Elevated nutrient availability enhances chondrocyte metabolism and biosynthesis in tissue-engineered cartilage.

OBJECTIVE: Chondrocytes, which typically rely on anaerobic metabolism, exhibit upregulated biosynthetic activity when subjected to conditions that elicit mixed aerobic-anaerobic metabolism. Previously, we observed that increasing media volume resulted in the transition from anaerobic to mixed aerobic-anaerobic metabolism. Maximal extracellular matrix (ECM) accumulation occurred at this transition as a result of changes in hypoxia-inducible factor 1α signaling and associated hypoxic gene expression. This study aimed to explore the effect of further increases in media availability on ECM synthesis and chondrocyte metabolism. METHODS: Primary bovine chondrocytes were grown in 3D high-density tissue culture under varying levels of media availability (4-16 mL/106 cells). Changes in ECM accumulation and metabolism were determined through biochemical assays and 13C-metabolic flux analysis (13C-MFA). RESULTS: Increasing media volumes resulted in higher accumulation of cartilaginous ECM (collagen and proteoglycans) and cellularity. Extracellular metabolite measurements revealed that elevated media availability led to increased glucose and glutamine metabolism, along with increased anaerobic activity. 13C-MFA utilizing [U-13C] glucose demonstrated that increased media availability significantly impacted central carbon metabolism, upregulating all glucose-related metabolic pathways (glycolysis, lactate fermentation, the tricarboxylic acid (TCA) cycle, hexosamine biosynthetic pathway, and the malate-aspartate shuttle). Furthermore, 13C-MFA indicated that glutamine was donating carbons to the TCA cycle, and additional studies involving [U-13C] glutamine tracing supported this notion. CONCLUSIONS: Elevated media availability upregulates ECM synthesis and leads to significant changes in metabolic phenotype. Glutamine plays an important role in chondrocyte metabolism and increases in glutamine metabolism correlate with increases in ECM accumulation.

Effect of nutrient metabolism on cartilaginous tissue formation.

A major shortcoming in cartilage tissue engineering is the low biosynthetic response of chondrocytes. While different strategies have been investigated, a novel approach may be to control nutrient metabolism. Although known for their anaerobic metabolism, chondrocytes are more synthetically active under conditions that elicit mixed aerobic-anaerobic metabolism. Here, we postulate this metabolic switch induces HIF-1α signaling resulting in improved growth. Transition to different metabolic states can result in the pooling of metabolites, several of which can stabilize HIF-1α by interfering with PHD2. Chondrocytes cultured under increased media availability accelerated tissue deposition with the greatest effect occurring at 2 ml/106 cells. Under higher media availability, metabolism switched from anaerobic to mixed aerobic-anaerobic. Around this transition, maximal changes in PHD2 activity, HIF-1α expression, and HIF-1 target gene expression were observed. Loss-of-function studies using YC-1 confirmed the involvement of HIF-1. Lastly, targeted metabolomic studies revealed that intracellular lactate and succinate correlated with PHD2 activity. This study demonstrates that cartilaginous tissue formation can be regulated by nutrient metabolism and that this response is mediated through changes in HIF-1α signaling. By harnessing this newly identified metabolic switch, engineered cartilage implants may be developed without the need for sophisticated methods which could aid translation to the clinic.

Implantation of scaffold-free engineered cartilage constructs in a rabbit model for chondral resurfacing.

Joint resurfacing techniques offer an attractive treatment for damaged or diseased cartilage, as this tissue characteristically displays a limited capacity for self-repair. While tissue-engineered cartilage constructs have shown efficacy in repairing focal cartilage defects in animal models, a substantial number of cells are required to generate sufficient quantities of tissue for the repair of larger defects. In a previous study, we developed a novel approach to generate large, scaffold-free cartilaginous constructs from a small number of donor cells (20 000 cells to generate a 3-cm(2) tissue construct). As comparable thicknesses to native cartilage could be achieved, the purpose of the present study was to assess the ability of these constructs to survive implantation as well as their potential for the repair of critical-sized chondral defects in a rabbit model. Evaluated up to 6 months post-implantation, allogenic constructs survived weight bearing without a loss of implant fixation. Implanted constructs appeared to integrate near-seamlessly with the surrounding native cartilage and also to extensively remodel with increasing time in vivo. By 6 months post-implantation, constructs appeared to adopt both a stratified (zonal) appearance and a biochemical composition similar to native articular cartilage. In addition, constructs that expressed superficial zone markers displayed higher histological scores, suggesting that transcriptional prescreening of constructs prior to implantation may serve as an approach to achieve superior and/or more consistent reparative outcomes. As the results of this initial animal study were encouraging, future studies will be directed toward the repair of chondral defects in more mechanically demanding anatomical locations.

