Modulating the mesenchymal stromal cell microenvironment alters exosome RNA content and ligament healing capacity.

Although mesenchymal stromal cell (MSC) based therapies hold promise in regenerative medicine, their clinical application remains challenging due to issues such as immunocompatibility. MSC-derived exosomes are a promising off-the-shelf therapy for promoting wound healing in a cell-free manner. However, the potential to customize the content of MSC-exosomes, and understanding how such modifications influence exosome effects on tissue regeneration remain underexplored. In this study, we used an in vitro system to compare the priming of human MSCs by 2 inflammatory inducers TNF-α and CRX-527 (a highly potent synthetic TLR4 agonist that can be used as a vaccine adjuvant or to induce anti-tumor immunity) on exosome molecular cargo, as well as on an in vivo rat ligament injury model to validate exosome potency. Different microenvironmental stimuli used to prime MSCs in vitro affected their exosomal microRNAs and mRNAs, influencing ligament healing. Exosomes derived from untreated MSCs significantly enhance the mechanical properties of healing ligaments, in contrast to those obtained from MSCs primed with inflammation-inducers, which not only fail to provide any improvement but also potentially deteriorate the mechanical properties. Additionally, a link was identified between altered exosomal microRNA levels and expression changes in microRNA targets in ligaments. These findings elucidate the nuanced interplay between MSCs, their exosomes, and tissue regeneration.

Exosome-educated macrophages and exosomes differentially improve ligament healing.

Recently, our group used exosomes from mesenchymal stromal/stem cells (MSCs) to simulate an M2 macrophage phenotype, that is, exosome-educated macrophages (EEMs). These EEMs, when delivered in vivo, accelerated healing in a mouse Achilles tendon injury model. For the current study, we first tested the ability of EEMs to reproduce the beneficial healing effects in a different rodent model, that is, a rat medial collateral ligament (MCL) injury model. We hypothesized that treatment with EEMs would reduce inflammation and accelerate ligament healing, similar to our previous tendon results. Second, because of the translational advantages of a cell-free therapy, exosomes alone were also examined to promote MCL healing. We hypothesized that MSC-derived exosomes could also alter ligament healing to reduce scar formation. Similar to our previous Achilles tendon results, EEMs improved mechanical properties in the healing ligament and reduced inflammation, as indicated via a decreased endogenous M1/M2 macrophage ratio. We also showed that exosomes improved ligament remodeling as indicated by changes in collagen production and organization, and reduced scar formation but without improved mechanical behavior in healing tissue. Overall, our findings suggest EEMs and MSC-derived exosomes improve healing but via different mechanisms. EEMs and exosomes each have attractive characteristics as therapeutics. EEMs as a cell therapy are terminally differentiated and will not proliferate or differentiate. Alternatively, exosome therapy can be used as a cell free, shelf-stable therapeutic to deliver biologically active components. Results herein further support using EEMs and/or exosomes to improve ligament healing by modulating inflammation and promoting more advantageous tissue remodeling.

TimeMeter assesses temporal gene expression similarity and identifies differentially progressing genes.

Comparative time series transcriptome analysis is a powerful tool to study development, evolution, aging, disease progression and cancer prognosis. We develop TimeMeter, a statistical method and tool to assess temporal gene expression similarity, and identify differentially progressing genes where one pattern is more temporally advanced than the other. We apply TimeMeter to several datasets, and show that TimeMeter is capable of characterizing complicated temporal gene expression associations. Interestingly, we find: (i) the measurement of differential progression provides a novel feature in addition to pattern similarity that can characterize early developmental divergence between two species; (ii) genes exhibiting similar temporal patterns between human and mouse during neural differentiation are under strong negative (purifying) selection during evolution; (iii) analysis of genes with similar temporal patterns in mouse digit regeneration and axolotl blastema differentiation reveals common gene groups for appendage regeneration with potential implications in regenerative medicine.

Extracellular Vesicle-Educated Macrophages Promote Early Achilles Tendon Healing.

