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Journal of Microencapsulation, May 2006; 23(3): 277–290
Wound dressings containing bFGF-impregnatedmicrospheres
SHA HUANG1, TIANZHENG DENG1, HONG WU2,
FAMING CHEN1, & YAN JIN1
1Department of Oral Histology and Pathology, College of Stomatology and 2Department of
Chemistry Faculty Preclinical Medicine, Fourth Military Medical University, Xi’an, PR China
(Received 30 May 2005; revised 11 August 2005; accepted 20 September 2005)
AbstractThe primary objective was to synthesize a novel wound dressing containing basic fibroblast growthfactor (bFGF)-loaded microspheres for promoting healing and tissue regeneration. Gelatin sponge waschosen as the underlying layer and elastomeric polyurethane membranes were used as the externallayer. To achieve prolonged release, bFGF addition was loaded in microspheres. The microsphereswere characterized for particle size, in vitro protein release and bioactivity. The bilayer dressings weretested in in vivo experiments on full-thickness skin defects created on pigs. Average size of themicrospheres was 14.36� 3.56 mm and the network sponges were characterized with an average poresize of 80–160 mm. Both the in vitro release efficiency and the protein bioactivity revealed that bFGFwas released in a controlled manner and it was biologically active as assessed by its ability to induce theproliferation of fibroblasts. It was observed that sustained release of bFGF provided a higher degreeof reduction in the wound areas. Histological investigations showed that the dressings werebiocompatible and did not cause any mononuclear cell infiltration or foreign body reaction. Thestructure of the newly formed dermis was almost the same as that of the normal skin. The applicationof these novel bilayer wound dressings provided an optimum healing milieu for the proliferating cellsand regenerating tissues in pig’s skin defect models.
Keywords: Basic fibroblast growth factor, wound dressing, sustained-release, microsphere, tissue regeneration
Introduction
The primary objective in wound care is the promotion of rapid wound healing with the best
functional and cosmetic results (Shibata et al. 1997). The dressing achieves the functions of
the natural skin by protecting the area from the loss of fluid and proteins, preventing
infection through bacterial invasion and subsequent tissue damage. In some cases, it
improves healing by providing a support for the proliferating cells (Yannas et al. 1980). For
an ideal wound dressing, materials should have flexibility, durability, adherence, a capability
Correspondence: Yan Jin, Department of Oral Histology and Pathology College of Stomatology Fourth Military Medical
University, Xi’an, 710032, PR China. Tel: þ86 029 83376471. Fax: þ86 029 83218039. E-mail: [email protected]
ISSN 0265–2048 print/ISSN 1464–5246 online � 2006 Taylor & Francis
DOI: 10.1080/02652040500435170
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of absorbing wound exudate and protecting the lesion from dehydration. From an
engineering viewpoint, it should also be easy to handle and to apply, comfortable when
in place and cost-effective.
It is a dynamic process in which a variety of cellular and matrix components act in concert
to re-establish the integrity of injured tissue. Therefore, an ideal wound dressing may play an
important role in this process (Matsuda et al. 1981). Growth factors play an important role
in cell growth and in the process of wound healing (Lynch et al. 1989). Among growth
factors, basic fibroblast growth factor (bFGF) plays a key role in development and
re-modelling and has the ability to regulate proliferation of endothelial cells and
development of angiogenesis (Kawai et al. 2000). BFGF also contributes to normal
wound healing by improving wound strength and the quality of the scar (Lu et al. 2000).
However, since the in vivo half-life of bFGF is very short, bFGF injected in soluble form
rapidly diffuses away from the site of injection and is also denatured and degraded
enzymatically (Azrin 2001). In recent years, the more possible way to enhance the in vivo
efficacy of bFGF is incorporation or encapsulation. Therefore, the drug delivery system of
bFGF was designed by the use of microspheres (Cortesi et al. 1997).
