Robot-assisted implantation of additively manufactured patient-specific orthopaedic implants: evaluation in a sheep model.

PURPOSE: Bone tumours must be surgically excised in one piece with a margin of healthy tissue. The unique nature of each bone tumour case is well suited to the use of patient-specific implants, with additive manufacturing allowing production of highly complex geometries. This work represents the first assessment of the combination of surgical robotics and patient-specific additively manufactured implants. METHODS: The development and evaluation of a robotic system for bone tumour excision, capable of milling complex osteotomy paths, is described. The developed system was evaluated as part of an animal trial on 24 adult male sheep, in which robotic bone excision of the distal femur was followed by placement of patient-specific implants with operative time evaluated. Assessment of implant placement accuracy was completed based on post-operative CT scans. RESULTS: A mean overall implant position error of 1.05 ± 0.53 mm was achieved, in combination with a mean orientation error of 2.38 ± 0.98°. A mean procedure time (from access to implantation, excluding opening and closing) of 89.3 ± 25.25 min was observed, with recorded surgical time between 58 and 133 min, with this approximately evenly divided between robotic (43.9 ± 15.32) and implant-based (45.4 ± 18.97) tasks. CONCLUSIONS: This work demonstrates the ability for robotics to achieve repeatable and precise removal of complex bone volumes of the type that would allow en bloc removal of a bone tumour. These robotically created volumes can be precisely filled with additively manufactured patient-specific implants, with minimal gap between cut surface and implant interface.

Matured Myofibers in Bioprinted Constructs with In Vivo Vascularization and Innervation.

For decades, the study of tissue-engineered skeletal muscle has been driven by a clinical need to treat neuromuscular diseases and volumetric muscle loss. The in vitro fabrication of muscle offers the opportunity to test drug-and cell-based therapies, to study disease processes, and to perhaps, one day, serve as a muscle graft for reconstructive surgery. This study developed a biofabrication technique to engineer muscle for research and clinical applications. A bioprinting protocol was established to deliver primary mouse myoblasts in a gelatin methacryloyl (GelMA) bioink, which was implanted in an in vivo chamber in a nude rat model. For the first time, this work demonstrated the phenomenon of myoblast migration through the bioprinted GelMA scaffold with cells spontaneously forming fibers on the surface of the material. This enabled advanced maturation and facilitated the connection between incoming vessels and nerve axons in vivo without the hindrance of a scaffold material. Immunohistochemistry revealed the hallmarks of tissue maturity with sarcomeric striations and peripherally placed nuclei in the organized bundles of muscle fibers. Such engineered muscle autografts could, with further structural development, eventually be used for surgical reconstructive purposes while the methodology presented here specifically has wide applications for in vitro and in vivo neuromuscular function and disease modelling.

Characterizing Bioinks for Extrusion Bioprinting: Printability and Rheology.

In recent years, new technologies based on 3D bioprinting have emerged as ideal tools with which to arrange cells and biomaterials in three dimensions and so achieve tissue engineering's original goals. The simplest and most widely used form of bioprinting is based on pneumatic extrusion, where 3D structures are built up by drawing patterns of cell-laden or non-cell-laden material through a robotically manipulated syringe. Developing and characterizing new biomaterials for 3D bioprinting (i.e., bioinks) is critical for the progress of the field. This chapter describes a series of protocols for developing, optimizing, and testing new bioinks for extrusion-based 3D bioprinting.

Design, Fabrication and Validation of a Precursor Pulsed Electromagnetic Field Device for Bone Fracture Repair.

Pulsed electromagnetic field (PEMF) stimulation has been utilized in the medical field since the early 20th century. A number of therapeutic devices have been developed for the treatment of bone fractures and other medical applications. Most of these devices are backed by limited quantitative evidence. In this paper we present the development of a PEMF device for the purposes of determining, through in vitro experimentation, the exposure parameters required to give the most optimal fracture repair. Following electromagnetic field characterization, the device was shown to match well with computational field simulations. The exposure system has been validated through adipose-derived stem cell viability studies with an exposure frequency of 5 Hz and an intensity of 1.1 mT, for a duration of seven days at 30 minutes per day. Under the specific field characteristics chosen, the fatty-tissue derived stem cell proliferation was not hindered and in fact was stimulated $( 0. 025 < P < 0.01)$ by the PEMF exposure. With continued experimentation of numerous exposure conditions at the cellular scale, it will be possible to quantitatively determine the optimal exposure conditions required to produce the most rapid fresh fracture repair. Following this, there is significant potential for development of an optimized wearable device suitable for enhancing repair of all types of bone fractures.

Biofabrication of human articular cartilage: a path towards the development of a clinical treatment.

