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EP-3529614-B1 - METHODS FOR IDENTIFYING A NON-HEALING SKIN WOUND AND FOR MONITORING THE HEALING OF A SKIN WOUND

EP3529614B1EP 3529614 B1EP3529614 B1EP 3529614B1EP-3529614-B1

Inventors

  • WOLFF-WINISKI, BARBARA
  • Stütz, Anton
  • DÖRFLER, Petra

Dates

Publication Date
20260506
Application Date
20171023

Claims (14)

  1. An in vitro method for identifying a skin wound in an individual as being a non-healing skin wound or healing skin wound, the method comprising: a) measuring i) the proliferation of primary fibroblast cells in the presence of a wound exudate sample or wound biofilm sample obtained from said skin wound, and/or ii) the fibroblast-derived matrix formation by primary fibroblast cells in the presence of a wound exudate sample or wound biofilm sample obtained from said skin wound, and b) identifying the skin wound as being a non-healing skin wound in case the value(s) obtained in i) and/or ii) is/are below a control value established in the absence of wound exudate or wound biofilm, or identifying the skin wound as being a healing skin wound in case the value(s) obtained in i) and/or ii) is/are equal to or above a control value established in the absence of wound exudate or wound biofilm, preferably wherein the value(s) in a) is/are measured at least in triplicate and/or a statistical significance is established.
  2. An in vitro method for monitoring the healing of a skin wound in an individual, the method comprising: a) measuring i) the proliferation of primary fibroblast cells in the presence of a wound exudate sample or wound biofilm sample obtained from said skin wound at a first time point, and/or ii) the fibroblast-derived matrix formation by primary fibroblast cells in the presence of a wound exudate sample or wound biofilm sample obtained from said skin wound at a first time point, b) optionally identifying the skin wound as being a non-healing skin wound at said first time point in case the value(s) obtained in a)i) and/or a)ii) is/are below a control value established in the absence of wound exudate or wound biofilm, or identifying the skin wound as being a healing skin wound at a first time point in case the value(s) obtained in a)i) and/or a)ii) is/are equal to or above a control value established in the absence of wound exudate wound biofilm, c) measuring i) the proliferation of primary fibroblast cells in the presence of a wound exudate sample or wound biofilm sample obtained from said skin wound at a second time point, and/or ii) the fibroblast-derived matrix formation by primary fibroblast cells in the presence of a wound exudate sample or wound biofilm sample obtained from said skin wound at a second time point, d) optionally identifying said skin wound as being a non-healing skin wound at said second time point, in case the value(s) obtained in c)i) and/or c)ii) is/are below a control value established in the absence of wound exudate or wound biofilm, or identifying the skin wound as being a healing skin wound at said second time point in case the value(s) obtained in c)i) and/or c)ii) is/are equal to or above a control value established in the absence of wound exudate or wound biofilm, e) A) identifying a skin wound at a second time point to exhibit improved healing in case the value obtained in c)i) at said second time point is higher than the value obtained in a)i) at said first time point, and/or the value obtained in c)ii) at said second time point is higher than the value obtained in a)ii) at said first time point, with the proviso that the value obtained in a)i) at said first time point and/or a)ii) at said first time point is equal to or below a control value established in the absence of wound exudate or wound biofilm, or B) identifying a skin wound at a second time point to exhibit worsened healing in case the value obtained in c)i) at said second time point is lower than the value obtained in a)i) at said first time point, and/or the value obtained in c)ii) at said second time point is lower than the value obtained in a)ii) at said first time point, with the proviso that the value(s) obtained in c)i) and/or c)ii) at said second time point is/are equal to or below 100% of a control value established in the absence of wound exudate or wound biofilm, and f) optionally repeating steps a) to e) at one or more later time points, thereby monitoring the healing of the skin wound, preferably wherein the first time point and the second time point are separated by between 6 hours and 12 months, and/or the values are measured at least in triplicate and/or a statistical significance is established.
  3. The method according to claim 1, wherein the method comprises a) measuring i) the proliferation of primary fibroblast cells in the presence of a wound exudate sample or wound biofilm sample obtained from said skin wound, and ii) the fibroblast-derived matrix formation by primary fibroblast cells in the presence of a wound exudate sample or wound biofilm sample obtained from said skin wound, and b) identifying the skin wound as being a non-healing skin wound in case the value obtained in a)i) is below a control value established in the absence of wound exudate or wound biofilm, and the value obtained in a)ii) is below a control value established in the absence of wound exudate or wound biofilm, preferably wherein the values obtained in a)i) and a)ii) are at least 10% below the respective control values, more preferably wherein the values obtained in a)i) and a) ii) are at least 15%, even more preferably are at least 20% below the respective control values, or identifying the skin wound as being a healing skin wound in case the value obtained in a)i) is equal to or above a control value established in the absence of wound exudate or wound biofilm, and the value obtained in a)ii) is equal to or above a control value established in the absence of wound exudate or wound biofilm, preferably wherein the values obtained in a)i) and a)ii) are at least 10%, more preferably at least 15%, even more preferably are at least 20%, above the respective control value, preferably wherein a combined value is established for the values obtained in a)i) and a)ii) and/or the values in a) are measured at least in triplicate and/or a statistical significance is established.
  4. The method according to claim 1 or 3, wherein step a) further comprises the following step: iiia) measuring the proliferation of HaCaT cells in the presence of a wound exudate sample or wound biofilm sample obtained from said skin wound, and wherein step b) comprises: b) identifying the skin wound as being a non-healing skin wound in case at least two, preferably three of the values obtained in i) to iiia) are below the respective control values established in the absence of wound exudate or wound biofilm, more preferably wherein the values obtained in i) and/or ii) and iiia) are at least 10%, more preferably at least 15%, below the respective control value, or identifying the skin wound as being a healing skin wound in case at least two, preferably three of the values obtained in i) to iii) are equal to or above the respective control values established in the absence of wound exudate or wound biofilm, more preferably wherein the values obtained in i) and/or ii) and iiia) are at least 10%, more preferably at least 15%, above the respective control value, preferably wherein a combined value is established for the values obtained in i) and/or ii) and iiia).
