Injectable Cryogels Research Articles

In-Bath 3D Printing of Anisotropic Shape-Memory Cryogels Functionalized with Bone-Bioactive Nanoparticles.

Cryogels exhibit unique shape memory with full recovery and structural stability features after multiple injections. These constructs also possess enhanced cell permeability and nutrient diffusion when compared to typical bulk hydrogels. Volumetric processing of cryogels functionalized with nanosized units has potential to widen their biomedical applications, however this has remained challenging and relatively underexplored. In this study, we report a novel methodology that combines suspension 3D printing with directional freezing for the fabrication of nanocomposite cryogels with configurable anisotropy. When compared to conventional bulk or freeze-dried hydrogels, nanocomposite cryogel formulations exhibit excellent shape recovery (>95%) and higher pore connectivity. Suspension printing, assisted with a prechilled metal grid, was optimized to induce anisotropy. The addition of calcium- and phosphate-doped mesoporous silica nanoparticles into the cryogel matrix enhanced bioactivity toward orthopedic applications without hindering the printing process. Notably, the nanocomposite 3D printed cryogels exhibit injectable shape memory while also featuring a lamellar topography. The fabrication of these constructs was highly reproducible and exhibited potential for a cell-delivery injectable cryogel with no cytotoxicity to human-derived adipose stem cells. Hence, in this work, it was possible to combine a gravity defying 3D printed methodology with injectable and controlled anisotropic macroporous structures containing bioactive nanoparticles. This methodology ameliorates highly tunable injectable 3D printed anisotropic nanocomposite cryogels with a user-programmable degree of structural complexity.

A self-healing hydrogel and injectable cryogel of gelatin methacryloyl-polyurethane double network for 3D printing.

Three-dimensional (3D) printing of soft biomaterials facilitates the progress of personalized medicine. The development for different forms of 3D-printable biomaterials can promotes the potential manufacturing for artificial organs and provides biomaterials with the required properties. In this study, gelatin methacryloyl (GelMA) and dialdehyde-functionalized polyurethane (DFPU) were combined to create a double crosslinking system and develop 3D-printable GelMA-PU biodegradable hydrogel and cryogel. The GelMA-PU system demonstrates a combination of self-healing ability and 3D printability and provides two distinct forms of 3D-printable biomaterials with smart functions, high printing resolution, and biocompatibility. The hydrogel was printed into individual modules through an 80µm or larger nozzle and further assembled into complex structures through adhesive and self-healing abilities, which could be stabilized by secondary photocrosslinking. The 3D-printed hydrogel was adhesive, light transmittable, and could embed a light emitting diode (LED). Furthermore, the hydrogel laden with human mesenchymal stem cells (hMSCs) was successfully printed and showed cell proliferation. Meanwhile, 3D-printed cryogel was achieved by printing on a subzero temperature platform through a 210µm nozzle. After secondary photocrosslinking and drying, the cryogel was deliverable through a 16-gage (1194µm) syringe needle and can promote the proliferation of hMSCs. The GelMA-PU system extends the ink pool for 3D printing of biomaterials and has potential applications in tissue engineering scaffolds, minimally invasive surgery devices, and electronic wound dressings. STATEMENT OF SIGNIFICANCE: The 3D-printable biomaterials developed in this work are GelMA-based ink with smart funcitons and have potentials for various customized medical applications. The synthesized GelMA-polyurethane double network hydrogel can be 3D-printed into individual modules (e.g., 11×11×5mm3) through an 80μm or larger size nozzle, which are then assembled into a taller structure over five times of the initial height by self-healing and secondary photocrosslinking. The hydrogel is adhesive, light transmittable, and biocompatible that can either carry human mesenchymal stem cells (hMSCs) as bioink or embed a red light LED (620nm) with potential applications in electronic skin dressing. Meanwhile, the 3D-printed highly compressible cryogel (e.g., 6×6×1mm3) is deliverable by a 16-gage (1194μm) syringe needle and supports the proliferation of hMSCs also.

Gelatin Methacryloyl Based Injectable Cryogels with Tunable Degradability for Cell Delivery.

