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Comparative Structure-Property Characterization of Poly(3-Hydroxybutyrate-Со-3-Hydroxyvalerate)s Films Under Hydrolytic And Enzymatic Degradation: Finding a Transition Point in 3-Hydroxyvalerate Content


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The hydrolytic and enzymatic degradation of polymer films of poly(3-hydroxybutyrate) (PHB) of different molecular mass and its copolymers with 3-hydroxyvalerate (PHBV) of different 3-hydroxyvalerate (3-HV) content and molecular mass, 3-hydroxy-4-methylvalerate (PHB4MV), and polyethylene glycol (PHBV-PEG) produced by the Azotobacter chroococcum 7B by controlled biosynthesis technique were studied under in vitro model conditions. The changes in the physicochemical properties of the polymers during their in vitro degradation in the pancreatic lipase solution and in phosphate-buffered saline for a long time (183 days) were investigated using different analytical techniques. A mathematical model was used to analyze the kinetics of hydrolytic degradation of poly(3-hydroxyaklannoate)s by not autocatalytic and autocatalytic hydrolysis mechanisms. It was also shown that the degree of crystallinity of some polymers changes differently during degradation in vitro. The total mass of the films decreased slightly up to 8–9% (for the high-molecular weight PHBV with the 3-HV content 17.6% and 9%), in contrast to the copolymer molecular mass, the decrease of which reached 80%. The contact angle for all copolymers after the enzymatic degradation decreased by an average value of 23% compared to 17% after the hydrolytic degradation. Young’s modulus increased up to 2-fold. It was shown that the effect of autocatalysis was observed during enzymatic degradation, while autocatalysis was not available during hydrolytic degradation. During hydrolytic and enzymatic degradation in vitro, it was found that PHBV, containing 5.7–5.9 mol.% 3-HV and having about 50% crystallinity degree, presents critical content, beyond which the structural and mechanical properties of the copolymer have essentially changed. The obtained results could be applicable to biomedical polymer systems and food packaging materials.
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Polymers 2020, 12, 728; doi:10.3390/polym12030728
Structure-Property Characterization of
Films Under Hydrolytic And Enzymatic Degradation:
Finding a Transition Point in 3-Hydroxyvalerate
Vsevolod A. Zhuikov
*, Yuliya V. Zhuikova
, Tatiana K. Makhina
, Vera L. Myshkina
Alexey Rusakov
, Alexey Useinov
, Vera V. Voinova
, Garina A. Bonartseva
Alexandr A. Berlin
, Anton P. Bonartsev
and Alexey L. Iordanskii
Research Center of Biotechnology of the Russian Academy of Sciences. 33, bld. 2 Leninsky Ave., Moscow
119071, Russia; (Y.V.Z.); (T.K.M.); (V.L.M.); (G.A.B.); (A.P.B.)
Federal State Budgetary Institution “Technological Institute for Superhard and Novel Carbon Materials”,
7a Tsentralnaya street, Troitsk, Moscow 108840, Russia; (A.R.); (A.U.)
Faculty of Biology, M.V. Lomonosov Moscow State University, Leninskie gory, 1-12, 119234, Russia;
Research Center of Chemical Physics the Russian Academy of Sciences. 4, Kosygin str., Moscow 119991,
Russia; (A.A.B.); (A.L.I.)
* Correspondence:; Tel.: +79153207380
Received: 20 February 2020; Accepted: 20 March 2020; Published: 24 March 2020
The hydrolytic and enzymatic degradation of polymer films of poly(3-hydroxybutyrate)
(PHB) of different molecular
mass and its copolymers with 3-hydroxyvalerate (PHBV) of different
3-hydroxyvalerate (3-HV) content and molecular
mass, 3-hydroxy-4-methylvalerate (PHB4MV),
and polyethylene glycol (PHBV-PEG) produced by the Azotobacter chroococcum 7B by controlled
biosynthesis technique were studied under in vitro model conditions. The changes in the
properties of the polymers during their in vitro degradation in the pancreatic
lipase solution and in phosphate-buffered saline for a long time (183 days) were investigated using
different analytical techniques. A mathematical model was used to analyze the kinetics of hydrolytic
degradation of poly(3-hydroxyaklannoate)s by not autocatalytic
and autocatalytic hydrolysis
mechanisms. It was also shown that the degree of crystallinity of some
polymers changes differently
during degradation in vitro. The total mass of the films decreased slightly up to 8–9% (for the high-
molecular weight PHBV with the 3-HV content 17.6% and 9%), in contrast to the copolymer
molecular mass, the decrease of which reached 80%. The contact angle for all copolymers after the
enzymatic degradation decreased by an average value of 23% compared to 17% after the hydrolytic
degradation. Young’s modulus increased up to 2-fold. It was shown that the effect of autocatalysis
was observed during enzymatic degradation, while autocatalysis was not available during
hydrolytic degradation. During hydrolytic and enzymatic degradation in vitro, it was found that
PHBV, containing 5.7–5.9 mol.% 3-HV and having about 50% crystallinity degree, presents critical
content, beyond which the structural and mechanical properties of the copolymer have essentially
changed. The obtained results could be applicable to biomedical polymer systems and food
packaging materials.
Polymers 2020, 12, 728 2 of 16
Keywords: poly(3-hydroxybutyrate), poly(3-hydroxybutyrate-со-3-hydroxyvalerate), poly(3-
hydroxybutyrate-со-4-methyl-3-hydroxyvalerate), biodegradation; hydrolysis; pancreatic lipase;
mechanical behavior
1. Introduction
Poly (3-hydroxybutyrate) (PHB), the main polymer of the polyhydroxyalkanoates family (PHA),
is the most well-known microbiological polyester, which is a promising alternative to biodegradable
synthetic thermoplastics [1–3] and other biocompatible polymers [4–11]. PHA are obtained
microbiologically. This production method allows varying the physicochemical properties of
polymers of this type over a wide range [12]. Because PHB has the ability to biodegrade and has high
biocompatibility, it is widely used for biomedical applications in regenerative medicine and tissue
engineering [13–22] and medicine forms [23–26]. It is able to create composites with synthetic
polymers, inorganic materials [19,27–29] and also as a new environmentally friendly material,
including application as a material for the packaging industry [15,24,30,31].
However, PHB has a number of disadvantages: fragility, lack of hydrophilicity, etc. In order to
improve the physicochemical properties, copolymers of PHB with other polyhydroxyalkanoates are
made. The copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) is made to compensate
disadvantages of PHB. Incorporation of HV into the PHB homopolymer chain considerably improves
the physicochemical properties, such as the melting temperature. The copolymer PHBV is more
plastic, extensible, and resilient due to a decrease in the value of Young’s modulus with an increase
in the HV molar fraction in PHB–HV polymer chain Reference [32] The copolymerization of PHB
with polyethylene glycol (PEG) increases water permeability and solubility due to the hygroscopicity
of PEG. In addition, biocompatibility and hydrophilicity are improved compared to a homopolymer
It is well known that the biodegradation of PHB both in living systems and in the environment
occurs through enzymatic and non-enzymatic processes that occur simultaneously in natural
conditions, compared to other biodegradable polymers (e.g., poly(lactic-co-glycolic) acid [33]). PHB
is considered to be moderately resistant to in vitro degradation, as well as to biodegradation in animal
tissues. The degradation rate is influenced by polymer characteristics, such as chemical composition,
crystallinity, morphology, and molecular mass [34,35].
Therefore, to develop novel medical devices and packing materials based on PHA and its
copolymers, it is necessary to know how the physicochemical properties of these polymers change
during their degradation. In order to understand what changes, occur with polymers in the human
body during degradation, it is necessary to study the kinetics of the change in the basic
physicochemical properties when they degrade in vitro under conditions simulating the internal
environment of the organism. Studies of degradation of PHA, especially on long periods, are rare
[36]. Thus, the purpose of this study is to obtain and compare the kinetic curves of the long-term
degradation of PHB and its copolymers. Considerable attention is paid to the change in the molecular
mass and degree of crystallinity of polymer films. In addition, at present in the literature, there are
no exact data on the effect of the monomer composition of the copolymer on the polymer
decomposition process. There is fragmentary evidence that the inclusion of > 10% 3-hydroxyvalerate
(3-HV) in the composition of the copolymer leads to a change in the structure of the crystalline
component of the polymer [37].
