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.

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.

Native human collagen type I provides a viable physiologically relevant alternative to xenogeneic sources for tissue engineering applications: A comparative in vitro and in vivo study.

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.

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.

Bioglass incorporated methacrylated collagen bioactive ink for 3D printing of bone tissue.

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.

Dual crosslinking strategy to generate mechanically viable cell-laden printable constructs using methacrylated collagen bioinks.

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., 0.5 mM (low dual) or 1 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 (p < 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.