the influence of hyperbaric oxygenation (hbo) on proliferation and differentiation of human...

Cell and Tissue Banking1: 261–269, 2000.© 2001Kluwer Academic Publishers. Printed in the Netherlands.

The influence of hyperbaric oxygenation (HBO) on proliferation anddifferentiation of human keratinocyte cultures in vitro

Dirk A. Hollander∗, Mohssen Y. Hakimi, Andre Hartmann, Kerstin Wilhelm & Joachim WindolfDepartment of Trauma and Reconstructive Surgery, Johann Wolfgang Goethe University Clinics,Theodor Stern-Kai 7, 60590 Frankfurt/Main, Germany∗Author for correspondence (Tel.:++49-69-6301-5082/6688; Fax:++49-69-6301-6439)

Key words:human keratinocyte cultivation, hyperbaric oxygenation, wound healing

Abstract

A drop in tissue oxygen partial pressure below 30 mm Hg as a result of reduced perfusion in an extensive area of acuteskin damage, or where a large number of chronic skin defects occur, inhibits collagen synthesis and neoangiogenesisin the various phases of wound healing. Subsequent granulation and epithelialisation are correspondingly impaired.

Hyperbaric oxygenation is now recognised as a valuable supplementary method of treatment for problematicwounds. Stimulation of fibroblast and endothelial cell proliferation through hyperbaric oxygenation has beendemonstrated in numerous studies.

The aim of this study was to investigate the effect of hyperbaric oxygen treatment on the proliferation anddifferentiation of human keratinocyte cultures.

The influence of hyperbaric oxygenation on the proliferation of human keratinocyte cultures was demonstratedusing flow-through cytometry and a fluorescence activated cell sorter, which detects fluorescence intensity followingincorporation of 5-bromo-2′-deoxyuridine in cell DNA.

The degree of cell differentiation was deduced from the expression of various components of the cytoskeleton, suchas cytokeratin 10 and involukrin, the production of which was quantified through the determination of monoclonalantibodies against cytokeratin 10 and involukrin from measurements of fluorescence activity in a flow-throughcytometer.

Hyperbaric oxygenation of cell culturesin vitro did not produce a significantly higher rate of cell proliferation,so that no increase in vitality was observed.

An interesting observation following exposure to hyperbaric oxygen was the marked increase in expression ofboth cytokeratin 10 and involukrin, as an indication of accelerated cell differentiation.

Introduction

For the normal, unimpaired healing of wounds it is par-ticularly important that neovascularisation, collagensynthesis, epithelialisation and resistance to infectioncan proceed under optimal conditions [1,2]. In order tomaintain its functions, whether in a state of activationor dormancy, every body cell depends on a continuoussupply of oxygen via the microcirculation [3,4]. Cellsare not able to store oxygen over an extended periodof time, so that a drop in the partial pressure of tissue

oxygen below 30 mmHg, as a result of reduced perfu-sion in an extensive area of acute skin damage, or wherea large number of chronic skin defects occur, results insevere impairment of wound tissue granulation [4–7].A critical stage of cutaneous wound regeneration fol-lowing wound creation is the development and matu-ration of the epidermis, which consists mainly (90%)of keratinocytes. This healing process can be disturbedby various pathological changes or through the ageingprocess itself. Common to all local and systemic factorsleading to wound healing problems, is an impairment

262 D.A. Hollanderet al.

of the complex interactions related to tissue oxygenperfusion [8].

Hyperbaric oxygenation (HBO) also offers a thera-peutic approach to the treatment of problematic softtissue defects, whereby perfusion of the tissue canbe directly influenced through the aspiration of pureoxygen under increased pressure. The resulting strongincrease in oxygen partial pressure (pO2) in the bloodis of decisive importance for the supply of oxygento tissue cells, since the difference in partial pressurebetween cell and blood plasma is the driving force foroxygen transportation. The supposed healing effect isaccounted for by a local increase in oxygen gradient andthe resulting increase in the rate of oxygen diffusion inan area of reduced perfusion. So far the only thera-peutic mechanism that could be established for HBOtreatment is the improved oxygen supply of oxygendeficient tissue, resulting in increased angioneogenesisand growth of connective tissue from increased colla-gen synthesis by fibroblasts [3,9–11]. An increase inlocal resistance to infection following HBO has alsobeen demonstrated [12].

Despite the very promising clinical results obtainedin treating problematic wounds, the effects of hyper-baric oxygen therapy are by no means well understoodat the cellular level. The present study thus examinesthe effect of HBO therapy on growth of keratinocytes;the most important epidermal cell type.

