Flow Behavior Prior to Crosslinking: The Need for Precursor Rheology for Placement of Hydrogels in Medical Applications and for 3D Bioprinting.

Hydrogels - water swollen cross-linked networks - have demonstrated considerable promise in tissue engineering and regenerative medicine applications. However, ambiguity over which rheological properties are needed to characterize these gels before crosslinking still exists. Most hydrogel research focuses on the performance of the hydrogel construct after implantation, but for clinical practice, and for related applications such as bioinks for 3D bioprinting, the behavior of the pre-gelled state is also critical. Therefore, the goal of this review is to emphasize the need for better rheological characterization of hydrogel precursor formulations, and standardized testing for surgical placement or 3D bioprinting. In particular, we consider engineering paste or putty precursor solutions (i.e., suspensions with a yield stress), and distinguish between these differences to ease the path to clinical translation. The connection between rheology and surgical application as well as how the use of paste and putty nomenclature can help to qualitatively identify material properties are explained. Quantitative rheological properties for defining materials as either pastes or putties are proposed to enable easier adoption to current methods. Specifically, the three-parameter Herschel-Bulkley model is proposed as a suitable model to correlate experimental data and provide a basis for meaningful comparison between different materials. This model combines a yield stress, the critical parameter distinguishing solutions from pastes (100-2000 Pa) and from putties (>2000 Pa), with power law fluid behavior once the yield stress is exceeded. Overall, successful implementation of paste or putty handling properties to the hydrogel precursor may minimize the surgeon-technology learning time and ultimately ease incorporation into current practice. Furthermore, improved understanding and reporting of rheological properties will lead to better theoretical explanations of how materials affect rheological performances, to better predict and design the next generation of biomaterials.

Sustained in vivo perfusion of a re-endothelialized tissue engineered kidney graft in a human-scale animal model.

Introduction: Despite progress in whole-organ decellularization and recellularization, maintaining long-term perfusion in vivo remains a hurdle to realizing clinical translation of bioengineered kidney grafts. The objectives for the present study were to define a threshold glucose consumption rate (GCR) that could be used to predict in vivo graft hemocompatibility and utilize this threshold to assess the in vivo performance of clinically relevant decellularized porcine kidney grafts recellularized with human umbilical vein endothelial cells (HUVECs). Materials and methods: Twenty-two porcine kidneys were decellularized and 19 were re-endothelialized using HUVECs. Functional revascularization of control decellularized (n = 3) and re-endothelialized porcine kidneys (n = 16) was tested using an ex vivo porcine blood flow model to define an appropriate metabolic glucose consumption rate (GCR) threshold above which would sustain patent blood flow. Re-endothelialized grafts (n = 9) were then transplanted into immunosuppressed pigs with perfusion measured using angiography post-implant and on days 3 and 7 with 3 native kidneys used as controls. Patent recellularized kidney grafts underwent histological analysis following explant. Results: The glucose consumption rate of recellularized kidney grafts reached a peak of 39.9 ± 9.7 mg/h at 21 ± 5 days, at which point the grafts were determined to have sufficient histological vascular coverage with endothelial cells. Based on these results, a minimum glucose consumption rate threshold of 20 mg/h was set. The revascularized kidneys had a mean perfusion percentage of 87.7% ± 10.3%, 80.9% ± 33.1%, and 68.5% ± 38.6% post-reperfusion on Days 0, 3 and 7, respectively. The 3 native kidneys had a mean post-perfusion percentage of 98.4% ± 1.6%. These results were not statistically significant. Conclusion: This study is the first to demonstrate that human-scale bioengineered porcine kidney grafts developed via perfusion decellularization and subsequent re-endothelialization using HUVEC can maintain patency with consistent blood flow for up to 7 days in vivo. These results lay the foundation for future research to produce human-scale recellularized kidney grafts for transplantation.

Assessment of electrospun cardiac patches made with sacrificial particles and polyurethane-polycaprolactone blends.

Congenital heart defects (CHDs) are the leading cause of death in live-born infants. Currently, patches used in the repair of CHDs are exclusively inert and non-degradable, which increases the risk of arrhythmia, follow-up surgeries, and sudden cardiac death. In this preliminary study, we sought to fabricate biodegradable scaffolds that can support cardiac regeneration in the repair of CHDs. We electrospun biodegradable scaffolds using various blends of polyurethane (PU) and polycaprolactone (PCL) with and without sacrificial poly(ethylene oxide) (PEO) particles and assessed the mechanical properties, cell infiltration levels, and inflammatory response in vitro (surface cell seeding) and in vivo (subcutaneous mouse implant). We hypothesized that a blend of the two polymers would preserve the low stiffness of PU as well as the high cell infiltration observed in PCL scaffolds. The inclusion of PU in the blends, even as low as 10%, decreased cell infiltration both in vitro and in vivo. The inclusion of sacrificial PEO increased pore sizes, reduced Young's moduli, and reduced the inflammatory response in all scaffold types. Collectively, we have concluded that a PCL patch electrospun with sacrificial PEO particles is the most promising scaffold for further assessment as in our heart defect model.

