Diagnostic method for connective tissue and its application

Abstract

The present invention relates to medicine and is used to evaluate conditions and to detect connective tissue pathology (and/or organ) by clonal analysis. The present invention relates to aesthetic medicine and is used for correction of aging skin changes. The method includes: cultivation of substrate-dependent cell colonies of analyzed tissue and/or organ, at conditions which provide formation of discrete colonies applicable for visualization, statistically reliable analysis of derived colonies, determination of at least one parameter which characterizes regenerative potential of population of substrate dependent cells, determination of at least one parameter which characterizes proliferative potential of population of substrate dependent cells, and processing of obtained results which allows to evaluate regenerative ability of patient's tissue and/or organ. The method provides objective qualitative characteristics of both the proliferative and regenerative potential which allows evaluating the regenerative potential of primary tissue (and/or organ) without complex and expensive instrumental studies. A computer based system for performing the present method is also provided.

1 - 5 . (canceled) 
     
     
         6 . A method for individual correction of aging skin changes in a subject, the method comprising:
 (i) cultivating skin fibroblast colonies from the subject at conditions which provide formation of discrete colonies applicable for visualization,   (ii) determining efficacy of colony formation as a parameter that defines regenerative potential of a fibroblast population,   wherein the efficacy of colony formation is calculated as a ratio of the formed colonies with the number of cells >20 to the total number of explanted cells, and if efficacy of colony formation is less than 45% for men and 36% for women, low regenerative potential (Rl) is determined; if efficacy of colony formation is in the range 45-49% for men and 36-45% for women, normal regenerative potential (Rn) is determined, and if efficacy of colony formation is more than 49% for men and 45% for women, high regenerative potential (Rh) is determined; and   (iii) determining percentages of dense and diffuse colonies in cell culture, as a parameter that defines proliferative cell potential,   wherein the dense colonies are characterized by mean of optical density ≧46 relative units (rel.un.), and the diffuse colonies are ≦25 rel.un. and if percentage of the dense colonies is less than 44%, and percentage of the diffuse colonies is more than 25% for men, and percentage of the dense colonies is less than 40%, and percentage of the diffuse colonies is more than 40% for women, low proliferative potential (PI) is determined, if percentage of the dense colonies is in the range 44-54%, and percentage of the diffuse colonies is in the range 20-25% for men, and percentage of the dense colonies is in the range of 40-50%, percentage of the diffuse colonies is in the range 30-40% for women, normal proliferative potential (Pn) is determined; if percentage of the dense colonies is more than 54%, and percentage of the diffuse colonies is less than 20% for men, and percentage of the dense colonies is more than 50%, and percentage of the diffuse colonies is less than 30% for women, high proliferative potential (Ph) is determined;   (iv) evaluating a morphofunctional status of a dermal fibroblast population of the subject and, optionally, entire dermal of the subject based on (ii) and (iii),   wherein a low regenerative potential and/or a low proliferative cell potential of the fibroblast population is indicative of abnormal tissue regenerative ability and a high regenerative potential and a high proliferative cell potential of the fibroblast population is indicative of quick tissue regenerative ability;   (v) designing an individual corrective program for correction of the aging skin changes of the subject based on the evaluation, and   (vi) conducting an individual skin correction according to the program,   wherein   when a Rl and PI are detected, injecting cultured autologous skin fibroblasts into the skin areas for correction at least once in one to two years,   when a Rl and Pn are detected, injecting cultured autologous skin fibroblasts into the skin areas for correction at least once in 1.5 to 2.5 years,   when a Rh and Pn are detected, injecting cultured autologous skin fibroblasts into the skin areas for correction at least once in 5 years or longer,   when Rn and Pn are detected, injecting cultured autologous skin fibroblasts into the skin areas for correction at least once in 3 to 5 years,   when Rn and Ph are detected, injecting cultured autologous skin fibroblasts into the skin areas for correction at least once in 4 to 5 years, and   when Rh and Ph are detected, no cell treatment is needed.   
     
