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.

Global Responses of Il-1ß-Primed 3D Tendon Constructs to Treatment with Pulsed Electromagnetic Fields.

Tendinopathy is accompanied by a cascade of inflammatory events promoting tendon degeneration. Among various cytokines, interleukin-1ß plays a central role in driving catabolic processes, ultimately resulting in the activation of matrix metalloproteinases and a diminished collagen synthesis, both of which promote tendon extracellular matrix degradation. Pulsed electromagnetic field (PEMF) therapy is often used for pain management, osteoarthritis, and delayed wound healing. In vitro PEMF treatment of tendon-derived cells was shown to modulate pro-inflammatory cytokines, potentially limiting their catabolic effects. However, our understanding of the underlying cellular and molecular mechanisms remains limited. We therefore investigated the transcriptome-wide responses of Il-1ß-primed rat Achilles tendon cell-derived 3D tendon-like constructs to high-energy PEMF treatment. RNASeq analysis and gene ontology assignment revealed various biological processes to be affected by PEMF, including extracellular matrix remodeling and negative regulation of apoptosis. Further, we show that members of the cytoprotective Il-6/gp130 family and the Il-1ß decoy receptor Il1r2 are positively regulated upon PEMF exposure. In conclusion, our results provide fundamental mechanistic insight into the cellular and molecular mode of action of PEMF on tendon cells and can help to optimize treatment protocols for the non-invasive therapy of tendinopathies.

The In Vivo Impact of Leukocyte Injections on Normal Rat Achilles Tendons: Potential Detriment to Tendon Morphology, Cellularity, and Vascularity.

In this study, we determine the in vivo effects of injecting sub-populations of leukocytes into normal rat Achilles tendons via a controlled laboratory study. Allogenic monocytes, granulocytes, or plasma were injected into 24 healthy rat Achilles tendons. Treated and contralateral un-treated control tendons then assessed for cellularity, histologic morphology, and vascularity after 7 and 14 days. Significant increases of 221% and 249% in cellularity (P = 0.014) were seen on day 14 within Achilles tendons injected with granulocytes as compared to plasma and monocytes, respectively. Also, significant improvement in morphology (P = 0.029) between days 7 and 14 was seen for the granulocyte injected Achilles tendons. Significant increases in cellularity after an injection of granulocytes, compared to monocytes and plasma, corresponds to a significant increase in inflammation within the tissue, suggesting that leukocyte-rich platelet-rich plasma (PRP) preparations are proinflammatory and potentially catabolic when injected into tendon tissue. The concentration and composition of white blood cells within PRP preparations is variable and needs to be better understood in order to optimize clinical utility of PRP injections.

Systemic corticosteroids improve tendon healing when given after the early inflammatory phase.

Inflammation initiates tendon healing and then normally resolves more or less completely. Unresolved inflammation might disturb the remodeling process. We hypothesized that suppression of inflammation during the early remodeling phase by systemic dexamethasone treatment can improve healing. 36 rats underwent Achilles tendon transection and were randomized to dexamethasone or saline on days 0-4 after surgery (early inflammatory phase), and euthanasia day 7. Another 54 rats received injections days 5-9 (early remodeling phase) and were euthanized day 12 for mechanical, histological and flow cytometric evaluation. Dexamethasone treatment days 0-4 reduced the cross-sectional area, peak force and stiffness by day 7 to less than half (p < 0.001 for all), while material properties (peak stress and elastic modulus) were not significantly affected. In contrast, dexamethasone treatment days 5-9 increased peak force by 39% (p = 0.002) and stiffness by 58% (p < 0.001). The cross-sectional area was reduced by 42% (p < 0.001). Peak stress and elastic modulus were more than doubled (p < 0.001 for both). Semi-quantitative histology at day 12 showed that late dexamethasone treatment improved collagen alignment, and flow cytometry revealed reduced numbers of CD8a+ cytotoxic T cells in the tendon callus. These results suggest that downregulation of lingering inflammation during the early remodeling phase can improve healing.

Brief Report: Group 3 Innate Lymphoid Cells in Human Enthesis.

