Engineered Regeneration 3 (2022) 24–40

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Engineered Regeneration
journal homepage: http://www.keaipublishing.com/en/journals/engineered-regeneration/

Polyhydroxyalkanoates in tissue repair and regeneration

Wentai Guo a,b,c, Keli Yang a,b, Xiusen Qin a,b, Rui Luo a,b, Hui Wang a,b,∗, Rongkang Huang a,b,∗
a
Department of Colorectal Surgery, Guangdong Provincial Key Laboratory of Colorectal and Pelvic Floor Diseases, The Sixth Affiliated Hospital of Sun Yat-sen
University, Guangzhou 510655, China
b
Institute of Biomedical Innovation and Laboratory of Regenerative Medicine and Biomaterials, Biomedical Material Conversion and Evaluation Engineering Technology
Research Center of Guangdong Province, Guangzhou 510655, China
c
School of Materials Science and Engineering, National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology,
Guangzhou 510006, China

a r t i c l e i n f o a b s t r a c t

Keywords: Developing advanced biocompatibility materials is of critical importance in biomedical engineering. Polyhydrox-
Polyhydroxyalkanoates yalkanoates (PHA), being famous for its flexible mechanical properties, thermal properties, biocompatibility, and
Biomedical engineering biodegradability, has been widely used in wound dressings, artificial blood vessels, heart valves, nerve conduits,
Tissue repair
bone and cartilage scaffolds, surgical sutures, and other fields. However, reports on the application of PHA in
Tissue regeneration
tissue repair and regeneration are often lacking in systematics. Here, a comprehensive and in-depth perception of
Biomaterial
the performance advantages and application value of PHA is provided. In this review, the following applications
of PHA in biomedical engineering are covered: i) soft tissue, ii) organ tissue, iii) vascular tissue, iv) heart valve
tissue, v) nerve conduit tissue, vi) bone tissue, vii) cartilage tissue and viii) others. Finally, an outlook on the
future research directions and challenges of PHA is presented.

1. Introduction
Compared to traditional petrochemical-based polymers, bio-sourced
polymers show many advantages and are often fermented from cheap
renewable resources. Moreover, biopolymers’ good biological proper-
ties and degradability make them widely used in biomedical engineer-
ing [1–3]. Among the many biopolymers, polyhydroxyalkanoates (PHA)
are linear polyesters synthesized by microorganisms and prototyped
as metabolic particles accumulated in the cytoplasm of prokaryotic
cells [4,5]. It is usually an energy and carbon storage material pro-
duced by bacterial microbiomes under the conditions of sufficient car-
bon sources but lacks nitrogen and phosphorus nutrients [6,7]. PHA
can be synthesized by most Gram-positive bacteria (Bacillus [8,9]),
Gram-negative bacteria (Pseudomonas [10,11], Alcaligenes [12,13], and
Aeromonas Bacillus [14,15]), anaerobic [16,17] and aerobic bacteria
[18,19], photosynthetic bacteria [20,21], and archaea [22,23]. These
microbiomes achieve PHA accumulation through a variety of carbon-
rich substrates. In addition, some bacteria have been shown to produce
PHA without any nutrient limitation, such as Alcaligenes latus strain IAM
12,664 T [24].
Currently, the PHA members produced by bacterial fermentation
vary considerably in structure and properties. This mainly depends on
microbial species, biosynthetic conditions, and carbon source substrate
in the production process, thereby changing the composition of PHA at
the hydroxyalkanoate (HA) monomer level [25–27]. Fig. 1 illustrates the
typical chemical structural of PHA, where each HA monomer has a side
chain attached to the main chain. It can be substituted by various func-
tional groups, including methyl (C1) to tridecyl (C13), aromatic, etc.
This variation makes PHA easily modified to synthesize polymers with
different properties [28,29]. According to the number of HA monomers
integrated into the polymerization chain, PHA can be divided into three
categories: the first category consists of short chain length PHA (short-
chain PHA) with 3 to 5 carbons, such as polyhydroxyvalerate (PHV)
and polyhydroxybutyrate (PHB); the second category consists of MCL-
PHA (medium chain length PHA) with 6 to 13 carbons, such as poly-
hydroxyhexanoates (PHHx) and polyhydroxyoctanoates (PHO); and the
third category of LCL-PHA (long chain length PHA) consisting of more
than 14 carbon units, such as 3-hydroxyoctadecanoic acid (3HOD) and
3-hydroxyhexadecanoic acid (3HHD) [30]. Among them, SCL-PHA has
typical thermoplastic properties with a high glass transition tempera-
ture (Tg); while MCL-PHA is similar to latex-like elastomers with a low
molecular weight.
Over the past decade, about 150 monomers of PHA poly-
mers have been identified, but only a few of them are in-
volved in the formation of PHA, including PHB, PHV, poly-4-
hydroxybutyrate (P4HB), PHO, poly-hydroxybutyrate-hydroxyvalerate
(PHBV), poly-hydroxybutyrate-co-hydroxyhexanoate (PHBHHx), and
poly-3-hydroxybutyrate-4-hydroxybutyrate (P3HB4HB) [31]. Due to its

∗
Corresponding author.
E-mail addresses: wang89@mail.sysu.edu.cn (H. Wang), huangrk3@mail.sysu.edu.cn (R. Huang).

https://doi.org/10.1016/j.engreg.2022.01.003
Received 30 December 2021; Received in revised form 18 January 2022; Accepted 19 January 2022
Available online 21 January 2022
2666-1381/© 2022 The Authors. Publishing Services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
W. Guo, K. Yang, X. Qin et al. Engineered Regeneration 3 (2022) 24–40

organ, vascular, nerve, bone, cartilage, drug carrier

skin, organ, vascular, nerve, bone, cartilage

skin, organ, vascular, nerve, bone, cartilage

skin, organ, vascular, bone, cartilage
heart valve, suture
Application

heart valve
–
Young’s modulus (GPa)
Fig. 1. The typical chemical structural of PHA. (a) PHB. (b) P4HB. (c) PHV. (d)

0.149 ∼ 0.180
PHO. (e) P3HB4HB. (f) PHBV. (g) PHBHHx.

1.1 ∼ 3.5

0.2 ∼ 2.9
0.0199

0.0236
biocompatibility and adaptability, these representative PHA has been

–
widely used in wound dressings [32], artificial blood vessels [33], heart

Elongation at break (%)

valves [34], bone [35] and cartilage scaffolds [36], nerve conduits [37],
and surgical sutures [38]. However, reports on the application of PHA
in tissue repair and regeneration are often lacking in systematics [39].

696.6 ∼ 1014
Therefore, in this review, we review the achievements of PHA in tissue

380 ∼ 850
268 ∼626
repair and regeneration over the past decade or so, and provide an out-

312.9
5∼6

look on the future application trends of PHA. The medical-engineering 14

50
multidisciplinary intersection has strongly promoted the progress of
PHA in personalized precision tissue repair and regeneration, while pro-
Tensile strength (MPa)

viding a new strategy for the development of biomedical engineering.

2. Properties of PHA
13.8 ∼ 104
40 ∼ 62

20 ∼ 38
9 ∼ 21
3 ∼16
31.2

PHA has good biodegradability, biocompatibility, non-

12

carcinogenicity, thermal and mechanical properties. Due to the

abundance of monomers, PHA also exhibits a high degree of structural
−50 ∼ −47

−45 ∼ −7
−8 ∼ −1
−2 ∼ −1

diversity. Based on these properties, this type of polymeric material

Characteristics of different types of PHA.

