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Histologic evaluation of critical size defect healing with natural and synthetic bone grafts in the pigeon (Columba livia) ulna.

Abstract: Fracture and bone segment loss are major clinical problems in birds. Achieving bone formation and clinical union in a fracture case is important for the survival of the bird. To evaluate the efficacy of bone grafts for defect healing in birds, 2 different bone grafts were investigated in the healing of a bone defect in 24 healthy pigeons (Columba livia). In each bird, a 1-cm critical size defect (CSD) was created in the left ulna, and the fracture was stabilized with external skeletal fixation (ESF). A graft of hydroxyapatite (HA) alone (n = 12 birds) or demineralized bone matrix (DBM) combined with HA (n = 12 birds) was implanted in the CSD. The CSD healing was evaluated at 3 endpoints: 3, 6, and 12 weeks after surgery. Four birds were euthanatized at each endpoint from each treatment group, and bone graft healing in the ulna CSD was evaluated by histologic examination. The CSD and graft implants were evaluated for quality of union, cortex development, and bone graft incorporation. Results showed no graft rejection in any bird, and all birds had connective tissue formation in the defect because of the bone graft application. These results suggest that bone defect healing can be achieved by a combination of osteoinductive and osteoconductive bone graft materials for clinical union and new bone regeneration in birds. The combination of DBM and HA resulted in a better quality bone graft (P < .05) than did HA alone, but there was no significant differences in cortex development or bone graft incorporation at 3, 6, or 12 weeks. From the results of this study, we conclude that HA bone grafts, alone or in combination with DBM, with external skeletal fixation is suitable and safe for bone defect and fracture treatment in pigeons.

Key words: critical size defect, ulna, histology, demineralized bone matrix, hydroxyapatite, new bone formation, avian, pigeon, Columba livia

Introduction

Bone grafting is a surgical procedure that is used to restore the function of bony structures. (1,2) The ideal bone graft contains osteoinductive, osteoconductive, and osteogenic properties. (3,4) Corticocancellous grafts are effective for new bone formation in birds. (5-7) Moreover, cortical grafts provide structural support in large bone defects in birds and have been grafted successfully. (8,9) Fracture repair in birds is challenging, (10) and bone defects remain a major clinical problem." If fracture complications are not managed properly, bone healing will not occur, resulting in nonunion, malunion, or delayed union. (10) Therefore, it is important to study the repair of bone fractures and segment loss with bone grafts in birds. Because of the lack of autografts in birds, alternate bone grafts can be used. (9-12) However, studies on the use of bone grafts in avian species are limited. (10)

Demineralized bone matrix (DBM) is a natural bone graft that possesses osteoinductive characteristics. (13) Its use was first reported by Senn (14) in 1889 for the treatment of skeletal defects. Demineralized bone matrix contains bone morphogenic protein 2 (BMP-2), which is in a unique group of proteins within the transforming growth factor [beta] super family. (15) Hydroxyapatite (HA) is a synthetic bone graft substitute that is commonly used for the treatment of osseous defects. (16-18) It has similar chemical properties to the mineral part of natural bone. (16-19) However, an HA graft has no ability to induce new bone formation; therefore, osteoinductive graft material, such as DBM, is mixed with the inorganic HA to induce new bone formation. (20-25) When combined, DBM and HA materials are able to provide new bone formation at the defect site because DBM contains growth factors (3) and HA has osteoconductive ability. Achieving bone formation and clinical union in bird fracture repair is important for the survival of the fractured bone. Therefore, there is a need for a safe and effective treatment with a suitable bone graft material for the bone defect healing process.

The purpose of this study was to investigate the efficacy of the combined use of DBM with HA graft material for bone union in a pigeon model. We hypothesized that the combination of DBM with HA graft would hasten osteogenesis and clinical union in critical size defect (CSD) healing in the pigeon ulna when used in combination with external skeletal fixation (ESF) pins for fracture stabilization. At present, there are few reports on the use of a mixture of DBM with HA for defect healing in birds. The information obtained from this study will be beneficial for treatment of bone defects and fracture fixation in clinical cases of fracture repair in birds.

Materials and Methods

Birds

This study was approved by the Animal Care and Use Committee of the Faculty of Veterinary Medicine, University Putra Malaysia. Six-month old pigeons (Columba livia), weighing (mean [SD]) 289 (12) g, were purchased from a local market and were housed in a well-ventilated room. Birds were allowed a 2-week acclimation period before the study. The pigeons were fed a commercial pigeon feed, and fresh tap water was available ad libitum. The room was cleaned, and feces were removed daily. The study was conducted at the University Veterinary Hospital. Faculty of Veterinary Medicine, University Putra Malaysia, Selangor, Malaysia.