OBJECTIVE: Group 3 innate lymphoid cells (ILC3s) play a pivotal role in barrier tissues such as the gut and the skin, two important sites of disease in spondyloarthritis (SpA). This study was undertaken to investigate whether normal or injured human enthesis, a key target tissue in early SpA, harbors ILC3s in entheseal soft tissue and adjacent perientheseal bone. METHODS: Interspinous ligament and spinous process bone from donors with no systemic inflammatory disease were collected, enzymatically digested, and immunophenotyped. The immunologic profile of entheseal cells was examined, and the transcriptional profile of sorted ILC3s was compared to that of ILC3s isolated from SpA synovial fluid (SF). To assess the ability of entheseal tissue to produce interleukin-17 (IL-17) and IL-22, entheseal digests were stimulated with IL-23 and IL-1ß. Osteoarthritic and ruptured Achilles tendon tissue was examined histologically. RESULTS: The proportion of ILCs in human entheseal soft tissue was higher than that in peripheral blood (P = 0.008); entheseal soft tissue and perientheseal bone both had a higher proportion of NKp44+ ILC3s (P = 0.001 and P = 0.043, respectively). Studies of retinoic acid receptor-related orphan nuclear receptor γt (RORγt), STAT3, and IL-23 receptor transcript expression validated the entheseal ILC3 phenotype. Cytokine transcript expression was similar in ILC3s isolated from enthesis and from SpA SF. Stimulation of normal entheseal digests with IL-23/IL-1ß led to up-regulation of IL-17A transcript, and histologic examination of injured/damaged entheses revealed the presence of RORγt-expressing cells. CONCLUSION: This work shows that human enthesis harbors a resident population of ILC3s, with the potential to participate in the pathogenesis of SpA.

Interleukin-23-Dependent γ/δ T Cells Produce Interleukin-17 and Accumulate in the Enthesis, Aortic Valve, and Ciliary Body in Mice.

OBJECTIVE: The spondyloarthritides (SpA) are a group of rheumatic diseases characterized by ossification and inflammation of entheseal tissue, the region where tendon attaches to bone. Interleukin-23 (IL-23) is involved in the pathogenesis of SpA by acting on IL-23 receptor (IL-23R) expressed on enthesis-resident lymphocytes. Upon IL-23 binding, CD3+CD4-CD8- tissue-resident lymphocytes secrete IL-17A and IL-22, leading to inflammation, bone loss, and ossification. Knowledge about enthesis-resident lymphocytes remains fragmentary, and the contribution of entheseal γ/δ T cells in particular is not clear. This study was undertaken to investigate the presence of γ/δ T cells in the enthesis. METHODS: We used 2-photon microscopy and flow cytometry to analyze entheseal lymphocytes from C57BL/6, Tcrd-H2BeGFP, Rorc-GFP, and IL-23R-eGFP mice. To analyze entheseal γ/δ T cells in IL-23-induced inflammation, Tcrd-H2BeGFP mice were crossed with mice of the susceptible B10.RIII background. Hydrodynamic injection of IL-23 minicircle DNA was performed for overexpression of IL-23 and induction of inflammation. Light-sheet fluorescence microscopy was used to visualize arthritic inflammation. RESULTS: Activated Vγ6+CD27- γ/δ T cells were abundant in uninflamed entheseal tissue and constituted the large majority of retinoic acid receptor-related orphan nuclear receptor γt (RORγt)+IL-23R+ enthesis-resident lymphocytes. Fetal thymus-dependent γ/δ T cells were the main source of IL-17A at the enthesis. Under inflammatory conditions, γ/δ T cells increased in number at the Achilles tendon enthesis, aortic root, and adjacent to the ciliary body. CONCLUSION: Entheseal γ/δ T cells are derived from fetal thymus and are maintained as self-renewing tissue-resident cells. As main IL-17A producers within tissues exposed to mechanical stress including enthesis, γ/δ T cells are key players in the pathogenesis of IL-23-induced local inflammation.

Chemokine expression of CCL2, CCL3, CCL5 and CXCL10 during early inflammatory tendon healing precedes nerve regeneration: an immunohistochemical study in the rat.

PURPOSE: Chemokines are major promoters of repair and may regulate nerve ingrowth that is essential in tendon healing. The purpose of this study was to assess the temporal occurrence of different chemokines during Achilles tendon healing in relation to sensory nerve regeneration. Chemokine presence in tendon healing has not been studied previously. METHODS: Chemokine expression, nerve regeneration, angiogenesis and inflammatory cell occurrence during healing of Achilles tendon rupture in the rat were studied by immunohistochemistry and histology including semiquantitative assessment. Markers for chemokines (CCL5, CCL2, CCL3, CXCL10), nerves (PGP-9.5) and sensory neuropeptide substance P (SP) were analysed at different time points (1 day-16 weeks) post-rupture. RESULTS: In intact tendons (controls) immunoreactivity to all chemokines, PGP-9.5 and SP were confined to the tendon surroundings. After rupture, there was rapid increase in the tendon proper of the chemokines studied, all exhibiting their peak expression at week 1. Subsequently, at weeks 2-6, emerging inflammatory cells and maximum sprouting of PGP-/SP-positive nerves were observed close to newly formed blood vessels within the tendon proper, while chemokine expression already decreased. During weeks 6-8, PGP-/SP-positive nerves withdrew from the rupture site and relocated together with the chemokines in the surrounding tendon. CONCLUSIONS: Early chemokine expression in the healing tendon precedes ingrowth of new nerves, angiogenesis and emergence of inflammatory cells. The fine-tuned temporal and spatial appearance of chemokines suggests a chemoattractant role for inflammatory cell migration and possibly also a role in angiogenesis and neurogenesis. Chemokines may thus exhibit vital targets for biological modulation of tendon repair.

