- Email: [email protected]
Serum-free cell culture medium induces acceleration of wound healing in guinea-pigs
E. S. Lindenbaum, Morphology
M. Tendler
and D. Beach
Research Unit, Bruce Rappaport Faculty of Medicine, Technion IIT, Haifa, Israel
Among fhe current methods employed in the treatment of wounds, a moist dressing is considered to be the optimal environment for the process of healing thereby avoiding de&cation of the wound bed. This study is based on the hypothesis that wound cell proliferation is dependent not only on moisture but also uponfhe composition of ihe moist microenvironment in the wound. That composition in turn is formed by di$.sion of nutrients from the existing vascular bed in and around the wound as well as by the wound cells’ cellular products. Since in woundsthe impaired vascular supply causes tissue deprivation, a conlinuous supply of nutrients and hormones will create an optimal subsfrafe for cellular milogenic achvify, synfhesis of matrix, growth factors and cytokines leading to wound healing. Mod$ied serum-free cell culture medium was supplemented with non-steroidal anabolic hormones: growth hormone, thyroxin and insulin, transfewin and sodium selenite. The medium was prepared in a 1 percenfalginate gel matrix. Under general anaeslhesia with ketamine (lmalgene 1000, Rhone Merieux,France) four 2 x z cm full-thickness skin patches were surgically extirpatedfrom the dorsum of Hartley-derived guinea-pigs. Each experimental group consisted of sevenanimals, i.e. 28 wounds lhat received the same treatment.Compositions of gelatin in saline, agarose in saline, agarose in medium and agarose in saline supplemented with the three hormones were compared to agarose in medium supplemented wifh the three hormones. After applicaiion of the gel (I mllcm2),the woun& weredressed with gauze, elasfic adhesive bandage and neffing. Change of bandage and administration of gel were pwformed every 48h under general anaesthesia, at which time all the wounds were washed wifh warm saline, measured, photographed and redressed as above. Computerized morphometric measurements of the photographs of each wound, in sequence, were made using ImageMeasure software. The dynamics of wound closure were quantified, analysed andplotted. The agarose in medium supplemented with fhe lhree anabolic hormones induce statistically significant (‘P < 0.001) acceleration of wound closure when compared to controls. No statistically sipificant difference was found amongthe controls.
Burns,Vol. 21, No. 2, 110-115,1995
Introduction The process of wound repair is complex. It involves the integration and control of growth of different types of tissuesand cells which are affected by both the systemic and local microenvironments. Currently, the moist wound
environment is considered by many to be the s&e-of-the art method for promoting wound healing. Occlusive dressingswhich keep the wound moist have been reported to increasere-epithelization and healing~ofchronic wounds and decreaseinflammation, pain, infection and scarring’. Moist dressings, which exert an important biological activity2, can be attained by using alginated gel dressings and calcium alginate dressings,currently used for absorbing exudate of dermal ulcers(Algosterile, J&J, Ascot, Berks, UK; Sorban, Dow, Midland, MI, USA ). Isotonic salinegels are utilized as moist protective dressingswhich promote healing (Normlgel, Scott Health Care), and copper chlorophyelin complex in gel (Chlorenin, Rystan Comp, Little Falls,NJ, USA) isusedfor its moist and bacteyiostatic effectOther hydrophilic hydrocolloid gels facilitate absorption of exudate (Gel-Syte, Baxter, Deerfield, IL, USA). Thus, it is generally acce ted that hydrated dressings provide an optimal woun dpenvironment to aid healing!..The blister” can be viewed as a hydrated occlusive biological dressing but it also contains elements released by damaged cells, which can be toxic. The rationale for using the hormone-supplemented serum-free medium in gel form as a wound dressing is to create an optimal microenvironment similar to that of an in vitro system for ‘culturing’ of the cells in situ within the wound space. The gelled media supplemented with nonsteroidal anabolic hormones provides a substrate for the cells in the wound similar to that used in tissue culture techniques. Wound healing is thus induced by promoting cellular activation thus leading to cell motility, mitogenicity and synthesis of extracellular matrix of cytokines and of growth factors.
Materials
and methods
Preparation of gel me&a The hormone-supplemented media in gel form was prepared under sterile conditions (patent filed). Delivery system One gram of Agarose Type III-A High EEO (Sigma Chemical Co. (St Louis, MO, USA) Cat. No. A-9793) was dissolved in 10 ml of 0.9 per cent NaCl saline.The mixture was autoclaved and then cooled down to 40°C. All preparations of the gels were .made using ~a final concentration of 1 per cent agarose or 4 per cent gelatin (Difco Lab. (Detroit, MI, USA) Cat. No. 0143-01).
Lindenbaum
et al.: Serum-free
cell culture medium
111
I.(
1.c
0.1
OS
0.8
0.6 & 2 t-
0.6
B 3
0.5
0.5
$ 0.4
0.3
0.2
0.1 i
1
0
4
6
8
10
12
14
Time t (days)
4
6
8
10
12
14
Time t (days)
Figure 1. Comparison of wound closure rates in experimental guinea-pigs treated with gelatin in saline (-•O-) and with agarose in saline (-H-).
Figure 2. Comparison of wound closure rates in experimental guinea-pigs treated with agarose in medium (-•-) and with agarose in saline (-W---).
