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148 Surface Modification of Magnesium and its Alloys for Biomedical Applications
The aim of histological preparation is to visualise reactions in tissue using an
appropriate stain. Use of different staining agents and suitable means of staining creates high-contrast images that allow structural analysis. A basic distinction can be
made between progressive and regressive techniques. It is assumed that the different
structures retai n the stain to differing extents (Lang, 2006). Other options are those of
indirect and direct staining and single or multiple staining (Lang, 2006). In histological examinations it is crucial that, following explantation of the sample, the materials, methods, and analysis used are comparable with other test materials, sites,
and research teams. A selection of staining agents and techniques are described
below.
4.5.3.1 Haematoxylin staining
The direct staining agent is haematein or oxidised haematoxylin (Romeis, 1989). The
haematoxylin stain is yellow brown in colour and is suited for progressive or regressive
staining of cell nuclei (Lang, 2006). Subsequent rinsing in water gives it its typical purple colour. Addition of eosin stains alkaline structures, such as cytoplasmic proteins, red.
4.5.3.2 Toluidine blue
This stain dyes cell nuclei a distinct blue colour and is suitable for making metachromasy visible (Schauer & Scheibe, 1959), a property that allows structures such as mast
cell granules and cartilage matrix to be stained purple. Toluidine blue provides a good
overall view and is particularly well suited for visualising bone tissue. Cells, cell
nuclei, osteoid seam, osteoclasts, and osteoblasts are stained different shades of
blue. Mineralised tissue appears pale blue.
4.5.3.3 Van Gieson’s stain
This is a triple stain suitable for viewing connective tissue. Cell nuclei appear blueblack; collagen, bright red; calcified bone, red; osteoid, muscle tissue, and cytoplasm,
yellow; and mast cell granules, red-brown. Amyloid, hyaline, and mucus are visible in
various tones between yellow and red (Romeis, 1989).
4.5.3.4 Masson-Trichrome-Goldner stain
Using this multiple stain, cells of mineralised and nonmineralised bone matrix show up
well. This property makes it the stain of choice for examining the morphology of noncalcified bone. The cell nuclei appear brownish black, and cytoplasmic staining makes
it possible to distinguish between osteoclasts and osteoblasts (Lang, 2006; Romeis,
1989; Schwarz et al., 2007).
4.5.3.5 Tartrate-resistant acid phosphatase
One of the group of enzymatic histological stains, this allows reliable detection of
osteoclasts,which appear a reddish-pink colour, and osteoclast progenitor cells
(Ballanti et al., 1997). It has been observed that, under certain conditions, other cells,
Bioabsorbable behaviour of magnesium alloys e an in vivo approach 149
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such as osteoblasts and osteocytes, may also be stained (Bianco et al., 1988; Nakano,
Toyosawa, & Takano, 2004).
4.3.5.6 Movat’s pentachrome stain
This is a multiple stain suitable for visualising different tissue types. Connective
tissue appears red; mineralised bone tissue, yellow ; mineralised cartilaginous tissue,
blue-green; and nonmineralised cartilaginous tissue, reddish-yellow. Collagen fi-
bres stain yellow; osteoid, dark red; cell nuclei, blue-black; and cytoplasm, a reddish colour.
4.5.4 Fluorescent microscopy
Another method of examining histological sections is fluore scen ce microscopy.
Monochromeor polychrome in vivo fluorescence labelling with flurochromes allows
the assessment of remodelling processes and the quantificationofbonegrowthover
time (Rahn, Bacellar, Trapp, & Perren, 1980; W itte et al., 2005). Rahn et al. (1980)
developed dosage regimens for animals and polychrom e labelling using five
different-coloured staining agents: xylenol orange, calcein green, tetracycline, alizarin complex, and calcein blue. These substances are generally applied subcutaneously or intravenously in an aqueous medium. The intravital staining dyes have
frequently been used in vivo in recent years; the only such stain to be used in
humans was tetracycline (An, 2003; Iwamoto, Takeda, Sato, & Yeh, 2004; Xu
et al. 2009).
