**3. Results**

*Tendons*

recorded.

**2.4 Biomechanical testing**

blinded observation methods.

adjacent tissue (**Table 1**).

**2.5 Histologic and histomorphometric analyses**

reconstructed using the 3D software. After thresholding, the BMD (mg/cm3

Rabbit knee joints including the tendon-bone tunnel site were collected. To analyze the tensile mechanical properties, tensile strength was measured by a universal testing machine. The specimen was fixed vertically on a 5000 N load-cell, and tensile strength was measured by pulling each specimen at a load displacement rate of 10 mm/min. The failure load and ultimate strength (N) were

Rabbit knee joints were collected and fixed in a neutralized formalin solution for 2 days and decalcified in 10% formic acid until being cut. The specimens were sliced into 4-um-thickness in an orientation parallel to the bone tunnel, and each section was stained with Masson's trichrome, and visualized using an optical microscope. Healing of the tendon in the bone tunnel was graded histomorphologically by two

Histomorphometric analysis was assessed for tendon healing in the bone tunnel. Quantitative histomorphometric analysis was performed by two blinded observations, which apportioned 0–3 points based on histomorphologic criteria, representing fibrocartilage formation, new bone formation, and tendon graft bonding to

*Histomorphometric analysis to assess healing of the tendon within the bone tunnel (full score = 9 points).*

mineralized tissue inside the tendon-bone tunnel junction was calculated.

) of the

**142**

**Table 1.**

The turbidity of collagen gel at 37°C was significantly increased between the time points of 10 (OD: 0.953) and 20 (OD: 4.099) minutes (**Figure 2**). The half release from the total rhBMP-2 quantity from the collagen gel was done within 5 days from incubation. And the rest half was released slowly for over 28 days when it was released 89.3% totally. The rhBMP-2 conjugated collagen gel showed a sustained release phase (**Figure 3**).

In the 3D-CT analysis, new bone formation was detected at the interface between the tendon and tibia bone tunnel in the rhBMP-2-collagen gel group after 3 weeks even though the normal distal epiphyseal plate of rabbits has a limited cancellous bone. However, the control and collagen gel groups did not show new bone formation (**Figure 4**). At 6 weeks after the surgery, the rhBMP-2-collagen gel group

**Figure 2.**

*Ultraviolet turbidity of 1% collagen gel at each time point, dependent on temperature; average of triplicates at each time point, which is 0, 10, 20, and 30 min at 37°C incubation.*

**Figure 3.** *Release profile of rhBMP-2 from 1% collagen solution; average of triplicates at each time point for 4 weeks.*

### **Figure 4.**

*3D CT images of the enthesis generated by transfer of the toe flexor or rhBMP-2<sup>+</sup> or rhBMP-2<sup>−</sup> bone complex to the proximal tibia at 3 and 6 weeks.*

**145**

**Figure 6.**

**Figure 5.**

*complex to the proximal tibia at 3 and 6 weeks.*

*complex to the proximal tibia at 3 and 6 weeks.*

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

slightly higher than the control group; however, it was not significant.

no significance between the collagen gel group and control group.

*Bone mineral density of the enthesis generated by transfer of the toe flexor or rhBMP-2+*

*Ultimate failure loads of the enthesis generated by transfer of the toe flexor or rhBMP-2+*

 *or rhBMP-2<sup>−</sup> bone* 

 *or rhBMP-2<sup>−</sup> bone* 

In the biomechanical test result, the ultimate failure load of the rhBMP-2-collagen gel group was significantly higher than the other groups at 3 and 6 weeks (**Figure 6**). After 3 weeks, the ultimate failure load of the rhBMP-2-collagen gel group was 2.5-fold higher than the control group. After 6 weeks, the thBMP-

showed higher new bone formation than the other groups. In addition, the BMD of the rhBMP-2-collagen gel groups was significantly higher than the control group at 3 and 6 weeks after the surgery (**Figure 5**). The BMD of the collagen gel group was

2-collagen gel group was 1.8-fold higher than the control group. However, there was

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

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

showed higher new bone formation than the other groups. In addition, the BMD of the rhBMP-2-collagen gel groups was significantly higher than the control group at 3 and 6 weeks after the surgery (**Figure 5**). The BMD of the collagen gel group was slightly higher than the control group; however, it was not significant.

In the biomechanical test result, the ultimate failure load of the rhBMP-2-collagen gel group was significantly higher than the other groups at 3 and 6 weeks (**Figure 6**). After 3 weeks, the ultimate failure load of the rhBMP-2-collagen gel group was 2.5-fold higher than the control group. After 6 weeks, the thBMP-2-collagen gel group was 1.8-fold higher than the control group. However, there was no significance between the collagen gel group and control group.