  5. The method according to claim 1, 3 or 4, wherein step a) further comprises one, two or three of the following steps iiib) to iiid) or one, two, three or four of the following steps iiib) to iiie): iiib) measuring the amount(s) of one or more M1 marker(s) and one or more M2 marker(s) in the supernatant of macrophages incubated with a wound exudate sample or wound biofilm sample obtained from said skin wound, wherein the macrophages are in co-culture with fibroblasts, wherein the one or more M1 markers are selected from CXCL10 and IL-23p19, and the one or more M2 markers are selected from CCL22 and CCL18, iiic) measuring the amount(s) and/or frequency distribution(s) of one or more M1 cell surface marker(s) and one or more M2 cell surface marker(s) on macrophages incubated with a wound exudate sample or wound biofilm sample obtained from said skin wound, wherein the macrophages are in co-culture with fibroblasts, wherein the one or more M1 cell surface markers are selected from CD38, CD64 and CD197, and wherein the one or more M2 cell surface markers are selected from CD200 receptor, CD206 and CD209, iiid) measuring the expression level(s) of one or more M1 marker mRNA(s) and one or more M2 marker mRNA(s) in macrophages incubated with a wound exudate sample or wound biofilm sample obtained from said skin wound, wherein the macrophages are in co-culture with fibroblasts, wherein the one or more M1 marker mRNA(s) are selected from CD38, CD64, CD197, CXCL10 and IL-23p19, and the one or more M2 marker mRNA(s) are selected from CD200 receptor (CD200R), CD206, CD209, CCL22 and CCL18, iiie) measuring the amount(s) of one or more cytokine markers in the supernatant of macrophages incubated with a wound exudate sample or wound biofilm sample obtained from said skin wound, wherein the macrophages are in co-culture with fibroblasts, and wherein the one or more cytokine markers are selected from IL-1alpha, IL-1beta and TNF-alpha, and wherein step b) comprises: b) identifying the skin wound as being a non-healing skin wound in case at least two, preferably three, four, five or six of (1) to (6) or at least two, preferably three, four, five, six or seven of (1) to (7) are fulfilled: (1) the value obtained in i) is below the respective control value established in the absence of wound exudate or wound biofilm, (2) the value obtained in ii) is below the respective control value established in the absence of wound exudate or wound biofilm, (3) the value obtained in iiia) is below the respective control value established in the absence of wound exudate or wound biofilm, (4) the ratio of amount(s) of one or more M1 marker(s) to the amount(s) of one or more M2 marker(s) obtained in iiib) is/are above a cut-off value, (5) the ratio of amount(s) and/or frequency distribution(s) of one or more M1 cell surface marker(s) to the amount(s) and/or frequency distribution(s) of one or more M2 cell surface marker(s) obtained in iiic) is/are above a cut-off value, in particular wherein the ratio is selected from a CD38/CD209 ratio, a CD197/CD209 ratio and a CD197/CD206 ratio, (6) the ratio of expression level(s) of one or more M1 marker mRNA(s) to the expression level(s) of one or more M2 marker mRNA(s) obtained in iiid) is/are above a cut-off value, (7) the value obtained in iiie) is above a cut-off value, or identifying the skin wound as being a healing skin wound in case at least two, preferably three, four, five or six of (1) to (6) or at least two, preferably three, four, five six or seven of (1) to (7) are fulfilled: (1) the value obtained in i) is equal to or above the respective control value established in the absence of wound exudate or wound biofilm, (2) the value obtained in ii) is equal to or above the respective control value established in the absence of wound exudate or wound biofilm, (3) the value obtained in iiia) is equal to or above the respective control value established in the absence of wound exudate or wound biofilm, (4) the ratio of amount(s) of one or more M1 marker(s) to the amount(s) of one or more M2 marker(s) obtained in iiib) is/are below a cut-off value, (5) the ratio of amount(s) and/or frequency distribution(s) of one or more M1 cell surface marker(s) to the amount(s) and/or frequency distribution(s) of one or more M2 cell surface marker(s) obtained in iiic) is/are below a cut-off value, in particular wherein the ratio is selected from a CD38/CD209 ratio, a CD197/CD209 ratio and a CD197/CD206 ratio, (6) the ratio of expression level(s) of one or more M1 marker mRNA(s) to the expression level(s) of one or more M2 marker mRNA(s) obtained in iiid) is/are below a cut-off value, (7) the value obtained in iiie) is below a cut-off value, preferably wherein a combined value is established for the values obtained in i), ii), iiia), iiib), iiic) iiid) and/or iiie).
