Improved suture pullout through genipin-coated sutures in human biceps tendons with spatially confined changes in cell viability.

BACKGROUND: The suture-tendon interface often constitutes the point of failure in tendon suture repair. In the present study, we investigated the mechanical benefit of coating the suture with a cross-linking agent to strengthen the nearby tissue after suture placement in human tendons and we assessed the biological implications regarding tendon cell survival in-vitro. METHODS: Freshly harvested human biceps long head tendons were randomly allocated to control (n = 17) or intervention (n = 19) group. According to the assigned group, either an untreated or a genipin-coated suture was inserted into the tendon. 24 h after suturing, mechanical testing composed of cyclic and ramp-to-failure loading was performed. Additionally, 11 freshly harvested tendons were used for short-term in vitro cell viability assessment in response to genipin-loaded suture placement. These specimens were analyzed in a paired-sample setting as stained histological sections using combined fluorescent/light microscopy. FINDINGS: Tendons stitched with a genipin-coated suture sustained higher forces to failure. Cyclic and ultimate displacement of the tendon-suture construct remained unaltered by the local tissue crosslinking. Tissue crosslinking resulted in significant cytotoxicity in the direct vicinity of the suture (<3 mm). At larger distances from the suture, however, no difference in cell viability between the test and the control group was discernable. INTERPRETATION: The repair strength of a tendon-suture construct can be augmented by loading the suture with genipin. At this mechanically relevant dosage, crosslinking-induced cell death is confined to a radius of <3 mm from the suture in the short-term in-vitro setting. These promising results warrant further examination in-vivo.

Focus on time: dynamic imaging reveals stretch-dependent cell relaxation and nuclear deformation.

Among the stimuli to which cells are exposed in vivo, it has been shown that tensile deformations induce specific cellular responses in musculoskeletal, cardiovascular, and stromal tissues. However, the early response of cells to sustained substrate-based stretch has remained elusive because of the short timescale at which it occurs. To measure the tensile mechanical properties of adherent cells immediately after the application of substrate deformations, we have developed a dynamic traction force microscopy method that enables subsecond temporal resolution imaging of transient subcellular events. The system employs a novel, to our knowledge, tracking approach with minimal computational overhead to compensate substrate-based, stretch-induced motion/drift of stretched single cells in real time, allowing capture of biophysical phenomena on multiple channels by fluorescent multichannel imaging on a single camera, thus avoiding the need for beam splitting with the associated loss of light. Using this tool, we have characterized the transient subcellular forces and nuclear deformations of single cells immediately after the application of equibiaxial strain. Our experiments reveal significant differences in the cell relaxation dynamics and in the intracellular propagation of force to the nuclear compartment in cells stretched at different strain rates and exposes the need for time control for the correct interpretation of dynamic cell mechanics experiments.

Tendon response to matrix unloading is determined by the patho-physiological niche.

Although the molecular mechanisms behind tendon disease remain obscure, aberrant stromal matrix turnover and tissue hypervascularity are known hallmarks of advanced tendinopathy. We harness a tendon explant model to unwind complex cross-talk between the stromal and vascular tissue compartments. We identify the hypervascular tendon niche as a state-switch that gates degenerative matrix remodeling within the tissue stroma. Here pathological conditions resembling hypervascular tendon disease provoke rapid cell-mediated tissue breakdown upon mechanical unloading, in contrast to unloaded tendons that remain functionally stable in physiological low-oxygen/-temperature niches. Analyses of the stromal tissue transcriptome and secretome reveal that a stromal niche with elevated tissue oxygenation and temperature drives a ROS mediated cellular stress response that leads to adoption of an immune-modulatory phenotype within the degrading stromal tissue. Degradomic analysis further reveals a surprisingly rich set of active matrix proteases behind the progressive loss of tissue mechanics. We conclude that the tendon stromal compartment responds to aberrant mechanical unloading in a manner that is highly dependent on the vascular niche, with ROS gating a complex proteolytic breakdown of the functional collagen backbone.

The Protein Mat(ters)-Revealing the Biologically Relevant Mechanical Contribution of Collagen- and Fibronectin-Coated Micropatterns.

Understanding cell-material interactions requires accurate characterization of the substrate mechanics, which are generally measured by indentation-type atomic force microscopy. To facilitate cell-substrate interaction, model extracellular matrix coatings are used although their tensile mechanical properties are generally unknown. In this study, beyond standard compressive stiffness estimation, we performed a novel tensile mechanical characterization of collagen- and fibronectin-micropatterned polyacrylamide hydrogels. We further demonstrate the impact of the protein mat on the tensile stiffness characterization of adherent cells. To our knowledge, our study is the first to uncover direction-dependent mechanical behavior of the protein coatings and to demonstrate that it affects cellular response relative to substrate mechanics.

The relationship between metastatic potential and in vitro mechanical properties of osteosarcoma cells.

