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.

A Feasibility Study on 3D Bioprinting of Microfat Constructs Towards Wound Healing Applications.

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.

In vitro characterization of xeno-free clinically relevant human collagen and its applicability in cell-laden 3D bioprinting.

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.

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.