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Tendon Healing: Mechanical Loading, Microdamage and Gene Expression
Linköping University, Department of Clinical and Experimental Medicine, Division of Surgery, Orthopedics and Oncology. Linköping University, Faculty of Medicine and Health Sciences.
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mechanical loading and the inflammatory response during tendon healing might be important for the healing process. Mechanical loading can improve the healing tendon but the mechanism is not fully understood. The aim of this thesis was to further clarify the effect of mechanical loading on tendon healing and how mechanical loading affects the inflammatory response during the healing process.

We used a rat Achilles tendon model to study healing. The rats were exposed to different degrees of loading by unloading methods such as paralysis of the calf muscles with Botox, tail suspension, and an orthosis (a boot). Full loading was achieved by free cage activity or treadmill walking. Microdamage in tendons, unloaded with Botox, was also investigated by needling. The healing tendons were evaluated in a materials testing machine (to analyze the mechanical properties), by gene expression analysis (microarray and PCR), or histology.

Our results show that moderate loading (unloading with Botox) improves the mechanical properties of healing tendons compared to minimal loading (unloading with Botox in combination with tail suspension or a boot), especially the material properties. In accordance to these findings, expression of extracellular matrix genes were also increased by moderate compared to minimal loading.

Full loading improved all mechanical properties and the expression of extracellular matrix genes was further increased compared to moderate loading. However, structural properties, such as the strength and the size of the healing tendon, were more affected by full loading. Full loading also affected the expression of inflammation-related genes during the early healing phase, 3 and 5 days after tendon injury, and increased the number of immune cells in the healing tendon tissue. Also microdamage of the healing tendon (detected by blood leakage) was increased by full loading compared to moderate loading during the early healing phase.

Induced microdamage by repeated needling in the healing tendon tissue increased the structural properties of the healing tendon. The gene expression after needling was similar to the gene expression after full loading.

The improvement of mechanical properties by loading in healing tendons was decreased by an anti-inflammatory drug called parecoxib, which decreases the production of prostaglandins by inhibiting COX-2 activity. The effect of parecoxib was reduced when loading was reduced but we could not confirm that the effect of parecoxib was related to the degree of loading. However, parecoxib abolished the stimulatory effect of microdamage.

In conclusion, these studies show that moderate loading improves the quality of the healing tendon whereas full loading also increases the quantity of the healing tendon tissue. Full loading creates microdamage and increases inflammation during the early healing phase. The strong effect of full loading on the structural properties might be due to microdamage. Indeed, the anti-inflammatory drug parecoxib seems to impair mechanical stimulation of healing tendons by reducing the response to microdamage.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2018. , p. 28
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1609
National Category
Biomaterials Science
Identifiers
URN: urn:nbn:se:liu:diva-145281DOI: 10.3384/diss.diva-145281ISBN: 9789176853610 (print)OAI: oai:DiVA.org:liu-145281DiVA, id: diva2:1184134
Public defence
2018-03-26, Belladonna, Campus US, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2018-02-20 Created: 2018-02-20 Last updated: 2018-02-28Bibliographically approved
List of papers
1. Low-level mechanical stimulation is sufficient to improve tendon healing in rats
Open this publication in new window or tab >>Low-level mechanical stimulation is sufficient to improve tendon healing in rats
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2012 (English)In: Journal of applied physiology, ISSN 8750-7587, E-ISSN 1522-1601, Vol. 113, no 9, p. 1398-1402Article in journal (Refereed) Published
Abstract [en]

Treatment of tendon injuries often involves immobilization. However, immobilization might not prevent mild involuntary isometric muscle contraction. The effect of weak forces on tendon healing is therefore of clinical interest. Studies of tendon healing with various methods for load reduction in rat Achilles tendon models show a consistent reduction in tendon strength by at least half, compared with voluntary cage activity. Unloading was not complete in any of these models, and the healing tendon was therefore still exposed to mild mechanical stimulation. By reducing the forces acting on the tendon even further, we now studied the effects of this mild stimulation. Rat Achilles tendons were transected and allowed to heal spontaneously under four different loading conditions: 1) normal cage activity; 2) calf muscle paralysis induced by botulinum toxin A (Botox); 3) tail suspension; 4) Botox and tail suspension, combined, to eliminate even mild stimulation. Healing was evaluated by mechanical testing after 8 days. Botox alone and suspension alone both reduced tendon callus size (transverse area), thereby impairing its strength compared with normal cage activity. The combination of Botox and suspension did not further reduce tendon callus size but drastically impaired the material properties of the tendon callus compared with each treatment alone. The peak force was only a fifth of that in the normal cage activity group. The results indicate that also the mild loading that occurs with either Botox or suspension alone stimulates tendon healing. This stimulation appears to affect mainly tissue quality, whereas stronger stimulation also increases callus size.

