Br. J. Surg. 1992, Vol. 79, September, 918-921
J. Slavin, J. A. Hunt*, J. R . Nasht, D. F. Williams* and A. N. Kingsnorth Departments of Surgery and tPathology and *Institute of Clinical Bioengineering, University of Liverpool, Liverpool, UK Correspondence to: Mr A. N. Kingsnorth, Department of Surgery, University of Liverpool, PO Box 147, Liverpool L69 3BX, UK
Recombinant basic fibroblast growth factor in red blood cell ghosts accelerates incisional wound healing The pharmacological manipulation of wound healing with locally applied growth factors is now a practical possibility. The effect of topical applications of recombinant basic jibroblast growth factor (bFGF) on the strength and cellularity of healing incisional rat skin wounds was investigated. Applications of bFGF in a simple vector (either a collagen suspension or saline) were not associated with any positive efSects on wound breaking load at 7 days after injury in comparison with vector-treated control wounds; at the highest dose of 50 p g per wound, breaking loads were signif;cantl-y decreased from a mean( s.e.m.) of 287(22) g / c m 2 in controls to 201(23) g / c m 2 ( P < 0.00.5). Increasing doses of appliedpeptide were paralleled by increasing wound cellularity. Delay of bFGF release at the site of application was achieved by encapsulation into red blood cell ghosts. Wounds treated with bFGF in such ghosts were 50 per cent stronger than paired control wounds (388(27)versus 256(28) g / c m 2 ,P < 0.002) 7days after injury. Treated wounds were sign2ficantly more cellular at 4 days than paired control wounds. Topical applications of bFGF applied at the time of injury exert a positive efSect on incisional wound strength only when a vector that delays release is used.
Over the past three decades a number of peptide growth factors present in serum or tissue extracts that support the growth of cells in culture have been identified. The response to injury is characterized by rapid cellular migration, division and differentiation; modulators of such processes would be expected to play a critical role. Recombinant biotechnology allows the production of specific peptides on a large scale, permitting examination of their pharmacological effects in uiuo and their clinical use as modulators of wound healing. An incisional skin wound healing primarily by first intention is representative of a typical surgical wound. The rate of healing in such a model has traditionally been assessed by the measurement of wound breaking strength, which rapidly increases in a well defined profile as granulation tissue containing collagen is deposited. Subsequent reorganization of collagen leads to a further increase in strength. Wound strength is therefore an indirect but objective measure of the healing process'. Local applications of transforming growth factor beta (TGF-P)2 and platelet-derived growth factor ( PDGF)3 in collagen suspensions accelerate the gain in strength of incisional wounds. A single application of epidermal growth factor4 has no positive effects on wound strength but, when release at the site of application is delayed by incorporation into liposomes, positive effects on wound strength between 7 and 14 days are seen. Applications of either P D G F or TGF-fi in a collagen suspension can be shown to reverse the deleterious effects of steroid therapy5, radiotherapy6 and cytotoxics' on the development of incisional wound strength. Basic fibroblast growth factor ( b F G F ) is a protein of 17000 Da molecular weight found throughout the body'. It is a potent angiogenic factor in viuog and in uitro,and is mitogenic and chemotactic for both fibroblasts and endothelial cells". New blood vessel growth and fibroblast proliferation are integral elements in the formation of granulation tissue. Injection of a blocking antibody to b F G F into a wound chamber impairs deposition of granulation tissue' When
incorporated in a gel within the centre of a polytetrafluoroethylene tube implanted subcutaneously in mice, b F G F increases cellular infiltration". Recombinant b F G F infiltrated into healing incisional wounds on the third day after operation has been shown to accelerate the gain in strength by about 35 per cent between the fifth and seventh days13. The present study further evaluates the effect of local applications of recombinant human b F G F applied at the time of wounding in an incisional rat skin wound model. Healing was assessed by the measurement of wound breaking load, together with the analysis of histological sections using a computerized image analysis system to provide quantitative measurements of wound cellularity .
