Tissue-Level Regeneration and Remodeling Dynamics are Driven by Mechanical Stimuli in the Microenvironment in a Post-Bridging Loaded Femur Defect Healing Model in Mice.

Bone healing and remodeling are mechanically driven processes. While the generalized response to mechanical stimulation in bone is well-understood, much less is known about the mechanobiology-regulating tissue-scale bone formation and resorption during the reparative and remodeling phases of fracture healing. In this study, we combined computational approaches in the form of finite element analysis and experimental approaches by using a loaded femoral defect model in mice to investigate the role of mechanical stimulation in the microenvironment of bone. Specifically, we used longitudinal micro-computed tomography to observe temporal changes in bone at different densities and micro-finite element analysis to map the mechanics of the microenvironment to tissue-scale formation, quiescence (no change in bone presence between time points), and resorption dynamics in the late reparative and remodeling phases (post bridging). Increasing levels of effective strain led to increasing conditional probability of bone formation, while decreasing levels of effective strain led to increasing probability of bone resorption. In addition, the analysis of mineralization dynamics showed both a temporal and effective strain level-dependent behavior. A logarithmic-like response was displayed, where the conditional probability of bone formation or resorption increased rapidly and plateaued or fell rapidly and plateaued as mechanical strain increased.

Individualized cyclic mechanical loading improves callus properties during the remodelling phase of fracture healing in mice as assessed from time-lapsed in vivo imaging.

Fracture healing is regulated by mechanical loading. Understanding the underlying mechanisms during the different healing phases is required for targeted mechanical intervention therapies. Here, the influence of individualized cyclic mechanical loading on the remodelling phase of fracture healing was assessed in a non-critical-sized mouse femur defect model. After bridging of the defect, a loading group (n = 10) received individualized cyclic mechanical loading (8-16 N, 10 Hz, 5 min, 3 × /week) based on computed strain distribution in the mineralized callus using animal-specific real-time micro-finite element analysis with 2D/3D visualizations and strain histograms. Controls (n = 10) received 0 N treatment at the same post-operative time-points. By registration of consecutive scans, structural and dynamic callus morphometric parameters were followed in three callus sub-volumes and the adjacent cortex showing that the remodelling phase of fracture healing is highly responsive to cyclic mechanical loading with changes in dynamic parameters leading to significantly larger formation of mineralized callus and higher degree of mineralization. Loading-mediated maintenance of callus remodelling was associated with distinct effects on Wnt-signalling-associated molecular targets Sclerostin and RANKL in callus sub-regions and the adjacent cortex (n = 1/group). Given these distinct local protein expression patterns induced by cyclic mechanical loading during callus remodelling, the femur defect loading model with individualized load application seems suitable to further understand the local spatio-temporal mechano-molecular regulation of the different fracture healing phases.

Real-time finite element analysis allows homogenization of tissue scale strains and reduces variance in a mouse defect healing model.

Mechanical loading allows both investigation into the mechano-regulation of fracture healing as well as interventions to improve fracture-healing outcomes such as delayed healing or non-unions. However, loading is seldom individualised or even targeted to an effective mechanical stimulus level within the bone tissue. In this study, we use micro-finite element analysis to demonstrate the result of using a constant loading assumption for all mouse femurs in a given group. We then contrast this with the application of an adaptive loading approach, denoted real time Finite Element adaptation, in which micro-computed tomography images provide the basis for micro-FE based simulations and the resulting strains are manipulated and targeted to a reference distribution. Using this approach, we demonstrate that individualised femoral loading leads to a better-specified strain distribution and lower variance in tissue mechanical stimulus across all mice, both longitudinally and cross-sectionally, while making sure that no overloading is occurring leading to refracture of the femur bones.

Application of subject-specific adaptive mechanical loading for bone healing in a mouse tail vertebral defect.

Methods to repair bone defects arising from trauma, resection, or disease, continue to be sought after. Cyclic mechanical loading is well established to influence bone (re)modelling activity, in which bone formation and resorption are correlated to micro-scale strain. Based on this, the application of mechanical stimulation across a bone defect could improve healing. However, if ignoring the mechanical integrity of defected bone, loading regimes have a high potential to either cause damage or be ineffective. This study explores real-time finite element (rtFE) methods that use three-dimensional structural analyses from micro-computed tomography images to estimate effective peak cyclic loads in a subject-specific and time-dependent manner. It demonstrates the concept in a cyclically loaded mouse caudal vertebral bone defect model. Using rtFE analysis combined with adaptive mechanical loading, mouse bone healing was significantly improved over non-loaded controls, with no incidence of vertebral fractures. Such rtFE-driven adaptive loading regimes demonstrated here could be relevant to clinical bone defect healing scenarios, where mechanical loading can become patient-specific and more efficacious. This is achieved by accounting for initial bone defect conditions and spatio-temporal healing, both being factors that are always unique to the patient.

