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Blue light-mediated photobiomodulation (PBM) is a promising approach to promote osteogenesis. However, the underlying mechanisms of PBM in osteogenesis are poorly understood. In this study, a human osteosarcoma cell line (i.e., Saos-2 cells) was subjected to intermittent blue light exposure (2500 µM/m2/s, 70 mW/cm2, 4.2 J/cm2, once every 48 h) and the effects on Saos-2 cell viability, metabolic activity, differentiation, and mineralization were investigated. In addition, this study addressed a possible role of blue light induced cellular oxidative stress as a mechanism for enhanced osteoblast differentiation and mineralization. Results showed that Saos-2 cell viability and metabolic activity were maintained upon blue light exposure compared to unilluminated controls, indicating no negative effects. To the contrary, blue light exposure significantly increased (p < 0.05) alkaline phosphatase activity and Saos-2 cell mediated mineralization. High-performance liquid chromatography (HPLC) assay was used for measurement of reactive oxygen species (ROS) activity and showed a significant increase (p < 0.05) in superoxide (O2â¢-) and hydrogen peroxide (H2O2) formed after blue light exposure. Together, these results suggest that the beneficial effects of blue light-mediated PBM on osteogenesis may be induced by controlled release of ROS.
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Terapia por Luz de Baja Intensidad , Osteogénesis , Humanos , Especies Reactivas de Oxígeno/metabolismo , Terapia por Luz de Baja Intensidad/métodos , Peróxido de Hidrógeno/farmacología , Proliferación Celular , Diferenciación CelularRESUMEN
Collagen methacrylation is a promising approach to generate photo-cross-linkable cell-laden hydrogels with improved mechanical properties. However, the impact of species-based variations in amino acid composition and collagen isolation method on methacrylation degree (MD) and its subsequent effects on the physical properties of methacrylated collagen (CMA) hydrogels and cell response are unknown. Herein, we compared the effects of three collagen species (bovine, human, and rat), two collagen extraction methods (pepsin digestion and acid extraction), and two photoinitiators (lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and Irgacure-2959 (I-2959)) on the physical properties of CMA hydrogels, printability and mesenchymal stem cell (MSC) response. Human collagen showed the highest MD. LAP was more cytocompatible than I-2959. The compressive modulus and cell viability of rat CMA were significantly higher (p < 0.05) than bovine CMA. Human CMA yielded constructs with superior print fidelity. Together, these results suggest that careful selection of collagen source and cross-linking conditions is essential for biomimetic design of CMA hydrogels for tissue engineering applications.
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Hidrogeles , Células Madre Mesenquimatosas , Bovinos , Animales , Humanos , Ratas , Hidrogeles/química , Colágeno/química , Ingeniería de Tejidos/métodos , Supervivencia CelularRESUMEN
A novel biofabrication modality, electrophoretic compaction with macromolecular alignment, was utilized to make collagen threads that mimic the native tendon's structure and mechanical properties. A device with kinematic electrodes was designed to fabricate collagen threads in continuous length. For the first time, a 3D-biotextile was woven purely from collagen. Mechanical properties and load-displacement behavior of the biotextile mimicked those of the native tendon while presenting a porosity of 80%. The open pore network facilitated cell seeding across the continuum of the bioscaffold. Mesenchymal stem cells (MSCs) seeded in the woven scaffold underwent tenogenic differentiation in the absence of growth factors and synthesized a matrix that was positive for tenomodulin, COMP and type I collagen. Up-regulation of tenomodulin, a tendon specific marker, was 11.6 ± 3.5 fold, COMP was up-regulated 16.7 ± 5.5 fold, and Col I was up-regulated 6.9 ± 2.7 fold greater on ELAC threads when compared to randomly oriented collagen gels. These results demonstrate that a bioscaffold woven by using collagen threads with densely compacted and anisotropically aligned substrate texture stimulates tenogenesis topographically, rendering the electrochemically aligned collagen as a promising candidate for functional repair of tendons and ligaments.
