More than 200,000 people are suffering from Anterior Cruciate Ligament (ACL) related injuries each year in the US. There is an unmet clinical demand for improving biological attachment between grafts and the host tissue in addition to providing mechanical support. For biological graft integration, it is important to provide a physiologically feasible environment for the host cells to enable them to perform their duties. However, behavior of cells during ACL healing and the mechanism of ACL healing is not fully understood partly due to the absence of appropriate environment to test cell behavior both in vitro and in vivo. This study aims at (i) investigating the change in fibril diameter of bovine ACL tissue upon injury and (ii) fabricating nanofiber-based scaffolds to represent the morphology and structure of healthy and injured ACL tissues. We hypothesized that distribution and mean diameter of ACL fibrils will be altered upon injury. Findings revealed that the collagen fibril diameter distribution of bovine ACL changed from bimodal to unimodal upon injury with subsequent decrease in mean diameter. Polycaprolactone (PCL) scaffold fiber diameter distribution exhibited similar bimodal and unimodal distribution behavior to qualitatively represent the cases of healthy and injured ACL, respectively. The native ACL tissue demonstrated comparable modulus values only with the aligned bimodal PCL scaffolds. There was significant difference between mechanical properties of aligned bimodal and unaligned unimodal PCL scaffolds. We believe that the results obtained from measurements of diameter of collagen fibrils of native bovine ACL tissue can serve as a benchmark for scaffold design.

The anterior cruciate ligament (ACL) tissue is a soft tissue connecting the femur and tibia at the knee joint and demonstrates a limited capacity for self-regeneration due to its low vascularity. The currently available clinical procedures are unable to fully restore damaged ACL tissue, and tissue engineering can offer options with a potential of restoring the torn/ruptured ACL by using biomimetic constructs that are similar to native tissue in terms of structure, composition, and functions. However, a model substrate to understand how the ACL cells regenerate the injured tissue is still not available. In this study, it is hypothesized that the nanofiber-based model substrate with bimodal and unimodal fiber diameter distributions will mimic the diameter distribution of collagen fibrils seen in healthy and injured sheep ACL, respectively. The aims were to (i) create an ACL injury in a sheep ACL by applying extensional force to rupture the healthy ACL tissue, (ii) measure the collagen fibril diameter distributions of healthy and injured ACL, (iii) fabricate polycaprolactone (PCL) nanofiber-based model constructs using electrospinning with diameter distributions similar to healthy and injured ACL tissue, and (iv) measure mechanical properties of ACL tissue and PCL electrospun constructs. The results showed that the fiber diameter distributions of PCL electrospun constructs and those of the healthy and injured ACL tissues were similar. The novelty in this investigation is that the collagen fibril diameter distribution of healthy and injured sheep ACL tissues was reported for the first time. The study is significant because it aims to create a model construct to solve an important orthopedic-related clinical problem affecting millions of people globally. The model construct fabricated in this work is expected to have an important impact on ACL regeneration efforts.

Fabrication of scaffolds via 3D printing is a promising approach for tissue engineering. In this study, we combined 3D printing with cryogenic crosslinking to create biocompatible gelatin/oxidized alginate (Gel/OxAlg) scaffolds with large pore sizes, beneficial for bone tissue regeneration. To enhance the osteogenic effects and mechanical properties of these scaffolds, we evaluated the impact of hydroxyapatite (HAp) on the rheological characteristics of the 2.86% (1:1) Gel/OxAlg ink. We investigated the morphological and mechanical properties of scaffolds with low, 5%, and high 10% HAp content, as well as the resulting bio- and osteogenic effects. Scanning electron microscopy revealed a reduction in pore sizes from 160 to 180 µm (HAp-free) and from 120 to 140 µm for both HAp-containing scaffolds. Increased stability and higher Young’s moduli were measured for 5% and 10% HAp (18 and 21 kPa, respectively) compared to 11 kPa for HAp-free constructs. Biological assessments with mesenchymal stem cells indicated excellent cytocompatibility and osteogenic differentiation in all scaffolds, with high degree of mineralization in HAp-containing constructs. Scaffolds with 5% HAp exhibited improved mechanical characteristics and shape fidelity, demonstrated positive osteogenic impact, and enhanced bone tissue formation. Increasing the HAp content to 10% did not show any advantages in osteogenesis, offering a minor increase in mechanical strength at the cost of significantly compromised shape fidelity.

Osteochondral (OC) tissue plays a crucial role due to its ability to connect bone and cartilage tissues. To address the complexity of structure and functionality at the bone–cartilage interface, relevant to the presence of the tidemark as a critical element at the bone–cartilage boundary, we fabricated graded scaffolds through sequential 3D printing. The scaffold’s bottom layer was based on a gelatin/oxidized alginate mixture enriched with hydroxyapatite (HAp) to create a rougher surface and larger pores to promote osteogenesis. In contrast, the upper layer was engineered to have smaller pores and aimed to promote cartilage tissue formation and mimic the physical properties of the cartilage. An electrospun ε-polycaprolactone (PCL) membrane with micrometer-range pores was incorporated between the layers to replicate the function of tidemark—a barrier to prevent vascularization of cartilage from subchondral bone tissue. In vitro cell studies confirmed the viability of the cells on the layers of the scaffolds and the ability of PCL mesh to prevent cellular migration. The fabricated scaffolds were thoroughly characterized, and their mechanical properties were compared to native OC tissue, demonstrating suitability for OC tissue engineering and graft modeling. The distance of gradient of mineral concentration was found to be 151 µm for grafts and the native OC interface.

Fabrication of scaffolds via 3D printing is a promising approach for tissue engineering. In this study, we combined 3D printing with cryogenic crosslinking to create biocompatible gela-tin/oxidized alginate (Gel/OxAlg) scaffolds with large pore sizes, beneficial for bone tissue re-generation. To enhance the osteogenic effects and mechanical properties of these scaffolds, we evaluated the impact of hydroxyapatite (HAp) on the rheological characteristics of the 2.86% (1:1) Gel/OxAlg ink. We investigated the morphological and mechanical properties of scaffolds with low, 5% and high 10% HAp content, as well as the resulting bio and osteogenic effects. Scanning electron microscopy revealed a reduction in pore sizes from 160-180 µm (HAp-free) to 120-140 µm for both HAp-containing scaffolds. Increased stability and higher Young’s moduli were measured for 5% and 10% HAp (18 and 21 kPa, respectively) compared to 11 kPa for HAp-free constructs. Biological assessments with mesenchymal stem cells indicated excellent cytocompati-bility and osteogenic differentiation in all scaffolds, with high degree of mineralization in HAp-containing constructs. Scaffolds with 5% HAp exhibit improved mechanical characteristics and shape fidelity, demonstrate positive osteogenic impact, and enhance bone tissue formation. Increasing the HAp content to 10% did not show any advantages in osteogenesis, offering a minor increase in mechanical strength at the cost of significantly compromised shape fidelity.