4.5.5 Scanning electron microscopy and energy-dispersive
X-ray spectroscopy
Scanning electron microscopy (SEM) is suitable for ultrastructural assessment between implant and tissue (An, 2003). (See Section 4.4.4 on biodegeneration.)
4.5.6 Micro-computed tomography and histomorphometry
Micro-CT allows both two- and three-dimensional visualisation of bone and provides
clear views of bone structure (Bernhardt et al., 2004). The two-dimensional images
can also be used to obtain morphometric parameters (Parfitt, 1988). Histomorphometry makes it possible to assess structural changes in the surrounding tissue and the
implantetissue interface (An, 2003). As well as providing options for quantit ative
analysis, this method has a further advantage, namely that the specimen is available
for further mechanical and histological investigations and large quantities can be
examined (Ruegsegger, K oller, & Mueller, 1996; Wachter et al., 2001). In recent
years, this time-consuming evaluation has been simplified by computer programs
(Huffer, Ruegg, Zhu, & Lepoff, 1994; Martin et al., 2002). To correctly evaluate
the histomorphometric findings, it is necessary t o apply the same scann in g parameters, but not the standardised sectional planes with the same threshold level
150 Surface Modification of Magnesium and its Alloys for Biomedical Applications
(Ruegsegger et al., 1996). Analysis is carried out using quantit ative and semiquantitative point systems. Parfitt (1988) standardised the nomenclature and unified the terminology (see Table 4.6). Analysis includes the measurement of bone mass
(expressed as a percentage), bone volume/total volume (Smet et al., 2006), trabecular
thickness, and trabecular number (Gabetetal.,2006). Wachter et al. (2001) postulate
that histomorphometry is, in terms of bone assessment, superior to histological examination. Admittedly, one disadvantage is the lack of information on the biological
characteristics of bone, particularly the evaluation of periosteal and endosteal remodelling. However, studies by a number of research teams show that the outcome of
micro-CT analysis closely matches that of histological findings (Butz, Ogawa,
Chang, & Nishimura, 2006; Stopie et al., 2007; van Oosterwyck et al., 2000)(see
Figure 4.15).
4.5.7 Chemical analysis (blood, other specimens)
Especially with biodegradable materials, the question arises as to whether degradation
products influence not only the implantetissue interface but also directly or indirectly
affect (or are stored in) other organs. One current means of evaluating this is to test the
blood of animal subjects for magnesium. Another option is to examine the regional
lymph nodes or the kidney s, the organs of elimination. Several authors report unchanged magnesium serum concentration (Li et al., 2008; Wong et al., 2010; Xu
et al., 2007) or changes in organ function (Witte et al., 2010; Zhang et al., 2010,
2009) (see Table 4.7).
4.6 Testing of magnesium alloy in or on bone e special
considerations
This section begins with a discussion of the advantages and disadvantages of implant
materials currently used in the vicinity of bone marrow, intended to highlight the significance of magnesium for the manufacture of implants to be used in bone.