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Anisotropically aligned collagen scaffolds mimic the microarchitectural properties of native tissue, possess superior mechanical properties, and provide the essential physicochemical cues to guide cell response. Biofabrication methodologies to align collagen fibers include mechanical, electrical, magnetic, and microfluidic approaches. Magnetic alignment of collagen was first published in 1983 but widespread use of this technique was hindered mainly due to the low diamagnetism of collagen molecules and the need for very strong tesla-order magnetic fields. Over the last decade, there is a renewed interest in the use of magnetic approaches that employ magnetic particles and low-level magnetic fields to align collagen fibers. In this review, the working principle, advantages, and limitations of different collagen alignment techniques with special emphasis on the magnetic alignment approach are detailed. Key findings from studies that employ high-strength magnetic fields and the magnetic particle-based approach to align collagen fibers are highlighted. In addition, the most common qualitative and quantitative image analyses methods to assess collagen alignment are discussed. Finally, current challenges and future directions are presented for further development and clinical translation of magnetically aligned collagen scaffolds.
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Biomimetic scaffolds provide the essential biophysical (e.g., surface topography, stiffness) and biochemical cues (e.g., composition) to guide cell morphology, proliferation, and differentiation. Although the effects of biomaterial-directed cues on cell response have been widely reported, few studies have sought to decouple these effects to better understand the interplay between the different physicochemical factors on tissue-specific cell function. Herein, beta-tricalcium phosphate (ß-TCP) was incorporated into electrochemically aligned collagen (ELAC) and random collagen threads, and the individual and interactive effects of collagen alignment (i.e., biophysical) and bioceramic incorporation (i.e., biochemical) on osteoblast cell morphology, proliferation, differentiation, and mineralization were investigated. Results showed that collagen alignment in ELAC threads was retained upon ß-TCP incorporation. Collagen alignment significantly improved (p < 0.05) the swelling capacity and stability of collagen threads, while ß-TCP incorporation showed no such effects. Tensile tests revealed that ß-TCP incorporation significantly decreased (p < 0.05) the strength and stiffness of ELAC threads. Significant increase (p < 0.05) in Saos-2 cell orientation and alkaline phosphatase (ALP) activity was observed on ELAC compared to random collagen threads indicating that aligned collagen serves as a key driving factor for osteogenesis. ß-TCP incorporation into random collagen threads had no effect on Saos-2 cell function. On the other hand, presence of ß-TCP significantly augmented (p < 0.05) Saos-2 cell metabolic activity, differentiation, and mineralization on ELAC threads. Together, these findings suggest that combining collagen alignment and ß-TCP incorporation can create robust tissue-mimicking scaffolds for bone regeneration applications.
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Collagen-based scaffolds have been widely used in tissue engineering. The alignment of collagen fibers and the degree of crosslinking in engineering tissue scaffolds significantly affect cell activity and scaffold stability. Changes in microarchitecture and crosslinking degree also impact the mechanical properties of collagen scaffolds. A clear understanding of the effects of collagen alignment and crosslinking degrees can help properly control these critical parameters for fabricating collagen scaffolds with desired mechanical properties. In this study, combined uniaxial mechanical testing and finite element method (FEM) were used to quantify the effects of fiber alignment and crosslinking degree on the mechanical properties of collagen threads. We have fabricated electrochemically aligned collagen (ELAC) and compared it with randomly distributed collagen at varying crosslinking degrees, which depend on genipin concentrations of 0.1% or 2% for crosslinking durations of 1, 4, and 24 h. Our results indicate that aligned collagen fibers and higher crosslinking degree contribute to a larger Young's modulus. Specifically, aligned fiber structure, compared to random collagen, significantly increases Young's modulus by 112.7% at a 25% crosslinking degree (0.1% (4 h), i.e., 0.1% genipin concentration with a crosslinking duration of 4 h). Moreover, the ELAC Young's modulus increased by 90.3% as the crosslinking degree doubled by changing the genipin concentration from 0.1% to 2% with the same 4 h crosslinking duration. Furthermore, verified computational models can predict mechanical properties based on specific crosslinking degrees and fiber alignments, which facilitate the controlled fabrication of collagen threads. This combined experimental and computational approach provides a systematic understanding of the interplay among fiber alignment, crosslinking parameters, and mechanical performance of collagen scaffolds. This work will enable the precise fabrication of collagen threads for desired tissue engineering performance, potentially advancing tissue engineering applications.
