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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 50  |  Issue : 2  |  Page : 57-62

Callus formation in bone fractures combined with brain injury in rat


1 Institute of Emergency and Critical Care Medicine, National Yang Ming University, National Taiwan University Hospital, Taipei, Taiwan
2 Institute of Emergency and Critical Care Medicine, National Yang Ming University, National Taiwan University Hospital; Department of Emergency, Taipei Veterans General Hospital, Taipei, Taiwan

Date of Submission14-Feb-2016
Date of Decision04-Apr-2016
Date of Acceptance06-Jun-2016
Date of Web Publication18-Apr-2017

Correspondence Address:
Hsin-Chin Shih
Department of Emergency, Taipei Veterans General Hospital, No. 201, Sector 2, Shih-Pai Road, Taipei
Taiwan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/fjs.fjs_20_17

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  Abstract 

Objective: The objective of this study was to determine the speed of bony union and the serum levels of biomarkers in the setting of bone fractures combined with brain injury.
Materials and Methods: In this study, Sprague–Dawley rats were randomized into four groups: sham, brain injury, bone fracture, and bone fracture plus brain injury groups. The serum levels of biochemical markers, namely, nerve growth factor (NGF), Wnt-3a, Dickkopf-related protein-1, receptor-activator of NF-κB ligand, and adrenocorticotropic hormone (ACTH), were measured on the days 1, 3, 7, and 14 following injury. Bony union was evaluated using radiographs every week for 6 weeks.
Results: Compared with the brain injury group and bone fracture group, the radiographs of the bone fracture plus brain injury group revealed enhanced callus formations in week 2. From week 3, the callus formation did not differ significantly among the groups. The serum levels of the biomarkers varied at different time points. The serum levels of NGF on days 1 and 3, Wnt-3a on days 3 and 14, and ACTH on days 1, 3, and 7 were significantly higher in the bone fracture plus brain injury group than in the bone fracture group.
Conclusions: Brain injury increases callus formation in simultaneous bone fracture. Considering the time point, early NGF, Wnt-3a, and ACTH elevation might be associated with early callus formation enhancement. The results indicate that these brain injury-induced biomarkers might play crucial role in accelerating bone healing.

Keywords: Bone fracture, brain injury, Callus


How to cite this article:
Chen YP, Shih HC. Callus formation in bone fractures combined with brain injury in rat. Formos J Surg 2017;50:57-62

How to cite this URL:
Chen YP, Shih HC. Callus formation in bone fractures combined with brain injury in rat. Formos J Surg [serial online] 2017 [cited 2019 Mar 21];50:57-62. Available from: http://www.e-fjs.org/text.asp?2017/50/2/57/204659


  Introduction Top


Bone fracture healing is a complex and systematic process. Clinical research has revealed that the rate of new bone formation around a fracture increases when patients have traumatic brain injury (TBI) and a bone fracture simultaneously.[1] The mechanisms of such phenomena have not been fully studied. Some researchers have suggested that certain osteogenic factors that cross the damaged blood–brain barrier (BBB) following TBI might be responsible.[2] The presence of systemically circulating osteogenic factors following TBI may promote osteogenesis and bone healing of associated fractures.[1],[2] According to molecular biological research, various growth factors are initially released around the fracture following injury.[2] The sequential release of growth factors acts on bone and cartilage cells, finally resulting in bone healing. However, the control mechanisms and interaction among these factors remain elusive.[3] Research has demonstrated that several factors potentially influencing mesenchymal or osteoprogenic proliferation are present or upregulated in the serum of patients following TBI; such factors include nerve growth factor (NGF), receptor-activator of NF-κB ligand (RANKL), Wnt, Dickkopf-related protein-1 (DKK-1), and adrenocorticotropic hormone (ACTH).[1],[2] In the present study, we investigated the speed of bone healing in a rat animal model with concomitant brain injury and explored the biomarkers responsible for osteogenesis.


