Veterinarni Medicina, 58, 2013 (7): 339–351 Review Article

A comparison of the microarchitecture of lower limb long between some animal models and humans: a review

V.J. Cvetkovic1, S.J. Najman2, J.S. Rajkovic1, A.Lj. Zabar1, P.J. Vasiljevic1, Lj.B. Djordjevic1, M.D. Trajanovic3

1Faculty of Sciences and Mathematics, University of Nis, Nis, Serbia 2Faculty of Medicine, University of Nis, Nis, Serbia 3Faculty of Mechanical Engineering, University of Nis, Nis, Serbia

ABSTRACT: Animal models are unavoidable and indispensable research tools in the fields of tissue engi- neering and experimental orthopaedics. The fact that there is not ideal animal model as well as the differences in the bone microarchitecture and physiology between animals and humans are complicate factors and make model implementation difficult. Therefore, the tendency should be directed towards extrapolation of the results from one animal model to another or from animal model to humans. So far, this is the first paper which provides an overview on the microarchitecture of lower limb long bones and discusses data related to diameter, osteon canal diameter and their orientation, as well as intracortical canals and trabecular tissue microarchitecture in commonly used animal models compared to humans depending on age, gender and anatomical location of the bone. Understanding the differences between animal model and human bone microarchitecture should enable a more accurate extrapolation of experimental results from one animal model to another or from animal models to humans in the fields of bone tissue engineering and experimental orthopaedics. Also, this should be helpful in making decisions on which animal models are the most suitable for particular preclinical testing.

Keywords: bone microarchitecture; lower limb; osteon; intracortical canals; trabeculae; animal models

Contents

1. Introduction 2.1.4. Vascular canals in the femoral cortex 2. structure 2.1.5. Lacuno-canalicular network 2.1. Microarchitecture of the cortical bone 2.1.6. Cortical bone thickness in the femur 2.1.1. Osteon and diameter 2.2. Microarchitecture of cancellous bone tissue 2.1.2. Osteon orientation 3. Concluding remarks 2.1.3. Haversian canal density 4. References

1. Introduction bone function аnd specific factors like age, gen- der, general condition and lifestyle (Jowsey 1966; Bone is a hierarchically organised tissue which Mavropoulos et al. 2007; Podshivalov et al. 2008). adapts and changes under the influence of the en- For example, human vertebrae cancellous bone is vironment and according to internal body factors. mostly made of rod-like trabeculae, while the femo- Bone structure is very variable among species but ral head mainly consists of plate-like trabeculae also in the body of one specimen depending on (Podshivalov et al. 2008). Bone structure can also

Supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Project No. III41017).