918
0007-1 323/92/090918-04 Q 1992 Butterworth-Heinemann Ltd
Materials and methods Sprague-Dawley rats weighing between 350 and 500 g were used. They were caged in groups of up t o four, fed standard rat chow, and maintained on a constant day-night cycle. Recombinant b F G F was a gift from Chiron, Emeryville, California, USA. Activity of growth factor in ilitro was ensured using a simple mitogenic assay using Swiss 3T3 fibroblasts. Mitogenicity was assessed by the uptake of tritiated thymidine. Growth factor was applied to wounds in a number of vectors: 0.15 mol/l saline, a dilute collagen suspension (Zyderm 11; Collagen Corporation, Palo Alto, California, USA) or red blood cell (RBC) ghosts. Wound niodel Six to eight rats were used for each variable. All experiments were conducted in a blinded manner after growth factor preparation. Under anaesthesia, each rat received bilateral dorsal longitudinal paired incisions 6.0 cm in length and 1.5 cm from the midline. One incision was treated with growth factor in a particular vector; the other acted as a coptrol and received vector alone. Incisions were treated and closed individually to prevent cross-contamination. Wounds were closed with five interrupted clips (Proximate; Ethicon, Edinburgh, UK ). After the animals were killed, the pelts were removed. Clips were removed and, using a standard cutting instrument, paired 1-cm strips cut from both
Basic fibroblast growth factor in wound healing: J. Slavin et al. wounds simultaneously. Typically three sets of paired strips per animal were taken for tensiometric analysis. Paired wound biopsies were also obtained for subsequent histological studies. For tensiometric analysis, each wound strip was mounted between pneumatic clamps on an Instron (High Wycombe, U K ) tensiometer. Each specimen was tested to disruption at a constant strain of 5 cm/min. A load cell was connected to a chart recorder and for each specimen a load-deformation curve produced from which the breaking load of each specimen was calculated. Histology Paired wound strips were fixed in 10 per cent formal saline for 48 h and processed together before embedding in paraffin wax. Specimens were orientated so that comparable transverse sections of a wound were obtained. Sections 4 pm thick were cut from each block using a microtome and stained with haematoxylin and eosin, a Van Gieson stain, or haematoxylin alone. Computerized image analysis of haematoxylin-stained nuclei in individual sections was used to produce a quantitative comparable measure of wound cellularity. Haematoxylinstained sections were examined with a Jeneval microscope (Carl Zeiss, Borehamwood, UK). A Hitachi KP140 solid-state camera (Joyce Loebl, Gateshead, U K ) produced a black-and-white image of a section, which was fed into the video interface of a Joyce Loebl Mini Magiscan image analysis system. Routines were generated using general-purpose image analysis software that enabled the automatic counting of the number of nuclei within a defined space. The field analysed consisted ofa rectangular area of 10 x 2 high-power fields ( H P F s ) (magnification x 200). The wound centre on an individual section was identified by examining both haematoxylin-eosin and Van Gieson stained sections. The rectangular counting area was orientated so that one long side corresponded to the wound centre and one short side was orientated along the outermost layer of epidermis. As the thickness of specimens was slightly greater than ten HPFs, all the nuclei within two HPFs of the wound centre were counted. Results are expressed as cells per 20 HPFs.
Results Seven animals were killed early, two because of haematoma formation and five because of self-induced wound disruption occurring in the first 24 h after injury. Applied in either saline or a collagen suspension to wounds at doses of between 0.5 and 50pg, b F G F did not increase mean breaking loads at 7 days in comparison with paired control incisions (Figure 1). At the highest dose used (50 pg b F G F in saline per wound), a significant decrease in breaking load was seen at 7 days (201(23) versus 287(22) g/cm2, P < 0.005). Wounds treated with 500 ng b F G F in collagen were less cellular than control wounds (Figure 2). As the dose of b F G F was increased, treated wounds became increasingly cellular in comparison with control ones (Figure 2 ) ; areas of highly vascularized granulation tissue were visible, sometimes disrupting the incision line. Cell types visible in wounds after 7 days were predominantly macrophages and fibroblasts. Analysis of cellular distribution within individual wounds treated with b F G F revealed that, although cellularity in the centre of the wound was comparable, higher cellular counts were seen further from the wound centre in wounds treated with growth factor compared with controls. The mean breaking load at 7 days of wounds treated with TGF-P ( 2 pg) in a collagen suspension was approximately 50 per cent greater than that of collagen-treated controls ( P < 0.01) (Figure 3 ) , a result in agreement with previous publication^^.^. No differences in cellularity were apparent between wounds treated with TGF-P and control wounds at 7 days (2440(244) versus 3308(450) cells per 20 HPFs).