An understanding of the processes of hydrolytic and enzymatic degradation of PHAs is very
important for the development of a new biodegradable and harmless packaging for meat, fish, dairy,
and vegetable food products [38–41].
Therefore, the goals of this work are to trace the changes in the physicochemical properties of
the copolymers and to search for the value of the included hydroxyvalerate in the PHV chain, which
has a key effect on the degradation mechanisms of the product.
Polymers 2020, 12, 728 3 of 16
2. Materials and Methods
2.1. Production of Films from PHAs
To study the in vitro biodegradation, a series of films 50 ± 10 μm in thickness and 90 mm in
diameter was made from PHB and its copolymers (Table 1) obtained by controlled biosynthesis using
producing strain Azotobacter chroococcum (Table S1, Figure S1–S3). The polymer (~400 mg) was
dissolved in chloroform (35 mL) overnight at room temperature. Films were prepared by casting from
a chloroform solution to the bottom of Petri dishes previously degreased. The films were dried until
the solvent was completely removed for at least 72 h at room temperature. Then, the plates were cut
from the resulting films with dimensions of 30 mm × 10 mm.
Table 1. The list of polymers used in the work. HV = hydroxyvalerate; PEG = polyethylene glycol;
PHB = poly(3-hydroxybutyrate); PHBV = poly(3-hydroxybutyrate-co-3-hydroxyvalerate); MV =
Substrate Molecular Mass,
The Content of 3-HV/(3-H4MV)/(PEG) in the
PHB 1095 1095 0
PHBV 2.5% 768 768 2.5
PHBV 5.9% 819 819 9.0
PHBV 9% 1010 1010 17.6
PHBV 17.6%
1190 1190 5.9
PHBV-PEG 290 290 4.69%
0.15% (PEG)
PHB-4MV 1340 1340 0.60 (3-H4MV)
2.2. Determination of MM
The molecular mass (MM) of PHB and its copolymers were determined by gel filtration
chromatography (GPC). Chloroform with the addition of 3% v/v methanol was used as a solvent. The
elution rate was 1 mL/min. Used as the detector was a Waters 2414 differential refractive index
detector and a UV detector and a waters 1525 pump. The sample was 100 μL with a concentration of
5 mg/ml. Four Waters styragel columns (Waters, Milford, MA, USA) (Styragel HT 6E, 4.6 mm × 300
mm) were used. Calibration was carried out using polystyrene reference samples having a narrow
distribution [31]. The data obtained by GPC were correlated with viscometric data estimated by
viscosimetry [40]. The viscosity was measured in a 30 °C solution of chloroform in a Ubbelohde
viscometer. Molecular mass was calculated using the Mark-Hauwink-Kuhn equation. The 6
specimens per sample were analyzed (Table S2).
A mathematical description for the not autocatalytic and autocatalytic degradation of aliphatic
polyesters mechanisms was proposed in Reference [42]. Assuming that the degree of degradation is
low, the authors proposed the following kinetic dependence based on the mean molecular mass of
polymers (1):
1/MM = 1/MM
+ kt, (1)
where MM and MM
are the mean molecular mass of the polymer component at time t and at the
initial time, respectively, and k is the rate constant. An equation that took autocatalysis into account,
which is the consequence of the appearance of the terminal groups of carboxylic acids, was also
proposed. The following equation can describe this process (2):
ln(MM) = –kt + lnMM
. (2)
Polymers 2020, 12, 728 4 of 16
2.3. Determination of Crystallinity of a Polymer
The crystallinity of PHA were measured by DSC (DSC 204 F1 Phoenix, Netzsch, Germany). The
samples were heated from 25 to 220 °C. at a heating rate of 10 K/min under an argon atmosphere. The
crystallinity of the PHA structure (Xc) can be calculated as follows (3):
Xc = (ΔHm/ΔH
m(PHB)) × 100%, (3)
where ΔHm is the enthalpy change caused by the melting of the test sample, respectively, and ΔH
(PHB) is the theoretical value for the thermodynamic enthalpy of melting that would have been
obtained for 100%-crystalline samples of the PHB (146.6 J/g) [43]. All calculations were carried out
for the second heating cycles (Figure S4). The 6 specimens per polymer sample were analyzed.
2.4. Exploration of Mechanical Properties
The mechanical properties of the films were studied using the nanoindentation method in
accordance with ISO 14577. The measurements were carried out using a NanoScan-4D scanning
nano-hardness tester (TISNCM, Troitsk, Moscow, Russia). Nanoindentation was performed on the
smooth side of the films. Films with dimensions of 2 mm × 2 mm were fixed with phenyl salicylate.
The load was carried out in a linear mode, the peak load on the sample was 5 mN. The load time was
equal to the unloading time and was 30 s. The peak load was maintained for 5 s. The average
penetration depth into the sample was not more than 10% of the film thickness (Figure S5). The 6
specimens per polymer sample were analyzed.
2.5. Contact Angle Measurement
The hydrophilicity of the polymer surfaces was evaluated by measuring the contact angle
between the drops of water and the “smooth” surface of the samples using the Contact Angle Meter
110 VAC (Cole-Parmer, Vernon Hills, IL, USA). For this purpose, a drop of distilled water (10 μL)
was applied to the surface of the films, and then the contact angle of wetting was measured. The
values were averaged over the corners obtained from 10 drops per film (Figure S6). The 6 films were
analyzed for each polymer sample.
2.6. In Vitro Degradation Experiment
The study of the degradation of PHA films was carried out as follows. The plates were incubated
in 15 mL of phosphate-buffered saline (PBS) and in 15 mL of a solution of porcine pancreatic lipase
in PBS (Sigma L3126) having a pH of 7.4 at 37 °C in a thermostat for 183 days. The lipase concentration
in the phosphate buffer solution was 0.25 mg/mL. The concentration of pancreatic lipase was selected
based on earlier obtained experimental data (Figure S7). The pH was monitored with an Orion 420 +
pH meter (Thermo Fisher Scientific, Waltham, MA, USA). To assess the changes in the mass of the
polymer plates, the test plates were withdrawn from the lipase solution after 1 week, 1 month, 3
months, and 6 months, dried, and weighed on a scale. The average mass of the plates was 15–25 mg.
The change in the mass of the plates during the degradation was determined gravimetrically on the
AL-64 scales (Max = 60 g, d = 0.1 mg, ACCULAB, Bohemia, NY USA). To prevent bacterial
contribution to the degradation of polymers, sodium azide (2 g/L) was added to the buffer solution,
and the buffer solution was replaced twice per week [37,44,45].
2.7. Statistical Analysis
For statistical analysis a one-way ANOVA was applied. In the tables and figures, the data were
presented as mean values and standard deviation (M ± SD) at a significance level of P < 0.05.
Polymers 2020, 12, 728 5 of 16
3. Results
3.1. The Decrease in Mass of PHA Films
Biodegradation of PHA occurs as a result of a combination of hydrolytic and enzymatic
degradation. This leads to a change in the mass of the samples and their physical and chemical
properties [1,2,12,44–46]. The analysis of the degradation kinetic curves (Figure 1) showed that during
the first week, the mass of all the samples under study was decreased.
Figure 1. The change in the mass of poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3-
hydroxyvalerate) (PHBV) (with a different molar content of hydroxyvalerate (HV)), PHB-4
methylvalerate (MV), PHBV-polyethylene glycol (PEG) films during degradation for 6 months in
phosphate buffer solution (A) and in the same phosphate buffer solution supplemented with the
pancreatic lipase (0.25 mg/mL) (B).
All the investigated PHA films did not show the visual erosion during the entire degradation
process. Subsequently, the dry mass of the PHA films placed in the lipase solution did not
demonstrate significant changes even after 180 days. The mass of all the samples did not decrease
more than to 92% of the initial mass, indicating a rather slow degradation in the lipase solution. The
greatest loss (~ 8–9%) of the mass was observed in the PHBV 17.6% 1190 and PHBV 9% 1010.