339 Review Article Veterinarni Medicina, 58, 2013 (7): 339–351 vary in different anatomical locations of the same 2004; Ajdukovic et al. 2005; Kalicanin et al. 2007; bone because of different functional requirements Janicijevic et al. 2008; Vasiljevic et al. 2009; Vukelic (Wolff 1892; Hert et al. 1994). This phenomenon et al. 2011). The goal of this review is to describe is partly defined by Wolff’s Law, which states: the differences in the microarchitecture of bone “Every change in the form and function of bone tissue primarily in the most frequently used mod- or of its function alone is followed by certain defi- els (i.e., rabbits and rats) and human lower limb nite changes in the bone internal architecture and long bones depending on gender, age and differ- equally definite alteration in its external conforma- ent lifestyles. As animal models represent the gold tion, in accordance with mathematical laws” (Wolff standard in preclinical studies, the tendency should 1892). Understanding bone microarchitecture is be towards an extrapolation of the results from one essential for understanding the functioning of animal model to another or from animal models healthy bone and also of the bone in the processes to humans. Extrapolation of results is very impor- of regeneration. tant because it reduces the number of experiments First steps towards the description of bone micro- on similar topics and contributes to the quality of architecture in animals and humans were made al​​ - interpretation of results. However, differences in ready in the 19th century (Jowsey 1964, 1966; Wolff bone microarchitecture and physiology between 1892). Recently, however, great progress has been animals and humans complicate and make it dif- made in this field (Hert et al. 1994; Bagi et al. 1997; ficult. Nevertheless, it is not impossible. Ardizzoni 2001; Sugawara et al. 2005; Tromp et al. 2006; Cooper et al. 2007; Mavropoulos et al. 2007; Martiniakova et al. 2008; Britz et al. 2009; Pazzaglia 2. Long bone structure et al. 2010; Schneider et al. 2010). Nevertheless, only a few papers comparing bone microarchi- Briefly, long bones of lower limbs consist of a tecture between animal models and humans have or shaft and or extended ends been published so far (Jowsey 1966; Bagi et al. 1997; whose surfaces are covered with articular carti- Pearce et al. 2007). Furthermore, there aren’t any lage that forms articulating surfaces. Between the papers which integrate data on bone microarchitec- diaphysis and epiphysis there are short, wide seg- ture of lower limbs long bones in frequently used ments – metaphyses that represent the transition animal models, i.e., rabbits and rats. between the epiphysis and diaphysis (Ross et al. In vivo research in experimental and veterinary 1989; Buckwalter et al. 1996). The , orthopaedics and bone tissue engineering is indis- which is filled with , is located in the pensable which implies the usage of different ani- diaphysis and surrounded by a wall of cortical bone. mal models. As all animal models have advantages Cancellous bone makes a very thin layer on the and disadvantages for their use in preclinical testing inner wall of the diaphysis of long bones which im- they must be considered only as an approximation proves the mechanical properties of cortical bone of humans. Pearce et al. (2007) concluded that dogs (Buckwalter et al. 1996). However, metaphyseal and have the most similar bone structure to humans epiphyseal parts are richer in cancellous bone tissue while rabbits have the least similar bone structure consisting of numerous trabeculae which are con- to humans. However, there are many ethical con- nected to build an irregular network with a system cerns related to the use of dogs as well as primates of canals and cavities filled with bone marrow. On a which also have the most similar bone structure cross section of long bones there are four different to humans. Considering ethical issues, handling, bone types: , cortical bone, housing, cost of cultivation and other factors it can and cancellous bone (Ross et al. 1989). be concluded that rabbits and rats are still the most According to Podshivalov et al. (2008) complex suitable for experimental orthopaedics and bone multilevel structure of bone tissue consists of five tissue engineering research. structural levels: Macro-structural (mm-μm) – tra- According to our experience with rats and rab- becular and cortical bone tissue diameter; Micro- bits as animal models in bone regeneration stud- structural (10–500 μm) – osteon and trabeculae; ies, there is a need for a systematic overview on Sub-microstructural (1–10 μm) – lamelae and indi- bone microarchitecture, especially in the femur vidual trabeculae; Nano-structural (100 nm–1 μm) and tibia which are some of the most common – and minerals deposited in the matrix and bone models in bone defect studies (Najman et al. Sub-nanostructural (less than 100 nm) – molecular 340 Veterinarni Medicina, 58, 2013 (7): 339–351 Review Article components of collagen and protein molecules. In canals are clearly different from Haversian because this paper, we will focus on the macro- and micro- they are not surrounded by concentric cacanals structural level of bone structure. are clearly different from Haversian because they are not surrounded by concentric lamellae like Haversian canals. The cement line represents the 2.1. Microarchitecture of the cortical bone outer osteon boundary; it contains less collagen than the surrounding tissue and is rich in sulphur, Bone tissue is divided into cortical and cancellous proteoglycans and glycoproteins (Martin et al. bone tissue. Proportions of cortical and cancellous 1998). tissues in bones vary between different mammalian Generally, it is accepted that osteon is formed by species as well as in different bones in the body of the remodelling effect of BMU (basic multicellular the same specimen. The porosity of the cortical unit) which consist of and , tissue is about 10% (Buckwalter et al. 1996). The due to the growth, repair and bone remodelling basic structural unit in the cortical bone of adult (Parfitt et al. 1994; Marks Hermey 1996; Martin mammals is osteon or the Haversian system (Hert et al. 1998; Mohsin et al. 2002). Osteon develop- et al. 1994; Schneider et al. 2010). Osteon consists ment in cortical bone occurs simultaneously with of concentric lamellae of bone matrix arranged the progression of the vascular network so that the around Haversian canals (Figure 1). Further, os- structure of the vascular network almost fits the teons are separated from each other by interstitial intracortical canal system (Lee et al. 1965; Harris lamellae and on the inner and outer surface of the et al. 1968; Pazzaglia et al. 2007). In humans, as cortical bone there are the inner and outer circum- in other mammalian species, osteon structure and ferential lamellae. Additionally, it was found that geometry depend on the age, sex, height and weight in humans consist of two different types of of individuals, genetic differences, differences in lamellae: compact lamellae that are rich in collagen lifestyle, and the direction of mechanical loads dur- and without (acellular lamellae), and the ing locomotion (Britz et al. 2009). loose lamellae that are poor in collagen but rich in osteocytes (cellular lamellae) (Marotti and Muglia 1988; Marotti 1996). Longitudinal Haversian canals 2.1.1. Osteon and Haversian canal diameter are linked with transverse Volkmann canals and make the system of canals in bone tissue. Nerves Table 1 presents data on the osteon and Haversian and blood vessels that supply the osteocytes and canal diameters in the cortex of the femur in humans, mature bone cells with nutrients and oxygen pass rhesus monkeys, rats and rabbits depending on sex, through this canal system (Levick 2004). Volkmann age and anatomical position. Osteon diameters in the cortical bone of the femur in humans from two different studies Jowsey (1966) and Britz et al. (2009) are match each other very closely (Table 1), but in the group of younger people it is larger than in the group of elderly people (Jowsey 1966). This could be explained by a reduced activity of cells involved in remodelling processes with age (Martin et al. 1980). In addition, osteon diameter decreases with age, not only in humans but also in monkeys and rats (Currey 1964; Singh and Gunberg 1971; Martin et al. 1980; Havill 2003). On the other hand, the diameter of the Haversian canal in younger people is smaller than in older individuals and tends to increase with age. It was found that the average Haversian canal diameter in younger people (under 50) is 156 μm and in the elderly (over 50 years) it is 212 μm (Jowsey 1966). A Figure 1. Osteon structure scheme. a = Haversian canal similar pattern of Haversian canal diameter increase diameter; b = osteon diameter; c = lacuno-canalicular net- is confirmed by recent research (Cooper et al. 2007). work; d = lacunae; e = concentric lamellae; f = cement line However, in the compact bone of ribs the diameter 341 Review Article Veterinarni Medicina, 58, 2013 (7): 339–351