  6. The method according to claim 4 or 5, wherein step b) comprises: b) identifying the skin wound as being a non-healing skin wound in case at least two, preferably three, four, five or six of (1) to (6) or at least two, preferably three, four, five six or seven of (1) to (7) are fulfilled: (1) the value obtained in i) is below the respective control value established in the absence of wound exudate or wound biofilm, (2) the value obtained in ii) is below the respective control value established in the absence of wound exudate or wound biofilm, (3) the value obtained in iiia) is below the respective control value established in the absence of wound exudate or wound biofilm, (4) the ratio of amount(s) of one or more M1 marker(s) to the amount(s) of one or more M2 marker(s) obtained in iiib) is/are above a cut-off value, wherein the one or more M1 markers are selected from CXCL10 and IL-23p19, and the one or more M2 markers are selected from CCL22 and CCL18, (5) the ratio of amount(s) and/or frequency distribution(s) of one or more M1 cell surface marker(s) to the amount(s) and/or frequency distribution(s) of one or more M2 cell surface marker(s) obtained in iiic) is/are above a cut-off value, wherein the one or more M1 cell surface markers are selected from CD38, CD64 and CD197, and wherein the one ore more M2 cell surface markers are selected from CD200 receptor, CD206 and CD209, in particular wherein the ratio is selected from a CD38/CD209 ratio, a CD197/CD209 ratio and a CD197/CD206 ratio, (6) the ratio of expression level(s) of one or more M1 marker mRNA(s) to the expression level(s) of one or more M2 marker mRNA(s) obtained in iiid) is/are above a cut-off value, wherein the one or more M1 marker mRNA(s) are selected from CD38, CD64, CD197, CXCL10 and IL-23p19, and the one or more M2 marker mRNA(s) are selected from CD200 receptor (CD200R), CD206, CD209, CCL22 and CCL18, (7) the value of amount(s) of one or more cytokine markers selected from IL-1alpha, IL-1beta and TNF-alpha obtained in iiie) are above a cut-off value, with the proviso that at least the value(s) obtained in i) and/or ii) is/are below the respective control value(s) established in the absence of wound exudate or wound biofilm, and/or identifying the skin wound as being a healing skin wound in case at least two, preferably three, four, five or six of (1) to (6) or at least two, preferably three, four, five six or seven of (1) to (7) are fulfilled: (1) the value obtained in i) is above the respective control value established in the absence of wound exudate or wound biofilm, (2) the value obtained in ii) is above the respective control value established in the absence of wound exudate or wound biofilm, (3) the value obtained in iiia) is above the respective control value established in the absence of wound exudate or wound biofilm, (4) the ratio of amount(s) of one or more M1 marker(s) to the amount(s) of one or more M2 marker(s) obtained in iiib) is/are below a cut-off value, (5) the ratio of amount(s) and/or frequency distribution(s) of one or more M1 cell surface marker(s) to the amount(s) and/or frequency distribution(s) of one or more M2 cell surface marker(s) obtained in iiic) is/are below a cut-off value, in particular wherein the ratio is selected from a CD38/CD209 ratio, a CD197/CD209 ratio and a CD197/CD206 ratio, (6) the ratio of expression level(s) of one or more M1 marker mRNA(s) to the expression level(s) of one or more M2 marker mRNA(s) obtained in iiid) is/are below a cut-off value, (7) the value of amount(s) of one or more cytokine markers selected from IL-1alpha, IL-1beta and TNF-alpha obtained in iiie) are below a cut-off value, with the proviso that at least the value(s) obtained in i) and/or ii) is/are equal to or above the respective control value(s) established in the absence of wound exudate or wound biofilm.
  7. The method according to claim 2, comprising a) measuring i) the proliferation of primary fibroblast cells in the presence of a wound exudate sample or wound biofilm sample obtained from said skin wound at a first time point, and ii) the fibroblast-derived matrix formation by primary fibroblast cells in the presence of a wound exudate sample or wound biofilm sample obtained from said skin wound at a first time point, b) optionally identifying the skin wound as being a non-healing skin wound at said first time point in case the value obtained in a)i) is below a control value established in the absence of wound exudate or wound biofilm, and the value obtained in a)ii) is below a control value established in the absence of wound exudate or wound biofilm, or identifying the skin wound as being a healing skin wound at said first time point in case the value obtained in a)i) is equal to or above a control value established in the absence of wound exudate or wound biofilm, and the value obtained in a)ii) is equal to or above a control value established in the absence of wound exudate or wound biofilm, c) measuring i) the proliferation of primary fibroblast cells in the presence of a wound exudate sample or wound biofilm sample obtained from said skin wound at a second time point, and ii) the fibroblast-derived matrix formation by primary fibroblast cells in the presence of a wound exudate sample or wound biofilm sample obtained from said skin wound at a second time point, d) optionally identifying the skin wound as being a non-healing skin wound at said second time point in case the value obtained in c)i) is below a control value established in the absence of wound exudate or wound biofilm, and the value obtained in c)ii) is below a control value established in the absence of wound exudate or wound biofilm, or identifying the skin wound as being a healing skin wound at said second time point in case the value obtained in c)i) is equal to or above a control value established in the absence of wound exudate or wound biofilm, and the value obtained in c)ii) is equal to or above a control value established in the absence of wound exudate or wound biofilm, e) A) identifying a skin wound at a second time point to exhibit improved healing in case the value obtained in c)i) at said second time point is higher than the value obtained in a)i) at said first time point, and the value obtained in c)ii) at said second time point is higher than the value obtained in a)ii) at said first time point, with the proviso that the values obtained in a)i) at said first time point and a)ii) at said first time point are equal to or below a respective control value established in the absence of wound exudate or wound biofilm, or B) identifying a skin wound at a second time point to exhibit worsened healing in case the value obtained in c)i) at said second time point is lower than the value obtained in a)i) at said first time point, and the value obtained in c)ii) at said second time point is lower than the value obtained in a)ii) at said first time point, with the proviso that the values obtained in c)i) and c)ii) at said second time point are equal to or below 100% of a respective control value established in the absence of wound exudate or wound biofilm, and f) optionally repeating steps a) to e) at one or more later time points.
