Oxygen-Generating Scaffolds for Cardiac Tissue Engineering Applications.

Homogeneous vascularization of implanted tissue constructs can extend to 5 weeks, during which cell death can occur due to inadequate availability of oxygen. Researchers are engineering biomaterials that generate and release oxygen in a regulated manner, in an effort to overcome this hurdle. A main limitation of the existing oxygen-generating biomaterials is the uncontrolled release of oxygen, which is ultimately detrimental to the cells. This study demonstrates the incorporation of calcium peroxide (CaO2) within a hydrophobic polymer, polycaprolactone (PCL), to yield composite scaffolds with controlled oxygen release kinetics sustained over 5 weeks. Oxygen-generating microparticles coencapsulated with cardiomyocytes in a gelatin-based hydrogel matrix can serve as model systems for cardiac tissue engineering. Specifically, the results reveal that the oxygen-generating microspheres significantly improve the scaffold mechanical strength ranging from 5 kPa to 35 kPa, have an average scaffold pore size of 50-100 µm, swelling ratios of 33.3-29.8%, and degradation with 10-49% remaining mass at the end of a 48 h accelerated enzymatic degradation. The oxygen-generating scaffolds demonstrate improvement in cell viability, proliferation, and metabolic activity compared to the negative control group when cultured under hypoxia. Additionally, the optimized oxygen-generating constructs display no cytotoxicity or apoptosis. These oxygen-generating scaffolds can possibly assist the in vivo translation of cardiac tissue constructs.

Advances in Targeting ACE2 for Developing COVID-19 Therapeutics.

Since the onset of the coronavirus pandemic in December 2019, the SARS-CoV-2 virus has accounted for over 6.3 million lives resulting in the demand to develop novel therapeutic approaches to target and treat SARS-CoV-2. Improved understanding of viral entry and infection mechanisms has led to identifying different target receptors to mitigate infection in the host. Researchers have been working on identifying and targeting potential therapeutic target receptors utilizing different candidate drugs. Angiotensin-converting enzyme-2 (ACE2) has been known to perform critical functions in maintaining healthy cardiorespiratory function. However, ACE2 also functions as the binding site for the spike protein of SARS-CoV-2, allowing the virus to enter the cells and ensue infection. Therefore, drugs targeting ACE2 receptors can be considered as therapeutic candidates. Strategies targeting the level of ACE2 expression have been investigated and compared to other potential therapeutic targets, such as TMPRSS2, RdRp, and DPP4. This mini review discusses the key therapeutic approaches that target the ACE2 receptor, which is critical to the cellular entry and propagation of the novel SARS-CoV-2. In addition, we summarize the main advantages of ACE2 targeting against alternative approaches for the treatment of COVID-19.

Oxygen generating scaffolds regenerate critical size bone defects.

Recent innovations in bone tissue engineering have introduced biomaterials that generate oxygen to substitute vasculature. This strategy provides the immediate oxygen required for tissue viability and graft maturation. Here we demonstrate a novel oxygen-generating tissue scaffold with predictable oxygen release kinetics and modular material properties. These hydrogel scaffolds were reinforced with microparticles comprised of emulsified calcium peroxide (CaO2) within polycaprolactone (PCL). The alterations of the assembled materials produced constructs within 5 ± 0.81 kPa to 34 ± 0.9 kPa in mechanical strength. The mass swelling ratios varied between 11% and 25%. Our in vitro and in vivo results revealed consistent tissue viability, metabolic activity, and osteogenic differentiation over two weeks. The optimized in vitro cell culture system remained stable at pH 8-9. The in vivo rodent models demonstrated that these scaffolds support a 70 mm3 bone volume that was comparable to the native bone and yielded over 90% regeneration in critical size cranial defects. Furthermore, the in vivo bone remodeling and vascularization results were validated by tartrate-resistant acid phosphatase (TRAP) and vascular endothelial growth factor (VEGF) staining. The promising results of this work are translatable to a repertoire of regenerative medicine applications including advancement and expansion of bone substitutes and disease models.

Eggshell Microparticle Reinforced Scaffolds for Regeneration of Critical Sized Cranial Defects.

