Jess G. Snedeker

Misalignment of total ankle components can induce high joint contact pressures

BACKGROUND A major cause of the limited longevity of total ankle replacements is premature polyethylene component wear, which can be induced by high joint contact pressures. We implemented a computational model to parametrically explore the hypothesis that intercomponent positioning deviating from the manufacturers recommendations can result in pressure distributions that may predispose to wear of the polyethylene insert. We also investigated the hypothesis that a modern mobile-bearing design may be able to better compensate for imposed misalignments compared with an early two-component design. METHODS Two finite element models of total ankle replacement prostheses were built to quantify peak and average contact pressures on the polyethylene insert surfaces. Models were validated by biomechanical testing of the two implant designs with use of pressure-sensitive film. The validated models were configured to replicate three potential misalignments with the most CLINICAL RELEVANCE version of the tibial component, version of the talar component, and relative component rotation of the two-component design. The misalignments were simulated with use of the computer model with physiologically relevant boundary loads. RESULTS With use of the manufacturers guidelines for positioning of the two-component design, the predicted average joint contact pressures exceeded the yield stress of polyethylene (18 to 20 MPa). Pressure magnitudes increased as implant alignment was systematically deviated from this reference position. The three-component design showed lower-magnitude contact pressures in the standard position (<10 MPa) and was generally less sensitive to misalignment. Both implant systems were sensitive to version misalignment. CONCLUSIONS In the tested implants, a highly congruent mobile-bearing total ankle replacement design yields more evenly distributed and lower-magnitude joint contact pressures than a less congruent design. Although the mobile-bearing implant reduced susceptibility to aberrant joint contact characteristics that were induced by misalignment, predicted average contact stresses reached the yield stress of polyethylene for imposed version misalignments of >5 degrees.

A novel concept for scaffold-free vessel tissue engineering: self-assembly of microtissue building blocks.

Current scientific attempts to generate in vitro tissue-engineered living blood vessels (TEBVs) show substantial limitations, thereby preventing routine clinical use. In the present report, we describe a novel biotechnology concept to create living small diameter TEBV based exclusively on microtissue self-assembly (living cellular re-aggregates). A novel bioreactor was designed to assemble microtissues in a vascular shape and apply pulsatile flow and circumferential mechanical stimulation. Microtissues composed of human artery-derived fibroblasts (HAFs) and endothelial cells (HUVECs) were accumulated and cultured for 7 and 14 days under pulsatile flow/mechanical stimulation or static culture conditions with a diameter of 3mm and a wall thickness of 1mm. The resulting vessels were analyzed by immunohistochemistry for extracellular matrix (ECM) and cell phenotype (von Willebrand factor, alpha-SMA, Ki67, VEGF). Self-assembled microtissues composed of fibroblasts displayed significantly accelerated ECM formation compared to monolayer cell sheets. Accumulation of vessel-like tissue occurred within 14 days under both, static and flow/mechanical stimulation conditions. A layered tissue formation was observed only in the dynamic group, as indicated by luminal aligned alpha-SMA positive fibroblasts. We could demonstrate that self-assembled cell-based microtissues can be used to generate small diameter TEBV. The significant enhancement of ECM expression and maturation, together with the pre-vascularization capacity makes this approach highly attractive in terms of generating functional small diameter TEBV devoid of any foreign material.

Evidence against proteoglycan mediated collagen fibril load transmission and dynamic viscoelasticity in tendon