Scaffold-based cell delivery can improve therapeutic effects of transplanted cells in cell therapy. Biomaterial scaffolds serveas niche for cell growth and proliferation which improves cell survival and overall function post cell delivery. In this study, gelatin methacryloyl based injectable scaffolds made using poly(ethylene)glycol as a sacrificial polymer and cryogelation as a technique, are demonstrated to have tunable degradability and porosity that is required for cell and drug delivery applications. The pore size (10-142µm) of these gels makes them suitable for loading different cell types as per the application. In vitro studies using mammalian cells confirm that these cryogels are cytocompatible. These cell-laden scaffolds are injectable and have a cell retention ability of up to 90% after injection. Rheology is done to evaluate stiffness and shape recovery property, and it is found that these gels can maintain their original shape even after applying 7 cycles of strain from 0.1% to 20%. Furthermore, their degradability can be modulated between 6 and 10 days by changing the overall polymer composition. Thus, injectability and degradability of these cryogels can circumvent invasive surgical procedures, thereby making them useful for a variety of applications including delivery of cells and bioactive factors.

Cryoprinting of nanoparticle-enhanced injectable hydrogel with shape-memory properties

Cryogels with interconnected macropores for cell seeding and shape-memory properties for non-invasion injection have shown great potential in tissue engineering. However, traditional manufacturing methods of cryogels are time-consuming and labor-intensive, which has limitations in rapidly fabricating customized cryogels. In this work, we developed a cryoprinting platform that combined 3D printing and cryogelation technologies to fabricate nanoparticle-enhanced injectable and shape-memory cryogels. After cryoprinting, the silk fibroin-based cryogels could be printed to a variety of shapes and sizes and allow rapid volumetric recovery after injection through a needle. These cryogels encapsulating laponite nanoparticles provided enhanced proliferation, spreading, and osteogenic differentiation of the seeded bone marrow-derived mesenchymal stem cells (BMSCs). In addition, the integration of implanted hydrogels with host tissues and the cell invasion within the hydrogel was found with no obvious inflammatory response after subcutaneous injection. These findings showed that the printed nanoparticle-enhanced cryogels could promote the seeded stem cells to actively participate in bone regeneration. In short, this work not only offered a feasible and effective platform to print customized cryogels, but also provided an injectable cryogel for bone tissue engineering via this advanced printing platform.

Shape memory injectable cryogel based on carboxymethyl chitosan/gelatin for minimally invasive tissue engineering: In vitro and in vivo assays.

Shape-memory cryogels have drawn attention as an injectable system to minimize the risks associated with surgical implantation in tissue engineering. To achieve shape memory behavior with hydration as an external stimulus, it is necessary to have a porous elastic network. To achieve this, it is crucial to control the crosslinking process at the time of pore formation, especially for natural-based polymers. In this study, a versatile method using a cryogelation method in the presence of chemical and physical crosslinkers is investigated to obtain an injectable super macroporous elastic structure based on a poly(ampholyte) (carboxymethyl chitosan) and a protein (gelatin). Mechanical, swelling, shape memorizing behavior, injectability, and in vitro and in vivo behavior of cryogels were studied. Cryogelation in a subzero temperature led to the formation of scaffolds with interconnected pores of the size of 350 μm which swelled completely after 3 min. Cryogels had crosslink density up to 22% and elastic modulus in the hydrated state up to 0.054 and 1.733 MPa at low and high strains, respectively, and low hysteresis (<30 kPa). Injectability studies confirmed the ability of the cryogels to be injected through a 16G needle. In vitro studies demonstrated good cellular penetration, cell adhesion, and high cell viability (>100%). In vivo studies using mice showed that the body's response was befitting without inflammation and any side effect for the liver and kidneys.

Impact of injectable chitosan cryogel microspherescaffolds on differentiation and proliferation of adiposederived mesenchymal stem cells into fat cells.

Difficulty in the clinical practice of stem cell therapy is often experienced in achieving desired target tissue cell differentiation and migration of stem cells to other tissue compartments where they are destroyed or die. This study was performed to evaluate if mesenchymal stem cells (MSCs) may differentiate into desired cell types when injected after combined with an injectable cryogel scaffold and to investigate if this scaffold may help in preventing cells from passing into different tissue compartments. MSCs were obtained from fat tissue of the rabbits as autografts and nuclei and cytoplasms of these cells were labeled with BrdU and PKH26. In Group 1, only-scaffold; in Group 2, only-MSCs; and in Group 3, combined stem cell/scaffold were injected to the right malar area of the rabbits. At postoperative 3weeks, volumes of the injected areas were calculated by computer-tomography scans and histopathological evaluation was performed. The increase in the volume of the right malar areas was more in Group 3. In histopathological evaluation, chitosan cryogel microspheres were observed microscopically within the tissue and the scaffold was only partially degraded. Normal tissue form was seen in Group 2. Cells differentiated morphologically into fat cells were detected in Groups 2 and 3. Injectable chitosan cryogel microspheres were used in vivo for the first time in this study. As it was demonstrated to be useful in carrying MSCs to the reconstructed area, help cell differentiation to desired cells and prevent migration to other tissue compartments, it may be used for reconstructive purposes in the future.