  8. The method according to claim 2, comprising: a) measuring i) the proliferation of primary fibroblast cells in the presence of a wound exudate sample or wound biofilm sample obtained from said skin wound at a first time point, and ii) the fibroblast-derived matrix formation by primary fibroblast cells in the presence of a wound exudate sample or wound biofilm sample obtained from said skin wound at a first time point, and one, two, three or four of iiia), iiib), iiic) and iiid), or one, two, three four or five of iiia), iiib), iiic) iiid) and iiie): iiia) the proliferation of HaCaT cells in the presence of a wound exudate sample or wound biofilm sample obtained from said skin wound at a first time point, iiib) the amount(s) of one or more M1 marker(s) and one or more M2 marker(s) in the supernatant of macrophages incubated with a wound exudate sample or wound biofilm sample obtained from said skin wound at a first time point, wherein the macrophages are in co-culture with fibroblasts, wherein the one or more M1 markers are selected from CXCL10 and IL-23p19, and the one or more M2 markers are selected from CCL22 and CCL18, iiic) the amount(s) and/or frequency distribution(s) of one or more M1 cell surface marker(s) and one or more M2 cell surface marker(s) on macrophages incubated with a wound exudate sample or wound biofilm sample obtained from said skin wound at a first time point, wherein the macrophages are in co-culture with fibroblasts, wherein the one or more M1 cell surface marker are selected from CD38, CD64 and CD197, and wherein the one or more M2 cell surface markers are selected from CD200 receptor, CD206 and CD209, iiid) the expression level(s) of one or more M1 marker mRNA(s) and one or more M2 marker mRNA(s) in macrophages incubated with a wound exudate sample or wound biofilm sample obtained from said skin wound at a first time point, wherein the macrophages are in co-culture with fibroblasts, iiie) the amount(s) of one or more cytokine markers in the supernatant of macrophages incubated with a wound exudate sample or wound biofilm sample obtained from said skin wound at a first time point, wherein the macrophages are in co-culture with fibroblasts, and wherein the one or more cytokine markers are selected from IL-1alpha, IL-1 beta and TNF-alpha, b) optionally identifying the skin wound as being a non-healing skin wound at said first time point or as a healing skin wound at a first time point pursuant to claim 3, 4 or 5, c) measuring i) the proliferation of primary fibroblast cells in the presence of a wound exudate sample or wound biofilm sample obtained from said skin wound at a second time point, and ii) the fibroblast-derived matrix formation by primary fibroblast cells in the presence of a wound exudate sample or wound biofilm sample obtained from said skin wound at a second time point, and one, two, three or four of iiia), iiib), iiic) and iiid), or one, two, three four or five of iiia), iiib), iiic) iiid) and iiie): iiia) the proliferation of HaCaT cells in the presence of a wound exudate sample or wound biofilm sample obtained from said skin wound at a second time point, iiib) the amount(s) of one or more M1 markers and one or more M2 markers in the supernatant of macrophages incubated with a wound exudate sample or wound biofilm sample obtained from said skin wound at a second time point, wherein the macrophages are in co-culture with fibroblasts, wherein the one or more M1 markers are selected from CXCL10 and IL-23p19, and the one or more M2 markers are selected from CCL22 and CCL18, iiic) the amount(s) and/or frequency distribution(s) of one or more M1 cell surface marker(s) and one or more M2 cell surface marker(s) on macrophages incubated with a wound exudate sample or wound biofilm sample obtained from said skin wound at a second time point, wherein the macrophages are in co-culture with fibroblasts, wherein the one or more M1 cell surface marker are selected from CD38, CD64 and CD197, and wherein the one or more M2 cell surface markers are selected from CD200 receptor, CD206 and CD209, iiid) the expression level(s) of one or more M1 marker mRNA(s) and one or more M2 marker mRNA(s) in macrophages incubated with a wound exudate sample or wound biofilm sample obtained from said skin wound at a second time point, wherein the macrophages are in co-culture with fibroblasts, wherein the one or more M1 marker mRNA(s) are selected from CD38, CD64, CD197, CXCL10 and IL-23p19, and the one or more M2 marker mRNA(s) are selected from CD200 receptor (CD200R), CD206, CD209, CCL22 and CCL18, iiie) the amount(s) of one or more cytokine markers in the supernatant of macrophages incubated with a wound exudate sample or wound biofilm sample obtained from said skin wound at a second time point, wherein the macrophages are in co-culture with fibroblasts, and wherein the one or more cytokine markers are selected from IL-1alpha, IL-1beta and TNF-alpha, d) optionally identifying the skin wound as being a non-healing skin wound at said second time point or as a healing skin wound at said second time point pursuant to claim 3, 4 or 5, e) A) identifying a skin wound at a second time point to exhibit improved healing in case at least two, preferably three, four, five or six of (1) to (6), or at least two, preferably three, four, five six or seven of (1) to (7) are fulfilled: (1) the value obtained in c)i) at said second time point is higher than the value obtained in a)i) at said first time point, (2) the value obtained in c)ii) at said second time point is higher than the value obtained in a)ii) at said first time point, (3) the value obtained in c)iiia) at said second time point is higher than the value obtained in a)iiia) at said first time point, (4) the ratio of amount(s) of one or more M1 marker(s) to the amount(s) of one or more M2 marker(s) obtained in c)iiib) at said second time point is lower than the ratio of amount(s) of one or more M1 marker(s) to the amounts of one or more M2 markers obtained in a)iiib) at said first time point, wherein the one or more M1 markers are selected from CXCL10 and IL-23p19, and the one or more M2 markers are selected from CCL22 and CCL18 (5) the ratio of amount(s) and/or frequency distribution(s) of one or more M1 cell surface marker(s) to the amount(s) and/or frequency distribution(s) of one or more M2 cell surface marker(s) obtained in c)iiic) at said second time point is lower than the ratio of amount(s) and/or frequency distribution(s) of one or more M1 cell surface marker(s) to the amount(s) and/or frequency distribution(s) of one or more M2 cell surface marker(s) obtained in a)iiic) at said first time point, wherein the one or more M1 cell surface markers are selected from CD38, CD64 and CD197, and wherein the M2 cell surface markers are selected from CD200 receptor, CD206 and CD209, in particular wherein the ratio is selected from a CD38/CD209 ratio, a CD197/CD209 ratio and a CD197/CD206 ratio, (6) the ratio of expression level(s) of one or more M1 marker mRNA(s) to the expression level(s) of one or more M2 marker mRNA(s) obtained in c)iiid) at said second time point is lower than the ratio of expression level(s) of one or more M1 marker mRNA(s) to the expression level(s) of one or more M2 marker mRNA(s) obtained in a)iiid) at