Journal: ACS biomaterials science & engineering
Tricalcium phosphate (TCP) ceramics are used as bone void fillers because of their bioactivity and resorbability, while their performance in bone regeneration and material resorption vary with their physical properties (e.g., the dimension of the crystal grain). Herein, three TCP ceramic bone substitutes (TCP-S, TCP-M, and TCP-L) with gradient crystal grain size (0.77 ± 0.21 μm for TCP-S, 1.21 ± 0.35 μm for TCP-M and 4.87 ± 1.90 μm for TCP-L), were evaluated in a well-established rabbit lateral condylar defect model (validated with sham) with respect to bone formation and material resorption up to 26 weeks. Surface structure-dependent bone regeneration was clearly shown after 4 weeks implantation with TCP-S having most mineralized bone (20.2 ± 3.4%), followed by TCP-M (14.0 ± 3.5%), sham (8.1 ± 4.2%), and TCP-L (6.6 ± 2.6%). Afterward, the amount of mineralized bone was similar in all the three groups, but bone marrow and material resorption varied. After 26 weeks, TCP-S induced most bone tissue formation (mineralized bone + bone marrow) (61.6 ± 7.8%) and underwent most material resorption (80.1 ± 9.0%), followed by TCP-M (42.9 ± 5.2% and 61.4 ± 8.0% respectively), TCP-L (28.3 ± 5.5% and 45.6 ± 9.7% respectively), and sham (25.7 ± 4.2%). Given the fact that the three ceramics are chemically identical, the results indicate that the surface structure (especially, the crystal grain size) of TCP ceramics can greatly tune their bone regeneration potential and the material resorption in rabbit condyle defect model, with the submicron surface structured TCP ceramic performing the best.
Bacterial adhesion to stainless steel 316L (SS316L), which is an alloy typically used in many medical devices and food processing equipment, can cause serious infections along with substantial healthcare costs. This work demonstrates that nanotextured SS316L surfaces produced by electrochemical etching effectively inhibit bacterial adhesion of both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus, but exhibit cytocompatibility and no toxicity toward mammalian cells in vitro. Additionally, the electrochemical surface modification on SS316L results in formation of superior passive layer at the surface, improving corrosion resistance. The nanotextured SS316L offers significant potential for medical applications based on the surface structure-induced reduction of bacterial adhesion without use of antibiotic or chemical modifications while providing cytocompatibility and corrosion resistance in physiological conditions.
In addition to a multitude of genetic and biochemical alterations, abnormal morphological, structural, and mechanical changes in cells and their extracellular environment are key features of tumor invasion and metastasis. Furthermore, it is now evident that mechanical cues alongside biochemical signals contribute to critical steps of cancer initiation, progression, and spread. Despite its importance, it is very challenging to study mechanics of different steps of metastasis in the clinic or even in animal models. While considerable progress has been made in developing advanced in vitro models for studying genetic and biological aspects of cancer, less attention has been paid to models that can capture both biological and mechanical factors realistically. This is mainly due to lack of appropriate models and measurement tools. After introducing the central role of mechanics in cancer metastasis, we provide an outlook on the emergence of novel in vitro assays and their combination with advanced measurement technologies to probe and recapitulate mechanics in conditions more relevant to the metastatic disease.
Fibroblast growth factor (FGF-2) is a multifunctional growth factor that has pleiotropic effects in different tissues and organs. In particular, FGF-2 has a special role in angiogenesis, an important process in development, wound healing, cell survival, and differentiation. Therefore, incorporating biological agents like FGF-2 within therapeutic biomaterials is a potential strategy to create angiogenic bioactivity for the repair of damaged tissue caused by trauma or complications that arise from age and/or disease. However, the use of growth factors as therapeutic agents can be costly and does not always bring about efficient tissue repair due to rapid clearance from the targeted site. An alternative would be a stable supramolecular nanostructure with the capacity to activate the FGF-2 receptor that can also assemble into a scaffold deliverable to tissue. We report here on peptide amphiphiles that incorporate a peptide known to activate the FGF-2 receptor and peptide domains that drive its self-assembly into supramolecular nanoribbons. These FGF2-PA nanoribbons displayed the ability to increase the proliferation and migration of the human umbilical vein endothelial cells (HUVECs) in vitro to the same extent as the native FGF-2 protein at certain concentrations. We confirmed that this activity was specific to the FGFR1 signaling pathway by tracking the phosphorylation of downstream signaling effectors such ERK1/2 and pH3. These results indicated the specificity of FGF2-PA nanoribbons in activating the FGF-2 signaling pathway and its potential application as a supramolecular scaffold that can be used in vivo as an alternative to the encapsulation and delivery of the native FGF-2 protein.
