early in human life , this tissue is populated by two cell types : notochordal cells ( ncs ) and np cells ( npcs ) . ncs naturally exist in clusters and contain large vesicles , whereas npcs are small , nonclustered , and chondrocyte - like cells . despite however , ncs can be distinguished from npcs by elevated brachyury ( t ) and cytokeratin 8 , 18 , and 19 expression . 
  in humans , ncs disappear before adolescence , and thereafter the onset of intervertebral disk degeneration , starting in the np , has been observed . certain species keep their ncs throughout adulthood and are known to experience the incidence and onset of disk degeneration much later in life . consequently , it has been proposed that ncs might be involved in the maintenance and regeneration of healthy np tissue . it has been shown that ncs can indeed increase the proteoglycan production by npcs in alginate coculture for up to 14 days . when npcs were cultured in medium conditioned by ncs ( for 4 days ) , however , it is yet unknown if ncs can have a direct regenerative potential on npcs in their native environment . this environment is characterized by low ph , oxygen , and glucose levels , as well as a high osmolarity . these factors , but also the composition of the extracellular matrix ( proteoglycans , collagens ) , can affect the cell response to an exogenous stimulation . the aim of this study was to investigate the effects of direct nc therapy in npc - populated bovine np explants , using an np culture model that was previously developed in our group . porcine spines ( 4 donors ; 8 weeks old ) were obtained from the slaughterhouse , in accordance with local regulations ( no approval from an ethical board required ) , and stored overnight at 4c . the thoracic and lumbar intervertebral disks were transversally opened to harvest the np tissue under sterile conditions . the tissue was pooled per donor and digested in 0.1% pronase protease ( calbiochem , darmstadt , germany ) in high glucose ( 4.5 g / l ) dulbecco 's modified eagle 's medium ( hgdmem ; invitrogen , bleiswijk , the netherlands )  +  1% penicillin / streptomycin ( pen / strep ; lonza , basel , switzerland ) for 1 hour at 37c and in 0.025% collagenase p ( roche , basel , switzerland ) in hgdmem  +  10% fetal bovine serum ( fbs ; invitrogen )  +  1% pen / strep overnight at 37c . after filtration on a 40-m cell strainer ( bd biosciences , breda , the netherlands ) , cells and cell clusters    40 m were stained with 5 m of carboxyfluorescein diacetate succinimidyl ester ( cfda - se ) according to the manufacturer 's protocol ( vybrant , invitrogen ) for cell tracking for the duration of the experiment . bovine tails ( 12 donors ; 24 months old ) were obtained from the slaughterhouse in accordance with local regulations ( no approval from an ethical board required ) . thirty - six np samples ( c2c5 ) were harvested , as described previously by van dijk et al . samples of different levels were equally distributed among the native and culture groups ( n   =  12 per group ) . the np samples from the culture groups were individually put in dialysis tubing ( 15-kda molecular weight cut off , spectra - por , rancho dominquez , california , united states ) and subsequently injected into the middle of the tissue with a 23-gauge needle ( hamilton , bonaduz , switzerland ) . the injection groups were : sham ( 10-l injection of phosphate - buffered saline [ pbs ] ) and nc ( 10-l injection of 250,000 cfda - se - stained porcine ncs in pbs ) . after the injection , the dialysis tubing was replaced and closed with a custom - made clip . the np samples were cultured in hypertonic polyethylene glycol ( peg ) medium ( 13.3% w / v 20 kda peg [ sigma , zwijndrecht , the netherlands ] ; 8.3 g / l dmem  +  3.7 g / l sodium bicarbonate  +  50 mg / l ascorbic acid  +  15.9 mg / l phenol red [ all sigma ]  +  1 g / l glucose  +  10% fbs [ both invitrogen ]  +  2% l - glutamine  +  1% sodium pyruvate  +  1% pen / strep [ all lonza ] ; ph 7.4 ) under hypoxic conditions ( 37c , 5% o2 , 5% co2 ) . samples were cultured for 42 days , and the medium was changed three times a week . native samples ( n   =  12 donors ) were harvested at day 0 , and each injection group was analyzed after 42 days of culture ( n   =  12 donors for each group ) . samples were cut in half , and for each of the four assays ( cell viability , biochemical assays and water content , gene expression , and histology ) , six half