Tg (°C)

−16.5
−37.2

has high application value in tissue repair and regeneration. As shown

4

in Table 1, we have summarized the properties and advantages of

common kinds of PHA.
104.2 ∼ 109.7
177 ∼ 180

152 ∼ 164
133 ∼ 170
120 ∼ 127
53 ∼ 61

2.1. Biodegradability
Tm (°C)

72.2

One of the unique biological properties of PHA is its degradability

in a variety of environments [40,41]. PHA can be degraded by mixed
Type of PHA

microbial populations existing in rivers, seawater, soil, and other en-

P3HB4HB

PHBHHx
Table 1

vironments under aerobic or anoxic conditions, and decomposed into

PHBV
P4HB

PHO
PHV
PHB

water and carbon dioxide [42,43]. These microorganisms degrade PHA

into low molecular weight monomers or oligomers by secreting PHA
depolymerase (hydrolases). The above oligomer and monomer compo-
nents are subsequently taken up by the cells as nutrients [44]. Qu et al.
tested the biodegradation of PHA in different environments and found
that PHB and PHBV can be wholly degraded under most conditions [45].
PHA is the currently known degradable biomaterial with a minor degra-
dation restriction. Its biodegradation rate mainly depends on the affinity
of PHA depolymerase for ester bonds in PHA polymers, which is influ-

25
W. Guo, K. Yang, X. Qin et al.
enced by polymer conformation, surface area, monomer type, monomer
composition, molecular weight, crystallinity, and environmental factors
(pH, humidity, and temperature) [46]. In general, the degradation rate
of polymers increases faster with decreasing crystallinity and melting
temperature (Tm). Therefore, MCL-PHA with low crystallinity and low
Tm degrades faster than SCL-PHA with high crystallinity and high Tm.
For example, P3HB4HB containing 4-hydroxybutyrate (4HB) and PHBV
copolymer containing 3-hydroxyvalerate (3HV) have lower crystallinity
and Tm than PHB homopolymer, so P3HB4HB and PHBV degrade faster
than PHB, and the degradation accelerates with increasing side-chain
monomer content [47].
2.2. Biocompatibility
Another unique biological property of PHA is biocompatibility,
which has attracted particular attention. In vitro experiments have
shown that PHA can support the growth of most tissue cells, including
epithelial cells, chondrocytes, and fibroblasts [48,49]. PHA will not trig-
ger a robust immune response when implanted in living tissues such as
rats, rabbits, and humans. This series of studies proved that PHA has ex-
cellent biocompatibility and can even promote cellular tissue adhesion
and proliferation [50,51]. Moreover, the biocompatibility of PHA can be
reflected in their immunological and physiological response after degra-
dation. Some monomers of the PHA polymerization chain, such as the
3-hydroxybutyric acid (3-HB), are natural metabolites produced by the
oxidation of fatty acids in the human liver [52]. It is present in human
blood at concentrations of 30–100 mg/L. 3-HB can reduce inflammation,
promote tissue regeneration [53], and act as an energy substance for the
body when necessary (starvation), eventually being excreted as carbon
dioxide [54,55]. In contrast, degradable polymers such as polylactic acid
(PLA) and polylactic-co-glycolic acid (PLGA) will cause accumulation of
lactic acid products after being implanted in the host, while PHA main-
tains pH stability during the degradation process, which undoubtedly
makes PHA more advantageous in terms of biocompatibility.
2.3. Non-carcinogenicity
Non-carcinogenicity is one of the critical properties of biopolymers
used in humans. Notably, few studies have reported whether PHA stim-
ulates cancer cell development while promoting cell proliferation [29].
In this case, Peng et al. found that rat fibroblasts cultured with PHB,
PHBV, P3HB4HB, and PHBHHx did not induce carcinogenesis, suggest-
ing that cells proliferating rapidly on PHA are regulated by the normal
cell cycle. In addition, primary rat osteosarcoma cells cultured in PHA
maintained normal morphology and did not transform into tumor mor-
phology [56]. The mechanism by which PHA promotes tissue regener-
ation may be related to the tissue cell response to 3-HB and microRNA-
mediated material-cell interactions [57]. Nevertheless, there is still no
clear evidence to confirm the non-oncogenic effect of PHA on all cell
lines. Promoting and enhancing the research of non-carcinogenicity of
PHA on whole-cell lines will undoubtedly be an important part of future
PHA applications research.
2.4. Thermal properties
PHA is a semi-crystalline polymer, and its thermodynamic proper-
ties are usually expressed by Tm of the crystalline phase and Tg of
the amorphous phase. Several studies have shown that Tm decreases
with increasing side chain carbon length, while Tg increases accord-
ingly [58,59]. Therefore, SCL-PHA and MCL-PHA often exhibit differ-
ent thermodynamic properties. For example, the crystallinity of PHB is
relatively high, about 60–80%, Tm is about 170°C, and the decomposi-
tion temperature (Td) is about 200°C. The close temperature threshold
leads to the poor thermal stability of PHB processing, which has to be
improved by adding second monomers such as 5-hydroxyvaleric acid
(5HV) and 3-hydroxypropionic acid (3HP) [60]. On the other hand, the
26
Engineered Regeneration 3 (2022) 24–40
crystallinity of MCL-PHA is relatively low, typically 40%, with Tg range
from −52°C to −25°C at room temperature and Tm range from 38°C to
80°C [61]. Since the Tg of MCL-PHA is lower than room temperature, it
can exhibit elastic properties similar to rubber at appropriate tempera-
tures [40]; when the external temperature is higher than Tm, MCL-PHA
will transform into an amorphous form with viscous.
2.5. Mechanical properties
The mechanical strength of PHA mainly depends on the carbon-chain
length, structure, and composition. PHB is the most rigid PHA family
member with a strength of over 45 MPa, and its physicochemical fea-
tures are similar to those of traditional plastics [29]. However, it has
poor processing performance, rapid aging speed, and low elongation at
break. It usually needs to be combined with other monomers to reduce
crystallinity, stiffness, and aging (4HB, 3HP, and 3HV). Another rep-
resentative member of SCL-PHA, P4HB, is a strongly thermoplastic, ex-
tensible ductile material with elongation at break up to 1,000%, making
it the most elastic PHA homopolymer produced to date. In 2007, P4HB
was first approved by the Food and Drugs Administration (FDA) to man-
ufacture surgical sutures [62,63]. Furthermore, in contrast to SCL-PHA,
MCL-PHA is more elastic, as well as a higher elongation at break and
lower tensile strength. MCL-PHA is a class of copolymer composed of
3HHx, 3H2O, 3HD, and 3HDD, with tensile strength between 5 MPa
−16.3 MPa and elongation at break of 88% - 356% [61]. Therefore, com-
pared with poly(𝜀-caprolactone) (PCL), PLA, PLGA, and other polymers,
PHA has wider applicability and the mechanical strength of PHA could
be adjusted by compounding or synthetic copolymerization to simulate
the final target environment [64,65].
3. Applications of PHA
Due to its structural diversity and favorable physical-chemical prop-
erties, PHA has received extensive attention in biomedical tissue engi-
neering. To date, many studies have reported partial application of PHA
in tissue repair and regeneration, including soft tissue engineering, or-
gan tissue engineering, vascular tissue engineering, heart valve tissue
engineering, nerve tissue engineering, bone tissue engineering, and car-
tilage tissue engineering (Fig. 2).
3.1. Soft tissue engineering
Soft tissue engineering covers many tissues, including skin, tendons,
blood vessels, nerves, and other tissues. As many researchers have re-
ported PHA applications in this area, a detailed description will be pre-
sented in the following chapters. In this section, we only introduce the
application of PHA in the repair of superficial soft tissues such as skin
and tendons.
In wound healing, basal cells surrounding the wound usually pro-
liferate and migrate to the center of the defect to form a single epithe-
lium layer and then differentiate into the squamous epithelium. Wound
dressings are the most effective way to accelerate wound healing, espe-
cially in large areas of skin tissue defects or infective wounds [66,67].
As shown in Table 2, the PHA engineering scaffolds prepared by tis-
sue engineering technology have obvious advantages in skin tissue re-
pair. This is mainly owing to the acceptable mechanical, biocompati-
ble, and repair-promoting properties of PHA. For example, Volova et al.
prepared P3HB4HB fibrous membranes by solvent casting and electro-
static spinning techniques as wound dressings. Their results showed
that P3HB4HB electrospun membranes were more conducive to stem
cell proliferation and differentiation. In contrast, these cells could pro-
mote the migration of peri‑wound epidermal cells to adjacent tissues
by producing an extracellular matrix (ECM). Therefore, PHA enhanced
angiogenesis, reduced inflammation, and ultimately promoted wound
healing [68,69]. Amirul et al. incorporated collagen peptides to fabri-
cate P3HB4HB nanofibrous membranes as a wound dressing to improve
W. Guo, K. Yang, X. Qin et al. Engineered Regeneration 3 (2022) 24–40

Fig. 2. Application of PHA in tissue repair and regeneration.

Table 2
PHA in soft tissue engineering.

