Evaluation of Autologous Bone Marrow Concentrate along with Hydroxyapatite-collagen for Management of Long Bone Fracture in Canines

Long bone fracture is the most common orthopaedic ailment encountered in routine canine practice. The goal of orthopedics and traumatology has been to return the patient to his pre-trauma state as quickly as possible. Internal fixation is preferred as associated with early immobilization and fast return to function. Bone healing success mainly depends on the presence of adequate mechanical stabilization and biological competence of the body, pro-osteogenic cells, scaffolds that allow bone growth (osteoconduction), growth factors (osteoinduction) and enough vascularization for an effective nutrient supply (Portal et al., 2012). Bone regeneration process may improve effectively and rapidly when stem cells are used. Stem cells are often employed with bone graft/ biomaterials/scaffolds and growth factors to accelerate bone healing at the fracture site (Iquanta et al., 2019).

The collagen-hydroxyapatite (HAp-col) composite is a synthetic bone graft has similar properties to the natural bone and has excellent osteoconductivity as a material for bone regeneration (Siswanto et al., 2020). The use of autologus bone marrow in fracture healing represents a promising method of application of tissue engineering in the orthopaedic field, which avoids many of the complications of the traditional bone grafting method commonly used so far (Sahu, 2018). Bone marrow contains osteoprogenitor stem cells which differentiate into chondroblasts and osteoblasts, cytokines and growth factors (Tseng et al., 2008). The transplantation of osteogenic cells in carrier system (Oryan et al., 2017) is an encouraging new approach to further enhance the process of bone reconstitution and remodeling (Perka et al., 2000). Biomechanical properties of titanium are often considered to be better than those of stainless steel with regard to biocompatibility, modulus of elasticity, osseointegration, corrosion resistance, and magnetic resonance imaging compatibility (Kitsugi et al., 1996).

Hence, the present study was planned to evaluate the efficacy of fracture healing using hydroxyapatite-collagen (HAp-Col) as a bone graft substitute and autologus bone marrow, after the internal fixation by titanium elastic pin.

Present study have been conducted in 12 clinical cases of dogs (above the 6 months of age) having the long bone fracture (femur, humerus and tibia), presented in Department of Veterinary Surgery and Radiology, VCC, Mhow. The study conducted from 2015 to June 2017. All the selected animals were randomly divided into two groups having six animals each. In group I (GI) fractures were immobilized by Internal fixation technique using appropriate size Titanium elastic intramedullary pin (Fig 1a). In group (GII), the stable internal fixation of fracture fragments was achieved as in GI and harvested autologous bone marrow concentrate along with Hydroxyapatite-collagenc (HAp-Col) granules was filled at the fracture gap (Fig 1b,c,d). The distribution of animals, bone involved and type of fracture were depicted in Table 1. A thorough clinical and radiological examination of the animals was performed to localize the fracture and to rule out any neurological deficit. Before planning of surgery health status of the animals was evaluated. All the animals were operated under general anesthesia in both the groups. Standard surgical approaches were made for femur, humerus and tibia fractures. After incision, the fractures ends were exposed, retrieved and immobilized by retrograde-normograde internal fixation technique using appropriate size of Titanium elastic pin in all the animals. In G II animals, 20-25 ml of bone marrow was harvested prior to the orthopedic procedure from iliac crests of same dog in a syringe having anticoagulant EDTA. The bone marrow was harvested using bone marrow needle (Fig 2a) and carefully layered on over equal volume of density gradient media (Hisep Fig 2b). After the centrifugation, the opaque buffy coat was collected which composed of mononuclear cells (lymphocyte, monocyte and mesenchymal stromal/stem cells) with platelets (Fig 3). This processed bone marrow concentrate was mixed with HAp-Col granules and used in G II animals, as described. A ready mead pack of Hap-Col granules (bovine origin) having a particle size of 0.5 to 0.9 mm were used as bone graft substitute as per size of fracture gap. All surgical wounds were closed as per standard. Post operative management followed by dressing of the surgical wound, immobilization of limb with Robert Jones bandage, parentral administration of antibiotic for 7 days and analgesic for 4 to 5 days. Surgical wound was assessed for wound swelling, exudation and healing on the basis of gross examination.



