Unlocking Potential: Low Bovine Serum Albumin Enhances the Chondrogenicity of Human Adipose-Derived Stromal Cells in Pellet Cultures.

Bovine serum albumin (BSA) plays a crucial role in cell culture media, influencing cellular processes such as proliferation and differentiation. Although it is commonly included in chondrogenic differentiation media, its specific function remains unclear. This study explores the effect of different BSA concentrations on the chondrogenic differentiation of human adipose-derived stromal/stem cells (hASCs). hASC pellets from six donors were cultured under chondrogenic conditions with three BSA concentrations. Surprisingly, a lower BSA concentration led to enhanced chondrogenesis. The degree of this effect was donor-dependent, classifying them into two groups: (1) high responders, forming at least 35% larger, differentiated pellets with low BSA in comparison to high BSA; (2) low responders, which benefitted only slightly from low BSA doses with a decrease in pellet size and marginal differentiation, indicative of low intrinsic differentiation potential. In all cases, increased chondrogenesis was accompanied by hypertrophy under low BSA concentrations. To the best of our knowledge, this is the first study showing improved chondrogenicity and the tendency for hypertrophy with low BSA concentration compared to standard levels. Once the tendency for hypertrophy is understood, the determination of BSA concentration might be used to tune hASC chondrogenic or osteogenic differentiation.

Scaffolded spheroids as building blocks for bottom-up cartilage tissue engineering show enhanced bioassembly dynamics.

Due to the capability of cell spheroids (SPH) to assemble into large high cell density constructs, their use as building blocks attracted a lot of attention in the field of biofabrication. Nevertheless, upon maturation, the composition along with the size of such building blocks change, affecting their fusiogenic ability to form a cohesive tissue construct of controllable size. This natural phenomenon remains a limitation for the standardization of spheroid-based therapies in the clinical setting. We recently showed that scaffolded spheroids (S-SPH) can be produced by forming spheroids directly within porous PCL-based microscaffolds fabricated using multiphoton lithography (MPL). In this new study, we compare the bioassembly potential of conventional SPHs versus S-SPHs depending on their degree of maturation. Doublets of both types of building blocks were cultured and their fusiogenicity was compared by measuring the intersphere angle, the length of the fusing spheroid pairs (referred to as doublet length) as well as their spreading behaviour. Finally, the possibility to fabricate macro-sized tissue constructs (i.e. cartilage-like) from both chondrogenic S-SPHs and SPHs was analyzed. This study revealed that, in contrast to conventional SPHs, S-SPHs exhibit robust and stable fusiogenicity, independently from their degree of maturation. In order to understand this behavior, we further analyze the intersection area of doublets, looking at the kinetic of cell migration and at the mechanical stability of the formed tissue using dissection measurements. Our findings indicate that the presence of microscaffolds enhances the ability of spheroids to be used as building blocks for bottom-up tissue engineering, which is an important advantage compared to conventional spheroid-based therapy approaches. STATEMENT OF SIGNIFICANCE: The approach of using SPHs as building blocks for bottom-up tissue engineering offers a variety of advantages. At the same time the self-assembly of large tissues remains challenging due to several intrinsic properties of SPHs, such as for instance the shrinkage of tissues assembled from SPHs, or the reduced fusiogenicity commonly observed with mature SPHs. In this work, we demonstrate the capability of scaffolded spheroids (S-SPH) to fuse and recreate cartilage-like tissue constructs despite their advanced maturation stage. In this regard, the presence of microscaffolds compensates for some of the intrinsic limitations of SPHs and can help to overcome current limitations of spheroid-based tissue engineering.

The p-rpS6-zone delineates wounding responses and the healing process.