Cell culture medium containing essential and non-essential amino acids, vitamins, glucose and organic constituents, major inorganic salts, trace elements and buffers was supplemented with three non-steroidal anabolic hormones - thyroxin, growth hormone and insulin/transferrin/sodium selenite combination. Ninety millilitres of the medium was warmed in a water-bath to 40°C. The media was then added to the agarosegel which had cooled to 40°C to form a homogeneousmixture which was aspirated into 10 and 20 ml syringes and refrigerated at 4°C.
the lumbar region. After washing the wounds with warm saline, the wounds were dressedwith the gel (1 ml/cm2), covered with gauze and anchoredwith elasticadhesiveand a Retelast netting (Medinet, s.p.a., Milano, Italy). Under general anaesthesia,the dressings were changed every 48 h at which time all wounds were washed with warm salineto remove any debris and any remaining gel within the wound. All wounds were then measuredand photographed, and fresh gel applied. The wounds in eachanimal were treated identically, i.e. with the same gel combination. The reasonfor this was to prevent any indirect effect of the gel constituents on the adjacent wounds. The macrophotographs of each wound in a sequenceat 48 h intervals were analysed using ImageMeasure (Phoenix Corp., Seattle, WA USA) cornput-erizedmorphometric program. The results were plotted asgraphs showing the time variation of fractional change in area(i.e. closure rate) (A, - AJA,, of the wounds treated with gelled media plus hormones versus the various controls. A, represents the initial wound area size and At the wound size at day t. Experiments in which various combinations of the components of the gel: gelatin in saline, agarosein saline and agarose in medium, three hormones in saline in agarose and three hormones in medium in agarose were performed on groups of seven animals each (i.e. 28 wounds/experiment) and the results were compared. Statistical analysisusing ANOVA (Analysis of Variance) was performed as well as the Scheffe procedure for multiple range test. No adjustment was made across the experiments.
The medium*
Animal
model
Hartley-derived Albino guinea-pigs weighing 300-400 g were used in this study. Following Ethical Committee approval of the protocols and 14 days of quarantine, the animalswere housed in individual cagesand fed regularly guinea-pig chow and water enriched with vitamin C ad libitum. All surgical procedures were performed under general anaesthesiausing ketamine HCl 150mg/kg. i.m. (Imalgene 1000, Rhone Merieux, Lyon, France). Four bilaterally symmetrical full-thickness skin segments measuring
2 x 2 cm were excised
from the dorsum
of eachanimal, two from the scapularregion and two from *At the time of acceptance of this manuscript for publication the exact composition of the medium has filed patent application status. Its composition cannot therefore be published. However interested readers should contact Professor Lindenbaum for further details on a confidential basis.
112
Burns: Vol. 21, No. 2, 1995 1s
Results The results of the five treatment groups were plotted and compared. Figures 1-6 show comparisons of the fractional change in area of the wounds. Comparisons of the following combinations were plotted: Efficacy of the gelling component Treatment with: gelatin in saline (n = 28) vs. agarose in saline (n = 28). Agarose in saline and gelatin in saline exerted similar rates of wound closure. There was no statistical difference between the two groups (Figure 7). Efficacy of the medium component of the gelled medium Treatment with: agarosein medium (n = 28) vs. agarosein saline (n = 28). The rate of wound closure in the presence of medium in agarose was similar to that observed with saline in agarose. The slightly slower closure rate in the medium treated wounds was not statistically significant (Figure2). Efficacy of the three hormones Treatment with: the three hormones in agarose in saline (n = 28) vs. agarosein saline(n = 28). The rate of closure of wounds treated with salinein the presenceand absenceof the three hormones was similar. The slightly slower closure rate in the hormone-treated wounds was not statistically significant. (Figure 3). Efficacy of the combination of three hormones in agarose in medium Treatment with: the three hormones in agarosein medium (n = 28) vs. agarosein medium (n = 28). The addition of the hormones to the medium induced statistically significant acceleration of wound closure, P < 0.001, when compared to the medium in the absenceof the three hormones. The hormones increasedthe transport of the medium components into the intracellular compartment and caused utilization of the medium by the cells in the wound
0.6 & r; q- 0.5 p 0.4
8 10 12 14 6 Time t (days) Figure 3. Comparisonof wound closureratesin experimental guinea-pigstreated with the three hormonesand agarosein saline(----0-) and with agarosein saline(---a---). 4
l.(IO.! 0.1I
(Figure 4).
0.7
Efficacy of the three hormones in agarose in medium (n = 28) vs. the three hormones in agarose in saline (n = 28) The presenceof the medium in combination with the three hormones yielded a statistically faster rate of wound closure when compared to treatment with the three hormones without medium. The increase in the rate of wound closure may be due to the utilization of the substrate, i.e. the medium, by the cells in the wound bed under the hormonal stimulation (Figure 5)
0.6
Efficacy of the three hormones in agarose in medium (n = 28) vs. gelatin in saline (n = 28), agarose in saline (n = 28) The presenceof the three hormones yielded a statistically significant faster rate of wound closure when compared to both gelatin in saline and agarosein saline (Figure 6). Statistical analysis using the Analysis of Variance (ANOVA) was performed to examine the statistical significance of rate of wound closure induced by the five combinations of treatment. Since this was found to be significant (TableI), the Scheffe test was used.
