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Inflammation in Cancellous and Cortical Bone Healing
Linköping University, Department of Clinical and Experimental Medicine, Division of Surgery, Orthopedics and Oncology. Linköping University, Faculty of Medicine and Health Sciences.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fractures in humans most commonly occur near the joints, in the metaphyseal bone area mainly consisting of cancellous bone. Despite this, mainly cortical fractures, located in the diaphyseal bone area, have been studied in experimental models of bone healing. It is known from previous studies that the diaphyseal fracture is sensitive to anti-inflammatory treatment, while metaphyseal bone healing is more resistant. The aim of this thesis is to study the inflammatory response to bone trauma in cancellous and cortical bone. A flow cytometric method was established for the purpose of examining the cellular composition of the inflammatory process in models of bone healing

In paper I the cellular composition of metaphyseal bone healing was studied with flow cytometry. The proximal tibia was traumatized and then studied at day 1, 3, 5 and 10 afterwards and compared to healthy mice. The contralateral proximal tibia was also studied at the same time points to delineate the trauma site specific inflammation. A few changes could be noted that seemed specific to the trauma site in macrophage phenotype development. However, the cellular composition was similar at the trauma site and in the contralateral proximal tibia. This notion of a general skeletal response was confirmed with analysis of the humerus at day 5.

In paper II a model of cortical bone healing apt for flow cytometry was developed and compared to cancellous bone healing. A furrow was milled along the femoral cortex and the healing bone tissue analyzed. The earliest time point that enough cells were present for flow cytometry was day 3. The cortical and cancellous model of bone healing was compared at day 3 and 5 to study how they evolve in comparison to each other. It was noted that they were similar in cellular composition at day 3, but had diverged at day 5. The cancellous model increased in neutrophilic granulocytes, whereas the cortical model increased in lymphocytes.

In paper III the cancellous and cortical model were compared under experimental intervention of indomethacin. It is known that indomethacin leads to weakened biomechanical properties in cortical bone healing, but not in cancellous bone healing. The effect on cellular composition with indomethacin was studied with flow cytometry and the extracellular protein profile in the healing bone tissue with mass spectrometry. Unexpectedly, inflammatory monocytes were increased in the cortical model at day 3 with indomethacin, but otherwise the models were similar in cell composition at day 3 and 5. In mass spectrometry there was a large increase in detected proteins at day 3 in the indomethacin exposed cortical model, but otherwise the models were similar. This points to an early and model specific effect of indomethacin. The observed lack of indomethacin-induced effects in cancellous bone healing is in line with the previously noted lack of indomethacin-induced effects on bone weakening. The apparently increased inflammatory activity in the cortical model with indomethacin exposure at day 3 might indicate the healing process to be disturbed and not able to progress from the early proinflammatory state to a more anabolic, anti-inflammatory state.

In paper IV the effect of macrophage depletion on healing of metaphyseal bone was studied. Clodronate was given for depletion at different time points prior to surgery and the pull-out force of a screw or tissue phenotyping of macrophages was performed a varying number of days after surgery. It was noted that metaphyseal bone healing was to a large extent inhibited by macrophage depletion up to two days after surgery, but not if depletion was done more than two days after surgery. Thus, macrophages seem to be most important during the first two days after trauma in cancellous bone healing. 

In summary this thesis provide insight to the natural development of bone healing. The findings emphasise that cancellous and cortical bone healing are different entities with differences in the inflammatory process leading to healing.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2019. , p. 49
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1668
National Category
Orthopaedics
Identifiers
URN: urn:nbn:se:liu:diva-156251DOI: 10.3384/diss.diva-156251ISBN: 9789176851128 (print)OAI: oai:DiVA.org:liu-156251DiVA, id: diva2:1303362
Public defence
2019-05-09, Belladonna, Campus US, Linköping, 13:00 (English)
Opponent
Supervisors
Note

En felaktig länk till posten förekom i den tryckta avhandlingen. Denna är ändrad i den elektroniska versionen / There was an icorrect link to this record in the printed version of the thesis. This is corrected in the electronic version

Available from: 2019-04-09 Created: 2019-04-09 Last updated: 2019-06-10Bibliographically approved
List of papers
1. Isolated metaphyseal injury influences unrelated bones A flow cytometric study of tibia and humerus in mice
Open this publication in new window or tab >>Isolated metaphyseal injury influences unrelated bones A flow cytometric study of tibia and humerus in mice
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2017 (English)In: Acta Orthopaedica, ISSN 1745-3674, E-ISSN 1745-3682, Vol. 88, no 2, p. 223-230Article in journal (Refereed) Published
Abstract [en]

Background and purpose - Fracture healing involves different inflammatory cells, some of which are not part of the traditional bone field, such as B-cells and cytotoxic T-cells. We wanted to characterize bone healing by flow cytometry using 15 different inflammatory cell markers in a mouse model of metaphyseal injury, and incidentally discovered a previously unknown general skeletal reaction to trauma. Material and methods - A bent needle was inserted and twisted to traumatize the cancellous bone in the proximal tibia of C57/Bl6 female mice. This is known to induce vivid bone formation locally in the marrow compartment. Cells were harvested from the injured region, the uninjured contralateral tibia, and the humerus. The compositions of the immune cell populations were compared to those in untraumatized control animals. Results - Tibial metaphyseal injury led to substantial changes in the cell populations over time. Unexpectedly, similar changes were also seen in the contralateral tibia and in the humerus, despite the lack of local trauma. Most leukocyte subsets were affected by this generalized reaction. Interpretation - A relatively small degree of injury to the proximal tibia led to systemic changes in the immune cell populations in the marrow of unrelated bones, and probably in the entire skeleton. The few changes that were specific for the injury site appeared to relate to modulatory functions.