Tendon healing follows a complex series of coordinated events, which ultimately produces a mechanically inferior tissue more scar-like than native tendon. More regenerative healing occurs when anti-inflammatory M2 macrophages play a more dominant role. Mesenchymal stromal/stem cells (MSCs) are able to polarize macrophages to an M2 immunophenotype via paracrine mechanisms. We previously reported that coculture of CD14+ macrophages (MQs) with MSCs resulted in a unique M2-like macrophage. More recently, we generated M2-like macrophages using only extracellular vesicles (EVs) isolated from MSCs creating "EV-educated macrophages" (also called exosome-educated macrophages [EEMs]), thereby foregoing direct use of MSCs. For the current study, we hypothesized that cell therapy with EEMs would improve in vivo tendon healing by modulating tissue inflammation and endogenous macrophage immunophenotypes. We evaluated effects of EEMs using a mouse Achilles tendon rupture model and compared results to normal tendon healing (without any biologic intervention), MSCs, MQs, or EVs. We found that exogenous administration of EEMs directly into the wound promoted a healing response that was significantly more functional and more regenerative. Injured tendons treated with exogenous EEMs exhibited (a) improved mechanical properties, (b) reduced inflammation, and (c) earlier angiogenesis. Treatment with MSC-derived EVs alone were less effective functionally but stimulated a biological response as evidenced by an increased number of endothelial cells and decreased M1/M2 ratio. Because of their regenerative and immunomodulatory effects, EEM treament could provide a novel strategy to promote wound healing in this and various other musculoskeletal injuries or pathologies where inflammation and inadequate healing is problematic. Stem Cells 2019;37:652-662.

Biomechanical Evaluation of a Minimally Invasive Fixation Method for Length Unstable Limb Injuries.

INFIX instrumentation has provided an alternative treatment option for anteriorly unstable pelvic injuries. In this study, we explore the biomechanical feasibility of using an INFIX construct in an unstable longbone model and present a unique clinical case of its use. The external fixation, locked plate and spinal implant constructs (n = 5 each) were applied to lengthunstable fracture models and tested under various loads. Analysis of variance and pairwise Ttests were performed with levels of significance adjusted by Bonferroni correction to account for multiple comparisons. The biomechanical stiffness of the INFIX was found to be intermediate between the other two constructs in axial loading and torsion and was equivalent to one of the other constructs in sagittal and lateral bending. It was never the most compliant construct in any testing mode. This study and case report demonstrate the biomechanical feasibility of using INFIX to treat limb injuries. (Journal of Surgical Orthopaedic Advances 29(1):1825, 2020).

Level-specific amputations and resulting regenerative outcomes in the mouse distal phalanx.

Mouse digit tip regeneration involves an intricate coordinated regrowth of the terminal phalanx, nail, dermis and epidermis. During this time, regenerating digits undergo wound healing, blastema formation, and differentiation. However, the regenerative response of the digit is dependent on the level of the amputation. Amputation of <30% of the distal phalanx (P3), with part of the base nail remaining, results in extensive digit regeneration. In contrast, >60% P3 removal results in no regeneration. This level-dependent regenerative ability of the mouse digit provides a comparative model between regeneration and non-regeneration that may enable identification of specific factors critical to regeneration. Although the ability to create regenerating and non-regenerating conditions has been well established, the regenerative response between these regions ("intermediate" zone) has received less scrutiny, and may add insight to the regenerative processes, including the degree of histolysis, and the level of blastema formation. The objective of this study is then to compare the regeneration capacity between amputation levels within the regenerating (<30%), intermediate (40-59%), and non-regenerating (>60%) regions. Results indicated that regenerative and intermediate amputations led to significant histolysis and blastema formation of the distal phalanx 14 days post-amputation. Unlike the regenerating digits, intermediate amputations led to incomplete regeneration whereby regrowth of the digits were not to the levels of the intact or regenerating digits. Non-regenerating amputations did not exhibit significant histolysis or blastema formation. Remarkably, the histolytic process resulted in day 14 P3 lengths that were similar regardless of the initial amputation over 19%. The differences in histolysis, blastema formation and injury outcomes were also marked by changes in the number of proliferating cells and osteoclasts. Altogether, these results indicate that although intermediate amputations result in histolysis and blastema formation similar to regenerating digits, the resulting cellular composition of the blastema differs, contributing to incomplete regeneration.

