**2. Materials and methods**

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

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 curve was plotted.

### **2.2 Animal study design and operative procedure**

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 IM; xylazine, 5 mg/kg IM.

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

**141**

**Figure 1.**

*medial tibia.*

*The Injectable rhBMP-2-containing Collagen Gel for Tendon Healing in a Rabbit…*

anterior tibia muscle was retracted laterally. A bone tunnel was created in the proximal tibia metaphysis with 30° angle relative to the long-bone axis using a drill (diameter: 2 mm). The finalized bone tunnel size after drilling was an average of 2.09 ± 0.04 mm-diameter and 5.13 ± 0.05 mm-length, which was measured by scanned microcomputed tomography images. The cut long digital extensor tendon was relocated. It was pulled manually through the bone tunnel and sutured to the periosteum and soft tissue at the medial proximal tibia with 3-0 nylon (**Figure 1**). Each 200μl of rhBMP-2-containing collagen gel was injected into the tendonbone tunnel junction. The joint capsule, fascia, and subcutaneous tissue were closed

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

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

*Operative procedure of the long digital extensor tendon sutured to the periosteum and soft tissue of rabbit* 

*DOI: http://dx.doi.org/10.5772/intechopen.82471*

with 3-0 Vicryl and skin was closed with 3-0 nylon.

**density (BMD)**

*The Injectable rhBMP-2-containing Collagen Gel for Tendon Healing in a Rabbit… DOI: http://dx.doi.org/10.5772/intechopen.82471*

anterior tibia muscle was retracted laterally. A bone tunnel was created in the proximal tibia metaphysis with 30° angle relative to the long-bone axis using a drill (diameter: 2 mm). The finalized bone tunnel size after drilling was an average of 2.09 ± 0.04 mm-diameter and 5.13 ± 0.05 mm-length, which was measured by scanned microcomputed tomography images. The cut long digital extensor tendon was relocated. It was pulled manually through the bone tunnel and sutured to the periosteum and soft tissue at the medial proximal tibia with 3-0 nylon (**Figure 1**).

Each 200μl of rhBMP-2-containing collagen gel was injected into the tendonbone tunnel junction. The joint capsule, fascia, and subcutaneous tissue were closed with 3-0 Vicryl and skin was closed with 3-0 nylon.