samples were used ( corresponding to six donors for each assay ) . np samples were incubated in 10 m calcein blue - am and 10 m propidium iodide ( both molecular probes , invitrogen ) in pbs for 2 hours . the viable cells (    =  730 nm ) , dead cells (    =  535 nm ) , and porcine nc location and morphology (    =  488  nm ; cfda - se staining ) were imaged with a confocal microscope ( clsm 510 meta , zeiss , mnchen , germany ) . the viability was assessed both near the center and the outer regions of the explant . the np samples were weighed ( wet weight ) and stored at 30c until further use . after overnight lyophilization ( freezone 2.5 , labconco , kansas city , missouri , united states ) , the sample weights were measured ( dry weight ) . the water content was calculated as ( wet weight    dry weight)/wet weight . the lyophilized samples were digested overnight at 60c in a papain solution ( 100  mm phosphate buffer , 5  mm l - cysteine , 5  mm ethylenediaminetetraacetic acid , and 140 g / ml papain , all from sigma ) . the amount of dna per sample was measured with a hoechst 33528 assay as described by cesarone et al , using a standard curve of double - stranded calf thymus dna ( sigma ) . the amount of sulfated glycosaminoglycans per sample relates to the proteoglycan content and was measured with a 1,9-dimethylmethylene blue assay as described by farndale et al , based on a standard curve of chondroitin - sulfate from shark cartilage ( sigma ) . the amount of hydroxyproline relates to the collagen content and was measured with a chloramine - t assay as described by huszar et al , based on a standard curve of trans-4-hydroxyproline ( sigma ) . bovine np samples were snap - frozen in liquid n2 and stored at 80c for rna isolation . briefly , samples were disrupted with a mikro - dismembrator ( sartorius , goettingen , germany ) . rna was isolated from the disrupted samples using trizol ( invitrogen ) and purified on a spin column ( rneasy mini kit , qiagen , venlo , the netherlands ) . the rna concentration and purity were measured on a spectrophotometer ( nd-1000 , isogen , de meern , the netherlands ) . of each sample , 75  ng of mrna was reverse transcribed to cdna ( superscript vilo kit , invitrogen ) , and gene expression was determined by quantitative polymerase chain reaction ( cfx384 , biorad , veenendaal , the netherlands ) . 18s was selected as the endogenous reference gene because it was found to be more stable than gapdh and rpl13a in all groups . the genes of interest were collagen type i ( col1a1 ) , collagen type ii ( col2a1 ) , and aggrecan ( acan ; table 1 ) . relative quantification was determined with the comparative 2 method , with the expression of the gene of interest relative to the expression of the reference gene 18s . list of primers for real - time quantitative polymerase chain reaction primer design ltd ( southampton , united kingdom ) . designed with beacon designer software ( premier biosoft , palo alto , california , united states ) and ordered from sigma ( zwijndrecht , the netherlands ) . compound ( sakuratek , zoeterwoude , the netherlands ) , slash frozen in isopentane at 80c , and stored at 30c until further use . from each sample , 10-m thick cryosections were cut ( microm hm 550 , thermo fisher scientific , kalamazoo , united kingdom ) and mounted on polysine adhesion slides ( thermo scientific ) . the slides were stained with weigert 's hematoxylin for cell nuclei and eosin for eosinophilic matrix . images were taken with a bright - field microscope ( observer , zeiss ) . with r - project software ( version 3.0.2 ) , levene test and shapiro - wilk test were used to test for homogeneity of variance and normality , respectively . the biochemical data , both homogeneous and normally distributed , was analyzed with a one - way analysis of variance followed by a bonferroni adjusted post hoc independent test . the gene expression data was found to be nonnormally distributed and was analyzed with kruskal - wallis test followed by a bonferroni corrected post hoc mann - whitney u test . statistical significance was assumed for p  <  0.05 . porcine spines ( 4 donors ; 8 weeks old ) were obtained from the slaughterhouse , in accordance with local regulations ( no approval from an ethical board required ) , and stored overnight at 4c . the thoracic and lumbar intervertebral disks were transversally opened to harvest the np tissue under sterile conditions . the tissue was pooled