Experimental Design

This study was performed with 24 birds randomly divided into 2 treatment groups, with 12 birds in each group. In group 1, birds (n = 12) were treated with HA, a synthetic bone graft material. In group 2, birds (n = 12) were treated with a combination of DBM, prepared from sacrificed pigeon bone, and HA. The bone grafts were implanted in the left ulna, and defects were stabilized with ESF pins. Four birds from each group were sacrificed at each of 3 endpoints, at 3, 6, and 12 weeks after surgery, respectively, for histologic assessment.

Surgical Procedure

For the bone graft procedure, pigeons were anesthetized with isoflurane administered by face mask at 4%-5%, in oxygen (1-1.5 L/min) for induction. (22) The pigeons were intubated with a semirigid, uncuffed endotracheal tube (2.5-4 mm inside diameter) and were maintained on isoflurane administered at 1.5%-2.5% by a modified Jackson Rees nonrebreathing anesthesia circuit system. (22) After induction, supplemental fluids (20 mL/kg SC lactated Ringer's solution) were administered, and the birds were maintained on a heating pad during the surgical procedure. The surgical site of the left ulna was aseptically prepared with topical chlorhexidine solution, and the surgical area was painted with povidone solution. To create a surgical defect, the wing was extended, and a dorsal approach was used to expose the left ulna. (23) A 2-cm longitudinal skin incision was created between the radius and ulna, and the bone was exposed. Next, a 1-cm long CSD was created at mid-shaft of the left ulna by inserting ESF pins multiple times around the cortex of the ulna with a minipin driver. The bone segment was then removed, resulting in a 1-cm bone defect. (24)

Fracture Fixation and Bone Graft Implantation

In all birds, the ulna fractures were stabilized with 4 type-1 ESF pins (size, 0.045 in; Imex Veterinary Inc, Longview, TX, USA). (25)

HA bone grafts: Twelve birds received synthetic HA grafts (GranuLab Sdn Bhd, Selangor, Malaysia) material. The HA granules (0.2 mL) were implanted in the CSD of the ulna (Fig 1).

DBM and HA combination bone grafts: In the remaining 12 birds, tubular DBM combined with HA (0.2 mL) granules were implanted in the 1-cm defect of the ulna (Fig 2). The DBM tubular graft was prepared from scarified pigeon bone as described by Jalila. (24)

After the ESF pins were placed, bone grafts were implanted, and the fascia, muscles, and skin were closed with 5-0 polyglycolic acid (Safil, Braun Aesculap, Barcelona, Spain). A section of latex Penrose drain tubing was inserted over the top of the ESF pins, parallel to and above the ulna. The latex tubing was then filled with 10 mL of acrylic material with a plastic syringe. Once the acrylic material had hardened completely, the ESF pins extending beyond the acrylic tubing were cut with a pin cutter. (25) After the procedure, butorphanol tartrate (0.2 mg/kg once SC; Torbugesic, Fort Dodge Animal Health, Fort Dodge, IA, USA) was administered. Oxytetracycline ointment (Terramycin, Zoetis, Inc, Kalamazoo, MI, USA) was applied on the incision site, and the wing was wrapped with a figure-of-8 bandage.

Clinical Monitoring and Sample Collection

Throughout the 12-week study, the birds were monitored, and the defect site, ESF pin sites, and incision sites were examined daily for evidence of healing. At the 3 endpoints--3, 6, and 12 weeks after surgery--4 pigeons from each group were euthanatized by administration of pentobarbital (0.3 mL IV). The ESF pins were removed from the ulna, and the bone specimens were collected and fixed in 10% formalin. The bones were decalcified into 5% formic acid for 48 hours. The specimens were then embedded in paraffin, cut into 4-[micro]m slices on the long axis, and stained with hematoxylin and eosin for examination of new bone formation at the defect site. (7) Assessment of the CSD healing with bone grafts was performed according to the modified scoring system by Ozturk et al, (3) as described in Table 1. The scoring data were reported as a mean (SE), and statistical analyses were performed by statistical software (SPSS version 21.0 software, IBM Inc, Chicago, IL, USA).

Histologic Results

HA graft healing-. At 3 weeks after graft implantation, the sections with HA showed no new bone formation. The HA graft was encapsulated with fibrous connective tissue; vascularity was present in the defect site and was contiguous with the host cortex. Of the 4 birds, 50% (2 of 4) of the ulna defects were healed with fibrous tissue covering the graft. No evidence of bone union was present (Fig 3). At 6 weeks after graft implantation, all birds showed 100% healing with fibrous connective tissue. The defects showed fibrous connective tissue surrounding the encapsulated HA graft particles, with some extension of immature bone at the defect site (Fig 4). At 12 weeks after graft implantation, graft particles were encapsulated with fibrous connective tissue in all 4 birds, and early bone growth was observed in the defect (Fig 5).