Place, publisher, year, edition, pages
TAYLOR & FRANCIS LTD, 2017
National Category
Orthopaedics
Identifiers
urn:nbn:se:liu:diva-137410 (URN)10.1080/17453674.2016.1274587 (DOI)000399484400018 ()28128005 (PubMedID)
Note

Funding Agencies|Swedish Research Council [VR 02031-47-5]; Linkoping University; Ostergotland County Council; European Communitys Seventh Framework Programme (FP7) [279239]

Available from: 2017-05-18 Created: 2017-05-18 Last updated: 2019-04-09
2. Different composition of leucocytes in cortical and cancellous bone healing in a mouse model
Open this publication in new window or tab >>Different composition of leucocytes in cortical and cancellous bone healing in a mouse model
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2018 (English)In: Bone and Joint Research, ISSN 2046-3758, E-ISSN 2352-1872, Vol. 7, no 12, p. 620-628Article in journal (Refereed) Published
Abstract [en]

Objectives Cortical and cancellous bone healing processes appear to be histologically different. They also respond differently to anti-inflammatory agents. We investigated whether the leucocyte composition on days 3 and 5 after cortical and cancellous injuries to bone was different, and compared changes over time using day 3 as the baseline. Methods Ten-week-old male C56/B16J mice were randomized to either cancellous injury in the proximal tibia or cortical injury in the femoral diaphysis. Regenerating tissues were analyzed with flow cytometry at days 3 and 5, using panels with 15 antibodies for common macrophage and lymphocyte markers. The cellular response from day 3 to 5 was compared in order to identify differences in how cancellous and cortical bone healing develop. Results Between day 3 and 5, the granulocytes increased in the cancellous model, whereas the lymphocytes (T cells, B cells, NK cells) and monocytes (CD11b+, 14/80+, CD206+, CD14+ ) increased in the cortical model. Conclusion These results suggest an acute type of inflammation in cancellous bone healing, and a more chronic inflammation in cortical healing. This might explain, in part, why cancellous healing is faster and more resistant to anti-inflammatory drugs than are diaphyseal fractures.

Place, publisher, year, edition, pages
BRITISH EDITORIAL SOC BONE JOINT SURGERY, 2018
Keywords
Metaphyseal; Diaphyseal; Cortical; Cancellous; Intramembranous; Fracture
National Category
Biomaterials Science
Identifiers
urn:nbn:se:liu:diva-154731 (URN)10.1302/2046-3758.712.BJR-2017-0366.R2 (DOI)000457234600001 ()30662708 (PubMedID)
Note

Funding Agencies|Swedish Research Council [VR 02031-47-5]; European Community [279239]

Available from: 2019-02-27 Created: 2019-02-27 Last updated: 2019-05-02
3. Temporal role of macrophages in cancellous bone healing
Open this publication in new window or tab >>Temporal role of macrophages in cancellous bone healing
2017 (English)In: Bone, ISSN 8756-3282, E-ISSN 1873-2763, Vol. 101, p. 129-133Article in journal (Refereed) Published
Abstract [en]

Macrophages are important phagocytosing and cytokine producing cells with effects on fracture healing. We used clodronate-containing liposomes to reduce the number of macrophages, in order to study their role in the early phases of cancellous bone healing. Holes were drilled bilaterally into the cancellous bone of the proximal metaphysis of the tibia of 60 mice. A screw was inserted in the hole in the right tibia. The day of surgery was day 0. Clodronate-containing liposomes were injected intraperitoneally as a single injection either day 4 or 1 (before surgery) or day 1 or 3 (after surgery). A control group underwent surgery as above, but received no clodronate. The mice were killed day 7. The mechanical quality of the new formed cancellous bone holding the screw was evaluated by a pull-out test. The contents of the drill hole in the left tibia was analyzed by microCT. Another set of 20 mice received a drill hole in the metaphysis of the right tibia, and were given either clodronate or saline injections days 3 and 2. The animals were killed day 1 and 3. Flow cytometry was used to analyze the composition of macrophage subpopulations in the regenerating tissue. Flow cytometry showed that clodronate injections day 3 and 2 led to a decrease in mature monocytes day 1 together with an increase in immature monocytes. On day 3 this effect had mostly disappeared, suggesting that the effect of the injections lasted 3 to 5 days. Mechanical testing revealed that the injections prior to surgery decreased the strength of the new formed bone, holding the screw, by about half. Bone density in the drill hole was similarly reduced. In contrast, the injections given day I and 3 had smaller and statistically insignificant effects. Since their depletion at later time points failed to produce a significant effect, it seems that the role of macrophages in cancellous bone is most crucial during the first two days after trauma. (C) 2017 Elsevier Inc. All rights reserved.

Place, publisher, year, edition, pages
ELSEVIER SCIENCE INC, 2017
Keywords
Macrophages; Bone healing; Flow cytometry; Cancellous; Metaphysis; Mechanical testing
National Category
Orthopaedics
Identifiers
urn:nbn:se:liu:diva-139383 (URN)10.1016/j.bone.2017.04.004 (DOI)000404319300016 ()28414141 (PubMedID)
Note

Funding Agencies|Swedish Research Council [K2013-52X-02031-47-5]; Linkoping University; Ostergotland County Council [LiO-619221]; European Communitys Seventh Framework Program (FP7) [279239]

Available from: 2017-08-07 Created: 2017-08-07 Last updated: 2019-04-09

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