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 two 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.

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.

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.

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.

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).

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.

Beyond the Core Suture: A New Approach to Tendon Repair.

Despite significant improvements in zone II flexor tendon repair over the last 2 decades, function-limiting complications persist. This article describes 2 novel repair techniques utilizing flexor digitorum superficialis (FDS) autografts to buttress the flexor digitorum profundus (FDP) repair site without the use of core sutures. The hypothesis being that the reclaimed FDS tendon autograft will redistribute tensile forces away from the FDP repair site, increasing overall strength and resistance to gapping in Zone II flexor tendon injuries compared with the current clinical techniques. METHODS: Two novel FDP repair methods utilizing portions of FDS have been described: (1) asymmetric repair (AR), and (2) circumferential repair. Ultimate tensile strength and cyclical testing were used to compare novel techniques to current clinical standard repairs: 2-strand (2-St), 4-strand (4-St), and 6-strand (6-St) methods. All repairs were performed in cadaveric sheep tendons (n = 10/group), by a single surgeon. RESULTS: AR and circumferential repair techniques demonstrated comparable ultimate tensile strength to 6-St repairs, with all 3 of these techniques able to tolerate significantly stronger loads than the 2-St and 4-St repairs (P < 0.0001). Cyclical testing demonstrated that AR and circumferential repair were able to withstand a significantly higher total cumulative force (P < 0.001 and P = 0.0064, respectively) than the 6-St, while only AR tolerated a significantly greater force to 2-mm gap formation (P = 0.042) than the 6-St repair. CONCLUSION: Incorporating FDS as an autologous graft for FDP repair provides at least a comparable ultimate tensile strength and a significantly greater cumulative force to failure and 2-mm gap formation than a traditional 6-St repair.

Tendon-to-Bone Healing in a Rat Extra-articular Bone Tunnel Model: A Comparison of Fresh Autologous Bone Marrow and Bone Marrow-Derived Mesenchymal Stem Cells.

BACKGROUND: Despite widespread acceptance of fresh autologous bone marrow (BM) for use in clinical practice, limited information exists to analyze if tendon-to-bone healing could be accelerated with local use of fresh autologous BM. PURPOSE: To investigate the effect of fresh autologous BM on tendon-to-bone healing with a novel rat model. STUDY DESIGN: Controlled laboratory study. METHODS: An extra-articular bone tunnel was created and filled with an autologous tendon graft in skeletally mature Sprague-Dawley rats (N = 60). They were then randomly divided into 3 groups: BM group (injection of fresh autologous BM into the tendon-bone interface, n = 20), BM-derived mesenchymal stem cell (BMSC) group (injection of allogenic cultured BMSCs, n = 20), and the control group (tendon-bone interface without injection of BM or BMSCs, n = 20). Biomechanical, histological, and immunohistochemical analyses were performed at 2 and 6 weeks after surgery. RESULTS: The BM group showed a relatively well-organized and dense connective tissue interface with better orientation of collagen fibers as compared with the BMSC group. At 2 weeks, the tendon-bone interface tissue thickness of the BMSC group was 140 ± 25 µm (mean ± SEM), which was significantly greater than the BM group (58 ± 15 µm). The BM group showed fewer M1 macrophages at the tendon-bone interface at 2 and 6 weeks (P < .001). In contrast, there were more M2 macrophages at the interface in the BM group 2 and 6 weeks postoperatively when compared with controls and the BMSC group (P < .001). Biomechanical tests revealed significantly higher stiffness in the BM group versus the control and BMSC groups at 2 and 6 weeks after surgery (P < .05). Load to failure showed similar trends to stiffness. CONCLUSION: These findings indicate that local delivery of fresh autologous BM enhances tendon-to-bone healing better than the alternative treatments in this study. This effect may be partially due to the observed modulation of inflammatory processes, especially in M2 macrophage polarization. CLINICAL RELEVANCE: Fresh autologous BM could be a treatment option for this disorder.

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.

Microparticles Locally Deliver Active Interleukin-1 Receptor Antagonist In Vivo.

