Anaphylatoxins and their corresponding receptors as potential drivers in cartilage calcification during osteoarthritis progression.

OBJECTIVE: The complement cascade as major fluid phase innate immune system is activated during progression of osteoarthritis (OA). Generated anaphylatoxins and the corresponding receptors C3aR and C5aR1 are associated with the calcification of blood vessels and involved in osteogenic differentiation. This study aims on elucidating whether complement activation products contribute to cartilage calcification of OA cartilage. METHOD: Human articular chondrocytes were osteogenically differentiated in vitro in the presence or absence of C3a, C5a, and bone morphogenetic protein (BMP) 2. Furthermore, macroscopically intact (OARSI grade ≤ 1) and highly degenerated human cartilage (OARSI grade ≥ 3) was used for C3aR and C5aR1 histochemistry. Calcification of the cartilage was assessed by Alizarin Red S and von Kossa staining. RESULTS: C3a and C5a amplified matrix mineralization during in vitro osteogenesis, while inhibition of the corresponding receptors impaired calcium deposition. Moreover, C3aR and C5aR1 expression was upregulated during osteogenic differentiation and also in degenerated cartilage. Additionally, anaphylatoxin receptor expression was positively associated with calcification of native cartilage tissue and calcium deposition during osteogenic differentiation. Finally, the pro-hypertrophic growth factor BMP2 induced the expression of C5aR1. CONCLUSIONS: Our findings indicate that anaphylatoxins and their receptors play a decisive role in cartilage calcification processes during OA progression.

In vivo ligamentogenesis in embroidered poly(lactic-co-ε-caprolactone) / polylactic acid scaffolds functionalized by fluorination and hexamethylene diisocyanate cross-linked collagen foams.

Although autografts represent the gold standard for anterior cruciate ligament (ACL) reconstruction, tissue-engineered ACLs provide a prospect to minimize donor site morbidity and limited graft availability. This study characterizes the ligamentogenesis in embroidered poly(L-lactide-co-ε-caprolactone) (P(LA-CL)) / polylactic acid (PLA) constructs using a dynamic nude mice xenograft model. (P(LA-CL))/PLA scaffolds remained either untreated (co) or were functionalized by gas fluorination (F), collagen foam cross-linked with hexamethylene diisocyanate (HMDI) (coll), or F combined with the foam (F + coll). Cell-free constructs or those seeded for 1 week with lapine ACL ligamentocytes were implanted into nude mice for 12 weeks. Following explantation, cell vitality and content, histo(patho)logy of scaffolds (including organs: liver, kidney, spleen), sulphated glycosaminoglycan (sGAG) contents and biomechanical properties were assessed.Scaffolds did not affect mice weight development and organs, indicating no organ toxicity. Moreover, scaffolds maintained their size and shape and reflected a high cell viability prior to and following implantation. Coll or F + coll scaffolds seeded with cells yielded superior macroscopic properties compared to the controls. Mild signs of inflammation (foreign-body giant cells and hyperemia) were limited to scaffolds without collagen. Microscopical score values and sGAG content did not differ significantly. Although remaining stable after explantation, elastic modulus, maximum force, tensile strength and strain at Fmax were significantly lower in explanted scaffolds compared to those before implantation, with no significant differences between scaffold subtypes, except for a higher maximum force in F + coll compared with F samples (in vivo). Scaffold functionalization with fluorinated collagen foam provides a promising approach for ACL tissue engineering. a Lapine anterior cruciate ligament (LACL): red arrow, posterior cruciate ligament: yellow arrow. Medial anterior meniscotibial ligament: black arrow. b Explant culture to isolate LACL fibroblasts. c Scaffold variants: co: controls; F: functionalization by gas-phase fluorination; coll: collagen foam cross-linked with hexamethylene diisocyanate (HMDI). c1-2 Embroidery pattern of the scaffolds. d Scaffolds were seeded with LACL fibroblasts using a dynamical culturing approach as depicted. e Scaffolds were implanted subnuchally into nude mice, fixed at the nuchal ligament and sacrospinal muscle tendons. f Two weeks after implantation. g Summary of analyses performed. Scale bars 1 cm (b, d), 0.5 cm (c). (sketches drawn by G.S.-T. using Krita 4.1.7 [Krita foundation, The Netherlands]).

Anatomical Tissue Engineering of the Anterior Cruciate Ligament Entheses.