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Biomimetic scaffolds composed of bioactive ceramic-based materials incorporated within a polymeric framework have shown immense promise for use in bone tissue engineering (BTE) applications. However, studies on direct comparison of the efficacy of different bioceramics on bone bioactivity and osteogenic differentiation are lacking. Herein, we performed an in vitro direct comparison of three different bioceramics-Bioglass 45S5 (BG), Laponite XLG (LAP), and ß-Tricalcium Phosphate (TCP)-on the physical properties and bone bioactivity of methacrylated collagen (CMA) hydrogels (10% w/w bioceramic:CMA). In addition, human MSCs (hMSCs) were encapsulated in bioceramic-laden CMA hydrogels and the effect of different bioceramics on osteogenic differentiation of hMSCs was investigated in two different culture medium-osteoconductive (without dexamethasone [DEX]) and osteoinductive (with DEX). Results showed that the stability of CMA hydrogels was maintained upon bioceramic addition. Compression testing revealed that BG incorporation significantly decreased (p < 0.05) the modulus of photochemically crosslinked CMA hydrogels. Incubation of TCP-CMA and LAP-CMA hydrogels in simulated body fluid showed deposition of hydroxycarbonate apatite layer on the surface indicating that these hydrogels may be more bone bioactive than BG-CMA and CMA only hydrogels. Cell cytoskeleton staining results showed greater cell spreading in TCP-CMA hydrogels. Furthermore, TCP incorporation significantly increased alkaline phosphatase activity (ALP; p < 0.05) in hMSCs. Together, these results indicate that TCP has superior osteogenic potential compared with BG and LAP and hence should be considered as a bioceramic of preferred choice for use in the biomimetic design of cell-laden hydrogels for BTE applications.
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Hidrogeles , Osteogénesis , Humanos , Hidrogeles/farmacología , Biomimética , Colágeno/farmacologíaRESUMEN
Collagen, along with proteoglycans, glycosaminoglycans, glycoproteins, and various growth factors, forms the extracellular matrix (ECM) and contributes to the complexity and diversity of different tissues. Herein, we compared the physicochemical and biological properties of ECM hydrogels derived from four different human tissues: skin, bone, fat, and birth. Pure human collagen type I hydrogels were used as control. Physical characterization of ECM hydrogels and assessment of cell response of cord-tissue mesenchymal stem cells (CMSCs) were performed. Decellularization efficiency was found to be >90% for all ECM. Hydroxyproline quantification assay showed that collagen content in birth ECM was comparable to collagen control and significantly greater than other sources of ECM. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the presence of γ, ß, α1 and α2 collagen chains in all ECMs. Gelation kinetics of ECM hydrogels was significantly slower than collagen control. Compressive modulus of skin ECM was the highest and birth ECM was the lowest. Skin and birth ECM hydrogels were more stable than bone and fat ECM hydrogels. CMSCs encapsulated in birth ECM hydrogels exhibited the highest metabolic activity. Rheological characterization revealed that all ECM-derived inks exhibited shear thinning properties, and skin-derived ECM inks were most suitable for extrusion-based bioprinting for the concentration and printing conditions used in this study. Overall, results demonstrate that the physicochemical and biological properties of ECM hydrogels vary significantly depending on the tissue source. Therefore, careful selection of tissue source is important for development of ECM-based biomimetic tissue constructs for regenerative medicine applications.
