Enforced Elongated Human Dermal Fibroblasts Exhibited Predominant Tenogenic Phenotype And Its Potential For Tendon Engineering

Objectives:In vitro expansion of tenocytes is likely to cause phenotype drift of cells.Previous report demonstrated that high density culture help to maintain the phenotype of in vitro cultured tenocytes.This study explored the possibility of tenogenic transdifferentiation of human dermal fibroblasts(HDFs)by high density monolayer culture.Methods:Cell density of 0.36×106 per dish(10 cm petri dish)for 4 passage culture(each for 4 days)was designated as a control of low density culture,whereas cell density of 2.5×106 per dish for 4 passage culture(each for 4 days)was employed as an experimental group to investigate high density culture mediated tenogenic transdifferentiation.At the time points of days 4,8 and 16 of in vitro culture,cells of both groups were photographed and collected for various analyses.Simultaneously,the potential mechanisms of this phenomenon was also investigated.Results:The results showed that when HDFs cultured at high density as opposed to low density after one passage culture(from day 1 to day 4),HDFs exhibited a preliminary tenogenic phenotype with higher gene expression levels of tenomodulin(TNMD),collagen type ?(COL3),decorin and tenascin-c(p<0.05)by qPCR.After two passage culture(day 5 to day 8),a relatively mature tenogenic phenotype was observed in high density cultured HDFs with significantly enhanced gene expression of TNMDy scleraxis(SCX),COL3,COL6,tenascin-c and decorin(p<0.05).At the fourth passage(day 12 to day 16),the expression of all tendon related markers was increased significantly in high density cultured fibroblasts(p<0.05).qPCR analysis also revealed that high density culture failed to induce other lineage differentiations(osteogenic,adipogenic and chondrogenic).It also failed to enhanced the gene expression of SOX9,COL2 and aggrecan and failed to induce tenogenic phenotype in high density cultured chondrocytes.In addition,high density culture of HDFs also significantly promoted the gene expression of transforming growth factor-?1(TGF?1)and growth differentiation factor(GDF)5,6,7 and 8,which are potent tenogenic inducing factors.The protein production of GDF8 and TGF ?1 was also increased by ELISA analysis.Cell morphology observation found that high density culture HDFs exhibited an apparent spindle cell shape,whereas the low density culture HDFs exhibited a spreading cell shape with outstretched pseudopodia.And treatment of cytochalasin D could disrupt cellular cytoskeleton,which resulted in cell shape change and disruption of high density induced expression of SCX and TNMD.Moreover,treatment of the inhibitor of TGF-? signaling(LY2109761)and the inhibitor of BMP signaling and GDF8 signaling(LDN193189)could abrogated high density-mediated tenogenic differentiation of HDFs.Conclusion:High-density culture is able to transiently induce the tenogenic phenotype of HDFs,but not their differentiation to other lineages.Although detailed mechanism study remains needed,the result of our preliminary study indicates that high density culture induced cell shape change and the release of pro-tenogenic factors may be involved in this phenotype switch.Objectives:Micropattern topography is widely investigated in mediating stem cell differentiation,but not explored for phenotype switch between mature cell types.This study aimed to examine the effect of microgroove topography on tenogenic transdifferentiation of human dermal fibroblasts.Methods:Silicone membrane with parallel microgroove of 10?m width and 3?m depth was employed to investigate cell morphology control mediated tenogenic transdifferentiation as an experimental group,whereas plain silicone membrane served as a control.After 4 and 12 days of in vitro culture of dermal fibroblasts onto two different silicone membranes,the tenogenic differentiation potential of induced cells was compared with qPCR,western blot and proteomics.The potential mechanisms involving in this phenomenon were also investigated.Results:Cells grown on microgrooved surface exhibited an elongated shape,whereas cells on plain membrane spread.qPCR analysis revealed significantly enhanced gene expression of scleraxis,tenomodulin,collagens ?,?,? and decorin in elongated cells.Western Blot showed enhanced protein production of tenomodulin and collagen ?.In addition,proteomics revealed enhanced production of collagen?A1,?A2,collagen ?A1,collagen ?A1,?A3 and decorin.By contrast,elongated and spread dermal fibroblasts expressed similar levels of chondrogenic,adipogenic and neurognic markers,although OCN and ALPL were relatively lower in elongated cells.Interestingly,elongated chondrocytes showed reduced expression of chondrogenic markers,but failed to induce tenogenic phenotype.Furthermore,elongated