Extracellular Matrix Characteristics Of Co-cultured Zonal Chondrocytes And Bone Marrow Mesenchymal Stem Cells In Vitro And In Vivo

Background: Articular cartilage is a heterogeneous tissue varying structure and composition with depth,and these differences are thought to be essential for optimal functioning of articular cartilage.ACI is the most commonly and effective therapy in treating cartilage defects.However,ACI does not take the natural varies of articular cartilage into account.More and more attention has been draw to engineer stratified articular cartilage rather than homogenous cartilage.Engineer cartilage with zonal chondrocytes is one of these approaches.Zonal chondrocytes were isolated successfully from different depth and could maintain their zonal differences during in vitro culture,and constructs that using zonal chondrocytes can minic the characteristics of native cartilage in vitro.Unfortunately,over the first few monolayer passages,zonal chondrocytes lose their zonal characteristics and chondrogenic phenotype which are hardly reacquired.Researches that employ zonal chondrocytes as seeds almost all used low passage or unexpanded zonal chondrocytes.However,primary chondrocytes always need to be passaged several times to get sufficient amount of chondrocytes in clinical scenarios.So we have to find a way to engineer stratified articular cartilage with less low passage or unexpanded zonal chondrocytes.Luckily,co-culture strategy was raised as a possible substitute of single chondrocytes therapy.Low passage or unexpanded chondrocytes isolated from no weight-bearing area co-culture with BMSCs induced similar chondrogenesis to single chondrocytes therapy.So this approach could reduce the need of chondrocytes,and that can diminish or even avoid dedifferentiation process to get sufficient amount of chondrocytes.Without dedifferentiation,zonal differences could be hold.Besides,with co-culture techniques,cartilage repair may become one-step procedure if chondrocytes expanding is not necessary.However,whether zonal chondrocytes replaced(partly)with BMSCs maintain zonal differences is still unclear.In this study,we employed a commonly used 3-dimensional(3D)culture system to investigate whether co-culture of zonal chondrocytes with BMSCs maintain their original differences in vitro and in vivo.Part one: Isolation fo zonal chondrocytes and zonal characteristics maintance of zonal chondrocyte cultured in 2D environmentObjective: 1.Isolation of zonal chondrocytes;2.Identification the zonal differences of primary zonal chondrocytes;3.Identification the zonal differences after passage;4.Isolation and identification of bone marrow mesenchymal stem cells.Methods: The 100 ?m of superficial zone of femur condylar cartilage was obtain with a dermatome and the middle and deep zone cartilage was obtained using a scalpel blade.The isolated cartilage tissues were digested with 0.2% type II collagenase for 6-8 hours.Bone marrow was aspirated into a 10 ml syringe and re-suspended onto a 10 cm flat plate in growth media.And attached cells were identified by multi-lineage differentiation.To determine the gene expression characteristics of zonal chondrocytes,total RNA was extracted from SZCs and MDZCs and the extracted RNA was reverse transcribed,then real-time PCR for glyceraldehyde-3-phosphate dehydrogenase(GAPDH),collagen type II(COL2A1),aggrecan(ACAN),proteoglycan 4(PRG4)was performed.Relative gene expression values were compared using the 2-?? Ct method.For the 2-dimensional(2D)proliferation assay,primary SZCs and MDZCs were plated in a 96-well plate in growth media.At days 3,5,7,and 10,proliferation rate was detected with Cell Counting Kit-8 reagent.Primary zonal chondrocytes were cultured on the plate and zonal chondrocytes reached 90% confluence were defined as P1.And the gene expression and proliferation rate of P1 and P2 were analysed again.Results: qRT-PCR performed using cDNA synthesized from RNA extracted from isolated primary zonal chondrocytes revealed that the expression levels of PRG4 and COL2 A were higher in the SZCs and that ACAN expression was higher in the MDZCs.The CCK-8 assay suggested that MDZCs proliferated faster than SZCs in 2D culture.However,there were no significant differences after one passage.The cells adhered to the plate showed a fibroblastic morphology.To identify the multi-lineage differentiation abilities,P3 BMSCs were induced to adipogenic,osteogenic and chondrogenic differentiation.The histology results suggested that the P3 BMSCs could differentiate into adipocyte,osteocyte and chondrocyte lineages.Conclusion: 1.Primary zonal chondrocytes isolated according to histology maintained zonal characteristics at P0;2.After only two passages these differences of gene and protein level and proliferation rate disappeared.Part two: Extracellular matrix characteristics of co-cultured zonal