  Materials and Methods Top


Animals

The research protocol was approved by the Institutional Animal Care and Use Committee of National Yang-Ming University, Taiwan. Sprague–Dawley rats were acclimated for at least 8 weeks before the experiments. During the study period, the rats were provided standard rat chow and water ad libitum; lighting was maintained on a 12 h cycle, and the rats were acclimated under standard laboratory conditions at 22°C ± 2°C and 50% ± 10% humidity. During the experiments, the rats were randomized into four groups: the control group (received anesthesia without injury manipulation), TBI group, bone fracture group, and bone fracture plus TBI group (n = 6 per group). All rats were handled at room temperature. After the experiments, the rats were cared for in the Laboratory Animal Center of National Yang-Ming University and were sacrificed on days 1, 3, 7, and 14 after injury.

Ethical approval

The study was conducted in accordance with the Declaration of Helsinki and was approved by the local ethics committee of the institute. Informed written consent was obtained from all patients prior to their enrollment in this study.

Traumatic brain injury

TBI was induced as previously reported.[4],[5] Briefly, the Sprague–Dawley rats were anesthetized using 30 mg/kg zoletil and 10 mg/kg xylazine, and the skull was exposed laterally. Punctures were made at the point between the lambda and bregma as well as between the central suture and left temporal ridge using a 5 mm trephine. After the brain injury procedure, the scalp was closed using 4-0 nylon sutures (Ethicon, Somerville, NJ). The rats were then returned to their cages and continuously monitored.

Femoral fracture model

Both femoral osteotomy and fixation were performed in the same manner as previously reported.[6] In brief, a transverse osteotomy was made at the midshaft of the left femur, and the fracture fragments were reduced and fixed using an intramedullary stainless steel wire (1.5 mm diameter, Synthes, Switzerland). The wire was cut on the surface of the intercondylar groove to avoid motion restriction of the knee joint. The rats were permitted unrestricted activity after recovery from anesthesia.

Radiological measurement of callus volume

We used the Perkins volume formula to evaluate the fracture callus volume.[7] The rats were anesthetized, and radiographs of the anterior–posterior and lateral positions of the fractures were taken. The callus volume was calculated using the following formula: 2π·R 1·(R 2 − R 1)·L, where R 1 = femur radius, R 2 = radius of the bone plus callus, and L = callus length. This formula provides a quantifiable estimation of the callus. The callus volume was evaluated on days 7, 14, 21, 28, 35, and 42 following injury.

Analysis of serum biochemical markers

For the measurement of serum biochemical markers, blood samples were collected at 1, 3, 7, and 14 days after injury. Peripheral blood (5 mL) was collected from the tail vein, and the serum was obtained by centrifugation of the blood at 1500 rpm for 10 min at 4°C for the removal of the cellular components. Finally, the serum was stored at −80°C until analysis. The serum DKK-1 level was measured using a commercially available ELISA assay kit (Enzo Life Sciences, USA). The serum levels of NGF, Wnt-3a, RANKL, and ACTH were analyzed using a Milliplex MAP Multiplex assay (MILLIPLEX, Merck KGaA, Darmstadt, Germany).

Statistical analysis

Analysis of variance with the nonparametric Kruskal–Wallis test was performed to determine the between-group differences. Results with P < 0.05 were considered statistically significant.


  Results Top


Radiology

The callus volumes were measured to determine whether TBI affects fracture healing. Callus formations were observed at the fracture sites from week 1 onward. The callus volume was significantly larger in the bone fracture plus TBI group than in the bone fracture group in week 2 (P < 0.05). Callus formation was identical among the experimental groups from week 3 onward [Figure 1].
Figure 1: (a) Radiographs of the fractures over 6 weeks. (b) Measurements of the callus volumes were conducted for the traumatic brain injury plus bone fracture group and the bone fracture group. Significant differences were observed in week 2 (*P < 0.05 traumatic brain injury plus bone fracture group vs. bone fracture group)