Table 1. Osteon and Haversian canal diameter in the femoral cortex depending on species, gender, age and ana- tomical location

Anatomical Osteon Haversian canal Species Gender Age References location diameter (µm) diameter (µm) Homo sapiens ♀/♂ 20–29 years shaft 252 ± 24 151 ± 25 Jowsey 1966 Homo sapiens ♀/♂ 50–59 years shaft 235 ± 13 195 ± 11 Jowsey 1966 Homo sapiens ♀/♂ 58 years shaft 220 _ Britz et al. 2009 Homo sapiens ♀/♂ 54.6 ± 21.2 years shaft _ 117.2 ± 89.6 Cooper et al. 2007 Homo sapiens ♀ 53.4 ± 23.6 years shaft _ 150.4 ± 118.9 Cooper et al. 2007 Homo sapiens ♂ 55.3 ± 19.9 years shaft _ 99.3 ± 63.4 Cooper et al. 2007 Rhesus sp. ♀/♂ _ shaft 216 ± 52 167 ± 46 Jowsey 1966 Martiniakova et al. Oryctolagus cuniculus ♂ 2,5 months shaft 199.83 ± 49.64 26.22 ± 5.73 2008 Martiniakova et al. Oryctolagus cuniculus ♀ 2,5 months shaft 231.38 ± 44.82 27.00 ± 5.94 2008 Oryctolagus cuniculus ♂ 8 months shaft _ 30.56 ± 9.91 Pazzaglia et al. 2010 of the Haversian canal remains approximately the aging about 1.5 μm (Ardizzoni 2001). Considering same size throughout lifespan, an average of 165 μm, that the loose and solid lamellae are alternately and does not vary significantly as confirmed by other shifting in the osteon wall, the space between solid studies, too (Takahashi et al. 1965; Jowsey 1966). lamellae decreases from cement lines towards the This example could be a proof that there is no identi- Haversian canal, and they get closer to each other cal pattern of Haversian canal diameter increasing in this direction. It is believed that the biological with age in different bones of the same organism. significance of such a structure is the secondary These differences exist probably because changes osteon strengthening from the cement line towards in the bone microarchitecture during lifespan occur the Haversian canal (Ardizzoni 2001). under the synergistic influence of different factors The diameter of the Haversian canal in rabbit but not under the influence of any single one. femur in two different studies by Martiniakova et Nevertheless, the trend of osteon diameter de- al. (2008) and Pazzaglia et al. (2010) are closely creasing and Haversian canals increasing with age matched despite the differences in age of the tested in lower limb long bones, or osteon wall thinning, animals (Table 1). The primate genus Rhesus has can be clearly seen from the data shown in Table 1. osteon and Haversian canal diameters most similar This clearly suggests that the osteon wall is thicker to humans (Table 1), probably because of genetic in young people, which is probably one of the rea- similarity and similar demands during locomotion. sons for the greater durability and stiffness in their It is clear from Table 1 that osteon wall thickness is bones. Besides this, it is interesting that in humans greater in rabbits than in humans and rhesus mon- it is observed that the osteon becomes more regular keys which can be associated with a different load (rounder) with age. In younger people osteon shape on the femur in these species determined by loco- at the cross section is rather irregular (Currey 1964; motion and lifestyle. In addition, in another study it Britz et al. 2009). Also, it was found that lighter was found that juvenile chimps have a larger diam- people have a much larger diameter of cortical ca- eter of the osteon then the macaque monkeys while nals than heavier individuals (Cooper et al. 2007). the size of the Haversian canal is approximately the It follows that heavier people have thicker osteon same in the two species (Mulhern and Ubelaker walls leading to proportionally stronger and more 2009). These data suggest that the osteon wall is durable bones which in turn can respond to higher thicker in chimpanzees because of the influence loads (Britz et al. 2009). of high mechanical forces which is the result of Inside the osteon, it was found that the average higher body weight in chimps and differences in thickness of the loose lamellae from the cement line locomotion and lifestyle (Schaffler and Burr 1984; direction to the Haversian canal is 5.5 μm, 4.5 μm Mulhern and Ubelaker 2009). However, chimpan- and 3 μm, respectively, while the thickness of the zees have significantly smaller osteon compared compact lamellae does not vary significantly, aver- with man (Mulhern and Ubelaker 2009). The bio-

