Cryogel scaffold-mediated delivery of adipose-derived stem cells promotes healing in murine model of atrophic non-union

Non-union is defined as the permanent failure of a bone to heal and occurs clinically in 5% of fractures. Atrophic non-unions, characterized by absent/minimal callus formation, are poorly understood and difficult to treat. We recently demonstrated a novel murine model of atrophic non-union in the 3.6Col1A1-tk (Col1-tk) mouse, wherein dosing with the nucleoside analog ganciclovir (GCV) was used to deplete proliferating osteoprogenitor cells, leading to a radiographic and biomechanical non-union after the mid-shaft femur fracture. Using this Col1-tk atrophic non-union model, we hypothesized that the scaffold-mediated lentiviral delivery of doxycycline-inducible BMP-2 transgenes would induce osteogenesis at the fracture site. Cryogel scaffolds were used as a vehicle for GFP+ and BMP-2+ cell delivery to the site of non-union. Cryogel scaffolds were biofabricated through the cross-linking of a chitosan-gelatin polymer solution at subzero temperatures, which results in a macroporous, spongy structure that may be advantageous for a bone regeneration application. Murine adipose-derived stem cells were seeded onto the cryogel scaffolds, where they underwent lentiviral transduction. Following the establishment of atrophic non-unions in the femurs of Col1-tk mice (4 weeks post-fracture), transduced, seeded scaffolds were surgically placed around the site of non-union, and the animals were given doxycycline water to induce BMP-2 production. Controls included GFP+ cells on the cryogel scaffolds, acellular scaffolds, and sham (no scaffold). Weekly radiographs were taken, and endpoint analysis included micro-CT and histological staining. After 2 weeks of implantation, the BMP-2+ scaffolds were infiltrated with cartilage and woven bone at the non-union site, while GFP+ scaffolds had woven bone formation. Later, timepoints of 8 weeks had woven bone and vessel formation within the BMP-2+ and GFP + scaffolds with cortical bridging of the original fracture site in both groups. Overall, the cell-seeded cryogels promoted osseous healing. However, while the addition of BMP-2 promoted the endochondral ossification, it may provide a slower route to healing. This proof-of-concept study demonstrates the potential for cellularized cryogel scaffolds to enhance the healing of non-unions

Facilitation and Competition among Invasive Plants: A Field Experiment with Alligatorweed and Water Hyacinth

Ecosystems that are heavily invaded by an exotic species often contain abundant populations of other invasive species. This may reflect shared responses to a common factor, but may also reflect positive interactions among these exotic species. Armand Bayou (Pasadena, TX) is one such ecosystem where multiple species of invasive aquatic plants are common. We used this system to investigate whether presence of one exotic species made subsequent invasions by other exotic species more likely, less likely, or if it had no effect. We performed an experiment in which we selectively removed exotic rooted and/or floating aquatic plant species and tracked subsequent colonization and growth of native and invasive species. This allowed us to quantify how presence or absence of one plant functional group influenced the likelihood of successful invasion by members of the other functional group. We found that presence of alligatorweed (rooted plant) decreased establishment of new water hyacinth (free-floating plant) patches but increased growth of hyacinth in established patches, with an overall net positive effect on success of water hyacinth. Water hyacinth presence had no effect on establishment of alligatorweed but decreased growth of existing alligatorweed patches, with an overall net negative effect on success of alligatorweed. Moreover, observational data showed positive correlations between hyacinth and alligatorweed with hyacinth, on average, more abundant. The negative effect of hyacinth on alligatorweed growth implies competition, not strong mutual facilitation (invasional meltdown), is occurring in this system. Removal of hyacinth may increase alligatorweed invasion through release from competition. However, removal of alligatorweed may have more complex effects on hyacinth patch dynamics because there were strong opposing effects on establishment versus growth. The mix of positive and negative interactions between floating and rooted aquatic plants may influence local population dynamics of each group and thus overall invasion pressure in this watershed

