Polymer Reviews
ISSN: 1558-3724 (Print) 1558-3716 (Online) Journal homepage: http://www.tandfonline.com/loi/lmsc20
Hyaluronic Acid-Based Biomaterials: A Versatile
and Smart Approach to Tissue Regeneration and
Treating Traumatic, Surgical, and Chronic Wounds
Zahid Hussain, Hnin Ei Thu, Haliza Katas & Syed Nasir Abbas Bukhari
To cite this article: Zahid Hussain, Hnin Ei Thu, Haliza Katas & Syed Nasir Abbas Bukhari
(2017): Hyaluronic Acid-Based Biomaterials: A Versatile and Smart Approach to Tissue
Regeneration and Treating Traumatic, Surgical, and Chronic Wounds, Polymer Reviews, DOI:
10.1080/15583724.2017.1315433
To link to this article: http://dx.doi.org/10.1080/15583724.2017.1315433
Accepted author version posted online: 17
Apr 2017.
Published online: 17 Apr 2017.
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Date: 12 May 2017, At: 18:25
POLYMER REVIEWS
https://doi.org/10.1080/15583724.2017.1315433
REVIEW
Hyaluronic Acid-Based Biomaterials: A Versatile and Smart
Approach to Tissue Regeneration and Treating Traumatic,
Surgical, and Chronic Wounds
Zahid Hussaina, Hnin Ei Thub, Haliza Katasc, and Syed Nasir Abbas Bukharid
a
Department of Pharmaceutics, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus,
Selangor, Malaysia; bDepartment of Pharmacology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan
Yaacob Latif, Kuala Lumpur, Malaysia; cCentre for Drug Delivery Research, Faculty of Pharmacy, Universiti
Kebangsaan Malaysia, Kuala Lumpur, Malaysia; dDrug and Herbal Research Centre, Faculty of Pharmacy,
Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
ABSTRACT
KEYWORDS
Wound healing is a multipart and dynamic process of replacing
devitalized and damaged cellular structures and tissue layers. Numerous
conventional wound dressings are employed for the management of
wounds but there is a lack of absolute and versatile choice. An ideal
wound healing modality should provide a moist environment, offer
protection from secondary infections, eliminate wound exudate, and
stimulate tissue regeneration. Hyaluronic acid (HA) has been known to
promote angiogenesis, granulation tissue formation, remodeling of
extracellular matrix (ECM), and wound healing. Accumulation and
turnover of ECM is a hallmark of tissue injury, repair, and remodeling in
wound healing. HA is a major component of ECM and plays an important
role in regulating tissue injury, accelerating tissue repair, and controlling
disease outcomes. A wide range of in vitro, in vivo, and clinical studies
have demonstrated the wound healing efficacy of HA-based biomaterials
not only in the treatment of wound in the tympanic membrane, skin, and
articular cartilage but also in tracheal and corneal wound healing. Recent
progress and improved therapeutic efficacy achieved through partial
modification and formation of HA-based biomaterials, including HAscaffolds, sponge-like hydrogels, anti-adhesive sheets, cultured dermal
substitutes, thin membranes, and dermal matrix grafts have been
discussed. The current review summarizes the evidence for the
therapeutic effectiveness of HA-based biomaterials in the treatment of
traumatic, surgical, and chronic wounds and tissue regeneration.
Biomaterials; hyaluronic acid;
wound healing; traumatic
and surgical wounds; chronic
wounds; tissue regeneration;
efficacy-upgradation
1. Introduction
Wound healing is an intricate process that involves the simultaneous actuation of blood
cells, soluble mediators, parenchymal cells, and extracellular matrix (ECM). The complex
CONTACT Zahid Hussain
zahidh85@yahoo.com
Nanopharmacy Unit and Transdermal Laboratories, Department of
Pharmaceutics, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, Bandar Puncak Alam 42300, Selangor,
Malaysia.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lmsc.
© 2017 Taylor & Francis Group, LLC
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wound healing process is completed through the well-organized collaboration of several
phases:
1) the inflammatory phase, which involves homeostasis/coagulation and inflammatory
cascades;
2) the proliferation phase, which involves rebuilding of new granulation tissues and the
de novo development of blood vessels (angiogenesis) followed by re-epithelialization
(resurfacing of the wound with epithelial cells); and
3) the maturation phase, which is the final phase and involves remodeling of collagen
from type III to type I.1,2
These phases are not typically associated with a rigorous or well-defined period of time
and may overlap and thus result in non-linear or chronic wounds.3–5 The transition between
phases usually depends on the maturation and differentiation of mast cells, fibroblasts, keratinocytes, and macrophages, which play key roles in the wound healing process.6–11 The
impaired functioning of macrophages, granulocytes, and chemotactic mediators as well as
deregulation of the neo-vascularization phase might prolong the healing process and result
in a chronic wound.12–14 The development of chronic wounds and their non-linear healing
process can also be associated with the excessive and persistent activity of matrix metalloproteinases MMPs and/or due to the chronic use of MMP inhibitors.15,16 Peripheral vascular
disease may also tend to reduce the healing capacity of an individual because of an insufficient supply of oxygen and nutrients to the wounded area.17 In addition, the development of
chronic wounds can be caused by the impaired functioning of nitric oxide, collagen deposition, anomalous proliferation and differen of fibroblasts and keratinocytes,13 and the accumulation of ECM and subsequent remodeling by MMPs.18 Thus, there is a higher
predisposition of patients with diabetic neuropathy or peripheral vascular diseases to transition from mild injuries to chronic wounds, which happen in response to the non-linear healing process. Cell migration, fixbroblastic differentiation, collagen remodeling, and
proliferation are decreased in impaired healing. This may be attributed not only to cellular
defects but also to changes in mediators associated with senescence19 and the diabetic process. The wound healing process in diabetic patients can also be adversely affected by concurrent underlying conditions, unrelieved pressure, and superinfection.
To date, numerous therapeutic strategies have been employed for the management of
acute and chronic wounds with the aim of accelerating the healing process, avoiding secondary complications, and improving patient compliance. Various wound healing approaches
include the following: conventional wound dressings such as hydrogel-, hydrocolloid-,
foam-, and film-based dressings and advanced standard procedures such as split-thickness
autografts, use of donor keratinocytes, cultured epithelial autografts, and Biobrane dressings.
However, in recent years, scientists have focused on the use of natural biopolymers as an
alternate therapy for optimizing therapeutic outcomes, patient adherence, and compliance
and minimizing off-target effects.
Hyaluronic acid (HA) is a natural polysaccharide and a major component of mammalian extracellular matrix (ECM). It consists of a linear polysaccharide comprised
alternating units of b-1,4-linked D-glucuronic acid and (b-1,3) N-acetyl-D-glucosamine20,21 (Chemical structure is shown in Fig. 1). HA is usually extracted from the
synovial fluid, umbilical cord, vitreous humor, rooster combs, or bacterial cultures in the
laboratory.22 HA has many important physiological functions such as structural and
space-filling, lubrication, tissue and ECM water absorption, and retention abilities.23,24 It
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is a non-allergenic, non-toxic, and biocompatible polymer25 with a wide range of biological functions including skin moisturizing,26 tissue regenerating,27 anti-wrinkle,28 inflammation moderating,29 cancer prognosing,30,31 and wound healing23,32,33 effects.
The mechanisms by which HA actively participates in wound healing remain unclear;
however, HA has been shown to affect cell functions by binding to cell surface receptors and
mediating a variety of downstream effects important in wound healing including increased
expression of pro-inflammatory cytokines (e.g., tumor necrosis factor a, interleukin 1b, and
interleukin-8), cell migration, cell proliferation, and organization of granulation tissue
Figure 1. (A) Repeating units in the chemical structure of hyaluronic acid and (B) different methods for the
synthesis of hyaluronic acid.
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matrix.20,34–36 In addition, HA influences cell behavior such as cell proliferation, differentiation, adhesion, and migration because of its unique hygroscopic, rheologic, and viscoelastic
properties.20,34–36
A number of in vitro and in vivo studies have documented the wound healing potential of
HA by promoting mesenchymal and epithelial cell migration and differentiation, enhanced
angiogenesis, and collagen deposition.29,32,37 In addition, the metabolic degradation products
of HA have also been reported to stimulate endothelial cell proliferation and migration,
modulate the inflammatory processes, and stimulate angiogenesis during various wound
healing stages.24,38,39 Because of its unique physical properties, HA creates an excellent
wound healing environment and has multifaceted roles in wound healing and scarring.40–43
The current review therefore aimed to precisely summarize the existing evidence for the
therapeutic superiority of HA-based dressings for the treatment of various types of wounds.
The promising roles of HA-mediated interventions in achieving greater therapeutic outcomes in the management of mild to severe, persistent wounds have been discussed. Recent
results from in vitro, in vivo, and clinical studies have been included to underline the unique
potential of HA-based modalities to optimize therapy outcomes and patient compliance.
