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3. Results

3.1. Generation of the Spindle Cell-Laden Constructs

The semi-spindle constructs were formed when the ACMS was rotated at a specific speed (30 r/min) [36]. During the combined mold rotation stage, the cell-laden fibrinogen solution moved around the internal branched mold according to the Weissenberg effect of non-Newtonian fluid. After the fibrinogen molecules were polymerized with the thrombin solution, cells (i.e., ADSCs and hepatocytes) were immobilized in different layers of the fibrin hydrogels and the cell-laden fibrin hydrogels took the shape of membranes (Figure 2c). The rotation speed had a significant effect on the semi-spindle cell-laden membrane structures. At the beginning (~10 r/min) most of the cell-laden hydrogels were deposited on the bottom of the mold lumen. When the rotation speed reached 20 r/min, the climbing pole phenomenon of the cell-laden fibrin hydrogels became more evident in the semi-spindle constructs. At 30 r/min, most of the internal branched molds were covered by the cell-laden fibrin hydrogels with a slim semi-spindle structure.

A spindle construct with two multi-branched vascular networks and two cell lines is shown in Figure 2. The vascular networks include one central artery-like channel, 7 branched arteriole-like channels, a middle capillary-like layer, 7 branched venule-like channels and a central vein-like channel. Each of the semi-spindle cell-laden construct contains a multi-branched vascular network (Figure 2a,e). When the two semi-spindle constructs were connected with a thin layer of ADSC-laden fibrin hydrogel, a complete vascular network (mimicking the natural arteries, arteriolae, capillaries, venulae and veins in a visceral organ) was constructed (Figure 2c,e). Tight connections between the two embodied semi-spindle constructs were achieved using a layer of middle ADSC-laden fibrin hydrogel, with a continuous PLGA overcoat extending from the two semi-spindle constructs. The 7 branched channels are not directly connected between the two halves in case of the “short circuit”. Thus, the vascular network includes one inlet (mimicking the artery), 7 branched arteriole-like channels, a capillary-like middle layer in between the two semi-spindle constructs, 7 branched venule-like channels and an outlet (mimicking the vein). The middle capillary-like layer can protect the fluid from “short circuit”, i.e., going directly from the arteriole-like channels to the venule-like channels, mimicking the real capillary structures in the liver.

3.2. Cell Viability in the Spindle Construct

Live/dead cells in the spindle construct were firstly detected using a LSCM after AO-PI staining. Dead cells appeared red for absorbing the PI dye, while living cells presented green with the AO. Figure 3 represents the detection results of the ADSCs and hepatocytes encapsulated in the fibrin membranes after 3, 6, and 9 days of in vitro culture. Before the fabrication stage, both the ADSCs and hepatocytes were distributed in the fibrinogen solution evenly. After being assembled, cells in the fibrin membranes were reunited as aggregates (Figure 3a,b). Large cell aggregates were prominent in the 9-day samples. During the 9 days in vitro culture, all the ADSCs were stained green, suggesting a very high level of stem cell survival capability. Meanwhile, a few red colored hepatocytes were dead. Hepatocyte viability was more than 98%. These results indicate that the cell assembling procedures, the rotational combined molding, PLGA coating and organic solvent extraction processes have no significant effects on the cell viability.

In Figure 4, the proliferation rates are determined by MTT tests. Both the ADSCs and hepatocytes proliferated during the 9 days of in vitro culture. The proliferation rates of the two types of cells were not linear with the cell densities and culture times. In particular, the proliferation graph of the ADSCs increased very sharply compared with that of the hepatocytes. These results indicate that the proliferation capability of the ADSCs was much higher than that of the hepatocytes over the 9 days culture. A significant difference was found between the two groups containing ADSCs and hepatocytes (p < 0.01).

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Figure 3. Live/Dead states of the adipose-derived stem cells (ADSCs) and hepatocytes in the spindle Figure 3. Live/Deadstatesoftheaadiposese--deriveded stem cells (ADSCs) and hepatocytes in the spindle

construct after different in vitro culture periods: (a) ADSCs 3 days; (b) ADSCs 6 days; (c) ADSCs 9 constructtafterdifferentferentininvitrovitrocultureperiods:(a)(ADSCsa) ADSCs3 days;3 days;(b) ADSCs(b) ADSCs6 days;6 days;(c) ADSCs(c) ADSCs9 days;9

days; (d) hepatocytes 3 days; (e) hepatocytes 6 days, a magnified image on the upper right corner to (days;) hepatocytes(d) hepatocytes3 days;3 (days;e) hepatocytes( ) hepatocytes6 days,6 days,magnifiedmagnifiedimageimageon theonupperthe upperright cornerright cornerto showto

show the living cells more clearly; (f) hepatocytes 9 days. Red color stands for dead cells, while green thes owlivingthe livingcells morecells clearly;more clearly;(f) hepatocytes(f) hepatocytes9 days9. daysRed color. Red standcolor standsfor deadfor cells,dead whilecells, whilegreengreencolor

color stands for living cells. standscolor tandsfor livingfor livingcells. cells.