dermal fibroblasts demonstrated enhanced gene and protein expression of TGF-?1.Moreover,treatment of recombinant TGF-?1 at low dose(2ng/ml)could enhance the gene expression of scleraxis and tenomodulin,but reduced a-SMA expression in elongated cells.By contrast,high dose TGF-?1(10ng/ml)inhibited tenogenic phenotype and enhance myofibroblast transformation of elongated cells.Mechanism study revealed that elongated shape also resulted in decreased RhoA activity and increased ROCK activity.Antagonizing TGF-? or inhibiting ROCK activity with Y27632 or depolymerization of actin with cytochalasin D could all significantly inhibited tenogenic phenotype induction particularly in elongated cells.Conclusion:This study demonstrated that elongated cell shape control can lead to tenogenic transdifferentiation of dermal fibroblasts,and this is likely mediated by the synergistic effect of TGF-?1 and cytoskeletal signals.Objectives:.Nanofiber materials are widely used in tissue regeneration due to the superiority of nanofiber scaffolds in better mimicing natural extracellular matrix(ECM)structure.It is reported that the well-aligned nanofibers have been used for inducing stem cells into tenogenic differentiation,but remain unexplored for tenogenic transdifferenitation of human dermal fibroblasts(HDFs).In this study,we investigated if well-aligned nanofibers would direct HDFs into tenogenic lineage,and if this particular pattern was also able to recruit endogenous fibroblasts into the scaffold and form tendon tissue de novo.Methods:The aligned and randomly-oriented nanofiber scaffolds were fabricated by SJES(stable jet electrospinning)technique.Then cells were seeded onto the aligned and random nanofiber scaffolds at the density of 1.5×103 cells/cm2 and the cell orientation was observed at different time points.The tenogenic transdifferen-tiation of human dermal fibroblasts(HDFs)culture onto the nanofiber scaffolds was analyzed by qPCR.The random and aligned nanofiber scaffolds were secured on a custom-made spring formed with stainless steel frame.The tenogenic differentiation of HDFs cultured in vitro in 3D scaffold was examined at 6 and 12 weeks.The effect of aligned nanofibers on HDFs ability to form engineered tendon was also investigated in a nude mouse model.In addition,the effect of aligned nanofibers on recruiting host fibroblasts into the scaffold and forming engineered tendon was investigated as well in a rat Achilles tendon defect model.Results:The well-aligned nanofibers could elongate HDFs and induce a tenogenic phenotype by up-regulating the gene expression of scleraxis,MKX,tenomodulin,collagens ?,?,?,decoriny BGN and FMOD in vitro in 2D nanofibers.The results of SEM examination demonstrated that HDFs seeded on parallel aligned nanofibers seemed to produce and deposit more extracellular matrices when compared to the cells on random nanofibers.Upon in vitro culture for 6 and 12 week,well-aligned nanofibers formed organized neo-tendon structure with significantly promoted tenongenic gene expression,compared to random nanofibers.In nude mouse model,cell-seeded aligned nanofiber scaffolds formed much more organized neo-tendon with stronger mechanical properties compared to random nanofibers after in vivo implantation for 3 months.Cell-free aligned nanofiber scaffolds were able to regenerate tendon de novo after 3 months of implantation at the site of rat Achilles tendon defect with more organized tissue structure and enhanced tenogenic gene expression when compared to cell-free random nanofibers.Conclusion:The aligned nanofibers were able to induce hDFs into a tenogenic phenotype in vitro.The aligned nanofibers were able to regenerate tendon in vivo via better recruiting host fibroblasts into the scaffold and inducing their tenogenic transdifferentiation.Aligned nanofibers integrated with other bioactive signals may become an optimized tenongenic inductive scaffold.Objectives:Our previous study of laboratory has demonstrated that autologous dermal fibroblasts was able to serve as the cell source for engineered tendon formation both in vitro and in vivo,whereas allogeneic cell based and cell-free scaffold based tendon engineering approaches have also been reported.Whether allogenic cell based or cell-free scaffold strategies are similar to autologous cell based strategy in tendon engineering remain unexplored.This study investigated the efficacy of engineered tendon repair among three groups using rabbit Achilles tendon repair as an animal model and dermal fibroblasts as the cell source.Methods:Autologous and allogeneic(GFP transgenic)rabbit fibroblasts were harvested from New Zealand White rabbit nuchal skin and the fifth passage cells were used in the experiments.Autologous dermal fibroblasts were seeded on the composite scaffold consisting of an inner part of PGA unwoven