Hypoxia Promotes Cartilage Regeneration in Cell-Seeded 3D-Printed Bioscaffolds Cultured with a Bespoke 3D Culture Device.

In this study, we investigated the effect of oxygen tension on the expansion of ADMSCs and on their differentiation toward their chondrocytic phenotype, regenerating a lab-based cartilaginous tissue with superior characteristics. Controversial results with reference to MSCs that were cultured under different hypoxic levels, mainly in 2D culturing settings combined with or without other biochemical stimulus factors, prompted our team to study the role of hypoxia on MSCs chondrogenic differentiation within an absolute 3D environment. Specifically, we used 3D-printed honeycomb-like PCL matrices seeded with ADMSCs in the presence or absence of TGF and cultured with a prototype 3D cell culture device, which was previously shown to favor nutrient/oxygen supply, cell adhesion, and infiltration within scaffolds. These conditions resulted in high-quality hyaline cartilage that was distributed uniformly within scaffolds. The presence of the TGF medium was necessary to successfully produce cartilaginous tissues with superior molecular and increased biomechanical properties. Despite hypoxia's beneficial effect, it was overall not enough to fully differentiate ADMSCs or even promote cell expansion within 3D scaffolds alone.

3D-Printed Antibacterial Scaffolds for the Regeneration of Alveolar Bone in Severe Periodontitis.

Current clinical treatment of periodontitis alleviates periodontal symptoms and helps to keep the disease under control for extended periods. Despite this, a significant destruction of the tooth's underlying bone tissue often takes place progressively. Herein, we present a two-way therapeutic approach for local delivery of antibacterial agents and bone tissue regeneration, incorporating ~1% w/w tetracycline hydrochloride (TCH) into a 3D-printed scaffold composed of poly(ε-caprolactone) (PCL). Samples were assessed for their morphological, physicochemical, pharmacokinetic, and antibacterial properties. Furthermore, osteoprecursor cells (MC3T3-E1) were employed to evaluate the osteoinductive potential of the drug-loaded scaffolds. Cell proliferation, viability, and differentiation were determined on all cell-seeded scaffolds. At the end of the culture, PCL-TCH scaffolds promoted abundant collagen organic matrix, demonstrating augmented alkaline phosphatase (ALP) activity and areas of accumulated mineralised bone tissue, despite their belayed cell proliferation. Based on the observed effectiveness of the PCL-TCH scaffolds to inhibit Staphylococcus aureus, these constructs could serve as an alternative bioactive implant that supports bacterial inhibition and favours a 3D microenvironment for bone tissue regeneration in severe periodontitis.

Acylcarnitines in Ophthalmology: Promising Emerging Biomarkers.

Several common ocular diseases are leading causes of irreversible visual impairment. Over the last decade, various mainly untargeted metabolic studies have been performed to show that metabolic dysfunction plays an important role in the pathogenesis of ocular diseases. A number of metabolites in plasma/serum, aqueous or vitreous humor, or in tears have been found to differ between patients and controls; among them are L-carnitine and acylcarnitines, which are essential for mitochondrial fatty acid oxidation. The metabolic profile of carnitines regarding a variety of diseases has attracted researchers' interest. In this review, we present and discuss recent advances that have been made in the identification of carnitines as potential metabolic biomarkers in common ocular diseases, such as age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, central retinal vein occlusion, primary open-angle glaucoma, rhegmatogenous retinal detachment, and dry eye syndrome.

The role of temperature in vitality and survival assessments of beam-trawled and discarded European plaice (Pleuronectes platessa).

Thermal stress can influence the recovery of fish released after capture. Vitality assessments using reflex and behavioural responses require that responses can be observed reliably, independent of temperature. Here, we tested whether reflex and behavioural impairment and survival of beam-trawled and discarded European plaice (Pleuronectes platessa) are independent from seasonal air and water temperature deviations. In total, 324 beam-trawled plaice (n = 196 in summer and n = 128 in winter) were exposed to two air temperature treatments and two water treatments (i.e. modified and ambient temperatures for both). The modified treatments (i.e. cooled in summer, warmed in winter) represent the thermal shock a fish may experience when being returned to the water. All reflexes and tested behaviours were affected by ambient temperature, with high impairment noted in summer. None of the reflexes were affected by temperature shocks alone, only body flex was. Body flex was highly impaired under every exposure combination. Fish size and duration of air exposure further influenced impairment of reflexes such as head complex and tail grab. More generally, post-release survival was assessed as 21% [95% CI: 16-28%] in summer and 99% [97-100%] in winter. Beam trawling in summer is likely to induce high reflex impairment and mortality in discarded plaice, and therefore spatial-temporal mitigation approaches should be prioritized over control of on-board temperatures.