Osteosarcoma is the most frequent primary tumor of bone and is characterized by its high tendency to metastasize in lungs. Although treatment in cases of early diagnosis results in a 5-yr survival rate of nearly 60%, the prognosis for patients with secondary lesions at diagnosis is poor, and their 5-yr survival rate remains below 30%. In the present work, we have used a number of analytical methods to investigate the impact of increased metastatic potential on the biophysical properties and force generation of osteosarcoma cells. With that aim, we used two paired osteosarcoma cell lines, with each one comprising a parental line with low metastatic potential and its experimentally selected, highly metastatic form. Mechanical characterization was performed by means of atomic force microscopy, tensile biaxial deformation, and real-time deformability, and cell traction was measured using two-dimensional and micropost-based traction force microscopy. Our results reveal that the low metastatic osteosarcoma cells display larger spreading sizes and generate higher forces than the experimentally selected, highly malignant variants. In turn, the outcome of cell stiffness measurements strongly depends on the method used and the state of the probed cell, indicating that only a set of phenotyping methods provides the full picture of cell mechanics.

Feasibility of the annulus fibrosus repair with in situ gelating hydrogels - A biomechanical study.

The surgical standard of care for lumbar discectomy leaves the annulus fibrosus (AF) defect unrepaired, despite considerable risk for a recurrent herniation. Identification of a viable defect repair strategy has until now been elusive. The scope of this ex vivo biomechanical study was to evaluate crosslinking hydrogels as potentially promising AF defect sealants, and provide a baseline for their use in combination with collagen scaffolds that restore disc volume. This study directly compared genipin crosslinked fibrin hydrogel (FibGen) as a promising preclinical candidate against a clinically available adhesive composed of glutaraldehyde and albumin (BioGlue). Forty-two bovine coccygeal functional spine units (FSU) were randomly allocated into four groups, namely untreated (control, n = 12), repaired with either one of the tested hydrogels (BioGlue, n = 12; FibGen, n = 12), or FibGen used in combination with a collagen hydrogel scaffold (FibGen+Scaffold, n = 6). All specimens underwent a moderate mechanical testing protocol in intact, injured and repaired states. After completion of the moderate testing protocol, the samples underwent a ramp-to-failure test. Lumbar discectomy destabilized the FSU as quantified by increased torsional range of motion (28.0° (19.1, 45.1) vs. 41.39° (27.3, 84.9), p<0.001), torsional neutral zone (3.1° (1.2, 7.7) vs. 4.8° (2.1, 12.1), Z = -3.49, p < 0.001), hysteresis(24.4 J (12.8, 76.0) vs. 27.6 J (16.4, 54.4), Z = -2.61, p = 0.009), with loss of both disc height (7.0 mm (5.0, 10.5) vs 6.1 mm (4.0, 9.3), Z = -5.16, p < 0.001) and torsional stiffness (0.76 Nmdeg-1 (0.38, 1.07) vs. 0.66 Nmdeg-1 (0.38, 0.97), Z = -3.98, p < 0.001). Most FibGen repaired AF endured the entire testing procedure whereas only a minority of BioGlue repaired AF and all FibGen+Scaffold repaired AF failed (6/10 vs. 3/12 vs. 0/6 respectively, p = 0.041). Both BioGlue and FibGen+Scaffold repaired AF partially restored disc height (0.47 mm (0.07, 2.41), p = 0.048 and 1.52 mm (0.41, 2.57), p = 0.021 respectively) compared to sham treatment (0.08 mm (-0.63, 0.88)) whereas FibGen-only repaired AF had no such effect (0.04 mm (-0.73, 1.13), U = 48.0, p = 1). The AF injury model demonstrated considerable change of FSU mechanics that could be partially restored by use of an AF sealant. While inclusion of a volumetric collagen scaffold led to repair failure, use of FibGen alone demonstrated clinically relevant promise for prevention of mechanical reherniation, outperforming an FDA approved sealant in this ex vivo test series.

Biomaterial surface energy-driven ligand assembly strongly regulates stem cell mechanosensitivity and fate on very soft substrates.

Although mechanisms of cell-material interaction and cellular mechanotransduction are increasingly understood, the mechanical insensitivity of mesenchymal cells to certain soft amorphous biomaterial substrates has remained largely unexplained. We reveal that surface energy-driven supramolecular ligand assembly can regulate mesenchymal stem cell (MSC) sensing of substrate mechanical compliance and subsequent cell fate. Human MSCs were cultured on collagen-coated hydrophobic polydimethylsiloxane (PDMS) and hydrophilic polyethylene-oxide-PDMS (PEO-PDMS) of a range of stiffnesses. Although cell contractility was similarly diminished on soft substrates of both types, cell spreading and osteogenic differentiation occurred only on soft PDMS and not hydrophilic PEO-PDMS (elastic modulus <1 kPa). Substrate surface energy yields distinct ligand topologies with accordingly distinct profiles of recruited transmembrane cell receptors and related focal adhesion signaling. These differences did not differentially regulate Rho-associated kinase activity, but nonetheless regulated both cell spreading and downstream differentiation.

Can Genipin-coated Sutures Deliver a Collagen Crosslinking Agent to Improve Suture Pullout in Degenerated Tendon? An Ex Vivo Animal Study.

