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Journal of Microencapsulation, May 2006; 23(3): 277–290 Wound dressings containing bFGF-impregnated microspheres SHA HUANG 1 , TIANZHENG DENG 1 , HONG WU 2 , FAMING CHEN 1 , & YAN JIN 1 1 Department of Oral Histology and Pathology, College of Stomatology and 2 Department 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) Abstract The primary objective was to synthesize a novel wound dressing containing basic fibroblast growth factor (bFGF)-loaded microspheres for promoting healing and tissue regeneration. Gelatin sponge was chosen as the underlying layer and elastomeric polyurethane membranes were used as the external layer. To achieve prolonged release, bFGF addition was loaded in microspheres. The microspheres were characterized for particle size, in vitro protein release and bioactivity. The bilayer dressings were tested in in vivo experiments on full-thickness skin defects created on pigs. Average size of the microspheres was 14.36 3.56 mm and the network sponges were characterized with an average pore size of 80–160 mm. Both the in vitro release efficiency and the protein bioactivity revealed that bFGF was released in a controlled manner and it was biologically active as assessed by its ability to induce the proliferation of fibroblasts. It was observed that sustained release of bFGF provided a higher degree of reduction in the wound areas. Histological investigations showed that the dressings were biocompatible and did not cause any mononuclear cell infiltration or foreign body reaction. The structure of the newly formed dermis was almost the same as that of the normal skin. The application of these novel bilayer wound dressings provided an optimum healing milieu for the proliferating cells and 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 ß 2006 Taylor & Francis DOI: 10.1080/02652040500435170 Journal of Microencapsulation Downloaded from informahealthcare.com by University of Connecticut on 10/29/14 For personal use only.

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Page 1: Wound dressings containing bFGF-impregnated microspheres

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

278 S. Huang et al.

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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

280 S. Huang et al.

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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.

282 S. Huang et al.

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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

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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

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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).

284 S. Huang et al.

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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.

Wound dressings containing bFGF-impregnated microspheres 285

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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)

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Figure 7. Continued.

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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).

References

Azrin M. 2001. Angiogenesis, protein and gene delivery. British Medicine Bulletin 59:211–225.

Baker PG, Haig G. 1981. Metronidazole in the treatment of chronic pressure sores and ulcers: A comparison with

standart treatments in general practice. Practitioner 225:569–573.

Choi YS, Hong SR, Lee YM, Song KW, Park MH, Nam YS. 1999. Study on gelatin-containing artificial skin:

I. Preparation and characteristics of novel gelatin-alginate sponge. Biomaterials 20(5):409–417.

Cortesi R, Esposito E, Osti M. 1999. Dextran cross-linked gelatin microspheres as a drug delivery system.

European Journal of Pharmaceutics & Biopharmaceutics 47(2):153–160.

Esposito E, Cortesi R, Nastruzzi C. 1996. Gelatin microspheres: Influence of preparation parameters and thermal

treatment on chemico-physical and biopharmaceutical properties. Biomaterials 17(20):2009–2020.

Kawai K, Suzuki S, Tabata Y. 2000. Accelerated tissue regeneration through incorporation of basic fibroblast

growth factor-impregnated gelatin microspheres into artificial dermis. Biomaterials 21(5):489–499.

Lu WW, Ip WY, Jing WM, Holmes AD, Chow SP. 2000. Biomechanical properties of thin skin flap after basic

fibroblast growth factor (bFGF) administration. British Journal of Plastic Surgery 53(3):225–229.

Lynch SE, Colvin RB, Antoniades HN. 1989. Growth factors in wound healing – single and synergistic effects on

partial thickness porcine skin wounds. Clinical Investigations 84:640–646.

Matsuda K, Suzuki S, Isshiki N, Yoshioka K, Wada R, Hyon SH, Ikada Y. 1981. Evaluation of a bilayer artifical

skin capable of sustained release of an antibiotic. Biomaterials 3(2):119–122.

Miyoshi M, Kawazoe T, Igawa HH, Tabata Y, Ikada Y, Suzuki SJ. 2005. Effects of bFGF incorporated into

a gelatin sheet on wound healing. Biomaterial Science: Polymer Edition 16(7):893–907.

Nastruzzi C, Pastesini C, Cortesi R, Esposito E, Gambari R, Menegatti E. 1994. Production and in vitro evaluation

of gelatin microspheres containing an antitumor tetra-amidine. Journal of Microencapsulation 11(3):249–260.

Nomi M, Atala A, Coppi PD. 2002. Principals of neovascularization for tissue engineering. Molecular Aspects of

Medicine 23(6):463–483.

Shibata H, Shioya N, Kuroyanagi Y. 1997. Development of new wound dressing composed of spongy collagen

sheet containing dibutyrylcyclic AMP. Biomaterial Science: Polymer Edition 8(8):601–621.

Wound dressings containing bFGF-impregnated microspheres 289

Jour

nal o

f M

icro

enca

psul

atio

n D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Con

nect

icut

on

10/2

9/14

For

pers

onal

use

onl

y.

Page 14: Wound dressings containing bFGF-impregnated microspheres

Ulubayram K, Hasirci N. 1992. Polyurethanes: Effect of chemical composition on mechanical properties and

oxygen permeability. Polymer 33(10):2084–2088.

Yannas IV, Burke JF. 1980. Design of an artificial skin. I. Basic design principles. Biomedical Materials Research

14:65–81.

Young S, Wong M, Tabata Y, Mikos AG. 2005. Gelatin as a delivery vehicle for the controlled release of bioactive

molecules. Journal of Control Release 109(1–3):256–274.

290 S. Huang et al.

Jour

nal o

f M

icro

enca

psul

atio

n D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Con

nect

icut

on

10/2

9/14

For

pers

onal

use

onl

y.