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.

Integration of mechanics and biology in computer simulation of bone remodeling.

Bone remodeling is a complex physiological process that spans across multiple spatial and temporal scales and is regulated by both mechanical and hormonal cues. An imbalance between bone resorption and bone formation in the process of bone remodeling may lead to various bone pathologies. One powerful and non-invasive approach to gain new insights into mechano-adaptive bone remodeling is computer modeling and simulation. Recent findings in bone physiology and advances in computer modeling have provided a unique opportunity to study the integration of mechanics and biology in bone remodeling. Our objective in this review is to critically appraise recent advances and developments and discuss future research opportunities in computational bone remodeling approaches that enable integration of mechanics and cellular and molecular pathways. Based on the critical appraisal of the relevant recent published literature, we conclude that multiscale in silico integration of personalized bone mechanics and mechanobiology combined with data science and analytics techniques offer the potential to deepen our knowledge of bone remodeling and provide ample opportunities for future research.

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.

Mechanical stimuli and matrix properties modulate cancer spheroid growth in three-dimensional gelatin culture.

Most patients who succumb to cancer have metastases to bone that contribute to their death. Cancer cells that metastasize to bone are regularly subjected to mechanical stimuli that may affect their proliferation, growth and protein expression. Understanding why some cancer cells thrive in this environment could provide insight into new approaches to prevent or treat metastasis to bone. We used 4T1 cells as a model of breast cancer cells, and implanted them in gelatin hydrogels with moduli of 1 or 2.7 kPa to mimic the properties of bone marrow. The constructs were subjected to either perfusion of media through the hydrogel or combined perfusion and cyclic mechanical compression for 1 h d-1 for 4 d. Controls were cultured in free-swelling conditions. The cells formed spheroids during the 4 d of culture, with larger spheroids in the statically cultured constructs than in perfusion or compressed constructs. In stiffer gelatin, smaller spheroids formed in compressed constructs than perfusion alone, while compression had no effect compared to perfusion in the softer gelatin. Immunostaining indicated that the spheroids expressed osteopontin, parathyroid hormone-related protein and fibronectin, which are all hallmarks of bone metastasis. The proliferative marker Ki67 was present in all spheroids on day 4. In the 1 kPa gelatin, Ki67 staining intensity was greater in the statically cultured, free-swelling constructs than in bioreactor culture, regardless of dynamic compression. By contrast, proliferation was higher in the compressed gelatins compared to perfusion alone in the 2.7 kPa constructs, although the spheroids were smaller, on average. This suggests the stiffer gelatin may restrict spheroid growth at the same time that it enhances mechanobiological signalling during compression. Taken together, 4T1 breast cancer cells are mechanically sensitive, and mechanical stimuli can alter their proliferation and protein expression within soft materials with mechanical properties similar to bone marrow. As such, both in vivo and in vitro models of cancer metastasis should consider the role of the mechanical environment in the bone.

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.

Mineral deposition and vascular invasion of hydroxyapatite reinforced collagen scaffolds seeded with human adipose-derived stem cells.

BACKGROUND: Collagen-based scaffolds reinforced with hydroxyapatite (HA) are an attractive choice for bone tissue engineering because their composition mimics that of bone. We previously reported the development of compression-molded collagen-HA scaffolds that exhibited high porosity, interconnected pores, and mechanical properties that were well-suited for surgical handling and fixation. The objective of this study was to investigate these novel collagen-HA scaffolds in combination with human adipose-derived stem cells (hASCs) as a template for bone formation in a subcutaneous athymic mouse model. METHODS: Collagen-HA scaffolds and collagen-only scaffolds were fabricated as previously described, and a clinically approved bone void filler was used as a control for the material. Constructs were seeded with hASCs and were pre-treated with either control or osteogenic media. A cell-free group was also included. Scaffolds were implanted subcutaneously in the backs of athymic nude mice for 8 weeks. Mineral deposition was quantified via micro-computed tomography. Histological and immunofluorescence images of the explants were used to analyze their vascular invasion, remodeling and cellularity. RESULTS: Cell-free collagen-HA scaffolds and those that were pre-seeded with osteogenically differentiated hASCs supported mineral deposition and vascular invasion at comparable rates, while cell-seeded constructs treated with the control medium showed lower mineralization after implantation. HA-reinforcement allowed collagen constructs to maintain their shape, provided improved cell-tissue-scaffold integration, and resulted in a more organized tissue when pre-treated in an osteogenic medium. Scaffold type and pre-treatment also determined osteoclast activity and therefore potential remodeling of the constructs. CONCLUSIONS: The results of this study cumulatively indicate that treatment medium and scaffold composition direct mineralization and angiogenic tissue formation in an ectopic model. The data suggest that it may be necessary to match the scaffold with a particular cell type and cell-specific pre-treatment to achieve optimal bone formation.