**Figure 5.**

*Tendons*

**Figure 3.**

**144**

**Figure 4.**

*the proximal tibia at 3 and 6 weeks.*

*3D CT images of the enthesis generated by transfer of the toe flexor or rhBMP-2+*

*Release profile of rhBMP-2 from 1% collagen solution; average of triplicates at each time point for 4 weeks.*

 *or rhBMP-2<sup>−</sup> bone complex to* 

*Bone mineral density of the enthesis generated by transfer of the toe flexor or rhBMP-2<sup>+</sup> or rhBMP-2<sup>−</sup> bone complex to the proximal tibia at 3 and 6 weeks.*

### **Figure 6.**

*Ultimate failure loads of the enthesis generated by transfer of the toe flexor or rhBMP-2+ or rhBMP-2<sup>−</sup> bone complex to the proximal tibia at 3 and 6 weeks.*

In the histology results, Masson's trichrome staining showed that collagen fibers and fibrous cartilage were widely detected in the tendon-bone interface of the rhBMP-2-collagen gel group at 3 weeks. The new bone was partly detected between the tendon and host bone (**Figure 7**). After 6 weeks, there was increased fibrous cartilage and new bone tissues between the tendon and the bone. Moreover, the new Sharpey-like fibers were detected in the rhBMP-2-collagen gel group (**Figure 8**).

### **Figure 7.**

*Masson's trichrome staining of the enthesis generated by transfer of the toe flexor or rhBMP-2+ or rhBMP-2<sup>−</sup> bone complex to the proximal tibia at 3 weeks: (A–C) control; (D–F) collagen only; (G–I) collagen with rhBMP-2; CF, collagen fibers; FC, fibrocartilage; HB, host bone; NB, new bone; S, Sharpey-like fibers; and T, tendon.*

### **Figure 8.**

*Masson's trichrome staining of the enthesis generated by transfer of the toe flexor or rhBMP-2+ or rhBMP-2<sup>−</sup> bone complex to the proximal tibia at 6 weeks: (A–C) control; (D–F) collagen only; (G–I) collagen with rhBMP-2; CF, collagen fibers; FC, fibrocartilage; HB, host bone; NB, new bone; S, Sharpey-like fibers; and T, tendon.*

**147**

**4. Discussion**

**Figure 9.**

cellular activity for new tissue formation [28, 29].

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

In the histomorphometric analysis results, the histologic score about enthesis formation in the rhBMP-2-collagen gel group was significantly higher than the other groups (**Figure 9**). Moreover, the results after 6 weeks were higher in score in the rhBMP-2-collagen gel group than the score in the other groups after 3 weeks.

 *or rhBMP-2<sup>−</sup> bone* 

*Histological score of the enthesis generated by transfer of the toe flexor or rhBMP-2<sup>+</sup>*

*complex to the proximal tibia at 3 and 6 weeks.*

The healing process after tendon or ligament reconstruction needs stable enthesis generation at the interface between the inserted tendon and drilled bone tunnel, which is one of the most important conditions [4, 24]. rhBMP-2 may be useful as a growth factor for new bone formation by inducing differentiation of osteoprogenitor cells to osteoblasts [25]. For the effective soft tissue healing, rhBMP-2 application will be the ideal method for new bone formation between the inserted tendon and the drilled bone tunnel. However, rhBMP-2 requires a carrier for embedding itself [26, 27]. It is important to establish the rhBMP-2 delivery system with immobilization for the sustained release in the surgical site. The immobilization of rhBMP-2 can enhance the host cell infiltration into the surgical site and stimulating

We used viscous and elastic collagen gel for the minimum loss and sustained release of rhBMP-2. Collagen gel is easy to inject and biocompatible for drug delivery. Collagen gel also has been employed as a scaffold in tissue engineering and regenerative medicine [30]. When the collagen gel is injected into the surgical site, it will be easy to use due to its viscous solution state. After its implantation, the gel becomes semisolid at body temperature. This thermos-sensitive state can make the injected gel stable and good for sustained release of a growth factor for soft tissue reconstruction. In this study, a rabbit extra-articular bone tunnel model was used to investigate the tendon/ligament healing in the drilled bone tunnel. We developed an advanced viscous rhBMP-2-conjugated collagen gel for the soft tissue reconstruction. This gel system will be useful as a void filler between the tendon graft and host bone tunnel. Collagen showed temperature-responsive gelation at the body temperature. This demonstrates that collagen gel can be effective for the stable filling into the surgical

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

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

### **Figure 9.**

*Tendons*

**Figure 7.**

*tendon.*

In the histology results, Masson's trichrome staining showed that collagen fibers

and fibrous cartilage were widely detected in the tendon-bone interface of the rhBMP-2-collagen gel group at 3 weeks. The new bone was partly detected between the tendon and host bone (**Figure 7**). After 6 weeks, there was increased fibrous cartilage and new bone tissues between the tendon and the bone. Moreover, the new Sharpey-like fibers were detected in the rhBMP-2-collagen gel group (**Figure 8**).