The purpose of our study was to investigate theeffects of HBO on proliferation and differentiation inhuman keratinocyte cultures.

On the one hand, it was hoped that the dataobtained might facilitate improvements in the qual-ity of in vivo cell transplants, for which a reducedcultivation period though accelerated proliferation ofkeratinocytes would be of importance in the treatmentof patients with severe burn injuries.

On the other hand, the study sought to clarifythe question of whether the promising clinical resultsobtained with HBO in the treatment of problem-atic wounds are related to changes in keratinocytebehaviour.

Methods and materials

Cell cultures

The keratinocytes cultivated for the experiments wereisolated from mammal skin left over from plasticreconstruction surgery.

Cells were isolated from a total of 10 mammal skinsamples according to a method described by Rheinwaldand Green. Samples were washed repeatedly withsterile, phosphate-buffered saline (PBS) free of Ca++

and Mg++ ions, following which thin ribbons of tis-sue were razored from stretched portions. These werethen placed in centrifuge tubes with 10 ml Hepes-buffered saline solution (HBSS) plus 0.25% trypsin and0.02% ethylenediamine tetraacetic acid (EDTA) andincubated overnight for 15–18 h in a refrigerator at 4◦C.

The following day, epidermis was separated fromdermis in 10 ml of cell culture medium with 20% foetalcalf serum (FCS), Gibco BRL, Cat. No. 10108-155.The epidermis so obtained, together with the culturemedium, was returned to the centrifuge tube with itscontent of HBSS, trypsin and EDTA and for 5–10 minviolently pipetted, in order separate it into its compo-nent cells. The resulting cell suspension was passedthrough a cell strainer into a another centrifuge tubeand subsequently centrifuged for 10 min at 1000 rpm.

Following centrifugation, the cell pellet was resus-pended in 1 ml of serum-free keratinocyte medium,10µl of which were mixed with 90µl of trypan blueprior to counting in a cell counting chamber. Follow-ing subsequent dilution with serum-free keratinocytemedium, the cells were seeded in culture bottles at adensity of 30,000 cells/cm2. At 60–80% confluency,passage of the keratinocytes proceeded according tothe following scheme: first the cells were washed withPBS, before being incubated in glucose/EDTA solu-tion for 10 min at 37◦C. The supernatant was thendrawn off by pipette and discarded, to be replacedwith trypsin/EDTA solution, with which the cells wereincubated for a further 5–10 min at 37◦C. After addingcell culture medium containing 20% KCS (to inacti-vate trypsin), the cell suspension was violently mixedusing a pipette and subsequently centrifuged for 10 minat 1000 rpm. Finally, following resuspension of thecell pellet in 1 ml of serum-free keratinocyte medium,the cells were counted. After counting, the cells wereeither used to seed cell culture bottles or the experimen-tal 6-well Falcon plates, whereby two wells per platewere used.

Hyperbaric oxygen treatment of cell cultures

In the experiments, keratinocytes from the primary cul-ture or from the first passage were used. The cells wereseeded at the subconfluent stage of development on 6-well plates at a cell density of 11,000–13,000 cells/cm2,

Human keratinocyte cultures and hyperbaric oxygenation 263

3 days prior to HBO. Two wells of each plate were used,so that for each skin sample there was a double trial.

The cells cultivated from each of the 10 skin samples,seeded on the double trail plates as described above,were subjected to different conditions in a hyperbaricoxygen chamber (Haux).

Plate 1: Negative control: keratinocytes incubatedoutside the chamber in normal room air atatmospheric pressure (1 bar).

Plate 2: keratinocytes incubated at 2 bar pressure innormal room air.

Plate 3: keratinocytes incubated at 2 bar pressure inpure oxygen.





The cells were supplied with oxygen via a closed ‘headtent’, of the type normally used on patients with, forexample, severe face injuries that prevent the use of anormal face mask.

Cell proliferation

In order to determine the effect on cell prolifera-tion, the individual plates were exposed to hyperbaricconditions for periods of 30 and 60 min.

Subsequently, proliferation can be determined byflow-through cytometry using a fluorescence activatedcell sorter (FACS-scan), which detects fluores-cence intensity following incorporation of 5-bromo-2′-deoxyuridine (BrdU) in cell DNA. BrdU is a synthetic,non-radioactive thymidine analogue, which is compet-itively incorporated into DNA in place of thymidine.BrdU was added in the form of BrdU labelling solu-tion immediately before the experiment was initiated.The prepared cell cultures were treated according tothe scheme described above. Evaluation of the exper-imental and control plates involved measurements offluorescence intensity by means of FACS scan (alsodescribed above), 24 h after the period of HBO.