said first time point, wherein the one or more M1 marker mRNA(s) are selected from CD38, CD64, CD197, CXCL10 and IL-23p19, and the one or more M2 marker mRNA(s) are selected from CD200 receptor (CD200R), CD206, CD209, CCL22 and CCL18, (7) the value obtained in c)iiie) at said second time point is lower than the value obtained in a)iiie) at said first time point, with the proviso that at least the value(s) obtained in c)i) and/or c)ii) at said second time point is/are higher at said second time point than the value(s) obtained in a)i) and/or a)ii) at said first time point, and with the proviso that the value obtained in a)i) at said first time point and/or a)ii) at said first time point is equal to or below a control value established in the absence of wound exudate or wound biofilm, or B) identifying a skin wound at a second time point to exhibit worsened healing in case at least two, preferably three, four, five or six of (1) to (6), or at least two, preferably three, four, five six or seven of (1) to (7) are fulfilled: (1) the value obtained in c)i) at said second time point is lower than the value obtained in a)i) at said first time point, (2) the value obtained in c)ii) at said second time point is lower than the value obtained in a)ii) at said first time point, (3) the value obtained in c)iiia) at said second time point is lower than the value obtained in a)iiia) at said first time point, (4) the ratio of amount(s) of one or more M1 marker(s) to the amount(s) of one or more M2 marker(s) obtained in c)iiib) at said second time point is higher than the ratio of amount(s) of one or more M1 marker(s) to the amounts of one or more M2 markers obtained in a)iiib) at said first time point, wherein the one or more M1 markers are selected from CXCL10 and IL-23p19, and the one or more M2 markers are selected from CCL22 and CCL18 in particular wherein the ratio is selected from a CD38/CD209 ratio, a CD197/CD209 ratio and a CD197/CD206 ratio, (5) the ratio of amount(s) and/or frequency distribution(s) of one or more M1 cell surface marker(s) to the amount(s) and/or frequency distribution(s) of one or more M2 cell surface marker(s) obtained in c)iiic) at said second time point is higher than the ratio of amount(s) and/or frequency distribution(s) of one or more M1 cell surface marker(s) to the amount(s) and/or frequency distribution(s) of one or more M2 cell surface marker(s) obtained in a)iiic) at said first time point, wherein the one or more M1 cell surface markers are selected from CD38, CD64 and CD197, and wherein the one or more M2 cell surface markers are selected from CD200 receptor, CD206 and CD209, (6) the ratio of expression level(s) of one or more M1 marker mRNA(s) to the expression level(s) of one or more M2 marker mRNA(s) obtained in c)iiid) at said second time point is higher than the ratio of expression level(s) of one or more M1 marker mRNA(s) to the expression level(s) of one or more M2 marker mRNA(s) obtained in a)iiid) at said first time point, wherein the one or more M1 marker mRNA(s) are selected from CD38, CD64, CD197, CXCL10 and IL-23p19, and the one or more M2 marker mRNA(s) are selected from CD200 receptor (CD200R), CD206, CD209, CCL22 and CCL18, with the proviso that at least the value(s) obtained in c)i) and/or c)ii) at said second time point is/are lower than the value(s) obtained in a)i) and/or a)ii) at said first time point, and with the proviso that the value(s) obtained in c)i) and/or c)ii) at said second time point is/are equal to or below 100% of a control value established in the absence of wound exudate or wound biofilm, (7) the value obtained in c)iiie) at said second time point is higher than the value obtained in a)iiie) at said first time point, and f) optionally repeating steps a) to e) at one or more later time points.
  9. The method according to any of the preceding claims, wherein - the individual is a mammal, preferably a human, and/or - the skin wound is selected from a wound of a diabetic patient, a wound which is infected by at least one microorganism, an ischemic wound, a wound in a patient suffering from deficient blood supply or venous stasis, an ulcer, such a diabetic ulcer, venous ulcer, arterial ulcer (e.g. ulcus cruris arteriosum), mixed ulcer, or pressure ulcer, a neuropathic wound, ulcus cruris, surgical wound, burn, dehiscence, neoplastic ulcer and rare ulcer, and/or - a non-healing skin wound is understood as a wound which does not close within 2 months under standard therapy, and/or - the individual exhibits further diseases and/or co-morbidities, and/or is treated with medication(s) for further diseases and/or co-morbidities, and/or - the skin wound is untreated or treated with one or more of the following: compression, wound dressings, surgical debridement, biological debridement, infection control, antibiotic therapy, negative pressure therapy, proteins, in particular growth factors, antibodies, peptides, sugars, cells or cell constituents, artificial skin, human blood-derived products, gene therapy or genetically engineered wound bed modifications, drugs, herbal medicines, plant extracts, and/or - the individual is identified to be treated with one or more of the therapies selected from compression, wound dressings, surgical debridement, biological debridement, infection control, antibiotic therapy, negative pressure therapy, proteins, in particular growth factors, antibodies, peptides, sugars, cells or cell constituents, artificial skin, human blood-derived products, gene therapy or genetically engineered wound bed modifications, drugs, herbal medicines, plant extracts, in case (i) a skin wound of the individual is identified as being a non-healing skin wound by a method according to claims 1 or 3 to 6, and/or a skin wound of the individual is identified to exhibit worsened healing at a second time point as compared to a first time point by a method according to claims 2, 7 or 8, and/or - the wound exudate sample is obtained by a physical or chemical method, in particular by applying negative pressure to the skin wound, in particular by using a negative pressure drainage device, a method using capillary forces, collecting wound exudate in a film dressing or membrane, collecting wound exudate in a syringe, applying an absorptive material, such as absorptive beads, or a filter, or by using a swab, such as a cotton swab, in particular wherein the film dressing or membrane is a cellulose layer and/or wherein the absorptive material is a cellulose layer, and/or - a healing skin wound is characterized by ongoing wound closure, granulation, absence of necrosis and/or absence of infections, and/or - a non-healing skin wound is characterized by a lack of wound closure, an increase of the area and/or depth of the wound, necrosis and/or infections of the skin wound, and/or lack of granulation, and/or - the fibroblast and/or monocyte cells used in the methods are human cells, preferably human cells obtained from healthy human individuals, from patients with comorbidities associated with impaired wound healing, such as diabetes, and/or from the individual patients providing the wound exudates or wound biofilms and/or - the wound exudate sample or wound biofilm sample is diluted between 1:2 to 1:1000, preferably between 1:10 and 1:200.
