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The avascular nature of cartilage makes it a unique tissue 1-4 , but whether and how the absence of nutrient supply regulates chondrogenesis remain unknown. Here we show that obstruction of vascular invasion during bone healing favours chondrogenic over osteogenic differentiation of skeletal progenitor cells. Unexpectedly, this process is driven by a decreased availability of extracellular lipids. When lipids are scarce, skeletal progenitors activate forkhead box O (FOXO) transcription factors, which bind to the Sox9 promoter and increase its expression. Besides initiating chondrogenesis, SOX9 acts as a regulator of cellular metabolism by suppressing oxidation of fatty acids, and thus adapts the cells to an avascular life. Our results define lipid scarcity as an important determinant of chondrogenic commitment, reveal a role for FOXO transcription factors during lipid starvation, and identify SOX9 as a critical metabolic mediator. These data highlight the importance of the nutritional microenvironment in the specification of skeletal cell fate.

Bone healing process is a complicated phenomenon regulated by biochemical and mechanical signals. Experimental studies have shown that ultrasound (US) accelerates bone ossification and has a multiple influence on cell differentiation and angiogenesis. In a recent work of the authors, a bioregulatory model for providing bone-healing predictions was addressed, taking into account for the first time the salutary effect of US on the involved angiogenesis. In the present work, a mechanobioregulatory model of bone solidification under the US presence incorporating also the mechanical environment on the regeneration process, which is known to affect cellular processes, is presented. An iterative procedure is adopted, where the finite element method is employed to compute the mechanical stimuli at the linear elastic phases of the poroelastic callus region and a coupled system of partial differential equations to simulate the enhancement by the US cell angiogenesis process and thus the oxygen concentration in the fractured area. Numerical simulations with and without the presence of US that illustrate the influence of progenitor cells' origin in the healing pattern and the healing rate and simultaneously demonstrate the salutary effect of US on bone repair are presented and discussed.

Three-dimensional (3D) bioprinting is a rapidly advancing tissue engineering technology that holds great promise for the regeneration of several tissues, including bone. However, to generate a successful 3D bone tissue engineering construct, additional complexities should be taken into account such as nutrient and oxygen delivery, which is often insufficient after implantation in large bone defects. We propose that a well-designed tissue engineering construct, that is, an implant with a specific spatial pattern of cells in a matrix, will improve the healing outcome. By using a computational model of bone regeneration we show that particular cell patterns in tissue engineering constructs are able to enhance bone regeneration compared to uniform ones. We successfully bioprinted one of the most promising cell-gradient patterns by using cell-laden hydrogels with varying cell densities and observed a high cell viability for three days following the bioprinting process. In summary, we present a novel strategy for the biofabrication of bone tissue engineering constructs by designing cell-gradient patterns based on a computational model of bone regeneration, and successfully bioprinting the chosen design. This integrated approach may increase the success rate of implanted tissue engineering constructs for critical size bone defects and also can find a wider application in the biofabrication of other types of tissue engineering constructs.

Although the field of bone regeneration has experienced great advancements in the last decades, integrating all the relevant, patient-specific information into a personalized diagnosis and optimal treatment remains a challenging task due to the large number of variables that affect bone regeneration. Computational models have the potential to cope with this complexity and to improve the fundamental understanding of the bone regeneration processes as well as to predict and optimize the patient-specific treatment strategies. However, the current use of computational models in daily orthopedic practice is very limited or inexistent. We have identified three key hurdles that limit the translation of computational models of bone regeneration from bench to bed side. First, there exists a clear mismatch between the scope of the existing and the clinically required models. Second, most computational models are confronted with limited quantitative information of insufficient quality thereby hampering the determination of patient-specific parameter values. Third, current computational models are only corroborated with animal models, whereas a thorough (retrospective and prospective) assessment of the computational model will be crucial to convince the health care providers of the capabilities thereof. These challenges must be addressed so that computational models of bone regeneration can reach their true potential, resulting in the advancement of individualized care and reduction of the associated health care costs., (© 2015 Wiley Periodicals, Inc.)

