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Regeneration of circuitous tissues, such as kidney, liver, and cartilage, continues to be a accurate and translational challenge. Adaptation of ex vivo cultured, crude beef in tissue grafts is amid one of the key barriers. Meniscus is a circuitous tissue consisting of collagen fibers and proteoglycans with acclivity phenotypes of fibrocartilage and functions to accommodate accordance of the knee joint, afterwards which the accommodating is acceptable to advance arthritis. Autogenous stem/progenitor beef regenerated the knee meniscus aloft spatially arise beastly affiliation tissue advance agency (CTGF) and transforming advance factor–β3 (TGFβ3) from a three-dimensional (3D)–printed biomaterial, enabling anatomic knee recovery. Sequentially activated CTGF and TGFβ3 were all-important and acceptable to actuate mesenchymal stem/progenitor cells, as a amalgamate citizenry or as single-cell progenies, into fibrochondrocytes that accordingly actinic procollagens I and IIα. Back arise from microchannels of 3D-printed, beastly meniscus scaffolds, CTGF and TGFβ3 induced autogenous stem/progenitor beef to differentiate and amalgamate zone-specific blazon I and II collagens. We again replaced sheep meniscus with anatomically correct, 3D-printed scaffolds that congenital spatially delivered CTGF and TGFβ3. Autogenous beef regenerated the meniscus with zone-specific cast phenotypes: primarily blazon I collagen in the alien zone, and blazon II collagen in the close zone, evocative of the built-in meniscus. Spatiotemporally delivered CTGF and TGFβ3 additionally adequate inhomogeneous automated backdrop in the regenerated sheep meniscus. Adaptation and directed adverse of autogenous beef in a tissue birthmark may accept implications in the about-face of circuitous (heterogeneous) tissues and organs.

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The meniscus in the knee aggregate is a crescent-shaped affiliation tissue amid the distal femoral and adjacent tibial condyles that provides structural accordance and absorbs automated armament (1). Meniscus injuries are advised with meniscus resection with or afterwards a cadaveric meniscus affix (2). Meniscectomy exposes femoral and tibial condyles to boundless abrasion and is a awful predisposing agency for osteoarthritis, the best accustomed account of abiding disabilities amid affection failure, analgesic bond injuries, respiratory disorders, and achievement (1). Meniscus backup with allografts or cadaveric tissue suffers from bound affix availability, antibody transmission, allowed rejection, and anatomical conflict (1, 2). Injuries and diseases of all fibrocartilage tissues, including the meniscus, the tendon-bone junctions, the intervertebral discs of the spine, and the temporomandibular joint, abort to alleviate spontaneously, arch to aggregate bloom affliction accountability estimated in assorted billions of dollars annually in the United States abandoned (1, 3).

A acutely insurmountable obstacle in meniscus regeneration, agnate to the about-face of best added circuitous tissues, is the absence of applicable beef able of regenerating assorted tissue phenotypes (1). Amalgamate populations of affiliation tissue beef aggregate the meniscus including fibroblast-like beef primarily in the alien area with abounding collagen blazon I, admitting the close area primarily consists of chondrocyte-like beef affluent in sulfate glycosaminoglycans (GAGs) and collagen blazon II (1, 5). The boilerplate area contains fibrochondrocytes, a corpuscle blazon that produces both blazon I and blazon II collagens (1, 5). Antecedent assignment on meniscus about-face has arise that no single-cell antecedent is acceptable to change amalgamate meniscus tissues (1, 6). Furthermore, the meniscus is bare of vascular accumulation except for the alien zone. The perceived absence of vascular-derived stem/progenitor beef is advised a hurdle in meniscus about-face (2).

Two acellular biomaterials austere for beastly use in Europe arise to abate affliction and affection but accept yielded inconsistent analytic aftereffect and appear adverse contest including affix shrinkage, ambiguous adapter and affiliation into host tissue, and myxoid decline (4, 5). Absent in antecedent assignment on the about-face of the meniscus is fibrocartilage phenotype that needs to be adequate for able meniscus activity to bear both compactness and compressive stresses (4, 5). The regenerated meniscus tissue should not abandoned restore corpuscle and cast backdrop but additionally accomplish automated functions, akin to added efforts to change the heart, lungs, kidney, bladder, and cartilage (7–10).

Here, we accept devised a three-dimensional (3D)–printed, anatomically actual biomaterial arch that can allure autogenous beef to change the meniscus with fibrocartilage tissues in specific zones. The arch releases two beastly proteins in a spatially and temporally controlled manner, arch to the address of assorted tissue phenotypes, acquiescent adorning clues for the about-face of circuitous tissues in a translational model. The present protein commitment access seems readily assertive for a beastly analytic balloon in which proteins are packaged in a acceptable accomplishment convenance (GMP) adeptness and alien to hospitals and clinics for accommodating use.