The role of mitochondrial reactive oxygen species in chondrocyte mechanotransduction.

Chondrocytes are mechanosensitive cells able to sense and respond to external mechanical stimuli through the process of mechanotransduction. Previous studies have demonstrated that mechanical stimulation causes mitochondrial deformation leading to mitochondrial reactive oxygen species (ROS) release in a dose-dependent manner. For this reason, we focused on elucidating the role of mitochondrial ROS as anabolic signaling molecules in chondrocyte mechanotransduction. Chondrocyte-seeded agarose gels were subjected to mechanical stimuli and the effect on matrix synthesis, ROS production, and mitogen-activated protein kinases (MAPK) signaling was evaluated. Through the use of ROS-specific staining, superoxide anion was the primary ROS released in response to mechanical stimuli. The anabolic effect of mechanical stimulation was abolished in the presence of electron transport chain inhibitors (complexes I, III, and V) and superoxide anion scavengers. Subsequent studies were centered on the involvement of MAPK pathways (ERK1/2, p38, and JNK) in the mechanotransduction cascade. While disruption of the ERK1/2 pathway had no apparent effect, the anabolic effect of mechanical stimulation was abolished in the presence of p38 and JNK pathway inhibitors. This suggest the involvement of apoptosis stimulating kinase 1 (ASK1), an upstream redox-sensitive MAP3K shared by both the JNK and p38 pathways. Future experiments will focus on the involvement of the thioredoxin-ASK1 complex which disassociates in the presence of oxidative stress, allowing ASK1 to phosphorylate several MAP2Ks. Overall, these findings indicate superoxide anion as the primary ROS released in response to mechanical stimuli and that the resulting anabolic effect on chondrogenic matrix biosynthesis arises from the ROS-dependent activation of the p38 and JNK MAPKs.

13C Metabolic Flux Analysis in Chondrocytes Reveals a Novel Switch in Metabolic Phenotype.

Chondrocytes are typically known for their anaerobic metabolism both in vivo and under culture conditions in vitro. However, chondrocytes have been shown to display greater biosynthetic activity when subjected to conditions that elicit aerobic metabolism. We have previously shown that tissue formation by chondrocytes can be upregulated by controlling nutrient availability and that this response arises from changes in glucose metabolism. The aim of the present study was to further characterize these changes through 13C-metabolic flux analysis (13C-MFA), as well as to determine the most optimal response. Primary bovine chondrocytes were grown in scaffold-free high-density tissue culture. [U-13C] glucose labeling experiments were combined with a tissue-specific metabolic network model to carry out 13C-MFA under varying levels of nutrient availability. 13C-MFA results demonstrated that when subjected to increasing nutrient availability, chondrocytes switch from a predominately anaerobic to a mixed aerobic-anaerobic phenotype. This metabolic switch was attributed to the saturation of the lactate fermentation pathway and metabolite overflow toward the tricarboxylic acid cycle. This effect appears to be similar to, but the inverse of, the Crabtree effect ("inverse Crabtree effect"). The relationships between metabolic flux and nutrient availability were then utilized to identify culture conditions that promote enhanced tissue formation. This novel metabolic effect presents a simple but effective approach for enhancing the biosynthetic response of chondrocytes-a key requirement to develop functional engineered cartilaginous tissue for joint resurfacing.

Expression of Chondrogenic Potential Markers in Cultured Chondrocytes from the Human Knee Joint.