Figure 4. Adipose-derived stem cell and hepatocyte proliferation rates in the fibrin hydrogel within

Figure 4. Adipose-derived stem cell and hepatocyte proliferation rates in the fibrin hydrogel within Figure 4. Adipose-derived stem cell and hepatocyte proliferation rates in the fibrin hydrogel within the spindle construct after different periods of in vitro cultures (p < 0.01).

the spindle construct after different periods of in vitro cultures (p < 0.01). the spindle construct after different periods of in vitro cultures (p < 0.01).

3.3. In Vitro Engagement Effects of ADSCs to ECs and Hepatocytes in the Spindle Constructs

3.3. In Vitro Engagement Effects of ADSCs to ECs and Hepatocytes in the Spindle Constructs 3.3. In Vitro Engagement Effects of ADSCs to ECs and Hepatocytes in the Spindle Constructs

As stated above, the two semi-spindle constructs were connected with a thin layer of ADSC-laden

As stated above, the two semi-spindle constructs were connected with a thin layer of ADSC- fibrinAshydrogelstated abov. Afte,r theADSCstwo insemithe-spindlefibrin hydrogelconstructswerewereengagedconnectedto ECs,witha vascularthin layernetworkof ADSCwas- laden fibrin hydrogel. After ADSCs in the fibrin hydrogel were engaged to ECs, a vascular network formedladen fibrwinthhydrogelgo-through. AfterbranchedADSCschannelsin the fibrininsidehydrogelthe spindlewereconstructsengaged to. FiguresECs, a vascular5 and 6 shownet orkthe was formed with go-through branched channels inside the spindle constructs. Figures 5 and 6 show immunofluorescencewas formed with go-throughand HEbrstanchedining resultschannelsof insidethe spindlethe spindleconstructsconstructswith low. Figures(1 105 and6 cells/mL)6 show the immunofluorescence and HE staining results of the spindle constructs with low (1 × 106 cells/mL) andthe immunofluorescencehigh (3 106 cells/mL)and HEinitistainingl ADSCresultsdensitiesof the atspindlediff rentconstructsengagementwith lowstages(1 × .10Along6 cells/mL)the and high (3 × 106 cells/mL) initial ADSC densities at different engagement stages. Along the two twoand multihigh -(3branched× 106 cells/mL)vascularinitialnetworksADSCnumerousdensitieshoneycombat different-likeengagementcapillary-likestagesstructures. Along theformedtwo multi-branched vascular networks numerous honeycomb-like capillary-like structures formed

(Figuremulti-branched5 –f). Especiallyvascularinnetworksthe middlenumerouscell-laden fibrinhoneycconnectionmb-like layer,capillarythe-likecapillastryuctures-like structuresformed (Figure 5d–f). Especially in the middle cell-laden fibrin connection layer, the capillary-like structures were(Figuremore5d–prominent,f). Especiallysimilarin the middleto thosecellat -theladendsfibrinof theconnectionmulti-branchedlayer, thearteriolescapillaryand-likevenulesstructuresin were more prominent, similar to those at the ends of the multi-branched arterioles and venules in a awererealmorenativepromiorganent,suchsimilaras thetoliverthose. Theat themicroporesends of thearemultimainly-branchedresultarteriolesof the highandwatervenulescontentin a real native organ, such as the liver. The micropores are mainly a result of the high water content fibrin fibrireal nativehydrogel,rgan,whichsuchbenefitsas the liverthe. capillaryThe micropores-like structureare mainlyformationresultalofngthethehighADSCwaterclusterscontent. Forfibrinthe hydrogel, which benefits the capillary-like structure formation along the ADSC clusters. For the hydrogel, which benefits the capillary-like structure formation along the ADSC clusters. For the

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middle cell-laden fibrin connection layer, sufficient nutrient, gas and waste exchanges take place when middle cell-laden fibrin connection layer, sufficient nutrient, gas and waste exchanges take place

the culture medium passes through the two separated multi-branched channel networks. when the culture medium passes through the two separated multi-branched channel networks.