The spatial boundaries of tissue response to wounding are unknown. Here, we show that in mammals, the ribosomal protein S6 (rpS6) is phosphorylated in response to skin injury, forming a zone of activation surrounding the region of the initial insult. This p-rpS6-zone forms within minutes after wounding and is present until healing is complete. The zone is a robust marker of healing as it encapsulates features of the healing process, including proliferation, growth, cellular senescence, and angiogenesis. A mouse model that is unable to phosphorylate rpS6 shows an initial acceleration of wound closure, but results in impaired healing, identifying p-rpS6 as a modulator but not a driver of healing. Finally, the p-rpS6-zone accurately reports on the status of dermal vasculature and the effectiveness of healing, visually dividing an otherwise homogeneous tissue into regions with distinct properties.

Wavelength-Dependent Effects of Photobiomodulation for Wound Care in Diabetic Wounds.

Photobiomodulation, showing positive effects on wound healing processes, has been performed mainly with lasers in the red/infrared spectrum. Light of shorter wavelengths can significantly influence biological systems. This study aimed to evaluate and compare the therapeutic effects of pulsed LED light of different wavelengths on wound healing in a diabetic (db/db) mouse excision wound model. LED therapy by Repuls was applied at either 470 nm (blue), 540 nm (green) or 635 nm (red), at 40 mW/cm2 each. Wound size and wound perfusion were assessed and correlated to wound temperature and light absorption in the tissue. Red and trend-wise green light positively stimulated wound healing, while blue light was ineffective. Light absorption was wavelength-dependent and was associated with significantly increased wound perfusion as measured by laser Doppler imaging. Shorter wavelengths ranging from green to blue significantly increased wound surface temperature, while red light, which penetrates deeper into tissue, led to a significant increase in core body temperature. In summary, wound treatment with pulsed red or green light resulted in improved wound healing in diabetic mice. Since impeded wound healing in diabetic patients poses an ever-increasing socio-economic problem, LED therapy may be an effective, easily applied and cost-efficient supportive treatment for diabetic wound therapy.

In vitro and Ex vivo Assessments of the Compatibility of Fibrin Sealant with Antimicrobial Compounds.

Background: Fibrin sealants are used as antimicrobial-releasing carriers for preventing surgical site infections; however, it is important to determine the release kinetics and antimicrobial effects of drugs added to fibrin sealants and the effects of drugs on clot/clotting properties. Materials and Methods: The antimicrobial and antibiofilm activity of cefazolin, colistin, gentamicin, oxacillin, tobramycin, and silver nitrate released from fibrin sealant were characterized using in vitro and ex vivo assays against bacteria commonly found on the skin. The effects of antimicrobial agents on the physical structure of the fibrin sealant were assessed with scanning electron microscopy (SEM) and on the clotting rate and strength of fibrin clots using run-off tests and rheology. Results: Generally, antibiotic agents were released gradually from fibrin sealant and were stable after release, with antimicrobial effects evident up to three days. Cefazolin, gentamicin, and oxacillin prevented biofilm formation of Staphylococcus aureus in porcine skin explants; gentamicin and colistin prevented biofilm formation of Pseudomonas aeruginosa. Gentamicin, cefazolin, colistin, and tobramycin did not affect the structural integrity or viscoelastic properties of fibrin sealant; changes were observed with oxacillin (SEM) and particularly silver nitrate (SEM and rheology). No antimicrobial agents caused deterioration of clotting time (run-off tests). Conclusions: From the antimicrobial agents tested, gentamicin and cefazolin showed prolonged release from fibrin sealant, sustained antimicrobial activity, and biofilm prevention properties against Staphylococcus aureus; similar results were observed for gentamicin and colistin against Pseudomonas aeruginosa. For each of these findings, the physical structure of the fibrin sealant, clotting rate, and strength of fibrin clots were unaffected.

Muscle-Specific Micro-Ribonucleic Acids miR-1-3p, miR-133a-3p, and miR-133b Reflect Muscle Regeneration After Single-Dose Zoledronic Acid Following Rotator Cuff Repair in a Rodent Chronic Defect Model.