The glycosaminoglycan (GAG) dermatan sulfate and chondroitin sulfate side-chains of small leucine-rich proteoglycans have been increasingly posited to act as molecular cross links between adjacent collagen fibrils and to directly contribute to tendon elasticity. GAGs have also been implicated in tendon viscoelasticity, supposedly affecting frictional loss during elongation or fluid flow through the extra cellular matrix. The current study sought to systematically test these theories of tendon structure-function by investigating the mechanical repercussions of enzymatic depletion of GAG complexes by chondroitinase ABC in a reproducible tendon structure-function model (rat tail tendon fascicles). The extent of GAG removal (at least 93%) was verified by relevant spectrophotometric assays and transmission electron microscopy. Dynamic viscoelastic tensile tests on GAG depleted rat tail tendon fascicle were not mechanically different from controls in storage modulus (elastic behavior) over a wide range of strain-rates (0.05, 0.5, and 5% change in length per second) in either the linear or nonlinear regions of the material curve. Loss modulus (viscoelastic behavior) was only affected in the nonlinear region at the highest strain-rate, and even this effect was marginal (19% increased loss modulus, p=0.035). Thus glycosaminoglycan chains of small leucine-rich proteoglycans do not appear to mediate dynamic elastic behavior nor do they appear to regulate the dynamic viscoelastic properties in rat tail tendon fascicles.

Biochemical and biomechanical gradients for directed bone marrow stromal cell differentiation toward tendon and bone.

Substrates with mechanical property gradients and various extracellular matrix ligand loadings were evaluated for their ability to direct bone marrow stromal cell differentiation along osteogenic and tenogenic lineages. After verifying reproducible mechanical compliance characteristics of commercial hydrogel gradient substrates, substrates were functionalized with whole length fibronectin or collagen, both of which are found in skeletal structures and are relevant to cell-matrix signalling. Bone marrow stromal cells were seeded onto the substrates in growth media and cultured first to examine cell attachment and morphology, indicating higher levels of attachment on collagen substrates after 1h, and increased spreading and organization trends after 24h. Differentiation studies showed an increase in osteoblast differentiation on fibronectin substrates while collagen substrates lacked osteogenic differentiation. Osteogenic differentiation decreased on substrates of lower stiffness and lower ligand density. Molecular investigations revealed an increase in relevant signalling molecules for osteoblasts that were consistent with differentiation studies, but detected the presence of tenoblast markers on collagen substrates within a narrow range of stiffness. Our results indicate that mechanovariant substrates do hold promise as a culture platform for directed differentiation to tendon and bone by altering gene level expression of relevant signalling molecules. This study aids in understanding the molecular mechanisms that drive differentiation from substrate based cues, and could aid the design of therapeutic biomaterials at the transition from tendon to bone.

Advanced glycation end-products diminish tendon collagen fiber sliding

Connective tissue aging and diabetes related comorbidity are associated with compromised tissue function, increased susceptibility to injury, and reduced healing capacity. This has been partly attributed to collagen cross-linking by advanced glycation end-products (AGEs) that accumulate with both age and disease. While such cross-links are believed to alter the physical properties of collagen structures and tissue behavior, existing data relating AGEs to tendon mechanics is contradictory. In this study, we utilized a rat tail tendon model to quantify the micro-mechanical repercussion of AGEs at the collagen fiber-level. Individual tendon fascicles were incubated with methylglyoxal (MGO), a naturally occurring metabolite known to form AGEs. After incubation in MGO solution or buffer only, tendons were stretched on the stage of a multiphoton confocal microscope and individual collagen fiber stretch and relative fiber sliding were quantified. Treatment by MGO yielded increased fluorescence and elevated denaturation temperatures as found in normally aged tissue, confirming formation of AGEs and related cross-links. No apparent ultrastructural changes were noted in transmission electron micrographs of cross-linked fibrils. MGO treatment strongly reduced tissue stress relaxation (p<0.01), with concomitantly increased tissue yield stress (p<0.01) and ultimate failure stress (p=0.036). MGO did not affect tangential modulus in the linear part of the stress-strain curve (p=0.46). Microscopic analysis of collagen fiber kinematics yielded striking results, with MGO treatment drastically reducing fiber-sliding (p<0.01) with a compensatory increase in fiber-stretch (p<0.01). We thus conclude that the main mechanical effect of AGEs is a loss of tissue viscoelasticity driven by matrix-level loss of fiber-fiber sliding. This has potentially important implications to tissue damage accumulation, mechanically regulated cell signaling, and matrix remodeling. It further highlights the importance of assessing viscoelasticity - not only elastic response - when considering age-related changes in the tendon matrix and connective tissue in general.

