human bone marrow cell culture
Bone marrow aspirates were obtained from three donors, 47, 57 and 69 years of age undergoing total hip arthroplasty after informed consent with approval of the local medical ethical committee (METC2004-142). The aspirates were plated as previously described .
To create a pellet, suspensions of 200,000 cells per 15 ml tube were centrifuged at 200 g for 8 minutes. For the scaffolds, suspensions of detached cells were seeded with 1*106 cells per scaffold, divided into 500,000 cells in 100 μl on each side of the Collagen-GAG scaffolds as described previously . The constructs were cultured for 7 days in medium as used for expansion (control medium). Afterwards all samples were either maintained in control medium or replaced with chondrogenic or osteogenic medium for 28 days. Half of the medium was replaced every 3 days.
Chondrogenic medium consisted of high-glucose DMEM containing 50 mg/mL of gentamicin and 1.5 mg/mL of Fungizone (Invitrogen) 25 μg/ml L-ascorbic acid 2-phosphate, 100 mM of sodium pyruvate (Invitrogen), 1:100 insulin-transferrin-selenium (ITS; BD Biosciences, Bedford, MA), 10 ng/mL of transforming growth factor beta-2 (TGF-b2), (R&D Systems, Abingdon, United Kingdom) and 100 nM dexamethasone (Sigma, St. Louis, MO). The osteogenic medium consisted DMEM containing 10% fetal calf serum (Gibco, selected batch), gentamicin and 1.5 mg/mL of Fungizone (Invitrogen) 0.1 mM L-ascorbic acid 2-phosphate, 10 mM beta-glycerol phosphate, 100 nM dexamethasone.
To investigate if bone formation in vivo can be enhanced by allowing mineralisation to occur before implantation, we have applied chondrogenic medium for 21 days and then switched to mineralizing medium conditions for the last 7 days of culture. For the switch 1 condition the chondrogenic medium was replaced after 21 days of culture with osteogenic medium for the remaining period of 7 days. For the switch 2 condition after 21 days of culture in chondrogenic medium, 10 mM beta-glycerol phosphate (as a source of phosphate to allow for mineralization) was added to the chondrogenic medium for the remaining period of 7 days.
Gene expression analysis
To confirm chondrogenic potential of MSCs prior to implantation, gene expression analysis of GAPDH, Sox9, cbfa1, collagen type II and collagen type X was performed as described previously  In addition, samples cultured as pellets were harvested from each MSC donor, fixed in 4% phosphate buffered formalin and embedded in paraffin for collagen type II immunohistochemistry (II-II6B3 antibody, 1:100; Developmental Studies Hybridoma Bank, Iowa City, IA, under contract N01-HD-6-2915 from the National Institute of Child Health and Human Development).
In vivo implantation of hMSC
To evaluate bone formation, cultured constructs were implanted subcutaneously in athymic mice (Balb/C nudes, CDL Nijmegen). For each donor, 3 constructs of each condition were implanted. Before surgery, the skin on both lateral sites of the spine was cleaned with 70% alcohol and 4 subcutaneous pockets were created in each mouse. The tissue engineered samples or pellets were inserted and the pockets closed. Three empty scaffolds were also implanted. Two of these were maintained for the duration of the culture period in expansion medium and one of these in chondrogenic medium. Eight and fourteen weeks after surgery, the animals were euthanized by CO2. The explanted samples were fixed in 4% paraformaldehyde, decalcified in formic acid and embedded in paraffin. The experiments were approved by the Dutch animal experiment committee.
Micro CT imaging
All samples were scanned using micro-CT (Skyscan model 1072, Kontich, Belgium) with a source of 50 kV/98mA without using a filter (resolution 8.1 μm per pixel). Each sample was rotated 180 degrees with a rotation step of 0.90 degrees, exposure time 2.9 seconds. 3D reconstruction, analysis and visualizations were made with NRecon version 1.6, CT-analyzer V1.9 (Skyscan) and 3D-Doctor™ (Able Software Corp., Lexington, United States).
Sections were stained with haematoxylin-eosin and evaluated for presence or absence of bone. A Fisher exact test was used to evaluate statistical significance. Histomorphometry was performed on 2-4 sections of each sample. From each section, low magnification digital images were made, images were pseudo colored and measurements were performed with image analyses techniques (Leica Qwin Pro-image analysis system, Wetzlar, Germany) to obtain the percentage of bone, bone marrow and other tissue.
Rat MSC isolation, culture and implantation
MSCs from 5 month-old inbred wild-type Fischer 344 (F344) rats were isolated and cultured according to standard procedures as described elsewhere . Culture and scaffold seeding was performed exactly as for the human MSCs as described above. The second switch condition was employed for the rat component of this study. Following 5 weeks in vitro, three constructs (scaffolds) of each condition were implanted subcutaneously into immunocompetent co-isogenic hPLAP-transgenic (human Placental Alkaline Phosphatase) F344 rats for 8 weeks. Animals were sacrificed by exsanguination under ketamine/xylazine anesthesia. Scaffolds were harvested and fixed in 40% ethanol at 4°C for 48 h, dehydrated and embedded in modified methylmetacrylate .
Immunohistochemistry for collagen type II
To analyze collagen type II expression, sections were incubated with 0.1% pronase (Sigma, St Louis, MO) for antigen retrieval and 1% hyaluronidase (Sigma, St Louis, MO). Sections were incubated for 2 h at room temperature with mouse monoclonal antibody against collagen type II (II-II6B3 antibody, 1:100; Developmental Studies Hybridoma Bank, Iowa City, IA, under contract N01-HD-6-2915 from the NICHD).
hPLAP immunohistochemical staining
For histochemical staining of the marker enzyme hPLAP deplastisized sections were rehydrated and heated at 65°C for 30 min in deionized water to block endogenous alkaline phosphatase activity. Cells expressing hPLAP were histochemically stained by incubation with an AP substrate (TRIS-HCl buffer (0.2 M, pH 8.5) containing Naphtol AS-MX 0.3 mg/ml (Sigma) and New Fuchsin 0.1 mg/ml (Chroma)) at room temperature for 1 hour and counterstained with haematoxylin.