WO2006097707A2

WO2006097707A2 - Skin composition treatment

Info

Publication number: WO2006097707A2
Application number: PCT/GB2006/000900
Authority: WO
Inventors: Penny Johnson; Damian Marshall; Gerard Markx
Original assignee: Intercytex Limited
Priority date: 2005-03-14
Filing date: 2006-03-14
Publication date: 2006-09-21
Also published as: GB0505203D0

Abstract

Compositions such as skin equivalents and methods for their manufacture. In particular, a method for modulating the tensile strength and/or elastic strength of a composition comprising living cells suspended in a 3-D support matrix, which method includes the step of subjecting the composition to ultrasound stimulation, characterised in that the ultrasound stimulation comprises alternating periods of exposure to ultrasound followed by rest periods, in a continuous cycle over a period of at least 21 days in which the duration of each period of exposure is about 1 s to 10 min, for example about 5 min or 2 min, preferably about 10 s, and in which the duration of each rest period is about 1 s to 30 min, for example about 15 min or 10 min, preferably 180 s, and in which the ultrasound stimulation has a pulse frequency of about 250 kHz to 50 MHz, for example about 500 kHz to 20 MHz, preferably 1 MHz.

Description

Skin Composition Treatment

The present invention relates to compositions such as skin equivalents and methods for their manufacture.

Bioengineered skin equivalents or skin substitutes have emerged over the past two decades, initially to replace autograft, allograft, and xenograft in burn applications. Skin equivalents have found even wider application in the treatment of chronic venous and chronic diabetic ulcers.

While autologous tissue transfers can be very effective in securing wound healing, such procedures are invasive, painful, and expensive, and cannot be performed by many wound care practitioners. In practical terms, skin equivalents represent artificial, off-the-shelf alternatives to skin grafts that avoid the pain and potential complications of harvesting, are always available in any quantity needed, and can be applied in an office setting. The ideal skin equivalent would adhere quickly to the wound and would once applied mimic the physiology and some of the mechanics of normal skin. It would be inexpensive, not subject to immune rejection by the host, and would be highly effective in accelerating tissue regeneration and wound repair. Unfortunately, this ideal skin equivalent has not yet been achieved. One of the limiting factors has been creating a skin equivalent with mechanical properties approximating that of natural skin, to allow ease of manipulation and better wound protection once applied.

Acoustic pulsed energy (ultrasound) is composed of pressure waves at a frequency above that of human audibility (approximately 20 kHz). The ultrasound wave is produced by a transducer, which transforms electrical energy into mechanical energy and produces a pressure wave with the same frequency as the electrical field applied to the transducer. The applications of ultrasound can be divided into diagnostic, disruptive and therapeutic ultrasound (1). Diagnostic ultrasound employs a frequency between 3 and 5 MHz at very low intensities to avoid tissue heating. Disruptive ultrasound uses low l frequencies (20 to 60 kHz) and high intensities, and therapeutic ultrasound uses mid-range frequencies and high intensity to exert and effect through heat generation.

Over recent years ultrasound has become increasingly used as a supplementary therapy to promote bone and wound healing (2, 3, 4, 5). It has been shown that ultrasound has the ability to promote calcium uptake (6) as well as increase DNA and protein synthesis in fibroblast cells (7).

The molecular mechanism by which low intensity pulsed ultrasound exerts an effect on fibroblast has recently been investigated (8). Zhou et al. showed that ultrasound uses integrins as mechanotransducer converting ultrasound stimuli into biochemical signals (8) in a similar manner to other forms of mechanical stress (9). In response to ultrasound, fibroblasts were shown to induce formation of F-actin stress fibers through activation of RhoA, and recruits paxillin to focal adhesions by a ROCK-dependent pathway (8). The Rho family of small GTPases and downstream targets such as ROCK regulate cytoskeletal organization (10) in particular the formation of stress fiber and focal adhesion complexes (11, 12). Furthermore, RhoA/ROCK acts as an upstream regulator of the ERK cascade to induce cell proliferation in response to ultrasound stimulation in primary human skin fibroblasts (8).

