Unraveling the influence of channel size and shape in 3D printed ceramic scaffolds on osteogenesis.

Bone has the capacity to regenerate itself for relatively small defects; however, this regenerative capacity is diminished in critical-size bone defects. The development of synthetic materials has risen as a distinct strategy to address this challenge. Effective synthetic materials to have emerged in recent years are bioceramic implants, which are biocompatible and highly bioactive. Yet nothing suitable for the repair of large bone defects has made the transition from laboratory to clinic. The clinical success of bioceramics has been shown to depend not only on the scaffold's intrinsic material properties but also on its internal porous geometry. This study aimed to systematically explore the implications of varying channel size, shape, and curvature in tissue scaffolds on in vivo bone regeneration outcomes. 3D printed bioceramic scaffolds with varying channel sizes (0.3 mm to 1.5 mm), shapes (circular vs rectangular), and curvatures (concave vs convex) were implanted in rabbit femoral defects for 8 weeks, followed by histological evaluation. We demonstrated that circular channel sizes of around 0.9 mm diameter significantly enhanced bone formation, compared to channel with diameters of 0.3 mm and 1.5 mm. Interestingly, varying channel shapes (rectangular vs circular) had no significant effect on the volume of newly formed bone. Furthermore, the present study systematically demonstrated the beneficial effect of concave surfaces on bone tissue growth in vivo, reinforcing previous in silico and in vitro findings. This study demonstrates that optimizing architectural configurations within ceramic scaffolds is crucial in enhancing bone regeneration outcomes. STATEMENT OF SIGNIFICANCE: Despite the explosion of work on developing synthetic scaffolds to repair bone defects, the amount of new bone formed by scaffolds in vivo remains suboptimal. Recent studies have illuminated the pivotal role of scaffolds' internal architecture in osteogenesis. However, these investigations have mostly remained confined to in silico and in vitro experiments. Among the in vivo studies conducted, there has been a lack of systematic analysis of individual architectural features. Herein, we utilized bioceramic 3D printing to conduct a systematic exploration of the effects of channel size, shape, and curvature on bone formation in vivo. Our results demonstrate the significant influence of channel size and curvature on in vivo outcomes. These findings provide invaluable insights into the design of more effective bone scaffolds.

Discovering an unknown territory using atom probe tomography: Elemental exchange at the bioceramic scaffold/bone tissue interface.

Here we report the first atom probe study to reveal the atomic-scale composition of in vivo bone formed in a bioceramic scaffold (strontium-hardystonite-gahnite) after 12-month implantation in a large bone defect in sheep tibia. The composition of the newly formed bone tissue differs to that of mature cortical bone tissue, and elements from the degrading bioceramic implant, particularly aluminium (Al), are present in both the newly formed bone and in the original mature cortical bone tissue at the perimeter of the bioceramic implant. Atom probe tomography confirmed that the trace elements are released from the bioceramic and are actively transported into the newly formed bone. NanoSIMS mapping, as a complementary technique, confirmed the distribution of the released ions from the bioceramic into the newly formed bone tissue within the scaffold. This study demonstrated the combined benefits of atom probe and nanoSIMS in assessing nanoscopic chemical composition changes at precise locations within the tissue/biomaterial interface. Such information can assist in understanding the interaction of scaffolds with surrounding tissue, hence permitting further iterative improvements to the design and performance of biomedical implants, and ultimately reducing the risk of complications or failure while increasing the rate of tissue formation. STATEMENT OF SIGNIFICANCE: The repair of critical-sized load-bearing bone defects is a challenge, and precisely engineered bioceramic scaffold implants is an emerging potential treatment strategy. However, we still do not understand the effect of the bioceramic scaffold implants on the composition of newly formed bone in vivo and surrounding existing mature bone. This article reports an innovative route to solve this problem, the combined power of atom probe tomography and nanoSIMS is used to spatially define elemental distributions across bioceramic implant sites. We determine the nanoscopic chemical composition changes at the Sr-HT Gahnite bioceramic/bone tissue interface, and importantly, provide the first report of in vivo bone tissue chemical composition formed in a bioceramic scaffold.

Design and evaluation of 3D-printed Sr-HT-Gahnite bioceramic for FDA regulatory submission: A Good Laboratory Practice sheep study.