3.2. Changes in Molecular Mass (MM)
MM is one of the most significant parameters of polymer degradation. The value of the
molecular mass of natural PHAs can be specified by the controlled bacterial biosynthesis and polymer
chemical processing [47]. When studying the biopolymers degradation, considerable attention is paid
to the MM, because it has a great effect on the other physicochemical parameters of biopolymers [48].
In addition, the MM values are the most indicative degradation parameter. It is extremely sensitive
to the polymer backbone destruction. It was found that with a slight change in the mass of the
samples, MM underwent more significant changes. The special features of MM changes during
biodegradation make it possible to understand the type of its mechanism: not autocatalytic or
autocatalytic [42,49].
Within 6 months of incubation in PBS and in lipase, the greatest loss of MM is observed in sample
PHB 1095 that was up to 80% of the initial MM (Figure 2). For the other polymer samples, a decrease
in MM was also observed, but no dependence on the molar content of HV was detected. Only two
polymers from this group in the first week increased their MM–PHBV 17.6% 1190 and PHBV 5.9%
Polymers 2020, 12, 728 6 of 16
Figure 2. The change in the molecular mass of PHB, PHBV (with a different molar content of HV), PHB-4MV,
PHBV-PEG films during hydrolytic (A) and enzymatic degradation (B) for 6 months.
To analyze the curves of the MM decrease during the degradation, the decomposition models of
partially crystalline polymers were applied [42,50]. To evaluate the applicability of the specific model,
the curves of degradation were calculated via the statistical correlation coefficients. For this object,
the plots of 1/MM and ln(MM) versus degradation time, reflecting not autocatalytic (not autocatalytic
for enzymatic degradation) and autocatalytic mechanism, respectively (Figure 3А,B).
Figure 3A shows that the analyzed curves are aligned with the increase in the molar content of
HV in the PHBV chain (from homopolymer (line #1) to copolymer with 17.6 mol% of HV (line #5).
Figure 3. Model of not autocatalytic degradation (A) and autocatalytic degradation (B), constructed
on the basis of the results of changes in the molecular mass of polyhydroxyalkanoates (PHA) in the
process of hydrolytic degradation. 1 – PHB 1095, 2 – PHBV 2.5% 768, 3 – PHBV 5.9% 819, 4 – PHBV
9% 1010, 5 – PHBV 17.6% 1190.
By presenting the tangent of the slope of the curves as a function of the HV content, the graph
of the slope angle dependence for the not autocatalytic model was obtained (Figure 4).
Polymers 2020, 12, 728 7 of 16
Figure 4. The graph of the slope angle on HV content for the not autocatalytic model of PHBV
hydrolytic degradation.
The intersection point for two lines corresponds to 5.9 mol. % HV in PHBV. These data confirm
that, at this molar content of PHBV, the polymer structure and corresponding mechanical properties
could be modified.
In accordance with the described models, correlation coefficients were determined for each
curve (Table 2). In Table 2, it is shown the correlation coefficients for hydrolytic and enzymatically
catalytic models.
Table 2. Correlation coefficients of a not-autocatalytic and autocatalytic degradation models.
Hydrolytic Degradation Enzymatic Degradation
(not-autocatalytic model)
(not -
PHB 1095 0.88 0.68 0.95 0.83
PHBV 2.5% 768
PHBV 5.9% 819 0.96 0.89 0.92 0.90
PHBV 9% 1010 0.96 0.90 0.99 0.97
PHBV 17.6% 1190 0.97 0.93 0.96 0.95
PHBV-PEG 290 0.92 0.83 0.99 0.98
PHB-4MV 1340 0.97 0.91 0.99 0.98
The correlation coefficients of the not-autocatalic model were quite high (>90%) for the both
hydrolytic and enzymatic degradation, while the correlation coefficients of the autocatalic model
were higher for the enzymatic degradation with the exception of polymers PHB 1095 and PHBV 2.5%
3.3. Degree of Crystallinity
The crystallinity degree of PHBV was calculated on the basis of the melting heat for the
completely crystalline PHB (146.6 J/g) [43]. Comparing the graphs, Figure 5A,B, you can see that in
the initial period of time the changes in crystallinity in different solutions are similar. In both cases,
in the period up to 1-month, strong changes in the degree of crystallinity were observed.
Polymers 2020, 12, 728 8 of 16
Figure 5. The change in the crystallinity of PHB, PHBV (with a different molar content of HV), PHB-
4MV, PHBV-PEG films during hydrolytic (A) and enzymatic degradation (B) for 6 months: 1 – PHB
1095, 2 – PHBV 2,5% 768, 3 – PHBV 5,9% 819, 4 – PHBV 9% 1010, 5 – PHBV 17,6% 1190, 6 – PHBV-
PEG 290, 7 – PHB-4MV 1340.
It was shown that the degree of crystallinity for the copolymers is less than for the sample of
PHB 1095. Thus, if the degree of crystallinity for intact PHB 1095 before incubation was 63%, the
degree of crystallinity of the PHBV copolymer with the highest molar HV content (17.6 mol. %) at the
initial moment was 34%. It is almost half that of PHB 1095. It can also be noted that the degree of
crystallinity decreases with increasing molar content of 3-hydroxyvalerate chains.
With further degradation of the polymers for 6 months, incubation of PHA films in PBS resulted
in a wave-shape change in the degree of crystallinity (Figure 5A). However, the degree of crystallinity
changed differently when incubated in a solution of phosphate buffer with pancreatic lipase. The
wave-shape change in crystallinity did not occur (Figure 5B), and its values were observed only in
PHB 1095 and PHBV 2.5% 768. It is also important to note that, unlike hydrolytic degradation, there
was a clear tendency (Figure 5B) to decrease the degree of crystallinity as the function of the molar
content of 3-HV in the homopolymer chain. In addition, the slope of the curves (the values of tgα in
Figure 5B) also changed. During degradation, the crystallinity of PHB 1095 increased over 6 months,
however, the crystallinity of the PHBV 17.6% 1090 decreased through 6 months. So, it is possible to
calculate the molar content of 3-HV in the PHBV chain, which begins to affect the structural and
mechanical properties of the entire polymer, which leads to a different character of polymer
decomposition (Figure 6).
Figure 6. The effect of the 3-HV molar content in the PHBV chain on the crystallinity degree of the
copolymers in the degradation process in the lipase solution for 6 months, in the coordinates of the
slope of the incubation time.
Polymers 2020, 12, 728 9 of 16
Figure 6 shows that after incubation in a lipase solution by the 6th month of degradation, the
degree of crystallinity was decreased if the percentage of 3-HV in the PHBV chain exceeded 5.7%.
This is very close to the value determined earlier in at the analysis of MM changes for the copolymer
with HV content equals to 5.9%.
3.4. The Change in Mechanical Properties of PHA Films
Changes in the mechanical properties of the copolymers as a result of degradation were
measured by the nanoindentation method (Figure 7). It should be noted that Young’s modulus of the
homopolymer (2.2 ± 0.06 GPa) before degradation was higher than that of the copolymers (~1 GPa).
This is due to the fact that the copolymers have a lower degree of crystallinity and, consequently,
Figure 7. (A) Graphs of the dependence of the young’s modulus on the content of 3-HV. The green
square corresponds to the PHB-4MV 1340 copolymer, and the blue square corresponds to the PHBV-
PEG 290 copolymer. (B) Changing Young’s modulus of the polymers in the process of enzymatic
During the first week, Young’s modulus of all polymers was sharply increased (Figure 7) The
largest value of Young’s modulus was observed for PHB 1095 kDa. Over a month, Young’s modulus
of the homopolymer increased to 4.7 ± 0.1 GPa and remained at this level throughout the entire
experiment (6 months). Young’s modulus of the other copolymers also increased during the first
week and did not change significantly in the future. However, the stiffness of the copolymers is still
less than that of the homopolymer, for comparison, Young’s modulus of the PHBV 17.6% 1190 for the
6-month degradation was 2.2 ± 0.07 GPa, which was half that of Young’s modulus of homopolymers.
