Role of the Peripheral Nervous System in Skeletal Development and Regeneration: Controversies and Clinical Implications.

PURPOSE OF REVIEW: This review examines the diverse functional relationships that exist between the peripheral nervous system (PNS) and bone, including key advances over the past century that inform our efforts to translate these discoveries for skeletal repair. RECENT FINDINGS: The innervation of the bone during development, homeostasis, and regeneration is highly patterned. Consistent with this, there have been nearly 100 studies over the past century that have used denervation approaches to isolate the effects of the different branches of the PNS on the bone. Overall, a common theme of balance emerges whereby an orchestration of both local and systemic neural functions must align to promote optimal skeletal repair while limiting negative consequences such as pain. An improved understanding of the functional bidirectional pathways linking the PNS and bone has important implications for skeletal development and regeneration. Clinical advances over the next century will necessitate a rigorous identification of the mechanisms underlying these effects that is cautious not to oversimplify the in vivo condition in diverse states of health and disease.

3D printed O2-generating scaffolds enhance osteoprogenitor- and type H vessel recruitment during bone healing.

Oxygen (O2)-delivering tissue substitutes have shown tremendous potential for enhancing tissue regeneration, maturation, and healing. As O2 is both a metabolite and powerful signaling molecule, providing controlled delivery is crucial for optimizing its beneficial effects in the treatment of critical-sized injuries. Here, we report the design and fabrication of 3D-printed, biodegradable, O2-generating bone scaffold comprising calcium peroxide (CPO) that once hydrolytically activated, provides long-term generation of oxygen at a controlled, concentration-dependent manner, and polycaprolactone (PCL), a hydrophobic polymer that regulate the interaction of CPO with water, preventing burst release of O2 at early time points. When anoxic conditions were simulated in vitro, CPO-PCL scaffolds maintained the survival and proliferation of human adipose-derived stem/stromal cells (hASCs) relative to PCL-only controls. We assessed the in vivo osteogenic efficacy of hASC-seeded CPO-PCL scaffolds implanted in a non-healing critical-sized 4-mm calvarial defects in nude mice for 8 weeks. Even without exogenous osteoinductive factors, CPO-PCL scaffolds demonstrated increased new bone volume compared to PCL-only scaffolds as verified by both microcomputed tomography analysis and histological assessments. Lastly, we employed a quantitative 3D lightsheet microscopy platform to determine that O2-generating scaffolds had similar vascular volumes with slightly higher presence of CD31hiEmcnhi pro-osteogenic, type H vessels and increased number of Osterix+ skeletal progenitor cells relative to PCL-only scaffolds. In summary, 3D-printed O2 generating CPO-PCL scaffolds with tunable O2 release rates provide a facile, customizable strategy for effectively treating, craniofacial bone defects. STATEMENT OF SIGNIFICANCE: Oxygen(O2)-delivering bone substitutes show promise in defect repair applications by supplying O2 to the cells within or around the graft, improving cell survivability and enhancing bone matrix mineralization. A novel O2-generating bone scaffold has been 3D printed for the first-time which ensures patient and defect specificity. 3D printed calcium peroxide-polycaprolactone (CPO-PCL) bone scaffold provides uninterrupted O2 supply for 22 days allowing cell survival in deprived O2 and nutrient conditions. For the first time, O2-driven bone regenerative environment in mice calvaria has been captured by light-sheet imaging which illuminates abundance of Osterix+ cells, angiogenesis at a single cell resolution indicating active site of bone remodeling and growth in the presence of O2.

3D Imaging Reveals Changes in the Neurovascular Architecture of the Murine Calvarium with Aging.

Calvarial nerves, along with vasculature, influence skull formation during development and following injury, but it remains unclear how calvarial nerves are spatially distributed during postnatal growth and aging. Studying the spatial distribution of nerves in the skull remains challenging due to a lack of methods to image and quantify 3D structures in intact bone. To visualize calvarial 3D neurovascular architecture, we imaged nerves and endothelial cells with lightsheet microscopy. We employed machine-learning-based segmentation to facilitate high-resolution characterization from post-natal day 0 (P0) to Week 80 (80wk). We found that TUBB3+ nerve density decreased with aging with the frontal bone demonstrating earlier onset age-related nerve loss than the parietal bone. In addition, nerves in the periosteum and dura mater exhibited similar yet distinct temporal patterns of nerve growth and loss. While no difference was observed in TUBB3+ nerves during skeletal maturation (P0 → 12wk), we did observe an increase in the volume of unmyelinated nerves in the dura mater. Regarding calvarial vasculature, larger CD31hiEmcn- vessel density increased with aging, while CD31hiEmcnhi vessel density was reduced. For all nerve markers studied, calvarial nerves maintained a preferential spatial association with CD31hiEmcnhi vessels that decreased with aging. Additionally, we used a model of Apert syndrome that demonstrates early coronal suture fusion to explore the impact of suture-related disease on neurovascular architecture. We identified a mild dysregulation of dural nerves and minor shifts in vessel populations. Collectively, this 3D, spatiotemporal characterization of calvarial nerves throughout the lifespan and provides new insights into age-induced neurovascular architecture.