& x To o*: s 0.C 0.: 0.2 0.1
Time t (days) Figure 4. Comparisonof wound closurerakesin eqerimental guinea-pigstreated with the- three hormonesand-agarosein medium(-O-) and with agarosein mediti (-W----f.
Lindenbaum et al.: Serum-free cell culture medium 1.c
0.9
0.8
0.7
0.6 Q 2 *-
a0 25 a I 25
0.5
g 0.4
0.3
0.2
0.1
I
0
4
2
6
8
10
12
14
Time t (days)
Time t (days) Figure 5. Comparison
of wound closure ratesin experimental guinea-pigs treated with three hormones and agarose in- saline (-0-) and with three hormones and agarose in medium (-m-g.
Table I. Statistical ANOVA Day
Gelatin in saline
Figure 6. Comparison of wound closure rates in experimental guinea-pigs treated with three hormones in agarose in medium (-R-), with gelatin in saline (--Cl--) and agarose in saline (--- . -).
of wound closure rates Agarose saline
in
Agarose in medium
Three hormones in Agarose in saline
Three hormones in Agarose in medium
P
4 Mean sd. PC
0.3725 0.2061 NS
0.3579 0.2025 NS
0.2870 0.1653 0.001
0.2249 0.1451 0.001
0.4844 0.1094 NA
0.001
Mean s.d. P<
0.5172 0.1970 0.05
0.5062 0.1854 0.05
0.4644 0.1669 0.001
0.4337 0.1390 0.001
0.6587 0.1017 NA
0.001
Mean s.d. PC
0.6668 0.1428 0.005
0.6942 0.1250 0.05
0.6167 0.1359 0.001
0.6382 0.1117 0.001
0.8034 0.0645 NA
0.001
0.7330 0.1356 0.001
0.7384 0.1419 0.001
0.7090 0.1113 0.001
0.7439 0.830 0.001
0.8802 0.0539 NA
0.001
sd. P<
6
8
10
NS, Not significant. NA, Not applicable. Mean, percentage changes in wound area. P-c using Scheffe test comparing M + 3H to all other
groups.
The Scheffe test, which compared the averages of all the groups, yielded a statistically significant difference, which emanated from the fact that the average in the group treated with the three hormones in agarosein medium was significantly higher than any of the other groups (TableI). The combination of the three hormonesand the agarose in medium induced a faster rate of wound closure which is
statistically significant when compared to all other experimental groups. The meanson days 4,6,8 and 10 for treatment with: the three hormones in agarosein medium were 0.484, 0.658, 0.803 and 0.880 respectively, and indicated a classical growth rate (TableI). These means were larger than the meansfor treatment with: (I) three hormones in agarosein
114
Burns:
Table II. Regression line equations for n = 5 points (P values, Pearson’s correlationcoefficientsr) Regression
A+S S+G
M+A BH+A+S 3H+M+A
line
0.0295+ 0.0767 0.0384 + 0.0749 0.0061+ 0.0731 0.0287 + 0.0780 0.0649 + 0.0893
i-
equation
P-f
x day
0.989
0.0015
x x x x
0.989 0.997 0.992 0.9814
0.0015 0.0002 0.001 0.003
day day day day
saline; (2) agarose in medium alone; (3) agarose in saline alone; (4) gelatin in salinealone, on day 4,6, 8 and day 10, respectively. Furthermore, the differences in the four latter groups were not statistically significant (TableI). To determine the growth rates, regression line equations for n = 5 points and Pearson’scorrelation coefficients Ywere calculated (TableII). This yielded a classicalgrowth rate as expected. A test for comparing the slopes of the regression lines was performed. Treatment with the three hormones in agarose in medium revealed a steeper, although not significant, slope of 0.089 compared to 0.073-0.078 found in the other groups (TableII). The presence of medium without the three hormones did not induce a significant acceleration in wound closure. Neither did the presence of the three hormones alone without the medium. Comparison of all experimental combinations to the experiment in which both the three hormones and the medium were present in agaroseshows that the latter had a significantly higher rate of wound closure when compared to all the other groups.
Discussion The hormone-enriched serum-free medium in gel form used in this study was selected to create an optimal microenvironment which could act as a substrate for the cells in the wound. It contains all the required nutrients such as essentialand non-essential amino acids, vitamins, organic constituents, inorganic salts, trace elements and hormones usually supplied by diffusion from the blood. It was hypothesized that the wound cells thus supplied would proliferate and synthesize the samegrowth factors asfound in conditioned media in vitro in the wound fluid in vivo. Gelatin derived from either acid- or lime-cured tissue, has been used elsewhere as a vehicle for wound healing preparations5. It has also been used as a component of surgicalglue9. Thus we compared the agarosegel with the gelatin gel as a vehicle for the serum-free medium preparation. Agarose (sepharose),a purified galactan hydrocolloid isolated from marine algae, was selected because of its special physical properties. It allows high diffusion and electrokinetic movement of biological molecules through it. It acts asa gelling substance,it adsorbsthe medium and at the sametime releasesit to the surface of the wound as the medium is consumed by the cells in the wound bed. The gel also createsa moist environment which is essential for the healing process.In addition, agarosewhich is used routinely in tissue culture, is compatible with living systems. Debridement and bacteriostatic effects are also achieved through the high osmotic action of the polysaccharide’.
