Purpose: Injury to articular cartilage found in knee joints is often disabling and affects both younger and older individuals. Injured cartilage rarely heals, leading to a progressive loss of normal function of the joint in many cases. Current treatments are far from satisfactory. One treatment is surgery (such as drilling through the cartilage to the bone, abrasion to remove diseased cartilage and microfracture). These procedures can result in the formation of a mixture of fibrous tissue and cartilage within the injury, which does not lead to a total recovery of joint function. Autologous human bone marrow stromal cell (hBMSC) transplantation maybe a potential avenue to regenerate articular cartilage. We previously determined that naive (untreated) hBMSC were able to form stable, non-hypertrophic cartilage when transplanted subcutaneously in conjunction with fibrin microbeads covalently coated with hyaluronic acid (FMBs). In our current study, we are investigating the ability of hBMSC/FMB constructs to generate native-like cartilage in an articular defect in immunocompromised rodents. Methods: hBMSCs were obtained from surgical bone waste and grown in culture for 2-3 passages. Approval for this study was given by the institutional Animal Care and Use Committee (ACUC). To create the articular cartilage defects in immunocompromised SRG rats (Hera/Charles River), the patella was laterally displaced and the distal femur was immobilized using a clamp. The defect was made in the trochlear groove of the distal femur using a 1.8mm diameter microdrill. The defect was either left unrepaired (sham), or subsequently filled with either FMBs alone (FMBs Alone), hBMSCs attached to FMBs (FMBs+cells), or with a pellet created by incubating BMSCs with FMBs for 10 days in chondrogenic medium. At the 4-week and 8-week timepoints following surgery, the rats were euthanized and femurs were harvested and fixed in 4% paraformaldehyde for 24 hours and then decalcified using a 10% EDTA solution for 30 days. After decalcification, the femurs were paraffin embedded and sectioned at a thickness of 6 μm. Histological staining was performed using hematoxylin and eosin, toluidine blue, and safranin O methods to assess cartilage formation. Immunohistochemistry for aggrecan within the cartilage was also performed to assess cartilage proteoglycan formation. Image analysis using Image J was performed on sections stained with safranin O. Cartilage thickness was approximated based on the thickness of the intact cartilage surrounding the defect, and a region of interest (ROI) was drawn surrounding the defect with the same depth as the intact cartilage. The image color channels were separated into red, green, and blue, and the blue channel was used to threshold for the stained proteoglycan. The “analyze particles” module of Image J was used to calculate the percentage of the ROI that stained positive for proteoglycan. Second-harmonic generation microscopy was also used to evaluate collagen fibril structure and organization. Results: Toluidine blue staining revealed cartilage formation in defects at the 4-week and 8-week timepoints in both the FMBs+cells and pellet groups. No cartilage was detected in the FMBs alone or sham groups (Figure 1). Percentages of defect area positive for cartilage proteoglycan in the FMBs+cells, pellet, FMBs alone, and sham group defect areas were 8.95%, 3.03%, 0.008%, and 0.094% respectively. A comparable ROI in healthy rat cartilage showed proteoglycan positivity of 58.43% of the area (Figure 2). Second harmonic generation imaging of the defect area showed that from sham to FMB alone to pellet to FMBs+cells groups, the organization of the collagen fibrils begin to more closely approximate healthy rat cartilage (Figure 3). Long-term time points are currently being analyzed. Conclusions: At the early timepoints presented in this abstract, there is evidence of cartilage formation in the drilled articular cartilage defects in both the FMBs+cells and pellet groups. This initial data shows the potential for our method to regenerate articular cartilage in vivo. Analysis of our long-term time points will reveal the extent and quality of healing induced by hBMSCs transplanted in conjunction with FMBs.
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