Anatomical Design and Production of a Novel 3-Dimensional Co-Culture System Replicating the Human Flexor Digitorum Profundus Enthesis

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.<br/

Optimizing an Intermittent Stretch Paradigm Using ERK1/2 Phosphorylation Results in Increased Collagen Synthesis in Engineered Ligaments

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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98478/1/ten%2Etea%2E2011%2E0336.pd

Engineering three-dimensional bone macro-tissues by guided fusion of cell spheroids

Introduction: Bioassembly techniques for the application of scaffold-freetissue engineering approaches have evolved in recent years towardproducing larger tissue equivalents that structurally and functionally mimicnative tissues. This study aims to upscale a 3-dimensional bone in-vitromodel through bioassembly of differentiated rat osteoblast (dROb) spheroidswith the potential to develop and mature into a bone macrotissue.Methods: dROb spheroids in control and mineralization media at differentseeding densities (1 × 104, 5 × 104, and 1 × 105 cells) were assessed for cellproliferation and viability by trypan blue staining, for necrotic core byhematoxylin and eosin staining, and for extracellular calcium by Alizarin redand Von Kossa staining. Then, a novel approach was developed tobioassemble dROb spheroids in pillar array supports using a customizedbioassembly system. Pillar array supports were custom-designed and printedusing Formlabs Clear Resin® by Formlabs Form2 printer. These supports wereused as temporary frameworks for spheroid bioassembly until fusionoccurred. Supports were then removed to allow scaffold-free growth andmaturation of fused spheroids. Morphological and molecular analyses wereperformed to understand their structural and functional aspects.Results: Spheroids of all seeding densities proliferated till day 14, andmineralization began with the cessation of proliferation. Necrotic core sizeincreased over time with increased spheroid size. After the bioassembly ofspheroids, the morphological assessment revealed the fusion of spheroidsover time into a single macrotissue of more than 2.5 mm in size with mineralformation. Molecular assessment at different time points revealed osteogenicmaturation based on the presence of osteocalcin, downregulation of Runx2(p &lt; 0.001), and upregulated alkaline phosphatase (p &lt; 0.01).Discussion: With the novel bioassembly approach used here, 3D bonemacrotissues were successfully fabricated which mimicked physiological osteogenesis both morphologically and molecularly. This biofabricationapproach has potential applications in bone tissue engineering,contributing to research related to osteoporosis and other recurrentbone ailments

Histomorphology of the subregions of the scapholunate ligament and its enthesis

Background  The scapholunate interosseous ligament (SLIL) has three subregions: dorsal, proximal, and volar. The SLIL enthesis has not previously been studied despite its important mechanical function in wrist joint biomechanics. Questions/Purposes  This study aims to compare the histomorphological differences between the SLIL subregions, including at their entheses. Three questions are explored: Do the gross dimensions differ between SLIL subregions? Does the enthesis qualitatively, and its calcified fibrocartilage (CF) quantitatively, differ between (a) SLIL subregions and (b) scaphoid and lunate attachments? Methods  Twelve fresh-frozen human cadaveric wrists were dissected and the gross dimensions of the SLIL subregions measured. Subregions were histologically processed for morphological and compositional analyses, including quantification of enthesis CF area. Results  The dorsal subregion was the thickest. The dorsal and volar subregions had fibrocartilaginous entheses, while the proximal subregion was attached to articular cartilage. The dorsal subregion had significantly more CF than the volar subregion. There was no significant difference in the enthesis CF between scaphoid and lunate attachments in the three subregions. Conclusions  There are significant morphological differences between the SLIL subregions. The dorsal subregion has the largest amount of CF, which is consistent with the greater biomechanical force subjected to this subregion. The similar histomorphology of the ligament at the scaphoid and lunate entheses suggests that similar biomechanical forces are applied to both attachments. Clinical Relevance  The histomorphological results confirm that the dorsal subregion is the strongest of the three subregions. The results from the entheseal region may have important implications in the study of graft incorporation during SLIL reconstruction

Design and Development of a Bioreactor System for Mechanical Stimulation of Musculoskeletal Tissue

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.</p

Engineering three-dimensional bone macro-tissues by guided fusion of cell spheroids