per donor and digested in 0.1% pronase protease ( calbiochem , darmstadt , germany ) in high glucose ( 4.5 g / l ) dulbecco 's modified eagle 's medium ( hgdmem ; invitrogen , bleiswijk , the netherlands )  +  1% penicillin / streptomycin ( pen / strep ; lonza , basel , switzerland ) for 1 hour at 37c and in 0.025% collagenase p ( roche , basel , switzerland ) in hgdmem  +  10% fetal bovine serum ( fbs ; invitrogen )  +  1% pen / strep overnight at 37c . after filtration on a 40-m cell strainer ( bd biosciences , breda , the netherlands ) , cells and cell clusters    40 m were stained with 5 m of carboxyfluorescein diacetate succinimidyl ester ( cfda - se ) according to the manufacturer 's protocol ( vybrant , invitrogen ) for cell tracking for the duration of the experiment . bovine tails ( 12 donors ; 24 months old ) were obtained from the slaughterhouse in accordance with local regulations ( no approval from an ethical board required ) . thirty - six np samples ( c2c5 ) were harvested , as described previously by van dijk et al . samples of different levels were equally distributed among the native and culture groups ( n   =  12 per group ) . the np samples from the culture groups were individually put in dialysis tubing ( 15-kda molecular weight cut off , spectra - por , rancho dominquez , california , united states ) and subsequently injected into the middle of the tissue with a 23-gauge needle ( hamilton , bonaduz , switzerland ) . the injection groups were : sham ( 10-l injection of phosphate - buffered saline [ pbs ] ) and nc ( 10-l injection of 250,000 cfda - se - stained porcine ncs in pbs ) . after the injection , the dialysis tubing was replaced and closed with a custom - made clip . the np samples were cultured in hypertonic polyethylene glycol ( peg ) medium ( 13.3% w / v 20 kda peg [ sigma , zwijndrecht , the netherlands ] ; 8.3 g / l dmem  +  3.7 g / l sodium bicarbonate  +  50 mg / l ascorbic acid  +  15.9 mg / l phenol red [ all sigma ]  +  1 g / l glucose  +  10% fbs [ both invitrogen ]  +  2% l - glutamine  +  1% sodium pyruvate  +  1% pen / strep [ all lonza ] ; ph 7.4 ) under hypoxic conditions ( 37c , 5% o2 , 5% co2 ) . samples were cultured for 42 days , and the medium was changed three times a week . native samples ( n   =  12 donors ) were harvested at day 0 , and each injection group was analyzed after 42 days of culture ( n   =  12 donors for each group ) . samples were cut in half , and for each of the four assays ( cell viability , biochemical assays and water content , gene expression , and histology ) , six half samples were used ( corresponding to six donors for each assay ) . np samples were incubated in 10 m calcein blue - am and 10 m propidium iodide ( both molecular probes , invitrogen ) in pbs for 2 hours . the viable cells (    =  730 nm ) , dead cells (    =  535 nm ) , and porcine nc location and morphology (    =  488  nm ; cfda - se staining ) were imaged with a confocal microscope ( clsm 510 meta , zeiss , mnchen , germany ) . the viability was assessed both near the center and the outer regions of the explant . the np samples were weighed ( wet weight ) and stored at 30c until further use . after overnight lyophilization ( freezone 2.5 , labconco , kansas city , missouri , united states ) , the sample weights were measured ( dry weight ) . the water content was calculated as ( wet weight    dry weight)/wet weight . the lyophilized samples were digested overnight at 60c in a papain solution ( 100  mm phosphate buffer , 5  mm l - cysteine , 5  mm ethylenediaminetetraacetic acid , and 140 g / ml papain , all from sigma ) . the amount of dna per sample was measured with a hoechst 33528 assay as described by cesarone et al , using a standard curve of double - stranded calf thymus dna ( sigma ) . the amount of sulfated glycosaminoglycans per sample relates to the proteoglycan content and was measured with a 1,9-dimethylmethylene blue assay as described by farndale et al , based on a standard curve of chondroitin - sulfate from shark cartilage ( sigma ) . the amount of hydroxyproline relates to the collagen content and was measured with a chloramine - t assay as described by huszar et al , based on a standard curve of trans-4-hydroxyproline ( sigma ) . bovine np samples were snap - frozen in liquid n2 and stored at 80c for rna isolation . briefly , samples were disrupted with a mikro - dismembrator ( sartorius , goettingen , germany ) . rna was isolated from the disrupted samples using trizol ( invitrogen ) and purified on a spin column ( rneasy mini kit , qiagen , venlo , the netherlands ) . the rna concentration and purity were measured on a spectrophotometer ( nd-1000 , isogen , de meern , the netherlands ) . of each sample , 75  ng of mrna was reverse transcribed to cdna ( superscript vilo kit , invitrogen ) , and gene expression was determined by quantitative polymerase chain reaction ( cfx384 , biorad , veenendaal , the netherlands ) . 