2. Synthesis of HA
On an industrial scale, HA is manufactured via two main processes:
1) extraction from the animal tissues, and 2) microbial fermentation.
Both methods are highly efficient and produce HA of polydisperse high molecular weight
(M.W. D 1 £ 106 Da, polydispersity ranging from 1.2 to 2.3) for various pharmaceutical and
cosmetic applications.44–46 The former process, despite having several advantages in industrial scale production, is hampered by several drawbacks. These drawbacks include significant degradation of the extracted HA caused by either:
(i) enzymatic hydrolysis of the HA polymer chain by the action of endogenous hyaluronidase activity in animal tissues or
(ii) the harsh conditions applied during the process of extraction.
Though the production of HA through extraction processes has improved over the past
years, there are still low yields due to the uncontrolled degradation during extraction. In
addition, the extraction of HA from animal tissues is also limited because of the potential
risk of contamination of extracted polymer with proteins and viruses; however, contamination can be minimized by sourcing animal tissues from healthy animals and controlling the
extraction environment. Nevertheless, concerns about viral (particularly avian) and protein
(particularly bovine) contamination have inspired investigation into the production of biotechnological products of HA.
In the last two decades, production of HA using bacterial fermentation on an industrial
scale has been employed as a prime production technique. Using this method, the percent
quality and production yield of HA can be improved dramatically by optimizing culture
media and cultivation conditions along with strain improvement in the early developmental
stages of bacterial fermentation using group A and C Streptococci. In doing so, HA yield has
reached 6–7 g L¡1, which is considered the highest yield range of a process with a mass
transfer limitation due to the high viscosity of the fermentation broth. Despite having several
advantages including higher yield, good quality, and purity of HA, risk of contamination
with bacterial endotoxins, proteins, nucleic acids, and heavy metals is a limiting factor.
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However, in recent years, the identification of the genes of bacterial strains involved in the
biosynthesis of HA and of the sugar nucleotide precursors have allowed industrialists to produce HA using safe and non-pathogenic recombinant strains.
Interestingly, in recent years, a new technology has been developed using isolated HA
synthase to catalyze the polymerization of UDP-sugar monomers. This newer enzymatic
technology for the synthesis of HA is versatile and allows for both the production of high
molecular weight HA and HA oligosaccharides with defined chain length and low polydispersity. Previously, the production of monodisperse HA oligosaccharides was demonstrated
by DeAngelis et al.47 using two single-action mutants of Pasteurella multocida HA synthase,
but large-scale production has not been achieved yet. The repeating unit of HA and a brief
schematic illustration of different methods employed in the synthesis of HA is presented
in Fig. 1.
3. Wound healing potential of HA-based biomaterials
Treatment of a wound is greatly dependent on several parameters such as the severity of the
wound, pathophysiological basis, patient health condition, and extent of tissue damage. The
selection of an appropriate wound dressing, together with the inclusion of therapeutic substances and healing enhancers (if employed), also plays a pivotal role in achieving therapeutic outcomes.
Typically, wounds can be treated using passive or hydro-active techniques;48 however, the
passive technique is normally employed for the management of acute wounds (as they
absorb reasonable amounts of exudate, and they can ensure good protection). On the other
hand, the hydro-active technique is usually employed for the management of chronic
wounds as they easily adapt to wounds and are able to maintain a moist environment that
can accelerate the healing process.49
3.1 Traumatic and surgical wound management
The skin serves its primary function as a protective barrier against environmental, physical,
or biochemical insults. A compromise in the structural integrity of the skin, either by acute
or chronic injuries, leads to multiple serious disarrays, which might result in morbidity and
mortality.19,50 To overcome these secondary impairments, the body tends to initiate a multidisciplinary and vibrant healing process at the site of injury, leading to partial restoration of
the skin’s barrier function, re-establishment of tissue integrity, and maintenance of internal
homeostasis.51 The natural process of wound healing comprises of four well-defined phases:
hemostasis, inflammation, proliferation, and remodeling. Hemostasis is a rapid phenomenon involving platelet aggregation and formation of a blood clot leading to a rapid cessation
of bleeding upon tissue injury52–54 as well as provision of ECM for cell migration.6 On the
other hand, the inflammatory phase involves the chemotaxis and migration of inflammatory
cells, such as macrophages and neutrophils, to the injured site.53 The proliferation phase is
induced by the inflammatory cytokines released from phagocytic cells, which involves proliferation and migration of fibroblasts at the end of the inflammatory phase.55 The re-epithelialization phase begins within hours of injury, is a part of the proliferative phase,6 and is
characterized by the formation of new blood vessels (angiogenesis or neovascularization),
which re-establishes perfusion to sustain the new tissues55 and the synthesis and deposition
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of fragments of ECM proteins such as collagen fibers and granulation tissues.53 Fibroblasts
are the prime cells to synthesize new ECM to support cell ingrowth using collagen as the
building blocks6 and thus play a key role in the wound healing process. The final phase
involves collagen remodeling and scar tissue formation.6 These vibrant phases are
complex and involve soluble mediators, ECM formation, and parenchymal cell
migration.50 Overall, the prime objectives of the wound healing process are quick relief of
pain, timely wound closure, and formation of an aesthetically acceptable scar. Wound healing not only involves the restoration of the skin barrier integrity and internal homeostasis,
but also diminishes the risk of infection and secondary complications.
Numerous studies have documented the therapeutic effects of both topical and systemic
delivery of HA (in various forms) to improve traumatic and surgical wound healing in laboratory animals.32,56–61 The topical application of HA tends to accelerate re-epithelialization
and thus diminish fibrosis and scar formation in mice and rats.59–63
In vivo studies evaluating the effects of chemically modified HA hydrogels and films are
limited; however, a preliminary study comparing healing rates in horses, rats, and dogs
found that cross-linked HA-based biomaterials (CMHAs) enhance wound healing.57 At
7 days post-treatment with CMHA hydrogel in rats, 17 days in dogs, and 26 days in horses,
the wound areas and sizes were significantly smaller than the control groups and the wound
beds of all species were grossly healthier in appearance in all CMHA-treated wounds.57
Based on the findings of this preliminary study, CMHA-based formulations (hydrogels and
films) were further tested in treating distal limb wounds in horses.64 In this study, three fullthickness skin wounds (6.25 cm2) were surgically created on the dorsomedial metacarpus
and metatarsus of each of four limbs of eight normal adult horses under general anesthesia.
Out of four treatment groups, the first group was the control group (without treatment), the
second group received a single application of CMHA gel, the third group received multiple
applications of CMHA gel, and the fourth group was treated with multiple applications of
CMHA film. Three days after surgery, wounds were photographed and assessed for wound
healing and tissue repair every 4 days throughout the study (47-days). Analysis of resulting
photomicrographs revealed a significantly faster healing rate, greater healing quality, and
less fragile epithelium in wounds treated with CMHA films, compared to other treatment
groups (Fig. 2).64
Besides gels and films, many other researchers have explored the wound healing efficacy
of HA-based hydrogels, sheets, sponges, and decellularized/porous scaffolds, alone or in
combination with other agents, for the treatment of traumatic and surgical wounds.65–73
Yan et al.69 fabricated a new dermal substitute comprised of silk fibroin (SF)/chondroitin
sulfate (CS)/hyaluronic acid (HA) ternary scaffolds (95–248 mm in pore diameter, 88–93%
in porosity). They investigated the wound healing efficacy of SF, SF/HA, and SF/CS/HA (80/
5/15) scaffolds in the treatment of dorsal full-thickness wounds using the Sprague-Dawley
rat model. There were no signs of empyema or infection between grafted scaffolds and the
surrounding tissue in all the experimental animals. The wound areas of SF/CS/HA ternary
scaffold-treated animals were smaller than those of the other groups and sharply reduced at
week 2 post-implantation (Fig. 3). At week 3, no wound was evident in the SF/CS/HA group,
while there were still small wounds at defect sites in the SF and SF/HA groups. Compared to
SF and SF/HA groups, the wound healing process was accelerated by SF/CS/HA scaffolds.69
Taken together, compared to SF and SF/HA, the SF/CS/HA scaffold-treated group showed
significant dermis regeneration and improved angiogenesis and collagen deposition.69
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Yan and coworkers69 also investigated the cellular basis of wound healing efficacy of SF/
CS/HA scaffolds. Histological examination revealed that granulation tissue formation and
angiogenesis were more obvious in the wounds treated with SF/HA and SF/CS/HA scaffolds
(Figs. 4e and 4i) at the end of week 1. They further observed that vasculogenesis in the defect
sites was evident in all experimental groups at week 2; however, the newly formed tissues had
filled nearly all the scaffold pores in the SF/CS/HA group (Fig. 4j). The vascularization and
granulation tissue formation was further increased at week 3, but the capillary network was
denser and more uniform in the SF/CS/HA group (Fig. 4k) compared to the other two
groups. All the scaffolds were degraded and replaced by neotissue by the fourth week
(Figs. 4d, 4h, and 4l); however, the ECM in the SF/CS/HA scaffolds group formed a uniform,
dense network (Fig. 4l). A significantly greater reduction in wound area, greater vascularization, and granulation tissue formation evidenced enhanced wound healing potential in the
SF/CS/HA group compared to control groups. The deposition of collagen integers was gradually increased from week 1 to week 4 in all the experimental groups; however, collagen
intensity was more prominent and uniformly distributed in the SF/CS/HA group. These
Figure 2. Representative photomicrographs of wounds from a single horse on day 31. (A) Control; (B) single gel; (C) multiple gel; and (D) multiple film. At this time point, the mean granulation tissue area
of the wounds treated with multiple films was significantly smaller than the controls.64 © Elsevier. Reproduced by permission of Elsevier. Permission to reuse must be obtained from the rightsholder.
