Long-bone fracture is a major orthopedic finding in dogs and cats, and the common causes of these fractures are traffic accidents or falls. Also, the location, type, and fragmentation are crucial factors affecting fracture repair. Therefore, thorough assessments of fractures before surgery are required to ensure successful recovery (10). The main goal of fracture treatment is to restore the normal function of the affected site by reconstructing the anatomic alignment and immobilizing bone fragments until the fracture has healed. Generally, the main treatment options for fracture reduction and fixation include an intramedullary pin, plate, and screw system or an external fixator system. However, in the case of complicated fractures such as non-union, delayed union, and non-reducible fractures, additional bone grafting can be used to promote bone healing (13). Bone grafting has been considered as a significant method supporting the high bone union rate in plate and screw fixation and stimulate bone healing (7).

Guided bone regeneration (GBR), which is indicated when using barrier membrane guides, has been extensively studied and is commonly used to treat bone defects in maxillofacial surgery (8). The main purpose of these methods is to prevent the ingrowth of undesirable soft tissue into the bone defect site, supporting new bone formation (20). Cell growth rates typically differ between soft tissues and bones; thus, without applying structural support such as a membrane, soft tissue may infiltrate the affected site and thereby prevent proper bone regeneration (17). Additionally, studies have shown that the membrane acting as a bioactive compartment during GBR, helping promote bone formation and remodeling through cellular and molecular mechanisms (19). GBR is a commonly used treatment in periodontal surgery in human dentistry, typically with barrier membranes. However, this technique has rarely been used for the repair of long-bone defects of the axial skeleton (8). In this study, by using the liquid form rhBMP-2 with a collagen membrane as a barrier membrane, we attempted to deliver soluble growth factors to the fractured site. The rhBMP-2 can be used in cases of comminuted fracture to accelerate healing. It is the rhBMP-2 that once soaked in collagen membrane. It can be placed in contact with the edges of bone fragments at fractures. The collagen membrane can absorb biological factors and has good permeability (2,4). In addition, the collagen membrane is absorbed after 4 weeks in body (1).

In these two cases, fracture sites were close to joints and considered complicated fractures that were difficult to treat using a traditional method. Thus, we applied bone grafts to fill the bone defect after performing arthrodesis using bridging plates. Additionally, we used collagen membranes with recombinant human bone morphogenetic protein-2 (rhBMP-2) to prevent BMP-2 leakage and to achieve successful bone regeneration using GBR.

Signalment

Case 1. A female mongrel dog aged two years and seven months weighing 6.3 kg was presented to the Veterinary Medical Teaching Hospital, Chonnam National University because of a left femoral fracture due to a car accident 7 months ago.

The lameness score was 4/5 (15), and intermittent non-weight-bearing walking was observed in a gait test. Orthopedic examination showed pain and abnormal range of motion on the left stifle and tarsal joints, and muscular atrophy. Additionally, radiographs of the left hind limb revealed a supracondylar fracture of the left femur (Fig. 1A). Blood biochemistry and complete blood count results were normal. Based on these results, we planned surgery for fracture repair using a plate and screw system.

Figure 1. Femur radiography before surgery (A) and after surgery (B-D) of case 1. (A) Left lateral view. Left femoral supracondylar fracture was confirmed. (B) Left lateral view one day after surgical application of the 2.0 plate-and-screw system. (C) Left lateral view four weeks after surgery. (D) Left lateral view four months after surgery.

Case 2. A spayed female Russian blue cat aged seven years and weighing 4.7 kg was referred to the Veterinary Medical Teaching Hospital, Chonnam National University because of a fracture of the left humerus after a fall from a cat tower (2 m height) five days ago.

In a physical examination, no significant problem was observed except for lameness of the left forelimb. However, radiographs revealed the left comminuted condylar fracture of the humerus (Fig. 2A). No abnormal results were observed in blood biochemistry and complete blood count. Therefore, we decided to perform arthrodesis using a plate and screw system.

Figure 2. Humerus radiography before surgery (A) and after surgery (B-D) of case 2. (A) Left lateral view. Left distal humerus fracture was confirmed. (B) Left lateral view one day after surgical application of the 2.0 plate-and-screw system. (C) Left lateral view four weeks after surgery. (D) Left lateral view four months after surgery.

Also, in both cases, we decided to use rhBMP-2-loaded bone grafts and a collagen membrane to promote bone healing.