Active osseointegration in an ex vivo porcine bone model.

Achieving osseointegration is a fundamental requirement for many orthopaedic, oral, and craniofacial implants. Osseointegration typically takes three to 6 months, during which time implants are at risk of loosening. The aim of this study was to investigate whether osseointegration could be actively enhanced by delivering controllable electromechanical stimuli to the periprosthetic bone. First, the osteoconductivity of the implant surface was confirmed using an in vitro culture with murine preosteoblasts. The effects of active treatment on osseointegration were then investigated in a 21-day ex vivo model with freshly harvested cancellous bone cylinders (n = 24; Ø10 mm × 5 mm) from distal porcine femora, with comparisons to specimens treated by a distant ultrasound source and static controls. Cell viability, proliferation and distribution was evident throughout culture. Superior ongrowth of tissue onto the titanium discs during culture was observed in the actively stimulated specimens, with evidence of ten-times increased mineralisation after 7 and 14 days of culture (p < 0.05) and 2.5 times increased expression of osteopontin (p < 0.005), an adhesive protein, at 21 days. Moreover, histological analyses revealed increased bone remodelling at the implant-bone interface in the actively stimulated specimens compared to the passive controls. Active osseointegration is an exciting new approach for accelerating bone growth into and around implants.

The Role of Cone Beam Computed Tomography in Periodontology: From 3D Models of Periodontal Defects to 3D-Printed Scaffolds.

The treatment of osseous defects around teeth is a fundamental concern within the field of periodontology. Over the years, the method of grafting has been employed to treat bone defects, underscoring the necessity for custom-designed scaffolds that precisely match the anatomical intricacies of the bone cavity to be filled, preventing the formation of gaps that could allow the regeneration of soft tissues. In order to create such a patient-specific scaffold (bone graft), it is imperative to have a highly detailed 3D representation of the bone defect, so that the resulting scaffold aligns with the ideal anatomical characteristics of the bone defect. In this context, this article implements a workflow for designing 3D models out of patient-specific tissue defects, fabricated as scaffolds with 3D-printing technology and bioabsorbable materials, for the personalized treatment of periodontitis. The workflow is based on 3D modeling of the hard tissues around the periodontal defect (alveolar bone and teeth), scanned from patients with periodontitis. Specifically, cone beam computed tomography (CBCT) data were acquired from patients and were used for the reconstruction of the 3D model of the periodontal defect. The final step encompasses the 3D printing of these scaffolds, employing Fused Deposition Modeling (FDM) technology and 3D-bioprinting, with the aim of verifying the design accuracy of the developed methodοlogy. Unlike most existing 3D-printed scaffolds reported in the literature, which are either pre-designed or have a standard structure, this method leads to the creation of highly detailed patient-specific grafts. Greater accuracy and resolution in the macroarchitecture of the scaffolds were achieved during FDM printing compared to bioprinting, with the standard FDM printing profile identified as more suitable in terms of both time and precision. It is easy to follow and has been successfully employed to create 3D models of periodontal defects and 3D-printed scaffolds for three cases of patients, proving its applicability and efficiency in designing and fabricating personalized 3D-printed bone grafts using CBCT data.

Electromechanical and biological evaluations of 0.94Bi0.5Na0.5TiO3-0.06BaTiO3 as a lead-free piezoceramic for implantable bioelectronics.