We aboriginal acquired fibrochondrocyte-like cells—meniscus-resident beef that accordingly produced both blazon I and blazon II collagens—from beastly cartilage basal and synovium mesenchymal stem/progenitor beef (MSCs) in vitro. Consecutive appliance of recombinant beastly affiliation tissue advance agency (rhCTGF) for 2 weeks in culture, followed by recombinant beastly transforming advance factor–β3 (rhTGFβ3) for 2 weeks (C→T in Fig. 1 and fig. S1), induced adverse of MSCs into beef that actinic procollagens I and IIα, suggesting that they were fibrochondrocyte-like cells, with a crop of 72.22% in monolayer adeptness and 76.92% in 3D fibrin hydrogels. This crop for C→T was decidedly college than that for the about-face (T→C) and the aggregate (T C), both in monolayer adeptness and in 3D fibrin (Fig. 1A and table S1) (Student’s t test: P < 0.000001 for C→T against T→C; P = 0.0032 for C→T against T C). C4 or T4 abandoned bootless to crop fibrochondrocyte-like cells. MSC adverse into fibrochondrocyte-like beef was attenuated aloft antipodal consecutive appraisal with TGFβ3 and CTGF (T→C) or either CTGF (C4) or TGFβ3 (T4) abandoned (Fig. 1A and table S1).

(A) Immunofluorescence appraisal of procollagen I alternation (proCOL-I) and procollagen IIα (proCOL-IIα) assembly by beastly MSCs advised in adeptness with C→T (rhCTGF for 2 weeks followed by rhTGFβ3 for 2 weeks), T→C (rhTGFβ3 for 2 weeks followed by rhCTGF for 2 weeks), C T (rhCTGF and TGFβ3 accordingly for 4 weeks), C4 (rhCTGF abandoned for 4 weeks), and T4 (rhTGFβ3 abandoned for 4 weeks). Corpuscle nuclei were decrepit with 4′,6-diamidino-2-phenylindole (DAPI). A absolute of bristles replicates were tested, with adumbrative images selected. (B to D) Announcement of (B) collagen blazon I (COL-I), (C) collagen blazon II (COL-II), and (D) aggrecan (AGC) mRNA afterwards MSC acknowledgment to treatments in (A), with the barring that circumstantial CTGF and TGFβ3 appliance (C T) was 4 weeks. Abstracts are averages ± SD (n = 5) normalized to GAPDH, again about to control. *P < 0.05 amid adumbrated groups; #P < 0.05 against control; P < 0.05 against absolute ascendancy (C4 for collagen blazon I or T4 for collagen blazon II and aggrecan), one-way appraisal of about-face (ANOVA) with post-hoc Tukey’s B test. Complete statistical appraisal is in table S2.

MSCs advised with C→T approved decidedly college collagen I, collagen II, and aggrecan mRNA than the ascendancy (untreated MSCs) (Fig. 1, B to D, and table S2). Moreover, C→T not abandoned induced MSC adverse but additionally yielded a fibrocartilaginous cast that decrepit absolutely for picrosirius red and alcian dejected in both monolayer adeptness (fig. S2A) and 3D fibrin gels (fig. S3A), advertence amalgam of both blazon I and blazon II collagens, respectively. C→T appraisal led to greater GAG agreeable than T→C (figs. S2B and S3B), but T→C produced added blazon II collagen than did C→T (figs. S2C and S3C). However, T→C bootless to crop a abundant cardinal of fibrochondrocyte-like cells, appropriately authoritative this appraisal access absurd for meniscus about-face in vivo (Fig. 1). We begin that C→T animated collagen I announcement by fibrochondrocytes primarily via focal adherence kinase (FAK) signaling, admitting collagen II and aggrecan announcement was apprenticed primarily via p38 signaling (fig. S4). Collectively, consecutive appliance of rhCTGF and rhTGFβ3 was all-important and acceptable to abet fibrochondrogenic adverse of beastly MSCs in vitro.

MSCs abandoned as mononucleated and adherent beef from cartilage basal and added sources are heterogeneous, and about represent a mix of complete beef such as osteoblasts, fibroblasts, and some stem/progenitor cells. We accordingly performed clonal appraisal to ascertain that fibrochondrocytes were acquired from progenies of a distinct MSC. Of 59 about called MSCs from beastly cartilage marrow, 29 (~53%) yielded clonal progenies, such as A5, B6, D7, and H1 (fig. S5A), by attached dilution. About 55% of clonal progenies, including A5 and B6, differentiated into fibrochondrogenic, osteogenic, chondrogenic, and adipogenic beef in agnate chemically authentic boilerplate (fig. S5B). Together, MSCs, as a amalgamate citizenry or as single-cell progenies, can differentiate into beef that accordingly aftermath blazon I and II collagens, arguably fibrochondrocytes aloft consecutive CTGF and TGFβ3 application, blame the charge for in vivo testing of fibrocartilage regeneration.

We chose to appraisal the activating aggregate of CTGF and TGFβ3 in an accustomed knee meniscus abrasion archetypal (11, 12), attributable to its abeyant generalizability in the about-face of complex, inhomogeneous tissues. Anatomically actual beastly meniscus scaffolds were bogus appliance 3D press (13). Briefly, poly-ε-caprolactone (PCL) was aqueous to assemble an anatomically actual meniscus arch modeled afterwards a reconstructed beastly body meniscus (Fig. 2A). The 3D-printed beastly meniscus arch was at ~40% of the absolute admeasurement of a beastly body meniscus (Fig. 2A), so that it fits in a corpuscle adeptness able-bodied for added experiments. Interlaid strands and abutting microchannels (diameters of 100 to 200 μm) were accumbent forth the circumferential administration as conduits for cellular ingrowth, artful the absolute meniscal collagen acclimatization (Fig. 2A, inset) (14) and acceptance for beef to be metabolically annoyed [within 200 μm of claret vessels, as apparent in (15)].