Current Concepts and Methods in Tissue Interface Scaffold Fabrication

Damage caused by disease or trauma often leads to multi-tissue damage which is both painful and expensive for the patient. Despite the common occurrence of such injuries, reconstruction can be incredibly challenging and often may focus on a single tissue, which has been damaged to a greater extent, rather than the environment as a whole. Tissue engineering offers an approach to encourage repair, replacement, and regeneration using scaffolds, biomaterials and bioactive factors. However, there are many advantages to creating a combined scaffold fabrication method approach that incorporates the treatment and regeneration of multiple tissue types simultaneously. This review provides a guide to combining multiple tissue-engineered scaffold fabrication methods to span several tissue types concurrently. Briefly, a background in the healing and composition of typical tissues targeted in scaffold fabrication is provided. Then, common tissue-engineered scaffold fabrication methods are highlighted, specifically focusing on porosity, mechanical integrity, and practicality for clinical application. Finally, an overview of commonly used scaffold biomaterials and additives is provided, and current research in combining multiple scaffold fabrication techniques is discussed. Overall, this review will serve to bridge the critical gap in knowledge pertaining to combining different fabrication methods for tissue regeneration without disrupting structural integrity and biomaterial properties

Tissue-Engineered Delivery of Manuka Honey

Manuka Honey (MH) contains the Unique Manuka Honey Factor (UMF), which is directly correlated to honey’s antibacterial efficiency and has a wide range of wound healing properties. In more recent years, MH has been incorporated into tissue-engineering scaffolds including cryogels, hydrogels, electrospun fibers, and electrosprayed nanoparticles. Tissue-engineered scaffolds can serve as alternative vehicles for delivery as compared to traditional wound dressings.The project objective was to investigate which of the four scaffold types can successfully incorporate MH (0, 1, 5, 10%) while retaining original parameters. We hypothesize that cryogels offer the best vehicle of delivery of MH because of increased durability (mechanical properties) compared to that of hydrogels and electrospun/electrosprayed scaffolds.https://digitalcommons.dartmouth.edu/wetterhahn_2023/1005/thumbnail.jp

In vitro characterization of MG-63 osteoblast-like cells cultured on organic-inorganic lyophilized gelatin sponges for early bone healing

The development of three-dimensional porous scaffolds with enhanced osteogenic and angiogenic potential would be beneficial for inducing early-stage bone regeneration. Previous studies have demonstrated the advantages of mineralized and nonmineralized acellular 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) cross-linked gelatin sponges enhanced with preparations rich in growth factors, hydroxyapatite, and chitin whiskers. In this study, those same scaffolds were mineralized and dynamically seeded with MG-63 cells. Cell proliferation, protein/cytokine secretion, and compressive mechanical properties of scaffolds were evaluated. It was found that mineralization and the addition of growth factors increased cell proliferation compared to gelatin controls. Cells on all scaffolds responded in an appropriate bone regenerative fashion as shown through osteocalcin secretion and little to no secretion of bone resorbing markers. However, compressive mechanical properties of cellularized scaffolds were not significantly different from acellular scaffolds. The combined results of increased cellular attachment, infiltration, and bone regenerative protein/cytokine secretion on scaffolds support the need for the addition of a bone-like mineral surface. Cellularized scaffolds containing growth factors reported similar advantages and mechanical values in the range of native tissues present in the early stages of bone healing. These results suggest that the developed composite sponges exhibited cellular responses and mechanical properties appropriate for promoting early bone healing in various applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2011–2019, 2016

A Comparison of Tissue Engineering Scaffolds Incorporated with Manuka Honey of Varying UMF