FigureFigure5. 5In. InvitrovitroengagementeffectsofofADSCs(lowcellldensity:11 × 101066cells/mL)totoECsECsat atdifferentdifferent culturecultureperiods:periods:(a()a)immunofluorescenceimmunofluorescence stainingg at the firstfirstdaybebeforeengagementshowingshowingthethe

purplish-blue ADSC nuclei withnegativeFacVIII marker (red);(b) immunofluorescence staining at purplish-blue ADSC nuclei with (b) immunofluorescence staining at

the fifth day showing that the dark red FacVIII marker emerged with reduced blue purplish-marker; the fifth day showing that the dark red FacVIII marker emerged with reduced blue purplish-marker;

(c) immunofluorescence staining at the tenth day showing that the FacVIII were positive (bright red)

(c) immunofluorescence staining at the tenth day showing that the FacVIII were positive (bright red)

with further reduced purplish-blue; (d) HE staining at the first day before engagement showing that with further reduced purplish-blue; (d) HE staining at the first day before engagement showing that the

the ADSCs encapsulated in the fibrin hydrogel randomly with a lot of micropores in the fibrin

ADSCs encapsulated in the fibrin hydrogel randomly with a lot of micropores in the fibrin hydrogel;

hydrogel; (e) HE staining at the fifth day showing that most of the ADSCs in the fibrin hydrogel were

(e) HE staining at the fifth day showing that most of the ADSCs in the fibrin hydrogel were elongated

elongated around the channels, with some large cell aggregates and macropores; (f) HE staining at around the channels, with some large cell aggregates and macropores; (f) HE staining at the tenth day,

the tenth day, showing that a thin layer of elongated ADSCs on the surface of the cell-laden fibrin showing that a thin layer of elongated ADSCs on the surface of the cell-laden fibrin hydrogel.

hydrogel.

At the beginning of engagement (0 day), ADSCs in the spindle construct were negative with the

At the beginning of engagement (0 day), ADSCs in the spindle construct were negative with the

FacVIII marker (Figures 5a and 6a). There was no FacVIII secretion around the ADSCs. The nuclei of the

FacVIII marker (Figures 5a and 6a). There was no FacVIII secretion around the ADSCs. The nuclei of

ADSCs expressed purplish-blue color with the fluorescence of DAPI. All the cells were encapsulated the ADSCs expressed purplish-blue color with the fluorescence of DAPI. All the cells were

in the fibrin membranes randomly with one or two (i.e., few or nearly no) large aggregates. Four days encapsulated in the fibrin membranes randomly with one or two (i.e., few or nearly no) large

after engagement, some of the ADSCs in both the constructs with low and high concentrations of aggregates. Four days after engagement, some of the ADSCs in both the constructs with low and high

fibrin were elongated with positive endothelial marker FacVIII (red color in Figures 5b and 6b). At the concentrations of fibrin were elongated with positive endothelial marker FacVIII (red color in Figures

tenth day of engagement, all the ADSCs along the multi-branched channels expressed endothelial

5b and 6b). At the tenth day of engagement, all the ADSCs along the multi-branched channels

marker FacVIII and organized into large cell clusters (Figures 5c and 6c,f,g). The purplish-blue colored expressed endothelial marker FacVIII and organized into large cell clusters (Figures 5c and 6c,f,g).

nuclei were encapsulated by thick endothelial marker FacVIII in red color (FiguresFacVIII5 and 6c). Some of

The purplish-blue colored nuclei were encapsulated by thick endothelial marker in red color the(Figurescells were5c aindproliferation6c). Some ofstageshe cellswithweredoublein proliferation-connected stagesnuclei.withEndothelialdouble-connectedcell morphologiesnuclei. dominaEndothelialed bothcellthemorphologieslow and highdominatedcell densitybothsamplesthe low. Inandparticular,high cell intimadensity-likesamplesstructure. In particular,mimicking theintimainner-layerlike structureof larg bloodmimickingvesselsthewasinnermuchlayermoreof largeprominentblood vesselsin the highwas muchcell densitymore prominent(i. ., low fibrin concthentration)high cell samplesdensity (i(Figure. ., low 6fibrinc,f,g).concentration)On the contrary,samplesit was(Figurehard for6c,f,g)the. elongatedOn the contrary,ECs initthewaslow cellharddensityfor thesampleselongatedto formECsthickin thecelllowsheetscell.densityThese resultssamplesindicateto formthatthickthecellADSCssheetsdiffer. Thentiatedse resultsinto

indicate that the ADSCs differentiated into ECs after the cocktail growth factor stimulation. The

ECs after the cocktail growth factor stimulation. The higher the ADSC density in the fibrin membrane

higher the ADSC density in the fibrin membrane was, the thicker the intima-like tissue formed. was, the thicker the intima-like tissue formed.