4.6.1 Metallic nonresorbable implants
Osteosynthesis materials generally used at present include high-allo yed stainless steel,
pure titanium, and titanium alloys (Disegi, 2000; Jain, Podworny, Hearn, Ande rson, &
Schemitsch, 1997; Pohler, 2000; Singh & Dahotre, 2007). Less expensive than tita-
nium, high-alloyed stainless steel is characterised not only by a high degree of mechanical strength and hardness, but also by good corrosion resistance (Disegi & Eschbach,
2000; Singh & Dahotre, 2007). Owing to their high density (almost double that of ti-
tanium), steel implants are substantially heavier than—and have considerably greater
elasticity than—titanium, which in turn is more elastic than the cortex of the bone (Dis-
egi & Eschbach, 2000; Pohler, 2000). Biocompatibility is affected by metal ions such
as nickel, chromium, and cobalt, which are released during the corrosion process
Bioabsorbable behaviour of magnesium alloys e an in vivo approach 151
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Table 4.6 Comparison of new with old terminology for selected
primary measurements (upper list) and derived indices (lower list)
on cancellous bone tissue; methods of calculating the latter are given
in original
Present
terminology
Trabecular
d
a
bone
Proposed
terminology
Bone volume
b,c
e
Abbreviation Units
e
BV/TV
volume (TBV)
(Relative) osteoid
Osteoid volume OV/BV %
volume (ROV)
(Absolute) osteoid
volume
f
(AOV)
(Relative) osteoid
Osteoid volume OV/TV %
Osteoid surface OS/BS %
surface (ROS)
g
(Active
) osteoblast
Osteoblast surface Ob.S/BS
surface (AOS)
i
(Mean
) osteoid seam
Osteoid thickness O.Th mcm
width (MOSW)
(Total) resorption
(Active
surface
j
(TRS)
k
) resorption
Eroded surface ES/BS %
Osteoclast surface Oc.S/BS
surface (ARS)
Osteoclast index (OI) Osteoclast number N.Oc/T.Ar
(Trabecular) specific
surface
n
(tSsp)
(Meani) wall
Bone surface BS/TV mm
Wall thickness W.Th. mcm
thickness (MWT)
o
surface
forming
p
(AFS)
Active
Mineralisation
(MF)
Calcification
(CR)
i
trabecular
Mean
plate thickness
r
q
rate
front
Mineralising
Mineralising
Mineral apposition
s
Trabecular
MS/BS %
surface
MS/OS %
surface
MAR mcm/d
rate
Tb.Th mcm
thickness
(MTPT)
%
h
l
m
%
%
/mm
2
2
/mm
3
Continued
152 Surface Modification of Magnesium and its Alloys for Biomedical Applications
Table 4.6 Comparison of new with old terminology for selected
primary measurements (upper list) and derived indices (lower list)
on cancellous bone tissue; methods of calculating the latter are given
in original e cont’d
Present
terminology
i
Mean
trabecular
a
Proposed
terminology
s
Trabecular number
b,c
Abbreviation Units
t
Tb.N /mm
plate density
(MTPD)
i
trabecular
Mean
plate separation
s
Trabecular
separation
t
Tb.Sp mcm
(MTPS)
Bone formation rate,
BMU level
(sV
Bone formation rate,
tissue level (
Bone formation rate,
volume referent
(BMU
u
)
S
Vf)
v
Adjusted apposition
rate
Bone formation rate BFR/BS mcm
Bone formation rate BFR/BV %/year
Aj.AR mcm/d
3
/mcm2/year
(vVf)
Mineralisation lag
time (MLT)
Sigma (duration of
Mineralisation lag
Mlt day
time
Formation period FP Day or year
formation) (of)
a
These are representative of current practice in different laboratories; it is not implied that all are used by any laboratory or
that any are used by most laboratories. Qualifying terms are in parentheses if their use is inconsistent between laboratories.
b
Measurement name only; need for inclusion of source and/or referent in name varies with context, as discussed in original.
c
Three-dimensional expression except where otherwise stated.
d
Source almost always included in name for this quantity, often omitted for others.
e
The full name and abbreviation would be cancellous bone volume/tissue volume (Cn-BV/TV); see notes b and d.
f
Also called osteoid volume density.
g
Designation usually based on morphology.
h
OS is another frequently used referent.
i
Including “mean” as part of the name should imply direct rather than indirect measurement and may lead to confusion with
the mean value in a group of subjects.
j
Also termed crenated or Howship’s lacunar surface.
k
Designation usually based on presence of osteoclasts.
l
ES sometimes used as an additional referent.
m
Bone perimeter is an alternative referent; note that expression must be 2D, not 3D.
n
Also called surface density.
o
Note wide variety of meanings presently given to the term “active.”
p
Often called “labeled surface” or “tetracycline surface” (double, single, or both).
q
Or calci fication.
r
Or mineralisation.
s
Note ambiguity between “trabecular” as a type of bone tissue and as a type of individual structural element.
t
Must specify whether calculated according to parallel plate or rod model or measured directly.
u
Many other synonyms given in original.
v
Equivalent to rate of bone turnover.