Scaffold permeability is a key parameter combining geometrical features such as pore shape, size and interconnectivity, porosity and specific surface area. It can influence the success of bone tissue engineering scaffolds, by affecting oxygen and nutrient transport, cell seeding efficiency, in vitro three-dimensional (3D) cell culture and, ultimately, the amount of bone formation. An accurate and efficient prediction of scaffold permeability would be highly useful as part of a scaffold design process. The aim of this study was (i) to determine the accuracy of computational fluid dynamics (CFD) models for prediction of the permeability coefficient of three different regular Ti6Al4V scaffolds (each having a different porosity) by comparison with experimentally measured values and (ii) to verify the validity of the semi-empirical Kozeny equation to calculate the permeability analytically. To do so, five CFD geometrical models per scaffold porosity were built, based on different geometrical inputs: either based on the scaffold's computer-aided design (CAD) or derived from 3D microfocus X-ray computed tomography (micro-CT) data of the additive manufactured (AM) scaffolds. For the latter the influence of the spatial image resolution and the image analysis algorithm used to determine the scaffold's architectural features on the predicted permeability was analysed. CFD models based on high-resolution micro-CT images could predict the permeability coefficients of the studied scaffolds: depending on scaffold porosity and image analysis algorithm, relative differences between measured and predicted permeability values were between 2% and 27%. Finally, the analytical Kozeny equation was found to be valid. A linear correlation between the ratio Φ(3)/S(s)(2) and the permeability coefficient k was found for the predicted (by means of CFD) as well as measured values (relative difference of 16.4% between respective Kozeny coefficients), thus resulting in accurate and efficient calculation of the permeability of regular AM scaffolds., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Despite a myriad of studies confirming the interaction between biology and mechanics, the exact nature of the main mechanical stimuli and their influence on the bone regeneration processes are still unclear. The hypothesis of this study was that the outcome of peri-implant healing under different implant loading regimens can be explained by the influence of fluid flow on the combination of angiogenesis and osteogenesis through its influence on cell proliferation and differentiation. To investigate this hypothesis a mathematical model of bone regeneration was applied to simulate the peri-implant healing in an in vivo repeated sampling bone chamber for different axial micromechanical implant loading regimes. When mechanical loading was modeled to influence both osteogenic and angiogenic processes, a good agreement was observed between simulations and experiments concerning the amount of bone in the bone chamber, its radial and longitudinal distribution, and the bone-implant contact for different implant displacement magnitudes.

Recently developed atrophic non-union models are a good representation of the clinical situation in which many non-unions develop. Based on previous experimental studies with these atrophic non-union models, it was hypothesized that in order to obtain successful fracture healing, blood vessels, growth factors, and (proliferative) precursor cells all need to be present in the callus at the same time. This study uses a combined in vivo-in silico approach to investigate these different aspects (vasculature, growth factors, cell proliferation). The mathematical model, initially developed for the study of normal fracture healing, is able to capture essential aspects of the in vivo atrophic non-union model despite a number of deviations that are mainly due to simplifications in the in silico model. The mathematical model is subsequently used to test possible treatment strategies for atrophic non-unions (i.e. cell transplant at post-osteotomy, week 3). Preliminary in vivo experiments corroborate the numerical predictions. Finally, the mathematical model is applied to explain experimental observations and identify potentially crucial steps in the treatments and can thereby be used to optimize experimental and clinical studies in this area. This study demonstrates the potential of the combined in silico-in vivo approach and its clinical implications for the early treatment of patients with problematic fractures.

When reconstructing a large segmental bone defect by means of a porous scaffold, a fixator is used to stabilize the reconstruction. The fixator stiffness is an important factor as it will influence the biomechanical environment to which scaffold and regenerating tissues are exposed. A finite element (FE) model can be used to predict the fixator stiffness. The goal of this study was to develop and validate a detailed 3D FE model of a custom-developed unilateral external fixator. In particular, it was hypothesized that the contact interfaces between the different fixator components play a major role for the prediction of the fixator stiffness. In vitro mechanical testing of the entire fixator as well as of separate fixator components was performed in order to measure the stiffness. The mechanical test set-ups were simulated by means of detailed FE models that considered different levels of refinement of the various contact interfaces. The error on predicted fixator stiffness in comparison to measured stiffness was reduced from 121% to 16% by refining the contact settings of the FE model. The individual sources of error between the measured and predicted fixator stiffness could be quantitatively assessed as well. In conclusion, this study warrants for a careful modelling of the geometry and contact settings, when using FE models for the prediction of fixator stiffness., (Copyright 2010 IPEM. Published by Elsevier Ltd. All rights reserved.)