(A) Anatomic about-face of beastly meniscus. Beastly meniscus scaffolds were 3D-printed with layer-by-layer degradation of PCL fibers (100-μm diameter), basic 100- to 200-μm channels. (B) Poly(lactic-co-glycolic acid) (PLGA) microspheres (μS) encapsulating rhCTGF and rhTGFβ3 were in concrete acquaintance with PCL microfibers. (C) Fluorescent dextrans assuming CTGF (green, 40 kD) and TGFβ3 (red, 10 kD) were delivered into the alien and close zones, respectively, of beastly meniscus scaffolds to appearance arch loading. Administration of dextrans was maintained from day 1 to day 8. (D) rhCTGF and rhTGFβ3 absolution from the PCL scaffolds over time in vitro. (E) Back the scaffolds were incubated aloft beastly synovium MSC monolayers for 6 weeks, spatiotemporally delivered rhCTGF and rhTGFβ3 induced beef to appraisal zone-specific collagen blazon I and II matrices, agnate to the built-in rat meniscus. (F) Arch with abandoned μS showed little cast accumulation afterwards 6 weeks of coculture with 1:1 admixture of fibrogenic and chondrogenic supplements (no advance factors in medium). Spatiotemporal commitment of rhCTGF and rhTGFβ3 induced fibrocartilaginous cast formation, consisting of alcian blue–positive, collagen II–rich cartilaginous cast in the close area and picrosirius red–positive, collagen I–rich coarse cast in the alien zone. A absolute of bristles replicates were tested, with adumbrative images called from the aforementioned scaffold.

rhCTGF and rhTGFβ3 were microencapsulated in PLGA μS, with rhCTGF in 50:50 PLA/PGA μS and rhTGFβ3 in 75:25 PLA/PGA μS (Fig. 2B). CTGF and TGFb3 μS were tethered to the alien and close regions of the meniscus scaffold, respectively. Concrete bonding amid protein-encapsulating μS and PCL microfibers was induced by ethanol. To validate multiphase μS distribution, fluorophore-conjugated dextrans with atomic weights agnate to rhCTGF (~40 kD) and rhTGFβ3 (~10 kD) were delivered to the scaffold’s alien and close zones, respectively, at day 1 (Fig. 2C). At day 8, the administration of 40-kD dextrans (green) and 10-kD dextrans (red) was maintained. Spheres with 50:50 arrangement showed controlled absolution of CTGF first, followed by TGFβ3. Relatively accelerated rhCTGF absolution from the alien band and slower absolution of rhTGFβ3 from the close band were abiding over 42 canicule (Fig. 2D).

Before in vivo appliance of the advance factor–loaded PCL scaffold, we absolute whether spatiotemporal commitment of rhCTGF and rhTGFβ3 induced zone-specific MSC adverse into beef that produced blazon I and II collagens. Absorptive meniscus scaffolds accumulation rhCTGF and rhTGFβ3 for spatiotemporally controlled absolution were placed aloft monolayer-cultured beastly synovium MSCs. Afterwards a 6-week evolution aeon in vitro, spatiotemporally arise rhCTGF and rhTGFβ3 induced zone-specific announcement of collagen types I and II, apparently from the differentiated synovium MSCs, with affinity to the built-in rat meniscus (Fig. 2E). Abounding collagen blazon I was present primarily in the alien area (picrosirius red–positive), admitting cartilaginous cast with collagen blazon II was primarily in the close area (alcian blue–positive) (Fig. 2F). A alloyed fibrocartilaginous cast developed in the boilerplate area (Fig. 2F).

We aimed to change the sheep meniscus in vivo by spatiotemporal absolution of rhCTGF and rhTGFβ3 in anatomically actual PCL scaffolds. Sheep is a broadly acclimated preclinical archetypal for meniscus about-face attributable to its affinity to bodies in vascular supply, anatomy, and biochemical agreement (16). The built-in sheep centermost meniscus was laser-scanned for the artifact of absolute or fractional meniscus arch (Fig. 3A), followed by spatial rhCTGF and rhTGFβ3 assimilation as declared above.

(A) Anatomic ambit of the centermost meniscus was acquired from 3D laser scanning of a sheep body meniscus and reconstructed in 3D. Anatomically actual meniscus scaffolds were bogus for absolute or fractional grafts by layer-by-layer degradation of PCL appliance 3D printing. Scale bar, 15 mm. (B) Activating modulus (E*), accumulator modulus (E′), and accident modulus (E″) of PCL scaffolds with preoptimized centralized microstructure (200-μm microstrands and 100-μm microchannels) compared with the built-in meniscus. (C) Abutment daybed backbone in scaffold-to-native meniscus alien rim, sutured with 2-0 Ethibond. (D) Calm abrasion accessory of the scaffold, abstinent beneath abiding frictional microburst compared with the built-in meniscus. Abstracts in (B) to (D) are agency ± SD (n = 5). P ethics in (B) to (D) were bent by one-way ANOVA (n = 5 per group). (E) A arch was able for article with 2-0 Ethibond suture. The sheep centermost meniscus was apparent by dislocating femoral condyle, followed by arch implantation. (F) Afterwards 12 weeks, the built-in arch from (E) absolutely integrated. Articular cartilages on femoral and tibia condyles showed little accident on hematoxylin and eosin (H&E) staining.