Cryogels: recent applications in 3D-bioprinting, injectable cryogels, drug delivery, and wound healing.

Cryogels are macroporous polymeric structures formed from the cryogelation of monomers/polymers in a solvent below freezing temperature. Due to their inherent interconnected macroporosity, ease of preparation, and biocompatibility, they are increasingly being investigated for use in biomedical applications such as 3D-bioprinting, drug delivery, wound healing, and as injectable therapeutics. This review highlights the fundamentals of macroporous cryogel preparation, cryogel properties that can be useful in the highlighted biomedical applications, followed by a comprehensive review of recent studies in these areas. Research evaluated includes the use of cryogels to combat various types of cancer, for implantation without surgical incision, and use as highly effective wound dressings. Furthermore, conclusions and outlooks are discussed for the use of these promising and durable macroporous cryogels.

Injectable Lignin-co-Gelatin Cryogels with Antioxidant and Antibacterial Properties for Biomedical Applications.

For several biomedical applications, it is essential to develop novel bioactive materials. Such biomaterials could potentially improve wound healing, prevent infections, or be used in immunoengineering. For example, bioactive materials that reduce oxidative stress without relying on antibiotics and other drugs could be beneficial. Hydrogel-based biomaterials, especially those derived from natural polymers, have been regarded as one of the most promising scaffolds for biomedical research. These multifunctional scaffolds can exhibit high water adsorption capacity, biocompatibility, and biomechanical properties that can match native tissues. Cryogels are a special type of hydrogels in which polymers are cross-linked around ice crystals. As a result, cryogels exhibit unique physical features, including a macroporous and interconnected network, flexibility, shape-memory properties, and syringe injectability. Herein, we developed a multifunctional, i.e., antibacterial, antioxidant, and injectable cryogel by combining lignin with gelatin. The cryogel with 0.2% lignin showed a compressive modulus of 25 kPa and a compressive stress of 140 kPa at 80% strain, which is, respectively, 1.8 and 7 times higher than those of the pure gelatin cryogels. Meanwhile, such a cryogel formulation could completely recover its shape after compression up to 90% and was needle-injectable. Additionally, the lignin-co-gelatin cryogel with 0.1-0.2 lignin showed 8-10 mm of inhibition zone against the most common surgical site infection-associated pathogenic bacteria. Furthermore, lignin-co-gelatin cryogel was found to scavenge free radicals and have good cytocompatibility, and the cryogels with up to 0.2% lignin minimally activate naïve mouse bone marrow-derived dendritic cells. Overall, the current approach shows great promise for the design of bioresource-based multifunctional cryogels for a wide range of biomedical applications.

Injectable Cryogels Associate with Adipose-Derived Stem Cells for Cardiac Healing After Acute Myocardial Infarctions.

Treatment of adipose-derived stem cells (ADSCs) provides support for novel methods of conveying baseline cell protein endothelial cells to promote acute myocardial infarction in gelatin sericin (GS) lamin-coated antioxidant systems (GS@L). The ratio of fixity modules, pores, absorption, and inflammation in the range of ka (65 ka), 149 ±39.8 μm, 92.2%, 42 ± 1.38, and 29 ± 1.9 were observed in the synthesized frames for GS. Herein, ADSC-GS@L was prepared, and the relevant substance for the development of cardiac regenerative applications was stable and physically chemical. In vitro assessments of ADSC-GS@L injectable cryogels established the enhanced survival rates of the cell and improved pro- angiogenic factors as well as pro-inflammatory expression, confirming the favorable outcomes of fractional ejections, fibro-areas, and vessel densities with reduced infraction dimensions. The novel ADSC-injecting cryogel method could be useful for successful heart injury therapies during acute myocardial infarction. Additionally, the method could be useful for successful heart injury therapies during coronary heart disease.

Injectable Cryogels in Biomedicine.