Cartilage extracellular matrix as a biomaterial for cartilage regeneration.

The extracellular matrix (ECM) of various tissues possesses the model characteristics that biomaterials for tissue engineering strive to mimic; however, owing to the intricate hierarchical nature of the ECM, it has yet to be fully characterized and synthetically fabricated. Cartilage repair remains a challenge because the intrinsic properties that enable its durability and long-lasting function also impede regeneration. In the last decade, cartilage ECM has emerged as a promising biomaterial for regenerating cartilage, partly because of its potentially chondroinductive nature. As this research area of cartilage matrix-based biomaterials emerged, investigators facing similar challenges consequently developed convergent solutions in constructing robust and bioactive scaffolds. This review discusses the challenges, emerging trends, and future directions of cartilage ECM scaffolds, including a comparison between two different forms of cartilage matrix: decellularized cartilage (DCC) and devitalized cartilage (DVC). To overcome the low permeability of cartilage matrix, physical fragmentation greatly enhances decellularization, although the process itself may reduce the chondroinductivity of fabricated scaffolds. The less complex processing of a scaffold composed of DVC, which has not been decellularized, appears to have translational advantages and potential chondroinductive and mechanical advantages over DCC, without detrimental immunogenicity, to ultimately enhance cartilage repair in a clinically relevant way.

Approaching the compressive modulus of articular cartilage with a decellularized cartilage-based hydrogel.

UNLABELLED: ECM-based materials are appealing for tissue engineering strategies because they may promote stem cell recruitment, cell infiltration, and cell differentiation without the need to supplement with additional biological factors. Cartilage ECM has recently shown potential to be chondroinductive, particularly in a hydrogel-based system, which may be revolutionary in orthopedic medicine. However, hydrogels composed of natural materials are often mechanically inferior to synthetic materials, which is a major limitation for load-bearing tissue applications. The objective was therefore to create an unprecedented hydrogel derived entirely from native cartilage ECM that was both mechanically more similar to native cartilage tissue and capable of inducing chondrogenesis. Porcine cartilage was decellularized, solubilized, and then methacrylated and UV photocrosslinked to create methacrylated solubilized decellularized cartilage (MeSDCC) gels. Methacrylated gelatin (GelMA) was employed as a control for both biomechanics and bioactivity. Rat bone marrow-derived mesenchymal stem cells were encapsulated in these networks, which were cultured in vitro for 6weeks, where chondrogenic gene expression, the compressive modulus, swelling, and histology were analyzed. One day after crosslinking, the elastic compressive modulus of the 20% MeSDCC gels was 1070±150kPa. Most notably, the stress strain profile of the 20% MeSDCC gels fell within the 95% confidence interval range of native porcine cartilage. Additionally, MeSDCC gels significantly upregulated chondrogenic genes compared to GelMA as early as day 1 and supported extensive matrix synthesis as observed histologically. Given that these gels approached the mechanics of native cartilage tissue, supported matrix synthesis, and induced chondrogenic gene expression, MeSDCC hydrogels may be promising materials for cartilage tissue engineering applications. Future efforts will focus on improving fracture mechanics as well to benefit overall biomechanical performance. STATEMENT OF SIGNIFICANCE: Extracellular matrix (ECM)-based materials are appealing for tissue engineering strategies because they may promote stem cell recruitment, cell infiltration, and cell differentiation without the need to supplement with additional biological factors. One such ECM-based material, cartilage ECM, has recently shown potential to be chondroinductive; however, hydrogels composed of natural materials are often mechanically inferior to synthetic materials, which is a major limitation for load-bearing tissue applications. Therefore, this work is significant because we were the first to create hydrogels derived entirely from cartilage ECM that had mechanical properties similar to that of native cartilage until hydrogel failure. Furthermore, these hydrogels had a compressive modulus of 1070±150kPa, they were chondroinductive, and they supported extensive matrix synthesis. In the current study, we have shown that these new hydrogels may prove to be a promising biomaterial for cartilage tissue engineering applications.

Chondroinduction from Naturally Derived Cartilage Matrix: A Comparison Between Devitalized and Decellularized Cartilage Encapsulated in Hydrogel Pastes.

Hydrogel precursors are liquid solutions that are prone to leaking after surgical placement. This problem was overcome by incorporating either decellularized cartilage (DCC) or devitalized cartilage (DVC) microparticles into traditional photocrosslinkable hydrogel precursors in an effort to achieve a paste-like hydrogel precursor. DCC and DVC were selected specifically for their potential to induce chondrogenesis of stem cells, given that materials that are chondroinductive on their own without growth factors are a revolutionary goal in orthopedic medicine. We hypothesized that DVC, lacking the additional chemical processing steps in DCC to remove cell content, would lead to a more chondroinductive hydrogel with rat bone marrow-derived mesenchymal stem cells. Hydrogels composed of methacrylated hyaluronic acid (MeHA) and either DCC or DVC microparticles were tested with and without exposure to transforming growth factor (TGF)-ß3 over a 6 week culture period, where swelling, mechanical analysis, and gene expression were observed. For collagen II, Sox-9, and aggrecan expression, MeHA precursors containing DVC consistently outperformed the DCC-containing groups, even when the DCC groups were exposed to TGF-ß3. DVC consistently outperformed all TGF-ß3-exposed groups in aggrecan and collagen II gene expression as well. In addition, when the same concentrations of MeHA with DCC or DVC microparticles were evaluated for yield stress, the yield stress with the DVC microparticles was 2.7 times greater. Furthermore, the only MeHA-containing group that exhibited shape retention was the group containing DVC microparticles. DVC appeared to be superior to DCC in both chondroinductivity and rheological performance of hydrogel precursors, and therefore DVC microparticles may hold translational potential for cartilage regeneration.