3.5. The Change in Hydrophobicity of PHA Films
The balance between hydrophobicity and hydrophilicity of the surface is one of the main
characteristics indicating biocompatibility of the surface. Biocompatibility is one of the most
important properties of polymers that can be used in medicine, so the degree of hydrophilicity of the
surface of the polymer affects the growth of cells [3].
During the biodegradation, the contact angle between the standard drop of water and the surface
of the polymer film decreased, which indicates that the hydrophobicity degree for the copolymers
decreases (Figure 8).
Polymers 2020, 12, 728 10 of 16
Figure 8. The changes of the contact angle at the water/film boundary of a PHA with a different HV
molar content in the PHB chain during enzymatic degradation (A) and hydrolytic degradation (B) for
6 months.
In general, during biodegradation, the contact angle for all copolymers contacted with lipase
decreased by an average value of 23% compared to 17% for hydrolytic degradation (the differences
are statistically significant, p ≤ 0.01).
4. Discussion
Thanks to their unique properties, PHAs are the most promising polymers for use in various
fields, such as ecology, biomedicine, and packaging. PHAs are biopolymer family that is obtained
using microorganisms. The Azotobacter chroococcum 7B strain was used for the biosynthesis of PHAs.
The polymers synthesized by this producing bacterial strain have low polydispersity, a widespread
spectrum of the 3-HB/3-HV monomer ratio in the PHB chain, and MM variability. By varying these
parameters during the synthesis of copolymers, their properties, such as Young’s modulus,
biodegradation rate, and biocompatibility, can also be varied.
The physicochemical parameters of PHAs are changed in different ways during degradation in
solutions of PBS and PBS with lipase. The initial mass of polymer films of various copolymers
remained virtually unchanged during the entire 183 days of the experiment (Figure 1). The decrease
in film mass during the first week can be explained by the dissolution of water-soluble oligomers and
monomers during their desorption from the film into aqueous media.
The change in MM is an important characteristic describing the degradation of polymers. As
mentioned earlier, using different methods of growing polymer producers, it is possible to vary the
molecular mass of the biosynthesized product. This procedure allows development of devices from
PHAs with a programmable rate of degradation. The articles presented recently [1,29,44,46] described
the hydrolytic degradation of polymers in the presence of various agents. But, in these works,
explanations of reaction mechanisms were not presented. This work is one of the first which to
attempt to compare the different behavior of biopolyesters in PBS and in the medium containing
pancreatic lipase as one of the most important components involved in the decomposition of PHA in
the human body. The study of molecular mass changes during degradation allows us to elucidate
this mechanism of the process.
Surprisingly, an increase in molecular mass was observed for PHBV 17.6% 1190 and PHBV 5.9%
819 (Figure 2A,B). The initial increase in molecular mass is associated with leaching of the low
molecular mass polymer fraction. This means that, in the manufacture of a polymer film, in its
volume, and on the surface, there are short-chain polymer residues that dissolve relatively well in
water. When studying the molecular mass of polymers prior to incubation in experimental solutions,
short-chain residues will affect the average molecular mass. Upon further placement of the polymer
films in aqueous solutions, these residues dissolve and are washed out of the polymer film. This leads
to a slight increase in molar mass during subsequent measurement. The hydrolysis of the amorphous
Polymers 2020, 12, 728 11 of 16
polymer component also occurs, which is located on the surface of the film, because, according to the
literature, the amorphous component decomposes 20 times faster than crystalline [48]. The
subsequent strong decrease in MM testified to the fact that the degradation of these polymers was
carried out not only on the surface of the polymer film but partially in volume. The absence of mass
reduction of polymer films and Young’s modulus change (due to the process of secondary
crystallization of polymers, and also, probably, to the elution of the amorphous component) (Figure
1,7) prompt the conclusion that our polymers will retain their structural and mechanical properties
over time and perform their function. Thus, the synthesized polymers can find application in the field
of bone tissue engineering for the manufacture of implants. The use of calculation models (Table 2)
suggested that, in the case of lipase addition, the process of autocatalysis began to effect the rate of
degradation, in contrast to hydrolysis in phosphate buffer. From the Table 2 data, it could be
concluded that all copolymers and homopolymer, PHB, are better described by a notautocatalytic
degradation model, since the values of R
correspond to it. The similar behavior of partially crystalline
polymers was presented in the study of Han et al. [46]. In addition, when constructing models and
calculating the slope of the curves, a transition point of 5.9% of 3-HV in the copolymer was obtained.
Probably, the presence of a more branched radical in the copolymer leads to the fact that it is more
difficult for water molecules to hydrolyze the ester bonds. And these steric hindrances begin to affect
the entire polymer precisely when the content of 3-HB is greater than 5.9%. In the future, taking this
factor into account can help to more correctly calculate the rate of degradation of medical materials
based on PHB and its homologues, PHA.
According to published data, the degree of crystallinity of PHA is quite high and varies between
40–70% [42,51]. These data are confirmed by our studies. The degree of crystallinity of the
homopolymer was 63%, and the PHBV copolymer 17.6% 1340 was almost two times less −34%.
Differences in the degree of crystallinity of polymer films during degradation in PBS and lipase
solutions were also demonstrated for the first time. A wave-like evolution in the degree of
crystallinity during degradation in PBS was proposed recently [44,45,50]. In these articles, it was
assumed that the changes would not go linearly. We showed that the degree of crystallinity during
hydrolysis will have a wave-shape change. All of this is due to crystallization and recrystallization
processes. This means the following. During degradation, the amorphous component decomposes
faster than the crystalline component 20 times [48]. This leads to an increase in the degree of
crystallinity in the polymer. However, hydrolysis occurs non-directionally, that is, the crystalline part
also decomposes. Upon decomposition of the crystalline part, an amorphous component is formed—
weaving—that leads to a decrease in the degree of crystallinity. Further, the decomposition of the
amorphous component or its secondary crystallization occurs, which again leads to an increase in the
degree of crystallinity. All of this will have a wave-like appearance, which we have shown in our
work. It should be noted that the PHB-4MV copolymer with a 4-MV content of 0.6%, in its physical
properties, in particular, crystallinity, is between the PHBV with a content of 5.9% and 9%. It seems
that 4-MV makes significant conformational changes in the three-dimensional structure of the
copolymer. At the same time, the addition of PEG to the composition of the copolymer does not
explicitly affect the polymer structure—the degree of crystallinity of PHBV-PEG is between the
copolymer with a 3-HV content of 2.5% to 5.9%. The 3-HV molar content in PHB-PEG is 4.86%.
Preliminary, we observed three types of morphological changes in ultrathin PHB films under
enzymatic degradation: the emergence of new lamellar structures, fragmentation of lamellar
structures, and the disappearance of lamellar structures (Figure S8–S10). However, the degree of
crystallinity of the polymers changed differently (from PBS) in the presence of a lipase. But, more
important is the result that describes the changes in the behavior of the degree of crystallinity during
degradation, depending on the content of HV included in the composition (Figure 9).
Polymers 2020, 12, 728 12 of 16
Figure 9. The model of in vitro degradation of PHB and PHBV. It was found that in the process of
degradation, the degree of crystallinity increases if the content of 3-HV in the copolymer is less than
5.9% and decreases if it is more than 5.9%.
Based on the obtained data, it can be concluded that when HV comonomer is included in the
molecular chain less than 5.9% of PHBV, there is the increase in degree of crystallinity for the films
during degradation. As the percentage of HV increases, the reverse process occurs—the decrease in
the degree of crystallinity. This is probably due to the influence of HV groups while the small HV
content does not prevent the crystallization of newly formed chains of PHB. Probably, HV
comonomer creates steric hindrances for folding into the perfect crystals, which leads to a decrease
in crystallinity. Owing to its importance, the above phenomenon requires further study since it can
affect the change in mechanical and transport (barrier) properties during degradation. In addition,
this phenomenon must be taken into account in the future when predicting the properties of products
based on PHA that are contacted with tissues during applications as implants or food packaging.
