**2.3 Analysis of three-dimensional computed tomography (CT) and bone mineral density (BMD)**

The BMD and mineralized tissue ingrowth inside the tendon-bone tunnel junction were quantified by using CT system. Specimens were scanned perpendicular to the long-bone axis covering the entry and exit of the bone tunnel. The sections were reconstructed using the 3D software. To quantify the amount of newly formed mineralized tissue over time, the regions of interest (ROI) was chosen and

**Figure 1.** *Operative procedure of the long digital extensor tendon sutured to the periosteum and soft tissue of rabbit medial tibia.*

*Tendons*

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is a well-known

growth factor for new bone formation [7–9]. This promotes the differentiation of undifferentiated mesenchymal cells into chondrogenic or osteogenic lineages that support new bone formation [10, 11]. This differentiation also needs in the reconstructed tendon-bone tunnel region for postnatal enthesis formation. There have been many studies about an effective rhBMP-2 delivery system using collagen-based materials such as sponge and gel for sustained release [12–15]. In the previous studies, rhBMP-2 was used for bone-tendon interface healing using a collagen sponge [16, 17]. However, the collagen sponge system had a limitation of rhBMP-2 localization to the only targeted region and minimizing leakage from the bone tunnel [18].For the enhanced delivery of rhBMP-2 into the targeted surgical sites, a viscous collagen gel may be useful. Another previous study had performed a comparative study of the osteogenic effects using two different rhBMP-2 delivery systems such as collagen sponge and collagen gel in a rat spinal fusion model [19]. The results showed that rhBMP-2 containing a collagen sponge had side effects of leakage BMP. To overcome this limitation, viscous collagen gel was applied to rabbit

The purpose of this translational study is to investigate whether the rhBMP-2-containing collagen gel can localize in the surgical site and improve new enthesis

1% (w/v) collagen gel originated from porcine skin was mixed with 50 μg/ml rhBMP-2. This concentration had been confirmed from our previous rat and rabbit animal studies [20–23]. To check temperature dependency of collagen's sol-gel phase transition, the optical density of 1% collagen gel at 37°C was read at 313 nm in an absorbance microplate reader at 10, 20, and 30-minute time points. For the release kinetics analysis of rhBMP-2-containing collagen gel, it was plated on 12-well plate and incubated in 1 ml phosphate-buffered saline (pH 7.4) at 37°C for 28 days. At each time point of 1, 3, 5, 7, 14, and 28-day time points, each supernatant was collected and stored at −80°C until reading. Then, the rhBMP-2 was quantitated using an enzyme-linked immunosorbent assay kit and a cumulative release

Healthy adult New Zealand White rabbits (n = 36, 3.0–3.5 kg) were used for this study. The animal treatment was followed by the Guidelines for Care and Use of Laboratory Animals, and this animal experiment was approved by the Committee of Experimental Animal Sciences. The rabbits were classified with three different groups: saline injection only (control group), collagen gel injection only without rhBMP-2 (collagen gel group), and rhBMP-2-conjugated collagen gel injection (rhBMP-2-collagen gel group). Rabbits were anesthetized with ketamine, 40 mg/kg

The rabbits underwent an operative procedure for an extra-articular tendonbone healing model at the rerouted long digital extensor tendon. The knee joint was accessed through a lateral para-patellar incision. The long digital extensor tendon was identified and then detached from its insertion at the lateral femoral condyle by sharp dissection. The free tendon was tied with 3-0 Vicryl. Then, the

formation within reconstructed tendon-bone tunnel after the surgery.

tendon-bone tunnel regions in this study.

**2.1 Conjugation of collagen gel and rhBMP-2**

**2.2 Animal study design and operative procedure**

**2. Materials and methods**

curve was plotted.

IM; xylazine, 5 mg/kg IM.

**140**

reconstructed using the 3D software. After thresholding, the BMD (mg/cm3 ) of the mineralized tissue inside the tendon-bone tunnel junction was calculated.