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Astrocytes, highly specialized glial cells, play a critical role in neuronal function. Variations in brain extracellular matrix (ECM) during development and disease can significantly alter astrocyte cell function. Age-related changes in ECM properties have been linked to neurodegenerative diseases such as Alzheimer's disease. The goal of this study was to develop hydrogel-based biomimetic ECM models with varying stiffness and evaluate the effects of ECM composition and stiffness on astrocyte cell response. Xeno-free ECM models were synthesized by combining varying ratios of human collagen and thiolated hyaluronic acid (HA) crosslinked with polyethylene glycol diacrylate. Results showed that modulating ECM composition yielded hydrogels with varying stiffnesses that match the stiffness of the native brain ECM. Collagen-rich hydrogels swell more and exhibit greater stability. Higher metabolic activity and greater cell spreading was observed in hydrogels with lower HA. Soft hydrogels trigger astrocyte activation indicated by greater cell spreading, high GFAP expression and low ALDH1L1 expression. This work presents a baseline ECM model to investigate the synergistic effects of ECM composition and stiffness on astrocytes, which could be further developed to identify key ECM biomarkers and formulate new therapies to alleviate the impact of ECM changes on the onset and progression of neurodegenerative diseases.
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BACKGROUND: Bone marrow plays a key role in bone formation and healing. Although a subset of marrow explants ossifies in vitro without excipient osteoinductive factors, some explants do not undergo ossification. The disparity of outcome suggests a significant heterogeneity in marrow tissue in terms of its capacity to undergo osteogenesis. QUESTIONS/PURPOSES: We sought to identify: (1) proteins and signaling pathways associated with osteogenesis by contrasting the proteomes of ossified and poorly ossified marrow explants; and (2) temporal changes in proteome and signaling pathways of marrow ossification in the early and late phases of bone formation. METHODS: Explants of marrow were cultured. Media conditioned by ossified (n = 4) and poorly ossified (n = 4) subsets were collected and proteins unique to each group were identified by proteomic analysis. Proteomic data were processed to assess proteins specific to the early phase (Days 1-14) and late phase (Days 15-28) of the culture period. Pathways involved in bone marrow ossification were identified through bioinformatics. RESULTS: Twenty-eight proteins were unique to ossified samples and eight were unique to poorly ossified ones. Twelve proteins were expressed during the early phase and 15 proteins were specific to the late phase. Several identified pathways corroborated those reported for bone formation in the literature. Immune and inflammatory pathways were specific to ossified samples. CONCLUSIONS: The marrow explant model indicates the inflammatory and immune pathways to be an integral part of the osteogenesis process.
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Médula Ósea/inmunología , Médula Ósea/patología , Mediadores de Inflamación/metabolismo , Osificación Heterotópica , Osteogénesis , Transducción de Señal , Animales , Médula Ósea/diagnóstico por imagen , Biología Computacional , Medios de Cultivo Condicionados/metabolismo , Proteómica/métodos , Ratas , Ratas Long-Evans , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Microtomografía por Rayos XRESUMEN
A rupture of the anterior cruciate ligament (ACL) is one of the most common knee ligament injuries affecting the young and active population. Tissue engineering strategies to reconstruct the damaged ACL have met with significant challenges mainly associated with poor graft integration at the bone-ligament interface (i.e., enthesis). In this study, a "design-build-validate" strategy was employed by combining 3D Raman spectral mapping and 3D printing to develop a tissue engineered scaffold that is compositionally similar to the ACL bone-ligament interface and can provide the essential biochemical cues to promote interface regeneration and facilitate functional graft to bone integration. Results showed that Raman spectroscopy is a highly efficient nondestructive technique to determine the biochemical composition of native ACL enthesis. 3D printing using combinatory inks consisting of different compositions of methacrylated collagen (CMA) and Bioglass (BG) allowed for the fabrication of BG gradient-incorporated collagen matrices (BioGIMs) with a transition region confirmed by Alizarin red S staining. Furthermore, Raman spectroscopy validated replication of ACL enthesis composition in BioGIMs. In addition, human mesenchymal stem cells (hMSCs) cultured on BioGIMs showed morphological differences along the length of the BioGIMs as evidenced by confocal microscopy of cell cytoskeleton-stained images indicating that the cells can sense the underlying differences in matrix composition. Overall, the "design-build-validate" strategy developed in this study has significant potential to generate biomimetic tissue constructs for use at the interface regions of synthetic grafts to promote better host integration and achieve full reconstruction of the ACL. Impact statement Poor graft integration at the bone-ligament interface (i.e., enthesis) is a significant clinical problem in anterior cruciate ligament (ACL) repair and reconstruction. In this study, Raman spectroscopy and 3D printing technologies were used in combination for the first time in a design-build-validate strategy to develop a continuous biomimetic Bioglass gradient-incorporated collagen matrix (BioGIM) that compositionally emulates the native ACL enthesis. These BioGIMs can be fused onto the ends of synthetic ACL grafts and have significant potential to provide the essential biochemical cues to guide tissue-specific cell differentiation, augment functional matrix reorganization, promote better graft integration, and achieve full reconstruction of damaged ACL.