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Parameters of the study
 
The weight bearing and lameness was analyzed clinically in both the groups and scored on day 0, 7, 14, 21, 30 and 60 as described by Cook et al., (1999) (Table 2). Post Radiographic examination (two orthogonal view) at 0 day post operative, 15^th, 30^th, 45^th, 60^th and 90^th day to evaluate apposition, alignment of fracture fragment, callus formation, ossification, organization of callus, reaction of bone to implant if any and stability of implant. The data was analysed using complete randomized design (CRD) as per described by Snedecor and Cochran (1994).

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All surgical wounds were healed by first intention healing in 7 to 15 day post- operatively in all the animals. Surgical wound showed no exudation, swelling and pain on palpation except in one animal of both the groups. These two animals self mutilated the wound resulted slight exudation on 3^rd and 5^th day respectively.  It was evident that titanium elastic nail alone and with HA-Col and autologous bone marrow was compatible to the animal’s body without any adverse effect. Similarly, Preethi et al., (2021) stated that none of the cases showed any adverse tissue reaction to bone graft, thus confirming its complete biocompatibility.

On 0 day before the operation, all the animals were having non weight bearing lameness (score 05). In GI, score was 5.00±0.00 on 3rd day with significant gradual reduction post operative days with 2.16±0.60 at 60^th day (Table 3, Fig 4). On 14^th day, four animals out of six showed toe touching (score - 04). On 30^th day four animals showed moderate weight bearing with noticeable change in gait, scored - 03.  On 60^th day, three animals (A2, A3, A4) scored 02, one animal (A6, A5, A1) scored 0, 01, 05 respectively. In GII, score was 4.66±0.21 on 3^rd day with significant gradual reduction of 1.00 ±0.44 at 60^th day  (Table 3, Fig 4). Five animals showed early limb use with toe touching (score 04) on 3^rd, 7^th and 14^th post operative days respectively. Two animals (C1, C2) scored 01 at 30^th day.  Two animals (C1, C2) scored 0 and 3 animals scored 01 and one animal (C6) scored 03 on 60^th day. The C3 animals was having much bone loss hence healing and weight wearing is delayed in this animal.





In present study GII animals, showed early weight bearing and no observable lameness  in  comparison  to GI animals, in spite of the fact, that few animals had spiral fractures and fractures with bone loss. These findings support radioghraphic finding also, which showed early callus formation. The improvement in limb use may be attributed to the use of HA-Col along with titanium nail which provide adequate stiffness, strain shielding and maintain the stability in load bearing bones (Wahl et al., 2006). Further use seeding of pluripotent cells trigger the patient’s own regenerative mechanism (Raulo et al., 2012). Similarly, Rathod et al., (2014) also reported good functional recovery of limb after using autologus bone marrow concentrate in six dogs. Dwivedi et al., (2008) also observed better functional outcome in groups of animals where bone graft substitute  was used.
 
Post Radiographic evaluation
 
Post surgery 0 day radiographs revealed proper opposition and alignment of fractured fragments with noticeable gap. The alignment was not perfect in two animals of group I (A1 and A5) and one animal (C6) of G II due to bone loss. In G II, interfragmentary gap was appreciated by granular opacity in most of the animals on immediate post surgery radioghaphs. This might be due to of the radiopaque granular structure of graft material at because of its mineral component. This was also in correlation with Tanaka et al., (2017) and Preethi et al., (2021).

Post operative radiographic examination (lateral and cranio-caudal view) in group I  showed moderate periosteal reaction and reduction of inter fragmentary gap in four animals (A1, A2, A5 and A6) except in two animals (A3 and A4-Fig 5) from 15^th to 30^th day. More proliferative external callus was observed in two animals (A1 and A6 -Fig 6) at 30^th day. On 45^th day, further reduction of fracture gap was observed in all animals. On 60^th day obliteration of fracture line in two animals  (A5 and A6) with moderate external callus in one animal (A5-Fig.07). Union on one side of cortex was observed in remaining four animals (A1, A2, A3 and A4) at 60th day. On 90th day complete fracture line was completely obliterated by external callus in one more animal (A2), while in the three animals (A1, A3 and A4) radiolucent line was visible and healing in progress.







