An insufficient fracture healing process causes bone nonunion at fracture sites, resulting in dysfunction. In racehorses, fractures can cause large economic losses because repair requires long-term rest; thus, affected horses are prevented from entering races. Fractures in horses are currently treated with external fixation by means of a cast or with internal fixation by use of metal plates and screws. These treatment strategies basically depend on the biogenic healing ability of bone. Bone regeneration methods that make use of various biogenic cytokines combined with surgical procedures have been proposed to enhance the process of fracture healing.1 Growth factors that enhance bone metabolism include acidic fibroblast growth factor,2–4 bFGF,5 bone morphogenic proteins,6–8 transforming growth factor-β1,4,9,10 insulin-like growth factor-1,1,11 and platelet-derived growth factor-BB.12,13 Among these factors, bFGF is recognized as a potent mitogen for various mesenchymal cells14,15 and reportedly induces vascularization11,16 and bone regeneration.17–19 This growth factor is also believed to regulate bone formation because of its ability to stimulate osteoblast cells to differentiate and proliferate.19 Therefore, treatment of horses with bFGF should theoretically shorten the rest period needed for fracture healing.
Generally, it is difficult for a saline (0.9% NaCl) solution containing a cell growth factor to exert the desired effect via injection into the target site because cell growth factors, including bFGF, are unstable and have a short intravital half-life. In fact, in a study20 in which rats were treated with bFGF, a solution of bFGF mixed with fibrin gel rapidly diffused away from the injection site and was metabolized. It has been suggested that drug delivery systems can be used to accurately deliver agents that have short intravital half-lives. With bFGF injection, a prolonged intravital effect can be achieved via sustained release of the growth factor. Various biodegradable polymers that act as carriers when combined with bFGF reportedly provide that sustained release.21–23 Cell growth factors combined with biodegradable carriers, such as collagen or gelatin, are continuously released by gradual hydrolysis of carriers, resulting in prolonged effects at the injection site.
Among available carriers, biodegradable hydrogel microspheres purified from gelatin have attracted great interest. Gelatin hydrogel microspheres do not degrade by simple hydrolysis but rather by proteolysis.23 Furthermore, speed of degradation of these microspheres is affected by their water content and can, therefore, be controlled.23 Use of gelatin hydrogel microspheres containing bFGF to treat bone fractures leads to vascularization24 and bone regeneration.19,25 The purpose of the study reported here was to evaluate the effect of intra-articular injection of gelatin hydrogel microspheres containing bFGF on experimentally induced defects in MC3s of horses, in vivo.
Materials and Methods
Animals—Six healthy Thoroughbred horses (1 male, 3 castrated males, and 2 females) were used. Horses were kept in stalls throughout the study period. They were fed 2 meals, each consisting of 3,500 g of dried grass, every day for 4 weeks after surgery, and 2 meals, each consisting of oats (900 g), bran (300 g), and dried grass (3,500 g), every day for the remainder of the study. Water intake was unrestricted throughout the study. The study protocol was approved by the Experimental Animal Committee of Obihiro University of Agriculture and Veterinary Medicine.
Preparation of gelatin hydrogel microspheres—Microspheres were prepared from gelatin isolated from bovine bone via an alkaline process.a First, 10 mL of an acqueous solution of gelatin (10% gelatin by weight) was prepared by dissolving gelatin in pure water. The solution was heated to 37°C while mixing. Olive oil (350 mL at 40°C) was added, and the mixture was stirred at 1,008 × g for 10 minutes at 40°C to form an emulsion. This emulsion was cooled in ice and stirred at 1,008 × g for 10 minutes to make gelatin spheres. The emulsion was then centrifuged at 12,000 × g for 10 minutes at 4°C to remove the supernatant and stirred at 1,008 × g for 10 minutes at 4°C to remove olive oil and wash the gelatin spheres. After the spheres were dispersed with a homogenizer, the washing process was repeated 3 times. The spheres were weighed after drying and cross-linked by use of a cold, cross-linking aqueous solution (0.1% Tween 80 and 25% glutaraldehyde). The spheres and the cross-linking solution were suspended in a homogenizer and stirred at 4°C for 24 hours. This process resulted in spheres with 95% water content.
