Decoupling the Effects of Collagen Alignment and Bioceramic Incorporation on Osteoblast Proliferation, Differentiation, and Mineralization.

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.

Magnetic Alignment of Collagen: Principles, Methods, Applications, and Fiber Alignment Analyses.

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.

Collagen and Beyond: A Comprehensive Comparison of Human ECM Properties Derived from Various Tissue Sources for Regenerative Medicine Applications.

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.

Xeno-Free Biomimetic ECM Model for Investigation of Matrix Composition and Stiffness on Astrocyte Cell Response.

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.

A comparative study of bone bioactivity and osteogenic potential of different bioceramics in methacrylated collagen hydrogels.

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.

Species-Based Differences in Mechanical Properties, Cytocompatibility, and Printability of Methacrylated Collagen Hydrogels.

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.

Design-Build-Validate Strategy to 3D Print Bioglass Gradients for Anterior Cruciate Ligament Enthesis Reconstruction.

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.