Therapeutic use of ultrasound as a promoter of wound healing involves the application of low intensity pulsed ultrasound applied to the wound for several minutes (2, 3, 4, 5), a technique that has been copied in vitro (7, 8). Zhou et al. used a .6 well plate format administering ultrasound simultaneously to all 6 wells at low frequency (1.5 MHz, 200μs pulse modulated at 1 kHz) in a single burst for either 6 or preferably 11 minutes, or in one experiment seven sequential bursts of either 6 or 11 minutes once per day for seven days, to elicit a response in fibroblasts (8). Zhou et al. found an increase I the total cell number upon sequential ultrasound stimulation, while a single 11 minute ultrasound stimulation resulted in an increase in bromodeoxyuridine incorporation and a promotion of cell proliferation via activation of integrin receptors and a Rho (a small G protein) / ROCK (Rho-associated coiled-coil- containing protein kinase) / ERK (extracellular signal-regulated kinase) signalling pathway (8). Reher et al. applied a transducer directly into the medium of a 6 well plate (each well individually) and applied ultrasound directly onto the cells for 5 minutes (7). Both these studies used a monolayer of fibroblast cells.

The present inventors have found that ultrasound can surprisingly have a marked effect on improving several mechanical properties important for compositions such as skin equivalents.

According to the present invention there is provided a method for modulating the tensile strength and/or elastic strength of a composition comprising living cells, in which the method includes the step of subjecting the composition to ultrasound stimulation.

Collagen in vivo has characteristic flexibility, strength and elasticity (12, 13). This has not yet been achieved in vitro through tissue culture to the extent of naturally occurring collagen in the body.

Compositions such as skin equivalents can be examined to assess their suitability to act as a skin replacement. Such compositions should possess characteristics and dynamics appropriate for example for the role of skin replacement in strength, elasticity and durability. The present invention provides a new method for improving such characteristics in compositions such as skin equivalents. The method may also be used to modulate (for example increase) other mechanical properties of the composition, for example mechanical properties such as durability, elasticity modulus, yield point, elastic limit, proportional limit, breaking point, lateral limit, permeability, compression, and/or hardness.

The tensile strength and/or elastic strength of the composition is preferably increased. The composition is preferably a connective tissue composition, for example a soft connective tissue composition such as a skin equivalent. The mechanical properties of the skin equivalent are therefore preferably modulated by means of the method of the invention to be more similar to natural skin.

The ultrasound stimulation may comprise alternating periods of exposure to ultrasound followed by rest periods. For example, the duration of each period of exposure may be about 1 s to 10 min, for example about 5 min or 2 min, preferably about 10 s. The duration of each rest period may be about 1 s to 30 min, for example about 15 min or 10 min, preferably 180 s. The duration of each exposure period is preferably proportional to the duration of each rest period. Particularly if longer exposure periods are used, the composition being stimulated by ultrasound may require cooling to avoid or minimise the effects of overheating. One example of an stimulation regime is 10 s of exposure to ultrasound followed by 180 s of no exposure.

It is well-known that collagen increases in cells subjected to physical stress. However, the signal supplied by a continuous stress event may diminish as cells adapt to that stress. Using ultrasound as the stress, the object of the invention can be subjected to increasing stress over a period whereby the stress is gradually "ramped-up" in either duration or frequency.

The composition may be subjected to ultrasound stimulation in cycles of increasing intensity for up to 50, 49 or 45 days, for example up to 30, 21, 15, or 8 days. In a preferred embodiment, the composition is subjected to a stimulation regime comprising 10 s of exposure to ultrasound followed by 180 s of no exposure, alternating continuously over a first period, of for example 7 days, followed by a stimulation regime comprising 20 s of exposure to ultrasound followed by 170 s of no exposure, alternating continuously over a second period , of for example 7 days, followed by a stimulation regime comprising 30 s of exposure to ultrasound followed by 160 s of no exposure, alternating continuously over a third period, of for example 7 days, and increasing exposure by 10 s and reducing the period of no exposure by 10 s in subsequent periods until by a seventh period, the exposure regime consists of 70 s of exposure followed by 120 s no exposure.

