Technique for puncture-aspiration of bone marrow from distal femoral and proximal tibial metaphysis. A. Entry point in femur and tibia in this study. B. Punctures made in line with a programmed total knee arthroplasty (TKA) skin incision. C. Preferred entry points for actual clinical practice. 

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... stem cells (MSCs) were first described in bone marrow by Friedenstein and Owen in 1988 [1] and have since been the subject of increasing research interest. MSCs can differentiate into many tissue types. They have paracrine and immunosuppressive effects and reparative and regenerative properties. These characteristics, and the ease with which they can be isolated, mean that MSCs are useful therapeutically and in tissue engineering [2-5]. One of the earliest and most common uses of MSCs is in the treatment of musculoskeletal pathologies, such as pseudarthrosis, osteochondral defects and avascular necrosis of the bone [6]. Several studies report the efficacy of MSCs in treating such pathologies, and the use of MSCs has extended to other fields of medicine [7-10]. Traditionally, the most common source of MSCs has been bone marrow from the iliac crest, but there are several reports showing these cells are also present in bone marrow from the vertebral body, humeral head and sternum [11-14]. Metaphyseal trabecular bone of the distal femur and proximal tibia has a similar structure to that in the previously mentioned locations [15]. The knee is a relatively straightforward and safe anatomical area for aspiration of bone marrow by bone puncture because of the ease of identification of anatomical landmarks and the well-established nature of the procedures involved. However, as far as we could determine after a thorough literature search, only a few studies investigate the existence of MSCs in this area [16,17]. The aim of this study was to compare aspirates from the metaphysis of the distal femur and proximal tibia with the assumed gold-standard aspirates of the iliac crest from the same patient. The study was approved by the Ethical Committee of our hospital. The primary hypothesis of the study was that MSCs with the same characteristics as those found in bone marrow from the iliac crest would be present in bone marrow from the metaphysis of the distal femur and proximal tibia. A secondary hypothesis was that the concentration of MNCs at the three locations would be similar. A further objective of this study was to determine whether bone marrow from the metaphysis of the distal femur and the proximal tibia represents a potential alternative source of MSCs. We obtained bone marrow aspirates through puncture of the iliac crest, distal metaphysis of the femur and proximal metaphysis of the tibia from a group of volunteer patients during total knee arthroplasty (TKA). MSCs were isolated from the aspirates, characterised and compared. Samples were taken from the limb undergoing surgery. The study was conducted on patients undergoing TKA to minimise iatrogenesis. Patients with knee osteoarthritis assigned to a TKA procedure were enrolled in a prospective, non-randomised way. Subjects were invited to participate and in all cases gave prior written informed consent. Exclusion criteria were age over 75 years, previous treatment with corticosteroids or cytostatic drugs, alcoholism, skin lesions in lower extremities, previous radiotherapy affecting the pelvis or knee, active infection, anaemia (Hb < 10.0 g/dl), leucopenia (< 4000/ml), and/or an active tumoural process. There were 20 subjects in the study: 4 males and 16 females, with a mean age of 70.9 years (range 64-75 years). Twenty bone marrow aspirates were collected from the iliac crest, 17 from the distal femur and 16 from the proximal tibia. In three patients, metaphyseal aspirates were obtained under tourniquet ischaemia to the limb; these samples were not taken in accordance with the protocol and so were not included in the study. In one patient, for unknown reasons, puncture and aspiration of the tibia failed to provide bone marrow. All samples were obtained by the same investigator in the operating room just before TKA surgery. Patients were supine, under spinal anaesthesia, and with deflated ischaemic tourniquet. The operating field was sterile. Puncture and bone marrow aspiration were performed with an 11-gauge cannulated trocar with lateral holes (Bone Marrow Aspiration System, Synthes. Umkirch, Germany). Puncture of the iliac crest was performed first. The skin puncture point was located over the iliac crest, approximately 5 cm posterior to the anterior superior iliac spine. The tip of the cannulated trocar was directed towards the widest part of the ilium. Local infiltration of this area with Bupivacaine (0.5%) reinforced postoperative analgesia. Puncture of the distal femoral metaphysis was made via the anterior face of the knee. The entry point was located 1 cm proximal to the superior pole of the patella. The trocar was directed through the quadriceps