BACKGROUND: The suture-tendon interface is often the weakest link in tendon-to-tendon or tendon-to-bone repair. Genipin is an exogenous collagen crosslink agent derived from the gardenia fruit that can enhance suture force to failure of the tendon-suture interface. Viable methods for intraoperative clinical delivery of genipin could be of clinical utility, but to our knowledge have not yet been extensively studied. QUESTIONS/PURPOSES: The purposes of this study were (1) to evaluate whether sutures precoated with genipin can augment the suture-tendon interface to improve force to failure, stiffness, and work to failure in healthy and degenerated tendons; and (2) to determine the effect of genipin on the extent and distribution of crosslinking. METHODS: Single-stitch suture pullout tests were performed ex vivo on 25 bovine superficial digital flexor tendons. To assess effects on native tissue, one group of 12 tendons was cut in proximal and distal halves and randomized to treatment (n = 12) and control groups (n = 12) in a matched-pair design. One simple stitch with a loop with either a normal suture or genipin-coated suture was applied to tendons in both groups. To simulate a degenerative tendon condition, a second group of 13 tendons was cut in proximal and distal halves, injected with 0.2 mL of collagenase D (8 mg/mL) and incubated for 24 hours before suturing with either a genipin-coated suture (n = 13) or their matched controls (n = 13). Sutures from all groups then were loaded to failure on a universal materials testing machine 24 hours after suturing. Suture pullout force, stiffness, and work to failure were calculated from force-displacement data and compared between the groups. Additionally, fluorescence was measured to determine the degree of crosslinking quantitatively and a qualitative analysis of the distribution pattern was performed by microscopy. RESULTS: In healthy tendon pairs, the median maximum pullout force was greater with genipin-coated sutures than with control sutures (median, 42 N [range, 24-73 N] versus 29 N [range, 13-48 N]; difference of medians, 13 N; p = 0.003) with corresponding increases in the required work to failure (median, 275 mJ [range, 48-369 mJ] versus 148 mJ [range, 83-369 mJ]; difference of medians, 127 mJ; p = 0.025) but not stiffness (median, 4.1 N/mm [range, 2.3-8.1 N/mm] versus 3.3 N/mm [range, 1.1-9.6 N/mm]; difference of medians, 0.8 N/mm; p = 0.052). In degenerated tendons, median maximum pullout force was greater with genipin-coated sutures than with control sutures (median, 16 N [range, 9-36 N] versus 13 N [range, 5-28 N]; difference of medians, 3 N; p = 0.034) with no differences in work to failure (median, 75 mJ [range, 11-249 mJ] versus 53 mJ [range, 14-143 mJ]; difference of medians, 22 mJ; p = 0.636) or stiffness (median, 1.9 N/mm [range, 0.7-13.4 N/mm] versus 1.6 N/mm [range, 0.5-5.6 N/mm]; difference of medians, 0.3 N/mm; p = 0.285). Fluorescence was higher in tendons treated with genipin-coated sutures compared with the control group, whereas higher fluorescence was observed in the treated healthy compared with the degenerated tendons (difference of means -3.16; standard error 1.08; 95% confidence interval [CI], 0.97-5.34; p = 0.006/healthy genipin: mean 13.04; standard error 0.78; 95% CI, 11.47-14.62; p < 0.001/degenerated genipin: mean 9.88; SD 0.75; 95% CI, 8.34-11.40; p < 0.001). CONCLUSIONS: Genipin-coated sutures improved force to failure of a simple stitch at the tendon-suture interface in healthy and degenerated tendons in an ex vivo animal model. Fluorescence was higher in tendons treated with genipin-coated sutures compared with the control group. CLINICAL RELEVANCE: A genipin-coated suture represents a potential delivery vehicle for exogenous crosslink agents to augment suture retention properties. In vivo animal studies are the next logical step to assess safety and efficacy of the approach.

High-resolution traction force microscopy on small focal adhesions - improved accuracy through optimal marker distribution and optical flow tracking.

The accurate determination of cellular forces using Traction Force Microscopy at increasingly small focal attachments to the extracellular environment presents an important yet substantial technical challenge. In these measurements, uncertainty regarding accuracy is prominent since experimental calibration frameworks at this size scale are fraught with errors - denying a gold standard against which accuracy of TFM methods can be judged. Therefore, we have developed a simulation platform for generating synthetic traction images that can be used as a benchmark to quantify the influence of critical experimental parameters and the associated errors. Using this approach, we show that TFM accuracy can be improved >35% compared to the standard approach by placing fluorescent beads as densely and closely as possible to the site of applied traction. Moreover, we use the platform to test tracking algorithms based on optical flow that measure deformation directly at the beads and show that these can dramatically outperform classical particle image velocimetry algorithms in terms of noise sensitivity and error. We then report how optimized experimental and numerical strategy can improve traction map accuracy, and further provide the best available benchmark to date for defining practical limits to TFM accuracy as a function of focal adhesion size.