-
*
N
-5-.. 300
Preparation of red blood cell ghosts RBC ghosts have been used by investigators in the past to delay release of drugs or proteins at a site of application in u i ~ o ' RBC ~ . ghosts were prepared according to a previously described method15. The Iodogen method (Pierce Chemicals, Chester, U K ) was used to iodinate bFGF. Standard pore size dialysis tubing (molecular weight cut-off 12000 D a ) was used throughout. Preliminary experiments indicated that retention of iodinated peptide separated from unreacted label by dialysis membrane was 92 per cent at 3 h and 60 per cent at 1 week. A high-efficiency dialysis entrapment method was used. Briefly, RBCs were washed several times in 0.15 mol/l saline and then packed. The haematocrit of the packed cell was accurately determined. Packed RBCs were placed in a dialysis bag with the volume of saline containing peptide theoretically necessary to shift the haematocrit to 80 per cent. The bag was sealed and placed in a hypo-osmolar lysis solution for 2 h at 4°C. It was then transferred to a sealing solution of 0.15 mol/l saline at room temperature for a further 30min. At all stages small amounts of '251-radiolabelled b F G F were included to assess uptake. Sealed blood cells were washed twice and the distribution of radioactivity between washings and cells used as an estimate of uptake. The mean(s.d.) uptake of b F G F from six experiments was 5 6 ( 6 ) per cent. RBC ghosts were used within 3 h of preparation. Stored at 4"C, 42 per cent of iodinated growth factor still remained within RBC ghosts at 2 weeks.
m
v
x
-0 200 m
.x
z
m
100
0
Dose 500ng Vector Collaaen
5 ug Col laaen u
5 ug Saline
50 ug Saline ~.
(n=i5)
(n=18)
(n=18)
(n=17)
Figure 1 Mean(s.e.m.) breaking load at 7 days of wounds treated with basicjibroblast growth factor in simple delivery vectors. 0, Control; 0, treated; n, number of paired wound strips. *P < 0,005 ( Wilcoxon signed rank test)
i 1 1 i
t
......
Experimental design The effects of b F G F on wound breaking load were initially examined at a number of different doses and in either collagen or saline vector at 7 days. As a positive control, the effect of TGF-/3 (2 pg in a collagen suspension) on wound breaking load at 7 days was examined. As no positive effects on breaking load were seen with b F G F delivered in either collagen or saline vectors, delaying release of b F G F at the site of application by incorporation into resealed RBC ghosts was then examined. This was done initially at 7 days and then at 4,12 and 21 days. Statistics All statistical comparisons were between paired wound strips; values are expressed as mean(s.e.m. ). Statistical analysis used the two-tailed Wilcoxon signed rank test. Statistical and probability values were calculated on a personal computer using a software package (Arcus Professional 1.4, Aughton, Lancs, U K ) .
Br. J. Surg., Vol. 79, No. 9, September 1992
......
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..i.:
.:.:.: ..::.:..:.: :.I:.:
.:.:.:.
....... :.:.:.
......
Dose 500ns Vector Collagen (n=6)
5ua
....... .... .:.:.:. .... .... .:.:.:. .... ....... .... ......: .:.:.:.
... ... ... ...... ... ... ...
.:.:_:.
.... ....... .... ... .:.:.:. ....
::j:: j::::
.......
...
Saline
Collagen
5Oug Saline
5ug RBC g h o s t s
(n=6)
(n=8)
(n=6)
(n=7)
5ug
Figure 2 Mean(s.e.m.) cellularity of wounds treated with basic fibroblast growth factor expressed as a percentage of cellularity ofpaired Control; 0, treated; n, number ofpaired wound control wounds. 0, biopsies; RBC, red blood cell. * P < 0.05: t P < 0.01 ( Wilcoxon signed rank test)
919
Basic fibroblast growth factor in wound healing: J. Slavin et al. 500
”
Dose Vector
1
*
Discussion t
2 ug TCF-B
5 ug bFGF RBC ghosts
Collagen (n = 20) Figure 3 Mean(s.e.m.) breaking load at 7 days of wounds treated with basic fibroblast growth,factor (bFGF) in red blood cell ( R B C ) ghosts or transforming growih factor beta ( T G F - p ) in a collagen suspension. 0, Control; 0 , treated; n, number of paired wound strips. * P < 0402; t P < 0.01 ( Wilcoxon signed rank trst) (n=24)
Table 1 Effect o f 5 p g per wound basic fibroblast growth factor in red bhod cell ghosts on wound breaking load ut dizerent times
Wound breaking load (g/cm’)
Control
Treated n P*
Day 7
Day 12
Day 21
256(28) 388(27) 24
