Abstract

The lateral ligaments of the ankle composed of the anterior talofibular (ATFL), calcaneofibular (CFL) and posterior talofibular ligaments (PTFL), are amongst the most commonly injured ligaments of the human body. Although treatment methods have been explored exhaustively, healing outcomes remain poor with high rates of re-injury, chronic ankle instability and pain persisting. The introduction and application of tissue engineering methods may target poor healing outcomes and eliminate long-term complications, improving the overall quality of life of affected individuals. For any surgical procedure or tissue-engineered replacement to be successful, a comprehensive understanding of the complete anatomy of the native structure is essential. Knowledge of the dimensions of ligament footprints is vitally important for surgeons as it guides the placement of bone tunnels during repair. It is also imperative in tissue-engineered design as the creation of a successful replacement relies on a thorough understanding of the native anatomy and microanatomical structure. Several studies explore techniques to describe ligament footprints around the body, with limited studies describing in-depth footprint dimensions of the ATFL, CFL and PTFL. Techniques currently used to measure ligament footprints are complex and require resources which may not be readily available, therefore a new methodology may prove beneficial.

While the COVID-19 pandemic highlighted the need for more accessible anatomy instruction tools, it is also well known that the time allocated to practical anatomy teaching has reduced in the past decades. Notably, the opportunity for anatomy students to learn osteology is not prioritised, nor is the ability of students to appreciate osteological variation. As a potential method of increasing accessibility to bone models, this study describes the process of developing 3D-printed replicas of human bones using a combination of structured light scanning (SLS) technology and 3D printing.

Human bones were obtained from the Anatomy Lab at the University of Edinburgh and were digitised using SLS via an Einscan H scanner. The resulting data was then used to print multiple replicas of varying materials, colours, scales and resolutions on an Ultimaker S3 3D printer. To gather opinion on these models and their variables, surveys were completed by anatomy students and educators (n=57). Data was collected using a Likert scale response, as well as free-text answers to gather qualitative information.

3D scans of the scapula, atlas (C1 vertebrae) and femur were successfully obtained. Plastic replicas were produced with defined variables in 4 separate stations e.g. different colours, to obtain results from survey respondents. For colour, 87.7% of survey respondents preferred white models, with 7% preferring orange and 5.3% preferring blue. For material, 47.4% of respondents preferred PLA (Polylactic acid), while 33.3% preferred ABS (Acrylonitrile butadiene styrene), 12.3% preferred Pet-G (Polyethylene terephthalate glycol), 3.5% preferred Glassbend and 3.5% had no preference. Additional results based on scale and resolution were also collected.

This initial study has demonstrated a proof-of-concept workflow for SLS technology to be combined with 3D printing to produce plastic replicas of human bones. Our study has provided key information about the colour, scale, material and resolution required for these models. Our future work will focus on determining accuracy of the models and their use as teaching aids for osteology education.

Jellyfish collagens exhibit auspicious perspectives for tissue engineering applications primarily due to their outstanding compatibility with a wide range of cell types, low immunogenicity and biodegradability. Furthermore, derived from a non-mammalian source, jellyfish collagens reduce the risk of disease transmission, minimising therefore the ethical and safety concerns. The current study aims to investigate the potential of 3-dimensional jellyfish collagen sponges (3D-JCS) in promoting bone tissue regeneration.

Both qualitative and quantitative analyses were performed in order to assess adhesion and proliferation of MC3T3 cells on 3D-JCL, as well as cell migration and bone-like ECM production. Histological and fluorescent dyes were used to stain mineral deposits (i.e. Alizarin Red S (ARS), Von Kossa, Tetracycline hydrochloride) while images were acquired using optical and confocal microscopy.

