Short summary

In Denmark, >1000 patients pr. year undergo surgical treatment due to segmental instability of the spine. Clinical outcome depends on the ability to obtain solid bone fusion. It is the goal to improve patients' pain and walking ability. This is obtained by creating sufficient space in the spinal canal and stabilize the affected spinal segment. Fusion of the affected site is important as clinical studies show superiority when obtaining solid fusion. In clinical practice, it would make a difference, if fusion rates were improved.

The aim for the project is to identify a cytokine profile of donor bone that will predict an anabolic bone response to select the best-suited bone allograft and thereby increase the success rate of allograft bone for spine surgery.

Rationale

Lumbar spinal stenosis (LSS), narrowing of the spinal column, leads to compression of the neural structures. It is typically caused by facet-joint arthrosis and bulging discs due to age related degenerative changes. In a more recent study, 13% of the population met criteria for symptomatic LSS. The prevalence is expected to increase over the next decades due to an aging population in industrialized countries. Clinical symptoms are often vague with pain in the back, buttocks, or lower extremities. The cardinal symptom of LSS is walking disability that can only be relieved when sitting or flexing the spine and the spectrum of symptoms range from mild to very severe.

Surgery is offered to regain walking ability and reduce pain but it can be difficult to obtaining adequate fusion in elderly patients. This is a challenge due to poorer osteoblast (OB) proliferation in elderly patients.

In an attempt to avoid peri- and postoperative donor site complications, nonstructural allograft has been used in Scandinavia for decades. Fusion rates when using allograft donor bone show considerable variations that might depend on different content and profiles of cytokines/growth factors within the donor material used. This has so far been sparsely investigated. Thus, a possibility to preselect bone allograft material with a high osteo inductivity and potential for successful healing rates would be clinically relevant.

The fusion rates when using allograft donor bone show considerable variation and might depend on the donor material used. A range of cytokines and growth factors present in bone tissue are known to affect bone remodeling. However, knowledge on the presence of such bioactive substances in allograft donor bone is sparse. In this project we will determine the cytokine/growth factor profile in allograft material and verify their effect on primary human osteoclasts (OCs, bone resorbing cells) and osteoblasts (OBs, bone forming cells) in cell cultures. We have documented that cells from donors in culture behave according to the unique age, menopause, bone turnover status etc. of the donors in vivo, making these cell culture models suited as surrogates to predict responses in vivo.

The fusion rates when using allograft donor bone show considerable variation and might depend on the donor material used. A range of cytokines and growth factors present in bone tissue are known to affect bone remodeling. However, knowledge on the presence of such bioactive substances in allograft donor bone is sparse

Description of the cohort

The experimental procedures are divided into 4 different tasks. Each task aims to study different hypothesis. With the combinations of the 4 tasks, we want to identify a profile of donor bone that will predict an anabolic bone response. In this project we will use 3 groups of participants.

The 1. Group will contain 50 bone allograft donors. This will be patients who in connection with hip prosthesis surgery choose to donate their femoral head to the bone bank at Vejle Hospital. The donated bone will be used for experimental-task 1, 2, 3 and 4.

Group 2 will contain 30 anonymous blood donors for generating OCs in the laboratory for tasks 2 and 4 and

Group 3 contains 15 anonymous osteoarthritis patients undergoing hip-surgery at Odense University Hospital. A piece of bone leftover from surgery is used for generating OBs in the laboratory for tasks 3 and 4.

Data and biological material

The donated bone specimens from Group 1 will be ground up to a paste-like substance. This will be centrifuged, and the supernatant will be collected - "bone liquid".

In task 1 the cytokine/growth factor levels in "bone liquid" will be measured by the Dept. of Clinical Immunology and Biochemistry, Vejle, Lillebaelt Hospital

In task 2 we want to investigate the effects of "bone liquid" on OC differentiation and bone resorption in vitro. By using an established procedure, monocytes will be differentiated into OCs within 9 days (12, 15). To investigate "bone liquid" effects on OC differentiation, CD14+ monocytes from anonymous blood donors in group 2 will be isolated and differentiate into OCs after 9 days. The mature OC will be seeded in well plates and treated with serial dilutions of "bone liquid" for 7 days. The TRAcP activity (marker of OC differentiation) will be determined and OCs will be stained to quantify the number of OCs and number of nuclei. In order to test the effects of "bone liquid" on bone resorption, some matured OCs will be reseeded onto bone slices and treated with serial dilutions of "bone liquid". After 3 days bone slices will be stained and analyzed. The conditioned media from these 3 days will be collected for analysis of the bone resorption marker, CTX.

In task 3 we will use the bone-specimens directly from patients undergoing hip surgery due to osteoarthritis. With this procedure we obtain OB lineage cells reflecting characteristics of early OBs in vivo. After 3 to 4 weeks in culture, these cells will be ready for the tasks. The early OBs will be lifted and reseed onto bovine cortical bone slices in order to best mimics the in vivo situation. The cells will be treated with serial dilutions of "bone liquid" for one week and with medium change twice. After a week, different analyses will be performed on the culture. Determination of cell growth by metabolic activity and number of OC will be done by.

In task 4 we want to investigate the effects of "bone liquid" on both OCs and OBs in vitro. Because we want to test the effects of "bone liquid" on both OCs and OBs, it is relevant to address the OCs and OBs in the same culture using a co-culture.

Collaborating researchers and departments

Clinical Cell Biology, Dept. of Pathology, Odense University Hospital & Dept. of Clinical Research, University of Southern Denmark o Analyze "bone liquid" in cell culture experiments.

• Jacob B. Olesen
• Louise Hjorth Lind

Dept. of Clinical Immunology and Biochemistry, Vejle, Lillebaelt Hospital & University Hospital of Southern Denmark, Department of Regional Health Research, University of Southern Denmark

• Jonna S. Madsen
• Dorte Olsen.

Spine Center of Region of Southern Denmark, Middelfart, Lillebaelt Hospital & University Hospital of Southern Denmark, Department of Regional Health Research, University of Southern Denmark

• Mikkel Østerheden Andersen