Mechano-Regulation of Trabecular Bone Adaptation Is Controlled by the Local in vivo Environment and Logarithmically Dependent on Loading Frequency.

It is well-established that cyclic, but not static, mechanical loading has anabolic effects on bone. However, the function describing the relationship between the loading frequency and the amount of bone adaptation remains unclear. Using a combined experimental and computational approach, this study aimed to investigate whether trabecular bone mechano-regulation is controlled by mechanical signals in the local in vivo environment and dependent on loading frequency. Specifically, by combining in vivo micro-computed tomography (micro-CT) imaging with micro-finite element (micro-FE) analysis, we monitored the changes in microstructural as well as the mechanical in vivo environment [strain energy density (SED) and SED gradient] of mouse caudal vertebrae over 4 weeks of either cyclic loading at varying frequencies of 2, 5, or 10 Hz, respectively, or static loading. Higher values of SED and SED gradient on the local tissue level led to an increased probability of trabecular bone formation and a decreased probability of trabecular bone resorption. In all loading groups, the SED gradient was superior in the determination of local bone formation and resorption events as compared to SED. Cyclic loading induced positive net (re)modeling rates when compared to sham and static loading, mainly due to an increase in mineralizing surface and a decrease in eroded surface. Consequently, bone volume fraction increased over time in 2, 5, and 10 Hz (+15%, +21% and +24%, p ≤ 0.0001), while static loading led to a decrease in bone volume fraction (-9%, p ≤ 0.001). Furthermore, regression analysis revealed a logarithmic relationship between loading frequency and the net change in bone volume fraction over the 4 week observation period (R 2 = 0.74). In conclusion, these results suggest that trabecular bone adaptation is regulated by mechanical signals in the local in vivo environment and furthermore, that mechano-regulation is logarithmically dependent on loading frequency with frequencies below a certain threshold having catabolic effects, and those above anabolic effects. This study thereby provides valuable insights toward a better understanding of the mechanical signals influencing trabecular bone formation and resorption in the local in vivo environment.

Optimization of mechanical stiffness and cell density of 3D bioprinted cell-laden scaffolds improves extracellular matrix mineralization and cellular organization for bone tissue engineering.

Bioprinting is an emerging technology in which cell-laden biomaterials are precisely dispersed to engineer artificial tissues that mimic aspects of the anatomical and structural complexity of relatively soft tissues such as skin, vessels, and cartilage. However, reproducing the highly mineralized and cellular diversity of bone tissue is still not easily achievable and is yet to be demonstrated. Here, an extrusion-based 3D bioprinting strategy is utilized to fabricate 3D bone-like tissue constructs containing osteogenic cellular organization. A simple and low-cost bioink for 3D bioprinting of bone-like tissue is prepared based on two unmodified polymers (alginate and gelatin) and combined with human mesenchymal stem cells (hMSCs). To form 3D bone-like tissue and bone cell phenotype, the influence of different scaffold stiffness and cell density of 3D bioprinted cell-laden porous scaffolds on osteogenic differentiation and bone-like tissue formation was investigated over time. Our results showed that soft scaffolds (0.8%alg, 0.66 ± 0.08 kPa) had higher DNA content, enhanced ALP activity and stimulated osteogenic differentiation than stiff scaffolds (1.8%alg, 5.4 ± 1.2 kPa). At day 42, significantly more mineralized tissue was formed in soft scaffolds than in stiff scaffolds (43.5 ± 7.1 mm3 vs. 22.6 ± 6.0 mm3). Importantly, immunohistochemistry staining demonstrated more osteocalcin protein expression in high mineral compared to low mineral regions. Additionally, cells in soft scaffolds exhibited osteoblast- and early osteocyte-related gene expression and 3D cellular network within the mineralized matrix at day 42. Furthermore, the results showed that cell density in 15 M cells/ml can promote cell-cell connections at day 7 and mineral formation at day 14, while 5 M cells/ml had the significantly higher mineral formation rate than 15 M cells/ml from day 14 to day 21. In summary, this work reports the formation of 3D bioprinted bone-like tissue using a simple and low-cost cell-laden bioink, which was optimized for stiffness and cell density, showing great promise for bone tissue engineering applications. STATEMENT OF SIGNIFICANCE: In this study, we presented for the first time a framework combining 3D bioprinting, bioreactor system and time-lapsed micro-CT monitoring to provide in vitro scaffold fabrication, maturation, and mineral visualization for bone tissue engineering. 3D bone-like tissue constructs have been formed via optimizing scaffold stiffness and cell density. The soft scaffolds had higher cell proliferation, enhanced alkaline phosphatase activity and stimulated osteogenic differentiation with 3D cellular network foramtion than stiff scaffolds. Significantly more mineralized bone-like tissue was formed in soft scaffolds than stiff scaffolds at day 42. Meanwhile, cell density in 15 M cells/ml can promote cell-cell connections and mineral formation in 14 days, while the higher mineral formation rate was found in 5 M cells/ml from day 14 to day 21.

