**7. Consolidation period**

When finished the distraction period, the external fixator is maintained in order to confer stability to the regenerated tissue and allow its mineralization and consolidation [3]. In this period, the FIZ starts to mineralize and the central region becomes radiographically sclerotic. During the following weeks, the columns of regenerated tissue become homogeneous as the primary bone tissue is replaced by Haversian bone. In small animals, it will take around 8–12 weeks to form a new cortex and medullary cavity [7, 10, 36, 37].

Over time, the longitudinally oriented trabeculae are transformed into transverse plaques, incorporating the collagen template [7, 8, 10, 36]. The bone microcolumns are covered with osteoblasts that actively produce osteoid. Each column is accompanied by a large vascular channel that preserves the ideal distance in order to allow a diffusion gradient between cells. The activity of bone cells during a DO is similar to what occurs during a fracture healing [8, 28, 38]. However, what happens at a tissue level, the continuous recruitment and activation of cells capable of producing and reabsorbing bone, significantly exceeds a fracture healing process [39]. Simultaneously during the extensive bone production, a remodeling occurs, producing *porosis* of the bone cortex and margins of the regenerated bone. After 2–3 months in animal and 4–6 months in humans, the Havers channels, through which blood vessels and nerves pass, are formed [11, 12, 38]. The bone marrow components, in the regenerated bone appear after 4 months. Bone remodeling is complete after 5–7 months in small animals and 12–24 months in humans. After this remodeling period, the mechanical integrity of the cortex is restored [1].

The consolidation period after a DO was investigated in a 20-year retrospective study, based on 115 animals submitted to a corticotomy and application of a circular external fixator [2]. The authors concluded that the radius requires less time to consolidate than the tibia and presented the hypothesis that this occurs due to during the march, the radius bears weight in a parallel axis and the tibia carries the weight through an oblique axis [40]. Another hypothesis is based on the fact that dogs bear around 60% of their weight on the thoracic limbs, therefore the weight carried by a radius is superior to what is supported by a tibia [41–43]. In experimental rabbit model, the effects of an angular osteotomy after a DO were studied, revealing that a 30° axis deviation at the distraction focus resulted in a 50% reduction of the regenerated bone [44]. In humans, the femur is referred to consolidate faster than the tibia [45]. Ilizarov's original technique described that the consolidation period before fixator removal should be 1 month/cm of new regenerated bone [46].

**99**

*Distraction Osteogenesis: Biological Principles and Its Application in Companion Animals*

The bone formation can be controlled through tomography, scintigraphy, ultrasound, and bone densitometry; however, radiographs continue to be the more practical method to determine the consolidation efficiency and when the bone is

Nowadays, numerous studies are focused on the molecular mechanism behind a DO, such as, the genetic expression of the bone morphogenic proteins (BMPs 2 and 4) which is induced by tension and mechanic stress [47, 48]. Other molecular signs, such as the insulin-like growth factor type 1 (IGF-1), transforming growth factor beta (TGFb) and fibroblastic growth factor 1 (FGF-1), associated with osteoblast proliferation and its differentiation from mesenchymal cells were identified at the distraction site [49, 50]. Another study identified that it is possible to accelerate the ossification process during a DO by administrating a recombinant homologous growth hormone [51].

Tissue engineering approaches have already been applied to promote bone regeneration at DO. The use of mesenchymal stem cells (MSCs) from autologous origin, isolated from the bone marrow or adipose tissue, or from human xenogeneic origin has been described in different animal models of DO like rats, rabbits, pigs, dogs, and sheep with promising outcomes; nonetheless many of the mechanism behind the process remain to be investigated, for example, the recruitment and activation of MSCs upon the initial stimulation by surgical trauma. Growth and differentiation factors, hormonal proteins, and pharmacological agents can be added in combination to the distraction site. The number of cells transplanted is measured in cell over DR (number of cells in millions divided by total distractions in millimeters) ranged from 0.03 to 5.00 M/mm. The cells can be injected after the distraction period, loaded into scaffolds and then transplanted to the distraction focus during the osteotomy or during the latency period [52]. Genetically modified MSCs have also been evaluated using growth and differentiation factors including bone morphogenetic protein-7 (BMP-7) [53, 54], BMP-2 [55, 56], basic fibroblast growth factor (bFGF) [57], transforming growth factor-β (TGFβ), and insulin-like growth factor-1 [58], as well as genes encoding transcription factors, such as osterix (Osx) [59, 60] and runt-related transcription factor 2 (Run×2) [61] with distinct