Parfitt (1988).
Bioabsorbable behaviour of magnesium alloys e an in vivo approach 153
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Figure 4.15 The histological sections (1) and their corresponding micro-CT images (2) for a
titanium implant placed in the trabecular bone of the condyle of the left tibia of the rabbit. The
left side of the implant corresponds to the ventral side of the tibia and the right side of the implant
to the dorsal side of thetibia (Stopie et al., 2007).
Table 4.7 Blood biochemical examination of rat before implantation
and 15 weeks post-implantation of MgeMneZn alloy in a bone
Before
implantation
Items
(n [ 2)
BUN (mmol/L) 7.07 0.32 8.47 0.51 5.99e14.99
CREA (mmol/L) 35.6 3.1 39.0 3.1 29.2e53.9
UA (mmol/L) 35 950 10 71.3e445.5
Kþ(mmol/L) 6.20 0.99 6.40 1.0 3.8e5.4
þ
Na
(mmol/L) 141.5 0.7 145.0 1.4 126e155
Cl
(mmol/L) 100.0 4.2 101.5 2.1 103.0e115.1
2þ
Ca
(mmol/L) 2.95 0.04 2.93 0.07 3.1e5.2
P (mmol/L) 2.93 0.08 2.99 0.42 1.0e3.55
2þ
Mg
(mmol/L) 1.18 0.05 1.28 0.13 1.32 0.03
Xu et al. (2007).
15 Weeks postimplantation
(n ¼ 2)
Recommended
13
level
14
14
14
154 Surface Modification of Magnesium and its Alloys for Biomedical Applications
(Hallab, Jacobs, & Black, 2000; Singh & Dahotre, 2007; Ungethuem & Winkler-
Gniewek, 1984), potentially resulting in allergies and in septic and aseptic reactions
(Singh & Dahotre, 2007). Titanium and its alloys exhibit excellent biocompatibility
and corrosion resistance, as well as favourable mechanical properties (Singh &
Dahotre, 2007) (see Table 4.8). Their only notable disadvantages are that their elastic-
ity is higher than that of bone and both their wear resistance and their resistance to
shearing forces is lower (Singh & Dahotre, 2007). Due to their high-elasticity module,
the above-mentioned metallic implants show substantially higher rigidity than bone,
leading to stress shielding and, in turn, to a delay in the healing process, to
Table 4.8 Characteristics of strategic orthopaedic metallic materials
Ti and Ti-base
Characteristics Stainless steels Cobalt-base alloys
alloys
Designation ASTM F-138 (316
LDVMO)
Principal alloying
elements (wt. %)
Advantages Cost, availability,
Disadvantages Long-term
Application Temporary devices
Fe (balance)
Cr (17e20)
Ni (10e14)
Mo (2e4)
processing
behaviour, high
modulus
(fracture plates,
screws, hip nails)
used for THRs
stems
ASTM F-75 ASTM
F-799 ASTM
F-1537 (cast
and wrought)
Co (balance)
Cr (19e30)
Mo (0e10)
Ni (0e37)
Wear resistance,
corrosion
resistance,
fatigue strength
High modulus Low wear
Dentistry casting,
prostheses stems
load-bearing
components in
TJR (wrought
alloys)
ASTM F-67 (ISO
5832/II) ASTM
F-136 (ISO
5832/II) ASTM
F-1295 (cast and
wrought)
Ti (balance) A1 (6)
V (4) Nb (7)
Biocompatibility
corrosion
resistance
minimum
modulus fatigue
strength
resistance, low
shear resistance
In THRs (with
modular Co-CrMo or ceramic)
femoral heads,
long-term
permanent
devices (nails,
pacemakers)
Singh and Dahotre (2007).