Type of PHA Combination Model Advantage Year Reference

PHB gelatin a diabetic rat wound model faster wound healing with higher content of hair follicles and sweat 2021 [72]
glands
PHBV GelMA/EGF a diabetic rat wound model rapid diabetic wound healing, favourable angiogenesis and cellular 2021 [74]
response
PHBV HMK/AgNPs a rat wound healing model better wound healing ability, good biocompatibility, and favorable 2020 [156]
antibacterial properties (Fig. 3b)
PHBV – a full-thickness mouse wound model mitigated excessive scar formation by regulating myofibroblast 2020 [71]
formation
PHBV nCeO2 – promoted the healing of diabetic wounds, enhanced vascularization 2020 [73]
and cell proliferation (Fig. 3c)
P3HB4HB BC third-degree skin burn model in rats promoted healing more effectively than VoskoPran (a commercial 2019 [69]
wound dressing)
PHBV AMPs/Tac a deep-wound mouse model accelerated wound healing by reducing the number of bacteria 2018 [78]
PHB DE a NOD/SCID mouse xenograft model greater angiogenic and M2 macrophage polarization properties 2018 [165]
P3HB4HB fibroblasts model skin defects in Wistar rats promoted wound healing, enhanced vascularization, and reduced 2016 [68]
inflammation
P3HB4HB FSCPs a rat wound healing model significant promotion on wound contractions (61%) 2016 [166]
PHB PCL/PEGMA – significant collagen deposition, enhanced vascularization and 2015 [167]
fibrosis
PHBV ASCs a rat wound healing model improved wound healing ability, reduced scarring, 2015 [168]
necessary bioactive
PHBV CS/USSCs – significantly promoted wound healing 2014 [169]
PHBV collagen/gelatin a rat wound healing model effective in promoting epidermal regeneration in the early stages of 2007 [170]
wound healing
HMK: high molecular keratin; AgNPs: Ag nanoparticles; DE: dermo-epidermal skin equivalents; FSCPs: fish-scale collagen peptides; USSCs: unrestricted somatic
stem cells.

surface wettability further while possessing biodegradability. Hence, in
vivo study using a Sprague Dawley (SD) rat model showed a positive ef-
fect of the P3HB4HB/collagen peptides composite membrane on wound
contraction with a maximum percentage of 79% [70]. To inhibit exces-
sive scarring in wound healing, Kim et al. prepared three kinds of PHBV
electrospun scaffolds with different 3HV contents (10, 30, and 60 mol%,
Fig. 3a). As the 3HV content increased, the crystallinity of PHBV poly-
mer decreased and the scaffolds became more elastic. The electrospun
scaffold group had thin granulation tissue generation in mouse full-skin
wound models without dehydration or nodular scar formation. These
results suggest that the higher 3HV content in PHBV makes the polymer
more elastic and maintains minimal dermal fibroblast/myofibroblast
transition to alleviate excessive scar proliferation, demonstrating its po-
tential as an ideal wound dressing [71]. For complex skin wounds, San-
hueza et al. prepared dual-size gelatin/PHB electrospun fibers for regen-
eration of diabetic wounds. The fibers treatment group exhibited higher
hair follicles, higher sweat gland content, and lower fibroblast content in
the wounds [72]. Robin and colleagues blended cerium oxide nanopar-
ticles (nCeO2 ) into PHBV electrospun fiber membranes. In their study,
the PHBV membranes incorporated with nCeO2 had a strong potential
to enhance cell proliferation and vascularization, which could promote
diabetic wound healing [73,74].
On the other hand, the long degradation cycle of PHA helps to de-
velop functional wound dressings with drug delivery capability. For
example, Marian et al. prepared chitosan (CS)/PHB composite mem-
branes loaded with kaempferol nanocrystals for infected wounds. The
membrane demonstrated sustained antibacterial properties with power-
ful killing effects against multi-drug resistant bacteria at low concentra-
tions, making it highly suitable for critically infected wounds healing
[75]. Marcano et al. obtained asymmetrically structured PHA fibrous
scaffolds containing anti-biofilm protein Dispersin B (DB) through phase
separation techniques. By optimizing the micro-nano structure of the
PHA substrate, the PHA/DB could be captured more effectively to in-
hibit the tissue surface biofilms or disrupt the formed biofilms [76,77].
Modification of implants with antimicrobial peptides (AMPs) could re-
duce antibiotic resistance and prevent infection, so Qiong et al. labeled
the AMPs, tachyplesin I (Tac) with PHA particle binding protein (PhaP)
and immobilized them on the PHBV scaffolds (Fig. 3d). The Tac-PhaP
coating enhanced the hydrophilicity of the PHBV scaffold and efficiently
inhibited the growth of harmful bacteria. Further experiments on deep
wound healing in mice verified the effect of the scaffold in inhibiting
wound bacterial population and accelerating wound healing in vivo [78].
In the field of tendon repair, Webb et al. investigated the practi-
cal repair effect of the PHBHHx/gel porous fiber scaffold in rat Achilles