Smart implantable electronic medical devices are being developed to deliver healthcare that is more connected, personalised, and precise. Many of these implantables rely on piezoceramics for sensing, communication, energy autonomy, and biological stimulation, but the piezoceramics with the strongest piezoelectric coefficients are almost exclusively lead-based. In this article, we evaluate the electromechanical and biological characteristics of a lead-free alternative, 0.94Bi0.5Na0.5TiO3-0.06BaTiO3 (BNT-6BT), manufactured via two synthesis routes: the conventional solid-state method (PIC700) and tape casting (TC-BNT-6BT). The BNT-6BT materials exhibited soft piezoelectric properties, with d33 piezoelectric coefficients that were inferior to commonly used PZT (PIC700: 116 pC/N; TC-BNT-6BT: 121 pC/N; PZT-5A: 400 pC/N). The material may be viable as a lead-free substitute for soft PZT where moderate performance losses up to 10 dB are tolerable, such as pressure sensing and pulse-echo measurement. No short-term harmful biological effects of BNT-6BT were detected and the material was conducive to the proliferation of MC3T3-E1 murine preosteoblasts. BNT-6BT could therefore be a viable material for electroactive implants and implantable electronics without the need for hermetic sealing.

Bioreactor analyses of tissue ingrowth, ongrowth and remodelling around implants: An alternative to live animal testing.

Introduction: Preclinical assessment of bone remodelling onto, into or around novel implant technologies is underpinned by a large live animal testing burden. The aim of this study was to explore whether a lab-based bioreactor model could provide similar insight. Method: Twelve ex vivo trabecular bone cylinders were extracted from porcine femora and were implanted with additively manufactured stochastic porous titanium implants. Half were cultured dynamically, in a bioreactor with continuous fluid flow and daily cyclic loading, and half in static well plates. Tissue ongrowth, ingrowth and remodelling around the implants were evaluated with imaging and mechanical testing. Results: For both culture conditions, scanning electron microscopy (SEM) revealed bone ongrowth; widefield, backscatter SEM, micro computed tomography scanning, and histology revealed mineralisation inside the implant pores; and histology revealed woven bone formation and bone resorption around the implant. The imaging evidence of this tissue ongrowth, ingrowth and remodelling around the implant was greater for the dynamically cultured samples, and the mechanical testing revealed that the dynamically cultured samples had approximately three times greater push-through fixation strength (p < 0.05). Discussion: Ex vivo bone models enable the analysis of tissue remodelling onto, into and around porous implants in the lab. While static culture conditions exhibited some characteristics of bony adaptation to implantation, simulating physiological conditions with a bioreactor led to an accelerated response.

The effect of nodal connectivity and strut density within stochastic titanium scaffolds on osteogenesis.

Modern orthopaedic implants use lattice structures that act as 3D scaffolds to enhance bone growth into and around implants. Stochastic scaffolds are of particular interest as they mimic the architecture of trabecular bone and can combine isotropic properties and adjustable structure. The existing research mainly concentrates on controlling the mechanical and biological performance of periodic lattices by adjusting pore size and shape. Still, less is known on how we can control the performance of stochastic lattices through their design parameters: nodal connectivity, strut density and strut thickness. To elucidate this, four lattice structures were evaluated with varied strut densities and connectivity, hence different local geometry and mechanical properties: low apparent modulus, high apparent modulus, and two with near-identical modulus. Pre-osteoblast murine cells were seeded on scaffolds and cultured in vitro for 28 days. Cell adhesion, proliferation and differentiation were evaluated. Additionally, the expression levels of key osteogenic biomarkers were used to assess the effect of each design parameter on the quality of newly formed tissue. The main finding was that increasing connectivity increased the rate of osteoblast maturation, tissue formation and mineralisation. In detail, doubling the connectivity, over fixed strut density, increased collagen type-I by 140%, increased osteopontin by 130% and osteocalcin by 110%. This was attributed to the increased number of acute angles formed by the numerous connected struts, which facilitated the organization of cells and accelerated the cell cycle. Overall, increasing connectivity and adjusting strut density is a novel technique to design stochastic structures which combine a broad range of biomimetic properties and rapid ossification.

In Vivo Cartilage Regeneration with Cell-Seeded Natural Biomaterial Scaffold Implants: 15-Year Study.

