Study Of 3D Bio-Printing Scaffolds With Cartilage Derived Decellularized Extracellular Matrix And Infrapatellar Fat Pad Mesenchymal Stem Cells For Repair Of Rabbit Cartilage Lesion

ObjectiveCartilage injury is one of the most important pathological processes in the pathogenesis and development of osteoarthritis with high clinical incidence.So far,there is no consensus on the optimal treatment.In this study,we aimed to prepare a cell-laden bio-ink containing infrapatellar fat pad adipose derived mesenchymal stem cells(IPFP-ADSC)and decellularized cartilage extracellular matrix(dCECM),and with which to bio-print 3D IPFP-ADSC-laden mimetic scaffolds with excellent capability of chondrogenesis in vitro and cartilage repair in vivo by means of advantageous precise stacking ability of 3D bio-printing.This study tried to throw out a minnow to catch a whale for tissue-engineered cartilage repair bioactive scaffolds to clinical translation.Methods and ResultsSection 1:Preparation and Biological Characteristics of IPFP-ADSC and dCECMMethods:IPFP-ADSC and subcutaneous fat adipose derived mesenchymal stem cell(SCF-ADSC)were extracted from the infrapatellar fat pad and subcutaneous adipose of rabbit with collagenase?,respectively.Multipotent differentiation ability(osteogenesis,chondrogenesis and adipogenesis)was identified with trilineage differentiation.Cell phenotypes were identified by flow cytometry(FC).Proliferation ability was assessed with CCK-8.Chondrogenic differentiation ability was further evaluated with real time polymerase chain reaction(RT-PCR)for gene expression of collagen?(COL2A1)and Sry Related HMG Box-9(Sox-9),as to find the ideal mesenchymal stem cell for cartilage regeneration.The porcine articular hyaluronic cartilage was treated with two different decellularization methods combined with physical,chemical and enzymatic processes.The residual chondrocytes,collagen,gycosaminoglycan(GAG)and double-stranded deoxyribonucleic acid(ds DNA)were evaluated with hematoxylin-eosin staining(HE staining)and corresponding essay-kits,respectively.Finally,the biocompatibility and chondrogenic effect of dCECM as a tissue-derived bio-active material on IPFP-ADSC was evaluated with Calcein AM/PI staining for live/dead cells,immunofluorescent staining(IF staining)for collagen?(COL-2)and aggrecan(ACAN),and RT-PCR for COL2A1 and Sox-9,respectively.Results:FC tests of IPFP-ADSC and SCF-ADSC showed that CD73?CD90 and CD105were highly expressed(>99%)while CD34?CD45 and HLA-DR were negatively expressed(<1%),matching the criteria referring to mesenchymal stem cells that proposed by the International Society for Stem Cell Therapy.Both IPFP-ADSC and SCF-ADSC could differentiate to osteocytes,chondrocytes and adipocytes.However,IPFP-ADSC was more powerful at chondrogenic differentiation,with relatively higher expression of COL2A1 and Sox-9(P<0.05).dCECM was successfully prepared by two methods,and with less than 5%residual ds DNA and few nucleuses in HE staining,indicating that chondrocytes were almost completely removed.Compared with method proposed in literature,more collagen was retained in the dCECM treated with modified method(P<0.05).IPFP-ADSC showed high viability after 1-week coculture with dCECM(>90%,P>0.05).The chondrogenesis specific maker protein(COL-2 and Sox-9)and gene(COL2A1 and Sox-9)expression level increased with the increase of dCECM concentration(P<0.05).Section 2:3D Bio-printing of IPFP-ADSC cell-laden dCECM-Hydrogel Scaffold and in vitro Study of Biological CharacteristicsPart 1:Preparation of Hybrid dCECM-Hydrogel Bio-ink and Characterization of3D Printing ScaffoldMethods:Methacrylic gelatin/alginate(GelMA/ALG)hydrogel bio-ink was prepared according to literatures and manufacturer's instructions,followed by mixture with different concentration of dCECM(A:3%,B:5%and C:7%).Cell viability,viscosity,storage and loss modulus of three types of dCECM/GelMA/ALG hybrid hydrogel bio-inks were evaluated for biocompatibility and printability.Three hydrogel scaffolds with different dCECM concentration were fabricated via Regenovo 3D Bio-Architect^?system,and the mechanical stiffness,porosity,swelling ratio,degeneration rate were evaluated to identify the optimal concentration of dCECM of the bio-ink for further cell-laden bio-scaffold experiment in vitro.Results:The rheological test showed that all three kinds of hybrid bio-inks represented the"shear thinning"and reversable sol-gel transition phenomenon.The gelatin temperatures of groups A and B were between 32?and 33?,which was higher than that of group C(27?29?),indicating that groups A and B were more suitable for printing at room temperature.After 72 hour's coculture,the cell viabilities of three bio-inks(un-crosslinked)were all higher than 80%while that in group C was the lowest(P<0.05).The addition of dCECM did not interfere the printability of hydrogel bio-ink.The scaffolds printed with 3%dCECM had better cell viability,mechanical strength,porosity and swelling ratio compared to those with 7%dCECM,while which had the lowest degenerative speed(P<0.05).Part 2:In vitro Study of Biological Characteristics of 3D Bio-printing IPFP-ADSC Cell-laden dCECM-Hydrogel Bio-ScaffoldMethods:The optimal concentration of dCECM was identified as 5%base on the results in the first part.Two types of IPFP-ADSC