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Mechanical evaluation of bone gap filled with rigid formulations castor oil polyurethane and chitosan in horses/Avaliacao mecanica do preenchimento de falhas osseas de equinos com formulacoes solidas de poliuretana de mamona e quitosana.

INTRODUCTION

Long bone fractures in adult horses are often comminuted. Resulting bone gaps interfere with force transmission through the fracture site, leading to orthopedic implant overload (NIXON, 1996).

Bone grafts are required to eliminate large bone gaps. Autografts are more commonly employed, but availability is limited and harvest morbidity must be considered, while allografts carry the risk of disease transmission and immune reactions (CALORI et al., 2011). Such concerns have fostered the search for synthetic, mechanically resistant bone substitutes for improved fracture stabilization in horses.

Over the last few years, the natural polymer chitosan has received increasing attention as a potential tissue gap filling alternative. Chitosan-based polymers have been successfully employed in treatment of periodontal bone lesions and bone defects and have been shown to stimulate new bone formation, as demonstrated in histological sections and in a wide variety of clinical tests (SPIN-NETO et al., 2008).

LISTONI (2013) evaluated bone marrowderived stem cell growth and osteogenic/chondrogenic differentiation following culture in chitosan/hyaluronic acid hybrid films and concluded that these membranes are able to support bone tissue repair.

Chitosan may also be added to inorganic biomaterials such as hydroxyapatite and beta-tricalcium phosphate; mechanical characteristics are enhanced and biocompatibility is maintained (GINALSKA & PRZEKORA, 2014).

A polymer containing beta-tricalcium phosphate, chitosan and platelet rich plasma (PRP) has been developed for bone bioengineering application; PRP did not interfere with biomechanical properties and the polymer was shown to induce stem cell proliferation and differentiation into in vitro osteoblasts (BI et al., 2010).

Hydroxyapatite, beta-tricalcium phosphate and chitosan have also been employed to develop a hybrid material with similar biocompatibility to chitosan and capable of withstanding load; the polymer was shown to act as a scaffold for bone cell culture and sufficiently high modulus of elasticity and load resistance have been demonstrated (ZHANG & ZHANG, 2002).

Castor oil polyurethane is a natural product derived from two basic castor oil components: polyol and pre-polymer. Castor oil polyurethane was shown to have similar characteristics to human bone permitting osteogenesis between fractured fragments, with no foreign body reaction (BOLSON et al., 2005).

Progressive connective and ostechondroid tissue formation at the bone/polyurethane interface has been described upon histological evaluation within 15, 30, 45 and 60 days of castor oil polyurethane implantation in murine femur, again with no foreign body reactions (MARTINELI JUNIOR, 2011).

Modulus of elasticity values of 328.265MPa and 1354.284MPa have been described following axial compression of both, porous and compact castor oil based polyurethane (with and without bubbles respectively). These values are lower than average values reported for the equine cortical bone, but similar to those documented in the trabecular bone of the equine third metacarpus (6093Mpa and 1174Mpa respectively) (VAN DE GRAAF, 2012).

A polyurethane/calcium mixture, alone or combined with bone marrow, has been tested in bone gap repair in rabbits; calcium has been shown to increase polyurethane porosity, with penetration of new bone tissue through the more superficial pores in the biomaterial (DEL CARLO et al., 2003).

Clinical and radiographic assessment of experimental bone gaps filled with castor oil polyurethane in horses revealed increased local inflammatory reaction and slower bone healing (DORNBUSCH et al., 2010).

MATERIALS AND METHODS

Five equine third metacarpal bone pairs were used in this study. Bone specimens were harvested from cadaver limbs and stored in a horizontal freezer until use. Specimens were thawed and the second, third and fourth metacarpal bones separated for locking compression plate (LCP) application. An 11-hole LCP was implanted at the dorsal aspect of the third metacarpal bone and a 1cm bone gap created in the mid diaphysis; proximal and distal bone ends were sawed off to produce flat, parallel articular surfaces amenable to attachment to a biomechanical testing machine.