Before in vivo implantation, the scaffold’s centralized microstructure was optimized to crop automated backdrop agnate to those of the built-in sheep meniscus. Amid 50- to 500-μm interlaid strands and interstrand microchannels, diameters of 200 and 100 μm were called because they yielded activating compressive modulus (E*), accumulator modulus (E′), and accident modulus (E″) agnate to the boilerplate area of the built-in sheep meniscus (Fig. 3B). Daybed tests were performed in harvested sheep cadavers afterwards fractional meniscectomy and abutment fixation (fig. S6). The best daybed backbone of the scaffold-to-outer rim of the built-in meniscus was decidedly college than the built-in fractional meniscus grafts-to-outer rims (54.5 N against 39.8 N) (Fig. 3C and fig. S6). The calm abrasion accessory on angular acceleration (0.01 to 1 rad/s) of the optimized arch was agnate to the built-in meniscus (Fig. 3D).

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A absolute of 11 skeletally complete (2- to 5-year-old) sheep were about allocated into ascendancy (scaffold abandoned with abandoned μS; n = 4) and appraisal (spatiotemporal rhCTGF and rhTGFβ3 delivery; n = 7) groups. We acclimated fractional meniscus grafting (Fig. 3A) attributable to aerial analytic ante of fractional meniscectomy (1, 2). Anatomically actual meniscus scaffolds were built-in afterwards fractional meniscectomy (Fig. 3E). Briefly, the centermost meniscus was apparent afterwards femoral condylectomy. With all ligaments preserved, ~80% centermost meniscus was resected including the inner, intermediate, and a aloft allocation of the alien zone, followed by backup with anatomically actual fractional meniscus scaffolds. The meniscus arch was sutured to the actual ~20% alien rim of the built-in meniscus. Twelve weeks afterwards implantation, the arch chip absolutely into the actual alien rim of the meniscus, with no notable accident to articular cartilage in either the femoral or tibial condyle (Fig. 3F).

The built-in sheep meniscus showed a adapted arrangement of extracellular cast administration with fibroblast-like beef amid collagen bundles in the alien zone, cartilage-like tissue in the close zone, and a alloyed coarse and cartilage tissue in the boilerplate area (Fig. 4A). Spatiotemporal commitment of CTGF and TGFβ3 from the built-in scaffolds yielded native-like fibrocartilage: a collagen-rich, accumbent coarse cast in the alien zone; a fibrocartilaginous cast in the boilerplate zone; and a cartilaginous cast in the close area (Fig. 4B), akin the zonal cast characteristics of the built-in sheep meniscus. Sheep menisci replaced with anatomically actual scaffolds encapsulating abandoned μS showed baggy coarse tissue throughout, afterwards zone-specific tissue phenotypes (Fig. 4C).

(A) Administration of coarse and cartilage cast in the built-in sheep meniscus. (B and C) Afterwards 12 weeks in vivo, zone-specific administration in the spatiotemporally arise rhCTGF and rhTGFβ3 accumulation (B) compared with the ascendancy accumulation (scaffolds with abandoned μS) (C). Tissue sections were decrepit for H&E, alcian dejected (AB) (aggrecan), and picrosirius red (PR) (collagens I and III). (D) Outer, intermediate, and close area phenotypes of beef clearing the regenerated meniscus (H&E staining). (E) Low-magnification images of retrieved meniscus grafts with spatiotemporal commitment of rhCTGF and rhTGFβ3 in allegory to abandoned μS afterwards 12-week in vivo implantation. Images are adumbrative of n = 4 for abandoned μS and n = 7 for C→T scaffolds.

The regenerated meniscus tissue showed zone-specific corpuscle phenotypes agnate to the built-in meniscus (Fig. 4D). The alien area of the regenerated meniscus was busy by fibroblast-like, spindle-shaped cells; chondrocyte-like beef in the close zone; and alloyed fibroblast- and chondrocyte-like beef in the boilerplate zone. Low-magnification images accepted the attendance of multiphase fibrocartilage throughout the scaffolds (Fig. 4E).

All sheep adeptness operation resumed weight address and locomotion as aboriginal as 3 weeks afterwards surgery. A sheep that accustomed a CTGF→TGFβ3 μS–laden arch in the centermost meniscus of the larboard knee approved absolutely adequate locomotion and weight address at 6 weeks post-op (movie S1), admitting a sheep that accustomed an anatomically actual arch but with abandoned μS is apparent in cine S2. Both sheep were able to resume locomotion and weight bearing. However, CTGF→TGFβ3 commitment not abandoned yielded zone-specific fibrocartilage tissue phenotype (Fig. 4B) but additionally adequate automated backdrop of the regenerated fibrocartilage tissue.

Instantaneous modulus (Ei), alleviation modulus (Er), and bendability (μ) were abstinent by applicable to a Kelvin solid viscoelastic archetypal (17, 18). Ei, Er, and μ of the built-in meniscus showed inhomogeneity forth the adorable zone, which includes the outer, intermediate, and close zones of meniscus (Fig. 5), acceptable attributable to multiphase administration of collagen and GAGs in the meniscus (17, 18). The CTGF→TGFβ3 commitment accumulation yielded Ei, Er, and μ agnate to the built-in meniscus [P > 0.05, one-way ANOVA; post-hoc atomic cogent aberration (LSD)] (Fig. 5A). Contrastingly, the viscoelastic backdrop of the abandoned μS accumulation were inferior to those of the CTGF→TGFβ3 μS accumulation or the built-in menisci (Fig. 5A).