The CXCL5/CXCR2 axis is sufficient to promote breast cancer colonization during bone metastasis.

Bone is one of the most common sites for metastasis across cancers. Cancer cells that travel through the vasculature and invade new tissues can remain in a non-proliferative dormant state for years before colonizing the metastatic site. Switching from dormancy to colonization is the rate-limiting step of bone metastasis. Here we develop an ex vivo co-culture method to grow cancer cells in mouse bones to assess cancer cell proliferation using healthy or cancer-primed bones. Profiling soluble factors from conditioned media identifies the chemokine CXCL5 as a candidate to induce metastatic colonization. Additional studies using CXCL5 recombinant protein suggest that CXCL5 is sufficient to promote breast cancer cell proliferation and colonization in bone, while inhibition of its receptor CXCR2 with an antagonist blocks proliferation of metastatic cancer cells. This study suggests that CXCL5 and CXCR2 inhibitors may have efficacy in treating metastatic bone tumors dependent on the CXCL5/CXCR2 axis.

Potassium channel activity controls breast cancer metastasis by affecting ß-catenin signaling.

Potassium ion channels are critical in the regulation of cell motility. The acquisition of cell motility is an essential parameter of cancer metastasis. However, the role of K+ channels in cancer metastasis has been poorly studied. High expression of the hG1 gene, which encodes for Kv11.1 channel associates with good prognosis in estrogen receptor-negative breast cancer (BC). We evaluated the efficacy of the Kv11.1 activator NS1643 in arresting metastasis in a triple negative breast cancer (TNBC) mouse model. NS1643 significantly reduces the metastatic spread of breast tumors in vivo by inhibiting cell motility, reprogramming epithelial-mesenchymal transition via attenuation of Wnt/ß-catenin signaling and suppressing cancer cell stemness. Our findings provide important information regarding the clinical relevance of potassium ion channel expression in breast tumors and the mechanisms by which potassium channel activity can modulate tumor biology. Findings suggest that Kv11.1 activators may represent a novel therapeutic approach for the treatment of metastatic estrogen receptor-negative BC. Ion channels are critical factor for cell motility but little is known about their role in metastasis. Stimulation of the Kv11.1 channel suppress the metastatic phenotype in TNBC. This work could represent a paradigm-shifting approach to reducing mortality by targeting a pathway that is central to the development of metastases.

Shear Stress in Bone Marrow has a Dose Dependent Effect on cFos Gene Expression in In Situ Culture.

INTRODUCTION: Mechanical stimulation of bone is necessary to maintain its mass and architecture. Osteocytes within the mineralized matrix are sensors of mechanical deformation of the hard tissue, and communicate with cells in the marrow to regulate bone remodeling. However, marrow cells are also subjected to mechanical stress during whole bone loading, and may contribute to mechanically regulated bone physiology. Previous results from our laboratory suggest that mechanotransduction in marrow cells is sufficient to cause bone formation in the absence of osteocyte signaling. In this study, we investigated whether bone formation and altered marrow cell gene expression response to stimulation was dependent on the shear stress imparted on the marrow by our loading regime. METHODS: Porcine trabecular bone explants were cultured in an in situ bioreactor for 5 or 28 days with stimulation twice daily. Gene expression and bone formation were quantified and compared to unstimulated controls. Correlation was used to assess the dependence on shear stress imparted by the loading regime calculated using computational fluid dynamics models. RESULTS: Vibratory stimulation resulted in a higher trabecular bone formation rate (p = 0.01) and a greater increase in bone volume fraction (p = 0.02) in comparison to control explants. Marrow cell expression of cFos increased with the calculated marrow shear stress in a dose-dependent manner (p = 0.002). CONCLUSIONS: The results suggest that the shear stress due to interactions between marrow cells induces a mechanobiological response. Identification of marrow cell mechanotransduction pathways is essential to understand healthy and pathological bone adaptation and remodeling.