Clinical implementation of novel products for tissue engineering and regenerative medicine requires a validated sterilization method. In this study, we investigated the effect of γ-irradiation and EtO degassing on material characteristics in vitro and the effect on template remodeling of hybrid tubular constructs in a large animal model. Hybrid tubular templates were prepared from type I collagen and Vicryl polymers and sterilized by 25 kGray of γ-irradiation or EtO degassing. The in vitro characteristics were extensively studied, including tensile strength analysis and degradation studies. For in vivo evaluation, constructs were subcutaneously implanted in goats for 1 month to form vascularized neo-tissue. Macroscopic and microscopic appearances of the γ- and EtO-sterilized constructs slightly differed due to additional processing required for the COL-Vicryl-EtO constructs. Regardless of the sterilization method, incubation in urine resulted in fast degradation of the Vicryl polymer and decreased strength (<7 days). Incubation in SBF was less invasive, and strength was maintained for at least 14 days. The difference between the two sterilization methods was otherwise limited. In contrast, subcutaneous implantation showed that the effect of sterilization was considerable. A well-vascularized tube was formed in both cases, but the γ-irradiated construct showed an organized architecture of vasculature and was mechanically more comparable to the native ureter. Moreover, the γ-irradiated construct showed advanced tissue remodeling as shown by enhanced ECM production. This study shows that the effect of sterilization on tissue remodeling cannot be predicted by in vitro analyses alone. Thus, validated sterilization methods should be incorporated early in the development of tissue engineered products, and this requires both in vitro and in vivo analyses.
Pulsatile chemotherapeutic delivery profiles may provide a number advantages by maximizing the anticancer toxicity of chemotherapeutics, reducing off-target side effects, and combating adaptive resistance. While these temporally dynamic deliveries have shown some promise, they have yet to be clinically deployed from implantable hydrogels, whose localized deliveries could further enhance therapeutic outcomes. Here, several pulsatile chemotherapeutic delivery profiles were tested on melanoma cell survival in vitro and compared to constant (flatline) delivery profiles of the same integrated dose. Results indicated that pulsatile delivery profiles were more efficient at killing melanoma cells than flatline deliveries. Furthermore, results suggested that parameters like the duration of drug “on” periods (pulse width), delivery rates during those periods (pulse heights), and the number/frequency of pulses could be used to optimize delivery profiles. Optimization of pulsatile profiles at tumor sites in vivo would require hydrogel materials capable of producing a wide variety of pulsatile profiles (e.g., of different pulse heights, pulse widths, and pulse numbers). This work goes on to demonstrate that magnetically responsive, biphasic ferrogels are capable of producing pulsatile mitoxantrone delivery profiles similar to those tested in vitro. Pulse parameters such as the timing and rate of delivery during “on” periods could be remotely regulated through the use of simple, hand-held magnets. The timing of pulses was controlled simply by deciding when and for how long to magnetically stimulate. The rate of release during pulse “on” periods was a function of the magnetic stimulation frequency. These findings add to the growing evidence that pulsatile chemotherapeutic delivery profiles may be therapeutically beneficial and suggest that magnetically responsive hydrogels could provide useful tools for optimizing and clinically deploying pulsatile chemotherapeutic delivery profiles.
Cells interact with and remodel their microenvironment, degrading large extracellular matrix (ECM) proteins (e.g., fibronectin, collagens) and secreting new ECM proteins and small soluble factors (e.g., growth factors, cytokines). Synthetic mimics of the ECM have been developed as controlled cell culture platforms for use in both fundamental and applied studies. However, how cells broadly remodel these initially well-defined matrices remains poorly understood and difficult to probe. In this work, we have established methods for widely examining both large and small proteins that are secreted by cells within synthetic matrices. Specifically, human mesenchymal stem cells (hMSCs), a model primary cell type, were cultured within well-defined poly(ethylene glycol) (PEG)-peptide hydrogels, and these cell-matrix constructs were decellularized and degraded for subsequent isolation and analysis of deposited proteins. Shotgun proteomics using liquid chromatography and mass spectrometry identified a variety of proteins, including the large ECM proteins fibronectin and collagen VI. Immunostaining and confocal imaging confirmed these results and provided visualization of protein organization within the synthetic matrices. Additionally, culture medium was collected from the encapsulated hMSCs, and a Luminex assay was performed to identify secreted soluble factors, including vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), basic fibroblast growth factor (FGF-2), interleukin 8 (IL-8), and tumor necrosis factor alpha (TNF-α). Together, these methods provide a unique approach for studying dynamic reciprocity between cells and synthetic microenvironments and have the potential to provide new biological insights into cell responses during three-dimensional (3D) controlled cell culture.