  10. The method according to any of the preceding claims, wherein i) measuring the proliferation of primary fibroblast cells in the presence of a wound exudate sample or wound biofilm sample obtained from a skin wound includes the following steps: (i) culturing primary human dermal fibroblast cells, (ii) incubating the cells on a solid support, thereby allowing the cells to adhere to the support, (iii) contacting the cells with the wound exudate sample or wound biofilm sample, which is optionally diluted, wherein the contacting may be performed before or after adherence of the cells occurs, (iv) determining the amount, preferably the cell number, including the formation of extracellular matrix, of the primary fibroblast cells, , preferably wherein the method is performed in 2D cell culture, and/or ii) measuring the fibroblast-derived matrix formation by primary fibroblast cells in the presence of a wound exudate sample or wound biofilm sample obtained from a skin wound includes the following steps: (i) seeding primary human dermal fibroblast cells on a support, which is preferably pre-coated with an adhesion enhancing agent, such as gelatin, (ii) culturing the cells on the support, preferably until confluence is reached, (iii) contacting the cells with (i) a matrix promoting supplement, and (ii) the wound exudate sample or wound biofilm sample, which is optionally diluted, wherein (i) and (ii) may be contacted simultaneously or sequentially, (iv) determining the amount of the fibroblast-derived matrix, preferably wherein the method is performed in 3D cell culture, and/or iii) measuring the proliferation of keratinocyte cells in the presence of a wound exudate sample or wound biofilm sample obtained from a skin wound includes the following steps: (i) culturing keratinocyte cells, (ii) incubating the cells on a solid support, thereby allowing the cells to adhere to the support, (iii) contacting the cells with the wound exudate sample or wound biofilm sample, which is optionally diluted, wherein the contacting may be performed before or after adherence of the cells occurs, (iv) determining the amount, preferably the cell number, of the keratinocyte cells, preferably wherein the method is performed in 2D cell culture, and/or iv) measuring the amount(s) of one or more M1 marker(s) and one or more M2 marker(s) in the supernatant of macrophages incubated with a wound exudate sample or wound biofilm sample obtained from a skin wound includes the following steps: (i) co-culturing primary human monocyte cells with (a) human dermal fibroblast cells in 2D cell culture or (b) fibroblast-derived matrices, (ii) incubating the cells until macrophage differentiation is reached, optionally wherein CD163 is used as a cell surface marker of macrophage differentiation, (iii) contacting the cells with a wound exudate sample or wound biofilm sample, which is optionally diluted, (iv) determining the amount of one or more M1 markers and one or more M2 markers in the cell culture supernatant, wherein the one or more M1 markers are selected from CXCL10 and IL-23p19, and the one or more M2 markers are selected from CCL22 and CCL18, more preferably wherein the markers are determined by using an immunological assay, even more preferably by using an ELISA assay, and/or v) measuring the amount(s) and/or frequency distribution(s) of one or more M1 cell surface marker(s) and one or more M2 cell surface marker(s) on macrophages incubated with a wound exudate sample or wound biofilm sample obtained from a skin wound includes the following steps: (i) co-culturing primary human monocyte cells with (a) human dermal fibroblast cells in 2D cell culture or (b) fibroblast-derived matrices, (ii) incubating the cells until macrophage differentiation is reached, optionally wherein CD163 is used as a cell surface marker of macrophage differentiation, (iii) contacting the cells with a wound exudate sample or wound biofilm sample, which is optionally diluted, (iv) determining the amount(s) and/or frequency distribution(s) of one or more M1 marker(s) and one or more M2 marker(s) on the cell surface of macrophages, wherein the one or more M1 cell surface markers are selected from CD38, CD64 and CD197, and the one or more M2 cell surface markers are selected from CD200 receptor (CD200R), CD206 and CD209, preferably wherein the amount(s) and/or frequency distribution(s) of the cell surface markers are determined by an immunological assay and/or a fluorescence assay, in particular by FACS analysis, more preferably wherein step iv) includes: contacting the macrophages with binding agents, preferably antibodies, which specifically recognize one or more M1 cell surface marker(s) and one or more M2 cell surface marker(s), wherein the binding agents are optionally labelled, in particular labelled with a fluorescent label, and determining the amounts of binding molecules bound to the macrophages, in particular by determining mean fluorescence intensity, thereby determining the amount(s) of the cell surface markers, and/or contacting the macrophages with binding agents, preferably antibodies, which specifically recognize one or more M1 cell surface marker(s) and one or more M2 cell surface marker(s), wherein the binding agents are optionally labelled, in particular labelled with a fluorescent label, and determining the percentages of cells which are positive for the one or more M1 cell surface marker(s) and the one or more M2 cell surface marker(s), respectively, within a cell population, in particular wherein FACS analysis is performed, thereby determining the frequency distribution(s) of the cell surface markers, and/or vi) measuring the expression level(s) of one or more M1 marker mRNA(s) and one or more M2 marker mRNA(s) in macrophages incubated with a wound exudate sample or wound biofilm sample obtained from a skin wound includes the following steps: (i) co-culturing primary human monocyte cells with (a) human dermal fibroblast cells in 2D cell culture or (b) fibroblast-derived matrices, (ii) incubating the cells until macrophage differentiation is reached, optionally wherein CD163 is used as a cell surface marker of macrophage differentiation, (iii) contacting the cells with a wound exudate sample or wound biofilm sample, which is optionally diluted, (iv) determining the expression level(s) of one or more M1 marker mRNA(s) and one or more M2 marker mRNA(s) in the macrophages, wherein the one or more M1 marker mRNA(s) are selected from CD38, CD64, CD197, CXCL10 and IL-23p19, and the one or more M2 marker mRNA(s) are selected from CD200 receptor (CD200R), CD206, CD209, CCL22 and CCL18, more preferably wherein the method comprises contacting a probe which specifically binds to a marker mRNA, wherein the probe is optionally labelled, with the macrophage RNA under conditions which are conducive to hybridization, and detecting the hybridized probe, and/or vii) measuring the amount(s) of one or more cytokine markers selected from IL-1alpha, IL-1beta and TNF-alpha in the supernatant of macrophages incubated with a wound exudate sample or wound biofilm sample obtained from a skin wound includes the following steps: (i) co-culturing primary human monocyte cells with (a) human dermal fibroblast cells in 2D cell culture or (b) fibroblast-derived matrices, (ii) incubating the cells until macrophage differentiation is reached, optionally wherein CD163 is used as a cell surface marker of macrophage differentiation, (iii) contacting the cells with a wound exudate sample or wound biofilm sample, which is optionally diluted, (iv) determining the amount of one or more cytokine markers selected from IL-1alpha, IL-1beta and TNF-alpha in the cell culture supernatant, preferably wherein the cytokine markers are determined by using an immunological assay, more preferably by using an ELISA assay.