After cross-linking, the spheres solution was transferred into centrifuge tubes, and the tubes were centrifuged at 12,000 × g for 10 minutes. The spheres were dispersed in 100mM glycine solution and stirred at room temperature (approx 25°C) to inactivate any remaining aldehyde or glutaraldehyde. The solution containing the spheres was then centrifuged at 12,000 × g for 10 minutes, and the spheres were washed with ultrapure water. Ultrapure water was added to the spheres solution, and the mixture was stirred and centrifuged at 12,000 × g for 10 minutes. This process was repeated 3 times. The spheres were finally dispersed in ultrapure water and lyophilized in liquid nitrogen to yield dry gelatin hydrogel microspheres (IEP, 5.0; water content, 95%).
Preparation of gelatin hydrogel microspheres containing bFGF—A sterilized filter tubeb was washed via addition of 500 μL of saline solution and centrifugation at 12,000 × g for 10 minutes at 4°C. Filtered liquid and saline solution that remained in the filter tube were removed by means of a pipette. The bFGFc (250 μg) was dissolved in 300 μL of saline solution, and the solution was transferred into another sterilized microtube in which the mixture was diluted further with saline solution to a final volume of 500 μL. The solution was transferred to the washed filter tube and centrifuged at 12,000 × g for 10 minutes at 4°C to achieve a mixture with a final concentration of 5 μg/μL. To prepare gelatin hydrogel microspheres containing bFGF, 20 μL of the final solution was added to 2 mg of microspheres, and the mixture was incubated at room temperature for > 1 hour.
Experimental induction of MC3 defects—Each horse was sedated with medetomidined (4 μg/kg, IV), and anesthesia was induced 5 minutes later via IV administration of diazepame (0.03 μg/kg) and ketaminef (2.2 mg/kg). Guaifenesing (25 to 50 mg/kg) was then infused rapidly until the horse became ataxic. The trachea was intubated, the horse was positioned on the surgical table, and anesthesia was induced with 1.5% halothaneh in oxygen. When necessary, dobutaminei was intermittently administered IV at 1 to 5 μg/kg/min, while a mean arterial pressure of approximately 75 mm Hg was maintained. At the beginning of surgery, cefalothin sodiumj (20 mg/kg) and flunixin meglumink (1.1 mg/kg) were administered IV.
Each horse was retained in right lateral recumbency, and the area between the midpoint of the MC3 and the coronary band in each forelimb was shaved, sterilized, and draped. A 20-gauge, 38.1-mm needle was inserted into each metacarpophalangeal joint from the point between the medial palmar aspect of the bone and the suspensory ligament. A 3-way stopcock was then attached to the needle, which was subsequently used to inject 20 mL of saline solution into the joint capsule to distend it. A drill pin (diameter, 2 mm), used as a guide, was inserted vertically into the longitudinal axis of the MC3 from the dorsal aspect proximal to the center of MC3. Next, a drill bit (diameter, 4.5 mm) was inserted vertically into the center of the dorsal surface of the medial condyle of MC3, parallel to the 2.0-mm drill pin. A hole was drilled to a depth of 40 mm. After drilling, the joint capsule and hypodermis were closed with 2-0 absorbable suturel and the skin was closed with 2-0 unabsorbable suture.m
Treatment with microspheres containing bFGF—Gelatin hydrogel microspheres containing bFGF were combined with 1 mL of saline solution and injected into the joint capsule of the right metacarpophalangeal joint (bFGF joint) via the inserted gauge needle that was used to distend the joint for surgery. Saline solution (1 mL) was injected into the joint capsule of the left metacarpophalangeal joint (control joint). Both joints were covered with wound-coating materials,n and a compressive bandage was applied by use of sterilized retractable, cotton, and self-adherento bandages.
Postoperative treatment—After surgery, cefalothin sodium (20 mg/kg, IV) was administered to horses twice a day for 5 days. In addition, flunixin meglumin (1.1 mg/kg, IV) and diclofenac sodiump (1.1 mg/kg, PO) were administered twice a day for 7 days. Bandages were replaced every 7 days, and skin sutures were removed 7 days after surgery. The distal portions of both forelimbs were bandaged for 2 weeks after surgery to prevent swelling.