Vol.
21,
No.
3,1995
The hypertonicity reduces oedema and thus promote\ diffusion of serum and nutrients from the surrounding vesselsinto the wound bed. Although our results did noi indicate that agaroseyielded faster rates of Lyound closurc~ when compared to gelatin, our preference MS basedon it:< unique physical properties Glucose, the main source of energy ior ceil&r mrtabolism, is present in all media since it is readily available for utilization by the cells. Both honey and sugar have beerr used in wound healing preparations since antiquity. Thr use of sugar, similar to agarose, was reported to enhance wound healing due to its osmotic action w-hi& acts as debriding and bactericidal agent. Its hypertonicity r&eves the oedema in and around the wound and thus allows for diffusion of nutrients and serumfrom the peripheral blood vessels.If or when the sugar undergoes fermentation, thr alcohol may act as an antiseptic”. In serum-free media, the use of insuim, transferrin and sodium selenite has been tested extensively on many cell lines in in vitro systems. Regardlessof other supplements needed in specific cell lines these three appear to bt~ mandatory for most.” Insulin stimulates growth, glucose incorporation into glycogen and fatty acids, and promotes the transport across the cell membrane. It is, therefore. essential For internalizing the constituents of the medium and making them available to the cells. Transferrin in culture acts asan iron transport protein, it binds metal ions and thus it is also a detoxifying agent. Sodium selenite participates in tile removal of free radicals of oxygen’“. It may play a role ir the control of oxygen tension in the wound. In an effort to keep the wounds free of infection frequent changes of bandage with concomitant washing of the gel from the wound resulted in a sponging effect. since the superficial bacteria were removed with the gel. Since experimentally induced surgical wounds in otherwise healthy animals would heal within 3-3 weeks regardless of the type of treatment, the efficac), of any treatment is the dynamics of wound closure under treatment compared to controls such as the crehicle, w components of the medicament. In the guinea-pig, contraction of the dermal paniculus carnosus muscle and the relatively high skin mobility join together to mask the initial phaseof wound closure. The attachment of the skin of the wound edges to the substratum is a prerequisite to the commencement of epithelialization. In the experimental group which was treated with the three hormones in medium in agarose,the effects of muscularcontraction and skin mobility were inhibited by day 6 becausethe wound edges adhered to the wound bed. Thus, from day ~7on in that experimental group, wound closure was due more to epithelialization than to skin contraction. Our results substantiate the claim that the combined effect of the media and the hormones is required for acceleration of the healing process. It clearly and statistically demonstrates that the combination of medium and hormones only, not the medium alone nor the hormones alone, can stimulate the proliferation of the wound cells in situ. Thus agarose was used successfullyas a vehicle for the serum-free medium treatment on wounds. The fastest wound closure rate was achieved using the media supplemented with non-steroidal anabolic hormones: insulin, transferrin, sodium selenite, growth hormones and thyroxin. Applications of this gel combination ifi clinical cases is currently underway.
Lindenbaum
et al.: Serum-free
cell culture
medium
115
Acknowledgements We gratefully Life Medical USA).
acknowledge the support of this work Sciences Inc. (214 Carnegie, Princeton,
by NJ
6
7
References Eaglstein W. Review - occlusive dressing. ] Dermatol Stlrg @d1993;19:716-720. Mulder G. Feasibility study on the use of an RGI-peptide hylurasic acid: artificial matrix for the healing of Venous Stasis Ulcer. In: 5th Annual Symposium on Wound Care, 1992; 73: p. 157. Alvarez 0, Rozint J, Meehan M. Principles of moist wound healing: indications for chronic wounds. In: Krasner D, ed. Chronic Wottnd Cure. Health Management Publication. 1990; pp 266-281. Winter GD. Formation of scab and the rate of epithelialization on superficial wounds in the skin of the domestic pig. Nature 1962; 193: 293-294. Kiyoharay Y, Komada F, Iwakawa S, Fuwat T, Okumura K.
8
9 10
Systemic effects of epidermal growth factor (EGF) ointment containing protease inhibitor or gelatin in rats with bums or open wounds. Biol Phamaceut Bull 1993; 16: 73-76. Fabiani JN, Jebara VA, Deloche A, Stephan Y, Carpentier A. Use of surgical glue without replacement in the treatment of type A aortic dissection. Circulation 1989; SO: 1264-8. Elaf Eriksson. Healing in a liquid environment. In: Proceedings of the 3rd Medical Research Forum on Wound Repair, San Diego, April 1993, p 141. Rund CR. Alternative treatment - alternative setting. In: Krasner D, ed. Chronic Wound Care. Health Management Publications, 1990; pp 309-3 17. Freshnery RI. Culture of Animal Cells, 2nd edn. New York: Alan R Liss, 1988; pp 77-81. McKeehan WL, Ham RG. Stimulation of colonal growth of normal fibroblasts with substrata coated with polymers. ] Cell Biol 1976; 71: 727-734.
Paper accepted
after revision
16 June 1994.
Correspondence should be addressed to: Professor Ella Lindenbaum, Faculty of Medicine, Technion POB 9649 Haifa, Israel.