OBJECTIVES: While substantial progress has been made in engineering cartilaginous constructs for animal models, further research is needed to translate these methodologies for human applications. Evidence suggests that cultured autologous chondrocytes undergo changes in phenotype and gene expression, thereby affecting their proliferation and differentiation capacity. This study was designed to evaluate the expression of chondrogenic markers in cultured human articular chondrocytes from passages 3 (P3) and 7 (P7), beyond the current clinical recommendation of P3. METHODS: Cultured autologous chondrocytes were passaged from P3 up to P7, and quantitative polymerase chain reaction (qPCR) was used to assess mRNA expression of chondrogenic markers, including collagen type I (COLI), collagen type II (COLII), aggrecan (AGG), bone morphogenetic protein 4 (BMP4), transcription factor SOX-9 (SOX9), proteoglycan 4 (PGR4), and transformation-related protein 53 (p53), between P3 and P7. RESULTS: Except for AGG, no significant differences were found in the expression of markers between passages, suggesting the maintenance of chondrogenic potential in cultured chondrocytes. Differential expression identified between SOX9 and PGR4, as well as between COLI and SOX9, indicates that differences in chondrogenic markers are present between age groups and sexes, respectively. CONCLUSIONS: Overall, expression profiles of younger and male chondrocytes exhibit conversion of mature cartilage characteristics compared to their counterparts, with signs of dedifferentiation and loss of phenotype within-group passaging. These results may have implications in guiding the use of higher passaged chondrocytes for engineering constructs and provide a foundation for clinical recommendations surrounding the repair and treatment of articular cartilage pathology in both sexes.

Passaged Articular Chondrocytes From the Superficial Zone and Deep Zone Can Regain Zone-Specific Properties After Redifferentiation.

BACKGROUND: Bioengineered cartilage is a developing therapeutic to repair cartilage defects. The matrix must be rich in collagen type II and aggrecan and mechanically competent, withstanding compressive and shearing loads. Biomechanical properties in native articular cartilage depend on the zonal architecture consisting of 3 zones: superficial, middle, and deep. The superficial zone chondrocytes produce lubricating proteoglycan-4, whereas the deep zone chondrocytes produce collagen type X, which allows for integration into the subchondral bone. Zonal and chondrogenic expression is lost after cell number expansion. Current cell-based therapies have limited capacity to regenerate the zonal structure of native cartilage. HYPOTHESIS: Both passaged superficial and deep zone chondrocytes at high density can form bioengineered cartilage that is rich in collagen type II and aggrecan; however, only passaged superficial zone-derived chondrocytes will express superficial zone-specific proteoglycan-4, and only passaged deep zone-derived chondrocytes will express deep zone-specific collagen type X. STUDY DESIGN: Controlled laboratory study. METHODS: Superficial and deep zone chondrocytes were isolated from bovine joints, and zonal subpopulations were separately expanded in 2-dimensional culture. At passage 2, superficial and deep zone chondrocytes were seeded, separately, in scaffold-free 3-dimensional culture within agarose wells and cultured in redifferentiation media. RESULTS: Monolayer expansion resulted in loss of expression for proteoglycan-4 and collagen type X in passaged superficial and deep zone chondrocytes, respectively. By passage 2, superficial and deep zone chondrocytes had similar expression for dedifferentiated molecules collagen type I and tenascin C. Redifferentiation of both superficial and deep zone chondrocytes led to the expression of collagen type II and aggrecan in both passaged chondrocyte populations. However, only redifferentiated deep zone chondrocytes expressed collagen type X, and only redifferentiated superficial zone chondrocytes expressed and secreted proteoglycan-4. Additionally, redifferentiated deep zone chondrocytes produced a thicker and more robust tissue compared with superficial zone chondrocytes. CONCLUSION: The recapitulation of the primary phenotype from passaged zonal chondrocytes introduces a novel method of functional bioengineering of cartilage that resembles the zone-specific biological properties of native cartilage. CLINICAL RELEVANCE: The recapitulation of the primary phenotype in zonal chondrocytes could be a possible method to tailor bioengineered cartilage to have zone-specific expression.

Injectable, high modulus, and fatigue resistant composite scaffold for load-bearing soft tissue regeneration.

