Obtaining healthy bone marrow and blood to put JUVENTAS in perspective

Progenitor cells and progenitor cell therapy have raised much interest in the
past decade. A wide variety of diseases, mainly of cardiovascular origin, have
been associated with reduced numbers and impaired function of (endothelial)
progenitor cells (EPC). Much of the EPC research in cardiovascular diseases has
focused on levels and function of blood-derived EPC, while the genuine EPC pool
resides in its niche in the bone marrow (BM). To draw mechanistic conclusions
on disturbance of mobilization from the BM to circulation and dysfunction of
the progenitor cells residing in the BM cells harvested from patients (with
critical limb ischemia [CLI]) should be placed in perspective to those derived
from healthy controls.

Primary Objective: The primary goal of this study is to obtain BM and PB from
healthy controls without overt cardiovascular diseases (ie patients that
undergo orthopaedic or vascular surgery [atherosclerotic disease excluded]) in
order to compare EPC levels (characterized and counted with flow cytometry) of
CLI patients and healthy controls in both BM and PB.
The BM and PB-borne progenitor cell levels and several plasma and serum factors
involved in neovascularization from controls without cardiovascular diseases
will be compared to patients with CLI included in the Juventas trial. The
ultimate goal is to provide conclusions on how mechanisms involved in EPC
mobilization from the BM to circulation, and progenitor cell numbers and
(dys)function in patients with CLI are changed compared to healthy controls and
the role of the BM herein.
Secondary Objective(s): To provide a foundation for further improvement of
cell- or cell-based therapies in patients with CLI (and cardiovascular diseases
in general) based on the findings of this study.

Cross-sectional observational study

Healthy controls included in this study will not be followed in time, since we
are not interested in the outcome of the controls. BM and PB are withdrawn and
a short health related checklist will be obtained from the healthy controls (to
exclude cardiovascular diseases and to be informed on medication use).
Measurements performed in the patient materials are performed at baseline and
will be compared to baseline measurements performed in the patients with CLI
included in the Juventas trial.

The BM will be harvested peroperatively by cannulation of the bone with a
needle in order to obtain approximately 20cc of BM by aspirating via a 50cc
syringe. In short we will adhere to the following procedure, after the
initiation of general or local anaesthesia, the BM puncture site is indicated
by the surgeon performing the intervention. The BM puncture site is the
location where active BM resides (iliac crest, caput femoris, acetabelum,
distal part of the femur or the proximal tibia) and will always be in the area
involved in the surgical procedure, thus no extra incisions have to be made.
After cannulation of the BM cavity with a BM aspiration needle (15G x 10-68mm),
regularly used for BM aspirations at the Department of Haematology and for the
BM aspirations performed in the Juventas trial, a 50cc syringe will be applied
at the tip of the BM needle and the proper amount (20cc) of BM will be aspired
and collected in two sodium-heparin coated 10cc tubes. Afterwards the BM needle
will be removed and the surgical procedure will be continued according to the
normal surgical routine.
BM harvesting has been performed with low complication rates in general
haemathology as well as in clinical trials (ie Juventas trial). In these cases
the bone is not directly visualized and hence chance for complicated procedures
seems even more likely. Additionally, in this study we only require a rather
small amount of BM when compared to the previously mentioned procedures
(100-1000cc). Besides this, a similar procedure is yet performed during
orthopaedic surgery in the UMC Utrecht without complications.
BM aspiration is generally considered to be safe and feasible. The majority of
the complications are related to the large amount of BM harvested (up to
1000cc) and the anaesthesia applied. BM aspiration during general surgical
procedures to obtain small amounts of BM has been reported previously by others
to be safe and feasible.
The subjects in this study do neither directly nor indirectly benefit from
participation. Nevertheless, the additional burden and risks are fairly small
and seem well tolerable and the results serve potential future mechanistic
insights and therapeutic developments for large amounts of patients suffering
from peripheral arterial disease. Moreover, the availability of blood and BM
from healthy controls would greatly enhance the information and strengthen the
potential mechanistic conclusions obtained from the patient materials obtained
in the Juventas trial (06/030).