The firm integration of anterior cruciate ligament (ACL) grafts into bones remains the most demanding challenge in ACL reconstruction, since graft loosening means graft failure. For a functional-tissue-engineered ACL substitute to be realized in future, robust bone attachment sites (entheses) have to be re-established. The latter comprise four tissue compartments (ligament, non-calcified and calcified fibrocartilage, separated by the tidemark, bone) forming a histological and biomechanical gradient at the attachment interface between the ACL and bone. The ACL enthesis is surrounded by the synovium and exposed to the intra-articular micromilieu. This review will picture and explain the peculiarities of these synovioentheseal complexes at the femoral and tibial attachment sites based on published data. Using this, emerging tissue engineering (TE) strategies addressing them will be discussed. Several material composites (e.g., polycaprolactone and silk fibroin) and manufacturing techniques (e.g., three-dimensional-/bio-printing, electrospinning, braiding and embroidering) have been applied to create zonal cell carriers (bi- or triphasic scaffolds) mimicking the ACL enthesis tissue gradients with appropriate topological parameters for zones. Functionalized or bioactive materials (e.g., collagen, tricalcium phosphate, hydroxyapatite and bioactive glass (BG)) or growth factors (e.g., bone morphogenetic proteins [BMP]-2) have been integrated to achieve the zone-dependent differentiation of precursor cells. However, the ACL entheses comprise individual (loading history) asymmetric and polar histoarchitectures. They result from the unique biomechanical microenvironment of overlapping tensile, compressive and shear forces involved in enthesis formation, maturation and maintenance. This review should provide a road map of key parameters to be considered in future in ACL interface TE approaches.

Co-Culture of Mesenchymal Stem Cells and Ligamentocytes on Triphasic Embroidered Poly(L-lactide-co-ε-caprolactone) and Polylactic Acid Scaffolds for Anterior Cruciate Ligament Enthesis Tissue Engineering.

Successful anterior cruciate ligament (ACL) reconstructions strive for a firm bone-ligament integration. With the aim to establish an enthesis-like construct, embroidered functionalized scaffolds were colonized with spheroids of osteogenically differentiated human mesenchymal stem cells (hMSCs) and lapine (l) ACL fibroblasts in this study. These triphasic poly(L-lactide-co-ε-caprolactone) and polylactic acid (P(LA-CL)/PLA) scaffolds with a bone-, a fibrocartilage transition- and a ligament zone were colonized with spheroids directly after assembly (DC) or with 14-day pre-cultured lACL fibroblast and 14-day osteogenically differentiated hMSCs spheroids (=longer pre-cultivation, LC). The scaffolds with co-cultures were cultured for 14 days. Cell vitality, DNA and sulfated glycosaminoglycan (sGAG) contents were determined. The relative gene expressions of collagen types I and X, Mohawk, Tenascin C and runt-related protein (RUNX) 2 were analyzed. Compared to the lACL spheroids, those with hMSCs adhered more rapidly. Vimentin and collagen type I immunoreactivity were mainly detected in the hMSCs colonizing the bone zone. The DNA content was higher in the DC than in LC whereas the sGAG content was higher in LC. The gene expression of ECM components and transcription factors depended on cell type and pre-culturing condition. Zonal colonization of triphasic scaffolds using spheroids is possible, offering a novel approach for enthesis tissue engineering.

Healing of Ligaments and Tendons: Tissue Engineering and Models.

The aim of this Special Issue is to summarize the latest developments in tendon/ligament research and tissue engineering (TE), providing helpful approaches for future tendon/ligament reconstruction (Figure 1) [...].

Cyclically stretched ACL fibroblasts emigrating from spheroids adapt their cytoskeleton and ligament-related expression profile.