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Serum biochemical markers

Nerve growth factor levels

The NGF levels increased following injury. The NGF levels were significantly higher in the TBI group and TBI plus bone fracture group than in the bone fracture group on days 1 and 3, and they were the same from day 7 onward [Figure 2].
Figure 2: Serum nerve growth factor levels increased in the traumatic brain injury group as well as the traumatic brain injury plus bone fracture group on days 1 and 3. The traumatic brain injury plus bone fracture group had significantly higher levels than the bone fracture group on days 1 and 3. The traumatic brain injury group had higher nerve growth factor levels than did the bone fracture group on day 3 (*P < 0.05, traumatic brain injury group or traumatic brain injury plus bone fracture group vs. the bone fracture group)

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Wnt-3a levels

The serum Wnt-3a levels increased after injury. The Wnt-3a levels were significantly higher in the TBI plus bone fracture group than in bone fracture group on days 3 and 14 [Figure 3].
Figure 3: Serum Wnt-3a levels were significantly higher in the traumatic brain injury plus bone fracture group than in the bone fracture group on days 3 and 14. The traumatic brain injury group had higher Wnt-3a levels than did the bone fracture group (*P < 0.05, traumatic brain injury group or traumatic brain injury plus bone fracture group vs. bone fracture group)

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Dickkopf-related protein-1 levels

Although serum DKK-1 protein levels tended to be higher in the TBI plus bone fracture group in comparison with the other groups, no significant difference was observed among the three groups at the different time points [Figure 4].
Figure 4: Serum Dickkopf-related protein-1 levels tended to be high in the traumatic brain injury plus bone fracture group. No significant difference was observed among three groups at the different time points

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Receptor-activator of NF-κB ligand levels

Although the serum RANKL levels tended to be lower in the TBI plus bone fracture group in comparison with the other groups, the nonparametric test revealed no significant difference among the groups at four time points [Figure 5].
Figure 5: Serum receptor-activator of NF-κB ligand levels tended low in the traumatic brain injury plus bone fracture group at the different time points. No significant difference was observed among three groups

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Adrenocorticotropic hormone levels

The serum ACTH levels increased following injury. The ACTH levels were significantly higher in the TBI plus bone fracture group than in the bone fracture group on days 1, 3, and 7. However, the ACTH levels decreased significantly on day 14 in the TBI plus bone fracture compared with those in the bone fracture group [Figure 6].
Figure 6: Serum adrenocorticotropic hormone levels after injury. Compared with the bone fracture group, the traumatic brain injury plus bone fracture group exhibited a significantly higher adrenocorticotropic hormone levels on days 1, 3, and 7 and exhibited lower adrenocorticotropic hormone levels on day 14 (*P < 0.05, traumatic brain injury plus bone fracture group vs. bone fracture group)

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  Discussion Top


Fracture healing is a complex event that involves the coordination of various processes.[8] Several clinical studies have indicated that the healing process is more rapid for fractures combined with brain injury than for fractures alone. However, the underlying mechanisms remain unclear. The BBB is a semipermeable barrier that separates the circulating blood from the extracellular fluid in the central nervous system (CNS).[9] The selective permeability of the BBB impedes the passage of many molecules. Hence, the BBB prevents possible infection and monitors the flow of CNS contents into the systemic circulation. When the BBB is damaged, molecules that are normally secluded within the CNS can cross the damaged BBB and diffuse into the systemic circulation. It is possible that any insult to the CNS is associated with BBB dysfunction and is responsible for the release of osteogenic factors from the CNS, which may cumulate in increased osteogenesis.[1],[2],[10] Several studies have detected the presence of humoral factors promoting osteogenesis, such as epidermal growth factor, NGF, arachidonic acid, leptin, neuron-specific enolase, and S100-B, in blood from TBI patients.[4],[9],[11],[12] In animal models of TBI and fracture, humoral factors released after TBI have been shown to play a role in the early development of heterotopic ossification and fracture healing through the expansion of mesenchymal progenitors.[2],[10] In addition to BBB damage, several reports have revealed that pituitary gland dysfunction induces the abnormal secretion of growth factors into the circulation, which may influence cell regeneration and promote fracture healing.[2],[11],[13]