There is an unmet clinical need for a spinal fusion implant material that recapitulates the biological and mechanical performance of natural bone. We have developed a bioceramic, Sr-HT-Gahnite, which has been identified as a potential fusion device material. This material has the capacity to transform the future of the global interbody devices market, with follow on social, economic, and environmental benefits, rooted in its remarkable combination of mechanical properties and bioactivity. In this study, and in line with FDA requirements, the in vivo preclinical systemic biological safety of a Sr-HT-Gahnite interbody fusion device is assessed over 26 weeks in sheep under good laboratory practice (GLP). Following the in-life phase, animals are assessed for systemic biological effects via blood haematology and clinical biochemistry, strontium dosage analysis in the blood and wool, and histopathology examination of the distant organs including adrenals, brain, heart, kidneys, liver, lungs and bronchi, skeletal muscle, spinal nerves close to the implanted sites, ovaries, and draining lymph nodes. Our results show that no major changes in blood haematology or biochemistry parameters are observed, no systemic distribution of strontium to the blood and wool, and no macroscopic or histopathological abnormalities in the distant organs when Sr-HT-Gahnite was implanted, compared to baseline and control values. Together, these results indicate the systemic safety of the Sr-HT-Gahnite interbody fusion device. The results of this study extend to the systemic safety of other Sr-HT-Gahnite implanted medical devices in contact with bone or tissue, of similar size and manufactured using the described processes. STATEMENT OF SIGNIFICANCE: This paper is considered original and innovative as it is the first that thoroughly reports the systemic biological safety of previously undescribed bioceramic material, Sr-HT-Gahnite. The study has been performed under good laboratory practice, in line with FDA requirements for assessment of a new interbody fusion device, making the results broadly applicable to the translation of sheep models to the human cervical spine; and also the translation of Sr-HT-Gahnite as a biomaterial for use in additional applications. We expect this study to be of broad interest to the readership of Acta Biomaterilia. Its findings are directly applicable to researchers and clinicians working in bone repair and the development of synthetic biomaterials.

Nondeterministic multiobjective optimization of 3D printed ceramic tissue scaffolds.

Despite significant advances in the design optimization of bone scaffolds for enhancing their biomechanical properties, the functionality of these synthetic constructs remains suboptimal. One of the main challenges in the structural optimization of bone scaffolds is associated with the large uncertainties caused by the manufacturing process, such as variations in scaffolds' geometric features and constitutive material properties after fabrication. Unfortunately, such non-deterministic issues have not been considered in the existing optimization frameworks, thereby limiting their reliability. To address this challenge, a novel multiobjective robust optimization approach is proposed here such that the effects of uncertainties on the optimized design can be minimized. This study first conducted computational analyses of a parameterized ceramic scaffold model to determine its effective modulus, structural strength, and permeability. Then, surrogate models were constructed to formulate explicit mathematical relationships between the geometrical parameters (design variables) and mechanical and fluidic properties. The Non-Dominated Sorting Genetic Algorithm II (NSGA-II) was adopted to generate the robust Pareto solutions for an optimal set of trade-offs between the competing objective functions while ensuring the effects of the noise parameters to be minimal. Note that the nondeterministic optimization of tissue scaffold presented here is the first of its kind in open literature, which is expected to shed some light on this significant topic of scaffold design and additive manufacturing in a more realistic way.

An experimental and numerical study of the microstructural and biomechanical properties of human peripheral nerve endoneurium for the design of tissue scaffolds.

Biomimetic design of scaffold architectures represents a promising strategy to enable the repair of tissue defects. Natural endoneurium extracellular matrix (eECM) exhibits a sophisticated microstructure and remarkable microenvironments conducive for guiding neurite regeneration. Therefore, the analysis of eECM is helpful to the design of bionic scaffold. Unfortunately, a fundamental lack of understanding of the microstructural characteristics and biomechanical properties of the human peripheral nerve eECM exists. In this study, we used microscopic computed tomography (micro-CT) to reconstruct a three-dimensional (3D) eECM model sourced from mixed nerves. The tensile strength and effective modulus of human fresh nerve fascicles were characterized experimentally. Permeability was calculated from a computational fluid dynamic (CFD) simulation of the 3D eECM model. Fluid flow of acellular nerve fascicles was tested experimentally to validate the permeability results obtained from CFD simulations. The key microstructural parameters, such as porosity is 35.5 ± 1.7%, tortuosity in endoneurium (X axis is 1.26 ± 0.028, Y axis is 1.26 ± 0.020 and Z axis is 1.17 ± 0.03, respectively), tortuosity in pore (X axis is 1.50 ± 0.09, Y axis is 1.44 ± 0.06 and Z axis is 1.13 ± 0.04, respectively), surface area-to-volume ratio (SAVR) is 0.165 ± 0.007 µm-1 and pore size is 11.8 ± 2.8 µm, respectively. These were characterized from the 3D eECM model and may exert different effects on the stiffness and permeability. The 3D microstructure of natural peripheral nerve eECM exhibits relatively lower permeability (3.10 m2 × 10-12 m2) than other soft tissues. These key microstructural and biomechanical parameters may play an important role in the design and fabrication of intraluminal guidance scaffolds to replace natural eECM. Our findings can aid the development of regenerative therapies and help improve scaffold design.