The association between mineralised tissue formation and the mechanical local in vivo environment: Time-lapsed quantification of a mouse defect healing model.

An improved understanding of how local mechanical stimuli guide the fracture healing process has the potential to enhance clinical treatment of bone injury. Recent preclinical studies of bone defect in animal models have used cross-sectional data to examine this phenomenon indirectly. In this study, a direct time-lapsed imaging approach was used to investigate the local mechanical strains that precede the formation of mineralised tissue at the tissue scale. The goal was to test two hypotheses: 1) the local mechanical signal that precedes the onset of tissue mineralisation is higher in areas which mineralise, and 2) this local mechanical signal is independent of the magnitude of global mechanical loading of the tissue in the defect. Two groups of mice with femoral defects of length 0.85 mm (n = 10) and 1.45 mm (n = 9) were studied, allowing for distinct distributions of tissue scale strains in the defects. The regeneration and (re)modelling of mineralised tissue was observed weekly using in vivo micro-computed tomography (micro-CT), which served as a ground truth for resolving areas of mineralised tissue formation. The mechanical environment was determined using micro-finite element analysis (micro-FE) on baseline images. The formation of mineralised tissue showed strong association with areas of higher mechanical strain (area-under-the-curve: 0.91 ± 0.04, true positive rate: 0.85 ± 0.05) while surface based strains could correctly classify 43% of remodelling events. These findings support our hypotheses by showing a direct association between the local mechanical strains and the formation of mineralised tissue.

Alginate dependent changes of physical properties in 3D bioprinted cell-laden porous scaffolds affect cell viability and cell morphology.

Three-dimensional (3D) cell-laden scaffolds are becoming more prevalent in bone tissue repair and regeneration. However, the influence of physical scaffold properties on cell behavior is still unclear. In this study, we fabricated four different alginate concentration (0.8, 1.3, 1.8 and 2.3%alg) composite cell-laden porous scaffolds using a 3D bioprinting technique. The aim was to investigate the changes of physical properties affected by the alginate concentration and the influences on cell behavior. The study showed that the different alginate concentration scaffolds had uniform macropores (500-600 µm) with compressive moduli ranging from 1.5 kPa (0.8%alg) to 14.2 kPa (2.3%alg). Long-term structural integrity of the printed scaffolds was achieved when cultured in cell culture media, but not when cultured in phosphate buffered saline (PBS). Scaffold structure, swelling behavior, and compressive moduli decreased with culturing time and higher alginate concentration lead to more stable physical scaffold properties. Meanwhile, human mesenchymal stem cell (hMSCs) laden non-printed and bioprinted composite scaffolds were fabricated. Bioprinting did not affect cell viability, but alginate concentration had a significant influence on cell viability and cell morphology. Lower alginate concentration scaffolds (0.8%alg) showed higher cell viability (84% ± 0.7% versus 68% ± 1.3%) compared to higher alginate concentration scaffolds (2.3%alg) at day 14. Live cell image in the 0.8%alg scaffolds demonstrated the formation of a 3D interconnected cellular network, while cells in the 1.8 and 2.3%alg scaffolds formed spheroids. In conclusion, this study broadens the design space for alginate-based bioinks for 3D bioprinting. Higher alginate concentration preserved better scaffold fidelity and mechanical properties. Better cell viability and cell spreading morphology was achieved in lower alginate concentration scaffolds, which is relevant for potential applications in bone tissue engineering.

Mechanical Stimuli in the Local In Vivo Environment in Bone: Computational Approaches Linking Organ-Scale Loads to Cellular Signals.

PURPOSE OF REVIEW: Connecting organ-scale loads to cellular signals in their local in vivo environment is a current challenge in the field of bone (re)modelling. Understanding this critical missing link would greatly improve our ability to anticipate mechanotransduction during different modes of stimuli and the resultant cellular responses. This review characterises computational approaches that could enable coupling links across the multiple scales of bone. RECENT FINDINGS: Current approaches using strain and fluid shear stress concepts have begun to link organ-scale loads to cellular signals; however, these approaches fail to capture localised micro-structural heterogeneities. Furthermore, models that incorporate downstream communication from osteocytes to osteoclasts, bone-lining cells and osteoblasts, will help improve the understanding of (re)modelling activities. Incorporating this potentially key information in the local in vivo environment will aid in developing multiscale models of mechanotransduction that can predict or help describe resultant biological events related to bone (re)modelling. Progress towards multiscale determination of the cell mechanical environment from organ-scale loads remains elusive. Construction of organ-, tissue- and cell-scale computational models that include localised environmental variation, strain amplification and intercellular communication mechanisms will ultimately help couple the hierarchal levels of bone.