In Group II postoperative day radiographs (lateral and cranio-caudal view) revealed mild periosteal reaction and progression of bridging callus with reduction of fracture gap in five  animals, while in  (C6), no periosteal reaction was noticed on 15^th  day.  On 30^th day, radioghraphs showed union of one aspect of cortex in two dogs (C2 - Fig 8 and C5), further reduction of gap with callus formation in  two animals (C1 and C4). In C3 animal union on cranial cortex was complete where as on other cortices  progression of collar of callus was observed (Fig 9). In C6 animal presence of periosteal reaction was noticed with visible fracture gap.





On 45^th day, there was complete consolidation of callus with thickening of cortex at fracture site in one dog (C2: Fig 8). On 60^th day, complete obliteration of fracture line was revealed in two animals (C1 and C5). In C3 animal union was complete on cranial cortex, with faint radiolucent line on caudal cortex (lateral view) and size of callus was bigger (Fig 9). In C6 animal union of the over ridden part was observed however a spur of bone on caudal cortex was noticed. In C4 animal radiolucent space on one aspect of bone while union on other aspect of bone was observed. On 90^th day, radiographs showed continuity of medullary cavity and remodeling in all five animals. Union was in progress in C6 animal with presence of radiolucent gap on one cortex.

Fracture union was earlier and with minimum periosteal callus formation in five animals in G II as compared to G I. This might be due to that bone marrow concentrate contains MSCs, osteoblast and osteoinductive growth factors such as bone morphogenic proteins (BMP), fibroblast growth factor (FGF) and transforming growth factor (TGF) which enhance the fracture healing (Knight and Hankenson, 2013). Further seeding of these elements on scaffolds, boost the osteogenic/inductive potential, may also used as alternative for autogenous bone grafting (Knight and Hankenson, 2013; Jaiswal et al., (2021). This was in corroboration with the findings of Rathod et al., (2014) and  Nnaji et al., (2017) who  observed that autologous bone marrow aspirates treated groups exhibited a more matured osteiod seams in terms of callus proliferation and mineralization in transverse tibia fractures in Nigeria indigenous dogs. It is speculated that bone marrow cells, most likely stem and progenitor cell, react vigorously and promote active bone formation around and inside the HAp-Col implant (Taniyama et al., 2015). The Histological examination of bone augmentation ability of  HAp-Col composite in a rat calvaria defect model revealed that the entire area of the gap was filled with prominent newly formed bone intermingled with the HAp-Col (Ozava et al., 2018). In addition to bone conductivity HAp/Col might have released the calcium ion for bone formation and growth factor (Nishikawa et al., 2005).       

In the present study ABMC was used, as it can bypass the time-consuming and difficult process of MSCs expansion, which must be expanded in vitro for several weeks to reach a sufficiently high number of cells for transplantation, which not only delays the treatment but also increases costs and contamination risk related to culture (Gali et al., 2016).

The case wise variation of fracture healing in the present study might be due to other contributing factors which also affect the bone regeneration. The factors such as age, weight, location, fractures gap, a shift that occurs in the bone, movement of the animal, post-operative immobilization, bandaging, post operative pain management, cooperation of veterinary patient and care by the pet owner are the factors which also affect the rate of healing in Veterinary patient.

The early radiographic healing and encouraging weight bearing at different intervals in treated group explains logically that titanium elastic nail as implant with HAp-Col graft and autologous bone marrow was compatible to the animal’s body without any adverse effect.  The faster fracture healing is always a need in Veterinary patient, the present study advocated good alternative to full fill better outcome of orthopaedic surgery. Hence it is concluded that Hydroxyapatite-collagen composite with autolologus bone marrow concentrate hasten the fracture healing in comparison to intramedullary pin alone in fracture cases of dog. This technique improves the early limb use by the patient and satisfy the owner.

The authors declare that they have no conflict of interest.