The enthesis, the specialized junction between tendon and bone, is a common site of injury. Although notoriously difficult to repair, advances in interfacial tissue engineering techniques are being ...developed for restorative function. Most notably are 3D in vitro co-culture models, built to recreate the complex heterogeneity of the native enthesis. While cell and matrix properties are often considered, there has been little attention given to native enthesis anatomical morphometrics and replicating these to enhance clinical relevance. This study focuses on the flexor digitorum profundus (FDP) tendon enthesis and, by combining anatomical morphometrics with computer-aided design, demonstrates the design and construction of an accurate and scalable model of the FDP enthesis. Bespoke 3D-printed mould inserts were fabricated based on the size, shape and insertion angle of the FDP enthesis. Then, silicone culture moulds were created, enabling the production of bespoke anatomical culture zones for an in vitro FDP enthesis model. The validity of the model has been confirmed using brushite cement scaffolds seeded with osteoblasts (bone) and fibrin hydrogel scaffolds seeded with fibroblasts (tendon) in individual studies with cells from either human or rat origin. This novel approach allows a bespoke anatomical design for enthesis repair and should be applied to future studies in this area.
The enthesis possesses morphological adaptations across the soft-hard tissue junction which are not fully restored during surgical avulsion repairs. This loss of anatomical structure, highly related ...to function, contributes to poor clinical outcomes. Investigating the native macro- and micro-structure of a specific enthesis can provide functional and biomechanical insights to develop specialised, novel tissue-engineered therapeutic options and potentially improve current surgical treatments for avulsion injuries.
This study examines the anatomy and histomorphology of the flexor digitorum profundus (FDP) enthesis in 96 fresh-frozen human cadaveric fingers, quantitatively and qualitatively analyzing the shape, size, angle of tendon fibres and histological architecture, and explores differences in sex, finger and distance along the enthesis using linear mixed effects models.
Macroscopically, results showed a consistent trapezoidal insertion shape of 29.29 ± 2.35 mm
mean surface area, but with significant morphometric size differences influenced primarily by the smaller dimensions of the little finger. Microscopically, a fibrocartilaginous enthesis was apparent with a 30.05 ± 0.72
mean angle of inserting tendon fibres, although regional variation in fibrocartilage and the angle change of tendon fibres before insertion existed.
The implication of these findings on native and specific FDP enthesis function is discussed whilst providing recommendations for optimal FDP enthesis recreation for interfacial tissue engineers and hand surgeons. The study emphasizes the importance of region-specific knowledge whilst also describing methods applicable to assessing any soft tissue insertion.
Bioassembly techniques for the application of scaffold-free tissue engineering approaches have evolved in recent years toward producing larger tissue equivalents that structurally and functionally ...mimic native tissues. This study aims to upscale a 3-dimensional bone
model through bioassembly of differentiated rat osteoblast (dROb) spheroids with the potential to develop and mature into a bone macrotissue.
dROb spheroids in control and mineralization media at different seeding densities (1 × 10
, 5 × 10
, and 1 × 10
cells) were assessed for cell proliferation and viability by trypan blue staining, for necrotic core by hematoxylin and eosin staining, and for extracellular calcium by Alizarin red and Von Kossa staining. Then, a novel approach was developed to bioassemble dROb spheroids in pillar array supports using a customized bioassembly system. Pillar array supports were custom-designed and printed using Formlabs Clear Resin
by Formlabs Form2 printer. These supports were used as temporary frameworks for spheroid bioassembly until fusion occurred. Supports were then removed to allow scaffold-free growth and maturation of fused spheroids. Morphological and molecular analyses were performed to understand their structural and functional aspects.
Spheroids of all seeding densities proliferated till day 14, and mineralization began with the cessation of proliferation. Necrotic core size increased over time with increased spheroid size. After the bioassembly of spheroids, the morphological assessment revealed the fusion of spheroids over time into a single macrotissue of more than 2.5 mm in size with mineral formation. Molecular assessment at different time points revealed osteogenic maturation based on the presence of osteocalcin, downregulation of Runx2 (
< 0.001), and upregulated alkaline phosphatase (
< 0.01).