High modulus, two-phase, bicontinuous scaffolds were prepared by photocross-linking an aqueous suspension of chondrocytes and N-methacrylate glycol chitosan with a hydrolyzable, hydrophobic, acrylated star-copolymer. Two acrylated star-copolymers were examined: poly(ε-caprolactone-co-d,l-lactide) (5446DLLACL) and poly(ε-caprolactone-co-trimethylene carbonate) (7030TMCCL). The scaffolds were assessed for injectability, two-phase interconnectivity, fatigue resistance, and long-term static culture behavior. The 7030TMCCL scaffolds demonstrated decreased moduli of 17% after 1 × 10(6) cycles at 30% strain and 5% after 56 days in culture, compared to the 5446DLLACL scaffolds, which exhibited decreases of 58 and 68%, respectively. The 7030TMCCL scaffolds accumulated more extracellular matrix after 56 days of culture (GAG: 20.1 ± 1, collagen: 35.5 ± 1.8 µg) compared to 5446DLLACL scaffolds (GAG: 13.2 ± 0.6, collagen: 6.2 ± 3.4 µg). Overall, the 7030TMCCL-based scaffolds were shown to be better suited for use as a load bearing soft tissue scaffold.

The effect of serial passaging on the proliferation and differentiation of bovine adipose-derived stem cells.

Adipose-derived stem cells (ASCs) represent an excellent cell source for the development of regenerative therapies for a broad variety of tissue disorders. Commonly, in vitro expansion is necessary to obtain sufficient cell populations for research purposes and clinical applications. Although it has been demonstrated that human ASCs can maintain their adipogenic, chondrogenic and osteogenic potential in long-term culture (up to 15 passages), it is not guaranteed that a satisfactory level of differentiation is achievable in later passages. In this study, we investigated the self-renewal and multilineage differentiation capacity of bovine ASCs, isolated from the interdigital fat pad, and explored how serial passaging influences the cells. A proliferation study examined the changes in growth kinetics from passage 1 to 5, and multilineage (adipogenesis, chondrogenesis and osteogenesis) differentiation studies were conducted to compare the potential between passage 2 (P2) and passage 5 (P5). From the proliferation study, a statistically significant change in the doubling time did not appear until P5. In the differentiation study, both P2 and P5 ASCs could be stimulated to undergo multilineage differentiation under specific culturing conditions. However, adipogenic and chondrogenic cultures showed significantly lower levels of differentiation in the P5-induced cultures. In contrast, P5-induced osteogenic cultures had higher alkaline phosphatase enzyme activity than P2-induced cultures, suggesting an increase in the osteogenic response with serial passaging. Overall, bovine ASCs are capable of self-renewal and multilineage differentiation; however, long-term in vitro expansion has a negative effect on adipogenic and chondrogenic differentiation, while potentially favoring osteogenesis.

Computer-assisted mosaic arthroplasty using patient-specific instrument guides.

PURPOSE: Success of mosaic arthroplasty requires that the transplanted plugs be positioned to reconstruct the curvature and height of the original articular surface. This case report demonstrates how to achieve correct plug positioning using patient-specific instrument guides manufactured on a 3D printer. METHODS: Using a 3D computer model of bone and cartilage, the harvesting of plugs and their placement at the defect site was planned on the computer. Instrument guides were manufactured in thermoplastic on a 3D printer; the bottom surface of the guides fit to the contour of the knee and the top surface contained holes to precisely position the surgical instruments. The instrument guides were used on a young female patient to repair a large articular cartilage defect in the left knee. RESULTS: The patient showed an increased range of motion in the knee and also a decrease in pain and discomfort at her 2-year follow-up. A CT arthrogram at 2 years postoperative showed a smooth and appropriate contour of the reconstructed cartilage over the defect. CONCLUSIONS: Image-based preoperative planning and the use of patient-specific instrument guides can yield a good patient outcome without requiring optically tracked intraoperative guidance.

Cell Cycle Synchronization of Primary and Cultured Articular Chondrocytes.

Cell cycle synchronization allows cells in a culture, originally at different stages of the cell cycle, to be brought to the same phase. It is normally performed by applying cell cycle arresting chemical agents to cells cultured in monolayer. While effective, isolated chondrocytes tend to dedifferentiate when cultured in monolayer and typically require 3D culturing methods to ensure phenotypic stability. Here, we describe both the conventional cell cycle synchronization method for cells in monolayer culture and an adapted method of synchronizing primary chondrocytes directly during the cell isolation process to limit potential dedifferentiation. Different methods including serum-starvation and treatment with thymidine, nocodazole, aphidicolin, and RO-3306 can synchronize the chondrocytes at different discrete phases. A cell purity of more than 90% in the S phase can be achieved with simultaneous cell isolation and synchronization using double thymidine treatment, generating a population of synchronized chondrocytes that show increased matrix synthesis when subsequently cultured in 3D.

