Demineralized bone matrix : A cheap solution for ulna defect healing in a Pigeon

Purpose of this study was to find out whether demineralized bone matrix (DBM) could be used for ulna defect healing in pigeons. For this study total 12 pigeons were purchased then divided into three groups and placing four birds per group. Study duration was 3, 6 and 12 weeks post bone grafting. A 1-cm bone defect was created in left ulna only then DBM graft was placed in between the ulna defect and graft was covered with host tissue. Surgical wound was closed with Vicryl 30 suture material. Then ulna fracture was fixed with four external skeletal fixation pins (type-1). The DBM graft was prepared from donor pigeon bone using hydrochloric acid (0.6M). DBM bone graft healing was evaluated with radiography and histology techniques. Results of radiographic assessment showed no bone graft healed after 3 and 6 weeks post-surgery, but after 12 weeks of healing, 50% birds showed bone union. While, histological observations showed DBM graft was successfully incorporated in defect site and formed some new bone formation after 3 and 6 weeks of surgery. After 12 weeks of graft healing there was increased new bone formation and good graft incorporation. DBM graft is easy to prepare and could be useful for defect healing in birds because it have bone morphogenic proteins (BMP-2). This study investigated that DBM graft showed potential effect for new bone formation and best graft for defect healing in pigeon.


Introduction
Most of the bone grafts are tested on animal model in medical and veterinary research.Rat, rabbit mouse and birds are commonly used as animal models.Ultimate goal of the bone graft study is to solve the bony defect problems in human and animal patients.Demineralized bone matrix (DBM) is a bone graft material used for reconstruction of bone defect healing.Segmental defect in birds is an orthopaedic problem and bony segment loss will not heal unless if treated by bone graft substitute.However, filling of bony defect could help to reduce the gap and provide environment for faster fracture healing.In bone grafting autogenous bone graft is considered as "gold standard" for repair of bone defects [1].But harvesting of the autogenous bone graft from another donor site is a problem.Therefore, DBM graft plays a key role in the replacement of allograft and is a best choice for defect healing On the source of mentioned results in various animals, it was decided to conduct an experiment on pigeons using DBM.Thus, DBM is one of the suitable bone graft for avian bone defect healing [4, 14] which has potential for bone defect repair.On the basis of the above reported facts it was decided to investigate the new bone formation capability of avian DBM graft for ulna defect healing in pigeons.It was hypothesized that DBM could be most suitable bone graft substitute for bird bone defect healing.Therefore, DBM implanted in a pigeon ulna defect.Bone graft healing was assessed by radiology and histology.Results of this study would be helpful for surgical management of bony defects using DBM graft in domestic and wild birds.

Methodology
This experimental study was conducted in the Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, University Putra Malaysia.The processed demineralized bone matrix (DBM) of avian source was tested using adult pigeons of both sex and ages ranging from 5-6 months old and mean body weight 295.5 gram body weight were used in this study.They were kept in an experimental house and two birds housed in a cage.Every day all birds were fed with commercial bird feed and water ad libitum.Food intake and water consumption was not recorded.During the study birds were ringed in legs and numbered from 1 to 12.All birds received thoroughly physical examination and observed daily for any disease.The experimental protocols performed on all pigeons were reviewed and approved by the animal care and use committee of the Faculty of Veterinary Medicine, University Putra Malaysia under approval number 10R118.