Cell differentiation

In order to obtain data on cell differentiation, cellcultures were subjected for 30 min to atmospheric

air or pure oxygen at different pressures accordingto the scheme described above. The degree of celldifferentiation can be deduced from the determinationof components of the cytoskeleton in the nucleus, suchas cytokeratin 10 and involukrin, which are synthe-sised during terminal differentiation. Production ofthese components, and thus the degree of cell dif-ferentiation, can be deduced from the determinationof monoclonal antibodies against cytokeratin 10 andinvolukrin by measurements of fluorescence activity ina flow-through cytometer.

Measurement of oxygen partial pressure inculture medium

In order to verify the effect of HBO on the cell cultureson the 6-well plates under their head tents (as describedabove) pO2 was determined in various random samplesof culture medium following exposure to increasedpressures of air and pure oxygen. A sample of culturemedium was taken and its pO2 measured, using a bloodgas analyser, and compared with the negative control(Plate 0).

Statistics

For statistical evaluation of the results the Wilcoxon–Mann–Whitney Test was used, which allowed a valueof p < 0.05 to be defined as statistically significant.

Results

The effect of oxygenation on the cell culture medium

Oxygenation of the cell culture medium followingexposure to hyperbaric oxygen was determined bymeasuring pO2 using a blood gas analyser. A slightincrease in pO2 was found following exposure to nor-mal air at increased pressures, while a more marked,though not linear, increase was found following expo-sure to pure oxygen at increased pressures (Table 1).

The effect of oxygenation on cell proliferation

In order to standardise the results, the mean valueobtained for fluorescence following exposure of thenegative control to normal room air at approximately1 bar pressure was taken as 100% for all 10 samples(plate 0).

Keratinocyte proliferation was determined after 30and 60 min exposure to hyperbaric oxygen from


Table 1. Verification of the effect of HBO on the cell cultures (6-well-plates under head tents) by determining pO2 of the culture mediumfollowing exposure using a blood gas analyser, comparison withnegative control (plate 1).

Exposed pressure Measured pO2

in culture medium:

n = 4

Negative control; room air 198.3± 2.3

(equivalent to 760 mm Hg)

Room air+ 1 bar pressure 254.9± 1.4


100% O2 + 1 bar pressure 793.5± 66.1




100% O2 + 2 bar pressure 1174.9± 41.5




100% O2 + 3 bar pressure 1396± 25.2


measurements of fluorescence intensity (FACS scan)24 h after BrdU incorporation in the cell nucleus usingmonoclonal antibodies (Figures 1 and 2).

Following 30 min treatment in room air at 2 bar (1bar overpressure), from the 10 samples a mean valueof 81% (standard deviation:±20) was obtained, whileafter 60 min treatment the mean value was 95% (±18).When pure oxygen was supplied at a pressure of 2 bara mean value of 83% (±25) was obtained after 30 minexposure, and after 60 min exposure a mean value of97% (±18).

Following 30 min treatment in room air at 3 bar (2bar overpressure), a mean value of 85% (±21), andfollowing 60 min, of 92% (±14) was obtained. Withpure oxygen at 3 bar a discrete increase to 102% (±24)was observed after 30 min exposure, while after 60 minthe mean value dropped back to 93% (±14).

Measurements with room air at 4 bar (3 bar over-pressure) produced a mean value of 99% (±10) after30 min, and 94% (±17) after 60 min exposure. Thecorresponding values for pure oxygen at 4 bar were95% (±14) and 93% (±14) respectively.

140

120

100

80

60

40

20

01 2

100 ± 0

81±19.783 ± 24.2 85 ± 21

102 ± 2499 ± 10

95 ± 14

3 4 5 6 7

Figure 1. Effect of HBO on keratinocyte proliferation: 30 min exposure. Measurements of fluorescence intensity (%) 24 h after BrdU incorpora-tion, mean values from 10 samples with standard deviation. 1: negative control, 2: room air, 1 bar overpressure, 3: 100% O2, 1 bar overpressure,4: room air, 2 bar overpressure, 5: 100% O2, 2 bar overpressure, 6: room air, 3 bar overpressure, 7: 100% O2, 3 bar overpressure.


140

120

100

80

60

40

20

01 2

100 ± 095 ± 18.1 97 ± 17.6

92 ± 13.8 93 ± 16.9 94± 16.6

93 ± 20.8

3 4 5 6 7

Figure 2. Effect of HBO on keratinocyte proliferation: 60 min exposure. Measurements of fluorescence intensity (%) 24 h after BrdU incorpora-tion, mean values from 10 samples with standard deviation. 1: negative control, 2: room air, 1 bar overpressure, 3: 100% O2, 1 bar overpressure,4: room air, 2 bar overpressure, 5: 100% O2, 2 bar overpressure, 6: room air, 3 bar overpressure, 7: 100% O2, 3 bar overpressure.

