3-D bioprinting constructs for cartilageregeneration23 September 2020, by Thamarasee Jeewandara

Schematic presentation of the study design and scaffoldconstruction. (A) Schematic Illustration of the studydesign with 3D bioprinted dual-factor releasing andgradient-structured MSC-laden constructs for articularcartilage regeneration in rabbits. Schematic diagram ofconstruction of the anisotropic cartilage scaffold andstudy design. (B) A computer-aided design (CAD) modelwas used to design the four-layer gradient PCLscaffolding structure to offer BMS for anisotropicchondrogenic differentiation and nutrient supply in deeplayers (left). Gradient anisotropic cartilage scaffold wasconstructed by one-step 3D bioprinting gradientpolymeric scaffolding structure and dual protein-releasing composite hydrogels with bioinksencapsulating BMSCs with BMP4 or TGF?3 ?S as BCSfor chondrogenesis (middle). The anisotropic cartilageconstruct provides structural support and sustainedrelease of BMSCs and differentiative proteins forbiomimetic regeneration of the anisotropic articularcartilage when transplanted in the animal model (right).Different components in the diagram are depicted at thebottom. HA, hyaluronic acid. Credit: Science Advances,doi: 10.1126/sciadv.aay1422

Cartilage injury is a common cause of jointdysfunction and existing joint prostheses cannotremodel with host joint tissue. However, it ischallenging to develop large-scale biomimeticanisotropic constructs that structurally mimic nativecartilage. In a new report on Science Advances, YeSun and a team of scientists in orthopedics,translational research and polymer science inChina, detailed anisotropic cartilage regenerationusing three-dimensional (3-D) bioprinting dual-factor releasing gradient-structured constructs. Theteam used the dual-growth-factor releasingmesenchymal stem cell (MSC)-laden hydrogels forchondrogenic differentiation (cartilagedevelopment). The 3-D bioprinted cartilageconstructs showed whole-layer integrity, lubricationof superficial layers and nutrient supply into deeperlayers. The scientists tested the cartilage tissue inthe lab and in animal models to show tissuematuration and organization for translation tohumans after sufficient experimental studies. Theone-step, 3-D printed dual-factor releasing gradient-structured cartilage constructs can assistregeneration of MSC- and 3-D bioprinted therapyfor injured or degenerative joints.

Chondrogenesis in the lab

Articular cartilage typically forms an elasticconnective tissue in the joint. Cartilage injury is anextremely common impairment although withlimited self-healing capacity due to the lowcellularity and avascular nature of the tissue.Damage to cartilage can be debilitating andcartilage or joint reconstruction is currentlyconsiderably challenging in the research lab. Inclinical practice arthritic joints can be replaced bytotal joint arthroplasty using metallic and syntheticprosthesis. However, the existing joint prosthesescannot remodel (or integrate) with host tissue,which can lead to long-term functional impairmentsthat can only be addressed via biologicalregeneration of the joint. Scientists have recentlydeveloped mesenchymal stem cell (MSC)

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transplants to stimulate directional differentiationinto chondrocytes as a new method for cartilagerepair. However, it is still challenging to mimic thegradient anisotropic structure and signalingapproaches in different layers to induce zonal-dependent chondrogenic differentiation andextracellular matrix (ECM) deposition to promoteosteochondral regeneration. In this work, Sun et al.developed a 3-D bioprinted, dual-factor releasing,gradient-structured MSC-laden construct to implantand establish whole-layer cartilage regeneration inan animal model.

3D bioprinted gradient cartilage scaffold for implantation.(A) Gross appearance of (a) human-scale and (b and c)rabbit-scale cartilage scaffold (b, NG with 150-?mspacing; c, NG with 750-?m spacing). Top view of therabbit cartilage scaffold is also shown (d, NG with 150-?mspacing; e, NG with 750-?m spacing; f, gradient scaffoldwith 150- to 750-?m spacing) atop of the SEM images (g,horizontal section; h, vertical section) taken for the150-?m NG scaffold to demonstrate the precisealignment of the PCL fibers in the printed scaffold. (B)Deconstruction of the gradient scaffold. The structure ofthe gradient scaffold was deconstructed into four layers.