The three-dimensional (3D) cultivation of intestinal cells and tissues in dynamic bioreactor systems to represent in vivo intestinal microenvironments is essential for developing regenerative medicine treatments for intestinal diseases. We have previously developed in vitro human intestinal tissue systems using a 3D porous silk scaffold system with intestinal architectures and topographical features for the adhesion, growth, and differentiation of intestinal cells under static culture conditions. In this study, we designed and fabricated a multifunctional bioreactor system that incorporates pre-epithelialized 3D silk scaffolds in a dynamic culture environment for in vitro engineering of human intestine tissues. The bioreactor system allows for control of oxygen levels in perfusion fluids (aerobic simulated intestinal fluid (SIF), microaerobic SIF, and anaerobic SIF), while ensuring control over the mechanical and chemical microenvironments present in native human intestines. The bioreactor system also enables 3D cell culture with spatial separation and cultivation of cocultured epithelial and stromal cells. Preliminary functional analysis of tissues housed in the bioreactor demonstrated that the 3D tissue constructs survived and maintained typical phenotypes of intestinal epithelium, including epithelial tight junction formation, intestinal biomarker expression, microvilli formation, and mucus secretion. The unique combination of a dynamic bioreactor and 3D intestinal constructs offers utility for engineering human intestinal tissues for the study of intestinal diseases and discovery options for new treatments.
Injectable hydrogels have gained popularity as a vehicle for the delivery of cells, growth factors, and other molecules to localize and improve their retention at the injection site, as well as for the mechanical bulking of tissues. However, there are many factors, such as viscosity, storage and loss moduli, and injection force, to consider when evaluating hydrogels for such applications. There are now numerous tools that can be used to quantitatively assess these factors, including for shear-thinning hydrogels because their properties change under mechanical load. Here, we describe relevant rheological tests and ways to measure injection force using a force sensor or a mechanical testing machine toward the evaluation of injectable hydrogels. Injectable, shear-thinning hydrogels can be used in a variety of clinical applications, and as an example we focus on methods for injection into the heart, where an understanding of injection properties and mechanical forces is imperative for consistent hydrogel delivery and retention. We discuss methods for delivery of hydrogels to mouse, rat, and pig hearts in models of myocardial infarction, and compare methods of tissue postprocessing for hydrogel preservation. Our intent is that the methods described herein can be helpful in the design and assessment of shear-thinning hydrogels for widespread biomedical applications.
The purpose of this study was to evaluate the effects of surface properties of bone implants coated with hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) on platelets and macrophages upon implant installation and compare them to grit-blasted Ti and Thermanox used as a control. Surface properties were characterized using scanning electron microscopy, profilometry, crystallography, Fourier transform infrared spectroscopy, and coating stability. For platelets, platelet adherence and morphology were assessed. For macrophages, morphology, proliferation, and polarization were evaluated. Surface characterization showed similar roughness of ∼2.5 μm for grit-blasted Ti discs, both with and without coating. Coating stability assessment showed substantial dissolution of HA and β-TCP coatings. Platelet adherence was significantly higher for grit-blasted Ti, Ti-HA, and Ti-β-TCP coatings compared to that of cell culture control Thermanox. Macrophage cultures revealed a decreased proliferation on both HA and β-TCP coated discs compared to both Thermanox and grit-blasted Ti. In contrast, secretion of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine TGF-β were marginal for grit-blasted Ti and Thermanox, while a coating-dependent increased secretion of pro- and anti-inflammatory cytokines was observed for HA and β-TCP coatings. The results demonstrated a significantly upregulated pro-inflammatory and anti-inflammatory cytokine secretion and marker gene expression of macrophages on HA and β-TCP coatings. Furthermore, HA induced an earlier M1 macrophage polarization but more M2 phenotype potency than β-TCP. In conclusion, our data showed that material surface affects the behaviors of first cell types attached to implants. Due to the demonstrated crucial roles of platelets and macrophages in bone healing and implant integration, this information will greatly aid the design of metallic implants for a higher rate of success in patients.