Characterization of PLA/Bovine Bone Composite

as A Candidate Material For Artificial Bone

Sigit Tri Wicaksono a), Adven FN Hutajulu b), Hosta Ardhyananta c), Amaliya
Rasyida d)

Department of Materials Engineering, Institut Teknologi Sepuluh Nopember

a)
sigittriw@gmail.com
b)
adven13@mhs.mat-eng.its.ac.id
c)
hostaa@mat-eng.its.ac.id
d)
amaliyarasyida@mat-eng.its.ac.id

Abstract-Artificial bone serves as a substitute for damaged bone that can be planted into human body without any
rejection. Polylactic acid (PLA) can be used as a material to produce artificial bone using 3D printing application. In this
study, PLA as receive (AR) was composited with bovine bone powder. The addition of bone powder was distinguished
by percentage that ranged as 10%wt, 20%wt, 30%wt, 40%wt, and 50%wt. FTIR analysis showed that bovine bone
powder was not chemically bonded with PLA-AR. Whereas XRD analysis proved that there was no new phase exist in
the composite material. SEM analysis proved that bovine bone powder was dispersed interstitially inside the pore of
PLA-AR. However, the bovine bone powder was agglomerated. Hardness testing proved that the addition of bone
powder resulted in the increasing of hardness value. Using Direct Mechanical Testing (DMA) analysis, it was found that
the strength of material decreased according to the decreasing of the value of tensile strength while the elongation at
break was fluctuated. The material was tested bacteria to show its resistance. It was tested to to positive gram bacteria
Bacillus Cereus and negative gram bacteria Eschericia Coli. From this evaluation, it was known that the composite
material PLA/Bovine bone powder has no anti-bacteria properties

INTRODUCTION

Bone is the most important part of the human structure. Bone serves as to support and shape human body.
Human bone consists of two main components which is 30 percent organic and 70 percent inorganic matters.
Organic matter consists of osteoblast cell, osteoid cell, and osteoclast cell. Inorganic bone components consist of
collagen proteins, glycosaminoglycans, and mineral that consists of calcium phosphate compounds that called
hydroxyapatite [1]. Compared with traditional autograph and allograph procedures, bone tissue engineering based on
autogenesis cells or tissue transplantation can reduce donor deficiency issue, transfer of pathogens, and immune
rejection. This has led to the research development since tissue engineering concept widespread [2].
The development and modification of bone has been done to achieve optimum interaction between the body and
the implantation material. This objective has been achieved by modification of implants’ surface topography,
chemical properties, energy, stress and material composition [3]. Instead of using steel material to support or replace
bone, polymeric materials can also be used in the production of artificial bone such as Polylactic acid (PLA). PLA is
biodegradable and bioactive aliphatic thermoplastic polyester derived from renewable natural resources such as
corn, tapioca, potatoes, starch and sugarcane. Synthetic polymers such as PLA has high mechanical properties,
however, its interactions with cells are very poor and produce acid degradation products [4].
This study aims to synthesize and characterize artificial bone candidate material from polylactic acid (PLA)
which is composed with cow bone by chemical blending process. From this process it is expected to obtain the
charactheristics of material and its interactions with bacteria. In this study, the percentage of bovine bone increase
was as much as 10wt%, 20wt%, 30wt%, 40wt% and 50wt%.