(A) Direct modulus (Ei), alleviation modulus (Er), and accessory of bendability (μ) were abstinent from stress-relaxation tests. Ei, Er, and μ of the built-in meniscus in outer, intermediate, and close zones were abstinent and compared with those of regenerated menisci. (B) Young’s modulus and ultimate backbone of the regenerated menisci by rhCTGF and rhTGFβ3 delivered from the arch compared with the abandoned μS. Abstracts in (A) and (B) are agency ± SD (n = 4 animals per group). P ethics for abandoned μS are compared with C→T (one-way ANOVA with post-hoc LSD).

Young’s modulus and ultimate backbone from compactness testing at 1%/s (17) were decidedly college in the CTGF→TGFβ3 arch accumulation and the built-in menisci than in the abandoned μS accumulation (Fig. 5B). The ultimate ache showed no cogent differences amid the regenerated and built-in menisci (Fig. 5B).

These allegation appearance that meniscus about-face with inhomogeneous tissue backdrop and anatomic accretion is accessible with a protein-releasing, acellular biomaterial scaffold. In adverse to corpuscle transplantation, the present activity of autogenous about-face aloft spatiotemporal absolution of two recombinant beastly proteins offers a ready-to-implant affix that may serve as a ameliorative prototype. About-face by corpuscle homing, now actuality sporadically approved in several tissue/organ systems (12, 13, 19), provides an another to corpuscle transplantation. Biologically, about-face by corpuscle transplantation or corpuscle homing is not mutually exclusive. Crude beef bury cytokines that advance the survival, corpuscle recruitment, or adverse of host autogenous beef (20). However, about-face by corpuscle homing circumvents the charge for ex vivo corpuscle culture. Autogenous beef that accept migrated to a healing anguish are acceptable those with the adeptness to survive in situ. Contrastingly, en accumulation crude beef are apparent to an ambiance that may not be abreast for corpuscle animation (20, 21). Further, transplantation of parenchymal cells, which is archetypal of all corpuscle commitment approaches, charge await on the application of autogenous stromal beef for the address of a complete tissue. Recently, advance agency bounden was apparent to be a primary apparatus of activity of fibrin in diabetic anguish adjustment afterwards corpuscle transplantation (22).

How recruited autogenous cells, including stem/progenitor cells, or crude parenchymal beef arrange stromal as able-bodied as vascular, neural, lymphatic, and allowed apparatus in a adequate tissue warrants added investigation. Here, a fibrochondrocyte phenotype was acquired from MSCs back CTGF and TGFβ3 were sequentially activated in vitro or spatiotemporally arise in vivo. FAK and p38 pathways accept been active in collagen and aggrecan synthesis, and appropriately were advised here. Our antecedent abstracts advance that C→T elevates collagen I mRNA announcement primarily via FAK signaling, admitting collagen II and aggrecan mRNA announcement was added primarily via p38 signaling. Such basic signaling studies could crop clues for added targets in acceptable meniscus regeneration.

Despite absolute and reproducible in vitro CTGF and TGFβ3 absolution kinetics that led to fibrochondrogenic phenotype, in vivo accomplishments of arise proteins and peptides are best advised by the affection of the tissue they are advised to change (23, 24), in this case, a constant crop of fibrocartilage in vivo. Meniscus about-face demands not abandoned apology of biological structures at corpuscle and cast levels but additionally the accommodation to apply automated functions, akin to the about-face of the heart, lungs, kidney, bladder, and claret argosy (7–9). Inhomogeneity is adapted of abounding biological tissues and all organs. No antecedent letters to our adeptness accept apparent anatomic about-face or apology of amalgamate meniscal tissue phenotypes (16, 25–29), although some studies accept apparent fractional healing with somewhat constant tissue or affliction abatement in ample beastly models or patients (4, 5).

The present abstraction letters about-face of an inhomogeneous, anatomic knee meniscus in a ample beastly archetypal with 3-month follow-up. Longer-term appraisal will be bare to appraise whether zone-specific tissue phenotypes are abiding with circadian function. Our antecedent assignment showed reconstitution of a single, compatible tissue—albeit coarse tissue—by in vivo commitment of CTGF abandoned (12) or articular cartilage aloft in vivo commitment of TGFβ3 abandoned (13). Corpuscle sources for the empiric meniscus about-face may acquire from synovium stem/progenitor beef in the anguish (30) and additionally from the actual ~20% native, vascularized meniscus tissue (2), suggesting that conceivably vascular-derived stem/progenitor beef are recruited by adapted molecules. Antecedent assignment has apparent the authority of bifold advance factors in the accumulation of a atypical neovascular arrangement (31).

A check of a ample beastly archetypal is its impossibility to abut corpuscle sources by corpuscle tracing/tracking approaches that are accessible in transgenic mice (32). The sheep archetypal additionally does not admittance ample immunophenotyping of regenerated tissues attributable to a absence of antibodies and probes. Although we were clumsy to clue corpuscle application in the sheep, host corpuscle application was approved in a 50% rat meniscus resection birthmark back LacZ beef in BMP7-treated Achilles bond allograft were crude (33, 34), which supports our antecedent of meniscus about-face by autogenous cells.

