recent results suggest that pore interconnections permeating the scaffold build material on the order of 2-20 µm (termed microporosity) drive bone growth into the macropore space at a faster rate and also provide a new space for bone growth, proliferating throughout the interconnected microporous network. The effects of microstructural features on bone growth has yet to be fully understood. This work presents the manufacture and characterization of novel combinatorial test scaffolds, scaffolds that test multiple microporosity and macroporosity designs within a single scaffold. Scaffolds such as this can efficiently evaluate multiple mechanical designs, with the advantage of having the designs colocated within a single defect site and therefore less susceptible to experimental variation. This paper provides the manufacturing platform, manufacturing control method, and demonstrates the manufacturing capabilities with three representative scaffolds.

The effect of BMP-2 on micro- and macroscale osteointegration of biphasic calcium phosphate scaffolds with multiscale porosity.

It is well established that scaffolds for applications in bone tissue engineering require interconnected pores on the order of 100 microm for bone in growth and nutrient and waste transport. As a result, most studies have focused on scaffold macroporosity (>100 microm). More recently researchers have investigated the role of microporosity in calcium phosphate -based scaffolds. Osteointegration into macropores improves when scaffold rods or struts contain micropores, typically defined as pores less than approximately 50 microm. We recently demonstrated multiscale osteointegration, or growth into both macropores and intra-red micropores (<10 microm), of biphasic calcium phosphate (BCP) scaffolds. The combined effect of BMP-2, a potent osteoinductive growth factor, and multiscale porosity has yet to be investigated. In this study we implanted BCP scaffolds into porcine mandibular defects for 3, 6, 12 and 24 weeks and evaluated the effect of BMP-2 on multiscale osteointegration. The results showed that given this in vivo model BMP-2 influences osteointegration at the microscale, but not at the macroscale, but not at the macroscale. Cell density was higher in the rod micropores for scaffolds containing BMP-2 compared with controls at all time points, but BMP-2 was not required for bone formation in micropores. In contrast, there was essentially no difference in the fraction of bone in macropores for scaffolds with BMP-2 compared with controls. Additionally, bone in macropores seemed to have reached steady-state by 3 weeks. Multiscale osteointegration results in bone-scaffold composites that are fully osteointegrated, with no 'dead space'. These composites are likely to contain a continuous cell network as well as the potential for enhanced load transfer and improved mechanical properties.

Micro-robotic deposition guidelines by a design of experiments approach to maximize fabrication reliability for the bone scaffold application.

This work aims to facilitate the transition of micro-robotic deposition (microRD) technology from the research bench to a mass manufacturing environment. The bone scaffolding application is targeted; however, the evaluation process developed is applicable to multiple colloidal material systems, length scales, and structure architectures. A design of experiments (DoE) approach is used to develop statistical correlations between three manufacturing treatments (material calcination time, nozzle size, and deposition speed) and defined reliability metrics. All three selected treatments have a significant effect on structure quality. A longer material calcination time improves the deposition of internal features. Logically, a larger nozzle size decreases structural defects. However, an unexpected result is revealed by this study. Higher deposition speeds are shown to either significantly improve or have no effect on structure quality, permitting a decrease in manufacturing time without adverse consequences.