Impact of Hydroxyapatite on Gelatin/Oxidized Alginate 3D-Printed Cryogel Scaffolds.

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.

Reasoning on Pore Terminology in 3D Bioprinting.

Terminology is pivotal for facilitating clear communication and minimizing ambiguity, especially in specialized fields such as chemistry. In materials science, a subset of chemistry, the term "pore" is traditionally linked to the International Union of Pure and Applied Chemistry (IUPAC) nomenclature, which categorizes pores into "micro", "meso", and "macro" based on size. However, applying this terminology in closely-related areas, such as 3D bioprinting, often leads to confusion owing to the lack of consensus on specific definitions and classifications tailored to each field. This review article critically examines the current use of pore terminology in the context of 3D bioprinting, highlighting the need for reassessment to avoid potential misunderstandings. We propose an alternative classification that aligns more closely with the specific requirements of bioprinting, suggesting a tentative size-based division of interconnected pores into 'parvo'-(d < 25 µm), 'medio'-(25 < d < 100 µm), and 'magno'-(d > 100 µm) pores, relying on the current understanding of the pore size role in tissue formation. The introduction of field-specific terminology for pore sizes in 3D bioprinting is essential to enhance the clarity and precision of research communication. This represents a step toward a more cohesive and specialized lexicon that aligns with the unique aspects of bioprinting and tissue engineering.

Biomimetic grafts from ultrafine fibers for collagenous tissues.

BACKGROUND: The ligament is the soft tissue that connects bone to bone and, in case of severe injury or rupture, it cannot heal itself mainly because of its poor vascularity and dynamic nature. Tissue engineering carries the potential to restore the injured tissue functions by utilization of scaffolds mimicking the structure of native ligament. Collagen fibrils in the anterior cruciate ligament (ACL) have a diameter ranging from 20 to 300 nm, which defines the physical and mechanical properties of the tissue. Also, the ACL tissue exhibited a bimodal distribution of collagen fibrils. Currently, the ability to fabricate scaffolds replicating this structure is a significant challenge. OBJECTIVE: This work aims at i) measuring the diameter of collagens of bovine ACL tissue, ii) investigating the fabrication of sub-100 nm fibers, and iii) fabricating aligned scaffolds with bimodal diameter distribution (with two peaks) resembling the healthy ACL structure. It is hypothesized that such scaffolds can be produced by electrospinning polycaprolactone (PCL) solutions. METHODS: To test the hypothesis, various PCL solutions were formulated in acetone and formic acid in combination with pyridine, and electrospun to generate sub-100 nm fibers. Next, this formulation was adjusted to produce nanofibers with a diameter between 100 nm and 200 nm. Finally, these solutions were combined in the co-electrospinning process, i.e., two-spinneret electrospinning, to fabricate biomimetic scaffolds with a bimodal distribution. RESULTS: Electrospinning of 8% and 15% PCL solutions, respectively, resulted in the production of fibers with diameters below and above 100 nm. The combined scaffold exhibited a bimodal distribution of aligned fibers with peaks around 80 and 180 nm, thus mimicking the collagen fibrils of healthy ACL tissue. CONCLUSION: This research is expected to have a society-wide impact because it aims to enhance the health condition and life quality of a wide range of patients.

Dual-Crosslinking of Gelatin-Based Hydrogels: Promising Compositions for a 3D Printed Organotypic Bone Model.

Gelatin-based hydrogels have emerged as a popular scaffold material for tissue engineering applications. The introduction of variable crosslinking methods has shown promise for fabricating stable cell-laden scaffolds. In this work, we examine promising composite biopolymer-based inks for extrusion-based 3D bioprinting, using a dual crosslinking approach. A combination of carefully selected printable hydrogel ink compositions and the use of photoinduced covalent and ionic crosslinking mechanisms allows for the fabrication of scaffolds of high accuracy and low cytotoxicity, resulting in unimpeded cell proliferation, extracellular matrix deposition, and mineralization. Three selected bioink compositions were characterized and the respective cell-laden scaffolds were bioprinted. Temporal stability, morphology, swelling, and mechanical properties of the scaffolds were thoroughly studied and the biocompatibility of the constructs was assessed using rat mesenchymal stem cells while focusing on osteogenesis. Experimental results showed that the composition of 1% alginate, 4% gelatin, and 5% (w/v) gelatine methacrylate, was found to be optimal among the examined, with shape fidelity of 88%, large cell spreading area and cell viability at around 100% after 14 days. The large pore diameters that exceed 100 µm, and highly interconnected scaffold morphology, make these hydrogels extremely potent in bone tissue engineering and bone organoid fabrication.