Biomechanical testings were performed using a universal testing machine (KRATOS[R], K5002) and a special device was designed to hold the distal and proximal bone epiphyses in place during biomechanical trials.

Castor oil polyurethane was prepared using an analytical. Following addition of 0.65g of polyol and 0.82 grams of calcium carbonate per gram of pre-polymer, components were mixed for approximately 6 minutes to obtain a firm solution. Resulting polyurethane was then placed in 6cm cylindrical Teflon molds of variable height. In order to prevent bubble formation during the polymerization process, expansion was controlled via a 590 gram pushing tube connected to a 5kg weight.

High component concentration in the formula (chitosan powder, distilled water, citric acid, dextrose and beta-calcium triphosphate; 1.36, 63.65, 60.08 and 104.78 grams respectively) required gradual addition of the different components to facilitate homogenization. Hence, the procedure involved four consecutive steps (VAN DE GRAAF, 2012), as follows:

First, a 2% chitosan hydrogel was prepared using chitosan, water and citric acid (1, 36.5 and 12.5 grams respectively). Then, chitosan, citric acid and dextrose (0.125, 1.023 and 1.2016 grams per gram of hydrogel respectively) were added to the mixture to form a paste-like substance within approximately 5 minutes. Calcium triphosphate was then added (0.65 grams per gram of paste) to yield a firm substance. Finally, the final substance was transferred to Petri dishes (16mm height x 60mm width) and incubated at 38[degrees]C for 24 hours.

This study involved compressive, nondestructive biomechanical testings. Two specimens were used for determination of the load to be employed; the first was submitted to destructive testing for elastic limit determination while the second was submitted to repeated non-destructive compression to evaluate potential changes between two consecutive testings. Changes were not observed; therefore, the maximum load was set at 1000 newtons (N) and applied to the central bone axis at a speed of 5mm/min.

This study involved three groups: Control (ND-C); castor oil polyurethane (ND-M) and chitosan (ND-Q). Using the crossing design described by ULIAN et al. (2008), all ten specimens were used in all three groups. Following mechanical testings in one group, the same specimens were used in the other group in a different order.

Strain gauges can be attached to the surface of target materials to provide site-specific strain readings. Strain gauges were used in this study to determine deformations at specific sites on the plate and bone. One uniaxial strain gauge was attached to the midpoint of the dorsal aspect of the plate and triaxial strain gauges were placed on the bone as follows: one medial and one lateral, 1cm distal to the gap; one on the palmar aspect, between the 3rd and 4th screws, approximately 5.5cm from the gap (Figures 1A and 4B). Uniaxial gauges were intended for assessment of the load transmitted by the plate and triaxial gauges to assess strains close to and away from the bone gap. In this manner, strains in the whole construct (i.e., on the plate, close to and away from to the bone gap) could be sampled during biomechanical testings.

RESULTS AND DISCUSSION

Time and procedural differences were noted in the preparation process of castor oil polyurethane and chitosan. Ease and speed of castor oil polyurethane preparation permits intraoperatively applicability, with proper fitting into gaps of different shapes (JACQUES, 2004); in contrast, chitosan required previous preparation in pre-defined shapes, translating into more difficult fitting.

This study involved non-destructive testings with loads up to 1000N, which are lower than the actual loads experienced by the limb of an adult horse at walk (7500N at the mid third metacarpal diaphysis; LES et al. 1998). However, such loads are believed to be close to those applied to the limb by smaller animals, or in the presence of a painful fracture.

Bearing these load magnitudes in mind, destructive testings, along with application of values corresponding to in vivo loads experienced by foals and adult horses during anesthetic recovery from long lasting procedures (e.g., orthopedic surgeries) would be required to determine maximal loads tolerated by bone gap filling materials such as those in this study.

