Controlled mechanical loading affects the osteocyte transcriptome in porcine trabecular bone in situ.

INTRODUCTION: Osteocytes modulate bone adaptation in response to mechanical stimuli imparted by the deforming bone tissue in which they are encased by communicating with osteoclasts and osteoblasts as well as other osteocytes in the lacuna-canalicular network through secreted cytokines and chemokines. Understanding the transcriptional response of osteocytes to mechanical stimulation in situ could identify new targets to inhibit bone loss or enhance bone formation in the presence of diseases like osteoporosis or metastatic cancer. We compared the mechanically regulated transcriptional response of osteocytes in trabecular bone following one or three days of controlled mechanical loading. METHODS: Porcine trabecular bone explants were cultured in a bioreactor for 48 h and subsequently loaded twice a day for one day or 3 days. RNA was isolated and sequenced, and the Tuxedo suite was used to identify differentially expressed genes and pathway analysis was conducted using Ingenuity Pathway Analysis (IPA). RESULTS: There were about 4000 differentially expressed genes following in situ culture relative to fresh bone. One hundred six genes were differentially expressed between the loaded and non-loaded groups following one day of loading compared to 913 genes after 3 d of loading. Only 45 of these were coincident between the two time points, indicating an evolving transcriptome. Clustering and principal component analysis indicated differences between the loaded and non-loaded groups after 3 d of loading. DISCUSSION: With sustained loading, there was a nine-fold increase in the number of differentially expressed genes, suggesting that osteocytes respond to loading through sequential activation of downstream genes in the same pathways. The differentially expressed genes were related to osteoarthritis, osteocyte, and chondrocyte signaling pathways. We noted that NFkB and TNF signaling are affected by early loading and this may drive downstream effects on the mechanobiological response. Moreover, these genes may regulate catabolic effects of mechanical disuse through their actions on pre-osteoclasts in the bone marrow niche.

Investigation of direction- and age-dependent prestretch in mouse cranial dura mater.

Cranial dura mater is a dense interwoven vascularized connective tissue that helps regulate neurocranial remodeling by responding to strains from the growing brain. Previous ex vivo experimentation has failed to account for the role of prestretch in the mechanical behavior of the dura. Here we aim to estimate the prestretch in mouse cranial dura mater and determine its dependency on direction and age. We performed transverse and longitudinal incisions in parietal dura excised from newborn (day [Formula: see text]4) and mature (12 weeks) mice and calculated the ex vivo normalized incision opening (measured width over length). Then, similar incisions were simulated under isotropic stretching within Abaqus/Standard. Finally, prestretch was estimated by comparing the ex vivo and in silico normalized openings. There were no significant differences between the neonatal and adult mice when comparing cuts in the same direction, but adult mice were found to have significantly greater stretch in the anterior-posterior direction than in the medial-lateral direction, while neonatal dura was essentially isotropic. Additionally, our simulations show that increasing curvature impacts the incision opening, indicating that flat in silico models may overestimate prestretch.

The effect of marrow secretome and culture environment on the rate of metastatic breast cancer cell migration in two and three dimensions.

Metastasis is responsible for over 90% of cancer-related deaths, and bone is the most common site for breast cancer metastasis. Metastatic breast cancer cells home to trabecular bone, which contains hematopoietic and stromal lineage cells in the marrow. As such, it is crucial to understand whether bone or marrow cells enhance breast cancer cell migration toward the tissue. To this end, we quantified the migration of MDA-MB-231 cells toward human bone in two- and three-dimensional (3D) environments. First, we found that the cancer cells cultured on tissue culture plastic migrated toward intact trabecular bone explants at a higher rate than toward marrow-deficient bone or devitalized bone. Leptin was more abundant in conditioned media from the cocultures with intact explants, while higher levels of IL-1ß, IL-6, and TNFα were detected in cultures with both intact bone and cancer cells. We further verified that the cancer cells migrated into bone marrow using a bioreactor culture system. Finally, we studied migration toward bone in 3D gelatin. Migration speed did not depend on stiffness of this homogeneous gel, but many more dendritic-shaped cancer cells oriented and migrated toward bone in stiffer gels than softer gels, suggesting a coupling between matrix mechanics and chemotactic signals.

Effects of mechanobiological signaling in bone marrow on skeletal health.

Bone marrow is a cellular tissue that forms within the pore space and hollow diaphysis of bones. As a tissue, its primary function is to support the hematopoietic progenitor cells that maintain the populations of both erythroid and myeloid lineage cells in the bone marrow, making it an essential element of normal mammalian physiology. However, bone's primary function is load bearing, and deformations induced by external forces are transmitted to the encapsulated marrow. Understanding the effects of these mechanical inputs on marrow function and adaptation requires knowledge of the material behavior of the marrow at multiple scales, the loads that are applied, and the mechanobiology of the cells. This paper reviews the current state of knowledge of each of these factors. Characterization of the marrow mechanical environment and its role in skeletal health and other marrow functions remains incomplete, but research on the topic is increasing, driven by interest in skeletal adaptation and the mechanobiology of cancer metastasis.