With the novel bioassembly approach used here, 3D bone macrotissues were successfully fabricated which mimicked physiological osteogenesis both morphologically and molecularly. This biofabrication approach has potential applications in bone tissue engineering, contributing to research related to osteoporosis and other recurrent bone ailments.
Abstract
Musculoskeletal tissue interfaces are a common site of injury in the young, active populations. In particular, the interface between the musculoskeletal tissues of tendon and bone is often ...injured and to date, no single treatment has been able to restore the form and function of damaged tissue at the bone–tendon interface. Tissue engineering and regeneration hold great promise for the manufacture of bespoke in vitro models or implants to be used to advance repair and so this study investigated the material, orientation and culture choices for manufacturing a reproducible 3D model of a musculoskeletal interface between tendon and bone cell populations. Such models are essential for future studies focussing on the regeneration of musculoskeletal interfaces in vitro. Cell-encapsulated fibrin hydrogels, arranged in a horizontal orientation though a simple moulding procedure, were shown to best support cellular growth and migration of cells to form an in vitro tendon–bone interface. This study highlights the importance of acknowledging the material and technical challenges in establishing co-cultures and suggests a reproducible methodology to form 3D co-cultures between tendon and bone, or other musculoskeletal cell types, in vitro.
Dynamic mechanical input is believed to play a critical role in the development of functional musculoskeletal tissues. To study this phenomenon, cyclic uniaxial mechanical stretch was applied to ...engineered ligaments using a custom-built bioreactor and the effects of different stretch frequency, amplitude, and duration were determined. Stretch acutely increased the phosphorylation of p38 (3.5±0.74-fold), S6K1 (3.9±0.19-fold), and ERK1/2 (2.45±0.32-fold). The phosphorylation of ERK1/2 was dependent on time, rather than on frequency or amplitude, within these constructs. ERK1/2 phosphorylation was similar following stretch at frequencies from 0.1 to 1 Hz and amplitudes from 2.5% to 15%, whereas phosphorylation reached maximal levels at 10 min of stretch and returned toward basal within 60 min of stretch. Following a single 10-min bout of cyclic stretch, the cells remained refractory to a second stretch for up to 6 h. Using the phosphorylation of ERK1/2 as a guide, the optimum stretch paradigm was hypothesized to be 10 min of stretch at 2.5% of resting length repeated every 6 h. Consistent with this hypothesis, 7 days of stretch using this optimized intermittent stretch program increased the collagen content of the grafts more than a continuous stretch program (CTL=3.1%±0.44%; CONT=4.8%±0.30%; and INT=5.9%±0.56%). These results suggest that short infrequent bouts of loading are optimal for improving engineered tendon and ligament physiology.
For musculoskeletal tissues that transmit loads during movement, the interfaces between tissues are essential to minimizing injury. Therefore, the reproduction of functional interfaces within ...engineered musculoskeletal tissues is critical to the successful transfer of the technology to the clinic. The goal of this work was to rapidly engineer ligament equivalents in vitro that contained both the soft tissue sinew and a hard tissue bone mimetic. This goal was achieved using cast brushite (CaHPO(4)·2H(2)O) anchors to mimic bone and a fibrin gel embedded with fibroblasts to create the sinew. The constructs formed within 7 days. Fourteen days after seeding, the interface between the brushite and sinew could withstand a stress of 9.51 ± 1.7 kPa before failure and the sinew reached a Young's modulus value of 0.16 ± 0.03 MPa. Treatment with ascorbic acid and proline increased the collagen content of the sinew (from 1.34% ± 0.2% to 8.34% ± 0.37%), strength of the interface (29.24 ± 6 kPa), and modulus of the sinew (2.69 ± 0.25 MPa). Adding transforming growth factor-β resulted in a further increase in collagen (11.25% ± 0.39%), interface strength (42 ± 8 kPa), and sinew modulus (5.46 ± 0.68 MPa). Both scanning electron and Raman microscopy suggested that the interface between the brushite and sinew mimics the in vivo tidemark at the enthesis. This work describes a major step toward the development of tissue-engineered ligaments for the repair of ligament ruptures in humans.