Another interesting phenomenon is that the engagement effects in the middle cell-laden fibrin

Another interesting phenomenon is that the engagement effects in the middle cell-laden fibrin

connection layer were obviously different from those around the multi-branched channels. In a HE connection layer were obviously different from those around the multi-branched channels. In a HE

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staining picture, macropore structurest res were detectetected at thee end of a brancheded channel (Figure 6i)6i).. There were no large cellll clusters (or spheroids) in the middle cell--laden fibrinfibrin connection layer.. However,plentyofofsmallsmallvortexesvortexesformedformedbybyendothelialelialce l-celllike-likeorganizationorganizationwerewerefoundfoundin thisinlayerthis. Thelayersizes. Theofsizestheofsmallthe smallvortexesvortexesin theinmiddlethe middlce el-ladencell-ladenfibrinfibrinconnectionconnectionlayerlayerrangedrangedfromfromoneoneto 20to 20m (Figureμm (Figure6h). Cells6h). withCellsredwithFacVIIIred mFacVIIIrker extendedmarker alongextendedthe smallalongvortexesthe small. Thevortexesmorphologies. The ofmorphologiesthe small vortexesof the smallwere similarvortexestowerethe capillarysimilar tostructuresthe capillaryin naturalstructuresliverin. a natural liver.

Figure 6. In vitro engagementteffectsofofADSCs(highcellcelldensity:33 × 106 cells/mL)toECss at different times: (a) immunoflfluorescence staining at the fifirst day before engagement showing that the nuclei of the ADSCs were purplish-blue with negative FacVIII marker; (b) immunoflfluorescence staining at the

fififth dayshowingthatthatthethepurpurplish-blue-bluemarkermarkerdecreaseddecreasedwhilewhilethe darktheredarkFacVIIIred mFacVIIIrker emergedarker andemergthedcellsandwerethe celongatedls were elongatedalong uniformalong adirection;uniform direction;( ) immunofluorescence( ) immunofluorescestainingceatstainingthe tenthat daythe tenthshowingdaythatshowingthe purplisthat th-ebluepurplishmarker-bluefurthemarkerdecreasedfurtherwithdecreasensed withlayerdenseof FacVIIIlayermarkedof FacVIIIred cellmarkedclusters;red cell(d) HEclusters;staining(d) atHEthestainingfirst dayatbeforethe firstengagementd y beforeshowingengagementthat stheowingADSCsthatencapsulatedthe ADSCs inencapsulatedthe fibrin hydrogelin the fibrinrandomlyhydrogelwithrandomlyfew smallwith acellfewaggregates;small cell aggregates;( ) HE staining(e) HEatstainingthe fifthatdaythe showingfifth daythatshowingmost ofthatthemostADSCsof thein theADSCsfibrininhydrogelthe fibrinwerehydrelongated;gel were(f)elongated;HE staining(f)atHEthestainingtenth dayat stheowingtenth andayelongatedshowing anADSCelongatsheedt onADSCthe surfacesheet onofthethesurfacecell-ladenof thefibrincell-hydrogel;laden fibrin(g)hydrogel;a SEM image( ) a ofSEMtheimageendothelialof -endotheliallike cell sheet-likealongcell sheeta branchedalong achannelbranchedat cthanneltenthatdaythe intenththedayspindletheconstructspindle withconstructsomewithfibrinsomefib rs;fibrin(h) immunofluorescencefibers; (h) immunofluorescencestaining showingstainingtheshowingendothelialendothelial-like cells in-likethe jointcells middlein the jointcell-middleladen fibrincell-ladenhydrogelfibrinlayerhydrogelwith alayerhigh orwiginalthhighcell densityoriginalatcellthedensitytenth dayat theengagement;tenth day (engagement;i) HE photo(i)showinga HE photothe cellshowing-ladenthefibrincell-hydrogelladen fibrinat theydrogelend ofat branchedthe end ofchannelbranchedat thechanneltenth

day engagement.

at the tenth day engagement.

In Figure 7a–c the mixed cells encapsulated in the fibrin membranes stained green, indicating that all the cells were alive after 6 days of in vitro culture. At the beginning of in vitro culture, two