24th to 28th November 1995 Phone: 91-141-376731 Fax: 91-141-514072
Dr Malti Gup O/6. Hospital Road Jaipur 302 001 Rajasthan, India
M. Tendler
and D. Beach
Research Unit, Bruce Rappaport Faculty of Medicine, Technion IIT, Haifa, Israel
Among fhe current methods employed in the treatment of wounds, a moist dressing is considered to be the optimal environment for the process of healing thereby avoiding de&cation of the wound bed. This study is based on the hypothesis that wound cell proliferation is dependent not only on moisture but also uponfhe composition of ihe moist microenvironment in the wound. That composition in turn is formed by di$.sion of nutrients from the existing vascular bed in and around the wound as well as by the wound cells’ cellular products. Since in woundsthe impaired vascular supply causes tissue deprivation, a conlinuous supply of nutrients and hormones will create an optimal subsfrafe for cellular milogenic achvify, synfhesis of matrix, growth factors and cytokines leading to wound healing. Mod$ied serum-free cell culture medium was supplemented with non-steroidal anabolic hormones: growth hormone, thyroxin and insulin, transfewin and sodium selenite. The medium was prepared in a 1 percenfalginate gel matrix. Under general anaeslhesia with ketamine (lmalgene 1000, Rhone Merieux,France) four 2 x z cm full-thickness skin patches were surgically extirpatedfrom the dorsum of Hartley-derived guinea-pigs. Each experimental group consisted of sevenanimals, i.e. 28 wounds lhat received the same treatment.Compositions of gelatin in saline, agarose in saline, agarose in medium and agarose in saline supplemented with the three hormones were compared to agarose in medium supplemented wifh the three hormones. After applicaiion of the gel (I mllcm2),the woun& weredressed with gauze, elasfic adhesive bandage and neffing. Change of bandage and administration of gel were pwformed every 48h under general anaesthesia, at which time all the wounds were washed wifh warm saline, measured, photographed and redressed as above. Computerized morphometric measurements of the photographs of each wound, in sequence, were made using ImageMeasure software. The dynamics of wound closure were quantified, analysed andplotted. The agarose in medium supplemented with fhe lhree anabolic hormones induce statistically significant (‘P < 0.001) acceleration of wound closure when compared to controls. No statistically sipificant difference was found amongthe controls.
Burns,Vol. 21, No. 2, 110-115,1995
Introduction The process of wound repair is complex. It involves the integration and control of growth of different types of tissuesand cells which are affected by both the systemic and local microenvironments. Currently, the moist wound
environment is considered by many to be the s&e-of-the art method for promoting wound healing. Occlusive dressingswhich keep the wound moist have been reported to increasere-epithelization and healing~ofchronic wounds and decreaseinflammation, pain, infection and scarring’. Moist dressings, which exert an important biological activity2, can be attained by using alginated gel dressings and calcium alginate dressings,currently used for absorbing exudate of dermal ulcers(Algosterile, J&J, Ascot, Berks, UK; Sorban, Dow, Midland, MI, USA ). Isotonic salinegels are utilized as moist protective dressingswhich promote healing (Normlgel, Scott Health Care), and copper chlorophyelin complex in gel (Chlorenin, Rystan Comp, Little Falls,NJ, USA) isusedfor its moist and bacteyiostatic effectOther hydrophilic hydrocolloid gels facilitate absorption of exudate (Gel-Syte, Baxter, Deerfield, IL, USA). Thus, it is generally acce ted that hydrated dressings provide an optimal woun dpenvironment to aid healing!..The blister” can be viewed as a hydrated occlusive biological dressing but it also contains elements released by damaged cells, which can be toxic. The rationale for using the hormone-supplemented serum-free medium in gel form as a wound dressing is to create an optimal microenvironment similar to that of an in vitro system for ‘culturing’ of the cells in situ within the wound space. The gelled media supplemented with nonsteroidal anabolic hormones provides a substrate for the cells in the wound similar to that used in tissue culture techniques. Wound healing is thus induced by promoting cellular activation thus leading to cell motility, mitogenicity and synthesis of extracellular matrix of cytokines and of growth factors.
Materials
and methods
Preparation of gel me&a The hormone-supplemented media in gel form was prepared under sterile conditions (patent filed). Delivery system One gram of Agarose Type III-A High EEO (Sigma Chemical Co. (St Louis, MO, USA) Cat. No. A-9793) was dissolved in 10 ml of 0.9 per cent NaCl saline.The mixture was autoclaved and then cooled down to 40°C. All preparations of the gels were .made using ~a final concentration of 1 per cent agarose or 4 per cent gelatin (Difco Lab. (Detroit, MI, USA) Cat. No. 0143-01).
Lindenbaum
et al.: Serum-free
cell culture medium
111
I.(
1.c
0.1
OS
0.8
0.6 & 2 t-
0.6
B 3
0.5
0.5
$ 0.4
0.3
0.2
0.1 i
1
0
4
6
8
10
12
14
Time t (days)
4
6
8
10
12
14
Time t (days)
Figure 1. Comparison of wound closure rates in experimental guinea-pigs treated with gelatin in saline (-•O-) and with agarose in saline (-H-).
Figure 2. Comparison of wound closure rates in experimental guinea-pigs treated with agarose in medium (-•-) and with agarose in saline (-W---).