Cartilage injuries cause pain and loss of function, and if severe may result in osteoarthritis (OA). 3D bioprinting is now a tangible option for the delivery of bioscaffolds capable of regenerating the deficient cartilage tissue. Our team has developed a handheld device, the Biopen, to allow in situ additive manufacturing during surgery. Given its ability to extrude in a core/shell manner, the Biopen can preserve cell viability during the biofabrication process, and it is currently the only biofabrication tool tested as a surgical instrument in a sheep model using homologous stem cells. As a necessary step toward the development of a clinically relevant protocol, we aimed to demonstrate that our handheld extrusion device can successfully be used for the biofabrication of human cartilage. Therefore, this study is a required step for the development of a surgical treatment in human patients. In this work we specifically used human adipose derived mesenchymal stem cells (hADSCs), harvested from the infra-patellar fat pad of donor patients affected by OA, to also prove that they can be utilized as the source of cells for the future clinical application. With the Biopen, we generated bioscaffolds made of hADSCs laden in gelatin methacrylate, hyaluronic acid methacrylate and cultured in the presence of chondrogenic stimuli for eight weeks in vitro. A comprehensive characterisation including gene and protein expression analyses, immunohistology, confocal microscopy, second harmonic generation, light sheet imaging, atomic force mycroscopy and mechanical unconfined compression demonstrated that our strategy resulted in human hyaline-like cartilage formation. Our in situ biofabrication approach represents an innovation with important implications for customizing cartilage repair in patients with cartilage injuries and OA.

Modal analysis of nanoindentation data, confirming that reduced bone turnover may cause increased tissue mineralization/elasticity.

It is widely believed that the activities of bone cells at the tissue scale not only govern the size of the vascular pore spaces (and hence, the amount of bone tissue available for actually carrying the loads), but also the characteristics of the extracellular bone matrix itself. In this context, increased mechanical stimulation (in mediolateral regions of human femora, as compared to anteroposterior regions) may lead to increased bone turnover, lower bone matrix mineralization, and therefore lower tissue modulus. On the other hand, resorption-only processes (in endosteal versus periosteal regions) may have the opposite effect. A modal analysis of nanoindentation data obtained on femurs from the Melbourne Femur Research Collection (MFRC) indeed confirms that bone is stiffer in endosteal regions compared to periosteal regions (E̅endost = 29.34 ± 0.75 GPa >E̅periost = 24.67 ± 1.63 GPa), most likely due to the aging-related increase in resorption modeling on endosteal surfaces resulting in trabecularization of cortical bone. The results also show that bone is stiffer along the anteroposterior direction compared the mediolateral direction (E̅anteropost = 28.89 ± 1.08 GPa >E̅mediolat = 26.03 ± 2.31 GPa), the former being aligned with the neutral bending axis of the femur and, thus, undergoing more resorption modeling and consequently being more mineralized.

Tailoring the mechanical properties of gelatin methacryloyl hydrogels through manipulation of the photocrosslinking conditions.

Photo-crosslinkable hydrogels, in particular gelatin methacryloyl (GelMa), are gaining increasing importance in biofabrication and tissue engineering. While GelMa is often described as mechanically 'tunable', clear relationships linking the photocrosslinking conditions to reaction rates, and the resulting mechanical properties, have not been described. Meanwhile the conditions employed in the literature are disparate, and difficult to compare. In this work, in situ rheological measurements were used to quantify the relative rate of reaction of GelMa hydrogels with respect to light intensity, exposure time and photo-initiator concentration. In addition the UV degradation of the photo-initiator Irgacure 2959 was measured by UV-vis spectroscopy, and used to estimate the rate of free radical production as a function of light exposure. Using these data an expression was derived which predicts the mechanical properties of GelMa hydrogels produced across a wide range of crosslinking conditions. The model was validated through fabrication of a GelMa gradient which matched predicted properties. Human mesenchymal stem cells encapsulated in crosslinked GelMa exhibited high (>90%) viability post encapsulation, however metabolic activity over one week was influenced by the intensity of light used during crosslinking. The expressions described may be used to aid rational choices of GelMa photocrosslinking conditions, especially in cell encapsulation experiments where minimising the cytotoxic elements in the reaction is a priority.

In situ handheld three-dimensional bioprinting for cartilage regeneration.