Lithium chloride-induced primary cilia recovery enhances biosynthetic response of chondrocytes to mechanical stimulation.

Mechanical stimulation is commonly used in cartilage tissue engineering for enhancing tissue formation and improving the mechanical properties of resulting engineered tissues. However, expanded chondrocytes tend to dedifferentiate and lose expression of their primary cilia, which is necessary for chondrocyte mechanotransduction. As treatment with lithium chloride (LiCl) can restore passaged chondrocytes in monolayer, in this study, we investigated whether this approach would be effective in 3D culture and restore chondrocyte mechanosensitivity. Chondrocytes at different passages (P0 to P2) were treated with 0-50 mM LiCl for 24 h, with different pre-culture durations (0 to 4 days). The primary cilia incidence and length were measured in α-tubulin-stained images. Treated chondrocytes were cultured with or without dynamic compression to evaluate the effect of LiCl-induced primary cilia expression on matrix synthesis by mechanically stimulated chondrocytes. LiCl treatment of chondrocytes in 3D agarose culture increased primary cilia incidence and length, with significant increases in incidence and length using 50 mM LiCl compared to other concentrations (P < 0.05). This effect was further optimized by including a 4-day pre-culture prior to the 24-h 50 mM LiCl treatment. Importantly, LiCl-induced primary cilia expression increased chondrocyte mechanosensitivity. When stimulated with dynamic compression, LiCl-treated P1 chondrocytes increased collagen (1.4-fold, P < 0.1) and proteoglycan (1.5-fold, P < 0.05) synthesis compared to untreated, unstimulated cells. The LiCl treatment method described here can be used to restore primary cilia in passaged chondrocytes, transforming them into a mechanosensitive cell source for cartilage tissue engineering.

TRPV4 activation enhances compressive properties and glycosaminoglycan deposition of equine neocartilage sheets.

Objective: To evaluate the effect of Transient Receptor Potential Vanilloid 4 (TRPV4) cation channel modulation on mesenchymal stromal cell (MSC)-derived neocartilage. Methods: RT-PCR was performed to evaluate mRNA levels of chondrogenic, hypertrophic and candidate mechanoresponsive genes in equine neocartilage sheets exposed to pulses of the TRPV4 agonist (GSK101) at different concentrations (N â€‹= â€‹10). Biochemical assays and mechanical tests (double indentation and unconfined compression) evaluated neocartilage properties (N â€‹= â€‹5). Results: GSK101 treatment (1 â€‹nM) increased ACAN levels after treatment for 1-h per day for 3 days. No increase was detected for hypertrophic markers RUNX2, MMP13, MMP1, ALP or COL10A1 at this concentration. This treatment regimen also increased sGAG content and enhanced compressive properties compared to untreated controls. GSK101 showed no effect on candidate mechanoresponsive genes at the time-point of analysis. Conclusions: Chemical activation of TRPV4 signalling can be used as a strategy to enhance matrix synthesis and maturation of MSC-derived engineered neocartilage and augment its load-bearing capacity.

Cell Cycle Synchronization of Primary Articular Chondrocytes Enhances Chondrogenesis.

OBJECTIVE: Although tissue engineering is a promising option for articular cartilage repair, it has been challenging to generate functional cartilaginous tissue. While the synthetic response of chondrocytes can be influenced by various means, most approaches treat chondrocytes as a homogeneous population that would respond similarly. However, isolated cells heterogeneously progress through the cell cycle, which can affect macromolecular biosynthesis. As it is possible to synchronize cells within discrete cell cycle phases, the purpose of this study was to investigate the effects of cell cycle synchronization on the chondrogenic potential of primary articular chondrocytes. DESIGN: Different methods of cell synchronization (serum starvation, thymidine, nocodazole, aphidicolin, and RO-3306) were tested for their ability to synchronize primary articular chondrocytes during the process of cell isolation. Cells (unsynchronized and synchronized) were then encapsulated in alginate gels, cultured for 4 weeks, and analyzed for their structural and biochemical properties. RESULTS: The double-thymidine method yielded the highest level of cell purity, with cells synchronized in S phase. While the cells started to lose synchronization after 24 hours, tissue constructs developed from initially S phase synchronized cells had significantly higher glycosaminoglycan and collagen II amounts than those developed using unsynchronized cells. CONCLUSIONS: Initial synchronization led to long-term changes in cartilaginous tissue formation. This effect was postulated to be due to the rapid auto-induction of TGF-ßs by actively dividing S phase cells, thereby stimulating chondrogenesis. Cell synchronization methods may also be applied in conjunction with redifferentiation methods to improve the chondrogenic potential of dedifferentiated or diseased chondrocytes.