The aim of this study is to design a novel wound dressing containing a bFGF delivery
system. A well-formed porous and biodegradable inner layer matrix that would serve as the
host for the proliferating cells and would degrade spontaneously without creating any
adverse effects, while the materials of the outer layer would enhance the intensity and
permeability of the wound dressing. And the tissue regenerates was planned to act as the
underlying dermal layer. Sustained-released bFGF was expected to activate cell prolifera-
tion, while the porous matrix would form the medium for these cells to adhere. Gelatin may
play a potential role to carry growth factor and have been used in wound dressings (Miyoshi
et al. 2005). As a denatured form of collagen, gelatin practically is known to have no
anti-genicity, while collagen expresses some in physiological conditions. Furthermore,
gelatin is far more economical and convenient than collagen (Choi et al. 1999). Gelatin is
biodegradable, therefore, it would degrade and be replaced by the newly regenerated tissue
and expected not to cause any damage to the wound area (Young et al. 2005). Polyurethane
membranes were used as the external layer because of their biocompatibility, highly
elastomeric (extensible) strength and permeability to gaseous substances (Nomi et al. 2002).
They are mechanically strong and protect the wound from the external effects (Ulubayram
et al. 1992). The study was concentrated on the synthesis of the wound dressing and on
in vivo testing for its effects on wounds experimentally created on the backs of york pigs.
Materials and methods
Materials
An aqueous solution of human recombinant bFGF was purchased from Sigma (USA),
gelatin was obtained from Sigma (USA), polyurethane (Tendra�) was obtained from
Molnlycke Health Care (Sweden). All other reagents or chemicals were purchased from
Sigma (USA) and were of analytical grade. All were used without any further treatment
or purification.
Preparation of bFGF-impregnated gelatin microspheres
Gelatin microspheres were prepared by a modified coacervation technique reported by
Nastruzzi (Nastruzzi et al. 1994). Briefly, an aqueous gelatin solution was added dropwise
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into paraffin oil while the mixture was mechanically stirred at 1000 rpm to form a water–oil
emulsion. Then the solution was rapidly cooled by immersing in ice–water medium. The
formed gelatin microspheres were filtered, washed with acetone and dried at room
temperature. Then the non-cross-linked microspheres prepared were placed in glutaralde-
hyde aqueous solution and gelatin cross-linking was allowed to proceed for 12 h. After
cross-linking the microspheres were placed in glycine aqueous solution for 30min to block
unreacted residual glutaraldehyde. The resulting microspheres were washed twice with
0.1wt.% Tween 80-solution, centrifuged and freeze-dried at room temperature.
In order to prepare the bFGF containing microspheres, bFGF (5 mg in 1ml phosphate
buffer, pH¼ 7.4) and heparin (5 ml) were added into the aqueous gelatin solution at the
beginning of microsphere preparation stage, respectively. The resultant microspheres were
designated as GM-bFGF.
Gelatin sponge preparation
Preparation of gelatin sponges. Aqueous gelatin solutions stirred at �2000 rpm for 30min at
room temperature and glutaraldehyde solutions were added to form cross-linkings. Then the
solutions were poured into moulds, frozen in liquid nitrogen and freeze-dried for 24 h.
The thickness of resultant sponges was 1 cm. The gelatin sponges were exposed to 60Co
to achieve sterilization prior to in vivo applications and labelled as GS.
Preparation of gelatin sponges with bFGF. BFGF (5mg bFGF in 1ml phosphate buffer,
pH¼ 7.4) and heparin (5ml) were added into the gelatin solutions (prepared as above) and
poured into moulds, respectively. The general procedure of sponge formation was followed.
These sponges were labelled as GS-bFGF.
Preparation of gelatin sponges with bFGF-loaded microspheres. Microspheres containing bFGF
were added into the gelatin solutions and the general procedure of sponge formation was
followed. These sponges were labelled as GS-GM-bFGF.
Preparation of bilayer wound dressing. The bilayer wound dressing was constructed at the
wound site, by initially applying the sponges and then covering with adhesive polyurethane
(Tendra�).