18s was selected as the endogenous reference gene because it was found to be more stable than gapdh and rpl13a in all groups . the genes of interest were collagen type i ( col1a1 ) , collagen type ii ( col2a1 ) , and aggrecan ( acan ; table 1 ) . relative quantification was determined with the comparative 2 method , with the expression of the gene of interest relative to the expression of the reference gene 18s . list of primers for real - time quantitative polymerase chain reaction primer design ltd ( southampton , united kingdom ) . designed with beacon designer software ( premier biosoft , palo alto , california , united states ) and ordered from sigma ( zwijndrecht , the netherlands ) . compound ( sakuratek , zoeterwoude , the netherlands ) , slash frozen in isopentane at 80c , and stored at 30c until further use . from each sample , 10-m thick cryosections were cut ( microm hm 550 , thermo fisher scientific , kalamazoo , united kingdom ) and mounted on polysine adhesion slides ( thermo scientific ) . the slides were stained with weigert 's hematoxylin for cell nuclei and eosin for eosinophilic matrix . np samples were incubated in 10 m calcein blue - am and 10 m propidium iodide ( both molecular probes , invitrogen ) in pbs for 2 hours . the viable cells (    =  730 nm ) , dead cells (    =  535 nm ) , and porcine nc location and morphology (    =  488  nm ; cfda - se staining ) were imaged with a confocal microscope ( clsm 510 meta , zeiss , mnchen , germany ) . the viability was assessed both near the center and the outer regions of the explant . the np samples were weighed ( wet weight ) and stored at 30c until further use . after overnight lyophilization ( freezone 2.5 , labconco , kansas city , missouri , united states ) , the sample weights were measured ( dry weight ) . the water content was calculated as ( wet weight    dry weight)/wet weight . the lyophilized samples were digested overnight at 60c in a papain solution ( 100  mm phosphate buffer , 5  mm l - cysteine , 5  mm ethylenediaminetetraacetic acid , and 140 g / ml papain , all from sigma ) . the amount of dna per sample was measured with a hoechst 33528 assay as described by cesarone et al , using a standard curve of double - stranded calf thymus dna ( sigma ) . the amount of sulfated glycosaminoglycans per sample relates to the proteoglycan content and was measured with a 1,9-dimethylmethylene blue assay as described by farndale et al , based on a standard curve of chondroitin - sulfate from shark cartilage ( sigma ) . the amount of hydroxyproline relates to the collagen content and was measured with a chloramine - t assay as described by huszar et al , based on a standard curve of trans-4-hydroxyproline ( sigma ) . bovine np samples were snap - frozen in liquid n2 and stored at 80c for rna isolation . briefly , samples were disrupted with a mikro - dismembrator ( sartorius , goettingen , germany ) . rna was isolated from the disrupted samples using trizol ( invitrogen ) and purified on a spin column ( rneasy mini kit , qiagen , venlo , the netherlands ) . the rna concentration and purity were measured on a spectrophotometer ( nd-1000 , isogen , de meern , the netherlands ) . of each sample , 75  ng of mrna was reverse transcribed to cdna ( superscript vilo kit , invitrogen ) , and gene expression was determined by quantitative polymerase chain reaction ( cfx384 , biorad , veenendaal , the netherlands ) . 