Alternatively, the composition may be subjected to ultrasound stimulation in cycles of increasing intensity for up to 50, 49 or 45 days, for example up to 30, 21, 15, or 8 days. In a preferred embodiment, the composition is subjected to a stimulation regime comprising 10 s of exposure to ultrasound followed by 180 s of no exposure, alternating continuously overa first period, of for example 7 days, followed by a stimulation regime comprising 10 s of exposure to ultrasound followed by 150 s of no exposure, alternating continuously over a second period, of for example 7 days, followed by a stimulation regime comprising 10 s of exposure to ultrasound followed by 120 s of no exposure over a third period, of for example 7 days, followed in a fourth period, of for example 7 days, by a stimulation regime consisting of 10 s of exposure to ultrasound followed by 90 s of no exposure, alternating continuously, followed in a fifth period, of for example 7 days, by a stimulation regime consisting of 10 s of exposure to ultrasound followed by 60 s of no exposure, alternating continuously, followed in a sixth period, of for example 7 days, by a stimulation regime consisting of 10 s of exposure to ultrasound followed by 30 s of no exposure alternating continuously, until by the seventh period, the exposure regime consists of 10 s of exposure followed by 10 s no exposure, in a continuous cycle for a further period, of for example 7 days, whereby the exposure period is 10 s and the period of no exposure (or rest period) decreases from 180 s to a minimum of 10s.

The increase in each period of exposure or frequency of exposure of the composition, or a combination of both, should be regulated so as not to cause irretrievable contraction of the composition.

The ultrasound stimulation may have a pulse frequency of about 250 KHz to 50 MHz, for example about 500 KHz to 20 MHz, preferably 1 MHz. A pulse frequency which is too low (for example below 250 MHz) may cause cavitation in the cells, where bubbles form and expand and implode to form holes in the cells. Conversely, particle manipulation may be complicated where the pulse frequency which is too high (for example above 50 MHz) because the wavelength of the ultrasound is smaller and high intensity generation of ultrasound is difficult.

The ultrasound may have an output intensity of about 5 to 1000 mW/cm².

In one embodiment, the ultrasound stimulation modulates (for example, increases) collagen and/or elastin production by the living cells.

The cell may be mammalian, for example human. Cells of the present invention may include fibroblasts, keratinocytes, stratum germinativum cells, and combinations or admixtures of such cells. The cells may be substantially fibroblasts, for example 90% to 100%, preferably 95% to 99.5%, and more preferably 97.5% to 99% fibroblasts. The fibroblasts are preferably dermal fibroblasts, for example human dermal fibroblasts.

The cells are preferably allogeneic, although autologous and/or xenogeneic cells may be used.

The cells are preferably actively synthetic or able to become actively synthetic rapidly.

The composition may comprise a support matrix. The support matrix may comprise a clottable or gelling substance such as fibrin, collagen, fibronectin, vitronectin, alginate, agar, collagen, PVA, hyaluronic acid, modified starches, carrageenans, carob, gelatine, pectin or gelling agent.

The cells are preferably suspended in the support matrix.

The matrix is preferably non-pyrogenic and/or sterile. In a preferred embodiment, the matrix is a fibrin matrix. The fibrin may have a concentration in the composition in the range of 3 to 12 mg.ml^"1, for example 7 to 12 mg.ml^"1 or 3 to 5 mg.ml^"1. The fibrin matrix is preferably formed by thrombin-mediated polymerisation of fibrinogen.

The composition may further comprise a protease inhibitor suitable for preventing breakdown of the matrix. The inhibitor may be a serine protease inhibitor, most preferably one or more selected from the list consisting of aprotinin, e-aminocaproic acid and tranexamic acid. Preferably, especially where the concentration of protein is in the range 7 to 12 mg.ml^"1, the protease inhibitor is aprotinin. Alternatively, especially where the concentration of protein is in the range 3 to 5 mg.ml^"1, the protease inhibitor may be tranexamic acid.

Also provided according to the present invention is a method of manufacturing a composition, for example a skin equivalent, comprising living cells and having modulated tensile strength and/or elastic strength, wherein the method includes the step of subjecting the composition to ultrasound stimulation as defined herein.

The composition and skin equivalent formed by the method of manufacture are also within the scope of the present invention.

Also provided is the use of ultrasound stimulation as defined herein in the manufacture of a composition for the treatment of a skin lesion.