Osteoporosis and muscle frailty are important health problems in elderly men and may be partly related to biological androgen activity. This androgen action can be mediated directly through stimulation of the androgen receptor (AR) or indirectly through stimulation of estrogen receptor-alpha (ERalpha) following aromatization of androgens into estrogens. To assess the differential action of AR and ERalpha pathways on bone and body composition, AR-ERalpha double-knockout mice were generated and characterized. AR disruption decreased trabecular bone mass, whereas ERalpha disruption had no additional effect on the AR-dependent trabecular bone loss. In contrast, combined AR and ERalpha inactivation additionally reduced cortical bone and muscle mass compared with either AR or ERalpha disruption alone. ERalpha inactivation--in the presence or absence of AR--increased fat mass. We demonstrate that AR activation is solely responsible for the development and maintenance of male trabecular bone mass. Both AR and ERalpha activation, however, are needed to optimize the acquisition of cortical bone and muscle mass. ERalpha activation alone is sufficient for the regulation of fat mass. Our findings clearly define the relative importance of AR and ERalpha signaling on trabecular and cortical bone mass as well as body composition in male mice.

Mechanical loading is one of the key factors that influence bone mass and the osseointegration of bone-anchored implants. From a clinical point of view, mechanical stimulation may be used to enhance bone strength and implant osseointegration. Among the many loading parameters that influence the response to mechanical loading, the effects of loading frequency and rate have been investigated in many studies. In this paper the most relevant animal studies that have addressed the effect of loading frequency, rate, and vibration on either bone adaptation or implant osseointegration are systematically reviewed. Apparently contradictory results are discussed and interpreted within the context of mechanotransduction and mechanoregulation of bone. A combined experimental and computational approach is suggested to address some of the remaining research questions.

The development of successful scaffolds for bone tissue engineering requires a concurrent engineering approach that combines different research fields. In order to limit in vivo experiments and reduce trial and error research, a scaffold screening technique has been developed. In this protocol seven structural and three biomechanical properties of potential scaffold materials are quantified and compared to the desired values. The property assessment is done on computer models of the scaffolds, and these models are based on micro-CT images. As a proof of principle, three porous scaffolds were evaluated with this protocol: stainless steel, hydroxyapatite, and titanium. These examples demonstrate that the modelling technique is able to quantify important scaffold properties. Thus, a powerful technique for automated screening of bone tissue engineering scaffolds has been developed that in a later stage may be used to tailor the scaffold properties to specific requirements.

A repeated sampling bone chamber methodology was developed for the study of the influence of the mechanical environment on skeletal tissue differentiation and bone adaptation around titanium implants. Via perforations, bone grows into the implanted outer bone chamber, containing an inner bone chamber with a central test implant. An actuator--easily mounted on the outer bone chamber--allows a controlled mechanical stimulation of the test implant. After each experiment, the inner bone chamber--with its content--can be harvested and analysed. A new inner bone chamber with a central implant can be inserted consecutively in the outer bone chamber and a new experiment can start. Pilot studies led to a reliable surgical protocol and showed the applicability of the methodology, offering the possibility to study skeletal tissue differentiation and adaptation around implants under well-controlled mechanical conditions, and this protected from external loading. Repeated sampling of the bone chamber allows conducting several experiments within the same animal at the same site, thereby excluding subject- and site-dependent variability and reducing the amount of experimental animals.

Although it is generally accepted that adverse forces can impair osseointegration, the mechanism of this complication is unknown. In this study, static and dynamic loads were applied on 10 mm long implants (Brånemark System, Nobel Biocare, Sweden) installed bicortically in rabbit tibiae to investigate the bone response. Each of 10 adult New Zealand black rabbits had one statically loaded implant (with a transverse force of 29.4 N applied on a distance of 1.5 mm from the top of the implant, resulting in a bending moment of 4.4 Ncm), one dynamically loaded implant (with a transverse force of 14.7 N applied on a distance of 50 mm from the top of the implant, resulting in a bending moment of 73.5 Ncm, 2.520 cycles in total, applied with a frequency of 1 Hz), and one unloaded control implant. The loading was performed during 14 days. A numerical model was used as a guideline for the applied dynamic load. Histomorphometrical quantifications of the bone to metal contact area and bone density lateral to the implant were performed on undecalcified and toluidine blue stained sections. The histological picture was similar for statically loaded and control implants. Dense cortical lamellar bone was present around the marginal and apical part of the latter implants with no signs of bone loss. Crater-shaped bone defects and Howship's lacunae were explicit signs of bone resorption in the marginal bone area around the dynamically loaded implants. Despite those bone defects, bone islands were present in contact with the implant surface in this marginal area. This resulted in no significantly lower bone-to-implant contact around the dynamically loaded implants in comparison with the statically loaded and the control implants. However, when comparing the amount of bone in the immediate surroundings of the marginal part of the implants, significantly (P < 0.007) less bone volume (density) was present around the dynamically loaded in comparison with the statically loaded and the control implants. This study shows that excessive dynamic loads cause crater-like bone defects lateral to osseointegrated implants.