5. Conclusion
Thus, a comprehensive study of the changes in the physicochemical properties of PHB and its
copolymers with 3-hydroxyvalerate with different monomer content during the long-term enzymatic
and hydrolytic biodegradation under in vitro model conditions was performed. In vitro
biodegradation of polymers was studied in PBS and in PBS in the presence of pancreatic lipase at 37
°C for 183 days. An insignificant drop in the mass of the polymers was revealed. However, the change
in molecular mass was more significant: a molecular mass decreases up to 80% was found in PHB
1095 kDa in both solutions. In addition, it was shown that, during the enzymatic degradation, the
effect of autocatalysis was observed, which was not observed during the hydrolytic degradation. It
was also found that Young’s modulus of the copolymers was lower than that of the homopolymer.
The incubation of polymer films in both solutions led to an increase in Young’s modulus by more 2-
fold. The changes in the degree of crystallinity of the polymers were wave-shape. The films based on
biopolymers became more hydrophilic during biodegradation. It was found that the value of ~5.7–
5.9% of HV content in the PHBV copolymer was a transition point for changes in the structural and
mechanical properties of the PHBV during its degradation.
Polymers 2020, 12, 728 13 of 16
Supplementary Materials: The following are available online at, Figure S1:
spectra of a PHBV copolymer with a content of HV 17% (A); HV 2.5% (B) and homopolymer PHB (C), I – CH(b),
(b), III – CH
(s)HV, IV – CH
(s)HВ, V – CH
(s) HV, s – side chain; b – polymer backbone. Figure S2:
NMR spectra of a PHBV copolymer with a content of PHB4MV (a) PHB chain: 1 – CH
(s), 2 – CH(b), 3 – CH
PHB-4MV chain (b): 4 – CH
(s), 5 – CH
(s), 6 –CH(b), 7 – CH
(b), s – side chain; b – polymer backbone. Figure S3:
H-NMR spectra of a PHBV-PEG: (a) – PHB chain: 1 – CH
(s), 2 – CH(b), 3 – CH
(b), PHBV chain: 4 – CH
(s), 5 –
(s), 6 – CH(b), 7 – CH
(b), s – side chain; b – polymer backbone.; enlarged plot of the graph is shown in the
inset (b); (b) PEG chain: «a» -O–CH
(4.24 ppm), «b» - CH
(3.73), «c» - common signal from the middle groups
-] (3.66 ppm), «e» and «d» end groups –CH
- (3.70 ppm) and –CH
-OH (3.61 ppm). Figure S4:
Thermogram of the PHBV copolymer 17.6% 1190 obtained by the DSC method. The thermogram shows two
heating curves (red and purple lines) and two cooling curves of the sample (white blue and deep blue lines).
Figure S5: Image of a characteristic curve in the coordinates of the dependence of the applied force on the
indenter displacement obtained using the nanoindentation method. Figure S6: The image of a drop of water on
the surface of a polymer film. Figure S7: Diagrams of changes in the weight of plates in (A) a buffer simulating
blood plasma (SBF) and in (B) phosphate buffer (PSB) depending on the concentration of pancreatic lipase for a
week and a month. Figure S8: The phase images of PHB film before (A) and after (B) hydrolysis in lipase solution.
Arrows indicate the new lamellas. Figure S9: The phase images of PHB film before hydrolysis in lipase solution
(A) and after (B). Arrows indicate the fragmentation of lamellae. Figure S10: The phase images of PHB film before
(A) and after (B) hydrolysis in lipase solution. Arrows indicate the disappearance of lamellar structures. Table
S1: Biosynthesis of copolymers of sodium phosphate buffer (PBS) with the A. chroococcum 7B producer strain
in the culture medium with sucrose as the main carbon source and salts of carboxylic acids as additional sources
of carbon and precursors for biosynthesis of the copolymers of poly(3-hydroxybutyrate) (PHB). HV =
hydroxyvalerate. PEG = polyethylene glycol. Table S2: Molecular mass of newly synthesized poly (3-
hydroxybutyrate) obtained by various purification methods.
Author Contributions: Conceptualization, V.A.Z., Y.V.Z.; methodology, V.A.Z., A.P.B., T.K.M., V.V.V., V.L.M.,
G.A.B., A.R., A.U.; validation, A.A.B., A.L.I. and A.P.B.; formal analysis, Y.V.Z.; investigation, V.A.Z.; resources,
T.K.M., V.L.M., G.A.B.; data curation, A.P.B.; writing—original draft preparation, V.A.Z.; writing—review and
editing, A.P.B, Y.V.Z., A.L.I., A.P.B.; supervision, A.P.B.; project administration, G.A.B. A.P.B. and A.L.I.
contributed equally to this work. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the Russian Science Foundation, project No 17-74-20104 (in part of PHBV
enzymatic degradation); by Russian Foundation of Basic Research, project No 18-29-09099 (in part of PHB
hydrolytic and enzymatic degradation) in all other parts in the framework of government assignment of the
Ministry of Science and Higher Education of the Russian Federation. The equipment used in this work was from
the User Facilities Center of M.V. Lomonosov Moscow State University and the User Facilities Center of Research
Center of Biotechnology of Russian Academy of Sciences.
Conflicts of Interest: The authors declare no conflict of interest.
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... The high biocompatibility of PHB is attributed to its lack of chemical activity. PHB is capable of hydrolytic and enzymatic degradation [30][31][32] In the body, PHB biodegradation occurs at the expense of phagocytic cells (macrophages, osteoclasts) [22]. Osteogenic differentiation of MSCs and osteoblast cells grown on scaffolds of PHB and its copolymers is confirmed by changes in cell morphology [26,[33][34][35], by suppressing their reproduction [25,36], increased calcium salt deposition and alkaline phosphatase activity [27,37], increased expression of markers of new bone formation [28,38]. ...
... This is important for medical devices because there is a relationship between crystallinity, mechanical properties and the biodegradation rate of materials. This means that they can be used to create products with a controlled degradation rate, which is particularly important for tissue-engineered implants [30]. At the same time, DSC images showed that the formation of bonds between polymer molecules does not occur, as the melting peak of PHB remains practically unchanged around 173 • C. Using the TGA method, it was found that chitosan influenced the thermal decomposition of the composites. ...
Poly(3-hydroxybutyrate) and chitosan are among the most widely used polymers for biomedical applications due to their biocompatibility, renewability and low toxicity. The creation of composite materials based on biopolymers belonging to different classes makes it possible to overcome the disadvantages of each of the components and to obtain a material with specific properties. Solving this problem is associated with difficulties in the selection of conditions and solvents for obtaining the composite material. In our study, acetic acid was used as a common solvent for hydrophobic poly(3-hydroxybutyrate) and chitosan. Mechanical, thermal, physicochemical and surface properties of the composites and homopolymers were investigated. The composite films had less crystallinity and hydrophobicity than poly(3-hydroxybutyrate), and the addition of chitosan caused an increase in moisture absorption, a decrease in contact angle and changes in mechanical properties of the poly(3-hydroxybutyrate). The inclusion of varying amounts of chitosan controlled the properties of the composite, which will be important in the future for its specific biomedical applications.
... Han et al. investigated biodegradation of copolymer P(3HB-co-3HV) films in a phosphate buffer solution with porcine pancreas lipase; after 180 days of incubation, the authors observed only slight weight deflections (remaining more than 95% of initial weight), while the molecular weight decrease reached 34-80% [24]. Zhuikov et al. reported similar results as various PHA copolymers incubated in the fluid containing pancreatic lipase, where the mass decreased by 8-9% and the molecular weight reduction reached up to 80% [25]. cine pancreas lipase [27]. ...
... Two possible explanations are offered: the leaching of the short polymer chains, Polymers 2022, 14, 1990 8 of 14 which is inconsistent with the mass decrease results; or spontaneous polycondensation, which is unfavorable in such an environment [30,31]. The leaching of low-molecular polymer fractions was previously discussed also by Zhuikov et al. [25]. ...