Bioabsorbable behaviour of magnesium alloys e an in vivo approach 155
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development of pseudoarthrosis and to pathological fractures following implant
removal (Gogolewski, 2000; Hoffmann, 1995).
Tissue metallosis around the implant was found to be another drawback of nonresorbable metallic implants, potentially leading to hypersensitivity, toxicity, and cancerogenicity (Agins et al., 1988; Mcdonald, Enneking, & Sundaram, 2002; Radhi,
Ibrahiem, & Al-Tweigeri, 1998; Ward, Thornbury, Lemons, & Dunham, 1990). The
generation of artefacts in CT (Link et al., 2000; Mahnken et al., 2003) and magnetic
resonance imaging (MRI) (Disegi & Eschbach, 2000; Pohler, 2000) is also
disadvantageous.
4.6.2 Resorbable polymer-based implants
PGAs, PLAs, and their copolymers are among the main substances used for
manufacturing osteosynthesis materials (Claes & Ignatius, 1998). Their elasticity
model resembles bone properties and thus prevents stress protection (Hofmann,
1995). However, rapid loss of strength and rigidity irrespective of the degradation pro-
cess presents a problem (Hofmann, 1995). This means that the implants are suitable
only for the treatment of non-load-bearing bones (Hofmann, 1995; Rehm et al.,
1997; von der Elst et al., 2000). Chemical composition, crystallinity, release of degra-
dation products, implant design, and surface properties are major determinants of
biocompatibility (Gogolewski, 2000; Hoffmann, Weller, Helling, Kre ttek, & Rehm,
1997; Wintermantel, 2002) (see Table 4.9). Several research teams investigating
biocompatibility regard foreign-body reactions as having negative effects ranging
from silent osteolysis to severe inflammation (B€ostman, 1991, 1992; Claes & Ignatius,
1998; Hoffmann et al., 1997; Suganuma and Alexander, 1993; Rehm, Helling, &
Claes, 1994). Another disadvantage is that the implants cannot be imaged using CT
and MRI (Hofmann, 1995; Rehm et al., 1997).
4.6.3 Suitability of magnesium and its alloys for metallic
implants
Magnesium and its alloys have an elasticity module that resembles that of the cortex of
the bone and generally possess similar mechanical properties (Kaese, 2002; Staiger
et al., 2006; Zhang et al., 2009). A large number of in vitro and in vivo studies postulate
that magnesium ions, magnesium, and degradation products (i.e., magnesium hydroxide) have a positive influence on the bone-remodelling processes and an osteoconductiveeffect (Castellani et al., 2011; Janning et al., 2010; Pietak, Mahoney, Dias, &
Staiger, 2008; Revell, Damien, Zhang, Evans, & Howlett, 2004; Rude et al., 2006;
Witte et al., 2005; Witte et al., 2007; Yamasaki et al., 2002; Yamasaki et al., 2003;
Zreiqat et al., 2002) (see Table 4.10 and Figure 4 .16). It has been shown that calcium
phosphate coating of implants has a positive effect on bone healing and thus ensures
better implant integration (Hayakawa, Yoshinari, Nemoto, Wolke, & Jansen, 2000).