Guided Growth Implant Failure is a Result of Cyclic Fatigue: Explant Analysis With Scanning Electron Microscopy.

BACKGROUND: Guided growth is often used to correct limb deformity and yet implant screw failure in modular systems has been reported. There have been no reports of plate failure and we do not know the exact mode of failure when screws do break. METHODS: We report the first published case of a fractured plate in a modular plate and screw construct that was used to correct Blount disease in a child through guided growth. The implants were removed and analyzed for method of failure using scanning electron microscopy. RESULTS: Scanning electron microscopy of the explant confirms that the mode of failure was not a result of static tension from growth. Rather, analysis confirms cyclic fatigue that led to crack propagation across the anterior side of the plate until overload caused complete plate failure. CONCLUSIONS: This analysis confirms an in vivo cyclic compression-relaxation of the growth plate presumably to weight-bearing, and that when excessive may lead to implant failure as seen here in this case. LEVEL OF EVIDENCE: Level V.

Articular cartilage zonal differentiation via 3D Second-Harmonic Generation imaging microscopy.

PURPOSE: The collagen structure throughout the patella has not been thoroughly investigated by 3D imaging, where the majority of the existing data come from histological cross sections. It is important to have a better understanding of the architecture in normal tissues, where this could then be applied to imaging of diseased states. METHODS: To address this shortcoming, we investigated the combined use of collagen-specific Second-Harmonic Generation (SHG) imaging and measurement of bulk optical properties to characterize collagen fiber orientations of the histologically defined zones of bovine articular cartilage. Forward and backward SHG intensities of sections from superficial, middle and deep zones were collected as a function of depth and analyzed by Monte Carlo simulations to extract the SHG creation direction, which is related to the fibrillar assembly. RESULTS: Our results revealed differences in SHG forward-backward response between the three zones, where these are consistent with a previously developed model of SHG emission. Some of the findings are consistent with that from other modalities; however, SHG analysis showed the middle zone had the most organized fibril assembly. While not distinct, we also report bulk optical property values for these different zones within the patella. CONCLUSIONS: Collectively, these results provide quantitative measurements of structural changes at both the fiber and fibril assembly of the different cartilage zones and reveals structural information not possible by other microscope modalities. This can provide quantitative insight to the collagen fiber network in normal cartilage, which may ultimately be developed as a biomarker for osteoarthritis.

Interleukin-1 receptor antagonist modulates inflammation and scarring after ligament injury.

Ligaments have limited regenerative potential and as a consequence, repair is protracted and results in a mechanically inferior tissue more scar-like than native ligament. We previously reported that a single injection of interleukin-1 receptor antagonist (IL-1Ra) delivered at the time of injury, decreased the number of M2 macrophage-associated inflammatory cytokines. Based on these results, we hypothesized that IL-1Ra administered after injury and closer to peak inflammation (as would occur clinically), would more effectively decrease inflammation and thereby improve healing. Since IL-1Ra has a short half-life, we also investigated the effect of multiple injections. The objective of this study was to elucidate healing of a medial collateral ligament (MCL) with either a single IL-1Ra injection delivered one day after injury or with multiple injections of IL-1Ra on days 1, 2, 3, and 4. One day after MCL injury, rats received either single or multiple injections of IL-1Ra or PBS. Tissue was then collected at days 5 and 11. Both single and multiple IL-1Ra injections reduced inflammatory cytokines, but did not change mechanical behavior. A single injection of IL-1Ra also reduced the number of myofibroblasts and increased type I procollagen. Multiple IL-1Ra doses provided no additive response and, in fact, reduced the M2 macrophages. Based on these results, a single dose of IL-1Ra was better at reducing the MCL-derived inflammatory cytokines compared to multiple injections. The changes in type I procollagen and myofibroblasts further suggest a single injection of IL-1Ra enhanced repair of the ligament but not sufficiently to improve functional behavior.