Novel model of biodegradable PHA copolymer films preparation was applied to evaluate biodegradability of various PHA copolymers and discuss its biomedical applicability. In this study, we illustrate the potential biomaterial degradation rate affectability by manipulation of monomer composition via controlling biosynthetic strategies. Within the experimental investigation, we have prepared two different copolymers of 3-hydroxybutyrate and 4-hydroxybutyrate – P(3HB-co-36 mol.% 4HB) and P(3HB-co-66 mol.% 4HB), by cultivating thermophilic bacterial strain Aneurinibacillus sp. H1 and further investigated its degradability in simulated body fluids (SBFs). Both copolymers revealed faster weight reduction in synthetic gastric juice (SGJ) and artificial colonic fluid (ACF) than simple homopolymer P3HB. In addition, degradation mechanisms differed across tested polymers, according to SEM micrographs. While incubated in SGJ, samples were fragmented due to fast hydrolysis sourcing from substantially low pH, which suggest abiotic degradation as the major degradation mechanism. On the contrary, ACF incubation indicated obvious enzymatic hydrolysis. Further, no cytotoxicity of the waste fluids was observed on CaCO-2 cell line. Based on these results in combination with high production flexibility, we suggest P(3HB-co-4HB) copolymers produced by Aneurinibacillus sp. H1 as very auspicious polymers for intestinal in vivo treatments.
... 489 Fragility and lack of hydrophilicity. 490 ...
... Flexibility and ease of processing are two advantages over PHB. 490 Structuring and easy to handle. 491 In compared to petroleum-based synthetic polymers, this material has a small processing window and a low strain-at-break. ...
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Smart biomaterials have been rapidly advancing ever since the concept of tissue engineering was proposed. Interacting with human cells, smart biomaterials can play a key role in novel tissue morphogenesis. Various aspects of biomaterials utilized in or being sought for the goal of encouraging bone regeneration, skin graft engineering, and nerve conduits are discussed in this review. Beginning with bone, this study summarizes all the available bioceramics and materials along with their properties used singly or in conjunction with each other to create scaffolds for bone tissue engineering. A quick overview of the skin-based nanocomposite biomaterials possessing antibacterial properties for wound healing is outlined along with skin regeneration therapies using infrared radiation, electrospinning, and piezoelectricity, which aid in wound healing. Furthermore, a brief overview of bioengineered artificial skin grafts made of various natural and synthetic polymers has been presented. Finally, by examining the interactions between natural and synthetic-based biomaterials and the biological environment, their strengths and drawbacks for constructing peripheral nerve conduits are highlighted. The description of the preclinical outcome of nerve regeneration in injury healed with various natural-based conduits receives special attention. The organic and synthetic worlds collide at the interface of nanomaterials and biological systems, producing a new scientific field including nanomaterial design for tissue engineering.
... PHB has a number of unique biological properties: complete biodegradability to non-toxic products, biocompatibility, non-carcinogenicity, and special diffusion properties that provide sustained drug release [19][20][21][22]. PHB undergoes hydrolytic, enzymatic, and cellular biodegradation, and the degradation time is highly dependent on the molecular weight, crystallinity degree, and device shape and microstructure [23]. PHB is also used as biomaterial to manufacture tissue-engineering scaffolds for cell cultivation, including growing mesenchymal stem cells (MSCs) [20]. ...
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Scaffold biocompatibility remains an urgent problem in tissue engineering. An especially interesting problem is guided cell intergrowth and tissue sprouting using a porous scaffold with a special design. Two types of structures were obtained from poly(3-hydroxybutyrate) (PHB) using a salt leaching technique. In flat scaffolds (scaffold-1), one side was more porous (pore size 100–300 μm), while the other side was smoother (pore size 10–50 μm). Such scaffolds are suitable for the in vitro cultivation of rat mesenchymal stem cells and 3T3 fibroblasts, and, upon subcutaneous implantation to older rats, they cause moderate inflammation and the formation of a fibrous capsule. Scaffold-2s are homogeneous volumetric hard sponges (pore size 30–300 μm) with more structured pores. They were suitable for the in vitro culturing of 3T3 fibroblasts. Scaffold-2s were used to manufacture a conduit from the PHB/PHBV tube with scaffold-2 as a filler. The subcutaneous implantation of such conduits to older rats resulted in gradual soft connective tissue sprouting through the filler material of the scaffold-2 without any visible inflammatory processes. Thus, scaffold-2 can be used as a guide for connective tissue sprouting. The obtained data are advanced studies for reconstructive surgery and tissue engineering application for the elderly patients.
... PHAs can be defined as homo-, co-, and terpolymers [9] and classified in relation to the number of carbon atoms in the monomer into: short-chain-length PHAs (scl-PHAs) with 3 to 5 carbons, medium-chain-length PHAs (mcl-PHAs) with 6 to 14 carbons, and long-chainlength PHAs (lcl-PHAs) with more than 14 carbons [10,11]. Among them, the copolymer PHBV has a strong potential for food packaging applications since it has considerably lower crystallinity and melting temperature (T m ), which diminish as the percentage of HV fraction in the polymer increases [12], exhibits enhanced flexibility, ductility, and elongation at break, and augmented tensile strength as a consequence of a reduction in the Young's modulus as the fraction of HV increases [13]. ...
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Food quality is mainly affected by oxygen through oxidative reactions and the proliferation of microorganisms, generating changes in its taste, odor, and color. The work presented here describes the generation and further characterization of films with active oxygen scavenging properties made of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) loaded with cerium oxide nanoparticles (CeO2NPs) obtained by electrospinning coupled to a subsequent annealing process, which could be used as coating or interlayer in a multilayer concept for food packaging applications. The aim of this work is to explore the capacities of these novel biopolymeric composites in terms of O2 scavenging capacity, as well as antioxidant, antimicrobial, barrier, thermal, and mechanical properties. To obtain such biopapers, different ratios of CeO2NPs were incorporated into a PHBV solution with hexadecyltrimethylammonium bromide (CTAB) as a surfactant. The produced films were analyzed in terms of antioxidant, thermal, antioxidant, antimicrobial, optical, morphological and barrier properties, and oxygen scavenging activity. According to the results, the nanofiller showed some reduction of the thermal stability of the biopolyester but exhibited antimicrobial and antioxidant properties. In terms of passive barrier properties, the CeO2NPs decreased the permeability to water vapor but increased the limonene and oxygen permeability of the biopolymer matrix slightly. Nevertheless, the oxygen scavenging activity of the nanocomposites showed significant results and improved further by incorporating the surfactant CTAB. The PHBV nanocomposite biopapers developed in this study appear as very interesting constituents for the potential design of new active organic recyclable packaging materials.
... polyesters and chitosan allows one to combine mechanical characteristics of PLA and PHB with the high sorption capacity of chitosan. During exploitation, these composition materials are exposed to action of such aggressive factors as hydrolysis, oxidation, ozonolysis and also enzymatic biodegradation via numerous microorganisms [14][15][16][17][18]. ...
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The film binary composites polylactide (PLA)–chitosan and poly(3-hydroxybutyrate) (PHB)–chitosan have been fabricated and their functional characteristics, such as hydrolysis resistance, biodegradation in soil, and ion sorption behavior have been explored. It was established that hydrolysis temperature and acidity of solutions are differently affected by the weight loss of these two systems. Thus, in the HCl aqueous solutions, the stability of the PHB-chitosan composites is higher than the stability of the PLA-chitosan one, while the opposite situation was observed for biodegradation in soil. The sorption capacity of both composites to Fe3+ ions was investigated and it was shown that, for PHB-chitosan composites, the sorption is higher than for PLA-chitosan. It was established that kinetics of sorption obeys the pseudo-first-order equation and limiting values of sorption correspond to Henry’s Law formalism. By scanning electron microscopy (SEM), the comparative investigation of initial films and films containing sorbed ions was made and the change of films surface after Fe3+ sorption is demonstrated. The findings presented could open a new horizon in the implementation of novel functional biodegradable composites.
... The molecular weight of poly(3-hydroxybutyrate) synthesized by wild-type bacteria ranges from 1 × 10 4 to 3 × 10 6 g/mol with a degree of polydispersity~2 [93,98,119]. The glass transition temperature of PHB is~4 • C, while the melting point is~180 • C [120,121]. A bifurcated peak of melting temperature is also sometimes observed in homopolymers. This phenomenon can be explained by the presence of crystallites of different degrees of perfection, which can include the thermal prehistory of the sample and the broad molecular weight distribution. ...