Articular cartilage can be easily damaged from human's daily activities, leading to inflammation and to osteoarthritis, a situation that can diminish the patients' quality of life. For larger cartilage defects, scaffolds are employed to provide cells the appropriate three-dimensional environment to proliferate and differentiate into healthy cartilage tissue. Natural biomaterials used as scaffolds, attract researchers' interest because of their relative nontoxic nature, their abundance as natural products, their easy combination with other materials, and the relative easiness to establish Marketing Authorization. The last 15 years were chosen to review, document, and elucidate the developments on cell-seeded natural biomaterials for articular cartilage treatment in vivo. The parameters of the experimental designs and their results were all documented and presented. Considerations about the newly formed cartilage and the treatment of cartilage defects were discussed, along with difficulties arising when applying natural materials, research limitations, and tissue engineering approaches for hyaline cartilage regeneration. Impact statement This review includes information from the literature and has never been complied before. The last 15 years were chosen to document and elucidate the developments on in vivo, articular cartilage treatment with cell-seeded natural biomaterials. The parameters of the experimental designs and their results were all documented and presented thoroughly. Natural materials overall seem to have a positive role in the recovery of cartilage defects. We expect that the knowledge from this review will be very useful to researchers organizing experimental protocols on critical analysis of advanced therapy medicinal products for human use.

Novel electrospun poly-hydroxybutyrate scaffolds as carriers for the wound healing agents alkannins and shikonins.

The aim of this study was to investigate the potential of novel electrospun fiber mats loaded with alkannin and shikonin (A/S) derivatives, using as carrier a highly biocompatible, bio-derived, eco-friendly polymer such as poly[(R)-3-hydroxybutyric acid] (PHB). PHB fibers containing a mixture of A/S derivatives at different ratios were successfully fabricated via electrospinning. Αs evidenced by scanning electron microscopy, the fibers formed a bead-free mesh with average diameters from 1.25 to 1.47 µm. Spectroscopic measurements suggest that electrospinning marginally increases the amorphous content of the predominantly crystalline PHB in the fibers, while a significant drug amount lies near the fiber surface for samples of high total A/S content. All scaffolds displayed satisfactory characteristics, with the lower concentrations of A/S mixture-loaded PHB fiber mats achieving higher porosity, water uptake ratios, and entrapment efficiencies. The in vitro dissolution studies revealed that all samples released more than 70% of the encapsulated drug after 72 h. All PHB scaffolds tested by cell viability assay were proven non-toxic for Hs27 fibroblasts, with the 0.15 wt.% sample favoring cell attachment, spreading onto the scaffold surface, as well as cell proliferation. Finally, the antimicrobial activity of PHB meshes loaded with A/S mixture was documented for Staphylococcus epidermidis and S. aureus.

Electrospun wound dressings containing bioactive natural products: physico-chemical characterization and biological assessment.

BACKGROUND: Current research on skin tissue engineering has been focusing on novel therapies for the effective management of chronic wounds. A critical aspect is to develop matrices that promote growth and uniform distribution of cells across the wound area, and at the same time offer protection, as well as deliver drugs that help wound healing and tissue regeneration. In this context, we aimed at developing electrospun scaffolds that could serve as carriers for the bioactive natural products alkannin and shikonin (A/S). METHODS: A series of polymeric nanofibers composed of cellulose acetate (CA) or poly(ε-caprolactone) (PCL) and varying ratios of a mixture of A/S derivatives, has been successfully fabricated and their physico-chemical and biological properties have been explored. RESULTS: Scanning electron microscopy revealed a uniform and bead-free morphology for CA scaffolds, while for PCL beads along the fibers were observed. The average diameters for all nanofibers ranged between 361 ± 47 and 487 ± 88 nm. During the assessment of physicochemical characteristics, CA fiber mats exhibited a more favored profile, while the assessment of the biological properties of the scaffolds showed that CA samples containing A/S mixture up to 1 wt.% achieved to facilitate attachment, survival and migration of Hs27 fibroblasts. With respect to the antimicrobial properties of the scaffolds, higher drug-loaded (1 and 5 wt.%) samples succeeded in inhibiting the growth of Staphylococcus epidermidis and S. aureus around the edges of the fiber mats. Finally, carrying out a structure-activity relationship study regarding the biological activities (fibroblast toxicity/proliferation and antibacterial activity) of pure A/S compounds - present in the A/S mixture - we concluded that A/S ester derivatives and the dimeric A/S augmented cell proliferation after 3 days, whereas shikonin proved to be toxic at 500 nM and 1 µM and alkannin only at 1 µM. Additionally, alkannin, shikonin and acetyl-shikonin showed more pronounced antibacterial properties than the other esters, the dimeric derivative and the A/S mixture itself. CONCLUSIONS: Taken together, these findings indicate that embedding A/S derivatives into CA nanofibers might be an advantageous drug delivery system that could also serve as a potential candidate for biomedical applications in the field of skin tissue engineering.