Evaluation of global load sharing and shear-lag models to describe mechanical behavior in partially lacerated tendons.

The mechanical effect of a partial thickness tear or laceration of a tendon is analytically modeled under various assumptions and results are compared with previous experimental data from porcine flexor tendons. Among several fibril-level models considered, a shear-lag model that incorporates fibril-matrix interaction and a fibril-fibril interaction defined by the contact area of the interposed matrix best matched published data for tendons with shallow cuts (less than 50% of the cross-sectional area). Application of this model to the case of many disrupted fibrils is based on linear superposition and is most successful when more fibrils are incorporated into the model. An equally distributed load sharing model for the fraction of remaining intact fibrils was inadequate in that it overestimates the strength for a cut less than half of the tendon's cross-sectional area. In a broader sense, results imply that shear-lag contributes significantly to the general mechanical behavior of tendons when axial loads are nonuniformly distributed over a cross section, although the predominant hierarchical level and microstructural mediators for this behavior require further inquiry.

Ultrasound assessment of ex vivo lung tissue properties using a fluid-filled negative pressure bath.

A relationship between tendon stress and strain and ultrasonic echo intensity has previously been defined in tendons, demonstrating a correlation between tissue stiffness and echo intensity. An analogous relationship between volume-dependent pressure changes and echo intensity changes in inflating lungs would indicate a correlation between lung compliance and echo intensity. Lung compliance is an important metric to diagnose pathologies which affect lung tissue mechanics, such as emphysema and cystic fibrosis. The goal of this study is to demonstrate a correlation between ultrasound echo intensity and lung tissue mechanics in an ex vivo model using a fluid-filled negative pressure bath design which provides a controlled environment for ultrasonic and mechanical measurements. Lungs from 4 male Sprague-Dawley rats were removed and mechanically tested via inflation and deflation in a negative pressure chamber filled with hetastarch. Specific volumes (1, 2, 3, and 4 mL) were removed from the chamber using a syringe to create negative pressure, which resulted in lung inflation. A pressure transducer recorded the pressure around the lungs. From these data, lung compliance was calculated. Ultrasound images were captured through the chamber wall to determine echo intensity (grayscale brightness in the ultrasound image), which was then related to mechanical parameters. Ultrasound images of the lung were successfully captured through the chamber wall with sufficient resolution to deduce echo intensity changes in the lung tissue. Echo intensity (0-255 scale) increased with volumetric changes (18.4 ± 5.5, 22.6 ± 5.1, 26.1 ± 7.5, and 42.9 ± 19.5 for volumetric changes of 1, 2, 3, and 4 mL) in a pattern similar to pressure (-6.8 ± 1.7, -6.8 ± 1.4, -9.4 ± 0.7, and -16.9 ± 6.8 cm H2O for 1, 2, 3, and 4 mL), reflecting changes in lung compliance. Measured rat lung tissue compliance was comparable to reported values from ex vivo lungs (0.178 ± 0.067, 0.378 ± 0.051, 0.427 ± 0.062, and 0.350 ± 0.160 mL/cm H20 for 1, 2, 3, and 4 mL), supporting proof of concept for the experimental method. Changes in echo intensity reflected changes in lung compliance in this ex vivo model, thus, supporting our hypothesis that the stiffness-related changes in echo intensity originally seen in tendon can be similarly detected in lung tissue. The presented ultrasound-based methods allowed measurement of local lung tissue compliance in a controlled environment, however, the methods could be expanded to facilitate both ex vivo and in vivo studies.

The influence of partial and full thickness tears on infraspinatus tendon strain patterns.