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Reconstrucción del Ligamento Cruzado Anterior , Ligamento Cruzado Anterior , Ligamento Cruzado Anterior/cirugía , Reconstrucción del Ligamento Cruzado Anterior/métodos , Cerámica , Colágeno/química , HumanosRESUMEN
Freeform reversible embedding of suspended hydrogels (FRESH) is a layer-by-layer extrusion-based technique to enable three-dimensional (3D) printing of soft tissue constructs by using a thermo-reversible gelatin support bath. Suboptimal resolution of extrusion-based printing limits its use for the creation of microscopic features in the 3D construct. These microscopic features (e.g., pore size) are known to have a profound effect on cell migration, cell-cell interaction, proliferation, and differentiation. In a recent study, FRESH-based 3D printing was combined with freeze-casting in the Freeze-FRESH (FF) method, which yielded alginate constructs with hierarchical porosity. However, use of the FF approach allowed little control of micropore size in the printed alginate constructs. Herein, the FF methodology was optimized for 3D printing of collagen constructs with greater control of microporosity. Modifications to the FF method entailed melting of the FRESH bath before freezing to allow more efficient heat transport, achieve greater control on microporosity, and permit polymerization of collagen molecules to enable 3D printing of stable microporous collagen constructs. The effects of different freezing temperatures on microporosity and physical properties of the 3D-printed collagen constructs were assessed. In addition, finite element (FE) models were generated to predict the mechanical properties of the microporous constructs. Further, the impact of different micropore sizes on cellular response was evaluated. Results showed that the microporosity of 3D-printed collagen constructs can be tailored by customizing the FF approach. Compressive modulus of microporous constructs was significantly lower than the non-porous control, and the FE model verified these findings. Constructs with larger micropore size were more stable and showed significantly greater cell infiltration and metabolic activity. Together, these results suggest that the FF method can be customized to guide the design of 3D-printed microporous collagen constructs.
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Hydrogels such as alginate and gelatin have shown potential as biomaterials in various three-dimensional (3D) bioprinting applications. However, parameters such as viscosity, porosity, and printability influence the performance of hydrogel-based biomaterials, and there are limited characterization studies conducted on the behavior of these constructs. In this work, a syringe-based extrusion bioprinter was used to print 3D constructs with bioink composed of various concentrations of alginate and gelatin along with fibrinogen and human umbilical vein endothelial cells. Instead of crosslinking the gelatin, the gelatin was left uncrosslinked to provide microporosity within the system that can impact the cellular response. Mechanical and biochemical characterization was performed to evaluate the structural stability and integrity of the printed constructs along with viability of embedded cells. Bioprinted constructs of a higher total concentration of alginate and gelatin yielded better stability and structural integrity after culture. More importantly, higher amounts of gelatin (i.e., 1:9 instead of 2:3 alginate:gelatin) were shown to improve printability, which is different than most studies that instead use alginate to improve printability. In addition, higher amounts of gelatin impacted the changes in surface morphological features of the constructs after incubation, and ultimately improved biocompatibility with our system. Overall, this study demonstrated that an uncrosslinked gelatin system can provide flexible printing parameters and surface morphologies, but careful control over the printing parameters may be required. The bioink concentration of 10% (w/v) with minimum alginate and higher gelatin concentration exhibited the best printability, cell survival, and viability.