Preparation bird deminaralized bone matrix (DBM)
For this study bone graft study DBM was prepared by sacrificing the two adult pigeons using I/V injection of sodium pentobarbital, where long bones were aseptically removed from carcass of pigeons.The collected bones were cleaned with sterilized distilled water and frozen at -80C for two days.All bones were washed then defatted in ethanol and decalcified in hydrochloric acid (HCL) 0.6M for three days with daily change.When pH of the decanting acid was lower than 0.9 then demineralized bones were removed from acid and bones were cut into 1-cm in a tubular shape.In next, DBM grafts were washed in de-ionized water for 5-minutes and neutralized in phosphate buffered saline to maintain the pH [6].DBM grafts were kept in sterile glass vials and lyophilized under freeze-drying machine and then stored at 6 °C till next application [8, 14, 15].Surgical procedure on birds For this study total 12 pigeons were randomly divided into three groups with four birds in each.While, ulna defects were implanted with a DBM graft only and graft healing was evaluated at 3, 6 and 12 weeks postoperatively.Anesthesia was performed in all birds using Isoflurane with the combination of oxygen.A face mask was used and oxygen was given first for 2 minutes afterwards general anesthesia was induced with 5% Isoflurane and oxygen flow of one litre /minute through a non-breathing circuit system.Then 2-mm sized un-cuffed endotracheal tube (Cole-type) was placed in trachea of the birds.After intubation aesthesia was maintained at 2.5% concentration of Isoflurane and mixed with Oxygen at 0.5 liters/minute [16].After anesthesia feathers were plucked around left ulna and operative area was scrubbed using chlorexidine solution and 10% Povidoneiodine.All pigeons were placed on dorsal recumbency and covered with surgical drape and operative site was exposed.All bones were approached dorsally to expose the ulna bone and similar procedure was performed by [17].A 2-cm longitudinal skin was incised and ulna bone located.After exposure of ulna bone a 1-cm ulna defect was made at mid shaft in each bird and fracture was fixed with four external skeletal fixations (ESF) positive profile threaded 0.045 size pins.All ESF pins were inserted on one side of ulna using Jecobs chuck.After fracture fixation DBM graft (1-cm) tubular piece was implanted in between the defects (Figure 1a).Lastly, skin and muscles was sutured with Vicry 3/0 in a simple interrupted pattern.Subsequently a Penrose tube 10-cm long was inserted over the ends of the ESF pins and tube was filled with acrylic mixture (acrylic powder 10 gram and5ml acrylic liquid; Jorvet™, Jorgensen, Inc., USA).Afterwards, Trans ESF pins over the acrylic column was then cut one by one using a pin cutter (Figure 1b).This study evaluated the birds for three endpoints i.e.3, 6 and 12 weeks post-surgery using radiology and histology techniques.

Bone graft healing assessment Radiology
For all birds radiograph of ulna was exposed on the day of DBM implantation and after every endpoint to assess the defect healing using scoring system.Presence of fracture line was scored as visible or not visible.Radiographic union was considered as a complete bone graft healing (Figure 2).

Statistics
The results from radiological and histological data were expressed as means and standard errors ( SE) from all birds and then compared by Kruskal-Wallis non-parametric ANOVA.All the tests were conducted at 95% confidence level and statistical analysis was conducted using SPSS 21.0 v (IBM ® SPSS ® Inc., USA).

Results
Present study was conducted on 12 birds for the duration of (3, 6 and 12 weeks) postoperative time.There was no any bird expired during observation time and no bird also showed DBM graft rejection.

Radiology findings
Radiographic results of defect healing (Table 1) showed that none of the birds showed bone union during 3 and 6 weeks of study.There was no significant difference found in radiological parameters in between any bird (p>0.05).Radiographic examination showed a radiolucent appearance in between defects and defect remained empty.As shown Figure 2d, DBM graft healing after 12 weeks, radiological examination have showed (2/4) birds showed clinical union and remaining two (50%) birds have no sign of clinical union.There was a significant difference of defect healing with DBM at 12 weeks (P0.05) as to compare 3 and 6 week respectively.
x, y, z . Means within column with different superscripts differed due to time (P0.05

Histology findings
Histological findings after 3 weeks of DBM healing (Table 2) showed that defect healed with the fibrous connective tissue.A small amount of new cortex had developed and there was some graft incorporation with the host bone.However, there was no significant difference (p>0.05) in terms of quality of union, cortex development and bone-graft incorporation at 3 weeks in any bird (Figure 3a).Over 6 weeks of DBM graft showed advanced defect healing, defect was covered with connective tissue and graft incorporation with the host bone in all birds.The defects healed with fibro-cartilage union and formation of new bone next to the bone graft.The graft was present and the matrix had achieved vascularisation at the defect site.However, there was no significant difference (p>0.05) in conditions of quality of union, cortex development and bone graft incorporation at 6 weeks of study and complete clinical union had not yet been achieved in this group (Figure 3b).After 12 weeks post-surgery period of DBM graft healing (Figure 3c) all the defects showed good bone graft incorporation with the host bone as compared to 3 and 6 weeks graft healing.A few sections revealed a cartilage area and some bone formation in the matrix.Mesenchymal cells had developed on the surface of the tubular graft.Histological findings of present study showed that the defects were healed by the newly developed bone with a trabecular form and DBM were covered with connective tissues, had formed fibro cartilaginous union and there was extension of new bone, and minute quantities of new bone were observed.No significant radiological and histological changes were observed during 3weeks and 6weeks of DBM healing in pigeons.Meanwhile, significant difference (P<0.05) in terms of the quality of bone union, cortex development and graft incorporation was observed after 12 weeks of the study.