Scaffold-based approaches for bone regeneration have been studied using a wide range of biomaterials as reinforcing agents to improve the mechanical strength and bioactivity of the 3D constructs. Eggshells are sustainable and inexpensive materials with unique biological and chemical properties to support bone differentiation. The incorporation of eggshell particles within hydrogels yields highly osteoinductive and osteoconductive scaffolds. This study reveals the effects of microparticles of whole eggshells, eggshells without a membrane, and a pristine eggshell membrane on osteogenic differentiation in protein-derived hydrogels. The in vitro studies showed that gels reinforced with eggshells with and without a membrane demonstrated comparable cellular proliferation, osteogenic gene expression, and osteogenic differentiation. Subsequently, in vivo studies were performed to implant eggshell microparticle-reinforced composite hydrogel scaffolds into critical-sized cranial defects in Sprague Dawley (SD) rats for up to 12 weeks to study bone regeneration. The in vivo results showed that the eggshell microparticle-based scaffolds supported an average bone volume of 60 mm3 and a bone density of 2000 HU 12 weeks post implantation. Furthermore, histological analyses of the explanted scaffolds showed that the eggshell microparticle-reinforced scaffolds permitted tissue infiltration and induced bone tissue formation over 12 weeks. The histology staining also indicated that these scaffolds induced significantly higher bone regeneration at 6 and 12 weeks as compared to the blank (no scaffold) and pristine gel scaffolds. The eggshell microparticle-reinforced scaffolds also supported significantly higher bone formation, remodeling, and vascularization over 6 and 12 weeks as confirmed by immunohistochemistry analysis. Collectively, our results indicated that eggshell microparticle-reinforced scaffolds facilitated significant bone regeneration in critical-sized cranial defects.

Composite Scaffolds from Gelatin and Bone Meal Powder for Tissue Engineering.

Bone tissue engineering offers versatile solutions to broaden clinical options for treating skeletal injuries. However, the variety of robust bone implants and substitutes remains largely uninvestigated. The advancements in hydrogel scaffolds composed of natural polymeric materials and osteoinductive microparticles have shown to be promising solutions in this field. In this study, gelatin methacrylate (GelMA) hydrogels containing bone meal powder (BP) particles were investigated for their osteoinductive capacity. As natural source of the bone mineral, we expect that BP improves the scaffold's ability to induce mineralization. We characterized the physical properties of GelMA hydrogels containing various BP concentrations (0, 0.5, 5, and 50 mg/mL). The in vitro cellular studies revealed enhanced mechanical performance and the potential to promote the differentiation of pre-osteoblast cells. The in vivo studies demonstrated both promising biocompatibility and biodegradation properties. Overall, the biological and physical properties of this biomaterial is tunable based on BP concentration in GelMA scaffolds. The findings of this study offer a new composite scaffold for bone tissue engineering.

Engineering calcium peroxide based oxygen generating scaffolds for tissue survival.

Oxygen supply is essential for the long-term viability and function of tissue engineered constructs in vitro and in vivo. The integration with the host blood supply as the primary source of oxygen to cells requires 4 to 5 weeks in vivo and involves neovascularization stages to support the delivery of oxygenated blood to cells. Consequently, three-dimensional (3D) encapsulated cells during this process are prone to oxygen deprivation, cellular dysfunction, damage, and hypoxia-induced necrosis. Here we demonstrate the use of calcium peroxide (CaO2) and polycaprolactone (PCL), as part of an emerging paradigm of oxygen-generating scaffolds that substitute the host oxygen supply via hydrolytic degradation. The 35-day in vitro study showed predictable oxygen release kinetics that achieved 5% to 29% dissolved oxygen with increasing CaO2 loading. As a biomaterial, the iterations of 0 mg, 40 mg, and 60 mg of CaO2 loaded scaffolds yielded modular mechanical behaviors, ranging from 5-20 kPa in compressive strength. The other controlled physiochemical features included swelling capacities of 22-33% and enzymatic degradation rates of 0.8% to 60% remaining mass. The 3D-encapsulation experiments of NIH/3T3 fibroblasts, L6 rat myoblasts, and primary cardiac fibroblasts in these scaffolds showed enhanced cell survival, proliferation, and function under hypoxia. During continuous oxygen release, the scaffolds maintained a stable tissue culture system between pH 8 to 9. The broad basis of this work supports prospects in the expansion of robust and clinically translatable tissue constructs.

Mineralized Hydrogels Induce Bone Regeneration in Critical Size Cranial Defects.