Optimal placement of fixation system for scaffold-based mandibular reconstruction.

A current challenge in bone tissue engineering is to create favourable biomechanical conditions conducive to tissue regeneration for a scaffold implanted in a segmental defect. This is particularly the case immediately following surgical implantation when a firm mechanical union between the scaffold and host bone is yet to be established via osseointegration. For mandibular reconstruction of a large segmental defect, the position of the fixation system is shown here to have a profound effect on the mechanical stimulus (for tissue regeneration within the scaffold), structural strength, and structural stiffness of the tissue scaffold-host bone construct under physiological load. This research combines computer tomography (CT)-based finite element (FE) modelling with multiobjective optimisation to determine the optimal height and angle to place a titanium fixation plate on a reconstructed mandible so as to enhance tissue ingrowth, structural strength and structural stiffness of the scaffold-host bone construct. To this end, the respective design criteria for fixation plate placement are to: (i) maximise the volume of the tissue scaffold experiencing levels of mechanical stimulus sufficient to initiate bone apposition, (ii) minimise peak stress in the scaffold so that it remains intact with a diminished risk of failure and, (iii) minimise scaffold ridge displacement so that the reconstructed jawbone resists deformation under physiological load. First, a CT-based FE model of a reconstructed human mandible implanted with a bioceramic tissue scaffold is developed to visualise and quantify changes in the biomechanical responses as the fixation plate's height and/or angle are varied. The volume of the scaffold experiencing appositional mechanical stimulus is observed to increase with the height of the fixation plate. Also, as the principal load-transfer mechanism to the scaffold is via the fixation system, there is a significant ingress of appositional stimulus from the buccal side towards the centre of the scaffold, notably in the region bounded by the screws. Next, surrogate modelling is implemented to generate bivariate cubic polynomial functions of the three biomechanical responses with respect to the two design variables (height and angle). Finally, as the three design objectives are found to be competing, bi- and tri-objective particle swarm optimisation algorithms are invoked to determine the most optimal Pareto solution, which represents the best possible trade-off between the competing design objectives. It is recommended that consideration be given to placing the fixation system along the upper boundary of the mandible with a small clockwise rotation about its posterior end. The methodology developed here forms a useful decision aid for optimal surgical planning.

A time-dependent mechanobiology-based topology optimization to enhance bone growth in tissue scaffolds.

Scaffold-based bone tissue engineering has been extensively developed as a potential means to treatment of large bone defects. To enhance the biomechanical performance of porous tissue scaffolds, computational design techniques have gained growing popularity attributable to their compelling efficiency and strong predictive features compared with time-consuming trial-and-error experiments. Nevertheless, the mechanical stimulus necessary for bone regeneration, which characterizes dynamic nature due to continuous variation in the bone-scaffold construct system as a result of bone-ingrowth and scaffold biodegradation, is often neglected. Thus, this study proposes a time-dependent mechanobiology-based topology optimization framework for design of tissue scaffolds, thereby developing an ongoing favorable microenvironment and ensuring a long-term outcome for bone regeneration. For the first time, a level-set based topology optimization algorithm and a time-dependent shape derivative are developed to optimize the scaffold architecture. In this study, a large bone defect in a simulated 2D femur model and a partial defect in a 3D femur model are considered to demonstrate the effectiveness of the proposed design method. The results are compared with those obtained from stiffness-based topology optimization, time-independent design and typical scaffold constructs, showing significant advantages in continuing bone ingrowth outcomes.

A machine learning-based multiscale model to predict bone formation in scaffolds.