18s was selected as the endogenous reference gene because it was found to be more stable than gapdh and rpl13a in all groups . the genes of interest were collagen type i ( col1a1 ) , collagen type ii ( col2a1 ) , and aggrecan ( acan ; table 1 ) . relative quantification was determined with the comparative 2 method , with the expression of the gene of interest relative to the expression of the reference gene 18s . list of primers for real - time quantitative polymerase chain reaction primer design ltd ( southampton , united kingdom ) . designed with beacon designer software ( premier biosoft , palo alto , california , united states ) and ordered from sigma ( zwijndrecht , the netherlands ) . bovine np samples were embedded in tissue - tek o.c.t . compound ( sakuratek , zoeterwoude , the netherlands ) , slash frozen in isopentane at 80c , and stored at 30c until further use . from each sample , 10-m thick cryosections were cut ( microm hm 550 , thermo fisher scientific , kalamazoo , united kingdom ) and mounted on polysine adhesion slides ( thermo scientific ) . the slides were stained with weigert 's hematoxylin for cell nuclei and eosin for eosinophilic matrix . with r - project software ( version 3.0.2 ) , levene test and shapiro - wilk test were used to test for homogeneity of variance and normality , respectively . the biochemical data , both homogeneous and normally distributed , was analyzed with a one - way analysis of variance followed by a bonferroni adjusted post hoc independent test . the gene expression data was found to be nonnormally distributed and was analyzed with kruskal - wallis test followed by a bonferroni corrected post hoc mann - whitney u test . after 42 days of culture , the distribution and morphology of the npcs in both culture groups were similar to native tissue ( fig . viable ncs were observed after culture ( figs . 1 and 2b ) , but the vacuolar structures were much smaller than before injection ( fig . 2a ) . staining of nucleus pulposus tissue on ( a ) day 0 and day 42 in the ( b ) sham or ( c ) notochordal cell  injected group . cytoplasm and matrix are stained pink ( eosin ) , cell nuclei black ( hematoxylin ) . notochordal cells ( ncs ) ( a ) prior to injection and ( b ) in nucleus pulposus tissue after 42 days of culture . cytoplasm of ncs is stained in green ( cell tracker carboxyfluorescein diacetate succinimidyl ester ( cfda - se ) ) , cytoplasm of all cells in blue ( calcein blue - am ; only in b ) , and nuclei of dead cells in red ( propidium iodide ) . as expected , dna content in the nc group was larger than in the native samples at day 0 due to the injection of ncs ( p   =  0.016 ) , but that of the sham - injected group was not significantly different ( fig . the collagen content remained unchanged , but gene expression data indicates that there was a shift in the type of collagen : col2a1 decreased in both groups ( p   =  0.003 nc , p   =  0.004 sham ) , whereas col1a1 increased statistically only in the sham group ( p   =  0.01 ; fig . the water content ( a ) per wet weight , and ( b ) sulfated glycosaminoglycans ( sgag ) , ( c ) dna , and ( d ) hydroxyproline ( hyp ) content per dry weight on day 0 ( native ) and day 42 ( sham or notochordal cell [ nc ] injection ) . values are mean    standard deviation ; n   =  6 ( sham group : n   =  5 ) . * p  <  0.05 . gene expression of ( a ) aggrecan ( acan ) , ( b ) collagen type ii ( col2a1 ) , and ( c ) collagen type i ( col1a1 ) relative to 18s on day 0 ( native ) and day 42 ( sham or notochordal cell [ nc ] injection ) . after 42 days of culture , the distribution and morphology of the npcs in both culture groups were similar to native tissue ( fig . viable ncs were observed after culture ( figs . 1 and 2b ) , but the vacuolar structures were much smaller than before injection ( fig . 2a ) . staining of nucleus pulposus tissue on ( a ) day 0 and day 42 in the ( b ) sham or ( c ) notochordal cell  injected group . cytoplasm and matrix are stained pink ( eosin ) , cell nuclei black ( hematoxylin ) . notochordal cells ( ncs ) ( a ) prior to injection and ( b ) in nucleus pulposus tissue after 42 days of culture . cytoplasm of ncs is stained in green ( cell tracker carboxyfluorescein diacetate succinimidyl ester ( cfda - se ) ) , cytoplasm of all cells in blue ( calcein blue - am ; only in b ) , and nuclei of dead cells in red ( propidium iodide ) . as expected , dna content in the nc group was larger than in the native samples at day 0 due to the injection of ncs ( p   =  0.016 ) , but that of the sham - injected group was not significantly different ( fig . the collagen content remained unchanged , but gene expression data indicates that there was a shift in the type of collagen : col2a1 decreased in both groups ( p   =  0.003 nc , p   =  0.004 sham ) , whereas col1a1 increased statistically only in the sham group ( p   =  0.01 ; fig . the water content ( a ) per wet weight , and ( b ) sulfated glycosaminoglycans ( sgag ) , ( c ) dna , and ( d ) hydroxyproline ( hyp ) content per