Chondroinductive Hydrogel Pastes Composed of Naturally Derived Devitalized Cartilage.

Hydrogel precursors are liquid solutions that are prone to leaking from the defect site once implanted in vivo. Therefore, the objective of the current study was to create a hydrogel precursor that exhibited a yield stress. Additionally, devitalized cartilage extracellular matrix (DVC) was mixed with DVC that had been solubilized and methacrylated (MeSDVC) to create hydrogels that were chondroinductive. Precursors composed of 10% MeSDVC or 10% MeSDVC with 10% DVC were first evaluated rheologically, where non-Newtonian behavior was observed in all hydrogel precursors. Rat bone marrow stem cells (rBMSCs) were mixed in the precursor solutions, and the solutions were then crosslinked and cultured in vitro for 6 weeks with and without exposure to human transforming growth factor ß3 (TGF-ß3). The compressive modulus, gene expression, biochemical content, swelling, and histology of the gels were analyzed. The DVC-containing gels consistently outperformed the MeSDVC-only group in chondrogenic gene expression, especially at 6 weeks, where the relative collagen II expression of the DVC-containing groups with and without TGF-ß3 exposure was 40- and 78-fold higher, respectively, than that of MeSDVC alone. Future work will test for chondrogenesis in vivo and overall, these two cartilage-derived components are promising materials for cartilage tissue engineering applications.

Decellularized cartilage may be a chondroinductive material for osteochondral tissue engineering.

Extracellular matrix (ECM)-based materials are attractive for regenerative medicine in their ability to potentially aid in stem cell recruitment, infiltration, and differentiation without added biological factors. In musculoskeletal tissue engineering, demineralized bone matrix is widely used, but recently cartilage matrix has been attracting attention as a potentially chondroinductive material. The aim of this study was thus to establish a chemical decellularization method for use with articular cartilage to quantify removal of cells and analyze the cartilage biochemical content at various stages during the decellularization process, which included a physically devitalization step. To study the cellular response to the cartilage matrix, rat bone marrow-derived mesenchymal stem cells (rBMSCs) were cultured in cell pellets containing cells only (control), chondrogenic differentiation medium (TGF-ß), chemically decellularized cartilage particles (DCC), or physically devitalized cartilage particles (DVC). The chemical decellularization process removed the vast majority of DNA and about half of the glycosaminoglycans (GAG) within the matrix, but had no significant effect on the amount of hydroxyproline. Most notably, the DCC group significantly outperformed TGF-ß in chondroinduction of rBMSCs, with collagen II gene expression an order of magnitude or more higher. While DVC did not exhibit a chondrogenic response to the extent that DCC did, DVC had a greater down regulation of collagen I, collagen X and Runx2. A new protocol has been introduced for cartilage devitalization and decellularization in the current study, with evidence of chondroinductivity. Such bioactivity along with providing the 'raw material' building blocks of regenerating cartilage may suggest a promising role for DCC in biomaterials that rely on recruiting endogenous cell recruitment and differentiation for cartilage regeneration.

Enabling Surgical Placement of Hydrogels Through Achieving Paste-Like Rheological Behavior in Hydrogel Precursor Solutions.

Hydrogels are a promising class of materials for tissue regeneration, but they lack the ability to be molded into a defect site by a surgeon because hydrogel precursors are liquid solutions that are prone to leaking during placement. Therefore, although the main focus of hydrogel technology and developments are on hydrogels in their crosslinked form, our primary focus is on improving the fluid behavior of hydrogel precursor solutions. In this work, we introduce a method to achieve paste-like hydrogel precursor solutions by combining hyaluronic acid nanoparticles with traditional crosslinked hyaluronic acid hydrogels. Prior to crosslinking, the samples underwent rheological testing to assess yield stress and recovery using linear hyaluronic acid as a control. The experimental groups containing nanoparticles were the only solutions that exhibited a yield stress, demonstrating that the nanoparticulate rather than the linear form of hyaluronic acid was necessary to achieve paste-like behavior. The gels were also photocrosslinked and further characterized as solids, where it was demonstrated that the inclusion of nanoparticles did not adversely affect the compressive modulus and that encapsulated bone marrow-derived mesenchymal stem cells remained viable. Overall, this nanoparticle-based approach provides a platform hydrogel system that exhibits a yield stress prior to crosslinking, and can then be crosslinked into a hydrogel that is capable of encapsulating cells that remain viable. This behavior may hold significant impact for hydrogel applications where a paste-like behavior is desired in the hydrogel precursor solution.