Articular cartilage injuries experienced at an early age can lead to the development of osteoarthritis later in life. In situ three-dimensional (3D) printing is an exciting and innovative biofabrication technology that enables the surgeon to deliver tissue-engineering techniques at the time and location of need. We have created a hand-held 3D printing device (biopen) that allows the simultaneous coaxial extrusion of bioscaffold and cultured cells directly into the cartilage defect in vivo in a single-session surgery. This pilot study assessed the ability of the biopen to repair a full-thickness chondral defect and the early outcomes in cartilage regeneration, and compared these results with other treatments in a large animal model. A standardized critical-sized full-thickness chondral defect was created in the weight-bearing surface of the lateral and medial condyles of both femurs of six sheep. Each defect was treated with one of the following treatments: (i) hand-held in situ 3D printed bioscaffold using the biopen (HH group), (ii) preconstructed bench-based printed bioscaffolds (BB group), (iii) microfractures (MF group) or (iv) untreated (control, C group). At 8 weeks after surgery, macroscopic, microscopic and biomechanical tests were performed. Surgical 3D bioprinting was performed in all animals without any intra- or postoperative complication. The HH biopen allowed early cartilage regeneration. The results of this study show that real-time, in vivo bioprinting with cells and scaffold is a feasible means of delivering a regenerative medicine strategy in a large animal model to regenerate articular cartilage.

Comparison of the moulding ability of Plaster of Paris and polyester cast material in the healthy adult forearm.

OBJECTIVES: To quantify the moulding ability of Plaster of Paris and polyester cast materials as assessed by the novel use of peripheral quantitative computed tomography. METHODS: A prospective crossover study was performed in 25 healthy volunteers aged 18-65 years. Participants' non-dominant wrist was immobilized using a synthetic polyester cast followed by a Plaster of Paris cast with three point moulding to simulate reduction of a dorsally angulated distal radius fracture. The novel use of peripheral quantitative computed tomography was used to measure the closeness of fit of each cast on an axial tomographic slice. RESULTS AND CONCLUSIONS: Plaster of Paris casts were able to achieve a closer mould than polyester when measured between the bone and the cast (p=0.002), as well as between the skin and the cast (p=0.001). There was no difference when stratified on BMI. Using pQCT assessment, a closely moulded fit was able to be more consistently achieved when using Plaster of Paris when compared to polyester casts of the distal radius. LEVEL OF EVIDENCE: III.

Handheld Co-Axial Bioprinting: Application to in situ surgical cartilage repair.

Three-dimensional (3D) bioprinting is driving major innovations in the area of cartilage tissue engineering. Extrusion-based 3D bioprinting necessitates a phase change from a liquid bioink to a semi-solid crosslinked network achieved by a photo-initiated free radical polymerization reaction that is known to be cytotoxic. Therefore, the choice of the photocuring conditions has to be carefully addressed to generate a structure stiff enough to withstand the forces phisiologically applied on articular cartilage, while ensuring adequate cell survival for functional chondral repair. We recently developed a handheld 3D printer called "Biopen". To progress towards translating this freeform biofabrication tool into clinical practice, we aimed to define the ideal bioprinting conditions that would deliver a scaffold with high cell viability and structural stiffness relevant for chondral repair. To fulfill those criteria, free radical cytotoxicity was confined by a co-axial Core/Shell separation. This system allowed the generation of Core/Shell GelMa/HAMa bioscaffolds with stiffness of 200KPa, achieved after only 10 seconds of exposure to 700 mW/cm2 of 365 nm UV-A, containing >90% viable stem cells that retained proliferative capacity. Overall, the Core/Shell handheld 3D bioprinting strategy enabled rapid generation of high modulus bioscaffolds with high cell viability, with potential for in situ surgical cartilage engineering.

Effect of Polyester and Plaster of Paris Casts on Determination of Volumetric Bone Mineral Density Assessed by Peripheral Quantitative Computed Tomography (pQCT).

Peripheral quantitative computed tomography (pQCT) is a non-invasive, low-radiation tool for measuring volumetric bone mineral density. It has potential for use in fracture healing applications; however, the unknown attenuation effects of cast material on peripheral quantitative computed tomography have contributed to its limited use in this area. The effect of two common cast materials, polyester and Plaster of Paris was investigated by performing both in vitro and in vivo studies. The in vitro study tested the effect of increasing layers of cast material on bone density measurements performed on a hydroxyapatite phantom. Cast thickness was directly associated with a reduction in bone mineral density, with twelve layers of polyester and Plaster of Paris resulting in a 0.55 and 2.21 % decrease in bone density measurements. Precision error in situ with polyester cast material was 0.71 %, and 2.31 % with Plaster of Paris cast material. The in vivo study comprised a prospective trial with 28 healthy adult participants to evaluate the effect of the two cast materials. Trabecular bone mineral density was increased by 0.5 % in the presence of a polyester cast and decreased by 4.22 % in the presence of a Plaster of Paris cast. Cortical bone mineral density was decreased by 3.46 and 5.54 % for polyester and Plaster of Paris, respectively. This study quantified the effects of orthopaedic casts on pQCT-derived bone parameters. The results suggest applicability of commonly utilised cast materials in combination with pQCT to assess fracture healing.

Patient-specific fracture risk assessment of vertebrae: A multiscale approach coupling X-ray physics and continuum micromechanics.