Assessment of healing—Radiography was conducted 1 day before and 4, 8, 12, and 16 weeks after surgery to evaluate the healing process of bone defect. Computed radiographyq (70 kV; exposure, 0.08 seconds; focus film distance, 70 cm) was performed to obtain lateromedial views of metacarpophalangeal joints.
Examination of metacarpophalangeal joints via MDRCT was performed on horses at 16 weeks after surgery. Horses were anesthetized as described previously. Multidetector-row computed tomography (135 kV; 150 mA; helical pitch, 5.5) was performed with each horse held on an examination table for large animals (1.8 × 3.2 m; height, 0.85 m) that was attached to the bed of the scanner.r
Data collected via MDRCT were processed by use of 3-dimensional, highly precise image-processing software.s The range measured with the analyzing software was a hexahedron located in the bone defect (Figure 1). A rectangle (width, 3.0 mm; height, 3.4 mm) superimposed on a circle (diameter, 4.5 mm) was divided into 3 rectangles that measured 1.0 mm in width and 3.4 mm in height. Next, 3 hexahedrons measuring 30 mm in depth were drawn from the upper and lower dorsal edge of the compact bone to the end edge of the column, and the rectangles that formed their bottom surfaces were gained (width, 1.0 mm; height, 3.4 mm; and depth, 30 mm). The CT value of each hexahedron was calculated in HUs, and the mean CT value of the 3 hexahedrons was considered to be the CT value of the whole bone defect.
Diagrams of the scheme for MDRCT measurement of experimentally induced defects in MC3s of horses. The range measured by MDRCT software was a hexahedron located in the bone defect. Panel A—Measured region of a bone defect in dorsal view, indicating three 1.0 × 3.4-mm squares. Panel B—Measured region of a bone defect in sagittal view. Data from 3 hexahedrons (width, 1.0 mm; height, 3.4 mm; and length, 30 mm) were acquired. The CT value of the 3 hexahedronal areas was considered to be the CT value of the whole bone defect.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1555
Diagrams of the scheme for MDRCT measurement of experimentally induced defects in MC3s of horses. The range measured by MDRCT software was a hexahedron located in the bone defect. Panel A—Measured region of a bone defect in dorsal view, indicating three 1.0 × 3.4-mm squares. Panel B—Measured region of a bone defect in sagittal view. Data from 3 hexahedrons (width, 1.0 mm; height, 3.4 mm; and length, 30 mm) were acquired. The CT value of the 3 hexahedronal areas was considered to be the CT value of the whole bone defect.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1555
Diagrams of the scheme for MDRCT measurement of experimentally induced defects in MC3s of horses. The range measured by MDRCT software was a hexahedron located in the bone defect. Panel A—Measured region of a bone defect in dorsal view, indicating three 1.0 × 3.4-mm squares. Panel B—Measured region of a bone defect in sagittal view. Data from 3 hexahedrons (width, 1.0 mm; height, 3.4 mm; and length, 30 mm) were acquired. The CT value of the 3 hexahedronal areas was considered to be the CT value of the whole bone defect.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1555
Statistical analysis—All summary data were expressed as mean ± SD. Differences in CT values for each horse before and after drug administration were determined by means of a Wilcoxon signed rank test. Values of P < 0.05 were considered significant.
Results
Analysis of lateral radiographs of both metacarpophalangeal joints of horses revealed increased radiopacity indicative of bone healing at 4 to 12 weeks after experimental induction of defects in joints treated with gelatin hydrogel microspheres containing bFGF (bFGF joints) and at 8 to 16 weeks after surgery in joints treated with saline solution (control joints; Figure 2). However, radiographs of control joints of 2 horses revealed no increased radiopacity at 16 weeks after surgery. Overall, bFGF joints had evidence of bone healing before or at the same time as control joints (Figure 3). The mean CT value for bFGF joints (411.7 ± 135.6 HU) was significantly higher than that for control joints (240.8 ± 133.1 HU).