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Figure 3. Macroscopic observations of skin wounds after implanting SF, SF/HA (80/20) and SF/CS/HA (80/
5/15) scaffolds for 1, 2, 3 and 4 weeks; scale bar D 10 mm.69 © Elsevier. Reproduced by permission of
Elsevier. Permission to reuse must be obtained from the rightsholder.
findings demonstrated that neotissue formation was accelerated by the SF/CS/HA group in
dermal tissue reconstruction.
The wound healing efficiency of HA in the form of a novel porous scaffold constructed in
conjunction with collagen and gelatin was investigated by Wang et al.68 using an
in vivo full thickness wound model. Full thickness excisions (2 cm in diameter) were surgically created on the backs of male Wistar rats. The wounds were immediately covered with
scaffold in treatment groups and compared to the control group in which wounds were left
open. The results showed that the healing rate was comparatively slower in the control group
compared with the wound covered with HA scaffold.68 The wound areas of the treatment
group at days 1, 2, 3, 4, 5, 7, and 10 after injury were 79.7 § 3.4%, 73.4 § 3.5%, 66.8 § 2.2%,
60.7 § 5.0%, 58.3 § 6.1%, 44.9 § 4.3%, and 24.0 § 2.1%, respectively. Their wound areas
were smaller than those of the control group at the same time intervals (97.4 § 5.5%, 82.7 §
2.2%, 75.3 § 3.7%, 71.4 § 3.8%, 67.9 § 8.0%, 62.1 § 9.4%, and 41.8 § 5.3%, respectively)
(Fig. 5I). Wounds treated with HA-scaffold showed more than 50% closure after 7 days and
almost 75% closure in 10 days. The superiority of HA-scaffolds for treating acute wounds
was also demonstrated by histological examination (Fig. 5II). The resulting micrographs
revealed that the epidermis in the HA-scaffold group was denser than in the control group,
which verified the skin-repairing efficacy of the HA-scaffold. Compared to the control
group, there was less neutrophil infiltration in the HA-scaffold group, which further accelerated wound closure. The faster wound closure rate, higher epidermis density, and diminished neutrophil infiltration demonstrated the superior ability of HA-scaffolds to accelerate
the healing of excisional wounds.68
In vitro phenotypic characterization of human epidermal and dermal cell cultures has also
shown superior cell proliferating and tissue regenerating ability following treatment with
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Figure 4. Histological examination of grafted scaffolds after transplantation at different time points indicating higher granulation tissue formation, vascularization, and collagen deposition in SF/CS/HA group
compared to control groups; (I) H & E stain (scale bar D 100 mm) and (II) Masson’s trichrome stains (scale
bar D 100 mm). 69 © Elsevier. Reproduced by permission of Elsevier. Permission to reuse must be obtained
from the rightsholder.
HA-based spongy-like hydrogels.71 The ability of HA-based hydrogels to stimulate proliferation and differentiation of human epidermal fractions was assessed. The resulting micrographs revealed a homogenous and cuboidal morphology characteristic of epidermal basal
cells (Fig. 6c). This observation was confirmed by the expression of the keratinocytes early
differentiation-associated marker, K5, of the cells adhered on the TCPS coverslips (Fig. 6a).
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Similarly, the ability of HA-based hydrogels to facilitate organization of endothelial cells was
investigated by the expression of typical vWF and CD31 markers, as identified on the TCPS
coverslips (Fig. 6b). After treating with HA-hydrogel, the endothelial cells displayed their
typical cobblestone morphology, and interestingly, despite the co-isolation with other dermal cells, they were capable of rearranging in vitro, forming their characteristic colonies
(Fig. 7d). Further, they expressed the epidermal and dermal phenotypic markers (Figs. 6c
and 6d).
Cerqueira et al.71 investigated the wound healing potential of HA-based spongy-like
hydrogels using an in vivo experimental model. Full thickness excisional wounds were surgically created on the skin of Swiss Nu/Nu male mice, and the wound closure rate was examined macroscopically throughout the implantation time. A progressive increase in the
wound closure rate was observed over time; however, significant differences between the
experimental and control groups were identified at earlier time points. Up to day 7, the post
implantation spongy-like HA hydrogel mice showed a significantly higher percentage of
wound closure (Fig. 7) compared with the control groups. These findings were also later confirmed with histological examination. The resulting photomicrographs revealed an accelerated wound closure rate and greater re-epithelialization, granulation tissue formation, and
tissue neovascularization in the HA hydrogel-treated groups compared with the control
group.71
HA has also established wound-healing effects in the form of decellularized scaffolds constructed along with the basic fibroblast growth factor (bFGF)73 and epidermal growth factor
(EGF).74,75
Wu et al.73 developed xenogeneic decellularized scaffolds using pig peritoneum after a
series of biochemical treatments to remove cells and antigenic components from the tissue.
These scaffolds were incorporated with HA and bFGF and were tested for the repair of skin
wounds.73 The wound healing efficacy of HA scaffolds was determined by analyzing the
thickness of the dermis layer and by measuring the wounded area. In this experiment, four
wounds were created on the dorsal body region of each rabbit. In each animal, wounds were
covered as follows: one wound was covered with Vaseline oil gauze, the second wound was
covered with decellularized scaffold alone, the third wound was covered with decellularized
scaffold containing HA and 1 mg/mL of bFGF, and the fourth wound was covered with
decellularized scaffold containing HA and 3 mg/mL bFGF. The results showed that Vaseline
oil gauzes or decellularized scaffolds gently peeled off the wounds on days 3, 6, 11, or 14
post-surgery. The lengths of the upside, downside, left side, and right side of each wound
were measured after the excision of the skin (original wound area) and on days 3, 6, 11, or
14 post-surgery. The size of the wound area and the healing rate were calculated for each
wound. The results showed that wounds covered with scaffold containing either 1 or 3 mg/
mL bFGF were significantly smaller than those covered with Vaseline oil gauzes or with scaffold alone; particularly, the wound covered with scaffold containing 1 mg/mL bFGF recovered the best among all four wounds. Further analysis revealed that higher healing rates of
47.24%, 74.69%, and 87.54% were observed on days 6, 11, and 14, respectively, in the groups
treated with scaffolds containing HA and bFGF compared with the wound healing rates of
24.84%, 42.75%, and 57.62% in the control groups (Fig. 8). Wounds covered with scaffolds
containing bFGF and HA showed more dermis regeneration than the other wounds and on
days 6, 11, and 14 post-implantation had dermis layers of 210.60, 374.40, and 774.20 mm
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Figure 5. Wound healing efficacy of HA-scaffold. (I) faster healing of excisional wound in scaffold
treated wounds (A) compared to control group (B) during treatment period, and wound contraction
ratios of scaffold and injury at different times (C), (II) histological examination of HA-scaffold treated
skin compared to control group suggested higher granulation tissue formation, increased epidermal
thickness, and less neutrophils infiltration in HA scaffold treated skin compared to control.68
© Wang et al. Reproduced by permission of Wang et al. Permission to reuse must be obtained
from the rightsholder.
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Figure 6. Phenotypic characterization by immunocytochemistry of cells from the epidermal and dermal
fractions after 2 days of culture (A, B) on tissue culture polystyrene coverslips and (C, D) entrapped in the
GG-HA spongy-like hydrogel. (A, C, D) Epidermal early-associated marker keratin 5 (K5, green) was
expressed by the cells from the epidermal fraction. (B, D) Endothelial cells were detected among dermal
cellular fraction through the expression of vWF (green) or CD31 (red). Nuclei were stained with DAPI, and
cytoskeleton F-actin fibers in GGHA spongy-like hydrogel constructs were stained with Phalloidin-TRITC.