Characterization of microspheres and bilayer wound dressing
Morphological analysis. The morphology of microspheres (bFGF-unloaded or bFGF-
loaded microspheres) and prepared bilayer wound dressing were examined using a light
microscope (Olympus BX-41) and a scanning electron microscope (SEM, HITACHI
S-2700) after coating the samples with a thin layer of gold under vacuum.
Microstructure analysis. The average sizes and size distribution of microspheres were
measured by a particle size analyser (Leica), using acetone as the solvent. The sponge
microstructures were investigated by geometrical measurement on SEM. The porosity and
average diameter of pores were measured using an Image Analyser (Leica).
Release studies and bFGF analysis by ELISA
Microspheres containing bFGF (GM-bFGF) and gelatin sponges with bFGF (GS-bFGF)
were placed in phosphate buffer (pH¼ 7.4) and incubated on a rotating incubator at 37�C,
Wound dressings containing bFGF-impregnated microspheres 279
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respectively. The buffer was replaced daily and the amount of bFGF in releasing media was
determined by an ELISA. ELISA was performed according to the procedure of the kit
(Quantikine� Human FGF Basic Immunoassay; R&D Systems, Minneapolis, MN).
Bioassay for bFGF
The biological activity of the released bFGF was determined by testing its ability to stimulate
the proliferation of cultured neonatal rat skin fibroblasts. The cells were isolated,
purified and cultured on 2-D culture dishes. The fibroblasts were plated at a density
of 5000 cells cm�2 on tissue culture dishes and overnight incubated in phosphate buffer
(pH¼ 7.4) medium at 37�C in 5% CO2. After cell adhesion, the culture supernatant was
replaced by a fresh medium and 100ml of the releasing medium was added into each well.
After incubation for 96 h, the cells were detached with EDTA and the cell number was
counted using a haemocytometer.
In vivo studies
Animal model and surgical procedure. Ten 1-month-old male york pigs, weighing
�10–15 kg, were used for in vivo experiments. The animals were fed with a standard diet
and housed individually in the animal care facilities of the University.
The pigs were anaesthetized by intra-peritoneal injection of a mixture (1 : 1) of nembutal
and atropine sulphate (3.5mg/0.07ml/york pig). After shaving and depilation, four
full-thickness skin defects (diameter¼ 3.2 cm, area¼ 8.0 cm2) were made, preserving the
panniculus carnosus, on the back of each york pig. On each animal, vaseline gauze was
applied to the first wound area and it was kept as control. The second wound was covered
with a gelatin sponge (GS). The third and fourth wounds were covered with free-bFGF
containing sponges (GS-bFGF) and bFGF-loaded microsphere containing sponges (GS-
GM-bFGF), respectively. Then the wounds were completely covered with a commercially
available adhesive polyurethane (Tendra�). Finally, the total wound area was covered with
gauze containing fradiomycin sulphate and the edges of the gauze were sutured to the skin
with a 4-0 nylon monofilament.
Everyday, the pigs were checked and the length and width of the lesions were measured.
To evaluate the biocompatibility and efficiency of the systems, specimens encompassing the
whole area were removed under general anaesthesia on the 7th, 14th and 21st days after the
operation. Specimens were fixed in formaldehyde to be processed for histology.
Wound area measurements. Length and width of the wound regions were measured by using
a microruler that was sterilized prior to every measurement. Assuming that the lesion is in
the shape of an ellipse (due to skin anisotropy), the following equation for the area of an
ellipse was used as suggested by Baker and Haig (1981):
Wound area ðcm2Þ ¼ ½long axis ðcmÞ � short axis ðcmÞ � ��=4:
Statistical analysis. Data are expressed as means�SD. Analysis of variance (ANOVA)
followed by the t-test was used to determine the significant differences among the groups
(p-values less than 0.05 were considered significant).
