The Effect of Low Molecular Weight Heparins on Fracture Healing

INTRODUCTION

The most common cause of death and morbidity in the trauma patient are the thromboembolic complications [1, 2]. Deep venous thrombosis occurs in 50-70% of patients submitted to acute fixation of proximal femur fractures, in multiple fractured patients, and in those presenting with spinal cord trauma when no prophylactic measure is performed. The most studied and used drug in prophylaxis of thrombosis is heparin [3]. Heparin- induced osteoporosis after long- term and high- dose usage of unfractioned heparin (UFH) has been considerably investigated too [4-6].

Low molecular-weight-heparin (LMWH) was developed in the decade of 80s to overcome some of the major disadvantages of unfractioned heparin. During these years, a new question has been raised as to whether LMWHs carries the same side effects compared to standard UFH [7]. Unfractioned heparin and LMWHs have been shown to have several harmful effects on bone, causing osteoporosis and enhancing the bone resorption. Moreover, both of them seems to increase calcium loss and reduce bone turnover [8-11]. However, there are few studies reporting the effects of LMWH on bone repair after fractures. Due to the fact that a great number of trauma patients with fractures regularly receive LMWH, it is essential to examine whether LMWH may have an adverse impact on fracture healing process.

LMWHs: HISTORICAL PERSPECTIVE AND OVERVIEW

Heparin, a highly sulfated glycosaminoglycan, was discovered to have antithrombotic properties by Mc Lean nearly 100 years before [12, 13]. Brinkhous et al. [14] then proved that heparin is an indirect anticoagulant, requiring a plasma cofactor- antithrombin (AT) III or simply referred as AT. The heparin/AT interaction causes a conformational change in AT. The activated AT then inactivates the coagulation enzyme thrombin (factor IIa) and other proteases involves in blood clotting most notably factor Xa and factor IXa. The molecular weight of heparin varies from 5000 to 30000 with a mean molecular weight of 15000 (approximately 50 monosacharide chains) [12].

LMWHs are polysulfated glycosaminoglycans which are almost one third the molecular weight of UFH and they are derived from UFH by chemical or enzymatic polymerization. LMWHs have an average molecular weight of 4000 to 5000 (about 15 monosacharide units per molecule). Due to the fact that LMWHs are prepared by different methods of depolymerization of heparin, they differ in many factors such as pharmacokinetic properties, anticoagulant profiles and they are not clinically interchangeable [13]. Thus, LMWHs are a group of similar but different drug agents. The various LMWHs approved for use are shown in (Table 1). It is estimated in a large number of studies that the use of LMWHs is the best way to prevent dangerous clinical complications like venous thrombosis or acute pulmonary embolism [13]. Especially in pregnancy the use of LMWHs is considered the golden rule in anticoagulant therapy. Also LMWHs are used in obstetrics for the prevention of firsttrimester loss or the placental dysfunction in women suffering from thrombophilia [15]. Finally, other advantages of LMWHs over standard heparin are the longer plasma half life and the fact that they have more predictable anticoagulant response. Also, it is of great importance that they require less intense laboratory monitoring [16].

FRACTURE HEALING PROCESS

Fracture healing is a complicated process that involves the coordination of a sequence of many biological events [17]. This process requires the action of appropriate cells such as osteoblasts, osteoclasts, fibroblasts, chondroblasts, macrophages, monocytes and lymphocytes. Moreover, a number of genes play an important role to this process including growth factors, transcription factors and genes that control matrix production and organization [18]. In 1975, Cruess and Dumont [19] suggested that fracture healing consisted of three phases the one following the other: an inflammatory phase, a reparative phase and a remodeling phase. In 1989, Frost [20, 21] suggested five stages of fracture healing: stage of hematoma, stage of granulation tissue, stage of callus formation, stage of modeling and stage of remodeling. For convenience, fracture healing will be described as recognized by Cruess and Dumont stressing the fact that the reparative phase is a combination of many processes (the stage of granulation tissue and the stage of callus formation) (Table 2). Moreover, although we can divide the fracture healing process into phases, we must draw attention to the fact that what we describe happened in one phase is noticed in the following one as well, and that events seen in phases that follow, were marked in a previous phase.