Scale bars correspond to 50 mm. GG-HA, gellan gum/hyaluronic acid; vWF, von Willebrand factor.71
© 2014 Mary Ann Liebert, Inc. Reproduced by permission of Mary Ann Liebert, Inc. Permission to reuse
must be obtained from the rightsholder.
compared to wounds with scaffolds alone having dermis layers of 82.60, 186.20, and
384.40 mm, respectively.73
The wound healing potential after treatment with decellularized scaffolds of HA was also
investigated in the presence of EGF.74 Analysis of the wounded area after the implantation
of HA scaffolds loaded with EGF indicated a significantly faster wound closure and smaller
wound compared with the control group. Histological examination also revealed a significantly thicker dermis layers in wounds treated with HA and EGF-embedded scaffolds compared to Vaseline oil gauze and scaffolds alone. The thicker dermis layers showed significant
regeneration of skin appendages on day 28 post-transplantation.74 These results clearly demonstrate the potential of HA-based scaffolds for the management of wounds.
These results were also in agreement with the results of the study by Su et al.75 in which
decellularized HA scaffolds loaded with EGF were developed and tested for wound healing.
The results showed that the wounds covered with scaffolds containing HA and EGF recovered best among all the 4 groups and had wound healing rates of 49.86%, 70.94%, and
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Figure 7. Evaluation of the effect of spongy-like hydrogel constructs over wound closure. (i) Representation of the percentage of wound closure in the experimental and control groups after 3, 7, 14, and
21 days. (ii) Representative macroscopic images of the wounds along the implantation time.71 © 2014
Mary Ann Liebert, Inc. Reproduced by permission of Mary Ann Liebert, Inc. Permission to reuse must be
obtained from the rightsholder.
87.41%, respectively, for days 10, 15, and 20 post-surgery compared to scaffolds alone with
wound healing rates of 29.26%, 42.80%, and 70.14%. Moreover, histological examination
revealed that thicker epidermis and dermis layers were observed in the wounds covered with
scaffolds containing HA and EGF than in the control groups and with EGF alone (Fig. 9).
These findings clearly identified the pharmacological importance of the presence of HA in
the scaffold for the treatment of human skin injuries.75
The incorporation of arginine and EGF into HA-sponges also improved healing efficacy
in Sprague-Dawley rats.76 Results showed that the experimental animals treated with
sponges containing arginine and EGF showed a significant decrease in the size of the fullthickness skin defect and an increase in the size of the intact skin island, when compared
with the control group. This suggests that EGF released from the HA spongy sheet serves to
promote re-epithelization. In the second experiment, each wound dressing was applied to a
full-thickness skin defect measuring 35 mm in diameter in the abdominal region of SpragueDawley rats, after removing necrotic skin caused by dermal burns. Polyurethane film
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Figure 8. Examination of wound healing on different days postsurgery. The wound treated with decellularised scaffold containing HA and 1 mg/mL bFGF had shown smallest wound size and faster healing rate
compared with control and other treatment groups.73 © Springer. Reproduced by permission of Springer
Science and Business Media, NY. Permission to reuse must be obtained from the rightsholder.
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dressing was applied to each wound dressing as a covering material. Both wound dressings
(Group I and Group II) potently decreased the size of the full-thickness skin defect and
increased re-epithelization from the wound margin compared to the control groups. These
findings indicated that wound dressings comprised of HA-based spongy sheets containing
arginine and EGF potently promoted wound healing by inducing moderate inflammation.76
Recently, a nanovesicle of HA (hyalurosome) was constructed using polyanion sodium
hyaluronate and loaded with curcumin to evaluate the healing efficacy of HA in the form of
nanovesicles.77 The physicochemical properties and in vitro/in vivo performances of the formulations were compared to those of liposomes having the same lipid and drug content.
The authors performed in vivo evaluation of curcumin-loaded hyalurosomes using a mouse
model to counteract 12-O-tetradecanoylphorbol (TPA)-produced inflammation and injuries, edema formation, infiltration of inflammatory cells, myeloperoxidase activity, and skin
re-epithelization.77 The resulting data demonstrated that the experimental animals treated
with TPA only showed a gradual increase in the area of underlined skin lesion over time, an
initial loss of skin was evident in the marginal zone at day 2, the skin damage was more diffuse at day 4, and the whole area was compromised with several crusts even in the central
area at day 6 (Fig. 10).77 However, upon visual inspection, the superior ability of curcuminloaded hyalurosomes to heal the wounds in comparison with controls and liposomes was
evident. In particular, using 0.5 hyalurosomes, the skin damaging effects of TPA were counteracted, thus moderating the skin lesions (only a slight loss of superficial skin was observed)
in the peripheral application zone (days 2 and 4) and favoring the complete re-epithelization
of the marginal area at day 6. The treated skin was similar to that of the healthy untreated
mice, except for being thinner and for the presence of some minor defects (Fig. 10).77 These
results revealed the therapeutic ability of curcumin-loaded hyalurosomes in avoiding damage and loss of superficial skin strata after daily TPA application. To confirm the beneficial
effect of curcumin-HA formulations, the inhibition of two biomarkers (edema and myeloperoxidase (MPO)) was also quantified. Edema and MPO are associated with skin inflammation, and their increase may inhibit the normal re-epithelization and re-establishment of
physiological conditions on the skin wound. Both hyalurosomes induced a strong inhibition
(> 80%, p < 0.05 in comparison with TPA) of the two biomarkers edema and MPO. Finally,
curcumin-loaded liposomes successfully inhibited edema (»80%), but they were not able to
counteract the MPO activity, showing an inhibition statistically similar to that obtained
using TPA (p > 0.05).77
A significant increase in wound healing efficacy of fibrin sheet (FS) was evidenced when
HA was incorporated into the FS (HA-FS).78 Skin incisions (0.8 cm2 area) were created on
the dorsal regions of the ears of New Zealand white rabbits. The skin specimens were collected at different time intervals (7, 14, and 28 days) and were analyzed visually and histologically. The resulting micrographs showed that healing was incomplete with plugs of exudates
with moderate numbers of necrotic tissue and inflammatory cells infiltrated into the
wounded sites at day 7 (Figs. 11a–11b). Interestingly, a complete wound closure and re-epithelization were achieved on day 14 (Figs. 11c–11d). Comparative analysis revealed significantly higher granulation tissues with sprouting capillaries in the experimental group treated
with HA-FS compared to the control group. At 28 days (Figs. 11e–11f), the researchers
observed a remarkable progression in healing with fully developed actively regenerating
granulation tissues in the HA-FS-treated group compared to the control. The wound healing
efficiency of HA-FS was further demonstrated by the observation of significantly more
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Z. HUSSAIN ET AL.
Figure 9. (A) Histological examination of wounded skin (H & E staining), (B) Thickness of dermis layer of
each wound. Results showed that wounds treated with scaffold embedded with HA and EGF showed significantly (p < 0.01) higher wound healing efficacy and compared to the control groups (n D 5).75
© Elsevier. Reproduced by permission of Elsevier. Permission to reuse must be obtained from the
rightsholder.
granulation tissue and neovascularization in the HA-FS treatment group. The regeneration
process was further supported by the development of an appendages-like structure in the
healing HA-FS-treated tissue (Fig. 11e), which was not seen in any of the tissues or sections
from the FS-treated wound (Fig. 11f).
HA has been shown to enhance healing potential after tracheal surgery.79 In one study,
twenty-two New Zealand white rabbits (11 experimental, 11 control) were used. A 2-mm
round surgical incision was created in the third tracheal ring of each experimental and control animal. During the treatment period, the experimental groups were dressed with 8 mm
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Figure 10. Pictures of dorsal skin lesions of mice treated with saline, curcumin dispersion, empty 0.5hyalurosomes and curcumin-loaded liposomes, 0.1 hyalurosomes or 0.5hyalurosomes at 2, 4 and 6 days, in
comparison with untreated skin.77 © Elsevier. Reproduced by permission of Elsevier. Permission to reuse
must be obtained from the rightsholder.
round sodium hyaluronate-based sponges (SEPRAPACKTM ) fixed over the wound surface
using fibrin glue (HemaseelTM ). The control groups were dressed with a plain 8-mm collagen
sponge in a similar fashion. For the purpose of analysis, the tracheal tissues were harvested at
week 4 post-operation and were histologically scored in regards to inflammation, connective
tissue organization, epithelial closure, chondrocyte death, and cartilage regeneration (clonal
cell formation) at the area of injury.79 The resulting data indicated that the number of
inflammatory cells that infiltrated the injury site in HA-treated wounds was significantly (p
< 0.05) lower than the control group. A lower percentage of animals treated with the HAbased sponge showed chondrocyte death at the wound edge (45.5% in HA vs. 81.8% in control) (p < 0.05), and a greater percentage of HA-treated animals revealed new clonal cell formation (72.7% HA vs 54.5% in the control) (p > 0.659) compared with the control. These
results suggest that HA can be a useful adjunct in improving postoperative tracheal wound
healing and repair.79
HA tissue engineered skin substitutes provided a feasible method to overcome the shortage of skin autografts by culturing keratinocytes and dermal fibroblasts in vitro.80 A study
was conducted with the aim of fabricating a bilayer of gelatin-chondroitin-6-sulfate-hyaluronic acid (gelatin-C6S-HA) biomatrices and evaluating their wound-promoting efficacy in
severe combined immunodeficiency (SCID) mice.80 The results showed that the human epidermis was well-developed with the expression of differentiated markers and basement
membrane-specific proteins at 4 weeks. After implantation, the percentages of skin graft take
were satisfactory, while the cell-seeded group performed better than the non-cell-seeded one.