Histological examination. Skin samples containing the whole wound area were removed
in the first, second and third weeks of post-operation and all specimens were immersed in
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10% buffer formalin. They were dehydrated in a graded series of ethanol and embedded
in paraffin. Five-to-seven micrometre thin sections were prepared and stained with
Haematoxylin and Eosin, Masson’s Trichrome and Gomori’s. Photomicrographs were
measured using an Olympus BH-7 light microscope.
Results and discussion
Characterization of microspheres
Morphological analysis results. Photomicrographs and SEM micrographs of microsphere
samples are given in Figures 1 and 2. The micrographs indicate that the microspheres did
not aggregate (Figure 1). From the SEM micrographs, the microspheres were quite
spherical. It is noticed that the samples of bFGF-unloaded had no cracks on the smooth
surfaces (Figure 2a), but loading of bFGF into microspheres caused roughness on the
surfaces (Figure 2b).
Particle size analysis of microspheres. It is possible to obtain different-sized microspheres by
changing the experimental conditions such as stirring speed, concentration of gelatin or by
addition of surface active materials to the reaction medium during preparation (Esposito
et al. 1996). In this study, microspheres prepared had mean diameters in the range
11.80–37.60 mm and the mean diameters of microsphere samples were 14.36� 3.56 mm.
Prior to in vivo applications, the microspheres were put through a sieve with an aperture of
16 mm to achieve uniformity.
Characterization of bilayer wound dressing
The SEM photographs of the bilayer wound dressing are shown in Figure 3. The gelatin
sponges show the porous and inter-connected network structures and they are characterized
Figure 1. Photomicrographs of microsphere samples. (� 40).
Wound dressings containing bFGF-impregnated microspheres 281
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by the porosity ranged between 40 and 70% with an average pore size of 80–160 mm. The
microspheres inside pores could be clearly seen in the sponge structure and did not interfere
with the pore structure of the sponge (Figure 3a). The full structure of a bilayer wound
dressing is composed of a thin polyurethane film (PU) over which the gelatin sponge (GS)
containing bFGF-loaded microspheres was attached. The polyurethane film did not affect
the porous structure of the gelatin sponges and the two layers adhered firmly to each other
(Figure 3b).
Kinetics of bFGF release and bioactivity
The release rate of bFGF from the microspheres (GM-bFGF) was fairly constant and the
cumulative release increased linearly with time, as judged by ELISA (Figure 4). By day 7 of
the release study, 92.9% of the impregnated bFGF was released to the external medium.
The release profile obtained from bFGF-containing microspheres incorporated into the
gelatin sponges was similar to that. In contrast, incorporating bFGF and heparin directly
(A)
(B)
Figure 2. SEM micrographs of microsphere samples. (� 2000) (a) The bFGF-unloaded sampleswere quite spherical and no crack on the smooth surfaces. (b) The loading of bFGF into microsphereswere spherical and roughness on the smooth surfaces.
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(A)
(B)
Figure 3. The SEM photographs of the gelatin sponges. (� 400) (a) The gelatin sponges show theporous and inter-connected network structures. The microspheres inside pores could be clearly seen.(arrow: GM). (b) The full structure of a bilayer wound dressing show that the polyurethane film (PU)did not affect the porous structure of the gelatin sponges and the two layers adhered firmly to eachother (arrow: PU).
00.5
11.5
22.5
33.5
44.5
5
1 2 3 4 5 6 7Times (Days)
Cum
ulat
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rele
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of b
FG
F (
mg)
GM-bFGFGS-bFGF
Figure 4. Kinetics of bFGF release judged by ELISA. The cumulative release from microspheresincreased linearly with time.
Wound dressings containing bFGF-impregnated microspheres 283
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into the gelatin sponges (GS-bFGF) resulted in a rapid release after an initial burst of �70%
from the sponges and 91.3% of the impregnated bFGF was released by day 2 of the study.