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Bioimpresión , Andamios del Tejido , Alginatos/química , Bioimpresión/métodos , Gelatina/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Hidrogeles/química , Impresión Tridimensional , Ingeniería de Tejidos/métodos , Andamios del Tejido/químicaRESUMEN
Xenogeneic sources of collagen type I remain a common choice for regenerative medicine applications due to ease of availability. Human and animal sources have some similarities, but small variations in amino acid composition can influence the physical properties of collagen, cellular response, and tissue remodeling. The goal of this work is to compare human collagen type I-based hydrogels versus animal-derived collagen type I-based hydrogels, generated from commercially available products, for their physico-chemical properties and for tissue engineering and regenerative medicine applications. Specifically, we evaluated whether the native human skin type I collagen could be used in the three most common research applications of this protein: as a substrate for attachment and proliferation of conventional 2D cell culture; as a source of matrix for a 3D cell culture; and as a source of matrix for tissue engineering. Results showed that species and tissue specific variations of collagen sources significantly impact the physical, chemical, and biological properties of collagen hydrogels including gelation kinetics, swelling ratio, collagen fiber morphology, compressive modulus, stability, and metabolic activity of hMSCs. Tumor constructs formulated with human skin collagen showed a differential response to chemotherapy agents compared to rat tail collagen. Human skin collagen performed comparably to rat tail collagen and enabled assembly of perfused human vessels in vivo. Despite differences in collagen manufacturing methods and supplied forms, the results suggest that commercially available human collagen can be used in lieu of xenogeneic sources to create functional scaffolds, but not all sources of human collagen behave similarly. These factors must be considered in the development of 3D tissues for drug screening and regenerative medicine applications.
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Colágeno Tipo I , Ingeniería de Tejidos , Animales , Colágeno/química , Colágeno/farmacología , Colágeno Tipo I/química , Colágeno Tipo I/farmacología , Matriz Extracelular/química , Humanos , Hidrogeles/química , Ratas , Ingeniería de Tejidos/métodos , Andamios del Tejido/químicaRESUMEN
In vitro models of osteogenesis are essential for investigating bone biology and the effects of pharmaceutical, chemical, and physical cues on bone formation. Osteogenesis takes place in a complex three-dimensional (3D) environment with cells from both mesenchymal and hematopoietic origins. Existing in vitro models of osteogenesis include two-dimensional (2D) single type cell monolayers and 3D cultures. However, an in vitro scaffold-free multicellular 3D model of osteogenesis is missing. We hypothesized that the self-inductive ossification capacity of bone marrow tissue can be harnessed in vitro and employed as a scaffold-free multicellular 3D model of osteogenesis. Therefore, rat bone marrow tissue was cultured for 28 days in three settings: 2D monolayer, 3D homogenized pellet, and 3D organotypic explant. The ossification potential of marrow in each condition was quantified by micro-computed tomography. The 3D organotypic marrow explant culture resulted in the greatest level of ossification with plate-like bone formations (up to 5 mm in diameter and 0.24 mm in thickness). To evaluate the mimicry of the organotypic marrow explants to newly forming native bone tissue, detailed compositional and morphological analyses were performed, including characterization of the ossified matrix by histochemistry, immunohistochemistry, Raman microspectroscopy, energy dispersive X-ray spectroscopy, backscattered electron microscopy, and micromechanical tests. The results indicated that the 3D organotypic marrow explant culture model mimics newly forming native bone tissue in terms of the characteristics studied. Therefore, this platform holds significant potential to be used as a model of osteogenesis, offering an alternative to in vitro monolayer cultures and in vivo animal models.
