Intra-articular delivery of purified mesenchymal stem cells from C57BL/6 or MRL/MpJ superhealer mice prevents posttraumatic arthritis

Abstract

Joint injury dramatically enhances the onset of osteoarthritis (OA) and is responsible for an estimated 12% of OA. Posttraumatic arthritis (PTA) is especially common after intra-articular fracture, and no disease-modifying therapies are currently available. We hypothesized that the delivery of mesenchymal stem cells (MSCs) would prevent PTA by altering the balance of inflammation and regeneration after fracture of the mouse knee. Additionally, we examined the hypothesis that MSCs from the MRL/MpJ (MRL) "superhealer" mouse strain would show increased multilineage and therapeutic potentials as compared to those from C57BL/6 (B6) mice, as MRL mice have shown exceptional in vivo regenerative abilities. A highly purified population of MSCs was prospectively isolated from bone marrow using cell surface markers (CD45-/TER119-/PDGFRα+/Sca-1+). B6 MSCs expanded greater than 100,000-fold in 3 weeks when cultured at 2% oxygen and displayed greater adipogenic, osteogenic, and chondrogenic differentiation as compared to MRL MSCs. Mice receiving only a control saline injection after fracture demonstrated PTA after 8 weeks, but the delivery of 10,000 B6 or MRL MSCs to the joint prevented the development of PTA. Cytokine levels in serum and synovial fluid were affected by treatment with stem cells, including elevated systemic interleukin-10 at several time points. The delivery of MSCs did not reduce the degree of synovial inflammation but did show increased bone volume during repair. This study provides evidence that intra-articular stem cell therapy can prevent the development of PTA after fracture and has implications for possible clinical interventions after joint injury before evidence of significant OA.

Figure 1

Cell sorting and expansion. A) Sorting strategy for representative MRL mesenchymal stem cell (MSC) isolation, with arrow indicating only CD45⁻,TER-119⁻ cells are shown in the second plot. B,E) MSCs plated down after sorting; C,F) Colonies expand rapidly; D,G) Cells retain morphology and rapid growth. Scale bars = 100 μm. H) Cumulative fold increase at 2% O_2; results averaged from 3 isolations.

Figure 2

Multi-lineage differentiation. A,B) Adipogenic lipid accumulation; D,E) Osteogenic Alizarin Red S staining; G,H) Chondrogenic Safranin-O/Fast Green staining. C,F,I) Quantification, results from ≥3 samples per group of one representative isolation with standard error of the mean displayed. Asterisk indicates significant effect by t-test. Scale bar = 100 μm (A,B,G,H) or 25 mm (D,E).

Figure 3

Evaluation of post-traumatic arthritis. A) Total joint modified Mankin score of joint degeneration, mean of ≥7 joints per group. Asterisk indicates significant effect by ANOVA, Fisher's post-hoc. B–D) Safranin-O/Fast-Green/Hematoxylin stained coronal section showing the articulation of the lateral femur (top) and lateral tibia (bottom) 8 weeks after fracture. Joint of each group with the highest structural Mankin scores on lateral side shown. Scale bar = 100 μm, arrow indicates fracture site.

Figure 4

Cell tracking. CM-DiI labeling of stem cells before injection. B6 MSCs were found at day 1 in A) bone marrow, B) synovium, and C) muscle. B6 MSCs were also found at D) day 3 in the lateral femoral synovium, E) day 7 near lateral ligamentous tissue, and F) 8 weeks in the tibial subchondral bone.

Figure 5

Systemic (serum) and local (synovial fluid from fractured knee) cytokine concentrations. A,B) Interleukin 1β (IL-1β); C–D) Interleukin 1 receptor antagonist (IL-1ra); E) Interleukin 10 (IL-10). Number of undetectable samples in parentheses. Asterisk indicates significant effect of treatment group at that time point by Kruskal-Wallis Test. Pre-fracture (Pre-Fx) and days 1, 3, 7 (n=3), and 8 weeks (n=8) after fracture.

Figure 6

Synovial response to fracture. A) Total joint synovitis score, mean of ≥7 joints per group. Significance to group shown (*) or all control groups (#) by ANOVA, Fisher's post-hoc. B) Hematoxylin/Eosin staining of lateral femur from a non-fractured control limb without synovial inflammation (mirror image shown). C) Hematoxylin/Eosin staining of lateral femur 8 weeks after fracture and injection of B6 MSCs. D) F4/80 staining for macrophages in the same joint as panel C. In all panels scale bar = 50 μm and arrows indicate synovium.

Figure 7

Morphological bone changes. A) Tibial bone volume; B) Tibial bone density; C) Bone volume in tibial metaphysis; D) Bone density in tibial metaphysis; E) Femoral bone volume / total volume; F) Femoral bone density. Significance to contralateral control (*), all control groups (#), or all groups (&) by ANOVA, Fisher's post-hoc; 8 joints per group.