Healing of fractures in osteoporotic rat mandible shown by the expression of bone morphogenetic protein-2 and tumour necrosis factor-α

doi:10.1016/j.bjoms.2004.10.018

British Journal of Oral and Maxillofacial Surgery

Volume 43, Issue 5, October 2005, Pages 383-391

https://doi.org/10.1016/j.bjoms.2004.10.018 Get rights and content

Abstract

We studied the healing process of mandibular closed fractures in osteoporotic rats using specific antibodies to bone morphogenetic protein-2 (BMP-2) and tumour necrosis factor-α (TNF-α). We confirmed the osteoporosis in rats after oophorectomy by micro-CT, and then caused unilateral closed fractures in the mandible and monitored the healing process after 7, 14, 21, and 28 days. Data were compared simultaneously with those from a group of rats that had a sham operation. During healing of the fracture in the osteoporotic group there was a prolonged phase of endochondral ossification, with an increased number of osteoclasts (p < 0.01). Expressions of BMP-2 and TNFα were more pronounced in the osteoporotic group and there was an increase in the number of osteoblasts and TNFα⁺ cells compared with the normal control (p < 0.01). BMP-2 was related to the differentiation of osteoblasts and the higher values of TNFα were correlated with the up-regulation of osteoclasts during the prolonged phase of bone turnover. We conclude that the healing of fractures in osteoporotic bone is delayed about a week compared with controls. In the healing of fractures in osteoporotic bone, there were more osteoblasts and osteoclasts but there was a predominance of osteoclasts probably induced by TNFα. The prolonged phase of bone turnover with osteoclast predominance in the osteoporotic group is suggestive of the cause of delay in the healing of the fracture.

Introduction

Fractures are common complications of osteoporosis in older people, but there have been few studies of the healing process of fractures in osteoporotic bones and most of these have been on long bones, such as tibia and femur, and have concentrated on the biomechanical properties of the phases of healing.1, 2, 3, 4, 5 The healing of a fracture involves a complex series of cellular events and these may be further complicated by osteoporosis. Namkung-Matthai et al. reported that osteoporosis influenced the early period of healing of fractures,³ whereas Kubo et al. suggested that osteoporosis influenced the late period of healing.⁴ Although it is well known that some cytokines and growth factors affect osteoporosis,6, 7, 8 their role in the healing of osteoporotic fractures has not been fully defined. Few reports are available regarding the expression of growth factors or cytokines, but animal studies have indicated that old age and oophorectomy impair both the formation of callus and the final mechanical strength.²

Bone morphogenetic proteins (BMPs) are the most potent osteoinductive growth factors. They are capable of initiating the development of osteoblasts and the formation of bone during embryonic life and during the healing of fractures.8, 9, 10, 11, 12, 13, 14 We have shown previously that BMP-2 participates in all stages of healing of fractures by causing the proliferation and differentiation of osteoprogenitor cells.11, 12 TNF-α is one of the most powerful stimulants of bone absorption and may contribute to osteoporosis by modulating the differentiation and function of osteoclasts.6, 7, 8, 15, 16, 17 Recent evidence suggests that TNF-α may be involved in the regulation of skeletal repair. Knowing their importance in remodelling of bone during skeletal homeostasis, we wanted to find out whether osteoporosis changes the pattern of expression of these cytokines in the coupling of cellular function during bone healing. We, therefore, undertook a study of the healing of fractures in osteoporotic mandibles in rats.

Section snippets

Preparation of osteoporotic rats

Forty female Wistar rats, aged 12 weeks (SLC Co. Ltd., Fukuoka, Japan), with a mean weight of 250–260 g had bilateral oophorectomy (Ovx) or a sham operation (Sx-as a control group) after intraperitoneal injection of pentobarbitone sodium 40 mg/kg (Nembutal, Abbot Laboratories Co. Ltd., Illinois, Chicago, USA) and were fed a low calcium diet (Oriental Bio-Service, Japan). At 24 weeks of age, osteoporosis was confirmed in the Ovx group at the sites of tibia and mandible by cone beam type micro

Confirmation of osteoporosis by three-dimensional micro-CT

Changes in the architecture of the bone were shown in the mandible and tibia of the oophorectomy group (Fig. 1). In the control group, the marrow space in the mandible was occupied mostly by the bony trabeculae with dense cortical bone (Fig. 1a). However, in the oophorectomy group, the mandible showed relatively large marrow spaces as a result of loss of bony trabeculae and more porous cortical bone (Fig. 1b). In the tibia of the control group, the marrow space was largely occupied by the