METHODS

The Bovine bone is synthesized to obtain powder material as the filler component to evaluate the effect of bovine
bone in PLA composite. The selection animal for includes several factors such as cost, availability, acceptance of
the social environment, and the ease of animal care [3]. In this case, this study decided to use bovine bone since this
material can be used as polyester to enhance the material properties in biomaterial application. The Bovine used in
this study was a 3 years adult bovine. The bones obtained were cranial skull consist of parietal bone, squamous
temporal bone, frontal bone, and occipital condyle. The skull was destroyed using compressive strength machine
into small pieces. It cleaned from soft tissue such as muscle and ligaments. The bone was boiled in 100 oC water for
4 hours to clean the fat. The boiling process was done repeatedly until the fat was clean.
Furthermore, the bone was rinsed with water repeatedly. Then, it immersed in 30 percent Hydrogen Peroxide for
4 hours. This solution oxidizes impurities on the surface layer and killed the bacteria. After that, the bone was rinsed
many times and soaked in water for 5 hours to cleans the hydrogen peroxide residue. The bone was the dried under
sun light for 12 hours aimed to obtain the properly dried bone. It then blended to minimize the powder size. The
powder filtered using vibrator sieving machine with frequency of 10Hz for 20 minutes. From this work, it obtained
bovine bone powder less than 40 um.
The process of synthesis and characterization of composite materials PLA/Bovine Bone began by dissolving
PLA as received (AR) filaments. The filament melting process was chosen since it was easier to perform and at the
cheaper than other. Material used was a white filament PLA-AR with diameter of 1.75 mm. This material was cut
into pieces into pellets with a length of approximately 1 cm. PLA-AR with a smaller size wass then immersed in
chloroform with a concentration of 99%. The mixing was using a liquid solvent. PLA-AR was generally soluble in
some liquid solvents such as dioxane, acetonitrile, chloroform, methylene chloride, trichloroethane, and
dichloroacetic acid [6].
PLA-AR was immersed for 30 minutes in a beaker glass and covered with aluminum foil to reduce the
chloroform evaporation. After the PLA-AR pellet had melted, PLA-AR was stirred in a magnetic stirrer with 50oC
temperature and 600 rpm for 30 minutes. After that, the bone powder was added into the solution. The mixing then
continued with a temperature of 65oC for 1 hour with 600rpm speed. The temperature was increased so that the
chloroform liquid evaporates faster to accelerate the polymerization of PLA-AR. After one hour, the mixture then
looked more viscous and formed paste. The paste was poured into a mold. There were two types of mold such as flat
glass to obtain thin and a box mold of aluminum to obtain block sample. The samples were dried in a room
temperature. The specimens were formed in accordance with ASTM D638 standard, which is standard for plastic
material tensile test.

(a) (b)
FIGURE 1. Specimen (a) film (b) square material with thickness minimum 6 mm.

Figure 1a shows the shape of the specimens that have been printed for the tensile test using DMA. For hardness
tests, the material was formed in accordance with ASTM D22400-00. This material was formed with minimum
thickness of 6mm. Hardness testing specimen was showed by Figure 1B.

RESULT AND DISCUSSION

The PLA-AR that has been synthesized by chemical blending was observed using Scanning Electron Microscopy
(SEM). Figure 2 shows the SEM result of PLA_AR with several magnifications. Figure 2a shows the morphology
structure of rough and clumpy PLA-AR. Figure 2d shows 5000 times magnification. This picture further clarified
the surface appearance of porous PLA-AR. This happens because during the synthesis process, the chloroform
evaporates slowly and unevenly that made the PLA-AR agglomerated into granular grains and attaches to each
other. Microscally this formed a lot of pores in the PLA-AR which is not appearing visually. PLA-AR has a pore
size of about 3.17 um [7].
All of samples with predetermined comparisons were characterized by FTIR, XRD, and SEM. From the FTIR
analysis, it was known that the material has several peaks which were described by stretching of CH3 groups in the
range 3000 - 2850 cm-1 with a wave peak of 2996.17 cm-1. This cluster belongs to the aromatic ring group.
Subsequently there was a weakened CH bonding group at the peak of 1995.02 cm-1 with 98.597 intensity. This
cluster also includes aromatic compounds. In addition there was also CH3 bending medium type with peak of
1382.22 cm-1 at the point of 85.442 intensity. At the peak wave of 1746.96 cm-1, there was a stretch of C=O group
with a strong intensity of 53.00. This indicated that in the PLA-AR material, there was a 6-membered lactone group
which is an ester group [8].

FIGURE 2. SEM PLA-AR material with magnification (a) 500x, (b) 1000x, (c)2500x, and (d)5000x

Further analysis showed that there was a very wide extent of C-C[O]-O groups. This characterized anhydride. To
the right of the graph there was CH bending with a wave peak height of 870.52 and 754.11. Both illustrated the
formation of the disubtitute and 1-2 disubtitute groups with strong absorption intensities. The FTIR test graph on
PLA-AR material could be seen in Figure 3.