The released bFGF was further assessed for its ability to stimulate fibroblasts proliferation
in vitro. The results presented in Figure 5 reveal that the releasing media collected from
bFGF containing matrices during the 7-day study stimulated the proliferation of fibroblasts
in vitro. The highest proliferation response was obtained when the cells were exposed to the
cumulative releasing media from days 3–5 of the experiment. This result is in accordance
with the ELISA results, which revealed the highest amount of released bFGF at middle days.
However, the highest proliferation response of media collected from GS-bFGF was obtained
at initial days (days 1–3). Comparison of the effective concentration of bFGF from the
proliferation assay with the amount released by ELISA shows that the released bFGF from
the microspheres (GM-bFGF) is more than 90% active at all times.
In vivo studies
The prepared dressings were applied on the full thickness skin defects created on the dorsal
regions of york pigs.
Macroscopic observations. One week after the operation, thick, depressed haemorrhagic
crusts existed on almost all these wounds with white-gray coloured centres. However, in
lesion 1, the wound areas were more depressed and covered with haemorrhagic crust with
a dark centre. Two weeks after the operation, the skin defects and crusts were partially
observed for lesions 1 and 2 in all animals. There was a thin haemorrhagic crust with a
thicker and dark-coloured centre in lesion 3. At lesion 4, skin defect and crust were hardly
observed and the periphery of the wounds were well vascularized. Three weeks after the
operation, the majority of skin defects seemed almost re-epithelialized. Macroscopically,
however, each case of groups are somewhat different in healing stage. For the control
wounds (lesion 1), haemorrhagic crusts hadn’t disappeared completely from all the lesions.
Compared with the control group, the decrease of wound area of lesions 2 and 3 were
remarkable. In lesion 4, the wound regions were covered with epidermis, the defect had
1.25±0.12
1.66±0.22
1.27±0.14
0.41±0.32
0.62±0.16
1.45±0.14
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Day 1-3 Day 3-5 Day 5-7
Rel
ativ
e ce
ll n
um
ber
(×1
0000
/cm
2 )
GM-bFGF
GS-bFGF
Figure 5. Bioactivity of the released bFGF. The bioactivity was determined using fibroblastproliferation assay. Y-axis is the relative cell proliferation resulting from the addition of bFGF releasedfrom the microspheres and the sponges at days 1–3, 3–5 and 5–7, as compared with controls. Valuesrepresent mean and standard deviation (n¼ 3). The difference between the two groups for each timeis significant (analysis of variance, p<0.05).
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disappeared and the wound area had closed. In this study, no infection was observed for any
of the animals throughout the experiments.
The area of the wounds originally produced was 8.0 cm2. At the 7th, 14th and 21st post-
operative day, the wound areas were calculated from the measured values and the average
values are given in Figure 6. And the decreased percentages in the wound areas of each
group were shown in Table I. In summary, there was a progressive decrease in the wound
areas with time. In this regard, the overall score of healing competency could be valued in
the order of GS-GM-bFGF, GS-bFGF, GS and Vaseline gauze. The difference between
GS-bFGF and GS-GM-bFGF was significant. However, the difference was not distinct
between GS and Vaseline gauze. Furthermore, the final difference of curative effect between
GS and GS-bFGF was not considered significant.
Histological findings. Seven days after the operation, the epithelium on the wound region
was missing in the control group. In other examined groups, the wound regions were
covered by thick crusts and epidermal parts were missing under the crust. Fourteen days
after the operation, the crust had disappeared and some of the wound were covered with a
Figure 6. Change in wound area of lesions treated with various dressings 1, 2, 3 weeks afteroperations. Lesion 1(vaseline gauze); Lesion 2(GS); Lesion 3(GS-bFGF); Lesion 4(GS-GM-bFGF).The healing competency could be valued in the order of GS-GM-bFGF, GS-bFGF, GS and Vaselinegauze.
Table I. The decrease percentages in the wound areas of each groups (%).
