INTRODUCTION: Flexor digitorum profundus (FDP) tendon injuries are very common in hand injuries and failure in repairing flexor tendon injuries can give rise to significant disability especially in young and working-age populations. With the advent of new suture materials, improved suture techniques and postoperative rehabilitation therapy, the clinical outcomes have made a significant progress. However, the healing process after tendon injury is slow and the regenerated tissue is inferior both in structure and function due to the hypocellular and hypovascular characteristics of tendon. The use of extracellular matrix (ECM) to promote tendon healing has gained particular interest in recent years. Although the ECM of most tissue has many conserved and common structural proteins, each tissue and organ has its unique biophysical characteristics tailored to its specific physiologic and biomechanical requirements. Tendon is rich in ECM components which have been verified to play significant role in the differentiation and organization of tendon. We hypothesized that the ECM derived from flexor tendon (fdpECM) would promote flexor tendon cell proliferation and enhance bone marrow stromal cells (BMSCs) tenogenesis.

METHODS: FDP tendons in Zone II were harvested from mixed-breed dogs (average weight, 20 kg) that were euthanized for other studies approved by Mayo Institutional Animal Care and Use Committee. FDP tendons were minced into small segments before frozen in PBS at -80 ºC and then thinly sliced at 40µm thickness using a cryotome. After pulverized in liquid nitrogen, the tissue powder was incubated in 1% Triton X-100 in PBS under continuous agitation for 24h at 4ºC for decellularization. After three washes (30 minutes per wash) in sterile PBS, the material was treated with 200U/ml DNase and 50U/ml RNase solution at 37 ºC for 12h, followed by washed in sterile PBS 6 times  (30 minutes per wash). The preparation was extracted with 3M urea in water with gentle agitation for 5 days at 4 ºC. The suspension was then centrifuged for 20min at 1500g to collect the extract supernatant. Urea was removed by dialysis of the extract contained in dialysis cassettes (3500MWCO) against deionized water for 2 days at 4 ºC. The dialyzed ECM extract was filtered through a 40 µm filter into a centrifugal filter tube (3000 MWCO) and spin-concentrated by 10 folds for 2h at 1500g. The final fdpECM solution was filter-sterilized through 0.22µm PVDF syringe filter units, and the protein concentration was determined using the BCA assay and then stored at 4ºC until further use. To observe the effect of fdpECM on the proliferation of FDP cells, the FDP cells (P3) were seeded in 96 well at a density of 2000 cells/well in Dulbecoco’s Modified Eagle’s Medium, 10% fetal bovine serum (FBS), and 100 units/ml penicillin and 100 mg/ml streptomycin as the control group. In the experimental group, the cells were cultured with media in control group plus fdpECM (50µg/mL). Before seeding, the FDP tendon cells were treated with IncuCyte® NucLight Green BacMam 3.0 Reagent for nuclear labeling according to the manufacturer protocol. The 96-well plate was incubated at 37°C with 5% CO2 at 100% humidity in the Incucyte S3. Both the phase and fluorescence images were taken very 12h. The proliferation data was processed using the Incucyte S3 software. In order to determine gene expression the FDP tendon cells and BMSCs were seeded in 12 well plate cultured with or without fdpECM (50µg/mL) separately. After 3 days culture, RNA was extracted using Trizol reagent, quantitated suing a NanoDrop 1000 spectrophotometer. Equal amount of total RNA from cell culture was used to synthesize cDNA. Real-time PCR was performed in triplicate and values were normalized using TABA-binding protein (TBP) as a control. Primers were purchased from Integrated DNA Technologies. Primers used for tenogenesis marker genes included scleraxis (SCX) and tenascin-C (TNC). ECM protein genes included collagen I (COL I) and collagen III (COL III). 

RESULTS: After decellularization, nuclei were removed from the FDP tendon (Figure 1A and B) and the DNA content significantly decreased compared to the original tissue (Figurer 1C, n=3, P<0.01). Compared to the control group, the cell number in phase metrics (Figurer 2A) and green metrics (Figure 2B) showed that the proliferation of FDP cells significantly increased in the fdpECM group. The TNC and Col III expression level of FDP cell in the fdpECM group was significantly higher than the control (Figure 3A). BMSCs showed higher expression of SCX than the control after cultured with fdpECM at 3 days (Figure 3B).

DISCUSSION: In this study, we investigated the response of ECM extracted from FDP tendon on the FDP tendon cell proliferation and gene expression of FDP cells and BMSCs. We hypothesize that fdpECM will promote FDP cell proliferation and enhance tenogenic differentiation of BMSCs. H&E staining and DNA content determination (Figure 1) indicated successful decellularization of FDP tendon. Before treating the cell with fdpECM, the final concentration used was calibrated to 50µg/ml. The FDP cells were labeled with green fluorescence, so the cell number could be calculated both in phase metrics and green metrics. After 12h seeded in the plate, the green fluorescence in FPD cell could be detected. Proliferation assays using both fluorescence and phase contrast showed fdpECM treatment had significantly increased FDP cell proliferation (Figure 2). The higher expression level of TNC and Col III in fdpECM group indicated that fdpECM may promote tendon differention (Figure 3A). The expression of SCX, a transcription factor specifically detected in tendon precursor cells was significantly higher in the fdpECM group, which revealed enhanced tenogenesis of BMSCs. Taken together, our study showed that the ECM derived from FDP tendons promoted flexor tendon cell proliferation and enhanced tenogenesis of BMSCs. Future work will explore multiple factors including the optimum dose of fdpECM and assessment of other non-tendon lineage markers including adipogenic and osteogenic markers.
 
SIGNIFICANCE: The ECM derived from FDP tendons may enhance FDP tendon regeneration and may ultimately improve tendon repair. 

ACKNOWLEDGEMENT:  This work was supported by a grant from the NIH/NIAMS (AR57745).

0556 - The Effect Of Extracellular Matrix Derived From Flexor Tendon On Flexor Tenocyte And Bone Marrow Stromal Cells: An In Vitro Study