An effective device and method for enhanced cell growth in 3D scaffolds: Investigation of cell seeding and proliferation under static and dynamic conditions.

Cell adhesion on 3D-scaffolds is a challenging task to succeed high cell densities and even cell distribution. We aimed to design a 3D-cell Culture Device (3D-CD) for static seeding and cultivation, to be used with any kind of scaffold, limiting cell loss and facilitating nutrient supply. 3D printing technology was used for both scaffold and device fabrication. Apart from testing the device, the purpose of this study was to assess and compare static and dynamic seeding and cultivation methods, of wet and dry scaffolds, under normoxic and hypoxic conditions and their effects on parameters such as cell seeding efficiency, cell distribution and cell proliferation. Human adipose tissue was harvested and cultured in 3D-printed poly(epsilon-caprolactone) scaffolds. Micro-CT scans were performed and projection images were reconstructed into cross section images. We created 3D images to visualize cell distribution and orientation inside the scaffolds. The group of prewetted scaffolds was the most favorable to cell attachment. The 3D-cell Culture Device (3D-CD) enhanced cell seeding efficiency with almost no cell loss. We suggest that the most favorable outcome can be produced with static seeding in the device for 24 h, followed either by static cultivation in the same device or by dynamic cultivation.

Assessment of cartilage regeneration on 3D collagen-polycaprolactone scaffolds: Evaluation of growth media in static and in perfusion bioreactor dynamic culture.

Efforts on bioengineering are directed towards the construction of biocompatible scaffolds and the determination of the most favorable microenvironment, which will better support cell proliferation and differentiation. Perfusion bioreactors are attracting growing attention as an effective, modern tool in tissue engineering. A natural biomaterial extensively used in regenerative medicine with outstanding biocompatibility, biodegradability and non-toxic characteristics, is collagen, a structural protein with undisputed beneficial characteristics. This is a study designed according to the above considerations. 3D printed polycaprolactone (PCL) scaffolds with rectangular pores were coated with collagen either as a coating on the scaffold's trabeculae, or as a gel-cell solution penetrating scaffolds' pores. We employed histological, molecular and imaging techniques to analyze colonization, proliferation and chondrogenic differentiation of Adipose Derived Mesenchymal Stem Cells (ADMSCs). Two different differentiation culture media were employed to test chondrogenic differentiation on gelated and non gelated PCL scaffolds in static and in perfusion bioreactors dynamic culture conditions. In dynamic culture, non gelated scaffolds combined with our in house TGF-ß2 based medium, augmented chondrogenic differentiation performance, which overall was significantly less favorable compared to StemPro™ propriety medium. The beneficial mechanical stimulus of dynamic culture, appears to outgrow the disadvantage of the "weaker" TGF-ß2 medium used for chondrogenic differentiation. Even though cells in static culture grew well on the scaffold, there was limited penetration inside the construct, so the purpose of the 3D culture was not fully served. In contrast dynamic culture achieved better penetration and uniform distribution of the cells within the scaffold.

Hyaline cartilage next generation implants from adipose-tissue-derived mesenchymal stem cells: Comparative study on 3D-printed polycaprolactone scaffold patterns.

We used additive manufacturing to fabricate 3D-printed polycaprolactone scaffolds of different geometry topologies and porosities. We present a comparative analysis of hyaline cartilage development from adipose-tissue-derived mesenchymal stem cells (ADMSCs) on three different, newly designed scaffold geometry patterns. The first scaffold design (MESO) was based on a rectilinear layer pattern. For the second pattern (RO45), we employed a 45° rotational layer loop. The design for the third scaffold (3DHC) was a three-dimensional honeycomb-like pattern with a hexagonal cellular distribution and small square shapes. We examined cell proliferation, colonization, and differentiation, in relation to the scaffold's structure, as well as to the mechanical properties of the final constructs. We gave emphasis on the scaffolds, both microarchitecture and macroarchitecture, for optimal and enhanced chondrogenic differentiation, as an important parameter, not well studied in the literature. Among the three patterns tested, RO45 was the most favourable for chondrogenic differentiation, whereas 3DHC better supported cell proliferation and scaffold penetration, exhibiting also the highest rate of increase onto the mechanical properties of the final construct. We conclude that by choosing the optimal scaffold architecture, the resulting properties of our cartilaginous constructs can better approximate those of the physiological cartilage.