FIGURE 3. FTIR of Poly-Lactic Acid as received. FIGURE 4. FTIR of bovine bone powder

Figure 3. shows the results of PLA-AR interpretation with FTIR and Figure 4 shows the interpretation of bovine
bine. From the analysis it was found that at peak of 3287.50 it could be seen that, at the intensity of 94.742 there was
a stretching of NH2. This cluster was hydrogen bonded and produced secondary amides. This group also allowed the
formation of organic acid compounds or phenols. Other organic acids may also formed like alanine, lysine and
hydroxyproline. These monomers bond to peptide among carboxyl groups and the amino groups. This bond formed
un-interrupted triple helix bond that represent 95 percent of tropocolagen molecule and two non-helical domains
(telopeptides) containing COOH-protein and -NH2 terminal [9].
Another absorption peak can be seen in the absorption position 2919.33 which showed the presence of CH3 bond
stretching. This bond was evident in the range 3000-2800 cm-1 which described the presence of methylene group
groups or aliphatic bonds. Another carbon bond was shown by the C≡N bond at 2360.90 cm-1 with peak intensity of
96,630. This indicated the presence of alkynes groups in the series of crushed bovine bones. Another peak was
observed in the range 1655 - 1610 cm-1 which was exactly in position 1636.96 cm-1 with an intensity of 90,751
indicating the presence of -O-NO2 groups with asymmetric stretching type. This indicated the presence of organic
nitro compounds. There were several peaks that were in the fingerprint. So the peak absorption peaks present in that
range were not discussed in this study. After observing the FTIR graph of PLA-AR and bovine bone powder,
follow-up observations were taken at PLA-AR composite material and bovine bone with addition of 10% Wt, 20%
Wt, 30% Wt, 40% Wt, and 50% wt bone powder.

FIGURE 5. FTIR of all samples

After the addition of bovine bone into PLA-AR, Figure. 5 showed the FTIR graph of PLA-AR sample, Bovine
bone powder and composite mixture of PLA-AR and bovine bone. From the mixing it could be observed that, it was
very difficult to see any changes in specimens with the addition of bovine bone. This was most likely due to the
absorption peaks of PLA-AR and bovine that bone covering each other. If observed, the position of the apex peak at
2919.33 on the cow bone is covered by the absorption peak at 2996.17 in the PLA. The same thing happened at the
peak of 1019.43 on cow bone, covered by peak 1080.86 at PLA-AR. From the testing above it could be concluded
that, PLA-AR and bovine bones did not form bonding chemically. This might be because PLA-AR was relatively
hydrophobic. This was because PLA-AR has a water contact angle of approximately 80o. This results in PLA-AR
having low cell affinity and difficult to bind with bovine bone.
PLA-AR, is chemically inert so that PLA-AR does not have a cluster chain with a reactive side [10]. This causes
the PLA-AR surface to not bind chemically to the surface of cow bone powder. The analysis using the X-ray
diffraction method as the initial step to identify phase formation in the sample was done by search-match with
standard data using High Score Plus. From the XRD analysis, there was a very high peak with 2theta position at
16.6874 with an intensity of 100 percent. The peak was adjacent to the peak of 16.5275 with 89.37 percent intensity.
Analysis using PDF card with ICCD reference code 00-054-1917, it was found that at that peak there was relatively
similar to the phase emerging on α-Poly (D-Lactide) with empirical formula of C3H5O3 and chemical formula
(C3H5O3). In the XRD test of bovine bone, obtained a very sharp 2theta peak with a position of 32.0949 with 100
percent intensity. The second highest peak was in the 2nd theta position of 26.1711 with 25.28 percent intensity.
Analysis using Highscore, showed that the compound which was contained in cow bone powder in this test was
Calcium Phosphate Hydroxide compound or commonly known as apatite with Ca5HO13P3 empirical formula
indexed with ICCD reference code 00-009-0432.
 FIGURE 6. XRD of addition bovine bone into PLA-AR
With the addition of bovine bone composition to the composite sample material, it could be noted that the
highest peak of PLA-AR was decreasing, while the highest peak of bovine bone was increased. Figure 6 shows the
association of adding bovine bone percentage to the composite mixture. From these figures it can be concluded that
the more bones were used in the composite material, the highest peak of PLA-AR would decrease, while the XRD
peak of bovine bone apatite would be more clearly visible along with the increasing of bovine bone powder
dissolved in the PLA-AR. It could be seen that the lower absorption of PLA-AR shows an increasingly amorphous
distribution [7].
From this analysis there was no new phase occurred which prove that PLA-AR and bovine bone have no
chemical bonding. This was similar with the result of FTIR test. The presence of bone powder in PLA-AR in these
specimens showed that the bovine bone powder only filled the pores of the PLA-AR by chemical blending process.
This is further clarified by the analysis by using (Scanning Electron Microscopy) SEM. Through SEM testing with
magnification of 5,000 times, it could be seen that there was no bone powder distribution in the PLA-AR composite
specimen with the addition of bone powder of 10% wt to 40% wt. This was shown clearly by Figure 7.