Local strain measurement reveals a varied regional dependence of tensile tendon mechanics on glycosaminoglycan content

Proteoglycans (PG) and their associated glycosaminoglycan (GAG) side chains are known to play a key role in the bearing of compressive loads in cartilage and other skeletal connective tissues. In tendons and connective tissues that are primarily loaded in tension, the influence of proteoglycans on mechanical behavior is debated due to conflicting experimental evidence that alternately supports or controverts a functional role of proteoglycans in bearing tensile load. In this study we sought to better reconcile these conflicting data by investigating the possibility that GAG content is differentially related to tensile tendon mechanics depending upon the anatomical subregion one considers. To test this hypothesis, we quantified the mechanical consequences of proteoglycan disruption within specific tendon anatomical subregions using an optical-mechanical measurement approach. Achilles tendons from adult mice were treated with chondroitinase ABC to obtain two groups consisting of native tendons and GAG-depleted tendons. All the tendons were mechanically tested and imaged with high-resolution digital video in order to optically quantify tendon strains. Tendon surface strains were locally analyzed in three main subregions: the central midsubstance, and the proximal and distal midsubstance near the muscle and bone insertions, respectively. Upon GAG digestion, the tendon midsubstance softened appreciably near the bone insertion, while elastic modulus in the central and proximal thirds was unchanged. Thus the contribution of PGs to tensile tendon mechanics is not straightforward and points to a heterogeneous and complex structure-function relationship in tendon. This study further highlights the importance of performing local strain analysis with regard to tensile tendon mechanics.

Equivalent stiffness after glycosaminoglycan depletion in tendon — an ultra-structural finite element model and corresponding experiments

The glycosaminoglycan (GAG) side-chains of small leucine-rich proteoglycans have been postulated to mechanically cross-link adjacent collagen fibrils and contribute to tendon mechanics. Enzymatic depletion of tendon GAGs (chondroitin and dermatan sulfate) has emerged as a preferred method to experimentally assess this role. However, GAG removal is typically incomplete and the possibility remains that extant GAGs may remain mechanically functional. The current study specifically investigated the potential mechanical effect of the remaining GAGs after partial enzymatic digestion. A three-dimensional finite element model of tendon was created based upon the concept of proteoglycan mediated inter-fibril load sharing. Approximately 250 interacting, discontinuous collagen fibrils were modeled as having a length of 400 μm, being composed of rod elements of length 67 nm and E-modulus 1 GPa connected in series. Spatial distribution and diameters of these idealized fibrils were derived from a representative cross-sectional electron micrograph of tendon. Rod element lengths corresponded to the collagen fibril D-Period, widely accepted to act as a binding site for decorin and biglycan, the most abundant proteoglycans in tendon. Each element node was connected to nodes of any neighboring fibrils within a radius of 100 nm, the slack length of unstretched chondroitin sulfate. These GAG cross-links were the sole mechanism for lateral load sharing among the discontinuous fibrils, and were modeled as bilinear spring elements. Simulation of tensile testing of tendon with complete cross-linking closely reproduced corresponding experiments on rat tail tendons. Random reduction of 80% of GAG cross-links (matched to a conservative estimate of enzymatic depletion efficacy) predicted a drop of 14% in tendon modulus. Corresponding mechanical properties derived from experiments on rat tail tendons treated in buffer with and without chondroitinase ABC were apparently unaffected, regardless of GAG depletion. Further tests for equivalence, conservatively based on effect size limits predicted by the model, confirmed equivalent stiffness between enzymatically depleted tendons and their native controls. Although the model predicts that relatively small quantities of GAGs acting as primary collagen cross-linking elements could provide mechanical integrity to the tendon, partial enzymatic depletion of GAGs should result in mechanical changes that are not reflected in analogous experimental testing. We thus conclude that GAG side chains of small leucine-rich proteoglycans are not a primary determinant of tensile mechanical behavior in mature rat tail tendons.

