ObjectiveTo assess the effect of microfracture and biomimetic hydrogel scaffold on tendon-to-bone healing in a rabbit rotator cuff tear model.MethodsGelatin and methacrylic anhydride were used to synthesize gelatin methacryloyl (GelMA). Then the GelMA were treated with ultraviolet rays and vacuum freeze-drying method to obtain a biomimetic hydrogel scaffold. The morphology of the scaffold was observed by gross observation and scanning electron microscope. Degradation of the scaffold was determined at different time points. Twenty-four adult New Zealand rabbits, weighting 2.8-3.5 kg and male or female, were surgically created the bilateral acute rotator cuff tear models. One shoulder was treated with microfractures on the footprint and transosseous suture (control group, n=24). The other shoulder was treated with the same way, except for putting the scaffold on the footprint before transosseous suture (experimental group, n=24). The general conditions of rabbits were observed postoperatively. Tendon-to-bone healing was evaluated by gross observation, Micro-CT, HE staining, and bio-mechanical testing at 4 and 8 weeks after operation.ResultsThe scaffold was white and has a porous structure with pore size of 31.7-89.9 μm, which degraded slowly in PBS solution. The degradation rate was about 95% at 18 days. All the rabbits survived to the completion of the experiment. Micro-CT showed that there was no obvious defect and re-tear at the tendon-to-bone interface in both groups. No difference was found in bone mineral density (BMD), tissue mineral density (TMD), and bone volume/total volume (BV/TV) between the two groups at 4 and 8 weeks postoperatively (P>0.05). HE staining showed that the fibrous scar tissue was the main component at the tendon-to-bone interface in the control group at 4 and 8 weeks postoperatively; the disorderly arranged mineralized cartilage and fibrocartilage formation were observed at the tendon-to-bone interface in the experimental group at 4 weeks, and the orderly arranged cartilage formation was observed at 8 weeks. Besides, the tendon maturation scores of the experimental group were significantly higher than those of the control group at 4 and 8 weeks (P<0.05). There was no significant difference in the ultimate load to failure and stiffness between the two groups at 4 weeks (P>0.05); the ultimate load to failure at 8 weeks was significantly higher in the experiment group than in the control group (t=4.162, P=0.009), and no significant difference was found in stiffness between the two groups at 8 weeks (t=2.286, P=0.071).ConclusionCompared with microfracture alone, microfracture combined with biomimetic hydrogel scaffold can enhance tendon-to-bone healing and improve the ultimate load to failure in rabbits.
Objective To explore the effect of chitosan (CS) hydrogel loaded with tendon-derived stem cells (TDSCs; hereinafter referred to as TDSCs/CS hydrogel) on tendon-to-bone healing after rotator cuff repair in rabbits. Methods TDSCs were isolated from the rotator cuff tissue of 3 adult New Zealand white rabbits by Henderson step-by-step enzymatic digestion method and identified by multidirectional differentiation and flow cytometry. The 3rd generation TDSCs were encapsulated in CS to construct TDSCs/CS hydrogel. The cell counting kit 8 (CCK-8) assay was used to detect the proliferation of TDSCs in the hydrogel after 1-5 days of culture in vitro, and cell compatibility of TDSCs/CS hydrogel was evaluated by using TDSCs alone as control. Another 36 adult New Zealand white rabbits were randomly divided into 3 groups (n=12): rotator cuff repair group (control group), rotator cuff repair+CS hydrogel injection group (CS group), and rotator cuff repair+TDSCs/CS hydrogel injection group (TDSCs/CS group). After establishing the rotator cuff repair models, the corresponding hydrogel was injected into the tendon-to-bone interface in the CS group and TDSCs/CS group, and no other treatment was performed in the control group. The general condition of the animals was observed after operation. At 4 and 8 weeks, real-time quantitative PCR (qPCR) was used to detect the relative expressions of tendon forming related genes (tenomodulin, scleraxis), chondrogenesis related genes (aggrecan, sex determining region Y-related high mobility group-box gene 9), and osteogenesis related genes (alkaline phosphatase, Runt-related transcription factor 2) at the tendon-to-bone interface. At 8 weeks, HE and Masson staining were used to observe the histological changes, and the biomechanical test was used to evaluate the ultimate load and the failure site of the repaired rotator cuff to evaluate the tendon-to-bone healing and biomechanical properties. Results CCK-8 assay showed that the CS hydrogel could promote the proliferation of TDSCs (P<0.05). qPCR results showed that the expressions of tendon-to-bone interface related genes were significantly higher in the TDSCs/CS group than in the CS group and control group at 4 and 8 weeks after operation (P<0.05). Moreover, the expressions of tendon-to-bone interface related genes at 8 weeks after operation were significantly higher than those at 4 weeks after operation in the TDSCs/CS group (P<0.05). Histological staining showed the clear cartilage tissue and dense and orderly collagen formation at the tendon-to-bone interface in the TDSCs/CS group. The results of semi-quantitative analysis showed that compared with the control group, the number of cells, the proportion of collagen fiber orientation, and the histological score in the TDSCs/CS group increased, the vascularity decreased, showing significant differences (P<0.05); compared with the CS group, the proportion of collagen fiber orientation and the histological score in the TDSCs/CS group significantly increased (P<0.05), while there was no significant difference in the number of cells and vascularity (P>0.05). All samples in biomechanical testing failed at the repair site during the testing process. The ultimate load of the TDSCs/CS group was significantly higher than that of the control group (P<0.05), but there was no significant difference compared to the CS group (P>0.05). Conclusion TDSCs/CS hydrogel can induce cartilage regeneration to promote rotator cuff tendon-to-bone healing.