Tears on the bursal and articular sides of the rotator cuff tendons are known to behave differently and strain is thought to play a role in this difference. This study investigates the effect of tear location on the changes in three strain measurements (grip-to-grip, insertion, and mid-substance tissue) in a sheep infraspinatus tendon model during axial loading. We introduced a 14 mm wide defect near the insertion from either the articular or bursal side of the tendon to three depths (3 mm, 7 mm & full) progressively. For each condition, tendons were sinusoidally stretched (4% at 0.5 Hz) while insertion and mid-substance strains were tracked with surface markers. For a fixed load, grip-to-grip strain increased significantly compared to intact for both cuts. Insertion strain increased significantly for the bursal-side defect immediately but not for the articular-side until the 66% cut. Mid-substance tissue strain showed no significant change for partial thickness articular-side defects and a significant decrease for bursal-side defects after the 66% cut. All full thickness cuts exhibited negligible mid-substance tissue strain change. Our results suggest that the tendon strain patterns are more sensitive to defects on the bursal side, and that partial thickness tears tend to induce localized strain concentrations in regions adjacent to the damaged tissue.

Mechanical compromise of partially lacerated flexor tendons.

Tendons function to transmit loads from muscle to move and stabilize joints and absorb impacts. Functionality of lacerated tendons is diminished, however clinical practice often considers surgical repair only after 50% or more of the tendon is lacerated, the "50% rule." Few studies provide mechanical insight into the 50% rule. In this study cyclic and static stress relaxation tests were performed on porcine flexor tendons before and after a 0.5, 1.0, 2.0, or 2.75 mm deep transverse, midsubstance laceration. Elastic and viscoelastic properties, such as maximum stress, change in stress throughout each test, and stiffness, were measured and compared pre- and post-laceration. Nominal stress and stiffness parameters decreased, albeit disproportionately in magnitude, with increasing percent loss of cross-sectional area. Conversely, mean stress at the residual area (determined using remaining intact area at the laceration cross section) exhibited a marked increase in stress concentration beginning at 47.2% laceration using both specified load and constant strain analyses. The marked increase in stress concentration beginning near 50% laceration provides mechanical insight into the 50% rule. Additionally, a drastic decrease in viscoelastic stress parameters after only an 8.2% laceration suggests that time-dependent mechanisms protecting tissues during impact loadings are highly compromised regardless of laceration size.

Feasibility and repeatability for in vivo measurements of stiffness gradients in the canine gastrocnemius tendon using an acoustoelastic strain gauge.

B-mode ultrasound is an established imaging modality for evaluating canine tendon injury. However, full extent of tendon injury often remains difficult to estimate, as small changes in sonographic appearance are associated with large changes in biomechanical strength. The acoustoelastic strain gauge (ASG) is an ultrasound-based tissue evaluation technique that relates the change in echo intensity observed during relaxation or stretching of tendons to the tissue's mechanical properties. This technique deduces stiffness gradient (the rate of change of normalized stiffness as a function of tissue strain) by analyzing the ultrasound dynamic images captured from gradually deforming tissue. ASG has been proven to accurately model strain and stiffness within tendons in vitro. To determine the feasibility and repeatability for in vivo ASG measurements of canine tendon function, stiffness gradients for the gastrocnemius tendons of 10 clinically normal dogs were recorded by two nonindependent observers at three sites (musculotendinous junction, mid tendon, and insertion). Average stiffness gradient indices (0.0132, 0.0141, 0.0136) and dispersion values (0.0053, 0.0054, 0.0057) for each site, respectively, were consistent with published mechanical properties for normal canine tendon. Mean differences of the average stiffness gradient index and dispersion value between observers and between limbs for each site were less than 16%. Using interclass coefficients (ICC), intra-observer (ICC 0.79-0.98) and interobserver (ICC 0.77-0.95) reproducibility was good to excellent. Right and left limb values were symmetric (ICC 0.74-0.92). Findings from this study indicated that ASG is a feasible and repeatable technique for measuring stiffness gradients in canine tendons.

Modulating Mesenchymal Stromal Cell Microenvironment Alters Exosome RNA Content and Ligament Healing Capacity.