Construct (bone/ plate/ gap with or without filling) deformation was observed in all groups in this study, with different intensities (Dunnett's test; P<0.05), with decreased construct length due to plate bending. Greatest and smallest deformations were documented in the control and chitosan group respectively (Figure 2). Lower levels of deformation following chitosan application possibly reflect the greater rigidity of this biomaterial (VAN DE GRAAF, 2012).

Mobility at the fracture site has a negative impact on fracture healing (HAK et al., 2010). In this study, reduction of 14% and 3% in deformation under loads up to 1000N following application of synthetic implants (castor oil polyurethane and chitosan respectively) was observed. Therefore, bone gap filling with these biomaterials was thought to be beneficial.

Biomaterial porosity enhances nutrient penetration and biomaterial incorporation at the expense of mechanical resistance (VAN DE GRAAF, 2012). Horses are heavy, irrational animals; hence biomaterial mechanical resistance should be prioritized over biological behavior in this species.

Loads applied to fractured bone epiphyses are transmitted through apposed fracture fragments and/or implants employed for fracture fixation. Therefore, the greater the load transmitted through the implant, the greater the deformation on the implant surface.

Deformations documented on the plate surface differed significantly between tested groups and between tested and control groups in this study (Dunnett's test; P<0.05), with 85% and 96% reduction in plate deformation under loads up to 1000N following application of castor oil polyurethane and chitosan implants respectively (Figure 3). Implant overload is one of the major problems in fracture treatment in horses (NIXON, 1996), and can be mitigated by load reduction via inclusion of biomaterial implants in osteosynthesis procedures. That doesn't mean that implants alone have the ability to prevent implant failure. Most implants used in horses are originally designed for humans; therefore, plaster casts and anesthetic recovery support are paramount for load reduction at the fracture site.

When loads are transmitted through apposed fracture fragments or biomaterial implants, contact surfaces will be loaded; the greater the force transmission through the fracture, the greater the deformation in bone tissues, which must be able to withstand such loads.

Strain gauges in gap vicinity revealed intergroup differences not only regarding deformation intensity, but also deformation direction (Figure 4). In contrast, readings sampled via palmar strain gauges placed 5.5cm away from the gap did not differ between groups (Figure 5). Therefore, increased loads resulting from filling of bone gaps with rigid biomaterials were limited to gap vicinity. Load distribution through the bone and plate screws likely prevented loads transmitted through the biomaterials to act at more distant sites.

Load transmission through biomaterials implants decreased total bone and plate deformation in this study, with increased loads on bone surfaces restricted to bone gap vicinity. Results presented suggested that bone gap filling with the biomaterials studied may bring biomechanical advantages to ostheosynthesis procedures.

The fact that it was used only mechanical testing suggests that the present study presented limitations for the immediate use of the evaluated materials, as clinical use are needed for other evaluations, such as behavior of these materials in biological systems. In this respect, the castor oil polyurethane presented in a more advanced stage, is already available for use in human orthopedics. However, chitosan formulation investigated differs from other formulations already employed and thus needs further evaluation for in vivo use.

These laboratory tested to assess the mechanical behavior of implants used in orthopedics and, in this study, the bone defect filling with biomaterials used brought Biomechanical osteosynthesis benefits to increase the strength and rigidity of the orthopedic implant / biomaterial / bone, to reduce the loads in which the implant was subjected due to the transmission of loads by biomaterial, which led to an increase in load on the bone surface next to the defect. Considering the mechanical behavior and the mechanical demands of osteosynthesis, it is possible to suggest that chitosan presents possibility of bringing greater benefit in filling bone of horse failures.

http://dx.doi.org/10.1590/0103-8478cr20150838

ACKNOWLEDGEMENTS

This work was funded by the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) with proces number 2010/17805-3.

