The Influence Of Chm-? On Ectopic Cartilage Regeneration And Homeostasis Maintenance

Bone marrow stromal stem cells(BMSCs),as a group of adult stem cells featured in rich sources,easy proliferation,and minimal injury of donor sites,have been proven to possess good ability of chondrogenesis and thus considered as an ideal seed cells for cartilage regeneration.However,ectopic ossification of BMSCs regenerated cartilage have greatly restricted its application in repairing subcutaneous cartilage defects(such as nasal or auricular).Different from BMSCs,chondrocytes can maintain stable chondrogenic phenotype in ectopic microenvironment,which was speculated to be related with the existence of anti-angiogenic factors.As a typical represent of anti-angiogenic factors in cartilage,Chondromodulin-?(Chm-?)has been widely reported in regulating cartilage growth,anti-angiogenesis,and anti-tumorigenesis.Therefore,elucidating the influence of Chm-? on characteristics and functions of chondrocyte as well as ectopic cartilage regeneration and its stability may help to solve the problem of MSC ectopic ossification.To address the above mentioned issue,our first part focused on the role of Chm-? in maintaining ectopic stability of chondrocyte regenerated cartilage.The current study demonstrated that Chm-? knockout did not obviously influence articular cartilage development in situ.However,native articular cartilage from Chm-? knockout(Chm-?^-/-,KO)but not wild type(WT)mice showed obvious ossification after subcutaneously implanted into nude mice for 16 d.Interestingly,cell morphology,cartilage specific matrix expression,and pellet culture demonstrated that Chm-? knockout had no obvious influence on phenotype,function,and chondrogenic ability of chondrocytes in vitro,except that cells in WT group proliferated a little faster than those in KO group.Nevertheless,Chm-? knockout directly interfered with in vivo ectopic cartilage regeneration when chondrocytes were subcutaneously injected into nude mice with matrigel.Moreover,Chm-? knockout obviously compromised ectopic stability of in vitro regenerated cartilage after subcutaneous implantation.These findings indicated that Chm-? was an indispensable factor for ectopic cartilage regeneration and the maintenance of cartilage homeostasis.Based on the above proved issue that Chm-? play an important in ectopic stability of chondrocyte regenerated cartilage,our next goal is to explore the role of Chm-? on ectopic stability of BMSCs regenerated cartilage.Mice is the most suitable model for investigating the problem,however,cells in bone marrow of mice are more complicated,contain lower portion of BMSCs.And there is no stable system for 3 D chondrogenesis.In the current study,we mixed bone slice with bone marrow for primary culture,after lineage magnetic selection,we obtained the first passage(P1)BMSCs which possessed relatively higher purity and ability of proliferation and differentiation;we compared different chondrogenic inducing protocol based on pellet model,and selected the best.Finally,stable culture and cartilage regeneration system of mice BMSCs were established.Based on the established protocol,we compared differences in histological structure and cartilage specific matrix expression of in vitro regenerated cartilage of BMSCs from WT and Chm-?^-/-mice,confirmed that Chm-? knockout did not obviously influence chondrogenic regeneration ability of BMSCs.Nevertheless,after implanted into subcutaneous environment of nude mice,in vitro long time regenerated cartilage from BMSCs of Chm-?^-/-mice ossified earlier.These findings indicated that Chm-? play an important role in maintaining ectopic stability of BMSCs regenerated cartilage.