FIGURE 7. SEM of composite materials with magnification 5000X (a) PLA-AR

(b) PLA/BB 10%wt (c) PLA/BB 20%wt (d) PLA/BB 30%wt (e)PLA/BB 40%wt (f)PLA/BB 50%wt.

In Fig. 7, the bovine bone powder was not visible on specimens with the addition of 10% wt, 20% wt, and
40%wt. it was because the number of bovine bone powder was very small compared with the composition of PLA-
AR. The bovine bone powder contained in the specimen was agglomerate on the bottom layer and was covered by
the PLA-AR layer. During the polymerization process, PLA-AR floated above the powder layer and the bone
precipitates coated under the specimen. This might be possible due to the density of the powder that greater than the
density of PLA-AR. The density of apatite is 3.19 g/cc, While the density of PLA-AR type is 1.25 g/cc.
Fig. 7f showed that the distribution of bovine bone powder in the PLA-AR specimen clearly showed that the
bovine bone powder filled the PLA-AR pore pores as indicated by the red arrows. However, bovine bone powder
looks clumped and bond to each other because it has a hydrophilic characteristic to bone grain of bone powder while
the PLA-AR was hydrophobic. This might be because the alanine content in bone was hydrophobic [11]. Bovine
bone powder seen on the specimen with a percentage of 50% addition seen in SEM testing because the level of bone
powder in the specimen is quite a lot so that there were some grains which came out of the cavity of the PLA-AR
material.
From the Dynamict Mechanical Analysis (DMA), obtained information that the strength of PLA-AR was 8.89
MPa. With the addition amount of cow bone powder, obtained the decreasing of material strength (tensile strength)
along with the addition of cow bone powder. This matter might occur due to the occurrence of agglomeration of
bovine bone powder that resulting in the declining of the physico-mechanical properties [12]. Young modulus and
tensile modulus was increase simultaneously with the increasing of bovine bone powder. PLA-AR in this research
has 1.98 GPa young modulus and 2.1 GPa Tensile Modulus. Human cortical bone has young modulus ranges from 3
yo 30 Gpa [12]. Table 2.1 showed mechanical properties of material using DMA analysis. From Table 2.1 could be
concluded that to meet the modulus young of cortical bone, it is necessary to add bone powder at least 50 percent of
the total weight of the specimen. The mechanical properties of the material could be seen from Table 1 and Figure 8.

TABLE 1. Mechanic properties of material using DMA testing

Material Tensile Young Elongation at Tensile
Composition Strength Modulus (GPa) break (%) Modulus (GPa)
(MPa)
PLA-AR 8.89 1.98213 0.2666 2.1
PLA-AR/BB 30%wt 5.44 2.69260 0.1436 3.5
PLA-AR/BB 50%wt 3.46 4.17656 0.0828 4.2