Djasim and collaborators created guidelines for craniofacial DO. They collected data from dog, rat, sheep, goat, rabbit, pig, and rhesus monkey models based on data from previous craniofacial DO studies. With the premise that intramembranous bones of the skull have a different vascular supply compared to long bones, therefore DO parameters suitable for orthopedic DO might be suboptimal for craniofacial DO. They concluded that a latency period may not be necessary in some animals such as sheep and pigs, and in others it produces far better-quality bone tissue as seen in rats and rabbits. They reaffirmed that the ideal distraction rate should be 1 mm/day, which should be halved when using rats and determined an ideal consolidation period of 6–8 weeks [62]. Another review in mandibular DO showed that the latency period ranged from 2 to 7 days. The distraction rate ranged from 0.4 to 2.4 mm/day. The total distraction gap obtained ranged from 3.2 to 20 mm, and

DO can also provide an option for limb sparing surgery upon resection of primary bone tumors, such as osteosarcomas. The bone transport osteogenesis (BTO),

*DOI: http://dx.doi.org/10.5772/intechopen.89157*

ready to remove the external fixation system [1, 38].

effects reported in the improvement of bone regeneration [52].

the consolidation period ranged from 4 days to 10 weeks [52].

**9. Distraction osteogenesis after oncologic surgery**

**8. Craniofacial distraction osteogenesis**

#### *Distraction Osteogenesis: Biological Principles and Its Application in Companion Animals DOI: http://dx.doi.org/10.5772/intechopen.89157*

The bone formation can be controlled through tomography, scintigraphy, ultrasound, and bone densitometry; however, radiographs continue to be the more practical method to determine the consolidation efficiency and when the bone is ready to remove the external fixation system [1, 38].

Nowadays, numerous studies are focused on the molecular mechanism behind a DO, such as, the genetic expression of the bone morphogenic proteins (BMPs 2 and 4) which is induced by tension and mechanic stress [47, 48]. Other molecular signs, such as the insulin-like growth factor type 1 (IGF-1), transforming growth factor beta (TGFb) and fibroblastic growth factor 1 (FGF-1), associated with osteoblast proliferation and its differentiation from mesenchymal cells were identified at the distraction site [49, 50]. Another study identified that it is possible to accelerate the ossification process during a DO by administrating a recombinant homologous growth hormone [51].

Tissue engineering approaches have already been applied to promote bone regeneration at DO. The use of mesenchymal stem cells (MSCs) from autologous origin, isolated from the bone marrow or adipose tissue, or from human xenogeneic origin has been described in different animal models of DO like rats, rabbits, pigs, dogs, and sheep with promising outcomes; nonetheless many of the mechanism behind the process remain to be investigated, for example, the recruitment and activation of MSCs upon the initial stimulation by surgical trauma. Growth and differentiation factors, hormonal proteins, and pharmacological agents can be added in combination to the distraction site. The number of cells transplanted is measured in cell over DR (number of cells in millions divided by total distractions in millimeters) ranged from 0.03 to 5.00 M/mm. The cells can be injected after the distraction period, loaded into scaffolds and then transplanted to the distraction focus during the osteotomy or during the latency period [52]. Genetically modified MSCs have also been evaluated using growth and differentiation factors including bone morphogenetic protein-7 (BMP-7) [53, 54], BMP-2 [55, 56], basic fibroblast growth factor (bFGF) [57], transforming growth factor-β (TGFβ), and insulin-like growth factor-1 [58], as well as genes encoding transcription factors, such as osterix (Osx) [59, 60] and runt-related transcription factor 2 (Run×2) [61] with distinct effects reported in the improvement of bone regeneration [52].

### **8. Craniofacial distraction osteogenesis**

Djasim and collaborators created guidelines for craniofacial DO. They collected data from dog, rat, sheep, goat, rabbit, pig, and rhesus monkey models based on data from previous craniofacial DO studies. With the premise that intramembranous bones of the skull have a different vascular supply compared to long bones, therefore DO parameters suitable for orthopedic DO might be suboptimal for craniofacial DO. They concluded that a latency period may not be necessary in some animals such as sheep and pigs, and in others it produces far better-quality bone tissue as seen in rats and rabbits. They reaffirmed that the ideal distraction rate should be 1 mm/day, which should be halved when using rats and determined an ideal consolidation period of 6–8 weeks [62]. Another review in mandibular DO showed that the latency period ranged from 2 to 7 days. The distraction rate ranged from 0.4 to 2.4 mm/day. The total distraction gap obtained ranged from 3.2 to 20 mm, and the consolidation period ranged from 4 days to 10 weeks [52].