Further provided is the use of ultrasound stimulation as defined herein in the manufacture of a skin equivalent for the treatment of a skin lesion.

In a further aspect there is provide the use of the composition or the skin equivalent of the invention in the manufacture of a medicament for the treatment of a skin lesion. In another aspect of the invention there is provided a method of treating a patient suffering from a skin lesion comprising topically applying of a composition or a skin equivalent of the invention.

Examples of skin lesions to which the present compositions can be applied include burns, venous ulcers, diabetic ulcers, pressure sores, and iatrogenic grating wounds.

Specific embodiments of the invention will now be described by way of example only with reference to the accompanying figures, in which:

Fig. 1 is a diagram showing administration of media in each well of experimental 6 well culture plates according to the experiments as described below;

Fig. 2 depicts an outline of ultrasound administration. (A) Electrical current is generated in a primary generator (PS), the current is then amplified using an amplifier (Amp) before being parsed through a timer controlled switch (S) to the transducers. (B) Photograph showing a close up of the transducer (arrow) and transducer housing, only the lower right hand plate receives current. (C and D) diagrams showing route of administration of ultrasound from the ultrasound plate to the cells for both monolayers (C) and cells in fibrin gels (D), in both cases the gap between the ultrasound plate and the culture well is filled using coupling gel to direct the ultrasound pulse;

Fig. 3 shows a standard curve of collagen concentration for collagen standards with line of best fit and equation for the line (Y=Mx+C) for a first experiment;

Fig. 4 is a graph showing differences in collagen concentration per lOOμl for each experimental sample in the first experiment;

Fig. 5 is a photograph showing that, after 23 days in culture under the different regimen, there is a visible difference in the structure of the gels tested in a second experiment;

Fig. 6 is a further standard curve of collagen concentration for the collagen standards with line of best fit and equation for the line (Y=Mx+C) for the second experiment; and

Fig. 7 is a graph showing differences in collagen concentration per lOOμl for each experimental sample in the second experiment.

Fig. 8 shows elastin protein expression in SKN constructs grown in TM or DMEM- 10 and treated or not treated with ultrasound for 49 days. Equal amounts of protein extracts from pepsin digested constructs grown in DMEM- 10, TM and in TM with ultrasound treatment were loaded onto the SDS PAGE gels and were transferred onto the membrane. The blot was probed with anti elastin antibodies raised in rabbit at concentration 1:200 and detected with swine anti rabbit HRP secondary at concentration 1 :2000. An increase in high molecular weight elastin was observed in the sample fed in TM for 49 days and treated with ultrasound in comaprison to the sample fed in TM but not exposed to ultrasound. There was no high molecular weight elastin detected in sample fed in DMEM- 10 control media.

Fig. 9 shows Masson Trichrome stained histology sections. SKN constructs grown in TM for 49 days and exposed to ultrasound, and SKN construct grown in TM alone both had good collagen deposition. SKN constructs grown in DMEM- 10 contained a lot a fibrin, indicating a lack of conversion from fibrin to collagen by the fibroblasts in these constructs

Fig. 10 shows the collagen immunohistochemistry of SKN constructs. (A) The high magnification photograph of SKN construct grown in TM and exposed to ultrasound for 49 days shows a good deposition of collagen (green). (B) The low magnification photograph of SKN construct grown in TM for 49 days shows good deposition of collagen (green). (C) The high magnification photograph of SKN construct grown in DMEM- 10 media for 49 days shows poor deposition of collagen (green).

Experimental

In the study outlined below we investigate the effects of ultrasound on collagen production in fibroblast cultures in both monolayer and in fibrin gels. In contrast to prior art studies, we have employed a stress-relaxation cycle of ultrasound administration and continue administration of ultrasound, specifically over a period of 21 days.

Materials and Methods Cell culture

For monolayer cultures, human dermal fibroblasts cells obtained from neonatal foreskin were seeded into the upper left hand wells, lower left hand wells and lower right hand wells of 6 well culture plates at a concentration of 1.5x10⁶ cells per well. The cells were incubated at 37⁰C, 5% CO₂ and allowed to grow to confluence with medium changes (DMEM-IO) every 3 days, before administration of ultrasound.