and into the anterior femoral cortex at a caudal angle of 30° (Figure 1). Finally, puncture of the proximal tibia metaphysis was conducted over the anterior tibial tubercle in a parallel direction to the articular surface. Entry points at the knee were in line with the planned skin incision for TKA surgery. A minimum of 5 ml bone marrow aspirate was obtained from each location. To avoid haemodilution of the aspirate, the depth and angle of the trocar was changed after each 2 ml of material had been aspirated [13-18]. Samples were placed in heparinised tubes, labelled and sent immediately to the laboratory. TKA surgery was then performed without modification to standard procedure. Postoperative analgesia and early rehabilitation for TKA were as per the hospital’s usual protocols. The minimal criteria described recently by the International Society for Cell Therapy (ISCT) were used to define MSCs in our study [19]. These criteria include adherence to plastic, specific surface antigen expression and potential to differentiate into adipocytes, osteoblasts and chondroblasts. Laboratory processing techniques followed Good Manufacturing Practice (GMP) [20]. Samples were filtered using a standard 100 μm filter to remove clots, fat and bone debris. Preliminary immunophenotypic analysis of bone marrow samples was conducted to rule out contamination with peripheral blood. Mononucleated cell (MNC) count was made with a Coulter counter. The MNC fraction was obtained by density gradient centri- fu gation in Ficoll-Paque (GE Healthcare. Freiburg, Germany). Centrifugation was performed at 400 g (1200 rpm) and 22°C for 30 minutes, after which the MNC layer was aspirated and washed twice in phosphate buffered saline (PBS) to remove remains of Ficoll. Osmotic shock was used to remove any remaining erythrocytes. MNCs were seeded at a density of 160,000-200,000 cells/ cm 2 in culture flasks and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with glutamine, 10% bovine foetal serum (BFS), and penicillin-streptomycin 1% (MACS medium, Miltenyi Biotec, Germany) and incubated. Expansion and cell viability were quantified by means of trypan blue staining and cell counting with a haemocytometer (Neubauer chamber). The maximum duration of cell culture was six weeks to preclude appreciable apparition of cells with abnormal phenotypes or mutagenic changes [21-24]. Cultures without appropriate cell expansion (or with conspicuous cell senescence, non plastic-adherent cells, or cell death) were recorded as a negative result in terms of MNC viability, considered as a failure and discarded. On completion of cell culture, MSCs were analysed by flow cytometry. The membrane antigens analysed included those proposed by the ISCT [19]: CD73, CD90, CD105, CD19, CD14, CD34, CD45 and HLA-DR. Other membrane markers were also included because of their proven utility in defining MSCs: VEGF, CD133, CD117, CD71 and CD271 [25-29]. Data were acquired with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) and analysed using MACS Quantify software (Miltenyi Biotech, Germany). As a control, and for purposes of calibration, an initial analysis was conducted with unmarked cells so that the natural levels of autofluorescence could be observed and thus the level of negativity established for each marker. MSCs were cultured in specific media for differentiation into osteoblasts, adipocytes and chondroblasts. For this purpose, we used commercial optimised differentiation media from Miltenyi Biotech (Bergisch Gladbach, Germany). For differentiation to osteoblasts, cells were incubated in NH OsteoDiff medium. Cells were seeded at 3×10 4 cells per slide flask. Medium was changed every three days. On day ten, cells were harvested and prepared for staining. After fixation with isopropanol, cells were stained with Alizarin Red and counterstained with haematoxylin. For differentiation to adipocytes, MSCs were incubated in NH AdipoDiff medium at 5×10 4 cells per slide flask, changing the medium every three days. On day 21, cells were prepared for staining. Adipocytes were fixed with methanol and stained with Oil Red O. Finally, for differentiation to chondroblasts, MSCs were incubated in NH ChondroDiff medium for 21 days, changing the medium every three days. A monolayer culture was used and cells were seeded at a density of 2.5×10 5 cells/ml. Chondroblasts were fixed with formaldehyde 4% and stained with toluidine blue. The following variables were evaluated in statistical analysis: MSC concentration and viability in bone marrow aspirates, the number of MSCs after cell culture, the viability of MSCs after culture, and the success rate of cultures. Results were compared using the Wilcoxon test for paired samples. A value of p<0.05 was taken to indicate statistical significance, with a 95% confidence interval. The technique used to obtain bone marrow samples from the knee was straightforward and reproducible in all patients. Anatomical landmarks were easily identified by palpation, even in obese patients. ...