Allogeneic Transplantation Of Extracellular Matrix Induced Ovary Endogenous Regeneration In Situ In Mice

The strategy of organ repair and regeneration in situ in the field of tissue engineering and regeneration medicine is the most potential treatment to organ failiure patients in clinical trials.Stimulating endogenous organ repair and regeneration in situ is not only recognized by proliferating and differentiating of niche cells,appropriate extracellular microenviroment is also essential for organ repair and regeneration.Comparing with other artificial biomaterials to mimic tissue or organ extracellular microenviroment,natural organ decellularied extracellular matrix(ECM)is the best choice to recapitulate the characteristics of organ ECM,which have the suitable biocompatibilities,the appropriate biophysical and biochemical properties and the whole organ 3D structure.Futhermore,organic ECM microenvironment also benefits to cell adheresion,proliferation and differentiation.Thus,we attempted to use allogeneic organic ECM to test whether it have the ability to induce endogenous organ regeneration in mammals.Here we use ovary organ-specific ECM from mice to verify this hypothesis.In our experiment groups,decellularized non-cell-residued ECM were allogeneic transplanted into bilaterally ovariectomised female mice recipients,while performing bilaterally ovariectomized surgical without transplantation of ovary extracellular matrix as control groups.After 30days,we found that there were regenerated ovaries in ovarian bursa of experiment mice,while an empty ovarian bursa appeared in control mice.According to statistical analysis of ovary regeneration rates,compared to the rate of 34.17%(205/552)in experiment group,there was significance difference with the rate of 0%(0/118)in control group.Further analysis of the morphology,the histology,the immunohistochemistry and the function of regenerated ovaries,we found that the regenerated ovaries was similar to normal ovaries and there were different glasses follicles in regenerated ovaries sections.Oocytes in regenerated ovaries could also mature in vitro and produce offsrings.In addition,regenerated ovaries also maintained the level of progesterone and estrogen in experimental mice.We further transplanted pre-acid stained ovary ECM into female mice recipients to test the inducible ability of ovary ECM and unfortunately discovered that the rate and extent of regeneration are significantly lower than transpalting non-treatment ovary ECM.Next,we assumed that the hardness artificial gelatin or collagen scaffolds,which contains single component also promote ovary regeneration.Contrarily,although there were cells migrated into scaffolds,neither gelatin nor collagen scaffolds can regenerate into ovary.Thus,we suggest that the natural oganic ovary ECM plays crucial role in ovary endogenous regeneration in situ.In addition,we found that the ovary endogenous regeneration in situ was extensively exsitent between the strains of mice,whereas transplantation of ovary ECM into kidney capsule failed to regenerate ovary.Furthermore,we choose the regenerated 2,5,8,10,15,20,25 and30days ovaries to explore the process of ovary regeneration and found that the cell numbers per 50?m~2 and follicle numbers in regenerated ovary sections were increased with the time of regeneration.With the emergence of the first follicle in regenerated 15days ovaries,the regenerated ovaries grandually contained different glasses of follicles during the following15days regeneration.Transcriptome sequence analysis of regenerated 10,15,30days ovaries and normal ovaires revealed that the results of regenerated 10days ovaries were similar to the results of regenerated15days ovaries,while the results of regenerated 30days ovaries were closed to the normal ovaires results.The significant difference highly expressed genes in regenerated 10,15days ovaries were clustered in the biological process of activation of immune response,ECM remodeling,angiogenesis,tissue migration and regeneration,while in regenerated 30days ovaries and normal ovaries were clustered in the biological process of follicle development,ovulation cycle,steroid biosynthetic process and reproductive system development.Meanwhile,we verified the results of transcriptome sequence analysis using PCR and quantitive-PCR technology.We next analysed the proteinomics between regenerated15days ovaries and normal ovaires and discovered that the significant difference highly expreseed proteins which clustered into the biological processe in regenerated 15days ovaries and normal ovaires,were similarily enriched in transcriptome sequence analysis.We proclaimed that ovary regeneration is a dynamic process which might include the migration of cells,tissue remodeling and the re-differentiation of cells.Thereafter,we demonstrated that the regeneration of oocyte was possibly de novo differentiated from endogenous precursor cells,which originated from bone marrow lin~-sca-1~-c-kit~+cells and enclosed by newly proliferating granolosa cells to form primodial follicles.We further revealed that lin~-cells differentiated into three germ cell cysts like structure when co-transplantation of gential ridge cells and lin~-cells into kidney capsules.In conclusion,we induced ovary endogenous regeneration in situ in mice using allogeneic ovary ECM.The dynamic process of ovary regeneration was witnessed the decellularized ovary ECM to the recellularized ovary,which contained lin~-sca-1~-c-kit~+cells differentiated into oocyte and reassembled follicles.We believe that the regeneration of functional ovaries has clinical promise both because of its simplicity and the possibility to regenerate ovary organ in mammals.