Total medium also used for cell culture contained the following ingredients (supplier indicated in brackets): 3 parts Dulbecco'sModified Eagle's Medium (DMEM) :1 part Hams F- 12 medium (BioWhittaker or Cambrex)) 2-10 % NBCS or FCS (InVitrogen)

4 mM GlutaMAX 2 mM L-glutamine (InVitrogen)

5 ng/ml epidermal growth factor (Sigma Corp.) 5 ng/ml TFG-beta (R&G Systems)

0.4 μg/ml hydrocortisone (Sigma)

IxIO^"4 M ethanolamine (Fluka, #02400 ACS grade or Sigma) +IxIO^"4 M o- phosphoryl-ethanolamine (Sigma) 5 μg/ml transferrin + 20 pM triiodothyronine (Sigma) 6.78 ng/ml selenium (Sigma Aldrich Fine Chemicals) 0.2 μg/ml L-proline (Sigma) and 0.1 μg/ml glycine (Sigma). For human dermal fibroblast cells suspended in fibrin gels, 6x10⁵ cells were mixed with 856μl Fibrinogen (5mg/ml) and 210μl Thrombin. The mixture was cast directly into the centre upper left hand wells, lower left hand wells and lower right hand wells of a 6 well culture plate in an approximately 2cm by 2cm square pattern and allowed to set before addition of medium.

Once the monolayer cultures were confluent and the fibrin gel cultures were set, fresh medium was added prior to administration of ultrasound as highlighted in Fig. 1.

Ultrasound administration

Ultrasound was administered using custom built apparatus as shown in Fig. 2A. Briefly, electrical current is generated in a primary generator (Fig. 2A "PS"), the voltage is then amplified to 80 Vpk-pk and a frequency of 1 MHz using an amplifier (Fig. 2A "Amp"). The current is then passed through a switch mechanism (Fig. 2A "S") controlled on a timer to the transducer located in the lower right hand portion of the transducer housing (Fig. 2B arrow). The transducer converts the electrical signal into an ultrasound wave which is channelled to the cell cultures through coupling medium (Figs 2C and 2D).

In all cases the lower right hand well of a 6 well culture plate was exposed to ultrasound at an exposure frequency of 1 MHz for 10 seconds followed by 180 seconds of no exposure (controlled through the timer switch) in continual cycles for at least 21 days. The samples were maintained at in an incubator at 37⁰C, 5% CO₂ for the duration of the experiment and the Medium was changed every 3-4 days.

Measurement of collagen

Collagen content of the samples after at least 21 days of ultrasound exposure was assessed using a soluble collagen assay (Sircol assay) according to the manufactures instructions. Briefly, ImI of 0.5M acetic acid containing 5mg/ml pepsin was added to the samples and incubated at room temperature for 1 hour. After 1 hour the samples were dissociated by drawing the sample into a syringe through progressively finer needles up to a maximum of 26G. Once a homogenous solution was achieved the samples were transferred to a 1.5ml micro-centrifuge tube and further incubated for at least 1 hour at 37⁰C on a rotating mixer, if after 1 hour the samples were not fully digested (clear solution) the digestion was allowed to proceed overnight. Meanwhile, collagen control samples were set up using various known concentration of collagen. Once the experimental samples were digested lOOμl was removed and placed into a fresh micro-centrifuge tube (the remaining samples was stored at -8O⁰C), ImI of Sircol dye reagent was added to all the samples (collagen control and experimental samples) and the sample were incubated at room temperature for 1 hour on a rotating mixer. After 1 hour the samples were centrifuged at i-iOjOOOxg for 10 minutes to pellet the collagen and the unbound dye was poured away, the inside of the tube was wiped to remove as much unbound dye as possible without disturbing the pellet. Next, ImI of Sircol alkali reagent was added to each tube and the samples were incubated for 30 minutes at room temperature on a rotating mixer. After the incubation was complete lOOμl of each sample was added in duplicate to a 96 well plate and read on a plate reader at a wavelength 540nm. A standard curve of collagen concentration was generated using the collagen control samples and this was used to assess collagen content in the experimental samples.