27
W. Guo, K. Yang, X. Qin et al.
tendon injury models (Fig. 4a). The composite scaffold showed excellent
tensile strength that matched the rat tendons, with full weight-bearing
and functional recovery at 20 days postoperatively. Moreover, no late-
onset inflammatory reaction was observed at 40 days after implanta-
tion, indicating the suitability of the PHBHHx scaffold for tendon repair
in vivo [79]. Next, Chen et al. prepared PHB membranes modified with
glutaraldehyde (GA), CS, dopamine (DA), and hydrogen peroxide. In
the rabbit anterior cruciate ligament (ACL) model, the functionalized
PHB membrane could enhance fixation and healing between tendon
and bone, providing a new approach to ACL reconstruction [80]. Be-
sides, Tashjian et al. utilized PHA scaffolds to enhance suture interface
tension in rotator cuff repair by investigating the performance of PHA
scaffolds on cyclic and fracture strength, which ultimately resulted in
more optimal mechanical support than the control group [81].
3.2. Organ tissue engineering
In organ replacement repair, biomedical engineering techniques are
used to construct bionic scaffolds that simulate the function and struc-
ture of organs. Subsequently, such scaffolds are implanted in the body
to replace original dysfunctional organs, potentially providing solutions
to some intractable diseases [82]. Recent studies reported the applica-
28
Engineered Regeneration 3 (2022) 24–40
Fig. 3. Examples of PHA in skin tissue engineering applications.
(a) PHBV fibrous meshes for preventing excessive scar formation
[71]. (b) Fabrication of PHBV/keratin composite membranes for
wound healing [156]. (c) GelMA/PHBV hybrid meshes loaded
with growth factors for diabetic wound healing [74]. (d) A PhaP-
Tac modified PHBV scaffold as anti-infective wound dressings
[78].
Fig. 4. Examples of PHA in organ tissue engineering applica-
tions. (a) A PHBHHx scaffold for tendon repair in rats [79]. (b)
PHBVHHx scaffolds applied to a mouse liver-injured model [83].
The scale bars are 5 mm, respectively. (c) The morphology of
PLA/PHB scaffolds [86]. (d) In vitro differentiation experiments
of PHB/CS/nBG scaffolds [87]. (e) CS/PHBV composite hydro-
gels for nucleus pulposus tissue engineering [157].
tion of PHA in organs such as liver, gastrointestinal tract, urinary tract,
teeth, and pancreatic islets (Table 3). For example, Su and colleagues
developed a PHBVHHx-based bionic scaffold loaded with mesenchymal
stem cells (MSCs); and then transplanted the scaffold into liver-injured
mice (Fig. 4b). The damaged liver showed a similar structure to normal
liver tissue after 28 days, with a significant decrease in total bilirubin
(TB) and alanine aminotransferase (ALT), indicating that the PHBVHHx
scaffold could promote the regeneration of damaged liver [83]. In the
field of gastrointestinal tract repair, Fan et al. constructed a composite
esophageal tissue engineering scaffold by using PHBHHx/poly(lactic-
co-glycolic acid) (PLGA) combined with decellularized porcine small
intestine submucosa (SIS). The mixture was successfully applied to re-
construct a half circumferential esophagus defect, suggesting that PHB-
HHx is an ideal artificial esophageal substrate [84]. In addition, El-
Khordagui et al. compared the anti-adhesion potential of PHB electro-
spun nanofibrous with methylene blue (MB) cast film. This PHB/MB
composite film effectively prevented visceral adhesions and enhanced
cecum wall healing without causing a significant inflammatory response
[85]. In the field of urethral repair, Findrik et al. successfully utilized a
3D printed PLA/ PHB composite scaffold as a tubular substitute for ure-
thral replacement (Fig. 4c). The scaffold provided stable support condi-
tions for urethral repair in dogs by adjusting the ratio between PLA and
PHB [86].
W. Guo, K. Yang, X. Qin et al.
Table 3
PHA in organ tissue engineering.
Type of PHA Combination Model
PHBV iPSCs – insulin, insulin protein, Pdx-1 and Glut-2 genes were
PHB CNTs – induced only a mild foreign body reaction and
PHB CS/nBG – increased expression of ALP, collagen type-I and DSPP
PHB PLA a dog urethral defect model
PHB MB a rat cecum model
PHBV gelatin – phenotypic marker proteins and ECM proteins
PHBVHHx MSCs liver-injured mouse models
PHBHHx PLGA/SIS full circumferential esophageal defects in rats
PHBHHx CS/insulin a diabetic rat model
PHBHHx/P3HB4HB PLA – construct spherical islet-like structures with
iPSCs: induced pluripotent stem cells.
In some other works, Forughi and colleagues investigated the effect
of PHB/CS/ nano-bioactive glass (nBG) composite fiber scaffolds on the
proliferation of exfoliated milk tooth stem cells (Fig. 4d). Their results
indicated that the composite scaffold increased cell proliferation rates
as well as calcium and phosphorus ion uptake rates. In addition, the ex-
pression of type I collagen and dentin sialic acid phosphoprotein (DSPP)
increased 6-fold, making it suitable for treating dental diseases [87].
Zarei et al. prepared PHB/carbon nanotubes (CNTs) scaffolds using elec-
trospinning technology, which showed that the scaffold induced only a
mild foreign body reaction and improved vascularization [88].
In the studies of islet transplantation for diabetes, insulin delivery
systems mixed with phospholipids were successfully prepared based on
PHBHHx nanoparticles, which significantly enhanced the controlled re-
lease of insulin and the therapeutic effect [89,90]. More interestingly,
Yang et al. used stem cell technology to construct islet-like spherical
structures on PHBHHx membranes, with significantly upregulated gene
expression and extracellular secretion of insulin [91]. In summary, PHB-
HHx proved to be an excellent fabrication material for islet transplan-
tation.
3.3. Vascular tissue engineering
Vascular grafts are in high demand for cardiovascular surgery such as
aortic replacement and coronary artery bypass graft surgery. Through
the division and proliferation of endothelial cells on both sides of the
anastomosis, they are interconnected with the help of vascular grafts to
restore the original endothelial structure. However, synthetic polymer
vessels lack growth potential and have a high risk of thromboembolism,
stenosis, and infection [92]. In recent years, PHA has become a sub-
strate material for developing functional tissue-engineered blood ves-
sels (TEBV) based on its modifiable elasticity, hemocompatibility, and
induction of elastin formation (Table 4). For example, Antonova et al.
tested the effect of PHBV/PCL vascular grafts containing basic fibrob-
last growth factor (bFGF) and vascular endothelial growth factor (VEGF)
in a sheep carotid artery interposition model, compared with expanded
polytetrafluoroethylene (ePTFE) artificial vessels (Fig. 5a). PHBV/PCL
vascular grafts showed a more stable long-term effect than ePTFE and
could effectively prevent aneurysm formation after 18 months [33].
Puppi et al. prepared PHBHHx vascular grafts with an inner diameter of
6 mm by wet electrostatic spinning to develop small-diameter polymeric
biodegradable vessels. Their results confirmed that these vessels could
29
Engineered Regeneration 3 (2022) 24–40
Advantage Year Reference
2021 [171]
significantly improved
2020 [88]
improved vascularization
2018 [87]
genes
a tubular urethral substitute with 2018 [86]
significant stability
prevented visceral adhesions and enhanced cecum 2016 [85]
wall healing without causing a significant
inflammatory response
2014 [172]
expressions were significantly improved
similar structure of normal livers; TB and ALT 2014 [83]
significantly decreased, and ALB significantly
increased
successfully used for reconstructing esophagus 2014 [84]
the bioavailability of insulin was significantly 2013 [90]
enhanced; novel ultralong-acting insulin injections
2010 [91]
significantly upregulated gene expression and
extracellular secretion of insulin
well withstand radial compressive stress, radial compliance, and circum-
ferential tensile stress, as well as strength maintenance during suturing.
Next, cytotoxicity and hemolysis tests compared the in vitro biocompat-
ibility of PHBHHx vascular grafts and commercial artificial vessels. The
developed scaffold could maintain the proliferation of human umbilical
vein endothelial cells (HUVECs) for two weeks; the scaffold could effec-
tively prevent thrombosis directly in contact with human blood [93].
Besides, Jayakumar and colleagues fabricated a tri-layer fiber scaffold
based on elastin nanofibers, polyvinyl alcohol (PVA) and PHBV by elec-
trostatic spinning technique (Fig. 5b and c). The in vitro biocompatibil-
ity shows that MSCs, smooth muscle cells (SMCs), and HUVECs exhib-
ited high growth rates on the composite scaffolds above. These results
emphasize the importance of fiber arrangement on a triple electrospun
scaffold, which is essential to mimic the multilayer structure of natural
vessels [94].
For tissue-engineered vascular applications, hemocompatibility is
another important biological feature. Qu et al. compared the platelet
adhesion of PHB, PHBV, and PHBHHx. Among them, PHBHHx signif-
icantly reduced hemolytic activity in vitro test contacted with erythro-
cytes and significantly reduced the number of adherent platelets com-
pared with other groups [95]. Li et al. investigated the platelet adhesion
of P3HB4HB and poly (propylene glycol)-poly (ethylene glycol)-poly
(propylene glycol) (PPG-PEG-PPG). The platelet adhesion number of the
composites could be changed by modifying the ratio of P3HB4HB to
PPG-PEG-PG [96]. These studies showed good biocompatibility of PHA
polymers in prolonged contact with blood. Besides, Arg-Gly-Asp (RGD)
peptides coating has been reported to improve the biocompatibility of
artificial vascular patches. Thus, Viktoriia et al. evaluated the hemo-
compatibility of RGD peptide modified PCL/PHBV patches and found
that the composite scaffolds could significantly promote the formation of
new vascular systems in vivo. RGD-modified PCL/PHBV patches showed
milder platelet aggregation, and fewer lysed red blood cells than com-
mercially available patches [97]. In another study, Larissa et al. incor-
porated VEGF and RGD peptides into small-caliber vascular grafts of
PHBV/PCL, which improved the graft compression strength and accel-
erated the formation of functional monolayer endothelial cells (Fig. 5d)
[98]. In addition, Cheng and colleagues further validated the induction
of P3HB4HB films on the elastin formation of intravascular RaSMCs.
Fastin elastin assay showed that 4HB monomer promoted the formation
and accumulation of elastin. Benefiting from its modifiable strength,
elasticity, and ability of elastin formation, P3HB4HB could be con-
W. Guo, K. Yang, X. Qin et al. Engineered Regeneration 3 (2022) 24–40

Table 4
PHA in vascular tissue engineering.

Type of PHA Combination Model Advantage Year Reference

PHBV PCL/VEGF/bFGF/SDF a sheep carotid artery interposition model higher primary patency, promoted vascular tissue 2021 [33]
regeneration
PHBV PCL/RGD peptides a rat abdominal aortas model facilitated the formation of neovasculature, resistance 2020 [97]
to calcification
PHBV PVA/elastin – mimicked natural arterial vascular structure 2018 [94]
PHBHHx PCL – good antithrombotic properties, sustained proliferation 2017 [93]
of HUVECs
PHBV PCL/VEGF a rat abdominal aortas model improved graft patency and accelerated monolayer 2016 [173]
endothelial cell formation
P3HB4HB PPG-PEG-PPG – controllable amount of platelet adhesion 2011 [96]
PHB collagen/CS a nude rat subcutaneous model promoted osteogenesis and vascularization 2010 [174]
P3HB4HB SMCs – ability to induce elastin formation, adjustable 2008 [99]
elasticity and strength
PHBHHx HUVECs/SMCs – enhanced vascular-related cellular affinity 2005 [95]

SDF: stromal cell-derived factor.