J. Slavin, J. A. Hunt*, J. R . Nasht, D. F. Williams* and A. N. Kingsnorth Departments of Surgery and tPathology and *Institute of Clinical Bioengineering, University of Liverpool, Liverpool, UK Correspondence to: Mr A. N. Kingsnorth, Department of Surgery, University of Liverpool, PO Box 147, Liverpool L69 3BX, UK
Recombinant basic fibroblast growth factor in red blood cell ghosts accelerates incisional wound healing The pharmacological manipulation of wound healing with locally applied growth factors is now a practical possibility. The effect of topical applications of recombinant basic jibroblast growth factor (bFGF) on the strength and cellularity of healing incisional rat skin wounds was investigated. Applications of bFGF in a simple vector (either a collagen suspension or saline) were not associated with any positive efSects on wound breaking load at 7 days after injury in comparison with vector-treated control wounds; at the highest dose of 50 p g per wound, breaking loads were signif;cantl-y decreased from a mean( s.e.m.) of 287(22) g / c m 2 in controls to 201(23) g / c m 2 ( P < 0.00.5). Increasing doses of appliedpeptide were paralleled by increasing wound cellularity. Delay of bFGF release at the site of application was achieved by encapsulation into red blood cell ghosts. Wounds treated with bFGF in such ghosts were 50 per cent stronger than paired control wounds (388(27)versus 256(28) g / c m 2 ,P < 0.002) 7days after injury. Treated wounds were sign2ficantly more cellular at 4 days than paired control wounds. Topical applications of bFGF applied at the time of injury exert a positive efSect on incisional wound strength only when a vector that delays release is used.
Over the past three decades a number of peptide growth factors present in serum or tissue extracts that support the growth of cells in culture have been identified. The response to injury is characterized by rapid cellular migration, division and differentiation; modulators of such processes would be expected to play a critical role. Recombinant biotechnology allows the production of specific peptides on a large scale, permitting examination of their pharmacological effects in uiuo and their clinical use as modulators of wound healing. An incisional skin wound healing primarily by first intention is representative of a typical surgical wound. The rate of healing in such a model has traditionally been assessed by the measurement of wound breaking strength, which rapidly increases in a well defined profile as granulation tissue containing collagen is deposited. Subsequent reorganization of collagen leads to a further increase in strength. Wound strength is therefore an indirect but objective measure of the healing process'. Local applications of transforming growth factor beta (TGF-P)2 and platelet-derived growth factor ( PDGF)3 in collagen suspensions accelerate the gain in strength of incisional wounds. A single application of epidermal growth factor4 has no positive effects on wound strength but, when release at the site of application is delayed by incorporation into liposomes, positive effects on wound strength between 7 and 14 days are seen. Applications of either P D G F or TGF-fi in a collagen suspension can be shown to reverse the deleterious effects of steroid therapy5, radiotherapy6 and cytotoxics' on the development of incisional wound strength. Basic fibroblast growth factor ( b F G F ) is a protein of 17000 Da molecular weight found throughout the body'. It is a potent angiogenic factor in viuog and in uitro,and is mitogenic and chemotactic for both fibroblasts and endothelial cells". New blood vessel growth and fibroblast proliferation are integral elements in the formation of granulation tissue. Injection of a blocking antibody to b F G F into a wound chamber impairs deposition of granulation tissue' When
incorporated in a gel within the centre of a polytetrafluoroethylene tube implanted subcutaneously in mice, b F G F increases cellular infiltration". Recombinant b F G F infiltrated into healing incisional wounds on the third day after operation has been shown to accelerate the gain in strength by about 35 per cent between the fifth and seventh days13. The present study further evaluates the effect of local applications of recombinant human b F G F applied at the time of wounding in an incisional rat skin wound model. Healing was assessed by the measurement of wound breaking load, together with the analysis of histological sections using a computerized image analysis system to provide quantitative measurements of wound cellularity .
