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Mechanisms in Tendon Healing: Pain, Biomarkers and the Role of Mast Cells
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Family Medicine and Preventive Medicine. Public Health and Caring Sciences. (Magnus Peterson and Gunnar Pejler)
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

Tendon injuries and tendinopathy are common disorders, but the underlying mechanisms are not well understood. The overall aim of this thesis was to better understand the mechanisms underlying tendon healing, pain, and inflammation.

The aim of the first study was to assess biomarkers of tendon healing, including procollagen type I (PINP) and type III (PIIINP) in relation to patient outcome in 65 patients with Achilles tendon rupture (ATR). At two weeks post-ATR, PINP and PIIINP-levels were quantified using microdialysis followed by ELISA. At one-year post-ATR patient outcome was assessed using the validated Achilles tendon Total Rupture Score. We found that higher ratio of PINP and PIIINP to total protein were significantly associated with less pain but more fatigue in the affected limb.

In the second study, we applied Intermittent Pneumatic Compression (IPC) therapy for two weeks to stimulate tendon healing. The patients received either adjuvant IPC treatment or treatment-as-usual in a plaster cast without IPC. We observed that IPC therapy significantly increased PINP levels in the injured tendon, suggesting enhanced healing response.

In our third study, we investigated healing response and the role of mast cells (MCs) in-vivo using an ATR rat model. Three weeks postoperatively, we demonstrated an increased number of MCs and a higher proportion of degranulated MCs in the injured tendon compared to the control. We further established that MCs in the injured tendon were positive for the glutamate receptor NMDAR1.

In our final study, we assessed the effect of glutamate stimulation on in-vitro-derived mouse bone marrow MCs. Mast cell degranulation was quantified through β-hexosaminidase release, immunofluorescence was used to quantify NMDARs at the protein level, and RT-qPCR/microarray was used to study the expression of NMDARs and associated genes. Glutamate induced a robust upregulation of glutamate receptors of both ionotropic and metabotropic type, both at the mRNA and at protein level. NMDAR1 co-localized with glutamate in the membrane of MCs, thereby confirming an interaction between glutamate and its receptor. Glutamate also induced expression of pro-inflammatory compounds such as IL-6 and CCL2 and transcription factors such as Egr2, Egr3 and FosB. Moreover, the NMDA-channel blocker MK-801 completely abrogated the response of MCs to glutamate, supporting a functional glutamate–glutamate receptor axis in MCs.

Together, findings presented in this dissertation reveal possible mechanisms of tendon healing in relation to pain and function, and establish a novel principle for how immune cells can communicate with nerve cells after ATR.

Abstract [sv]

Senskador och tendinopati är vanliga problem, men de underliggande mekanismerna otillräckligt undersökta. Målet med denna avhandling är att öka kunskapen om mekanismer under läkningen av senor, och hur dessa relaterar till smärta och inflammation.

Syftet med den första studien var att kvantifiera biomarkörer för läkning av Achillessena, genom analys av prokollagen typ I (PINP) och typ III (PIIINP) hos 65 patienter efter ruptur av Achillessenan. Två veckor efter achillesruptur kvantifierades PINP och PIIINP-nivåerna med mikrodialys följt av ELISA-analys. Ett år efter achillesruptur bedömdes patienternas upplevda besvär med användning av ett validerat formulär, Achilles Tendon Total Rupture Score. Ökad andel PINP och PIIINP av totala mängden protein vid två veckor var signifikant associerat med mindre smärta men ökad fatigue i skadat ben efter ett år.

I nästa studie utvärderade vi effekten av intermittent pneumatisk kompression (IPC) under två veckor av senans läkning efter achillesruptur. Patienterna fick antingen tilläggsbehandling med IPC eller vanlig behandling i gipsskena utan IPC. Vi kunde visa att behandling med IPC signifikant ökade nivån av PINP i den skadade senan, vilket tyder på förbättrad läkning.

I den tredje studien undersökte vi mastcellers roll under läkningen av Achillessena efter ruptur i en råttmodell. Tre veckor postoperativt visade vi ett ökat antal mastceller och en högre andel degranulerade av mastceller i den läkande senan jämfört med senan på den andra (den friska) sidan. Vi kunde också påvisa glutamatreceptorn NMDAR1 hos mastceller i den läkande senan.

I den fjärde studien bedömde vi effekten av glutamatstimulering in-vitro, på mastceller från benmärg hos mus. Degranulering av mastceller kvantifierades genom frisättning av β-hexosaminidas. För att kvantifiera NMDAR på proteinnivå använde vi immunfluorescens, och för att studera uttrycket av NMDAR och associerade gener använde vi RT-qPCR/mikroarray. Vi kunde visa att glutamat inducerar uppreglering av glutamatreceptorer av både jonotropisk och metabotropisk typ i mastceller, både på mRNA- och proteinnivå. NMDAR1 samlokaliserade med glutamat i membranet på mastceller, vilket därmed bekräftar en interaktion mellan glutamat och dess receptor. Glutamat inducerade också uttryck av pro-inflammatoriska proteiner såsom IL-6 och CCL2, samt transkriptionsfaktorer såsom Egr2, Egr3 och FosB. Dessutom upphävde NMDA-kanalblockeraren MK-801 fullständigt effekten av glutamat på mastceller, vilket talar för en funktionell betydelse av interaktionen mellan glutamat och glutamatreceptorer i mastceller.