The basement membrane proteins including laminin, type IV collagen, type VII collagen,
integrin a6, and integrin b4 were all detected at the dermal-epidermal junction, which
showed a continuous structure in the 4 weeks after grafting. This bilayer gelatin-C6S-HA
skin substitute not only has a positive effect on promoting wound healing but also has a high
rate of graft take.80
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Figure 11. Histological examination of wounded rabbit ear skin after treating with HA incorporated fibrin
sheet (HA-FS) in comparison with fibrin sheet (FS) as control at various time intervals; (A) day 7, (B) day 14
and (C) day 28. Histological features of these monographs showed remarkably higher healing efficiency in
HA-FS group compared to control (FS) at various time points.78 © Elsevier. Reproduced by permission of
Elsevier. Permission to reuse must be obtained from the rightsholder.
The addition of exogenous HA to beta-tricalcium phosphate (CP) has been reported to
promote osseous tissue healing of apical lesions following peri-radicular surgery in an experimental dog model.81 The results showed signs of regeneration with newly formed bone tissue and fibrovascular connective tissue within the treated cavity sites and complete
resorption of the implemented materials. The newly formed bone consisted mainly of osteoid bone trabeculae with some more mature dense bone present at the periphery of the cavity site. There was no significant difference in the percentage of newly formed bone tissues
(p > 0.05) and bone trabeculae thickness (p > 0.05) between the two study groups; however,
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a slight increase in osseous tissue healing in the HA-based CP group revealed an additional
pharmacological and therapeutic aspect of HA and is a future prospect to further explore
the mechanism of HA in promoting osseous tissue healing.81
Previously, Turley and Torrance found that the biodegradation of HA produces byproducts that aid in epithelial cell proliferation and migration. A number of reported studies
found that enzymes involved in the degradation of HA stimulate cell proliferation, providing
further evidence that HA must be broken down in order to promote cell growth.82,83 The
molecular structure of HA itself also facilitates the movement of cells within the ECM, providing a substrate for cell migration and proliferation and thus enhancing dermal repair.82
Interestingly, HA may also play a significant role in angiogenesis and the inflammatory
response, further supporting cellular growth.84,85 However, there was no significant difference between the thicknesses of the epidermis treated with vitronectin growth factor alone
and vitronectin growth factor together with HA delivery vehicle. The addition of HA did
not enhance all the cellular responses to vitronectin growth factor examined.86
The molecular weight of HA is also an important factor when considering its role in
wound regeneration.59,87–90 However, there is no consensus regarding the superiority of a
specific molecular weight of HA for wound healing. Some studies demonstrated that HA
with high molecular weight (HMW-HA) showed better wound healing efficacy by promoting keratinocyte proliferation, granulation tissue formation, and collagen deposition.89,91
However, other studies revealed that LMW-HA exhibited superior healing efficacy of incisional and excisional wounds. Authors demonstrated that application of LMW-HA to the
skin stimulates keratinocytes to release b-defensin-2, which improves self-defense of the
skin and protects vulnerable cutaneous tissue from infections.92 On the other hand, authors
have demonstrated that superior wound healing efficacy of LMW-HA is owing to its promising antimicrobial potential against the pathogenic bacteria which could diminish formation
of new granulation tissue at the site of injury. Thus, their strong antimicrobial efficacy enables them to promote wound healing processes.93 The application of LMW-HA has also
been shown to prevent oxygen free radical-based damage to the granulation tissues during
the wound healing process. These findings suggest greater ability of LMW-HA-based biomaterials in promoting healing of incisional and excisional wounds.94 Several other experimental models reported that LMW-HA significantly promotes angiogenesis during wound
healing processes compared to MMW-HA and HMW-HA.95,96 In contrast, a recent study
conducted by Ghazi et al.89 revealed that the application of medium molecular weight HA
(MMW-HA, 100–300 kDa) significantly enhanced wound healing compared to low molecular weight HA (LMW-HA, 50–100 kDa) and high molecular weight HA (HMW-HA, 1000–
1400 kDa).89 They demonstrated that higher wound healing efficacy of MMW-HA was due
to the up-regulation of junctional adhesion molecules at the epidermal diffusion border.89
In addition to the above reports, the summary of therapeutic efficacy of HA-based biomaterials for the treatment of traumatic and surgical wounds is presented in Table 1.
3.2 Chronic wound management
A chronic wound is a wound that does not heal in an orderly set of stages and in a predictable amount of time the way most wounds do. Chronic wounds are characterized by an
excessive persistent inflammatory phase, prolonged infection, and the failure of defense cells
to respond to environmental stimuli.1 There are numerous complications which could
Scaffolds constructed of silk fibroin,
chondroitin sulfate and HA
Scaffold features
Wound healing/ Dermal tissue
regeneration
Study parameters
Major outcomes
References
1. Superior dermis regeneration (smaller wound area)
[69]
2. Improved angiogenesis and collagen deposition compared
to scaffold lacking collagen;
3. Significant reduction in positive expression of growth
factors with progression of wound
healing.
Male Wistar rats (250–285 g) (Full
Porous scaffold constructed of
Wound closure rate/ Epidermal tissue 1. Faster wound closure rate
[68]
thickness skin wounds)
collagen, HA and gelatin
reconstruction
2. Accelerated epidermal tissue regeneration
3. Diminished neutrophils infiltrates
Swiss Nu/Nu male mice (Full
Human dermal/epidermal cell
Wound closure rate/ Wound healing 1. Accelerated wound closure rate
[71]
thickness excisional wounds)
fractions entrapped directly within
progression
2. Faster re-epithelialization and neovascularization
a gellan gum/HA spongy-like
3. Accelerated tissue remodeling
hydrogel
Rabbit (Full thickness skin wounds) Xenogeneic decellularized scaffold
Wound closure rate/ Dermis
1. Faster would healing rates
[73]
constructed of HA C bFGF
regeneration
2. Superior dermis regeneration with scaffold, regardless of
basic fibroblast growth factor concentration.
Rabbit (Full thickness skin wounds) Decellularized scaffold constructed
Wound closure rate/ Dermis
1. Faster wound closure
[74]
of HA C EGF
regeneration
2. Superior dermis regeneration
Rats (Full thickness skin wounds)
Xenogeneic decellularized scaffold
Wound closure rate/ Dermis
1. Faster would healing rates
[75]
constructed of HA C EGF
regeneration
2. Significant dermis regeneration with scaffold
Sprague-Dawley Rats (Full
HA sponge containing arginine and
Wound closure rate/ Dermis
1. Faster healing rate
[76]
thickness abdominal incision)
EGF
regeneration
2. Superior dermis regeneration
Rat (Midline abdominal incision)
Hydrogel of HA embedded in mildly Peritoneal tissue adhesion
1. Efficient shielding to the wound area
[66]
crosslinked alginate
2. Prevention of peritoneal tissue adhesion
3. Facilitated wound healing
[72]
In vitro inter-tissue model
Anti-adhesive spongy sheet
Release of vascular endothelial
1. Significant release of VEGF and hepatocyte
growth factors.
composed of HA and collagen
growth factor and hepatocyte
containing EGF.
growth factor – in vitro model
Sprague-Dawley Rats (Full thickness
Wound healing
2. Faster wound healing
abdominal incision)
3. Superior dermis regeneration
4. Preventing surgically excised tissue from adhering to
surrounding tissue
Diabetic mice (Full thickness skin
HA and collagen spongy sheet
Cytokine production by fibroblasts/
1. 3 times higher VEGF release and 3.6 times higher HGF
[99]
defect)
containing EGF and vitamin C
Granulation tissue formation/
compared to control
Angiogenesis
2. Significantly higher rate of granulation tissue formation
3. Faster angiogenesis
Sprague-Dawley Rats (Full thickness HA and collagen spongy sheet
Production of VEGF and HGF/ Wound 1. Significantly higher production of VEGF and HGF
[43]
skin)
containing EGF
size/ Re-epithelialization and
2. Higher rate of granulation tissue formation
granulation tissue formation
3. Faster angiogenesis
Sprague–Dawley Rats (Full
thickness skin wounds)
Experimental model
Table 1. Wound healing and skin regeneration efficacy of HA scaffolds for the treatment of traumatic and surgical wounds.