For translation, we anticipate a 3D-printed beastly meniscus-shaped actual arch area both recombinant beastly proteins, rhCTGF and rhTGFβ3, are tethered in the scaffold’s microchannels and packaged in a GMP/GLP (good class practice) facility. The packaged articles are alien to hospitals and clinics area meniscus backup surgeries are performed. The surgeons alter afflicted menisci appliance the absolute arthroscopy action with an off-the-shelf meniscus arch rather than the accepted allograft from body meniscus. Reproducible ancestry of fibrochondrocytes from postnatal MSCs by CTGF and TGFβ3 may accept ample implications in the about-face of added fibrocartilage tissues, including the intervertebral disc of the spine, the tendon-bone junction, and the temporomandibular joint, anniversary of which, back diseased, is a astringent bloom affliction burden.

The all-embracing cold and architecture of the abstraction was to actuate whether autogenous cells, including stem/progenitor cells, can be differentiated in situ to change circuitous tissues, accurately the knee meniscus. The primary ambit of abstraction architecture included (i) extracellular cast amalgam and announcement of aggrecan, GAGs, and collagen types I and II, which collectively are macromolecules in the built-in meniscus; (ii) clonogenicity of cartilage basal and synovium MSCs; (iii) adverse accommodation of cartilage basal and synovium MSCs at citizenry and clonal levels; (iv) automated backdrop of arch actual including daybed tests; and (v) automated backdrop of regenerated tissues.

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The adeptness of the acellular, advance factor–releasing actual to change fibrocartilage was advised in vivo in an ovine model. A ability appraisal was performed afore our preclinical, ample beastly study, as declared below. The aboriginal sample admeasurement was bristles for the ascendancy accumulation and eight for the advised group. A absolute of 13 sheep were operated on, with 11 extensive end credibility (n = 4 for control; n = 7 for treated). Two sheep were absent because of anesthesia and surgical complications. The appraisal was randomized and addled from aggregation associates who performed the surgeries and postsurgical care. End credibility were predefined as the attendance or absence of statistically cogent differences of the above-listed parameters. No outliers were eliminated.

Human MSCs were abandoned from commercially available, beginning accomplished cartilage basal samples or surgically removed synovium of bearding developed donors (age range, 20 to 25 years) per our antecedent methods (12, 13). Access 2/3 MSCs (100,000 beef per well) were argent in six-well adeptness plates, or 3D pellets were formed by centrifuging 2 × 106 MSCs in 15-ml conical tubes. Monolayered beef or corpuscle pellets were advised with (i) CTGF (100 ng/ml) (BioVendor) for 2 weeks followed by TGFβ3 (10 ng/ml) (R&D Systems) (C→T); (ii) TGFβ3 (10 ng/ml) for 2 weeks followed by CTGF (100 ng/ml) for 2 weeks (T→C); (iii) admixture of CTGF (100 ng/ml) and TGFβ3 (10 ng/ml) for 4 weeks (C T); or (iv) CTGF for 4 weeks (C4), TGFβ3 for 4 weeks (T4), and advance boilerplate as a ascendancy (Fig. 1). Fibrochondrogenic consecration supplement [ascorbic acids (50 μg/ml)] and chondrogenic consecration supplements [1% 1× ITS 1 solution, sodium pyruvate (100 μg/ml), l-ascorbic acerbic 2-phosphate (50 μg/ml), l-proline (40 μg/ml), 0.1 μM dexamethasone] were included in the CTGF and TGFβ3 treatments, appropriately (12).

After 4 weeks of treatment, harvested samples (monolayers and pellet sections) were decrepit with alcian dejected and picrosirius red per our antecedent methods (12). GAGs and types I and II collagen were quantitatively assayed (Biocolor) and normalized to DNA agreeable (12). Immunofluorescence was performed to analyze beef cogent proCOL-I (ab64409) and/or proCOL-IIα (ab17771), both from Abcam.

MSC clones were accustomed by attached concoction (12). Briefly, access 0 MSCs were abeyant at a absorption of one corpuscle per 200 μl and seeded in 96-well plates with 200 μl of approved advance boilerplate per well. Afterwards 24 hours, wells with abandoned one corpuscle were apparent (fig. S5) and maintained in adeptness at 37°C and 5% CO2, with boilerplate change every 3 to 4 days. Afterwards 3 to 4 weeks, single-cell–derived clonal progenies were advised with 0.25% trypsin-EDTA and seeded into 24-well plates. Clonal progenies were transferred to six-well plates aloft ~80% assemblage and maintained with boilerplate change every 3 to 4 days. Accustomed clones were induced for multilineage adverse into osteogenic, chondrogenic, and adipogenic lineages per our antecedent methods (12), and fibrochondrogenic birth as declared aloft (fig. S5).

Anatomically actual meniscus scaffolds were bogus appliance 3D-Bioplotter (EnvisionTEC) per our antecedent assignment (13). Briefly, the anatomic ambit of the centermost meniscus of beastly or skeletally complete sheep was captured by multislice laser scanning and reconstructed by computer-aided design. PCL (molecular weight, ~65,000; Sigma) was aqueous at 120°C and dispensed, afterward the band aisle dictated by 3D architecture of centralized microstructures. For the beastly meniscus scaffold, interlaid strands and abutting microchannels had a bore of 100 μm, and circumferentially accumbent fibers were added to epitomize absolute collagen acclimatization in the built-in meniscus (Fig. 2A). For the sheep meniscus arch (Fig. 3A), 300-μm microstrands and 100-μm microchannels were bent to be optimal (Fig. 3A) to almost automated backdrop of the built-in meniscus.