NGF is a growth factor that promotes peripheral nerve regeneration in rats. Thus, NGF is crucial for the regeneration of injured peripheral nerves. In the present study, the serum NGF levels were significantly higher in the TBI plus bone fracture group than in the single fracture groups on days 1 and 3, which is similar to the results of Zhuang et al.[11] NGF may be induced by brain injury and secreted into the peripheral blood. NGF promotes the phosphorylation of osteoblasts and accelerates fracture healing. Previous studies have also shown that NGF receptors are expressed in bone-forming cells and are involved in the regulation of bone formation in vivo.[3],[14]

A recent study revealed that the Wnt signaling pathway is activated during postnatal bone regenerative events such as ectopic endochondral bone formation and fracture repair.[15] Furthermore, Wnt-3a – a member of the Wnt gene family, which is activated during bone formation and fracture repair – can influence bone mass and bone development.[15],[16] Moreover, NGF may regulate myelination and activate the Wnt/β-catenin cell signaling pathway in Schwann cells.[17] In the current study, the Wnt-3a levels increased significantly on the day 3 and in week 2 in the TBI plus bone fracture group. However, we found no report that provides direct evidence that NGF activates osteoblasts through the Wnt-3a pathway.

The DKK-1 gene encodes the Dkk-1 protein, which is a member of the dickkopf family and acts as an antagonist of Wnt. Dkk-1 plays a crucial role in skeletal development and bone remodeling. DKK- 1 binds to the Wnt receptor of the osteoblast and blocks Wnt-3a signaling. Inhibition of DKK-1 may enable the progress of bone cell differentiation.[18],[19] In the present study, the DKK-1 levels tended to be upregulated in the TBI group but did not reach statistical significance. Moreover, no correlation was observed between the Wnt-3a levels and DKK-1 levels.

RANKL, a factor produced by mature osteoblasts, is responsible for the coordination of bone formation and bone resorption.[19] Osteoprotegerin is a secreted decoy receptor that is a key regulator of RANKL signaling, and this receptor antagonizes osteoclast differentiation.[8],[19] Bone turnover is a balance between bone absorption by osteoclasts and bone formation by osteoblasts. This balance may be reflected by the RANKL levels. RANKL signaling is one of the signaling pathways responsible for the differentiation of osteoblasts into osteoclasts. In the present study, although the nonparametric test revealed no significant statistical differences, the RANKL levels decreased in the TBI plus bone fracture group, potentially indicating lower osteoclast activity, which contributes in part to enhanced callus formation. However, the RANKL levels were not decreased in the groups ofsingle injury. Some unknown factors may be released in the combined injury (bone fractures combined with brain injury). Additional studies should be conducted to clarify this finding.

Circulating ACTH is secreted by the hypothalamus. ACTH has been reported to induce osteoblasts.[20] In the present study, the serum ACTH levels were significantly high in the TBI plus bone fracture group. In addition, a previous study observed elevated ACTH levels after trauma, surgical stress, inflammatory response, and increased metabolism.[21]

The present study has certain limitations. First, the expression of biomarkers in local tissues, such as in the brain, callus, or soft tissue surrounding the fracture site, was not measured. Such measurements might have provided more direct evidence of their contributions. Second, although our injury model is consistent among rats, we did not measure the injury degree in the brain using a microscope, which may reflect the level of BBB impairment.


  Conclusion Top


Brain injury increases callus formation in simultaneous bone fracture. Early NGF, Wnt-3a, and ACTH elevation are associated with early callus formation enhancement. These results indicate the possible importance of specific biomarkers induced by brain injury in accelerating bone healing.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]



 

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