918
0007-1 323/92/090918-04 Q 1992 Butterworth-Heinemann Ltd
Materials and methods Sprague-Dawley rats weighing between 350 and 500 g were used. They were caged in groups of up t o four, fed standard rat chow, and maintained on a constant day-night cycle. Recombinant b F G F was a gift from Chiron, Emeryville, California, USA. Activity of growth factor in ilitro was ensured using a simple mitogenic assay using Swiss 3T3 fibroblasts. Mitogenicity was assessed by the uptake of tritiated thymidine. Growth factor was applied to wounds in a number of vectors: 0.15 mol/l saline, a dilute collagen suspension (Zyderm 11; Collagen Corporation, Palo Alto, California, USA) or red blood cell (RBC) ghosts. Wound niodel Six to eight rats were used for each variable. All experiments were conducted in a blinded manner after growth factor preparation. Under anaesthesia, each rat received bilateral dorsal longitudinal paired incisions 6.0 cm in length and 1.5 cm from the midline. One incision was treated with growth factor in a particular vector; the other acted as a coptrol and received vector alone. Incisions were treated and closed individually to prevent cross-contamination. Wounds were closed with five interrupted clips (Proximate; Ethicon, Edinburgh, UK ). After the animals were killed, the pelts were removed. Clips were removed and, using a standard cutting instrument, paired 1-cm strips cut from both
Basic fibroblast growth factor in wound healing: J. Slavin et al. wounds simultaneously. Typically three sets of paired strips per animal were taken for tensiometric analysis. Paired wound biopsies were also obtained for subsequent histological studies. For tensiometric analysis, each wound strip was mounted between pneumatic clamps on an Instron (High Wycombe, U K ) tensiometer. Each specimen was tested to disruption at a constant strain of 5 cm/min. A load cell was connected to a chart recorder and for each specimen a load-deformation curve produced from which the breaking load of each specimen was calculated. Histology Paired wound strips were fixed in 10 per cent formal saline for 48 h and processed together before embedding in paraffin wax. Specimens were orientated so that comparable transverse sections of a wound were obtained. Sections 4 pm thick were cut from each block using a microtome and stained with haematoxylin and eosin, a Van Gieson stain, or haematoxylin alone. Computerized image analysis of haematoxylin-stained nuclei in individual sections was used to produce a quantitative comparable measure of wound cellularity. Haematoxylinstained sections were examined with a Jeneval microscope (Carl Zeiss, Borehamwood, UK). A Hitachi KP140 solid-state camera (Joyce Loebl, Gateshead, U K ) produced a black-and-white image of a section, which was fed into the video interface of a Joyce Loebl Mini Magiscan image analysis system. Routines were generated using general-purpose image analysis software that enabled the automatic counting of the number of nuclei within a defined space. The field analysed consisted ofa rectangular area of 10 x 2 high-power fields ( H P F s ) (magnification x 200). The wound centre on an individual section was identified by examining both haematoxylin-eosin and Van Gieson stained sections. The rectangular counting area was orientated so that one long side corresponded to the wound centre and one short side was orientated along the outermost layer of epidermis. As the thickness of specimens was slightly greater than ten HPFs, all the nuclei within two HPFs of the wound centre were counted. Results are expressed as cells per 20 HPFs.
Results Seven animals were killed early, two because of haematoma formation and five because of self-induced wound disruption occurring in the first 24 h after injury. Applied in either saline or a collagen suspension to wounds at doses of between 0.5 and 50pg, b F G F did not increase mean breaking loads at 7 days in comparison with paired control incisions (Figure 1). At the highest dose used (50 pg b F G F in saline per wound), a significant decrease in breaking load was seen at 7 days (201(23) versus 287(22) g/cm2, P < 0.005). Wounds treated with 500 ng b F G F in collagen were less cellular than control wounds (Figure 2). As the dose of b F G F was increased, treated wounds became increasingly cellular in comparison with control ones (Figure 2 ) ; areas of highly vascularized granulation tissue were visible, sometimes disrupting the incision line. Cell types visible in wounds after 7 days were predominantly macrophages and fibroblasts. Analysis of cellular distribution within individual wounds treated with b F G F revealed that, although cellularity in the centre of the wound was comparable, higher cellular counts were seen further from the wound centre in wounds treated with growth factor compared with controls. The mean breaking load at 7 days of wounds treated with TGF-P ( 2 pg) in a collagen suspension was approximately 50 per cent greater than that of collagen-treated controls ( P < 0.01) (Figure 3 ) , a result in agreement with previous publication^^.^. No differences in cellularity were apparent between wounds treated with TGF-P and control wounds at 7 days (2440(244) versus 3308(450) cells per 20 HPFs).