  11. The methods according to any of the preceding claims, wherein the following method steps are performed simultaneously: i) measuring the proliferation of primary fibroblast cells in the presence of a wound exudate sample or wound biofilm sample obtained from a skin wound, and ii) measuring the fibroblast-derived matrix formation by primary fibroblast cells in the presence of a wound exudate sample or wound biofilm sample obtained from a skin wound, and optionally iiia) measuring the proliferation of keratinocyte cells in the presence of a wound exudate sample or wound biofilm sample obtained from a skin wound, and/or iiib) measuring the amount(s) of one or more M1 marker(s) and one or more M2 marker(s) in the supernatant of macrophages incubated with a wound exudate sample or wound biofilm sample obtained from a skin wound, wherein the macrophages are in co-culture with fibroblasts, wherein the one or more M1 markers are selected from CXCL10 and IL-23p19, and the one or more M2 markers are selected from CCL22 and CCL18, and/or iiic) measuring the amount(s) and/or frequency distribution(s) of one or more M1 cell surface marker(s) and one or more M2 cell surface marker(s) on macrophages incubated with a wound exudate sample or wound biofilm sample obtained from a skin wound, wherein the macrophages are in co-culture with fibroblasts, wherein the one or more M1 cell surface markers are selected from CD38, CD64 and CD197, and wherein the one or more M2 cell surface marker are selected from CD200 receptor, CD206 and CD209, and/or iiid) measuring the expression level(s) of one or more M1 marker mRNA(s) and one or more M2 marker mRNA(s) in macrophages incubated with a wound exudate sample or wound biofilm sample obtained from a skin wound, wherein the macrophages are in co-culture with fibroblasts, wherein the one or more M1 marker mRNA(s) are selected from CD38, CD64, CD197, CXCL10 and IL-23p19, and the one or more M2 marker mRNA(s) are selected from CD200 receptor (CD200R), CD206, CD209, CCL22 and CCL18, and/or iiie) measuring the amount(s) of one or more cytokine markers in the supernatant of macrophages incubated with a wound exudate sample or wound biofilm sample obtained from a skin wound, wherein the macrophages are in co-culture with fibroblasts, and wherein the one or more cytokine markers are selected from IL-1alpha, IL-1beta and TNF-alpha, preferably wherein the method steps are performed on a single support, more preferably wherein the support is a chip, array, such as a microarray or nanoarray, a plate, such as a multiwell plate, or a dish.
  12. A kit comprising the agents for performing the method steps i) to iiid) or i) to iiie) of claim 11, wherein the kit comprises: a) primary fibroblast cells, b) keratinocyte cells, c) a support having a plurality of defined areas or cavities, wherein a subset of areas or cavities are (i) coated with adhesion enhancing agent, preferably gelatin, and/or (ii) are filled with fibroblast-derived matrix (FDM), d) optionally a matrix promoting supplement, and e) optionally monocyte cells, and f) binding agents, preferably antibodies, which specifically recognize one or more M1 cell surface marker(s) and one or more M2 cell surface marker(s), and, optionally: binding agents, preferably antibodies, which specifically recognize one or more M1 marker(s) and one or more M2 marker(s), and/or probes which specifically recognize one or more M1 marker mRNA(s) and one or more M2 marker mRNA(s), wherein the one ore more M1 cell surface markers are selected from CD38, CD64 and CD197, and wherein the one or more M2 cell surface markers are selected from CD200 receptor, CD206 and CD209, and wherein the one or more M1 markers are selected from CXCL10 and IL-23p19, and the one or more M2 markers are selected from CCL22 and CCL18, and wherein the one or more M1 marker mRNA(s) are selected from CD38, CD64, CD197, CXCL10 and IL-23p19, and the one or more M2 marker mRNA(s) are selected from CD200 receptor (CD200R), CD206, CD209, CCL22 and CCL18, and g) binding agents, preferably antibodies, which specifically recognize one or more one or more cytokine markers selected from IL-1alpha, IL-1beta and TNF-alpha.
  13. A method for screening for compounds suitable for modulating skin wound healing, comprising the following steps: A) measuring the proliferation of primary fibroblast cells in the presence of (i) a wound exudate sample or wound biofilm sample obtained from a skin wound of at least one individual, and (ii) at least one candidate compound, and B) performing one, two, three, four five or six of the following method steps B1) to B6) in case the value obtained in A) is at least 10% above or at least 10% below a control value established in the absence of the at least one candidate compound: B1) measuring the fibroblast-derived matrix formation by primary fibroblast cells in the presence of (i) a wound exudate sample or wound biofilm sample obtained from a skin wound of at least one individual and (ii) said at least one candidate compound, B2) measuring the proliferation of keratinocyte cells in the presence of (i) a wound exudate sample or wound biofilm sample obtained from a skin wound of at least one individual and (ii) said at least one candidate compound, B3) measuring the amount(s) of one or more M1 marker(s) and one or more M2 marker(s) in the supernatant of macrophages incubated with (i) a wound exudate sample or wound biofilm sample obtained from a skin wound of at least one individual and (ii) said at least one candidate compound, wherein the macrophages are in co-culture with fibroblasts, wherein the one or more M1 markers are selected from CXCL10 and IL-23p19, and the one or more M2 markers are selected from CCL22 and CCL18, B4) measuring the amount(s) and/or frequency distribution(s) of one or more M1 cell surface marker(s) and one or more M2 cell surface marker(s) on macrophages incubated with (i) a wound exudate sample or wound biofilm sample obtained from a skin wound of at least one individual and (ii) said at least one candidate compound, wherein the macrophages are in co-culture with fibroblasts, wherein the one or more M1 cell surface markers are selected from CD38, CD64 and CD197, and wherein the one or more M2 cell surface markers are selected from CD200 receptor, CD206 and CD209, B5) measuring the expression level(s) of one or more M1 marker mRNA(s) and one or more M2 marker mRNA(s) in macrophages incubated with (i) a wound exudate sample or wound biofilm sample obtained from a skin wound of at least one individual and (ii) said at least one candidate compound, wherein the macrophages are in co-culture with fibroblasts, wherein the one or more M1 marker mRNA(s) are selected from CD38, CD64, CD197, CXCL10 and IL-23p19, and the one or more M2 marker mRNA(s) are selected from CD200 receptor (CD200R), CD206, CD209, CCL22 and CCL18, B6) measuring the amount(s) of one or more cytokine markers in the supernatant of macrophages incubated with (i) a wound exudate sample or wound biofilm sample obtained from a skin wound of at least one individual and (ii) said at least one candidate compound, wherein the macrophages are in co-culture with fibroblasts, and wherein the one or more cytokine markers are selected from IL-1alpha, IL-1 beta and TNF-alpha. wherein the compound is identified as being suitable for modulating skin wound healing, in case at least one value obtained in B1) to B6) is at least 10% above or at least 10% below a control value established in the absence of the candidate compound, preferably wherein the method steps pursuant to A) and B1) to B6) are performed according to claim 10.