342 Veterinarni Medicina, 58, 2013 (7): 339–351 Review Article logical significance of smaller osteon formation al. 2008). In Bos aurus, Ovis aries, Sus scrofa domes- lies in a response to a specific lifestyle (jumping, tica, and Equus caballus, it was concluded that there using all four limbs during locomotion etc.) which were no significant differences between the sexes determined that weight-bearing bones require in- in bone microarchitecture (Urbanova and Novotny creasing resistance to micro and macro fractures 2005). (Frost 1990; Mohsin et al. 2006, van Oers et al. 2008). In rabbit femoral cortical bone, it was shown that Haversian canals are not continuous straight 2.1.2. Osteon orientation tunnels but successive shifts of straight and de- viant segments that occur at branching points In the cortex of diaphysis of the long bones in man (Pazzaglia et al. 2009). Branching angles typically (femur, tibia, humerus, radius) osteons are predomi- range from 10° to 30° and 150° to 170° and there nantly orientated longitudinally but they are not abso- is also a branching angle of 90°, while nodes at lutely parallel to the longitudinal axis of the bone. The branching points are usually composed of three, most prevalent opinion is that the osteons are oriented four or five branches with the largest proportion of parallel to the direction of the loading force (Fiala and nodes with three branches (Pazzaglia et al. 2009). Hert 1993; Hert et al. 1994; Baca et al. 2007). Most of There are a lot of data about differences in the the osteons are oriented in a direction skewed from structure of bone tissue between the sexes, both in the longitudinal axis and the inclination angle varies, humans and experimental mammals (Thompson depending on the type of bone, from 0° to 15°. For ex- 1981; Burr et al. 1990; Scheuer 2002; Cooper et ample the mean inclination angle in the human femur al. 2007; Martiniakova et al. 2008; Britz et al. is 8.1° (Hert et al. 1994). Further, osteons are set to 2009). Data about the difference in osteon and form two helical systems which are placed opposite Haversian canal size between men and women one another at opposite sides of the diaphysis. Also, one vary greatly. Some suggest that the diameter of the helical system is larger than the other due to specific osteon and diameter of Haversian canal in women effects of torque moments and due to maximum princi- is greater than in men, while other studies sug- pal strain in everyday lifestyle (Lanyon and Bourn 1979; gest the opposite. Less frequently there are claims Hert et al. 1994). In addition, a similar osteon arrange- that there are no significant differences between ment was found in the sheep radius where osteons are them (Thompson 1981; Burr et al. 1990; Pfeiffer slightly twisted orientated from the bone axis and in 1998; Cooper et al. 2007; Britz et al. 2009). In the dog femur where osteons are orientated in a helical the primate species Macaca mulatta it was found way around the bone shaft in a clockwise direction in that osteon densities are higher in females than in the right femur, and in the same pattern but counter males (Havill 2003). Also, in rabbits the diameter clockwise in the left femur (Cohen and Harris 1958; of the osteon is significantly different between the Mohsin et al. 2002). sexes; it is larger in females than in males, which the authors explain with the different lengths of the femur between sexes. However, this is not 2.1.3. Haversian canal density the case with a Haversian canal diameter which is approximately the same size in different sexes Table 2 shows the values of Haversian canal density without significant differences (Martiniakova et in the cortex of the femur at different anatomical

Table 2. Haversian canal density in the femoral cortex depending on species, gender, age and anatomical location

Haversian canal Species Gender Age Anatomical location References density (mm2) Homo sapiens ♀/♂ 54.6 ± 21.2 years shaft-subperiosteal area 12.3 ± 2.4 Cooper et al. 2007 Homo sapiens ♀ 53.4 ± 23.6 years shaft-subperiosteal area 11.5 ± 2.5 Cooper et al. 2007 Homo sapiens ♂ 55.3 ± 19.9 years shaft-subperiosteal area 12.7 ± 2.2 Cooper et al. 2007 Oryctolagus cuniculus ♂ 8 months middle shaft 111.3 ± 14.48 Pazzaglia et al. 2010 Oryctolagus cuniculus ♂ 8 months distal shaft 58.87 ± 16.93 Pazzaglia et al. 2010 Oryctolagus cuniculus ♂ 8 months shaft-subperiosteal area 125.1 ± 13.43 Pazzaglia et al. 2010 Oryctolagus cuniculus ♂ 8 months Shaft-subendosteal area 87.82 ± 46.98 Pazzaglia et al. 2010