-
*
N
-5-.. 300
Preparation of red blood cell ghosts RBC ghosts have been used by investigators in the past to delay release of drugs or proteins at a site of application in u i ~ o ' RBC ~ . ghosts were prepared according to a previously described method15. The Iodogen method (Pierce Chemicals, Chester, U K ) was used to iodinate bFGF. Standard pore size dialysis tubing (molecular weight cut-off 12000 D a ) was used throughout. Preliminary experiments indicated that retention of iodinated peptide separated from unreacted label by dialysis membrane was 92 per cent at 3 h and 60 per cent at 1 week. A high-efficiency dialysis entrapment method was used. Briefly, RBCs were washed several times in 0.15 mol/l saline and then packed. The haematocrit of the packed cell was accurately determined. Packed RBCs were placed in a dialysis bag with the volume of saline containing peptide theoretically necessary to shift the haematocrit to 80 per cent. The bag was sealed and placed in a hypo-osmolar lysis solution for 2 h at 4°C. It was then transferred to a sealing solution of 0.15 mol/l saline at room temperature for a further 30min. At all stages small amounts of '251-radiolabelled b F G F were included to assess uptake. Sealed blood cells were washed twice and the distribution of radioactivity between washings and cells used as an estimate of uptake. The mean(s.d.) uptake of b F G F from six experiments was 5 6 ( 6 ) per cent. RBC ghosts were used within 3 h of preparation. Stored at 4"C, 42 per cent of iodinated growth factor still remained within RBC ghosts at 2 weeks.
m
v
x
-0 200 m
.x
z
m
100
0
Dose 500ng Vector Collaaen
5 ug Col laaen u
5 ug Saline
50 ug Saline ~.
(n=i5)
(n=18)
(n=18)
(n=17)
Figure 1 Mean(s.e.m.) breaking load at 7 days of wounds treated with basicjibroblast growth factor in simple delivery vectors. 0, Control; 0, treated; n, number of paired wound strips. *P < 0,005 ( Wilcoxon signed rank test)
i 1 1 i
t
......
Experimental design The effects of b F G F on wound breaking load were initially examined at a number of different doses and in either collagen or saline vector at 7 days. As a positive control, the effect of TGF-/3 (2 pg in a collagen suspension) on wound breaking load at 7 days was examined. As no positive effects on breaking load were seen with b F G F delivered in either collagen or saline vectors, delaying release of b F G F at the site of application by incorporation into resealed RBC ghosts was then examined. This was done initially at 7 days and then at 4,12 and 21 days. Statistics All statistical comparisons were between paired wound strips; values are expressed as mean(s.e.m. ). Statistical analysis used the two-tailed Wilcoxon signed rank test. Statistical and probability values were calculated on a personal computer using a software package (Arcus Professional 1.4, Aughton, Lancs, U K ) .
Br. J. Surg., Vol. 79, No. 9, September 1992
......
5::::: ...... ...
.:.:.:. .:.:.>
....... .. ..... .::..:.:.: .::..:> : .:.:.:. :.y;; '0:. .:.:.. ..... ...... :.:.:. ...... ... .:.:.: ..a,.. .,..
..i.:
.:.:.: ..::.:..:.: :.I:.:
.:.:.:.
....... :.:.:.
......
Dose 500ns Vector Collagen (n=6)
5ua
....... .... .:.:.:. .... .... .:.:.:. .... ....... .... ......: .:.:.:.
... ... ... ...... ... ... ...
.:.:_:.
.... ....... .... ... .:.:.:. ....
::j:: j::::
.......
...
Saline
Collagen
5Oug Saline
5ug RBC g h o s t s
(n=6)
(n=8)
(n=6)
(n=7)
5ug
Figure 2 Mean(s.e.m.) cellularity of wounds treated with basic fibroblast growth factor expressed as a percentage of cellularity ofpaired Control; 0, treated; n, number ofpaired wound control wounds. 0, biopsies; RBC, red blood cell. * P < 0.05: t P < 0.01 ( Wilcoxon signed rank test)
919
Basic fibroblast growth factor in wound healing: J. Slavin et al. 500
”
Dose Vector
1
*
Discussion t
2 ug TCF-B
5 ug bFGF RBC ghosts
Collagen (n = 20) Figure 3 Mean(s.e.m.) breaking load at 7 days of wounds treated with basic fibroblast growth,factor (bFGF) in red blood cell ( R B C ) ghosts or transforming growih factor beta ( T G F - p ) in a collagen suspension. 0, Control; 0 , treated; n, number of paired wound strips. * P < 0402; t P < 0.01 ( Wilcoxon signed rank trst) (n=24)
Table 1 Effect o f 5 p g per wound basic fibroblast growth factor in red bhod cell ghosts on wound breaking load ut dizerent times
Wound breaking load (g/cm’)
Control
Treated n P*
Day 7
Day 12
Day 21
256(28) 388(27) 24