Mechanical stress of ligaments varies; hence, ligament fibroblasts must adapt their expression profile to novel mechanomilieus to ensure tissue resilience. Activation of the mechanoreceptors leads to a specific signal transduction, the so-called mechanotransduction. However, with regard to their natural three-dimensional (3D) microenvironment cell reaction to mechanical stimuli during emigrating from a 3D spheroid culture is still unclear. This study aims to provide a deeper understanding of the reaction profile of anterior cruciate ligament (ACL)-derived fibroblasts exposed to cyclic uniaxial strain in two-dimensional (2D) monolayer culture and during emigration from 3D spheroids with respect to cell survival, cell and cytoskeletal orientation, distribution, and expression profile. Monolayers and spheroids were cultured in crosslinked polydimethyl siloxane (PDMS) elastomeric chambers and uniaxially stretched (14% at 0.3 Hz) for 48 h. Cell vitality, their distribution, nuclear shape, stress fiber orientation, focal adhesions, proliferation, expression of ECM components such as sulfated glycosaminoglycans, collagen type I, decorin, tenascin C and cell-cell communication-related gap junctional connexin (CXN) 43, tendon-related markers Mohawk and tenomodulin (myodulin) were analyzed. In contrast to unstretched cells, stretched fibroblasts showed elongation of stress fibers, cell and cytoskeletal alignment perpendicular to strain direction, less rounded cell nuclei, increased numbers of focal adhesions, proliferation, amplified CXN43, and main ECM component expression in both cultures. The applied cyclic stretch protocol evoked an anabolic response and enhanced tendon-related marker expression in ACL-derived fibroblasts emigrating from 3D spheroids and seems also promising to support in future tissue formation in ACL scaffolds seeded in vitro with spheroids.

Complement regulation in tenocytes under the influence of leukocytes in an indirect co-culture model.

INTRODUCTION: The present in vitro study was undertaken to learn about the effects of leukocytes on tenocytes in respect to complement regulation simulating an inflammatory scenario of the traumatized tissue. METHODS: Human hamstring tendon-derived tenocyte monolayers were co-cultured indirectly with human leukocytes (either Peripheral Blood Mononuclear Cells [PBMCs] or neutrophils) using a transwell system with/without (+ /wo) 10 ng/ml tumor necrosis factor α (TNFα) for 4 and 24 h. Tenocyte and leukocyte cell survival was assessed by live-dead assay. Tenocyte gene expression of TNFα, the anaphylatoxin receptor C5aR and the cytoprotective complement regulatory proteins (CRP) CD46, CD55 and CD59 was monitored using qPCR. TNFα was detected in the culture supernatants using ELISA. RESULTS: C5aR gene expression was significantly induced by TNFα after 4 h, but impaired in the presence of leukocytes + TNFα after 24 h. At 4 h, PBMCs activated by TNFα induced the CRP CD46 gene expression. However, CD55 was significantly suppressed after 24 h by neutrophils + /woTNFα. Leukocytes activated by TNFα decreased also significantly the gene expression of the more downstream acting CRP CD59 after 4 h. TNFα gene expression and ELISA analysis revealed an amplified TNFα expression/release in tenocyte co-cultures with PBMC + /woTNFα, probably contributing to complement regulation. CONCLUSION: TNFα might represent a crucial soluble mediator exerting diverse time-dependent effects on tenocyte complement regulation.

Minispheroids as a Tool for Ligament Tissue Engineering: Do the Self-Assembly Techniques and Spheroid Dimensions Influence the Cruciate Ligamentocyte Phenotype?

Spheroid culture might stabilize the ligamentocyte phenotype. Therefore, the phenotype of lapine cruciate ligamentocyte (L-CLs) minispheroids prepared either by hanging drop (HD) method or by using a novel spheroid plate (SP) and the option of methyl cellulose (MC) for tuning spheroid formation was tested. A total of 250 and 1000 L-CLs per spheroid were seeded as HDs or on an SP before performing cell viability assay, morphometry, gene expression (qRT-PCR) and protein immunolocalization after 7 (HD/SP) and 14 (SP) days. Stable and viable spheroids of both sizes could be produced with both methods, but more rapidly with SP. MC accelerated the formation of round spheroids (HD). Their circular areas decreased significantly during culturing. After 7 days, the diameters of HD-derived spheroids were significantly larger compared to those harvested from the SP, with a tendency of lower circularity suggesting an ellipsoid shape. Gene expression of decorin increased significantly after 7 days (HD, similar trend in SP), tenascin C tended to increase after 7 (HD/SP) and 14 days (SP), whereas collagen type 1 decreased (HD/SP) compared to the monolayer control. The cruciate ligament extracellular matrix components could be localized in all mini-spheroids, confirming their conserved expression profile and their suitability for ligament tissue engineering.

Complement Regulation in Human Tenocytes under the Influence of Anaphylatoxin C5a.