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Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Modelos Biológicos , Osteogénesis/fisiología , Animales , Células de la Médula Ósea/ultraestructura , Técnicas de Cultivo de Célula , Células Cultivadas , Citometría de Flujo , Inmunohistoquímica , Masculino , Microscopía Electrónica , Modelos Animales , Ratas , Ratas Long-Evans , Espectrometría por Rayos X , Espectrometría RamanRESUMEN
Chronic wounds affect over 400,000 people in the United States alone, with up to 60,000 deaths each year from non-healing ulcerations. Tissue grafting (e.g., autografts, allografts, and xenografts) and synthetic skin substitutes are common treatment methods, but most solutions are limited to symptomatic treatment and do not address the underlying causes of the chronic wound. Use of fat grafts for wound healing applications has demonstrated promise but these grafts suffer from low cell viability and poor retention at the wound site resulting in suboptimal healing of chronic wounds. Herein, we report on an innovative closed-loop fat processing system (MiniTCTM) that can efficiently process lipoaspirates into microfat clusters comprising of highly viable regenerative cell population (i.e., adipose stromal cells, endothelial progenitors) preserved in their native niche. Cryopreservation of MiniTCTM isolated microfat retained cell count and viability. To improve microfat retention and engraftment at the wound site, microfat was mixed with methacrylated collagen (CMA) bioink and 3D printed to generate microfat-laden collagen constructs. Modulating the concentration of microfat in CMA constructs had no effect on print fidelity or stability of the printed constructs. Results from the Alamar blue assay showed that the cells remain viable and metabolically active in microfat-laden collagen constructs for up to 10 days in vitro. Further, quantitative assessment of cell culture medium over time using ELISA revealed a temporal expression of proinflammatory and anti-inflammatory cytokines indicative of wound healing microenvironment progression. Together, these results demonstrate that 3D bioprinting of microfat-laden collagen constructs is a promising approach to generate viable microfat grafts for potential use in treatment of non-healing chronic wounds.
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Collagen type I, commonly derived from xenogenic sources, is extensively used as a biomaterial for tissue engineering applications. However, the use of xenogenic collagen is typically associated with species specific variation in mechanical, structural, and biological properties that are known to influence cellular response and remodeling. In addition, immunological complications and risks of disease transmission are also major concerns. The goal of this study is to characterize a new xeno-free human skin-derived collagen and assess its applicability as a bioink for cell-laden 3 D bioprinting. Four different concentrations of human collagen (i.e., 0.5 mg/mL, 1 mg/mL, 3 mg/mL and 6 mg/mL) were employed for the synthesis of collagen hydrogels. In addition, bovine collagen was used as a xenogenic control. Results from SDS-PAGE analysis showed the presence of α1, α2, and ß chains, confirming that the integrity of type I human collagen is maintained post isolation. Polymerization rate and compressive modulus increased significantly with increase in the concentration of human collagen. When comparing two different sources of collagen, the polymerization rate of xenogenic collagen was significantly faster (p < 0.05) than human collagen while the compressive modulus was comparable. Raman spectroscopy showed a large peak in the Amide I band around 1600 cm-1, indicating a dense and supraorganized fibrillar structure in human collagen hydrogels. Conversely, Amide I band intensity for xenogenic collagen was comparable to that of Amide II and Amide III bands. Further, the use of 6 mg/mL human collagen as a bioink yielded 3 D printed constructs with high shape fidelity and cell viability. On the other hand, xenogenic collagen failed to yield stable 3 D printed constructs. Together, the results from this study provides an impetus for using human-derived collagen as a viable alternative to xenogenic sources for 3 D bioprinting of clinically relevant scaffolds for tissue engineering applications.
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Bioimpresión , Colágeno/química , Impresión Tridimensional , Animales , Materiales Biocompatibles , Bovinos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Colágeno/farmacología , Colágeno/fisiología , Colágeno/ultraestructura , Fuerza Compresiva , Humanos , Hidrogeles/química , Polimerizacion , Ingeniería de Tejidos , Andamios del Tejido/químicaRESUMEN
Bioactive three-dimensional (3D) printed scaffolds are promising candidates for bone tissue engineering (BTE) applications. Here, we introduce a bioactive ink composed of Bioglass 45S5 (BG) and methacrylated collagen (CMA) for 3D printing of biomimetic constructs that resemble the organic and inorganic composition of native bone tissue. A uniform dispersion of BG particles within the collagen network improved stability and reduced swelling of collagen hydrogels. Rheological testing showed significant improvement in the yield stress and percent recovery of 3D printed constructs upon BG incorporation. Further, addition of BG improved the bone bioactivity of 3D printed constructs in stimulated body fluid. BG incorporated CMA (BG-CMA) constructs maintained high cell viability and enhanced alkaline phosphatase activity of human mesenchymal stem cells. In addition, cell-mediated calcium deposition was significantly higher on BG-CMA constructs, compared to CMA alone. In conclusion, 3D printed BG-CMA constructs have significant potential for use in BTE applications.