Radiological characteristics of the calcaneal spurs in psoriatic arthritis.

OBJECTIVES: Inflammation at the entheses is a distinguishing feature of psoriatic arthritis (PsA). Enthesitis at the heel is the most common location at the Achilles and plantar fascia insertions on the calcaneus. This study aimed to 1) describe the morphological features and measurements of plantar calcaneal spurs in subjects with PsA and controls and 2) determine radiological features that differentiate between inflammatory and non-inflammatory calcaneal spurs. METHODS: Weight bearing lateral foot radiographs of 101 subjects with PsA and 38 control subjects without inflammatory arthritis were examined for plantar calcaneal and Achilles spurs. Three measurements were taken from each radiograph: plantar spur base, mid-segment, and length in millimeters. The differences in radiographic measurements, and the presence of fluffy periostitis of the plantar spurs were then compared between PsA patients and controls. RESULTS: Of the 101 subjects with PsA, 76 (75%) had at least one plantar calcaneal spur and 32 (31.5%) had at least one Achilles tendon spur, compared to 18 (47%) and 3 (8%) respectively in control group (p=0.004). Fluffy plantar periostitis was identified in 14 PsA subjects and none of the controls (p=0.01). The dimensions of plantar spurs were significantly different between groups - longer mid-segment distinguished patients with PsA from controls. CONCLUSIONS: Calcaneal spurs are more common in subjects with PsA than controls. Longer mid-segment measurement was associated with PsA. This study indicates that the presence of fluffy plantar periostitis and broad based and longer mid-segment dimensions are radiological features for inflammatory spurs.

Experimental diabetes induces structural, inflammatory and vascular changes of Achilles tendons.

This study aims to demonstrate how the state of chronic hyperglycemia from experimental Diabetes Mellitus can influence the homeostatic imbalance of tendons and, consequently, lead to the characteristics of tendinopathy. Twenty animals were randomly divided into two experimental groups: control group, consisting of healthy rats and diabetic group constituted by rats induced to Diabetes Mellitus I. After twenty-four days of the induction of Diabetes type I, the Achilles tendon were removed for morphological evaluation, cellularity, number and cross-sectional area of blood vessel, immunohistochemistry for Collagen type I, VEGF and NF-κB nuclear localization sequence (NLS) and nitrate and nitrite level. The Achilles tendon thickness (µm/100g) of diabetic animals was significantly increased and, similarly, an increase was observed in the density of fibrocytes and mast cells in the tendons of the diabetic group. The average number of blood vessels per field, in peritendinous tissue, was statistically higher in the diabetic group 3.39 (2.98) vessels/field when compared to the control group 0.89 (1.68) vessels/field p = 0.001 and in the intratendinous region, it was observed that blood vessels were extremely rare in the control group 0.035 (0.18) vessels/field and were often present in the tendons of the diabetic group 0.89 (0.99) vessels/field. The immunohistochemistry analysis identified higher density of type 1 collagen and increased expression of VEGF as well as increased immunostaining for NFκB p50 NLS in the nucleus in Achilles tendon of the diabetic group when compared to the control group. Higher levels of nitrite/nitrate were observed in the experimental group induced to diabetes. We conclude that experimental DM induces notable structural, inflammatory and vascular changes in the Achilles tendon which are compatible with the process of chronic tendinopathy.

Characteristics of donor and host cells in the early remodeling process after transplant of Achilles tendon with and without live cells for the treatment of rotator cuff defect--what is the ideal graft for the treatment of massive rotator cuff defects?