A central part of the complement system, the anaphylatoxin C5a was investigated in this study to learn its effects on tenocytes in respect to understanding the potential expression of other crucial complement factors and pro-inflammatory mediators involved in tendinopathy. Human hamstring tendon-derived tenocytes were treated with recombinant C5a protein in concentrations of 25 ng/mL and 100 ng/mL for 0.5 h (early phase), 4 h (intermediate phase), and 24 h (late phase). Tenocytes survival was assessed after 24 h stimulation by live-dead assay. The gene expression of complement-related factors C5aR, the complement regulatory proteins (CRPs) CD46, CD55, CD59, and of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 was monitored using qPCR. Tenocytes were immunolabeled for C5aR and CD55 proteins. TNFα production was monitored by ELISA. Tenocyte survival was not impaired through C5a stimulation. Interestingly, the gene expression of C5aR and that of the CRPs CD46 and CD59 was significantly reduced in the intermediate and late phase, and that of TNFα only in an early phase, compared to the control group. ELISA analysis indicated a concomitant not significant trend of impaired TNFα protein synthesis at 4 h. However, there was also an early significant induction of CD55 and CD59 mediated by 25 ng/mL anaphylatoxin C5a. Hence, exposure of tenocytes to C5a obviously evokes a time and concentration-dependent response in their expression of complement and pro-inflammatory factors. C5a, released in damaged tendons, might directly contribute to tenocyte activation and thereby be involved in tendon healing and tendinopathy.

Maintenance of Ligament Homeostasis of Spheroid-Colonized Embroidered and Functionalized Scaffolds after 3D Stretch.

Anterior cruciate ligament (ACL) ruptures are usually treated with autograft implantation to prevent knee instability. Tissue engineered ACL reconstruction is becoming promising to circumvent autograft limitations. The aim was to evaluate the influence of cyclic stretch on lapine (L) ACL fibroblasts on embroidered scaffolds with respect to adhesion, DNA and sulphated glycosaminoglycan (sGAG) contents, gene expression of ligament-associated extracellular matrix genes, such as type I collagen, decorin, tenascin C, tenomodulin, gap junctional connexin 43 and the transcription factor Mohawk. Control scaffolds and those functionalized by gas phase fluorination and cross-linked collagen foam were either pre-cultured with a suspension or with spheroids of LACL cells before being subjected to cyclic stretch (4%, 0.11 Hz, 3 days). Stretch increased significantly the scaffold area colonized with cells but impaired sGAGs and decorin gene expression (functionalized scaffolds seeded with cell suspension). Stretching increased tenascin C, connexin 43 and Mohawk but decreased decorin gene expression (control scaffolds seeded with cell suspension). Pre-cultivation of functionalized scaffolds with spheroids might be the more suitable method for maintaining ligamentogenesis in 3D scaffolds compared to using a cell suspension due to a significantly higher sGAG content in response to stretching and type I collagen gene expression in functionalized scaffolds.

Growth characteristics of human juvenile, adult and murine fibroblasts: a comparison of polymer wound dressings.

OBJECTIVE: Fibroblasts are important for the successful healing of deep wounds. However, the influence exerted by Cuticell, a natural polymer on fibroblasts and by the synthetic polymer, Suprathel, made of poly-L-lactic acid, is not sufficiently characterised. This study compared the survival and growth characteristics of human juvenile and adult dermal fibroblasts as well as murine fibroblast cell line L929, on a natural polymer with those of a synthetic polymer using different culture models. METHOD: Murine, juvenile and adult human fibroblasts were seeded on both the natural and synthetic polymers using statical slide culture or the medium air interface and dynamical rotatory culture. Cell adherence, viability, morphology and actin cytoskeleton architecture were monitored for 1-7 days. Biomaterial permeability was checked with a previously established diffusion chamber. RESULTS: The majority of the murine and adult human fibroblasts survived in slide and rotatory cultures on both wound dressings. The fibroblasts seeded on the synthetic polymer exhibited phenotypically a typical spread shape with multiple cell adhesion sites earlier than those on the natural polymer. The highest survival rates in all tested fibroblast species over the entire observation time were detected in rotatory culture (mean: >70%). Nevertheless, it led to cell-cluster formation on both materials. In the medium air interface culture, few adult fibroblasts adhered and survived until the seventh day of culture on both the natural and synthetic polymers, and no viable juvenile and L929 fibroblasts could be found by day seven. Apart from a significant higher survival rate of L929 in slide culture on the natural polymer compared with the synthetic polymer at the end of the culturing period (p<0.0001), and a higher cell survival of L929 on the natural polymer in medium air interface culture, only minor differences between both materials were evident. This suggested a comparable cytocompatibility of both materials. Permeability testing revealed slightly higher permeance of the natural polymer compared with the synthetic polymer. CONCLUSION: Cell survival rates depended on the culture system and the fibroblast source. Nevertheless, the juvenile skin fibroblasts were the most sensitive. This observation suggests that wound dressings used in treating children should be tested beforehand with juvenile fibroblasts to ensure the dressing does not compromise wound healing. Future experiments should also include the response of compromised fibroblasts, for example, from burn patients.

