Introduction
Segmental defects in long bones are challenging. Defects may arise due to resection of neoplasia, debridement of osteomyelitis or atrophic non-union following fracture repair. Limb-salvage options include distraction osteogenesis, vascularised bone grafts, combinations of demineralised bone matrix, autogenous or allogenous grafts with or without use of bone morphogenic proteins, similar growth factors or stem cells, and staged repair following induction of an ectopic membrane around the defect.
Description of the case
A 9y7m old entire female Coton de Tulear presented with a 4-month history of left coxofemoral and right femorotibial joint luxation following a road traffic accident. A concurrent comminuted left tibial fracture had been managed with a plate, lag screws and cerclage. Radiographs revealed atrophic non-union of the tibial fracture with a central diaphyseal defect of ca. 22% of the tibial length. The owner declined euthanasia or amputation as options. Coxofemoral joint stabilisation and femorotibial joint arthrodesis were performed, and a grafting procedure planned for management of the tibial defect.

Following a caudomedial approach to the tibia, loose implants were removed and the muscle tissue filling the defect bluntly separated to create space for grafting. The proximal and distal bone ends were opened. Fresh cancellous autograft from the ipsilateral iliac crest was mixed with a commercial surface-modified allograft (Fortigen-P 1cc, Veterinary Transplant Services) and diced clotted platelet-rich fibrin (A-PRF, Puremed) before liquid platelet-rich fibrin (i-PRF, Puremed) was added to form the graft into a flexible solid implant. The surgical sites were closed routinely.

Postoperative radiographs showed good filling of the defect with a mixture of radiopaque and radiolucent material. Due to unrelated health problems, the patient was euthanased 5 weeks postoperatively. Radiographs obtained post-mortem showed maintenance of the graft volume with a reduction in radiopacity. Ultrasonography identified a hyperechoic graft structure extending out from the level of the proximal and distal cortices. On dissection, a solid, flexible tissue was found joining the proximal and distal fragments with the gross appearance of fibrocartilaginous tissue.

Following preservation in formalin, the tibia was decalcified for 10 weeks in 22% formic acid before cutting into sagittal sections and embedding in paraffin wax. Representative sections were stained with HE, Masson Trichrome, Safranin O, Gram and von Willebrands stains.
Non-mature osteoid with a woven collagen structure dominated the bone ends towards the segmental defect. Cartilage formation was also observed in this area. Within the defect, multiple necrotic bone pieces, with empty lacuna, were seen surrounded by fibrosis. Despite the massive fibrotic response a regular pseudoarthrosis formation was not observed. No evidence of infection was observed. Angiogenesis and vascularization was observed between the allograft fragments.
Conclusions
Modern low-speed platelet-rich fibrin preparations contain large numbers of activated platelets and leukocytes (1), and slowly release growth factors such as PDGF, VEGF, TGF and IGF-1 over a period of 10 days (2). Platelet-rich fibrin can improve soft-tissue healing (3) and has been shown to speed bone healing in oral surgery (4).

Normally, a segmental bone defect will heal due to new bone formation coming from differentiation of pluripotent stem cells located in the end- and periosteum. However, a reduced amount of oxygen will result in differentiation towards chondrocytes and, thereby, cartilage formation which over time will be remodeled to bone tissue by endochondral ossification. However, this is a slower healing process in comparison to direct intramembranous ossification. Thus, the presence of cartilage indicates non-optimal vascularization, which may reflect delayed vascular ingrowth into the relatively large graft volume.

There are limited data available on healing of large mixed grafts in vivo; however, the initial loss of radiopacity seems consistent with other case reports. The maintenance of graft volume and evidence of vascularization suggests that progression to full healing may have occurred with more time. Potentially, this mixed platelet-rich fibrin and allograft/autograft approach may permit simple, single-stage repair of segmental or other large defects.
Bibliography
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2. Kobayashi E, Flückiger L, Fujioka-Kobayashi M, Sawada K, Sculean A, Schaller B, Miron RJ. Comparative release of growth factors from PRP, PRF, and advanced-PRF. Clin Oral Investig. 2016 Dec;20(9):2353-2360. doi: 10.1007/s00784-016-1719-1. Epub 2016 Jan 25. PMID: 26809431.
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4. Choukroun J, Diss A, Simonpieri A, Girard MO, Schoeffler C, Dohan SL, Dohan AJ, Mouhyi J, Dohan DM. Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part V: histologic evaluations of PRF effects on bone allograft maturation in sinus lift. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006 Mar;101(3):299-303. doi: 10.1016/j.tripleo.2005.07.012. PMID: 16504861.

Address for Correspondence
Dr. James Miles - Faculty Of Health And Medical Sciences, University Of Copenhagen Department Of Veterinary Clinical Sciences, Dyrlægevej 16, 1870 Frederiksberg C, Denmark - Phone 20570840 - E-mail jami@sund.ku.dk