Mechanical strength testing

Assays to test mechanical strength include stretching and stretch cycling of constructs to determine elasticity modulus, yield point, elastic limit, proportional limit and breaking point. These can be calculated using the computer software, for example as provided by Mecmesin.

The 'boring' or 'dough inflation' test (for example using equipment provided by Stable Micro Systems) of constructs can be used to determine lateral stretch and lateral deformation values. This is carried out by either inserting a probe laterally through the constructs (boring) or by weighing down the edges and applying air pressure through the middle of the construct (dough inflation). Permeability may also be assessed in a test similar to inflation assay.

Compression and hardness may be assessed using probing techniques.

Constructs are placed on a strength-testing device, either alone or on a supportive backing material, such as glass slides. Constructs may also be sutured at the corners to maintain shape until clamped. The constructs are grasped between the two clamps on a machine, which will be oriented horizontally or vertically. Software supplied with Stable Micro Systems machines (for example) allows for a range of variables, such as mm/minute, Newton/gram force, cycling regimes and so on.

Results

Experiment 1 - Ultrasound on monolayer cultures Once the monolayer cultures had reached confluence the ultrasound regimen was initiated. The experiment was performed in triplicate with three culture receiving DMEM- 10 medium, 3 cultures receiving Total medium and 3 cultures receiving Total medium and ultrasound for 10 seconds followed by 180 seconds of no ultrasound in cycles for 21 days. During this 21 day period one of the ultrasound plates became disconnected and so the samples were removed from the study (6 samples remained). Furthermore, on day 16 one of the samples growing in Total medium contracted, and so was also removed from the study leaving 2 DMEM-IO cultures, 1 Total medium culture and 2 Total medium plus ultrasound cultures remaining. On day 21 both ultrasound cultures contracted, however, as this was the end of the experiment both samples were used for assessment of collagen content.

Collagen content was assessed using the Sircol assay and a standard curve of collagen concentration was derived using the absorbance of the samples at 540nm (Fig. 3). From the collagen standards absorbance's a line of best fit (Y=Mx+C) was ascertained and this equation was re-organised (x=(Y-C)/M) to give the collagen concentration (x) of the experimental samples using their absorbances (Y) at 540nm. For example, the line of best fit for the collagen standards (Fig. 3) is:

Y=0.0045x+0.0145

Therefore the average absorbance (Y) of the DMEM-10 (1) sample (0.028, Table 1) gives the equation:

X=(0.028-0.0145)/0.0045

This gives a collagen concentration per lOOμl of 4.089μg or 40.9μg of collagen in the original ImI digested construct (Table 2).

The absorbances and collagen concentrations of the experimental samples are shown in Table 1 and represented graphically in Fig. 4, total collagen concentration for experimental samples are shown in Table 2.

DMEM-10 D DMMEEMM-10 Total Medium Ultrasound Ultrasound

(1) (2) (D (2) A Abbss ll 0 0..002277 0 0..003333 0.187 0.304 0.216

Abs 2 0.028 0.033 0.190 0.316 0.233

Ave. 0.028 0.033 0.189 0.310 0.225

DMEM DMEM Total Medium Ultrasound Ultrasound

((11)) ((22)) (1) (2)

Collagen 4.089 5.311 39.867 66.867 47.867 per lOOμl

Table 1 - The duplicate absorbance reading (and average reading) for each experimental sample. Also shown are the collagen concentration per lOOμl for each sample. Sample Total collagen

DMEM-IO (1) 40.9 μg

DMEM-IO (2) 53.1 μg

Total Medium 398.7 μg

Ultrasound (1) 668.7 μg

Ultrasound (2) 478.7 μg

Table 2 - Total collagen content of each ImI digested sample.

Experiment 2 - Ultrasound on cultures in fibrin gels

Fibroblast cultures cast in fibrin gels were transferred into either DMEM-IO or Total medium once the gel had set. 3 cultures were grown in DMEM- 10 medium, 3 cultures were grown in Total medium and 3 cultures were grown in Total medium plus ultrasound, 10 seconds on followed by 180 seconds of no ultrasound in cycles for 23 days. After 23 days the gels (Fig. 5) were digested with Pepsin and digestible collagen content was ascertained. Again a standard curve of collagen concentration was devised (Fig. 6) and the equation for the line of best fit was used to ascertain the collagen concentration per lOOμl of each of the experimental samples (Table 3 and Fig. 7). The total concentration of pepsin digestible collagen per sample is shown in Table 4.