dry weight on day 0 ( native ) and day 42 ( sham or notochordal cell [ nc ] injection ) . values are mean    standard deviation ; n   =  6 ( sham group : n   =  5 ) . gene expression of ( a ) aggrecan ( acan ) , ( b ) collagen type ii ( col2a1 ) , and ( c ) collagen type i ( col1a1 ) relative to 18s on day 0 ( native ) and day 42 ( sham or notochordal cell [ nc ] injection ) . values are mean    standard deviation ; n   =  6 . * p  <  0.05 this study is the first to show that translating the results of in vitro coculture of ncs / npcs to in situ repair in tissue is not straightforward . whereas previous studies have shown that ncs were able to stimulate extracellular matrix production and related gene expression by isolated npcs , this effect was not observed when ncs were injected in np explants . this discrepancy may be explained by two mechanisms : the different ratio of nc to npc used or the initial presence of a proteoglycan - rich extracellular matrix . first , the nc - to - npc ratio in the current study was approximately 20:80 , whereas this ratio was 50:50 in previous direct cell cocultures . in a therapeutic perspective , however , a ratio of 50:50 is not realistic : due to the nutrient limitations of the intervertebral disk in vivo , multiplying the local cell density by 2 may lead to the death of both endogenous and exogenous cell populations . moreover , a ratio of 30:70 , close to what was used here , was also reported to have a positive , even if milder , effect on npcs . second , in the in vitro studies , the npcs were isolated from their extracellular matrix prior to coculture in alginate . because the matrix produced during culture is compared with the initial amount , which is small ( or even nonexistent ) in alginate cultures after cell isolation , production of proteoglycans will appear as large increases in matrix production . contrarily , the initial amount of matrix in the explants is high , and the matrix production per cell has to increase to a great extent to change the total matrix content . because of this difference , it is unclear how the matrix production rates by npcs in alginate cocultures relate to those in tissue . however , it has been observed that the expression of acan and col2a1 decreased significantly after npc isolation , but gradually increased in three - dimensional culture ( agarose ) until reaching native gene expression levels by day 25 of culture . this result indicates that the isolation procedure and three - dimensional culture of npcs have an effect on their phenotype and metabolism . the proteoglycan production by npcs in three - dimensional culture ( alginate and agarose ) also increased in a time - dependent manner , but was not compared with the native proteoglycan production rate . the native expression of brachyury ( t ) , cytokeratins 8 and 18 , acan , and col2a1 by ncs decreased significantly after 1 day of culture in alginate beads . in addition , ncs are sensitive to the culture conditions : ascorbic acid , physiologic osmolarity , medium type , and hypoxia contribute to maintenance of their phenotype , whereas a ph  <  7.2 , both very low and high glucose concentrations , and fbs in the culture medium ( unpublished data from our group ) are detrimental to the phenotype . the presence of fbs in the medium during the 42 days of culture could explain the disappearance of nc vesicles by the end of the culture . however , the morphologic changes can also be attributed to the isolation of ncs from their natural environment and culture in a mature np . the change in morphology is probably coupled to a change in functionality of the ncs . if the morphology and gene expression profiles of ncs change gradually during culture , this result could explain that the regulatory effect of ncs is stronger in short - term ( 3 days ) than in long - term ( > 14 days ) coculture . in this respect , the use of nc - conditioned medium may be a solution for long - term in situ cultures . medium conditioned by ncs for 4 days has indeed been shown to increase the proteoglycan production by npcs in in vitro cultures , in levels even higher than in cocultures with direct cell  cell contact . . the administration of soluble factors in fact could be more attractive than exogenous cell injection as smaller - gauge needles can be used . needle punctures of 25 gauge ( required for cell injection ) have been shown to have a detrimental effect on disk biomechanics . the np explants