140

120

100

80

60

40

20

01 2

100 ± 0

108 ± 24.6104 ± 17.5

107 ± 18.3

95 ± 17.4

104 ± 19.9

102 ± 19.8

3 4 5 6 7

Figure 3. Effect of HBO on keratinocyte differentiation by cytokeratin 10 synthesis: 30 min exposure. Measurements of fluorescence intensity(%) 24 h after exposure, mean values from 10 samples with standard deviation. 1: negative control, 2: room air, 1 bar overpressure, 3: 100%O2, 1 bar overpressure, 4: room air, 2 bar overpressure, 5: 100% O2, 2 bar overpressure, 6: room air, 3 bar overpressure, 7: 100% O2, 3 baroverpressure.


160

140

120

100

80

60

20

40

01 2

100 ± 0

119 ± 32.8

109 ± 22.3 108 ± 14.5

100 ± 16.8

107 ± 29.7

110 ± 21

3 4 5 6 7

Figure 4. Effect of HBO on keratinocyte differentiation by involucrin synthesis: 30 min exposure. Measurements of fluorescence intensity (%)24 h after exposure, mean values from 10 samples with standard deviation. 1: negative control, 2: room air, 1 bar overpressure, 3: 100% O2, 1 baroverpressure, 4: room air, 2 bar overpressure, 5: 100% O2, 2 bar overpressure, 6: room air, 3 bar overpressure, 7: 100% O2, 3 bar overpressure.

Apart from a statistically not significant increase incell proliferation (p > 0.05) following 30 min expo-sure to pure oxygen at 2 bar, all the other measurementsshowed a decrease in growth rate.

The effect of oxygenation on cell differentiation

The cell cultures were exposed to atmospheric air andpure oxygen at different pressures for 30 min. Thesamples were tested 24 h following treatment for dif-ferences in the expression of the cytoskeleton com-ponents, cytokeratin 10 and involukrin, which aresynthesised during terminal differentiation (Figures 3and 4).

In order to standardise the evaluation, the mean val-ues for fluorescence in the negative controls was takento be 100%.

In room air at 2 bar (1 bar overpressure) the meanvalue for cytokeratin 10 expression was 108% (±25),and for involukrin expression 119% (±33). The cor-responding values in pure oxygen at 2 bar were 104%(±18) and 109% (±22), respectively.

In room air at 3 bar (2 bar overpressure) the meanvalue for cytokeratin 10 expression was 107% (±18),and for involukrin expression 108% (±15). The corre-sponding values in pure oxygen at 2 bar were 95%

(±17) and 100% (±17), respectively. These werelowest observed levels of expression.

In room air at 4 bar (3 bar overpressure) the meanvalue for cytokeratin 10 expression was 104% (±20),and for involukrin expression 107% (±30). The cor-responding values in pure oxygen at 4 bar were 102%(±20) and 110% (±21), respectively.

A smaller, though statistically not significant (p >0.05), degree of differentiation compared with the neg-ative control, indicated by a reduced mean involukrin10 expression, was only found in keratinocyte cul-tures exposed to pure oxygen at 3 bar. Under the sameconditions, involukrin expression remained unaltered.

Evaluation of the remaining data showed a gen-eral increase in cell differentiation, which, however,according to the Wilcoxon–Mann–Whitney Test, wereonly statistically significant (p > 0.05) for the expres-sion of cytokeratin 10 following exposure to room airat pressures of 2 and 3 bar.

Discussion

The cultivation and transplantation of autologoushuman keratinocytes on various kinds of support-ing material is becoming an established method of