BACKGROUND: Zoledronic acid improves bone microarchitecture and biomechanical properties after chronic rotator cuff repair (RCR) in rats. Besides the positive effects of zoledronic acid on bone mineral density and bone microarchitecture, bisphosphonates have positive effects on skeletal muscle function. PURPOSES/HYPOTHESIS: The purposes of this study were to (1) longitudinally evaluate circulating bone- and muscle-specific serum micro-ribonucleic acids (miRNAs) and (2) investigate supraspinatus muscle tissue after tenotomy and delayed RCR in a rat model. It was hypothesized that zoledronic acid would improve muscle regeneration after chronic RCR in rats. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 34 male Sprague-Dawley rats underwent unilateral (left) supraspinatus tenotomy (time point 1) with delayed transosseous RCR after 3 weeks (time point 2). All rats were sacrificed 8 weeks after RCR (time point 3). Animals were randomly assigned to 2 groups. One day after RCR, the control group was given 1 mL of subcutaneous saline solution, and the intervention group was treated with a subcutaneous single-dose of 100 µg/kg body weight of zoledronic acid. All 34 study animals underwent miRNA analysis at all 3 time points. In 4 animals of each group, histological analyses as well as gene expression analyses were conducted. RESULTS: Circulating miRNAs showed significantly different expressions between both study groups. In the control group, a significant downregulation was observed for muscle-specific miR-1-3p (P = .004), miR-133a-3p (P < .001), and miR-133b (P < .001). Histological analyses showed significantly higher rates of regenerating myofibers on the operated side (left) of both study groups compared with the nonoperated side (right; P = .002). On the nonoperated side, significantly higher rates of regenerating myofibers were observed in the intervention group compared with the control group (P = .031). The myofiber cross-sectional area revealed significantly smaller myofibers on both sides within the intervention group compared with both sides of the control group (P < .001). Within the intervention group, significantly higher expression levels of muscle development/regeneration marker genes embryonal Myosin heavy chain (P = .017) and neonatal Myosin heavy chain (P = .016) were observed on the nonoperated side compared with the operated side. CONCLUSION: An adjuvant single-dose of zoledronic acid after RCR in a chronic defect model in rats led to significant differences in bone- and muscle-specific miRNA levels. Therefore, miR-1-3p, miR-133a-3p, and miR-133b might be used as biomarkers for muscle regeneration after RCR. CLINICAL RELEVANCE: Adjuvant treatment with zoledronic acid may improve muscle regeneration after chronic RCR in humans, thus counteracting fatty muscle infiltration and atrophy.

Novel 3D-Printed Cell Culture Inserts for Air-Liquid Interface Cell Culture.

In skin research, widely used in vitro 2D monolayer models do not sufficiently mimic physiological properties. To replace, reduce, and refine animal experimentation in the spirit of '3Rs', new approaches such as 3D skin equivalents (SE) are needed to close the in vitro/in vivo gap. Cell culture inserts to culture SE are commercially available, however, these inserts are expensive and of limited versatility regarding experimental settings. This study aimed to design novel cell culture inserts fabricated on commercially available 3D printers for the generation of full-thickness SE. A computer-aided design model was realized by extrusion-based 3D printing of polylactic acid filaments (PLA). Improvements in the design of the inserts for easier and more efficient handling were confirmed in cell culture experiments. Cytotoxic effects of the final product were excluded by testing the inserts in accordance with ISO-norm procedures. The final versions of the inserts were tested to generate skin-like 3D scaffolds cultured at an air-liquid interface. Stratification of the epidermal component was demonstrated by histological analyses. In conclusion, here we demonstrate a fast and cost-effective method for 3D-printed inserts suitable for the generation of 3D cell cultures. The system can be set-up with common 3D printers and allows high flexibility for generating customer-tailored cell culture plastics.

ESWT Diminishes Axonal Regeneration following Repair of the Rat Median Nerve with Muscle-In-Vein Conduits but Not after Autologous Nerve Grafting.