were cultured in hypertonic peg , as described by van dijk et al . this culture system previously showed a stable biochemical content and col2a1 gene expression during long - term culture ( 42 days ) , but native acan and col1a1 expression were not maintained . in the current study , the expression of the latter genes did not decrease , which may be due to two differences in culture conditions : ( 1 ) injection of pbs prior to culture , or ( 2 ) the ph of the culture medium , which was not adjusted to 7.1 in the current study . it is possible that the injection of pbs in the tissue triggered a change in gene expression , but an anabolic response would not be expected . furthermore , it has been described that the proteoglycan synthesis by npcs is higher at ph 7.1 than at 7.4 , which is not reflected in the proteoglycan content and related acan gene expression in the current study compared with the previous one . improved np culture systems are currently available for future experiments . 
  although the results from this study show that direct injections of ncs are not effective for np regeneration , this finding should be interpreted with care for translation to the human situation . first of all , the np tissue used in this study corresponded to human grade ii on the thompson grading scale . although the mechanical loading of bovine caudal tissue may be more similar to that in the human cervical rather than lumbar spine , the biochemical composition and cell population of bovine and human grade ii np tissue have been shown to be similar . although early stage regeneration would theoretically be ideal , the regenerative potential of these cells may be less than for cells of a higher grade of degeneration . second , although the issue of interspecies size differences was avoided by using an np explant culture , such a system also introduced drawbacks . interplay with the surrounding tissues was not possible , and certain physiologic factors ( i.e. , dynamic loading , limited nutrient supply ) could not be simulated in the current system . molecular - based regenerative therapies are aimed for treatment of nps with moderate degeneration . in this stage , the resident cells are still chondrocytic in nature , able to produce matrix , and the degenerative changes are not yet insurmountable . cell - based regenerative therapies are considered applicable for more advanced degeneration , where the depleted , senescent , or dedifferentiated resident cell population would need additional help to replenish the extracellular matrix . however , the sensitivity of detection methods for moderate disk degeneration is currently insufficient , and more importantly , our inability to provide accurate prognosis for aggressive symptomatic disk degeneration is actually a challenge to introduce early preventive treatments . certainly , as more sensitive diagnostic tools will be developed , the selection of patients for disk regeneration will become clearer . contrary to alginate coculture , no anabolic response was observed when ncs were injected into np explants , which may be due to the observed changed morphology of the ncs during the 42 days of culture where they became more similar in appearance to resident npcs . therefore , considering the results with nc - conditioned medium , it may be a more promising alternative for in situ stimulation of npcs .
study designin vitro disk explant culture.objectivenotochordal cells ( ncs ) have been shown to upregulate matrix production by nucleus pulposus ( np ) cells in coculture . to examine the translation of these in vitro results to a nativelike setting , 
 the regenerative potential of ncs injected into np tissue was assessed in this study.methodsnp explants were cultured after injection with ncs in phosphate - buffered saline ( pbs ) or with pbs alone ( sham ) . at days 0 and 42 , cell viability and morphology , water , dna , sulfated glycosaminoglycan and hydroxyproline content , and gene expression of anabolic markers were analyzed.resultsncs remained viable during culture , but their morphology changed . 
 the biochemical content remained unchanged , except for the dna content in the nc group . 
 overall acan expression remained unchanged , whereas col2a1 decreased during culture.conclusionsno overall anabolic response was observed when ncs were injected into np explants . 
 ncs were found to survive but did not display the typical nc morphology by the end of the culture period .