Cell culture medium containing essential and non-essential amino acids, vitamins, glucose and organic constituents, major inorganic salts, trace elements and buffers was supplemented with three non-steroidal anabolic hormones - thyroxin, growth hormone and insulin/transferrin/sodium selenite combination. Ninety millilitres of the medium was warmed in a water-bath to 40°C. The media was then added to the agarosegel which had cooled to 40°C to form a homogeneousmixture which was aspirated into 10 and 20 ml syringes and refrigerated at 4°C.
the lumbar region. After washing the wounds with warm saline, the wounds were dressedwith the gel (1 ml/cm2), covered with gauze and anchoredwith elasticadhesiveand a Retelast netting (Medinet, s.p.a., Milano, Italy). Under general anaesthesia,the dressings were changed every 48 h at which time all wounds were washed with warm salineto remove any debris and any remaining gel within the wound. All wounds were then measuredand photographed, and fresh gel applied. The wounds in eachanimal were treated identically, i.e. with the same gel combination. The reasonfor this was to prevent any indirect effect of the gel constituents on the adjacent wounds. The macrophotographs of each wound in a sequenceat 48 h intervals were analysed using ImageMeasure (Phoenix Corp., Seattle, WA USA) cornput-erizedmorphometric program. The results were plotted asgraphs showing the time variation of fractional change in area(i.e. closure rate) (A, - AJA,, of the wounds treated with gelled media plus hormones versus the various controls. A, represents the initial wound area size and At the wound size at day t. Experiments in which various combinations of the components of the gel: gelatin in saline, agarosein saline and agarose in medium, three hormones in saline in agarose and three hormones in medium in agarose were performed on groups of seven animals each (i.e. 28 wounds/experiment) and the results were compared. Statistical analysisusing ANOVA (Analysis of Variance) was performed as well as the Scheffe procedure for multiple range test. No adjustment was made across the experiments.
The medium*
Animal
model
Hartley-derived Albino guinea-pigs weighing 300-400 g were used in this study. Following Ethical Committee approval of the protocols and 14 days of quarantine, the animalswere housed in individual cagesand fed regularly guinea-pig chow and water enriched with vitamin C ad libitum. All surgical procedures were performed under general anaesthesiausing ketamine HCl 150mg/kg. i.m. (Imalgene 1000, Rhone Merieux, Lyon, France). Four bilaterally symmetrical full-thickness skin segments measuring
2 x 2 cm were excised
from the dorsum
of eachanimal, two from the scapularregion and two from *At the time of acceptance of this manuscript for publication the exact composition of the medium has filed patent application status. Its composition cannot therefore be published. However interested readers should contact Professor Lindenbaum for further details on a confidential basis.
112
Burns: Vol. 21, No. 2, 1995 1s
Results The results of the five treatment groups were plotted and compared. Figures 1-6 show comparisons of the fractional change in area of the wounds. Comparisons of the following combinations were plotted: Efficacy of the gelling component Treatment with: gelatin in saline (n = 28) vs. agarose in saline (n = 28). Agarose in saline and gelatin in saline exerted similar rates of wound closure. There was no statistical difference between the two groups (Figure 7). Efficacy of the medium component of the gelled medium Treatment with: agarosein medium (n = 28) vs. agarosein saline (n = 28). The rate of wound closure in the presence of medium in agarose was similar to that observed with saline in agarose. The slightly slower closure rate in the medium treated wounds was not statistically significant (Figure2). Efficacy of the three hormones Treatment with: the three hormones in agarose in saline (n = 28) vs. agarosein saline(n = 28). The rate of closure of wounds treated with salinein the presenceand absenceof the three hormones was similar. The slightly slower closure rate in the hormone-treated wounds was not statistically significant. (Figure 3). Efficacy of the combination of three hormones in agarose in medium Treatment with: the three hormones in agarosein medium (n = 28) vs. agarosein medium (n = 28). The addition of the hormones to the medium induced statistically significant acceleration of wound closure, P < 0.001, when compared to the medium in the absenceof the three hormones. The hormones increasedthe transport of the medium components into the intracellular compartment and caused utilization of the medium by the cells in the wound
0.6 & r; q- 0.5 p 0.4
8 10 12 14 6 Time t (days) Figure 3. Comparisonof wound closureratesin experimental guinea-pigstreated with the three hormonesand agarosein saline(----0-) and with agarosein saline(---a---). 4
l.(IO.! 0.1I
(Figure 4).
0.7
Efficacy of the three hormones in agarose in medium (n = 28) vs. the three hormones in agarose in saline (n = 28) The presenceof the medium in combination with the three hormones yielded a statistically faster rate of wound closure when compared to treatment with the three hormones without medium. The increase in the rate of wound closure may be due to the utilization of the substrate, i.e. the medium, by the cells in the wound bed under the hormonal stimulation (Figure 5)
0.6
Efficacy of the three hormones in agarose in medium (n = 28) vs. gelatin in saline (n = 28), agarose in saline (n = 28) The presenceof the three hormones yielded a statistically significant faster rate of wound closure when compared to both gelatin in saline and agarosein saline (Figure 6). Statistical analysis using the Analysis of Variance (ANOVA) was performed to examine the statistical significance of rate of wound closure induced by the five combinations of treatment. Since this was found to be significant (TableI), the Scheffe test was used.