Investigations reporting positive effects of extracorporeal shockwave therapy (ESWT) on nerve regeneration are limited to the rat sciatic nerve model. The effects of ESWT on muscle-in-vein conduits (MVCs) have also not been investigated yet. This study aimed to evaluate the effects of ESWT after repair of the rat median nerve with either autografts (ANGs) or MVCs. In male Lewis rats, a 7 mm segment of the right median nerve was reconstructed either with an ANG or an MVC. For each reconstructive technique, one group of animals received one application of ESWT while the other rats served as controls. The animals were observed for 12 weeks, and nerve regeneration was assessed using computerized gait analysis, the grasping test, electrophysiological evaluations and histological quantification of axons, blood vessels and lymphatic vasculature. Here, we provide for the first time a comprehensive analysis of ESWT effects on nerve regeneration in a rat model of median nerve injury. Furthermore, this study is among the first reporting the quantification of lymphatic vessels following peripheral nerve injury and reconstruction in vivo. While we found no significant direct positive effects of ESWT on peripheral nerve regeneration, results following nerve repair with MVCs were significantly inferior to those after ANG repair.

Effects of Extracorporeal Shockwave Therapy on Functional Recovery and Circulating miR-375 and miR-382-5p after Subacute and Chronic Spinal Cord Contusion Injury in Rats.

Extracorporeal shockwave therapy (ESWT) can stimulate processes to promote regeneration, including cell proliferation and modulation of inflammation. Specific miRNA expression panels have been established to define correlations with regulatory targets within these pathways. This study aims to investigate the influence of low-energy ESWT-applied within the subacute and chronic phase of SCI (spinal cord injury) on recovery in a rat spinal cord contusion model. Outcomes were evaluated by gait analysis, µCT and histological analysis of spinal cords. A panel of serum-derived miRNAs after SCI and after ESWT was investigated to identify injury-, regeneration- and treatment-associated expression patterns. Rats receiving ESWT showed significant improvement in motor function in both a subacute and a chronic experimental setting. This effect was not reflected in changes in morphology, µCT-parameters or histological markers after ESWT. Expression analysis of various miRNAs, however, revealed changes after SCI and ESWT, with increased miR-375, indicating a neuroprotective effect, and decreased miR-382-5p potentially improving neuroplasticity via its regulatory involvement with BDNF. We were able to demonstrate a functional improvement of ESWT-treated animals after SCI in a subacute and chronic setting. Furthermore, the identification of miR-375 and miR-382-5p could potentially provide new targets for therapeutic intervention in future studies.

Occurrence of Lymphangiogenesis in Peripheral Nerve Autografts Contrasts Schwann Cell-Induced Apoptosis of Lymphatic Endothelial Cells In Vitro.

Peripheral nerve injuries pose a major clinical concern world-wide, and functional recovery after segmental peripheral nerve injury is often unsatisfactory, even in cases of autografting. Although it is well established that angiogenesis plays a pivotal role during nerve regeneration, the influence of lymphangiogenesis is strongly under-investigated. In this study, we analyzed the presence of lymphatic vasculature in healthy and regenerated murine peripheral nerves, revealing that nerve autografts contained increased numbers of lymphatic vessels after segmental damage. This led us to elucidate the interaction between lymphatic endothelial cells (LECs) and Schwann cells (SCs) in vitro. We show that SC and LEC secretomes did not influence the respective other cell types' migration and proliferation in 2D scratch assay experiments. Furthermore, we successfully created lymphatic microvascular structures in SC-embedded 3D fibrin hydrogels, in the presence of supporting cells; whereas SCs seemed to exert anti-lymphangiogenic effects when cultured with LECs alone. Here, we describe, for the first time, increased lymphangiogenesis after peripheral nerve injury and repair. Furthermore, our findings indicate a potential lymph-repellent property of SCs, thereby providing a possible explanation for the lack of lymphatic vessels in the healthy endoneurium. Our results highlight the importance of elucidating the molecular mechanisms of SC-LEC interaction.

Gelatin methacryloyl as environment for chondrocytes and cell delivery to superficial cartilage defects.