Bone marrow mechanotransduction in porcine explants alters kinase activation and enhances trabecular bone formation in the absence of osteocyte signaling.

Bone is a dynamic tissue that can adapt its architecture in response to mechanical signals under the control of osteocytes, which sense mechanical deformation of the mineralized bone. However, cells in the marrow are also mechanosensitive and may contribute to load-induced bone adaptation, as marrow is subjected to mechanical stress during bone deformation. We investigated the contribution of mechanotransduction in marrow cells to trabecular bone formation by applying low magnitude mechanical stimulation (LMMS) to porcine vertebral trabecular bone explants in an in situ bioreactor. The bone formation rate was higher in stimulated explants compared to unloaded controls which represent a disuse condition (CNT). However, sclerostin protein expression in osteocytes was not different between groups, nor was expression of osteocytic mechanoregulatory genes SOST, IGF-1, CTGF, and Cyr61, suggesting the mechanoregulatory program of osteocytes was unaffected by the loading regime. In contrast, c-Fos, a gene indicative of mechanical stimulation, was upregulated in the marrow cells of mechanically stimulated explants, while the level of activated c-Jun decreased by 25%. The activator protein 1 (AP-1) transcription factor is a heterodimer of c-Fos and c-Jun, which led us to investigate the expression of the downstream target gene cyclin-D1, a gene associated with cell cycle progression and osteogenesis. Cyclin-D1 gene expression in the stimulated marrow was approximately double that of the controls. The level of phosphorylated PYK2, a purported inhibitor of osteoblast differentiation, also decreased in marrow cells from stimulated explants. Taken together, mechanotransduction in marrow cells induced trabecular bone formation independent of osteocyte signaling. Identifying the specific cells and signaling pathways involved, and verifying them with inhibition of specific signaling molecules, could lead to potential therapeutic targets for diseases characterized by bone loss.

Mechanically induced bone formation is not sensitive to local osteocyte density in rat vertebral cancellous bone.

Osteocytes play an integral role in bone by sensing mechanical stimuli and releasing signaling factors that direct bone formation. The importance of osteocytes in mechanotransduction suggests that regions of bone tissue with greater osteocyte populations are more responsive to mechanical stimuli. To determine the effects of osteocyte population on bone functional adaptation we applied mechanical loads to the 8th caudal vertebra of skeletally mature female Sprague Dawley rats (6 months of age, n = 8 loaded, n = 8 sham controls). The distribution of tissue stress/strain within cancellous bone was determined using high-resolution finite element models, osteocyte distribution was determined using nano-computed tomography, and locations of bone formation were determined using three-dimensional images of fluorescent bone formation markers. Loading increased bone formation (3D MS/BS 10.82 ± 2.09% in loaded v. 3.17 ± 2.05% in sham control, mean ± SD). Bone formation occurred at regions of cancellous bone experiencing greater tissue stress/strain, however stress/strain was only a modest predictor of bone formation; even at locations of greatest stress/strain the probability of observing bone formation did not exceed 41%. The local osteocyte population was not correlated with locations of new bone formation. The findings support the idea that local tissue stress/strain influence the locations of bone formation in cancellous bone, but suggest that the size of the osteocyte population itself is not influential. We conclude that other aspects of osteocytes such as osteocyte connectivity, lacunocanilicular nano-geometry, and/or fluid pressure/shear distributions within the marrow space may be more influential in regulating bone mechanotransduction than the number of osteocytes. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:672-681, 2018.

Intraoperative biomechanics of lumbar pedicle screw loosening following successful arthrodesis.

Pedicle screw loosening has been implicated in recurrent back pain after lumbar spinal fusion, but the degree of loosening has not been systematically quantified in patients. Instrumentation removal is an option for patients with successful arthrodesis, but remains controversial. Here, we quantified pedicle screw loosening by measuring screw insertion and/or removal torque at high statistical power (beta = 0.02) in N = 108 patients who experienced pain recurrence despite successful fusion after posterior instrumented lumbar fusion with anterior lumbar interbody fusion (L2-S1). Between implantation and removal, pedicle screw torque was reduced by 58%, indicating significant loosening over time. Loosening was greater in screws with evoked EMG threshold under 11 mA, indicative of screw misplacement. A theoretical stress analysis revealed increased local stresses at the screw interface in pedicles with decreased difference in pedicle thickness and screw diameter. Loosening was greatest in vertebrae at the extremities of the fused segments, but was significantly lower in segments with one level of fusion than in those with two or more. CLINICAL SIGNIFICANCE: These data indicate that pedicle screws can loosen significantly in patients with recurrent back pain and warrant further research into methods to reduce the incidence of screw loosening and to understand the risks and potential benefits of instrumentation removal. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2673-2681, 2017.