& x To o*: s 0.C 0.: 0.2 0.1
Time t (days) Figure 4. Comparisonof wound closurerakesin eqerimental guinea-pigstreated with the- three hormonesand-agarosein medium(-O-) and with agarosein mediti (-W----f.
Lindenbaum et al.: Serum-free cell culture medium 1.c
0.9
0.8
0.7
0.6 Q 2 *-
a0 25 a I 25
0.5
g 0.4
0.3
0.2
0.1
I
0
4
2
6
8
10
12
14
Time t (days)
Time t (days) Figure 5. Comparison
of wound closure ratesin experimental guinea-pigs treated with three hormones and agarose in- saline (-0-) and with three hormones and agarose in medium (-m-g.
Table I. Statistical ANOVA Day
Gelatin in saline
Figure 6. Comparison of wound closure rates in experimental guinea-pigs treated with three hormones in agarose in medium (-R-), with gelatin in saline (--Cl--) and agarose in saline (--- . -).
of wound closure rates Agarose saline
in
Agarose in medium
Three hormones in Agarose in saline
Three hormones in Agarose in medium
P
4 Mean sd. PC
0.3725 0.2061 NS
0.3579 0.2025 NS
0.2870 0.1653 0.001
0.2249 0.1451 0.001
0.4844 0.1094 NA
0.001
Mean s.d. P<
0.5172 0.1970 0.05
0.5062 0.1854 0.05
0.4644 0.1669 0.001
0.4337 0.1390 0.001
0.6587 0.1017 NA
0.001
Mean s.d. PC
0.6668 0.1428 0.005
0.6942 0.1250 0.05
0.6167 0.1359 0.001
0.6382 0.1117 0.001
0.8034 0.0645 NA
0.001
0.7330 0.1356 0.001
0.7384 0.1419 0.001
0.7090 0.1113 0.001
0.7439 0.830 0.001
0.8802 0.0539 NA
0.001
sd. P<
6
8
10
NS, Not significant. NA, Not applicable. Mean, percentage changes in wound area. P-c using Scheffe test comparing M + 3H to all other
groups.
The Scheffe test, which compared the averages of all the groups, yielded a statistically significant difference, which emanated from the fact that the average in the group treated with the three hormones in agarosein medium was significantly higher than any of the other groups (TableI). The combination of the three hormonesand the agarose in medium induced a faster rate of wound closure which is
statistically significant when compared to all other experimental groups. The meanson days 4,6,8 and 10 for treatment with: the three hormones in agarosein medium were 0.484, 0.658, 0.803 and 0.880 respectively, and indicated a classical growth rate (TableI). These means were larger than the meansfor treatment with: (I) three hormones in agarosein
114
Burns:
Table II. Regression line equations for n = 5 points (P values, Pearson’s correlationcoefficientsr) Regression
A+S S+G
M+A BH+A+S 3H+M+A
line
0.0295+ 0.0767 0.0384 + 0.0749 0.0061+ 0.0731 0.0287 + 0.0780 0.0649 + 0.0893
i-
equation
P-f
x day
0.989
0.0015
x x x x
0.989 0.997 0.992 0.9814
0.0015 0.0002 0.001 0.003
day day day day
saline; (2) agarose in medium alone; (3) agarose in saline alone; (4) gelatin in salinealone, on day 4,6, 8 and day 10, respectively. Furthermore, the differences in the four latter groups were not statistically significant (TableI). To determine the growth rates, regression line equations for n = 5 points and Pearson’scorrelation coefficients Ywere calculated (TableII). This yielded a classicalgrowth rate as expected. A test for comparing the slopes of the regression lines was performed. Treatment with the three hormones in agarose in medium revealed a steeper, although not significant, slope of 0.089 compared to 0.073-0.078 found in the other groups (TableII). The presence of medium without the three hormones did not induce a significant acceleration in wound closure. Neither did the presence of the three hormones alone without the medium. Comparison of all experimental combinations to the experiment in which both the three hormones and the medium were present in agaroseshows that the latter had a significantly higher rate of wound closure when compared to all the other groups.
Discussion The hormone-enriched serum-free medium in gel form used in this study was selected to create an optimal microenvironment which could act as a substrate for the cells in the wound. It contains all the required nutrients such as essentialand non-essential amino acids, vitamins, organic constituents, inorganic salts, trace elements and hormones usually supplied by diffusion from the blood. It was hypothesized that the wound cells thus supplied would proliferate and synthesize the samegrowth factors asfound in conditioned media in vitro in the wound fluid in vivo. Gelatin derived from either acid- or lime-cured tissue, has been used elsewhere as a vehicle for wound healing preparations5. It has also been used as a component of surgicalglue9. Thus we compared the agarosegel with the gelatin gel as a vehicle for the serum-free medium preparation. Agarose (sepharose),a purified galactan hydrocolloid isolated from marine algae, was selected because of its special physical properties. It allows high diffusion and electrokinetic movement of biological molecules through it. It acts asa gelling substance,it adsorbsthe medium and at the sametime releasesit to the surface of the wound as the medium is consumed by the cells in the wound bed. The gel also createsa moist environment which is essential for the healing process.In addition, agarosewhich is used routinely in tissue culture, is compatible with living systems. Debridement and bacteriostatic effects are also achieved through the high osmotic action of the polysaccharide’.