Cartilage damage typically starts at its surface, either due to wear or trauma. Treatment of these superficial defects is important in preventing degradation and osteoarthritis. Biomaterials currently used for deep cartilage defects lack appropriate properties for this application. Therefore, we investigated photo-crosslinked gelatin methacryloyl (gelMA) as a candidate for treatment of surface defects. It allows for liquid application, filling of surface defects and forming a protective layer after UV-crosslinking, thereby keeping therapeutic cells in place. gelMA and photo-initiator lithium phenyl-2,4,6-trimethyl-benzoylphosphinate (Li-TPO) concentration were optimized for application as a carrier to create a favorable environment for human articular chondrocytes (hAC). Primary hAC were used in passages 3 and 5, encapsulated into two different gelMA concentrations (7.5 wt% (soft) and 10 wt% (stiff)) and cultivated for 3 weeks with TGF-ß3 (0, 1 and 10 ng/mL). Higher TGF-ß3 concentrations induced spherical cell morphology independent of gelMA stiffness, while low TGF-ß3 concentrations only induced rounded morphology in stiff gelMA. Gene expression did not vary across gel stiffnesses. As a functional model gelMA was loaded with two different cell types (hAC and/or human adipose-derived stem cells [ASC/TERT1]) and applied to human osteochondral osteoarthritic plugs. GelMA attached to the cartilage, smoothened the surface and retained cells in place. Resistance against shear forces was tested using a tribometer, simulating normal human gait and revealing maintained cell viability. In conclusion gelMA is a versatile, biocompatible material with good bonding capabilities to cartilage matrix, allowing sealing and smoothening of superficial cartilage defects while simultaneously delivering therapeutic cells for tissue regeneration.

A Microfluidic Multisize Spheroid Array for Multiparametric Screening of Anticancer Drugs and Blood-Brain Barrier Transport Properties.

Physiological-relevant in vitro tissue models with their promise of better predictability have the potential to improve drug screening outcomes in preclinical studies. Despite the advances of spheroid models in pharmaceutical screening applications, variations in spheroid size and consequential altered cell responses often lead to nonreproducible and unpredictable results. Here, a microfluidic multisize spheroid array is established and characterized using liver, lung, colon, and skin cells as well as a triple-culture model of the blood-brain barrier (BBB) to assess the effects of spheroid size on (a) anticancer drug toxicity and (b) compound penetration across an advanced BBB model. The reproducible on-chip generation of 360 spheroids of five dimensions on a well-plate format using an integrated microlens technology is demonstrated. While spheroid size-related IC50 values vary up to 160% using the anticancer drugs cisplatin (CIS) or doxorubicin (DOX), reduced CIS:DOX drug dose combinations eliminate all lung microtumors independent of their sizes. A further application includes optimizing cell seeding ratios and size-dependent compound uptake studies in a perfused BBB model. Generally, smaller BBB-spheroids reveal an 80% higher compound penetration than larger spheroids while verifying the BBB opening effect of mannitol and a spheroid size-related modulation on paracellular transport properties.

Cytotoxicity, Retention, and Anti-inflammatory Effects of a CeO2 Nanoparticle-Based Supramolecular Complex in a 3D Liver Cell Culture Model.

Both cerium oxide (CeOx) nanoparticles and mefenamic acid (MFA) are known anti-inflammatory agents with hepatoprotective properties and are therefore prescribed for one of the major diseases in the world, nonalcoholic fatty liver disease (NAFLD). To study the potential cytotoxicity and anti-inflammatory effects as well as drug retention of a potential therapeutic CeOx/MFA supramolecular complex, a well-standardized hepatic (HepG2) spheroid model was used. Results showed that the highest cytotoxicity for the CeOx/MFA supramolecular complex was found at 50 µg/mL, while effective doses of 0.1 and 1 µg/mL yielded a significant decrease of TNF-α and IL-8 secretion. Time-resolved analysis of HepG2 spheroids revealed a spatiotemporal distribution of the supramolecular complex and limited clearance from the internal microtissue over a period of 8 days in cultivation. In summary, our results point at rapid uptake, distribution, and biostability of the supramolecular complex within the HepG2 liver spheroid model as well as a significant anti-inflammatory response at noncytotoxic levels.