Despite significant research in therapeutic protein delivery, localized and sustained delivery of active therapeutic proteins remains a challenge. Delivery is a particular challenge for therapeutic proteins with a short half-life. Herein, localized delivery of interleukin-1 receptor antagonist (IL-1Ra) by mineral coated microparticles (MPs) is assessed in a healing rat medial collateral ligament (MCL). The local tissue concentration and systemic serum concentration of IL-1Ra, the anti-inflammatory activity of IL-1Ra delivered with MPs, and whether IL-1Ra loaded MPs (IL-1Ra MPs) are immunogenic in a healing ligament are also examined. IL-1Ra MPs significantly increase the local concentration of IL-1Ra compared to soluble IL-1Ra at 7 and 14 days after treatment but do not elevate the systemic concentration of IL-1Ra at these time points, indicating localized delivery of IL-1Ra. IL-1Ra MPs significantly reduce inflammation caused by the MPs themselves, indicating the IL-1Ra is active. Finally, IL-1Ra MPs do not induce a foreign body response and decrease the immunogenicity of human IL-1Ra in a healing rat MCL. Overall, mineral coated microparticles have the ability to locally deliver active therapeutic proteins for an extended period of time.

In vivo evaluation of effects of sedation on results of acoustoelastography of the superficial digital flexor tendons in clinically normal horses.

OBJECTIVE To assess the effects of sedation on results of acoustoelastography of the superficial digital flexor tendons (SDFTs) in clinically normal horses. ANIMALS 27 clinically normal horses. PROCEDURES For each horse, the pathology index (PI) for the SDFT of each thoracic limb was determined by use of acoustoelastography at 4 locations (5, 10, 15, and 20 cm distal to the accessory carpal bone). Horses were evaluated before and after they were sedated with a combination of detomidine hydrochloride (0.01 mg/kg, IV) and butorphanol tartrate (0.01 mg/kg, IV). A repeated-measures ANOVA was used for statistical analysis. RESULTS Overall, the PI was lower after sedation than before sedation. In addition, the PI was lower at more distal locations than at more proximal locations. There was not a significant effect of limb (left or right). Differences among individual horses accounted for the largest variance effect. CONCLUSIONS AND CLINICAL RELEVANCE Sedation with detomidine and butorphanol facilitated acoustoelastography; however, it decreased the SDFT PI in clinically normal horses and should be used consistently in prospective studies. Variance associated with each individual horse in the sample population had the greatest effect on the PI.

Chiral behavior in rat tail tendon fascicles.

Ex vivo tendon mechanical behavior has been well described under rotationally constrained uniaxial tensile testing. During standard loading of rat tail tendon (RTT) fascicles, apparent axial twist has been observed. To quantify this behavior, we designed a custom testing setup, utilizing magnetic suspension, to allow unconstrained axial rotation during tensile loading. We characterized the rotational behavior of single and paired RTT fascicles under cyclic loading. We also measured stress relaxation across loading cycles as well as "rotational relaxation". Single fascicle nonlinear stretch-twist coupling is well described by the asymptotic function Δθ=A(1-e-Bε) in which fascicles rotated a mean ±51.1° within about 1% applied axial strain. On average, paired fascicles rotated just over 10° less. Specimen cross-sectional diameter had a noticeable effect on the measured mechanical properties, particularly effective elastic modulus. Such stretch-twist coupling and size dependence cannot be understood via classical elasticity but is predicted by Cosserat (micropolar) elasticity. The current study demonstrates RTT fascicles are chiral based on observed axial load-induced twist. Additionally, our findings support existing research that suggests a helical fascicle structure. Potential consequences of helical substructures, mechanical and biological, merit further investigation.

Impacts of Interleukin-17 Neutralization on the Inflammatory Response in a Healing Ligament.

In this study, we sought to improve ligament healing by modulating the inflammatory response after acute injury through the neutralization of Interleukin-17 (IL-17), which we hypothesized would decrease inflammatory cell infiltration and cytokine production. Administration of an Interleukin-17 neutralizing antibody (IL-17 NA) immediately following a rat medial collateral ligament (MCL) transection resulted in alterations in inflammatory cell populations and cytokine expression within the healing ligament, but did not reduce inflammation. Specifically, treatment resulted in a decrease in M2 (anti-inflammatory) macrophages, an increase in T cells, and an increase in the levels of IL-2, IL-6, and IL-12 in the MCL 7 days post injury. IL-17NA treatment, and subsequent immunomodulation, did not result in improved ligament healing, as measured by collagen composition and wound size.

Mesenchymal Stem Cell Therapy on Tendon/Ligament Healing.