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One of the important directions in the development of modern medical devices is the search and creation of new materials, both synthetic and natural, which can be more effective in their properties than previously used materials. Traditional materials such as metals, ceramics, and synthetic polymers used in medicine have certain drawbacks, such as insufficient biocompatibility and the emergence of an immune response from the body. Natural biopolymers have found applications in various fields of biology and medicine because they demonstrate a wide range of biological activity, biodegradability, and accessibility. This review first described the properties of the two most promising biopolymers belonging to the classes of polyhydroxyalkanoates and polysaccharides—polyhydroxybutyrate and chitosan. However, homopolymers also have some disadvantages, overcome which becomes possible by creating polymer composites. The article presents the existing methods of creating a composite of two polymers: copolymerization, electrospinning, and different ways of mixing, with a description of the properties of the resulting compositions. The development of polymer composites is a promising field of material sciences, which allows, based on the combination of existing substances, to develop of materials with significantly improved properties or to modify of the properties of each of their constituent components.
... Polyhydroxyalkanoates (PHAs), like polyesters, are one of the most common biopolymers in regenerative medicine due to their biocompatibility and biodegradation properties [2]. Poly(3-hydroxybutyrate) (PHB) is one of the most common forms of PHA homopolymers [3]. PHB consists of D-3-hydroxybutyric acid monomers and is an isotactic polyester with regular units, which can be expressed by the degree of crystallinity of the polymer [4]. ...
Various variants of poly(3-hydroxybutyrate) (PHB) were synthesized by varying three factors (sucrose concentration, phosphate concentration, and aeration level) while growing the producer Azotobacter vinelandii 12 using full-factorial design. Bacteria grown at elevated sucrose concentration, low phosphate concentration, and high aeration level (C+/P−/O+) showed the maximum PHB yield (0.49 g/L). Molecular weights (MW) of PHB samples obtained under diverse conditions differed by more than 30 times (from 49 to 1698 kDa). Low molecular weight PHBs were observed at low sucrose levels and high aeration (C–/O+). All other PHB samples had MW over 1200 kDa. The crystallinity of all PHB samples was determined by differential scanning calorimetry and was within the range of 62–68%. These results show that, with optimization, it will be possible to synthesize PHBs with different physicochemical properties for a wide range of biomedical problems, including tissue engineering.
... There was a negative linear correlation between T c and 3HV content (R 2 = 0.54), and also between T c and X c of the extracted PHBVs (R 2 = 0.54). An Increase in 3HV content results in an increase in the amorphous phase and thus leads to a reduction in X c and T c [48]. A lower T c indicates that the complete crystallization takes a longer time [49]. ...
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Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with various 3-hydroxyvalerate (3HV) contents biosynthesized by mixed microbial consortia (MMC) fed fermented dairy manure at the large-scale level was assessed over a 3-month period. The thermal, mechanical, and rheological behavior and the chemical structure of the extracted PHBV biopolymers were studied. The recovery of crude PHBV extracted in a large Soxhlet extractor with CHCl3 for 24 h ranged between 20.6% to 31.8% and purified to yield between 8.9% to 26.9% all based on original biomass. 13C-NMR spectroscopy revealed that the extracted PHBVs have a random distribution of 3HV and 3-hydroxybutyrate (3HB) units and with 3HV content between 16% and 24%. The glass transition temperature (Tg) of the extracted PHBVs varied between −0.7 and −7.4 °C. Some of the extracted PHBVs showed two melting temperatures (Tm) which the lower Tm1 ranged between 126.1 °C and 159.7 °C and the higher Tm2 varied between 152.1 °C and 170.1 °C. The weight average molar mass of extracted PHBVs was wide ranging from 6.49 × 105 g·mol−1 to 28.0 × 105 g·mol−1. The flexural and tensile properties were also determined. The extracted polymers showed a reverse relationship between the 3HV content and Young’s modulus, tensile strength, flexural modulus, and flexural strength properties.
This is the first comprehensive study of the impact of biodegradation on the structure, surface potential, mechanical and piezoelectric properties of poly(3‐hydroxybutyrate) (PHB) scaffolds supplemented with reduced graphene oxide (rGO) as well as cell behavior under static and dynamic mechanical conditions. There was no effect of the rGO addition up to 1.0 wt% on the rate of enzymatic biodegradation of PHB scaffolds for 30 days. The biodegradation of scaffolds led to the depolymerization of the amorphous phase, resulting in an increase in the degree of crystallinity. Because of more regular dipole order in the crystalline phase, surface potential of all fibers increased after the biodegradation, with a maximum (361 ± 5 mV) after the addition of 1 wt% rGO into PHB as compared to pristine PHB fibers. By contrast, PHB‐0.7rGO fibers manifested the strongest effective vertical (0.59 ± 0.03 pm/V) and lateral (1.06 ± 0.02 pm/V) piezoresponse owing to greater presence of electroactive β‐phase. In vitro assays involving primary human fibroblasts revealed equal biocompatibility and faster cell proliferation on PHB‐0.7rGO scaffolds compared to pure PHB and nonpiezoelectric polycaprolactone scaffolds. Thus, the developed biodegradable PHB‐rGO scaffolds with enhanced piezoresponse are promising for tissue‐engineering applications. This article is protected by copyright. All rights reserved
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In this study, the effects of material thickness and processing method on the degradation rate and the changes in the mechanical properties of poly(lactic-co-glycolic acid) material during simulated physiological degradation were investigated. Two types of poly(lactic-co-glycolic acid) materials were considered: 0.12 mm solvent-cast films and 1 mm compression-moulded plates. The experimental results presented in this study were compared to the experimental results of Shirazi et al. (Acta Biomaterialia 10(11):4695-703, 2014) for 0.25 mm solvent-cast films. These experimental observations were used to validate the computational modelling predictions of Shirazi et al. (J Mech Behav Biomed Mater 54: 48-59, 2016) on critical diffusion length scale and also to refine the model parameters. The specific material processing methods considered here did not have a significant effect on the degradation rate and the changes in mechanical properties during degradation; however, they influenced the initial molecular weight and they determined the stiffness and hardness of the poly(lactic-co-glycolic acid) material. The experimental observations strongly supported the computational modelling predictions that showed no significant difference in the degradation rate and the changes in the elastic modulus of poly(lactic-co-glycolic acid) films for thicknesses larger than 100 μm.
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The design of new synthetic grafted poly(3-hydroxybutyrate) as composite 3D-scaffolds is a convenient alternative for tissue engineering applications. The chemically modified poly(3-hydroxybutyrate) is receiving increasing attention for use as biomimetic copolymers for cell growth. As of yet, these copolymers cannot be used efficiently because of the lack of good mechanical properties. Here, we address this challenge, preparing a composite-scaffold of grafted poly(3-hydroxybutyrate) polyurethane for the first time. However, it is unclear if the composite structure and morphology can also offer a biological application. We obtained the polyurethane by mixing a polyester hydroxylated resin with polyisocyanate and the modified polyhydroxyalkanoates. The results show that the poly(3-hydroxybutyrate) grafted with poly(vinyl alcohol) can be successfully used as a chain extender to form a chemically-crosslinked thermosetting polymer. Furthermore, we show a proposal for the mechanism of the polyurethane synthesis, the analysis of its morphology and the ability of the scaffolds for growing mammalian cells. We demonstrated that astrocytes isolated from mouse cerebellum, and HEK293 can be cultured in the prepared material, and express efficiently fluorescent proteins by adenoviral transduction. We also tested the metabolism of Ca2+ to obtain evidence of the biological activity.