REFERENCES

BI, L. et al. Reconstruction of goat tibial defects using an injectable tricalcium phosphate/chitosan in combination with autologous platelet-rich plasma. Biomaterials, v.31, n. 12, p.3201-3211, 2010. Available from: <http://www.cusabio.cn/ wenxian/167.pdf>. Accessed from: jul. 22, 2016. doi: 10.1016/j. biomaterials.2010.01.038.

BOLSON, J. et al. Clinical, radiological, macroscopical and histological analysis of domestic quail (Coturnix japonica) humerus submitted to implant of polyurethane from castor oil polymer (Ricinnus communis). Ciencia Rural, v.35, n.5, p.11231130, 2005. Available from: <http://www.studiocalori.it/images/ pdf/pubblicazioni/087.pdf>. Accessed from: jul. 22, 2016.

CALORI, G.M. et al. The use of bone-graft substitutes in large bone defects: Any specific needs? Journal of Care Injured, v.42, p.56-63, 2011. Available from: <http://www.scielo.br/pdf/cr/ v35n5/a21v35n5.pdf>. Accessed from: jul. 22, 2016. doi:10.1016/j. injury.2011.06.011.

DORNBUSCH, P.T. et al. Avaliacao radiografica da aplicacao do polimero de mamona em falhas osseas induzidas em equinos. Archives of Veterinary Science, v.15, n.1, p.01-08, 2010. Available from: <http://repositorio.unesp. br/bitstream/handle/11449/72212/2-s2.0-78649646540. pdf?sequence=1&isAllowed=y>. Accessed from: jul. 22, 2016.

PRZEKORA, A.; GINALSKA, G. Biological properties of novel chitosan-based composites for medical application as bone substitute. Central European Journal of Biology, v.9, n.6, p.634-641, 2014. Available from: <http://www.degruyter.com/ downloadpdf/j/biol.2014.9.issue-6/s11535-014-0297-y/s11535014-0297-y.xml>. Accessed from: jul. 22, 2016. doi: 10.2478/ s11535-014-0297-y.

JACQUES, J.W. et al. The role of the ricin polyurethane as substitute of the bone autogenous graft in rabbits. Revista Colegio Brasileiro Cirurgioes, v.31, n.4, p.236-241, 2004. Available from: <http://www.scielo.br/pdf/rcbc/v31n4/ v31n4a04>. Accessed from: jul. 22, 2016.

LISTONI, A.J. Estudo do potencial de diferenciacao de celulastronco mesenquimais equinas oriundas do sangue da medula ossea cultivadas sobre um filme multicamadas de biopolimeros: quitosna, HA e CMC. 2013. 59f. Dissertacao (Mestrado em que especialidade?)--Instituto de Biociencias de Botucatu, Universidade Estadual Paulista Julio de Mesquita Filho, SP.

MARTINELI JUNIOR, W. Estudo histologico dos tecidos conjuntivo e osteoide em enxertos osseos com polimero de mamona: estudo experimental em ratos. 2011. 93f. Dissertacao (Mestrado em Ciencias)--Faculdade de Ciencias Medicas Universidade de Campinas, Campinas, SP.

MOREIRA, R.C. Estudo biomecanico do terceiro metacarpiano de equinos. 2008. 55f. Dissertacao (Mestrado em Medicina Veterinaria)--Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sao Paulo, SP. Available from: <http://www. teses.usp.br/teses/disponiveis/10/10137/tde-22042009-100210/>. Accessed: Jul. 13, 2010.

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SPIN-NETO, R. et al. Biomateriais a base de quitosana com aplicacao medica e odontologica: revisao de literatura. Revista de Odontologia da UNESP, v.37, n.2, p.155-161, 2008. Available from: <http://www.revodontolunesp.com.br/files/v37n2/v37n2a09. pdf>. Accessed from: jul. 22, 2016.

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VAN DE GRAAF, G.M.M. Avaliacao da resistencia mecanica a compressao axial de diferentes formulacoes de poliuretana de mamona com carbonato de calcio e de quitosana com fosfato de calcio. 2012. 84f. Dissertacao (Mestrado em Ciencias)--Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sao Paulo, SP.