chondrocytes and bone marrow mesenchymal stem cells in vitroObjective: 1.Co-culture BMSCs and zonal chondrocytes in alginate gel in chondrogenic media;2.Measured the COL2A?ACAN and PRG4 gene expression at day 1?7 and 21 of alginate beads;3.Measured the DNA and GAG content at day 1?7 and 21 of alginate bead.Methods: Primary zonal chondrocytes and P3 BMSCs were re-suspended in sterile,low viscosity alginate gel alone or mixed at ratio of 1:2.The alginate/cell suspension was slowly dropped into a 102 mM CaCl2 gelation solution through a 23-gauge needle.The newly formed alginate beads were cultured in chondrogenic media.Samples were harvested at days 1,7,and 21 for gene expression analysis and for the DNA and GAG quantification.Measure Lubricin in the supernatant of day 1,7 and 21 after 24 hours culture with ELISA.Results: The MDZC co-culture group had the higher expression of ACAN than the SZC co-culture group,and had the lower expression of PRG4 than the SZC co-culture group at day 1 and 7.And the COL2 A expression level of co-culture groups had no difference at day 1 and 7.The same trend was found in the MDZC and SZC groups.In addition,the MDZC co-culture and MDZC groups showed higher GAG content(normalized to DNA content)than the SZC co-culture and SZC groups,respectively,at day 7 in vitro.At day 21 in vitro,no differences in gene-expression and GAG content 8 were observed between the co-culture groups nor among the single chondrocyte groups.Although the single zonal chondrocyte groups had higher GAG contents(normalized to DNA content)than the respective co-culture groups,when normalized to the initial seeded zonal chondrocytes,the co-culture groups had GAG contents similar to the respective single zonal chondrocyte groups in vitro at day 7 and higher GAG contents than the respective single zonal chondrocyte groups at day 21.The same trend was found with Lubricin.The concentration of Lubricin in SZC group and SZC co-culture group were higher than MDZC group and MDZC co-culture group at day 1 and 7,respectively.However,SZC,MDZC,SZC co-culture and MDZC co-culture groups had the similar concentration of Lubricin at day 21.Conclusion: 1.During 3D culture in vitro,zonal chondrocytes co-cultured with BMSCs showed zonal extracellular matrix at earlier stage(day1 and day7);2.However,these zonal extracellular matrix differences disappeared at later stage(day 21).Part three: Extracellular matrix characteristics of co-cultured zonal chondrocytes and bone marrow mesenchymal stem cells in vivoObjective: 1.Implanted alginate beads into 6-week-old nu/nu mice;2.After 2 and 8 weeks,the animals were killed,and the samples were used for histologic and biochemical analyses(GAG and DNA).Methods: For in vivo experiments,alginate beads fabricated according to the protocols described above.The beads were then immediately implanted into subcutaneous dorsal pockets of 14 male 6-week-old nu/nu mice.After 2 and 8 weeks,the animals were killed,and the samples were used for histologic and biochemical analyses.Results: In according to the in vitro study,the MDZC and MDZC co-culture groups deposited more GAG than the SZC and SZC co-culture groups,respectively,after 2 weeks in vivo.Although the single zonal chondrocyte groups had higher GAG contents(normalized to DNA content)than the respective co-culture groups,when normalized to the initial seeded zonal chondrocytes,the co-culture groups had GAG contents similar to the respective single zonal chondrocyte groups in vitro at day 7 and higher GAG contents than the respective single zonal chondrocyte groups in vivo at week 2.Furthermore,the SCZ and SZC co-culture groups had higher levels of lubricin than the MDZC and MDZC co-culture groups,respectively,at week 2 in vivo,which was confirmed by immunofluorescence staining.In vivo,GAG synthesis(normalized to DNA content)was similar at the end of 8 weeks.The single zonal chondrocyte groups exhibited greater GAG content(normalized to DNA content)than the respective co-culture groups in vitro.Unlike the in vitro culture,the co-culture group and the single zonal chondrocyte groups had similar GAG contents(normalized to DNA content)at week 8.However,when normalized to the initial seeded zonal chondrocytes,the co-culture groups had much more GAG content than the respective single zonal chondrocyte groups both in vitro and in vivo.Moreover,there were no significant differences in lubricin and col2 immunofluorescence signals between the co-culture groups,nor among the single zonal chondrocyte groups at week 8.Interestingly,the cells in all groups in vivo showed more direct cell-to-cell contact than the in vitro culture.Conclusion: 1.Zonal chondrocytes co-cultured with BMSCs in vivo showed zonal extracellular matrix at earlier stage(week 2);2.And same as in vitro,these zonal extracellular matrix differences disappeared at later stage(week 8)in vivo.