Elastography: modality-specific approaches, clinical applications, and research horizons

Manual palpation has been used for centuries to provide a relative indication of tissue health and disease. Engineers have sought to make these assessments increasingly quantitative and accessible within daily clinical practice. Since many of the developed techniques involve image-based quantification of tissue deformation in response to an applied force (i.e., “elastography”), such approaches fall squarely within the domain of the radiologist. While commercial elastography analysis software is becoming increasingly available for clinical use, the internal workings of these packages often remain a “black box,” with limited guidance on how to usefully apply the methods toward a meaningful diagnosis. The purpose of the present review article is to introduce some important approaches to elastography that have been developed for the most widely used clinical imaging modalities (e.g., ultrasound, MRI), to provide a basic sense of the underlying physical principles, and to discuss both current and potential (musculoskeletal) applications. The article also seeks to provide a perspective on emerging approaches that are rapidly developing in the research laboratory (e.g., optical coherence tomography, fibered confocal microscopy), and which may eventually gain a clinical foothold.

Tendon glycosaminoglycan proteoglycan sidechains promote collagen fibril sliding-AFM observations at the nanoscale.

The extracellular matrix of tendon is mainly composed of discontinuous Type-I collagen fibrils and small leucine rich proteoglycans (PG). Macroscopic tendon behaviors like stiffness and strength are determined by the ultrastructural arrangement of these components. When a tendon is submitted to load, the collagen fibrils both elongate and slide relative to their neighboring fibrils. The role of PG glycosaminoglycan (GAG) sidechains in mediating inter-fibril load sharing remains controversial, with competing structure-function theories suggesting that PGs may mechanically couple neighboring collagen fibrils (cross-linking them to facilitate fibril stretch) or alternatively isolating them (promoting fibril gliding). In this study, we sought to clarify the functional role of GAGs in tensile tendon mechanics by directly investigating the mechanical response of individual collagen fibrils within their collagen network in both native and GAG depleted tendons. A control group of Achilles tendons from adult mice was compared with tendons in which GAGs were enzymatically depleted using chondroitinase ABC. Tendons were loaded to specific target strains, chemically fixed under constant load, and later sectioned for morphological analysis by an atomic force microscope (AFM). Increases in periodic banding of the collagen fibrils (D-period) or decreases in fibril diameter was considered to be representative of collagen fibril elongation and the mechanical contribution of GAGs at the ultrascale was quantified on this basis. At high levels of applied tendon strain (10%), GAG depleted tendons showed increased collagen stretch (less fibril sliding). We conclude that the hydrophilic GAGs seem thus not to act as mechanical crosslinks but rather act to promote collagen fibril sliding under tension.

Supraspinatus tendon load during abduction is dependent on the size of the critical shoulder angle: A biomechanical analysis.

Shoulders with supraspinatus (SSP) tears are associated with significantly larger critical shoulder angles (CSA) compared to disease‐free shoulders. We hypothesized that larger CSAs increase the ratio of joint shear to joint compression forces (defined as “instability ratio”), requiring substantially increased compensatory supraspinatus loads. A shoulder simulator with simulated deltoid, supraspinatus, infraspinatus/teres minor, and subscapularis musculotendinous units was constructed. The model was configured to represent either a normal CSA of 33° or a CSA characteristic of shoulders with rotator cuff tears (38°), and the components of the joint forces were measured. The instability ratio increased for the 38° CSA compared with the control CSA (33°) for a range of motion between 6° to 61° of thoracohumeral abduction with the largest differences in instability observed between 33° and 37° of elevation. In this range, SSP force had to be increased by 13–33% (15–23 N) to stabilize the arm in space. Our results support the concept that a high CSA can induce SSP overload particularly at low degrees of active abduction.