Computational modeling methods combined with non-invasive imaging technologies have exhibited great potential and unique opportunities to model new bone formation in scaffold tissue engineering, offering an effective alternate and viable complement to laborious and time-consuming in vivo studies. However, existing numerical approaches are still highly demanding computationally in such multiscale problems. To tackle this challenge, we propose a machine learning (ML)-based approach to predict bone ingrowth outcomes in bulk tissue scaffolds. The proposed in silico procedure is developed by correlating with a dedicated longitudinal (12-month) animal study on scaffold treatment of a major segmental defect in sheep tibia. Comparison of the ML-based time-dependent prediction of bone ingrowth with the conventional multilevel finite element (FE2) model demonstrates satisfactory accuracy and efficiency. The ML-based modeling approach provides an effective means for predicting in vivo bone tissue regeneration in a subject-specific scaffolding system.

A modular design strategy to integrate mechanotransduction concepts in scaffold-based bone tissue engineering.

Repair or regeneration of load-bearing bones has long been an incentive for the tissue engineering community to develop a plethora of synthetic bone scaffolds. Despite the key role of physical forces and the mechanical environment in bone regeneration, the mechanotransduction concept has rarely been incorporated in structural design of bone tissue scaffolds, particularly those made of bioactive materials such as hydrogels and bioceramics. Herein, we introduce a modular design strategy to fabricate a load bearing device that can support a wide range of hydrogel- and ceramic-based scaffolds against complex in-vivo loading conditions to induce desirable mechanical strains for bone regeneration within the scaffolds. The device is comprised of a fenestrated polymeric shell and ceramic structural pillars arranged in a sophisticated configuration to provide ample internal space for the scaffold, also enabling it to purposely regulate the levels of strains and stresses within the scaffolds. Utilizing this top-down design approach, we demonstrate that the failure load of alginate hydrogels increases 3200-fold in compression, 300-fold in shear and 75-fold in impact, achieving the values that enable them to withstand physiological loads in weight-bearing sites, while allowing generation of osteoinductive strains (i.e., 0.2-0.4%) in the hydrogel. This modular design approach opens a broad range of opportunities to utilize various bioactive but mechanically weak scaffolds for the treatment of load-bearing defects and exploiting mechanobiology strategies to improve bone regeneration.

A Novel Bone Substitute with High Bioactivity, Strength, and Porosity for Repairing Large and Load-Bearing Bone Defects.

Achieving adequate healing in large or load-bearing bone defects is highly challenging even with surgical intervention. The clinical standard of repairing bone defects using autografts or allografts has many drawbacks. A bioactive ceramic scaffold, strontium-hardystonite-gahnite or "Sr-HT-Gahnite" (a multi-component, calcium silicate-based ceramic) is developed, which when 3D-printed combines high strength with outstanding bone regeneration ability. In this study, the performance of purely synthetic, 3D-printed Sr-HT-Gahnite scaffolds is assessed in repairing large and load-bearing bone defects. The scaffolds are implanted into critical-sized segmental defects in sheep tibia for 3 and 12 months, with bone autografts used for comparison. The scaffolds induce substantial bone formation and defect bridging after 12 months, as indicated by X-ray, micro-computed tomography, and histological and biomechanical analyses. Detailed analysis of the bone-scaffold interface using focused ion beam scanning electron microscopy and multiphoton microscopy shows scaffold degradation and maturation of the newly formed bone. In silico modeling of strain energy distribution in the scaffolds reveal the importance of surgical fixation and mechanical loading on long-term bone regeneration. The clinical application of 3D-printed Sr-HT-Gahnite scaffolds as a synthetic bone substitute can potentially improve the repair of challenging bone defects and overcome the limitations of bone graft transplantation.

Nondestructive characterization of bone tissue scaffolds for clinical scenarios.

OBJECTIVES: This study aimed to develop a simple and efficient numerical modeling approach for characterizing strain and total strain energy in bone scaffolds implanted in patient-specific anatomical sites. MATERIALS AND METHODS: A simplified homogenization technique was developed to substitute a detailed scaffold model with the same size and equivalent orthotropic material properties. The effectiveness of the proposed modeling approach was compared with two other common homogenization methods based on periodic boundary conditions and the Hills-energy theorem. Moreover, experimental digital image correlation (DIC) measurements of full-field surface strain were conducted to validate the numerical results. RESULTS: The newly proposed simplified homogenization approach allowed for fairly accurate prediction of strain and total strain energy in tissue scaffolds implanted in a large femur mid-shaft bone defect subjected to a simulated in-vivo loading condition. The maximum discrepancy between the total strain energy obtained from the simplified homogenization approach and the one obtained from detailed porous scaffolds was 8.8%. Moreover, the proposed modeling technique could significantly reduce the computational cost (by about 300 times) required for simulating an in-vivo bone scaffolding scenario as the required degrees of freedom (DoF) was reduced from about 26 million for a detailed porous scaffold to only 90,000 for the homogenized solid counterpart in the analysis. CONCLUSIONS: The simplified homogenization approach has been validated by correlation with the experimental DIC measurements. It is fairly efficient and comparable with some other common homogenization techniques in terms of accuracy. The proposed method is implicating to different clinical applications, such as the optimal selection of patient-specific fixation plates and screw system.

