Study Of The Role Of ECM In Mice Amputated Digit Regeneration Model With Preserved Soft Tissue

Objectives: Previously,we found that porcine bladder ECM could induce bone regeneration in mice with P2 digit amputations by Micro CT assay,and ECM+ BMP2 could induce excessive bone regeneration.In this study,we further explored the morphological characteristics and macrophage change patterns during ECM-induced bone regeneration.This study is expected to provide new ideas and methods for the clinical treatment of bone damage.Methods: The 2nd and 4th digit of the left and right hind limbs of adult Kunming mice were amputated at Phalange 2.The left "index finger" and the right"ring finger" were implanted with ECM as the ECM group,while the left "ring finger" and the right "index finger were amputated only and served as the control group.Digit samples were processed with transparent specimens,histological staining,and Micro CT to observe the structural changes of bone tissue;immunohistochemistry was performed to observe the aggregation and distribution of macrophages.The digits of macrophage-depleting mice were amputated as same in the above mice,and the left paw was depleted of the macrophage,while the right paw of the same mouse was left untreated for the contralateral group.Results: There was bone regeneration in both the ECM and control groups,and there was no difference in the bone regeneration process: their cartilage proliferated on the surface of the backbone toward the distal end,and was gradually replaced by bone tissue through endochondral ossification;there was also bone regeneration in the control group,which was a new finding.There was a significant difference in the amount of regeneration between the ECM and control groups: bone volume was higher in the ECM group than in the control group;the free bone was only present in the ECM group;the ECM group failed to restore bone density to pre-amputated digit levels within 70 d.The number of CD68 and CD206 macrophages in the ECM group was higher than that in the control group.The number of CD68 and CD206 macrophages in the ECM group peaked twice at 7 and 21 days and stabilized after 28 days.In the control group,CD68 macrophages peaked at about 7 d after breakage and stabilized after 14 d.The number of CD206 always showed no significant fluctuation,and the distribution of CD68 was accompanied by the chondrogenesis/ossification process.At 0 d of digit amputation,the macrophages were depleted,and after 35 d the P2 of the control remained in the form of freshly amputated digits,but a small amount of new bone production was found in the P2 of the ECM group.The number of macrophages in the contralateral digits was also decreased,but bone regeneration was not affected.When macrophages were depleted 10 d after digit amputation,cartilage proliferation was inhibited and cartilage ossification was delayed,cartilage still existed until 35 days after digit amputation.Conclusions: regenerative responses were found in both ECM and control groups,and the volume of regenerated bone was lower in the control group than in the ECM group.ECM promoted and induced chondrogenesis,but chondrogenesis and ossification were accompanied by severe damage to the original bone stem.ECM promoted aggregation of macrophages,and CD68 macrophages traveled with new bone,which was not observed with CD206 macrophages.Macrophages may play an important role in both chondrogenesis and ossification.