Altered architecture and cell populations affect bone marrow mechanobiology in the osteoporotic human femur.

Age-related increases in trabecular bone porosity, as seen in osteoporosis, not only affect the strength and stiffness, but also potentially the mechanobiological response of bone. The mechanical interaction between trabecular bone and bone marrow is one source of mechanobiological signaling, as many cell populations in marrow are mechanosensitive. However, measuring the mechanics of this interaction is difficult, due to the length scales and geometric complexity of trabecular bone. In this study, a multi-scale computational scheme incorporating high-resolution, tissue-level, fluid-structure interaction simulations with discrete cell-level models was applied to characterize the potential effects of trabecular porosity and marrow composition on marrow mechanobiology in human femoral bone. First, four tissue-level models with different volume fractions (BV/TV) were subjected to cyclic compression to determine the continuum level shear stress in the marrow. The calculated stress was applied to three detailed models incorporating individual cells and having differing adipocyte fractions. At the tissue level, compression of the bone along its principal mechanical axis induced shear stress in the marrow ranging from 2.0 to 5.6 Pa, which increased with bone volume fraction and strain rate. The shear stress was amplified at the cell level, with over 90% of non-adipocyte cells experiencing higher shear stress than the applied tissue-level stress. The maximum shear stress decreased by 20% when the adipocyte volume fraction (AVF) increased from 30%, as seen in young healthy marrow, to 45 or 60% AVF typically found in osteoporotic patients. The results suggest that increasing AVF has similar effects on the mechanobiological signaling in bone marrow as decreased volume fraction.

Comparison of solid and fluid constitutive models of bone marrow during trabecular bone compression.

The mechanical environment and mechanobiology of bone marrow may play essential roles in bone adaptation, cancer metastasis, and immune cell regulation. However, the location of marrow within the trabecular pore space complicates experimental measurement of marrow mechanics. Computational models provide a means to assess the shear stress and pressure in the marrow during physiological loading, but they rely on accurate inputs for the marrow and the physics assumed for the interaction of bone and marrow. Elastic, viscoelastic, and fluid constitutive properties have all been reported from experimental measurements of marrow properties. It is unclear whether this ambiguity reflects the various length-scales, loading rates, and boundary conditions of the experiments, or if the material models are sufficiently similar as to be interchangeable. To address this question, we analyzed both the mean shear stress and its spatial distribution induced in marrow during compression of trabecular bone cubes when using linear elastic, neo-Hookean, viscoelastic, and power-law fluid constitutive models. Experimentally reported parameters were initially applied for all four constitutive models, resulting in poor agreement. The parameters of the soft solid models were calibrated by linear interpolation so that the volume averaged shear stress agreed with the fluid model for each, but this could only be accomplished on a specimen-by-specimen basis. Following calibration, the root-mean-squared (RMS) difference between the solid and fluid constitutive models was still greater than 26% even when the overall mean shear stress was in close agreement, indicating that the spatial distribution of stress is also sensitive to the constitutive model. As such, the choice of constitutive model should be backed by a strong rationale, and results should be interpreted with care.

The roles of architecture and estrogen depletion in microdamage risk in trabecular bone.