Post-operation
Groups 1 week 2 week 3 week
Control group 65� 0.43 70� 0.38 74� 0.38
GS group 69� 0.42 72� 0.35 80� 0.32
GS-bFGF group 72� 0.19* 76� 0.30* 82� 0.21*
GS-GM-bFGF group 78� 0.21* 89� 0.19* 95� 0.17*
Values are expressed as mean�SD for 10 animals.*Compared with control groups, the decrease of areas of wounds on which GS-bFGF and GS-GM-bFGF appliedwas higher and the difference between them was considered significant, p<0.05.
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continuous epidermis. In the GS and GS-bFGF applied wounds, the stratified epithelium
that formed the epidermis was much thicker than that of the normal skin and the wound
regions were filled with the connective tissue of the dermis. The junctions of the normal
dermis and the dermis in the wound regions were very prominent in all groups examined.
In the GS-GM-bFGF applied wounds, epithelization was mostly complete and appeared to
be thick at the periphery of the wound region. Thick, coarse collagen fibres of the normal
dermis continued horizontally with the newly formed thin collagen fibres. Twenty-one days
(A)
(B)
Figure 7. Histological findings after 21 days post operation. (A) Gomori’s (� 10), (D) HE (� 4):Wound region of the control groups after three weeks. The surface of the wound is covered with a thinepidermis. Dermis consists of thin collagen fibers. (B) Gomori’s (� 10) (E) HE (� 4); Wound regionof the GS-bFGF group after three weeks. The epidermis and dermis appear to be almost the same asthe normal skin except the collagen fibers are parallel to the surface and less organized. (C) Masson’sTrichrome (� 10) (F) Gomori’s (� 10): Wound region of the GS-GM-bFGF group after three weeks.Epithelization is completed. Dermis is infiltrated with collagen fibers in a mesh structure.
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after the operation, the structure of the epidermis in the experimental groups became almost
the same as that of the normal in appearance and thickness. In the control group, the
junctions of the normal and newly formed dermis were still prominent though not so distinct
as the 14th day groups (Figure 7a). In the GS and GS-bFGF applied group, the junction was
less well demarcated, with some horizontal fibres extending between the two regions
(Figure 7b). In the GS-GM-bFGF group, the wound had healed completely and was
covered with a newly formed dermis. The collagen fibres of the two regions continued and
intermingled gently with each other (Figure 7c).
Conclusion
The aim of this work was to prepare a bilayer wound dressing containing bFGF delivery
system as a novel covering in healing skin defects. The compatibilities of the dressings were
designed and tested with a series of preliminary in vitro protein release and bioactivity
(C)
(D)
Figure 7. Continued.
Wound dressings containing bFGF-impregnated microspheres 287
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(E)
(F)
Figure 7. Continued.
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experiments and in vivo experiments by applying them on full thickness skin defects created
on york pigs.
As a result, microspheres can serve as delivery vehicles for controlled release of bFGF and
released bFGF can enhance fibroblasts proliferation in vitro. The well-formed porous
structure of gelatin allowing growth of fibroblasts and capillaries into the matrix, with good
affinity, adhesiveness and non-irritability for tissues, make it used as the inner layer material
of an ideal wound dressing. Furthermore, the materials of the outer layer enhanced the
tensile strength of the wound dressing. Besides the repair time of epidermis was the shortest
in all groups, the wound cavity was filled with a dermis that appeared to have a structure
resembling the normal dermis, without any scar tissue formation in the GS-GM-bFGF
applied lesions.
In this study, the GS-GM-bFGF group was found to show the best properties as a wound
dressing from all the macroscopic observations and histological findings. The other
properties of these materials are as follows: the preparation process is relatively simple, there
are no toxic substances among the constituents, they have a high body fluid absorption
capacity, the release rate of growth factors from them are controllable and they are very soft
and therefore cause no disturbances during the application.
Acknowledgements
This work was supported by the Hi-tech Research and Development Program of China
(863 Program: 2002AA205041, 2005AA205241).
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