LMWHs AND BONE BIOLOGY

The high dose and long term use of heparin has been recognized by scientists as a risk factor for the development of osteoporosis and osteoporotic human fractures [22-24]. Data on osteoporosis associated with LMWHs are contradictory [25, 26]. Most of the reported cases of symptomatic osteoporosis with spontaneous fractures occurred in pregnant women treated with UFH recurrent thromboembolism [27]. Dalhman et al. [4] reported that a rate 2,2% of patients receiving UFH had osteoporotic spinal fractures. Several factors can cause heparin induced osteoporosis. Some of the most important are the variations in the metabolism of vitamin D and the high rate of bone resorption. Another factor is the decreased activity of osteoblast either the overactivation of osteclasts [28, 29].

Osteblasts and osteclasts are responsible for bone homeostasis (bone formation and resorption) respectively. The use of UFH may disturbs the maintain balance between these two major cell types and causes heparin- induced osteoporosis.

Moreover, osteoblasts number and function is very important for the integration of endoprosthetic implants, the permanent remodeling processes of bone but also for the process of fracture healing. Recent studies prove that osteoblasts arise from mesenchymal stem cells [30]. Mesenchymal stem cells (MSCs) are extremely proliferative stromal cells that have the ability of forming bone and cartilage. They play an important role in bone homeostasis because they are an appropriate source for osteoblasts [31, 32]. Furthermore, except from osteoblasts, mesenchymal stem cells may differentiate in vitro or in vivo, into variety of cell type’s inclunding: chondryoblasts, adipocytes and muscle cells [30]. Which factors are responsible for regulating the differentiation of mesenchymal stem cells are to large extent under investigation. However, current studies suggest that a number of cytokines are responsible for the physiological differentiation of (MSCs) into mature osteblasts including interleukin (IL), IL 1, IL-6, IL-11 and tumor necrosis factor (TNF) α [30]. It is possible therefore, that heparin alters the expression of one or more of these cytokines [33]. By the same mechanism maybe LMWHs impair bone metabolism.

In addition, osteoblast proliferation in humans is affected by many growth factors, insulin-like growth factors (IGFs) I and II included [34]. In human osteoblasts we can find a surface binding protein for IGFs (IGF binding protein 5) to which heparin could bind [35]. Heparin binding happens competitively with the binding of other IGFs and that might explain the impaired regulation of osteoblast differentiation and possible changes in the formation of bones after long-term treatment with heparin [36].

Finally, osteoclasts play an important role on bone biology. Osteoclasts are multinucleated cells that resorb bone tissue and they are developed from macrophages. They are regulated by several factors including 1, 25 dihydroxyvitamin D3, parathormone (PTH), interleukin 11 (IL-11). Also osteoclasts formation requires the presence of receptor activator of nuclear factor kB (RANKL) and macrophage colony stimulating factor (M-CSF) [37-39].

This review presents the studies about the effects of the LMWHs on bone biology (bone cells and bone metabolism) and highlights the underlying mechanisms by which LMWHs may impair fracture healing (Fig. 1a, b).

LITERATURE REVIEW

DISCUSSION

It is difficult to assess the true effect of LMWH on fracture healing process. Based on literature research, there are no studies on the role of LMWHs on fracture healing in humans. The difficulties to evaluate the effects of LMWHs on the skeletal system of humans becomes from two serious reasons. First because the number of patients receiving LMWHs for long periods is limited. Second because experiments on fracture healing in humans contains many risks and complications. For these reasons, the most important evidence for the role of LMWHs on fracture healing comes from in vitro and animal studies.

The results of the animal studies (rats, rabbits) vary from no impairment of LMWHs on healing [43-46] to impairment on the healing process [41]. The fracture healing process in animal studies was assessed by histological, radiological and mechanical methods. Histological methods performed in all animal studies. But the gold standard method for evaluating fracture healing process, the mechanical tests, performed in only three studies [41, 44, 64]. In that way, because of the small number of animal studies, the different methodology used and the disagreement in results, no conclusive results can be drawn. Moreover, it has been suggested that animal fracture models do not offer any applicability to human fracture healing process. In most animals the cell biology, biochemistry, healing process and therapeutics needs, differ from those of humans [58].