In vitro evaluation of novel titania-containing borate bioactive glass scaffolds.

Titanium-containing borate bioactive glass scaffolds (0, 5, 15, and 20 mol %, identified as BRT0, BRT1, BRT3, and BRT4) with a microstructure similar to that of human trabecular bone were prepared and evaluated in vitro for potential bone loss applications in revision total knee arthroplasty (rTKA). Methyl thiazolyl tetrazolium (MTT) cell viability assays of scaffold ion release extracts revealed that BRT0 scaffolds (0 mol % titanium) inhibited cell proliferation and activity at day 14. At day 30, all scaffold extracts decreased cell proliferation and activity significantly. However, live/dead cell assay results demonstrated that degradation products from all the scaffolds had no inhibitory effect on cell viability. Significant bactericidal efficacies of BRT3 extracts against Escherishia coli (Gram-negative) and BRT1 extracts against Staphylococcus aureus and Staphylococcus epidermidis (both Gram-positive bacteria) were demonstrated. Finally, evaluation of the cell/bioactive glass surface interactions showed well-spread cells on the surface of the BRT3 glass discs and BRT1 and BRT3 scaffolds, when compared to BRT0 and BRT4 scaffolds. The results indicate that by changing the Ti4+ :B3+ ratio, the ion release and consequently cell proliferation could be improved. in vitro results in this study demonstrate that BRT3 scaffolds could be a promising candidate for addressing bone loss in rTKAs; however, in vivo studies would be required to evaluate the effect of a dynamic environment on the cell and tissue response to the fabricated scaffolds.

Characterization of Mechanical and Dielectric Properties of Silicone Rubber.

Silicone rubber's silicone-oxygen backbones give unique material properties which are applicable in various biomedical devices. Due to the diversity of potential silicone rubber compositions, the material properties can vary widely. This paper characterizes the dielectric and mechanical properties of two different silicone rubbers, each with a different cure system, and in combination with silicone additives. A tactile mutator (Slacker™) and/or silicone thickener (Thi-vex™) were mixed with platinum-cured and condensation-cured silicone rubber in various concentrations. The dielectric constants, conductivities, and compressive and shear moduli were measured for each sample. Our study contributes novel information about the dielectric and mechanical properties of these two types of silicone rubber and how they change with the addition of two common silicone additives.

Comparative Evaluation of Two Glass Polyalkenoate Cements: An In Vivo Pilot Study Using a Sheep Model.

Poly(methyl methacrylate) (PMMA) is used to manage bone loss in revision total knee arthroplasty (rTKA). However, the application of PMMA has been associated with complications such as volumetric shrinkage, necrosis, wear debris, and loosening. Glass polyalkenoate cements (GPCs) have potential bone cementation applications. Unlike PMMA, GPC does not undergo volumetric shrinkage, adheres chemically to bone, and does not undergo an exothermic setting reaction. In this study, two different compositions of GPCs (GPCA and GPCB), based on the patented glass system SiO2-CaO-SrO-P2O5-Ta2O5, were investigated. Working and setting times, pH, ion release, compressive strength, and cytotoxicity of each composition were assessed, and based on the results of these tests, three sets of samples from GPCA were implanted into the distal femur and proximal tibia of three sheep (alongside PMMA as control). Clinical CT scans and micro-CT images obtained at 0, 6, and 12 weeks revealed the varied radiological responses of sheep bone to GPCA. One GPCA sample (implanted in the sheep for 12 weeks) was characterized with no bone resorption. Furthermore, a continuous bone-cement interface was observed in the CT images of this sample. The other implanted GPCA showed a thin radiolucent border at six weeks, indicating some bone resorption occurred. The third sample showed extensive bone resorption at both six and 12 weeks. Possible speculative factors that might be involved in the varied response can be: excessive Zn2+ ion release, low pH, mixing variability, and difficulty in inserting the samples into different parts of the sheep bone.

Tantalum-containing mesoporous bioactive glass powder for hemostasis.