Graft healing with the combination of DBM and HA: At 3 weeks after implanting the DBM-HA graft combination, the ulna defects in all 4 birds were healed completely with fibrous tissue union (Fig 6). Both grafts were present in the defect site, but there was little graft incorporation between the 2 materials. There was no significant difference between the DBM-HA approach and the HA alone in the quality of union, cortex development, or bone graft incorporation at the 3-week endpoint. At 6 weeks after graft implantation, all ulna defects were healed with fibrocartilaginous union. Graft material was present with some graft incorporation and defect healing (Fig 7). There was no evidence of bone graft rejection in this combination in any bird. At 12 weeks after graft implantation, both grafts were well incorporated with each other and with the host bone in all birds (Fig 8). The tubular DBM graft had formed an extension of the cortex, but the quality of union was not achieved with this combination. At 6 and 12 weeks, the combination DBM with HA showed some progressive graft incorporation when compared with HA alone, but there was no significant difference in mean score values at 12 weeks. At all endpoints of 3, 6, and 12 weeks, no significant differences were observed between the HA group and DBM with HA group in the quality of the union (Table 2). All the defects healed with fibrous tissue union. The HA group showed no significant bone formation or cortex development at any of the 3 endpoints, although the graft was covered by layers of fibrous tissue union and vascularity was observed. There was no rejection of the HA synthetic bone grafts when used alone or in combination with DBM for the repair of ulna defects in the bird models.

Discussion

Reports on the use of bone graft in avian species are few, and specific descriptions of fracture fixation and defect treatment in domestic and wild birds are scarce. (10) The results of this study indicate that fracture fixation and bone graft application, particularly DBM with HA as used in this study, are useful for bone defect treatment in birds when combined with an ESF. Although our findings are similar to the results previously reported in a pigeon model, (6,7) the major finding of our study is the CSD healing with bone grafts forming new bone at the defect site.

The combination of DBM as a natural graft and HA a synthetic graft is a common treatment method but has shown differing results. (3) In our study, the results of the combined DBM and HA, did not show significantly faster bone formation or clinical union in any group at any time. The results of this study are in agreement with the findings of the study by Ozturk et al. (3) In that study, the combined DBM-HA implanted in radial defect repair of a rat model did not show improved defect healing. However, in our study, immature bone formation and defect filling because of the HA granules were observed.

Ideally, bone graft material should have osteogenic, osteoinductive, and osteoconductive properties (26) and should be cost effective. Osteogenic graft material (autograft) contains bone cells, known as osteoblasts, which facilitate the regeneration of new bone. Autogenous bone grafts are believed to be the "gold standard" because they contain the 3 ideal properties. (26) However, because of the limitations of autogenous bone grafts in birds, alternative bone graft materials are needed, such as DBM and HA. (3,6,10) In the DBM-HA group, the osteoinductive (allograft) material induced bone formation because DBM contains bone morphogenic proteins that have the ability to induce new bone formation. (27-28) In 1965, Urist (28) first reported that DBM had osteoinductive properties and contained bone morphogenic proteins. Demineralized bone matrix is suitable as an alternative bone graft that induces bone formation. (24) Hydroxyapatite is a synthetic bone graft substitute that has a structure similar to that of human bone. It provides a scaffold and possesses osteoconductive properties. (29) Osteoconductive bone grafts provide a framework that stimulates bone deposition, but they do not induce new bone formation. (26) In the current study, the histologic findings showed that DBM prepared from pigeon bone and used in conjunction with HA induced some bone formation in the pigeon ulna model. In addition, the quality of the bone union, cortical development, and graft incorporation observed were a result of the osteoinductive efficacy exerted by the grafted tubular matrix in the pigeon model. The findings of bone formation and cortical development with the DBM in the pigeon model in this study are supported by those of Urist et al. who reported that bone induction was a result of the influence of BMPs in the DBM. However, Aspenberg et al (31) disagreed and reported that DBM had no effect on bone formation in skeletal defects in the rabbit model. Jalila (24) found similar histologic confirmation of new bone formation at 6 weeks in a pigeon model and proved that bone formation occurred by endochondral ossification. Additionally, DBM encouraged new bone formation and bridging of bone defects in various animal studies. (32)

In this study, HA, a synthetic bone graft material that is very slowly resorbed, was tested alone for efficacy in CSD healing of the pigeon ulna. Our results showed the HA graft filled the defect area and provided a framework; however, granules were covered by fibrous connective tissue only. If the duration of the study had been extended, the bone graft possibly may have improved the quality of union of the defect healing. (3)