OMEM(I) DMEM (2) DMEM (3) Total (1) Total{2) Total (3) Ultrasound (1) Ultrasound (2) Ultrasound (3)

Reading 1 ; ^"ooi6 0012 0015 ^"θO38 002 0009 0054 0034 009

Reading 2 ¹ 0017 0011 0013 0037 0019 0008 0052 003 0089

Avetaαe 00165 00115 0014 00375 00195 00085 0053 0032 00895

DMEM(I) DMEM (2) DMEM (3) Total (1) Total (2) Total (3) ' Ultrasound (1) Ultrasound (2) Ultrasound (3) collagen per IQQuI 835 788 812 1033 864 760 if 79 981 1522

Table 3 - The duplicate absorbance reading (and average reading) for each experimental sample. Also shown are the collagen concentration per lOOμl for each sample.

Table 4 - Total pepsin digestible collagen content of each ImI digested sample.

Conclusions

Experiment 1 — monolayer cultures

After 21 days in culture the monolayer cultures grown in Total medium and exposed to ultrasound had a much higher pepsin digestible collagen content than both the culture grown in total medium alone and the cultures grown in DMEM-IO medium. This suggests that exposure of the cells to the ultrasound regime has a positive effect on collagen production by the cells.

There is significantly more collagen in the cultures grown in Total medium (with and without ultrasound) than in the cultures grown DMEM- 10 medium. This results confirms published results that the Total medium has a collagen boosting effect on fibroblast cells.

Experiment 2 — Fibroblasts in fibrin gels After 23 days in culture the samples grown in Total medium and exposed to ultrasound had an increase in pepsin digestible collagen compared to the cultures grown in Total medium and the cultures grown in DMEM-10 medium. The differences in collagen concentration between all the samples were smaller than observed in the monolayer cultures. This may be due to how the collagen is laid down by the cells in the fibrin matrix. Also, the technique used in this experiment only measures collagen that can be digested by pepsin and other collagen present in the gel samples may therefore be missed using this assay. It remains to be seen if other collagen assays detect different collagen levels in the samples.

The samples grown in Total medium and exposed to ultrasound were more difficult to break down during the pepsin digestion step and required more mechanical disruption using syringes with progressively finer needles. The samples grown in DMEM- 10 medium on the other hand required no mechanical disruption and broke down during the initial 1 hour pepsin digestion step. This would suggest an increase in tensile strength of the sample grown in Total medium.

The samples grown in Total medium and the samples grown in Total medium with ultrasound were very malleable and could be folded and unfolded without loss of shape or integrity. This was not true for the samples grown in DMEM- 10 medium. This again would indicate increased tensile strength.

Mechanical and/or tensile strength can be quantified using techniques known in the prior art, for example as described above.

References

(1) Ziskin MC. Applications of ultrasound in medicine - comparison with other modalities. In: Ultrasound: medical applications biological effects and hazard potential. New York Press: 49-61, 1987

(2) Rubin, C, Bolander, M., Ryaby, J. P., and Hadjiargyrou, M. J Bone Joint Surg Am. Feb;83-A(2):259-70, 2001

(3) ByI NN, McKenzie AL, West JM, Whitney JD₅ Hunt TK, Sheuenstuhl HA. Arch Phys Med Rehabil.;73:656-64. 1992 (4) Taskan L Ozyazgan I, Tercan M, Kardas HY, Balkanli S, Saraymen R, Zorlu U, Ozugul Y. Plast Reconstr Surg.;100(4):966-72. 1997

(5) ByI NN, McKenzie A, Wong T, West J, Hunt TK. J Orthop Sports Phys Ther. Nov;18(5):619-28. 1993

(6) Mortimer AJ, Dyson M. Ultrasound Med Biol; 14(6):499-506., 1988 (7) Reher P, Doan N, Bradnock B, Meghji S and Harris M. Cytokine 11: 416- 423, 1999