343 Review Article Veterinarni Medicina, 58, 2013 (7): 339–351 locations in humans and rabbits, depending on sex middle shaft than in the distal shaft cortex (Pazzaglia and age. Haversian canal density in the femoral et al. 2007). The density of Haversian canals is also middle shaft in rabbits is higher than in the distal greater in the femoral middle shaft (Table 1). Since shaft, which is accordant with the fact that the pres- the Haversian canal is occupied by blood vessels ence of blood vessels in the middle shaft is higher these two facts are in agreement. Differences in vas- than in the distal shaft (Pazzaglia et al. 2008, 2010). cular canal densities in older and younger specimens Additionaly, Haversian canal density is significantly occur due to remodelling processes that take place higher in the subperiosteal than the subendosteal during aging. Vascular canal density is therefore part of the cortex which is probably the result of greater in older specimens (Pazzaglia et al. 2007). intensive remodelling in the subperiosteal area Also, the vascular canal diameter in femoral cortical (Pazzaglia et al. 2010). bone in rabbits corresponds to the average diameter Haversian canal density in rabbits is up to 10 times of Haversian canals because the blood vessels are higher than in humans (Table 2). Since each osteon mostly situated there (Table 1 and 3). has one Haversian canal, a high density of Haversian The number of intracortical canal openings on canals in rabbit cortical bone indicates that the den- cortical bone surface is directly correlated to the sity of the osteon is large. Further, Haversian canal distribution of blood vessels located inside or out- density in humans does not differ significantly with side the bone. Openings represent spots where regard to sex (Table 2), which is consistent with the blood vessels enter or exit bone. Scanning Electron fact that the distance between Haversian canals Microscopy analysis showed that the number of does not vary significantly between the sexes in canal openings on the endosteal surface in the mid- humans being 337.2 ± 38.3 μm in men and 53.1 ± dle shaft is larger than in the distal shaft which is 3.33 μm in women (Cooper et al. 2007). not the case on the periosteal surface (Pazzaglia et al. 2009). This is probably because the inner side, which is closer to bone marrow, has more 2.1.4. Vascular canals in the femoral cortex blood vessels that are entering the bone tissue, compared to the periosteal surface (Ohtani et al. Table 3 presents data about the density of vascular 1982; Skawina et al. 1994). The density of vascular canal openings, vascular canal density and diameter canal openings is the highest in the middle shaft in rabbits with respect to age, gender and anatomical and this is logical because canal density is also location. The same age and gender specimens have highest in this part of the bone. Canal openings vascular canal densities much greater in the femoral on the endosteal surface in the middle shaft have

Table 3. Density of vascular canal openings, vascular canal density and diameter in the femoral cortex depending on species, gender, age and anatomical location

Age Anatomical Density of vascular Vascular canal Vascular canal Species Gender References (months) location canal openings (mm2) density (mm2) diameter (µm) Oryctolagus Pazzaglia ♂ 8 middle shaft _ 142.3 ± 34.41 39 ± 2.4 cuniculus et al. 2007 Oryctolagus Pazzaglia ♂ 8 distal shaft _ 84.13 ± 22.13 26.3 ± 5.0 cuniculus et al. 2007 Oryctolagus Pazzaglia ♂ 1.5 middle shaft _ 112 ± 25.22 27 ± 1.46 cuniculus et al. 2007 Oryctolagus Pazzaglia ♂ 1.5 distal shaft _ 91.25 ± 15.41 26.71 ± 2.89 cuniculus et al. 2007 Oryctolagus middle shaft: Pazzaglia ♂ 8 25.86 ± 4.84 _ _ cuniculus periosteal surface et al. 2009 Oryctolagus distal shaft: Pazzaglia ♂ 8 22.2 ± 5.6 _ _ cuniculus periosteal surface et al. 2009 Oryctolagus middle shaft: Pazzaglia ♂ 8 44.84 ± 19.6 _ _ cuniculus endosteal surface et al. 2009 Oryctolagus distal shaft: Pazzaglia ♂ 8 27.56 ± 4.41 _ _ cuniculus endosteal surface et al. 2009