Sequential mineralization enables the integration of minerals within the 3D structure of hydrogels. Hydrolyzed collagen-based hydrogels are sequentially mineralized over 10 cycles. One cycle is defined as an incubation period in calcium chloride dihydrate followed by incubation in sodium phosphate dibasic dihydrate. Separate cycles are completed at 30-minute and 24-hour intervals. For the gels mineralized for 30 min and 24 h, the compressive moduli increases from 4.25 to 87.57 kPa and from 4.25 to 125.47 kPa, respectively, as the cycle number increases from 0 to 10. As indicated by X-ray diffraction (XRD) and Fourier transform infrared analysis (FTIR) analyses, the minerals in the scaffolds are mainly hydroxyapatite. In vitro experiments, which measure mechanical properties, porous structure, mineral content, and gene expression are performed to evaluate the physical properties and osteoinductivity of the scaffolds. Real time-quantitative polymerase chain reaction (RT-qPCR) demonstrates 4-10 fold increase in the expression of BMP-7 and osteocalcin. The in vivo subcutaneous implantation demonstrates that the scaffolds are biocompatible and 90% biodegradable. The critical size cranial defects in vivo exhibit nearly complete bone regeneration. Cycle 10 hydrogels mineralized for 24 h have a volume of 59.86 mm3 and a density of 1946.45 HU. These results demonstrate the suitability of sequentially mineralized hydrogel scaffolds for bone repair and regeneration.

Mineralized paper scaffolds for bone tissue engineering.

Mineralized polymer scaffolds have proven to be effective biomaterials for inducing osteoinductivity in bone tissue engineering. Sequential mineralization is a promising technique for depositing minerals in three-dimensional (3D) scaffolds. Paper, which is made of cellulose fibers, can be used as a tissue scaffold due to its highly porous structure and flexibility, as well as its excellent ability to wick fluids and support the growth of bone cells. In this study, paper-based, mineralized scaffolds were fabricated using sequential mineralization. We conducted experiments with two groups of scaffolds based on different incubation times in the mineralization solutions (30 min and 24 h). Ten cycles of mineralization were performed for each group. We found that the mineral content increased as the cycle number increased and that the 24-h group scaffolds consistently had more mineralization than did the 30-min group scaffolds when measured at the same cycle number. A quantitative reverse transcription-polymerase chain reaction was performed for two osteogenic differentiation markers of the preosteoblasts that were grown on the mineralized paper scaffolds. The gene expression results for bone-specific markers revealed that the mineralized scaffolds were osteoinductive. Subcutaneous implantation of the scaffolds in rats demonstrated favorable biocompatibility, high vascularization, and non-immunogenicity in vivo. The overall results suggest that the sequentially mineralized paper scaffolds are promising materials for use in bone tissue engineering.

Synthesis and characterization of photocrosslinkable albumin-based hydrogels for biomedical applications.

Protein-based biomaterials are widely used to generate three-dimensional (3D) scaffolds for tissue regeneration as well as compact delivery systems for drugs, genes, and peptides. Specifically, albumin-based biomaterials are of particular interest for their ability to facilitate controlled delivery of drugs and other therapeutic agents. These hydrogels possess non-toxic and non-immunogenic properties that are desired in tissue engineering scaffolds. This work employs a rapid ultraviolet (UV) light induced crosslinking to fabricate bovine serum albumin (BSA) hydrogels. Using four different conditions, the BSA hydrogel properties were modulated based on the extent of glycidyl methacrylate modification in each polymer. The highly tunable mechanical behavior of the material was determined through compression tests which yielded a range of material strengths from 4.4 ± 1.5 to 122 ± 7.4 kPa. Pore size measurements also varied from 7.7 ± 1.7 to 23.5 ± 6.6 µm in the photocrosslinked gels. The physical properties of materials such as swelling and degradation were also characterized. In further evaluation, 3D scaffolds were used in cell encapsulation and in vivo implantation studies. The biocompatibility and degradability of the material demonstrated effective integration with the native tissue environment. These modifiable chemical and mechanical properties allow BSA hydrogels to be fine-tuned to a plethora of biomedical applications including regenerative medicine, in vitro cancer study models, and wound healing approaches.

Hydroxyapatite-Incorporated Composite Gels Improve Mechanical Properties and Bioactivity of Bone Scaffolds.