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Background: Poly(hydroxyalkanoates) (PHA) have recently attracted increasing attention due to their biodegradability and high biocompatibility, which makes them suitable for the development of new prolong drug formulations. Objective: This study was conducted to develop new prolong paclitaxel (PTX) formulation based on poly(3- hydroxybutyrate) (PHB) microparticles. Method: PHB microparticles loaded with antitumor cytostatic drug PTX were obtained by spray-drying method using Nano Spray Dryer B-90. The PTX release kinetics in vitro from PHB microparticles and their cytotoxity on murine hepatoma cell line MH-22a were studied. Microparticles antitumor activity in vivo was studied using intraperitoneally (i.p.) transplanted tumor models: murine Lewis lung carcinoma and xenografts of human breast cancer RMG1. Results: Uniform PTX release from PHB-microparticles during 2 months was observed. PTX-loaded PHB microparticles have demonstrated a significant antitumor activity versus pure drug both in vitro in murine hepatoma cells and in vivo when administered i.p. to mice with murine Lewis lung carcinoma and xenografts of human breast cancer RMG1. Conclusion: The developed technique of PTX sustained delivery from PHB-microparticles has therapeutic potential as prolong anticancer drug formulation.
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Poly(3-hydroxybutyrate)-poly(ethylene glycol) (PHB-PEG) copolymer is a novel member of poly-hydroxyalkanoates (PHAs) produced by biotechnological PEGylation with improved biocompatibility and biodegradability. We used the PHB-PEG to produce the porous 3D scaffolds for bone tissue engineering. The PHB-PEG scaffolds were made by gas porous formation technique using ammonium carbonate as a porogen. The 3D-scaffolds on the base of homopolymer poly(3-hydroxybutyrate) (PHB) and its blend with PEG 70:30 w/w (PHB+PEG) were used for comparison. In this study morphology (e.g., porosity), hydrophilicity, thermal properties and protein adsorption of PHB-PEG 3D scaffolds were examined. Bone marrow stromal cells (BMSCs) and 3T3 fibroblast cells were cultured on PHB-PEG 3D scaffolds. Cell viability, growth and adhesion on polymer 3D scaffolds were investigated by XTT test and scanning electron microscopy. The obtained data showed that the PHB-PEG copolymer scaffolds demonstrated lower hydrophobicity and better biocompatibility in comparison with the PHB homopolymer scaffolds and these indicators were not inferior to the PHB+PEG blend. It was also shown that PHB-PEG 3D scaffolds are suitable substrate for cell growth and could be applied for bone tissue engineering.
Cited By :1, Export Date: 9 March 2018, References: Narayan, R., (2001) Orbit Journal, 1, p. 1;
The effect of microwave heating wheat grains (700 W for 0–60 s) on gluten, farinograph, pasting properties and baking (steamed bread and biscuit) of flour was studied. The lipase (LA) and lipoxygenase (LOX) activities of the microwave-treated wheat were monitored, and the accelerated storage at 35 °C of whole wheat flour was also investigated. The results showed that the gluten, farinograph properties and viscosity were influenced to a small extent when microwave treatment time was less than or equal to 20 s and the temperature of the grains was less than or equal to 56 °C. Texture profile analysis indicated that steamed bread made from wheat treated by microwave for 20 s was softer and of better quality. Microwave treatment for longer periods (≥30 s) increased the temperature ≥68 °C, that damaged the gluten and made wheat unsuitable for making steamed bread; however, suitable for making food with lower gluten requirements, such as biscuits. The results obtained from enzyme activity and accelerated storage experiments demonstrated that microwave treatment could inactivate LA and LOX and extend the shelf-life.
We previously reported that the tailor-made random poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (R-PHBHV) and higher-order PHBHV (O-PHBHV) produced by haloarchaea possessed unique material properties to meet biomedical application-specific requirements. Here, we further investigated the biocompatibility and biodegradation of these novel materials. Cell biocompatibility of solution-cast films, assessed using rat fibroblast and osteoblast cells, revealed that R-PHBHV and O-PHBHV exhibited better support for cell attachment and proliferation compared with the bacteria-produced poly-3-hydroxybutyrate (PHB) and PHBHV or polylactic acid (PLA). In vitro and in vivo biodegradation of these materials were evaluated in lipase-containing phosphate buffered solution (LPBS) at pH 7.4 and by implantation in the rabbit dorsal subcutis, respectively. As expected, the R-PHBHV and O-PHBHV films degraded much faster in vivo than those observed in vitro, as demonstrated by obvious weight loss, heavy surface erosion, and fast molecular weight drop under implantation condition. These films showed diverse in vivo degradation rates. Among them, the O-PHBHV-1 film degraded fastest and even faster than PLA. Generally, the tissue response was mild for R-PHBHV and O-PHBHV compared with the controls during the implantation period. Taken together, these data revealed that R-PHBHV and O-PHBHV copolyesters had a wild range of biodegradation profiles and excellent biocompatibility. Thus, haloarchaea-produced PHBHV materials would have great potential for use in different biomedical applications.
Conducting polyaniline can be prepared and modified using several procedures, all of which can significantly influence its applicability in different fields of biomedicine or biotechnology. The modifications of surface properties are crucial with respect to the possible applications of this polymer in tissue engineering or as biosensors. Innovative technique for preparing polyaniline films via in-situ polymerization colloidal dispersion mode using four stabilizers (poly-N-vinylpyrrolidone; sodium dodecylsulfate; Tween 20 and Pluronic F108) was developed. The surface energy, conductivity, spectroscopic features, and cell compatibility of thin polyaniline films were determined using contact-angle measurement, the van-der Pauw method, Fourier-transform infrared spectroscopy, and assay conducted on mouse fibroblasts, respectively. The stabilizers significantly influenced not only the surface and electrical properties of the films but also their cell compatibility. Sodium dodecylsulfate seems preferentially to combine both the high conductivity and good cell compatibility. Moreover, the films with sodium dodecylsulfate were non-irritant for skin, which was confirmed by their in‐vitro exposure to the 3D-reconstructed human tissue model.
Orthopedic implants containing biodegradable magnesium have been used for fracture repair with considerable efficacy; however, the underlying mechanisms by which these implants improve fracture healing remain elusive. Here we show the formation of abundant new bone at peripheral cortical sites after intramedullary implantation of a pin containing ultrapure magnesium into the intact distal femur in rats. This response was accompanied by substantial increases of neuronal calcitonin gene-related polypeptide-α (CGRP) in both the peripheral cortex of the femur and the ipsilateral dorsal root ganglia (DRG). Surgical removal of the periosteum, capsaicin denervation of sensory nerves or knockdown in vivo of the CGRP-receptor-encoding genes Calcrl or Ramp1 substantially reversed the magnesium-induced osteogenesis that we observed in this model. Overexpression of these genes, however, enhanced magnesium-induced osteogenesis. We further found that an elevation of extracellular magnesium induces magnesium transporter 1 (MAGT1)-dependent and transient receptor potential cation channel, subfamily M, member 7 (TRPM7)-dependent magnesium entry, as well as an increase in intracellular adenosine triphosphate (ATP) and the accumulation of terminal synaptic vesicles in isolated rat DRG neurons. In isolated rat periosteum-derived stem cells, CGRP induces CALCRL- and RAMP1-dependent activation of cAMP-responsive element binding protein 1 (CREB1) and SP7 (also known as osterix), and thus enhances osteogenic differentiation of these stem cells. Furthermore, we have developed an innovative, magnesium-containing intramedullary nail that facilitates femur fracture repair in rats with ovariectomy-induced osteoporosis. Taken together, these findings reveal a previously undefined role of magnesium in promoting CGRP-mediated osteogenic differentiation, which suggests the therapeutic potential of this ion in orthopedics.
Hydrolysis of poly(3-hydroxybutyrate) (PHB) was studied using acidic functionalized ionic liquid 1-methyl-3-(3-sulfopropyl)-immidazolium hydrogen sulfate ([HSO3-pmim][HSO4]) as catalyst. The influences of experimental parameters, such as reaction temperature, reaction time, the amount of catalyst and mole ratio of water to PHB were investigated. Under the conditions of reaction temperature 160 °C, reaction time 4.0 h, m([HSO3-pmim][HSO4]):m(PHB) = 0.06:1 and n(H2O):n(PHB) = 7:1, the conversion of PHB was over 98%. The ionic liquid could be reused up to 6 times without apparent decrease in the conversion of PHB. In addition, the kinetics of the reaction was investigated, the result indicated that hydrolysis of PHB in [HSO3-pmim][HSO4] was a first-order kinetic reaction and the activation energy was 171.1 kJ/mol.