344 Veterinarni Medicina, 58, 2013 (7): 339–351 Review Article an oval shape and diameters of between 30 µm and et al. 2004). In several different animal species and 1000 µm (Pazzaglia et al. 2009). in humans, it was found that the number of cana- Five different shapes of canals were found in rab- liculi is higher on the side of the lacunae that face bit femoral cortical bone: cylindrical 39.2%, coni- the Haversian canal than on the side that face the cal with proximal openings 9.4%, conical with distal cement line, so this might be a pattern (Remaggi openings 35.1%, elliptical 12.2% and hyperbolic 4.1% et al. 1998). This way of arrangement of canaliculi (Pazzaglia et al. 2010). Cooper et al. (2006) reported is probably caused by different distance from the that cylindrical canals are located in primary osteons blood vessels (Schneider et al. 2010). In mice, the and in secondary osteons with lamelar apposition. average diameter of canaliculi is 259 nm while the Elliptical and hyperbolic canals are located at spots average length is 104 nm (You et al. 2004). where irregular absorption occurs. Canals that are still in the process of maturing have conical shapes and the position of the widest opening is in the di- 2.1.6. Cortical bone thickness in the femur rection of the cutting cone (Cooper et al. 2006). The total volume of all canals compared to the total vol- Table 4 shows data regarding cortical bone thick- ume of bone tissue (in rabbit femoral cortical bone) ness in the femur in humans, rabbits and rats re- is 5.88 ± 2.21% (Pazzaglia et al. 2010). garded to age, gender and anatomical location. Sex hormones have a significant impact on cortical bone thickness as well as on the bone microarchitecture 2.1.5. Lacuno-canalicular network (Bagi et al. 1997; Tromp et al. 2006). The average thickness of cortical bone located in the femoral Osteocytes are found in cavities, called lacunae, neck region in postmenopausal women is about of the osteons lamellae (Figure 1) and they commu- 10 times greater compared to ovariectiomised fe- nicate with each other via dendritic-like extensions male rats (Bagi et al. 1997). Also, cortical thickness that pass through micro-canals called canaliculi in intact women is approximately 10 times greater (Doty et al. 1981). Each has dozens of than in intact rabbits. Since, cortical bone thick- thin dendritic extensions; for example, there are 41 ness represents a macro-structural entity it is not in humans and 115 in horse (Beno et al. 2006). The appropriate to compare these data because humans Lacuno-canalicular network is the finest network have bigger femurs than rats and rabbits, so corti- in the cortical bone tissue which orientation and cal bone thickness is also proportionally greater. morphology depends on the species, age, anatomi- However, it was shown that cortical bone on cross cal location on the same bone, and arrangement of section occupies 72.5% of the total bone area in collagen fibres in the bone matrix and on effects the rat femur (Figure 2), while in humans it is only of mechanical loads (Schneider et al. 2010). For 12.5% (Bagi et al. 1996, 1997). Differences in corti- example, it was found that the density of lacunae was reduced in older rats while the number of cana- liculi increased and they became more properly organized (Okada et al. 2002). Some data have been reported regarding lacuno-canalicular geometry; we present some of these data below. In mice, lacunae in long bones are generally ellip- tical in shape but they can be round also, while their volume varies from 191 μm3 to 274 μm3 and their mean space is 26 μm (Wang et al. 2005; Schneider et al. 2010) However, in rabbit long bones lacunar volumes are about 350 μm3 (Remaggi et al. 1998). Lacunae are parallel to the axis of the bone and it is believed that this is due to the influence of me- chanical loads (Vatsa et al. 2008). Canaliculi are cylindrical in shape and they are Figure 2. Femoral cross section in rat Rattus norvegicus arranged asymmetrically along the lacunar canal, (Wistar strain). P = periosteum; C = cortex; E = endos- gradually increasing its diameter towards it (You teum. Scanning Electron Micrography; bar 100 µm 345 Review Article Veterinarni Medicina, 58, 2013 (7): 339–351

Table 4. Cortical bone thickness in the femur depending on species, gender, age and anatomical location

Cortical bone Species Gender Age Anatomical location References thickness (mm) Homo sapiens ♀ 66.5 years neck 3.3 ± 1.4 Bagi et al. 1997 (postmenopasual) Homo sapiens ♀/♂ 20–29 years shaft 6.06 ± 1.0 Jowsey 1966 Homo sapiens ♀/♂ 50–59 years shaft 5.92 ± 0.8 Jowsey 1966 Homo sapiens ♀/♂ 54.6 ± 21.2 years shaft 4.68 ± 1.18 Cooper et al. 2007 Homo sapiens ♀ 53.4 ± 23.6 years shaft 4.01 ± 1.23 Cooper et al. 2007 Homo sapiens ♂ 55.3 ± 19.9 years shaft 5.06 ± 0.97 Cooper et al. 2007 Ratus norvegicus ♀ 12 months neck 0.3 ± 0.01 Bagi et al. 1997 (ovariectomized) Oryctolagus cuniculus ♂ 8 months middle shaft 0.456 ± 0.06 Pazzaglia et al. 2007 Oryctolagus cuniculus ♂ 8 months distal shaft 0.606 ± 0.12 Pazzaglia et al. 2007 Oryctolagus cuniculus ♂ 1.5 months middle shaft 0.584 ± 0.09 Pazzaglia et al. 2007 Oryctolagus cuniculus ♂ 1.5 months distal shaft 3.3 ± 1.4 Pazzaglia et al. 2007 cal bone abundance are due to a specific response to There is a significant difference in trabecular ge- different mechanical loads and strains in humans and ometry between ovariectomised and intact female rats bones during locomotion (Martens et al. 1983). rats. Trabeculae lose their thickness and become The femur in humans, especially the femoral neck, less abundant while the trabecular space increases. is exposed to higher mechanical load during bipedal Aging is one of the factors that contribute to this locomotion than the femur in rats. Because of that process, too (Bagi et al. 1996). In humans trabecular cancellous tissue occupies a bigger part than cortical thickness and space is very different between the bone in the human femur and vice versa in rats, which tibia and femur, while the abundance of trabeculae will be discussed later. Further, in rabbits and rats is similar, which is not the case in rats (Table 5). differences between cortical bone thickness in distal Trabeculae in the femoral neck are approximately and middle femoral parts are much smaller; this is not four times thicker in humans than in rats, and be- the case in humans (Table 4) (Pazzaglia et al. 2007). cause of that there are less trabeculae per 1 mm2 in humans than in rats. This difference occurs also because of the unequal femoral size among these 2.2. Microarchitecture of cancellous bone tissue two species meaning that macro-structural entities cannot be compared. However, it was shown that Cancellous bone is a sponge-like bone and it in rats cancellous bone tissue occupies a propor- is made out of elements called trabeculae, which tionally smaller area than in humans (Bagi et al. look like honeycomb, and differ in size and shape 1997). Despite this, humans have more cancellous (Buckwalter et al. 1996). Cancellous bone is the bone tissue which increases the total strength of most abundant in the metaphyseal area of long the femur and acts like a load conductor, allowing bones and it is surrounded by cortical bone. The bipedal locomotion (Wolff 1892; Pugh et al. 1973; porosity of trabecular bone in intact rats is be- Martens et al. 1983; Parfitt 1984 1987). In addition, tween 50 to 90%; the same is broadly true in hu- humans have two kinds of cancellous bone tissue: man (Buckwalter et al. 1996; Sikavitsas et al. 2001; peripheral and central part of the cancellous bone. Yang et al. 2011). Osteocyte density in cancellous Peripheral cancellous bone is organised as a net bone in the femoral head (caput femoris) in rats of plate-like trabeculae which are oriented in the is 93 000 mm−3, and in mice from 50 000 mm−3 to direction of body weight forces, and are connected 66 000 mm−3 in the femoral middle shaft (Mullender with rod-like perpendicular trabeculae. The cen- et al. 1996; Schneider et al. 2007) tral part of spongy bone consists mostly of rod-like Data on the thickness, space and number of tra- trabeculae (Bagi et al. 1997). beculae in humans and rats with respect to age, gen- Changes in sex hormone levels in an organism der and anatomical location are shown in Table 5. are one of the most common causes of trabecular