Bone quantity, or density, has insufficient power to discriminate fracture risk in individuals. Additional measures of bone quality, such as microarchitectural characteristics and bone tissue properties, including the presence of damage, may improve the diagnosis of fracture risk. Microdamage and microarchitecture are two aspects of trabecular bone quality that are interdependent, with several microarchitectural changes strongly correlated to damage risk after compensating for bone density. This study aimed to delineate the effects of microarchitecture and estrogen depletion on microdamage susceptibility in trabecular bone using an ovariectomized sheep model to mimic post-menopausal osteoporosis. The propensity for microdamage formation in trabecular bone of the distal femur was studied using a sequence of compressive and torsional overloads. Ovariectomy had only minor effects on the microarchitecture at this anatomic site. Microdamage was correlated to bone volume fraction and structure model index (SMI), and ovariectomy increased the sensitivity to these parameters. The latter may be due to either increased resorption cavities acting as stress concentrations or to altered bone tissue properties. Pre-existing damage was also correlated to new damage formation. However, sequential loading primarily generated new cracks as opposed to propagating existing cracks, suggesting that pre-existing microdamage contributes to further damage of bone by shifting load bearing to previously undamaged trabeculae, which are subsequently damaged. The transition from plate-like to rod-like trabeculae, indicated by SMI, dictates this shift, and may be a hallmark of bone that is already predisposed to accruing greater levels of damage through compromised microarchitecture.

The sensitivity of nonlinear computational models of trabecular bone to tissue level constitutive model.

Microarchitectural finite element models have become a key tool in the analysis of trabecular bone. Robust, accurate, and validated constitutive models would enhance confidence in predictive applications of these models and in their usefulness as accurate assays of tissue properties. Human trabecular bone specimens from the femoral neck (n = 3), greater trochanter (n = 6), and lumbar vertebra (n = 1) of eight different donors were scanned by µ-CT and converted to voxel-based finite element models. Unconfined uniaxial compression and shear loading were simulated for each of three different constitutive models: a principal strain-based model, Drucker-Lode, and Drucker-Prager. The latter was applied with both infinitesimal and finite kinematics. Apparent yield strains exhibited minimal dependence on the constitutive model, differing by at most 16.1%, with the kinematic formulation being influential in compression loading. At the tissue level, the quantities and locations of yielded tissue were insensitive to the constitutive model, with the exception of the Drucker-Lode model, suggesting that correlation of microdamage with computational models does not improve the ability to discriminate between constitutive laws. Taken together, it is unlikely that a tissue constitutive model can be fully validated from apparent-level experiments alone, as the calculations are too insensitive to identify differences in the outcomes. Rather, any asymmetric criterion with a valid yield surface will likely be suitable for most trabecular bone models.

Pressure and shear stress in trabecular bone marrow during whole bone loading.

Skeletal adaptation to mechanical loading is controlled by mechanobiological signaling. Osteocytes are highly responsive to applied strains, and are the key mechanosensory cells in bone. However, many cells residing in the marrow also respond to mechanical cues such as hydrostatic pressure and shear stress, and hence could play a role in skeletal adaptation. Trabecular bone encapsulates marrow, forming a poroelastic solid. According to the mechanical theory, deformation of the pores induces motion in the fluid-like marrow, resulting in pressure and velocity gradients. The latter results in shear stress acting between the components of the marrow. To characterize the mechanical environment of trabecular bone marrow in situ, pore pressure within the trabecular compartment of whole porcine femurs was measured with miniature pressure transducers during stress-relaxation and cyclic loading. Pressure gradients ranging from 0.013 to 0.46 kPa/mm were measured during loading. This range was consistent with calculated pressure gradients from continuum scale poroelastic models with the same permeability. Micro-scale computational fluid dynamics models created from computed tomography images were used to calculate the micromechanical stress in the marrow using the measured pressure differentials as boundary conditions. The volume averaged shear stress in the marrow ranged from 1.67 to 24.55 Pa during cyclic loading, which exceeds the mechanostimulatory threshold for mesenchymal lineage cells. Thus, the loading of bone through activities of daily living may be an essential component of bone marrow health and mechanobiology. Additional studies of cell-level interactions during loading in healthy and disease conditions will provide further incite into marrow mechanobiology.

Hydroxyapatite reinforced collagen scaffolds with improved architecture and mechanical properties.