Discussion
Fracture due to trauma is a common surgical problem in domestic and wild birds.In this study demineralized bone matrix (DBM) avian source was used for ulna defect healing.
Present study was planned to investigate the DBM and fixed in between the bone defects of pigeon ulna.On the basis of evaluating the DBM for defect healing, this study was divided in three endpoints (3, 6 and 12 weeks) post-operatively.DBM graft has proved to be useful to achieve bone union in birds.This study showed that defect treated with DBM is useful and found a good bone graft substitute on radiological examination after 12 weeks of graft healing.However, there was no significant bone formation found during 3 and 6 weeks post-surgery in any bird.None of the birds showed any inflammatory reaction but improved bone graft healing was observed when the time was extended.There was a significant [1].DBM is one of the bio-materials is used for bone formation [2].It is prepared in powder, chipped and tubular form that has been used in animal and in bird model experiments [3-4].It is a cheap bone graft and prepared by removing mineral from bone using hydrochloric acid (0.6N) and remaining matrix has ability to induce new bone formation [5].When DBM graft is implanted in defect site, healing occurs by three biological process such as ostogenesis, osteoinductionand osteoconduction [6].The osteoinductive property of DBM supports the undifferentiated mesenchymal cells into the osteoblast cells, osteoconduction provide frame work and osteogenesis is the process of bone generation from bone forming cells [7].It is well known that fracture repairing is a challenge in birds [6] and there is limited literature on the use of bone grafts in birds.The main reason for a bone graft study is to provide the best treatment in cases of bone fragment loss due to fracture in birds.It is useful bone graft substitute because it induces new bone formation and provides mechanical support for fracture healing [8].With the implantation of DBM in defect site it reduces the healing time.Many studies reported that DBM provides a framework for bone formation and also advances the differentiation of mesenchymal cells into new bone forming cells by a five-day process [9-10].DBM is used in humans for bone graft surgery [11].Furthermore, in birds the bones such as the humours and femur are pneumatised because of such a unique structure, the bones are brittle and therefore many birds are at risk of fractures [12].In short, the sources of autogenous bone grafts in birds are limited [13].

Figure 1 .
Figure 1.(a) Arrow shows implanted DBM graft in between the ulna defect of a pigeon (b) Postoperative photograph shows acrylic column and Trans ESF pins were cut in a pigeon ulna

Figure 2 .
Figure 2. Radiological findings of DBM for pigeon ulna defect healing (a) Postoperative VD view showing there was good bone alignment and reduction with ESF (b) Arrow showing no evidence of bone union at 3-weeks post-surgery (c) Arrow showing some evidence of callus formation and extension of new bone at 6-weeks post-surgery (d) Arrow showing visible evidence of bone union at 12-weeks post-surgery and ESF removed (e) Circle showing complete graft attachment with host and bone unionPost-mortem and histologyFor histological examination birds were euthanized using I/V injection of sodium pentobarbital and post-mortem examination was conducted.Implant was movable at 3 and 6 weeks of healing.However, ulna was stable in 12 weeks period of healing.Bone graft along with soft tissue covering were removed from sacrificed birds and processed for histology.All specimens were fixed in 10% neutral buffered formalin solution for four days on daily change [19] then decalcified using 5% formic acid for three days with daily changes till complete removal of calcium [14].Decalcified bones

Figure 3 .
Figure 3. Histological findings of DBM graft healing in a pigeon ulna (a) DBM healing at 3weeks, Arrow showing graft DBM graft incorporation.(b) DBM healing at 6weeks.Arrow showing implant incorporation with host bone (c) DBM graft incorporation at 12 weeks post implantation.Arrow showing DBM graft and new bone formation.BM=Bone marrow formation in DBM.HB=Host bone, SM=Skeletal muscle.(10x, H & E stain) Swartz SM, Bennett MB & Carrier DR  (1992).Wing bone stresses in free flying bats and the evolution of skeletal design for flight.Nature 359: 726-729.