The purpose of this study was to investigate novel bone formation with bone graft substitutes for the successful clinical treatment of fractures and fracture nonunion in birds. Two limitations of this study were the few birds and the duration of study, which decreased its statistical power. This was, in part, because of the costs associated with the birds used and the study material. Although the DBM graft was prepared easily with negligible cost, the HA graft material is expensive. The CSD healing was assessed based on a scoring system, and the criteria for bone graft healing were adopted from a histologic assessment of defect healing in rats. (3) Because this is the first study of this type, to our knowledge, there is no standard scoring system for bone graft healing in bird models. The advantages of DBM when used alone or in combination with other bone graft materials are that it is easy to prepare, is cost effective, and is suitable for bone grafting in bird fracture management. Pigeons are an excellent avian model for bone graft studies and fracture stabilization techniques. Importantly, ESF provides excellent stabilization of fractures in bird models. (33)

The histologic results of our study did not show faster clinical union with the DBM and HA combination compared with HA alone. However, results showed that DBM is effective for CSD healing in the pigeon ulna. Thus, DBM may be an ideal bone graft substitute for repairing fracture and segment loss in caged and wild birds. This study of bone graft healing of CSD in ulnas repaired with an ESF was conducted over 12 weeks, and data were collected only for that limited time. Therefore, a further study should be conducted in birds over a longer period to determine the quality of new bone formation and clinical union in CSD repaired with this technique.

References

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Ahmed Tunio, DVM, MSc, Abu Jalila, DVM, MSc, PhD, Yong Meng Goh, DVM, PhD, Shameha-Intan, DVM, PhD, and Ganabadi Shanthi, DVM, PhD

From the Departments of Veterinary Clinical Studies (Tunio, Jalila) and Veterinary Preclinical Studies (Goh, Shameha-Intan, Shanthi), Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400UPM. Serdang, Selangor, Malaysia.

Table 1. Modified histologic assessment criteria for critical size
defect healing in the pigeon ulna model. (a)

Parameters                        Description                    Score

Quality of        No evidence of fibrous or bone union             0
  union           Fibrous tissue union                             1
                  Fibrocartilaginous union or cartilage union      2
                  Mineralized cartilage and immature bone          3
                    union
                  Bone union                                       4
Cortex            No cortex formed                                 0
  development     New bone formed along external cortex            1
                  Recognizable formation of both outer cortex      2
                    and medullary space
                  Cortex formed and but incomplete bridging        3
                  Complete formation of cortex and bridging        4
                    of defect
Bone-graft        Graft present; no incorporation and no new       0
  incorporation     bone formation
                  Graft present; some incorporation with new       1
                    bone formation; minimal amount of new
                    bone formation
                  Graft present; some incorporation with new       2
                    bone formation; moderate amount of new
                    bone formation
                  Graft present; some incorporation with new       3
                    bone formation. continuous with host bone
                  Good incorporation, ample new bone and           4
                    remodeling
                  Excellent incorporation and advanced             5
                    remodeling

Table 2. Histologic scores (mean [SE]) from critical-size defect
healing across treatment groups in the model of bone graft healing in
the pigeon ulna (n = 24).

                                             HA graft
                           End-point   (n = 12 birds), (a-c)
Parameters                 time, wk          mean (SE)

Quality of union               3           1.0 (0.0) AX
                               6          1.50 (0.28) AX
                              12          2.25 (0.25) AY
Cortex development             3           1.0 (0.0) AX
                               6           1.0 (0.0) AY
                              12           1.0 (0.0) AY
Bone-graft incorporation       3           1.0 (0.0) AX
                               6          1.25 (0.25) BY
                              12          1.50 (0.28) AY

                                           DBM-HA grafts
                           End-point   (n = 12 birds), (a,b)
Parameters                 time, wk          mean (SE)

Quality of union               3          1.75 (0.25) AX
                               6           2.0 (0.0) AY
                              12           2.0 (0.0) AY
Cortex development             3           1.0 (0.0) AX
                               6           1.0 (0.0) AX
                              12           2.0 (0.0) BY
Bone-graft incorporation       3          1.50 (0.28) AX
                               6           2.0 (0.0) AY
                              12          1.75 (0.25) AX

Abbreviations: HA indicates hydroxyapatite; DBM, demineralized bone
matrix.

(a) 4 birds per subgroup at each endpoint time.

(b) Means within row with different (A or B) letters are
significantly different because of treatments.

(c) Means within column with different (X or Y) letters are
significantly different because of the time after surgery.
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Title Annotation:Original Study
Author:Tunio, Ahmed; Jalila, Abu; Goh, Yong Meng; Shameha-Intan; Shanthi, Ganabadi
Publication:Journal of Avian Medicine and Surgery
Article Type:Report
Date:Jun 1, 2015
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