     
         7 . A method for individual correction of aging skin changes in a subject, the method comprising:
 (i) cultivating skin fibroblast colonies of the subject at conditions which provide formation of discrete colonies applicable for visualization,   (ii) determining efficacy of colony formation as a parameter that defines regenerative potential of a fibroblast population,   wherein the efficacy of colony formation is calculated as percentage of the formed colonies with the number of cells >20 to the total number of explanted cells, and if efficacy of colony formation is less than 45% for men and less than 36% for women, low regenerative potential is determined; if efficacy of colony formation is in the range 45-49% for men and 36-45% for women, normal regenerative potential is determined; if efficacy of colony formation is more than 49% for men and more than 45% for women, high regenerative potential is determined; and   (iii) determining percentages of dense, mixed and diffuse colonies in cell culture and a proliferation index, as parameters that define proliferative cell potential,   wherein proliferation index is defined by formula PI=[1(PD)+2(PM)+3(PS)]/100(%), wherein where PI is a proliferation index; PD is the percentage of the diffuse colonies, (%); PM is the percentage of mixed colonies, (%); PS is the percentage of the dense colonies, (%), wherein the dense colonies are characterized by mean optical density ≧46 relative units (rel.un.), and the diffuse colonies are ≦25 rel.un., and if proliferative index is less than 2.0 for men and less than 1.8 for women, low proliferative potential is determined; if proliferation index is in the range 2.0-2.4 for men and 1.8-2.0 for women, normal proliferative potential is determined; if proliferation index is more than 2.4 for men and more than 2.0 for women, high proliferative potential is determined; (iv) evaluating a morphofunctional status of a dermal fibroblast population of the subject and, optionally, entire dermal of the subject based on (ii) and (iii),   (iv) evaluating a morphofunctional status of a dermal fibroblast population of the subject and, optionally, entire dermal of the subject based on (ii) and (iii),   wherein a low regenerative potential and/or a low proliferative cell potential of the fibroblast population is indicative of abnormal tissue regenerative ability and a high regenerative potential and a high proliferative cell potential of the fibroblast population is indicative of quick tissue regenerative ability;   (v) designing an individual corrective program for correction of the aging skin changes of the subject based on the evaluation, and   (vi) conducting an individual skin correction according to the program,   wherein   when a Rl and PI are detected, injecting cultured autologous skin fibroblasts into the skin areas for correction at least once in one to three two years,   when a Rl and Pn are detected, injecting cultured autologous skin fibroblasts into the skin areas for correction at least once in 1.5 to 2.5 years,   when a Rh and Pn are detected, injecting cultured autologous skin fibroblasts into the skin areas for correction at least once in 5 years or longer,   when Rn and Pn are detected, injecting cultured autologous skin fibroblasts into the skin areas for correction at least once in 3 to 5 years,   when Rn and Ph are detected, injecting cultured autologous skin fibroblasts into the skin areas for correction at least once in 4 to 5 years, and   when Rh and Ph are detected, no cell treatment is needed.   
     
     
         8 . The method according to  claim 6 , wherein analysis is carried out by using a computer and mathematic modeling to define parameter values which characterize the fibroblast colonies. 
     
     
         9 . The method according to  claim 7 , wherein analysis is carried out by using a computer and mathematic modeling to define parameter values which characterize fibroblast colonies. 
     
     
         10 . The method according to  claim 6 , wherein ability of substrate-dependent cells to form colonies is used as a parameter which characterizes the regenerative potential. 
     
     
         11 . The method according to  claim 6 , wherein a proportion of cells with various density is used as a parameter which characterizes the proliferative potential of the cells. 
     
     
         12 . The method according to  claim 6 , wherein processing of obtained results provides a comparison of parameters with normal values. 
     
     
         13 . The method according to  claim 7 , wherein ability of substrate-dependent cells to form colonies is used as a parameter which characterizes the regenerative potential. 
     
     
         14 . The method according to  claim 7 , wherein a proportion of cells with various density is used as a parameter which characterizes the proliferative potential of the cells. 
     
     
         15 . The method according to  claim 7 , wherein processing of obtained results provides a comparison of parameters with normal values. 
     
     
         16 . A method for individual correction of aging skin changes in a subject, the method comprising:
 conducting an individual skin correction according to an individual corrective program for correction of aging skin changes in the subject, said conducting comprises:   injecting cultured autologous skin fibroblasts into skin areas for correction at least once in one to two years, when a Rl and PI are detected,   when a Rl and Pn are detected, injecting cultured autologous skin fibroblasts into the skin areas for correction at least once in 1.5 to 2.5 years,   injecting cultured autologous skin fibroblasts into skin areas for correction at least once in 5 years or longer, when a Rh and Pn are detected,   injecting cultured autologous skin fibroblasts into skin areas for correction at least once in 3 to 5 years, when Rn and Pn are detected,   injecting cultured autologous skin fibroblasts into skin areas for correction at least once in 4 to 5 years, when Rn and Ph are detected, or   no cell treatment is needed, when Rh and Ph are detected,
 wherein the program for correction of aging skin changes in the subject is based on evaluation comprising: 
   (i) cultivating skin fibroblast colonies from the subject at conditions which provide formation of discrete colonies applicable for visualization,   (ii) determining efficacy of colony formation as a parameter that defines regenerative potential of a fibroblast population,   wherein the efficacy of colony formation is calculated as a ratio of the formed colonies with the number of cells >20 to the total number of explanted cells, and if efficacy of colony formation is less than 45% for men and 36% for women, low regenerative potential (Rl) is determined; if efficacy of colony formation is in the range 45-49% for men and 36-45% for women, normal regenerative potential (Rn) is determined, and if efficacy of colony formation is more than 49% for men and 45% for women, high regenerative potential (Rh) is determined; and   (iii) determining percentages of dense and diffuse colonies in cell culture, as a parameter that defines proliferative cell potential,   wherein the dense colonies are characterized by mean of optical density ≧46 relative units (rel.un.), and the diffuse colonies are ≦25 rel.un. and if percentage of the dense colonies is less than 44%, and percentage of the diffuse colonies is more than 25% for men, and percentage of the dense colonies is less than 40%, and percentage of the diffuse colonies is more than 40% for women, low proliferative potential (PI) is determined, if percentage of the dense colonies is in the range 44-54%, and percentage of the diffuse colonies is in the range 20-25% for men, and percentage of the dense colonies is in the range of 40-50%, percentage of the diffuse colonies is in the range 30-40% for women, normal proliferative potential (Pn) is determined; if percentage of the dense colonies is more than 54%, and percentage of the diffuse colonies is less than 20% for men, and percentage of the dense colonies is more than 50%, and percentage of the diffuse colonies is less than 30% for women, high proliferative potential (Ph) is determined; and   (iv) evaluating a morphofunctional status of a dermal fibroblast population of the subject and, optionally, entire dermal of the subject based on (ii) and (iii),   wherein a low regenerative potential and/or a low proliferative cell potential of the fibroblast population is indicative of abnormal tissue regenerative ability and a high regenerative potential and a high proliferative cell potential of the fibroblast population is indicative of quick tissue regenerative ability.   
     