Qualitative data indicated successful adhesion and proliferation of MC3T3 cells on the 3D-JCS as well as cell migration along with ECM production both on the inner and outer surface of the scaffolds. Moreover, quantitative analyses indicated a four-fold increase of ARS uptake between 2- and 3-dimensional cultures (N=3) as well as an eighteen-fold increase of ARS uptake for the 3D-JCS (N=3) when cultured in osteogenic conditions compared to control. This suggests the augmented osteogenic potential of MC3T3 cells when cultured on 3D-JCS. Nevertheless, the cell-mediated mineral deposition appeared to alter the mechanical properties of the jellyfish collagen sponges that were previously reported to exhibit low mechanical properties (compressive modulus: 1-2 kPa before culture).

The biocompatibility, high porosity and pore interconnectivity of jellyfish collagen sponges promoted adhesion and proliferation of MC3T3 cells as well as cell migration and bone-like ECM production. Their unique features recommend the jellyfish collagen sponges as superior biomaterial scaffolds for bone tissue regeneration. Further studies are required to quantify the change in mechanical properties of the cell-seeded scaffolds and confirm their suitability for bone tissue regeneration. We predict that the 3D-JCS will be useful for future studies in both bone and bone-tendon interface regeneration.

Acknowledgments

This research has been supported by a Medical Research Scotland Studentship award (ref: -50177-2019) in collaboration with Jellagen Ltd.

To ensure clinical relevance, the in vitro engineering of tissues for implantation requires artificial replacements to possess properties similar to native anatomy. Our overarching study is focussed on developing a bespoke bone-tendon in vitro model replicating the anatomy at the flexor digitorum profundus (FDP) tendon insertion site at the distal phalanx. Anatomical morphometric analysis has guided FDP tendon model design consisting of hard and soft tissue types. Here, we investigate potential materials for creation of the model's bone portion by comparison of two bone cements; brushite and genex (Biocomposites Ltd).

3D printed molds were prepared based on anatomical morphometric analysis of the FDP tendon insertion site and used to cast identical bone blocks from brushite and genex cements. Studies assessing the suitability of each cement type were conducted e.g. setting times, pH on submersion in culture medium and interaction with fibrin gels. Data was collected using qualitative imaging and qualitative measurements (N=3,n=6) for experimental conditions.

Both brushite (BC) and genex (GC) cements could be cast into bespoke molds, producing individual blocks and were mixed/handled with appropriate setting times. On initial submersion in culture medium, BC caused a reduction in pH values (7.49 [control]) to 6.85) while GC remained stable (7.59). Reduction in pH value also affected fibrin gel interaction where gel was seen to be detaching/not forming around BC and medium discolouration was noted. This was not observed in GC. While GC outperformed BC in initial tests, repeated washing of BC led to pH stabilisation (7.5,3xwashes), consistent with their further use in this model.

This study has compared BC and GC as materials for bone block production. Both materials show promise, and current work assessing material properties and cell proliferation are needed to inform our choice for use in our FDP-tendon-bone interface model.

This research was supported by an ORUK Studentship award (ref:533). Genex was kindly provided by Biocomposites, Ltd.

3D spheroid culture is a bridge between standard 2D cell culture and in vivo research which mimics the physiological microenvironment in scaffold-free conditions. Here, this 3D technique is being investigated as a potential method for engineering bone tissue in vitro. However, spheroid culture can exhibit limitations, such as necrotic core formation due to the restricted access of oxygen and nutrients. It is therefore important to determine if spheroids without a sizeable necrotic core can be produced. This study aims to understand necrotic core formation and cell viability in 3D bone cell spheroids using different seeding densities and media formulations.

Differentiated rat osteoblasts (dRObs) were seeded in three different seeding densities (1×10⁴, 5×10⁴, 1×10 cells) in 96 well U-bottom cell-repellent plates and in three different media i.e., Growth medium (GM), Mineralisation medium 1 (MM1) and MM2. Spheroids were analysed from day 1 to 28 (N=3, n=2). Cell count and viability was assessed by trypan blue method. One way ANOVA and post-hoc Tukey test was performed to compare cell viability among different media and seeding densities. Histological spheroid sections were stained with hematoxylin and eosin (H&E) to identify any visible necrotic core.