Reinforcing polymeric scaffolds with micro/nanoparticles improve their mechanical properties and render them bioactive. In this study, hydroxyapatite (HA) is incorporated into 5% (w/v) gelatin methacrylate (GelMA) hydrogels at 1, 5, and 20 mg mL-1 concentrations. The material properties of these composite gels are characterized through swelling, degradation, and compression tests. Using 3D cell encapsulation, the cytocompatibility and osteogenic differentiation of preosteoblasts are evaluated to assess the biological properties of the composite scaffolds. The in vitro assays demonstrate increasing cell proliferation and metabolic activity over the course of 14 d in culture. Furthermore, the scaffolds support osteogenic differentiation of the microencapsulated preosteoblasts. For the in vivo study, the composite scaffolds are subcutaneously implanted in rats for 14 d. The histological staining of the explanted in vivo samples exhibits the functional advantages of the scaffold's biocompatibility, biodegradability, and integration into the existing host tissue. This work demonstrates the enhanced mechanical and biological performance of HA-gelatin composite hydrogels for bone tissue engineering applications.

Nanophosphor-Based Contrast Agents for Spectral X-ray Imaging.

Lanthanide-based nanophosphors (NPhs) are herein developed as contrast agents for spectral X-ray imaging, highlighting the chemical, macromolecular and structural differences derived from ligand exchange on computed tomography (CT) and solvent dispersibility. Taking advantage of the ability of spectral X-ray imaging with photon-counting detectors to perform image acquisition, analysis, and processing at different energy windows (bins), enhanced signal of our K-edge materials was derived, improving sensitivity of CT imaging, and differentiation between water, tumor-mimic phantoms, and contrast materials. Our results indicate that the most effective of our oleic acid-stabilized K-edge nanoparticles can achieve 2-4x higher contrast than the examined iodinated molecules, making them suitable for deep tissue imaging of tissues or tumors. On the other hand, ligand exchange yielding poly(acrylic acid)-stabilized K-edge nanoparticles allows for high dispersibility and homogeneity in water, but with a lower contrast due to the high density of the polymer grafted, unless further engineering is probed. This is the first well-defined study that manages to correlate NPh grafting density with CT numbers and water dispersibility, laying the groundwork for the development of the next generation CT-guided diagnostic and/or theranostic materials.

Eggshell particle-reinforced hydrogels for bone tissue engineering: an orthogonal approach.

Hydrogel-based biomimetic scaffolds have generated broad interest due to their tunable physical, chemical, and biological properties for bone tissue engineering applications. We fabricated eggshell microparticle (ESP) reinforced gelatin-based hydrogels to obtain mechanically stable and biologically active three-dimensional (3D) constructs that can differentiate pre-mature cells into osteoblasts. Physical properties including swelling ratio, degradation, and mechanical properties of the composite hydrogels were investigated. Pre-osteoblasts were encapsulated within the ESP-reinforced hydrogels to study their differentiation and evaluate mineral deposition by these cells. The ESP-reinforced gels were then subcutaneously implanted in a rat model to determine their biocompatibility and degradation behaviors. The composite hydrogels have shown outstanding tunability in physical and biological properties holding substantial promise for engineering mineralized tissues (e.g. bone, cartilage, tooth, and tendon). These 3D scaffolds enabled the differentiation of pre-osteoblasts without the use of specialized osteogenic growth medium. The ESP-reinforced gels exhibited significant enhancement in mineralization by pre-osteoblasts. These behaviors are positively correlated with increasing concentrations of ESP. Findings suggest that our novel composite hydrogel exhibits superior mechanical properties and indicates a favorable in vivo response by subcutaneous implantation in a rat model.

Synthesis and characterization of photocrosslinkable hydrogels from bovine skin gelatin.

Hydrogels that mimic native tissues chemically and structurally have been increasingly sought for a wide variety of tissue engineering applications. Gelatin can be naturally derived from different sources and functionalized to fabricate hydrogels that exhibit high cytocompatibility and favorable biodegradable properties. The amino groups on the gelatin backbone can be substituted by adding varying proportions of methacrylic anhydride (MAA) to create biomimetic hydrogels which can be used as tissue engineering scaffolds. Gelatin from different sources yields hydrogels with distinctive physical, chemical, and biological properties. In this work, gelatin from bovine skin was used to fabricate hydrogels with varying degrees of crosslinking content using 1, 4, 7, and 10 mL MAA. The material properties of these hydrogels were characterized. The cytocompatibility of the gelatin-based hydrogels was studied using L6 rat myoblasts. The hydrogels from bovine skin gelatin exhibit mechanical properties that are conducive for applications which require substrates to propagate cell growth, migration, and proliferation rapidly. These hydrogels exhibit exceptional tunability behavior which makes them useful and applicable to culture different cell types.