A normal healing response after ligament and tendon rupture results in scar formation and an inferior tissue that fails to emulate its original structure, composition, and function. More regenerative healing (closer to the original) can be obtained through early suppression of inflammatory cells and associated cytokines. Examination of the immune mediated response of mesenchymal stem/stromal cells (MSCs) during healing indicates that MSCs reprogram macrophages from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype. Based on these studies our objective was to treat ligament and tendon injuries with MSCs in order to modulate their inflammatory response. Our initial studies using allogeneic cells demonstrated an in vivo dose dependency of MSCs on ligament healing. Medial collateral ligaments (MCLs) treated with 1 × 106 (low dose) MSCs exhibited less inflammation and a reduced number of M1 macrophages compared to ligaments treated with 4 × 106 (high dose) MSCs. Strength of ligament was also improved with the low dose treatment. We then examined the in vivo effects of MSCs that had been preconditioned to be more anti-inflammatory. Treatment with these preconditioned MSCs was compared with normally processed (unconditioned) MSCs using the rat Achilles tendon and MCL healing models. Pre-conditioned MSCs significantly reduced inflammation by increasing the M2 macrophages and decreasing the M1 macrophages. Most importantly, treatment with pre-conditioned MSCs improved tissue strength to levels comparable to intact tissue. Overall, pre-conditioned MSC-treatment out-performed unconditioned MSCs to improve ligament and tendon healing by stimulating a more robust, paracrine-mediated immunosuppressive response.

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.

Immune modulation with primed mesenchymal stem cells delivered via biodegradable scaffold to repair an Achilles tendon segmental defect.

Tendon healing is a complex coordinated series of events resulting in protracted recovery, limited regeneration, and scar formation. Mesenchymal stem cell (MSC) therapy has shown promise as a new technology to enhance soft tissue and bone healing. A challenge with MSC therapy involves the ability to consistently control the inflammatory response and subsequent healing. Previous studies suggest that preconditioning MSCs with inflammatory cytokines, such as IFN-γ, TNF-α, and IL-1ß may accelerate cutaneous wound closure. The objective of this study was to therefore elucidate these effects in tendon. That is, the in vivo healing effects of TNF-α primed MSCs were studied using a rat Achilles segmental defect model. Rat Achilles tendons were subjected to a unilateral 3 mm segmental defect and repaired with either a PLG scaffold alone, MSC-seeded PLG scaffold, or TNF-α-primed MSC-seeded PLG scaffold. Achilles tendons were analyzed at 2 and 4 weeks post-injury. In vivo, MSCs, regardless of priming, increased IL-10 production and reduced the inflammatory factor, IL-1α. Primed MSCs reduced IL-12 production and the number of M1 macrophages, as well as increased the percent of M2 macrophages, and synthesis of the anti-inflammatory factor IL-4. Primed MSC treatment also increased the concentration of type I procollagen in the healing tissue and increased failure stress of the tendon 4 weeks post-injury. Taken together delivery of TNF-α primed MSCs via 3D PLG scaffold modulated macrophage polarization and cytokine production to further accentuate the more regenerative MSC-induced healing response. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:269-280, 2017.

Advanced quantitative imaging and biomechanical analyses of periosteal fibers in accelerated bone growth.