*Masson's trichrome staining of the enthesis generated by transfer of the toe flexor or rhBMP-2+*

*Masson's trichrome staining of the enthesis generated by transfer of the toe flexor or rhBMP-2+*

*2<sup>−</sup> bone complex to the proximal tibia at 6 weeks: (A–C) control; (D–F) collagen only; (G–I) collagen with rhBMP-2; CF, collagen fibers; FC, fibrocartilage; HB, host bone; NB, new bone; S, Sharpey-like fibers; and T,* 

*2<sup>−</sup> bone complex to the proximal tibia at 3 weeks: (A–C) control; (D–F) collagen only; (G–I) collagen with rhBMP-2; CF, collagen fibers; FC, fibrocartilage; HB, host bone; NB, new bone; S, Sharpey-like fibers; and T,* 

 *or rhBMP-*

 *or rhBMP-*

**146**

**Figure 8.**

*tendon.*

*Histological score of the enthesis generated by transfer of the toe flexor or rhBMP-2<sup>+</sup> or rhBMP-2<sup>−</sup> bone complex to the proximal tibia at 3 and 6 weeks.*

In the histomorphometric analysis results, the histologic score about enthesis formation in the rhBMP-2-collagen gel group was significantly higher than the other groups (**Figure 9**). Moreover, the results after 6 weeks were higher in score in the rhBMP-2-collagen gel group than the score in the other groups after 3 weeks.

### **4. Discussion**

The healing process after tendon or ligament reconstruction needs stable enthesis generation at the interface between the inserted tendon and drilled bone tunnel, which is one of the most important conditions [4, 24]. rhBMP-2 may be useful as a growth factor for new bone formation by inducing differentiation of osteoprogenitor cells to osteoblasts [25]. For the effective soft tissue healing, rhBMP-2 application will be the ideal method for new bone formation between the inserted tendon and the drilled bone tunnel. However, rhBMP-2 requires a carrier for embedding itself [26, 27]. It is important to establish the rhBMP-2 delivery system with immobilization for the sustained release in the surgical site. The immobilization of rhBMP-2 can enhance the host cell infiltration into the surgical site and stimulating cellular activity for new tissue formation [28, 29].

We used viscous and elastic collagen gel for the minimum loss and sustained release of rhBMP-2. Collagen gel is easy to inject and biocompatible for drug delivery. Collagen gel also has been employed as a scaffold in tissue engineering and regenerative medicine [30]. When the collagen gel is injected into the surgical site, it will be easy to use due to its viscous solution state. After its implantation, the gel becomes semisolid at body temperature. This thermos-sensitive state can make the injected gel stable and good for sustained release of a growth factor for soft tissue reconstruction.

In this study, a rabbit extra-articular bone tunnel model was used to investigate the tendon/ligament healing in the drilled bone tunnel. We developed an advanced viscous rhBMP-2-conjugated collagen gel for the soft tissue reconstruction. This gel system will be useful as a void filler between the tendon graft and host bone tunnel.

Collagen showed temperature-responsive gelation at the body temperature. This demonstrates that collagen gel can be effective for the stable filling into the surgical

site and rhBMP-2 will be effectively and slowly released from the stable gel without any loss by irrigation during the surgery. The phase transformation will also affect the degradation rate of the collagen gel and the time course of stimulation of osteogenesis [18].

In vivo test results showed that the rhBMP-2-collagen gel group increased the fusion rate between the grafted tendon and host bon tunnel. BMD analysis results also showed the enhanced new bone formation by rhBMP-2.

In conclusion, the injectable rhBMP-2-containing collage gel induced earlier and more new bone formation at the tendon-bone tunnel interface. This study demonstrated that the mixture of the rhBMP-2 and collagen gel can accelerate the healing process of the grafted tendon in the host bone tunnel. The clinical use of the injectable rhBMP-2-collagen gel will be promising for the enhancement of tendon/ ligament reconstruction in the future.