     
         17 . The method according to  claim 16 , further comprising designing the individual corrective program for correction of aging skin changes of the subject based on the evaluation prior to the correction. 
     
     
         18 . A method for individual correction of aging skin changes in a subject, the method comprising:
 conducting an individual skin correction according to an individual corrective program for correction of aging skin changes in the subject, said conducting comprises:   injecting cultured autologous skin fibroblasts into the skin areas for correction at least once in one to two years, when a Rl and PI are detected,   injecting cultured autologous skin fibroblasts into the skin areas for correction at least once in 1.5 to 2.5 years, when a Rl and Pn are detected,   injecting cultured autologous skin fibroblasts into the skin areas for correction at least once in 5 years or longer, when a Rh and Pn are detected,   injecting cultured autologous skin fibroblasts into the skin areas for correction at least once in 3 to 5 years, when Rn and Pn are detected,   injecting cultured autologous skin fibroblasts into the skin areas for correction at least once in 4 to 5 years, when Rn and Ph are detected, or   no cell treatment is needed, when Rh and Ph are detected,
 wherein the program for correction of aging skin changes in the subject is based on evaluation comprising: 
   (i) cultivating skin fibroblast colonies of the subject at conditions which provide formation of discrete colonies applicable for visualization,   (ii) determining efficacy of colony formation as a parameter that defines regenerative potential of a fibroblast population,   wherein the efficacy of colony formation is calculated as percentage of the formed colonies with the number of cells >20 to the total number of explanted cells, and if efficacy of colony formation is less than 45% for men and less than 36% for women, low regenerative potential is determined; if efficacy of colony formation is in the range 45-49% for men and 36-45% for women, normal regenerative potential is determined; if efficacy of colony formation is more than 49% for men and more than 45% for women, high regenerative potential is determined; and   (iii) determining percentages of dense, mixed and diffuse colonies in cell culture and a proliferation index, as parameters that define proliferative cell potential,   wherein proliferation index is defined by formula PI=[1(PD)+2(PM)+3(PS)]/100(%), wherein where PI is a proliferation index; PD is the percentage of the diffuse colonies, (%); PM is the percentage of mixed colonies, (%); PS is the percentage of the dense colonies, (%), wherein the dense colonies are characterized by mean optical density ≧46 relative units (rel.un.), and the diffuse colonies are ≦25 rel.un., and if proliferative index is less than 2.0 for men and less than 1.8 for women, low proliferative potential is determined; if proliferation index is in the range 2.0-2.4 for men and 1.8-2.0 for women, normal proliferative potential is determined; if proliferation index is more than 2.4 for men and more than 2.0 for women, high proliferative potential is determined; (iv) evaluating a morphofunctional status of a dermal fibroblast population of the subject and, optionally, entire dermal of the subject based on (ii) and (iii), and   (iv) evaluating a morphofunctional status of a dermal fibroblast population of the subject and, optionally, entire dermal of the subject based on (ii) and (iii),   wherein a low regenerative potential and/or a low proliferative cell potential of the fibroblast population is indicative of abnormal tissue regenerative ability and a high regenerative potential and a high proliferative cell potential of the fibroblast population is indicative of quick tissue regenerative ability.   
     
     
         19 . The method according to  claim 18 , further comprising designing the individual corrective program for correction of aging skin changes of the subject based on the evaluation prior to the correction.