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[65]
1. HA C ACS showed superior bone filling potential
vs. control (p < 0.05) and ACS (p D 0.017)
(Continued on next page)
[102]
[101]
[100]
[67]
1. Significant new bone formation,
[70]
2. Larger bone formation area,
3. Larger bone volume and bone mineral density in
treatment conditions with larger GDF-5 concentration
1. Re-epithelization occurred progressively from the periphery
to the center of the wound
[72]
1. Significant increase in the release of VEGF and HGF
Retrospective study (n D 29);
Tissue-engineered dermis composed Healing time/ Scar condition/ Patient
Patients with removal of basal
of autologous cultured dermal
compliance
cell carcinoma from face
fibroblasts seeded on HA sheet
Ò
Generation of skin-like tissue
1. Superior integration of graft within the
Fresh wounds from surgical
Hyalomatrix 3D HA scaffolding C
autologous skin grafting
surrounding tissues
procedure (n D 6); Patients with
2. Regenerated dermis with extracellular matrix rich in type I
inveterate disabling scar
collagen and elastic fibers, and with reduced type III
retraction, with soft tissue defect
collagen
resulting from surgical scar
removal
Rabbit (wound surface)
HA C porcine acellular dermal matrix Expression of collagen/ Generation of 1. Exogenous HA relieves graft contracture on rabbit wound
grafts and autologous skin
CD44 receptors
surfaces
2. Significant increase of collagen I and III expression
3. Stimulates the generation of more CD44 receptors to
strengthen its enzymolysis.
4. Promote the vascularisation of the wound surface
Japanese white rabbits (Full
HA C acellular dermal matrix grafts
Collagen expression
1. Significant boost in the expression of collagens
thickness skin defects)
I and III and decrease the ratio of collagen I/
collagen III.
2. Faster wound healing and basilar membrane remodeling
3. Mitigation of the contraction of skin transplant
Japanese white rabbits (Full
HA C porcine acellular dermal matrix Collagen expression/ Biomechanical
1. Faster wound healing and basilar membrane remodeling
thickness skin defects)
grafts
performance of transplant skin
2. Decreased contraction of skin transplant
Two-layered cultured dermal
Production of VEGF and HGF
substitute: Upper layer is of HA
and collagen spongy sheet with or
without EGF. The lower layer is a HA
spongy sheet and Collagen gel
containing fibroblasts.
Adult Wistar rat (Critical-size calvaria HA: 1.0% HA; HA C ACS: 1.0% HA C Connective tissue formation
defects)
absorbable collagen sponge; ACS:
absorbable collagen sponge;
Control: no treatment
Rabbit (Critical-size calvaria defects) HA hydrogels loaded with GDF-5
Proliferation and differentiation/
Osteogenesis
In vitro: Wound surface model
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Study parameters
Major outcomes
3. Significant boost in the expression of collagens I and III
and decrease the ratio of collagen I/collagen III.
HA C porcine acellular dermal matrix Collagen expression/ Angiogenesis/
1- Significant basilar membrane remodeling
grafts C thin skin autograft
Dermal matrix
2- Decreased contraction of skin transplant
3- Increased expression of collagens I and III and decrease
the ratio of collagen I/collagen III.
HA C Iodine (Hyiodine)
Wound contraction/ Granulation
1- Significant acceleration of wound healing in first 5 days of
treatment.
2- Larger thickness of proliferating epidermis in Hyiodine
treated wounds compared with saline treated wounds.
3- Significantly lesser wound exudate on the top of wounds
treated with Hyiodine.
Wound closure/ Inflammatory cells
1- Significantly lower percentage of inflammatory
Sodium hyaluronate-based sponge
infiltration/ Cartilage regeneration
cells infiltration in HA treated animals.
(SEPRAPACKTM )
2- Lower percentage of chondrocyte death in HA based
sponge treated animals.
3- Greater percentage of clonal cells formation in HA treated
animals.
HA produced from microbial
Wound healing/ Wound contraction
1- Significantly faster healing in 16 days.
fermentation: Streptococcus
2- Significant wound contraction in HA treated wounds
Zooepidemicus: MTCC 3523
compared with control group.
Scaffold features
[105]
[79]
[104]
[103]
References
Abbreviations: HA, hyaluronic acid; GDF, growth and differentiation factor 5; HGF, hepatocyte growth factor; VEGF, vascular endothelial growth factor; EGF, epidermal growth factor; bFGF, basic
fibroblast growth factor.
Wistar rats
New Zealand white rabbits (Full
thickness surgical incision)
Rat (Full thickness skin wounds)
Japanese white rabbits (Full
thickness skin defects)
Experimental model
Table 1. (Continued )
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Z. HUSSAIN ET AL.
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impair the normal wound healing process and lead to the transformation of acute wounds
(minor injury) into chronic wounds (non-linear wounds). Chronic wounds are those that
have failed to restore the anatomical and functional integrity of the skin over a period of
three months.55 Chronic wounds seem to be detained in one or more of the phases of wound
healing. These wounds provide a significant burden to patients, health care professionals,
and healthcare systems with 5.7 million patients affected in the US alone costing an estimated 20 billion dollars annually.55,97 Moreover, chronic wounds have also been proven to
show a high bacterial bio-burden, which can further complicate wound restoration.98
Chronic wounds usually manifest as secondary complications of other disease processes i.
e., diabetic foot ulcers (DFUs) as a response of diabetes mellitus, pressure ulcers as a result
of spinal cord injuries, neurodegenerative processes like Pick’s disease, and venous ulcers in
response to reduced blood perfusion to certain tissues owing to improper functioning of
venous valves.55 These causative diseases significantly impact various vital mechanisms of
normal wound healing such as biochemical signaling, ECM deposition, or cell migration,
which could result in impaired wound healing and transformation to chronic wounds. For
example, hyperglycemia in diabetic patients may inhibit ECM deposition by upregulating
the proteolytic action of matrix metalloproteinases (MMPs) via increased levels of tumor
necrosis factor-alpha (TNF-a) and interleukins (IL-1b).106 In addition, DFUs may also
impair keratinocyte migration and leukocyte function leading to infection. Moreover,
depleted levels of inorganic phosphate within diabetic patients could also result in low levels
of adenosine triphosphate (ATP), leading to a significant attenuation of the immune
response.106 Signaling molecules like epidermal growth factor (EGF) function normally to
stimulate proliferation and migration of keratinocytes during wound closure; however,
aging, disease, and sun damage inhibit keratinocyte ability to respond to EGF and other
growth-promoting mitogens.107 The above mechanisms contribute to impaired wound healing and are the focus of new therapeutic modalities that center on incorporating both ECM
and various signaling molecules within chronic wounds in order to promote regeneration
and wound repair.
Unlike acute wounds, chronic non-healing wounds impose a substantial challenge to the
conventional wound dressings and demand the development of novel and advanced wound
healing modalities. An efficient management plan for a chronic stubborn wound remains a
challenge. However, a better understanding of the molecular biology and pathophysiology of
chronic wounds could result in more efficient and improved therapeutic paradigm for the
management of chronic wounds. Researchers believe that the wound healing process can be
actively promoted by amending the expression of prime biological mediators, which are key
parameters for the healing process.108 In general, it is already well-established that an efficient and well-controlled healing process can be achieved by adopting standard guidelines
such as the following:
(1) the wounded area should be dressed with adequate biomaterials which can prevent the
contamination/infection of the affected area over the long-term duration of wound
management,
(2) an ideal moist environment needs to be provided to potentiate the wound healing rate
(wound closure) and prevent wound dissection,
(3) use of a medicated dressing which can provide a sustained and proficient release of
fabricated pharmacological moieties and biomolecules, and
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Z. HUSSAIN ET AL.
(4) preventing a rapid degradation of medicated dressing and irregular releasing pattern of
fabricated drugs during the healing process.109,110
The management plan for chronic wounds is defined based on the severity and range of
tissues affected. This can be assessed by classifying the chronic wounds according to
Wagner’s system.111,112 Accordingly, the chronic wounds can be classified into various
grades as follows:
(1) “grade 0” indicates no ulcers in affected tissues with high-risk of secondary
complications,
(2) “grade-1” refers to a partial and/or full thickness ulcer,
(3) “grade-2” indicates a deep ulcer which has penetrated down to the ligaments and muscle but with no bone involvement,
(4) “grade-3” refers to a deep ulcer with cellulitis or abscess formation,
(5) “grade-4” indicates a localized gangrene, and
(6) “grade-5” indicates an extensive and deep gangrene of the whole tissue.113
The classification of chronic wounds is important as it may facilitate selection of an
appropriate dressing depending on the wound type, severity, and phase of wound
development.114
To date, numerous polymer-based wound dressings are employed for the management of
chronic wounds; among them, HA is a well-recognized and versatile choice. Preliminary
clinical data supported that composite Laserskin graft with a fibroblast cell layer was a powerful tool with respect to its durability, biocompatibility, graft take rate, low infection rate,
and seeding efficacy. This study was conducted by Lobmann et al.87 with 14 patients having
diabetic foot lesion. The chronic wounds of these diabetic patients were grafted with autologous human keratinocytes cultured on membranes composed of benzyl ester of HA. Results
demonstrated that 79% of DFUs treated with HA template grafts fully healed between 7 and
64 days post-procedure. Interestingly, 3 of the grafts that failed to survive had been grafted
in patients with considerable arterial occlusive disease or with concomitant infection. Based
on the findings, the authors proposed that the transplantation of autologous human keratinocytes with HA may allow for faster closure of diabetic foot lesions and subsequently
reduce the length of hospitalization.87
The composite Laserskin graft is an HA derivative consisting of micropores that support
cell growth. Its use as a template for cultured epithelial cell grafts has been studied extensively. The efficiency of seeding the template with cultured epithelial cells is dependent upon
the use of a fibroblast feeder layer.