Recombinant beastly CTGF and TGFβ3 were encapsulated in 50:50 and 75:25 (PLA/PGA) μS, respectively, appliance bifold chrism (12). Two altered PLGA ratios were activated to crop controlled CTGF and TGFβ3 release. Briefly, a absolute of 250 mg of PLGA was attenuated into 1 ml of dichloromethane. CTGF (10 μg) or TGFβ3 (2.5 μg) was adulterated to 50 μl afterward the manufacturer’s protocols and added to the PLGA solution, basic a admixture (primary emulsion) that was blurred for 1 min (water-in-oil). The primary chrism was again added to 2 ml of 1% polyvinyl booze (PVA; atomic weight, 30,000 to 70,000), followed by 1-min bond [(water-in-oil)-in-water]. Aloft abacus 100 ml of PVA, the admixture was afflicted for 1 min at allowance temperature. A absolute of 100 ml of 2% isopropanol was added to the final chrism and continuously afflicted for 2 hours to abolish the solvent. Ascendancy microspheres (empty, afterwards CTGF or TGFβ3, absorption a admixture of both 75:25 and 50:50 PLA/PGA) were bogus with 50 μl of distilled water. Absolution kinetics of CTGF and TGFβ3 from PLGA μS were abstinent by incubating microspheres (10 mg/ml) encapsulating CTGF [in phosphate-buffered acrid (PBS)] or TGFβ3 (0.1% bovine serum albumin) at 37°C with balmy agitation for up to 42 canicule (12). Aloft centrifugation at 2500 rpm for 5 min, afloat of the PLGA μS evolution band-aid was collected. Arise CTGF and TGFβ3 absorption was abstinent appliance enzyme-linked immunosorbent appraisal (ELISA) kits afterward the manufacturer’s protocols.

PLGA μS (10 mg/ml) absolute either CTGF or TGFβ3 were again congenital on scaffold’s microstrands by booze appraisal for 1 hour (35). CTGF-encapsulated μS were activated in the microchannels in the scaffold’s outer/middle zones, admitting TGFβ3-encapsulated μS were activated in inner/middle zones. Multiphase administration of CTGF and TGFβ3 μS was accurate by accumulation μS-encapsulating fluorescein-conjugated dextran (40 kD) and Alexa Fluor 546 dextran (10 kD), apery CTGF and TGFβ3, respectively. Dextran administration was visualized appliance Maestro (PerkinElmer). Aloft fibrin gel beverage into microchannels, scaffolds with dextran-encapsulated μS were incubated at 37°C for 8 canicule and beheld with Maestro.

The anatomically actual meniscus scaffolds with spatiotemporal commitment of CTGF-TGFβ3 were placed on top of monolayer able beastly synovium MSCs (100,000 per well) in a six-well plate. Afterwards 6 weeks of adeptness with a admixture of fibrogenic and chondrogenic supplemented medium, the scaffolds were harvested and analyzed for corpuscle application and zone-specific tissue formation. Corpuscle application was accepted by DAPI staining in the scaffolds, and tissue accumulation was evaluated by assay and immunofluorescence.

A absolute of 11 sheep were about allocated into advised (n = 7) and ascendancy (n = 4) groups. A centermost parapatellar access was fabricated through the bark and subcutaneous tissue. A crabbed parapatellar arthrotomy was performed, and the patella was luxated medially. Afterwards coil of the aggregate and acknowledgment of the centermost meniscus, condylectomy was performed appliance an aquiver saw from the axial ancillary of the centermost femoral condyle against the distal centermost femoral metaphysis. All the associated aggregate ligaments were preserved. Then, a fractional meniscectomy was performed by resecting all but ~20% of the meniscus’ alien area and anon grafted with anatomically actual meniscus scaffold, followed by suturing (2-0 Ethibond Excel, Braided). Condylectomy was repaired with 2- or 4.5-mm-diameter cortical cartilage screws. The aggregate abridged and subcutaneous tissue were bankrupt alone in connected absorbable suture. The bark was staple-closed and covered with a antiseptic bandage.

The operated sheep were bedfast to baby pens (4 × 4 ft2) to abbreviate abrupt or continued movements. Sheep were in absolute visual, vocal, and nasal acquaintance with anniversary other, but were in abstracted pens to abbreviate careless trauma. Affliction medication (narcotics and nonsteroidal anti-inflammatory drugs) was administered intramuscularly for a minimum of 2 weeks post-op. Aloft complete condylectomy healing on radiographs, sheep were group-housed in a baby paddock (six sheep per 12 × 12 ft2 paddock) for 1 anniversary for acclimation and again transferred to a ample paddock (96 × 96 ft2) until euthanasia. All in vivo samples were harvested at 12 weeks post-op. Harvested tissue was anchored in 4% formaldehyde, anchored in paraffin, and cut in 5-μm sections.

Four of seven advised samples were called for automated testing. The added three samples were acclimated for tissue-based analyses.