346 Veterinarni Medicina, 58, 2013 (7): 339–351 Review Article

Table 5. Trabecular thickness, space and number in the femur and tibia depending on species, gender, age and anatomical location

Trabecular Anatomical Trabecular Trabecular Species Gender Age number References location thickness (µm) space (µm) (mm) femoral Nogueira Homo sapiens ♀/♂ _ 128.6–201.0 313.5–619.4 1.34–1.94 methaphysis et al. 2010 Hildebrand Homo sapiens ♀/♂ 67.3 ± 15.5 years femoral head 127–284 454–940 1.092–2.387 et al. 1999 Ulrich et al. Homo sapiens ♀/♂ 69 ± 15 years femoral head 120–257 480–984 0.98–1.91 1999 tibial Nogueira Homo sapiens ♀/♂ _ 379.5–372.4 211.7–131.1 1.69–1.99 methaphysis et al. 2010 Ratus norvegicus femoral Bagi et al. ♀ 12 months 62.7 ± 3.2 386.3 ± 34.2 4.1 ± 0.4 (ovariectomized) methaphysis 1997 Ratus norvegicus femoral Tromp et al. ♀ 12 weeks 102 ± 12 130 ± 39 4.4 ± 0.6 (ovariectomized) methaphysis 2006 femoral Tromp et al. Ratus norvegicus ♀ 12 weeks 91 ± 14 93 ± 20 5.5 ± 0.7 methaphysis 2006 Hanson and Ratus norvegicus ♀/♂ _ proximal tibia 48.2–64.1 158.3–188.1 5.2–5.8 Bagi 2004 Mavropoulos Ratus norvegicus ♀ 6 months proximal tibia 50 _ 4.01 et al. 2007 structural variation (Bagi et al. 1996; Mavropoulos Additionally, mechanical load produced by backpack et al. 2007; Yang et al. 2011). Ovariectomy in female loads increased bone mineral content and bone min- rats causes a loss in trabecular tissue volume of 82% eral density by 27% and 11% respectively, compar- and reduces the number of trabeculae by 63% in ing to control groups (Tromp et al. 2006). The same proximal tibia during the first 17 weeks after the conclusion was made in experiments dealing with ovariectomy (Mavropoulos et al. 2007). In ovariec- astronauts in zero gravity conditions and in other tomised females trabecular tissue is four times less similar studies (Vico et al. 2000; Shackelford et al. abundant than in control groups and mineral density 2004). Also, it was shown that intense physical activ- in trabeculae significantly decreases (Bagi et al. 1996; ity in humans preserves trabecular bone microarchi- Yang et al. 2011). Also, a loss of bone volume of 76% tecture by increasing the number of trabeculae in and trabecular abundance of 56% in proximal tibia tibia bone by 12.7% compared to the control group region was reported in rats that were on a protein- (Nilsson et al. 2010). restricted diet (Mavropoulos et al. 2007). However, low levels of sex hormones and a protein-restricted diet has more negative effects on cancellous tissue 3. Concluding remarks volume and the number of trabeculae in proximal tibia bone than in rat mandibulae (Mavropoulos et In conclusion, we can say that the structure of al. 2007). One explanation is that during mastication bone tissue is very specific and varies during life, cancellous bone tissue in the mandibula is exposed and in different functional and physiological stages. to a higher mechanical load than the tibial bone There is no single factor which independently af- during locomotion. In addition, a control group, fects bone structure and observed effects are rath- which was fed with soft food, had greater loss in er due to a specific combination of many factors. cancellous tissue of the mandibula compared to the However, it is our opinion that mechanical loading group which was fed with solid food (Mavropoulos has a stronger impact on bone microarchitecture et al. 2007). Similar parameters of cancellous bone at all levels in comparison with other factors. For in female rats were investigated, and it was shown example osteon wall thickness is greater in younger that loads in backpacks compensate the negative ef- persons than in older ones, since Haversian canal fects of a lack of sex hormones on cancellous bone. diameter is smaller and osteon diameter is bigger. 347 Review Article Veterinarni Medicina, 58, 2013 (7): 339–351