#### **9. Distraction osteogenesis after oncologic surgery**

DO can also provide an option for limb sparing surgery upon resection of primary bone tumors, such as osteosarcomas. The bone transport osteogenesis (BTO),

*Clinical Implementation of Bone Regeneration and Maintenance*

**7. Consolidation period**

cortex and medullary cavity [7, 10, 36, 37].

are appropriate, and that the ideal DR must be based on individual characteristics

The distraction rhythm (DRy), the number of lengthening times made per day, influences the quality and quantity of the regenerated bone tissue and is important in the preservation of the soft tissue integrity during the procedure [7, 10, 34]. Ilizarov observed, using a canine model, that by using an automatic distractor capable of performing a DRy of 60 times per day, would produce a significantly better quality of bone when compared with DRy of 1–4 times per day. A DR of 1 mm a day with a DRy of 4 times a day was determined as ideal [7, 10]. In a goat model of tibia lengthening, DRy of 1, 4, and 720 times per day would not affect the strength, rigidity, and histomorphometric characteristic of the regenerated bone and would not affect the somatosensory potential of the peripheral nerves [34, 36]. Another study using the same animal model concluded that increasing the DRy would result in less muscular degeneration [39]. In Veterinary Medicine, it is recommended DRy of 2–4 times per day [9, 18, 31, 33].

When finished the distraction period, the external fixator is maintained in order to confer stability to the regenerated tissue and allow its mineralization and consolidation [3]. In this period, the FIZ starts to mineralize and the central region becomes radiographically sclerotic. During the following weeks, the columns of regenerated tissue become homogeneous as the primary bone tissue is replaced by Haversian bone. In small animals, it will take around 8–12 weeks to form a new

Over time, the longitudinally oriented trabeculae are transformed into transverse plaques, incorporating the collagen template [7, 8, 10, 36]. The bone microcolumns are covered with osteoblasts that actively produce osteoid. Each column is accompanied by a large vascular channel that preserves the ideal distance in order to allow a diffusion gradient between cells. The activity of bone cells during a DO is similar to what occurs during a fracture healing [8, 28, 38]. However, what happens at a tissue level, the continuous recruitment and activation of cells capable of producing and reabsorbing bone, significantly exceeds a fracture healing process [39]. Simultaneously during the extensive bone production, a remodeling occurs, producing *porosis* of the bone cortex and margins of the regenerated bone. After 2–3 months in animal and 4–6 months in humans, the Havers channels, through which blood vessels and nerves pass, are formed [11, 12, 38]. The bone marrow components, in the regenerated bone appear after 4 months. Bone remodeling is complete after 5–7 months in small animals and 12–24 months in humans. After this

remodeling period, the mechanical integrity of the cortex is restored [1].

The consolidation period after a DO was investigated in a 20-year retrospective study, based on 115 animals submitted to a corticotomy and application of a circular external fixator [2]. The authors concluded that the radius requires less time to consolidate than the tibia and presented the hypothesis that this occurs due to during the march, the radius bears weight in a parallel axis and the tibia carries the weight through an oblique axis [40]. Another hypothesis is based on the fact that dogs bear around 60% of their weight on the thoracic limbs, therefore the weight carried by a radius is superior to what is supported by a tibia [41–43]. In experimental rabbit model, the effects of an angular osteotomy after a DO were studied, revealing that a 30° axis deviation at the distraction focus resulted in a 50% reduction of the regenerated bone [44]. In humans, the femur is referred to consolidate faster than the tibia [45]. Ilizarov's original technique described that the consolidation period before fixator removal should be 1 month/cm of new regenerated bone [46].

such as age, osteotomy site, and need for angular correction [12, 35].