Treatment and results

Case 1. Before general anesthesia, a fentanyl patch (12 µg/h; Fentanyl patch 12 µg/h; Myungmoon Pharm, Seoul, Korea) was applied. Before surgery, the patient intravenously received cimetidine (5 mg/kg; H-2^®AMP; JW Pharmaceutical, Seoul, Korea) and cefazolin (20 mg/kg; CKD INJ 1 g; Chong Kun Dang Pharm, Seoul, Korea). After that, the patient was premedicated with glycopyrrolate (0.005 mg/kg; Glycopyrrolate Reyon AMP 1 mL; Reyon Pharm, Seoul, Korea) by subcutaneous injection and butorphanol (0.3 mg/kg; Butophan INJ 1 mg/mL; Myungmoon Pharm, Seoul, Korea) and midazolam (0.3 mg/kg; Vascam INJ 5 mg/mL; Hana Pharm, Seoul, Korea) by intravenous injection. Anesthesia was induced by intravenous administration of propofol (4 mg/kg; Provive INJ 1% 10 mg/mL; Myungmoon Pharm, Seoul, Korea). General anesthesia was maintained using 1.0-2.5% isoflurane (Forane^®; JW Pharmaceutical, Seoul, Korea), and 100% pure oxygen was supplied after tracheal intubation.

Under general anesthesia, the stifle joint was surgically prepared. A lateral incision was performed to gain access to the fracture site (Fig. 3A). Severe muscular atrophy and stiff joint were found due to the non-treated distal femoral fracture. Moreover, the femoral condyle was fragile; thus, the planned cross-pinning application was not possible. Therefore, the distal part of the left femur was cut, and stifle joint arthrodesis was performed using the 2.0 plate and screw system (Jeil medical, Seoul, Korea) (Fig. 3B). After aligning bone fragments with a plate, an autograft, alloplast, and a collagen membrane (Lyoplant; B. Braun, Melsungen, Germany) loaded with rhBMP-2 (0.25 mg; 1 mg/mL) was grafted to the bone defects (Fig. 3C). The rhBMP-2 and alloplast used in this study were NOVOSIS^® (Daewoong Pharmaceutical Company, Seoul, Korea). After the surgery, the patient received cimetidine (5 mg/kg) and amoxicillin hydrate/diluted potassium clavulanate (12 mg/kg; Amocla INJ 0.6 g; Kuhnil Pharm, Seoul, Korea) intravenously for 3 days and then carprofen (2.2 mg/kg; Rimadyl Tab 25 mg/tab; Zoetis Inc., Parsippany, NJ, USA) and amoxicillin hydrate/potassium clavulanate (12 mg/kg; Lactamox Tab amoxicillin 50 mg/tab; clavulanate 125 mg/tab; Aprogen pharm, Sungnam, Korea) were administrated orally twice per day for 12 days. Bandages with a splint were maintained for six weeks after surgery to limit movement in the fracture site and to support bone alignment because of the patient’s vigorous activity. One day after surgery, radiographic evaluation confirmed good bone alignment and showed that implantation material was maintained well. Four weeks after the surgery, radiographs revealed that bone fragments began to unite, and the stifle arthrodesis site was almost healed four months after the surgery.

Figure 3. Intraoperative photographs of fractures (A, D) and modified arthrodesis (B, E) with recombinant human bone morphogenetic protein-2 (rhBMP-2) application and collagen membrane (C, F) in two cases. (A) Left distal femoral fracture. (B) Plating for modified arthrodesis. (C) Grafted site covered using a collagen membrane. (D) Left distal humerus fracture. (E) Plating for modified arthrodesis. (F) Grafted site covered with a collagen membrane.

Case 2. Twenty-four hours before surgery, a fentanyl patch (12 µg/h; Fentanyl patch 12 µg/h) was applied. Before surgery, the patient was intravenously administrated famotidine (0.5 mg/kg; Gaster INJ 20 mg; Dong-a, Seoul, Korea) and cefazolin (20 mg/kg). After that, the patient was premedicated with glycopyrrolate (0.005 mg/kg) by subcutaneous injection, and morphine (1 mg/kg; Morphine HCl INJ 10 mg/mL; Hana Pharm, Seoul, Korea) and midazolam (0.3 mg/kg) by intravenous injection. Anesthesia was induced by intravenous injection with alfaxalone (3 mg/kg; Alfaxan INJ 10 mg/mL; Jurox, Rutherford, Australia). After tracheal intubation, anesthesia was maintained using 1.0-2.2% isoflurane, and 100% pure oxygen was supplied.