Fig. 5. Examples of PHA in vascular and heart valve tissue engineering appli-
cations. (a) A PHBV/PCL tissue-engineered scaffold for carotid artery repair
[33]. (b-c) The construction process of engineered vascular scaffolds based on
PHBV/PVA [94]. (d) The process of implanting PHBV/PCL grafts containing
RGD peptides into rat abdominal aortas [98]. (e) A tri-leaflet heart valve scaf-
fold based on P4HB polymer [158].

sidered as a valuable material for artificial vascular engineering [99].
Similarly, P4HB has also been studied for the manufacture of vascular
scaffolds. For example, sheep autologous pulmonary artery epithelial
cells were inoculated onto a P4HB/polyglycolic acid (PGA) scaffold to
strengthen pulmonary arteries in vivo. The up-regulation of matrix met-
alloproteinases (MMPs) expression demonstrated the effectiveness of the
P4HB scaffold in pulmonary artery remodeling [100].
3.4. Heart valve tissue engineering
Surgical replacement of artificial biological or mechanical valves is
the most effective therapeutic modality for heart valve disease. How-
ever, these valves lack growth and durability property, leading to throm-
botic complications in patients due to irregular blood contact surfaces.
Therefore, heart valve tissue engineering (HVTE) based on dynamic con-
ditions has emerged as an important strategy for treating heart valve
diseases [101]. Recent studies have shown that PHA is an excellent sub-
strate for HVTE (Table 5). Sodian et al. fabricated a tri-leaflet heart valve
scaffold based on P3HB4HB and inoculated it with ovarian arterial vas-
cular cells. As made of thermoplastic PHA polymers, these valve scaf-
folds could be molded into the desired valve shape using stereolithogra-
phy and ensure synchronized beating of the tri-leaflet valves. The cells
could survive and produce ECM efficiently after pulsatile flow exposure
[102,103]. Subsequently, Sodian et al. inoculated sheep carotid artery
vascular cells (ScAVCs) onto the P4HB heart valve scaffold, which was
implanted and replaced the original pulmonary valve in lamb models.
The results demonstrated that the P4HB heart valve scaffold was covered
by normal tissue without thrombosis, and the valve showed no signifi-
cant regurgitation up to 120 days after the operation. The pathological
data suggested that the surrounding area of the scaffold was mainly lam-
inar fibrous tissue with glycosaminoglycans as ECM [104].
In addition, PHA polymers can be used as heart valve coatings or
patches. For example, the decellularized porcine aortic valves coated
with PHBHHx showed better resistance, increasing tensile strength and
reducing calcification in vivo [105]. Another example is a high porosity
tri-leaflet heart valve prepared using polyglycolic acid (PGA) and coated
with P4HB. In this study, Perry et al. isolated and extracted BMSCs from
the sternal bone marrow of adult sheep. The cells (2 × 106 cells/mL)
were delivered onto the PGA/P4HB composite scaffold to form a "tis-
sue" with biomechanical properties similar to those of the natural heart
valve. After implantation in sheep for 20 weeks, the diameter of the
valve scaffold increased from 19 mm to 23 mm as sheep grew up [106].
Their results suggest that the P4HB composite valve may have growth
potential. On the one hand, due to the inherent elasticity and ductility of

30
W. Guo, K. Yang, X. Qin et al.
Table 5
PHA in heart valve tissue engineering.
Type of PHA Combination Model
MCL-PHA PCL/CPCs the postmortem murine heart models
PHO NVRMs – no negative effect on cardiomyocyte
PHB PCL/PLA/PA an infarcted rat heart model
PHBV PLLA/PGS – formation of myocardial patches with a
PHBHHx decellularized porcine aortic valve pulmonary position in sheep without
cardiopulmonary bypass
MCL-PHA/P4HB PGA/vascular cells 2 cm defect in the pulmonary artery of
sheep
CPCs: cardiac progenitor cells; NVRMs: neonatal ventricular rat myocytes; PGS: poly(glycerol sebacate); PLLA: poly(L-lactide).
P4HB could adapt to tissue growth; on the other hand, stem cells prolif-
erate and differentiate on the scaffold surface, forming a homogeneous
layer of fused cells and extracellular matrix. This newly formed valve
tissue thickens the PGA/P4HB scaffold to gradually accommodate the
development of cardiovascular system defects in children with congeni-
tal heart disease. For cardiac tissue patches, Baghdadi et al. fabricated a
multifunctional cardiac patch of PHO, whose mechanical strength was
close to myocardial tissue [107]. Castellano et al. compared PHB with
PCL, PLA, and polyamide (PA) scaffolds for cardiac repair in infarcted
rat hearts. The PHB composite scaffold could attenuate the inflamma-
tory response in cardiac tissue. Although PHB and PCL materials could
reduce negative ventricular remodeling, only PHB could induce signif-
icant angiogenesis [108]. These results suggested that PHB was more
beneficial for repairing and remodeling cardiac tissue. Besides, Duver-
noy et al. used the PHB cardiac patch for the first time in human patients.
Computerized tomography (CT) showed a 27% reduction in adhesions
between PHB patch and heart surface compared to the non-patch group
[109].
3.5. Nerve conduit tissue engineering
The recovery of motor, sensory and autonomic function caused by
peripheral nerve injury is difficult in nerve repair. Traumatic neuromas
will form if the nerve is severed too far apart (more than 2.5 cm) or scar-
ring between the severed ends. Currently, nerve catheter implantation
surgery is often performed clinically for nerve repair and functional re-
construction [110]. The nerve sheath cells in the injured area proliferate
to form ribbon-shaped syncytial cells, and then the proximal axon grad-
ually grows at a rate of about 1 mm per day through the nerve conduit.
After reaching the terminal axon, the sheath cells produce myelin to en-
circle the axon to form a myelin sheath. This regenerative process often
takes more than several months to complete. PHA has been extensively
employed for nerve repair due to its biocompatibility and piezoelectric
properties (Table 6). For example, PHB could promote differentiation
of Schwarm cells (SCs) and support axonal regeneration within the host
sciatic nerve (Fig. 6c) [111]. Mohanna et al. tested the function of the
PHB/glial growth factor (GGF) conduits in bridging long nerves using
a rabbit gastrocnemius nerve defect model. At 60 days postoperatively,
the PHB composite conduits significantly accelerated nerve regeneration
in the defect and reinformed the motor organ. At 120 days postopera-
tively, the number of SCs and axonal regeneration were higher in the
PHB composite conduits group than in the blank control group [112]. In
another study, Schaakxs et al. inoculated PHB catheters with differenti-
ated adipose-derived stem cells (dASCs) and primary SCs. Their results
found that the catheter promoted rapid nerve regeneration during the
early stage of sciatic nerve repair in rats. In the later stage, a superior
31
Engineered Regeneration 3 (2022) 24–40
Advantage Year Reference
maximize regeneration of myocardial 2018 [34]
infarction, controlled CPCs delivery
2018 [107]
contraction, enhanced cell proliferation
and adhesion
induced significant angiogenesis in 2014 [108]
cardiac tissue, attenuated the
inflammatory expression of M2
macrophage phenotype
2010 [175]
structure similar to that of native tissue
promoted the proliferation of cells 2007 [105]
associated with valve tissue and inhibited
calcification
provided proper lung function in lambs 2000 [104]
within 120 days
functional recovery could be observed in the host, with more axons con-
necting the neural distal stump and a reduction in muscle atrophy [113].
However, the hydrophobicity, brittleness, and crystallization ten-
dency of PHB may hinder the ductility and flexibility of the conduit. To
eliminate these effects, Yu et al. prepared porous nerve conduits with
PHBHHx and successfully applied them to repair sciatic nerve defects
in rats (Fig. 6d) [114]. In addition, PHBHHx showed better biocom-
patibility with neuronal progenitor cells (NPCs) and stem cells (NSCs);
and thus promoted neuronal differentiation and synaptogenesis of these
cells in vitro. Wang et al. implanted NSCs-loaded PHBHHx films into a rat
traumatic brain injury (TBI) model. Their results revealed that PHBHHx
films were able to support the long-term survival of transplanted NSCs
in vivo; subsequently, NSCs could migrate into damaged brain areas, dif-
ferentiate into astrocytes and neurons without additional reactive glial
proliferation [37]. On the other hand, Wang et al. evaluated the repair
capacity of PHBHHX, P3HB4HB, and PHB for the central nervous sys-
tem (CNS) and their differentiation on 3D or 2D matrices. In particular,
the 3D nanofiber structure of PHBHHX could facilitate CNS synaptogen-
esis and has great potential in promoting CNS differentiation into bene-
ficial neurons [115,116]. Sakar et al. showed that nerve grafts made
of PHBHHx with 3D oriented nanofibers significantly strengthen the
nerve regeneration, either alone or in conjunction with hMSCs [117].
Zhang et al. incorporated poly(ethylene oxide) (PEO) into the PHBV
nerve conduits and found that the aligned nanofiber scaffolds provided
topographic guidance for directed cell growth and extension, and suc-
cessfully repairing sciatic nerve defects in rats. [118]. Their results sug-
gested that PHBV was a promising manufacturing material for neural
repair.
3.6. Bone tissue engineering
Bone repair is a progressive and intersecting process, usually divided
into three phases: the hematoma mechanization phase, the primary bone
scab formation phase, the bone scab remodeling and shaping phase. Al-
though bone tissue can regenerate naturally, large bone defects (usually
>2 cm) are difficult to self-healing. Bone tissue engineering provides me-
chanical support to promote new bone regeneration by inducing bone-
associated cell growth and differentiation [119]. Table 7 summarizes
the applications of PHA in bone tissue engineering in recent years. Doyle
et al. reported that PHB implants could rapidly form bone scabs around
bone defects, which subsequently shaped into ordered new bone tissue
connected to the surrounding normal bone tissue. No inflammation was
found at 12 months after implantation [120]. However, compared to
the natural tissue of the host, PHA lacks the sufficient bioactive func-
tion to generate a physiological apatite coating around the material sur-
face. Therefore, to improve the osteogenic properties of PHA, some inor-
W. Guo, K. Yang, X. Qin et al. Engineered Regeneration 3 (2022) 24–40