Place, publisher, year, edition, pages
American Physiological Society, 2012
Keywords
Achilles tendon, mechanical stress, wound healing, hindlimb unloading, immobilization
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-85847 (URN)10.1152/japplphysiol.00491.2012 (DOI)000310649200007 ()
Note

Funding Agencies|Swedish Research Council|2009-6725|Linkoping University||Ostergotland County Council||King Gustaf V and Queen Victoria Free Mason Foundation||Swedish National Center for Research in Sports||

Available from: 2012-11-30 Created: 2012-11-30 Last updated: 2018-02-20
2. Different gene response to mechanical loading during early and late phases of rat Achilles tendon healing
Open this publication in new window or tab >>Different gene response to mechanical loading during early and late phases of rat Achilles tendon healing
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2017 (English)In: Journal of applied physiology, ISSN 8750-7587, E-ISSN 1522-1601, Vol. 123, no 4, p. 800-815Article in journal (Refereed) Published
Abstract [en]

Mechanical loading stimulates tendon healing both when applied in the inflammatory phase and in the early remodeling phase of the process, although not necessarily via the same mechanisms. We investigated the gene response to mechanical loading in these two phases of tendon healing. The right Achilles tendon in rats was transected, and the hindlimbs were unloaded by tail suspension. The rats were exposed to 5 min of treadmill running 3 or 14 days after tendon transection. Thereafter, they were resuspended for 15 min or 3 h until euthanasia. The controls were suspended continuously. Gene analysis was first performed by microarray analysis followed by quantitative RTPCR on selected genes, focusing on inflammation. Fifteen minutes after loading, the most important genes seemed to be the transcription factors EGR1 and C-FOS, regardless of healing phase. These transcription factors might promote tendon cell proliferation and differentiation, stimulate collagen production, and regulate inflammation. Three hours after loading on day 3, inflammation was strongly affected. Seven inflammation-related genes were upregulated according to PCR: CCL20, CCL7, IL-6, NFIL3, PTX3, SOCS1, and TLR2. These genes can be connected to macrophages, T cells, and recruitment of leukocytes. According to Ingenuity Pathway Analysis, the recruitment of leukocytes was increased by loading on day 3, which also was confirmed by histology. This inflammation-related gene response was not seen on day 14. Our results suggest that the immediate gene response after mechanical loading is similar in the early and late phases of healing but the late gene response is different. NEW amp; NOTEWORTHY This study investigates the direct effect of mechanical loading on gene expression during different healing phases in tendon healing. One isolated episode of mechanical loading was studied in otherwise unloaded healing tendons. This enabled us to study a time sequence, i.e., which genes were the first ones to be regulated after the loading episode.

Place, publisher, year, edition, pages
AMER PHYSIOLOGICAL SOC, 2017
Keywords
tendon healing; gene expression; inflammation; mechanical loading
National Category
Microbiology
Identifiers
urn:nbn:se:liu:diva-143094 (URN)10.1152/japplphysiol.00323.2017 (DOI)000414037800002 ()28705996 (PubMedID)
Note

Funding Agencies|Swedish Research Council [K2013-52X-02031-47-5]; Swedish National Centre for Research in Sports

Available from: 2017-11-22 Created: 2017-11-22 Last updated: 2018-04-03
3. Microtrauma stimulates rat Achilles tendon healing via an early gene expression pattern similar to mechanical loading
Open this publication in new window or tab >>Microtrauma stimulates rat Achilles tendon healing via an early gene expression pattern similar to mechanical loading
2014 (English)In: Journal of applied physiology, ISSN 8750-7587, E-ISSN 1522-1601, Vol. 116, no 1, p. 54-60Article in journal (Refereed) Published
Abstract [en]