The increase in pH value during degradation may also lead to osteoblast stimulation
(Kaese, 2002). As soon as the capacity for resorption of the surrounding tissue has
been exceeded, gas bubbles form (Witte, Hort, et al., 2008). These are resorbed
156 Surface Modification of Magnesium and its Alloys for Biomedical Applications
Table 4.9 Degradation rates of various resorbable polymeric implants
Complete
Polymer (implant
form)
Retained strength
(%/week)
Total strength loss
(months)
resorption time
(months)
Polydioxanone
(sutures)
Poly(glycolide-
co-trimethylene
carbonate)
(sutures)
Polyglycolide
(sutures)
PoIy(glycolide-co-
lactide)(sutures)
Poly(
L-lactide)
(solid, nonoriented)
L-lactide)
Poly(
(solid, oriented)
Poly(L/DL-lactide)
70/30% (solid,
nonoriented)
Poly(L/DL-lactide)
80/20% (solid,
nonoriented)
Poly (L/DL-lactide)
80/20% (porous
membranes)
60/4 (40/6) 26
55/4 (14/7) 2.5 6
30/2 1 4
30/3 1 2
40/8 3 1e72
80/12 (65/25) 1e736e72
a
40/12
50/12
20/12
a
a
324e36
424e36
412e18
a
a
a
a
Values to be proven by further experiments.
Gogolewski (2000).
over a period of several weeks; no negative effect on the surrounding tissue could be
demonstrated (Erdmann et al., 2011; Hampp et al., 2012; Kraus et al., 2012; Li et al.,
2008; Witte et al., 2005; Zhang, Xu, et al., 2009). Several research teams suggest that
compact implantation material may hinder the closure of a borehole in the cortex by
preventing an influx of osteoprogenitor cells (Henslee et al., 2011). The rabbit animal
model is favoured and recommended for in vivo studies (Meyer-Lindenberg et al.,
2007; Tsai et al., 2008; Carranza-Bencano et al., 1999; Rudert, 2002; ISO 10993-6:
2007).
It is essential that bone implants are sufficiently stable, especially during the
first few weeks. Based on the assumption t hat fractures of the thigh take around
Bioabsorbable behaviour of magnesium alloys e an in vivo approach 157
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Table 4.10 Maximum push-out force (F
) and energy absorption to failure (EA) for each implantation
(s
u
), ultimate shear strength
max
period and implant type (median [first to third quartiles],
ManneWhitney U-test)
Implantation
period Mg-alloy Ti-alloy P
4 weeks n 12 13
F
(N) 49.35 (37.63e55.53) 23.58
max
(N/
s
u
mm
2.43 (1.80e2.81) 1.12 (0.57e1.50) 0.002
2
)
EA (mJ) 1.07 (0.92e1.40) 0.39 (0.17e0.79) 0.004
12 weeks n 12 7
(n) 151.87
f
max
(133.30e185.33)
(N/
s
u
mm
6.17 (5.29e7.23) 4.14 (3.25e4.55) 0.002
2
)
EA (mJ) 12.45 (9.08e18.70) 3.24 (2.23e7.41) 0.002
24 weeks n 89
(N) 185.16
F
max
(157.73e221.9S)
(N/
s
u
mm
7.65 (6.61e8.71) 2.14 (1.26e3.53) 0.001
2
)
EA (mJ) 22.64 (11.05e36.86) 0.70 (0.36e1.46) 0.004
(11.55e30.99)
100.83
(80.10e109.59)
44.78
(30.57e90.10)
0.001
0.003
0.001
Castellani et al. (2011).
12 weeks to heal (Gu et al., 2011), Staiger et al. (2006) advocate that biodegradable
implants should have sufficient mechanical properties for at least this length of
time. Hutmacher (2000) recommends that osteosynthesis materials used in bone
should have biomechanical stability of around 2 months prior to the expected onset
of controlled degradation. The degeneration rate of magnesium implants differs
depending on their location in the bone; it is greater in the medullary cavity than
in the cortical bone (Erdmann et al., 2011; Xu et al., 2007; Zhang, Xu, et al.,
2009)(seealsoSection 4.2). In a study by Xu et al. (2007), the magnesium alloy
in rat femurs degraded by 10e17% over the first 9 weeks, with only 50% of t he
original amount remaining after a total of 18 weeks. However, in the treatment
of iatrogenic bone/cartilage defects with magnesium sponges made of the AZ91
alloy, the formation of degradation products prevented the onset of the h oped -for
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