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Rodrigo Crispim Moreira (I) * Guilherme Maia Mulder Van De Graaf (I) Cesar Augusto Pereira (II) Andre Luis do Valle De Zoppa (III)

(I) Programa de Pos-graduacao em Veterinaria e Cirurgia (VCI), Faculdade de Veterinaria e Zootecnia (FMVZ), Universidade de Sao Paulo (USP), 15053-794, Sao Jose do Rio Preto, SP, Brasil. E-mail:crispimr@hotmail.com. Corresponding author.

(II) Instituto de Ortopedia (IOT), Faculdade de Medicina e Zootecnia (FMVZ), Universidade de Sao Paulo (USP), Sao Paulo, SP, Brasil.

(III) Departamento de Cirurgia, Faculdade de Medicina Veterinaria e Zootecnia (FMVZ), Universidade de Sao Paulo (USP), Sao Paulo, SP, Brasil.

Received 06.10.15 Approved 06.16.16 Returned by the author 09.30.16 CR-2015-0838.R2

Figure 2--Graphic representation of construct deformations sampled by
the mechanical testing machine.

Deformation under loads up to 1000N   Deformation (mm).

ND-C                                  1.41
ND-M                                  0.48
ND-Q                                  0.35

Bar chart showing mean deformation values sampled by the mechanical
testing machine in the control (ND-C), castor oil polyurethane
(ND-M) and chitosan (ND-Q) groups. Source: (MOREIRA, R. C., 2014).

Note: Table made from bar graph.

Figure 3--Deformation readings given by uniaxial strain gauges
attached to the plate mid-point, in each group.

Plate Strain Gauge ([micro]d)   Relative Deformantion ([micro]d)

ND-C                            1906ral
ND-M                            311.10
ND-Q                            1900ral

Bar chart showing mean plate deformation values sampled via a uniaxial
strain gauge (mm), in the control (ND-C), castor oil polyurethane
(ND-M) and chitosan (ND-Q) groups. Note positive values (i.e., tension
on the plate surface). Source:
(MOREIRA, R. C., 2014).

Note: Table made from bar graph.

Figure 4--Deformation readings given by triaxial strain gauges
attached to the lateral and medial aspects of the distal fragment
during maximum load (1000N) non-destructive compressive testings.

Realative Deformation ([micro]d)   Lateral gages   Medial gages

ND-C                               52.63           27.23
ND-M                               -69.04          -67.1
ND-Q                               -33.35          5.9

Bar chart showing largest deformation values ([micro]d) sampled via
triaxial strain gauges attached to the lateral and medial aspects
of the distal fragment during maximum load non-destructive
testings, in the control (ND-C), castor oil polyurethane (ND-M) and
chitosan (ND-Q) groups. Positive and negative values indicate
tension and compression respectively. Source: (MOREIRA, R. C.,
2014).

Note: Table made from bar graph.

Figure 5--Deformation readings given by a triaxial strain gauge
attached to the palmar aspect of the distal fragment during maximum
load (1000N) non-destructive compressive testings.

Palmar Strain Gauge ([micro]d)   Relative Deformation ([micro]d)

ND-C                             -95.04
ND-M                             -78.68
ND-Q                             -96.45

Bar chart showing largest mean deformation values ([micro]d) sampled via
a triaxial strain gauge attached to the palmar aspect of the distal
fragment during maximum load non-destructive testing, in the
control (ND-C), castor oil polyurethane (ND-M) and chitosan (ND-Q)
groups. Negative values indicated compression. Source: (MOREIRA, R.
C., 2014).

Note: Table made from bar graph.
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Title Annotation:texto en ingles
Author:Moreira, Rodrigo Crispim; Van De Graaf, Guilherme Maia Mulder; Pereira, Cesar Augusto; De Zoppa, And
Publication:Ciencia Rural
Date:Dec 1, 2016
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