cell-laden bio-printing scaffolds were fabricated via Regenovo 3D Bio-Architect^?:IPFP-ADSC/dCECM(+)/GelMA/ALG and IPFP-ADSC/dCECM(-)/GelMA/ALG,with a cell density of 10⁷/mL.Cell viabilities were evaluated with Calcein AM/PI staining on days 3/7/14/28.Dissociative COL-2 and ACAN in culture medium supernatant were detected at different timepoints(days 3/7/10/14/21/28).The chondrogenesis specific marker protein(COL-2 and SOX-9)and gene expression(COL2A1 and SOX-9)were further evaluated with Western Blot and RT-PCR,respectively,to identify the chondrogenic ability of dCECM and 3D culture.Results:IPFP-ADSC viabilities in scaffolds were higher than 90%on 14^th day and 85%on 28^th day post-printing,respectively(P>0.05).IF sating,ELISA assay,Western Blot and Gene expression of specific markers of chondrogenesis,gradually increased with time in both groups.Significant higher expression of COL-2,ACAN and Sox-9 were detected in dCECM(+)scaffolds than that in dCECM(-)ones on 4^th week after printing weather in 2D plane culture or 3D scaffolds(P<0.05).Meanwhile,weather in dCECM(+)or dCECM(-)group,3D stereoscopic cultivation system showed higher expression of these chondrogenic specific markers than 2D system did on 4th week(P<0.05),indicating that dCECM and 3D stereoscopic cultivation system had synergetic chondrogenic effect on IPFP-ADSC.Section 3:In vivo Study of 3D Bio-printing IPFP-ADSC Cell-laden dCECM-Hydrogel Bio-Scaffold for Rabbit Cartilage RepairMethods:The immunogenicity of IPFP-ADSC and IPFP-ADSC/dCECM/GelMA/ALG bio-scaffolds were evaluated by subcutaneously injection or embedding to rabbits.Four intervention groups were as follows:PBS,IPFP-ADSC,dCECM/GelMA/ALG scaffold and IPFP-ADSC/dCECM/GelMA/ALG scaffolds were subcutaneously injected or embedded.The specimens were taken on the 2^nd and 4^th weeks after surgery for macroscopic observation and HE staining for foreign body reaction.Further,healthy New Zealand rabbits were randomly allocated into four groups(with 6 rabbits in each group):group A:PBS intraarticular injection;group B:IPFP-ADSC intraarticular injection;group C:dCECM/GelMA/ALG scaffolds implantation combined with IPFP-ADSC intraarticular injection;and group D:IPFP-ADSC/dCECM/GelMA/ALG scaffolds implantation.Bilateral femoral trochlear cartilage defects were created and treated with interventions mentioned above.Macro-and micro-scoring system(International Cartilage Repair Society Score and The Modified O'Driscoll Histological Score,respectively)were used to assess the newly generated tissue on the 6^th and the 12^th week after intervention.Results:Two weeks after subcutaneous injection or scaffold implantation in rabbits,no adhesion or neoplasma were observed in groups PBS and IPFP-ADSC.Peri-scaffold hyperaemia and mild adhesion were observed.With high-magnification microscopy observation,neutrophil infiltration existed,while no significant difference was detected between groups((3.6±0.5 vs 3.4±0.5,P>0.05).On 4^th week after intervention,neither adhesion nor hyperaemia was observed,neutrophil infiltration alleviated(1.4±1.14 vs 1.8±0.8,P>0.05),foreign body reaction in both groups decreased(P<0.05).Rabbit femoral trochlea chondral defect models were successfully established.In group D,the defect sites were almost totally filled with neo-generated cartilage tissue,and no crack was observed at the interface of repaired tissue and surrounding native tissue.The articular surface of the newly formed tissue was smooth and had more collagen and proteoglycan deposition than any other intervention group.Accordingly,the histological score of group D was significantly higher than any other group,followed by groups C,B and A(P<0.05).ConclusionIPFP-ADSC can be successfully extracted from IPFP by collagenase?,and was confirmed to have multipotent differentiation ability.Compared to SCF-ADSC,it represented stronger chondrogenic differentiation ability and might be the ideal seed cells for tissue engineering of cartilage repair.Modified decellularization method with more physical chondrocytes wall-broken process and less enzyme usage could retain more extracellular components like collagen while prepare qualified dCECM.dCECM represented good biocompatibility and chondrogenic bio-activity when co-cultured with IPFP-ADSC.dCECM/GelMA/ALG hybrid hydrogel bio-ink had good biocompatibility and printability,and with the advantage of 3D bio-printing,it could be printed with IPFP-ADSC homogeneously to fabricate biomimetic scaffold.IPFP-ADSC and IPFP-ADSC/dCECM/GelMA/ALG scaffolds showed mild immunogenicity and excellent histocompatibility.Under the synergistic effect of dCECM and 3D cultivation,the IPFP-ADSC/dCECM/GelMA/ALG bio-scaffolds with homogeneously ladened IPFP-ADSC can repair the chondral lesion faster and better than either the IPFP-ADSC intraarticular injection alone or with unordered combination of dCECM/GELMA/ALG scaffolds implantation.With the advantages of 3D bio-printing technology,the IPFP-ADSC cell-laden dCECM bio-scaffolds achieved the goal of dynamic integration of seed cells,bioactive factor and scaffold materials,which has good chondrogenic differentiation ability in vitro and cartilage repair effect in vivo.It might be a promising tissue engineering technique for cartilage repair with great clinically translational value.