Sammantaget visar de fynd som presenteras i denna avhandling möjliga mekanismer för läkning av Achillessena i relation till smärta och funktion och introducerar en ny princip för hur immunceller kan kommunicera med nervceller efter achillesruptur.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. , p. 47
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1611
Keywords [en]
Tendon Healing, Pain, Biomarkers, Mast Cells, Inflammation, Microdialysis, Neurotransmitters, Glutamate Receptors, Procollagen, Transcriptional factors
National Category
Immunology in the medical area Cell and Molecular Biology Orthopaedics
Research subject
Biology with specialization in Molecular Immunology; Biology with specialization in Molecular Biology; Neuroscience
Identifiers
URN: urn:nbn:se:uu:diva-394741ISBN: 978-91-513-0801-2 (print)OAI: oai:DiVA.org:uu-394741DiVA, id: diva2:1365126
Public defence
2019-12-13, Room C8:305, BMC, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2019-11-22 Created: 2019-10-23 Last updated: 2019-11-22
List of papers
1. Procollagen markers in microdialysate can predict patient outcome after Achilles tendon rupture.
Open this publication in new window or tab >>Procollagen markers in microdialysate can predict patient outcome after Achilles tendon rupture.
2016 (English)In: BMJ open sport & exercise medicine, ISSN 2055-7647, Vol. 2, no 1, article id e000114Article in journal (Refereed) Published
Abstract [en]

OBJECTIVE: Patients who sustain acute Achilles tendon rupture (ATR) exhibit variable and mostly impaired long-term functional, and patient-reported outcomes. However, there exists a lack of early predictive markers of long-term outcomes to facilitate the development of improved treatment methods. The aim of this study was to assess markers of tendon callus production in patients with ATR in terms of outcome, pain, and fatigue.

STUDY DESIGN AND SETTING: Prospective cohort study; level of evidence 2. Outpatient orthopaedic/sports medicine department.

PATIENTS: A total of 65 patients (57 men, 8 women; mean age 41±7 years) with ATR were prospectively assessed.

ASSESSMENTS: Markers of tendon callus production, procollagen type I N-terminal propeptide (PINP) and procollagen type III N-terminal propeptide (PIIINP), were assessed 2 weeks postoperatively using microdialysis followed by enzymatic quantification. Normalised procollagen levels (n-PINP and n-PIIINP) were calculated as the ratio of procollagen to total protein content. Pain and fatigue were assessed at 1 year using reliable questionnaires Achilles tendon Total Rupture Score (ATRS).

RESULTS: Patients exhibited fatigue (77.6%) and pain (44.1%) to some extent. Higher levels of n-PINP (R=0.38, p=0.016) and n-PIIINP (R=0.33, p=0.046) were significantly associated with less pain in the limb. Increased concentrations of PINP (R=-0.47, p=0.002) and PIIINP (R=-0.37, p=0.024) were related to more self-reported fatigue in the leg. The results were corroborated by multiple linear regression analyses.

CONCLUSIONS: Assessment of procollagen markers in early tendon healing can predict long-term patient-reported outcomes after ATR. These novel findings suggest that procollagen markers could be used to facilitate the development of improved treatment methods in patients who sustain ATR.

TRIAL REGISTRATION NUMBERS: NCT01317160: Results. NCT02318472: Pre-results.

Place, publisher, year, edition, pages
London, UK: , 2016
Keywords
Achilles, Chronic, Collagen, Injuries, Tendon
National Category
Orthopaedics
Research subject
Orthopaedics
Identifiers
urn:nbn:se:uu:diva-395018 (URN)10.1136/bmjsem-2016-000114 (DOI)27900179 (PubMedID)
Available from: 2019-10-11 Created: 2019-10-11 Last updated: 2019-10-23
2. Achilles tendon rupture healing is enhanced by intermittent pneumatic compression upregulating collagen type I synthesis
Open this publication in new window or tab >>Achilles tendon rupture healing is enhanced by intermittent pneumatic compression upregulating collagen type I synthesis
Show others...
2018 (English)In: Knee Surgery, Sports Traumatology, Arthroscopy, ISSN 0942-2056, E-ISSN 1433-7347, Vol. 26, no 7, p. 2021-2029Article in journal (Refereed) Published
Abstract [en]

PURPOSE AND HYPOTHESIS: Adjuvant intermittent pneumatic compression (IPC) during leg immobilization following Achilles tendon rupture (ATR) has been shown to reduce the risk of deep venous thrombosis. The purpose of this study was to investigate whether IPC can also promote tendon healing.