Microscopic appearance of the hydrogel-PCL compositestructure in each layer demonstrated goodinterconnectivity and delicate, orderly aligned structurefor each layer. (C and D) Good cell viability is shownrespectively for superficial and deep layers after printingwith live/dead assay (green, live cells; red, dead cells)(C) under a microscope and (D) under a confocalmicroscope. DAPI, 4?,6-diamidino-2-phenylindole. (E)Cell spreading in superficial and deep layers withcytoskeleton staining. (F) Immunostaining for cartilagemarkers in superficial and deep layers. Expression ofCOL2A1 and PRG4, the lubrication markers, wassignificantly higher in the superficial layers with smallpore size (a and b), while the chondrogenic cells in thedeep layers (c and d) mostly presented with hypertrophicphenotype (COL10A1 expression). Photo credit: Ye Sun,First Affiliated Hospital of Nanjing Medical University.Credit: Science Advances, doi: 10.1126/sciadv.aay1422

3-D bioprinting cartilage constructs

The team used 3-D bioprinting to develop differentjoint tissue constructs for joint reconstruction. Theymimicked the native cartilage by includingbiochemical stimulus (BCS) with diverse growthfactor releasing constructs and biomechanicalstimulus (BMS) with small pore sizes to induce chondrogenesis. They then created a third cartilageconstruct as a double stimulus (DS) group toinclude the two versions of stimuli. For growthfactors, the team chose a combination of bonemorphogenetic protein (BMP4) and the transforming growth factor ?3 (TGF?3) in thecartilage construct to regenerate complexinhomogeneous joint tissues. Sun et al. thendeveloped a hydrogel to deliver the growth factorsand used poly(lactic-co-glycolic acid) (PLGA)microspheres as a carrier/vehicle. The teammaintained constant fiber spacing for the BMS(biomechanical stimulus) group and BCS(biochemical stimulus) group to develop the non-gradient scaffolds, while introducing graduallyvarying fiber spacing for scaffolds in the DS (doublestimulus) group. The scientists also used poly(?-caprolactone) (PCL) polymers and integrated themto the biomimetic scaffold construct. In this way,they developed rabbit cartilage constructs using 4 x4 x 4 mm scaffolds and human cartilage constructsusing 14 x 14 x 14 mm scaffolds.

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The 3D printing process using OPUS system showing thehydrogel-PCL patterning and gradient microchannels ofevery layer and the whole cartilage construct. Credit:Science Advances, doi: 10.1126/sciadv.aay1422

Testing the effects of the scaffolds andcartilaginous matrix formation in the lab

To test the impact of the growth factors on bonemarrow stromal cell (BMSC) viability andproliferation, Sun et al. cultured BMSCs inhydrogels for seven days. The microspheres firstreleased sustained and controlled volumes ofgrowth factors in the lab. Then the scientistsfollowed the experiment with cell viability andproliferation assays on printed scaffolds to note thesurvival of BMSCs at 60 minutes (day zero), sevendays and 21 days after bioprinting. The live cellsshowed increased cell viability on day zero,followed by sustained growth from days three to 21.After 21 days, Sun et al. noted good 3-D cellanchorage on the scaffold and the work showed thedevelopment of a favorable microenvironment forBMSC growth and differentiation to formchondrocytes in the lab.

Before translating the 3-D bioprinted scaffold intoan animal model, Sun et al. tested if the delivery ofgrowth factors could induce layer-specific BMSCdifferentiation into chondrocytes. They followed anexperimental protocol and observed aggrecan (aglial marker protein) and type II collagen to formhyaline articular chondrocyte-like cells. The teamthen transplanted the cartilage scaffolds into an animal model and examined their functionality for12 weeks in vivo. The enhanced mechanical

properties of the resulting articular cartilage showedpromising regeneration to provide structural supportfor the newly formed cartilage tissue. After 12weeks, Sun et al. conducted immunofluorescenceimaging to show resemblance of the implantedcartilage constructs to native joint cartilagesurrounded by the cartilage matrix.