Dynamic compression. Afore in vivo implantation, activating compression was performed to bout scaffold’s automated backdrop with those of the built-in meniscus (13). Briefly, annular plugs were punched from the axial regions of scaffolds and built-in meniscus (5 × 5 mm2) (n = 5 per group). Compressive testing was performed beneath 10% circadian ache at 2 Hz, and activating compressive backdrop were characterized as circuitous compressive modulus E*, accumulator modulus E′, and accident modulus E″. All automated tests were performed appliance ElectroForce BioDynamic testing arrangement (Bose).

Pull-out strength. About 75% of the sheep centermost meniscus was longitudinally cut with a scalpel. A absolute of four captivation sutures (2-0 Ethibond Excel, Braided) were placed amid the close and alien portions of the meniscus and the scaffold. The best daybed force was abstinent afterwards ascent the samples appliance stainless animate active on ElectroForce compactness grips and pulled at 0.5 mm/s (36).

Friction coefficient. Calm abrasion coefficients, μeq, on angular acceleration were abstinent to appraisal the scaffold’s frictional acreage (19). Annular samples (10-mm bore × 3-mm thickness), able from the boilerplate arena of menisci and scaffolds, were anchored to an closed basal platen appliance cyanoacrylate cement and absorbed in a PBS bath. Afterwards 5% compressive tare ache for 1800 s, a alternation of consecutive angular velocities (0.01, 0.1, and 1 rad/s) were activated for 120 s each. Abrasion accessory (μeq) was affected as abrasion force (F) disconnected by accustomed force (N). With an acceptance of aught alien frictional microburst at the centermost and beeline administration forth with the adorable direction, the boilerplate frictional force, F, was acquired as F = 4T/3r0, area T is the frictional torque and r0 was the specimen’s radius.

Stress relaxation. Disc-shaped samples (5-mm bore × 2-mm thickness) were able from the inner, middle, and alien regions of the built-in and regenerated menisci. Appliance great compression, samples were preconditioned with 15 cycles of 0 to 5% compressive strain, followed by accent alleviation with 20% compressive strain. A Kelvin accepted solid viscoelastic archetypal was activated to account direct modulus (Ei), modulus of alleviation (Er), and bendability (μ) (19, 24).

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Tensile test. Circumferential strips of the boilerplate arena of the built-in and regenerated menisci were cut into dog cartilage shapes 25 mm in breadth and 1 mm in boilerplate array per antecedent methods (19, 24). Afterwards preconditioning of 15 cycles of 0 to 2% strain, compactness tests were performed at 1% ache per second. The beeline allocation of anniversary stress-strain ambit was acclimated to actuate the Young’s modulus (Ey). The ultimate backbone and ache was bent from anniversary curve.

Sample sizes for all quantitative abstracts were bent by ability appraisal with one-way ANOVA appliance an α akin of 0.05, ability of 0.8, and aftereffect admeasurement of 1.50 called to appraise cast synthesis, gene expressions, and automated backdrop in the regenerated meniscus tissues and controls aloft assay of accustomed abstracts distribution. In case of skewed abstracts distribution, a nonparametric test, Kruskal-Wallis one-way ANOVA, will be performed appliance an α akin of 0.05, ability of 0.8, and aftereffect admeasurement of 1.50. Appraisal appliance PASS (NCSS) adumbrated all-important sample sizes of four to seven to accomplish a ability amount of 0.8 for the in vitro and in vivo ambit acclimated in the present study. Expected SD and agency were entered on the base of our antecedent assignment for meniscus tissue about-face and MSC differentiations (13). Briefly, f appraisal was performed with estimated agency and SDs of ascendancy and appraisal groups appliance the afterward equation:where pi = ni/N; ni = cardinal of ascertainment in accumulation i; N = absolute cardinal of observations; μi = estimated beggarly of accumulation i; μ = admirable mean; σ2 = absurdity about-face aural the group. For all quantitative data, one-way ANOVA with post-hoc LSD or Tukey’s B tests were used.


Fig. S1. Fibrochondrogenic adverse of beastly cartilage basal MSCs in 3D fibrin.

Fig. S2. Fibrochondrogenic cast assembly by fibrochondrocytes acquired from beastly MSCs in monolayer culture.

Fig. S3. Fibrochondrogenic cast assembly by fibrochondrocytes acquired from beastly MSCs in 3D fibrin gel.

Fig. S4. FAK and p38 signaling plays a role in collagen and aggrecan announcement afterwards fibrochondrocyte adverse in vitro.

Fig. S5. Single-cell progenies of beastly cartilage basal MSCs differentiate into fibrochondrocytes.

Fig. S6. Automated testing of arch to meniscus and partially resected meniscus.

Table S1. Fibrochondrocyte adverse from beastly cartilage basal MSCs in monolayer and 3D cultures.

Table S2. Statistical significances for comparisons in amid groups in Fig. 1, fig. S2, and fig S3.

Movie S1. Sheep movement afterwards centermost meniscus backup with 3D-printed arch with spatial CTGF and TGFβ3 delivery.

Movie S2. Sheep movement afterwards centermost meniscus backup with 3D-printed arch with abandoned μS.

K. A. Athanasiou, J. Sanchez-Adams, Engineering the Knee Meniscus (Morgan and Claypool Publishers, San Rafael, CA, 2009).

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