Table 6
PHA in nerve conduit tissue engineering.

Type of PHA Combination Model Advantage Year Reference

MCL-PHA PCL 10 mm transected sciatic nerve rat model excellent neuroregenerative properties and 2021 [160]
bioresorption rates (Fig. 6b)
PHBHHx NSCs a rat traumatic brain injury model promoted the survival and proliferation of 2019 [37]
transplanted NSCs in TBI rats
PHB chitosan/hMSCs a rat sciatic nerve injury model guided damaged axons across the injured region 2018 [176]
PHBV PEO/laminin 12 mm transected sciatic nerve rat model promoted peripheral nerve regeneration 2018 [118]
PHB SCs/dASCs – greater axon myelination and less muscle atrophy 2017 [113]
PHBHHx hMSCs 10 mm nerve gaps in rats the nerve grafts with 3D oriented nanofibers guided 2014 [117]
nerve regeneration
PHBV SCs 30 mm transected sciatic nerve rat model restored nerve continuity and myelination of nerve 2014 [177]
fibers
PHB – – facilitated recovery of motor and sensory functions of 2009 [178]
peripheral nerves
PHBHHx – 10 mm transected sciatic nerve rat model rapid recovery of severed nerve function 2009 [114]
PHB SCs a rat cervical spinal cord injury model significantly promoted the survival of SCs and axonal 2008 [179]
regeneration
PHB GGF 2–4 cm peroneal nerve gaps in rabbits stimulated regeneration of long interstitial nerves 2003 [180]

Fig. 6. Examples of PHA in nerve tissue engineering applica-

tions. (a) A PHBV electrospun fiber conduit for sciatic nerve re-
pair in rats [159]. (b) Nerve guidance conduits based on MCL-
PHA/PCL blend [160]. The scale bar is 100 μm. (c) Peptide func-
tionalized PHB nanofibrous scaffolds with enhanced activity of
SCs [111]. (d) In vivo morphological changes of PHBHHx nerve
conduits [114].

ganic/organic biological materials such as hydroxyapatite (HA), bioac-
tive glass (BG), and alginate (ALG) were incorporated with PHA. As HA
contributes up to 70% of the bone matrix, incorporation of HA could
improve the osteoinductivity and osteoconductivity of PHB composites
[121,122]. The PHB/HA composite scaffolds could induce a stable bone
tissue adaptation response without foreign body inflammation. In vitro
studies have also shown that the PHB/HA scaffold supports the adhesion
and differentiation of hMSCs and human maxillary osteoblasts (HOBs).
In particular, PHB/HA scaffolds coated with type I collagen effectively
induced ectopic bone formation in nude mice models. After a period of
implantation, high expression of osteocalcin confirmed the osteogenic
characteristics of the cell-seeded PHB/HA scaffolds. In contrast, no sig-
nificant osteocalcin expression or osteoinduction was found in the con-
trol group [123]. On the other hand, the addition of a specific concentra-
tion of nBG to the PHB scaffold significantly improved human osteosar-
coma cells (MG63) proliferation, induced better osteoconductivity and
alkaline phosphatase (ALP) activity [124,125]. Hajiali and colleagues
evaluated the feasibility of using PHB/nBG nanocomposites as bone tis-
sue engineering scaffolds. Immersion of PHB/nBG scaffolds in simulated
body fluid (SBF) for one month could form a thin layer of HA mineraliza-
tion on its surface, which provided augmented repairability for osseoin-
tegration and bone reconstruction [126]. Bioactive osteogenic scaffolds
can be prepared by mixing PHB with hydrogels; therefore, Volkov et al.
fabricated ALG hydrogel-based PHB/HA scaffold by salt leaching tech-
nique using three-dimensional printing (Fig. 7d). The produced scaffolds
provided high strength support for the hydrogels; on the other hand, the
large number of interconnected pore structures inside the scaffolds pro-
vided space for the growth of MSCs and induced osteogenic differentia-
tion of the cells. On day 28, the hydrogel-based PHB/HA composite scaf-
folds loaded with MSCs achieved a very high regeneration rate (94%)
of radial bone defects in rat parietal bone, demonstrating the potential
of bioactive polymer scaffolds in bone tissue engineering [35].
PHBV, another commonly used PHA polymer, was widely mixed
with bioactive compounds for bone tissue engineering. PHBV exhib-
ited comparable mechanical compressive strength as human bone and
improved the in vitro bioactivity of the PHA scaffold. For instance, an
animal experiment reported that the PHBV/HA composite was acceler-
ated the thickness of the new bone increasing from the initial 130 𝜇m
to 770 𝜇m within six months, indicating this scaffold had the supe-
rior property of promoting bone structural reconstruction [127]. Adeno-
sine, another bioactive compound mixed with PHBV, can promote os-
teogenic differentiation of MSCs and osteoprogenitor cell. Zhong and
colleagues systematically investigated the osteogenic ability of the com-
posite nanofibers by doping adenosine molecules in PHBV electrospun
nanofibers in vitro and in vivo. The adenosine-loaded PHBV electrospun
fibers exhibited strong bone regenerative potential in rabbit skull de-
fect repair [128]. Furthermore, Cool et al. compared the osteogenic
properties of PHBV mixed with calcined hydroxyapatite (CHA), and
𝛽-tricalcium phosphate (𝛽-TCP), respectively. It was shown that the
nanoscale reinforced phase incorporated in PHBV improved osteogenic
properties while decreasing the inflammatory response [129]. In ad-
dition, Chernozem et al. proposed a new scaffold that combined PHB
and PHBV with calcium carbonate mineralized piezoelectric bodies.
The presence of minerals could significantly promote apatite formation

32
W. Guo, K. Yang, X. Qin et al. Engineered Regeneration 3 (2022) 24–40

Table 7
PHA in bone tissue engineering.