Lam et al.115 performed studies comparing the efficacy of seeding composite Laserskin
grafts with cultured keratinocytes alone and with an allogenic fibroblast cell layer. Laserskin’s
micropores are laser-produced perforations that are 40 mm in diameter that can deliver keratinocytes which are roughly 20 mm thick. A 10 cm by 10 cm sheet of Laserskin (along with
a fibroblast feeding layer) can plate about 4 million keratinocyte cells.115 Laser skin, when
accompanied by allogenic fibroblasts, is a highly effective human skin substitute, which can
be used for wound resurfacing. The comparative study reported by Lam and colleagues demonstrated that the seeding efficacy of human keratinocytes on plain Laserskin was 75% while
Laserskin with the fibroblast layer boasted a 95% efficacy. The difference was even more pronounced in rat keratinocytes, which increased from 36% to 88% with the addition of the
feeder layer.115
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Al Bayaty et al.116 also investigated the wound healing efficacy after topical application of
hyaluronate gel in a streptozotocin (STZ)-induced diabetic rat model. Four groups of adult
male Sprague-Dawley rats were used in this study. A 2 cm full-thickness skin wound was
experimentally created on the posterior neck area of each rat. Group 1 animals were topically
treated with the vehicle (gum acacia in normal saline) as a placebo control group. Group 2
animals served as a reference standard and were treated topically with Intrasite gel (a clinically proven amorphous hydrogel wound dressing containing 2.3% of carboxymethylcellulose together with 20% propylene glycol). Animals of Group 3 and 4 were treated topically
with Aftamed (high molecular weight hyaluronic acid, 240 mg/100 g gel) and Gengigel 0.8%
(hyaluronic acid gel 0.8% w/w), respectively. The wound healing activity was evaluated
blindly by an observer unaware of the experimental groups and the test protocol. It was
observed that experimental animals dressed with Intrasite gel, Aftamed high molecular
weight HA (240 mg/100 g gel), and Gengigel 0.8% HA gel showed faster healing rates compared to the placebo control group.116 Further comparative analysis between tested groups
showed significantly faster healing in the Aftamed (HA-incorporated gel)-treated animals
compared to other treatment groups. Moreover, HA-based gel (Aftamed)-treated animals
showed fewer scars at wound enclosures, fewer numbers of inflammatory cells, and significantly higher angiogenesis and integers of collagen fibers deposition compared to the control
and other treatment groups.116 These findings suggested that HA-based dressings/formulations/scaffolds exhibit promising wound healing efficacy.
Clinical significance of the HA-iodine complex, Hyiodine, has been studied on 22 patients
suffering from complicated diabetic foot wound for the complete healing of their infected
diabetic wounds.117 Hyiodine was either spread directly over the wound, or (more frequently) gauze was immersed in Hyiodine and then put on/into the wound. Then, several
layers of dry gauze covered the wound. Results showed that complete healing was evident in
18 out of 22 patients with DFUs within 6–20 weeks after the start of treatment, depending
on the wound character, localization, and extent.117 Two patients were treated with Hyiodine, and significant wound improvement was apparent. Treatment was not successful in
two subjects with ischemic defects due to simultaneous arterial occlusion. Another study
was conducted on 18 more patients suffering from complicated diabetic foot wounds by
Sobotka et al.118 to further confirm the wound healing activity of a unique system for wound
treatment, which was based on a combination of high molecular weight sodium hyaluronate
with an iodine complex-Hyiodine. Wound healing was monitored daily, and wound pictures
were taken each second week. Clinical improvement was observed in the majority of
patients. This suggests that the HA-iodine complex dressing has potential that needs to be
developed from controlled studies.
Researchers have also highlighted the clinical significance of HA-embedded hydrofiber
dressings (HyalofillÒ ) in the management of chronic diabetic wounds. HyalofillÒ is an absorbent, soft, and conformable fibrous wound dressing which is purely composed of HYAFFÒ ,
an ester of HA. The advantages of using HYAFFÒ as wound dressing include:
1) efficient absorption of exudate at the wound site,
2) creating a moist environment to promote wound healing,
3) promoting granulation tissue formation, and
4) enhanced efficacy of wound repair by accelerating the wound healing process.
Vazquez et al.38 evaluated the therapeutic efficacy of HA-embedded hydrofiber dressing
(HyalofillÒ , Convatec, USA) in treating neuropathic diabetic foot wounds. This study was
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Z. HUSSAIN ET AL.
conducted with 36 patients with complicated DFUs. All patients received surgical debridement for their diabetic foot wounds, were continuously treated with HA-based dressing, and
dressing changes took place every other day. The assessment criteria for the wound healing
efficacy included the complete wound closure time and the percentage of the patients achieving completed wound closure within 20 weeks. Therapy was then followed by a moistureretentive dressing until complete epithelialization was achieved. Results obtained indicated
that 75% of wounds were healed within the 20-week evaluation period. The average duration
of HyalofillÒ therapy in all patients was 8.6 § 4.2 weeks. Deeper wounds were over 15 times
less likely to be healed than shallow wounds. Thus, based on the findings, it was revealed
that a therapeutic regimen entailing moist hyaluronan-containing dressings can be a suitable
adjunct to treat diabetic foot ulcers.38
Silver has been extensively used to control infections since ancient times. The use of silver
nanoparticles has been well documented because of its wide-spread applications in antibacterial.119,120 anti-inflammatory,119 wound healing,121,122 and tissue engineering activities.123
Silver-based medical products have been proven to be effective in retarding and preventing
bacterial infections.119,120 It is worth reporting that there is an increasing interest in using silver nanoparticles technology in the development of bioactive biomaterials. Recently, the
effect of different molecular weights of HA, with or without silver nanoparticles, on the
wound healing efficacy was studied by Fouda et al.93 using an STZ-induced diabetic rat
model. The experimental animals were segregated into five groups: group 1 was the baseline
group treated with saline, group 2 was treated with low molecular weight HA without silver
nanoparticles (HA1), group 3 was treated with low molecular weight (50 and 100 kDa) HA
without silver nanoparticles (HA2), group 4 was treated with medium molecular weight
(100 and 300 kDa) HA without silver nanoparticles (HA3), and group 5 was treated with
high molecular weight (1000 and 1400 kDa) HA with silver nanoparticles (HA4). The assessment of wound healing efficacy was carried out by visualizing the morphology of wounds
daily throughout the investigational period. To observe the improvements in wound healing,
morphometric signals were assessed at two day intervals.93 The size of the wound opening
was significantly greater in sections from older rats in contrast to those from the young rats.
The wound openings of older rats treated with HA4 were gradually minimized and became
totally closed at day 8 after wounding. Moreover, the number of new blood vessels and the
depth of the dermal tissue in the wounded area were significantly lower in older rats in comparison to the younger ones, but HA4 was found to significantly stimulate angiogenesis and
the assembly of dermal constituents (Fig. 12).93
Further, they evaluated the healing potential of HA-based formulations via histological
examination (Fig. 13).93 Histological examination identified greater healing potential of the
HA4-treated group compared to other treatment and control groups. In the HA4 group,
greater amounts of symmetrically distributed integers of collagen fibers were observed compared to other treatment groups. In addition, a well-defined, fully constructed epidermis and
proliferated hair follicles, which are characteristic features of normal healthy skin, were also
observed in the HA4 group (Fig. 13). These findings suggested a promising wound healing
potential of HA-based formulations.93
An evidence-based review was conducted also by Hancı et al.124 exploring the significance
of HA in post-tonsillectomy pain relief and wound healing. Fifty patients (20 males, 30
females mean age of 13.56 years) were included in this prospective, double-blind, and controlled clinical review. HA was applied to one side and the other side was used as a control
POLYMER REVIEWS
27
during tonsillectomy. Therefore, the same patient was evaluated and the post-tonsillectomy
pain was scored, excluding individual bias.124 Post-tonsillectomy throat pain was measured
twice a day (in the morning and in the afternoon) during the period of 14 postoperative
days using the visual analog scale (VAS) on a scale of 0–10 after 2 h of analgesic intake.