Cryogels are interconnected macroporous materials that are synthesized from a monomer solution at sub-zero temperatures. Cryogels, which are used in various applications in many research areas, are frequently used in biomedicine applications due to their excellent properties, such as biocompatibility, physical resistance and sensitivity. Cryogels can also be prepared in powder, column, bead, sphere, membrane, monolithic, and injectable forms. In this review, various examples of recent developments in biomedical applications of injectable cryogels, which are currently scarce in the literature, made from synthetic and natural polymers are discussed. In the present review, several biomedical applications of injectable cryogels, such as tissue engineering, drug delivery, therapeutic, therapy, cell transplantation, and immunotherapy, are emphasized. Moreover, it aims to provide a different perspective on the studies to be conducted on injectable cryogels, which are newly emerging trend.

Hyaluronic Acid-Based Shape-Memory Cryogel Scaffolds for Focal Cartilage Defect Repair

Traumatic joint injuries can result in significant cartilage defects, which can greatly increase the risk of osteoarthritis development. Due to the limited self-healing capacity of avascular cartilage, tissue engineering approaches are required for filling defects and promoting cartilage regeneration. Current approaches utilize invasive surgical procedures for extraction and implantation of autologous chondrocytes; therefore, injectable biomaterials have gained interest to minimize the risk of infection as well as patient pain and discomfort. In this study, we engineered biomimetic, hyaluronic acid (HA)-based cryogel scaffolds that possess shape-memory properties as they contract and regain their shape after syringe injection to noninvasively fill cartilage defects. The cryogels, fabricated with HA and glycidyl methacrylate at -20°C, resulted in an elastic, macroporous, and highly interconnected network that provided a conducive microenvironment for chondrocytes to remain viable and metabolically active after injection through a syringe needle. Chondrocytes seeded within cryogels and cultured for 15 days exhibited enhanced cell proliferation, metabolism, and production of cartilage extracellular matrix glycosaminoglycans compared with HA-based hydrogels. Furthermore, immunohistochemical staining revealed production of collagen type II from chondrocyte-seeded cryogels, indicating the maintenance of cell phenotype. These results demonstrate the potential of chondrocyte-seeded, HA-based, injectable cryogel scaffolds to promote regeneration of cartilage tissue for nonsurgically invasive defect repair. Impact statement Hyaluronic acid-based shape-memory cryogels provide a conducive microenvironment for chondrocyte adhesion, proliferation, and matrix biosynthesis for use in repair of cartilage defects. Due to their sponge-like elastic properties, cryogels can fully recover their original shape back after injection while not impacting metabolism or viability of encapsulated cells. Clinically, they provide an opportunity for filling focal cartilage defects by using a single, minimally invasive injection of a cell encapsulating biocompatible three-dimensional scaffold that can return to its original structure to fit the defect geometry and enable matrix regeneration.

Injectable drug loaded gelatin based scaffolds as minimally invasive approach for drug delivery system: CNC/PAMAM nanoparticles

Drug delivery systems have been employed to increase efficiency of scaffolds designed for tissue engineering targets, especially remediating an injury. One step even further is utilizing injectable materials to eliminate the need for a second injury in a less invasive manner. The aim of this study is preparation and evaluation of preformed injectable cryogel to overcome fluid leakage of in situ formed hydrogels. For this reason, gelatin was chosen as the main component to resemble the natural extracellular matrix. When it comes to drug nomination, betamethasone sodium phosphate was chosen since severe inflammation response is one of the main reasons for prolong healing. For drug delivery part, two different carriers namely cellulose nanocrystal (CNC) and polyamidoamine (PAMAM) dendrimer were used. It was hypothesized that these nanocarriers not only provide a sustained release of the drug but also boost up mechanical properties. Therefore, prepared samples were characterized chemically, physically and biologically. Based on the results, highly branched nanocarrier (i.e. PAMAM) showed better behavior in mechanical properties (E = 14 kPa) and drug delivery (loading efficiency = 70% and sustain release in 24 h) rather than nanorod carrier (i.e. CNC) with E = 11 kPa and 65% loading efficiency. However, both nanocarriers significantly decrease the rate of degradation almost two times. It seems likely the above mentioned properties as well as the advantage of high cell viability (99% and 105% for CNC and PAMAM incorporated samples respectively) makes the prepared samples a useful alternatives for minimally invasive drug delivery as a tool for tissue engineering applications.

Autoclavable and Injectable Cryogels for Biomedical Applications.