METHODS: One hundred and fifty patients with surgical repair of acute ATR were post-operatively leg immobilized and prospectively randomized. Patients were allocated for 2 weeks of either adjuvant IPC treatment (n = 74) or treatment-as-usual (n = 74) in a plaster cast without IPC. The IPC group received 6 h daily bilateral calf IPC applied under an orthosis on the injured side. At 2 weeks post-operatively, tendon healing was assessed using microdialysis followed by enzymatic quantification of tendon callus production, procollagen type I (PINP) and type III (PIIINP) N-terminal propeptide, and total protein content. 14 IPC and 19 cast patients (control group) consented to undergo microdialysis. During weeks 3-6, all subjects were leg-immobilized in an orthosis without IPC. At 3 and 12 months, patient-reported outcome was assessed using reliable questionnaires (ATRS and EQ-5D). At 12 months, functional outcome was measured using the validated heel-rise test.

RESULTS: At 2 weeks post-rupture, the IPC-treated patients exhibited 69% higher levels of PINP in the ruptured Achilles tendon (AT) compared to the control group (p = 0.001). Interestingly, the IPC-treated contralateral, intact AT also demonstrated 49% higher concentrations of PINP compared to the non-treated intact AT of the plaster cast group (p = 0.002). There were no adverse events observed associated with IPC. At 3 and 12 months, no significant (n.s.) differences between the two treatments were observed using patient-reported and functional outcome measures.

CONCLUSIONS: Adjuvant IPC during limb immobilization in patients with ATR seems to effectively enhance the early healing response by upregulation of collagen type I synthesis, without any adverse effects. Whether prolonged IPC application during the whole immobilization period can also lead to improved long-term clinical healing response should be further investigated. The healing process during leg immobilization in patients with Achilles tendon rupture can be improved through adjuvant IPC therapy, which additionally prevents deep venous thrombosis.

LEVEL OF EVIDENCE: Randomized controlled trial, Level I.

Keywords
Achilles tendon rupture, Intermittent pneumatic compression devices, Microdialysis, Procollagen, Regeneration
National Category
Surgery
Identifiers
urn:nbn:se:uu:diva-395017 (URN)10.1007/s00167-017-4621-8 (DOI)28668970 (PubMedID)
Available from: 2019-10-11 Created: 2019-10-11 Last updated: 2019-10-23
3. Increased mast cell degranulation and co-localization of mast cells with the NMDA receptor-1 during healing after Achilles tendon rupture
Open this publication in new window or tab >>Increased mast cell degranulation and co-localization of mast cells with the NMDA receptor-1 during healing after Achilles tendon rupture
Show others...
2017 (English)In: Cell and Tissue Research, ISSN 0302-766X, E-ISSN 1432-0878, Vol. 370, no 3, p. 451-460Article in journal (Refereed) Published
Abstract [en]

The role of inflammation and the mechanism of tendon healing after rupture has historically been a matter of controversy. The purpose of the present study is to investigate the role of mast cells and their relation to the NMDA receptor-1 (a glutamate receptor) during healing after Achilles tendon rupture. Eight female Sprague Dawley rats had their right Achilles tendon transected. Three weeks after rupture, histological quantification of mast cell numbers and their state of degranulation was assessed by histochemistry. Co-localization of mast cell tryptase (a mast cell marker) and NMDA receptor-1 was determined by immunofluorescence. The intact left Achilles tendon was used as control. An increased number of mast cells and a higher proportion of degranulated mast cells were found in the healing Achilles tendon compared to the intact. In addition, increased co-localization of mast cell tryptase and NMDA receptor-1 was seen in the areas of myotendinous junction, mid-tendon proper and bone tendon junction of the healing versus the intact tendon. These findings introduce a possible role for mast cells in the healing phase after Achilles tendon rupture.

Place, publisher, year, edition, pages
Berlin Heidelberg: , 2017
Keywords
Achilles tendon healing, Mast cells, NMDA, Rats, Tryptase
National Category
Cell and Molecular Biology Medical and Health Sciences
Research subject
Orthopaedics; Immunology
Identifiers
urn:nbn:se:uu:diva-395522 (URN)10.1007/s00441-017-2684-y (DOI)
Available from: 2019-10-20 Created: 2019-10-20 Last updated: 2019-10-23
4. Glutamate Triggers the Expression of Functional Ionotropic and Metabotropic Glutamate Receptors in Mast Cells
Open this publication in new window or tab >>Glutamate Triggers the Expression of Functional Ionotropic and Metabotropic Glutamate Receptors in Mast Cells
Show others...
(English)Manuscript (preprint) (Other academic)
Keywords
Glutamate, glutamate receptors, mast cells, NMDA-receptors, tryptase
National Category
Cell and Molecular Biology Immunology in the medical area
Research subject
Immunology; Neuroscience
Identifiers
urn:nbn:se:uu:diva-394714 (URN)
Available from: 2019-10-09 Created: 2019-10-09 Last updated: 2019-10-23

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