Uremic Toxins and Ciprofloxacin Affect Human Tenocytes In Vitro.

Tendinopathy is a rare but serious complication of quinolone therapy. Risk factors associated with quinolone-induced tendon disorders include chronic kidney disease accompanied by the accumulation of uremic toxins. Hence, the present study explored the effects of the representative uremic toxins phenylacetic acid (PAA) and quinolinic acid (QA), both alone and in combination with ciprofloxacin (CPX), on human tenocytes in vitro. Tenocytes incubated with uremic toxins +/- CPX were investigated for metabolic activity, vitality, expression of the dominant extracellular tendon matrix (ECM) protein type I collagen, cell-matrix receptor ß1-integrin, proinflammatory interleukin (IL)-1ß, and the ECM-degrading enzyme matrix metalloproteinase (MMP)-1. CPX, when administered at high concentrations (100 mM), suppressed tenocyte metabolism after 8 h exposure and at therapeutic concentrations after 72 h exposure. PAA reduced tenocyte metabolism only after 72 h exposure to very high doses and when combined with CPX. QA, when administered alone, led to scarcely any cytotoxic effect. Combinations of CPX with PAA or QA did not cause greater cytotoxicity than incubation with CPX alone. Gene expression of the pro-inflammatory cytokine IL-1ß was reduced by CPX but up-regulated by PAA and QA. Protein levels of type I collagen decreased in response to high CPX doses, whereas PAA and QA did not affect its synthesis significantly. MMP-1 mRNA levels were increased by CPX. This effect became more pronounced in the form of a synergism following exposure to a combination of CPX and PAA. CPX was more tenotoxic than the uremic toxins PAA and QA, which showed only distinct suppressive effects.

Different Frequency of Cyclic Tensile Strain Relates to Anabolic/Catabolic Conditions Consistent with Immunohistochemical Staining Intensity in Tenocytes.

Tenocytes are mechanosensitive cells intimately adapting their expression profile and hence, their phenotype to their respective mechanomilieu. The immunolocalization and expression intensity of tenogenic, anabolic and catabolic markers in tenocytes in response to in vitro mechanical loading have not been monitored by immunohistochemical staining (IHC). Thus, we investigated the association between IHC intensities, different stimulation frequencies, and tenogenic metabolism using a versatile mechanical stretcher. Primary tenocytes obtained from murine Achilles tendons were transferred to poly(dimethylsiloxane) (PDMS) elastomeric chamber. Chambers were cyclically stretched by 5% in uniaxial direction at a variation of tensile frequency (1 or 2 Hz) for 3 h. After stretching, cell physiology, IHC intensities of tendon-related markers, and protein level of the angiogenesis marker vascular endothelial growth factor (VEGF) were evaluated. Cell proliferation in tenocytes stimulated with 1 Hz stretch was significantly higher than with 2 Hz or without stretch, while 2 Hz stretch induced significantly reduced cell viability and proliferation with microscopically detectable apoptotic cell changes. The amount of scleraxis translocated into the nuclei and tenomodulin immunoreactivity of tenocytes treated with stretch were significantly higher than of non-stretched cells. The collagen type-1 expression level in tenocytes stretched at 1 Hz was significantly higher than in those cultivated with 2 Hz or without stretching, whereas the matrix metalloproteinase (MMP)-1 and MMP-13 immunoreactivities of cells stretched at 2 Hz were significantly higher than in those stimulated with 1 Hz or without stretching. The secreted VEGF-protein level of tenocytes stretched at 2 Hz was significantly higher than without stretching. Our IHC findings consistent with cell physiology suggest that appropriate stretching can reproduce in vitro short-term tenogenic anabolic/catabolic conditions and allow us to identify an anabolic stretching profile.