Wound healing was assessed by direct visual examination of the area of slough in each tonsillar fossa at days 7, 10, and 14 post-operation and scored on a scale of 0 to 5 (0 D
completely healed wound, 5 D not healed wound). The wound healing score was evaluated
by the method described by Magdy et al.125 The resulting data showed that scores of posttonsillectomy throat pain were significantly (p < 0.001) lower in HA-treated patients compared to the control group both in the mornings and in the afternoons at all times during
the postoperative care. Thus, the results reveal that HA was effective in reducing the posttonsillectomy pain. Similarly, HA significantly (p < 0.001) enhanced wound healing in HAtreated patients compared to non-treated (control) patients at all times. At the end of the
two-week follow-up period, the wound in the HA-treated operation side was almost
completely healed, indicating that the rate of healing was comparatively faster with HA treatment.124 A plethora of other studies involving the use of HA, with or without bioactive moieties, in the management of chronic wounds are summarized in Table 2.
Figure 12. (A) Representative external photographs of full thickness skin wounds in control untreated and
different treated groups. Photographs were taken from different rat groups two weeks post-wounding
(Wound was rectangle, 10 £ 20 mm). Resulting photographs showed that the wound size becomes
smaller in groups II–IV in comparison to the control wound size or the group I wound size. (B) The total
count of the pathogenic bacteria grown on the full thickness wounded skin from older untreated (Control)
and treated rats. Full thickness skin samples were taken from different rat groups one day post-wounding
(Wound was rectangle, 10 £ 20 mm). Values shown are means § SD. Shows the significance in comparison with the control group. (C) Agar bacterial culture from different groups.93 © Elsevier. Reproduced by
permission of Elsevier. Permission to reuse must be obtained from the rightsholder.
28
Z. HUSSAIN ET AL.
4. Summary
Conventional wound dressings have gained recognition in the management of mild-tomoderate acute wounds; however, chronic non-healing wounds impose a substantial
challenge to the conventional wound dressings and there is a demand for the development of novel and advanced wound healing techniques. Among several natural biopolymers, hyaluronic acid has long been recognized as a versatile wound healing modality.
Figure 13. Representative Masson Trichrome staining of full thickness wounded skin from older untreated
(control) and treated rats (group HA1-HA4) (X 100). Full thickness skin samples were taken from different
rat groups two weeks post-wounding (Wound was rectangle, 10 £ 20 mm). Epidermal cells (Ep); Collagen
fibers (Coll); Epidermal tongues (black arrows); Bubbles (red arrows); Hair follicles (HF).93 © Elsevier. Reproduced by permission of Elsevier. Permission to reuse must be obtained from the rightsholder.
Bioactive substance
Keratinocyte stem cell therapy
Hyiodine
Hyiodine
Full thickness excisional
wound model
22 human patients with
diabetic foot wounds
18 human patients with
diabetic foot wounds
14 human patients with
Hyaluronan – iodine Complex
19 non-healing diabetic
wounds
8 human patients with sternal Hyaluronate – iodine Complex
wounds
36 human patients with non- Hyalofill
healing DFUs
Poly-N-acetyl glucosamine
(pGlcNAc) membrane
db/db diabetic mice
14 human patients with non- Autologous human
healing diabetic foot
keratinocytes
lesions
db/db diabetic mice
Poly-N-acetyl glucosamine
(pGlcNAc)
Experimental model
Major outcomes
Wound healing
Hyaluronan – iodine soaked Healing rate/ Amount of
gauze
exudate formed
[130]
[129]
[38]
[118]
[117]
[128]
[127]
[126]
[87]
References
(Continued on next page)
1. Complete healing of DFU in 20 weeks of treatment.
2. Deeper wounds were over 15 times less likely to
heal than superficial wounds
1. Significant healing rate.
2. Fourteen wounds progressed to complete healing
with a mean healing time of 18.1 § 15.1 weeks.
1. The mean (SD) length of treatment was 136 days.
2. Complete healing was achieved in 7 patients, and 1
patient underwent a reconstructive operation for
wound closure.
1. Significant faster healing of 79% of diabetic
foot lesions between 7 and 64 days.
2. Length of hospitalization was reduced.
1. Faster rate of healing (90% closure in 16.6 days).
2. Accelerated proliferation and vascularization of
granulation tissue.
Wound closure/ Granulation 1. Faster wound closure achieved by re-epithelialization
tissue formation
and increased keratinocyte migration.
2. Accelerated granulation tissue formation, cell
proliferation, and vascularization.
3. Up-regulated levels of VEGF, uPAR and MMP3,
MMP9.
Wound contraction
1. More-complete wound closure resulted from edge reepithelialization and contraction
2. Higher ratio of granulation tissue formation
3. Significant dermal regeneration
Wound closure/ Complete
1. Significant improvement in the size of diabetic ulcers.
healing
2. Complete healing was evident in 18 patients within
6–20 weeks after the start of treatment
Wound closure/ Healing
1. Significant improvement in the size of diabetic
rate
wounds was observed in majority of patients.
Healing of diabetic foot
lesions/ length of
hospitalization
Wound closure/
Angiogenesis
Therapeutic factors
Hyaluronan – iodine soaked Healing rate/ Amount of
gauze
exudate formed
High molecular weight
sodium hyaluronateiodine complex
Hyaluronan –containing
wound dressing
Hyaluronan (HyalomatrixÒ )
- esterified hyaluronan
scaffold beneath a
silicone membrane
Sodium hyaluronate-iodine
complex
Nanofiberous material
Nanofibers
HA benzyl ester films
Delivery system
Table 2. Summary of wound healing efficacy of HA for the management of chronic non-healing wounds.
POLYMER REVIEWS
29
Arginine and EGF
Mixture of amino acids
STZ diabetic rats
Human patients with
neuropathic ulcers
STZ diabetic rats
—
Cross-linked high and low
molecular weight HA
foam
HA gel (Vulcamin)
High molecular weight HA
gel
Hyalograft-3D autograft C
Laserskin autograft
Delivery system
Ulcer area/ predisposed
infection
Wound area/ angiogenesis
Wound size/ Epithelization
Wound healing/ ECM
formation and
reepithelization
Wound healing/ Ulcer size
Therapeutic factors
1. A 50% reduction in ulcer area was achieved
significantly faster in the treatment group.
2. Weekly percentage ulcer reduction was consistently
higher in the treatment group.
3. Complete ulcer healing was evident in 12 weeks.
1. Significant reduction in wound size.
2. Remarkable increased in number of macrophages
and fibroblast.
3. Accelerated collagen deposition and reepithelization of the wounds.
1. Accelerated wound healing.
2. Significant reduction in wound size.
3. Increased re-epithelization.
1. After 3 month, the ulcer area and the number
of infective complications were clearly decreased.
1. Significant improvement in wound healing
rate.
2. Greater angiogenesis and granulation tissue
formation.
3. Numbers of pathogenic bacteria grown on full
thickness wound were significantly reduced.
Major outcomes
[133]
[132]
[76]
[116]
[131]
References
Abbreviation: STZ, streptozotocin; HA, hyaluronic acid; VEGF, vascular endothelial growth factor; uPAR, urokinase-type plasminogen activator; MMP3, metalloproteinases-3; MMP9, metalloproteinases-9; EGF, epithelial growth factor; DFUs, diabetic foot ulcer.
Different molecular weight HA
(low, medium, high) – with
or without silver
nanoparticles
—
Autologous tissue-engineered
graft – a 2-step HYAFF
autograft
Bioactive substance
STZ diabetic rats
180 human patients with
dorsal and plantar DFUs
Experimental model
Table 2. (Continued )
30
Z. HUSSAIN ET AL.
POLYMER REVIEWS
31
Critical analysis of the literature revealed that hyaluronic acid, alone or in combination
with other agents (fibrin, collagen, gelatin, growth factors, curcumin, silver, etc.) has
shown superior healing efficiency in the treatment of the tympanic membrane, skin
articular cartilage, trachea, and corneal wounds. Hyaluronic acid has been proven to be
an efficacious wound healing agent in various forms such as HA-scaffolds, -sponge-like
hydrogels, -anti-adhesive sheets, hydrogels, films, cultured dermal substitutes, -thin
membranes, and -dermal matrix grafts. A wide range of in vitro, in vivo, and clinical
studies summarized in this review provided substantial evidence for the superior wound
healing efficacy of hyaluronic acid-based biomaterials in the treatment of acute and
chronic wounds. The superior wound healing efficacy of hyaluronic acid-based biomaterials is evident via upregulating keratinocytes proliferation, enhanced granulation tissue formation, improved angiogenesis, increased epidermal thickness, and accelerated
subcellular tissues regeneration. Thus, the current review aimed to summarize the available convincing evidence for the therapeutic and clinical dominance of hyaluronic acidbased biomaterials for the management of acute and chronic wounds. Therefore, in the
near future, research will certainly focus on the development of more efficient and less
expensive hyaluronic acid-based medicated wound dressings that can improve therapeutic outcomes and patient quality of life.
Acknowledgements
The authors are very thankful to the Faculty of Pharmacy, Universiti Teknologi MARA, Puncak
Alam Campus, Malaysia, for their support and for providing the resources needed to accomplish
this review.
Funding
A grant from the Institute of Research Management & Innovation (IRMI) (600-IRMI/DANA 5/3/LESTARI (0007/2016)) is gratefully acknowledged.
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