**98**

an adaption of the DO technique, is used to preserve limb function after resection of large segmental bone defects. Briefly, after the tumor excision, an osteotomy is performed on the proximal bone segment, creating a distraction focus and resulting on a small portion of healthy bone which will act as the transport segment. Then using an external ring fixator, this segment is slowly distracted in the defect direction, creating regenerated tissue resulting in bone union and a bridged effect. The distraction should continue until 3 days after the segments touch in order to compress the distal healthy bone, turning it metabolically active, this process is called docking. Successful docking is achieved when the transport segment heals with the adjacent bone. It is possible to predict the timing of docking by measuring the distance between the two bones on radiographs and calculating the number of days required to achieve contact based upon the DR. The surgeon should consider grafting when the transport segment is approximately less than 0.5 cm from contact with the docking site. When owners strongly wish to avoid further surgery, autologous bone marrow graft, obtained from the patient, could be mixed with canine demineralized bone matrix (DBM) into the docking site, acting as a vehicle of mesenchymal stem cells and osteoinductive signals [63, 64].

BTO surged as an alternative to cadaveric allograft bone transports, which was seen as the main limb salvage procedure in alternative to amputation; however, complications such as non-union, graft fracture, and infection are referred in the literature. One study reported that nearly one half of the patients develop infection may be associated with the lack of intrinsic blood supply surrounding the allograft and tumor resection area [65]. This high complication rate could lead to soft tissue lost, chronic pain, non-weight-bearing lameness, multiple surgeries, and even amputation [66–68].

The extent of the needed tissue resection can be planned based on detailed radiographs, scintigraphy, or ideally, a preoperative magnetic resonance imaging (MRI) which will be essential to help build the fixator frame, and assess the extent of the tumor involvement within the bone marrow, as it commonly exceeds the extent detected on radiographs. The surgeon should plan to excise at least 2 cm of bone proximal to the most proximal extent of tumor identified [63]. The patients with better outcome in DO upon oncologic surgery are those whose tumors are located in the distal radius or ulna, due to bigger pancarpal arthrodesis success [64]. The best candidates for limb salvage are those whose tumors involve less than 50% of the bone and have minimal soft tissue involvement. In theory, the extension of tumor treatable with this technique is limited to by the ability to achieve appropriate margins. There must be at least enough bone remaining in the proximal radius to create a transport segment and to place three wires above the transport segment. Dogs with infected allografts after prior limb salvage surgery are suitable candidates for bone transport, unless they have had recent radiation therapy. Patients with pathologic fracture, multicentric neoplasia, metastasis or severe intercurrent health conditions should not be considered as favorable candidates [63].

After the tumor resection, BTO is similar to a conventional DO, a latency period of 3 days is sufficient unless the dog is receiving chemotherapy treatment. In those cases, a longer latency period of up to 7 days should be applied. Afterwards, the distraction should consist on a DR of 1 mm/day and DRy of 2–4 times a day. Immediately after a chemotherapy session, distraction should be ceased for 3 days before being restarted. This waiting period can be eliminated if the patient shows signs of premature consolidation. Radiographic reassessment should be made every 10–14 days during bone transport and every 3–4 weeks after docking. Some animals may require higher DR to prevent premature consolidation, while other may require occasional "resting" period of 2–5 days. If the regenerated bone begins to be progressively thinning, ductile, and with "hourglass" shape in radiographs, it is

**101**

*Distraction Osteogenesis: Biological Principles and Its Application in Companion Animals*

mineralization of regenerate bone, and time of docking [63].

2–3 months to evaluate for metastasis or local tumor recurrence [63].

osteoblast may counteract the inhibitory effects of chemotherapy [75].

likely that distraction osteogenesis is negatively affected by radiation [75].

The complication associated with a DO include muscular contractures, subluxations, vascular and nerve lesions, premature or delayed consolidation, and even bone non-union. The placement of intramedullary pins near a nerve or large caliber blood vessel can lead to damage on those structures during the lengthening [1].

Neuromuscular lesions are rarely associated with lengthening phases unless they exceed 30% of the limb size [33, 82, 83]. Subluxations are associated with muscular contractures and can occur in substantially excessive lengthening. Premature consolidations can be prevented with an adequate DR and DRy. Delayed consolidations are multifactorial but are more commonly reported cases where an excessive DR was applied. Bone non-union is on its own associated to an infectious process. A strict radiographic protocol allows a control, assessment, and readjustment in order

One of the limitations of this technique is the long period necessary for the newly formed bone to mature, mineralize, and consolidate. The external fixators must be kept until the end of the consolidation period in order to confer the stabil-

**10. Complications of distraction osteogenesis**

to avoid these complications [1].

ity necessary to obtain better quality bone [3].

