Adipose derived pericytes rescue fractures from a failure of healing--non-union

Abstract

Atrophic non-union is attributed to biological failure of the fracture repair process. It occurs in up to 10% of fractures, results in significant morbidity to patients, and treatment often requires complex reconstructive procedures. We tested the ability of human bone derived marrow mesenchymal stem cells (MSC), and human adipose derived pericytes (the native ancestor of the MSC) delivered percutaneously to the fracture gap to prevent the formation of atrophic non-union in a rat model. At eight weeks, 80% of animals in the cell treatment groups showed evidence of bone healing compared to only 14% of those in the control group. Radiographic parameters showed significant improvement over the eight-week period in the cell treatment groups, and histology confirmed bone bridges at the fracture gap in the both treatment groups. The quality of bone produced and its biomechanical properties were significantly enhanced in both treatment groups. The results from this study demonstrate that MSC and pericytes have significant bone regeneration potential in an atrophic non-union model. These cells may have a role in the prevention of atrophic non-union and could enable a paradigm shift in the treatment of fractures at high risk of failing to heal and developing non-union.

Figure 1. Pericytes at the origin of MSC.

(A) Pericytes expressing CD146 reside on the abluminal surface of endothelial cells expressing vWF. (B) αSMA expressing pericytes co-express MSC markers including CD90. (C) In-vitro pericytes express MSC markers including CD105. Cultured pericytes are multipotent as seen by their ability to differentiate into bone (D) (Alizarin red staining ×10), fat (E) (Oil Red O staining × 10), cartilage (F) (Alcian blue staining × 5).

Figure 2. Assessment of fracture healing using serial radiography.

(A) Serial radiographs demonstrating the development of bony non union (top row) and the progression to atrophic non-union in our model, (B) Quantitative assessment of callus in the two experimental groups (pericytes and MSC injection) and the control group, (C) Radiographic evidence of union (top) versus persistent non-union (bottom) at 8 weeks.

Figure 3. Multi-parametric analysis of fracture healing.

(A) Quantitative Micro-CT analysis, (B) Histological assessment of the components present at the fracture gap, (C) Mechanical testing using 4 point bending, (D) Cell tracing reveals no transplanted cells in the fracture gap at 8 weeks.

Figure 4. Purification and analysis of pericytes by flow cytometry.

(A) Pericytes can be purified from the SVF of adipose tissue by FACS. Cells (circled in red) are selected on an initial FSC v SSC dot plot. (B) Following removal of dead cells, haematopoietic (CD45+) and endothelial cells (CD31+), pericytes (red box) can be selected based on their unique phenotype (CD146+, CD34−, CD31−, CD45−). (C,D) Pericytes maintain a stable phenotype over extended periods of culture (CD146+, CD31−).