  14. The method for screening for compounds of claim 13, wherein a) the at least one compound is selected from a small molecule, a hormone, sugar, protein, peptide, polymer, biological, such as a protein, a peptide, an antibody or derivative thereof, or a conjugate thereof, a nucleic acid, such a viral agent, or one or more cell(s), such as one or more genetically modified cell, and/or b) the at least one compound is selected from an immunomodulatory agent, more preferably an immunosuppressive agent, an antibiotic, an antiinfective, a growth factor, a cytokine, an antiproliferative agent and an agent stimulating proliferation, and/or c) the at least one compound is a single compound, or 2, 3, 4, 5, or more different compounds, wherein the 2, 3, 4, 5, or more different compounds may be present in a single composition or in 2 or more separate compositions, and/or d) the values are measured at least in triplicate and/or a statistical significance is established in B), more preferably, wherein p≤0,05, p≤0,001 or p≤0,001, and/or the compound is identified as being suitable for modulating skin wound healing, in case at least one value obtained in B1) to B6) is at least 10% above or at least 10% below a control value established in the absence of the candidate compound with statistical significance, more preferably, wherein p≤0,05, p≤0,001 or p≤0,001.

Description

The present invention relates to an in vitro method for identifying a skin wound in an individual as being a non-healing skin wound or healing skin wound, in vitro methods for monitoring the healing of a skin wound in an individual, methods for screening for compounds suitable for modulating skin wound healing, as well as kits related thereto. Chronic wounds are a major health issue worldwide with 5.7 million affected patients in the US alone and an expected increase due to the aging population and growing incidence of metabolic diseases. Chronic wounds have a multifactorial etiology and are dependent on different variables: a) underlying disease, e.g. diabetes, arterial or venous insufficiency, b) pressure, c) age and nutritional status and d) microbial environment. Chronic wounds are generally understood as those wounds that have not healed within 2 months. They are a major health issue worldwide. In developed countries, including the US and the EU, it has been estimated that 1 to 2% of the total population will experience a chronic wound during their lifetime [Gottrup F (2004) Am J Surg 187:38S-43S]. In the US alone, approximately 5.7 million patients are affected. This number is expected to increase due to the aging population and growing incidence of metabolic diseases. The major chronic wound indications are venous ulcers, pressure ulcers and diabetic foot ulcers. Venous ulcers are defects in pathologically altered tissue on the lower leg based on chronic venous insufficiency, often accompanied by deep venous thrombosis. Pressure ulcers are the results of severe tissue hypoxemia in immobilized patients. Diabetic foot ulceration can affect up to 25% of patients with diabetes throughout their lifetime and often results in lower limb amputation. The standard of care for all of these wounds, as recommended by the German Society for Dermatology [Dissemond J et al (2014) JDDG 1610-03791201411207:541-554] includes wound dressings, surgical and biological (maggot) debridement, infection control and negative pressure therapy. Regranex® (PDGF: platelet-derived growth factor) is the only registered pharmacological treatment, but its therapeutic efficacy is minor, as is the success of cell-based therapies. Recurrence is a problem in one third of all chronic wounds, regardless of their treatment. Even though they are anti-inflammatory in other settings, topical corticosteroids cannot be used because one of their side effects is actually delayed wound healing [Hengge UR (2006) J Am Acad Dermatol 54:1-15]. Non-steroidal anti-inflammatory drugs, e.g. ibuprofen, are only effective in ameliorating wound pain [Dissemond j et al (2014)]. Mendez et al., J Vascular Surgery (1999), 30(4):734-743 discloses that venous ulcer wound fluids inhibit the proliferation of fibroblasts. However, it is very often difficult to assess whether a skin wound in an individual is or develops into a non-healing, chronic wound and/or whether the healing of a skin wound improves or worsens in future. A plurality of markers is known in the context of wound healing. Furthermore, it has been described in the past for certain markers, that some markers are elevated or decreased in groups of non-healing wounds in general (either pooled or separately, determining average values) as compared to healing wounds in general (again either pooled or separately, determining average values). However, when analyzing individual wound samples, the absolute values of such markers in single individuals often vary tremendously and do not allow for monitoring or diagnosing a single skin wound in a single individual reliably. As an example, Myeloperoxidase, Matrix-Metalloproteinase and Elastase enzyme activities are found to be generally lower in healing skin wound exudates as compared to the non-healing wounds. However, the variability of the enzyme levels determined in the individual wound exudate sample is very high and therefore do not allow as such for a reliable identification of non-healing skin wounds (Figures 8 to 10). Furthermore, IL-1α, IL-1β and TNF-α cytokine levels are elevated in general in non-healing skin as compared to healing skin wounds (Figure 11). However, the individual values again vary strongly. For example, cytokine levels are in the same range as in non-healing wound exudates for some of the healing wound exudates (Figure 13), and only about 50% of the wound exudate samples from non-healing wounds induce cytokine secretion by macrophages (Figure 15). Methods for reliably identifying non-healing skin wounds and/or for identifying that the healing of a skin wound worsens would provide an opportunity to further treat such skin wounds in time for the individual affected, thereby avoiding or attenuating recurrence and/or worsening of the healing status. Therefore, there is a need for methods, kits and devices allowing for accurate diagnosis and monitoring of non-healing skin wounds in an individual, as well as methods which allow for predicting