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Células Madre Mesenquimatosas , Andamios del Tejido , Huesos , Cerámica , Colágeno , Humanos , Tinta , Impresión Tridimensional , Ingeniería de Tejidos/métodosRESUMEN
Photopolymerization of methacrylated collagen (CMA) allows for 3D bioprinting of tissue scaffolds with high resolution and print fidelity. However, photochemically crosslinked CMA constructs are mechanically weak and susceptible to expedited enzymatic degradation in vivo. The goal of the current study was to develop a dual crosslinking scheme for the generation of mechanically viable cell-laden printable constructs for tissue engineering applications. Dual crosslinking was performed by first photochemical crosslinking of CMA hydrogels using VA-086 photoinitiator and UV exposure followed by chemical crosslinking with two different concentrations of genipin (i.e., 0.5â¯mM (low dual) or 1â¯mM (high dual)). The effect of dual crosslinking conditions on gel morphology, compressive modulus, stability and print fidelity was evaluated. Additionally, human MSCs were encapsulated within CMA hydrogels and the effect of dual crosslinking conditions on viability and metabolic activity was assessed. Uncrosslinked, photochemically crosslinked, and genipin crosslinked CMA hydrogels were used as controls. SEM results showed that gel morphology was maintained upon dual crosslinking. Further, dual crosslinking significantly improved the compressive modulus and degradation time of cell-laden and acellular CMA hydrogels. Cell viability results showed that high cell viability (i.e., >80%) and metabolic activity in low dual crosslinked CMA hydrogels. On the other hand, cell viability and metabolic activity decreased significantly (pâ¯<â¯0.05) in high dual crosslinked CMA hydrogels. Quantitative fidelity measurements showed the measured parameters (i.e., line widths, pore size) were comparable between photochemically crosslinked and dual crosslinked constructs, suggesting that print fidelity is maintained upon dual crosslinking. In conclusion, application of low dual crosslinking is a viable strategy to yield mechanically superior, cell compatible and printable CMA hydrogels.
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Bioimpresión/métodos , Colágeno , Metacrilatos , Impresión Tridimensional , Andamios del Tejido , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colágeno/química , Colágeno/farmacología , Humanos , Hidrogeles/química , Células Madre Mesenquimatosas/efectos de los fármacos , Metacrilatos/química , Metacrilatos/toxicidad , Ingeniería de Tejidos/métodosRESUMEN
Irgacure 2959 (I2959) is widely used as a photoinitiator for photochemical crosslinking of hydrogels. However, the free radicals generated from I2959 have been reported to be highly cytotoxic. In this study, methacrylated collagen (CMA) hydrogels were photochemically crosslinked using two different photoinitiators (i.e., I2959 and VA086) and the effect of photoinitiator type, photoinitiator concentration (i.e., 0.02 and 0.1%) and crosslinking time (1 and 10 min) on gel morphology, compressive modulus, and stability were investigated. In addition, Saos-2 cells were encapsulated within the hydrogels and the effect of photochemical crosslinking conditions on cell viability, metabolic activity, and osteoblast functionality was assessed. Scanning electron microscopy imaging showed that photochemical crosslinking decreased the porosity of the hydrogels resulting in decrease in water retention ability compared to uncrosslinked hydrogels. On the other hand, photochemical crosslinking improved the stability of CMA hydrogels (p < 0.05). Uniaxial compression tests showed that increasing the photoinitiator concentration significantly improved the compressive modulus of CMA hydrogels (p < 0.05). Results from the live-dead assay showed that VA086 crosslinked hydrogels exhibited higher cell viability compared to I2959 (p < 0.05) crosslinked hydrogels indicating that VA086 is more cytocompatible compared to I2959. Furthermore, Alizarin Red S staining revealed a significantly more pronounced cell-mediated mineralization on VA086 crosslinked hydrogels (p < 0.05) indicating that Saos-2 cells retain their normal functionality in the presence of VA086. In summary, these results indicate that VA086 is a more biocompatible photoinitiator compared to I2959 for the generation of photochemically crosslinked CMA hydrogels for tissue engineering applications. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2019.