Antimicrobial photodynamic therapy fighting polymicrobial infections - a journey from in vitro to in vivo.

Rapidly evolving multidrug resistance renders conventional antimicrobial strategies increasingly inefficient. This urges the exploration of alternative strategies with a lower potential of resistance development to control microbial infections. A promising option is antimicrobial photodynamic therapy (aPDT), especially in the setting of wound infections. In this study its effectiveness was tested as a treatment option for polymicrobially infected wounds in both in vitro and in vivo models. First, aPDT was applied to wound-relevant Gram-positive and Gram-negative bacteria in planktonic culture as the standard in vitro test system and compared different media to show a possible dependency of the therapy on the surrounding environment. In a second step, aPDT was investigated in an in vitro model mimicking the wound bed conditions using fibrin-coated culture plates. Finally, we tested aPDT in vivo in a polymicrobial infected wound healing model in immunocompromised BALB/c mice. In vitro, it was shown that the bactericidal effectiveness of aPDT was strongly dependent on the surrounding environment of the phototoxic reaction. In vivo, the significant delay in wound healing induced by polymicrobial infection was drastically diminished by a two-times application of aPDT using 100 µM methylene blue (generally regarded as safe for topical application on human skin) and 24 J cm-2 pulsed red LED light. Our experiments suggest that aPDT is capable of significantly improving wound healing also in complicated polymicrobially infected wound situations.

The response of gingiva monolayer, spheroid, and ex vivo tissue cultures to collagen membranes and bone substitute.

Collagen membranes and bone substitute are popular biomaterials in guided tissue regeneration for treatment of traumatized or diseased periodontal tissue. Development of these biomaterials starts in monolayer cell culture, failing to reflect in vivo tissue organization. Spheroid cultures potentially mimic in vivo tissues in structure and functionality. This study aims to compare gingiva cell (GC) monolayers and spheroids to ex vivo gingiva. Human GC monolayers, spheroids and gingiva ex vivo tissues were cultured on plastic surfaces, collagen membranes or bone substitute. Hematoxylin-eosin (HE) staining, immunohistochemistry for KI67 and caspase 3 (CASP3), resazurin-based toxicity assays, quantitative polymerase chain reaction for collagen I (COL1A1), vascular endothelial growth factor (VEGF), angiogenin (ANG), interleukin (IL)6 and IL8 and ELISA for COL1A1, VEGF, ANG, IL6 and IL8 were performed in all cultures. Morphology was different in all culture set-ups. Staining of KI67 was positive in monolayers and staining of CASP3 was positive in spheroids. All culture set-ups were viable. COL1A1 production was modulated in monolayers and ex vivo tissues at mRNA levels, VEGF in monolayers and ex vivo tissues at mRNA levels and in spheroids at protein levels, ANG in spheroids at mRNA levels and in monolayers and spheroids at protein levels, IL6 in monolayers and spheroids at mRNA levels and in spheroids and ex vivo tissues at protein levels and IL8 in monolayers and ex vivo tissues at mRNA levels. Modulations were surface-dependent. In conclusion, each culture model is structurally and functionally different. Neither GC monolayers nor spheroids mimicked gingiva ex vivo tissue in all measured aspects.

Stiffness Matters: Fine-Tuned Hydrogel Elasticity Alters Chondrogenic Redifferentiation.