treatment for chronic, poorly healing wounds [13–45]. Numerous studies have demonstrated that HBOtreatment can stimulate the proliferation of fibroblastsand endothelial cells [3,9,46,47]. Hehenbergeret al.[47] stimulated fibroblasts for an hour with air orpure oxygen at pressures between 106 and 300 kPa,whereby a maximum increase in growth was achievedat 250 kPa. Tompachet al. [46] found a significantincrease in fibroblast growth only after 120 min stim-ulation, while endothelial cells required only 15 minexposure in an oxygen pressure chamber. In January1999 the first data on keratinocyte cultures exposedto hyperbaric oxygen were published. Dimitrijevichet al. [48] showed that daily 90-min exposures overa period of several days resulted in a marked reductionin cell growth. They also demonstrated that fibroblastsneed to be exposed over a period of several days ifthey are to show a significant increase in prolifera-tion. Karasek [49], who studied the relationship ofkeratinocyte growth to different oxygen concentrationsat normal pressure, observed optimal proliferation ratesat concentrations between 20% and 40%, while con-centrations of more than 50% or below 10% resultedin a marked reduction in proliferation. Reavenet al.[50] showed in 1986 that keratinocytes grow optimallyin an atmosphere containing 40% oxygen. In contrast,a publication by Horikoshiet al. [51] reports a markedproliferation optimum for keratinocytes with a pO2 inthe medium of 133 mmHg, which corresponds to anoxygen concentration of 18%.

Damaged tissue that is regenerating itself in the pro-cess of healing requires more oxygen than healthy,undamaged tissue. Oxygen is an essential nutrientfor normal tissue homeostasis, and during the heal-ing process large amounts are consumed in fibroblastdifferentiation and in the synthesis of collagen [3,7].

HBO, due to the pressure-related increase in oxygensolubility (Henry–Dalton Law), increases tissue pO2

and thus the oxygen supply to the individual cells [10].As Hunt et al. [3] reported back in 1972, repeatedincreases in tissue oxygen tension result in an increasein fibroblast activity and in higher levels of collagensynthesis. Also, the formation of granulation tissueis stimulated in the presence of hyperbaric oxygenthrough increased capillary angiogenesis [3,9]. HBOalso results in a general improvement in resistance toinfection by increasing leucocyte numbers and phago-cytosis [12]. The experimental approach in this presentstudy was to elucidate the effect of hyperbaric oxygentreatment on the growth of human keratinocyte culturesin respect to proliferation and differentiation.

The cultivation and transplantation of autologouskeratinocytes has been used for the past 20 years in thetreatment of problematic wounds [52–55]. However,there is still a lot of experimental work to be done in thevery promising field, much of which is being directedtowards improving the transplants. This work includesthe development of biomaterials as cell supports, andefforts to improve laboratory techniques, for example,by optimising culture media [45,56–58].

Based on the results already obtained from stud-ies of growth in endothelial cells and fibroblasts byTompachet al. [46] and Hehenbergeret al. [47], anincrease in keratinocyte proliferation following HBOwas expected to be found. This would offer the possibil-ity of reducing the time needed for their cultivation, aswell as of increasing vitality of the cells, which in a stateof semiconfluency have not yet begun to differentiate,but are still largely in the proliferation phase.

However, the results of this study generally indi-cate a reduction in keratinocyte proliferation in FACSscan following HBO, so that one can hardly speak ofthis parameter being favourably effected and likely toimprove the quality of autologous transplants.

Nevertheless, a very interesting observation wasthat HBO resulted in a marked increase in cytoker-atin 10 and involukrin expression, as an indication ofaccelerated cell differentiation. For the healing processitself this is not considered to be positive.In vivo thekeratinocytes are formed in the stratum basale, fromwhere they migrate, undergoing increasing differenti-ation (flattening and keratinisation), towards the sur-face of the skin. Since keratin synthesis and cornifi-cation are the main characteristics of keratinocytes, itmight be worth considering HBOin vivofollowing skincell transplantation, so as to promote differentiation ofthe cells responsible for cornification, thus thickeningthe epidermis and providing better protection againstexternal influences.

Summarising, it can be said thatin vitro hyperbaricoxygen treatment, as already reported by Dimitrijevich[48], does not have a toxic effect on cultures of humankeratinocytes. However,in vitro oxygenation of cellcultures prior toin vivo transplantation was not foundto have a favourable effect on cell proliferation oron increasing vitality of the transplant. According tothe evaluation of our results, a reduction in the timerequired for transplant cultivation cannot be achievedby this method.

However, since the observed increase in the ratedifferentiation is an interesting discovery, we arecurrently studying the possibility of stimulating a


protective function through hyperbaric oxygen ther-apy in vivo following the transplantation of autolo-gous keratinocyte cultures to problematic soft-tissuewounds.

Acknowledgements

The authors thank Professor D. Boehmer, Center ofHyperbaric and Diving Medicine at the OrthopedicUniversity Clinics in Frankfurt/Main, Germany, forhis kind collaboration. The authors thank K. Exner,MD, Director of the Department of Plastic Surgery,St. Markus-Hospital, Frankfurt/Main, Germany, forthe supply of adult human skin.

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the influence of hyperbaric oxygenation (hbo) on proliferation and differentiation of human...

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