fibers and an outer part of PLA-PGA(2:4)net scaffold knitted as autologous group,whereas allogenic group was similarly performed using allogeneic cells and the composite scaffold alone was used as the cell-free scaffold group.After cell-seeding,both cell-free and cell-seeded scaffolds were wrapped with 2%(20 mg/ml)cross-linked sodium hyaluronate gel(HA).All constructs were cultured in an incubator for 2h,and then in vivo implanted to repair Achilles tendon defect.Specimens were respectively harvested at 7 and 13 months post operation for gross,histological and mechanical analysis.Results:Our preliminary results demonstrate that tissue samples harvested at 7 months post-implantation revealed similar gross view and histological outcome,but there were some difference in the degradation rate of implanted scaffold among three groups;and tissue samples harvested at 13 months post-implantation also revealed similar gross view and histological outcome among three groups.The engineered tendons at 13 months post-implantation were relatively more mature than those of 7 months post-implantation with more material degradation.Mechanical analysis showed that there was no significant difference among three group(p>0.05).The diameter of collagen fibrils was slightly higher in autologous group than in cell-free group at 7 months time point(p<0.05),and slightly higher than allogeneic group and cell-free groups at 13 months time point.There was no significant difference between allogeneic group and cell-free group.Conclusion:These results suggest that dermal fibroblasts are likely to serve as the cell source for engineered tendon repair in vivo.And this study demonstrated that the feasibility of autologous,allogeneic and cell-free approaches in tendon regeneration,although there were some differences in histological structure of formed tissue,degradation rate of implanted scaffold and the successful rate.Objectives:Our previous work found that cell-free scaffold wrapped with cross-linked sodium hyaluronic acid gel(HA)could reconstruct tendon-like tissue in a partial defect model of rabbit Achilles tendon.However,this model failed to provide full loaded mechanical stimulation due to partial connection of unsectioned tendon.In this study,we further investigated if full-loaded mechanical stimulation and HA coating could promote tendon regeneration by cell-free composite scaffold in model of full sectioned Achilles tendon defect.Methods:Hyaluronic acid gel(HA,0.5%)was injected into the inner part of PGA unwoven fibers and the cross-linked HA gel(2%)was used to wrap the outer part of knitted net scaffold.Cell-free composite scaffold was implanted into the dorsal subcutaneous layer and tendon defect respectively as cell-free scaffold subcutaneous group and cell-free scaffold tendon group.Cell-free composite scaffold with HA was implanted into the dorsal subcutaneous layer and tendon defect respectively as cell-free scaffold-HA subcutaneous group and cell-free scaffold-HA tendon group.Specimens were respectively harvested at 3 and 6 months post-implantation for gross,histological,TEM,mechanical analysis and gene analysis.Results:Our preliminary results demonstrated that at 3 and 6 months post-implantation,the formed tissues in tendon defect were much better developed with more organized tissue structure and stronger mechanical properties when compared to the formed tissue in the dorsal subcutaneous environment(p<0.05).qPCR analysis revealed significantly enhanced gene expression of teno,nodulin,collagens ?,?,?,tenascin-c,decorin and fibromodulin in the formed tissues in tendon defect(p<0.05).At 3 months post-implantation,the mechanical properties of the formed tissues in cell-free scaffold-HA tendon group were stronger than those of cell-free scaffold tendon group(p<0.05),but there was no significant difference between two groups at 6 months post-implantation(p>0.05).At 3 months post-implantation,the histological structure of the engineered tendon in cell-free scaffold-HA tendon groupwas better than that of the engineered tendon in cell-free scaffold tendon group and the diameter of collagen fibrils was slightly higher than in cell-free scaffold tendon group(p<0.05).But at 6 months post-implantation,the engineered tendon in two groups showed similar histological structure and the same diameter of collagen fibrils(p>0.05).Conclusion:In repair of full sectioned Achilles tendon,cell-free scaffold could form better tendon-like tissue via the effect of full mechanical loading.In cell-free scaffold-HA tendon group,HA could better recruit host cells in the early time of tendon formed and HA was degratated with increased time.Then in the later time,the engineered tendons of composite scaffold and composite scaffold-HA were not apparently difference.HA play an important role in the engineered tendon.