Prior to any clinical application, terminal sterilization of biomaterials is a critical process imposed by the Food and Drug Administration. Of all the methods available for sterilization, high-pressure steam sterilization such as autoclaving is the most widely used. While autoclave sterilization minimizes pathogen contamination, it can dramatically impact both structural and biological properties of biomaterials. It has recently been reported that injectable cryogels with shape memory properties hold great promises as 3D macroporous biomimetic scaffolds for biomedical applications including tissue engineering. In this study, the impact of autoclave sterilization on properties of a series of cryogels is measured. Unlike conventional hydrogels, cryogels made of natural polymers demonstrate a strong resilience to autoclave sterilization. This process does not alter either their macrostructural or unique physical properties including syringe injectability. The scaffolds' bioactive sites are preserved and autoclaved cryogels retain their excellent cytological compatibility post-autoclaving. Furthermore, autoclaved cryogels do not trigger a notable activation of primary murine bone marrow-derived dendritic cells suggesting a minimal risk for biomaterial-induced inflammation, which is further confirmed by an in vivo histologic analysis. In summary, these results further demonstrate the huge potential of cryogels in the biomedical field and their capacity to be translated into clinical applications.

Injectable Hyaluronic Acid-co-Gelatin Cryogels for Tissue-Engineering Applications.

Polymeric scaffolds such as hydrogels can be engineered to restore, maintain, or improve impaired tissues and organs. However, most hydrogels require surgical implantation that can cause several complications such as infection and damage to adjacent tissues. Therefore, developing minimally invasive strategies is of critical importance for these purposes. Herein, we developed several injectable cryogels made out of hyaluronic acid and gelatin for tissue-engineering applications. The physicochemical properties of hyaluronic acid combined with the intrinsic cell-adhesion properties of gelatin can provide suitable physical support for the attachment, survival, and spreading of cells. The physical characteristics of pure gelatin cryogels, such as mechanics and injectability, were enhanced once copolymerized with hyaluronic acid. Reciprocally, the adhesion of 3T3 cells cultured in hyaluronic acid cryogels was enhanced when formulated with gelatin. Furthermore, cryogels had a minimal effect on bone marrow dendritic cell activation, suggesting their cytocompatibility. Finally, in vitro studies revealed that copolymerizing gelatin with hyaluronic acid did not significantly alter their respective intrinsic biological properties. These findings suggest that hyaluronic acid-co-gelatin cryogels combined the favorable inherent properties of each biopolymer, providing a mechanically robust, cell-responsive, macroporous, and injectable platform for tissue-engineering applications.

An in vitro analysis of injectable methacrylated alginate cryogels incorporated with PRP targeting minimally invasive treatment of bone nonunion

The macroporous structure and high interconnectivity of cryogels supports fluid flow, cell mobility, and angiogenesis throughout the scaffold. Additionally, a pre-formed injectable cryogel allows the potential for minimally invasive treatment of bone nonunions that would normally require surgical intervention. Such a defect is due to compromised signaling pathways by biological or biomechanical disturbance during fracture healing. The addition of powdered platelet-rich plasma (PRP) introduces soluble factors normally found in the early inflammatory stages of bone healing, allowing the bone to restart the healing process with the cryogel scaffold as a template for bone formation. A physical characterization of methacrylated alginate (MAA) and PRP-loaded cryogels was performed to determine the most suitable fabrication methods for bone applications. Further investigations were performed to assess cell proliferation and infiltration of MG-63 cells on the scaffolds, the rate of PRP elution, and the bioactive molecules being eluted at various time points. In this study, it was found that higher concentrations of PRP and MAA caused an increase in mechanical stiffness; compromising the shape memory properties of the cryogel. However, the addition of freeze-thaw cycles increased the porosity of the matrix while providing a more compressible scaffold that eluted PRP at a faster rate.

Heparin Functionalized Injectable Cryogel with Rapid Shape-Recovery Property for Neovascularization.

Cryogel based scaffolds have high porosity with interconnected macropores that may provide cell compatible microenvironment. In addition, cryogel based scaffolds can be utilized in minimally invasive surgery due to its sponge-like properties, including rapid shape recovery and injectability. Herein, we developed an injectable cryogel by conjugating heparin to gelatin as a carrier for vascular endothelial growth factor (VEGF) and fibroblasts in hindlimb ischemic disease. Our gelatin/heparin cryogel showed gelatin concentration-dependent mechanical properties, swelling ratios, interconnected porosities, and elasticities. In addition, controlled release of VEGF led to effective angiogenic responses both in vitro and in vivo. Furthermore, its sponge-like properties enabled cryogels to be applied as an injectable carrier system for in vivo cells and growth factor delivery. Our heparin functionalized injectable cryogel facilitated the angiogenic potential by facilitating neovascularization in a hindlimb ischemia model.