Biomechanical cues such as shear stress, stretching, compression, and matrix elasticity are vital in the establishment of next generation physiological in vitro tissue models. Matrix elasticity, for instance, is known to guide stem cell differentiation, influence healing processes and modulate extracellular matrix (ECM) deposition needed for tissue development and maintenance. To better understand the biomechanical effect of matrix elasticity on the formation of articular cartilage analogs in vitro, this study aims at assessing the redifferentiation capacity of primary human chondrocytes in three different hydrogel matrices of predefined matrix elasticities. The hydrogel elasticities were chosen to represent a broad spectrum of tissue stiffness ranging from very soft tissues with a Young's modulus of 1 kPa up to elasticities of 30 kPa, representative of the perichondral-space. In addition, the interplay of matrix elasticity and transforming growth factor beta-3 (TGF-ß3) on the redifferentiation of primary human articular chondrocytes was studied by analyzing both qualitative (viability, morphology, histology) and quantitative (RT-qPCR, sGAG, DNA) parameters, crucial to the chondrotypic phenotype. Results show that fibrin hydrogels of 30 kPa Young's modulus best guide chondrocyte redifferentiation resulting in a native-like morphology as well as induces the synthesis of physiologic ECM constituents such as glycosaminoglycans (sGAG) and collagen type II. This comprehensive study sheds light onto the mechanobiological impact of matrix elasticity on formation and maintenance of articular cartilage and thus represents a major step toward meeting the need for advanced in vitro tissue models to study both re- and degeneration of articular cartilage.

Contraction dynamics of dental pulp cell rod microtissues.

OBJECTIVES: The factors that contribute to the morphological changes of dental pulp cell-derived microtissues are unknown. Here, we investigated the contraction dynamics of rod-shaped microtissues derived from dental pulp cells and examined the underlying cell signaling pathways. METHODS: Human dental pulp cells were seeded into agarose molds to assemble into rod-shaped microtissues. Resazurin- and tetrazolium-based cytotoxicity assays, Live/Dead staining, and hematoxylin and eosin staining for histological evaluation of rods were performed. Rod contraction was evaluated and measured for a period of 10 days. The role of TGF-ß, phosphoinositide 3-kinase (PI3K)/AKT, and mitogen-activated protein kinase (MAPK) signaling pathway was analyzed. RESULTS: Dental pulp cells readily assembled into rods, maintaining the geometric shape for 48 h. Following this period, they condensed to form stable spheroidal structures that remained vital for 10 days from seeding. Inhibition of phosphoinositide 3-kinase signaling pathway by LY294002 significantly prolonged the diminution in the length of rods formed by dental pulp cells. TGF-ß and pharmacological inhibition of TGF-ß signaling did not show pronounced effects. CONCLUSION: Overall, dental pulp cells readily formed rod-shaped patterns of microtissues which, over a period of time, condensed into more stable spheroidal structures. Hence, technologies like bioprinting, using direct fabrication of microtissues need to consider the contraction dynamics. CLINICAL RELEVANCE: The field of regenerative endodontology will benefit from our findings as it can be applied as a novel platform to test the impact of pharmacological agents, biomaterials, and regenerative approaches including bioprinting.

The impact of collagen membranes on 3D gingival fibroblast toroids.

BACKGROUND: Development in guided tissue regeneration requires biomaterial testing. 3D cell constructs represent a new approach to bridge the gap between cell culture and animal models. Following the hypothesis that attachment behavior of cells could be observed in toroidal 3D cell constructs, the aim of this study was to evaluate 3D gingival fibroblast (GF) toroids as a simple and feasible in vitro assay to test attachment of oral fibroblasts to collagen membranes. METHODS: 3D ring-like structures (toroids) were formed from human GF. Hematoxylin-eosin staining was performed with formed GF toroids. Produced GF toroids were seeded onto plastic surfaces or collagen membranes. The morphology was documented at 24 h, 48 h and 72 h after seeding with light and fluorescence microscopy. Toroid vitality was assessed at same time points with a resazurin-based toxicity assay. RESULTS: GF showed normal morphology in toroid hematoxylin-eosin staining. Over 72 h, GF toroids on plastic surfaces stayed unchanged, while GF toroids on collagen membranes showed dilatation. GF toroids on plastic surfaces and collagen membranes were metabolically active over the observed period. CONCLUSIONS: Depending on the surface material, 3D GF toroids show different attachment behavior. Thus, GF toroids are suitable as simple assay to study attachment behavior to various biomaterials.