The growing use of online education tools, combined with an increasing demand for anatomical education from non-traditional student cohorts, prompted us to develop a postgraduate Anatomical Sciences ...degree programme. Taught entirely online, this programme of study allows students from anywhere in the world to study anatomy and related subjects at the University of Edinburgh. Here, the initial three years of programme delivery is described, including an examination of the student cohorts, their geographical location, their background on application and an exploration of qualitative feedback on their programme experience.
Data collected from the applications of student cohorts from academic year 2015/16 through to 2017/18 were anonymised and analysed to assess student personal characteristics. Also, qualitative student feedback data were obtained from evaluation questionnaires collected anonymously at the end of each course of the programme.
The majority of the Anatomical Sciences students were female (72%) and within the age range 20–29. In addition, despite being accessible all over the world, the majority of the student cohort resided in the UK (50%). Qualitative feedback suggested that online teaching in anatomy was well-received and enjoyed, particularly in the form of video lectures and bespoke online resources. Challenges such as the modes of assessment and the lack of exposure to anatomical material were noted.
Investigating the backgrounds and opinions of the initial Anatomical Sciences students is a useful method to ensure a high level of delivery as the programme continues. Here, data and feedback for the first three years of the programme have been analysed and have highlighted specific challenges of teaching anatomy online. Finally, the future directions of online education within the anatomical sciences field is discussed.
•Anatomical Sciences is a new postgraduate anatomy programme delivered entirely online.•Postgraduate online education in anatomy is a new, previously unexplored topic.•Students enrolled on the programme are from a variety of subject backgrounds.•Specific successes include the inclusion of interactive tools to help consolidate learning.•Challenges include building an online community and the lack of tactile learning approaches.
We report on the development of a bioreactor system for mechanical stimulation of musculoskeletal tissues. The ultimate object is to improve the quality of medical treatment following injuries of the ...enthesis tissue. To this end, the tissue formation process through the effect of mechanical stimulation is investigated. A six-well system was designed, 3D printed and tested. An integrated actuator creates strain by applying a force. A contactless position sensor monitors the travels. An electronic circuit controls the bioreactor using a microcontroller. An IoT platform connects the microcontroller to a smartphone, enabling the user to alter variables, trigger actions and monitor the system. The system was stabilised by implementing two PID controllers and safety measures. The results show that the bioreactor design is suited to execute mechanical stimulation and to investigate the tissue formation and regeneration process. The bioreactor reported here can now be implemented in tissue engineering applications including tissue specimen.
Ligaments and tendons have previously been tissue engineered. However, without the bone attachment, implantation of a tissue-engineered ligament would require it to be sutured to the remnant of the ...injured native tissue. Due to slow repair and remodeling, this would result in a chronically weak tissue that may never return to preinjury function. In contrast, orthopaedic autograft reconstruction of the ligament often uses a bone-to-bone technique for optimal repair. Since bone-to-bone repairs heal better than other methods, implantation of an artificial ligament should also occur from bone-to-bone. The aim of this study was to investigate the use of a poly(ethylene glycol) diacrylate (PEGDA) hydrogel incorporated with hydroxyapatite (HA) and the cell-adhesion peptide RGD (Arg-Gly-Asp) as a material for creating an in vitro tissue interface to engineer intact ligaments (i.e., bone-ligament-bone). Incorporation of HA into PEG hydrogels reduced the swelling ratio but increased mechanical strength and stiffness of the hydrogels. Further, HA addition increased the capacity for cell growth and interface formation. RGD incorporation increased the swelling ratio but decreased mechanical strength and stiffness of the material. Optimum levels of cell attachment were met using a combination of both HA and RGD, but this material had no better mechanical properties than PEG alone. Although adherence of the hydrogels containing HA was achieved, failure occurs at about 4 days with 5% HA. Increasing the proportion of HA improved interface formation; however, with high levels of HA, the PEG HA composite became brittle. This data suggests that HA, by itself or with other materials, might be well suited for engineering the ligament-bone interface.
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