This study evaluates the hemostatic properties of tantalum-containing mesoporous bioactive glasses (Ta-MBGs) through a suite of in-vitro methods: hemolysis percentage, zeta potential, blood coagulation assays (Activated Partial Thromboplastin Time - APTT and Prothrombin Time - PT) and cytotoxicity tests. Five compositions of Ta-MBG, with x mol% Ta2O5 added to the glass series (80-x)SiO2-15CaO-5P2O5-xTa2O5 where x=0 (0Ta), x=0.5 (0.5Ta), x=1 (1Ta), x=5 (5Ta), and x=10 (10Ta) mol%, were synthesised. The hemostatic potential of all the Ta-MBGs was confirmed by their negative zeta potential (-23 to -31 mV), which enhances the intrinsic pathway of blood coagulation. The hemolysis percentages of all Ta-MBGs except 10Ta showed statistically significant reductions compared to the same experiments carried out both in the absence of a sample ('no treatment' group) and in the presence of 10Ta. These observations validate the consideration of Ta-MBGs as hemostatic agents as they do not cause significant lysis of red blood cells. Cytotoxicity analysis revealed that Ta-MBGs had no effect on bovine fibroblast viability. Furthermore, a reduction in both APTT (a test to evaluate the intrinsic pathway of coagulation) and PT (a test to evaluate the extrinsic pathway) signified enhancement of hemostasis: 5Ta caused a significant reduction in APTT compared to 'no treatment', 1Ta and 10Ta and a significant reduction in PT compared to 0Ta. Therefore, we conclude that 5mol% of Ta optimised the hemostatic properties of these mesoporous bioactive glasses.

Human-engineered auricular reconstruction (hEAR) by 3D-printed molding with human-derived auricular and costal chondrocytes and adipose-derived mesenchymal stem cells.

Microtia is a small, malformed external ear, which occurs at an incidence of 1-10 per 10 000 births. Autologous reconstruction using costal cartilage is the most widely accepted surgical microtia repair technique. Yet, the method involves donor-site pain and discomfort and relies on the artistic skill of the surgeon to create an aesthetic ear. This study employed novel tissue engineering techniques to overcome these limitations by developing a clinical-grade, 3D-printed biodegradable auricle scaffold that formed stable, custom-made neocartilage implants. The unique scaffold design combined strategically reinforced areas to maintain the complex topography of the outer ear and micropores to allow cell adhesion for the effective production of stable cartilage. The auricle construct was computed tomography (CT) scan-based composed of a 3D-printed clinical-grade polycaprolactone scaffold loaded with patient-derived chondrocytes produced from either auricular cartilage or costal cartilage biopsies combined with adipose-derived mesenchymal stem cells. Cartilage formation was measured within the constructin vitro, and cartilage maturation and stabilization were observed 12 weeks after its subcutaneous implantation into a murine model. The proposed technology is simple and effective and is expected to improve aesthetic outcomes and reduce patient discomfort.

Self-crimping, biodegradable, electrospun polymer microfibers.

Semicrystalline poly(l-lactide-co-ε-caprolactone) (P(LLA-CL)) was used to produce electrospun fibers with diameters on the subcellular scale. P(LLA-CL) was chosen because it is biocompatible and its chemical and physical properties are easily tunable. The use of a rotating wire mandrel as a collection device in the electrospinning process, along with high collection speeds, was used to align electrospun fibers. Upon removal of the fibers from the mandrel, the fibers shrunk in length, producing a crimp pattern characteristic of collagen fibrils in soft connective tissues. The crimping effect was determined to be a result of the residual stresses resident in the fibers due to the fiber alignment process and the difference between the operating temperature (T(op)) and the glass-transition temperature (T(g)) of the polymer. The electrospun fibers could be induced to crimp by adjusting the operating temperature to be greater than that of the polymer glass-transition temperature. Moreover, the crimped fibers exhibited a toe region in their stress-strain profile that is characteristic of collagen present in tendons and ligaments. The crimp pattern was retained during in vitro degradation over 4 weeks. Primary bovine fibroblasts seeded onto these crimped fibers attached, proliferated, and deposited extracellular matrix (ECM) molecules on the surface of the fiber mats. These self-crimping fibers hold great promise for use in tissue engineering scaffolds for connective tissues that require fibers similar in structure to that of crimped collagen fibrils.