But this regularity was not noticed in the cortical For successful treatment of bone defects it is very bones of ribs so it could not be the underlying rea- important to integrate the basic elements in bone son for why these parameters are changing. This is tissue engineering – cells and biomaterial scaf- probably because the mechanical loading of ribs is folds – with patient gender, age, health, anatomical constant during life, which is not the case in weight- shape, position and microarchitecture of the bone bearing bones like the femur or tibia. In addition, defect. Therefore, all parameters mentioned above, overweight persons in the same age group, have including morphological ones, must be considered thicker osteon walls. Higher loads also trigger os- in such treatments. This is especially important teon diameter reduction due to physical activity or when bone defects are being treated with bone sub- higher body weight. The biological significance of stituents which must be compatible in every sense such processes and changes is an increase in bone with the bone itself. Bone substituents with ad- strength, and also increasing resistance to micro- equate microarchitecture enable angiogenesis and and macro-fractures. Also, osteon wall thickness osteogenic cell propagation, which is very impor- and osteon density are higher in rabbits compared tant for osteoreparative and osteoregenerative pro- to rhesus monkeys and humans because of different cesses as we have already shown (Ajdukovic et al. ways of locomotion and lifestyles. Therefore, rab- 2005; Janicijevic et al. 2008; Vasiljevic et al. 2009). bits have a more solid and stronger cortical bone In that sense this overview could be of benefit in that resists high impact force acting upon the femur further studies and for the fabrication of artificial during jumping. Furthermore, it was shown that a bone substituents for bone tissue engineering. significant correlation exists between morphologi- Commonly used animal models (i.e. rats and cal parameters of bone tissue and its mechanical rabbits) are very important in preclinical stud- properties – strength and stiffness (Voide et al. ies of various bone tissue defect healing. Because 2008). there is not an ideal animal model, an understand- Osteon orientation in long bones is also affected ing of the differences in bone microarchitecture by mechanical loads and their orientation is paral- between humans and animal models is needed lel with direction of the maximum principal stress for extrapolation and interpretation of the results and strain (Lanyon and Bourn 1979; Fiala and Hert from in vivo studies. We hope that in this regard 1993; Hert et al. 1994; Baca et al. 2007). They form the present review will be of benefit. Also, a proper two helical systems, opposite one another, located experimental set up and choice of suitable animal at opposite sides of the diaphysis of the long bone. models is crucial. Therefore, this overview should One of these systems is always bigger than the other be helpful in facilitating decisions on which ani- one because of torque moments. This osteon ori- mal models are most suitable for different pre- entation relates to loading forces during lifetime clinical studies. mostly during motion and other life activities (Hert et al. 1994). This pattern is seen to be widespread in long bones of lower limbs in mammals, which 4. REFERENCES was confirmed by many different studies (Cohen and Harris 1958; Fiala and Hert 1993; Hert et al. Ajdukovic Z, Najman S, Dorđevic L, Savic V, Mihailovic 1994; Mohsin et al. 2002; Baca et al. 2007). V, Petrovic D, Ignjatovic N, Uskokovic D (2005): Repair Differences in cortical bone tissue and cancellous of bone tissue affected by osteoporosis with hy- bone tissue between humans and rats are great. The droxyapatite-poly-L-lactide (HAp-PLLA) with and femur is heavily loaded bone in humans, especially without blood plasma. Journal of Biomaterials Appli- in the metaphyseal region; consequently cancellous cations 20, 179–190. bone tissue is more developed in this region com- Ardizzoni A (2001): Osteocyte lacunar size-lamellar pared to rats, because cancellous bone conducts thickness relationships in human secondary osteons. mechanical forces better than cortical bone and Bone 28, 215–219. acts like an armature and load absorber. Variations Baca V, Kachlik D, Horak Z, Stingl J (2007): The course in bone tissue structure are strongly influenced by of osteons in the compact bone of the human proximal different levels of sex hormones, but intense load femur with clinical and biomechanical significance. could preserve bone tissue volume despite low level Surgical and Radiologic Anatomy 29, 201–207. of sex hormones (Tromp et al. 2006; Mavropoulos Bagi C, DeLeon E, Ammann P, Rizzoli R, Miller S (1996): et al. 2007). Histo anatomy of the proximal femur in rats, Impact 348 Veterinarni Medicina, 58, 2013 (7): 339–351 Review Article

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Corresponding Author: Vladimir J. Cvetkovic, University of Nis, Faculty of Sciences and Mathematics, Department of Biology and Ecology, Visegradska, 33, 18000 Nis, Serbia Tel. +381 1835 5015, E-mail: [email protected]

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