Mechanical loading increases the strength of healing tendons, but also induces small localized bleedings. Therefore, it is unclear if increased strength after loading is a response to mechanotransduction or microtrauma. We have previously found only five genes to be up-regulated 15 min after a single loading episode, of them four were transcription factors. These genes are followed by hundreds of genes after 3 h, many of them involved in inflammation. We now compared healing in mechanically unloaded tendons with or without added microtrauma induced by needling of the healing tissue. Nineteen rats received Botox into the calf muscle to reduce loading, and the Achilles tendon was transected. Ten rats were randomized to needling days 2-5. Mechanical testing on day 8 showed increased strength by 45% in the needling group. Next, another 24 rats were similarly unloaded, and 16 randomized to needling on day 5 after transection. Nineteen characteristic genes, known to be regulated by loading in this model, were analyzed by qRT-PCR. Four of these genes were regulated 15 min after needling. Three of them (Egr1, c-Fos, Rgs1) were among the five regulated genes after loading in a previous study. Sixteen of the 19 genes were regulated after 3 h, in the same way as after loading. In conclusion, needling increased strength, and there was a striking similarity between the gene expression response to needling and mechanical loading. This suggests that the response to loading in early tendon healing can, at least in part, be a response to microtrauma.

Place, publisher, year, edition, pages
American Physiological Society, 2014
Keywords
qRT-PCR; unloading; mechanical testing; early response genes; inflammation
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-103870 (URN)10.1152/japplphysiol.00741.2013 (DOI)000329196100008 ()
Available from: 2014-01-31 Created: 2014-01-30 Last updated: 2018-02-20
4. COX-2 inhibition impairs mechanical stimulation of early tendon healing in rats by reducing the response to microdamage
Open this publication in new window or tab >>COX-2 inhibition impairs mechanical stimulation of early tendon healing in rats by reducing the response to microdamage
2015 (English)In: Journal of applied physiology, ISSN 8750-7587, E-ISSN 1522-1601, Vol. 119, no 5, p. 534-540Article in journal (Refereed) Published
Abstract [en]

Early tendon healing can be stimulated by mechanical loading and inhibited by cyclooxygenase (COX) inhibitors (nonsteroidal anti-inflammatory drugs). Therefore, we investigated if impairment of tendon healing by a COX-2 inhibitor (parecoxib) is related to loading. Because loading might infer microdamage, which also stimulates healing, we also investigated if this effect is inhibited by parecoxib. The Achilles tendon was transected in 114 rats. Three degrees of loading were used: full loading, partial unloading, and unloading (no unloading, Botox injections in the plantar flexor muscles, or Botox in combination with tail suspension). For each loading condition, the rats received either parecoxib or saline. In a second experiment, rats were unloaded with Botox, and the tendon was subjected to microdamage by needling combined with either saline or parecoxib. Mechanical testing day 7 showed that there was a significant interaction between loading and parecoxib for peak force at failure (P less than 0.01). However, logarithmic values showed no significant interaction, meaning that we could not exclude that the inhibitory effect of parecoxib was proportionate to the degree of loading. Microbleeding was common in the healing tissue, suggesting that loading caused microdamage. Needling increased peak force at failure (P less than 0.01), and this effect of microdamage was almost abolished by parecoxib (P less than 0.01). Taken together, this suggests that COX-2 inhibition impairs the positive effects of mechanical loading during tendon healing, mainly by reducing the response to microdamage.

Place, publisher, year, edition, pages
AMER PHYSIOLOGICAL SOC, 2015
Keywords
tendon healing; COX-2; NSAIDs; mechanical stimulation; microdamage
National Category
Physiology Pharmacology and Toxicology
Identifiers
urn:nbn:se:liu:diva-122063 (URN)10.1152/japplphysiol.00239.2015 (DOI)000360694300013 ()26159755 (PubMedID)
Note

Funding Agencies|Swedish Research Council [K2013-52X-02031-47-5]; Swedish National Centre for Research in Sports; King Gustaf V and Queen Victoria Free Mason Foundation

Available from: 2015-12-18 Created: 2015-10-19 Last updated: 2018-02-20

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