PURPOSE: We examined the characteristics of donor and host cells in the early remodeling process after transplant of Achilles tendon with and without live cells to repair rotator cuff defects. We also clarified which graft with or without live cells was superior in the early remodeling process. MATERIALS AND METHODS: Sprague-Dawley (SD) rats and green fluorescent protein (GFP) rats were used; they were divided into 3 groups: in group SD, the Achilles tendons of GFP rats were transplanted into the defects of SD rats; in group GFP, the Achilles tendons of SD rats were transplanted into GFP rats; in group GFP-Fr, frozen Achilles tendons of SD rats were transplanted into GFP rats. At 3 and 7 days after surgery, these sections were examined histologically and immunohistochemically with anti-heat shock protein (HSP) 47 and anti-macrophage antibodies. RESULTS: Donor cells gradually decreased, but HSP47-positive donor cells were detected at 3 days in group SD. Host cells infiltrated into the graft from the surrounding tissue, and their numbers in groups SD and GFP gradually increased more significantly than in group GFP-Fr. Macrophages derived from the donor tissue were absent in all groups. The remodeling process of the frozen graft was slower than that in the case of the graft that was not frozen. CONCLUSION: These results demonstrate that live donor cells have a positive effect on the remodeling process. Therefore, autografts with live cells considered to be preferred to frozen allografts or synthetic materials without live cells for transplant for rotator cuff defects.

Histology and immunohistochemistry study of ovine tendon grafted with cBMSCs and BMMNCs after collagenase-induced tendinitis.

OBJECTIVES: The aim of this study was to compare the regeneration abilities of cultured bone marrow mesenchymal cells (cBMSC) and bone marrow mononuclear cells (BMMNC) with fibrin glue, saline solution and sham control in collagenase-induced tendinitis of the Achilles tendon in sheep. METHODS: Six sheep were recruited randomly to each group: cBMSC, BMMNC, fibrin, saline and sham control. Each group received the relative treatment two weeks after inducing lesions (T(0)). After eight weeks (T(8)) of treatment, the tendons were harvested and evaluated for histomorphology, Collagen type I, III, Cartilage Oligomeric Matrix Protein (COMP) and CD34 positive cells expression. RESULTS: Histology and immunohistochemistry showed similar capabilities of cBMSC and BMMNC to restore the architecture of fibres and Extra Cellular Matrix (ECM), with a high expression of collagen type I and COMP and a very low expression of collagen type III in treated tendons. The complete architectural disruption of fibres, dramatic reduction of collagen Type I and COMP expression and increase collagen type III expression were commonly observed in tendons treated with fibrin or saline only. The presence of CD34 positive cells was appreciable in the BMMNC group while few cBMSC showed this cluster of differentiation, not expressed in tendons treated with fibrin or saline. CLINICAL SIGNIFICANCE: The data in this study show the efficacy of cBMSC and BMMNC in regenerating tendon tissue after collagenase-induced tendinitis.

Nerve-related characteristics of ventral paratendinous tissue in chronic Achilles tendinosis.

Ultrasound and Doppler examination has shown high blood flow-neovascularisation inside and outside the ventral Achilles tendon in chronic painful tendinosis, but not in pain-free normal Achilles tendons. In patients with Achilles tendinosis, injections with the sclerosing substance polidocanol, targeting the areas with increased blood flow, have been demonstrated to give pain relief. A drawback when interpreting these findings is the fact that the pattern of nerve supply in the target area, i.e. the ventral area of the tendon, is so far unknown. In this study, therefore, tissue specimens from this area, obtained during surgical treatment of patients with chronic painful midportion Achilles tendinosis, were examined. In the examined area, containing loose connective tissue, the general finding was a presence of large and small arteries and nerve fascicles. The nerve fascicles were distinguished in sections processed for the pan-neural marker protein gene-product 9.5. The nerve fascicles contain sensory nerve fibers, as shown via staining for the sensory markers substance P (SP) and calcitonin gene-related peptide, and sympathetic nerve fibers as seen via processing for tyrosine hydroxylase. In addition, there were immunoreactions for the SP-preferred receptor, the neurokinin-1 receptor, in blood vessel walls and nerve fascicles. Some of the blood vessels were supplied by an extensive peri-vascular innervation, sympathetic nerve fibers being a distinct component of this innervation. There was also a marked occurrence of immunoreactions for the alpha1-adrenoreceptor in arterial walls as well as in the nerve fascicles. Altogether, these findings suggest that the area investigated is under marked influence by the nervous system, including sympathetic and sensory components. Thus, sympathetic/sensory influences may be involved in the pain mechanisms from this area. In conclusion, the nerve-related characteristics of the area targeted by the polidicanol injection treatment for Achilles tendinosis, are shown here for the first time.