As it concerns the effect of LMWHs to osteoblasts in vitro studies the results are contradictory. Many in vitro studies have reported a reduced osteoblast- inhibition by LMWH compared to UFH [31, 49]. On the other side, Muir JM et al. demonstrated that both heparin and LMWH had the tendency to decrease bone formation by decreasing osteoblast number and activity, but that only heparin increases osteoclast differentiation and activity [48]. As it concerns the effect of LMWHs to osteoclasts it seems from the limited number of studies that LMWHs increase the number and the activity of osteoclasts, stimulating bone resorption [39]. It is certain that in vitro studies have the disadvantage that can only mimic in vivo conditions but not entirely describe them.

On the basis of the results from the current study, two other factors seem to play a crucial role on fracture healing process. The hematoma at the inflammatory phase of fracture and the angiogenesis. Street et al. [41] found that LMWH increased interfragmentary hematoma. Also in another study Street et al. has shown that the high potassium concentration of fracture site hematoma is cytoxic to endothelial cells and osteoblasts [59]. Therefore, increased fracture site hematoma volume may have deleterious effects on fracture healing process. On the other side, it is unclear whether the hematoma improves fracture healing by increasing the supply of osteoprogenitor cells [60, 61].

But not only fracture hematoma can be affected by LMWHs also angiogenesis can be influenced [62]. Vascular endothelial growth factor (VEGF) plays a major role in the process of angiogenesis during the fracture repair especially at the early phase. Norby et al. reported that the use of LMWHs suppressed the VEGF- induced angiogenesis [63]. Furthermore, the study of Sarahrudi et al. was the first comparative systematic measurement of VEGF serum levels in patients receiving enoxaparin. In this study no significant difference of the VEGF expression was observed [57].

Moreover, effects of LMWHs on mechanical properties of unfractured bones have not been intensively studied. There are only a few experimental reports on their effect on bone strength [55].

In clinical practice, pregnancy is one of the few situations in therapeutics where LMWHs are recommended for prolonged use, but it is not clear from the data if LMWHs are responsible for osteoporosis and osteoporotic fractures in pregnant women. That happens because osteoporosis could arise in pregnant women due to other risk factors including pre- pregnancy low BMI or low dietary calcium intense [15, 23, 25].

Furthermore, it is important to understand that the heterogeneous pharmacologic profile of each LMWH, results from the different methods of manufacturing. The mechanisms responsible for the differences between the effects of different LMWHs could be the result of their different ability to bind different proteins. These proteins in their turn affect bone metabolism in different ways. For sure, these mechanisms need to be elucidated. Most studies in literature review were designed to investigate the effect of only one LMWH. The most studied LMWHs were enoxaparin and dalteparin (Table 5).

Furthermore, most studies differ for the animal’s kind, number, fractured bone, fracture control and mostly for the bony callus evaluation period (Table 6). The number of animals is important for estimating the magnitude of the effect of LMWHs. The average number of animals used in these studies was about thirty. For assessing the real effect of LMWHs on fracture healing process large clinical studies are needed, designed to compare different types of LMWHs, different dosages and in different patient groups. Until then no safe conclusions can be made and no effect of LMWH is evidence- based.

Finally, despite there is no statistically significant results for the effects of LMWHs on fracture healing process, with some studies to report deleterious effect, it is our fair evaluation that daily LMWH administration should continue to be the golden rule for prophylaxis of DVT in trauma patients (risk: benefit ratio) [69-71]. Especially in patients with a reduced bone mineral density, e.g. after steroid therapy or because of renal insufficiency, hyperparathy-roidism or idiopathic osteoporosis, prophylaxis of thrombo-embolism with a smaller osteocatabolic potential than heparin is decisive.

REFERENCES