FIGURE 8. Strain vs stress of material PLA-AR and its composites

From Table 1 it could be concluded that elongation at break increased simultaneously with the increasing of the
bovine bone powder weight. This showed that material elasticity decreased with the increasing of bovine bone
powder since bovine bone is brittle and the collagen has been removed from preparation process [5]. According to
ASTM D2240, the hardness test was taken to the materials. Figure 9 showed the effect of the increasing of bovine
bone powder to the hardness of the specimen. Testing was taken to six materials and obtained the hardness is
increased according to the increasing weight of the bone powder. Figure 9 shows the hardness of the material PLA-
AR that reach 62.1D. The hardness of the specimen felt due to the addition of bone powder as much as 10% wt.
However, the hardness of the specimens increased with the increase in the percentage of bovine bone powder. This
was due to the uneven distribution of cow bone powder throughout the surface of the material. This was indicated by
the SEM test results that confirmed the data that the bone powder did not spread evenly.
 70 63,9 65,5
62,1

Materials Hardness (Shore D)

60 54,5 56,1

50 44,2
40
30
20
10
0
0 10 20 30 40 50
Percentage weight addition of bovine bone powder

FIGURE 9. Hardness of the materials

Observations of bacterial reactions to the material had been successfully performed on the material. This test was
performed on PLA-AR materials and composite materials to analyze the source of bacterial reactions by using
diffusion method. The diffusion method occurred because the anti-bacterial compound diffused into the agar
medium so that there would be no bacteria survive around the around the pit. It was marked by the formation of
inhabitation zone. The width of the inhibitory diameter, indicating the inhibition of the antibacterial compound
[13,14]. Figure 10 shows the activity of E.Coli and Bacillus Aureus bacteria on PLA-AR/ Bovine Bone composite
materials. Table 2 shows the measurements of bacterial growth diameter on the agar specimens. The diameter of the
bacterial inhibition zone could be calculated by measurement. If the inhibition zone diameter of 0-15mm, it was
called resistant bacteria. Whereas if the diameter of 15-20 called bacteria are intermediate and if larger than 20 mm
was called bacteria were sensitive. From Table 2 it could be concluded that PLA-AR composite material and bovine
bone were sensitive to bacteria. This might occur because the gram-positive cell wall was a peptidoglycan which
one of its constituents was a hydrophobic amino acid D-alanine [11].

(a) (b)
FIGURE 10. Materials reaction to the growth of bacteria (a) E.Coli (b) B. Aureus

TABLE 2 Measurement of bacteria inhibition

Inhibition Zone Diameter (mm)

Specimen Escherichia Coli Bacillus Aurelus
I II Average Property I II Average Property
CHCl3 9 10 9.5 R 14 6 10 R
PLA/CHCl3 10 0 5 R 9 7 8 R
HCl 17 19 18 R 31 26.5 28.75 R
BB/HCl 15 19 17 R 29 20 24.5 R
PLA/BB /CHCl3 0 7 3.5 R 12 8 10 R
 *I: Repeating 1 II: Repeating 2 R:Resisten

Alanine was also formed in bovine bone powder. This made the bacteria quite familiar with bovine bone powder
and could survive under normal conditions of bovine bone. This resulted in the composite material of PLA-AR/BB
that had not have bacterial resistance. PLA-AR in this study did not have antibacterial properties. This was proven
by the presence of bacteria that grew on the sample of PLA-AR. To obtain antibacterial properties, PLA-AR should
be developed in order to release allyl isothiocyanate to fight bacteria [15].

CONCLUSION

Based on the results of testing and analysis of data that has been done on the composite PLA-AR and bone
powder, the following conclusions could be drawn: Synthesis of bone candidate material could be done using PLA-
AR material and bone powder using chemical blending method. The addition of bone powder in composite material
has an effect on material characteristics. With the addition of cow bone powder, the strength of the material was
reduced by the marked decrease in the value of tensile strength. Material hardness decreased when the mass of cow
bone was increased 10% then increased as the weight of bovine bone increased. The addition of bone powder to
PLA-AR by chemical blending method did not produce chemical bond. Both materials bind mechanically so that
there was no new phase appeared in SEM, XRD, and FTIR analysis. PLA-AR/BB composite materials did not have
resistance to bacteria. If this material were in a bacterial contaminated environment, bacteria could grow and
multiplied in the environment around this material.

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