(8) Zhou S, Schmelz A, Seufferlein T, Li Y, Zhao J, Bachem MG. J Biol Chem. Dec 24;279(52):54463-9, 2004

(9) Pavalko, F. M., Chen, N. X., Turner, C. H., Burr, D. B., Atkinson, S., Hsieh, Y. F., Qiu, J., and Duncan, R. L. Am. J. Physiol. 275, C1591-C1601, 1998

Hall, A. Science 219, 509-514, 1998

(10) Clark, E. A., King, W. G., Brugge, J. S., Symons, M., and Hynes, R. O. J. Cell Biol. 142, 573-586, 1998

(11) D'Addario, M., Arora, P. D., Fan, J., Ganss, B., Ellen, R. P., and McCulloch, C. A. J. Biol Chem. 276, 31969-31977, 2001

(12) Neidert et al. Biomaterials 23, 3717-3731, 2002

(13) Henry et al. Technique to Characterise the Mucosal Layer of Large Intestine. NUI, Galway Faculty of Engineering Research Day (2004), pi -2 (see http://corrib.it.nuigalway.ie/ResearchDay2004/Mechanical/HenryJerome.pdf).

Claims

1. A method for modulating the tensile strength and/or elastic strength of a composition comprising living cells suspended in a 3-D support matrix, which method includes the step of subjecting the composition to ultrasound stimulation, characterised in that the ultrasound stimulation comprises alternating periods of exposure to ultrasound followed by rest periods, in a continuous cycle over a period of at least 21 days in which the duration of each period of exposure is about 1 s to 10 min, for example about 5 min or 2 min, preferably about 10 s, and in which the duration of each rest period is about 1 s to 30 min, for example about 15 min or 10 min, preferably 180 s, and in which the ultrasound stimulation has a pulse frequency of about 250 kHz to 50 MHz, for example about 500 kHz to 20 MHz, preferably 1 MHz.

2. The method according to claim 1 wherein the ultrasound stimulation comprises alternating periods of exposure to ultrasound followed by rest periods, in a continuous cycle over a period of at least 49 days.

3. The method according to claim 1 or 2, in which the composition is a skin substitute.

4. The method according to claim 1 or 2, in which the composition is a skin equivalent.

5. The method according to any preceding claim, in which the tensile strength and/or elastic strength of the composition is increased.

6. The method according to any preceding claim, in which the ultrasound stimulation modulates (for example, increases) collagen and/or elastin production by the living cells.

7. The method according to any preceding claim, in which the cells are mammalian, for example human.

8. The method according to any preceding claim, in which the cells are fibroblasts, for example neonatal dermal fibroblasts.

9. The method according to any preceding claim, in which the support matrix comprises a clottable or gelling substance such as fibrin, collagen, fibronectin, vitronectin, alginate, agar, collagen, PVA, hyaluronic acid, modified starches, carrageenans, carob, gelatine, pectin or gelling agent.

10. A method of manufacturing a composition comprising living cells and having modulated tensile strength and/or elastic strength, wherein the method includes the step of subjecting the composition to ultrasound stimulation as defined in any of claims 1 to 9.

11. The method of claim 10₅ in which the composition is a skin substitute.

12. The method of claim 10, in which the composition is a skin equivalent.

13. A composition formed by the method of claim 10.

14. A skin substitute formed by the method of claim 11.

15. A skin equivalent formed by the method of claim 12.

16. Use of ultrasound stimulation as defined in any of claims 1 to 9 in the manufacture of a composition for the treatment of a skin lesion.

17. Use of ultrasound stimulation as defined in any of claims 3 and 5 to 9 in the manufacture of a skin substitute for the treatment of a skin lesion.

18. Use of ultrasound stimulation as defined in any of claims 4 to 9 in the manufacture of a skin equivalent for the treatment of a skin lesion.

19. Use of the composition of claim 13 or the skin substitute of claim 14 or the skin equivalent of claim 15 in the manufacture of a medicament for the treatment of a skin lesion.

20. A method of treating a patient suffering from a skin lesion comprising topically applying the composition of claim 13 or the skin substitute of claim 14 or the skin equivalent of claim 15 to the skin lesion.