Three-dimensional, label-free cell viability measurements in tissue engineering scaffolds using optical coherence tomography.

In the field of tissue engineering, 3D scaffolds and cells are often combined to yield constructs that are used as therapeutics to repair or restore tissue function in patients. Viable cells are often required to achieve the intended mechanism of action for the therapy, where the live cells may build new tissue or may release factors that induce tissue regeneration. Thus, there is a need to reliably measure cell viability in 3D scaffolds as a quality attribute of a tissue-engineered medical product. Here, we developed a noninvasive, label-free, 3D optical coherence tomography (OCT) method to rapidly (2.5 min) image large sample volumes (1 mm3 ) to assess cell viability and distribution within scaffolds. OCT imaging was assessed using a model scaffold-cell system consisting of a polysaccharide-based hydrogel seeded with human Jurkat cells. Four test systems were used: hydrogel seeded with live cells, hydrogel seeded with heat-shocked or fixed dead cells and hydrogel without any cells. Time series OCT images demonstrated changes in the time-dependent speckle patterns due to refractive index (RI) variations within live cells that were not observed for pure hydrogel samples or hydrogels with dead cells. The changes in speckle patterns were used to generate live-cell contrast by image subtraction. In this way, objects with large changes in RI were binned as live cells. Using this approach, on average, OCT imaging measurements counted 326 ± 52 live cells per 0.288 mm3 for hydrogels that were seeded with 288 live cells (as determined by the acridine orange-propidium iodide cell counting method prior to seeding cells in gels). Considering the substantial uncertainties in fabricating the scaffold-cell constructs, such as the error from pipetting and counting cells, a 13% difference in the live-cell count is reasonable. Additionally, the 3D distribution of live cells was mapped within a hydrogel scaffold to assess the uniformity of their distribution across the volume. Our results demonstrate a real-time, noninvasive method to rapidly assess the spatial distribution of live cells within a 3D scaffold that could be useful for assessing tissue-engineered medical products.

Measuring dimensionality of cell-scaffold contacts of primary human bone marrow stromal cells cultured on electrospun fiber scaffolds.

The properties and structure of the cellular microenvironment can influence cell behavior. Sites of cell adhesion to the extracellular matrix (ECM) initiate intracellular signaling that directs cell functions such as proliferation, differentiation, and apoptosis. Electrospun fibers mimic the fibrous nature of native ECM proteins and cell culture in fibers affects cell shape and dimensionality, which can drive specific functions, such as the osteogenic differentiation of primary human bone marrow stromal cells (hBMSCs), by. In order to probe how scaffolds affect cell shape and behavior, cell-fiber contacts were imaged to assess their shape and dimensionality through a novel approach. Fluorescent polymeric fiber scaffolds were made so that they could be imaged by confocal fluorescence microscopy. Fluorescent polymer films were made as a planar control. hBSMCs were cultured on the fluorescent substrates and the cells and substrates were imaged. Two different image analysis approaches, one having geometrical assumptions and the other having statistical assumptions, were used to analyze the 3D structure of cell-scaffold contacts. The cells cultured in scaffolds contacted the fibers in multiple planes over the surface of the cell, while the cells cultured on films had contacts confined to the bottom surface of the cell. Shape metric analysis indicated that cell-fiber contacts had greater dimensionality and greater 3D character than the cell-film contacts. These results suggest that cell adhesion site-initiated signaling could emanate from multiple planes over the cell surface during culture in fibers, as opposed to emanating only from the cell's basal surface during culture on planar surfaces.

S-nitrosylation of cytoskeletal proteins.

Nitric oxide has pronounced effects on cellular functions normally associated with the cytoskeleton, including cell motility, shape, contraction, and mitosis. Protein S-nitrosylation, the covalent addition of a NO group to a cysteine sulfur, is a signaling pathway for nitric oxide that acts in parallel to cyclic guanosine monophosphate (cGMP), but is poorly studied compared to the latter. There is growing evidence that S-nitrosylation of cytoskeletal proteins selectively alters their function. We review that evidence, and find that S-nitrosylation of cytoskeletal targets has complementary but distinct effects to cyclic-GMP in motile and contractile cells-promoting cell migration, and biasing muscle contraction toward relaxation. However, the effects of S-nitrosylation on a host of cytoskeletal proteins and functions remains to be explored.