Enhanced Growth of Lapine Anterior Cruciate Ligament-Derived Fibroblasts on Scaffolds Embroidered from Poly(l-lactide-co-ε-caprolactone) and Polylactic Acid Threads Functionalized by Fluorination and Hexamethylene Diisocyanate Cross-Linked Collagen Foams.

Reconstruction of ruptured anterior cruciate ligaments (ACLs) is limited by the availability and donor site morbidity of autografts. Hence, a tissue engineered graft could present an alternative in the future. This study was undertaken to determine the performance of lapine (L) ACL-derived fibroblasts on embroidered poly(l-lactide-co-ε-caprolactone) (P(LA-CL)) and polylactic acid (PLA) scaffolds in regard to a tissue engineering approach for ACL reconstruction. Surface modifications of P(LA-CL)/PLA by gas-phase fluorination and cross-linking of a collagen foam using either ethylcarbodiimide (EDC) or hexamethylene diisocyanate (HMDI) were tested regarding their influence on cell adhesion, growth and gene expression. The experiments were performed using embroidered P(LA-CL)/PLA scaffolds that were seeded dynamically or statically with LACL-derived fibroblasts. Scaffold cytocompatibility, cell survival, numbers, metabolic activity, ultrastructure and sulfated glycosaminoglycan (sGAG) synthesis were evaluated. Quantitative real-time polymerase chain reaction (QPCR) revealed gene expression of collagen type I (COL1A1), decorin (DCN), tenascin C (TNC), Mohawk (MKX) and tenomodulin (TNMD). All tested scaffolds were highly cytocompatible. A significantly higher cellularity and larger scaffold surface areas colonized by cells were detected in HMDI cross-linked and fluorinated scaffolds compared to those cross-linked with EDC or without any functionalization. By contrast, sGAG synthesis was higher in controls. Despite the fact that the significance level was not reached, gene expressions of ligament extracellular matrix components and differentiation markers were generally higher in fluorinated scaffolds with cross-linked collagen foams. LACL-derived fibroblasts maintained their differentiated phenotype on fluorinated scaffolds supplemented with a HMDI cross-linked collagen foam, making them a promising tool for ACL tissue engineering.

SV40 Transfected Human Anterior Cruciate Ligament Derived Ligamentocytes-Suitable as a Human in Vitro Model for Ligament Reconstruction?

Cultured human primary cells have a limited lifespan undergoing dedifferentiation or senescence. Anterior cruciate ligaments (ACL) are hypocellular but tissue engineering (TE) requires high cell numbers. Simian virus (SV) 40 tumor (T) antigen expression could extend the lifespan of cells. This study aimed to identify cellular changes induced by SV40 expression in human ACL ligamentocytes by comparing them with non-transfected ligamentocytes and tissue of the same donor to assess their applicability as TE model. Human ACL ligamentocytes (40-year-old female donor after ACL rupture) were either transfected with a SV40 plasmid or remained non-transfected (control) before monitored for SV40 expression, survival, and DNA content. Protein expression of cultured ligamentocytes was compared with the donor tissue. Ligamentocyte spheroids were seeded on scaffolds embroidered either from polylactic acid (PLA) threads solely or combined PLA and poly (L-lactide-co-ε-caprolactone) (P(LA-CL)) threads. These scaffolds were further functionalized with fluorination and fibrillated collagen foam. Cell distribution and survival were monitored for up to five weeks. The transfected cells expressed the SV40 antigen throughout the entire observation time, but often exhibited random and incomplete cell divisions with significantly more dying cells, significantly more DNA and more numerous nucleoli than controls. The expression profile of non-transfected and SV40-positive ligamentocytes was similar. In contrast to controls, SV40-positive cells formed larger spheroids, produced less vimentin and focal adhesions and died on the scaffolds after 21 d. Functionalized scaffolds supported human ligamentocyte growth. SV40 antigen expressing ligamentocytes share many properties with their non-transfected counterparts suggesting them as a model, however, applicability for TE is limited.

Human nasoseptal chondrocytes maintain their differentiated phenotype on PLLA scaffolds produced by thermally induced phase separation and supplemented with bioactive glass 1393.