Discussion

Osteoporosis is a major health problem in elderly people and often results in spontaneous fractures of the skeleton with minimal trauma. In recent years, there have been several studies of fracture healing in osteoporotic animals and most of them focused on the long bones. To understand the influence of osteoporosis on the healing of a fracture of the mandible, we used a rat model of osteoporosis resulting from oophorectomy and a low calcium diet. Osteoporosis was confirmed by micro-CT during

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In addition to baseline decreases in bone mineral density, which predispose post-menopausal females to an increased fracture risk, estrogen depletion is also associated with a blunted reparative response to osseous injury. A murine model of osteoporosis demonstrated that the repair tissue had a higher ratio of undifferentiated mesenchymal cells, a less robust chondrogenic response, and a weaker callus at seven days after injury [74]. At 14 and 21 days following the injury, the osteoporotic mice had delayed bone formation and also had a greater presence of immature woven, rather than lamellar bone, compared to controls.
This review presents a summary of basic science evidence examining the influence of tumor necrosis factor-alpha (TNF-α) on secondary fracture healing. Multiple studies suggest that TNF-α, in combination with the host reservoir of peri-fracture mesenchymal stem cells, is a main determinant in the success of bone healing. Disease states associated with poor bone healing commonly have inappropriate TNF-α responses, which likely contributes to the higher incidence of delayed and nonunions in these patient populations. Appreciation of TNF-α in fracture healing may lead to new therapies to augment recovery and reduce the incidence of complications.
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As the procedure of bone fracture healing is a complex cascade of cellular events and regulation of it still remains to be poorly understood, thus the relationship between fracture healing and osteoporosis is unclear [2]. Evidence have been accumulating and have suggested that osteoporosis does impair the healing procedure of fractures as judged by the formation of callus, mineralization and mechanical properties of healed bones tissues [3–8]. As a result, to improve the regenerated bone tissue quality and to shorten the healing time for bone fracture patient with osteoporosis are two of the most important issues to be addressed by clinicians and researchers, although there are some arguments on the healing of osteoporotic fracture, alterations in bone turnover [9,10].
Treatment of weight-bearing bones fractures with defects is critical for patients with osteoporosis's rehabilitation. Although various tissue engineering methods were reported, the best treating strategy for tissue engineering remains to be identified as the limitation of enhancing the ability of the osteogenetic differentiation potential of seed cell is one of the cardinal issues to be solved. The objective of this study is to investigate the feasibility of applying licochalcone-A (L-A) and bone marrow mesenchymal stem cells (BMSC)-aggregate in bone repairing tissue engineering and further study the biological effects of L-A on the cell aggregate formation and osteogenic properties. 80 female Sprague Dawley rats underwent bilateral ovariectomy were made with a 3.5 mm femurs bone defects without any fixation. These rats were then randomly assigned to five different treatment groups: (1) empty defect (n = 16), (2) CA-LA (n = 16), (3) CA-N (n = 16), (4) CA-L (n = 16), (5) CA-S (n = 16) and 16 female SD rats were treated as a control. Data showed that L-A administrated cell aggregate had a stronger osteogenic differentiation and mineralized formation potential than non-administrated group both in vitro and in vivo. For in vitro study, L-A administrated group had a more significant expression of ECM, osteogenic associated maker in addition with more mineralized area and higher ALP activity compared with the control group. For in vivo study, 3D reconstruction of micro-CT, HE staining and bone strength results showed that newly formed bone in groups administrated by L-A was significant higher than that in Sham group at 2, 4, 8 and 12 weeks after transplantation, especially for groups which was systematically injected with L-A at 8 weeks. Results of our study demonstrated that LA could positively affect cell behavior in cell-aggregate engineering and could be a promising strategy in treating osteoporotic weight-bearing bones fractures with defects.
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Osteoporotic fractures show reduced callus formation and delayed bone healing. Cellular sources of fracture healing are mesenchymal stem cells (MSC) that differentiate into osteoblasts by stimulation with osteoinductive cytokines, such as BMP-2. We hypothesized that impaired signal transduction and reduced osteogenic differentiation capacity in response to BMP-2 may underlie the delayed fracture healing.
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Healing of fractures in osteoporotic rat mandible shown by the expression of bone morphogenetic protein-2 and tumour necrosis factor-α

Abstract

Introduction

Section snippets

Preparation of osteoporotic rats

Confirmation of osteoporosis by three-dimensional micro-CT

Discussion

J. Orthop. Res.

J. Orthop. Res.

Bone

J. Steroid Biochem. Mol. Biol.

Bone

Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod.

Br. J. Oral Maxillofac. Surg.

Br. J. Oral Maxillofac. Surg.

Bone