Although mesenchymal stromal cell (MSC) based therapies hold promise in regenerative medicine, their applications in clinical settings remain challenging due to issues such as immunocompatibility and cell stability. MSC-derived exosomes, small vesicles carrying various bioactive molecules, are a promising cell-free therapy to promote tissue regeneration. However, it remains unknown mainly regarding the ability to customize the content of MSC-derived exosomes, how alterations in the MSC microenvironment influence exosome content, and the effects of such modifications on healing efficiency and mechanical properties in tissue regeneration. In this study, we used an in vitro system of human MSC-derived exosomes and an in vivo rat ligament injury model to address these questions. We found a context-dependent correlation between exosomal and parent cell RNA content. Under native conditions, the correlation was moderate but heightened with microenvironmental changes. In vivo rat ligament injury model showed that MSC-derived exosomes increased ligament max load and stiffness. We also found that changes in the MSCs' microenvironment significantly influence the mechanical properties driven by exosome treatment. Additionally, a link was identified between altered exosomal microRNA levels and expression changes in microRNA targets in ligaments. These findings elucidate the nuanced interplay between MSCs, their exosomes, and tissue regeneration.

Time-dependent ultrasound echo changes occur in tendon during viscoelastic testing.

The viscoelastic behavior of tendons has been extensively studied in vitro. A noninvasive method by which to acquire mechanical data would be highly beneficial, as it could lead to the collection of viscoelastic data in vivo. Our lab has previously presented acoustoelasticity as an alternative ultrasound-based method of measuring tendon stress and strain by reporting a relationship between ultrasonic echo intensity (B mode ultrasound image brightness) and mechanical behavior of tendon under pseudoelastic in vitro conditions [Duenwald, S., Kobayashi, H., Frisch, K., Lakes, R., and Vanderby Jr, R., 2011, "Ultrasound Echo is Related to Stress and Strain in Tendon," J. Biomech., 44(3), pp. 424-429]. Viscoelastic properties of the tendons were not examined in that study, so the presence of time-dependent echo intensity changes has not been verified. In this study, porcine flexor tendons were subjected to relaxation and cyclic testing while ultrasonic echo response was recorded. We report that time- and strain history-dependent mechanical properties during viscoelastic testing are manifested in ultrasonic echo intensity changes. We also report that the patterns of the echo intensity changes do not directly mimic the patterns of viscoelastic load changes, but the intensity changed in a repeatable (and therefore predictable) fashion. Although mechanisms need further elucidation, viscoelastic behavior can be anticipated from echo intensity changes. This phenomenon could potentially lead to a more extensive characterization of in vivo tissue behavior.

Tendon strain measurements with dynamic ultrasound images: evaluation of digital image correlation.

Strain is an essential metric in tissue mechanics. Strains and strain distributions during functional loads can help identify damaged and pathologic regions as well as quantify functional compromise. Noninvasive strain measurement in vivo is difficult to perform. The goal of this in vitro study is to determine the efficacy of digital image correlation (DIC) methods to measure strain in B-mode ultrasound images. The Achilles tendons of eight male Wistar rats were removed and mechanically cycled between 0 and 1% strain. Three cine video images were captured for each specimen: (1) optical video for manual tracking of optical markers; (2) optical video for DIC tracking of optical surface markers; and (3) ultrasound video for DIC tracking of image texture within the tissue. All three imaging modalities were similarly able to measure tendon strain during cyclic testing. Manual/ImageJ-based strain values linearly correlated with DIC (optical marker)-based strain values for all eight tendons with a slope of 0.970. DIC (optical marker)-based strain values linearly correlated with DIC (ultrasound texture)-based strain values for all eight tendons with a slope of 1.003. Strain measurement using DIC was as accurate as manual image tracking methods, and DIC tracking was equally accurate when tracking ultrasound texture as when tracking optical markers. This study supports the use of DIC to calculate strains directly from the texture present in standard B-mode ultrasound images and supports the use of DIC for in vivo strain measurement using ultrasound images without additional markers, either artificially placed (for optical tracking) or anatomically in view (i.e., bony landmarks and/or muscle-tendon junctions).

Biomechanical comparison between 2 guided-growth constructs.