Cell number increased from day 1 to 28 in all three seeding densities with a notable decrease in cell viability. 1×10⁴ cells proliferated faster than 5×10⁴ and 1×10⁵ cells and had proportionately similar cell death. The necrotic core area was relatively equivalent between all cell seeding densities. The larger the spheroid size, the larger is the size of the necrotic core.

This study has demonstrated that 3D spheroids can be formed from dRobs at a variety of seeding densities with no marked difference in necrotic core formation. Future studies will focus on utilising the bone cell spheroids for engineering scalable scaffold-free bone tissue constructs.

The COVID-19 pandemic necessitated a pivot to online learning for many traditional, hands-on subjects such as anatomy. This, coupled with the increase in online education programmes, and the reduction of time students spend in anatomy dissection rooms, has highlighted a real need for innovative and accessible learning tools. This study describes the development of a novel 3-dimensional (3D), interactive anatomy teaching tool using structured light scanning (SLS) technology. This technique allows the 3D shape and texture of an object to be captured and displayed online, where it can be viewed and manipulated in real-time.

Human bones of the upper limb, vertebrae and whole skulls were digitised using SLS using Einscan Pro2X/H scanners. The resulting meshes were then post-processed to add the captured textures and to remove any extraneous information. The final models were uploaded into Sketchfab where they were orientated, lit and annotated. To gather opinion on these models as effective teaching tools, surveys were completed by anatomy students (n=35) and anatomy educators (n=8). Data was collected using a Likert scale response, as well as free text answers to gather qualitative information.

3D scans of the scapula, humerus, radius, ulna, vertebrae and skull were successfully produced by SLS. Interactive models were produced via scan data in Sketchfab and successfully annotated to provide labelled 3D models for examination. 94% of survey respondents agreed that the interactive models were easy to use (n=35, 31% agree and 63% strongly agree) and 97% agreed that the 3D interactive models were more useful than 2D images for learning bony anatomy (n=35; 26% agree and 71% strongly agree).

This initial study has demonstrated a suitable proof-of-concept for SLS technology as a useful technique for producing 3D interactive online tools for learning and teaching bony anatomy. Current studies are focussed on determining the SLS accuracy and the ability of SLS to capture soft tissue/joints. We believe that this tool will be a useful technique for generating online 3D interactive models to study orthopaedic anatomy.

Abstract

Abstract

Entheses are the anchorage sites of tendons to bones in the musculoskeletal system. They have a unique microanatomy that allow smooth transfer of mechanical load through tendon to bone. However, entheses are prone to injury due to their small surface area^1,2. The overall success rate of the current gold standard treatment (directly attaching the tendon to bone) is small^3,4. Consequently, the aim of this study was to evaluate different hydrogels and their suitability for developing an in-vitro co-culture system to manufacture 3D tissue interfaces.

To create a 3D in-vitro tissue interface, half-well plugs were created by pouring silicone in wells of a 24-well plate. When set, it was cut into halves to be used as half-well plugs, blocking one side of a culture well. A tendon-cell-encapsulated hydrogel was poured into the exposed half and, when set, the plug was removed and a bone-cell-encapsulated gel was added. Cells were fluorescently labelled to enable identification of cell types under fluorescent microscopy (Tendon – green, bone – red). The suitability of different hydrogels to form an in vitro tissue interface was evaluated: fibrin, agarose and gellan.

This study demonstrates that 3D co-cultures can be manufactured in-vitro. The novel system enabled the culture of two cell types (bone/tendon) in direct contact, creating an in-vitro interface. In addition, this study shows that fibrin gel supports cell morphology, while both cell types failed to show normal morphology in agarose and gellan. Further studies evaluating cell viability in these hydrogels are currently underway.