The patient was prepared in the same manner as case 1 and was positioned in the right lateral recumbency. The fracture site was exposed through a lateral incision (Fig. 3D). Small bone fragments were removed and the edges of the fractured bone segments were trimmed with a piezoelectric surgery unit (Surgystar Plus, Dmetec, Gyeonggi, Korea). Then, arthrodesis was performed using a plate and screw system (Fig. 3E).

The bone defect site was treated in the same way as that of case 1 (Fig. 3F). After surgery, famotidine (0.5 mg/kg) and cefazolin (20 mg/kg) were administrated intravenously for 2 days, and gabapentin (10 mg/kg, Neurontin Cap 100 mg/cap; Pfizer, Freiburg, Germany), amoxicillin hydrate/potassium clavulanate (12 mg/kg), and meloxicam (0.05 mg/kg, Metacam 0.5 mg/mL; Boehringer Ingelheim, Rhein, Germany) were prescribed orally for 11 days. A splint was used to increase elbow joint stability for four weeks after surgery and was replaced with a soft padded bandage after that. Radiographs showed that the bone healing period was similar to that of case 1.

In dentistry, combinations of barrier membranes and grafting materials have been used to promote proper alveolar bone healing in cases of bone defects by providing sufficient space and establishing osteoconductive properties (14,17). Several preclinical and clinical studies demonstrated that the use of a mixture of membranes and graft materials could support the healing of alveolar bone or tissue. Additionally, variable types of allografts or membranes have been used for alveolar bone healing (18). GBR procedures are commonly used in maxillofacial surgery or with dental implants; however, this method has also been applied to defects of long-bones in recent studies (3,8,16).

Membranes that are used in GBR are non-resorbable or bio-resorbable (18). Among bio-resorbable membranes, collagen membranes are commonly used owing to their low immunogenicity and biodegradability (17). Also, collagen can be an excellent delivery carrier for other drugs such as antibiotics because it can provide a natural extracellular environment that enhances the activity of drugs and antimicrobial agents (6). We used BMP-2 to promote bone healing in these cases because of the difficulty of bone healing at the fracture sites. Moreover, BMP is one of the most commonly used growth factors for osteoinduction, and it has been demonstrated in several studies as promoting bone regeneration. However, when the BMP leaks to other sites around the fracture site, various side effects, such as ectopic bone formation, osteolysis, bone-cyst formation, and inflammatory complications, became more frequent in humans (9,12). In a clinical trial, rhBMP-2 was applied to the lumbar area in patients with disc disease or spondylolisthesis, and ectopic bone formation resulting in recurrent back pain was confirmed using a CT scan (5).

Therefore, in this study, we used a collagen membrane to maintain sufficient space for bone formation and prevent rhBMP-2 leakage as it worked as a delivery carrier for BMP-2. To assess healing status, radiographic images were taken one day, four weeks, and four months after surgery (Fig. 1 and Fig. 2). Typically, bone union begins about seven weeks after surgery (11). In these two cases, we performed arthrodesis using an autograft, alloplasts, and a collagen membrane loaded with rhBMP-2. Follow-up radiographs showed initiating union of bone fragments at four weeks after surgery and complete bone union was observed after four months. In both cases, bone healing was faster than expected bone regeneration process, and no ectopic ossification was found in radiographs. We also confirmed that using rhBMP-2 in addition to bone grafting facilitated new bone formation in the process of fracture healing in these cases. Additionally, the rhBMP-2 loaded collagen membrane supported proper bone regeneration by preventing soft tissue invasion and continuously maintaining rhBMP-2 at the application site without leakage.

Grafting of bone substitutes that maintain sufficient space is necessary for morphological preservation of bone alignment in the healing of fractures with bone defects. In this study, spatial retention capability was provided by using bone grafting, and the use of rhBMP-2 and a collagen membrane supported bone healing based on the concept of the GBR. Furthermore, this method will be helpful in the management of complicated fractures that cannot be treated easily.

This study was supported by the National Research Foundation (NRF) grant funded by the Korea government (MSIT) (No. 2020R1C1C1009798).

The authors have no conflicting interests.