Hydroxyapatite (HA) reinforced collagen scaffolds have shown promise for synthetic bone graft substitutes and tissue engineering scaffolds. Freeze-dried HA-collagen scaffolds are readily fabricated and have exhibited osteogenicity in vivo, but are limited by an inherent scaffold architecture that results in a relatively small pore size and weak mechanical properties. In order to overcome these limitations, HA-collagen scaffolds were prepared by compression molding HA reinforcements and paraffin microspheres within a suspension of concentrated collagen fibrils (∼ 180 mg/mL), cross-linking the collagen matrix, and leaching the paraffin porogen. HA-collagen scaffolds exhibited an architecture with high porosity (85-90%), interconnected pores ∼ 300-400 µm in size, and struts ∼ 3-100 µm in thickness containing 0-80 vol% HA whisker or powder reinforcements. HA reinforcement enabled a compressive modulus of up to ∼ 1 MPa, which was an order of magnitude greater than unreinforced collagen scaffolds. The compressive modulus was also at least one order of magnitude greater than comparable freeze-dried HA-collagen scaffolds and two orders of magnitude greater than absorbable collagen sponges used clinically. Moreover, scaffolds reinforced with up to 60 vol% HA exhibited fully recoverable elastic deformation upon loading to 50% compressive strain for at least 100,000 cycles. Thus, the scaffold mechanical properties were well-suited for surgical handling, fixation, and bearing osteogenic loads during bone regeneration. The scaffold architecture, permeability, and composition were shown to be conducive to the infiltration and differentiation of adipose-derive stromal cells in vitro. Acellular scaffolds were demonstrated to induce angiogenesis and osteogenesis after subcutaneous ectopic implantation by recruiting endogenous cell populations, suggesting that the scaffolds were osteoinductive.

Development of an in vivo rabbit ulnar loading model.

Ulnar and tibial cyclic compression in rats and mice have become the preferred animal models for investigating the effects of mechanical loading on bone modeling/remodeling. Unlike rodents, rabbits provide a larger bone volume and normally exhibit intracortical Haversian remodeling, which may be advantageous for investigating mechanobiology and pharmaceutical interventions in cortical bone. Therefore, the objective of this study was to develop and validate an in vivo rabbit ulnar loading model. Ulnar tissue strains during loading of intact forelimbs were characterized and calibrated to applied loads using strain gauge measurements and specimen-specific finite element models. Periosteal bone formation in response to varying strain levels was measured by dynamic histomorphometry at the location of maximum strain in the ulnar diaphysis. Ulnae loaded at 3000 microstrain did not exhibit periosteal bone formation greater than the contralateral controls. Ulnae loaded at 3500, 4000, and 4500 microstrain exhibited a dose-dependent increase in periosteal mineralizing surface (MS/BS) compared with contralateral controls during the second week of loading. Ulnae loaded at 4500 microstrain exhibited the most robust response with significantly increased MS/BS at multiple time points extending at least 2weeks after loading was ceased. Ulnae loaded at 5250 microstrain exhibited significant woven bone formation. Rabbits required greater strain levels to produce lamellar and woven bone on periosteal surfaces compared with rats and mice, perhaps due to lower basal levels of MS/BS. In summary, bone adaptation during rabbit ulnar loading was tightly controlled and may provide a translatable model for human bone biology in preclinical investigations of metabolic bone disease and pharmacological treatments.

The in situ mechanics of trabecular bone marrow: the potential for mechanobiological response.

Bone adapts to habitual loading through mechanobiological signaling. Osteocytes are the primary mechanical sensors in bone, upregulating osteogenic factors and downregulating osteoinhibitors, and recruiting osteoclasts to resorb bone in response to microdamage accumulation. However, most of the cell populations of the bone marrow niche,which are intimately involved with bone remodeling as the source of bone osteoblast and osteoclast progenitors, are also mechanosensitive. We hypothesized that the deformation of trabecular bone would impart mechanical stress within the entrapped bone marrow consistent with mechanostimulation of the constituent cells. Detailed fluid-structure interaction models of porcine femoral trabecular bone and bone marrow were created using tetrahedral finite element meshes. The marrow was allowed to flow freely within the bone pores, while the bone was compressed to 2000 or 3000 microstrain at the apparent level.Marrow properties were parametrically varied from a constant 400 mPas to a power law rule exceeding 85 Pas. Deformation generated almost no shear stress or pressure in the marrow for the low viscosity fluid, but exceeded 5 Pa when the higher viscosity models were used. The shear stress was higher when the strain rate increased and in higher volume fraction bone. The results demonstrate that cells within the trabecular bone marrow could be mechanically stimulated by bone deformation, depending on deformation rate, bone porosity, and bone marrow properties. Since the marrow contains many mechanosensitive cells, changes in the stimulatory levels may explain the alterations in bone marrow morphology with aging and disease, which may in turn affect the trabecular bone mechanobiology and adaptation.