Vol.
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3,1995
The hypertonicity reduces oedema and thus promote\ diffusion of serum and nutrients from the surrounding vesselsinto the wound bed. Although our results did noi indicate that agaroseyielded faster rates of Lyound closurc~ when compared to gelatin, our preference MS basedon it:< unique physical properties Glucose, the main source of energy ior ceil&r mrtabolism, is present in all media since it is readily available for utilization by the cells. Both honey and sugar have beerr used in wound healing preparations since antiquity. Thr use of sugar, similar to agarose, was reported to enhance wound healing due to its osmotic action w-hi& acts as debriding and bactericidal agent. Its hypertonicity r&eves the oedema in and around the wound and thus allows for diffusion of nutrients and serumfrom the peripheral blood vessels.If or when the sugar undergoes fermentation, thr alcohol may act as an antiseptic”. In serum-free media, the use of insuim, transferrin and sodium selenite has been tested extensively on many cell lines in in vitro systems. Regardlessof other supplements needed in specific cell lines these three appear to bt~ mandatory for most.” Insulin stimulates growth, glucose incorporation into glycogen and fatty acids, and promotes the transport across the cell membrane. It is, therefore. essential For internalizing the constituents of the medium and making them available to the cells. Transferrin in culture acts asan iron transport protein, it binds metal ions and thus it is also a detoxifying agent. Sodium selenite participates in tile removal of free radicals of oxygen’“. It may play a role ir the control of oxygen tension in the wound. In an effort to keep the wounds free of infection frequent changes of bandage with concomitant washing of the gel from the wound resulted in a sponging effect. since the superficial bacteria were removed with the gel. Since experimentally induced surgical wounds in otherwise healthy animals would heal within 3-3 weeks regardless of the type of treatment, the efficac), of any treatment is the dynamics of wound closure under treatment compared to controls such as the crehicle, w components of the medicament. In the guinea-pig, contraction of the dermal paniculus carnosus muscle and the relatively high skin mobility join together to mask the initial phaseof wound closure. The attachment of the skin of the wound edges to the substratum is a prerequisite to the commencement of epithelialization. In the experimental group which was treated with the three hormones in medium in agarose,the effects of muscularcontraction and skin mobility were inhibited by day 6 becausethe wound edges adhered to the wound bed. Thus, from day ~7on in that experimental group, wound closure was due more to epithelialization than to skin contraction. Our results substantiate the claim that the combined effect of the media and the hormones is required for acceleration of the healing process. It clearly and statistically demonstrates that the combination of medium and hormones only, not the medium alone nor the hormones alone, can stimulate the proliferation of the wound cells in situ. Thus agarose was used successfullyas a vehicle for the serum-free medium treatment on wounds. The fastest wound closure rate was achieved using the media supplemented with non-steroidal anabolic hormones: insulin, transferrin, sodium selenite, growth hormones and thyroxin. Applications of this gel combination ifi clinical cases is currently underway.
Lindenbaum
et al.: Serum-free
cell culture
medium
115
Acknowledgements We gratefully Life Medical USA).
acknowledge the support of this work Sciences Inc. (214 Carnegie, Princeton,
by NJ
6
7
References Eaglstein W. Review - occlusive dressing. ] Dermatol Stlrg @d1993;19:716-720. Mulder G. Feasibility study on the use of an RGI-peptide hylurasic acid: artificial matrix for the healing of Venous Stasis Ulcer. In: 5th Annual Symposium on Wound Care, 1992; 73: p. 157. Alvarez 0, Rozint J, Meehan M. Principles of moist wound healing: indications for chronic wounds. In: Krasner D, ed. Chronic Wottnd Cure. Health Management Publication. 1990; pp 266-281. Winter GD. Formation of scab and the rate of epithelialization on superficial wounds in the skin of the domestic pig. Nature 1962; 193: 293-294. Kiyoharay Y, Komada F, Iwakawa S, Fuwat T, Okumura K.
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Systemic effects of epidermal growth factor (EGF) ointment containing protease inhibitor or gelatin in rats with bums or open wounds. Biol Phamaceut Bull 1993; 16: 73-76. Fabiani JN, Jebara VA, Deloche A, Stephan Y, Carpentier A. Use of surgical glue without replacement in the treatment of type A aortic dissection. Circulation 1989; SO: 1264-8. Elaf Eriksson. Healing in a liquid environment. In: Proceedings of the 3rd Medical Research Forum on Wound Repair, San Diego, April 1993, p 141. Rund CR. Alternative treatment - alternative setting. In: Krasner D, ed. Chronic Wound Care. Health Management Publications, 1990; pp 309-3 17. Freshnery RI. Culture of Animal Cells, 2nd edn. New York: Alan R Liss, 1988; pp 77-81. McKeehan WL, Ham RG. Stimulation of colonal growth of normal fibroblasts with substrata coated with polymers. ] Cell Biol 1976; 71: 727-734.
Paper accepted
after revision
16 June 1994.
Correspondence should be addressed to: Professor Ella Lindenbaum, Faculty of Medicine, Technion POB 9649 Haifa, Israel.
24th to 28th November 1995 Phone: 91-141-376731 Fax: 91-141-514072
Dr Malti Gup O/6. Hospital Road Jaipur 302 001 Rajasthan, India