sj-docx-1-cll-10.1177_09636897241226546 – Supplemental material for Effect of High BMI on Human Bone Marrow–Derived Mesenchymal Stromal Cells

Supplemental material, sj-docx-1-cll-10.1177_09636897241226546 for Effect of High BMI on Human Bone Marrow–Derived Mesenchymal Stromal Cells by Qiang Zong, Katrin Bundkirchen, Claudia Neunaber and Sandra Noack in Cell Transplantation</p

Towards the development of osteochondral allografts with reduced immunogenicity

Nowadays, repair and replacement of hyaline articular cartilage still challenges orthopedic surgery. Using a graft of decellularized articular cartilage as a structural scaffold is considered as a promising therapy. So far, successful cell removal has only been possible for small samples with destruction of the macrostructure or loss of biomechanics. Our aim was to develop a mild, enzyme-free chemical decellularization procedure while preserving the biomechanical properties of cartilage. Porcine osteochondral cylinders (diameter: 12 mm; height: 10 mm) were divided into four groups: Native plugs (NA), decellularized plugs treated with PBS, Triton-X-100 and SDS (DC), and plugs additionally treated with freeze-thaw-cycles of - 20 ◦C, - 80 ◦C or shock freezing in nitrogen (N2) before decellularization. In a nondecalcified HE stain the decellularization efficiency (cell removal, cell size, depth of decellularization) was calculated. For biomechanics the elastic and compression modulus, transition and failure strain as well as transition and failure stress were evaluated. The - 20 ◦C, - 80 ◦C, and N2 groups showed a complete decellularization of the superficial and middle zone. In the deep zone cells could not be removed in any experimental group. The biomechanical analysis showed only a reduced elastic modulus in all decellularized samples. No significant differences were found for the other biomechanical parameters.</p

Percentage of regulatory T cells after HEK cell application.

<p>Relative expression of Foxp3 in CD4⁺CD25⁺ lymphocytes was significantly increased in the group treated with vitHEK cells compared to endotoxemic mice, which received homHEK cells, saline-treated positive-controls (+) and non-endotoxemic negative-controls (−).</p

Mouse model of sublethal endotoxemia (study design).

<p>Male C3H/HeN mice were randomly assigned to either application of vital AFS cells (vitAFS), homogenized AFS cells (homAFS) or saline only (positive-control). Sublethal endotoxemia was induced by intraperitoneal injection of LPS. Mice distributed to the vitAFS group received 10⁶ vital AFS cells in 0.7 ml sterile phosphate buffered saline (PBS) intraperitoneally 2 h after LPS challenge. The same amount of cells and PBS was used for treatment of the homAFS group, but prior to injection vital cells were disrupted using the Sonopulse cell disperser (BANDELIN electronic, Berlin, Germany). The positive-control group received 0.7 ml sterile PBS without cells or disrupted cell material. Mice were sacrificed 24 h after LPS challenge and blood as well as tissue specimens were harvested. A group of 6 animals was sacrificed without any treatment (negative-control) to determine basal cytokine levels, T-cell subtype populations and lung polymorphonuclear neutrophil (PMN) infiltration.</p

Pulmonary PMN infiltration after AFS cell application.

<p>Pulmonary infiltration with PMNs was significantly increased in positive-controls (+) compared to negative-controls (−) 24 h after LPS injection. Mice treated with hom AFS cells showed significantly decreased pulmonary neutrophil influx compared to positive-controls (+).</p

Plasma cytokine response 24 h after LPS challenge (AFS cells).

*<p>p<0.05 vs. Control.</p

Pulmonary PMN infiltration after HEK cell application.

<p>Pulmonary infiltration with PMNs was significantly increased in positive controls (+) and homHEK (p<0.05 vs. [−]) treated endotoxemic mice compared to negative-controls (−) 24 h after LPS injection. Mice treated with vitHEK cells showed significantly decreased pulmonary neutrophil influx compared to positive-controls (+) and homHEK treated endotoxemic mice.</p

Percentage of regulatory T cells after AFS cell application.

<p>The percentage of Foxp3⁺ lymphocytes was significantly increased in the pool of CD4⁺CD25⁺ lymphocytes after application of homAFS compared to saline-treated positive-controls (+) and non-endotoxemic negative-controls (−).</p

Preparation of vital AFS cells and homogenized AFS cells.

<p>10⁶ vital AFS cells (vitAFS) were prepared in 0.7 ml sterile phosphate buffered saline (PBS) for intraperitoneal injection in mice randomly assigned to the vitAFS group (2 h after LPS challenge). For the preparation of homogenized AFS cells, 10⁶ vital AFS cells underwent 5 min cell-disruption in 0.7 ml PBS prior to injection by sonication.</p

IL-2 plasma levels after HEK application.

<p>The IL-2 plasma levels were significantly increased in the group treated with vitHEK compared to saline-treated positive-controls (+).</p