Architectural Design of 3D Printed Scaffolds Controls the Volume and Functionality of Newly Formed Bone.

The successful regeneration of functional bone tissue in critical-size defects remains a significant clinical challenge. To address this challenge, synthetic bone scaffolds are widely developed, but remarkably few are translated to the clinic due to poor performance in vivo. Here, it is demonstrated how architectural design of 3D printed scaffolds can improve in vivo outcomes. Ceramic scaffolds with different pore sizes and permeabilities, but with similar porosity and interconnectivity, are implanted in rabbit calvaria for 12 weeks, and then the explants are harvested for microcomputed tomography evaluation of the volume and functionality of newly formed bone. The results indicate that scaffold pores should be larger than 390 µm with an upper limit of 590 µm to enhance bone formation. It is also demonstrated that a bimodal pore topology-alternating large and small pores-enhances the volume and functionality of new bone substantially. Moreover, bone formation results indicate that stiffness of new bone is highly influenced by the scaffold's permeability in the direction concerned. This study demonstrates that manipulating pore size and permeability in a 3D printed scaffold architecture provides a useful strategy for enhancing bone regeneration outcomes.

Fracture behaviors of ceramic tissue scaffolds for load bearing applications.

Healing large bone defects, especially in weight-bearing locations, remains a challenge using available synthetic ceramic scaffolds. Manufactured as a scaffold using 3D printing technology, Sr-HT-Gahnite at high porosity (66%) had demonstrated significantly improved compressive strength (53 ± 9 MPa) and toughness. Nevertheless, the main concern of ceramic scaffolds in general remains to be their inherent brittleness and low fracture strength in load bearing applications. Therefore, it is crucial to establish a robust numerical framework for predicting fracture strengths of such scaffolds. Since crack initiation and propagation plays a critical role on the fracture strength of ceramic structures, we employed extended finite element method (XFEM) to predict fracture behaviors of Sr-HT-Gahnite scaffolds. The correlation between experimental and numerical results proved the superiority of XFEM for quantifying fracture strength of scaffolds over conventional FEM. In addition to computer aided design (CAD) based modeling analyses, XFEM was conducted on micro-computed tomography (µCT) based models for fabricated scaffolds, which took into account the geometric variations induced by the fabrication process. Fracture strengths and crack paths predicted by the µCT-based XFEM analyses correlated well with relevant experimental results. The study provided an effective means for the prediction of fracture strength of porous ceramic structures, thereby facilitating design optimization of scaffolds.

L-2-hydroxyglutaric aciduria: a report of six cases and review of the literature.

L-2-hydroxyglutaric aciduria is a rare and novel autosomal recessive inherited neurometabolic disorder. Since its first description by Duran in 1980, less than 100 cases have so far been reported. Occurring mostly in childhood, it is characterized by slowly progressive neurological dysfunction with cerebellar ataxia, pyramidal signs, intellectual decline, seizure, and extrapyramidal symptoms. MRI scanning is highly characteristic and screening for organic acid (L-2-hydroxyglutaric acid) in urine, serum, and cerebrospinal fluid is diagnostic. We investigated six Iranian children, aged 4, 14, 16, and 16 years, (the last one had two affected brothers and both of them died of similar illness at the ages of 20 and 22), by urinary organic acids assay and MRI scanning with suspicion of this rare disorder. Symptoms were suspicious for one of the leukoencephalopathies accompanied by macrocephaly. Affected cases were evaluated because of mild to moderate psychomotor retardation and regression. Head circumferences were above 2 standard deviations. Urine levels of L-2-hydroxyglutaric acid were strongly increased. MRI scanning of the brain showed hyperintense signal on T2W images of the subcortical white matter and basal ganglia in all of them. Because of its inheritance pattern (autosomal recessive) and the high rate of consanguineous marriages in Iran, the prevalence of this disorder might be high among the mentally-handicapped patients, especially those with macrocephaly. Therefore, this entity should be considered in the differential diagnosis of mentally-retarded patients with macrocephaly.