recommended to slow or stop distraction for a few days. Conversely, if the wires in the transport segment begin to bend in the direction of distraction, the DR should increase for 2–3 days (1.5–2 mm/day) to help preventing premature consolidation. The radiographs and fixator should also be regularly evaluated to adjust DR and DRY, document broken wires, evidence of tumor recurrence, progression of

Fixators should be removed when peripheral bridging of the central radiolucent zone within the regenerate tissue is evident on radiographs, the columns of new bone are mineralized, and when the docking site union has been achieved. It can be difficult to evaluate the stability of the docking site before removing the fixator frame, due to the concentration of metal hardware. If doubt exists regarding effective union, the fixator removal can be delayed provided the patient is not having substantial soft-tissue problems. Osteosarcoma patients should be restaged every

Negative effects of systemic chemotherapy and radiotherapy are reported in distraction osteogenesis [69–71]. Chemotherapy likely impedes the osteoblasts to cope with the increased functional demand and compromises bone callus formation during a DO. High dose chemotherapy reduces colony forming unit fibroblast by 50% in the bone marrow, by 10% on cortical bone, and 20% in trabecular bone [72]. However, two studies compared patients who underwent DO with and without chemotherapy and it did not demonstrate any difference in the bone healing process between patient groups [73, 74]. The hypothesis proposed is that DO's effect on

In humans, some bone sarcomas, most commonly Ewing sarcoma, adjuvant radiotherapy is a treatment option, being reported adverse effects of radiation therapy on up to 74% of patients [76–78], namely wound healing problems, infection, muscle and joint contracture, ankylosis, osteitis, non-union, pathological fractures, tendon adhesion, and radiation induced sarcoma [76–79]. In a rabbit tibia model, it was demonstrated that radiation exposure decreases the quantity and quality of regenerate and angiogenesis during a DO [80]. Also, in a rabbit mandible model, it was found that osteogenesis is delayed after a 60-Gy dose of radiation, even though viable osteoblast and osteocytes may still be present [81]. It is therefore

*DOI: http://dx.doi.org/10.5772/intechopen.89157*

#### *Distraction Osteogenesis: Biological Principles and Its Application in Companion Animals DOI: http://dx.doi.org/10.5772/intechopen.89157*

recommended to slow or stop distraction for a few days. Conversely, if the wires in the transport segment begin to bend in the direction of distraction, the DR should increase for 2–3 days (1.5–2 mm/day) to help preventing premature consolidation. The radiographs and fixator should also be regularly evaluated to adjust DR and DRY, document broken wires, evidence of tumor recurrence, progression of mineralization of regenerate bone, and time of docking [63].

Fixators should be removed when peripheral bridging of the central radiolucent zone within the regenerate tissue is evident on radiographs, the columns of new bone are mineralized, and when the docking site union has been achieved. It can be difficult to evaluate the stability of the docking site before removing the fixator frame, due to the concentration of metal hardware. If doubt exists regarding effective union, the fixator removal can be delayed provided the patient is not having substantial soft-tissue problems. Osteosarcoma patients should be restaged every 2–3 months to evaluate for metastasis or local tumor recurrence [63].

Negative effects of systemic chemotherapy and radiotherapy are reported in distraction osteogenesis [69–71]. Chemotherapy likely impedes the osteoblasts to cope with the increased functional demand and compromises bone callus formation during a DO. High dose chemotherapy reduces colony forming unit fibroblast by 50% in the bone marrow, by 10% on cortical bone, and 20% in trabecular bone [72]. However, two studies compared patients who underwent DO with and without chemotherapy and it did not demonstrate any difference in the bone healing process between patient groups [73, 74]. The hypothesis proposed is that DO's effect on osteoblast may counteract the inhibitory effects of chemotherapy [75].

In humans, some bone sarcomas, most commonly Ewing sarcoma, adjuvant radiotherapy is a treatment option, being reported adverse effects of radiation therapy on up to 74% of patients [76–78], namely wound healing problems, infection, muscle and joint contracture, ankylosis, osteitis, non-union, pathological fractures, tendon adhesion, and radiation induced sarcoma [76–79]. In a rabbit tibia model, it was demonstrated that radiation exposure decreases the quantity and quality of regenerate and angiogenesis during a DO [80]. Also, in a rabbit mandible model, it was found that osteogenesis is delayed after a 60-Gy dose of radiation, even though viable osteoblast and osteocytes may still be present [81]. It is therefore likely that distraction osteogenesis is negatively affected by radiation [75].