While in clinical settings, bone mineral density measured by computed tomography (CT) remains the key indicator for bone fracture risk, there is an ongoing quest for more engineering mechanics-based approaches for safety analyses of the skeleton. This calls for determination of suitable material properties from respective CT data, where the traditional approach consists of regression analyses between attenuation-related grey values and mechanical properties. We here present a physics-oriented approach, considering that elasticity and strength of bone tissue originate from the material microstructure and the mechanical properties of its elementary components. Firstly, we reconstruct the linear relation between the clinically accessible grey values making up a CT, and the X-ray attenuation coefficients quantifying the intensity losses from which the image is actually reconstructed. Therefore, we combine X-ray attenuation averaging at different length scales and over different tissues, with recently identified 'universal' composition characteristics of the latter. This gives access to both the normally non-disclosed X-ray energy employed in the CT-device and to in vivo patient-specific and location-specific bone composition variables, such as voxel-specific mass density, as well as collagen and mineral contents. The latter feed an experimentally validated multiscale elastoplastic model based on the hierarchical organization of bone. Corresponding elasticity maps across the organ enter a finite element simulation of a typical load case, and the resulting stress states are increased in a proportional fashion, so as to check the safety against ultimate material failure. In the young patient investigated, even normal physiological loading is probable to already imply plastic events associated with the hydrated mineral crystals in the bone ultrastructure, while the safety factor against failure is still as high as five. Copyright © 2016 John Wiley & Sons, Ltd.

A multiscale analytical approach for bone remodeling simulations: linking scales from collagen to trabeculae.

Bone is a dynamic and hierarchical porous material whose spatial and temporal mechanical properties can vary considerably due to differences in its microstructure and due to remodeling. Hence, a multiscale analytical approach, which combines bone structural information at multiple scales to the remodeling cellular activities, could form an efficient, accurate and beneficial framework for the prognosis of changes in bone properties due to, e.g., bone diseases. In this study, an analytical formulation of bone remodeling integrated with multiscale micromechanical models is proposed to investigate the effects of structural changes at the nanometer level (collagen scale) on those at higher levels (tissue scale). Specific goals of this study are to derive a mechanical stimulus sensed by the osteocytes using a multiscale framework, to test the accuracy of the multiscale model for the prediction of bone density, and to demonstrate its multiscale capabilities by predicting changes in bone density due to changes occurring at the molecular level. At each different level, the bone composition was modeled as a two-phase material which made it possible to: (1) find a closed-form solution for the energy-based mechanical stimulus sensed by the osteocytes and (2) describe the anisotropic elastic properties at higher levels as a function of the stiffness of the elementary components (collagen, hydroxyapatite and water) at lower levels. The accuracy of the proposed multiscale model of bone remodeling was tested first by comparing the analytical bone volume fraction predictions to those obtained from the corresponding µFE-based computational model. Differences between analytical and numerical predictions were less than 1% while the computational time was drastically reduced, namely by a factor of 1 million. In a further analysis, the effects of changes in collagen and hydroxyapatite volume fractions on the bone remodeling process were simulated, and it was found that such changes considerably affect the bone density at the millimeter scale. In fact, smaller tissue density induces remodeling activities leading to finally higher overall bone density. The multiscale analytical model proposed in this study potentially provides an accurate and efficient tool for simulating patient-specific bone remodeling, which might be of importance in particular for the hip and spine, where an accurate assessment of bone micro-architecture is not possible.

Intravoxel bone micromechanics for microCT-based finite element simulations.

While micro-FE simulations have become a standard tool in computational biomechanics, the choice of appropriate material properties is still a relevant topic, typically involving empirical grey value-to-elastic modulus relations. We here derive the voxel-specific volume fractions of mineral, collagen, and water, from tissue-independent bilinear relations between mineral and collagen content in extracellular bone tissue (J. Theor. Biol. 287: 115, 2011), and from the measured X-ray attenuation information quantified in terms of grey values. The aforementioned volume fractions enter a micromechanics representation of bone tissue, as to deliver voxel-specific stiffness tensors. In order to check the relevance of this strategy, we convert a micro Computer Tomograph of a mouse femur into a regular Finite Element mesh, apply forces related to the dead load of a standing mouse, and then compare simulation results based on voxel-specific heterogeneous elastic properties to results based on homogeneous elastic properties related to the spatial average over the solid bone matrix compartment, of the X-ray attenuation coefficients. The element-specific strain energy density illustrates that use of homogeneous elastic properties implies overestimation of the organ stiffness. Moreover, the simulation reveals large tensile normal stresses throughout the femur neck, which may explain the mouse femur neck's trabecular morphology being quite different from the human case, where the femur neck bears compressive forces and bending moments.