Damage of hyaline cartilage such as nasoseptal cartilage requires proper reconstruction, which remains challenging due to its low intrinsic repair capacity. Implantation of autologous chondrocytes in combination with a biomimetic biomaterial represents a promising strategy to support cartilage repair. Despite so far mostly tested for bone tissue engineering, bioactive glass (BG) could exert stimulatory effects on chondrogenesis. The aim of this work was to produce and characterize composite porous poly(L-lactide) (PLLA)/1393BG scaffolds via thermally induced phase separation (TIPS) technique and assess their effects on chondrogenesis of nasoseptal chondrocytes. The PLLA scaffolds without or with 1, 2.5, 5% BG1393 were prepared via TIPS technique starting from a ternary solution (polymer/solvent/non-solvent) in a single step. Scaffolds were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD) and differential scanning calorimetric analysis (DSC). Human nasoseptal chondrocytes were seeded on the scaffolds with 1 and 2.5% BG for 7 and 14 days and cell survival, attachment, morphology and expression of SOX9 and cartilage-specific extracellular cartilage matrix (ECM) components were monitored. The majority of chondrocytes survived on all PLLA scaffolds functionalized with BG for the whole culture period. Also inner parts of the scaffold were colonized by chondrocytes synthesizing an ECM which contained glycosaminoglycans. Type II collagen and aggrecan gene expression increased significantly in 1% BG scaffolds during the culture. Chondrocyte protein expression for cartilage ECM proteins indicated that the chondrocytes maintained their differentiated phenotype in the scaffolds. BG could serve as a cytocompatible basis for future scaffold composites for osteochondral cartilage defect repair. Abbreviations: AB: alcian blue ACAN: gene coding for aggrecan; BG: Bioactive glass; 2D: two-dimensional; 3D: three-dimensional; COL2A1: gene coding for type II collagen; DAPI: 4',6-diamidino-2-phenylindole; DMEM: Dulbecco's Modified Eagle's Medium; DMMB: dimethylmethylene blue; DSC: Differential scanning calorimetric analysis; ECM: extracellular matrix; EDTA: ethylenediaminetetraacetic acid; EtBr: ethidium bromide; FCS: fetal calf serum; FDA: fluorescein diacetate; GAG: glycosaminoglycans; HDPE: high density polyethylene; HE: hematoxylin and eosin staining; HCA: hydoxylapatite; PBE: phosphate buffered EDTA100 mM Na2HPO4 and 5 mM EDTA, pH8; PBS: phosphate buffered saline; PFA: paraformaldehyde; PG: proteoglycans; PI: propidium iodide; PLLA: Poly-L-Lactic Acid Scaffold; RT: room temperature; SD: standard deviation; SEM: scanning electron microscopy; sGAG: sulfated glycosaminoglycans; SOX9/Sox9: SRY (sex-determining region Y)-box 9 protein; TBS: TRIS buffered saline; TIPS: Thermally Induced Phase Separation; XRD: X-ray diffraction analysis.

Migrating Myofibroblastic Iliotibial Band-Derived Fibroblasts Represent a Promising Cell Source for Ligament Reconstruction.

The iliotibial band (ITB) is a suitable scaffold for anterior cruciate ligament (ACL) reconstruction, providing a sufficient mechanical resistance to loading. Hence, ITB-derived fibroblasts attract interest for ligament tissue engineering but have so far not been characterized. This present study aimed at characterizing ITB fibroblasts before, during, and after emigration from cadaveric ITB explants to decipher the emigration behavior and to utilize their migratory capacity for seeding biomaterials. ITB and, for comparison, ACL tissues were assessed for the content of alpha smooth muscle actin (αSMA) expressing fibroblasts and degeneration. The cell survival and αSMA expression were monitored in explants used for cell isolation, monolayer, self-assembled ITB spheroids, and spheroids seeded in polyglycolic acid (PGA) scaffolds. The protein expression profile of targets typically expressed by ligamentocytes (collagen types I-III, elastin, lubricin, decorin, aggrecan, fibronectin, tenascin C, CD44, ß1-integrins, vimentin, F-actin, αSMA, and vascular endothelial growth factor A [VEGFA]) was compared between ITB and ACL fibroblasts. A donor- and age-dependent differing percentage of αSMA positive cells could be detected, which was similar in ITB and ACL tissues despite the grade of degeneration being significantly higher in the ACL due to harvesting them from OA knees. ITB fibroblasts survived for several months in an explant culture, continuously forming monolayers with VEGFA and an increased αSMA expression. They shared their expression profile with ACL fibroblasts. αSMA decreased during the monolayer to spheroid/scaffold transition. Using self-assembled spheroids, the migratory capacity of reversible myofibroblastic ITB cells can be utilized for colonizing biomaterials for ACL tissue engineering and to support ligament healing.