Type of PHA Combination Model Advantage Year Reference

PHB HA/ALG/MSCs critical size parietal bone defect in rats enhanced regeneration of critical-sized bone defects 2020 [35]
PHBV aloe vera-derived gel – higher expression of ALP, mineralized bone-related 2020 [181]
genes and proteins
PHBHHx rhBMP-2 critical sized bone defect (15 mm) in rabbits enhanced bone regeneration and facilitated 2020 [133]
vascularization
PHB BC critical-size mouse calvaria defects support preadipocytes proliferation and osteoblast 2020 [182]
differentiation, consequently inducing new bone
formation
P3HB4HB – a calvarial defect model in rats excellent biocompatibility and bone regeneration 2019 [183]
ability
PHBV adenosine critical-sized rabbit cranial defects excellent biocompatibility and osteogenic capacity 2019 [128]
PHBV PEG – activation of the ALP activity, increased gene 2019 [184]
expression of bone specific markers, and mineral
nodules formation
P3HB4HB GO critical-sized calvarial defect of rats enhanced biomechanical properties, and fast 2017 [163]
osteogenic capability (Fig. 7c)
PHBV 𝜅-CG – significant bioactive and osteogenic properties 2017 [185]
PHB – critical size parietal bone defect in rats good biocompatibility and an osteoconductive 2015 [186]
properties
PHBHHx SMC critical defect of 15 mm in length in rabbits excellent biodegradability and biocompatibility for 2014 [187]
more effective osteogenesis in vivo
PHBHHx MBG critical-size rat calvarial defects stimulated bone regeneration, improved the 2014 [132]
osteogenic activity and bone defect restoration
PHBV HA critical-sized rabbit radius defects significant repair effects on bone defects in critical 2013 [164]
areas
PHBV 𝛽-Ca2 SiO4 – stimulated the transcription of TGF-𝛽1 and BMP-7 2013 [188]
genes, promoted the activity of ALP
PHB HAP/HOBs athymic rnu/run rats induced ectopic bone formation in nude rats 2006 [123]
PHBV CaP-Gelfix femoral defect model in rats increased bone mineral density 2004 [189]

𝜅-CG: 𝜅-carrageenan; HAP: hydroxyapatite; CaP: calcium phosphate.

Fig. 7. Examples of PHA in bone tissue engineering applications. (a) Optical

image of 3D PHBV/CS porous scaffolds fabricated by laser sintering [161].
The scale bar is 500 μm. (b) A novel PHBV composite for infection treat-
ment and bone defect filling [162]. (c) Electrospun P3HB4HB/graphene oxide
(GO) scaffolds for promoting bone repair in rats [163]. (d) PHB/HA/ALG scaf-
folds seeded with MSCs enhanced the regeneration of bone defects [35]. (e)
SEM morphology and micro-CT of PHBHHx scaffolds [133]. The scale bars
are 1 mm, respectively. (f) Procedure for the preparation of implantable 3D
HA/PHBV scaffolds [164].

around PHB/PHBV scaffolds, thus stimulating bone tissue growth [130].
These studies provided a new therapeutic strategy for PHBV polymers
for bone tissue regeneration.
Apart from PHB and PHBV, PHBHHx has been used for bone tis-
sue engineering due to its biological properties, including increased
ALP activity and calcium deposition. Even under in vitro culture con-
ditions, hMSCs could maintain good morphology and differentiate into
osteoblasts on PHBHHx membranes [131]. The porous structure of the
scaffold provided sufficient space for vascular growth during bone de-
fect repair. Hence, Zhao et al. prepared PHBHHx/mesoporous bioactive
glass (MBG) composite porous scaffolds with high compressive strength
through 3D printing technology. Compared with MBG scaffolds, PHB-
HHx exhibited enhanced bioactivity and osteogenic properties, includ-
ing hBMSCs proliferation, ALP elevation, rapid apatite formation, and
enhanced expression of bone-related genes. As a result, subsequent im-
plantation of PHBHHx/MBG porous scaffolds into critical-sized rat cra-
nial defects could greatly promote bone regeneration [132]. Moreover,
Yang et al. designed three kinds of PHBHHx-based porous structure scaf-

33
W. Guo, K. Yang, X. Qin et al. Engineered Regeneration 3 (2022) 24–40

Table 8
PHA in cartilage tissue engineering.

Type of PHA Combination Model Advantage Year Reference

PHBV quercetin a cartilage defect nude mice model reduced oxidative stress in chondrocytes and inhibited 2021 [138]
apoptosis
PHB chitosan a sheep knee cartilage defect model reconstructed subchondral calcification layer and 2021 [36]
improved bone tissue integration
PHB/PHBHHx SVFCs/hASCs cartilage defects in rabbit knee joints promoted cell proliferation and migration for more 2019 [190]
desirable cartilage healing
P3HB4HB ASCs/chondrocytes cartilage defects in rabbit knee joints significant improvement in cartilage damage repair 2015 [191]
PHBHHx PhaP-RGD – promoted chondrogenic differentiation of MSCs seeded 2015 [139]
on PHBHHx films
PHBV BG cartilage defects in rabbits higher cartilage matrix content and enhanced 2014 [137]
biomechanical properties
PHBV PLCL a cartilage defect nude mice model enhanced the compressive modulus of scaffolds and 2013 [192]
cartilaginous tissue formation
PHBV meniscal cells meniscal defects in rabbit knee joints regenerative tissue with structure similar to normal 2011 [193]
meniscal fibrocartilage
PHB/PHBHHx hASCs a cartilage defect nude mice model formation of cartilage-like tissue with positive 2009 [136]
toluidine blue staining, safranin O, and collagen type-II
PHBHHx chondrocytes cartilage defects in rabbits good subchondral bone connection with surrounding 2008 [135]
chondrocyte filling
PHBHHx/PHB chondrocytes – chondrocytes were able to proliferate and maintain 2002 [194]
their phenotype within 28 days
SVFCs: stromal vascular fraction cells; PLCL: poly (l-lactide-co-caprolactone).

Fig. 8. Examples of PHA in cartilage tissue engineering applications. (a) Eval-

uation of 3D scaffolds prepared from PHBHHx for cartilage repair in rab-
bits [135]. The scale bars are 4 mm, respectively. (b) The morphology of
hASCs/PHB/PHBHHx constructs after subcutaneous implantation [136]. The
scale bars are 1 cm, respectively. (c) A CHIT/PHB scaffold for treating osteo-
chondral defects [36].

folds and investigated the effect of hierarchical porous scaffolds on HU-
VECs (Fig. 7e). The PHBHHx scaffolds with large micropores were ver-
ified to promote vascularization and bone regeneration through rabbit
bone defects [133].
3.7. Cartilage tissue engineering
Unlike bone tissue, cartilage is poorly self-repairing because of the
lack of vascular and lymphatic distribution, and fibrous tissue is of-
ten involved in repairing larger defects. Traditional surgical procedures
such as joint lavage cleanup, microfracture, and cartilage grafting can-
not overcome the functional deficits caused by cartilage tissue injury.
In recent years, cartilage tissue engineering techniques have provided
new ideas for cartilage repair and regeneration. Table 8 summarizes the
applications of PHA polymers in cartilage tissue engineering. Deng et al.
inoculated rabbit articular chondrocytes on a PHBHHx/PHB porous scaf-
fold, which showed better growth than pure PHB. The level of type II
collagen anchored on the scaffold was significantly increased through-
out the culture cycle (collagen II can be used as a marker of chon-
drocyte differentiation and maturation), indicating that PHBHHx/PHB
obviously promoted chondrocytes differentiation [134]. Subsequently,
PHBHHx/PHB/hASCs composite scaffolds were implanted into a rabbit
cartilage defect model (Fig. 8a). The arrows point to the defects surgi-
cally created on the femoral patellar groove of the knee joints and filled
with a PHBHHx cartilage scaffold in the lower defect area. Due to the
pre-seeded chondrocytes in the scaffold, the engineered cartilage tis-
sue achieved better surface integrity and more ECM accumulation than
the control group, which demonstrates that PHBHHx could be an ex-
cellent material for cartilage tissue engineering [135]. To investigate
the potential value of PHBHHx/PHB, Ye et al. inoculated hASCs onto
PHBHHx/PHB porous scaffolds and implanted them into subcutaneous
layers of nude mice. Six months after implantation, significant cartilage-
like tissue could be observed in the composite scaffold group, with pos-
itive staining for type II collagen, saffron O, and toluidine blue (Fig. 8b)
[136]. Zhou et al. inoculated rabbit chondrocytes on the PHBV/BG com-
posite scaffold and could observe the same repair effect in a rabbit car-
tilage defect repair model [137].
On the other hand, to address the poor bioactivity of PHBV, Chen
et al. prepared a PHBV fibrous scaffold grafted with quercetin (QUE)
by surface modification method. This composite scaffold significantly
promoted cartilage regeneration by regulating relevant gene transcrip-
tion, reducing chondrocyte oxidative stress, and inhibiting chondrocyte
apoptosis [138]. Li et al. coated the surface of PHBHHx with arginine
glycosyl aspartate RGD peptides as well as PhaP. In vitro cellular exper-
iments demonstrated that the PHBHHx scaffold coated with PhaP-RGD
could promote adhesion, proliferation, and chondrogenic differentiation