Representative, sequential lateral radiographic views of metacarpophalangeal joints of a horse in which a defect (arrows) in each MC3 had been experimentally induced. The joint capsule of the right metacarpophalangeal joint was injected with gelatin hydrogel microspheres containing bFGF (top row); that of the left metacarpophalangeal joint was injected with saline (0.9% NaCl) solution (bottom row). Radiography was performed 1 day before and 4, 8, 12, and 16 weeks after surgery. In this horse, the bone defect is represented by increased radiopacity at 12 weeks (top row) and 16 weeks (bottom row) after induction of the defect.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1555
Representative, sequential lateral radiographic views of metacarpophalangeal joints of a horse in which a defect (arrows) in each MC3 had been experimentally induced. The joint capsule of the right metacarpophalangeal joint was injected with gelatin hydrogel microspheres containing bFGF (top row); that of the left metacarpophalangeal joint was injected with saline (0.9% NaCl) solution (bottom row). Radiography was performed 1 day before and 4, 8, 12, and 16 weeks after surgery. In this horse, the bone defect is represented by increased radiopacity at 12 weeks (top row) and 16 weeks (bottom row) after induction of the defect.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1555
Representative, sequential lateral radiographic views of metacarpophalangeal joints of a horse in which a defect (arrows) in each MC3 had been experimentally induced. The joint capsule of the right metacarpophalangeal joint was injected with gelatin hydrogel microspheres containing bFGF (top row); that of the left metacarpophalangeal joint was injected with saline (0.9% NaCl) solution (bottom row). Radiography was performed 1 day before and 4, 8, 12, and 16 weeks after surgery. In this horse, the bone defect is represented by increased radiopacity at 12 weeks (top row) and 16 weeks (bottom row) after induction of the defect.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1555
Representative MDRCT images of the metacarpophalangeal joints of a horse in which a defect (rectangle) in each MC3 had been experimentally induced with a drill. Notice that the amount of defect refilling in the joint treated with gelatin hydrogel microspheres containing bFGF (right; rectangle area, 3.0 mm × 3.4 mm) is substantially greater than that in the control joint (left; rectangle area, 3.0 × 3.4 mm). In this horse, the CT value for the treated joint was 324.3 HU and that for the control joint was 73.2 HU.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1555
Representative MDRCT images of the metacarpophalangeal joints of a horse in which a defect (rectangle) in each MC3 had been experimentally induced with a drill. Notice that the amount of defect refilling in the joint treated with gelatin hydrogel microspheres containing bFGF (right; rectangle area, 3.0 mm × 3.4 mm) is substantially greater than that in the control joint (left; rectangle area, 3.0 × 3.4 mm). In this horse, the CT value for the treated joint was 324.3 HU and that for the control joint was 73.2 HU.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1555
Representative MDRCT images of the metacarpophalangeal joints of a horse in which a defect (rectangle) in each MC3 had been experimentally induced with a drill. Notice that the amount of defect refilling in the joint treated with gelatin hydrogel microspheres containing bFGF (right; rectangle area, 3.0 mm × 3.4 mm) is substantially greater than that in the control joint (left; rectangle area, 3.0 × 3.4 mm). In this horse, the CT value for the treated joint was 324.3 HU and that for the control joint was 73.2 HU.
Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1555
Discussion
In the study reported here, results of radiography and MDRCT indicated that intra-articular injection of metacarpophalangeal joints with gelatin hydrogel microspheres containing bFGF expedited bone healing in horses with experimentally induced defects of MC3s. Overall, MC3s in joints treated with bFGF healed faster, as revealed via radiographic analysis, than did MC3s in control joints. In addition, the mean CT value obtained via MDRCT analysis was significantly higher in bFGF joints, compared with the value for control joints.