IL-10 Could Play a Role in the Interrelation between Diabetes Mellitus and Osteoarthritis.

The association between osteoarthritis (OA), obesity and metabolic syndrome suggests an interrelation between OA and diabetes mellitus (DM). Little is known about the role of anti-inflammatory cytokine interleukin (IL)-10 in the interrelation between OA and DM. Hence, the effects of IL-10 under hyperglycemia (HG) and hyperinsulinemia (HI) in human articular chondrocytes (hAC) and chondrosarcoma cell line Okayama University Medical School (OUMS)-27 were examined. HAC and OUMS-27, cultured in normoglycemic (NG) and HG conditions were stimulated with insulin and/or IL-10. Cell survival, metabolic activity, proliferation and extracellular matrix (ECM) synthesis were immunocytochemically examined. No significant differences in vitality of hAC neither in pure NG (NGw/o) nor HG (HGw/o) conditions were found. Applying HI and/or IL-10 in both conditions reduced significantly the vitality of hAC but not of OUMS-27. HG impaired significantly hAC metabolism. When combined with HI + IL-10 or IL-10 alone it decreased also significantly hAC proliferation compared to NGw/o. In OUMS-27 it induced only a trend of impaired proliferation compared to NGw/o. hAC but not OUMS-27 reduced significantly their collagen type (col) I, SOX9 and proteoglycan (PG) synthesis in HG combined with HI +/- IL-10 compared to NGw/o. IL-10 could not moderate HI and HG effects. In contrast to hAC OUMS-27 showed limited sensitivity as DM model.

Viscoelastic Behavior of Embroidered Scaffolds for ACL Tissue Engineering Made of PLA and P(LA-CL) After In Vitro Degradation.

A rupture of the anterior cruciate ligament (ACL) is the most common knee ligament injury. Current applied reconstruction methods have limitations in terms of graft availability and mechanical properties. A new approach could be the use of a tissue engineering construct that temporarily reflects the mechanical properties of native ligament tissues and acts as a carrier structure for cell seeding. In this study, embroidered scaffolds composed of polylactic acid (PLA) and poly(lactic-co-ε-caprolactone) (P(LA-CL)) threads were tested mechanically for their viscoelastic behavior under in vitro degradation. The relaxation behavior of both scaffold types (moco: mono-component scaffold made of PLA threads, bico: bi-component scaffold made of PLA and P(LA-CL) threads) was comparable to native lapine ACL. Most of the lapine ACL cells survived 32 days of cell culture and grew along the fibers. Cell vitality was comparable for moco and bico scaffolds. Lapine ACL cells were able to adhere to the polymer surfaces and spread along the threads throughout the scaffold. The mechanical behavior of degrading matrices with and without cells showed no significant differences. These results demonstrate the potential of embroidered scaffolds as an ACL tissue engineering approach.

The interrelation of osteoarthritis and diabetes mellitus: considering the potential role of interleukin-10 and in vitro models for further analysis.

INTRODUCTION: Today, not only the existence of an interrelation between obesity/adipositas and osteoarthritis (OA) but also the association of OA and diabetes mellitus (DM) are widely recognized. Nevertheless, shared influence factors facilitating OA development in DM patients still remain speculative up until now. To supplement the analysis of clinical data, appropriate in vitro models could help to identify shared pathogenetic pathways. Informative in vitro studies could later be complemented by in vivo data obtained from suitable animal models. MATERIALS AND METHODS: Therefore, this detailed review of available literature was undertaken to discuss and compare the results of currently published in vitro studies focusing on the interrelation between OA, the metabolic syndrome and DM and to propose models to further study the molecular pathways. RESULTS: The survey of literature presented here supports the hypothesis that the pathogenesis of OA in DM is based on imbalanced molecular pathways with a putative crucial role of antiinflammatory cytokines such as IL-10. CONCLUSION: Future development of versatile micro-scaled in vitro models such as combining DM and OA on chip could allow the identification of common pathogenetic pathways and might help to develop novel therapeutic strategies.