34
W. Guo, K. Yang, X. Qin et al. Engineered Regeneration 3 (2022) 24–40

of hMSCs [139]. In addition, biopolymers based on decellularized chi- of PHA fermentation, the instruments for fermentation can only pro-
tosan (CHIT) have been effective in treating osteochondral defects and vided by a few manufactures and require high production conditions.
traumatic cartilage. A sheep knee cartilage defect repair model con- In addition, the technical barriers to PHA fermentation are high and
firmed the superior repair effect of porous CHIT/PHB scaffolds (Fig. 8c) usually rely on professionals for production and monitoring. From an
[36]. Ching et al. prepared PHB/PHO composite scaffolds with different economic point of view, the market competitiveness of PHA is insuf-
mixing ratios. When the ratio of PHB to PHO was 1:0.25, the composite ficient. Currently, numerous studies are attempting to reduce the cost
scaffolds matched the stiffness of natural articular cartilage, and the ori- of PHA fermentation processes and to improve the extraction of indus-
ented fibrous structure mimicked the collagen fiber network in cartilage trial processes by appropriate methods. More importantly, the physi-
tissue. Their results suggested that these scaffolds were expected to be cal properties of PHA have not exceeded or even reached the level of
used to repair cartilage tissue in clinical practice and reduce the risk of existing plastic products. The number of PHA monomers exceeds 150,
secondary osteoarthritis [140]. making it difficult for fermentation products to maintain complete con-
sistency with each other. The inherent physical aging, embrittlement,
slow crystallization, and low nucleation density limit its extension in
3.8. Others
many practical applications. Therefore, it is necessary to further con-
trol the monomer structure and properties of PHA through metabolic
In other fields, PHA has been used to manufacture medical sutures
engineering to improve the applicability of PHA and its derivative ma-
with sufficient strength to make them suitable for hemostasis, wound
terials. These fermentation and modification technologies are technical
healing, and ligature tissue attachment. After being implanted into ex-
hurdles that must be overcome in the future development of PHA. In
perimental animals, monofilament sutures made of PHB and PHBV
addition, there are no standards or policy support for technical review
showed significantly lower inflammatory and other reactions than those
guidelines of PHA production and application. In the future, with the
of conventional intestinal sutures. Shishatskaya et al. compared the re-
growing problem of "white plastic" and the promotion of the concept of
sponse of PHB and PHBV sutures to surrounding tissues, and found that
sustainable development, the acceptance of bioplastics by society will
both had enough required strength for suturing myofascial wounds; no
increase, which is conducive to the market of PHA and biodegradable
adverse effects such as necrosis, septic inflammation, or fibrocystic cal-
bioplastics industry.
cification were observed during up to one year of implantation [38,141].
In recent years, PHA has been applied in more and more fields, in-
In addition, PHBHHx exhibited better biocompatibility and hemocom-
cluding biological medicine, daily chemical, food packaging, agricul-
patibility compared with PHB and PHBV. Its excellent fracture strength
ture, and other areas. Among them, the biomedical field is undoubt-
and flexibility made the use of PHBHHx as a medical suture more fa-
edly the focal point for the future production and application of PHA.
vorable. He et al. prepared two types of fibers: a multifilament made
Therefore, innovative purification technologies such as synthetic bi-
of PHBV/PLA and a monofilament made of PHBHHx. The mechani-
ology and bioreactor system design, are urgently needed to remove
cal properties, biocompatibility, and degradability of these two fibers
lipopolysaccharides (LPS), proteins and other bacterial impurities re-
were evaluated. It was found that these fibers maintained a high ten-
maining from the fermentation process; and to ensure the stability of
sile strength even after one year of implantation [142]. PHA fibers have
molecular weight, composition, and purity of PHA polymers. Over-
great potential for tissue suturing because they can degrade into bene-
all, the unique properties such as elastic mechanical properties, ther-
ficial 3-HB monomers and reduce scar tissue formation.
mal properties, biocompatibility, biodegradability, nontoxic degrada-
Moreover, highly open porous microspheres with PHBHHx could
tion products, and non-carcinogenic effects make PHA more attractive
also be synthesized as injectable cell-laden scaffolds to protect cells from
and applicable than other biopolymers. In addition, PHA can be modi-
stress during injection and safely transport cells to defect tissues [143].
fied by blending with other organic or inorganic materials to meet the
PHA can also be used as carriers for drug delivery, such as antibiotics
needs of various applications. Therefore, PHA is widely used in biomed-
[144,145], anticancer drugs [146,147] and hormones [148,149]. Zhu
ical engineering, especially in tissue repair and regeneration, includ-
et al. prepared composite scaffolds of MBG and PHBHHx for the treat-
ing wound dressings, artificial blood vessels, heart valves, nerve con-
ment of osteoarthritic tuberculosis by three-dimensional printing tech-
duits, bone and cartilage scaffolds, surgical sutures, etc. However, only
nology. High doses of isoniazid (INH)/rifampicin (RFP) antituberculosis
an exceptionally few commercialized PHA products have been applied
drugs were preloaded into the chemically modified MBG. The compos-
to clinics, and the vast majority of research is still at the experimental
ite scaffold showed long-lasting drug release in vitro. In a rabbit femoral
stage with many technical hurdles. In the short-term future, given the
defect model, the antitubercular drug loading scaffold could achieve in-
high production cost of PHA, the direction of product development will
tratubular new bone production and long-lasting antitubercular effects
mainly focus on high-value medical consumables such as interventional
[150]. In the treatment of chronic osteomyelitis, PHBV and P3HB4HB
valves, artificial blood vessels, and artificial bone joints. By capturing
biopolymer rods loaded with antibiotics (Duocid, Sulfadiazine) were
the high-end medical market, it will catch up with other degradable
manufactured to prevent and treat associated infections [151]. More-
polymer materials in terms of research and development technologies.
over, 3HB and its derivative methyl 3-hydroxybutyrate (3HBME) could
In the long-term development trend, as the production cost of PHA de-
promote osteoblast growth and differentiation by regulating rapid cal-
creases, the product development direction will be extended to low-
cium deposition and serum ALP activity, leading to the prevention of os-
value medical consumables such as wound dressings and surgical su-
teoporosis [152,153]. Similarly, the PHBV/PLGA nanoparticles loaded
tures. With the performance advantages of PHA, it is further expected
with the recombinant protein teriparatide exhibit a stable and syner-
to achieve comprehensive coverage of the medical device market. In the
gistic effect on promoting bone resorption and have been used in the
future, with the benefits of medical-engineering multidisciplinary inter-
treatment of osteoporosis [154,155].
section, promoting and strengthening PHA research in personalized and
precise tissue repair and regeneration could provide new strategies for
4. Prospects and conclusion developing high-performance biomaterials.

PHA is a highly promising alternative to petroleum-derived plastics Author contributions

as a degradable biopolymer. Since PHA fermenting bacteria were first
identified, significant breakthroughs have been made in applying PHA,
but its high production costs and poor processability hinder the commer-
cialization. According to statistics, the production cost of PHA is about
ten times higher than that of petroleum-derived plastics. In the process
W.T.G. conducted conceptualization, data curation, and original
draft; K.L.Y. conducted data curation and formal analysis; X.S.Q. con-
ducted software and visualization; L.R. conducted investigation and
validation; H.W. conducted supervision, funding acquisition, and re-

35
W. Guo, K. Yang, X. Qin et al.
sources; R.K.H. conducted project administration, methodology, and re-
view & editing.
Declaration of Competing Interest
We declare that we have no financial and personal relationships with
other people or organizations that can inappropriately influence our
work, there is no professional or other personal interest of any nature
or kind in any product, service, and/or company that could be con-
strued as influencing the position presented in, or the review of, the
manuscript entitled, “Polyhydroxyalkanoates in tissue repair and re-
generation”.
Acknowledgments
This work was supported by the National Key Research and
Development Program of China (2021YFC2101700), and the Sun
Yat-sen University Clinical Research 5010 Program (No. 2017008).
Part of the materials for Fig. 2 were required from Servier
Medical Art (http://smart.servier.com/), licensed under a Creative
Common Attribution 3.0 Generic License (https://creativecommons.
org/licenses/by/3.0/).
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