PURPOSE: The accepted mechanism explaining the accelerated growth following periosteal resection is that the periosteum serves as a mechanical restraint to restrict physeal growth. To test the veracity of this mechanism we first utilized Second Harmonic Generation (SHG) imaging to measure differences of periosteal fiber alignment at various strains. Additionally, we measured changes in periosteal growth factor transcription. Next we utilized SHG imaging to assess the alignment of the periosteal fibers on the bone both before and after periosteal resection. Based on the currently accepted mechanism, we hypothesized that the periosteal fibers adjacent to the physis should be more aligned (under tension) during growth and become less aligned (more relaxed) following metaphyseal periosteal resection. In addition, we measured the changes in periosteal micro- and macro-scale mechanics. METHODS: 30 seven-week old New Zealand White rabbits were sacrificed. The periosteum was imaged on the bone at five regions using SHG imaging. One centimeter periosteal resections were then performed at the proximal tibial metaphyses. The resected periosteal strips were stretched to different strains in a materials testing system (MTS), fixed, and imaged using SHG microscopy. Collagen fiber alignment at each strain was then determined computationally using CurveAlign. In addition, periosteal strips underwent biomechanical testing in both circumferential and axial directions to determine modulus, failure stress, and failure strain. Relative mRNA expression of growth factors: TGFß-1, -2, -3, Ihh, PTHrP, Gli, and Patched were measured following loading of the periosteal strips at physiological strains in a bioreactor. The periosteum adjacent to the physis of six tibiae was imaged on the bone, before and after, metaphyseal periosteal resection, and fiber alignment was computed. One-way ANOVA statistics were performed on all data. RESULTS: Imaging of the periosteum at different regions of the bone demonstrated complex regional differences in fiber orientation. Increasing periosteal strain on the resected strips increased periosteal fiber alignment (p<0.0001). The only exception to this pattern was the 10% strain on the tibial periosteum, which may indicate fiber rupture at this non-physiologic strain. Periosteal fiber alignment adjacent to the resection became less aligned while those adjacent to the physes remained relatively unchanged before and after periosteal resection. Increasing periosteal strain on the resected strips increased periosteal fiber alignment (p<0.0001). The only exception to this pattern was the 10% strain on the tibial periosteum, which may indicate fiber rupture (and consequent retraction) at this non-physiologic strain. Increasing periosteal strain revealed a significant increase in relative mRNA expression for Ihh, PTHrP, Gli, and Patched, respectively. CONCLUSION: Periosteal fibers adjacent to the growth plate do not appear under tension in the growing limb, and the alignments of these fibers remain unchanged following periosteal resection. SIGNIFICANCE: The results of this study call into question the long-accepted role of the periosteum acting as a simple mechanical tether restricting growth at the physis.

3D FSE Cube and VIPR-aTR 3.0 Tesla magnetic resonance imaging predicts canine cranial cruciate ligament structural properties.

Estimation of cranial cruciate ligament (CrCL) structural properties in client-owned dogs with incipient cruciate rupture would be advantageous. The objective of this study was to determine whether magnetic resonance imaging (MRI) measurement of normal CrCL volume in an ex-vivo canine model predicts structural properties. Stifles from eight dogs underwent 3.0 Tesla 3D MRI. CrCL volume and normalized median grayscale values were determined using 3D Fast Spin Echo (FSE) Cube and Vastly under-sampled Isotropic PRojection (VIPR)-alternative repetition time (aTR) sequences. Stifles were then mechanically tested. After joint laxity testing, CrCL structural properties were determined, including displacement at yield, yield load, load to failure, and stiffness. Yield load and load to failure (R(2)=0.56, P <0.01) were correlated with CrCL volume determined by VIPR-aTR. Yield load was also correlated with CrCL volume determined by 3D FSE Cube (R(2)=0.32, P <0.05). Structural properties were not related to median grayscale values. Joint laxity and CrCL stiffness were not related to MRI parameters, but displacement at yield load was related to CrCL volume for both sequences during testing (R(2)>0.57, P <0.005). In conclusion, 3D MRI offers a predictive method for estimating canine CrCL structural properties. 3D MRI may be useful for monitoring CrCL properties in clinical trials.

Primed Mesenchymal Stem Cells Alter and Improve Rat Medial Collateral Ligament Healing.

Cell therapy with mesenchymal stem cells (MSCs) can improve tissue healing. It is possible, however, that priming MSCs prior to implantation can further enhance their therapeutic benefit. This study was then performed to test whether priming MSCs to be more anti-inflammatory would enhance healing in a rat ligament model, i.e. a medial collateral ligament (MCL). MSCs were primed for 48 h using polyinosinic acid and polycytidylic acid (Poly (I:C)) at a concentration of 1 µg/ml. Rat MCLs were surgically transected and administered 1 × 10(6) cells in a carrier solution at the time of injury. A series of healing metrics were analyzed at days 4 and 14 post-injury in the ligaments that received primed MSCs, unprimed MSCs, or no cells (controls). Applying primed MSCs beneficially altered healing by affecting endothelialization, type 2 macrophage presence, apoptosis, procollagen 1α, and IL-1Ra levels. When analyzing MSC localization, both primed and unprimed MSCs co-localized with endothelial cells and pericytes suggesting a supportive role in angiogenesis. Priming MSCs prior to implantation altered key ligament healing events, resulted in a more anti-inflammatory environment, and improved healing.