IntroductionBioassembly 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 in-vitro model through bioassembly of differentiated rat osteoblast (dROb) spheroids with the potential to develop and mature into a bone macrotissue.MethodsdROb spheroids in control and mineralization media at different seeding densities (1 × 104, 5 × 104, and 1 × 105 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.ResultsSpheroids 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 (p &lt; 0.001), and upregulated alkaline phosphatase (p &lt; 0.01).DiscussionWith 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

Exploiting cell-mediated contraction and adhesion to structure tissues in vitro

Progress in tissue engineering is now impacting beyond the field of regenerative medicine. Engineered tissues are now used as tools to evaluate the toxicity of compounds or even to enable the modelling of disease. While many of the materials that are used to facilitate tissue growth are designed to enable cell attachment, many researchers consider that the contraction and modification of these matrices by attached cells is not desirable and take measures to prevent this from occurring. Where substantial alignment of the molecules within tissues, however, is a feature of structure the process of contraction can be exploited to guide new matrix deposition. In this paper, we will demonstrate how we have used the cell contraction process to generate tissues with high levels of organization. The tissues that have been grown in the laboratory have been characterized using a suite of analytical techniques to demonstrate significant levels of matrix organization and mechanical behaviour analogous to natural tissues. This paper provides an overview of research that has been undertaken to determine how tissues have been grown in vitro with structuring from the molecular, right through to the macroscopic level

Nanoscale crystallinity modulates cell proliferation on plasma sprayed surfaces

Calcium phosphate coatings have been applied to the surface of metallic prostheses to mediate hard and soft tissue attachment for more than 40 years. Most coatings are formed of high purity hydroxyapatite, and coating methods are often designed to produce highly crystalline surfaces. It is likely however, that coatings of lower crystallinity can facilitate more rapid tissue attachment since the surface will exhibit a higher specific surface area and will be considerably more reactive than a comparable highly crystalline surface. Here we test this hypothesis by growing a population of MC3T3 osteoblast-like cells on the surface of two types of hip prosthesis with similar composition, but with differing crystallinity. The surfaces with lower crystallinity facilitated more rapid cell attachment and increased proliferation rate, despite having a less heterogeneous surface topography. This work highlights that the influence of the crystallinity of HA at the nano-scale is dominant over macroscale topography for cell adhesion and growth. Furthermore, crystallinity could be easily adjusted by without compromising coating purity. These findings could facilitate designing novel coated calcium phosphate surfaces that more rapidly bond tissue following implantation

AVID: An integrative framework for discovering functional relationships among proteins

BACKGROUND: Determining the functions of uncharacterized proteins is one of the most pressing problems in the post-genomic era. Large scale protein-protein interaction assays, global mRNA expression analyses and systematic protein localization studies provide experimental information that can be used for this purpose. The data from such experiments contain many false positives and false negatives, but can be processed using computational methods to provide reliable information about protein-protein relationships and protein function. An outstanding and important goal is to predict detailed functional annotation for all uncharacterized proteins that is reliable enough to effectively guide experiments. RESULTS: We present AVID, a computational method that uses a multi-stage learning framework to integrate experimental results with sequence information, generating networks reflecting functional similarities among proteins. We illustrate use of the networks by making predictions of detailed Gene Ontology (GO) annotations in three categories: molecular function, biological process, and cellular component. Applied to the yeast Saccharomyces cerevisiae, AVID provides 37,451 pair-wise functional linkages between 4,191 proteins. These relationships are ~65–78% accurate, as assessed by cross-validation testing. Assignments of highly detailed functional descriptors to proteins, based on the networks, are estimated to be ~67% accurate for GO categories describing molecular function and cellular component and ~52% accurate for terms describing biological process. The predictions cover 1,490 proteins with no previous annotation in GO and also assign more detailed functions to many proteins annotated only with less descriptive terms. Predictions made by AVID are largely distinct from those made by other methods. Out of 37,451 predicted pair-wise relationships, the greatest number shared in common with another method is 3,413. CONCLUSION: AVID provides three networks reflecting functional associations among proteins. We use these networks to generate new, highly detailed functional predictions for roughly half of the yeast proteome that are reliable enough to drive targeted experimental investigations. The predictions suggest many specific, testable hypotheses. All of the data are available as downloadable files as well as through an interactive website at . Thus, AVID will be a valuable resource for experimental biologists