Angiopoietin-like 4 production upon treatment with hypoxia and L-mimosine in periodontal fibroblasts.

BACKGROUND AND OBJECTIVE: A key factor in the modulation of angiogenesis as well as in bone resorption is angiopoietin-like 4. However, the role of angiopoietin-like 4 in periodontal tissue is unknown. Here, we hypothesized that hypoxia and the hypoxia mimetic agent L-mimosine can induce the production of angiopoietin-like 4 in periodontal fibroblasts. METHODS: Human periodontal ligament fibroblasts (PDLF) were cultured in monolayer and spheroid cultures. The cultures were incubated in the presence of hypoxia or L-mimosine. Angiopoietin-like 4 mRNA and protein levels were measured by qPCR and ELISA, respectively. Also, the impact of Lipopolysaccharides of E. coli and P. gingivalis, interleukin (IL)-1ß and tumor necrosis factor (TNF)α was evaluated. Furthermore, we tested dependency on hypoxia-inducible factor (HIF)-1 activity by Western blotting for HIF-1 and inhibitor studies with echinomycin. Potential autocrine effects were assessed by exposure of PDLF to recombinant angiopoietin-like 4 in full length, C-terminal and N-terminal fragments. The impact on viability, DNA synthesis, alkaline phosphatase, and matrix mineralization was evaluated. RESULTS: Both hypoxia and L-mimosine elevated angiopoietin-like 4 mRNA and protein levels in monolayer cultures of PDLF. HIF-1 was elevated after both hypoxia and L-mimosine treatment. LPS, IL-1ß, and TNFα did not modulate angiopoietin-like 4 levels significantly. Addition of echinomycin in the cultures inhibited the production of angiopoietin-like 4. In spheroid cultures of PDLF, the increase did not reach the level of significance at mRNA and protein levels. Angiopoietin-like 4 in full length, C-terminal, and N-terminal fragments did not modulate viability, DNA synthesis, alkaline phosphatase, and matrix mineralization. CONCLUSION: Overall, we found that hypoxia and the hypoxia mimetic agent L-mimosine can stimulate angiopoietin-like 4 production in monolayer cultures of PDLF. This increase depends on HIF-1 activity. Future studies will reveal how the modulation of angiopoietin-like 4 in the periodontium contributes to periodontal disease and regeneration.

Angiogenin production in response to hypoxia and l-mimosine in periodontal fibroblasts.

BACKGROUND: A major mediator of angiogenesis is angiogenin, which is expressed in the early phase of healing in oral tissue engineering strategies. It is unclear how angiogenin is regulated in the periodontal tissue. The objective of this study was to reveal the regulation of angiogenin in response to hypoxia and the hypoxia mimetic agent l-mimosine in periodontal fibroblasts. METHODS: Human fibroblasts of the periodontal ligament (PDLF) and the gingiva (GF) in monolayer and spheroid cultures were exposed to hypoxia or l-mimosine. The production of angiogenin was evaluated at mRNA and protein levels with reverse transcription quantitative polymerase chain reaction and enzyme-linked immunosorbent assays, respectively. Echinomycin, an inhibitor of hypoxia-inducible factor (HIF)-1 activity, was used to test the involvement of HIF-1. RESULTS: Our data show that hypoxia and l-mimosine can increase angiogenin mRNA and protein levels in PDLF monolayer cultures. In GF monolayer cultures, we found an increase of angiogenin at the mRNA level in response to hypoxia. The increase of angiogenin can be blocked by inhibition of HIF-1 signaling via echinomycin. In PDLF and GF spheroid cultures, the impact of hypoxia and l-mimosine did not reach the level of significance. CONCLUSION: Hypoxia and the hypoxia mimetic agent l-mimosine can increase the production of angiogenin via HIF-1 signaling in PDLF monolayer cultures but not in spheroid cultures. GF were less sensitive to the impact of hypoxia and l-mimosine. Overall, these results suggest a link between hypoxia, HIF-1 signaling and angiogenin in the periodontium.