Collagen Fibril Diameter Distribution of Sheep Anterior Cruciate Ligament.

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.

Osteochondral Interface: Regenerative Engineering and Challenges.

Osteochondral (OC) defects are debilitating for patients and represent a significant clinical problem for orthopedic surgeons as well as regenerative engineers due to their potential complications, which are likely to lead to osteoarthritis and related diseases. If they remain untreated or are treated suboptimally, OC lesions are known to impact the articular cartilage and the transition from cartilage to bone, that is, the cartilage-bone interface. An important component of the OC interface, that is, a selectively permeable membrane, the tidemark, still remains unaddressed in more than 90% of the published research in the past decade. This review focuses on the structure, composition, and function of the OC interface, regenerative engineering attempts with different scaffolding strategies and challenges ahead of us in recapitulating the native OC interface. There are different schools of thought regarding the structure of the native OC interface: stratified and graded. The former assumes the cartilage-to-bone interface to be hierarchically divided into distinct yet continuous zones of uncalcified cartilage-calcified cartilage-subchondral bone. The latter assumes the interface is continuously graded, that is, formed by an infinite number of layers. The cellular composition of the interface, either in respective layers or continuously changing in a graded manner, is chondrocytes, hypertrophic chondrocytes, and osteoblasts as moved from cartilage to bone. Functionally, the interface is assumed to play a role in enabling a smooth transition of loads exerted on the cartilage surface to the bone underneath. Regenerative engineering involves, first, a characterization of the native OC interface in terms of the composition, structure, and function, and, then, proposes the appropriate biomaterials, cells, and biomolecules either alone or in combination to eventually form a structure that mimics and functionally behaves similar to the native interface. The major challenge regarding regeneration of the OC interface appears to lie, in addition to others, in the formation of tidemark, which is a thin membrane separating the OC interface into two distinct zones: the avascular OC interface and the vascular OC interface. There is a significant amount of literature on regenerative approaches to the OC interface; however, only a small portion of them consider the importance of tidemark. Therefore, this review aims at highlighting the significance of the structural organization of the components of the OC interface and increasing the awareness of the orthopedics community regarding the importance of tidemark formation after clinical interventions or regenerative engineering attempts.

A biomimetic synthetic nanofiber-based model for anterior cruciate ligament regeneration.

Reconstructed ACL cannot completely restore its functions due to absence of physiologically viable environment for optimal biomaterial-cell interaction. Currently available procedures only mechanically attach grafts to bone without any biological integration. How the ACL cells perform this biological attachment is not fully understood partly due to the absence of appropriate environment to test cell behavior both in vitro and in vivo. Availability of biomimetic models would enable the scientists to better explore the behavior of cells at health and during tissue healing. In this study, it is hypothesized that the collagen fibril diameter distribution in rat ACL changes from a bimodal distribution in the healthy ACL to a unimodal distribution after injury, and that this change can be mimicked in synthetic nanofiber-based constructs. This hypothesis was tested by first creating an injured rat ACL model by applying a mechanical tensile force to the healthy ACL tissue until rupture. Secondly, the collagen fibril diameter distributions of healthy and injured ACL tissue were determined, and polycaprolactone (PCL) constructs were created to mimic the distributions of collagen fibrils in healthy and injured tissues. Findings reveal that the fiber diameter distribution of aligned bimodal PCL constructs were similar to that of the collagen fibrils in native ACL tissue. This study is significant because suggested bimodal and unimodal fibrous model constructs, respectively, represent a healthy and injured tissue environment and the behavior of ACL cells cultured on these constructs may provide significant input on ACL regeneration mechanism.

Change in Collagen Fibril Diameter Distribution of Bovine Anterior Cruciate Ligament upon Injury Can Be Mimicked in a Nanostructured Scaffold.

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.

Chemical Composition of the Essential Oil of the Boreal Relict of Pyrola rotundifolia L. from Northern Kazakhstan.

In Kazakhstan Pyrola rotundifolia L. is the plant-relict in the flora of insular pine forests of the region of low hillocks and declivities in Kazakhstan - a group of insular pine forests of Kokshetau, Bayanaul and Karkaralinsk. In this study, the essential oils from dried aerial parts of P. rotundifolia, collected in natural habitats of the State National Natural Park "Burabay" (Akmola oblast, Northern Kazakhstan), were extracted by hydrodistillation and analyzed by gas chromatography - mass spectrometry. The yield of the essential oil amounted to 0.057 % in relation to the mass of the air-dry raw material. The major components in dried plant oil were 2,6-dimethyl-1,4-naphthoquinone (12.99-93.49%) and dibutyl phthalate (4.42-40.48%), depending on the growth conditions.