Cell viability and anchoring in the printed anisotropicscaffold. (A) Schematic of anisotropic cartilage scaffoldconstruction with fabrication of gradient scaffoldingstructure (left) and large-scale printing of aligned protein-releasing BMSC-laden hydrogel (right). Scale bar, 1 mm.(B) Gross appearance of PLGA ?S–encapsulated BMSC-laden hydrogel under a microscope (top). Printed cell-laden hydrogel causes cell alignment in a longitudinaldirection of the printed paths, forming a reticular networkwith cell interaction (bottom). (C) Live/dead cell assaysshowed ?95% cell viability maintained through day 1 to21 for all four layers with gradient spacing (4th row,150-?m spacing; 3rd row, 350-?m spacing; 2nd row,550-?m spacing; 1st row, 750-?m spacing).Immunostaining of cytoskeleton (rightmost column)showed cell spreading both in the hydrogel and on thePCL fibers throughout the four layers of the construct.Scale bar, 500 ?m. (D and E) Quantified cell viability andproliferation in the printed scaffolds. (F) Cell anchoring inthe scaffolds. (a to c) At day 21, good 3D anchoring tothe PCL fiber cylinder was observed for the MSC cellsreleased from the hydrogel. (d to f) Similar cell anchoringwas observed for PCL fibers in adjacent layers. (b), (c),(e), and (f) are 3D demonstration of cell anchoring in (a)and (d), respectively. Scale bars, 100 ?m. Photo credit:Ye Sun, First Affiliated Hospital of Nanjing MedicalUniversity. Credit: Science Advances, doi:

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10.1126/sciadv.aay1422

Better repairing effects of cartilage implants ina rabbit knee cartilage defect model

Sun et al. used rabbit experimental models to testthe capacity of cartilage scaffolds during kneerepair. They constructed the scaffolds using one-step 3-D bioprinting to provide structural supportand sustained release of cells. The experimentfacilitated biomimetic regeneration of the nativearticular cartilage and the implant showed betterintegration at the site of defect by 24 weeks for thedual stimuli (DS) experimental group comparedwith both BMS and BCS groups. The teammonitored the site of transplantation with magneticresonance imaging (MRI) for notable healing of thearticular cartilage after 24 weeks (within sixmonths). The results showed better cartilage repairand joint management with the DS cohortcompared to the BCS or BMS animal groups.

Dual-factor releasing, and gradient-structured cartilagescaffold demonstrated better repairing effect ofanisotropic cartilage in rabbit knee cartilage defect modelin vivo. (A) Scaffold implantation process and grossappearance of the repair cartilage at 8, 12, and 24weeks. MRI was made for the operated knee joint (fifthrow), demonstrating significant better resolution ofsubchondral edema and healing of the articular surface(white arrowheads) for joint transplanted with DSscaffolds. (B to F) Chondroprotective effects of the

scaffolds were compared by (B) histological scoringevaluation of the repaired cartilage tissue during in vivoimplantation. (C) Mankin score and (D) ICRS(International Cartilage Repair Society) histological scoreof articular cartilage in the femoral condyle (FC) and tibialplateau (TP) in both groups with scaffold implantation. *P

The team observed articular cartilage transitions from thesuperficial zone to deeper zones and tested theproperties of the generated cartilage comparatively withthe native cartilage. As before, the DS-scaffold showedingrown microvessels with improved growth compared tothe other groups (BCS and BMS). In this way, Ye Sunand colleagues generated 3-D bioprinted anisotropicconstructs with structural integrity for joint reconstruction,articular cartilage regeneration and functional knee articular cartilage construction in a rabbit model. The 3-Dbioprinted functional constructs acted as prosthesisduring joint replacement or cartilage repair to heal injuredor degenerate joints in animal models. The scientists willconduct further experiments to understand the functionalsustenance of joint construction in animal models. Afterfurther experiments in pre-clinical animal models, theteam envision translating the 3-D bioprinted constructsvia mini-invasive arthroscopy to replace damaged ordegenerative joints within humans.

More information: Ye Sun et al. 3D bioprinting dual-factor releasing and gradient-structured constructs readyto implant for anisotropic cartilage regeneration, ScienceAdvances (2020). DOI: 10.1126/sciadv.aay1422

Chang H Lee et al. Regeneration of the articular surfaceof the rabbit synovial joint by cell homing: a proof ofconcept study, The Lancet (2010). DOI:10.1016/S0140-6736(10)60668-X

April M Craft et al. Generation of articular chondrocytesfrom human pluripotent stem cells, Nature Biotechnology(2015). DOI: 10.1038/nbt.3210

Benjamin R. Freedman et al. Biomaterials to Mimic andHeal Connective Tissues, Advanced Materials (2019). DOI: 10.1002/adma.201806695

© 2020 Science X Network

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APA citation: 3-D bioprinting constructs for cartilage regeneration (2020, September 23) retrieved 23December 2021 from https://medicalxpress.com/news/2020-09-d-bioprinting-cartilage-regeneration.html

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