Purpose. Manuka honey (MH) is an antibacterial agent specific to the islands of New Zealand containing both hydrogen peroxide and a Unique Manuka Factor (UMF). Although the antibacterial properties of MH have been studied, the effect of varying UMF of MH incorporated into tissue engineered scaffolds have not. Therefore, this study was designed to compare silk fibroin cryogels and electrospun scaffolds incorporated with a 5% MH concentration of various UMF. Methods. Characteristics such as porosity, bacterial clearance and adhesion, and cytotoxicity were compared. Results. Pore diameters for all cryogels were between 51 and 60 µm, while electrospun scaffolds were 10 µm. Cryogels of varying UMF displayed clearance of approximately 0.16 cm for E. coli and S. aureus. In comparison, the electrospun scaffolds clearance ranged between 0.5 and 1 cm. A glucose release of 0.5 mg/mL was observed for the first 24 hours by all scaffolds, regardless of UMF. With respect to cytotoxicity, neither scaffold caused the cell number to drop below 20,000. Conclusions. Overall, when comparing the effects of the various UMF within the two scaffolds, no significant differences were observed. This suggests that the fabricated scaffolds in this study displayed similar bacterial effects regardless of the UMF value

Investigating Manuka Honey Antibacterial Properties When Incorporated into Cryogel, Hydrogel, and Electrospun Tissue Engineering Scaffolds

Honey is well-known for its wound healing capability and Manuka honey (MH) contains a unique Manuka factor, providing an additional antibacterial agent. Previously, there has not been a practical way to apply MH to a wound site, which renders treatment for an extended period extremely difficult. Tissue-engineered scaffolds offer an alternative treatment method to standard dressings by providing varying geometries to best treat the specific tissue. MH was incorporated into cryogels, hydrogels, and electrospun scaffolds to assess the effect of scaffold geometry on bacterial clearance and adhesion, as well as cellular adhesion. Electrospun scaffolds exhibited a faster release due to the nanoporous fibrous geometry which led to a larger partial bacterial clearance as compared to the more three-dimensional cryogels (CG) and hydrogels (HG). Similarly, the fast release of MH from the electrospun scaffolds resulted in reduced bacterial adhesion. Overall, the fast MH release of the electrospun scaffolds versus the extended release of the HG and CG scaffolds provides differences in cellular/bacterial adhesion and advantages for both short and long-term applications, respectively. This manuscript provides a comparison of the scaffold pore structures as well as bacterial and cellular properties, providing information regarding the relationship between varying scaffold geometry and MH efficacy

Direct Integration of 3D Printing and Cryogel Scaffolds for Bone Tissue Engineering

Cryogels, known for their biocompatibility and porous structure, lack mechanical strength, while 3D-printed scaffolds have excellent mechanical properties but limited porosity resolution. By combining a 3D-printed plastic gyroid lattice scaffold with a chitosan–gelatin cryogel scaffold, a scaffold can be created that balances the advantages of both fabrication methods. This study compared the pore diameter, swelling potential, mechanical characteristics, and cellular infiltration capability of combined scaffolds and control cryogels. The incorporation of the 3D-printed lattice demonstrated patient-specific geometry capabilities and significantly improved mechanical strength compared to the control cryogel. The combined scaffolds exhibited similar porosity and relative swelling ratio to the control cryogels. However, they had reduced elasticity, reduced absolute swelling capacity, and are potentially cytotoxic, which may affect their performance. This paper presents a novel approach to combine two scaffold types to retain the advantages of each scaffold type while mitigating their shortcomings

Embracing ethical research: implementing the 3R principles into fracture healing research for sustainable scientific progress

As scientific advancements continue to reshape the world, it becomes increasingly crucial to uphold ethical standards and minimize the potentially adverse impact of research activities. In this context, the implementation of the 3R principles-Replacement, Reduction, and Refinement-has emerged as a prominent framework for promoting ethical research practices in the use of animals. This article aims to explore recent advances in integrating the 3R principles into fracture healing research, highlighting their potential to enhance animal welfare, scientific validity, and societal trust. The review focuses on in vitro, in silico, ex vivo, and refined in vivo methods, which have the potential to replace, reduce, and refine animal experiments in musculoskeletal, bone, and fracture healing research. Here, we review material that was presented at the workshop "Implementing 3R Principles into Fracture Healing Research" at the 2023 Orthopedic Research Society (ORS) Annual Meeting in Dallas, Texas