Alginate,21 amylopectin,26 fibrin gel,20 collagen minipellet,22 and gelatin hydrogel microspheres have all been evaluated as carriers for sustained release of bFGF. Because physical stimulation of carrier is weak in the synovial fluid, we decided to use gelatin hydrogel microspheres (IEP, 5.0) as a carrier and injected microspheres containing bFGF into metacarpophalangeal joints to treat horses with experimentally induced bone defects. In another study,24 gelatin hydrogel microspheres containing bFGF were also used for a similar purpose in rabbits. Microspheres with an IEP value of 5.0 have an intravital negative charge (pH, 7.0) and can electrostatically interact with bFGF, which has a positive charge, to make a complex.23,27,28 As the microsphere complex is degraded by intravital hydrolysis, bFGF bound to the microspheres is gradually liberated, resulting in sustained release over a long period. In the present study, we prepared gelatin hydrogel microspheres with a water content of 95%. Results of another study23 indicated that SC administration of bFBF-containing gelatin hydrogel microspheres that had a water content of 96.9% into the dorsum of mice produces effects for > 21 days. Therefore, we believe that the bFGF-containing microspheres used in the present study remained active for > 21 days. In addition, results regarding bone repair in the present study suggested that sustained release of bFGF was more effective in healing bone defects in horses than the biogenic healing ability of bone.
In the study reported here, radiographic analysis revealed earlier signs of bone healing in horses that received bFGF versus horses that received saline solution. This indicated that the refilling of experimentally induced holes in bone was faster in bFGF joints than it was in the control joints. In addition, MDRCT examination revealed significantly higher CT values (HUs) in bFGF joints, compared with values in control joints. This CT value can be used to evaluate the degree of bone filling by calculating x-ray absorbance in the area of interest. Therefore, the results of this study suggested that gelatin hydrogel microspheres containing bFGF can stimulate refilling of bone defects.
Reportedly, bFGF stimulates differentiation and proliferation of immature mesenchymal cells and inhibits differentiation and synthesis of osteoblast cell matrix.13,29,30 In addition, osteoblast progenitor cells stimulate bone resorption and activate the bone regeneration cascade and bone remodeling via generation of intrinsic transforming growth factor-β1 and activation of alkaline phosphatase.31 Therefore, it is believed that bFGF accelerates fracture healing by stimulating all steps in the fracture-healing process.
To support the clinical use of gelatin hydrogel microspheres containing bFGF, additional studies are needed that make use of the same experimental protocol as that reported here and incorporate histologic examination of bone defects, evaluation of long-term effects of microsphere administration, and evaluation of the strength of any regenerated bone tissue. In addition, the use of gelatin hydrogrel microspheres containing bFGF at other fracture sites needs to be investigated. Because expression of growth factors differs during the bone healing process,32,33 multiple growth factors have been used to regulate or stimulate tissue regeneration in vitro.34 Thus, additional studies of bone healing that make use of different growth factors are also warranted.
ABBREVIATIONS
| bFGF | Basic fibroblast growth factor |
| CT | Computed tomography |
| HU | Hounsfield unit |
| IEP | Isoelectric point |
| MC3 | Third metacarpal bone |
| MDRCT | Multidetector-row computed tomography |
Isoelectric point, version 5.0, Nitta Gelatin Co, Kyoto, Japan.
Ultrafree-0.5, Millpore, Kyoto, Japan.
Trafermin Fiblast Spray 250, Kaken Pharmaceutical Co Ltd, Tokyo, Japan.
Domitor (0.1%), Nippon Zenyaku Kougyou Co Ltd, Osaka, Japan.
Horizon (0.5%), Astellas Inc, Tokyo, Japan.
Veterinary Ketalar 50, Sankyo Yell Yakuhin Co Ltd, Tokyo, Japan.
ALPS Pharmaceutical Industries Co Ltd, Tokyo, Japan.
Furosen, Takeda Pharmaceutical Industry Co Ltd, Tokyo, Japan.
Retamex (2%), Sankyo Yell Yakuhin Co Ltd, Tokyo, Japan.
Coaxin, Tobishi Pharmaceutical Co Ltd, Tokyo, Japan.
Banamine (5%), Dainippon Sumitomo Pharma Co Ltd, Osaka, Japan.
2-0 Vicryl, Johnson & Johnson, Tokyo, Japan.
2-0 Ethilon, Johnson & Johnson, Tokyo, Japan.
Melolin, Smith & Nephew, Tokyo, Japan.
Coban, 3M, Tokyo, Japan.
Blesin tablet, Sawai Pharmaceutical Co Ltd, Tokyo, Japan.
XG-1V, Fuji Film Co, Tokyo, Japan.
Asterion 4, Toshiba, Tokyo, Japan.
Virtual Place Advance, AZE, Tokyo, Japan.
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