BACKGROUND: Correction of deformity using guided growth with plate and screw constructs has shown good results in the correction of angular deformities in children. Some recent reports have shown device failure, perhaps because of increased patient weight as seen in Blount disease. The purpose of our study was to compare the strength to failure between 2 similar devices, the Orthofix 8-plate manufacturer 1 (M-1) and Biomet Peanut plate manufacturer 2 (M-2), using 2 different screw types: solid and cannulated. METHODS: A model of bone was developed using 30-pcf solid polyurethane foam as cancellous bone and high-density polyethylene as cortical bone. A 10.0-mm defect was created through the polyurethane foam and was spanned by a plate and screw system. Under the assumption that device failure is caused by cyclical loading, each device underwent fatigue testing on an MTS Bionix machine with a 4-Hz micromotion of 5.0 mm at -500-N compression, and the number of cycles to failure was recorded. RESULTS: All devices failed at the screw shaft; plates did not break under any circumstances. The highest mean number of cycles to failure was seen with the M-2 device using solid, stainless-steel screws (22,614 cycles; SD, 6885). On comparing with titanium screws, solid screws were significantly stronger in both the M-1 (P=0.002) and M-2 devices (P=0.013). The M-2 device with cannulated screws was noted to be significantly stronger than the M-1 device with cannulated screws (P=0.036). CONCLUSIONS: This study reveals a significant increase in strength in one titanium cannulated guided-growth system over another. Solid screws are also shown to be significantly stronger than cannulated screws. Long-term clinical data will be required to determine whether this difference results in lower failure rates. CLINICAL RELEVANCE: Use of a stronger guided-growth device may be of benefit for correction of deformity in children who are heavier, such as those with Blount disease. Comparative clinic trials will be needed to confirm the advantage of one device over another.

Periostin modulates extracellular matrix behavior in tendons.

Periostin, originally named osteoblast-specific factor 2 (OSF-2) has been identified primarily in collagen rich, biomechanically active tissues where its role has been implicated in mechanisms to maintain the extracellular matrix (ECM), including collagen fibrillogenesis and crosslinking. It is well documented that periostin plays a role in wound healing and scar formation after injury, in part, by promoting cell proliferation, myofibroblast differentiation, and/or collagen fibrillogenesis. Given the significance of periostin in other scar forming models, we hypothesized that periostin will influence Achilles tendon healing by modulating ECM production. Therefore, the objective of this study was to elucidate the effects of periostin during Achilles tendon healing using periostin homozygous (Postn -/-) and heterozygous (Postn +/-) mouse models. A second experiment was included to further examine the influence of periostin on collagen composition and function using intact dorsal tail tendons. Overall, Postn -/- and Postn +/- Achilles tendons exhibited impaired healing as demonstrated by delayed wound closure, increased type III collagen production, decreased cell proliferation, and reduced tensile strength. Periostin ablation also reduced tensile strength and stiffness, and altered collagen fibril distribution in the intact dorsal tail tendons. Achilles tendon outcomes support our hypothesis that periostin influences healing, while tail tendon results indicate that periostin also affects ECM morphology and behavior in mouse tendons.

The influence of macrophage depletion on ligament healing.

Despite a complex cascade of cellular events to reconstruct damaged extracellular matrix (ECM), ligament healing results in a mechanically inferior, scar-like tissue. During normal healing, the number of macrophages significantly increases within the wound site. Then, granulation tissue expands into any residual, normal ligamentous tissue (creeping substitution), resulting in a larger region of healing, greater mechanical compromise, and an inefficient repair process. To study the effects of macrophages on the repair process, bilateral, surgical rupture of their medial collateral ligaments (MCLs) was done on rats. Treatment animals received liposome-encapsulated clodronate, 2 days before rupture to ablate phagocytosing macrophages. Ligaments were then collected at days 5, 11, and 28 for immunohistochemistry (IHC) and/or mechanical testing. Clodronate treatment reduced both the M1 and M2 macrophages at day 5 and altered early healing. However, the macrophages effectively returned to control levels after day 5 and reinitiated a wound-healing response. Our results suggest that an early macrophage response, which is necessary for debridement of damaged tissue in the wound, is also important for cytokine release to mediate normal repair processes. Additionally, nonspecific inhibition of macrophages (without regard to specific macrophage populations) can control excessive granulation tissue formation but is detrimental to early matrix formation and ligament strength.